Volume 171 Number 1 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board WALLIS H. CLARK, JR., Bodega Marine Laboratory, GEORGE D. PAPPAS, University of Illinois at Chicago University of California SIDNEY K. PIRCE, University of Maryland C. K. GOVIND, Scarborough Campus, University of Toronto LlONEL L REBHUN, University of Virginia JUDITH P. GRASSLE, Marine Biological Laboratory HERBERT SCHUEL, State University of New York at Buffalo MICHAEL J. GREENBERG, C. V. Whitney Marine Laboratory, University of Florida V '*GINIA L. SCOFIELD, University of California at Los Angeles School of Medicine MAUREEN R. HANSON, Cornell University LAWRENCE B. SLOBODKIN, State University of New RONALD R. HOY, Cornell University York at Stony Brook LIONEL JAFFE, Marine Biological Laboratory JOHN D. STRANDBERG, Johns Hopkins University HOLGER W. JANNASCH, Woods Hole Oceanographic JOHN M. TEAL, Woods Hole Oceanographic Institution Institution WILLIAM R. JEFFERY, University of Texas at Austin DONALD P. WOLF, University of Texas Health Sciences Center CHARLOTTE P. MANGUM, The College of William and Mary SEYMOUR ZIGMAN, University of Rochester Editor: CHARLES B. METZ, University of Miami AUGUST, 1986 Printed and Issued by LANCASTER PRESS, Inc. PRINCE & LEMON STS. LANCASTER, PA CONTENTS No. 1, AUGUST 1986 Annual Report of the Marine Biological Laboratory 1 INVITED REVIEW MARKL, JURGEN Evolution and function of structurally diverse subunits in the respiratory protein hemocyanin from arthropods 90 DEVELOPMENT AND REPRODUCTION MARTIN, VICKI J., AND WILLIAM E. ARCHER A scanning electron microscopic study of embryonic development of a marine hydrozoan 116 SAFRANEK, Louis, CLAYTON R. SQUIRE, AND CARROLL M. WILLIAMS Precocious termination of diapause in neck- and abdomen-ligated pupal preparations of the tobacco hornworm, Manduca sexta 126 ECOLOGY AND EVOLUTION HAIRSTON, NELSON G., JR., AND EMILY J. OLDS Partial photoperiodic control of diapause in three populations of the freshwater copepod Diaptomus sanguineus 135 LEVIN, LISA A. Effects of enrichment on reproduction in the opportunistic polychaete Streblospio benedict i (Webster): a mesocosm study 143 MCFADDEN, CATHERINE S. Laboratory evidence for a size refuge in competitive interactions between the hydroids Hydractinia echinala (Hemming) and Podocoryne carnea (Sars) " '. 161 GENERAL BIOLOGY ANDERSON, O. ROGER, NEIL R. SWANBERG, J. L. LINDSEY, AND PAUL BENNETT Functional morphology and species characteristics of a large, solitary radiolarian Physematium muelleri 175 RUPPERT, EDWARD E., AND ELIZABETH J. BALSER Nephridia in the larvae of hemichordates and echinoderms 188 TELFORD, MALCOLM, AND RICH Mooi Resource partitioning by sand dollars in carbonate and siliceous sedi- ments: evidence from podial and particle dimensions 197 PHYSIOLOGY CARIELLO, L., L. ZANETTI, A. SPAGNUOLO. AND L. NELSON Effects of opioids and antagonists on the rate of sea urchin sperm pro- gressive motility 208 CARLSSON, KARL-HEINZ, AND GERD GADE Metabolic adaptation of the horseshoe crab, Limulus polyphemus, during exercise and environmental hypoxia and subsequent recovery 217 CONTENTS HEBRANK, MARY REIDY, AND JOHN H. HEBRANK The mechanics of fish skin: lack of an "external tendon" role in two teleosts 236 MANGUM, CHARLOTTE P., AND Louis E. BURNETT, JR. The CO 2 sensitivity of the hemocyanins and its relationship to Cl~ sen- sitivity 248 MATTSON, MARK P., AND EUGENE SPAZIANI Evidence for ecdysteroid feedback on release of molt-inhibiting hormone from crab eyestalk ganglia 264 POWELL, M. A., AND G. N. SOMERO Adaptations to sulfide by hydrothermal vent animals: sites and mech- anisms of detoxification and metabolism 274 No. 2, OCTOBER 1986 INVITED REVIEW SCHULTZ, GILBERT A. Molecular biology of the early mouse embryo 29 1 BEHAVIOR BARLOW, ROBERTS., JR., MAUREEN K. POWERS, HEIDI HOWARD, AND LEON- ARD KASS Migration of Limulus for mating: relation to lunar phase, tide height, and sunlight 310 CELL BIOLOGY CANNON, GAIL W., MASAKAZU TSUCHIYA, DAN RITTSCHOF, AND JOSEPH BONAVENTURA Magnesium dependence of endotoxin-induced degranulation of Limulus amebocytes 330 COHEN, WILLIAM D. Association of centrioles with the marginal band in skate erythrocytes 338 KITAMURA, AKIO Attachment of mating reactive Paramecium to polystyrene surfaces: IV. Comparison of the adhesiveness among six species of the genus Para- mecium 350 NAGAI, MAKOTO, NORIKO OSHIMA, AND RYOZO FUJII A comparative study of melanin-concentrating hormone (MCH) action on teleost melanophores 360 DEVELOPMENT AND REPRODUCTION AMANO, SHIGETOYO Larval release in response to a light signal by the intertidal sponge Hal- ochondria panicea 371 ECOLOGY AND EVOLUTION BLACKSTONE, NEIL W. Variation of cheliped allometry in a hermit crab: the role of introduced periwinkle shells 379 YOUNG, CRAIG M., PAUL G. GREENWOOD, AND CYNTHIA J. POWELL The ecological role of defensive secretions in the intertidal pulmonate Onchidella boreal is . 391 CONTENTS GENERAL BIOLOGY CLARK, STEVEN D., AND CLAYTON B. COOK Inhibition of nematocyst discharge during feeding in the colonial hydroid Halocordvle disticha (= Pennaria tiarelld): the role of previous prey- killing ." 405 PARASIT^ { LEE, C. G. L., AND Y. K. IP Effect of host fasting and subse efeeding on the glycogen metab- olizing enzymes in Hymenole dnuta (Cestoda) 417 BIOLOGY CROLL, ROGER P., AND RAYMU, /. S. Lo Distribution of serotonin-like immunoreactivity in the central nervous system of the periwinkle, Littorina littorea (Gastropoda, Prosobranchia, Mesogastropoda) 426 EMERY, SCOTT H., AND ANDREW SZCZEPANSKI Gill dimensions in pelagic elasmobranch fishes 44 1 O'BRIEN, JACK J., DONALD L. MYKLES, AND DOROTHY M. SKINNER Cold-induced apolysis in anecdysial brachyurans 450 SHORT REPORT VAN DOVER, CINDY LEE, AND ROBERT W. LICHTWARDT A new trichomycete commensal with a galatheid squat lobster from deep-sea hydrothermal vents 46 1 ABSTRACTS ABSTRACTS OF PAPERS PRESENTED AT THE GENERAL SCIENTIFIC MEETINGS OF THE MARINE BIOLOGICAL LABORATORY 469 Cell motility and cytoskeleton 469 Developmental biology 471 Ecology 477 Fertilization 485 Neurobiology 489 Physiology 500 No. 3, DECEMBER 1986 GROSS, PAUL R. Alberto Monroy (1913-1986) 507 INVITED REVIEW MONROY, ALBERTO A centennial debt of developmental biology to the sea urchin 509 DEVELOPMENT AND REPRODUCTION KAWAMURA, KAZUO, AND MITSUAKI NAKAUCHI Development of spatial organization in palleal buds of the compound ascidian, Symplegma reptans 520 CONTENTS ECOLOGY AND EVOLUTION BOULDING, ELIZABETH GRACE, AND MICHAEL LABARBERA Fatigue damage: repeated loading enables crabs to open larger bivalves 538 CONNOR, VALERIE M. The use of mucous trails by intertidal limpets to enhance food resources 548 LA BARRE, STEPHANE C, JOHN C. COLL, AND PAUL W. SAMMARCO Defensive strategies of soft corals (Coelenterata: Octocorallia) of the Great Barrier Reef. II. The relationship between toxicity and feeding deterrence 565 LOWELL, RICHARD B. Crab predation on limpets: predator behavior and defensive features of the shell morphology of the prey 577 PETERSON, CHARLES H., AND STEPHEN R. FEGLEY Seasonal allocation of resources to growth of shell, soma, and gonads in Mercenaria mercenaria 597 SMILEY, SCOTT Metamorphosis of Stichopus californicus (Echinodermata: Holothuro- idea) and its phylogenetic implications 611 GENERAL BIOLOGY COON, STEVEN L., AND DALE B. BONAR Norepinephrine and dopamine content of larvae and spat of the Pacific oyster, Crassostrea gigas 632 ROE, PAMELA Parthenogenesis in Carcinonemertes spp. (Nemertea: Hoplonemertea) 640 PHYSIOLOGY BELLON-HUMBERT, CHANTAL, FRANCOIS VAN HERP, AND HUGO SHOONE- VELD Immunocytochemical study of the red pigment concentrating material in the eyestalk of the prawn Palaemon serratus Pennant using rabbit antisera against the insect adipokinetic hormone 647 MATTISSON, ARTUR, AND RAGNAR FANGE The cellular structure of lymphomyeloid tissues in Chimaera monstrosa (Pisces, Holocephali) 660 MICHIBATA, H., T. TERADA, N. ANADA, K. YAMAKAWA, AND T. NUMAKUNAI The accumulation and distribution of vanadium, iron, and manganese in some solitary ascidians 672 O'DELL, STEVEN J., AND GROVER C. STEPHENS Uptake of amino acids by Pareurythoe californica: substrate interaction modifies net influx from the environment 682 SHORT REPORT STRATHMANN, RICHARD R., AND LARRY R. MCEDWARD Cyphonautes' ciliary sieve breaks a biological rule of inference 694 INDEX to VOLUME 171 701 THE BIOLOGICAL BULLETIN THE BIOLOGICAL BULLETIN is published six times a year by the Marine Biological Laboratory, MBL Street, Woods Hole, Massachusetts 02543. Subscriptions and similar matter should be addressed to THE BIOLOGICAL BULLETIN, Marine Biological Laboratory, Woods Hole, Massachusetts. Single numbers, $20.00. Subscription per volume (three issues), $50.00 ($100.00 per year for six issues). Communications relative to manuscripts should be sent to Dr. Charles B. Metz, Editor, or Pamela Clapp, Assistant Editor, at the Marine Biological Laboratory, Woods Hole, Massachusetts 02543. POSTMASTER: Send address changes to THE BIOLOGICAL BULLETIN, Marine Biological Laboratory, Woods Hole, MA 02543. Copyright 1986, by the Marine Biological Laboratory Second-class postage paid at Woods Hole, MA, and additional mailing offices. 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HANSON, Cornell University LAWRENCE B. SLOBODKJN, State University of New RONALD R. HOY, Cornell University York at Stony Brook LIONEL JAFFE, Marine Biological Laboratory JOHN D. STRANDBERG, Johns Hopkins University HOLGER W. JANNASCH, Woods Hole Oceanographic JOHN M. TEAL, Woods Hole Oceanographic Institution Institution WILLIAM R. JEFFERY, University of Texas at Austin DONALD P. WOLF, University of Texas Health Sciences Center CHARLOTTE P. MANGUM, The College of William and Mary SEYMOUR ZIGMAN, University of Rochester Editor: CHARLES B. METZ, University of Miami AUGUST, 1986 Printed and Issued by LANCASTER PRESS, Inc. PRINCE & LEMON STS. LANCASTER, PA. 111 The BIOLOGICAL BULLETIN is issued six times a year at the Lancaster Press, Inc., Prince and Lemon Streets, Lancaster, Penn- sylvania. Subscriptions and similar matter should be addressed to The Biological Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts. Single numbers, $20.00. Subscription per volume (three issues), $50.00 ($100.00 per year for six issues). Communications relative to manuscripts should be sent to Dr. Charles B. Metz, Editor, or Pamela Clapp, Assistant Editor, Marine Biological Laboratory, Woods Hole, Massachusetts 02543. THE BIOLOGICAL BULLETIN (ISSN 0006-3185) POSTMASTER: Send address changes to THE BIOLOGICAL BULLETIN, Marine Biological Laboratory, Woods Hole, MA 02543. Second-class postage paid at Woods Hole, MA, and additional mailing offices. LANCASTER PRESS, INC., LANCASTER, PA IV THE MARINE BIOLOGICAL LABORATORY EIGHTY-EIGHTH REPORT, FOR THE YEAR 1985 NINETY-EIGHTH YEAR I. TRUSTEES AND STANDING COMMITTEES 1 II. MEMBERS OF THE CORPORATION 6 1 . LIFE MEMBERS 6 2. REGULAR MEMBERS 7 3. ASSOCIATE MEMBERS 26 III. CERTIFICATE OF ORGANIZATION 30 IV. ARTICLES OF AMENDMENT 31 V. BYLAWS 31 VI. REPORT OF THE DIRECTOR 36 VII. REPORT OF THE TREASURER 40 VIII. REPORT OF THE LIBRARIAN 50 IX. EDUCATIONAL PROGRAMS 50 1. SUMMER 50 2. SPRING 60 3. SHORT COURSES 62 X. RESEARCH AND TRAINING PROGRAMS 63 1 . SUMMER 63 2. YEAR-ROUND 72 XI. HONORS 79 XII. INSTITUTIONS REPRESENTED 83 XIII. LABORATORY SUPPORT STAFF . 87 I. TRUSTEES Including Action of the 1985 Annual Meeting OFFICERS PROSSER GIFFORD, Chairman of the Board of Trustees, Woodrow Wilson International Center for Scholars, Smithsonian Building, Washington, DC 20560 DENIS M. ROBINSON, Honorary Chairman of the Board of Trustees, High Voltage Engineering Corporation, Burlington, Massachusetts 01830 DAVID L. CURRIER, Treasurer, PO Box 2476, Vineyard Haven, Massachusetts 02568 PAUL R. GROSS, President of the Corporation and Director of the Laboratory, Marine Biological Laboratory, Woods Hole, Massachusetts 02543 DAVID D. POTTER, Clerk, Harvard Medical School, Cambridge, Massachusetts 02138 Copyright 1986, by the Marine Biological Laboratory Library of Congress Card No. A38-518 (ISSN 0006-3185) 2 MARINE BIOLOGICAL LABORATORY EMERITI JOHN B. BUCK, National Institutes of Health AURIN CHASE, Princeton University SEYMOUR S. COHEN, Woods Hole, Massachusetts ARTHUR L. COL WIN, University of Miami LAURA HUNTER COL WIN, University of Miami D. EUGENE COPELAND, Marine Biological Laboratory SEARS CROWELL, Indiana University ALEXANDER T. DAIGNAULT, Boston, Massachusetts TERU HAYASHI, Miami, Florida HOPE HIBBARD, Oberlin College LEWIS KLEINHOLZ, Reed College MAURICE KRAHL, Tucson, Arizona DOUGLAS MARSLAND, Cockysville, Maryland CHARLES B. METZ, University of Miami HAROLD H. PLOUGH, Amherst, Massachusetts (Deceased 1 1/85) C. LADD PROSSER, University of Illinois JOHN S. RANKIN, Ashford, Connecticut MERYL ROSE, Waquoit, Massachusetts GEORGE T. SCOTT, Woods Hole, Massachusetts MARY SEARS, Woods Hole, Massachusetts CARL C. SPEIDEL, University of Virginia (no mailings) ALBERT SZENT-GYORGYI, Marine Biological Laboratory W. RANDOLPH TAYLOR, University of Michigan GEORGE WALD, Woods Hole, Massachusetts CLASS OF 1989 GARLAND E. ALLEN, Washington University PETER B. ARMSTRONG, University of California, Davis ROBERT W. ASHTON, Gaston Snow Beekman and Bogue JELLE ATEMA, Marine Biological Laboratory HARLYN O. HALVORSON, Brandeis University JOHN G. HILDEBRAND, University of Arizona THOMAS J. HYNES, JR., Meredith and Grew, Inc. ROBERT MAINER, The Boston Company BIRGIT ROSE, University of Miami CLASS OF 1988 CLAY ARMSTRONG, University of Pennsylvania JOEL P. DAVIS, Seapuit, Inc. ELLEN R. GRASS, The Grass Foundation JUDITH GRASSLE, Marine Biological Laboratory HOLGER JANNASCH, Woods Hole Oceanographic Institution GEORGE M. LANGFORD, University of North Carolina ANDREW SZENT-GYORGYI, Brandeis University KENSAL VAN HOLDE, Oregon State University RICHARD YOUNG, Wellesley Hills, Massachusetts CLASS OF 1987 EDWARD A. ADELBERG, Yale University JAMES M. CLARK, Shearson/American Express HAROLD GAINER, National Institutes of Health TRUSTEES AND STANDING COMMITTEES WILLIAM GOLDEN, New York, New York HANS KORNBERG, University of Cambridge LASZLO LORAND, Northwestern University CAROL REINISCH, Tufts University HOWARD A. SCHNEIDERMAN, Monsanto Company SHELDON J. SEGAL, The Rockefeller Foundation CLASS OF 1986 GEORGE H. A. CLOWES, JR., Cancer Research Institute GERALD FISCHBACH, Washington University JOHN E. HOBBIE, Ecosystems Center EDWARD A. KRAVITZ, Harvard Medical School RODOLFO LLINAS, New York University D. THOMAS TRIGG, Wellesley, Massachusetts GERALD WEISSMANN, New York University NANCY SABIN WEXLER, New York, New York J. RICHARD WHITTAKER, Marine Biological Laboratory STANDING COMMITTEES EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES PROSSER GIFFORD* PAUL R. GROSS* DAVID L. CURRIER* JUDITH GRASSLE, 1988 JOHN G. HILDEBRAND, 1988 CAROL REINISCH, Chairman DANIEL ALKON EDWARD JASKUN JOHN HOBBIE, 1986 EDWARD KRAVITZ, 1986 ANDREW SZENT-GYORGYI, 1988 KENSAL VAN HOLDE, 1987 ANIMAL CARE COMMITTEE ROXANNA SMOLOWITZ RAYMOND STEPHENS J. RICHARD WHITTAKER BUILDINGS AND GROUNDS COMMITTEE KENYON TWEEDELL, Chairman LAWRENCE B. COHEN A. FARMANFARMAIAN ALAN FEIN DANIEL GILBERT CIFFORD HARDING, JR. FERENC HAROSI FRANCIS HOSKIN DONALD B. LEHY* THOMAS MEEDEL PHILIP PERSON ROBERT D. PRUSCH THOMAS REESE EVELYN SPIEGEL CAPITAL DEVELOPMENT COMMITTEE RICHARD W. YOUNG, Chairman PROSSER GIFFORD* WILLIAM T. GOLDEN HARLYN O. HALVORSON ANN Moss, Chairman EDWARD ENOS WILLIAM EVANS JOHN HELFRICH EMPLOYEE RELATIONS COMMITTEE ROGER HOBBS GEORGE LILES JOHN MACLEOD 4 MARINE BIOLOGICAL LABORATORY FELLOWSHIPS COMMITTEE THORU PEDERSON, Chairman EDUARDO MACAGNO JUDITH GRASSLE CAROL REINISCH JOAN HOWARD* J. RICHARD WHITTAKER GEORGE LANGFORD FINANCIAL POLICY AND PLANNING COMMITTEE GEORGE H. A. CLOWES, JR., Chairman ROBERT MAINER ROBERT ASHTON W. NICHOLAS THORNDIKE DAVID L. CURRIER* J. RICHARD WHITTAKER THOMAS HYNES HOUSING, FOOD SERVICE AND DAY CARE COMMITTEE JELLE ATEMA, Chairman STEPHEN HIGHSTEIN DANIEL ALKON DARCY KELLEY NINA S. ALLEN THOMAS REESE ROBERT B. BARLOW, JR. BRIAN SALZBERG GAIL BURD HOMER P. SMITH* RONALD CALABRESE SUSAN SZUTS MONA GROSS INSTITUTIONAL BIOSAFETY RAYMOND STEPHENS, Chairman JOSEPH MARTYNA PAUL DE WEER ANDREW MATTOX* PAUL ENGLUND BARRY T. O'NEIL* HARLYN O. HALVORSON AL SENFT DONALD LEHY INSTRUCTION COMMITTEE JUDITH GRASSLE, Chairman BRUCE PETERSON RANDALL S. ALBERTS JOAN RUDERMAN ALAN FEIN BRIAN SALZBERG HARLYN O. HALVORSON HERBERT SCHUEL JOHN G. HILDEBRAND ANDREW SZENT-GYORGYI JOAN HOWARD* INVESTMENT COMMITTEE THOMAS TRIGG, Chairman WILLIAM T. GOLDEN DAVID L. CURRIER* MAURICE LAZARUS PROSSER GIFFORD* ROBERT MAINER LIBRARY JOINT MANAGEMENT COMMITTEE PAUL R. GROSS, Chairman JOSEPH KEIBALA EDWARD A. ADELBERG JOHN SPEER GEORGE GRICE JOHN STEELE TRUSTEES AND STANDING COMMITTEES LIBRARY JOINT USERS COMMITTEE EDWARD A. ADELBERG, Chairman LIONEL JAFFE GARLAND ALLEN LAURENCE P. MADIN WILFRED BRYAN JOHN SCHLEE A. FARMANFARMAIAN FREDERIC SERCHUK JANE FESSENDEN* OLIVER ZAFIRIOU MARINE RESOURCES COMMITTEE SEARS CROWELL, Chairman JACK LEVIN CARL J. BERG, JR. RODOLFO LLINAS JUNE HARRIGAN ANNE F. O'MELIA WILLIAM JEFFERY JOHN S. RANKJN IZJA LEDERHENDLER JOHN VALOIS* Louis LEIBOVITZ JONATHAN WITTENBERG RADIATION SAFETY COMMITTEE PAUL DE WEER, Chairman ANTHONY LIUZZI RICHARD L. CHAPPELL ANDREW MATTOX* SHERWIN COOPERSTEIN HARRIS RIPPS DANIEL GROSCH RAYMOND STEPHENS Louis KERR* WALTER VINCENT RESEARCH SERVICES COMMITTEE RAYMOND STEPHENS, Chairman BRYAN NOE ROBERT B. BARLOW, JR. BARRY C/NEIL* ROBERT GOLDMAN BRUCE PETERSON JOHN G. HlLDEBRAND BlRGIT ROSE SAMUEL S. KOIDE JOEL ROSENBAUM RAYMOND LASEK SIDNEY TAMM RESEARCH SPACE COMMITTEE J. RICHARD WHITTAKER, Chairman EDUARDO MACAGNO CLAY ARMSTRONG JERRY MELILLO ROBERT GOLDMAN JOSEPH SANGER JOAN HOWARD* ROGER SLOBODA DAVID LANDOWNE EVELYN SPIEGEL HANS LAUFER STEVEN TREISTMAN RODOLFO LLINAS IVAN VALIELA LASZLO LORAND SAFETY COMMITTEE JOHN HOBBIE, Chairman DONALD LEHY DANIEL ALKON ANDREW MATTOX* D. EUGENE COPELAND BARRY T. O'NEIL EDWARD ENOS EDWARD SADOWSKI ALAN FEIN RAYMOND STEPHENS Louis KERR PAUL STEUDLER ALAN KUZIRIAN FREDERICK THRASHER ex officio 6 MARINE BIOLOGICAL LABORATORY II. MEMBERS OF THE CORPORATION Including Action of the 1985 Annual Meeting LIFE MEMBERS ABBOTT, MARIE, 259 High St., RFD 2, Coventry, CT 06238 ADOLPH, EDWARD F., University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642 BEAMS, HAROLD W., Department of Zoology, University of Iowa, Iowa City, IA 53342 BEHRE, ELLINOR, Black Mountain, NC 2871 1 BERNHEIMER, ALAN W., New York University, College of Medicine, Charlottesville, VA 22908 BERTHOLF, LLOYD M., Westminster Village #21 14, 2025 E. Lincoln St., Bloomington, IL 61701 BISHOP, DAVID W., Department of Physiology, Medical College of Ohio, C.S. 10008, Toledo, OH 43699 BOLD, HAROLD C., Department of Botany, University of Texas, Austin, TX 78712 BRIDGMAN, A. JOSEPHINE, 715 Kirk Rd., Decatur, GA 30030 BURBANCK, MADELINE P., Box 15134, Atlanta, GA 30333 BURBANCK, WILLIAM D., Box 1 5 1 34, Atlanta, GA 30333 CARPENTER, RUSSELL L., 60 Lake St., Winchester, MA 01890 CHASE, AURIN, Professor of Biology Emeritus, Princeton University, Princeton, NJ 08540 CLARKE, GEORGE L., Address unknown COHEN, SEYMOUR S., 10 Carrot Hill Rd., Woods Hole, MA 02543 COLWIN, ARTHUR, 320 Woodcrest Rd., Key Biscayne, FL 33149 COLWIN, LAURA HUNTER, 320 Woodcrest, Key Biscayne, FT 33149 COPELAND, D. E., 41 Fern Lane, Woods Hole, MA 02543 COSTELLO, HELEN M., 507 Monroe St., Chapel Hill, NC 27514 CROUSE, HELEN, Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306 DILLER, IRENE C., Rydal Park Apartment 660, Rydal, PA 19046 DILLER, WILLIAM F., Rydal Park Apartment 660, Rydal, PA 19046 (deceased 2/86) ELLIOTT, ALFRED M., 428 Lely Palm Ext., Naples, FL 33962-8903 FERGUSON, JAMES K. W., 56 Clarkehaven St., Thornhill, Ontario L4J 2B4, Canada FISHER, J. MANERY, Department of Biochemistry, University of Toronto, Ontario, Canada M5S 1A8 FRIES, ERIK F. B., 41 High Street, Woods Hole, MA 02543 OILMAN, LAUREN C., Department of Biology, University of Miami, PO Box 24918, Coral Gables, FL 33 124 GREEN, JAMES W., 409 Grand Ave., Highland Park, NJ 08904 HAMBURGER, VIKTOR, Professor Emeritus, Washington University, St. Louis, MO 63130 HAMILTON, HOWARD L., Department of Biology, University of Virginia, Charlottesville, VA 22901 HIBBARD, HOPE, 143 East College St., Apt. 309, Oberlin, OH 44074 HISAW, F. L., 5925 SW Plymouth Drive, Corvallis, OR 97330 HOLLAENDER, ALEXANDER, Council for Research Planning, 1717 Massachusetts Ave., NW, Washington, DC 20036 HUMES, ARTHUR, Marine Biological Laboratory, Woods Hole, MA 02543 JOHNSON, FRANK H., Department of Biology, Princeton University, Princeton, NJ 08540 KAAN, HELEN W., Royal Megansett Nursing Home, Room 205, PO Box 408, N. Falmouth, MA 02556 KAHLER, ROBERT, PO Box 423, Woods Hole, MA 02543 (deceased 10/85) KILLE, FRANK R., 1 1 1 1 S. Lakemont Ave. #444, Winter Park, FL 32792 KINGSBURY, JOHN M., Department of Botany, Cornell University, Ithaca, NY 14853 KLEINHOLZ, LEWIS, Department of Biology, Reed College, Portland, OR 97202 LAUFFER, MAX A., Department of Biophysics, University of Pittsburgh, Pittsburgh, PA 15260 LEFEVRE, PAUL G., 15 Agassiz Rd., Woods Hole, MA 02543 LEVINE, RACHMIEL, 2024 Canyon Rd., Arcadia, CA 91006 LOCHHEAD, JOHN H., 49 Woodlawn Rd., London SW6 6PS, England, U. K. MEMBERS OF THE CORPORATION LYNN, W. GARDNER, Department of Biology, Catholic University of America, Washington, DC 200 17 MAGRUDER, SAMUEL R., 270 Cedar Lane, Paducah, KY 42001 MANWELL, REGINALD D., Syracuse University, Lyman Hall, Syracuse, NY 13210 MARSLAND, DOUGLAS, Broadmead N12, 13801 York Rd., Cockeysville, MD 21030 MILLER, JAMES A., 307 Shorewood Drive, E. Falmouth, MA 02536 MILNE, LORUS J., Department of Zoology, University of New Hampshire, Durham, NH 03824 MOORE, JOHN A., Department of Biology, University of California, Riverside, CA 92521 MOUL, E. T., 43 F. R. Lillie Rd., Woods Hole, MA 02543 NACE, PAUL F., 5 Bowditch Road, Woods Hole, MA 02543 PAGE, IRVING H., Box 516, Hyannisport, MA 02647 PLOUGH, HAROLD H., 31 Middle St., Amherst, MA 01002 (deceased 1 1/85) POLLISTER, A. W., 313 Broad Street, Harleysville, PA 19438 POND, SAMUEL E., PO Box 63, E. Winthrop, ME 04343 (deceased 10/85) PROSSER, C. LADD, Department of Physiology and Biophysics, Burrill Hall 524, University of Illinois, Urbana, IL61801 PROVASOLI, LUIGI, Haskins Laboratories, 165 Prospect Street, New Haven, CT 06510 PRYTZ, MARGARET MCDONALD, 21 McCouns Lane, Oyster Bay, NY 1 1771 RANKIN, JOHN S., JR., Box 97, Ashford, CT 06278 RENN, CHARLES E., Route 2, Hempstead, MD 21074 RICHARDS, A. GLENN, 942 Cromwell Ave., St. Paul, MN 551 14 RICHARDS, OSCAR W., Pacific University, Forest Grove, OR 97462 SCHARRER, BERTA, Department of Anatomy, Albert Einstein College of Medicine, 1 300 Morris Park Avenue, Bronx, NY 10461 SCHLESINGER, R. WALTER, University of Medicine and Dentistry of New Jersey, Department of Microbiology, Rutgers Medical School, PO Box 101, Piscataway, NJ 08854 SCHMITT, F. O., Room 16-512, Massachusetts Institute of Technology, Cambridge, MA 02139 SCOTT, ALLAN C., 1 Nudd St., Waterville, ME 04901 SCOTT, GEORGE T., 10 Orchard St., Woods Hole, MA 02543 SHEMIN, DAVID, Department of Biochemistry and Molecular Biology, Northwestern University, Evanston, IL 6020 1 SONNENBLICK, B. P., Department of Zoology and Physiology, Rutgers University, 195 University Ave., Newark, NJ07102 SPEIDEL, CARL C., 1873 Field Rd., Charlottesville, VA 22903 (no mailings) STEINHARDT, JACINTO, 1 508 Spruce St., Berkeley, CA 94709 STUNKARD, HORACE W., American Museum of Natural History, Central Park West at 79th St., New York, NY 10024 TAYLOR, W. RANDOLPH, Department of Biology, University of Michigan, Ann Arbor, MI 48109 TAYLOR, W. ROWLAND, 152 Cedar Park Road, Annapolis, MD 21401 TEWINKEL, Lois E., 4 Sanderson Ave., Northampton, MA 01060 TRACER, WILLIAM, The Rockefeller University, 1230 York Ave., New York, NY 10021 WALD, GEORGE, 67 Gardner Road, Woods Hole, MA 02543 WEISS, PAUL A., Address unknown WICHTERMAN, RALPH, 3 1 Buzzards Bay Ave., Woods Hole, MA 02543 WIERCINSKI, FLOYD J., Prof. Emeritus, Department of Biology, Northwestern Illinois University, Chicago, IL 60625 YOUNG, D. B., 1 137 Main St., N. Hanover, MA 02357 ZINN, DONALD J., PO Box 589, Falmouth, MA 02541 ZORZOLI, ANITA, 18 Wilbur Blvd., Poughkeepsie, NY 12603 ZWEIFACH, BENJAMIN W., % Ames, University of California, La Jolla, CA 92037 REGULAR MEMBERS ACHE, BARRY W., Whitney Marine Laboratory, University of Florida, Rt. 1 Box 121, St. Au- gustine, FL 32086 ACHESON, GEORGE H., 25 Quissett Ave., Woods Hole, MA 02543 8 MARINE BIOLOGICAL LABORATORY ADAMS, JAMES A., Department of Biological Sciences, Tennessee State University, 3500 John Merritt Blvd., Nashville, TN 37203 ADELBERG, EDWARD A., Department of Human Genetics, Yale University Medical School, PO Box 3333, New Haven, CT 06510 AFZELIUS, BJORN, Wenner-Gren Inslitute, University of Stockholm, Stockholm, Sweden ALBERTE, RANDALL S., University of Chicago, Barnes Laboratory, 5630 S. Ingleside Ave., Chi- cago, IL 60637 ALBRIGHT, JOHN T., 7 Siders Pond Rd., Falmouth, MA 02540 (deceased 10/85) ALKON, DANIEL, Seclion on Neural Systems, Laboratory of Biophysics, NIH, Marine Biological Laboratory, Woods Hole, MA 02543 ALLEN, GARLAND E., Department of Biology, Washington University, St. Louis, MO 63130 ALLEN, NINA S., Department of Biology, Wake Forest University, Box 7325, Reynolds Station, Winston-Salem, NC 27109 ALLEN, ROBERT D., Departmenl of Biology, Dartmouth College, Hanover, NH 03755 (deceased 3/86) ALSCHER, RUTH, Department of Biology, Manhatlanville College, Purchase, NY 10577 (deceased 7/85) AMATNIEK, ERNEST, 4797 Boston Post Rd., Pelham Manor, NY 10803 ANDERSON, EVERETT, Departmenl of Anatomy, LHRBB, Harvard Medical School, Boston, MA 021 15 ANDERSON, J. M., 110 Roat St., Ithaca, NY 14850 ARMET-KJBEL, CHRISTINE, Biology Department, University of Massachusetts Boston, Boston, MA 02 125 ARMSTRONG, CLAY M., Department of Physiology, Medical School, University of Pennsylvania, Philadelphia, PA 19174 ARMSTRONG, PETER B., Departmenl of Zoology, Universily of California, Davis, CA 95616 ARNOLD, JOHN M., Pacific Biomedical Research Cenler, Universily of Hawaii, 41 Ahui St., Honolulu, HI 96813 ARNOLD, WILLIAM A., 102 Balsam Rd., Oak Ridge, TN 37830 ASHTON, ROBERT W., Gaslon Snow Beekman and Bogue, 14 Wall St., New York, NY 10005 ATEMA, JELLE, Marine Biological Laboratory, Woods Hole, MA 02543 ATWOOD, KIMBALL C., PO Box 673, Woods Hole, MA 02543 AUGUSTINE, GEORGE, JR., Seclion of Neurobiology, Department of Biological Sciences, Uni- versity of Soulhern California, Los Angeles, CA 90089-037 1 AUSTIN, MARY L., 506'/2 N. Indiana Ave., Bloominglon, IN 47401 BACON, ROBERT, PO Box 723, Woods Hole, MA 02543 BAKER, ROBERT G., New York Universily Medical Cenler, 550 Firsl Ave., New York, NY 10016 BALDWIN, THOMAS O., Department of Biochemistry and Biophysics, Texas A&M University, College Stalion, TX 77843 BANG, BETSY, 76 F. R. Lillie Rd., Woods Hole, MA 02543 BARKER, JEFFERY L., Nalional Institutes of Health, Bldg. 36 Room 2002, Bethesda, MD 20892 BARLOW, ROBERT B., JR., Inslitute for Sensory Research, Syracuse University, Merrill Lane, Syracuse, NY 13210 BARTELL, CLELMER K., 2000 Lake Shore Drive, New Orleans, LA 70122 BARTH, LUCENA J., 26 Quissetl Ave., Woods Hole, MA 02543 BARTLETT, JAMES H., Departmenl of Physics, Box 1921, Universily of Alabama, Universily, AL 35486 BASS, ANDREW H., Seely Mudd Hall, Departmenl of Neurobiology and Behavior, Cornell Uni- versily, Ithaca, NY 14853 BATTELLE, BARBARA-ANNE, Whitney Laboralory, Rl. 1 , Box 1 2 1 , St. Augustine, FL 32086 BAUER, G. ERIC, Departmenl of Anatomy, University of Minnesota, Minneapolis, MN 55455 BEAUGE, Luis ALBERTO, Inslilulo de Invesligacion Medica, Casilla de Correo 389, 5000 Cordoba, Argenlina BECK, L. V., School of Experimental Medicine, Department of Pharmacology, Indiana Uni- versity, Bloomington, IN 47401 MEMBERS OF THE CORPORATION BEGG, DAVID A., LHRRB, Harvard Medical School, 45 Shattuck St., Boston, MA 021 15 BELL, EUGENE, Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139 BENJAMIN, THOMAS L., Department of Pathology, Harvard Medical School, 25 Shattuck St., Boston, MA 021 15 BENNETT, M. V. L., Albert Einstein College of Medicine, Department of Neuroscience, 1300 Morris Park Ave., Bronx, NY 10461 BENNETT, MIRIAM F., Department of Biology, Colby College, Waterville, ME 04901 BERG, CARL J., JR., Marine Biological Laboratory, Woods Hole, MA 02543 BERNE, ROBERT W., University of Virginia, School of Medicine, Charlottesville, VA 22908 BEZANILLA, FRANCISCO, Department of Physiology, University of California, Los Angeles, CA 90052 BIGGERS, JOHN D., Department of Physiology, Harvard Medical School, Boston, MA 021 15 BISHOP, STEPHEN H., Department of Zoology, Iowa State University, Ames, IA 50010 BLAUSTEIN, MORDECAI P., Department of Physiology, School of Medicine, University of Mary- land, 655 W. Baltimore Street, Baltimore, MD 21201 BODIAN, DAVID, Address unknown BODZNICK, DAVID A., Department of Biology, Wesleyan University, Middletown, CT 06457 BOETTIGER, EDWARD G., 29 Juniper Point, Woods Hole, MA 02543 BOGORAD, LAWRENCE, The Biological Laboratories, Harvard University, Cambridge, MA 02 1 38 BOOLOOTIAN, RICHARD A., Science Software Systems, Inc., 1 1 899 W. Pico Blvd., W. Los Angeles, CA 90064 BOREI, HANS G., Long Cove, Stanley Point Road, Minturn, ME 04659 BORGESE, THOMAS A., Department of Biology, Lehman College, CUNY, Bronx, NY 10468 BORISY, GARY G., Laboratory of Molecular Biology, University of Wisconsin, Madison, WI 53715 BOSCH, HERMAN F., PO Box 542, Woods Hole, MA 02543 BOTKIN, DANIEL, Department of Biology, University of California, Santa Barbara, CA 93106 BOWLES, FRANCIS P., PO Box 674, Woods Hole, MA 02543 BOYER, BARBARA C, Department of Biology, Union College, Schnectady, NY 12308 BRANDHORST, BRUCE P., Biology Department, McGill University, 1205 Ave. Dr. Penfield, Montreal, Quebec, Canada H3A 1B1 BRINLEY, F. J., Neurological Disorders Program, NINCDS, 716 Federal Building, Bethesda, MD 20892 BROWN, JOEL E., Department of Ophthalmology, Box 8096 Sciences Center, Washington Uni- versity, 660 S. Euclid Ave., St. Louis, MO 631 10 BROWN, STEPHEN C., Department of Biological Sciences, SUNY, Albany, NY 12222 BUCK, JOHN B., NIH, Laboratory of Physical Biology, Room 112, Building 6 Bethesda, MD 20892 (Life Member 10/85) BURD, GAIL DEERIN, Department of Molecular and Cellular Biology, Biosciences West, Room 305, University of Arizona, Tucson, AZ 85721 BURDICK, CAROLYN J., Department of Biology, Brooklyn College, Brooklyn, NY 1 1210 BURGER, MAX, Department of Biochemistry, Biocenter, Klingelbergstrasse 70, CH-4056 Basel, Switzerland BURKY, ALBERT, Department of Biology, University of Dayton, Dayton, OH 45469 BURSTYN, HAROLD LEWIS, 523 National Center, U. S. Geological Survey, Reston, VA 22092 BURSZTAJN, SHERRY, Neurology Department Program in Neuroscience, Baylor College of Medicine, Houston, TX 77030 BUSH, LOUISE, 7 Snapper Lane, Falmouth, MA 02540 CALABRESE, RONALD L., Department of Biology, Emory University, 1555 Pierce Drive, Atlanta, GA 30322 CANDELAS, GRACIELA C., Department of Biology, University of Puerto Rico, Rio Piedras, PR 00931 CARIELLO, Lucio, Stazione Zoologica, Villa Comunale, Naples, Italy CARLSON, FRANCIS D., Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218 10 MARINE BIOLOGICAL LABORATORY CASE, JAMES, Department of Biological Sciences, University of California, Santa Barbara, CA 93106 CASSIDY, J. D., St. Albert's Priory, University of Dallas, Irving, TX 75061 CEBRA, JOHN J., Department of Biology, Leidy Labs, G-6, University of Pennsylvania, Phila- delphia, PA 19174 CHAET, ALFRED B., University of West Florida, Pensacola, FL 32504 CHAMBERS, EDWARD L., Department of Physiology and Biophysics, University of Miami, School of Medicine, PO Box 016430, Miami, FL 33101 CHANG, DONALD C, Department of Physiology, Baylor College of Medicine, 1 200 Moursund, Houston, TX 77030 CHAPPELL, RICHARD L., Department of Biological Sciences, Hunter College, Box 210, 695 Park Ave., New York, NY 10021 CHAUNCEY, HOWARD H., 30 Falmouth St., Wellesley Hills, MA 02181 CHARLTON, MILTON P., Physiology Department MSB, University of Toronto, Toronto, Ontario, Canada M5S 1A8 CHILD, FRANK M., Department of Biology, Trinity College, Hartford, CT 06106 CHISHOLM, REX L., Department of Cell Biology and Anatomy, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL 6061 1 CITKOWITZ, ELENA, 410 Livingston St., New Haven, CT 0651 1 CLARK, A. M., 48 Wilson Rd., Woods Hole, MA 02543 CLARK, ELOISE E., Vice President for Academic Affairs, Bowling Green State University, Bowling Green, OH 43403 CLARK, HAYS, 26 Deer Park Drive, Greenwich, CT 06830 CLARK, JAMES M., Shearson Lehman/American Express, 55 Water Street, 42nd Floor, New York, NY 10041 CLARK, WALLIS H., JR., Bodega Marine Lab, PO Box 247, Bodega Bay, CA 94923 CLAUDE, PHILIPPA, Primate Center, Capitol Court, Madison, WI 53706 CLAYTON, RODERICK K., (resigned 10/85) CLOWES, GEORGE H. A., JR., The Cancer Research Institute, 194 Pilgrim Rd., Boston, MA 02215 CLUTTER, MARY, Senior Science Advisor, Office of the Director, Room 518, National Science Foundation, Washington, DC 20550 COBB, JEWELL P., President, California State University, Fullerton, CA 92634 COHEN, ADOLPH I., Department of Ophthalmology, School of Medicine, Washington University, 660 S. Euclid Ave., St. Louis, MO 631 10 COHEN, CAROLYN, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02 154 COHEN, LAWRENCE B., Department of Physiology, Yale University, 333 Cedar St., New Haven, CT06510 COHEN, ROCHELLE S., Department of Anatomy, University of Illinois at Chicago, 808 S. Wood Street, Chicago, IL 60612 COHEN, WILLIAM D., Department of Biological Sciences, Hunter College, 695 Park Ave., New York, NY 10021 COLE, JONATHAN J., Institute for Ecosystems Studies, Cary Arboretum, Millbrook, NY 12545 COLEMAN, ANNETTE W., Division of Biology and Medicine, Brown University, Providence, RI 02912 COLLIER, JACK R., Department of Biology, Brooklyn College, Brooklyn, NY 1 1210 COLLIER, MARJORIE McCANN, Biology Department, Saint Peter's College, Kennedy Boulevard, Jersey City, NJ 07306 COOK, JOSEPH A., The Edna McConnell Clark Foundation, 250 Park Ave., New York, NY 10017 COOPERSTEIN, S. J., University of Connecticut, School of Medicine, Farmington Ave., Far- mington, CT 06032 CORLISS, JOHN O., Department of Zoology, University of Maryland, College Park, MD 20742 CORNELL, NEAL W., 6428 Bannockburn Drive, Bethesda, MD 208 1 7 MEMBERS OF THE CORPORATION 1 1 CORNMAN, IVOR, 10A Orchard St., Woods Hole, MA 02543 CORNWALL, MELVIN C, JR., Department of Physiology L714, Boston University School of Medicine, 80 E. Concord St., Boston, MA 021 18 CORSON, DAVID WESLEY, JR., Laboratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole, MA 02543 CORWIN, JEFFREY T., Bekesy Lab of Neurobiology, 1993 East- West Road, University of Hawaii, Honolulu, HI 96822 COSTELLO, WALTER J., College of Medicine, Ohio University, Athens, OH 45701 COUCH, ERNEST F., Department of Biology, Texas Christian University, Fort Worth, TX 76129 CREMER-BARTELS, GERTRUD, Universitats Augenklinik, 44 Munster, West Germany CROW, TERRY J., Department of Physiology, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261 CROWELL, SEARS, Department of Biology, Indiana University, Bloomington, IN 47405 CURRIER, DAVID L., PO Box 2476, Vineyard Haven, MA 02568 DAIGNAULT, ALEXANDER T., 280 Beacon St., Boston, MA 021 16 DAN, KATSUMA, Professor Emeritus, Tokyo Metropolitan Union, Meguro-ku, Tokyo, Japan D'AVANZO, CHARLENE, School of Natural Science, Hampshire College, Amherst, MA 01002 DAVID, JOHN R., Seeley G. Mudd Building, Room 504, Harvard Medical School, 250 Longwood Ave., Boston, MA 021 15 DAVIS, BERNARD D., 23 Clairemont Road, Belmont, MA 02170 DAVIS, JOEL P., Seapuit, Inc., PO Box G, Osterville, MA 02655 DAW, NIGEL W., 78 Aberdeen Place, Clayton, MO 63105 DEGROOF, ROBERT C., RR#1 Box 343, Green Lane, PA 18054 DEHAAN, ROBERT L., Department of Anatomy, Emory University, Atlanta, GA 30322 DELANNEY, Louis E., Institute for Medical Research, 2260 Clove Drive, San Jose, CA 95128 DEPHILLIPS, HENRY A., JR., Department of Chemistry, Trinity College, Hartford, CT 06106 DETERRA, NOEL, Marine Biological Laboratory, Woods Hole, MA 02543 DETTBARN, WOLF-DIETRICH, Department of Pharmacology, School of Medicine, Vanderbilt University, Nashville, TN 37127 DE WEER, PAUL J., Department of Physiology, School of Medicine, Washington University, St. Louis, MO 63 110 DISCHE, ZACHARIAS, Eye Institute, College of Physicians and Surgeons, Columbia University, 639 W. 165 St., New York, NY 10032 DIXON, KEITH E., School of Biological Sciences, Flinders University, Bedford Park, South Aus- tralia DOWDALL, MICHAEL J., Department of Biochemistry, University Hospital and Medical School, Nottingham N672 UH, England, U. K. DOWLING, JOHN E., The Biological Laboratories, Harvard University, 16 Divinity St., Cambridge, MA 02 138 DuBoiS, ARTHUR BROOKS, John B. Pierce Foundation Laboratory, 290 Congress Ave., New Haven, CT 065 19 DUDLEY, PATRICIA L., Department of Biological Sciences, Barnard College, Columbia University, New York, NY 10027 DUNCAN, THOMAS K., Department of Environmental Science, Nichols College, Dudley, MA 01570 DUNHAM, PHILIP B., Department of Biology, Syracuse University, Syracuse, NY 13210 EBERT, JAMES D., Office of the President. Carnegie Institution of Washington 1530 P St., NW, Washington, DC 20008 ECKBERG, WILLIAM R., Department of Zoology, Howard University, Washington, DC 20059 ECKERT, ROGER O., Department of Zoology, University of California, Los Angeles, CA 90024 EDDS, KENNETH T., Department of Anatomical Sciences, SUNY, Buffalo, NY 14214 EDER, HOWARD A., Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 EDWARDS, CHARLES, Rm. 9N222, Bldg. 10, IADDK/NIH, Bethesda, MD 20892 EGYUD, LASZLO G., 18 Skyview, Newton. MA 02150 12 MARINE BIOLOGICAL LABORATORY EHRENSTEIN, GERALD, NIH, Bethesda, MD 20892 EHRLICH, BARBARA E., Department of Physiology, Albert Einstein College of Medicine, 1 300 Morris Park Ave., Bronx, NY 10461 EISEN, ARTHUR, Z., Chief of Division of Dermatology, Washington University, St. Louis, MO 63110 EISENMAN, GEORGE, Department of Physiology, University of California Medical School, Los Angeles, CA 90024 ELDER, HUGH YOUNG, Institute of Physiology, University of Glasgow, Glasgow, Scotland, U.K. ELLIOTT, GERALD F., The Open University Research Unit, Foxcombe Hall, Berkeley Rd., Boars Hill, Oxford, England, U. K. EPEL, DAVID, Hopkins Marine Station, Pacific Grove, CA 93950 EPSTEIN, HERMAN T., Department of Biology, Brandeis University, Waltham, MA 02154 ERULKAR, SOLOMON D., 318 Kent Rd., Bala Cynwyd, PA 19004 ESSNER, EDWARD S., Kresege Eye Institute, Wayne State University, 540 E. Canfield Ave., Detroit, MI 48201 FAILLA, PATRICIA M., 2149 Loblolly Lane, Johns Island, SC 29455 (Life Member 12/85) FARMANFARMAIAN, A., Nelson Biology Lab., Busch Campus, Rutgers University, Piscataway NJ 08854 FAUST, ROBERT G., Department of Physiology, Medical School, University of North Carolina, Chapel Hill, NC27514 FEIN, ALAN, Laboratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole, MA 02543 FELDMAN, SUSAN C, Department of Anatomy, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 100 Bergen St., Newark, NJ 07103 FERGUSON, F. P., National Institute of General Medical Science, NIH, Bethesda, MD 20892 FESSENDEN, JANE, Marine Biological Laboratory, Woods Hole, MA 02543 FESTOFF, BARRY W., Neurology Service (127), Veterans Administration Medical Center, 4801 Linwood Blvd., Kansas City, MO 64128 FINKELSTEIN, ALAN, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 FISCHBACH, GERALD, Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 631 10 FISCHMAN, DONALD A., Department of Cell Biology and Anatomy, Cornell University Medical College, 1300 York Ave., New York, NY 10021 FISHMAN, HARVEY M., Department of Physiology, University of Texas Medical Branch, Gal- veston, TX 77550 FLANAGAN, DENNIS, Editor, Scientific American, 415 Madison Ave., New York, NY 10017 Fox, MAURICE S., Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02 138 FRANZINI, CLARA, Department of Biology G-5, School of Medicine, University of Pennsylvania, Philadelphia, PA 19174 FRAZIER, DONALD T., Department of Physiology and Biophysics, University of Kentucky Med- ical Center, Lexington, KY 40536 FREEMAN, ALAN R., Department of Physiology, Temple University, 3420 N. Broad St., Phil- adelphia, PA 19140 FREEMAN, GARY L., Department of Zoology, University of Texas, Austin, TX 78172 FREINKEL, NORBERT, Center for Endocrinology, Metabolism & Nutrition, Northwestern Uni- versity Medical School, 303 E. Chicago Avenue, Chicago, IL 6061 1 FRENCH, ROBERT J., Department of Biophysics, University of Maryland, School of Medicine, Baltimore, MD 21201 FREYGANG, WALTER J., JR., 6247 29th St., NW, Washington, DC 20015 FULTON, CHANDLER M., Department of Biology, Brandeis University, Waltham, MA 02154 FURSHPAN, EDWIN J., Department of Neurophysiology, Harvard Medical School, Boston, MA 02115 MEMBERS OF THE CORPORATION 1 3 FUSELER, JOHN W., Department of Biology, University of Southwestern Louisiana, Lafayette, LA 70504 FUTRELLE, ROBERT P., College of Computer Science, Northeastern University, 360 Huntington Avenue, Boston, MA 02 1 1 5 FYE, PAUL, PO Box 309, Woods Hole, MA 02543 GABRIEL, MORDECAI, Department of Biology, Brooklyn College, Brooklyn, NY 11210 GADSBY, DAVID C, Laboratory of Cardiac Physiology, The Rockefeller University, 1230 York Avenue, New York, NY 10021 GAINER, HAROLD, Section of Functional Neurochemistry, NIH, Bldg. 36 Room 2A21, Bethesda, MD 20892 GALATZER-LEVY, ROBERT M., 180 N. Michigan Avenue, Chicago, IL 60601 GALL, JOSEPH G., Carnegie Institution, 115 West University Parkway, Baltimore, MD 21210 GASCOYNE, PETER, Marine Biological Laboratory, Woods Hole, MA 02543 GELFANT, SEYMOUR, Department of Dermatology, Medical College of Georgia, Augusta, GA 30904 GELPERIN, ALAN, Department of Biology, Princeton University, Princeton, NJ 08540 GERMAN, JAMES L., Ill, The New York Blood Center, 310 East 65th St., New York, NY 1002 1 GIBBS, MARTIN, Institute for Photobiology of Cells and Organelles, Brandeis University, Wal- tham, MA 02 154 GIBLIN, ANNE E., Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543 GIBSON, A. JANE, Wing Hall, Cornell University, Ithaca, NY 14850 GIFFORD, PROSSER, The Wilson Center, Smithsonian Building, 1000 Jefferson Drive, SW, Washington, DC 20590 GILBERT, DANIEL L., NIH, Laboratory of Biophysics, NINCDS, Bldg. 36, Room 2A-29, Bethesda, MD 20892 GIUDICE, GIOVANNI, Via Archirafi 22, Palermo, Italy GLUSMAN, MURRAY, Department of Psychiatry, Columbia University, 722 W. 168th St., New York, NY 10032 GOLDEN, WILLIAM T., 40 Wall St., New York, NY 10005 GOLDMAN, DAVID E., 63 Loop Rd., Falmouth, MA 02540 GOLDMAN, ROBERT D., Department of Cell Biology and Anatomy, Northwestern University, 303 E. Chicago Ave., Chicago, IL 6061 1 GOLDSMITH, PAUL K., 551 1 Oakmont Avenue, Bethesda, MD 20034 GOLDSMITH, TIMOTHY H., Department of Biology. Yale University, New Haven, CT 06520 GOLDSTEIN, MoiSE H., JR., EE & CS Department, Johns Hopkins University, Baltimore, MD 21218 GOODMAN, LESLEY JEAN, Department of Biological Sciences, Queen Mary College, Mile End Road, London, El 4NS, England, U. K. GOTTSCHALL, GERTRUDE Y., 315 E. 68th St., 9-M, New York, NY 10021 (deceased 7/85) GOUDSMIT, ESTHER M., Department of Biology, Oakland University, Rochester, MI 48063 GOULD, ROBERT MICHAEL, Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Rd., Staten Island, NY 10314 GOULD, STEPHEN J., Museum of Comparative Zoology, Harvard University, Cambridge, MA 02138 GOVIND, C. K., Department of Zoology, Scarborough Campus, University of Toronto, 1265 Military Trail, West Hill, Ontario, Canada, MIC 1A4 GRAHAM, HERBERT, 36 Wilson Rd., Woods Hole, MA 02543 GRANT, PHILIP, Department of Biology, University of Oregon, Eugene, OR 97403 GRASS, ALBERT, The Grass Foundation, 77 Reservoir Rd., Quincy, MA 02170 GRASS, ELLEN R., The Grass Foundation, 77 Reservoir Rd., Quincy, MA 02170 GRASSLE, JUDITH, Marine Biological Laboratory, Woods Hole, MA 02543 GREEN, JONATHAN P., Department of Biology, Roosevelt University, 430 S. Michigan Avenue, Chicago, IL 60605 GREENBERG, EVERETT PETER, Department of Microbiology, Stocking Hall, Cornell University, Ithaca, NY 14853 14 MARINE BIOLOGICAL LABORATORY GREENBERG, MICHAEL J., C. V. Whitney Lab, Rt. 1 Box 121, St. Augustine, FL 32086 GREIF, ROGER L., Department of Physiology, Cornell University, Medical College, New York, NY 10021 GRIFFIN, DONALD R., The Rockefeller University, 1230 York Ave., New York, NY 10021 GROSCH, DANIEL S., Department of Genetics, Gardner Hall, North Carolina State University, Raleigh, NC 27607 GROSS, PAUL R., President and Director, Marine Biological Laboratory, Woods Hole, MA 02543 GROSSMAN, ALBERT, New York University, Medical School, New York, NY 10016 GUNNING, A. ROBERT, PO Box 165, Falmouth, MA 02541 GWILLIAM, G. P., Department of Biology, Reed College, Portland, OR 97202 HALL, LINDA M., Department of Genetics, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 HALL, ZACK W., Department of Physiology, University of California, San Francisco, CA 94143 HALVORSON, HARLYN O., Rosenstiel Basic Medical Sciences Research Center, Brandeis Uni- versity, Waltham, MA 02 1 54 HAMLETT, NANCY VIRGINIA, Department of Biology, Swarthmore College, Swarthmore, PA 19081 HANNA, ROBERT B., College of Environmental Science and Forestry, SUNY, Syracuse, NY 13210 HARDING, CLIFFORD V., JR., Kresege Eye Institute, Wayne State University, 540 E. Canfield, Detroit, MI 48201 HAROSI, FERENC I., Laboratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole, MA 02543 HARRIGAN, JUNE F., Laboratory of Biophysics, Marine Biological Laboratory, Woods Hole, MA 02543 HARRINGTON, GLENN W., Department of Microbiology, School of Dentistry, University of Missouri, 650 E. 25th St., Kansas City, MO 64108 HARRIS, ANDREW L., Department of Biophysics, Johns Hopkins University, 34th & Charles Sts., Baltimore, MD21218 HASCHEMEYER, AUDREY E. V., Department of Biological Sciences, Hunter College, 695 Park Ave., New York, NY 10021 HASTINGS, J. W., The Biological Laboratories, Harvard University, Cambridge, MA 02138 HAUSCHKA, THEODORE S., RD1, Box 781, Damariscotta, ME 04543 HAYASHI, TERU, 7105 SW 112 Place, Miami, FL 33173 HAYES, RAYMOND L., JR., Department of Anatomy, Howard University, College of Medicine, 520 W St., NW, Washington, DC 20059 HENLEY, CATHERINE, 5225 Pooks Hill Rd., #1 127 North, Bethesda, MD 20034 HERNDON, WALTER R., University of Tennessee, Department of Biology, Knoxville, TN 37996-1100 HESSLER, ANITA Y., 5795 Waverly Ave., La Jolla, CA 92037 HEUSER, JOHN, Department of Biophysics, Washington University, School of Medicine, St. Louis, MO 63 110 HIATT, HOWARD H., Brigham and Women's Hospital, 75 Francis Street, Boston, MA 021 15 HIGHSTEIN, STEPHEN M., Department of Otolaryngology, Washington University, St. Louis, MO 63110 HILDEBRAND, JOHN G., Arizona Research Laboratories, Division of Neurobiology, 603 Gould- Simpson Science Building, University of Arizona, Tucson, AZ 85721 HILLIS-COLINVAUX, LLEWELLYA, Department of Zoology, The Ohio State University, 484 W 12th Ave., Columbus, OH 43210 HILLMAN, PETER, Department of Biology, Hebrew University, Jerusalem, Israel HINEGARDNER, RALPH T., Division of Natural Sciences, University of California, Santa Cruz, CA 95064 HINSCH, GERTRUDE W., Department of Biology, University of South Florida, Tampa, FL 33620 HOBBIE, JOHN E., Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543 HODGE, ALAN J., Marine Biological Laboratory, Woods Hole, MA 02543 MEMBERS OF THE CORPORATION 1 5 HOFFMAN, JOSEPH, Department of Physiology, School of Medicine, Yale University, New Haven CT06510 HOLLYFIELD, JOE G., Baylor School of Medicine, Texas Medical Center, Houston, TX 77030 HOLTZMAN, ERIC, Department of Biological Sciences, Columbia University New York NY 10017 HOLZ, GEORGE G., JR., Department of Microbiology, SUNY, Syracuse, NY 13210 HOSKIN, FRANCIS C. G., Department of Biology, Illinois Institute of Technology, Chicago, IL 60616 HUGHTON, RICHARD A., Ill, Ecosystems Center, Marine Biological Laboratory Woods Hole MA 02543 HOUSTON, HOWARD E., 2500 Virginia Ave., NW, Washington, DC 20037 HOWARD, JOAN E., Marine Biological Laboratory, Woods Hole, MA 02543 HOWARTH, ROBERT, Section of Ecology & Systematics, Corson Hall, Cornell University, Ithaca, NY 14853 HOY, RONALD R., Section of Neurobiology and Behavior, Cornell University, Ithaca, NY 14850 HUBBARD, RUTH, 67 Gardner Road, Woods Hole, MA 02543 HUFNAGEL, LINDA A., Department of Microbiology, University of Rhode Island, Kingston, RI 02881 HUMMON, WILLIAM D., Department of Zoology, Ohio University, Athens. OH 45701 HUMPHREYS, SUSIE H., Kraft Research and Development, 801 Waukegan Rd., Glenview, IL 60025 HUMPHREYS, TOM D., University of Hawaii, PBRC, 41 Ahui St., Honolulu, HI 96813 HUNTER, BRUCE W., Box 321, Lincoln Center, MA 01773 HUNTER. ROBERT D., Department of Biological Sciences, Oakland University, Rochester, NY 48063 HUNZIKER, HERBERT E., Esq., PO Box 547, Falmouth, MA 02541 HURWITZ, CHARLES, Basic Science Research Laboratory, Veterans Administration Hospital, Albany, NY 12208 HURWITZ, JERARD, Memorial Sloan Kettering Institute, 1275 York Avenue, New York NY 11021 HUXLEY, HUGH E., Medical Research Council, Laboratory of Molecular Biology, Cambridge, England, U. K. HYNES, THOMAS J., JR., Meredith and Grew, Inc., 125 High Street, Boston, MA 021 10 ILAN, JOSEPH, Department of Anatomy, Case Western Reserve University, Cleveland, OH 44106 INGOGLIA, NICHOLAS, Department of Physiology, New Jersey Medical School, 100 Bergen St., Newark, NJ 07 103 INOUE, SADUYKI, McGill University Cancer Centre, Department of Aantomy, 3640 University St., Montreal, Quebec, Canada, H3A 2B2 INOUE, SHINYA, Marine Biological Laboratory, Woods Hole, MA 02543 ISSADORIDES, MARIETTA R., Department of Psychiatry, University of Athens, Monis Petraki 8, Athens, 140, Greece ISSELBACHER, KURT J., Massachusetts General Hospital, Boston, MA 021 14 IZZARD, COLIN S., Department of Biological Sciences, SUNY, Albany, NY 12222 JACOBSON, ANTONE G., Department of Zoology, University of Texas, Austin, TX 78712 JAFFE, LIONEL, Marine Biological Laboratory, Woods Hole, MA 02543 JAHAN-PARWAR, BEHRUS, Center for Laboratories & Research, New York State Department of Health, Empire State Plaza, Albany, NY 12201 JANNASCH, HOLGER W., Woods Hole Oceanographic Institution, Woods Hole, MA 02543 JEFFERY, WILLIAM R., Department of Zoology, University of Texas, Austin, TX 78712 JENNER, CHARLES E., Department of Zoology, University of North Carolina, Chapel Hill, NC 27514 JONES, MEREDITH L., Division of Worms, Museum of Natural History, Smithsonian Institution, Washington, DC 20560 JOSEPHSON, ROBERT K., School of Biological Sciences, University of California, Irvine, CA 92664 16 MARINE BIOLOGICAL LABORATORY KABAT, E. A., Department of Microbiology, College of Physicians and Surgeons, Columbia University, 630 West 168th St., New York, NY 10032 KALEY, GABOR, Department of Physiology, Basic Sciences Building, New York Medical College, Valhalla, NY 10595 KALTENBACH, JANE, Department of Biological Sciences, Mount Holyoke College, South Hadley, MA 01075 KAMINER, BENJAMIN, Department of Physiology, School of Medicine, Boston University, 80 East Concord St., Boston, MA 021 18 KAMMER, ANN E., Division of Biology, Kansas State University, Manhatten, KS 66506 KANE, ROBERT E., University of Hawaii, PBRC, 41 Ahui St., Honolulu, HI 96813 KANESHIRO, EDNA S., Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221 KAO, CHIEN-YUAN, Department of Pharmacology (Box 29), SUNY, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 1 1203 KAPLAN, EHUD, The Rockefeller University, 1230 York Ave., New York, NY 10021 KARAKASHIAN, STEPHEN J., Apt. 16-F, 165 West 91st St., New York, NY 10024 KARLIN, ARTHUR, Department of Biochemistry and Neurology, Columbia University, 630 West 168th St., New York, NY 10032 KARUSH, FRED, 183 Summit Lane, Bala-Cynwyd, PA 19004 (Life Member 8/85) KATZ, GEORGE M., Fundamental and Experimental Research, Merck, Sharpe and Dohme, Rahway, NJ 07065 KEAN, EDWARD L., Department of Ophthalmology and Biochemistry, Case Western Reserve University, Cleveland, OH 44101 KELLEY, DARCY BRISBANE, Department of Biological Sciences, 1018 Fairchild, Columbia Uni- versity, New York, NY 10032 KELLY, ROBERT E., Department of Anatomy, College of Medicine, University of Illinois, PO Box 6998, Chicago, IL 60680 KEMP, NORMAN E., Department of Zoology, University of Michigan, Ann Arbor, MI 48104 KENDALL, JOHN P., Faneuil Hall Associates, One Boston Place, Boston, MA 02108 KEYNAN, ALEXANDER, Hebrew University, Jerusalem, Israel KIEHART, DANIEL P., Department of Cellular and Developmental Biology, Harvard University, 16 Divinity Ave., Cambridge, MA 02138 KIRSCHENBAUM, DONALD, Department of Biochemistry, SUNY, 450 Clarkson Ave., Brooklyn, NY 11 203 (deceased 10/85) KLEIN, MORTON, Department of Microbiology, Temple University, Philadelphia, PA 19122 KLOTZ, I. M., Department of Chemistry, Northwestern University, Evanston, IL 60201 KOIDE, SAMUEL S., Population Council, The Rockefeller University, 66th St. and York Ave., New York, NY 10021 KONIGSBERG, IRWIN R., Department of Biology, Gilmer Hall, University of Virginia, Char- lottesville, VA 22903 KORNBERG, SIR HANS, Department of Biochemistry, University of Cambridge, Tennis Court Rd., Cambridge, CB2 7QW, England, U. K. KOSOWER, EDWARD M., Ramat-Aviv, Tel Aviv, 69978 Israel KRAHL, M. E., 2783 W. Casas Circle, Tucson, AZ 85741 KRANE, STEPHEN M., Massachusetts General Hospital, Boston, MA 021 14 KRASSNER, STUART M., Department of Developmental and Cell Biology, University of Cali- fornia, Irvine, CA 927 1 7 KRAUSS, ROBERT, FASEB, 9650 Rockville Pike, Bethesda, MD 20205 KRAVITZ, EDWARD A., Department of Neurobiology, Harvard Medical School, 25 Shattuck St., Boston, MA 021 15 KRIEBEL, MAHLON E., Department of Physiology, B.S.B., Upstate Medical Center, 766 Irving Ave., Syracuse, NY 13210 KRIEG, WENDELL J. S., 1236 Hinman, Evanston, IL 60602 KRISTAN, WILLIAM B., JR., Department of Biology B-022, University of California, San Diego, CA 92093 MEMBERS OF THE CORPORATION 1 7 KUHNS, WILLIAM J., University of North Carolina, 5 12 Faculty Lab Office, Bldg. 23 1-H, Chapel Hill, NC27514 KUSANO, KIYOSHI, Illinois Institute of Technology, Department of Biology, 3300 South Federal St., Chicago, IL60616 KUZIRIAN, ALAN M., Laboratory of Biophysics, NINCDS-NIH, Marine Biological Laboratory, Woods Hole, MA 02543 LADERMAN, AIMLEE, PO Box 689, Woods Hole, MA 02543 LAMARCHE, PAUL H., Eastern Maine Medical Center, 489 State St., Bangor, ME 04401 LANDIS, DENNIS M. D., Department of Developmental Genetics and Anatomy, Case Western Reserve Medical School, 2119 Abington Road, Cleveland, OH 44106 LANDIS, STORY C, Department of Phamacology, Case Western Reserve University Medical School, 2119 Abington Road, Cleveland, OH 44106 LANDOWNE, DAVID, Department of Physiology, University of Miami, R-430, PO Box 016430, Miami, FL33101 LANGFORD, GEORGE M., Department of Physiology, Medical Sciences Research Wing 206H, University of North Carolina, Chapel Hill, NC 27514 LASER, RAYMOND J., Case Western Reserve University, Department of Anatomy, Cleveland, OH 44 106 LASTER, LEONARD, University of Oregon, Health Sciences Center, Portland, OR 97201 LAUFER, HANS, Biological Sciences Group U-42, University of Connecticut, Storrs, CT 06268 LAZAROW, PAUL B., The Rockefeller University, 1230 York Avenue, New York, NY 10021 LAZARUS, MAURICE, Federated Department Stores, Inc., 50 Cornhill, Boston, MA 02108 LEADBETTER, EDWARD R., Department of Molecular and Cell Biology, U-131, University of Connecticut, Storrs, CT 06268 LEDERBERG, JOSHUA, President, The Rockefeller University, 1230 York Ave., New York, NY 10021 LEDERHENDLER, IZJA I., Laboratory of Biophysics, Marine Biological Laboratory, Woods Hole, MA 02543 LEE, JOHN J., Department of Biology, City College of CUNY, Convent Ave. and 138th St., New York, NY 10031 LEIBOVTTZ, LOUIS, Laboratory for Marine Animal Health, Marine Biological Laboratory, Woods Hole, MA 02543 LEIGHTON, JOSEPH, 1201 Waverly Rd., Gladwyne, PA 19035 LEIGHTON, STEPHEN, NIH, Bldg. 13 3W13, Bethesda, MD 20892 LERMAN, SIDNEY, Laboratory for Ophthalmic Research, Emory University, Atlanta, GA 30322 LERNER, AARON B., Yale University, School of Medicine, New Haven, CT 06510 LESTER, HENRY A., 156-29 California Institute of Technology, Pasadena, CA 91 125 LEVIN, JACK, Clinical Pathology Service, VA Hospital- 1 13A, 4150 Clement St., San Francisco, CA 94121 LEVINTHAL, CYRUS, Department of Biological Sciences, Columbia University, 435 Riverside Drive, New York, NY 10025 LEVITAN, HERBERT, Department of Zoology, University of Maryland, College Park, MD 20742 LINCK, RICHARD W., Department of Anatomy, Jackson Hall, University of Minnesota, 321 Church Street, S.E., Minneapolis, MN 55455 LING, GILBERT, 307 Berkeley Road, Merion, PA 19066 LIPICKY, RAYMOND J., Department of Cardio-Renal/HFD 110, FDA Bureau of Drugs, Rm. 16B-45, 5600 Fishers Lane, Rockville, MD 20857 LISMAN, JOHN E., Department of Biology, Brandeis University, Waltham, MA 02154 Liuzzi, ANTHONY, Department of Physics, University of Lowell, Lowell, MA 01854 LLINAS, RODOLFO R., Department of Physiology and Biophysics, New York University Medical Center, 550 First Ave., New York, NY 10016 LOEWENSTEIN, WERNER R., Department of Physiology and Biophysics, University of Miami, PO Box 016430, Miami, FL 33101 LOEWUS, FRANK A., Institute of Biological Chemistry, Washington State University, Pullman, WA99164 18 MARINE BIOLOGICAL LABORATORY LOFTFIELD, ROBERT B., Department of Biochemistry, School of Medicine, University of New Mexico, 900 Stanford, NE, Albuquerque, NM 87131 LONDON, IRVING M., Massachusetts Institute of Technology, Cambridge, MA 02139 LONGO, FRANK J., Department of Anatomy, University of Iowa, Iowa City, IA 52442 LORAND, LASZLO, Department of Biochemistry and Molecular Biology, Northwestern University, Evanston, IL 60201 LUCKENBILL-EDDS, LOUISE, 12907 Crookston Lane, #28, Rockville, MD 20851 LURIA, SALVADOR E., Massachusetts Institute of Technology, Department of Biology, Cambridge, MA 02 139 LYNCH, CLARA J., 4800 Fillmore Ave., Alexandria, VA 2231 1 MACAGNO, EDUARDO R., 1003B Fairchild, Columbia University, New York, NY 10022 MACNiCHOL, E. F., JR., 45 Brewster Street, Cambridge, MA 02138 MAGLOTT, DONNA R. S., 1014 Baltimore Road, Rockville, MD 20851 MAIENSCHEIN, JANE ANN, Department of Philosophy, Arizona State University, Tempe, AZ 85281 MAINER, ROBERT, The Boston Company, One Boston Place, Boston, MA 02108 MALKIEL, SAUL, Allergic Diseases, Inc., 130 Lincoln St., Worcester, MA 01605 MANALIS, RICHARD S., RR #10, 400N, Columbia City, IN 47625 MANGUM, CHARLOTTE P., Department of Biology, College of William and Mary, Williamsburg, VA 23185 MARGULIS, LYNN, Department of Biology, Boston University, 2 Cummington St., Boston, MA 02215 MARINUCCI, ANDREW C, Department of Civil Engineering, Princeton University, Princeton, NJ 08544 MARSH, JULIAN B., Department of Biochemistry and Physiology, Medical College of Pennsyl- vania, 3300 Henry Ave., Philadelphia, PA 19129 MARTIN, LOWELL V., Marine Biological Laboratory, Woods Hole, MA 02543 MASER, MORTON, PO Box EM, Woods Hole, MA 02543 MASTROIANNI, LuiGl, JR., Department of Obstetrics and Gynecology, University of Pennsyl- vania, Philadelphia, PA 19174 MATHEWS, RITA W., Department of Medicine, New York University Medical Center, 550 First Ave., New York, NY 10016 MATTESON, DONALD R., Department of Physiology, G4, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 MAUTNER, HENRY G., Department of Biochemistry and Pharmacology, Tufts University, 136 Harrison Ave., Boston, MA 02 1 1 1 MAUZERALL, DAVID, The Rockefeller University, 1230 York Ave., New York, NY 10021 MAZIA, DANIEL, Hopkins Marine Station, Pacific Grove, CA 93950 MAZZELLA, LUCIA, Laboratorio di Ecologia del Benthos, Stazione Zoologica di Napoli, P.ta S. Pietro 80077, Ischia Porto (NA), Italy McCANN, FRANCES, Department of Physiology, Dartmouth Medical School, Hanover, NH 03755 McCLOSKEY, LAWRENCE R., Department of Biology, Walla Walla College, College Place, WA 99324 MCLAUGHLIN, JANE A., PO Box 187, Woods Hole, MA 02543 McMAHON, ROBERT F., Department of Biology, Box 19498, University of Texas, Arlington, TX76019 MEEDEL, THOMAS, Boston University Marine Program, Marine Biological Laboratory, Woods Hole, MA 02543 MEINERTZHAGEN, IAN A., Department of Psychology, Life Sciences Center, Dalhousie Uni- versity, Halifax, Nova Scotia, Canada B3H 451 MEINKOTH, NORMAN A., 43 1W Woodland Avenue, Springfield, PA 19064 MEISS, DENNIS E., 462 Solano Avenue, Hayward, CA 94541 MELILLO, JERRY A., Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543 MELLON, RICHARD P., PO Box 187, Laughlintown, PA 15655 MEMBERS OF THE CORPORATION 19 MELLON, DEFOREST, JR., Department of Biology, University of Virginia, Charlottesville, VA 22903 MENZEL, RANDOLF, Institut fir Tierphysiologie, Free Universitat of Berlin, 1000 Berlin 41, Federal Republic of Germany METUZALS, JANIS, Department of Anatomy, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada KIN 9A9 METZ, CHARLES B., Institute for Molecular and Cellular Evolution, University of Miami, 521 Anastasia Ave., Coral Gables, FL 33134 MILKMAN, ROGER, Department of Zoology, University of Iowa, Iowa City, IA 52242 MILLS, ERIC L., Institute of Oceanography, Dalhousie University, Halifax, Nova Scotia, Canada MILLS, ROBERT, 10315 44th Avenue, W 12 H Street, Bradenton, FL 33507-1535 MITCHELL, RALPH, Pierce Hall, Harvard University, Cambridge, MA 02138 MIYAMOTO, DAVID M., Department of Biology, Seton Hall University, South Orange, NJ 07079 MIZELL, MERLE, Department of Biology, Tulane University, New Orleans, LA 701 18 MONROY, ALBERTO, Stazione Zoologica, Villa Comunale, Naples, Italy MOORE, JOHN W., Department of Physiology, Duke University Medical Center, Durham, NC 27710 MOORE, LEE E., Department of Physiology and Biophysics, University of Texas, Medical Branch, Galveston, TX 77550 MORIN, JAMES G., Department of Biology, University of California, Los Angeles, CA 90024 MORRELL, FRANK, Department of Neurological Sciences, Rush Medical Center, 1753 W. Con- gress Parkway, Chicago, IL 60612 MORRILL, JOHN B., JR., Division of National Sciences, New College, Sarasota, FL 33580 MORSE, RICHARD S., 193 Winding River Rd., Wellesley, MA 02181 MORSE, ROBERT W., Box 574, N. Falmouth, MA 02556 MORSE, STEPHEN SCOTT, The Rockefeller University, 1230 York Ave., Box 2, New York, NY 10021-6399 MOSCONA, A. A., Department of Biology, University of Chicago, 920 East 58th St., Chicago, IL 60637 MOTE, MICHAEL I., Department of Biology, Temple University, Philadelphia, PA 19122 MOUNTAIN, ISABEL, Vinson Hall #1 12, 6251 Old Dominion Drive, McLean, VA 22101 MUSACCHIA, XAVIER J., Graduate School, University of Louisville, Louisville, KY 40292 NABRIT, S. M., 686 Beckwith St., SW, Atlanta, GA 30314 NAKA, KEN-!CHI, National Institute for Basic Biology, Okazaki, 444, Japan NAKAJIMA, SHIGEHIRO, Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 NAKAJIMA, YASUKO, Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 NARAHASHI, TOSHIO, Department of Pharmacology, Medical Center, Northwestern University, 303 East Chicago Ave., Chicago, IL 6061 1 NASATIR, MAIMON, Department of Biology, University of Toledo, Toledo, OH 43606 NELSON, LEONARD, Department of Physiology, Medical College of Ohio, Toledo, OH 43699 NELSON, MARGARET C, Section on Neurobiology and Behavior, Cornell University, Ithaca, NY 14850 NICHOLLS, JOHN G., Biocenter, Klingelbergstr 70, Basel 4056, Switzerland NICOSIA, SANTO V., Department of Pathology, University of South Florida, College of Medicine, Box 1 1, 12901 North 30th St., Tampa, FL 33612 NIELSEN, JENNIFER B. K., Merck, Sharp & Dohme Laboratories, Bldg. 50-G, Room 226, Rahway, NJ 07065 NOE, BRYAN D., Department of Anatomy, Emory University, Atlanta, GA 30345 OBAID, ANA LIA, Department of Physiology and Pharmacy, University of Pennsylvania, 4001 Spruce St., Philadelphia, PA 19104 OCHOA, SEVERO, 530 East 72nd St., New 'ork, NY 10021 ODUM, EUGENE, Department of Zoology, University of Georgia, Athens, GA 30701 20 MARINE BIOLOGICAL LABORATORY OERTEL, DONATA, Department of Neurophysiology, University of Wisconsin, 283 Medical Science Bldg., Madison, WI 53706 O'HERRON, JONATHAN, Lazard Freres and Company, One Rockefeller Plaza, New York, NY 10020 OLINS, ADA L., University of Tennessee Oak Ridge, Graduate School of Biomedical Sciences, Biology Division ORNL, PO Box Y, Oak Ridge, TN 37830 OLINS, DONALD E., University of Tennessee Oak Ridge, Graduate School of Biomedical Sci- ences, Biology Division ORNL, PO Box Y, Oak Ridge, TN 37830 O'MELIA, ANNE F., Department of Anatomy, New York Medical College, Valhalla, NY 10595 OSCHMAN, JAMES L., 9 George Street, Woods Hole, MA 02543 PALMER, JOHN D., Department of Zoology, University of Massachusetts, Amherst, MA 01002 PALTI, YORAM, Department of Physiology and Biophysics, Israel Institute of Technology, 12 Haaliya St., BAT-GALIM, POB 9649, Haifa, Israel PANT, HARISH C, Laboratory of Preclinical Studies, National Institute on Alcohol Abuse and Alcoholism, 12501 Washington Ave., Rockville, MD 20852 PAPPAS, GEORGE D., Department of Anatomy, College of Medicine, University of Illinois, 808 South Wood St., Chicago, IL 60612 PARDEE, ARTHUR B., Department of Pharmacology, Harvard Medical School, Boston, MA 02115 PARDY, ROSEVELT L., School of Life Sciences, University of Nebraska, Lincoln, NE 68588 PARMENTIER, JAMES L., Duke University Marine Laboratory, Beaufort, NC 28516 PASSANO, LEONARD M., Department of Zoology, Birge Hall, University of Wisconsin, Madison, WI 53706 PEARLMAN, ALAN L., Department of Physiology, School of Medicine, Washington University, St. Louis, MO 63 110 PEDERSON, THORU, Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545 PERKINS, C. D., 400 Hilltop Terrace, Alexandria, VA 22301 PERSON, PHILIP, Special Dental Research Program, Veterans Administration Hospital, Brooklyn, NY 11219 PETERSON, BRUCE J., Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543 PETHIG, RONALD, School of Electronic Engineering Science, University College of N. Wales, Dean St., Bangor, Gwynedd, LL57 IUT, U. K. PETTIBONE, MARIAN H., Division of Worms, W-213, Smithsonian Institution, Washington, DC 20560 PFOHL, RONALD J., Department of Zoology, Miami University, Oxford, OH 45056 PIERCE, SIDNEY K., JR., Department of Zoology, University of Maryland, College Park, MD 20740 POINDEXTER, JEANNE S., Public Health Research Institute, 455 First Avenue, New York, NY 10016 POLLARD, HARVEY B., NIH, F. Building 10, Room 10B17, Bethesda, MD 20892 POLLARD, THOMAS D., Department of Cell Biology and Anatomy, Johns Hopkins University, 725 North Wolfe St., Baltimore, MD 21205 POLLOCK, LELAND W., Department of Zoology, Drew University, Madison, NJ 07940 PORTER, BEVERLY H., 13617 Glenoble Drive, Rockville, MD 20853 PORTFR, KEITH R., Biosciences Department, University of Maryland, Baltimore County, Wilkens Av< nue, Catonsville, MD 21228 PORTER. ARY E., Department MCD Biology, Campus Box 347, University of Colorado, Boulder, CO 80309 POTTER, DAVIL>, Department of Neurobiology, Harvard Medical School, Boston, MA 021 15 POTTER, H. DAVSD, PO Box 2286, Bloomington, IN 47401 (resigned 7/2/85) POTTS, WILLIAM T., Department of Biology, University of Lancaster, Lancaster, England, U.K. POUSSART, DENIS, Department of Electrical Engineering, Universite Laval, Quebec, Canada PRATT, MELANIE M., Department of Anatomy and Cell Biology, University of Miami School of Medicine (R124), PO Box 016960, Miami, FL 33101 MEMBERS OF THE CORPORATION 2 1 PRENDERGAST, ROBERT A., Department of Pathology and Ophthalmology, Johns Hopkins University, Baltimore, MD 21205 PRICE, CARL A., Waksman Institute of Microbiology, Rutgers University, PO Box 759, Pisca- taway, NJ 08854 PRICE, CHRISTOPHER H., Biological Science Center, Boston University, 2 Cummington St., Boston, MA 02215 PRIOR, DAVID J., Department of Biological Sciences, University of Kentucky, Lexington, KY 40506 PRUSCH, ROBERT D., Department of Life Sciences, Gonzaga University, Spokane, WA 99258 PRZYBYLSKI, RONALD J., Case Western Reserve University, Department of Anatomy, Cleveland, OH 44 104 PURVES, DALE, Department of Physiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 631 10 QUIGLEY, JAMES, Department of Microbiology and Immunology Box 44, SUNY Downstate Medical Center, 450 Clarkson Ave., Brooklyn, NY 1 1203 RABIN, HARVEY, DuPont Biomed. Prod.-BRL-2, 331 Treble Cove Road, No. Billerica, MA 01862 RAFF, RUDOLF A., Department of Biology, Indiana University, Bloomington, IN 47405 RAKOWSKJ, ROBERT F., Department of Physiology and Biophysics, UHS/ The Chicago Medical School, 3333 Greenbay Rd., N. Chicago, IL 60064 RAMON, FIDEL, Dept. de Fisiologia y Biofisica, Centrol de Investigacion y de, Estudius Avanzados del Ipn, Apurtado Postal 14-740, Mexico, D. F. 07000 RANZI, SILVIO, Sez Zoologia Sc Nat, Via Coloria 26, 120133, Milano, Italy RATNER, SARAH, Department of Biochemistry, Public Health Research Institute, 455 First Ave., New York, NY 10016 REBHUN, LIONEL I., Department of Biology, Gilmer Hall, University of Virginia, Charlottesville, VA 22903 REDDAN, JOHN R., Department of Biological Sciences, Oakland University, Rochester, MI 48063 REESE, THOMAS S., Marine Biological Laboratory, Woods Hole, MA 02543 REINER, JOHN M., Department of Biochemistry, Albany Medical College of Union University. Albany, NY 12208 REINISCH, CAROL L., Tufts University School of Veterinary Medicine, 203 Harrison Avenue, Boston, MA 02 1 1 5 REUBEN, JOHN P., Department of Biochemistry, Merck, Sharp and Dohme, PO Box 2000, Rahway, NJ 07065 REYNOLDS, GEORGE T., Department of Physics, Jadwin Hall, Princeton University, Princeton, NJ 08540 RICE, ROBERT V., 30 Burnham Dr., Falmouth, MA 02540 RICKLES, FREDERICK R., University of Connecticut, School of Medicine, VA Hospital, New- ington, CT 06 1 1 1 RIPPS, HARRIS, Department of Ophthalmology, University of Illinois at Chicago, College of Medicine, 1855 W. Taylor Street, Chicago, IL 6061 1 ROBERTS, JOHN L., Department of Zoology, University of Massachusetts, Amherst, MA 01002 ROBINSON, DENIS M., High Voltage Engineering Corporation, Burlington, MA 01803 ROCKSTEIN, MORRIS, 335 Fluvia Ave., Miami, FL 33134 RONKIN, RAPHAEL R., 3212 McKinley St., NW, Washington, DC 20015 (Life Member 10/85) ROSBASH, MICHAEL, Rosenstiel Center, Department of Biology, Brandeis University, Waltham, MA 02 154 ROSE, BIRGIT, Department of Physiology R-430, University of Miami School of Medicine, PO Box 016430, Miami, FL 33152 ROSE, S. MERYL, Box 309 W, Waquoit, MA 02536 ROSENBAUM, JOEL L., Department of Biology, Kline Biology Tower, Yale University, New Haven, CT 06520 ROSENBERG, PHILIP, School of Pharmacy, Division of Pharmacology, University of Connecticut, Storrs, CT 06268 22 MARINE BIOLOGICAL LABORATORY ROSENBLUTH, JACK, Department of Physiology, New York University School of Medicine, 550 First Ave., New York, NY 10016 ROSENBLUTH, RAJA, 3380 West 5th Ave., Vancouver 8, British Columbia, Canada V6R 1R7 ROSLANSKY, JOHN, Box 208, Woods Hole, MA 02543 ROSLANSKY, PRISCILLA F., Box 208, Woods Hole, MA 02543 Ross, WILLIAM N., Department of Physiology, New York Medical College, Valhalla, NY 10595 ROTH, JAY S., Division of Biological Sciences, Section of Biochemistry and Biophysics, University of Connecticut, Storrs, CT 06268 ROWLAND, LEWIS P., Neurological Institute, 710 West 168th St., New York, NY 10032 RUDERMAN, JOAN V., Department of Anatomy, Harvard Medical School, Boston, MA 021 15 RUSHFORTH, NORMAN B., Case Western Reserve University, Department of Biology, Cleveland, OH 44 106 RUSSELL-HUNTER, W. D., Department of Biology, Lyman Hall 029, Syracuse University, Syr- acuse, NY 13210 SAFFO, MARY BETH, Center for Marine Studies, 273 Applied Sciences, University of California, Santa Cruz, CA 95064 SAGER, RUTH, Sidney Farber Cancer Institute, 44 Binney St., Boston, MA 021 15 SALAMA, GUY, Department of Physiology, University of Pittsburgh, Pittsburgh, PA 15261 SALMON, EDWARD D., Department of Zoology, University of North Carolina, Chapel Hill, NC 27514 SALZBERG, BRIAN M., Department of Physiology, University of Pennsylvania, 4010 Locust St., Philadelphia, PA 19174 SANDERS, HOWARD, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 SANGER, JEAN M., Department of Anatomy, School of Medicine, University of Pennsylvania, 36th and Hamilton Walk, Philadelphia, PA 19174 SANGER, JOSEPH, Department of Anatomy, School of Medicine, University of Pennsylvania, 36th and Hamilton Walk, Philadelphia, PA 19174 SATO, EIMEI, Department of Animal Science, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan SATO, HIDEMI, Sugashima Marine Biological Laboratory, Nagoya University, Sugashima-cho, Toba-chi, Mie-Ken 5 1 7, Japan SATTELLE, DAVID B., AFRC Unit-Department of Zoology, University of Cambridge, Downing St., Cambridge CB2 3EJ, U. K. SAUNDERS, JOHN, JR., Department of Biological Sciences, SUNY, Albany, NY 12222 SAZ, ARTHUR K., Medical and Dental Schools, Georgetown University, 3900 Reservoir Rd., NW, Washington, DC 20051 SCHACHMAN, HOWARD K., Department of Molecular Biology, University of California, Berkeley, CA 94720 SCHIFF, JEROME A., Institute for Photobiology of Cells and Organelles, Brandeis University, Waltham, MA02154 SCHMEER, ARLENE C., Mercene Cancer Research Hospital of Saint Raphael, New Haven, CT 06511 SCHNAPP, BRUCE J., Marine Biological Laboratory, Woods Hole, MA 02543 SCHNEIDER, E. GAYLE, University of Nebraska Medical Center, Department of Biochemistry, 42nd and Dewey Ave., Omaha, NE 68105 SCHNEIDERMAN, HOWARD A., Monsanto Company, 800 North Lindberg Blvd., Dl W, St. Louis, MO 63166 SCHOTTE, OSCAR E., Department of Biology, Amherst College, Amherst, MA 01002 SCHUEL, HERBERT, Department of Anatomical Sciences, SUNY, Buffalo, NY 14214 SCHUETZ, ALLEN W., School of Hygiene and Public Health, Johns Hopkins University, Balti- more, MD21205 SCHWARTZ, JAMES H., Center for Neurobiology and Behavior, New York State Psychiatric Institute Research Annex, 722 W. 168th St., 7th Floor, New York, NY 10032 SCHWARTZ, MARTIN, Department of Biological Sciences, University of Maryland, Baltimore County, Catonsville, MD 21228 (deceased 8/85) SCOFIELD, VIRGINIA LEE, Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90024 MEMBERS OF THE CORPORATION 23 SEARS, MARY, PO Box 152, Woods Hole, MA 02543 SEGAL, SHELDON J., Population Division, The Rockefeller Foundation, 1133 Avenue of the Americas, New York, NY 10036 SELIGER, HOWARD H., Johns Hopkins University, McCollum-Pratt Institute, Baltimore, MD 21218 SELMAN, KELLY, Department of Anatomy, College of Medicine, University of Florida, Gaines- ville, FL 32601 SENFT, JOSEPH, 378 Fairview St., Emmaus, PA 18049 SHANKLIN, DOUGLAS R., PO Box 1267, Gainesville, FL 32602 SHAPIRO, HERBERT, 6025 North 13th St., Philadelphia, PA 19141 SHAVER, GAIUS R., Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543 SHAVER, JOHN R., Apt. 602, Cond. Castillo del Mar, Isla Verde, San Juan, PR 00913 SHEETZ, MICHAEL P., Department of Cell Biology and Physiology, Washington University Medical School, 606 S. Euclid Ave., St. Louis, MO 631 10 SHEPARD, DAVID C, PO Box 44, Woods Hole, MA 02543 SHEPRO, DAVID, Department of Biology, Boston University, 2 Cummington St., Boston, MA 02215 SHERIDAN, WILLIAM F., Biology Department, University of North Dakota, Grand Forks, ND 58202 SHERMAN, I. W., Division of Life Sciences, University of California, Riverside, CA 92502 SHILO, MOSHE, Department of Microbiological Chemistry, Hebrew University, Jerusalem, Israel SHOUKIMAS, JONATHAN J., Marine Biological Laboratory, Woods Hole, MA 02543 SIEGEL, IRWIN M., Department of Ophthalmology, New York University Medical Center, 550 First Avenue, New York, NY 10016 SIEGELMAN, HAROLD W., Department of Biology, Brookhaven National Laboratory, Upton, NY 11973 SILVER, ROBERT B., Laboratory of Molecular Biology, University of Wisconsin, 1525 Linden Drive, Madison, WI 53706 SJODIN, RAYMOND A., Department of Biophysics, University of Maryland, Baltimore, MD 21201 SKINNER, DOROTHY M., Oak Ridge National Laboratory, Biology Division, Oak Ridge, TN 37830 SLOBODA, ROGER D., Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 SLUDER, GREENFIELD, Cell Biology Group, Worcester Foundation for Experimental Biology, 22 Maple Ave., Shrewsbury, MA 01545 SMITH, HOMER P., Marine Biological Laboratory, Woods Hole, MA 02543 SMITH, MICHAEL A., Jl Sinabung, Buntu #7, Semarang, Java, Indonesia SMITH, PAUL F., PO Box 264, Woods Hole, MA 02543 SMITH, RALPH I., Department of Zoology, University of California, Berkeley, CA 94720 SORENSON, MARTHA M., Depto de Bioquimica-RFRJ, Centre de Ciencias da Saude-I.C.B., Cidade Universitaria-Fundad, Rio de Janeiro, Brasil 21.910 SPECK, WILLIAM T., Case Western Reserve University, Department of Pediatrics, Cleveland, OH 44 106 SPECTOR, A., College of Physicians and Surgeons, Columbia University, Black Bldg., Room 1516, New York, NY 10032 SPEER, JOHN W., Marine Biological Laboratory, Woods Hole, MA 02543 SPIEGEL, EVELYN, Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 SPIEGEL, MELVIN, Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 SPRAY, DAVID C., Albert Einstein College of Medicine, Department of Neurosciences, 1300 Morris Park Avenue, Bronx, NY 10461 STEELE, JOHN HYSLOP, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 STEINACHER, ANTOINETTE, Department of Otolaryngology, Washington University, School of Medicine, 491 1 Barnes Hospital, St. Louis, MO 631 10 STEINBERG, MALCOLM, Department of Biology, Princeton University, Princeton, NJ 08540 STEPHENS, GROVER C., Department of Developmental and Cell Biology, University of California, Irvine, CA 927 17 24 MARINE BIOLOGICAL LABORATORY STEPHENS, RAYMOND E., Marine Biological Laboratory, Woods Hole, MA 02543 STETTEN, DEWITT, JR., Senior Scientific Advisor, NIH, Bldg. 16, Room 118, Bethesda, MD 20892 STETTEN, JANE LAZAROW, 2 West Drive, Bethesda, MD 20814 STEUDLER, PAUL A., Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543 STOKES, DARRELL R., Department of Biology, Emory University, Atlanta, GA 30322 STOMMEL, ELIJAH W., 766 Palmer Avenue, Falmouth, MA 02540 STRACHER, ALFRED, Downstate Medical Center, SUNY, 450 Clarkson Ave., Brooklyn, NY 11203 STREHLER, BERNARD L., 2235 25th St., #217, San Pedro, CA 90732 STUART, ANN E., Department of Physiology, Medical Sciences Research Wing 206H, University of North Carolina, Chapel Hill, NC 27514 SUGIMORI, MUTSUYUKI, Department of Physiology and Biophysics, New York University Medical Center, 550 First Avenue, New York, NY 10016 SUMMERS, WILLIAM C., Huxley College, Western Washington University, Bellingham, WA 98225 SUSSMAN, MAURICE, Department of Life Sciences, University of Pittsburgh, Pittsburgh, PA 15260 SZABO, GEORGE, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115 SZENT-GYORGYI, ALBERT, Marine Biological Labortory, Woods Hole, MA 02543 SZENT-GYORGYI, ANDREW, Department of Biology, Brandeis University, Waltham, MA 02154 SZENT-GYORGYI, EVA SZENTKJRALY, Department of Biology, Brandeis University, Waltham, MA 02 154 SZUTS, ETE Z., Laboratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole, MA 02543 TAKASHIMA, SHIRO, Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19 174 (resigned 6/8 5) TAMM, SIDNEY L., Boston University Marine Program, Marine Biological Laboratory, Woods Hole, MA 02543 TANZER, MARVIN L., Department of Biochemistry, Box G, Medical School, University of Con- necticut, Farmington, CT 06032 TASAKI, ICHIJI, Laboratory of Neurobiology, Bldg. 36, Rm. 2D10, NIMH, NIH, Bethesda, MD 20892 TAYLOR, DOUGLASS L., Biological Sciences, Mellon Institute, 440 Fifth Avenue, Pittsburgh, PA 15213 TAYLOR, ROBERT E., Laboratory of Biophysics, NINCDS, NIH, Bethesda, MD 20892 TEAL, JOHN M., Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 TELFER, WILLIAM H., Department of Biology, University of Pennsylvania, Philadelphia, PA 19174 THORNDIKE, W. NICHOLAS, Wellington Management Company, 28 State St., Boston, MA 02109 TRAVIS, D. M., Veterans Administration Medical Center, Fargo, ND 58102 TREISTMAN, STEVEN N., Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545 TRIGG, D. THOMAS, 125 Grove St., Wellesley, MA 02181 TRINKAUS, J. PHILIP, Osborn Zoological Labs, Department of Zoology, Yale University, New Haven. CT 065 10 TROLL, WALTER, Department of Environmental Medicine, College of Medicine, New York University, ? / York, NY 10016 TROXLER, ROBERT F., Department of Biochemistry, School of Medicine, Boston University, 80 East Concord St., Boston, MA 021 18 TUCKER, EDWARD B.. Biology Department, Vassar College, Poughkeepsie, NY 12601 TURNER, RUTH D., Mollusk Department, Museum of Comparative Zoology, Harvard University, Cambridge, MA 02 138 TWEEDELL, KENYON S., Department of Biology, University of Notre Dame, Notre Dame, IN 46656 MEMBERS OF THE CORPORATION 25 TYTELL, MICHAEL, Department of Anatomy, Bowman Gray School of Medicine, Winston- Salem, NC27103 UENO, HIROSHI, Laboratory of Biochemistry, The Rockefeller University, 1230 York Ave., New York, NY 10021 URETZ, ROBERT B., Division of Biological Sciences, University of Chicago, 950 East 59th St., Chicago, IL 60637 VALIELA, IVAN, Boston University Marine Program, Marine Biological Laboratory, Woods Hole, MA 02543 VALOIS, JOHN, Marine Biological Laboratory, Woods Hole, MA 02543 VAN HOLDE, KENSAL, Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331 VILLEE, CLAUDE A., Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115 VINCENT, WALTER S., School of Life and Health Sciences, University of Delaware, Newark, DE 19711 WAINIO, WALTER W., 331 State Road, Princeton, NJ 08540 (Life Member 10/85) WAKSMAN, BYRON, National Multiple Sclerosis Society, 205 East 42nd St., New York, NY 10017 WALL, BETTY, 9 George St., Woods Hole, MA 02543 WALLACE, ROBIN A., Whitney Marine Laboratory, Rte. 1, Box 121, St. Augustine, FL 32086 WANG, AN, Wang Laboratories, Inc., Bedford Road, Lincoln, MA 01773 WARNER, ROBERT C, Department of Molecular Biology and Biochemistry, University of Cal- ifornia, Irvine, CA 927 1 7 WARREN, KENNETH S., The Rockefeller Foundation, 1 1 33 Avenue of the Americas, New York, NY 10036 WARREN, LEONARD, Department of Therapeutic Research, School of Medicine, Anatomy- Chemistry Building, University of Pennsylvania, Philadelphia, PA 19174 WATERMAN, T. 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RICHARD, Marine Biological Laboratory, Woods Hole, MA 02543 WIGLEY, ROLAND L., 35 Wilson Road, Woods Hole, MA 02543 WILBER, CHARLES G., Department of Zoology, Colorado State University, Fort Collins, CO 80523 WILSON, DARCY B., Medical Biology Institute, 1 1077 North Torrey Pines Road, La Jolla, CA 92037 WILSON, EDWARD O., Museum of Comparative Zoology, Harvard University, Cambridge, MA 02138 WILSON, T. HASTINGS, Department of Physiology, Harvard Medical School, Boston, MA 02 1 1 5 WILSON, WALTER L., 743 Cambridge Drive, Rochester, MI 48063 WITKOVSKY, PAUL, Department of Ophthalmology, New York University Medical Center, 550 First Ave., New York, NY 10016 WITTENBERG, JONATHAN B., Department of Physiology and Biochemistry, Albert Einstein College, 1300 Morris Park Ave., New York, NY 10016 26 MARINE BIOLOGICAL LABORATORY WOLFE, RALPH, Department of Microbiology, 131 Burrill Hall, University of Illinois, Urbana, IL 61801 WOODWELL, GEORGE M., 64 Church Street, Woods Hole, MA 02543 WORGUL, BASIL V., Department of Ophthalmology, Columbia University, 630 West 168th St., New York, NY 10032 Wu, CHAU HsiUNG, Department of Pharmacology, Northwestern University Medical School, 203 E. Chicago Ave., Chicago, IL 6061 1 WYTTENBACH, CHARLES R., Department of Physiology and Cell Biology, University of Kansas, Lawrence, KS 66045 YEH, JAY Z., Department of Pharmacology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 6061 1 YOUNG, RICHARD, Mentor O & O, Inc., South Shore Park, Hingham, MA 02043 ZACKROFF, ROBERT, 66 White Horn Drive, Kingston, RI 0288 1 ZIGMAN, SEYMOUR, School of Medicine and Dentistry, University of Rochester, 260 Crittenden Blvd., Rochester, NY 14620 ZIGMOND, RICHARD E., Department of Pharmacology, Harvard Medical School, 250 Longwood Ave., Boston, MA 02 115 ZIMMERBERG, JOSHUA J., Bldg. 12A, Room 2007, NIH, Bethesda, MD 20892 ZUCKER, ROBERT S., Department of Physiology, University of California, Berkeley, CA 94720 ASSOCIATE MEMBERS ACKROYD, DR. FREDERICK W. ADELBERG, DR. AND MRS. EDWARD A. AHEARN, MR. AND MRS. DAVID ALDEN, MR. JOHN M. ALLEN, Miss CAMILLA K. ALLEN, DR. NINA S. ANDERSON, MR. J. GREGORY ANDERSON, DRS. JAMES L. 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HOBBIE, DR. AND MRS. JOHN HOCKER, MR. AND MRS. LON HODGE, MRS. STUART HOFFMAN, REV. AND MRS. CHARLES HOKIN, MR. RICHARD HORNOR, MR. TOWNSEND HORWITZ, DR. AND MRS. NORMAN H. HOSKIN, DR. AND MRS. FRANCIS HOUSTON, MR. AND MRS. HOWARD E. HOWARD, MR. AND MRS. L. L. HUETTNER, DR. AND MRS. ROBERT J. HUTCHISON, MR. ALAN D. HYNES, MR. AND MRS. THOMAS J., JR. INOUE, DR. AND MRS. SHINYA ISSOKSON, MR. AND MRS. ISRAEL 28 MARINE BIOLOGICAL LABORATORY JACKSON, Miss ELIZABETH B. JAFFE, DR. AND MRS. ERNST R. JANNEY, MRS. F. WISTAR JEWETT, G. F., FOUNDATION JEWETT, MR. AND MRS. G. F., JR. JONES, MR. AND MRS. DEWITT C, III JONES, MR. AND MRS. FREDERICK, II JORDAN, DR. AND MRS. EDWIN P. KAAN, DR. HELEN W. KAHLER, MR. AND MRS. GEORGE A. KAHLER, MRS. ROBERT W. KAMINER, DR. AND MRS. BENJAMIN KARPLUS, MRS. ALAN K. KARUSH, DR. AND MRS. FRED KELLEHER, MR. AND MRS. PAUL R. KENDALL, MR. AND MRS. RICHARD E. KEOSIAN, MRS. JESSIE KETCHUM, MRS. PAUL KlEN, MR. AND MRS. PlETER KJNNARD, MRS. L. RICHARD KISSAM, MR. WILLIAM M. KIVY, DR. AND MRS. PETER KOHN, DR. AND MRS. HENRY I. KOLLER, DR. LEWIS R. KORGEN, DR. BEN J. KUFFLER, MRS. STEPHEN W. LAFFERTY, Miss NANCY LASTER, DR. AND MRS. LEONARD LAUFER, DR. AND MRS. HANS LAVIGNE, MRS. RICHARD J. LAWRENCE, MR. FREDERICK V. LAWRENCE, MR. AND MRS. WILLIAM LEATHERBEE, MRS. JOHN H. LEBLOND, MR. AND MRS. ARTHUR LEESON, MR. AND MRS. A. Dix LEHMAN, Miss ROBIN LEMANN, MRS. LUCY B. LENHER, DR. AND MRS. SAMUEL LEVINE, DR. AND MRS. RACHMIEL LEVY, MR. STEPHEN R. LINDNER, MR. TIMOTHY P. LITTLE, MRS. ELBERT LOEB, MRS. ROBERT F. LOVELL, MR. AND MRS. HOLLIS R. LOWE, DR. AND MRS. CHARLES W. LOWENGARD, MRS. JOSEPH MACKEY, MR. AND MRS. WILLIAM K. MACLEISH, MRS. MARGARET MACNARY, MR. AND MRS. B. GLENN MACNlCHOL, DR. AND MRS. EDWARD F., JR. MAHER, Miss ANNE CAMILLE MAHLER, MRS. HENRY MARSH, Miss JULIA MARSLAND, DR. DOUGLAS MARTYNA, MR. AND MRS. JOSEPH C. MARVIN, DR. DOROTHY H. MASTROIANNI, DR. AND MRS. LUIGI, JR. MATHER, MR. AND MRS. FRANK J., III. MATHERLY, MR. AND MRS. WALTER MATTHIESSEN, DR. AND MRS. G. C. McCoy, DR. AND MRS. FLOYD W. MCCUSKER, MR. AND MRS. PAUL T. MCELROY, MRS. NELLA W. MCILWAIN, DR. SUSAN G. MCLANE, MRS. T. THORNE MEIGS, MR. AND MRS. ARTHUR MEIGS, DR. AND MRS. J. WISTER MELILLO, DR. AND MRS. JERRY M. MELLON, RICHARD KING, TRUST MELLON, MR. AND MRS. RICHARD P. MENDELSON, DR. MARTIN METZ, DR. AND MRS. CHARLES B. MEYERS, MR. AND MRS. RICHARD MILLER, DR. DANIEL A. MIXTER, MR. AND MRS. WILLIAM J., JR. MIZELL, DR. AND MRS. MERLE MONROY, DR. AND MRS. ALBERTO MONTGOMERY, DR. AND MRS. CHARLES H. MONTGOMERY, DR. AND MRS. RAYMOND B. MOOG, DR. FLORENCE MOORE, DR. AND MRS. JOHN A. MORSE, MRS. CHARLES L., JR. MORSE, DR. M. PATRICIA MOUL, DR. AND MRS. EDWIN T. MURRAY, DR. DAVID M. MYLES-TOCHKO, DR. CHRISTINA J. NACE, DR. AND MRS. PAUL NELSON, MRS. LEONARD NELSON, DR. PAMELA NEWTON, MR. WILLIAM F. NlCKERSON, MR. AND MRS. FRANK L. NORMAN, MR. AND MRS. ANDREW E. NORMAN FOUNDATION NORRIS, MR. AND MRS. JOHN A. NORRIS, MR. WILLIAM O'HERRON, MR. AND MRS. JONATHAN ORTINS, MR. AND MRS. ARMAND O'SULLIVAN, DR. RENEE BENNETT PAPPAS, DR. AND MRS. GEORGE D. PARK, MRS. FRANKLIN A. PARK, MR. AND MRS. MALCOLM S. PARMENTER, Miss CAROLYN L. PENDERGAST, MRS. CLAUDIA PENDLETON, DR. AND MRS. MURRAY E. PENNINGTON, Miss ANNE H. PERKINS, MR. AND MRS. COURTLAND D. PERSON, DR. AND MRS. PHILIP MEMBERS OF THE CORPORATION 29 PETERSON, MR. AND MRS. E. GUNNAR PETERSON, MR. AND MRS. E. JOEL PETERSON, MR. RAYMOND W. POINTE, MR. ALBERT PORTER, DR. AND MRS. KEITH R. PRESS, DRS. FRANK. AND BILLIE PROSKAUER, MR. RICHARD PROSSER, DR. AND MRS. C. LADD PSALEDAKIS, MR. NICHOLAS PSYCHOYOS, DR. ALEXANDRE PUTNAM, MR. ALLAN RAY PUTNAM, MR. AND MRS. WILLIAM A., Ill RAYMOND, DR. AND MRS. SAMUEL REYNOLDS, DR. AND MRS. GEORGE REZNIKOFF, MRS. PAUL RICCA, DR. AND MRS. RENATO A. RIGHTER, MR. HAROLD RIINA, MR. AND MRS. JOHN R. ROBB, MRS. ALISON A. ROBERTS, Miss JEAN ROBERTSON, MRS. C. W. ROBINSON, DR. DENIS M. ROOT, MRS. WALTER S. ROSENTHAL, MlSS HlLDE ROSLANSKY, DRS. JOHN AND PRISCILLA Ross, DR. AND MRS. DONALD Ross, DR. VIRGINIA ROTH, MR. STEPHEN ROWE, MR. DON ROWE, MR. AND MRS. WILLIAM S. RUBIN, DR. JOSEPH RUGH, MRS. ROBERTS RUSSELL, MR. AND MRS. HENRY D. RYDER, MR. AND MRS. FRANCIS C. SAGER, DR. RUTH SALGUERO, MRS. CAROL G. SARDINHA, MR. GEORGE H. SAUNDERS, DR. AND MRS. JOHN W. SAUNDERS, MRS. LAWRENCE SAUNDERS, LAWRENCE, FUND SAWYER, MR. AND MRS. JOHN E. SAZ, MRS. RUTH L. SCHLESINGER, DR. AND MRS. R. WALTER SCOTT, DR. AND MRS. GEORGE T. SCOTT, MR. AND MRS. NORMAN E. SEARS, MR. AND MRS. HAROLD B. SEARS, MR. HAROLD H. SEGAL, DR. AND MRS. SHELDON J. SENFT, DR. AND MRS. ALFRED SHAPIRO, MRS. HARRIET S. SHEMIN, DR. AND MRS. DAVID SHEPRO, DR. AND MRS. DAVID SIMMONS, MR. TIM SMITH, DRS. FREDERICK E. AND MARGUERITE A. SMITH, MRS. HOMER P. SMITH, MR. VAN DORN C. SNYDER, MR. ROBERT M. SOLOMON, DR. AND MRS. A. K. SPECHT, MRS. HEINZ SPIEGEL, DR. AND MRS. MELVIN STEELE, MRS. JOHN H. STEINBACH, MRS. H. BURR STETSON, MRS. THOMAS J. STETTEN, DR. AND MRS. DEWITT, JR. STEWART, MR. AND MRS. PETER STONE, MR. ANDREW G. STREHLER, DR. AND MRS. BERNARD STUNKARD, DR. HORACE SWANSON, DR. AND MRS. CARL P. SWOPE, MR. AND MRS. GERARD L. SWOPE, MRS. GERARD, JR. TAYLOR, MRS. MARGERY G. TAYLOR, DR. AND MRS. W. RANDOLPH TIETJE, MR. AND MRS. EMIL D., JR. TIMMINS, MRS. WILLIAM TODD, MR. AND MRS. GORDON F. TOLKAN, MR. AND MRS. NORMAN N. TRACER, MRS. WILLIAM TRIGG, MR. AND MRS. D. THOMAS TROLL, DR. AND MRS. WALTER TULLY, MR. AND MRS. GORDON F. ULBRICH, MRS. MARY STEINBACH VALOIS, MR. AND MRS. JOHN VEEDER, MRS. RONALD A. VINCENT, DR. WALTER S. WAKSMAN, DR. AND MRS. BYRON H. WARD, DR. ROBERT T. WARE, MR. AND MRS. J. LINDSAY WARREN, DR. HENRY B. WATT, MR. AND MRS. JOHN B. WEEKS, MR. AND MRS. JOHN T. WEINSTEIN, Miss NANCY B. WEISBERG, MR. AND MRS. ALFRED M. WHEELER, MR. AND MRS. PAUL S. WHITNEY, MR. AND MRS. GEOFFREY G., JR. WlCHTERMAN, DR. AND MRS. RALPH WlCKERSHAM, MR. AND MRS. A. A. TlLNEY WIESE, DR. CONRAD WILHELM, DR. HAZEL S. WILSON, MR. AND MRS. T. HASTINGS WINN, DR. WILLIAM M. WOLFINSOHN, MRS. WOLFE WOODWELL, DR. AND MRS. GEORGE M. YNTEMA, MRS. CHESTER L. ZINN, DR. AND MRS. DONALD J. ZIPF, DR. ELIZABETH ZWILLING, MRS. EDGAR 30 MARINE BIOLOGICAL LABORATORY III. CERTIFICATE OF ORGANIZATION (On File in the Office of the Secretary of the Commonwealth) No. 3170 We. Alpheus Hyatt, President, William Stanford Stevens, Treasurer, and William T. Sedgwick, Edward G. Gardiner, Susan Mims and Charles Sedgwick Minot being a majority of the Trustees of the Marine Biological Laboratory in compliance with the requirements of the fourth section of chapter one hundred and fifteen of the Public Statutes do hereby certify that the following is a true copy of the agreement of association to constitute said Corporation, with the names of the subscribers thereto: We, whose names are hereto subscribed, do, by this agreement, associate ourselves with the intention to constitute a Corporation according to the provisions of the one hundred and fifteenth chapter of the Public Statutes of the Commonwealth of Massachusetts, and the Acts in amend- ment thereof and in addition thereto. The name by which the Corporation shall be known is THE MARINE BIOLOGICAL LAB- ORATORY. The purpose for which the Corporation is constituted is to establish and maintain a laboratory or station for scientific study and investigations, and a school for instruction in biology and natural history. The place within which the Corporation is established or located is the city of Boston within said Commonwealth. The amount of its capital stock is none. In Witness Whereof we have hereunto set our hands, this twenty seventh day of February in the year eighteen hundred and eighty-eight, Alpheus Hyatt, Samuel Mills, William T. Sedgwick, Edward G. Gardiner, Charles Sedgwick Minot, William G. Farlow, William Stanford Stevens, Anna D. Phillips, Susan Mims, B. H. Van Vleck. That the first meeting of the subscribers to said agreement was held on the thirteenth day of March in the year eighteen hundred and eighty-eight. In Witness Whereof, we have hereunto signed our names, this thirteenth day of March in the year eighteen hundred and eighty-eight, Alpheus Hyatt, President, William Stanford Stevens, Treasurer, Edward G. Gardiner, William T. Sedgwick, Susan Mims, Charles Sedgwick Minot. (Approved on March 20, 1888 as follows: / hereby certify that it appears upon an examination of the within written certificate and the records of the corporation duly submitted to my inspection, that the requirements of sections one, two and three of chapter one hundred and fifteen, and sections eighteen, twenty and twenty-one of chapter one hundred and six, of the Public Statutes, have been complied with and I hereby approve said certificate this twentieth day of March A.D. eighteen hundred and eighty-eight. CHARLES ENDICOTT Commissioner of Corporations) BYLAWS 3 1 IV. ARTICLES OF AMENDMENT (On File in the Office of the Secretary of the Commonwealth) We. James D. Ebert, President, and David Shepro, Clerk of the Marine Biological Laboratory, located at Woods Hole, Massachusetts 02543, do hereby certify that the following amendment to the Articles of Organization of the Corporation was duly adopted at a meeting held on August 15, 1975, as adjourned to August 29, 1975, by vote of 444 members, being at least two-thirds of its members legally qualified to vote in the meeting of the corporation: VOTED: That the Certificate of Organization of this corporation be and it hereby is amended by the addition of the following provisions: "No Officer, Trustee or Corporate Member of the corporation shall be personally liable for the payment or satisfaction of any obligation or liabilities incurred as a result of, or otherwise in connection with, any commitments, agreements, activities or affairs of the corporation. "Except as otherwise specifically provided by the Bylaws of the corporation, meetings of the Corporate Members of the corporation may be held anywhere in the United States. "The Trustees of the corporation may make, amend or repeal the Bylaws of the corporation in whole or in part, except with respect to any provisions thereof which shall by law, this Certificate or the bylaws of the corporation, require action by the Corporate Members." The foregoing amendment will become effective when these articles of amendment are filed in accordance with Chapter 180, Section 7 of the General Laws unless these articles specify, in accordance with the vote adopting the amendment, a later effective date not more than thirty days after such filing, in which event the amendment will become effective on such later date. In Witness whereof and Under the Penalties of Perjury, we have hereto signed our names this 2nd day of September, in the year 1975, James D. Ebert, President; David Shepro, Clerk. (Approved on October 24, 1975, as follows: I hereby approve the within articles of amendment and, the filing fee in the amount of $10 having been paid, said articles are deemed to have been filed with me this 24th day of October, 1975. PAUL GUZZI Secretary of the Commonwealth) V. BYLAWS OF THE CORPORATION OF THE MARINE BIOLOGICAL LABORATORY (Revised August 16, 1985) I. (A) The name of the Corporation shall be The Marine Biological Laboratory. The Cor- poration's purpose shall be to establish and maintain a laboratory or station for scientific study and investigation, and a school for instruction in biology and natural history. (B) Marine Biological Laboratory admits students without regard to race, color, sex, national and ethnic origin to all the rights, privileges, programs and activities generally accorded or made 32 MARINE BIOLOGICAL LABORATORY available to students in its courses. It does not discriminate on the basis of race, color, sex, national and ethnic origin in employment, administration of its educational policies, admissions policies, scholarship and other programs. II. (A) The members of the Corporation ("Members") shall consist of persons elected by the Board of Trustees, upon such terms and conditions and in accordance with such procedures, not inconsistent with law or these Bylaws, as may be determined by said Board of Trustees. Except as provided below, any Member may vote at any meeting either in person or by proxy executed no more than six months prior to the date of such meeting. Members shall serve until their death or resignation unless earlier removed with or without cause by the affirmative vote of two-thirds of the Trustees then in office. Any member who has attained the age of seventy years or has retired from his home institution shall automatically be designated a Life Member provided he signifies his wish to retain his membership. Life Members shall not have the right to vote and shall not be assessed for dues. (B) The Associates of the Marine Biological Laboratory shall be an unincorporated group of persons (including associations and corporations) interested in the Laboratory and shall be organized and operated under the general supervision and authority of the Trustees. III. The officers of the Corporation shall consist of a Chairman of the Board of Trustees, President, Director, Treasurer and Clerk, elected or appointed by the Trustees as set forth in Article IX. IV. The Annual Meeting of the Members shall be held on the Friday following the Second Tuesday in August in each year at the Laboratory in Woods Hole, Massachusetts, at 9:30 a.m. Subject to the provisions of Article VIII(2), at such meeting the Members shall choose by ballot six Trustees to serve four years, and shall transact such other business as may properly come before the meeting. Special meetings of the Members may be called by the Chairman or Trustees to be held at such time and place as may be designated. V. Twenty five Members shall constitute a quorum at any meeting. Except as otherwise required by law or these Bylaws, the affirmative vote of a majority of the Members voting in person or by proxy at a meeting attended by a quorum (present in person or by proxy) shall constitute action on behalf of the Members. VI. (A) Inasmuch as the time and place of the Annual Meeting of Members are fixed by these Bylaws, no notice of the Annual Meeting need be given. Notice of any special meeting of Members, however, shall be given by the Clerk by mailing notice of the time and place and purpose of such meeting, at least 1 5 days before such meeting, to each Member at his or her address as shown on the records of the Corporation. (B) Any meeting of the Members may be adjourned to any other time and place by the vote of a majority of those Members present or represented at the meeting, whether or not such Members constitute a quorum. It shall not be necessary to notify any Member of any ad- journment. VII. The Annual Meeting of the Trustees shall be held promptly after the Annual Meeting of the Corporation at the Laboratory in Woods Hole, Massachusetts. Special meetings of the Trustees shall be called by the Chairman, the President, or by any seven Trustees, to be held at such time and place as may be designated. Notice of Trustees' meetings may be given orally, by telephone, telegraph or in writing; and notice given in time to enable the Trustees to attend, or in any case notice sent by mail or telegraph to a Trustee's usual or last known place of residence, at least one week before the meeting shall be sufficient. Notice of a meeting need not be given to any Trustee if a written waiver of notice, executed by him before or after the meeting is filed with the records of the meeting, or if he shall attend the meeting without protesting prior thereto or at its commencement the lack of notice to him. BYLAWS 33 VIII. (A) There shall be four groups of Trustees: ( 1 ) Trustees (the "Corporate Trustees") elected by the Members according to such procedures, not inconsistent with these Bylaws, as the Trustees shall have determined. Except as provided below, such Trustees shall be divided into four classes of six, one class to be elected year to serve for a term of four years. Such classes shall be designated by the year of expiration of their respective terms. (2) Trustees ("Board Trustees") elected by the Trustees then in office according to such procedures, not inconsistent with these Bylaws, as the Trustees shall have determined. Except as provided below, such Board Trustees shall be divided into four classes of three, one class to be elected each year to serve for a term of four years. Such classes shall be designated by the year of expiration of their respective terms. It is contemplated that, unless otherwise determined by the Trustees for good reason. Board Trustees shall be individuals who have not been considered for election as Corporate Trustees. (3) Trustees ex officio, who shall be the Chairman, the President, the Director, the Treasurer, and the Clerk. (4) Trustees emeriti who shall include any Member who has attained the age of seventy years (or the age of sixty five and has retired from his home institution) and who has served a full elected term as a regular Trustee, provided he signifies his wish to serve the Laboratory in that capacity. Any Trustee who qualifies for emeritus status shall continue to serve as a regular Trustee until the next Annual Meeting whereupon his office as regular Trustee shall become vacant and be filled by election by the Members or by the Board, as the case may be. The Trustees ex officio and emeriti shall have all the rights of the Trustees, except that Trustees emeriti shall not have the right to vote. (B) The aggregate number of Corporate Trustees and Board Trustees elected in any year (excluding Trustees elected to fill vacancies which do not result from expiration of a term) shall not exceed nine. The number of Board Trustees so elected shall not exceed three and unless otherwise determined by vote of the Trustees, the number of Corporate Trustees so elected shall not exceed six. (C) The Trustees and Officers shall hold their respective offices until their successors are chosen in their stead. (D) Any Trustee may be removed from office at any time with or without cause, by vote of a majority of the Members entitled to vote in the election of Trustees; or for cause, by vote of two-thirds of the Trustees then in office. A Trustee may be removed for cause only if notice of such action shall have been given to all of the Trustees or Members entitled to vote, as the case may be, prior to the meeting at which such action is to be taken and if the Trustee so to be removed shall have been given reasonable notice and opportunity to be heard before the body proposing to remove him. (E) Any vacancy in the number of Corporate Trustees, however arising, may be filled by the Trustees then in office unless and until filled by the Members at the next Annual Meeting. Any vacancy in the number of Board Trustees may be filled by the Trustees. (F) A Corporate Trustee or a Board Trustee who has served an initial term of at least two years duration shall be eligible for re-election to a second term, but shall be ineligible for re- election to any subsequent term until two years have elapsed after he last served as Trustee. IX. (A) The Trustees shall have the control and management of the affairs of the Corpo- ration. They shall elect a Chairman of the Board of Trustees who shall be elected annually and shall serve until his successor is selected and qualified and who shall also preside at meetings of the Corporation. They shall elect a President of the Corporation who shall also be the Vice Chairman of the Board of Trustees and Vice Chairman of meetings of the Corporation, and who shall be elected annually and shall serve until his successor is selected and qualified. They shall annually elect a Treasurer who shall serve until his successor is selected and qualified. They shall elect a Clerk (a resident of Massachusetts) who shall serve for a term of four years. Eligibility for re-election shall be in accordance with the content of Article VIII(F) as applied to corporate or Board Trustees. They shall elect Board Trustees as described in Article VIII(B). They shall appoint a Director of the Laboratory for a term not to exceed five years, provided 34 MARINE BIOLOGICAL LABORATORY the term shall not exceed one year if the candidate has attained the age of 65 years prior to the date of the appointment. They may choose such other officers and agents as they may think best. They may fix the compensation and define the duties of all the officers and agents of the Corporation and may remove them at any time. They may fill vacancies occurring in any of the offices. The Board of Trustees shall have the power to choose an Executive Committee from their own number as provided in Article X, and to delegate to such Committee such of their own powers as they may deem expedient in addition to those powers conferred by Article X. They shall from time to time elect Members to the Corporation upon such terms and conditions as they shall have determined, not inconsistent with law or these Bylaws. (B) The Board of Trustees shall also have the power, by vote of a majority of the Trustees then in Office, to elect an Investment Committee and any other committee and, by like vote, to delegate thereto some or all of their powers except those which by law, the Articles of Or- ganization or these Bylaws they are prohibited from delegating. The members of any such committee shall have such tenure and duties as the Trustees shall determine; provided that the Investment Committee, which shall oversee the management of the Corporation's endowment funds and marketable securities, shall include the Chairman of the Board of Trustees, the Trea- surer of the Corporation, and the Chairman of the Corporation's Budget Committee, as ex officio members, together with such Trustees as may be required for not less than two-thirds of the Investment Committee to consist of Trustees. Except as otherwise provided by these Bylaws or determined by the Trustees, any such committee may make rules for the conduct of its business; but, unless otherwise provided by the Trustees or in such rules, its business shall be conducted as nearly as possible in the same manner as is provided by these Bylaws for the Trustees. X. (A) The Executive Committee is hereby designated to consist of not more than ten members, including the ex officio Members (Chairman of the Board of Trustees, President, Director and Treasurer); and six additional Trustees, two of whom shall be elected by the Board of Trustees each year, to serve for a three-year term. (B) The Chairman of the Board of Trustees shall act as Chairman of the Executive Com- mittee, and the President as Vice Chairman. A majority of the members of the Executive Com- mittee shall constitute a quorum and the affirmative vote of a majority of those voting at any meeting at which a quorum is present shall constitute action on behalf of the Executive Com- mittee. The Executive Committee shall meet at such times and places and upon such notice and appoint such sub-committees as the Committee shall determine. (C) The Executive Committee shall have and may exercise all the powers of the Board during the intervals between meetings of the Board of Trustees except those powers specifically withheld from time to time by vote of the Board or by law. The Executive Committee may also appoint such committees, including persons who are not Trustees, as it may from time to time approve to make recommendations with respect to matters to be acted upon by the Executive Committee or the Board of Trustees. (D) The Executive Committee shall keep appropriate minutes of its meetings and its action shall be reported to the Board of Trustees. (E) The elected Members of the Executive Committee shall constitute as a standing "Com- mittee for the Nomination of Officers," responsible for making nominations, at each Annual Meeting of the Corporation, and of the Board of Trustees, for candidates to fill each office as the respective terms of office expire (Chairman of the Board, President, Director, Treasurer, and Clerk). XI. A majority of the Trustees, the Executive Committee, or any other committee elected by the Trustees shall constitute a quorum; and a lesser number than a quorum may adjourn any meeting from time to time without further notice. At any meeting of the Trustees, the Executive Committee, or any other committee elected by the Trustees, the vote of a majority of those present, or such different vote as may be specified by law, the Articles of Organization or these Bylaws, shall be sufficient to take any action. XII. Any action required or permitted to be taken at any meeting of the Trustees, the Executive Committee or any other committee elected by the Trustees as referred to under Article IX may be taken without a meeting if all of the Trustees or members of such committee, BYLAWS 35 as the case may be, consent to the action in writing and such written consents are filed with the records of meetings. The Trustees or members of the Executive Committee or any other com- mittee appointed by the Trustees may also participate in meeting by means of conference telephone, or otherwise take action in such a manner as may from time to time be per- mitted by law. XIII. The consent of every Trustee shall be necessary to dissolution of the Marine Biological Laboratory. In case of dissolution, the property shall be disposed of in such a manner and upon such terms as shall be determined by the affirmative vote of two-thirds of the Board of Trustees then in office. XIV. These Bylaws may be amended by the affirmative vote of the Members at any meeting, provided that notice of the substance of the proposed amendment is stated in the notice of such meeting. As authorized by the Articles of Organization, the Trustees, by a majority of their number then in office, may also make, amend, or repeal these Bylaws, in whole or in part, except with respect to (a) the provisions of these Bylaws governing (i) the removal of Trustees and (ii) the amendment of these Bylaws and (b) any provisions of these Bylaws which by law, the Articles of Organization or these Bylaws, requires action by the Members. No later than the time of giving notice of the meeting of Members next following the making, amending or repealing by the Trustees of any Bylaw, notice thereof stating the substance of such change shall be given to all Corporation Members entitled to vote on amending the Bylaws. Any Bylaw adopted by the Trustees may be amended or repealed by the Members entitled to vote on amending the Bylaws. XV. The account of the Treasurer shall be audited annually by a certified public accountant. XVI. Except as otherwise provided below, the Corporation shall, to the extent legally per- missible, indemnify each person who is, or shall have been, a Trustee, director or officer of the Corporation or who is serving, or shall have served, at the request of the Corporation as a Trustee, director or officer of another organization in which the Corporation directly or indirectly has any interest, as a shareholder, creditor or otherwise, against all liabilities and expenses (including judgments, fines, penalties and reasonable attorneys' fees and all amounts paid, other than to the Corporation or such other organization, in compromise or settlement) imposed upon or incurred by any such person in connection with, or arising out of, the defense or disposition of any action, suit or other proceeding, whether civil or criminal, in which he or she may be a defendant or with which he or she may be threatened or otherwise involved, directly or indirectly, by reason of his or her being or having been such a Trustee, director or officer. The Corporation shall provide no indemnification with respect to any matter as to which any such Trustee, director or officer shall be finally adjudicated in such action, suit or proceeding not to have acted in good faith in the reasonable belief that his or her action was in the best interests of the Corporation. The Corporation shall provide no indemnification with respect to any matter settled or compromised, pursuant to a consent decree or otherwise, unless such settlement or compromise shall have been approved as in the best interests of the Corporation, after notice that indemnification is involved, by (i) a disinterested majority of the Board of Trustees or of the Executive Committee or, (ii) a majority of the Corporation's Members. Indemnification may include payment by the Corporation of expenses in defending a civil or criminal action or proceeding in advance of the final disposition of such action or proceeding upon receipt of an undertaking by the person indemnified to repay such payment if it is ultimately determined that such person is not entitled to indemnification under the provisions of this Article XVI, or under any applicable law. As used in this Article, the terms "Trustee," "director" and "officer" include their respective heirs, executors, administrators and legal representatives, and an "interested" Trustee, director or officer is one against whom in such capacity the proceeding in question or another proceeding on the same or similar grounds is then pending. To assure indemnification under this Article of all persons who are determined by the Corporation or otherwise to be or to have been "fiduciaries" of any employee benefit plan of 36 MARINE BIOLOGICAL LABORATORY the Corporation which may exist from time to time, this Article shall be interpreted as follows: (i) "another organization" shall be deemed to include such an employee benefit plan, including, without limitation, any plan of the Corporation which is governed by the Act of Congress entitled "Employee Retirement Income Security Act of 1974," as amended from time to time ("ERISA"); (ii) "Trustee" shall be deemed to include any person requested by the Corporation to serve as such for an employee benefit plan where the performance by such person of his or her duties to the Coiporation also imposes duties on, or otherwise involves services by, such person to the plan or participants or beneficiaries of the plan; (iii) "fines" shall be deemed to include any excise taxes assessed on a person with respect to an employee benefit plan pursuant to ERISA; and (iv) actions taken or omitted by a person with respect to an employee benefit plan in the performance of such person's duties for a purpose reasonably believed by such person to be in the interest of the participants and beneficiaries of the plan shall be deemed to be for a purpose which is in the best interests of the Corporation. The right of indemnification provided in this Article shall not be exclusive of or affect any other rights to which any Trustee, director or officer may be entitled under any agreement, statute, vote of members or otherwise. The Corporation's obligation to provide indemnification under this Article shall be offset to the extent of any other source of indemnification or any otherwise applicable insurance coverage under a policy maintained by the Corporation or any other person. Nothing contained in this Article shall affect any rights to which employees and corporate personnel other than Trustees, directors or officers may be entitled by contract, by vote of the Board of Trustees or of the Executive Committee or otherwise. VI. REPORT OF THE DIRECTOR I have the pleasure of writing a Director's Report which is concerned in large part with plans for the future, rather than with analysis of the past. My pleasure implies neither a lack of interest in the past nor any uneasy omen. It comes, rather, from my review of those tall stacks of paper recording the events, the finances, the problems, and the accomplishments scientific and administrative of the past year, a review that always precedes the writing of these reports. There it becomes evident that on most fronts of MBL operations there are meetings, studies, and planning exercises directed toward the post-Centennial decade. How fundamental a change this represents in the state of the place can be appreciated only by those who know something of the MBL's past. Such activity means that we see no threats or difficulties, in things as they are, grave enough to inhibit creative anticipation. It means that MBL people are sufficiently confident of the health of the enterprise to give time to imagining an exciting future, rather than to considering how to survive. It was not always thus. I am able therefore to write of plans and goals, confident that the remaining content of this volume will be at least reassuring, and possibly even a source of pride, to members of the Cor- poration. Committees 1985 was MBL Centennial Year minus three, none too soon to begin to plan for the events of that celebration. The Trustees having reviewed and approved a proposed structure for a committee to plan and execute the events, the structure was built, with people, in the course of the year. Overseeing all Centennial-related activities will be a Centennial Committee, which includes among its distinguished members James D. Ebert as Chairman. We are doubly fortunate in Ebert's acceptance of the responsibility: he is not only a devoted Corporation member and the Laboratory's President-Director emeritus, but also a scientist and administrator of international stature. The oversight committee includes, inter alia, the Chairmen of four subcommittees, each of which has been empaneled and is hard at work. In charge of the Subcommittee on History is Garland Allen. Raymond E. Stephens chairs the Subcommittee on Events, and "events" is meant to cover not only the necessary celebratory occasions of 1988, but scientific meetings of international significance, beginning in 1987. An expert and REPORT OF THE DIRECTOR 37 enthusiastic Subcommittee on Public Information is headed by author and friend of the Laboratory John Pfeiffer. And finally, financial management of the Centennial celebrations, in extenso, is the responsibility of a Finance Subcommittee headed by D. Thomas Trigg. I like to believe that it is a measure of the MBL's future, as well as of its remarkable history, that we are able to assemble so talented a group of volunteers to work for an institution that is not, after all, among those listed as best-endowed, nor is it constantly in the public eye. By the reorganization of old committees and the creation of several new ones, the Trustees have signaled a new determination to manage the MBL seriously and well as though, in fact, it were itself well and to be taken seriously, in the way that large and complex intellectual enterprises must today be taken if they are to succeed. Thus we have in the course of the year sharpened the charge of the Investment Committee, expanded and systematized the work of the Compensation Committee, created a new and professionally qualified Audit Committee, and empaneled a Committee on Lab- oratory Goals. Investment, the first of these, has been in existence for a good many years and it has given the MBL invaluable service. But with our investments now in excess of $1 1 million, it is important for all levels of decision-making to be identified and monitored, and for the many choices that have to be made each year to be made on a reliable schedule, recorded promptly, and implemented by timely actions of the MBL admin- istration and the investment management firm. All these needs are dealt with under the revised committee charge. The members have accepted it. As the MBL's still-skeletal administrative capability is enhanced, it becomes nec- essary for the compensation of managers a category that includes all the Department heads to be reviewed with as much care, knowledge of market forces, and attention to individuals as has always been the case for non-administrative employees. The new Compensation Committee will see to that. As recently as 1978, when the business and academic worlds were well into the second generation of administrative data processing by electronic means, the MBL was still doing all its business with pencil and paper. It did not take much effort to convince the community that the time had come for conversion to electronic data processing. The conversion was made coincident with reorganization of the accounting functions under the Controller's office. Subsequent improvements in financial man- agement have been evident to nearly everyone associated with the Laboratory. We are, however, at the junction of two very fast-flowing streams: the technology itself, and the increasingly complex financial operations of the MBL. In the future we will require not only computer-based accounting, but excellent accounting at that, and incisive data analysis for management and planning. The MBL must also have regular and more expert liaison with its external auditors, those of government and foun- dation offices as well as those we employ for the annual review of our books. The new Audit Committee is superbly qualified for these purposes. It will work with the Controller, henceforth, as well as with the Director and all the Trustees, to whom it will ultimately report. The last comprehensive plan for the Laboratory was written in 1978 and adopted by the Trustees in the winter of 1979. It was a comprehensive plan in the sense that it set goals in each of the MBL missions: summer research, year-round research, instruction; and it did so in the context of existing resources and reasonable projections of their growth. It was a plan meant to serve, with modification and amendment as needed, up to the Centennial, give or take a year. As it happens, this was the first, as well as the last, such long-term plan for the institution. The 1979 plan, and all the work of review, data analysis, and feasibility testing that went into it, were done by the then-new Director. It could not have been otherwise: 38 MARINE BIOLOGICAL LABORATORY differences of opinion within the Corporation, even on fundamental issues, were too great, and organizational and administrative resources too small, to have allowed the Corporation, speaking via its Trustees, to do the job. We have succeeded with what is, by any reasonable standard, a remarkable fraction of the 1979 plan. It is a fair expectation that the essentials, save perhaps a Marine Resources Center, will have been realized by 1988. To be sure, some of the elements have had to be modified. Seven years is a very long time in modern biology, and in the history of relations between government and independent research laboratories. But most of the modifications have been upward in scale, not downward, and most of those, too, have been achieved or will be achieved in the near future. It is time for a new MBL plan. Even if we were in a field less dynamic and more predictable than biomedical science, we should need one: seven years is an ancient guideline for sticking to an undertaking. Thereafter, in response to the inevitable itch, there must be some scratching. Sensible people and institutions anticipate the itch and regulate the scratch. The MBL is now strong enough. There is sufficient loyalty and unanimity on what were recently divisive issues to allow the next plan to be made by those who should be its architects: the Corporation and the Trustees themselves. It is not that the Director is alien to either body: he is one of both, and by the nature of things will always, whoever he or she may be, have unlimited access to their wisdom and their beliefs (not all of which are necessarily wise). But in an institution as horizontal as the MBL, and in so explosively growing a business as biomedical science, no comprehensive plan should be authored by one person. Machinery for making the next comprehensive plan was established during the 1985-86 year. It begins with a new Trustees' Committee, the Committee on Laboratory Goals, chaired by Gerald Fischbach. In its initial form the Committee consists entirely of Corporation members: as its inquiries are broadened during the summer of 1986, members and consultants will be added according to need. This Committee has a simple but very weighty charge: to recommend to the Trustees directions and standards for all MBL scientific programs in the decade following the Centennial. The recom- mendations are to be based upon exhaustive analysis of current resources, probable future resources, special strengths and opportunities in the existing configuration, actual or potential weaknesses, and, insofar as is possible, an adherence to the founding principles of the Laboratory. It is perhaps well to list the relevant ones here: Independence; Avoidance of identification with any single biological discipline; Emphasis upon intellectual quality, as opposed to mere utility; Maximum utilization of resources of the sea for research, but no exclusive com- mitment to marine biology for its own sake; Instruction and scientific communication as important as private, individual re- search. Dr. Fischbach's carefully assembled and highly qualified committee is at work: every Corporation member will be hearing from and of it before long. In the end, the product of its arguments and eventual agreements will change the shape of the MBL as much as, perhaps more than, did the Director's plan of 1979. This is as it should be: a biological institution whose spectrum of interests does not change within a de- cade in these waning decades of the twentieth century cannot be a part of the action of life science. All the rest Nothing else needs to be said in this year's Director's Report. The facts are in the book. Value judgments are implicit in the continued fluorishing of MBL programs, REPORT OF THE DIRECTOR 39 even though we are in times and circumstances such that other institutions, better- endowed and with missions more easily featured in Sunday Supplements, have fallen into deep trouble. I can afford to report in pastel a set of strokes meant to suggest reality, rather than to render it. Year-round research: Shinya Inoue's superb book, product of his years as a resident MBL scientist fully engaged in research, is published. It is the definitive text of the new technology of video microscopy, and that, in turn is an MBL story. Tom Reese will give a Friday Evening Lecture in 1986: its title will be Kinesin an MBL Project. The Ecosystems Center, now led by John Hobbie, installs a sophisticated mass spec- trometry facility for its own and general use, reflecting increased exploitation of stable isotope methods in ecological research. The facility is funded by generous grants from the NSF and the A. W. Mellon Foundation, and to some extent by the Center's own reserve funds. The productivity of Louis Leibovitz's Laboratory of Marine Animal Health continues to rise: a science of marine animal medicine to include invertebrates as well as vertebrates is in the making. The Laboratories of Sensory Physiology and of Biophysics continue to be identified by various forms of recognition as excellent. Serious planning for the future of the Boston University Marine Program appears to have begun: searches for a new Director and additional faculty are underway. The program continues to attract excellent graduate students. In these times of insanely chancy funding, such distinguished MBL investigators as J. R. Whittaker, R. E. Ste- phens, and O. Shimomura continue to be solidly supported. L. JafiVs National Vi- brating Probe facility provides more and more, not only for Jaffe's important research, but for others who discover how useful is the unique field-mapping capability of the instrument. The whole year-round scientific community seems, despite the peculiar arrangements by which it exists at the MBL, to be productive, mutually cooperative, even, possibly, happy. Summer research and instruction: The merest vignette, but one that should suffice for Corporation members, most of whom have a sense of how things are in this era of federal deficit reduction: in 1986 the MBL will again be full. At least at the time of writing (May, 1986), there is no suggestion of a decline of demand for research space, instruction, conference facilities. Applications for places in summer courses are up by more than 30% over 1985. The Friday Evening Lectures will be as distinguished as ever; there will be more fellows and scholars; more short-term visitors to courses and to the scientific community as a whole. Not unimportant: there will be twenty new, very handsome rental cottages: every one will be filled: there is a waiting list already. The departments: Here is the most attenuated vignette of all. Hurricane Gloria. In September of 1985, with barely adequate warning, the storm was upon us, and at a time of year when there is an understandable relaxation a slight let-down of the guard among MBL employees, catching a communal breath after the frenetic sum- mer. To be sure, Gloria could have been much nastier with the northeast USA than she was. But there was nevertheless a great deal of damage everywhere along the coast, and Woods Hole was within the target area. On an already wet and evil morning, not long after dawn: I find the MBL a beehive. B & G staff, Aparatus staff, John Valois and the Captains, research assistants everyone with responsibility (including, of course, a cadre of anxious scientists, torn between fears for the supercold freezer in the laboratory and worries about the food freezer and the children at home). A beehive literally. What might be taken for a lot of senseless milling about is in fact the worker bees in a schwdntzetanz: moving pur- posefully, communicating, putting forward the imperative of the hive. All vessels afloat are secured; heavy storm shutters are fixed in place; scores of sandbags are brought and emplaced; emergency power provided for; glass area protected. Not long after, the first great blasts strike ... 50 knots . . . and there are creakings and the groans 40 MARINE BIOLOGICAL LABORATORY of tied-down structure under strain. Water Street begins to live up to its name as waves break green and white over the sea wall. We are lucky: the tide is falling and will continue to fall through the peak of wind velocity, which ought to be at about noon. At noon in fact it seems the late evening of a vicious winter's day. My hand anemometer, borrowed from the nav-station of UCA, my boat (a pugnacious fiddler- crab is UCA), registers 75 knots. The long day ends. It is still blowing crazily, trees bending as though they were mere sticks; but it is clear that the MBL will be all right. The next day is partly sunny and warm. The sea is innocent and seems to know nothing about violence and danger. Practically no words were said, before or after, by anyone behaving as a drill- sergeant. MBL people simply did what they know how to do, when it had to be done. The damage was, in the end and when assessed, minor. As is always the case here, we learned a little about how to do things better the next time. VII. REPORT OF THE TREASURER In February of 1986, 1 discussed with Dr. Gifford my wish to be replaced as Trea- surer of the Marine Biological Laboratory. This decision on my part was reached after thoughtful inquiry, one year "on the job," and my judgment that the responsibilities of the office of the Treasurer, stated and implied, require full-time "hands-on" attention and management. Today the Laboratory operates year-round. It has financial transactions with entities both private and public. Its financial health depends on its continuing ability to attract grants and gifts for the purchase and maintenance of its facilities and equipment. To meet the current demand, the Laboratory engages in many "businesses." The financial consequences of these businesses and the time it takes to manage them varies widely. Many of these responsibilities should not be delegated nor should they be managed on a "knee jerk" reaction basis by a part-time volunteer Treasurer. I have decided to step aside in support of this judgment. My review of prior years' Treasurer's reports to the Corporation shall be my guide in the preparation of this report. First, I shall comment on the Statement of Assets and Liabilities (including fund balances). Then I shall comment briefly on the Schedule of Support, Revenues, Ex- penses, and Changes in the Fund Balance. Net current assets (Working Capital) decreased $482,000 occasioned by a decrease in cash and cash equivalents of $600,000, of which $450,000 was transferred to the Pooled Investment portfolio; the increase in Receivables of $441,000 mostly in grants, includes $21 1,000 of billings for which payment was received subsequent to the year- end closing of the books. Efforts to reduce the grants and other receivables have been underway and I am pleased to report that these receivables have been reduced from $1,1 15,000 to $497,000 as of the end of March, 1986. Accounts payable increased $174,000. Note Payable of $100,000 represents the proceeds of a temporary unsecured loan. These proceeds were used for the Memorial Circle construction pending the recording of and repayment from the permanent secured loan. This occurred in January of 1986. Two new items were added to the schedule in S985; a contingent liability of $50,000 for the over-recovery of overhead costs and an accrual of $75,000 for wages earned during the latter part of the year, which were paid in 1986. Investments (including retirement fund securities) increased $861,000 (cost value) principally reflecting the first increment of $500,000 from the Mellon Match com- mitment. MBL books its investments at cost. (Not all non-profit corporations do so.) Given the remarkable advance in stock and bond values in the last quarter of 1985, the year- REPORT OF THE TREASURER 41 end book value of the endowment (including quasi-endowments) and the retirement funds is understated by $2,254,000. The Statement of Support, Revenues, Expenses, and Changes in Fund Balance shows a significant increase in expenses of $1,000,000 an increase of 10%, while income to support these expenses increased $300,000 or 2.8%. I am advised that the 1985 Budget prepared and approved in 1984 anticipated the increased expense. In summary, if one includes the positive influence of the securities market on the financial position of the MBL as of year-end 1985, it was on average a very good year for the MBL. The financial control unit under the direction of John Speer and with the assistance of Coopers & Lybrand, our independent auditors, and other counsel, have been engaged for some time in developing improvements in the accounting, personnel management, and cost control systems of the MBL. During 1985, three significant recommendations were presented to the Executive Committee and received their approval. They were: (a) An improved overhead assignment formula that will ensure the recovery of a fair division of these costs from the various users of the facilities and equipment. (b) A personnel management system which includes individual job descriptions and evaluations. These will be most useful in providing standards for logical and equitable management and compensation to our staff. (c) The establishment of separate funds in the financial accounting system for housing and for food services. These new restricted accounts will appear in the 1986 financial reports. Under "Other but Necessary Business": the twenty units of additional housing at Memorial Circle were authorized and commenced in the fall of 1985 with completion scheduled for June, 1986. I am pleased to report that they have been finished on time and within budget. The placement of these new units has been the subject of favorable trustee comment. Credit for this excellent work belongs to our engineers, Holmes & McGrath, who developed the site plan and utility design; Woodside Park Corporation for construction of the buildings, roads, etc.; to Homer Smith for his continuing as- sistance at all levels including the procurement of the furnishings for the cottages; and to Donald Lehy, the owner's representative at the site. In 1985, Dr. Gross sought and obtained multiple year gifts for the support of the instruction program. These gifts are sufficient to balance instruction costs through 1987. This was a most necessary life sustaining transfusion for this function. From where and from whom will funds be obtained to replace those funds when they are expended as planned? When will our endowments increase in principal value so they will generate sufficient income to replace our dependency on crisis funding for on- going functions? These are questions that should and must be answered by the senior management, Trustees, Corporators, and the Associates of MBL. This brings me full circle to my opening remarks. Times have changed. Financial planning and its execution is a full-time, year-round responsibility of the MBL. The corporate office charged with this responsibility is and has been that of the Treasurer, when in fact, the duties have been performed on a daily basis by a non-corporate officer, the Controller. It is my conviction that this arrangement should be changed and that the MBL must have a full-time senior financial officer. While I have been preparing these comments, I have learned that Robert D. Manz recently of Coopers & Lybrand and for the past several years the auditor in charge of the MBL audit, has recently established himself as a financial consultant in private practice. Bob has agreed to become MBL's Treasurer and will bring to the office a board knowledge of the financial affairs of non-profit, scientific research organizations and in the specific, a unique knowledge of the strengths and weaknesses of MBL's financial affairs. 42 MARINE BIOLOGICAL LABORATORY Coopers &Lybrand certified public accountants To the Trustees of Marine Biological Laboratory Woods Hole, Massachusetts We have examined the balance sheet of Marine Biological Laboratory as of December 31, 1985 and the related statement of support, revenues, expenses and changes In fund balances for the year then ended. Our examination was made In accordance with generally accepted auditing standards and, accordingly, Included such tests of the accounting records and such other auditing procedures as we considered necessary In the circumstances. We previously examined and reported upon the financial statements of the Laboratory for the year ended December 31, 1984, which condensed statements are presented for comparative purposes only. In our opinion, the financial statements referred to above present fairly the financial position of Marine Biological Laboratory at December 31, 1985 and Its support, revenues, expenses and changes In fund balances for the year then ended, In conformity with generally accepted accounting principles applied on a basis consistent with that of the preceding year. C-OOptni> T Boston, Massachusetts April 18, 1986 REPORT OF THE TREASURER 43 o\ (N ON ON o r- NO ro O V) oo o oc oo m ON O rf r4 ON O NO ON NO NO' 00 NO ON O ^ ON OO O n in NO s * ON Tt NO' _' ^^ NO "- * r^ o r i/~j ^^ OO O-J f**) f*") O ri ri Tf OO oo m TT rTj Tt NO Tj- ^sO OO NO ^* ON oo OO <0 oo NO ^ -~ r*^ CN| ro p_T ^ , ,' , ' fee "" " ri Z $ tw c/5 c S <3 p^ 3 X J s: U o OJ - 2 X c *l B C/3 u ^5 o '*-! 03 T3 f*l Liabilities an Accounts payable and ac Deferred income X i> Z u x r3 >^ S 3 O Z Total current liabili Construction accounts p Current unrestricted fun f~* mv+ # - ^ - i j~t+ t\rl f i r> 1 3 C 3 * S-8 S" ^ H - X = u -> c 3=> J V) c /a T3 O T3 X "LI C P Unexpended income ( Endowment funds: Unrestncted purposes Restricted purposes Quasi-endowment funds Unrestricted purposes Restricted purposes ON g ON OO VI OO VI fS oo TT O Tf NO O ^t NO m ^ o in r oo NO ~~. ^, "", Tt , Cl 5 Tt" vl oo' vT ^ oo' O NO O NO in' ON . ri f-* ON fN fNj r*"^ ON *^" ON i. r^i oo O r-' >n ON O) o r<-> NO ON O i oo r- NO ' ' fee trt U. of allowance foi C 3 incurred on en PU O i! Z H- (U a c ,g 2 i 'J75 D. c/) n ^ OJ C O a i ^ Z 1 'C e o. it 3 O uJ X) 03 OJ X; 1 C! i o o 03 o T3 C 1 > '5 ^ t/i c 3 uncoiled u -a t/5 < u o 03 1 fc O 13 ;tments, at ., buildings accumulate hjJ 1 t/5 C S O O O < 'C O OJ OJ J3 6 in u > c T3 J % 3 44 MARINE BIOLOGICAL LABORATORY 2 - tS . O oo vi so fN r*i oo fN_ so^ sO TT n fN p " ,3 rs . so o so fN "! W f 1 Cti O >/^ 1 o 5 H a oo ^ ^ 1 LABORA ES AND CP " oo m "^ VH ^^ o> ci l-a C cd ment Funds Restricted GICAL EXPENS <^ t; O QJ ^ Q "8'fl Quasi-endov, Unrestricted 3 - i 1 m T3 UH C ^ O D " E "S S % 03 o & -3 1 C z - S ftj * 3 ^ << 1 P4 ^ S s E ^ ^^^/ 1 ? i i 0- ^ D u. 1 H -<3 2 Z a; u ^ ^ c ^ * H H 1 C ^ > /^ <^T OO ^ i/^ I in f1 CM Tt a % o c a c 1 o '-> -c ej e o V wrt and revenues: rant reimbursemen costs: Instruction Research scovery of indirect related to resear instruction e i I E 3 2 5b o a a c o M > Instruction c o a; C Dormitory 15 oo c c Q Library Biological bulletin Research services Manne resources Other festment income alized gains (losses investments -o c o c .0 scellaneous revenu Total support and f at H D C/5 c V at. < o S 9. REPORT OF THE TREASURER 45 vO MO O *f^> O OO rs Vi oo rs ON SO 5 g r* r- oo sO ON oo oo r- r~ OO ON ON VI V) *N 00 VJ rs - so r- so T VI OC V) OO oo '""* "1 M rs rs ^ " sO f*) OO OO oo "* ON I I /1 f*1 ^ t O Tf 0s O sq r-^ > f f*} oo' ON so* sO T Os v> O O <^1 ' * I I s o Ov I i 8 a sO O o' OO V> rs *} QN O ON f*"i O oo oo o -> \o r- r- sO sor-oov~ii^-,Troo rsrs ^^ O TT oo o Tt sO ON V S v> rs oo O f^i O v^ ^o v> f*i rs r- r- TJ- Tt SO o ) T- O. ooooocv>rsr-oo sors rsi OO OO w-1 V) r^ r*^m^^r-rooN o r-- T Tt* *"^ v= c c a u u E , E n u rt c % 8 T3 S o S c 3 c ^ V O i_ ^ c S. s c '; (*- ^ S c ^rt -2 ^ 4> flj flj d l|| f 1 1 1 1 1 1 % * Pit! iKitiL i .s * i s * -1 & -| I s :1 1 o HIHIIJIIIIII 1 !!.! oit/^a.i & ^ o x . n ,i> c nun [ o > *" B | f^l 111 'CcEggc^c ^9>i;|Ece sa Sil>' K s S ^ FT T3 o -c u C *s Q a < S LU O Total transfers among 3 1 o c B efl r: u E > -s 1 1 tff (/ flj a. O C C C rt n "i The accompanying ?ir proportionate share at market value adjusted for any additions or disposals to pooled funds. REPORT OF THE TREASURER 47 C. Land, Buildings, and Equipment: The following is a summary of the unrestricted plant fund assets: 7955 Land Construction in progress Buildings Equipment Less accumulated depreciation $ 689,660 140,826 14,861,244 2,113,321 17,805,051 6,579,654 $11,225,397 1984 $ 689,660 14,772,449 1,974,495 17,436,604 6,090,140 $11,346,464 Depreciation is computed using the straight-line method over estimated useful lives of fixed assets. D. Retirement Fund: The Laboratory has a noncontributory pension plan for substantially all full-time employees, which complies with the requirements of the Employee Retirement Income Security Act of 1974. The Lab- oratory's policy is to fund pension costs accrued, as determined under the projected unit credit method. The actuarially determined pension expense charged to operations in 1985 was $146,253. As of the latest valuation date, based on benefit information at December 31, 1984, the actuarial present values of vested and nonvested benefits, assuming an investment rate of return of 7%, were approximately $1,300,175 and $60,833, respectively. At December 31, 1985, net assets of the plan available for benefits were approximately $1,687,072. In addition, the Laboratory participates in the pension program of the Teachers Insurance and Annuity Association. E. Pledges and Grants: As of December 31, 1985 and 1984, the following amounts remain to be received on gifts and grants for specific research and instruction programs, and are expected to be received as follows: 1986 1987 1988 Decembers], 1985 Unrestricted $15,000 10,000 10,000 $35,000 Restricted $ 703,128 550,240 15,000 $1,268,368 December 31, 1984 Unrestricted $5,000 $5,000 Restricted $ 8,000 6,651 $14,651 In February 1979, the Laboratory initiated the MBL Second Century Fund, a phased effort, to secure $23,000,000 in support of capital rehabilitation, new construction, and endowment funds. As of December 31, 1985, the Laboratory has received pledges related to this effort of approximately $8,550,129, of which a substantial portion has been collected. 48 MARINE BIOLOGICAL LABORATORY fN o oc. oo SO ^. ^ m r~ >n 3> rt~. o (*rj t^ <-, On < [- ~o sq^ rn^ fN o *\ l> (N j-^. ,-r. , , fN [^^ (N *n C .* C Oo "~* oo t^- CxO i oo r^- ~-> 2; ^ ^t 00 TT 1 oo' Os r^' ~ O fN cs ^-^ " in C c 1> e > ^ oo ^ o r Os in c SO t LTl so ' "" >j. Os SO r~~ OS r<-j r- ^ o oo m [ p^. n i/'i |y- ( in -2^ Ov t^- t KM r^ -2 Os oo " fN^ 'f oo' f 1 C3 ^ OO -^ KM iy> <^l OO 'Os o 'OO KM SO >n tN f- m 1 1 S q Os Os OO _- 1/ _ 1 fN ' so" ' OO ~ Os m 1 rj so sC Os r; Os oo so ^J- r ~ Os 00 "V"| r^, f"^ u O fN' o--' rn o o Os O fN p j V, K-' QJ O o\ r Os rj ^ OO 3 ^^ ~~i ro Os OO oo "5 ~ "* oo" fil i> nj Q TD Q JH (U un "^s C <_. u, g; ^0 S s^S +-> ^ ^ ^ W5 S) ^J Ji E "= - 'C o tn 6 c 3 t * : - ~ u C Os fN r~- fN SO^ OO SO o' fN r-' O i 00 fN fN so (A fN fN so fN fN c o orations CT o C rrent operatioi o u 'I a. o fc c/5 T3 u -Q V3 c S _3 K a 3 O c a _c K; "^ u ^5 o 3 T3 T3 i 3 4-^ investm 1 P*. current current ~-> A \ restricte owmeni T3 ^ N *- 1 U 1 c I JH 2 f2 c T3 (U *- ^u c a a> N -> Availablt 13 T3 C OJ rement fi 1 O * *! S 1 1 a; D '*_* u Qi C D REPORT OF THE TREASURER 49 oo oo t~- oo oo \o oo o oo r~ i-~ oo c- >o i o ri EH- ^ r-_ o jj e ^ J3 ' ' rr, 3 4J >. 5 g ^ " eg a> *j > c t- C O f O 1> *- T3 > s i 1 g- I i il g S T3 ? W ' C C O 3 '2 "^ ijj "O O" ^ 111 111 "T" C/l p < t/1 flj * i 2 o c c S D D oJ OS D ai a: I 3 ! 8 Q i 03 u u , TD X5 . d K .9 Q 8 P Q 50 MARINE BIOLOGICAL LABORATORY VIII. REPORT OF THE LIBRARIAN The National Marine Fisheries Service in Woods Hole has placed its library col- lection in the MBL Library. We now provide full library services to the scientists at the Fisheries making the MBL Library the main library for all four institutions in Woods Hole. This move has been discussed for years and it became official when the MBL was awarded the contract by NOAA early this year. Our collection will be in- creased by approximately 1 500 books. Most of the journal sets held at the Fisheries are duplicated here and their volumes will be sent to other Fishery libraries. A few new titles will be added to the periodical collection. The scientists at the Fisheries have used the MBL Library since the founding of the Laboratory. The Minutes of the MBL Trustee meeting in 1889 record that the Fisheries contributed both books and pamphlets to the original collection compiled in 1888. Over the years their scientists have been helpful with advice on collection development in their specialized subjects. Everyone benefits from such an arrangement. The Fisheries will save space and money while at the same time contributing financially to the MBL Library operation. The Reprint collection (Stack One) was cleared this year for the final move of the periodical collection. We say "final move" as we are committed to a collection of no larger than 200,000 volumes. The new laser disc technology makes it possible to keep adding new material to the collection without requiring more shelf space. The Library Users Committee and the librarians are studying all new developments of this method of electron storage. During August, when a large number of scientists were in residence, we had a Library Week. A number of companies exhibited their products for three days and an evening meeting was held for discussion with all involved. For three months we moved the periodical collection, using the stack floor gained by the reprint move. All journals are now housed in alphabetical order on the five floors of stacks. The Reprints have been placed in storage until time permits for rear- rangement in another area of the library. Ninety percent of the reprints are available in the published volumes in the stacks and all are cataloged in the main catalog by author so the major part of the collection is still available. This year we started placing our serial collection of nearly 5000 titles into the OCLC database. It's an expensive project but we are fortunate to have all expenses covered by a Title III Grant from the Massachusetts Library Commissioners. Catherine Norton, Assistant Librarian, wrote the grant working with the Librarian at the Falmouth Public Library and 42 other Public libraries on the Cape and Islands. All serial titles held in southeastern Massachusetts will be placed in this database. The information will be available on-line to all libraries. Catherine is the coordinator of the project and a new IBM-PC is in our Library for the specific use of the people involved in entering the material. The computer will remain the property of the Library when the project is completed. IX. EDUCATIONAL PROGRAMS SUMMER BIOLOGY OF PARASITISM Course directors ENGLUND, PAUL, Johns Hopkins School of Medicine SHER, ALAN, NIAID/NIH EDUCATIONAL PROGRAMS 51 Other faculty, staff, and lecturers ALLISON, ANTHONY C., Syntex ANDERS, ROBIN, Walter and Eliza Hall Institute, Australia BANGS, JAMES D., Johns Hopkins University BEVERLY, STEPHEN, Harvard Medical School BLOOM, BARRY, Albert Einstein College of Medicine BROWN, KIM H., University oflowa BURAKOFF, STEVEN J.. Harvard Medical School CARTER, RICHARD, NIAID/NIH CANTOR, CHARLES, Columbia University CERAMI, ANTHONY, Rockefeller University CHEEVER, ALLEN, NIAID/NIH CLEVELAND, DON, Johns Hopkins School of Medicine DALTON, JOHN, Johns Hopkins School of Medicine DAVIS, MARK, Stanford University DINTZIS, HOWARD, Johns Hopkins School of Medicine DONELSON, JOHN, University of Iowa DWYER, DENNIS, M., NIH GEARHART, PATRICIA, Johns Hopkins School of Medicine HART, GERALD W., Johns Hopkins School of Medicine HOWARD, RUSSELL, NIAID/NIH HOWARD, JAMES, Wellcome Laboratories, U.K. JAMES, STEPHANIE, George Washington School of Medicine JOINER, KEITH, NIAID/NIH KNOPF, PAUL, Brown University KRAKOW, JESSICA L., Johns Hopkins School of Medicine LENARDO, MICHAEL, University of Iowa MCGUIRE, MARIELENA VALEZ, NIAID/NIH Moss, BERNARD, NIAID/NIH MARSDEN, PHILIP, Federal University of Brasilia, Brazil NASH, THEODORE, NIAID/NIH NEVE, FRANKLIN A., NIH NELSON, GEORGE, University of Liverpool, U.K. NUSSENZWEIG, VICTOR, New York University School of Medicine PFEFFERKORN, ELMER, Dartmouth Medical School VAN DER PLOEG, LEX, Columbia University RAVDIN, JONATHAN, University of Virginia School of Medicine SACKS, DAVID, NIAID/NIH SCOTT, PHILLIP, NIAID/NIH SHER, KATHY, Columbia University SHEVACH, ETHAN, NIH SNARY, DAVID, Wellcome Laboratories, U.K. SPIELMAN, ANDREW, Harvard Medical School STRAND, METTE, Johns Hopkins School of Medicine TURNER, MERVYN J., Merck Sharp & Dohme ULLMAN, BUDDY, University of Kentucky Medical Center WANG, CHING C, University of California WARREN, KENNETH, Rockefeller Foundation WASSOM, DAVID L., Cornell University WARD, DAVID, Yale University Students ANDREWS, NORMA W., New York University ARGUELLO, CARLOS LOPEZ, National Polytechnic Institute, Mexico 52 MARINE BIOLOGICAL LABORATORY BARRAL, ALDINA, M. P., Federal University of Bahia, Brasil BENNETT, KAREN L., Carnegie Institution BURNS, JAMES M., JR., Hahnemann University CORDOVA, JOSE L., Boston University DOERING, TAMARA L., Johns Hopkins School of Medicine FEBBRAIO, MARIA, Cornell University Medical School FOLEY, MICHAEL, University of Dundee, U.K. JOHNSON, ROLLIN BREESE, Cambridge University, U.K. LAFAILLE, JUAN JOSE, Institute de Quimica, Brasil LiMO, MOSES KiPROP, International Laboratory for Research on Animal Diseases, Kenya MOGYOROS, MYRIAM K., Weizmann Institute of Science, Israel OSOTIMEHIN, BABATUNDE, University of Ibadan, Nigeria WOOLLETT, GILLIAN, University of Edinburgh, U.K. ZENTELLA-DEHESA, ALEJANDRO, Rockefeller University CELLULAR NEUROBIOLOGY IN THE LEECH Course director NiCHOLLS, JOHN, University of Basel, Switzerland Other faculty, staff, and lecturers CALABRESE, RONALD, Harvard University FRIESEN, W. OTTO, University of Virginia KRISTAN, WILLIAM, University of California, San Diego MACAGNO, EDUARDO, Columbia University MULLER, KENNETH, University of Miami Medical School PAYTON, BRIAN, Memorial University of Newfoundland, Canada Ross, WILLIAM, New York Medical College SALZBERG, BRIAN M., University of Pennsylvania STENT, GUNTHER, University of California, Berkeley THOMPSON, WESLEY J., University of Texas WEISBLAT, DAVID A., University of California WILSON, DARCY B., Scripps Clinic and Research Institute ZIPSER, BIRGIT, NIH Students ACKLIN, SUSAN, University of Basel, Switzerland HYSON, RICHARD L., University of Colorado LAW, MARGARET I., University of California, Berkeley LEV-RAM, VARDA, Weizmann Institute of Science, Israel MAROM, SHIMON, Israel Institute of Technology, Israel MIDTGAARD, JENS, Kobenhavns University, Denmark MONETA, MARIA EUGENIA, University of Chile, Chile PESSIN, MELISSA S., Johns Hopkins School of Medicine WILHERE, GEORGE F., Northwestern University WILSON, GISELA F., University of Wisconsin EMBRYOLOGY: A MODERN COURSE IN DEVELOPMENTAL BIOLOGY Course directors BRANDHORST, BRUCE, McGill University, Canada JEFFERY, WILLIAM, University of Texas EDUCATIONAL PROGRAMS 53 Other faculty, staff, and lecturers AXEL, RICHARD, Columbia University BATES, WILLIAM, University of Texas BEACH, REBECCA, University of Texas BORISY, GARY G., University of Wisconsin BRINKLEY, B. R., University of Alabama CHIKARAISHI, DONA, Tufts University CONDEELIS, JOHN, Albert Einstein College of Medicine CRAIN, WILLIAM, Worcester Foundation of Experimental Biology CRAIN, CALEB, Worcester Foundation of Experimental Biology DAWID, IGOR, NIH DOHMEN, M. R., University of Utrecht, Netherlands DWORKJN, MARK, Columbia University DWORKIN-ROSTL, EVA, Columbia University FALKENTHAL, SCOTT, Ohio State University FIRTEL, RICHARD A., University of California GEHRING, WALTER, University of Basel, Switzerland GERHART, JOHN C, University of California GOLDHAMMER, DAVID, Ohio State University GOUDEAU, HENRI, Cen Saclay, France GOUDEAU, MARIE, University of Marie Curie, France GURNEY, M., University of Chicago GURDAN, JOHN, Cambridge University, U.K. HARRIS, ALBERT K., University of North Carolina HILLE, MERRILL B., University of Washington HOLLAND, LINDA, Scripps Clinic and Research Institute JACKLE, HERBERT, Max Planck Institute, FRG JACOBSON, ANTONE, University of Texas JAFFE, LAURINDA, University of Connecticut KADO, RAYMOND, Centre National/Recherche Scientifique, France KLINE, DOUGLAS, University of California LAPUK, SETH, University of Connecticut LENGYEL, JUDITH, University of California LEVINE, M., Columbia University MAUL, GERD, The Wistar Institute MAXSON, ROBERT E., JR., University of Southern California MYLES, DIANE, University of Connecticut MORROW, LAURA, University of Texas NEWMAN, STUART A., Cornell University Medical School OLINS, JOSH, Earlham College ORDAHL, CHARLES, University of California RANKIN, MARY ANN, University of Texas RUBIN, L., Rockefeller University SANGER, JOSEPH, University of Pennsylvania SATER, AMY, University of Texas SCHATTEN, GERALD, Florida State University SCHATTEN, HEIDI, Florida State University SCHULTZ, GILBERT A., University of Calgary, Canada SHUR, BARRY, University of Texas SHRUTKOWSKI, ANTHONY, Columbia University SMITH, L. DENNIS, Purdue University SZELLOSI, DANIEL, Stazione Centrale Physiologic Animale, France GRANT-TAKEDA, NANCY J., University of Strasbourg, France TURNER, PAUL, University of Connecticut Health Center UZMAN, AKIF, University of Texas 54 MARINE BIOLOGICAL LABORATORY Students ALLEN, PHILIP G., JR.**, Harvard University BASOLO, ALEXANDRA*. University of Texas BEIS, ANDRONIKI. University of Thessalonki, Greece BLAIR, SETH S.*, University of Washington BRAULT, ELISABETH C.*, Centre National/Recherche Scientifique, France CHISHOLM, JULIA CLARE*, University of Cambridge, U.K. COLE, ERIC S., University of Iowa COFFE, GERARD*, University of Paris, France DIKE, LAURA E.*, Boston University DUBE, FRANCOIS, Stanford University DUBIN, ROBERT A., City University of New York EPSTEIN, HELEN*, Columbia University FARQUHARSON, ANDREW*, University of Glasgow, U.K. GOLSTEYN, ROY M.*, University of Calgary, Canada HAFNER, MATHIAS, German Cancer Research Center, FRG HOWARD, KENNETH RAMSEY, University of London, U.K. IGLESIAS, ANA, University of Connecticut Health Center KAO, KENNETH R.**, University of Toronto, Canada MIOTTA, KAREN A., University of Colorado MOURY, JOHN DAVID*, University of Texas RANSICK, ANDREW J.*, University of Texas RAYSON, THOMAS C., University of Hawaii SPEKSNIJDER, JOHANNA E.*, University of Utrecht, Netherlands SWALLA, BILLIE J.**, University of Iowa TELFER, ABBY*, Harvard University TURNER, PAUL R.**, University of Connecticut Health Center WELCH, JEFFREY E.*, University of North Carolina MARINE ECOLOGY Course director FRANK, PETER W., University of Oregon Other faculty, staff, and lecturers ALBERTE, RANDALL, University of Chicago BERT, THERESA M., Yale University Buss, LEO, Yale University CARLTON, JAMES, Williams College, Mystic Seaport CARACO, NINA, Mary Flager Cary Arboretum, New York CASWELL, HAL, Woods Hole Oceanographic Institution CONNELL, JOSEPH, University of California COLE, JON, Mary Flager Cary Arboretum, New York FOREMAN, KENNETH, Marine Biological Laboratory FROST, B. W., University of Washington GALLAGHER, EUGENE, University of Massachusetts GIBLIN, ANN, Marine Biological Laboratory GRASSLE, J FREDERICK, Woods Hole Oceanographic Institution GRASSLE, JUDITH, Marine Biological Laboratory HARBISON, G. R. /oods Hole Oceanographic Institution HOBBIE, JOHN, Marine Biological Laboratory HOWARTH, ROBERT, Marine Biological Laboratory * Post-course Participants ** Advanced Research Training Program Participants EDUCATIONAL PROGRAMS 55 JANNASCH, HOLGER, Woods Hole Oceanographic Institution JEFFERIES, ROBERT L., University of Toronto, Canada KELLY, JACK, Cornell University KOEHL, MIMI, University of California, Berkley MANN, KENNETH H., Bedford Institute of Oceanography, Canada ODUM, WILLIAM, University of Virginia OSMAN, RICHARD, Academy of Natural Sciences of Philadelphia PETERSON, C. H., University of North Carolina POSEY, MARTIN, University of Oregon RHOADS, DONALD, Yale University RIETSMA, CAROL, SUNY, New Paltz SCHELTEMA, RUDOLPH, Woods Hole Oceanographic Institution SEBENS, KENNETH, Northeastern University SMITH, ROBERT, University of Chicago STOECKER, DIANE, Woods Hole Oceanographic Institution VALIELA, IVAN, Boston University VITTOR, BARRY, B. Vittor and Associates WELSHMEYER, NICHOLAS, Harvard University WOODWELL, GEORGE, Woods Hole Research Center Students AVOL, SANDRA L.*, Yale University School of Medicine BARNETT, BRUCE A.*, North Dakota University BOCCA, ADRIAN HORACIO*, Universidad Nacional del Comahue, Argentina BURGOS, GUILLERMO E.*, National Institute/Fishery Research, Argentina FEE, ELISABETH J.*, Ohio State University HOHMAN, WILLIAM L., University of Minnesota IRIBARNE, OSCAR OSVALDO*, Universidad Nacional del Comahue, Argentina MARTINEZ, DANIEL EDUARDO*. Universidad Nacional Bahia Blanca, Argentina MOREK, MICHELE M., Brescia College MULSOW, SANDOR G.*, Universidad Austral de Chile, Chile RAY, ANDREA!.*, University of Chicago RENZI, MARTA ALICIA*. National Institute/Fishery Research, Argentina SCOTT, DILLON*. University of Massachusetts TRESIERRA-AGUILAR, ALVARO*. National University of Trujillo, Peru VERAZAY, GUILLERMO A., National Institute/Fishery Research, Argentina ZEA, SVEN E.*, University of Texas MICROBIOLOGY: MOLECULAR ASPECTS OF CELLULAR DIVERSITY Course directors GREENBERG, PETER, Cornell University WOLFE, RALPH, University of Illinois Other faculty, staff, and lecturers BLAKESMORE. RICHARD, University of New Hampshire DILLING, WALTRAUD, University of Konstanz, FRG FRANKEL, RICHARD, Massachusetts Institute of Technology HARRIGAN, JUNE, Marine Biological Laboratory HARTZELL, PATRICIA, University of Illinois HARWOOD, CAROLINE, Cornell University KAPLAN, HEIDI, Cornell University LEIGH, JOHN, Massachusetts Institute of Technology LEADBETTER, JARED, Marine Biological Laboratory SCHINK, BERNARD, University of Konstanz, FRG * Post-course Participants 56 MARINE BIOLOGICAL LABORATORY Students BOBIK, THOMAS A.. University of Illinois BRYDEN, CYNTHIA G., Boston University Marine Program COUTINHO, JOHN B., Queen's University, Canada DAMERVAL, THIERRY, Institute Pasteur, France DUNLAP, PAUL V., Cornell University GRAY, KENDALL M., University of California LEWITUS, ALAN!., Woods Hole Oceanographic Institution MARINELARENA, ALEJANDRO J., Universidad Nacional de la Plata, Argentina MARTIN, MICHAEL M., University of Michigan O'BRIEN, WENDY A., Dana-Farber Cancer Institute PASCUAL-SPADA, MARIA MERCEDES, Universidad de Santa Ursula, Argentina PRATT, CHARLES W., University of Illinois ROUVIERE, PIERRE E., University of Illinois SCHWARZENBACH, RENE P., Swiss Federal Institute for Water Resources, Switzerland SMITH, CLIFFORD MARK, Harvard University STANTON, ALICIA M., SUNY, Geneseo SUMMERS, LISA CAROL, Swarthmore College TRENT, JONATHAN D., Scripps Institution of Oceanography WARSHAW, JANE E., University of Massachusetts ZEYER, JOSEF A., Swiss Federal Institute for Water Resources, Switzerland MOLECULAR AND CELLULAR IMMUNOLOGY Course director REINISCH, CAROL, Tufts University School of Veterinary Medicine Other faculty, staff, and lecturers ACUTO, ORESTE, Dana-Farber Cancer Institute ALPER, CHESTER, Center for Blood Research BARBOSA, JAMES, Dana-Farber Cancer Institute SELLER, DAVID, Harvard Medical School BEVAN, MICHAEL, Scripps Clinic and Research Institute Boss, JERRY, Harvard University CAPRA, J. DONALD, University of Texas CEBRA, JOHN J., University of Pennsylvania DATTA, SYAMAL, Tufts University EISENBARTH, GEORGE S., Joslin Diabetes Clinic FINK, PAMELA, University of California Cancer Center, San Diego GROOPMAN, JEROME E., Deaconess Hospital HOCHMAN, PAULA, Tufts University HOGG, NANCY. Imperial Cancer Research Fund, England, U.K. JOINER, KEITH, NIAID/NIH KINCADE, PAUL W., Oklahoma Medical Research Foundation LESKOWITZ, SIDNEY, Tufts University McCLUSKY, ROBERT, Massachusetts General Hospital MosiLR DON, Medical Biological Institute NADLER, LEE , Dana-Farber Cancer Institute PARKER, KENNETH, Harvard University PARKER, DAVID C, University of Massachusetts Medical School RICHARD, FRANK, Yale school of Medicine ROCKLIN, Ross, Tufts New England Medical Center SCHLOSSMAN, STUART, Dana-Farber Cancer Institute SCHWARTZ, ROBERT, Tufts New England Medical Center SPRENT, JONATHAN, Scripps Clinic and Research Institute EDUCATIONAL PROGRAMS 57 STROM, TERRY, Massachusetts General Hospital SUNSHINE, GEOFFREY H., Tufts University THEOHARIDES, THEOHARIS, Tufts University WALL, RANDOLPH, University of California, Los Angeles WILSON, DARCY B., Medical Biological Institute Students BAKST, MURRAY R., U. S. Department of Agriculture BALAZS, TIBOR, U. S. Food and Drug Administration CINQUINA, CARMELA L., West Chester University CONWAY, CAROLYN M., Virginia Commonwealth University GRAZE, KATHLEEN K., New York State Psychiatric Institute DE HOLL, JOHN DAVID, Washington and Lee University JACOBS, JEROME B., St. Vincent Hospital KOMMINENI, CHOUDARI, Mobil Oil Corporation LIGAS, JAMES R., University of Connecticut MAMUYA, WILFRED, Boston University MCCARTHY, JAMES G., University of Connecticut MCFARLAND, WALTER G., University of Louisville MEYDANI, SIMIN N., Tufts University MIRANDA, MARIA T., University of Puerto Rico PHILLIPS, FREDRICJ., John F. Kennedy University RAO, POTU N., University of Texas SABA, THOMAS, Albany Medical College SMITH, JESSICA L., Ohio University TROESCH, CLAUDIA D., University of Southern California NEURAL SYSTEMS AND BEHAVIOR Course directors CAREW, THOMAS, Yale University KELLEY, DARCY, Columbia University Other faculty, staff, and lecturers BASS, ANDREW, Cornell University BURD, GAIL, Rockefeller University BYRNE, JOHN, University of Texas Medical School CALABRESE, RONALD, Harvard University GROBER, MATTHEW, University of California, Los Angeles HAERTER, URSULA, Cornell University HARRIS-WARRICK, RONALD, Cornell University HOSKJNS, SALLY, Columbia University JOHNSON, BRUCE, Boston University KARTEN, HARVEY, SUNY, Stony Brook MACAGNO, EDUARDO, Columbia University MARDER, EVE, Brandeis University MARLER, PETER, Rockefeller University MARCHATERRE, MARTIN, Marine Biological Laboratory MCROBERT, SCOTT, Temple University MENZEL, RANDOLPH, Free University of Berlin, FRG PEINADO, ALEX, Columbia University RUSAK, BENJAMIN, Dalhousie University, Canada SCHAFER, SABINE, Free University of Berlin, FRG SEGIL, NEIL, Columbia University SIMPSON, BLAIR, Rockefeller University THOMPSON, RICHARD, Stanford University 58 MARINE BIOLOGICAL LABORATORY TOMPKINS, LAURIE, Temple University TOBIAS, MARTHA, Columbia Universi ty WALSH, JOHN, University of Texas Medical School WEEKS, JANIS, University of California ZIPSER, BIRGIT, NIH Students BAADER, ANDREAS, University of Berlin, FRG CRONER, LISA J.. Duke University DUBAS, FRANCOISE, University of Texas FEIGENBAUM, JANET D., Stanford University FRIEDMAN, ROBERT M., C. B. Wilson Center HODGSON, TRACY M., Wayne State University INGRAM, DONNA A., University of Texas KATAYAMA, AKIKO, University of Hawaii LOHMANN, KENNETH J., University of Washington MARCUS, EMILIE A., Yale University McPHERSON, DUANE, University of Texas Medical Branch MONTEMAYOR, MICHELLE E., University of Illinois PELUFFO, LUCAS, University of California, San Diego READ, HEATHER L., University of Texas REIMER, KATRIN, University of Munich, FRG RiOULT-PEDOTTi, MARC GUY, Swiss Federal Institute of Technology, Switzerland RiOULT-PEDOTTi, MENGIA SERAINA, University of Zurich, Switzerland SCHARFF, CONSTANCE, Rockefeller University SCHOLZ, KENNETH P., University of Texas STENGL, MONIKA, Columbia University NEUROBIOLOGY Course director KARLIN, ARTHUR, Columbia University Other faculty, staff, and lecturers ADAMS, PAUL, SUNY, Stony Brook ANDREWS, BRIAN, NINCDS/NIH ANDERSON, DAVID, Columbia University BAKALYAR, HEATHER, William Smith College BLUM, MARIANNE, Columbia University BRETT, ROGER, SUNY, Stony Brook FISHBACH, GERALD, Washington University School of Medicine GREENGARD, PAUL, Rockefeller University GURNEY, ALISON, California Institute of Technology HALL, LINDA, Albert Einstein College of Medicine HEUSER, JOHN, Washington University School of Medicine HESS, PETER, Yale University HUSE, WILLIAM, Yale School of Medicine HUDSON, RICHARD, University of Toledo, Canada INOUE, TED, Cornell University JONES, STEVEN, SUNY, Stony Brook KAO, PETER, Columbia University KANDEL, ERIC, Columbia University LANDIS, DENNIS, Massachusetts General Hospital LANDIS, STORY, Harvard Medical School LEVITAN, IRWIN, Brandeis University LESTER, HENRY, California Institute of Technology EDUCATIONAL PROGRAMS 59 LISMAN, JOHN, Brandeis University MARDER, EVE, Brandeis University MARLER, JENNIFER, Yale University MATSUMOTO, STEVEN, Harvard Medical School MILLER, CHRISTOPHER, Brandeis University RA VIOLA, ELIO, Harvard Medical School REESE, THOMAS, NINCDS/NIH/Marine Biological Laboratory REEDY, MICHAEL, Duke University Medical School RICHARDS, FREDERIC M., Yale University ROBERTS, JAMES, Columbia University ROWLAND, LEWIS, Columbia University SIGWORTH, FRED, Yale University School of Medicine SILMAN, ISRAEL, Weizmann Institute, Israel SPUDICH, JOHN, Albert Einstein College of Medicine Students BALICE-GORDON, RITA J., University of Texas BREZINA, VLADIMIR, University of California, Los Angeles FALLS, DOUGLAS L., Ellis Hospital ITAGAKI, HARUHIKO, University of Alabama IVENS, INGEBORG, Ruhr-Universitat Bochum, FRG KOROSHETZ, WALTER JOSEPH, Harvard Medical School MONTAGUE, P. READ, University of Alabama, Birmingham OWENS, JESSE L., University of Alaska REGAN, LAURA J., Harvard University RICH, MARK M., Washington University SAEZ, JUAN, Albert Einstein College of Medicine SUTTON, FEDORA, Howard University PHYSIOLOGY: CELL AND MOLECULAR BIOLOGY Course director GOLDMAN, ROBERT, Northwestern University Other faculty, staff, and lecturers ALBRECHT-BUEHLER. G., Northwestern University BENDER, WELCOME, Harvard Medical School BLOOM, KERRY, University of North Carolina CANDE, W. ZACHEUS, University of California CHISHOLM, REX, Northwestern University DUTCHER, SUSAN, University of Colorado EARNSHAW, WILLIAM, Johns Hopkins University FUKUI, YOSHIO, Osaka University, Japan GETHING, MARY JANE, Cold Spring Harbor Laboratory GLASER, Luis, Washington University School of Medicine GREEN, KATHLEEN, Northwestern University HUBBARD, ANN, Johns Hopkins University HYAMS, JEREMY, University College, U.K. JAMIESON, JAMES, Yale University School of Medicine JONES, JONATHAN, Northwestern University KABACK, D., University of New Jersey LEINWAND, LESLIE, Albert Einstein Medical School LUCA, FRANK, Worcester Foundation of Experimental Biology MAYRAND, SANDRA, Worcester Foundation of Experimental Biology McGovERN, KAREN J., Dana-Farber Cancer Institute MCNALLY, ELIZABETH, Albert Einstein Medical School 60 MARINE BIOLOGICAL LABORATORY MURRAY, ANDREW, Massachusetts General Hospital PEDERSON, THORU, Worcester Foundation of Experimental Biology ROEDER, ROBERT G., Rockefeller University RUSHFORTH, ALICE, Earlham College SCHWARTZ, LAWRENCE M., University of North Carolina SOLL, DAVID, University of Iowa SPUDICH, JAMES, Stanford University STEINERT, PETER, NIH UTKU, SHERMIN, Earlham College VALLEE, RICHARD, Worcester Foundation of Experimental Biology WEBEN, ERIC D., Mayo Foundation WITMAN, GEORGE, Worcester Foundation of Experimental Biology YEH, ELAINE, CIBA-GEIGY Students BARBE, MARY F., Wake Forest University BARISH, MICHAEL E., University of California, Los Angeles BLACKMON, RONALD H., Howard University BOCKMAN, RICHARD S., Cornell University Medical College BYERS, TIMOTHY J., Harvard University CAMPBELL, COLIN R., Boston University School of Medicine CASSIMERIS, LYNNE U., University of North Carolina CHEN, TUNG-LING L., University of Maryland DANOWSKI, BARBARA A., University of North Carolina EDSON, KATHRYN J., University of Minnesota FREEMAN, MARK E., University of Virginia GOODWIN, ELIZABETH, Brandeis University HALUSKA, FRANK G., University of Pennsylvania HANNA, MAYA, Harvard University HARMON, GARY L., Howard University HUNNICUTT, GARY R., University of Texas Health Science Center LACEY, MERRI LYNN, University of California, Riverside LANS, DEBORAH A., University of California, Berkeley LENART, THOMAS D., Johns Hopkins University NOORILY, STUART W., Boston University OBAR, ROBERT A., Boston University C/HALLORAN, THERESA J., University of North Carolina PINES, JONATHON N., Cambridge University, U.K. PRET, ANNE-MARIE, Wesleyan University SCHUMAKER, KAREN S., University of Maryland TROESCH, CLAUDIA D., University of Southern California TUTTLE, REBECCA, Purdue University VIKSTROM, KAREN LYNN, University of Michigan WALTHER, ZENTA, Yale University WANG, GORDON V. L., University of Hawaii WARNER, CECILIA A., Northwestern University WESTON, WAYDE M., University of Pennsylvania Wu, BEI-YUE, Wayne State University Wu, JULIAN K., Tufts University YABOWITZ, RACHEL, University of Miami SPRING BIOPHYSICS OF NEURAL FUNCTION Course director ALKON, DANIEL L., NINCDS/NIH, Marine Biological Laboratory EDUCATIONAL PROGRAMS 6 1 Other faculty, staff, and lecturers BRODWICK, MALCOLM, University of Texas BRIGHTMAN, MILTON, NINCDS/NIH CHARLTON, MILTON, University of Toronto, Canada CLAPHAM, DAVID, Harvard Medical School CONNOR, JOHN, Bell Laboratories DEFELICE, Louis, Emory University School of Medicine DELORENZO, ROBERT, Yale University School of Medicine DIONNE, VINCENT E., University of California, San Diego FINKELSTEIN, ALAN, Albert Einstein College of Medicine FOREMAN, ROBIN, Marine Biological Laboratory FRENCH, ROBERT, University of Maryland School of Medicine GILBERT, CHARLES, Rockefeller University GOVIND, C. K., University of Toronto, Canada GRUOL, DONNA L., Research Institute of Scripps Clinic HOCHBERGER, PHILLIP, Bell Laboratories JOHNSON, BRUCE, Boston University KAPLAN, EHUD, Rockefeller University LECAR, HAROLD, NIH MATSUMOTO, HIRO, Purdue University MCDERMOTT, AMY, NIH MORRIS, CATHERINE, University of Ottawa, Canada MORAN, NAVA, NINCDS/NIH PALOTTA, BARRY, University of North Carolina PAPPAS, GEORGE, University of Illinois Medical Center POLLARD, HARVEY, NIADDKD/NIH RASMUSSEN, HOWARD, Yale University School of Medicine REESE, THOMAS, NINCDS/NIH/Marine Biological Laboratory ROJAS, EDUARDO, NIADDKD/NIH SCHNAPP, BRUCE, Marine Biological Laboratory SZUTS, ETE, Marine Biological Laboratory WEISS, THOMAS F., Massachusetts Institute of Technology YEO, CHRISTOPHER, Medical Research Council, U.K. ZBICZ, KERRY, NIAAA/NIH Students BREW, HELEN M., University College, London, U.K. CHANDRAMANI, NINA, Syracuse University CHEN, CHONG, University of Rhode Island GALENO, THERESA M., New York State Department of Health GARG, AJAY P., Albert Einstein College of Medicine MAGUIRE, GREGORY W., University of Miami MAZZANTI, MICHELE, Emory University School of Medicine MCCLINTOCK, TIMOTHY S., University of Florida OWENS, JESSE L., University of Alaska PARK, SUSANNA S., Yale University RICE, PETER J., West Virginia University Medical Center SATTER, RUTH L., University of Connecticut SILVA, NANCY L., Yale University School of Medicine SREBRO, BOLEK, University of Bergen, Norway TAHMOUSH, ALBERT J., Hahnemann University ZUFALL, FRANK, Institute of Animal Physiology, FRG 62 MARINE BIOLOGICAL LABORATORY SHORT COURSES BASIC IMMUNOC YTOCHEMICAL TECHNIQUES IN TISSUE SECTIONS AND WHOLE MOUNTS September 30-October 5, 1985 Course directors BELTZ, BARBARA, Harvard Medical School BURD, GAIL D., Rockefeller University Course assistants KOBIERSKI, LINDA, Harvard Medical School O'LouGHLiN, BARBARA, Rockefeller University Students BEVAN, JOHN A., University of Vermont CALLARD, IAN P., Boston University CHIKARAISHI, DONA, Tufts University School of Medicine COPELAND, JONATHAN, Swarthmore College HAYASHI, JON H., University of North Carolina JENSEN, KARL, U. S. Environmental Protection Agency MEKEEL, HAROLD E., Harvard Medical School OAKLEY, BRUCE, University of Michigan OLAND, LYNNE A., Georgetown University STUART, ANN E., University of North Carolina TALAMO, BARBARA R., Tufts Medical School TIEMAN, SUZANNAH BLISS, SUNY, Albany YAHR, PAULINE, University of California, Irvine MARICULTURE: CULTURE OF MARINE INVERTEBRATES FOR RESEARCH PURPOSES May 19-25, 1985 Course director CARL BERG, JR., Marine Biological Laboratory Other faculty and assistants ADEY, WALTER, Smithsonian Institute BOWER, CAROL, Eastern Connecticut State University CAPO, THOMAS, Marine Biological Laboratory CAPUZZO, JUDITH, Woods Hole Oceanographic Institution DOYLE, ROGER, Dalhousie University, Canada GARIBALDI, Louis, New York Aquarium GUILLARD, BOB, Bigelow Laboratories HANLON, ROGER, University of Texas HARRIGAN, JUNE, NIH/Marine Biological Laboratory LANGDON, CHRISTOPHER, University of Delaware TURNER, DAVID, Eastern Connecticut State University Students ABRANT, RONALD J., Loyola University BACHAND, ROBERT G., Long Island Sound Task Force Oceanic Society GARFIELD, NINA H., Western Psychiatric Institute GIRALDO, JORGE A.. Washington University HAWES, ROBERT O., University of Maine HERZIG, CHRIS, Occidental College HINES, JOHN L., Massachusetts General Hospital KOCHANE, IDA, Newtonville, MA KUTZ, STEVEN L., George Mason University EDUCATIONAL PROGRAMS 63 STABILE, JOSEPH, City College, CUNY WARD, STEPHEN H., U. S. Environmental Protection Agency THREE INTEGRATED SHORT COURSES IN QUANTITATIVE IMAGE AND SIGNAL ANALYSIS FOR BIOLOGISTS December 9- 19, 1985 Faculty BUSCHMANN, ROBERT J., VA Medical Center, Chicago HASELGROVE, JOHN C., University of Pennsylvania JONES, JUDSON P., University of Pennsylvania PALMER, LARRY A., University of Pennsylvania PEACHEY, LEE D., University of Pennsylvania Students ARIANO, MARJORIE, University of Vermont College of Medicine ASTION, MICHAEL, University of Pennsylvania School of Medicine BEUERMAN, ROGER, Louisiana State University Eye Center BOSCH, ELIZABETH, University of California, Los Angeles BRAVO, MARY, Northwestern University CROPPER, ELIZABETH, Columbia University ECKERT, BARRY S., SUNY, Buffalo EISENMAN, LEONARD M., Jefferson Medical College FAMIGLIETTI, EDWARD V., JR., Wayne State University FRANZ, THOMAS P., Procter and Gamble Co. FRIEDMAN, MARC M., Georgetown University GRADY, PATRICIA A., University of Maryland School of Medicine HEATH, JULIAN P., University of Pennsylvania JACOBS, MYRON S., New York University Dental Center KETTEN, DARLENE R., Massachusetts Eye and Ear Infirmary LA VIA, LYNN A., University of Kansas School of Medicine LUCZKA, CAROL JEAN, Cornell University McCAULEY, BRIGID, University of Missouri, St. Louis MCEACHRON, DONALD LYNN, Drexel University MILLARD, PAUL J., Cornell University MOHR, CHARLES, Cornell University MOSES, RANDY L., Louisiana State University Medical Center NELSON, GINA M., University of Colorado, Denver PAINE, PHILIP L., Michigan Cancer Foundation PARK, JANIE C., Florida Institute of Technology PECK, CAROL K., University of Missouri, St. Louis POOLE, MAX, East Carolina University PREVETTE, DAVID M., Bowman Gray School of Medicine ROYE, DAVID B., University of North Carolina, Wilmington SENS, MARY ANN, Medical University of South Carolina SPOSITO, NADINE M., SUNY Stony Brook THORNBURG, KENT L., Oregon Health Sciences University WHEELER, DAVID A., Brandeis University YANO, BARRY L., Dow Chemical, U.S.A. X. RESEARCH AND TRAINING PROGRAMS SUMMER PRINCIPAL INVESTIGATORS ALLEN, ROBERT DAY, Dartmouth College ANDERSON, WINSTON A., Howard University 64 MARINE BIOLOGICAL LABORATORY ARMSTRONG, CLAY M., University of Pennsylvania ARMSTRONG, PETER B., University of California, Davis AUGUSTINE, GEORGE, University of Southern California BACIGALUPO, JUAN, University of Chile, Chile BARLOW, ROBERT B., Syracuse University BARRY, DANIEL, University of Michigan BARRY, SUSAN R., University of Michigan BEAUGE, Luis ALBERTO, Institute de Investigacion Medica, Argentina BENNETT, MICHAEL V. L., Albert Einstein College of Medicine BEZANILLA, FRANCISCO, University of California, Los Angeles BODZNICK, DAVID, Wesleyan University BORGESE, THOMAS A., Lehman College BORON, WALTER F., Yale University BOYER, BARBARA C, Union College BRADY, SCOTT T., Case Western Reserve University BREHM, PAUL, Tufts University School of Medicine BROWN, JOEL E., Washington University BURDICK, CAROLYN J., Brooklyn College BURGER, MAX M., University of Basel, Switzerland CHANG, DONALD C., Baylor College of Medicine CHAPPELL, RICHARD L., Hunter College CHARLTON, MILTON P., University of Toronto, Canada CHOW, ROBERT H., University of Pennsylvania COHEN, LAWRENCE B., Yale University School of Medicine COHEN, ROCHELLE S., University of Illinois COHEN, WILLIAM D., Hunter College COOPERSTEIN, SHERWIN J., University of Connecticut Health Center COSTELLO, WALTER J., Ohio University CORWIN, JEFFREY T., University of Hawaii COTANCHE, DOUGLAS A., University of South Carolina COURTOIS, YVES, National Medical Research Laboratory, France CRAIG, CATHERINE, Cornell University DEARRY, C. ALLEN, University of California, Berkeley DE WEER, PAUL, Washington University School of Medicine DUNHAM, PHILIP B., Syracuse University DUNLAP, KATHLEEN, Tufts Medical School ECICBERG, WILLIAM R., Howard University FISHMAN, HARVEY M., University of Texas Medical Branch GILBERT, DANIEL L., NINCDS/NIH GIUDITTA, ANTONIO, Western Psychiatric Institute GOVIND, C. K., University of Toronto, Canada GRAF, WERNER M., Rockefeller University HALVORSON, HARLYNO., Brandeis University HARRINGTON, JOHN P., University of Alaska HIGHSTEIN, STEPHEN M., Washington University School of Medicine HOSKIN, FRANCIS C. G., Illinois Institute of Technology HUMPHREYS, TOM, University of Hawaii INGOGLIA, NICHOLAS A., New Jersey Medical School KALMIJN, ADRIANUS, Scripps Institution of Oceanography KAMINER, BENJAMIN, Boston University KAPLAN, EHUD, Rockefeller University KELLY, ROBERT E., University of Illinois KEYNAN, ALEX, Memorial Sloan Kettering KHAN, SHAHID M. M., University of Punjab, Pakistan KNIER, JULIE A., University of Minnesota KORNBERG, HANS, University of Cambridge, United Kingdom LANDOWNE, DAVID, University of Miami LANGFORD, GEORGE M., University of North Carolina School of Medicine RESEARCH AND TRAINING PROGRAMS 65 LASER, RAYMOND J., Case Western Reserve University LAUFER, HANS, University of Connecticut LEVIN, JACK, University of California LEVIS, RICHARD A., Rush Medical Center LICHTMANN, JEFF, Washington University LIPICKY, RAYMOND JOHN, Food and Drug Administration LISMAN, JOHN, Brandeis University LLINAS, RUDOLFO R., New York University Medical Center LOEWENSTEIN, WERNER R., University of Miami LORAND, LASZLO, Northwestern University MALBON, CRAIG C, SUNY, Stony Brook MANCILLAS, JORGE R., Scripps Clinic & Research Institute MARCUM, JAMES A., Massachusetts Institute of Technology MATTESON, DONALD R., University of Pennsylvania MATSUMURA, FUMIO, Michigan State University METUZALS, J., University of Ottawa, Canada MOORE, JOHN W., Duke University MULLINS, LORIN J., University of Maryland, Baltimore NAGEL, RONALD L., Albert Einstein College NAKA, KEN-!CHI, National Institute of Basic Biology, Japan NARAHASHI, TOSHIO, Northwestern University Medical School NASI, ENRICO, Boston University NELSON, LEONARD, Medical College of Ohio NOE, BRYAN D., Emory University OSSES, Luis, Institute Venezolanode Investigaciones Cientifican, Venezuela OXFORD, GERRY S., University of North Carolina PALMER, JOHN, University of Massachusetts PETERSON, RUSSELL L., Howard University PIERCE, SIDNEY K., University of Maryland PINKHASOV, ELIZABETH, Hunter College PRATT, MELANIE M., University of Miami School of Medicine PUMPLIN, DAVID W., University of Maryland School of Medicine PURVES, DALE, Washington University QUIGLEY, JAMES P., SUNY, Downstate Medical Center RAKOWSKI, ROBERT F., Chicago Medical School RANDO, THOMAS A., Harvard Medical School REBHUN, LIONEL I., University of Virginia REED, CHRISTOPHER G., Dartmouth College REYNOLDS, GEORGE T., Princeton University RICKLES, FREDERICK R., University of Connecticut RIPPS, HARRIS, New York University School of Medicine RUDERMAN, JOAN V., Harvard Medical School RUSSELL, JOHN M., University of Texas Medical Branch SAKURANGA, MASAMORI, Nippon Medical School, Japan SALZBERG, BRIAN M., University of Pennsylvania SANGER, JEAN, University of Pennsylvania SANGER, JOSEPH W., University of Pennsylvania SATTELLE, DAVID B., University of Cambridge, England, U.K. SCHUEL, HERBERT, SUNY, Buffalo SCOFIELD, VIRGINIA L., University of California, Los Angeles SEGAL, SHELDON J., Rockefeller Foundation SHEETZ, MICHAEL?., University of Connecticut Health Center SILVER, ROBERT B., University of Wisconsin SJODIN, RAYMOND A., University of Maryland, Baltimore SLOBODA, ROGER D., Dartmouth College SLUDER, GREENFIELD, Worcester Foundation for Experimental Biology SMITH, STEPHEN J., Yale School of Medicine SMOLOWITZ, ROXANNA, Marine Biological Laboratory 66 MARINE BIOLOGICAL LABORATORY SPECK, WILLIAM T., Rainbow Babies and Childrens Hospital SPIEGEL, EVELYN, Dartmouth College SPIEGEL, MELVIN, Dartmouth College STEINACKER, ANTOINETTE, Washington University STEPHENS, PHILIP!., ViHanova University STEWART, RANDALL R., Columbia University STRACHER, ALFRED, SUNY, Downstate Medical Center STRUMWASSER, FELIX, Boston University STUART, ANN E., University of North Carolina TAKEDA, KENNETH, Universite Louis Pasteur, France TASAKI, ICHIJI, NIMH/DHHS TASHIRO, JAY SHIRO, Kenyon College TAYLOR, ROBERT E., NINCDS/NIH TILNEY, LEWIS, University of Pennsylvania TORRES, RAFAEL M., University of California, Los Angeles TREISTMAN, STEVEN N., Worcester Foundation for Experimental Biology TRINKAUS, JOHN PHILIP, Yale University TROLL, WALTER, New York University Medical Center TSACOPOULOS, MARCO, University of Geneva, Switzerland TYTELL, MICHAEL, Wake Forest University VERGARA, CECILIA, University of Chile, Chile VERSELIS, VYTAUTAS, Albert Einstein College of Medicine VINCENT, WALTER S., University of Delaware WAGENBACH, GARY, Carleton College WALZ, BERND, University of Ulm, West Germany WEBB, CHRISTINA, University of California, Los Angeles WEISS, DIETER G., Universitat Munich, West Germany WEISSMAN, GERALD, New York University Medical Center YEH, JAY Z, Northwestern University Medical School ZIGMAN, SEYMOUR, University of Rochester ZlMMERBERG, JOSHUA, NIADDKD/NIH ZOTTOLI, STEVEN J., Williams College LIBRARY READERS ADELBERG, EDWARD A., Yale Medical School ALBRIGHT, JOHN T., Harvard School of Dental Medicine ALKON, DANIEL, NIH/Marine Biological Laboratory ALLEN, GARLAND, Washington University ANGELETTI, RUTH HOGUE, University of Pennsylvania Medical School ARMSTRONG, MARGARET, University of California BABITSKY, STEVEN, Kisten, Babitsky, Latimer & Beitman BANG, BETSY, Marine Biological Laboratory BEAN, CHARLES P.. General Electric Company BERNHEIMER, ALAN W., New York University College of Medicine BOETTIGER, JULIE, Temple University BOURNE, DONALD, Marine Biological Laboratory BOYER, JOHN F., Union College BURSYTAJN, SHERRY, Baylor College of Medicine CANDELAS, GRACIELA C, University of Puerto Rico CARLSON, FRANCIS, Johns Hopkins University CARRIERS, RITA M., S ?NY, Downstate Medical Center CHABOT, JENNIFER L., Kenyon College CHAPPELL, RICHARD L., Hunter College, CUNY CHILD, FRANK, Trinity College CHINARD FRANCIS P., New Jersey Medical School RESEARCH AND TRAINING PROGRAMS 67 CLARK, ARNOLD M., Marine Biological Laboratory COBB, JEWEL PLUMME, California State University COHEN, LEONARD A. C, American Health Foundation COHEN, SEYMOUR S., Marine Biological Laboratory COLLIER, JACK R., Brooklyn College CONWAY, A. F., Randolph-Macon College CRAIG, CATHERINE L., Cornell University DAVIS, BERNARD D., Harvard Medical School DUNCAN, THOMAS K., Nichols College EDER, HOWARD A., Albert Einstein College of Medicine EISEN, HERMAN N., Massachusetts Institute of Technology ELLNER, JERROLD J., Case Western Reserve University FANTINI, BERNARDINO, Stazione Zoologica, Naples, Itlay FARMANFARMAIAN, A., Rutgers University FEINGOLD, DAVID S., New England Medical Center FELDMAN, SUSAN C., New Jersey Medical School FREINKEL, NORBERT, Northwestern University Medical School FRENKEL, KRYSTYNA, New York University Medical Center FRIEDLER, GLADYS, Boston University School of Medicine GABRIEL, ABRAM, Johns Hopkins Hospital GALATZER-LEVY, ROBERT, University of Chicago GARCIA-DE LA TORRE, IGNACIO, Hospitale General de Occidente, Mexico GARDNER, ELIOT LA WREN, Albert Einstein College of Medicine GERMAN, JAMES L., The New York Blood Center GOLDSTEIN, MOISE H., Johns Hopkins University GOODGAL, SOL H., University of Pennsylvania School of Medicine GOTTLIEB, LEONARD S., Boston University School of Medicine GRANT, PHILIP, University of Oregon GROSCH, DANIEL S., North Carolina State University GROSS, PAUL, Marine Biological Laboratory GUTTENPLAN, JOSEPH, New York University Dental Center GWILLIAM, G. FRANK, Reed College HARDING, CLIFFORD, Kresge Eye Institute HAYASHI, TERU, Papanicolaou Cancer Institute, University of Miami HEMPLING, HAROLD G., Medical University of South Carolina HERSKOVITS, THEODORE T., Fordham University HILDEBRAND, JOHN G., Columbia University HILL, ROBERT B., University of Rhode Island ILAN, JOSEPH, Case Western Reserve University ILAN, JUDITH, Case Western Reserve University INOUE, SHINYA, Marine Biological Laboratory INOUE, SADAYUKI, McGill University, Canada JAFFE, LIONEL, University of Pittsburg JOSEPHSON, ROBERT K., University of California, Irvine KACZMAREK, LEONARDK., Yale University School of Medicine KAPLAN, ILENE M., Union College KLEIN, DAVID L., University of California Medical Center, San Francisco KOULISH, SASHA, College of Staten Island, CUNY KRANE, STEPHEN, Massachusetts General Hospital LADERMAN, AIMLEE D., Smithsonian Institute LAZAROW, PAUL B., Rockefeller University LEFEVRE, MARIAN E., Brookhaven National Laboratory LEACH, BERTON J., Rockville, MD LEE, JOHN J., City College, CUNY LEIGHTON, JOSEPH, Medical College of Pennsylvania LEVINE, RACHMIEL, City of Hope Medical Center LEVITAN, IRWIN B., Brandeis University 68 MARINE BIOLOGICAL LABORATORY LEVITZ, MORTIMER, New York University Medical Center LIDDLE, LARRY B., Southampton College LLOYD, DANIEL, University of California LOCKWOOD, ARTHUR H., Albert Einstein Medical Center LONGO, FRANK, University of Iowa LURIA, SALVADOR E., Massachusetts Institute of Technology MARFEY, PETER, SUNY, Albany MAUNTER, HENRY, Tufts University School of Medicine MAUZERALL, DAVID, Rockefeller University McCANN-COLLiER, MARJORIE, Saint Peters College MESELSON, MATTHEW, Harvard University METUZALS, JAMS, University of Ottawa, Canada MITCHELL, RALPH, Harvard University MIZELL, MERLE, Tulane University MONROY, ALBERTO, Stazione Zoologica, Naples, Italy MORRELL, FRANK, Rush Presbyterian, St. Lukes Medical Center MORRELL-DETOLEDO, LEYLA, Rush Presbyterian, St. Lukes Medical Center MOSHE, SHILO, Hebrew University, Israel NICKERSON, PETER A., SUNY, Buffalo OLINS, DONALD, University of Tennessee OLINS, ADA L., University of Tennessee OSCHMAN, JAMES L., Levity Corporation PARSEGIAN, V. ADRIAN, NIH PERSON, PHILIP, VA Medical Center, Brooklyn PFIEFFER, JOHN, New Hope, PA POOLE, ALAN, Marine Biological Laboratory PROVASOLI, LUIGI, Yale University PRUSCOTT, LAURA A., Gonzaga University REINER, JOHN M., Albany Medical College RICE, ROBERT V., Carnegie-Mellon University ROSENBLUTH, RAJA, Simon Fraser University ROTH, EUGENE, JR., Mt. Sinai School of Medicine ROTH, LORRAINE, Brigham and Women's Hospital ROWLAND, LEWIS P., Neurological Institute RUSHFORTH, NORMAN B., Case Western Reserve University RUSSELL-HUNTER, W. D., Syracuse University SALEM, DAVID, Princeton University SANTOS-SACCHI, JOSEPH, New Jersey Medical School SCHLESINGER, R. W., Rutgers Medical School SCHWARTZ, JAMES H., Center for Neurobiology and Behavior SCHWARTZ, MARTIN, University of Maryland, Baltimore County SEAVER, GEORGE A., Seaver Associates SHAPLEY, ROBERT, Rockefeller University SHEMIN, DAVID, Northwestern University SHEPRO, DAVID, Boston University SHERMAN, IRWIN W., University of California SHIH, YiNG-HSfEN, Institute of Developmental Biology, China SHRIFTMAN, MOLLIE STARR, North Nassau Mental Health Center SHUB, D/. 1UNY, Albany SIGEL, ELIZABETH K., Kenyon College SONNENBLICK, BENJAMIN, Rutgers University SPECTOR, ABRAHAM, Columbia University SPOTTE, STEPHEN, Mystic Marinelife Aquarium STEINBERG, MALCOLM, Princeton University STEPHEN, MICHAEL J., Rutgers University STEPHENSON, WILLIAM K., Earlham College RESEARCH AND TRAINING PROGRAMS 69 SUSSMAN, MAURICE, University of Pittsburg SZENT-GYORGYI, ANDREW G., Brandeis University SzENT-GvORGYi, EVA M., Brandeis University TASHIRO, JAY SHIRO, Kenyon College TRACER, WILLIAM, Rockefeller University TRUE, MERRILL ALLA, Bio-Oceanic Research TRUE, RENATE, College of the Mainland TWEEDELL, KENYON S., University of Notre Dame VAN HOLDE, K. E., Oregon State University WAINIO, WALTER, Rutgers University WALBORN, PATRICIA ANN, Kenyon College WARREN, LEONARD, Wistar Institute WEBB, H. MARGUERITE, Marine Biological Laboratory WEIDNER, EARL, Louisiana State University WHEELER, GEORGE, Brooklyn College WlCHTEREMAN, RALPH, Woods Hole, MA WILBUR, CHARLES G., Colorado State University WILSON, DONELLA J., Whitehead Institute WITTENBERG, BEATRICE, Albert Einstein College of Medicine WITTENBERG, JONATHAN, Albert Einstein College of Medicine WOLKEN, JEROME J., Carnegie-Mellon University WORGUL, BASIL V., Columbia University WORTHINGTON, C. R., Carnegie-Mellon University YOUNG, LILY, New York University Medical Center YOUNG, WISE, New York University Medical Center Yow, F. W., Kenyon College ZACKS, SUMNER I., The Miriam Hospital/Brown University OTHER RESEARCH PERSONNEL AHLUWALIA, BALWANT, Howard University ALLIEGRO, MARK, SUNY, Buffalo ALLEN, NINA STROMGREN, Wake Forest University ALTAMIRANO, ANIBAL, University of Texas ARMSTRONG, CLARA FRANZINI, University of Pennsylvania ARNOLD, JOHN M., University of Hawaii AZARAFF, LENORE, Princeton University BAKER, ROBERT, New York University Medical Center BAKER, Ross, Boston University BATES, HISLA, Hunter College BAUER, G. ERIC, University of Minnesota BENNETT, ELENA, Connecticut College BEARCE, CATHERINE, Dartmouth College BOYLE, RICHARD, Washington University School of Medicine BORDEN, PAULA, University of Hawaii BOWLES, ELIZABETH, Wake Forest University BROWN, LESLEE DODD, Northwestern University BREITWEISER, GERDA E., University of Texas Medical Branch BREMMER, THEODORE, Howard University BUCHANAN, JO-ANNE, Yale University BYRNE, PAUL, National Institute of Mental Health/Department of Health and Human Services CATTARELLI, MARLINE, Universite Claude Bernard, France CAPUTO, CARLO, Institute de Investigacion Medica, Argentina CARIELLO, Lucio, Zoological Station, Naples, Italy 70 MARINE BIOLOGICAL LABORATORY CHEN, ERIC, Northwestern University CHANDLER, ROBERT, University of Maryland CLARKE, MARGARET, Albert Einstein College COHEN, GEORGE, Kenyon College COLTON, CAROL, NINCDS/NIH CORONADO, ROBERT, University of North Carolina COTA-PENUELAS, GABRIEL, University of Pennsylvania COUCH. ERNEST, Texas Christian University COUCH, JOHN, Boston University CRONKJTE, DONALD, Hope College CZINN, STEVEN, Rainbow Babies & Childrens Hospital DAVIDSON, SARAH, Columbia University DEWEILLE, JAN, University of Utrecht, Netherlands DEFILIPPIS, ANTHONY, Howard University DENTLER, WILLIAM L., University of Kansas DIXON, ROBERT, College of The Holy Cross DIPOLO, REINALDO, Institute de Investigacion Medica, Argentina DINSMORE, JONATHAN H., Dartmouth College EHRLICH, BARBARA, Albert Einstein College EISENMANN, GEORGE, University of California, Los Angeles ESTUS, STEVEN, Rainbow Babies & Childrens Hospital PATH, KARL, Case Western Reserve University FENNELLY, G. J., University of New Jersey FEINMAN, RICHARD, SUNY, Downstate Medical Center FINK, RACHEL A., Yale University FISHER, GREGORY, Carnegie Mellon University FLIMETA, JEFF, Carleton College FONG, C. N., University of Toronto, Canada FORT, EDDIE, A., University of Texas Medical Branch FORSCHER, PAUL, Yale University FRANK, DOROTHY, Rainbow Babies and Childrens Hospital GADSBY, DAVID C., Rockefeller University GAINER, HAROLD, NICHD/NIH GALBRAITH, JIM, University of Pennsylvania GILBERT, SUSAN P., Dartmouth College GLABACH, RICHARD K., Syracuse University GONZALEZ, HUGO, University of Maryland GOULD, ROBERT, New York Institute For Basic Research in Mental Retardation GOWER, DAVID, Bowman Gray Medical School GRIFFITH, C., McGill University, Canada GRUNER, JOHN A., New York University HARRIS, E. M., Duke University HERLANDS, Louis, Rockefeller Foundation HILL, DAVID, Bowman Gray Medical School HINES, MICHAEL, Duke University HIRIART, MARCIA, University of Pennsylvania HORST. CYNTHIA, Emory University HOUGHTON, SUSAN, Marine Biological Laboratory HOWARD, HEIDI, Marine Biological Laboratory HUG, CHRISTOPHER, University of Cincinnati HUNT, TIM, University of Cambridge, United Kingdom INOUE, TOMO, Boston University IWASA, KUNIHIKO, NINCDS/NIH JACKSON, Lo VERNE, University of Ottawa, Canada JiA-XiN, Liu, Baylor College JOHNSTON, KAREN, University of Toronto, Canada RESEARCH AND TRAINING PROGRAMS 7 1 KAMINO, KOHTARO, Tokyo Medical & Dental University, Japan KASS, LEONARD, Eastern Virginia Medical School KLEIN, KAREN, University of Illinois KLEIN, DEBORAH S., Brown University KNAKAL, ROGER C, Yale University KNIER, BRUCE, Kenyon College KoiDE, SAMUEL S., Population Council, The Rockefeller University KONNERTH, ARTHUR, University of Pennsylvania LANDOLFA, MICHAEL A., Union College LEGIER, ROJAS, University of Puerto Rico LEHMAN, HERMAN, Rockefeller University Liu, JESSICA, Harvard University LLANO, MARIA ISABEL, University of Pennsylvania LONDON, JILL, Yale University LOPEZ-BARNEO, JOSEPH, University of Pennsylvania LORAND, JOYCE, Northwestern University MAHAJAN, DAMODAR, Rainbow Babies and Children Hospital MACHAN, TERRY E., University of California, Berkeley MACKJN, JULIE, Emory University MACDONALD, BELINDA, Howard University MATSUMURA, ICHIRO, Massachusetts Institute of Technology MCCARTHY, ROBERT ALAN, University of Basel, Switzerland McGuiNNESS, THERESA, New York University MEAROW, KAREN, University of Toronto, Canada MELLON, DEFOREST, University of Virginia MILLER, ROBERT, Case Western Reserve University MISEVIC, GRADIMIR, University of Basel, Switzerland NAKAYE, TOSHIO, National Institute of Mental Health, Department of Health and Human Services NASI, PATRICIA, Worcester Foundation for Experimental Biology NEIGEL, JOSEPH, University of California, Los Angeles NEVINS, MARK, University of Connecticut OBAID, ANA LIA, University of Pennsylvania OBRIST, KARIN, University of Basel, Switzerland OHKI, SHINPEI, SUNY, Buffalo OLIVKA, GABRIELE, University of California, Los Angeles ORKAND, RICHARD K., University of Pennsylvania PALAZZO, ROBERT, University of Virginia PANT, HARISH, NIAAA/NIH-NIMH/DHHS PAXHIA, TERESA M., University of Rochester School of Medicine PETERKIN, DARRYL, Yale University PERRY, GAVIN, Washington University PEREZ, ROSA, Howard University PINTO, MARIA, University of California, Los Angeles POCHAPIN, MARK B., Cornell University POTTER, DEBORAH, University of Toronto, Canada QUINN, RICHARD, University of Maryland REQUENA, JAIME, I.D.E.A., Venezuela RESGADO, HECTOR, University of Maryland RICCIO, ROBERT, New Jersey Medical School RIEDER, CONLY L., New York State Department of Public Health RONAN, MARK C., Wesleyan University ROSE, BIRGIT, University of Miami School of Medicine SAHNI, MUKESH, Rockefeller Foundation SANTELLA, LUIGIA, Naples Zoological Station, Italy SCHOLL, MICHAEL JEFFREY, University of Miami 72 MARINE BIOLOGICAL LABORATORY SCHROEDER, BARBARA M., University of Miami SCHROER, TRINA, University of California, San Francisco SCHUEL, REGINA, SUNY, Buffalo SEARS, TED, Dartmouth College SEITZ, ROCHELLE, Colgate University SHILO, MOSHE, Hebrew University, Israel SIMPSON, MARCIA, Amherst College SINKUS, MELISSA, University of Connecticut SOODEEN, CLAUDIA, Howard University SPECK, ANASTASIA, Mount Holyoke College SPECK, STEPHANIE, Syracuse University STANDART, NANCY M., University of Cambridge STEUER, ERIC R., University of Connecticut STIMERS, JOSEPH R., University of California, Los Angeles STEWART, SEAN, McGill University STRACHER, ADAM, Amherst College SUGIMORI, MUTSUYUKI, New York University School of Medicine SWENSON, KATHERINE, Harvard Medical School SZEBENYI, GYORGYI, Hunter College TANGUY, JOELLE, University of Paris, France TILLOTSON, DOUGLAS, Boston University TSUCHIDA, CRAIG, University of California, Los Angeles UENO, HIROSHI, Rockefeller University VALE, RONALD, Stanford University VADASZ, ANDREW, University of Guelph, Canada VIELE, DANIEL P., Boston University WALTON, ALAN J., Oxford University, United Kingdom WESTENDORF, JOANNE, Harvard Medical School WHITAKER, MICHAEL, University College, London, England, U.K. WHITTEMBURY, JOSE, University of Pennsylvania WILLIAMS, ERROL, Princeton University YANG, PAUL, New York University YADA, TOSHIHIKO, University of Miami ZAKEVICIUS, JANE, New York University School of Medicine ZECEVIC, DEJAN, Yale School of Medicine ZHAO, ZHAE-YiONG, Baylor College YEAR-ROUND PROGRAMS BOSTON UNIVERSITY MARINE PROGRAM (BUMP) Directors VALIELA, IVAN WHITTAKER, J. RICHARD Staff (of Boston University unless otherwise indicated) ALLEN, SARAH ATEMA, JELLE BUCHSBAUM, ROBERT CROWTHER, ROBERT DZIERZEWSKI, MICHELLE FREADMAN, MARVIN HAHN, DOROTHY HUMES, ARTHUR G. LOESCHER, JANE RESEARCH AND TRAINING PROGRAMS 73 LOHMANN DENAH MEEDEL,THOMAS MULSOW, SANDOR RIETSMA, CAROL TAMM, SIDNEY L. TAMM, SIGNHILD TAYLOR, MARGERY TIERNEY, ANN JANE VALIELA, IVAN VAN ETTEN, RICHARD VOIGHT, RAINER, University of Gottingen, Denmark WHITTAKER, J. RICHARD Graduate students ALBER, MERRYL JOHNSON, BRUCE BANTA, GARY LAVALLI, KARI BARSHAW, DIANA MERCURIC, KIM BORRONI, PAOLA MERRILL, CARL BRYANT, DONALD Moss, ANTHONY COSTA, JOSEPH PAYNE, MICHAEL COULTER, DOUGLAS SCOTT, MARSHA COWAN, DIANE TAMSE, ARMANDO ELLIS, SARAH TROTT, THOMAS FABRICANT, ROBIN WEBB, JACQUELINE FOREMAN, KENNETH WHITE, DAVID GLICK, STEPHEN WOOD, SUSAN HANDRICH, LINDA Undergraduates FIELDS, DAVID HARRISON, REBECCA Me BRIDE, JAMES OPPENHEIMER, JILL SUNBURY, SUSAN Visiting investigators COGSWELL, CHARLOTTE, University of Connecticut D'AVANZO, CHARLENE, Hampshire College POOLE, ALAN, Boston University RIETSMAN, CAROL, SUNY, New Paltz Wu, SHAN-CHIN, Institute of Oceanology, Peoples' Republic of China DEVELOPMENTAL AND REPRODUCTIVE BIOLOGY LABORATORY Director GROSS, PAUL R. LABORATORY OF BIOPHYSICS Director ADELMAN, WILLIAM J., JR. Staff (of NINCDS/NIH unless otherwise indicated) 74 MARINE BIOLOGICAL LABORATORY Section on Neural Membranes CLAY, JOHN R. FOHLMEISTER, JuRGEN F., University of Minnesota GOLDMAN, DAVID E., SUNY, Binghamton HODGE, ALAN J., Marine Biological Laboratory LAVOIE, ROBERT, Marine Biological Laboratory MARTIN, DOROTHY L. MUELLER, RUTHANNE, Marine Biological Laboratory RICE, ROBERT V., Carnegie-Mellon University STANLEY, ELIS F. TYNDALE, CLYDE L., Marine Biological Laboratory WALTZ, RICHARD B., Marine Biological Laboratory Section on Neural Systems ALKON, DANIEL L., Chief BANK, BARRY, University of Toronto COLLIN, CARLOS COULTER, DOUGLAS, Boston University DISTERHOFT, JOHN, Northwestern University Medical School HARRIGAN, JUNE, Marine Biological Laboratory HOPP, HANS-PETER KUBOTA, MlCHINORI KUZIRIAN, ALAN M. KUZIRIAN, JEANNE LEDERHENDLER, IZJA, Marine Biological Laboratory LEIGHTON, STEPHEN, Biomedical Engineering and Instrumentation Branch, NIH MCPHEE, DONNA NAITO, SHIGETAKA NEARY, JOSEPH, Marine Biological Laboratory SAKAKIBARA, MANABU LABORATORY OF CARL J. BERG, JR. Director BERG, CARL J., JR. Staff ADAMS, NANCY ORR, KATHERINE S. Visiting investigators FARMER, MARY, Sea Education Association WARD, JACK, Division of Fisheries, Government of Bermuda LABORATORY OF CAROL L. REINISCH Director REINISCH, CAROL L., Tufts University School of Veterinary Medicine Staff MIOSKY, DONNA SMOLOWITZ, ROXANNA RESEARCH AND TRAINING PROGRAMS 75 LABORATORY OF D. EUGENE COPELAND Director COPELAND, D. EUGENE LABORATORY OF HOWARD HUGHES MEDICAL INSTITUTE Staff (of Howard Hughes Medical Institute unless otherwise indicated) BIDWELL, JOSEPH CAPO, THOMAS GAGOSIAN, SUSAN GOOD, MICHAEL NADEAU, LLOYD PAIGE, JOHN A. SCHWARTZ, JAMES H., Columbia University LABORATORY OF DEVELOPMENTAL GENETICS Director WHITTAFLER, J. RICHARD Staff CROWTHER, ROBERT LOESCHER, JANE L. MEEDEL, THOMAS H. MERCURIO, KIMBERLY Visiting investigators COLLIER, J. R., Brooklyn College Wu, SHAN-CHIN, Institute of Oceanology, Peoples' Republic of China LABORATORY OF JUDITH P. GRASSLE Director GRASSLE, JUDITH P. Staff GELFMAN, CECILIA E. MILLS, SUSAN W. SHIMETA, JEFFREY S. WAGENBACH, GARY, Carleton College LABORATORY FOR MARINE ANIMAL HEALTH Director LEIBOVITZ, Louis, Cornell University Staff ABT, DONALD A., University of Pennsylvania HADDEN, SCHUYLER, Cornell University 76 MARINE BIOLOGICAL LABORATORY McCAFFERTY, MICHELLE, Cornell University MONIZ, PRISCILLA C, Marine Biological Laboratory LABORATORY OF NOEL DE TERRA Director DE TERRA, NOEL LABORATORY OF OSAMU SHIMOMURA Director SHIMOMURA, OSAMU, Boston University School of Medicine Staff SHIMOMURA, AKEMI Visiting investigator MUSICKI, BRANISLAV, Harvard University LABORATORY OF RAYMOND E. STEPHENS Director STEPHENS, RAYMOND E., Marine Biological Laboratory/Boston University School of Medicine Staff OLESZKO-SZUTS, SUSAN, Marine Biological Laboratory CORSON, D. WESLEY, Marine Biological Laboratory REED, CHRISTOPHER, Dartmouth College STOMMEL, ELIJAH W., Marine Biological Laboratory/Boston University School of Medicine LABORATORY OF ROBERT V. ZACKROFF Director ZACKROFF, ROBERT V. LABORATORY OF SENSORY PHYSIOLOGY Directors FEIN, ALAN MACNICHOL, EDWARD F., JR. Staff COLLINS, BARBARA A. COOK, PATRICIA B. CORSON, D. WESLEY FUKUROTANI, KENKiCHI HAROSI, FERENC I. OLESZKO-SZUTS, SUSAN PAYNE, RICHARD RESEARCH AND TRAINING PROGRAMS 77 REID, MARTHA STEWART, CHARLES SZUTS, ETE Z. THOMAS, LAURA WOOD, SUSAN ZAHAJSZKY, TIBOR Visiting investigators CORNWALL, CARTER, Boston University School of Medicine HAWRYSHYN, CRAIG W., Cornell University PETRY, HEYWOOD M., SUNY, Stony Brook TSACOPOULOS, MARCO, University of Geneva, Switzerland WALZ, BERND, University of Ulm, West Germany LABORATORY OF SHINYA INOUE Director INOUE, SHINYA, Marine Biological Laboratory/University of Pennsylvania Staff AKINS, ROBERT, University of Pennsylvania WOODWARD, BERTHA M. LABORATORY OF NEUROBIOLOGY Director REESE, THOMAS S. Staff (of NINCDS/NIH unless otherwise indicated) ANDREWS, S. BRIAN BURGER, TINA, Marine Biological Laboratory CHENG, TONI P. O. EVENDEN, PHYLLIS PELS, GREGOR, Max Planck Institut, FRG FROKJAER-JENSEN, JORGEN, University of Copenhagen, Denmark HAMMAR, KATHARINE KHAN, SHAHID, Marine Biological Laboratory MICHAUD, JAYNE MURPHY, JOHN C, Marine Biological Laboratory PHILBIN, LINDA M. REESE, BARBARA F. SHEETZ, MICHAEL P., Washington University SCHNAPP, BRUCE J. VALE, RONALD D. WALROND, JOHN P. WISGIRDA, MARY, Marine Biological Laboratory Woo, HEIDE, Marine Biological Laboratory NATIONAL FOUNDATION FOR CANCER RESEARCH Director SZENT-GYORGYI, ALBERT 78 MARINE BIOLOGICAL LABORATORY Staff GASCOYNE, PETER R. C. MCLAUGHLIN, JANE A. MEANY, RICHARD A. PETHIG, RONALD, University College of North Wales, U. K. Student PRICE, JONATHAN A.. University College of North Wales, U. K. NATIONAL VIBRATING PROBE FACILITY Director JAFFE, LIONEL, Marine Biological Laboratory Staff SCHEFFEY, CARL SHIPLEY, ALAN WISGIRDA, MARY Visiting investigators ALLEN, NINA, Wake Forest University ALLEN, ROBERT D., Dartmouth College BJORKMAN, THOMAS, Cornell University CRAWFORD, KAREN, University of Illinois, Urbana DEMAREST, JEFFERY, University of California, Los Angeles ETTENSOHN, CHARLES, Duke University FLUCK, RICHARD, Franklin & Marshall College KATZ, URI, Israel Institute of Technology, Haifa, Israel KUNKEL, JOSEPH, University of Massachusetts LEVY, SIMON, Boston University MACHEN, TERENCE, University of California, Los Angeles MASTROIANNI, LuiGi, University of Pennsylvania PAYNE, RICHARD, Marine Biological Laboratory Qui, TiNG-Hu, Xiamen Fisheries College, Fujian, China RUTTEN, MICHAEL, Harvard University SARDET, CHRISTIAN, Station Marine Villefranche sur Mer, France STRAUSS, JERRY, University of Pennsylvania TROXELL, CYNTHIA, University of Colorado, Boulder TURRECK, RICHARD, University of Pennsylvania WALSBY, ANTHONY E., Bristol University, England, U. K. WEIJER, KEES, University of Munich, FRG THE ECOSYSTEMS CENTER Director HOBBIE, JOHN E. Staff and consultants BANTA, GARY FRUCI, JEAN BERGQUIST, BERIT FRY, BRIAN D. DISOSWAY, MASON GIBLIN, ANNE E. FERRY, ELIZABETH GRIFFIN, ELISABETH A. RESEARCH AND TRAINING PROGRAMS 79 HELFRICH, JOHN V. K. OPPENHEIMER, JILL HOUGHTON, RICHARD A. PETERSON, BRUCE J. HOWARTH, ROBERT W. PLUMMER, NANCY JOHNSON, STEPHEN REGAN, KATHLEEN JORDAN, MARILYN SEMINO, SUZANNE J. LAUNDRE, JAMES SHAVER, GAIUS R. LEFKOWITZ, DANIEL STEUDLER, PAUL A. MANN, ALICIA STONE, THOMAS MARINO, ROXANNE TUCKER, JANE MATHERLY, WALTER TURNER, ANDREA R. MELILLO, JERRY M. WHITE, DAVID NADELHOFFER, KNUTE YANDOW, TIMOTHY Trainees RUDNICK, DAVID, University of Rhode Island Visiting investigator LUTHER, GEORGE, Kean College of New Jersey XI. HONORS FRIDAY EVENING LECTURES ALLEN, ROBERT D., Dartmouth College, 28 June, "Microtubules, Motility, and Cytoplasmic Transport" HILDEBRAND, JOHN G., Columbia University, 5 July, Lang Lecture, "Explorations of a Miniature Brain " LEDER, PHILIP, Harvard Medical School, 12 July, "Misplacing Genes: Genetic Engineering and the Cancer Problem" RAFF, MARTIN, University College, London, 18, 19 July, Forbes Lectures, "An Antibody and Cell Culture Approach to Mammalian Neurodevelopment: I. Cell-Cell Interactions in the Developing Peripheral Nervous System; II. Cell Lineages and Cell Differentiation in the Developing Central Nervous System " EDELMAN, GERALD, The Rockefeller University, 26 July, "Cell Adhesion Molecules and the Regulation of Animal Form" GEHRING, WALTER, Biozentrum, University of Basel, 2 August, "Homeotic Genes and the Control of Development" BENACERRAF, BARUJ, Harvard Medical School, 9 August, "Immunology or an Insight into Nature's Identity System" ALLEN, GARLAND E., Washington University, 16 August, "T. H. Morgan and the MBL: A Tale of Embryos and Genes" MASTROIANNI, LUIGI, JR., University of Pennsylvania, 23 August, C. Lalor Burdick Lecture, "Human in vitro Fertilization: The New Frontier in Gamete Physiology" ASSOCIATES' LECTURE GROSS, PAUL R., Marine Biological Laboratory, 17 August, "Builders and Science of the MBL" FREDERICK B. BANG FELLOWSHIP MARCUM, JAMES A., Massachusetts Institute of Technology CHARLES ULRICK AND JOSEPHINE W. BAY FOUNDATION FELLOWSHIP SMOLOWITZ, ROXANNA, Marine Biological Laboratory 80 MARINE BIOLOGICAL LABORATORY ERNEST EVERETT JUST FELLOWSHIPS IN BIOLOGY JOSIAH MACY, JR., FOUNDATION PETERSON, RUSSELL LEON, Howard University STEPHEN W. KUFFLER FELLOWSHIPS X KHAN, SHAHID M. M., University of Punjab, Pakistan \TAKEDA, KENNETH, Universite Louis Pasteur, France FRANK R. LILLIE FELLOWSHIP - GHERING WALTER, University of Basel, Switzerland MBL SUMMER FELLOWSHIPS AUGUSTINE, GEORGE, University of Southern California BARRY, SUSAN R., University of Michigan COSTELLO, WALTER, Ohio University MATTESON, DONALD, University of Pennsylvania ^NASI, ENRICO, Boston University REED, CHRISTOPHER, Dartmouth College SATTELLE, DAVID B., University of Cambridge, England, U.K. SILVER, ROBERT B., University of Wisconsin WEISS, DIETER G., Universitat Munich, ERG HERBERT W. RAND FELLOWSHIPS GEIGER, BENJAMIN, Weizmann Institutes, Israel NAKA, KEN-ICHI, National Institute of Basic Biology, Japan TINKER FOUNDATION FELLOWSHIPS BACIGALUPO, JUAN, University of Chile, Chile BEAUGE, Luis, Institute de Investigacion Medica, Argentina TORRES, RAFAEL, University of California, Los Angeles BIOLOGY CLUB OF NEW YORK FEE, ELIZABETH J., Ohio State University WARSHAW, JANE E., University of Massachusetts FATHER ARSENIUS BOYER SCHOLARSHIP FEE, ELIZABETH J., Ohio State University GARY N. CALKJNS MEMORIAL SCHOLARSHIP WARSHAW, JANE E., University of Massachusetts FRANCES S. CLAFF MEMORIAL SCHOLARSHIP HOMAN, WILLIAM L., University of Minnesota EDWIN GRANT CONKLIN MEMORIAL SCHOLARSHIP HOMAN, WILLIAM L., University of Minnesota HONORS 8 1 FOUNDERS SCHOLARSHIP BURGOS, GUILLERMO E., National Institute for Fishery Research, Argentina ALINE D. GROSS SCHOLARSHIPS DIKE, LAURA, Boston University MiOTTO, KAREN A., University of Colorado MERKEL H. JACOBS SCHOLARSHIP SCOTT, DILLON, University of Massachusetts ERNEST EVERETT JUST SCHOLARSHIPS IN BIOLOGY HARMON, GARY L., Howard University SUTTON, FEDORA, Howard University ARTHUR KLORFEIN FUND SCHOLARSHIPS BEIS, ANDRONIKI, University of Texas BRAULT, ELISABETH-CECILE, CNRS CHISHOLM, JULIA CLARE, University of Cambridge, U. K. FARQUHARSON, ANDREW, University of Glasgow, U. K. GOLSTEYN, ROY M., University of Calgary, Canada LUCILLE P. MARKEY CHARITABLE TRUST SCHOLARSHIPS BAADER, ANDREAS, University of Berlin, FRG BRYDEN, CYNTHIA G., Boston University Marine Program COUTINHO, JOHN B., Queen's University, Canada DUBAS, FRANCOISE, University of Texas DUBE, FRANCOIS, Stanford University FRIEDMAN, ROBERT M., C. B. Wilson Center GRAY, KENDALL M., University of California HAFNER, MATHIAS, German Cancer Research Center, FRG HALUSKA, FRANK G., University of Pennsylvania HANNA, MAYA, Harvard University HOWARD, KENNETH RAMSEY, University of London, U.K. HYSON, RICHARD L., University of Colorado KATAYAMA, AKIKO, University of Hawaii LACEY, MERRI LYNN, University of California, Riverside LANS, DEBORAH A., University of California, Berkeley LENART, THOMAS D., Johns Hopkins University LEV-RAM, VARDA, Weizmann Institute of Science, Israel LEWITUS, ALAN J., Woods Hole Oceanographic Institution MARCUS, EMILIE A., Yale University MAROM, SHIMON, Israel Institute of Technology, Israel MIDTGAARD, JENS, Kobenhavns University, Denmark O'HALLORAN, THERESA J., University of North Carolina PASCUAL-SPADA, MARIA MERCEDES, Universidad Santa Ursula, Argentina PELUFFO, LUCAS, University of California, San Diego PESSIN, MELISSA S., Johns Hopkins School of Medicine PINES, JONATHON N., Cambridge University, U.K. REIMER, KATRIN, University of Munich, FRG RIOULT-PEDOTTI, MARC GUY, Swiss Federal Institute of Technology, Switzerland 82 MARINE BIOLOGICAL LABORATORY SCHARFF, CONSTANCE, Rockefeller University SPEKSNIJDER, JOHANNA E., University of Utrecht, Netherlands STENGL, MONIKA, Columbia University SUMMERS, LISA CAROL, Swarthmore College TROESCH, CLAUDIA D., University of Southern California TUTTLE, REBECCA, Purdue University WALTHER, ZENTA, Yale University WARNER, CECILIA A., Northwestern University WARSHAW, JANE E., University of Massachusetts WESTON, WAYDE M., University of Pennsylvania WILSON, GiSELA F., University of Wisconsin ZEYER, JOSEF A., Swiss Federal Institute for Water Resources, Switzerland S. O. MAST MEMORIAL SCHOLARSHIP BLAIR, SETH S., University of Washington DUBE, FRANCOIS, Stanford University TURNER, PAUL R., University of Connecticut Health Center ALLEN R. MEMHARD SCHOLARSHIP SCOTT, DILLON, University of Massachusetts MARJORIE W. STETTEN SCHOLARSHIPS IGLESIAS, ANA, University of Connecticut Health Center SPEKSNIJDER, JOHANNA E., University of Utrecht, Netherlands SURDNA FOUNDATION SCHOLARSHIPS BARNETT, BRUCE A., North Dakota University KAO, KENNETH R., University of Toronto, Canada RAY, ANDREA J., University of Chicago SCOTT, DILLON, University of Massachusetts TURNER, PAUL R., University of Connecticut Health Center VERAZAY, GUILLERMO A., National Institute for Fishery Research, Argentina SOCIETY OF GENERAL PHYSIOLOGISTS SCHOLARSHIPS LACEY, MERRI LYNN, University of California, Riverside MONTAGUE, P. REED, University of Alabama, Birmingham OWENS, JESSE L., University of Alaska SPEKSNIJDER, JOHANNA E., University of Utrecht, Netherlands TINKER FOUNDATION SCHOLARSHIPS ANDREWS, NORMA, New York University Medical Center ARGUELLO, CARLOS, National Polytechnic Institute, Mexico BARRALL, ALDINA, Federal University of Bahia, Brasil MARINLARENA, ALEJANDRO JORGE, Institute de Limnologia, Argentina MARTINEZ, DANIEL EDUARDO, Universidad Nacional Bania Blanca, Argentina MONETA, MARIA EUGENIA, University of Chile, Chile SAEZ, JUAN, Albert Einstein College of Medicine TRESIERRA, ALVARO, National University of Trujillo, Peru ZEA, SVEN E., University of Texas, Austin HONORS 83 XII. INSTITUTIONS REPRESENTED U.S.A. Academy of Natural Sciences of Philadelphia Alabama, University of Alaska, University of Albert Einstein College of Medicine American Health Foundation American Museum of Natural History Amherst College Arizona, University of Atlantex and Zieler Instrument Corporation Axon Instruments, Inc. Bausch & Lomb Baylor College of Medicine Beckman Instruments, Inc. Bell Laboratories Bellco Biotechnology Bethesda Research Labs Bigelow Laboratories Bio-Oceanic Research Bio-Rad Bioanalytical Systems Biodyne Electronics Biomedical Research and Development Boston University Boston University Medical School Bowling Green State University Bowman Gray Medical School Brandeis University Brescia College Brigham and Women's Hospital Brinkmann Instruments Brookhaven National Laboratory Brooklyn College Brown University Buffalo, University of. School of Medicine California Institute of Technology California State University, Fullerton California State University, Northridge California, University of California, University of, Berkeley California, University of, Davis California, University of, Irvine California, University of, La Jolla California, University of, Los Angeles California, University of, Los Angeles, Medical School California, University of. Riverside California, University of, San Diego California, University of, San Francisco California, University of, Santa Barbara Cambridge Hospital Carleton College Carnegie Institution of Washington Carnegie-Mellon University Case Western Reserve University Catholic University of America C. B. Wilson Center Cedars-Sinai Medical Center Center for Blood Research Chicago, University of Chicago, University of. Medical School Cincinnati, University of, College of Medicine City of Hope Medical Center Clark University Colgate University Cold Spring Harbor Laboratory College of the Holy Cross College of the Mainland Colorado State University Colorado, University of Colorado Video Columbia University Columbia University College of Physicians and Surgeons Connecticut College Connecticut, University of Connecticut, University of, Health Center Cornell University Cornell Medical School Coulter Electronics Crimson Camera Technical Sales, Inc. Dagan Corporation DAGE-MTI Damon Biotech, Inc. Dana-Farber Cancer Institute Dartmouth College Dartmouth Medical School Deaconess Hospital Delaware, University of Dow Chemical Drexel University DonSanto Corporation Duke University Duke University Medical Center Dupont Corporation EG&G Earlham College East Carolina University Eastern Connecticut State University Eastern Virginia Medical School Eastman Kodak Company Ellis Hospital 84 MARINE BIOLOGICAL LABORATORY Emory University Emory University School of Medicine Environmental Protection Agency Ethicon, Inc. Fairleigh Dickinson University Florida Institute of Technology Florida State University Florida, University of Florida, University of, College of Medicine Flow Laboratory Flushing Hospital Medical Center Fordham University General Electric Company General Scanning George Mason University Georgetown University George Washington School of Medicine Georgia Institute of Technology Georgia, University of Gilford Gilson Medical Electronics Gonzaga University Grass Foundation Grass Instrument Company Hacker Instruments Hampshire College Hahnemann University Harbor Branch Foundation, Inc. Harvard Medical School Harvard School of Dental Medicine Harvard University Harvard University School of Public Health Hawaii, University of Hoefer Science Instruments Hopkins Marine Station Horn Point Laboratory Howard Hughes Medical Institute Howard University Hunter College IBI IDEA ISCO Illinois Institute of Technology Illinois, University of, Chicago Illinois, University of, College of Medicine Illinois, University of, Medical Center Imperial College Indiana University Institute for Marine and Aquarium Studies Instrumentation Marketing Corporation Interactive Video Systems International Business Machines Iowa State University Iowa, University of Jackson Laboratories Jefferson Medical College John F. Kennedy University Johns Hopkins Hospital Johns Hopkins University Johnson Research Foundation Joslin Diabetes Clinic Kansas State University Kansas, University of Kean College of New Jersey Kentucky, University of Kenyon College Kip & Zonen Kisten, Babitsky, Latimer & Beitman Kresge Eye Institute LKB Instruments, Inc. Lab Line Instruments, Inc. Lander College, South Carolina Lehman College Leitz, E. Inc. Levity Corporation Long Island Sound Task Force Oceanic Society Louisiana State University Louisville, University of Lowell, University of Loyola University of Chicago META Systems, Inc. Maine, University of Marine Biomedical Institute Marlboro College, Vermont Mary Flagler Gary Arboretum, NY Maryland, University of Maryland, University of, School of Medicine Massachusetts Eye and Ear Infirmary Massachusetts General Hospital Massachusetts Institute of Technology Massachusetts, University of Massachusetts, Univerity of, Medical School Mayo Foundation Medical College of Ohio Medical College of Pennsylvania Medical Systems Corporated Medical University of South Carolina Memorial Sloan Kettering Mental Health Research Institute of Michigan Merck, Sharp and Dohme Research Laboratories Miami, University of Miami, University of, School of Medicine Michigan Cancer Foundation Michigan State University Michigan, University of Millhauser Laboratory INSTITUTIONS REPRESENTED 85 Millipore Millsaps College, Mississippi Minnesota, University of Minnesota, University of, Medical School Miriam Hospital Missouri, University of Mobile Oil Corporation Montana State University Moravian College, Pennsylvania Mount Holyoke College Mount Sinai School of Medicine Mystic Marinelife Aquarium National Eye Institute/NIH National Foundation for Cancer Research National Institute for Basic Biology National Institute of Aging/NIH National Institute of Alcohol Abuse and Alcoholism/NIH National Institute of Mental Health/NIH National Institutes of Health National Institute of Neurological and Communicative Disorders and Stroke/ NIH National Marine Fisheries Service Naylor Dana Institute for Disease Prevention Nebraska, University of Neurological Research Institute, New York New Alchemy Institute New Brunswick Scientific, Inc. New England Medical Center New Hampshire, University of New Jersey Medical School New York Aquarium New York Blood Center New York, City College of New York, City University of New York, City University of, Herbert Lehman College New York Institute for Basic Research in Mental Retardation New York Medical College New York State Department of Public Health New York, State Psychiatric Institute New York, State University of, Albany New York, State University of, Binghamton New York, State University of, Buffalo New York, State University of, Downstate Medical Center New York, State University of, Geneseo New York, State University of. New Paltz New York, State University of, Pottsdam New York, State University of. Stony Brook New York University College of Dentistry New York University Medical Center New York University School of Medicine Nichols College Nikon, Inc. North Carolina State University North Carolina, University of North Carolina, University of, School of Medicine North Dakota, University of North Nassau Mental Health Center Northeastern University Northwestern University Medical school Notre Dame, University of Oberlin College Occidental College Ocean Pond Corporation Ohio, Medical College of Ohio State University Ohio, University Oklahoma Medical Research Foundation Oklahoma, University of Olympus Corporation of America Optiquip OPTRA, Inc. Oregon Health Sciences University Oregon State University Oregon, University of PMI-Strang Clinic Papanicolaou Cancer Institute Pennsylvania, University of Pennsylvania, University of, School of Dental Medicine Pennsylvania, University of. School of Medicine Pennsylvania, University of. Veterinary Medicine Pharmacia, Inc. 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LABORATORY SUPPORT STAFF Including Persons Who Joined or Left the Staff During 1985 Biological Bulletin METZ, CHARLES B., Editor MOUNTFORD, REBECCA J. CLAPP, PAMELA L. MARINE BIOLOGICAL LABORATORY Buildings and Grounds LEHY, DONALD B., Superintendent ANDERSON, LEWIS B. AVERETT, JUDITH BALDIC, DAVID P. BOURGOIN, LEE E, CARINI, ROBERT J, COLLINS, PAUL J. CONLIN, HENRY P. DUART, VICTORIA DUTRA, STEVEN J. ENOS, GLENN R. EVANS, FRANCES G. FUGLISTER, CHARLES K. GEGGATT, RICHARD E. GONSALVES, WALTER W., JR. GRINNELL, KAREN M. HAINES, KEVIN M. ILLGEN, ROBERT F. JOHNSON, FRANCES N. KUIL, ELISABETH LEWIS, RALPH H. Controller's Office SPEER, JOHN W., Controller BINDA, ELLEN F. CAMPBELL, RUTH B. CARREIRO, LORRAINE F. DAVIS, DORIS C. Director's Office GROSS, PAUL R., President and Director ASHMORE, JILL M. General Manager's Office SMITH, HOMER P., General Manager GEGGATT, AGNES L. GEGGATT, CYNTHIA C. Grants and Educational Services HOWARD, JOAN E., Coordinator DWANE, FLORENCE FERZOCO, SUSAN J. 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MONIZ, PRISCILLA MURPHY, CHARLES F. PARKER, FLORIA J. C. TASSINARI, EUGENE MAClNNES, ARCH Rioux, MARGARET A. PIMENTAL, MELANIE B. PORAVAS, MARIA SADOWSKI, EDWARD A. SYLVIA, FRANK E. COPELAND, D. EUGENE, Special Consultant to Electron Microscope Laboratory KERR, Louis M. MATTOX, ANDREW H., Safety Officer LECUYER, DOUGLAS LUNN, JEFFREY R. MARTYNA, GWENDOLYN MARTYNA, JONATHAN W. MEYER, LINNEA MONTROLL, CHARLES MYETTE, VINCENT C. NELSON, CHRISTEN L. PARMELEE, ALLEN PEAL, RICHARD W. PLUMMER, SEAN L. PORAVAS, CHRISTOS G. POTHIER, JAHN A. REMSEN, DAVID RUSHFORTH, LORNA ANN SANGER, RICHARD H., JR. SWOPE, STEPHEN P., JR. VALOIS, FRANCIS X. VANALSTYNE, MARK DEVEER, LISA ANN WETZEL, ERNEST D. WINSPEAR, DAVID A. WYTTENBACH, ANN G. Reference: Biol. Bull. 171: 90-1 15. (August, 1986) EVOLUTION AND FUNCTION OF STRUCTURALLY DIVERSE SUBUN1TS IN THE RESPIRATORY PROTEIN HEMOCYANIN FROM ARTHROPODS JURGEN MARKL Zoologisches Institut, Universitdt Miinchen, Luisenstr. 14, D-8000 Miinchen 2, Federal Republic of Germany ABSTRACT Native aggregation level and subunit composition of the hemocyanins from 86 adult chelicerates and crustaceans, and from the larval stages of 2 crabs, were analyzed by means of electron microscopy, polyacrylamide electrophoresis, immuno blotting, and crossed immunoelectrophoresis, supported by a variety of preparative separation techniques. The up to eight immunologically discernible subunit types were interspe- cifically correlated, classified, and evolution lines derived. Phylogenetic consequences are discussed, and are particularly aggravating in spiders. A single subunit suffices for the formation of hexamers (1 X 6). In the architecture of higher-ordered hemocyanins, the various subunits act as building-blocks of distinct specification. This was studied in 2 X 6 molecules from a hunting spider and several crustaceans, and in 4 X 6 hemocyanin from a tarantula. The various subunits are present in constant proportions. The total set is required to reorganize the original aggregate from subunit mixtures. Stable oligomeric segments of native hemocyanin particles revealed the gross distribution of the diverse subunits. Immuno electron microscopy of the native hemocyanins decorated with monospecific Fab fragments showed the exact topographic position of each subunit type, and detailed models of the quaternary structure could be derived. The oxygen binding function of 4 X 6 hemocyanin from the tarantula Eurypelma californicum is excessively modulated by subunit interaction phenomena. We measured native, reassembled, and mercury-blocked 4 X 6-mers, oligomeric segments, single subunits, and reassembled 4 X 6-mers with one subunit type chemically modified. The spatial range of allosteric interaction, and specific contributions of the diverse subunits are outlined. INTRODUCTION Hemocyanin, a blue copper-protein, functions as an oxygen carrier in the blood of arachnids, horseshoe crabs, crustaceans, and centipedes. Another hemocyanin occurs in inkfishes, chitons, many snails, and some primitive bivalves; however, despite certain similarities, molluscan hemocyanin differs decisively from arthropod hemocyanin in its main structural features, and therefore will not be discussed here. Most certainly, both pigments have evolved independently from tyrosinase, an ancestral precursor. Arthropod hemocyanins are multi-subunit proteins with a molecular mass of about 75,000 per polypeptide chain. The polypeptides or subunits are arranged as cubic hexamers (1 : 6), or multiples of hexamers (2 X 6, 4 X 6, 6 X 6, 8 X 6); the native aggregation level is species-specific. (Ghiretti, 1968; Van Holde and van Bruggen, 1971; Van Holde and Miller, 1982; van Bruggen et at, 1982; Ellerton el at, 1983; Received 4 February 1986; accepted 29 May 1986. Present address: Institute of Cell and Tumor Biology, German Cancer Research Center, D-6900 Hei- delberg, Federal Republic of Germany. 90 HEMOCYANIN SUBUNITS IN ARTHROPODS 91 Huber and Lerch, 1986; Mangum et ai, 1985; Morse et al, 1986). In molecular mass, the 8 X 6-mer (48 subunits) exceeds human hemoglobin 50-fold, and measures about 25 nanometers across, corresponding to the size of a ribosome. The three-dimensional structure of the basic hexamer recently was elucidated in detail (Gaykema et al, 1984). All hemocyanin aggregates are clearly visible in the electron microscope (Fig. 3); the arrangement of hexamers in the higher-ordered molecules, as shown in Figure 1 , was partially derived by computer image analysis (Van Heel and Frank, 1981; Bijlholt et al, 1982). Hemocyanin is not incorporated in cells, due to physical problems (Mangum, 1985), but floats freely in the blood in concentrations up to 120 mg/ml (scorpions), meaning that it fills up to '/ 10 of the hemolymph space; the average distance from one molecule to the next equals its own diameter. Arthropod hemocyanin particles dis- sociate at alkaline pH into their subunits, which are capable still of reversibly binding oxygen. By dialysis of the obtained subunit mixtures against neutral pH, a self-assembly at least of hexamers, and frequently also of higher-ordered molecules occurs. Each subunit is a delicate structure of around 10,000 atoms: 620 amino acids, which form three domains containing the binuclear copper site, 20 alpha helices, and a large seven-stranded beta barrel (Gaykema et al, 1984; Linzen et al, 1985). At first glance this appears to be a considerable luxury placing two metal atoms in the position to bind one molecule of oxygen in the respiratory organs, and to free it again in an oxygen-consuming tissue. The red hemoglobin and a derivative, the green chloro- cruorin, achieve this with only 140 amino acids per polypeptide chain and thus attain, with the same effort of protein material, a four-fold oxygen binding capacity. The third known group of respiratory proteins, the pink hemerythrins, with a five-fold oxygen transport capacity, does an even better job in this respect. Therefore, one might suggest that hemocyanin, which in most groups was inefficient to compete with the more economical pigments, is a dead-end product of evolution. However, looking at their distribution among the Invertebrata, we observe that hemocyanin occurs exces- sively in highly complex animals whereas the more primitive phyla dispose of either hemoglobin, chlorocruorin, or hemerythrin (Prosser, 1973; Bonaventura and Bon- aventura, 1980). This is surprising because extracellular hemoglobins as well as red blood cell hemoglobins occur sporadically in molluscs as well as in arthropods (foremost in lower taxa), and thus have been available phylogenetically. What possible advantage made hemocyanin successful over hemoglobin in presumably 100,000 highly advanced animal species, despite its considerably lower oxygen binding capacity? This can be speculated at least for arthropods. As with hemoglobin, the reversible oxygen binding of the blue protein is enhanced, modulated, and adapted by so-called "allosteric" phenomena, based on complex interactions between subunits. Arthropod hemocyanins display this functional plasticity to an extent unknown for hemoglobin or any other allosteric protein (Loewe, 1978; Mangum, 1985). Moreover, no hemoglobin found in a possible candidate for the ancestral arthropod or mollusc exhibits an appreciable allosteric behavior. Among the Invertebrata, the heme proteins with more than token cooperativity are, with a single exception (the bivalve Scapharca inaequivalvis: Ascoli et al., 1986), the extracellular ones, which invariably occur in the more specialized groups in a particular taxon. So the ancestors of the hemocyanin-containing molluscs and arthropods were really choosing between a functionally inflexible intracellular hemoglobin (high to moderate oxygen affinity, essentially uncooperative and pH-, inorganic ion-, and organic cofactor-insensitive) and a highly plastic (with respect to oxygen affinity and pH dependence), moderately to highly cooperative, inorganic ion and organic cofactor modulable extracellular hemocyanin. To make the decision easier, it is harder to push a red blood cell-containing hemolymph around in an arthropodan or molluscan circulatory system that lacks capillaries (Mangum, 1985). 92 J. MARKL Vertebrates evolved a special intracellular hemoglobin which is modulable to some degree, and which compensates for its limited functional plasticity with strong ven- tilation and circulatory systems which continuously create high gas diffusion rates. Moreover, their interior regulation is efficient enough to embed the hemoglobin in permanent homeostasis, which protects it against drastic changes of the milieu. As a result, their blood gained the extremely high oxygen carrying capacity of intracellular hemoglobin. The higher arthropods display sensory and locomotory activities com- parable to those of many vertebrates. As in vertebrates, these activities require a con- tinuous, abundant oxygen supply of the involved tissues, despite the more limited vegetative control mechanism in these animals and despite their gills and lungs being lined with chitin that hinders gas diffusion. Their respiratory protein thus plays a much more decisive role as a molecular interface between body tissues and environment. Extremely flexible in function, arthropod hemocyanin compensates for environmental and physiological changes during the life-cycle of the organism (Mangum, 1980; Man- gum, 1983), and it also has demonstrated flexibility in the evolution of countless crabs, crayfishes, shrimps, isopods, scorpions and spiders, each adapted to a special aquatic or terrestrial situation. The "clue" to arthropod hemocyanins is their multigenicity: they are composed of several structurally and functionally different types of subunits. This has been studied intensively worldwide during the last decade (for review: Linzen, 1983). The phylogeny of those subunits, and of the various oligomers, is now well understood. In addition we learned that the native aggregation level, and also most probably the oxygen binding characteristics, are ultimately dependent on the special subunit composition. MATERIALS AND METHODS Animal sources, and the specificity of the applied antisera (raised in rabbits) are described elsewhere (Markl et ai, 1986a). Other methods are described in detail in the cited literature. However, for a better understanding of the following discussion, a brief description of the major immunochemical approaches may be useful. Immu- nochemists use very powerful and sensitive techniques (e.g., radioimmunoassay, en- zyme-linked immunoassay) which enables the calculation of immunological differences between proteins in 1% steps by successively quantitating the immune reaction between each of the proteins and a suited antiserum. Unfortunately, in our case those methods were inadequate because the hemocyanin subunit samples, especially from crustaceans, were uncontrollably contaminated by self-reassembled aggregates (if the dissociation conditions were too gentle), or by partially denatured subunits (if we chose more drastic conditions). Those contaminants display special immune reactions: compared to subunits, aggregates react at least ten-fold stronger, and denaturation, (e.g., with urea) spirited away intraspecific as well as interspecific immunological differences between hemocyanin subunits (Lamy et ai, 198 la; Stocker, 1984; Kempter et al, 1985). Therefore our data would have been seriously falsified had we used a "blind" quantitative method. Crossed imrn unoelectrophoresis We instead applied the crossed immunoelectrophoresis which, though only being semi-quantitative and less sensitive (soluble immune complexes escape the detection), allows a selective comparison of the desired components in a particular sample. This two-dimensional technique (Weeke, 1973) in the first dimension separates proteins in an agarose gel according to charge differences. In the second dimension the proteins HEMOCYANIN SUBUNITS IN ARTHROPODS 93 migrate, again electrophoretically, into an antiserum-containing agarose gel. Each pro- tein which reacts with the antiserum forms a curved precipitation line; intersecting lines allow the structural comparison of the respective proteins. In all the patterns discussed here, the anode was on the left in the first dimension. Variations of this technique (tandem-crossed and crossed-line immunoelectrophoresis) enable an inter- section of proteins from two different samples. Immuno blotting Especially for comparisons of phylogenetically distant subunits, the more sensitive immuno blotting ("Western blotting") technique was used (Towbin et al, 1979; Bur- nette, 198 1 ). The native subunit patterns obtained by polyacrylamide gel electrophoresis were transferred by diffusion (the currently used electro transfer caused denaturation!) onto an immobilizing nitrocellulose sheet, and then treated with subunit-specific rabbit antibodies. A second goat antiserum against rabbit antibodies was applied subsequently. The second antibodies carry pick-a-back horseradish peroxidase molecules. This en- zyme catalyzes a reaction with added diamino benzidine, resulting in a brownish color. Specifically, only those subunit bands which had reacted with the first antibody are stained, whereas other subunits remain invisible. The weak peroxidase activity of hemocyanin (Ghiretti, 1968) was low. This technique is advantageous as soluble im- mune complexes also are recorded. However, immuno blotting could not serve us exclusively because it was unable to distinguish slight differences between closely related subunits; for this, crossed immunoelectrophoresis was the better tool. HEMOCYANINS OF THE ARACHNIDA AND OF ALLIED GROUPS Extent of subunit diversity in tarantula hemocyanin Ten years ago, when we decided to study the blue blood protein of the North American tarantula, Eurypelma californicum, a 4 X 6-mer, few and very contradictory data were available on the number of subunit types present in any one hemocyanin. Polyacrylamide gradient slabgel electrophoresis is an extremely powerful technique used to separate a mixture of very similar proteins. In a buffer system which separates proteins primarily according to charge differences (molecular mass differences played an additional, but minor role), the subunit mixture of tarantula hemocyanin yielded 6 distinct bands: 5 monomers (= single polypeptides) and a dimer. Using a combination of gel chromatography, ion exchange chromatography, and preparative polyacrylamide electrophoresis (today we use immuno affinity chromatography in one single step), each of the six components could be preparatively isolated. In a detergent-containing polyacrylamide gel, which separates polypeptides according to molecular mass differ- ences, we detected that the dimeric subunit is composed of two different monomers (Schneider et al., 1977; Mark! et al., 1979a). Thus, the final result was a complex pattern of seven polypeptide chains, which we later have designated as a through g (Fig. 1 ). Also a third method visualized the marked diversity of those subunits, namely the crossed immunoelectrophoresis (Fig. 1). No immunological cross-reactivities be- tween any two subunits could be detected (Lamy et al., 1979a). This indicated that the surface structures must be significantly different, which implied major differences in function. Distribution of the 4 X 6-mer and its derivatives We found no hemocyanin in the sun spider ("wind scorpion") Galeodes sp., and in the watermite Hydrachna geographica (Markl et al., 1986a). Both animals possess 94 J. MARKL Bathynomus FIGURE 1. Topographical models of the quaternary structure of four arthropodan hemocyanins. The phylogenetic tree shows the relationships between the various subunit types (Kempter et al., 1985; Markl el al, 1986a). The subunits are visualized in their respective patterns of crossed immunoelectrophoresis. Subunit topographies are based on electron microscope analyses of the overall morphology (Van Holde and van Bruggen, 1971; Markl, 1980; Bijlholt el al, 1982; van Bruggen, 1983), and were derived from the combined results of four different approaches: (i) determination of subunit compositions and stoichiometries, (ii) analysis of oligomeric dissociation fragments in comparison with the whole molecule, (iii) reassembly experiments with various subunit combinations, (iv) direct observation in the electron microscope after having decorated the native particles with subunit-specific antibodies (respectively antibody fragments). Beyond the 1 X 6 level, for the formation of the original aggregate the presence of all subunit types is ultimately required. This reflects perfectly the distinct structural roles played by these subunits. *: Subunit h is restricted to scorpion hemocyanin (see Fig. 2). 1 X 6 hemocyanin of the deep sea isopod Bathynomus giganteus (a gift of M. Brenowitz) is exclusively composed of alpha subunits (Van Holde and Brenowitz, 1981, and unpub. data). 2X6 hemocyanin of the spider Cupiennius salei is composed of a disulfide-bridged dimer d-d and 10 monomers /(Markl, 1980). 2X6 hemocyanin of the crab Cancer pagurus consists of a non-covalent dimer alpha'-alpha', a central 4-beta cluster, and, more peripheral, 2 alpha and 4 gamma subunits. A certain flexibility in substituting gamma subunits for alpha appears to exist. 2X6 hemocyanin from the freshwater crayfish Astacus leptodactylus is constructed correspondingly, but contains a disulfide-bridged dimer. The crab Callinectes sapidus and the lobster Homarus americanus fit into the scheme, although in those cases no dimer could be identified (Markl et al., 1983; Stocker et al., 1986). 4x6 hemocyanin of the tarantula Eurypelma californicum is constructed of a central tetrameric bc- bc ring, symmetrically surrounded by 20 monomers: one a, d, e, f, and g in each basic hexamer. Most probably, peripheral bridges between the two 2 X 6-meric halves are formed via// According to Mark! et al. (198 Id). Comparable results stem from scorpion and xiphosuran hemocyanins (Lamy et al., 1981b, 1983b). a well-developed trachea system and therefore probably have no need of a respiratory pigment. However, as indicated in Figure 2, 4 X 6-mers composed of multiple subunits are present in whip scorpions and whip spiders (Markl et al., 1978, 1979b). A break- through like that made with Eurypelma hemocyanin was achieved in the analysis of the 4X6 hemocyanin of another arachnid, the scorpion Androctonus australis, and the 8 X 6 hemocyanin of a xiphosur, the horseshoe crab Limulus polyphemus (Hoylaerts et al., 1979; Lamy et al., 1979b, c; Markl et al, 1979b; Brenowitz et al, 1981). An- droctonus hemocyanin contains an eighth subunit type, designated by us as h, and Limulus hemocyanin shows two variations of subunit g, but for the rest both molecules are composed like Eurypelma hemocyanin (Fig. 2). This correspondence was recently confirmed by comparative immunochemistry: each subunit of the tarantula has a HEMOCYANIN SUBUNITS IN ARTHROPODS 95 Hydrachno geographica g(4] Mastigoproctus brasilianus Tnchodamon froesi Tarantuta palmata Androctonus australis Leiurus quinquestriatus Colossendeis spec L.mulus Polyphemus Tachypleus tndentatus Eurypelma Androctonus Colossendeis ' Cupienmus SDS PAGE FIGURE 2. Distribution of hemocyanin among the subphylum Chelicerata. The patterns of crossed immunoelectrophoresis show the respective subunit composition; hatched squares symbolize the native aggregation states (8 X 6, 4 X 6, 2 X 6; small squares correspond to hexamers *). Immunologically homologous subunits are identically labeled using the designations a-h established for spider hemocyanins. Scorpion and xiphosuran subunits are additionally labeled according to their original designations (Lamy et ai, 1979a, b, c; Markl, 1980). The results from Tachypleus and Leiurus (Lamy et ai, 1979c; Markl et ai. 1984) are not discussed in the text, but fit well into the scheme. Whip spider and whip scorpion hemocyanin subunits were studied only by electrophoresis (Markl et ai, 1979b). Opilionids possess 2x6 hemocyanin (Kempter et ai, 1985), whereas no hemocyanin was found in Galeodes, Hydrachna, and Colossendeis (Markl et ai, 1986a). The figure is taken from Markl et ai (1986a). Left insert: polyacrylamide gel electrophoresis (PAGE) of hemolymph proteins in the presence of a detergent (sodium dodecyl sulphate = SDS), showing that the sea spider Colossendeis possesses no hemo- cyanin (He), but it does possess the typical arachnidan non-respiratory protein (NRP). This was con- firmed by immuno blotting. Comparable unpublished results were recently obtained with the pycnogonid Nymphon sp. Right insert: phylogenetic relationships between the eight cheliceratan subunits according to the combined information of Lamy et ai, 1983a, Markl et ai, 1984, and Kempter et ai, 1985. Broader lines indicate that with anti-crustacean alpha antiserum, subunits a, d, and /are recognized best. * For better comparison with the current literature: the sedimentation coefficients of the four aggregates are 60S (8X6 = 48-mer), 35S (4X6 = 24-mer), 24S (2x6= 12-mer), and 16S (1 X 6 = 6-mer). homologous subunit in scorpion and horseshoe crab hemocyanin (Lamy et al, 1983a; Markl et al, 1984; Kempter et al., 1985). Moreover, a phylogenetic tree of the subunits could be derived: the pairs d/f, e/g, b/c, and a/h. respectively, are phylogenetically closely connected (Fig. 1 , and right insert in Fig. 2). Although at that point the various subunits still could be neutral features that have been inherited because they weren't selected against, the heterogeneity appeared as a basic structural design worth conserving in evolution at least since that time when the progenitors of arachnids and xiphosurs diverged from each other. Fossils show that this happened during the Siluran era, more than 400 million years ago (Tiegs and Manton, 1958; Bergstrom et al., 1980). All the more surprisingly, 4X6 hemocyanin 96 J. MARKL was discarded twice in rather advanced arachnidan groups, and replaced by relatively simple 1 X 6 and 2X6 hemocyanins (Fig. 2). This occurred in the harvestmen (daddy- long-legs; Kempter et a/., 1985) and, independently, in certain spiders (Wibo, 1966; Markl et al, 1976, 1983, I986a). This phenomenon has been studied intensively, especially in spiders. The blood of the large Central American hunting spider Cupien- nius salei contains both 1 X 6 and 2X6 hemocyanin. The 1 X 6-mers showed electrophoretically 5 different subunit bands. However, to our suprise, these compo- nents were completely identical immunologically (Markl and Kempter, 198 la). All five could be correlated immunologically with subunit / of Eurypelma (Markl et al., 1984; Kempter et al., 1985). 2 X 6-mers, present as a main component of Cupiennius blood, are also composed of / : homologons, but also contain a dimeric subunit (Fig. 1 ). This dimer is immunologically related to, but somewhat distinct from the monomers (Markl, 1980) and, as revealed recently by immuno blotting, predominantly related to Eurypelma subunit d (Markl et al., 1986a). Thus, the 2 X 6 hemocyanin of Cu- piennius can be derived phylogenetically from a 4 X 6-mer progenitor. The architecture o/Cupiennius hemocyanin Parallel to the comparative studies described so far, we attempted from the begin- ning to analyze hemocyanin subunits with respect to their reassembly behavior, native stoichiometry, and topologic position in the respective oligo-hexamer. Our goal was to reveal principles of the blue protein's architecture. We used a variety of analytical methods, but especially polyacrylamide gel electrophoresis, immunochemistry, and electron microscopy. Each of the monomeric/isozyms of Cupiennius salei hemocyanin formed regular homo-hexamers in reassembly experiments; to reorganize the dode- camer, the disulfide-bridged dimer dd also was required (Markl, 1980; Markl and Kempter, 198 la). Stoichiometrically, each 2X6 particle contained one copy of dd and ten copies of/ Electron microscopy indicated that a one-point contact between the two hexameric halves could be assumed (Fig. 1). Reducing agents, which cleaved the isolated dimer into single d monomers, also cut the 2 X 6-mer down to hexamers. Together, these techniques strongly indicated, but only indirectly, that the dimer was an inter-hexamer bridge how could this be tested more rigorously? Immuno labeling experiments, as described below, were unsuccessful because of the marked immuno- logical similarity between Cupiennius ddandf. The solution came unexpectedly: when we monitored the dissociation of a 2 X 6 fraction electrophoretically, we observed that the process passed over a stable oligomeric fragment of unusual size. Molecular mass determinations characterized it as heptamer. In a carefully directed electron microscopic survey we indeed detected a strange, yet undescribed molecule: a regular hexamer with a protruding particle (Fig. 3). We isolated this heptamer and, by reducing agents, cut off the protrusion obviously a fragment of the molecular bridge. The products of this cleavage were hexamers and single subunits d. Thus indeed, the pro- trusion was identified as the half of the dimer. The resulting model of quaternary structure (Fig. 1 ) was, among all hemocyanin models, the first which showed a directly localized subunit type in its topologic position (Markl, 1980). The 4 / -mer a symmetrical mosaic Computer analysis of electron microscope images revealed the general morphology of Eurypelma californicum hemocyanin particles (Bijlholt et al., 1982): the two basic hexamers within a half-structure are rotated against each other in a right angle and come in close contact; the 2 X 6-meric halves keep more distance and are slightly tilted with respect to each other (Fig. 1 ). Since two subunit types form a 2 X 6-mer, the necessity of five more different components to make the protein twice as large was difficult to see, and we asked HEMOCYANIN SUBUNITS IN ARTHROPODS 97 - ^*+2Z' V r> ' ' &w % Jl " A -'' ,-' t .-*.* 35 S 305 245 18S 165 FIGURE 3. Above: electron micrographs of hemocyanin molecules from the spider Cupiennins salei, negatively stained with 1% unbuffered uranyl acetate. Besides 1 X 6 and 2x6 aggregates, heptameric intermediate structures are visible (arrows). The bar represents 50 nm. The left magnified image shows a heptamer with its protruding particle, which could be identified as half-dimer d. The right enlargement shows a 2 x 6 molecule in the same orientation. From Markl (1980). Below: electron micrographs of negatively stained hemocyanin particles from the tarantula Eurypelma californicum. The molecules were obtained after 3 hours dialysis of native 4 x 6-mers against pH 9.6, which caused a partial dissociation into oligomeric fragments (upper panel, the bar represents 50 nm). The magnified images (lower panel) have the position and orientation of the heterodimeric subunit be indicated, and are from left to right: 4 x 6-mer (= 24-mer, sediments in the analytical ultracentrifuge with 35S); 3 x 6-mer with one additional subunit protruding in the gap (= 19-mer, 30S); 2 X 6-mer (= dodecamer, 24S); 1 X 6- mer with an additional subunit protruding (= heptamer; 18S); 1 x 6-mer (= hexamer, 16S). The figure is taken from Markl et al, 1981c. ourselves whether all of these components are really incorporated in the same he- mocyanin particle. Using various analytical methods, we tried to detect a heterogeneity of the 4X6 molecules of Eurypelma; finally we were convinced that indeed only a single type of 4 X 6-mer exists (Markl et al., 1980). The 7 subunits, designated a through g, are present in constant proportions: 4a + 2b + 2c + 4d + 4e + 4/+ 4g = 24 (Markl et al, 1980, 198 la). The self-assembly process of the 4 X 6-mer is char- acterized by a striking specificity: reassembled molecules equal native molecules in the stoichiometry of their subunit composition (Decker et al., 1980). None of the subunits was able to self-assemble to homo-hexamers, in contrast to certain horseshoe crab and scorpion hemocyanin components (Lamy et al., 1977; Bijlholt et al., 1979; Brenowitz et al., 1983). A series of reassembly experiments with all possible subunit combinations revealed that indeed each subunit type is required to build up the oligo- 98 J. MARKL meric architecture. A substitution by another subunit is not successful: the reassembly process will stop at a certain level, which is typical for each subunit combination (Markl et al, 1981b, 1982). Most of the intermediate structures obtained morpholog- ically corresponded to one of the dissociation fragments described below (Fig. 3). Stable 4X6 particle -vith a "normal" appearance in the electron microscope are only reorganized in the presence of all seven components. After this, our goal was to localize each subunii within the native 4X6 particle. Again, an essential part was the investigation of stable oligomeric dissociation fragments. We attempted a carefully directed search for heptamers, which we indeed found (Fig. 3), together with "con- ventional" dodecamers and hexamers (Markl et al., 198 Ic). Additionally, a new frag- ment appeared: a 19-mer, composed of 3 hexamers and a small protrusion in the gap (Fig. 3). Isolation and analysis of the 4 fragments revealed that the various copies of the 7 subunits are symmetrically distributed among the 4X6 particle (Markl et al., 198 la). The exact topology was determined by direct localization of subunits in the electron microscope after having decorated the 4 X 6-mer with subunit-specific antibody fragments (Markl et al., 1 98 1 d). A ringlike bc-bc tetramer forms the central core which connects the hexameric quarter-structures. Presumably, two f-j homodimers achieve more peripheral contacts between the 2 X 6-halves. Within each 2 X 6-mer, subunits a and d are involved in the inter-hexamer connection, whereas subunits e and g are arranged peripherally. This model of quaternary structure (Fig. 1 ) is in agreement with a model which was published in the same year for the 4X6 hemocyanin of the scorpion Androctonus australis (Lamy et al., 1981b). 8X6 hemocyanin of the horseshoe crab Limulus polyphemus is dissociable into 4X6 half-molecules, which morphologically correspond in detail with native An- droctonus and Eurypelma 4 X 6-mers (Bijlholt et al, 1982), and consist of an im- munologically related set of subunits (Lamy et al., 1983a; Kempter et al., 1985). Hybrid hemocyanin molecules ("protein chimaeras"), which we obtained by co-reas- sembly of subunits from the three species, further underlined this structural relationship (van Bruggen et al., 1980). The recently published subunit topology of this largest of all arthropod hemocyanins indeed confirmed its far-reaching homology with tarantula and scorpion hemocyanin (Lamy et al., 1983b). Hemocyanin subunit structure, and the evolution of spiders The striking conservatism of the 7 subunit/4 X 6 structure raised the question, what happened with Cupiennius to cause such a dramatic change in its design? To find an answer we attempted a broad survey including 40 species of spiders from 25 families (Markl et al., 1983, 1986a). Previous taxonomic schemes for the higher clas- sification of spiders at the family level have been based on adult morphology or be- havior, a topic on which there is currently substantial disagreement (Eberhard, 1982). H. W. Levi stated in 1978: "Spider classification at the present is in chaos." (Personal remark, cited in: R. F. Foelix. 1979. Biologie der Spinnen. Thieme, Stuttgart). To use spider hemocyanin as a taxonomic character should be advantageous, because in con- trast to most other tried characters the direction of its evolution seems clear: the 2 X 6-mer is a derivative of the 4 X 6-mer and not vice versa, because the 4 X 6-mer also occurs in other Chelicerata. According to current textbook taxonomy, the order of spiders (Aiapeae) contains four suborders: Mesothelae (which were not studied by us), Orthognatha (e.g., Eurypelma), Cribellata, and Labidognatha. The Labidognatha are further subdivided in Haplogynae (with simple sex organs) and Entelegynae (with complex sex organs). Cupiennius is an entelegyne spider. The four orthognath families studied all possess 4 X 6-mers with a subunit com- position corresponding to that of Eurypelma hemocyanin; a homologous structure which also occurs in seven entelegyne families (Fig. 4). Eleven other entelegyne families. HEMOCYANIN SUBUNITS IN ARTHROPODS 99 however, dispose of hemocyanins which are clearly homologous to that ofCupiennius (Fig. 4). Their extreme immunological similarity strongly supports the hypothesis that the last group is a natural, monophyletic taxon. We therefore propose to designate this taxon as "Neo-Entelegynae," and the remaining entelegyne spiders, denned by their 4X6 hemocyanin, as "Arch-Entelegynae" (Markl and Runzler, 1986). It was very interesting for us to detect in a cribellate spider an intermediate step in the transition from the 7-subunit to the 2-subunit particle: Filistata insidiatrix possesses only hexameric hemocyanin (This contradicts Wibo, 1966, who reported 4X6 hemocyanin for this species ). The patterns of crossed immunoelectrophoresis showed five subunit peaks (Fig. 4). Absent was heterodimer be, which makes the presence of only hexamers in this spider understandable: in Eurypelma californicum 4X6 hemocyanin, be forms a tetrameric core (Fig. 1), which in reassembly experiments was indispensable to exceed the hexamer level (van Bruggen et a!., 1980; Markl et ai, 1982). Amaurobius fenestralis, the second cribellate spider that we investigated, pos- sesses a 2 X 6 hemocyanin with the typical subunit composition of our Neo-Entelegynae (Fig. 4). Alternatively, the spider Uroctea durandi, which morphologically appears to be a very close relative of certain cribellate species (Kullmann and Zimmermann, 1976), belongs to our Arch-Entelegynae according to its hemocyanin structure. The Cribellata possess two unique devices: a typical spinning plate, the cribellum and, at their fourth pair of legs, a special comb to brush silk, the calamistrum. Because a repeated, independent evolution of these structures is highly improbable, most experts treat the Cribellata as a natural, monophyletic taxon (e.g., Bristowe, 1938; Levi, 1966; Lehtinen, 1967). However, the striking morphological similarity between various Cri- bellata and particular entelegyne species stimulated the idea that cribellate characters have been repeatedly and independently lost in spider evolution (Lehtinen, 1967). Our data strongly support this hypothesis, and indicate that at least all the Neo-En- telegynae, and probably the entire labidognath suborder, stem from cribellate progen- itors. Compared to the various schemes based on other characters, our results agree substantially with Lehtinen (1967, 1978), despite minor differences which predomi- nantly are refinements. The haplogyne spider Dysdera crocata possesses a hexameric hemocyanin like Filistata, but composed of only two different subunits in comparable proportions; immunologically they correspond to Eurypelma f and d (Fig. 4). The close relationship to Cupiennius hemocyanin is obvious; however, the specific structural role of subunit das hexamer linker (Markl, 1980) is not (yet?) established in Dysdera. Thus Dysdera, and probably the entire group of haplogyne spiders (although their monophyletic nature is doubted) are either late descendents of a neo-entelegyne spider family, or indeed have conserved molecular features of the transition phase between the two hemocyanins. It should be noted that Lehtinen (1967) postulated a close relationship between Filistatidae and Haplogynae, which supports the second assumption. Although presently we are unable to decide with lasting certainty, whether Filistata and Dysdera hemocyanin represent true "missing links," they can serve as evolutionary models. It may well be that an ancestral cribellate spider, for whatever reason, lost its ability to genetically express the heterodimer, and thus was restricted to hexameric hemocyanin with possibly drastic (though obviously not lethal!) negative consequences: drop of functional plasticity, increase of colloid osmotic pressure, and blood viscosity. In the descendents of this "genetic cripple," three more subunits (probably unnecessary to bring a hexamer to function) disappeared from the phenotype, and later a new mode of inter-hexamer bridging was invented. This was the starting signal for the evolution of a large variety of highly advanced trappers, hunters, and jumpers (Fig. 4). Whether this new hemocyanin, possibly the result of an evolutionary accident, conceals any functional advantage over the 4 X 6-mer remains an interesting, unanswered question. 100 J. MARKL RGURE 4. Phylogenetic tree of spiders as deduced from their hemocyanin subunit composition. Typical subunit patterns of crossed immunoelectrophoresis, and symbols for the respective native aggregation state (1 X 6, 2 X 6, 4 X 6) are shown. The 7 subunit/4 x 6-mer occurs throughout the Orthognatha and, im- munologically somewhat different though doubtlessly related, in 7 labidognath families, which cover orb- weavers and other stationary trionychan (= three-clawed) species. The 2 subunit/2 X 6-mer is typical for another 1 1 labidognath families, including besides some net spiders (Agelenidae) a large variety of free hunters: lurking seizers, sudden jumpers, and tenacious runners. Due to the extremely close immunological relatedness of the subunits of all 2 X 6-mers, these three-clawed (Trionycha) and two-clawed (Dionycha) spiders most probably represent a monophyletic taxon. The transition of 4 X 6 in 2 X 6 hemocyanin is conserved, or at least modeled, within the cribellate family Filistatidae (Filistata insidiatrix) and the haplogyne family Dysderidae (Dysdera crocata), which both possess 1 X 6 hemocyanin, but with a different subunit composition. Note that Filistata hemocyanin lacks subunit be which in Eurypelma californicum reassembly experiments was indispensable to exceed the 1 X 6 level (Markl el al. 1982). A second cribellate spider, Amaurobius fenestralis, possesses 2x6 hemocyanin. According to our data, Labidognatha and Cribellata are not separable, but represent phylogenetically intersecting, nested groups. Entelegynae is a polyphyletic taxon, containing two natural groups, which are disconnected by a haplogyne/cribellate cluster. We have therefore proposed to substitute "Entelegynae" for two new monophyletic terms, namely "Arch-Entelegynae" and "Neo-Entelegynae" (Markl and Runzler, HEMOCYANIN SUBUNITS IN ARTHROPODS 101 The sea spiders a phylogenetic mystery? Besides the aquatic xiphosurans and the terrestrial arachnids, in current taxonomy the subphylum Chelicerata includes as a third class the marine sea spiders (Pycnogonida = Pantopoda), although the phylogenetic position of these indeed spider-like, often incredibly tiny animals is uncertain. Among experts it is still debated whether they are arachnids, non-arachnidan chelicerates, or a completely independent arthropod subphylum (Bergstrom et al, 1980). Also remarkably, they have no respiratory or- gans very unusual among arthropods, especially aquatic species, in which cutaneous gas exchange is blocked by an impermeable exoskeleton. Hoping to detect a phylo- genetically interesting new version of the blue protein, we have studied the minute Nymphon sp. from the North Sea and the huge Antarctic Colossendeis sp. (Markl et al., 1986a; and unpub. data). Unfortunately, polyacrylamide electrophoresis of their blood yielded no trace of hemocyanin-like protein chains. Earlier, Redmond and Swanson (1968) reported that spectral-absorption curves of the blood of another giant Antarctic sea spider, Ammothea striata, showed no evidence of a blood respiratory pigment. Nymphon with its size below 1 cm, and body and extremities as thin as threads, is probably qualified for cutaneous gas exchange. However, the absence of an oxygen carrier in Colossendeis and Ammothea, which both exceed in size a large tropical spider, but live in the oxygen-poor aquatic envi- ronment, is surprising. On the other hand, many cold-blooded animals found in polar seas lack respiratory pigments because of the high oxygen solubility in icy-cold body fluids. Instead of hemocyanin as desired, in both species we observed electrophoretically two prominent polypeptide chains with molecular masses around 1 10,000 the typical range of the second major blood protein of spiders and other arachnids (left insert in Fig. 2). This protein was described by us in earlier studies and designated as a "non- respiratory protein" (Markl et al., 1976, 1979b; Linzen et al., 1977). In case of the tarantula Eurypelma californicum, it was recently further characterized as lipoprotein associated with a carbonic anhydrase (Stratakis and Linzen, 1984). By immuno blotting, indeed a structural relationship between the non-respiratory protein of the tarantula, and the pycnogonid protein was shown (Markl et al, 1986a). According to the available data this protein is restricted to scorpions, whip scorpions, whip spiders, and spiders, and was detected in neither xiphosuran nor crustacean blood; non-respiratory blood proteins of those two groups are composed of considerably smaller polypeptide chains (Markl et al., 1979b, c). Thus, the occurrence of this protein is a good argument in favor of a close phylogenetic relationship between the sea spiders and the arachnids. SUBUNIT DIVERSITY IN CRUSTACEAN HEMOCYANINS At least 600 million years ago, the branch leading to the Chelicerata was separated from the line leading to the Crustacea (Tiegs and Manton, 1958; Schram, 1982). Since today both groups have hemocyanins based on a hexameric architecture, this hemo- 1986). Our results agree in principal with the evolution scheme of Lehtinen (1967), which is based on morphological characters. The figure was slightly modified from Markl et al. (1986a). The following species were employed: Agelena labvrinthica, Amaurobius fenestralis, Araneus diadematus, Araneus umbriaticus, Argiope aurantia, Argiope bruennichi, Atrax formidabilis, Atypus affinis, Callilepis nocturna, Chiracanthium elegans. Clubiona terrestris, Cupiennius salei, Cvrtophora citricola. Dolomedes fimbriatus, Dysdera crocata, Eurypelma cali- fornicum, Filistata insidiatrix, Haplodrassus signifer, Heriaeus hirtus, Linyphia marginala, Liocranum rup- icola, Menemems taeniatus, Mela segmentata. Micrommata rosea, Nemesia sp., Oxyopes lineatus, Pardosa amentata. Philodromus collinus, Pholcus phalangioides, Pisaura mirabilis, Salticus scenius. Scotophaeus quadripunctatus, Synaema globosum, Tarentula fabrilis. Tegenaria atrica, Tetragnatha extensa, Theridion varians, Thomisus onustus, Uroctea durandi, Xysticus bifasciatus. 102 J- MARKL cyanin undoubtedly belonged to their common ancestor. Thus it was of considerable interest to study the subunit composition of crustacean hemocyanins, which are com- monly 1 X 6 or 2 X 6 aggregates (the only known exception are the thalassinid shrimps, which possess a tetrahedrical 4 X 6-mer: Miller et ai, 1977; van Bruggen, 1983). We applied high resolution electrophoresis techniques, and revealed complex patterns of subunit heterogeneity in various crustacean hemocyanins: between three and eight distinct polypeptides were detected throughout (Markl et al, 1978, 1979c). It was impossible, however, to recognize a common scheme except for the fact that in 2 X 6- mers the heterogeneity was somewhat more excessive than in native single hexamers. To monitor evolutionary trends, we needed a tool to sort all these subunits into groups of similar function. Because protein function is a matter of surface structure, the tool employed was immunochemistry: each antibody is specifically directed against a fragment of the protein surface, and refuses binding if this epitope is altered. The outcome of a comparative immunochemical analysis of 4 1 crustacean hemocyanins is described below. Subunit composition of 2 X 6 hemocyanins from brachyuran crabs The typical hemocyanin of a brachyuran crab, like the blue crab Callinectes sapidus, or the green shore crab Carcinus maenas, is a 2 X 6-mer, composed of two immu- nologically discernible subunit fractions (Rochu and Fine, 1980; Markl and Kempter, 198 la, b; Ghidalia et al., 1985). We have designated them as alpha and beta (Fig. 5). Beta is immunologically unrelated to alpha, which indicates substantial differences in function. Alpha subunits from different crab species are immunologically similar; they remained relatively unchanged after the worldwide radiation of the Brachyura, and therefore were classified by us as "phylogenetically conservative." In a striking contrast, beta subunits differ greatly immunologically among the species, and therefore were classified as "phylogenetically variable" (Markl and Kempter, 198 la). Obviously, dur- ing crab evolution, these two subunit types endured very different selection pressures, which again indicates fundamental differences in function. All in all, we studied 32 species covering a broad environmental and activity range, from shallow water, intertidal, fresh water, and land, and from most agile to rather clumsy (Markl and Kempter, 1981b; Markl et al, 1986a). The representatives of 10 out of 1 1 crab families possessed alpha and beta subunits (Fig. 5), regardless of how diverse their subunit patterns were electrophoretically. Many Brachyura also have a third hemocyanin subunit, designated as gamma. Gamma appears to be a late off- spring of alpha, because their immunological reactions are similar. The only exception from this general scheme was detected in the family Ocypo- didae: the hemocyanins of a fiddler crab ( Uca urvillei) and of three species of ghost crabs (Genus Ocypode} are exclusively composed of alpha subunits (Fig. 5). It was very interesting to detect that Uca hemocyanin particles are only single hexamers, which indicates that the absence of beta may be correlated with a restriction to the hexameric level. However, much to our surprise, all three Ocypode species possess mainly : ( 6-meric hemocyanin (Stocker, 1984; Markl et al., 1986a; B. Johnson, pers. cor :-m.), In contrast to Uca, Ocypode hemocyanin contains two electrophoretically distinct alpha isozymes (Fig. 5). The cathodic of these immunologically identical pro- teins migrates in the range of beta, but carries no beta-typical antigen determinants. One could assume that the situation within the Ocypodidae mirrors an ancient trend, leading from single alpha hexamers via alpha/alpha 2 X 6-mers to alpha/beta 2X6- mers. However since among the Brachyura, Uca and Ocypode belong to a rather specialized family, other possibilities had to be considered as well. To clarify this point we had to answer the question of whether beta is an invention of the comparatively modern crabs. HEMOCYANIN SUBUNITS IN ARTHROPODS 103 OCYPODIDAE Uca urvillei OCYPODIDAE Ocypode quadrata Ocypode nobili Ocypode ceratophthalma GECARCINIDAE Gecarcoidea lalandn Cardisoma carnifex XANTHIDAE: Etisus laevimanus Cymo quadrilobatus Atergatis roseus Enphia sebana PORTUNIDAE Portunus pelagicus Macropipus holsatus Charybdis cruciata Podophthalmus vigil Scylla serrata Callmectes sapidus Thalamita crenata Carcmus maenas GECARCINUCIDAE Phricothelphusa limula GRAPSIDAE Grapsus albolmeatus Grapsus tenuicrustatus Pachygrapsus marmoratus Plagusia depressa Parasesarma lepidum PARATHELPHUSIDAE: Somanniathelphusa sexpunctata CALAPPIDAE: Calappa hepatica Matuta lunans CANCRIDAE Cancer pagurus MAJIDAE Maja squmado Camposcia retusa Hyas araneus DROMIIDAE Dromia personata FIGURE 5. Brachyuran crabs arranged according to their hemocyanin subunit composition as analyzed by crossed immunoelectrophoresis. The native aggregation states (1 x 6, 2 X 6) are symbolized. Immuno- logically corresponding subunits are identically designated. It should be noted that immunologically ho- mogeneous subunit peaks may contain several electrophoretically separated isozymes. Thus, the electrophoretic heterogeneity is generally higher than indicated here (for review: Linzen, 1983). The tropical species were collected on the Malayan Peninsula. The animals cover a broad environmental range from fairly constant to permanently varying marine milieus, from icy to tropic temperatures, and through all stages of land-life: dependent on moist burrows during lowtide (Etisits), sun-exposed on rocks (Plagusia), in cold freshwater falls (Phricothelphusa), in warm stuffy rice fields (Somanniathelphusa), in mangrove mud (Scylla), in soil burrows (Cardisoma), in palmtree forests (Gecarcoidea), on hot sandy beaches (Ocypode). The smallest (Cymo) had a carapace width of less than 2 cm, the largest (Scylla) of more than 20 cm. Their activities range from clumsy dwelling (Dromia), slow rambling (Hyas), hectic rowing (Matuta), tenacious waving ( Uca), fast swimming (Charybdis), swift walking (Carcinus), and sudden jumping (Grapsus), to extremely speedy sand running (Ocypode). The figure is taken from Markl et a/. ( 1986a). Spiny lobster 1X6 hemocyanin a possible predecessor? According to convincing palaeontological records the brachyuran crabs, the latest appearing decapod group, evolved from ancestors related to spiny lobsters (Schram, 1982). Interestingly, spiny lobsters possess 1 X 6 hemocyanin. We investigated the European species Palinurus vulgaris, and its American relative Pamdirus interruptus (Markl et ai, 1979c, 1983, 1986a; Stocker, 1984). Their hemocyanins are composed of an immunologically homogeneous subunit fraction, which corresponds to brach- yuran alpha (Fig. 6). Panulirus hemocyanin contains a second, cathodic subunit in 104 J. MARKL addition (designated as c by Neuteboom et al, 1986) which, to a certain degree, is immunologically related to alpha. Beta-typical antigen determinants were not detected. This is consistent with our definition of the behavior of a gamma subunit, and the component was designated accordingly, although we have no further evidence that Panulirus and brachyuran gamma components are really homologous. From its subunit composition, the heniocyanin of a scyllarid (Scyllarus arctus), a close relative of spiny lobsters, fits we)' into the scheme (Fig. 6); however, surprisingly it forms 2X6 particles (unpub.). Although the phylogeny of subunit beta, and also the structural requirements for a 2 X 6 formation remained unclear, the situation found within the Palinura at least further supported the hypothesis that alpha subunits were the basic hexamer formers. Hemocyanins from lobsters, freshwater crayfishes, and shrimps Geological strata document that, when the first Brachyura appeared 200 million years ago (their great radiation started 140 million years later upon a worldwide for- mation of shallow water seas), the Astacura were already established (Schram, 1982). Astacura also possess 2X6 hemocyanin. In the case of the European freshwater crayfish Astacus leptodactylus it is composed of four distinct subunits: a disulfide bridged dimer and three monomers (MarkI et al., 1979c; Pilz et al., 1980; Markl and Kempter, 1981b). Without major difficulties, the dimer and two of the three monomers could be assigned to the alpha/gamma cluster of the Brachyura (Fig. 6). Again, the conservative nature of alpha-typical antigen determinants was confirmed (Stocker, 1984; Markl et al., 1986a). What remained was the question of a possible relationship between the third Astacus monomer and brachyuran beta. By crossed immunoelectrophoresis, anti-Astacus antiserum did not precipitate Cancer pagur us beta, and vice versa. There- fore, for a comparison of structurally distant antigens we applied the immuno blotting technique, which is more efficient. This sensitive method revealed that anli-Astacus antibodies specific for the third monomeric subunit preferentially bind to brachyuran beta and vice versa, whereas alpha components are only weakly recognized (Markl et al., 1986a). Comparable data stem from the 2X6 hemocyanin of the lobster Homarus americanus, although in this case the alpha fraction was immunologically homogeneous and no dimer was present; therefore we could not define a subunit alpha' in this animal (Fig. 6). Hemocyanin from another decapod, the caridean shrimp Palaemon elegans, contains only alpha and gamma subunits (Stocker, 1984; Markl et al., 1986a); however, its native aggregation level is 1 X 6 (Fig. 6)! These results indicated: first, beta is not an invention of the Brachyura, but phylogenetically considerably older, and second, the presence of beta is correlated with the appearance of 2 X 6 particles. The second conclusion is implicated by the exceptions Ocypode and Scyllarus, which will be dis- cussed below. It should be noted that in shrimps and crayfishes, gamma subunits are only defined according to their close relationship with alpha subunits; in contrast to alpha and beta, the homology of astacuran or caridean gamma with the respective palinuran and brachyuran components is uncertain. Gamma could have evolved independently from alpha several times. The origin of subunit beta It was found recently that 2X6 hemocyanin also occurs in a fourth decapodan group, namely the Anomura (Stocker, 1984; Markl et al, 1986a). Surprisingly, despite a marked electrophoretic heterogeneity, the subunits of 2 X 6 hemocyanins from a galatheid shrimp (Galathea squamiferd), a hermit crab (Pagurus bernhardus), and the coconut crab (Birgus latro) were immunologically completely homogeneous (Fig. 6). HEMOCYANIN SUBUNITS IN ARTHROPODS 105 a Callmectes sapidus Carcmus maenas Grapsus Uca Ocypode tenuicrustatus ^urvillei quadrata a-a Panulirus interruptus Bathy nornus giganteus Pagurus bernhardus Galathea squamifera Birgus latro FIGURE 6. The distribution of immunologically corresponding hemocyanin subunits among the Crus- tacea. The various subunit patterns of crossed immunoelectrophoresis are shown, and the native aggregation states (1 X 6, 2 X 6) are indicated. It appears that decapodan hemocyanins evolved from an ancient alpha hexamer; the first step was the invention of beta-typical antigen determinants (arrow), which is correlated with the occurrence of 2 X 6 aggregates. Beta was repeatedly lost in later appearing groups (Caridea, Palinura, Uca/Ocypode). All alpha subunits are homologous proteins as are all beta subunits. Gamma differs, however: except for their partial identity with alpha, we were unable to identify typical immune reactions which interpose between more distant gamma subunits. Thus, rather than being homologous, they could represent convergent offsprings of different alpha progenitors. It should be noted that our results, although useful in explaining the phylogenetic coherence between the various subunits, in most cases are insufficient to refine the current phylogenetic tree derived from morphological characters and palaeontological records (Schram, 1982), which is the basis for this scheme. As an exception, our data strongly support an early branch of the Anomura as indicated, which was not derived from classical approaches. The figure was modified from Markl el al. (1986a). 106 J- MARKL Thus, in terms of immunochemistry, only one single subunit type is present. We obtained clear immunologica! cross-reactions with brachyuran and astacuran alpha but also, and this was the second surprise, with astacuran (not with brachyuran!) beta. The common presence of alpha- and beta-typical antigen determinants on a single subunit cannot be explained as the result of a gene fusion, because the size of the anomuran polypeptide chains is quite within the expected range (Mr - 76,000-83,000). Provided that hemocyanin genes are arranged in a single cluster, one could expect a current rearrangement by unequal crossing-over, which may well explain the anomuran result. However, the marked divergence between alpha and beta in crabs and crayfishes strongly indicates the existence of two different gene clusters like, for example, the case with the mammalian globin genes. It is therefore much more likely to assume that in the Anomura the blue protein has preserved an ancient feature, and that in later appearing species, alpha and beta antigen determinants have been separated by the independent evolution of isozymes. This strongly suggests that the evolution of the decapod 2 X 6-mer indeed began with the appearance of subunit beta, although later beta was repeatedly lost (Palinura, Caridea, Ocypodidae). This, however, was not necessarily tantamount to a permanent restriction to the hexameric level, because alternative, beta-free modes of 2 X 6-mer formation evolved in specialized groups (Ocypode, Scyllarus). Krill and isopod 1X6 hemocyanin: models of an ancient design We tried to demonstrate this by examining the situation outside of the Decapoda. The widespread hemocyanin aggregate of the other Malacostraca is the 1 X 6-mer, which we studied from the Antarctic krill Euphausia superba and from the giant deep sea isopod Bathynornus giganteus (Fig. 6). The subunits of both proteins exhibited a completely homogeneous peak in crossed immunoelectrophoresis, and corresponded immunologically to subunit alpha of decapodan hemocyanins. Thus, those 1 X 6- mers are entirely composed of alpha subunits (Fig. 1; the different information of Markl el al, 1983, was due to denaturation). It appears, moreover, that Euphausia hemocyanin has maintained a considerably ancient surface structure: by immuno- electrophoresis, it was the only crustacean hemocyanin which could be precipitated (in its oligomeric form not as subunits: see below) by an antiserum raised against tarantula hemocyanin (Van Holde and Brenowitz, 1981; M. Brenowitz, pers. comm.; Stocker, 1984; Markl et al., 1986a, and unpub. data). This definitely showed that, in arthropods, alpha-typical antigen determinants form the ancestral design of the blue protein's surface. Mirrors larval crab hemocyanin the blue protein 's evolution? The subunit composition of crab hemocyanins have endured ontogenetic changes (Terwilliger and Terwilliger, 1982). We therefore studied various larval stages of the spider crab Hyas araneus and the shore crab Carcinus maenas. Hemocyanin from the planktonic zoea, and from the benthonic megalops of both species was composed of a single subunit type, which clearly cross-reacted with adult alpha subunits (Markl et al., 1986a), although electrophoretically it behaved somewhat differently. The native aggregation level of those larval hemocyanins is mainly hexameric, but also 2X6- mers have been observed in the electron microscope (unpub.). Thus, the loss of subunit beta in the evolution of some decapoda, and an alternative mode of 2 X 6-mer for- mation (Ocyi ode, Scyllarus), could have occurred to preserve larval characters. Recently, we have further monitored the following events in Carcinus maenas: after metamorphosis, during the following molting cycles subunit beta appears, al- though its proportion in the first juvenile crab is still rather low. 2X6 hemocyanin particles can be seen in the electron microscope, but the main aggregation level is still HEMOCYANIN SUBUNITS IN ARTHROPODS 107 hexameric. The second juvenile crab clearly contains mostly 2X6 hemocyanin with a considerable proportion of subunit beta. Subunit gamma appears several stages later when the carapace measures 1 2 mm in width. The typical, stoichiometrically correct subunit pattern of the adults is established when the carapace width measures 22 mm or more (L. E. Precht, B. Steiff, and J. Markl, unpub. data). It is interesting that not only phylogenetically, but also ontogenically, alpha subunits appear first. Also our above presumption that gamma may be a rather late evolutionary product is mirrored in ontogenesis. A model of crustacean 2X6 hemocyanins The results described above indicate that particular subunits play distinct roles in the formation of 2 X 6 structures. This was closely investigated in two astacuran and two brachyuran species. The overall morphology of these particles, especially the one- point contact between the two hexamers as shown in Figure 1, is described by van Bruggen (1983). Reassembly experiments with isolated subunits alpha, beta, or gamma of the 2 X 6 hemocyanins from the crayfishes Astacus leptodactylus and Homarus americanus, and from the crabs Cancer pagitrus and Callinectes sapidus showed that in most of the cases, homo-hexamers can be formed (Markl and Kempter, 198 la; Stocker et al., 1986). However, all three immunologically defined classes of subunits are required to reach the 2X6 level (Stocker et al., 1986). Immunologically identical isozymes are in some cases capable of substituting for each other, especially in Homarus and Callinectes. Heptameric segments of native 2 X 6-mers were observed in Astacus, whereas the 2X6 particles of crabs and Homarus dissociate via hexamers (Markl et al., 198 Ic). The inter-hexamer bridge was identified in the case of Astacus and Cancer. Surprisingly the bridge is not formed by subunit beta as expected. In Cancer a particular alpha isozyme (designated as alpha') has a tendency to dimerize, and connects the two hexamers (Markl et al., 1983). Correspondingly, in Astacus the disulfide-bridged dimer is a correlate of alpha. This makes the existence of beta-free 2X6 structures as present in Ocypode, Scyllarus, and in larval crabs, at least structurally understandable. It should also be noted that 2X6 hemocyanins were reported for two species outside of the Decapoda: the isopod Ligia pallasii, and the stomatopod Sqnilla mantis (Ter- williger, 1982; van Bruggen, 1983). From our data described above, we would expect a beta-free mode of 2 X 6 formation also in those hemocyanins. Our next step was to analyze the respective subunit stoichiometries. Finally, labeling of the dodecamers with subunit-specific antibody fragments, or with intact antibody molecules, and observation of the resulting complexes in the electron microscope led to a uniform topological model (Fig. 1). It shows that one dimer alpha'-alpha' and four copies of beta form a central cluster. Although beta may not be involved directly in the bridge, these four subunits are clearly in the topographical position to play a functional key-role. One alpha occupies the extreme outer edge of each hexamer, and four copies of gamma fill the periphery (Stocker et al., 1986). Though a preliminary topological model of a crustacean 2X6 hemocyanin was already published by Jeffrey (1979), this is the first detailed conception of its architecture. The connection to the cheliceratan subphylum We were particularly interested in detecting immunological relationships between crustacean hemocyanin subunits and those from the Chelicerata. By immunoelectro- phoresis, a precipitation of crustacean hemocyanins with an anti-chelicerate antiserum, or vice versa, was only successful if the hemocyanin was either present in its oligomeric form (see above: Euphausia), or denatured in 8 M urea (Stocker, 1984). Therefore, we performed immuno blotting experiments with electrophoretic patterns of native hemocyanin subunits from the horseshoe crab Limuhis, the scorpion Androctonus, the tarantula Eurypelma, and the hunting spider Cupiennius against anti-alpha antisera 108 J. MARKL from crustaceans. In each case, the total set of chelicerate subunits was recognized (Markl et al, 1986a). Anti-beta and anti-gamma antisera give similar, but much weaker reactions. As judged semiquaniJtatively, chelicerate subunits related to Eurypelma a, d, and /are recognized best. This is illustrated in the right insert of Figure 2. It should be interesting to analyze comparatively the 6 X 6 hemocyanin of the myriapod Scutigera coleoptrata described by Mangum et al. (1985). Our results do not mean that there is one particular chelicerate subunit which corresponds to alpha, and another subunit which corresponds to beta; those specializations are certainly late developments. We showed that despite their impressive diversity in recent species, the hemocyanin sub- units of all arthropods have maintained to a different extent some common an- cient surface structures. These features were inherited, unchanged, over at least 600 million years! INTERACTION OF SUBUNITS IN THE OXYGEN BINDING PROCESS Recently, several complete amino acid sequences of chelicerate and crustacean hemocyanin subunits were published, the structure of the active copper-site was further elucidated, the conformation of a crustacean 1 X 6-mer was studied in a 3.2 Angstrom resolution, and the topologic models of Limulus and Androctonus hemocyanin were considerably refined (Solomon, 1981; Sizaret et al., 1982; Schartau et al., 1983; Schneider et al., 1983; Eyerie and Schartau, 1985; Gaykema et al., 1985; Lamy et al., 1985; Linzen et al., 1985). This information, in the context of the results described here, has demystified, to a considerable degree, the architecture and the evolution of arthropod hemocyanins. Moreover, a wealth of data exists on their function as oxygen carriers (e.g., Van Holde and van Bruggen, 1971; Bonaventura and Bonaventura, 1980; Mangum, 1980, 1983, 1985; Van Holde and Miller, 1982; Antonini et al., 1983; Bridges et al, 1983; Ellerton et al, 1983). However, the processes which determine the typical oxygen binding, and the specific contribution of each subunit type, are still obscure. Presently, Eurypelma californicum hemocyanin is the best understood example. The 4 X 6-mer in action, and the abilities of isolated subunits Hemocyanin from the tarantula Eurypelma californicum is characterized by a relatively low oxygen affinity, a strong normal Bohr effect (= pH sensitivity of oxygen affinity), and an extreme cooperativity (sigmoidity of the oxygen binding curve); ad- ditionally, the cooperativity depends considerably on pH (Fig. 7). Upon direct mea- surements of blood pH and blood P 02 in different tissues during exercise and rest, these properties can be interpreted as highly adaptive with respect to the animal's environment, behavior, and physiology. (Linzen et al, 1977; Loewe, 1978; Angersbach, 1978; Decker^ al, 1983a,b;Fincketfa/., 1986; Paul, 1986). In contrast, as illustrated in Figure 7, isolated subunits are non-cooperative (hyperbolic binding curve), show a high oxygen affinity, and indicate a complete absence of any Bohr effect. All seven subunits, studied individually, behaved rather uniformly in those aspects (Decker et al, 1979: Markl et al, 198 Ib, e). Thus, the 4 X 6-mer acts as a system; all its vital abilities are qualitatively new and unexpected, and cannot be predicted from the prop- erties of the subunits. The systemic characters are created by an interaction of the 24 constituents. With subunits in the blood instead of 4 X 6 particles, the animals could impossibly survive. An interesting question was now, at what structural level the re- spective function occurs? Events within and beyond the native 1X6 fragment Tarantula hemocyanin again was an exceptionally useful molecule, because the four oligomeric dissociation fragments described above (Fig. 3) could be stabilized in their respective structure over a broad pH range a fundamental requirement for a HEMOCYANIN SUBUN1TS IN ARTHROPODS 109 half - saturation oxygen pressu re (Pso) 8.0 cooperativity ( n 50 ) FIGURE 7. Illustration of the functional plasticity of native 4 X 6-meric hemocyanin of the tarantula Eurypelma califomicum, compared to the functional inflexibility, and the very different behavior, of the isolated subunits. Subunits (small black areas) show a low p50 around 5 mm Hg, a high oxygen affinity. This behavior is independent of pH (= no Bohr effect). Moreover, they are non-cooperative (hyperbolic oxygen binding curve: n50 = 1). All subunits behave similarly (Decker et al. 1979; Mark! et ai, 1981e). In contrast, the 4 x 6-mer (large white rectangles) shows p50 values up to 30 mm Hg (at pH 7.5), or low oxygen affinities. Moreover, it exhibits impressive cooperativities (sigmoid oxygen binding curves); the maximum at pH 8.0 goes up to n50 = 8 and more. Oxygen affinity, and also cooperativity, are both strongly pH dependent (Loewe, 1978), which is highly adaptive with respect to the animal's physiology and behavior (Angersbach, 1978). We recently detected that, although the natural resting blood pH of the tarantula is 7.5 (Angersbach, 1978), the in vivo behavior of the 4 X 6-mer should be like that illustrated here for pH 8.0; calcium and magnesium ions in native concentrations (4 mAl each) modulate the function correspondingly (B. Markl and J. Markl, unpub.). All in all, this aggregate of 24 functionally limited components displays completely novel physiological properties. These systemic characters are created by subunit interaction phenomena, and cannot be predicted from the behavior of the isolated subunits. To study the molecular organization of those interactions, one attempt is to analyze the abilities of oligomeric dissociation fragments (Savel et al., 1983, 1986); for example in the 1 x 6 quarter-structure (areas on the left of the dashed lines), the 4 X 6-mer's oxygen affinity is already fully established, but cooperativity reaches only 'A of the final values. Another attempt is to analyze reassembled 4 x 6-mers which have a chemically modified subunit incorporated. Modification was done, for example, with mercury(II) ions. A treatment of the whole molecule blocks the interaction processes entirely: the morphologically intact 4 X 6-mer functions like isolated subunits (Markl et al., 1986b). Hemocyanins composed of one mercury-labeled and six unmodified subunit types show a reduced functional plasticity (spotted areas), which is believed to be due to the encoupling of the modified subunit from the interaction processes (Markl et al., 1986b). detailed comparison of their function. Another chelicerate, Limulus polyphemus, un- fortunately could not fulfill this demand (Brenowitz et al., 1984). In contrast, 1 X 6 hemocyanin fragments of certain crustaceans, like the lobster Homarus americanus, the thalassinid shrimp Callianassa californiensis, and in the mangrove crab Scylla serrata, are stable (Tai and Kegeles, 1971; Arisaka and Van Holde, 1979; Herskovits et al., 1983; Decker et ai, 1986a). An oxygen binding analysis of the isolated quarter-structure (1 X 6-mer) of Eu- rypelma hemocyanin revealed that already on this level, the typical oxygen affinity and the full Bohr effect of the 4 X 6-mer are established; however, cooperativity reaches only 25% of the native value (Fig. 7; Savel et al., 1983). The heptamer equals the hexamer in these properties, but in the half-molecule (2 X 6-mer) cooperativity abruptly 110 J. MARKL rose to 50% of the end value. The 19-meric fragment provides no further enhancement; for the jump to 100% the entire 4 X 6-mer was required (Savel et al, 1986). These results are consistent with a recently introduced theoretical description of the oxygen binding process, the so-called "nesting model," an extension of the classical "MWC" model (Decker et a!., I986b). This new model is based on the idea of Wyman (1984) that cooperative oxygen binding manifests itself by a hierarchy of nested allosteric units a theoretical approach which was experimentally proved here not only for the first hemocyanin, but generally for the first allosteric macromolecule. Moreover it was interesting that the ultimate creator of all studied systemic characters is indeed the hexamer; the higher structural levels only quantitatively improve effects. Installed subunits act as amplifiers and transmitters We have started to characterize specific roles of the various subunits in the overall oxygen binding process, because their uniformity in the isolated state does not nec- essarily mean that they behave identically when incorporated in the 4X6 particle. Our current project deals with the analysis of reassembled 4 X 6-mers which have one chemically modified subunit type. First, we had to establish the gentle immuno affinity chromatography for subunit purification to prevent the loss of important aspects of function due to uncontrolled protein damage later in the reassembled molecules. We have already analyzed reassembly products with incorporated "apo-subunits" (copper-free), or "met-subunits" (copper oxydized), but most of our data stem from experiments with "mercury-subunits": Mercury(II) ions undialyzably bind to tarantula hemocyanin in an amount of 1-2 atoms per subunit. The effect of a mercury treatment of the whole 4 X 6-mer is dramatic: it is still a 4 X 6-mer, and still binds oxygen, but the binding properties correspond to those of single subunits (Markl et al., 1986b). This means that all subunit interactions are totally blocked. Comparable behavior is exhibited by Limulus polyphemus and Callinectes sapidus hemocyanin (Brouwer et al., 1983). Correspondingly, the treatment of a subunit with mercury, followed by its reincorporation into the 4 X 6-mer, should result in an uncoupling of the interaction processes. In such experiments indeed in the case of all subunits, alterations of oxygen affinity, cooperativity, and Bohr effect were monitored (Fig. 7). According to the data, a relatively uniform role as amplifiers can be ascribed to the various monomeric sub- units. Heterodimer be also amplifies, and additionally seems to function as a molecular transmitter between the four hexamers (Markl et al., 1 986b). This is the first information about the specific contribution of individual subunit types to the allostery of any one hemocyanin molecule. FUTURE ASPECTS Despite our new understanding of the structure, function, and evolution of subunit diversity in arthropod hemocyanins, there remain many challenging questions. For example, tarantula hemocyanin displays its cooperativity maximum at pH 8.0, but the resting blood pH of the animal as measured in vivo is 7.5, and drops to 7.0 after activity (Angersbach, 1978; Loewe, 1978). In this range, however, cooperativity does not convincingly exceed the level already achieved (at least at pH 8.0) by the isolated 1X6 Ler-structure (Fig. 7). For what reason does a complicated, ^-controlled interaction \ sf ween the four hexamers actually exist, if it is not, or not fully, utilized by the anims Very recently, we discovered that the whole problem was due to our incomplete knowledge of the actual situation: calcium and magnesium ions in their native blood concentrations (4 mAf each: Schartau and Leidescher, 1983) enhance, at pH 7.5, operativity to the maximum level (B. Markl and J. Markl, unpub.). It should therefore be interesting to investigate the influence of these ions, and of other modulators, on particular subunits. Other important problems related to subunit diversity are the biological significance of changes in subunit composition while crustaceans adapt to different environments HEMOCYANIN SUBUNITS IN ARTHROPODS 1 1 1 (Mason el al., 1983), the significance of ontogenetic changes, the significance of the interspecific variability of subunit beta, and similar projects. Another open field is the observation of conformational changes in certain subunits during oxygen binding, which presently is being attempted in our laboratory using fluorecent probes (Leidescher and Linzen, 1986). Although hemocyanin c-DNA already could be cloned, and se- quenced (Voit and Schneider, 1986), the cytoplasmatic events in the hemocyanin synthesizing cells are still unknown. Those "cyanocytes" (Fahrenbach, 1970) proliferate in crabs from lymphocytogenic nodules in the outer gizzard wall and in spiders from the inner heart wall (Ghiretti et al., 1977; Kempter, 1983). One of our most important goals is to delve into the cyanocytes, to analyze the location, structure, and organization of hemocyanin genes, which are still entirely concealed, and to reveal how the expres- sion of the diverse subunits is regulated. ACKNOWLEDGMENTS The described projects were supported by the Deutsche Forschungsgemeinschaft (grants Ma 843 to myself, and grants Li 107 to B. Linzen). I thank my various co- workers and colleagues for their splendid and friendly cooperation, particularly Drs. Anette Savel, Walter Stocker, Bernhard Kempter, Michael Brenowitz, and Heinz Decker. Especially with the last one I shared many hours of fruitful discussions. Co- workers of special quality were Angelika Markl and Barbara Markl. The skilled tech- nical assistance of Maria Brenzinger and Heide Storz is gratefully acknowledged. I am thankful to Prof. Ernst F. J. van Bruggen (Rijksuniversiteit Groningen, The Nether- lands), and to Profs. 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ARCHER Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556 ABSTRACT An indepth three-dimensional investigation examined the surface morphological changes associated with the development of a typical marine hydrozoan early cleavage embryo into a mature planula larva. During hydrozoan embryogenesis the arrangement and distribution of surface microvilli change; cilia of some epitheliomuscle cells appear before embryos gastrulate; and the embryo undergoes dramatic changes in body shape. Early cleaving embryos are bizarre in morphology, however, by the end of late cleavage the embryos have rounded to form a sphere. Gastrulation is characterized by the appearance of a blastopore at the future posterior end of the planula and by the migration of cells over the margins of the blastopore to the inside of the embryo. The product of gastrulation is a young planula which elongates and decreases in overall diameter to form a mature planula that eventually attaches via its anterior end to a substrate and undergoes metamorphosis. INTRODUCTION The cnidarians display a simple architecture and exhibit exceptional morphogenetic plasticity and adaptability. The phylum is unusual in that its postembryonic devel- opment has been much more thoroughly studied than its embryogenesis. Most previous investigations of cnidarian embryogenesis have concentrated on the internal mor- phology of the planula larva. These studies primarily utilized the techniques of light microscopy and transmission electron microscopy (Martin and Thomas, 1977, 1980, 198 la, b, 1983a; Martin and Chia, 1982; Martin et ai, 1983; Walch el al, 1986). Hotchkiss et al. (1984) used a scanning electron microscopic cryofracture technique to examine both the surface morphology and the internal morphology of a hydrozoan planula and attempted to correlate their findings with other studies employing trans- mission electron microscopy. Although the previous work on planulae has contributed valuable information concerning the morphology of the larval form of the cnidarians, major gaps still exist in our basic knowledge of early development in this lower animal phylum. As a result of this lack of information, the following scanning electron mi- croscopic study was undertaken to examine the surface morphological changes asso- ciated with the development of a typical marine hydrozoan early cleavage embryo into a mature planula. Such an indepth three-dimensional investigation of the cnidarian embryonic life cycle has never been done. This study provides important information concerning the external morphological changes of embryos during their development and sheds some insight into the morphogenetic shaping of the hydrozoan planula. MATERIALS AND METHODS Mature colonies of the marine hydrozoan Pennaria tiarella (McCrady) were col- lected from pier pilings at the North Carolina Institute of Marine Sciences, Morehead Received 18 February 1986; accepted 28 May 1986. 116 SEM OF A HYDROZOAN 1 1 7 City, North Carolina. Fronds from mature male and female colonies were placed together in large finger bowls of filtered seawater. Bowls were placed in the dark at 6:00 p.m., and at 9:00 p.m. they were returned to the light. Embryos in early cleavage stages were observed in the bottoms of the dishes. These embryos were transferred to small dishes of filtered seawater and reared at 23C to various developmental ages (Table I). Early cleavage embryos (2-4 hours old), late cleavage embryos (6 hours old), 8-hour embryos (gastrulae), and 10-, 36-, and 48-hour planulae were prepared for scanning electron microscopy (SEM). Animals were fixed for 1 hour in 2.5% glutar- aldehyde, pH 7.4, in 0.2 M phosphate buffer. They were postfixed for 1 hour in 2% osmium tetroxide in 1.25% sodium bicarbonate, pH 7.2. Samples were dehydrated through a graded series of ethanols and then critical point dried from CO 2 using a Denton critical point dryer equipped with a Tousimis liquid CO 2 water/particulate filter. Animals were coated with gold palladium in a Denton sputter coater. Stubs were examined with a JEOL JSM-T300 SEM operated at 15 kV. Individual cell types were identified according to developmental age of the embryo and surface specializa- tions as previously described in transmission electron microscopic studies (Martin and Thomas, 1977, 1980, 198 la, b). Embryos undergoing gastrulation were contin- uously examined under a Zeiss light microscope until immature 10-hour planulae were formed. RESULTS Cleavage in Pennaria tiarella embryos is holoblastic, unequal, and asynchronous (Figs. 1-4) (Martin and Thomas, 1977). The random cleavage pattern results in the formation of blastomeres of unequal size. A period of early cleavage extends from 2 hours postfertilization to the beginning of 6 hours postfertilization. During this time no one embryo cleaves in exactly the same fashion and the embryos exhibit numerous bizarre shapes and reach the 128-256 cell stage (Figs. 1, 2). Early cleavage blastomeres have numerous cytoplasmic blebs extending from their surfaces (Fig. 3). These blebs are transient and disappear in the later cleavage stages. Microvilli arranged in distinct patches project from the surface of each blastomere (Fig. 5). The tips of microvilli in each clump appear to come together at their apical ends to form a point. Early cleavage is very rapid and by 6 to 8 hours postfertilization a solid blastula (late cleavage) is formed (Fig. 6). The blastomeres are more uniform in size than during early cleavage and the embryo assumes the shape of a sphere. The embryo measures ca. 230 yum in diameter. During this stage a few cilia appear which are associated with the blastomeres that will give rise to the epitheliomuscle cells of the young planula (Fig. 7). By the end of 6 hours postfertilization the large blebs found in association with the early cleavage blastomeres have disappeared and a mucous-like sheath is seen covering parts of the surface of the late cleaving embryo (Figs. 6, 7). The microvilli of the late cleavage stage are shorter than those seen in earlier stages and are organized into clumps which are not as distinct as in earlier stages (Fig. 8). The microvilli are found on certain regions of the blastomere, while other regions of the same blastomere are devoid of these structures (Fig. 8). Some blastomeres possess few if any microvilli (Fig. 8). By 8 hours postfertilization the blastomeres are of equal size. The surface of the embryo is smooth, the contours are regular, and a single indentation is present at one pole (Figs. 9, 10). This indentation corresponds to a blastopore, and the pole at which it forms corresponds to the future posterior end of the planula (Fig. 11) (Martin and Thomas, 1983b). Some of the cells at the surface appear to migrate in a radial fashion toward the blastopore, roll over the margins of the pore, and disappear to the inside FIGURE 1 . Early cleavage embryo (4 hours postfertilization). Cleavage is bizarre and unequal resulting in the formation of small and large blastomeres. X233. FIGURE 2. Early cleavage embryo (4 hours postfertilization). The irregular cleavage pattern in this embryo is very different from that in Figure 1. X290. FIGURE 3. Early cleavage blastomeres. Numerous cytoplasmic blebs (arrow) project from the early blastomeres. XI 163. FIGURE 4. Asynchronous early cleavage embryo (2 hours postfertilization). The right side of the embryo is cleaving while the left is not. X260. 118 ??3&S \?tfWr. /? ',tXi * *& i *L FIGURE 5. Microvilli associated with the blastomeres of an early cleavage embryo (4 hours). Each blastomere is covered with distinct patches of microvilli. The microvilli of each patch converge in their apical regions to form a common focal point. X5425. FIGURE 6. Late cleavage (6 hours old). During late cleavage the embryo assumes the shape of a round sphere and the blastomeres are becoming more equal in size. X290. FIGURE 7. Late cleavage embryo (just before gastrulation). A few short cilia and mucous appear at the surface before the animal undergoes gastrulation. X6000. 119 Microvilli of the late cleavage embryo (early 8 hours old). Patches of short microvilli cover certain regions ,:;' the blastomeres while other areas are bare. The microvilli associated with the patches do not come to i in their apical regions as seen in early cleavage (Fig. 5). X7750. FIGURE 9. Lute 8-hour gastrula. The blastomeres are relatively uniform in size and the surface is smooth. An indentation (blastopore) forms at one end of the animal. X290. FIGURE 10. Blastopore region. Cells on the outside of the embryo move toward this pore, roll over the margins of the pore, and disappear to the inside. X 1650. 120 SEM OF A HYDROZOAN 121 (Martin and Archer, submitted). This stage represents gastrulation. The gastrula is ca. 250 jim long and 190 pm wide. During gastrulation surface specializations characteristic of mucous cells appear (see Fig. 16 for gland cell specialization). These specializations consist of a single cilium surrounded by a low disorganized collar of microvilli. The cilium and microvilli sit within a small ectodermal surface depression (Fig. 16). Thus by the end of gastrulation surface projections of mucous cells and epitheliomuscle cells are distinguishable (see Figs. 15, 16). The epitheliomuscle cell possesses a single cilium that projects from its surface. These cilia are short when they first appear and later become longer as the embryo matures to the planula stage (Figs. 7, 15). Very few if any microvilli are associated with this cilium. Each epitheliomuscle cell and mucous cell is monociliated. The surface specializations of the epitheliomuscle cells and mucous cells do not change as the embryo matures, except for the elongation of the cilium. Blastomeres located closest to the blastopore possess short cilia while those farthest from the blastopore have longer cilia. Each blastomere corresponds to either an epitheliomuscle cell or a mucous cell. Between 8 and 10 hours postfertilization the gastrula elongates in an anterior- posterior direction to form a young planula (Fig. 1 1 ). This 10-hour planula measures ca. 350 /urn long, 180 /j.m wide in the anterior region, 170 nm wide in the mid area, and 120 ^m wide in the tail region. A distinct anterior pole and posterior pole are visible (Fig. 1 1). The blastopore is located at the posterior pole of the planula and is nearly closed (Fig. 1 1 ). The surface cells are numerous, small, and uniform in size. Epitheliomuscle cells and mucous cells comprise the ectoderm. Microvilli of the 10-hour embryo are numerous and are not arranged in distinct patches as seen during early development. They are uniformly distributed over the surfaces of the ectodermal cells. This distribution pattern persists until the planula begins to metamorphose. The 10-hour planula elongates to form the mature planula which is anywhere from 24 to 96 hours old depending upon temperature (Figs. 12, 14). During the elongation period the planula grows in length and becomes narrow in diameter; surface specializations of neurosensory cells and nematocytes appear (Figs. 17, 18); and numbers of cells increase. By 36 hours the planula is ca. 700 /urn long, 1 50 ^m wide in the anterior region, 100 Mm wide in the mid area, and 80 ^m wide in the tail (Fig. 12). The ectoderm of the 36-hour planula consists of epitheliomuscle cells, mucous cells, and nematocytes (see Figs. 15, 16, and 18 for distinguishing surface projections of these cells). The epitheliomuscle cells comprise the majority of the ectodermal cells and are found along the entire length of the planula. Mucous cells are the second major type and are mostly concentrated in the anterior third of the animal (Martin and Archer, sub- mitted). Numerous surface specializations of these cells are abundant in the head region. Nematocytes are located in a region extending from the anterior end of the planula to the lower third of the planula and are most abundant in the anterior end. Apical projections of nematocytes are characterized by a single cilium projecting from a basal collar of long microvilli (Fig. 18). The cilium is displaced to one side of the collar. All cilia at this stage are long (Fig. 13). By 36 hours an indentation is visible at the anterior end of the planula (Fig. 1 3). This indentation is not the same pore formed by the blastopore during gastrulation. Long cilia project from this indentation. This indentation appears to help the planula attach to a substrate. At 48 hours the planula is ca. 800 urn long, 120 ^m wide in the head region, 60 Mm wide in the mid area, and 40 ^m wide at the tail (Fig. 14). Five types of surface specializations are present by 48 hours (Figs. 15-18). Microvilli are scattered over all the ectodermal cell surfaces. Apical regions of epitheliomuscle cells are characterized by a solitary cilium (Fig. 15). Mucous cells possess a single cilium surrounded by a FIGURE 1 Ten-hour planula. The animal has elongated and has a distinct anterior end and posterior end. The anterior end is directed down in this micrograph. Microvilli are scattered over the surfaces of all ectodermal cells. Cilia are numerous. The blastopore (arrow) is located at the posterior tip of the planula. X280. FIGURE 12. Thirty-six-hour planula. The planula is long and narrow. Cilia are abundant, long, and scattered over the entire surface of the animal. The anterior and posterior regions are much narrower than seen in the 10-hour planula (Fig. 1 1). The anterior end is down. X165. FIGURE 13. Thirty-six-hour planula. An indentation is present in the middle of the anterior end. This pit is not the same indentation formed by the blastopore. Long cilia project out from the pit. XI 100. FIGURE 14. Forty-eight-hour planula. Anterior end is directed down. X145. SEM OF A HYDROZOAN 123 loose collar of microvilli, all of which arise from a small surface depression (Fig. 16). Apical extensions of neurosensory cells are visible by this stage (Fig. 1 7). These sensory cells possess a single cilium surrounded by a bulbous cluster of microvilli. The cilium is located in the center of the cluster. Neurosensory cells occur all along the length of the planula but are most concentrated in the anterior head region of the planula. Nematocyte projections identical to those described for the 36 hour planula are also seen (Fig. 18). The nematocyte specializations are more numerous than those seen at 36 hours, however, they do have the same distribution pattern. Planulae become competent to metamorphose anytime between 24 hours and 96 hours postfertilization depending on temperature. Planulae may metamorphose naturally or they can be induced to metamorphose by treating with cesium chloride (Martin and Archer, in prep.). Shortly after planulae attach to a substrate, their cells lose microvilli and cilia. For a summary of the time sequence of developmental events in embryos ofPennaria tiarella at 23C see Table I. DISCUSSION The planula larva is the best described representative of the cnidarian embryonic life cycle (Martin and Thomas, 1977, 1980, 198 la, b, 1983a, b; Freeman, 1981; Martin and Chia, 1982; Martin et al., 1983; Berking, 1984; Walch et ai, 1986). The mor- phological events prior to planula formation have been largely ignored in the past, and as a result information on early development in the cnidarians is lacking (Martin and Thomas, 1977, 1983b; Martin et al., 1983). In an attempt to understand better embryonic morphogenesis in the cnidarians we used scanning electron microscopy to examine the development of a typical marine hydrozoan beginning with early cleavage and ending with the mature planula. During the development ofPennaria tiarella the embryo undergoes extensive changes in body shape. Early cleaving embryos have a bizarre morphology, however, by the end of late cleavage the embryos have rounded to form spheres. Such a rounding of the embryo may be essential if an organized form of gastrulation is to follow. In Pennaria tiarella gastrulation is organized with the formation of a blastopore at the future posterior end of the planula and the migration of cells over the margins of the blastopore to the inside of the embryo (Martin and Archer, in prep.). Such movements of cells resemble the morphogenetic process of invagination, a type of gastrulation not pre- viously reported for the Hydrozoa (Tardent, 1978). A similar pattern of gastrulation FIGURE 1 5. Surface projections of epitheliomuscle cells. A single cilium extends from each cell. X6500. FIGURE 16. Surface projection of a mucous cell. A single cilium surrounded by a disorganized clump of microvilli extends from a slight surface depression. X6000. FIGURE 17. Surface extension of a neurosensory cell. A single cilium projecting from the center of a collar of microvilli characterizes this cell type. X6000. FIGURE 18. Surface extension of a nematocyte. A single cilium projecting from the side of a collar of long microvilli distinguishes the nematocyte. X6000. 124 V. J. MARTIN AND W. E. ARCHER TABLE I Developmental time table for embryos of Penriaria tiarella Developmental age Stage (hours postfertilization) Distinguishing characteristics Early cleavage 1-6 Bizarre shape; irregular cleavage; holoblastic cleavage; asynchronous cleavage Late cleavage 6-8 Holoblastic cleavage; more regular cleavage; more synchronous cleavage; spherical shape; cilia appear Gastrulation 8-10 Appearance of blastopore; axial elongation; localization of embryonic tissue types; separation of germ layers with the formation of the mesoglea Early planula 10 Anterior, posterior axis established; closure of blastopore; presence of cilia of epitheliomuscle and mucous cells Mature planula 24-96 Axial elongation; formation of anterior depression; appearance of surface specializations of neurosensory cells and nematocytes; attachment; metamorphosis has been observed for embryos of the hydrozoan Podocoryne carnea, in which a blas- topore is also present (Martin and Archer, submitted). The product of gastrulation is a short fat planula. As the young animal grows into a mature planula capable of attaching and metamorphosing, planular length increases while planular diameter in the anterior, mid, and tail regions decreases. During development of Pennaria tiarella the distribution patterns and numbers of microvilli and cilia change. In early and late cleavage the microvilli are abundant and are found in distinct patches. Cleavages are very rapid in these embryos and the arrangement of microvilli during this period of intense mitotic activity may play an important role in the ability of these blastomeres to adhere together and form stable contacts. The patch arrangement of microvilli may increase the surface area of these projections and hence provide sites for numerous adhesion molecules. As development proceeds through late cleavage cell division slows and the arrangement and number of microvilli change. The number of microvilli per animal increases through the mature planula stage and they are not arranged in patches. The microvilli disappear shortly after the mature planula attaches. At the beginning of gastrulation, the microvilli appear as single entities. Because cell division is slowed, pretty much synchronous, and of a more ordered pattern by gastrulation, perhaps not as many adhesion molecules are needed for the initial sticking together of these cells as were needed earlier. The presence of a single microvilli on the surfaces of cells from early gastrulation through the mature planula stage may be a reflection of the need for fewer adhesion factors during later development. Cilia are not found on early cleavage embryos. Only after the blastomeres have rounded to form a late cleavage sphere do a few short cilia appear. These cilia are associated with blastomeres that will form differentiated epitheliomuscle cells after gastrulation. The presence of cilia on these late cleavage blastomeres may indicate that some of ;se early cells are predetermined during cleavage before the actual separation of the two germ layers occurs during gastrulation. As development continues to the mature planula stage cilia grow in length and increase in number. Surface specializations characteristic of mucous cells, neurosensory cells, and nematocytes form. The appearance of these specializations at particular developmental ages as SEM OF A HYDROZOAN 125 determined from scanning electron microscopy corresponds well to their developmental appearance noted using transmission electron microscopy (Martin and Thomas, 1977, 1980, 1981b, 1983a). Sexual reproduction and embryogenesis have never been a primary focal point in cnidarian research. This is surprising because the cnidaria offer excellent material for the study of the evolution of embryogenesis. Embryogenesis in the simpler forms at times appears almost "anarchic," whereas, in the more advanced cnidaria highly com- plex mosaic patterns are seen (Metschnikoff, 1886; Carre, 1969). The present study examines embryogenesis in a marine hydrozoan. Findings such as the arrangement and distribution of microvilli during development, the presence of cytoplasmic blebs on early blastomeres, the presence of cilia during late cleavage, the formation of a blastopore, the migration and invagination of surface cells during gastrulation, and the changes in body shape as the hydrozoan embryo progresses from early cleavage to the mature planula have not been reported previously. Additional studies of em- bryogenesis in the phylum are needed to fill in the major gaps that exist in our basic knowledge of morphogenesis in this lower animal phylum. LITERATURE CITED BERKING, S. 1984. Metamorphosis of Hydractinia echinata insights into pattern formation in hydroids. Roux'sArch. Dev. Biol. 193: 370-378. CARRE, D. 1 969. Etude du developpement larvaire de Sphaewnectes gracilis (Claus, 1 873) et de Sphaeronectes irregularis (Claus, 1873), Siphonophores Calycophores. Cah. Biol. Mar. 10: 31-34. FREEMAN, G. 1981. The role of polarity in the development of the hydrozoan planula larva. Roux's Arch. Dev. Biol. 190: 168-184. HOTCHKJSS, A., V. MARTIN, AND R. APKARIAN. 1984. A scanning electron microscopic surface and cryo- fracture study of development in the planulae of the hydrozoan, Pennaria tiarella. Scanning Electron Microsc. 11:717-727. MARTIN, V., AND F. CHIA. 1982. Ultrastructure of a scyphozoan planula, Cassiopeia xamachana. Biol. Bull. 163: 320-328. MARTIN, V., AND M. THOMAS. 1977. A fine-structural study of embryonic and larval development in the gymnoblastic hydroid Pennaria tiarella. Biol. Bull. 153: 198-218. MARTIN, V., AND M. THOMAS. 1980. Nerve elements in the planula of the hydrozoan Pennaria tiarella. J. Morphol. 166: 27-36. MARTIN, V., AND M. THOMAS. 198 la. The origin of the nervous system in Pennaria tiarella as revealed by treatment with colchicine. Biol. Bull. 160: 303-310. MARTIN, V., AND M. THOMAS. 1981b. Elimination of the interstitial cells in the planula larva of the marine hydrozoan Pennaria tiarella. J. Exp. Zool. 217: 303-323. MARTIN, V., AND M. THOMAS. 1983a. Establishment and maintenance of morphological polarity in epithelial planulae. Trans. Am. Microsc. Soc. 102: 18-24. MARTIN, V., AND M. THOMAS. 1983b. An SEM analysis of early development in Pennaria tiarella. Am. Zool. 23: 1014. MARTIN, V., F. CHIA, AND R. Koss. 1983. A fine-structural study of metamorphosis of the hydrozoan Mitrocomella polydiademata. J. Morphol. 176: 261-287. METSCHNIKOFF, E. 1886. Embryologische studien an Medusen. Ein Beitrag :ur Genealogie der Primitiv- organe. Alfred Holder, Vienna. TARDENT, P. 1978. Coelenterata, Cnidaria. Pp. 199-302 in Morphogenese der Tiere. Gustav Fischer Verlag, Stuttgart. WALCH, E., V. MARTIN, AND W. ARCHER. 1986. Evidence of a microtrabecular cytoskeletal lattice in glandular cells of hydrozoan planulae. J. Morphol. 187: 353-362. Reference: Biol. Bull. 171: 126-134. (August, 1986) PRECOCIOUS TERMINATION OF DIAPAUSE IN NECK- AND ABDOMEN LIGATED PUPAL PREPARATIONS OF THE TOBACCO HORNWORM, MANDUCA SEXTA LOUIS SAFRANEK, CLAYTON R. SQUIRE, AND CARROLL M. WILLIAMS Department of Cellular and Developmental Biology, Harvard University, Cambridge, Massachusetts 02138 ABSTRACT When ligatures were placed between the head and thorax of freshly pupated horn- worms, the resulting brainless preparations initiated adult development weeks earlier than intact diapausing controls and months earlier than similar preparations from which the brain had been surgically extirpated. This phenomenon could not be re- produced by removal of any single recognized cephalic neural or endocrine organ or any combination of organs. A similar accelerated development took place in many isolated pupal abdomens prepared by ligature or surgical section between the thorax and abdomen. Extirpation of the prothoracic glands of either diapause or non-diapause pupae resulted in only a very slight delay in the onset of adult development. Nevertheless, this development remained highly dependent on the brain, since preparations lacking brains as well as prothoracic glands underwent a prolonged developmental arrest. Preparations lacking prothoracic glands demonstrated elevated levels of ecdysone at the outset of adult development, although these levels were slightly lower than those of intact individuals at a similar stage. These findings suggest that sources of ecdysone outside the prothoracic glands can respond to a hormonally active brain and contribute significantly to the elevated liters of ecdysone that accompany much of normal adult development. In addition, the present results direct attention to the possible existence of a head-centered mechanism for maintenance of the low ecdysone liter necessary for the persistence of pupal diapause. INTRODUCTION Insect pupal diapause is classically conceived as a developmental hiatus attributable to the failure of the prothoracic glands (PG) to secrete ecdysone a failure considered in turn to reflect the diapausing brain's inability to secrete the prothoracicotropic hormone (PTTH). But in the tobacco hornworm, Manduca sexta, the diapause-like arrest that ensues after surgical excision of the pupal brain is eventually terminated, albeit with substantial delay relative to intact pupae (Judy, 1972; Wilson and Larsen, 1974; Safranek and Williams, 1980). Possible mechanisms for this outcome include autonomous secretion of ecdysone by the PG or by additional tissues not subject to the classical type of regulation by the brain. Received 6 January 1986; accepted 19 May 1986. Abbreviations: PG - prothoracic gland; PTTH = prothoracicotropic hormone; LD = long-day; SD = short-day; SEG = subesophageal ganglion; PTG = prothoracic ganglion; FG = frontal ganglion; MTG = mesothoracic ganglion; VNC = ventral nerve cord segment; CC-CA = corpora cardiaca-corpora allata complexes. 126 DIAPAUSE REGULATION IN THE HORNWORM 127 For forty years the PG have been considered the principal source of ecdysone. Nevertheless, ecdysteroid production in at least certain species has been shown to proceed outside the PG in sources that include the testes, ovaries, and oenocytes (for reviews, see Hoffman and Hetru, 1983; Rees, 1985). The physiological significance of these abdominal sources is supported by instances in which ecdysone-dependent de- velopment takes place in the absence of the PG-for example, after surgical extirpation of the PG or in isolated abdomens (Chadwick, 1955; Ichikawa, 1962; Hsaio et al, 1975; Delbecque et al, 1978; Delbecque and Slama, 1980; Slama, 1983). But most studies to date fail to clarify whether these sources contribute significantly to the rising levels of ecdysone that accompany molting in intact insects. The brain is known to be an important and sometimes necessary organ for the generation of molt-inducing levels of ecdysone. But again, numerous instances of ecdysone-dependent development in the absence of the brain have been described (see Safranek and Williams, 1980, for review). The current study focuses on the pupal stage of the tobacco hornworm where we have encountered the paradoxical finding that decapitation of freshly pupated diapause-destined individuals actually accelerates the initiation of adult development. The present experiments document this finding and probe its endocrine basis, drawing attention to previously unsuspected aspects of the regulation of normal adult development. MATERIALS AND METHODS Hornworms were reared as described previously (Safranek and Williams, 1980) at 25C under either a short-day (SD, 12L: 12D) or long-day (LD, 17L:7D) photoperiod. SD pupae were derived from larvae reared under SD conditions and normally un- derwent a pupal diapause. LD pupae were derived from larvae under LD conditions; these did not diapause but initiated development typically within five days after pu- pation. The day of pupation is termed "Day 1" of the pupal stage, and the first seven days of the pupal stage "Week 1 ." Operations and ligations were carried out as described previously (Safranek and Williams, 1980; Safranek et al, 1980) and as described below. All operated preparations were maintained under SD conditions at 25 C, the initiation of development being recognized by detachment and retraction of the trachea from the overlying pupal cuticle of the forewings. Removal of the subesophageal ganglion (SEG) or the prothoracic or mesothoracic ganglia (PTG, MTG) was accomplished through a small ventral midline incision be- ginning at the base of the pupal proboscis; in many instances the proboscis was still quite flexible and could be bent slightly from the midline to facilitate the surgical approach. Removal of the prothoracic glands (PG) was accomplished after removal of a rectangular section of the dorsal thoracic cuticle. The main body of each PG was identified nested in its characteristic position just medial to the large tracheal trunk adjoining the prothoracic spiracle. It was gently teased free of its fine connections to surrounding tissues and then withdrawn by the aid of two forceps used in a hand over hand fashion to draw the remainder of the gland slowly from more anterior regions. Abdomens were prepared by ligation within 6 h of larval-pupal ecdysis shortly after the pupal wings attained their final size and position. Older abdomens were surgically isolated using a razor blade to section the pupal cuticle and body wall behind the thorax. The gut was either ligated with a fine sterile thread or draped over the side of the exposed edge of the abdomen. A sterile glass cover slip was applied over the anterior end of the abdomen and sealed with melted wax (Tackiwax, CENCO). Radioimmu- noassays of ecdysteroids were performed as described elsewhere (Carrow et al, 1981). 128 L. SAFRANEK ET AL. RESULTS Effects of brain removal and kcad-ligation on the development of pupae In the course of studies on the regulation of pupal diapause we noted that pupae head-ligated promptly after eclosion to the pupal stage initiated development much sooner than would have been expected of brainless pupae. To document this we ligated 100 SD pupae within 2-6 h after pupation. An additional 100 SD pupae were set aside as unoperaied controls. All individuals were examined at weekly intervals for the onset of tracheal apolysis. As documented in Figure 1 , the ligated preparations initiated development an average of one month prior to intact diapausing pupae and approximately four months earlier than expected for pupae whose brains had been surgically extirpated (Safranek and Williams, 1980). We repeated the experiment on approximately 50 freshly ecdysed SD pupae and 50 similar LD pupae, with 50 intact SD pupae serving as controls. Here again over 50% of the neck-ligated preparations had initiated adult development by the fifth week, irrespective of LD or SD status; by the ninth week over 90% had done so. By contrast, 50% of the intact SD controls initiated development only after 1 1 weeks and 90% only after 13 weeks. The development of the LD preparations thus was delayed by more than a month relative to that of intact LD pupae, which, as previously men- tioned, initiate development within a week of pupation. But both LD and SD prep- arations developed in this instance over a month before intact diapausing pupae and several months earlier than would have been expected of pupae whose brains had been excised rather than removed by neck-ligation. 100- 345678 Weeks After Pupation 10 12 FIGURE 1. Acceleration of diapause termination by neck-ligation of diapause pupae. Diapause pupae were ligated 2-6 h after ecdysis. Initiation of development was recognized by apolysis of the wing epidermis. Ligated preparations are indicated by dark circles, intact controls by clear triangles. DIAPAUSE REGULATION IN THE HORNWORM 129 Two explanations for these results seemed possible. Either a developmental inhibitor existed in the head which could be removed by ligation but not by brain excision or else the ligation itself provided an ecdysiotropic stimulus to the headless preparations. We examined these possibilities in the following experiments. Effects of removal of the brain plus other cephalic organs We attempted to identify a cephalic inhibitor by surgical extirpation of known cephalic neuroendocrine centers. Because head-ligated preparations lacked the brain as well as all other cephalic organs and because surgical extirpation of the brain alone produced a greatly prolonged diapause that could be readily distinguished from the abbreviated diapause of head-ligated preparations, most of our experiments involved simultaneous removal of the brain in addition to one or more of the other cephalic organs. Thus, the brain was removed in tandem with the subesophageal ganglion (SEG), the frontal ganglion (FG), the prothoracic ganglion (PTG), the mesothoracic ganglion (MTG), or the corpora cardiaca-corpora allata complexes (CC-CA). In ad- dition, brain removal was performed in combination either with severance of all neural connections to the SEG, which was thereby left wholly free in the head, or with sev- erance of the neural connection between the SEG and PTG only. Additional groups of brainless pupae with appropriate sham operations were established as controls. These experiments included operations on the PTG and MTG because of the proximity of these ganglia to a neck-ligature. All these experiments were performed on diapause- destined SD pupae 6 to 1 2 h after pupal ecdysis. The results (Table I) demonstrated no significant differences between the various groups of pupae. Irrespective of the surgical procedures performed, all groups entered an extended diapause averaging over 30 weeks. We considered in addition the possibility that the brain itself might be the source of an inhibitor. We were driven to entertain this possibility because for reasons of technical ease we routinely head-ligated pupae about 2 to 6 h after pupal ecdysis but normally surgically removed the brain 6 to 12 h after ecdysis. Thus, the brain could potentially have released an inhibitor during the initial 6 h after ecdysis. To investigate this possibility we removed the brains from a group of larvae 1-2 days prior to the wandering period; as controls we sham-operated additional larvae. Subsequently late on the day of pupal ecdysis we removed the brains of half of the previously sham- operated individuals. In addition, we sham-operated all the other individuals, both those that had previously lost their brains and also the other half of the previously sham-operated group. We subsequently monitored all the preparations for the initiation of development at weekly intervals. All of the 23 twice sham-operated preparations initiated adult development after 1-4 months. By contrast, brainless pupae entered an extended diapause regardless of whether the brain had been removed before (n = 17) or after pupation (n == 23). At 4 months less than 25% of either group had initiated development, and after 7 months more than 50% of each group still remained in diapause. Thus, evidence for a brain-derived inhibitor was entirely negative. Effects ofPG removal on adult development All these results failed to define an inhibitory center in the head. This raised the possibility that head-ligation itself might produce an injury reaction which elicited development through a stimulatory effect on the PG. To examine this possibility we implanted into brainless, diapausing 1 -day-old pupae the PG removed from 3-day- old SD pupal preparations that had previously been head-ligated on Day 1 . As controls 130 L. SAFRANEK ET AL. we implanted the PG from intact Day 3 SD pupae into brainless 1 -day-old pupal hosts and thereafter examined a!) the preparations for development at weekly intervals. During the subsequent 6 weeks, only 1 of the 1 5 pupae in each group initiated de- velopment. This contrasted greatly with the rapid development of a further control group of 18 heo i preparations in which half had initiated development by 6 weeks. These indicated that head-ligation did not generate within three days of ligation versible, significant increase in PG activity. In the preceding experiment, after removal of the PG from the head-ligated pupae, the thorax of each was resealed and the preparation observed at weekly intervals for the initiation of development. Much to our surprise, extirpation of the PG failed to block development of these decapitated preparations. At six weeks similar percentages of head-ligated pupae (9 of 18) and head-ligated pupae lacking PG (8 of 15) had terminated diapause, suggesting that development of head-ligated pupae did not require ecdysone secretion by the PG. We initially considered that our surgical procedure must not have removed the entire PG. But repeated, detailed dissection of pupae after PG extirpation failed to reveal any retained portion of the PG. Comparison of the extirpated PG with whole PG carefully dissected from sacrificed pupae did not suggest that our routine operation left behind any portion of the PG. These concerns were rendered moot by additional experiments. We prepared over 300 isolated pupal abdomens either by ligation within 4 h of the larval-pupal ecdysis or by surgical isolation of slightly older pupal abdomens as described under Materials and Methods. Although abdomens prepared by either approach had a very low mor- tality within the first week, preparations of both types experienced a sharply increasing mortality thereafter. Few preparations exhibited spontaneous motion beyond eight TABLE I Effects of surgical extirpation of cephalic and thoracic organs on diapause termination Organs removed 1 Number of preparations % Developed at 50 weeks Average time to development 2 Brain plus sham 3 181 67 35 12 Brain plus SEG 41 85 34 10 Brain plus PTG 27 81 31 10 Brain plus EG 21 38 33 12 Brain plus MTG 11 73 32 11 Brain (SEG loose) 22 73 34 12 Brain plus VNC 4 17 65 39 12 Brain plus CC-CA 16 78 36 10 1 Abbreviations: SEG: = subesophageal ganglion; PTG = prothoracic ganglion; EG = frontal ganglion; MTG = mesothoracic ganglion; VNC = ventral nerve cord; CC-CA = corpora cardiaca-corpora allata complexes. 2 Averages are standard deviation and are calculated only for those preparations which had developed by 50 weeks. Thus the average time to development for the entire group of preparations would have been longer than >hat listed here. 3 A group of sham-operated pupae was established for each experimental group of preparations listed below. No experimental group developed at a rate significantly different from its control group or from the pooled set of sham-operated preparations as listed on this line. Shams included brain removal plus a sham operation on the ventral thorax except in the control group for preparations without the brain and CC-CA complex, in which instance only the brain of the sham preparations was removed through a cephalic incision and no ventral incision was placed. 4 The segment of the ventral nerve cord between the SEG and PTG was severed. DIAPAUSE REGULATION IN THE HORNWORM 131 weeks. Nevertheless we repeatedly witnessed the spontaneous development of both male and female abdomens prepared by either technique: the earliest fully scaled and pigmented adult abdomen was obtained five weeks after pupation, others after as long as six months. Overall we have witnessed the adult development of 27 pupal abdomens, with approximately equal numbers of males and females and an average time from the isolation of the abdomens to the completion of adult development of 1 2 weeks. If survival could be enhanced, our experience suggests that many more, and perhaps all, of these preparations would be able to undergo adult development. The time course of development of these abdomens suggested that an abdominal source of a molting hormone could account for the spontaneous development of head-ligated pupal preparations: although we could not witness the initiation of adult development in isolated abdomens, the first individuals to complete adult development among either the head-ligated preparations or the isolated abdomens did so after about five weeks. Development took place in abdomens isolated from both LD and SD pupae; although the sample size was relatively small, no differences were noted in the times at which development was completed within the two groups. What might this unusual source of molting hormone contribute to normal devel- opment? As we have seen here and elsewhere (Safranek and Williams, 1980), surgical extirpation of either the LD or SD pupal brain results in a developmental arrest of several months duration. By contrast, extirpation of the PG fails to block development of otherwise intact pupae. This was demonstrated in the case of LD pupae after removal of the PG from 12 Day 2 pupae: all of these pupae subsequently initiated development within 6 weeks of the operation, the earliest at 1 week, and 75% by 2.5 weeks. This represented a delay of only about 1 week relative to 1 2 control pupae whose PG had been extirpated and then replaced into the thoracic cavity, all of which initiated adult development within 6-1 1 days of the operation. PG removal also failed substantially to alter the duration of diapause in SD pupae. In this instance PG were removed from 1 8 Day 3 SD pupae; 1 5 similar pupae served as controls after receiving sham operations. Half of the 1 5 controls had initiated development at 9 weeks, as had half of the preparations lacking PG. The controls had an average diapause duration of 12 weeks, the experimentals, 1 3 weeks. Neither preparations subjected to PG extirpation nor sham-operated controls eclosed successfully; nevertheless, the course of adult devel- opment appeared grossly similar for both groups and essentially normal in its character and duration. These experiments demonstrated the ability of an ecdysteroid source outside the PG to initiate and support adult development. In addition, the more rapid development of LD preparations relative to their SD counterparts suggested that these sources could respond to a hormonally active brain. Further to demonstrate this phenomenon we implanted brains from Day 1 LD pupae into Day 3 SD pupae from half of which both the brain-CC-CA complex and PG were removed, from the other half, the brain- CC-CA complex only. Both groups developed promptly. Of 10 preparations that had retained their PG, all commenced development within 6 weeks, the average being 2 weeks. Among the group without PG 3 of 14 developed only after several months whereas the remaining 1 1 initiated development within 5 weeks, the average of these being 3 weeks. Manifestly, the absence of the PG failed to block or even to delay substantially the rapid onset of development in response to a LD brain. In an additional control group of 15, the brain-CC-CA complex and the PG were removed but a LD brain was not implanted. All of these preparations underwent a prolonged diapause of at least three months and more than half remained in diapause after six months. As indicated in the previous experiments, these data again demonstrate that devel- opment can be stimulated by a hormonally active brain even in the absence of PG. 1 32 L. SAFRANEK ET AL. Is the development of preparations lacking PG accompanied by elevated ecdysteroid liters? We inquired whether the onset of adult development in pupae lacking PG was accompanied by a rise in the ecdysteroid level. To this end we collected hemolymph samples from 3 groups of pupae on the day of tracheal apolysis namely, intact LD pupae, LD pupae whose PG had been removed 24-48 h after pupation, and SD pupae whose PG had been removed at 2 weeks after pupation and which at that time had also received an implantation of a Day 1 LD pupal brain. We also measured the ecdysteroid levels of a group of 2-4 week old diapausing SD pupae. Samples were analyzed in a RIA for ecdysteroids. The results are summarized in Table II. Manifestly, both sets of preparations lacking PG had ecdysteroid levels markedly greater than those typically found in the course of diapause. Nevertheless, these liters were only about one-fourth those occurring in the intact LD pupae at a similar develop- mental stage. DISCUSSION The present results describe a peculiar abbreviation of the hornworm pupal dia- pause when head-ligation rather than surgical excision was employed for removal of the brain promptly after pupal ecdysis. Adult development was advanced by months relative to surgically debrained preparations and by weeks relative to intact diapausing pupae. Yet we were unable to identify a cephalic source of a molting inhibitor among recognized neural or endocrine organs in the head. We nevertheless continue to favor an inhibitor as an explanation of this phenom- enon. Although we cannot rule out the involvement of a powerful stimulatory injury effect as can sometimes be seen after surgical manipulation of mature pupae (Wilson and Larsen, 1974; pers. obs.), several arguments oppose this explanation and favor an inhibitor. First, the considerable surgical injuries resulting from combined brain and SEG-PTG removal had no stimulatory effect on development, nor did we find evidence of any irreversible activation of the PG after ligation. Indeed, our experience suggests that injury shortly after pupation delays development: this is the case with young non-diapausing pupae where even a small cephalic cuticular wound will delay development by 1-2 days. Second, the developmental stimulation witnessed following neck-ligation of pupae does not occur promptly as one might expect from injury; rather the stimulation becomes apparent only after at least two weeks and often much longer. Third, the active involvement of an inhibitor during diapause would account for the ability of hornworm pupae to develop in the absence of the brain: the eventual disappearance of the hypothetical inhibitor would permit resumption of development even in the absence of a positive stimulus from the brain. These considerations lead TABLE II Effects of proth.oracic gland extirpation on the ecdysteroid liter at the outset of adult development Procedure Stage Ecdysteroid titer ' SD pupa-PG + LD brain Tracheal apolysis 5.9 1.9 LDpupa-PG Tracheal apolysis 4.9 1.8 LD pupa, intact Tracheal apolysis 223 SD pupa, intact Pupal diapause, 2 weeks 0.21 0.03 The ecdysteroid titer is expressed in ng/ml 0-ecdysone equivalents. DIAPAUSE REGULATION IN THE HORNWORM 133 us to suggest that in the normal course of the horn worm's pupal diapause an inhibitor is elaborated by the cephalic region that is responsible for the maintenance of the diapausing condition. Over time this inhibitor is eliminated by breakdown and/or by cessation of its production, thereby permitting the initiation of adult development. This model, consistent with the present findings, finds some support in the literature. Results similar to those recorded here have been noted in Anther ea pernyi (Waku, 1959) wherein pupae head-ligated promptly after pupation experienced an abbreviated diapause relative to both brainless preparations and intact unchilled diapause pupae. So also, in Pieris brassicae removal of the brain during diapause accelerated diapause termination; moreover, abdomens isolated from chilled diapausing pupae underwent spontaneous development in contrast to the persistent diapause of abdomens isolated from young, unchilled individuals (Kono, 1977). The acknowledged preeminent role of a molt-inhibiting hormone in the development of Crustacea illustrates the potential for inhibitory control of the ecdysteroid titer among arthropods (Highnam and Hill, 1977). These examples, as well as our own, do not preclude a contributing role for the brain in diapause termination. Indeed, we suggest that pupal-adult development may reflect an interplay of both stimulatory and inhibitory factors. The final noteworthy finding of our study is the ability of isolated pupal abdomens to initiate adult development. This propensity is unique to this stage, since isolated larval abdomens, even when implanted with brains, fail to undergo a molt (Safranek and Williams, 1980). Development of isolated abdomens or of preparations lacking prothoracic glands has previously been noted in late larval and pupal stages of diverse orders of insects (Chadwick, 1955; Hsiao et al, 1975; Delbecque eta/., 1978; Delbecque and Slama, 1980) including the Lepidoptera (Ichikawa, 1962; Kono, 1977; Slama, 1983). Production of ecdysteroids outside the PG has been even more widely docu- mented and has been shown to derive from testes (Loeb et al., 1982), ovaries (see Hoffman and Hetru, 1983, for review), or oenocytes (Romer et al., 1974; Studinger and Willig, 1975). We do not presently know the source of molting hormone driving the development of isolated pupal hornworm abdomens, but since this development occurred in both male and female abdomens on a similar time scale, we favor an extra-gonadal source. The contribution of these abdominal sources to normal adult development is un- certain. The present experiments make clear that sources of ecdysteroids outside the PG are more active in the presence of a non-diapausing brain. Moreover, in either diapause or non-diapause pupae these sources can support development in the absence of PG at a rate very nearly that of intact pupae. When development ensues in the absence of PG, ecdysteroid levels rise into a range near that of intact pupae. We note that development of preparations without PG does typically lag that of controls by several days and that the ecdysteroid liters at the outset of tracheal apolysis are uni- formly slightly lower than those of controls at the same stage of development. Nev- ertheless the extra-PG sources clearly respond to an active brain and can generate high developmentally effective levels of ecdysteroids at least 25-fold greater than those found during the course of diapause. Thus, our observations indicate that at least one novel source of ecdysone plays a definite and possibly important role in the endocrine events normally associated with the onset of adult development. Although the ecdysteroid source outside the PG responds to an active brain, it may differ from the PG in other aspects of its regulation. We note in particular that the time courses of development initiation in head-ligated preparations with or without PG as well as of isolated abdomens all appeared quite similar. Thus, the peculiar accelerated development of neck-ligated diapausing pupae could potentially reflect the activity largely of an abdominal ecdysteroid source. This source might be especially responsive to the disappearance of an inhibitory influence emanating from the head. 134 L. SAFRANEK ET AL. Manifestly the picture presented here differs substantially from that derived through classic studies of the Cecropia silkworm. Whether one or the other picture will prove paradigmatic we cannot yet say. But certainly the literature portrays a complex picture even among the silkworms. We have already noted the work of Waku in Antherea pernyi. In addition, Ichikawa (1962) described the development of isolated abdomens of Samia cynfhfa in the presence of an active brain. And the regulation of diapause termination in unchilled or inadequately chilled Cecropia or in Cecropia that fail to develop promptly after termination of chilling but which ultimately develop all remain to be investigated. Although the present experiments along with those of others make clear that the regulation of pupal diapause termination in the Lepidoptera will very likely not make for a simple yarn, even now we see evidence of a common thread. ACKNOWLEDGMENTS We acknowledge early support of these investigations by the N.I.H. and the sub- sequent generous support of Rohm and Haas. LITERATURE CITED CARROW, G. M., R. L. CALABRESE, AND C. M. WILLIAMS. 1981. Spontaneous and evoked release of pro- thoracicotropin from multiple neurohemal organs of the tobacco hornworm. Proc. Natl. Acad. Sci. USA 78: 5866-5870. CHADWICK, L. E. 1955. Molting of roaches without prothoracic glands. Science 121: 435. DELBECQUE, J.-P. A., J. DELCHAMBRE, M. HIRN, AND M. DEREGGI. 1978. Abdominal production of ec- dysterone and pupal-adult development in Tenebrio molitor (Insecta, Coleoptera). Gen. Comp. Endocrinol. 35: 436-444. DELBECQUE, J-P., AND K. SLAMA. 1980. Ecdysteroid litres during autonomous metamorphosis in a Dermestid beetle. Z. Naturforsch. 35c: 1066-1080. HIGHNAM, K. C., AND L. HILL. 1977. The Comparative Endocrinology of the Invertebrates. Edward Arnold, London. 357 pp. HOFFMAN, J. A., AND C. HETRU. 1983. Ecdysone. Pp. 65-68 in Endocrinology of Insects, R. G. H. Downer and H. Laufer, eds. Alan R. Liss, Inc., New York. HSIAO, T. H., C. HSIAO, AND J. DEWILDE. 1975. Moulting hormone production in the isolated larval abdomen of the Colorado beetle. Nature 255: 727-728. ICHIKAWA, M. 1962. Brain and metamorphosis of Lepidoptera. Gen. Comp. Endocrinol. Suppl. 1: 331-336. JUDY, K. J. 1972. Diapause termination and metamorphosis in brainless tobacco hornworms (Lepidoptera). Life Sci. 11:605-611. KONO, Y. 1977. Ultrastructural changes of neurosecretory cells in the pars intercerebralis during diapause development in Pieris rapae. J. Insect Physiol. 23: 1461-1473. LOEB, M. J., C. W. WOODS, E. P. BRANDT, AND A. B. BORKOVEC. 1982. Larval testes of the tobacco budworm: a new source of insect ecdysteroids. Science 218: 896-898. REES, H. H. 1985. Biosynthesis of ecdysone. Pp. 249-293 in Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 7, G. A. Kerkut and L. I. Gilbert, eds. Pergamon Press, New York. ROMER, F., H. EMMERICH, AND J. NOVOCK. 1974. Biosynthesis of ecdysones in isolated prothoracic glands and oenocytes of Tenebrio molitor in vitro. J. Insect Physiol. 20: 1975-1987. SAFRANEK, L., AND C. M. WILLIAMS. 1980. Studies of the prothoracicotropic hormone in the tobacco homworm, Manduca sexta. Biol. Bull. 158: 141-153. SAFRANEK, L., B. CYMBOROWSKI, AND C. M. WILLIAMS. 1980. Effects of juvenile hormone on ecdysone- dependent development in the tobacco hornworm, Manduca sexta. Biol. Bull. 158: 248-256. SLAMA, i< i. Illusive functions of the prothoracic gland in Galleria. Ada Entomol. Bohemoslov. 80: 161-176. STUDINGER. G.. AND A. WILLIG. 1975. Biosynthesis of a-ecdysone and /3-ecdysone in isolated abdomens of larvae of Musca domestica. J. Insect Physiol. 21: 1793-1798. WAKU, Y. 1959. Studies on the hibernation and diapause in insects III Further notes on the O 2 -uptake change and breaking of pupal diapause in the Chinese oak-silkworm. Sci. Rep. Tohoku Univ. 25: 1-12. WILSON, G. R., AND J. R. LARSEN. 1974. Debraining and diapause development in Manduca sexta pupae. J. Insect Physiol. 20: 2459-2473. Reference: Biol. Bull. 171: 135-142. (August, 1986) PARTIAL PHOTOPERIODIC CONTROL OF DIAPAUSE IN THREE POPULATIONS OF THE FRESHWATER COPEPOD DIAPTOMUS SANGUINEUS NELSON G. HAIRSTON, JR. 1 AND EMILY J. OLDS 2 1 Section of Ecology and Systematics, Cornell University, Ithaca, New York 14853: and 2 Entomology Department, Michigan State University, East Lansing, Michigan 48824 ABSTRACT Populations of the freshwater calanoid copepod Diaptomus sanguineus inhabiting three Rhode Island ponds switch from making subitaneous (immediately hatching) to diapausing eggs on different dates. From results of previous research the timing of diapause appears to correspond closely to the individual causes of seasonally harsh conditions in each pond. The results of rearing copepods from each pond in controlled laboratory environments indicate that each population possesses a unique spectrum of sensitivity to photoperiod. The responses obtained, however, fail to describe ade- quately either the rapidity with which the onset of diapause occurs in natural popu- lations, or the differences in diapause timing between ponds. In initiating diapause, the copepods must respond to seasonal environmental cues other than critical pho- toperiod. INTRODUCTION Diapause is a physiological and developmental response adopted by a broad variety of animals as a means of avoiding seasonally uninhabitable periods in the environment. Annual climatic changes are frequently responsible for the onset of harsh conditions, and examples of temperate zone animals that enter diapause prior to winter are espe- cially well documented (see Danilevskii, 1965; Beck, 1980; and Tauber et ai, 1985 for reviews). When a species' geographical range covers a broad spectrum of latitudes, intolerable winter conditions for local populations may begin at markedly different times of year. Often these populations exhibit responses to diapause-controlling cues correlated with the climatic gradient. For example, animals collected at northern lat- itudes initiate diapause earlier in the autumn or at longer photoperiods than those collected further south (e.g.. Showers et al, 1975; Holtzer et al, 1976; Lumme and Oikarinen, 1977; Marcus, 1984). Several investigators have shown that such variation is genetically based indicating that populations are specifically adapted to the latitudes at which they reside (Masaki, 1963, 1967; Showers et al, 1975;Istock, 1981; Marcus, 1984). At the same time, there is evidence that some local populations retain additive genetic variance for diapause traits (Hoy, 1977; Istock, 1981; Lumme, 1982; Hairston and Walton, 1986), presumably maintained by local variation in selection pressures. A third potential source of variation in diapause is that between populations residing at the same latitude. For such variation to exist, however, local habitats must be sufficiently isolated or represent sufficiently different selective regimes to overcome the homogenizing force of dispersal. The planktonic freshwater copepod Diaptomus sanguineus is a resident of small lakes and ponds in northeastern North America Received 28 February 1986; accepted 19 May 1986. 135 136 N. G. HAIRSTON, JR. AND E. J. OLDS (Wilson, 1959). Within any given geographical area, many of these bodies of water differ in potentially important ways such as basin depth with its accompanying effect on the annual water ' ature cycle, seasonal permanency, and the types of plank- tivores present. Three populations of D. sanguineus residing in three ponds in southern Rhode Island exhibit distinct seasonal patterns of egg diapause (Fig. 1A). In Bullhead Pond the copepods switch from production of subitaneous (immediately hatching) eggs to production of diapausing eggs in late March; in Pond C diapause is initiated at the end of April, and in Pond A diapause begins principally in mid-May (Hairston and Munns, 1984; Hairston et ai, 1985; Hairston, 1986). These patterns are regularly repeated in successive years and represent real differences between reproductive phe- nologies of the three populations even though the ponds in which they live are geo- graphically quite close to each other (Ponds A and C are <200 m apart and ca. 27 km from Bullhead Pond). We showed previously (Hairston and Olds, 1984) that female copepods reciprocally transplanted between Bullhead Pond and Pond A began pro- duction of diapausing eggs at the time of year appropriate to their home pond rather than the pond to which they were transferred, and concluded that D. sanguineus was physiologically unable to alter its reproduction in response to changes in pond type. What then is the basis for the differences in timing of diapause observed be- tween ponds? One obvious possibility is that the copepods have distinct adaptations suitable to the local habitat in which they live. Hairston and Munns (1984) showed that D. san- guineus in Bullhead Pond began production of diapausing eggs at the appropriate time to avoid a spring increase in planktivory by resident sunfish. Ponds A and C, on the other hand, are temporary bodies of water and contain no fish. In Pond C the copepods switch to making diapausing eggs at the correct time of year to avoid sea- sonally intense predation by the dipteran larvae Chaoborus and Mochlonyx (Hairston et al, 1985; Hairston, 1986). Neither of these predatory flies is abundant in Pond A, where D. sanguineus appears to produce diapausing eggs as an adaptation to survive periods of pond drying (Hairston et al., 1985; Hairston and Olds, in prep.). If timing of diapause is a distinct adaptation of the copepods to the separate conditions found in each pond, then the differing seasonal patterns of reproduction must have a genetic basis. Photoperiod is the environmental cue initiating diapause in a broad variety of animals including insects (e.g.. Beck, 1980; Tauber and Tauber, 1981), mammals (Flint et al., 1981), and crustacean zooplankton (Einsle, 1964; Stress and Hill, 1965; Stress, 1969;Spindler, 1971; Watson and SmaUman, 1971; Marcus, 1980). As a result, it is logical to ask if the different D. sanguineus populations switch from production of subitateous eggs to diapausing eggs at different critical day lengths. The data in Figure 1 A can be replotted as the fraction of each population carrying subitaneous eggs (as opposed to diapausing eggs) at the day length prevailing when the copepods were collected (Fig. IB). If the interpopulation differences in the timing of diapause initiation result from the copepods being sensitive to different critical day lengths, then animals collected at each of the three ponds and reared in the laboratory under a : iries of appropriately chosen photoperiods should respond with distinct pat- terns of subitaneous and diapausing egg production. Specifically, at day lengths shorter than about ', 1 hours all copepods should make subitaneous eggs, at intermediate day lengths of around 1 3 hours Bullhead Pond copepods should make diapausing eggs while copep , i'rom Ponds A and C should make subitaneous eggs, and at day lengths longer than at nit S4.5 hours all copepods should make diapausing eggs. Here we test this hypothesis as a route to understanding the causes underlying the distinct repro- ductive phenologies of D. sanguineus living in Bullhead Pond, Pond A, and Pond C. DIAPAUSE IN A FRESHWATER COPEPOD 137 100 80 60 CO o o CO Z> o 40 20 Bullhead Pond Pond C "Pond A FEB. MAR. APR. MAY 11 12 13 14 PHOTOPERIOD (hr light) 15 FIGURE 1. The fractions of female Diaptomus sanguineus from three Rhode Island ponds making subitaneous (immediately hatching) eggs, as opposed to diapausing eggs, as a function of time of year. A. Egg type versus date of collection. The lines are summaries of data from Hairston and Munns (1984), Hairston el al. (1985), and Hairston ( 1986) showing the annual variation in timing of diapause. Data points after the switch to production of diapausing eggs are omitted here to facilitate illustration. Copepods in temporary Pond A have been shown in some years to return to production of subitaneous eggs for brief periods in June after the pond refilled with water from heavy rains. These reversals, discussed in detail elsewhere (Hairston and Olds, 1984; in prep.), have been omitted here to simplify illustration. B. Egg type versus prevailing day length (sunrise to sunset) on the date of collection. Photoperiodic responses are calculated from mean fractions of females making subitaneous eggs for the years illustrated in A. Closed circles denote photoperiods tested in laboratory experiments. MATERIALS AND METHODS The study sites, Bullhead Pond and Ponds A and C, are described in detail elsewhere (Hairston, 1980; Hairston et al., 1983; Hairston and Olds, 1984). Diaptomus sanguineus has 1 2 separate instars: six naupliar and six copepodid stages, plus an egg stage which the female copepods carry in a sac attached to their urosome. The animals are active in the water column during winter and spring, and make two or three generations per year before producing diapausing eggs (Hairston and Olds, 1984; Hairston et al., 1985). Live first and second stage nauplii were collected during March through May from each of the three Rhode Island ponds using a 75 jum mesh net. The nauplii were isolated in 2 L glass jars filled with 1 L of filtered pond water; 200 nauplii per jar. These cultures were fed twice weekly from algal stocks, and maintained with approx- imately 1 X 1 7 cells -ml ' of Chlam ydomonas sp. and 1 X 10 5 cells- ml' 1 ofEuglena sp. The copepods were reared at 9C and at a series of photoperiods in controlled environment chambers. Survival under these laboratory conditions was 40% to 60% from nauplius to adult. Raising the animals from early naupliar instars ensured more than sufficient time for the copepods to respond to the photoperiod treatments. Else- where we have shown that upon exposure to a change of day length, female D. san- guineus can alter the type of egg they produce within three clutches (Hairston and 138 N. G. HAIRSTON, JR. AND E. J. OLDS Olds, in prep.). Thus, unlike many species of insects (Beck, 1980), there is no early sensitive instar that irreversibly programs the copepod's diapause phenology. It seems likely that egg type in D. sanguineus is determined principally by females because diapausing eggs have a thick, highly structured chorion laid down by the mother whereas subitaneous eggs have a thin homogeneous chorion, and because the two egg types are provisioned differently (Hairston and Olds, 1984). Probably due to these structural differences and the physiological adjustments they imply, clutches are com- posed of either subitaneous or diapausing eggs. Mixed clutches are never made. Females do not store sperm and mating is required before each clutch can be produced. We do not know, however, what role, if any, males play in determining egg type. The photoperiods were chosen to conform with the design described in the Intro- duction (Fig. IB). Day length 9:55 corresponds to the date 25 January (sunrise to sunset), well before the switch to diapause in any of the populations. Day length 13:05 corresponds to 10 April when the Bullhead Pond population has almost com- pletely shifted to making diapausing eggs but the Ponds A and C populations still produce principally subitaneous eggs, and 14:45 corresponds to 28 May when all populations produce mainly diapausing eggs. Bullhead Pond copepods also were reared at a fourth daylength, 1 1:40 corresponding to 10 March shortly before they switch from subitaneous to diapausing eggs. The copepods were allowed to mature in the 2 L jars. Females carrying their first clutches were isolated individually in 15 ml of medium in 6-well tissue culture plates, and observed until either their eggs had hatched or they had dropped their egg sacs. The females were then placed in new 2 L jars with males and allowed to produce a second clutch. Copepods carrying their second egg clutches were reisolated in the tissue culture wells and egg hatching was again monitored. The process was continued until all females had produced three clutches. Females were pooled in 2 L jars because mating success was substantially higher in this arrangement than when males and females were isolated in small volumes of water. The procedure, however, does not permit a determination of the sequence of clutches made by individual females. Sub- itaneous and diapausing eggs cannot be distinguished under light microscopy, but we have shown previously (Hairston and Olds, 1984; Hairston and Munns, 1984) that in contrast to diapausing eggs, subitaneous eggs hatch rapidly, usually within one week after being laid. As in our earlier research, eggs that hatched within two weeks after production were designated subitaneous eggs, whereas those that had not hatched during this period were designated diapausing eggs. By these methods we established the fractions of subitaneous and diapausing eggs made by D. sanguineus from each of the three ponds when reared under the three or four experimental photoperiods. In addition, the fractions of egg types produced as first, second, and third clutches were determined independently in each treatment. RESULTS Populations of Diaptomus sanguineus from the three ponds have distinct photo- periodi responses (Fig. 2). In each case, copepods reared under short day lengths produceo significantly greater fractions of subitaneous clutches than those reared under longer phoioperiods (Ponds A and C, x 2 = 1 16.2 and 94.9 respectively, df = 2, P < .001; Bullhead Pond, x 2 = 21 1.1, df = 3, P < .001). Figure 2 illustrates both the results from the treatments described here, and those from a second experiment in which Bullhead Pond and Pond A copepods reared under winter conditions (8L: 16D, 4 1 C) produced only subitaneous eggs and those reared under summer conditions (16L:8D, 20 1C) made only diapausing eggs (Hairston and Olds, in prep.). DIAPAUSE IN A FRESHWATER COPEPOD 139 100 Bullhead Pond 10 11 12 13 14 PHOTOPERIOD (hr light) 15 16 FIGURE 2. The fractions of subitaneous (immediately hatching) clutches of eggs produced by female Diaptomus sanguineus reared at the photoperiods illustrated. Data are for all clutches regardless of order of production. For sequence of clutch production see Table I. Experiments at intermediate day lengths were run at 9 1C, whereas those at 8L:16D and 16L:8D were run at 4 1C and 20 1C respectively. The levels of response differed significantly among D. sanguineus drawn from different ponds. At day length 9:55 hours Bullhead Pond females made a greater fraction of subitaneous eggs than either Pond A or Pond C females (x 2 = 14.67 and 41.68 respectively, df =: 1, P < .001), and Pond A females made a greater fraction of subitaneous eggs than Pond C females (x 2 := 6.37, df = 1, P < .02). At longer pho- toperiods where subitaneous egg production was generally low. Bullhead Pond results differed significantly from Pond C at day length 13:05 hours (x 2 = 5.44, df : 1, P < .02), and from Pond A at day length 14:45 hours (x 2 := 3.91, df - 1, P < .05). Although others have found that diaptomid copepods tend to switch from making subitaneous eggs to diapausing eggs as they grow older (Roen, 1957; Champeau, 1970; Gehrs and Martin, 1974; Walton, 1985), and such a reproductive pattern is expected theoretically in highly variable environments (Hairston et ai, 1985; Hairston and Olds, in prep.), no clear trend of this sort is present in our results (Table I). Under a photoperiod of 9:55L both Pond A and Pond C females reduced the fraction of sub- itaneous eggs they produced in second compared to first clutches (x 2 == 6.61 and 7.89 respectively, df = 1 , P < .02), but in each case they returned to making more subitaneous eggs in their third clutches than in their second clutches. DISCUSSION Our hypothesis was that the distinct reproductive phenologies of Diaptomus san- guineus living in three Rhode Island ponds (Fig. 1 A) reflect unique adaptations to the seasonal events in those habitats. Specifically we proposed that copepods taken from different populations begin production of diapausing eggs at different times of year because they respond proximately to different photoperiods. In a broad sense the hypothesis is born out (Fig. 2), in that (1) the copepods make principally subitaneous eggs at short photoperiods and principally diapausing eggs at long photoperiods and (2) significant differences in this response pattern exist between populations. However, the change from subitaneous (nondiapausing) eggs to diapausing eggs occurs more gradually and over a much wider range of photoperiods than is consistent with the field data (cf.. Fig. 2 and Fig. IB). Furthermore, the specific photoperiods at which 140 N. G. HAIRSTON, JR. AND E. J. OLDS TABLE I Fractions of subitaneous (immediately hatching) clutches of eggs as opposed to diapausing clutches, produced by female Diaptomus sanguineus reared at four photoperiods, and the change in this fraction as the females aged /"i.e. 1st, 2nd, and 3rd clutches) Clutch Photoperiod sequence Pond Bullhead A C 9:55L-14:05D 1st 2nd 3rd 0.85 (72)' 0.82 (50) 0.90 (20) 0.62 (69) 0.42 (24) 0.50 (4) 0.53(53) 0.22 (32) 0.78 (9) 11:40L-12:20D 1st 2nd 3rd 0.38(131) 0.36(75) 0.50 (2) 13:05L-10:55D 1st 2nd 3rd 0.14(98) 0(47) 0(6) 0(55) 0(43) 0.21 (24) 0.03 (72) 0(41) 0.10(10) 14:45L-9:15D 1st 2nd 3rd 0.04 (48) 0(9) - (0) 0(70) 0(28) 0(12) 0.02(58) 0.03(32) 0(5) The values in parentheses give the total numbers of reproducing females in each treatment. the switches take place in the field populations differ markedly from those recorded in the laboratory. Female copepods collected from Bullhead Pond carry principally subitaneous eggs until mid-March when day length exceeds 1 1.5 hours. The switch in egg types then occurs rapidly and by the end of March, at day lengths of 12.5 hours, nearly all have switched to making diapausing eggs (Fig. 1). In contrast, Bullhead Pond copepods cultured in the laboratory make a small, but significant, fraction of diapausing eggs when exposed to only about 10 hours of light (comparable to the day length on 25 January), and yet continue to make a significant fraction of subitaneous eggs at a photoperiod greater than 1 3 hours of light (comparable to the day length on 10 April). Half of the females in the field have switched to carrying diapausing eggs at a day length of 12.2 hours (or 24 March), whereas in the laboratory this point is reached much earlier at exposure to about 1 1.2 hours of light (comparable to 27 February). For Ponds A and C, the differences between the populations in nature and their behavior in the laboratory are even more striking. One half of the female copepods in Pond A have switched to making diapausing eggs when day length has reached about 14.3 hours (14 May), but in the laboratory 50% diapause lies at 10.6 hours of light (com- parable to 12 March). In Pond C, one half of the females have switched to diapause at a day length of 1 3.9 hours (29 April), whereas in the laboratory 50% diapause occurs at less than 9.9 hours of day light (comparable to 25 January). Not only do laboratory-reared D. sanguineus exhibit photoperiod responses in- consistent in timing with the patterns of reproduction recorded in the field, but the order of the responses is also incongruous (cf. Fig. IB and Fig. 2). In the natural populations, Bullhead Pond females are the first to switch to producing diapausing eggs, followed by Pond C females, and finally by those in Pond A. In the laboratory, however, Pond C copepods switch to diapause at the shortest day lengths followed by Pond A and Bullhead Pond copepods. The fact that the differences in laboratory photoperiod responses between the three populations are statistically significant (Re- DIAPAUSE IN A FRESHWATER COPEPOD 141 suits) indicates that the dissimilarities are inherent. Since all the copepods were cultured under identical laboratory conditions, environmental sources of variation were pre- sumably removed, and the populations from the three ponds apparently differ genet- ically. It is clear that the natural populations of D. sangiiineus must use environmental cues other than critical photoperiod to set the timing of diapause. The behavior observed in the laboratory, although real, represents only a single component of a more complex response pattern. Without further experimentation it is difficult to say what factors might be involved in a complete characterization of the environmental cue for the onset of diapause. The photoperiodic response of many animals is modifiable to some extent by temperature (e.g.. Beck, 1980), including the marine calanoid copepod La- bidocera aestiva studied by Marcus (1982). As spring progresses into summer, D. sangiiineus experiences both an increase in day length and an increase in water tem- perature, and one might suppose that each population is adapted to respond to a unique combination of these two environmental variables. Two difficulties with such a suggestion are ( 1 ) that exposing Pond A and C animals to a temperature abnormally low for the photoperiod used should have enhanced the production of subitaneous over diapausing eggs, but instead had the opposite effect, and (2) that even the appro- priate temperature and photoperiod combination did not produce the correct response by Bullhead Pond copepods. A third type of environmental information available to the copepods at all three ponds is the direction and rate of change of photoperiod. Tauber and Tauber (1970) have shown for a lacewing (Neuroptera) that autumnal diapause can be induced at relatively long photoperiods if the insects are exposed to decreasing rather than constant day lengths. Sensitivity to such a change in day length, were it to exist in D. sangiiineus, could explain both the rapidity and the timing of the onset of diapause. Whatever the environmental cue or cues used by the copepods to time the switch to diapause, discovering their precise nature will permit more detailed investigation of the evolutionary bases of interpopulation differences in reproductive phenology. ACKNOWLEDGMENTS We thank C. A. Tauber, M. J. Tauber, and two referees for their comments on the manuscript; R. P. Clark and J. E. O'Brien for access to Bullhead Pond; and G. H. Wheatley for help at the University of Rhode Island, W. Alton Jones Research Campus. The research was generously supported by NSF Grant BSR-8307350. LITERATURE CITED BECK., S. 1980. Insect Photoperiodism. 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S. 1959. Free-living Copepoda: Calanoida. Pp. 738-8 1 5 in Freshwater Biology, W. T. Edmondson, ed. Wiley, New York. Reference: Biol. Bull. 171: 143-160. (August, 1986) EFFECTS OF ENRICHMENT ON REPRODUCTION IN THE OPPORTUNISTIC POLYCHAETE STREBLOSPIO BENEDICT! (WEBSTER): A MESOCOSM STUDY LISA A. LEVIN Department of Marine, Earth and Atmospheric Sciences, North Carolina State University. Raleigh, North Carolina 27695 ABSTRACT The influence of organic enrichment on growth and planktotrophic development of the spionid polychaete Streblospio benedicti Webster was examined in two mesocosm experiments conducted at the MERL facility. University of Rhode Island. Specimens of S. benedicti were collected and their reproductive traits monitored near the con- clusion of a two-year eutrophication experiment, and in the middle of a sludge addition experiment. Nutrient (N, P, and Si) enrichments at 8X and 32X the average aerial input into Narragansett Bay, Rhode Island, resulted in increases in body length, segment number, and length per segment, and a doubling of brood size in S. benedicti females. These increases were substantially higher during May (12C) than August (20C). Enrichment effects were stronger in the 8X than 32X nutrient treatment. In the sewage sludge experiment body size increased 20% over control values at the highest (8X) sludge treatment level (nitrogen loading equivalent to the 8X nutrient treatment) but no significant increase was noted at the 4X sludge level, which received half as much nitrogen as the 8X sludge treatment. Mean brood size increased by a factor of 4.6 over controls in the 8X sludge treatment and by a factor of 2.3 in the 4X sludge treatment. Within the range of adult body sizes observed, brood size enhancement occurred independent of increased length or segment number in both nutrient and sludge en- richment treatments. The ability to translate elevated food supply directly into increased reproductive output may underly opportunistic dynamics in macrobenthos. Brood size enhancement of the magnitude observed probably contributes to the high S. be- nedicti densities found in polluted or organically enriched settings. INTRODUCTION Increased concern over pollution of harbors and bays during the 1960's and 1970's spawned interest in community structure and succession of estuarine macrofauna (reviewed in Pearson and Rosenberg, 1978). Many estuarine studies revealed life history characteristics and population dynamics in polychaetes, crustaceans, and bivalves which are defined as opportunistic (e.g., Grassle and Grassle, 1974; McCall, 1975). Life history traits associated with opportunism in macrobenthos include small size, rapid colo- nization ability, short generation time, high reproductive rate (r), and high mortality rate (Grassle and Grassle, 1974). Brood protection is also a common feature among many opportunistic polychaetes, including Capitella spp., Polydora ligni, and Stre- blospio benedicti (Grassle and Grassle, 1974; Levin, 1984a), and among peracarid crustaceans (Barnes, 1980). However, not all opportunists brood young; the bivalve Mulinia lateralis has completely planktonic development (Chanley and Andrews, 1971). Received 5 February 1986; accepted 28 May 1986. 143 144 L. A. LEVIN Opportunistic species colonize and dominate benthic assemblages during or fol- lowing bottom disturbances, such as those caused by release of sewage or industrial wastes (Reish, 1971; Boesch, 1973), dredging and spoil disposal (Oliver, 1979), nutrient additions and subsequent eutrophication (Nixon et al, 1984), oil spills (Grassle and Grassle, 1974; Sanders et al., 1980), or severe storms and hurricanes (Boesch et al, 1976; Rhoads and Boyer, 1983). Biological disturbance of sediment, such as that caused by digging activities of crabs, bottom fish, rays, or whales may also lead to colonization and persistence of opportunistic taxa (Virnstein, 1977; Van Blaricom, 1982; Levin, 1984a, Oliver and Slattery, 1985). Pearson and Rosenberg (1978) make a distinction between disturbance and en- richment opportunists. They cite Capitella spp., Streblospio benedict i, Scolelepis fu- liginosa, and dorvilleids as examples of species which colonize disturbances specifically resulting from organic enrichment. Species in the genus Streblospio, common in North America and Europe, are often numerically dominant in polluted or enriched estuarine habitats. S. benedicti recruits are typically found in caged settlement containers which trap fine organic particulates. Densities of over 100,000 individuals/m 2 have been reported in settling containers and clearings exposed for 2 weeks (McCall, 1975; Virn- stein, 1977; Levin, 1984a). Enhanced recruitment may have resulted from either active habitat selection or passive entrainment (Hannan, 1984). Streblospio is not limited to enriched or disturbed habitats. S. benedicti has been reported from most of the shallow estuaries, marshes, and mudflats studied in the United States, with the exception of some in Washington and Hawaii (Levin, 1984b). This species is often a moderate component of the background community but pop- ulation size can grow exponentially in response to disturbance. Similar behavior is documented for Capitella spp. and Polydora ligni (Pearson and Rosenberg, 1978). The mechanisms underlying opportunistic responses are just now being investi- gated. High population growth rates are thought to derive from life history character- istics while high mortality rates have been attributed to competition from later stages (Grassle and Grassle, 1974; Pearson and Rosenberg, 1978; Gallagher et al., 1983; Gallagher, pers. comm.). The dynamics of Capitella sp. I fed Gerber's cereal, was studied in the laboratory by Chesney and Tenore (1985a, b). They observed high amplitude population fluctuations in the presence and absence of induced mortality (artificial predation) over 90 weeks. High mortality occurred and they concluded that Capitella sp. I was unlikely to achieve equilibrium dynamics on its own. Based on these observations Chesney (1985) suggests that inherent life history traits (e.g., re- productive lags and a tendency to overshoot carrying capacity) rather than competition are probably responsible for population declines of opportunists observed during succession in soft sediments. However, the meiofaunal community present in these experiments (Alongi, 1985) was not considered in the interpretation of Capitella sp. I dynamics and may have influenced resource availability. The ability to rapidly translate increases in food availability to increased repro- ductive output should lead to opportunistic population dynamics, particularly if off- spring remain to further utilize the enhanced food supply. Production of more gametes and offspring, independent or instead of concomitant somatic growth (which uses energy ai> :akes time), should also enhance rapid population growth in opportunists. Eckelbarger ! n press) suggests that opportunism among polychaetes is limited to species with brief game togenic cycles, which permit numerical response to specific conditions before those conditions disappear. Spionid and capitellid polychaetes can produce mature gametes only a few weeks after initiating gametogenesis (Eckelbarger, in press). This paper examines the reproductive responses by Streblospio benedicti to different forms of organic enrichment. S. benedicti was studied in two experiments conducted STREBLOSPIO RESPONSE TO ENRICHMENT 145 in mesocosms at the Marine Ecosystems Research Laboratory (MERL), University of Rhode Island. One was a eutrophication experiment involving nutrient additions, the other involved addition of secondary sewage sludge. In an earlier paper (Levin, 1984b) I suggested that the widespread success of S. benedict! may be attributable to the occurrence of both planktotrophic and lecithotrophic modes of development in this species. The research presented here considers only 5". benedict i with planktotrophic development and examines the plasticity of reproductive traits, such as egg and brood sizes. Streblospio benedicti background Streblospio benedicti Webster (Spionidae) inhabits the upper 2-3 cm of muddy sediments and constructs ephemeral tubes of fine-grain particles. This species is both a suspension feeder and surface-deposit feeder on plankton, organic aggregates, and sediments. Rod-shaped fecal pellets are deposited outside the tube (Levin, 1981). S. benedicti exhibits both planktotrophic and lecithotrophic modes of development in North America, but only planktotrophic development has been reported for S. benedicti in Narragansett Bay and in the MERL tanks (Levin, 1984b., J. P. Grassle, pers. comm.). Oogenesis takes place in paired ovaries in anterior segments (Eckelbarger, 1980) and fertilized ova enter paired dorsal brood pouches where development proceeds. Larvae are brooded to a 3-5 setiger stage, approximately 220 /um in length. Following release from brood pouches larvae usually feed in the plankton for 10-2 1 days before settlement (Levin, 1984b). Females can release hundreds of planktotrophic larvae per brood (Levin, 1984b) and may produce as many as 14 broods in a lifetime (Levin, DePatra, and Creed, in prep.). MATERIALS AND METHODS Streblospio benedicti was sampled from the MERL mesocosms, located at the University of Rhode Island on the lower West Passage of Narragansett Bay, RI. Sam- pling was conducted on 12 May and 17 August 1983, towards the conclusion of a 2-year eutrophication experiment and on 26 July 1984 during a 3-month sewage sludge enrichment experiment. The design of the MERL mesocosms is described in Pilson et al. (1979). System-wide results of the first year of the eutrophication exper- iment are presented in Nixon et al. (1984) and preliminary results of the sludge ex- periment are described in Oviatt (1984). Methodology critical to this investigation will be reviewed here. The MERL mesocosms were 5.5m high cylindrical tanks ( 1 .83 m diameter). The tanks received seawater from Narragansett Bay at a rate sufficient to completely replace the water every 27 days. The mesocosms were mixed four times a day with rotating plungers on a schedule designed to mimic tidal currents and to resuspend bottom sediments to similar levels as in the Bay. The walls of the tanks were brushed twice a week to prevent fouling. Each tank contained a tray 2.52 m 2 X 40 cm deep filled with sediments. These sediments were collected intact from Narragansett Bay (using a 0.25 m 2 box core) between 28 April and 8 May 1981 for the eutrophication experiment. Fresh sediments were collected from central Narragansett Bay in October 1983, prior to the start of the sludge experiment. On 7 June 1984, surface sediments (to 2 cm depth) were removed from all tanks to eliminate a bloom of the tunicate Mogula manhattensis and fresh sediments from the Bay were added. Narragansett Bay sedi- ments were muddy, containing roughly 83% silt-clay at the collection site (Hunt and Smith, 1983). The dominant taxa in these sediments at the start of the eutrophication 146 L. A. LEVIN experiment were the polychaetes Mediomastus ambiseta, Poly dor a ligni, Streblospio benedicti, and Chaetozone sp., and the bivalves Nucula annulata and Yoldia limatula (Grassier/., 1985). Eutroph icatic leriment The eutrophication experiment involved daily additions of inorganic nitrogen, phosphorus, and silicon (Molar ratio 12.80:1.00:0.91) at IX, 2X, 4X, 8X, 16X, and 32X the average areal input (of sewage and runoff) to Narragansett Bay (Table I). Each of six tanks received a different enrichment level and three tanks remained as unen- riched controls. Nutrient additions began on 1 June 198 1 and continued daily through 26 September 1983. Specimens of Streblospio benedicti were collected from 2 control tanks (Nos. 5 and 8), the 8X enrichment tank (No. 1), and the 32X enrichment tank (No. 7) on 12 May and 17 August 1983, approximately 2 and 2| years from the start of the experiment. The S. benedicti adults collected must have entered the tank as larvae from Narragansett Bay or were produced by adults established within the tank sediments. They could not have been individuals collected directly from the bay at the start of the experiment, since the lifespan of S. benedicti is <12 months (Levin, DePatra, and Creed, in prep.). Average temperature in the MERL mesocosm tanks was 12-13CinMay 1 98 3 and 20-2 1C in August 1983. Because this large temperature difference could have significant effects on reproduction, I consider the two sampling dates to represent two temperature treatments rather than replicate samplings. Streblospio benedicti was collected from the upper 4 cm of MERL tank sediments using 1" diameter cylindrical cores (5.067 cm 2 , n = 5 cores/tank on each sampling date). Sediments were sieved through a 500-yum screen and all S. benedicti were sorted live under a dissecting microscope. Stage of maturity and sex were noted for all spec- imens. Females brooding young were isolated in petri dishes, anesthetized in 5% MgCl 2 , and the following reproductive traits were measured: length (mm), number of setigers, position of the first gametogenic setiger, number of ova/ovary, ovum diameter (Mm), number of brood pouches, number of larvae/brood pouch, and number of larvae/ brood. TABLE I Experimental enrichments in the MERL eutrophication and sewage sludge experiments Eutrophication experiment 1 June 1981-26 September 1983 Daily additions (millimoles/m 2 ) Treatment level N P Si Ox - - 8x 23.04 1.798 1.643 32x 92.03 7.19 6.570 e sludge experiment 18 June 1984-21 September 1984 Daily additions (millimoles/m 2 ) Treatment level C N P Si Ox - - - 4x sludge 105 14.5 2.07 0.38 8x sludge 210 29.0 4.13 0.77 STREBLOSPIO RESPONSE TO ENRICHMENT 147 Sewage sludge experiment The sewage sludge experiment consisted of seven treatments. Nutrients (N, P, and Si) were added daily to 3 tanks at IX, 4X, and 8X enrichment levels, as in the eutro- phication experiment. Primary and secondary sewage sludge from the Cranston, Rhode Island, Water Pollution Control Facility was added daily at IX, 4X, and 8X levels to generate 3 treatments which received total nitrogen at loading rates equivalent to the nutrient treatments. Three unenriched tanks served as controls. The daily enrichments began on 12 June 1984 and were terminated on 22 September 1984. Streblospio benedicti was sampled in 2" diameter cores ( 1 7.57 cm 2 X 4 cm deep) from the 4X and 8X nutrient tanks, the 4X and 8X sludge tanks, and 3 control tanks on 26 July 1984. Five to eleven cores were collected from each tank though time did not permit pro- cessing of all cores. Sediments were sieved and S. benedicti was sorted and measured as described for the eutrophication experiment. J. P. Grassle provided density data for S. benedicti collected the preceding day using 1" diameter cores (5.067 cm 2 X 4 cm deep) and a 300-jum screen (Grassle and Grassle, 1984; Grassle et al, 1985). Few reproductive S. benedicti were found in the 1984 nutrient enrichment tanks, so only the sludge enrichment and control treatments are considered here. Statistical analyses Statistical tests were carried out with SAS software (Ray, 1 982). All analyses except for regressions of brood size on adult length and setiger number were performed on untransformed data. One-way ANOVAs were used to analyze effects of enrichment level on S. benedicti densities in the eutrophication and sludge experiments. A two- way ANOVA and a SNK a posteriori test were performed to evaluate effects of en- richment level and month on female length, segment number, the first gametogenic setiger, ovum diameter, and numbers of ova/ovary, paired brood pouches, larvae/ brood pouch, and larvae/brood in the eutrophication experiment. Effects of sludge level on these same parameters were analyzed using a one-way ANOVA and SNK test. A comparison was made of all 9 enrichment treatments across experiments, (May 1983, August 1983, and July 1984) using a one-way ANOVA and a SNK test for each reproductive character. Data from the eutrophication and sludge experiments were combined to determine Pearson product-moment correlations between reproductive traits. Brood size data from the eutrophication and sludge experiments were log trans- formed to satisfy least-squares assumptions prior to performing regressions of brood size on adult length and on setiger number. Homogeneity of variances was examined using F tests to compare error mean squares from regressions for individual treatments. No departures from homogeneity were observed within each experiment. Regressions of log brood size on both adult length and on setiger number were performed across enrichment levels (separately in the eutrophication and sludge experiments), allowing for different intercepts and slopes. RESULTS Eutrophication experiment Streblospio benedicti densities in each enrichment treatment are shown in Table II for samples collected in May and August 1983. In May S. benedicti exhibited no density difference among enrichment levels (F 3 16 = 1.92, P > 0.05). Mean densities in all treatments ranged from 2.8 to 9.6 individuals/5.07 cm 2 core (5,600 to 19,200 148 L. A. LEVIN TABLE II Density of Streblospio benedicti in the MERL eutrophicatwn experiment Food level x Do (#/core) S.D. n (# of cores) Core size (cm 2 ) x Density (#/m 2 ) % Females brooding 12 May 1983 Ox (Tank 5) 9.60 6.20 5 5.07 19,200 80 Ox (Tank 8) 2.80 1.48 5 5.07 5,600 55 8x(Tank 1) 4.00 4.38 5 5.07 8,000 75 32x (Tank 7) 4.80 4.70 5 5.07 9,600 58 17 August 1985 Ox (Tank 5) 4.60 2.94 5 5.07 9,200 50 Ox (Tank 8) 0.00 0.00 4 5.07 0,000 8x(Tank 1) 25.60 16.33 5 5.07 51,200 50 32x (Tank 7) 31.00 11.64 4 5.07 62,000 58 (Mesh size = 500 ^. One-way ANOVA on density. May:F 316 = 1.92, P> 0.05. August: F 3 . 14 = 7.8 \,P< 0.005. individuals/m 2 ). Three months later, in August, the 8X and 32X enrichment tanks exhibited mean densities equivalent to 25.6 and 31.0 individuals/core respectively (51,200 and 62,000 individuals/m 2 ). These values were significantly higher than control values in August, but not different from each other (F 3 , 14 == 7.81, P < 0.005). On both sampling dates at least 50% of the females collected in each treatment were brooding larvae (Table II). The two control (OX) tanks, sampled in May, exhibited between-tank differences in number of ova/ovary (x == 6.2 in tank 8 vs. x = 4.2 in tank 5, one-way ANOVA, P = 0.007). However, there was no significant difference in brood size. May reproductive data from the two control tanks were pooled in analyses of enrichment level effects. Mean values of female reproductive traits are given for S. benedicti at OX, 8X, and 32X enrichments in May and at 8X and 32X enrichments in August in Table III. Unfortunately, only two reproductive females were found in the control (OX) tanks in August, so analysis of food level effects for August does not include the OX treatment. During May, female body length was significantly greater in the 8X and 32X enrichment tanks than in the controls (P = 0.0087), but in both May and August, body length did not differ between the 8X and 32 X treatments. The number of setigers was significantly greater in 8X than control treatments during May (P = 0.0304), but no difference in setiger number was observed between the 32X treatment and controls in May or between 8X and 32 X treatments in both May and August. Between-month comparisons of body size, for 8X and 32X treatments, suggest that in August there was a decrease in body length (P = 0.0090) but no accompanying change in segment number (Table III, Fig. 1 ). Length per segment was greater in May than August in enrichec .anks. Females sampled in August may have been younger, summer recruits, while the j ?,er individuals sampled in May probably overwintered in the tanks. As one spends to treatments other traits may be constrained to follow, thus correlations among reproductive characters must be considered in interpretation of treatment eftec Measures of body size (length and setiger number) were positively correlated with numbers of ova per ovary (r = .55 for length, r = .44 for setiger number), total number of paired brood pouches (r = .73, r = .82), number of larvae per brood pouch (r ; = .52, r = .36), and number of larvae per brood (r = .69, r = .61) STREBLOSPIO RESPONSE TO ENRICHMENT TABLE III Reproductive traits of Streblospio benedicti in the A1ERL eutrophication experiment 149 12 May 1983 17 August 1985 2-way ANOVA Ox 8x 32x 8x 32x x(S.D.) x(S.D.) x(S.D.) x(S.D.) x(S.D.) Food Month Adult length (mm) No. of setigers First gametogenic setiger Ovum diameter (urn) 5.2(1.4) n = 36 7.2(1.9) 6.1(1.6) n= 11 n= 18 5.7(1.2) n= 17 4.8(1.0) n= 19 P=.0087 P=.0090 P = .0304 NS NS NS NS NS b 42.6 (6.0) n = 35 a 49.4(6.1) 46.2(5.8) n= 12 n= 18 45.4(5.8) n= 17 41.6(4.4) n= 19 a 10.6(1.1) n= 16 10.6 (0.8) n= 15 b 10.1 (0.6) n = 36 b 9.8(0.8) 10.0(0.6) n= 13 n= 18 56.1 (23.2) n = 38 60.0(21.7) 61.3(27.3) n= 13 n= 19 50.8(18.3) n= 12 56.7(19.5) n= 15 No. of ova/ovary 5.8(1.9) 8.1(2.8) 5.9(1.9) 6.1(1.9) 4.1(1.0) P=.0002 P=.QQ\\ n = 37 n = 9 n=15 n = 1 1 n = 1 3 a No. of paired brood 9.0(2.8) 14.0(3.6) 11.8(2.6) 9.2(2.7) 9.5(2.2) NS P=.0228* pouches n = 37 n=13 n=17 n=17 n=17 No. of larvae/brood 6.8(2.3) 10.4(3.4) 7.4(1.7) 7.4(1.6) 5.0(1.4) P=.0011 />=.0021 pouch n=18 n=ll n = 14 n = 10 n = 2 No. of larvae/brood 103.9(54.7) 278.2(143.3) 184.3(90.1) 131.6(45.6) 67.6(41.7) />=.0001 P = .0002 n = n= 13 n= 15 n= 14 n = 21 * Significant food-month interaction P= .0177. (Lines show differences among means analyzed for each month separately.) NS = not significant. (Table IV). These correlations suggest that changes in body size may also lead to changes in fecundity. Numbers of ova and larvae were also highly correlated with one another (r = .6 1 ), thus we might expect to see them respond in identical fashion to food and temperature (month) treatments. Body size was not correlated with egg size or with position of the first gametogenic setiger (Table IV). Both enrichment level and month (temperature) had significant effects on ovum number (P = 0.0002 for food, P = 0.001 1 for month), numbers of larvae per brood pouch (P = 0.001 1, P = 0.0021) and brood size (P = 0.0001, P = 0.0002) (Table III). Ovum size and position of the 1st gametogenic setiger were unaffected (Table III). During May the mean number of larvae produced per brood was 167% higher than 150 L. A. LEVIN 90-, 80- E 40- 40 42 44 46 48 50 52 NUMBER OF SETIGERS 56 58 FIGURE 1. Least-square regression of Streblospio benedicli segment number on length in the MERL eutrophication experiment. May 1983; August 1983. May OX (control): y = -3. 11 +0.196x P<.0001 8X :y = -6.59 + 0.277x /><.0001 32X :y = -5.65+0.255x /><.0001 August 8X :y= 2.65 + 0.183x /><.0001 32X :y= 2.99 + 0.187x f<.0001 Slopes for May 8X and 32X treatments differ significantly from the others. the controls (x : 104) in the 8X enrichment (x = 278) and 77% higher than the controls in ihe 32X enrichment (x = 184). However, only the 8X and OX treatments differed sigi icantly from one another (P = 0.0001). Seven females produced broods > 300 larvae (the largest was 548); all were large individuals from enriched treatments. Brood size ;as highly correlated with adult length (r = .69, P < 0.0001) and segment number (r : .61, P < 0.0001). Scatter plots of May brood sizes as a function of length and setiger number are given for each enrichment level in Figures 2a and b. Although correlation does not necessarily indicate a causal relationship, the well known STREBLOSPIO RESPONSE TO ENRICHMENT 151 TABLE IV Correlation coefficients of Streblospio benedicti reproductive traits in the MERL eutrophication and sludge experiments combined First No. of No. of Adult No. of gametogenic Ovum No. of paired brood larvae/brood No. of length setigers setiger diameter ova/ovary pouches pouch larvae/brood Adult length 1.0 .86 NS NS .55 .73 .52 .69 No. of setigers 1.0 NS NS .44 .82 .36 .61 First gametogenic setiger 1.0 NS -.20 NS NS -.21 Ovum diameter 1.0 .21 NS NS NS No. of ova/ovary 1.0 .40 .54 .61 No. of paired brood pouches 1.0 .50 .70 No. of larvae/brood pouch 1.0 .71 No. of larvae/brood 1.0 All values are significant at P < .05. NS = not significant. association between body size and fecundity raises the possibility that the increases in brood size observed in May enrichment treatments (8X and 32 X) resulted solely from increases in body size. To examine this possibility, log brood size was regressed on both adult length and on setiger number, across enrichment levels. Both regressions yielded r 2 values > 0.995. Tests for differences among slopes across enrichment levels were significant (P < 0.0001 ) for regressions of log brood size on setiger number (F 3 46 = 24.5) and length (F 3i47 = 23.5). However, fitted lines for the three enrichment levels did not intersect within the range of observed setiger numbers (8X values > 32X > OX). The OX and 32X treatment lines crossed only at the very largest adult lengths observed. Predicted brood sizes for the 8X treatment were greater than those for the OX and 32X treatments within the entire range of body sizes observed. Thus, though the regression lines for each enrichment level were not parallel, their intersection took place at biologically meaningless (unrealistically large) body and brood sizes. A between-month comparison shows significantly (P == 0.0002) smaller broods were produced in August (Table III, Fig. 3). Brood sizes were less than half those observed in May; mean brood size was 132 for the 8X treatment and 68 for the 32X treatment. Sewage sludge experiment The mean density of S. benedicti was considerably lower in the control (OX en- richment) tanks [x = 4.63 individuals/ 17 cm 2 core (2,635/m 2 )] than in the 4X_sludge treatment [x = 60 individuals/core (34,140/m 2 )] or the 8X sludge treatment [x = 22 individuals/core (12,51 5/m 2 )] (F 4 , 10 =: 17.01, P< 0.001) on 26 July 1984, one month into the sewage experiment (Table V). Density estimates based on two 1" cores per tank provided by J. P. Grassle were 4-5 times higher than those from the 2" diameter cores (due to use of a finer mesh for processing sediment), but yielded consistent differences among treatments (Table V). One control tank (No. 3) produced all except one of the control females brooding young. Fifty-five percent of females were brooding in that control tank, 88% were brooding in the 4X sludge treatment, and 85% were brooding in the 8X sludge treatment (Table V). 152 L. A. LEVIN W j 600- O o 500 J O -i go 0> _ 400- z 300- 200- 100- O o o o o A A O 6 7 LENGTH (mm) 10 ? 700 n i LJ | 600- Q 5 500 ~ 40 " 300- 2OO- k B O O O A A OA 34 36 38 40 42 44 46 48 50 52 NUMBER Of SETIGERS 54 56 56 60 62 64 FIGURE 2. Streblospio benedicti brood size as a function of: A) length and B) setiger number in the MERL eutrophication experiment, 12 May 1983. = controls (Tanks 5 and 8); O = 8X enrichment (Tank 1); A = 32X enrichment (Tank 7). Evaluation of enrichment level effects on female traits indicate that female length (P = 0.0319) and number of setigers (P = 0.0244) were significantly greater in the 8X sludge treatment than in the 4X and OX treatments, which did not differ from one another (Table VI). Females in the 8X sludge treatment also produced more ova/ ovary (P = 0.0002), more larvae per brood pouch (P = 0.0005), and more larvae per brood (P = 0.0001) than the other two treatments, which did not differ significantly from one another (Table VI). Mean brood size in the 8X sludge treatment (x = 462) was almost twice that of the 4X sludge tank (x = 235) and over 4 times that of the control^ : 100). The only trait for which the 4X sludge treatment differed from the control wa in the total number of paired brood pouches (P - 0.0004). As in the eutrophication experiment, position of the first gametogenic segment and ovum size were not affected by sludge level. A scatter plot of brood size as a function of length (Fig. 4a) and segment number (Fig. 4b) for each sludge enrichment level reveals an exponential increase in brood size with body size. Five individuals in the 8X sludge treatment produced over 500 STREBLOSPIO RESPONSE TO ENRICHMENT 153 700-, y X. 600-1 to 8 9 o 5OO- t 40O- 300- 200- 100- O 34 36 38 40 42 44 46 48 50 NUMBER OF SETIGERS 52 54 56 58 60 62 64 FIGURE 3. Streblospio benedict i brood size as a function of setiger number in the enriched treatments of the MERL eutrophication experiment. 12 May 1983: O = 8X enrichment, A = 32X enrichment. 17 August 1983: = 8X enrichment, A = 32X enrichment. The numbers indicate identical data points. larvae/brood (Fig. 4). The maximum brood size observed, 1058, far surpassed any values reported for S. benedicti in a laboratory or field situation. Regressions of log brood size on adult length and on setiger number, across sludge enrichment levels, yielded r 2 values > 0.994. Tests for differences among slopes across sludge levels were significant for regressions of log brood size on setiger number (F 3 35 = 14.59, P < 0.0001) and on length (F 3 , 36 = 20.50, P < 0.0001). However, fitted lines for the three sludge levels did not intersect within the range of observed adult segment numbers or body lengths. For all sizes of worms capable of reproducing, predicted brood sizes were greater in the 8X sludge treatment than in the 4X sludge treatment, and both were greater than those in the OX sludge treatment. TABLE V Density of Streblospio benedicti in the MERL sewage sludge experiment (26 July 1984) Food level x Density (#/core) S.D. n (# of cores) x Density (#/m 2 ) % Females brooding Ox (Tank 3) 5.64 (4.0) 2.90 11 (2) 3,209 (7,905) 55 Ox (Tank 5) 3.00 (4.0) 1.41 3 (2) 1,707 (7,905) No mature females Ox (Tank 8) 1.50 (4.5) 1.50 2 (2) 854 (8,893) 100 (one mature female) 4x (Tank 6) > 60.00 (84.0) 1 (2) > 34, 140 (165,019) 88 8x (Tank 2) 22.00 (25.0) 10.42 4 (2) 12,518 (49,407) 85 ( ) indicates data from two cores/tank collected on 25 July 1984 by J. P. Grassle. Screen Size = 300 ; Core Size = 5.07 cm 2 . (Screen Size = 500 ^m. Core Size = 17.57 cm 2 ); One-way ANOVA on density: F 4 , IO = 17.01, P< 0.001. 54 L. A. LEVIN TABLE VI Reproductive traits of S. benedicti in the MERL sludge experiment 26 July 1984 Sludge enrichment level One-way ANOVA Ox 4x 8x x (S.D.) x(S.D.) x(S.D.) P Adult length (mm) No. of setigers First gametogenic setiger Ovum diameter (urn) No. of ova/ovary No. of paired brood pouches No. of larvae/brood pouch No. of larvae/brood 5.3(1.4) n= 11 5.8(1.0) n= 19 6.8 (2.0) n= 14 F 2 .4i = 3.75 .0319 F 2 , 40 = 4.08 .0244 F 2 ,4o = 0.39 NS F 2 , 39 = 0.2 1 NS F 2 , 37 = 7.14 .0002 F 2 , 42 = 9.44 .0004 F 2 , 32 = 9.86 .0005 F 2 ,4o=12.78 .0001 47.9(6.7) n= 14 41.4(5.5) n= 11 45.4(4.8) n= 18 9.5 (0.9) n= 13 9.3 (0.7) n= 12 9.6 (0.5) n = 18 60.9 (25.0) n= 11 55.0(23.6) n= 17 58.6 (23.8) n= 14 6.2(1.3) n= 11 5.4(1.7) n= 16 8.4 (3.0) n= 13 9.0 (2.6) n= 12 12.4(3.3) n= 19 14.3 (3.3) n= 14 7.1 (1.7) n= 12 9.0(1.4) n= 10 11.7(3.7) n= 13 100.0(54.0) n= 11 235.1 (107.1) n= 19 462.8 (296.4) n= 13 Lines show significant differences among means. NS = not significant. Comparison of the eutrophication and sewage sludge experiments A comparison was made of all nine treatments (3 nutrient levels in May 1983, 2 nutrient levels in August 1983, and 3 sludge levels in July 1984) for each reproductive character (Table VII). May 1983 and July 1984 unenriched controls showed no sig- nificant difference in any trait except position of the first gametogenic setiger (Table VII). S. benedicti from the 8X nutrient addition in May 1983, were most similar to the < sludge addition in July 1984. These treatments produced adults which were significantly longer, had more segments, more brood pouches, and greater brood size than the May 1983 and July 1984 controls. The 4X sludge treatment and May 32X nutrient addition generally exhibited the next highest values for these traits, but did not always fer significantly from control values. Females produced significantly larger broods in the 8X sludge treatment than in any other treatment. DISCUSSION The sacrifice of replication for an increased number of treatments seems to be a common difficulty in the design of mesocosm and other large-scale experiments (see STREBLOSPIO RESPONSE TO ENRICHMENT 155 IIOO- _ I000 ~ A A * 900- N * OT 5 800- o O -J g s 7oo- 2 | 600- A A A A 2 500- A A 400- 300- o 9 o o 200- A fi 100- 0- A A Q ' s 3 4 5 4 8 9 10 ll LENGTH (mm) I IOO-, 1000- B A 900- 8OO- A 1 70 " A A LJ j 600- * 1 8 3 500 ~ & m T J 40 " 1 o oo o t. 300- 200- & ft 100- 0- 32 J4 36 38 40 42 44 46 48 50 5Z 54 56 58 60 62 64 NUMBER OF SETIOERS FIGURE 4. Streblospio benedicti brood size as a function of: A) length and B) setiger number in the MERL sewage sludge experiment. 26 July 1984. = OX control (Tank 3); O = 4X sludge addition (Tank 6); A = 8X sludge addition (Tank 2). Hurlbert, 1984; Smith et ai, 1984 for discussion). The lack of tank replication of enrichment treatments in both the MERL nutrient and sewage experiments must temper the interpretation of the data. The fact that the control (OX) tanks behaved 156 L. A. LEVIN TABLE VII Comparison of Streblospio benedicti reproductive traits among all MERL treatments 1.= Ox Nutrients May '83 2. = 8x Nutrients May '83 3. = 32x Nutrients May '83 4. = 8x Nutrients August '83 5. = 32x Nutrients August '83 6. = Ox Sludge July '84 7. = 4x Sludge July '84 8. = 8x Sludge July '84 One-way ANOVA Student-Neumann-Keuls Test Adult length No. of setigers First gametogenic setiger Ovum diameter (^m) No. of ova/ovary No. of paired brood pouches No. of larvae/brood pouch No. of larvae/brood F 7 .,37= 4.97, P=. 0001 28374615 F 7 .,36= 4.04, P=. 0005 28374156 F 7m = 6.39, P=. 0001 54132786 F 713 i = 0.32, P=NS 36285174 F 7 .,, 7 = 6.19, P=. 0001 82463175 F 7 .,, g = 10.10, P=. 0001 82735416 F 782 = 7.12, P=. 0001 82734615 F 7 . 126 = 17.61. P=. 0001 82734165 NS = not significant. similarly lends little confidence that single tanks are an adequate reflection of all possible outcomes of particular levels of enrichment. Each treatment exhibited its own benthic dynamics throughout the experiments. Some tanks evolved oxygen stress in response to nutrient loadings or high densities of competitors or predators while others did not (J. P. Grassle, pers. comm.; Frithsen et al, 1985). Within a treatment, ma- crofaunal community composition varied greatly from year to year (Frithsen et al., 1985). However, several benthic species exhibited essentially linear responses to level of nutrient additions after the first summer of the eutrophication experiment (Grassle and Grassle, 1984; Grassle et al., 1985). To understand responses of S. benedicti it is necessary to consider both biotic and abiotic influences on reproduction. During the eutrophication experiment in both May and August 1983, the length, segment number, egg production, and brood size of S. benedicti in the 32X enrichment treatment fell below those of the 8X enrichment tank (Table III). Despite a four-fold increase in nutrient loading, it appears that 5". bened 1 id not respond in the 32X treatment. Intraspecific competition has been shown iv. luence somatic growth and reproductive allocation in benthic invertebrates (Peterson, 1 : Zajac, 1985). However, there was no detectable difference between S. benedicti dc, ssties in the 8X and 32X treatments throughout the spring and summer of 1983 (Tabli . P. Grassle, pers. comm.; J. Frithsen, pers. comm.). The density of Polydora ligni, a potential competitor, was extremely high in the 32X treatment during that period. P. ligni abundance was 5.7 times higher than S. benedicti in April (x = 56,246 vs. 9,868/rn 2 ), 10.6 times higher in May (x = 1 14,860 vs. 10,854/m 2 ), 8.7 STREBLOSPIO RESPONSE TO ENRICHMENT 157 times higher in June (x = 1 86,698 vs. 2 1 ,5 1 2/m 2 ), and 4.2X higher in July (x = 262,482 vs. 62,167/m 2 ). P. ligni densities were consistently lower than S. benedicti in the 8X enrichment tank during those months (J. P. Grassle, pers. comm.; J. Frithsen, pers. comm.). The 32X enrichment tank experienced anoxic conditions between 29 July and 3 August 1983 (Frithsen et al, 1985). P. ligni densities in this tank declined. Following this event, S. benedicti increased to 346,359 individuals/m 2 by September, the highest density observed for this species during the 3-year experiment. Mulinia lateralis was consistently abundant in this treatment throughout the summer and fall of 1983 (Frithsen, pers. comm.). P. ligni and 5. benedicti were among the species which demonstrated the greatest numerical response to nutrient enrichment in 1983 and sewage enrichment in 1984. Both species filter organic particles from the water column and feed on surface deposits. Frithsen and Doering (submitted) showed that these two species can increase net sedimentation when they reach high abundances. P. ligni is larger than S. benedicti, has longer palps (pers. obs.), is more aggressive (Levin, 1981; Whitlatch, pers. comm.), and could easily outcompete S. benedicti for food at the densities observed in the 32 X enrichment tank. These interactions may have contributed to the small size and lower fecundity exhibited by S. benedicti in the 32 X treatment relative to the 8X enrichment. In addition, oxygen stress might have produced decreases in reproductive output. A similar anoxic event occurred in mid September, 1984, when the 8X sludge treatment went anoxic and all benthos died. Just prior to the anoxia S. benedicti had been the numerical dominant. Unfortunately, no reproductive data were collected at that time. Temperature may influence the magnitude of the reproductive response given by 6". benedicti to organic enrichment. At low temperatures in May 1983 ( 12C), increased food supply led to increases in size of reproducing females (Fig. 1) and to increased brood size independent of body size (Figs. 2a, b). In August, when temperatures had risen to 20C, enrichment effects were reflected in level of reproductive activity. Few or no reproductive females were collected in control tanks. In the 8X and 32X en- richments, reproductive activity was high but body size and brood size were comparable to May control levels (Table II). Younger ages of females, due to recruitment during the summer, and response to increased metabolic demands at higher temperature or lower oxygen levels, may have been responsible. In addition, six- to seven-fold increases in S. benedicti density (Table II) may have intensified intraspecific competition and resulted in lower brood sizes than observed in May. In laboratory manipulations of temperature and food regimes. Levin and Creed (in press) found that body size and brood size of North Carolina 5". benedicti with planktotrophic development increased in response to cooler temperatures. In that study food level had no effect on quantitative reproductive characters but lower food levels decreased the proportion of females which reproduced. The experiment did not examine competition for food. S. benedicti appears to have the ability to translate enhanced food supplies directly into increased reproductive output. Utilization of heterosynthetic yolk sources during vitellogenesis, by sequestering materials from the circulatory system (Eckelbarger, 1980), may be one means by which S. benedicti can transfer food rapidly and directly into eggs and offspring. This capability may be essential to opportunists which depend on ephemeral resources and which, through utilization of these resources, may even contribute to their own demise. S. benedicti with planktotrophic development mature rapidly. They disperse their young during a 10-2 1 day larval planktonic phase (Levin, 1984b) and thus are not dependent on temporal persistence of a specific habitat. Food quality, particularly nitrogen content, may regulate polychaete growth and reproduction (Tenore, 1977; Tenore and Chesney, 1985). Nutrient addition treatments in the eutrophication experiment and sewage treatments in the sludge experiment 158 L. A. LEVIN exhibited elevated chlorophyll levels, and enhanced phytoplankton concentrations (Nixon et al, 1984; J. Maugham, pers. comm.). The combination of sewage sludge and a rich phytoplankton supply clearly represents a high quality food source for S. benedicti. The 2-5-fold increases in brood size exhibited in the 8X and 4X sludge tanks (relative to controls) far surpass the increases observed in the nutrient enrichment treatments the previous year. In studies of the benthic dynamics in the nutrient and sludge treatments during summer 1984, Maughan (in prep.) found that S. benedicti densities increased in the sludge treatments but not in the nutrient treatments. Phy- toplankton production was also lower in the nutrient treatments during summer 1984 and Maughan (in prep.) suggests that filtering activities of the abundant amphipods, molluscs, and polychaetes in these tanks were responsible. By August 1984 5". benedicti had also attained very high densities in the IX sewage treatment (J. Maugham, in prep.). Percent organic carbon in the top cm of sediment of the 1 X, 4X, and 8X sludge treatments during August (3.58, 2.52, and 2.92, respectively) was higher than in control tanks (2.16-2.26) or the IX, 4X, and 8X nutrient treatments (2.05, 2.19, and 2.58, respectively). These data suggest that at equivalent loadings, the sewage yielded more food for deposit feeders than the nutrient additions. Other constituents of the sewage sludge, which were not present in the nutrient addition tanks, may have stimulated recruitment, growth, or reproduction in S. benedicti. Streblospio benedicti response to organic enrichment in the form of rapid, large increases in brood size is not surprising, and may demonstrate one reproductive tactic underlying opportunistic population dynamics. The calanoid copepods Acartia tonsa and A. hudsonica responded to nutrient enrichments with increases in daily egg pro- duction, maximum length and dry weight (Sullivan and Ritacco, 1985). However, these increases were often not reflected in zooplankton abundance while increased brood size in 5". benedicti was accompanied by elevated benthic densities. The poly- chaete response to food may also explain why we often see strong seasonal cycles in S. benedicti populations which actively reproduce all year (e.g., Levin, 1984a). Increases in brood size during periods of increased food availability or cooling temperatures (Levin and Creed, in press) may cause dramatic recruitment peaks. It is not known whether the increased brood size exhibited by the small individuals in the sewage sludge tanks were accompanied by a decrease in reproductive output later in life. Release of larvae earlier rather than later in life is certainly adaptive for an opportunistic species. More work is needed to determine exactly what triggers population explosions in opportunistic species and how these explosions are achieved. In the case of enrich- ment opportunists such as S. benedicti, organic particulates are rapidly converted into both somatic tissue and offspring and order of magnitude increases in larval production may result. ACKNOWLEDGMENTS I wish to thank the University of Rhode Island and those responsible for the MERL facility, especially C. Oviatt, for providing access to their experiments, sup- porting data, and laboratory facilities. J. Maughan, J. P. Grassle, J. Frithsen, D. Rud- nick, J. Hughes, R. Zajac, C. Gelfman, S. Mills, and S. Brown-Leger provided invaluable assistance with the sampling and sorting of Streblospio. J. Maugham, J. Frithsen, and J. P. Grassle provided data and information on various aspects of the MERL exper- iments and critiqued earlier drafts of this manuscript. Special thanks are extended to J. P. and J. F. Grassle for introducing me to the MERL facility and for their enthusiastic support of my endeavors. C. Brownie, E. L. 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MCFADDEN* Department of Biology, Yale University, New Haven, Connecticut 06511 ABSTRACT Size refugia from competition, whereby one organism may grow too large to sub- sequently be overgrown by a superior spatial competitor, have rarely been documented in marine benthic communities. Size-symmetrical and size-asymmetrical competitive interactions were established between colonies of two hermit crab-associated hydroids, Hydractinia echinata and Podocoryne carnea, to assess the outcome of competition for space between the two species and its possible size-dependence. In size-symmetrical interspecific contests, P. carnea overgrew and killed H. echinata in 100% of 74 observed encounters. In size-asymmetrical contests in which H. echinata was always the larger colony, P. carnea was able to overgrow H. echinata in only 55% of 76 contests. H. echinata reaches a size refuge from overgrowth by P. carnea, but this "safe" size depends on the position occupied by a colony of H. echinata on a substrate with respect to physical or biological barriers to growth. The outcome of intraspecific competition for space between P. carnea colonies depends on the relative growth rates of the competitors. In 23 intraspecific contests, the P. carnea colony with the highest rate of stolonal growth was always competitively dominant, and also overgrew H. echinata most rapidly in the size-symmetrical inter- specific encounters. The ability of P. carnea to overgrow H. echinata in size-asym- metrical contests, however, did not depend on the growth rate of the P. carnea colony. Data on the distribution and abundance of these two species suggest that P. carnea recruits to hermit crab shells at a low frequency and is thus a rare member of the hermit crab epifaunal community. The observed differences in interspecific competitive ability may reflect asymmetry in the frequencies with which these species encounter one another. The probability that a colony of H. echinata will encounter P. carnea is low, hence there will be little selection for interspecific competitive ability in H. echin- ata. The probability that a colony of P. carnea will encounter the common H. echinata is high; P. carnea, therefore, should maintain a mechanism for recognizing and ov- ergrowing this important spatial competitor. INTRODUCTION The outcome of interactions between organisms does not remain constant throughout their lives. Encounters between competitors or between predator and prey may be sensitive to the relative sizes of the interacting individuals, and the effect one species has on another consequently may change throughout the ontogeny of those species (Buss, 1980; Werner and Gilliam, 1984). The dependence of many predator- prey interactions on prey size has been well documented in marine benthic commu- nities: prey organisms which by chance escape predation when small may eventually Received 4 February 1986; accepted 6 May 1986. * Present address: Department of Zoology NJ-15, University of Washington, Seattle, Washington 98195. 161 162 C. S. MCFADDEN grow too large to be consumed by a given predator (Ebling et ai, 1964; Paine, 1965, 1976; Dayton, 1971; Birkeland, 1974; Connell, 1975). The ability of some individuals in a population t :ch a size refuge from predation may contribute significantly to the maintenance : community diversity and structure (Connell, 1975; Paine, 1976, 1977). Few studies have examined how the outcome of competitive interactions between species may change as the relative sizes of the competing individuals vary. In several examples with colonial marine invertebrates, the direction of interspecific overgrowth has been shown to depend on the size (thickness) of the competing colonies: species A usually overgrows species B when A is larger, and vice versa (Day, 1977; Buss, 1980; Russ, 1982). Size refugia, where an individual of species B eventually reaches a size at which an individual of species A will be unable to overgrow it, may occur when the outcome of competition between two species is size-dependent (Buss, 1980). Size refugia from competition in benthic marine communities have been demonstrated by Buss (1980), in a system involving two bryozoans and a coralline alga in Panama, and by Sebens (1982), in a study of competition between the soft coral, Alcyonium siderium, and the compound tunicate, Aplidiwn pallidum, in the Gulf of Maine. In this study, I provide evidence for another example of an inferior spatial competitor, the athecate hydroid Hydractinia echinata (Hemming), reaching a size at which it can no longer be overgrown by an otherwise competitive dominant, the closely related hydroid, Podocoryne carnea (Sars). In Long Island Sound and other Atlantic soft-bottom coastal areas, pagurid hermit crabs are extremely abundant, and the gastropod shells they occupy represent a source of hard substratum which supports a unique encrusting fauna (Karlson and Cariolou, 1982; Karlson and Shenk, 1983). Hydractinia echinata is one of the most common epifaunal species found on pagurid shells in Long Island Sound; Podocoryne carnea is present at much lower frequencies in this community. H. echinata and P. carnea display aggression towards one another, and in a previous examination of interspecific competitive ability P. carnea was shown to overgrow H. echinata consistently (Gallien and Govaere, 1974). However, all interactions examined were grossly size-asymmet- rical, with a small explant of H. echinata placed in contact with a large P. carnea colony. This particular combination of colony sizes is only one of many conditions under which colonies may contact one another in natural encounters. The outcome of interspecific competition for space between H. echinata and P. carnea is examined further here, in both size-symmetrical and Hydractinia-biased size-asymmetrical in- teractions, to determine if P. carnea remains the superior spatial competitor across a range of size-specific encounters. Natural history Although the zooid morphology differs little between the genera (Goette, 1916; Mills, 1976), P. carnea and H. echinata display different patterns of basal tissue growth across a substratum. Growth of a colony of//, echinata is regulated by two interacting processes elongation of stolons and expansion of ectodermal mat tissue (Fig. la). Stolons branc h and anastomose to form intricate networks adherent to the substratum. Mat tissue, \ ch consists of interconnecting gastrovascular canals and interstitial cells sandwiL i between ectoderm, grows as a continuous sheet. As the mat tissue expands, it inc /orates existing stolons into its structure. The interaction of the growth rates of mat tis i r- and stolons, combined with factors such as stolon branching fre- quency, give each colony of H. echinata a characteristic growth morphology during ontogeny (McFadden et ai, 1984). There is considerable genetic variation in stolon SIZE REFUGE FROM COMPETITION 163 FIGURE 1. Camera lucida tracings of 17-day-old colonies, comparing the growth morphology of H. echinata (A) with that of P. earned (B). H. echinata produces mat tissue (stippled area) and stolons during colony ontogeny, while P. carnea covers the substratum with a stolon network only. Asterisks indicate the positions of feeding polyps. Scale bars = 5 mm. production: colony morphologies range from "stolonless" colonies which produce little or no stolon tissue as they grow, to "stoloniferous" colonies which form extensive stolon networks throughout colony ontogeny (Schijfsma, 1939; McFadden et al, 1984). Unlike H. echinata, P. carnea produces no mat tissue, but covers the substratum with an extensive stolon network (Fig. Ib). This network increases in density by con- tinued proliferation of stolons; adjacent stolons eventually fuse laterally to form a basal crust analogous to the mat of//, echinata (Braverman, 1963, 1971, 1974; Brav- erman and Schrandt, 1966, 1969). The hyperplastic growth reaction which occurs when genetically unrelated colonies of H. echinata contact one another is described in detail elsewhere (Hauenschild, 1954, 1956; Mueller, 1964;Ivker, 1972; Buss et al., 1984). Briefly, nematocyst-bearing hyperplastic stolons arising from both colonies intertwine to form an extensive tangle in the area of contact (Buss et al., 1984), and one colony will eventually overgrow and kill the other (Ivker, 1972). Competitive dominance is strictly transitive (Ivker, 1972), and highly correlated with growth morphology; in size-symmetrical encounters, col- onies with high stolonal growth rates ("stoloniferous") predictably defeat colonies with slowly growing or no stolons ("stolonless") (Buss and Grosberg, in prep.). The competitive overgrowth reaction of P. carnea is very similar to that of //. echinata (Tardent and Buhrer, 1982). Upon contact with a conspecific, stolons raise off the substratum and arch over the neighboring colony, producing a tangle of hyperplastic stolons in the area of contact. Scanning electron micrographs of P. carnea hyperplastic stolons show numerous discharged nematocyst threads, indicating that intraspecific overgrowth occurs by the same mechanism in the two genera. Transitivity and morphological correlates of intraspecific competitive ability between colonies of P. carnea will be examined briefly here, prior to discussion of interspecific competition. MATERIALS AND METHODS The colonies of //. echinata and P. carnea used in laboratory competition exper- iments were collected in August 1981 from the shallow subtidal (-3 m) gravel-mud bottom adjacent to No Man's Island, Old Quarry Harbor, Guilford, Connecticut. Individuals of Pagurus longicarpus with hydroid-encrusted shells were collected as 164 C. S. MCFADDEN encountered using SCUBA and were transported to the laboratory, where small pieces of ectodermal tissue c ,v,-, mining 1-3 feeding polyps were excised from each shell. These tissue explants v, ced on Plexiglas culture slides and held down by a loop of thread tied arou> ihe slide (Ivker, 1972). After 2-3 days, explants attached to the Plexiglas, and th< thread was removed. This technique was used for all clonal prop- agations reiern j to in this paper. Colonies were maintained at approximately 20C in recirculating natural seawater, and were fed for 2 h daily with day-old Anemia nauplii. Colonies were returned to clean seawater immediately after each feeding. Each slide was brushed weekly with a fine camePs-hair paintbrush to prevent the accumulation of growth-inhibiting detritus. The use of clonal organisms facilitates an examination of size-specific competitive interactions, because encounters between individuals of the same genotype can easily be replicated over a wide range of size relationships. The competitive ability of an individual which is killed by a competitor when small can nonetheless be examined when the same individual is large by using a clonal replicate, whereas the competitive ability of a non-clonal organism which is killed when small can never be assessed at a larger size. In addition, the degree to which the outcome of a competitive interaction is due to genotypic variation in the competitive ability of the individuals involved can be separated from strictly size-dependent effects by pairing any one individual (ge- notype) with numerous competitor genotypes. Int rasped fie competition between colonies of P. carnea Size-symmetrical competitive interactions were initiated between all possible pair- wise combinations of four genotypes of P. carnea (labeled PI, P2, P3, P4). [The as- sumption has been made that each field-collected colony represents a unique genotype; potential difficulties with this assumption are discussed in detail in McFadden et al. (1984).] Single polyp explants of each of two colonies were established approximately 2 cm apart on Plexiglas slides and allowed to grow into contact with one another. The number of replicates of each pairwise combination varied from 2 to 6, due to difficulties experienced getting explants of some genotypes to attach successfully to slides. The interactions were observed at approximately weekly intervals until tissue of one of the two colonies could no longer be discerned on the slide. In addition, three replicate clones of each of the four genotypes were established as controls to determine colony morphology and growth rate in the absence of competitive interactions. Each control colony was traced at 3-day intervals over a period of 1 7 days, using a camera lucida attachment to a Wild dissecting microscope at 7.5 X. Drawings were digitized using an image analysis system [Measuronics Corp., Linear Measuring Set (LMS)] to de- termine total length of stolons present at each date. Cumulative growth curves were plotted for each colony, and the slope of the linear regression of the log-transformed curve [log (mm stolon) = m log (time)] was used as an index of stolon growth rate. For further discussion of this method for fitting growth curves, see McFadden et al. (1984). Interspet Competition: size-symmetrical contests Size-symmetrical contests were initiated between 20 genotypes of H. echinata (labeled H1-H20) and 4 genotypes of P. carnea (P1-P4) in all 80 possible pairwise combinations. Single polyp explants of each species were established 1 cm apart on Plexiglas slides and the interaction of the two colonies was observed at approximately weekly intervals until one colony had completely overgrown the other. Overgrowth SIZE REFUGE FROM COMPETITION 165 was considered complete when no polyps remained in one of the two colonies. Several colonies died before contact had occurred, including all four replicates of HI 4; these pairs have been eliminated from the results. Size-asymmetrical contests Size-asymmetrical contests were established between clones of the same 20 ge- notypes of H. echinata and 4 genotypes of P. carnea. Single polyp explants of H. echinata were established on Plexiglas slides and allowed to grow undisturbed for a period of six weeks. At this time they were photographed to record size and general growth morphology, and a single polyp explant of P. carnea was then established approximately 1 cm from the periphery (mat edge or outermost stolons) of the H. echinata colony. All four clones of genotype H10 appeared unhealthy at the time P. carnea was introduced onto the slides and they were therefore eliminated from the experiment. The interspecific interactions were observed over a period of seven months, at which time the experiment was terminated. Photographs taken of the H. echinata colonies at the time of attachment of the P. carnea explants were converted to line drawings using a camera lucida on a Wild dissecting microscope at 7.5 X (McFadden et ai, 1984). The area of mat tissue and area covered by stolon network (the polygon determined by connecting the free tips of all stolons) were then digitized using an Apple II graphics tablet to yield estimates of the size and morphology of each H. echinata colony just prior to contact with P. carnea. RESULTS Intraspecific competition between P. carnea colonies The outcome of intraspecific competition between colonies of P. carnea is highly transitive, and can be predicted by stolon growth rate (Table I). P3, the colony with the fastest stolon growth rate, is the competitive dominant; PI, the colony with the slowest growth rate, is consistently overgrown by all other genotypes. Out of 23 contests, the only outcome which deviates from this dominance hierarchy is one of the 3 contests between genotypes P3 and P4. It is possible that the identities of the two colonies on TABLE I Mean duration (in days) of intraspecific contests between four genotypes of P. carnea Winning genotype Growth rate* PI P4 P2 P3 PI (1.007) 86(2) S.D. = 42 67(4) S.D. = 17 70(6) S.D. = 24 P4 Losing genotype P2 (1.062) (1.319) 122(2) S.D. = 6 131 (2) S.D. = 19 179(6) S.D. = 30 P3 (1.622) 118(1) * Slope of linear regression fit to the log-transformed cumulative growth curve [log(mm)/log(day)]. Numbers in parentheses indicate number of replicates. 166 C. S. MCFADDEN this slide were reversed during the experiment; all other contests involving either P3 or P4 yielded results consistent with predictions based on a transitive hierarchy. The amount c j > ne required for one P. carnea colony to overgrow another depends only on the identity of the losing colony in the contest (Table I; Kruskal-Wallis, within rows, P < .0 1 }. nd not on the identity of the winner (Table I; Kruskal-Wallis, within columns, P '- 2). For instance, there was no difference in the number of days required for genotypes P2, P3, and P4 to overgrow inferior competitor PI, while the amount of time P3, the competitive dominant, took to overgrow PI, P2, and P4 was highly variable (Table I). Interspecific competition H. echinata exhibits little or no hyperplastic growth upon contact with P. carnea. Occasionally the growing tips of//, echinata stolons swell and rise off the substratum slightly in response to contact with P. carnea, but further hyperplastic development rarely occurs. Stolons of P. carnea rapidly overgrow H. echinata stolons without be- coming hyperplastic, but begin hyperplastic growth immediately upon contact with mat tissue. P. carnea stolons are unable to grow across mat tissue, and consequently, mounds of hyperplastic stolon up to 5 mm in height accumulate at the periphery of the mat area at every point of contact between P. carnea stolons and H. echinata mat tissue (Fig. 2). These mounds may extend out over the surrounded mat tissue for as much as 2 cm, but they are not anchored to the underlying mat tissue and are easily broken off. The underlying H. echinata polyps are resorbed subsequent to overgrowth FIGURE 2 A contest between P. carnea (P) and H. echinata (H). P. carnea has produced hyperplastic stolons (S) where it is in contact with H. echinata mat tissue. SIZE REFUGE FROM COMPETITION 167 by P. earned hyperplastic stolons, but if polyps remain alive elsewhere in the colony, the overgrown mat tissue can remain alive. If hyperplastic tissue is removed from the overgrown areas of mat, new polyps may be regenerated on the exposed mat tissue, as is also possible in cases of intraspecific overgrowth in H. echinata (Ivker, 1972). Death of the overgrown H. echinata colony occurs only when P. carnea hyperplastic stolons cover the entire surface of the colony and all the polyps have been resorbed, presumably curtailing nutrient intake and leading to starvation. Size-symmetrical interspecific contests In every one of the 74 size-symmetrical contests, P. carnea overgrew and killed H. echinata (Table II). The mean time for complete overgrowth was 37 days, although several H. echinata colonies survived for considerably longer. Genotype H12 withstood overgrowth by P4 for 161 days, eventually with just a single polyp protruding through P. carnea hyperplastic stolons. There is a significant association between the duration of a contest and the genotype of the P. carnea colony (Kruskal-Wallis, P < .02). P3, the fastest growing P. carnea genotype, overgrew H. echinata in the least time (X = 26 days), whereas P4, one of the two P. carnea genotypes with the slowest growth rates, took the longest time to overgrow H. echinata (X = 46 days) (Table II). The growth morphology of the H. echinata colony does not affect the rate at which it is overgrown by P. carnea. Genotypes of H. echinata were categorized into three groups based on growth morphology: stolonless, stoloniferous, or intermediate. Mor- phological category was determined by previous quantitative measures of growth of these genotypes (McFadden et al., 1984), as well as evaluation of the growth of each clone during the experiment. The duration of a contest between P. carnea and H. echinata did not differ significantly between these three morphological groups (Kruskal- Wallis, P> .21). Size-asymmetrical interspecific contests Small P. carnea successfully overgrew and killed large H. echinata in 42 of the 76 size-asymmetrical contests (Table III). In no cases did H. echinata overgrow P. carnea. The other 34 contests were terminated after approximately 235 days, at which time both species still occupied space on each slide, but the boundary between colonies, delineated by P. carnea hyperplastic stolons, had remained static for 2-3 months in all cases. All of these "standoffs" reflected identical situations: P. carnea had covered all areas of the slide which were initially vacant or occupied by H. echinata stolons, but was unable to overgrow living H. echinata mat tissue. Both colonies were substrate- limited by the presence of the other, and further growth could take place only if one colony died or resorbed tissue in the zone of contact. Unlike the symmetrical contests, there was no significant relationship between the genotype of the P. carnea colony and the length of time it took to overgrow H. echinata in those asymmetrical contests which ended in the death of the H. echinata colony (Kruskal-Wallis, P > .47). There was also no significant difference between H. echinata morphological types when genotypes were pooled into the categories stolonless, sto- loniferous, and intermediate (Kruskal-Wallis, P > .75). However, if absolute size of the H. echinata colony is examined instead of this qualitative measure of morphology, a significant trend is apparent. Colony size was broken down into two separate mea- surements, total area covered by mat tissue and total area covered by stolon networks; colony thickness does not change as colony area increases. It has been shown that the growth rates of mat tissue and stolons are largely independent of one another (Me- 168 C. S. MCFADDEN q o^ - r \ u^) X ^ - so tN r- X J X 2 ?! oi S g. 01 CN| 04 >n _o o O PC LJJ a a PC r- 01 NC OO r*"t ^ ' ^ oa -5 H * o PC __ oj m o NO o~i r<- oo s X i ON r- O r"> Osl f 0) "a oo ffi 04 r~ o r- fi rO f^j fxj ^ 1 PC ^ fN <^j ^o r- r^i \o ^^ ro 1 1 X _^ (^J /~i ^O 1^1 r^ r^t -* r^} 'erous. i X ^ t~~ 01 ON 01 fN| r^i c^ co 'c o ~0 5 X ry^ r^ o\ o */" ^ r^ r^ r*- II GO 2 1 X ry-) ^O "/^ */^ rN r^i m o 04 2 3 U 2 X , , 5 - S Pi II PC % |. _0 ~c _o ^ >> "o 00 O o .. 1 b "O Nil II -O g a J3 & a CM m NW 0- CU CU CU J SIZE REFUGE FROM COMPETITION 169 1 o X < < < < X < < fN, < oo OO E C/2 >n < < < ^ r-i n X ~ - X ^ < < < < c/i 3 O I/-. ON r^ 01 vii X -J r- < 'c o a ^T r- r- o T H" X ^J ^^ ^O O*^ /" _ oo 2 X r- r- \^2 ^O ^O V) 5 | OJ ^ CN r- \o ri c c _o X nj SO VI t/~ ' II o M o r ^s oo Ifi _u X c "Jjj o jj "tj o N^ Su o t/5 U4 oa fej X ~ II _) H * ' &3 X ] ^\ O OO Vj ^^ & x; "sj ij C r- X c^ < < < < *- llM O 1 o 1 sO X | | O^ "~ */~i O^ u 4 n .N l/~l c^ X ^ < < < < 0> c ' -"* 13 *J > X on < < oo 1 'E 2 c i X 01 0) OO . "o 00 O "3 c 1^ "o ' " -o Q. ^ .15, n = 42), a!t' ; ; gh survival time appears to be generally longer for colonies with large stolon netw s (Fig. 3). There is, however, a significant correlation between the length of time a genotype of H. echinata withstood overgrowth and the mat area of the colony at the onset of the interaction (Kendall tau, r = .74, P < .001, n = 42) (Fig. 4). There appears to be a survival threshold at a mat area of approximately 100 mm 2 . The frequency with which H. echinata colonies were overgrown by P. carnea is significantly higher among colonies with an initial mat area less than 100 mm 2 than among those in two larger size classes, 100-200 mm 2 and >200 mm 2 ( x 2 = 17.8, df = 2, P < .005) (Table IV). The survival rate of colonies does not differ between the two upper size classes (x 2 = 0.03, df = 1 , P > .80). H. echinata colonies with small initial mat areas were usually killed after P. carnea had completely surrounded them and built up enough hyperplastic stolons around their periphery to extend completely across the mat. H. echinata colonies with large initial mat areas could not be surrounded by P. carnea due to their size and to the confines of the culture slides, and hence could not be overgrown. DISCUSSION The position occupied by a H. echinata colony on a limited substratum may significantly affect the outcome of competition between a colony of this species and ^ 250 ooaDO 9 200' 4- CO o> 150 O w 100 f r=0.12 1 s D 50^ f \'. p>0.15 Q V 100 200 300 400 500 Stolon Area on Day (mm 2 ) FIGURE 3. Co velation of the area covered by H. echinata stolons at the onset of an asymmetrical interspecific contest and the length of time the colony survived before being overgrown by P. carnea. Open circles represent colonies which remained alive at the end of the experiment. Numbers indicate multiple observations. SIZE REFUGE FROM COMPETITION 171 CO > CO Q 0) 0> +* O O O O i 250 O O OOD O 200 W 150 ' > 100 r. * * * r=0.74 50- /*** !A A p<0.001 100 200 300 Mat Area on Day (mm 2 ) FIGURE 4. Correlation of the area of//, echinata mat tissue at the onset of an asymmetrical interspecific contest and the length of time the colony survived before being overgrown by P. cornea. Open circles represent colonies which were still alive at the end of the experiment. Numbers indicate multiple observations. Dashed lines demarcate the three size classes compared in Table IV. one of P. carnea. The results presented above suggest that the ability of P. carnea to overgrow large H. echinata may depend on P. carnea first surrounding its competitor. Whether this condition can be met will be a function not only of the size of the H. echinata colony at the onset of the interaction, but also of the size of the free substratum, the position of the H. echinata colony relative to substrate boundaries, and the dif- ferential growth rates of the two species. Colonies with mat areas greater than 100 mm 2 at the time P. carnea was introduced reached the edges of the culture slides before P. carnea was able to surround them with its stolon network, and hence survived. Colonies initially smaller than 100 mm 2 were not growing rapidly enough to reach the limits of the slide before being surrounded by P. carnea. The mat area threshold of 100 mm 2 above which P. carnea overgrows H. echinata is significant only in the context of the particular substrate employed in this study. In a substrate patch wider than 26 mm (the width of a Plexiglas slide), P. carnea should be able to surround and overgrow H. echinata colonies which are larger than 100 mm 2 at the onset of interspecific contact. A smaller substrate area, or the proximity of a TABLE IV Initial mat area (mm 2 ) Number of H. echinata colonies overgrown by P. carnea Number of H. echinata colonies surviving interaction <100 100-200 >200 21 16 5 2 23 8 172 C. S. MCFADDEN colony to a substrate boundary, will lower the size at which H. echinata colonies become safe from o^ growth. A colony which cannot be surrounded due to its prox- imity to a phv boundary will become safe from overgrowth at a size at which it could be ovei were it instead surrounded by P. carnea. There will be a lower size limit to su :i a positional refuge: a very small H. echinata colony is likely to be overgrown by P. carnea regardless of its position on the substratum. The interaction of colony size with position on the substratum may provide a refuge from competition on natural substrata. Many of the hermit crab shells on which H. echinata and P. carnea settle offer considerably less surface area than the slides used in this laboratory study. In addition, both the shell aperture and apex may operate as effective substrate barriers; a colony positioned along the aperture or around the apex of a shell will be difficult for another colony to surround. H. echinata planulae do recruit preferentially to points around the aperture of a shell (Teitelbaum, 1966). If new recruits of both species recruit simultaneously and in similar locations on the same shell it is likely that P. carnea will overgrow H. echinata and monopolize the substratum. If P. carnea recruits to a shell with an already established H. echinata colony, the result will be either overgrowth of the H. echinata colony and monopol- ization of the entire shell by P. carnea, or, alternatively, a "standoff" between the two species with maintenance of a static boundary between them (see Connell, 1976; Karl- son, 1980). The results of interspecific encounters in the laboratory suggest that, were all else equal, P. carnea should eventually competitively exclude H. echinata on hermit crab shells. However, data on the distribution and frequency of occurrence of these two species in Long Island Sound do not support this prediction. In all seasons, H. echinata is much more abundant than P. carnea: from June through October, 1982, only ap- proximately 7% of all hydroid-encrusted shells collected were occupied by P. carnea, while Hydractinia colonies occupied the remaining 93% (Buss, Yund, and Harrison, in prep.). Competition, evidenced by hyperplastic stolons, occurs frequently between colonies of H. echinata which occupy the same shell. However, contact between H. echinata and P. carnea was observed only once from over 1000 hydroid-occupied shells collected (Buss, Yund, and Harrison, in prep.). This low rate of encounter between P. carnea and H. echinata is a product of the low frequency of occurrence of P. carnea combined with the probability that individuals of both species will colonize the same shell. Early competitive exclusion of H. echinata by P. carnea when both recruit to the same shell may also contribute to the low observed encounter rate. Models of a two-species community in which one species is the dominant com- petitor predict that a population of the inferior species can be maintained if there is a concomitant difference in the recruitment ability of the two species such that the inferior competitor is able to recruit to unoccupied substrata more quickly or more reliably than the dominant competitor (Armstrong, 1976). In a system in which a size refuge from competition is in operation, a difference in rate of recruitment, or in the seasonal timing of recruitment, may enable the inferior competitor to grow to a safe size before the dominant competitor can recruit onto the open substratum (Sebens, 1 982). i igher rates of post-recruitment mortality of the superior competitor, or frequent interfere, with its overgrowth ability by events such as partial predation, are addi- tional met; i isms which would allow an inferior competitor to reach a size refuge before being ;. 'ergrown. There is some evidence that the failure of P. carnea to displace H. echinata on hermit crab shells, despite its competitive superiority, may be due to a low rate of recruitment of this species to hermit crab-inhabited shells. In Long Island Sound, recruits of P. carnea (small colonies occupying less than 30% of the shell surface) were SIZE REFUGE FROM COMPETITION 173 found on only 0.2% of all shells collected from June to October, 1982, (n = 1663), whereas H. echinata recruits were present on 22% of these shells (Buss, Yund, and Harrison, in prep.). Because recruitment of P. carnea is apparently a rare event in this community, most H. echinata colonies will reach a size at which they are safe from overgrowth without ever encountering P. carnea. Yet the competitive superiority of P. carnea in interspecific contests ensures that an individual which does successfully recruit to a hermit crab shell can acquire and maintain space on that shell even if it is already occupied, or subsequently invaded, by H. echinata. The asymmetry in the degree of interspecific recognition displayed by these two species can be interpreted in light of the rate at which they are likely to encounter one another in the field. H. echinata is one of the most abundant members of the hermit crab epifaunal community (Karlson and Shenk, 1983), and there is a high rate of encounter of this species with conspecifics and with other common epifaunal species, such as the bryozoan, Alcyonidium polyoum (Karlson and Shenk, 1983). However, the probability that an individual of H. echinata will encounter P. carnea during its lifetime is relatively low; consequently, there should be little selective pressure acting to maintain its ability to recognize this uncommon species as a potential competitor. As shown, H. echinata does not produce hyperplastic stolons in response to contact with P. carnea. P. carnea, on the other hand, has a high probability of encountering a H. echinata colony upon recruiting to a hermit crab shell, due to the high percentage of all shells which are occupied by this species. Selection should act on P. carnea to maintain a mechanism for recognition and subsequent deployment of competitive structures against this important interspecific competitor. ACKNOWLEDGMENTS I am greatly indebted to L. Buss for his advice, support, and encouragement throughout all stages of this study. I also thank R. Lerner and L. Boyer for technical assistance, and T. Daniel, R. Grosberg, D. Harvell, T. Hughes, R. 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SWANBERG, J. L. LINDSEY, AND PAUL BENNETT Biological Oceanography, Lamont-Doherty Geological Observatory oj Columbia University. Palisades, New York 10964 ABSTRACT Physematium spp. and related genera of radiolaria (e.g., Thalassolampe and Actissd) are characterized by a large, limpid spherical cell body varying in cytoplasmic com- pactness, but characteristically possessing numerous small (ca. 3 nm dia.) symbionts held in fine radiating axopodia surrounding the large central capsulum. An analysis of the cytoplasmic organization of Physematium muelleri suggests that this organism has adapted to a pelagic existence by increasing surface area to enhance prey capture while conserving biomass through the development of a large internally alveolate, spheroidal cell possessing fluid-filled spaces. The thin capsular wall is supported by a network of cytoplasmic strands emanating from the perinuclear region of the intra- capsulum. The fine structural organization of the cytoplasm, composition and thickness of the central capsular wall, and the amount and kind of material deposited within the perinuclear envelope appear to be more significant taxonomic discriminating char- acteristics than the number or kind of siliceous spicules produced surrounding the central capsulum. The possible phylogenetic relationships among some genera related to Physematium and the functional morphology of the large, fluid-filled central capsule of close relatives are presented. INTRODUCTION Radiolaria are among the most abundant of biomineralizing Sarcodina occurring widely in the world's oceans. Their diverse and elegant siliceous skeletons have long attracted biological interest and have provided the main taxonomic characteristics used to classify radiolaria. In general, taxonomic criteria distinguishing species have included the overall shape of the skeleton, whether spherical, spiral, oval, or occurring as scattered spicules, etc. Further distinctions have been made on the number of concentric hollow spheres in the skeleton, or the geometry and arrangement of pores. Number and arrangement of surface spicules and spines also have been used widely in making taxonomic discriminations. The extensive use of skeletons as taxonomic indicators may be attributed to their elegant, geometric regularity and abundance in the sedimentary record (e.g., Haeckel, 1887; Riedel, 1971). However, some species lack siliceous deposits or produce only few or scattered spicules in their peripheral cytoplasm making cytoplasmic morphology more significant in their taxonomy. Ap- plication of light and electron microscopic analyses of radiolarian cytoplasm has clearly contributed to finer taxonomic distinctions (e.g., Hollande and Enjumet, 1953; Cachon andCachon, 1977, 1985; Anderson, 1976, 1978a,b, 1983). The larger species producing little or no siliceous deposits have recently been investigated more extensively for their physiological and fine structural characteristics toward a more exact understanding Received 27 February 1986; accepted 13 May 1986. 175 176 O. R. ANDERSON ET AL. of their functional morphology and phylogenetic affinities (e.g., Cachon and Cachon, 1977; Anderson, 197; , 1983; Swanberg and Harbison, 1980; Swanberg and Anderson, 1981; Anderson iotfield, 1983). Some of these gelatinous, larger radiolaria reach diameters of s J millimeters (Fig. 1 ), and are easily observed with the unaided eye and appear as oscent, pale-green, or yellowish spheroidal bodies suspended in seawater. Among these larger, gelatinous, solitary species of radiolaria, Haeckel (1887) de- scribed ten genera and forty-two species. The major diagnostic generic features he used were the presence or absence of alveoli within the large central capsule or in the peripheral envelope of cytoplasm known as the extracapsulum, and the occurrence and form of spicules in the extracapsulum. Of these species, Physematiwn atlanticum (Meyen, 1834) was one of the earliest of radiolaria identified. We have found this organism in great abundance near Barbados in December and January, and in the southern Sargasso Sea especially during the months of February and March, where it is often the commonest large solitary spumellarian in the surface waters. Unfortunately, the original description is rather ambiguous. Schneider's description (1858) of P. muelleri is unambiguous, however, and as it has priority over Haeckel's taxa, we have assigned our material to this species. P. muelleri is characterized by a large, somewhat opalescent, spheroidal cell body (1-5 mm dia.) surrounded by a hyaline gelatinous layer making the total diameter of the organism 3-6 mm. Fine axopodia radiate peripherally through this gelatinous sheath. The nucleus was described by Haeckel as possessing a thick membrane sur- rounded by an alveolate intracapsulum with a thin capsular wall, though Cachon and Cachon (1977) have shown that the intracapsulum is not alveolate, based on fine structure evidence. The presence of C- or S-shaped, siliceous spicules in the extracap- sulum distinguish P. muelleri from its close relative Thalassolampe margarodes based on the classical description. Brandt (1902) maintained that the presence of such isolated siliceous spicules were of limited systematic use at higher taxonomic categories, and then only when the soft body parts could be considered to be definitively different. He revised Haeckel's systematic scheme to place these genera together in the family Physematidae. He further observed that the cytoplasmic structure of Physematium muelleri and Thalassolampe margarodes agreed so closely as to make the latter genus superfluous; hence he recommended that the skeletonless genera be grouped with the older established genus Physematium. Modern researchers (Hollande and Enjumet, 1953; Cachon and Cachon, 1977; 1985) have accepted Brandt's familial designation but followed Haeckel's scheme in retaining the genus Thalassolampe for non-spiculate organisms. Although the spicules constitute a salient feature for systematic categorization, potentially, one of the most biologically significant characteristics of Physematium is the organization of the cytoplasm into a large gelatinous, spheroidal form with intra- capsular lobes. These lobes vary in arrangement from a loosely packed anastomosing network with large peripheral vacuoles (as in Physematium) to more closely spaced lobes with numerous small peripheral vacuoles near the capsular wall (as in Actissd). The functional significance of this organization is examined in relation to host-algal symbiotic v >ciations, prey apprehension, buoyancy functions, and possible phyletic relationship: yrnong related genera. To further elucidate the contribution of spicule number and moi ;;>hology to the systematics of these large, gelatinous solitary radiolaria, we have examined the variation in spicule abundance and morphology in specimens collected by divers in open ocean locations near Barbados and in the Atlantic Ocean. PHYSEMATIUM MORPHOLOGY AND CHARACTERISTICS 177 MATERIALS AND METHODS Individual organisms were collected into hand-held glass jars by divers in the Ca- ribbean Sea at a location approximately 1 mile from the west coast of Barbados, West Indies, during the month of January and at open ocean locations near Bermuda in the months of May and June, or during collection at sea in the North Atlantic Ocean (research cruises, OCEANUS 1 15 and 170, KNORR 53 and 94, ISELIN 83-1, and 83-1 1, CALANUS 85-5, and JOHNSON SEA-LINK). Specimens collected near Bar- bados were fixed for electron microscopy (Anderson, 1976), embedded in epoxy (LX 112), sectioned with a diamond knife, and ultrathin sections collected on uncoated copper grids. Sections were post-stained with Reynold's lead citrate and observed with a Philips EM 200 or EM 201 electron microscope operated at 60 kV. Other specimens were fixed immediately after capture using cacodylate-buffered 3% glutaraldehyde (pH = 7.8) and refrigerated for later examination by light microscopy and preparation for scanning electron microscopy. Specimens for scanning electron microscopic exami- nation were rinsed in distilled water, immersed in 10% v/v ethanol to prevent large ice crystal formation during freezing, deposited on scanning microscope stubs, frozen in liquid nitrogen, and freeze-dried under vacuum. A Cambridge Stereoscan 250 Mk2 scanning electron microscope was used to examine the specimens. RESULTS Light-microscopic views of the whole organism (Fig. 1) exhibit the large centrally located nucleus, widely spaced lobes, and alveolate intracapsulum, enclosed by a thin capsular wall adorned with a thin layer of loose spicules (Fig. 2) of varying morphology but typically appearing as C- or S-shaped structures (ca. 100-150 nm in length). An overview of the organization of the central capsule is presented in the composite line drawing (Fig. 3) and low-magnification electron microscopic perspective (Fig. 4). The organization and position of the nucleus (N), the surrounding radially arranged lobes and their relationship to the thin peripheral capsular wall (CW) is illustrated in Figures 4 and 5. Numerous cytoplasmic strands (fusules) occur in the capsular wall and connect the intracapsular cytoplasm with the radiating rhizopodia penetrating the peripheral jelly layer. The intracapsular lobes are widely spaced and interconnected by thin strands of cytoplasm. Alveoli formed by large vacuoles surrounded by a thin layer of cytoplasm within the intracapsular lobes occur sporadically within the central capsular cytoplasm and more commonly at the perimeter near the capsular wall. Higher magnifications of the nucleus (Figs. 6, 7) exhibit the perinuclear perforated organic wall and extensions of the nucleus through the pores into the surrounding cytoplasmic lobes. The peri- nuclear organic wall exhibits a finely fibrous texture and is enclosed within a cisterna produced by cytoplasmic extensions of the intracapsular lobes (Arrow, Fig. 7). The nuclear membranous envelope is separate from the cisternal membrane enclosing the fibrous matter of the perinuclear wall and exhibits a structure typical for eukarayotic cells with transmembranous pores. The intracapsular lobes are richly supplied with mitochondria, Golgi bodies (Fig. 8), and electron-dense granules, frequently grouped with peroxisomes in clusters (Fig. 9). Groups of mitochondria are commonly observed encircling a cluster of electron-dense granules and smaller mitochondria (arrow, Fig. 4). Occasional peroxisomes are distributed at the periphery of the ring of mitochondria, and some of the peroxisomes encircle the mitochondria (Fig. 9) indicating a close structural and perhaps functional association. Large, less densely stained lipid droplets 178 O. R. ANDERSON ET AL. 2 FIGURE 1 . A living Physematium muelleri with prominent intracapsular lobes and a centrally located nucleus. Bar = 1 mm. FIGURE 2. Siliceous spicules on the surface of the central capsular wall of P. muelleri. Bar = 20 nm. are also observed in the regions of the cytoplasmic lobes where the ensembles of encircling mitochondria and dense granules are abundant. The capsular wall possesses numerous fusules (strands of cytoplasm) directed out- ward, forming continuity between the intracapsular lobes and the extracapsular rhi- zopodial assembly (Fig. 5). The fusule structure resembles that in other large species of solitary radiolaria (e.g., Anderson, 1976, 1983, p. 1 14), consisting of a thin strand of cytoplasm emerging from a tip of an intracapsular lobe, penetrating the capsular wall to which it is attached, and emerging on the extracapsular side as an electron- dense segment surrounded by a collar-like rim. The rim is perforated by micropores, giving a sieve-like quality to the wall of the rim. Distal portions of the extracapsulum exhibit rhizopodia and digestive vacuoles of varying diameter. Scanning electron mi- croscopic views of the surface of the central capsule (Figs. 10, 11) show that the fusules occur in small clusters distributed over the surface of the central capsule. This is consistent with the transmission electron microscopic evidence showing long spaces of capsular membrane separating groups of fusules. As a contribution to the comparative fine structure of Physematium and its relatives, we have examined the capsular organization of Actissa sp. collected at the same location near Barbados (Fig. 12). The peripheral capsular cytoplasm is more dense than that of Physe, ! um, and possesses large peripheral vacuoles near the capsular wall as described earlier by light microscopic investigations (e.g., Haeckel, 1887). These fine structural data confirm one of the distinguishing characteristics of the two species reported by Haeckel, i.e.. the presence of a thickened organic wall and large intracap- sular spheroidal vacuoles in Actissa, and their absence in Physematium. Prominent outward directed collars (asterisk, Fig. 12) in the capsular wall of Actissa enclose the PHYSEMATIUM MORPHOLOGY AND CHARACTERISTICS 179 CW FIGURE 3. A line drawing illustrating a segment of the central capsular cytoplasm including the nucleus (N) with a thick organic wall (OW), radially arranged, widely spaced lobes with large vacuoles (V), and a thin peripheral capsular wall (CW) bearing the fusules (F) connecting the intracapsulum with extra- capsulum. fusule cytoplasmic strands. Numerous mitochondria and occasional segments of en- doplasmic reticulum occur in the region proximal to the vacuolar layer. Symbiont fine structure We have observed very small yellow-green to yellow-brown symbionts (ca. 3-4 Mm dia.), similar to those figured by Hollande and Enjumet (1953), in the extracapsular cytoplasm of both Physematium and Actissa. The fine structure of the symbionts (Fig. 1 3) indicates they are one of the Chrysophycophyta (eukaryotic yellow-green pigmented flagellates) with plastids composed of lamina with three thylakoids. The double mem- brane of the nuclear envelope encloses the plastids which are found occasionally in a parietal position within the cytoplasm. A granular pyrenoid within the plastid is pen- etrated by thylakoid membranes (Py, Fig. 13) and is prominently displayed in longi- tudinal sections. Mitochondrial lobes with tubular cristae and profiles of Golgi bodies 180 O. R. ANDERSON ET AL. J 4 6 FIGURE - ;itage of representative transmission electron microscopic sections along a radial dimension from i is (N) to the capsular wall (CW) exhibits the thick, porous, nuclear wall, surrounding cytoplasmic lobes, a s the peripheral thin capsular wall. Clusters of densely stained lipid deposits and mitochondria (arrow) occur abundantly in the intracapsular lobes. Bar = 5 nm. FIGURE 5. Fusule detail showing the protrusion of the capsular wall (CW) surrounding the cytoplasmic strand (F) projecting distally from the central capsule. Bar = 1 ^m. FIGURE 6. An enlarged view of the thickened wall (OW) surrounding the nucleus (N) with lobate projections of the nucleus in the pores and extending into the perinuclear space. Bar = 1 urn. 7 ' FIGURE 7. A detailed view of the organic wall surrounding the nucleus, showing the fine fibrillar quality of the organic substance in the wall and the enclosing living membrane (arrow) extending from a nearby cytoplasmic lobe. Bar = 0.5 nm. FIGURE 8. Golgi apparatus in a segment of an intracapsular lobe of Physemalium muelleri. Bar = 0.5 ^m. FIGURE 9. A close spatial association occurs frequently between mitochondria (M) and peroxisomes (P) which sometimes encircle the mitochondria. Bar = 0.5 ^m. FIGURE 10. A scanning electron microscopic view of the surface of the central capsular wall showing the arrangement of fusules. Bar = 50 /^m. FIGURE 11. A higher magnification view of a fusule and surrounding rhizopodia on the central capsular wall. Bar = 1 181 Fic.(. The peripheral intracapsular organization in Actissa sp. exhibits the large peripheral vacuoles (\) i ihe cytoplasm and thickened capsular wall (CW) with short fusules (asterisk) directed pe- ripherally. Bar = 1 pm. FIGURE 1 3. V section of a yellow-green pigmented algal symbiont (ca. 3 /xm diameter) associated with Physematium mui'ieri and Actissa sp. The parietal plastids with internal pyrenoids (Py) are enclosed within the double membranes surrounding the nucleus (N). Storage vacuoles (V) are commonly observed in the symbionts. Bar = O.S ^m. FIGURE 14. The fine structure of the pyrenoid (Py) and its surrounding starch sheath within a dino- flagellate symbiont associated with P. muelleri. Bar = 0.5 ^m. 182 PHYSEMATIUM MORPHOLOGY AND CHARACTERISTICS 183 are scattered throughout the central cytoplasm. Some symbionts possess a large ex- centrically located vacuole which is electron luscent or sometimes contains amorphous matter or densely staining granules (V, Fig. 13). We have not observed flagella, probably owing to the coccoid state of the algal cells as is typically observed in a symbiotic association with radiolaria (e.g., Anderson, 1976, 1983; Anderson et al, 1983). Phy- sematium sp. also possess dinoflagellate symbionts (Fig. 14) resembling those previously observed in radiolaria (Anderson, 1976, 1983). In some cells, we have observed as many as three pyrenoids, while in previous observations of dinoflagellate symbionts (identified as Amphidinium sp.) there were either one or two pyrenoids. Spicule abundance and morphological diversity A sample of 60 specimens exhibiting a gross morphology of Physematium was examined to determine the abundance of spicules within the cytoplasm immediately surrounding the central capsular membrane. The abundance varied from no spicules to a few to hundreds and thousands per organism, suggesting an intergradation in spicule density among specimens. We suspect that spicule number may not be a good characteristic to distinguish species and therefore suggest that additional research is needed to evaluate Haeckel's assumption that spicule presence or absence is a species- specific trait. The absence of spicules may be due to a physiological state of the organism, rather than a genetic difference. A survey of 121 specimens of SCUBA-collected Phy- sematium muelleri was made to determine the morphology of the spicules. The shape of the spicules was categorized as (1) straight needles, (2) C-shaped, (3) mixture of C-shaped and S-shaped, (4) mixture of C-shaped and straight, or (5) a mixture of shapes including all of the above and C-shaped forms with a small side branch. Fifty- four had simple straight spicules, 34 had C-shaped spicules, 5 had a mixture of C- shaped and S-shaped, 14 had a mixture of straight and C-shaped, and 14 had a mixture of heterogeneous shapes mentioned above. These data indicate that gradations in mixtures of spicule shape occur in specimens collected from the same locality, and that spicule type is probably not a good criterion for erecting separate species. The general intergradation of form of the spicules also exemplifies the remarkable heter- omorphic variability in spicule composition of Radiolaria and raises the more general issue of the merit of using fine skeletal details in setting species boundaries. DISCUSSION There is a component of arbitrariness inherent in all taxonomic criteria and the hierarchy of relative importance of various features is inescapably anthropocentric; this is particularly predominant in the systematics of radiolaria because of the salient aesthetic properties of their cytoplasmic and skeletal morphology. In recent publica- tions, we have partially addressed the issue of taxonomic criteria and the appropriate kinds of attributes that may be most productive in developing a phylogenetically sound and heuristically valid taxonomic paradigm (Anderson, 1983, pp. 82-84; Swanberg and Anderson, 1985; Swanberg ?/ al, 1985, 1986). Our research on solitary and colonial radiolarian physiology (e.g., Anderson, 1978b; Anderson and Botfield, 1983; Swanberg, 1983; Anderson et al., 1985; Swanberg and Anderson, 1985) of Spongodrymus sp. and related spongiose skeletal solitary Spumellaria has given us an opportunity to examine in some detail a number of the larger, gelatinous Spumellaria including Actissa, Physematium, Thalassolampe, and Thalassicolla. A summary of our current under- standing of the major morphological and fine structural features distinguishing these four genera is presented in Table I. 184 O. R. ANDERSON ET AL. to t .2 6 Q. O _o "= "S ^> > f*1 "O y^ , _2 Q 10 c^ ^.' Ci "S ; ii g^ o 'i _2 a S 11 1 S & 3-3.2 o .a ca y C oo > to fc c j; O 13 H | d 2 "8 -| i t/5 > "s "' 2 1 oo 2 = y 1^ i) S 5; c > U > OJ s- c .^ =0 3'c -2 Q^T: < idiolarian - S to 'C b > (U l- C J3 O OJ Q. III g _ C i I 3 ^3 / 52 O "3 t 11 3 > 1 ^> 13 o ^3 'f. ^ *0 Q> r~ r^ II * OJ 5 ts x *^ ~- u K 00 - J = a-S 1 | 1 ||i tf, t< O p J= C o ^ "o 5 a C/5 ds pa QJ U* 1 i 5 1 ^ ^ ? "3 I 1 E | c rf ^S 1-B g '3 S d a g J 'S S'" 5 f' s S o o u z Intracapsular 1 0* 03 e . ^ oo p E M 5 tP _o ^- ^ - o o c *-e - a o g 6 ! S - g "S c c 2 " ci ^i i/> D. "=^ .% ^ a 5 3 Si -E 2 a| o g ^. .S S 5 o c = 1 1 a 11 S-l X uu 1 I 00 o ^o a o. c 00 o { PHYSEMATWM MORPHOLOGY AND CHARACTERISTICS 185 Our evaluation of the fine structure of the cytoplasm of these organisms in relation to skeletal spicule variation has been informed by the observations of Hollande and Enjumet (1960, p. 66) decrying the poor systematic value of some skeletal variations, especially the significance of lattice versus spongiose skeletal morphology, and has led us to re-evaluate the importance of spicule abundance and morphology in erecting generic categories. This critical re-appraisal seems especially relevant to P. mueUeri because of its unusual delicate spicules and the unique features of its large spheroidal central capsule including the very thin capsular membrane and the substantial peri- nuclear wall. We consider these features to be significant phyletically and taxonomically and representative of a functional morphology adapted to enhance buoyancy, algal symbiont associations, and possibly prey apprehension. We present the first observation of a yellow-green pigmented chrysophyte-type alga in association with radiolaria. It is not immediately clear, however, why some individuals possess the yellow-green pigmented algal associates while others have di- noflagellate symbionts. Similar thin-walled cytoplasmic sheaths of host cytoplasm sur- round both kinds of algae, but we do not know if the physiology of the association, including kind and translocation rate of photosynthates from alga to host, is similar for the two types of algae. The cytoplasmic organization of the larger gelatinous Spumellaria suggests a phy- logenetic pattern of development progressing from an ancestral form resembling Phy- sematium with a thick perinuclear wall and delicate vacuolated capsular cytoplasm toward Actissa with a thickened capsular wall, rather closely packed cytoplasmic lobes bearing numerous peripheral alveolate vacuoles, but still lacking extracapsular alveoli. At a more advanced stage, an organization more characteristic of modern Thalassicolla sp. may have emerged, with densely packed intracapsular lobes of cytoplasm, a thick- ened porous capsular wall, and a massive array of extracapsular alveoli. Our fine structural analyses of P. mueUeri show that the delicate intracapsular cytoplasm supporting the thin central capsular membrane provides a large increase in cell volume with moderate cytoplasmic elaboration. This delicate construction, while increasing surface area and conserving cytoplasmic mass, also leaves the nucleus relatively unprotected. The thickened perinuclear wall may provide protection for the nucleus suspended within the delicate web of anastomosing intracapsular lobes. The adaptive value of the radially arranged lobes with large intra-lobular spaces is not obvious. Observations of living specimens floating in the open ocean and in laboratory culture indicate that, like many gelatinous Spumellaria, these organisms are neutrally buoyant. This buoyancy may be attained by secretion of low density fluids within the free space among the lobes. The presence of a fluid within the central capsule has been confirmed by piercing the organisms in laboratory culture. In most cases, the pierced organisms exude the fluid, but do not burst. The capsular membrane eventually heals and the large inflated form is re-established. The functional morphological significance of these features appears to be profound. An hypothetical spherical organism relying on density-dependent predation for food is under selective pressure to increase its ratio of surface area to volume with minimum expenditure of energy and maximum utilization of cytoplasmic mass (Anderson, 1985, Swanberg el al, 1985). One way to accomplish this is to protrude thin lobes of cytoplasm either supported on delicate, elongate skeletal elements if present (Anderson, 1983, pp. 178-180, 1985; Swanberg et al., 1985) or attached to a thin lamina such as the delicate capsular membrane to increase the associative strength while simultaneously keeping the amount of supporting surface cytoplasm at a minimum. Few skeletonless organisms appear to have employed this option as observed in P. mueUeri. A large surface area for improved symbiont holding capacity and greater probability of prey 1 86 O. R. ANDERSON ET AL. apprehension, is produced while the large fluid-filled spaces between the delicate cy- toplasmic lobes volume at low metabolic expense. Although the capsule con- tour remains sph- cal, the total cytoplasmic surface area is large relative to the smaller, metabolicall v ; volume. The radiating delicate axopodia surrounding the globose central capsule are efficiently disposed to provide a large prey apprehending area. HaeckeS < onsidered Actissa to be the most "primitive" of the radiolaria. Our observations suggest that in its ultrastructure Physematium muelleri is actually closer to a simple spherical cell and more primitive in its organization of the capsular cy- toplasm than many Spumellaria. The adaptation to increased surface area at low metabolic cost is a major strategy for a number of groups of gelatinous metazoan predators such as Coelenterata and Ctenophora in the open ocean. If, indeed, Phy- sematium is a primitive form of radiolarian, the evolution of a light-weight, large- surface-area morphology may have been an early adaptation that preceded massive skeletal deposition as a means of supporting and enhancing large cytoplasmic surfaces in these symbiont-bearing, opportunistic planktonic predators. ACKNOWLEDGMENTS We express appreciation to the staff of the Bellairs Research Institute, St. James, Barbados, and to the staff of the Bermuda Biological Station, St. Georges, Bermuda. This work was supported by the Biological Oceanography Division of the National Science Foundation (OCE 84-08137). This is Bermuda Biological Station Contribution No. 1094 and Lamont-Doherty Geological Observatory Contribution No. 4017. LITERATURE CITED ANDERSON, O. R. 1976. Ultrastructure of a colonial radiolarian Collozoum inerme and a cytochemical determination of the role of its zooxanthellae. Tissue Cell 8: 195-208. ANDERSON, O. R. 1978a. Light and electron microscopic observations of feeding behavior, nutrition, and reproduction in laboratory cultures of Thalassicolla nucleata. Tissue Cell 10: 401-412. ANDERSON, O. R. 1978b. Fine structure of a symbiont-bearing colonial radiolarian Collosphaera globularis and I4 C isotopic evidence for assimilation of organic substances from its zooxanthellae. J. Ultrastruct. Res. 62: 181-189. ANDERSON, O. R. 1983. Radiolaria. Springer- Verlag, New York. 352 pp. ANDERSON, O. R. 1985. An hypothetical analysis of the phylogenetic and functional significance of spherical skeletons in some spumellarian Radiolaria. Radiolaria: International Newsletter for Radiolaria Researchers 9: 32-36. ANDERSON, O. R., AND M. BOTFIELD. 1983. Biochemical and fine structure evidence for cellular specialization in a large spumellarian radiolarian Thalassicolla nucleata. Mar. Biol. 72: 235-241. ANDERSON, O. R., N. R. SWANBERG, AND P. BENNETT. 1983. Fine structure of yellow-brown symbionts (Prymnesiida) in solitary radiolaria and their comparison with similar acantharian symbionts. J.Protozool. 30:718-722. ANDERSON, O. R., N. R. SWANBERG, AND P. BENNETT. 1985. Laboratory studies of the ecological significance of host-algal nutritional associations in solitary and colonial Radiolaria. J. Afar. Biol. Assoc. UK 65: 263-272. BRANDT, K. 1902. Beitrage zur Kenntnis der Colliden. Archiv Protist. 1: 59-88. CACHON, J., AND M. CACHON. 1977. Le systeme axopodial des Collodaires (radiolaires Polycystines) 2. Thalassolampe margarodes Haeckel. Archiv Protist. 119: 401-406. CACHON, , AND M. CACHON. 1985. Class Polycystinea. Pp. 283-302 in An Illustrated Guide to the Protozoa, J. ce, S. H. Hutner, and E. Bovee, eds. Society of Protozoologists, Lawrence, Kansas. HAECKEL, E. '. Report on the Radiolaria collected by HMS Challenger during the years 1873-1876. Pp. I in Report of the Voyage of the Challenger, Vol. 18, C. W. Thomson and J. Murray, eds. Her /iajest's Stationery Office, London. HOLLANDE, A., AND M. F.NJUMET. 1953. Contribution a Tetude biologique des sphaerocollides (Radiolaires collodaires et radiolaires polycyttaires) et de leurs parasites. Ann. Sci. Nat. Zoo/. 2: 99-183. HOLLANDE, A., AND M. ENJUMET. 1960. Cytologie, evolution, et systematique des Sphaeroides (Radiolaires). Arch. Mus. Nut. Hist. Nat. (7 eme Serie) 7: 1-134. PHYSEMATIUM MORPHOLOGY AND CHARACTERISTICS 187 MEYEN, F. 1834. Beitrage zur Zoologie, gesammelt auf einer Reise um die Erde. Nova Act. Acad. Leap. Carol. 5: 125-218. RIEDEL, W. R. 1971. Systematic classification of polycystine Radiolaria. Pp. 649-66 1 in The Micropaleontology of Oceans. M. Funnell and W. R. Riedel, eds. Cambridge University Press, Cambridge. SCHNEIDER, A. 1858. Ueber 2 neue Thalassicollen von Messina. Arch. Anal. Physiol. Wiss. Med. Jahre 1858, pp. 38-42. SWANBERG, N. R. 1983. The trophic role of colonial Radiolaria in oligotrophic oceanic environments. Limnol. Oceanogr. 28: 655-666. SWANBERG, N. R., AND G. R. HARBISON. 1980. The ecology of Collozoum longiforme, sp. nov. a new colonial radiolarian from the equatorial Atlantic Ocean. Deep-Sea Res. 27: 715-731. SWANBERG, N. R., AND O. R. ANDERSON. 1981. Collozoum caiidatum sp. nov.: a giant colonial radiolarian from equatorial and Gulf Stream waters. Deep-Sea Res. 28A: 1033-1047. SWANBERG, N. R., AND O. R. ANDERSON. 1985. The nutrition of radiolarians: trophic activity of some solitary spumellaria. Limnol. Oceanogr. 30: 646-652. SWANBERG, N. R., P. BENNETT, J. L. LINDSEY, AND O. R. ANDERSON. 1986. The biology of a coelodendrid: a mesopelagic phaeodarian radiolarian. Deep-Sea Res. 33: 15-25. SWANBERG, N. R., O. R. ANDERSON, AND P. BENNETT. 1985. Spongiose spumellarian Radiolaria: the functional morphology of the radiolarian skeleton with a description of Spongostaurus, a new genus. Mar. Micropaleontol. 9: 455-464. Reference: Biol. Bull. 171: 188-196. (August, 1986) NEPt DIA IN THE LARVAE OF HEMICHORDATES AND ECHINODERMS EDWARD E. RUPPERT AND ELIZABETH J. BALSER Department of Biological Sciences, Clemson University, Clemson, South Carolina 29634-1903 ABSTRACT The pore canal-hydropore complex in the larvae of echinoderms and hemichordates has long been recognized as an important character establishing a close phylogenetic relationship between the two phyla. An experimental and ultrastructural analysis of this complex in a tornaria and a bipinnaria larva indicates that it is a functional nephridium. The ciliated pore canal drives a constant, unidirectional efflux of coelomic fluid out of the hydropore. Two percent and 14% of the body volume are cleared per hour at the hydropore by a tornaria of Schizocardium brasiliense and a bipinnaria of Asterias forbesi, respectively. Fluid recovery by the coelom is from the blastocoel, the presumptive blood vascular space, across basal lamina and podocytes lining the coe- lomic cavity suggesting that the discharged fluid is formed by ciliary-driven ultrafil- tration. Although invertebrate deuterostomes are believed to lack discrete excretory organs, an analysis of the metamorphosis of the larval nephridia suggests that adult echinoderms and hemichordates possess functional metanephridial systems. INTRODUCTION A signal achievement of classical morphology was the recognition of a close re- lationship between hemichordates and echinoderms based on structural similarities of their larvae (Gemmill, 1914; van der Horst, 1939). The most striking of these similarities is the pore canal-hydropore complex. It consists of a duct leading from a coelomic cavity to an external pore situated asymmetrically to the left of the dorsal midline as described in larvae of Enteropneusta and all five extant classes of echino- derms (Hyman, 1955, 1959). Yet despite the careful attention paid to this organ by morphologists of the nineteenth and early twentieth centuries, only one, T. H. Morgan, commented on its function. He speculated that "The whole structure is suggestive of an excretory arrangement [in the tornaria] . . ." (Morgan, 1894). Invertebrate deuterostomes are generally believed to lack discrete nephridial organs and nowhere is the belief more frequently advanced than in the Echinodermata (Bin- yon, 1966; Barnes, 1980). We have been studying the generality of a model that predicts the occurrence of functional metanephridia in organisms where there is the potential for ultrafiltration of vascular fluid into a coelomic cavity, viz., in coelomates with a blood vascular system (Ruppert and Smith, 1985, material in prep.). Our attention was drawn to larval echinoderms and hemichordates because of reports of a contractile eating rhythmically adjacent to the coelom and pore canal of definitive larvae in the L^ uroidea (Gemmill, 1918), Echinoidea (Bury, 1896), Asteroidea (Gemmill, 1914), and F nteropneusta (Morgan, 1 894). This organization suggested that blastocoelic fluid could 1 e pressure filtered across the wall of the coelom and pore canal, modified by the lining cells, and released at the hydropore. Received 4 February 1986; accepted 6 May 1986. HEMICHORDATE AND ECHINODERM NEPHRIDIA 189 Objectives of this investigation were to determine the pattern of fluid flow in the pore canal in the larvae of the asteroid, Asterias forbesi, and the enteropneust, Schi- zocardium brasiliense, and to identify potential sites of ultrafiltration using transmission electron microscopy (TEM). Because it soon became apparent that the larval complexes functioned as nephridia, another objective was to re-examine the adult derivatives of the larval structures to determine if adult enteropneusts and echinoderms might express functional nephridia. MATERIAL AND METHODS Collections of adult animals were made in March 1985 from a rock jetty at Murrell's Inlet, South Carolina (Asterias}, and from a mudflat at North Inlet near Georgetown, South Carolina (Schizocardiwri) (Fox and Ruppert, 1985). Tornaria larvae of Schi- zocardium and bipinnaria larvae of Asterias were reared from eggs spawned in the laboratory. Cultures were maintained under constant agitation at 16C for approxi- mately 6 months with thrice weekly changes of natural seawater (33%o) and the addition of a few squirts from a Pasteur pipet of Dunaliella salina and Isochrysis galbana, as food. Flow direction and flow rates in the ciliated pore canal were determined by mi- croperfusion of the adjoining coelom with 0.45 ^m latex microspheres (Polysciences), stabilized with BSA (Sigma), and suspended in seawater. These were introduced through the hydropore without damage to the larva. Particle movements were observed directly under a compound microscope (Zeiss Photomicroscope I) and recorded using cinemi- crography. Cinemicrographic sequences were obtained on 16 mm film (Kodak Plus- X, Double-X, and Ektachrome 7240 color positive film) at 16 fps. Analyses of particle movement were made from prints of selected sequences and with the aid of a motion analyzer (Athena model NT-O). Volumes of internal cavities of both larvae were calculated from digitized tracings (HIPAD digitizer, Houston Instruments, Austin, Texas) of serial cross sections of specimens embedded in Polybed 812 (Polysciences). The computer analysis (Victor 9000, Victor Technologies, Scotts Valley, California) was based on an algorithm for the area of a polygon with vertices (Pearson, 1974). Morphological data were obtained from living larvae and adults, plastic embedded specimens (Rieger and Ruppert, 1978), and thick sections drawn or photographed on a dissecting (Wild M-5) or compound microscope (Zeiss Photomicroscope I plus drawing tube). Specimens were fixed for thick sectioning and TEM (Philips EM 300) in 2.5% glutaraldehyde in 0.2 M Millonig's phosphate buffer for 1 h. Postfixation, after a brief buffer rinse, was in 1.0% OsO 4 in 0.2 M buffer for 1 h. After standard alcohol dehydration, the specimens were embedded in Polybed 812. RESULTS Development of the complex Within 72 hours of fertilization at 16C, the larvae of both species are swimming and possess a ciliated pore canal and dorsal hydropore joining a coelomic cavity to the exterior (Figs. 1A, 2A). A spherical vesicle develops beside the hydropore in the tornaria and near the right coelom in the bipinnaria about 3 weeks into larval life. The vesicle, a coelomic cavity, increases in size as it migrates through the blastocoelic jelly to graft, along two of its sides, near the junction of the pore canal and its coelom (Figs. 1C, 2C). An open cavity, continuous with the blastocoel, remains between one 190 E. E. RUPPERT AND E. J. BALSER **> D JX, >* co f I w be B c5 CO J " - -- be IVv axs \\ : i -"f'-v / ecm < ecm be ~" r^ , ^K , / -tf'<*. A '' -.^ " : - - -- , , -. -. - ,, ^ tw- --^ o,|f -jiflfSi*!/ w^7- ' ' ;> 1; * : M% vtfeMl ; HEMICHORDATE AND ECHINODERM NEPHRIDIA 191 wall of the vesicle and the pore canal coelom (Fig. 2C). Once in place, the vesicle undergoes a contraction every 5-10 seconds. Fluid transport Direct microscopic examination and cinemicrography revealed a continuous, cil- iary-driven efflux of particles from the coelom and pore canal at the hydropore (Fig. 3). All cells forming the pore canal are ciliated as are most of the peritoneal cells lining the coelomic cavities. Table I contains morphometric data for the two larvae. The calculated average particle velocity was 46 ^m-s" 1 (6.25 SE, n = 4, tornaria) and 88 /urn s" 1 (8.9 SE, n = 2, bipinnaria) in the ectodermal portion of the pore canal. From the dimensions of these cylindrical canals, calculations were made of the fluid volume cleared at the hydropore. Assuming a parabolic velocity profile across the pore canal, the volume cleared is 5.4 nl-hr 1 by the tornaria and 27 nl-h ' by the bipinnaria. At the calculated rate of discharge through the hydropore, the tornaria will replace its coelomic volume once every 1 3 min and the bipinnaria every 56 min. Because the coeloms do not collapse with the constant hydroporic efflux, fluid must be recovered from the blastocoel across the coelomic epithelium. Such a flux across the coelomic epithelium raises the possibility of a ciliary-driven ultrafiltration of blas- tocoelic fluid. Given blastocoelic volumes of 0.27 /A and 0.14 /nl in the tornaria and bipinnaria, the entire blastocoelic volume could be cleared in 50 h and 5 h, respectively. The percent body water discharged per hour is approximately 2% for the tornaria and 14% for the bipinnaria. infrastructure of the filtration surface Transmission electron microscopy of the coelomic lining of the anterior coelom and mesodermal part of the pore canal in the early tornaria reveals that the entire lining is composed of podocytes lying on a thin basal lamina except for a few myo- epithelial cells forming the apical muscle band (Fig. 2B, D, E, F). In the definitive tornaria, the podocytic lining is restricted to the region of contact between the coelom and pulsatile vesicle, and along the proximal portion of the pore canal (Fig. 2C). The remainder of the lining differentiates as myoepithelial cells that will contribute to the musculature of the adult proboscis. In the early and definitive bipinnaria, portions of the left coelom at the level of the hydropore are also lined by podocytes, especially along the medial and mediodorsal walls (Fig. ID, E). Other coelomic regions have not been surveyed. FIGURE 1 . Hydropore-pore canal complex of the bipinnaria larva of Asterias forbesi (Asteroidea). (A) Left lateral view of living early larva showing left coelom (co), blastocoel (be), and pore canal (arrow; bar = 200 urn). (B) Lateral view of pore canal (pc) traversing blastocoel (be) from left coelom (co) and opening dorsally at the hydropore (hp). Arrows define limit of ectodermal part of canal (bar = 50 nm). (C) Dorsal view of pore canal (pc), hydropore (hp), and pulsatile vesicle (pv) in the blastocoel (be). Complex is not yet fully organized. Top of micrograph is anterior (bar = 30 ^m). (D) TEM of peritoneal podocytes from medial wall of left coelom at the level of the pore canal. The basal lamina (ecm) is the only continuous barrier between the blastocoelic (be) and coelomic (co) compartments (fp, foot processes of podocytes; bar = 1.0 Mm). (E) TEM of podocyte foot processes (fp) from same anatomical region as in D (be, blastocoel; co, left coelom; ecm, basal lamina; bar = 0.5 p.m). (F) TEM of a portion of an axial gland blood vessel (bvs) of a juvenile Asterias (see Fig. 4; axs, axial sinus coelom; ecm, basal lamina; fp, foot processes of peritoneal podocytes; bar = 0.5 ^m). (G) TEM of columnar cell from ectodermal part of larval pore canal (pc). Each cell bears a single cilium (not shown) projecting into the pore canal (arrow = coated pit; mv, microvilli; tw, terminal web; bar = 1.0 192 E. E. RUPPERT AND E. J. BALSER : < '- V '-"' ' c -' ' * - , \\V^ . ":-*.." A , s * /\ < **r^ -^ ^_, Ji&#x.*V^f>..S>^ " ^ -, 'H, ^t-^r-rvx- ^ HEMICHORDATE AND ECH1NODERM NEPHRIDIA 193 FIGURE 3. Ciliary transport of microbeads in the pore canal of Schi:oc ardium brasiliense (Enterop- neusta). (A-F) Cinemicrographs of a clump of microbeads in the mesodermal part of the pore canal (arrows; 0.25 s interval between frames). Velocity measurements for the calculations in the text were obtained only for the ectodermal part of the canal (hp, hydropore; Ib, latex microbeads in anterior coelom; Ip, plume of microbeads expelled at hydropore; pc, pore canal; bar = 50 The ectodermally derived part of the pore canal adjoining the hydropore in both species is organized as a cuboidal to columnar, ciliated epithelium (Figs. 1G, 2H). The microvillar density and vesicular content of these cells are significantly higher than those associated with the adjacent, overlying, squamous larval epidermis. The metamorphosis of the larval nephridia is described in Figure 4. DISCUSSION The results suggest that larval enteropneusts, asteroids, and perhaps larval echi- noderms of the remaining four classes, all of which develop pore canal complexes (Hyman, 1955), possess a functional nephridium probably involved in extracellular volume regulation. The calculated values of 2% and 14% of the body volume cleared FIGURE 2. Hydropore-pore canal complex of the tornaria larva of Schizocardium brasiliense (Enter- opneusta). (A) Left lateral view of living early larva (be, blastocoel; co, anterior coelom; mb, muscle bands; pc, pore canal; bar = 200 /^m). (B) Lateral view of pore canal (pc) traversing blastocoel (be) and opening dorsally at the hydropore (hp). Arrows indicate ectodermal portion of canal (co, anterior coelom; pn, podocyte nuclei; bar = 40 ^m). (C) Dorsal view of definitive complex (anterior is toward top). The spherical pulsatile vesicle (pv) is joined laterally to the wall of the anterior coelom (co) enclosing a small cavity (arrow) that is continuous with the blastocoel (be; hp, hydropore; mb, muscle band; pc, pore canal; bar = 100 jim). (D) TEM of peritoneal podocytes (pn) from proximal pore canal. The podocyte basal lamina (ecm) is the only continuous barrier between the blastocoelic (be) and coelomic (co) compartments (fp, foot processes; bar = 1.0 ^m). (E-F) Transverse and grazing TEM sections of larval podocytes (bars = 0.5 ^m). TEM of podocyte foot processes (fp) on small blood vessels (bvs) in the glomerulus of the enteropneust Saccoglossus kowalevskii (see Fig. 4; ecm, basal lamina; bar = 0.5 ^m). (H) TEM of columnar cell from ectodermal part of larval pore canal. Each cell bears more than one cilium (not shown) projecting into the pore canal (arrow = coated pit; mv, microvilli; bar = 1.0 194 E. E. RUPPERT AND E. J. BALSER TABLE I Morphomethc da''/ irvae t^Schizocardium brasiliense and Asterias forbesi (volumes in Larva Tornaria Bipinnaria Total -i (mm) 1.1 1.6 Body vol. 2.9 X 10-' 1.9 X 10"' Blastocoel vol. 2.7 x 10-' 1.3 X 10~' Protocoel vol. 1.2X 10~ 3 Pulsatile vesicle vol. 1.1 X 10~ 4 1.3 X 10-" Enterocoel vol. 2.6 X 10~ 2 Left mesocoel vol. 2.8 X JO' 4 Right mesocoel vol. 4.0 X 10~ 4 Left metacoel vol. 6.6 X 10-" Right metacoel vol. 7.2 X 10~ 4 Gut vol. 1.5 X 10~ 2 3.0 x 10~ 2 Ectodermal pore canal length (^m) 45 35 Ectoderm pore canal diameter (^m) 9 15 per hour by these larvae fall within or close to the general range of 1% to 10% given by Kirschner (1967) for aquatic animals. It should be noted, however, that the values presented here are rough estimates based on measurements of the few particles that remained in the focal plane of the microscope while in transit along the pore canal. Given the constant hydroporic efflux, fluid recovery by the adjoining coelom must be from the surrounding blastocoel across the layer of continuous basal lamina and peri- toneal podocytes. The mechanism of fluid recovery across the body wall has not been addressed in this study. We speculate that it is driven by an osmotic gradient (Oglesby, 1981), perhaps established by proteins in the blastocoelic jelly. A protonephridium can be denned functionally as an excretory organ where ni- tration occurs on the nephridium (terminal cell) and is driven by cilia whereas, in metanephridial systems, nitration occurs on blood vessels or their analogues and ni- tration pressure is muscular in origin (Ruppert and Smith, 1985, material in prep.). If invertebrate nephridia are so denned, then the pore canal-hydropore complex of these larvae can be considered as a functional protonephridium with the ciliated cells of the pore canal forming the pump driving nitration. It is possible that, when the pulsatile vesicle is positioned, the system functions as a metanephridium, with the ciliary pump being augmented by a muscular pump, the pulsatile vesicle. Supersedure of a larval protonephridium by a later metanephridium is a common feature of rep- resentatives in several phyla, e.g., Annelida (Goodrich, 1945), Phoronida (Emig, 1982), and Mollusca (Brandenburg, 1966). By following the transformation of the larval nephridium during metamorphosis (summary Fig. 4), it is possible to predict that adult asteroids and enteropneusts also possess discrete, functional nephridia. The heart complex, situated in the proboscis of adult enteropneusts, has the structural components of a metanephridial system, including a capillary bed overlain with podocytes associated with the heart ("glomer- ulus"; i 2G; Wilke, 1971). Modification of the ultrafiltrate could occur in the pro- boscis cotiorn, thus delivering nutrients to the proboscis musculature, a tissue with a poorly developed vascular supply. Some modification may also occur along the pro- boscis duct before a fluid is discharged by the ciliary excurrent at the proboscis pore (Balser, 1985, material in prep.). The axial complex in adult asteroids, associated with the madreporite, also has the structure of a metanephridial system. We speculate that vascular fluid in the cap- FIGURE 4. Comparison of larval nephridia with adult derivatives. The following conventions apply throughout: small arrows = presumed direction of filtration, larger arrows = observed direction of ciliary beat, cross hatching = pulsatile vesicles in larvae and their derivatives in the adults [madreporic vesicle or dorsal sac (ds) in Asterias, pericardium (pe) in Schizocardium], dashed lines = coelomic epithelia composed of podocytes, loose stipple = anterior larval coeloms and their adult derivatives (axial sinus (axs) of Asterias and proboscis coelom (pr) of Schizocardium), fine stipple = blastocoel of larvae and their adult derivative, the blood vascular system, bold lines = larval ectodermal pore canals and adult derivatives [pore canals of the madreporite (md) of Asterias and the proboscis duct (pd) of Schizocardium]. (A) Lateral view of the bipinnaria of Asterias showing nephridial organs. (B) Disproportionately enlarged apical portion of axial complex of adult Asterias illustrating anatomical relationships of major components as viewed in vertical section: madreporite (md), madreporic vesicle (ds), stone canal (sc), axial sinus (axs), axial gland (axg), gastric haemal tuft (ght), genital coelom (gc), and perivisceral coelom (pvc). The madreporic vesicle is contractile like its ontogenetic precursor, the pulsatile vesicle. Both the gastric haemal tuft (ght) and axial gland (axg) are tangles of small blood vessels. The gastric haemal tuft spans the pervisceral coelom (pvc) to join the major vessels of the gut while the axial gland vessels join the hyponeural sinus vessels, a portion of the blood vascular system (BVS) and its surrounding coelom that parallels the distribution and extent of the nervous system. The genital coelom (gc) and the BVS it encloses form a ring at the aboral surface of the disc. Vessels from the gut, genital, and peripheral parts of the BVS communicate directly (asterisk) with the vascular cavity ("head process" of axial gland) enclosed by the contractile madreporic vesicle (ds). Pressure for filtration of vascular fluid in the axial gland vessels across the ECM and podocytes of the axial sinus coelom (axs) may be generated by the madreporic vesicle or perhaps by contractions of vessels within the axial gland itself. (C) Lateral view of the tornaria of Schizocardium showing nephridial organs. (D) Disproportionately enlarged proboscis organs of adult Schizocardium illustrating anatomical relationships of major components: proboscis duct (pd), pericardium (pe), glomerulus (gl), proboscis coelom (pr), and stomochord (st). The pericardium (pe) is contractile like its ontogenetic precursor, the pulsatile vesicle. Pressure for filtration of vascular fluid from the heart and glomerular vessels across the ECM and podocytes of the proboscis coelom may be generated by the pericardium or by contraction of vessels entering the central sinus of the heart. 195 196 E. E. RUPPERT AND E. J. BALSER illary bed of the axi; ;! i .Sand is pressure filtered across the layer of peritoneal podocytes (Fig. IF; Bare and von Hehn, 1968) into the axial sinus (a coelomic cavity) where modific could occur. A fluid could be discharged by the ciliary excurrent from the pon > f the madreporite. The rf< ion of discrete excretory organs in larval hemichordates and echi- noderms was prompted by a consideration of a general model for nephridial design and function (Ruppert and Smith, 1985, in prep.). We anticipate that further exper- imental investigations of other predictions in the model will lead to similar discoveries and an enhanced understanding of renal function and nutrient translocation in animals. ACKNOWLEDGMENTS Thanks to D. G. Heckel for writing the computer programs and to S. A. Gauthreaux for use of the motion analyzer. Discussions with J. M. Colacino, J. E. Doeller, J. D. Ferraris, D. W. Kraus, J. M. Lawrence, E. B. Pivorun, S. Smiley, P. R. Smith, J. M. Turbeville, A. P. Wheeler, and J. P. Wourms were much appreciated. The research was supported by NSF Grant No. BSR-8408500 to E. E. Ruppert. LITERATURE CITED BALSER, E. J. 1985. Ultrastructure and function of the proboscis complex of Saccoglossus (Enteropneusta). Am. Zool. 25: 41 A. BARGMANN, W., AND G. VON HEHN. 1968. Uber das Axialorgan ("mysterious gland") von Asterias rubens L. Z. Zellforsch. 88: 262-277. BARNES, R. D. 1980. Invertebrate Zoology, 4th Ed. Saunders College, Philadelphia. PA. 1089 pp. BINYON, J. 1966. Salinity tolerance and ionic regulation. Pp. 359-378 in Physiology of Echinodermata, R. A. Boolootian, ed. Interscience Publ. New York, NY. BRANDENBURG, J. 1966. Die Reusenformen der Cyrtocyten. Zool. Beitr. N. F. 12: 345-417. BURY, H. 1896. The metamorphosis of echinoderms. Q. J Microsc. Sci. 38: 45-131. EMIG, C. C. 1982. The biology of Phoronida. Adv. Mar. Biol. 19: 1-89. Fox, R. S., AND E. E. RUPPERT, 1985. Shallow-Water Marine Benthic Macroinvertebrates of South Carolina. U. So. Carolina Press, Columbia, SC. 329 pp. GEMMILL, J. F. 1914. The development and certain points in the adult structure of the starfish Asterias rubens. L. R. Soc. Lond. Phil. Trans. B 205: 213-294. GEMMILL, J. F. 1918. Rhythmic pulsation in the madreporic vesicle of young ophiuroids. Q. J. Microsc. Sci. 10: 239-278. GOODRICH, E. S. 1945. The study of nephridia and genital ducts since 1895. Q. J. Microsc. Sci. 86: 113- 392. HORST, C. J. VAN DER. 1939. Hemichordata. Kl. Ordn. Tierreichs 4: 1-737. HYMAN, L. H. 1955. The Invertebrates: Echinodermata. McGraw-Hill Book Co., New York, NY. 763 pp. HYMAN, L. H. 1959. The Invertebrates: Smaller Coelomate Groups. McGraw-Hill Book Co., New York, NY. 783 pp. KJRSCHNER, L. B. 1967. Comparative physiology: invertebrate excretory organs. Ann. Rev. Phvsiol. 29: 169-196. MORGAN, T. H. 1894. The development of Balanoglossus. J. Morphol. 9: 1-86. OGLESBY, L. C. 1981. Volume regulation in aquatic invertebrates. J. Exp. Zool. 215: 289-301. PEARSON, C. E. 1974. Handbook of Applied Mathematics, van Nostrand Reinhold, New York, NY. 1265 PP. Rn . M.. AND E. E. RUPPERT. 1978. Resin embedments of quantitative meiofauna for ecological and tural studies description and application. Mar. Biol. 46: 223-235. RUPPERI , AND P. R. SMITH. 1985. A model to explain nephridial diversity in animals. Am. Zool. 25: 41 WILKE, U. 1 i )ie Feinstruktur des Glomerulus von Glossobalanus minutus Kowalevsky (Enteropneusta). Cyi- 5:439-447. Reference: Biol. Bull. 171: 197-207. (August, 1986) RESOURCE PARTITIONING BY SAND DOLLARS IN CARBONATE AND SILICEOUS SEDIMENTS: EVIDENCE FROM PODIAL AND PARTICLE DIMENSIONS MALCOLM TELFORD AND RICH MOOI Department of Zoology, University of Toronto, Ontario, Canada M5S 1A1 ABSTRACT The sand dollars, Leodia sexiesperforata (Leske) and Encope michelini L. Agassiz, have overlapping geographical ranges and may co-occur in mixed flocks. Leodia is restricted entirely to biogenic carbonate sediments. Mellita quinquiesperforata (Leske), which has a similar geographical range to Leodia, occurs only on siliceous terrigenous substrates and the two species never co-exist. Encope michelini L. Agassiz occurs on both types of substrate. All three species are podial particle pickers, and use barrel- tipped podia, especially the long type surrounding the geniculate spine fields of the oral surface, for food collection. A typical mellitid of 100 mm diameter can have up to one million barrel-tipped podia. These podia have the same mean diameters in Leodia (71.6 5.62 ^m) and Mellita (71.8 3.59 urn). The diversity of sizes is significantly greater in Leodia. The barrel-tipped podia of E. michelini are very much larger (104.4 11.1 ^m). The substrates inhabited by the three species have approx- imately 90% of their particles in the 100-400 urn range. Whereas Mellita is non- selective in collecting food particles, Leodia clearly selects small particles (50-200 fj.m) and shuns those above 200 nm. Encope michelini includes 26% of particles over 200 ^m in its food grooves, but does not take those below 100 nm. Differences in feeding behavior thus provide a basis for resource partitioning between these sympatric species. They are discussed in relation to podial dimensions and spination, and compared with feeding behavior in Mellita quinquiesperforata. INTRODUCTION The feeding activities of sand dollars have been extensively investigated in recent years. It has become clear that many of them rely on oral surface podia for the selection of food material from the substrate. The tiny fibulariid, Echinocyamus pusillus (O. F. Muller), picks up diatoms, debris, or sediment particles and conveys them by podia to the mouth where they are chewed or scraped by the lantern teeth (Telford el al, 1983). Echinarachnius parma (Lamarck) (Echinarachniidae) ingests sediment material without apparent selection of any particular size range, although diatoms are actively selected (Filers and Telford, 1984). The lunulate sand dollar, Mellita quinquiesperforata (Leske) (Mellitidae), similarly collects sediment particles by means of oral surface podia and fractures them by action of the lantern teeth as they are ingested (Telford et al., 1985). These studies all suggest that sand dollars feed in exactly the way that should be expected of an echinoid, despite their curious shape. These studies have directly challenged the former hypothesis of an aboral sieve mechanism proposed by Goodbody (1960). In this paper we exaTiine the feeding mechanisms of two more mellitid sand dollars, Leodia sexiesperforata (Leske) and Encope michelini L. Agassiz, which occur Received 13 March 1986; accepted 14 May 1986. 197 198 M. TELFORD AND R. MOOI in mixed flocks. Ou : purpose was to determine whether other members of this family use the same po: ding mechanism and to explore the functional significance of differences in \ r>iiology of podia and spines. Two species living together might be expected to s Jifferences in their use of the common food resource. Mooi ( 1986a, b) has descri* ' some differences between the podia of these species. In this paper we examine the relationship between podial dimensions and the size of sediment particles collected du ing the feeding process. This is the first published description of feeding in any species of Encope and the first re-examination of Leodia sexiesperforata since Goodbody (1960) proposed the sieve mechanism for that species. A direct comparison will be made between these two species, which live on biogenic carbonate sediments, and Mellita quinquiesperforata, which lives on terrigenous siliceous sediments. MATERIALS AND METHODS Specimens of L. sexiesperforata and E. michelini, 50-110 mm in length, were collected in shallow water (3-15 m) off Long Key, Florida, during July, 1984, and maintained in running seawater with natural substrate in the laboratory. Observations of feeding and ciliary currents were made on live specimens using methods described elsewhere (Telford et ai, 1985). Several specimens were fixed in the field, in 10% formalin buffered in seawater, for analysis of gut and food groove contents. Six sediment samples of approximately 500 ml were collected from the surface (top 20 mm) in different places among the sand dollar flock. These were fixed in 10% buffered formalin to preserve living organisms and organic material. Larger samples were collected from time to time and kept fresh for use in holding tanks and for feeding observations. Additional specimens of L. sexiesperforata and substrate samples obtained from Eleuthera, Bahamas, in February, 1983, were preserved in the same way. Measurements of podia and distribution of podial pores in specimens of E. michelini and L. sexies- perforata collected at Torch Key, Florida (1982) were compared with specimens of Mellita quinquiesperforata, collected at Atlantic Beach, North Carolina (1982). Specimens of the three species, from personal collections and those of the United States National Museum (USNM), were examined as follows (USNM catalog numbers in parentheses): Leodia sexiesperforata: personal collections from Bahamas, Barbados, Florida Keys and Panama; USNM collections from Bermuda (El 4495), Bahamas (El 4892, E9009, #32651), Cuba (E10384), Dominican Republic (E14559), Puerto Rico (#19656), St. Thomas (El 183), St. Kitts (#7000), Windward Islands (E14560), Belize (#18932), Panama (#14579), Colombia (E14561), and Brazil (#5388). Mellita quinquiesperforata: personal collections from North and South Carolina, Georgia, Atlantic and Gulf coasts of Florida; USNM collections from Virginia (#4980), Alabama (El 59 12, El 59 14, El 59 18, #25416-22), Louisiana (E6797), Texas (E6581-2, E5350), Panama (E14584), Colombia (E8091-2), Trinidad (El 4062), Puerto Rico (E6608-1 1), and Brazil (El 7 195). Encode michelini: personal collections from Long Key, Pigeon Key and Torch Key, Florida; USNM collections from South Carolina (E30005), Georgia (E29843- 6), Gu, coast of Florida (#2185), Gulf of Mexico (E26711, E26714) and Brazil (E26706). Podia (Mooi, 1986a, b) and spines (Telford et al, 1985) were classified and the distribution of different types on the sand dollars was mapped. Isolated spines were measured by ocular micrometer; inter-spine distances were similarly estimated from SAND DOLLAR RESOURCE PARTITIONING 199 live and freshly killed specimens under a binocular microscope. Distribution of cilia on different spine types was examined by light microscopy of isolated spines. Tip diameters of barrel-tipped podia were measured by eye-piece micrometer. Tissue was scraped away from the oral surface in ambulacra I, II, and III from four different specimens of each species, and mounted on a microscope slide. Diameters of 20 podia from each ambulacrum were determined. The data were pooled for each species and means and standard deviations calculated for the combined 240 mea- surements. Large specimens were chosen so that differences between species would not be obscured by size differences. Specimens of E. michelini ranged in size from 93.3 X 94.0 to 104.0 X 105.8 mm; L. sexiesperforata from 91.1 X 90.8 to 101.6 X 108.0 mm and M. quinquiesperforata from 92.8 X 97.8 up to 98.0 X 99.6 mm. Numbers of pores within the geniculate spine fields of these specimens were determined by cutting out small pieces of test and dissolving soft tissues in 5.25% sodium hypochlorite (commercial strength bleach). Cleaned pieces were dry-mounted on a slide and ex- amined with transmitted light. The total number of podial pores in each of five fields of view in each ambulacrum (I, II, and III) was determined. The data were pooled for each specimen and the means calculated as number of podial pores per square mil- limeter of geniculate spine field. Estimates of total oral surface area and of the different spine fields were made by cutting out photographic reproductions of the sand dollars and weighing the individual areas. Analyses of natural substrate, food groove material, and gut contents followed the methods developed by Ellers and Telford (1984) and by Telford et al, (1985). Very small samples of substrate were strewn on glass microscope slides. All particles within several fields of view were drawn in outline by camera lucida after which length and width of at least 1000 from each sample were measured. Material from the food grooves was treated in the same way, but the sample sizes were somewhat smaller. Sediment grains were assigned to size classes 0-24, 25-49, 50-99, 100-199, 200-399, 400-799, and >800 ^m and size-frequency histograms were constructed. The units used by geologists were not used because our interest centered on the numbers and size of sand grains and diameters of podial tips, not on sieve analyses of mass. However, our sediment analysis program also calculated the size-frequency distributions ac- cording to size classes for a standard sieve series, for purposes of comparison with other published data. Mean, standard deviation, and elongation (width/length) was calculated and the degree of angularity of the grains was estimated (Leeder, 1982). The significance of differences between particle size-frequency distributions of indi- vidual substrate samples and food groove samples was tested by Chi-square analysis, and between means by the t-statistic. All statistical procedures followed Sokal and Rolf (1981). Acid soluble carbonate was determined gravimetrically following digestion in HC1 or EDTA. RESULTS Ciliary currents among the spines of L. sexiesperforata and E. michelini are similar to those already described for M. quinquiesperforata (Telford et al, 1985). On the aboral surface two quite distinct patterns of flow occur. Within the petaloids, there is a central area of centrifugal flow. Between the respiratory podia, flow is directed from the center of the petaloid (ambulacrum) towards the outside (interambulacrum). In- ternally, coelomic fluid and cells flow from the outer to the inner pore of each podium, exactly counter to the external current. Around the petaloids, in the interambulacra, the respiratory flow is entrained in a general centrifugal current. Centrifugal flow appears to be diverted close to the lunules, so that their walls are washed by a downward flow within the depth of the spine field. It is easy to exaggerate the current towards 200 M. TELFORD AND R. MOOI the lunules. Row is usually visualized by means of suspended particles which may occur natural!) (< ais, etc.), or be introduced (Anemia eggs, black ink, etc.). Particles never describe ; : aight centrifugal trajectory. The spines are placed somewhat like the pins in a r -ul machine. Suspended particles may be carried around a spine, or be driven t right or left. Thus a group of particles will tend to fan out in the centrifugal iow and, inevitably, some will be carried passively towards the lunules. As in - uinquiesperforata, most particles reaching the ambitus or passing via the lunules, are deposited into the sediment. There is a complex pattern of flow across the oral surface, which must be visualized in three dimensions. In the centers of the locomotory spine fields and the pressure drainage channels (Fig. 1 ) flow is centripetal but strongly divergent towards the margins of those zones, where it is deflected down- wards. At the peristome small centripetal components remain and these unite in a downward flow beneath the mouth. There are some small differences in the flow patterns of M. quinquiesperforata and other species treated in this paper and they can be best attributed to differences in the relative sizes of the spine fields. Estimates of 5.0cm FIGURE 1 . Distribution of spine types (left half) and ciliary currents (right half) on (a) Leodia sexies- perforata and (b) Encope michelini. For left half: f, fringe spines; g, geniculate spines; p, pressure drainage channel (pdc) spines; c, circum-oral spines; a, anal spines; fg, food grooves. For right half: arrows indicate direction of ciliary current flow; circles with central dot show convergence of currents with resulting downward flow to the substrate; locomotory areas stippled. SAND DOLLAR RESOURCE PARTITIONING 201 particle velocities in the ciliary currents were alike in all three species, ranging from 0.50 to 0.95 mm-s' 1 . In both E. michelini and L. sexiesperforata, aboral spination is very similar to that of M. quinquiesperforata (Telford et al, 1985), but those of Leodia are more slender, those of E. michelini generally larger and more robust. The spines have the same orientations with respect to body axis, anterior to posterior size gradations and ratios of miliary to club-shaped spines. Spines of the oral surface are equally diversified into locomotory, geniculate, pdc spines, but in Leodia and Encope, the locomotory areas are relatively smaller and the geniculate areas correspondingly larger. In M. quinquiesperforata the locomotory spines occupy more than 30% of the aboral surface area, whereas in E. michelini and L. sexiesperforata they occupy only 25 and 20% of the surface, respectively. Interspine distances for Leodia and Encope are given in Table I. Locomotory spines in these two species are equally spaced but there are some dif- ferences in the spacing of geniculate and other spines. The ciliary currents described above are powered by diametrically opposed bands of cilia which extend for about 100 ^m along the shaft of each spine from its base. Barrel-tipped podia occur between the geniculate spines in all three species. In E. michelini the tip diameter is 104.4 1 1.08 ^m (n = 240); in L. sexiesperforata it is 71.6 5.62 ^m (n = 240) and in M. quinquiesperforata it is almost identical (71.8 3.59), but Leodia has a significantly greater range of podial sizes (P < 0.001) (Fig. 2). The difference between E. michelini and the other two species is statistically significant (P <^ 0.001). There are highly significant differences between the densities of podial pores in these three species (P <^ 0.001) (Table II). Geniculate spine fields occupy approximately 75% of the total oral surface in Leodia, about 70% in E. michelini, and 65% in Mellita. Pressure drainage channels represent only about 5% of oral surface area in all three species. Leodia sexiesperforata and E. michelini were collected from mixed flocks in shallow water, 5-10 m depth. They were always found on biogenic, carbonate sediments con- TABLE I Interspine spacing (^m) in Leodia sexiesperforata and Encope michelini L. sexiesperforata E. michelini Spine types Space S.D. Space Locomotory-locomotory 303.0 30.2 297.8 38.2 Locomotory-miliary 190.4 26.5 180.0 35.7 Pdc-pdc 498.5 57.8 448.2 70.0 Pdc-miliary 214.8 33.8 240.7 63.3 Geniculate-geniculate 181.5 22.0 223.7 46.9 Anterior fringe-fringe 277.0 37.2 317.0 50.5 Posterior fringe-fringe 186.7 31.1 281.5 43.8 Anterior club-club 286.7 41.5 263.0 37.8 Anterior club-miliary 134.8 30.5 144.4 19.3 Anterior miliary-miliary 79.3 20.4 129.6 17.7 Posterior club-club 321.1 35.1 201.5 37.5 Posterior club-miliary 80.5 13.8 88.2 20.2 Posterior miliary-miliary 62.2 14.0 58.5 14.8 Anal lunule marginals 233.3 39.3 252.6 35.4 Lunular club-miliary 88.2 18.7 99.3 22.0 Lateral lunule marginals 189.6 33.5 Spaces given are mean distances between spine shafts immediately above the basal collar. For all mea- surements, n = 25. 202 M. TELFORD AND R. MOOI i Leodia Encope 60 80 100 120 PODIAL TIP DIAMETER (urn) 140 FIGURE 2. Mean (vertical bar), standard deviation (box) and range of sizes (urn) (horizontal line) of short barrel-tipped podia in the geniculate spine fields ofMellita quinquiesperforata. Leodia sexiesperforata, and Encope michelini. sisting of fragments of coralline algae (Halimeda etc.), shell, and coral debris. Ap- proximately 90% of the particles were in the 100-400 nm range (Fig. 3), with a mean size of 213 68.4 /urn. The Eleuthera (Bahamas) sample, where only Leodia was found, had a lower mean grain size (158 43.1) due to larger numbers of particles (23%) between 50 and 100 /urn. Mellita quinquiesperforata occurs on terrigenous, siliceous sediments with a similar percentage of particles in the 100-400 /um range (Fig. 3) and a mean size of 186 63.9. The remaining 10% of the Mellita substrate was less than 100 um. In the biogenic sediment, 6% of the particles exceeded 400 /an. The mean elongation of the rather angular quartz sand grains was 0.70. The biogenic TABLE II Density ofpodial pores per mm 2 in the geniculate spine fields o/~Mellita quinquiesperforata, Leodia sexiesperforata, and Encope michelini Ambulacrum 1 Ambulacrum 2 Ambulacrum 3 Mean S.D. Mellita 1 140 168 180 Mellita 2 136 173 167 Mellita 3 147 170 178 Mellita 4 144 168 183 163 16.5 Leodia 1 115 139 143 Lem 111 136 146 Leoc. 120 135 150 Leodia - 109 134 140 132 14.1 Encope 1 100 102 103 Encope 2 98 107 99 Encope 3 91 104 105 Encope 4 95 99 102 100 4.4 SAND DOLLAR RESOURCE PARTITIONING 203 O o LU O cr LU 90. D Siliceous sediment ED Mellita D Carbonate sediment D Leodia ,_, 80_ B Encope 70. I 60. : 50. I 40. 30. | [-, 20_ P IS ill 10. | Hi i <^ | - Tin 4567 PARTICLE 1234 SIZE CLASSES FIGURE 3. Particle size distributions in natural siliceous and carbonate sediments and food grooves of sand dollars inhabiting them. Mean particle size in the siliceous sediment was 186.2 63.91 nm (n = 3109), and in the carbonate sediment 212.9 68.43 nm (n = 3362). Within the food grooves, mean particle sizes were: Mellita, 181.7 54.23 ^m (n = 2735); Leodia, 140.8 34.64 ^m (n = 2969); Encope, 183.0 88.52 M m (n = 2418). grains were more rounded, distinctly subangular, with mean elongation of 0.72. Dis- solution of small subsamples showed that this sediment was 100% carbonate. However, under the microscope occasional non-carbonate particles were observed. When the biogenic particles were dissolved carefully in 1-2% HC1 or in EDTA, delicate organic "ghosts" of the grains became visible. Leodia and Encope, like Mellita, are podial particle pickers. During feeding long barrel-tipped podia around the geniculate spine fields pick up individual sand grains which are then passed from podium to podium in an orderly progression towards the food grooves. As the particles are passed along, they can be held beneath the tips of the geniculate spines, in the space above the sediment, or they may pass between the tips of the spines, which are in constant motion during feeding. Upon arrival at the food grooves, the particles are moved centripetally towards the mouth by action of the food groove podia. Adhesion of particles and podia is at least partially due to a sticky glue-like substance (Thomas and Hermans, 1985). As the particles become coated with this material they readily adhere to each other and form cohesive strings in the food grooves. Analysis of the size-frequency distributions of particles in the food grooves (Fig. 3), shows that Mellita is non-selective: it collects particles in essen- tially the same proportions as they occur in the substrate. In the food grooves of Leodia, 87% of the particles were in the size range 100-200 yum, and the mean was 141 34.6. Particles in the food grooves of E. michelini had a greater mean size (183 88.5 nm). Differences between the mean sizes and the frequency distributions for these two species were statistically significant (P <^ 0.00 1 ). In both species, food groove material included a slightly higher proportion of forams and diatoms than observed in the native sediment. Particle size-frequency analysis of gut contents from Leodia and E. michelini was not possible by the methods used here. The contents included a large proportion of well pulverized sand grains, among a few larger particles. Some fragments were large enough to identify as broken forams, diatoms, and shell debris, but most of the material was amorphous and cohesive. 204 M. TELFORD AND R. MOOI Fine material, such as particles of carmine and black ink (used for flow visualiza- tion), were depo m the sediment around the ambitus. Those particles which were included in on ' Kice flow were brought to the edges of the locomotory spine fields where thev v. Jien deposited by downward currents. Some of this fine material adhered to r. a and sand grains. As a consequence, some was included in the food groove mai al, still clinging to larger particles. We saw no evidence that this fine materi Jeliberately selected. In fact, most of it was left in the sediment. DISCUSSION Leodia sexiesperforata and Encope michelini have partially overlapping distri- butional ranges and often occur together in mixed flocks. Leodia ranges from North Carolina to Uruguay, including the Florida Keys, Bahamas, Greater and Lesser Antilles, and the Gulf of Mexico. Encope michelini is distributed from North Carolina south to the Florida Keys and throughout the Gulf of Mexico, but not in the Bahamas (Serafy, 1979). Mellita quinquiesperforata extends from Massachusetts to Florida, throughout the Gulf, Caribbean, Central and South America to Brazil, as well as all the Antilles (Serafy, 1979). From our own field experience and examination of museum material, it is apparent that Leodia occurs only on biogenic sands and that Mellita is restricted to terrigenous sediments. It is curious that this very striking feature of dis- tribution has not been remarked upon previously. Encope michelini appears to inhabit both sediment types. In our own field studies we have only found this species on the biogenic sediments of the Florida Keys. However, examination of museum specimens from South Carolina, Georgia, elsewhere in Florida, and the Gulf of Mexico, showed mixtures of shell debris and substantial amounts of quartz grains in the food grooves. The sediment particle sizes at the different collection sites were very similar. The mean particle size of siliceous material of Atlantic Beach (Mellita) is remarkably close to previously reported values for Bird Shoal, (180.8 59.74) (Telford et al., 1985) and for Florida (Serafy, 1979) ". . . fine quartz sand with modal grain size of 0.18 mm . . ." They are also quite comparable with the data reported by Weihe and Gray (1968) for their collecting sites in North Carolina. The mixed flocks of Leodia and E. michelini occur on a substrate incorporating a small but significant number of particles over 400 ^m (6%), but otherwise very like the Mellita substrate. Many authors, in- cluding those already cited, have remarked on the scarcity of fine particles (<50 ^m) in sand dollar habitats. The sediment analyses provided by Lane and Lawrence ( 1 982) for a Mellita population near Tampa (Florida), showed 92% of the grains in the 125- 250 fj.m size class, 5% in the 62.5-125 class, and the rest smaller than that. Our Mellita substrates did include 10% of the particles below 100 ^m, but there was a substantial proportion over 250 ^m (Fig. 3). There are small differences in spination between the three species. On the aboral surface, the miliary sacs which fill the spaces between the tips of club spines, preventing the entry of particles during burrowing (Mooi, in press), are largest in Mellita and smallest in E. michelini. The generally centripetal ciliary currents remove the few small particles which drop through the protective canopy (Mooi, in press; Telford et al, 1985 } 'nterspine spacing on the aboral surface is wider in Leodia and Encope and the latter i 3 wider spacing between the ambital fringe spines. On the oral surface, spacing bei ^ en locomotory spines is widest in Mellita (460 86 ^m) but all other inter-spine ci-r ?r,ces are somewhat smaller. Within the geniculate spine fields Mellita has significantly more barrel-tipped podia per mm 2 (Table II) and Leodia likewise has more than E. michelini. These are the food gathering podia, which adhere to collected particles. Mean podial diameters reported here for Mellita (72 ^m), based on large SAND DOLLAR RESOURCE PARTITIONING 205 numbers of measurements, are somewhat smaller than those reported by Phelan ( 1 977) (84 yum) and much smaller than our own earlier reported value of 120 ^m (Telford et ai, 1985) which, in retrospect, appears to represent an extreme and not the typical size of food gathering podia. In fact, these suckered podia in Mellita rarely approach 100 Aim, even close to the peristome. Encope michelini has very much larger podia, none of them as small as the mean sizes for Mellita and Leodia (Fig. 2). All three species use the impressively large numbers of podia on the oral surface for the collection and transport of food. Leodia has the most extensive geniculate spine areas and we estimate that individuals 100 mm in length have approximately 1 X 10 6 barrel-tipped podia, of which 150 X 10 3 are food collecting (long b-t) podia. Mellita has some 0.85 X 10 6 b-t podia (125 X 10 3 long) and E. michelini 0.70 X 10 6 (100 X 10 3 long). According to our estimates, the total area of the suckered podial tips in Mellita and Leodia represents over 40% of the total oral surface area and about 15% of this is the actual food collecting, long barrel-tipped podia. In E. michelini the total is even higher, about 60% of the total oral surface area, with a similar proportion of long b-t podia. Ciliary currents do not contribute significantly to the feeding process. As reported earlier (Telford et ai, 1985), Mellita is non-selective in its feeding. Both Leodia and E. michelini (Fig. 3) include disproportionately high percentages of the 100-200 Atm fraction in their food grooves. Leodia also appears to select from the 50- 100 /urn class and to shun particles greater than 200 /urn. Encope michelini takes very few particles less than 100 A*m and includes significant amounts (26%) above 200 /j.m. Although one would not expect sand to be a limiting resource for these cohabiting sand dollars, some divergence of feeding and, hence, partitioning of the food resource appears to occur. Hammond (1982) concluded that sympatric holothuroids and echi- noids in a similar habitat in Jamaica, did not show any resource partitioning: they all ingested sediment mixtures very similar to the composition of the surrounding sand. Although they are nonselective for particle sizes, Hammond (1983) did find evidence that some holothuroids and echinoids were selective for the organic content of grains. Ellers and Telford (1984) and Telford et al. (1985) found that feeding in clypeasteroids could be stimulated by presentation of diatom-enriched material, and both Leodia and E. michelini included relatively high proportions of forams and diatoms in the food grooves, suggesting that these were being actively selected. Scheibling (1980) found that microphagous feeding in the asteroid Oreaster reticulatus was similarly stimulated by the presence of diatoms, suggesting that selection of particles for nutrient content might, indeed, be widespread in deposit feeding echinoderms. The small dif- ferences in spination and the larger differences in podial dimensions and distribution, do not supply a ready mechanical explanation for resource partitioning by Leodia and E. michelini. It might be expected that species collecting larger particles would have wider spaces between the geniculate spines. This does not appear to be borne out by observation. The space between spine tips is constantly changing as the spines move and it is clear that Mellita, at least, is able to collect a mean particle size ( 1 80 Aim) which is greater than the mean stationary inter-spine distance (150 urn)- With podia very much like those of Leodia, Mellita nonetheless collects food particles more like the size range taken by E. michelini. It is possible that spacing between geniculate spines and podial tip dimensions together provide an upper limit to the size of particle handled. We suspect also that the podia are poorly suited to adhesion on particles substantially smaller than themselves and that this sets a lower limit. Certainly, the mean food groove particle sizes are 1.5 to 2.5 times the podial tip dimensions. In a comparison of E. aberrans and E. michelini, Phelan (1972) speculated that they might prefer different particle size ranges and noted that the dimensions of the food grooves and peristome differed. Encope michelini has wider, less distinct food grooves ( 750 206 M. TELFORD AND R. MOOI jum) than either Leodia or Mellita (~600 pm). Peristome diameters of Mellita and E. michelini (~3.75 mm) are significantly larger than Leodia (2.25 mm), in similar sized individuals. T<" e dimensions are 1 5-20 times the mean linear dimensions of ingested particles difficult to see how these peristome diameters might influence the size of parti- :' elected as food. We would suggest that E. michelini and Leodia might tolerate ^r sediment size ranges and particle size-frequency distributions. Both have gr podial size diversity than Mellita, which might allow them to handle a wider div of food particles. We suspect that Leodia overlaps the distribution of E. mi'.-:, rti only towards the upper and lower extremes of their respective particle tolerance . We can offer no explanation at present for the separation of Mellita and Leodia on different substrate types, both of which seem to be acceptable to Encope species. When Goodbody (1960) examined stomach contents in L. sexiesperforata he was unable to identify much of the material but remarked on the small size of the particles. Until recently this has been one of the mainstays of the sieve hypothesis but Telford et al. (1985) concluded that the lantern of Mellita quinquiesperforata fragmented sand grains as they were ingested. Carbonate sand grains contain an organic matrix in which the calcite is originally deposited and, in addition, they are extensively penetrated by fine filaments of algae and sponges. It is this organic material which becomes visible under the microscope as an organic "ghost" of the sand grain following slow dissolving. They may thus be a more rewarding nutrient source than siliceous grains bearing only surface organics. The penetration by algae and sponges also makes these grains much more readily breakable by the lantern teeth. For this reason, sand grains ingested by Leodia and E. michelini are thoroughly pulverized into an unrecognizable paste, whereas the siliceous grains ingested by Mellita are broken into smaller but still rec- ognizable granules. ACKNOWLEDGMENTS This work was supported by the Natural Sciences and Engineering Research Council of Canada through Operating Grant #A4696. We are indebted to the Director and Staff of the Sea World Shark Institute, Long Key, Florida, for the use of research and boat facilities. We also thank our colleagues Tony Harold and Hugh Griffith for their entertaining company, help in field work, and many discussions. LITERATURE CITED ELLERS, O., AND M. TELFORD. 1984. Collection of food by oral surface podia in the sand dollar, Echinar- achnius parma (Lamarck). Biol Bull. 166: 574-585. GOODBODY, I. 1960. The feeding mechanism in the sand dollar, Mellita sexiesperforata (Leske). Biol. Bull. 119:80-86. HAMMOND, L. S. 1982. Analysis of grain-size selection by deposit-feeding holothurians and echinoids (Echi- nodermata) from a shallow reef lagoon, Discovery Bay, Jamaica, West Indies. Mar. Ecol. Prog. Ser. 8: 25-36. HAMMOND, L. S. 1983. Nutrition of deposit feeding holothuroids and echinoids (Echinodermata) from a reef lagoon. Discovery Bay, Jamaica, West Indies. Mar. Ecol. Prog. Ser. 10: 297-306. LANE, J. J. M. LAWRENCE. 1982. Food, feeding and absorption efficiencies of the sand dollar, Mt. "iinquiesperforata (Leske). Estuarine Coastal Shelf Sci. 14: 421-431. LEEDER, M. 1- . Sedimentology: Process and Product. George Allen and Unwin, London. 334 pp. MOOI, R. 1986a. : piratory podia of clypeasteroids (Echinodermata, Echinoides): I. Functional anatomy. Zoomotj 106:21-30. MOOI, R. 1986b. K< spiratory podia of clypeasteroids (Echinodermata, Echinoides). II. Diversity. Zoo- morphology 10 , ^ -90. MOOI, R. Structure and function of clypeasteroid miliary spines (Echinodermata, Echinoides). Zoomorphology (In press). SAND DOLLAR RESOURCE PARTITIONING 207 PHELAN, T. F. 1972. Comments on the echinoid genus Encope and a new subgenus. Proc. Biol. Soc. Wash. 85: 109-130. PHELAN, T. H. 1977. Comments on the water vascular system, food grooves, and ancestry of the clypeasteroid echinoids. Bull. Mar. Sci. 27: 400-422. SERAFY, D. K. 1979. Memoirs of the Hourglass cruises. V(III): Echinoids (Echinodermata: Echinoidea). Florida Department of Natural Resources, St. Petersburg, Florida. 1 20 pp. SCHEIBLING, R. E. 1980. The microphagous feeding behavior of Oreaster reticulatus (Echinodermata: As- teroidea). Mar. Behav. Physiol. 7: 225-232. SOKAL, R. R., AND F J. ROHLF. 1981. Biometry. 2nd ed., W. H. Freeman and Co., San Francisco. 859 pp. TELFORD. M., A. S. HAROLD, AND R. Mooi. 1983. Feeding structures, behavior and microhabitat ofEchin- ocyamus pusillus (Echinoidea: Clypeasteroida). Biol. Bull. 151: 745-757. TELFORD, M., R. Mooi, AND O. ELLERS. 1985. A new model of podial deposit feeding in the sand dollar, Mellita quinquiesperforata (Leske): the sieve hypothesis challenged. Biol. Bull. 169: 431-448. THOMAS, L. A., AND C. O. HERMANS. 1985. Adhesive interactions between the tube feet of a starfish, Leptasterias hexactis, and substrata. Biol. Bull. 169: 675-688. WEIHE, S. C., AND I. E. GRAY. 1968. Observations on the biology of the sand dollar Mellita quinquiesperforata (Leske). J. Elisha Mitchell Sci. Soc. 84: 315-327. Reference: Biol. Bull. Ill: 208-216. (August, 1986) EFFECTS OF OPIOIDS AND ANTAGONISTS ON THE RATE OF SEA URCHIN SPERM PROGRESSIVE MOTILITY L. CARIELLO 1 , L. ZANETTI 1 , A. SPAGNUOLO 1 , AND L. NELSON 2 * l Stazione Zoologica, Naples, Italy; ^Department of Physiology, Medical College of Ohio, Toledo, Ohio 43699; and Marine Biological Laboratory, Woods Hole, Massachusetts ABSTRACT Opioids exert a profound effect on the motility of sea urchin sperm cells suspended in artificial seawater (ASW). With prolonged exposure to narcotic agents, the rate of progression increased in a time- and dose-dependent manner up to an optimum con- centration. Methionine enkephalin acetate (metenkephalin) caused a doubling in the rate of progression while the opiate antagonist, ( )-naloxone caused the sperm cells to increase their rate of forward motion by up to 75% above the control rate. The metenkephalin increase occurred at lower concentrations than the naloxone optimum. In combination, a subthreshold dose of (-)-naloxone, completely abolished the stim- ulatory effects of metenkephalin. Plus-naloxone, the inactive isomer of ( )-naloxone did not have any effect by itself on sea urchin sperm motility nor did it alter the metenkephalin-induced effect. Even though the magnitude and the nature of the sperm cells' responses to the opioids and antagonists appear somewhat atypical compared to the responses of neurobiological systems, the lack of effect of (+)-naloxone confirms the specificity of the response and is consistent with the presence of opioid receptors in the sperm cell. INTRODUCTION Neuroactive agents cause sperm cells to alter their swimming behavior and, in some cases may affect their fertilizing capacity (Atherton et al., 1978; Bavister et a!., 1979; Cornett and Meizel, 1978; Nelson, 1978; Sastry et al., 1981). The sperm cells of mussels, sea urchins, and starfish modulate their behavior when exposed to cholinergic agents (Nelson, 1978). Spermatozoa of vertebrate species from rams (Stewart and Forrester, 1978), bulls (Egbunike, 1982), boars (Sekine, 1951), and rabbits ( Bishop et al., 1976) to humans (Zeller and Joel, 1941; Sastry et al., 1981) also tested positively for the presence of acetylcholine, its receptor, acetylcholinesterase, and choline acetyltransferase. The rate of propulsion of sea urchin spermatozoa varies biphasically both when exposed to acetylcholine and to nicotine (Nelson, 1972). The presence of a nicotinic receptor in the sea urchin sperm was confirmed when it was shown that both d- tubocurarine (Nelson, 1973) and a-bungarotoxin (Nelson, 1976) inhibited their mo- tility. Low concentrations of decamethonium, which interacts with the cholinergic receptor at neural synapses and neuromuscular junctions, caused a slight transitory increase in the rate of motile progression but depressed the motility at slightly higher concentrations (Nelson, 1973). Cholinergic agents have been postulated to affect the transmembrane and intra- cellular transport of calcium into and within the sperm cell (Nelson, 1978). This Received 10 March 1986; accepted 6 May 1986. * Address for correspondence and reprint requests. 208 OPIOIDS AFFECT SEA URCHIN SPERM 209 interdependence resembles the acetylcholine-calcium relationship in brain cell syn- aptosomes demonstrated by the inhibition of the release of acetylcholine due to the blocking of calcium uptake when the synaptosomes are incubated in the presence of morphine (Sanfacon et al., 1977). The action of 0-endorphin on rat brain synaptosomes was even more potent (Guerrro-Munoz et al., 1979). It was found that after a delay period, morphine-treated spermatozoa of the Med- iterranean sea urchins, Arbacia lixula, Paracentrotus lividus, and Sphaerechinus gran- ularis, appeared to swim more rapidly and for longer periods than untreated control cells under microscopic examination (Cariello and Nelson, 1984). The present exper- iment was designed to test whether the opioid effect on sperm cell behavior could be interpreted in terms of the effects of the narcotics on the brain synaptosome model. MATERIALS AND METHODS To quantify the effects of the treatment, spermatozoa of the purple sea urchin, Arbacia punctiilata, (the sea urchins, kept in running seawater aquaria were replenished several times weekly as needed during June, July and August) were aligned and oriented under gentle centrifugation in the horizontal rotor of an IEC Model CL centrifuge. Male sea urchins were induced to spawn by intracoelomic injection of 0.5 M KC1. One-tenth ml of the concentrated sperm cells was diluted in 20 ml of artificial seawater (ASW) (The turbidity of the suspension was adjusted to an optical density of 0.5-0.7, equivalent to 7-9 X 10 6 cells/ml.) One liter of ASW contains: NaCl, 24.72 g; KC1, 0.67 g; CaCl 2 -2H 2 O, 1.36 g; MgCl 2 -6H 2 O, 4.66 g; MgSO 4 -7H 2 O, 6.29 g; and NaHCO 3 , 0.18 g. Two ml of the sperm cell suspension were pipetted into each of 6 round, 1-cm diameter cuvettes. The control cuvette contained 0.2 ml of ASW; the second cuvette contained 0.2 ml of 10% formaldehyde; the remaining 4 cuvettes con- tained 0.2 ml of a given concentration of the test reagent. Each cuvette was inverted 2X to disperse the contents. The suspensions were preincubated for 0, 5, 10, and 15 minute periods at room temperature (22-25 C) and the sperm cells were aligned at 120 X g for 4 min. This centrifugal force orients the cells while displacing the form- aldehyde-killed and non-motile cells only minimally or not at all (Nelson, 1972). Treated cells then swim more rapidly or more slowly than the control cells toward the bottom of the cuvettes and the optical densities (O.D.) at 540 nm are measured in a Bausch and Lomb model 340 spectrophotometer, before and after orientation. Readings were corrected for any displacement of the killed cells and forward motility was expressed as percent of the change in O.D. of the control suspension normalized to 100 (Nelson, 1972). The effect of each concentration of the opioid or antagonist was determined on at least six different samples for each incubation period. The sperm cells were exposed to methionine enkephalin acetate (Sigma) over a concentration range of 0.0016 to 1.23 mM and to (-)-naloxone-HCl (Endo) over a range of 0.0016 to 4.87 mM(Figs. 1, 2). The effect of competition between the opioid and the antagonist was tested at a fixed concentration of 0.005 mMnaloxone over the entire concentration series of metenkephalin (Fig. 3). Plus-naloxone, (kindly supplied by NIDA), the en- antiomer of (-)-naloxone, which presumably has much less ability to interact with the opiate receptor (lijima et al., 1978), was diluted to concentrations of 0.0016 mM to 1 .09 mM in ASW. Plus-naloxone was tested both alone and in combination with the metenkephalin for the sea urchin sperm motility responses (Fig. 4). RESULTS The Arbacia sperm responded maximally to metenkephalin at a concentration between 0. 1 and 0.2 millimolar whether or not the cells had been preincubated in the 210 L. CARIELLO ET AL. 200 150 1 100 50 -30 -2.0 -10 log CONCENTRATION (mM) 00 FIGURE 1 . Motility ofArbacia sperm suspended in ASW containing 0.00 1 6 to 1 .23 mM metenkephalin. Abscissa: log cone, in mM. Ordinate: percent change in motility (control taken as 100%) after orientation at 120 X g. Symbols represent periods of preincubation before orientation. Solid circles, min; open circles, 5 min; open triangles, 10 min; solid triangles, 15 min. opioid before centrifugal orientation. While the brief exposure of the "zero-time" cells during centrifugation elicited only about a 15% increase in progressive rate at that concentration, less than 5 micromoles per liter appeared to suffice as a threshold concentration for the metenkephalin preincubated for 1 5 minutes before alignment. Concentrations of the opioids greater than 1 mM/1 ASW were inhibitory (Fig. 1 ). The response to the opiate antagonist, (-)-naloxone, began to develop at about 25 micromolar while maximal stimulation occurred at 0.5 millimolar and lesser in- creases in forward motility appeared at slightly higher concentrations, inhibition oc- curring at 2 millimolar and above (Fig. 2). Metenkephalin caused a doubling at its optimum while naloxone stimulated the sperm to a 75% increase over the control rate at its optimum when the sperm cells were preincubated for 1 5 minutes. Nearly complete inhibition with 1 5 minutes exposure occurred at about 1 millimolar metenkephalin but it required about 5 times that amount of (-)-naloxone to cause nearly complete cessation of movement. Since both metenkephalin and (anomalously), (-)-naloxone appeared to exert delayed stimulatory effects on the spermatozoa to somewhat different though over- lapping concentrations, it was important to test for interactive effects of the opioid and the so-called antagonist. This is illustrated in Figure 3, a composite of the graphs OPIOIDS AFFECT SEA URCHIN SPERM 211 200 150 100 50 -30 -2.0 -1.0 0.0 log CONCENTRATION ( mM) 1.0 FIGURE 2. Motility ofArbacia sperm suspended in ASW containing 0.0016 to 4.87 mM (-)-naloxone- HC1. Abscissa: log cone, in mM. Ordinate: percent change in motility after 4 min orientation at 1 20 X g. Symbols, as in Figure 1 . of the results of the 15-minute preincubation periods for metenkephalin, open circles; ( )-naloxone, solid circles; and the mixture of the two compounds, open squares. In the combined drug study, the sperm cells were incubated at room temperature in suspensions containing about 1/5 the minimally effective concentration of ( )-nal- oxone, 5 ^mole/1, over the whole concentration range of metenkephalin assayed (from 0.0016 to 1.23 millimolar). Alone, metenkephalin depressed motility below the control rate at about 0.8 mM and increased the motile rate by about 15% at 7 nM. The subliminal concentration, 5 /J.M, of (-)-naloxone completely blocked all stimulatory responses to the metenkephelin, although the rate of decline in motility induced by supraoptimal concentrations of either the opioid or antagonist alone was less pro- nounced in the combined drug medium (Fig. 3). Since the ( )-naloxone exhibited atypical pharmacological action in the sea urchin sperm system, the inactive enantiomer, ( + )-naloxone, was tested for its effects in the motility assay. Figure 4 shows the sperm cell responses to this drug, both alone and in combination with metenkephalin. By itself, (+)-naloxone (triangles) had no effect on motility at concentrations between micromolar and millimolar. Similarly, the in- creased motility due to metenkephalin (circles) was unaffected when the incubation medium contained 5 micromolar (+)-naloxone along with the metenkephalin (x's), in contrast to the reversing effect of 5 micromolar (-)-naloxone on metenkephalin shown in Figure 3. The data in Figure 4 depict the effects after a 15 min incubation. Similarly, the curve for the (-)-naloxone alone (squares) measured concurrently shows comparable characteristics with the previous experiment in Figure 3 (The data for Figs. 1, 2, and 3 were collected in July, 1984, those for Fig. 4 in July, 1985.) In preliminary experiments morphine sulfate exerted a depressant effect on Spha- erechinus sperm during brief exposure, but following prolonged incubation, up to 45 212 L. CARIELLO ET AL. 200 150 _J P O 100 50 -30 -20 -10 log CONCENTRATION (mM) 00 FIGURE 3. Drug interaction effect on Arbacia sperm. Incubation mixture contains (-)-naloxone at a fixed concentration of 0.005 mM and varying amount of metenkephalin. Ordinate: percent change in motility after 15 min incubation. Abscissa: log cone, in mM. Open squares 0.005 mM (-)-naloxone and 0.0016 to 4.87 mM metenkephalin; open circles, metenkephalin alone, 0.0016 mM to 1.23 mM: solid circles, ( )- naloxone alone 0.0016 to 4.87 mM. Note that 0.005 mM (-)-naloxone completely blocks stimulation due to metenkephalin. minutes, the rate of sperm progression increased to 70% above that of the controls (Cariello and Nelson, 1984). DISCUSSION Responsiveness to the action of the pharmacological agents by sperm cells is quan- titatively demonstrated in terms of changes in their rate (or pattern) of motile pro- gression. We have here presented evidence that opioids and opiate antagonists elicit both time- and dose-dependent alterations in Arbacia sperm swimming rate. But it is quite evident that suspensions of living sperm cells of marine invertebrates, while sharing some physiological properties with the sperm of vertebrates yet exhibit char- acteristic behavioral responses that differ both qualitatively and quantitatively from other excitable tissues. Intact sea urchin sperm cells respond to narcotics and antagonists. These experi- ments were undertaken following our preliminary observations that morphine had a pronounced effect in the sperm cells of the Mediterranean sea urchin, Sphaerechinus granulahs (Cariello and Nelson, 1984). Quantitative determinations on the motility OPIOIDS AFFECT SEA URCHIN SPERM 213 200 150 O _/ O 100 O O 50 -i 1 1 r -3.0 -20 -1.0 log CONCENTRATION (mM) 0.0 FIGURE 4. Plus-naloxone and Arbacia sperm. Abscissa: log cone, in mM. Ordinate: percent change in motility after 15-min incubation in ASW containing ( + )-naloxone alone (open triangles); (-)-naloxone alone (open squares); metenkephalin alone (open circles); and metenkephalin and ( + )-naloxone 0.005 mM (X X). of sperm cells of Arbacia punctidata suspended in ASW showed that metenkephalin acetate caused a biphasic dose-dependent response (Fig. 1 ). Metenkephalin doubled the rate of progression measured after a 1 5-minute preincubation period, with lesser degrees of motility enhancement after shorter exposure. At concentrations greater than optimum, metenkephalin depressed motility more acutely with increasing ex- posure time. This aspect is brought into focus in the drug-interaction depicted in Figure 3 in which the subthreshold concentration of (-)-naloxone, having reversed metenkephalin's stimulatory effect at suboptimal amounts, becomes overridden al- lowing motility to decline as the metenkephalin was applied in supraoptimal doses. Under these conditions (-)-naloxone fulfills its putative role as a competitive opiate antagonist even though by itself it acts atypically in the sea urchin sperm system. Usually (-)-naloxone does not exhibit agonistic effects and so is considered to be a relatively pure antagonist which may be taken as an indicator for opioid receptor- mediated physiological processes and, biochemically, in ligand-displacement assays of naturally occurring morphine-like factors. While ( )-naloxone is almost devoid of agonistic effects in whole animal or organ-receptor assay, nevertheless it appears by virtue of the blocking of metenkephalin stimulation to be of the class of substances referred to either as partial agonists or agonist-antagonists in the context of the sea urchin sperm system. The specificity of the sperm opioid receptor appears to be attested not only by ( )- naloxone's blocking of metenkephalin's agonist action, but further confirmed by the inability of (+)-naloxone to influence metenkephalin's stimulation as well as by the fact that (+)-naloxone itself is without discernible effect on Arbacia sperm motility at concentrations below 1 millimolar. According to lijima et al. (1978), who synthesized 214 L. CARIELLO ET AL. it from thebaine, the (+)-naloxone had no more than 1/1000 to 1/10,000 the phar- macological action of ( )-naloxone and can therefore "serve to test the stereospecificity of the biochemical and pharmacological actions of (-)-naloxone." These investigators tested the (+)-naloxone on three bioassay systems including the rat brain membrane receptor binding assay, guinea pig ileum, and the reversal of morphine inhibition of the adenylate cyclase activity of a neuroblastoma X glioma hybrid. In the Arbacia sperm motility assay the threshold for metenkephalin stimulation fell in the micromolar range, that due to (-)-naloxone commenced at an order of magnitude higher, while above one millimolar, (+)-naloxone showed only a slightly depressant effect but did not reverse the metenkephalin effect at all. By these criteria, the Arbacia sperm may be considered to be endowed with opiate receptors that may bind endogenous opioids and also the opiate antagonist (-)-naloxone. Sastry and co- workers (1982) have extracted enkephalin-like and substance P-like compounds from human, rat, and bull spermatozoa, and from human seminal plasma and from rat male accessory glands which they detected by radioimmunoassay. The seminal plasma contained higher levels of met- and leu-enkephalins than did the sperm, while the distribution of substance P in these tissues was the reverse. These investigators point to the role of the opioid peptides in regulation of acetylcholine-induced Ca 2+ fluxes which are essential for sperm motility and the acrosome reaction. Immunoreactive /3-endorphin-like material was detected in Leydig cells, epidy- dymal epithelia, seminal vesicles, and vas deferens of rat, mouse, guinea pig, hamster, and rabbit (Shu-Dong et ai, 1982). Fravioli et al. (1984) investigated the possible role of j8-endorphin, met-enkephalin, and calcitonin in human sperm motility regulation. In their study, they report that metenkephalin does not affect the motility at concen- trations between nanomolar and millimolar, although /3-endorphin starts to depress motility at about 0.5 micromolar. On the other hand, calcitonin, in seminal fluid at about three times its concentration in peripheral blood plasma, markedly depresses human sperm motility in the nanomolar range and completely inhibits movement at micromolar concentration. It is not clear at this time why (-)-naloxone behaves anomalously in stimulating Arbacia sperm, nor why metenkephalin's stimulatory effect on the sea urchin sperm should differ from its reported lack of effect on human sperm. Similarly unanticipated results with opioids have been reported by Zagon and McLaughlin (1983, 1986). Heroin inhibition of the growth of transplanted neuro- blastoma in mice was blocked by concomitant administration of naloxone. Paradox- ically, when naloxone was tested alone at concentrations sufficient to interact with opiate receptors, this opiate antagonist was extremely effective in prevention or retar- dation of the tumor growth. According to these investigators, the antagonist naltrexone can promote tumorigenesis at a dose that prevents the analgesic action of morphine, but it exerts antineoplastic effects at concentrations that only temporarily block an- tinociception by morphine. Conclusions To meet criteria for pharmacological action, a given agent must elicit a characteristic response ! om sperm cells with appropriate dose- and time-dependent parameters. These ma> uTer from responses observed in neurobiological systems: the dose required, the magnitude of altered action, the time course for the optimum expression should provide insights that could suggest the locus and number, as well as the accessibility, of specific receptors. Similar peculiarities have been documented in the behavior of sperm cells challenged by other well-defined agonists and antagonists. The central action of narcotics and endogenous opioids has been attributed to OPIOIDS AFFECT SEA URCHIN SPERM 215 interference with neurotransmission since both morphine and /3-endorphin inhibit the release of acetylcholine from brain synaptosomes by preventing Ca 2+ uptake (San- facon et al, 1977; Guerrero-Munoz el al, 1979) and naloxone blocks these effects. This test system would serve as a relevant model for interprobation of sperm cell behavior assuming that the spermatozoa possessed opioid receptors with which ap- propriate ligands would interact to affect the calcium-dependent acetylcholine release mechanism. We have shown that: ( 1 ) the endogenous opioid metenkephalin in ASW alters the sperm cell swimming rate biphasically; (2) (-)-naloxone, the specific opiate antagonist, in a subthreshold dose reverses metenkephalin stimulation even though by itself, ( )-naloxone also exerts a dose-dependent biphasic response; and (3) (+)- naloxone is without effect in concentrations below 1 millimolar, both by itself and when combined with metenkephalin. Thus, it appears that Arbacia sperm cells may indeed be equipped with opioid receptors. Alternatively, according to recently published reports by Haynes and Smith (1982) and Haynes et al. (1984), the opioid peptide /3-endorphin and to a somewhat lesser extent, methionine enkephalin, inhibit motor endplate-specific acetylcholinesterase in skeletal muscle of embryonic and newborn rats. In previous studies, it has been established that the sperm cells of Arbacia punctulata and other species possess a functional nicotonic cholinergic system which participates in regulation of motile performance. Calmodulin-antagonists, calcium-chelators, and calcium channel blockers also have been shown to alter the rate of motile progression (Cariello and Nelson, 1985). Moreover, eserine, nicotine, and decamethonium affect the uptake and intracellular distribution of calcium in sperm cells (Nelson et al., 1982). In summary, the evidence is consistent with the presence in sperm cells of opioid receptors, although somewhat atypical, and also an interdependent acetylcho- line-modulated calcium-uptake system. The nature and extent of interaction of these two systems in spermatozoa remain to be elucidated. ACKNOWLEDGMENTS This work has been partially supported by National Science Foundation Research Grants PCM8302582 and INT8300181 and by the Consiglio Nazionale di Ricerche. 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(August, 1986) METABOLIC ADAPTATION OF THE HORSESHOE CRAB, LIMVLUS POLYPHEMUS, DURING EXERCISE AND ENVIRONMENTAL HYPOXIA AND SUBSEQUENT RECOVERY* KARL-HEINZ CARLSSON 1 AND GERD GADE 2 ** l lnstitutfur Pharmakologie und Toxikologie, Universitat des Saarlandes, D-6650 Homburg/Saar, Federal Republic of Germany, and 2 Institut fur Zoologie IV der Universitat Dusseldorf, Universitdtsstr. 1, D-4000 Dusseldorf 1, Federal Republic of Germany ABSTRACT Metabolic responses to exercise, to exposure to environmental anaerobiosis, and to subsequent recovery were investigated in muscle, hepatopancreas, and hemolymph of the horseshoe crab, Limulus polyphemus. Exercise caused a considerable decline in arginine phosphate and the formation of D-lactate in muscle tissue, whereas the adenylate energy charge was maintained. Some of the D-lactate appears to have been transported from muscle tissue into the hemolymph. This occurred, apparently, during exercise as well as during recovery. Hemolymph postbranchial P 02 , which decreased during exercise, and tissue phosphagen stores were rapidly restored to aerobic control values upon recovery, while D-lactate oxidation was protracted, especially in muscle. Environmental anaerobiosis for 48 h was fueled by the breakdown of arginine phosphate (considerable only in muscle tissue) and glycogen, resulting in the accu- mulation of arginine, D-lactate, and alanine in both muscle and hepatopancreas. Al- anine production may occur via glutamate-pyruvate transaminase and glutamate de- hydrogenase, which take over the role of D-lactate dehydrogenase to maintain redox balance during the later phases of anaerobiosis. Recovery from anaerobiosis was char- acterized by a rapid replenishment of the phosphagen, a rapid drop in alanine con- centration, and a protracted time-course for the decline in D-lactate levels, which was somewhat faster in the hepatopancreas than in muscle tissue. INTRODUCTION Hypoxia and even anoxia are encountered by many marine species. The horseshoe crab Limulus polyphemus, has successfully occupied microhabitats such as the estuarine intertidal zone and shallow tidal marshes, where low oxygen partial pressures may occur daily as well as a seasonally. This species is capable of extensive locomotion with its walking legs; smaller animals often swim by rhythmically moving their legs and gill leaflets (see Shuster, 1982). During excessive locomotory activity, oxygen consumption by these muscles probably exceeds the rate at which oxygen can be delivered by the circulatory system. Thus, under these conditions energy provisions are met by anaerobic metabolism (see Ga'de, 1983). Metabolic adaptations to both environmental and functional hypoxia have been studied to some extent in invertebrates, mostly molluscs, annelids, and crustaceans (for reviews see Schottler, 1980; Ga'de, 1983). During functional hypoxia, energy is typically provided by the breakdown of phosphagens (mostly arginine phosphate) and Received 7 November 1985; accepted 25 April 1986. * The experimental work in this study was performed at the Institut fur Zoophysiologie der Universitat Bonn (FRG). ** To whom reprint requests should be sent. 217 218 K. CARLSSON AND G. GADE by anaerobic glycolysis, resulting in the production of octopine (or other opine com- pounds) or lactate (for reviews see Ga'de, 1980; Ga'de and Grieshaber, 1986). During environmental hypoxia, the phosphagen arginine phosphate again is important for ATP production. Additionally, Crustacea ferment glycogen and accumulate the clas- sical end product L-lactate. However, in most annelids and molluscs, there is a si- multaneous fermentation of glycogen and aspartate resulting in the accumulation of succinate and alanine during the first hours of hypoxia (de Zwaan and Dando, 1984). Later, when the aspartate pool is depleted, succinate production is derived exclusively from glycogen. This may be achieved by regulation at the phosphoenolpyruvate branchpoint (de Zwaan, 1977). The bulk of carbon flow is through phosphoenolpyr- uvate carboxykinase (PEPCK) rather than pyruvate kinase (PK). During prolonged periods of hypoxia, succinate is further metabolized to propionate. Only a few investigations have examined the metabolic events immediately fol- lowing environmental or functional anaerobiosis (see Ellington, 1983). These events include the recharging of the high energy phosphates, ATP and arginine phosphate, oxidation of end products, and resynthesis of anaerobic substrates. Surprisingly, only scarce and scattered data on the above processes are available for the horseshoe crab. Arginine kinase is present in muscle (Blethen, 1972), indicating that arginine phosphate might contribute to energy production. The horseshoe crab possesses a D-lactate dehydrogenase (Long and Kaplan, 1968) which is present in a system of kinetically distinct, tissue-specific isoenzymes (Carlsson and Ga'de, 1985). PK and PEPCK have been investigated in the tissues of L. polyphemus (Falkowski, 1974; Zammit and Newsholme 1978; Zammit et al, 1978). The kinetic data suggest that the bulk of carbon flow is through PK rather than PEPCK (Zammit and Newsh- olme, 1978). Fields (1982) found that the bulk of label from anaerobic 14 C-glucose degradation by in vitro heart preparations was accumulated in lactate. Only trace amounts of radioactivity appeared in succinate. Therefore in the present study we wanted to evaluate the overall capacity for anaerobic metabolism in muscle and hepatopancreas of Limulus polyphemus by es- timating the relative maximal activities of enzymes of the intermediary metabolism. We were also interested in learning which metabolic events take place during exercise in muscle and hemolymph, and which take place during various periods of environ- mental hypoxia in muscle, hepatopancreas, and hemolymph. Finally, following the exercise stress and environmental hypoxia, we analyzed the metabolic changes in the three tissue compartments after recovery under aerobic conditions. MATERIALS AND METHODS Animals and tissues Specimens of the adult horseshoe crab, Limulus polyphemus, (20-25 cm prosoma width) were obtained live from the Marine Biological Laboratory (Woods Hole, MA), shipped by air freight to Bonn (FRG), and kept at 15C 2C, in artificial seawater (33%o salinity), in aquaria of 300 1. Until four days before experimentation, the animals were fed frozen fish or mussel meat once a week. Musci tissue from two different sites were used in this study. First, for the exercise experiments, we analyzed the muscle that moves the gill leaflets. Second, due to its small mass and its possible contribution to contractions even under hypoxic conditions, the telson levator muscle was chosen for the experiments dealing with environmental anoxia and recovery. Furthermore, the activities of key enzymes were also measured in the levator muscle, but preliminary experiments indicated that they were very similar in the gill-muscle. L. POLYPHEMUS METABOLIC ADAPTATION 219 In addition, a portion of the hepatopancreas from the dorsal anterior part of the animal was analyzed for enzymatic activities and changes of metabolites during en- vironmental anoxia and recovery. Materials Biochemicals were purchased from Sigma Chemical Company (Taufkirchen, FRG) and Boehringer GmbH (Mannheim, FRG). All other chemicals were of reagent grade quality and came from Merck (Darmstadt, FRG). D-lactate dehydrogenase from muscle tissue ofLimuluspolyphemus was purified as described elsewhere (Carlsson and Ga'de, 1985) and was used to assay D-lactate concentrations in tissues and hemolymph. Experimental procedure Metabolic responses to work and recovery. Animals were removed from the aquaria for different experiments, at zero time, and subjected to various periods of exercise. Horseshoe crabs held vertically in water by their telsons attempt to right themselves by making swimming movements using their legs and gills; under the conditions chosen, about 45 to 55 gill movements were made per min. After a given period of exercise (24, 47, 96, 132, 161, and 240 s), animals were removed from the tank, and the muscles that move the gill leaflets were excised and frozen in liquid nitrogen. This procedure took about 20 s. After 4 min of exercise, some animals were left to recover in well-aerated seawater for various intervals, and treated as above. Also, a zero time group was selected that had done no work at all. Other animals were exercised for 2, 4, and 10 min, and the D-lactate concentration in the hemolymph (postbranchial, taken through the arthrodial membrane between the prosoma and the opisthosoma) was measured before and after exercise as well as at various time intervals during recovery. In some of these animals, the P 02 of the postbranchial hemolymph was determined with an oxygen electrode (E 5046, Radi- ometer; Copenhagen, Denmark) housed in a thermostated (12 2C) cell (D 616, Radiometer). The electrode was calibrated with air-saturated seawater and with water completely depleted of oxygen by the addition of Na 2 SO 3 to give a 7% solution. All tissues were stored at -25 C. Metabolic responses to environmental hypoxia, exposure to air, and recovery. Horseshoe crabs were incubated in Plexiglas respiration chambers filled with about 15 1 seawater at 12C which had been gassed with pure nitrogen until P 02 (monitored with an oxygen electrode) reached almost zero mm Hg. After the animals were inserted, the chamber was flushed with nitrogen for another 15 to 30 min before it was closed. The chamber was placed in a constant temperature room ( 1 2 1 C) for the remainder of the experiment. At the end of different incubation periods, animals were removed, a postbranchial hemolymph sample was taken, and portions of the telson levator muscle and hepatopancreas were dissected, blotted on filter paper, and immediately frozen in liquid nitrogen. An aliquot of the hemolymph sample was immediately used for P 02 determination (see above), and the rest was blown into perchloric acid (see below). Another group of horseshoe crabs was incubated in the Plexiglas respiration cham- ber without any seawater at 12C (air exposure). A third group of animals was exposed to air with their gill leaflets mechanically prevented from ventilating. This was achieved by binding the gill leaflets firmly onto the body with the help of metal strips fastened with rubber bands. 220 K. CARLSSON AND G. GADE Biochemical analyses of tissue and hemolymph samples Tissue sample.-, were fragmented with a mortar and pestle chilled in liquid nitrogen. For each ana! is, approximately 200 mg of tissue of an individual muscle was weighed and homog r:.,ed in 10 volumes (w:v) 6% perchloric acid (0-4C). The homogenates were centr aged at 19,000 X g for 20 min and the supernatant fluids neutralized with 5 M KHCO 3 . The neutralized extract was centrifuged as above and the supernatant immediately used for the determination of the adenylates, arginine, and arginine- phosphate. Other metabolites were determined after storage of the extracts at -25 C. ATP was determined according to Lamprecht and Trautschold (1974), ADP and AMP after Jaworeck et al. (1974), arginine by the method of Ga'de and Grieshaber (1975), arginine phosphate according to Grieshaber and Ga'de (1976), alanine after D. H. Williamson (1974), succinate after J. R. Williamson (1974), and D-lactate ac- cording to Gawehn and Bergmeyer (1974) using the D-lactate dehydrogenase from Limulus polyphemus. Using conspecific D-LDH is advantageous because of its much higher affinity for D-lactate than the commercially available enzyme preparation from Lactobacillus (Carlsson and Ga'de, 1985). No interference with heavy metal ions was observed in our lactate assay as outlined previously (Ga'de, 1984). Hemolymph samples (100-300 mg) were blown into pre-weighed vials containing 0.5 ml 6% perchloric acid; the vials were weighed again, centrifuged as above, and the supernatants were neutralized with 5 A/K 2 CO 3 . After centrifugation, the supernatant was used for the D-lactate determination. All metabolite data were analyzed for significant changes by Student's /-test using confidence limits of P ^ 0.05. Profile of muscle and hepatopancreas enzyme activities Activities of key enzymes of the intermediary metabolism were estimated in crude, desalted, cell-free extracts of the telson levator muscle and hepatopancreas of L. po- lyphemus. Approximately 1 g of tissue was cut into small pieces, resuspended in a 5- fold volume (w:v) of extraction buffer [100 mA/ triethanolamine-HCl (TRA) buffer containing 1 mA/ 2-mercaptoethanol at pH 7.6], homogenized by sonification (Branson sonifier), and centrifuged at 20,000 X g for 20 min. The supernatant was passed through a Sephadex G-25 column (Deutsche Pharmacia GmbH, Freiburg, FRG) to remove low molecular weight compounds. The enzymes were assayed by standard procedures reported in the literature. The assay conditions have been outlined in detail previously (Meinardus-Hager and Ga'de, 1986). Exceptions are the reaction mixtures (final concentration in a 1 ml cuvette) of the following enzymes: Alcohol dehydrogenase (EC 1.1.1.1): 85.5 m M sodium pyrophosphate buffer pH 9, 19 mM glycine, 6.2 mA/ semicarbazid-hydrochloride, 0.6 A/ethanol, 1.8 mM NAD + , 1 mM glutathion. D-lactate dehydrogenase (LDH, EC 1.1.1.28): 100 mA/ potassium phosphate buffer pH 7.0, 0.15 mA/ NADH, start with 2.5 mA/ pyruvate (1 mA/ pyruvate in case of extracts from hepatopancreas). Gluta.iine synthetase (EC 6.3.1.2): 200 mA/ Tris/HCl pH 7.7, 100 mA/ hydrox- ylamrnoniun chloride, 10 mA/Na 2 HAsO4, 0.5 mA/MnSO 4 , 1.5 mA/ADP, 100 mA/ glutamine. After 30 min the reaction was stopped by addition of an acidic solution of FeCl 3 X 6 H 2 O and the extinction measured at 546 nm. All assays were conducted in a filter photometer (Vitatron DCP 4) or a LKB Ultrospec recording spectrophotometer at 25 C. Assays were initiated by the addition of substrate. L. POLYPHEMUS METABOLIC ADAPTATION 221 RESULTS Profile of the activities of key enzymes in the intermediary metabolism The activities of some enzymes involved in the energy metabolism in muscle and hepatopancreas tissue of L. polyphemus are listed in Table I. Glycogen phosphorylase in the muscle was only 6% in the a-form, while in the hepatopancreas the a-form of the enzyme represented 46% of the total enzyme activity. In the muscle tissue, the activity of the hexokinase was about 7-fold higher than that of the activated phos- phorylase, whereas no significant difference between these two enzymes was observed in the hepatopancreas. Glyceraldehyde-3-phosphate dehydrogenase of the muscle had the highest activity of the enzymes of the Embden-Meyerhof pathway followed by pyruvate kinase and phosphofructokinase. This was true also for hepatopancreas, but TABLE I Activity of various enzymes in telson levator muscle and hepatopancreas from Limulus polyphemus Enzyme activity (yumoles substrate conversion per min and g wet weight) Enzyme (n) Telson levator muscle Hepatopancreas Enzymes involved in glycolysis: Phosphorylase a-form (2) 0.022 0.005 0.13 0.03 a + b-form 0.34 0.10 0.28 0.06 Hexokinase (4) 2.32 0.45 0.39 + 0.16 Phosphofructokinase (3) 29.9 6.2 1.220.12 Glyceraldehydephosphate dehydrogenase (3) 136+12 10.5 2.9 Pyruvate kinase (3) 73.3 13 5.6+ 1.4 Lactate dehydrogenase (4) 102 1 29.3 5.1 Octopine dehydrogenase (3) n.d.* n.d. Strombine dehydrogenase (3) n.d. n.d. Alanopine dehydrogenase (3) n.d. n.d. Alcohol dehydrogenase (3) n.d. n.d. Enzyme involved in phosphagen utilization: Arginine kinase (3) 616+ 17 34.0 9.6 Citric acid cycle enzymes: Citrate synthase (2) 2.05 0.28 1.49 0.08 Malate dehydrogenase (4) 156 15 91 7 Enzymes involved in amino acid metabolism: Glutamate dehydrogenase NADH-dependent (4) 1.74 0.24 0.69 + 0.12 NADPH-dependent (4) n.d. n.d. Glutamine synthetase (2) 0.027 0.008 0.081 0.013 in the presence of 10 mM alanine (2) 0.01 8 0.005 0.005 + 0.016 Glutamate-oxaloacetate transaminase (3) 28.9 + 3.7 19.1 7.7 Glutamate-pyruvate transaminase (3) 13.5 1.0 16. 7 2.4 Enzymes involved in gluconeogenesis: Fructose diphosphatase (3) 0.740.19 2.04 0.1 8 Phosphoenolpyruvate carboxykinase (3) 1.50.3 2.1 0.5 * n.d. = not detectable. 222 K. CARLSSON AND G. GADE the maximum activities were 10- to 20-fold lower than in muscle tissue. Of the four terminal pyrm ai- reductases measured in both tissues, only D-lactate dehydrogenase was present, liere was no activity at all of octopine-, alanopine-, and strombine dehydroge ase. The activity of D-lactate dehydrogenase in muscle tissue was almost as high a.- Uie activity of glyceraldehyde-3-phosphate dehydrogenase and, in hepato- pancreas, 3-fold of the latter activity. Fermentation of glycogen to ethanol occurs in anoxic muscle of fishes (Shoubridge and Hochachka, 1980) and in tissues of larvae of the insect Chironomus thumii thumii (Wilps and Zebe, 1976). Due to the absence of activity of alcohol dehydrogenase in tissues of L. polyphemus, this type of fermentation cannot take place. Arginine kinase activity was very high in muscle tissue, but the hepatopancreas displayed only a fraction of this activity. Malate dehydrogenase activity was very high in both tissues, and there was a significant activity of citrate synthase in both tissues. Activity of phosphoenolpyruvate carboxykinase, which catalyses the fixation of CO 2 , could readily be measured in both tissues, but activity was slightly higher in the hepatopancreas. This was also true for the enzyme fructose-biphosphatase, an enzyme thought to operate during gluconeogenesis. Enzymes involved in amino acid metabolism were also assayed in both tissues. The transaminases showed the highest activities. Glutamate dehydrogenase in both tissues was only active with NADH, and not NADPH, as the co-substrate. Very low activities of glutamine synthetase were found in both tissues (but they were higher in hepatopancreas); this enzyme was inhibited by the addition of 10 mM alanine to the reaction mixture. Metabolic responses to exercise and recovery When specimens of L. polyphemus were subjected to exercise, there was a linear relationship between the numbers of gill movements and time (Fig. 1, inset). The level of D-lactate in the gill-muscle tissue increased linearly until up to 3 min and then plateaued (Fig. 1). After 20 and 30 min of recovery from 4 min of exercise, the D- lactate levels were significantly lower than the exercise value, but the control levels were not attained. There was no change in the alanine concentration during work (Fig. 1). Exercise also had no significant effect on the adenylates in the muscle tissue and, consequently, the adenylate energy charge did not change (Table II). In contrast, arginine phosphate levels fell significantly during exercise (Fig. 2). The most pronounced change occurred during the first 47 s of exercise. The increase in free arginine levels was a mirror image of the pattern of change in arginine phosphate levels (Fig. 2). During recovery, arginine phosphate levels rose rapidly and reached initial levels after about 20 to 30 min. At the same time there was a decrease in the arginine concentration. There were no changes in aspartate and succinate levels in any of these experimental treatments (results not shown). In the hemolymph, D-lactate concentrations did not change during 2 min of ex- ercise, whereas concentrations rose significantly during recovery (Fig. 3): the highest ictate concentrations were observed after 5 min of recovery. After 90 min of re- hemolymph D-lactate concentrations were not different from controls. After 4 and 10 ~iin of exercise, the D-lactate concentrations in the hemolymph were sig- nificantly increased above control concentrations, however, the concentrations at the two times e not significantly different from each other. During recovery from 4 and 10 min of work, there was an apparent large increase of the D-lactate concentration in the hemolymph; however, due to the inherent variability of the measurement, this increase was not significantly different from the values obtained just after work. L. POLYPHEMUS METABOLIC ADAPTATION 223 Ul cn 0) 2 o E 200 r " /f o /I * E / / UO *- > X/ /I I xx T^ CD O x 80 40 ^> p i^ E - > i%/ *^ X f * I 1 2 min 4 lac y 2 min U * \A/nrl^. . ^ 10 20 min 30 FIGURE 1 . Alterations in the levels of D-lactate (D D) and alanine (O O) in muscle that moves the gill leaflets of Limulus polyphemus during exercise and recovery. Each value is a mean SD (n = 4). The inset shows the relationship of the numbers of gill movements versus time. P 02 levels of the hemolymph decreased after 4 min of exercise, but were already back to control levels after 10 min of recovery (Fig. 4). Metabolic responses to environmental hypoxia, exposure to air, and recovery In a first series of experiments we analyzed the oxygen partial pressure of post- branchial hemolymph and the blood D-lactate concentration after subjecting specimens TABLE II Alterations in the levels ofaden ylates (nmoles per g wet weight) and the calculated energy charge in muscle that moves gill leaflets r*"l MM -r ~^- oo 's o i ' fN fN s d d d d d d d 2 60 * * * Oi cs '/~, -^- -JT, -f fN S, ^^ O o O - CM H d d +\ d +1 d +1 d +1 d +1 d +1 1 m r^ 1^ i r^) NO 2 2 d d d d d d d MM jC' PJ ^> _) "X3 00 W) KM sC r-- NO r- ^i Tf S "^ * oo ON HI o r- ON r- o r" ^ /"} NO NO r- NO 4-* a.S d d d d d d d o c ^ o o go o ^ " f-. *-t SSI V) _- __^ ^ NO o PI m ON oo c +l 3 >. NO fN fN ^~ ^f ' ' u C Er^/-| j~~ CM d d d d d d d & s Jj H +1 +1 +1 -f-l -[-1 +1 +1 c . ~ "S " "3 '.c 13 S "g o.S 00 J= "H 1 -! 7T | O d d O r-i O OO O O OO * UJ ^ ""3 E w fN Tf k? P'S 228 K. CARLSSON AND G. GADE 8 16 Time (h) FIGURE 6. Effect of incubating L//WM/MS polyphemus in oxygen-free seawater for various times on the levels of arginine phosphate and arginine in the telson levator muscle and hepatopancreas. The time course of changes occurring during recovery in muscle is also shown; onset of recovery is indicated by the arrow. Each value is a mean SD (n = 5). was some breakdown of arginine phosphate in the hepatopancreas during hypoxia, quantitatively this did not contribute much to overall energy production due to the low initial levels of this compound (Fig. 6). During recovery after hypoxia, arginine phosphate levels in muscle returned to initial values within 1 h. There were significant changes in the levels of D-lactate and alanine during hypoxia in both muscle and hepatopancreas tissue. After an initial increase in the first hour, D-lactate levels in muscle tissue as well as in hepatopancreas seemed to increase slightly vith time, but due to the high variability these changes were not significant (Fig. 7). Until 2 h of recovery had passed, D-lactate levels in muscle remained high, and they ere still above control levels after 8 h of recovery. In the hepatopancreas, the decrease >-iactate levels was somewhat faster, yet control values were not reached after 8 h oi overy. lanine levels increased with time of hypoxia in both tissues (Fig. 8). However, in muscle lissue most of the alanine was produced until 24 h of hypoxia, whereas there was a further increase in alanine levels between 24 and 48 h of hypoxia in the hepatopancreas. Succinate production was minimal during the first 10 h of hypoxia in the muscle tissue and was absent in hepatopancreas (Fig. 8). Aspartate levels of L. POLYPHEMUS METABOLIC ADAPTATION 229 (muscle) 8 16 Time (h) (hepatopancreas) J L FIGURE 7. Effect of incubating Limn/us polyphemus in oxygen-free seawater for various times on the level of D-lactate in the telson levator muscle and hepatopancreas. The time course of changes occurring during recovery is also shown; onset of recovery is indicated by the arrow. Each value is a mean SD (n = 4). muscle and hepatopancreas were 0.29 0. 1 1 and 0.24 0.06 ^moles/g wet weight, respectively, in control animals and 0. 1 3 0.06 and 0.07 0.04 /umoles/g wet weight, respectively, after 48 h of hypoxia (for all data: n = 4). -6 icn the longitudinal axis while maintaining the hoop axis at a constant stress. Ahile monitoring strains of both the longitudinal and hoop axes simulta- neously. 1 he constant hoop stress applied in these tests was 0.12 MN/m 2 , a value choser 3 correspond with the more extensible lower range of the longitudinal stress- strain curves, and therefore a value likely to fall within the in vivo range of stresses occurring during normal swimming movements. The purpose of these biaxial stressing tests was to determine the relative contributions of the crossed-fiber system and the interfibrillar matrix material to the mechanical properties of the whole skin. If the extensibility of the skin is controlled solely by the helical fibers, the ratio of the hoop to longitudinal stresses applied is always equal to the tangent of the fiber angle resulting from these stresses times the tangent of the initial fiber angle. Using this relationship a longitudinal stress-strain curve can be constructed for any constant hoop stress ap- plied, which will predict skin properties if they are due only to a set of continuous fibers. Comparison with the experimentally obtained stress-strain curve for whole skin should allow the roles of the fibers and the matrix to be assessed. RESULTS Structure of the skin The skin of the Norfolk spot is typical of most teleosts, consisting of a covering of ctenoid scales that are anchored to the pigmented epidermis and are surrounded by a clear gelatinous material. Beneath the epidermis is the stratum compactum, or the collagenous layer of the skin. Microscopy reveals that alternating sheets of parallel fibers comprise this layer. Figure 1 is a polarized light micrograph of a section cut ; FIGURE i. Polar: :i.o light micrograph of a radial section of spot skin cut parallel to one set of fibers; 12 fibers (or fiber layers) can be seen in long section. MECHANICS OF FISH SKIN 239 perpendicular to the skin and parallel to one set of fibers; twelve fibers (one per layer) can be seen in long section. The fibers are generally smaller in diameter in the outermost layers of the skin, and range in thickness from about 4 to 10 yum. The total skin thickness for animals of this size class (about 20 cm standard length) examined ranged from about 220 to 300 /mi. Fiber angles were determined at numerous locations on the fish and those obtained from one specimen are shown in Figure 2. In general the angles of the forward leaning fibers were lower than those of the corresponding backward leaning fibers, with the former ranging from 45 to 62 and the latter from 62 to 80. The fibers of the dorsal half of the caudal peduncle, however, were exceptional, with the forward leaning fibers forming an angle of 75 with the long axis of the fish and the backward leaning fibers forming an angle of only 30. In all cases, the fiber angles of the forward leaning fibers were examined within a few degrees of the pitch of the scale rows that overlay the collagenous layer of the skin. The skin of the skipjack tuna, like other tunas, differs from the skin of most teleosts in that it is devoid of scales over most of its surface. Scales are present only in an irregularly shaped region just behind the opercular opening; this scaled region is known as the "corselet." A thin layer of pigmented epidermis covers the rest of the fish, and this is easily abraided to reveal the fine collagen fibers below. The stratum compactum is only a thin, nearly transparent layer in this fish, and once the pigmented epidermis is abraided the axial musculature is readily visible through the collagenous layer. Microscopy reveals that alternating sheets of parallel fibers comprise this layer, just as they do in the spot. Figure 3 is a polarized light micrograph of a section cut perpendicular to the skin and parallel to one set of fibers. Three fibers (one per layer) can be seen in long section and between these, fibers of the alternating three layers can be seen in oblique end section. The fibers near the upper and lower boundaries of the skin are the thinnest, about 20 nm in diameter, while those occupying the center of the skin's thickness are much thicker, about 80 /mi in diameter. The total skin thickness for skipjack tunas of this size class (about 45 cm fork length) ranged from about 280 to 350 /urn. Fiber angles varied widely with respect to position on the fish, and like the spot, were generally not the same in the forward and backward leaning directions at the same point on the fish, as shown in Figure 4. In the middle regions of the fish fiber FIGURE 2. Fiber angles (in degrees) measured at various locations on one Norfolk spot specimen. 240 M. R. HEBRANK AND J. H. HEBRANK FIGURE 3. Polarized light micrograph of a radial section of skipjack tuna skin cut parallel to one set of fibers. Three fibers (or fiber layers) can be seen in long section alternating with three layers of fibers shown in oblique end section. angles generally fell between 55 and 75, but near the mid-dorsal and mid- ventral lines fiber angles became as low as 20 and as high as 87. Fiber angles could be seen to change by several degrees as the mid-dorsal and mid-ventral lines were traversed and also changed slightly as the lateral line was traversed. In some cases fibers could be seen to curve as they suddenly changed pitch near the dorsal and ventral midlines. Mechanical testing In Figures 5 and 6 the results of uniaxial tensile tests are shown in the form of stress-strain curves for skin stretched in the direction of one set of fibers and in the hoop and longitudinal directions for each of the two species. For the skin of the Norfolk spot stretched in the direction of the fibers very low extensions are obtained FIGURE 4. Fiber angles (in degrees) measured at several locations on one skipjack tuna specimen. MECHANICS OF FISH SKIN 241 15- 1-0- CNJ E CO UJ QL \ (ft 05- on-fiber hoop longitudinal O 1O STRAIN FIGURE 5. Typical stress-strain curves obtained for spot skin stretched uniaxially in the on-fiber, hoop, and longitudinal directions. at high stresses. The fibers therefore appear to be reasonably inextensible, especially within the range of stresses applied to the hoop and longitudinal directions. The elastic modulus (the slope of the stress-strain curve in its steep region) obtained as an average of fifteen on-fiber pulls is 75.0 MN/m 2 , as shown in Table I. The stress-strain curves for skin of the spot subjected to uniaxial stretching in the hoop and longitudinal directions reveal anisotropy in the mechanical properties of the skin. Skin stressed in the hoop direction exhibits a J-shaped curve in which the skin extends by several percent while the stress remains low, then following this initial extension the stress-strain curve becomes steeper as the skin deforms less freely under the applied load. In contrast, skin stressed in the longitudinal direction exhibits a more linear stress-strain curve as it extends fairly uniformly over the range of stresses applied. The mean terminal elastic modulus for spot skin pulled in the longitudinal direction is 2.4 MN/m 2 , which is significantly lower (F (K23) = 14.4, P < 0.001 ) than that of skin pulled in the hoop direction, a value of 16.4 MN/m 2 . In addition, the skin is significantly stiffer in the on-fiber direction than in the hoop direction (F (U 5) = 13.9, P < 0.001). Like the skin of the spot, that of the skipjack tuna is relatively inextensible in the direction of the fibers, as shown in Figure 6. However, in contrast to the spot, skin of the tuna stressed in the hoop direction is about as stiff as skin stressed in the on-fiber direction. The mean elastic modulus of the skin stressed in the hoop direction is 60.2 MN/m 2 , while that of skin stressed in the on-fiber direction is 36.2 MN/m 2 , as shown in Table I, although these differences are not significant (F (1 2 9) = 1.96, P = 0.20). The skin of the tuna stressed uniaxially in the longitudinal direction is similar to 242 M. R. HEBRANK AND J. H. HEBRANK 15- 10- LO LO LJ Ct CO 05- on-fiber hoop longitudinal 10 /o STRAIN 15 20 FIGURE 6. Typical stress-strain curves obtained for skipjack tuna skin stretched uniaxially in the on- fiber, hoop, and longitudinal directions. that of the spot in that its stress-strain curve is more linear in shape and the skin is quite extensible. The differences in stiffness for skin stressed in the longitudinal direction compared to skin stressed in both the hoop and on-fiber directions are significant: for longitudinal versus hoop, F (U6) = 17.16, P < 0.001, and for longitudinal versus on- fiber, F (1>2 9) = 27.51, P < 0.001. The mean elastic modulus for skipjack tuna skin stressed in the longitudinal direction is 6.9 MN/m 2 , an order of magnitude lower than those of the other two directions tested. TABLE I Comparison of mean terminal elastic moduli for skin of the Norfolk spot and the skipjack tuna stressed uniaxially in three directions Norfolk spot elastic modulus Skipjack tuna elastic modulus On-fiber Lori, 7.50 X 10 7 N/m 2 (S.D. = 4.99X10 7 ) (n=15) 1.64X 10 7 N/m 2 (S.D. =0.63 X 10 7 ) (n=12) 2.41 x 10 6 N/m 2 (S.D. = 2.26 x 10 6 ) (n=13) 3.62 X 10 7 N/m 2 (S.D. = 2.30 X 10 7 ) (n=12) 6.02 X 10 7 N/m 2 (S.D. = 5.44X 10 7 ) (n= 19) 6.92 x 10 6 N/m 2 (S.D. =4.25 X 10 6 ) (n=19) MECHANICS OF FISH SKIN 243 02Ch O *10 % STRAIN 20 FIGURE 7. Biaxial stress-strain curve obtained for spot skin pulled in the longitudinal direction while the hoop direction was held at a constant stress of 0.12 MN/m 2 . A theoretical curve for a pure fiber model subjected to the same ratio of stresses is shown as a dashed line. The bar labeled H shows the range of strains recorded in the hoop direction. Breaking stresses and strains could not be determined for skin from either fish pulled in any direction. On application of high loads (approximately 50% higher than those shown in Figures 5 and 6 for each skin direction) the skin always failed at the clips attaching the skin to the testing device. In general, skin samples pulled in each of the three directions did not return to 0-251 -10 *1O /o STRAIN FIGURE 8. Biaxial stress-strain curve obtained for skipjack tuna skin subjected to the same conditions as in Figure 7. 244 M. R. HEBRANK AND J. H. HEBRANK V OJ i/> c 4- O cny- Q. C O O o (A 0) o T ID n> T3 C 3 C CT fl3 -D 0) 3 a. -t-j i/i O D O en o -C C 1- o o t A IB l/> C D 1/1 O T3 4) U 3 O O *- U 14- O tny- Q. C o o o in V O I. O o MECHANICS OF FISH SKIN 245 their original lengths once the load was removed, and the unloading curves always fell below the corresponding loading curves. However, this hysteresis may be exag- gerated by frictional losses inherent in the testing device. Biaxial stressing tests demonstrate that the skins of the spot and the skipjack do not behave as simple crossed-fiber systems. Stress-strain curves for tests performed in which the hoop axes were maintained at a constant stress of 0.12 MN/m 2 are shown for both fishes in Figures 7 and 8. Also shown in these figures are graphs of the predicted curve, based on the continuous fiber model described previously, subjected to the same ratio of stresses applied to the skin and having an initial fiber angle of 65. It can be seen from these graphs that the skin of neither the spot nor the skipjack behaves in a manner similar to the model. Both are considerably stiffer than the model, having steeper curves than predicted, yet both require lower stresses applied to the longitudinal axes to achieve large initial extensions prior to crossing the predicted curve. It is important to note that negative strains obtained for both the hoop and lon- gitudinal directions were very small. Under these biaxial conditions much larger neg- ative strains should have been observed, since the crossed-fiber system should allow both shrinking and stretching to occur simultaneously in the orthogonal sides of the skin. Instead, for both the spot and the skipjack, it appears that other material com- ponents within the skin dominate over the crossed-fiber system, as shown diagram- matically in Figure 9. In this figure, row A depicts the behavior of a crossed-fiber model and row B depicts that of spot and tuna skin. When hoop forces are applied that exceed the longitudinal forces, the skin shown in B2 extends in the hoop direction but fails to contract in the longitudinal direction. In contrast, the model shown in A2 does contract in the longitudinal direction, and it therefore maintains a constant area. When lon- gitudinal forces that equal the still-present hoop forces are applied to the model (A3), it extends in the longitudinal direction while contracting in the hoop direction. (Because forces are equal in both directions, it now has the same dimensions as it had in Al, before any forces were applied.) The skin shown in B3, however, does not contract in the hoop direction and extends very little in the longitudinal direction. (Its area has increased.) Much greater longitudinal forces are required (B4) to obtain both hoop contraction and longitudinal extension in the skin. The same longitudinal forces applied to the model (A4) result in greater degrees of both hoop contraction and longitudinal extension, with no increase in area. DISCUSSION The collagenous layers within the skin of both the Norfolk spot and the skipjack tuna occupy the major part of the skin's thickness, and this relatively thick collagenous layer accounts for the skin's strong construction. In this respect the skins of these fishes are similar to those of both the eel and the shark, however, relative to total body size the spot and the tuna both have skin that is comparatively thin. Mechanical tests of the skins of these fishes reveal other similarities and differences: while the terminal elastic moduli are similar in cases of uniaxial stressing in the hoop and longitudinal directions, the skin of the spot and skipjack both become stiffer at lower extensions than does eel skin. In the directions of the fibers themselves the skin is an order of magnitude less stiff than that of the eel, indicating that either the fibers themselves are not continuous over the lengths of the skin samples tested, or else the fine fibrils that comprise the fibers pull apart from each other when loaded directly along their axes. 246 M. R. HEBRANK AND J. H. HEBRANK More impo, ?, r ;, however, the skin of the spot and the skin of the skipjack do not undergo the - changes expected of a crossed-fiber system, thus the skin is not capable mitting forces down the length of the fish. In a fish such as the eel or the shark uscle contraction in the anterior region bends the fish, and so the skin on the ex side is extended in the longitudinal direction. This extension, however, produce contraction in the hoop direction as the fiber angle decreases, until these dimens oual changes are resisted by pressurization of the body fluids beneath the skin. Now the skin becomes stiff, and further longitudinal force applied to the skin by the anterior muscles results in tension transmitted to the tail by the skin. In this way the skin of the eel or the shark can act as an external tendon, as suggested by Wainwright et al (1978). The results of biaxial tests of spot and skipjack skin reported here demonstrate that contraction of one side of the skin does not occur concomitant with extension of the orthogonal direction. Without this contraction tension cannot be transmitted by the skin down the length of the fish during swimming movements. Although the skin appears to have helically arrayed fibers suitable for an external tendon, it cannot function as one. Instead, for the spot and the skipjack, the crossed-fiber array of collagen seems to function primarily to keep the tough exterior surface of the fish smooth and free of kinks during swimming movements. A smooth surface is an important factor promoting hydrodynamic performance. It is interesting to note that while eels and sharks possess skin capable of acting as an external tendon, this study suggests that teleosts in general probably do not. Eels and sharks share one feature of the skin that may relate to the ability to transmit forces: they have extremely thick skin. A thick skin is clearly beneficial to these fishes in consideration of some of their peculiar behaviors; some sharks have been observed to bite each other during courtship and eels spend much of their time burrowed beneath the substrate. In contrast, most teleosts have skin that is much thinner in proportion to their body size and with a reduction in skin thickness apparently comes a loss in the ability to transmit forces. Webb and Skadsen (1979) recently suggested that a reduction in skin mass is related to a fish's ability to accelerate rapidly during prey capture, and so it seems likely that those fishes that rely on this "stalk and sprint" method of feeding have skins with mechanical properties similar to the Norfolk spot and the skipjack tuna. There is another morphological feature common to the eel and the shark but not to most advanced teleosts, and this is the arrangement of the axial musculature. Al- exander (1969) described two patterns of the axial musculature of fishes, which he termed the "selachian" and "teleost" arrangements. Relevant to this study is the fact that the selachian pattern is found in the sharks, eels, Amia, Acipenser, and Salmo, while the teleost pattern is found in virtually all other teleosts that swim using lateral undulations of the body. In addition, Willemse (1972) noted that in the eel and the shark the myosepta are thickened at the periphery of the fish where they attach to the skin: such thickenings are not found in the spot or the tuna. Finally, those fishes having ^lachian arrangement of the axial musculature tend to have relatively high vertebral eels and sharks have over 100 vertebrae, Amia has about 80, Acipenser has about , and Salmo has about 60. In contrast, tunas in general have about 35-40 i the spot has only 24. Parities and differences suggest that perhaps those fishes in which the selachian - arrangement and high vertebral numbers are found also have skin capable s an external tendon, although the functional relationships between these three c ..nents are unknown. In his functional analyses of the selachian and teleost muscle arrangements Alexander (1969) concludes that the selachian arrange- MECHANICS OF FISH SKIN 247 ment allows for the development of greater bending forces than does the teleost, but the teleost arrangement allows the fish to bend more quickly than the selachian. It is also important to recognize that anguilliform swimmers (eels and sharks) must transmit forces over a longer portion of the propulsive wave compared to the subcarangiform swimmers (e.g., the spot) and the thunniform swimmers (tunas). It may prove possible, then, that fishes utilize one of two methods of swimming: contractions of the axial musculature, which bend the fish more slowly, do so in such a way as to generate forceful bending moments and the forces generated may be transmitted by the skin; or by contractions of the axial musculature that bend the fish more rapidly at the expense of weaker bending moments, and the forces developed by the muscles cannot be transmitted by the skin. Examination of the skins ofAmia, Acipenser, and Salmo, as well as additional advanced teleosts, are needed before such positive correlations between muscle, skin, and backbone types can be established. ACKNOWLEDGMENTS The authors extend their thanks to S. A. Wainwright for helpful advice and dis- cussions throughout this study, and to an anonymous reviewer for suggestions to improve the manuscript. This work was supported by a Training Grant in Morphology to M. R. H. from the Cocos Foundation. LITERATURE CITED ALEXANDER, R. M. 1969. Orientation of muscle fibres in the myomeres of fishes. J. Marine Biol. Assoc. t/tf 49: 263-290. BROWN, G. A., AND S. R. WELLINGS. 1970. Electron microscopy of the skin of the teleost, Hippoglossides elassodon. Z. Zellforsch. Mikrosh. Anal. 103: 149-169. CLARK, R. B. 1964. Dynamics in Metazoan Evolution. Clarendon Press, Oxford. 313 pp. FUJII, R. 1968. Fine structure of the collagenous lamella underlying the epidermis of the goby Chasmichthys gulosus. Annot. Zool. Jpn. 41: 95-106. HAWKES, J. W. 1974. The structure offish skin. I. General organization. Cell Tissue Res. 149: 147-158. HEBRANK, M. R. 1980. Mechanical properties and locomotor functions of eel skin. Biol. Bull. 158: 58-68. MOTTA, P. J. 1977. Anatomy and functional morphology of dermal collagen fibers in sharks. Copeia 1977(3): 454-464. NADOL, J. B., JR., J. R. GIBBONS, AND K. R. PORTER. 1969. A reinterpretation of the structure and devel- opment of the basement lamella: an ordered array of collagen in fish skin. Dev. Biol. 20: 304-331. VIDELER, J. J. 1974. On the interrelationships between morphology and movement in the tail of the cichlid fish Tilapia nilotica (L). Neth. J. Zool. 25: 143-194. WAINWRIGHT, S. A., F. VOSBURGH, AND J. H. HEBRANK. 1978. Shark skin: function in locomotion. Science 202: 747-749. WEBB, P. W., AND SKADSEN, J. M. 1979. Reduced skin mass: an adaptation for acceleration in some teleost fishes. Can. J. Zool. 57: 1570-1575. WILLEMSE, J. J., 1972. Arrangement of connective tissue fibres in the musculus lateralis of the spiny dogfish, Squalus acanthias L. (Chondrichthyes). Z. Morphol. Tierel2: 231-244. Reference: Biol. Bull. 111. 248-263. (August, 1986) SENSITIVITY OF THE HEMOCYANINS AND ITS RELATIONSHIP TO Cl~ SENSITIVITY CHARLOTTE P. MANGUM AND LOUIS E. BURNETT, JR.* Department of Biology, College of William and Mary. Williamsburg. Virginia 23185 ABSTRACT The effect of CO 2 on hemocyanin-oxygen binding is not generally related to the effect of Cl~. Some hemocyanins respond to both and some to either one alone. The direction of the responses of O 2 affinity of the various hemocyanins to CO 2 is poorly correlated with the direction of responses to other effectors. The influence of CO 2 on Busycon and Lirnulus hemocyanins reaches its maximum at high pH. Since the effect can be abolished by restoring divalent cation activities to the control levels prior to the addition of CO 2 , we suggest that the effect is not specific but rather indirect, by the pairing of the allosteric effectors Ca +2 and Mg +2 with the CO 2 anions. In contrast the effect of CO 2 on crustacean hemocyanins is greater at low pH and it can be enhanced by maintaining HCO 3 ~ levels within narrow limits and permitting PCO 2 to vary by a large factor. This finding suggests that the effective species is molecular CO 2 . While Cl~ influences only oxygen affinity, CO 2 may influence cooperativity as well. In different species the effects of both Cl and CO 2 may or may not be great enough to be phys- iologically important. INTRODUCTION A site on the hemocyanin (He) molecule which is linked to the active site com- petitively binds divalent cations and H + . Its molecular features and their physiological importance have been explored in detail (Arisaka and Van Holde, 1979; reviewed by Mangum, 1980; Miller and Van Holde, 1981). The nature and significance of anion binding to the Hcs are less clear. While the effects of the organic anion L-lactate on the crustacean Hcs are beginning to be elu- cidated by a number of investigators (reviewed by Bridges and Morris, 1986), the influence of CT is not as well understood. Cl~ specifically influences HcO 2 binding in the chelicerate arthropod Lirnulus (Sullivan et al, 1974; Diefenbach and Mangum, 1983) and the gastropod mollusc Busycon (Mangum and Lykkeboe, 1979), but its effect on crustacean Hcs ranges from strong (Brouwer et al., 1978) to absent (Truchot, 1975; Mason et al., 1983). Brix and Torensma ( 1981) and Torensma and Brix (1981) concluded that the effects of Cl~ and CO 2 on gastropod Hcs are both allosteric and linked to one another, suggesting a d-HCO 3 ~ site analogous to the divalent cation- H + site. In fact, the effects of CO 2 in whatever form are even less clear than those of Cl~. nted out earlier (Burnett and Infantine, 1984), only one of the several investi- gation, -> the subject convincingly demonstrates a significant and specific effect of CO 2 on C ffinity of the crustacean Hcs (Truchot, 1973). In contrast, a number of negative rep )rts have appeared (reviewed by Burnett and Infantine, 1984; see also ruary 1986; accepted 25 April 1986. * Permanent address: Department of Biology, University of San Diego, Alcala Park, San Diego, California 92110. 248 C0 2 SENSITIVITY OF HEMOCYANINS 249 Morris and Bridges, 1985). After the paper by Burnett and Infantine (1984) was in press a large specific effect of CO 2 on a crab He was reported by Greenaway et al. (1983) but, curiously, the response is opposite to that found in Carcinus (Truchot, 1973). An effect of very low levels of CO 2 on O 2 affinity of gastropod He has been found in three species (Mangum and Lykkeboe, 1979; Torensma and Brix, 1981) but the mechanism remains unclear. Mangum and Lykkeboe (1979) suggested that it may be either direct and specific, viz. by means of CO 2 binding to a site linked to the active site, or indirect and non-specific, viz. by means of ion-pair formation of HCO 3 and CO 3 ~ 2 with the divalent cations, thus inhibiting the action of other allosteric effectors. Brix and Torensma (1981) and Torensma and Brix (1981) described the CO 2 effect as allosteric, but presented as supporting evidence only the fit of their data to the Monod-Wyman-Changeux model. However, the model, which describes a particular form of allosteric behavior, would not distinguish between direct and indirect allosteric actions. We have investigated the effect of CO 2 and Cl~ on HcO 2 binding in a variety of species chosen because they are either the same as or closely related to those studied earlier and also because they may represent various combinations of CO 2 and Cr sensitivity. We have investigated the mechanisms of CO 2 effects on gastropod (and also chelicerate) Hcs, by controlling the levels of divalent cation activity while varying those of CO 2 anions. If the divalent cations are the immediate effectors, we would expect to see no effect of CO 2 . We have investigated the mechanism of the effect on crustacean Hcs by controlling (in large part) the levels of the CO 2 anions while allowing CO 2 to vary by a large factor. In this case we would expect the CO 2 effect to vary if molecular CO 2 is the immediate effector. MATERIALS AND METHODS Collection and holding of animals The crustaceans Cal/inectes sapidus Rathbun and Palaeomonetes pugio Holtuis, the chelicerate Limulus polyphemus (Linnaeus), and the gastropod mollusc Busycon canaliculatum Linnaeus were collected locally. The crustacean Penaeus duorarum Burkenroad was collected offshore of Beaufort, North Carolina and Carcinus maenas (Linnaeus) at Mt. Desert Island, Maine. The polyplacophoran mollusc Cryptochiton stelleri Middendorff was purchased from commercial sources. Animals were held at 16-25C, depending on origin, and salinities (20-35%o) either identical to those at the collection sites or approximating those specified in previous investigations. The ex- perimental temperature was chosen similarly. Procurement and preparation of samples If the ionic composition of the blood of a species was unknown it was determined with ion-selective electrodes (Mangum and Lykkeboe, 1979). Blood samples were obtained by syringe sampling. The samples were centrifuged to remove debris, in the case of the arthropods after first declotting with a tissue grinder. The sera were then either used for O 2 binding measurements without modi- fication or first dialyzed against the desired saline (4C, 24 h). O 2 binding O 2 binding was determined by two methods: ( 1 ) for measurements in the presence of different levels of CO 2 a spectrophotometric method was employed (Burnett, 1979; 250 C. P. MANGUM AND L. E. BURNETT, JR. Burnett and lino, 1984). Mixtures of N 2 (99.9995%, scrubbed with Oxisorb), CO 2 (99.5% 'epending on PO 2 , either air (scrubbed with soda lime and Dri- Rite) or O ; fe) were prepared with Wosthoff pumps, humidified in a gas washing bottle, ; .ii over samples incubated in a thermostatically controlled shaker bath. Th >ples had been diluted by factors ranging from 13.5 to 101 with 0.05 M Tris M ate buffered saline. Changes in absorbance ( 1 cm light path) were determined at : 5 nm, depending on species, with a Bausch & Lomb Spectronic 20 color- imetei , These data are illustrated with open symbols. (2) The cell respiration method (Mangum and Lykkeboe, 1979) was used to de- termine the effects of inorganic ions. Samples were diluted by 10% with buffered saline or buffered test solution (final concentration 0.05 M Tris Maleate buffer), and pH (Fisher Accumet with Ross electrode) and PCO 2 (Radiometer electrode, calibrated with 1.05 and 3.03% CO 2 ) were measured at the end of an experiment. These data are illustrated with closed symbols. In this procedure PO 2 is lowered at a constant rate by respiring yeast cells, which also excrete CO 2 . To ascertain that PCO 2 in a buffered preparation does not change during an experiment, a second measurement was per- formed in parallel, but PCO 2 was measured instead of PO 2 . Following equilibration of the electrode with the sample, no change was detected (Fig. 1). O : carrying capacity The total O 2 capacity of polyplacophoran blood is known from a single observation on a single individual (Redmond, 1962). After first subtracting the absorbance of deoxyHc to eliminate light scatter, an estimate was made from the spectrophotometric data using the extinction coefficient for gastropod He (Nickerson and Van Holde, 1970). Total O 2 was also obtained from the records made during the cell respiration procedure as follows (Fig. 2): The area under the (dashed) line representing a constant rate of O 2 depletion describes the volume of free O 2 , which can be evaluated knowing 6.9 4.6 2.3 I E E "^2.3 O tL . I ^MMMQ 5 10 TIME (MIN) 15 FIGURE 1. ,ng of PCO 2 during depletion of O 2 in: A. Saline, with no He and no exogenous buffer. B. Saline y enaeus duorarum He but no exogenous buffer. C. Saline + P. duorarum He and 0.05 M Tris Maleate buffer (pH 7.6). 20C. CO 2 SENSITIVITY OF HEMOCYANINS 251 FIGURE 2. O 2 depletion by yeast cells in presence of Cryptochiton stelleri He. pH 7.43, 1 5C. Hatched area shows volume of free O 2 ; stippled area shows volume of HcO 2 . temperature and solute concentration. The area under the curve representing the de- viation from that line describes the volume of He-bound O 2 , which can be evaluated as a fraction or, more often, a multiple of the free O 2 area. Data analysis When pH, CO 2 , and Cl were either controlled or when all three had no detectable effect, the significance of differences between mean values of P 50 or n 50 was estimated by Student's Mest; the probability of a significant difference is specified below. When pH and CO 2 were varied simultaneously and Cr controlled, the data were described by semilogarithmic (log Y) regression lines and the overlap (if any) between 95% confidence intervals around the lines used as the criterion of significance. When NaCl 252 C. P. MANGUM AND L. E. BURNETT, JR. was varied w >H and PCO 2 were controlled, the P 50 data were described by semi- logarithmic re ion lines and the difference of the slope from zero (at P - .05) used as the criten : inally, when HCO 3 ~ and NaCl were controlled while pH and PCO 2 were van utaneously, the data were described by semilogarithmic regression lines a -verlap (if any) between 95% confidence intervals around the slopes was used as i ; criterion. If a substance is said below to have an effect these criteria were exceeded. RESULTS Busycon canaliculatum Mangum and Lykkeboe (1979) showed that in the channeled conch Cl~ has a specific effect on HcO 2 binding and that, at high pH, the addition of molecular CO 2 lowers the oxygen affinity of low salinity samples and raises the oxygen affinity of high salinity samples, but only above pH 7.9 where the reverse Bohr shift is quenched by divalent cations. They suggested that, if the CO 2 effect were indirect (viz. by means of ion-pair formation with the divalent cations), the difference between high and low salinity could be explained quantitatively by their findings on the effects of individual inorganic ions: at high salinity the addition of CO 2 immobilizes some of the divalent cations that normally lower oxygen affinity but enough Ca +2 and Mg +2 remains to quench the reverse Bohr shift; the net result is an increase in oxygen affinity. At low salinity, where Ca +2 and Mg +2 are already scarce, their immobilization by CO 2 does not leave enough free divalent cations to quench the reverse Bohr shift. Na + and Cl~ become the dominant inorganic ions, and they lower oxygen affinity, which is in fact the net result. The present findings confirm the effect of CO 2 at high salinity, only above about pH 7.9 (Fig. 3). To more directly demonstrate the mechanism the following experiment was per- formed: first a sample of serum was dialyzed against buffered physiological saline for 24 h and O 2 binding determined. Then 25 mA/ NaHCO 3 ~ was added to the stirred preparation in which a Ca +2 selective electrode was immersed, and the change in Ca activity noted. O 2 binding was determined again. While the sample was stirred with the Ca electrode immersed, Ca(NO 3 ) 2 was then added in sufficient quantity to restore the original level of Ca activity; O 2 binding was measured a third time. This experiment controls for pair-formation of the CO 2 anions with Ca +2 but not Mg +2 . It could not be performed with a total divalent cation selective electrode for a variety of reasons, the most important of which are the lower sensitivity and resolution of the electrode. Normally these problems are mitigated by chelating Ca with EGTA and then measuring pMg alone; the remedy would not have been possible in the present context without eliminating an important effector of O 2 binding. Since the percent pair formation of 1% and Mg with the CO 2 anions is the same (Kester and Pytkowicz, 1969) and since Fects of Ca +2 and Mg +2 on O 2 binding are very nearly so (Mangum and Lykkeboe, i, the pair formation with Mg was calculated and additional Ca(NO 3 ) 2 was added late the original Mg^ 2 activity using Ca +2 instead. Changes in monovalent ose small magnitudes have no detectable effect (Mangum and Lykkeboe, inding was determined a fourth time. ^d earlier (Mangum and Lykkeboe, 1979), the addition of NaHCO 3 to the blood ra s the O 2 affinity of high salinity blood and lowers the O 2 affinity of low salinity t I). It does not clearly influence cooperativity (P = .09-0.70). The third and fourth Eeps of the experiment were performed on high salinity blood. When the original actiuty of only Ca was restored, O 2 affinity dropped slightly though sig- C0 2 SENSITIVITY OF HEMOCYANINS 253 14 A A ^ 10 _ 8 A , *<* ^ (4) O E 6 E IM O E * * 00* O 4 m 0. o O o 2 o 1 1 1 1 1 1 1 7. 2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 PH n 50 I r B ao 7.2 7.6 8.0 8.4 8.8 PH FIGURE 3. Effect of CO 2 on Busvcon canaliculatum HcO 2 binding. Tonometric method. (O) PCO 2 = 0; (A) PCO 2 = 0.74 mm Hg; ( C ) PCO 2 = 7.4 mm Hg; (D) PCO 2 = 14.8 mm Hg. 20C. Serum was diluted with 0.05 M Tris maleate buffered high salinity saline (from Mangum and Lykkeboe, 1979). A. Oxygen affinity. B. Cooperativity. nificantly, but still differed from the control value. When additional Ca was added to simulate the change in total divalent cations, O 2 affinity decreased further to a value that is indistinguishable from the control. Cryptochiton stelleri The He of the giant sea cradle is clearly cooperative and it has a small (though significant) normal Bohr shift (Fig. 4). The present results essentially agree with earlier 254 C. P. MANGUM AND L. E. BURNETT, JR. TABLE I Effect ofNaHi '' ' a(NO 3 ) 2 on Busycon canalicalatum HcO? binding HS LS so r>5o "50 n 50 Control, PCO 2 1.50- 1.59 mm Hg 10.75 0.31 (8) 1.31 0.04 (8) 9.70 0.40 (6) 1.36 0.13 (6) + 25 mM NaHCO 3 PCO 2 5.5-5.7 mm Hg 9.31 0.17 (6) 2 1.40 0.03 (6) 10.70 0.30 (8) 1.43 0.25 (8) Same, + Ca +2 to Equal pCa in control. PCO 2 5.7 mm Hg 9.95 0.20 (9) 3 1.33 + 0.05(9) Same, + Ca +2 to Equal pCa + pMg in control 10.72 0.20 (8) 4 1.33 0.06 (8) 1 Cell respiration method:serum was dialyzed against 0.05 AI Tris maleate buffered high (HS) or low (LS) salinity saline (from Mangum and Lykkeboe, 1979). 1 9.7-20. 1C, pH 8.18-8.23. Mean S.E. (N). 2 P = 0.0035 vs control. 3 P = 0.025 vs control, 0.0375 vs + NaHCO 3 alone. 4 P = 0.04 vs + Ca +2 to equal pCa in control, 0.97 vs control. findings (Manwell, 1958), although the comparison is made somewhat difficult by the different pH ranges investigated. The slope of a regression line describing the two sets of control data for P 50 in Figure 4A is -0.20 0.04 (95% C. I.). Also in the combined control data, there is a small but significant increase in cooperativity with pH. The O 2 affinity data for and 14.8 mm Hg PCO 2 differ significantly in the pH range 7.2-7.6 but not in the range 7.7-7.85 (Fig. 4A). At high PCO 2 cooperativity is lower (P = 0.002) and there is a small but significant decrease with pH. Neither HcO 2 affinity (Fig. 4B) nor cooperativity (1.87 0.09 S. E., N == 6) respond to NaCl. The data in Figure 4 were obtained using blood taken from the hemocoel directly into a hypodermic needle passed through two adjacent dorsal shell plates. When an initial attempt to perform the operation was unsuccessful, a blood sample was obtained by slitting the foot and draining the hemocoel. No other organs were damaged although some mucus was produced. The sample formed a blue precipitate and, after centrif- ugation at low speed, the supernatant fluid did not combine reversibly with oxygen. When the pellet was washed with saline, it did not go into solution but it still combined reversibly with oxygen. Measured with the cell respiration technique, oxygen affinity was higher than that of the syringe sample, but only by about 25%; cooperativity was lower but still easily detected (n 50 = 1.72 0.19 S. E., N = 12). Apparently the native ier order structure is not critical to cooperativity and a relatively low O 2 affinity, c difference in absorbance between oxygenated and deoxygenated samples, cor- rects the dilution factor indicates that the native He concentration in one animal was aboi .74 g/100 ml, yielding an HcO 2 capacity of only about 0.33 (total O 2 capacity < 93) ml/ 100 ml. The integrals obtained from the records made during the cell res; tion procedure indicate an HcO 2 carrying capacity in another animal of 0.22 ml/. I : vii or a total blood O 2 carrying capacity of 0.80 ml/ 100 ml. The results support the ; .mce of an extremely low O 2 carrying capacity of the blood in this class (Redmond, 1962). CO 2 SENSITIVITY OF HEMOCYANINS 255 25 0> I 20 o m o_ 15 I 72 _L 74 76 PH 78 80 o t02 u o D Q, OD 1 1 1 1 1 71 73 7.5 7.7 7.9 8.1 PH 20 I 15 o Q_ B J. 100 200 NaCI (mM) 300 400 500 FIGURE 4. A. Effect of CO 2 on Cryptochiton stelleri HcO 2 binding. Tonometric method: serum was diluted with 0.05 M Tris maleate buffered, filtered seawater (35%o) in which the animals had been shipped. (O) PCO 2 = 0, (D) PCO 2 = 14.8 mm Hg. 15C. Cell respiration method: after dialysis against seawater, Tris maleate buffer (final concentration 0.05 M) was added to the serum. () PCO 2 1.32-5.41 mm Hg. B. Effect of NaCI on HcO 2 affinity. Cell respiration method: serum was dialyzed against 10 mMCa(NO 3 ) 2 + 0.05 M Tris maleate buffer, pH 7.62-7.65. PCO 2 3.0-3.3 mm Hg. 15C. 256 C. P. MANGUM AND L. E. BURNETT, JR. Penaeus duoran At pH '. h is likely to approximate the physiological value, the He of the pink shn, 1 ' a moderate oxygen affinity and pronounced cooperativity. In the pH range i it also has a large normal Bohr shift (Fig. 5); the slope of a regression line des g the two sets of control data is -1.09 0.19 (95% C. I.). The data are virtualh indistinguishable from those reported earlier for P. setiferus (Brouwer et ai, 1978). As also in P. setiferus (Brouwer et ai, 1978), P. duorarum HcO 2 affinity clearly responds to NaCl (Fig. 5B). However, it responds identically to NaNO 3 , Na 2 SO 4 (plotted as mA/ cation), and choline Cl~. There is a significant decrease in cooperativity (P - 0.05-0.001, Fig. 5B). Neither property responds to CO 2 , although the trend in coop- erativity is the same as that in the data for CO 2 sensitive Hcs (P = 0. 15, Fig. 5 A). Palaeomonetes pugio At putative physiological pH the He of the grass shrimp has an extremely low O 2 affinity and fairly little cooperativity (Fig. 6). The slope of a semilogarithmic regression line describing the control data for P 50 is - 1 .33 (0.56 95% C. I.). HcO 2 affinity clearly responds to CO 2 (Fig. 6A), which does not appear to influence cooperativity (P = 0.18). The pH dependence of P 50 at high PCO 2 appears to be about the same as that at low PCO 2 , but the estimate made by regression analysis is much greater (-1.75 0.1 1). In view of the far greater variability in the control data (r 2 = 0.756) the numerical estimate of the Bohr shift at high PCO 2 (r 2 = 0.998), which shows extremely great pH dependence, is probably the more accurate of the two. NaCl clearly raises HcO 2 affinity but does not change cooperativity (Fig. 6B). Due to the great difficulty of obtaining ample volumes of blood from these always small animals, which were even smaller than usual at the time of the experiment on NaCl sensitivity because the population had just reproduced, we did not investigate the specificity of this response. Callinectes sapidus The He of the blue crab does not respond specifically to Cl~ (Mason et al, 1983). Oxygen affinity (but not cooperativity) does respond to CO 2 (Fig. 7). Because it was concluded earlier that the He of a species belonging to the same genus (C. bellicosus) is not sensitive to CO 2 within the range 1.5-7.4 mm Hg, the response of C. sapidus was examined in more detail. While there may appear to be little or no difference in P 50 at and 1 .5 mm Hg (Fig. 7), the two sets of data are in fact significantly different. When the data for C. bellicosus are analyzed similarly, however, an effect of CO 2 can be demonstrated only at 23C and only at pH 7.5 and above (Burnett and Infantino, 1984). Of the other four species examined by Burnett and Infantino (1984) a similar trend may be present in the data for Pachygrapsus crassipes (though P > .05); however, no sign of an effect can be perceived in the remaining three (Burnett and Infantino, 1984). Thus we suggest that our original conclusion, viz. that CO 2 is not an important effector of HCO 2 binding in the five species, was essentially correct (Burnett and In- fantino, 1984). Regardless, only at much higher PCO 2 is there an appreciable response of HCO 2 affinity in C. sapidus; cooperativity does not respond even at that level In a attempt to identify the CO 2 species responsible for the effect, we dialyzed aliquots s sample against either (1) saline containing no exogenous CO 2 , or (2) saline t mning high levels of NaHCO 3 . When pH was adjusted by adding Tris maleate buffered saline (final concentration 0.05 M), PCO 2 varied by more than an order of magnitude while HCO 3 ~ varied by only about 25% (Fig. 7B). The oxygen affinity of this sample was depressed even more at low pH and high PCO 3 than at high pH and low PCO 2 . If this result were due to the greater change in pCa at high CO 2 SENSITIVITY OF HEMOCYANINS 257 o (0 o o~- 02 ^ E i Jo O b. - O + 4 I U 1 . o C T3 c\j (B H UJLU) og e_ 00 10 u _ ft o O ^ *^ .'S cu ^--^ "^ Scr ^ .= ? H Q .y ^ o C C rn 4 % o O O O (6n ujuu) 2 Sx o CVJ to 09 U CVJ 00 CVJ o K o c O u-i H u q -k r~. CU O S 2 r>*-. e B^II ^J"S5 W cd 3 C K .S ^. 2 'S T3 O ^ 258 C. P. MANGUM AND L. E. BURNETT, JR. 80 60 40 20 1 1 1 1 7.4 7.6 PH 7.8 8.0 o o o 7.4 7.6 7.8 8.0 PH A - 3 - 2 o T in - Q. * 30 * * li i i i !__ 100 200 300 NoCI (mM) 400 500 . Effect of CO 2 on Palaemonetes pugio HcO 2 binding. Tonometric method: serum was dialyzed again ,e containing 261 mAf NaCl, 8.1 mA/ KC1. 6.3 mAf CaCl 2 , 4.4 mA/ MgCl : , and 29.5 mA/ Na 2 SO 4 intel and Farmer, 1983), and diluted with 0.05 M Tris maleate buffered saline. (O) PCO 2 = 0, (. M.8 mm Hg. 17C. B. Effect of NaCl on HcO 2 binding. Cell respiration method: serum was diah-.. si 10 mA/Ca(NO 3 ) 2 + 0.05 mA/ Tris maleate buffer, pH 7.65-7.68, PCO 2 1.3-1.5 mm Hg. 25 C. (} P 50; (A) n so . CO 2 SENSITIVITY OF HEMOCYANINS 259 ~ 20 o> x E E o "> 10 , A Q 7.4 7.6 7.8 PH 73 7.5 7.7 7.9 P H 60 40 20 1,0 ~ 8 o 6 in 0. 4 f- 70 7.2 74 76 PH 78 8.0 82 4 i- 2 7 t ' * i i i i m A A l P V A 7.2 7.4 7.6 7.8 PH 8.0 8.2 FIGURE 7. A. Effect of CO 2 on Callinectes sapidus HcO 2 binding. Tonometric method: serum was diluted with 0.5 M Tris maleate buffered high salinity saline (from Mason el al.. 1983). 25C. (O) PCO 2 = 0, (D) PCO 2 = 1.5 mm Hg; (A) PCO 2 = 14.8 mm Hg. () For comparison, unpublished data collected earlier by cell respiration method: serum was dialyzed against same Tris maleate buffered saline. 25 C, PCO 2 unknown. B. Effect of high NaHCO 3 . Cell respiration method: in controls (), PCO 2 and [NaHCO 3 ] vary from 0.32 mm Hg and 4.09 mM at pH 8.25 to 6.98 mm Hg and 1 .69 mM at pH 7.24. In experiments (A), PCO 2 and [NaHCO 3 ] vary from 1.2 mm Hg and 13.7 mM at pH 8.25 to 1.4 mm Hg and 10.61 mM at pH 7.00. 25C. Dashed lines, which differ significantly, were fitted by regression analysis. 260 C. P. MANGUM AND L. E. BURNETT, JR. than at low pH, O 2 affinity should have been lowered at high pH and unchanged at low pH rather tha n the other way around. Carcini Truci, / :i973, 1975) showed earlier that, in European members of this species, Cr ha> no effect whereas CO 2 clearly raises O 2 affinity. Our data confirm the CO 2 effect. ich occurs in about the same magnitude in the North American population (Fig. 8). They also show that CO 2 significantly lowers cooperativity and its pH de- pendence. Limulus polyphemus Cl~ has a specific effect on HcO 2 affinity (but not cooperativity) in the horseshoe crab (Sullivan et al, 1974; Diefenbach and Mangum, 1983). CO 2 has a significant effect on O 2 affinity as well; an apparent decrease in cooperativity is not quite significant (P = 0.11; Fig. 9). The experiment designed to identify the mechanism of the CO 2 effect on Busycon He was also performed on Limulus He, with the exception that the step simulating the effect of changing only pCa was omitted. The addition of NaHCO 3 significantly raises O 2 affinity (Table II) but not cooperativity (P = 0.19) and the addition of Ca(NO 3 ) 2 to restore the original levels of free Ca +2 and Mg +2 lowers O 2 affinity back again to a value that does not differ from the control. DISCUSSION Various combinations of Cl~ and CO 2 sensitivity of the Hcs can be found. In any particular species HcO 2 affinity may be sensitive to both (e.g., Busycon, Limulus, and Palaeomonetes), to Cl" but not CO 2 (Penaeus) or to CO 2 but not Cr (Cryptochiton, 20r ^^- I 10 E E 8 ID 6 7.2 7.4 7.6 7.8 pH o 6 o 04 00 .00 - o a a A 2 7.2 7.4 7.6 7.8 PH FIGURE 8. Effect of CO 2 on Carcinus maenas HcO 2 binding. Tonometric method: serum was diluted with 0.05 A/Tris raaleate buffered saline (from Robertson, 1960). 16C, (O) PCO 2 = 0, (A) PCO 2 = 14.8 mm Hg. CO 2 SENSITIVITY OF HEMOCYANINS 261 20 I E E o 10 tr> _L _L 74 7.6 7.8 pH 8.0 4r I 7.3 7.5 7.7 7.9 8.1 PH FIGURE 9. Effect of CO 2 on Limulus polyphemus HcO 2 binding. Tonometric method: serum was diluted with 0.05 A/Tris maleate buffered high salinity saline (from Towle and Mangum, 1982). (O) PCO 2 = 0, (A) PCO 2 = 14.8 mm Hg. 25C. Carcinus, and Callinectes). In contrast the effect of CO 2 (if it occurs) is poorly correlated with sensitivity to other ionic effectors. While CO 2 consistently raises the O 2 affinity of Hcs (Busycon, Limulus) with reverse Bohr shifts (and, more importantly, positive divalent cation responses), it may either raise (Callinectes, Carcinus, and Palaeomo- netes), lower (Cryptochitori) or have no effect on (Penaeus) the O 2 affinity of Hcs with normal Bohr shifts. Different combinations of CO 2 sensitivity of HcO 2 affinity and cooperativity can also be found. CO 2 may affect O 2 affinity but not cooperativity (e.g., Busycon, Limulus, and Callinectes), both (Carcinus, Cryptochiton, and perhaps Pa- laeomonetes) or perhaps neither (Penaeus). If CO 2 does have an effect, it generally lowers cooperativity and reduces its pH dependence. Our results suggest that the chemical species responsible for the effect of CO 2 on portunid crab HcO 2 affinity is molecular carbon dioxide. The effect is greater at low than at high pH and the effector changes O 2 affinity in the same direction as the divalent cations. We can think of no indirect mechanism that would produce such a response. Moreover, the experiment in which HCO 3 " was held within a relatively narrow range while PCO 2 was allowed to change by a large factor produced a much greater effect of CO 2 at low pH, also implicating the molecular species. Our results also support the hypothesis that the mechanism responsible for the effect on Busycon and Limulus Hcs is the action of the CO 2 anions on the allosteric effectors free Ca +2 and Mg +2 rather than a direct effect. It occurs only at high pH, it TABLE II Effect ofNaHCO 3 and Ca(NO } ) 2 on Limulus polyphemus HcO 2 binding^ Pso 150 Controls PCO 2 1.06 mm Hg + 25 mA/ NaHCO 3 PCO 2 5.05 mm Hg + Ca (NO 3 ) 2 to equal pCa + pMg in control 17.1 0.2(5) 15.4 0.3 (5) 2 16.9 0.5 (6) 3 1.89 0.03 (5) 1.82 0.05 (5) 1.38 0.05 (6) ' Cell respiration method:serum was dialyzed against 0.05 A/Tris maleate buffered high salinity saline (from Towle and Mangum, 1982). 19.7C. pH 8.21-8.23. Mean S.E. (N). 2 P < 0.001 vs control. 3 P = 0.05 vs + NaHCO 3 , 0.75 vs control. 262 C. P. MANGUM AND L. E. BURNETT, JR. requires more CO : sugh than at low salinity and it can be quantitatively explained by the restorati of total divalent cation activity to levels that existed prior to the addition c There is no reason to postulate a binding site linked to the active site for ' inions compete. The similar responses in these two species also support the con : >n drawn earlier that the fundamentally different quaternary structures of the iascan and arthropod Hcs do not mandate different O 2 binding properties (Ma urn et al, 1985). The surprising effect of CO 2 on the He of the freshwater crab Holthuisana (Green- away. Bonaventura, and Taylor, 1983; Greenaway, Taylor, and Bonaventura, 1983) may be due to the same phenomenon; this He may not be directly sensitive to carbon dioxide. Unlike the response of other crustacean Hcs, the O 2 affinity of this molecule decreases with the addition of molecular CO 2 , (9.9 mm Hg), and the effect is slightly greater at pH 7.6 than at 7.22-7.30. Moreover, the levels of divalent cations in the blood of this freshwater crab are presumably relatively low (Greenaway and MacMillen, 1978), even at low levels of CO 2 . On the other hand, relatively few of the CO 2 anions would be formed by the addition of 9.9 mm Hg PCO 2 in that pH range (Greenaway, Bonaventura, and Taylor, 1983). Its He would have to be especially sensitive to divalent cations. Typically the slopes of Hill plots of HcO 2 equilibria increase with oxygenation. Since these data are often collected by a tonometric procedure that involves the stepwise addition of unscrubbed room air, we suggest that at least a fraction of the slope change may result from an increase in O 2 affinity with CO 2 rather than oxygenation, especially in species in which cooperativity is not very sensitive to CO 2 . The Cl~ sensitivity of Penaeus He may be either ( 1 ) specific and mimicked exactly by a specific effect of Na + , at least within the physiological range, or (2) a general effect of ionic strength. The information presently available does not decide the question. Since CO 2 sensitivity is not related to the sensitivity of inorganic anions (and since the nature of the binding sites is totally unknown), it is not possible at present to predict the effects of CO 2 (or Cl~) on the O 2 affinity of an unknown He. Regrettably, if the knowledge is needed it must be acquired in each case. Since it may be quite large, the effect of CT may be an important factor in understanding how a HcO 2 transport system works. In Penaeus, for example, a change from 330 to 560 mM CT~, well within the physiological range in many crustaceans (though perhaps not in these offshore species), causes a 50% change in P 50 (pH 7.6). Though more often small, the CO 2 effect may become similarly large at 25 C and physiological pH and Cl~, the maximum being 46% in the range 0-14.8 mm Hg (Palaemonetes). This is considerably greater than the effect of CO 2 on mammalian hemoglobin at the same temperature and at 100 mA/ Cl~ and pH 7.4 (calculated from data shown by Imai, 1982). The effect of CO 2 on He cooperativity is probably even more important. At pH 7.6 the maximum is a change of about 100% (Carcinus), which could radically alter oxygen- ation states at both the gill and the tissues. A more quantitative assessment awaits ixtensive measurements of physiological extremes of PCO 2 made simultaneously with surements of HcO 2 binding and its other determinants. ACKNOWLEDGMENT al by NSF DCB 84-14856 (Regulatory Biology). LITERATURE CITED ARISAKA, F., A VAN HOLDE. 1979. Allosteric properties and the association equilibria of hemocyanin from ( fa californiensis. J. Mol. Biol. 134: 41-73. C0 2 SENSITIVITY OF HEMOCYANINS 263 BRIDGES, C. R., AND S. MORRIS. 1986. Modulation of haemocyanin-oxygen affinity by L-lactate a role for other cofactors. In Invertebrate Oxygen Carriers, B. Linzen, Ed. Springer- Verlag, Heidelberg. (In press.) BRIX, O., AND R. TORENSMA. 1981. The molecular basis for the reversed Root effect in the blood of the marine prosobranch, Buccinnm undatum. Mol. Physiol. 1: 209-212. BROUWER, M., C. BONA VENTURA, AND J. BONA VENTURA. 1978. Analysis of the effect of three different allosteric ligands on oxygen binding by hemocyanin of the shrimp, Penaeus setiferus. Biochemistry 17:2148-2154. BURNETT, L. E. 1 979. The effects of environmental oxygen levels on the respiratory functions of hemocyanin in the crabs Libinia emarginata and Ocypode quadrala. J. Exp. Zool. 200: 289-300. BURNETT, L. E., AND R. L. INFANTINO. 1984. The CO 2 -specific sensitivity of hemocyanin oxygen affinity in the decapod crustaceans. J. Exp. Zool. 232: 59-65. DIEFENBACH, C. O. DA C., AND C. P. MANGUM. 1983. The effects of inorganic ions and acclimation salinity on oxygen binding of the hemocyanin of the horseshoe crab, Limulus polvphemus. Mol. Phvsiol. 4: 197-206. GREENAWAY, P., AND R. E. MACMILLEN. 1978. Salt and water balance in the terrestrial phase of the inland crab Holthuisiana (Austrothelphiisa) transversa Martens (Parathelphusoidea: Sundathelphusidae). Physiol. Zool. 51:217-229. GREENAWAY, P., J. BONAVENTURA, AND H. H. TAYLOR. 1983. Aquatic gas exchange in the freshwater/ land crab, Holthuisiana transversa. J. Exp. Biol. 103: 225-236. GREENAWAY, P., H. H. TAYLOR, AND J. BONAVENTURA. 1983. Aerial gas exchange in Australian freshwater/ land crabs of the genus Holthuisiana. J. Exp. Biol. 103: 237-251. IMAI, K. 1982. Allosteric Effects on Haemoglobin. Cambridge University Press, Cambridge. 275 pp. KESTER, D. R., AND R. M. PYTCOWICZ. 1969. Sodium, magnesium and calcium sulfate-ion pairs in seawater at 25C. Limnol. Oceanogr. 14: 686-692. MANGUM, C. P. 1980. Respiratory function of the hemocyanins. Am. Zool. 20: 19-38. MANGUM, C. P., AND G. LYKKEBOE. 1979. The influence of inorganic ions and pH on oxygenation properties of the blood in the gastropod mollusc Busycon canaliculatitm. J. Exp. Zool. 207: 417-430. MANGUM, C. P., J. L. SCOTT, R. E. L. BLACK, K. I. MILLER, AND K. E. VAN HOLDE. 1985. Centipedal hemocyanin: its structure and its implications for arthropod phylogeny. Proc. Natl. Acad. Sci. USA 82:3721-3725. MANTEL, L. H., AND L. L. FARMER. 1983. Osmotic and ionic regulation. Pp. 54-161 in The Biology of Crustacea. L. H. Mantel, ed. Academic Press, New York. MANWELL, C. 1958. The oxygen-respiratory pigment equilibrium of the hemocyanin and myoblogin of the amphineuran mollusc Cryptochiton stelleri. J. Cell. Comp. Physiol. 52: 341-358. MASON, R. P., C. P. MANGUM, AND G. GODETTE. 1983. The influence of inorganic ions and acclimation salinity on hemocyanin-oxygen binding in the blue crab Callinectes sapidus. Biol. Bull. 164: 104- 123. MILLER, K. I., AND K. E. VAN HOLDE. 1981. The effect of environmental variables on the structure and function of hemocyanin from Callianassa californiensis. I. Oxygen binding. /. Comp. Phvsiol. 143: 253-260. MORRIS, S., AND C. R. BRIDGES. 1985. An investigation of haemocyanin oxygen affinity in the semi- terrestrial crab Ocypode saratan Forsk. J. Exp. Biol. 117: 1 19-132. NICKERSON, K. W., AND K. E. VAN HOLDE. 1971. A comparison of molluscan and arthropod hemocyanin. I. Circular dichroism and absorption spectra. Comp. Biochem. Physiol. 39B: 855-872. REDMOND, J. R. 1962. The respiratory characteristics of chiton hemocyanins. Physiol. Zool. 35: 304-313. ROBERTSON, J. D. 1960. Ionic regulation in the crab Carcinus maenas (L.) in relation to the moulting cycle. Comp. Biochem. Physiol. 1: 183-212. SULLIVAN, B., J. BONAVENTURA, AND C. BONAVENTURA. 1974. Functional differences in the multiple hemocyanins of the horseshoe crab, Limulus polvphemus L. Proc. Nat. Acad. Sci. 71: 2558-2562. TORENSMA, R., AND O. BRIX. 1981. Oxygen binding of Neptunea antiqua hemocyanin. The significance of allosteric ligands. Mol. Physiol. 1: 213-221. TRUCHOT, J-P. 1973. Action specifique de carbone sur Taffinite pour Toxygene de 1'hemocyanin de Carcinus maenas (L.) (Crustace Decapode Brachyoure). Comptes Rendus Acad. Sci. Paris 276: 2965-2968. TRUCHOT, J-P. 1975. Factors controlling the in vivo and in vitro oxygen affinity of the hemocyanin in the crab Carcinus maenas (L.). Respir. Physiol. 24: 173-179. Reference: Biol. Bull. 171: 264-273. (August, 1986) EVIDEN* ECDYSTEROID FEEDBACK ON RELEASE OF MOLT- IN TING HORMONE FROM CRAB EYESTALK GANGLIA MARK P. MATTSON 1 AND EUGENE SPAZIANI Department of Biology, University of Iowa, Iowa City, Iowa 52242 ABSTRACT The content and release of molt-inhibiting hormone activity (MIH) in isolated eyestalk ganglia of crabs (Cancer antennarius S.) were measured in vitro as a function of exposure in vivo to elevated or reduced hemolymph ecdysteroid levels. Ecdysteroid liters of intermolt crabs injected with 20-hydroxyecdysone (two 45 /ug injections/24 h) rose 8- to 10-fold; MIH released from subsequently isolated ganglia was significantly less than that released from ganglia of saline-injected controls, while MIH content of ganglia from treated crabs was increased. The hemolymph ecdysteroid level of intermolt crabs was low (6 ng/ml) and was further reduced by 40% 6 days after Y-organectomy. MIH release from ganglia of both Y-organectomized and sham-operated control crabs was high, similar to that of unoperated controls, but MIH content of ganglia from both Y-organectomized and sham groups was significantly reduced relative to controls. The results indicate a negative feedback regulation of MIH release but not synthesis by ecdysteroids and are discussed in relation to the patterns of ecdysteroid liters ob- served in the normal cruslacean moll cycle. INTRODUCTION In vertebrales, sleroids produced by neuropeplide-regulaled glands exert feedback aclions on produclion and/or release of Ihe Iropic neurohormones. Feedback effects are generally negative as in adrenal glucocorticoid inhibition of corticolropin-releasing factor and adrenocorticolropin (ACTH) release from the hypothalamus and Ihe pi- luilary, respeclively (see Keller- Wood and Dallman, 1984 for review); feedback of gonadal sleroids on gonadolropin release is predominanlly negalive allhough posilive effecls are seen under certain physiological circumslances (Baldwin et at., 1974; Martin et al, 1974; Labrie et al, 1978). Insecl neurosecrelion of brain prolhoracicolropic hormone (PTTH) is apparenlly subject to inhibilion by Ihe ecdysleroid moiling hor- mone, ecdysone, which is produced by Ihe targel prolhoracic glands (Steel, 1975). In these cases neuropeplide regulation of targel cell sleroidogenesis is positive, and negative feedback provides a homeoslalic mechanism for maintaining circulating sleroid hor- mone levels wilhin a relalively narrow range. The ecdysteroidogenic glands of crus- taceans (Y-organs), on the other hand, are subject lo negalive regulalion by Ihe eyeslalk neuropeplide moll-inhibiling hormone (MIH; cf. Skinner, 1985); Ihe exislence of a "spending feedback arrangemenl is probable bul has nol been explored. cruslacean moll-conlrolling syslem consisls of eyeslalk neurosecrelory cells (X-: XO) wilh enlarged axonal endings conlained in Ihe neurohemal sinus gland ?bruary 1986; accepted 23 April 1986. Abbrev! GCS, eyestalk ganglia-conditioned saline; MIH, molt-inhibiting hormone; PTTH, pro- thoracicolropu u>; SG, sinus gland; XO, X-organ. 1 Present address (and to whom reprint requests should be sent): Department of Anatomy, Colorado State University, Fort Collins, CO 80523. 264 ECDYSTEROID FEEDBACK ON MIH RELEASE 265 (SG) and paired peripheral steroidogenic Y-organs which produce ecdysone (Chang et al, 1976; Cooke and Sullivan, 1982; Watson and Spaziani, 1985a). MIH is a heat- stable, trypsin-sensitive peptide (Rao, 1965; Quackenbush and Herrnkind, 1983) which by immunological and functional criteria appears to be related to neurohypophysial peptides of the vasopressin family (Mattson and Spaziani, 1985a). Serotonergic neurons mediate release of MIH (Mattson and Spaziani, 1 985b, 1 986a) and one of the conditions governing the release of MIH is stress (Mattson and Spaziani, 1985c, 1986a). Recent advances in Y-organ culture methods have allowed demonstration of a direct suppres- sion of Y-organ ecdysteroid production by MIH activity in eyestalk extracts (Watson and Spaziani, 1985b; Mattson and Spaziani, 1985d, Webster, 1986), sinus gland-con- ditioned saline (Soumoff and O'Connor, 1982; Mattson and Spaziani, 1985b, Webster, 1986), and eyestalk ganglia-conditioned saline (GCS; Mattson and Spaziani, 1985b). In vitro methods also show that cyclic AMP mediates this inhibition of steroidogenesis (Mattson and Spaziani, 1985a, e) and that calcium-calmodulin antagonizes the MIH effect by activating Y-organ cAMP phosphodiesterase (Mattson and Spaziani, 1986b). Thus the effects of c AMP on Y-organ steroidogenesis are opposite to those of the tropic hormones on steroidogenic glands of both vertebrates (Schimmer, 1980; Sala et al., 1979) and insects (Smith et al., 1984). Recent development of a formal MIH bioassay based upon in vitro suppression of Y-organ ecdysteroid production (Mattson and Spaziani, 1985d) allowed assessment of neurotransmitter regulation of release of MIH activity from isolated crab eyestalk ganglia (Mattson and Spaziani, 1985b). The present study employs the MIH bioassay and isolated ganglia techniques to assess possible feedback regulation of MIH release by ecdysteroids. Circulating ecdysteroid liters were artificially elevated or suppressed by injection of 20-hydroxyecdysone or Y-organectomy, respectively; eyestalk ganglia were subsequently isolated and ganglionic content and release of MIH was quantified by bioassay. MATERIALS AND METHODS Animals and experimental treatment Female rock crabs Cancer ant ernnarius Stimpson (Marinus Inc., Westchester, Cal- ifornia; Pacific Biomarine, Venice, California) were used in all experiments; animals were in intermolt upon sacrifice at the end of experiments (staged by examination of the continuity of endodermis and carapace; cf., Skinner, 1985). Crabs were maintained individually in water table compartments containing constantly-recirculating, charcoal- filtered, reconstituted seawater at 16-17C. A 12/12 h light/dark photocycle was maintained; crabs were fed fish three times weekly and were allowed to acclimate to their environment for at least one week prior to experimentation to reduce possible stress effects on the XO-Y-organ axis (Mattson and Spaziani, 1985c). Hemolymph samples were withdrawn in 300 n\ volumes from the sinus at the base of the fourth walking leg, while treatments were administered by injection (300 n\ volumes) through the periarthrodal membrane at the base of the first walking leg. Twenty (20)-hydrox- yecdysone was obtained from Sigma Chemical Co. (St. Louis, Missouri) and injected at a dose of 45 jug/300 n\\ this concentration ( 10 3 A/) was estimated to give an initial hemolymph concentration of 10~ 5 M based upon an average hemolymph volume/ crab of 30 ml. Y-organ ablation was carried out on animals chilled for 1 h at 4C. A 3-5 mm diameter hole was made through the ventral carapace using a portable dental drill with a rough burr bit. Y-organs were ablated by cautery and the carapace opening sealed with paraffin wax. For sham operations, holes were drilled 1 cm distolateral to 266 M. P. MATTSON AND E. SPAZIANI the Y-organ site and underlying tissue was cauterized. Crabs were examined at sacrifice with the aid of ssecting microscope to verify Y-organ destruction. Eyestalk ; Jicubation and extract preparation At the end of treatment periods eyestalks were extirpated, placed in Pantin's saline (Pantin. 1934), and the entire optic ganglia complexes (including the intact X-organ- sinus g'and system) were removed to saline as reported previously (Mattson and Spa- ziani, 1985b). Ganglia were incubated (1 ganglion complex/ 100 /A saline) for 2 h at 20C in an atmosphere of 50% O 2 /50% room air with constant rotary shaking at 60 rpm; we previously found that 2-h incubations resulted in release of MIH activity which was intermediate to shorter (30-min) or longer (8-h) incubations and thus allowed for greater sensitivity in detection of changes in release in response to experimental treatments (Mattson and Spaziani, 1985b). After incubations, the ganglia-conditioned saline (GCS) was removed, placed in a boiling water bath for 2 min, and centrifuged at 1000 X g for 10 min. The supernatant volume was adjusted to the preincubation volume with glass distilled water and stored frozen for MIH bioassay. Ganglia extracts were prepared by homogenization in saline (2 ganglia/ 100 ^1) followed by heat treat- ment in a boiling water bath as previously described (Mattson and Spaziani, 1985d). A dose of two eyestalk equivalents of extract was used for bioassay of MIH; this dose was previously determined to be near the ED 50 for inhibition of Y-organ steroidogenesis (Mattson and Spaziani, 1985d) and thus allowed for maximal sensitivity of the bioassay to changes in MIH content of ganglia due to experimental treatments. Bioassay of MIH activity The MIH bioassay has been previously described (Mattson and Spaziani, 1985b, d). Briefly, activated Y-organs from 48-h de-eyestalked crabs were removed, quartered, and placed in 0.5 ml of fetal bovine serum-supplemented Medium 199. To the in- cubation medium was then added either 100 ^1 of saline (for determination of basal ecdysteroid production), eyestalk extract, (2 eyestalk equivalents) or GCS ( 1 eyestalk equivalent). Incubations were for 24 h, after which incubation medium was removed and stored at 4C for ecdysteroid RIA, while tissue was processed for protein quan- tification. The relative ability of GCS or eyestalk extracts (see figure legends) to inhibit Y-organ steroidogenesis was used as a measure of MIH activity (Mattson and Spaziani, 1985b, d). The ecdysteroid contents of GCS and ganglia extracts (at concentrations used for the MIH bioassay) per se were below the limit of detection of the ecdy- steroid RIA. Ecdysteroid and protein Quantification Serum and incubation medium were assayed directly for ecdysteroids by RIA as iously described (Mattson and Spaziani, 1985c, d). The RIA utilized 3 H-ecdysone (6C i/mmol; New England Nuclear, Bedford, MA), ecdysone antiserum (antibody Horn et al, 1976) which was a gift from Dr. W. E. Bollenbacher (Dept. of Biology University of North Carolina, Chapel Hill, NC), and ecdysone standards (Research ' 'us, Bayonne, NJ). Inter- and intra-assay coefficients of variation were 8% and xtively. Ecdysone antiserum H-21B has a 10-fold greater affinity for ecdysone 1 20-hydroxyecdysone (Horn et al., 1976; Watson and Spaziani, 1985b); as crab hemoiyrnph contains predominantly 20-hydroxyecdysone (Chang et al., 1976) actual ecdystero i titers for hemolymph in this study are likely an order of magnitude higher than the values presented. The Y-organs of C. antennarius in vitro secrete, in ECDYSTEROID FEEDBACK ON MIH RELEASE 267 addition to ecdysone, an unknown, less polar, ecdysteroid (structural analysis is in progress); this unknown appears in quantities 5-fold greater than ecdysone but has an affinity for antiserum H-2 1 B 100-fold less than ecdysone (Watson and Spaziani, 1985b). Thus, ecdysone levels that we report in the medium of MIH bioassay runs are un- derrepresented by well under an order of magnitude. Protein was quantified by the Bradford (1976) method. Statistics were done by Students Mest and all values are expressed as mean and standard error of the mean (SEM). RESULTS Serum ecdysteroid tilers of crabs given injections of 45 jug of 20-hydroxyecdysone at 0-h and 1 8 h later were elevated 3- and 8-fold 1 8- and 24-h, respectively, after the initial injection (Fig. 1). Y-organectomy reduced hemolymph ecdysteroid levels over a 6-day period to 65% of levels in sham-operated controls (Fig. 2). Eyestalk ganglia removed from crabs with elevated ecdysteroid levels (24 h after initial 20-hydroxyec- dysone injection; cf. Fig. 1) released significantly less MIH activity during a 2-h in- cubation in saline than did control ganglia; ganglia-conditioned saline from controls inhibited basal Y-organ steroidogenesis by 42%, while that from crabs with elevated ecdysteroid tilers did not affecl ecdysleroid produclion significanlly (Fig. 3). Isolaled ganglia from Y-organeclomized animals wilh reduced ecdysleroid lilers (cf., Fig. 2) released MIH activily al levels similar lo ganglia from sham-operaled conlrols or from unoperaled conlrols (Y-organ sleroidogenesis was suppressed 40-50%; Fig. 3). Exlracls of eyeslalk ganglia from Ihe ecdysleroid-lreated crabs contained significantly more MIH activily lhan ganglia exlracls from conlrol crabs (Fig. 4); Y-organ sleroidogenesis was inhibiled 65% and 80% by exlracls from conlrol and 20-hydroxyecdysone-injecled crabs, respeclively. Figure 4 also shows lhal MIH aclivily conlained in exlracls of ganglia from Y-organeclomized crabs and from sham operales was significanlly less lhan lhal in ganglia from unoperaled conlrols (40% suppression of Y-organ ecdysle- i eo \ cr> CO 40 CO >> o o CO 20 I 20-hydroxyecdysone 18 24 Time (hr) FIGURE 1 . Hemolymph ecdysteroid liters after 20-hydroxyecdysone administration. Crabs were injected with saline (control) or 45 ng of 20-hydroxyecdysone (arrows) after serum sampling at the given times. Ecdysteroids were quantified by RIA. Points and lines are the mean and SEM of samples from 4 crabs from 1 of 2 duplicate experiments. Values for 20-hydroxyecdysone-injected crabs > control at 1 8- and 24-h (P < 0.01); 24-h 20-hydroxyecdysone value > 18-h 20-hydroxyecdysone value (P < 0.05). 268 M. P. MATTSON AND E. SPAZIANI ; . U Sham YOE Time (days) FIGURE 2. Effects of Y-organ removal on hemolymph ecdysteroid levels. Y-organectomy or sham operations were performed after serum sampling at time 0; hemolymph ecdysteroid levels were monitored at the given times thereafter. Points and lines are the mean and SEM from 4 to 5 crabs. Values for Y- organectomized crabs < corresponding sham values at days 4-6 (P < 0.05); combined values for sham days 4 and 6 < sham day 2 value (P < 0.05). roidogenesis for ganglia extracts from Y-organectomized and sham groups; 65% suppression for ganglia extracts from unoperated controls). DISCUSSION We found that injection of 20-hydroxyecdysone increased circulating ecdysteroid liters. The same was found by Adelung (1967) after injecting intermolt crabs with a single dose of ecdysone, followed by extraction of whole animals at intervals and measuring ecdysteroid by the Calliphora assay. He also observed that the ecdysteroid level fell rapidly within the first 4 hours after injection, to 10% of the administered dose, and then, surprisingly, rose again over the subsequent 18 hours to the 50% level before finally declining. These events were interpreted to result first from a rapid clearing of injected ecdysone and then endogenous secretion of ecdysone by the Y- organs, directly stimulated by the injected hormone through a positive feedback mech- anism. That the source of the secondary rise in ecdysteroid was endogenous was un- equivocally demonstrated: an injection of 3 H-ecdysone was cleared rapidly over 4 hours as before but levels of the tracer continued downward over the subsequent time period (Adelung, 1967). In the present study, we show that elevated ecdysteroid levels in crab hemolymph inhibit release of MIH activity from subsequently isolated eyestalk ganglia (Fig. 3). It appears that the better interpretation of Adelung's results, based on present !- owledge, is that the secondary rise in ecdysteroids he observed was due to transient in -ibition of MIH release induced by the ecdysteroid injection. Jology similar to that employed in the present study was used to dem- onstrate i k inhibition of ACTH release from rat pituicytes in vitro (Mulder and Smelik, 1977); ACTH release from pituicytes of corticosterone-treated rats was mea- sured with a b say based on stimulation of steroidogenesis by cultured adrenal cells. In that experiment pituitary cells exhibited the feedback effects for several hours after ECDYSTEROID FEEDBACK ON MIH RELEASE 269 CO f MIH activity released from control ganglia in 2-h incubations elicited deu-i e suppression of Y-organ steroidogenesis; MIH release from feedback- inhibited nglia was therefore likely less than 1% of levels released from control ganglia. idition, ganglia from crabs with elevated ecdysteroids contain significantly more Mi I activity than ganglia from control intermolt crabs (Fig. 4). The combined results suggest a primary negative feedback effect on MIH release relative to synthesis. Comparable effects were seen in adrenal corticoid feedback on ACTH release; Jones et al. (1977) found that corticosteroids inhibited release of ACTH from cultured pi- tuitary cells without affecting its synthesis. Y-organectomized crabs showed only a 40% reduction in circulating ecdysteroid levels (Fig. 2). Other studies in which Y-organs were ablated and serum ecdysteroid levels were monitored yielded similar results (see Skinner, 1985 for review); one study suggested that ecdysteroids released by ovaries may account for the maintenance of low levels of ecdysteroids in the absence of Y-organs (Lachaise and Hoffman, 1977). While the reduction of ecdysteroid levels caused by Y-organectomy did not affect MIH release (Fig. 3), MIH content of ganglia from Y-organectomized and from sham- operates was reduced (Fig. 4). In light of previous studies indicating that stressors reduce ecdysteroid liters by promoting MIH release (Mattson and Spaziani, 1985c, 1986a) and prevent molting (Aiken, 1969), we propose that stress due to surgery may have caused a large release of MIH prior to post-surgery day 6, resulting in depletion of ganglionic MIH content and low amounts of MIH available for further release. Serum ecdysteroid levels during crab molting cycles have been determined in several species (Adelung, 1967, 1969; Chang, et al. , 1976; Hopkins, 1 983; Soumoff and Skinner, 1983; Skinner, 1985) and follow a consistent pattern. Associated with the transition from intermolt to premolt is one or more relatively small, transient rises in hemolymph ecdysteroid liters (3- to 10-fold over intermolt levels). There follows a dramatic rise (100- to 300-fold) that precedes, and presumably initiates, ecdysis. Tilers then fall sharply just prior lo ecdysis and relurn shortly Ihereafler lo or below intermolt levels. Results of Ihe presenl sludy and of previous work in Ihis and olher laboralories suggesl inleraclions of Ihe XO-SG- Y-organ neuroendocrine syslem lhal may accounl for Ihe observed changes in hemolymph lilers. The small, pre-ecdysial rises may resull from preliminary reduclion in MIH secrelion due lo inlernal or exlernal environmenlal cues (e.g., changes in pholoperiod and/or lemperalure, Bliss and Boyer, 1964, Aiken, 1969; inlrinsic neuronal oscillalors lhal are species specific, Arechiga et al., 1985; reduclions in slressful inpuls. Malison and Spaziani, 1985c, 1986a). Once inilialed, Ihe small ecdysleroid peaks may creale changes in aclivily of eyeslalk neurosecrelory cells or Iheir inpuls resulling in feedback inhibilion of MIH release (cf. Fig. 5). The large secondary rise in ecdysleroids is consequenlly permilled, causally linked lo ac- cenlualed feedback inhibilion of MIH release (bul nol synlhesis). The magnilude of this rise in ecdysleroids may be explained in part by our recenl findings (Malison and 'iani, 1986b) lhal calcium anlagonizes MIH suppression of Y-organ aclivily. Il is /cumenled (cf. Greenaway, 1985) lhal hemolymph calcium lilers rise sharply prio the moll and fall agin, more or less coincidenl wilh Ihe changes in ecdysleroids. the pre-ecdysial rise and fall in ecdysleroids also may be calcium-linked. In any cast with ecdysleroid levels finally depressed, feedback inhibilion on neurose- crelory ould be relieved and MIH release reinslaled lo dominale Ihe poslmoll and inlermo lormonal environmenls. However, while Ihis scenario may be plausible for crusta*. t is nol apparenlly consislenl wilh all evenls in insecls, which exhibil Ihe same geucrai pattern of cyclic changes in ecdysteroid levels (cf., Bollenbacher et al., 1975). Steel (1 75} provided evidence of negative feedback effecls of ecdysleroids MIH Serotonergic Neuron Neurosecretory tt\ Neuron ^T-^ 20 hydroxyecdysone Environmental Inputs Eyestalk Ganglia MIH \ R cholesterol ATP AC cAMP Ca 5' AMP ecdysone ecdysone Y organ FIGURE 5. Model of neuroendocrine regulatory interactions of the X-organ-sinus gland- Y-organ system. Environmental inputs including stress activate serotonergic eyestalk neurons which stimulate MIH-containing neurosecretory cells of the X-organ (XO)-sinus gland (SG) complex to release MIH. MIH in hemolymph binds to putative Y-organ cell surface receptors (R) resulting in activation of adenylate cyclase (AC) and generation of cAMP. cAMP suppresses production of ecdysone from cholesterol, an effect antagonized by calcium (Ca) which activates a calcium-calmodulin-sensitive cAMP-phosphodiesterase. Thus with continued release of MIH, ecdysone liters remain low and the intermolt state is maintained. A reduction of MIH release (due to a transient increase in hemolymph ecdysteroid levels or to reduced peripheral neural input) releases Y-organs from inhibition (decreased Y-organ cAMP levels) and ecdysone production is increased. Ecdysone is converted in peripheral tissues to 20-hydroxyecdysone. The latter eventually exerts feedback inhibition on release of MIH from XOSG cells, permitting the large rise in hemolymph ecdysteroid tilers prior to the molt. Y-organ activity subsequently declines (cause unknown), ecdysteroid liters fall, and MIH is again released reinslaling the intermolt slage. See text for further discussion and delails. 271 272 M. P. MATTSON AND E. SPAZIANI on release of the insect tropin, PTTH. The problem of consistency in model arises from the fact that the insect tropin stimulates ecdysteroid secretion whereas the crus- tacean tropi ohibitory. Thus, the initial rises in ecdysteroids in insects, and the subsequr n : :ge increase, would be expected to result from a stimulation, not suppres- sion, of release. In further contrast with crustaceans, the subsequent pre-ecdysial fall in ec iysteroids is more satisfactorily explained in insects by the feedback inhibition hypothc ;is. Clearly, resolution of these questions must await the development of a sensitive means for measuring arthropod tropic hormone levels in hemolymph as a function of the molting cycle. ACKNOWLEDGMENTS This work was supported by National Institutes of Health Institutional Grants 2T32MH1 5 172-07 (M.P.M.) and AM25295-06 (E.S.) to the University of Iowa, and National Science Foundation Grant DCB 8408957 (E.S.). LITERATURE CITED ADELUNG, D. 1967. Die Wirkung von Ecdyson bei Carcinus maenas L. und der Crustecdysontiter wahrend eines Hautungszyklus. Zoo/. An:. Suppl. 30: 264-272. ADELUNG, D. 1969. 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A rapid and sensitive method for quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 72: 248-253. CHANG, E. S., B. A. SAGE, AND J. D. O'CONNOR. 1976. The qualitative and quantitative determination of ecdysones in tissues of the crab. Pachygrapsus grassipes, following molt induction. Gen. Comp. Endocrinol. 30:21-33. COOKE, I. M., AND R. E. SULLIVAN. 1982. Hormones and neurosecretion. Pp. 205-290 in The Biology of Crustacea, Vol. 3, H. L. Atwood and D. C. Sandeman, eds. Academic Press, New York. GREENAWAY, P. 1985. Calcium balance and moulting in the Crustacea. Biol. Rev. 60: 425-454. HOPKINS, P. M. 1983. Patterns of serum ecdysteroids during induced and uninduced proecdysis in the fiddler crab, Uca pugilator. Gen. Comp. Endocrinol. 52: 350-356. HORN, D. S., J. S. WILKJE, B. S. SAGE, AND J. D. O'CONNOR. 1976. A high affinity antiserum specific for the ecdysone nucleus. J. Insect Physiol. 22: 901-905. JONES, M. T., E. W., HILLHOUSE, AND J. L. BURDEN. 1977. Dynamics and mechanics of corticosteroid feedback at the hypothalamus and pituitary gland. /. Endocrinol. 73: 405-414. KELLER- WOOD, M. E., AND M. F. DALLMAN. 1984. Corticosteroid inhibition of ACTH secretion. Endocrine Rev. 5: 1-24. ABRIE, F., J. DROUIN, L. FERLAND, L. LAGACE, M. BEAULIEU, A. DE LEAN, P. A. KELLY, M. G. CARON, AND V. RAYMOND. 1978. Mechanism of action of hypothalamic hormones in the anterior pituitary gland and specific modulation of their activity by sex steroids and thyroid hormones. Recent Prog. \orrn. Res. 34: 25-93. LACHAISE, F., AND J. A. HOFFMAN. 1977. Ecdysone et development ovarien chez un Decapode, Carcinus s. C. R. Acad. Sci. Paris 285: 701-704. MARTIN, J. E.. TYREY, J. W. EVERETT, AND R. E. FELLOWS. 1974. Estrogen and progesterone modulation of the pituitary response to LRF in the cyclic rat. Endocrinology 95: 1664-1673. MATTSON, M. -D E. SPAZIANI. 1985a. Functional relations of crab molt-inhibiting hormone and neuro; lophysial peptides. Peptides 6: 635-640. MATTSON, M. P., . SPAZIANI. 1985b. 5-hydroxytryptamine mediates release of molt-inhibiting hormone activity from isolated crab eyestalk ganglia. Biol. Bull. 169: 246-255. ECDYSTEROID FEEDBACK ON MIH RELEASE 273 MATTSON, M. P., AND E. SPAZIANI. 1985c. Stress reduces hemolymph ecdysteroid levels in the crab: mediation by the eyestalks. J. Exp. Zool. 234: 319-323. MATTSON, M. P., AND E. SPAZIANI. 1985d. Characterization of molt-inhibiting hormone (MIH) action on crustacean Y-organ segments and dispersed cells in culture, and a bioassay for MIH activity. J. Exp. Zool. 236:93-101. MATTSON, M. P., AND E. SPAZIANI. 1985e. Cyclic AMP mediates the negative regulation of Y-organ ec- dysteroid production. Mol. Cell. Endocrinol. 42: 185-189. MATTSON, M. P., AND E. SPAZIANI. 1986a. Regulation of the stress-responsive X-organ-Y-organ axis by 5- hydroxytryptamine in the crab. Cancer antennarius. Gen. Comp. Endocrinol. 62: 419-427. MATTSON, M. P., AND E. SPAZIANI. 1986b. Calcium antagonizes cAMP-mediated suppression of crab Y- organ steroidogenesis in vitro: evidence for activation of cAMP-phosphodiesterase by calcium- calmodulin. Mol. Cell. Endocrinol. In press. MULDER, G. H., AND P. G. SMELIK. 1977. A superfusion technique for the study of the site of action of glucocorticoids in the rat hypothalamus-pituitary-adrenal system in vitro. I. pituitary cell superfusion. Endocrinology 100: 1 143-1 152. PANTIN, C. F. A. 1934. On the excitation of crustacean muscle. J. Exp. Biol. 11:1 1-27. QUACKENBUSH, L. S., AND W. F. HERRNKIND. 1983. Partial characterization of eyestalk hormones controlling molt and gonadal development in the spiny lobster Panulirus argus. J. Crust. Biol. 3: 34-43. RAO, K. R. 1965. Isolation and partial characterization of the molt-inhibiting hormone of the crustacean eyestalk. Experientia 21: 593-594. SALA, G. B., M. L. DUFAU, AND K. J. CATT. 1979. Gonadotropin action in isolated ovarian luteal cells. J. Biol. Chem. 254: 2077-2083. SCHIMMER, B. P. 1980. Cyclic nucleotides in hormonal regulation of adrenocorticotropin functions. Adv. Cyclic Nucleotide Res. 13: 181-214. SKINNER, D. M. 1985. Molting and Regeneration. Pp. 43-145 in The Biology of Crustacea, Vol. 9, D. Bliss, ed. Academic Press, New York. SMITH, W. A., L. I. GILBERT, AND W. E. BOLLENBACHER. 1984. The role of cyclic AMP in the regulation of ecdysone synthesis. Mol. Cell. Endocrinol. 37: 285-291. SOUMOFF, C., AND J. D. O'CONNOR. 1982. Repression of Y-organ secretory activity by molt-inhibiting hormone in the crab, Pachygrapsus crassipes. Gen. Comp. Endocrinol. 48: 432-439. SOUMOFF, C., AND D. M. SKINNER. 1983. Ecdysteroid titers during the molt cycle of the blue crab resemble those of other Crustacea. Biol. Bull. 165: 321-329. STEEL, C. G. H. 1975. A neuroendocrine feedback mechanism in the insect molting cycle. Nature 253: 267- 269. WATSON, R. D., AND E. SPAZIANI. 1985a. Effects of eyestalk removal on cholesterol uptake and ecdysone secretion by the crab (Cancer antennarius) Y-organs in vitro. Gen. Comp. Endocrinol. 57: 360- 370. WATSON, R. D., AND E. SPAZIANI. 1985b. Biosynthesis of ecdysteroids from cholesterol by crab Y-organs, and eyestalk suppression of cholesterol uptake and secretory activity, in vitro. Gen. Comp. Endocrinol. 59: 140-148. WEBSTER, S. G. 1986. Neurohormonal control of ecdysteroid biosynthesis by Carcinus maenas Y-organs in vitro, and preliminary characterization of the putative molt-inhibiting hormone (MIH). Gen. Comp. Endocrinol. 61: 237-247. Reference: Biol. Bull 111: 274-290. (August, 1986) ADAPT/ n of these enzymes will be reported in a subsequent paper (Powell and Somero. prep.). Activities of enzymes of energy metabolism We surveyed the tissues of the three species for the activities of enzymes that serve to diagnose the types of pathways of energy metabolism operating to generate ATP. CytOx activity is an indicator of the capacity for electron transport chain activity, i.e., for aerobic respiration; citrate synthase (CS) is an indicator of citric acid (Krebs) cycle activity. Malate dehydrogenase (MDH) is involved in several metabolic functions [citric acid cycle activity; the transfer of reducing equivalents across the mitochondrial membrane; and in the anaerobic pathway involving the channeling of phosphoenol- pyruvate to succinate, via oxaloacetate, malate, and fumarate (Hochachka and Somero, 1984)]. CS activities were uniformly higher in tissues of Riftia than in the two bivalves, indicating a higher capacity for citric acid cycle function in the tube worm than in the clam or mussel. CytOx activities also were much higher on the average in Riftia than in the bivalves, suggesting that the tube worm has a more aerobic poise to its energy metabolism than the clam or mussel. MDH activities varied by less than an order of magnitude among all of the tissues examined (Table II). The finding that a much higher ratio of MDH to CS (and CytOx) characterized the tissues of the two bivalves relative to Riftia, suggests that the MDH in the bivalve tissues may be prin- cipally important in the context of anaerobic metabolism. In trophosome of Riftia there was no systematic variation in MDH or CS activities with position, unlike the pattern noted for SOx activity (Table I). Protection of CytOx activity by blood In addition to a detoxification strategy that relies on rapid oxidation of sulfide when it first enters the body, protection of aerobic respiration also may depend on mechanisms for detoxifying sulfide that passes through the body surface and enters the circulation. We earlier showed (Powell and Somero, 1983) that the blood of Riftia was capable of offsetting the inhibition of CytOx activity caused by sulfide. To inves- tigate protection by blood in more detail we further characterized the protective effects of Riftia blood and also studied blood of the clam, Calyptogena. The importance of mechanisms for protecting CytOx activity from inhibition by sulfide is emphasized by the data in Figure 2, which show that the CytOx systems of Riftia and Bathymodiolus are highly sensitive to sulfide, as is the CytOx of Calyptogena (see Fig. 3). The CytOx systems of Riftia and Bathymodiolus show identical sensitivities to sulfide (pH 6.0 data), with virtually complete inhibition occurring by sulfide con- centrations of 20-50 \iM. The sensitivity of CytOx to sulfide is strongly dependent on pH, an- t > O 80 60 40 20 SULFIDE I 5 SULFIDE EQUILIBRATION (WIN) 10 10 BLOOO EQUILIBRATION (WIN) FIGURE 4. Time-dependent effects of sulfide on Riftia pachyptila plume CytOx activity, and time- dependent reversal of inhibition by blood. Sulfide was added to CytOx preparations, and activity was measured at the times indicated. The filled circles represent time of pre-incubation of enzyme preparations with sulfide prior to initiation of assay. The open diamond symbols represent addition of 10 /xl of blood at t = 0, and immediate assay. Open circles represent addition of blood after 10 min pre-incubation of enzyme preparations with sulfide; assays were then initiated 0, 5, and 10 min after blood addition. Filled squares represent CytOx activity of enzyme preparations without sulfide added. of sulfide reduced or eliminated the ability of the blood to protect CytOx activity, indicating a saturation of the sulfide-binding capacity of the blood at higher sulfide concentrations. The finding that the protective effects of blood could be reduced or 100 0.2 0.4 0.6 0.8 SULFIDE in BLOOD (mM) FIGURE 5. Effects of pH and blood sulfide concentration on protection by blood of CytOx activity. Ten n\ of blood containing known amounts of sulfide were added to CytOx assays inhibited by 2 pM sulfide (pH 6.0), and 10 pM sulfide (pH 7.4). Sulfide-free blood (10 n\) provided 100% protection (equal to control activity without sulfide added) under the assay conditions used. 284 M. A. POWELL AND G. N. SOMERO eliminated through exposure of the blood to sulfide prior to adding a blood sample to the CytOx assay mixture shows that the protective factors are not oxidizing sulfide, in which case the effects of sulfide would be reduced during equilibration of blood and sulfide Instead, the protective effects are due to a binding of sulfide that leads to protection of the sulfide from oxidation. A TP synthesis studies To test the hypothesis that sulfide and other reduced sulfur compounds are used in these symbioses as important sources of energy, we measured the abilities of sulfide, thiosulfate, and sulfite to stimulate ATP synthesis in freshly prepared tissue homog- enates of symbiont-containing and symbiont-free tissues. We used two types of ho- mogenates: ones which were prepared in iso-osmotic buffer (buffered seawater) to avoid osmotic lysis of the bacteria ("intact" studies), and ones prepared in dilute Hepes buffer, in which the bacteria were lysed. Lysis was checked by examining the homogenates microscopically. The data in Figure 6 show that at least one of the reduced sulfur compounds tested was effective in stimulating ATP synthesis with each species. For homogenates of trophosome, only lysed bacteria were capable of synthesizing ATP, and sulfite and sulfide were the only two reduced sulfur compounds effective in stimulating ATP production. In homogenates of Calyptogena gill, only sulfite was able to stimulate ATP production and, as with trophosome, only lysed bacteria were effective in syn- thesizing ATP under our experimental conditions. Homogenates of gill of Bathymo- diolus differed in three respects from those of the other species. First, only intact bacteria exhibited ATP production in response to reduced sulfur compounds. Second, thiosulfate was the most effective reduced sulfur compound in driving ATP synthesis. Third, the amount of ATP produced was much lower in Bathymodiolus preparations than in the other species. Stimulation of ATP synthesis by reduced sulfur compounds was not observed for any of the symbiont-free tissues tested (Riftia body wall, foot and mantle of Calyptogena and Bathymodiolus; data not shown). Bacterial contents of tissues To determine if the differences in sulfide oxidizing activities and ATP synthesizing capacities were correlated with differences in the contribution of the bacterial endo- symbionts to total tissue mass, we counted bacteria in homogenates of the symbiont- containing tissues, measured the sizes and volumes of the bacteria, and computed the percent of tissue mass due to bacteria (Table III). The contribution of bacterial cells to total tissue mass was highest in trophosome, next highest in clam gill, and lowest in the mussel gill, the same ranking found for sulfide oxidizing and ATP synthesizing activities. DISCUSSION dies were designed to elucidate some of the mechanisms by which the three domir vent animals, Riftia pachyptila, Calyptogena magnifica, and Bathy- modiolus hilus, succeed in exploiting the energy of the sulfide molecule while avoiding i effects on aerobic respiration. Our results indicate that the three symbioses sha< ;rt.ain mechanisms in common for metabolizing and detoxifying sulfide, yet dit orne aspects of their sulfide relationships. SULFIDE METABOLISM IN VENT ANIMALS 285 0.8 - Riftw-lysed CONTROL O SULFITE SULFIDE D THIOSULFATE 20 40 60 80 Seconds 100 120 0.8 r Riftio-intact 40 60 80 Seconds 100 120 CLAM-lysed 40 60 80 Seconds 100 120 CLAM -intact 100 150 Seconds 200 250 0.04 at o E c 0.02 MUSSEL- intact 0.04 Q. I < O E 0.02 MUSSEL- lysed 50 100 150 Seconds 200 250 50 100 150 Seconds 200 250 FIGURE 6. ATP concentrations (nmoles ATP/gFW tissue) in homogenates incubated with reduced sulfur compounds [sulfide (), sulfite (O), or thiosulfate (IH)] at 1 mM final concentration or no sulfur compound [control ()]. Lysed samples were homogenized and assayed in low osmolarity buffer; intact samples were homogenized and assayed in buffered artificial seawater. Intactness of bacteria was judged by phase-contrast microscopy. Each frame presents data gathered with a different tissue homogenate. Each symbol represents the average of duplicate samples. Protection of aerobic respiration from poisoning by sulfide The occurrence of CytOx activity in all three species (Table II) and the observation that Riftia (Childress el al, 1984) and Calyptogena (Arp et ai, 1984) consume oxygen at rates comparable to those reported for shallow-living species, at comparable mea- 286 M. A. POWELL AND G. N. SOMERO TABLE III Bacterial com of symbiont-containing tissues of vent animals Size of bacteria # Bacteria/gFW (avg, range) % Bacteria Riftw tila trophosome (top) (bottom) 4.37 10.3 1.05 X 10 5.6 x 10 9 9 (3) (3) 4.0, 2-6 4.0, 2-6 15 35 Calyptogena magnifica gill 3.30-3 .64 X 10" (2) 0.75, 0.5-1.0 7, 7 Bathymodiolus thermophilus gill 1.70-1 .81 X 10" (2) 0.75, 0.5-1.0 4.0 Bacterial composition of tissues. Measurements and calculations are described in Materials and Methods. For R. pachyptila, mean standard deviation (n) are given, and for the bivalves range and (n) are given. surement temperatures, suggest that the vent organisms are likely to possess mecha- nisms for protecting themselves from poisoning by sulfide. The needs for these pro- tective mechanisms are shown by the sensitivities of the CytOx enzymes of these species to sulfide (Figs. 2, 3), sensitivities which are as high as those of CytOx systems of animals from low-sulfide environments (National Research Council, 1979). Two types of mechanisms for protecting aerobic respiration from poisoning by sulfide were identified in this study. First, in all three species, a "peripheral defense" strategy that employs a zone of sulfide oxidizing activity in cells of the body surface may be important in detoxifying rapidly any sulfide that enters the cells. The histo- chemical localization studies (Fig. 1 ) indicate that the sulfide oxidizing activities of body wall muscle of Riftia and of foot and mantle tissues of Calyptogena (and Bath- ymodiolus; data not shown) are restricted to the superficial cell layer(s) of these tissues. The reported SOx activities in terms of gFW of tissue thus may grossly under-represent the specific activities of SOx in the superficial cell layers of the symbiont-free tissues. This "peripheral defense" type of sulfide detoxification strategy was previously reported in Solemya reidi (Powell and Somero, 1985), and we propose that it may be a generally occurring mechanism in soft-bodied marine invertebrates living in sulfide- rich habitats. For these organisms, there appears to be no impermeable barrier to exclude sulfide from the outer body surface. Arthropods, e.g., the vent crustaceans, appear to lack this "peripheral defense" mechanism, and instead detoxify any sulfide that has entered their circulation by oxidizing it in the hepatopancreas (Vetter el al, 1986). Arthropods may be highly impermeable to sulfide, except at areas where the exoskeleton is very thin, so most of the body surface may not be threatened by sulfide entry and poisoning of respiration. ond mechanism for preventing poisoning of respiration by sulfide exists in Riftia anc Calyptogena. High molecular weight factors in the bloods of the tube worm (Arp and Cl Iress, 1983; Fisher and Childress, 1984) and the clam (Arp et al, 1984) bind sul ide lightly, and appear capable of extracting sulfide from the environment and transpcj g it via the circulation to the bacterial symbionts. These binding factors, which are pr ; in Riftia (Arp and Childress, 1983; Childress et al, 1984) and probably also in Calyptogena (Arp et al, 1984) appear to be important in holding free SULFIDE METABOLISM IN VENT ANIMALS 287 sulfide concentrations in blood to low values. We suggest that as long as the sulfide- binding proteins remain unsaturated, too little free sulfide exists in solution in the blood of these species to pose a significant threat to aerobic respiration. Bathymodiolus does not have a sulfide-binding protein in its blood (Drs. A. J. Arp and J. J. Childress, University of California, Santa Barbara, pers. comm.), so this type of defense against sulfide poisoning does not play a part in the mussel's strategies for protecting respiration from sulfide. The role of pH in establishing the effects of sulfide on CytOx activity bears close examination. As shown for CytOx of Riftia (Fig. 2), the inhibitory effects of sulfide on CytOx activity increased as pH was reduced. This observation suggests that H 2 S, not HS~ or S = , is the inhibitory form of sulfide. A similar conclusion has been reached in studies of other organisms (cf. Environmental Protection Agency, 1976). Near pH 7, roughly equal amounts of H 2 S and HS~ are present. When pH is increased above 7, the amount of the inhibitory species, H 2 S, falls rapidly and, as shown by our results (Fig. 5) the protection by blood increases as well. The enhanced protection by blood at higher pH values in the physiological pH range is consistent with the findings of Childress et al. (1984) that sulfide binding by blood of Riftia increased between pH values of approximately 5.5 and 7.5. The finding that sulfide binding by blood and protection by blood of CytOx are high at the average blood pH of Riftia, approximately 7.5, (Childress et al., 1984), suggests that protection of respiration by the sulfide- binding protein is highly effective under physiological pH conditions. Intracellular pH values for Riftia have not been determined, but it seems reasonable to assume that Riftia, like other animals, maintains its intracellular pH approximately 0.4-0.5 pH units acidic to blood (cf, Reeves, 1977). Thus, any free sulfide entering the cells would pose a significant threat to CytOx activity since at pH 7.0-7. 1 substantial concentrations of the inhibitory form of sulfide, H 2 S, would be present. Several questions about the mechanisms used to defend against poisoning by sulfide remain. One concerns the means by which sulfide passes from the ambient vent water into the animals' circulatory systems without poisoning mitochondria! respiration. For example, in the plume of Riftia, which is thought to serve as the major entry route for sulfide (Childress et al., 1984), large amounts of sulfide must cross a highly aerobic (Table II) tissue. The low sulfide oxidase activities in plume (Table I) suggest that little oxidation of sulfide takes place during the transport process. Oxidation of sulfide during passage across a transport surface into the circulatory system, where it is complexed in a non-toxic form by the sulfide-binding proteins, is undesirable, of course. The maximal energy yield from sulfide will be attained only if sulfide, not some partially oxidized sulfur compound, is delivered to the site of sulfur metabolism, e.g., the bacteria in the trophosome. We suggest that sulfide transport in plume may be pericellular, a route which would avoid the problems of sulfide inhibition of res- piration and the partial oxidation of this energy resource. A second unanswered question about the interactions of aerobic respiration with sulfide concerns the mussel, Bathymodiolus, which appears to lack sulfide binding proteins in its blood. The activities of CytOx present in tissues of the mussel may be protected entirely by sulfide oxidase enzymes serving a detoxification role. However, these sulfide oxidizing enzymes could reduce the amount of sulfide reaching the bac- terial symbionts (see below). Reduced sulfur compounds as energy sources Discussions of the symbioses between sulfide biome invertebrates and their bacterial symbionts have emphasized the potential contributions that reduced sulfur compounds 288 M. A. POWELL AND G. N. SOMERO like sulfide mi; nake to the energy needs of these organisms (cf., Felbeck and Somero, 1982; Janna.s and Mottl, 1985). It has been emphasized that carbon dioxide fixation via the C: i-Benson cycle could be driven by the energy released in bacterial sulfide oxidation, id that the reduced carbon compounds synthesized in the bacterial sym- bionts c I be translocated to the host to satisfy some fraction of its nutritional requirements. In the clam, Solemya velum, Cavanaugh (1983) demonstrated that sulfide and thiosulfate were effective in stimulating carbon dioxide fixation. Belkin et al. (1986) showed that sulfide, but not thiosulfate, stimulated the fixation of carbon dioxide in homogenates of Rift ia trophosome; thiosulfate, but not sulfide, was effective in stim- ulating carbon dioxide fixation in homogenates of gill from Bathymodiolus. To our knowledge, these are the only studies that have demonstrated directly the roles of reduced sulfur compounds in driving endergonic processes in these symbioses. To gain additional understanding of the efficacy of reduced sulfur compounds in supplying energy for these symbioses, we reasoned that it would be especially important to determine how these compounds affected the ATP synthesis of the organisms. Because ATP is the dominant "energy currency" of the cell, changes in ATP concen- trations in response to exposure to different reduced sulfur compounds could be a very sensitive indicator of the importance of these compounds to the generation of biologically useful forms of energy in the symbioses. Prior to our studies the capacities of these symbioses to trap the energy released during the oxidation of reduced sulfur compounds in the form of ATP had not been investigated. Neither had there been study of the relative abilities of different reduced sulfur compounds, e.g., sulfide, sulfite, and thiosulfate, to supply energy in these symbioses. The results of our experiments suggest that the symbiont-containing tissues of Riftia, Calyptogena, and Bathymodiolus can exploit the energy of reduced sulfur com- pounds. Because we used un fractionated homogenates in our studies, i.e., animal tissue and bacteria were present, our results do not demonstrate unambiguously that the symbionts were solely or primarily responsible for the ATP production driven by reduced sulfur compounds. The correlation between ATP production and bacterial contribution to tissue mass is consistent with a dominant role for the symbionts in this process (Table III); however, a role for animal-localized ATP synthesis mechanisms cannot be excluded (see below). The reduced sulfur compounds most effective in driving ATP synthesis differed among the three symbioses (Fig. 6). Also, the total amount of ATP that was produced by oxidation of reduced sulfur compounds differed among the three symbioses studied (Fig. 6). In Riftia trophosome homogenates, sulfide and sulfite were most effective in stimulating ATP production, while in Calyptogena only sulfite was effective, and in Bathymodiolus, thiosulfate was most effective (Fig. 6). In Riftia and Calyptogena, only homogenates containing lysed bacteria exhibited the capacity to exploit reduced sulfur compounds, while in Bathymodiolus intact bacteria had to be present for ATP synthesis to occur. Riftia trophosome had the highest ATP synthesizing capacity, followed by f Calyptogena and Bathymodiolus. finding that sulfide was effective only in the case of Riftia, where the highest activi Ox were found, and where SOx was clearly localized in the bacterial symbiont ~ig. 1), suggests that the tube worm may be the only one of these three pply sulfide directly to its bacterial symbionts. valves, where sulfide was not effective as an energy source for driving ATP pro der our experimental conditions, the abilities of sulfite and thio- sulfate to s synthesis suggest that only oxidation products of sulfide may be delivered L animal to its symbionts. The sulfide oxidizing activities found in SULFIDE METABOLISM IN VENT ANIMALS 289 the gills of the two vent bivalves could not be localized histochemically due to the very small sizes of the bacteria (Table III). Therefore, it is not clear whether the measured SOx activities are animal or bacterial. However, the finding that in gills of Solemya reidi all of the SOx activity is found in the animal compartment (Powell and Somero, 1985) is an interesting precedent in this context. Perhaps in the vent bivalves as well the initial step(s) in sulfide oxidation occur in the animal tissue. Belkin et al. (1986) found sulfide to be the most effective energy source for driving net CO 2 fixation in trophosome of Riftia, and thiosulfate to be the most effective energy source in Bathymodiolus. Our findings on the relative abilities of reduced sulfur compounds to drive ATP synthesis agree with their results, and we suggest oxidation of sulfide by the animal tissues as the source of thiosulfate for the symbionts of Bath- ymodiolus. If in the bivalves the initial step(s) in sulfide oxidation occur in the animal com- partment of the symbiosis, not in the bacteria, then the possibility exists that mito- chondrial ATP production might be driven by the energy released in sulfide oxidation. This is the case for mitochondria of Solemya reidi (Powell and Somero, 1986). Because the homogenization procedures we used in the studies of the three vent symbioses would have ruptured mitochondria, we cannot exclude the possibility that mitochon- dria in both symbiont-containing and symbiont-free tissues are able to exploit the energy released in mitochondria! sulfide oxidation to drive ATP synthesis. We plan to examine this possibility during future studies of the vent animals. In conclusion, our studies show that, despite many similarities, the three symbioses studied also exhibit important differences, e.g., in preferred reduced sulfur compounds for driving ATP synthesis, in the absolute capacity for ATP synthesis in the symbiont- containing tissues, and in mechanisms for transporting and detoxifying sulfide. The three symbioses may also differ in the relative roles played by the bacterial symbionts in the nutritional needs of the animal. This is suggested by both anatomical and biochemical results. Riftia entirely lacks a digestive system (Jones, 1981), and the unusual carbon isotope ratios of its tissues, ratios which are the same in symbiont- containing and symbiont-free tissues (Rau, 1981), suggest a strong reliance on the reduced carbon compounds translocated from bacterial to animal cells. Calyptogena has a greatly reduced digestive system (Boss and Turner, 1980), and has more bacterial endosymbionts per mass of gill than Bathymodiolus. The activities of SOx and ATP synthesis are higher in the clam than in the mussel as well. Calyptogena is found exclusively at sites of active venting (see Arp et al., 1984), whereas Bathymodiolus is found in active venting sites and at sites peripheral to the main venting regions (see Hessler and Smithey, 1983). Bathymodiolus has a well-developed digestive system, and a ciliary-mucus net feeding capacity (Kenk and Wilson, 1985). Thus, where Riftia and Calyptogena may depend absolutely on a symbiotic source of reduced carbon compounds, Bathymodiolus may be able to exploit a greater variety of microhabitats in the vent ecosystem. ACKNOWLEDGMENTS These studies were supported by National Science Foundation grants OCE83- 1 1259 (to G. N. Somero and H. Felbeck), and OCE83-1 1256 (Facilities support grant for the Galapagos '85 program, co-principal investigators, Drs. J. J. Childress and K. L. Johnson, University of California, Santa Barbara), and by training grant PMS GM 073 1 3. We gratefully acknowledge the assistance of the captains and crew members of the research vessels R/V Melville, R/V Atlantis II, and DSRV Alvin. We acknowledge helpful discussions of these findings with Ms. Amy Anderson, and Drs. A. J. Arp, J. J. Childress, H. Felbeck, C. Fisher, and R. D. Vetter. 290 M. A. POWELL AND G. N. SOMERO LITERATURE CITED ARP, A. J., A }. J. CHILDRESS. 1983. Sulfide binding by the blood of the hydrothermal vent tube worm, P -iila. Science 219: 295-297. ARP, A. J., J ui DRESS, AND C. R. FISHER, JR. 1984. Metabolic and blood gas transport characteristics Irydrothermal vent bivalve Calyptogena magnified. Physiol. Zool. 57: 648-662. BELKIN, S . NELSON, AND H. W. JANNASCH. 1 986. Symbiotic assimilation of CO 2 in two hydrothermal vent animals, the mussel Bathymodiolus thermophilus. and the tube worm, Riftia pachyptila. Biol. Bull. 170: 110-121. Boss, K. J., AND R. D. TURNER. 1980. The giant white clam from the Galapagos Rift, Calyptogena magnified species novum. Malacologia 20: 161-194. CAVANAUGH, C. M. 1983. Symbiotic chemoautotrophic bacteria in sulfide-habitat marine invertebrates. Nature 302: 58-61. CAVANAUGH, C. M., S. L. GARDINER, M. L. JONES, H. W. JANNASCH, AND J. B. WATERBURY. 1981. Prokaryotic cells in the hydrothermal vent tube worm, Riftia pachyptila Jones: possible chemoau- totrophic symbionts. Science 213: 340-342. CHILDRESS, J. J., A. J. ARP, AND C. R. FISHER, JR. 1984. Metabolic and blood characteristics of the hydro- thermal vent tube-worm, Riftia pachyptila. Mar. Biol. 83: 109-124. CHILDRESS, J. J., AND T. J. MICKEL. 1980. A motion compensated shipboard precision balance system. Deep-Sea Res. 27A: 965-970. Environmental Protection Agency. Ecological Research Series. 1976. Effect of Hydrogen sulfide on fish and invertebrates. II. Hydrogen sulfide determination and relationship between pH and sulfide toxicity. EPA-600/3-76-062b. Environmental Protection Agency, Washington, DC. FELBECK, H. 1981. Chemoautotrophic potential of the hydrothermal vent tube worm, Riftia pachyptila Jones (Vestimentifera). Science 213: 336-338. FELBECK, H. 1983. Sulfide oxidation and carbon fixation by the gutless clam Solemya reidi: an animal- bacteria symbiosis. J. Comp. Physiol. 152: 3-11. FELBECK, H., J. J. CHILDRESS, AND G. N. SOMERO. 1981. Calvin-Benson cycle and sulphide oxidation enzymes in animals from sulphide-rich habitats. Nature 292: 291-293. FELBECK, H., AND G. N. SOMERO. 1982. Primary production in deep-sea hydrothermal vent organisms: role of sulfide-oxidizing bacteria. Trends Biochem. Sci. 1: 201-204. FISHER, C. R., JR., AND J. J. CHILDRESS. 1984. Substrate oxidation by trophosome tissue from Riftia pachyptila Jones (Phylum Pogonophora). Mar. Biol. Lett. 5: 171-183. FISHER, M. R., AND S. C. HAND. 1984. Chemoautotrophic symbionts in the bivalve Lucinafloridana from seagrass beds. Biol. Bull. 167: 445-459. HAND, S. C., AND G. N. SOMERO. 1983. Energy metabolism pathways of hydrothermal vent animals: adaptations to a food-rich and sulfide-rich deep sea environment. Biol. Bull. 165: 167-181. HESSLER, R. R., AND W. SMITHEY. 1983. The distribution and community structure of megafauna at the Galapagos rift hydrothermal vents. NATO Conf. Ser. (Mar. Sci.) 12: 735-770. HOCHACHKA, P. W., AND G. N. SOMERO. 1984. Biochemical Adaptation. Princeton, NJ. 537 pp. JANNASCH, H. W., AND M. J. MOTTL. 1985. Geomicrobiology of deep-sea hydrothermal vents. Science 229:717-725. JONES, M. L. 1981. Riftia pachyptila. new genus, new species, the vestimentiferan worm from the Galapagos rift geothermal vents (Pogonophora). Proc. Biol. Soc. Wash. 93: 1295-1313. KARL, D. M., AND O. HOLM-HANSEN. 1978. Methodology and measurement of adenylate energy charge ratios in environmental samples. Mar. Biol. 48: 185-197. KENK, V. C., AND B. R. WILSON. 1985. A new mussel (Bivalvia, Mytilidae) from hydrothermal vents in the Galapagos Rift zone. Malacologia 26: 253-271. National Research Council, Division of Medical Science, Subcommittee on Hydrogen Sulfide. 1979. Hydrogen Sulfide. University Park Press, Baltimore. 183 pp. POWELL, M. A., AND G. N. SOMERO. 1983. Blood components prevent sulfide poisoning of respiration of the hydrothermal vent tube worm, Riftia pachyptila. Science 219: 297-299. WELL, M. A., AND G. N. SOMERO. 1985. Sulfide oxidation occurs in the animal tissue of the gutless clam, 'emya reidi. Biol. Bull. 169: 164-181. A., AND G. N. SOMERO. 1986. Hydrogen sulfide oxidation is coupled to oxidative phosphorylation ii; ; :>chondria of Solemya reidi. Science (in press). Hydrothermal vent clam and tube worm 13 C/ I2 C: Further evidence of non-photosynthetic ;. Science 213: 338-340. The interaction of body temperature and acid-base balance in ecothermic vertebrates. tint 39: 559-586. VETTER, ! WELLS. A. L. KURTSMAN, AND G. N. SOMERO. 1986. Sulfide detoxification by the crab Bythograea thermydron and other decapod crustaceans. Physiol. Zool. (in press). HEBRANK, MARY REIDY, AND JOHN H. HEBRANK The mechanics of fish skin: lack of an "external tendon" role in two teleosts .... ,-r.V A , 236 MANGUM, CHARLOTTE P., AND Louis E. BURNETT, JR. The CO 2 sensitivity of the hemocyanins and its relationship to Cl~ sen- sitivity 248 MATTSON, MARK P., AND EUGENE SPAZIANI Evidence for ecdysteroid feedback on release of molt-inhibiting hormone from crab eyestalk ganglia 264 POWELL, M. A., AND G. N. SOMERO Adaptations to sulfide by hydrothermal vent animals: sites and mech- anisms of detoxification and metabolism 274 \ CONTENTS Annual Report of the Marine Biological Laboratory 1 ,fr. . ; >V- ,~ - -A J ' INVITED REVIEW "'- ^'] MARKL, JURGEN Evolution and function of structurally diverse subunits in the respiratory protein hemocyanin from arthropods 90 / i DEVELOPMENT AND REPRODUCTION MARTIN, VICKJ J., AND WILLIAM E. ARCHER A scanning electron microscopic study of embryonic development of a marine hydrozoan - " 1 1 6 SAFRANEK, Louis, CLAYTON R. SQUIRE, AND CARROLL M. WILLIAMS Precocious termination of diapause in neck- and abdomen-ligated pupal preparations of the tobacco homworm, Manduca sexta 1 26 1 ECOLOGY AND EVOLUTION ) , /4 HAIRSTON, NELSON G., JR., AND EMILY J. OLDS Partial photoperiodic control of diapause in three populations of the freshwater copepod Diaptomus sanguineus :./.'.. ^y.. . . 135 LEVIN, LISA A. Effects of enrichment on reproduction in the opportunistic polychaete Streblospio benedicti (Webster): a mesocosm study ''..i .;V- -,\ ^3 MCFADDEN, CATHERINE S. Laboratory evidence for a size refuge in competitive interactions between the hydroids Hvdractinia echinata (Flemming) and Podocoryne carnea (Sars) ,'J. . ... r,\. . . ^ . . . .'.(.V. ( .4 : ( '. .7, -^<%- l61 r ( X" GENERAL BIOLOGY ^ ; ANDERSON, O. ROGER, NEIL R. SWANBERG, J. L. LINDSEY, AND PAUL BENNETT Functional morphology and species characteristics of a large, solitary radiolarian Physematium muelleri . . 175 RUPPERT, EDWARD E., AND ELIZABETH J. BALSER Nephridia in the larvae of hemichordates and echinoderms ........ 188 TELFORD, MALCOLM, AND RICH Mooi Resource partitioning by sand dollars in carbonate and siliceous sedi- ments: evidence from podial and particle dimensions 197 i.\ y " /. \. ^ ^ [> \ [ PHYSIOLOGY ^' -' - ^\ - 15 , (' ' CARIELLO, L., L. ZANETTI, A. SPAGNUOLO, AND L. NELSON Effects of opioids and antagonists on the rate of sea urchin sperm pro- gressive motility i j y^O: 208 CARLSSON, KARL-HEINZ, AND GERD GADE Metabolic adaptation of the horseshoe crab, Limulus polyphemus, during exercise and environmental hypoxia and subsequent recovery 217 Continued on Cover Three \ Volume 171 Number 2 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board WALLIS H. CLARK, JR., Bodega Marine Laboratory, GEORGE D. PAPPAS, University of Illinois at Chicago University of California SIDNEY K. PIERCE, University of Maryland C. K. GOVIND, Scarborough Campus, University of Toronto LIONEL I. REBHUN, University of Virginia JUDITH P. GRASSLE, Marine Biological Laboratory HERBERT SCHUEL, State University of New York at Buffalo MICHAEL J. GREENBERG, C. V. Whitney Marine Laboratory, University of Florida VIRGINIA L. SCOFIELD, University of California at Los Angeles School of Medicine MAUREEN R. HANSON, Cornell University LAWRENCE B. SLOBODKIN, State University of New RONALD R. HOY, Cornell University York at Stony Brook LIONEL JAFFE, Marine Biological Laboratory JOHN D. STRANDBERG, Johns Hopkins University HOLGER W. JANNASCH, Woods Hole Oceanographic JOHN M. TEAL, Woods Hole Oceanographic Institution Institution WILLIAM R. JEFFERY, University of Texas at Austin DONALD P. WOLF, University of Texas Health Sciences CHARLOTTE P. 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PAPPAS, University of Illinois at Chicago University of California SIDNEY K. PIERCE, University of Maryland C. K. GOVIND, Scarborough Campus, University of Toronto LIONEL I. REBHUN, University of Virginia JUDITH P. GRASSLE, Marine Biological Laboratory HERBERT SCHUEL, State University of New York at Buffalo MICHAEL J. GREENBERG, C. V. Whitney Marine Laboratory, University of Florida VIRGINIA L. SCOFIELD, University of California at Los Angeles School of Medicine MAUREEN R. HANSON, Cornell University LAWRENCE B. SLOBODKJN, State University of New RONALD R. HOY, Cornell University York at Stony Brook LIONEL JAFFE, Marine Biological Laboratory JOHN D. STRANDBERG, Johns Hopkins University HOLGER W. JANNASCH, Woods Hole Oceanographic JOHN M. TEAL, Woods Hole Oceanographic Institution Institution WILLIAM R. JEFFERY, University of Texas at Austin DONALD P. WOLF, University of Texas Health Sciences CHARLOTTE P. MANGUM, The College of William and Mary SEYMOUR ZIGMAN, University of Rochester Editor: CHARLES B. METZ, University of Miami OCTOBER, 1986 Printed and Issued by LANCASTER PRESS, Inc. PRINCE & LEMON STS. LANCASTER, PA. m The BIOLOGICAL BULLETIN is issued six times a year at the Lan- caster Press, Inc., Prince and Lemon Streets, Lancaster, Pennsylvania. Subscriptions and similar matter should be addressed to The Bio- cai Bulletin, Marine Biological Laboratory, Woods Hole, Mas- sachusetts. Single numbers, $20.00. Subscription per volume (three issues), $50.00 ($100.00 per year for six issues). Communications relative to manuscripts should be sent to Dr. Charles B. Metz, Editor, or Pamela Clapp, Assistant Editor, Marine Biological Laboratory, Woods Hole, Massachusetts 02543. THE BIOLOGICAL BULLETIN (ISSN 0006-3185) POSTMASTER: Send address changes to THE BIOLOGICAL BULLETIN, Marine Biological Laboratory, Woods Hole, MA 02543. Second-class postage paid at Woods Hole, MA, and additional mailing offices. LANCASTER PRESS, INC., LANCASTER, PA IV Reference: Biol. Bull. 171: 291-309. (October, 1986) MOLECULAR BIOLOGY OF THE EARLY MOUSE EMBRYO GILBERT A. SCHULTZ Department of Medical Biochemistry. Health Sciences Centre, The University of Calgary, 3330 Hospital Drive N. W., Calgary, Alberta T2N4N1 Canada ABSTRACT The transition from maternal to embryonic control of development in the early mouse embryo occurs during the 2-cell stage. By the 2-cell stage, all classes of RNA are transcribed from the embryonic genome. Most of the changes in protein synthetic pattern that occur during the first cleavage are post-transcriptionally regulated. Later events, including compaction and blastocyst formation, require transcription from the embryonic genome, but some elements of post-transcriptional regulation are also involved. The result of the preimplantation developmental period is the formation of the blastocyst with two distinct cell types, the trophectoderm and inner cell mass cells. Each cell type is committed to a different developmental pathway and exhibits specific patterns of protein synthesis and DNA methylation. INTRODUCTION In the past decade, significant progress has been made in the manipulation of reproduction and development in mammalian species. Artificial insemination and embryo transfer are now widely used in the livestock industry and in the treatment of human infertility. While the length of time a mammalian embryo spends in the preimplantation period and the cell number achieved prior to implantation varies from one species to another, early development in every case is characterized by the formation of a blastocyst containing two morphologically and biochemically distin- guishable cell types. On this basis the mouse is a convenient and inexpensive model for basic research. Moreover, genetic uniformity and the ability to use genetic ap- proaches are important considerations in any biological study. This consideration is satisfied in mice by the many inbred strains or Fl hybrids available. The acquisition of the background knowledge and technical achievements (see Daniel, 1971; Daniel, 1978) necessary for experimental manipulation of mammalian eggs and embryos has largely occurred within the mouse system. For example, it was in the mouse that it was determined that early blastomeres are totipotent (Tarkowski and Wroblewska, 1 967) and that parthenogenetically activated eggs can develop up to post-implantation stages (Graham, 1970; Tarkowski et al, 1970; Surani and Barton, 1983). Similarly, the first reliable in vitro culture methods (Brinster, 1963; Whitting- ham, 197 1) and embryo freezing and thawing techniques (Whittingham et al, 1972) were developed for mouse embryos. Successful constructions of chimaeric mouse embryos of tetraparental heritage have been included in the developments (Mintz, 1965). Other experimental manipulations for nuclear transfer (Surani and Barton, 1983; McGrath and Solter, 1984) and gene transfer by microinjection (Gordon and Ruddle, 1981; Costantini and Lacy, 1981; Wagner et al., 1981) have also been pio- neered in the mouse system. Elegant experiments with appropriate gene constructs to study expression of exogenously supplied genes ("transgenes") have followed (Pal- miter et al, 1982; Krumlauf et al, 1985). All of these recent developments have Received 6 May 1986; accepted 29 July 1986. 291 292 G. A. SCHULTZ depended on general knowledge about macromolecular synthesis and gene expres- sion in th J early embryo. This re v begins with an outline of general procedures for handling mice, recov- ering earl? embryos, and assessment of the major morphological changes accompa- nying d velopment to the blastocyst stage. An evaluation of maternal control of early development is made prior to consideration of transcription from the embryonic ge- nome. Patterns of protein synthesis influenced by post-transcriptional processes dur- ing maternal control of the first cleavage are included along with a summary of pro- tein synthetic activity dependent upon transcription of template RNAs from the zy- gote genome. The review ends with a discussion of the appearance of cell-type specific patterns of protein synthesis and DNA methylation at the blastocyst stage. Where possible, relevant comparisons to parallel developmental features in eggs and em- bryos of the well-studied marine organisms and other comparative systems have been included. OBTAINING EGGS AND EMBRYOS Successful embryological studies, especially at the molecular level, require a large supply of eggs and embryos. In outbred strains of mice, natural cycles and mating lead to the generation of 12 to 14 embryos per female. Inbred strains, which are less vigorous in reproductive capacity, have smaller litter sizes. Hence, in many labora- tories, female mice are hormonally stimulated to cause an increase in the number of eggs that are ovulated. Hormonal manipulation also confers the further advantage of being able to regulate the time of ovulation (within certain limits) such that the collec- tion of eggs or embryos at specific stages of development coincides with work sched- ules and convenience of the investigator. Procedures for the superovulation of mice and the timing of ovulation and mating have been described by Gates (1971) and Whitten and Champlin ( 1 978), respectively. The highest yield of eggs (average of 89.5 per female; Gates, 1971) is obtained when prepuberal females of three weeks of age are superovulated, because this is a time when a wave of follicle maturation is occurring at the ovarian level. The immature mouse cannot, however, be used to derive embryos that have developed in vivo be- yond the 2-cell stage (e.g., morulae or blastocysts). Normal preimplantation develop- ment probably does not occur because eggs and embryos are transported too rapidly through the oviduct to the uterus (Gates, 1971). Fertilized eggs and 2-cell embryos from appropriate strains of prepuberal females will develop to blastocysts (with nor- mal attrition rates) if subjected to in vitro culture. Because of high numbers at the outset, this can be a good source of in v/7ra-derived material. In all superovulation procedures, a larger proportion of preimplantation embryos (about 12%) have sister- chromatid exchange chromosomal abnormalities than do embryos (about 3.5%) de- rived from normal matings (Elbling and Colot, 1985). To obtain embryos developed in utero, many investigators use random-bred mice that have reached reproductive age at 7 to 8 weeks. Superovulation at this stage leads to egg yields (30 to 35) that are approximately three times that of normal cycling females. The females are stimulated by intraperitoneal injection of 5 to 10 I.U. of pregnant mare serum gonadotrophin (PMSG). This preparation has a relatively sta- ble half-life (and therefore requires only a single injection to maintain stimulation of follicles over the 2 ! /2 day period to ovulation) and is a relatively inexpensive source of FSH (follicle-stimulating hormone) activity. Induction of ovulation is stimulated 44 to 48 hours later by the intraperitoneal injection of 5 to 10 I.U. of hormone with LH (luteinizing hormone) activity. The usual commercial source is human chorionic gonadotrophin (HCG). Rupture of matured Graafian follicles and release of ova to MOLECULAR BIOLOGY OF MOUSE EMBRYOS 293 Meiotic Maturation Oocyte J Growth N Primordial Germ Cells MET n ) Ovulation and Fertilization (12 HOURS POST-HCG) 1-cell Stage (Day 1) 2-cell Stage (Day 2) 8-cell Stage, Compaction, Embryo Enters Uterus (Day 3) Blastocyst Growth and Expansion (Day 4) Implantation (Day 5) TROPHECTODERM FIGURE I. Diagrammatic outline of early development in the mouse. Abbreviations are as follows: PMSG, pregnant mare serum gonadotrophin; HCG, human chorionic gonadotrophin; GVBD, germinal vesicle breakdown; MET I, first meiotic metaphase; MET II, second meiotic metaphase; ICM, inner cell mass. the fimbrial end of the oviduct occurs approximately 1 2 hours after HCG administra- tion (Fig. 1 ). The light-dark sequence is a major modulator in polyestrous mammals like mice. Endogenous LH surges lead to an ovulation time about 3 h after the mid-point of the dark period ( Whitten and Champlin, 1 978). Hence, it is common to employ a system in which the mouse room is maintained with 12 to 14 hours of light and 10 to 12 hours of dark with lights on at 5:00 to 7:00 hours and lights off at 19:00 hours. Injec- tions of PMSG are often scheduled between 1 6:00 to 20:00 hours, and HCG is admin- 294 G. A. SCHULTZ istered at 12:00 to 16:00 hours two days later. A 4:00 p.m. (16:00 hours) HCG injec- tion leads to ovulation at about 4:00 a.m., three hours after the mid-point of a 7:00 o'clock to 7:00 o'clock dark cycle, and coincides with any endogenous LH surge. If females are placed with fertile active males at the time of the HCG injection, copula- tion occurs and fertilization of eggs takes place in the fimbrial end of the oviduct shortly after ovulatory release. Of course, if it is desirable to study ovulation and fertilization events during normal working hours, the cycle can be delayed by chang- ing the dark-light cycle of the mouse room and the injection schedule. The presence of a copulation or vaginal plug (a coagulation of seminal proteins) on the morning after mating is the criterion commonly used to infer that fertilization occurred and that preimplantation embryogenesis was initiated. With hormonal ma- nipulation of females and selection of reproductively active males, plug rates of 75 to 80% are commonly achieved. This aids the investigator in obtaining sets of staged pregnant females from which to derive embryos at a particular stage of development. Random matings of spontaneously cycling females invariably yield low plug rates. Yet, a peak of fertile mating (up to 50% of females) often takes place on the third night after females are paired with males, due to acceleration of estrous cycling under the influence of male pheromones (Whitten and Champlin, 1978). Alternatively, fe- males in proestrous can be identified by microscopic examination of cells from vagi- nal smears of mice or, for the experienced eye, the changes in vaginal size and color- ation (Champlin et ai, 1973). These pre-selected females from a natural cycling pop- ulation will yield high plug rates upon mating. TIME COURSE OF MORPHOLOGICAL CHANGES DURING PREIMPLANTATION DEVELOPMENT During early embryonic life of mice destined for production of preimplantation embryos, primordial germ cells arise and migrate via the hindgut and dorsal mesen- tery to the dorsal body wall and the genital ridges (Fig. 1 ). In female embryos oogonia transform into oocytes, which progress through early meiotic prophase during fetal life and reach the diplotene stage at about the time of birth. During prepuberal and reproductive life, sets of oocytes begin to grow from a diameter of about 1 2 ^m to a diameter of about 85 nm over a two- week period (Fig. 1). During this period, the acellular zona pellucida that surrounds the plasma membrane of the ovulated egg (Fig. 2b) is laid down (Bleil and Wassarman, 1980). Under appropriate hormonal influence, these oocytes can be induced to undergo meiotic maturation and to be ovulated. At the time of ovulation, the unfertilized mouse egg, like that of Xenopus, is ar- rested at second meiotic metaphase and has extruded one polar body (Fig. 2b). This state differs from that in sea urchin eggs which have completed meiosis prior to fertil- ization as well as the situation in Spisula and Asterias, where eggs are arrested at first meiotic prophase at the time of sperm penetration (Browder, 1984). Within 1 to 3 hours after fertilization, mouse eggs complete meiosis and generate a second polar body (Fig. 2c). The sperm head decondenses to form the male pronucleus 4 to 8 hours after sperm entry, and the female pronucleus forms between 5 to 9 hours after sperm penetration (Hewlett and Bolton, 1985). The pronuclei move to the center of the egg, DNA replication occurs, pronuclei fuse, and the first cleavage division takes place about 20 hours after the time of sperm entry (Hewlett and Bolton, 1985) or roughly 32 to 34 hours after the program is initiated by administration of HCG to induce ovulation (Fig, I), Subsequent cleavages occur at roughly 12-hour intervals but syn- chrony among blastomeres is lost early. It is interesting to note that the first polar body often degenerates during the first cell cycle. The second polar body becomes MOLECULAR BIOLOGY OF MOUSE EMBRYOS 295 aligned in the plane of the first cleavage furrow (Hewlett and Bolton, 1985) and is nearly always positioned between the two blastomeres at the 2-cell stage (Fig. 2d and 2e). Following successive cleavages to the 8-cell stage (Fig. 2h), a morphological reor- ganization of the embryo called compaction (Fig. 2i) occurs at about 72 hours post- HCG administration. The distinct outline of individual blastomeres is lost and cells flatten tightly against one another. Gap junctions form in concert with the tight inter- cellular contacts and membrane and other cellular components rearrange from a nonpolarized to a radially polarized pattern (Ducibella and Anderson, 1975; Duci- bella et al, 1977; Lo and Gilula, 1979; Ziomek and Johnson, 1980; Reeve and Zio- mek, 1981). The development of cell contacts fixes cells in position and the polarity which occurs during compaction establishes an "inside" and "outside" orientation to blastomeres. Polarity is maintained as cell division continues in the embryo and the embryo is transported from the oviduct to the uterus (Fig. 1 ). Cells of the embryo tend to differentiate along different lineages depending upon their relative position as "inside" or "outside" in the compacted morula. Within the 64- to 128-cell blastocyst (fourth day of development), an outer layer of trophectoderm cells surrounds the inner cell mass (ICM) cells in the blastocyst cavity (Fig. 21). Just prior to implantation, the ICM differentiates into primitive endoderm and primitive ectoderm. The troph- ectoderm and primitive endoderm gives rise to extraembryonic structures and the embryonic contribution to the placenta, whereas the primitive ectoderm becomes the embryo proper (Papaioannou, 1982; Gardner, 1982). MATERNAL MESSENGER RNA UTILIZATION AFTER FERTILIZATION During oocyte development in the mouse, all classes of RNA are synthesized and accumulated in the egg. Polyadenylated RNA [poly(A)+ RNA], as a marker of puta- tive mRNA, also accumulates and has a high stability. The information available on RNA and protein synthesis during oogenesis in the mouse will not be reviewed here because it has been covered recently in other publications (Bachvarova, 1985; Schultz, 1986). Suffice it to say that during oocyte growth and meiotic maturation, several changes in the two dimensional patterns of newly synthesized protein occur, and during meiotic maturation about half of the accumulated polyadenylated RNA becomes either deadenylated or degraded (Bachvarova, 1985). The newly ovulated mouse egg contains about 23 ng of proteins (Brinster, 1967), between 0.35 to 0.50 ng of total RNA (Olds et al., 1973; Piko and Clegg, 1982), and about 20 pg of poly(A)+ RNA (Piko and Clegg, 1982). The first cleavage of the mouse zygote is controlled largely, if not exclusively, by informational macromolecules accumulated in the egg. From the 2-cell stage onward, transcription from the zygote genome is necessary for normal development to con- tinue. These conclusions are derived from several lines of experimentation. First, 30 to 40% of the bulk of maternal RNA (Bachvarova and De Leon, 1980), 70% of the total poly(A)+ RNA (Levey et al., 1978; Piko and Clegg, 1982), and as much as 90% of the histone (Graves et al., 1985a) and actin (Giebelhaus et al, 1985) messenger RNA is degraded within the first 24 hours after fertilization as development proceeds to the 2-cell stage. In this regard it is interesting to note that exogenous globin mRNA injected into newly fertilized mouse eggs is actively translated 1 5 to 1 7 hours later but is also eliminated on a functional basis by the 4-cell stage (Brinster et al, 1980). Second, while some RNA synthesis has begun within pronuclei of the mouse zygote, the rate of RNA synthesis increases markedly up to the late 2-cell stage such that the synthesis of all classes of RNA is readily detectable (Young et al, 1978; Piko and Clegg, 1982; Clegg and Piko, I983a, b). Third, a large number of changes in the 296 G. A. SCHULTZ "v.- FIGURE 2. Photomicrographs of mouse eggs and preimplantation stages of development. Panels a, d, g, and j show low power views: a, unfertilized eggs at 16 hours post-HCG; d, 2-cell embryos at 42 hours post-HCG; g, uncompacted 8-cell embryos at 66 hours post-HCG; and j, blastocysts at 96 hours post-HCG. The scale bar in j is 50 /im. For higher power photographs, the scale bar in k represents 50 tan. Stages are as follows: b, newly ovulated unfertilized egg with one polar body at 14 hours post-HCG; c, fertilized egg with two polar bodies at 1 8 hours post-HCG; e, 2-cell embryo at 42 hours post-HCG; f, 4-cell stage at 54 hours post-HCG; h, uncompacted 8-cell embryo at 66 hours post-HCG; i, compacted 8-cell embryo at 72 hours post-HCG; k, early cavitating blastocyst at 90 hours post-HCG; 1, mid-blastocyst stage at 98 hours post-HCG. Abbreviations are as follows: p, polar body; zp, zona pellucida; ICM, inner cell mass; t, trophec- toderm. All photomicrographs were taken with a Zeiss IM-35 inverted phase contrast microscope. MOLECULAR BIOLOGY OF MOUSE EMBRYOS 297 polypeptide synthetic pattern occur as fertilized eggs develop to the 2-cell stage (van Blerkom and Brockway, 1975; Levinson et al, 1978; Braude el al, 1979; Howe and Solter, 1979; Cullen et al, 1980). These changes appear to be regulated exclusively at the post-transcriptional level utilizing maternal components, since they also occur in physically enucleated eggs (Petzholdt et al., 1980) or in eggs treated with the tran- scriptional inhibitor a-amanitin (Braude et al., 1979; Flach et al., 1982; Bolton et al., 1984). Fourth, fertilized eggs treated with transcriptional inhibitors like a-amanitin and actinomycin D are markedly inhibited in development beyond the 2-cell stage (Golbus et al, 1973; Warner and Versteegh, 1974). Finally, genetic variants of pro- teins have been used to advantage to identify paternal gene expression in embryos derived from crosses of appropriate mouse lines. Paternally derived 0-glucuronidase (Wudl and Chapman, 1976) and /3 2 -microglobulin (Sawicki et al, 1982) are both detectable by the late 2-cell stage. Since spermatozoa do not contribute mRNA to the egg, this genetic evidence, along with the biochemical studies already listed, confirms that some newly derived transcripts from the embryonic genome are utilized at least by the 2-cell stage. Taken together, the work presented above indicates that the transition from ma- ternal to embryonic control in the mouse embryo occurs at the 2-cell stage. The time course of this first cell division covers approximately 24 hours. In the same period of time, a fertilized Xenopus egg, under normal conditions, would have developed to the 10,000-cell gastrula stage and a sea urchin embryo would have developed to a 500-cell hatching blastula stage (Davidson, 1976). Similarly, the period of maternal control extends to the gastrula stage in Xenopus embryos and to the blastula stage in sea urchin embryos (Davidson, 1976). It is possible that the long cell-cycle for the first division in the mouse is sufficient to allow chromatin re-organization, new tran- scription, and early transition of genetic control from maternally derived to zygote genome-derived messenger RNA molecules. Biochemical characterization of the maternal RNA in sea urchin and Xenopus eggs has revealed that as much as 70% of the mass of poly(A)+ RNA contains inter- spersed repetitive sequence elements (Costantini et al, 1980; Anderson et al, 1982). These molecules are large (often 5 to 1 5 kilobases in length) and similar in structure to nuclear RNA. Similar transcripts are not present in the poly(A)+ mRNA associ- ated with polysomes of later gastrulae stages of development. This RNA with inter- spersed repeats is not functional when added to in vitro translation systems (Richter et al, 1984). Analysis of poly(A)+ RNA in the mouse egg, however, suggests the absence of such non-functional transcription products containing interspersed repeti- tive elements (Kaplan et al, 1985). One of the major repetitive elements in human and mouse DNA is the Alu sequence family (Jelinik and Schmid, 1982). RNA from full-grown oocytes and ovulated eggs does not contain any larger proportion of se- quences complementary to Alu repetitive elements than does poly(A)+ RNA from differentiated cell types such as liver and brain (Kaplan et al, 1985). Moreover, the molecular weight distribution of molecules containing sequences complementary to Alu DNA probes is similar in mouse egg, liver, and brain cytoplasmic RNA, a finding one would not expect if there were large unprocessed RNA transcripts in mouse egg cytoplasm (Kaplan et al., 1985). Fertilization of the sea urchin egg triggers a series of events that results in a marked increase in the rate of protein synthesis within only a few minutes of sperm entry (Denny and Tyler, 1964; Epel, 1967). Part of this is mediated by mobilization of stored maternal mRNA components (Winkler et al, 1985) into polysomes (Monroy and Tyler, 1963) and part by an increase in translation activity and elongation rate (Brandeis and Raff, 1979; Hille and Albers, 1979; Winkler^ al, 1985). Accompany- ing the increase in protein synthetic rate during the first cleavage is the doubling of 298 G - A - SCHULTZ the amount of poly(A)+ RNA due to cytoplasmic polyadenylation of pre-existing mRNA species (Slater et al, 1973; Wilt, 1973). The significance of this event is un- known since the rise in protein synthetic rate occurs even when cytoplasmic polyade- nylation is blocked in sea urchin embryos treated with cordycepin (Mescher and Humphreys, 1974). When [ 3 H]-adenosine is used as a precursor, a higher rate of incorporation of adenosine into poly(A) tails versus internal locations in the RNA of the 1-cell mouse embryo is observed (Young and Sweeney, 1979; Clegg and Piko, 1983a, b). This cytoplasmic polyadenylation process results in about a 40% increase in the number of poly(A)+ RNA molecules in the newly fertilized 1-cell zygote compared to the ovulated egg (Clegg and Piko, 1983a, b). The net increase involves polyadenylation of previously stored non-adenylated RNA molecules, but there is also a component involving degradation of some pre-existing poly(A)+ RNA. For example, actin mRNA is partially deadenylated in fertilized eggs (Bachvarova et al, 1985). Thus, there appears to be a turnover in the poly(A)+ RNA population in the 1-cell embryo and this could lead to changes in the amounts of individual mRNAs and, in part, to the observed changes in protein synthetic pattern that occur in the 1-cell to 2-cell transition period. As documented earlier, 70% or more of the egg poly(A)+ RNA is eliminated through turnover by the late 2-cell stage (Piko and Clegg, 1982; Clegg and Piko, 1983a). Post-transcriptional modification of maternal mRNA through capping has also been studied. Addition of [ 2 H]-methyl cap structures to maternally derived histone mRNAs following fertilization of the sea urchin egg has been reported (Caldwell and Emerson, 1985). The mRNAs coding for these early cleavage type histones are stored in the female pronucleus (De Leon et al., 1 983) and have no access to the translational machinery of the egg until released after the first nuclear division. The capping pro- cess accompanies the translation of these histone mRNAs. It has been suggested that capping of maternal mRNA may be a mechanism by which stored mRNA in sea urchin eggs is activated because it has been shown that, contrary to mammalian cells, sea urchin eggs lack the ability to initiate translation of uncapped mRNAs and have an absolute requirement for 5' cap structures (Winkler et al., 1983). The incorporation of a low level of [ 3 H]-guanosine into 5'-terminal m 7 G struc- tures of RNA in 1-cell mouse embryos has also been reported (Young, 1977). Since more radioactivity was observed in m 7 G structures than in Gp derived from internal positions, it was postulated that capping of pre-existing RNA molecules may occur in newly fertilized mouse eggs (Young, 1977). In other studies which assessed the degree of capping by translational inhibition with cap analogues and end-labeling of mRNA molecules after enzymatic removal of cap structures with tobacco acid phosphatase, no differences in unfertilized and fertilized egg mRNA were observed (Schultz et al, 1980). Although post-transcriptional regulation of maternal mRNA in the 1-cell mouse embryo via capping is an appealing proposal, such a mechanism should be viewed with caution since uncapped mRNAs in mammalian cells are un- stable (Banerjee, 1980) and since cap structures are required on pre-mRNAs of mam- malian cells for correct splicing and excision of intervening sequences (Konarska et al, 1984; Kramer era/., 1984). RNA SYNTHESIS DURING PREIMPLANTATION DEVELOPMENT Early attempts to measure incorporation of [ 3 H]-uridine into RNA in the 1-cell mouse embryo yielded inconclusive results. Low levels of incorporation were ob- served, but the significance of the findings was complicated by the fact that failure to detect RNA polymerase activity in 1-cell mouse embryos (Moore, 1975) suggested MOLECULAR BIOLOGY OF MOUSE EMBRYOS 299 that the embryonic genome was not active until after the first cleavage. In studies on absolute rates of synthesis based on specific activities of precursor pools, Clegg and Piko (1977) discovered that [ 3 H]-adenosine was taken up and converted to ATP in mouse embryos about 1000 times more readily than the parallel conversion of uri- dine to UTP. Using [ 3 H]-adenosine as a labeled precursor, experiments to re-examine RNA synthesis in the 1 -cell embryo were conducted (Clegg and Piko, 1 983a, b). Some [ 3 H]-adenosine was observed to be incorporated into tRNA in the 1-cell embryo, but the majority was due to turnover of the 3'-terminal AMP (Clegg and Piko, 1983b). The synthesis of some heterogeneous RNA devoid of poly(A) tracts was also ob- served, but at a low rate (0.3 pg-cell/h). There was also a low rate of synthesis of internally labeled poly(A)+ RNA but the majority of [ 3 H]-adenosine incorporation in this class of RNA molecules was associated with turnover of 3'-terminal poly(A) tails (Clegg and Piko, 1983a, b). By the 2-cell stage, the rate of poly(A)+ RNA synthe- sis was measured to have increased five-fold over the 1-cell rate and ribosomal RNA synthesis was occurring at the rate of 0.4 pg/embryo/h (Clegg and Piko, 1983b). The capacity for transcription of RNA polymerase II genes in the 1-cell fertilized egg is demonstrated conclusively by plasmid microinjection experiments. Both the herpes simplex virus (HSV) thymidine kinase (TK) gene and a hybrid gene in which the HSV TK gene is fused to a mouse metallotheionein promotor are transcribed and expressed as enzymatic activity (translation) in fertilized mouse eggs (Brinster et al., 1982). In contrast to RNA polymerase type III genes (Brinster et al., 1981), the TK genes are transcribed much more effectively in fertilized eggs than in growing oocytes and other pre-fertilization stages (Brinster etal., 1982; Chen etal, 1986). An interest- ing observation on RNA processing also emerges from these studies. By comparison of expression of the HSV-TK gene (no introns), the SV40 TK gene (one intron), and the chicken TK gene (six introns), the presence of introns is associated with decreased ex- pression (Chen et al. , 1986). This may reflect a limited capacity for RNA splicing at this early stage of development. Nonetheless, the experiments demonstrate an enhanced capacity for transcription in fertilized eggs compared to the unfertilized ovum. On the basis of specific activities of uridine pools, Clegg and Piko ( 1 977) measured the absolute rates of RNA synthesis from the 2-cell stage to the blastocyst and ob- served a fifty- fold increase on an embryonic basis. However, if rates are calculated on a cellular basis, the value changes more modestly from about 1 .25 pg/cell/h at the 2- to 4-cell stage, to 2.5 pg/cell/h in the 8-cell embryo to about 5 pg/cell/h in the blasto- cyst. These rates are not very much lower than the rate of total RNA synthesis (5.7 pg/cell/h) reported for exponentially growing HeLa cells (Brandhorst and McCon- key, 1974). The rates are greater than those measured for heterogeneous RNA synthe- sis in cleavage and blastula stages of sea urchin embryos (Brandhorst and Humphreys, 1971; Wu and Wilt, 1974). In summary, all classes of RNA, including poly(A)+ RNA, are actively synthesized from the 2-cell stage and onwards throughout the pre- implantation period ( Levey etal., 1978; Piko and Clegg, 1982). Whereas maternal oocyte mRNA is largely degraded by the late 2-cell stage (see previous section), turnover rates of newly synthesized RNA have also been studied. The average half-life of mRNA measured in mouse morulae is 8 to 1 1 hours, while that in blastocysts is 14 to 26 hours (Kidder and Pedersen, 1982). As in HeLa cells (Singer and Penman, 1973) and rabbit blastocysts (Schultz, 1974), the mRNA decay profile in mouse blastocytes also has been observed to be biphasic with short-lived (about 6 hours) and long-lived (24 hours or more) components (Kidder and Pedersen, 1982). Such decay curves, however, probably represent an average of a continuum of decay of different classes of mRNA with different half-lives. The mixture of short- lived and long-lived components is consistent with studies on the continued synthesis 300 G. A. SCHULTZ and disappear?. ace of certain polypeptides when morulae are cultured to blastocyst stages in the ! ; . -sence of the transcriptional inhibitor, a-amanitin (Braude, 1 979a, b). One | if mRNAs that has been studied in detail in the early mouse embryo is the set i uanscripts derived from histone genes. The histone genes of mammals are part a small multi-gene family with 10 to 20 different genes for each histone protein. Four H3 genes, three H2b genes and two H2a genes have been isolated from three separate mouse genomic clones (Sittman et al, 1983; Graves et al, 1985b). Two of the gene clusters are localized on chromosome 1 3, and the third is on chromo- some 3. An SI nuclease mapping technique has been developed to measure expres- sion of the individual genes (Graves et al, 1985b). When this technique is applied to maternal mRNA derived from unfertilized mouse eggs and zygote-genome mRNA derived from the blastocyst stage, a number of changes are observed. A large amount (40 to 50%) of the histone H3 mRNA in the egg is complementary to the H3 gene located on chromosome 3 (H3.614) whereas only 14% of the histone H3 mRNA in the blastocyst is derived from the H3.6 1 4 gene (Graves et al., 1 985a). Similarly, nearly all the H2a mRNA in the egg is derived from the H2a.614 gene on chromosome 3, whereas only 30% of the H2a mRNA in blastocysts is complementary to the H2a.6 14 sequence. These and other data demonstrate that the same set of histone genes seem to be expressed in eggs and early embryos, but there are large differences in the relative abundance of certain histone mRNA types. Changes in histone gene sets expressed during sea urchin development are also well-documented (Newrock et al, 1978; Maxson et al, 1 983), but just as in the mouse embryo, the significance of these changes with respect to control of gene expression is not known. PATTERNS OF PROTEIN SYNTHESIS IN THE EARLY EMBRYO The ovulated mouse egg contains the machinery to synthesize proteins due to accumulation of rRNA, mRNA, tRNA, and ribosomes during oogenesis (Bachvar- ova, 1985). However, many of the ribosomes in the egg may not be functional when judged by their ability to form initiation complexes in vitro with a synthetic messenger RNA (Bachvarova and De Leon, 1977). Indeed, spare translational capacity of the fertilized mouse egg for injected globin mRNA is extremely limited (Ebert and Brins- ter, 1983). During the first 24 hours of post-fertilization development, there is little change in net protein synthetic rate, protein turnover, or total protein content (Brins- ter et al, 1976; Merz et al, 1981). During this same interval, there is a loss of 70 to 90% of the mRNA (Piko and Clegg, 1982; Giebelhaus et al, 1983). It follows that much of the mRNA in the unfertilized egg is in a form where it is not used for transla- tion or is used at a very low efficiency. From a qualitative point of view, the changes in the pattern of protein synthesis during the early cleavage period are very marked. In a previous section we have al- ready documented that many of the changes in the 1-cell to 2-cell transition period are post-transcriptionally controlled. It is important to note that some of the changes in protein pattern and cytoplasmic structure (e.g., mitochondrial translocation; van Blerkom and Runner, 1984) that occur up to the stage of pronuclear fusion appear to be contr -lied by post-translational processes such as glycosylation and phosphory- lation rath r than translation of stored maternal mRNA (van Blerkom, 1981, 1985; Howlett anc Bolton, 1 985). Other changes up to the 2-cell stage do seem to depend on sequential activation of selected mRNAs by some translational control mechanism (Braude et a!., 19 >; Cascio and Wassarman, 1982). In summary, there are fertiliza- tion-independent, fertilization-accelerated, and fertilization-dependent changes in polypeptide synthesis during the first cleavage. Some are due to post-translational MOLECULAR BIOLOGY OF MOUSE EMBRYOS 301 modification, some are due to differential mRNA activation and others are due to differential polypeptide turnover (Howlett and Bolton, 1985). All of the latter mecha- nisms are involved in producing the well-described series of changes in polypeptide synthetic pattern that occur within a protein complex with a molecular mass of about 35,000 during the first 24 hours after fertilization (Levinson et al, 1978; Braude et al, 1979; Cullen et al., 1980; van Blerkom, 1981; Flach et al, 1982; Howlett and Bolton, 1985). Changes in patterns of protein synthesis following fertilization or egg activation also occur in other systems. In the sea urchin embryo, there is a large increase in protein synthetic rate at fertilization (Grainger et al, 1979) with accompanying changes in the qualitative pattern of protein synthesis (Evans et al, 1983). For exam- ple, maternal mRNAs which code for four proteins (whose synthesis is barely detect- able in the unfertilized egg) become actively translated to yield abundant products of synthesis after activation. Some of these proteins (termed cyclins) are destroyed every time the cell divides (Evans et al, 1983). In addition, mRNAs for early histone vari- ants are stored in the female pronucleus (Venezky et al., 1981) but are not translated until after the first cleavage (Wells et al, 1981). With continued development, a switch to late embryonic histone variants occurs (Maxson et al, 1983). In the surf clam, Spisula solidissima, there is only a small increase in protein synthetic rate upon fertilization but a major change in the classes of proteins that are synthesized due to selective activation of maternal mRNAs also occurs (Rosenthal et al, 1980; Tansey and Ruderman, 1983). Perhaps these changes are so dramatic because the Spisula egg has not yet undergone meiotic maturation. In any regard, striking examples of translational control of changing patterns of protein synthesis are documented in these studies. An interesting set of polypeptides which appears at the early 2-cell stage of mouse development is a complex with approximate molecular weight of 67,000 to 70,000. Synthesis of these polypeptides is dependent upon new transcription since they do not appear in fertilized eggs cultured to the 2-cell stage in the presence of a-amanitin (Rach etal, 1982; Bensaudeefa/., 1983; Bolton et al, 1984). One-dimensional pep- tide maps of the 68,000 (68K) and 70,000 (70K) components are not distinguishable from two heat shock proteins, hsp 68 and hsp 70 derived from cultured mouse F9 cells (Bensaude et al., 1983). The developmental regulation of such heat shock or stress proteins is not unique to 2-cell mouse embryos. For example, hsp 70 mRNA accumulates in Xenopus oocytes (Bienz and Gurdon, 1982) but is translated in the oocyte only after heat shock treatment. After fertilization, translation of the hsp 70 mRNA cannot be induced by hyperthermia and cleavage stage embryos lack measur- able hsp 70 mRNA and have no detectable hsp 70 synthesis (Bienz, 1984; Heikkila et al, 1985). A number of hsp mRNAs also accumulate in Drosophila oocytes (Zim- merman et al, 1983) and larval and pupal stages (Cheney and Shearn, 1983). It is interesting to note, however, that in spite of the developmental regulation of the hsp 68-70 genes, the cleaving mouse embryo is refractory to induction of additional hsp synthesis by exogenous stress-inducing stimuli. A heat-shock "incompetent" period, in which hsp 68-70 synthesis is not induced in response to environmental stress, has also been observed prior to the blastoderm stage of Drosophila embryos (Dura, 1981), the blastula stage in sea urchin embryos (Roccheri et al, 1981), and the mid-blastula stage in Xenopus embryos (Bienz, 1984; Heikkila et al, 1985). The ability to respond to stress by the synthesis of hsp 68-70 is acquired by the blastocyst stage in both the mouse (Wittig et al, 1983; Morange et al, 1984) and the rabbit embryo (Heikkila and Schultz, 1 984). Failure of induction during cleavage may be related to the presence of constitutive levels of hsp 70 protein in the early embryos (Morange et al, 1984) al- 302 G. A. SCHULTZ though the d? ; ial response to heat shock in the preimplantation embryo remains unexpSairv .waits further study. Alth >i patterns of protein synthesis during the first cell division have been shown to . derived from the translation of both maternal and zygote-genome de- rived ';ies, there is no evidence for the translation of oogenetic mRNA in the mouse embryo after the 4-cell stage. Processes such as compaction, cavitation, and blastocyst formation require transcription from the embryonic genome, although an element of post-transcriptional regulation is also involved (Kidder and McLachlin, 1985). For example, the transcription of templates necessary for the critical events in compaction of the 72-hour post-HCG 8-cell embryo are completed by the 4-cell stage (Kidder and McLachlin, 1985). Conversely, in the process of cavitation and develop- ment of the morula to the blastocyst, transcriptional and translational processes are tightly coupled (Braude, 1979a, b; Kidder and McLachlin, 1985). From a qualitative point of view, two-dimensional gel patterns of proteins synthe- sized by 72-hour post-HCG morulae have been compared to those from embryos cultured in vitro to the blastocyst stage and to patterns from embryos arrested by culture in the presence of a-amanitin (Braude, 1979a, b). The major feature of these patterns is that the majority of the polypeptides show little change between the mor- ula and blastocyst stages and are translated from messenger RNAs of relatively high stability, since they continue to be synthesized 24 hours later despite the presence of a transcriptional inhibitor. A small set of polypeptides (Braude, 1979b) fails to appear or to increase in intensity in the presence of the inhibitor and a similar number per- sists in the presence of the inhibitor when normally synthesis would have ceased prior to the blastocyst stage. The normal events ultimately do lead to the appearance of a number of specific polypeptides in both the trophectoderm and ICM cell lineages at the blastocyst stage (van Blerkom et al, 1976; Handyside and Johnson, 1978; Brulet etal, 1980; Howe et al, 1980). Quantitatively, the rate of protein synthesis remains at a relatively low level from fertilization to the 8-cell stage. Once the 8-cell stage is reached, there is a progressive increase in synthetic rate accompanying the transition of the morula to the blastocyst (Epstein and Smith, 1973; van Blerkom and Brockway, 1975; Brinster et al, 1976; Abreu and Brinster, 1978). Ribosome numbers increase progressively with develop- mental stage due to active synthesis of rRNA and ribosomal proteins (La Marca and Wassarman, 1979). These rates of synthesis are sufficient for the production of about 2.5 X 10 6 ribosomes/embryo/h and can account for both the increase in ribosomal content and increased protein synthetic rate in the blastocyst (Piko and Clegg, 1982). An additional component may involve a shift in the pool of poly(A)+ RNA in the subribosomal fraction of morulae to the polysome fraction in blastocysts (Kidder and Conlon, 1985). DNA METHYLATION IN EARLY DEVELOPMENT Considerable interest has surrounded the field of DNA methylation in cell differ- entiation, because methylation of DNA sequences, for at least some genes in eukary- otes, is associated with inhibition of transcription (see Ehrlich and Wang, 1981, and Doerfle 33 for reviews). Five (5)-methyl cytosine appears as a minor base in the DNA ; organisms. It is produced by enzymatic (methylase) addition to some of the cytos sidues that are adjacent to guanine (m 5 CpG) in genomic DNA. During DNA r cation, the cytosine of the newly synthesized strand is usually sym- metrically meth ; ?.ted as soon as a sequence is made. If a CpG pattern is not methyl- ated, it will remain that way (Ehrlich and Wang, 1981). An alteration of the methyla- tion pattern of a eel?, (hyper- or under-methylation) relative to a parental cell line can MOLECULAR BIOLOGY OF MOUSE EMBRYOS 303 potentially lead to a heritable change that will be passed on from one cell generation to the next, generating a distinct cell lineage at the DNA level. In mammalian cells, initial studies indicated that genes were undermethylated in tissues in which they were expressed during development or cell differentiation and hypermethylated when inactive. This appears to be true for about one-third of the thirty genes analyzed to date (Kolata, 1985). In addition, many of the so-called "housekeeping genes," which are active in all cell types are undermethylated at their 5'-terminal initiation sequences. In another 20% of the genes, there is no correlation between methylation and gene activity (Kolata, 1985). Since there is a lesser degree of methylation of cytosine residues in lower vertebrates and essentially no methyla- tion in Drosophila DNA (Ehrlich and Wang, 1981), association of methylation with gene expression may be restricted to mammalian cells. In addition, methylation may be associated only in a secondary way with respect to expression of genes rather than being the primary event in turning off genes. Nonetheless, interesting patterns of DNA methylation are associated with formation of the first distinct cell lineages in the mouse embryo. At about 4.5 days of development, just before implantation of the blastocyst, ICM cells become either primitive ectoderm or primitive endoderm. Along with the trophectoderm layer, the primitive endoderm gives rise to extraembryonic structures while the primitive ectoderm contributes to the three germ layers of the embryo (Pa- paioannou, 1982; Gardner, 1982). On the basis of methyl-sensitive restriction endo- nuclease digestion patterns of mouse satellite DNA and dispersed repetitive sequence, it has been shown that these sequences are undermethylated in all derivatives of the extraembryonic lineages compared to those in primitive ectoderm or DNA of adult tissues (Chapman et ai, 1984). Similarly, the DNA of the trophoblast component of rabbit blastocysts has been shown to be undermethylated (Manes and Menzel, 1981). Recently it also has been demonstrated that the undermethylation of DNA in extra- embryonic structures of the mouse embryo is not restricted to repetitive sequences and includes alpha-fetoprotein, albumin, and major urinary protein structural gene sequences (Rossant et al., 1986). The same sequences are heavily methylated in em- bryonic tissues as early as 7.5 days of development (Rossant et ai, 1986). These find- ings along with other studies (Razin et al, \ 984; Young and Tilghman, 1 984) confirm that major differences in DNA methylation occur as cell lineages are established in the early embryo. In the process of establishing different methylation patterns in the early cell line- ages, both de novo methylation and demethylation probably occur. In general, sperm DNA is highly methylated with respect to structural genes, although satellite DNA sequences are less methylated in sperm than in adult tissues (Waalwijk and Flavell, 1978; Sanford et al., 1985). Oocyte DNA is undermethylated with respect to repeti- tive DNA sequences (Sanford et al., 1985). Retroviral sequences introduced into the preimplantation embryo become de novo methylated, but sequences introduced into post-implantation stages (8-day mouse embryos) escape methylation (Jahner et al., 1982). Taken together with the fact that the DNA of cleavage-stage rabbit (Manes and Menzel, 1981) and mouse embryos (Singer et al. , 1979) has levels of methylation similar to that of adult tissues, it is possible that the de novo methylation of sperm and egg DNA may occur early. If this is so, it follows that the differentiating trophec- toderm and primitive endoderm lineages must undergo extensive demethylation pro- cesses. There are insufficient data to make firm conclusions about the timing of de novo methylation events in the embryo, and the possibility that de novo methylation does not occur until the primitive ectoderm lineage is established must also be enter- tained (Sanford et al., 1985). Although it is clear that the methylation pattern of the alternate lineages is distinct, the significance of this observation with respect to 304 G. A. SCHULTZ subsequent < erstiation and gene expression in each line remains to be established through furl esearch. CONCLUDING REMARKS ysis of the molecular biology of the early mouse embryo is difficult because of its small size and limited numbers of available embryos. Nonetheless, the develop- ment of recombinant DNA techniques of high sensitivity recently has allowed ap- proaches to problems of gene expression in early mouse embryos that previously could be studied only in systems where embryological material was more abundant. Using S 1 mapping techniques, it has been possible to measure histone mRNA tran- scripts in mouse blastocysts that are present in as few as 600 copies per cell (Graves et ai, 1985a). In situ hybridization methods have been developed for sea urchin em- bryos that can detect mRNAs in defined cell types in as few as 50 copies or less per cell (Cox et al, 1984; Angerer and Davidson, 1984). This molecular cytological ap- proach can be applied to a small number of embryos and is potentially well-suited to investigation of early mammalian embryos. Although laborious to construct, cDNA libraries from mRNA from mouse oocytes and blastocyts have now been made (Mc- Connell and Watson, 1986). In time these libraries will aid the identification of genes expressed at particular stages of early development. A key feature of development in the mouse that requires future emphasis is the identification of marker genes that are activated in time and space during the period of early implantation, germ line formation in the embryo proper, and early morpho- genesis and organogenesis. The techniques now exist to study patterns of gene expres- sion during this critical phase. I purposely have not included extensive discussions of the use of genetically engineered transgenic mice in this review because, to date, this approach has yielded information primarily on tissue specificity of gene expression in fetal or adult organs. 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POWERS 2 , HEIDI HOWARD 3 , AND LEONARD KASS 4 Marine Biological Laboratory, Woods Hole. Massachusetts 02543 ABSTRACT In the spring, horseshoe crabs (Limulus polyphemus) along the eastern coast of North America migrate toward shore to build nests close to the water's edge. In 1984 the mating season near Woods Hole, Massachusetts, extended from 14 May to 7 July. Mating activity during this period fluctuated with the phase of the moon, the height of the tide, and diurnal changes in daylight. As the moon approached new and full phases, large numbers of animals migrated into the intertidal zone to mate and build nests. They appeared 1 -2 h before high tide, and returned to deep water about 2 h after high tide. No mating activity occurred during low tides. The two daily high tides in this region are unequal in height. The inequality is greatest during new and full moons. At these times most animals migrated toward shore on the higher tide, which occurred in the late afternoon and throughout the night. As the moon passed through quadrature the tidal inequality diminished and reversed. Mating activity changed accordingly: shore migration diminished, becoming nearly equal on the two equally high tides, and in several days shifted to the higher high tide. The cue for shifting appears to be the first light of dawn and not the height of the tide itself. We suggest that the migration of Limulus involves several sensory modalities including vision. INTRODUCTION Much has been learned about vision from research on the horseshoe crab, Limu- lus polyphemus. The classic studies of the Limulus lateral eye by H. K. Hartline and his colleagues revealed basic mechanisms of retinal function common to many ani- mals (Hartline, 1 969; Ratliff, 1 974). More recent studies show that the Limulus visual system is an excellent example of the central modulation of retinal sensitivity (Barlow et al, 1977; Barlow, 1983; Barlow et al, 1985). Efferent nerve signals generated by a circadian clock located in the brain increase the sensitivity of the lateral eyes at night. Laboratory studies show that the circadian increases in retinal sensitivity can be de- tected behaviorally (Powers and Barlow, 1985), and field studies indicate that vision mediates some aspects of mating behavior (Barlow et al, 1 982). Further studies of the visual component of Limulus mating behavior should enhance our understanding of the functional organization of the visual system (Barlow et al., 1987). As a first step we carried out a detailed investigation of mating activity itself. Limulus mates in intertidal zones along the eastern coast of North America and along much of the coast surrounding the Gulf of Mexico (Shuster, 1979). These coastal regions are dynamic environments. Their physical characteristics undergo large, pei xlic changes due to movements of the earth and moon. Although such Received 21 April 1986; accepted 30 July 1986. Permanent add esses: 'Institute for Sensory Research, Syracuse University, Syracuse, NY 13244- 5290; 2 Departmem of Psychology, Vanderbilt University, Nashville, TN 37240; 3 2 Potter Park, Cam- bridge, MA 02138; and 4 Department of Zoology, University of Maine, Orono, ME 04469. 310 MIGRATION OF MATING LIMULUS 311 Mashnee Dike Research Site -o OHW. V/;Vv.y;;'v:. : .v':; Access Road ^;-^::;::^{^^:^\:^y^^ : ^ Camera East Site Pole 8+0 6+0 Riprap M.L.W. 10.01 33 Meters FIGURE 1 . Map of the research site on Mashnee Dike. We denned the East Pole as the steel pipe located in 1933 by the U. S. Army Corps of Engineers at 7037'46"W and 4146'34"N. Other pipes placed at 100 ft intervals mark the main baseline. All counts were taken in transects 1, 2, and 3 as denned by the shoreline and the East Pole of the main baseline. Broken lines indicate sectors of the transects. The water elevation at mean high water (M.H.W.) is 4.0 feet above mean low water (M.L.W.). The access road on the dike is lined with utility poles that support overhead wires (OHW). The camera site 100 ft from the East Pole was used in a concurrent study of visually guided behavior of Limit/us (Barlow el al. , 1 984, 1 986). dramatic fluctuations would appear to produce a forbidding environment, many ma- rine and terrestrial species either inhabit the intertidal zones permanently or migrate into them for important biological activities (review: Neumann, 1981). Many such animals synchronize their activities with the major environmental rhythms of the intertidal zone. Preliminary observations indicated that lunar phase, tide height, and sunlight influence the mating activity ofLimulus (Howard el al., 1984). We report here a quantitative study of the imigration ofLimulus to the tidal zone for mating. We examine the temporal relationships between migratory behavior and the seasonal, lunar, tidal, and daily light cycles. MATERIALS AND METHODS We quantified mating activity as the number of animals appearing within the intertidal zone. This definition is based on prior observations that large numbers of adult horseshoe crabs regularly appear along the coastline in conjunction with breed- ing activities (Schuster, 1957, 1958, 1979;Cavanaugh, 1975; Rudloe and Herrnkind, 1976;Rudloe, 1978, 1979, 1980; Barlow et al., 1982, 1984; Cohen and Brockmann, 1983; Howard et al, 1984). The data were gathered between 13 May and 12 July 1984. Observations were also made throughout the 1985 and 1986 mating seasons. Research site Figure 1 shows the location of the research site at Mashnee Dike, Cape Cod, Mas- sachusetts. Mashnee Dike is a narrow strip of sandy beach constructed by the U. S. Army Corps of Engineers in 1 933 to connect Mashnee Island with Cape Cod. Perma- nent landmarks from the original survey include iron pipes that define the main base- line of the survey. One of the pipes, designated the "East Pole," stands about 2 ft. high at longitude 7037'46" West and latitude 4 1 46'34" North. No houses or artificial lights are located within 0.8 km of the research site. 312 R. B. BARLOW, JR., ET AL. TABLE I Number of I '. : <. ounted in 1 984 Pairs Single Total Moving Nesting Male Female animals Near 601 1378 2866 2 6826 Middle 1000 550 2621 1 5722 Far 614 129 958 3 2447 Females 2215 2057 6 4,278 (29%) Males 2215 2057 6445 10,717(71%) Total animals 4430 4114 6445 6 14,995 Counting procedures To sample animal numbers in the area, we established three 10 m X 10m tran- sects along the shoreline, using the East Pole and the water's edge as reference points. This technique is similar to one used by C. M. Cavanaugh (pers. comm.). The west side of Transect 1 (see Fig. 1 ) was defined by a line perpendicular to the shoreline and passing through the East Pole. The north side of all three transects was defined by the water's edge. The transects were separated from each other by 10m. Two reflective posts were driven into the sand several meters above mean high water (M.H.W.) along the continuation of the east and west borders of each transect. This allowed us to determine their boundaries for each counting session. The position of the transects thus remained fixed in the longshore direction, shifting along the onshore-offshore direction as the tide flooded and ebbed (Fig. 1 ). Most counts were taken around the times of the two daily high tides because previous observations indicated that few animals appeared at this site during low tides (Barlow et al., 1982; Howard et al, 1984). On three occasions we counted animals every half hour around the clock, to confirm that Limulus appeared in the intertidal zone only during high tides. At all other times counts were made every half hour for a period of up to 3.5 h beginning about 1 h before predicted high tide. In each counting session the observer began at the west side of Transect 1 and waded toward the east side counting the animals within the "near" sector, the third of the transect adjacent to the water's edge. Upon reaching the east side, determined by lining up the reflective posts on shore, the observer moved offshore about 4 m, waded toward the west side, and counted the animals in the "middle" sector (the middle third of the transect). The observer then moved to about 8 m offshore, walked across the transect, and counted the animals in the "far" sector (the outer third of the transect; depth < 1 m). This procedure was repeated for Transects 2 and 3 with the entire session requiring about 10 min. "irnals were categorized as they were counted, and the number in each category was noted for the three sectors of each transect. The categories were "moving pairs" (female and male in amplexus and freely swimming), "nesting pairs" (female-male pair had burrowed into the sand), and unpaired "single males." Frequently we noted how many gJe males were "freely swimming" and how many were associated with a nest ("sate males''). With this counting technique, no animals were overlooked in the transects during the day. We estimate that less than 5% were overlooked at night. The an nove slowly enough that counting the same animal twice would seldom occur. Because unpaired females are rarely found on mating beaches (Cavanaugh, 1975; MIGRATION OF MATING LIMULUS 313 100r 09 ** 0) c - 50 o CD .0 E Near Middle / o .-o - A 345 Weeks of season 6 FIGURE 2. The relative number of nests counted in three regions of the transects varied across the mating season. The three symbols give the distribution of nests in the sectors as percentages of the total number of nests counted during a given week. In the early part of the season most animals nested in the near sector of the transects, closest to the water's edge. By the end of the season most animals were nesting in the middle sector. Few nests were ever observed in the far sector of the transects. Sectors are indicated in Figure 1 . R. B. Barlow, Jr., unpub. obs.), we did not examine every free-swimming animal. However when we suspected that a free-swimming animal might be female because of its large size, we picked it up to identify its gender. Only six were females. In several counting sessions in 1985 and 1986 we identified the gender of all unpaired animals and each time found about 0.5% were females. Tide measurements To estimate the times of high tides in planning our counting sessions we used the Cape Cod Canal 1 984 Tide Tables, published by the New England division of the Full moon New moon Full moon New moon O 200 E 'c Q 100 3 c X CD 2 o 15 May 20 25 30 1 June FIGURE 3. Maximum number ofLimulns counted in the transects each day of the 1 984 mating season at Mashnee Dike. The height of each bar shows the largest total number of males and females recorded in a single half-hourly counting session during the solar day. The open and filled circles indicate the full and new moons of 14 and 30 May and 13 and 28 June (Eldridge, 1984). 314 R B. BARLOW, JR., ET AL. U. S. Army Corps of Engineers (1983). The most appropriate tables were those for Wings Neck, jocated 0.6 nautical miles southwest of Mashnee Dike. Except during foul weather, the predicted time of high tide at Wings Neck differed from that at Mash ;f , Dike by less than 5 min. The actual time of high tide at Mashnee Dike was determined by the observer during each counting session by noting the distance from the East Pole to the water's edge. Tide height was measured by the U. S. Army Corps of Engineers with a nitrogen gas gauge submerged in the approach channel to the New Bedford-Fairhaven Harbor. Although the approach channel is located 1 1 .6 nautical mi. from our observation site, the tide heights measured in the channel are within 1-2 cm of those at Mashnee Dike (F. Morris, pers. comm.). RESULTS Mating season The mating season of Limulus polyphemus at Mashnee Dike in 1984 lasted 55 days, from 14 May to 7 July (Fig. 3). Table I gives the numbers of males, females, and pairs counted in the transects during this period. Because we did not tag individual animals, the values provide information on the relative proportions of animals in the different categories but not their absolute numbers. Absolute values would be influenced by animals that remained in a transect for more than one counting period, moved from one transect to another, or returned to the mating area on subsequent days. When all transects are considered together the numbers of moving and nesting pairs were approximately equal (2215 and 2057, respectively), and were less than the total number of single males (6445). If each pair is counted as 1 male and 1 female, more than twice as many males ( 10,7 17) as females (4,278) were sighted in the tran- sects. For the sessions during which we divided single males into free-swimming and satellite, their ratio was approximately 80:20. As anticipated, very few single females were seen. Limulus were not uniformly distributed across all three sectors of the transects (Table I). More moving pairs were counted in the middle sector than in either the near or far sectors. More nesting pairs were counted in the near sector, with progres- sively smaller numbers in the middle and far sectors. Single males were concentrated in the near and middle sectors, where the number of pairs was greatest. The distribution of the nesting pairs within the transects changed systematically as the season progressed. Relatively more nests were found in the near sector along the water's edge in the beginning of the season, and in the middle sector later in the season (Fig. 2). Few were seen in the far sector. Nesting pairs were not observed in the transects until 19 May, five days after the first moving pairs appeared. Nests ap- peared first in the near sector (on 19 May), then in the middle sector (on 21 May), and finally in the far sector, sporadically after 26 May. The shift of nests away from the shoreline as the season progresses may reflect a change in predation or an influ- ence of temperature. Semilunar rhythm Mating activity was correlated with phases of the moon (Fig. 3). Large numbers of animals were counted each day before and during the new moon on 30 May and around the full moon on 1 3 June. A smaller peak occurred near the new moon on 28 June. The total number of nests counted in the transects each solar day followed MIGRATION OF MATING LIMULUS 315 May 15 20 25 30 June 1 10 15 20 25 July 1 Midnight Noon Midnight FIGURE 4. Daily mating activity at Mashnee Dike between 14 May and 7 July 1984. The bars indicate the times during which at least 10 Limulus were counted in the transects. Refer to Figure 5 for the duration of daily counting sessions during the peak of the season. The lines connect the times of each daily high tide as it advanced through the solar day. The lines have slopes of about 50 min day 1, indicating that each tide crests at intervals of 24.9 h, the length of the lunar day. 316 R. B. BARLOW, JR., ET AL. May 20 21 22 23 24 Noon Midnight Noon Midnight MIGRATION OF MATING LIMULUS 317 the same pattern. The fluctuations in migratory activity thus had a period of about 1 5 days, indicative of a semilunar rhythm. A thorough quantitative study was not achieved in 1985 because many animals were removed from the area by fisherman during the height of the season. Nonethe- less, the mating activity of the residual population exhibited pronounced fluctuations comparable to those in 1984. The first large peak of activity coincided with the new moon on 23 May and subsequent peaks occurred during the full moon on 3 June and the new moon on 1 7 June. Again the fluctuation in mating activity had a period of about 1 5 days and the season lasted for about 50 days. Tidal rhythm Mating activity occurred only during high tides (Fig. 4). The bars give the periods during each day when 10 or more animals were observed in the three transects. Mashnee Dike is subjected to a semidiurnal tidal regime, and the diagonal lines con- nect the actual times at which each of the two tides reached maximum height throughout the season. The close correlation between the lines and bars shows that significant numbers of animals appeared in the transects only at the high tides. The tidal component in the daily pattern of mating activity persisted throughout the season. A line through the middle of the periods of activity (bars) associated with either high tide would have a slope of about 50 min per day. This equals the slope of the line representing the times of the high tides. Thus, daily episodes of mating activ- ity advance through the solar day along with the high tides each with periods approxi- mating 24.9 h, the length of the lunar day. Figure 5 shows the distribution of animals during the half month of greatest mat- ing activity (20 May to 3 June). Small vertical arrows indicate the times of high tides during this period. High tides occurred near midnight and noon on 20 May and progressed through the day to noon and midnight respectively by 3 June. Mating activity over this period occurred during both daily high tides with peak activity gen- erally observed about 1-2 h after tidal crests. The animals then began leaving the nesting area to migrate offshore. On 23, 28, and 30 May we counted animals in the transects every half hour for 30 hours or more. These long-term counts clearly show that no animals were present in the transects during low tides. In addition, underwater observations using SCUBA diving gear revealed very few moving and no buried Limulus within 100 m of the shoreline during low tide. Instead, numerous animals, both paired and single, were found on the bottom at depths up to 8 m between 100 and 400 m offshore. Some were swimming, some were stationary, but none were nesting. More animals were observed during one of the two daily high tides than during the other. From 20 May to 24 May more appeared on the early morning tide than on the midday tide. These differences were large, approaching 200: 1 . On 24 May, the difference declined and on 25 May it reversed, with more Limulus appearing on the afternoon tide. On successive days, most animals continued to migrate to shore on FIGURE 5. Daily distributions of horseshoe crabs in the transects between 20 May and 3 June 1984. Counts were taken only during the hours marked by baselines. For example, animals were counted around the clock on 22-23 May, 27-28 May, and 30-3 1 May and near the times of high tides (vertical arrows) on other days. Large arrows indicate the higher daily tides. The flanks of some distributions (about 10%) have been extrapolated when full counts were not available. Note that more animals appeared in the transects during the early morning tides from 1 9 May to 24 May and during the evening tides after 25 May. Dark bars and vertical lines indicate darkness (see Fig. 9). The data are double-plotted to emphasize the periodicity of the mating activity. Vertical scale equals 100 animals. 318 R. B. BARLOW, JR., ET AL. this tide as it progressed through the late afternoon and into the night. The animals abandoned i:hc tide only after it moved past dawn on 7 June (not included in Fig. 5). They then shifted to the opposite (afternoon) tide and repeated the cycle. In the fol- lowing sec f.ons we present evidence that these tidal preferences are related to the relative heights of the tides. Semidiurnal inequality of tide height On most days during the mating season one tide was clearly higher than the other. Such inequalities in tide height are caused at certain latitudes by the moon's monthly declination north and south of the plane of the ecliptic. This declination produces asymmetries in the system of tide-generating forces such that two tidal waves of different heights occur each day at the northern and southern latitudes whenever the moon is not over the equator (Wylie, 1979). During the spring the pattern of tidal inequality in the Cape Cod area produces maximal differences in the heights of suc- cessive tides twice monthly at full and new moons. These diminish over the following two weeks and reverse twice monthly during the first and last quadratures. Figure 6 shows the actual (top) and predicted (bottom) tide heights at Mashnee Dike from 12 May to 3 June 1984. This period includes the full moon of 15 May, the new moon of 30 May, and the 15-day period covered in Figure 5. One of the two daily tides is indicated by a stippled bar, the other by a black bar. The predicted changes in tides follow a relatively smooth function, whereas the actual changes do not because they are influenced by unpredictable weather conditions. However, both exhibit maximal flood and ebb tides at the full and new moons, with neap tides at the lunar quadrature on 23 May. The daily low tides were nearly equal in height but the high tides were not. The inequality in high tides was greatest during the full moon and minimal near quadrature on 23 May when it reversed and became large again at new moon. Mating activity on Mashnee Dike was closely related to the magnitude of the inequality between high tides. Figure 7 compares actual tide measurements with mating activity over the 8-day period from 20 to 27 May. On 20, 21, and 22 May, the daily high tides differed in height and more animals were counted within the transects during the higher tides, which occurred in the early morning hours before dawn. This was also the case on 25, 26, and 27 May when the majority of animals again appeared during the higher high tides, which occurred in the afternoon on those days. The animals' preference for the afternoon tide grew as the tidal inequal- ity increased, and continued as this tide progressed through the evening and into the early morning hours. Limulus did not, however, always prefer the higher high tide. Even though the tidal inequality diminished and reversed, the majority of animals continued to populate the early morning tides, which were slightly lower than the afternoon tides. They switched to the higher afternoon tide on 25 May, three days after tidal -ersal. This behavior was repeated during the next tidal cycle, when they again ched to the afternoon high tide three days after reversal of the tidal inequality at the lunar quadrature on 6 June. We believe these exceptional days are significant for understanding factors that control Limulus mating activity, and we will consider them in more detail below. Not onl> ere more animals generally counted during the higher high tides, but the relative number appearing in association with any two consecutive high tides was roughly proportional to the relative difference in the heights of the two tides. This point is illustrated in Figure 8. In general, when the difference in the heights of the two high tides was large, as on 29 May, the degree of preference was also large. When MIGRATION OF MATING LIMULUS 319 Full moon O New moon CD ^ 2 *- .c O) '0 O -1 4r s * '\- \ 3 x-s :|: :| : : :| X ** 0) 0) ~ 2 - 0) ^r 0) 1 ^ +i - 1 T3 Q) o T3 CD L -1 - -2 _ 12 15 20 25 30 May 1984 June FIGURE 6. Predicted (bottom) and actual (top) heights of tides at Mashnee Dike during the first half of the 1984 mating season (Army Corps of Engineers). One of the two semidiurnal tides is indicated by black bars and the other by stippled bars. The tidal excursions were maximal during full and new moons with one tide (stippled bars) higher during the full moon and the other (black bars) during the new moon. Excursions were minimal during the lunar quadrature on 23 May when the inequality in heights reversed. Note that the heights of the low tides did not differ much over this period. 320 R. B. BARLOW, JR., ET AL. May 20 May 21 May 24 May 25 200 .2 co 'c CO o 100 <- Q. 0> -1 Midnight Noon Midnight Noon Midnight FIGURE 7. Comparison of tide height with the number of horseshoe crabs counted in the transects during the week of 20 to 27 May. From 20 to 22 May more animals appeared on the early morning tide, which was the higher of the two daily tides. On 23 May most animals remained with the early morning tide even hough the inequality had reversed and the afternoon tide was higher. This behavior persisted until 25 May. when the animals' tide preference switched to the evening tide. MIGRATION OF MATING LIMULUS 321 I 1 1 1 1 I 1 1 1 1 1 1 5 20 25 30 5 10 15 20 25 30 5 May June 1984 July FIGURE 8. The relative proportion of animals observed on a given high tide (top) corresponds to the relative height of the tide (bottom). The difference in proportion of animals is equal to the difference between the total number of animals observed on two semidiurnal high tides divided by the sum observed on both tides. Each point on the bottom graph represents the difference in heights of the pair of semidiurnal high tides in feet. A sinusoid with a period of one lunar month (29.4 days) and an amplitude of 0.85 was fitted to the tidal data by eye. The same function shifted by 3.2 days is plotted in the top half of the figure. the difference in tide height was small, as on 6 June, the degree of preference was small. But the correlation was not perfect. The sinusoidal function with a period of 29.4 days that describes the tidal data in the bottom half of Figure 8 must be shifted to the right by 3.2 days to fit the behavioral data. This shift implies a phase lag of nearly 3 days between changes in relative tide height and changes in the animals' preference for one tide over the other. Sunlight Mating activity was more strongly correlated with the tidal inequality than with light or darkness. For example, from 20 May to 3 June most animals (77%) migrated into the transects during the highest high tides indicated in Figure 5 by large arrows. These tides occurred from 1 530 to 0330 h as shown by the stippled bars in Figure 9. Over this 1 5-day period half of the animals appeared in the transects during light ( 1 530 to 2 100 h) and half during darkness (2 100 to 0330 h). We conclude that Limu- lus generally prefer to mate on the highest tides regardless of when they occur. 322 R. B. BARLOW, JR., ET AL. Sunset Sunrise Sunset Sunrise Highest tides 600 CO E 400 c CO CD E 200 L Noon Midnight Noon Midnight Noon FIGURE 9. Comparison of mating activity with daylight, darkness, and the tidal inequality. The num- ber of animals counted from 20 May to 3 June was plotted as a function of time of day by summing every half hour the counts shown in Figure 5. The data are double-plotted. The times of sunset (2010 h) and sunrise (05 10 h) are given at the top with dusk (2 1 10 h) and dawn (04 10 h) indicated by cross hatching and darkness by black bars. Because these times changed slightly with each day the data are shown for 27 May, which is in the middle of the period. The stippled bars give the times of occurrence of the higher of the two daily tides over the 1 5-day period. Note that the peak number of animals was observed before sunset. DISCUSSION mating activity of Limulus polyphemus is related to the environmental changes iuced by periodic motions of the earth and moon. The yearly rotation of the earth about the sun produces seasonal changes in daylength and in ocean tem- perature iaily rotation of the earth about its axis produces diurnal changes in daylight and semidiurnal changes in the levels of most oceans. The monthly rotation of the moon about the earth modulates the tidal flows of the oceans and produces periodic inequalities in the daily tides in the northern and southern hemispheres. All of these astronomical events affect the physical environment of intertidal zones, and MIGRATION OF MATING LIMULUS 323 this study indicates that all appear to influence Limulus mating behavior within the intertidal zones of Cape Cod, Massachusetts. Time of year Limulus mates seasonally. The animals entered and built nests in the intertidal zone at Mashnee Dike for about 8 weeks in 1984, beginning in mid-May and ending in early July (Fig. 3). This was also the case for the neighboring shores of Buzzards Bay, Narragansett Bay, Vineyard Sound, and Nantucket Sound (R. B. Barlow Jr., unpub. obs.). The time of the year and duration of the mating season in 1984 at Mashnee Dike was similar to that of other years from 1982 to 1986. The mating season is longer in the Gulf of Mexico and farther south along the eastern coast of North America, generally beginning earlier in the spring and ending later in the sum- mer or even late fall (Schuster, 1979; Rudloe and Herrnkind, 1976). The choice of spring for mating undoubtedly reflects periodic environmental fac- tors caused by the earth's rotation about the sun. The most prominent factors are an increase in the duration of daily sunlight and a warming of the waters in the northern hemisphere. At more southern latitudes, the waters warm sooner and the days are longer in early spring. These differences may explain the earlier mating seasons in the southern latitudes if Limulus initiated mating when a particular ocean temperature and/or daylength was achieved. Either mechanism appears plausible. Limulus is a poikilotherm and its overall locomotor activity is influenced by ambient water tem- perature (R. B. Barlow, Jr., unpub. obs.). Also, the Limulus visual system possesses a circadian clock which maintains an accurate record of seasonal changes in photope- riod (Barlow, 1982). Lunar phase Mating activity of a variety of intertidal invertebrates is associated with the lunar syzygies (Klapow, 1972; Enright, 1975; Neumann, 1976; Christy, 1978; Zucker, 1978; Saigusa, 1981). Limulus appears to be no exception: mating activity was maxi- mal during new and full moons throughout the season. In both 1 984 and 1985, Limu- lus began migrating to the beach after the full moon, in early May. In 1984, mating activity increased significantly in the last week of May as new moon approached, decreased rapidly in early June after the new moon, and then peaked twice again at the full and new moons in June. No activity was detected during the full moon in July. The pattern of mating activity in 1985 was similar in every respect to that in 1984 except that the season began about 10 days earlier in the year. This shift coin- cides exactly with the earlier appearance of the new moon in 1 985. Two previous studies report that Limulus mating activity on the Gulf Coast of Florida was also coordinated with new and full moons. In contrast to our results, both studies found maximal mating activity associated with full moons. Rudloe (1980) counted Limulus appearing at Mashes Sand, Florida, between March and November, 1977. Mating apparently began with the new moon of 19 March. Peak numbers of animals were observed in association with high tides between early April and early June. Although daytime tides were monitored throughout, night tides were only monitored from 18 May. Rudloe reported maximal mating during high tides at full moons, with no breeding during neap tides. However, substantial mating also occurred during new moons, especially at the beginning of the season when overall numbers were largest. Cohen and Brockmann (1983) also observed more animals in association with the full moon at Seahorse Bay, Florida, between 12 July and 10 September 1980. The reasons for the differences between our observations and those of Rudloe (1980) and Cohen and Brockmann (1983) are not known. 324 R- B. BARLOW, JR., ET AL. In the nesing area of Mashnee Dike, unpaired males far outnumber paired males (Table IV -suit is consistent with observations in Florida (Rudloe, 1 980; Cohen and Br n n, 1 983). As these authors also noted, many males mill about a nesting pair ;iitly attempting to dislodge the clasping male or to get close enough to depos. ; ,perm where they might be effective. Although in our studies satellite males never dislodged a clasping male (see also Cohen and Brockmann, 1983), they did release sperm around the nesting pair and in the nest itself after the pair had left. Since fertilization is external, sperm release by satellite males may increase the possi- bility that their sperm will fertilize some eggs. Tide height Limulus migrated in great numbers into the intertidal zones during new and full moons, but only on high tides (Figs. 3, 4, 5). None migrated toward shore during low tides. Although significant numbers of animals mated on neap tides at the lunar quadrature, maximal activity was always observed during the highest high tides asso- ciated with the new and full moons. Numerous species synchronize their mating ac- tivity in the intertidal zone with the tides (for reviews see DeCoursey, 1976; Naylor, 1976; Neumann, 1981). For example, grunion bury fertilized eggs at the high-water mark during spring high tides and the larvae hatch about two weeks later when the next series of spring tides washes the upper beach (see Neumann, 1981). Limulus appears to have adapted the same strategy in Florida (Rudloe, 1980). In other areas along the East Coast of North America, however, Limulus nests subtidally in up to 1 m of water (see Cohen and Brockmann, 1983). Indeed, we observed nests being built in areas of the intertidal zone that are inundated by high tides twice a day every day during the lunar month (see Table I). The preferred region for nesting moved away from the high-water mark as the season progressed (Fig. 2). The location of nesting sites within the intertidal zone may be related to the relative risks of predation in different geographical regions and seasonal changes in such risks in a given region. In this regard, it is interesting that geographically separate populations of Clunio, a ma- rine insect, appear to have adapted to the local conditions of the semimonthly tidal regime (see Neumann, 1976). Semidiurnal inequality of tide height Mating activity on Mashnee Dike was directly related to the tidal inequality (Fig. 8). Most Limulus individuals (77%) appeared in the transects on the higher high tides which occurred between 1530 and 0330 h (Figs. 5, 9). The greatest mating activity was observed on the highest spring tides in the early evening ( 1 800 to 2400 h) during the new and full moons. In sum, tide height is a powerful predictor of mating activity at Mashnee Dike. A study along the Gulf Coast of Florida also reported a positive correlation be- tween the number of Limulus mating and the highest high tides (Cohen and Brock- mann, 1983). However, another Florida study in a nearby area reported the opposite result: more mated on the lower high tides (Rudloe, 1980). Cav; taugh (1975) reported that Limulus mated only after dark at Mashnee Dike. Our study does not support this conclusion. From 20 May to 3 June, half of the animals were counted in the transects during darkness (2100 to 0330 h), and half were counted d uring light. We conclude that Limulus generally favor the highest high tides whether they occur during the day or at night. Coordination of behavior with the inequality of tide height is not unique to Limu- lus. In an elegant laboratory study, Enright ( 1 972) showed that the endogenous loco- MIGRATION OF MATING LIMULUS 325 motor activity of the intertidal isopod Excirolana chiltoni was directly correlated with the height of the daily high tides in its natural habitat along the shores of southern California. The region has a semidiurnal tidal regime of the mixed type, and the endogenous activity of the isopod in isolation was maximal during the higher high tide when the tidal inequality was also maximal. When Figures 1 and 2 of Enright's paper are replotted in the format of Figure 8 of the present paper, their similarity is striking: the ratio of Excirolana locomotor activity on consecutive high tides appears to be directly related to the ratio of tide heights. Activity decreased as the tidal in- equality decreased, and increased again as the inequality increased. This circasemilu- nar rhythm is endogneous in Excirolana, but it may not be in Limulus. Under diurnal lighting Limulus occasionally exhibits nocturnal locomotor activity in the laboratory, and under constant darkness the locomotor activity sometimes exhibits an endoge- nous rhythm (T ~ 24 h) for several days (Barlow and Palfai, 1971; J. Turnbull and R. B. Barlow, Jr., unpub. obs.). We have never observed an endogenous lunar or semilunar rhythm for Limulus under laboratory conditions. How does Limulus detect the tidal inequality? A variety of cues are potentially available, particularly when the inequality is large. Indeed, the degree of preference for the highest tides was maximal around the times of maximal tidal inequality. How- ever, as the tidal inequality diminished near lunar quadrature, more animals began to migrate to the intertidal zone during the lower high tides. This behavior would be understandable if the discrimination between the two high tides became progressively more difficult as their heights approached equality. The phase lag of 2-3 days between tidal inequality and animal inequality suggests that Limulus persisted in migrating to the beach on alternate high tides until some environmental factor signaled them to shift to the opposite high tide. As discussed below, we believe the signal to shift tides may be the first light of dawn. Possible role of sunlight Three aspects of our results suggest that Limulus may use only environmental cues to synchronize its shoreward migration with tidal inequalities. ( 1 ) As already noted, there is a 2-3 day phase lag between the reversal of the tidal inequality and the shift of animal preference to the opposite high tide (Fig. 7). (2) With few excep- tions, the maximum number of animals observed in association with a high tide oc- curred after tide crest (Fig. 5). The exceptional tides were all close to dawn when the animals' preference for one high tide over the other was changing. To see this, com- pare the early morning distribution of 24 May with the afternoon distribution of 23 May in Figure 5. Note that near dawn the maximum number of animals occurred before tide crest. (3) On several occasions we observed a mass exodus of animals from the nesting area as the first light of dawn appeared on the horizon. The rapid movement of animals away from shore was quite unlike the normal decline of mating activity observed on ebbing tides at other times of the day. We suggest that the first light of dawn may serve as a cue for the animals to shift to the opposite high tide after it has become the higher tide. The Limulus visual system is well equipped to detect the first light of dawn. In the early hours of the morning, the animals' visual sensitivity begins to undergo an endogenous transition from a highly sensitive nighttime state to a much less sensitive daytime state (Barlow, 1983). Although visual sensitivity is declining, it can still de- tect single photons (Kaplan and Barlow, 1976). Another important factor may be the massive turnover of photoreceptor membrane which is triggered at dawn by the first rays of light (Chamberlain and Barlow, 1 979, 1 984). Within minutes about 70% of the photosensitive rhabdom structure of every photoreceptor cell is torn down, leaving a 326 R B. BARLOW, JR., ET AL. small vo!?: jodopsin-containing membrane to transduce light, and after one half hour th dom structure is fully reassembled. Although the process of rhab- dom reot: is analogous to the outer segment renewal of vertebrate rods and cones (Ye /6), its function is not understood. However, such a massive metabolic eveni may disrupt vision, which males use to locate potential mates (Barlow et al.. 198 The change in migratory behavior near dawn may be triggered by a daily environ- mental event other than dawn. One possibility is the solar component of the tide, but an analysis of tidal components shows that the solar component does not peak near dawn at Mashnee Dike (R. Gregory-Allen, pers. comm.). The relationship between migratory behavior, dawn, and tide could be further tested by observing a population that nests in an area with a different tidal regime. Several such areas exist near Woods Hole but thus far we have not found one with a mating population of sufficient size. Our data are consistent with the idea that the animals prefer the highest tides but are incapable of detecting the reversal of tide heights perhaps because the difference in tide height is smallest when the reversal occurs. Because the tide reversal at Mashnee Dike always occurs before dawn (0300 h), we suggest that the animals stay with this early morning tide until it occurs after dawn, 2-3 days later. Then they shift their shoreward migration to the higher high tide. They continue to follow the afternoon high tide as it progresses through the night until it again occurs after dawn. Such a strategy does not require an endogenous rhythm of the sort detected in the isopod by Enright (1972). However, it does require information about the timing of the two flooding tides. An endogenous circatidal oscillator would provide this infor- mation, but studies with other animals indicate that exogenous factors such as water flow and bottom vibration alone are sufficient (Hastings, 1981; Neumann, 1978). It is also possible that Limulus possesses an hour-glass timing system which signals the animal at a fixed interval after a preceding event. Such hour-glass timing systems have been detected in other animals (Neumann, 1981). They do not require an en- dogenous oscillator. Summary of Limulus mating activity in 1984 Figure 10 summarizes our findings during the 1984 mating season at Mashnee Dike. It shows the heights of the two high tides as "paths" through time. Limulus symbols indicate which high tide attracted the majority of animals on any given day. At the beginning of the season we counted more animals during the higher tide that occurred in darkness. The animals continued to prefer that tide until after the tidal reversal on 23 May, changing to the higher tide on 25 May (Fig. 7). On this day both tides occurred in daylight. Once the animals switched to the afternoon tide they again continued to prefer this tide until after the tidal reversal on 6 June, switching their preference after both high tides occurred during the day. This pattern was re- peated following the final tidal reversal of the season on 21 June. The animals thus showed a clear preference for the higher tide: more were seen in association with the higher tide in 44 of 5 1 observation intervals (lunar days). The seven exceptions oc- curred after the three reversals in tide height. One interpretation is that the first light of dawn signaled the time to switch to the opposite tide. A model for the regulation of mating activity in Limulus From the evidence presented here, and our knowledge about the visual system of Limulus polyphemus, we propose the following model for the regulation of mating activity in this species. The initiation of mating activity at the beginning of the season MIGRATION OF MATING LIMULUS 327 m o n m CM o CM c 3 ~3 o eo m CVJ o - CM - in CD in in l> T3 ^ If || o .S *i ni a - 'S ">> :s.s o c i o s ^ s !a>^ -C JS . 4J <- S? C ;> O 3 fa CO O ^ ^ J*s 13 "O >> o (|dA9| eas ueaiu dAoqe jeej) m6;9ij eo co a OS - oo-O S c 5 w .2-a > c CO CO ^MJ 1J 2 -o .|| 6 <*- (U "S o - x> ^c E CO '.Q > E S-o E v V) OT Jj ' -S S U eo "3 01 a g D >. O -o ^ 328 R. B. BARLOW, JR., ET AL. is determine^ by changes in daylength and/or water temperature that occur in the spring. D , P. J., ed. 1976. Biological Rhythms in the Marine Environment. University of South Carolina Press, Columbia. Eldridge Tide and Pilot Book. 1984. Robert Eldridge. White, Publisher. Boston, MA. ENRIGHT, J. T. 1972. A virtuoso isopod. Circa-lunar rhythms and their tidal fine structure. /. Comp. Physio!. IT. 141-162. MIGRATION OF MATING LIMULUS 329 ENRIGHT, J. T. 1975. Orientation in time: endogenous clocks. Pp. 917-944 in Physiological Mechanisms Vol. II, Part 2, 0. Kinne, ed. John Wiley & Sons, London. HARTLINE, H. K. 1969. Visual receptors and retinal interaction. Pp. 242-259 in Les Prix Nobel en 1967, The Nobel Foundation. HASTINGS, M. H. 1981. The entraining effect of turbulence on the circatidal activity rhythm and its semi- lunar modulation in Eurydice pulchra. J. Mar. Biol. Assoc. U. K. 61(1): 151-160. HOWARD, H. A., R. W. FIORDALICE, M. D. CAMARA, L. KASS, M. K. POWERS, AND R. B. BARLOW, JR. 1984. Mating behavior of Limulus: relation to lunar phase, tide height and sunlight. Biol. Bull. 167: 527. KAPLAN, E., AND R. B. BARLOW, JR. 1976. Energy, quanta, and Limulus vision. Vision Res. 16: 745-75 1 . KLAPOW, L. A. 1972. Fortnightly molting and reproductive cycles in the sandbeach isopod Excirolana chiltom. Biol. Bull. 143: 568-591. NAYLOR, E. 1976. Rhythmic behavior and reproduction in marine animals. Adaptation to the Environ- ment: Essays on the Physiology of Marine Animals, R. C. Newell, ed. Butterworths, London. NEUMANN, D. 1976. Adaptations of chironomids to intertidal environments. Ann. Rev. Entomol. 21: 387- 414. NEUMANN, D. 1978. Entrainment of a semi-lunar rhythm by simulated tidal cycles of mechanical distur- bance. J. Exp. Biol. Ecol. 35(1): 73-86. NEUMANN, D. 1981. Tidal and lunar rhythms. In Handbook of Behavioral Neurobiology, Vol. 4: Biological Rhythms, J. Aschoff, ed. Plenum Press, New York. POWERS, M. K., AND R. B. BARLOW, JR. 1985. Behavioral correlates of circadian rhythms in the Limulus visual system. Biol. Bull. 169: 578-591. RATLIFF, F., ed. 1974. Studies on Excitation and Inhibition in the Retina, a Collection of Papers from the Laboratory ofH. K. Hart line. Rockefeller Press, New York. RUDLOE, A. 1978. Some ecologically significant aspects of the behavior of the horseshoe crab, Limulus polyphemus. Ph.D. thesis, The Florida State University, Tallahassee, Florida. RUDLOE, A. 1979. Limulus polyphemus: A review of the ecologically significant literature. Pp. 27-35 in Biomedical Applications of the Horseshoe Crab (Limulidae), E. Cohen, ed. Alan R. Liss, Inc. New York. RUDLOE, A. 1980. The breeding behavior and patterns of movement of horseshoe crabs, Limulus polyphe- mus, in the vicinity of breeding beaches in Apalachee Bay, Florida. Estuaries 3: 1 77- 1 83. RUDLOE, A., AND W. F. HERRNKJND. 1976. Orientation of Limulus polyphemus in the vicinity of breeding beaches. Mar. Behav. Physiol. 4: 75-89. SAIGUSA, M. 1 98 1 . Adaptive significance of a semilunar rhythm in the terrestrial crab Sesarma. Biol. Bull. 160:311-321. SCHUSTER, C. N., JR. 1957. Xiphosura (with special reference to Limulus polyphemus). Geol. Soc. Am. Mem.61: 1171-1174. SCHUSTER, C. N., JR. 1958. On morphometric and serological relationships within the Limulidae with particular reference to Limulus polvphemus (L.). Ph.D. dissertation abstract, New York Diss. Abstr. 18:11 1-312. SCHUSTER, C. N., JR. 1979. Distribution of the American horseshoe "crab," Limulus polyphemus (L.). Pp. 3-26 in Biomedical Applications of the Horseshoe Crab (Limulidae), E. Cohen, ed. A. R. Liss, New York. U. S. ARMY CORPS OF ENGINEERS, New England Division Cape Cod Canal Field Office, Buzzards Bay, Massachusetts. 1983. Cape Cod Canal 1984 Tide Tables. U. S. Government Printing Office 198360001410. WYLIE, F. E. 1979. Tides and the Pull of the Moon. The Stephen Greene Press, Brattleboro, Vermont. YOUNG, R. W. 1976. Visual cells and the concept of renewal. Invest. Ophthalmol. 15: 700-725. ZUCKER, N. 1978. Monthly reproductive cycles in three sympathetic hood-building tropical fiddler crabs (genus Uca). Biol. Bull. 155: 410-424. Reference: Biol. Bud. 171: 330-337. (October, 1986) NESIUM DEPENDENCE OF ENDOTOXIN-INDUCED DEGRANULATION OF LIMULUS AMEBOCYTES GAIL W. CANNON, MASAKAZU TSUCHIYA*, DAN RITTSCHOF, AND JOSEPH BONAVENTURA Marine Biomedical Center, Duke University Marine Laboratory, Fivers Island, Beaufort, North Carolina 28516, and *Wako Pure Chemical Industries, Ltd., Osaka Research Laboratory, Hyogo, 661 Japan ABSTRACT Amebocytes are blood cells that function in the defense system of the horseshoe crab Limulus polyphemus. When they are withdrawn from the animal, they flatten and degranulate within hours. We have found, however, that when a small amount of blood is drawn into a syringe containing a large amount of 3% NaCl, free of diva- lant cations, and further diluted (to a final concentration of approximately 1:500) in 3% NaCl into tissue-culture-treated plates, amebocytes do not flatten or degranulate, even in the presence of endotoxin. This dilution technique was used to determine direct effects of Ca ++ and Mg ++ in the flattening and degranulation of amebocytes. Ca ++ or Mg ++ added at normal hemolymph concentration to 3% NaCl caused rapid flattening. Degranulation did not occur for several days. In the presence of nanogram quantities of endotoxin, cells in Ca ++ fortified saline remained granulated, while those in Mg ++ fortified saline completely degranulated within several hours. Experiments with EGTA and EDTA confirmed that Ca ++ is not essential for degran- ulation of amebocytes in the presence of serum. Although Ca ++ and Mg ++ are both influential in the in vitro flattening of Limulus amebocytes, Mg ++ is essential for the process of degranulation. INTRODUCTION Granular amebocytes are the major blood cells of Limulus polyphemus and play an important role in the defense system. In the presence of endotoxin, factors of the extracellular gelation system are released from amebocytes. The resultant extracellu- lar gel presumably immobilizes bacteria (Bang, 1956; Levin and Bang, 1964, 1968; Levin, 1976; Armstrong and Rickles, 1982). In the absence of endotoxin, amebocytes in culture are motile (Armstrong, 1977, 1979, 1980) and are able to phagocytose (Armstrong and Levin, 1978). In the absence of anti-aggregating chemicals such as JV-ethylmaleimide (Solem, 1 970), EDTA (Kenney et at, 1 972; Armstrong, 1 980) pro- pranolol (Murer et al, 1975), and methylxanthine derivatives (theophilline, caffeine, theobromine) (Kobayachi and Yamamoto, 1974), amebocytes in vitro flatten and degranulate (Armstrong and Levin, 1978; Armstrong 1977, 1979, 1980). In order to understand better the activation of amebocytes, a modified dilution technique that prevents flattening and degranulation of amebocytes in vitro without the necessity for the addition of anti-aggregating chemicals was developed. Using this technique, we observed the independent effects of Mg ++ and Ca ++ on flattening and endotoxin- induced degranulation of amebocytes. We found that, although both Mg ++ and Ca + Received 13 May 1986; accepted 28 July 1986. 330 MG ++ IN LIMULUS AMEBOCYTE DEGRANULATION 331 caused flattening of amebocytes, only Mg ++ was essential for endotoxin-induced de- granulation. MATERIALS AND METHODS Animals Limulus polyphemus were obtained throughout the year by trawl from high salin- ity waters (ranging near 1000 kOs) from 10C to 29C within three miles of the Beau- fort Inlet on the eastern shore of North Carolina. The animals were maintained in outdoor, tidal-exchanged, natural-bottomed, flow-through pens, (25 feet by 25 feet, containing a maximum of 200 animals at any given time), where they were able to feed at will. No exogenous food was added. One to three days before experimentation, the animals were moved to indoor tanks of flowing seawater and maintained unfed until use. Reagents Water used in experiments was sterile for injection (Abbott Laboratories, North Chicago, Illinois). Endotoxin was Escherichia coli UKT-B lipopolysaccharide (WAKO Pure Chemical Industries, Ltd., Osaka, Japan). Potency of the endotoxin was confirmed with U.S.P. Reference Standard Endotoxin EC-5 by the Limulus Amebocyte Lysate test. The potency of this preparation was 25 EU/ng. One thousand EU of this preparation contained less than 0.005 ^g of Mg ++ and less than 0.02 ^g of Ca ++ as measured by atomic absorption. Sterile Trypan Blue (Gibco, Grand Island, Maine) was made isotonic by addition of sodium chloride. Other chemicals were reagent grade. Inorganic chemicals were rendered pyrogen-free by baking at 1 80C- 2lOCfor4h. Equipment Sterile Linbro tissue-culture-treated, multi-well plates, (Cat. No. 76-033-05), ster- ile Linbro non-tissue-culture-treated, multi-well plates (Cat. No. 76-258-05, Flow Laboratories, McLean, Virginia), plastic, 60 X 1 5 mm non-tissue-culture-treated Pe- tri dishes (Falcon #1007, Oxnard, California), 16 gauge, 25.4 mm needles (#5197), and 60 cc Plastipak syringes (#5663) (Becton-Dickenson, Rutherford, New Jersey) were used. Glassware, spatulas, and forceps were rendered pyrogen-free by baking at temperatures of 180C-210C for 4 h. Differential interference contrast, phase, and brightfield photomicrography was employed; results were recorded on Kodak Pana- tomic-X 32 film (Eastman Kodak, Rochester, New York). Bleeding animals To obtain amebocytes, we cleansed the flexure of the animals' prosoma and opis- thosoma with 70% ethanol and withdrew 1 to 2 ml of hemolymph by cardiac punc- ture with sterile 16 g needles attached to sterile 60 cc syringes containing 49 ml of pyrogen-free 3% NaCl. Preparation of serum Bulk hemolymph for serum preparation, obtained by cardiac puncture with a 1 3 gauge needle, was allowed to clot overnight at 4C and was centrifuged at 2000 rpm 332 G. W. CANNON ET AL. for 1 h to remove clotted amebocytes and cellular debris. Serum was stored until use at4C. P; of amebocytes Amebocytes were prepared for tissue culture experimentation as follows. Immedi- ately after bleeding, approximately 50 to 100 ^1 hemolymph, diluted 1:50 with 3% NaCl, were added dropwise to tissue-culture-treated, multi-well plates containing an additional ml of 3% NaCl. Final cell concentration, determined by direct cell count with a hemocytometer, was approximately 4 to 5 X 10 4 per ml. Cells settled undis- turbed for at least 1 h before experimentation. Amebocytes were prepared for photomicrography by placing pyrogen-free cover- slips in plastic Petri dishes. Cells settled onto the coverslips, which then could be removed for more effective photomicrography. Surface effects and viability study To determine the effects of various surfaces on amebocytes, cultures in 3% saline were tested on tissue-culture-treated, multi-well plates, non-tissue-culture-treated plastic Petri dishes, glass Petri dishes, and siliconized-glass Petri dishes. Trypan blue was used as a viability stain (Merchant et al, 1964). Cells were placed in 3% NaCl into all the wells of a tissue-culture-treated, multi-well plate. Cells were kept unfed at ambient temperature and conditions. One well of cells per day was tested for viability. Ca ++ andMg ++ studies Amebocytes were placed into multi-well, tissue-culture-treated plates as above. After the cells had settled, supernatant was removed with a sterile pipet and was re- placed with isotonic saline containing calcium chloride at 0.24 mM lo 15.0 mM or magnesium sulfate at 3.30 mMto 75 mM. Endotoxin was added at final concentra- tions of 10-2500 EU/ml to cells at all of the concentrations of Mg ++ and Ca ++ stud- ied. Within 24 h of addition of Mg ++ and Ca ++ , coverslips with experimental cells were removed from dishes and placed uncovered on slides. Results were recorded by photomicrography with differential interference contrast optics. EDTA and EOT A studies Amebocytes were placed into tissue-culture-treated, multi-well plates. EDTA at 20 mM and 40 mM or EOT A at 5 mM and 10 mM was added to the serum. NaCl was removed from prepared cells and replaced with serum alone or serum containing either EDTA or EGTA at the above concentrations. Results were recorded within 24 h by photomicrography. RESULTS Development of dilution technique Substrates were tested for their effects on amebocyte morphology. Amebocytes attached to tissue-culture-treated, multi-well plates but did not flatten or degranulate MG ++ IN LIMULUS AMEBOCYTE DEGRANULATION 333 * e - o FIGURE 1 . Effects of surfaces on Limulus amebocytes cultured for 1 h in 3% NaCl. (a) Sterile Linbro tissue-culture-treated, multi-well plates; cells are ovoid and granulated, (b) Sterile Linbro non-tissue-cul- ture-treated, multi-well plates; intact granules released from lysed cells. Brightfield photomicrographs, X200. until death (Fig. la). Trypan blue exclusion tests indicated that these cells remained alive for approximately one week. In non-tissue-culture-treated plastic dishes, many cells disintegrated upon contact with the surface and others attached but contracted and became generally smaller and more rounded. Granules from cells that disinte- grated remained intact (Fig. Ib). On siliconized glass surfaces, amebocytes attached and contracted with some disintegration similar to non-tissue-culture-treated plastic plates. Cells on non-siliconized glass Petri dishes attached and remained ovoid with some slight contraction and rounding similar to those in tissue-culture-treated, multi- well plates. Tissue-culture-treated, multi-well plates were, therefore, used experimen- tally because they affected cellular morphology least. Seasonal variation was observed in the reactivity of amebocytes and their responses to surfaces. During summer months, amebocytes appeared to respond to stimuli (se- rum or Mg ++ ) more quickly and looked "healthier" (more regularly shaped) than did amebocytes obtained during winter months. This phenomenon did not alter the re- sults reported. Experiments Amebocytes cultured in 3% NaCl remained ovoid and granulated. Amebocytes cultured in 3% NaCl and exposed to 50 mA/Mg ++ (Fig. 2a) flattened completely and remained granulated for 24 h. The process of flattening is begun in the presence of 6.25 mM Mg ++ and is complete at concentrations of 25 mM Mg ++ and above. A similar reaction was observed with 10 mM Ca ++ . The process of flattening is begun in the presence of 0.5 mA/Ca ++ and is complete at concentrations of 10 mMCa ++ 334 G. W. CANNON ET AL. b FIGURE 2. Effects of endotoxin on Limulus amebocytes cultured for 6 h in 3% NaCl containing 50 mM Mg ++ on glass coverslips. (a) 3% NaCl containing 50 roM Mg ++ without endotoxin; cells are flat and granulated, (b) 3% NaCl containing 50 mM Mg ++ in the presence of 125 EU/ml endotoxin; cells are flat and degranulated. Differential interference contrast photomicrographs, X400. and above. Various transitional stages of flattening are seen between 6.25 and 25 mM Mg ++ and between 0.5 mM and 10 mMCa ++ . Endotoxin at 2500 EU/ml added to amebocytes cultured in 3% NaCl did not cause degranulation. Amebocytes remained ovoid and granulated. When 125 EU/ ml endotoxin were added to amebocytes in the presence of 50 mM Mg ++ , degranula- tion was complete within 24 h (Fig. 2b). Mg ++ -facilitated degranulation as a function of endotoxin concentration is shown in Table I. The effects of varying concentrations of Mg ++ in the presence of constant amounts of endotoxin are shown in Table II. The amount of endotoxin necessary to produce degranulation was dependent on Mg ++ concentration. A minimum of 25 mM Mg ++ was required for the degranula- tion of amebocytes in the presence of 2500 EU/ml of endotoxin. When endotoxin in concentrations ranging from 10 to 2500 EU/ml was added to amebocytes in the pres- ence of 0.1 to 15 mA/Ca ++ , the amebocytes remained granulated. TABLE I supported degranulation of amebocytes in the presence of endotoxin Endotoxin (EU/ml) Mg ++ (mM) 0.0 15.6 31.2 62.5 125 250 1000 2500 + 50 + = granuiated. = partially granulated, - = degranulated. MG ++ IN LIMULUS AMEBOCYTE DEGRANULATION 335 TABLE II Endotoxin-induced degranulation of amebocytes in the presence ofMg ++ l ++ (mM) Endotoxin (EU/ml) 0.0 0.5 3J 62 12.5 25.0 50.0 + + + + + + + 1000 + + + + 2500 + + + - -_ + = granulated, = partially granulated, = degranulated. Amebocytes cultured in 10 to 100% Limulus serum flattened and also degranu- lated within 24 h. EDTA at 20 mM and 40 mM in hemolymph serum prevented amebocytes from flattening and degranulating. However, amebocytes exposed to 5 mM and 10 mM EGTA-supplemented serum flattened and degranulated within 24 h. DISCUSSION Amebocytes, when placed on negatively charged surfaces, (glass and tissue-cult- ure-treated, multi-well plates), in 3% NaCl, attached but retained the in vivo charac- teristics of ovoid shape and granule retention for one week (Fig. la). The phenome- non of amebocytes disentegrating with intact granule release when placed on hydro- phobic surfaces (siliconized glass and non-tissue-culture-treated, plastic dishes) (Fig. Ib) may be a result of interaction between these surfaces and cell membranes. The technique of obtaining amebocytes with few morphological changes in the absence of anti-aggregating chemicals will be useful for future studies on amebocyte activation. Partial flattening and spontaneous degranulation of amebocytes on artificial sur- faces have been reported (Armstrong, 1980). Divalent cations and unknown hemo- lymph components have been considered to be causal factors in these cellular modi- fications (Armstrong, 1980). In this study, flattening of amebocytes cultured in 3% NaCl resulted from the addition of Mg ++ , Ca ++ , or hemolymph serum. Spontaneous degranulation followed the addition of hemolymph serum. To prevent or delay these reactions without anti-aggregating chemicals, immediate dilution by a factor of at least 500 was necessary. Amebocytes obtained by this method, although not in an in vivo state, had not begun the processes of flattening and degranulation. Amebocytes cultured in 3% NaCl began to flatten in the presence of greater than 12.5 mM Mg ++ or 1.0 mM Ca ++ . In the absence of endotoxin these amebocytes re- tained their granules for more than 24 h. Endotoxin causes the gelation of Limulus hemolymph (Levin and Bang, 1964). Gelation follows the exocytosis of amebocyte granules, which contain all of the fac- tors of the gelation system (Murer et al, 1975; Ornberg and Reese, 1981; Armstrong and Rickles, 1982). No triggering mechanism for exocytosis has been reported. As a result of the dilution technique, endotoxin could be added in amounts as great as 2500 EU/ml with no resultant degranulation of amebocytes cultured in 3% NaCl. This failure to degranulate indicates that degranulation requires the presence of a factor other than endotoxin. Our studies indicate that Mg ++ is that factor. In the presence of 50 mM Mg ++ , degranulation occurred rapidly among amebocytes when more than 125 EU/ml endotoxin were added (Table I). Higher concentrations of 336 G. W. CANNON ET AL. endotoxin required less Mg ++ for degranulation to occur (Table II). Degranulation of amebocytes did not occur in the presence of calcium even at a concentration of 2500 I 1 endotoxin. Therefore, Mg ++ , but not Ca ++ , is essential for endotoxin- inducc aegranulation. Although magnesium-dependent exocytosis may not be un? iature, we have found no similar reports. This presents a challenge to examine more closely the role of Mg ++ as a trigger for biological events. The observation that EDTA inhibited spontaneous degranultion of amebocytes in serum and EGTA did not inhibit degranulation supports the assertion that Ca +H is not necessary for the degranulation of amebocytes. EDTA chelates both Mg ++ and Ca ++ while EGTA chelates Ca ++ but does not chelate Mg ++ . Spontaneous degranula- tion occurs within hours in amebocytes cultured in hemolymph (Armstrong, 1980, 1982). When cells were cultured by the dilution technique, complete degranulation was delayed. Eventual degranulation may be due to residual amounts of Mg ++ and other serum factors added as part of the original innoculum. Aggregation of amebocytes and flattening and degranulation of amebocytes are separable processes. There is a serum factor that causes aggregation even in the pres- ence of EDTA (Kenney et al, 1972). Our observations indicate that EDTA inhibits both flattening and degranulation even in the presence of hemolymph that contains this aggregating factor. Limulus hemolymph contains 46 mMMg ++ and 10 mM Ca ++ (Prosser, 1973), sufficient amounts to cause flattening of amebocytes in culture. However, amebocytes not only flatten but also degranulate in the presence of serum even in the absence of endotoxin; and since Mg ++ and Ca ++ do not cause degranulation, the presence of a "degranulation-promoting" factor in hemolymph serum is indicated. It appears that spontaneous degranulation, described by Armstrong ( 1 982), is caused by this "degranulation-promoting" factor present in hemolymph serum. This factor may be released from tissues or amebocytes when Limulus has been injured or irritated. Our observations that endotoxin in the presence of Mg ++ causes degranulation and that degranulation occurs in the presence of serum in the absence of endotoxin suggest two possible series of events. The degranulation-promoting factor may have binding sites analogous to those of the lipopolysaccharide molecule and, therefore, may activate amebocytes to degranulate through the same pathway or mechanism. Alternatively, there may be two separate mechanisms that initiate amebocyte degran- ulation. One pathway may involve the reaction of Mg ++ with lipopolysacharride lead- ing to degranulation since the presence of Mg ++ appears to be necessary for degranu- lation to occur. A second pathway may involve separate reactions, involving the de- granulation-promoting factor, that may lead to a link with the lipopolysaccharide/ Mg ++ pathway. Further research is needed to test these hypotheses. Another area of possible future research involves the question of whether Mg ++ acts intracellularly or extracellularly. ACKNOWLEDGMENTS orted by Wako Pure Chemical Industries, Ltd. and NIH Grant ESO-1908. LITERATURE CITED ARMSTRONG . B. 1977. Interaction of the motile blood cells of the horseshoe crab, Limulus. Studies on contact pseudopodial activity and cellular overlapping in vitro. Exp. Cell Res. 107: 127-138. ARMSTRONG, P. B., AND J. LEVIN. 1978. In vitro phagocytosis by Limulus blood cells. / Invert. Pathol. 34: 145-151., MG ++ IN LIMULUS AMEBOCYTE DEGRANULATION 337 ARMSTRONG, P. B. 1979. Motility of the Limulus blood cell. J. CellSci. 37: 169-180. ARMSTRONG, P. B. 1980. Adhesion and spreading of Limulus blood cells on artificial surfaces. /. Cell Sci. 44: 243-262. ARMSTRONG, P. B., AND F. R. RJCKLES. 1982. Endotoxin-induced degranulation of the Limulus amebo- cyte. Exp. Cell Res. 140: 15-24. BANG, F. B. 1956. A bacterial disease of Limulus polyphemus. Bull Johns Hopkins Hosp. 98: 325-35 1. KENNEDY, D. M., F. A. BELAMARICH, AND D. SHEPRO. 1972. Aggregation of horseshoe crab (Limulus polyphemus) amebocytes and reversible inhibition of aggregation by EDTA. Biol. Bull. 143: 548- 567. KOBAYASHI, M., AND M. YAMAMOTO. 1974. Preparation of pre-gel from the amebocytes of Japanese horseshoe crab (Tachvpleus tridentatus) with the anti-aggregating effect of methylxanthines. II. Yakugaku Zasshi 94(3): 198-303. LEVIN, J., AND F. B. BANG. 1964. The role of endotoxin in the extracellular coagulation of Limulus blood. Bull. Johns Hopkins Hosp. 115: 265-274. LEVIN, J., AND F. B. BANG. 1968. Clottable protein in Limulus: its localization and kinetics of coagulation by endotoxin. Thromb. Diath. Haemorrh. 19: 186-197. LEVIN, J. 1976. Blood coagulation in the horseshoe crab (Limulus polyphemus): a model for mammalian coagulation and hemostasis. Pp. 87-102 in Animal Model of Thrombosis and Hemorrhagic Dis- eases. DHEW Publication No. (NIH) 76-982, Washington, DC. MERCHANT, D. J., R. H. KAHN, AND W. H. MURPHEY, JR. 1964. Handbook of Cell and Organ Culture. Pp. 155-210. Burgess Publishing Co., Minneapolis. MURER, E. H., J. LEVIN, AND R. HOLME. 1975. Isolation and studies of the granules of the amebocytes of Limulus polyphemus, the horseshoe crab. J. Cell Physiol. 86: 533-542. ORNBERG, R. L., AND T. S. REESE. 1981. Beginning of exocytosis captured by rapid-freezing of Limulus amebocytes. / Cell Biol. 90: 40-54. PROSSER, C. L. 1973. Comparative Animal Physiology, 3rded. p. 85. Saunders, Philadelphia. SOLEM, N. O. 1970. Some characteristics of the clottable protein of Limulus polyphemus blood cells. Thromb. Diath. Haemorrh. 23: 1 70. Reference: Biol. Bull. 171: 338-349. (October, 1986) 3CIATION OF CENTRIOLES WITH THE MARGINAL BAND IN SKATE ERYTHROCYTES WILLIAM D. COHEN Department of Biological Sciences, Hunter College of C.U.N.Y., 695 ParkAve., New York, New York 10021, and the Marine Biological Laboratory, Woods Hole, Massachusetts 02543 ABSTRACT Centrioles are associated with the marginal bands (MBs) in certain invertebrate erythrocytes, functioning as organizing centers for MB reassembly (Nemhauser et al, 1983). However, a similar association has not been observed previously in erythro- cytes of vertebrates. Detergent-lysed erythrocytes ("cytoskeletons") of the skate Raja erinacea contain centrioles visible as paired dense dots in phase contrast. In uranyl acetate-stained whole mounts and in thin sections (TEM) they exhibit typical right- angle orientation and 9-triplet ultrastructure. Although the centriole pairs in some cytoskeletons are distant from the MB, surveys of their distribution in preparations from different animals indicates that it is non-random, with the majority adjacent to the MB or less than 1 ^m from it. Many of the centriole pairs appear to be attached to MB microtubules, or have microtubules extending from them toward the MB. In rare instances, pointed cytoskeletons are observed with the centrioles at the apex from which fibers radiate, suggesting a morphogenetic function. The observations support the possibility that centrioles function during MB biogenesis in differentiating verte- brate erythrocytes, with loss of functional location as the cells mature. INTRODUCTION Comparative studies of cytoskeletal structure in blood cells of vertebrates and invertebrates have shown that marginal bands (MBs) of microtubules are prominent components of erythrocytes and clotting cells throughout the animal kingdom (Meves, 1 9 1 1 ; Fawcett and Witebsky, 1964;Behnke, 1970;Goniakowska-Witalinska and Witalinksi, 1976; Cohen and Nemhauser, 1985). In erythrocytes, MBs first ap- pear during cellular morphogenesis and are believed to function in bringing about the transformation from spherical to flattened discoid or elliptical cell shape (Barrett and Scheinberg, 1972; Small and Davies, 1972; Barrett and Dawson, 1974; Yama- moto and luchi, 1975). Recent experiments on vertebrate and invertebrate erythro- cytes indicate that MBs continue to function in mature cells, resisting shape changes and/or restoring cell shape after deformation by external forces (Joseph-Silverstein and Cohen, 1984, 1985). Although the circumferential location of MB microtubules in the plane of cell flattening is of considerable interest with respect to spatial control of microtubule arrays, relatively little is known of the mechanisms involved in MB formation. In mature chicken erythrocytes there may be structural or molecular "tracks" along the inner cell surface which guide the growth of MB microtubules during experimentally Received i 3 June 1986; accepted 30 July 1986. Abbreviations: PIPES = piperazine-n-n'-bis (2-ethane sulfonic acid), EGTA = ethyleneglycol-bis-(b- aminoethyl ether) n n'-tetraacetic acid, TAME = p-tosyl arginine methyl ester HC1, MB = marginal band, SAC = cell surface-associated cytoskeleton, PMSF = phenylmethylsulfonyl fluoride. 338 CENTRIOLES IN SKATE ERYTHROCYTES 339 induced MB reassembly (Granger and Lazarides, 1982; Miller and Solomon, 1984), and in chick bone marrow erythroblasts centrosomes may play a role at an early stage of MB formation (Murphy et al, 1986). In certain invertebrate ("blood clam") erythrocytes, MB-associated centrioles serve as organizing centers during tempera- ture or taxol-induced MB reassembly (Cohen and Nemhauser, 1980; Nemhauser et al, 1983; Joseph-Silverstein and Cohen, 1985), and MB-associated centrioles have also been observed in sea cucumber erythrocytes (Fontaine and Lambert, 1973). However, a similar structural relationship has not been reported previously for ma- ture (circulating) erythrocytes of any vertebrate. The purpose of this paper is to show that, for one vertebrate at least, such an association does exist. In the skate, Raja erinacea, most of the erythrocytes contain readily identifiable centriole pairs, a majority of which are adjacent to the MB. MATERIALS AND METHODS Skates (Raja erinacea} were provided by the Department of Marine Resources of the Marine Biological Laboratory, Woods Hole, Massachusetts. The animals were maintained in running seawater at MBL or in cooled ( 1 0C), aerated tanks of "Instant Ocean" artificial seawater (Aquarium Systems, Inc., Eastlake, Ohio) at Hunter Col- lege. In the latter case "trace elements" (Hawaiian Marine Imports, Inc., Houston, Texas) were added to the water periodically. Small samples of blood were obtained by snipping the tail tip, and larger ones by heart puncture after anesthesthetizing the animals in 0.04% tricaine in seawater. "Cytoskeletons" for routine examination and scoring of centriole location under phase contrast, were prepared by dilution of blood or cell suspensions approximately 1:10 into Triton lysis medium consisting of 100 mM PIPES, 5 mM EGTA, 1 mM MgCl 2 , pH 6.8 (=PEM) containing 10 mM TAME and 0.4% Triton X-100. This medium had been used previously for studies of cytoskeletal structure in blood cells of diverse species (Cohen, 1978; Cohen et al., 1982; Cohen and Nemhauser, 1985). In one experiment, 0. 1% glutaraldehyde was included in the lysis medium to achieve simultaneous lysis and fixation (Cohen and Nemhauser, 1980). Cell morphology was observed in phase contrast in both living cells and cells fixed in Elasmobranch Ring- er's (Cavanaugh, 1975) containing 0.1% glutaraldehyde so as to avoid artifacts due to contact with glass slides and coverslips. Cytoskeleton whole mounts for transmission electron microscopy were prepared on Formvar-coated grids. The Formvar surface was pre-treated with polylysine (1% solution of MW > 400,000, followed by water washes and air drying) to enhance retention of material (Mazia et al., 1975). Cytoskeleton suspensions in Triton lysis medium were placed on grids for 5 min, followed by a wash in PEM and 10 min fixation in PEM containing 2% glutaraldehyde. Subsequently, grids were washed in PEM and in water, stained with 1% aqueous uranyl acetate, and air-dried. Material was prepared for thin sectioning as follows: 0.3 ml packed washed cells were suspended in 6 ml Brij lysis medium, consisting of PEM containing 0.6% Brij 58, 10 mM TAME, and 0.1 mM PMSF (freshly added). Brij was used because it produced less twisting of cytoskeletons than Triton, and also permitted easy resuspen- sion of cytoskeletons. The material was centrifuged for 1 minute at top speed in the International Clinical centrifuge (2250 X g), resuspended as before and centrifuged again. The cytoskeletons were resuspended and fixed 1 h at room temperature in 6 ml PEM containing 2.5% glutaraldehyde. They were sedimented, washed once in 6 ml PEM by resuspension and centrifugation, postfixed for 1 h in 1% OsO 4 in PEM, washed three times in PEM, dehydrated in ethanol, and embedded in Epon. 340 W. D. COHEN FIGURE 1 . Erythrocytes of the skate, Raja erinacea, exhibiting the nucleated, flattened, elliptical morphology typical of all non-mammalian vertebrates (a). On rare occasion, cells with single or double- pointed shape are observed (b). Phase contrast. Thin sections were cut with diamond knives on the Sorvall MT-2 ultramicrotome (DuPont Instruments, Newtown, Connecticut), and stained with saturated uranyl acetate in 50% ethanol followed by Reynold's lead citrate. Whole mounts and thin sections were examined in the Hitachi HS-8 (50 kV) or Zeiss EM IOC (80 kV) trans- mission electron microscopes. RESULTS In both fresh blood samples and samples of washed, fixed cells, the erythrocytes of the skate, Raja erinacea, are found to be morphologically similar to those of most non-mammalian vertebrates (Fig. la). They are nucleated, flattened, and, with rare exception, elliptical, with the long axis in the 20-25 nm range. This is a relatively large size for fish in general, but typical of elasmobranchs as compared with teleosts (Andrew, 1966; Nemhauser et al, 1979). Cells with single or double-pointed shape are rarely observed morphological variants (Fig. Ib). Kate erythrocyte cytoskeletons, prepared by lysis of the cells with Triton X-100 undei editions previously observed to stabilize MBs, are shown in Figure 2. In phase c >ntrast the centrioles, verified as such by electron microscopy (see below), appear as paired dense "dots," and usually they are readily visible in flat (untwisted) cytoskeletons because there is little competing cytoplasmic structure. In many cases the centrioles ar very close to and possibly in contact with the MB. At higher magni- fications, with flattening of cytoskeletons under the coverslip to enhance viewing, CENTRIOLES IN SKATE ERYTHROCYTES 341 FIGURE 2. Skate erythrocyte cytoskeletons (Triton X- 1 00 lysis), as observed in phase contrast under oil immersion. Centrioles appear as paired, phase-dense "dots" (arrowheads), (a) One of the centrioles very close to or in contact with MB; N = nucleus, (b) Centriole pair adjacent to, but not in direct contact with major part of MB; (c) centriole pair appearing to touch MB, with a "fiber" (f) extending away from it. some of the centriole pairs are found to be attached to fibers (microtubule bundles) which are part of the MB, or which extend from the centrioles toward a distant point on the MB (Fig. 3). Confirmation of the attachment of centrioles to the MB, including some which appeared separated from the main body of the MB, was obtained by examination of cytoskeleton whole mounts in TEM. In Figure 4 the centrioles are observed to be attached to only one or possibly a few microtubules of the spread MB. Figure 5 shows centrioles which, in phase contrast, would appear to be close to, but not touching the MB, but which are actually connected to it by radiating microtubules. Examination of the centrioles in underexposed prints revealed their cylindrical shape, microtubu- lar substructure, and sometimes (as in Fig. 5) their approximately orthogonal orienta- FIGURE 3. Cytoskeletons flattened under the coverslip to provide improved higher magnification views of centriole-associated fibers in phase contrast. Such fibers appear to be part of the MB (a, b; arrow- heads), or to extend from at least one of the centrioles toward a distant point on the MB (c; arrowhead). Flattening also generally produced an artifactual increase in area of nucleus, as in b. 342 W. D. COHEN - *' * r% : '.A . - -itS ' i'-: |* . 1 n.m FIGURE 4. Skate erythrocyte cytoskeleton whole mount, uranyl acetate staining, TEM. (a) Survey view; the MB in this region has spread against the substratum; centrioles = ce. The "membrane skeleton," or "cell surface-associated cytoskeleton" (SAC), has collapsed onto the substratum and is visible as a back- ground network, (b) Higher magnification view of centrioles in (a), revealing that the centriole pair is attached to one, or at most a few, MB microtubules. (c) Underexposed print of centrioles in b, in which centriolar substructure can be detected. tion. Thin sections confirmed that they were typical centrioles, about 0.2 X 0.35 with "9 + 0" substructure (Fig. 6). While most cytoskeletons contained centrioles adjacent to or near the MB, in some the centrioles were located between MB and nucleus, while in others they were adjacent to the nucleus. Figure 7 illustrates a case in which the centriole pair was closer to nucleus than to MB. In such cases, radiating microtubules usually were not evident. In all of the whole mounts examined by TEM, the cell "membrane skeleton" or "surface-associated cytoskeleton" (SAC) was visible as a background network throughout the region between nucleus and MB (Figs. 4, 5, 7). wo cytoskeletons out of several thousand examined in phase contrast were poin ; md incomplete at one end, with a curved MB at the other end. In these, the centrio were located within the pointed tip, in a region from which fibers radiated toward ihe closed end of the MB (Fig. 8). Though rare, these cytoskeletons gave the impression that the centrioles were active at one "pole" as organizing centers for a forming MB, Upon casual inspection, the spatial distribution of centrioles appeared to be non- random, with most centriole pairs close to or touching the MB. This was confirmed CENTRIOLES IN SKATE ERYTHROCYTES 343 0.2 FIGURE 5. Skate erythrocyte cytoskeleton whole mount, uranyl acetate staining, TEM. (a) Survey view, the MB in this region is compact (not spread as in Fig. 4), with radiating microtubules connecting the centriole pair to the MB. The cell surface-associated cytoskeletal network (SAC) also is visibl