Volume 173
Number 1
THE
BIOLOGICAL BULLETIN
PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
RUSSELL F. DOOLITTLE, University of California at
San Diego
WILLIAM R. ECKBERG, Howard University
ROBERT D. GOLDMAN, Northwestern University
C. K. GOVIND, Scarborough Campus, University
ofToronto
JUDITH P. GRASSLE, Marine Biological Laboratory
MICHAEL J. GREENBERG, C. V. Whitney Marine
Laboratory, University of Florida
MAUREEN R. HANSON, Cornell University
JOHN E. HOBBIE, Marine Biological Laboratory
LIONEL JAFFE, Marine Biological Laboratory
WILLIAM R. JEFFERY, University of Texas at Austin
GEORGE M. LANGFORD, University of North
Carolina at Chapel Hill
GEORGE D. PAPPAS, University of Illinois at Chicago
SIDNEY K. PIERCE, University of Maryland
HERBERT SCHUEL, State University of New York at
Buffalo
VIRGINIA L. SCOFIELD, University of California at
Los Angeles School of Medicine
LAWRENCE B. SLOBODKIN, State University of New
York at Stony Brook
JOHN D. STRANDBERG, Johns Hopkins University
DONALD P. WOLF, Oregon Regional Primate Center
HOLGER W. JANNASCH, Woods Hole Oceanographic SEYMOUR ZIGMAN, University of Rochester
Institution
Editor: CHARLES B. METZ, University of Miami
AUGUST, 1987
Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE &. LEMON STS.
LANCASTER, PA
Marine Biological Laboratory
^LIBRARY
:-: SEP 141987 •!§
Woods Hole, Mass.
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 Bio-
logical 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 © 1987, by the Marine Biological Laboratory
Second-class postage paid at Woods Hole, MA, and additional mailing offices.
ISSN 0006-3 185
INSTRUCTIONS TO AUTHORS
The Biological Bulletin accepts outstanding original research reports of general interest to biologists
throughout the world. Papers are usually of intermediate length (10-40 manuscript pages). Very short
papers (less than 10 manuscript pages including tables, figures, and bibliography) will be published in a
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after formal review. A paper will usually appear within four months after its acceptance.
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scripts which do not conform will be returned to authors for correction before review.
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STRACTS, with the minor differences set out below. The most generally useful list of biological journal titles
is that published each year by BIOLOGICAL ABSTRACTS (BIOSIS List of Serials; the most recent issue). For-
eign authors, and others who are accustomed to using THE WORLD LIST OF SCIENTIFIC PERIODICALS, may
find a booklet published by the Biological Council of the U.K. (obtainable from the Institute of Biology,
4 1 Queen's Gate, London, S. W.7, England, U.K.) useful, since it sets out the WORLD LIST abbreviations for
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A. Journal abbreviations, and book titles, all underlined (for italics)
B. All components of abbreviations with initial capitals (not as European usage in WORLD LIST e.g.
J. Cell. Comp. Physiol. NOT/ cell. comp. Physiol.)
C. All abbreviated components must be followed by a period, whole word components must not (i.e.
J. Cancer Res.)
D. Space between all components (e.g. J. Cell. Comp. Physiol., not J.Cell.Comp.Physiol.)
E. Unusual words in journal titles should be spelled out in full, rather than employing new abbrevi-
ations invented by the author. For example, use Rit Visindafjelags Islendinga without abbreviation.
F. All single word journal titles in full (e.g. Veliger, Ecology, Brain).
G. The order of abbreviated components should be the same as the word order of the complete title
(i.e. Proc. and Trans, placed where they appear, not transposed as in some BIOLOGICAL ABSTRACTS
listings).
H. A few well-known international journals in their preferred forms rather than WORLD LIST or
USASI usage (e.g. Nature, Science, Evolution NOT Nature, Lond., Science, N.Y.; Evolution, Lancaster,
Pa.)
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date. Authors (or delegates or foreign authors) will receive page proofs of articles shortly before publication.
They will be charged the current cost of printers' time for corrections to these (other than corrections of
printers' or editors' errors).
„- -*••*
THE
BIOLOGICAL BULLETIN
PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
RUSSELL F. DOOLITTLE, University of California
at San Diego
WILLIAM R. ECKBERG, Howard University
ROBERT D. GOLDMAN, Northwestern University
C. K. GOVIND, Scarborough Campus, University
ofToronto
JUDITH P. GRASSLE, Marine Biological Laboratory
MICHAEL J. GREENBERG, C. V. Whitney Marine
Laboratory, University of Florida
MAUREEN R. HANSON, Cornell University
JOHN E. HOBBIE, Marine Biological Laboratory
LIONEL JAFFE, Marine Biological Laboratory
HOLGER W. JANNASCH, Woods Hole Oceanographic
Institution
WILLIAM R. JEFFERY, University of Texas at Austin
GEORGE M. LANGFORD, University of
North Carolina at Chapel Hill
GEORGE D. PAPPAS, University of Illinois at Chicago
SIDNEY K. PIERCE, University of Maryland
HERBERT SCHUEL, State University of New York at
Buffalo
VIRGINIA L. SCOFIELD, University of California at
Los Angeles School of Medicine
LAWRENCE B. SLOBODKJN, State University of New
York at Stony Brook
JOHN D. STRANDBERG, Johns Hopkins University
DONALD P. WOLF, Oregon Regional Primate Center
SEYMOUR ZIGMAN, University of Rochester
Editor: CHARLES B. METZ, University of Miami
AUGUST, 1987
Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE & LEMON STS.
LANCASTER, PA.
111
Marine Biological Laboratory
LIBRARY
SEP 141987
Woods Hole, Mass.
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-NINTH REPORT, FOR THE YEAR 1 986 — NINETY-NINTH YEAR
I. TRUSTEES AND STANDING COMMITTEES 1
II. MEMBERS OF THE CORPORATION 6
1 . LIFE MEMBERS 6
2. REGULAR MEMBERS 8
3. ASSOCIATE MEMBERS 28
III. CERTIFICATE OF ORGANIZATION 32
IV. ARTICLES OF AMENDMENT 33
V. BYLAWS 34
VI. REPORT OF THE DIRECTOR 39
VII. REPORT OF THE TREASURER 43
VIII. REPORT OF THE LIBRARIAN 55
IX. EDUCATIONAL PROGRAMS 55
1 . SUMMER 55
2. SPRING 64
3. SHORT COURSES 65
X. RESEARCH AND TRAINING PROGRAMS 67
1 . SUMMER 67
2. YEAR-ROUND 76
XI. HONORS 82
XII. INSTITUTIONS REPRESENTED 85
XIII. LABORATORY SUPPORT STAFF 89
I. TRUSTEES
Including Action of the 1986 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, 200 Ocean Lane, Key
Biscay ne,FL 33 149
ROBERT MANZ, Treasurer, 1 Spafford Road, Milton, MA 02186
PAUL R. GROSS, President of the Corporation, Marine Biological Laboratory, Woods Hole,
MA 02543
J. RICHARD WHITTAKER, Director of the Laboratory, Marine Biological Laboratory, Woods
Hole, MA 02543
DAVID D. POTTER, Clerk, Harvard Medical School, Cambridge, MA 02138
Copyright © 1987, by the Marine Biological Laboratory
Library of Congress Card No. A38-5 1 8
(ISSN 0006-3 185)
2 MARINE BIOLOGICAL LABORATORY
EMERITI
JOHN B. BUCK, National Institutes of Health
AURIN CHASE, Princeton University
GEORGE H. A. CLOWES, JR., The Cancer Research Institute
SEYMOUR S COHEN, Woods Hole, Massachusetts
ARTHUR L. COLWIN, University of Miami
LAURA HUNTER COLWIN, 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
CHARLES B. METZ, University of Miami
KEITH PORTER, University of Maryland
C. LADD PROSSER, University of Illinois
JOHN S. RANKIN, Ashford, Connecticut
MERYL ROSE, Waquoit, Massachusetts
JOHN SAUNDERS, JR., SUNY, Albany
GEORGE T. SCOTT, Woods Hole, Massachusetts
MARY SEARS, Woods Hole, Massachusetts
HOMER P. SMITH, Woods Hole, Massachusetts
CARL C. SPEIDEL, University of Virginia (no mailings)
ALBERT SZENT-GYORGYI, Marine Biological Laboratory (deceased 10/22/86)
W. RANDOLPH TAYLOR, University of Michigan
GEORGE WALD, Woods Hole, Massachusetts
CLASS OF 1990
JOHN E. DOWLING, Harvard University
GERALD FISCHBACH, Washington University School of Medicine
ROBERT D. GOLDMAN, Northwestern University
JOHN E. HOBBIE, Marine Biological Laboratory
RICHARD KENDALL, Massachusetts Governor's Office
JOAN V. RUDERMAN, Duke University
ANN E. STUART, University of North Carolina
D. THOMAS TRIGG, Wellesley, 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 M. ARMSTRONG, University of Pennsylvania
JOEL P. DAVIS, Seapuit, Inc.
ELLEN R. GRASS, The Grass Foundation
TRUSTEES AND STANDING COMMITTEES
JUDITH GRASSLE, Marine Biological Laboratory
HOLGER W. JANNASCH, Woods Hole Oceanographic Institution
GEORGE M. LANGFORD, University of North Carolina
ANDREW SZENT-GYORGYI, Brandeis University
KENSAL VAN HOLDE, Oregon State University
RICHARD W. YOUNG, Wellesley Hills, Massachusetts
CLASS OF 1987
EDWARD A. ADELBERG, Yale University
JAMES M. CLARK, Shearson/ American Express
HAROLD GAINER, National Institutes of Health
WILLIAM T. GOLDEN, New York, New York
HANS KORNBERG, University of Cambridge
LASZLO LORAND, Northwestern University
CAROL L. REINISCH, Tufts University
HOWARD A. SCHNEIDERMAN, Monsanto Company
SHELDON J. SEGAL, The Rockefeller Foundation
STANDING COMMITTEES
EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES
PROSSER GIFFORD* JUDITH GRASSLE, 1 988
PAUL R. GROSS* HARLYN O. HALVORSON, 1989
J. RICHARD WHITTAKER* JOHN G. HILDEBRAND, 1989
ROBERT MANZ* ANDREW SZENT-GYORGYI, 1988
JOHN E. DOWLING, 1990 KENSAL VAN HOLDE, 1988
GERALD FISCHBACH, 1990
ANIMAL CARE COMMITTEE
CAROL L. REINISCH, Chairman ROXANNA SMOLOWITZ
DANIEL ALKON RAYMOND E. STEPHENS
EDWARD JASKUN J. RICHARD WHITTAKER
BUILDINGS AND GROUNDS COMMITTEE
KENYON S. TWEEDELL, Chairman DONALD B. LEHY*
LAWRENCE B. COHEN THOMAS H. MEEDEL
RICHARD D. CUTLER* PHILIP PERSON
ALAN FEIN LIONEL I. REBHUN
DANIEL L. GILBERT THOMAS S. REESE
CIFFORD V. HARDING, JR. EVELYN SPIEGEL
FERENC I. HAROSI
CAPITAL DEVELOPMENT COMMITTEE
RICHARD W. YOUNG, Chairman WILLIAM T. GOLDEN
PROSSER GIFFORD* HARLYN O. HALVORSON
EMPLOYEE RELATIONS COMMITTEE
JOHN V. K. HELFRICH, Chairman EDWARD ENOS
JUDITH ASHMORE WILLIAM A. EVANS
FLORENCE DWAYNE JOHN B. MACLEOD
4 MARINE BIOLOGICAL LABORATORY
FELLOWSHIPS COMMITTEE
THORU PEDERSON, Chairman EDUARDO MACAGNO
JUDITH GRASSLE CAROL L. REINISCH
JOAN E. HOWARD* J. RICHARD WHITTAKER*
GEORGE M. LANGFORD
FINANCIAL POLICY AND PLANNING COMMITTEE
GEORGE H. A. CLOWES, JR., Chairman ROBERT MAINER
ROBER i W. ASHTON W. NICHOLAS THORNDIKE
DAVID L. CURRIER* J. RICHARD WHITTAKER
THOMAS J. HYNES, JR.
HOUSING, FOOD SERVICE AND DAY CARE COMMITTEE
JELLE ATEMA, Chairman LouANN KING*
ROBERT B. BARLOW, JR. THOMAS S. REESE
GAIL D. BURD JOAN RUDERMAN
RONALD L. CALABRESE BRIAN M. SALZBERG
STEPHEN M. HIGHSTEIN SUSAN SZUTS
INSTITUTIONAL BIOSAFETY
RAYMOND E. STEPHENS, Chairman DONALD B. LEHY*
PAUL J. DE WEER JOSEPH MARTYNA
PAUL T. ENGLUND ANDREW H. MATTOX*
HARLYN O. HALVORSON* AL SENFT
PAUL LEE
INSTRUCTION COMMITTEE
JUDITH GRASSLE, Chairman* HANS LAUFER
BRIAN FRY JOAN V. RUDERMAN
HARLYN O. HALVORSON* BRIAN M. SALZBERG
JOHN G. HILDEBRAND* ROGER D. SLOBODA
JOAN E. HOWARD* ANDREW SZENT-GYORGYI*
INVESTMENT COMMITTEE
D. THOMAS TRIGG, Chairman ROBERT MANZ*
PROSSER GIFFORD* JOHN W. SPEER*
WILLIAM T. GOLDEN W. NICHOLAS THORNDIKE
MAURICE LAZARUS J. RICHARD WHITTAKER*
LIBRARY JOINT MANAGEMENT COMMITTEE
J. RICHARD WHITTAKER, Chairman* JOHN W. SPEER*
GARLAND E. ALLEN JOHN H. STEELE
GEORGE D. GRICE
LIBRARY JOINT USERS COMMITTEE
GARLAND E. ALLEN, Chairman LAURENCE P. MADIN
WILFRED B. BRYAN JOHN SCHLEE
A. FARMANFARMAIAN FREDERIC SERCHUK
JANE FESSENDEN* OLIVER C. ZAFIRIOU
LIONEL F. JAFFE
TRUSTEES AND STANDING COMMITTEES
MARINE RESOURCES COMMITTEE
ROBERT D. GOLDMAN, Chairman GEORGE D. PAPPAS
WILLIAM D. COHEN ROGER D. SLOBODA
RICHARD D. CUTLER* MELVIN SPIEGEL
Louis LEIBOVITZ ANTOINETTE STEINACHER
TOSHIO NARAHASHI JOHN VALOIS*
RADIATION SAFETY COMMITTEE
PAUL J. DE WEER, Chairman ANDREW H. MATTOX*
RICHARD L. CHAPPELL HARRIS RIPPS
SHERWIN J. COOPERSTEIN RAYMOND E. STEPHENS
DANIEL S. GROSCH WALTER S. VINCENT
RESEARCH SERVICES COMMITTEE
BIRGIT ROSE, Chairman RAYMOND J. LASEK
ROBERT B. BARLOW, JR. BRYAN D. NOE
RICHARD D. CUTLER* BRUCE J. PETERSON
ROBERT D. GOLDMAN JOEL L. ROSENBAUM
JOHN G. HILDEBRAND RAYMOND E. STEPHENS
JOAN E. HOWARD* SIDNEY L. TAMM
SAMUEL S. KOIDE
RESEARCH SPACE COMMITTEE
JOSEPH SANGER, Chairman LASZLO LORAND
CLAY M. ARMSTRONG EDUARDO MACAGNO
ROBERT D. GOLDMAN JERRY A. MELILLO
JOAN E. HOWARD* ROGER D. SLOBODA
DAVID LANDOWNE EVELYN SPIEGEL
HANS LAUFER STEVEN N. TREISTMAN
RODOLFO R. LLINAS IVAN VALIELA
SAFETY COMMITTEE
JOHN E. HOBBIE, Chairman ALAN M. KUZIRIAN
DANIEL L. ALKON DONALD B. LEHY*
D. EUGENE COPELAND ANDREW H. MATTOX*
RICHARD D. CUTLER* EDWARD A. SADOWSKI
EDWARD ENOS RAYMOND E. STEPHENS
ALAN FEIN PAUL A. STEUDLER
LOUIS M. KERR
TRUSTEES' COMMITTEES
AUDIT COMMITTEE
ROBERT MAINER, Chairman D. THOMAS TRIGG
ROBERT MANZ* KENSAL VAN HOLDE
SHELDON J. SEGAL RICHARD W. YOUNG
INVESTMENT COMMITTEE
D. THOMAS TRIGG, Chairman ROBERT MANZ*
WILLIAM T. GOLDEN W. NICHOLAS THORNDIKE
MAURICE LAZARUS
6 MARINE BIOLOGICAL LABORATORY
COMPENSATION COMMITTEE
GEORGE H. A. CLOWES, JR., Chairman HARLYN O. HALVORSON
JAMES M. CLARK THOMAS J. HYNES, JR.
COMMITTEE ON LABORATORY GOALS
GERALD FISCHBACH, Chairman JOHN E. HOBBIE
MICHAEL V. L. BENNETT DAVID D. POTTER
HARLYN O. HALVORSON JOAN V. RUDERMAN
JOHN G. HILDEBRAND J. RICHARD WHITTAKER*
CENTRAL CENTENNIAL COMMITTEE
JAMES D. EBERT, Chairman JOHN PFEIFFER
PAMELA CLAPP, Assistant KEITH R. PORTER
GARLAND E. ALLEN C. LADD PROSSER
ROBERT B. BARLOW JOHN REED
RICHARD KENDALL D. THOMAS TRIGG
II. MEMBERS OF THE CORPORATION
Including Action of the 1986 Annual Meeting
LIFE MEMBERS
ABBOTT, MARIE, c/o Katherine Y. Hutchinson, Bunker Hill Road, Andover, CT 06232
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 #2114, 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, 7 1 5 Kirk Rd., Decatur, GA 30030
BUCK, JOHN B., NIH, Laboratory of Physical Biology, Room 1 12, Building 6, Bethesda, MD
20892
BURBANCK, MADELINE P., Box 1 5 1 34, Atlanta, GA 30333
BURBANCK, WILLIAM D., Box 15134, Atlanta, GA 30333
CARPENTER, RUSSELL L., 60-H Lake St., Winchester, MA 01890
CHASE, AURIN, Professor of Biology Emeritus, Princeton University, Princeton, NJ 08540
CLARKE, GEORGE L., Address Unknown
CLOWES, GEORGE H. A., JR., The Cancer Research Institute, 194 Pilgrim Rd., Boston, MA
02215
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 Rd., Key Biscayne, FL 33149
COPELAND, D. E., 41 Fern Lane, Woods Hole, MA 02543
COSTELLO, HELEN M., Carolina Meadows, Villa 1 37, Chapel Hill, NC 275 14
CROUSE, HELEN, Institute of Molecular Biophysics, Florida State University, Tallahassee, FL
32306
* ex-officio
MEMBERS OF THE CORPORATION 7
DILLER, IRENE C, Rydal Park, Apartment 660, Rydal, PA 19046
DILLER, WILLIAM F., Rydal Park, Apartment 660, Rydal, PA 19046 (deceased 2/8/86)
ELLIOTT, ALFRED M., 428 Lely Palm Ext., Naples, FL 33962-8903
FAILLA, PATRICIA M., 2149 Loblolly Lane, Johns Island, SC 29455
FERGUSON, JAMES K. W., 56 Clarkehaven St., Thornhill, Ontario, Canada L4J 2B4
FISHER, J. MANERY, Department of Biochemistry, University of Toronto, Toronto, Ontario,
Canada M5S 1 A8 (deceased 9/9/86)
FRIES, ERIK F. B., 4 1 High Street, Woods Hole, MA 02543
OILMAN, LAUREN C., Department of Biology, University of Miami, PO Box 24918, Coral
Gables, FL 33 134
GREEN, JAMES W., 409 Grand Ave., Highland Park, NJ 08904
HAMBURGER, VIKTOR, Professor Emeritus, Washington University, St. Louis, MO 63 1 30
HAMILTON, HOWARD L., Department of Biology, University of Virginia, Charlottesville, VA
22901
HIBBARD, HOPE, c/o Jeanne Stephens, 374 Morgan St., 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
KARUSH, FRED, 183 Summit Lane, Bala-Cynwyd, PA 19004
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., 1 5 Agassiz Road, Woods Hole, MA 02543
LEVINE, RACHMIEL, 2024 Canyon Rd., Arcadia, CA 91006
LOCHHEAD, JOHN H., 49 Woodlawn Rd., London SW 6 6PS, England, U. K.
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 N 1 2, 1 380 1 York Rd., Cockeysville, MD 2 1 030 (deceased
8/17/86)
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
POLLISTER, A. W., 313 Broad Street, Harleysville, PA 19438
PROSSER, C. LADD, Department of Physiology and Biophysics, Burrill Hall 524, University of
Illinois, Urbana, IL 6 1801
PROVASOLI, LUIGI, Haskins Laboratories, 165 Prospect Street, New Haven, CT 065 10
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 55 1 14
RICHARDS, OSCAR W., Pacific University, Forest Grove, OR 97462
RONKIN, RAPHAEL R., 3212 McKinley St., NW, Washington, DC 20015
SCHARRER, BERTA, Department of Anatomy, Albert Einstein College of Medicine, 1 300 Mor-
ris Park Avenue, Bronx, NY 10461
8 MARINE BIOLOGICAL LABORATORY
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 02 1 39
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 Univer-
sity, Evanston, IL 60201
SMITH, HOMER P., 8 Quissett Ave., Woods Hole, MA 02543
SONNENBLICK, B. P., Department of Zoology and Physiology, Rutgers University, 195 Univer-
sity Ave., Newark, NJ 07 102
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
WAINIO, WALTER W., 331 State Road, Princeton, NJ 08540
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., Department of Biology, Northwestern Illinois University, Chicago, IL
60625
WILBER, CHARLES G., Department of Zoology, Colorado State University, Fort Collins, CO
80523
YOUNG, D. B., 1 1 37 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., c/o Ames, University of California, La Jolla, CA 92037
REGULAR MEMBERS
ACHE, BARRY W., Whitney Marine Laboratory, University of Florida, Rt. 1, Box 121, St.
Augustine, FL 32086
ACHESON, GEORGE H., 25 Quissett Ave., Woods Hole, MA 02543
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 065 10
AFZELIUS, BJORN, Wenner-Gren Institute, University of Stockholm, Stockholm, Sweden
ALBERTE, RANDALL S., University of Chicago, Barnes Laboratory, 5630 S. Ingleside Ave.,
Chicago, IL 60637
ALKON, DANIEL, Section on Neural Systems, Laboratory of Biophysics, NIH, Marine Biologi-
cal 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 Sta-
tion, Winston-Salem, NC 27109
ALLEN, ROBERT D., Department of Biology, Dartmouth College, Hanover, NH 03755 (de-
ceased 3/23/86)
AMATNIEK, ERNEST, 4797 Boston Post Rd., Pelham Manor, NY 10803
ANDERSON, EVERETT, Department of Anatomy, LHRBB, Harvard Medical School, Boston,
MA 02115
ANDERSON, J. M., 1 10 Roat St., Ithaca, NY 14850
MEMBERS OF THE CORPORATION
ARMET-KiBEL, CHRISTINE, Biology Department, University of Massachusetts-Boston, Bos-
ton, MA 02 125
ARMSTRONG, CLAY M., Department of Physiology, Medical School, University of Pennsylva-
nia, Philadelphia, PA 19174
ARMSTRONG, PETER B., Department of Zoology, University of California, Davis, CA 95616
ARNOLD, JOHN M., Pacific Biomedical Research Center, 209 Snyder Hall, 2538 The Mall
Honolulu, HI 96822
ARNOLD, WILLIAM A., 102 Balsam Rd., Oak Ridge, TN 37830
ASHTON, ROBERT W., Gaston 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., Section of Neurobiology, Department of Biological Sciences, Uni-
versity of Southern California, Los Angeles, CA 90089-037 1
AUSTIN, MARY L., 506'/2 N. Indiana Ave., Bloomington, IN 47401
AYERS, DONALD E., Marine Biological Laboratory, Woods Hole, MA 02543
BACON, ROBERT, PO Box 723, Woods Hole, MA 02543
BAKER, ROBERT G., New York University Medical Center, 550 First Ave., New York, NY
10016
BALDWIN, THOMAS O., Department of Biochemistry and Biophysics, Texas A&M University,
College Station, TX 77843
BANG, BETSY, 76 F. R. Lillie Rd., Woods Hole, MA 02543
BARKER, JEFFERY L., National Institutes of Health, Bldg. 36, Room 2002, Bethesda, MD
20892
BARLOW, ROBERT B., JR., Institute for Sensory Research, Syracuse University, Merrill Lane,
Syracuse, NY 13210
BARRY, DANIEL T., Department of Physical Medicine and Rehabilitation, ID204, University
of Michigan Hospital, Ann Arbor, MI 48109-0042
BARRY, SUSAN R., Department of Physical Medicine and Rehabilitation, ID204, University
of Michigan Hospital, Ann Arbor, MI 48109-0042
BARTELL, CLELMER K., 2000 Lake Shore Drive, New Orleans, LA 70122
BARTH, LUCENA J., 26 Quissett Ave., Woods Hole, MA 02543 (deceased 7/26/86)
BARTLETT, JAMES H., Department of Physics, Box 1921, University of Alabama, Tuscaloosa,
AL 35489
BASS, ANDREW H., Seely Mudd Hall, Department of Neurobiology and Behavior, Cornell
University, Ithaca, NY 14853
BATTELLE, BARBARA-ANNE, Whitney Marine Laboratory, Rt. 1, Box 121, St. Augustine, FL
32086
BAUER, G. ERIC, Department of Anatomy, University of Minnesota, Minneapolis, MN 55455
BEAUGE, Luis ALBERTO, Institute de Investigacion Medica, Casilla de Correo 389, 5000 Cor-
doba, Argentina
BECK, L. V., School of Experimental Medicine, Department of Pharmacology, Indiana Uni-
versity, Bloomington, IN 47401
BEGENISICH, TED, Department of Physiology, University of Rochester, Rochester NY 14642
BEGG, DAVID A., LHRRB, Harvard Medical School, 45 Shattuck St., Boston, MA 02 1 1 5
BELL, EUGENE, Department of Biology, Massachusetts Institute of Technology, 77 Massachu-
setts Ave., Cambridge, MA 02 1 39
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 M., 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
10 MARINE BIOLOGICAL LABORATORY
BISHOP, STEPHEN H., Department of Zoology, Iowa State University, Ames, IA 50010
BLAUSTEIN, MORDECAI P., Department of Physiology, School of Medicine, University of
Maryland, 655 W. Baltimore Street, Baltimore, MD 21201
BLOOM, KERRY S., Department of Biology, University of North Carolina, Chapel Hill, NC
27514
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 (resigned 8/8/86)
BOOLOOTIAN, RICHARD A., Science Software Systems, Inc., 3576 Woodcliff Rd., Sherman
Oaks, CA 9 1403
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
(resigned 3/86)
BOWLES, FRANCIS P., PO Box 674, Woods Hole, MA 02543
BOYER, BARBARA C, Department of Biology, Union College, Schenectady, NY 12308
BRANDHORST, BRUCE P., Biology Department, McGill University, 1205 Ave. Dr. Penfield,
Montreal, P. Q., Canada H3A 1 B 1
BREHM, PAUL, Department of Physiology, Tufts Medical School, Boston, MA 021 1 1
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 63 1 10
BROWN, STEPHEN C., Department of Biological Sciences, SUNY, Albany, NY 12222
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 11210
BURGER, MAX, Department of Biochemistry, Biocenter, Klingelbergstrasse 70, CH-4056 Ba-
sel, Switzerland
BURKY, ALBERT, Department of Biology, University of Dayton, Dayton, OH 45469
BURSTYN, HAROLD LEWIS, 216 Bradford Parkway, Syracuse, NY 13224
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, At-
lanta, GA 30322
CANDELAS, GRACIELA C., Department of Biology, University of Puerto Rico, Rio Piedras, PR
00931
CAREW, THOMAS J., Department of Psychology, Yale University, PO Box 1 1 A, Yale Station,
New Haven, CT 06520
CARIELLO, Lucio, Stazione Zoologica, Villa Comunale, Naples, Italy
CARLSON, FRANCIS D., Department of Biophysics, Johns Hopkins University, Baltimore, MD
21218
CASE, JAMES, Department of Biological Sciences, University of California, Santa Barbara, CA
93106
CASSIDY, REV. J. D., St. Rose Priory, Springfield, KY 40069
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
MEMBERS OF THE CORPORATION 1 1
CHANG, DONALD C., Department of Physiology and Molecular Biophysics, Baylor College of
Medicine, One Baylor Plaza, 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 02 1 8 1
CHARLTON, MILTON P., Physiology Department MSB, University of Toronto, Toronto, On-
tario, Canada M5S 1 A8
CHILD, FRANK M., Department of Biology, Trinity College, Hartford, CT 06 106
CHISHOLM, REX L., Dept. 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 065 1 1
CLARK, A. M., 48 Wilson Rd., Woods Hole, MA 02543
CLARK, ELOISE E. Vice President for Academic Affairs, Bowling Green State University, Bowl-
ing Green, OH 43403
CLARK, HAYS, Property Management Ltd., 125 Mason St., Greenwich, CT 06830
CLARK, JAMES M., Shearson Lehman Brothers Inc., Two World Trade Center, 105th Floor,
New York, NY 10048
CLARK, WALLIS H., JR., Bodega Marine Lab, PO Box 247, Bodega Bay, CA 94923
CLAUDE, PHILIPPA, Primate Center, Capitol Court, Madison, WI 53706
CLAY, JOHN R., Marine Biological Laboratory, Woods Hole, MA 02543
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 5 1 8, National Science
Foundation, Washington, DC 20550
COBB, JEWELL P., President, California State University, Fullerton, CA 92634
COHEN, ADOLPH L, Department of Ophthalmology, School of Medicine, Washington Univer-
sity, 660 S. Euclid Ave., St. Louis, MO 631 10
COHEN, CAROLYN, Rosenstiel Basic Medical Sciences Research Center, Brandeis University,
Waltham, MA02154
COHEN, LAWRENCE B., Department of Physiology, Yale University School of Medicine, B-
106 SHM, PO Box 3333, New Haven, CT 065 10-8026
COHEN, MAYNARD, Department of Neurological Sciences, Rush Medical College 600 South
Paulina, Chicago, IL 606 1 2
COHEN, ROCHELLE S., Department of Anatomy, University of Illinois at Chicago, 808 S.
Wood Street, Chicago, I L 606 1 2
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,
RI02912
COLLIER, JACK R., Department of Biology, Brooklyn College, Brooklyn, NY 11210
COLLIER, MARJORIE McCANN, Biology Department, Saint Peter's College, Kennedy Boule-
vard, 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
CORNMAN, IVOR, 10A Orchard St., Woods Hole, MA 02543 (resigned 12/4/86)
CORNWALL, MELVIN C., JR., Department of Physiology L714, Boston University School of
Medicine, 80 E. Concord St., Boston, MA 02 1 1 8
CORSON, DAVID WESLEY, JR., 1034 Plantation Lane, Mt. Pleasant, SC 29464
CORWIN, JEFFREY T., Bekesy Lab of Neurobiology, 1993 East- West Road, University of Ha-
waii, Honolulu, HI 96822
COSTELLO, WALTER J., College of Medicine, Ohio University, Athens, OH 45701
12 MARINE BIOLOGICAL LABORATORY
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
CROWTHER, ROBERT, Marine Biological Laboratory, Woods Hole, MA 02543
CURRIER, DAVID L., PO Box 2476, Vineyard Haven, MA 02568
DAIGNAULT, ALEXANDER T., 280 Beacon St., Boston, MA 021 16
DAN, KATSUMA, 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 Long-
wood Ave., Boston, MA 02 1 1 5
DAVIDSON, ERIC H., Division of Biology, California Institute of Technology, Pasadena, CA
91125
DAVIS, BERNARD D., 23 Clairemont Road, Belmont, MA 02 1 78
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 95 128
DEPHILLIPS, HENRY A., JR., Department of Chemistry, Trinity College, Hartford, CT 06 106
DETERRA, NOEL, 2 1 5 East 1 5th St., New York, NY 1 0003
DETTBARN, WOLF-DIETRICH, Department of Pharmacology, School of Medicine, Vanderbilt
University, Nashville, TN 37 127
DE WEER, PAUL J., Department of Physiology, School of Medicine, Washington University,
St. Louis, MO 63 110
DISCHE, ZACH ARIAS, Eye Institute, College of Physicians and Surgeons, Columbia University,
639 W. 165 St., New York, NY 10032 (dropped 3/86)
DIXON, KEITH E., School of Biological Sciences, Flinders University, Bedford Park, South
Australia
DONELSON, JOHN E., Department of Biochemistry, University of Iowa, Iowa City IA 52242
DOWDALL, MICHAEL J., Department of Zoology, School of Biological Sciences, University of
Nottingham, University Park, Nottingham N672 UH, England, U. K.
DOWLING, JOHN E., The Biological Laboratories, Harvard University, 16 Divinity St., Cam-
bridge, MA 02 1 38
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 Uni-
versity, 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
DUNLAP, KATHLEEN, Department of Psychology, Tufts Medical School, Boston, MA 021 1 1
EBERT, JAMES D., Office of the President, Carnegie Institute 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
(deceased 6/1 8/86)
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, NIAADK/NIH, Rm. 403, Bldg. 10, Bethesda, MD 20892
EGYUD, LASZLO G., 1 8 Skyview, Newton, MA 02 1 50
EHRENSTEIN, GERALD, NIH, Bethesda, MD 20892
MEMBERS OF THE CORPORATION 13
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.
ENGLUND, PAUL T., Department of Biological Chemistry, Johns Hopkins School of Medicine,
Baltimore, MD 2 1205
EPEL, DAVID, Hopkins Marine Station, Pacific Grove, CA 93950
EPSTEIN, HERMAN T., Department of Biology, Brandeis University, Waltham, MA 02254
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
FARMANFARMAIAN, A., Department of Biological Sciences, Nelson Biological Laboratory,
Rutgers University, PO Box 1059, Piscataway, NJ 08854
FEIN, ALAN, Laboratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole,
MA 02543
FEINMAN, RICHARD D., Box 8, Department of Biochemistry, SUNY Health Science Center,
Brooklyn, NY 11203
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 ( 1 27), Veterans Administration Medical Center, 480 1
Linwood Blvd., Kansas City, MO 64128
FINKELSTEIN, ALAN, Albert Einstein College of Medicine, 1 300 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 Medi-
cal College, 1 300 York Ave., New York, NY 1002 1
FISHMAN, HARVEY M., Department of Physiology, University of Texas Medical Branch, Gal-
veston,TX 77550
FLANAGAN, DENNIS, 12 Gay St., New York, NY 10014
Fox, MAURICE S., Department of Biology, Massachusetts Institute of Technology, Cambridge,
MA 02 138
FRANK, PETER W., Department of Biology, University of Oregon, Eugene, OR 97403
FRANZINI, CLARA, Department of Biology G-5, School of Medicine, University of Pennsylva-
nia, Philadelphia, PA 19174
FRAZIER, DONALD T., Department of Physiology and Biophysics, University of Kentucky
Medical Center, Lexington, KY 40536
FREEMAN, ALAN R., Department of Physiology, Temple University, 3420 N. Broad St., Phila-
delphia, PA 19140 (resigned 3/86)
FREEMAN, GARY L., Department of Zoology, University of Texas, Austin, TX 78 172
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 Medical Physiology, University of Calgary, 3330 Hospital
Dr., NW, Calgary, Alberta T2N 4N1 Canada
FREYGANG, WALTER J., JR., 6247 29th St., NW, Washington, DC 20015
FRY, BRIAN, Marine Biological Laboratory, Woods Hole, MA 02543
FUKUI, YOSHIO, Department of Cell Biology and Anatomy, Northwestern University Medical
School, Chicago, IL 60201
14 MARINE BIOLOGICAL LABORATORY
FULTON, CHANDLER M, Department of Biology, Brandeis University, Waltham, MA 02 1 54
FURSHPAN, EDWIN J., Department of Neurophysiology, Harvard Medical School, Boston, MA
02115
FUSELER, JOHN W., Department of Biology, University of Southwestern Louisiana, Lafayette,
LA 70504
FUTRELLE, ROBERT P., College of Computer Science, Northeastern University, 360 Hunting-
ton 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, 1 230 York
Avenue, New York, NY 1002 1
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, 1 1 5 West University Parkway, Baltimore, MD 21210
GALLANT, PAUL E., Laboratory of Preclinical Studies, Bldg. 36, NIAAA/NIH, 1250 Washing-
ton Ave., Rockville, MD 20892
GASCOYNE, PETER, Department of Experimental Pathology, Box 85E, University of Texas
System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Texas Medical
Center, 6723 Bertner Avenue, Houston, TX 77030
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 67th 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. 1 68th 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. 55 1 1 Oakmont Avenue, Bethesda, MD 20034
GOLDSMITH, TIMOTHY H., Department of Biology, Yale University, New Haven, CT 065 10
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.
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., Zoology Department-Scarborough, University of Toronto, 1265 Military
Trail, West Hill, Ontario, Canada, MIC 1A4
GRAF, WERNER, Rockefeller University, New York, NY 10021
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 02 1 70
MEMBERS OF THE CORPORATION 15
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 Ave-
nue, Chicago, IL 60605
GREENBERG, EVERETT PETER, Department of Microbiology, Stocking Hall, Cornell Univer-
sity, Ithaca, NY 14853
GREENBERG, MICHAEL J., Whitney Marine Laboratory, 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 1002 1
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 1046 1
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., 7415 Makaa Place, Honolulu, HI 96825
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, MD 2 12 18
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 1 12 Place, Miami, FL 33173
HAYES, RAYMOND L., JR., Dept. 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
HEPLER, PETER K., Department of Botany, University of Massachusetts, Amherst, MA 01003
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,
MO63110
HILDEBRAND, JOHN G., Arizona Research Laboratories, Division of Neurobiology, 603
Gould-Simpson Science Building, University of Arizona, Tucson, AZ 85721
16 MARINE BIOLOGICAL LABORATORY
HILL, SUSAN D., Department of Zoology, Michigan State University, E. Lansing, MI 48824
HILLIS-COLINVAUX, LLEWELLYA, Department of Zoology, The Ohio State University, 484 W
1 2th Ave., Columbus, OH 432 1 0
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
HoBBUi, JOHN E., Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543
HODGE, ALAN J., Marine Biological Laboratory, Woods Hole, MA 02543
HOFFMAN, JOSEPH, Department of Physiology, School of Medicine, Yale University, New
Haven, CT 065 10
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
HOUGHTON, 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, Ith-
aca, 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 968 13
HUNTER, BRUCE W., Box 32 1 , Lincoln Center, MA 0 1 773
HUNTER, ROBERT D., Department of Biological Sciences, Oakland University, Rochester, NY
48063
HUNZIKER, HERBERT E., Esq., PO Box 547, Falmouth, MA 0254 1
HURWITZ, CHARLES, Basic Science Research Lab, 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 02 110
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 Anatomy, 3640 Univer-
sity St., Montreal, PQ, 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, 32 Fruit Street, Boston, MA 02 1 1 4
IZZARD, COLIN S., Department of Biological Sciences, SUNY, Albany, NY 12222
JACOBSON, ANTONE G., Department of Zoology, University of Texas, Austin, TX 78712
MEMBERS OF THE CORPORATION 17
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 Institu-
tion, Washington, DC 20560
JOSEPHSON, ROBERT K., School of Biological Sciences, University of California, Irvine, CA
92664
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 Col-
lege, Valhalla, NY 10595
KALTENBACH, JANE, Department of Biological Sciences, Mount Holyoke College, South Had-
ley,MA01075
KAMINER, BENJAMIN, Department of Physiology, School of Medicine, Boston University, 80
East Concord St., Boston, MA 02 1 1 8
KAMMMER, ANN E., Department of Zoology, Arizona State University, Tempe, AZ 85281
KANE, ROBERT E., University of Hawaii, PBRC, 41 Ahui St., Honolulu, HI 96813
KANESHIRO, EDNA S., Department of Biological Sciences, University of Cincinnati, Cincin-
nati, OH 45221
KAO, CHIEN-YUAN, Department of Pharmacology (Box 29), SUNY, Downstate Medical Cen-
ter, 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 9 1st St., New York, NY 10024
KARLIN, ARTHUR, Department of Biochemistry and Neurology, Columbia University, 630
West 168th St., New York, NY 10032
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
University, 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
KENDALL, RICHARD, 26 Green Harbor Rd., East Falmouth, MA 02536
KEYNAN, ALEXANDER, Hebrew University, Jerusalem, Israel
KJEHART, DANIEL P., Department of Cellular and Developmental Biology, Harvard Univer-
sity, 16 Divinity Avenue, Cambridge, MA 02138
KLEIN, MORTON, Department of Microbiology, Temple University, Philadelphia, PA 19103
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 8574 1
KRANE, STEPHEN M., Massachusetts General Hospital, Boston, MA 02 1 14
KRASSNER, STUART M., Department of Developmental and Cell Biology, University of Cali-
fornia, Irvine, CA 927 1 7
18 MARINE BIOLOGICAL LABORATORY
KRAUSS, ROBERT, FASEB, 9650 Rockville Pike, Bethesda, MD 20814
KRAVITZ, EDWARD A., Department of Neurobiology, Harvard Medical School, 25 Shattuck
St., Boston, MA 02 115
KRIEBEL, MAHLON E., Department of Physiology, B.S.B., Upstate Medical Center, 766 Irving
A ve., Syracuse, NY 13210
KRIEG, WENDELL J. S., 1236 Hinman, Evanston, IL 60602 (resigned 3/86)
KRISTAN, WILLIAM B., JR., Department of Biology B-022, University of California San Diego,
San Diego, CA 92093
KUHNS, WILLIAM J., University of North Carolina, 512 Faculty Lab Office, Bldg. 231-H,
Chapel Hill, NC 275 14
KUSANO, KJYOSHI, Illinois Institute of Technology, Department of Biology, 3300 South Fed-
eral St., Chicago, IL 606 1 6
KUZIRIAN, ALAN M., Laboratory of Biophysics, NINCDS-NIH, Marine Biological Labora-
tory, 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 Pharmacology, Case Western Reserve University Medical
School, 21 19 Abington Road, Cleveland, OH 44106
LANDOWNE, DAVID, Department of Physiology, Yale University School of Medicine, 333 Ce-
dar St., New Haven, CT 065 10
LANGFORD, GEORGE M., Department of Physiology, Medical Sciences Research Wing 206H,
University of North Carolina, Chapel Hill, NC 275 14
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, 1 230 York Avenue, New York, NY 1002 1
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
LEHY, DONALD B., Marine Biological Laboratory, Woods Hole, MA 02543
LEIBOVITZ, 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
LEINWAHN, LESLIE ANN, Department of Microbiology and Immunology, 1300 Morris Park
Ave., Bronx, NY 10461
LERMAN, SIDNEY, Laboratory for Ophthalmic Research, Emory University, Atlanta, GA
30322
LERNER, AARON B., Yale University, School of Medicine, New Haven, CT 065 10
LESTER, HENRY A., 156-29 California Institute of Technology, Pasadena, CA 91 125
LEVIN, JACK, Clinical Pathology Service, VA Hospital- 1 1 3A, 4 1 50 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
MEMBERS OF THE CORPORATION 19
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 (dropped 9/1/86)
LIPICKY, RAYMOND J., Department of Cardio-Renal/HFD 1 10, FDA Bureau of Drugs, Rm.
16B-45, 5600 Fishers Lane, Rockville, MD 20857
LISMAN, JOHN E., Department of Biology, Brandeis University, Waltham, MA 02 1 54
Liuzzi, ANTHONY, Department of Physics, University of Lowell, Lowell, MA 01854
LLINAS, RODOLFO R., Department of Physiology and Biophysics, New York University Medi-
cal Center, 550 First Ave., New York, NY 10016
LOEWENSTEIN, WERNER R., Department of Physiology and Biophysics, University of Miami,
PO Box 0 1 6430, Miami, FL 33 1 0 1
LOEWUS, FRANK A., Institute of Biological Chemistry, Washington State University, Pullman,
WA99164
LOFTFIELD, ROBERT B., Department of Biochemistry, School of Medicine, University of New
Mexico, 900 Stanford, NE, Albuquerque, NM 87 1 3 1
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 Uni-
versity, Evanston, IL 60201
LUCKENBILL-EDDS, LOUISE, 1 55 Columbia Ave., Athens, OH 4570 1
LURIA, SALVADOR E., 48 Peacock Farm Rd., Lexington, MA 02173
MACAGNO, EDUARDO R., 1003B Fairchild, Columbia University, New York, NY 10022
MACNiCHOL, E. F., JR., 45 Brewster Street, Cambridge, MA 02 1 38
MAGLOTT-DUFFIELD, 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 02 108
MALBON, CRAIG CURTIS, Department of Pharmacological Sciences, Health Sciences Center,
SUNY, Stony Brook, Stony Brook, NY 1 1794-865 1
MALKIEL, SAUL, Allergic Diseases, Inc., 130 Lincoln St., Worcester, MA 01605
MANALIS, RICHARDS., Department of Biological Sciences, Purdue University, 2101 Coliseum
Blvd., East, Ft. Wayne, IN 46805
MANGUM, CHARLOTTE P., Department of Biology, College of William and Mary, Williams-
burg, VA 23 185
MARGULIS, LYNN, Department of Biology, Boston University, 2 Cummington St., Boston,
MA 022 15
MARINUCCI, ANDREW C., 26 Woodlawn Ave., North Brunswick, NJ 08902
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
MARTINEX-PALOMO, ADOLFO, Seccion de Patologia Experimental, Cinvesav-ipn, 17000
Mexico, D. F. A. P., 14-740, Mexico
MASER, MORTON, PO Box EM, Woods Hole, MA 02543
MASTROIANNI, LUIGI, 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, 1 36
Harrison Ave., Boston, MA 021 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
20 MARINE BIOLOGICAL LABORATORY
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 Univer-
sity, Halifax, Nova Scotia B3H 45 1 , Canada
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
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, Ot-
tawa, Ontario KIN 9A9, Canada
METZ, CHARLES B., 7220 SW 124th St., Miami, FL 33156
MILKMAN, ROGER, Department of Zoology, University of Iowa, Iowa City, IA 52242
MILLS, ERIC L., Oceanography Dept., Dalhousie University, Halifax, Nova Scotia B3H 4J1,
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, Drew University, Madison NJ 07940
MIZELL, MERLE, Department of Biology, Tulane University, New Orleans, LA 701 18
MONROY, ALBERTO, Stazione Zoologica, Villa Comunale, Naples, Italy (deceased 8/23/86)
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.
Congress Parkway, Chicago, IL 606 1 2
MORRILL, JOHN B., JR., Division of National Sciences, New College, Sarasota, FL 33580
MORSE, RICHARD S., 1 93 Winding River Rd., Wellesley, MA 02 1 8 1
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
MULLINS, LORIN J., University of Maryland, School of Medicine, Baltimore MD 21201
MUSACCHIA, XAVIER J., Graduate School, University of Louisville, Louisville, KY 40292
NABRIT, S. M., 686 Beckwith St., SW, Atlanta, GA 30314
NADELHOFFER, KNUTE, Marine Biological Laboratory, Woods Hole, MA 02543
NAKA, KEN-ICHI, National Institute for Basic Biology, Okazaki, Japan 444
NAKAJIMA, SHIGEHIRO, Department of Biological Sciences, Purdue University, West Lafay-
ette, IN 47907
NAKAJIMA, YASUKO, Department of Biological Sciences, Purdue University, West Lafayette,
IN 47907
MEMBERS OF THE CORPORATION 21
NARAHASHI, TOSHIO, Department of Pharmacology, Medical Center, Northwestern Univer-
sity, 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, 119 Forest Home Drive, Ithaca, NY 14850
NICHOLLS, JOHN G., Biocenter, Klingelbergstr 70, Basel 4056, Switzerland
NICOSIA, SANTO V., Department of Pathology, University of South Florida, College of Medi-
cine, Box 1 1, 12901 North 30th St., Tampa, FL 33612
NIELSEN, JENNIFER B. K., Merck, Sharp & Dohme Laboratories, Bldg. 50-G, Room 226, Rah-
way, 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 York, NY 10021
ODUM, EUGENE, Department of Zoology, University of Georgia, Athens, GA 30701
OERTEL, DONATA, Department of Neurophysiology, University of Wisconsin, 283 Medical
Science Bldg., Madison, WI 53706
O'HERRON, JONATHAN, Lazard Freres and Company, 1 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., 16 Evergreen Lane, Chappaqua, New York 10514
OSCHMAN, JAMES L., 9 George Street, Woods Hole, 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 606 1 2
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., Becton Dickinson, PO Box 12016, Research Triangle Park, NC
27709
PASSANO, LEONARD M., Department of Zoology, Birge Hall, University of Wisconsin, Madi-
son, 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 0 1 545
PERKINS, C. D., 400 Hilltop Terrace, Alexandria, VA 22301
PERSON, PHILIP, Oral Health Director, Research Testing Labs, Inc., 167 E. 2nd St., Hunting-
ton Station, NY 11746
PETERSON, BRUCE J., 82 Hillcrest Dr., Falmouth, MA 02540
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., Science Division, Long Island University, Brooklyn Campus,
Brooklyn, NY 11201
POLLARD, HARVEY B., NIH, F Building 10, Room 10B17, Bethesda, MD 20892
22 MARINE BIOLOGICAL LABORATORY
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
POOLE, ALAN F., 1 14 Metoxit Road, Waquoit, MA 02536
PORTER, BEVERLY H., 13617 Glenoble Drive, Rockville, MD 20853
PORTER, KEITH R., 4009 St. John's Lane, Ellicott City, MD 2 1043
PORTER, MARY E., Department MCD Biology, Campus Box 347, University of Colorado,
Boulder, CO 80309
POTTER, DAVID, Department of Neurobiology, Harvard Medical School, Boston, MA 021 15
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 (R 124), PO Box 016960, Miami, FL 33101
PRENDERGAST, ROBERT A., Department of Pathology and Ophthalmology, Johns Hopkins
University, Baltimore, MD 21205
PRESLEY, PHILLIP H., Carl Zeiss, Inc., 1 Zeiss Drive, Thornwood, NY 10594
PRICE, CARL A., Waksman Institute of Microbiology, Rutgers University, PO Box 759, Piscat-
away,NJ 08854
PRICE, CHRISTOPHER H., Biological Science Center, Boston University, 2 Cummington St.,
Boston, MA 022 1 5
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, Cleve-
land, OH 44 104
PURVES, DALE, Department of Anatomy, 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
RAKOWSKI, ROBERT F., Department of Physiology and Biophysics, UHS/The Chicago Medi-
cal School, 3333 Greenbay Rd., N. Chicago, IL 60064
RAMON, FIDEL, Dept. de Fisiologia y Biofisca, Centrol de Investigacion y de, Estudius Avan-
zados del Ipn, Apurtado Postal 14-740, Mexico, D. F. 07000
RANZI, SILVIO, Sez Zoologia Sc Nat, Via Coloria 26, 120 1 33, 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, Charlottes-
ville,VA 22901
REDDAN, JOHN R., Department of Biological Sciences, Oakland University, Rochester, MI
48063
REESE, BARBARA F., Marine Biological Laboratory, Woods Hole, MA 02543
REESE, THOMAS S., Marine Biological Laboratory, Woods Hole, MA 02543
REINER, JOHN M., Albany Medical College of Union University, Department of Biochemistry,
Albany, NY 12208
REINISCH, CAROL L., Tufts University School of Veterinary Medicine, 203 Harrison Avenue,
Boston, MA 02 115
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,CT06111
MEMBERS OF THE CORPORATION 23
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., 200 Ocean Lane Drive, Key Biscayne, FL 33149
ROCKSTEIN, MORRIS, 335 Fluvia Ave., Miami, FL 33 1 34
ROSBASH, MICHAEL, Rosenstiel Center, Department of Biology, Brandeis University, Wal-
tham, MA 02 154
ROSE, BIRGIT, Department of Physiology R-430, University of Miami School of Medicine, PO
Box 016430, Miami, FL 33149
ROSE, S. MERYL, Box 309W, 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 Connecti-
cut, Storrs, CT 06268
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 V6R 1R7, Canada
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, Uni-
versity of Connecticut, Storrs, CT 06268
ROWLAND, LEWIS P., Neurological Institute, 710 West 168th St., New York, NY 10032
RUDERMAN, JOAN V., Department of Zoology, Duke University, Durham, NC 27706
RUSHFORTH, NORMAN B., Case Western Reserve University, Department of Biology, Cleve-
land, OH 44 106
RUSSELL-HUNTER, W. D., Department of Biology, Lyman Hall 029, Syracuse University,
Syracuse, NY 13210
SAFFO, MARY BETH, Center for Marine Studies, 273 Applied Sciences, University of Califor-
nia, Santa Cruz, CA 95064
SAGER, RUTH, Sidney Farber Cancer Institute, 44 Binney St., Boston, MA 02 1 1 5
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
SANBORN, RICHARD C., 5862 North Olney St., Indianapolis, IN 46220
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 517, Japan
SATTELLE, DAVID B., AFRC Unit-Department of Zoology, University of Cambridge, Down-
ing St., Cambridge CB2 3EJ, England, 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 2005 1
SCHACHMAN, HOWARD K., Department of Molecular Biology, University of California,
Berkeley, CA 94720
SCHATTEN, GERALD P., Integrated Microscopy Facility for Biomedical Research, University
of Wisconsin, 1 1 17 W. Johnson St., Madison, WI 53706
24 MARINE BIOLOGICAL LABORATORY
SCHATTEN, HEIDI, Department of Zoology, University of Wisconsin, Madison WI 53706
SCHIFF, JEROME A., Institute for Photobiology of Cells and Organelles, Brandeis University,
Waltham, MA 02 1 54
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, Department of Obstetrics and Gynecology, Yale University School of
Medicine, 333 Cedar St., New Haven, CT 065 10
SCHNEIDERMAN, HOWARD A., Monsanto Company, 800 North Lindberg Blvd., D1W, St.
Louis, MO 63 166
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, MD 2 1205
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
SCOFIELD, VIRGINIA LEE, Department of Microbiology and Immunology, UCLA School of
Medicine, Los Angeles, CA 90024
SEARS, MARY, PO Box 152, Woods Hole, MA 02543
SEGAL, SHELDON J., Population Division, The Rockefeller Foundation, 1 133 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,
Gainesville, 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., 6 1 5 Jones St., Lansing, MI 489 1 2- 1 7 1 8
SHEETZ, MICHAEL P., Department of Cell Biology and Physiology, Washington University
Medical School, 606 S. Euclid Ave., St. Louis, MO 63 1 10
SHEPARD, DAVID C., PO Box 44, Woods Hole, MA 02543
SHEPRO, DAVID, Department of Biology, Boston University, 2 Cummington St., Boston, MA
02215
SHER, F. ALAN, Immunology and Cell Biology Section, Laboratory of Parasitic Disease, NI-
AID, Building 5, Room 1 14, NIH, Bethesda, MD 20892
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
MEMBERS OF THE CORPORATION 25
SLUDER, GREENFIELD, Cell Biology Group, Worcester Foundation for Experimental Biology,
22 Maple Ave., Shrewsbury, MA 0 1 545
SMITH, MICHAEL A., J 1 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 2 1 .9 10
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, Dept. of Otolaryngology, Washington University, School of Med-
icine, 49 1 1 Barnes Hospital, St. Louis, MO 63 1 10
STEINBERG, MALCOLM, Department of Biology, Princeton University, Princeton, NJ 08540
STEPHENS, GROVER C., Department of Developmental and Cell Biology, University of Cali-
fornia, Irvine, CA 927 1 7
STEPHENS, RAYMOND E., Marine Biological Laboratory, Woods Hole, MA 02543
STETTEN, DEWITT, JR., Senior Scientific Advisor, NIH, Bldg. 16, Room 1 18, Bethesda, MD
20892
STETTEN, JANE LAZAROW, 2 W Drive, Bethesda, MD 208 14
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 25 th St., #217, San Pedro, CA 90732
STRUMWASSER, FELIX, Department of Physiology, Boston University School of Medicine,
Boston, MA 02 118
STUART, ANN E., Department of Physiology, Medical Sciences Research Wing 206H, Univer-
sity of North Carolina, Chapel Hill, NC 275 14
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 Laboratory, Woods Hole, MA 02543 (deceased
10/22/86)
SZENT-GYORGYI, ANDREW, Department of Biology, Brandeis University, Waltham, MA
02154
SZENT-GYORGYI, EVA SZENTKIRALY, Department of Biology, Brandeis University, Waltham,
MA 02 154
SZUTS, ETE Z., Laboratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole,
MA 02543
26 MARINE BIOLOGICAL LABORATORY
TAMM, SIDNEY L., Boston University Marine Program, Marine Biological Laboratory, Woods
Hole, MA 02543
TANZER, MARVIN L., Department of Oral Biology, Medical School, University of Connecti-
cut, 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
TRACER, WILLIAM, Rockefeller University, 1230 York Ave., New York, NY 10021
TRAVIS, D. M., Veterans Administration Medical Center, Fargo, ND 58102
TREISTMAN, STEVEN N., Worcester Foundation for Experimental Biology, Shrewsbury, MA
01545
TRIGG, D. THOMAS, 1 25 Grove St., Wellesley, MA 02 1 8 1
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, New York, NY 10016
TROXLER, ROBERT F., Department of Biochemistry, School of Medicine, Boston University,
80 East Concord St., Boston, MA 02 1 1 8
TUCKER, EDWARD B., The City University of New York, Baruch College, Box 502, 17 Lexing-
ton Ave., New York, NY 10010
TURNER, RUTH D., Mollusk Department, Museum of Comparative Zoology, Harvard Uni-
versity, Cambridge, MA 02 1 38
TWEEDELL, KENYON S., Department of Biology, University of Notre Dame, Notre Dame, IN
46656
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
VALLEE, RICHARD, Cell Biology Group, Worcester Foundation for Experimental Biology,
Shrewsbury, MA 01 545
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
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
WANG, CHING CHUNG, University of California, School of Pharmacy, San Francisco, CA
94143
MEMBERS OF THE CORPORATION 27
WARNER, ROBERT C, Department of Molecular Biology and Biochemistry, University of Cal-
ifornia, Irvine, CA 927 1 7
WARREN, KENNETH S., The Rockefeller Foundation, 1133 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. H., Yale University, Biology Department, Box 6666, 610 Kline Biology
Tower, New Haven, CT 065 10
WATSON, STANLEY, Woods Hole Oceanographic Institution, Woods Hole, MA 02543
WEBB, H. MARGUERITE, Marine Biological Laboratory, Woods Hole, MA 02543
WEBER, ANNEMARIE, Department of Biochemistry, School of Medicine, University of Penn-
sylvania, Philadelphia, PA 19174
WEBSTER, FERRIS, Box 765, Lewes, DE 19958
WEIDNER, EARL, Department of Zoology and Physiology, Louisiana State University, Baton
Rouge, LA 70803
WEISS, LEON P., Department of Animal Biology, School of Veterinary Medicine, University
of Pennsylvania, Philadelphia, PA 19174
WEISSMANN, GERALD, New York University, 550 First Avenue, New York, NY 10016
WERMAN, ROBERT, Neurobiology Unit, The Hebrew University, Jerusalem, Israel
WESTERFIELD, R. MONTE, The Institute of Neuroscience, University of Oregon, Eugene, OR
37403
WEXLER, NANCY SABIN, 1 5 Claremont Avenue, Apt. 92, New York, NY 10027
WHITE, ROY L., Department of Neuroscience, Albert Einstein College, 1300 Morris Park Ave-
nue, Bronx, NY 10461
WHITTAKER, J. RICHARD, Marine Biological Laboratory, Woods Hole, MA 02543
WIGLEY, ROLAND L., 35 Wilson Road, Woods Hole, MA 02543
WILSON, DARCY B., Medical Biology Institute, 1 1077 North Torrey Pines Road, La Jolla, CA
92037
WILSON, EDWARD O., Museum, Comparative Zoology, Harvard University, Cambridge, MA
02138
WILSON, T. HASTINGS, Department of Physiology, Harvard Medical School, Boston, MA
02115
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, 1 300 Morris Park Ave., New York, NY 1 00 1 6
WOLFE, RALPH, Department of Microbiology, 1 3 1 Burrill Hall, University of Illinois, Urbana,
IL61801
WOODWELL, GEORGE M., 64 Church Street, Woods Hole, MA 02543 (resigned 5/86)
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 606 1 1
WYTTENBACH, CHARLES R., Department of Physiology and Cell Biology, University of Kan-
sas, Lawrence, KS 66045
YEH, JAY Z., Department of Pharmacology, Northwestern University Medical School, 303 E.
Chicago Ave., Chicago, IL 6061 1
YOUNG, RICHARD W., Mentor O & O, Inc., 3000 Longwater Dr., Norwell, MA 0206 1-1610
ZACKROFF, ROBERT, 66 White Horn Drive, Kingston, RI 0288 1
ZIGMAN, SEYMOUR, School of Medicine and Dentistry, University of Rochester, 260 Critten-
den Blvd., Rochester, NY 14620
ZIGMOND, RICHARD E., Department of Pharmacology, Harvard Medical School, 250 Long-
wood Ave., Boston, MA 02 1 1 5
ZIMMERBERG, JOSHUA J., Bldg. 12A, Room 2007, NIH, Bethesda, MD 20892
ZOTTOLI, STEVEN J., Department of Biology, Williams College, Williamstown MA 01267
ZUCKER, ROBERT S., Department of Physiology, University of California, Berkeley, CA 94720
28
MARINE BIOLOGICAL LABORATORY
ASSOCIATE MEMBERS
ACKROYD, DR. FREDERICK W.
ADAMS, DR. PAUL
ADELBERG, DR. AND MRS. EDWARD A.
AHEARN, MR. AND MRS. DAVID
ALDEN, MR. JOHN M.
ALLEN, Miss CAMILLA K.
ALLEN, DR. NINA S.
AMON, MR. CARL H. JR.
ANDERSON, MR. J. GREGORY
ANDERSON, DRS. JAMES L. AND
HELENE M.
ARMSTRONG, DR. AND MRS. SAMUEL C.
ARNOLD, MRS. Lois
ATWOOD, DR. AND MRS. KJMBALL C., Ill
AYERS, MR. DONALD
BAKER, MRS. C. L.
BALL, MRS. ERIC G.
BALLANTINE, DR. AND MRS. H. T., JR.
BANG, MRS. FREDERIK B.
BANG, Miss MOLLY
BANKS, MR. AND MRS. WILLIAM L.
BARKIN, MR. AND MRS. MEL A.
BARROWS, MRS. ALBERT W.
BAUM, MR. RICHARD T.
BEERS, DR. AND MRS. YARDLEY
BELESIR, MR. TASOS
BENNETT, DR. AND MRS. MICHAEL V. L.
BERG, MR. C. JOHN
BERNHEIMER, DR. ALAN W.
BERNSTEIN, MR. AND MRS. NORMAN
BERWIND, MR. DAVID McM.
BICKER, MR. ALVIN
BIGELOW, MRS. ROBERT O.
BIRD, MR. WILLIAM R.
BLECK, DR. THOMAS B.
BOCHE, MR. DAVID
BODEEN, MR. AND MRS. GEORGE H.
BOETTIGER, DR. AND MRS. EDWARD G.
BOETTIGER, MRS. JULIE
BOLTON, MR. AND MRS. THOMAS C.
BONN, MR. AND MRS. THEODORE H.
BORGESE, DR. AND MRS. THOMAS
BOWLES, DR. AND MRS. FRANCIS P.
BRADLEY, DR. AND MRS. CHARLES C.
BRADLEY, MR. RICHARD
BROWN, MRS. FRANK A., JR.
BROWN, MR. AND MRS. HENRY
BROWN, MR. AND MRS. JAMES
BROWN, MRS. NEIL
BROWN, DR. AND MRS. THORNTON
BROYLES, DR. ROBERT H.
BUCK, DR. AND MRS. JOHN B.
BUCKLEY, MR. GEORGE D.
BUNTS, MR. AND MRS. FRANK E.
BURT, MRS. CHARLES E.
BUSH, DR. LOUISE
BUXTON, MR. AND MRS. BRUCE E.
BUXTON, MR. E. BREWSTER
CALKINS, MR. AND MRS. G. N., JR.
CAMPBELL, DR. AND MRS. DAVID G.
CASE, DR. AND MRS. JAMES
CARLSON, DR. AND MRS. FRANCIS
CARLTON, MR. AND MRS. WINSLOW G.
CHANDLER, MR. ROBERT
CHASE, MR. TOM H.
CHILD, DR. AND MRS. FRANK M.
CHURCH, DR. WESLEY
CLAFF, MR. AND MRS. MARK
CLARK, DR. AND MRS. ARNOLD
CLARK, MR. AND MRS. HAYS
CLARK, MR. AND MRS. JAMES McC.
CLARK, MRS. LEONARD B.
CLARK, MR. AND MRS. LEROY, JR.
CLARKE, DR. BARBARA J.
CLEMENT, MRS. ANTHONY
CLOWES FUND, INC.
CLOWES, DR. AND MRS. ALEXANDER W.
CLOWES, MR. ALLEN W.
CLOWES, DR. AND MRS. G. H. A., JR.
COBURN, MR. AND MRS. LAWRENCE
COLEMAN, DR. AND MRS. JOHN
CONNELL, MR. AND MRS. W. J.
COOK, DR. AND MRS. PAUL W., JR.
COPELAND, DR. AND MRS. D. EUGENE
COPELAND, MR. FREDERICK C.
COPELAND, MR. AND MRS. PRESTON S.
COSTELLO, MRS. DONALD P.
CRABB, MR. AND MRS. DAVID L.
CRAIN, MR. AND MRS. MELVIN C.
CRAMER, MR. AND MRS. IAN D. W.
CRANE, MRS. JOHN O.
CRANE, JOSEPHINE B., FOUNDATION
CRANE, MR. THOMAS S.
CROSS, MR. AND MRS. NORMAN C.
CROSSLEY, Miss DOROTHY
CROSSLEY, Miss HELEN
CROWELL, DR. AND MRS. SEARS
CURRIER, MR. AND MRS. DAVID L.
DAIGNAULT, MR. AND MRS.
ALEXANDER T.
DANIELS, MR. AND MRS. BRUCE G.
DAVIDSON, DR. MORTON
DAVIS, MR. AND MRS. JOEL P.
DAY, MR. AND MRS. POMEROY
DECKER, DR. RAYMOND F.
DEMELLO, MR. JOHN
DiBERARDiNO, DR. MARIE A.
DICKSON, DR. WILLIAM A.
MEMBERS OF THE CORPORATION
29
DIEROLF, DR. SHIRLEY H.
DRUMMEY, MR. AND MRS. CHARLES E.
DRUMMEY, MR. TODD A.
DuBois, DR. AND MRS. ARTHUR B.
DUDLEY, DR. PATRICIA
DUPONT, MR. A. FELIX, JR.
DUTTON, MR. RODERICK L.
EBERT, DR. AND MRS. JAMES D.
EGLOFF, DR. AND MRS. F. R. L.
ELLIOTT, MRS. ALFRED M.
ENOS, MR. EDWARD, JR.
EPPEL, MR. AND MRS. DUDLEY
ESTABROOK, MR. GORDON C.
EVANS, MR. AND MRS. DUDLEY
FARLEY, Miss JOAN
FARMER, Miss MARY
FAULL, MR. J. HORACE, JR.
FERGUSON, DR. AND MRS. JAMES J., JR.
FISHER, MRS. B. C.
FISHER, MR. FREDERICK S., Ill
FISHER, DR. AND MRS. SAUL H.
FORBES, MR. JOHN M.
FORD, MR. JOHN H.
FRANCIS, MR. AND MRS. LEWIS W., JR.
FRENKEL, DR. KRYSTINA
FRIBOURGH, DR. JAMES H.
FRIENDSHIP FUND
FRIES, DR. AND MRS. E. F. B.
FYE, DR. AND MRS. PAUL M.
GABRIEL, DR. AND MRS. MORDECAI L.
GAGNON, MR. MICHAEL
GAISER, DR. AND MRS. DAVID W.
GALLAGHER, MR. ROBERT O.
GARFIELD, Miss ELEANOR
GARREY, DR. WALTER E.
GELLIS, DR. AND MRS. SYDNEY
GEPHARD, MR. STEPHEN
GERMAN, DR. AND MRS. JAMES L., Ill
GEWECKE, MR. AND MRS. THOMAS H.
GlFFORD, DR. AND MRS. CAMERON
GIFFORD, MR. JOHN A.
GlFFORD, DR. AND MRS. PROSSER
GILBERT, DRS. DANIEL L. AND CLAIRE
GILBERT, MRS. CARL J.
GILDEA, DR. MARGARET C. L.
GILLETTE, MR. AND MRS. ROBERT S.
GLAD, MR. ROBERT
GLASS, DR. AND MRS. H. BENTLEY
GLAZEBROOK, MR. JAMES
GLAZEBROOK, MRS. JAMES R.
GOLDMAN, MRS. MARY
GOLDRING, MR. MICHAEL
GOLDSTEIN, DR. AND MRS. MOISE H., JR.
GOODWIN, MR. AND MRS. CHARLES
GOULD, Miss EDITH
GRACE, Miss PRISCILLA B.
GRANT, DR. AND MRS. PHILIP
GRASSLE, MRS. J. F.
GREEN, MRS. DAVIS CRANE
GREEN, Miss GLADYS M.
GREER, MR. AND MRS. W. H., JR.
GRIFFITH, DR. AND MRS. B. HEROLD
GROSCH, DR. AND MRS. DANIEL S.
GROSS, MRS. MONA
GUNNING, MR. AND MRS. ROBERT
HAAKONSEN, DR. HARRY O.
HAIGH, MR. AND MRS. RICHARD H.
HALL, MR. AND MRS. PETER A.
HALL, MR. WARREN C.
HALVORSON, DR. AND MRS. HARLYN O.
HAMSTROM, Miss MARY ELIZABETH
HARVEY, DR. AND MRS. RICHARD B.
HASSETT, MR. AND MRS. CHARLES
HASTINGS, DR. AND MRS. J. WOODLAND
HAUBRICH, MR. ROBERT R.
HAY, MR. JOHN
HAYS, DR. DAVIDS.
HEDBERG, MRS. FRANCES
HEDBERG, DR. MARY
HENLEY, DR. CATHERINE
HERSEY, MRS. GEORGE L.
HIATT, DR. AND MRS. HOWARD
HICHAR, MRS. BARBARA
HILL, MRS. SAMUEL E.
HlRSCHFELD, MRS. NATHAN B.
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 C. G.
HOUSTON, MR. AND MRS. HOWARD E.
HOWARD, MR. AND MRS. L. L.
HOYLE, DR. MERRILL C.
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
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., Ill
JONES, MR. AND MRS. FREDERICK, II
JONES, MR. FREDERICK S., Ill
JORDAN, DR. AND MRS. EDWIN P.
KAAN, DR. HELEN W.
KAHLER, MR. AND MRS. GEORGE A.
30
MARINE BIOLOGICAL LABORATORY
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
KEOUGHAN, Miss PATRICIA
KETCHUM, MRS. PAUL
KlEN, MR. AND MRS. PlETER
KINNARD, MRS. L. RICHARD
KISSAM, MR. WILLIAM M.
KIVY, DR. AND MRS. PETER
KOHN, DR. AND MRS. HENRY I.
ROLLER, DR. LEWIS R.
KORGEN, DR. BEN J.
KUFFLER, MRS. STEPHEN W.
LAFFERTY, Miss NANCY
LARMON, MR. JAY
LASTER, DR. AND MRS. LEONARD
LAUFER, DR. AND MRS. HANS
LAVIGNE, MRS. RICHARD J.
LAWRENCE, MR. FREDERICK V.
LAWRENCE, MR. AND MRS. WILLIAM
LAZAROW, DR. PAUL
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
LEPROHON, MR. JOSEPH
LEVINE, MR. JOSEPH
LEVINE, DR. AND MRS. RACHMIEL
LEVY, MR. STEPHEN R.
LINDNER, MR. TIMOTHY P.
LITTLE, MRS. ELBERT
LIVINGSTONE, MR. AND MRS. ROBERT
LOEB, MRS. ROBERT F.
LOVELL, MR. AND MRS. HOLLIS R.
Low, Miss DORIS
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
MAHLER, MRS. SUZANNE
MANSWORTH, Miss MARIE
MARSH, DR. AND MRS. JULIAN
MARTYNA, MR. AND MRS. JOSEPH C.
MASON, MR. APPLETON
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, MRS. Lois
MCCUSKER, MR. AND MRS. PAUL T.
MCELROY, MRS. NELLA W.
MCILWAIN, DR. SUSAN G.
MCLARDY, DR. TURNER
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.
MILLER, MR. AND MRS. PAUL
MIXTER, MR. AND MRS. WILLIAM J., JR.
MIZELL, DR. AND MRS. MERLE
MONROY, MRS. ALBERTO
MONTGOMERY, DR. AND MRS.
CHARLES H.
MONTGOMERY, DR. AND MRS.
RAYMOND B.
MOOG, DR. FLORENCE
MOORE, DRS. JOHN AND BETTY
MORGAN, Miss AMY
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
NACE, MR. PAUL F., JR.
NELSON, DR. AND 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. BARRY
NORRIS, MR. AND MRS. JOHN A.
NORRIS, MR. WILLIAM
O'HERRON, MR. AND MRS. JONATHAN
OLSZOWKA, Miss JANICE S.
O'NEiL, MR. AND MRS. BARRY T.
C/RAND, MR. AND MRS. MICHAEL
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, DR. CHARLES
PARMENTER, Miss CAROLYN L.
PELTZ, MR. AND MRS. WILLIAM L.
MEMBERS OF THE CORPORATION
31
PENDERGAST, MRS. CLAUDIA
PENDLETON, DR. AND MRS. MURRAY E.
PENNINGTON, Miss ANNE H.
PERKINS, MR. AND MRS. COURTLAND D.
PERSON, DR. AND MRS. PHILIP
PETERSON, MR. AND MRS. E. GUNNAR
PETERSON, MR. AND MRS. E. JOEL
PETERSON, MR. RAYMOND W.
PETTY, MR. RICHARD F.
PETTY, MR. WILLIAM
PFEIFFER, MR. AND MRS. JOHN
PLOUGH, MR. AND MRS. GEORGE H.
POINTE, MR. ALBERT
POINTE, MR. CHARLES
POTHIER, DR. AND MRS. AUBREY
PORTER, DR. AND MRS. KEITH R.
PRESS, DRS. FRANK AND BILLIE
PROSKAUER, MR. RICHARD
PROSKAUER, MR. JOSEPH H.
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
REESE, Miss BONNIE
REINGOLD, MR. STEPHEN C.
REYNOLDS, DR. AND MRS. GEORGE
REYNOLDS, MR. ROBERT M.
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. ROBERT
Ross, DR. VIRGINIA
ROTH, DR. AND MRS. STEPHEN
ROWE, MR. DON
ROWE, MR. AND MRS. WILLIAM S.
RUBIN, DR. JOSEPH
RUGH, MRS. ROBERTS
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. CLAYTON C.
SEARS, MR. AND MRS. HAROLD B.
SEARS, MR. HAROLD H.
SEAVER, MR. GEORGE
SEGAL, DR. AND MRS. SHELDON J.
SENFT, DR. AND MRS. ALFRED
SHAPIRO, MRS. HARRIET S.
SHAPLEY, DR. ROBERT
SHEMIN, DR. AND MRS. DAVID
SHEPRO, DR. AND MRS. DAVID
SIMMONS, MR. TIM
SINGER, MR. AND MRS. DANIEL M.
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
SPOTTE, MR. STEPHEN
STEELE, MRS. JOHN H.
STEIN, MR. RONALD
STEINBACH, MRS. H. BURR
STETSON, MRS. THOMAS J.
STETTEN, DR. AND MRS. DEWITT, JR.
STETTEN, DR. GAIL
STEWART, MR. AND MRS. PETER
STONE, MR. ANDREW G.
STREHLER, DR. AND MRS. BERNARD
STUNKARD, DR. HORACE
SUDDITH, MR. WILLIAM
SWANSON, DR. AND MRS. CARL P.
SWOPE, MRS. GERARD, JR.
SWOPE, MR. AND MRS. GERARD L.
SZENT-GYORGYI, DR. AND MRS. ANDREW
TABOR, MR. GEORGE H.
TAYLOR, MR. JAMES K.
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
TUCKER, Miss RUTH
TULLY, MR. AND MRS. GORDON F.
ULBRICH, MRS. MARY STEINBACH
VALOIS, MR. AND MRS. JOHN
VAN BUREN, MRS. HAROLD
32
MARINE BIOLOGICAL LABORATORY
VAN HOLDE, MRS. KENSAL E.
VEEDER, MRS. RONALD A.
VINCENT, DR. WALTER S.
WAGNER, MR. MARK
WAKSMAN, DR. AND MRS. BYRON H.
WARD, DR. ROBERT T.
WARE, MR. AND MRS. J. LINDSAY
WARREN, DR. HENRY B.
WARREN, DR. AND MRS. LEONARD
WATT, MR. AND MRS. JOHN B.
WEEKS, MR. AND MRS. JOHN T.
WEINSTEIN, Miss NANCY B.
WEISBERG, MR. AND MRS. ALFRED M.
WEISS, MR. AND MRS. MALCOLM
WHEELER, DR. AND MRS. PAUL S.
WHITEHEAD, MR. AND MRS. FRED
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.
WINSTEN, DR. JAY A.
WITTING, Miss JOYCE
WOLFINSOHN, MRS. WOLFE
WOODWELL, DR. AND MRS. GEORGE M.
YNTEMA, MRS. CHESTER L.
YOUNG-WALLACE, Miss NINA L.
ZINN, DR. DONALD J.
ZIPF, DR. ELIZABETH
ZWILLING, MRS. EDGAR
III. CERTIFICATE OF ORGANIZATION
(On File in the Office of the Secretary of the Commonwealth)
No. 3 1 70
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 Trust-
ees 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 amendment thereof and in addition thereto.
The name by which the Corporation shall be known is THE MARINE BIOLOGICAL LABO-
RATORY.
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.
ARTICLES OF AMENDMENT 33
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, 1 888 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)
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, activi-
ties or affairs of the corporation.
"Except as otherwise specifically provided by the Bylaws of the corporation, meet-
ings 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.
34 MARINE BIOLOGICAL LABORATORY
(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 sation 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, na-
tional and ethnic origin to all the rights, privileges, programs and activities generally accorded
or made available to students in its courses. It does not discriminate on the basis of race, color,
sex, national and ethnic origin in employment, administration or its educational policies, ad-
missions 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 proce-
dures, not inconsistent with law or these Bylaws, as may be determined by said Board of Trust-
ees. 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 Trust-
ees 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.
BYLAWS 35
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 Members of any
adjournment.
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.
VIII. (A) There shall be four groups of Trustees:
( 1 ) Trustees (the "Corporate Trustees") elected by the Members according to such proce-
dures, 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
each 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 Trea-
surer, 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
36 MARINE BIOLOGICAL LABORATORY
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 rilled 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 Corpora-
tion. 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
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 condi-
tions 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 Organi-
zation or these Bylaws they are prohibited from delegating. The members of any such commit-
tee shall have such tenure and duties as the Trustees shall determine; provided that the Invest-
ment 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 Treasurer
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
BYLAWS 37
Committee 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
Committee. 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 Execu-
tive 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,
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
committee 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 permitted
by law.
XIII. The consent of every 1 rustee shall be necessary to dissolution of the Marine Biologi-
cal 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 meet-
ing, 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 mak-
ing, 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.
38 MARINE BIOLOGICAL LABORATORY
XV. The account of the Treasurer shall be audited annually by a certified public ac-
countant.
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 indi-
rectly has any interest, as a shareholder, creditor or otherwise, against all liabilities and ex-
penses (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 proceed-
ing 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 ulti-
mately 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 respec-
tive 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
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, includ-
ing, 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 Corporation 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 indemnifica-
tion 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.
REPORT OF THE DIRECTOR 39
VI. REPORT OF THE DIRECTOR
"The cook was a good cook, as cooks go; and as cooks go she went.'
— Saki
Transition
It may be a violation of the best taste, but I believe that I should begin this report,
my last from the Director's office of the MBL, on a personal note. I do so not because
personal matters are worthy of the first position, but because a change of the Director-
ship of the MBL is. Since a good many Corporation Members have only the Annual
Report in The Biological Bulletin as a source of comprehensive information on the
Laboratory's work of the prior year, this seems to me the proper place and position
for a statement on the change.
I wrote the Director's Report for 1 985-6 in the Candle House. The window before
which the computer monitor was placed overlooks an angle of Great Harbor and
Vineyard Sound that I know as well as I know the shape of my hand. This report is
being written in Charlottesville, Virginia, overlooking Thomas Jefferson's Academi-
cal Village, the heart of the University of Virginia. In June of 1986 I announced
to the Executive Committee, and later that summer to the Trustees and the MBL
community as a whole, that I would be leaving the Directorship and the Laboratory
at the end of October, 1986. I had accepted, just prior to the first announcement,
appointment in November as Vice President and Provost at the University of
Virginia.
My effort was at the time, as it is now for readers not reached by the earlier one,
to urge that this decision was made for reasons wholly positive as regards the MBL
and my relationship with it — as of course it has been for Virginia. I found the Labora-
tory in — and no self-congratulation is intended — a much stronger position that
might have been expected at the time of my coming to the Directorship. It now had
enhanced administrative resources, vastly better physical facilities, a development
and public relations program that was the envy of larger and richer institutions,
heightened national and international recognition for the quality of its research and
instructional programs, and a renewed loyalty of its Corporation membership.
I had never intended to remain a full-time (or better, double-time) administra-
tor—for that is what, given the limits of my talents, the MBL job quickly became—
beyond the end of a second term, which would have been in 1988. But I had no
reason to be ashamed of what had been accomplished in nine years of tenure of the
office. I would have stayed out the term and no more. But the offer from Virginia was
a very attractive one, and it came from a University I had already served in other
capacities and for whose intellectual life, Jeffersonian traditions, and physical place I
have deep admiration. It had always been my intention to return to university life
after fulfilling a deeply felt obligation to the MBL (where, in reality, I had become a
biologist). In those circumstances the result of a cost/benefit analysis was clear: no
great harm would be done to the Laboratory by my leaving it at that time, provided
that a good transition of leadership could be managed and the Centennial planning
could be kept on track; and the chances of another university place like as the one at
Virginia appearing would diminish after 1988.
Thus it is that I am here and the MBL is there. I miss the Laboratory much more
than I miss being its Director. There is every reason to believe that, given good health
and some luck, opportunities to serve it in other ways will arise. I shall take advantage
of them.
40 MARINE BIOLOGICAL LABORATORY
Key to the decision was a judgment as to the effectiveness with which a transition
to new leadership could be made in 1 986-7. Although I had as yet no way of identify-
ing the eventual actors therein, my judgment was that the thing could and would be
done. And so it proved. Fellow embryologist Richard Whittaker agreed generously
to an infringement of his productive research in order to serve as acting Director. No
better-qualified person could have been found for this role.
A distinguished search committee was quickly empaneled, fulfilled its responsibil-
ity for an exhaustive national search, and was able eventually to recruit as the MBL's
next Director Harlyn O. Halvorson. His contributions to microbiology are recog-
nized worldwide, and his activities at the MBL — as Trustee, Executive Committee
Member, Course Director, and advisor to earlier Directors — comprise an unmatched
record of dedicated and effective service.
The Centennial Committee, already in place at the time of my decision, was obvi-
ously off to a good start, and its programs for the Centennial year, which begins now
(August, 1 987), are a fine amalgam of high scientific standards, good taste, and poten-
tial outreach to a larger public.
I miss very much, in short, the view out my window from Candle House 301, but
I feel justified in having had no fears for the health of the institution to which I had
devoted so much physical and emotional energy in the vicinity of that view.
Management
Changing governance and management in an essentially academic institution
such as the MBL is difficult in principle. It is made more difficult when there is a
hundred-year history of outstanding achievement; and the difficulties multiply fur-
ther when the organization is as idiosyncratic (or "horizontal," as Chairman Gifford
likes to describe it) as is ours. Change was and remains necessary, nevertheless, as I
have urged in earlier Director's Reports and before what seem to me now numberless
meetings of Trustees and committees. Nothing that has happened during the last,
transition-preoccupied year has interfered — I am delighted to report — with the pro-
cesses of orderly management change set in motion several years ago.
The Trustees — Board and Corporate — have been brought much more closely
into touch with operations management and decision making than ever before. In-
deed, this change has accelerated since the last Director's Report was published. The
new Audit Committee, and other Trustees' committees functioning under revised
charges, have rendered invaluable service to the Laboratory and its paid administra-
tion. As a consequence, our financial and political positions have been strengthened
visibly, even since August of 1986. The Committee on Laboratory Goals, chaired
conscientiously by Gerald Fischbach and charged a year ago, has produced a short
but forceful report on those goals — on what they should be, and on what steps should
be taken toward their accomplishment.
I support their recommendations with enthusiasm. This was an accomplished,
critical, and independent committee: it is a great pleasure to note that had I written
the report (to succeed my very solo effort of 1979), it might have been in a different
prose style, but its content would have been indistinguishable from what is now be-
fore the MBL community for analysis, debate, and — action.
Treasurer David Currier, who served the Laboratory so handsomely in that posi-
tion and whose banking skills made possible the splendid new MBL cottage develop-
ment, retired with the well-earned thanks of the community and has been replaced
by Robert Manz. Mr. Manz brings to the treasurer's responsibilities more than out-
standing education, technical, and personal credentials: he is a former officer of Coo-
REPORT OF THE DIRECTOR 41
pers and Lybrand, our external auditors for many years, and was a leading member
of the team assigned to the MBL account. He knows the Laboratory as well as—
perhaps better than — any full-time employee. The Treasurer's being another of those
critical jobs for which the MBL must depend upon volunteers, i.e., upon good will,
we are doubly lucky to have the good will of Robert Manz.
Two new management positions were designed, funded, and filled in the course
of the year. Richard D. Cutler became the Laboratory's first Facilities, Project, and
Services Manager, and LouAnn D. King accepted appointment as Coordinator of
Conferences and Housing. These attractive and experienced people have taken on
challenging responsibilities subdivided and redefined from among the host of such
carried by former General Manager Homer Smith.
Mr. Smith's retirement could certainly be described by the cliche, "The End Of
An Era," and a pretty long era at that. But the description would fail. Homer has left
his job, to be sure, but he has not left Woods Hole nor the MBL. There is no reason
to believe that the "era" of his involvement with the Laboratory has ended; and we
wish him and Cynthia Smith a retirement — if that word may be used — of continued
good health and undiminished activity. That, I am sure, is the way they want it to be.
Donald Ayers, finally, now directs the fully functional Development Office, and
we can be confident that his programs, aided and additionally monitored by Lisa
Thimas (formerly Assistant to the Director), will flourish during the Centennial Year
before us.
Remarkably, these are a mere sample, not the totality of appointments and
change processes that took place during the transition year. Let no one be concerned
about a let-down of effort or a diminution of those management skills of which the
MBL has been so much in need. All that we need to be concerned about is the means
of increasing further the rate of positive change.
Systems
In that connection it is noteworthy that three of the critical systems upon which
those who must operate, and make decisions for, the MBL have been changed and
improved during the year past. Most important, the many roadblocks, internal as
well as external, impeding progress toward a rational system of overhead recovery
have been eliminated. The system is now in operation, and although it works no
better than those of our peer institutions, it is no worse. The high optimism implicit
in such a negative statement will be understood by all who have grappled in decades
past with the problem of reimbursing the Laboratory for its costs in housing and
supporting research.
A job classification system was designed, discussed widely, and put in place for all
MBL employees. This had been the majority wish for many years, and its fulfillment
has indeed brought a measure of regularity and central accountability to the manage-
ment of operations and personnel. The system includes not only a set of objective
job descriptions and grades, but also processes for the hearing and adjudication of
disputes and grievances. Already called upon for service in that connection has been
the Classification Review Board established for the purpose and chaired, ably as al-
ways, by Joan Howard.
Last but not least, the Controller's department now has a greatly improved system
of accounts and data management, aided by the appointment of an Assistant Control-
ler with excellent accounting skills and a resident specialist in electronic data process-
ing. It is not amiss to note that these changes are a direct consequence of the enhanced
Trustee oversight initiated two years ago, to which I have referred above.
42 MARINE BIOLOGICAL LABORATORY
Research and education
Summer research activities and accomplishments were at their now accustomed
high levels in the summer of 1986 and, as will be evident to readers of this report by
the time it has been published, so will they be in 1 987. There are very few laboratories
or library accommodations unspoken for in summer at the MBL. The only concern
I have heard expressed from time to time is that not enough applicants are rejected,
or, to put it in the way it is usually phrased, that the MBL ought to have more of a
choice among potential summer scientists. Perhaps so; but the financial realities and
demography of those disciplines practiced by MBL summer investigators speak
differently to me. I believe that we do very well to have a significant percentage of the
most honored neuroscientists, cell biologists, developmental biologists, microsco-
pists, and the like here at the MBL every summer, and it is insurance against stagna-
tion that we can admit most of those others, not yet so honored perhaps, who have
legitimate research to perform here. And lest the reader believe what is sometimes —
unfairly and untruthfully — asserted, that the MBL is a club for its "regulars" and
nothing more, let him inspect the roster of last summer's MBL Fellows and indepen-
dent investigators for the new and the still-young. The evidence is heartening. No
other organization can boast so large and so diverse an assembly of biologists engaged
upon serious research for a meaningful part of the year.
The year-round research program, focus of the plan presented by the Committee
on Laboratory Goals, ended the year in about the same configuration as at the begin-
ning. It was as large an enterprise as the MBL can accommodate within existing build-
ings; it was well-funded; and it was steadily productive. As I have said elsewhere, there
is no university for which such a group, collected together as a Biology Department,
would not be a prize.
There are, however, changes imminent. The National Institute for Neurological
and Communicative Diseases and Stroke has decided, for what are considered in
Bethesda to be good reasons, on an eventual recall of at least the bulk of its on-
location program at the MBL. It is possible that a part of this effort will remain for
several years, but under the existing directives, most of the program will revert to
permanent residence on the NIH campus, with summer research at the MBL. Balanc-
ing this, there have been selections from among excellent applicant investigators for
year-round accommodation, so that the size of the resident research program will not
change very much in the near term, nor will its very high quality overall.
If, however, the Corporation and Trustees elect to implement the recommenda-
tions of the Fischbach committee, there will be major change indeed, and it will have
to be accompanied by new methods of funding and supporting year-round scientists,
new programs, a net increase in size, and large additions to the inventory of research
space and general-use facilities.
The instructional program, whose unique contribution to the scientific manpower
of this nation has been recognized by astonishingly (for these times) generous support
from government and private agencies, fared well in 1 986 and will clearly continue
thus in 1987. In no small part this is due to the far-sighted support of such private
donors as the Markey Trust, the Pew Memorial Trust, the Grass Foundation, and
the Klingenstein Fund. But success and a tough-minded maintenance of the highest
standards of quality are also testimony to the very spirit of the MBL, inherited and
still vigorous, as established in its teaching programs by the Founders. In no small
measure the smooth operation of our complex instructional enterprise — much more
complex, in important ways, than the mounting of courses in a university — has de-
pended, and will probably continue to depend, upon three things: ( 1 ) the flow of
financial support for indirect as well as direct costs from private donors; (2) the collab-
REPORT OF THE DIRECTOR 43
oration, especially via equipment loans, of the world's leading manufacturers of sci-
entific instruments; and (3) wise, artful, and minimally intrusive management of the
courses and their people, as exemplified in the work of Joan Howard and her ever-
helpful staff in the Office of Sponsored Programs.
Successful a story as this is, it will not remain so without continuous effort. It was
a principle of the last administration, as it was of the MBL's first, that the instructional
program shares the first priority with research, and that the teaching is as much on
the moving frontier of our science as is the research of any particular laboratory. The
principle will continue, I hope, to be held. Thus far the signs are good. By way of
example, I might cite an unusual and already noteworthy collaboration of the MBL
and the University of Georgia in the teaching of Plant Cell and Molecular Biology.
And if the current plans for the next version of Embryology are implemented, it will
be a further signal to the effect that curricular and organizational adventurousness
need not be absent from teaching by an eminent faculty, to the world's most able
classes of young biologists.
If all the above has the scent of hyperbole, I apologize but also deny any such
purpose. The MBL is a very remarkable organism: the bare facts upon which this
summary is based are printed in the accompanying pages of the Annual Report and
in other MBL publications. I have simply written a summary, with a few laudatory
adjectives where they are clearly justified. We tend easily to forget, as intimates of
quality, how special a thing it is in the world. We do not forget problems with the
same celerity: those, like the crying of a baby, evolve a sound that cannot be ignored.
Albert Szent-Gyorgyi
Among the score of Nobelists associated with the Laboratory during the two or
three decades past, none made such a mark upon the day-to-day life of the place as
did Albert Szent-Gyorgyi, who died at a ripe old age during the year. Of course that
mark was to some extent a consequence of Szent-Gyorgyi's year-round residence in
Woods Hole and at the MBL, from a time when his Institute for Muscle Research
was virtually the only active group during the non-summer months until a few years
ago, when it was well-eclipsed, in size and in visibility, by other year-round labora-
tories. But his influence did not depend solely upon presence. Prof's was a mind and
a personality of most unusual strength. His intellectual power and personal charm
were merely facets of the whole man, who was also possessed of moral power, a re-
markable persuasiveness, an abundance of love and good cheer, and overlying all, an
aesthetic imperative that drove him to the still-unexplored for its beauty first, and is
utility second. During the height of his career, when I was a student here, he was for
many of us the paradigm of the scientist as the spearhead of culture. Throughout his
long life, even at the very end, when he and I could converse only in shouts, he was
for me and, I know, for many others, the very model of a man.
Coda
The MBL nurtures us all, as men and women, as extenders of culture, as investiga-
tors, as teachers of the best science to the best students. Long may it flourish; and to
it a happy hundredth birthday!
VII. REPORT OF THE TREASURER
This is my first report to you as Treasurer. Although I have been at the job for
almost a year now, this is my first chance to say that I am privileged and honored to
have an opportunity to contribute to this excellent and exciting institution. I look
44 MARINE BIOLOGICAL LABORATORY
forward to working with the staff, Executive Committee, Trustees, and Corporation
as we move into the Laboratory's Second Century.
The financial statements of the Laboratory for the year ended December 31,1 986,
follow this report. Before I comment on the financial results for the year, I must
discuss some changes in presentation that have been made to the financial statements
to reflect actions taken by the Corporation and the Executive Committee in the last
year and to more clearly present the financial position of the Laboratory.
In the Statement of Support, Revenues, Expenses, and Changes in Fund Balances
(Statement of Support), a separate column has been provided for the newly estab-
lished Housing Enterprises Fund within the Current Unrestricted Fund. The Balance
of Operations for this fund of $ 1 12,294 has been transferred to a Reserve for Repairs
and Replacements restricted to expenditure on housing. This is a major step forward
for the Laboratory in providing for its financial future; I applaud the wisdom of your
decision to establish this fund.
The financial statements no longer show the assets or results of operations of the
Retirement Funds. These funds are not actually assets of the MBL but are owned by
the Laboratory's pension plan, so they should not appear in the Laboratory's financial
statements but in those of the Plan. To date there have been no separately issued
financial statements of the Retirement Plan because of its size. The Plan has now
grown large enough that provisions of the Employees' Retirement Income Security
Act require separate financial statements of the Plan; these have been prepared, and
the Retirement Fund has been removed from the Laboratory's financial statements.
Also affecting pension accounting, the Laboratory has adopted the new require-
ments of the Financial Accounting Standards Board on accounting for pension ex-
pense of the Laboratory (i.e., its obligation to contribute to the plan). The new re-
quirements seek to more accurately reflect the impact of changes in investment and
annuity market conditions as well as employment and actuarial expectations of the
employer's pension obligation. For the Laboratory this has meant a reduction in pen-
sion expense from $ 1 42,833 in 1985 to $82,682 in 1 986, chiefly because of the favor-
able performance of the Retirement Plan's investment portfolio.
The Balance Sheet now reflects the market value of the Laboratory's invested
funds, rather than their book value. I have recommended this change so that you will
always have before you the actual market value of the endowment rather than a book
number which reflects only the timing of past donations and the results of past invest-
ment activity.
I trust that these comments will assist you as you examine the Laboratory's Finan-
cial statements.
The results of operations for 1986 show a picture of continued current operating
strength, with some fluctuations that bear watching. They suggest some significant
improvements in the long-term strength of the Laboratory — if trends begun this year
can be sustained and expanded — and they point to an agenda for future actions.
Total support and revenues increased from 1985 to 1986 by $272,488 while ex-
penses increased by $269,648.
Within support and revenues, gains in gifts, recovery in indirect costs from the
summer program (laboratory fees), and dormitory and dining fees were offset by de-
clines in direct support for year-round research and the associated recovery of indirect
costs, and slight declines in the income of Research Services and Marine Resources.
It is encouraging to see some strength in fee income — it suggests that the Laboratory
renders a measurable service which can be directly supported by the users of that
service — but for the same reason it is discouraging to see the declines in Research
Services and Marine Resources income. I have recommended to the Director that a
REPORT OF THE TREASURER 45
careful examination of all the fee for service income of the Laboratory be made in
order to make the best possible match between service and income, and to guarantee
the future economic viability of our services.
Gifts increased by more than $200,000 in 1986. We have continued to receive
strong support from the Pew and Markey foundations for the instructional program;
we made significant strides in the Mellon match gift for Library endowment; and
we received a $200,000 gift from the Monsanto Corporation in anticipation of our
Centennial celebration. Such dedicated support allows your laboratory to maintain
the excellence of its programs. As your Treasurer, I must point out, however, that we
are perilously dependent on the generosity of donors of gifts for current use. In order
to achieve the assurance of continued excellent programs, we need to seek a dramatic
increase in our endowment.
Within the expense categories, I call your attention to the increases in housing and
depreciation expense, that in aggregate, amount to almost $250,000. Of this amount,
approximately $133,000 is attributable to the additional housing units at Memorial
Circle and an additional $76,000 was expensed as administrative costs, thus complet-
ing our planned "full costing" of the separate Housing Enterprises Fund. More sig-
nificantly, if you look down the column entitled "Housing Enterprises Fund," you
will note that for the first time, the MBL, as projected and previously mentioned, has
funded $ 1 12,294 in depreciation costs associated with the housing enterprise. These
funds are being set aside to help finance major future capital improvements in the
housing facilities.
We again ended the year with an excess of revenues over expenses in the current
unrestricted fund ($70,590). You should thank your Controller, John Speer, for his
role in achieving this result. The Laboratory is well served by John's vigilance over
the operating budget and his ability to see clouds on the financial horizon and recom-
mend a course long before the storm strikes.
As your Treasurer, I will never use the word "surplus" in connection with an
excess of revenues over expenses until the Laboratory has been able to use that excess
to fund depreciation on its plant. As I told the Trustees last winter and hope to dem-
onstrate to the rest of the Corporation, the annual "surpluses" of the Laboratory are
wiped out when depreciation is taken into account. We have in fact been able to
maintain the quality of our physical plant through the generosity of our donors, but
this means that the heroic capital fund raising efforts of our directors in the last ten
years have been required to maintain rather than to improve the quality of our plant.
I believe we must set as a goal the funding of a significant portion of our deprecia-
tion expense from operating revenues. This year we took a modest first step towards
that goal and transferred $33,650 from the current unrestricted fund to a reserve for
replacements. We must do much more in the future.
In this report I have tried to indicate those aspects of our financial condition that
merit our attention and will require concerted action. I have no doubt left some of
your questions on the financial performance of 1986 unanswered here, but I welcome
them directly and will do my best to respond.
46 MARINE BIOLOGICAL LABORATORY
certified public accountants One Post Office Square in principal areas ot the world
Boston, Mass 02109
telephone (617) 574-5000
TWX 710-321-0489
telex 6817018
&Lybrand
To the Trustees of
Marine Biological Laboratory
Woods Hole, Massachusetts
We have examined the balance sheet of Marine Biological
Laboratory as of December 31, 1986 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 • 1985, 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, 1986 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, except for the changes, with which we concur, in the
method of accounting for investments as described in Note C, the method of
accounting for pension expense as described in Note E and the method of
accounting for pension funds as described in Note J.
Very truly yours,
o
Boston, Massachusetts V—OOOCTO f,
April 18, 1987
REPORT OF THE TREASURER
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MARINE BIOLOGICAL LABORATORY
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REPORT OF THE TREASURER
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50 MARINE BIOLOGICAL LABORATORY
NOTES TO FINANCIAL STATEMENTS
A. Purpose of the Laboratory:
The purpose of Marine Biological Laboratory (the "Laboratory") is to establish and maintain a
laboratory or station for scientific study and investigations, and a school for instruction in biology
and nature history.
B. Significant Account ing Policies:
Basis of Presentation — Fund A ccounting
In order to ensure observance of limitations and restrictions placed on the use of resources available
to the Laboratory, the accounts of the Laboratory are maintained in accordance with the principles
of "fund accounting." This is the procedure by which resources are classified into separate funds in
accordance with specified activities or objectives.
Externally restricted funds may only be utilized in accordance with the purposes established by the
donor or grantor of such funds. However, the Laboratory retains full control over the utilization of
unrestricted funds. Restricted gifts, grants, and other restricted resources are accounted for in the
appropriate restricted funds. Restricted current funds are reported as revenue when received and as
related costs are incurred. Unrestricted current funds are reported as revenue when earned.
Endowment funds are subject to restrictions requiring that the principal be invested with income
available for use for restricted or unrestricted purposes by the Laboratory. Quasi-endowment funds
have been established by the Laboratory for the same purposes as endowment funds; however, the
principal of these funds may be expended for various restricted and unrestricted purposes.
Fixed Assets
Fixed assets are recorded at cost. Depreciation is computed using the straight-line method over esti-
mated useful lives of fixed assets.
Reclassifications
The financial statements for 1986 reflect certain changes in classification of revenue, expenses and
changes in fund balances. Similar reclassifications have been made to amounts previously reported
in order to provide consistency of the financial statements. In addition, the financial statements
reflect in 1986 the segregation of the current unrestricted fund balance into two components: the
current unrestricted and the housing enterprise fund balances.
Contracts and Grants
Revenues associated with contracts and grants are recognized in the statement of support, revenues,
expenses and changes in fund balances when received and as related costs are incurred. The Labora-
tory reimbursement of indirect costs relating to government contracts and grants is based on negoti-
ated indirect cost rates with adjustments for actual indirect costs in future years. Any over- or under-
recovery of indirect costs is recognized through future adjustments of indirect cost rates.
Investments
Investments purchased by the Laboratory are carried at market value (Note C). Money market secu-
rities are carried at cost which approximates market value. Investments donated to the Laboratory
are carried at fair market value at the date of the gift. For determination of gain or loss upon disposal
of investments, cost is determined based on the average cost method. The Laboratory is the benefi-
ciary of certain endowment investments which are held in trust by others. These investments are
reflected in the financial statements. Every ten years the Laboratory's status as beneficiary is reviewed
to determine that the Laboratory's use of these funds is in accordance with the intent of the funds.
REPORT OF THE TREASURER 5 1
Investment Income and Distribution
The Laboratory follows the accrual basis of accounting except that investment income is recorded
on a cash basis. The difference between such basis and the accrual basis does not have a material
effect on the determination of investment income earned on a year-to-year basis.
Investment income includes income from the investments of specific funds and from the pooled
investment account. Income from the pooled investment account is distributed to the participating
funds on the basis of their proportionate share at market value adjusted for any additions or disposals
to pooled funds.
C. Change in Accounting Met hod for Investments:
Effective January 1, 1986, the Laboratory adopted the accounting policy of reporting investments
and the related fund balances at market value to more clearly reflect the financial impact of the
Laboratory's investment policies. Investments and the related fund balances in prior years were
reported at cost. The cumulative increase in the fund balances at December 31,1 986 and 1 985 is as
follows:
Current Restricted Funds: 1986 1 985
Unexpended gifts $ 11,782
Endowment funds:
Unrestricted $ 80,070 329,166
Restricted 81,780 215,763
Quasi-endowment funds:
Unrestricted 31,438 126,333
Restricted 142,527 436,913
Increase in unrealized appreciation and related
fund balances $335,815 $1.119,957
This change has been retroactively applied to the fund balances as of the beginning of the year ended
December 3 1 , 1985 as follows:
Unexpended gifts $ (364)
Endowment funds:
Unrestricted 263,969
Restricted 311,892
Quasi-endowment funds:
Unrestricted (3,747)
Restricted 104.458
Cumulative unrealized gain/loss $ 676,208
D. Land, Buildings, and Equipment:
The following is a summary of the unrestricted plant fund assets:
1986 1985
Land $ 689,660 $ 689,660
Construction in progress 140,826
Buildings 16,333,358 14,861,244
Equipment 2,170,878 2.113.321
19,193,896 17,805,051
Less accumulated depreciation (7,143.565) (6,579.654)
$12,050,331 $11,225,397
52
MARINE BIOLOGICAL LABORATORY
E. Retirement Fund:
During 1986, the Laboratory elected early application of Statement of Financial Accounting Stan-
dard No. 87, "Employer's Accounting for Pensions." This Statement establishes standards of finan-
cial accounting and reporting for an employer that offers pension benefits to its employees and super-
cedes earlier standards. The early election reduced the actuarially determined pension expense from
$132,866 to $82,682.
The Laboratory has a noncontributory defined benefit pension plan for substantially all employees.
Contributions are intended to provide for benefits attributed to the service date, but also those ex-
pected to be earned in the future.
Actuarial present value of benefit obligations:
Accumulated benefit obligation including vested benefits of
$1,484,283
Projected benefit obligation
Plan assets at fair value
Projected benefit obligation less than plan assets
Unrecognized net (gain) or loss
Prior service cost not yet recognized in net periodic pension cost
Unrecognized net obligation at March 1, 1986
Prepaid pension cost (pension liability) recognized in the statement
of financial position
Net pension cost for fiscal year ending December 31,1 986:
Service cost — benefits earned during the period
Interest cost on projected benefit obligation
Actual return on plan assets
Net amortization and deferral
Net periodic pension cost
1,686.685
2.561,619
2.608,987
47,368
186,054
(316.104)
$ (82.682)
138,391
142,381
(319,877)
121.787
$ 82,682
The actuarial present value of the projected benefit obligation was determined using a discount rate
of 7.3% and rates of increase in compensation levels of 6%. The expected long-term rate of return
on assets was 8%.
In addition, the Laboratory participates in the defined contribution pension program of the Teachers
Insurance and Annuity Association. Expenses amounted to $106,535 in 1986 and $95,858 in 1985.
F. Pledges and Grants:
As of December 31, 1986 and 1985, 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:
December 31. 1986
December 31, 1985
1987
1988
1989
Unrestricted
$20,000
10,000
$30,000
Restricted
$615,027
40,764
5.764
$661,555
Unrestricted
$10,000
10,000
$20,000
Restricted
$550,240
15,000
$565.240
G. Interfund Borrowings:
Interfund balances at December 3 1 are as follows:
Current Funds
Due to plant funds
Due to endowment funds
Due to restricted quasi-endowment funds
1986
$(169,615)
(115,909)
(200.750)
$(486,274)
1985
$ (56,669)
(156,622)
$(213,291)
REPORT OF THE TREASURER 53
H. Mortgages and Notes Payable:
The mortgage note payable with a term of 26 years is in the amount of $ 1 .3 million bearing interest
based on the bank's prime rate plus three quarters percent (.75%) on a floating basis for the initial
five year period with a floor of 7.50% and a ceiling of 1 3.00%. The interest rate at December 31,1 986
was 9.00%. The mortgage loan is collateralized by a first mortgage on the land and properties known
as Memorial Circle, with recourse in the event of default limited to this land and property and the
related revenue. Principal and interest payments of $ 1 5,000 are due and payable monthly commenc-
ing January 19, 1987.
Other notes payable consist of the following:
Unsecured note with interest at 7.90% with monthly principal
payments of $22 1.20 plus interest $ 9,194
Unsecured note with interest at 6.90% with monthly principal
payments of $394. 71 plus interest 12.990
$22,184
At December 31, 1986, these mortgages and notes payable had aggregate future annual principal
payments as follows:
Amount
1987 $ 73,001
1988 78.654
1989 83.899
1990 86,492
1991 94,127
Thereafter 906.010
1,322,183
Less current portion 73.001
$1,249,182
54
MARINE BIOLOGICAL LABORATORY
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REPORT OF THE LIBRARIAN 55
VIII. REPORT OF THE LIBRARIAN
Our serial titles are now all entered into the On-Line OCLC catalog which is based
in Ohio. The number of requests for copies of articles in our periodical collection has
doubled since this project was completed. Over 350 libraries and laboratories sent
requests during 1986, and we now serve a larger scientific community than we did
before our collection was included in this database.
In preparation for the Centennial we have prepared a computer program for the
records in the Archives. Lists of all scientists, students, lecturers, employees, and oth-
ers who have been at the MBL since 1 888 will be placed in an archival database. This
information will be valuable to science historians, our public information office, and
the library reference staff. Most of this material will be entered by Ruth Davis and
her volunteer staff. Photographs held in the Rare Books and Archives area are being
cataloged. Negatives are being made of a number of the rare ones. Many of these
photographs will be used in a Centennial book that is being planned for 1988.
We gave one-day tours of the Library in June to two groups of librarians. One was
a group from the Boston meeting of the Special Librarians Association and the other
was a group named the "Rte. 1 28 Librarians" from the Hi-Tech libraries in that area.
Binding increased this year since we picked up a number of volumes where one
or two issues were missing. We bound these noting the "lacks" on the spine. Over
3000 volumes were sent to the binders during the winter months.
IX. EDUCATIONAL PROGRAMS
SUMMER
BIOLOGY OF PARASITISM
Course directors
ENGLUND, PAUL, Johns Hopkins University
SHER, ALAN, NIAID/NIH
Other faculty, staff, and lecturers
ALDRITT, SUSAN, Harvard University
BEVERLY, STEPHEN, Harvard Medical School
BLOOM, BARRY, Albert Einstein College of Medicine
BROWN, KIM, University of Iowa
BURAKOFF, STEVEN, J., Dana-Farber Cancer Institute
BURNS, JAMES, Hahnemann University
BUTTERWORTH, ANTHONY, University of Cambridge, UK
CANTOR, CHARLES, Columbia University
CARTER, RICHARD, NIAID/NIH
CERAMI, ANTHONY, Rockefeller University
CLEVELAND, DON, Johns Hopkins University
DINTZIS, HOWARD, Johns Hopkins University
DONELSON, JOHN, University of Iowa
DOOLITTLE, RUSSELL, University of California, San Diego
DVORAK, JAMES, NIAID/NIH
GEARHART, PATRICIA, Johns Hopkins University
GOTTLIEB, MICHAEL, Johns Hopkins University
HART, GERALD W., Johns Hopkins University
HERELD, DALE, Johns Hopkins University
HOWARD, JAMES, Wellcome Laboratories
56 MARINE BIOLOGICAL LABORATORY
HOWARD, RUSSELL, NIAID/NIH
JAMES, STEPHANIE, George Washington School of Medicine
JOINER, KEITH, NIAID/NIH
KNOPF, PAUL, Brown University
MARTINEZ-PALOMO A., Center for Advanced Research
McMAHON-PRATT, D., Yale University Medical School
Moss, BERNARD. NIAID/NIH
NELSON, GEORGE, University of Liverpool, UK
NEVA, FRANKLIN A., NIAID/NIH
NUSSENZWEIG, VICTOR, New York University Medical Center
OTTESON, ERIC, NIAID/NIH
PEREIRA, MIERCIO, Tufts University School of Medicine
PFEFFERKORN, ELMER, Dartmouth Medical School
VAN DER PLOEG, LEX, Columbia University
RIBEIRO, JOSE, Harvard University
ROCK, THEODORE, Howard Hughes Medical Institute
SACKS, DAVID, NIAID/NIH
SCOTT, PHILLIP, NIAID/NIH
SHARKEY, ANDREW, University of Edinburgh, UK
SHEVACH, ETHAN, NIAID/NIH
SPIELMAN, ANDREW, Harvard University School of Public Health
STRAND, METTE, Johns Hopkins University
SUPLICK, KATHY, Hahnemann University
TURNER, MERVYN J., Merck Sharp and Dohme Research Laboratories
WALLIKER, D., University of Edinburgh, UK
WANG, CHING C., University of California, San Francisco
WARD, DAVID, Yale University
WARD, SAMUEL, Carnegie Institute
WARREN, KENNETH, Rockefeller Foundation
WASSOM, DONALD, University of Wisconsin
Students
ALANO, PIETRO, University of Milan, Italy
ANDERSEN, BIRGITTE JYDING, Statens Serum Institute, Denmark
DOSHI, PARULD., UMDNJ-Rutgers Medical School
EID, JOSIANE E., Johns Hopkins University
HARYANA, SOFIA M., Gadjah Mada University, Indonesia
LOMBARDI, GEORGE V., Washington University
Lucius, RICHARD H. C., University of Heidelberg, FRG
MORZARIA, SUBHASH P., International Laboratory for Research on Animal Diseases, Kenya
ROSALES, JOSE Luis E., Centre de Investigacion y de Estudios Avanzados del IPN, Mexico
SAMARAS, NICHOLAS, Walter and Eliza Hall Institute of Medical Research, Australia
SHONEKAN, OPEOLU A., University of Ibadan, Nigeria
SINNIS, PHOTINI, Dartmouth Medical School
STUCKY, PAMELA D., University of California, San Francisco
TALAMAS, PATRICIA R., Centro de Investigacion y de Estudios Avanzados del IPN, Mexico
WEIDANZ, WILLIAM P., Hahnemann University School of Medicine
ZIMMERMAN, RONALD J., Vanderbilt University
EMBRYOLOGY: A MODERN COURSE IN DEVELOPMENTAL BIOLOGY
Course directors
BRANDHORST, BRUCE, McGill University, Canada
JEFFERY, WILLIAM, University of Texas
EDUCATIONAL PROGRAMS 57
Other faculty, staff, and lecturers
ARNOLD, JOHN M, University of Hawaii
CHILDS, GEOFFREY, Albert Einstein College of Medicine
CLARK, WALLIS, Bodega Marine Station
COSTANTINI, FRANK, Columbia University
DAVIDSON, ERIC, California Institute of Technology
ELDON, ELIZABETH, M. D. Anderson Hospital
EMERSON, CHARLES, University of Virginia
GERHART, JOHN, University of California, Berkeley
GIMLICH, ROBERT, University of California, Berkeley
GOLSTEYN, ROY, University of Calgary, Canada
GROSS, PAUL, Marine Biological Laboratory
HAFNER, MATHIAS, German Cancer Research Center, FRG
HILLE, MERRILL, University of Washington
JAENISCH, RUDOLF, Massachusetts Institute of Technology
JAFFE, LAURINDA, University of Connecticut Health Center
JAFFE, LIONEL, Marine Biological Laboratory
KADO, RAYMOND, Centre National Recherche Scientifique, France
KEMPHUES, KENNETH, Cornell University
KLEIN, WILLIAM, M. D. Anderson Hospital
KLINE, D., University of Connecticut
LEE, JAMES, California Institute of Technology
MARZLUFF, WILLIAM, Florida State University
MEEDEL, THOMAS H., Marine Biological Laboratory
RICHTER, JOEL, Worcester Foundation for Experimental Biology
ROBERTS, JAMES, Hutchinson Cancer Center
ROSBASH, MICHAEL, Brandeis University
RUDERMAN, JOAN, Duke University
SCHATTEN, GERALD, University of Wisconsin
SCHATTEN, HEIDI, Florida State University
SLUDER, GREENFIELD, Worcester Foundation for Experimental Biology
WESSEL, GARY, M. D. Anderson Hospital
WHITTAKER, J. RICHARD, Marine Biological Laboratory
WILT, FRED, University of California, Berkeley
WINKLER, MATTHEW, University of Texas, Austin
WORMINGTON, MICHAEL, Brandeis University
Students
ANDERSON, MARYDILYS S., Yale University
BERG, CELESTE A., Carnegie Institution/Yale University
BICKEL, SHARON E., Baylor College of Medicine
BLOOM, THEODORA L., University of Cambridge, England, UK
BURSDAL, CAROL A., Duke University
FORRESTER, WILLIAM C, University of Washington
HAFNER, MATHIAS**, German Cancer Research Center, FRG
HARDIN, PAUL E., Indiana University
HARDIN, SUSAN H.. Indiana University
HOULISTON, EVELYN, University of Cambridge, England, UK
JURSNICH, VICTORIA A., University of California, Irvine
KIRBY, COLLEEN M., Cornell University
KOENIG, GERD, Max Planck Institut fur Entwicklungsbiologie, FRG
KUBIAK, JACEKZ., Warsaw University, Poland
RUBACHA, ALICE, Rice University
** Advanced Research Training Program participant.
58 MARINE BIOLOGICAL LABORATORY
SAAVEDRA, CAROL, McGill University, Canada
SCHOLER, ANNE-MARIE, Han/ard University
SCHROETER, SALLY J., University of Michigan
SMOLICH, BEVERLY D., University of Virginia
SYMES, KAREN, National Institute of Medical Research, England, UK
TALEVI, RICCARDO, University of Naples, Italy
VARNUM, SUSAN M., Brandeis University
VELLECA, MARK A., Yale University
VITES, ANA M., University of Connecticut Health Center
WHARTON, L. LYNN, University of Massachusetts Medical School
MARINE ECOLOGY
Course director
FRANK, PETER W., University of Oregon
Other faculty, staff, and lecturers
ANDERSON, DONALD M., Woods Hole Oceanographic Institution
Buss, LEO, Yale University
CAPUZZO, JUDITH, Woods Hole Oceanographic Institution
CARACO, NINA, Mary Flagler Cary Arboretum
CARLTON, JAMES, Williams College
CARON, DAVID A., Woods Hole Oceanographic Institution
CASWELL, HAL, Woods Hole Oceanographic Institution
CAVANAUGH, COLLEEN, Harvard University
COLE, JON, Mary Flagler Cary Arboretum
DAVIS, CABELL, Woods Hole Oceanographic Institution
DELANO, M., Environmental Protection Agency
DEUSER, WERNER G., Woods Hole Oceanographic Institution
FOREMAN, KENNETH, Marine Biological Laboratory/BUMP
FREADMAN, MARVIN, Marine Biological Laboratory/BUMP
FROST, BRUCE W., University of Washington
GALLAGHER, EUGENE, University of Massachusetts
GAINES, ARTHUR G., JR., Woods Hole Oceanographic Institution
GIBLIN, ANN, Marine Biological Laboratory
GRASSLE, J. FREDERICK, Woods Hole Oceanographic Institution
GRASSLE, JUDITH, Marine Biological Laboratory
HARBISON, G. RICHARD, Woods Hole Oceanograpic Institution
HOBBIE, JOHN E., Marine Biological Laboratory
HUSTON, MICHAEL, Oak Ridge National Laboratory
JEFFERIES, ROBERT L., University of Toronto, Canada
MANN, KENNETH H., Bedford Institute of Oceanography, Canada
MARCY, MARIBEL, Smith College
MURCHELLANO, ROBERT, National Marine Fisheries Service
OSMAN, RICHARD, Academy of Natural Sciences of Philadelphia
PASCUAL-DUNLAP, M. MERCEDES, Cornell University
PETERSON, CHARLES HENRY, University of North Carolina
PETERSON, BRUCE R., Marine Biological Laboratory
PREGNALL, MARSHALL, University of Massachusetts
REX, MICHAEL, University of Massachusetts
RHOADS, DONALD, Yale University
RICE, DONALD, Chesapeake Biological Laboratory
RUBLEE, PARKE A., Whitman College
SANDERS, HOWARD L., Woods Hole Oceanographic Institution
SEBENS, KENNETH, Northeastern University
EDUCATIONAL PROGRAMS 59
SHELLEY, PETER, Conservation Law Foundation
VALIELA, IVAN, Boston University
WALLACE, GORDON, University of Massachusetts
WEINBERG, JAMES R., Woods Hole Oceanographic Institution
WELSCHMEYER, NICHOLAS, Harvard University
WIEBE, PETER H., Woods Hole Oceanographic Institution
Students
BROWN, ANNE C., University of Oregon
COMIN, FRANCISCO A., University of Barcelona, Spain
DICKENS, VIRGINIA A., Goucher College
DIOGENE, GEORGES F., University of Barcelona, Spain
DUBILIER, NICOLE, University of Hamburg, FRG
FALK, KATHLEEN, University of Massachusetts, Boston
FREY, IRIS J. F., Philipps-University Marburg, FRG
HART, ROBERTA., University of California, Berkeley
KASMER, JOHN M., University of Vermont
MORUCCI, CARLO, University La Sapienza of Rome, Italy
MYERS, PHILIP E., University of South Carolina
O'HARA, ELLEN MARGARET, Villanova University
SPANO, ANNAMARIA, Istituto Superiore de Sanita of Rome, Italy
SVENDSEN, BETTY-ANN E., University of Dallas
THIVAKARAN, ALAGIRI G., Annamalai University, India
THOMAS, CECELIA R., Hinds Jr. College
ZAPATA, FERNANDO A., University of Arizona
MICROBIOLOGY: MOLECULAR ASPECTS OF CELLULAR DIVERSITY
Course directors
GREENBERG, PETER, Cornell University
WOLFE, RALPH, University of Illinois
Other faculty, staff, and lecturers
ARMITAGE, JUDITH, Oxford University, UK
BLAKEMORE, RICHARD, University of New Hampshire
BOBIK, THOMAS, University of Illinois
DILLING, WALTRAUD, University of Konstanz, FRG
DiMARCO, ANTHONY, University of Illinois
DUNLAP, PAUL, Cornell University
FRANKEL, RICHARD, Massachusetts Institute of Technology
JEFFERYS, JUDITH, Oxford University, UK
KAPLAN, SAMUEL, University of Illinois
KAISER, DALE, Stanford University
KROPINSKI, ADAM, Marine Biological Laboratory
KROPINSKI, ANDREW, Queen's University, Canada
MACNAB, ROBERT, Yale University
PFENNIG, NORBERT, Universitat Konstauz, FRG
ROUVIERE, PIERRE, University of Illinois
SPUDICH, JOHN, Albert Einstein College of Medicine
WIDDEL, FRIEDRICH, University of Illinois, Urbana-Champaign
WRAIGHT, COLIN A., University of Illinois
Students
ANDERSON, KAREN L., University of Iowa
DOBBS, FREDC, Florida State University
60 MARINE BIOLOGICAL LABORATORY
GALLO, MARK A., Cornell University
GIBSON, SUSAN A., University of Oklahoma
KHANDEKAR, SANJAY S., Portland State University
KING, STAGG L., U< isity of Washington
KOT, MARK, ?. uty of Arizona
KUTZ, Sus ' Diversity of Arizona
LANE, DA. .. Indiana University
Liu, SHU M., University of Oregon
MANGIN. KATRINA L., University of Arizona
MARCIKJK, DOUGLAS A., Hope College
MICHEL, TOMAST., University of California, Davis
PADGITT, PATRICIA J., Creighton University
SILVERSTONE, SARA E., University of California, Davis
SPORMANN, ALFRED MICHAEL, Philipps Universitat, FRG
STEPHENS, CRAIG MICHAEL, University of Virginia
STODDARD, STEVEN F., University of Wisconsin, Madison
WEISS, DAVIDS., University of California, Berkeley
ZHAO, HONGXUE, University of Illinois
NEURAL SYSTEMS AND BEHAVIOR
Course directors
CAREW, THOMAS, Yale University
KELLEY, DARCY, Columbia University
Other faculty, staff, and lecturers
AVITABLE, ELENA, Columbia University
BASS, ANDREW, Cornell University
BORST, AXEL, Max Planck Institut fur Cell Biologic, FRG
BURD, GAIL, University of Arizona
BYRNE, JOHN, University of Texas Medical School
CALABRESE, RONALD, Harvard University
CARROLL, LESLIE, Thomas Jefferson University
CASAGRANDE, VIVIAN, Vanderbilt University
CLEARY, LEONARD, University of Texas
ELLIOT, ELLEN, University of North Carolina
FRANK, JILLIAN, New York University
GOLDMAN-RAKJC, PATRICIA, Yale University Medical School
GORLICK, DENNIS, Columbia University
HARRIS- WARRICK, RONALD, Cornell University
HOSKINS, SALLY, Columbia University
JACOBS, GWEN, University of California, Berkeley
JOHNSON, BRUCE, Cornell University
LEVINE, RICHARD, Rice University
MACAGNO, EDUARDO, Columbia University
MARDER, EVE, Brandeis University
McROBERT, SCOTT, Temple University
MOISEFF, ANDREW, University of Connecticut
NORTHCUTT, GLENN, University of Michigan
NUSBAUM, MICHAEL, Brandeis University
PEARSON, KEIR, University of Alberta
SIMMONS, JAMES, Brown University
SQUIRE, LARRY, University of California, San Diego
TOBIAS, MARTHA, Columbia University
EDUCATIONAL PROGRAMS
TOMPKINS, LAURIE, Temple University
WEEKS, JANIS, University of California, Berkeley
Students
APPLEGATE, APRIL V., Johns Hopkins School of Medicine
GLOWER, ROBERT P., Cornell University
DODD, FRANK, Cornell University
EDMONDS, BRIAN W., University of Virginia
ELIOT, LISE SUZANNE, Columbia University
GARCIA CABRERA, INMACULADA, University of Bergen, Norway
HAMMER, MARTIN, Freie Universitat Berlin, FRG
HARRINGTON, MARY E., Dalhousie University, Canada
HIRANO, ARLENE A., The Rockefeller University
KIEHN, OLE, The Panum Institute, Denmark
KNOWLTON, BARBARA, Stanford University
LoTuRCO, JOSEPH J., Yale University
LUSTIG, CORNEL, Weizmann Institute, Israel
MENCIO, TRACEY L., Rutgers University
MORGAN, MICHAEL M., University of California, Los Angeles
NIRENBERG, SHEILA, Harvard Medical School
NISSANOV, JONATHAN, University of Colorado
ROBERTS, SETH D., University of California, Berkeley
STREICHERT, LAURA D., Stanford University
WEAVER, DEBORAJ., University of Maryland, Baltimore County
NEUROBIOLOGY
Course director
KARLIN, ARTHUR, Columbia University
Other faculty, staff, and lecturers
ADAMS, PAUL, SUNY, Stony Brook
AGNEW, WILLIAM, Yale University
ANDERSON, DAVID, Columbia University
ANDREWS, BRIAN, NINCDS/NIH
ARMSTRONG, KATIE, Rice University
BRETT, ROGER, SUNY, Stony Brook
CLAUDIO, TONI, Yale University Medical School
CONNER, JOHN, Bell Laboratories
DiPAOLA, MARIO, Columbia University College of Physicians and Surgeons
EHRLICH, BARBARA, Albert Einstein College of Medicine
FISCHBACH, GERALD, Washington University School of Medicine
GURNEY, ALISON, California Institute of Technology
HALL, LINDA, Albert Einstein College of Medicine
HATTEN, MARY E., New York University Medical Center
HESS, PETER, Harvard University
HEUSER, JOHN, Washington University
INOUE, TOMO, McGill University, Canada
JESSELL, THOMAS, Columbia University
JONES, STEVEN, SUNY, Stony Brook
KHAN, SAHID, Marine Biological Laboratory
LANDER, ARTHUR, Columbia University
LANDIS, DENNIS, Massachusetts General Hospital
LESTER, HENRY, California Institute of Technology
62 MARINE BIOLOGICAL LABORATORY
LEVITAN, IRWIN, Brandeis University
MACKINNON, RODERICK, Brandeis University
MARGULIES, DAVID, Columbia University College of Physicians and Surgeons
MATSUMOTO, STEV ,;, Harvard University
McNiVEN, !V! J niversity of Maryland
MILLER, O ;R, Brandeis University
MOOSEK.E- , MARK, Yale University
MURR . University of Pennsylvania
PAULSEN, HENRY, Yale University Medical School
RA VIOLA, ELIO, Harvard Medical School
REFSE, THOMAS S., NINCDS/NIH/Marine Biological Laboratory
ROLE, LORNA, Columbia University
ROSENBLUTH, JOHN, New York University
ROWLAND, L., Columbia University
SCHNAPP, BRUCE, Marine Biological Laboratory
SHEETZ, MICHAEL, Washington University School of Medicine
SILMAN, ISRAEL, Weizmann Institute of Science, Israel
SPUDICH, JOHN, Albert Einstein College of Medicine
STERNWEIS, PAUL, University of Texas Health Center
WILLARD, ALAN L., University of North Carolina
Students
BLEY, KEITH R., Yale University
CARLBERG, MATS, University of Lund, Sweden
FEDEROFF, HOWARD J., Massachusetts General Hospital
FERNANDEZ- VALLE, CRISTINA, University of Miami
HALPERN, MARNIE E., Yale University
HORRIGAN, FRANK T., Stanford University
LEW, DANIEL J., The Rockefeller University
PLEASURE, SAMUEL J., University of Pennsylvania
PORTER, DEVRA, Vanderbilt University
SCHWEIZER, FELIX E., Universitat Basel, Switzerland
SUPATTAPONE, SuRACHAi, Johns Hopkins School of Medicine
ZUMBROICH, THOMAS J., University of Oxford, Oxford, UK
PHYSIOLOGY
Course director
GOLDMAN, ROBERT, Northwestern University
Other faculty, staff, and lecturers
ALBRECHT-BUEHLER, G., Northwestern University
BECKERLE, MARY, University of North Carolina
BENDER, WELCOME, Harvard University
BLOOM, KERRY, University of North Carolina
CHISHOLM, REX, Northwestern University
DEROSIER, DAVID, Brandeis University
DESSEV, GEORGE N., Northwestern University
FILETI, LISA, Boston University
FUKUI, YOSHIO, Osaka University, Japan
GOLDMAN, ANNE, Northwestern University
GOLDSTEIN, LARRY, Harvard University
HAN, PETER, Earlham College
HAY, ELIZABETH D., Harvard University
HOLM, CONNIE, Harvard University
EDUCATIONAL PROGRAMS 63
HOPKINSON, SUSAN, Northwestern University Medical School
HORVITZ, H. ROBERT, Massachusetts Institute of Technology
HYAMS, JEREMY, University College, UK
JONES, JONATHAN, Northwestern University
KIEHART, DAN, Harvard University
LEINWAND, LESLIE, Albert Einstein College of Medicine
LINDBERG, UNO, University of Stockholm, Sweden
MAYRAND, SANDRA, Worcester Foundation for Experimental Biology
MATTOX, ANDREW, Marine Biological Laboratory
MCNALLY, ELIZABETH, Albert Einstein College of Medicine
MORELAND, ROBERT, Dana Farber Cancer Institute
OLMSTED, JOANNA, University of Rochester
PARYSEK, LINDA, Northwestern University Medical School
PEDERSON, THORLJ, Worcester Foundation for Experimental Biology
PTASHNE, MARK, Harvard University
RICH, ALEXANDER, Massachusetts Institute of Technology
RUDERMAN, JOAN, Duke University
RUSHFORTH, ALICE, Earlham College
RUSKIN, BARBARA, Harvard University
SCHWARTZ, LAWRENCE M., University of North Carolina
SHAPIRO, LUCY, Albert Einstein College of Medicine
SINGER, REBECCA, Albert Einstein College of Medicine
SOHN, REGINA LEE, Albert Einstein College of Medicine
SPUDICH, JAMES, Stanford University
STEVENSON, BRUCE, Yale University
SZENT-GYORGYI, ANDREW, Brandeis University
TARDIFF, JILL, Albert Einstein College of Medicine
TAYLOR, MARK, Northwestern University Medical School
VALE, RON, Marine Biological Laboratory
VALLEE, RICHARD, Worcester Foundation for Experimental Biology
WARNER, CECELIA, Northwestern University Medical School
WARNER, JONATHAN, Albert Einstein College of Medicine
WEINSTEIN, RONALD, Rush Medical Center
WHITMAN, GEORGE, Worcester Foundation for Experimental Biology
WIEBEN, ERIC D., Mayo Foundation
YEH, ELAINE, CIBA-GEIGY
Students
AKINS, JR., ROBERT E., University of Pennsylvania
BEEMAN, ANNE M., Dartmouth Medical School
BISWAS, SURAJIT K., University of Pennsylvania
BLACK, KRISTIN, University of California, Berkeley
BRADLEY, DAVID, University of Pennsylvania
CAULEY, KEITH A., University of Michigan
DABORA, SANDRA L., University of Connecticut
DAHL, STEPHEN C, Wesleyan University
DASSO, MARY C., Cambridge University, England, UK
DEYST, KATHERINE A., Tufts University
ERIKSSON, ULF J., Uppsala University, Sweden
FOLTZ, KATHLEEN R., Purdue University
GANNON, PAMELA M., Tufts University-Sackler School
GELLES, JEFF, California Institute of Technology
GUDEMAN, DAVID M., Kansas University
HARPER, DAVIDS., University of Illinois, Chicago
HORNE, MARY C., University of California, San Francisco
KATZ, KENNETH S., Amherst College
64 MARINE BIOLOGICAL LABORATORY
KENNA, MARGARET A.. University of North Carolina
KENNEY, LINDA!., University of Pennsylvania
LAUERMAN, TOD V '• ' .>hns Hopkins University
MEINHOF, C/ «3, University of California, San Diego
MELUH, P\\fi University of Maryland
MOHL, ViP , Washington State University
REGINATO. ANTONIO M., University of Pennsylvania
RUDOLFS KAREN M., Dartmouth College
SARDET, CLAUDE C. S., Centre National de la Recherche Scientifique, France
SEGRK. GINO V., Massachusetts General Hospital/Harvard Medical School
SYMONS, MARC H. C., Weizmann Institute of Science, Israel
THALER, CATHERINE D., University of California, Riverside
TROXELL, CYNTHIA L., University of Colorado
WADSWORTH, WILLIAM G., University of Missouri
WATSON, CORNELIUS A., Wesleyan University
YANAGIHARA, RICHARD, National Institute of Neurological and Communicative Disorders
and Strokes/NIH
YORK, KAREN PICKWICK, University of Pennsylvania
ZAND, MARTIN S., Northwestern University Medical School
SPRING
ANALYTICAL AND QUANTITATIVE LIGHT MICROSCOPY IN BIOLOGY,
MEDICINE, AND MATERIALS SCIENCE
April 3- 10, 1986
Course director
INOUE, SHINYA, Marine Biological Laboratory
Other faculty, staff, and lecturers
AKINS, ROBERT, University of Pennsylvania
ELLIS, GORDON W., University of Pennsylvania
KNUDSON, ROBERTA., Marine Biological Laboratory
LANNI, FREDERICK, Carnegie Mellon University
LUBY-PHELPS, KATHERINE, Carnegie Mellon University
LUTZ, DOUGLAS, Harvard University
SALMON, EDWARD A., University of North Carolina
TAYLOR, D. LANSING, Carnegie Mellon University
WALKER, RICHARD, University of North Carolina
Commercial faculty
ABROMOWITZ, MORTIMER, Olympus Corporation of America
ALEXANDER, SCOTT, Nikon, Inc.
BEACH, DAN, Carl Zeiss, Inc.
BREEN, BILL, Interactive Video Systems
DEMIAN, JEFFREY, Nikon, Inc.
ESSER, HERMAN, Ikegami Electronics (USA), Inc.
FOSTER, BARBARA, Carl Zeiss, Inc.
GRACE, JOHN, Crimson Camera Technical Sales, Inc.
HANNAWAY, WYNDHAM, G. W. Hannaway Associates
HINSCH, JAN, E. Leitz, Inc.
JONES, JEFFREY, Olympus Corporation of America
KELLER, ERNST, Carl Zeiss, Inc.
KIMURA, T., Olympus Corporation of America
EDUCATIONAL PROGRAMS 65
KLEIFGEN, GERRY, Dage-MIT
KNUTRUD, PAUL, Interactive Video Systems
ORWELL, PATTY, E. Leitz, Inc.
PACKARD, MEL, Quantex Corporation
PRESLEY, PHIL, Carl Zeiss, Inc.
REGAN, ANN, Ikegami Electronics (USA), Inc.
RUBINOW, JERRY, Universal Imaging Corporation
SCHEIRER, KURT, Nikon Inc.
SCOTT, ERIC, Ikegami Electronics (USA), Inc.
TAYLOR, RICHARD, Colorado Video
THOMAS, PAUL, Dage-MIT
VRATNEY, MELANA, Nikon Inc.
Students
ANDERSON, DONALD M., Woods Hole Oceanographic Institution
ARONSON, JOHN F., Wistar Institute
BARI, DANIEL, Universidad Nacional de Cuyo-Conicet, Argentina
CAVICCHIA, JUAN CARLOS, Universidad Nacional de Cuyo-Conicet, Argentina
CHAMBERS, EDWARD L., University of Miami School of Medicine
DUBINSKY, JANET M., Washington University School of Medicine
FEIGENSON, GERALD W., Cornell University
GALLAGER, SCOTT M., Woods Hole Oceanographic Institution
GLANZMAN, DAVIDL., Howard Hughes Medical Institute
KHAN, SHAHID M. M., Albert Einstein College of Medicine
KLEITMAN, NAOMI, Washington University School of Medicine
McCuLLOH, DAVID H., University of Miami School of Medicine
MILLER, PAUL, Bell Laboratories
NORRIS, CAROLYN, Bardeen Labs
PALAZZO, ROBERT E., University of Virginia
REESE, TOM, Marine Biological Laboratory
RUSSELL, JAMES T.,NIH
SCHULZE, ERIC, University of California, San Francisco
SHEEHY, PAULA., NIH
VALE, RONALD D., Marine Biological Laboratory
VOYVODIC, JAMES, Washington University School of Medicine
WIGSTON, DONALD, Emory University School of Medicine
WOMACK, MARY, Howard Hughes Medical Institute
YEAGER, MARK D., Cornell University
SHORT COURSES
CELL AND MOLECULAR BIOLOGY OF PLANTS
August 4- 14, 1986
Coordinators
DURE, LEON, The University of Georgia
KEY, JOE L., The University of Georgia
Lecturers
AUSUBEL, FRED, Massachusetts General Hospital
CHUA, NAM, Rockefeller University
CLEGG, MICHAEL, University of California, Riverside
CROUCH, MARTHA, Indiana University
FRALEY, ROB, Monsanto Company
66 MARINE BIOLOGICAL LABORATORY
HEPLER, PETER K., University of Massachusetts
LEVINGS, C. S. III. North Carolina State University
MALMBERG, RUSSEI.L, The University of Georgia
MEAGHER, RICHARD, The University of Georgia
PALEVITZ, BARRY, The University of Georgia
QUAIL, PFTI R, University of Wisconsin
SOMMe : ; v LE, CHRIS, Michigan State University
STROI. i, JUDITH, The University of Georgia
TLM; /.E, BILL, University of California, Davis
VARNER, JOE, Washington University
YODER, OLIN, Cornell University
Students
AGARWAL, MUNNA LAI, Centre National de la Recherche Agronomiue, France
ARMOUR, SUSAN, CIBA-GEIGY Corp.
ARMOUR, TOBY, Edgartown, Massachusetts
BECK, JAMES J., CIBA-GEIGY Corp.
CAROZZI, NADINE, CIBA-GEIGY Corp.
CHENEY, DONALD P., Northeastern University
CURRY, L. JEANNE, University of Massachusetts
ELLIOTT, WILLIAM, Hartwick College
GOLDMAN, PEG, New Haven, Connecticut
HANLEY, SUSAN, BioTechnical International Inc.
HAUGE, BRIAN, Massachusetts General Hospital
LOTSTEIN, RICHARD, CIBA-GEIGY Corp.
McCABE, BRIAN, Bloomington, Indiana
MINEO, LORRAINE, Lafayette College
MULCARE, DONALD J., Southeastern Massachusetts University
NAM, HONG GIL, Massachusetts General Hospital
NOBLE, REGINALD D., Bowling Green State University
ROSE, VIRGINIA, Concord, Massachusetts
SAXENA, INDER MOHAN, University of Texas, Austin
WILLIAMS, SHERICCA C., CIBA-GEIGY Corp.
BASIC IMMUNOCYTOCHEMICAL TECHNIQUES IN
TISSUE SECTIONS AND WHOLE MOUNTS
October 19-25, 1986
Course directors
BELTZ, BARBARA, Harvard Medical School
BURD, GAIL D., University of Arizona
Course assistants
KENT, CARLA, University of Arizona
KOBIERSKI, LINDA, Harvard Medical School
Students
AKANA, SUSAN FONG, University of California, San Francisco
BRADFUEHRER, PETER D., Cornell University
FOERSTER, ANNE, McMaster University, Canada
GETCHELL, MARILYN L., Wayne State University
HAMILTON, KATHRYN A., New England Medical Center
HAMMAR, KATHERINE M., Marine Biological Laboratory/NIH
HELLUY, SIMONE, The University of Alberta, Canada
EDUCATIONAL PROGRAMS 67
KRUSZEWSKA, BARBARA, University of Texas, Austin
LAUFER, HANS, University of Connecticut
Ross, LINDA S., University of Texas, Austin
SASAVAGE, NANCY L., Bethesda Research Laboratories
TiLSON, HUGH A., National Institute of Environmental Health Sciences
WHITE, JOEL, The Florida State University
WOOD, SUSAN F., Marine Biological Laboratory/BUMP
ZIGMOND, RICHARD E., Harvard Medical School
X. RESEARCH AND TRAINING PROGRAMS
SUMMER
PRINCIPAL INVESTIGATORS
ALLEN, NINA S., Wake Forest University
ALLEN, LABORATORY, Dartmouth College
ANDERSON, WINSTON A., Hunter College
ARMSTRONG, CLAY M., University of Pennsylvania
ARMSTRONG, PETER B., University of California, Davis
ATWOOD, KIM, Marine Biological Laboratory
AUGUSTINE, GEORGE, University of Southern California
BARRY, DANIEL, University of Michigan
BARLOW, ROBERT B., Syracuse University
BARRY, M., Albert Einstein College of Medicine
BARRY, SUSAN R., University of Michigan
BEAUGE, Luis ALBERTO, Institute de Investigacion Medica, Argentina
BEGENISICH, TED, University of Rochester Medical Center
BENNETT, MICHAEL V. L., Albert Einstein College of Medicine
BEZANILLA, FRANCISCO, University of California, Los Angeles
BLUNDON, JAY A., University of Maryland
BODZNICK, DAVID, Wesleyan University
BORGESE, THOMAS A., Lehman College
BORON, WALTER F., Yale University
BOYER, BARBARA C, Union College
BRADY, SCOTT T., University of Texas Health Science Center
BREHM, PAUL, Tufts University School of Medicine
BROWN, JOEL E., Washington University
BURDICK, CAROLYN J., Brooklyn College
BURGER, MAX M., University of Basel, Switzerland
CARMELIET, PETER, University of Leuven, Belgium
CHANG, DONALD C., Baylor College of Medicine
CHAPPELL, RICHARD L., Hunter College
CHARLTON, MILTON P., University of Toronto, Canada
CLEVELAND, MARK V. B., Braintree Laboratories
COHEN, LAWRENCE B., Yale University
COHEN, WILLIAM D., Hunter College
CONDOURIS, GEORGE A., New Jersey Medical School
COOPERSTEIN, SHERWIN J., University of Connecticut
CORNWALL, M. CARTER, Boston University School of Medicine
D'AVANZO, CHARLENE, Hampshire College
DEWEER, PAUL, Washington University
DUBAS, FRANCOISE, Whitney Laboratory of Marine Biology
DUBE, FRANCOIS, University of Quebec
DUNLAP, KATHLEEN, Tufts University School of Medicine
68 MARINE BIOLOGICAL LABORATORY
ECKBERG, WILLIAM R.. Howard University
EHRLICH, BARBARA, Albert Einstein College
FEINMAN, RICHARD, SUN Y Health Sciences Center
FESTOFF, BARRY W.. University of Kansas Medical Center
FISHMAN, HARVFV M., University of Texas Medical Branch
FREADMAN, MARVIN, Marine Biological Laboratory
FULKERSON, JOHN PRYOR, University of Connecticut School of Medicine
GADSBY. DAVIDC., Rockefeller University
GAINER, HAROLD, NICHD/NIH
GARBER, SARAH S., Brandeis University
GEORGE, EDWIN B., Case Western Reserve University
GILBERT, DANIEL L., NINCDS/NIH
GIUDITTA, ANTONIO, University of Naples, Italy
GOULD, ROBERT, New York Institute for Basic Research
GOVIND, C. K., University of Toronto
GRAF, WERNER M., Rockefeller University
HALVORSON, HARLYN O., Brandeis University
HEPLER, PETER K., University of Massachusetts
HIGHSTEIN, STEPHEN M., Washington University
HILL, ROBERT B., University of Rhode Island
HILL, SUSAN DOUGLAS, Michigan State University
HOSKIN, FRANCIS C. G., Illinois Institute of Technology
HORN, RICHARD, University of California Medical School
HUMPHREYS, TOM, University of Hawaii
JOHNSON, KENNETH A., Pennsylvania State University
JOSEPHSON, ROBERT K., University of California, Irvine
KALTENBACH, JANE C., Mount Holyoke College
KAMINER, BENJAMIN, Boston University
KAO, PETER, Columbia University
KAPLAN, EHUD, Rockefeller University
KEYNAN, ALEXANDER, Memorial Sloan Kettering Cancer Center
KEM, WILLIAM R., University of Florida
KORNBERG, HANS, University of Cambridge, UK
LANDOWNE, DAVID, University of Miami
LANGFORD, GEORGE M., University of North Carolina
LASER, RAYMOND J., Case Western Reserve University
LAUFER, HANS, University of Connecticut
LEVIS, RICHARD A., Rush Medical Center
LINDGREN, CLARK, Duke University Medical Center
LIPICKY, RAYMOND JOHN, Food and Drug Administration
LISMAN, JOHN, Brandeis University
LLINAS, RUDOLFO R., New York University
LOEWENSTEIN, WERNER R., University of Miami
MALBON, CRAIG C., State University of New York, Stony Brook
MATTESON, DONALD R., University of Pennsylvania
METUZALS, J., University of Ottawa, Canada
MORRELL, FRANK, Rush-Presbyterian-St. Luke's Medical Center
MORRELL, LEYLA DE TOLEDO, Rush-Presbyterian-St. Luke's Medical Center
MULLINS, LORIN J., University of Maryland
NARAHASHI, TOSHIO, Northwestern University
NELSON, LEONARD, Medical College of Ohio
NOE, BRYAN D., Emory University
NOLEN, THOMAS G., Yale University
OHKI, SHINPEI, State University of New York, Buffalo
PALEVITZ, BARRY A., University of Georgia
RESEARCH AND TRAINING PROGRAMS 69
PARSONS, THOMAS D., University of Pennsylvania School of Dental Medicine
PALMER, JOHN D., University of Massachusetts, Amherst
PEROZO, EDUARDO, Institute Venezolano de Investigaciones Cientificas, Venezuela
PIERSON, BEVERLY K., University of Puget Sound
POOLE, THOMAS J., Upstate Medical Center
PUMPLIN, DAVID W., University of Maryland
PURVES, DALE, Washington University
QUIGLEY, JAMES P., SUNY, Downstate Medical Center
RAKOWSKI, ROBERT F., Chicago Medical School
REBHUN, LIONEL I., University of Virginia
REID, JOHN, Hampshire College
RENDER, JOANN, Hamilton College
REYNOLDS, GEORGE T., Princeton University
RICKLES, FREDERICK R., University of Connecticut Health Center
RIPPS, HARRIS, University of Illinois College of Medicine
ROME, LAWRENCE C, University of Tennessee
Ross, WILLIAM, New York Medical College
RONAN, MARK, Wesleyan University
RUDERMAN, JOAN V., Duke University
RUSSELL, JOHN M., University of Texas
SALZBERG, BRIAN M., University of Pennsylvania
SANGER, JOSEPH W., University of Pennsylvania
SARDET, CHRISTIAN, Station Zoologique, France
SCHROER, TRINA, University of California, San Francisco
SEGAL, SHELDON J., Rockefeller Foundation
SILVER, ROBERT B., University of Wisconsin
SJODIN, RAYMOND A., University of Maryland School of Medicine
SLOBODA, ROGER D., Dartmouth College
SMITH, STEPHEN J., Yale University School of Medicine
SPECK, WILLIAM T., Rainbow Babies & Childrens Hospital
SPIEGEL, EVELYN, Dartmouth College
SPIEGEL, MELVIN, Dartmouth College
STANLEY, ELisF., NINCDS/NIH
STEPHENS, PHILIP J., Villanova University
STOCKBRIDGE, NORMAN, University of Alberta, Canada
STRACHER, ALFRED, SUNY Health Sciences Center
STRUMWASSER, FELIX, Boston University
STUART, ANN E., University of North Carolina
TAKEDA, KENNETH, Universite Louis Pasteur, France
TASHIRO, JAY SHIRO, Kenyon College
TAYLOR, ROBERT E., NINCDS/NIH
TILNEY, LEWIS, University of Pennsylvania
TRAVIS, JEFFREY L., Vassar College
TREISTMAN, STEVEN N., Worcester Foundation
TRINKAUS, JOHN PHILIP, Yale University
TROLL, WALTER, New York University
TUCKER, EDWARD B., Vassar College
VINCENT, WALTER S., University of Delaware
WAITE, MOSELEY, Bowman Gray School of Medicine
WEIDNER, EARL, Louisiana State University
WEISS, DIETER G., Institute of Zoology, FRG
WEISSMAN, GERALD, New York University Medical Center
WHITE, ROY L., Albert Einstein College of Medicine
YEH, JAY Z., Northwestern University
ZIGMAN, SEYMOUR, University of Rochester School of Medicine
70 MARINE BIOLOGICAL LABORATORY
LIBRARY READERS
ADELBERG, EDWARD, Yale Medical School
AKINS, KATHLEEN. Tufts University
ALKON, DANIEL, NIH/NINCDS
ALLEN, GARLAND E., Washington University
ANDERSON. EVERETT, Harvard Medical School
APOSHIAN, H. VASKEN, University of Arizona
BABITSKY, STEVEN, Kistin, Babitsky, Latimer & Beitman
BANG, BETSY, MBL
BARRETT, DENNIS, University of Denver
BEAN, CHARLES, Rensselaer Polytechnic Institute
BEMIS, WILLY, University of Massachusetts
BOETTIGER, JULIE, Temple University
BOYER, JOHN, Union College
BROWNE, ROBERTA., Wake Forest University
BUCK, JOHN, NIH
BURSZTAJN, S., Baylor College of Medicine
CANDELAS, GRACIELA C., University of Puerto Rico
CARRIERS, RITA, Downstate Medical Center
CASAGRANDE, VIVIEN A., Vanderbilt University
CHAMBERS, EDWARD L., University of Miami School of Medicine
CHEN, CHONG, Boston University Marine Program
CHILD, FRANK, Trinity College
CLARK, ARNOLD, MBL
COBB, JEWEL PLUMME, California State University
COHEN, LEONARD A., American Health Foundation
COHEN, SEYMOUR S., MBL
COLEN, B. D., Newsday
D'ALESSIO, GIUSEPPE, University of Naples
DETTBARN, WOLF-D., Vanderbilt University
DIPEOLU, OLUSEGUN O., Tuskegee University
DUNCAN, THOMAS K., Nichols College
EBERT, JAMES D., Carnegie Institution of Washington
ECKBERG, WILLIAM D., Howard University
ELLNER, JEROLD, Case Western Reserve University
FARB, DAVID, SUNY
FARMANFARMIAN, A., Rutgers University
FEINGOLD, DAVID S., New England Medical Center
FELDMAN, SUSAN, New Jersey Medical School
FIELD, GEORGE, Center for Astrophysics
FISHER, SAUL H., Milhauser Laboratory
FRIENKEL, KRYSTYNA, NYU Medical Center
FRIEDLER, GLADYS, Boston University School of Medicine
FRENKEL, NORBERT, Northwestern University Medical School
FUSSELL, CATHARINE P., Pennsylvania State University
GERMAN, JAMES L., New York Blood Center
GEWURZ, HENRY, Evanston, Illinois
GOLDSTEIN, MOISE H., JR., Johns Hopkins University
GOODGAL, SOLH., University of Pennsylvania School of Medicine
GOUNARIS, ANNE D., Vassar College
GRANT, PHILIP, University of Oregon
GROSSMAN, ALBERT, NYU
GUTTENPLAN, JOSEPH B., NYU Dental Center
HARDING, CLIFFORD V., Kresge Eye Institute
HAZEL, LEA, Kenyon College
HERSKOVITS, THEODORE T., Fordham University
RESEARCH AND TRAINING PROGRAMS 7 1
HILDEBRAND, JOHN G., University of Arizona
HILL, RICHARD W., Michigan State University
HILLMAN, PETER, Hebrew University
HILTS, PHILIP J., Washington Post
ILAN, JOSEPH, Case Western Reserve University
ILAN, JUDITH, Case Western Reserve University
INOUE, SADAYUKI, McGill University
JACOBS, LISA, Kenyon College
KALAT, JAMES W., North Carolina State University
KALTENBACH, JANE C, Mt. Holyoke College
KARUSH, FRED, University of Pennsylvania
KARUSH, WILLIAM, California State University
KELLY, ROBERT, University of Chicago, College of Medicine
KLEIN, DAVID, University of California, San Francisco
KLEIN, MORTON, Temple University Medical School
KLEMOW, KENNETH M., Wilkes College
KRANE, STEPHEN M., Massachusetts General Hospital
LADERMAN, AIMLEE, MBL
LAZAROW, PAUL B., Rockefeller University
LEE, JOHN J., City College of CUNY
LEIGHTON, JOSEPH, The Medical College of Pennsylvania
LEVITZ, MORTIMER, NYU Medical Center
LLOYD, DAN, Simmons College
LONG, CAROLE A., Hahnemann University School of Medicine
LORAND, LASZLO, Northwestern University
LYNCH, ELIZABETH, Kenyon College
MACKENZIE, DEBORA OLIVIA, New Scientist
MACLEISH, WILLIAM H., Houghton-Mifflin
MAIENSCHEIN, JANE, Arizona State University
MARFEY, ANNE, Danish Writers Guild
MARINE RESEARCH, INC.
MASER, MORTON, Woods Hole Educational Associates
MATSUMURA, FUMIO, Michigan State University
MAUTNER, HENRY G., Tufts University School of Medicine
MAUZERALL, DAVID, Rockefeller University
MCCANN-COLLIER, MARJORY, St. Peter's College
McCoY, FLOYD W., Lamont-Doherty Geological Observatory
MELE, SUZANNAH, Kenyon College
MILLER, JULIE ANN, Science News
MILLER, MELISSA, Kenyon College
MILLS, ERIC L., Dalhousie University
MITCHELL, RALPH, Harvard University
MIZELL, MERLE, Tulane University
MONROY, ALBERTO, Naples Zoological Station
MOORE, JOHN W., Duke University Medical Center
MORSE, PATRICIA M., Northeastern University
MUSACCHAI, X. J., University of Louisville
NAGEL, RONALD L., Albert Einstein College of Medicine
NICKERSON, PETER A., SUNY, Buffalo
OLINS, ADA L., University of Tennessee, Oak Ridge
OLINS, DONALD E., University of Tennessee, Oak Ridge
OLSZOWKA, ALBERT J., SUNY, Buffalo
OTT, KAREN, University of Evansville
PEISACH, JACK, Albert Einstein College of Medicine
PERSON, PHILIP, VA Medical Center, Brooklyn, New York
POOLE, ALAN F., MBL
PRICE, BILL, Kenyon College
72 MARINE BIOLOGICAL LABORATORY
PROVASOLI, LUIGI, Yale University
PRUSCH, ROBERT, Gonzaga University
RAEBURN, PAUL, Associated Press
REINER, JOH^' M. Vibany Medical College
RINGER, STEPHKV. Childrens Hospital
ROBINSON VI BL
ROTH, Ei
RUSSELL, H., University of Arizona College of Medicine
Ru HUNTER, W. D., Syracuse University
SCMit SINGER, R. WALTER, University of Medicine and Dentistry of New Jersey
SCHMIDT, SUSANNE, Cape Cod Planning and Economic Development
SEAVER, GEORGE, Seaver Assoc.
SHAPLEY, ROBERT, Rockefeller University
SHEMIN, DAVID, Northwestern University
SHEPARD, FRANK, Woods Hole Data Base
SHEPRO, DAVID, Boston University
SHRIFTMAN, MOLLIE STARR, North Nassau Mental Health Center
SLUDER, GREENFIELD, Worcester Foundation for Experimental Biology
SOHN, JOEL, Joel Sohn Seafood
SPECTOR, ABRAHAM, Columbia University
SPOTTE, STEPHEN, Mystic Marinelife Aquarium
STEINBERG, MALCOLM S., Princeton University
STEPHENS, MICHAEL J., Rutgers University
STEPHENSON, WILLIAM K., Earlham College
STEVENS, CHARLES F., Yale Medical School
SZENT-GYORGYI, ANDREW G., Brandeis University
SZENTKIRALYI-SZENT-GYORGYI, EVA M., Brandeis University
TONE, JEFFERSON, Kenyon College
TRACER, WILLIAM, Rockefeller University
TUTTLE, FRANK, Kenyon College
TWEEDEL, KENYON S., University of Notre Dame
VAN HOLDE, K. E., Oregon State University
WAGNER, ROBERT R., University of Virginia
WAINIO, WALTER, Rutgers University
WANGH, LAWRENCE, Brandeis University
WARREN, LEONARD, Wistar Institute
WEBB, H. MARGUERITE, MBL
WEINER, JONATHAN, Doylestown, Pennsylvania
WHEELER, GEORGE E., Brooklyn College
WHITTENBERG, BEATRICE, Albert Einstein College of Medicine
WHITTENBERG, JONATHAN, Albert Einstein College of Medicine
WlCHTERMAN, RALPH, MBL
WILBUR, CHARLES G., Colorado State University
WOLKEN, JEROME J., Carnegie Mellon University
WORGUL, BASIL V., College of Physicians and Surgeons, Columbia University
YATHIRAJ, SANJAY, Kenyon College
YOUNG, WISE, NYU Medical Center
Yow, F. W., Kenyon College
ZACKS, SUMNER I., The Miriam Hospital
ZIMMERMAN, MORRIS, Merck Sharp & Dohme Research Laboratory
ZOTTOLI, STEVEN J., Williams College
OTHER RESEARCH PERSONNEL
ABRAHAMIAN, LORI, University of Connecticut Health Center
ABRAMSON, CHARLES, State University of New York Health Sciences Center
RESEARCH AND TRAINING PROGRAMS 73
ALEXANDER, R. MCNEILL, University of Leeds, UK
ALTAMIRANO, ANIBAL, University of Texas
ARMSTRONG, SANDRA, Lower Merion High School
ASHLEY, C. C, Oxford University
BAKER, ROBERT, New York University
BAKER, Ross, University of Connecticut Medical Center
BATES, HISLA, Hunter College
BENNETT, ELENA P., Connecticut College
BISIER, ENRIQUE FONT, University of Tennessee
BLECK, THOMAS P., Rush Medical College
BLUMER, JEFFREY L., Rainbow Babies and Children's Hospital
BORST, DAVID, Illinois State University
BOYLAN, JEANETTE, Michigan State University
BREITWEISER, GERDA E., University of Texas Medical Branch
BROSIUS, D., Albert Einstein College of Medicine
BROWN, LESLEE DODD, Northwestern University Medical School
BROWNE, CAROL, Wake Forest University
BUTNAM, JOHN A., Washington University School of Medicine
CAPUTO, CARLO, Institute Venezolano de Investigaciones Cient
CARIELLO, Lucio, Naples Zoological Station, Italy
CATTARELLI, MARTINE H., Yale University School of Medicine
CALLAWAY, JAY, University of Washington
CATANEO, RENE, NYS Institute for Basic Research in Developmental Disabilities
CHANDLER, ROBERT, University of Maryland
CHOW, ROBERT H., University of Pennsylvania
CHEN, ERIC, Northwestern University
CLARK, GEOFF, Braintree Laboratories
COLTON, CAROL, Georgetown University Medical School
COHEN, AVRUM, University of Chicago
COTA-PENUELAS, GABRIEL, University of Pennsylvania
COUCH, ERNEST, Texas Christian University
COTE, RICK, University of Wisconsin
CZINN, STEVEN, Rainbow Babies and Childrens Hospital
DAVIDSON, DAVID, New York University Medical School
DAVIDSON, SARAH, Columbia University
DEWEILLE, JAN, University of Utrecht, Netherlands
DIPOLO, REINALDO, Institute de Investigacion Medica, Argentina
DIXON, ROBERT, College of the Holy Cross
DOME, JEFF, University of Pennsylvania School of Medicine
DOWLING, JOHN E., Harvard University
DOUGHERTY, KATHLEEN, University of Delaware
DUAX, J. B., SUNY, Buffalo
DULDULAO, MARLYN, University of Hawaii
EATON, D. C., University of Texas Medical Branch
EHRENSTEIN, DAVID, Oberlin College
EHRENSTEIN, G., NINCDS/NIH
FINK, RACHEL D., Mount Holyoke College
FLACKER, JONATHAN M., Emory University
FONG, C. N., University of Toronto, Canada
FRANK, DOROTHY, Rainbow Babies and Childrens Hospital
GILBERT, SUSAN P., Dartmouth College
GONSALVES, NEIL, Rhode Island College
GONZALEZ, HUGO, University of Maryland
GRAUBARD, KATHERINE, University of Washington
GRAY, DAVID A., University of Southern California
GREINER, FRANCINE, Emory University
74 MARINE BIOLOGICAL LABORATORY
GRIFFITHS, PETER J., University Laboratory of Physiology
GRUNER, JOHN A., New York University
GREEN, WENDY B., Amherst College
HANTAI, DANIEL, 1 M.S.E.R.M.
HEITHAUS, E, R., Kenyon College
HERLANDS, Louis, Population Council
HIRIART, MARCIA, University of Pennsylvania
HOCSON. DANIAL, University of Puget Sound
HOLBROOK, PAMELA G., Massachusetts Institute of Technology
HOLDER, DAVID, City University of New York
HOUGHTON, SUSAN, Marine Biological Laboratory
HOMOLA, ELLEN, University of Connecticut
HUNT, TIM, University of Cambridge, UK
HUNT, JOHN R., Baylor College of Medicine
IVENS, KEITH, Howard University
JACKSON, LOVERNE, University of Ottawa, Canada
JOCKUSCH, BRIGITTE M., University of Bielefeld, FRG
JOHNSON, EDWIN, Brandeis University
KASS, LEONARD, University of Maine
KAHLER, CHERYL, Kansas City Veterans Administration Medical Center
KAHN, TERRI, Rainbow Babies and Childrens Hospital
KEM, ELAINE S., Fairleigh Dickinson University
KEM, JAMES, University of Florida
KISHIMOTO, YASUA, Johns Hopkins School of Medicine
KNAKAL, ROGER C., Yale University
KNIER, JULIE A., University of Minnesota
KOIDE, SAMUEL S., Population Council
KONZELMANN, DANIEL J., Eastern Illinois University
KOSIK, K. S., Harvard University Medical School
LANDOLFA, MICHAEL A., Union College
LANDAU, MATTHEW, University of Connecticut
LEECH, COLIN A., University of Cambridge, UK
LEHMAN, HERMAN, Rockefeller University
LEOPOLD, PHILIP LUTZ, Georgetown University
LONDON, JILL, Yale University School of Medicine
LOPEZ-BARNEO, JOSE, University of Seville Medical School, Spain
LOPICCOLO, DANIEL, Medical College of Ohio
LUCA, FRANK, Duke University Medical Center
LUTZ, GORDON, University of Tennessee
MACK, ERIN, University of Puget Sound
MAMUYA, WILFRED, Boston University School of Medicine
MASSEY, ERIC, University of North Carolina
MASSIOTTE, J. MATHIEU, University of Connecticut Health Center
MCCARTHY, ROBERT ALAN, University of Basel, Switzerland
McGuiNNESS, T., Rockefeller University
MELLO, ANIBEL, Rhode Island College
MENICHINI, ENRICO, Northwestern University
MERRITT, MARIA, Wake Forest University
MEYER, MONICA A., Vassar College
MILLER, ROBERT, Case Western Reserve University
MILLS, VAN, The University of North Carolina
MISEVIC, GRADIMIR, University of Basel, Switzerland
MOCHEL, SUSAN, Tufts University
MURRAY, SANDRA, University of Pittsburgh
NAKA, KEN-!CHI, National Institute of Basic Biology, Japan
NICHOLAS, CRAIG JOHN, Syracuse University
RESEARCH AND TRAINING PROGRAMS 75
NISHIO, MATOMO, Northwestern University Medical School
OBAID, ANA LIA, University of Pennsylvania
ORTIZ, ROSALEE, Howard University
OSSES, Luis, University of California, Los Angeles
PALAZZO, ROBERT, University of Virginia
PANT, HARISH, NIAAA/NIMH/DHHS
PAXHIA, TERESA M., University of Rochester
PAXSON, CHERYL, University of Chicago Medical School
PAULSEN, REINHARD, Ruhr University, FRG
PEREZ, ROSA, Hunter College
RALPH, WALTER, City University of New York
RASGADO-FLORES, HECTOR, University of Maryland
RENDER, TIMOTHY JOHN, University College, Oxford, UK
REQUENA, JAIME, I.D.E.A., Venezuela
RIESEN, WILLIAM J., Yale University
ROSE, BIRGIT, University of Miami
ROBINSON, JoHNT., University of North Carolina
ROBINSON, PHYLLIS, Brandeis University
ROSENBAUM, ROBERT, Vassar College
RUDOLPH, REBECCA, University of Puget Sound
SANDS, VICKJ, University of Puget Sound
SANGER, JEAN, University of Pennsylvania
SAHNI, MUKESH, Rockefeller Foundation
SAK.AI, HIROKO, National Institute for Basic Biology, Japan
SAWYER, PAM, University of Ottawa, Canada
SCHLUP, VERENA, University of Basel, Switzerland
SCHNEIDER, ERIC, Wesleyan University
SCHIMINOVICH, DAVID, Yale University
SCHNEIDER, MELISSA, Hamilton College
SEITZ-TUTTER, DIETER, Institute fur Zoologie, FRG
SHEETZ, JENNIFER, Duke University
SHEN, JOANNE, University of Southern California
SIEGAL, NINA, Case Western Reserve University
SIMPSON, MARCIA, Amherst College
SOLOMON, JOEL, Washington University School of Medicine
SPIRES, SHERRILL, University of Rochester Medical Center
STEINACKER, ANTOINETTE, Washington University School of Medicine
STOCKBRIDGE, LISA, University of Alberta, Canada
STOKES, DARRELL, Emory University
STRONG, JOHN C., University of Maryland, Baltimore
SUGIMORI, MUTSUYUKI, New York University
SWANDULLA, DIETER, University of Pennsylvania
SWENSON, KATHERINE, Harvard University School of Medicine
TAKEDA, KIMIHISA, Tottori University, Japan
TAKLA, NORA, Washington University School of Medicine
TANGUY, JOELLE, Laboratoire de Neurobiologie, France
TEDESCHI, BRUCE, Louisiana State University
TELFER, JANICE, Wake Forest University
THIBAULT, LAWRENCE, University of Pennsylvania
TOTH, JOSEPH, Hunter College
TRICAS, TIMOTHY, Washington University School of Medicine
TWERSKY, LAURA, Hunter College
TYTELL, MICHAEL, Wake Forest University
UENO, HIROSHI, Rockefeller University
UGORETZ, JOHN, La Jolla High School
VERSELIS, VYTALITAS, Albert Einstein College of Medicine
76 MARINE BIOLOGICAL LABORATORY
WALTON, ALAN J., Oxford University, UK
WANG, XIN-SHANG, Vassar College
WEBB, CHRISTINA, University of California, Los Angeles
WESTENDORF, Jcv NNE M., Duke University
WHITTAKER, Josi Howard University
WHITTEM; SE, University of Pennsylvania
WILLI A ' :OME, Hunter College
Wool I • .-•; C., California State University, Los Angeles
ZAKEV ;, JANE, University of Illinois
z, JOSEPH, Albert Einstein College of Medicine
ZEA. SVEN E., University of Texas
ZECEVIC, DEJAN, Institute of Biological Research, Yugoslavia
ZHAO, ZHAE-YIONG, Baylor College
YEAR-ROUND PROGRAMS
BOSTON UNIVERSITY MARINE PROGRAM (BUMP)
Director
STRICKLER, J. RUDI
Faculty (of Boston University unless otherwise indicated)
ATEMA, JELLE TAMM, SIDNEY L.
FREADMAN, MARVIN TAMM, SIGNHILD
HUMES, ARTHUR G. (Emeritus) TIERNEY, ANN JANE
SUMAN, DANIEL VALIELA, IVAN
Staff (of Boston University unless otherwise indicated)
CROMARTY, STUART SUNLEY, DANIEL
DZIERZEWSKI, MICHELLE TAYLOR, MARGERY
HAHN, DOROTHY VAN ETTEN, RICHARD
LOHMANN, DENAH WOODWARD, HELEN
Graduate students
ALBER, MERRYL COULTER, DOUGLAS
BANTA, GARY COWAN, DIANE
BARSHAW, DIANA ELLIS, SARAH
BORRONI, PAOLA ELSKUS, ADRIA
CHEN, CHONG GALLAGER, SCOTT
CORROTO, FRANK CLICK, STEPHEN
COSTA, JOSEPH HANDRICH, LINDA
Undergraduates
BRAN, TERRENCE MULSOW, SANDOR
BROWN, SIDNEY SCOTT, MARSHA
CARLON, DAVID TAMSE, ARMANDO
MURPHY, TARA TROTT, THOMAS
HAHN, JILL WEBB, JACQUELINE
HERSH, DOUGLAS WHITE, DAVID
KRIEGER, YUTTA WOODS, SUSAN
LAVALLI, KARI SAPONARO, STEPHEN
MERCURIC, KIM SHAPIRO, RACHEL
MERRILL, CARL WALLACE, RICHARD
MOORE, PAUL
RESEARCH AND TRAINING PROGRAMS 77
Visiting investigators
D'AVANSO, CHARLENE, Hampshire College
POOLE, ALAN, Boston University
RIETSMA, CAROL, SUNY, New Paltz
SARDA, RAFAEL, University of Barcelona
VOIGT, RAINER, University of Gottingen
DEVELOPMENTAL AND REPRODUCTIVE BIOLOGY LABORATORY
Director
GROSS, PAUL R.
LABORATORY OF BIOPHYSICS
Director
ADELMAN, WILLIAM J., JR.
Staff (of NINCDS/NIH unless otherwise indicated)
Section on Neural Membranes
CLAY, JOHN R.
FOHLMEISTER, JuRGEN R., University of Minnesota
GOLDMAN, DAVID E., SUNY, Binghamton
HODGE, ALAN J., Marine Biological Laboratory
KRAMER, JUDITH A., University of Cincinnati College of Medicine
LAVOIE, ROBERT, Marine Biological Laboratory
MARTIN, DOROTHY L.
McMAHON, WILLIAM E., Marine Biological Laboratory
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
CHEN, CHONG
COLLIN, CARLOS
COULTER, DOUGLAS, Boston University
DISTERHOFT, JOHN, Northwestern University Medical School
HARRIGAN, JUNE, Marine Biological Laboratory
HOPP, HANS-PETER
IKENO, HIDETOSHI
KUBOTA, MlCHINORI
KUZIRIAN, ALANM.
KUZIRIAN, JEANNE
LEDERHENDLER, IZJA, Marine Biological Laboratory
LEIGHTON, STEPHEN, Biomedical Engineering and Instrumentation Branch, NIH
LOTURCO, JOSEPH
McPHiE, DONNA
NAITO, SHIGETAKA
NEARY, JOSEPH, Marine Biological Laboratory
SAKAKIBARA, MANABU
78 MARINE BIOLOGICAL LABORATORY
LABORATORY OF CARL J. BERG, JR.
Director
BERG, CARL J, JR.
Staff
ADAMS, NANCY
ORR, KATKERINE 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
LABORATORY OF D. EUGENE COPELAND
Director
COPELAND, D. EUGENE
LABORATORY OF DEVELOPMENTAL GENETICS
Director
WHITTAK.ER, J. RICHARD
Staff
CROWTHER, ROBERT
LOESCHER, JANE L.
MEEDEL, THOMAS H.
MERCURIO, KIMBERLY
Visiting investigators
COLLIER, J. R., Brooklyn College
Summer intern (undergraduate)
ZELLER, ROBERT, Boston University
LABORATORY OF JUDITH P. GRASSLE
Director
GRASSLE, JUDITH P.
Staff
GELFMAN, CECILIA E.
MILLS, SUSAN W.
RESEARCH AND TRAINING PROGRAMS 79
LABORATORY FOR MARINE ANIMAL HEALTH
Director
LEIBOVITZ, Louis, Cornell University
Staff
ABT, DONALD A., University of Pennsylvania
HAMILTON, HEATHER A., Cornell University
JENNER, JENNIFER L., Cornell University
McCAFFERTY, MICHELLE, Cornell University
MONIZ, PRISCILLAC., Marine Biological Laboratory
LABORATORY OF OSAMU SHIMOMURA
Director
SHIMOMURA, OSAMU, Boston University School of Medicine
Staff
SHIMOMURA, AKEMI
Visiting investigators
MUSICKI, BRANISLAV, Harvard University
NAKAMURA, HIDESHL Harvard University
LABORATORY OF RAYMOND E. STEPHENS
Director
STEPHENS, RAYMOND E., Marine Biological Laboratory/Boston University School of
Medicine
Staff
GOOD, MICHAEL J., Marine Biological Laboratory
OLESZKO-SZUTZ, SUSAN, Marine Biological Laboratory
STOMMEL, ELIJAH W., Marine Biological Laboratory/Boston University School of Medicine
LABORATORY OF SENSORY PHYSIOLOGY
Director
FEIN, ALAN
Staff
HAROSI, FERENC I.
PAYNE, RICHARD
SZUTS, ETE Z.
WOOD, SUSAN
ZAHAJSZKY, TIBOR
Visiting investigators
CORNWALL, CARTER, Boston University School of Medicine
HAWRYSHYN, CRAIG W., Cornell University
PETRY, HEYWOOD M., SUNY, Stonybrook
80 MARINE BIOLOGICAL LABORATORY
TSACOPOULOS, MARCO, University of Geneva, Switzerland
WALZ, BERND, University of Ulm, West Germany
LABORATORY OF SHINYA INOUE
Director
INOUE, SHFNYA, Marine Biological Laboratory, University of Pennsylvania
Staff
ANNIBALLI, DYON, Cornell Engineering School
BOYD, STEVEN, Cornell Engineering School
GREEN, DANIEL, Cornell Engineering School
INOUE, THEODORE, Cornell Engineering School
RUBINOW, JERRY, Cornell Engineering School
SHIMOMURA, SACHI
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
CHLUDZINSK.I, JOHN, Marine Biological Laboratory
CRISE, BRUCE, Marine Biological Laboratory
EVENDEN, PHYLLIS
FROKJAER-JENSEN, JORGEN, University of Copenhagen
GALLANT, PAUL
GARBUS-GOOCH, CYNTHIA, Marine Biological Laboratory
HAMMAR, KATHERINE
JAROCHE, DEANNA, Marine Biological Laboratory
KHAN, SHAHID, Marine Biological Laboratory
MCCUSKER, ELIZABETH
MURPHY, JOHN C., Marine Biological Laboratory
REESE, BARBARA F.
SHEETZ, MICHAEL P., Washington University
SCHNAPP, BRUCE J.
TATSUOKA, HOZUMI
TERASAKJ, MARK
VALE, RONALD D.
WALROND, JOHN P.
WISGIRDA, MARY, Marine Biological Laboratory
NATIONAL FOUNDATION FOR CANCER RESEARCH
Director
SZENT-GYORGYI, ALBERT
RESEARCH AND TRAINING PROGRAMS 8 1
Staff
GASCOYNE, PETER R. C.
MCLAUGHLIN, JANE A.
MEANY, RICHARD A.
PETHIG, RONALD, University College of North Wales, UK
Student
PRICE, JONATHAN A., University College of North Wales, UK
NATIONAL VIBRATING PROBE FACILITY
Director
JAFFE, LIONEL, Marine Biological Laboratory
Staff
DIXON, STEVEN
SHIPLEY, ALAN
STEWART, MARY
WILLIAMS, PHILLIP C.
Visiting investigators
ALLEN, NINA, Wake Forest University
BJORKMAN, THOMAS, Cornell University
BOWDAN, ELIZABETH, University of Massachusetts, Amherst
DURHAM, JOHN, Mt. Sinai Hospital, New York
ETTENSOHN, CHARLES, Duke University
FINK, RACHEL, Mount Holyoke College
FLUCK, RICHARD, Franklin & Marshall College
KATZ, URI, Israel Institute of Technology, Haifa, Israel
KUNKEL, JOSEPH, University of Massachusetts
LEVY, SIMON, Boston University
PAYNE, RICHARD, Marine Biological Laboratory
RUBIN, CLINTON, Tufts Medical School
SARDET, CHRISTIAN, Station Marine Villelfranche sur Mer, France
SKADHAUGE, ERIC, Royal Veterinary University, Copenhagen
SPEKSNEIJDER, J. H., Marine Biological Laboratory
TRINKAUS, JOHN, Yale University
TROXELL, CYNTHIA, University of Colorado, Boulder
WEIJER, KEES, University of Munich, FRG
WEISENSEEL, MANFRED, University of Karlsruhe, FRG
ZIVKOVIC, DANA, University of Utrecht
THE ECOSYSTEMS CENTER
Director
HOBBIE, JOHN E.
Staff and consultants
BANTA, GARY GIBLIN, ANNE
BOWLES, FRANCIS P. GRIFFIN, ELISABETH A.
FERRY, ELIZABETH HELFRICH, JOHN V. K.
GARRITT, ROBERT HOUGHTON, RICHARD A.
82 MARINE BIOLOGICAL LABORATORY
JOHNSON, STEPHEN POVIA, SANDRA
LAUNDRE, JAMES RAY, ANDREA
LEFKOWITZ, DANIEL REGAN, KATHLEEN
MATHERLY, WALTHZR SEMINO, SUZANNE
MCKERROW, ALFXA SHAVER, GAIUS R.
MELILLO, JERRY M. STEUDLER, PAUL A.
MICHENER, ROBERT STONE, THOMAS A.
NADELHOFFER, KNUTE J. TUCKER, JANE
OPPENHEIMER, JILL TURNER, ANDREA R.
PETERSON, BRUCE J. WHITE, DAVID
PLUMMER, NANCY YANDOW, TIMOTHY
Trainees
RASTETTER, EDWARD, University of Virginia
RUDNICK, DAVID, University of Rhode Island
Visiting scientists
JORDAN, MARILYN J.
O'BRIEN, W. JOHN
RUBLES, PARKE
WARING, RICHARD
XI. HONORS
FRIDAY EVENING LECTURES
SIMBERLOFF, DANIEL, Florida State University, 27 June, "Academic Ecology and Environ-
mental Problems: Red Scales, Vampire Bats, and Spotted Owls"
ALBERSHEIM, PETER, University of Georgia, 4 July, "Oligosaccharins — A New Class of Regu-
latory Molecules in Plants and Animals"
BROWN, DONALD D., Carnegie Institution of Washington, 1 1 July, "The Molecular Basis of
Differential Gene Expression"
STEVENS, CHARLES F., Yale University School of Medicine, 17, 1 8 July, Forbes Lectures, "Mo-
lecular Basis for the Brain's Electrical Activity: I. Electrical Excitability of Neurons:
II. Communication between Neurons"
KAISER, DALE, Stanford University School of Medicine, 25 July, "Cell-Cell Interactions in a
Simple Developmental Pathway"
REESE, THOMAS S., NINCDS, NIH, and Marine Biological Laboratory, 1 August, "Kinesin—
An MBL Project"
DOOLITTLE, RUSSELL F., University of California, San Diego, 8 August, "Evolution of the
Vertebrate Plasma Proteins "
KANDEL, ERIC R., College of Physicians & Surgeons of Columbia University and the Howard
Hughes Medical Institute, 1 5 August, Lang Lecture, "The Long and Short of Memory"
SELA, MICHAEL, The Weizmann Institute of Science, 22 August, "From Synthetic Antigens to
Synthetic Vaccines"
TRINKAUS, J. P., Yale University, 29 August, "Metazoan Cell Movements: Invasion and Mor-
phogenesis"
CHARLES ULRICK AND JOSEPHINE W. BAY FOUNDATION FELLOWSHIP
SMOLOWITZ, ROXANNA, Marine Biological Laboratory
ERNEST EVERETT JUST FELLOWSHIPS IN BIOLOGY
JOSIAH MACY, JR., FOUNDATION
WHITE, ROY L., Albert Einstein College of Medicine
HONORS 83
MBL SUMMER FELLOWSHIPS
DLIBE, FRANCOIS, Universite du Quebec a Rimouski, Canada
EHRLICH, BARBARA, University of Connecticut Health Center
PIERSON, BEVERLY K., University of Puget Sound
POOLE, THOMAS L., SUNY, Syracuse
ROME, LAWRENCE C., University of Tennessee
TAKEDA, KENNETH, University Louis Pasteur, France
TRAVIS, JEFFERY, Vassar College
BIOLOGY CLUB OF NEW YORK
KASMER, JOHN M., University of Vermont
FATHER ARSENIUS BOYER SCHOLARSHIP
KASMER, JOHN M., University of Vermont
GARY N. CALKINS MEMORIAL SCHOLARSHIP
DIOGENE, GEORGE F., University of Barcelona, Spain
FRANCES S. CLAFF MEMORIAL SCHOLARSHIP
FREY, IRIS J. F., Philipps-University Marburg, FRG
EDWIN GRANT CONKLIN MEMORIAL SCHOLARSHIP
C/HARA, ELLEN M., Villanova University
LUCRETIA CROCKER SCHOLARSHIP
FALK, KATHLEEN, University of Massachusetts
HART, ROBERTA., University of California, Berkeley
MORUCCI, CARLO, University of La Sapienza of Rome, Italy
ZAPATA, FERNANDO A., University of Arizona
FOUNDERS-OTTO LOEWI
AKINS, ROBERT E., JR., University of Pennsylvania
FOUNDERS- WALTER E. GARREY
C/HARA, ELLEN M., Villanova University
FOUNDERS-S. O. MAST
SMOLICH, BEVERLY, University of Virginia
ALINE D. GROSS SCHOLARSHIP
RENDER, JoANN, Hamilton College
MERKEL H. JACOBS SCHOLARSHIP
KASMER, JOHN M., University of Vermont
84 MARINE BIOLOGICAL LABORATORY
ARTHUR KLORFEIN FUND SCHOLARSHIPS
HAMMER, MARTIN, Institut fuer Tierphysiologie, FRG
HARRINGTON, MARY E,, Dalhousie University, Canada
JURNISCH, VK ; . A.. University of California, Irvine
LUSTIG, CORN'. I miann Institute, Israel
SUPATTAPONF, MAI, Johns Hopkins University
LUCILLE P. MARKEY CHARITABLE TRUST SCHOLARSHIPS
BISWAS, SURAJIT, University of Pennsylvania
BLOOM, THEODORA L., University of Cambridge, England
BRADLEY, DAVID, University of Pennsylvania
BROWN, ANNEC, University of Oregon
CAULEY, KEITH A., University of Michigan
DAHL, STEPHEN C., Wesleyan University
DASSO, MARY C., Cambridge University, UK
DEYST, KATHERINE A., Tufts University
DIOGENE, GEORGE F., University of Barcelona, Spain
DUBILIER, NICOLE, University of Hamburg, FRG
FALK, KATHLEEN, University of Massachusetts
FOLTZ, KATHLEEN R., Purdue University
FREY, IRIS J. F., Philipps-University Marburg, FRG
GANNON, PAMELA M., Tufts University
GUDEMAN, DAVID M., Kansas University
HAFNER, MATHIAS, German Cancer Research Center, FRG
HART, ROBERTA., University of California
HOULISTON, EVELYN, University of Cambridge, UK
KOENIG, GERD, MPI fur Entwicklungsbiologie, FRG
KUBIAK, JACEK Z., Warsaw University, Poland
SAAVEDRA, CAROL, McGill University, Canada
SMOLICH, BEVERLY, University of Virginia
SVENDSEN, BETTY-ANN E., University of Dallas
SYMES, KAREN, National Institute of Medical Research, UK
TALEVI, RICCARDO, University of Naples, Italy
THIVAKARAN, ALAGIRI G., Annamalai University
VELLECA, MARK A., Yale University
VITES, ANA M., University of Connecticut Health Center
ZAPATA, FERNANDO A., University of Arizona
ALLEN M. MEMHARD SCHOLARSHIP
BROWN, ANNEC., University of Oregon
JAMES S. MOUNTAIN MEMORIAL FUND, INC. SCHOLARSHIPS — 1986
DAHL, STEPHEN, Wesleyan University
DASSO, MARYC., Cambridge University, UK
FOLTZ, KATHLEEN R., Purdue University
GUDEMAN, DAVID M., Kansas University Medical Center
KATZ, KENNETH S., University of Massachusetts
SYMONS, MARC H. C., Weizmann Institute, Israel
HONORS
85
JAMESS. MOUNTAIN MEMORIAL FUND, INC. SCHOLARSHIPS — 1985*
CHEN, TUNG-LING, University of Maryland
GOODWIN, ELIZABETH B., Brandeis University
HANNA, MAYA, Harvard University
PRET, ANNE-MARIE, Wesleyan University
WALTHER, ZENTA, Yale University
Wu, BEI-YUE, Wayne State University
SOCIETY OF GENERAL PHYSIOLOGISTS SCHOLARSHIPS
BLOOM, THEODORA L., University of Cambridge, UK
HOULISTON, EVELYN, University of Cambridge, UK
SURDNA FOUNDATION SCHOLARSHIPS
SPANO, ANNAMARIA, Institute Superiore di Sanitz, Rome, Italy
SVENDSEN, BETTY-ANN E., University of Dallas
MARJORIE W. STETTEN SCHOLARSHIP
SCHWEIZER, FELIX E., Biozentrum/Universitat Basel, Switzerland
XII. INSTITUTIONS REPRESENTED
U.S.A.
Academy of Natural Sciences of
Philadelphia
Albany Medical Center
Albert Einstein College of Medicine
American Health Foundation
Amherst College
Arizona Research Laboratory
Arizona State University
Arizona, University of
Arizona, University of, College of Medicine
Atlantex and Zieler Instrument
Corporation
Axon Instruments, Inc.
Bardeen Laboratory
Bausch & Lomb
Baylor College
Baylor College of Medicine
Beckman Instruments, Inc.
Bell Laboratories
Bethesda Research Labs
Bigelow Laboratories
BioTechnical International Inc.
Biodyne Electronics
Biomedical Engineering and
Instrumentation Branch, NIH
Bodega Marine Station
Boston University
Boston University Marine Program
Boston University Medical School
Bowling Green State University
Bowman Gray Medical School
Braintree Laboratories
Brandeis University
Brinkmann Instruments
Brooklyn College
Brown University
California Institute of Technology
California State University
California State University, Los Angeles
California, University of
California, University of, Berkeley
California, University of, Davis
California, University of, Irvine
California, University of, Los Angeles
California, University of, Riverside
California, University of, San Diego
California, University of, San Francisco
Carnegie Institution of Washington
Carnegie-Mellon University
Case Western Reserve University
Center for Advanced Research
Center for Astrophysics
* The Marine Biological Laboratory regrets the omission of 1985 scholarship recipients in the 1985 An-
nual Report [Biol. Bull. 171(1)].
86
MARINE BIOLOGICAL LABORATORY
Chesapeake Biological Laboratory
Chicago, University of
Chicago, University of, Medical School
Childrens Hospital
Cincinnati, University of, College of
Medicine
College of the Holy Cross
Colorado, U mversity of
Colorado, University of, Boulder
Colorado Video
Columbia University
Columbia University College of Physicians
and Surgeons
Connecticut, University of
Connecticut, University of. Health Center
Connecticut, University of. Medical Center
Connecticut, University of. School of
Medicine
Conservation Law Foundation
Cornell Engineering School
Cornell University
Coulter Electronics
Creighton University
Crimson Camera Technical Sales, Inc.
Dagan Corporation
DAGE-MTI
Dalhousie University
Dallas, University of
Damon Biotech, Inc.
Dana-Farber Cancer Institute
Dartmouth College
Dartmouth Medical School
Delaware, University of
Denver, University of
Dow Chemical
Duke University
Duke University Medical Center
Dupont Corporation
Earlham College
Eastern Illinois University
Eastman Kodak Company
Emory University
Emory University School of Medicine
Environmental Protection Agency
Ethicon, Inc.
Evansville, University of
Fairleigh Dickinson University
Florida State University
Florida, University of
Flow Laboratory
Fordham University
Franklin and Marshall College
General Electric Company
General Scanning
Georgetown University Medical School
George Washington School of Medicine
Georgia, University of
Gilson Medical Electronics
Gonzaga University
Goucher College
Grass Instrument Company
Hacker Instruments
Hampshire College
Hahnemann University
Hahnemann University School of
Medicine
G. W. Hannaway Associates
Hartwick College
Harvard Medical School
Harvard University
Harvard University School of Public
Health
Hawaii, University of
Hinds Jr. College
Hoefer Science Instruments
Hope College
Howard Hughes Medical Institute
Howard University
Hunter College
Hutchinson Cancer Center
IBI
IDEA
I.N.S.E.R.M.
Ikegami Electronics Inc.
ISCO
Illinois Institute of Technology
Illinois, University of, Chicago
Illinois, University of. College of Medicine
Illinois, University of, Urbana-Champaign
Indiana University
Instrumentation Marketing Corporation
Interactive Video Systems
International Business Machines
Iowa, University of
Johns Hopkins School of Medicine
Johns Hopkins University
Kansas City Veterans Administration
Medical Center
Kansas, University of
Kansas, University of, Medical School
Kenyon College
Kip & Zonen
Kisten, Babitsky, Latimer & Beitman
Kresge Eye Institute
LKB Instruments, Inc.
Lab Line Instruments, Inc.
LaFayette College
Lamont-Doherty Geological Observatory
Lander College, South Carolina
Lehman College
Leitz, E. Inc.
Levity Corporation
INSTITUTIONS REPRESENTED
87
Liberty Mutual Research Center
Louisiana State University
Louisville, University of
META Systems, Inc.
Maine, University of
Mary Flagler Gary Arboretum, NY
Maryland, University of
Maryland, University of, Baltimore
Massachusetts General Hospital
Massachusetts Institute of Technology
Massachusetts, University of
Massachusetts, University of, Amherst
Massachusetts, University of. Medical
School
Mayo Foundation
Medical College of Ohio
Medical College of Pennsylvania
Memorial Sloan Kettering
Merck, Sharp and Dohme Research
Laboratories
Miami, University of
Miami, University of. School of Medicine
Michigan State University
Michigan, University of
Millhauser Laboratory
Minnesota, University of
Miriam Hospital
Missouri, University of
Monsanto Company
Mount Holyoke College
Mount Sinai Hospital
Mystic Marinelife Aquarium
National Institute of Child Health and
Human Development
National Institute of Environmental Health
Sciences
National Institute of Mental Health/NIH
National Institutes of Health
National Institute of Neurological and
Communicative Disorders and Stroke/
NIH
National Marine Fisheries Service
New Alchemy Institute
New Brunswick Scientific, Inc.
New England Medical Center
New Hampshire, University of
New Jersey Medical School
New Jersey, University of. Medicine and
Dentistry
New York Blood Center
New York, City University of
New York Institute for Basic Research in
Developmental Disabilities
New York Institute for Basic Research in
Mental Retardation
New York Medical College
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. Health
Sciences Center
New York, State University of. New Paltz
New York, State University of. Stony
Brook
New York University
New York University College of Dentistry
New York University Medical Center
New York University School of Medicine
Nichols College
Nikon, Inc.
North Carolina, University of
North Nassau Mental Health Center
Northeastern University
Northwestern University
Northwestern University Medical School
Notre Dame, University of
Oak Ridge National Laboratory
Oberlin College
Ocean Pond Corporation
Oklahoma, University of
Olympus Corporation of America
Optiquip
OPTRA, Inc.
Oregon State University
Oregon, University of
Pennsylvania State University
Pennsylvania, University of
Pennsylvania, University of. School of
Dental Medicine
Pennsylvania, University of. School of
Medicine
Pharmacia, Inc.
Photonic Microscopy
Pittsburg, University of
Portland State University
Princeton University
Procter and Gamble Company
Puerto Rico, University of
Puget Sound, University of
Purdue University
Quantex Corporation
R & M Biometrics, Corp.
Radiomatic Instruments
Rainbow Babies and Children's Hospital
Rainin Instrument Company
Reed College
Rensselaer Polytechnic Institute
Rhode Island College
Rhode Island, University of
Rice University
MARINE BIOLOGICAL LABORATORY
Rochester, University of
Rochester, University of. Medical Center
Rochester, University of, School of
Medicine and Dentistry
Rockefeller Foundation
Rockefeller University
Rush Mecli .-;.;! Center
Rush-pvc' ::>tcnan, St. Luke's Medical
Center
Rutgers University
Rutgers University Medical School
Savant Instruments
Sea Education Association
Simmons College
Smith College
Smithsonian Institution
Sorvall Instruments
South Carolina, University of
Southeastern Massachusetts University
Southern California, University of
Stanford University
St. Peter's College
Swift Instruments
Syntex
Syracuse University
Technical Products International, Inc.
Temple University
Temple University Medical School
Tennessee, University of
Tennessee, University of, Oak Ridge
Texas Christian University
Texas, University of
Texas, University of, Austin
Texas, University of. Health Center
Texas, University of. Medical Branch
Texas, University of. Medical School
Texas, University of, Medicine and
Dentistry
Thomas Jefferson University
Trinity College
Tufts University
Tufts University, Sackler School
Tufts University School of Medicine
Tufts University, School of Veterinary
Medicine
Union College
United States Food and Drug
Administration
Upjohn Company
Universal Imaging Corporation
Upstate Medical Center
VWR Scientific
Vanderbilt University
Vassar College
Vermont, University of
Veterans Administration Hospital, San
Francisco
Veterans Administration Medical Center
Villanova University
Virginia, University of
B. Vittor and Associates
Wake Forest University
Washington and Lee University
Washington State University
Washington University
Washington, University of
Washington University School of Medicine
Wayne State University
Wesleyan University
Whitman College
Whitney Marine Laboratory
Wilkes College
William and Mary, College of
Williams College
Wisconsin, University of
Wisconsin, University of, Madison
Wistar Institute
Woods Hole Data Base
Woods Hole Education Associates
Woods Hole Oceanographic Institution
Woods Hole Research Center
Worcester Foundation for Experimental
Biology
World Precision Instruments
Yale University
Yale University Medical School
Carl Zeiss, Inc.
FOREIGN INSTITUTIONS
Alberta, University of, Canada
Annamala University, India
Barcelona, University of, Spain
Basel, University of, Switzerland
Bedford Institute of Oceanography, Canada
Bergen, University of, Norway
Calgary, University of, Canada
Cambridge University, UK
Centre National de la Recherche
Scientifique, France
Centra de Investigacion y de Estudios
Avanzados del IPN, Mexico
Copenhagen, University of, Denmark
Dalhousie University, Canada
Division of Fisheries, Bermuda
Edinburgh, University of, Scotland, UK
INSTITUTIONS REPRESENTED
89
Free University of Berlin, FRG
Gadjah Mada University, Indonesia
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Reference: Biol. Bull. 173: 92-109. (August, 1987)
PHYSIOLOGICAL ROLES OF PROSTAGLANDINS AND OTHER
EICOSANOIDS IN INVERTEBRATES
DAVID W. STANLEY-SAMUELSON
Dcj'ii.'-nnent of Entomological Sciences, University of California, Berkeley, California 94720
ABSTRACT
Prostaglandins and other biologically active derivatives of polyunsaturated fatty
acids have been detected in a large number of invertebrate species. A brief summary
of the mammalian background of arachidonic acid metabolism is provided, and the
physiological significance of these compounds in invertebrates is reviewed. Topics
include regulation of ion flux, temperature regulation, reproductive biology, cell ag-
gregation, and host-parasite interactions. Finally, perspectives on current and possi-
ble future research are offered.
INTRODUCTION
The term eicosanoid was introduced and used by Corey et al. (1980) to describe
the various biologically active derivatives of eicosapolyenoic fatty acids, especially
arachidonic acid. So far, we know of four major groups of eicosanoids: the prostaglan-
dins (PCs), the hydroperoxy- and hydroxyeicosatetraenoic acids (HPETEs and
HETEs), the leukotrienes (LTs), and the lipoxins (LXs). Interest in the significance
of eicosanoids in the biology of mammals stems from physiological studies conducted
in the early twentieth century. In the earliest reference to one group of eicosanoids,
the PGs, Jappelli and Scafa (1906) noted that extracts of dog prostrate glands caused
paralysis of central respiratory control and changed heart rates when injected into
dogs and rabbits. The discovery of PG pharmacological activity in human seminal
fluids (Kurzrok and Lieb, 1930) probably marks the beginning of the detailed studies
of the clinical significance of these compounds. Elucidation of the chemical structures
of PGs in the early 1960's (Bergstrom et al., 1962a, b) greatly increased the pace of
research and discovery, hindered in that decade mainly by the limited availability of
working quantities of purified compounds. It is now known that PGs are present and
play important roles in almost all mammalian tissues and fluids (Horrobin, 1978).
Examples of PG action include pathophysiological actions such as mediation of the
inflammatory response (which we commonly block by ingestion of aspirin) and par-
ticipation in the blood-clotting cascade, as well as physiological actions such as con-
traction of smooth muscle.
The growth of PG research began with initial physiological observations, along
with isolation and structural determinations of individual PGs. This was followed by
the development of techniques to produce PGs in a commercially profitable way for
clinical and biological studies. Commercial production of PGs evolved from biosyn-
thesis from appropriate precursor fatty acids using large-scale enzyme preparations,
through the discovery of naturally occurring sources of PGs and of intermediates in
chemical synthesis to economical total synthesis. Hence, the first report of PGs in
Received 15 April 1987; accepted 26 May 1987.
Abbreviations: PG == prostaglandin, LT = leukotriene, HETE = hydroxyeicosatetraenoic acid,
HPETE = hydroperoxyeicosatetraenoic acid, LX = lipoxin.
92
PHYSIOLOGICAL ROLES OF PROSTAGLANDINS 93
an invertebrate animal, the gorgonian coral Plexaura homomella (Weinheimer and
Spraggins, 1969), met with tremendous interest, not as a zoological discovery, but as
a commercial source of PG for laboratory study. In the years between this first discov-
ery of a potentially economical source of PG and the development of appropriate
synthetic strategies, the search for other biological sources of PGs turned up many
examples of their occurrence in marine invertebrates, albeit at tissue concentrations
far below the point of commercial interest.
One of the PGs in greatest abundance in the coral. 1 5-epi-PGA2 , is not pharmaco-
logically active in the usual mammalian biological assays for PG activity (Nakano,
1969). Chemical modification of the naturally occurring form to clinically useful
structures, as well as commercial, ecological, and environmental aspects of sustained
PG yield from coral have been reviewed (Theoder, 1977; Berte, 1981; Bundy, 1985).
Other papers describe evidence for the occurrence of PGs in over one hundred inver-
tebrate species. Christ and van Dorp (1972) detected PG-biosynthesis activity in five
invertebrates — including two coelenterates, a mollusc, an annelid and an arthro-
pod— but not in two insect species. Using a classical bioassay for the pharmacological
effect of PG on contraction of mammalian smooth muscle, Nomura and Ogata
(1976) detected PGs in a procordate, and in representatives of Echinodermata, Mol-
lusca, Annelida, Coelenterata, and Arthropoda (including an insect). PGs were also
detected by bioassay in saliva of another terrestrial arthropod, the tick Boophilus
microplus (Dickinson et al., 1976; Higgs et ai, 1976). Using radioimmunoassay,
Shemesh et al. (1979) found PGs in reproductive organs and salivary glands of an-
other tick. Since all PGs are formed from a common intermediate, prostaglandin-
endoperoxide, PG synthesis could be inferred from activity of prostaglandin-endo-
peroxide synthetase. Morse et al. (1978) detected this enzyme activity in 41 species
of coelenterates collected in the Caribbean Sea and the Pacific Ocean. Gromov et al.
(1982) used radioimmunoassay to estimate amounts of two PGs in a snail. Korot-
chenko et al. (1983) found smooth muscle-contracting activity in 10 echinoderm
species; they also refer to finding PG activity in 40 other invertebrates.
Aside from detection of PGs in a large number of invertebrate species, certain
reports suggest that eicosanoids play fundamental physiological roles in representa-
tives of many invertebrate phyla. Such findings are interesting because they provide
insights into the details of regulatory physiology. Interest extends to an evolutionary
axis because discovery of eicosanoid physiology especially in the very early phyla
suggests that the significance of these compounds is not limited to vertebrate and
clinical physiology, but was established early in metazoan evolution.
Evolutionary interest may eventually extend to plants, as well. Gregson et al.
(1979) described the occurrence of two PGs in the red alga Gracilaria lichenoides,
and Janistyn (1982) reported chemical identification of PGF2a in the flowering plant
Kalanchoe blossfeldiana. A prostaglandin-like compound was produced from lin-
olenic acid by an enzyme preparation of flaxseed (Zimmerman and Feng, 1978). The
physiological significance of these compounds in plants is not clear, but compounds
that inhibit PG-biosynthesis in mammals inhibited growth in four fungus species
(Herman and Herman, 1985; Kerwin et al., 1986). Earlier inhibitor studies showed
inhibition of flowering in Pharbitis nil (Groenewald and Visser, 1974). Although
these findings are preliminary, they suggest that eicosanoids may be of broad biologi-
cal significance.
The goal of this review is to provide an appreciation of the physiological signifi-
cance of eicosanoids in invertebrate animals. Since the appropriate nomenclature
and physiological background comes from decades of work on various mammal sys-
tems, it is useful to begin with a background from mammal studies.
94 D. W. STANLEY-SAMUELSON
A BACKGROUND FROM MAMMALIAN STUDIES
Upon stimulation by various agonists, many mammal cells hydrolyze polyunsat-
urated fatty acids (PUFAs), by action of phospholipase A2, from the beta carbon of
membrane phospholipids. Three C20 PUFAs — dihomo-7-linolenic (C20:3n6), ara-
chidonic (C20:4n6), and eicosapentaenoic (C20:5n3) acids — may be metabolized by
one of two major pathways into biologically active molecules. In the cyclooxygenase
pathway, PUFAs are transformed into prostaglandins and thromboxanes, whereas
the lipoxygenase pathway leads to hydroperoxy- and hydroxypolyenoic fatty acids
which are themselves biologically active as well as further metabolized into lipoxins
and leukotrienes. Since these are all derivatives of C20 PUFAs, they may be collec-
tively referred to as eicosanoids. The following description of the biosynthesis and
physiological roles of these compounds in mammals is assembled from several re-
views and books (Horrobin, 1978; Samuelsson el a/., 1978; Hansson el al, 1983;
Samuelsson, 1983; Serhan el al., 1985), and is presented with minimum referencing.
PGs are C20 carboxylic acids with a five-membered ring variously substituted at
C9 and Cl 1, and two aliphatic chains featuring a substitution at C15 and one, two,
or three double bonds. The structures of the principle PGs are shown in Figure 1 . PGs
are designated as lettered and numbered series. The numbers indicate the number of
aliphatic double bonds, giving rise to the one-, two-, and three-series PGs. The letters
are associated with the particular pattern of substitutions on the five-membered ring:
PGE features C9 keto, Cl 1 hydroxyl substitutions; PGF a C9,C1 1 dihydroxyl pat-
tern; PGD a C9 hydroxyl, Cl 1 keto arrangement. PGs of the A, B, D, E, and F series
are so distinguished.
Biosynthesis of PGs is a multistep operation beginning with formation of the pros-
taglandin endoperoxides — first PGG — by action of microsomal prostaglandin endo-
peroxide synthetase. The same enzyme also cleaves the hydroperoxy group of PGG
to form PGH. PGH is the root intermediate in the synthesis of the primary PGs: PGD
is formed by a glutathione-S-transferase, PGE requires prostaglandin endoperoxide
E isomerase and PGF prostaglandin endoperoxide reductase; PGI is formed by pros-
taglandin endoperoxide I isomerase and thromboxane A (TxA) by prostaglandin
endoperoxide thromboxane A isomerase.
PGs have been detected in most mammalian tissue systems where they are in-
volved in many well-catalogued (Horrobin, 1978) physiological activities. Examples
of PG action include contraction of smooth muscle (i.e., uterine, gut, and blood ves-
sel), attenuating cellular response to hormones, and release of digestive acid in the
stomach. Thromboxane A2 is a potent inducer of platelet aggregation; its name is
taken from its origin, the thrombocytes.
Lipoxygenase pathways first lead to hyproperoxy fatty acids which can be reduced
by peroxidases, and possibly by non-enzymatic reactions, to corresponding hydroxy
fatty acids (Fig. 2). Arachidonic acid is the best-studied lipoxygenase substrate in
mammals, and oxygen can be added at various positions, leading to 5-, 8-, 9-, 1 1-,
12-, and 1 5-hydroxyeicosatetraenoic acids (the various HETEs). Di- and tri-hydroxy
fatty acids also can be formed by lipoxygenase acting on the same fatty acid substrate
more than once; another route to trihydroxy acids is by way of an epoxy-hydroxy
acid. While PGs are involved in various physiological as well as pathophysiological
actions, the lipoxygenase products apparently are involved in pathophysiological ac-
tions such as bronchial constriction. The lipoxygenase reactions are found in defense
systems such as the various leukocytes, macrophages, monocytes, lung, and spleen.
HETEs are biologically active in defense roles. For example, 5-, 9-, and 1 1-HETE are
all active in inducing the chemokinesis and chemotaxis associated with migration of
eosinophils into the site of certain hypersensitivity reactions.
PHYSIOLOGICAL ROLES OF PROSTAGLANDINS 95
PROSTAGLANDINS
homo-tf-LINOLENIC OH
OH
ARACHIDONIC
EICOSAPENTAENOIC OH
RING FEATURES OF PROSTAGLANDINS
PGA
FIGURE 1 . Relationship between the 1 -, 2-, and 3-series prostaglandins and their parental polyunsat-
urated fatty acids, respectively C20:3n6, C20:4n6, and C20:5n3, is indicated by the arrows. X indicates
cyclooxygenase activity. Ring features of five prostaglandins are shown in the lower panel where R stands
for the aliphatic chains shown on the complete structures.
The leukotrienes (LTs; Fig. 3) were discovered during work on rabbit polymor-
phonuclear leukocytes, and take their names from this and the conjugated triene
structure they have in common. The following description of LTs comes from the
review by Samuelsson (1983). There are two classes of leukotrienes: the cysteine-
containing group (LTC4, LTD4, and LTE4), and LTB4, which is not substituted.
Biosynthesis of the LTs begins with formation of 5-hydroperoxy-6,8,l 1,14-eicosa-
tetraenoic acid (5-HPETE) by action of lipoxygenase followed by conversion to LTA4
by abstraction of a hydrogen and elimination of a hydroxyl anion, catalyzed by a
soluble enzyme, dehydrase. LTA4 is converted to LTB4 by hydrolase, or into the
parental cysteine-containing LT (LTC4) by a glutathione-S-transferase. The cysteine-
containing LTs feature a thioether linkage at C6 to cysteine; LTC4 is 7-glutamyl-
cysteinyl-glycyl substituted; glutamyl transpeptidase elimination of the glutamine
residue forms cysteinylglycyl LTD4 which can be metabolized into cysteinyl LTE4.
LTs have been identified in several cell systems including rabbit, human, mouse,
and rat leukocytes; mouse and rat macrophages; and human and guinea pig lung.
The biological significance of these compounds lies in their identification as the slow-
reacting substance of anaphylaxis (SRS-A). This material is a mediator in asthma and
other mammalian hypersensitivity reactions; SRS-A is released with other mediators
96
D. W. STANLEY-SAMUELSON
0(0)H
COOH
,COOH
0(0)H
15-H(P)ETE
6-ri(P)ETE
H(0)0
H(0)
.COOH
COOH
12-H(P)ETE
,COOH
9-H(P)ETE 0(0)H
I1-H(P)ETE
FIGURE 2. Structures of lipoxygenase metabolites of arachidonic acid, hydroxyeicosatetraenoic and
hydroperoxyeicosatetraenoic acids.
,COOH
Arachidonic acid
OOH
LTA,
,COOH
5-HPETE
Addition of,
glutathione/
, Enzymatic
^hydrolysis
LTC
A k=
LTD,
LTE,
COOH
CHCONHCH,COOH
I i
NHCOCH,CH9CHCOOH
1 L I
NH0
COOH
CHCONHChUCOOH
I i
NH-,
COOH
CHCOOH
.COOH
LIB,
FIGURE 3. Structures of leukotrienes.
PHYSIOLOGICAL ROLES OF PROSTAGLANDINS 97
,COOH
Arachidonic acid
COOH
15-HPETE
HO OH H OH
'COOH \W rOOH
H OH HO OH
Lipoxin A (LX-A) Lipoxin B ( LX-Bl
FIGURE 4. Structures of lipoxins.
after interaction of antigens such as pollen with immunoglobulin. SRS-A is a mixture
of the cysteine-containing LTs. LTB4, which does not contain cysteine, stimulates
enzyme release, adhesion of neutrophils to endothelial cells, and movement of fluids
through vessel walls in microcirculation. Lindgren el al. (1985) showed that LTs oc-
cur in the rat brain — most prominently in the hypothalamus and median emi-
nence— and that they may be involved in hormone release by brain cells.
The lipoxins (Lx; Fig. 4) are the most recently discovered metabolites of arachi-
donic acid. They share the characteristic feature of a conjugated tetraene structure.
Two major LXs, LXA and LXB, were formed by human leukocytes; LXA stimulated
oxygen metabolism and generation of active oxygen species in human neutrophils.
The action of LXA in neutrophils differs from the action of leukotriene B4 and may
represent another physiological mechanism of host defense. Lipoxins appear to be
formed by 5-lipoxygenase activity on a substrate formed by 1 5-lipoxygenase metabo-
lism of arachidonic acid. (The trivial name lipoxins is an abbreviation of lipoxygenase
interaction products.)
The PGs, LTs, and LXs are involved in basic physiological processes at the cellu-
lar level and appear to be especially important in various pathophysiological re-
sponses such as inflammation, blood-clotting, asthma, and tumor growth. Due to
their clinical significance, much effort is directed toward appreciating the regulation
of arachidonic acid metabolism and developing specific inhibitors of PG, LT, and
LX biosynthesis. Specific compounds will be mentioned in the contexts of biological
studies in various invertebrate systems. Here it should be mentioned that within a
given mammalian system there is considerable tissue variation in the effects of vari-
ous inhibitors; moreover, there is variation between mammalian species. In light of
tissue and specific variations in cyclooxygenase and lipoxygenase systems in mam-
mals, one notes that the considerable literature on mammals should not be taken as
a set of rules of the biochemistry of fatty acids in invertebrates. It is more appropriate
to interpret the background as a loose set of guidelines, likely to be misleading at
98 D. W. STANLEY-SAMUELSON
crucial points in our consideration of the physiological significance of eicosanoids in
invertebrates.
PHYSIOLOGICAL SIGNIFICANCE OF EICOSANOIDS IN INVERTEBRATES
Regulation of ion flux
Like other freshwater bivalves, Ligumia subrostrata maintains its body fluids hy-
perosmotic to the aquatic medium, largely by regulating the flux of sodium, its major
blood cation (Dietz, 1977, 1979). PGE2 appears to be a component of the sodium
regulation system because inhibition of endogenous PG-biosynthesis by injection of
indomethacin, a potent cyclooxygenase inhibitor in mammals, increased sodium
flux. The effect lasted about 15 hours, approximately doubling the control values.
Alternatively, when PGE2 was injected in parallel experiments, sodium influx de-
clined about 5-fold from control values. Since chloride concentrations and sodium
outflux remained unchanged during these experiments, Graves and Dietz (1979)
concluded that PGE2 participates in ion regulation by specifically controlling so-
dium influx. A tissue specificity may also exist because indomethacin modified the
activity of the epithelial cells involved in sodium uptake without changing urinary
sodium loss.
Indomethacin stimulated sodium influx in a dose-dependent way over the con-
centration range of 0.05 to 0.25 ^mol/g dry wt. Other PG-synthetase inhibitors in
mammals — meclofenamate (a cyclooxygenase inhibitor), and dexamethasone
(which inhibits phospholipase A2, and hence, regulates substrate availability) — also
stimulate sodium uptake (Saintsing and Dietz, 1983). The stimulatory effect of PG-
synthetase inhibitors was neutralized by co-injection of PGE2, supporting the view
that PG is part of the system regulating epithelial sodium flux. PGE2 reduces influx;
reduction of PGE2 biosynthesis may increase influx by attenuating the PG inhibition
of uptake, but positive stimulation seems to depend on a biogenic amine, 5-hydroxy-
tryptamine (5-HT, or serotonin), rather than on another PG since PGF2a acts much
like PGE2 (Saintsing and Dietz, 1983). Cyclic AMP (cAMP) also stimulates sodium
uptake (Graves and Dietz, 1982), which suggests that PG inhibition and 5-HT stimu-
lation of sodium flux may both function via antagonistic effects on adenyl cyclase
activity.
Arachidonic acid injections apparently increased renal outflux of sodium without
changing epithelial uptake. Graves and Dietz (1979) suggested that the arachidonic
acid may initially alter renal function, and be metabolized too quickly to allow forma-
tion of inhibitory levels of PGE2 in epithelial tissue. Another possibility is that ion
regulation is more complex (Graves and Dietz, 1982). If, as in mammals, arachidonic
acid is potentially metabolized into a variety of prostanoid compounds, then we can
imagine one metabolite, PGE2 , inhibiting epithelial uptake while others, not yet iden-
tified, modify renal ion flux in ways still unknown.
The idea that PGs regulate epithelial sodium uptake in a freshwater mussel is
based mostly on pharmacological treatments with appropriate compounds. Saintsing
et al. (1983) showed the presence of PGs in L. subrostrata extracts by RIA, lending
further support to natural occurrence and biological activity in an aquatic inver-
tebrate.
PGE2 is also involved in ion regulation in the marine bivalve Modiolus demissus
(Freas and Grollman, 1 980). When isolated gills were subjected to hypoosmotic stress
by incubation for 60 minutes in 25% seawater, there was a 10-fold increase in PGE2
released into the medium, suggesting an increase in biosynthesis and release of the
PHYSIOLOGICAL ROLES OF PROSTAGLANDINS 99
PG. In addition to this osmotic action on PG release, there is a specific ionic effect.
To test for possible ionic effects, gills were incubated in artificial seawater of fixed
osmotic concentration, but selectively free of sodium, calcium, potassium, or magne-
sium. Only the magnesium-free artificial seawater stimulated gills to increase PGE2
release. However, the apparent osmotic effect is not due solely to depletion of envi-
ronmental magnesium because gills incubated in hypoosmotic seawater with normal
magnesium concentrations also induced increased PG release. Hence, gill tissues of
this marine bivalve respond to changes in osmotic and ionic concentrations.
In mammals, the physiological activities of many PGs are mediated by specific
cellular receptor sites. Freas and Grollman (1981) showed the existence of specific
PGA2 binding sites in homogenates of gills, mantles, siphons, adductor, and upper
and lower visceral masses. In gills, these sites were ionic, pH dependent, and revers-
ible. To date this is the only study of PG binding sites in invertebrate tissues; such a
finding adds considerable verisimilitude to physiological propeties of PGs.
Mediation of behavioral thermoregulation and fever
PGE, appears to mediate febrile response to infection in a number of mammals,
including monkeys (Crawshaw and Still, 1975), sheep (Hales et ai, 1973), rabbits
(Stitt, 1973; Lin, 1978), cats (Milton and Wendlandt, 1970; 1971), and guinea pigs
(Szekely and Komaroni, 1978). Fever also occurs in non-mammalian vertebrates,
although the increased body temperatures appear to be mediated by behavioral as
opposed to endogeneous physiological mechanisms. Behavioral fever has been ob-
served in frogs (Casterlin and Reynolds, 1977a, Myhre et ai, 1977), a lizard (Bern-
heim and Kluger, 1976), and several fishes (Reynolds et al., 1976).
Some aquatic invertebrates express behavioral fever in response to bacterial infec-
tion by moving into a zone of warmer water. The freshwater crayfish Cambarus bar-
toni exhibited a 2°C behavioral fever after innoculation with a suspension of killed
bacteria (Aeromonas hydrophila) by choosing higher temperatures in a gradient
trough (Casterlin and Reynolds, 1977b). This behavioral response to infection may
be mediated by endogenous formation of PGE, because increasing doses of the PG
also induced 1 to 3.5°C fevers when injected over the range of 50 to 500 ^/individual
(Casterlin and Reynolds, 1978). Three marine arthropods — the American lobster
Homarus americanus, the pink shrimp Penaeus duorarum, and the horseshoe crab
Limulus polyphemus — similarly increased their temperature preferenda by more
than 4°C in response to 100 ^g injections of PGE, (Casterlin and Reynolds, 1979).
Two terrestrial arthropods, the scorpions Bathus occitanus and Androctonnus
australis, regulated their body temperatures by selecting appropriate positions along
temperature gradients in a sand box. A. australis increased temperature preferences
by 15°C and B. occitanus by 20°C after treatment with physiological doses of PGE,
(Cabanac and Le Guelte, 1980). Although it is not known whether these species gen-
erate fever due to bacterial infection, it appears that PGs may be involved in some
aspect of behavioral thermoregulation.
Together, these reports suggest that PGs may be some part of the thermoregula-
tory physiology of many invertebrates. The idea is based on the observation of in-
creased body temperatures in response to individual doses of a single compound,
namely PGE,. Important detailed biochemical questions remain unanswered: do
PGs naturally occur in these species? Does PG biosynthesis increase after infection,
but before the febrile response? Do all PGs induce fever, or is a more specific set of
these compounds involved? Research in this area may assume ecological interest, as
suggested by remarks below.
100 D. W. STANLEY-SAMUELSON
Among terrestrial invertebrates, many medium to large size insects regulate tho-
racic temperaiur <o a set point suited to the high metabolic demands of powered
flight by belr ! (Casey, 1981) or physiological (Kammer, 1981) means. In addi-
tion to flyin,: >, thermoregulation has been studied in caterpillers of two sphinx
moths, // •:/ and Manduca sexta (Casey, 1976, 1977). H. lineata appears to
sustai x>dy temperatures and correspondingly high rates of feeding by basking
in appro e postures; M. sexta does not maintain high temperatures even though
feeding and growth rates are reduced considerably at cooler temperatures. These
dirll-ivnt behaviors appear to be linked to differences in predator defense mechanisms
and in seasonal availability of their host plants. Other caterpillers, including the but-
terflies Vanessa io and V. urtica, huddle in groups, resulting in increased body tem-
perature and development rates (Mosebach-Pukowski, 1938). Similarly, the larvae of
wax moths thermoregulate, partly, by huddling or scattering (Smith, 1941).
Many insect species are resistant to viral infection when maintained at higher
temperatures (Tanada, 1967). Watanabe and Tanada (1972) reviewed several lepi-
dopteran cases of insect viruses which do not cause lethal infections at higher temper-
atures, including larvae of the armyworm Pseudaletia unipunctata, the cabbage
looper Trichoplusia ni, and the corn ear worm Heliothis zea. Hence, behavioral ther-
moregulation in invertebrates may effect such biological parameters as feeding and
development rates, and resistance to disease. PGs may be an important biochemical
mediator in this area of physiological ecology.
Control of hatching
In the barnacle Balanus balanoides, full egg-laying involves passing eggs along
oviducts into ovisacs produced by oviducal glands. Fully formed egg masses are fi-
nally released into the mantle cavity, where they remain until hatching which corre-
sponds with spring algal blooms (Crisp, 1962; Clare et a/., 1985). The synchrony of
spring bloom and egg hatching could be related to a component in the nutrition of
adult barnacles. However, Crisp and Spenser (1958) showed that seawater extracts of
unfed and fed adults were equally effective in inducing hatching. They proposed a
barnacle hatching substance, endogenously produced by adults, and showed that the
substance acts upon the musculature of mature embryos, not on the egg case.
The hatching substance appeared to be a PG (Clare et a/., 1982, 1985). The sub-
stance is extractable in a system optimized for PGs, it behaves like a PG on thin layer
chromatography, and extracts of the dried cortex of a commercial source of PG (the
gorgonian Plexura homomalla) acted biologically and chemically like barnacle hatch-
ing substance. Extracts made in the presence of aspirin — a PG-synthetase inhibitor
in mammals — did not induce hatching. Clare et al (1985) concluded that barnacle
hatching substance is either a PG or a PG-like compound.
Subsequent work underscores the importance of rigorous chemical methodolo-
gies in indentification of biologically active compounds. Holland et al (1985) ex-
tracted 50 kg of barnacles, then processed the extracts through four sequential systems
of thin layer chromatography. The active compound was detected by bioassay at
each stage. The purified compound was derivatized for gas chromatography-mass
spectroscopy (GC-MS), which yielded a single major GC peak. Mass spectra of de-
rivatized hatching factor and hydrogenated derivatized hatching factor were consis-
tent, not with a PG, but with another eicosanoid, 10,11,1 2-trihydroxy-5,8, 11,17 eico-
satetraenoic acid (Fig. 5). This compound is probably a lipoxygenase derivative of
C20:5n3, an abundantly available fatty acid in marine invertebrates and also the pre-
cursor of the 3-series PGs.
PHYSIOLOGICAL ROLES OF PROSTAGLANDINS 101
OH OH
OH
FIGURE 5. Structure of barnacle hatching factor, 10, 1 l,12-trihydroxy-5,8,l 1,17-eicosatetraenoic
acid.
Reproduction in Mollusca
PGs appear to stimulate egg production in the freshwater snail Helisoma durgi
(Kunigelis and Saleuddin, 1986). When injected directly into the haemocoel of
adults, ng quantities of PGE2 produced apparent discomfort in all individuals and
even death in isolated cases with no increase in egg masses or in eggs per mass. But
when introduced into the female genital opening in a viscous fluid designed to ap-
proximate semem, PGE2 treatments stimulated a long-term increase in egg produc-
tion. Four weeks after treatment of virgin snails with 25, 50, and 100 ng doses of
PGE2, cumulative egg production was about 200, 425, and 650 eggs per animal, re-
spectively.
Reproductive tissues from virgin and mated snails, the ovotestis, seminal vesicle,
bursa copulatrix, and oothecal gland presented substantial PG-biosynthetic activity
in vitro. Mating significantly altered the activity in two of the tissues. In ovotestis,
synthesis of PGE2 decreased while PGA2 synthesis increased with no change in syn-
thesis of PGF2(V. Synthetic activity changed in the bursa copulatrix, with PGE2 and
PGA2 reduced to effective zero after mating; PGF2(V was again unchanged. Differences
in PG-synthetic activity did not occur in seminal vesicle or oothecal gland (Kunigelis
and Saleuddin, 1986). These two lines of evidence — the effects of PG treatments on
egg production and alterations in PG-synthetic activity — suggest that PGs play im-
portant reproductive roles in this snail.
PGs are also produced by accessory sex glands of another snail, Lymnaea stag-
nalis (Clare et ai, 1986). Homogenates of the albumen gland, bursa copulatrix, pros-
trate gland, and seminal vesicles converted radioactive arachidonic acid into labelled
products that co-eluted with 6-keto-PGE,, PGE2, PGA2/B2 (not resolved), throm-
boxane B2 (TxB2), and several unknown compounds. Whole organs also converted
arachidonic acid into these compounds, although in proportions different from the
homogenates of the same organs. Effects of mating on PG-biosynthetic activity were
not tested, nor were effects of PG administration on reproductive functions; nonethe-
less, the PGs formed in the reproductive organs eventually may be shown to play a
still undefined role.
PGs induce spawning in two other molluscs, the abalone Haliotis refescens and
the mussel Mytilus califorianus. When added to seawater cultures at 3 X 10"12 M,
PGE induced about a third and PGF about a half of male and female abalone to
spawn (Morse et ai, 1977). Although the physiological mechanisms remain unclear,
important biochemical insights have emerged. Addition of hydrogen peroxide to sea-
water tanks induced synchronous spawning in H. refescens and M. califorianus. This
observation is connected to the biochemistry of PG biosynthesis as understood in
mammals. The first step in the conversion of arachidonic acid to the 2-series PGs is
catalyzed by fatty acid cyclooxygenase (also known as prostaglandin endoperoxide
synthetase). This involves first activation of the enzyme by a hydroperoxy group, then
elimination of a hydrogen atom from C13 of arachidonic acid, leaving a free radical.
This is followed by adding a peroxy radical in a bridge across C9 and Cl 1 , formation
of the 8, 12 carbon-carbon bond (required for the cyclopentane ring in the final prod-
102 D. W. STANLEY-SAMUELSON
uct), isomerization of the 11,12 double bond to 1 2, 1 3, and addition of another peroxy
radical to C15, with concomitant isomerization of the 12,13 double bond to 13,14.
These final electron shifts generate PGG2, a short-lived intermediate in the conver-
sion of an acid to PG. The hydrogen peroxide effect is pH dependent, with
lower cone is releasing spawning at higher alkalinity. Morse et al. ( 1 977) sug-
gested alkaline conditions (pH 9. 1 ) favored decomposition of hydrogen per-
oxide highly reactive hydroperoxy free radical. Since a hydroperoxy group
enzyme and peroxy radicals are added in two steps in the formation of
PGG- . the free radicals derived from hydrogen peroxide may enhance overall conver-
sion of precursor fatty acids to PGs.
PGs appear to be important in basic physiological functions in molluscs, includ-
ing ion regulation, possible renal function, and reproductive biology. This prelimi-
nary work sets the stage for important questions of the precise physiological activity,
and offers the possibility of gaining greater understanding of invertebrate physiology
and appreciation of PGs in these systems.
Oocyte maturation in starfish
Starfish oocytes develop to the first meiotic prophase, then await the spawning
period. Maturation, or meiosis reinitiation, is induced by a hormone produced and
released by the follicle cells surrounding the oocytes, 1-methyladenine. Once stimu-
lated by the hormone, the oocytes complete the developmental path leading to fertil-
izable cells.
Arachidonic and eicosapentaenoic acids also induce oocyte maturation in three
species of starfish: Asterias rubens, Marthosterius glacialis, and Luidia ciliaris
(Meijer et al., 1984). The PUFA-induced maturation is specific to these two fatty
acids because 35 other fatty acids, ranging from C4:0 to C24:l and including satu-
rated, monounsaturated, and polyunsaturated fatty acids, did not induce maturation.
The maturation effect is dependent upon extracellular calcium and occurs at physio-
logical concentrations (i.e., 50% maturation dose = 0.65 yuM arachidonic acid). The
fatty acids stimulate the complete maturation program, including germinal vesicle
breakdown, fertilization, and development into normal larvae. Fatty acids endoge-
nous to the oocytes are able to stimulate maturation because addition of phospholi-
pase A2 , an enzyme that hydrolyses the fatty acid from the beta-carbon of phospholip-
ids, also stimulated maturation. The phospholipase effect was calcium-dependent,
and specific because phospholipases C and D did not bring on maturation.
The hormone effect probably proceeds through release and metabolism of PU-
FAs. Two phospholipase A2 inhibitors in mammals, quinacrine and bromophenacyl
bromide, inhibit hormone-stimulated maturation, which can be overcome by in-
creasing 1 -methyl adenine concentrations. Five PGs did not stimulate maturation,
and three cyclooxygenase inhibitors — acetylsalicylic acid, indomethacin, and tolazo-
line — did not inhibit maturation. On the other hand, three lipoxygenase inhibitors
in mammals — quercetin, eicosatetraynoic acid and butylated hydroxytoluene — did
inhibit hormone-induced maturation. Four products of lipoxygenase metabolism of
arachidonic acid, 12- and 1 5-hydroxyeicosatetranoic acids (HETE) and their corre-
sponding hydroperoxyeicosatetraenoic acids (HPETE) stimulated maturation.
Oocytes convert radioactive arachidonic acid into HETEs (Meijer et al., 1986a).
Conversion of arachidonic acid does not occur in the absence of calcium, nor are
oocytes stimulated to maturation. Following incubation with radioactive arachidonic
acid, fractions with chromatographic behavior of HETEs were recovered and found
to stimulate oocyte maturation. The lipoxygenase inhibitor eicosatetraynoic acid in-
PHYSIOLOGICAL ROLES OF PROSTAGLANDINS 103
hibited both conversion of arachidonic acid and stimulation of oocytes. It would
appear, then, that 1-methyladenine acts by release of PUFA, followed by conversion
to a biologically active HETE, which induces maturation of the oocytes.
Injection studies suggested that 12- and 15-HETE and corresponding HPETEs
stimulated oocyte maturation (Meijer et a/., 1984). Upon re-evaluation, it was found
that the tested compounds were contaminated with 5% of 8-HETE, the active com-
pound in maturation (Meijer et al., 1986a). Meijer et al. (1987) showed that (8R)-
HETE, but not (8S)-HETE, is produced by starfish oocytes. The R isomer is the only
active compound when tested in pure form, and other lipoxygenase products, includ-
ing other HETEs and leukotrienes are not active.
A survey of eight starfish species shows that while the hormone 1-methyladenine
stimulates maturation in all species, the stimulatory effect of arachidonic acid and 8-
HETE occurs in only three of them (Meijer et al., 1986b). Species differences in re-
sponse to various eicosanoids also have been observed in various physiological set-
tings in mammals. At this early period of appreciating the possible physiological ac-
tivities of these compounds in invertebrates systems, species differences underscore
the hazards inherent in forming generalizations.
Cercarial penetration of skin
Eggs of the blood fluke Schistosoma mansoni leave their mammalian hosts in
urine or feces, and continue larval development in snails. Free-swimming larvae
called cercariae reinfect mammalian hosts by burrowing through the skin or by inges-
tion with drinking water (Storer and Usinger, 1965). It has been known for a number
of years that skin surface lipids stimulate cercarial penetration of animal membranes
(Stirewalt, 1971). Among the skin surface lipids, free fatty acids, especially polyunsat-
urated fatty acids, appeared to be most efficacious in stimulating penetration (Austin
et al., 1972). Salafsky et al. ( 1984a) looked at the effect of certain fatty acids on two
cercarial behaviors in vitro, namely cessation of swimming and initiation of penetra-
tion. Their results show that certain PUFAs attracted cercariae to the center of their
test membranes while monounsaturated fatty acids did not. A few fatty acids gave
intermediate results because two monounsaturated fatty acids were as stimulatory as
the PUFAs, and two other monounsaturates were less stimulatory than the PUFAs
but were clearly more stimulatory than controls. Cyclooxygenase metabolites, rather
than the PUFAs per se, may alter cercarial behavior. Two inhibitors of cyclooxygen-
ase — ibuprofen and, to a lesser degree, aspirin — inhibited cercarial response to
PUFA. 1 3-Azaprostanoic acid, thought to specifically antagonize the platelet throm-
boxane/endoperoxide receptor in mammals, was also inhibitory.
PUFAs and certain of their metabolites may affect cercarial penetration as well as
modify behaviors that precede penetration. When Salafsky et al. (1984b) compared
cercarial penetration into skin membranes prepared from essential fatty acid (EFA)
deficient and EFA replete adult rats, they found about three times less penetration in
the preparations from EFA deficient rats. Again, the inhibition may be related to
formation of eicosanoids. Interperitoneal injections of ibuprofen led to a time-depen-
dent accumulation of the drug in the skin of EFA replete rats. Cercarial penetration of
the drug-treated skin was reduced. The percent inhibition increased with increasing
amount of ibuprofen accumulated in the skin, up to a maximum inhibition of
about 84%.
When cercariae were incubated with radioactive linoleic acid, radioactivity could
be recovered in high-pressure liquid chromatography fractions that eluted with
PGE2, PGD2, LTC4, LTB4 and 5-HETE. These data suggest that cyclooxygenase and
104 D. W. STANLEY-SAMUELSON
lipoxygenase sy unction within the cercariae. Radioimmunoassays of extracts
from cercarf; oated with linoleic acid were also consistent with these products.
Fusco et al. concluded that formation of eicosanoids is an essential step in
penetn '^kin by cercariae of Schistosoma mansoni. If this can be sup-
ported 1 °»'ork, it may present a rather interesting situation in which the
P7 e provided by a vertebrate host is metabolized into biologically active
a parasite.
yet known how the eicosanoids alter the behavior of the cercariae or
•enetration of mammalian skin. Fusco et al. (1985) suggest that vasodila-
nch is induced by certain PGs, may help the parasite find and infiltrate the
,/d system. It would appear that the eicosanoids, in this mode, would be usurped
by the parasites to alter the host physiology. In this case, the finding by Rumjanek
and Simpson ( 1 980) that adult worms do not synthesize PGE or PGF may be appreci-
ated in terms of host physiology. On the other hand, the behavioral effects of cessation
of swimming and initiation of penetration (Salafsky et al., 1984a), also induced by
skin lipids, suggest a direct effect on the cercariae.
Sponge cell aggregation
Rich et al. (1984) suggest that the calcium dependent aggregation of marine
sponge cells of Microcione prolifera is stimulated by leukotriene B4 (LTB4). LTB4
induced rapid cell aggregation in a dose-dependent way at 0.2 and 1 .2 ^M treatments.
The effect appears to be specific for LTB4 because eight PGs of A, B, D, E, and F
series and eight lipoxygenase products failed to induce aggregation.
The calcium ionophore A23187 and the species-specific aggregation factor
(MAF) stimulate cell aggregation. The aggregating effects of these compounds can
be inhibited by cyclooxygenase inhibitors including nordihydroquaiaretic acid and
indomethicin, which also interfere with calcium flux. These data show that those
agents which inhibit calcium flux also inhibit aggregation while those that promote
calcium movement also promote aggregation. Interpretation is difficult because while
a specific lipoxygenase product promotes aggregation, inhibitors of cyclooxygenase
metabolism inhibit it. Perhaps both pathways are involved in cell aggregation, with
LTB4 stimulating PG formation, which then acts in concert with the LTB4.
Egg-laying behavior in crickets
The roles of PGs in insect reproduction were reviewed by Stanley-Samuelson and
Loher (1986), from which the following summary is drawn. PGs were detected in
extracts of various tissues from over a dozen species of insects. The most well under-
stood physiological role of PGs is releasing egg-laying behavior in the field cricket
Teleogryllus commodus. Adult females undergo sexual maturation, during which the
abdomen becomes filled with hundreds of mature eggs. Certain behaviors that are
likely to bring females into contact with males also develop. Insemination is achieved
by transfer of a spermatophore to the genital organ of a female from where its contents
migrate into the female's spermathecae. Cyclooxygenase activity is associated with
the spermatophore contents, and once in the spermathecae of newly mated females,
arachidonic acid is converted into PG.
It is not known how the PG formed in the spermatheca releases egg-laying behav-
ior, but increases in spermathecal and hemolymph PG titer after mating suggest that
the PG acts at some site distant from the source. The observations that PGE2 does
not stimulate contraction of oviduct muscles in T. commodus (Loher, 1984) nor in a
PHYSIOLOGICAL ROLES OF PROSTAGLANDINS 105
cockroach (Cook et a/., 1984) and that oviposition behavior is a complex activity
directed by the central, rather than peripheral, nervous system (Loher, 1984) support
the hypothesis that the PGs function at the level of the central nervous system.
Using egg-laying to assay structure-function relationships among a range of eico-
sanoids, Stanley-Samuelson et al. (1986) found that highest egg-laying activity was
associated with E-series PGs. The A-, B-, D- and F-series induced zero to intermediate
egg-laying. Structures that departed from the basic PG structure, represented by 15-
HETE and prostacyclin, were inactive. The 2-series PGs were more active than their
1 -series analogues; hence, there may be a biological specificity for PGE2 in releasing
egg-laying behavior in that particular cricket species.
Highest egg-laying activity was induced by 15-keto-PGE2. In mammalian sys-
tems, this compound is formed by the action of prostaglandin dehydrogenase, located
mainly in lungs, but also in liver and kidney. Biologically active PGE is rapidly
cleared from the circulation of mammals by the activity of this enzyme. The observa-
tion that a biologically inactive compound, in the usual mammal assays, was associ-
ated with the greatest increase in egg-laying behavior marks a potentially important
point in comparative physiology. Several features of the biology of eicosanoids appear
to uniformly occur in the vertebrate and invertebrate systems as understood to date.
For example, many compounds that inhibit the action of cyclooxygenase in mam-
mals similarly inhibit the activity in invertebrates. On the other hand, as shown here,
while the mammalian background will be important and useful in work on inverte-
brate systems, fundamental differences are to be expected.
PERSPECTIVES
Various eicosanoids appear to be involved in the regulation of a variety of physio-
logical and behavioral areas in representatives of many invertebrate phyla. In some
cases (such as mediation of behavioral thermoregulation), the evidence for an eicosa-
noid function is based on treatment of animals with a single compound and observa-
tion of the response. At this level of observation, it remains to be established that
eicosanoids are physiologically involved. Given a good base of preliminary observa-
tions, important research goals would be to firmly show that, in the case at hand, PGs
do mediate thermoregulatory behavior. In still other cases, such as the role of PG in
releasing egg-laying behavior in crickets, there is sufficient evidence to accept that
certain PGs do release egg-laying, although some details of the physiological mecha-
nism— where in the central nervous system PGs act and how they alter behavior —
are not yet understood. Research in this area could usefully be aimed, not at re-
affirming the role of the eicosanoid, but at aquiring more details of the action. In study
areas where considerable biochemical details are established — as in starfish oocyte
maturation — cellular events remain unknown. Again, understanding how eicosa-
noids act remains a major research goal.
We are aware of eicosanoid roles in particular physiological areas in a given inver-
tebrate organism. We know, for example, that PG releases egg-laying behavior in
females of the cricket T. commodus. PGs are also detected in salivary glands, endo-
crine glands, Malpighian tubules, testes, and ventral nerve cords. Aside from the
known role in altering behavior, what do PGs contribute to the other tissue systems
in which they appear? Are they involved in regulating ion flux in Malpighian tubules,
secretion in salivary glands, and neural function in the nerve cord, within the same
organism?
Eicosanoids appear to be produced and to act at local, tissue, or cellular levels in
mammals. PGE, produced by adipocytes functions within the same cells to modulate
106 D. W. STANLEY-SAMUELSON
the lipid mobilizing effect of certain hormones. Moreover, there are mechanisms that
block PG circulation. The global circulation of PGE2, for example, is checked by the
action of pro Jin dehydrogenase, located mainly in lungs, which converts the
active com into a biologically inactive product. However, in the cricket T.
comn ! dated that the release of egg-laying behavior by PGE2 is mediated
in i. >ike a broadly circulating hormone (Stanley-Samuelson and Loher,
198' -Samuelson et ai, 1986). This point can be extended to research in the
iction, at the whole-organism level, of eicosanoids in invertebrates. The
xyeicosatetraenoic acid that functions as hatching substance in the barnacle
oe an example of a compound produced in one organ system with its action
observed elsewhere, again suggesting hormone action.
With several likely roles of eicosanoids set forth, general research areas include
establishing more firmly the activities, elucidating cellular details, and appreciating
the possible modes of action of these compounds. One can assume that as details of
eicosanoid action become known they will contribute greatly to our understanding
of invertebrate physiology.
ACKNOWLEDGMENTS
I am grateful to Drs. W. Loher, R. H. Dadd, M. O. Theisen, and R. A. Jurenka
for reading and making useful comments on the paper. The author and the work on
T. commodus were supported by NIH grant RO 1 HD036 1 9 to W. Loher.
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INTERSPECIFIC AGGRESSIVE BEHAVIOR OF THE
LIMORPHARIAN CORYNACTIS CALIFORNICA
vIDARIA: ANTHOZOA): EFFECTS ON SYMPATRIC
CORALS AND SEA ANEMONES
NANETTE E. CHADWICK
Department of Zoology, University of California, Berkeley, California 94720
ABSTRACT
Corallimorpharians are sessile cnidarians that are morphologically similar to the
actiniarian sea anemones and scleractinian corals. This study describes for the first
time the behavioral mechanism and effects of aggression by a corallimorpharian. Pol-
yps of the temperate clonal corallimorpharian Corynactis californica extruded their
mesenteries and associated filaments onto members of certain species of sea anemo-
nes and corals. They did not exhibit this behavior intraspecifically, and members
of different clones of C. californica remained expanded upon contact. In contrast,
members of four species of corals and zoanthids responded to contact with C. califor-
nica by contracting their tentacles, and members of three sea anemone species bent
or moved away, detached from the substrate, or attacked using their aggressive struc-
tures. When interspecific contact was prolonged, individuals of C. californica ex-
truded filaments onto, and killed polyps of, the sea anemones Anthopleura elegantis-
sima and Metridium senile within 3 weeks, and the corals Astrangia lajollaensis and
Balanophyllia elegans within 4-10 months under laboratory conditions. The use of
extruded mesenterial filaments by C. californica to attack members of other antho-
zoan species is similar to the aggressive behavior exhibited by many scleractinian reef
corals. Field observations suggest that C. californica may use this agonistic behavior
during interspecific competition for space on hard marine substrate.
INTRODUCTION
Some of the most striking behaviors exhibited by members of the class Anthozoa
(Phylum Cnidaria) are the aggressive behaviors of certain actiniarian sea anemones
and scleractinian corals. Corals may attack competitors using sweeper tentacles
(Richardson et al., 1979; Wellington, 1980; Bak et ai, 1982; Chornesky, 1983; Hi-
daka and Yamazato, 1984), sweeper polyps (Sheppard, 1982), extruded mesenterial
filaments (Lang, 1973;Glynn, 1974;Loya, 1976; Cope, 1981; Bak?/ al., 1982; Logan,
1984), or nematocysts discharged from the colony surface (Rinkevich and Loya,
1983), and actiniarian sea anemones may use elongated catch tentacles (Williams,
1975; Purcell, 1977) or marginal vesicles called acrorhagi (Bonnin, 1964; Francis,
1973b;Ottoway, 1978; Bigger. 1980; Brace, 1981;Ayre, 1982;Sebens, 1984). These
aggressive responses are complex. They often involve the induced morphogenesis and
directed application of specialized structures packed with nematocysts (Purcell, 1 977;
Chornesky, 1983; Watson and Mariscal, 1983; Hidaka and Yamazato, 1984;Hidaka,
1985), and may be initiated upon recognition of other genotypes or species of antho-
zoans(Lang, 1973; Bigger, 1980).
Received 2 March 1987; accepted 20 May 1987.
110
CORALLIMORPHARIAN BEHAVIOR 1 1 1
However, little is known about the aggressive behavior of another group of antho-
zoans, the corallimorpharians. Sebens (1976) reported the effects of competitive in-
teractions between corallimorpharians and other anthozoans on the Caribbean coast
of Panama, but did not specify the behaviors they used. The only other study relating
to corallimorpharian behavior is that of Hamner and Dunn (1980), who described
the unique feeding mechanism of some tropical Pacific corallimorpharians in which
prey are enfolded in the oral disk.
Corallimorpharians occur throughout the world (Carlgren, 1949) and may be
abundant on temperate rocky shores (Hand, 1955; Forster, 1958; Pequegnat, 1964;
Castric-Fey el a!., 1978; Foster and Schiel, 1985), as well as on tropical coral reefs
(Fishelson, 1970; den Hartog, 1980). Certain members of this group form clonal ag-
gregations that cover large areas of hard substrate, and are the dominant sessile organ-
isms in some temperate marine communities (Forster, 1958; Castric-Fey el ai, 1978).
Thus, interactions of corallimorpharians with other sessile organisms may have im-
portant consequences for the structure of these communities.
Corallimorpharians superficially resemble the actiniarian sea anemones in that
they lack a calcareous skeleton (Carlgren, 1949). However, they are more like the
stony corals in most other aspects of their morphology: they lack basilar muscles, may
have tissue connections between adult polyps, lack ciliated tracts on their mesenterial
filaments, and their cnidae are similar to those of corals (Carlgren, 1949; Schmidt,
1 974; den Hartog, 1 980). In light of the morphological relationships among members
of these three anthozoan groups, a comparison of their aggressive behaviors is of
interest.
This study describes the interspecific aggressive behavior of the temperate clonal
corallimorpharian Corynactis californica. This behavior was first recorded in Chao's
(1975) unpublished student paper. He observed that C. californica extruded mesen-
terial filaments to damage the sea anemones, 4 nthopleura elegantissima and Melrid-
ium senile during interspecific interactions in the laboratory. This is aggression,
which is defined by Webster's Third New International Dictionary as "an offensive
action or attack," and in this instance is elicited upon contact with the anemones.
Haderlie el al. (1980) briefly mentioned this behavior in their account of the natural
history of C. californica. The present paper expands on these reports by presenting a
quantitative analysis of mesenterial filament extrusion by C. californica, the specific-
ity of this aggressive response, and its effect on the behavior and survival of some
common sea anemones and corals under laboratory conditions.
Natural history
Corynactis californica Carlgren 1936 is the only species of corallimorpharian to
occur along the west coast of North America, where it ranges from Washington State
(Birkeland, 1971) to San Benitos Island in central Baja California (J. Engel, Tatman
Foundation, pers. comm.). Members of this species reproduce asexually by fission
(Hand, 1955) and budding (pers. obs.) to form aggregations on hard substrate (Fager,
1971; Haderlie et al., 1980; Foster and Schiel, 1985), from the lower intertidal zone
(Hand, 1955) to at least 50 meters depth (Birkeland, 1971; Schmieder, 1984, 1985).
C. californica polyps are common on the vertical faces of subtidal rock reefs where
they attain densities of up to 3000 polyps per square meter (Pequegnat, 1964). In
southern California, field experiments show that the presence of aggregations of C.
californica may increase the abundance of rock oysters (Vance, 1978) and mussels
(Landenberger, 1967; Wolfson et ai, 1979) by protecting them from predation by sea
stars. Groups of this corallimorpharian form interspecific boundaries with clones of
112 N. E. CHADWICK
the sea anemones Anlhopieiira elegantissima and Metridium senile on artificial sub-
strates such as wharf pilings (Chao, 1975; Haderlie and Donat, 1978) and offshore oil
platforms (Carlisle el al, 1964). Groups of C californica also co-occur on subtidal
rock reefs in ke >is with several species of corals, most commonly the colonial
coral As i -ant ;<v/.s7.v (Pequegnat, 1964) and the solitary corals Balanophyllia
elegcu; • nhus stearnsii (Pearse and Lowry, 1974; Lewbel el al. , 1981; Fos-
, 1985; North et al., 1985).
:cs greatly between different clonal aggregations of C californica. Clones
rn. pink, orange, or occasionally blue or purple. Members of each aggrega-
tion asexually produce polyps of the same color in both the laboratory (pers. obs.)
and the field (Turner et a I., 1 969). Thus, color in this species appears to be genetically
controlled, and in the present study polyps from different, distinctively colored aggre-
gations were assumed to be genetically different (non-clonemates).
MATERIALS AND METHODS
Collection and maintenance of organisms
Specimens of C. californica and the other organisms used in this study were col-
lected at four sites along the coast of central California (Table I). Laboratory experi-
ments were conducted between June 1984 and July 1986 at three facilities of the
University of California: Bodega Marine Laboratory. Joseph M. Long Marine Labo-
ratory, and in cold rooms on the Berkeley campus. Organisms were maintained in
plastic trays supplied with flowing seawater at ambient sea temperature (13-15°C),
or in closed refrigerated aquaria filled with aerated natural seawater. All tanks were
cleaned and animals fed adult brine shrimp (Anemia salina) weekly.
Mechanism and specificity of aggressive behavior
The first set of experiments focused on a description of mesenterial filament extru-
sion by C californica, and determination of the stimuli that elicit this response. Only
fully expanded, undamaged individuals of C. californica were used, and all within
two weeks of collection. Polyps were brought into contact with a range of physical
and biological stimuli (Table I) to elicit extrusion. Polyps were observed continuously
for the first hour of contact, then once each hour for at least 12 h, and then intermit-
tently for several days. A different individual of C. californica was used for each obser-
vation; Table I shows the number of replicate observations with each stimulus. Data
were collected on the diameter and behavior of each polyp, occurrence of mesenterial
filament extrusion, interval from the start of contact to extrusion, duration of extru-
sion, and the origin and maximal length of any extruded filaments.
Effects ofC. californica on selected anthozoans
During the above contacts between polyps of C. californica and seven other spe-
cies of anthozoans (Table I), data were also collected on the behavioral response of
each anthozoan. Their responses to C. californica were categorized as: contracted,
expanded, bent the column away, moved away on the pedal disk, detached from the
substrate, or attacked C. californica. During trials between C. californica and mobile
anthozoans such as the actiniarian sea anemones, the latter were repeatedly moved
back into contact with C. californica to allow adequate time for a response.
The second set of experiments examined effects of C. californica on the survival
of selected anthozoans over several weeks in the laboratory. To test the effect of C.
CORALLIMORPHARIAN BEHAVIOR 113
californica on actiniarian sea anemones, individuals of the clonal anemones An-
ihopleura elegant issima and Mctridium senile were placed in the center of groups of
C. californica that were attached to shells or rocks. This method prevented movement
away from contact by the anemones. Control anemones were placed on rocks that
were interspersed in the same tray with experimental groups, but not in contact with
C. californica. Data were then collected on the behavior and condition of the anemo-
nes once a week for three weeks.
Effects of prolonged contact with C. californica also were examined in two species
of scleractinian corals, Astrangia lajollaensis and Balanophyllia elegans. Individual
corals were attached to glass microscope slides or shells using H. A. Calahan's Ma-
rinepoxy (Davis Instruments, San Leandro, CA 94578). This epoxy has been used on
anthozoans for several years in the laboratory without apparent harm (J. S. Pearse,
University of California, Santa Cruz, pers. comm.). Barnacle shells bearing aggrega-
tions of C. californica were broken into small bits, and each piece of shell bearing a
single polyp of C. californica was cemented adjacent to a coral. Empty shells with no
C. californica were glued next to other corals as controls. Experimental and control
plates of corals were then intermingled in trays of seawater, and the condition and
behavior of each polyp was recorded once each month for 1 2 months. During this
time, polyps of the asexually reproducing species budded off new individuals, and
each month these were counted and the degree to which they had overgrown other
polyps was determined.
RESULTS
Description of aggressive behavior in C. californica
Upon contact, the tentacles of individual C californica adhered to those of polyps
of certain other anthozoans. Then the interacting polyps often contracted slightly and
their tentacles retracted. Over the next few minutes, the two polyps went through
several cycles of expansion, contact, contraction, and re-expansion. If they main-
tained fairly constant tentacular contact, a mass of highly convoluted mesenteries
and their associated filaments eventually appeared at the mouth or through a break
in the body wall of the C. californica polyp (Fig. la). These filaments were withdrawn
into the coelenteron at the end of each extrusion. Of 214 C. californica individuals
observed, most extruded filaments through the mouth (69%), through openings in
the column (7%), or along the junction of column and base (24%) of the polyp. One
polyp put out filaments through the tips of its tentacles. These openings in the body
wall were temporary and healed soon after the mesenterial filaments were withdrawn.
C. californica individuals almost always directed filaments laterally toward the
side on which they had been stimulated (in 98% of cases, n == 214). These filaments
then adhered to the source of stimulation and spread over its surface. They appeared
highly extensible (Fig. Ib), and if the stimulus source was pulled away, the filaments
could be stretched up to four times the diameter of the polyp to which they belonged.
Extruded filaments ranged in length from 1 to 42 mm (median == 3 mm). However,
most polyps extruded filaments only 1-10 mm in length (91% of polyps, n =: 190),
or about 0.1-1.5 times polyp diameter. Extrusion length did not vary with polyp size;
small (5 mm diameter) polyps often extruded filaments at least 10 mm in length,
while many large (>15 mm diameter) polyps put out filaments only 2-4 mm long.
Often several mesenteries with their attached filaments were extruded by a single
polyp, and they frequently spread to cover the organism that was the source of stimu-
lation.
114
N. E. CHADWICK
FIGURE 1 . A. Side view of extrusion of mesenterial filaments by an individual of the corallimorphar-
ian Corynactis californica onto a contracted polyp of the actiniarian sea anemone Metridium senile (left).
B. Top view of mesenterial filament extrusion by two polyps of C. californica (center) onto a retreating
individual of the actiniarian sea anemone Anthopleura elegantissima (upper right). Photo by Galen Rowell.
Note that in both photographs the filaments extend toward the actiniarians, and that in B they adhere to
the anemone as it moves away. Scale bars = 1 cm.
CORALLIMORPHARIAN BEHAVIOR
115
30r
(fl
<a
•g
'>
TO
Q)
O
<D
CL
10 -
time (hours)
FIGURE 2. Time from start of contact to start of mesenterial filament extrusion by individuals of
Corynactis californica upon contact with members of other anthozoan species and large food items (n
= 190, median = 2.5 h, range = 0.5-72 h).
The interval from the start of contact to the start of extrusion ranged from 0.5
to 72 hours, but most individuals began to extrude filaments within a few hours of
application of an appropriate stimulus (Fig. 2). At least 0.5 h of continuous contact
was necessary to elicit extrusion; when contact was intermittent, extrusion often be-
gan only after several days. Most extruded filaments reached their maximal length
1-12 h from the start of contact (median = 7 h, range =: 1-72 h, n = 129), and then
were slowly withdrawn back into the coelenteron. The duration of extrusion varied
greatly (median = 7 h, range =~- 1-144 h, n = 149); when contact with an appropriate
stimulus was continuous, the filaments of some polyps remained extruded for up to
six days.
Specificity of filament extrusion by C. californica
C. californica polyps extruded mesenterial filaments most frequently upon con-
tact with certain types of biological stimuli (Table I). They did not respond to conspe-
cifics, and instead, both clonemate and nonclonemate polyps remained expanded
and intermingled their tentacles during contact. In contrast, a large percentage of C.
californica individuals extruded mesenterial filaments onto members of three species
of actiniarian sea anemones and the scleractinian coral Astrangia lajollaensis (Table
I). Extrusion onto the solitary corals Paracyathus stearnsii and Balanophyllia elegans
was less frequent and often occurred only after 12 or more hours of contact. All ten
polyps of C californica that extruded filaments onto P. stearnsii did so 1 3-50 h from
the start of contact, and extrusion was observed onto B. elegans only after several
days or weeks from the start. Few C. californica individuals responded to the zoanthid
Epizoanthus scotinus (Table I).
C. californica polyps rarely used mesenterial filaments to attack other sessile or-
ganisms such as hydroids, colonial tunicates, sponges, or algae (Table I). However,
they did extrude filaments onto food items that were too large to ingest (Table I). To
assess the size threshold for ingestion of large food items, expanded individuals of C.
californica were offered pieces of fish that were less than, equal to, or slightly greater
than their own polyp volume (by visual estimate). The polyps injested food items
that were smaller than or equal to their own volume in 38/49 cases (78%). When
offered larger prey, however, they almost always extruded filaments over the food
(Table I).
116
N. E. CHADWICK
TABLE I
Collection sites, sti» •' percent of Corynactis californica that extruded mesenterial
filaments onto ea> ' • .-•rimulns
% C. californica
Collection
that extruded
site Stimulus
Common name filaments
ANTHOZOANS
BCHM Corynactis californica
clonemates
non-clonemates
MB Anlhopleura elegantissima
MB Metridiwn senile
B Epiactis prolifera
H Astrangia lajollaensis
CH Paracyatluts stearnsii
H Balanophyllia elegans
C Epiioanthns scot inns
NON-ANTHOZOAN SESSILE ORGANISMS
C A/lopora californica
C Garveia annulata
H Acarnns erilhicii.s
H Diaperoecia californica
H Archidistoma psammion
H Cystodytes lobata
H Rhodymenia pacifica
LARGE FOOD ITEMS
MB Mytilns ednlis
H Sebasles spp.
PHYSICAL STIMULI
Puncture with a glass needle**
Contact with a sterile glass rod
Corallimorpharian
Actiniarian sea anemone
Actiniarian sea anemone
Actiniarian sea anemone
Scleractinian coral
Scleractinian coral
Scleractinian coral
Zoanthid
Hydrocoral
Hydroid
Sponge
Bryozoan
Colonial tunicate
Colonial tunicate
Red alga
Bay mussel
Rock fish
0 (28)
0 (40)
97. 7* (44)
89.4* (38)
100* (17)
88.9* (27)
43.5 (23)
6.5 (31)
13.6 (22)
23.5
0
0
11.1
4.8
13.3
0
(17)
(10)
(28)
(18)
(21)
(15)
(21)
100* (21)
83.0* (53)
4.8 (21)
2.4 (42)
Numbers in parentheses indicate the number of polyps of C californica exposed to each stimulus.
Collection sites: B = Breakwater at Doran Beach Park, Bodega Bay, Sonoma County, CA, on intertidal
boulders, C = Cordell Bank, Marin County, CA, on rock pinnacles at 40-50 m depth, H = Hopkins Marine
Life Refuge, Monterey County, CA, on rock reefs at 10 m depth, M = Monterey Municipal Wharf #2,
Monterey County, CA, intertidaily on wharf pilings.
* Responses significantly greater than those to all other stimuli, G-test for homogeneity of replicates,
G= 14.08, P<. 05.
** The column of each polyp of C. californica was punctured with a sterile glass needle, which was left
in place for at least 1 2 h.
Differences were observed in the quality of extrusion onto food items versus an-
thozoans. When presented with large pieces of fish or mussel, most C. californica
expanded, pressed their oral disks and tentacles onto the food, and extruded filaments
out through their mouths (Table II). In contrast, when contacting anthozoans such
as sea anemones or corals, C californica often contracted and/or put out filaments
laterally through openings in the body wall (Table II). Filaments extruded onto an-
thozoans also were significantly longer than those extruded onto prey items (Fig. 3).
In response to physical contact with an inert glass rod, or physical damage to the
column wall, C. californica rarely extruded filaments (Table I). However, polyps did
put out filaments when subjected to extreme physical stress, such as when they were
accidentally crushed or became desiccated. Individuals also occasionally extruded
filaments in the absence of any apparent stimuli.
CORALLIMORPHARIAN BEHAVIOR
117
TABLE II
Comparison of behavioral responses to different stimuli (food items versus anthozoans)
by Corynactis californica during extrusion ofmesenterial filaments
Number of C. californica with each type of response
during extrusion
Posture:
Expanded Contracted
Filament
Stimulus origin:
Mouth
Body wall Mouth
Body wall
Large food items (fish, mussel)
63
1 1
0
Anthozoans
(corals, zoanthids, sea anemones)
24
29 54
23
See Table 1 for species of stimuli used.
The distribution of the responses is dependent upon the type of stimulus contacted (R X C test of
independence using G-test, G = 62.61, P < .0 1 ).
Effects of C. californica on the behavior and sunival of other anthozoans
Contact with polyps of C. californica caused strong avoidance or attack responses
by most of the anthozoans tested (Table III). However, conspecific C. californica of
different genotypes (non-clonemates) did not avoid each other, and most remained
expanded during contact. Non-mobile anthozoans of other species, such as sclerac-
tinian corals and zoanthids, contracted their tentacles and often their entire polyps
within minutes when placed in contact with C. californica (Table III). Two individu-
als of the coral Paracyathus stearnsii extruded their mesenterial filaments at 7 h but
these did not extend far enough to contact or damage C. californica polyps. The
actiniarian sea anemones varied in response depending upon whether they were sur-
rounded by C. californica polyps. When not surrounded, most individuals of An-
thopleura elegantissima and Metridium senile bent away, moved away via pedal loco-
motion, or attacked the corallimorpharian (Table III). Three polyps of A. elegantis-
sima inflated their specialized aggressive structures called acrorhagi and applied them
to C. californica at 0.5-2 h. These attacks left acrorhagial peels that caused localized
V)
.C
<*-
o
40
30
20
10
•
123456789 10 y>10
length of filaments extruded (mm)
FIGURE 3. Comparison of length of mesenterial filaments extruded by Corynactis californica in re-
sponse to large food items (shaded bars, n = 65, median = 2 mm, range =1-10 mm) versus anthozoans
(striped bars, n = 123, median = 5 mm, range = 1-42 mm). A significant difference exists between the two
populations (normal approximation to the Wilcoson rank sum test. Z = 6.83, P < .01). See Table I for
species used.
118
N. E. CHADWICK
TABLE III
Variation in the bei responses of selected anthozoans to contact with polyps of the
corallimorphar,a.- lactis californica
Number with each behavioral response
Total
number
p.nthozoan tested Expand Contract
Bend
away
Move
away
Detach
base
Attack
, CLONEMATE
CONSPECIFICS
OF C. californica 40 38
CORALS 106 12
ZOANTHIDS 20 0
SEA ANEMONES
Anthopleura
elegantissima
not surrounded 34 0
surrounded 1 5 4
Metridium senile
not surrounded 22 0
surrounded 18 3
Epiactis prolifera
not surrounded 14 0
2
92
20
0
6
1
1
0
0
0
0
2
0
2
0
1
0
0
0
29
0
8
0
0
0
0
0
5
1
5
0
2a
0
3h
0
5c + 5d
9c
0
Types of attack: a = extrusion of mesenterial filaments by the coral Paracyathus stearnsii at 7 h; b
= acrorhagi; c = extruded acontia; d = catch tentacles, used by 5/10 individuals that possessed them. See
Table I for species of corals and zoanthids used.
Not surrounded/surrounded indicates whether or not each anemone was surrounded by polyps of C.
californica during the interaction.
The distribution of responses was dependent upon the type of anthozoan involved (R X C test of
independence using G-test, P < .0 1 ).
damage to C. californica polyps, but the damaged areas healed within a few days.
Five out of ten polyps of Metridium senile that possessed well-developed aggressive
structures (catch tentacles) also inflated and applied them to polyps of C. californica
within 2-1 1 h of contact. However, none of these catch tentacles adhered to the coral-
limorpharians, and they did not appear to cause damage. Metridium senile also fre-
quently extruded acontia onto C. californica, both when surrounded and not sur-
rounded (Table III). The acontia adhered strongly to and killed some C. californica
individuals. Polyps of the actiniarian sea anemone Epiactis prolifera were tested only
when not surrounded, and most avoided contact within 3 h by moving away on the
substrate or detaching their pedal disks and then rolling or floating away (Table III).
Individuals of the sea anemones M. senile and A. elegantissima were killed within
one to three weeks (Fig. 4a) during prolonged contacts with surrounding groups of C.
californica polyps. These anemones often detached from the substrate, but adhered to
the tentacles of the surrounding C californica polyps and were unable to escape. They
were then repeatedly attacked by the extruded mesenterial filaments of C. californica,
and their tissues became necrotic within a few days. Control anemones that did not
contact C. californica remained expanded and firmly attached to the substrate
throughout the experiment.
Corynactis californica had a much slower but fatal effect on members of two spe-
cies of scleractinian corals. Within two weeks from initial contact, C. californica had
caused tissue damage to most individuals of the corals Astrangia lajollensis (57/74
polyps damaged, =77%), and Balanophyllia elegans (12/19 polyps damaged, =63%).
CORALLIMORPHARIAN BEHAVIOR
119
A. sea anemones
_a>
!5
—
o
100-
75-
50-
25-
T
T
contact
Corynactls
no
contact
1 2
time (weeks)
-f- 1001
C
0)
o
fe 7S
a.
50-
25-
0J
B. corals
contact
Corynactis
no
contact
2468
time (months)
10
12
FIGURE 4. Effect of contact with the corallimorpharian Corynactis californica on the survival of
selected sea anemones and corals. Bars represent 95% confidence limits. See text for details. A. Effect on
polyps of the actiniarian sea anemones A nthopleura elegantissirna (solid lines, n = 21 contact, n = 20 no-
contact) and Metridium senile (dashed lines, n = 17 contact, n = 17 no-contact). At three weeks, the
proportions of anemones killed in the experimental (contact) versus control (no-contact) groups were sig-
nificantly different for both species (G-test of independence for proportions, P < .0 1 ). B. Effect on polyps
of the scleractinian corals Astrangia lajollaensis (solid lines, n = 69 contact, n = 3 1 no-contact) and Balano-
phyllia elegans (dashed lines, n = 19 contact, n = 21 no-contact). At twelve months the proportions of
corals killed in the experimental (contact) versus control (no-contact) groups were significantly different
for both species (G-test of independence for proportions, P < .01 ).
During the ensuing months, C. californica polyps asexually produced many new indi-
viduals which eventually grew over and around the corals. In 6 months on one plate,
10 C. californica individuals produced over 80 polyps that killed and covered the
original 10 A lajollaensis polyps. After 12 months of contact, corallimorpharian pol-
yps had killed most of the corals on the experimental plates (Fig. 4b).
120 N. E. CHADWICK
C. californica individuals appeared to affect only the corals that they touched. In
several cases, tissue was damaged and calcareous skeleton was exposed only on the
side of a coral that faced toward a C. californica polyp. In cases where asexually pro-
duced po! alifornica grew away from and ceased to contact the experimental
corals, the latter remained healthy and undamaged. Control corals that were isolated
from contact with C. californica also remained alive (Fig. 4b), and during the year
produced many new polyps, presumably both sexually (via brooded planulae) in Ba-
lanophvllia elegans, and asexually (via clonal budding) in Astrangia lajollaensis.
DISCUSSION
This report is the first detailed description of aggressive behavior in a coralli-
morpharian. The type of aggression exhibited by C californica, extrusion of mesente-
rial filaments, is very similar to the attack behavior of many tropical scleractinian
corals (Lang, 1973; Glynn, 1974; Loya, 1976; Wellington, 1980; Cope, 1981; Bak et
ai, 1982; Logan, 1984). C. californica and certain corals readily extrude their mesen-
terial filaments onto members of other anthozoan species and onto large food items
(Table I; Yonge, 1930a; Lang, 1973). The timing of the extrusion response is also
remarkably similar in corals and C. californica. Lang (1973) reported that Jamaican
reef corals extruded their filaments 0.5-12 h after initiation of contact with certain
coral species, and Glynn (1974) noticed extrusion by eastern Pacific corals 8-12 h
after contact with competing corals. In the present study, most C. californica individ-
uals also put out their filaments within 12 h (Fig. 2). The extruded filaments of both
C. californica and scleractinian corals cause extensive damage to and eventually kill
other anthozoans if contact is prolonged and if, in corals, the other colony is small
enough (Lang, 1973; Fig 4). Since corals and corallimorpharians are morphologically
very similar (den Hartog, 1 980), one might expect to see this similarity in their aggres-
sive behaviors as well. This type of aggression, via mesenterial filament extrusion,
differs from the competitive behavior of some of the actiniarian sea anemones that
coexist with C. californica and use their marginal spherules (Francis, 1973b) or catch
tentacles (Purcell, 1977) to attack competitors. These differences in behavior under-
score the major morphological differences between a corallimorpharian such as
C. californica, and actiniarian sea anemones. They also support the idea that coralli-
morpharians are more closely related to scleractinian corals than they are to sea
anemones.
Unlike the specialized aggressive structures of actiniarian sea anemones that are
used only during competitive interactions (Bonnin, 1964; Francis, 1973b; Williams,
1975; Purcell, 1977; Watson and Mariscal, 1983), the mesenterial filaments of C
californica appear to serve a variety of functions. In all anthozoans studied thus far,
the mesenterial filaments are the major sites for digestion and absorption of food in
the coelenteron (Yonge, 1930b; Nicol, 1959; Van-Praet, 1985). These filaments con-
tain gland cells that secrete strong proteolytic enzymes, as well as nematocysts that
may inject cytolytic toxins into prey (Van-Praet, 1985). Special areas on the filaments
and adjacent mesenteries then absorb the partially digested foodstuffs (Yonge, 1 930b;
Van-Praet, 1985). Corynactis californica also extrudes mesenterial filaments onto
food that is too large to take into the coelenteron (Table I), presumably to digest it
externally. This behavior allows polyps to consume a large range of prey sizes. Certain
tropical Pacific corallimorpharians envelope prey in the oral disk, and then extrude
filaments out of the mouth to digest them (Hamner and Dunn, 1980). Many species
of reef-building corals also consume prey externally via extruded filaments (Carpen-
ter, 1910; Yonge, 1930a, 1968; Goreau et ai, 1971). Thus, two major functions of
CORALLIMORPHARIAN BEHAVIOR 121
mesenterial filaments in these organisms appear to be the internal breakdown and
absorption of food, and external consumption of large prey. Mesenterial filaments
also are used during physical stress. Some corals extrude their filaments when oil is
introduced into their coelenterons (Bak and Elgershuizen, 1976), when they are ex-
posed to intense light (Lang, 1973), or when they are handled roughly (Duerden,
1 902). C. californica polyps also exhibit extrusion when stressed (see Results). Finally,
divers observed C californica polyps extruding their mesenterial filaments onto one
of their major predators, the sea star Dermasterias imbricata (Annett and Pierotti,
1984; pers. obs.). Thus, the lobed filaments along the edges of anthozoan mesenteries
may serve multiple functions in certain corals and corallimorpharians.
An interesting aspect of the aggressive/defensive use of mesenterial filaments by
C. californica is the complete lack of response to conspecifics (Table I). Members of a
given clonal aggregation presumably would benefit from damaging and overgrowing
those of a different, genetically distinct aggregation (as discussed by Francis, 1973b).
However, in the field distinctly colored groups of C. californica often intermingle and
show no evidence of aggression or damage along their interacting borders (pers. obs.).
The wide, anemone-free zones that are visible between aggregations in other species
known to show interclonal aggression (Francis, 1973a; Purcell, 1977) do not occur
in this species. Most reef corals that use mesenterial filaments to attack competitors
also only extrude them interspecifically (Lang, 1973; Cope, 1981 ). One exception has
been reported: the Caribbean coral Montastrea annularis appears to extrude fila-
ments onto conspecific colonies to damage them (Logan 1984, 1986).
The present study demonstrates that under laboratory conditions C. californica
strongly affects both the behavior and survival of certain other anthozoans (Table III,
Fig. 4). These results have several ecological implications. In shallow subtidal habitats
along the coast of California where C. californica occurs, hard surfaces are often com-
pletely covered with organisms (Pequegnat, 1964; Haderlie and Donat, 1978; Vance,
1 978; Schmieder, 1 984, 1 985), and space for settlement and growth may be a limiting
resource for sessile animals. The species of sea anemones tested in this study often
moved away from or otherwise avoided contact with C. californica (Table III); where
they co-occur in the field, this behavior might free space for growth along the interspe-
cific borders of C. californica aggregations. The avoidance responses of these sea
anemones are the same behaviors used to effectively escape attack by conspecifics
(Francis, 1973b; Purcell, 1977) and predators (Waters, 1973; Edmunds el ai, 1976).
However, the specialized aggressive structures ofAnthopleura elegantissima and Me-
tridium senile apparently were not effective against C. californica (see Results). The
acontia of M. senile caused the most damage to C. californica, and in the field may
allow the former to kill polyps of the latter along their interacting borders. L. Harris
(University of New Hampshire, pers. comm.) has observed that, in the laboratory,
M. senile also uses acontia to attack individuals of the sea anemones A elegantissima.
Actinia equina, and Urticina (= Tealia) piscivora.
The results of the present study differ somewhat from those presented by Chao
(1975). He described an aggressive hierarchy in which C. californica was dominant
over A. elegantissima, while M. senile was dominant over both of the former species.
The present results confirm that C. californica causes tissue damage to A. elegantis-
sima (Table III, Fig. 4a), but show that C. californica and M. senile damage each
other, with no clear competitive outcome. A clear dominance ranking of these three
cnidarian species remains to be determined.
Field observations also suggest that C. californica damages sea anemones and cor-
als under natural conditions. Chao (1975) noticed that interspecific boundary areas
about 2-3 cm wide occurred between aggregations of C. californica and the sea anem-
122 N. E. CHADWICK
ones Anthopleura elegantissima and Metridium senile found on intertidal pilings at
the Monterey Wharf. Francis (1973b) and Purcell (1977) showed that anemone-free
zones between :<ups within the latter two species are maintained by aggression be-
tween cio^ orridors along their boundaries with C. californica could be main-
tained by oidance behaviors of the anemones (Table III), or by the death of
anemone1 ;hat have been repeatedly attacked by the mesenterial filaments of C. cali-
for> i-a).
subtidal rock reefs, certain scleractinian corals also appear to be negatively
cted by contact with C. californica. Fadlallah (1981) observed a polyp of C. califor-
nica extruding filaments onto and killing an individual of the solitary coral Balano-
phvllia elegans in the kelp forest at Hopkins Marine Life Refuge (HMLR) in Monte-
rey. Polyps of B. elegans and the colonial coral Astrangia lajollaensis that occur adja-
cent to C. californica on subtidal boulders at HMLR often show damaged tissues and
exposed skeletons (pers. obs.). In addition, the vertical distribution of C. californica
and A. lajollaensis on large subtidal reefs suggests some sort of negative interaction.
Pequegnat (1964) found that C. californica was most abundant near the top of a
subtidal reef in southern California, and became more sparse with depth. In contrast,
A. lajollaensis formed large colonies near the base of the reef and decreased in abun-
dance with height, occurring at low densities near the reef top. These inverse patterns
of abundance also can be observed on large (2-5 m high) subtidal reefs at HMLR
in central California (pers. obs.). Efforts are currently underway to document the
distributions of these anthozoans at HMLR, and to test the ecological effects of their
behavioral interactions in the field.
Members of the genus Corynactis produce clonal aggregations in tropical and
temperate marine habitats throughout the world (Carlgren, 1949). Corynactis viridis
is the most abundant sessile organism on shallow subtidal rocks walls at the Glenan
Archipelago on the Atlantic coast of France (Castric-Fey et ai, 1978), and at Plym-
outh, England (Forster, 1958); C. parvula occurs on Caribbean reefs where it may
interact with a variety of corals and sea anemones (den Hartog, 1980). These conge-
ners may show aggressive behavior similar to that of C. californica, as well as use
other competitive mechanisms (den Hartog, 1 977), thus affecting the abundance and
distribution of co-occuring sessile organisms.
Many so-called lower animals exhibit complex aggressive behaviors associated
with resource defense. Such behaviors are observed in polychaete worms (Evans,
1973; Dimock, 1974; Roe, 1975), chitons (Chelazzi et ai, 1983), limpets (Stimson,
1970; Branch, 1975; Wright, 1982), sea urchins (Schroeter, 1978; Maier and Roe,
1983), and sea stars (Menge and Menge, 1974; Wobber, 1975), as well as in the many
anthozoans discussed in this paper. Yet the behaviors of these organisms are rarely
considered in theoretical works on aggression and territoriality, most of which focus
on birds and mammals ( Waser and Wiley, 1 979; Murray, 1981; Davies and Houston,
1984), nor are they included in recent texts on animal behavior (Huntingford, 1984;
Ridley, 1986). Because marine invertebrates often are sessile or slow-moving, and
may be clonal as well, their aggressive behaviors have developed under a different set
of constraints than have those of most vertebrates. More extensive consideration of
aggression in the lower invertebrates may lead to important new insights into the
evolution and ecology of animal conflict.
ACKNOWLEDGMENTS
I thank C. Hand, R. Caldwell, W. Sousa, J. Pearse, D. Fautin, B. Rinkevich, A.
Johnson, and T. Hunter for constructive criticism throughout this research and for
CORALLIMORPHARIAN BEHAVIOR 123
comments on the manuscript. I am also indebted to members of Cordell Bank Expe-
ditions, especially R. Schmieder, who collected the specimens from Cordell Bank.
Research facilities were provided by Bodega Marine Laboratory, Joseph M. Long
Marine Laboratory, and Hopkins Marine Station. Financial support was generously
provided by the Lerner-Gray Fund of the American Museum of Natural History, the
Chancellor's Discretionary Fund and a Regent's Fellowship from the University of
California at Berkeley, Sigma Xi, and the intercampus program of the Institute of
Marine Resources at Scripps Institution of Oceanography. This research was com-
pleted in partial fulfillment of the requirements for the doctoral degree at the Univer-
sity of California, Berkeley.
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DEVELOPMENT, METAMORPHOSIS, AND SEASONAL ABUNDANCE
OF EMB >S AND LARVAE OF THE ANTARCTIC SEA URCHIN
STERECHINUS NEUMAYERI
HO BOSCH, KATHERINE A. BEAUCHAMP, M. ELIZABETH STEELE,
AND JOHN S. PEARSE
Institute of Marine Science, University oj California, Santa Cruz, California 95064
ABSTRACT
The development to metamorphosis of the shallow-water antarctic sea urchin,
Sterechinus neumayeri, is described for the first time. Developmental stages are sim-
ilar to those of closely related temperate species with feeding larvae, but the rate of
development is extremely slow. Hatching of ciliated blastulae occurs approximately
140, 128, and 1 10 hours after fertilization at -1.8, -1.0, and -0.5°C, respectively,
more than twice the time required for closely related temperate species near their
normal ambient temperature. Larvae reared at — 1 .8 to — 0.9°C are capable of feeding
20 days after fertilization and are competent to metamorphose after 1 1 5 days. Early
cleavage embryos, blastulae, gastrulae, and prism larvae of this species were collected
from the plankton adjacent to McMurdo Station, Antarctica, in early November and
December, 1984 and 1985. Echinoplutei were not found during this study, but they
have been collected from the plankton in other years; there is no evidence that the
larvae are demersal. The timing of spawning ensures that feeding larvae are in the
plankton during the abbreviated summer peak of phytoplankton abundance in Mc-
Murdo Sound. Recruitment of juveniles into the benthos most likely occurs in syn-
chrony with the subsequent period of high levels of benthic chl a concentrations.
INTRODUCTION
The perception that brooding is the prevalent mode of development among spe-
cies of antarctic echinoderms has been firmly established over the past century
(Thomson, 1876; Thorson, 1950, Mileikovsky, 1971; Dell, 1972; White, 1984).
Brooding is most apparent within the shallow-water echinoid faunas (Arnaud, 1974;
Picken, 1980). Three families in three separate orders (Cidaridae, order Cidaroidea;
Schizasteridae, order Spatangoida; and Echinidae, order Echinoidea) represent the
antarctic echinoids. Two of the three families are dominated by species that brood.
Fell (1976) reported that 12 of 19 known species of antarctic cidarids are known
brooders, and 3 others almost certainly brood. In addition, females of all 21 known
species of antarctic schizasterids brood their young in specialized sunken aboral petal-
oids known as marsupia (Fell, 1976).
It is unclear whether the high incidence of brooding species among these two
families is a consequence of ongoing selection in the antarctic environment or of
phylogenetic history (Dell 1972; Fell, 1976; Arnaud, 1977). Fell (1976) hypothesized
that the ancestral forms of antarctic cidarids (ancestral goniocidarids) brooded their
young, and he suggested that cidarids colonized the antarctic as brooders. No ances-
tral form has been clearly established for antarctic schizasterids. Since extant non-
Received 25 September 1986; accepted 27 May 1987.
126
ANTARCTIC ECHINOID REPRODUCTION 127
antarctic representatives of this group have unprotected development, the brooding
habit of antarctic species may have evolved subsequent to their colonization of the
antarctic (Fell, 1976). In either case, the numerical success (i.e., number of species)
of cidarids and schizasterids in the antarctic apparently is related — at least in part —
to their brooding habits.
In contrast to the cidarids and schizasterids, the antarctic echinids are represented
by only five species, all within a single genus, Sterechinus (Fell, 1976). Individuals of
one species, S. neumayeri, are the most abundant echinoids in shallow-water sur-
rounding the antarctic continent. The relatively small maximum egg sizes reported
for three antarctic species of Sterechinus (0.15 in S. neumayeri, and 0.25 mm S.
agassizii and .S. antarcticus) are indicative of a free-swimming mode of development
(Mortensen, 1909, 1910; Pearse and Giese, 1966). Moreover, despite frequent collec-
tions, brooding has not been reported for any of the six species of the genus (Fell,
1976). The absence of post-spawning parental care among antarctic representatives
of this group is in sharp contrast with the predominant mode of development in other
antarctic echinoids.
Little is known about the embryonic and larval stages of non-brooding antarctic
echinoids. Mortensen (1913) described echinoplutei from plankton samples collected
by the German South Polar Expedition ( 1 90 1 - 1 903). Mortensen assigned the larvae
to 5". neumayeri because it was a common species, had very small eggs, and was not
known to brood. A pair of echinoplutei was collected from midwater in McMurdo
Sound by the British National Antarctic Expedition (MacBride and Simpson, 1908).
Mortensen (1913) also assigned these to S. neumayeri. Since the publication of these
reports over 70 years ago, little additional information on the developmental stages
of non-brooding antarctic echinoids has been obtained. Pearse and Giese (1966) de-
scribed the reproductive cycle of a population of S. neumayeri in McMurdo Sound,
and suggested that the larvae of this species are demersal and not pelagic because they
have been taken from the plankton so rarely; however, the larval development of
antarctic echinoids had not been observed or described.
The present paper describes the development through metamorphosis of Sterechi-
nus neumayeri, and draws special attention to the slow rates of embryonic and larval
development. In addition, we present information on the seasonal abundance of em-
bryos and larvae of this species in the near-shore waters of McMurdo Sound, Ant-
arctica.
MATERIALS AND METHODS
Individuals of Sterechinus neumayeri were collected by scuba divers from 15-25
m depth beneath the annual sea ice adjacent to McMurdo Station, Antarctica (77°
51' S, 166° 40' E). In November 1983, immediately after collection, approximately
two dozen animals were transported to the University of California, Santa Cruz,
where gametes were fertilized and the larvae were reared through metamorphosis in
an ice bath (-0.5 to 0.5°C) kept in a 4°C refrigerated unit. Additional studies of the
developmental stages and developmental rate of S. neumayeri were carried out at
McMurdo Station; ripe animals were collected in November, 1984, and larvae were
reared through metamorphosis and early juvenile stages to December, 1985. The
running seawater system at McMurdo Station maintains aquarium seawater temper-
atures between —1.8 (winter) and —0.9 (summer)°C, which allowed us to rear em-
bryos and larvae close to their ambient temperatures.
128 I- BOSCH ET AL.
Spawning ami >n of gametes
Spavv .'ed by intracoelomic injection of 0.5 M KC1 solution. Eggs
collected f spawning females were washed in clean 5 ^m filtered seawater
an<j a few drops of dilute sperm suspension in a 4 liter polycarbonate
• ter approximately 20 minutes, eggs were filtered off with 20 /urn nitex
ncl placed in 4 liter culture vessels with clean 5 /urn filtered seawater.
Rearing of embryos and larvae
Embryos and larvae were reared in gently stirred and unstirred cultures (Hine-
gardner, 1 969; Strathmann, 1971). The water in the culture vessels was changed every
four days using a 20 ^m mesh nitex strainer to retain the embryos and larvae.
At McMurdo Station, larvae were fed semi-daily with equal amounts of bacterized
cultures of Isochrysis galbana and Phaeodactylum tricornutum (total concentration
10,000-15,000 cells/ml), which were grown at 15°C in continuous light using half
strength F medium (Guillard and Ryther, 1962). Algal samples to be used as food
were centrifuged for 10 minutes at 5000 rpm and resuspended in clean filtered seawa-
ter (- 1 .5°C). Phytoplankton concentrations were measured using a Palmer Maloney
counting chamber.
Initially, at Santa Cruz, several phytoplankton species (including both temperate
and antarctic forms) were tested as potential sources of food for the larvae. Among
five temperate species tested (Amphidinium carter! , Dunaliella tertiolecta, Isochrysis
galbana, Phaeodactylum tricornutum and Rhodomonas sp.), I. galbana and P. tricor-
nutum were most resistant to low temperatures. These phytoplankton appeared to be
healthy, even after being in larval cultures for two days, and were readily consumed
by the larvae. Cells of the antarctic diatom Thalassiosira antarctica were not readily
ingested by early stage plutei.
Settlement and metamorphosis of larvae reared in Santa Cruz was induced by
adding echinoplutei to glass dishes containing pieces of PVC pipe covered with a
bacterial-algal film (Hinegardner and Tuzzi, 1971). The bacterial-algal film was pre-
pared by placing the PVC pipe in a large dish that was held in a running seawater
table for several days. Competent echinoplutei reared at McMurdo Station were suc-
cessfully induced to settle and metamorphose with sediment samples collected from
various depths ( 1 2, 20, 25, and 30 m) within the adult habitat.
Embryonic developmental rates
Time of development to hatching at different temperatures was determined for
embryos reared at McMurdo Station by holding them in culture vials that were ( 1 )
in a refrigerated unit at -0.5 (-0.7 to -0.3)°C, (2) in a running seawater table in the
laboratory at - 1 .0 (- 1 .2 to -0.9)°C, and (3) submerged in the sea 5 m below the level
of the sea ice at ambient temperature, - 1 .8 (- 1 .9 to - 1 .7)°C (a small heated hut with
a hole in the floor and through the sea ice was used as a staging area). Approximately
50 newly spawned eggs from a pair of females and a single drop of dilute sperm
suspension were mixed in each of 3 sets of 10, 5 ml capacity vials filled with 5 ^m
filtered seawater at the appropriate temperature. Progress of development and incu-
bation temperature were monitored every 12-16 hours during early cleavage stages
and every 2 hours near the time of hatching. Because agitation and small changes in
temperature may adversely affect rates of embryonic development, only previously
undisturbed culture vials were used for observations of developing embryos. The time
ANTARCTIC ECHINOID REPRODUCTION 129
of hatching was defined as the time when at least 10% of the ciliated blastulae in a
particular incubation vial were released from the fertilization membrane.
Field collection of embryos and larvae
Plankton samples were collected on a weekly or bimonthly basis from September,
1 984 to December, 1 985 using both diver-towed and stationary current-fed plankton
nets (240 ^m mesh) at various locations in McMurdo Sound. The conical, stationary
nets measured 2 m in length with a circular mouth opening of 0.3 m. The diver-towed
net was 2 m long and had a rectangular mouth of 0.1 X 0.3 m. Each of the current-
fed nets was held open continually by a steel frame; net bouyancy was regulated with
a float. Two or three nets were attached to a weighted steel cable and suspended by
scuba divers from the undersurface of the sea ice for 24 to 48 hours. At the points of
attachment to the cable, the nets had a ball bearing swivel which allowed them to
orient to the shifting directions of the prevailing currents.
Because the larvae may be demersal, 5 replicate bottom cores of 8 cm diameter
were taken monthly from October, 1984 through October, 1985 at 10, 20, 25, and
30 m depth adjacent to McMurdo Station.
All samples were sorted for larvae and other organisms within two days of collec-
tion. Early developmental stages that were not readily identifiable were isolated from
field samples and reared in the laboratory until they reached a recognizable larval
stage. Sizes of embryonic and larval stages as well as larval skeletal morphology of
the field-collected specimens were noted and compared to those of embryos and lar-
vae reared in the laboratory from fertilization.
RESULTS
Sequence oj development
Development of Sterech inns neumayeri was followed through metamorphosis at
Santa Cruz (-0.5 to 0.5°C) and McMurdo Station (-1.8 to -0.9°C) (Table I). The
eggs are small (mean diameter = 0.179 mm; n = 55) and negatively buoyant. Early
development yields a typical sea urchin prism larva. Stomadeal breakthrough occurs
20 days after fertilization at approximately — 1.5°C, and soon thereafter the larvae
begin to feed. By the 2 1st day, the postoral and anterolateral paired arms of the echi-
nopluteus are formed. The larval epithelium is now sparsely covered with red pig-
ment granules, more or less randomly distributed. Formation of the posterodorsal
and the much shorter preoral pair of arms begins at approximately 43 and 56 days
after fertilization, respectively. The onset of the eight-arm pluteus stage is closely
timed with the formation of the anterior epaulettes as well as the appearance of the
five lobes of the hydrocoel (Fig. 1 ). At this stage of development the larvae are similar
to those previously described from collections of earlier antarctic expeditions (Mac-
Bride and Simpson, 1908; Mortensen, 1913).
Further thickening of the ciliary band along the posterior margin of the larva
results in the formation of the posterior epaulettes. By approximately the 80th day of
development at - 1 .8 to — 0.9°C, the tube feet primordia are formed. Soon thereafter,
a variable number (1-3) of triradiate spines appear on the external surface of approxi-
mately 40% of the larvae. The most conspicuous of the spines is located in a medial
position at the posterior end of the larva, while the other two are formed on the right
side, near the bases of the postoral and posterodorsal rods of the larval skeleton.
Metamorphosis is relatively slow, lasting 2-3 hours before the non-feeding ben-
thic juvenile is formed. Newly metamorphosed juveniles retain many of the pigment
1 30 I. BOSCH ET AL.
TABLE I
Develop^. .nm.ximate sizes of developmental stages of Sterechinus neumayeri reared
in Santa > (-0.5 to 0.5 °C) and McMurdo Station, A ntarctica (-1.8 to- 0. 9°C)
First appearance
(days)
Size
)pmerital stage
(mm)
-1.8to-0.9°C
-0.5to0.5°C
, /edegg
.18-.19
—
—
blastula
.21
2.1
1.7
Hatching
—
5.1
3.7
Gastrula
.22
10
8
Prism
.32
16
15
Early pluteus
.35
21
17
Six-arm pluteus
.54
43
29
Early eight-arm pluteus
.80
56
42
Late eight-arm pluteus
1.20
103
100
Juvenile
.44
115
107
Sizes represent the diameter of ova, blastulae and juveniles, maximum length of gastrulae and prism
larvae, and length from the aboral apex to the tips of postoral arms of echinoplutei.
granules characteristic of larval stages, but otherwise have a pale, whitish appearance.
They have a single set of well developed tube feet as well as 1 0 juvenile and 1 5 primary
spines. The triradiate spines which appeared on the surface of echinoplutei are re-
tained on the aboral surface of juveniles.
Duration of embryonic development
Embryos reared below the sea ice (-1.9 to -1.7°C), in a seawater table (-1.2 to
-0.9)°C, and in a refrigerator (-0.7 to -0.3°C) at McMurdo hatched at 140, 122, and
1 10 hours, respectively. Time to first hatching for embryos reared in an ice bath at
Santa Cruz (—0.5 to 0.5°C) was approximately 88 hours.
Occurrence of eggs, embryos and larvae in the plankton
One hundred and twenty (120) plankton samples were taken from McMurdo
Sound between September, 1984 and December, 1985. Of these, 56 were taken from
near the undersurface of the ice or, in the absence of sea ice, near the surface of the
water. Fourteen were taken from midwater (10-20 m depth), and 50 were collected
from near the bottom at 1 5-30 m depth.
Large numbers (500-600) of embryos, free-swimming blastulae, and gastrulae
that closely resembled those of laboratory reared Sterechinus neumayeri were col-
lected from the plankton at all depths sampled using both stationary and diver-held
plankton nets. Eggs and early stage embryos were collected predominantly during the
third and fourth weeks of November, 1 984 and 1 985. Hatched blastulae and gastrulae
at various stages of development were predominant during the first week of Decem-
ber, although several un hatched and newly hatched blastulae were collected from
surface waters on the 9th of November, 1985. Four prism larvae were identified from
midwater samples taken in mid to late December, but no echinoplutei were collected
during this study. No sea urchin eggs, embryos, or larvae were found in the 240 bot-
tom cores collected and examined.
ANTARCTIC ECHINOID REPRODUCTION
131
100 (jm
FIGURE 1 . Early eight-arm pluteus of Sterechinus neumayeri shortly after the formation of the pre-
oral pair of arms (indicated by arrow). Scale bar = 100
DISCUSSION
Embryonic and larval development
Compared to other species that have been studied, the developmental stages of
Sterechinus neumayeri are most similar in shape and size to those of the temperate
echinoid, Echinus esculentus (MacBride, 1903). However, the formation of spines
on the external surface of the larvae, separate from the juvenile rudiment, clearly
distinguishes the larvae ofS. neumayeri from those of E. esculentus and other species
studied within the family Echinidae (MacBride, 1903; Arrau, 1958; Cram, 1971).
Morphologically similar spines reportedly develop on the echinoplutei of several
other species of regular echinoids, including both euechinoid and cidaroid forms
(Onoda, 1931, 1936; Fukushi, 1960; R. Emlet, pers. comm.).
The time of development for the entire period from fertilization to metamorpho-
sis of Sterechinus neumayeri is extremely long. Within the family Echinidae, the tern-
132
I. BOSCH ET AL.
UJ
S
90-
60-
30-
10
1 5
20
25
30
TEMPERATURE (°C)
FIGURE 2. Duration of embryonic development to hatching as a function of temperature for seven
species of echinids and strongylocentrotids with indirect development. Hatching occurs at the ciliated
blastula stage. Mean diameter of ova ranges between 80 (Strongylocentrotus purpuratus) to 179 ^m (Stere-
chinus neumaveri). 4 Strongylocentrotus droebachiensis reared at 0, 4, 8°C (Stephens, 1980) and 9-10°C
(Strathmann, 1974); • S.frantiscanw reared at 10, and 12-13°C (Strathmann, 1974); A S. pulcherrimus
reared at 25-27°C (Onoda, 1 936); O S. purpuratus reared at 1 0°C (Strathmann, 1 974); D Loxechinus a/bus
reared at 13-14°C (Arrau, 1955); • Parechinus angitlosus reared at 15°C (Cram, 1971); 0 Sterechinus
neumaveri reared at - 1 .9 to - 1 .7, - 1 .2 to -0.9, -0.7 to -0.3, and -0.5 to 0.5°C (this study).
perate species Parechinus angulosus and Psammechinus miliaris are competent to
metamorphose 60 days after fertilization at ambient temperatures (10-1 6°C) (Shearer
el al, 1913; Cram, 1971), less than half the time required for S. neumaveri near
their normal ambient temperature (- 1 .5°C). This observation agrees with the general
trend noted by Emlet el al. (in press) between decreased temperatures (and increased
latitudes) and increased time to metamorphosis for echinoids with planktotrophic
larvae.
Because factors unrelated to temperature may influence rates of post-embryonic
development (e.g., larval food and density, Kume and Dan, 1968; Hinegardner,
1969), we critically compared the rates of embryonic development to the hatched
blastula stage at different temperatures, both of Sterechinus neumaveri and other sea
urchin species with planktotrophic larvae within the families Echinidae and Strongy-
locentrotidae. Time to hatching ranged from a minimum of 13 hours at 25-27°C in
the tropical species Strongylocentrotus pulcherrimus to a maximum of 140 hours at
-1.9 to — 1.7°C for S. neumaveri, and was intermediate for temperate species near
their normal ambient temperatures. The duration of embryonic development to
hatching for these seven echinoid species is a curvilinear function of temperature,
with increased sensitivity at lower temperatures (Fig. 2). A direct relationship be-
tween the duration of embryonic development and temperature has been found with
interspecific comparisons among other poikilotherm groups, including asteroids
(Pearse, 1969), amphipods (Bregazzi, 1972), barnacles (Patel and Crisp, 1960), cope-
pods (McClaren el al., 1969), and rotifers (Herzig, 1983b). Moreover, studies on sin-
gle species or physiological races reveal the same function, describing the immediate
thermodynamic effect of temperature on developmental processes [See for example.
ANTARCTIC ECHINOID REPRODUCTION 133
Bougis (1971), Stephens ( 1 972), and McEdward (1985) for temperate echinoids; Her-
zig(1983a) forcopepods; Herzig(1983b) for rotifers; and Ross and Quetin (1986) for
antarctic krill]. The direct relationship between temperature and duration of embry-
onic development, both within a single species and among groups of related species,
suggests that there is little or no temperature compensation for developmental rates
in poikilotherms, resulting in the observed general trend of increasingly longer peri-
ods of development with greater latitude.
The tendency for increased lecithotrophic development among high latitude ma-
rine invertebrates was well documented by Thorson (1950) who proposed that the
combination of low temperatures — which act to increase development time — and a
short season of phytoplankton abundance in high latitude environments select
against planktotrophic larvae. Thorson's (1950) explanation has been challenged by
several authors. In particular. Underwood ( 1 974) and Clarke ( 1 982, 1 983) argue that
there should be no a priori reason to expect ontological processes to be rate-limited
by temperature because all poikilotherms have evolved the capability to modify those
processes for the effects of temperature. However, although numerous mechanisms
for metabolic temperature compensation have been identified (Hochachka and Som-
ero, 1984), there are few examples of developmental rate compensation for tempera-
ture in any previous work (Clarke, 1 982). Development is a complex, highly synchro-
nized process involving many biochemical and structural changes. As suggested by
Patel and Crisp ( 1 960), basic patterns of temperature-developmental rate interactions
may not be readily modified in evolution.
Seasonal abundance and distribution
The presence of embryonic and early larval stages of Sterechinus neumayeri in
the plankton during early to mid November and December, 1984 and 1985 is in
accordance with previous estimates of the spawning time of this species in McMurdo
Sound (Pearse and Giese, 1 966). Observations of spawning urchins further substanti-
ate this conclusion: males spawned in shallow water near McMurdo Station on two
occasions during the first week of November, 1984 (B. Gullikson and T. Klinger,
pers. comm.). Coupled with known development times of laboratory-reared embryos
and larvae, this evidence suggests that larvae of S. neumayeri feed between late De-
cember and early March, coinciding with the summer peak of phytoplankton abun-
dance in McMurdo Sound (Bunt, 1964; Rivkin et a/., 1986). Consequently, settle-
ment of larvae onto the benthos will occur predominantly during late February and
March, in synchrony with the annual period of high benthic chl a concentration that
occurs during the austral Fall (Berkman et al. 1986).
Twenty-five plankton tows and 16 bottom cores were collected and examined
between late December and early March, 1 984- 1 985, yet no echinoplutei of Sterechi-
nus neumayeri were found. Littlepage (1966, 1968, and pers. comm.) collected and
analyzed 547 plankton samples taken throughout the year from McMurdo Sound
but found no echinoderm larvae. The conspicuous absence of echinoplutei from
plankton samples taken over areas where adult 5". neumayeri are abundant led Pearse
and Giese ( 1 966) to suggest that the embryos and larvae of this sea urchin are demer-
sal. However, large numbers of S. neumayeri embryos and early larvae were collected
from the water column during this study. Moreover, echinoplutei of this species have
been taken from the antarctic plankton in other years: all 48 specimens recorded by
MacBride and Simpson (1908) and Mortensen (1913) were taken from the water
column; in addition, four echinoplutei of S. neumayeri were collected from near-sur-
face waters, over approximately 300 m of water, in early January, 1986 (Rivkin et
1 34 I- BOSCH ET AL.
al, 1986). Thi otrates that embryos and larvae of S. neumayeri are
readily car bottom by currents. Given the active swimming behavior
of echi; ory cultures (I. Bosch, pers. obs.), it is unlikely that develop-
ment of th is demersal. More extensive, multi-annual sampling is needed to
provide ; e evidence on the larval distribution of S. neumayeri.
ACKNOWLEDGMENTS
We thank R. L. Britton and B. Marinovic for assistance in the field, J. S. Oliver
for collecting and transporting urchins to Santa Cruz, and G. Fryxell for providing
stock cultures of antarctic phytoplankton; the Antarctic Services Inc., of ITT, espe-
cially J. Wood and S. Ackely, the Antarctic Support Services of the National Science
Foundation, and the U. S. Navy Antarctic Support Force for their logistic support;
W. T. Doyle, Director of the Institute of Marine Sciences, University of California,
Santa Cruz and R. T. Hinegardner for encouragement and support; and E. Bay-
Schmith, R. B. Emlet, R. T. Hinegardner, J. B. McClintock and J. Ott for suggestions
on the manuscript. Supported by NSF Grant No. DPP-83 17082.
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Reference: Biol. Bull. 173: 136-159. (August, 1987)
FEEDING >l: \TIONS OF THE FORAMINIFERAN CIBICIDES
REF; NG EPIZOICALLY AND PARASITICALLY ON THE
ARCTIC SCALLOP ADAMUSSIUM COLBECKI
STEPHEN P. ALEXANDER AND TED E. DELACA*
A-002, Marine Biology Research Division, Scripps Institution of Oceanography,
La Jolla, California 92093
ABSTRACT
The calcareous foraminifer Cibicides refulgens is a conspicuous and abundant
component of the epifaunal community living on the valves of the free-swimming
Antarctic scallop, Adamussium colbecki. Examination of this association using light
microscopy, scanning electron microscopy, radiotracer, and resin-casting/sectioning
techniques, demonstrates that the foraminifer possesses a combination of morpho-
logical and physiological adaptations, unique among benthic calcareous foramini-
fera, which enhance its ability to acquire nutrients in an otherwise oligotrophic and
seasonal environment. Three distinct modes of nutrition are employed: (1) grazing
the algae and bacteria living upon the scallop shell surface, (2) suspension feeding
through the use of a pseudopodial net deployed from a unique superstructure of ag-
glutinated tubes which form an extension to the calcareous test, and (3) parasitism
by eroding through the scallop's shell, and using free amino acids from the highly
concentrated pool in the extrapallial cavity.
INTRODUCTION
A variety of benthic foraminiferal species are known to live most, or part of their
lives, epizoically on a wide range of organisms. Examples include Rosalina globularis
d'Orbigny, R. anomala Terquem, R. carnivora Todd, Cibicides refulgens Montfort,
C. lobatulus Walker and Jacob, C. pseudoungerians (Cushman), Discorbis wrighti,
and Discorbinnella sp. which firmly attach to macroscopic algae and metazoans such
as hydroids, bryozoans, tunicates, crustaceans, isopods, amphipods, decapods, pyc-
nogonids, brachiopods, gastropods, and bivalves (Nyholm, 1961; Todd, 1965; De-
Laca and Lipps, 1972; Hayward and Haynes, 1976; Zumwalt and DeLaca, 1980;
Mullineaux and DeLaca, 1984; Alexander, 1985; Moore, 1985).
The most important association in terms of numbers of foraminifera appears to
involve filter feeding invertebrates, particularly free-swimming bivalves. Hayward
and Haynes ( 1 976) reported 998 individual foraminifers on one specimen of the com-
mercial scallop Clamys opercularis (Linneaus) of which 765 were Cibicides lobatulus.
Similarly, Mullineaux and DeLaca (1984) noted an average of 1 386 foraminifers on
21 specimens of the Antarctic scallop Adamussium colbecki, 901 of which were C.
refulgens.
For the majority of associations between filter feeding invertebrates and epizoic
Received 27 March 1987; accepted 22 May 1987.
* Presently: Division of Polar Programs, National Science Foundation, 1800 G Street, Washington,
DC 205 50.
136
C. REFULGENS, MORPHOLOGY AND ECOLOGY 137
foraminifers, the host shell may provide not only a firm substrate for attachment, but
also the added advantage of a relatively silt-free environment. In motile bivalves liv-
ing in areas of strong currents and wave action, the shell may provide further protec-
tion against sand shifting and possible burial with fatal consequences (Dobson and
Haynes, 1973; Hayward and Haynes, 1976). Even sessile molluscs such as Mytilm
can provide a relatively silt-free substrate (Allen, 1 953), and Notocorbula living in the
silt-laden Mississippi delta, offers a preferred habitat for Hanzawaina sp. by crawling
above the layers of accumulating flocculent material (Bock and Moore, 1969). Fur-
thermore, life activities of the host can enhance the availability of nutrients to the
epizoic foraminifers; for example seven species living on the shell of the brachiopod
Tichosina floridensis Cooper, are thought to benefit from suspended food material
transported by the inhalant and exhalent feeding currents (Zumwalt and De-
Laca, 1980).
The means by which foraminifera attach themselves to the shells of their hosts
varies considerably. Some adhere simply, with no detectable effect or marking on
the substrate (Bock and Moore, 1969; Zumwalt and DeLaca, 1980), while others
extensively pit and erode the subsurface layers (DeLaca and Lipps, 1972) and even
penetrate the entire shell to reach the mantle cavity (Todd, 1965).
In this paper we use radiotracer techniques, light and scanning electron micros-
copy, resin casting, and sectioning methods to describe the remarkable morphological
and physiological adaptations of a large Orbitoidacean, Cibicides refulgens, to its spe-
cialized epizoic habit on the free-swimming Antarctic scallop, Adamussium colbecki.
The study site
The study site at Explorers Cove (approximately 77.6 S, 163.5 E), McMurdo
Sound, Antarctica, was used previously by Stockton (1984) and Mullineaux and De-
Laca (1984).
The sediment is homogeneous fine silt mainly deposited from the late austral
summer/early autumn freshwater input to the locality via streams originating from
the Commonwealth and Wales glaciers (DeLaca, unpub. obs.). The virtual absence
of currents (Mullineaux and DeLaca, 1984) prevents any reworking of the sediments
and results in a seasonal accumulation of fine silt. Adamussium colbecki occurs in
densities of up to 90 m 2 and forms 90% of the available hard substrate in the area
(Stockton, 1984); it resides within depressions in the sediment which are caused by
the light, non-locomotory flapping action of its valves (Mullineaux and DeLaca,
1984; Stockton, 1984).
The benthic community of Explorers Cove is thought to resemble the deep sea in
species diversity, abundance of individual organisms, sediment relief, and long-term
stability of temperature, salinity, and oxygen (Dayton and Oliver, 1977). The austral
summer is accompanied by an increase in primary productivity by ice algae living
within the lower 10 cm of sea ice, and is followed in late summer by release of the
algae into the water column from the melting ice. Thus a seasonal pulse of organic
material is contributed to the developing in situ productivity in shallow-water (De-
Laca, unpub. data).
MATERIALS AND METHODS
Living specimens of A. colbecki were collected from 20 to 27 m by scuba diving
through holes blasted in the 3 m thick sea ice, and maintained in an aquarium at in
situ temperature and salinity (- 1 .8°C and 34%o S).
138 S. P. ALEXANDER AND T. E. DELACA
Most mate >r scanning electron microscopy (SEM) was coated with gold
and/or carbon £ ;>ined in a Cambridge Stereoscan MK 2 operated at 20 kV;
images were \\ford FP4 35mm film developed in Microphen.
Scallr-: :pifaunal communities were embedded in Spurr's low viscosity
resjn j Inc.), then ground and polished using carborundum, diamond,
aiK! (,.ie abrasives until the desired plane of section was reached. Azure
jiue, and methylene blue in borax (Richardson et al, 1960) was used
,.:nic material. Other scallop shells, cleared of epifauna by boiling in 20
i. n peroxide, were gradually embedded in Spurr's over three days to pene-
trate the fine cavities of the shell material. After polymerization, the block was frac-
tured along the plain of the shell so that the upper block retained only a thin translu-
cent layer of calcite. Alternating treatments of 0. 1 N HC1 at room temperature, and
3% aq. sodium hypochlorite at 60°C, removed calcite and organic layers, respectively;
the exposed face of the lower block forms a perfect cast of the scallop shell dorsal
surface and the canals and cavities within the calcite itself. Observations were made
with an ETEC Autoscan SEM at the Wadsworth Center for Laboratories and Re-
search, Albany, New York.
For SEM examination of substrate pitting, agglutinated tube morphology, and
pseudopodial deployment, six scallops with epifauna were fixed for 2 h in 6% glutaral-
dehyde buffered with 0. \M sodium cacodylate at pH 7.4. Dorsal valves were rinsed
five times with distilled water, rapidly frozen at -40°C, and freeze dried in an inverted
position. Other C. refulgens were picked from all size classes of A. colbecki (Stockton,
1984) and examined for gross morphology, aperture, and spiral face detail; attach-
ment zones were fractured and the exposed calcite laminae studied for evidence of
pitting or tunneling. Etched (using 0.1 N HC1) and non-etched inner valve surfaces
were examined for perforations.
To determine the rate of substrate pitting and agglutinated tube formation, fora-
minifera were picked from the dorsal valves of A. colbecki, cleaned of extrathalamous
material, and placed in semi-enclosed, transparent plastic chambers attached to areas
of non-pitted dorsal valves from recently killed scallops (50% of which had most of
their microflora removed). It was not possible to use living A colbecki since the frantic
flapping of collected specimens prevented the introduction and subsequent attach-
ment of C. refulgens to the upper valves. Such violent movements are not usual in
the normal habit of A. colbecki (Mullineaux and DeLaca, 1984; Stockton, 1984).
The valves with experimental foraminifers were returned to the collection site for 3
months; the containing chambers did not alter ambient light levels and a loose fitting
lid prevented silt accumulation but permitted exchange of dissolved materials.
ATP analysis (DeLaca, 1986) was used to distinguish living from dead foramini-
fera and to measure foraminiferal biomass. A carbon to ATP ratio of 300 was tenta-
tively assumed for application to ATP values from C. refulgens, since recent work
shows that this ratio is remarkably constant between the two taxonomically distant
rhizopod species, Gromia oviformis (order Testacida) and Astrammina rara (order
Foraminiferida) (DeLaca, 1 986). Cellular nucleotides were extracted with phosphate/
citrate buffer at 100°C, and data were used for normalizing experimental results.
Labeled amino acids (14C) in the same proportions as a typical algal protein hydro-
lysate (Amersham corporation, product CFB.25; see table I), were used to demon-
strate uptake through the pecten shell by individual foraminifers. Plastic containers
(12 ml volume) were sealed to the inner valve surface with silicon vacuum grease,
and the seawater within enriched with 2.5 nCi radio-labeled amino acids at 100 nM
final concentration. This concentration of amino acids is approximately 25 times
lower than that recorded for free amino acids within the extrapallial cavity (see Re-
C. REFULGENS, MORPHOLOGY AND ECOLOGY 139
suits) and 14.7 times greater than at the sediment/water interface (DeLaca, 1982).
Incubations lasted up to four days, and controls with containers on both faces of the
shell (one contained label; the other retained any diffused label) measured leaching
through non-pitted shell and passive diffusion into heat killed (30°C for 30 min),
attached foraminifers. To establish the viability of animals harvested, 10 specimens
from each experimental group were analyzed for ATP content.
To measure influx rates of dissolved amino acids, individual animals were re-
moved from pecten shells, cleared of all extraneous materials, and allowed to recover
from handling for 24 hours prior to experimentation. These animals were incubated
in experimental medium [10 ml filter sterilized seawater (FSSW) with labeled and
unlabeled compounds (depending upon experiment)]. Incubated specimens were
washed in 5-6 serial baths of FSSW (~ 1 min each) until wash water registered no
significant radioactivity over background levels. Influx was determined by measuring
the level of accumulated radioactivity in experimental animals (homogenized in
Aquasol 2) with a Beckman LS 6800 liquid scintillation counter. "Time zero" and
heat killed controls were used.
To measure grazing rates of C. refulgens, epiflora of the dorsal valves were labeled
in situ with [I4C] sodium bicarbonate in light at temperatures between —1.8 and 0°C
for 1 2 hours; individual cleaned and heat killed C. refulgens were placed on these
shells prior to labelling. After incubation, the scallop shells with foraminifers were
washed in serial baths of FSSW until the radioactivity of wash water was not signifi-
cantly over background levels. Twenty individual diatoms, living foraminifers, and
heat killed foraminifers were selected as time-zero samples and extracted in 1 .0 ml of
hot ( 100°C) phosphate/citrate buffer. After removal of 10 ^1 of supernatant for ATP
analyses, the extract was dried and digested with 0.3 ml of 0.2 TV perchloric acid prior
to adding Aquasol 2. Subsequent specimens were sampled at three 6-h intervals, and
similarly processed. Radiation counts obtained from the time zero specimens were
subtracted, and the results used to calculate the number of cells ingested. Experimen-
tal protocols for isolating, washing, and determining 14C uptake by single algal cells
were taken after Rivkin and Seliger (1981).
Suspension feeding was investigated using radiolabeled bacteria and diatoms.
Bacteria were isolated from the sediments of New Harbor and further isolated on
2216 Marine Agar (Difco). Selected cultures were then labeled with [I4C] leucine
(ICN) at log growth in Marine Broth (2216 Difco). Labeled bacteria were washed
free of extraneous label by repeated centrifugation and resuspension in FSSW, until
supernatant radioactivity was not significantly over background levels. Cell concen-
trations were determined with a Petroff-Hauser counting cell, and disintegrations per
cell measured by liquid scintillation using Aquasol 2. Nitzchia cylindricus cultures,
provided by Dr. C. W. Sullivan (University of Southern California), were grown with
Alga-grow media (Carolina Biol. Suppl. Co.) in FSSW and labeled with [I4C] sodium
bicarbonate. These cultures were concentrated and washed on nitex screen and resus-
pended to the desired concentration (measured using a plankton counting cell).
Four dorsal valves of living Adamussium colbecki were removed, and the aggluti-
nated portions of 50 C. refulgens were gently but thoroughly removed from the test,
leaving the foraminifers securely attached to the shells. Two of the shells were main-
tained at temperatures between - 1 .8 and 0°C, and the others were warmed to 30°C
for 30 min (heat killed controls); shells were suspended upside down for 6 h in a
culture vessel with a suspension (maintained with a small stream of air) of labeled
diatoms in seawater. This configuration was duplicated to measure bacterial capture.
At to and hourly intervals, samples of both diatom and bacterial suspensions were
taken (by centrifugation and filtration with nitex screen, respectively) to check for
140 P ALEXANDER AND T. E. DeLACA
dissolved label a asure cell concentration. Following incubation, 30 foramin-
ifers with <, -"s, and 30 foraminifers without, were detached, cleaned
of extranet rial i. if necessary), washed through 10 serial washings of FSSW,
extractec phosphate buffer, and processed as described above.
were measured using light microscopy. Approximate cell vol-
umes -'<ed by appropriate geometric formulae corresponding to the cell
content was also calculated (Strathmann, 1967).
/olumes of fluid were sampled from the extrapallial cavity of A. colbecki
een the mantle and inner surface of the shell) by passing a blunt cannula at-
iied to a syringe through a window cut in the 0.5-0.7 mm thick shell. Separation,
identification, and quantification of free amino acids in selected samples were accom-
plished using high pressure liquid chromatography (HPLC) after fluorescence derivi-
tization with ortho-pthaldialdehyde (see Stephens, 1982).
RESULTS
The dorsal surfaces ofAdamussium colbecki living in sedimentary environments
are encrusted with attached foraminifera (Fig. 1) including C. refulgens. The force
required to dislodge an individual of C. refulgens from the surface of a bivalve shell
increases with size of the individual, as does the extent and depth of substrate erosion.
Juvenile C. refulgens (Fig. 2) are easily dislodged using a fine needle, whereas adult
specimens (Figs. 3-6) must be pried off with a stout microprobe. A random sample
of shell surfaces under Cibicides refulgens demonstrated that only 45% (n = 200 on
each of 5 shells) of foraminifers had caused etching. Similar sampling near the umbo
revealed that 92% of the foraminifers resided in etched concavities whereas only 12%
of those foraminifers nearer the shell margins were attached over etched shell (100
foraminifers examined on each of 5 shells). No significant etching was detected after
three months on shells artificially infested with C. refulgens (Fig. 7).
A progression in the extent of substrate pitting caused by increasing sizes of fora-
minifers and the age of the shell is clearly visible using a dissecting microscope. Use
of the SEM demonstrated that pits caused by younger C. refulgens generally extend
no deeper than the uppermost laminae of the valve. At this stage the striations visible
on the shell surface (Stockton, 1984) and the smaller perpendicular 'ribs' connecting
them (Fig. 8) are removed completely in the area of the pit, and the exposed calcite
is eroded to appear as irregular granules with multitudinous 'micro-canals' (Figs. 9,
10). Further pitting results in enlargement of the microcanals to form distinct canal
openings which penetrate several calcite layers, and are to some extent guided by
planes of weakness within, or between, the layers (Figs. 11, 13). This phenomenon is
dramatically illustrated by resin casts which show the pit erosion in reverse, produc-
ing a 'cathedral effect' from the pattern of channels within the scallop shell material
(Fig. 12). The distributions of canal openings within the substrate pits as a whole do
not exhibit any noticeable pattern.
The bond between the test wall of the umbilical face of C. refulgens and the upper-
most calcite layer in the pit is sufficiently strong so that when a specimen is forcefully
detached from the shell, a layer of shell material will often remain attached to the
foraminifer. It is then possible to observe deeper canals ramifying through the shell;
these canals are generally fewer in number than the more superficial canals, but are
larger in diameter ( 10-14 ^m) and more conspicuous.
Fracturing a scallop shell directly through a substrate pit allows for detailed SEM
study of groups of canals in the middle and lower layers (Fig. 22). Scanning electron
micrographs (Figs. 13, 14, 16) demonstrate conclusively that the canals do not funda-
C. REFULGENS, MORPHOLOGY AND ECOLOGY
14
• *"
FIGURE la. The free swimming Antarctic scallop Adamussium colbccki with characteristic epizoic
growth. Conspicuous attached faunal components include the agglutinated tube of a large polycheate,
hydrozoans, bryozoans, and commonly four or more species of benthic foraminifera. The most abundant
and conspicuous species is Cibicides refulgens with its agglutinated tubes. Scale bar = 1 cm. b. Oblique
view of the dorsal valve of A. colbecki with attached C. refulgens and associated agglutinated tubes reaching
into the overlying water. Vertical tubes may extend to 5 mm and exhibit three orders of branching. Scale
bar = 5 mm.
mentally follow lines of weakness within the shell, and therefore it appears that the
foraminifer's cytoplasm can control both the extent and direction of the dissolution
process. The thick resin cross-sections of C. refulgens attached to the valve surface
revealed many visible canals extending from the base of the pit through most of the
calcite layers perpendicular to the plane of the laminae (Figs. 17, 18). However as a
142
S P. ALEXANDER AND T. E. DELACA
FIGURE 2. Juvenile Cibicides refulgens attached to surface of A. colbecki dorsal valve. A rudimentary
peripheral agglutinated tube has been built (white arrow). Vertical tubes are not present. Two juveniles
have been removed to show the shallow surface etching of the shell (black arrows). Scale bar = 163 /mi.
FIGURE 3. Plan view of an attached adult with a well developed agglutinated tube system (large
arrows) and net of pseudopodia on the scallop shell surface (small arrows). Scale bar = 200 ^m.
FIGURE 4. Oblique view of specimen in Figure 3. Pseudopodia (small arrows) can be seen traversing
the space between the agglutinated tubes (large arrows) and the substrate. Scale bar = 143 Mm.
FIGURE 5. Detail of Figure 3 showing composition of agglutinated tubes and the presence of a fine
pseudopod (arrows) radiating away from the foraminifer, across the shell surface. Scale bar = 102 /nm.
FIGURE 6. Oblique view of two attached adult Cibicides refulgens. An agglutinated tube can be seen
clearly raised away from the scallop shell surface (arrow). Scale bar = 1 54
C. REFULGENS. MORPHOLOGY AND ECOLOGY 143
result of the curvature of the canals, and the limited depth of field, it was not possible
to photograph a single element traversing the complete shell thickness without inter-
ruption. The canal walls are significantly smoother than the adjacent calcite exposed
at the fracture zone (Figs. 16, 22), but there is no evidence of an actively secreted
lining.
The inner valve surface is generally lined with overlapping, angular, tile-like cal-
cite crystals (Fig. 23), between which are many naturally occurring pores leading to
the lamina beneath (Fig. 24). Upon careful scrutiny of this inner layer in the SEM,
circular areas (approximately 15-50 ^m in diameter) with significantly enlarged
pores (Fig. 23) are evident marking the area above which an individual foraminiferan
is attached on the outer valve surface. After etching for 5 to 10 minutes with 0.1 TV
HC1, the outermost calcite layer is removed and those salient markings are revealed
more clearly (Figs. 19, 20). High magnification detail shows them to be closely spaced
canal openings (Fig. 2 1 ), and there remains little doubt that these openings are contin-
uous with the canals which originate in the surface pit, and penetrate deep into the
bivalve's shell.
Examination of the exposed face of an adult C. refulgens detached from the sub-
strate (Fig. 25, 26) reveals that the spiral face is not adhered to the shell material over
its complete area due to a pattern of grooves radiating from the primary aperture to
the peripheral test margins. The roof of each groove is the spiral test face, and the
floor is formed by the etched shell material of the pit floor. Typically, four to five such
grooves of approximately 30 to 50 nm width connect areas immediately adjacent to
the primary aperture (Figs. 25, 29) with those more remotely situated on the opposite
test margins. Etched bivalve shell which forms the base of the grooves often exhibits
a pattern of fine channels (5-10 /urn wide) running parallel with the main trend of the
groove (Fig. 30), giving the impression of the streamlines oriented with the direction
of the main cytoplasmic flow within the grooves. In addition, the lumina of the radial
grooves are continuous with that of the peripheral tube encircling the test at its point
of contact with the substrate (Figs. 25, 26).
Cibicides refulgens secondarily forms an elaborate agglutinated tube system
around, and extending from, its attached test. The agglutinated tube system typically
is comprised of: ( 1 ) a peripheral tube encircling most, if not all, of the lateral test
margin at the point of contact between it and the substrate (Figs. 2, 3, 25, 26), and
(2) radial tubes attached to the substrate and test, extending over the shell surface and
vertically away from it (Figs. Ib, 3, 4, 27). These tubes often form several branches
(Fig. 3).
All radial tubes originate from the peripheral tube, either dividing from it without
any observable thickening, or arising from a distinct node at a particular point. Tubes
extending horizontally along the shell surface and vertically into the overlying water
may branch from the same nodes. There is no obvious organization of the branching
patterns of C. refulgens. Typically, C. refulgens has from 1 to 6 (x == 3, n = 50) aggluti-
nated tubes extending vertically up to 5 millimeters (x ---- 2.5, n ---- 50) from their
points of origin at the peripheral tube. Vertical tubes may exhibit up to three orders
of branching, and tube diameter does not vary consistently with length or distance
from the test/substrate; thickenings or nodes can occur at any point along a tube. The
interior tube surface is smoother than the outer surface (Fig. 26), and in freeze dried
specimens it is partially covered with a layer of cytoplasm.
Intact tubes, when viewed in the SEM, do not show clearly defined apertures;
openings are inferred by the presence of pseudopodia which extend from many points
along the tubes to either the shell surface, test surface, or other tubes. Apparent aper-
144
S. P. ALEXANDER AND T. E. DeLACA
••'
. Ti
• . . .w».i£
It; • -;,%:
FIGURE 7. Substrate markings caused by an adult Cibicides refulgens after three months of attach-
ment. There is no visible etching of the shell surface, but adhesion was great enough to break away some
test material upon removal of the foraminifer. Scale bar = 200 /urn.
FIGURE 8. 'Early stages' of substrate pitting. The striae of the scallop shell have been removed, and
from two to three laminae have been eroded. There is no evidence of boring to form canals. Scale bar
= 110 urn.
FIGURE 9. Detail of peripheral region of an early stage pit. The surface lamina has been etched away
(lower left) and the calcite beneath has been partially eroded to form fine, angular granules. Scale bar = 1 1
/urn.
C. REFULGENS. MORPHOLOGY AND ECOLOGY 145
tures such as that shown in Figure 3 1 are caused by tube breakage during collecting
or transport of the scallops.
Removal of specimens from the water causes the vertical tubes to collapse against
and adhere to the substrate, forming what then appears to be a system of surface tubes
which resemble polycheate worm tubes. However, cytologically fixed and freeze dried
tubes are able to partially support themselves thereby almost maintaining their natu-
ral positions (Figs. 4-6), and thus facilitating examination in the SEM.
The walls of all tubes are clearly agglutinated and comprise fine (silt- and clay-
sized) mineral particles, diatom frustules, fine organic detritus, and occasional sponge
spicules (Figs. 5, 26-28, 3 1 ). The cementing material is not clearly distinguished from
the agglutinated particles and does not cover their outer surfaces. Wall thickness var-
ies considerably but is generally from one to four particles thick with no evidence of
layering or selection of specific particle size for construction. Particle recruitment
by foraminiferal cytoplasm during tube construction seems to be dependent on the
availability of sedimentary material on the scallop shell surface. Similarly, the incor-
poration of specific diatom frustules into the tubes is related to the dominant flora
growing upon the scallop shell, and perhaps, the diet of the foraminifer.
Extrathalamous cytoplasm and pseudopodia were studied for gross morphology
using both living and freeze dried specimens attached to pectin shells. An extensive
pseudopodial net was observed spread over most of the shell surface in areas densely
populated by C. refulgem (Fig. 32). The dorsal test surface of C. refulgens usually is
partially covered with cytoplasm in the form of tangled strands (Figs. 32, 33). From
this, randomly branching and anastomosing networks of pseudopodia emanate and
connect with neighboring tests, agglutinated tubes, and/or clumps of algal or detrital
material. Trunk pseudopods are usually found closer to the substrate, originating
from peripheral or vertical agglutinated tubes, and traversing portions of the shell
while remaining suspended above it (Figs. 3, 4, 5). Fine pseudopodial elements
branch at apparently random points along trunk pseudopods and connect with others
nearby (Fig. 34 ), or attach to the substrate beneath. These elements often merge with
a finer net system attached to the substrate at raised points such as striae, and spread
over most of the shell surface in the vicinity of the adult C. refulgens (Fig. 35). The
rectilinearity and patterning of elements forming the fine nets and the limited extent
of sagging when they are bearing dense mineral particles is indicative of tension
within the system. The larger trunk pseudopods often visibly sag when crossing spaces
between neighboring foraminifers and clumps of detrital material.
Vertical agglutinated tubes also give rise to trunk pseudopods and finer branching
elements suspended freely in the water space immediately surrounding and above
the tubes. Relative movement of the water in this space causes the pseudopods to
bend and wave freely, demonstrating extreme flexibility in response to water
movement.
All of the pseudopodia have a sticky quality when touched with single hair brushes
or steel microprobes; once adhered they stretch considerably under tension before
breaking. Diatoms, sedimentary particles, and organic debris are commonly observed
attached to pseudopodia (Figs. 36, 37), and large clumps of detrital material were
FIGURE 10. Detail of pit base in Figure 9. Note the fine 'pores' visible between the angular calcite
granules. Scale bar = 2.8 ^m.
FIGURE 11. A well developed substrate pit (bottom half of micrograph) with characteristic deep
borings visible (arrows) penetrating several laminae of the scallop shell. Scale bar = 62 urn.
146
S. P. ALEXANDER AND T. E. DnLACA
FIGURE 12. A resin cast of the central portion of a substrate pit formed by an adult Cibicides reful-
gens; the raised central area represents channels within the scallop shell which were originally occupied by
foraminiferal cytoplasm. Scale bar = 20 nm.
FIGURE 13. Irregular etching of calcite at the pit edge. A bored hole in upper calcite layer (X) has
been undercut by subsequent dissolution of lower layers (arrow). Scale bar = 3 1 nm.
FIGURE 14. Peripheral area of a deep substrate pit showing transition from scallop shell surface
(bottom right) to extensively etched pit base (top left) and a circular vertical boring (X). Scale bar = 18 Mm.
FIGURE 1 5. Transition from normal scallop shell surface (left) to a deep pit (right) eroded by a large
adult Cibicides refulgens. Removal of the foraminifer has torn away the uppermost calcite layer, exposing
C. REFULGENS. MORPHOLOGY AND ECOLOGY 147
often observed suspended above the substrate within pseudopodial nets. Such inclu-
sions may be partially engulfed by cytoplasm and/or suspended by a pattern of reticu-
lar 'subnets' formed between main pseudopodial elements (Fig. 36).
Figure 40 presents the results of three experiments to further examine the sources
of particulate organic material used as a nutrient source by Cibicides refulgens.
Though patchy in distribution, benthic diatoms (primarily Cocconeis sp. approxi-
mately 1 5 /urn in length) represent a potentially significant resource to grazing organ-
isms living on the surface of the bivalve. Time course studies monitoring the corn-
sumption of radio-labeled benthic algae by C. refulgens demonstrated average grazing
rates of 54.5 diatoms mg~' h ' (n = 60, min = 14, max =: 1030). The relatively large
differences in uptake rate can be accounted for by the proximity of the foraminifer's
attachment site to benthic diatoms on the surface of the bivalve shell. Two other
experiments examined the rate of capture of suspended bacteria and diatoms. These
experiments were additionally designed to determine the relative importance of the
agglutinated tubes in suspension feeding. In both of these experiments half of the
attached foraminifers were cleaned of all agglutinated tubes to evaluate the impor-
tance of these structures to suspension feeding efficiency. While suspended cultures
ofNitzchia cylindricus (5-10 fj.m at concentrations of 8 X 104 cells ml"1) were taken
at rates of 153 cells mg ' h~' (n = 15, min. = 82, max. = 284) by foraminifers with
their arborescent agglutinated tube structures intact, those without this superstruc-
ture averaged rates of only 62 cells mg"1 h"1 (n = 15, min. = 32, max. = 141). Sim-
ilarly, suspended bacteria (unidentified gram negative rods 1.2 X 2 ^m at 2 X 106
cells ml"1) were consumed in greater numbers by C. refulgens with agglutinated tubes
(x = 4.2X 102 cells mg"1 h"',n == 15, min - 1.1 X 102, max. = 6.7 X 102) than those
without agglutinated tubes (x = 69 cells mg"1 h"1, n = 15, min. = 21, max. = 1.7
X 102).
The discovery of pronounced etching channels penetrating through the shell
clearly placed foraminiferal cytoplasm in contact with the nutrient-rich extrapallial
space formed between the mantel and the inner surface of the shell, and suggested a
parasitic relationship. Our studies using radio-labeled amino acids demonstrated the
0.5-0.7 mm thick unetched bivalve shell is not permeable to free amino acids. How-
ever, when the inner surface of the bivalve shell opposite attached C. refulgens was
bathed with radio-labeled amino acids (100 p.M), the foraminifers consistently be-
came radioactive within a few hours. These experiments were duplicated with heat
(30°C for 30 min) killed C. refulgens and no radioactivity was detected.
Figure 39 presents the uptake of uniformly 14C labeled mixture of amino acids
from seawater at various concentrations. This curve is clearly hyperbolic and suggests
that the transport system for amino acids in Cibicides refulgens can be described by
the Michaelis-Menten equation. The data have therefore been analyzed by a Hanes-
Woolf plot (where substrate concentration divided by rate of influx is plotted against
substrate concentration). As shown in Figure 40, Jmax is 3.59 X 10"3 ^moles mg"1
(wet weight of protoplasm) IT1 and the Kt (substrate concentration at which the rate
of uptake = Jmax/2) is 10.43 \iM.
Analysis of the fluid filling the extrapallial cavity were conducted using high pres-
unetched laminae beneath and canals penetrating deeper into the shell material (arrows). Part of the agglu-
tinated peripheral tube remains secured to the shell surface (large arrowheads). Scale bar = 36 nm.
FIGURE 16. Detail of Figure 1 5 demonstrating the distinct canal borings (X) in shell material beneath
the attached foraminifer. Scale bar = 6.7
148
17
S. P. ALEXANDER AND T. E. DeLACA
FIGURE 1 7. Thick cross section through a resin-embedded adult Cibicides refidgens attached to Ad-
amussium colbecki. Groups of canals are discernible originating from the base of the pit and passing
through most of the shell thickness (arrows). Scale bar = 200 ^m.
FIGURE 18. Detail of Figure 17 showing the canals to be continuous through to the inner-most
laminae of the scallop shell (arrows). Scale bar = 480 ^m.
FIGURE 19. Acid etched inner shell surface with foraminifers attached to the opposing face. Each
mark (arrows) corresponds to groups of canals penetrating the shell from the surface pits above. Scale bar
= 250 /im.
C. REFULGENS. MORPHOLOGY AND ECOLOGY 149
sure liquid chromatography. The results (Table I) revealed concentrations of 2527
nM (2.527 mM) free amino acids with extremely high concentrations of glycine
(2066.3 /uM).
DISCUSSION
The rarity of loosely attached or 'roaming' C. refulgens on the surfaces of scallop
shells strongly suggests that the sessile habit is preferred by this species. The poorly
eroded pits beneath juveniles and the extensive pits associated with adults, leads to
the assumption that pitting progresses with growth at least until the adult stage is
reached (data on the life span of C refulgens are unavailable). Specimens experimen-
tally placed on a previously unmarked scallop shell became firmly attached to the
substrate and began to construct agglutinated tubes. However, the lack of significant
etching after three months raises several interesting questions: is this the typical rate
of etching which would be observed by those specimens attached to live A. colbecki?
Alternatively, is it significantly lower in response to the absence of specific cues? We
have demonstrated that amino acids do not normally leach through the shell mate-
rial, thus it seems unlikely that this would act as a cue to initiate excavation; cues
could conceivably come from a variety of stimuli, such as rates of sediment accumu-
lation on the shell surface, the presence or absence of organic materials from scallop
excretion, and the presence/absence of water movements over the shell surface. Ex-
tensive further studies are required to investigate the role (if any) of environmental
cues in initiating substrate erosion by C. refulgens.
Parasitism
Our investigations have shown that 50% of attached C. refulgens significantly
erode the surface of the scallop's shell and excavate channels to the extrapallial cavity.
Though the scallop shell normally is not permeable to dissolved amino acids, radiola-
beled studies have consistently shown uptake of amino acids by attached foramini-
fers. This uptake could only have been mediated by pseudopodia penetrating through
the shell. The nutritional significance of dissolved amino acids to several marine in-
vertebrate species has been discussed by other workers (Southward and Southward,
1972; Stephens, 1981) including foraminifera (DeLaca el ai, 1981; DeLaca, 1982).
Cibicides refulgens has the ability to absorb free amino acids at relatively low sub-
strate concentrations (K, = 10.43 nM). However, concentrations of free amino acids
within the extrapallial space are more than two hundred times higher (2527 nM,
[2.527 mA/]) than the half saturation concentration (Kt). Therefore, a logical assump-
tion that the foraminifer has little difficulty realizing its maximal rate of influx (Jmax
= 3.59 X 10 3Mg, mg~1,h"')from the scallop, and presumably this source of material
would be available to the foraminifer year-round.
A wide range of associations between individuals of different species in which one
FIGURE 20. Detail of Figure 1 9 showing salient features directly beneath a substrate pit on the oppos-
ing shell surface. Scale bar = 60 ^m.
FIGURE 21. Detail of Figure 20 showing fine perforations present in laminae exposed by etching
with HC1. Scale bar = O.9 /mi.
FIGURE 22. Detail of canals exposed during fracture of the shell through a pit. These canals are
approximately midway through the dorsal shell material and contain precipitated material, most probably
cytoplasm. Scale bar = 23
150
S. P. ALEXANDER AND T. E. DeLACA
FIGURE 23. Inner surface of dorsal scallop valve showing marking which is often observed when the
opposing surface is heavily colonized with Cibicides refulgens. Scale bar = 6.2 nm.
FIGURE 24. Detail of Figure 23 demonstrating enlarged 'pore' between calcite plates (X) and numer-
ous 'micropores' located peripherally (arrows). Scale bar = 1 .0 ^m.
FIGURE 25. Umbilical view of an adult Cibicides refulgens removed from the valve surface. The
primary aperture (arrow head) opens into (a) the lumen of the peripheral agglutinated tube (dashed line)
and (b) grooves created between the umbilical face and the base of the pit (dotted lines). Scale bar = 150
C. REFULGENS, MORPHOLOGY AND ECOLOGY 151
or both derive benefit from the other have been described in the literature. They range
from being obligate to being facultative (each partner being able to live without the
involvement of the other), and the grades of association within this range often are
not distinct. For convenience the relationships are frequently termed commensal and
parasitic. By definition, a parasite always lives to the detriment of its host. Parasitic
life styles are frequently specialized and lead to development of morphological as well
as physiological adaptations which ensure efficiency. The relationship between C.
refulgens and A. colbecki is very similar to that described by Todd ( 1 965 ) for Rosalina
carnivora and Lima angolensis. Unlike Todd's work however, the present study pres-
ents unambiguous evidence that C. refulgens does derive nourishment from the man-
tel of its host. Whether the cumulative affects of approximately 900 attached C. reful-
gens (~400 [45%] of which may have created channels through the shell) have a
detrimental affect on the bivalve in this marginal environment remains unknown,
but seems likely.
Grazing
Morphological test elaboration in the form of a constructed horizontal tube sys-
tems on the scallop shell surface effectively increases the distance that pseudopodia
can gather food without severely increasing risk of cytoplasmic loss to predation or
other causes. For example, we have observed tanaid crustaceans living in tubes on the
scallop shell and feeding on unprotected cytoplasm of extended pseudopodia from C.
refulgens. Of course, cytoplasm within the agglutinated tubes of the foraminifer is
contiguous with cytoplasm in the lumen of the last formed chamber and thus the
tubes are regarded as an extension of that chamber. Whereas most calcareous fora-
minifera are compelled to withdraw all extrathalamous cytoplasm into the test when
unfavorable conditions or predators are encountered, C. refulgens individuals need
only withdraw pseudopodia into the agglutinated tubes for protection. Thus the total
volume of cytoplasm deployed, and therefore the total area grazed, is vastly increased
without much risk of cytoplasmic loss. Most calcareous foraminifera use only the
cytoplasm present in the last formed, penultimate and sometimes the antepenulti-
mate chamber for pseudopodia and extrathalamous cytoplasmic activity (Anderson
and Be, 1978; Anderson, 1983; Alexander and Banner, 1984; Alexander, 1985), and
therefore may gather food at limited distances from the test without considerable risk
to cytoplasm.
Adamussium colbecki shells typically are colonized by benthic diatoms and bacte-
ria, but their concentrations, diversity, and percentage of surface coverage, however,
vary from specimen to specimen. This heterogeneity is typical in New Harbor both
spatially and temporally on large and small scales, and organic productivity in this
portion of McMurdo Sound is extremely seasonal; our observations indicate that
pronounced shallow-water productivity may be limited to as little as 2'/2 months.
(DeLaca, unpub. data). Although approximately six months of continuous sunlight
FIGURE 26. Detail of Figure 25, demonstrating continuity of the primary aperture (arrow head),
with the lumen of the peripheral agglutinated tube (arrows) and that of a vertical agglutinated tube (V).
Scale bar = 67 ^m.
FIGURE 27. Surface morphology of a typical agglutinated tube (in this case, radial and in contact
with the substrate). Scale bar = 67 /urn.
FIGURE 28. Detail of typical agglutinated material forming tube walls. Arrows = diatom frustules.
Scale bar = \2
152
S. P. ALEXANDER AND T. E. DELACA
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" ->-• r> ' ~''V: '
' * . ' ^* "^ - V JW **•
. •** -+ * ~
FIGURE 29. Detail of Figure 25 showing a radial umbilical 'groove' (G) which exists between the
substrate surface in the pit and the umbilical test wall. A = primary aperture; C = Calcite broken away
from pit base. Scale bar = 48 ^m.
FIGURE 30. A typical eroded 'channel' (Ch) commonly observed on calcite which forms the base of
umbilical 'grooves' in attachment pits. Scale bar = 4.2 jum.
FIGURE 31. Detail of agglutinated tubes (T) formed by juvenile Cibicides refulgens in Figure 2. D
= Diatom; W = test wall of last formed chamber. Scale bar = 18 ^m.
FIGURE 32. Oblique view of adult Cibicides refulgens attached to dorsal scallop valve. An extensive
net of pseudopodia (arrow heads) is visible over the substrate and extending from the dorsal test surface
(arrows). Scale bar = 83
C REFULGENS. MORPHOLOGY AND ECOLOGY 153
is available annually, the combination of low angle of incident radiation, sea ice, and
snow cover reduces the period of primary' productivity further (see Dayton and Oli-
ver, 1977).
Benthic diatoms on the scallop shell surface, as well as amorphous organic mate-
rial and sediment, were attached to and transported by pseudopodia. Our experi-
ments using in situ l4C-labeled attached, and motile benthic diatoms, demonstrate
that the foraminifers graze upon the naturally occurring 'lawns' of algae, and that
relatively high numbers (x = 54 diatoms mg~' h~') are consumed. Through the ap-
proximation of biomass and conversion to carbon content (Strathmann, 1967), it is
estimated that if rate of harvest remained constant, those benthic diatoms would have
contributed approximately 1 X 10~3 Mg C mg~' h '. That value is approximately one-
half the amount of carbon obtained through the uptake of dissolved amino acids;
thus grazing microorganisms from the surface of the scallop shell appears to be an
important factor in C. refulgem nutrition. The discorbid foraminifer, Rosalina globu-
laris, also forms deep pits in its preferred substrate (DeLaca and Lipps, 1972), and
grazes upon algae in the immediate vicinity; however, in conditions of low food con-
centration it roams in search of algae (Sliter, 1965). In view of the patchy distribution
of algae on A. col beck i, the selection of a sedentary habit by C. refulgens seems to
have potentially reduced its grazing abilities. This disadvantage is more than compen-
sated for by the permanent, or semi-permanent attachment between the scallop shell
and the foraminifer, which virtually eliminates the risk of being swept off the shell
during swimming movements of the scallop, and enables further morphological and
physiological adaptations to the epizoic habit.
The radial grooves existing between the spiral side of the foraminifer' s test and the
surface of the pits increases the efficiency of this greater cytoplasmic volume through
provision of a short line of communication between the primary aperture and the
lumen of the peripheral tube on the opposite test side; this facilitates rapid exchange
of cytoplasmic organelles and inclusions such as mitochondria, phagocytosed mate-
rial, and energy substrates between the most distal pseudopodia and the intrathala-
mous cytoplasm. These internal-external lines of communication between deeply
situated cytoplasm and the external milieu, are considered important in foramini-
feran cell systems (Brasier, 1982).
Suspension feeding
Using the vertical agglutinated tubes as conduits for streaming pseudopodia and
as anchors for pseudopodial nets, C. refulgens exploits suspension feeding as a third
trophic mechanism. Figure 38 depicts the most typical arrangement of pseudopodia
in an undisturbed living specimen; free pseudopodia are not rigid structures, but yield
to water movement. Pseudopodial nets are randomly arranged and thus form a wide
range of mesh sizes. Construction of the nets is initiated by the extension of pseudopo-
dia from apertures along the vertical tubes, followed by contact with other tubes or
nearby structures, and elaboration through bi-directional cytoplasmic flow. While
unsupported pseudopodia of C. refulgens also have been seen projecting into the
water, the construction of an agglutinated tube system provides scaffolding for further
suspended pseudopodia within the water column, as well as a reservoir of protected
FIGURE 33. Dorsal test surface (TS) of an adult Cibicides refulgens showing cytoplasmic strands
(arrow heads) reaching to adjacent detrital material (far right). Scale bar = 3.2 nm.
154
. P. ALEXANDER AND T. E. DELACA
FIGURE 34. Trunk pseudopodia (TP) crossing the scallop shell surface and radiating away from a
large adult Cibicides refulgens. Fine pseudopodia can be seen branching from the main trunk and attaching
to the substrate (arrows). D = diatoms. Scale bar = 16.6 /on.
FIGURE 35. Finely branching pseudopodia (arrows) forming a net above the substrate. Detrital mate-
rial (De) and diatoms (D) are entrained by pseudopodia. Scale bar = 1 7.2 ^m.
FIGURE 36. Diatom (D) and attached detritus suspended above substrate by a fine anastomosing
pseudopodia (arrows). Scale bar = 6.9 ^m.
FIGURE 37. Cytoplasm (Cy) of pseudopod which has adhered to several diatoms (D) on the substrate
surface. Note fine cytoplasmic threads (arrow heads). Scale bar = 8
C REFULGENS, MORPHOLOGY AND ECOLOGY
155
39
1
J.
3.0-
2.0-
1.0-
40
-3
I
K =10.43/11 M
3 300 1
200
100
10
50
100
.y E
•5 2
c ^
<U ^
CD D
11
£.2
5 Q
-o
c
FIGURE 38. Cibicides refulgens attached to the shell of Adamussium colbecki, with pseudopodia
deployed from agglutinated structures. (Not to scale.)
FIGURE 39. Velocity of uptake as a function of the concentration of dissolved amino acids in seawa-
ter. The values for Jmax (ngrams/mg h) and K., were obtained from a Hanes- Wolff linear transformation of
the data. Each point is the mean of 10 replicates; bars represent the range of measurements.
FIGURE 40. Numbers of bacteria or diatoms taken (organisms/mg h) by C. refulgens through grazing
(first bar) or suspension feeding. All agglutinated material was removed from half of the attached foramini-
fers (diagonal lines) while the remaining subpopulation was undisturbed (stippling) in order to determine
if suspension feeding was facilitated by the agglutinated test extensions. Bar heights represent mean values
(15 replicates for each suspension experiment and 60 replicates for the grazing experiment); error bars
depict the range of values for each experiment.
cytoplasm. Agglutinated tubes radiate away from the substrate and have been mea-
sured to heights of five millimeters, and pseudopodia have been measured to extend
an additional three millimeters from the tips of these tubes. The resulting canopy of
branching tubes and pseudopodia potentially increases the volume of water available
to suspension feeding by a factor of 10 to 20. Suspension feeding efficiency is further
enhanced by the near proximity of other foraminifers and their tubes.
Our experiments using radio-labeled prey demonstrated that C. refulgens cap-
156 S. P. ALEXANDER AND T. E. DELACA
TABLE I
Free amino acu!> />.' />'< extrapallial space o/Adamussium colbecki
Concentration of A A
in extrapallial cavity Percent AA in
nM 14C protein hydrolysate*
87.1
9.3
! ne
165.5
6.3
Asparagine
11.0
0.0
Glycine
2066.3
4.6
Histidine
0.0
4.0
Phenylalanine
26.5
6.7
Proline
—
5.6
Serine
76.8
4.8
Tyrosine
11.8
3.6
Glutamic acid
24.1
11.8
Valine
13.1
6.8
Isoleucine
8.9
4.8
Leucine
12.7
11.8
Lysine
17.3
5.1
* An analysis given by Amersham Corporation for its product CFB.25 ([U-'4C]algal protein hydroly-
sate).
tured both suspended diatoms and bacteria, and that capture efficiency was enhanced
by agglutinated tubes by factors of 2.5 and 6. 1 for diatoms and bacteria, respectively.
It is relevant to note here that to facilitate the experiment, concentrations of both
suspended food sources were higher than the foraminifers would experience
naturally.
Several basic mechanisms of filter feeding are involved in systems such as that
used by C. refulgens. According to Rubenstein and Koehl (1977), true sieving would
not be an important contribution to particle capture in the nets of C. refulgens be-
cause the average mesh size far exceeds the diameter of particles most likely to be
encountered in the fine detritus of New Harbor. 'Direct interception' of particles by
the sticky pseudopodia would form a large part of the filtering process, as would
'motile particle deposition' of, for example, motile algae and bacteria. 'Gravitational
deposition' of particles resuspended by the non-locomotory flapping motion of the
scallops, is likely to provide an important source of captured material. The require-
ment for water movement to allow filter feeding through the pseudopodial sieve must
be satisfied almost entirely by movements of scallops, either locomotory (which oc-
curs infrequently; see Mullineaux and DeLaca, 1984), non-locomotory, or by low
velocity localized turbidity currents observed by DeLaca et al. (1980).
How material is entrapped by pseudopodia of C. refulgens and transported to the
agglutinated tubes or the cell body, has not been previously investigated. However,
detailed information is available on the ultrastructural aspects of particle/prey entrap-
ment and transport by pseudopodia of the Antarctic foraminifer Astrammina rara
(see Bowser and DeLaca 1985a, b; Bowser et al., 1986), and Allogromia sp. (Bowser
and McGee-Russell, 1982). In Allogromia sp. attachment is mediated by 'ultrami-
crospikes' and 'ultramicrowebs' which possess special adhesive properties (Fig. 3, in
Bowser and McGee-Russell, 1982; McGee-Russell et al., 1982). Structures almost
identical to the ultramicrowebs of Allogromia sp. were observed in C. refulgens. These
structures were found at the points of pseudopodial bifurcation, and in areas where
particles were suspended (see Fig. 37) by the pseudopodia. That these structures sur-
C. REFULGENS, MORPHOLOGY AND ECOLOGY 157
vived the crude freeze-drying techniques available to us in Antarctica, without low
calcium treatment and critical point drying, suggests that they may be even more
extensive than our results indicate. These two taxonomically distant species appear
to employ a similar mechanism for particle entrapment.
Suspension feeding has been reported for a number of benthic foraminiferal gen-
era, most of which possess elevated stalk-like tests anchored at one end to the sub-
strate (See Christiansen, 1971; and Lipps, 1982; 1983 for reviews); in addition, two
species of the benthic rotaliid Elphidium have been observed with three-dimensional
pseudopodial networks extending into the seawater medium, which would be effi-
cient collectors of free-floating food particles (Jepps, 1942; Sheehan and Banner,
1972). In addition, the arborescent foraminifer Notodendrodes antarctikos DeLaca,
living in New Harbor, captures bottom sediments which are brought into suspension
by activities of larger benthic invertebrates (DeLaca el ai, 1980) and, as with C. reful-
gens, this specialized mode of feeding is regarded as being an adaptation to an unusual
oligotrophic environment. The availability of resuspended organic material could
provide a more consistent source of food during the dark austral winter when benthic
diatom productivity is low, and as such would compliment nutrients obtained by
other mechanisms such as the uptake of free amino acids from the sediment and
surrounding seawater in the case of N. antarctikos, and parasitism in the case of C.
refulgens.
The incorporation of agglutinated material into the test of calcareous foraminifera
is an uncommon phenomenon which appears to be restricted to the suborder Milio-
lina. Within the family Miliolidae, three genera (Sigmoilopsis, Ammomasilina, and
Schlumbergerind) reportedly have test walls composed of agglutinated material
bound by a calcareous cement; the genus Denstostomina has an external agglutinated
layer of grains (Loeblich and Tappan, 1964). Within the Nubecularidae, Nubeculina
has much coarse agglutinated material on the exterior of its chambers, and Nodobacu-
laria incorporates occasional sand grains into the test (opera, cita.). However, among
the suborder Rotaliina, test construction involving both agglutinated and calcareous
material is rare. Nyholm ( 1 96 1 ) described a coniform stage of Cibicides lobatulus
(which he regards as having developed from a zygote) with associated "tube-shaped
structures composed of agglutinated material" extending vertically from the apex of
the test, or occasionally, basally branching and leading from the aperture. Nyholm
( 1 96 1 ) noted that an interspace of a few microns exists between the cytoplasm and
the agglutinated wall of the coniform test; this may be morphologically analogous to
the lumen of the circular agglutinated tube at the periphery of the test in C. refulgens.
Indeed, regardless of which part of C. lobatulus ' life cycle these agglutinated tubes are
associated, it is apparent that they are, in some respects, structurally similar. Nyholm
(1961) did not suggest a function for the agglutinated tubes which project into the
water above the attached sarcode (although he states that the agglutinated coniform
test determines the form of the outer calcareous chambers), but in light of our findings
with C. refulgens, it is tempting to speculate that they are also concerned with the
deployment of pseudopodia into the surrounding water as a mechanism for suspen-
sion feeding.
The taxonomic and phylogenetic significance of extensive agglutinated additions
to the tests of members of the genus Cibicides is not understood and remains an
interesting question for taxonomists.
ACKNOWLEDGMENTS
This paper was made possible by the opportunity to use the SEM facility at the
Department of Botany and Microbiology, University of Canterbury, Christchurch,
158 S. P. ALEXANDER AND T. E. DELACA
New Zealand, and e thank Mrs. K. Card for assistance. In addition, SEM work was
performed .? /adsworth Center for Laboratories and Research, New York State
Dept. of ) '.'•>•', and preparations were made in the laboratories of Dr. C.
Rieder ' RR02157) with assistance from Dr. S. Bowser; the final manu-
scrir neftted considerably from the comments of Dr. Bowser. Dr. S. M. Mc-
:ridly provided resin-grinding facilities at the State University of New
ny, for which we are grateful. Field and logistical support was provided
National Science Foundation Division of Polar Programs, and research was
upported by NSF grant DPP 83-05475.
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Reference: Biol. Bull. 173: 160-168. (August, 1987)
TIDAL H > GAMETOGENESIS: REPRODUCTIVE VARIATION
G POPULATIONS OF GEUKENSIA DEMISSA*
FRANCISCO J. BORRERO
/ /artment of Biology and Belle W. Baruch Institute. University of South Carolina,
Columbia, South Carolina 29208
ABSTRACT
High tidal populations of the mussel Geukensia demissa experience reduced filter
feeding time as a result of aerial exposure. This study tested the hypothesis that such
populations exhibit a temporal delay in their gametogenic cycle compared to popula-
tions from the low intertidal. As predicted, quantitative estimations of gametogenic
condition of mussels from 10 high tidal populations were lower than those of mussels
from 1 1 low intertidal populations in May 1986. A two-fold difference in potential
feeding time was accompanied by a delay of about two months in the reproductive
activity of high tidal mussels. This study demonstrates that temporal reproductive
variation among populations of (7. demissa across the intertidal zone may be as large,
or larger than variation among latitudinally separated populations of this species.
Site-to-site variation in timing of reproduction within the North Inlet Estuary may
also be as large as temporal latitudinal variation. Level of occurrence in the intertidal
zone and hence length of submersion and potential feeding time exert profound
influence in the timing of the reproductive cycle of the ribbed mussel, Geukensia
demissa.
INTRODUCTION
Variation in the timing of gametogenesis and spawning has been documented
among populations of a number of marine invertebrates (Giese and Pearse, 1974, for
a review). Such variation is common among populations of bivalve molluscs (Lubet
et ai, 1981; Newell et al., 1982; Bayne and Newell, 1983), but the causes of this
variability are not understood. Differences of environmental temperature (Orton,
1920; Sastry, 1970) and seasonality of abundance and composition of food (Newell
et al., 1982; Rodhouse et al., 1984) are thought to affect the reproductive cycles of
marine bivalves. Latitudinally separated populations of the same species may exhibit
large differences in the timing of reproductive activities (Sastry, 1970; Lubet et al.,
1981; Barber and Blake, 1983; Brown, 1984), but comparable variation also may be
observed among populations separated by much smaller distances (Seed and Brown,
1977; Newell et al., 1982). Microgeographic variation in the timing of reproductive
activities may be due to site-specific differences in food supply (Bayne and Worrall,
1980; Newell et al., 1982; Worrall et ai, 1983; Rodhouse et al., 1984). However, the
causes of variation in timing and/or intensity of reproduction have been established
Received 6 March 1987; accepted 1 April 1987.
Abbreviation: GVF, Gamete Volume Fraction.
* Contribution 679 from the Belle W. Baruch Institute for Marine Biology and Coastal Research,
University of South Carolina.
160
TIDAL HEIGHT AND GAMETOGENESIS 161
in only a few cases (Mann, 1 979; Velez and Epifanio, 1981; MacDonald and Thomp-
son, 1986).
Variation in height along the intertidal zone poses a dramatic gradient of food
availability for filter feeding organisms. Since feeding can only occur during submer-
sion, the potential feeding time is limited by the length of submersion. The effect of
differences in potential feeding time on the energy balance of filter feeding animals is
not clearly understood, but food availability and nutritional condition may strongly
influence reproductive activity (Barber and Blake, 1983; Bayne and Newell, 1983).
Therefore, differences in feeding time may have important consequences on the ga-
metogenic cycles.
This study examines the reproductive cycles of populations of the ribbed mussel
Geukensia demissa (Dillwyn) (Bivalvia:Mytilidae) that occupy different tidal levels
in the same salt marsh habitat. Previous studies suggested that diminishing the nutri-
tional status of bivalve molluscs may delay their gametogenic cycle (Mann, 1979;
Velez and Epifanio, 1981). Therefore, the specific hypothesis that the reproductive
cycle of G. demissa will be delayed in high intertidal mussels which experience de-
creased potential feeding time relative to low tidal mussels, was tested.
Although G. demissa is a dominant secondary producer in salt marsh ecosystems
of the eastern United States (Kuenzler, 1961; Fell et al., 1982; Jordan and Valiela,
1982; Bertness, 1984), information on its reproductive biology is scarce. Ribbed mus-
sel populations from New England and Connecticut exhibit a single spawning period
from June through September (Brousseau, 1982; Jordan and Valiela, 1982), whereas
in North Carolina and Georgia, peak reproductive activity might occur later, and/or
gametogenic activity extend longer, in the year (McDougall, 1943; Kuenzler, 1961).
This study provides a quantitative comparison of the reproductive cycles of popula-
tions of G. demissa from South Carolina and describes temporal reproductive varia-
tion associated with level of occurrence in the intertidal zone.
MATERIALS AND METHODS
During 1983-84, monthly samples of mussels were collected from a low tidal site
(Site 1, Up Clambank) and a high tidal site (Site 2, Ely Creek), less than 1 km apart,
within the North Inlet Estuary, South Carolina (Fig. 1 ). The high-tidal site is a short-
form Spartina alt ern (flora marsh habitat, which is covered by water approximately 4
hours per day. The low tidai site is an intertidal oyster (Crassostrea virginica) bed,
where mussels occur among the oysters. This site is covered by water for about 8
hours per day. Each sample consisted of approximately 25 mussels ranging from 25
to 100 mm in shell length.
Mussels were brought to the laboratory, scrubbed clean, and their shell lengths
measured. A 1-cm2 section was cut from the same area of the right mantle lobe of
each animal and processed as follows. Serial sections 5 microns thick were prepared
from paraffin embedded tissues and stained with hematoxylin and eosin. The frac-
tional area of the mantle section that is composed of gametes (Gamete Volume Frac-
tion, GVF ) was determined using stereology (Lowe et al, 1 982), and expressed as the
mean GVF for each sample. GVF values were arcsine transformed to assure normal
distribution. Two-way ANOVA (Sokal and Rohlf, 1981) was used to examine the
effect of date and site on reproductive condition of mussels. The effects of date and
sex were determined separately for each site, using two-way ANOVA. Mussels were
separated into six shell length classes, and the effect of shell length on reproductive
condition was determined using ANCOVA (Sokal and Rohlf, 1981). Mussel shell
length was used as the covariate. Mean GVF values were detransformed for illustra-
162
F. J. BORRERO
r
+jT
^'GEORGETOWN
f;(/CHARLESTON
•>*-
1 <j ' <?r r_ J ;/ _: .;••
C-«" ^JM^-^^ <T' ; U
/i^^^es^^^-. \\ .J
*\ ^s>- "A I A *^
^si-^r*0"""*,
'«' - .•** -X2W,* S.
:*' BAY fex/
^^^ rd
. H?) r ^ ^
/ — " *^>c \ \ i\ (\3/ / a i -x"1* / /
*«. iS^ '\U; >-•- / //v*) / ; P
TOWN
-N v NORTH
\\ INLST
. » /
FIGURE 1. Map of the North Inlet Estuary, South Carolina, indicating the ten sites studied. 1 = Up-
Clambank, 2 = Bly Creek, 3 = Debidue Creek, 4 = Old Man Creek, 5 = Town Creek, 6 = Oyster Landing,
7 = Oyster Island, 8 = Goat Island, 9 = Debidue Island, 10 = Jones Creek.
tion in figures. Data on water temperatures at sites within the North Inlet were avail-
able from the Long Term Ecological Research (LTER) program of the Baruch Insti-
tute, University of South Carolina.
RESULTS
Temporal differences in the reproductive cycles of the high and low tidal popula-
tions of G. demissa were observed. At the low tidal site, mussels had relatively con-
TIDAL HEIGHT AND GAMETOGENESIS
163
QC
U.
UJ
h-
UJ
O
1.0
0.8
0.6
0.2
0.0
30
m
TJ
20 m
DO
10
DO
m
O
NDJFMAMJJASO
1983 1984
FIGURE 2. Reproductive cycles of Geukensia demissa during 1 983-84, from a low tidal population
(Site I. Up-Clambank: •), and a high tidal population (Site 2, Ely Creek: O). Mean values of total GVF
for approximately 25 individuals are plotted each month. Vertical lines indicate the standard error of the
mean. Asterisks indicate significant differences (P < 0.05) between the two sites. Seasonal variation in
surface water temperature (•) at Bread and Butter Creek (see Fig. 1). Each point represents a single mea-
surement of temperature.
stant and low GVF values from November to March, after which a rapid increase of
GVF occurred, reaching a peak of about 0.57 by June (Fig. 2). By late April, no
animals with sexually undifferentiated gonads were found. The increase of mean
GVF closely followed the pattern of increasing water temperature in North Inlet,
showing a significant (P < 0.05) and positive correlation (r = 0.776). This population
maintained relatively high values of GVF for about four months (May to early Au-
gust). The highest GVF value was observed in August (0.65), and spawning probably
took place in this month and proceeded through September. It is possible that another
spawning event of lesser intensity occurred at this site in June, which is supported by
the observation of a significant (P < 0.05, Tukey a-posteriori test) decline of about
20% in the mean GVF values between June and July.
At the high tidal site, mussels exhibited a different reproductive cycle (Fig. 2).
Gametogenesis started later, possibly between May and June. A rapid increase of
GVF occurred between June and September, reaching an apparent peak of about
0.65 in September. Spawning occurred in September, shortly after gametogenic de-
velopment (Fig. 2). Since information on the reproductive condition of this popula-
tion was available only from June to October, a correlation analysis between GVF
and temperature is not very meaningful. However, the reproductive cycle of mussels
at this site was delayed 2-3 months with respect to the pattern of increase in water
temperature (Fig. 2).
The ANOVA procedure indicated temporal differences (P < 0.05) in the repro-
ductive cycle of the high and low tidal populations of Geukensia. The pattern of
gametogenic development of male and female mussels was similar in the magnitude
of the GVF values attained. However, male mussels appeared to start maturing earlier
than females in both populations, since the sex ratio was skewed toward males in
164 F J BORRERO
samples colV n November and February at the low tidal site, and in June
at the high U iie gametogenic cycle proceeded, the sex ratio did not differ
signiJicar- • later samples from either site. No evidence of hermaphrodit-
jsm -, -d from 429 mussels examined. Sexually undifferentiated animals
Wei' ^ April at the low tidal site, and until as late as July at the high tidal
urther evidence for temporal displacement of the onset of gameto-
:en the two populations. ANCOVA did not indicate a significant effect
i length on the GVF values observed on animals from either population
0.05).
In summary, the reproductive cycles of the two populations differed in three ma-
jor aspects: timing of the onset of gametogenesis; time of occurrence of spawning;
and length of time mussels remained in a mature reproductive condition prior to
spawning. Interestingly, despite the above temporal differences in reproduction, the
highest GVF values observed on mussels from the two sites were similar (Fig. 2).
A sampling program involving mussels from high and low tidal levels at a number
of sites including the two original populations was conducted to determine whether
the observed temporal differences in the reproductive cycles were restricted to the
two sites studied, or whether they represented a general phenomenon among mussel
populations across the intertidal zone. Since the 1983-84 samples indicated that the
difference in reproductive condition was greatest at the end of the spring (Fig. 2),
sampling was conducted on 24 May 1986. Based upon the results of the earlier sam-
pling, the prediction that in May high tidal mussels should exhibit lower GVF values
than mussels from the low intertidal was made. To test this hypothesis, ten sites were
chosen such that low and high tidal mussel populations could be found (Fig. 1 ). Ap-
proximately 15 mussels, 40-90 mm long, were collected from low and high tidal
levels at each site. The high tidal level was Spartina marsh, similar at all sites, but the
substrate at low tidal levels was marsh at some sites, and intertidal oyster beds at
others. Therefore, the consistent difference among sites was tidal height and not habi-
tat type. The samples were treated as described earlier. The effects of site, tidal level,
and sex on reproductive condition were ascertained by ANOVA. Potential differ-
ences in GVF among mussels of different sizes were examined using ANCOVA.
The gametogenic condition of mussels from 10 sites in May 1986 is presented in
Figure 3. Significant site-to-site variation in reproductive condition was observed (P
< 0.0001, ANOVA). Despite this variation among sites, high tidal populations had
lower GVF values than low tidal populations at all sites. These tidal-related differ-
ences in gametogenic condition were highly significant (P < 0.0001, ANOVA). A
Bonferoni procedure was used to assure 95% confidence in all statements regarding
simultaneous pair-wise comparisons of GVF at each tidal level within sites. These
comparisons indicate that the differences in GVF of high and low tidal mussels were
significant in all but sites 4 (Old Man Creek) and 5 (Town Creek) (Figs. 1, 3). This
analysis also suggests that tidal level has a larger effect on reproductive condition than
does habitat type. All three habitat types were available at only one site (site 9, De-
bidue Island). At this site, GVF values of low tidal marsh and low tidal oyster bed
mussels were similar, and significantly different (P < 0.05) from that of high intertidal
mussels (Fig. 3). The evidence for a larger effect of tidal level is further supported by
the fact that regardless of habitat type, high tidal mussels had lower GVF values than
mussels from the low intertidal at all sites. The observed differences in reproductive
activity among tidal levels cannot be explained solely by temperature. Observations
at the two primary sites indicate that no major temperature difference occurs between
the two tidal levels, and that this difference is not systematic through time (Borrero
and Hilbish, unpub. obs.). The temperature variation among sites within the estuary
is also very small (LTER Data-base).
TIDAL HEIGHT AND GAMETOGENESIS
165
1-0 r
O
«
^—
•
\—
i *
0 0.8
•i * »
DC
LL
UJ °6
A + J * 4 *
? 1
D
I Jk
-1 0.4
O
'll ' 1*
t\ ^ >
W 0.2
•I \ *
LU
<r n n
<J <} <j> 0
1 1 1 1 1 I ^^^^ !H^^ J
O
123456 789 10
SITE
FIGURE 3. Reproductive condition of mussels from high and low tidal populations at ten sites in the
North Inlet Estuary, on 24 May 1986. Mean values of total GVF for approximately 1 5 individuals and the
standard error of the mean are plotted for each site and tidal level. Symbols represent mussels from high
tidal marsh (O), low tidal marsh (•), and low tidal oyster bed (A) substrates. The order of sites on the x-
axis was established at random. Asterisks indicate significant (P < 0.05) differences between tidal levels at
each site.
Within the range of sizes considered, ANCOVA did not indicate a significant
effect of mussel shell length on reproductive condition, and no difference was found
in the reproductive condition of male and female mussels (P > 0.05, ANOVA). Ex-
amination of the frequency of sexually active (male, female) versus undifferentiated
mussels indicates again that the onset of gametogenesis is delayed at high tidal loca-
tions, compared to populations from the lower intertidal. While all mussels were
sexually active at most low tidal populations in May 1 986, undifferentiated animals
were found at seven of the high tidal populations. The reproductive condition of
mussels from the two original populations in the 1983-84 samples was very similar
to that obtained from the same populations in 1986.
DISCUSSION
Current understanding of the feeding physiology of suspension-feeding bivalves
indicates that the reduction in feeding time experienced by intertidal populations is
directly proportional to the duration of aerial exposure. Bivalve molluscs exhibit a
limited capacity to compensate for this reduction in feeding time (Bayne et al, in
press). The reduction in energy intake by individuals at different tidal levels should
be reflected in their productivity patterns. Tidal-related differences in productivity
may result in variability of energy allocation to reproduction and timing of reproduc-
tive activities.
The results of this study demonstrate that level in the intertidal zone and hence
length of submersion and potential feeding time affect the timing of the reproductive
cycle of mussel populations. High tidal mussels in the North Inlet Estuary exhibited
166 F. J. BORRERO
delayed gonadal ;sc \ elopment compared to mussels from the lower intertidal. Spawn-
ing was d ted only for the two populations from sites 1 and 2, but temporal
difference onset of gametogenesis and reproductive condition of mussels in
May i 9 e additional evidence that the delay in reproductive activity of high
; is a general phenomenon. Studies on the reproduction of littoral
1 other bivalve species indicate this may be a common pattern. Differ-
>ductive maturity ofCardium edule from low intertidal and high shore
? interpreted as due to the difference in synchrony of spawning at varying
e levels (Boyden, 197 1 ). Spawning by intertidal Modiolus modiolus occurred be-
tween late autumn and winter while it extended through most of the year in a subtidal
population from Ireland (Seed and Brown, 1977). Hackney (1983) observed varia-
tions in the timing of gonadal activity and spawning between well-flooded and irregu-
larly flooded intertidal populations of Polymesoda caroliniana from Mississippi and
Florida. Not surprisingly, this pattern seems to apply to other invertebrate groups.
Palmer (1980) reported a temporal delay in the reproductive maxima of intertidal
Microarthridion littorale (Copepoda), compared to a subtidal population at the same
site. Similarly, the highest percentage of individuals with egg masses was associated
with longer submergence time among two species of barnacles, interpreted as an effect
of time available for feeding on reproductive activity (Page, 1984).
A complete comparison of the effects of substrate type upon reproductive condi-
tion cannot be achieved with the data from the present study. However, the effect of
tidal height was apparent at all sites despite the heterogeneous nature of the substrates.
This suggests that tidal height explains a major portion of the overall variance in
reproductive condition.
Microgeographic variation in the timing of reproductive activity documented in
this study may be as great as that observed among latitudinally separated populations.
This is evident from a comparison of the reproductive cycle of populations of
G. demissa from the east coast of the United States. In Massachusetts, ripe mussels
were observed in June-July, and spawning may occur in August-September (Jordan
and Valiela, 1982). In Connecticut, gonadal development began in March, fully ripe
individuals were observed June through September, and spawning occurred during
the summer months (Brousseau, 1982). Spawning took place between August and
September in North Carolina ( McDougall, 1 943 ), and Kuenzler (1961) indicated that
spawning proceeded during July-August and into September in Georgia. No latitudi-
nal pattern of variation appears to exist in the reproductive cycle of G. demissa. The
results of the present study demonstrate temporal variation in reproductive activity
within the North Inlet Estuary, as large as that reported from latitudinally separated
localities. Furthermore, temporal variation in reproductive condition among tidal
levels at a single site may be larger than variation between localities at greatly different
latitudes.
Similar results were obtained by Newell et al. (1982) in a study on reproductive
variation ofMytilus edulis. A latitudinal pattern in the timing of reproductive activity
of this species could not be established along the eastern coast of the United States.
Site-to-site differences in food quantity and/or quality, and not temperature were
identified as the major determinants of the timing of gametogenesis and spawning of
Mytilus populations (Newell et al., 1982). The present study supports these conclu-
sions. The two mussel populations described here differed two-fold in the time avail-
able for feeding, and a similar difference in length of submersion applied for the high
and low tidal populations at all sites sampled.
TIDAL HEIGHT AND GAMETOGENESIS 167
ACKNOWLEDGMENTS
I thank Drs. T. J. Hilbish, F. J. Vernberg, D. Lincoln, S. A. Woodin, R. J. Feller,
D. Edwards, D. S. Wethey, S. E. Stancyk, W. K. Michener, and three anonymous
reviewers for discussion and criticism and for substantially improving the original
manuscript with their critical reviews and comments. L. Barker provided water tem-
perature information. Drs. R. I. E. Newell and V. S. Kennedy instructed on the use
of stereology. Technical assistance by M. Walker and C. Cook is appreciated. Funds
were provided by the Department of Biology and the Baruch Institute of the Univer-
sity of South Carolina, a summer scholarship from the Southeast Chapter of the Ex-
plorers Club, and the Aquaculture Fellowship from the South Carolina Wildlife and
Marine Resources Division.
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(Lamarck) at its southern distributional limit. J. Exp. Mar. Biol. Ecol. 66: 247-256.
BAYNE, B. L., ANDC. M. WORRALL. 1980. Growth and production of mussels Mytilus edulis from two
populations. Mar. Ecol. Prog. Ser. 3: 317-328.
BAYNE, B. L., AND R. C. NEWELL. 1983. Physiological energetics of marine molluscs. Pp. 407-5 15 in The
Mollusca. Vol. 4, A. S. M. Saleuddin and K. M. Wilbur, eds. Academic Press, New York.
BAYNE, B. L., A. J. S. HAWKINS, AND E. NAVARRO. Feeding and digestion in suspension feeding bivalve
molluscs: the relevance of physiological compensations. Am. Zool. (in press).
BERTNESS, M. D. 1 984. Ribbed mussels and Spartina alterniflora production in a New England salt marsh.
Ecology 65(6): 1794-1807.
BOYDEN, C. R. 1971. A comparative study of the reproductive cycles of the cockles Cerastoderma edule
and C. glaucum. J. Mar. Biol. Assoc. U.K. 51: 605-622.
BROUSSEAU, D. J. 1 982. Gametogenesis and spawning in a population ofGeukensia demissa (Pelecypoda:
Mytilidae) from Westport, Connecticut. The Veliger 24(3): 247-25 1 .
BROWN, R. A. 1984. Geographical variation in the reproduction of the horse mussel, Modiolus modiolus
(Mollusca:Bivalvia). / Mar. Biol. Assoc. U.K. 64: 75 1-770.
FELL, P. E., N. C. OLMSTEAD, E. CARLSON, W. JACOB, D. HITCHCOCK, ANDG. SILBER. 1982. Distribution
and abundance of macroinvertebrates on certain Connecticut tidal marshes, with emphasis on
dominant molluscs. Estuaries 99( 1 ): 2 1-28.
GIESE, A. C., ANDJ. S. PEARSE. 1974. Introduction: general principles. Pp. 1-49 in Reproduction of Marine
Invertebrates, Vol. 1, A. C. Giese and J. S. Pearse, eds. Academic Press, New York.
HACKNEY, C. T. 1 983. A note on the reproductive season of the Carolina marsh clam Polymesoda carolini-
ana (Bosc) in an irregularly flooded Mississippi marsh. Gulf Res. Rep. 7(3): 281-284.
JORDAN, T. E., AND I. VALIELA. 1982. A nitrogen budget of the ribbed mussel, Geukensia demissa, and
its significance in nitrogen flow in a New England salt marsh. Limnol. Oceanogr. 27( 1 ): 75-90.
KUENZLER, E. J. 1961. Structure and energy flow of a mussel population in a Georgia salt marsh. Limnol.
Oceanogr. 6: 191-204.
LOWE, D. M., M. N. MOORE, AND B. L. BAYNE. 1982. Aspects of gametogenesis in the marine mussel
Mytilus edulis L. J. Mar. Biol. Assoc. U.K. 62: 1 33-145.
LTER Data-base. Long Term Ecological Research Program, Belle W. Baruch Institute for Marine Biology
and Coastal Research, University of South Carolina, Columbia. F. J. Vernberg, Director.
LUBET, P., J.-P. GIMAZANE, ANDG. PRUNUS. 1981. Etude du cycle de reproduction de Mytilus gallopro-
vincialis (Lmk) (Moll. Lamellibranche) a la limite meridionale de son aire de repartition. Com-
paraison avec les autres secteurs de cette aire. Haliotis 11:1 57- 1 70.
MACDONALD, B. A., AND R. J. THOMPSON. 1986. Influence of temperature and food availability on the
ecological energetics of the giant scallop Placopecten magellanicus. III. Physiological ecology, the
gametogenic cycle and scope for growth. Mar. Biol. 93: 37-48.
MANN, R. 1979. The effect of temperature on growth, physiology, and gametogenesis in the Manila clam,
Tapes philippinarum (Adams & Reeve, 1 850). /. Exp. Mar. Biol. Ecol. 38: 1 2 1 - 1 33.
McDouGALL, K. D. 1943. Sessile marine invertebrates of Beaufort, North Carolina. Ecol. Monogr. 13:
321-374.
NEWELL, R. I. E., T. J. HILBISH, R. K. KOEHN, AND C. J. NEWELL. 1982. Temporal variation in the
reproductive cycle of Mvtilus edulis L. (Bivalvia, Mytilidae) from localities on the east coast of
the United States. Biol. Bull. 162: 299-310.
168 F. J- BORRERO
ORTON, J. H. 1920. oerature, breeding and distribution in marine animals. J. Mar. Biol. Assoc.
U.K. 1' K56.
PAGE, H. variation in reproductive patterns of two species of intertidal barnacles, Pollicipes
;->y and Chthamalus fissus Darwin. /. Exp. Mar. Biol. Ecol. 74: 259-272.
PA; • anation in life-history patterns between intertidal and subtidal populations of the
ae copepodMicroarthridion littorale. Mar. Biol. 60: 159-165.
. C. M. RODEN, G. M. BRUNNELL, M. P. HENSEY, T. MCMAHON, B. OTTWAY, AND
i. RYAN. 1984. Food resource, gametogenesis and growth ofMytilus edulis on the shore and
-:pended culture: Killary Harbour, Ireland. J. Mar. Biol. Assoc. U.K. 64: 5 1 3-529.
, A. N. 1970. Reproductive physiological variation in latitudinally separated populations of the
bay scallop, Aequipecten irradians Lamarck. Bio. Bull. 138: 56-65.
SEED, R., AND R. A. BROWN. 1977. A comparison of the reproductive cycles ofModiolus modiolus (L.),
Ceraslodema (= Cardium) edule (L.), and Mytilus edulis L. in Strangford Lough, Northern Ire-
land. OecologiaM: 173-188.
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VELEZ, A., AND C. E. EPIFANIO. 198 1 . Effects of temperature and ration on gametogenesis and growth in
the tropical mussel Perna perna (L.). Aquaculture 22: 2 1 -26.
WORRALL, C. M., J. WIDDOWS, AND D. M. LOWE. 1 983. Physiological ecology of three populations of the
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Reference: Biol. Bull. 173: 169-177. (August, 1987)
DIFFERENCES IN THE DURATION OF EGG DIAPAUSE OF
LABIDOCERA AESTIVA (COPEPODA: CALANOIDA) FROM THE
WOODS HOLE, MASSACHUSETTS, REGION
NANCY H. MARCUS*
Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543
ABSTRACT
The duration of diapause ofLabidocera aestiva eggs collected from the field and
reared in the laboratory was determined at 5°C. A clear seasonal trend was observed.
Diapause eggs produced in the early fall required a much longer exposure to cold to
yield a 50% hatch (CT50) (i.e., the duration of diapause was longer) than eggs pro-
duced later in the fall. Eggs produced by laboratory animals that were reared at 14°C,
8L-16D, required a shorter period of chilling to terminate diapause than the eggs of
animals reared at 19°C, 12L-12D. Considerable variation in the CT50 value was also
observed among laboratory cultures that were all reared under identical conditions,
but which differed in terms of selection history. The results indicate that both the
genotype of the egg and the conditions prevailing during oocyte formation influence
the duration of diapause. Eggs that were stored at 5°C for periods longer than 300 days
no longer hatched upon warming. It is suggested that the variation in the duration of
diapause is an adaptation that promotes synchronization of hatching by ensuring
that all individuals terminate diapause at approximately the same time, and survival
during the winter by conferring cold-hardiness. Synchronizing the onset of post-dia-
pause development is also discussed as an alternative mechanism for achieving syn-
chronous hatching.
INTRODUCTION
The calanoid copepod, Labidocera aestiva, is a seasonal member (summer and
fall) of the planktonic community in the Woods Hole region. In this area most
L. aestiva females have the genetic potential to produce two types of eggs: subitaneous
and diapause (Marcus, 1982). Subitaneous eggs are produced during the summer
and fall; diapause eggs are produced during the fall. Both egg types begin to develop
following their release by females. Subitaneous eggs typically hatch within 1 to 4 days
at 2 1 to 23°C (Marcus, 1 979). Diapause eggs enter a refractory phase after 24 to 48 h
of development. During the refractory phase, further embryogenesis is not apparent
(Marcus, pers. obs.) and diapause eggs cannot be induced to hatch even if conditions
are favorable. The duration of diapause (i.e., the length of the refractory period) is
positively related to the temperature at which eggs are held (Grice and Gibson, 1975;
Marcus, 1979). Once the refractory phase is completed, post-diapause development
and hatching occurs if conditions (e.g., temperature) are favorable. For instance, at
21 to 23°C hatching typically occurs within 1 to 2 days (Marcus, 1979). Several field
and laboratory studies (Marcus, 1979, 1980, 1 984) support the claim that the perpet-
Received 2 March 1987; accepted 14 May 1987.
* Present address: Department of Oceanography, Florida State University, Tallahassee, FL 32306.
169
170 N. H. MARCUS
uation of L. year after year in the Woods Hole region is due to the diapause
eggs whid rater on the sea-bottom and hatch in the spring.
:-A~td laboratory studies on L. aestiva I observed that many diapause
egj-, <_d to hatch at 19°C following a chilling period of 4 weeks at 5°C.
eggs would hatch with a shorter period of chilling while others re-
n longer exposure to cold. A comparison of diapause eggs obtained from
collected in the field showed that the period of chilling that would result in a
hatch at 19°C was longer for the diapause eggs of females collected early in the
fail (Marcus, 1986). This study examines in more detail the seasonal variation in the
duration of diapause of eggs of freshly caught animals from the field and compares
the results to values obtained for diapause eggs of females reared in the laboratory.
The results indicate that the genotype of an egg and the environmental factors acting
during oocyte formation influence the duration of diapause. Based on the results, I
suggest that the variation in the duration of diapause is an adaptation that promotes
synchronization of hatching in the field by ensuring that all individuals terminate
diapause at approximately the same time, and survival during the winter by confer-
ring cold-hardiness.
MATERIALS AND METHODS
Diapause eggs were obtained from animals collected at one to two week intervals
over a period of 2 years from October 1 98 1 to October 1 983. For each sampling date,
adult females were collected from Vineyard Sound by towing a 3/» m diameter, 243 ^
mesh plankton net for 10 min. Water temperature was determined on a surficial
bucket sample for all but two collection dates. For these dates, water temperature
was estimated based on the daily temperature record for water off the Woods Hole
Oceanographic Institution dock. A comparison of several dates showed that the
WHOI values were typically about 1 °C less than Vineyard Sound values. Field sam-
pling dates and surface water temperature at the time of collection are shown in Table
I. In the laboratory females were transferred to 100 ml dishes containing 5 /im-filtered
seawater and the dinoflagellate Gymnodinium nelsoni (500 cells/ml). The dishes were
incubated overnight at 19°C. The next day eggs were collected by pipette, pooled in
a separate dish of filtered seawater, and returned to the incubator for 2-4 days to
allow the subitaneous eggs to hatch. Unhatched eggs that appeared to be diapause
eggs (i.e., the interiors were green, with a clear perimeter) were distributed into 75 ml
glass screw capped jars (20-25 eggs/jar) containing filtered seawater, and refrigerated
at 5°C. Eggs that were obviously non-viable (i.e., the interiors were brownish, granu-
lar, and disintegrating) were discarded. Every 2 to 4 days, a jar was removed (except
for the 2 collections in 1981 for which duplicate jars were removed), warmed to 1 9°C,
and held at that temperature. After 4 to 5 days the proportion of hatched eggs was
ascertained. The average hatch of the duplicates was recorded for the two 1 98 1 collec-
tion dates.
Eggs of laboratory-reared animals were from 1 2 different cultures that were reared
either at a temperature of 14 or 19°C (±1°C), and a photoperiodic regimen of 8L-
1 6D or 1 2L- 1 2D. The eggs collected from each culture were 1 -2 days old. The adults
were approximately 2 weeks past reproductive maturity. The cultures represented
specific generations of three inbred lines that were being perpetuated as part of a long-
term selection experiment designed to assess the potential for evolutionary change in
the diapause response threshold (i.e., the necessary conditions for the expression of
diapause). Each line was initiated from 500-1000 nauplii that were derived from
pooled batches of either subitaneous or diapause eggs produced by 60 females col-
DURATION OF EGG DIAPAUSE 171
TABLE I
Collection dates, surface water temperature (°C). chilling lime (days) required for initial hatch, CTSO values
(days), and regression parameters pertaining to diapause eggs of field collected females
Date °C Initial CT50 r2 Slope
9/20/82
19
14
23.19
.88
7.31
9/26/83
21
18
28.82
.80
9.07
9/27/82
19
12
28.61
.79
4.14
10/04/82
18
14
27.40
.88
6.03
10/11/83
18
23
26.13
.90
13.69
10/12/82
17
16
23.02
.78
10.11
10/19/81
14
4
15.44
.89
3.66
10/21/82
14
6
24.14
.74
3.71
10/24/83
16
20
22.37
.84
8.61
11/01/82
14
8
17.30
.91
5.25
11/10/82
13
6
16.53
.90
4.81
11/23/82
11
4
15.36
.80
3.22
11/29/82
10
2
8.98
.89
3.44
12/01/81
7
6
10.99
.99
3.16
12/08/82
10
2
10.17
.96
2.71
lected from the field. Two of the lines were termed subitaneous. Each generation was
perpetuated from 500-1000 nauplii derived from just the subitaneous eggs that were
produced by the preceding generation of animals. A third diapause line was perpetu-
ated in a similar manner, but from just diapause eggs. The specific rearing conditions
and selection histories are shown in Table II. The proportion of subitaneous and
diapause eggs produced by each generation of animals varied within and between the
lines. The diet for all cultures consisted of a standard mix of four dinoflagellates.
General methods for rearing of L. aestiva have been described previously (Marcus,
1980). Eggs from each culture were incubated at 19°C for 4 to 5 days after which
the diapause eggs were distributed (20 to 30 eggs/jar) into 75 ml jars. The jars were
refrigerated at 5°C. At 2 to 4 day intervals the jars were removed, warmed to 19°C,
and held at that temperture. The proportion of eggs that hatched after 4 to 5 days was
determined.
For each field sampling date and laboratory culture, values of percent hatch were
transformed to probit values (Finney, 1952). A regression analysis was performed
with these values versus the number of days chilled (Iog10) to derive an estimate of
the days of chilling required to promote a 50% hatch (CT50). Calculations were done
with an IBM PC and the statistical software package, STATPRO.
The effect of long-term storage at 5°C on egg viability and hatching was examined
for the 2 sets of diapause eggs obtained from culture 339. After the initial analysis
period, jars of eggs were removed at intervals of up to 4 weeks for more than a year.
The hatch of these eggs after warming to 1 9°C was ascertained as described above.
RESULTS
In general, a shorter period of chilling was necessary to promote initial and 50%
hatching of eggs produced by females collected from the field later in the fall (Table
I, Fig. 1 ). The results of the Probit transformation and regression analysis permit a
quantified comparison of these differences and the derivation of the median effective
chilling period (i.e., the number of days of chilling that promote a 50% hatch). The
172
N. H. MARCUS
, -s.
-
E
N
T
H
A
T
C
H
lOOf
75"
50"
25"
o-Lt
DAYS CHILLED
FIGURE 1. Percent hatch of diapause eggs, from field-collected females, at 19°C after chilling at 5°C
for the designated number of days. Each set of connected points represents a specific sampling date. Dates
for each month are grouped by the indicated symbols.
regression parameters (slope, r2) and CT50 values are shown in Table I. The coefficient
of determination values (r2) ranged from .74 to .99 indicating that the linear regres-
sion relationship was a good one for estimating the CT50. For the diapause eggs of
field-collected females, the CT50 values ranged from 8.98 to 28.82 days. The slope
values of the regression ranged from 2.71 to 13.69 probit value/days (log,0). This
latter parameter provides an indication of the time spread of diapause duration
around the median. A high value corresponds to a very short interval for the time
from initial to maximal hatching. The highest values tended to occur during Septem-
ber and October, and the lowest during November and December. This same pattern
was found for the CT50 values. Further analysis revealed that a very good positive
correlation (r2 = .84) existed between CT50 values and surface water temperature at
the time of sampling (Fig. 2).
The median effective duration of chilling also differed among the laboratory
reared groups although the range of values was not as great as observed for the field
group. The regression parameters (slope, r2) and CT50 values are shown in Table II.
As for the field group the r2 values were high (.71 to .94). The CT50 values ranged
from 5.82 to 21.09 days. The slope values of the regression ranged from 1 .70 to 5.23
probit value/days (log,0). The lowest CT50 values were obtained for the 3 cultures
(370, 371, 374) that were reared at 14°C and 8L-16D. For 2 pairs of cultures, 370
and 372, and 371 and 373, the within pair cultures were established from the same
pool of eggs in the 23rd generation, but were reared at the two alternative sets of
conditions. In both cases the CT50 values were lower for the cultures reared at 14°C,
and 8L-16D. A third unpaired culture (374) was reared at 14°C and 8L-16D and
also yielded the third lowest CT50 value.
The two sets of eggs that were obtained from culture 339 were collected on differ-
ent days and the CT50 values differed by almost 5 days. The long term response to
chilling was also different for the two sets (Fig. 3). The hatch after chilling increased
more rapidly during the first 30 days for 339b, but a high hatch was maintained for
DURATION OF EGG DIAPAUSE
173
CT50 VALUES VS FIELD TEMP.
30"
C
T
5
0
20"
10"
0
5 10 15 20
TEMPERATURE ( °C)
FIGURE 2. Linear regression analysis of CT50 value of each field sample and the surface water temper-
ature at the time of collection.
only 150 to 200 days whereas a high hatch was maintained by the eggs of 339a for
almost 300 days. Although the hatch of both sets dropped off to near 0% levels after
300 days, many of the eggs in both sets still appeared viable.
DISCUSSION
This study shows that the median effective number of days of chilling at 5°C de-
creased as the fall season progressed for the diapause eggs of field-collected females.
TABLE II
Culture # (generation, subitaneous-s or diapause-d line), rearing conditions (photoperiod and
temperature), CT50 values (days), and regression parameters pertaining
to diapause eggs of laboratory-reared females
Culture #
Conditions
CT
50
* Eggs collected from same culture, but on two different days.
Slope
370 (23s)
14°C, 8L-16D
10.43
.85
2.18
371 (23s)
14°C, 8L-16D
5.82
.71
1.70
374 ( 6s)
14°C, 8L-16D
10.74
.90
2.92
375 ( 3s)
19°C, 12L-12D
12.16
.91
2.59
372 (23s)
19°C. 12L-12D
11.77
.78
3.11
373 (23s)
19°C, 12L-12D
14.33
.74
2.37
376 ( 7d)
19°C, 12L-12D
19.33
.78
2.41
323 ( 7s)
19°C 12L-12D
17.92
.83
4.42
329 (15s)
19°C, 12L-12D
13.64
.87
3.04
325 ( 4d)
19°C 12L-12D
21.09
.78
3.27
339a( 5d)*
19°C, 12L-12D
16.59
.93
5.23
339b( 5d)*
19°C, 12L-12D
11.81
.94
3.73
330 (15s)
19°C, 8L-16D
15.66
.84
3.54
174
N. H. MARCUS
E
R
C
N
T
H
A
T
C
H
100-
X: 339a
: 339b
100
200
300
400
DAYS CHILLED
FIGURE 3. Percent hatch of diapause eggs, produced by culture 339 on different days (a and b), at
19°C after chilling at 5°C for the designated number of days.
The range of values spanned 20 days. Similar seasonal trends have been reported for
the diapause stages of insects (Burdick, 1937; Church and Salt, 1952). During the
collecting period of L. aestiva, water temperatures ranged from 20.5 to 6.9°C, a
difference of approximately 14.0°C. The two temperatures at which the laboratory
animals were reared differed by 5°C and the number of days of chilling required to
achieve a 50% hatch differed by as much as 15.5 days. At a constant 19°C, the range
in CT50 values was about 10 days for the eggs of laboratory-reared animals. Thus for
the laboratory-reared animals considerable variation in the median effective days of
chilling was obtained, despite the fact that the environmental conditions were the
same. This variation must reflect genetic differences. Further evidence for genetic
variation are the different responses observed for the eggs of culture 339 that were
collected on different days. Since the eggs all came from the same culture, the only
possible explanation is that the eggs collected on the different days were produced by
different mixes of parents. Although the cultures of animals that were used for the
analyses represented different generations of three genetically distinct lines, no obvi-
ous association was observed between diapause duration and generation number or
selection history.
Although genetic differences appear to be important, environmental factors may
also have an effect. The three shortest times to achieve a 50% hatch were obtained for
the cultures that had been reared at 14°C and 8L-16D (Table II). This same relation-
ship with temperature and short-daylengths was evident for the eggs of the field col-
lected animals. The work of Denlinger and Bradfield (1981) on the tobacco horn-
worm provides a possible explanation for these trends. They showed that the duration
of diapause was influenced by the number of short day cycles perceived by individu-
als. As the number of short day cycles experienced by an individual increased, the
duration of diapause decreased. They concluded that in the field the duration of dia-
pause is shorter for individuals entering diapause late in the fall because declining
temperatures lead to slower development and therefore a longer exposure to short
daylengths. If this mechanism characterizes L. aestiva it is unlikely that the oocyte
DURATION OF EGG DIAPAUSE 175
itself could perceive the number of short day cycles. Hence, the effect would have to
be mediated through the parent female as a "maternal effect." In the case of L. ae-
stiva, it is also possible that declining temperatures directly influence the physiologi-
cal state of the female and, in turn, oogenesis. The differences observed for the eggs
of field-collected animals could also result from variation in maternal age. Animals
collected late in the fall might be older than ones collected early in the fall. However,
this would not explain the variation expressed by the eggs of laboratory-reared fe-
males since all of the animals were similar in age. Krysan and Branson (1977) con-
ducted specific crosses with the corn rootworm and showed that the duration of dia-
pause was affected both by the genotype of the embryo and a maternal component.
Further experiments are needed to assess the relative importance of these compo-
nents in L. aestiva.
The long term response to chilling observed for the eggs of culture 339 (Fig. 3) is
very similar to patterns observed for several insect species (Hussey, 1955; Cranham,
1972; Lees, 1955). For these species, the percent of individuals terminating diapause
increased to a maximum with increasing length of exposure to cold, then remained
high with longer exposure to cold, and finally declined with excessive time of expo-
sure to cold. In each case the percent terminating diapause remained low after exces-
sive exposure, but the interpretation of the results differed among authors. Hussey
(1955) suggested that the decline was part of an annual cycle and that given enough
time the percent terminating diapause would increase again. Hussey believed that
there was an internal gating rhythm that controlled emergence from diapause. How-
ever, since he did not carry the experiments through for another year it cannot be
certain that death had not occurred. The rhythm concept was not discussed by the
others despite similar results. They concluded that the eggs had lost viability and
would never hatch. It would certainly be advantageous for an egg of L. aestiva to
remain viable beyond one season. L. aestiva eggs that are buried do not hatch despite
favorable temperatures (Marcus and Schmidt-Gengenbach, 1986). The probability
of completing development should be higher for a diapause egg (from the previous
fall) that is uncovered no later than the summer than for an egg which is not uncov-
ered until October or November. If hatching occurred only in October and Novem-
ber, the likelihood of completing development should be diminished due to declining
temperatures. A gating rhythm that controlled the onset of post-diapause develop-
ment would reduce the probability of eggs hatching at an inappropriate time. How-
ever, this study does not support such an hypothesis. After 300 days of chilling, the
percent hatch of L. aestiva eggs after warming declined. After more than 400 days of
chilling, hatching has not increased again though many of the eggs look viable. Thus
this study indicates that eggs cannot survive more than 300 days of constant exposure
to 5°C in the laboratory. However, this life span may be quite different in the field,
where eggs probably experience long periods of anoxia and exposure to hydrogen
sulfide. Although the effect of such parameters on the viability of L. aestiva eggs is
not known, it has been reported (Uye et al, 1984) that exposure to organic pollution
reduces the viability of resting eggs of neritic marine copepods.
From studies of insects and freshwater copepods, I suggested (Marcus, 1979) that
L. aestiva eggs terminate diapause at different times during the winter in the field,
and are held at a stage of pre-hatch readiness because water temperatures are below
the threshold for post-diapause development and hatching. This study supports that
hypothesis, although the duration of diapause under field conditions appears to differ
from that observed in the laboratory where temperature was held constant at 5°C.
The data (Marcus, 1984) for eggs collected from bottom sediments in Buzzards Bay,
Massachusetts, indicated that the refractory phase was not completed by all eggs in
176 N. H. MARCUS
December. It was suggested that the eggs which failed to hatch at this time were pro-
duced late 2 fail and had not completed the refractory period. By February all
eggs apix ve completed the refractory phase as evidenced by the high hatch
of eggs i red at 19°C. The present study does not support that suggestion. Be-
cauc' sonai variation in the duration of diapause it is possible that eggs
cted from sediments in December and did not hatch in the labora-
warrning, were produced in September as well as in December,
•i ature is not the only environmental parameter that affects the transition
»m diapause to development. Hatching is also affected by light and oxygen concen-
tration (reviewed by Grice and Marcus, 198 1). Although the effect of these parame-
ters on the termination of diapause and the onset of post-diapause development in
marine copepods has not been clarified. Brewer (1964) reported that exposure to re-
duced oxygen concentrations was necessary to terminate egg diapause in the freshwa-
ter copepod, Diaptomus stagnalis, and Watson and Smallman ( 197 1) suggested that
photoperiod was a necessary cue for the resumption of development in Diacyclops
navus. The transition from dormancy to development is mediated by pH in brine
shrimp (Busa and Crowe, 1983).
Diapause is an important factor in the synchronization of life cycles (Tauber et
al, 1986). Two possible ways in which synchronization can be achieved are synchro-
nizing the termination of diapause and synchronizing the onset of post-diapause de-
velopment and hatching. Both mechanisms characterize L. aestiva in Woods Hole
waters. Since diapause eggs are produced over a span of several months, the longer
diapause of eggs produced early in the fall ensures that they do not terminate diapause
until winter temperatures have declined below the threshold for post-diapause devel-
opment. Conversely, the shorter diapause of eggs produced late in the fall ensures
that diapause will be completed prior to the time when water temperature exceeds
the threshold in the spring. Since diapause in the field terminates by February or
March in Woods Hole waters (Marcus, 1 984) and hatching does not occur until May
(Grice and Gibson, 1975) the precise coincidence of diapause termination should be
less important than the coincidence of the onset of post-diapause development in the
promotion of synchronous hatching. Thus, as long as the refractory phase is com-
pleted before the threshold for post-diapause development or hatching is exceeded,
synchronization of hatching should still occur. The coincidence of diapause termina-
tion among overwintering eggs may be more critical at more southern latitudes where
water temperatures at the time of diapause termination are more likely to exceed the
threshold and thus individuals would resume development as soon as the refractory
period ended. The results also suggest that diapause is important because it promotes
the survival of individuals by conferring cold-hardiness. Diapause eggs which com-
plete their refractory period by January (Marcus, 1 984), can tolerate exposure to cold
winter temperatures. However, subitaneous eggs do not survive extended exposure
to such temperatures (Grice, unpub.). I suggest that the variation in diapause duration
expressed by L. aestiva is an adaptation that promotes synchronization by ensuring
that all individuals terminate diapause at approximately the same time, and survival
by conferring cold-hardiness.
ACKNOWLEDGMENTS
I thank C. Fuller and P. Alatalo for their valuable assistance in the field and labora-
tory. J. Schmidt-Gengenbach, S. Twombly, and two anonymous reviewers provided
helpful criticism of the manuscript. Supported by NSF Grants OCE82- 14882 and
OCE85-09863.
DURATION OF EGG DIAPAUSE 177
LITERATURE CITED
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the hatching of the eggs stage. Physiol. Zoo/. 37: 1-20.
BURDICK, H. 1937. The effects of exposure to low temperature on the developmental time of embryos of
the grasshopper Melanoplus differentially (Orthoptera). Physiol. Zoo/. 10: 156-170.
BUSA, W., AND J. CROWE. 1983. Intracellular pH regulates transitions between dormancy and develop-
ment of brine shrimp (Anemia salina) embryos. Science 221: 366-368.
CHURCH, N., AND R. SALT. 1952. Some effects of temperature on development and diapause in eggs of
Melanoplus bivittatus (Say) (Orthoptera: Acrididae). Can. J. Zoo/. 30: 99-1 7 1 .
CRANHAM, J. 1972. Influence of temperature on hatching of winter eggs of fruit-tree red spider mite,
Panonychus ulmi (Koch). Ann. App. Biol. 70: 1 19-1 37.
DENLINGER, D., AND J. BRADFIELD. 1981. Duration of pupal diapause in the tobacco hornworm is deter-
mined by number of short days received by the larva. /. Exp. Biol. 91: 33 1-337.
FINNEY, D. 1952. Probit Analysis: A Statistical Treatment of the Sigmoid Response Curve. Cambridge
University Press, London.
GRICE, G., AND V. GIBSON. 1975. Occurrence, viability, and significance of resting eggs of the calanoid
copepod Labidocera aestiva. Mar. Biol. 31: 335-337.
GRICE, G., AND N. MARCUS. 1981. Dormant eggs of marine copepods. Oceanogr. Mar. Biol. Ann. Rev.
19: 125-140.
HUSSEY, N. 1955. The life histories of M^ay//gw/«s;?mHO/ro/7/n/.9 Wachtel(Hymenoptera:Chalcidoidea)
and its principal parasite with descriptions of the developmental stages. Trans. R. Entomol. Soc.
Lond. 106: 133-151.
KRYSAN, J., ANDT. BRANSON. 1977. Inheritance of diapause intensity in Diabrotica virgifera. J. Hered.
68:415-417.
LEES, A. 1953. Environmental factors controlling the evocation and termination of diapause in the fruit
tree red spider mite Metatetranychus ulmi Koch (Acarina:Tetranychidae). Ann. Appl. Biol. 40:
449-486.
MARCUS, N. 1979. On the population biology and nature of diapause of Labidocera aestiva (Copepoda:
Calanoida). Biol. Bull. 157: 297-305.
MARCUS, N. 1980. Photoperiodic control of diapause in the marine calanoid copepod Labidocera aestiva.
Biol. Bull. 158:311-318.
MARCUS, N. 1982. The reversibility of subiatneous and diapause egg production by individual females of
Labidocera aestiva (Copepoda:Calanoida). Biol. Bull. 162: 39-44.
MARCUS, N. 1984. Recruitment of copepod nauplii into the plankton: importance of diapause eggs and
benthic processes. Mar. Ecol. Prog. Ser. 15: 47-54.
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Zoo/. 26: 469-477.
MARCUS, N., AND!. SCHMIDT-GENGENBACH. 1986. Recruitment of individuals into the plankton: the
importance of bioturbation. Limnol. Oceanogr. 31: 206-210.
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New York.
UYE, S., M. YOSHIYA, K. UEDA, ANDS. KASAHARA. 1984. The effect of organic sea-bottom pollution on
survivability of resting eggs of neritic calanoids. Crustacean Suppl. 1: 390-403.
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Reference: Biol. Bull. 173: 178-187. (August, 1987)
'HEMICAL FEATURES OF SHRIMP HEMOCYTES
JO ELLEN HOSE, GARY G. MARTIN, VAN ANH NGUYEN,
JOHN LUCAS, AND TEDD ROSENSTEIN
Department of Biology. Occidental College, Los Angeles, California 90041
ABSTRACT
Morphological studies suggest that there are several types of decapod hemocytes;
however, distinguishing criteria based on conventional staining techniques are often
subtle or ambiguous. Cytochemical features of ridgeback prawn (Penaeidae: Sicyonia
ingentis) hemocytes were studied using specific stains for lysosomes, cytoplasmic con-
tents, and granule enzymes. This approach facilitates the differentiation of cell types
in the ridgeback prawn and provides information on the functions of and relation-
ships among different cell types.
Agranular hemocytes and a subgroup of small granule hemocytes contain exten-
sive cytoplasmic glycoprotein deposits which display smudgy, intense staining with
Sudan black B. As previously shown, coagulogen — the clotting material in deca-
pods— stains with Sudan black B when extracted from lysed hemocytes. Other hemo-
cyte types display light staining limited to granule membranes.
Lysosomes are not observed in agranular cells and are rarely present in small
granule hemocytes with glycoprotein deposits. Small granule hemocytes without de-
posits and large granule hemocytes contain numerous lysosomes as shown by the
presence of acid phosphatase, /3-glucuronidase, and nonspecific esterase. Acid phos-
phatase is observed in the Golgi body of these cells, within small vesicles, and in
small granules. The granules in large granule hemocytes rarely show acid phosphatase
reaction, yet small acid phosphatase-positive vesicles fuse with the large granules. The
acid phosphatase in the large granules may exist in an inactive form. Prophenoloxi-
dase activity is localized only in large granules. The physiological significance of he-
mocyte cytochemistry is also discussed.
INTRODUCTION
Crustacean hemocytes perform a variety of physiological and pathological func-
tions including coagulation (Ravindranath, 1980), phagocytosis, recognition of for-
eign material, carbohydrate transport, and encapsulation (Bauchau, 1981). Several
hemocyte categories have been recognized in decapod crustaceans based on morpho-
logical criteria. Morphological features are often subtle and ambiguous, and are not
readily recognized by other investigators. In addition, morphological criteria are
rarely based on properties that facilitate the differentiation between stages in hemo-
cyte maturation or among cells with different physiological functions. Hence, previ-
ous investigations have failed to define a clear correspondence between various cell
types and their functions.
In an attempt to develop a comprehensive description of crustacean hemocyte
formation and function, cytochemical techniques were used to complement our pre-
vious morphological description of shrimp hematopoietic tissue (Martin et al, 1987)
and circulating hemocytes (Martin and Graves, 1985). Electron microscopic exami-
Received 2 January 1987; accepted 19 May 1987.
178
CYTOCHEMISTRY OF SHRIMP HEMOCYTES 179
nation of hemocytes from the ridgeback prawn (Penaeidae: Sicyonia ingentis) shows
the existence of four cell types: agranular, small granule with cytoplasmic deposits,
small granule without cytoplasmic deposits, and large granule hemocytes. Agranular
hemocytes are small cells with a high nucleus:cytoplasm ratio (Martin et al, 1987).
Their cytoplasm contains little other than aggregations of electron-dense deposits. A
subset of small granule hemocytes contains similar electron-dense cytoplasmic de-
posits, one to six round striated granules, and occasional electron-dense granules. In
contrast, a distinct subset of small granule hemocytes and the large granule hemocytes
lack cytoplasmic deposits and striated granules. These hemocytes have many (> 10)
electron-dense, electron-lucent, or punctate granules which range in diameter from
0.4 nm in small granule hemocytes to 0.8 ^m in large granule cells. Intermediate
stages were observed between agranular hemocytes and small granule hemocytes with
deposits and between small granule hemocytes without deposits and large granule
hemocytes, suggesting the existence of two distinct hemocyte lines.
In view of the difficulty in accurately identifying certain hemocyte categories at
the light microscope level, various enzymatic and cytochemical methods were evalu-
ated for use in hemocyte classification. The goals of this study are to (1) identify
cytochemical stains which can be used to differentiate specific hemocyte types, and
(2) provide useful information on the function of the various cell types.
MATERIALS AND METHODS
Animals
Ridgeback prawns were collected and maintained as previously described (Martin
et al., 1987). Shrimp averaged 14.5 g and were in molt stages C and D (Ander-
son, 1985).
Tissue collection and preparation
Hemolymph (usually 0.2 cc) was withdrawn from the ventral sinus or heart into
a 1 cc syringe containing anticoagulant (Martin and Graves, 1985). Hemocyte smears
were then prepared on glass microscope slides, allowed to air dry, and used for light
microscopy.
Hemocytes and hematopoietic nodules to be examined at the electron micro-
scopic (EM) level were fixed in 2.5% glutaraldehyde in 0. 1 M sodium cacodylate (pH
7.8) containing 12% glucose for 1 h at room temperature. Following a 30 min wash
in 0.1 M sodium cacodylate (pH 7.8) containing 24% sucrose, the tissues were post-
fixed in 1% OsO4 in 0.1 M sodium cacodylate for 1 h at room temperature, dehy-
drated in a graded series of ethanol, and infiltrated and embedded in Spurrs' (1969)
low viscosity plastic.
Epigastric hematopoietic nodules were dissected from shrimp as described by
Martin et al. (1987). Touch preparations of sagittally cut nodules were air-dried prior
to the cytochemical demonstration of prophenoloxidase (Ppo). Frozen sections (7
Aim thick) were cut using a Tissue Tek II cryostat for the demonstration of lysosomal
enzymes. Thin sections (7 /urn) were also prepared using formalin-fixed, par-
affin-embedded tissue.
Demonstration of cytoplasmic constituents
Following a two-minute fixation in absolute ethanol, smears were stained with
bromphenol blue or periodic acid-Schiff(PAS). Smears to be stained with Best's car-
1 80 J. E. HOSE ET AL.
mine (Sheehan and Hrapchak, 1980) were fixed in ethanol for 30 minutes. PAS and
carmine were compared with and without prior digestion by a-amylase. For the diges-
tions, hemocytes were suspended in 0.5% aqueous amylase for 1 h, then pelleted by
a 5 min centrifugation at 500 X g in a table top centrifuge before preparation of the
smear. Sections of hematopoietic nodule were stained with bromphenol blue or PAS.
Enzymatic extractions to demonstrate composition of cytoplasmic deposits and
granules were also examined using EM. Hemocytes were fixed in 2.5% glutaraldehyde
in 0.1 M sodium cacodylate (pH 7.8) containing 12% glucose for 1 h, then washed in
cacodylate buffer and kept at 4°C for 12 to 18 h. The hemocyte pellet was then dehy-
drated through a graded ethanol series, infiltrated and embedded in Spurrs' (1969)
low viscosity plastic. Thin sections were cut on a Porter Blum MT2B ultramicrotome,
picked up on gold grids, and floated on one of the following solutions for 2 to 20 h at
37°C: (A) 0.5% protease in 0.2 M phosphate buffer (pH 7.4) or (B) 0.5% a-amylase in
0.2 M phosphate buffer (pH 7.4). These sections and control sections (floated on
distilled water for an equivalent period of time) were examined unstained and stained
(0.5% uranyl acetate in 0.05 M Tris-maleate for 1 h at room temperature) using a
Hitachi HU1 1A transmission electron microscope.
Lipids were demonstrated in hemocyte smears and nodule touch preparations
using a commercial Sudan black B kit (Sigma Chemical Co. Kit #380) according to
provided directions.
Prophenoloxidase activity
To test for the presence of prophenoloxidase (Ppo), hemocytes and hematopoietic
tissue touch preparations were fixed in 2.5%. glutaraldehyde in 0. 1 M phosphate buffer
(pH 7.4) for 1 h at 4°C. The cells were given three 1 5-min rinses in phosphate buffer,
incubated in 0. 1 % L-DOPA in phosphate buffer for 1 6 h at room temperature (Soder-
hall and Smith, 1976), and examined by light microscopy.
Lysosomal enzymes
The presence of acid phosphatase (Sigma Chemical Co. Kit #386), jfr-glucuroni-
dase (Kit #180), and a-aryl naphthyl esterase — a nonspecific esterase (Kit #90) —
were demonstrated at the light microscopic level using commercial research kits
(Sigma Chemical Co.). Hemocyte smears and frozen sections of hematopoietic nod-
ule were fixed in glutaraldehyde and incubated according to provided directions.
Staining patterns for each enzyme were quantified at 1000X by estimating the num-
bers and sizes of positive areas in 10 cells from each of the 4 hemocyte categories
described by Martin el al. ( 1 987).
To localize acid phosphatase at the EM level, fixed hemocytes and hematopoietic
nodules were washed thoroughly and then incubated in a medium consisting of
40 mM Tris-maleate buffer (pH 5), 1 1.5 mM sodium /3-glycerophosphate, 2.4 mM
lead nitrate, and 5% sucrose at 37°C for 2 h. Hemocytes were then processed as de-
scribed above.
The same procedure was followed for glucose-6-phosphatase, alkaline phospha-
tase, and peroxidase except for the use of different incubation media. For glucose-6-
phosphatase, the fixed cells were incubated in a medium composed of 25 mg glucose-
6-phosphate, 27 ml distilled water, and 20 ml of 0.3 M Tris-maleate buffer (pH 9.7).
The incubation medium for alkaline phosphatase consisted of 4 ml 1.25% sodium (3-
glycerophosphate, 4 ml of 0.2 M Tris-maleate buffer (pH 9), 9.4 ml distilled water,
and 2.6 ml of 1% lead nitrate. The peroxidase medium contained 5 mg 3,3-diamino-
CYTOCHEMISTRY OF SHRIMP HEMOCYTES 181
benzidine tetrahydrochloride, 10 ml of Tris-maleate buffer (pH 7.6), and 0.1 ml of
1%H2O2.
RESULTS
Cytoplasmic constituents
The abundant cytoplasmic deposits of agranular hemocytes and a subset of small
granule hemocytes are composed of glycoproteins as evidenced by positive reactions
with PAS, carmine, and bromphenol blue. Digestion with a-amylase prior to applica-
tion of PAS and carmine reduced but did not completely remove the staining of these
cytoplasmic deposits. Tissue sections of the epigastric hematopoietic nodule stained
with PAS or bromphenol blue yields results similar to those in free hemocytes.
Sudan black B produces a distinctive staining pattern in agranular hemocytes and
small granule hemocytes with cytoplasmic deposits (Figs. 1, 2). These cells appear
smudgy, with the heavy dark stain obscuring nuclear characteristics. Only a thin clear
zone adjacent to the plasma membrane is occasionally present. Staining of the cy-
toplasmic deposits by Sudan black B indicates the presence of a lipid moiety associ-
ated with the glycoprotein. In small granule hemocytes lacking deposits (Fig. 3) and
in large granule hemocytes (Fig. 4), delicate staining is evident only around granule
and nuclear membranes, producing a diffuse pattern. Maturing hemocytes from the
hematopoietic nodule display identical staining patterns.
Granule histochemistry
Granules in free and maturing hemocytes are stained with PAS, carmine, and
bromphenol blue, indicating the presence of glycoproteins. Prior amylase digestion
removes granular staining by PAS and carmine.
Prophenoloxidase activity is visualized following incubations of fixed hemocytes
and hematopoietic tissue touch preparations with L-DOPA (Figs. 5-8). Ppo activity
is limited to granules of small granule hemocytes lacking glycoprotein deposits and
large granule hemocytes. In some animals (molt stage D), almost 100% of these cell
types display intense activity (> 10 positive granules each) while in intermolt shrimp,
less than 1% of these cells are positive. Similar results are obtained using hemocyte
smears and tissue touch preparations.
No peroxidase activity is observed in any of the hemocyte categories.
The glycoprotein content of large and small granules is also seen in sectioned
tissues that were subsequently treated with protease or «-amylase. Figure 9 shows a
large granule from a hemocyte viewed after standard preparation. Figures 10 and 1 1
show granules in sections treated with a-amylase ( 12 h) and protease (6 h). At these
times, the core of the granules has been extracted, however, with longer incubations
(20 h), the entire granule is extracted by both enzymes.
Lysosomal enzymes
Three hydrolases (acid phosphatase, /i-glucuronidase, and nonspecific esterase)
were used to demonstrate the presence of lysosomes in hemocytes at the light micro-
scope level (Table I). These stains yield similar cytochemical information for each
specific hemocyte type although individual hydrolases produce slightly different
staining patterns. Agranular hemocytes do not contain any of the lysosomal enzymes.
Glycoprotein-rich small granule hemocytes exhibit between zero and three focally
positive areas consistent with the size of lysosomes. These cells occasionally contain
182
J. E. HOSE ET AL.
10
11
FIGURES I -4. Light micrographs of agranular, small granule hemocyte with deposits, small granule
hemocyte without deposits, and large granule hemocyte, respectively, treated to show sites of prophenoloxi-
dase. The first two cells have no reaction product. The granules (arrows) in the small granule hemocyte
without deposits react as does the entire cytoplasm of the large granule hemocyte. All figures 2500X.
FIGURES 5-8. Light micrographs of same cell types as in Figures I -4, treated with Sudan black B.
Agranular and small granule hemocytes with deposits show dense reaction products in the cytoplasm which
obscure the nucleus. The latter two cell types have minimal staining and the nucleus (N) is clearly observed.
All figures 2500X.
FIGURE 9. Transmission electron micrograph showing homogeneous and electron-dense granules
(G) from a large granule hemocyte fixed with both glutaraldehyde and osmium and stained with uranyl
acetate and lead citrate. 43,OOOX.
FIGURE 10. Transmission electron micrograph showing a granule from a large granule hemocyte
that was fixed only with glutaraldehyde. Thin sections were floated on a protease solution for 6 h and
examined without stain. Note the low electron density and extraction of the granule core (C). 43,OOOX.
FIGURE 1 1 . Transmission electron micrograph of a granule from a large granule hemocyte prepared
as in Figure 10 and then floated on a solution of a-amylase for 12 h. Note the low electron density of the
granule and extraction of its core (C). 43,000x.
a few acid phosphatase-positive granules as well. In contrast, small granule hemocytes
lacking cytoplasmic deposits have from three to eight positive foci consistent with
lysosomes. Half of these cells have only a few (0-3) positive granules while the re-
maining small hemocytes contain over 30 positive granules. A few of the latter group,
presumably transitional to large granule hemocytes, also exhibit a few large acid phos-
phatase-positive granules. In the large granule hemocytes, up to three focally positive
CYTOCHEMISTRY OF SHRIMP HEMOCYTES
183
TABLE I
Distribution oflysosomal enzymes in shrimp hemocytes
Hemocyte type
Acid phosphatase
/8-glucuronidase
Glucose-6- Non-specific
phosphatase esterase
Alkaline
phosphatase
Agranular
None
None
None None
None
Small granule
hemocyte with
deposits
Rare(l-3RS*/
Cell)
Rare(l-3RS/
Cell)
None Few(l-10RS/
Cell)
None
Small granule
hemocyte without
deposits
Mixed (50% of cells
have>30RS/
Cell 50% of cells
Many
(>10RS/Cell)
None I ntermediate ( 1 0-
30 RS/Cell)
None
Large granule
hemocytes
have 1-10RS/
Cell)
Mixed (50%. of cells
have 0-1 RS/Cell
and nuclei are
pycnotic 50% of
cells have 4-8
RS/Cell)
Many
(> 10 RS/Cell)
None
Many
(> 30 RS/Cell)
None
* RS stands for reaction sites.
areas consistent with lysosomes were observed. From zero to five small granules are
positive as well as from zero to two large granules. Among the large granule hemo-
cytes, the largest cells which contain eccentrically placed, pycnotic nuclei were usually
acid phosphatase-negative or contain only one positive focus.
Electron microscopy localization of acid phosphatase yields similar results with
no reaction product detected in agranular hemocyte (Fig. 12). Staining is infrequently
observed in small granule hemocytes containing glycoprotein deposits and is re-
stricted to small vesicles and granules of the non-striated variety (Fig. 13). Heavy
staining is found in the granules of the small granule hemocyte lacking deposits (Fig.
14). Large granule hemocytes have reaction product dispersed throughout the cell in
vesicles and the smaller granules. Only a few of the large granules stain positive al-
though these were morphologically indistinguishable from non-reactive granules
(Fig. 15). In Figure 16, acid phosphatase-positive trans cisternae and small vesicles
are shown budding from a Golgi body. These vesicles (Fig. 16, inset) appear to pro-
gressively coalesce, forming larger reaction vesicles (Fig. 1 7), then small granules, and
finally large granules (Fig. 1 8).
Nonspecific esterase is observed only in granulated cells. Glycoprotein-rich small
granule hemocytes are completely negative or contain up to 10 tiny positive areas
consistent with the size of vesicles. In contrast, from 10 to over 30 positive vesicles
are observed in small granule hemocytes without cytoplasmic deposits. Large granule
hemocytes contain numerous (>30) positive vesicles. Patterns of /3-glucuronidase
staining in granulated cells are similar to those of nonspecific esterase in addition
to the presence of a few (<3) positive foci of lysosomal size in the small granule
hemocytes.
Maturing hemocytes from frozen sections of the hematopoietic nodule were ex-
amined for the presence of acid phosphatase and /3-glucuronidase. Staining patterns
of acid phosphatase are identical between maturing hemocytes and those described
above for free hemocytes. 0-glucuronidase activity is not observed in touch prepara-
tions of the hematopoietic nodule.
All hemocytes are negative for alkaline phosphatase and glucose-6-phosphatase.
184
J. E. HOSE ET AL.
15
G
16
17
18
CYTOCHEMISTRY OF SHRIMP HEMOCYTES 185
DISCUSSION
Results of cytochemical tests support the morphological classification of ridge-
back prawn hemocytes previously developed in our laboratory (Martin et al., 1987)
and yield information on the physiological functions performed by the various hemo-
cyte types. A combination of two or three cytochemical tests is suggested for classifi-
cation of shrimp hemocytes. Sudan black B produces a distinctive smudgy staining
pattern in agranular hemocytes and small granule hemocytes with cytoplasmic de-
posits. Acid phosphatase can be used to differentiate agranular cells, which are nega-
tive for lysosomal enzymes. Prophenoloxidase activity is limited to small granule
hemocytes without cytoplasmic deposits and large granule hemocytes; however, sig-
nificant activity may only be demonstrable during the D stage of the molt cycle (Bau-
chau, 1981).
The glycoprotein deposits in the cytoplasm of agranular hemocytes and a sub-
group of small granule hemocytes are distributed in linear arrays throughout the en-
tire cell and are evident in all molt stages (unpub. obs.). In contrast, glycogen — which
it resembles ultrastructurally — is typically confined to one area of decapod hemocytes
and does not have a linear arrangement (Johnston et al., 1973; Bauchau, 1981). Gly-
cogen has been shown to be transported by hemocytes (Johnston et al., 1973; Bau-
chau, 1981) and may involve the enzyme glucose-6-phosphatase (Johnston and Da-
vies, 1 972). This enzyme, however, was not detected in shrimp hemocytes. The glyco-
protein may contain a lipid moiety since the deposits are intensely stained by Sudan
black B. Such chemical properties are consistent with those of the primary coagula-
tion protein, coagulogen (Durliat, 1985). In decapod hemocytes, intracellular coagu-
logen does not appear to be localized in granules although granules are necessary
for coagulation to occur (Ravindranath, 1980; Durliat, 1985). Shrimp small granule
hemocytes with lipoglycoprotein deposits contain granules with a striated or concen-
tric substructure (Martin et al., 1987). Similar granules have been observed in Limu-
lus (Copeland and Levin, 1 985), crabs (Bodammer, 1 978), lobsters (Hearing and Ver-
nick, 1967; Goldenberg et al., 1986), and crayfish (Unestam and Nylund, 1972), and
alterations in the striated granules of shrimp have been observed early in the process
of hemolymph coagulation (unpub. obs.).
Lysosomes were observed in each cell type except for agranular hemocytes. Small
FIGURES 12 AND 13. Transmission electron micrographs of an agranular hemocyte (Fig. 12) and a
small granule hemocyte with deposits (Fig. 13) treated to display sites of acid phosphatase activity. No
reaction sites are present in agranular cells. In small granule hemocytes with deposits, reaction sites (X) are
rare and then localized to granules of the electron-dense variety. Striated granules (S) are never labelled. In
both cells, note the small amount of cytoplasm which contains the deposits (arrows). Both figures 20.000X.
FIGURE 1 4. Transmission electron micrograph of a small granule hemocyte that lacks deposits show-
ing a few reaction sites for acid phosphatase in granules (G) and vesicles (V). 20.000X. Inset shows a higher
magnification (43,OOOX) micrograph of the small granules.
FIGURE 15. Transmission electron micrograph of a large granule hemocyte showing reaction sites
for acid phosphatase in vesicles throughout the cytoplasm (arrows) and some of the granules (G). Other
granules (X) show no reaction product. 20.000X.
FIGURE 16. Transmission electron micrograph showing a Golgi body in a large granule hemocyte.
The trans-cisternae contains the acid phosphatase reaction product. 42,000x. Inset shows a vesicle with
reaction product. Similar vesicles are commonly seen around Golgi bodies as well as throughout the cyto-
plasm. 42.000X.
FIGURE 1 7. Transmission electron micrograph showing acid phosphatase reaction product in three
vesicles of increasing diameter. Note how the smallest vesicle appears to be fusing (arrow) with the medium
sized vesicle and that the contents of the vesicles are not as electron dense as fully mature granules. 32,000x.
FIGURE 18. Transmission electron micrograph showing acid phosphatase reaction product in one
large vesicle (V) and not in two adjacent granules (G). 32,OOOX.
186 J- E. HOSE ET AL.
granule hemocytes with glycoprotein deposits contained only one to three lysosomes
per cell. In small granule hemocytes without deposits and in large granule hemocytes,
many lysosome e identified using LM and EM cytochemistry. Although the gran-
ules in thes s are morphologically indistinguishable, they may be enzymatically
heteroge s (see Bauchau, 1981). Using TEM, acid phosphatase was localized in
some iot all of the granules of the shrimp. The same results were observed in
5 of the clam Mercenaria mercenaria (Yoshino and Cheng, 1976) and
interpreted to indicate a heterogeneous population of granules or a non-synchronized
cycle of granule production, perhaps with the final enzyme stored in an inactive form.
The presence of numerous lysosomes in the large and small granulocytes which lack
deposits supports the suggestion that these cells are phagocytic (Bauchau, 1981; Sod-
erhall et al, 1986) and that the granules are available for intracellular degradation
processes. However, because of the large number of granules in a single hemocyte, it
is unlikely that granules could be exclusively reserved for phagocytosis. Other re-
searchers suggested extracellular functions for these granules, including recognition
of foreign material (Soderhall and Smith, 1983) and agglutinin sequestration (Stang-
Voss, 1971).
The recognition of foreign material in arthropods is mediated by the propheno-
loxidase system which is located in the granules (Soderhall and Smith, 1983). Results
of the present study show that only large granule hemocytes and small granule hemo-
cytes without deposits contain prophenoloxidase activity. Soderhall and Smith
( 1 983) obtained similar conclusions of Ppo activity within granular hemocytes in the
crab, Carcinus maenus. Ppo activation and exocytosis in response to endotoxin or @-
glucan exposure can initiate the coagulation cascade and serves as the crustacean
equivalent to the alternate complement and properdin pathways in mammals (Dur-
liat, 1985). The proposed existence of the Ppo system in the small granule hemocytes
without deposits-large granule hemocyte line and the coagulation enzymes in the
other hemocyte line suggests cooperativity among shrimp hemocytes during endo-
toxin-mediated coagulation analogous to that observed for defense reactions in in-
sects (Ratcliffe et al., 1984) and crustaceans (Soderhall et al., 1986).
Based upon results of morphological (Martin et al., 1987) and cytochemical stud-
ies, shrimp hemocytes can be divided into two cell lines, the deposit line (composed
of agranular and striated granule hemocytes) and the granulocyte line (small and
large granule hemocytes). Although the lysosomal enzyme data presented here are
consistent with maturing stages of a single cell line, the following arguments support
our theory: ( 1 ) striated granules are never found in granulocytes (The striated granule
shown in Fig. 7B of Martin and Graves, 1985, was taken from a small granule hemo-
cyte containing cytoplasmic deposits. However, at that time, no distinction was
made between the deposit and granulocyte lines.); (2) glycolipoprotein deposits are
never observed in granulocytes; (3) mitosis is observed both in agranular hemocytes
and in small granule hemocytes which lack cytoplasmic deposits; (4) cells are present
as a continuum of differentiation between agranular and striated granule hemocytes
and between small and large granule hemocytes; and (5) clusters of deposit cells and
granulocytes are usually segregated within the hematopoietic tissue. The utility of this
classification scheme must now be determined by functional studies identifying the
role of the various hemocyte types in crucial biological processes such as coagulation,
defense reactions, wound healing, and exoskeleton hardening.
ACKNOWLEDGMENTS
We thank Terri Fu and Sidne Omori for their technical help. The project was
supported by NSF grant DCB-8502 1 50 to GM and JEH.
CYTOCHEMISTRY OF SHRIMP HEMOCYTES 187
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BAUCHAU, A. G. 1981. Crustaceans. Pp 386-420 in Invertebrate Blood Cells, Vol. 2. Academic Press, New
York.
BODAMMER, J. E. 1978. Cytological observations on the blood and hemopoietic tissue in the crab, Calli-
nectes sapidus. I. The fine structure of hemocytes from intermoult animals. Cell. Tissue Res. 187:
79-86.
COPELAND, D. E., AND J. LEVIN. 1985. The fine structure of the amoebocyte in the blood of Limitlus
polyphemus. I. Morphology of the normal cell. Biol. Bull. 169: 449-457.
DURLIAT, M. 1985. Clotting processes in Crustacea Decapoda. Biol. Rev. 60: 473-498.
GOLDENBERG, P. Z., A. H. GREENBERG, AND J. M. GERRARD. 1986. Activation of lobster hemocytes:
cytoarchitectural aspects. J. Invertebr. Pathol. 47: 143-154.
HEARING, V. J., AND S. H. VERNICK. 1967. Fine structure of the blood cells of the lobster, Homarus
americanus. Ches. Sci. 8: 1 70.
JOHNSTON, M. A., AND P. S. DAVIES. 1972. Carbohydrates of the hepatopancreas and blood tissues of
Carcinus. Comp. Biochem. Physio/. 41 B: 433-443.
JOHNSTON, M. A., H. Y. ELDER, AND P. S. DAVIES. 1983. Cytology of Carcinus haemocytes and their
function in carbohydrate metabolism. Comp. Biochem. Physiol. 46A: 569-581.
MARTIN, G. G., AND B. L. GRAVES. 1985. Fine structure and classification of shrimp hemocytes. /. Mor-
phol. 185: 339-348.
MARTIN, G. G., J. E. HOSE, AND J. J. KIM. 1987. Structure of hematopoietic nodules in the ridgeback
prawn, Sicvonia ingentis: light and electron microscopic observations. J. Morphol. 192: 193-204.
RATCLIFFE, N. A., C. LEONARD, AND A. F. ROWLEY. 1984. Prophenoloxidase activation: nonself recogni-
tion and cell cooperation in insect immunity. Science 226: 557-559.
RAVINDRANATH, M.H.I 980. Haemocytes in haemolymph coagulation of arthropods. Biol. Rev. 55: 1 39-
170.
SHEEHAN, D. C., AND B. B. HRAPCHAK. 1 980. Theory and Practice ofHistotechnology. C. V. Mosby Co.,
St. Louis. 48 1 pp.
SODERHALL, K., AND V. J. SMITH. 1983. Separation of haemocyte populations of Carcinus maenus and
other marine decapods, and prophenoloxidase distribution. Dev. Comp. Immunol. 7: 229-239.
SODERHALL, K., V. J. SMITH, AND M. W. JOHANSSON. 1986. Exocytosis and uptake of bacteria by isolated
haemocyte populations of two crustaceas: evidence for cellular co-operation in the defence reac-
tions of arthropods. Cell Tissue Res. 245: 43-49.
SPURRS, A. 1969. A low viscosity epoxy embedding medium for electron microscopy. J. Ultrastrucl. Res.
26:31-43.
STANG-Voss, C. 1971. Zur ultrastruktur der blutzellen wirbelloser tiere. V. Liber die hamocyten von Asta-
cus astacus (L.) (Crustacea). Z. Zellforsch. 122:68-75.
UNESTAM, T., AND J.-E. NYLUND. 1972. Blood reactions in vitro in crayfish against a fungal parasite,
Aphanomyces astaci. J. Invertebr. Pathol. 19: 94-106.
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Reference: Biol. Bull. 173: 188-204. (August, 1987)
IMPULSE PROPAGATION AND CONTRACTION IN THE TUNIC
OF A COMPOUND ASCIDIAN
G. O. MACKIE AND C. L. SINGLA
Department of Biology, University of Victoria, Victoria. British Columbia, Canada V8W 2Y2
ABSTRACT
Diplosoma listerianum and D. macdonaldi (Earn. Didemnidae) have a network
of cells ("monocytes") in the tunic which contain high concentrations of microfila-
ments and react positively with NBD-phallacidin, indicating the presence of F-actin.
The tunic is contractile, especially in the areas around the cloacal apertures, which
can be closed completely. Myocytes are concentrated in sphincter-like bundles
around these openings, but also are found throughout the tunic. Electrophysiological
recordings reveal a diffuse conduction system in the tunic propagating all-or-none
impulses ("tunic potentials," TPs) through all parts with a conduction velocity of
< 1 .5 cm • s ', and a refractory period of 1 .6 s. TPs correlate one-for-one with contrac-
tions. The system is excitable to the touch, but is also spontaneously active, showing
steady patterns of potentials as well as regular, 'parabolic1 bursts. The evidence sug-
gests that the myocyte net itself conducts the impulses triggering the contractions. In
the absence of conventional nerves and muscles, the system provides the colony with
a way of regulating the effluent water current and hence the volume of a common
cloacal space.
The TP system is not 'wired in' to the ascidiozooids either as a sensory or as a
motor pathway. The tunic acts as an independent behavioral entity.
INTRODUCTION
The ascidian tunic or test is "an outer covering which completely surrounds the
individual zooid in solitary ascidians or forms a common groundwork in which the
zooids are embedded in colonial species." (Goodbody, 1974). It is a secretion product
of the body wall epithelium and consists of a matrix of proteins and carbohydrates
(including cellulose) into which cells migrate from the hemocoel during develop-
ment. Blood vessels often penetrate the tunic, and sensory processes from receptors
whose cell bodies lie in the underlying epithelium may also extend into the tunic
(references in Bone and Mackie, 1982) but muscles and nerves' are absent. The vari-
ous cells present may be concerned with secretion of tunic materials, phagocytosis,
self-nonself discrimination, coloration, and some other less well understood func-
tions. Some tunic cells are capable of movement and have contractile pseudopodia or
filopodia, but the contractions reported are very slow (< 1 14 ^m per hour in Botryllus
according to Izzard, 1974). Several authors (e.g., Saint-Hilaire, 1931;Godeaux, 1964)
have likened the tunic to mesenchyme. Brien ( 1 966) calls it "a living envelope, equiv-
alent to a sort of peripheral mesenchyme." Unlike mesenchyme, however, it is not
Received 9 April 1987; accepted 26 May 1987.
' There appears to be only one report of nerve cells in the tunic of an ascidian, that of Das (1936). No
later study on tunic histology supports this claim.
188
TUNIC RESPONSE SYSTEM 189
covered by epithelium but is exposed to the environment, and in this respect it more
resembles a cuticular or exoskeletal tissue.
Given the absence of nerves and muscles from the tunic, it is not surprising that
there have been no reports that the structure responds to stimulation, contracts, or
'behaves' in the usual sense, although in several cases it is composed of a fairly plastic,
viscous material capable of short-term conformational changes (Delia Valle, 1908;
Godeaux, 1964). During observations on Diplosoma listerianum, however, it became
clear that this species has a tunic in which electrical signals propagate on an all-or-
nothing basis, mediating contractions of the tunic itself. In this report, the electro-
physiological characteristics of this conduction system are described, along with an
account of the activities performed and of the cells likely responsible for conduction
and contraction. The evidence implicates a novel type of cell ("rnyocyte") as the basis
for both conduction and contraction. These cells seem to combine the properties of
conventional nerves and muscles including the ability to function as pacemakers.
They are distributed throughout the whole tunic in the form of a dense network
which, it is proposed, constitutes the structural basis for the behavioral action system
whose electrical correlates are picked up with recording electrodes.
MATERIALS AND METHODS
Two species of Diplosoma were used in this study. D. listerianum Milne-Edwards,
1841, was obtained at the Stazione Zoologica in Naples, Italy. A species tentatively
identified as D. macdonaldi Herman, 1 886 was obtained at the Friday Harbor Labo-
ratories of the University of Washington, and at the Bamfield Marine Station, Barn-
field, British Columbia, Canada. D. macdonaldi and D. listerianum are very similar
and may be conspecific (Monniot, 1 974). The specimens collected at Naples grew on
the walls of the public display aquarium and elsewhere in the seawater system, where
they appear to be endemic. D. macdonaldi specimens were collected from rocks and
pilings in the intertidal zone. Following the method of Delia Valle ( 1 908 ), specimens
were removed from their natural substrates and transferred to glass slides or petri
dishes. There they attached after a few hours, subsequently resumed growth, ex-
panded and put out new attachment structures ("crampons"1). All the experiments
reported in this paper were performed on transplanted specimens maintained in run-
ning seawater in the laboratory. The bulk of the work was done at Naples, and
D. listerianum was used for all the illustrations except Figures 2, 8, and 9.
For histological study, pieces of tunic were dissected out and mounted as whole
mounts either fresh or after fixation and examined by phase contrast or Nomarski
differential interference contrast microscopy. NBD-phallacidin (from Molecular
Probes Inc., 24750 Lawrence Road, Junction City, Oregon 97448) was used as a
specific fluorescent stain for F-actin. Material was embedded in Epon 8 1 2 for electron
microscopy after standard fixation and processing.
Electrophysiological study was carried out on small, whole colonies which had
become well established on their glass or plastic substrates. A slow flow of water was
maintained through the preparation dish during the experiments to ensure that the
colonies behaved as nearly as possible as in nature. Thus, temperatures in the prepara-
tion dish were kept close to those in the seawater systems at the laboratories where the
animals were maintained ( 17-19°C at Naples, 1 1-1 3°C at Friday Harbor). A simple
thermistor flow meter (Mackie et ai, 1983) was used to record changes in water flow
velocity out of the cloacal apertures. For stimulation and recording, polyethylene
suction electrodes were used. Signals were amplified and displayed on an oscilloscope
or on a chart recorder. For consistency with our earlier papers on tunicate electro-
190
G. O. MACKIE AND C. L. SINGLA
cloacal aperture
— zooid
common cloaca
crampon "
FIGURE 1 . Diplosoma listerianium, cut-away drawing after Lahille ( 1 890). The zooids hang by their
oral siphons from the upper tunic layer and are anchored below by strands of tunic drawn up from the
basal tunic layer, which is attached to the substrate by "crampons." Arrows show water flow.
physiology (Bone and Mackie, 1982) the polarity of the electrical records is arranged
so that negative events go up, positive down.
General description of Diplosoma and its activities
In Diplosoma and other didemnids the tunic is drawn out into thin sheets — an
upper sheet from which the zooids are suspended and a lower (basal) sheet which
attaches to the substrate (Fig. 1 ). The tunic material composing these sheets is directly
exposed to the seawater on both sides, and lacks an epithelial covering. A thin layer
of tunic encases the zooids (depicted by Carlisle, 1953) and this continues down into
an attachment strand ("stalk") which anchors the zooid to the basal tunic sheet. A
retractor muscle and fine blood vessels (30 /^m diam.) pass down the stalk from the
zooid. It is incorrect to refer to the stalk as the retractor muscle (e.g., Berrill, 1950) as
it is composed primarily of tunic, and the muscle penetrates it for only a short dis-
tance. The blood vessels entering the stalk, typically four (Pizon, 1905), enter the
basal sheet and run out into it, terminating in vascular ampullae. The ampullae con-
tract and expand, pulsating rhythmically as in other ascidians, but never swell to
more than 250 ^m in diameter. Contrary to the arrangement in colonial styelids such
as Botryllus, the blood vessels of different zooids are not interconnected. The vascular
ampullae are responsible for the formation of 'crampons' (ramponi, Wurzeln}: spe-
cialized patches of tunic material 180-240 ^m in diameter by which the basal tunic
adheres to the substrate. The ampullae, along with their blood vessels, may withdraw
after the crampons are complete, leaving behind an attachment strand of pure tunic
material. These strands are most conspicuous around the edges of the colony (Fig.
2). When elongated, they resemble the guy-ropes of a tent (Carlisle, 1 96 1 ). Crampons
are also present underneath the colony, roughly four per zooid stalk.
Water enters the colony through the oral siphons of the ascidiozooids. As the
zooids lack atrial siphons, water passes directly out into the common cloacal cavity
from which it finally exits via large cloacal apertures, which are often more than 1
mm in diameter. The exhalent water forms a plume that may rise to a considerable
height above the surface of the colony. Small apertures (<150 ^m) are also present
in the basal tunic (Fig. 1) and water passes through them into the narrow space be-
tween the tunic and the substrate and then to the exterior. The cloacal apertures are
simply holes in the tunic and should not be referred to as siphons, as they are not
parts of zooids. A single large cloacal aperture may serve as the exhalent water route
TUNIC RESPONSE SYSTEM
191
FIGURE 2. Diplosoma macdonaldi. A. Portion of a colony seen from above, showing a cloacal aper-
ture (ca), crampons (cr), and zooids, some with their oral siphons (os) in focus. B. Enlargement of edge,
showing two crampons, both containing vascular ampullae. The one on the left (am) is expanded, while
the one on the right — which comes from another zooid — is contracted. Arrows show the blood vessel of
the ampulla on the right.
for some 50 zooids. Stimulation of the tunic at any point results in slow closure of
the cloacal apertures, a response discussed in detail below.
A well-maintained colony which is actively feeding and growing in undisturbed
conditions tends to be flat, the stalks of its zooids very short (<100 /urn), and the
common cloacal space relatively small. The blood vessels passing down the zooid
stalks extend well out into the basal tunic. Around the edges of the colony these
vessels push out and form crampons (Fig. 2B). In colonies which are not feeding and
growing so vigorously or which have been kept in stagnant water for a few hours, the
blood vessels retract and retreat up the stalks into their zooids. trailing their ampullae
behind them. At the same time, the stalks elongate and are drawn out into thin
strands 1 mm or more in length. Elongation of the stalks accompanies swelling of the
cloacal space with exhalent water, and the whole colony expands. These changes,
documented in part by Delia Valle (1908, and earlier papers cited), seem to be a
response to changed water conditions, but it is interesting to learn that in Diplosoma
virens expansion and contraction are periodic events exhibited according to a diurnal
rhythm (J. S. Ryland, pers. comm.).
Didemnid colonies are known to be capable of locomotion (e.g., Delia Valle,
1908; Carlisle, 1961; Ryland et al, 1984). The exact mechanism of locomotion has
never been properly analyzed, but it involves the projection of finger-like tunic pro-
cesses containing blood vessels, whose ampullae form new crampons at attachment
sites ahead of the colony in the direction of movement. At the rear end of the moving
colony these attachment processes, vacated by their blood vessels, are stretched out
thin and eventually detach or break off. There is some evidence of positive phototaxis:
Delia Valle (1908) found that colonies tended to move upward in the public display
tanks at Naples — which are lit from above — stopping only when they reached the
surface. Carlisle (1961) found that Diplosoma moved sideways when illuminated
from the side. Crampons once formed cannot be lifted up and moved to another site,
so the movement cannot be thought of as a type of 'walking'; rather, it resembles the
192 G. O. MACKIE AND C. L. SINGLA
motion of a tracked vehicle, a slow flowing over fixed points which presumably re-
quires secretion of new tunic at the advancing end. The process requires further study.
The asckUozooids ofDiplosoma behave like solitary ascidians (reviewed by Bone
and Mackie, 1982), pumping water continuously when not disturbed, and contract-
ing their oral siphons and arresting their cilia in response to mechanical interference,
as for instance with the entry of an excessively large food particle. Stronger mechani-
cal stirnuSation causes retraction of the whole zooid by the retractor muscle which
runs down into the proximal part of the stalk. These activities are carried on indepen-
dently by the zooids. Stimulation does not cause the spread of zooid contractions or
ciliary arrests across the colony. This is in marked contrast to the situation in Bortryl-
lus and its relatives, where signals propagate through the colonial network of blood
vessels triggering behavioral events in the zooids (Mackie and Singla, 1983).
Histology
The living tunic is soft, pliable, and transparent. The ground substance shows no
regional differentiation except at the surfaces, where there is a thin (50 nm) cuticular
layer comparable to the "outermost cuticle" ofdona tadpole larvae (Gianguzza and
Dolcemascolo, 1984), but bearing a fuzzy surface coating 200 nm thick. There ap-
pears to be no counterpart to the subcuticular zone seen in adults of this and other
solitary ascidian species (De Leo et ai, 1981; D'Ancona Lunetta, 1983), but a layer
about 200 nm deep underlying the cuticle is more densely fibrous than in other re-
gions. Calcareous spicules are present (Carlisle, 1953) but are extremely small (<10
^m) and far apart. Conspicuous in all parts of the tunic are the large, spherical, vacuo-
lated cells termed "kalymmocytes" by Salensky ( 1 892) which are probably the coun-
terparts of the bladder cells (Blasenzelleri) or Saint-Hilaire (1931) and the cellules
vesiculeuses of Godeaux (1964). Peres (1948) — one of the few authors to study post-
larval Diplosoma — calls them "lacunae," which is clearly inappropriate, as they are
cells, not spaces. Also present are cells resembling the granulocytes, morula cells,
phagocytes, and other immigrant blood cells described in various tunicates by various
authors. Much uncertainty surrounds the identification of such cells, but this is irrele-
vant to the present discussion. Bacteria are usually present in the tunic ground sub-
stance.
Of particular interest in the context of the present investigation are two cell types,
both with processes interconnecting to form networks. Neither of these is clearly iden-
tifiable on the basis of previous descriptions, so they will be given new names: filo-
podial cells and myocytes. Filopodial cells (Fig. 3A) are restricted to the surface layer
of the tunic, while the myocytes lie deeper. Filopodial cells are flattened in the plane
of the surface layer, with three or more broad cytoplasmic expansions resembling
neuronal growth cones, each of which subdivides into numerous fine filopodia. The
filopodial cells form a fairly regular network, and are spaced out so that the filopodia
just make contact. The cells termed myocytes (Fig. 3B, C) are usually bi-, tri- or
multipolar, with thicker, much longer processes than the filopodial cells. Their pro-
cesses show few branches, and rarely subdivide to form filopodia. They are fairly
straight, and run for considerable distances through the territories of adjacent myo-
cytes, making numerous contacts with other such processes. The myocyte layer is
thick, not two-dimensional like that of the filopodial cells. The myocytes are present
in all parts of the tunic but are concentrated into sphincter-like bundles around the
cloacal apertures (Fig. 3C) and around the necks of the ascidiozooids. Their presence
and circular orientation in these places strongly implicates them in the role of the
contractile elements responsible for constricting the cloacal apertures and for pulling
TUNIC RESPONSE SYSTEM 193
in the tunic over the ascidiozooids when retracted, hence the designation "myocyte."
The filopodial cells seem less likely to fulfill such a role, as they show no such concen-
trations around the openings, and because their processes seem too delicate to be
effective as contractile elements.
Material stained with NBD-phallacidin and examined under a fluorescence mi-
croscope at 460 nm showed the myocytes as uniformly fluorescent objects, indicating
the presence of F-actin (Fig. 4A). Kalymmocytes also reacted positively, but other
cells in the tunic showed little response. The filopodial cells showed a very weak fluo-
rescence, and only their thicker processes could be seen at all.
Under the electron microscope (Fig. 5), the myocytes are characterized by dense
masses of rather loosely arranged fine microfilaments. True smooth muscle in ascidi-
ans by contrast shows thick and thin myofilaments arranged in strictly parallel arrays
(Nevitt and Gilly, 1 986). Further, using NBD-phallacidin, true muscle from the man-
tles of the ascidiozooids in Diplosoma showed a much stronger fluorescent reaction
(Fig. 4B) than was apparent in myocytes in the same preparations. For these reasons,
and because of their arrangement in the form of a diffuse plexus, it seems appropriate
to recognize the myocytes as a new cell type distinct from conventional smooth
muscle.
As noted, the filopodial cells and the myocytes lie in different layers of the tunic,
and show few points of contact; therefore, while it is conceivable that the filopodial
cells could represent a primitive, neuroid conduction network mediating responses
of the myocytes, the likelihood of this seems remote. However, we do not know what
function the filopodial cells serve.
RESULTS
The tunic pulse system: basic properties
Colonies growing in good condition on slides sometimes show no electrical activ-
ity in the tunic. Usually, however, it is possible to detect spontaneous patterns of
small electrical impulses (tunic pulses, TPs) which propagate without decrement
through all parts of the tunic, showing no alteration in wave form even when recorded
at distances of several centimeters from the site of initiation. They can be conducted
through narrow bridges of tunic less than 0.5 mm wide. They are exhibited in newly
settled colonies with only two or four zooids. TPs may be evoked by tactile and elec-
trical stimulation of the tunic as well as appearing spontaneously. Their characteris-
tics may be summarized as follows:
Wave form. When the electrode is first attached it may be impossible to detect
any signal above the noise level, as a dense plug of tunic must first fill the tip of the
electrode. However, once the electrode is well attached, and usually after 30 minutes,
signals can be recorded without difficulty for hours or even days. With fine suction
electrodes (ca., 50 yum I.D.) attached to the surface of the tunic, the signals are re-
corded as initially positive-going events rarely exceeding 100 v\ in amplitude, with
a small but long-lasting negative after-potential (Fig. 6A), and a total duration of
about 2.5s. With larger-bore electrodes the events are more symmetrically biphasic.
Recordings from the inner and outer surfaces show similar TP wave forms and ampli-
tudes. Attempts to record intracellularly from the myocytes failed, so interpretation
of these extracellularly recorded events is difficult, but they are probably compound
action potentials representing the summed depolarizations of many conducting ele-
ments. Somehow, the topography of the electrode attachment area converts these
summed negative events into a predominantly positive-going signal. With a fine elec-
194
G. O. MACKJE AND C. L. SINGLA
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TUNIC RESPONSE SYSTEM
195
FIGURE 4. NBD-phallacidin: A. Fluorescent reaction in myocyte net (arrowheads), and in shrunken
kalymmocytes (k); B. In conventional muscle from mantle wall of a zooid.
trode there would be relatively few conducting elements contributing to the signal,
and they would tend to fire in synchrony so the wave form shown in Figure 6A may
closely approximate the fundamental event recorded d.c. from a single cell.
Slow conduction. Conduction velocity is 1.0-1.5 cm-s ' at 19°C (Fig. 6B). No
.
0-2 (Jm
2 pm
FIGURE 5. Electron micrographs of a myocyte (A) and its process enlarged (B), showing fibrillar
contents. Bacteria (b) are often present in the tunic ground substance.
196 G. O. MACKIE AND C. L. SINGLA
J
FIGURE 6. Tunic pulses (TPs). A. Spontaneous TP recorded under optimal conditions with a fine
(50 Mm I.D.) extracellular suction electrode (scale bars: 1 s, 100 /*V). B. A TP recorded sequentially from
the inside of the basal sheet of the tunic (upper trace) and from the outside of the upper sheet (lower trace)
following a shock (*) on the basal sheet. Recording electrodes were 3 mm apart, conduction velocity 1.3
cm-s ' (scale bars: 100ms, 50 ^V). C. With two shocks (*) 1.6s apart, a response was elicited only to the first
shock (upper trace). When the interval between shocks was increased to 1.8 s, both shocks were followed by
TPs (scale bars: 0.5 s, 50 ^V). D. A mechanical stimulus (arrowhead) elicited a burst of TPs (scale bars: 10
s, 200 j/V).
significant variations in conduction velocity were observed in different parts of the
tunic. Conduction time increases markedly with successive shocks. With shocks at 7
s intervals, conduction time increased by 50% of its initial value after only 6 pulses.
It is not clear if increasing conduction time is due to slower conduction or to passage
of impulses via less direct routes.
Long refractory period. At 19°C, the absolute refractory period was 1 .6 s (Fig. 6C).
In the figure, a second response was obtained with two shocks 1.8 s apart, but the
amplitude of the second TP was considerably reduced, and showed a longer latency.
A long refractory period would be expected if the action potential has a long duration,
as proposed above.
Mechanical and electrical excitability. TPs can be evoked by pinching or pricking
the tunic (Fig. 6D) or by delivering electrical shocks through a suction electrode at-
tached to it. As with the recording electrodes, a plug of tissue must fill the tip of the
stimulating electrode firmly before experiments can begin. Large shocks are needed,
undoubtedly due to current shunting through the aqueous component of the tunic.
Responses can usually be obtained with stimulator settings of 30-50 V, 2-5 ms, using
an electrode with an internal tip diameter of about 120 /^m. Chemical sensitivity was
not examined in detail, but the mucus and body fluids of a small keyhole limpet
(species undetermined) which is the most obvious predator ofDiplosoma colonies in
the seawater system at the Stazione Zoologica had no effect on spontaneous pulse
patterns recorded from the tunic. The TP system does not appear to be affected by
changes in light intensity, but this aspect also needs further study.
Spontaneity. Specimens studied in as near to natural conditions as attachment of
electrodes would allow showed either (a) absence of all electrical activity, (b) steady,
almost metronomic pulse trains going on for periods of hours in some cases, typically
with TPs 7-10 seconds apart (Fig. 7 A), or (c) bursts of TPs repeated at regular inter-
TUNIC RESPONSE SYSTEM
197
t
1
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J
i
1
!
I
|
B
FIGURE 7. Spontaneous TP patterns. A. Steady pulse pattern (scale: 10 s). B. 'Parabolic' burst (scale:
5 min). C. Resetting of steady TP pattern by delivering shocks (*) to produce premature firing of the system.
Note that the TP elicited by the shock and the one following are both of reduced amplitude (scale: 1 min.)
D. Termination of steady, spontaneous TP pattern by electrical stimulation (*), causing a high frequency,
artificial TP burst (6 TPs, 6 seconds apart, scale: 30 s).
vals. These sequences vary considerably, but in typical long-term burst patterns, the
bursts last about 10-18 minutes (Fig. 7B), comprise 20-35 individual TPs, and are
followed by 10-18 minutes of silence before the next burst. Spike frequency increases
during the early part of the burst and decreases toward the end. If spike frequencies
are plotted graphically, the curve approximates to a parabola. Parabolic bursting is
typical of many pacemakers e.g., many molluscan neurons (Strumwasser, 1968). In
a preparation exhibiting a steady TP pattern, delivering a shock slightly before the
next predicted spontaneous event resets the pacemaker (Fig. 7C). A steady TP rhythm
can be terminated or interrupted by stimulating the preparation at a frequency greater
than the rhythm frequency (Fig. 7D).
The ability to produce pulse trains and burst patterns is not restricted to any par-
ticular part of the tunic. Small pieces of tunic with no zooids in them from various
parts of the upper and basal sheets produced rhythms similar to those seen in intact
colonies.
Effect of elevated Mg2+. TP rhythms continued unaffected in 81 mM Mg2+. In
105 rrLMMg2+, spontaneous TP patterns ceased, but the system could still be excited
electrically. In 1 50 mMMg2+, all TP activity ceased. These findings suggest that either
conduction, contraction, or junctional transmission in the myocyte network is de-
pendent on extracellular calcium, as magnesium ions block calcium channels (Hagi-
wara and Takahashi, 1 967).
Effect of curare and acetylcholine. Tubocurarine chloride had no effect on the
wave forms of TPs nor on their spontaneous patterns when used at concentrations
up to 5 X 10~5 g-ml ' over 24 hours. Addition of acetylcholine chloride to the same
final concentration had no detectable effect. These findings suggest that nerves are
not involved in the tunic responses, as peripheral nerves in tunicates typically operate
through cholinergic synapses (e.g., Florey, 1967; Mackie et ai, 1974).
Electrical activity ofascidiozooids
Recordings from the zooids show ciliary arrest potentials (CAPs) like those de-
scribed in numerous other tunicates (reviewed by Bone and Mackie, 1982). As re-
ported by Mackie (1974) for another compound ascidian, Distaplia occidentalis, the
CAP patterns of different zooids in the colony show no coordination. Attenuated
198
G. O. MACKIE AND C. L. SINGLA
FIGURE 8. Cloacal aperture before (A) and after (B) stimulation of the tunic. Three TPs were elicited
10 seconds apart, leading to reduction of the circumference of the aperture by 17%.
CAPs can be recorded a short distance down the zooid stalk and in the upper sheet
of the tunic close to the zooids; these signals are probably picked up electrotonically,
rather than being conducted events.
Recordings from the vascular ampullae show small potentials similar to those
recorded from the ampullae of colonial styelids and Perophora, and like them exhib-
ited in a rhythm coinciding with the contractions which propel blood through the
system (Mackie and Singla, 1983). Ampullae belonging to the same zooid are coordi-
nated, but those of different zooids are not. The two ampullae shown in Figure 2B
belong to different zooids and are out of phase. Cycle time is about 140 s and, as in
Botrylloides, the potentials typically occur in doublets.
Effector correlates of tunic pulses
So far as we know, tunic pulses have no relationship to the electrical pulse patterns
recorded from the zooids, and vice versa; nor do TPs seem to be involved in the
locomotory process. Locomotion has been observed in colonies showing no TP pat-
terns as well as in those showing such patterns. In fact, it seems unlikely that locomo-
tion is controlled by any colony-wide coordinating system. The pulsatile movements
of the blood vessels and vascular ampullae certainly play a part in locomotion but
they are not coordinated on a colonial basis.
The only clearly demonstrable effect of TP activity is the contraction of the cloacal
apertures (Fig. 8). Constriction of the aperture results in an increase in the rate of
water flow through the opening. This occurs in a stepped manner, with each step
corresponding to a single TP (Fig. 9). Following cessation of TPs, the aperture relaxes
slowly. This effect of TPs can be observed both during experimentally induced and
spontaneous TP activity, given repetitive firing at a sufficiently high frequency.
For more detailed study, given the sluggish nature of the response, it was conve-
TUNIC RESPONSE SYSTEM
199
FIGURE 9. Change in rate of water flow through a cloacal aperture following stimulation of the tunic.
A stimulating electrode (S) on the tunic evokes TPs, picked up with a recording electrode (R) and shown
as small events following large stimulus artifacts on upper trace. Lower trace shows stepped increase in
flow rate accompanying the stimulus train, recorded with a glass based thermistor flow meter (F). Following
the stimulus train, flow rate returns to normal as the cloacal aperture dilates.
nient to monitor changes in the cloacal apertures visually, using a scalar eyepiece to
measure diameters, from which changes in circumference could be calculated. (The
myocytes are arranged in circular arrays around the openings, so changes in circum-
ference represent length changes in the contractile tissue. ) As expected, long TP bursts
produce more contraction than short TP bursts at any given pulse frequency. With
shocks set to evoke TPs at intervals of six seconds, summation of contractions is
approximately linear until the preparation has shortened to about two-thirds of its
resting length, when the curve flattens out (Fig. 10). Pulses more than about 15 sec-
onds apart do not usually produce a summing response. It was observed that relax-
ation following contraction generally involves a period of hyperextension, after which
the preparation returns to its resting length (Fig. 1 1 ), but no TP activity accompanies
this final phase. Finally, it was shown that with long duration pulse trains at any
given frequency, the preparation fails to maintain the level of contraction exhibited
initially, but lengthens to a plateau level which is maintained indefinitely (Fig. 12).
100
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8
u
50
5 10
Number of pulses at 10 p.p.m.
15
FIGURE 10. Summation of contractions during TP trains evoked by stimulation at 10 pulses per
minute.
200
G. O. MACKIE AND C. L. SINGLA
o>
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last pulse
a 3 pulses
« 1 5 pulses
-I
time (mins)
FIGURE 1 1 . Changes in circumference of a cloacal aperture following TP trains of 3 pulses and 1 5
pulses, both at 10 pulses per minute.
For the experiments reported above, stimulation parameters were deliberately
kept at a moderate level, so that each shock produced a single propagated TP.
Stronger stimulation which causes multiple firing of the TP system, or repetition of
normal stimuli at higher frequencies can produce almost complete closure of the
apertures. Under these circumstances, the whole upper surface of the tunic has con-
tracted to some extent, and the cloacal space has diminished. Therefore, although no
attempt was made to quantify these observations, it seems clear that the contraction
of the cloacal apertures is only part of an overall contractile response involving the
whole tunic.
Re-examination of Botryllus
The discovery of a tunic conduction system in Diplosoma raised questions about
our earlier results with Perophora and with Botryllus and its relatives (Mackie and
E
o
5
• — control
Time (mins)
FIGURE 12. Changes in circumference of a cloacal aperture as observed over a five minute period
with stimulation at two different frequencies, and with an unstimulated control. Each shock produced a
single TP.
TUNIC RESPONSE SYSTEM 201
Singla, 1983), where we found that coordination of colonial activities occurred by
epithelial conduction in the blood vessels connecting the zooids. It is conceivable that
in these cases conduction also involves myocytes in the tunic itself. Therefore, the
earlier investigation was repeated using B. schlosseri, which grows on the walls of the
storage tanks at the Naples aquarium. The earlier results were correct. The propa-
gated signals in Botryllus can be recorded only from the vascular ampullae and blood
vessels. There is no sign of conduction in parts of the tunic where there are no blood
vessels. It was confirmed that the blood vessel impulses ("network potentials," NPs)
cause ciliary arrests in the zooids, as earlier claimed. Therefore, this NP system in
Botryllus is distinct from the TP system in Diplosoma. It is interesting that Diplosoma
has a version of the NP system, but it operates only within the confines of individual
zooids, and presumably functions to coordinate the contractions of the four vascular
branches which run out into the tunic from each zooid. Thus, the NP system occurs
in Aplousobranchs (Diplosoma), Phlebobranchs (Perophora), and Stolidobranchs
(various Botryllinae) and must be regarded as a basic ascidian action system. To date
the TP system has been identified in only one family of aplousobranchs, the Didemni-
dae, represented in Diplosoma, and may be peculiar to this group.
DISCUSSION
The evidence presented here demonstrates the ability of the tunic of a didemnid
ascidian to conduct all-or-none propagated impulses in response to electrical stimula-
tion and for these signals to cause contractions of the tunic. No such findings have
been reported for other species, and it seems probable that the properties of conduc-
tion and contraction are not widespread in the Ascidiacea, and may indeed prove to
exist only in the family Didemnidae. The system enables the colony to control its
exhalent water stream, a function performed in most ascidians at the individual zooid
level, by muscles in the walls of the atrial siphons. The zooids in didemnids lack atrial
siphons, and the only way of controlling water outflow is by regulating the size of the
openings in the tunic (the common cloacal apertures). Therefore it seems possible
that the properties of conduction and contraction in the didemnid tunic evolved in
parallel with the reduction and loss of the atrial siphons of the zooids, primarily as a
way of allowing the organism to control its exhalent water currents.
It is not clear exactly what benefits would be associated with the ability to regulate
water flow through the colony. Strong stimulation can produce almost complete clo-
sure of the cloacal apertures, which might be advantageous in the presence of a preda-
tor. Less strong stimulation causes constriction of the apertures and produces narrow,
high velocity water plumes, which rise to a greater height above the colony; this would
reduce the amount of water recycled through the colony and increase advection of
fresh water from the surroundings. Contractility also allows the colony to regulate
the volume of water in its cloacal cavity thereby enabling it to expand or contract, an
adaptation that might be put to a variety of uses. As noted earlier, Diplosoma virens,
which possesses photosynthetic symbionts (Prochloron) in its tunic, expands and con-
tracts on a diurnal basis (J. S. Ryland, pers. comm.).
We have searched in vain for evidence that the tunic conduction pathway medi-
ates protective responses of the zooids. The majority of colonial animals have some
means of coordinating their defensive responses, and this is true of ascidian colonies
like Perophora and Botryllus, whose zooids are coordinated by signals transmitted
through the colonial vascular system (Mackie and Singla, 1983). But Diplosoma ap-
pears to be an exception. Here there is no colonial vascular network and impulses
propagated in the tunic conduction system seem to have no effect on the zooids.
202 G. O. MACKIE AND C. L. SINGLA
Indeed, as Delia Valle (1908) remarked in the context of locomotory behavior, the
tunic has its own 'individuality,' meaning that it has a life of its own, functioning
without reference to the zooids contained in it.
Re; sg the cellular basis for conduction and contraction in the tunic, there
can be little doubt that the cells termed myocytes are responsible for the contractions.
It alsc >eems likely that these cells conduct the electrical signals for their own contrac-
he only other cells arranged in a net-like configuration — the filopodial cells —
lie in a different layer of the tunic and make few contacts with the myocytes, so they
are probably not the conducting elements. There is nothing inherently unlikely in
the idea of a primitive contractile system which conducts its own impulses. Vertebrate
cardiac muscle and many sorts of smooth muscle show this ability. However, we are
hestitant to call the cells in question muscle cells because they exhibit a lower level
of differentiation than true smooth muscle cells in tunicates, both in terms of their
general morphology and of their ultrastructure. The term 'myocyte' seems best for
these actin-loaded cells which lack thick myofilaments, are arranged in a loose net-
work, conduct impulses very slowly, and show very long contraction latencies.
Non-muscle contractility is well developed in ascidians. Tail resorption in ascid-
ian tadpoles involves the rapid transformation of squamous epithelial cells into tall,
flask-shaped cells during which actin microfilaments become aligned in the apical
(Distaplia) or basal (Botryllus) cytoplasm. Discussing these findings, Cloney (1982)
states that "the caudal epidermis clearly provides the driving force in tail resorption."
Sperm release in Ciona involves contraction of the sperm duct epithelium, again by
organization of actin microfilaments. The assembly of the filaments is triggered by
light (Woollacott and Porter, 1977). Microfilaments are also involved in the contrac-
tions of the vascular ampullae of colonial styelids like Botryllus, Botrylloides, and
Metandrocarpa (DeSanto and Dudley, 1969; Katow and Watanabe, 1978; Mackie
and Singla, 1983). The epithelial cells in these cases communicate via gap junctions,
which presumably provide intercellular pathways for transmission of the impulses
which coordinate the contractions of the ampullae. A similar mechanism may apply
to the myocyte network in the tunic of Diplosoma, but intracellular recordings and
demonstration of coupling between myocytes are required to prove this. The possibil-
ity that the myocytes communicate via chemical synapses cannot be ruled out, espe-
cially in view of the system's sensitivity to magnesium.
Thus we believe the tunic myocyte net is a system evolved de novo in Diplosoma
and probably in other didemnids to bring about coordinated contractions of the ex-
halent water openings, thus bringing water flow under colonial control. Contractions
are slow, conduction velocity is the slowest on record for any animal with the excep-
tion of hexactinellid sponges (Mackie el ai, 1983), the system has a limited carrying
capacity in terms of impulse frequency, and it appears to fatigue very quickly. Nerves
and muscles probably would allow the animal to respond with much more alacrity;
however, there are no nerves or muscles in the tunic of any ascidian, so it would
seem that there was no evolutionary starting point for a conventional neuro-muscular
system and a completely new type of cell — the myocyte — had to be evolved, albeit
as a rather inefficient substitute.
These findings emphasize the unusual versatility of the Tunicata in developing
mechanisms of colonial coordination without ever using simple, direct nervous inter-
connections. Diplosoma uses an excitable myocyte network in the tunic, Botryllus
uses its excitable vascular epithelium, the zooids in a colony of Pyrosoma signal to
each other by responding visually to each others' biolurninescent flashes (Mackie and
Bone, 1978), and salps relay signals between zooids by excitable epithelial pathways
arranged in series with nerves (Bone et al, 1980; Anderson and Bone, 1980). It is
unlikely that these examples exhaust the list of possible mechanisms.
TUNIC RESPONSE SYSTEM 203
ACKNOWLEDGMENTS
The bulk of the experimental work reported here was performed during a visit by
G.O.M. to the Stazione Zoologica, Naples, Italy, aided by travel and operating grants
from the Natural Sciences and Engineering Research Council of Canada (NSERC).
We are most grateful to the Director and staff of the Stazione Zoologica, and particu-
larly to Dr. Amedeo de Santis, for making facilities and a complete set of electronic
recording equipment available for this study. Some follow-up work was performed
at the Friday Harbor Laboratories of the University of Washington, Friday Harbor,
Washington, and we thank the Director, Dr. A. O. D. Willows for providing space and
facilities there. C.L.S. thanks NSERC for salary and funding for use of the electron
microscope facilities of the University of Victoria. Ms. Joan Glazier of the Bamfield
Marine Station, British Columbia, collected and shipped to us specimens of Diplo-
soma macdonaldi.
During the preparation of this work, we have had helpful correspondence or dis-
cussions with Dr. Stuart Arkett, Dr. Michael Cavey, Dr. Richard Cloney, Ms. Sarah
Cohen, Dr. Jean Godeaux, Dr. John Ryland, and Dr. Thomas Schroeder.
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Reference: Biol. Bull. 173: 205-221. (August, 1987)
BIVALVE HEMOCYANIN: STRUCTURAL, FUNCTIONAL, AND
PHYLOGENETIC RELATIONSHIPS
C. P. MANGUM1, K. I. MILLER2, J. L. SCOTT', K. E. VAN HOLDE2,
AND M. P. MORSE3
Department of Biology. College of William and Mary, Williamsburg, Virginia 23185;2Department of
Biochemistry and Biophysics, Oregon State University, Con'allis, Oregon 97331; and* Marine Science
Center and Biology Department, Northeastern University. Nahant. Massachusetts 01980
ABSTRACT
The hemocyanin-like molecule found in the blood of the most primitive bivalves
(protobranchs) reversibly binds O2. Its respiratory properties and its sedimentation
behavior are both distinctive. Although its electron-dense image looks like that of the
gastropod hemocyanins, its molecular weight differs from those of all other molluscan
Hcs and is more consistent with the concept of bivalve hemocyanin as a pair of octo-
pod hemocyanins. Bivalve hemocyanin occurs in the solemyoids as well as the nucu-
loids, which argues for the integrity of the Protobranchia as a natural taxon. The
ancestral bivalve O2 carrier was previously believed to be a simple intracellular hemo-
globin, which is found in the less primitive Pteriomorpha. The most obvious interpre-
tation of the present results, however, is that hemocyanin is the primitive bivalve O2
carrier and that it was replaced by the red blood cell, which originated at least twice:
once in the pteriomorph bivalves and at least once in other taxa.
INTRODUCTION
Recently Morse el al. (1986) reported the presence of electron dense images that
resemble molluscan hemocyanins (Hcs) in the blood of two nuculoid bivalves. In
addition, the denatured subunits exhibited the same anomalous behavior during so-
dium dodecyl sulfate polyacrylamide electrophoresis as those of some other mollus-
can Hcs, viz., they migrated to a position corresponding to a lower molecular weight
than expected from other aspects of quaternary structure (Van Holde, 1983; Ryan et
al., 1985). Finally, copper electrons were identified in the blood of one species by X-
ray spectroscopy of sections of the auricle. Thus the evidence indicates the existence
of a molecule that closely resembles molluscan He in the most primitive members of
the class Bivalvia, which was formerly believed to use either heme proteins or no O2
carrier at all (Mangum, 1 980a; Terwilliger and Terwilliger, 1985).
This finding has considerable phylogenetic importance. First, the Hcs found in
the various molluscan classes are believed to exhibit differences, albeit quite subtle
ones, in quarternary structure (Ryan et al, 1985). Therefore a comparison may eluci-
date evolutionary relationships between them. Second; it has been suggested that red
blood cells (RBCs), which occur in the somewhat less primitive pteriomorphs, may
represent the ancestral condition among the bivalves (e.g. , Mangum, 1 980a). Third, if
instead He is the primitive O2 carrier in the class, then the RBC must have originated
independently on at least two occasions, within the bivalves and in other taxa.
In the present contribution we report evidence of reversible O2 binding, which
demonstrates that the molecule in the blood of both groups (solemyoid as well as
Received 2 March 1987; accepted 1 May 1987.
205
206 C. P. MANGUM ET AL.
nuculoid) of protobranch bivalves is a typical O2 carrier, not a He-like precursor. We
also describe additional aspects of molecular structure and respiratory function that
enable comparison of the protobranch blood O2 carrier with the Hcs found in other
molluscs. Finally, we explore the implications of our findings for RBC phylogeny.
MATERIALS AND METHODS
Acila castrensis (Hinds) and Cyclocardia (=Venericardia) ventricosa (Gould)
were collected near San Juan Island, Washington. Yoldia limatida (Say), Nucula
proximo. Say, and Solemya velum Say were purchased from commercial sources in
Massachusetts.
O2 uptake (VO2) of intact animals was determined as the depletion of O2 in the
PO2 range 120-159 mm Hg, measured with a self-stirring polarographic electrode
(Yellow Springs Instrument Co. Model 54). To prevent extraneous O2 uptake by shell
epibiota, the animals were disinfected by immersion for a few seconds in benzalkoni-
um chloride (0.13%). Vacant shells given this treatment do not take up appreciable
volumes of O2.
Blood was obtained by first inducing the animals to empty their mantle cavities
and then prying open the valves about 1 mm or less with a jeweler's screwdriver. The
valves were reflected backwards about 270° and the animals placed in a small funnel
draining into an Eppendorf tube. When the blood ceased to drain, additional volumes
were obtained by centrifuging the animals at a very low speed. After repeated prob-
lems with apparent proteolysis during sedimentation analysis, we collected the blood
of Y. limatida by draining it directly into a mixture of protease inhibitors, which
appeared to enhance the stability of the preparation. About 1 ml blood was drained
into a 60 n\ solution containing 30 pg leupeptin, 30 pg pepstatin A, and 3 yumoles
phenylmethylsulfonyl fluoride.
The gills of Y. limatida were dissected and extracted with 0.5% Na2CO3. The
extract was diluted by 10% with pyridine, reduced with a few grains of sodium dithio-
nite, and, due to its very small volume, examined with Zeiss micro- and Hartridge
reversion spectroscopes (Mangum and Dales, 1965).
The bloods were centrifuged and immediately prepared for electron microscopy
(Zeiss EM- 109) by negative staining with uranyl acetate (Mangum etai, 1985; Morse
et al., 1986). In the present investigation the blood was diluted with 0.05 M Tris
maleate buffer + 10 mMCaC!2 (pH 7.63) by factors ranging from 1:9 to only 1:50,
because its appearance suggested a low He concentration. The sample and the stain
were applied to the grid with an atomizer.
O2 binding was determined on fresh (never frozen) blood samples from A. cas-
trensis, S. velum, and Y. limatida by the cell respiration method (Mangum and Lyk-
keboe, 1979). Due to the size of the N. proxima individuals (2-5 mm), it was neces-
sary to stockpile frozen material until the requisite volume (300 n\) accumulated.
About half of the material on which the measurements were performed had been
frozen. The samples were diluted by 10% with Tris maleate (final concentration 0.05
M) buffered seawater (32%o) containing commercial yeast cells. An attempt to first
concentrate A. castrensis blood by membrane filtration was unsuccessful due to pre-
cipitation of some of the material, which was also noted during the O2 binding mea-
surements. No precipitation of the Y. limatula, S. velum, or N. proxima material was
observed.
The O2 affinities of heme proteins are often concentration dependent. Because
most experimental procedures require dilute solutions, the results do not accurately
reflect in vivo values. To obtain physiologically meaningful data for the branchial
BIVALVE HEMOCYANIN 207
heme protein of Y. limatula, O2 binding in the present investigation was also deter-
mined using whole gills, dissected intact. An absorption spectrum of the bathing me-
dium indicated that there was no loss from the gills during the measurement. It was
necessary to modify the cell respiration procedure because the method requires that
the rate of free O2 depletion be linear. This is achieved by lowering the PO2 with
particles such as isolated mitochondria or yeast cells which are so small that the diffu-
sion distance is not limiting. If the O2 uptake of whole unperfused gills had been
allowed to make an appreciable contribution to total O2 uptake, this condition would
clearly have been violated and the apparent O2 affinity would have been erroneously
low due to an extraneous departure from linearity. The problem was circumvented
by first determining the VO2 of the gills and then adding large numbers of yeast cells
so that yeast VO2 was more than 10 times gill VO2. The cell respiration method also
requires that the rate of O2 depletion be slow enough to permit equilibration of the
electrode at any PO2. If this condition had been violated, the result would have been
an erroneously high O2 affinity, because an apparent oxygenation state would have
coincided with a PO2 that actually had obtained earlier, at a higher oxygenation state.
The possibility was eliminated by ascertaining equilibration under the following ex-
perimental conditions: using the He of the crab Cancer magister, whose respiratory
properties are well known (e.g., Graham el ai, 1983), the rate of O2 depletion was
increased until an erroneously high O2 affinity was obtained. The period for depletion
of free O2 from 100 to 0% air saturation was considerably less than 25 s. In the mea-
surements on gills, much longer periods (87-233 s) were employed.
Absorption spectra of the medium and of fresh Hcs were determined with Beck-
man DK-2A and Varian 2200 spectrophotometers. To eliminate light scatter, the He
samples were first diluted with dissociating buffer (0.05 MTris HC1, pH 8.95 + 0.05
M EDTA) by 50 to 97% depending on color intensity.
All sedimentation experiments were performed in a Beckman Model E analytical
ultracentrifuge equipped with scanner optics. Wavelengths in the vicinity of the He
bands at 280 and 345 nm were used. Temperature was controlled to <0.1°C. Sedi-
mentation coefficients were measured from the midpoints of the well-defined bound-
aries and corrected to S20.w in the usual way. The sedimentation equilibrium experi-
ment was conducted at 1 500 rpm, using the heavy J rotor. Equilibrium was attained
when no difference could be noted between scans approximately 6 h apart. After
equilibrium the rotor was accelerated to 6000 rpm and a baseline recorded approxi-
mately 4 h later.
RESULTS
O2 uptake
Intact individuals of Acila castrensis take up O2 measurably, but VO2 is more
than two orders of magnitude lower than in the heterodont Cyclocardia ventricosa
(Table I), which was collected from the same bottom on the same occasion and held
in the laboratory in the same container for the same period. The difference in body
size can account for only a small fraction of the difference in O2 uptake. Moreover,
VO2 is also orders of magnitude lower in A. castrensis than in the pterimorph Noetia
ponderosa, a much larger animal measured at a slightly lower temperature (Table I).
Absorption
The dissociated subunits of the four bivalve Hcs absorb at 280 and 345 nm (Fig.
2). Other molluscan Hcs absorb in the same regions (Nickerson and Van Holde,
208
C. P. MANGUM ET AL.
TABLE I
Oxygen uptake in bivalves with specialized oxygen carriers
Species
O2 carrier
VO2
(Ail/g dry wt-h)
Dry wt.
(mg)
Temp.
(°C)
Source
Ad la cti
Vene
••' "iricosa
He
extracellular Hb
Noeliaponderosa intracellular Hb
Glycimeris
nummaria intracellular Hb
2.0 ± 0.9(6) 107.9-122.8 11.5 present data
269.5 ± 15.5(6) 33.5-34.9
148 5 X 103
ca. 49. 5a 2.7X103 +16-20 Kruger, 1957
11.5 present data
Deaton and Mangum,
10 1976
1 Converted from original data assuming that dry wt. = 20% wet wt. Mean ± SE (n).
1971). R. C. Terwilliger kindly communicated data for polyplacophoran He, which
have not been reported in the literature. Observations on the He of the chiton Chae-
topleura apliculata were also made together with those reported here. The peak at
345 nm disappears in the presence of sodium borohydride (e.g., Fig. 1).
Extracts of the gill of Y. limatula clearly form a pyridine hemochromagen with
absorption bands at 542 and 556 nm. High concentrations of red granules also were
observed in the nerve ganglia and connectives of this species but not in A. castrensis or
N. proxima, which appeared to lack branchioglobin (Bb) as well. However, a seawater
extract of the whole bodies of N. proxima appeared to form a pyridine hemochroma-
320
340
3 50
FIGURE 1. Absorption spectra of protobranch Hcs. (A) Acila castrensis, (N) Nucula proxima, and
(S) Solemya velum. Abscissa is wavelength in nm and ordinate is absorbance.
BIVALVE HEMOCYANIN
209
B
100 „
10
80
60
4.0
e 2.0
E
1.0
0.8
0.6
10
\
0.1
70
72
7.4 76
PH
7.8
8.0
0.01
• o
• o
s
70
7.2
7.4
7.6
PH
78
80
82
1
1
1
0.1
1
10
po2
FIGURE 2. A. PH dependence of O2 binding by Acila castrensis (•), Nucula proximo. (D), Solemya
velum (<>), and Yoldia limatula (O) Hcs. 20°C, 0.05 M Tris maleate buffered blood. B. Hill plot of 62
equilibrium of Yoldia limulata (•, pH 8. 10.) and Acila castrensis (O, pH 7.78) Hcs.
gen, although the visual observation could not be confirmed even by microspectro-
scopic observation due to the very small volume (ca., 10 n\) obtained.
O2 binding of the bloods
Unlike other Hcs, A. castrensis He binds O2 non-cooperatively (e.g., Fig. 2). The
Hill coefficient («50), which is independent of pH, is 1 .03 (±0.03 SE, n == 11). Among
the Hcs, A. castrensis also has an unprecedentedly high O2 affinity (Fig. 2), thus re-
sembling tissue O2 carriers more than most blood O2 carriers. Unlike tissue O2 carri-
ers, however, A. castrensis He has a small but significant normal Bohr shift. The slope
of the regression line describing the data in Figure 2, or A log P5oM pH, is -0.23
(±0.08 95% C.I.)- HcO2 binding in A", proxima (which belongs to the same family
as A. castrensis) is also non-cooperative (0.94 ± 0.09 SE; n =: 7) and it has a similar
Bohr shift (-0. 16 ± 0.07 95% C.I.) although its O2 affinity is somewhat lower. The
He of Y. limatula (which belongs to a different family) has a much lower O2 affinity,
though still fairly high for a molluscan He, and it is moderately cooperative (Fig. 2).
Its Bohr shift is indistinguishable from that of the other nuculoid Hcs (-0.24 ± 0.05).
210 C. P. MANGUM ET AL.
FIGURE 3. Electron micrographs of bivalve Hcs. A. Yoldia limatula. B. Solemya velum. Scale bar
50 nm.
The He of S. velum (which belongs to a different order) resembles Y. limatula He in
terms of O2 affinity and cooperativity, but its Bohr shift is much larger (-0.6 1 ± 0. 1 7).
O2 carrying capacity of the bloods (HcO2 + O2) was estimated from absorbance
at the active site, using the extinction coefficient for Busycon He (Nickerson and Van
Holde, 1971). At 1 1.5°C and 32%o salinity the value for A. castrensis blood is 1.05
ml/ 100 ml, for S. velum is 1.00 ml/ 100 ml, and for one sample from Y. limatula is
0.96 ml/ 100 ml. N. proxima blood, which is much bluer than the others, carries 2.85
ml/ 1 00 ml. The figure for A. castrensis should be regarded as low due to precipitation
in the sample. However, a similarly low figure (0.76 ml/ 100 ml) for another sample
from Y. limatula was obtained from integrals of the curves describing deoxygenation
(procedure detailed by Mangum and Burnett, 1986). Moreover, the difference be-
tween N. proxima, S. velum, and Y. limatula cannot be due to starvation of the latter
two in the laboratory (which, in fact, has not been reported for molluscan Hcs) since
they were held for the same period (<2 days).
O2 binding by gills
Two determinations of O2 binding by intact Y. limatula gills, which should pro-
vide physiologically meaningful information, gave P50 values of 0.43 and 0.46 mm
Hg and n values of 0.98 and 1 .02 (20.3°C, ambient pH 8.0 1 ).
Electron microscopy
Since N. proxima is so closely related to A. castrensis, the small amount of mate-
rial available was used for other purposes. The shapes of S. velum and Y. limatula
Hcs (Fig. 3) are indistinguishable from that of A. castrensis He, which was described
earlier (Morse el al, 1 986). All three molecules are six-tiered cylinders and, like many
gastropod Hcs, appear as circles in top view and as squares in side view (see Ghiretti-
Magaldi et al., 1979, van Bruggen el al, 1981). They lack the "belt," or unequal
spacing of the six tiers, found in one species (van der Laan el al., 1981). The width
(31 nm) of the Y. limatula squares appears to be slightly but significantly (P < .001
according to Student's / test) smaller than that of 12 tiered cylinders found in the
blood of the gastropod Busycon canlicutatum. These dimensions were determined by
mixing a small volume of B. canaliculatum blood with a large volume of Y. limatula
BIVALVE HEMOCYANIN
211
CcTMg*
EDTA
YOLDIA
T T T
15S 96S
AC I LA
FIGURE 4. Scanner traces showing dissociation and reassociation of bivalve Hcs at pH 7.65, 20°C.
Yoldia limanda: ( 1 ) in 0.05 M Tris-HCl, 50 mM MgCL, 10 mM CaCl2; (2) dialyzed against 0.05 A/Tris-
HC1 + 10 mM EDTA; (3) dialyzed back again against the original buffer. Acila castrensis: (4) as in 1; (5)
as in 2; (6) as in 3. In 5 and 6 the middle boundary sediments at about 55S.
blood and then measuring the width of all (24) of the 12-tiered cylinders observed
and a sample of 100 6-tiered cylinders. The bivalve circles have a five-fold rotational
symmetry and a collar and a cap. When dissociated to halves, the molecule looks like
a three-tiered rectangle, which absorbs more stain at one end than the other, and as
circles, only some of which have collars and caps (see Fig. 3 in Morse et ai, 1986). The
images of half molecules, which have also been described for Helix He, are believed to
reflect the absence of collars and caps at the broken surfaces (van Bruggen et ai,
198 1 ). Like gastropod Hcs, the bivalve squares are about 35 nm long.
Physical characterization
Sedimentation velocity experiments with A. castrensis and Y. limatula Hcs were
performed at room temperature and under a variety of solvent conditions. The results
are summarized as follows:
(1) In 0.05 M Tris-HCl buffer (pH 7.65) containing 50 mMCaCl2 and 10 mM
MgCl2, both Hcs exhibited single, sharp boundaries. The sedimentation coefficients
(S°2o,w), when corrected to standard conditions and extrapolated to zero He concen-
tration, were 95.8 for A. castrensis He and 88.8 for Y. limatula He.
(2) When the two He solutions were dialyzed exhaustively against 10 mMEDTA
in 0.05 M Tris-HCl (pH 7.65), they behaved differently (Fig. 4). Y. limatula He disso-
ciated completely to yield a single boundary with S20.w : 15.9S. Under the same
conditions A. castrensis He showed incomplete dissociation, yielding two compo-
nents with S20,w ~-~- 54 and 18S. Attempts at reassociation also gave quite different
results. Upon dialysis of the Tris EDTA treated material back to Tris Ca+:: + Mg+2,
Y. limatula He quantitatively reassociated to the 89S component. Under the same
conditions only partial reassociation could be attained with A. castrensis He.
(3) When Y. limatula He was dialyzed against a series of dilutions of the Tris
buffer in which the divalent cations were reduced to '/10, '/20, '/to, and finally Vioo of
212
C. P. MANGUM ET AL.
1001
50-
5.0 mM MgCl2
1.0 mM CaCI2
FIGURE 5. Relative amounts of Yoldia limatula He of three aggregation states when equilibrated to
0.05 A/Tris buffers (pH 7.65; 20°C) containing varying amounts of MgCl: and CaCl2 (see text for details).
their original concentrations, the resultant dissociation yielded a mixture of three
components: the 89S He (the whole molecule), another with a sedimentation coeffi-
cient of 55S (probably a half molecule), and a third with a sedimentation coefficient
of about 15S (Fig. 5). The present data do not indicate just what multiple of the
polypeptide chain this smallest product represents, but the sedimentation coefficient
corresponds to that of the dissociation product in the presence of EDTA.
The reversible dissociation behavior of Y. limatula He strongly suggests that, like
Octopus He (Van Holde and Miller, 1985), it is composed of a single type of subunit.
In contrast, the incomplete reassociation of A. castrensis He is more like that of other
molluscan Hcs(Van Holde and Miller, 1982). Furthermore Y. limatula He, like Octo-
pus He, dissociates in the presence of EDTA at a much lower pH than normally
required for other molluscan Hcs. Although divalent cation levels must be reduced
to extremely low levels before dissociation begins, Y. limatula He dissociates at pH
7.65, which is probably close to the physiological value. In all likelihood, at higher
pH it would dissociate at higher divalent cation levels.
DISCUSSION
Respiratory properties and their relationships to protobranch biology
Allen (1978) suggested that protobranch bivalves are able to exist with their small
and, in his view, relatively inefficient feeding organs because they have low metabolic
rates. The present findings support his suggestion, at least with respect to aerobic
metabolism. However, we should point out that, relative to bivalves that both use the
gill as a filter-feeding organ and lack an O2 carrier, the branchial surface area is also
small in thepteriomorph Noetia ponderosa, whose feeding has not been investigated
and whose VO2 is not especially low (Mangum, 1980a). Almost certainly VO2 is in-
fluenced by other factors in addition to feeding efficiency. While it is believed that
conventional feeding in Solemya is supplemented or perhaps even supplanted by
a symbiotic relationship with chemoautotrophic bacteria (Cavanaugh, 1980, 1983;
Felbeck, 1983; Doeller, 1984; Fisher and Childress, 1984; Reid and Brand, 1986), no
sign of bacteria can be found in electron micrographs of the gills of protobranchs such
as A. castrensis (mentioned by Reid and Brand, 1986) and Y. limatula (M. P. Morse,
unpub. obs.).
BIVALVE HEMOCYANIN 213
The uniformly normal Bohr shift of the bivalve Hcs resembles those of polyplaco-
phoran and cephalopod Hcs. Gastropod Hcs have either reversed Bohr shifts (proso-
branch), a combination of reversed and normal Bohr shifts (prosobranch and pulmo-
nate), or none at all (opisthobranch). As indicated above, the extremely high O2
affinity and lack of cooperativity of A. castrensis He is unique. The moderate cooper-
ativity and O2 affinity of S. velum and Y. limatida Hcs are common among the mol-
luscs, although examples of much greater cooperativity are known (Mangum,
1980b). O2 carrying capacity appears distinctively low, at least in S. velum and Y.
limatida, but typical of molluscan HcO2 transport systems in N. proxima. Why the
nuculoids, with such similar respiratory and cardiovascular systems, should have Hcs
with such different respiratory properties remains to be elucidated.
The anatomical relationships between the protobranch O2 carriers also are in-
triguing. At least in Y. limatida and S. velum, O2 must move from the environmental
source into the heme protein-containing branchial epithelium. From there the O2
moves into the He-containing blood, where it is carried by convection to the meta-
bolic sink. But in both species the O2 affinity of the branchioglobin (Bb) is higher than
that of the He (see Doeller et ai, 1983, for values for S. velum Bb). The arrangement
violates the fundamental design principle of an O2 transfer system, which mandates
the highest O2 affinity in the compartment most remote from the environmental
source. Bb must actually be a barrier to O2 influx as long as it is not fully oxygenated.
The physiological question is complicated by uncertainty surrounding the func-
tion of bivalve Bb and other tissue heme proteins. Doeller (1984) suggested that S.
velum Bb transports sulfide to the chemoautotrophic bacteria in the gills; the sulfide-
oxidizing bacteria are believed to serve as key components of a newly discovered
mode of animal nutrition. As pointed out by Dando et al. (1985), this function does
not preclude the possibility of others, such as facilitated diffusion or O2 storage.
We noted that the period from onset to completion of nonlinearity of O2 uptake
by Y. limatida gills (in the absence of yeast cells) was only 162 s. This period includes
both the diffusion-limited and BbO2-supplied components. The molecule cannot be
an O2 store of significant longevity. We suggest that BbO2 carrying capacity also be
considered in the continuing debate on functions of tissue heme proteins.
Structural properties
The sedimentation coefficients observed for the bivalve Hcs are surprisingly low.
The Hcs that would seem to resemble bivalve He in shape are the six-tiered cylinders
found in the prosobranch whelk Kelletia and the pulmonate snail Helix (van Bruggen
et al., 1981). Opisthobranch and the other prosobranch Hcs studied tend to form
larger aggregates or have special features such as unequal spacing of the six tiers.
Cephalopod and polyplacophoran Hcs are three-tiered cylinders.
As Table II shows, however, almost all reliable measurements of the sedimenta-
tion coefficients of the gastropod six-tiered multiple yield values of S°20,w between 100
and 105S. We were struck by the value for Y. limatida He, which is 10-15% lower.
A lower value might be explained by either a looser quaternary structure, greater
hydration, or a lower molecular weight. The molecular weight obtained from Figure
6 depends on the value assumed for the partial specific volume (i>) (see Van Holde,
1985). Unfortunately we have neither an experimentally determined value for i» for
Y. limatida He nor an amino acid composition, from which it might be estimated.
Values reported for molluscan Hcs range from about 0.73 (gastropod) to 0.74 (cepha-
lopod). The former yields a molecular weight of 6.5 X 106 and the latter yields 6.8
X 106. Either is much lower than the values reported for the gastropod 100-105
S Hcs (Table II).
214
C. P. MANGUM ET AL.
TABLE II
Comparative properties of native hemocyanin molecules oj bivalves and gastropods
Species
S°
20,w
(svedbergs)
M
(g/molx 106)
V
(cm-Vg)
Class: Gastropoda1
A rchachalina marginata
102.3
9.1
—
Buccinitni undatum
101.1
9.0
—
Bus\ -con canal iculat um
103.2
8.8
.727
Helix pomatia (at)
104.3
8.7
.727
Helix pomatia (ft)
105.8
9.0
—
Murex trunculus
102.7
8.9
—
Paludina vivipara
102.5
8.7
—
Pila leopoldvillensis
101.2
8.7
—
Class: Bivalvia2
Acila castrensis
95.8
—
—
Yoldia limatula
88.8
6.5-6.8
—
' Data from Van Holde and Miller ( 1 982). Original references are given therein.
2 Present data. The value of M for Yoldia hemocyanin depends upon whether a value of 0.73 (as for
gastropods) or 0.74 (as for cephalopods) is assumed.
The data are more consistent with the concept of bivalve He as a pair of cephalo-
pod Hcs. A pair of polyplacophoran Hcs would have a much higher molecular weight
(Ryan et ai, 1985; Herskovits et ai, 1986). The cephalopod 51-60S particles, how-
ever, do not pair to form six-tiered cylinders. If they did, they might give rise to a
particle with a sedimentation coefficient of about 90S and an electron-dense image
much like that of Y. limatula He. If we assume that native Y. limatula He is a 20-
mer of polypeptide chains, like other 6-tiered molluscan Hcs, then the chain weight
must be approximately 3.4 X 105. This is very close to the weight of octopod chains
In A
49
5O
51
FIGURE 6. Determination of molecular weight of Yoldia limatula He by sedimentation equilibrium.
A represents concentration in arbitrary units of A345 nm; r is the distance from center of rotation.
BIVALVE HEMOCYANIN 215
(Gielens et al, 1986; Lamy et ai, 1986), and substantially smaller than that of gastro-
pod chains (4.0-4.5 X 105). Such a conclusion seemingly contradicts the observation
that bivalve chains (Morse et ai, 1 986) run more slowly than cephalopod chains (Van
Holde and Miller, 1 982) on SDS gels. However, SDS gel electrophoresis is notoriously
unreliable for glycoproteins such as Hcs. It has been frequently reported that esti-
mates of subunit molecular weight are in substantial error for these proteins.
On the basis of the available information we suggest that the bivalve Hcs may
resemble octopod Hcs in having a small (relative to other molluscan Hcs) subunit,
but share with gastropod Hcs the capacity to associate to 20-mers. This conclusion
is supported by the apparently smaller width of the bivalve cylinders. In possible
contradiction, however, we should mention that Ellerton and Lankovsky (1983) re-
ported a 26-30 nm wide and 28-34 nm long He in the primitive archaeogastropod
Haliotis iris.
Phylogeny of the molluscan Hcs
The most recent discussions of molluscan phylogeny suggest two major phyletic
lines leading from the postulated ancestor, which is in turn descended from an acoelo-
mate animal at the turbellarian-nemertine level of organization (Runnegar and Po-
jeta, 1986; Salvini-Plawen, 1986). One of these phyletic lines is an aplacophoran-
polyplacophoran lineage and the other leads through the monoplacophorans to the
gastropods, the rostroconch-bivalve-scaphopods, and the cephalopods (Fig. 7). In
view of the equally recent conclusion that the nemertines are, in fact, descendants of
an annelid-like coelomate (and, we suggest, RBC-containing) animal (Turbeville and
Ruppert, 1 986), the condition of the coelom in the ancestral mollusc probably should
be reconsidered. Regardless, the present findings emphasize the importance of ascer-
taining the properties of O2 carriers (if any) in the poorly known molluscan classes
such as the monoplacophorans and the scaphopods. We also look forward to the
results of the sedimentation equilibrium studies of archaeogastropod He which were
underway at the time of Ellerton and Lankovsky's 1983 report. The elucidation of
the structures of the Hcs (if any) in these three groups and also additional members
of other molluscan groups may have important implications for molluscan phylog-
eny. In our view, the detail available at present does not permit a very confident
conclusion concerning the evolutionary relationships of the molluscan Hcs.
Origins of the red blood cell and its simple hemoglobins
If the status of postulated transitional group is disregarded, there is some consen-
sus among molluscan systematists concerning the ancestry of the bivalves. Along with
the Gastropoda, Scaphopoda, and Cephalopoda, the class Bivalvia is believed to be
descended from the Monoplacophora (Cox et al., 1969; Newell, 1969; Stasek, 1972;
Pojeta, 1978; Runnegar, 1978; Runnegar and Pojeta, 1986; Salvini-Plawen, 1986).
There is considerable disagreement, however, on the relationships of the different
groups of bivalves. Newell (1969) described six subclasses and assigned the solemy-
oids and the nuculoids to separate ones. However, Allen (1986) argued for the integ-
rity of the Protobranchia as a subclass that includes all bivalves with simple, pectinate
gills and described only one other subclass, the Lamellibranchia. Our findings
strongly support Allen's (1986) view. While we cannot provide evidence for the ab-
sence of He in all lamellibranchs, we can provide evidence of the absence of any O2
carrier in the blood of one pteriomorph (Modiolus demissus) and two heterodonts
(Crassostrea virginica and Rangia cuneata): When measured with a Lexington In-
216
C. P. MANGUM ET AL.
chordates
(o)
arthropods
echinoderms
(o)
hemichordates
Dhoronids brachiopods
nematodes
rotifers
common / /
ancestor// gastrotrichs
platyhelminths
ctenophores
cnidarians
ancestral
eumetazoan
B
opisthobranchs pulmonates coleoids nautiloids
7"
prosobranchs
ammonoids
T
higher lamellibranchs
O
pteriomorphs
I o
protobranchs
primitive gastropods primitive cephalopods primitive bivalves
scaphopods
?
polyplacophorans.
monoplacophorans
?
aplacophorans
,o
ancestral mollusc
?o
FIGURE 1. Phylogeny of: A. The red blood cell; B. The molluscan O: carriers. (©) symbolizes RBCs
and (••••) symbolizes molluscan He. Question mark indicates uncertainty.
BIVALVE HEMOCYANIN 217
struments Co. analyzer, the total O2 contents of these bloods did not differ from that
of the seawater to which the animals were acclimated. Moreover, these bloods also
lacked absorption maxima in the region of 345 nm, as did plasma of the RBC-con-
taining pteriomorph Noetia ponderosa (C. P. Mangum, unpub. obs).
The higher bivalve taxa, including the Heterodonta (which contains most of the
familiar species), are regarded as suspect (Newell, 1969). But the Pteriomorpha, a
relatively primitive group consisting of the anisomyarians, the extinct cyrtodonts,
and the RBC-containing arcoids, appears to be a natural taxon. In addition, there is
general agreement that the protobranch bivalves are even more primitive than the
pteriomorphs (Newell, 1969; Allen, 1978, 1986). This relationship has several im-
plications for the question of the origin and further evolution of O2 transport systems,
because it is among the pteriomorphs that one finds RBCs resembling counterparts
and containing Hbs similar to those in other phyla at comparable levels of organiza-
tion. The subject is of such importance that it is discussed in detail below.
Nucleated RBCs containing either monomeric or oligomeric Hbs are found in
seven animal phyla (summarized by Mangum and Mauro, 1985), including five
(Phoronida, Annelida, Echiura, Nemertina, and Mollusca) that are often regarded as
not too distantly related to one another and at an intermediate stage of phylogenetic
development (Fig. 7A). While the limited anatomical information indicates the possi-
bility of some distinctly different features of the RBCs in each group, it also indicates
many similarities. The physiological information, also limited, indicates a similar
metabolic organization of at least annelid and molluscan RBCs, which differs from
that of avian and mammalian RBCs and, possibly, the sipunculid pink blood cell
(Mauro and Isaacks, 1984; Mangum and Mauro, 1985).
Within the molluscs, RBCs occur widely in one order of pteriomorph bivalves (as
well as in a single species of heterodonts; Terwilliger et al., 1983), and they almost
certainly occur in the Aplacophora, which was regarded by Hyman (1967; p. 69) as
the "genuinely primitive" molluscan class (Fig. 7B). Hyman (1967; p. 65) noted that
"the coelomic fluid has a reddish hue invested in the corpuscles, except in the Chae-
todermidae, where the red substance, not proved to be hemoglobin, is dissolved in
the fluid itself." Despite the caveat, it is highly likely that this pattern reflects yet
another instance of O2 transport by RBCs (see also Baba, 1940) as the primitive con-
dition and of multidomain, extracellular Hb as the derived condition. Well known
examples include the annelids as well as the lamellibranch bivalves. RBCs contain-
ing simple heme proteins are found in more primitive species and extracellular Hbs
that differ fundamentally from one another as well as from the simple heme proteins
occur in more advanced taxa. One pteriomorph bivalve, believed to represent the
transitional stage, has both kinds of Hbs in its RBCs (Grinich and Terwilli-
ger, 1980).
There is a strong possibility that the nemertines exhibit the same trend. Hyman
(1951; p. 490) believed that the red color of the blood "resides in the corpuscles,"
which is true of a few marine species (Vernet, 1979). In support of this contention
Hyman ( 195 1 ) cited the 1 872 report by Lankester, whose words indicate otherwise:
"the colour is due to Haemoglobin diffused in the liquid" (p. 73, italics ours). Polu-
howich (1970; also pers. comm.), who reported Hb in freshwater (and therefore not
primitive) nemertines, did not detect RBCs. Outside of the vertebrates, RBCs are
unknown in freshwater animals, which is believed to be due to their osmotic fragility
(Mangum, 1980a).
On the basis of the distribution of the RBC summarized above and illustrated in
Figure 7, there is no compelling reason to postulate more than one origin of the
RBC and its simple hemoglobins. One need only to suppose that the RBC originated
218 C. P. MANGUM ET AL.
shortly after circulating body fluids arose (Fig. 7). In more advanced groups it was
repeatedly replaced by extracellular O2 carriers due to the greater viscosity of the large
bore tubes that dominate primitive cardiovascular systems (Mangum, 1976), and it
was inherited by two more advanced deuterostome groups: the echinoderms and the
chordates.
The most obvious interpretation of the existence of He in the blood of proto-
branch bivalves is that the hypothesis of a common origin of the RBC is incorrect.
This interpretation has the following implications: Protobranchs represent the ances-
tral bivalve condition and their HcO2 transport system was either lost (anisomy-
arians) or replaced (arcoids) in the pteriomorphs by an intracellular HbO2 transport
system of de novo origin. This interpretation is consistent with the presence of neuro-
and branchioglobin in the same individuals that contain He. It is also consistent with
the recent finding that the tertiary structures of the simple Hbs found in the annelids,
bivalves, and primitive vertebrates are similar to one another and to mammalian
myoglobin, and different from that of higher vertebrates Hbs (Perutz, 1985; Royer et
ai, 1985; W. E. Love, pers. comm.). According to this multiple origin hypothesis,
structural similarity of the simple Hbs of the lower animals is due to two separate
origins from their tissue heme proteins, which also have the same tertiary structure
(presently unknown, but probable), not a common origin.
The weaknesses of this interpretation include uncertainty about the integrity of
the class Bivalvia(McAlester, 1966;Cox^/. ai, 1969; Newell, 1969; Runnegar, 1978)
and the absence of a clear selection pressure for the replacement of He with RBCs.
The simple heme proteins found in pteriomorph RBCs would seem to offer no clear
advantages over protobranch Hcs. Indeed, their respiratory properties are not nearly
as plastic, at least under physiological conditions (Mangum, 1980a), and therefore
the selection pressure would seem to be negative.
Two alternative interpretations seem so unlikely that they can be dismissed with
some confidence. ( 1 ) The hypothesis that the RBC and its simple Hbs had a common
origin and that protobranch RBCs were lost in favor of a HcO2 transport system
after the divergence of the protobranchs and the pteriomorphs entails at least two
independent origins of molluscan He culminating in similar quaternary structures
with very different functional properties. As pointed out in detail earlier (Mangum el
ai, 1985), this absence of correlation between known aspects of quaternary structure
and respiratory function fails to provide a selection pressure for convergent evolution
of those aspects of structure and it implies that they are conservative, ancestral charac-
ters. The point is strengthened by the present finding of a similar quaternary structure
of bivalve and other molluscan Hcs, with a strikingly different combination of respi-
ratory properties. (2) The notion that the RBC had a common origin and that the
pteriomorphs, not the protobranchs, are the more primitive bivalves can also be dis-
missed. Abundant morphological evidence indicates otherwise.
A third alternative, the hypothesis that the RBC had a common origin and that
the Protobranchia and the Pteriomorpha did not have a common ancestor, is some-
what more difficult to reject. While the possibility of a di- and even polyphyletic
origin of the bivalves is frequently mentioned (e.g., Cox et ai, 1969; Newell, 1969;
Runnegar, 1978), the position has been advocated positively and forcefully only with
respect to a separate origin of the Lucinacea (McAlester, 1966), not separate origins
of the Protobranchia and at least one other lineage containing the Pteriomorpha and
the (infrequently) Hb-containing Heterodonta. The strongest supporting evidence
may be the results reported by Purchon ( 1 978), who employed a matrix analysis based
on set theory to cluster and thus to gauge the degree of relatedness between the 40
recent bivalve superfamilies whose taxonomic integrity is uncontroversial. Using the
BIVALVE HEMOCYANIN 219
nine (of 1 7) characters that Newell ( 1 969) had designated as diagnostic of the super-
families and that were either practical or suitable for the analysis, Purchon (1978)
identified only two major clusters of bivalves, the nuculoids and the rest. His conclu-
sion is reflected in Allen's (1986) diagnosis of the two quite different bivalve sub-
classes. With the stipulation that the nuculoid cluster should include the solemyoids,
as in Allen's (1986) scheme, the present findings identify a tenth character that sup-
ports the notion of one and only one major "taxochasm" among the bivalves (Pur-
chon, 1978). We mention the possibility of diphyly less in advocacy of it than as an
alternative that circumvents the weaknesses of the hypothesis of multiple origins of
the RBC and which, therefore, should not be ignored.
Further progress in understanding the evolution of O2 transport systems awaits
further elucidation of the structure and function of tissue heme proteins and also
further understanding of how bivalve O2 transport systems work. When details such
as blood gas levels, pH, responses to hypoxia, respiratory and cardiovascular design
constraints, etc. are known in protobranchs as well as additional Hb-containing bi-
valves, then the selection pressures favoring the evolution of systems with particular
properties will become clearer.
ACKNOWLEDGMENTS
Supported by NSF DCB 84-14856 (Regulatory Biology), BSR 83-07714 (System-
atic Biology), and DMB 17310 (Biochemistry). CPM is grateful to M. J. Greenberg
for leads to the literature on bivalve phylogeny and to R. D. Barnes for helpful discus-
sion. For MPM this is Contribution No. 153 from the Marine Science Center of
Northeastern University.
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Reference: Biol. Bull. 173: 222-229. (August, 1987)
PARTICLE SIZE AND FLOW VELOCITY INDUCE AN INFERRED
SWITCH IN BRYOZOAN SUSPENSION-FEEDING BEHAVIOR
BETH OKAMURA1
Smithsonian Marine Station at Link Port, 5612 Old Dixie Highway, Fort Pierce, Florida 33450
ABSTRACT
The feeding rates of two bryozoan species varied with particle size and flow veloc-
ity. In one species, increased flow reduced feeding on larger particles. The anoma-
lously high capture rate of the largest particles by the smaller of the two species indi-
cates a switch in feeding by ciliary currents to feeding that involves a high degree of
tentacular activity. This is the first quantification of feeding under alternate modes
in a benthic invertebrate and suggests that tentacular feeding may provide a signifi-
cant source of nutrition for bryozoans.
INTRODUCTION
It is increasingly clear that switches in feeding strategies are common in benthic
marine organisms. Some polychaetes, clams, and amphipods switch from deposit to
suspension feeding with increases in flow or suspended material; some corals both
entrap zooplankters with their tentacles and use mucus to entangle suspended par-
ticles; and some active suspension feeders feed passively under certain conditions
(e.g., barnacles, ascidians, brachiopods, and sponges) (Lewis and Price, 1975; Taghon
et at, 1980; Dauer et at, 1981; LaBarbera, 1984; Olafsson, 1986). There is also evi-
dence for alternate feeding modes in zooplanktonic suspension feeders. Copepods
have been argued to filter feed on small particles and to actively grasp particles of
larger size (e.g., Conover, 1966; Richman and Rogers, 1969; Poulet, 1974) [but see
more recent clarification of copepod feeding by Koehl and Strickler (1981)]. In ad-
dition, many suspension feeders take up dissolved organic matter, although the pro-
cess is presumably continuous and would entail no switch in feeding behavior
(Stephens and Schinske, 1961; JoYgensen, 1976; DeBurgh and Fankboner, 1978;
Stewart, 1979).
In bryozoans, feeding currents produced by cilia lining the tentacles of the lopho-
phore can be accompanied by a high degree of tentacular activity ranging from simple
individual tentacle-flicking to encaging particles with all of the tentacles (Winston,
1978). Encagement activity is generally observed when particles are large; however,
there has been no explicit test of the factors promoting greater tentacular versus ciliary
feeding in bryozoans. In addition, the amount of food ingested under alternate modes
has not been determined for any benthic invertebrate. This study compares the effects
of particle size and ambient flow velocity on the feeding of the two closely related
arborescent bryozoans, Bugula neritina and B. stolonifera.
Bryozoans are exclusively colonial animals common in both modern and fossil
marine habitats. A variety of colony morphologies are found among bryozoan spe-
Received 16 March 1987; accepted 27 May 1987.
1 Present address: Department of Oceanography, Dalhousie University, Halifax, Nova Scotia, Canada
B3H4J1.
222
SWITCHING IN BRYOZOAN FEEDING MODES 223
TABLE I
Dimensions* of Bugula neritina andB. stolonifera
Biigula neritina Bugula stolonifera
Maximum size of colony (cm) 8 3-4
Number of tentacles 23-24 13-14
Mean length of tentacles (cm) 6.16X10"2 4.47 X 10~2
Mean diameter of lophophore (cm) 7.64 X 1CT2 4.41 x 1(T2
Mean diameter of mouth (cm) 7.4X10"3 4.9 X 10~3
1 Values from Ryland and Hayward ( 1977) and Winston (1978, 1982).
cies. These include encrusting, arborescent, and massive colonies. The zooids (mod-
ules) that compose bryozoan colonies also vary substantially in form and function
both among species and within colonies of the same species. Feeding zooids ingest
suspended particles using the ciliated crown of tentacles, the lophophore. Gut con-
tents and laboratory rearing experiments indicate that flagellates and diatoms can be
important food items (Winston, 1977, and references therein). However, little is
known of the food sources for field populations since plankton is inherently patchy
in nature, the partial digestion of gut contents hinders identification of ingested mate-
rial, and the small size of feeding zooids and the even smaller size of their prey make
direct observation in the field difficult. The effect of flow on bryozoan feeding
has received some attention (Okamura, 1984, 1985), but the effect of particle size
is unknown.
MATERIALS AND METHODS
Colonies of the arborescent, anascan cheilostomes Bugula stolonifera and B. neri-
tina co-occur in fouling communities that develop on submerged structures in ports
and harbors. In Florida, colonies occur on seagrasses, oyster shells, docks, canal walls,
floats, rotting wood, algae, coastal rock ledges, and inlet breakwaters (Winston, 1 982).
Both species are widespread. B. stolonifera is the smaller species (see Table I) and
often colonizes and grows within B. neritina colonies. In this study, B. neritina and
B. stolonifera colonies were collected from the floating docks of the Harbor Branch
Oceanographic Institution at Link Port, Florida. Laboratory observations of bryo-
zoan feeding behavior confirm that high degrees of tentacular activity and encage-
ment of particles occur in several Bugula species (including B. neritina) and seem to
be associated with particle size and motility (Winston, 1978). However, these obser-
vations were made in still water.
Feeding experiments were performed by submerging colonies in a recirculating
flow tank (Vogel and LaBarbera, 1978) in which currents of known mean velocities
could be created in the working section (13 cm X 13 cm X 13 cm). The flow tank
contained a suspension of latex particles (polystyrene divinylbenzene calibration par-
ticles: Duke Scientific Corporation, Palo Alto, CA). (Initial observations showed that
the bryozoans would ingest these particles.) Two flow velocities were created in the
flow tank: a relatively slow flow (1-2 cm/s) and a relatively fast flow (10-12 cm/s).
Flow measurements taken with an electromagnetic flow probe (Marsh McBirney No.
523) in the field at the branch tip level of Bugula stolonifera (Okamura, 1 984) indicate
that both species encounter flow velocities in the experimental range (measurements
224 B. OKAMURA
were made in a habitat where both species occurred). Feeding on three sizes of latex
particles was assessed at each flow velocity. Particle diameters were 9.6 (SD = 0.5),
14.6 (SD =: 1.0), and 19.1 (SD =1.1) microns. At the outset of experiments, particle
concentrations in the flow tank were set at 100 particles/ml by adding appropriate
volumes of stock suspensions of known concentrations to a known volume of filtered
seawater in the flow tank. Concentrations of 100 particles/ml lie well within the range
of concentrations of flagellates measured in the field (e.g., Jtfrgensen, 1966; Bullivant,
1968). Control runs in the flow tank indicated that latex particles do not settle out of
suspension over time at either flow velocity employed (Okamura, 1984).
Up to three replicate colonies were placed in the flow tank during a given experi-
ment. Only portions of colonies were used in all experiments. [Clipping colonies does
not affect feeding activity (Okamura, 1984)]. Colonies were allowed to feed for 20
min and then were removed from the flow tank and placed in dilute sodium hypo-
chlorite. This treatment dissolves the organic contents of colonies but leaves intact
the exoskeleton, membranous material, and latex particles. Ingested latex particles
(that can initially be discerned only poorly in the gut before the gut wall dissolves)
remain trapped within the zooidal exoskeleton and membranes and can be counted
easily. These counts provided an estimate of the mean number of particles ingested
per feeding zooid per colony (range of zooids sampled per colony = 5-100, mean
= 64.4, SD = 35.2) (range of colonies replicated per treatment = 8-16). The effects
of flow velocity and particle size on the mean number of particles ingested per zooid
per colony were then analyzed with two-way analyses of variance for each species.
RESULTS
Bugida neritina ingested few small particles at both flow velocities (see Fig. 1A).
More large particles were consumed than medium-sized particles in slow flow. This
pattern reversed itself in fast flow (note the nearly significant interaction term). The
smaller B. stolonijera showed greatest ingestion of medium-sized particles in slow
flow (see Fig. 1 B). Feeding on medium-sized and small particles was inhibited in fast
flow; however, large particles were captured in great numbers.
DISCUSSION
Feeding patterns and their causal explanations
Rubenstein and Koehl (1977) used aerosol models of filtration to clarify suspen-
sion-feeding mechanisms, however these models can only be applied to passive sus-
pension feeders. Because bryozoans and other active suspension feeders generate
feeding currents, complex three-dimensional flow patterns arise between self-gener-
ated feeding currents and local currents near the feeding structures (Jtfrgensen, 1980).
For feeding to occur, particles must be transferred from local currents into the self-
generated feeding currents, and in doing so they must pass through a boundary zone
characterized by steep velocity gradients (Jtfrgensen, 1 980). The behavior of particles
that enter steep velocity gradients is uncertain (Strathmann, 1 982). With this in mind,
several factors may explain the patterns of feeding from different flows on particles
of varying size by Bugula stolonijera and B. neritina.
Large-sized particles travel with greater momentum and thus may be carried fur-
ther downstream before crossing flow lines in velocity gradients. Strathmann ( 197 1 ,
1982) argued that no evidence indicates that momentum carries particles across flow
lines so that they will impinge upon the ciliary tracts of echinoderm larvae and lopho-
SWITCHING IN BRYOZOAN FEEDING MODES
225
8.0-
6.0-
J> 2.0
i
TJ
O
O
N
O
(0
a
6
c
c
CO
0)
E
1 0.0-
8.0-
6.0-
4.0-
2.0-
neritina
slow fast
FLOW
—I—-
slow
FLOW
fast
FIGURE I. Mean number of 9.6 (S), 14.6 (M), and 19.1 (L) micron particles captured per feeding
zooid per colony (±2 SE) by Biigula ncrilina (A) and B stolonifera (B) in slow and fast flow. Two-way
analysis of variance for B. neritina: F, 55 (velocity term) = 1.347, P = 0.251; F2.5? (particle size term)
= 18.120, P < 0.001; F2 55 (velocity X particle size interaction) = 2.931, P = 0.062. Two-way analysis of
variance for B. stolonifera: F, 59 (velocity term) = 0.035, P = 0.0851; F2.59 (particle size term) = 1 1.183, P
< 0.00 1 ; F.,59 (velocity X particle size interaction) = 42.679, P < 0.00 1 .
phorates, and hence that momentum does not play a role in the suspension feeding
of these organisms. However, the role of momentum in the transport of particles out
of local currents and into feeding currents is unknown. Alternatively, the relatively
greater drag experienced by large-sized particles may act to sweep them further down-
stream before crossing flow lines in velocity gradients. Both momentum and drag
increase with ambient flow velocity.
The larger lophophores of Bugula neritina create stronger feeding currents, and
these may account for its greater effectiveness in capturing large particles from slow
flow. For B. stolonifera, highest ingestion rates in slow flow were on medium-sized
particles. In fast flow, B. neritina was hindered in feeding on large particles and
showed highest ingestion rates on intermediate-sized particles. The greater momen-
tum of or drag on large particles in faster flow may make their ingestion more difficult.
However, anomalously high feeding rates on large particles in fast flow were observed
for B. stolonifera. The most likely explanation for this is a switch in feeding from
mainly ciliary currents to feeding that involves a high degree of tentacular activity.
Unfortunately, a switch in feeding technique was not anticipated and, consequently,
lophophore behavior was not observed with a microscope during the experiments.
226 B. OKAMURA
Furthermore, colonies were fixed and sampled when time permitted. When the feed-
ing patterns were eventually discerned, there were no colonies available for observing
lophophoral behavior (both species are highly seasonal in Florida). However, as men-
tioned earlier, laboratory observations indicate that feeding in many Bugula species
does involve high degrees of tentacular activity and encagement (Winston, 1978).
Only moderate levels of tentacular activity were observed in B. stolonifera, but since
Winston's observations were made in still water and the sizes of suspended particles
were unspecified, conditions that would have invoked high degrees of tentacular ac-
tivity or encagement by B. stolonifera may not have been present.
The inferred switch in feeding mode by Bugula stolonifera is induced by increased
flow rate and depends on particle size. The larger B. neritina showed no evidence of
a switch in feeding behavior. However, if particles of larger size or perhaps if faster
flow velocities had been employed, a switch in feeding would be expected. High de-
grees of tentacular activity were observed for B. neritina, including the formation of
cages with its tentacles (Winston, 1978). The reduction in feeding on small and medi-
um-sized particles with increased flow by B. stolonifera is in accord with previous
results (Okamura, 1985).
Best and Thorpe ( 1 983, 1 986) provide evidence that bryozoans are capable of altering
the strength of their feeding currents and do so in response to particle concentration. An
alternate explanation for the present results is that the feeding patterns are produced by
feeding currents of different strengths. If this is so, the greater flux of large particles in fast
flow would induce Bugula stolonifera, but not B. neritina, to produce stronger feeding
currents. While this is a possibility, it is considered unlikely since B. stolonifera fed dispro-
portionately on large particles present in mixtures (composed of equal proportions of all
three particle sizes) in fast flow (Okamura, in prep). In this case, if stronger feeding cur-
rents were produced, particles of all three sizes would be expected to be ingested in equal
proportions. It is more likely that the disproportionate ingestion of large particles from
mixtures was a result of selective tentacular feeding.
It is notable that the apparent switch in feeding by Bugula stolonifera entails such
a large increase in capture. Tentacular feeding may involve a much greater energetic
expenditure than ciliary feeding. Only when the gain is great (i.e., many large particles
per unit time) will feeding that involves a high degree of tentacular activity be a worth-
while strategy. Note that a switch to tentacular feeding results in a much greater
amount of "biomass" captured [mean mass of large particles captured by B. stoloni-
fera in fast flow - 30.80 X 19~9 g (SD == 8.50), of medium-sized particles = 6.21
X 10"9g(SD= 1.87), and of small particles = 1.09X 10~9 g(SD = 0.42)]. However,
the costs of ciliary and tentacular feeding are unknown. The reduced surface area
offered by small particles for tentacular contact may preclude tentacular feeding on
particles below a minimum size, or perhaps particles must exceed a certain relative
size to be perceived individually.
Plasticity in feeding behavior and its implications
It is evident that many suspension feeders are capable of great plasticity in feeding
behavior. Alternate feeding techniques are invoked by variations in the suspension
from which they capture their food. These variations may be characteristics of the
prey items (e.g., size, motility, chemistry) or physical properties of the medium itself
(e.g., temperature, density, and the patterns of fluid flow). Since suspension feeders
will regularly encounter suspensions that vary in both physical properties and prey
items, plasticity in feeding response is expected. The study of suspension feeding in
SWITCHING IN BRYOZOAN FEEDING MODES 227
still water on uniform particles may often provide an incomplete picture of the feed-
ing of many organisms. This is exemplified by the studies of Best and Thorpe (1983,
1986). They argue that tentacular flicking and the more localized ciliary reversal
mechanism that Strathmann (1982) proposes to account for particle capture during
ciliary feeding are not the main methods of feeding employed by bryozoans. They
suggest that, overall, the bulk of particles ingested are those that feeding currents carry
down the center of the lophophore towards the mouth. The importance of ciliary
reversal and tentacular activity in feeding are rejected on the basis of calculating the
number of reversals and tentacular flicks required to explain the ingestion rates they
observed. However, their evidence may be biased due to their use of extremely high
particle concentrations (50-200 cells M! '), small particle sizes, and the absence of
ambient currents in their experiments. Their study suggests that very high particle
concentrations may swamp contributions to feeding by mechanisms other than the
bulk flow of particles through the center of the lophophore, while this study suggests
that high degrees of tentacular activity depend on both particle size and ambient flow.
Many investigators have studied the relationship between bryozoan colony form
and the patterns of self-generated feeding currents through colonies (Cowen and
Rider, 1972; McKinney, 1977, 1986a, b; Taylor, 1979; Anstey, 1981; McKinney et
ai, 1986). Results reported here indicate that feeding currents may not always be of
primary importance in particle capture. The potential for alternate feeding behaviors
should be appreciated when interpreting colony morphology solely in terms of feed-
ing current patterns. Both stenolaemate and gymnolaemate bryozoans display high
degrees of tentacular activity even in still water (Winston, 1978). Tentacular feeding
from faster ambient flow and/or on certain types of prey may provide a significant
source of nutrition for a variety of bryozoans.
Optimal foraging theory attempts to explain and predict many aspects of the for-
aging behavior of animals by assessing foraging tactics in terms of maximizing net
rates of energy gain and therefore fitness (e.g., Schoener, 1971; Pyke 1977, 1984;
Hughes, 1980). Which prey will be the "best" is determined by the energy content of
the prey and the energetic cost to the predator of searching for and handling the
prey. Thus, understanding patterns of prey selection, prey vulnerability, and feeding
behaviors is crucial in the interpretation of foraging strategies. Particle size appears
to relate to prey vulnerability in bryozoan feeding. Flow velocity induces a switch in
feeding behavior that results in a shift in the size of particles captured. Furthermore,
flow velocity appears to control the vulnerability of particles of certain size ranges
even when feeding under one mode (note greater feeding on large particles in slow
flow but on medium-sized particles in fast flow by Bugula neritind). Prey vulnerabil-
ity and patterns of prey capture are thus determined by both the constraints imposed
by flow and by the flow-induced change in feeding tactics. This suggests that the role
of flow on particle size selection and the behavior of suspension feeders merits further
investigation. In addition, a switch in feeding tactics by bryozoans implies that these
organisms perceive and assess prey availability and subsequently adopt the most
efficient feeding mode (i.e., the one that maximizes net energy gain). It appears that
predictions of optimal foraging theory may be applicable to benthic suspension feed-
ers despite their seemingly simple sensory capabilities and sessile existence.
ACKNOWLEDGMENTS
I thank A. H. Cheetham, M. A. R. Koehl, F. K. McKinney, M. E. Rice, P. D.
Taylor, and two referees for suggesting improvements to the manuscript. This is con-
tribution #1 77 of the Smithsonian Marine Station at Link Port.
228 B. OKAMURA
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Reference: Biol. Bull. 173: 230-238. (August, 1987)
EPITHELIAL WATER PERMEABILITY IN THE EURYH ALINE MUSSEL
GEUKEi 'SIA DEMISSA: DECREASE IN RESPONSE TO HYPOOSMOTIC
MEDIA AND HORMONAL MODULATION1
LEWIS E. DEATON2
Whitney Marine Laboratory, University oj Florida, Route I, Box 121, St. Augustine, Florida 32084
ABSTRACT
The diffusional water permeability of isolated mantles from the mussel Geukensia
demissa was reduced by incubation of the tissues in hypoosmotic media. The perme-
ability of mantles from 1000 mOsm seawater (SW)-accli mated animals was 6 X 10"'
cm/s. A four-hour incubation in 500 mOsm SW or 250 mOsm SW reduced the water
permeability by 2 X 10~5 cm/s and 4 X 10 5 cm/s, respectively. A half-hour exposure
to the hypoosmotic medium was sufficient to induce the decrease in permeability.
The water permeability of mantles incubated in isosmotic SW containing acetone
extracts of ganglia from 1000 mOsm SW-acclimated mussels or of mantle from 500
mOsm SW-acclimated mussels was significantly reduced. Extracts of gill had no
effect.
Ovine prolactin (50 mg/ml) decreased the water permeability of mantles in isos-
motic seawater. Cortisol ( 10~4 M), arginine vasopressin ( 10~6 M), and the molluscan
neuropeptide FMRFamide ( 10 6 M) had no effect.
These results show that the epithelial water permeability of euryhaline bivalves
varies with changes in the ambient salinity, and that these permeability changes may
be modulated by factors of neural origin.
INTRODUCTION
A number of specific physiological mechanisms facilitate the survival of euryha-
line marine animals in habitats characterized by variations in salinity. These mecha-
nisms include changes in urinary output, regulation of the extracellular fluid compo-
sition, volume regulation, and changes in epithelial permeability to water and ions.
A reduced epithelial permeability to water in dilute media has been reported in
several invertebrates, including a number of arthropods (Rudy, 1967; Smith, 1970a;
Capen, 1972; Smith and Rudy, 1972; Cornell, 1973; Hannen and Evans, 1973; Lock-
wood et ai, 1973; Roseijadi et ai, 1976; Thuet, 1978), three polychaetes (Smith,
1964, 1970b; Fletcher, 1974), and a bivalve (Prusch and Hall, 1978). The phenome-
non has been observed in osmoconformers (e.g., Mytilus edulis, Libinia emarginatd)
and in osmoregulators (e.g., Rhithropanopeus harrisi, Nereis limnicola). Nearly all of
these data were collected by measuring changes in the fluxes of water into or out of
whole animals exposed to dilute media. As indicated by Cornell (1979), part of the
observed decreases in the flux of water across the epithelium of an intact animal could
be effected by changes in circulation or ventilation. This criticism does not apply to
Received 9 February 1987; accepted 26 May 1987.
' This is contribution number 265 from the Tallahassee, Sopchoppy and Gulf Coast Marine Biological
Association.
2 Current address: Department of Biology, University of Southwestern Louisiana, Lafayette, Louisi-
ana 70504.
230
EPITHELIAL WATER PERMEABILITY IN G. DEMISSA 231
studies of the response of the epithelial permeability of isolated tissues to changes in
salinity.
Isolated tissues from only a few marine invertebrates have been used to examine
changes in water permeability in response to dilution of the medium. Cantelmo
(1977) found that the water permeability of gut epithelia and gills from the crabs
Cancer irroratus and Callinectes sapidus was lower in tissues isolated from animals
acclimated to 40% seawater than in tissues from animals acclimated to 100% seawa-
ter. However, exposure of isolated tissues to hypoosmotic media for 2 h did not
change their water permeability. Acclimation of the mussel Mytilus edulis to reduced
salinity caused a decrease in the diffusional permeability of water across isolated man-
tle tissues (Prusch and Hall, 1978).
Neurohormones have been implicated in the modulation of water permeability
in a variety of invertebrates. For example, extracts of the thoracic ganglion decrease
the water flux across isolated crab gut and gills (Mantel, 1968; Berlind and Kamem-
oto, 1977), and the injection of a brain homogenate reduces the rate of water ex-
change of earthworms (Carely, 198 1 ). Two lines of evidence suggest a possible modu-
lation of water balance by neural products in gastropod molluscs. Injection of syn-
thetic thyrotropin releasing hormone into the freshwater snail Lymnaea stagnalis
causes a slight loss in wet weight (Grimm-Jorgenson, 1979). Similarly, injection of
homogenized R-15 cells from the abdominal ganglion of the opisthobranch Aplysia
brasiliensis resulted in a 5% gain in wet weight (Kupfermann and Weiss, 1976).
The present study was undertaken to determine the response of the epithelial
water permeability of isolated mantle tissue from the euryhaline mussel Geukensia
demissa to decreases in the ambient osmotic concentration. This tissue was also used
as a bioassay system to determine the effects of extracts of the ganglia and other tissues
on water permeability.
The results show that the diffusional water permeability (Pd) of the mussel de-
creases in response to the dilution of the external medium, and that water permeabil-
ity may be modulated by a factor of neural origin.
MATERIALS AND METHODS
Animals
Atlantic ribbed mussels, Geukensia demissa granosissima, were collected from a
salt marsh near St. Augustine Beach, Florida. The animals were kept unfed in running
seawater (30%o) at ambient temperature. All animals were used within 3 weeks of
collection.
Histology
The adductor muscles of individual mussels were cut, the animals opened, and
pieces of the central portion of the mantle dissected free. The tissue was fixed in
filtered seawater containing 2% glutaraldehyde, dehydrated and cleared in a graded
series of water/ethanol/t-butyl alcohol, and embedded in paraffin. Sections (10 ^m)
were cut on a microtome, stained with hematoxylin eosin, and mounted on glass
slides.
Measurement of diffusional water permeability
The adductor muscles of individual mussels were cut and the valves carefully
pried apart. Each mussel provided two tissues, the mantle covering the inside of the
232 L. E. DEATON
right valve and the mantle covering the inside of the left valve. The left and right
mantle halves were cut away from the visceral mass and detached from the margins
of their respective valves. The isolated tissues were placed in small covered dishes
containing 5 ml of 1000 mOsm seawater (SW) which was aerated via small bore
tubing. After 60 minutes of incubation, one mantle half was mounted over the aper-
ture of one half of a diffusion chamber and secured with a soft rubber o-ring. The
jes were always mounted in the chamber so that the movement of tritated water
was from the extrapallial cavity side to the mantle cavity side. Five ml of medium
were placed in the chamber to provide a hydrostatic pressure head sufficient to check
the mounted tissue for obvious leaks. The chamber was then assembled and both
sides filled with medium by alternate additions of 2-3 mis. The total volume of each
compartment of the chamber was 1 4 ml; the area of exposed tissue was 1 cm2. Mixing
and aeration were provided by gas lift pumps powered by water-saturated air. About
1 /uCi of tritiated water was added to one compartment of the chamber (the "hot"
side), and following 10 minutes equilibration, 100 yul samples of the medium in the
other compartment (cold side) were removed at 1 5 minute intervals for 60-90 min-
utes. These samples were mixed with 10 ml scintillation cocktail and counted on a
liquid scintillation counter. In experiments using paired left and right mantles, the
flux across one mantle was measured while the matching tissue was transferred to a
dish containing either control (1000 mOsm SW) or experimental medium. These
matching tissues were further incubated from one half to four hours and then their
Pd values determined in the same rinsed and dried chamber used for the matching
control measurement.
The water flux across the tissue and the diffusional water permeability were calcu-
lated from equations 1 and 2, respectively:
/ 1 \ Q* / 1 \ Q*/Qi = specific activity in compartment 1
1- Ji2 2 = I " I ~ I A I Q? = amount of isotope in compartment 2
VUQf/Q, W -
-time
JPH2° A = area of tissue
— *a Cw = molar concentration of water
The differences in Pd values between paired left and right mantles were averaged
and differences among treatment means assessed by Student's / test.
Tissue extracts
The pedal, visceral, and cerebral ganglia from 250 mussels acclimated to 1000
mOsm SW were dissected from the animals and pooled in a large volume of cold
acetone to extract putative hormones and inactivate proteolytic enzymes; acetone
extracts were also made of the gills and mantles from these mussels. The mantles of
200 mussels acclimated to 500 mOsm SW were also extracted in acetone. The extracts
were evaporated to dryness on a rotary evaporator and the water soluble portion of
the residue taken up in a minimal volume of distilled water. The dose added to the
incubation media was approximately 1 animal equivalent. All tissue extracts and
hormones were added to the incubation media and to the fluid in both compartments
of the diffusion chamber.
RESULTS
Histology
Figure 1 shows a cross section of the central portion of the mantle. This complex
tissue separates the extrapallial space from the mantle cavity. Both surfaces are lined
EPITHELIAL WATER PERMEABILITY IN G. DEMISSA
233
MC
EPS
FIGURE 1. Cross-section through the central portion of the mantle ofGenkensia demissa. A. Section
from the mantle cavity (MC) to the extrapallial space (EPS) showing the epithelia (epi) on both surfaces
with underlying muscle layers (m). The epithelia are separated by connective tissue which encompasses
many hemolymph vessels (*), few genital follicles or canals (g), and transverse muscle bundles (tmb) con-
necting the two subepithelial muscle layers. Bar = 200 ^m. B. Columnar extrapallial epithelium with thin
underlying muscle layer. Bar = 100 ^m. C. Squamous mantle cavity epithelium with thick underlying
muscle layer. Fibers from the transverse muscle bundles splay out to join the subepithelial muscle layer
(arrows in C2). Bar = 100
with an epithelium underlain by a muscle layer (Fig. 1 A). The extrapallial epithelial
cells are much taller than those lining the mantle cavity, but the subepithelial muscle
layer associated with the extrapallial space is much thinner than that on the mantle
cavity side of the tissue (compare Figs. 1 A, ICi). The bulk of the mantle is occupied
by connective tissue in which are found numerous hemolymph vessels and, in these
non-reproductive specimens, occasional genital canals (Fig. 1A). Bundles of muscle
fibers traverse the mantle joining the two subepithelial muscle layers (Figs. 1A,
1C^). Similar structures have been described for the mantles of other species (Beed-
ham, 1958).
Diffusional water permeability
Preliminary experiments showed that the accumulation of counts in the cold
compartment of the diffusion chamber was linear with time for over six hours, indi-
cating that the 10 minute equilibration with labelled water was sufficient for attain-
ment of a steady-state flux across the tissue. The diffusional water permeabilities of
234 L. E. DEATON
TABLE I
Changes in diffisional water permeability (Pd) of mantles from 1000 mOsm seawater-acclimated
Geukensia demissa after a four hour incubation in various seawaters
Treatment medium lOOOmOsm 500 mOsm 250 mOsm
Change in Pd 0.5 x 10 5 ***-2.3 X 10~5 ***_4.! x 10-s
SD 1.1X10'5 3.0X105 1.3X10'5
n_ ^8 9 7
Values are in cm/s and represent differences in Pd between paired left and right mantles: one mantle
of each pair was incubated in 1000 mOsm SW for 1 h; the other was incubated in the treatment medium
for 4 h. Values significantly different from the 1000 mOsm treatment are marked with *** (P < .00 1 ).
mantles from animals acclimated to 1000 and 500 mOsm for three weeks were, re-
spectively, 7.9 ± 3.3 X 10"5 (n = 10) and 4.3 ± 0.7 X 10 5 cm/s). These values are
higher than the mean (2.2 X 10"5 cm/s obtained by Prusch and Hall (1978) for man-
tles of G. demissa acclimated to 1000 mOsm SW. Their animals, collected near
Woods Hole, Massachusetts, were undoubtedly the subspecies G. demissa demissa.
Differences in chamber design and differences between the two G. demissa subspecies
probably account for the discrepancy.
There were no significant differences between the mean Pd values of mantles incu-
bated for four hours in 1000 mOsm SW (6. 1 ± 2.9 X 10"5 cm/s) and the mean Pds of
the matching control (incubated in 1 000 mOsm S W for 1 h) tissues (6.5 ± 2.9 X 10~f
cm/s), nor was the mean of the differences between paired tissues (0.5 ± 1.1 X 10~f
cm/s) significantly different from zero. In contrast, the water permeability of mantles
incubated in 500 or 250 mOsm SW for four hours was decreased by '/3 and %, respec-
tively, compared to paired controls (Table I). The data from a representative experi-
ment are shown in Figure 2. The movement of labelled water across both tissues is
linear with time; the movement of water across the tissue incubated in 500 mOsm
SW is slower. The magnitude of the reduction in flux is constant throughout the
experiment.
The time course of the reduction of epithelial water permeability (Pd) in 500
mOsm SW is summarized in Table II. A thirty minute incubation in dilute seawater
was sufficient to induce a decrease of about 1 X 10~5 cm/s in the Pd value. Longer
incubations further reduce the permeability, but these values were not significantly
different from that induced by a thirty minute incubation (Table II).
When mantles were incubated in isosmotic medium containing an extract of gan-
glia from 1000 mOsm SW-acclimated mussels or an extract of mantles from 500
mOsm SW-acclimated mussels, the water permeabilities were significantly reduced.
Extracts of other tissues had no effect (Table III).
Hormones which affect the water permeability of vertebrate tissues were tested
for effects on the bivalve mantle. Ovine prolactin significantly reduced the Pd value
of mantle tissues in isosmotic media. Neither arginine vasopressin nor cortisol
changed the permeability of mantles in isosmotic SW (Table IV). The small reduction
in the Pd value of tissues incubated in isosmotic media with the molluscan neuropep-
tide FMRFamide was not significant (Table IV). Colchicine did not prevent the de-
crease in Pd induced by exposure to dilute media (Table V).
DISCUSSION
The diffusional water permeability (Pd) of isolated mantles from the euryhaline
mussel Geukensia demissa decreases when the tissues are exposed to hypoosmotic
EPITHELIAL WATER PERMEABILITY IN G. DEM1SSA
235
30
25-
20
c pm
xlO3 l5
10
.0
15 30 45
Time (min)
60
FIGURE 2. The unidirectional movement of Initiated water across a piece of isolated mantle ofGeuken-
sia demissa. Total counts per minute appearing in the "cold" side of a diffusion chamber are plotted as a
function of time. The data are from paired mantle tissues from one mussel: the left mantle was incubated
for 1 h in 1000 mOsm seawater (solid circles); the right mantle was incubated in 500 mOsm seawater (open
circles) prior to measurement of the tritiated water flux in a diffusion chamber containing the same media.
media; the decrease in permeability is proportional to the magnitude of the decrease
in the ambient osmotic concentration. Furthermore, the Pd of the isolated tissue incu-
bated in isosmotic medium is reduced by a vertebrate hormone and by an endoge-
nous factor of neural origin.
The reduction in permeability induced by a 30 min exposure to 500 mOsm seawa-
ter is less than that resulting from long-term acclimation of the mussels to 500 mOsm
SW. The reduction of water permeability by ganglion extracts from 1000 mOsm-SW
acclimated mussels and mantle extracts from 500 mOsm-SW acclimated mussels
suggests that the putative factor is produced in the ganglia and released to the periph-
TABLE II
Time course of change in water permeability (PJ of mantles from WOO mOsm seawater-acclimated
Geukensia demissa during incubation in 1000 mOsm or 500 mOsm seawater
Treatment medium
lOOOmOsm
500 mOsm
Incubation duration (h)
0.5
1
2
3
4
0.5
1
2
3
4
Pd change (XlO~5cm/s)
0.9
0.4
0.3
0.4
0.2
*-0.9
***-!.
0
**-1.4
***-!. 9
***-!. 9
SD
1.1
0.4
1.4
0.9
0.9
0.6
0.
9
1.4
1.8
1.4
n
4
3
4
8
5
4
10
9
10
7
Values are differences in Pd between paired left and right mantles: one mantle was incubated in 1000
mOsm SW for 1 h; the other was incubated in the treatment medium for 0.5 to 4 h. Values marked with
asterisks are significantly different from the corresponding 1000 mOsm treatment value (* = P < .05, **
= /><. 01, *** = /><. 001)
236 L. E. DEATON
TABLE III
The effect of various tissue extracts on the water permeability (PJ of mantles from WOO mOsm seawater
acclimated Geukensia demissa incubated four hours in 1000 mOsm seawater
Treatment medium
1000SW
1000SW
1000SW
1000SW
+ 1000
+ 1000
+ 1000
+ 500
1000SW
mantle ext.
gill ext.
ganglia ext.
mantle ext.
Pd change
0.5
1.3
0.6
*-1.4
**-1.5
SD
1.4
1.1
1.6
1.2
1.9
n
18
6
5
3
10
Values are xlO 5 cm/s and represent differences in Pd between paired left and right mantles: one
mantle was incubated in 1000 mOsm SW for 1 h; the other was incubated in the treatment medium for 4
h. Values marked by asterisks are significantly different from that for 1000SW(* = P< .05;** = P< .01).
ery during acclimation to low ambient salinity. Prusch and Hall (1978) observed a
67% reduction in the water permeability of mantles isolated from the mussel Mytilus
edulis during four weeks of acclimation to 70% seawater. Thus, in the intact animal,
continuous release of neural factors may facilitate a larger decline in permeability
than occurs in isolated tissues. As yet there are no data on the size, structure, or
chemical nature of this putative neurohormone. However, it is not FMRFamide (Ta-
ble III).
Prolactin reduced the water permeability of G. demissa mantles in isosmotic me-
dia (Table IV). Prolactin also reduces the permeability of teleost epithelia to water
and ions (Doneen and Bern, 1974; Foskett et al, 1983). While the presence of prolac-
tin has been demonstrated by immunocytochemical methods in ascidians (Pestarino,
1984), it has not been reported in any other invertebrate. Therefore it is unlikely that
the active substance in G. demissa ganglia is prolactin.
Khan and Salueddin (1979; 198 1 ) associated changes in the anatomy of the sep-
tate junctions between kidney cells in the snail Helisoma duryi with increased water
permeability. Extracts of the visceral ganglia induce these changes which occur within
TABLE IV
The effects of selected hormones on the change in diffusional water permeability (PJ of mantles from WOO
mOsm seawater-acclimated Geukensia demissa incubated four hours in WOO mOsm seawater
Treatment medium
1000SW
1000SW
1000SW
1000SW
+ Arg
+ Prolactin
+ FMRFamide
+ cortisol
vasopressm
1000SW
(50Mg/ml)
(\0~6M)
( 10~4 M)
(10-6M)
Pd change
0.5
***-2.3
-0.4
-0.5
-0.4
SD
1.1
1.4
0.9
1.5
2.0
n
18
6
4
4
5
Values are XlO 5 cm/s and represent differences in Pd between paired left and right mantles: one
mantle was incubated in 1000 mOsm SW for 1 h; the other was incubated in the treatment medium for 4
h. The value marked by *** is significantly different from that for 1000 SW (P < .001).
EPITHELIAL WATER PERMEABILITY IN G. DEMISSA 237
TABLE V
The effect of colchicine on the water permeability (Pd) of mantles from 1000 mOsm seawater-acclimated
Geukensia demissa incubated four hours in 500 mOsm seawater
Treatment medium
500 SW + colchicine
500 SW (2X10~4A/)
Pd change -2.3 -2.0
SD 3.0 3. 1
n 9 3
Values are X 10~5 cm/s and represent differences in Pd between paired left and right mantles: one mantle
was incubated in 1000 mOsm SW for 1 h; the other was incubated in the treatment medium for 4 h.
30 minutes. Neurohormones, then, can alter the water permeability of molluscan
epithelia by causing changes in the structure of the tissues, thereby changing the resis-
tance of the paracellular pathway to water movement. The failure of colchicine to
prevent a decrease in the Pd of mantles exposed to dilute media (Table V) suggests
that microfilament activity is not involved in the process.
If the major route of water movement across the G. demissa mantle is paracellu-
lar, osmotic swelling of the epithelial cells could contribute to a decrease in the water
permeability of the tissue during exposure to hypoosmotic media. Isolated G. demissa
ventricles exposed to hypoosmotic seawater stop beating, but the mechanical activity
of the ventricle recovers within 90-120 minutes (Pierce and Greenberg, 1972). Re-
covery of the mechanical activity of the ventricle apparently is due to cellular volume
regulation. If the time course of recovery of cellular volume by the mantle cells is
similar to that of the myocardial cells, osmotic swelling cannot account for the reduc-
tion in water permeability induced by 2-4 h incubations in hypoosmotic seawater.
However, in the absence of data on the time course of changes in the volume of
mantle cells exposed to hypoosmotic stress, the possibility that cell swelling accounts
for some or all of the decrease in epithelial permeability cannot be ruled out.
The mantle of G. demissa is vascularized (Fig. 1 ) and therefore well-perfused by
the circulation. Mounting the tissues in the diffusion chamber precluded perfusion,
and therefore the effects of delivery of the tissue extracts and other drugs via the
circulation cannot be assessed.
Extracts of various nervous tissues affect epithelial water permeability of crusta-
ceans and annelids (Mantel, 1968; Tullis and Kamemoto, 1974; Berlind and Ka-
memoto, 1977; Carely, 1981). While none of these factors has yet been identified, it
is clear that neural factors modulate water permeability in euryhaline invertebrates.
In summary, the epithelial water permeability of euryhaline molluscs changes
during acclimation to changes in the ambient salinity. These changes in permeability
may be modulated by one or more neural factors of unknown structure. The mecha-
nisms responsible for increases or decreases in water permeability apparently involve
changes in the junctional complexes between the epithelial cells, but factors affecting
transcellular water permeability, such as the insertion or removal of water channels
or changes in the composition of the membrane lipid bilayer, cannot be ruled out.
ACKNOWLEDGMENTS
I thank Dr. Michael J. Greenberg for critical readings of the manuscript. This
work was supported by a grant (PCM 83093 14) to MJG and LED from the National
Science Foundation.
238 L. E. DEATON
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METAL REGULATION AND MOLTING IN THE BLUE CRAB,
CALLINECTES SAPIDUS: METALLOTHIONEIN FUNCTION
IN METAL METABOLISM
DAVID W. ENGEL1 AND MARIUS BROUWER2
' National Marine Fisheries Service, NOAA, Southeast Fisheries Center, Beaufort Laboratory, Beaufort,
North Carolina 28516-9722 and 2Duke University Marine Laboratory, Marine Biomedical Center,
Beaufort, North Carolina 28516-9722
ABSTRACT
We recently demonstrated that zinc, copper, and hemocyanin metabolism in the
blue crab varies as a function of the molt cycle. To extend these observations, and
better delineate metal metabolism in marine crustaceans, we have conducted experi-
ments to determine if environmental temperature and season of the year affect con-
centrations of hemocyanin and copper in the hemolymph and copper and zinc in the
digestive gland. Overwintering, cold water crabs (6°C) had decreased hemocyanin
and copper in the hemolymph and normal zinc and copper in the digestive gland
with respect to summer crabs collected at 20-30°C. When these crabs were warmed
to 20°C and fed fish for three weeks, they showed increases in the concentrations of
copper in the digestive gland, and copper and hemocyanin in the hemolymph. In
addition, a change from a zinc to a copper-dominated metallothionein was found in
a majority of the warmed crabs, suggesting the involvement of copper metallothio-
nein in the resynthesis of hemocyanin. Based on these observations and previous data
(Engel, 1987) a conceptual model of copper and zinc partitioning in the blue crab
has been constructed. In this model, metallothionein has an important role in metal
regulation both during molting and in the changes related to season of the year. Met-
allothionein-bound copper and zinc appear to be regulated at the cellular level for
the synthesis of metalloproteins, such as hemocyanin (copper) and carbonic anhy-
drase (zinc), both of which are necessary for normal growth and survival. Finally, we
present evidence showing that copper metallothionein can directly transfer its metal
to the active site of apohemocyanin. Copper insertion seems to precede the formation
of viable oxygen binding sites.
INTRODUCTION
Studies of the reputed function of metallothionein in marine organisms have been
concerned primarily with its role in detoxifying elevated concentrations of trace-met-
als accumulated from polluted environments (Roesijadi, 1981; Engel and Brouwer,
1984; and George and Viarengo, 1985). These reviews discussed the potential consti-
tutive or regulatory function of metallothionein in metal metabolism, but empha-
sized its role in detoxifying metals.
Previously we alluded to the possibility that metallothioneins may play a role in
organismal and cellular metal metabolism in marine species (Engel and Brouwer,
Received 9 February 1987; accepted 19 May 1987.
239
240 D. W. ENGEL AND M. BROUWER
1984; Engel and Roesijadi, 1987). Such suggestions also have been made concerning
zinc and copper metabolism in mammals. Cousins (1982, 1985) discussed the role
of metallothionein in zinc metabolism in rats. The observation that glucocorticoid
hormones can significantly alter zinc metabolism and increase metallothionein syn-
thesis in the liver without the administration of exogenous zinc also supports the
hypothesis that metallothionein is active in normal metal metabolism (Karin, 1985).
More recently Petering and Fowler (1986) discussed the normal or constitutive as-
pects of metallothionein synthesis and turnover in mammals, and correlations also
were made with non-mammalian organisms. There is a growing body of evidence,
therefore, that metallothioneins are indeed involved in the regulation of metal me-
tabolism.
Recently it was demonstrated that blue crabs collected from unpolluted environ-
ments significantly alter tissue metal concentrations and the metal composition of
metallothionein during the molt cycle (Engel, 1987). These studies clearly demon-
strated that metallothionein in a marine crustacean is actively involved in normal
physiological and biochemical processes of metal regulation at the cellular level that
control growth and reproduction. Additionally, blue crab metallothionein also is as-
sociated with cellular metal detoxification and sequestration (Brouwer et ai, 1984;
Engel and Brouwer, 1984).
Two series of experiments were performed to explore further the role of metallo-
thionein in metal metabolism. The first series of experiments examined the effect of
overwintering on the metal metabolism of the blue crab at both the tissue and cyto-
solic level. The second series of experiments measured the ability of metallothionein
to donate copper for activation of apohemocyanin in vitro. In addition, we discuss
how metallothionein and the metals bound to it relate to the physiological and bio-
chemical changes that occur during molting. We also propose a model for the direct
involvement of metallothionein as a metal donor in the synthesis of hemocyanin and
zinc enzymes.
MATERIALS AND METHODS
All crabs used in these experiments were captured in the vicinity of Beaufort,
North Carolina, by commercial fishermen. A group often intermolt (C4) male blue
crabs (Callinectes sapidus) were obtained in February 1986, and were maintained
in the laboratory at ambient temperature and salinity (6°C, 30%o). After a week of
acclimation, five crabs were taken for hemolymph and tissue samples. The remaining
five crabs were held for an additional three weeks, and water temperature was allowed
to increase to about 20°C in 10 days. During the three week period the crabs were fed
chopped fish every other day. At the end of three weeks the remaining five crabs were
killed and hemolymph and digestive gland samples were taken.
Tissue metal measurements
The concentrations of copper and zinc were determined in samples of digestive
gland and hemolymph from individual blue crabs. The hemolymph samples were
collected by severing the fifth pereiopod at the meropodite and collecting the fluid in
a polyethylene vial. A portion of the hemolymph was taken for metal analysis and
the remainder was used for determination of hemocyanin concentration. The crabs
were killed by removing the carapace, and the digestive gland was dissected out and
used for total metal measurements and cytosolic metal determinations. The tissue
BLUE CRAB METAL METABOLISM
241
TABLE I
Amino acid composition of blue crab and lobster metallothionein (Residues/6500 Daltons)
Blue crab
Lobster
CdMTagill
CdMTh
digestive gland
ZnMTb
digestive gland
CuMTc
digestive gland
Cysteine
18
17
18
18
Asp/Asn
4
4
4
3
Thr
3
5
5
4
Ser
7
5
6
5
Glu/Gln
8
7
6
4
Pro
5
6
5
6
Gly
5
7
6
5
Ala
2
3
3
3
Val
1
1
1
—
Met
—
—
—
—
He
—
—
—
—
Leu
—
1
1
—
Tyr
—
—
—
—
Phe
—
—
—
—
His
1
—
—
—
Lys
7
7
7
8
Arg
1
1
1
1
62
64
63
57
3 Brouwer el at. 1 984.
h Brouwer unpub. results.
c Brouwer el al 1986.
that was used for determination of cytosolic distribution of metals was frozen rapidly
and stored in a freezer at -70°C.
Tissue samples used for metal analysis were oven dried at 100°C for 48 h and wet
ashed with concentrated HNO3 at 90°C. Residue was dissolved in 0.25 N HC1 and
concentrations of copper and zinc were measured using flame atomic absorption
spectrophotometry. Preparative and measurement techniques were calibrated against
the National Bureau of Standards, Oyster Reference Material #1566.
Apohemocyanin reconstitution experiments
We have shown that the digestive gland of the American Lobster, Homarus ameri-
canus, contains an abundant supply of copper-metallothionein (Engel and Brouwer,
1986). The amino acid composition of the purified metallothionein from lobster is
similar to that of the blue crab (Table I). In view of this similarity, and the relative
ease with which it can be isolated from the lobster, we have used lobster digestive
gland as the source of copper metallothionein in our apohemocyanin reconstitution
experiments.
Hemocyanin and copper metallothionein, to be used in copper transfer experi-
ments, were prepared as described previously (Brouwer et al., 1986). Hemocyanin
concentration was calculated from the optical density at 280 nm, using E}*m =: 14.3
and a value of 75,000 for the molecular weight of a single oxygen-binding site carrying
subunit (Nickerson and Van Holde, 197 1 ). Apohemocyanin was prepared by mixing
242 D. W. ENGEL AND M. BROUWER
hemocyanin in 50 mA/ Tris pH 8, 10 mMCaCl2, with an equal volume of buffer
containing 20 rnM KCN. To prepare hemocyanin samples with different amounts of
bound copper, the protein was either incubated with KCN for 10 minutes at room
temperature, or dialyzed for 30 minutes against 20 mM KCN, followed by removal
of the KCN on Sephadex G-25. Reconstitution of apohemocyanin was performed by
mixing the apoprotein with purified copper metallothioneins in 50 mM Tris pH 8,
10 mA/CaC^, in the absence of oxygen.
Copper insertion into the active site of the apoprotein was measured by fluores-
cence spectroscopy. Apohemocyanin was excited at 280 nm and the quenching of
the tryptophan fluorescence, which accompanies copper incorporation, was moni-
tored at 340 nm with a SPEX Fluorolog fluorescence spectrophotometer in the ratio
mode. The concentration of functionally active oxygen binding sites was determined
from the intensity of the copper-oxygen charge transfer band at 340 nm after addition
of O2 to the degassed incubation mixture.
RESULTS
Effect of overwintering on metal partitioning
Differences were observed in the concentrations of copper in hemolymph and
digestive gland samples among the three groups of intermolt C4 crabs that were exam-
ined (summer, 1985; winter-cold, 1986; and winter-warmed, 1986). In the hemo-
lymph there was a correlation between the physiological condition of crabs and the
concentrations of hemocyanin and copper (Fig. 1). Both summer and warmed hard
crabs had hemocyanin and copper concentrations that were higher than the cold
crabs, but only the difference between the copper concentrations in summer and cold
hard crabs was significant (P < .05). Zinc concentrations did not change significantly
(P > .05) among the three groups of crabs (Fig. 1 ), and did not appear to be positively
correlated with hemocyanin concentration. In the digestive glands there was no sig-
nificant difference (P > .05) in concentrations of copper between the summer and
cold water crabs, but there was a significant (P < .05) increase in the crabs that were
warmed (Fig. 2). Once again zinc concentrations did not show significant changes (P
> .05). The large increase in copper concentration in the warmed crabs is correlated
with the observed increase in hemocyanin in the hemolymph.
The elution profiles obtained after gel-permeation chromatography of the cytosol
from digestive glands of cold and warmed crabs showed differences in metals bound
to metallothionein. Among the cold water crabs four of five had metallothionein
peaks that contained primarily zinc, while three of four (i.e., one chromatographic
sample lost) of the warmed crabs had metallothioneins that contained primarily cop-
per (Fig. 3-II and III). Thus, the majority of cold water crabs had Cu/Zn ratios associ-
ated with metallothionein that were reminiscent of premolt animals (i.e., high zinc
low copper) while the majority of warmed crabs had patterns similar to those of sum-
mer intermolt crabs (i.e., high copper low zinc) (Fig. 3-1). These data show that envi-
ronmental conditions, physiological state, and feeding can affect tissue metal concen-
trations and the cytosolic distributions of copper and zinc in blue crabs.
Apohemocyanin reconstitution experiments
Removal of copper from the active site of hemocyanin results in an increase of
the intrinsic tryptophan fluorescence of the protein (Fig. 4). This observation allowed
us to make a distinction between copper insertion and formation of native functional
BLUE CRAB METAL METABOLISM
243
HEMOLYMPH
o>
E
O
O
2
LLJ
I
60
.
50
-
I
1
40
30
-
I
--
1
20
-
10
-
n
o
K
QC ^
16.0
14.0
O o 10.0
O »E 8.0
'o
X
o
LU
Q.
a.
O
O
6.0
4.0
2.0
Z
O
LLI
O
Z
N
3.0
o n
2.0
1.0
0
I
II III
l-Summer Hard Crab
ll=Winter Hard Crab (cold)
Ill-Winter Hard Crab (warmed)
FIGURE 1. Concentrations of hemocyanin, copper, and zinc in the hemolymph of blue crabs col-
lected in the summer (Engel, 1987) and winter. The winter crabs all were collected at the same time. Half
(5) were sampled at ambient temperature (6°C) and the other half (5) were warmed to 20°C and fed fish
every other day for three weeks. Each histogram represents a mean of five individual crabs plus or minus
standard error of the mean.
oxygen binding sites. Both processes can be experimentally followed by fluorescence
and absorbance spectroscopy as shown in Figure 5. The data demonstrated that incu-
bation of apohemocyanin with copper metallothionein leads to fluorescence quench-
ing before viable oxygen binding sites are formed, suggesting that copper insertion
precedes the formation of biologically active oxygen binding sites (see Discussion).
DISCUSSION
As indicated earlier (Brouwer et ai, 1986), one of the proposed functions of cop-
per metallothionein is as a Cu+1 donor for hemocyanin synthesis. The present experi-
ment, with dormant and warmed crabs, shows that the predominance of copper on
metallothionein among the warmed crabs is associated with the increased levels of
hemocyanin in the hemolymph. Our earlier work with blue crabs also suggested a
strong correlation between molting, copper metallothionein, and hemocyanin syn-
244
D. W. ENGEL AND M. BROUWER
DIGESTIVE GLAND
)NCENTRATiC
mol/kg)
•^ en 0) -si
b b b b
-
0 b 3.0
-
£ 5 2.0
fc 1.0
8
*-
T
_L
1
1
II
III
0 7.0
< *5 6.0
fE^ 5.0
-
T
I
I
1
1
I
5 i 4.o
-
O T
ZQ 3.0
-
8x 2.0
-
o" 1.0
-
iTi n
l=Summer Hard Crab
ll=Winter Hard Crab (cold)
lll=Winter Hard Crab (warmed)
FIGURE 2. Concentrations of copper and zinc in the digestive glands of blue crabs collected in the
summer and winter. Further information on handling of the crabs is in Figure 1 .
thesis (Engel, 1987). The studies reported in the present paper support that observa-
tion and demonstrate that environmentally induced changes and nutrition also can
cause changes in the copper/zinc ratios associated with metallothionein. This obser-
vation is important because it further emphasizes the possible constitutive role of
metallothioneins in normal metabolism.
In the following section we will develop a model of the regulation of copper and
zinc partitioning in the blue crab, based on studies by us (Engel, 1987; also present
paper), Soumoffand Skinner ( 1 983), and Henry and Kormanik ( 1 985).
The diagrams in Figure 6 display the physiological and biochemical processes
involved in crustacean molting and the cyclic and chronological nature of these
events. The first of these diagrams (Fig. 6 A) depicts the relative duration of the differ-
ent portions of the molt cycle. The actual timing of events is dependent upon both
environmental temperature and the size of the crab (Johnson, 1980). This type of
presentation emphasizes the fact that the most dramatic/traumatic changes in the
crabs occur over a relatively short period of time. The changes in concentrations of
copper and zinc associated with metallothionein are dramatic and provide further
evidence as to the dynamic nature of the molting process (Fig. 6B). If it is assumed
that metallothionein-bound copper and zinc are associated with metalloprotein and
metalloenzyme synthesis, we can predict when hemocyanin and zinc enzyme synthe-
sis occurs during the cycle (Fig. 6C, D). These predictions are tentative and will need
to be confirmed in future studies, since there are no direct data available in the litera-
ture on these aspects of crustacean physiology.
Copper metallothionein levels during the molt cycle are related directly to hemo-
cyanin concentrations in hemolymph, and inversely to ecdysteroid concentrations in
the hemolymph (Fig. 7). The decrease in digestive gland copper metallothionein is
BLUE CRAB METAL METABOLISM
245
l-Summer Crab
Q
o
111
o
z
<
CO
<r
O
CO
m
Absorbance
oCopper
FRACTION NUMBER
60
O)
3-
o
z
N
O
cc
LU
CL
CL
O
O
FIGURE 3. Sephadex G-75 elution profiles of digestive gland cytosol prepared from blue crabs col-
lected during the summer and winter. For further information on the crabs see Figure I. Protein separations
were made using 60 mM Tris buffer, pH 7.9 with 2 mA/|tf-mercaptoethanol with a 2.6 X 60 cm column at
a flow rate of 30 ml/h in all three groups of crabs (I, II, III).
correlated with an increase in ecdysteroid liter in blue crab hemolymph (Soumoffand
Skinner, 1 983). The ecdysteroid concentrations followed the same general pattern for
males and immature females throughout the molt cycle. After molt the ecdysteroid
level decreases rapidly with concomitant decreases in hemocyanin concentrations.
Coincident with these decreases is an increase in copper metallothionein during the
A2 and B, stages, followed by an increase in hemocyanin during B, . Such interrela-
tionships suggest a possible association between the molting hormone ecdysteroid,
and the regulation of hemocyanin synthesis and levels of cytosolic copper. It is rele-
vant to emphasize that synthesis of constitutive metallothioneins in mammals is un-
der the control of steroid hormones (Karin et al, 1 980 a, b; Karin el ai, 1981). No
such information exists for the invertebrate metallothioneins. The effect of the molt-
ing hormone 20-hydroxyecdysone on metallothionein synthesis in the blue crab is
presently under investigation.
Comparisons of zinc metallothionein (Engel, 1987) and ecdysteroid concentra-
tions (Soumoffand Skinner, 1 983) and carbonic anhydrase activity (Henry and Kor-
manik, 1985) during the molt cycle suggest an inter-relationship between these three
246
D. W. ENGEL AND M. BROUWER
9. 45X10-
CO
HI
LU
o
LU
O
CO
LU
cc
o
13
7.00X10-
315.00
335.00
WAVELENGTH (nm)
355.00
FIGURE 4. Fluorescence intensity of deoxygenated lobster hemocyanin in 50 mM Tris pH 8.0 + 10
mMCaC\2 as a function of the percentage copper remaining in the active site after dialysis against 20 mM
Cyanide for 0, 5, 10, and 30 min. ( 1 ) 100%, (2) 60%, (3) 30%, (4) 8%. Excitation is at 280 nm.
2.48
CO
z
LU
LU
O
z
LU
O
CO
LLJ
rr
O
2.40
2.32
2.24
.5
3 «
0
16
24
36
TIME (hours)
FIGURE 5. Change in fluorescence intensity and oxygen binding capacity (A Y) of partial apohemo-
cyanin (5 nM) in 50 mM Tris pH 8.0 + 10 mM CaCl2 still containing 35% of its original copper, as
a function of incubation time with copper-metallothionein ( 10 ^M Cu) in the absence of oxygen. The
fluorescence change (copper insertion) precedes the formation of viable oxygen binding sites.
BLUE CRAB METAL METABOLISM
247
Hemocyanln
Synthesis
and
Turnover
Zinc Enzyme
Synthesis
(Carbonic
Anhydrase)
Activity Increasing
FIGURE 6. Diagrammatic representation of the physiological and biochemical events occurring dur-
ing the molt cycle of the blue crab. (A) the molt cycle of the blue crab with the duration of each portion
indicating time. The designations of the molt stages are: C, — * C4, hard crab; D, — • D4; premolt; E, ecdysis;
A,-A2, soft crab; B,-B2, papershell crab (Mangum, 1985). (B) The relative concentrations of copper and
zinc on metallothionein are represented by the size of the copper, Cu and zinc, Zn symbols. (C) and (D)
These two figures represent predicted hemocyanin and zinc enzyme synthesis activities generated from
previously collected data (Engel, 1987). The degrees of shading are indications of the proposed activities
of the biochemical pathways involved in hemocyanin synthesis and turnover, and zinc enzyme synthesis
(carbonic anhydrase).
components (Fig. 8). During the premolt period (D!-D3) when both zinc metallothio-
nein and ecdysteroid are at their peaks, the new epidermis is being synthesized be-
neath the existing exoskeleton. At molt both zinc metallothionein and ecdysteroid
decrease, and between stages A, and A2 there is an abrupt increase in carbonic anhy-
drase activity in the newly formed exoskeleton epidermis (Henry and Kormanik,
1985). This rapid increase, which occurs over a period of hours, suggests that the
enzyme may be synthesized and present in the new epidermis as an apo-protein, and
is not activated by zinc until after molt. Even though the decrease in zinc-metallothio-
nein occurs in the digestive gland and the increase of carbonic anhydrase in the epi-
dermis, these two events may be linked. Possibly some of the zinc bound to metallo-
thionein at the time of molt could be mobilized via the hemolymph to activate the
apo-carbonic anhydrase. This hypothesis is attractive since preliminary results from
our laboratory have shown the release of zinc from zinc metallothionein during stages
A, and A2 (D. W. Engel, unpub. data).
The proposed cycles for copper and zinc-metallothionein (Figs. 7, 8) are specula-
tive, but they are based upon the best available information on the physiological and
248
D. W. ENGEL AND M. BROUWER
> z
o o
2 <
at oc
> o
i- z
< O
-1 o
111
or
Ecdysteroid*
Hemocyanin
.Copper Metallothionein
J I
i L
I I
I I
C D, D2 D3 D4 E A, A2 B, B2 C
MOLT STAGE
ill
x a:
i- i-
O z
-I LU
is
til O
Q. cr
Q. LLJ
O H
O tO
-I Q
£Z
DC <
*Soumoff and Skinner (1983)
FIGURE 7. A diagrammatic representation of the processes involved in hemocyanin synthesis and
turnover, and the interactions between ecdysteroids and copper metallothionein. The data on hemocyanin
and copper metallothionein concentrations are from Engel ( 1987) and for ecdysteroid from SoumorTand
Skinner (1983).
biochemical events controlling metal partitioning during molt. These changes are
reproducible, and our experiments concerning the effects of thermal changes on
metal distributions give further support to the hypothesis that metallothionein is a
constitutive metal-binding protein in blue crabs.
LLJ
to
oc
Q
CD h-
cr o
o
LLJ
LLJ
Lt
Carbonic Anhydrase*
Zinc Metallothionein
Ecdysteroid**
""r" V
D2 D3 D4 A,
MOLT STAGE
A2
Bi
LLJO
ILJ
LU
DC
tULLJ
LLJO
DC HI
B2
*Henry and Kormanik (1985) **Soumoff and Skinner (1983)
FIGURE 8. A diagrammatic representation of the processes involved in the synthesis of zinc-depen-
dent enzymes and in particular carbonic anhydrase, and how zinc metallothionein and ecdysteroid interact
to affect enzyme activity. The data on zinc metallothionein are from Engel (1987), on ecdysteroid from
SoumorTand Skinner (1983). and carbonic anhydrase from Henry and Kormanik (1985).
BLUE CRAB METAL METABOLISM 249
The events and processes described here and in an earlier publication (Engel,
1987) do not address the question of control of the molt cycle and metal turnover.
During the molt cycle there are pronounced changes in the cytosolic distribution
and tissue concentrations of metals and accompanying changes in the hemolymph
ecdysteroid concentrations. Studies by Singer and Lee (1977) suggest that the hemo-
lymph hormonal levels are modulated by changing MFO (mixed function oxygen-
ases) activity in the antennal gland. These authors demonstrated that MFO activity
in the gland also varies with stages of the molt cycle. This activity is negatively corre-
lated with the ecdysteroid levels (Soumoffand Skinner, 1983), suggesting that the
MFO system controls steroid/hormonal concentrations in molting blue crabs, which
in turn may affect metal partitioning as described in this paper.
Further evidence for metallothionein's metal regulatory function comes from the
in vitro hemocyanin reconstitution experiments. Apohemocyanin can only be recon-
stituted with Cu+1 (Konings et a/., 1969; Lontie and Witters, 1973). Since copper
binds to metallothionein as Cu+l, and since copper metallothionein levels and hemo-
cyanin biosynthesis seem to be linked in vivo, we initiated a study of hemocyanin-
copper metallothionein interaction in vitro. The data presented in Figure 4 show that
the intrinsic tryptophan fluorescence of lobster hemocyanin strongly depends on the
amount of Cu+1 bound to the active site. This is in line with the observations that
several crustacean hemocyanins contain tryptophan residues in close proximity to
the binuclear copper site (Gaykema et al., 1984; Linzen et ai, 1985). This property
allowed us to make a distinction betwen Cu+1 incorporation into the active site of
apohemocyanin and the formation of native functional oxygen binding sites. It is
evident from Figure 5 that the quenching of tryptophan fluorescence, observed when
apohemocyanin is incubated with copper metallothionein, precedes the formation of
biologically active oxygen binding sites. This strongly suggests that the copper transfer
process is followed by a slow reordering of the tertiary structure of the copper sites to
the native configuration. Similar sequences have been demonstrated for the reconsti-
tution process of many Cu+2 proteins where binding of copper to the active site is
followed by a slow return of the protein to its biologically active state (Kertesz et al.,
1972; Morpurgo et al., 1972; Rigo et al., 1978; Marks et al., 1979; Blaszak et al.,
1 983). This observation may also explain why reconstitution of apohemocyanin with
copper metallothionein can only be accomplished under anaerobic conditions
(Brouwer et al., 1986). The Cu+1 in the distorted sites is not capable of combining
reversibly with oxygen. Interaction of oxygen with Cu41 under these conditions re-
sults in oxidation of metal. These Cu+2-sites will not bind oxygen and are lost for
detection by absorbance spectroscopy. This hypothesis is presently under further in-
vestigation.
The studies described in this paper have demonstrated that marine Crustacea are
excellent model systems to study the role of metallothionein in copper/zinc metabo-
lism on an organismal, cellular, and molecular level. Only when this function of met-
allothionein is fully understood will it be possible to assess its value as a metal-detoxi-
fying protein.
ACKNOWLEDGMENTS
The authors thank Mr. William J. Bowen III, and Lt. (jg) Debra Davis of our
Laboratory for their assistance during this investigation. Hooper Family Seafood,
Smyrna, NC; and Pittman Seafood, Merrimon, NC, supplied the crabs used in this
investigation. The authors also thanks Dr. Bruce A. Fowler, National Institute of
250 D. W. ENGEL AND M. BROUWER
Environmental Health Sciences; Dr. G. Roesijadi, Chesapeake Biological Labora-
tory, University of Maryland; and Drs. Brenda Sanders and Kenneth Jenkins, Cali-
fornia State University, Long Beach, for reviewing this manuscript.
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crab, Callinectes sapidus: involvement of hemocyanin and characterization of cadmium-binding
proteins. Mar. Environ. Res. 14: 71-88.
BROUWER, M., P. WHALING, AND D. W. ENGEL. 1986. Copper-metallothioneins in the American lobster,
Homarus americanus: potential role as Cu(I) donors to apohemocyanin. Environ. Health Per-
spect. 65:93-100.
COUSINS, R. J. 1982. Relationship of metallothionein synthesis and degradation to intracellular zinc me-
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Holland, New York.
COUSINS, R. J. 1985. Absorption, transport, and hepatic metabolism of copper and zinc: special reference
to metallothionein and ceruloplasmin. Physiol. Rev. 65: 238-309.
ENGEL, D. W. 1987. Metal regulation and molting in the blue crab, Callinectes sapidus: copper, zinc, and
metallothionein. Biol. Bull. 172: 69-82.
ENGEL, D. W., AND M. BROUWER. 1984. Trace metal-binding proteins in marine molluscs and crusta-
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ENGEL, D. W., ANDM. BROUWER. 1986. Copper and cadmium metallothioneinsin the American lobster,
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FREE D-AMINO ACIDS IN THE TISSUES OF MARINE BIVALVES
HORST FELBECK AND SANDRA WILEY
University of California San Diego, Scripps Institution oj Oceanography, Marine Biology
Research Division, A-002, La Jo/la, California 92093
ABSTRACT
Seventeen species of marine bivalves were surveyed for the presence of free D-
alanine, D-aspartate, and D-valine in their tissues. D-aspartate was found in several
species in concentrations approaching those of L-aspartate. D-alanine was detected —
particularly in lucinid and vesicomyid clams — at levels exceeding manyfold those of
L-alanine. D-valine was absent in all cases. A test of a hydrolysate of bulk soluble
proteins of Lucinoma aequizonata, a species characterized by extremely high levels
of D-alanine, showed no major incorporation of D-alanine into proteins. The im-
plications of these results, for previously published analytical data and for human
nutrition, are discussed.
INTRODUCTION
D-amino acids generally are viewed as natural oddities. They are usually not
found in proteins, and they occur only occasionally in sizable quantities, either freely
dissolved, or incorporated into peptides and metabolites in the tissues of animals and
plants (Robinson, 1976; Bodanszky and Perlman, 1969; Corrigan, 1969). However,
it has been suggested that the D-isomers of amino acids were as common as the L-
forms in ancient prebiotic times on earth. Since the L-isomers were used exclusively
by various life forms, they were removed from this natural equilibrium. The supply
of L-amino acids was maintained by chemical racemization from the D-isomers left
behind (Aono and Yuasa, 1977). Even today relatively large amounts of D-amino
acids can be identified in oceanic waters, where apparently they have been formed
by chemical racemization from the large pool of dissolved L-amino acids (Lee and
Bada, 1977).
Widespread attention was first focused on D-amino acids when they were pro-
posed to be causes and indicators of cancer. Proteins in tumor tissues were thought
to contain high levels of D-glutamic acid, thus distinguishing them from normal tis-
sue (Koegl and Erxleben, 1939). This theory was rejected, however, after years of
controversy (Miller, 1950).
Recently, the occurrence of D-aspartate instead of the L-form in some proteins
has been a focus of investigation for molecular repair mechanisms (McFadden and
Clarke, 1982). It was reported that methylated aspartyl residues in erythrocyte mem-
brane proteins had been converted to the D-form. According to the theory, the ap-
pearance of D-aspartate in proteins is a first sign of degradation. These proteins are
either tagged for disposal, or the D-aspartyl residue can be reversed to the L-form
after methylation.
A similar mechanism, time-dependent chemical racemization of aspartate within
Received 9 September 1986; accepted 28 May 1987.
252
D-AMINO ACIDS IN BIVALVES 253
proteins with a low turnover rate, like eye lens or dental proteins, has been used to
date these proteins by measuring the ratio of the D- to the L-form of aspartate (Mas-
ters, 1983; Bada and Brown, 1980). Exactly the same principle of racemization has
also been used extensively to determine, with great accuracy, the ages of fossils, since
the normally present L-aspartate racemizes chemically at a constant rate after the
death of an organism (Bada and Schroeder, 1975).
D-aspartate may also be a neurotransmitter (Wiklund et ai, 1982). Presently, it
is being used experimentally as a non-metabolizabie replacement for L-glutamate
and L-aspartate in neurotransmitter research, since it can use the same uptake sites
(Drejer<Y0/.. 1983; Taxt and Storm-Mathisen, 1984).
Since finding that the artificial sweetener aspartame produces D-aspartate when
heated (e.g., during cooking) another line of research has been initiated (Boehm and
Bada, 1 984). Thus humans may be exposed nutritionally to considerable amounts of
a D-amino acid due to increased consumption of aspartame worldwide.
Only a few publications have focused on the metabolic role of free D-amino acids
in animal and plant tissue. D-alanine is present in some molluscs (Matsushima et a/.,
1984) and crustaceans (D'Aniello and Giuditta, 1980), and it has been demonstrated
to be synthesized during anaerobic metabolism in annelids (Felbeck, 1 980; Schoettler
et ai, 1983). Recently, a study of D-amino acids, as indicated by the reaction with
D-amino acid oxidase, in a variety of marine invertebrates was published (Preston,
1987a). D-amino acids were found in 18 of the 43 species of the 8 phyla surveyed.
The presence and metabolism of D-aspartate has been investigated in the tissues of
the bivalve Solemya reidi (Felbeck, 1985) and of some cephalopods (D'Aniello and
Giuditta, 1 977, 1 978). In all cases, the D- and the L-forms were present in about equal
concentrations. The concentrations of D- and L-alanine in the polychaete Arenicola
marina are approximately the same, and the concentrations of the two isomers in-
crease similarly in response to metabolic stress (Felbeck, 1 980; Schoettler et ai, 1 983).
The bivalve Solemya reidi takes up D-aspartate from environmental seawater and
metabolizes it just as quickly as it does the L-form. Initially the D-form is converted
into the L-isomer before further metabolism takes place (Felbeck, 1985). The uptake
and metabolism of D-alanine from seawater has been described recently for coelomo-
cytes of the annelid Glycera dibranchiata (Preston, 1987b).
Marine invertebrates commonly have extremely high concentrations of free
amino acids which, in the event of osmotic stress, form the largest share of the pool
of intracellular osmolytes (Bishop et ai, 1983). Therefore, D-amino acids in this pool
might serve as important metabolic reserves, sinks, or regulatory factors.
Several investigators have described either isolated occurrences of individual D-
amino acids in some invertebrates or have measured the unspecified presence of D-
amino acids. No known publication has surveyed organisms for individual D-amino
acids. The recent availability of chromatographic screening techniques for some
amino acid isomers prompted our investigation of a number of marine bivalves for
the presence of specific D-amino acids. We chose to study the Bivalvia because their
physiology is well known, a variety of species is readily available, and their tissues
contain high concentrations of free amino acids.
MATERIALS AND METHODS
Animals
Animals were purchased live at fish markets or collected from a variety of loca-
tions (Table I). The animals from the Santa Barbara channel were collected shipboard
254
H. FELBECK AND S. WILEY
TABLE I
Collection areas of animals
Animal
Collection area
Bathymodiolus thennophilus
Calyptogena elongata
Cliione californiensis
Chione stiitchburyi
Codakia obicularis
Codakia tigerina
Corbicula (liiminea
Crassostrea virginica
Hiatel/a pholadis
Hi unites multirugosus
Lima hemphilli
Lucinoma aequizonata
Mercenaria mercenaria
Modiohis capax
Mytilus edulis
Solemya reidi
Tapes japonic a
Pacific, Galapagos hydrothermal vents
Pacific, Santa Barbara channel
Pacific, Gulf of California
Fish market, San Diego, California
Atlantic, Bahamas, intertidal
Fish market, Phillipines
Fish market, San Diego
Fish market, San Diego
Pacific, La Jolla, California
Pacific, San Diego
Pacific, San Diego
Pacific, Santa Barbara channel
Fish market, San Diego
Pacific, San Diego
Fish market, San Diego
Pacific, Santa Monica Bay
Fish market, San Diego
by otter-trawl. Solemya reidi was collected shipboard in Santa Monica Bay by Van
Veen grab. Most other species were collected by divers and were maintained alive in
flow-through seawater tanks, at approximate in situ temperatures, for a maximum of
ten days before being sacrificed. Bathymodiolus thermophilus specimens were col-
lected by the submarine DSRV "Alvin" during a cruise to the Galapagos hydrother-
mal vents. The animals were frozen upon retrieval. Codakia tigerina was purchased
alive at a fish market in the Philippines and then shipped by air freight in 70% alcohol.
Sample preparation
To account for the presence of symbiotic bacteria in the gills of some of the bi-
valves used in this study (Felbeck el a/., 1981; Felbeck, 1983), all bivalves were
opened, and the gills were removed and analyzed separately from the remaining
soft parts.
The tissue, frozen with liquid nitrogen, was first pulverized in a mortar. The ho-
mogenization was then completed in 1 N HC1O4 with an Ultra-Turrax homogenizer.
The homogenate was centrifuged at 12,000 X g for 15 min, and the supernatant was
neutralized with 3 M KHCO3. The resulting precipitate was removed by centrifuga-
tion. An aliquot of this extract was derivatized with o-phthaldialdehyde (OPA) and
N-acetyl-L-cystein (NAC), according to the method described by Aswad (1984). The
amino acid isomers were then separated on a CIS reverse phase column with a gradi-
ent of 50 mA/ sodium acetate, pH 5.8, containing 8% methanol (Sol. A) to methanol
(Sol. B). The gradient was (in % of solution B): 0 min, 0%; 4 min, 0%; 10 min 25%;
20 min 27%; 34 min, 52%; and 50 min, 52%. Using this gradient — which was modi-
fied from the one described by Aswad (whose sole purpose was to separate D-and L-
aspartate) — the two alanine and valine isomers could also be separated completely.
Using a Gilson Datamaster integrator, standards for the D- and L-isomers of
aspartate, valine, and alanine were used to determine standard response curves.
When samples were analyzed, the area under each individual peak was used to deter-
mine concentration and, subsequently, the ratio of the individual stereoisomers.
D-AMINO ACIDS IN BIVALVES 255
To determine whether D-alanine was present in the proteins of L. aequi-onata,
tissue of a whole animal was homogenized in distilled water with an Ultra Turrax.
After centrifugation, the pellet was twice resuspended and rehomogenized in water.
The combined supernatants were dialyzed against multiple changes of distilled water
for five days to remove all free amino acids. The resulting solution of mixed soluble
proteins of L. aequizonala was then precipitated with perchloric acid, centrifuged,
and the pellet hydrolyzed overnight with HC1. The hydrolyzate was then analyzed
for D-amino acids as described above.
RESULTS
Significant concentrations of D-aspartate and D-alanine and their L-isomers were
detected (Table II); no D-valine was found. All Lucinidae showed high concentra-
tions of D-alanine — concentrations much higher than those of L-alanine. D-alanine
also was detected in Mercenaria mercenaria, both species ofChione, Hinnites gigan-
teus, Lima hemphilli (gills), Bathymodiolus thermophilus, Crassostrea virginica
(gills). Tapes japonica, Hiatella pholadis, and Corbiculafluminea. In all of these spe-
cies, the concentration ratios of D- to L-isomer was below one. In the Mytilidae Myti-
lus edluis and Modiolus capax, no D-alanine was detected, but D-aspartate was found
in concentrations approaching those of the L-isomer. D-aspartate was also detected
in the gills of Bathymodiolus thermophilus.
No D-alanine was detected in the hydrolyzed soluble protein fraction of Luci-
noma aequi~onata.
Because amino acid levels among individual animals of the same species are typi-
cally highly variable, we did not attempt to establish average concentrations for a
large number of bivalves but instead focused on the presence of D-amino acids. We
postulate that the detection of D-amino acids in any individual organism is significant
for the species in general.
DISCUSSION
The lucinids contained the highest D- to L-ratio of alanine. The extremely high
level of free alanine in Codakia ohicularis tissues has been measured only by ion-
exchange amino acid analysis and, therefore, has been attributed entirely to "generic"
alanine acting as an osmoregulatory agent or an end-product of anaerobic metabo-
lism (Berg and Alatalo, 1984). In fact, most of this alanine is in the D-form, prompt-
ing us to question the function of the D-alanine in this bivalve as well as in all lucinid
clams. One possibility is that the D-alanine is entirely made by the symbiotic bacteria
inside the cells of the gill. Gram negative bacteria, like the symbiotic species found in
the gill (see Schweimanns and Felbeck, 1 985, for review), often contain D-alanine in
their cell walls (Katz and Detrain, 1977). Therefore, extraction of the cell wall could
yield significant amounts of the D-isomer of alanine. In addition, these bacteria are
thought to provide a major share of the bivalves' nutritional needs by fixing CCK from
the seawater and transferring reduced organic compounds, possibly including D-ala-
nine, to the host. It is unlikely that the D-amino acids originate in bacteria, however,
since tissues lacking bacteria have a D- to L-isomer ratio similar to that of gills densely
populated with bacteria. If the bacteria produce and export D-alanine, then the gill
preparations should show a larger share of the D-isomer. In addition, some bivalve
species (Table II) without symbiotic bacteria also have high concentrations of D-
alanine.
Another peculiar aspect of the large proportion of D-alanine in the free amino
256
H. FELBECK AND S. WILEY
TABLE II
Concentrations ofD- and L-amino acids in the tissues of marine bivalves
Animal
Tissue
n
L-alanine
(umol/g
fw)
(x±SD)
D-alanine
(umol/g
fw)
(x + SD)
Ratio
D/L
L-aspartate
(umol/g
fw)
(x + SD)
D-aspartate
( umol/g fw)
(x±SD)
Ratio
D/L
SOLEMYIDAE
Solemya reidi
foot
2
14.2 ±6.9
0.1 ±0.1
0.01
13. 3 ±2.4
12.4 ± 1.6
0.93
gill
2
3.9 ±0.7
1.4 ±0.6
0.36
7.6 ±2.0
5.4 ± 2.2
0.71
MYTILIDAE
Mytilus edulis
foot
2
4.4 + 0.2
—
—
4.6 ± 1.8
1.4 ±0.5
0.3
gill
2
2.8 ±0.9
—
—
3.9 ±0.8
3.5 ±0.7
0.9
Modiolus capax
foot
2
2.7 ± 1.4
4 ±5.7
1.5
6.9 + 2.7
2.6 ±0.01
0.38
gill
1
0.5
0.7
1.4
2.9
2.3
0.79
Bathymodiolus
foot
1
12.3
0.9
0.08
2.4
—
—
thermophilus
gill
1
11.4
3.2
0.28
2.2
0.7
0.31
OSTREIDAE
Crassostrea virginica
mantle
1
19.4
—
—
3.5
—
—
gill
2
9.9 ±2.6
1.1 ±0.1
0.12
5. 3 ±0.5
—
—
PECTINIDAE
Hinnites
foot
2
1.4+ 1.2
1.1+0.9
0.79
1.0± 1.3
0.4 ±0.6
0.4
multirugosus
gill
2
0.5 ±0.2
0.7 ±0.9
1.4
0.5 ±0.2
0.2 ±0.2
0.4
LIMIDAE
Lima hemphilli
VENERIDAE
gill
1.0 + 0.5
0.5 ±0.3
0.5
0.8 ±0.1
Tapes japonica
foot
1
13.4
11.2
0.84
15.5
—
—
gill
1
4.7
3.5
0.74
1.7
0.2
0.12
Chione califomiensis
foot
1
3.0
2.1
0.7
14.6
—
—
gill
3
3.8 ±2.6
1.8+ 1.5
0.47
4.5 ±0.9
—
—
Chione stutchburyi
foot
1
4.6
2.0
0.44
6.6
—
—
gill
2
3.3 ± 1.5
1.3 ±0.7
0.39
3. 3 ±2.8
—
—
Mercenaria
foot
2
17.7 + 3.1
16.2 ± 1.9
0.92
7.8 ± 3.5
—
—
mercenaria
gill
3
4.2 ± 2.2
2.8 ± 1.4
0.67
4.1 + 1.4
—
—
CORBICULIDAE
Corhiaila /Iiiminea
foot
2
1.9 ±0.4
0.9 ± 1.2
0.47
0.7 ±0.2
—
—
gill
3
3.8 ±0.8
0.6 + 0.1
0.16
0.4 ±0.3
—
—
HIATELLIDAE
Hiatella pholadis
gill
2
4.4+ 1.0
1.4 ±0.2
0.32
2.3 ± .01
—
—
VESICOMYIDAE
Calyptogena
foot
1
1.1
11.3
10.45
11.5
—
—
elongata
gill
1
0.3
2.7
9.0
1.4
—
—
LUCINIDAE
Codakia obicularis
foot
1
13.5
187.2
13.9
0.3
0.2
0.67
gill
1
2.4
26.6
li.l
0.3
0.1
0.33
Codakia ligerina
foot
1
0.4
22.4
56.0
0.6
0.1
0.17
gill
1
0.4
21.5
53.7
0.4
—
—
Lucinoma
foot
1
3.8
84.2
22.2
0.4
0.3
0.8
aequizonala
gill
3
3.1 ±0.5
26.1 ±4.1
8.4
1.0 ±0.9
0.1
0.1
The levels are given in ^mol/gram fresh weight with the standard deviation. When only gills were tested, the foot was
too small to be easily dissected and analysed. (" — " below 0. 1 ^mol/g fw)
acids of Lucinidae is that the enzyme most commonly responsible for the formation
of D-amino acids — amino-acid racemase — would cause an equal distribution be-
tween the two isomers (Barman, 1969). The fact that up to 98% of the free alanine
pool is in the D-form suggests that: (a) another specialized enzyme is responsible
for the metabolism of the D-isomer; and (b) the D-isomer is not metabolized after
conversion to the L-form by a racemase, but used separately. Aside from the lucinid
clams, however, other examples were found where the ratio of D- to L-alanine was
lower: below one. Here we assume that a racemase interconverts the two isomers.
The occurrence of D-aspartate can be explained more easily by the presence of a
D-AMINO ACIDS IN BIVALVES 257
racemase, for the maximal ratio of the D- to the L-isomer was around one. Indeed,
this specific racemase has already been demonstrated in Solemya reidi (Felbeck,
1 985). The aspartate-racemase does not catalyze the conversion of alanine.
In spite of the high D-alanine concentration in Lucinoma aequizonata, no detect-
able quantities of D-alanine were found in the proteins of this animal. Therefore, the
selection for the L-isomer of alanine in protein synthesis is significant. Since the
method used only provides a crude overview of a selected group of proteins — those
soluble in distilled water — we cannot exclude the possibility that some minor fraction
of the soluble or insoluble proteins would include D-alanine; neither of these would
have been detected by the method used.
Wide ranging surveys for the presence of D-amino acids are rarely in the literature.
The review article by Corrigan (1969) and Preston's (1987a) recent results are the
only known examples. Certainly, one reason is that simple, quick methods to deter-
mine the concentrations of D- and L-isomers of individual amino acids have been
published only recently. Before this, either both isomers were detected as a sum (e.g.,
in HPLC with OPA/mercaptoethanol derivatization or with the classical ion ex-
change amino acid analyzer) or just the L-isomer was detected in typically stereospe-
cific enzymatic determinations. Since it was always assumed that no D-amino acids
were present, the results obtained by these methods were taken as representative for
"all" amino acids. The D-amino acids concentrations found by Preston ( 1 987a) were
obtained unspecifically with a test using D-amino acid oxidase and, therefore, were
only applicable as indicator of the general presence of most D-amino acids ( D-aspar-
tate and D-glutamate do not react with the D-amino acid oxidase).
Our survey includes the amino acids alanine and aspartate, both of which are
commonly found in high concentrations in marine invertebrates, and shows the fre-
quent occurrence of both stereoisomers.
This result is significant for "standard" experimental research organisms like Myt-
ilus edulis (Bishop et ai, 1 983). In this species, the pool of free aspartate is used as an
initial substrate for anaerobic metabolism (see de Zwaan and Putzer, 1985, for re-
view). Whenever the concentration of this amino acid was tested using enzymatic
methods, only about half of the available amino acid was detected; i.e., the pool of
aspartate was actually higher than measured. This may explain the apparent lack of
enough initial substraate for anaerobic energy metabolism, as recently reviewed by
de Zwaan and Putzer (1985). Similarly, in other organisms such as the polychaete
Arenicola marina, the initial depletion of the (enzymatically measured) L-aspartate
is not large enough (Felbeck, 1980; Schoettler et ai, 1983). We think it is possible
that D-aspartate, as well as the L-isomer, occurs in Arenicola, and that it serves there
as additional substrate not detected by enzymatic analysis which after rapid racemiza-
tion also can be used as metabolic substrate.
We conclude that many published results where amino acid levels in invertebrates
have been used as indicators for metabolic pathways, or to calculate metabolic rates,
will have to be reassessed because D-amino acids may be present in the tissues used.
Currently, we can only speculate what the metabolic role of D-amino acids is in
marine invertebrates. Amino acids are usually used as osmolytes in the tissues of
marine invertebrates and, therefore, are often present in very high concentrations
(Bishop, 1983; Yancey et al, 1983). The exchange of part or most of the L-isomer
for the D-isomer may influence regulatory mechanisms involving these amino acids.
Glutamate-pyruvate-transaminase is inhibited by high levels of L-alanine (Barman,
1969); the D-isomer may not have this effect on this enzyme.
Finally, large quantities of free D-amino acids in tissues of common marine bi-
258 H. FELBECK AND S. WILEY
valves may affect human health. Some of our test species were obtained from com-
mercial fish markets. For example, large quantities of D-alanine were found in Co-
dakia tigerina specimens bought in a fish market in the Philippines. These fish are
routinely consumed by humans. Even Mytilus edulis, one of the most common bi-
valves cultured and consumed in large quantities, contains a concentration of D-
aspartate, equal to that of L-aspartate, which is usually between 3 and 14 ^mol/g
fresh weight (de Zwaan and Putzer, 1985). Little research has been done on the me-
tabolism of D-amino acids in humans or on the effect of long-term exposure to D-
amino acids. D-amino acids can cause analgesia in humans, and some D-amino acids
are powerful inhibitors of some enzymes involved in regular metabolic pathways
(Koyuncuoglu and Berkman, 1982). The result presented in this paper — that some
D-amino acids exist sometimes in extremely high concentrations in commonly con-
sumed shellfish — should prompt a closer examination of the effects of D-amino acids
on humans.
ACKNOWLEDGMENTS
This research was funded by NSF grant OCE83- 1 1 259 to HF and George Somero.
We thank Ron McConnaughey and John O'Sullivan from SIO, and Mr. Colin Higgs
of the Department of Fisheries (Nassau, Bahamas) for collecting and providing speci-
mens. Spencer Luke for identifying most of the species, and Drs. Patricia Masters
and JeffBada for help in hydrolyzing a protein sample and assistance with the HPLC.
Mr. Robert Yin kindly provided Codakia tigerina from the Philippines.
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Reference: Biol. Bull. 173: 260-276. (August, 1987)
TROPHOSOME ULTRASTRUCTURE AND THE CHARACTERIZATION
OF ISOLATED BACTERIOCYTES FROM INVERTEBRATE-SULFUR
BACTERIA SYMBIOSES
STEVEN C. HAND
Department of Environmental, Population and Organismic Biology, University of Colorado, Campus Box
B-334. Boulder, Colorado 80309. and Department of Biology. University of
Southwestern Louisiana, Lafayette, Louisiana 70504
ABSTRACT
Electron microscopy of trophosome tissue from the vestimentiferan tubeworm
Riftia pachyptila clearly indicates that the bacterial symbionts are enclosed within
animal cells (bacteriocytes). The structure of this lobular tissue is complex. Each lob-
ule consists of an outer layer of trophochrome cells (devoid of symbionts, but with
numerous pigmented granules), an inner region of bacteriocytes, and a central hemo-
lymph space. Sulfur deposits within bacteria decrease in size and number with in-
creasing distance of the bacteria from the hemolymph space. Bacteria located toward
the center of the lobule appear smaller than those nearer the periphery, suggesting
that metabolic and developmental gradients exist. Trophochrome cells and free bac-
teria were enriched from the trophosome ofR. pachyptila.
A procedure is described for the isolation of bacteriocytes from gill tissue of the
bivalves Calyptogena magnified and Lucina floridana. Numerous bacteria reside in
vacuoles within the bacteriocyte cytoplasm, as do large (5- 10 micron), heterogeneous
granules. Maximum CO2 fixation rate at 20°C for bacteriocytes from C. magnified is
13.2 nmoles CO2/mg protein/h, compared to 21.6 nmoles CO2/mg protein/h for L.
floridana bacteriocytes. Fixation by bacteriocytes from C. magnified is inhibited by
sulfide, and to a lesser extent thiosulfate, at 0.1-1.0 mA/. Thiosulfate increases CO2
fixation two-fold in L. floridana bacteriocytes.
C. magnified bacteriocytes incubated for 1 h in 0.5 mA/ sulfide maintain higher
intracellular ATP concentrations (3.3 nmoles/million cells; 1.01 mA/) than do con-
trol cells without sulfide (1.02 nmoles/million cells; 0.31 mA/). These results and
comparable observations suggest that the identities of exogenous sulfur compounds
exploited for chemical energy by the symbiosis may depend on the structural integrity
and organization of the experimental preparation.
INTRODUCTION
In symbiosis between sulfur bacteria and marine invertebrates, various metabolic
features are critically dependent on the cellular integrity of each participant. To study
these characteristics without disrupting cellular structure, we developed a procedure
for isolating intact bacteriocytes (eucaryotic cells that contain large numbers of bacte-
rial endosymbionts) from gill tissues of the hydrothermal vent clam, Calyptogena
magnified, and the shallow-water bivalve Lucina floridana, an inhabitant of seagrass
beds. An ultrastructural description of the intact trophosome of the hydrothermal
Received 16 April 1987; accepted 29 May 1987.
260
INVERTEBRATE-BACTERIA SYMBIOSES 261
vent tubeworm Riftiapachyptila (Pogonophora) provides new information about cel-
lular arrangements and metabolic potentials in this symbiont-containing tissue. Fi-
nally, using bacteriocyte suspensions prepared from the bivalves, we measured intra-
cellular ATP levels and the capacity for carbon fixation in the presence of various
sulfur compounds.
The primary advantage of using bacteriocytes for metabolic studies is that the
symbiotic bacteria are retained in their natural microenvironment. As a conse-
quence, the bacteria receive chemical signals (sulfur compounds, dissolved gases, etc.)
via the cytoplasm of the host cell. Furthermore, all bacteriocyte surfaces are in direct
contact with medium constituents, so that the effects of slowly exchanging compart-
ments such as connective tissue spaces are minimized. Thus individual bacteriocytes
are considered functional symbiotic units, and their response to various stimuli quan-
tified on a cellular basis.
Until now, isolated invertebrate cells have not been used to study physiological
and biochemical relationships between sulfur oxidizing bacteria and the host. Rather,
previous studies have focused on other levels of biological organization and complex-
ity. Data have been obtained using ( 1 ) the intact symbiosis at the whole-organism
level (e.g., Anderson, 1986; Arp et aL 1984; Childress et al., 1984; Felbeck, 1983,
1985; Fiala-Medioni et al.. 1986), (2) excised tissues (e.g., Cavanaugh, 1983; Dando
etal, 1985; Felbeck, 1983; Powell and Somero, 1983), (3) variously prepared homog-
enates of tissues (e.g., Felbeck, 1981; Felbeck etal., 1981; Fisher and Childress, 1984;
Fisher and Hand, 1984; Hand and Somero, 1983; Powell and Somero, 1985, 1986a),
and (4) isolated bacteria and cellular organelles (e.g., Belkin et al., 1986; Powell and
Somero, 1986b). Depending on the degree of tissue disruption, significant variation
was observed in the metabolism of sulfur compounds and the rates and characteristics
of carbon fixation. For example, using homogenates of gill tissue from C. magnifica,
Powell and Somero (1986b) reported that stimulation of ATP synthesis by sulfur
compounds occurred only when bacteria contained therein were lysed.
Experimental preparations that maintain the bacteria in more biologically realis-
tic surroundings offer new opportunities for assessing their metabolic potential. This
possibility was the impetus for the present study. In addition to isolating bacteriocytes
from gill tissue of C. magnifica and L. floridana, we isolated trophochrome cells
(green pigmented cells) and free bacteria from trophosome tissue of R. pachyptila.
Although electron micrographs presented herein indicate bacteriocytes within the
trophosome, we were unsuccessful in isolating them intact from this source.
MATERIALS AND METHODS
Experimental animals and reagents
Specimens of Rift i a pachyptila and Calyptogena magnifica were collected in
March 1985 during the hydrothermal vent expedition to the Galapagos Rift with the
submersible DSRV Alvin at the "Rose Garden"1 site (Hessler and Smithey, 1983).
The live animals on board the RV Melville were handled as described by Powell and
Somero ( 1986a). Tissue samples were dissected from specimens ofR. pachyptila and
C. magnifica typically within 3 h of their arrival on board ship. Tissue weights were
determined with the motion compensated shipboard balance developed by Childress
and Mickel( 1980).
Specimens of the eulamellibranch bivalve Lucina floridana were collected from
the sulfide-rich sediments of Thalassia and Ruppia seagrass beds in St. Joseph's Bay,
Florida. Animals were maintained in the laboratory as described by Fisher and Hand
( 1 984) for no more than three weeks prior to tissue dissection.
262 S. C. HAND
Hyaluronidase (Type 1-S), collagenase (Type IV), DNAase I (Type IV), chymo-
trypsin (Type II), soybean trypsin inhibitor, and Percoll were purchased from Sigma
Chemical Co. NaH'4CO3 was obtained from New England Nuclear. All other chemi-
cals were reagent grade. Solutions of sodium sulfide were prepared fresh before each
experiment (Powell and Somero, 1986a) and maintained under nitrogen until use
(1-3 h). To reduce mechanical damage to isolated cells, siliconized glassware was
used in all steps described below, and all pipets were firepolished.
Tissue dissociation
C. magnified gill tissue was placed on a chilled glass plate and minced into cubes
varying in size from 0.5 mm to 2 mm. The tissue was rinsed briefly in Ca++-Mg++
free salt solution (CMF solution) (508 mM NaCl, 10 mM KC1, 8.7 mM NaHCO3,
28.6 mM Na2SO4, 0.1 mM EGTA, 4 mM glucose, pH 7.2) to remove mucus. The
tissue was then transferred to 50 ml flasks and incubated for 1 5 min at 20°C in 20 ml
of CMF solution on a rotary shaker (1 10 cycles/min). This initial medium was re-
placed with 10 ml of artificial seawater (4 1 1 mM NaCl, 9.6 mM KC1, 54 mM MgCl2,
10.5 mMCaCl2, 8.8 mMNaHCO3, 23.6 mM Na2SO4, glucose 4 mM, pH 7.2) con-
taining hyaluronidase (400 U/ml), collagenase (500 U/ml), and chymotrypsin (70 U/
ml), and the tissue was incubated for 1.5 h at 20°C. At the end of this period, the
tissue was rinsed with CMF solution and incubated 1 5 min in 10ml of CMF solution
containing bovine serum albumin ( 1 mg/ml), trypsin inhibitor (0.4 mg/ml), and
DNAase 1(15 U/ml). The tissue in this solution was flushed 20-30 times through a
siliconized Pasteur pipet, a procedure that released large numbers of cells. The cellu-
lar suspension was filtered sequentially through 250 micron and 100 micron nylon
mesh (Tetko, Inc.; Elmsford, New York) to remove undissociated tissue.
Isolated cells from Riftia trophosome were prepared similarly, except the tissue
incubation with enzymes was shortened to one hour at 20°C. The concentrations of
enzymes were all reduced 50%, compared to the levels used for C. magnifica.
The procedure for dissociation of gill tissue from L. floridana differed from the
above protocol for C. magnifica in several respects. The concentration of the artificial
seawater was 40 ppt (470 mM NaCl, 11 mM KC1, 62 mM MgCl2 , 12mMCaCl2, 10
mM NaHCO3, 27 mM Na2SO4, 0.1 mM EGTA, 5 mM glucose, pH 7.2), and the
CMF solution consisted of 581 mM NaCl, 1 1.4 mM KC1, 10 mM NaHCO3, 32.7
mM Na2SO4, 0.1 mM EGTA, 5 mM glucose, pH 7.2. The minced gill tissue was
incubated for 15 min at 37°C in CMF solution, and the concentrations of enzymes
used in the subsequent incubation (1 h at 37°C) were 50% of those used for C. mag-
nifica tissue.
Cell isolation
The cellular suspension from C. magnifica gill was divided into two 5-ml portions,
each of which was layered onto a Percoll gradient at 4°C. This gradient separated
bacteriocytes from other cell types and from acellular and subcellular debris. Cellular
suspensions ofR. pachyptila trophosome were treated similarly. The 40-ml discontin-
uous gradient consisted of four steps of 10% (density, 1.042), 30% (1.065), 50%
(1.089), and 70% (1.111) Percoll. Each step was prepared by adding appropriate
amounts of Percoll and deionized water to 2 ml of a concentrated CMF stock (5X).
The cells settled without centrifugation for 3 h at 4°C, and cells that had accumulated
at each interface were collected and rinsed twice with artificial seawater to remove
Percoll (which interferes with the assay for CO2 incorporation).
INVERTEBRATE-BACTERIA SYMBIOSES 263
The Percoll gradient was changed to 30%, 50%, 70%, and 90% (density, 1 . 1 20) for
separation of L. fioridana bacteriocytes. Each step was prepared in the 40 ppt CMF
solution (final concentration).
Cell concentrations were determined with a hemocytometer. The distinguishing
features used to identify bacteriocytes under light microscopy were their granular
appearance, lack of cilia, and relatively large diameter (20 microns, C. magnified; 40
microns, L. fioridana).
Transmission electron microscopy
Isolated cells to be fixed for electron microscopy were transferred to Beem cap-
sules and centrifuged at low speed (500 X g) to concentrate the cells. The supernatant
was removed, and glutaraldehyde (4% in 0.3 M PIPES buffer, pH 7.2 at room temper-
ature) was layered over the cells. Intact tissue for fixation was dissected into small
blocks ( 1 mm diameter) and placed in plastic specimen trays containing glutaralde-
hyde solution. After 30-60 min, the glutaraldehyde was removed, and the Beem cap-
sule (or specimen tray) was filled with warmed agar (1.5% in 0.3 M PIPES, pH 7.2).
After the agar solidified, the capsules were given three 15-min washes in buffer and
then placed in 1% osmium tetroxide (prepared in 0.2 M potassium phosphate buffer,
pH 7.4) for 2-3 h. The preparations were washed thoroughly with deionized water,
dehydrated in a graded acetone series, and embedded in Spurr's low-viscosity media.
All the steps above were completed on board ship. Sections were cut with a Sorvall
MT 5000 Ultramicrotome and stained with 4% uranyl acetate followed by lead ci-
trate. Cells were viewed with a Hitachi H-600 electron microscope.
CO 2 fixation studies
Isolated cells (100,000-500,000 for each assay) were incubated for up to 30 min
in 0.5 ml of artificial seawater (pH 8.2) containing 1 microcurie of NaH14CO3, with
and without various concentrations of sulfide and thiosulfate. All incubations were
performed at 20°C. Incorporation of CO2 was stopped by vigorously mixing 0.1 ml
of 1 2 N HC1 with each sample. Samples were transferred to plastic counting vials
and heated for 2 h at 90°C. Radioactivity remaining in the acid soluble fraction was
quantified with liquid scintillation counting. Values for duplicate samples stopped at
time 0 were subtracted from all treatments.
A TP measurements
Experiments to determine the influence of sulfur compounds on cellular ATP
levels were performed similarly to those above, but the radioactive bicarbonate was
omitted from the incubation medium. At the end of the incubation, cells were sedi-
mented with low speed centrifugation (500 X g, 5 min) at 4°C, and the incubation
medium was decanted. Cells were then resuspended in 0.5 ml of ice-cold 0.6 M per-
chloric acid and homogenized. The homogenate was neutralized (and perchlorate
salts precipitated) with 0. 1 5 ml of a solution containing 0.2 7VKOH, 0.4 Mimidazole,
and 0.4 M KC1. The supernatant was initially stored in liquid nitrogen on board ship
and later transferred to a -80°C freezer until ATP analyses could be performed.
ATP was measured with an enzyme-coupled fluorometric assay (modified from
Lowry and Passonneau, 1972). All solutions were filtered through Gelman TCM-450
(0.45 micron) filters before use. The 1 .2 ml assay mixture contained 50 mMTris-HCl
buffer pH 8.1, 1.0 mM MgCl2, 0.2 mM dithiothreitol, 0.05 mM NADP, 0.1 mM
glucose, and 50 microliters of sample. First, 0.07 units of glucose-6-phosphate dehy-
264 S. C. HAND
drogenase were added to eliminate endogenous G-6-P, and then 0.34 units of hexoki-
nase were added for the quantification of ATP. The excitation wavelength was 365
nm, and the emission monochromator was set at 460 nm. The increase in fluores-
cence was measured with a Turner Model 430 spectrofluorometer, and the fluores-
cence signal was adjusted so that 0. 1 nmole of ATP gave a 25% full scale deflection.
Protein measurements
Total protein was analyzed following the procedure of Peterson (1977).
RESULTS
Morphology oftrophosome tissue
Fresh trophosome from R. pachyptila is a gelatinous, pulpy, dark iridescent-green
tissue. If the tissue is suspended in saline, numerous finger-like lobules project into
the medium producing a villous appearance. Each lobule is approximately 0. 1 5 mm
in diameter and has a complex ultrastructure that is revealed by examining cross
sections with electron microscopy (Fig. 1 A).
The cells composing the outer pigmented layer of the lobule are tightly packed
with membrane-bound inclusions of at least three morphological types. One type of
granule (Fig. IB, 2A) is homogeneous in composition, weakly electron-dense, and
similar in appearance to mucus droplets or mucigen granules found in goblet cells of
intestinal epithelia (e.g.. Porter and Bonneville, 1973). In contrast, the darker osmio-
philic granules (Fig. IB, 2B) contain highly organized, crystalline arrays of material
(probably proteinaceous) that may be responsible for the intense green color of the
trophosome. Indeed, if this outer cellular layer is osmotically lysed and removed from
fresh trophosome tissue, the underlying tissue is white (R. Vetter, pers. comm.). The
third type of inclusion (Fig. IB) has an electron density intermediate to that of the
previous two granules, and its appearance indicates a heterogeneous composition.
Thus, based on the internal morphology, these pigmented cells comprising the outer
layer oftrophosome tissue are referred to hereafter as trophochrome cells.
Nuclei are visible in the trophochrome cells, but other common organelles (e.g.,
mitochondria, endoplasmic reticulum) are infrequent. The granules described above
occupy the vast majority of the intracellular space. Even though it is possible that
osmotic swelling could have accentuated the size of these inclusions, the structural
integrity of both the limiting and internal cell membranes does not suggest extensive
swelling.
Subtending the trophochrome cell layer are numerous bacterial endosymbionts
(Fig. 3A, B). The bacteria housed in trophosome tissue are roughly spherical, often
with irregular cell envelopes, and are approximately 3-5 microns in diameter. As one
moves toward the center of the lobule, the morphology of the bacteria changes. Sulfur
deposits within bacteria increase in both size and number, and ribosomes in the bac-
terial cytoplasm are less distinct compared to bacteria located toward the periphery
of the lobule (Fig. 3 A, B). (Note: sulfur deposits are identified as vacuoles where sulfur
was extracted during tissue dehydration and embedding procedures.) Bacteria located
toward the center of the lobule also appear smaller. Concentric membrane whorls
within the bacteria are occasionally visible (Fig. 4 A, upper left).
Although it is difficult to fully trace eucaryotic cell membranes, the following evi-
dence indicates that the vacuole-enclosed bacteria are located within animal cells
(bacteriocytes). There are nuclei, mitochondria, and other organelles interspersed
among the bacteria (Fig. 4A, B). In Figure 4A, it is possible to delineate (moving
INVERTEBRATE-BACTERIA SYMBIOSES
265
FIGURE 1 . A. Cross section of a trophosome lobule from Riftia pachyptila viewed at low magnifica-
tion with transmission EM. The outer trophochrome cell layer contacts the coelomic fluid space (cf).
Subtending this pigmented cell layer are numerous bacteria (b). At the center of the lobule is a hemolymph
space (hs). Scale bar = 10 microns. B. Higher magnification of the trophochrome cells showing a nucleus
(n) and the tight intracellular packaging of diverse types of granules. Scale bar = 3 microns.
266
S. C. HAND
\
FIGURE 2. Two types of vacuole-enclosed granules present in trophochrome cells from Riftia pa-
c/iyptila. A. Nondescript granules of uniform density, similar in appearance to mucigen granules. Scale bar
= 1 micron. B. Electron-dense granule containing crystalline arrays (ca) of material that is proteinaceous in
appearance. Scale bar = 2 microns.
outward from the center of the bacterium labeled "b") the bacterial cell envelope, the
peribacterial membrane, and immediately adjacent, a nuclear envelope.
At the very center of the trophosome lobule, there is a hemolymph space or sinus
extending longitudinally (Fig. 4B). We were unable to discern a basal lamina separat-
ing the bacteriocytes from the hemolymph space of the lobule.
Since the morphology and fine structure of the gill tissue from C. magnified (Fiala-
Medioni and Metivier, 1986) and L. floridana (Fisher and Hand, 1984) have already
been described, we will not redescribe them here.
Isolated cell preparations
The cell purity for bacteriocytes isolated from bivalve gill tissue was approxi-
mately 70-80 percent (Table I). The amount of cellular debris present was not quanti-
fied, but was generally low. The yield of bacteriocytes was higher from L. floridana
gill tissue than from C. magnified. The reason may be that, while dissociation of L.
floridana tissue was performed at 37°C, the incubation temperature for C. magnifica
tissue had to be reduced to 20°C because of its temperature sensitivity. Although
using 37°C incubations with the vent clam tissue improved the yield, the resulting
bacteriocytes were not viable, as judged by the lack of ability to fix CO2. Prior to the
Percoll gradient step, the bacteriocytes represented 1 1% of the total cells in suspension
from L. floridana gill. Thus, the density gradient fractionation achieved a 7-fold en-
richment of bacteriocytes.
Attempts to isolate intact bacteriocytes from trophosome tissue were unsuccess-
ful, suggesting that these cells are very fragile and are unable to withstand the isolation
INVERTEBRATE-BACTERIA SYMBIOSES
267
f.Vf£. J *»
• : *V
•*•••-
' .
•1v-.';-;.-
• • •
I " -
FIGURE 3. Bacteria (b) of Riftia pachyptila trophosome tissue. A. Located toward the periphery of
the lobule, these bacteria are granular and contain very few sulfur deposits (observed as holes in the section
where leaching has occurred). Scale bar = 1 micron. B. Bacteria located toward the center of the lobule
contain more sulfur vacuoles (v), and their cytoplasm is less granular. Scale bar = 1 micron.
268
S. C. HAND
FIGURE 4. A. Bacterium (b) immediately adjacent to an animal cell nucleus (n). Scale bar = 1 mi-
cron. B. Micrograph showing hemolymph spaces (hs) located at the center of the lobule. Scale bar = 5
microns.
procedures. In contrast, trophochrome cells were enriched to a purity of 80%, and
free bacteria were isolated in high yield and at a comparable purity to the pigmented
trophosome cells. The primary contaminants of the trophochrome cell preparation
were free bacteria.
INVERTEBRATE-BACTERIA SYMBIOSES 269
TABLE I
Yield and purity of isolated cell preparations
Gradient interface Yield Purity Protein
Cell source and type (% Percoll) (million cells/g tissue) (% of total) (mg/million cells)
C. magnified
Bacteriocytes 1.16±0.08a 72 ± 5.6 0.90 ±0.1 4
30-50%, 50-70% (n = 3) (n = 5) (n = 3)
Non-bacteriocyte
epithelial cells 10-30% 10.3 97 0.16
L. floridana
Bacteriocytes
50-70%
3.0 81 ±0.8
(n = 3)
—
R. paehyptila
Bacteria
10-30%
— 79, 70
0.014
Trophochrome cells
30-50%, 50-70%
— 78,82
1.09
a Mean ± standard error.
One prominent ultrastructural feature of isolated bacteriocytes from C. magnified
is wide-spread fields of bacteria — easily the most numerous subcellular structures in
the cytoplasm (Figs. 5A, C; 6A). Compared to the bacteria present in trophosome
tissue, these bacteria are much smaller (0.5-0.7 micron diameter). They are clearly
contained in vacuoles within the bacteriocyte, and nuclear regions are evident
(Fig. 6A).
Large granules (approximately 5- 1 0 micron diameter) are a second salient feature
of bacteriocytes from both C. magnified (Fig. 5 A, C) and L. floridana (Fig. 5B). Some
of the C magnified granules are irregularly shaped and quite electron-dense, and
others are more circular and have a stippled appearance. The electron-dense granules
are morphologically similar to those of bacteriocytes from L. floridana (Fig. 5B, and
Fisher and Hand, 1984). When viewed with Nomarski differential-interference-con-
trast microscopy, the granules of L. Floridana are striking and are certainly the domi-
nant inclusion of the bacteriocyte (Fig. 5B). Previous work indicated the presence of
iron in these granules (Fisher and Hand, 1984; cf., Wittenberg, 1985), but whether
the bacteriocytes' granules in C. magnified are chemically similar is unknown. As
judged by the sedimentation behavior of bacteriocyte populations in Percoll gradi-
ents, L. floridana bacteriocytes have a greater density, which may be a consequence
of differences between these pigment granules. Bacteriocytes from L. floridana also
are over two-fold larger in diameter than those from C. magnified (Fig. 5A, B).
The morphology of isolated trophochrome cells (Fig. 6B) is essentially unchanged
from that seen for intact tissue (Fig. IB). On the other hand, isolated bacteria look
more irregular in overall shape compared to those viewed in situ and occasionally
were more vacuolated, suggesting that the isolation procedure for this cell type needs
improvement.
Metabolic properties of isolated cells
The capacity for carbon dioxide fixation of C. magnified bacteriocytes and iso-
lated bacteria from Rift id trophosome is presented in Table II. In the absence of sulfur
compounds, incorporation of CO2 by bacteriocytes proceeds at a rate of 13 nmoles
CO2/mg protein/h. Fixation is inhibited by sulfide and, to a lesser degree, thiosulfate
270
S. C. HAND
FIGURE 5. A. Isolated bacteriocytes from the gill tissue of Calyptogena magnified viewed with trans-
mission EM. Two distinctly different granules (g) are observable in the cytoplasm. Scale bar = 1 5 microns.
B. Nomarski light micrograph of bacteriocytes from Lucinafloridana gill tissue, emphasizing the promi-
nent granules in these very large cells. Scale bar = 60 microns. C. Higher magnification of C. magnified
bacteriocytes illustrating the expansive fields of symbiotic bacteria (b). Nucleus (n), granules (g). Scale bar
= 5 microns.
INVERTEBRATE-BACTERIA SYMBIOSES
271
FIGURE 6. A. High magnification (78,000 x) of the symbiotic bacteria present in the cytoplasm of
isolated bacteriocytes from Calyptogena magnified. Note the peribacterial membrane encompassing each
bacterium, and the distinct nuclear regions. The bacteria are approximately an order of magnitude smaller
than those in Riftia trophosome. Scale bar = 0.5 micron. B. Isolated trophochrome cell from Riftia pachyp-
tila. Scale bar = 5 microns.
across the range of concentrations used here. The rate of fixation by L. floridana
bacteriocytes is approximately doubled by the addition of 0. 1 mM thiosulfate; higher
thiosulfate concentrations do not appreciably alter the fixation rate. Incorporation
by non-bacteriocyte epithelial cells of C. magnified is approximately one third the
rate of C. magnified bacteriocytes. The incorporation by the non-bacteriocyte prepa-
ration is presumably due to eucaryotic enzymes like pyruvate carboxylase and phos-
phoenolpyruvate carboxykinase, both known to occur in marine bivalves (e.g., Fel-
beck, 1983; Meinardus-Hagar and Gade, 1986). Contamination by bacteriocytes in
this preparation is low (Table I).
The positive influence of sulfide on the intracellular ATP levels of C. magnified
bacteriocytes contrasts with the inhibitory effect seen on CO2 fixation (Table III).
After 60 min, bacteriocytes incubated in 0.5 mM sulfide contain 3.30 nmoles ATP/
million cells (approximately 1 .0 1 mM) , compared to 1 .02 nmoles/million cells (0.3 1
mM) with no added sulfur, and 1.74 nmoles/million cells (0.54 mM) with 0.5 mM
thiosulfate.
DISCUSSION
The primary objectives of this study were ( 1 ) to describe ultrastructural features of
Riftia trophosome tissue, particularly those related to the distribution and subcellular
location of bacteria and the nature of the pigmented layer of trophochrome cells, (2)
to prepare suspensions of isolated cells from invertebrate tissues that contain bacterial
endosymbionts, and (3) to use these cellular preparations for characterizing meta-
bolic aspects of the symbioses.
Morphological evidence supports the conclusion that symbiotic bacteria present
in the trophosome of R. pachyptila are contained within animal cells (i.e., bacterio-
272 S. C. HAND
TABLE II
Incorporation of carbon dioxide in isolated celt suspensions at 20°C
Preparation
Sulfur compound
present
CPM/million
cells/h
n moles CO2/
million cells/h
nmoles CO2/
mg protein/h
C. magnified
Bacteriocytes
none
5632
5976
13.9
14.8
13.0
13.3
Na2S: 0. 1 mM
4590
11.4
4272
10.6
9.5
0.5 mM
3696
9.1
—
4224
10.5
9.5
1 .0 mM
2676
6.6
—
Na2S2O3:0.1 mM
4488
5904
11.1
14.6
13.2
0.5 mM
3084
7.6
3684
9.1
8.1
1.0 mM
3792
9.4
8.4
Non-bacteriocyte
epithelial cells
none
Na2S: 0. 1 mM
0.5 mM
292
404
302
0.72
0.10
0.75
4.5
6.2
4.6
1.0 mM
318
0.79
4.8
1.5 mM
348
0.86
5.3
2.0 mM
136
0.34
2.1
Na.S.OvO.l mM
0.5 mM
292
266
0.72
0.66
4.5
4.0
1.0 mM
246
0.61
3.8
1.5mA/
274
0.68
4.2
2.0mM
292
0.72
2.2
L. floridana
Bacteriocytes
none
Na2S2O,:0.1 mM
0.2 mM
3440
7776
7176
8.4
19.0
17.6
—
0.5 mM
8728
21.4
—
0.75 mM
7912
19.4
—
1.0 mM
8824
21.6
—
R. pachyptila
free bacteria
none
—
—
13.5
cytes). Electron micrographs show that nuclei and other eucaryotic organelles are
frequently interspersed among the vacuole-enclosed bacteria, and eucaryotic cell
membranes appear to enclose bacteria and such organelles within the same cell. Cava-
naugh (1985) suggested that an intracellular location for the bacteria was probable,
but previous evidence has been inconclusive (Cavanaugh et al, 1981; Cavanaugh
1983, 1985).
A second notable point regarding trophosome fine structure is the distribution of
sulfur deposits among the bacteria within a trophosome lobule. The bacteria located
closer to the outer trophochrome layer clearly have fewer deposits than do bacteria
located nearer the hemolymph space at the center of the lobule. This spatial distribu-
tion could reflect greater access of the latter bacteria to high sulfide concentrations in
INVERTEBRATE-BACTERIA SYMBIOSES
TABLE III
Intracellular A TP levels of Calyptogena magninca hacteriocvtes in the presence
of sulfur compounds at 20°C
273
Calculated intracellular
Incubation time1
Sulfur
nmoles ATP/
ATP concentration
(min)
compound
million cells
(mM)2
0
none
1.34
0.41
30
none
0.83
0.25
0.5mAfNa2S
1.34
0.41
0.5mA/Na2S2O;,
0.88
0.28
60
none
1.02
0.31
0.5mA/Na2S
3.30
1.01
0.5mA/Na2S2O3
1.74
0.54
90
none
2.03
0.62
0.5mMNa2S
3.75
1.15
0.5 mAl Na2S2O3
1.29
0.40
'Cells were incubated at 20°C in artificial seawater (pH 8.2) containing the indicated concentrations
of sulfide and thiosulfate.
2 The intracellular concentrations of ATP were calculated using an average bacteriocyte diameter of
19.8 ± 0.8 microns (SE, n = 25), as determined from transmission electron micrographs. Cellular water
content was assumed to be 80%.
Rift ia vascular blood (Childress et ai, 1984). Indeed, Vetter (1985) showed that in
the absence of sulfide, sulfur globules are lost from bacterial symbionts in the gill
tissue of Lucinoma anmdata; presumably the presence of sulfide would stimulate
deposition of these elemental sulfur stores. The occurrence and possible roles of sulfur
deposits in free-living sulfur bacteria recently have been reviewed (Vetter, 1985). It
should be noted that the coelomic fluid ofRiftia also contains sulfide, but it is unclear
whether this sulfide pool is available directly to the symbionts, or must first be trans-
ferred to the vascular circulation (Childress et ai, 1 984). If the density of sulfur depos-
its is related to the level of sulfur-based metabolism occurring in a given bacterium,
then a gradient of metabolic activity could exist, with higher metabolic potential be-
ing possessed by bacteria closer to the central hemolymph space of the lobule. Bacte-
ria near the hemolymph space also appear smaller, which is consistent with them
being younger than the larger bacteria toward the outer trophochrome layer.
The morphology of the pigmented layer of trophosome tissue was far more com-
plex than originally anticipated. First, the trophochrome layer does not contain bacte-
rial endosymbionts, although the cells do contain extensive numbers of cytoplasmic
inclusions (Figs. 1 B, 2A, B). The function(s) of these cells is not understood presently,
but their internal structure suggests several possibilities. Some of the intracellular
droplets or granules are structurally similar to those found in mucus-secreting cells
(e.g., intestinal goblet cells). What benefit the secretion of mucus-like material (if it
were to occur) by trophochrome cells would confer to the tubeworm is unclear, other
than possibly lubricating the external surface of the trophosome that is in direct appo-
sition to the internal surface of the body wall of the worm. Another possible function
of trophochrome cells, that of phagocytosis and degradation of aging bacteria, is sug-
gested by the heterogeneous contents of other intracellular granules (Fig. 1 B). Future
experiments measuring levels of protease activity in isolated trophochrome cells
could be enlightening in this context. Finally, trophochrome cells also contain elec-
tron-dense material that is deposited as crystalline arrays in some granules (Fig. 4B).
274 S. C. HAND
This material is likely proteinaceous and may be the pigment responsible for the deep
green color of the trophosome.
The region of the trophosome containing the bacteriocytes is a diffuse, loosely
associated tissue, which suggests that its dissociation into suspensions of single bacte-
riocytes should have been relatively straight forward. Apparently however, the cells
are very fragile, and we were unable to obtain intact bacteriocytes from trophosome
even when mechanical agitation was kept to a minimum.
Thus, the isolated cellular preparation that may have the most potential for im-
proving our understanding of the mechanisms involved in metabolic utilization of
sulfur by the symbiosis is the bacteriocytes from C. magnified and L. floridana. The
ultrastructure of these isolated cells is comparable to the structure as viewed in situ
(Fisher and Hand, 1984; Fiala-Medioni and Metivier, 1986), with the exception that
bacteria in isolated bacteriocytes from C magnified appear a little more spherical
than those in intact tissue (pers. obs., and Fiala-Medioni and Metivier, 1986). The
size of C magnifica bacteria differs markedly from those of Rifiia trophosome, and
data from 5S rRNA sequencing suggest the bacteria from the two sources have mini-
mal affiliation (Stahl et ai, 1984; Lane et ai, 1985).
The temperature sensitivity of C. magnifica bacteriocytes closely parallels the ob-
servations of Belkin et ai (1986) in their studies with gill homogenates of the vent
mussel Bathymodiolus thermophilus. In both cases, the capacity for CO2 fixation de-
clines dramatically at temperatures above 20°C. Thus, the bacterial endosymbionts
from C. magnifica are comparable to those of B. thermophilus in that both appear to
be sulfur-oxidizing bacteria with psychrophilic characteristics (cf., Belkin et al, 1 986).
The detrimental effect of warm temperature was not observed for bacteriocytes from
the shallow-water bivalve L. floridana, and in general this cellular preparation seemed
hardier.
In the absence of an exogenously added sulfur source, the rate of CO2 fixation by
C. magnifica bacteriocytes was 1 3 nmoles/mg protein/h. Rates obtained with homog-
enates of B. thermophilus gill ranged between 1.7 and 9.4 nmoles/mg protein/h, de-
pending on temperature and the thiosulfate concentrations present (Belkin et al.,
1986). Using "purified" bacteria from B. thermophilus, Belkin et al, (1986) reported
maximum CO2 fixation rates of approximately 40 nmoles/mg protein/h in the pres-
ence of thiosulfate. In the present study, CO2 fixation by C. magnifica bacteriocytes
was inhibited by sulfide across the range of 0.1 to 1.0 mM. Less inhibition was seen
with thiosulfate. Similarly, Anderson (1986) showed that CO2 fixation by whole speci-
mens ofSolemya reidi was inhibited at sulfide concentrations above 0. 1 mM in sur-
rounding seawater. This protobranch bivalve has a chemoautotrophic metabolism
based on the presence of sulfur bacteria localized in its gill tissue (Felbeck, 1983).
Unfortunately, we do not have measurements of CO2 fixation by bacteriocytes at
sulfide concentrations below 0.1 mM. Considering Anderson's results (1986), such
sulfide levels could well stimulate the process.
CO2 fixation by L. floridana bacteriocytes was increased approximately two-fold
by thiosulfate concentrations between 0. 1 and 1 .0 mM, a pattern virtually identical
to that seen for gill homogenates of B. thermophilus (Belkin et al., 1 986). Exogenously
added sulfide was minimally effective in stimulating the process in L. floridana bac-
teriocytes (preliminary observations) and in B. thermophilus homogenates (Belkin et
ai, 1986). The rate of CO2 fixation by isolated bacteria from R. pachyptila tropho-
some was 13.5 nmoles/mg protein/h in the absence of added sulfur. The response to
sulfide and thiosulfate was highly variable, and thus the results are not reported here.
This variability could be related to the general instability of the CO2 fixation capacity
exhibited by trophosome preparations (Belkin et ai, 1986).
INVERTEBRATE-BACTERIA SYMBIOSES 275
Although sulfide (0. 1 mA/and above) inhibited CO2 fixation by isolated bacterio-
cytes from C. magnified, the presence of 0.5 mM sulfide promoted higher intracellu-
lar ATP concentrations than control incubations without any added sulfur. The op-
posing effects of sulfide on the two processes may be related to the sulfide concentra-
tions used; as discussed earlier, if lower sulfide levels had been tried, CO2 fixation
might also have been stimulated. Thiosulfate was not as effective in enhancing intra-
cellular ATP levels. Powell and Somero (1986a), using lysed bacterial preparations
from C. magnified gill, showed that sulfite stimulated ATP synthesis while thiosulfate
and sulfide did not. In their study, ATP was measured using the firefly luciferase
technique, and incubations were of shorter duration. Most likely, components of the
animal cell contributed to sulfide utilization in our study with isolated bacteriocytes.
Although some degree of spontaneous oxidation is possible, Powell and Somero
(1985) localized sulfide oxidizing bodies in the animal cell cytoplasm of S. reidi gill
tissue. These workers also have conclusively demonstrated that mitochondria iso-
lated from Solemya reidi gill tissue can couple sulfide oxidation to the formation of
ATP via the electron transport system and oxidative phosphorylation (Powell and
Somero, 1986b).
Thus, the initial step(s) of sulfide oxidation could occur in bacteriocytes prior to
the sulfur compound reaching the endosymbiont, and some of the ATP synthesis in
bacteriocytes could reflect mitochondrial processing. Consequently, the identities of
sulfur compounds exploited for chemical energy by the symbiosis may depend on
the structural organization and integrity of the biological preparation under study.
Additional metabolic characteristics of invertebrate-sulfur bacteria symbioses may
display similar dependencies in ways yet to be identified.
ACKNOWLEDGMENTS
Appreciation is extended to the captains and crew members of the R/V Melville,
R/V Atlantis II, and DSRV Alvin. The expedition was supported by NSF grant
OCE83- 1 1 256 (facilities support grant for the Galapagos '85 program; Drs. J. J. Chil-
dress and K. L. Johnson, University of California, Santa Barbara, co-principal inves-
tigators). Travel funds were supplied to SCH by the University of Southwestern Loui-
siana. Helpful discussions of these findings with Drs. J. Pickett-Heaps and L. A.
Staehelin (MCD Biology, University of Colorado) and Dr. S. Schmidt (EPO Biology)
are gratefully acknowledged. Technical assistance was provided by Mr. V. Bullman,
Mr. M. Fisher and Mr. L. Harwood, and advice concerning the fixation and embed-
ding procedures for isolated cells was provided by Drs. R. C. Brown and B. E. Lem-
mon (University of Southwestern Louisiana).
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ENERGETICS OF CONTRACTILE ACTIVITY IN ISOLATED RADULA
PROTRACTOR MUSCLES OF THE WHELK BUSYCON CONTRARIUM:
ANAEROBIC END PRODUCT ACCUMULATION AND RELEASE
ROBERT W. WISEMAN AND W. ROSS ELLINGTON1
Department of Biological Science, The Florida State University. Tallahassee, Florida 32306-3050
ABSTRACT
Anaerobic energy metabolism during contractile activity was investigated in the
isolated radula protractor muscle of the whelk Busycon contrarium. Spectrophoto-
metric assay of enzyme activities in crude tissue extracts revealed particularly high
pyruvate reductase activities with octopine dehydrogenase displaying the highest ac-
tivity. During electrically induced isotonic contractions of the radula protractor mus-
cles, the following end products, listed in order of increasing level, accumulated in
the tissue: strombine, octopine and alanopine (the "opines"), and D-lactate. Pyruvate
levels increased three-fold during muscle contraction, suggesting that pyruvate plays
a key role in the regulation of the pyruvate reductases. The muscle released lactate,
but none of the opines, into the incubation medium, with rates exceeding 3 ^moles
• min ' -g wet wt '. During the later phases of contraction, more D-lactate was re-
leased into the medium than accumulated in the muscle. We conclude that transport
of D-lactate permits sustained flux through lactate dehydrogenase because of a reduc-
tion in product inhibition. Furthermore, we hypothesize that D-lactate transport may
be coupled to H+ export or OH import, which would then serve to regulate the
extent of accumulation of glycolytically produced protons.
INTRODUCTION
The muscles of marine molluscs possess two distinctly different mechanisms of
energy production during periods of reduced oxygen availabiliy. During environmen-
tal anaerobiosis — i.e., whole-organism exposure to anoxia — aspartate and glycogen
are cofermented, yielding succinate and alanine as end products (Gade, 1983). The
succinate pathway typically occurs at relatively low rates and is associated with a
reverse Pasteur effect in these muscles (Storey, 1985). A number of molluscs may
undergo functional anaerobiosis, where oxygen demand exceeds delivery. In this in-
stance, only certain tissues are rendered anoxic (Gade, 1 983). Under these conditions,
the glycolytic flux is several orders of magnitude higher than under conditions of
environmental anaerobiosis (Livingstone, 1982; Gade, 1983). The higher energy out-
puts necessary for burst activity are provided by glycogen fermentation and the shunt-
ing of pyruvate through pyruvate reductases such as lactate and opine dehydroge-
nases (Gade and Grieshaber, 1 986), resulting in the accumulation of D-lactate, octo-
pine, alanopine, or strombine.
Opine dehydrogenases catalyze the reductive condensation of pyruvate and an
amino acid according to the following general reactions:
Received 22 September 1986; accepted 18 May 1987.
' To whom reprint requests should be sent.
277
278 R. W. WISEMAN AND W. R. ELLINGTON
arginine + pyruvate + NADH ^ octopine + NAD
(ODH, octopine dehydrogenase)
alanine + pyruvate + NADH ^ alanopine + NAD
(ADH, alanopine dehydrogenase)
glycine + pyruvate + NADH ^ strombine + NAD
(SDH, strombine dehydrogenase)
Many molluscan muscles have the enzymatic potential for producing several opine
end products as well as D-lactate since high activities of opine and D-lactate dehy-
drogenases may occur in the same tissue (Zammit and Newsholme, 1976; Living-
stone el al., 1983). For example, the pedal retractor (Baldwin et al, 1981; Baldwin
and England, 1982) and radula (Ellington, 1982) muscles of gastropods contain sig-
nificant activities of all opine dehydrogenases as well as D-lactate dehydrogenase. The
relative contribution of these enzymes to the maintenance of glycolytic flux during
contractile activity, not yet fully explored, is considered in this paper.
The metabolic disposition of end products of anaerobic metabolism in molluscs
(Ellington, 1983b) is poorly known. Propionate is released into the hemolymph of
Mytilus edulis (Zurburg et al., 1982), whereas succinate appears to accumulate in
the hemolymph of the clam Mercenaria mercenaria (Korycan and Storey, 1983).
Octopine is not released into the hemolymph during contractile activity in the giant
scallop, Placopecten magellicanus (de Zwaan et al., 1980). However, hemolymph
octopine levels are slightly elevated after contractile activity or hypoxia in the cepha-
lopods Sepia officinalis and Loligo vulgaris (Storey and Storey, 1979; Gade, 1980).
Alanopine and strombine appear not to be released from molluscan muscles. Octo-
pine, alanopine, and strombine levels fall during recovery, indicating oxidation in
situ (Ellington, 1983b).
The present study focuses on the metabolism of the radula protractor muscle of
the large marine gastropod Busycon contrarium. This muscle possesses relatively high
activities of lactate and opine dehydrogenases. The presence of several pyruvate re-
ductases in the radula protractor muscle poses questions about the control of these
enzymes and the disposition of their products. We show that electrical stimulation of
this muscle while it is subjected to anoxia induces the formation of all opines as well
as D-lactate. Interestingly, formation of D-lactate is much greater than that of the
other end products, even though the activities of the opine dehydrogenases are much
higher. Further, D-lactate is released from the exercising muscle into the medium
while the opines are retained. End-product removal may enhance the formation of
additional D-lactate and, as a result, large amounts of carbon can be shunted through
lactate dehydrogenase allowing for higher, sustained glycolytic fluxes during anoxia.
MATERIALS AND METHODS
Animals
Specimens of the whelk Busycon contrarium were collected off Alligator Point in
Franklin County, Florida, and were maintained in the flowing-seawater system at the
Florida State University Marine Laboratory near St. Theresa. Individuals used in
experiments were transferred to the Florida State University campus, where they were
maintained for brief periods in a recirculating seawater system.
ANAEROBIC METABOLISM OF RADULA MUSCLES 279
Biochemicals
Biochemicals were purchased from Boehringer-Mannheim (Indianapolis) and
Sigma Chemical Company (St. Louis). D-Lactate dehydrogenase, used to determine
D-lactate, was purified from the muscle of the horseshoe crab Limulus polyphemus.
Octopine dehydrogenase, used to assay for octopine, arginine, and arginine phos-
phate, was purified from the adductor muscles of the scallop Argopecten irradians
concentricus. Succinyl Co A synthase, used in succinate assays, was a gift from Dr.
William Bridger, Department of Biochemistry, University of Alberta, Edmonton.
Experimental procedure
Intact radula protractor muscles, dissected from the proboscis apparatus, were
ligated at both ends with surgical silk and placed in a 5 X 75-mm muscle bath filled
with 1.5 ml of MBL (Marine Biological Laboratory) formula artificial seawater
buffered with 5 mA/hydroxyethylpiperazine ethanesulfonic acid (pH = 7.8). One end
of the muscle was fastened to a hook electrode and pulled into a rubber sleeve at the
bottom of the muscle bath. The other end was attached to a Narco Biosystems iso-
tonic myograph transducer with the silk suture. Muscles were suspended in the bath
at 1.5 times their resting length (measured upon excision). Temperature was main-
tained by immersion of the bath in a larger water-filled vessel which was jacketed and
controlled by a Brinkmann model RM 6 recirculating water bath (20°C). Contrac-
tions were recorded with the isotonic transducer connected to a Narco Biosystems
model MK IV physiograph. A second electrode, inserted in the bath, delivered
square-wave pulses (60 volts, 40 ms) at 2.5-s intervals from a Grass model SD9 stimu-
lator. The bath was gassed with normocapnic nitrogen (0.05% CO2) through a 75-
mm, 22-gauge Luer lock syringe needle.
Each muscle preparation was fastened in the bath and bubbled with nitrogen for
1 5 min. Control experiments were terminated at this point, and the tissues were re-
moved, blotted, and frozen in liquid nitrogen. Experimental groups were gassed as
the controls were, and then subjected to various periods of electrical stimulation (2.5,
5, 10, and 15 min) in the presence of normocapnic nitrogen before being frozen.
In all cases, the medium was decanted from the bath and stored at -70°C for later
analysis.
Enzyme assays
Freshly dissected muscles were homogenized in 24 volumes of extraction buffer
(50 mA/triethanolamine, pH 7.4, 1 mA/EDTA, 20 mM mercaptoethanol, 20% glyc-
erol) with a Tekmar UltraTurrax tissue homogenizer. The homogenate was centri-
fuged at 1 2,000 X g for 20 min at 4°C. The supernatant was passed through a Sepha-
dex G-25 column (1.5 X 14 cm) equilibrated with extraction buffer less glycerol,
which removed low-molecular-weight compounds. The proteins in the void volume
were used as the source of enzyme activities. The activities were determined spectro-
photometrically with a Gilford model 252-1 spectrophotometer according to the
methods outlined by Ellington (1982).
Metabolite assays
Nuetralized perchloric acid extracts were prepared from the radula protractor
muscles frozen at -70°C according to the methods of Graham and Ellington (1985).
Arginine phosphate was assayed spectrophotometrically by the method of Grieshaber
280 R. W. WISEMAN AND W. R. ELLINGTON
TABLE I
Profile ofpvruvate reductase activities in desalted tissue extracts ofradula protractor muscles from
Busycon contrarium
Enzyme Activity
D-Lactate dehydrogenase 36.46 ± 9.7 1
Strombine dehydrogenase 6 1 .98 ± 1 4.88
Alanopine dehydrogenase 96.87 ± 19.66
Octopine dehydrogenase 509.83 ± 40.73
Activities are expressed in //moles • min" ' g wet wt~ ' and were measured at 25°C. Data represents mean
1 SD, n = 4.
and Gade (1976). Pyruvate was assayed fluorometrically in a Farrand Optical model
A-4 fluorometer by the method of Lowry and Passonneau (1972). Both pyruvate
and arginine phosphate levels were determined immediately after neutralization to
eliminate sample loss.
Aspartate, succinate, arginine, and malate were determined spectrophotometri-
cally according to the method of Williamson and Corkey ( 1 969), Williamson ( 1 974),
Grieshaber and Gade ( 1 976), and Williamson and Corkey ( 1 969), respectively. Octo-
pine and lactate were assayed fluorometrically essentially as outlined by Graham and
Ellington (1985).
Concentrations of the free amino acids alanine and glycine were determined by
HPLC on a Dionex amino acid analyzer with a Pierce amino acid column and buffers
(Pierce Chemical Company). Alanopine and strombine concentrations were also de-
termined by HPLC methods (Fiore el ai, 1984).
RESULTS
Enzyme activities
Freshly prepared extracts of the radula protractor muscles of Busycon contrarium
displayed high activities of all four pyruvate reductases (Table I). Octopine dehy-
drogenase (ODH) had the highest activity of the enzymes assayed. Alanopine dehy-
drogenase (ADH) displayed somewhat lower activity followed by strombine (SDH)
and D-lactate (LDH) dehydrogenases (Table I).
Contractile activity
Contractile activity under nearly anoxic conditions was maintained within 98%
of initial values for the first 5 min of electrical stimulation (Fig. 1 ). There was a general
trend towards a decline in force thereafter. This pattern was evident in all muscle
preparations tested.
Metabolite levels in the tissue and the medium
Arginine phosphate levels declined at the onset of muscular activity, reached a
minimum after 10 min, and remained relatively constant thereafter (Table II). Free
arginine levels increased in the first 2.5 min of contractile activity, then fell to near
control levels at the end of the experiment (Table II). Within 10 min, alanine levels
were three times greater than levels measured initially, but returned to near control
levels after 15 min (Fig. 2). Glycine levels did not change significantly during the 15
ANAEROBIC METABOLISM OF RADULA MUSCLES
281
100.
80.
0)
£ 60 J
g
I 40 -
2?
20 .
10
15
mm
FIGURE 1. Changes in contractile force for electrically stimulated isotonic contractions of isolated
radula protractor muscles from Busycon contrarium versus time. Units are percent change of initial force.
Points represent means ± 1 SD, n = 5.
min of contractile activity (Fig. 2). Aspartate levels in the muscle declined during
contractile activity, with the bulk of the change taking place in the first 5 min (Fig.
2). Succinate and malate levels in muscles stimulated for 15 min were less than 0.5
)umoles-g wet wlT1.
After 2.5 min of stimulation, pyruvate levels in the muscle increased dramatically
(Fig. 3). Strombine was not a major glycolytic end product, as this compound accu-
mulated to levels approaching only 1 /zmole-g wet wt"1 (Fig. 3). In contrast, octopine
accumulated linearly during the experiment (Fig. 3, Table II). The sum of octopine,
free arginine, and arginine phosphate levels was constant (Table II), indicating no
net change in the total arginine pool in the tissue. Alanopine showed the highest
accumulation of all the opines, but formation did not begin immediately as this com-
pound was not detectable in the first 2.5 min of stimulation (Fig. 3). D-lactate was
the predominant end product formed in the muscle, exhibiting a dramatic increase
during the later periods of contraction (Fig. 3).
Octopine, alanopine, and strombine were not released into the medium by the
muscle preparations. In contrast, a significant amount of D-lactate was found in the
TABLE II
Total arginine pool (arginine phosphate, arginine. octopine. and total arginine) for neutralized perchloric-
acid extracts ofisotonically contracting radula protractor muscles isolated from Busycon contrarium
Metabolite
Time
(min)
Free arginine
Arginine phosphate
Octopine
Total
0
2.37 ±2.41
10.69 ±3.77
0.46 ± .29
13.47 ±4.39
2.5
4.44 ± 1.17
7.35 ±2.83
1.11 ± .85
12.55 + 3.34
5
3.75 ± 1.04
6.98 ± 1.50
1.71 ± .38
12.44± 1.18
10
2.71 ± .83
4.93 ±2. 75
3.47 ± 1.64
10.76 ±2.84
15
2.44 ± .48
6.98 ±2.64
4.72+ 1.73
14.14± 1.26
Levels are expressed in /umoles - g wet wt ' . Data represent means
± 1 SD, n = 5.
282
R. W. WISEMAN AND W. R. ELLINGTON
12.
11.
10.
9.
8.
7.
en ..
o> 5.
o
I
3.
2.
1 .
0
ALA
0
I
2.5
ASP
10
15
0 2.5
10
15
mm
FIGURE 2. Changes in levels of the amino acids alanine (ALA), glycine (GLY), and aspartate (ASP)
as determined by HPLC of neutralized perchloric-acid extracts of electrically stimulated radula protractor
muscles from Busvcon contrarium over time. Units are ^moles-g wet wt '. Points represent means ± 1
SD, n = 5.
medium (Fig. 4). In fact, during the last 5 min, more D-lactate was released into the
medium than accumulated in the muscle (Fig. 4).
DISCUSSION
The high activities of all four pyruvate reductases in the radula protractor muscle
of B. contrarium are similar to those observed in the radula retractor muscles of this
species (Ellington, 1982) and in other gastropod muscles (Baldwin and England,
1982; Livingstone et ai, 1983). The highest activity, exhibited by octopine dehy-
drogenase, was comparable to activities observed in cephalopod molluscs (Baldwin
and England, 1980). The simultaneous accumulation of D-lactate, alanopine, octo-
pine, and to a lesser extent strombine, which occurs in the radula protractor system
during muscular activity, indicates that all four of these reductases operate under
functional anoxia. The accumulation of these end products is temporally correlated
with changes in pyruvate levels. Pyruvate levels increased dramatically during the
time course of muscle contraction.
Pyruvate reductases are thought to be equilibrium enzymes (de Zwaan and
Dando, 1984; Ga'de and Grieshaber, 1986) and are thus regulated by changes in the
concentrations of substrates and products. Pyruvate is the common substrate for all
four reductases. The observed elevations in pyruvate levels in B. contrarium radula
ANAEROBIC METABOLISM OF RADULA MUSCLES
283
mm
FIGURE 3. Pyruvate (PYR), D-lactate (D-LAC), octopine (OCT, alanopine (ALN), and strombine
(STR) levels in electrically stimulated radula protractor muscles from Busycon contrarium over time, as
determined by HPLC and fluorometry. Units are ^moles- g wet wt '. Points represent means ± 1 SD, n
c
protractor would undoubtedly enhance all pyruvate reductase activities. In the case
of opine dehydrogenases, the concentrations of amino acid co-substrates are also im-
portant. In the radula protractor muscle, we found that alanine, glycine, and free
arginine levels were in the 2-8 /umole-g~' range. Finally, pyruvate reductases are
influenced by accumulation of their respective products. Opine dehydrogenases seem
to be particularly sensitive to product inhibition (Gade and Grieshaber, 1986).
D-Lactate was the dominant glycolytic end product even though the maximal
LDH activity measured in crude tissue extracts was the lowest. Isolated radula muscle
of B. contrarium displayed the highest levels of D-lactate accumulation yet observed
during functional anoxia in molluscs. Meinardus and Gade (1981) observed a rela-
284
R. W. WISEMAN AND W. R. ELLINGTON
mm
FIGURE 4. Distribution of lactate in the incubation media (M) and tissue (Tis) as well as the total
(To) lactate produced during electrically induced isotonic contractions of the radula protractor muscles
from the whelk Busycon contrarium as determined by fluorometric analysis of neutralized perchloric-acid
extracts of each over time. U nits are /imole-g wet wt '. Points represent means ± 1 SD, n = 5. Where error
bars are absent, the error bar was less than the size of the symbol used to mark points.
lively modest accumulation of D-lactate in electrically stimulated preparations of the
foot muscle of the cockle Cardium edule. The preferential production of D-lactate
versus the opines in B. contrarium radula protractor muscle may be related to a higher
binding capacity for pyruvate. The apparent Kms for pyruvate of molluscan LDHs
are considerably lower than corresponding Kms of ODHs and somewhat lower than
pyruvate Kms for strombine and alanopine dehydrogenases (Gade and Griesha-
ber, 1986).
Isolated radula muscle preparations of B. contrarium did not release octopine,
alanopine, or strombine into the medium. In addition, the total arginine pool (free
arginine, arginine phosphate, and octopine) remained constant during contractile
activity. The decline in aspartate levels during anoxia probably reflects transdeamina-
tion to alanine because, at the end of 1 5 min, the alanopine-alanine pool size was
roughly equivalent to the decrease in aspartate plus initial alanine levels. This conser-
vation of nitrogen in both the arginine (arginine, arginine phosphate, octopine) and
alanine (alanine, alanopine, aspartate difference) pools is consistent with the observed
absence of transport of opine end products out of the muscle.
In contrast to that of the opines, release of D-lactate from the muscle was very
large. In fact, D-lactate export was greater than D-lactate accumulation during the
10-15-min period of observation. D-lactate release could have at least two major
functional advantages. First, removal of the product of this reaction would change
the mass action ratio ([lactate]/[pyruvate]) in favor of more product formation. Sec-
ond, if lactate were transported out in a symport system with a proton (H+) or in an
antiport system (OH"), this process would help the muscle cells regulate intracellular
pH
ANAEROBIC METABOLISM OF RADULA MUSCLES 285
The total D-lactate produced during the 1 5 min of contractile activity approached
50 Mmoles-g wet wt '. On the basis of established proton stoichiometries ofglycolysis
(Portner el al, 1984) — i.e., one mole of protons (H+) per mole lactate (or opine)
produced — lactate and also opine production clearly impose a significant acid load
on the muscle. Buffering capacities of whelk radular and ventricular muscle, as deter-
mined by the homogenate titration method (Castellini and Somero, 1981), ranged
from 30.7 to 39.5 Slykes-g wet wt"1 (Eberlee and Storey, 1985; Graham and Elling-
ton, 1985). However, the buffering capacity of B. contrarium ventricles, as deter-
mined by imposing an acid load (Ellington, 1985) and measuring pH, by phosphorus
nuclear magnetic resonance (3IP-NMR) spectroscopy, yielded a value approaching
24 Slykes-g wet wt ' (Ellington, unpub. obs.). Regardless of the exact position of the
buffering capacity, the acid load imposed on the muscle clearly could not be offset by
purely passive means such as buffering.
Lactate transport has been studied extensively in erythrocytes. Three mechanisms
of lactate transport have been identified: (1) non-ionic diffusion, (2) classical anion
transport, and (3) monocarboxylate carrier (H+ symport or OhT antiport) mediated
transport. Deuticke el al. (1982) reported at least three parallel pathways of lactate
transport in erythrocytes. Lactate transport in erythrocytes has become the paradigm
on which mitochondrial (Palmieri el al., 1971), hepatocyte(Fafournoux ?/#/., 1985),
and whole-muscle (Mainwood and Worsley-Brown, 1975; Seo, 1984) lactate trans-
port have been modeled. The efflux of lactate from the radula protractor muscle of
B. contrarium is probably caused by one or several of these mechanisms. Because the
pKa of lactic acid is 3.86, and the intracellular pH of molluscan tissues under a variety
of conditions ranges from 7.1 to 6. 6 (Ellington, 1983a; Graham and Ellington, 1985),
almost all the acid would dissociate into anions; therefore, the rates of non-ionic
diffusion should be low. A lactate: H+ symport (or lactate:OH antiport), if present in
the radula protractor muscle of B. contrarium, could be of critical importance in
regulating pHj, especially during periods of elevated glycolytic rates.
In contrast to lactate, the opines are not released into the medium by the radula
protractor muscle of B. contrarium. In fact, there is no direct evidence for release of
opines from any molluscan tissue, although the increase in hemolymph levels during
contractile activity in cephalopods (Storey and Storey, 1979; Gade, 1980) and the
decline in the total arginine pool in the mantle muscle during swimming in S. offici-
nalis (Storey and Storey, 1979) and Loligo vulgaris (Grieshaber and Gade, 1976)
suggest indirectly that octopine is released in these cases. No other study of contractile
activity in molluscs has revealed a decline in the total arginine pool or release of
opines from the muscle (de Zwaan and Dando, 1984).
Why is lactate readily exported from molluscan muscle cells while opines appear
to be retained? Specific transporters for amino acids are present in a wide range of
cell types (Preston and Stevens, 1 982); thus there is no fundamental impediment with
respect to transport of these compounds. Opine formation results in no net increase
in the number of osmotically active particles because the amino acid condenses with
pyruvate derived from a large polymer (glycogen). In contrast, lactate formation re-
sults in an increase in osmotically active particles, as glycogen is broken down into
smaller fragments. The lack of disturbance of internal osmolarity has been used as a
potential functional explanation for the use of opine dehydrogenases rather than
LDH in certain molluscan muscles (Zandee el al., 1980; Fields, 1983). A logical deriv-
ative of the argument is that it would be disadvantageous to transport out opine end
products. However, accumulated end products represent only a small fraction (<5%)
of the pool of osmotically active substances. Thus, removal of end products, or lack
thereof, for the sole purpose of cell volume regulation seems unlikely. We favor the
286 R. W. WISEMAN AND W. R. ELLINGTON
possibility that the lack of export of opines is related to the lack of a mechanism for
coupling this movement with regulation of pH,. In fact, since octopine, alanopine,
and strombine have both positive and negative charges at prevailing pHj conditions,
a transport mechanism that could couple H+ (symport) or OFT (antiport) movement
with opine export would be difficult to envision.
The absence of significant amounts of succinate or malate accumulation makes
the fermentation of aspartate during functional anoxia unlikely. Although the carbon
skeleton may be unaccounted for, nitrogen is balanced through alanine formation.
Most probably, the amino group of aspartate is transaminated ultimately to pyruvate
to yield alanine, with the carbon skeleton of aspartate entering the Krebs cycle as
malate or oxaloacetate. This hypothesis entails the assumption that there is enough
oxygen available to the cells to sustain a significant level of aerobic metabolism, at
least during the early phases of contractile activity. The myoglobin content of radula
muscle is high (Ball and Meyerhof, 1940; Fange and Mattisson, 1958), and molluscan
myoglobins typically have low P50 values (Read, 1 966). Glycolytic rates, as evidenced
by end product accumulation, are low in the early phases of contractile activity, sug-
gesting that energy production is largely aerobic during this period. Presumably, the
oxygen used in this period could be derived from an internal oxygen store such as
myoglobin. Contractile force decreases in the later portions of the time course while
pyruvate concentrations increase, suggesting that there is a transition from aerobic
to anaerobic processes. Thus, aspartate may be an aerobic substrate during the early
phases of muscle contraction.
To sum up, our studies have shown that during contractile activity in the radula
protractor muscle of B. contrarium, high glycolytic rates prevail with pyruvate being
shunted through all the major pyruvate reductases. Strombine, octopine, alanopine,
and D-lactate accumulated in order of increasing levels. D-lactate was the major end
product although D-lactate dehydrogenase displayed the lowest in vitro activity of all
pyruvate reductases. D-Lactate, but none of the opines, was released from the muscle
into the incubation medium, with rates exceeding 3 ^moles-min"1 -g wet wt~'. The
removal of lactate from the muscle enhances the mass action ratio in favor of lactate
formation, thereby increasing carbon flux through this enzyme. The removal of lac-
tate from muscle cells during contractile activity may also help to regulate pH;
through a H+ symport or OH~ antiport system. This potential role of lactate transport
in the regulation of pH; is currently the subject of intensive investigation in this labo-
ratory.
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phosphofructokinase, glycerol phosphate dehydrogenase, lactate dehydrogenase, octopine dehy-
drogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphate kinase, glutamate-oxa-
loacetate transaminase, and arginine kinase in relation to carbohydrate utilization in muscles
from marine invertebrates. Biochem. J. 160: 447-462.
ZANDEE, D. I., D. R. HOLWERDA, AND A. DE ZWAAN. 1980. Energy metabolism in bivalves and cephalo-
pods. Pp. 185-206 m Animals and Environmental Fitness, Vol. 1, R. Gilles, ed. Pergamon Press,
Oxford.
ZURBURG, W., A. M. T. DE BONT, AND A. DE ZWAAN. 1982. Recovery from exposure to air and the
occurrence of strombine in different organs of the sea mussel Mytilus edulus. Mol. Phvsiol. 2:
135-147.
DE ZWAAN, A., AND P. R. DANDO. 1984. Phosphoenolpyruvate metabolism in bivalve molluscs. Mol.
Physiol. 5:285-310.
DE ZWAAN, A., R. J. THOMPSON, AND D. R. LIVINGSTONE. 1980. Physiological and biochemical aspects
of the valve snap and valve closure responses in the giant scallop, Placopecten magellanicus. II.
Biochemistry./ Comp. Physiol. 137: 105-1 15.
PHYSIOLOGY
DEATON, LEWIS E.
Epithelial water permeability in the euryhaline mussel Geukensia
demissa: decrease in response to hypoosmotic media and hormonal
modulation +. . . .,.;;. 230
ENGEL, DAVID W., AND MARIUS BROUWER
Metal regulation and molting in the blue crab, Callinectes sapidus: met-
allothionein function in metal metabolism 239
FELBECK, HORST, AND SANDRA WILEY
Free D-amino acids in the tissues of marine bivalves .yY. '. 252
HAND, STEVEN C.
Trophosome ultrastructure and the characterization of isolated bacte-
riocytes from invertebrate-sulfur bacteria symbioses . ;"':% .- . /T^A 260
WISEMAN, ROBERT W., AND W. Ross ELLINGTON
Energetics of contractile activity in isolated radula protractor muscles
of the whelk Busycon contrarium: anaerobic end product accumulation
and release . 277
,.-• ••'•?.- .:•
CONTENTS - •;?
i
'• • ' *
Annual Report of the Marine Biplp^jpal Laboratory . . •, .-. . .-.1 .............. 1
INVITED REVIEW
STANLEY-SAMUELSON, DAVID W.
Physiological roles of prostaglandins and other eicosanoids in inverte-
brates ____ ........................... . ....... . . . ........ 92
BEHAVIOR
CHADWICK, NANETTE E.
Interspecific aggressive behavior of the corallimorpharian Corynactis
californica (Cnidaria: Anthozoa): effects on sympatric corals and sea
anemones ..................... -* ........... . ......... .... 1 1 0
DEVELOPMENT AND REPRODUCTION
BOSCH, ISIDRO, KATHERINE A. BEAUCHAMP, M. ELIZABETH STEELE, AND
JOHN S. PEARSE
Development, metamorphosis, and seasonal abundance of embryos and
larvae of the antarctic sea urchin Sterechinus neumayeri ........... 126
ECOLOGY AND EVOLUTION
ALEXANDER, STEPHEN P., AND TED E. DELACA
Feeding adaptations of the foraminiferan Cibicides refulgens living epi-
zoically and parasitically on the antarctic scallop Adamussium colbecki 1 36
BORRERO, FRANCISCO J.
Tidal height and gametogenesis: reproductive variation among popula-
tions of Geukensia demissa ................................. 1 60
MARCUS, NANCY H.
Differences in the duration of egg diapause of Labidocera aestiva (Co-
pepoda: Calanoida) from the Woods Hole, Massachusetts, region .... 1 69
GENERAL BIOLOGY
HOSE, Jo ELLEN, GARY G. MARTIN, VAN ANH NGUYEN, JOHN LUCAS, AND
TEDD ROSENSTEIN
Cytochemical features of shrimp hemocytes .................... 178
MACKIE, G. O., AND C. L. SINGLA
Impulse propagation and contraction in the tunic of a compound
ascidian ............................................... 188
MANGUM, C. P., K. I. MILLER, J. L. SCOTT, K. E. VAN HOLDE, AND M. P.
MORSE
Bivalve hemocyanin: structural, functional, and phylogenetic rela-
tionships ....... , ....................................... 205
OKAMURA, BETH
Particle size and flow velocity induce an inferred switch in bryozoan
suspension-feeding behavior .......... . ..................... 222
Continued on Cover Three
Volume 173
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Reference: Biol. Bull. 173: 289-298. (October, 1987)
ORIENTATION OF THE HORSESHOE CRAB, LIMULUS POLYPHEMUS,
ON A SANDY BEACH
MARK L. BOTTOM' AND ROBERT E. LOVELAND2
^Fordham University, Divisional' Science and Mathematics, College at Lincoln Center, New York, New
York 10023, and2 Rutgers — The State University, Department of Biological Sciences and Bureau of
Biological Research, Nelson Biological Laboratories, Piscataway, New Jersev 08854
ABSTRACT
Adult horseshoe crabs (Limulus polyphemus) spawn on sandy intertidal beaches
and then return toward the water. Field experiments demonstrated that beach slope
was more significant than vision in this orientation behavior. Both blinded and nor-
mally sighted crabs showed rapid seaward orientation on beaches with a seaward
slope of approximately 6°. Orientation performance was poor on a flat beach, al-
though sighted crabs slightly out-performed blinded crabs. The observed orientation
behavior was correlated with the large numbers of horseshoe crabs which failed to
return to the water after spawning on sand bars or similar habitats lacking a slope
gradient.
INTRODUCTION
Adult horseshoe crabs (Limulus polyphemus L.) migrate every spring into Dela-
ware Bay and other Atlantic coast estuaries to spawn on sandy beaches (Shuster,
1982; Shuster and Botton, 1985). These sublittoral animals find, and amplex with, a
mate, migrate to the intertidal zone to deposit and fertilize the eggs, and then return
to the sea. This implies the existence of spatial orientation mechanisms at each critical
stage of the reproductive cycle. Although little is known about the mechanism of long
distance migrations from the continental shelf to estuarine spawning grounds (Botton
and Ropes, 1987), Rudloe and Herrnkind (1976) showed that submerged crabs near
breeding beaches can orient in response to wave surge. Barlow et al. (1982) found
that visual cues are important during mating, and Barlow et al. (1986) suggest that
light may be a significant environmental factor associated with seasonal and diurnal
variability in mating activity. Visual stimuli may elicit other behavioral responses
including direction and speed of locomotion (Cole, 1923; Northrup and Loeb, 1923;
Ireland and Barlow, 1 978) and telson and gill movements (Powers and Barlow, 1 985).
The orientation cues which enable horseshoe crabs to return to the water after
mating have not been previously considered. This behavior has important ecological
consequences because animals "stranded" on an exposed intertidal beach are sub-
jected to physiologically stressful conditions, including high temperatures, desicca-
tion, and osmotic imbalance (Herrnkind, 1 983). Among shore zone arthropods, both
visual cues (e.g., sun, moon, polarized light, landmark orientation) and nonvisual
cues (such as beach slope) are known (Herrnkind, 1972, 1983;Schone, 1984). In 1985
during a preliminary beach survey of Delaware Bay in the area of Fortescue, New
Jersey, we observed large numbers of live horseshoe crabs burrowed on a relatively
flat section of exposed intertidal beach at low tide. We hypothesized that this aberrant
Received 27 April 1987; accepted 22 July 1987.
289
290 M. L. BOTTON AND R. E. LOVELAND
behavior resulted from the inability of the animals to orient seaward in the absence
of beach slope: in this report, we present an experimental test of that hypothesis.
Orientationa! behavior of sighted and blinded individuals on sloped and flat beaches
were quantified to evaluate the importance of beach slope and light for adult horse-
shoe crabs.
MATERIALS AND METHODS
All experiments were conducted between 26 May and 7 June 1986 at the New
Jersey Oyster Research Laboratory (NJORL), located on the Delaware Bayshore in
Cape May County, New Jersey. The study area was located just north of the labora-
tory. One site had a slope and sediment composition typical of this relatively uniform
and undisturbed beach (Shuster and Botton, 1 985). During spring, these beaches have
a slope of some 6.4 degrees in approximately a westerly direction. The second site
was a flat sand bar formed by the outlet of a salt marsh creek. It was located approxi-
mately 100 m north of the sloped beach site, also with the bay toward the west. Horse-
shoe crabs spawned in large numbers in this and adjacent areas during full moon high
tides. Both sites within the study area provided similar visual fields: an open horizon
in the seaward direction and a line of vegetation (mainly Phragmites) above the high
water line in the landward (easterly) direction.
Orientation was studied within "arenas" modeled after Mrosovsky and Shettle-
worth (1968). On each beach, a 4-m radius circle was inscribed and a 30-cm trench
dug along its perimeter. Stranded horseshoe crabs and any large pieces of debris were
removed from the arena before use. The arena was divided into 16 equal sectors of
22.5° each. Sector 1, beginning at compass point north, was arbitrarily designated as
0°, sector 2 began 22.5° in a clockwise direction, et seq.
Adult male horseshoe crabs were collected from the bay immediately before the
study of their behavior. Crabs with missing appendages, or with damaged, missing,
or heavily encrusted eyes, were discarded. Those crabs kept out of water for more
than 3 minutes were thoroughly wetted down before the trial. The animals used in
the blinding experiments were prepared by drying the carapace around the lateral
and median eyes using a paper towel, and then placing patches of opaque adhesive
duct tape over the eyes.
Each of the four combinations of beach slope and vision, i.e., sloped beach/
sighted, sloped beach/blinded, flat beach/sighted and flat beach/blinded, were run on
at least two separate days, with a total sample size of not less than 4 1 individuals per
combination (range, 8-23 crabs per run). Crabs were tested individually to avoid
behavioral interactions because they often pause or change direction when other
crabs are encountered along the beach (pers. obs.). To begin a trial, the top few cm
of sand in the arena was smoothed using a wooden board to remove the track of
the previous animal; this procedure also disrupted any possible gradients in surface
sediment moisture. The crab was placed in the center of the arena facing away from
the water and on a line perpendicular to the shoreline. If the animal burrowed in
place, it was lifted out of the sand and re-started; if it burrowed twice in succession, it
was rejected.
A trial was completed when the animal's prosoma crossed the perimeter of the
arena. Animals failing to complete a trial, or which burrowed, or which remained
motionless on the surface of the sediment for 5 consecutive minutes were recorded
but not used in the statistical analysis of this behavioral data set. Investigators re-
corded the elapsed time of the test, the section of the arena from which the crab
exited, and the number of pauses longer than 30 s. The linear distance of the path
was measured by placing a metered string along the animal's track; a sketch of the
path for each trial was made.
ORIENTATION OF INTERTIDAL LIMULUS 291
TABLE I
Comparison of the number of "disoriented" (live, rightside-up) Limulus polyphemus on flat and sloped
beaches in Delaware Bay on each of three days during Spring 1986
Mean no. disoriented crabs
per 1 5-m transect
Date Flat Sloped n
23 May
55.2
12.2
5
4.552**
24 May
93.3
9.8
4
3.342*
25 May
101.5
4.5
4
3.623*
n = number of replicate 15-m transects counted on each type of beach, t = t-statistic comparing mean
number of crabs on each type of beach. Means on flat beach significantly greater than mean number on
sloped beach at P < .05 (*) or P < .005 (**).
The performance of a group of crabs was assessed using a number of variables
including percent crossing the perimeter, time required to leave the arena, linear dis-
tance travelled, and the number of pauses and circles made by the animals. We calcu-
lated each animal's "meandering score" as the linear distance of the path divided by
the radial distance (4.0 m) (Mrosovsky and Kingsmill, 1985). We computed mean
vectors, mean angles of orientation, and 95% confidence intervals for each group of
animals (Zar, 1984, p. 428). All animals were assumed to cross at the midpoint angle
in the appropriate sector. The Rayleigh test (Zar, 1984, p. 443) examined the null
hypothesis that there was no mean population direction. Differences between mean
angles were analyzed using the Watson-Williams procedure (Zar, 1984, p. 446).
We surveyed the "disoriented" crabs in transects on the flat and sloped beaches
on 23-25 May. Disoriented crabs were denned as those live animals remaining right-
side-up on the breeding beach at low tide, as distinguished from the normal behavior,
which is to follow the receding tide and spend the low tide period on the intertidal
sand flat. Live upside-down crabs, "stranded" by wave action, were not counted.
RESULTS
Surveys of disoriented crabs
The number of disoriented (live, rightside-up) stranded crabs was significantly
higher on the flat beach than the sloped beach on each of the three days (Table I).
Since similar, or perhaps slightly lower numbers of mating horseshoe crabs actually
approached the flat beach compared with nearby sloped beaches (based on Botton,
1982 and personal observations during the 1986 field season), the accumulation of
disoriented crabs on the flat beach is not likely to be a numerical artifact. Similar
dense concentrations of disoriented crabs were observed near creek mouths along
Delaware Bay north of our study area during a survey in 1 985. In contrast, only about
20-25% of live crabs stranded on sloped beaches during May, 1986 were rightside-
up (disoriented) animals; the remainder were stranded upside-down as a result of
wave action and/or telson abnormalities (R. E. Loveland and M. L. Botton, unpub.
data). Approximately 24-34% of the disoriented individuals on the flat beach were
stranded as mated pairs (males amplexed to females). By comparison, only 46 disori-
ented mated pairs (of a total of 4247 stranded crabs) were found on two sloped 90-m
study beaches between 15 May and 21 June 1986 (R. E. Loveland and M. L. Botton,
unpub. data).
292
M. L. BOTTON AND R. E. LOVELAND
N
N
FIGURE 1 . Orientation behavior of horseshoe crabs with normal vision on a sloped beach. Run A
held 26 May, 1610-1850, bright sunlight, wind W less than 5 mph. Run B held 2 June, 1057-1323, cloudy
with occasional drizzle, wind NNE 15 mph. Length and direction of mean vector r shown by solid arrow;
Rayleigh's Test indicated significant mean population direction in both runs. Seaward direction indicated
by open arrow. Typical "fish hook" path followed by a crab is shown in A.
Orientation on sloped beach. All sighted crabs showed strong seaward orientation
(Fig. 1). The path followed by nearly all crabs in these trials was the "fish hook"
pattern illustrated in Figure la. Typically, a crab first walked in the direction it was
placed (in this case, uphill) before turning to its left or right. More crabs turned left
(n = 26) than right (n = 16) but the difference was non-significant (x2 - 2.38, .25
< P < .10). There was no significant difference in mean angle between a group of
crabs run in late afternoon (n = 23) and a second group (n = 23) run in midday
(Watson-Williams test, F = 0.229, n.s.). In both trials, 22 animals (96%) completed
the test and there was no significant difference in the time it took to complete the test
(Mann-Whitney U-test, z = 0.493, n.s.). The meandering score was significantly
lower in the late day group (U = 365.0, P < .005). Pausing and circling behaviors
were noted only five times each (Table II).
Blinded crabs also showed strong seaward orientation on the sloped beach (Fig.
2). Mean angles of crabs tested in early morning and mid-afternoon were virtually
identical. In both the morning and afternoon experiments, 20 animals (87%) com-
pleted each trial. Animals in the morning trial took significantly longer (Mann- Whit-
ney U-test, U = 352.5, P < .001) and had a higher meandering score (U = 316, P
< .002) than the afternoon group. This difference was probably related to a steepening
of the beach slope before the afternoon trial, which was caused by strong wave action
several tidal cycles earlier. Blinded animals followed the typical fish hook path de-
scribed above; the direction of turning was random (20 to the left, 21 to the right).
No blinded animal circled in either trial and pausing was infrequent (Table II).
Orientation on flat beach. Horseshoe crabs with normal vision had difficulty ori-
ORIENTATION OF INTERTIDAL LIMULUS
293
TABLE II
Measures of orientation performance (means and standard errors = SE) of horseshoe crabs
on sloped and flat beaches
Sloped
beach
Flat beach
Normal vision
Blinded
Normal vision
Blinded
Variable
A
B
A
B
A
B
C
A
B
C
Time to completion
88.9
103.3
134
,7
58.1
271.9
128.8
192.1
183.2
395.7
374.9
SE
11.8
16.4
19
.1
5.6
53.5
37.6
22.0
61.3
137.2
64.8
Meandering score
1.21
1.43
1,
.48
1.37
2.36
2.15
2.01
1.72
2.04
3.45
SE
0.03
1.13
0.04
0.07
0.49
0.32
0.15
0.27
0.28
0.37
No. of pauses
0.04
0.26
0
22
0.17
0.50
0.50
0.13
0.75
1.87
1.06
SE
0.04
0.12
0
.09
0.10
0.20
0.31
0.07
0.41
0.62
0.36
No. of circles
0.09
0.13
0
.00
0.00
1.00
0.60
1.17
0.75
0.67
2.33
SE
0.06
0.07
0
.00
0.00
0.35
0.31
0.31
0.31
0.21
0.52
Number completing
">T
22
20
20
11
8
21
6
6
13
Number burrowing
0
0
1
1
4
1
0
1
4
2
Number stopped
without burrowing
1
1
2
2
1
1
->
1
5
3
Total number in run
23
23
23
23
16
10
23
8
15
18
Time to completion (in seconds) and meandering scores based on only those animals crossing the
perimeter of the 4-m radius testing arena. Letters at top of column designate individual runs.
enting on the flat beach. Three separate runs were conducted. In afternoon runs un-
der both overcast and sunlight conditions, values of Rayleigh's z indicated that crabs
were not significantly oriented in any direction (Fig. 3a, b). In the third run, on a
sunlit morning, there was significant orientation in a seaward direction (Fig. 3c).
However, four animals which exited through a seaward-facing sector were actually
travelling parallel to the shoreline when they crossed the perimeter.
Sighted crabs on the flat beach had a 10-fold increase in pauses and number of
circles, and higher meandering scores, compared with animals on the sloped beach
(Table II). Nine of the 40 animals (22.5%) tested in the three runs either burrowed or
stopped moving for 5 minutes, thus failing to complete the test.
Horseshoe crabs behaved differently on the flat beach than on the sloped beach,
although variability was high among those tested. Very few paths formed the fish
hook pattern described earlier. Initially, crabs moved rapidly in the direction they
were placed, but typically they turned and slowed down within the first meter. Many
crabs reared up on their pedipalps and moved slightly from side to side. This behavior
was often followed by circling, which in most cases began within 1 m of the release
point, although some animals made wider loops, "figure 8's," or both (Fig. 3a).
Blinded crabs were even more disoriented on the flat beach than were the sighted
individuals. The percentage of crabs completing the test was the lowest of the four
experimental combinations (Table II). Seven of the 41 crabs (17%) burrowed before
crossing the perimeter and another 9 (22%) stopped moving without burrowing. Of
those crossing the perimeter, the direction was random (Fig. 4). There was no signifi-
cant difference in the time to complete a trial between the three groups; however,
crabs in the third run had a significantly higher meandering score than those in the
first two.
In comparing sighted and blinded crabs on _the flat beach, the blinded animals
took longer (x = 333.8 s) than sighted animals (x = 201.4 s) to complete the test (U
= 663, P < .05). The meandering scores were not significantly different between the
294
M. L. BOTTON AND R. E. LOVELAND
N
N
7
7
FIGURE 2. Orientation behavior of blinded horseshoe crabs on a sloped beach. Run A held 28 May,
0710-1038, heavy cloud cover with mist, wind WSW 5 mph. Run B held 4 June, 1250-1600, bright sun,
wind S 10 mph; slightly steeper slope than Run A because of strong wave action during previous 48 hours.
Length and direction of mean vector r shown by solid arrow; Rayleigh's Test indicated significant mean
population direction in both runs.
sighted crabs and the first two groups of blinded crabs, but both were significantly
lower than the third group of blinded crabs (Kruskal-Wallis test, H = 11.54, P
< .005).
DISCUSSION
As noted by Herrnkind (1983), orientation by shore zone arthropods could poten-
tially involve visual and/or nonvisual guideposts. Among arthropods, visual cues are
important in various amphipods, isopods, decapods, insects, and wolf spiders (Herrn-
kind, 1 972). In contrast, beach slope has been demonstrated to be involved in orienta-
tion far less frequently (Hamner et ai, 1 968; Craig, 1 973). At our Delaware Bay study
area, there appears to be a strong visual contrast between the dark horizon in a land-
ward direction and the open horizon in the bayward direction; therefore, we consid-
ered the possibility that horseshoe crabs on sandy beaches might be employing vision.
There is considerable precedent in the literature concerning the behavioral re-
sponses of Limulus to light. Cole (1923) found that asymmetrically blinded animals
20-60 mm in diameter (which he erroneously considered to be adults) circled most
frequently in the direction of the remaining lateral eye. Northrup and Loeb (1923)
found that young crabs (ca. 1 5 cm length) were negatively phototropic. Limulus can
also detect polarized light (Waterman, 1950), but at present there is no experimental
evidence implicating this in any known behavioral response. More recently, Barlow
and collaborators (e.g., Barlow et ai, 1980) have shown the presence of a circadian
ORIENTATION OF INTERTIDAL L1MULUS
295
N
N
N
/
FIGURE 3. Orientation behavior of horseshoe crabs with normal vision on a flat beach. Run A held
21 May, 1555-1930, heavy cloud cover, wind calm. Run B held 28 May, 1622-1747, bright sun, wind
WSW 5 mph. Run C held 5 June, 0825-1200, bright sun, wind S 5 mph. Length and direction of mean
vector r shown by solid arrow; Rayleigh's Test rejected the null hypothesis of no mean population direction
in Run C only. Seaward direction indicated by open arrow. Typical path followed by a crab is shown in A.
296
M. L. BOTTON AND R. E. LOVELAND
N
FIGURE 4. Orientation behavior of blinded horseshoe crabs on a flat beach. Runs A (30 May, 1314-
1450, wind W 5 mph) and B (31 May, 0748-1205, wind W 5 mph) were combined (open circles) because
of the small number of animals completing each trial. Run C (7 June, 0730- 1 245, mostly sunny, wind S 5
mph) shown with filled circles; length and direction of mean vector r (solid arrow) based on Run C data.
Population showed random orientation. Seaward direction indicated by open arrow.
rhythm in ommatidial morphology and visual sensitivity, modulated by a clock in
the animal's brain. Visual sensitivity is higher at night, enabling males to recognize
female "models" even during the evening (Barlow et ai, 1 982). Blinded crabs released
in the subtidal environment were more disoriented than sighted crabs (Ireland and
Barlow, 1978). However, both blinded and sighted submerged crabs were capable of
orienting in the vicinity of the breeding beach when wave surge was present (Rudloe
and Herrnkind, 1976).
Experiments with blinded and sighted crabs on both sloped and flat beaches indi-
cate that beach slope, not visual stimuli, is the primary cue used by horseshoe crabs
to return to the water. Both blinded and sighted animals showed rapid seaward orien-
tation on a sloped beach. In contrast, orientational performance was severely im-
paired on a spawning beach lacking slope. Sighted and blinded crabs showed pausing
and circling behaviors on the flat beach far more frequently than on the sloped beach.
Circling, accompanied frequently by the animal's rearing up on their pedipalps, may
have been an attempt to obtain directional information from gravitational cues. Since
these directional cues were lacking in the flat arena, it is not surprising that meander-
ing scores on the flat beach were higher, and many animals either burrowed, stopped
moving entirely, or left the arena on a heading that took them away from the water.
The physiological basis for the observed response to beach slope is not clear. De-
spite the extensive use of Limulus in neurophysiological research, no statocyst or
other balance organ has been described. However, proprioreceptors have been identi-
fied from the gnathobases and joints of the walking legs (Barber, 1956, 1960; Pringle,
1956; Barber and Hayes, 1964). We hypothesize that stimulation of these receptors
in a crab walking "uphill" may elicit a turning response which then directs crabs
down the slope, toward the water. A crab can presumably detect when it has started
ORIENTATION OF INTERTIDAL LIMULUS 297
"downhill," since once on this heading, turns are infrequent. The information regis-
tered by these proprioreceptors would be constant on a flat beach, which may under-
lie their lack of orientation. It seems unlikely that mechanoreceptors on the dorsal
carapace (Kaplan el al, 1976) or lateral spines (Eagles, 1973) are involved in beach
orientation, although they may provide positional information under different cir-
cumstances.
The comparison between the responses of blinded and sighted animals on the flat
beach suggests a possible secondary role for vision in sandy beach orientation. One
of three groups of sighted crabs showed significant seaward orientation (Fig. 3), but
why this group did, and the other two did not, is not apparent. Overall, sighted crabs
on the flat beach exhibited somewhat better orientation (as measured by percent fin-
ishing, number of pauses, and meandering scores) than the blinded crabs on the same
arena, although their performance was much poorer than the blinded crabs on the
sloped beach.
A comparable and ecologically similar orientation problem confronts female sea
turtles after egg laying. In these animals, in contrast to Limulus, the primary cues are
visual. Females, as well as hatchlings, assess the brightness differential between the
open, seaward horizon and the darker, vegetation-lined landward horizon (Ehrenfeld
andCarr, 1967; Mrosovsky and Carr, 1967; Mrosovsky and Shettleworth, 1968;Mro-
sovsky, 1970). In comparison, there is some evidence for positive geotropism among
hatchlings (Parker, 1922; Van Rhijn, 1979), although Mrosovsky and Kingsmill
(1985) point out that on many turtle nesting areas, slope is very irregular and not as
good a predictor of seaward direction as is the open horizon. This configuration is
markedly different at the Cape May shore of Delaware Bay, where undisturbed
beaches consistently have a seaward slope. A positive geotaxis is therefore a reliable
orientation behavior for adult horseshoe crabs, whereas for sea turtles, it is not. How-
ever, it might be adaptive for a species to have a secondary orientation mechanism
should the primary system fail. For sea turtles, geotaxis may function, albeit weakly,
when vision is impaired (Van Rhijn, 1979); a similar hierarchy of cues exists in the
amphipod, Orchestoidea corniculata (Herrnkind, 1983). For horseshoe crabs, vision
may be of some limited value when the crabs become stranded on a flat beach, but
many animals which spawned on this type of beach are unable to find the water
only a few meters away (Table I). Under these circumstances, it seems adaptive for
horseshoe crabs to burrow because it conserves energy and keeps their book-gills in
contact with cooler, moist sand. If high tide during the next day is of equal or greater
amplitude than the one on which they were stranded, the crabs will have a good
chance of survival, but if tidal heights are declining, there may be localized, large-
scale mortality.
ACKNOWLEDGMENTS
We are grateful to Marine Biologicals, Inc., and the Fordham University Research
Council for financial support, and to Dr. R. A. Lutz for the use of laboratory facilities.
We are also grateful to K. A. Becker, P. Claxon, and P. Jones for their assistance in
running the behavioral trials.
LITERATURE CITED
BARBER, S. B. 1956. Chemoreception and proprioreception in Limulus. J. E.\p. Zool. 131: 5 1-69.
BARBER, S. B. 1960. Structure and properties of Limulus articular proprioreceptors. J. Exp. Zool. 143:
283-321.
BARBER, S. B., AND W. F. HAYES. 1964. A tendon receptor organ in Limulus. Cornp. Biochem. Physiol.
11: 193-198.
298 M. L. BOTTON AND R. E. LOVELAND
BARLOW, R. B., JR. S. C. CHAMBERLAIN, AND J. Z. LEVINSON. 1980. Limulus brain modulates the struc-
ture and function of the lateral eyes. Science 210: 1037-1039.
BARLOW, R. B., JR., L. C. IRELAND, AND L. KASS. 1982. Vision has a role in Limulus mating behaviour.
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BARLOW, R. B., M. K. POWERS, H. HOWARD, AND L. KASS. 1986. Migration of Limulus for mating:
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BOTTON, M. L. 1982. Predation by adult horseshoe crabs, Limulus polyphemus (L.) and its effect on ben-
thic intertidal community structure of breeding beaches in Delaware Bay, New Jersey. Ph.D.
thesis, Rutgers University. 466 pp.
BOTTON, M. L., AND J. W. ROPES. 1987. Populations of horseshoe crabs on the northwestern Atlantic
continental shelf. Fish. Bull, (in press).
COLE, W. H. 1923. Circus movements of Limulus and the tropism theory. /. Gen. Physiol. 5: 4 1 7-426.
CRAIG, P. C. 1973. Orientation of the sand-beach amphipod, Orchestoidea corniculata. Anim. Behav. 21:
699-706.
EAGLES, D. A. 1973. Lateral spine mechanoreceptors in Limulus polyphemus. Comp. Biochem. Phvsiol.
44A: 557-575.
EHRENFELD, D. W., AND A. CARR. 1967. The role of vision in the sea-finding orientation of the green
turtle (Chelonia rnydas). Anim. Behav. 15: 25-36.
HAMNER, W. M., M. SMYTH, AND E. D. MULFORD JR. 1968. Orientation of the sand-beach isopod Tylos
punctatus. Anim. Behav. 16: 405-409.
HERRNKIND, W. F. 1972. Orientation in shore-living arthropods, especially the sand fiddler crab. Pp. 1-
59 in Behavior of Marine Animals, Vol. I, Invertebrates, H. E. Winn and B. L. Olla, eds. Plenum
Press, New York.
HERRNKJND, W. F. 1983. Movement patterns and orientation. Pp. 41-105 in The Biology of Crustacea,
Vol. 7, Behavior and Ecology. F. J. Vernberg and W. B. Vernberg, eds. Academic Press, New
York.
IRELAND, L. C., AND R. B. BARLOW JR. 1978. Tracking normal and blindfolded Limulus in the ocean by
means of acoustic telemetry. Biol. Bull. 155: 445-446.
KAPLAN, E., R. B. BARLOW JR., S. C. CHAMBERLAIN, AND D. J. STELZNER. 1976. Mechanoreceptors on
the dorsal carapace of Limulus. Brain Res. 109: 6 1 5-622.
MROSOVSKY, N. 1 970. The influence of the sun's position and elevated cues on the orientation of hatchling
sea turtles. Anim. Behav. 18: 648-65 1 .
MROSOVSKY, N., AND A. CARR. 1967. Preference for light of short wavelengths in hatchling green sea
turtles, Chelonia mydas. tested on their natural nesting beaches. Behaviour 28: 2 1 7-23 1 .
MROSOVSKY, N., AND S. F. KINGSMILL. 1985. How turtles find the sea. Z. Tierpsychol. 67: 237-256.
MROSOVSKY, N., AND S. J. SHETTLEWORTH. 1968. Wavelength preferences and brightness cues in the
water finding behavior of sea turtles. Behaviour 27: 21 1-257.
NORTHRUP, J. H., AND J. LOEB. 1923. The photochemical basis of animal heliotropism. J. Gen. Phvsiol.
5:581-595.
PARKER, G. H. 1922. The crawling of young loggerhead turtles toward the sea. J. Exp. Zool. 36: 323-33 1 .
POWERS, M. K., AND R. B. BARLOW JR. 1985. Behavioral correlates of circadian rhythms in the Limulus
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SHUSTER, C. N. JR. 1982. A pictorial review of the natural history and ecology of the horseshoe crab
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Reference: Biol. Bull. 173: 299-310. (October, 1987)
THE FEEDING BEHAVIOR OF PAR.4NOPHRYS CARNIVORA
(CILIATA, PHILASTERIDAE)
DAVID KAHAN.1 THEODORA BAR-EL.1 NORBERT WILBERT,3
SAMSON LEIKEHMACHER,2 AND SAMUEL OMAN2
1 Department of Zoology, Hebrew University of Jerusalem, Jerusalem, Israel, 2 Department of Statistics,
Hebrew University oj Jerusalem, Jerusalem, Israel, and *Zoologisches Institut,
Poppelsdorfer Schloss, Bonn, West Germany
ABSTRACT
The marine ciliate Paranophrys carnivora, isolated from the Mediterranean coast
of Israel, was found to feed on a varied diet of bacteria, algae, and living and non-
living tissues. Chlorococcum sp. and Duualiella parva, the algal species on which P .
carnivora grew best, did not elicit a chemosensory response; tissues and bacteria did.
In experiments on stationary phase ciliates, betaine, choline, L-histidine, and tri-
methylamine oxide elicited a positive chemosensory response at concentrations as
low as 10~6Mto 10"3A/.
INTRODUCTION
Most ciliates feed on particulate matter consisting mainly of microorganisms
(bacteria and algae) of a size appropriate to their buccal apparatus. The particles,
whether suspended or settled, are collected via specialized cilia near the oral opening
(Corliss, 1979).
In Tetrahymena, one of the ciliates most studied, particulate matter seems obliga-
tory for feeding. An autoclaved 2% proteose peptone medium which supports a
flourishing culture, loses this capability when the particles are removed by millipore
filtration. Addition of inert particles lacking any nutritive value to the filtered me-
dium restores its growth potential (Rasmussen and Kludt, 1970; Rasmussen and
Modeweg-Hansen, 1973). Extensive experiments performed by Fenchel ( 1980a, b, c)
with inert "latex" particles indicate that various ciliates select their food primarily by
particle size.
When offered different combinations of algal species of the same size range, the
ciliate Favella ehrenbergii showed a preference for one species, indicating that food
selection is also based on factors other than size (Stoecker el al., 1981). Selective
feeding has been attributed to chemical stimuli in various other ciliates e.g., Nassula
(Poilvert, 1959), Stentor coeruleus (Tartar, 1961; Rapport et al., 1972), and Podo-
phrya calkinsi (Hull, 1 96 1 ). In addition, particle movement affects the feeding behav-
ior of ciliates (Karpenko et al., 1977).
Most studies on the feeding behavior of ciliates focused on those feeding on micro-
organisms e.g., Parameeiiim, Tetrahymena (Levandowsky and Hauser, 1978; Van
Houten et al., 1981, 1982; Antipa et al., 1983; Levandowsky et al., 1984; Leick and
Hellung-Larsen, 1985; Hellung-Larsen et al., 1986). Studies on ciliates that feed on
tissues are scarce (Levandowsky and Hauser, 1978; Van Houten et al., 1981). The
marine ciliate Paranophrys carnivora, which was recently described, feeds on tissues
of living or dead organisms (Czapik and Wilbert, 1986). The feeding behavior of this
organism is described in this paper.
Received 9 February 1987; accepted 22 July 1987.
299
300 D. KAHAN ET AL.
MATERIALS AND METHODS
Cultivation and morphology of Paranophrys carnivora
P. carnivora was isolated from samples collected from the Mediterranean Sea at
the coast of Dor, Israel. The samples were rich in various protozoans: Ciliata e.g.,
Euplotes, and Flagellata, mostly autotrophic ones, including Dunaliella. Initial obser-
vations showed that P. carnivora fed on algae. They were also observed to gather in
the vicinity of freshly injured invertebrates (crustaceans) and feed on their tissues.
Several P. carnivora clones were prepared and grown on Dunaliella parva as well
as on a strain of the bacterium Enterobacter aerogenes which can grow at a salinity
of 35%. No attempt was made to eliminate the original bacterial flora. The most suc-
cessful clone was further cultured on E. aerogenes for growth curve and feeding be-
havior studies. These bacteria were grown on brain heart Agar (Difco) slants at 28°C,
and then harvested in sterile water to give a suspension having an absorbance of 1 . 1-
1.3 at 430 nm, as measured with a Bausch and Lomb "Spektronic 20" spectropho-
tometer. A 2-ml inoculum of a 3-4 day-old culture of Paranophrys and 0.4 ml of the
bacterial suspension were added to a test tube (3 cm diameter, 20 cm length) contain-
ing 30 ml of sterile (autoclaved) 35%o artificial seawater (Instant Ocean salts from
Aquarium Systems, Mentor, Ohio, in filtered water, hereafter referred to as ASW).
The culture was then incubated at 28°C in a temperature-controlled chamber. For
growth curve studies, 0.5- or 1-ml samples were removed at various intervals from
each of three cultures which had been inoculated simultaneously from the same
source, and preserved in a 10% Bouin solution. All of the ciliates in each sample were
counted in a glass chamber at 60X with a hand tally. Size determinations were made
at 600X using an ocular micrometer.
The morphological description and identification was based on the same clone
which was kept in 15 ml of ASW in covered glass vials at 20°C and fed every 14
days on 3 pin-head sized bits of either oligochaete or crustacean meat. Biometric
measurements were made using a light microscope on ciliates stained by the protargol
(Wilbert, 1975) and silver nitrate (Chatton and LwofF, 1930) methods. For scanning
electron microscopy, cells were fixed instantaneously by rapid addition of a large
volume of 2.7%. OsO4 in ASW. After 10-15 minutes in fixative at room temperature,
the cells were washed with 2% glutaraldehyde in ASW. After 10 minutes they were
dehydrated in a graded ethanol series, dried by the critical point method, coated with
gold-palladium, and viewed in a Joel 840 scanning electron microscope.
Feeding and growth experiments with marine algae
The species of marine algae whose names are given in Table II were cultivated in
test tubes in a medium of ASW enriched with Walne solution (Walne, 1966) in a
temperature-controlled room at 18°C and under continuous illumination. Young,
flourishing week-old cultures were inoculated with Paranophrys carnivora and fur-
ther incubated under continuous illumination at 25°C. The cultures were examined
with a dissecting microscope at 40X — both initially and at various intervals during a
week — to appraise the ciliate population growth. Samples were also examined under
higher power of the light microscope, while alive and after they were killed with a 1%
formalin solution. At 60X, the dimensions of the algae (length/width or diameter)
were determined using an ocular micrometer. Ciliates were also examined for vacu-
oles containing algae.
Behavior experiments
Capillary tube assay. The amino acids tested were purchased from Sigma (L-
leucine, L-isoleucine, L-proline, hydroxy-L-proline, L-arginine, D- and L-histidine,
FEEDING BEHAVIOR OF P. CARNIl'OR.4 301
L-cysteine); Nutritional Biochemical Corporation (DL-phenylalanine, DL-a-alanine.
DL-serine, L-methionine, L-threonine, DL-asparagine); British Drug House, Ltd.
(glycine, L-aspartic acid); Merck & Co., Inc. (L-tyrosine); Light & Co., Ltd. (L-cys-
tine); Fluka (DL-valine); and CHR (L-tryptophan). Betaine hydrochloride, choline
chloride, and trimethylamine oxide (TMAO) were also purchased from Sigma, and
proteose peptone and brain heart infusion from Difco Laboratories.
The substances to be tested were dissolved in distilled water and the pH of the
solution was adjusted, if necessary, to the pH of ASW (pH 8). Glass capillary tubes
(Modulohm of Helver, Denmark) 5-8 mm long and 0.7-1.0 mm in external diame-
ter, were filled with the test solution and dried. In a previous study with another
species of the same family, Porpostoma not at um, many ciliates would enter a control
capillary containing fresh medium even when the medium they were swimming in
was only half-an-hour old (Kahn et ai, 1981). Therefore we modified the capil-
lary assay by drying the test solutions and then, during the experiment, allowing the
test substances to become dissolved in the same medium the ciliates were swimming
in, rather than use fresh medium as a solute. Control capillary tubes were not filled
with any chemicals. Tissue culture dishes, 35 X 10 mm (Falcon), were each filled with
2 ml of ciliates from the stationary phase of culture, which had been diluted with fresh
ASW to a density ranging from 100 to 300 ciliates per ml. For each concentration and
substance to be tested, a test and a control tube were placed in different halves of each
experimental dish. When both test and control dry capillaries were immersed in the
ciliate suspension at the start of each experiment, they became filled with the medium
in which the ciliates were suspended. The test substances were dissolved within the
experimental period (the substances found later to elicit a positive chemosensory re-
sponse were further tested separately in a series of identical capillaries. These sub-
stances dissolved completely within a five-minute period). In each experimental run,
a test and control capillary pair were tested in each of three dishes i.e., in triplicate.
Betaine, found earlier to elicit a chemosensory response from Paranophrys carnivora
(mistakenly identified as P. magna: Kahan et ai, 1985), was used at a concentration
of 10~' M (together with a control capillary) as a standard in each experimental run
to verify the responsiveness of the ciliates. These betaine and control pairs were also
run in triplicate. Using a dissecting microscope, the number of ciliates in each tube
(up to about 100) was recorded with a hand tally at intervals during a 30-minute
period. Levandowsky et al. (1984) preferred using flat capillaries to eliminate diffi-
culties encountered in viewing Tetrahymena ciliates through cylindrical capillaries.
We did not experience difficulty in counting moving Paranophrys carnivora in the
cylindrical tubes. In the initial screening, most of the amino acids were tested at a
concentration of 10 ' M, with the exception of L-glutaminc acid and L-tryptophan,
at 5 X 10"2 M, and L-tyrosine, at 2 X 10~3 M. L-histidine was the only amino
acid which elicited a positive response at least as strong as that of betaine. This amino
acid, as well as betaine, choline, and trimethylamine oxide (TMAO), was further
tested in at least four experimental runs at concentrations from 10~6 or 10~'
to 10" 'A/.
The chemosensory response was computed at each time interval as the ratio of
the number of ciliates in the tube containing the test substance to the total number
of ciliates in both the test and control tubes. Since statistical analysis (via Mests) of
the differences in response at different times during each half-hour experimental run
showed no consistent effect of observation times, the index of chemosensory response
for a given experimental run was defined as the maximum of the chemosensory re-
sponses at the time intervals measured. To adjust for variation in chemotactic respon-
siveness over the different days of the experimental runs, a relative index of chemotac-
tic activity for a given substance at a given concentration, was defined as the ratio of
302 D. KAHAN ET AL.
its chemotactic activity to the index of the standard (betaine, at a concentration of
10"1 M) for the same experimental run.
Both the index and relative index of chemosensory response of P. carnivora, for
the various substances at different concentrations, were analyzed by two-way analysis
of variance (Scheffe, 1959). Effects on the index due to substances or concentrations
were analyzed using the S-method of multiple comparisons (Scheffe, 1959).
Dialysis experiments. Dialysis bags, 20 cm in length and 1 .6 cm in diameter ( Visk-
ing Tubing, The Scientific Instrument Center, Ltd.), were filled with 10 ml of either
test solution (5% proteose peptone in ASW) or control (ASW alone). They were im-
mersed in separate finger bowls each containing 150 ml of ciliate suspension at a
density of 40 per ml. This was prepared by diluting a stationary phase culture with
fresh ASW. To evaluate the behavioral effect, the tubing was first examined along
its entire length — using the low magnification of the dissecting microscope — for the
greatest congregation of ciliates. This section was further examined under 40X and
the number of ciliates on both the test and control bags was compared at consecutive
time intervals for up to 2 hours.
RESULTS
The growth curve of Paranophrys carnivora and associated morphological changes
The growth curve of P. carnivora fed on Enterobacter aerogenes at 28°C is shown
in Figure 1. Figure 1 shows that the logarithmic growth phase continues for up to
about 30 hours with a generation time of 7-8 hours. The stationary phase which
follows is short, and after 48 hours there is a moderate decline in the number of
ciliates. This phase continues until the experiments are terminated at the end of the
fifth day. During the growth experiments the shape of the cell changed from ovoid
(the "trophic" form, having a length to width ratio of 1.8 in the logarithmic phase)
to more elongated (the "swimming" form, with a ratio of 2.2 or more in the stationary
and decline phases). More pronounced differences between the two forms were ob-
tained with cultures fed on either oligochaete or crustacean meat and, rarely, from
cultures fed on algae. Scanning electron micrographs of the two forms from cultures
fed on Dunaliella parva are shown in Figures 2 and 3. The biometric data presented
in Table I are of silver stained specimens from cultures fed on oligochaete or crusta-
cean meat, as are the light micrographs given in Figures 4 and 5. In Figure 5, note
the marked appearance of the stained kinetosomes and the protrichocysts, another
characteristic of the swimming form.
Feeding and growth experiments with marine algae
Table II shows that Paranophrys carnivora ingested most of the algae offered.
However, different results were obtained with the various algal species ingested. The
best growth was obtained with Chlorococcum sp. and Dunaliella parva; no growth
occurred with Chlorella saccharophila and Dunaliella primolecta. As might be ex-
pected, those algal species that were not ingested did not support good ciliate cultures.
Chemosensory response
In the laboratory, P. carnivora fed on either Enterobacter aerogenes, various algae,
or wounded Anemia, dead or alive, when each of these diets was offered individually.
Differences in chemosensory responses were obtained when capillary tubes contain-
ing one of the diets was offered with a capillary containing no food (control), and the
number of ciliates in each of the two tubes compared after 10 minutes. As shown in
FEEDING BEHAVIOR OF P. CARNIl'OR.4
303
10
ou
O
o>
CL
CD
0.5
o
i_
CL>
_Q
E
13
20 40 60 80 100 120
Hours
FIGURE 1 . Growth curve of Paranophrys carnivora. Results are based on three replicate cultures.
Table III, Artemia homogenate and the E. aerogenes suspension elicited a positive
chemosensory response, whereas the alga Dunaliella parva elicited none. These re-
sults were obtained with ciliates that had been previously cultivated on each of the
diets indicated. When Chlorococcum sp. was offered instead of D. parva, the same
results were obtained.
Since Enterobacter was routinely cultivated on brain heart agar, it was thought
that its effect could have been due to the presence of some dissolved ingredients from
the growth medium in the suspension. Indeed, brain heart infusion did elicit a posi-
tive chemosensory response in capillary experiments. To determine whether the bac-
teria themselves are effective, they were washed by centrifugation and offered to the
ciliates in a capillary tube. After repeated rinsings in ASW, neither the bacterial pellet
nor the supernatant gave a positive result. However, when the washed pellet was
suspended in fresh ASW, incubated for up to 48 hours at 1 8°C, and then centrifuged
again, the resulting supernatant elicited a chemosensory response. This indicates that
washed bacteria excrete with time an effective substance or release such a factor upon
disintegration.
Positive results were obtained with other microbiological growth media, such as
proteose peptone and casein hydrolysate. In dialysis experiments with proteose pep-
tone, Paranophrys was found to be attracted to those molecules which were able to
pass through the cellophane membrane, e.g., amino acids. In further capillary tests
to screen individual amino acids, only L-histidine, of the various amino acids tested,
was as effective as betaine, a substance previously found to elicit a chemosensory
response from P. carnivora (Kahan et al, 1985). Choline and trimethylamine oxide
(TM AO), compounds with a chemical structure similar to that of betaine, also elic-
ited a positive response at least as strong as that of betaine. The four substances (beta-
304
D. KAHAN ET AL.
•
FIGURES 2-5 are to the same scale and view the ventral side (note buccal cavity).
FIGURE 2. Scanning electron micrograph of trophic form ofParanophrys carnivora (arrowhead indi-
cates part of an algal cell, Dunaliella parva, engulfed by the ciliate).
FIGURE 3. Scanning electron micrograph of swimming form of P. carnivora (arrowheads indicate
algal cells from culture which have adhered to the ciliate).
FIGURE 4. Photomicrograph of silver-stained trophic form of P. carnivora.
FIGURE 5. Photomicrograph of silver-stained swimming form of P. carnivora.
FEEDING BEHAVIOR OF P. CARNIVORA 305
TABLE I
Cell dimensions of the trophic and swimming forms of Paranophrys carnivora (given in micrometers)
Length
Width
Distance from
anterior pole
to end of UM
Form
Range
x± S.E.
Range
.v±S.E.
Range
;c±S.E.
Trophic
36-56
47.08 ± 1.3
18-35
25.18± 1.07
18-25
20.82 ± 1.17
(17)
(22)
(17)
Swimming
40-60
49.44+ 1.47
13-22
16. 67 ±0.62
24-29
26.4 + 0.52
(18)
(15)
(15)
Numbers in parenthesis indicate the number of observations.
ine, L-histidine, choline, and TM AO) were offered to P. carnivora at various concen-
trations; the results were analyzed as described previously. The index averaged over
the experimental runs is shown in Figure 6 for each substance at the various concen-
trations. The average index of chemosensory response elicited by betaine, choline,
and TMAO was significantly greater than 0.5, at the 5% level or more, at all the
concentrations examined, and by L-histidine for concentrations of at least 10~3 M
(concentrations of 10~4, 10 5, and 10"6 M were also tested, but the response was not
significantly greater than 0.5).
Two-way analysis of variance of the index of chemosensory response showed sig-
nificant effects due to material (P-value : : 0.028) and concentration (f-value :
0.000). The means of the relative index of chemosensory response (the index, pre-
viously defined under Materials and Methods as "capillary tube assay," which adjusts
for the level of response to the betaine standard during the same experimental run)
exhibited the same behavior as the nonadjusted index, indicating that the differences
in the index of chemosensory response represented in Figure 6 are not due to varying
levels of overall chemosensory responsiveness (as measured by the response to the
standard, betaine at 10~' A/) on the different days of experimental runs. The analysis
of variance is based on the nonadjusted chemosensory response, as opposed to the
adjusted response, because the data on the former satisfied the required statistical
assumptions on the error terms more closely (see Scheffe, 1959, p. 5).
Figure 6 suggests that choline, betaine, and TMAO are similar in the response
they elicit from P. carnivora, the main differences in the chemosensory response in-
dex being due to different concentrations. This was confirmed for betaine and choline
using the S-method (Scheffe, 1959) of multiple comparisons to compare effects on
the chemosensory response index due to materials or concentrations. The average
index for betaine and choline at the concentration of 10~' M was significantly differ-
ent (at the 5% level) from the average at the 10~6 M level; and the differences between
the averages at the 10~' M and 10~5 M levels, and at the 10 2 M and 10"6 M levels,
were almost significant at the 10% level.
DISCUSSION
Although previously thought (Czapik and Wilbert, 1986) to feed on fresh and
decomposing tissues, the present study establishes that Paranophrys carnivora can
feed on a more varied diet. This diet includes algae and bacteria in addition to tissues.
In this respect it seems to be closer in its dietary spectrum to P. thompsoni and P.
magna than to the other species of the genus. P. thompsoni was reported to live on
bacteria and heterotrophic flagellates which developed in hatched gelatinous egg
306
D. KAHAN ET AL.
TABLE II
Growth on and ingestion of different algal species by Paranophrys carnivora
Algal class:
Species
Source
Algal size"
Ingestion Ciliates'
of algae growth6
Baciilariophyceae
Amphora sp. 12
J.L.,CUNYC
35-40/2-4
no +
Phaeodactylum
tricornutum
CMBRDG CCd
20-30/2.5
yes ++
Chlorophyceae
Chlamydomonas
provasoli
J.L.,CUNY
4-8
no +
Chlamydomonas
hedleyi
J.L.,CUNY
5-10/3-9
yes + +
Chlorella
stigmatophora
IOLRe
5-6/3-4
yes +
Chlorella
saccharophila
IOLR
3
yes
Chloroccoccum sp.
J.L..CUNY
2-3
yes ++++
Dunaliella
primolecta
CMBRDG CC
6-14/4-13
yes
Dunaliella sp.
Strain C9AA
B.G.,HUr
10-18/8-13
no +
Dunaliella sp.
Strain El
B.G., HUf
14-18/8-10
no +
Dunaliella sp.
Strain 1644
B.C., HUf
11-21/8-15
no +
Dunaliella sp.
Strain L10
B.C., HUf
9-14/8-13
no +
Dunaliella
tertiolecta
B.G.,HUf
8-12/4-8
yes ++ +
Dunaliella parva
B.G., HUf
6-12/3-8
yes ++++
Dunaliella sp.
Strain 14
E.G., HUf
6-10/3-8
yes + + +
Dunaliella sp.
Strain E4
B.C., HUf
5-10/3-8
yes + + +
Dunaliella sp.
Strain Iran 6
B.C., HUf
5-8/3-8
yes + + +
Nannochloris sp.
Strain W5 15
J.L.,CUNY
8-12/6-8
no + +
Cyanophyceae
Anacystissp.
Houde8
2-3
yes + +
Prasinophyceae
Tetraselmis chuii
IOLR
12-13/8-9
no
Prymnesiophyceae
Isochrysis galbana
IOLR
4-6
yes ++
a The dimensions (length/width or diameter) are given in ^m.
"Rating code: ++++, excellent; +++,good; ++, fair; +, poor; -, no growth.
c John Lee, City University of New York.
d Cambridge Culture Collection.
e Institute of Oceanographic and Limnological Research, Haifa.
f B. Ginzburg, Hebrew University (Ginzburgand Ginzburg, 1985).
8 E. D. Houde, University of Miami, Florida.
masses of dipterans (Didier and Wilbert, 1976), while P. magna was cultivated in
cultures to which split peas had been added (Borror, 1972) and presumably fed on
the bacterial flora. Nevertheless, comparative dietary experiments on the above-men-
tioned species should be further extended in order to establish their feeding pattern.
FEEDING BEHAVIOR OF P. CARNIVOR.4
307
TABLE III
Chemosensory response of Paranophrys carnivora to different diets as determined by capillary assay
Test diet offered in capillary
Diet cultivated on
Dunaliella
parva
Enterobacter
aerogenes
Anemia
homogenate
Algae (Dunaliella parva)
Bacteria
(Enterobacter aerogenes)
Fresh meat
(wounded Anemia)
Cultures of the ciliates were grown each on a different diet as indicated. The "+" indicates a positive
response and "— ", no response.
Our attempts (unpub.) to introduce P. carnivora as a symbiont living in the coelenter-
ates Cordylophora sp., Cassiopea sp., and Aiptasia as well as in the crustaceans Ar-
ternia salina and Macrobrachium rosenbergii, did not succeed. Two other species of
the genus, P. marina and P. carcini, were found inside coelenterates (Thompson and
x
CD
CD
CO
C
o
CL
tO
CD
co
C.
CD
CO
O
£
CD
-C
0
C
D
CD
.0
0.9
0.8
0.7
0.6
0.5
10"
10'
,-2
10"- 10 ' 10"- 10
Molar concentration
_c
FIGURE 6. Dose-response curves for substances which elicit a positive chemosensory response from
Paranophrys carnivora by the capillary assay. Each figure represents the mean of the values of the index
of chemosensory response obtained from all of the observations for a particular substance at a certain
concentration. The results obtained with betaine are indicated by circles, with trimethylamine oxide by
triangles, with choline chloride by squares, and with L-histidine by diamonds. A full (black) figure indicates
that the mean index of chemosensory response is significantly greater than 0.5 at the 1% level, and a half-
full figure, at the 5% level. L-histidine was also tested at 10 4, 10~5, and 10~6 M. but the results were not
significantly greater than 0.5.
308 D. KAHAN ET AL.
Berger, 1965) and in the hemolymph of crustaceans (Groliere and Leglise, 1977),
respectively.
Like many other ciliates (Fenchel, 1980a, b, c), P. carnivora ingests suspended
inert particles such as polystyrene beads (unpub.) and living microorganisms. Here
size seerns to be a limiting factor in food ingestion. The largest food vacuoles observed
did not exceed 7 yum in diameter, and algal species having size ranges above this
limit were not ingested i.e., Amphora sp., Dunaliella strains C9AA, El, 1644, L10,
Nannochloris sp., and Tetraselmis chuii. However, some of the large species (the four
strains of Dunaliella mentioned above and Nannochloris sp.) did sustain growth. This
could be due to the ciliates' feeding on bacteria contaminating the algal cultures and/
or on disintegrating algal cells in aged cultures. The same explanation could be offered
for the ciliates' growth on Phaeodactylum triconutum. While C. provasoli was in the
size range of algae that could be engulfed, it was not ingested. This is probably due to
the tendency of the latter algal cells to form bigger sized aggregates, or to their having
a chemoinhibitory effect on phagocytosis by Paranophrys. Those species of algae that
were ingested by Paranophrys (Table II) gave growth results that varied in their rating
from no growth, i.e., Chlorella saccharophila and Dunaliella primolecta, to excellent
growth i.e., Dunaliella parva and Chlorococcum sp. However, these latter two species
did not elicit a positive chemosensory response from Paranophrys carnivora in our
experiments. Although algae are known to release assimilated carbon into the culture
medium (Hunstman, 1972; Fogg, 1977; Saks, 1982), Dunaliella parva and Chlorococ-
cum sp. evidently do not release a substance eliciting a chemosensory response from
Paranophrys carnivora.
Betaine, choline, L-histidine, and trimethylamine oxide, the substances found to
elicit a positive chemosensory response from Paranophrys carnivora, are known to
affect feeding behavior in various other organisms (Lindstedt, 1971; Levandowsky
and Hauser, 1978; Heinen, 1980; Caprio, 1984). They are also widely distributed in
various organisms including bacteria and algae (Bell and Mitchell, 1972; Levandow-
sky and Hauser, 1978; Edwards, 1982; Galinski and Truper, 1982; Abe, 1983; Ko-
nosuetal.. 1983; Shirani et a/., 1983; Imhoffand Rodriguez-Valera, 1984; Morihiko
et al, 1984) and therefore could be indicators of a wide variety of food sources for
Paranophrys carnivora. There may be other effective substances as yet untested for
Paranophrys carnivora, which have recently been found to elicit a chemosensory re-
sponse from other ciliates such as Parameciwn (Antipa and Norton, 1985) and Tetra-
hymena (Leick and Hellung-Larsen, 1985; Hellung-Larsen et al., 1986). Paranophrys
carnivora responds to the D-isomer of histidine, which does not occur in nature, and
in this respect resembles Tetrahymena thermophila ( Almagor et al., 1 98 1 ), which also
responds to both the L and D forms of an amino acid (methionine).
Another characteristic Paranophrys carnivora shares with Tetrahymena is its
body transformation. The morphological differences in body proportions between
the ovoid feeding form and elongated swimming form of P. carnivora appear more
pronounced when the ciliate is cultivated on tissues and on rare occasions when
grown on algae, after depletion of the food organisms. A similar transformation in
form appears after starvation in Tetrahymena thermophila. In the latter, the transfor-
mation is accompanied by several other changes i.e., oral replacement, caudal cilium
appearance, and increase in number of somatic basal bodies and cilia, as well as in
speed (Nelsen, 1978; Nelsen and DeBault, 1978). In P. carnivora, significant changes
in body proportion and an increase in the somatic basal bodies have been noticed.
Greater control of culture conditions of the ciliates (as may be obtained with an axe-
nic culture) would enable further discerning and understanding of this phenomenon
in P. carnivora.
FEEDING BEHAVIOR OF P. CARNIVOHA 309
ACKNOWLEDGMENTS
D. K. gratefully acknowledges the Schonbrunn Foundation for funding the re-
search performed at the Hebrew University of Jerusalem. The assistance of Eli Hatab,
M. Devorachek, and A. Nevo in photographing SEM pictures is greatly appreciated.
We are grateful to M. Levandowsky for reading the manuscript and making valuable
comments.
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INTRACELLULAR pH DECREASES DURING THE IN VITRO
INDUCTION OF THE ACROSOME REACTION IN
THE SPERM OF SICYONIA INGENTIS
FRED J. GRIFFIN, WALLIS H. CLARK JR., JOHN H. CROWE, AND LOIS M. CROWE
Department of Zoology, University of California, Davis, California 95616 and Bodega Marine
Laboratory, Bodega Bay, California 94923
ABSTRACT
Activation of the sperm of many invertebrate and some vertebrate species to un-
dergo an acrosome reaction is accompanied by an increase in intracellular pH (pH;).
In each of these instances the pHj of the unactivated cell is relatively low (6.9-7.4).
Unactivated sperm of the marine shrimp, Sicyonia ingentis, possess an elevated pHj
(8.5). Induction of the acrosome reaction (exocytosis of the acrosomal vesicle and
generation of an acrosomal filament) is accompanied by a decrease in pH, (7.8). Low
external pH elicits acrosomal filament formation in sperm that have undergone acro-
somal exocytosis, but does not induce exocytosis in unreacted sperm. The ionophore,
nigericin, enhances the percent of sperm that form filaments in low pH seawater (pH
< 8.0), but does not elicit filament formation at external pHs > 8.0. Valinomycin
induces filament formation in sperm that have undergone exocytosis over a wide
range of external pHs (5.75-8.5). The ability of valinomycin to induce filament for-
mation in the upper portion of this pH range (8.0) declines as the extracellular K+
concentration rises. These results demonstrate that the sperm of S. ingentis undergo
a pHj decrease as a result of the acrosome reaction and that the decrease is associated
with acrosomal filament formation. In addition, they also suggest that an efflux of K+
ions is connected to the pHi decrease.
INTRODUCTION
As a prerequisite to fertilization, most sperm must first undergo acrosomal alter-
ations, termed the acrosome reaction (Dan, 1952). Among the majority of inverte-
brate sperm and sperm of a few select vertebrates, the acrosome reaction (AR) is
composed of the exocytosis of the acrosomal vesicle and generation of an acrosomal
filament (reviewed by Dan, 1967; Austin, 1968). The AR occurs when a sperm con-
tacts an egg-derived inducer. The inducer, a component of one of the egg investments,
interacts with the plasma membrane of the sperm and initiates a series of ionic and
biochemical sperm-associated events that lead to, among other sperm-associated
changes, the AR (see Shapiro and Eddy, 1980; Lopo, 1983 for reviews). For example,
the ionic changes associated with the AR in the sperm of Strongylocentrotus purpura-
tus include: ( 1 ) an uptake of extracellular Ca++ which is thought to be involved in the
exocytosis of the acrosomal vesicle (Tilney et at, 1978; Shackman et at, 1981); (2) a
Na+ associated H+ efflux which is necessary for the polymerization of actin filaments
Received 29 May 1987; accepted 28 July 1987.
Abbreviations: ASW (artificial sea water), DMO ( l4C-dimethyloxazolidine 2,4-dione), EW (egg water),
pH, (intracellular pH), pH0 (extracellular pH).
Reprint requests to: W. H. Clark, P.O. Box 247, Bodega Bay, CA 94923.
311
312 F. J. GRIFFIN ET AL.
and thus, the formation of the acrosomal filament; and (3) a K+ efflux that leads to a
depolarization of the sperm membrane potential (Shackman el ai, 198 1 ). Although
not thoroughly documented, it appears that the ARs of many motile invertebrate
sperm involve the same ionic changes.
Unlike the sperm of most invertebrates, the sperm of the natantian decapod, Sicy-
onia ingentis, are nonmotile. These cells possess an anterior spike (contained within
an acrosomal vesicle), a subacrosome, and a posteriorly located main body which
houses the nucleus (Kleveet al, 1 980; Shigekawa and Clark, 1986). S. ingentis sperm
do not possess flagella and also lack organized mitochondria. Sperm are transferred
to female seminal receptacles during mating and stored until spawning, which may
occur several weeks to months later (Anderson el al., 1985). Thus, these sperm re-
main in an unactivated state for extended periods after transfer from the male. At
spawning, ova are released from paired ovopores and mixed with sperm ejected from
the seminal receptacles. Sperm bind spike first to ova and become activated to un-
dergo a biphasic AR (Clark el al., 1984). Within seconds bound sperm undergo the
first phase of the AR, acrosomal exocytosis (which includes the loss of the spike),
and 10-20 minutes later they complete the AR by generating an acrosomal filament
(second phase). Thus, in vivo the two phases of the AR are temporally separated.
Previous work demonstrates that the first phase, acrosomal exocytosis, depends upon
external Ca++ (Clark el al., 198 1 ) as is true in other systems. These experiments were
conducted with sperm taken from males, which are not competent to form acrosomal
filaments (Clark el al., 1984). The ionic requirements for acrosomal filament forma-
tion have not been investigated.
The ability to induce a complete AR in sperm removed from female seminal
receptacles and incubated in isolated egg products enabled us to investigate the ionic
requirements of the AR's second phase, acrosomal filament formation. The present
paper: ( 1 ) describes the in vitro induction of a complete AR using egg components;
(2) presents data suggesting that the sperm possess a high intracellular pH (pHj) prior
to undergoing the AR and that a pH, decrease is associated with the second phase of
the AR (formation of the acrosomal filament); and (3) provides data indicating that
the outward movement of K+ ions is involved in the pHj drop.
MATERIALS AND METHODS
Collection and maintenance of animals
Specimens ofSicyonia ingentis were collected using an otter-trawl in 60-100 me-
ters of water off San Pedro, California. Live animals were transported in chilled sea-
water (8-10°C) to the Bodega Marine Laboratory and maintained in flow-through
seawater tanks at ambient temperatures ( 10-14°C). Gravid females were isolated and
kept under constant light in a 500 gallon flow-through tank. The lights were turned off
to initiate spawning. Animals were monitored for spawning under a red light (Kodak
Wratten #2 filter).
Collection of egg water
Spawning animals were removed from the tank and held over 50 ml glass beakers
containing chilled (4°C) artificial seawater (ASW) prepared according to Cavanaugh
(1956). After the negatively buoyant ova (1-2.5 X 103) had settled to the bottom
of the beaker, approximately 3/4 of the seawater was drawn off. The ova were then
resuspended by swirling and kept in suspension for five minutes. The remaining ASW
A pH, DECREASE AT SPERM ACTIVATION 313
containing egg-derived components was then pipeted out of the beakers, cleared by
centrifugation (100,000 X g, 15 min), and divided into 1 ml aliquots. The protein
concentration of each egg water (EW) batch was determined after the method of
Lowry et al. (1951). EW was stored in liquid nitrogen if not used immediately.
Collection of sperm
In S. ingentis, only sperm that have been transferred to a female and stored in the
female's seminal receptacles are competent to: (1) undergo the acrosome reaction
(AR) in response to egg derived components; and (2) form an acrosomal filament as
part of the AR, regardless of the manner of induction (Clark et al., 1984). As a result,
only sperm taken from seminal receptacles of the female were used. Seminal recepta-
cles from ten or more females were pooled, homogenized in ASW using a Wheaton
5 ml tissue grinder to free sperm, and hand centrifuged to remove fragments of empty
receptacles. Free sperm were pelleted from the supernatant at 200 X g for five min-
utes. Pelleted sperm were resuspended in ASW and used within one hour of isolation.
Induction of the acrosome reaction with egg water
Isolated sperm ( 106 cells) were incubated in 1 ml of experimental (containing EW)
and control (containing ASW) solutions. Aliquots of cells in each experiment were
fixed (with a drop of 5% glutaraldehyde in ASW) at appropriate times and scored with
phase microscopy (400X) for: ( 1 ) percent unreacted; (2) percent that had undergone
acrosomal exocytosis but had not formed acrosomal filaments; and (3) percent fully
reacted (sperm which possessed acrosomal filaments). For each experimental run (n),
duplicate 20 n\ aliquots were removed and 100 sperm were scored in each for acroso-
mal status.
Intracellular pH determinations
Isolated sperm (2. 1 X 108) were divided into three equal samples. One sample was
incubated for 10 min in 1.6 ml of ASW (pH 8.0); this sample was used to measure
the pHj of unreacted sperm. A second sample was incubated in 1 .6 ml of EW (pH 7.8)
for 10 min; these sperm were used to measure the pHj of sperm that had undergone
acrosomal exocytosis. The last sample was incubated in 1 .6 ml of EW for 50 min;
these sperm were used to measure the pHj of fully reacted sperm. Intracellular pH
determinations were made with 14C-dimethyloxazolidine 2,4-dione (DMO) using a
modification of the technique described by Waddell and Butler ( 1 959). At the conclu-
sion of the initial incubations, each of the three samples was divided in half: ( 1 ) 25 /A
of 3H2O and 50 iA of 14C inulin were added to one; and (2) to the other, 25 n\ of 3H2O
inulin and 50 n\ of 14C DMO (final concentration of 33 ^M) were added. After a 20
min equilibration period: (1) triplicate 20 ^1 aliquots (controls) were transferred to
scintillation vials containing 15 ml of ACS scintillation fluid (Beckman); (2) 10 /A
samples were removed, fixed, and scored for acrosomal status; and (3) triplicate 200
/tl samples were microfuged (Fisher Model #235B) for 90 seconds through a 95 vol-
ume percent silicone oil (Dow Corning 704)-5 volume percent hexane solution, the
supernatants were removed, and the tips of the microfuge tubes (containing the sperm
pellets) were cut off and placed in scintillation fluid. The samples sat overnight and
were then counted on a Beckman LSI 00 scintillation counter. Calculation of internal
water space and pH{ determinations followed those described by Shackman et
al. (1981).
314 F. J. GRIFFIN ET AL.
pH induction experiments
Sperm (107/ml) were incubated for 5 min in either ASW pH 8.0 or EW pH 8.0
after which 100 ,ul aliquots were added to 900 ^1 of ASW at the following pHs: 5.75,
6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 9.5. Reactions were halted 10 min later with a
drop of 5% glutaraldehyde (in ASW) and acrosomal status was scored. In experiments
using sperm that had spontaneously undergone acrosomal exocytosis in ASW alone,
sperm were scored until 100 reacted cells had been observed in each duplicate. In
those using EW to induce acrosomal exocytosis, counts were performed as described
above. The pH of ASW was determined on an Orion (EA920) pH meter. Above pH
8.0, ASW was adjusted with 0.1-1.0 TV NaOH; below pH 8.0 it was adjusted with
0.1-1.0 A^HCl or 0.2 Macetate buffer. All ASWs were pH adjusted just prior to use.
lonophore induction experiments
In separate experiments, sperm (107/rnl) were induced to undergo acrosomal
exocytosis in EW as described above and 100 n\ aliquots were added to 900 jul of
ASW at the pHs described in the previous section. Immediately after the addition of
sperm, 5 ^1 of nigericin (0.5 mA/in 100% DMSO) or valinomycin (1.0 mMin 100%
DMSO) were added, with mixing, to the sperm suspensions. Control samples con-
tained 0.5% DMSO. Aliquots of each treatment were fixed with a drop of 5% glutaral-
dehyde (in ASW) 5 minutes after the addition of the ionophores or DMSO and sperm
were scored for acrosomal status. In addition, samples were removed and fixed to
determine levels of acrosomal exocytosis prior to introduction into pH ASWs and
the addition of the ionophores.
ASWs of different [K+] were obtained by adding or deleting equal molar amounts
of KC1 and NaCl from the MBL formula for ASW (Cavanaugh, 1956).
RESULTS
Induction of the acrosome reaction by egg water
Acrosomal status of S. ingentis sperm is easily scored with phase microscopy.
Figure 1 illustrates an unreacted sperm, a sperm that has undergone acrosomal
exocytosis, and a fully reacted sperm (possessing an acrosomal filament). Sperm re-
moved from the seminal receptacles of females and incubated in 50 ng/m\ (protein)
of egg water (EW) undergo a complete AR (acrosomal exocytosis and formation of
an acrosomal filament) in which the temporal separation between the two phases is
maintained. Within 1 min of exposure to EW, S. ingentis sperm underwent acroso-
mal exocytosis at levels greater than ASW controls (41% as compared to 7.3%) and
by 5 min, greater than 75% of the sperm had undergone acrosomal exocytosis (Fig.
2). These sperm (after a 1 5 min incubation in EW) had only undergone acrosomal
exocytosis; they did not possess acrosomal filaments. Sperm that had undergone acro-
somal exocytosis did begin to form filaments, commencing approximately 30 min
after introduction into EW (Fig. 3). At 45 min, more than 50% of all sperm counted
possessed acrosomal filaments; this translates into more than two-thirds of exocy-
tosed sperm possessing filaments. By 60 min, approximately 60% of all sperm (85%
of exocytosed sperm) had formed filaments. The sperm that formed acrosomal fila-
ments were those that had undergone acrosomal exocytosis. The total percent of
sperm that had either undergone only exocytosis or undergone a complete AR re-
mained constant through 60 min (Fig. 3).
A pH, DECREASE AT SPERM ACTIVATION
315
FIGURE 1. Phase micrographs of the three activational states of Sicyonia ingentis sperm; (A) an
unreacted sperm possessing an anterior spike, (B) a sperm that has undergone acrosomal exocytosis and
has lost the spike, and (C) a fully reacted sperm possessing an acrosomal filament.
A small but consistent number of sperm (8-10%) isolated in artificial seawater
(ASW), and not transferred to EW, underwent acrosomal exocytosis (Fig. 2). This
percent not only remained constant with increased incubation times, but exocytosis
was the only portion of the AR that occurred. Sperm isolated to ASW have been
observed for up to 1 80 min without seeing acrosomal filaments.
Intracellular pH measurements
Based on the accumulation ratios of the DMO uptake experiments we have calcu-
lated an average pH; for unreacted and reacted S. ingentis sperm. Unreacted sperm
100,
80-
CO
o 60
o
o
x 40 -
Ld ^U ~
20-
0
0
-4-
5 10
TIME (MIN)
15
FIGURE 2. EW induction of acrosomal exocytosis; response over time. Sperm were incubated in 50
^g/ml EW (O) or ASW (•), fixed at the times designated above, and scored for acrosomal status. Data
points are means; vertical lines are standard deviations (n = 4). Each replicate utilized a separate batch of
sperm and EW.
316
F. J. GRIFFIN ET AL.
t/2
in
o
i—
o
o
X
LJ
100-
80-
60-
40-
20-
0
0 -I — • — • — •
15 30
TIME (MIN)
45
r 100
-80
-60
-40
-20
0
60
m
z
—i
CO
FIGURE 3. Acrosomal filament formation as a function of time. Sperm were incubated in 50
of EW. Aliquots of sperm were removed, fixed, and scored for percent exocytosis (O) and percent formed
filaments (•) at each time point. Data points are the means of four replicates; each replicate was conducted
with different batches of sperm and EW. Vertical lines are standard deviations.
removed from seminal receptacles and placed in ASW (pH 8.0) possessed a pH; of
8.47 ± 0.27. In these samples, greater than 91% of the sperm were unreacted and
none of the reacted sperm possessed acrosomal filaments (Table I). Sperm incubated
in EW(pH 7.8) for 10 min prior to the addition of the DMO possessed a significantly
lower pHj of 7.81 ± 0.13 (P < 0.05). Greater than 74%. of these sperm had undergone
acrosomal exocytosis and approximately 3% possessed acrosomal filaments at the
end of the equilibration period (30 min after sperm had been introduced into EW).
The pH, of sperm incubated in EW for 50 min, 8.01 ± 0.06, was also lower than that
of unreacted sperm (P < 0.05), but was not significantly different from the 10 min
EW samples. Seventy-one percent of the sperm incubated in EW for 50 min possessed
filaments at the end of the equilibration period (70 min after introduction into EW).
TABLE I
of Sicyonia ingentis sperm
Sample
Exocytosed'
Filaments2
pH,
ASW
EW,o
EW50
8.3 ±2. 5
72. 3 ±3.1
6.0 ±2.0
0
2.7 ± 1.5
71.0 ±2.0
8.47 ±0.27
7.81 ±0.13
8.01 ±0.06
Isolated sperm were reacted in EW for 10 min (EW,0) and 50 min (EW50) and used to measure pH, of
sperm that had undergone only acrosomal exocytosis and sperm that had fully reacted, respectively. The
pH, of unreacted sperm (ASW) was measured after incubating isolated sperm for 10 min in ASW.
' Percent of sperm which had undergone acrosomal exocytosis only at the time of disruption.
2 Percent of sperm which had undergone a complete AR at the time of disruption.
A pH, DECREASE AT SPERM ACTIVATION 317
Effect of external pH on theAR
External pH (pH0), within the range examined, does not elicit the first phase of
the AR, acrosomal exocytosis, in S. ingentis sperm (Fig. 4 A). The percentages of
sperm that were unreacted after transfer to the pH ASWs did not vary significantly
with pH0. This was true for those sperm that had been preincubated in ASW alone,
as well as for those sperm that had been preincubated in EW. In those experiments
where sperm had been preincubated in EW, the percent that did not undergo acro-
somal exocytosis averaged 26. 1 for all pH0s with no observable pH-dependent trend.
The same held true for those sperm that were not exposed to EW (incubated in ASW
only); the percent of these sperm that did not react averaged 90.7 for all pH0 treat-
ments. Such was not the case with regard to acrosomal filament formation.
Exposure of sperm to low pH0 did elicit the formation of acrosomal filaments.
Sperm induced to undergo exocytosis with EW and subsequently transferred to pH0s
of less than 7 underwent filament formation within 10 min of transfer (Fig. 4B). This
represents a reduction of the temporal separation between the two phases of 20-35
min. The pH0 optimum for filament induction was between pH 5.75 and 6.5. Below
pH 5.75, exocytosed sperm were disrupted and above pH 7.0, sperm did not form
acrosomal filaments within the reduced temporal window. Similar results were ob-
tained with sperm that had spontaneously undergone exocytosis in ASW (not incu-
bated in EW). The percentages of these sperm that formed filaments after exposure
to low pH0 were somewhat less than the sperm treated with EW, but the effect of pH0
was similar (Fig. 4).
Induction of acrosomal filament formation by ionophores
The response of sperm to low pH0 was enhanced with the addition of either nigeri-
cin or valinomycin, however, the pH0 optima were different for both ionophores (Fig.
5). Greater than 50% of sperm that had undergone acrosomal exocytosis in EW and
were subsequently exposed to nigericin for 5 min underwent filament formation in
pH0 6.0-7.5. At pH0 5.75, 31.5 ± 8.2% of such sperm possessed filaments, however,
the number of unreacted sperm was twice that of the other pH0 treatments, suggesting
that at pH0 5.75 reacted sperm were disrupting (Fig. 5A). With the addition of nigeri-
cin, not only were the percentages of filament formations increased at low pH0s (6.0-
7.5), but the range for filament induction was shifted 0.5-1.0 units basic.
Valinomycin not only elicited more filament formations than any of the other
treatments, but it was also effective over a broader range of pH0s than the other treat-
ments (Fig. 5). Greater than 80% of exocytosed sperm underwent filament forma-
tion in pH0 5.75-8.0. At pH0s 9.0 and 9.5 the percentages of filaments were dramati-
cally reduced. As in low pH0 inductions in the absence of ionophores, there was no
observable effect on acrosomal exocytosis (Fig. 5A).
The ability of the ionophore valinomycin to induce formation of acrosomal fila-
ments was pH0 dependent when extracellular K+ was elevated (Fig. 6). Filament for-
mation in pH 6.0 ASW was not reduced by increasing concentrations of extracellular
K+, however, a steady decline in the percentage of filaments was observed in pH 8.0
ASW as the K+ level was increased. When the [K+] was increased from 10 mA/to 20
mM, filament formation at pH0 8.0 decreased by approximately 50%. At 30 mM K+
filament formation declined by another 50%, and at 40 mM K+ no filaments were
observed. In pH 9.0 ASW, filaments were only formed in low K+ (K+-free ASW).
318
F. J. GRIFFIN ET AL.
Q
LJ
I—
O
LJ
rr
I
o
100n
80-
60-
40
20
0
9 — °
8
10
CO
h-
LJ
100n
80-
60-
B
PH
FIGURE 4. Effects of external pH upon the AR. (A) Acrosomal exocytosis. (B) Acrosomal filament
formation. Sperm ( 107/ml) were incubated for 5 min in either ASW pH 8.0 (•) or EW pH 8.0 (O) after
which 100 ^1 aliquots were added to 900 ^1 of ASW at the pHs indicated above. Reactions were halted 10
min later with a drop of 5% glutaraldehyde (in ASW) and acrosomal status was scored. Data points in (B)
represent mean % of exocytosed sperm that formed filaments.
DISCUSSION
Induction of the two phases (acrosomal exocytosis and acrosomal filament forma-
tion) of the AR in S. ingentis sperm is temporally separated and sequential in vivo
(Clark et al., 1984). Upon binding to ova, sperm undergo acrosomal exocytosis and
some 10-20 min later undergo acrosomal filament formation. The present report has
demonstrated that the in vitro induction of this AR in sperm removed from female
A pH, DECREASE AT SPERM ACTIVATION
319
O
<
Ld
o:
I
z
o
80-
60-
40-
20-
0
8
10
CO
i—
z:
UJ
B
PH
FIGURE 5. The effects of pH0 on valinomycin and nigericin induction of the AR. (A) Acrosomal
exocytosis. (B) Acrosomal filament formation. Sperm (107/ml) were first induced to undergo acrosomal
exocytosis in EW, 100 n\ aliquots were added to 900 ^1 of ASW at the pHs indicated above, and then
exposed to either 1 /uA/ nigericin (O) or 5 nM valinomycin (•). Sperm were fixed after 5 min and scored for
reactions. Data points in (B) represent mean percent of exocytosed sperm that formed acrosomal filaments;
vertical lines are standard deviations, n = 3. Each n in each experimental batch represents sperm pooled
from different females, different ASWs, and different EW and nigericin solutions.
seminal receptacles and incubated in solutions containing isolated egg components
(EW) is also temporally separated and sequential. In vitro, acrosomal exocytosis is
achieved within 2.5-5 min, yet sperm that have undergone acrosomal exocytosis do
not form acrosomal filaments for an additional 30-45 min. Thus the temporal sepa-
ration that is observed on the surface of an ovum is preserved albeit lengthened under
320
F. J. GRIFFIN ET AL.
100-1
PH 6.0
20
K + (mM)
FIGURE 6. Acrosomal filament formation and external K+. Sperm (107/ml) were induced to un-
dergo acrosomal exocytosis in EW (5 min). One hundred (100) ^1 samples were then transferred to 900 n\
of ASWs containing from 0 to 40 mM K+ at pH0 6.0 (A), pH0 8.0 (•), and pH0 9.0 (A). Samples were fixed
at 5 min and scored for acrosomal status. Control samples (O) were preincubated in ASW (not exposed to
EW), added to ASW (pH 8.0) containing the described [K+], and exposed to 0.5% DMSO. Data points are
mean percent of exocytosed sperm that formed filaments (n = 3); vertical lines are standard deviations.
in vitro conditions. The ability to elicit a complete AR in vitro and the fact that the
two phases are separated has allowed the dissection of the two phases with respect to
the controls of activation. Based upon direct measurements of pHj, low pH0 induc-
tions of the AR, and the effects of both low pH0 and external [K+] on ionophore
inductions, we propose that formation of the acrosomal filament in 5". ingentis sperm
is associated with a pH, decrease.
Measurements of intracellular pH in S. ingentis sperm suggest that: ( 1 ) unreacted
sperm possess a high intracellular pH; (2) prior to formation of the acrosomal fila-
ment these cells undergo a pHj decrease; and (3) subsequent to filament formation
they do not return to the unactivated pH,. Although DMO is a widely used probe for
determining pHj, it does have limitations (Roos and Boron, 1981; Busa and Nucci-
telli, 1 984). These include: ( 1 ) DMO measurements reflect an average pH for the cell
and do not provide information on the pH of subcellular compartments (e.g., the
acrosomal vesicle or the subacrosome); and (2) alkaline membrane-bound organelles
can sequester DMO, giving an erroneous picture of the pH of other subcellular com-
partments (Roos and Boron, 1981; Busa and Nuccitelli, 1984). For example,
Grinstein el al. (1984) have reported that a DMO measured pHj increase at lympho-
cyte proliferation is in fact not an activational pHj change, but rather an increase in
the number of mitochondria (which results in an increased DMO uptake by the cells).
The structural organization and the direction of the measured pHj change in S.
ingentis sperm, however, allowed us to entertain the supposition that the pHj change
was real and was associated with filament formation. Unreacted S. ingentis sperm
possess three subcellular regions: a nucleus, a subacrosome, and an acrosomal vesicle;
A pH, DECREASE AT SPERM ACTIVATION 321
mature sperm do not possess mitochondria (Shigekawa and Clark, 1986). As a result,
any pH; changes would be expected to be associated with one of these compartments
and two of them are involved in the AR. We would not expect, a priori, an overall
pHj decrease to occur simply as a result of acrosomal exocytosis; the acrosomal vesicle
is an acidic organelle (Kleve et al, 1980) and therefore its loss at exocytosis might be
expected to yield an increase in average pH, . It was therefore reasonable to expect the
pHj changes to be associated with the subacrosome.
Results of the low pH0 induction experiments correlate well with the pH, measure-
ments and delineate at which phase of the AR the pH; drop occurs. Neither low
pH (<7.5) alone nor low pH in conjunction with nigericin or valinomycin induce
unreacted sperm to undergo acrosomal exocytosis. All three do induce acrosomal
filament formation in sperm that have undergone exocytosis. It follows that the pH;
decrease is associated with the second phase of the AR, formation of the acrosomal
filament. Furthermore, low pH0 elicits filament formation in sperm that have exocy-
tosed in ASW and have not been exposed to EW. This indicates that the pH0 is not
acting through a pH alteration of EW, rather, it is directly influencing filament forma-
tion. These observations are in contrast to previous studies demonstrating that a net
rise in pH; occurs during the AR in sperm of other species (Shackman et al., 1981;
Working and Meizel, 1983; Matsui et #/.,1986). By contrast with the sperm of S.
ingentis, these cells have been reported to possess depressed pH,s prior to activation.
For example, the pH; of unreacted S. piirpuratus sperm is between 6.6 and 7.3, based
upon measurements obtained with weak bases (Shackman et al., 1981). Using 9-
aminoacridine, the pH, of the sperm of the starfish Aster ias amurensis and A. pectini-
fera was reported to be 7.4-7.5 (Matsui et al., 1986). In the hamster sperm, the intra-
crosomal pH has been measured to <5, also using 9-aminoacridine (Meizel and
Deamer, 1978).
The ionophore nigericin exchanges K+ or Na+ for H+ (the selectivity for K+ over
Na+ is more than an order of magnitude), thus it is an electroneutral ionophore that
dissipates proton gradients across cell membranes (Pressman, 1976; Johnson and
Scarpa, 1976). As such, the pH, of sperm in the presence of nigericin should more
closely parallel the pH0 of the ASW than in the low pH0 experiments conducted
without ionophore. The results of the nigericin induction experiments agree well with
the measured pH; changes that occur during the AR. Based on the DMO measure-
ments, sperm decrease pHj from 8.5 to between 7.8-8.0 as a result of the AR. Nigeri-
cin elicits filament formation at pH0s 6.0-8.0 in sperm that have undergone
exocytosis. Since the pH0/pHj gradient at pH0s above 8.0 would not favor a nigericin
induced pHj decrease, filament formation would not be expected. Conversely, as the
pH0 is decreased, it would be expected that at some pH0 an acid overload in the
presence of nigericin would occur. This occurs between pH 5.75 and 6.0 in S. ingen-
tis sperm.
Valinomycin, like low pH ASW and nigericin, does not elicit acrosomal ex-
ocytosis, but will induce acrosomal filament formation. However, unlike the other
two, valinomycin is pH-independent over a wide pH0 range (pH 5.75-8.0) at normal
extracellular K+ concentrations (10 mA/). The ability of valinomycin to elicit fila-
ment formation does become sensitive to pH0 at elevated extracellular K+ concentra-
tions. In 10 mA/ K+ ASW filament formation proceeds at pH0 6.0 and 8.0; no fila-
ments are seen at pH0 9.0. As the [K+] is increased to 40 mM(in 10 mA/ increments),
filament formation declines approximately 50% at each incremental rise in [K+] in
pH 8.0 ASW. At pH0 9.0, filament formation is inhibited in [K+] > 10 mA/, however,
filament formation will proceed if the K+ concentration is below 10 mA/. Valinomy-
322 F. J. GRIFFIN ET AL.
tin transports only K+ (the selectivity over Na+ is greater than three orders of magni-
tude) across r ::mbranes and therefore is electrogenic (Johnson and Scarpa, 1976;
Pressman, 1 976). The results of the valinomycin/pH/K+ experiments suggest that the
ionophr re is facilitating a K+ efflux, however, they also suggest that the K+ efflux
does not in itself elicit filament formation. The fact that filament formation in pH
8.0 ASW is very sensitive to small changes in the extracellular K+ concentration leads
us to suggest that the pHj decrease elicits acrosomal filament formation. The iono-
phore facilitates a K+ efflux which results in an alteration of the sperm membrane
potential (hyperpolarization?) and this change in membrane potential drives a proton
influx.
This study has demonstrated that unactivated sperm of S. ingentis possess an
unusually high resting pHj, that they undergo a decrease in pH, as a result of the AR,
and that the pHj decrease is associated with formation of the acrosomal filament. The
pHj measurements and shifts that occur during the AR in S. ingentis sperm must be
viewed within the context of this unique system. These cells, after transfer to a female,
are stored for several weeks or more in exoskeletal seminal receptacles during which
time they undergo maturational and/or capacitational changes (Clark el ai, 1984).
During storage they are separated from the seawater (pH ca. 8.0-8.2) by only the
seminal plasm in which they are embedded. Thus, these cells probably maintain a
pH; in the same region as that found in their environment (seawater). At least two
possibilities arise that would functionally explain why these sperm possess such a high
unactivated pH,: ( 1 ) the energetic costs of maintaining an elevated pHs are less than
if pHj were depressed below physiological levels (ca. 7.0-8.0); or (2) since sperm un-
dergo maturational/capacitational changes while in the seminal receptacles of the
female, the elevated pH; might be associated with these processes (e.g., in the preven-
tion of premature filament formation). These, of course, are not all inclusive nor are
they mutually exclusive; rather, they are questions that await investigation.
ACKNOWLEDGMENTS
We thank C. Hand, J. Shenker, and R. Nuccitelli for their critical comments and
discussion. This work was supported by grants from Sea Grant (NA85AA-D-SG140
R/A-6 1 ) to WHC, and Sea Grant (NA85 AA-D-SG 1 40 R/A 62) and NSF (DMB85-
18194)toJHCandLMC.
LITERATURE CITED
ANDERSON, S. L., W. H. CLARK JR., AND E. S. CHANG. 1985. Multiple spawning and molt synchrony in
a free spawning shrimp (Sicyonia ingentis: Penaeoidea). Biol. Bull. 168: 377-394.
AUSTIN, C. R. 1968. Ultrastructure of Fertilization. Holt, Rinehart and Winston, New York. 145 pp.
BUSA, W. B., AND R. NUCCITELLI. 1984. Metabolic regulation via intracellular pH. Am. J. Phvsiol. 246:
R409-R438.
CAVANAUGH, G. M. ed. 1956. Formulae and Methods of the Marine Biological Laboratory Chemical
Room. Woods Hole. Pp. 55-56.
CLARK, W. H., JR., M. G. KLEVE, AND A. I. YUDIN. 1981. An acrosome reaction in natantian sperm. J.
Exp. Zool. 218:279-291.
CLARK, W. H., JR., A. I. YUDIN, F. J. GRIFFIN, AND K. SHIGEKAWA. 1984. The control of gamete activa-
tion and fertilization in the marine Penaeidae, Sicyonia ingentis. Pp. 459-472 in Advances in
Invertebrate Reproduction 3, W. Engels et a/., eds.
DAN, J. C. 1952. Studies on the acrosome reaction. I. Reaction to egg-water and other stimuli. Biol. Bull.
107: 54-66.
DAN, J. C. 1967. Acrosome reaction and lysins. Pp. 237-293 in Fertilization, Vol. 1, C. B. Metz and A.
Monroy, eds. Academic Press, New York.
A pH, DECREASE AT SPERM ACTIVATION 323
GRINSTEIN, S., S. COHEN, H. M. LEDERMAN, AND E. W. GELFAND. 1984. The intracellular pH of quies-
cent and proliferating human and rat thymmic lymphocytes. J. Cell. Physiol. 121: 87-95.
JOHNSON, R. G., AND A. SCARPA. 1976. Internal pH of isolated chromaffin vesicles. J. Biol. Chem. 251:
2189-2191.
KLEVE, M. G., A. I. YUDIN, AND W. H. CLARK JR. 1980. Fine structure of the unistellate sperm of the
shrimp, Sicyonia ingentis (Natantia). Tissue Cell 12: 29-49.
LOPO, A. C. 1983. Sperm-egg interactions in invertebrates. Pp. 269-324 in Mechanism and Control of
Fertilization, J. F. Hartmann, ed. Academic Press, New York.
LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with
the Folin-phenol reagent. J. Biol. Chem. 193: 265-275.
MEIZEL, S., AND D. W. DEAMER. 1978. The pH of the hamster sperm acrosome. J. Histochem. Cvtochem.
26:98-105.
MATSUI, T., I. NISHIYAMA, A. HINO, AND M. HOSHI. 1986. Intracellular pH changes of starfish sperm
upon the acrosome reaction. Dev. Growth Differ. 28: 359-368.
PRESSMAN, B. C. 1976. Biological applications of ionophores. Ann. Rev. Biochem. 45: 501-530.
Roos, A., AND W. F. BORON. 198 1 . Intracellular pH. Physiol. Rev. 61: 296-434.
SHACKMAN, R. W., R. CHRISTEN, AND B. M. SHAPIRO. 1981. Membrane potential depolarization and
increased intracellular pH accompany the acrosome reaction of sea urchin sperm. Proc. Natl.
Acad. Sci. 78: 6066-6070.
SHAPIRO, B. M., AND E. M. EDDY. 1980. When sperm meets egg: biochemical mechanisms of gamete
interaction. Int. Rev. Cytol. 66: 257-302.
SHIGEKAWA, K., AND W. H. CLARK JR. 1986. Spermiogenesis in the marine shrimp, Sicvonia ingentis.
Dev. Growth Differ. 28: 95-1 12.
TILNEY, L. G., D. P. KIEHART, C. SARDET, AND M. TiLNEY. 1978. Polymerization of actin iv . Role of
Ca++ and H+ in the assembly of actin and membrane fusion in the acrosome reaction of echino-
derm sperm. J. Cell Biol. 77: 536-550.
WADDELL, W. J., AND T. C. BUTLER. 1959. Calculation of intracellular pH from the distribution of 5,5-
dimethyl-2,4-oxazolidinedione (DMO). J. Clin. Invest. 38: 720-729.
WORKING, P. K., AND S. MEIZEL. 1983. Correlation of increased intracrosomal pH with the hamster
sperm acrosome reaction. J. E.\p. Zoo/. 227: 97-107.
Reference: Biol. Bull. 173: 324-334. (October, 1987)
A MORPHOLOGICAL EXAMINATION OF GASTRULATION IN A
MARINE ATHECATE HYDROZOAN
VICKI J. MARTIN
Department of Biological Sciences, University of Notre Dame. Notre Dame, Indiana 46556
ABSTRACT
The early embryonic development of the marine hydrozoan Halocordyl disticha
is examined via light histology and transmission electron microscopy. Particular em-
phasis is devoted to the gastrula and the mode of gastrulation. Cleavage in Halocordyl
disticha is irregular, total, and asynchronous resulting in the production of stereoblas-
tulae. Each stereoblastula forms a blastopore at the future posterior end of the larva
and gastrulates via invagination to produce a lecithotrophic planula larva. During
gastrulation spherical surface cells radially migrate toward the blastopore, become
cuboidal-shaped in the region of the pore, and disappear to the interior of the embryo.
Gastrulation requires 2 h to complete, during which time the ectoderm becomes sepa-
rated from the endoderm by a mesoglea, interstitial cells arise in the central endo-
derm, and the embryo elongates to form a planula larva. This study presents the first
documented example of invagination in the Hydrozoa.
INTRODUCTION
Cnidarians represent an early phase of metazoan evolution. Their simple architec-
ture combined with their exceptional morphogenetic plasticity and adaptability make
them popular animals for examining developmental processes and principles. The
phylum is unusual in that its postembryonic development has been more thoroughly
investigated than its embryogenesis. This is surprising because the cnidarians offer
excellent material for the study of the evolution of embryogenesis. In the simpler
cnidarians embryogenesis may appear "anarchic," whereas in the more advanced
forms one sees complex mosaic patterns of embryogenesis (Metschnikoff, 1886;
Carre, 1969).
Since the work of Metschnikoff in the late 1 80(Ts a few papers dealing with embry-
onic development of the Hydrozoa have been published (Van de Vyver, 1964, 1967,
1980; Bodo and Bouillon, 1968; Mergner, 1972; Freeman, 1981; Martin and Archer,
1986). Mergner (1972) attempted to provide a general overview of the processes in-
volved in cleavage, germ layer formation, and postembryonic development and con-
cluded that there was great diversity in hydrozoan developmental modes. Van de
Vyver (1980) analyzed via light histology modes of cleavage, germ layer formation,
and postembryonic development of several species of hydrozoans and concluded that
the modes of embryonic development in the Hydrozoa are restricted to a very few
which are commonly distributed in the animal kingdom. She stated that two types of
cleavage commonly occur in the Hydrozoa and that cleavage is dependent upon yolk
quantity of the egg. As a general rule, cleavage for eggs adequately supplied with
yolk is radial, total, and adequal. Such cleavage is characteristic of eggs of Filifera or
Received 21 April 1987; accepted 27 July 1987.
324
GASTRULATION IN A HYDROZOAN 325
Capitata corynoidea developing in a gonophore or spawned into the water by free-
swimming medusae. Large yolk-filled eggs such as those of Capitata tubularoidea
undergo irregular cleavage. Van de Vyver (1980) further concluded that the most
important difference between the types of cleavage in the hydrozoans is the presence
or absence of a blastocoele since its occurrence will determine the mode of germ
layer formation. MetschnikorT( 1886) proposed that eggs released by free-swimming
medusae form coeloblastulae while others developing inside gonophores form ste-
reoblastulae. Van de Vyver (1980) suggested that although Metschnikoffs first point
might be true, his second is certainly not. Furthermore, Van de Vyver (1980) stated
that within the Hydrozoa the processes of gastrulation are numerous and may vary
from species to species. Within the Hydrozoa gastrulation has been shown to occur
via either ingression, multipolar ingression, delamination, or simple cellular rear-
rangements (Jagersten, 1972; Tardent, 1978: Van de Vyver, 1980). Invagination has
not yet been reported in the Hydrozoa although it is common in anthozoans and
scyphozoans (Tardent, 1978; Van de Vyver, 1980). Van de Vyver (1980) reported
that polar ingression, multipolar ingression, and delamination are characteristic of
coeloblastulae, whereas in stereoblastulae the cells which occupy the periphery of the
embryo simply become progressively different from those situated in the center. She
claimed that no movements of cells occur in stereoblastulae.
From the above discussion it is quite clear that our basic knowledge concerning
embryonic morphogenesis in the cnidarians is sketchy and that additional studies of
embryogenesis in this phylum are needed. In this study the early development of a
marine athecate hydrozoan, Halocordyl disticha, is analyzed via light histology and
transmission electron microscopy. Particular emphasis is devoted to the gastrula. Ha-
locordyl disticha is a member of the suborder Capitata and forms free-swimming
medusae which release eggs and sperm into seawater where fertilization is external.
Cleavage is irregular, asynchronous, and total resulting in the formation of stereoblas-
tulae which gastrulate via invagination to produce lecithotrophic planula larvae. This
study presents the first documented example of invagination in the Hydrozoa.
MATERIALS AND METHODS
Mature colonies of the marine hydrozoan Halocordyl disticha were collected from
pier pilings at the University of North Carolina Institute of Marine Sciences in More-
head City, North Carolina. Fronds from mature male and female colonies were
placed together in large finger bowls of filtered seawater. Subsequently, the bowls
were placed in the dark at 6:00 pm and returned to the light at 9:00 pm. Within 1
hour after exposure to light early cleavage stages were found in the bottoms of the
dishes. These embryos were transferred to small finger bowls of filtered seawater and
reared at 23°C to the planula stage.
Early cleavage embryos, late cleavage embryos, blastulae, gastrulae, and young
planulae were prepared for either light histology or transmission electron microscopy.
Animals for light microscopy were fixed for 1 hour in 10% formalin in seawater,
dehydrated in an ethanol series, and embedded in Paraplast Plus paraffin. Serial sec-
tions, 10 nm thick, were mounted on glass slides and stained with either Azure B or
the SchifFs reagent (nucleal feulgen reaction). Live embryos and prepared histological
sections were photographed with a Zeiss standard research microscope. Embryos un-
dergoing cleavage and gastrulation were also continuously examined under the mi-
croscope until young planulae were formed.
Samples for electron microscopy were fixed for 1 h in 2.5% glutaraldehyde, pH
326 V. J. MARTIN
7.4, in 0.2 M phosphate buffer. They were postfixed for 1 h in 2% osmium tetroxide,
pH 7.2, in 1.25% sodium bicarbonate. Specimens were dehydrated in an ethanol
series, infiltrated, and embedded in Spurr's embedding media. Blocks were serially
secti. •..',' on a Porter-Blum MT-2B ultramicrotome, placed on 150-mesh copper
grid , and stained with 3.5% uranyl acetate in ethanol followed by lead hydroxide.
ids were examined and photographed with a Hitachi H-600 transmission electron
microscope.
Surface cells of live embryos at various stages of gastrulation were marked with
Nile Blue and subsequently monitored for their movement. The marking technique
involved using a 0.01% solution of Nile Blue in seawater. The dye was drawn into
microcapillary pipettes with varying bore diameters. Embryos were immobilized for
marking by placing them in a tiny groove in the bottom of a Falcon small plastic petri
dish. The dye-containing micropipettes were gently touched to the surfaces of the
embryos for 30-60 seconds producing small blue patches of marked cells of varying
diameter (depending upon pipette bore size) along the animal surface. Previous
marking studies using planulae indicate that Nile Blue is nontoxic at the 0.01% con-
centration and embryos stained with Nile Blue retain the dye for several days. The
dye will not diffuse into unstained tissue (Martin, unpub.). After marking the gastrula
cells, half of the animals were removed from the grooves and returned to small dishes
containing filtered seawater. The other half were left immobilized in the grooves and
their dishes placed in a moist chamber to prevent samples from drying. The marked
cells of the immobilized and free-moving animals were continuously examined
throughout gastrulation for change in axial position.
RESULTS
Cleavage in embryos ofHalocordyldisticha is holoblastic, unequal, and asynchro-
nous (Figs. 1-4). Cleavage begins 1 h after fertilization and results in the formation
of blastomeres of unequal size. A period of early cleavage extends to the beginning
of 6 h postfertilization during which time no one embryo cleaves in exactly the same
fashion. Such embryos assume numerous bizarre shapes and sizes and reach the 128-
256 cell stage (Martin and Archer, 1986). Early cleavage is rapid and by 6 to 8 h
postfertilization a stereoblastula (late cleavage) is formed (Figs. 3, 4). The stereoblas-
tula assumes the shape of a sphere and the blastomeres are more uniform in size than
during early cleavage. The stereoblastula is ca. 230 nm in diameter and consists of an
outer layer of small spherical blastomeres surrounding an inner layer of larger spheri-
cal blastomeres (Fig. 4).
By 8 h postfertilization the surface of the embryo is smooth and a single indenta-
tion appears at one pole of the embryo (Figs. 5, 6, 10-12). This indentation corre-
sponds to a blastopore, and the pole at which it forms marks the future posterior pole
of the planula (Figs. 7, 8, 13, 14). This stage represents gastrulation and the young
gastrula is ca. 250 p.m long and 190 ^m wide (Fig. 1 1). Gastrulation requires 2 hours
to complete and during this time a number of events occur (Figs. 5-20). The initial
blastopore indentation will deepen to form a groove (Figs. 5, 6, 10-12). Some of
the cells on the surface migrate in a radial fashion toward the deepening blastopore,
invaginate over the lips of the pore, and disappear to the inside. Such movement of
cells is easily visualized using the Nile Blue marking procedure. Marked patches of
blue cells move toward the blastopore, briefly inhabit the lips of the pore, and eventu-
ally disappear from the surface of the animal. Hence on a marked animal a blue patch
of cells can be traced from the surface to the blastopore region, and ultimately to the
GASTRULATION IN A HYDROZOAN
327
B-J
EN
B- • ?
{
4
m
B-
6
FIGURES 1 -9. Histological sections of developing embryos of Halocordyl disticha.
FIGURE 1 . Early cleavage embryo (3 h postfertilization) X200.
Early cleavage embryo (3 h postfertilization) X200.
Late cleavage 7-h embryo (stereoblastula). Note the absence of a mesoglea and interstitial
FIGURE 2.
FIGURE 3.
cells. X200.
FIGURE 4.
FIGURE 5.
Stereoblastula (7 h postfertilization) X200.
Early 8-h gastrula. An early blastopore (B) is visible. Separation of the two germ layers is
apparent and interstitial cells (arrow) appear in the central endoderm. E, ectoderm; EN, endoderm. X200.
FIGURE 6. Mid-gastrula stage. The blastopore (B) has deepened to form a groove. A mesoglea (black
arrow) is seen as are numerous interstitial cells (white arrow). X200.
FIGURE 7. Nine-hour gastrula which has begun to elongate. The blastopore (B) is located at the
posterior pole of the embryo and a mesoglea is present (black arrow). Cells migrating over the lips of the
blastopore to the inside are visible (white arrows). X200.
FIGURE 8. Elongating 9-h gastrula. Central endodermal interstitial cells (white arrow) are distin-
guishable from the outer endodermal gastrodermal cells (black arrow). B, blastopore. X200.
FIGURE 9. Ten-hour planula. The ectoderm is separated from the endoderm by an acellular meso-
glea (M). White arrow, interstitial cells; Black arrow, gastrodermal cells. X200.
328
V. J. MARTIN
10
11
12
13
15
FIGURES 10-15. Gastrulation in Halocordyl disticha.
FIGURE 10. Early 8-h gastrula with a slight indentation (blastopore) at the future posterior pole. X75.
FIGURE 1 1 . Mid-8-h gastrula with a deepening blastopore. x75.
FIGURE 1 2. Late 8-h gastrula with a prominent blastopore. Lips of the blastopore are visible and the
embryo has begun to elongate. X75.
FIGURE 1 3. Elongating 9-h gastrula. The embryo has a distinct anterior end and a posterior end. The
blastopore is visible at the posterior end. X75.
FIGURE 14. Late 9-10-h gastrula which has elongated. The blastopore is still visible at the posterior
pole. X75.
FIGURE 15. Ten-hour planula. The blastopore has completely closed producing a 2 germ layered
planula larva. X75.
GASTRULATION IN A HYDROZOAN
329
B
FIGURE 1 6. Longitudinal section through the blastopore region of an 8-h embryo. As cells move into
the region of the pore they change from a spherical shape to a cuboidal shape (arrows). Microvilli and cilia
from cells forming the lips of the blastopore project into the space of the pore (B). X4900.
animal interior. Time required for such patch movement (i.e., from the initial
marked surface position to the disappearance at the blastopore) varies anywhere from
1 5-30 minutes. The shape of the migrating cells changes as they move inward.
Examination of the blastopore region via transmission electron microscopy illus-
330
V. J. MARTIN
FIGURE 17. Enlargement of a portion of the blastopore region of an 8-h embryo. Cuboidal-shaped
cells form the lips of the pore. B, groove of the blastopore; N, nuclei of cells in the blastopore area. X5000.
trates the true nature of the indentation (Figs. 16-18). In the region of the blastopore
groove, spherical surface cells become cuboidal (Figs. 16, 17). Hence the cells forming
the lips of the blastopore are cuboidal. Such cuboidal-shaped cells possess cilia and
microvilli that project into the groove of the pore (Figs. 16-18). The cuboidal cells of
the blastopore eventually disappear to the interior of the embryo. As cells invaginate
a clear separation of the ectoderm and endoderm becomes visible with the formation
of an acellular mesoglea (Figs. 5-8).
During gastrulation there is localization of embryonic tissue types within the en-
doderm. The presumptive gastrodermal cells become distinguishable from the mes-
enchymal-like interstitial cells (Figs. 5-8, 19, 20). The interstitial cells appear as an
aggregate of cells in the central endodermal core of the embryo during invagination.
At this time some cytodifferentiation has begun since interstitial cells stain more
darkly with azure B than do the more peripheral gastrodermal cells (Fig. 8). In the
early gastrula (just prior to blastopore formation) the central blastomeres consist of
large yolk-filled masses (Fig. 19). Such blastomeres appear to be loosely packed in
the center of the embryo as indicated by the large intercellular spaces between the
blastomeres (Fig. 19). At this stage interstitial cells are not yet present. Once invagina-
tion begins the loose arrangement of the central blastomeres is lost (intercellular
spaces disappear) and clusters of small round interstitial cells appear in the center of
the embryo (Fig. 20). Such interstitial cells become clearly segregated from the outer
forming columnar gastrodermal cells.
Between 8 and 10 h postfertilization the gastrula elongates in an anterior-posterior
GASTRULATION IN A HYDROZOAN
33
FIGURE 18. Cross-section through the blastopore region of an 8-h embryo. Microvilli and cilia of
migrating cells extend into the space of the blastopore (B). N, nucleus of cell in region of the blastopore.
X6700.
direction to form a young planula (Figs. 7-9, 12-15). The 10-h planula is ca. 350
long, 180 /urn wide in the anterior region, 170 ^m wide in the mid region, and 120
^um wide in the tail (Fig. 15). By 10 h the planula has a distinct anterior end and
posterior end. The blastopore is located at the posterior pole of the planula and will
soon close (Figs. 14, 15). No gastrovascular cavity or mouth is found in the planula
at any stage of its development. The 10-h planula will elongate to form a mature
planula (24-96 h old depending on temperature) which will attach via its anterior
end to a substrate and undergo metamorphosis.
.
DISCUSSION
Within the Cnidaria the processes of gastrulation are numerous and vary widely
from species to species (Tardent, 1 978). Among the anthozoans gastrulation has been
reported to occur via either invagination, delamination, multipolar ingression, or a
combination of invagination and polar ingression (Tardent, 1978). In scyphozoans
gastrulation may occur via invagination, polar ingression, multipolar ingression, or
invagination plus polar immigration (Tardent, 1978). In hydrozoans examples of
gastrulation by polar ingression, multipolar ingression, and delamination have been
reported. However, until now no examples of invagination have been documented
(Jagersten, 1972; Tardent, 1978).
Jagersten (1972) provided a brief overview of gastrulation in the cnidarians and
stated that within the phylum a connection existed between the mode of gastrulation
and whether the formed larva was lecithotrophic or planktotrophic. In species which
gastrulate via either delamination, multipolar ingression, or unipolar ingression, the
332
V. J. MARTIN
FIGURE 1 9. Central inner blastomeres of an early 8-h embryo. These central endoblast cells are filled
with yolk and are separated from each other by large intercellular spaces. No distinguishable interstitial
cells are yet present. N, nucleus of central endoblast cell. X4000.
FIGURE 20. Central endoblast region of a 9-h embryo. Clusters of young interstitial cells are visible.
As the interstitial cells increase in number the intercellular space decreases and the central region of the
embryo assumes a more compact appearance. The interstitial cells are completely set apart from the outer
gastrodermal cells during gastrulation. X4000.
GASTRULATION IN A HYDROZOAN 333
derived larvae exhibit lecithotrophy and never planktotrophy. In species which pro-
duce actively feeding larvae (planktotrophic) the mode of gastrulation is via invagina-
tion. Jagersten (1955, 1959) presented arguments supporting the ideas that the origi-
nal method of gastrulation in the cnidarians was via invagination, that the plankto-
trophic larval life was the primitive condition, and that lecithotrophy was a secondary
trait which arose independently on different occasions within the phylum. Further-
more he stated that lecithotrophy is dominant among the Cnidaria. Jagersten (1972)
and Widersten (1968) proposed that the most primitive features of the phylum are
found within anthozoans and the most altered within the hydrozoans. Jagersten
(1972) further said that lecithotrophy may occur in larvae which exhibit invagination
(e.g., PachycerinatKus). Despite the moderate quantity of yolk in the eggs of these
animals, invagination persists.
This study documents the occurrence of invagination in the Hydrozoa. Embryos
ofHalocordyl disticha form stereoblastulae which gastrulate via invagination to pro-
duce lecithotrophic planula larvae. Marking studies clearly indicate that surface cells
migrate to the blastopore, occupy the lips of the pore, and eventually disappear to the
interior of the gastrula. Neither a mouth nor a gastrovascular cavity form in these
planulae. The absence of a mouth in cnidarian embryos which gastrulate via invagi-
nation is not uncommon, as examples also exist among the scyphozoans (Amelia,
Cyanea) (Jagersten, 1972).
Jagersten (1972) claimed that the common ancestor of the Metazoa included a
Gastrea form, a creature with both an alimentary cavity and a mouth. He proposed
that the almost universal distribution of the invagination gastrula was conclusive evi-
dence for the Gastrea theory. The hydrozoans can now be added to this universal list
as invagination gastrulae are found within this class. Furthermore, if Widersten
(1968) and Jagersten (1972) are correct in their assumptions that invagination is the
primitive condition within the Cnidaria and that the anthozoans are the more primi-
tive class, then the invagination process as described in this paper for a marine hydro-
zoan may illustrate a stubborn retention of this original primitive condition. Clearly,
further investigations of early embryogenesis in the Hydrozoa concentrating on
modes of gastrulation are needed to complement the work presented for Halocordyl
disticha.
ACKNOWLEDGMENTS
I thank Margaret Martin for her help in collecting animals and William Archer
for his technical assistance. This research was supported in part by a grant from the
National Science Foundation, DCB-8702212.
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ionemus vertens Aqassiz. Cah. Biol. Afar. 9: 69-104.
CARRE, D. 1969. Etude du developpement larvaire de Sphaeronectes gracilis (Claus. 1873) et de Sphaero-
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JAGERSTEN, G. 1955. On the early phylogeny of the Metazoa. The Bilaterogastrea-theory. Zool. Bidr.
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JAGERSTEN, G. 1959. Further remarks on the early phylogeny of the Metazoa. Zool. Bidr. Upps. 33: 79-
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JAGERSTEN, G. 1972. Cnidaria. Pp. 13-22 in Evolution of the Metazoan Life Cycle A Comprehensive
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MARTIN, V., AND W. ARCHER. 1986. A scanning electron microscopic study of embryonic development
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Reference: Biol. Bull. 173: 335-344. (October, 1987)
VARIABILITY IN THE PATTERN OF SEXUAL REPRODUCTION OF
THE CORAL STYLOPHOR.4 PISTILLATA AT EILAT, RED SEA:
A LONG-TERM STUDY
B. RINKEVICH* AND Y. LOYA
Department of Zoology. The George S. Wise Faculty of Life Sciences, Tel Aviv University,
Ramat Aviv 69978, Israel
ABSTRACT
Sexual reproduction of the Red Sea coral Stylophora pistillata was followed at
Eilat in a long-term study (1974-1984). Field examination of over 9000 colonies
through 1 19 months indicated that S. pistillata had a reproductive season of approxi-
mately 8 months (varying from 6 to 9 months). Premature planulae and eggs were
aborted following winter storms, resulting in a lowering of the planular index and the
number of female gonads per polyp. Histological examinations of tissue from 20 large
colonies which were studied for several years, until they were found dead in situ,
indicated that either sexuality (reproductive states) and/or fecundity could be com-
pletely altered from one reproductive season to the next: i.e., hermaphroditic colonies
exhibiting high fecundity in one season became male or even sterile thereafter, and
vice versa. In addition, great variability in reproduction between successive years was
recorded in sexuality and in the fecundity of shallow water populations. Shallow wa-
ter colonies (5 m) possessed up to 5 times more female gonads per polyp and shed 5
to 20 times more planulae than deep water colonies (25 to 45 m) in which the repro-
ductive season is 2 to 3 months shorter.
We suggest that the changes in the hermaphroditic, male, or sterile modes of re-
production in S. pistillata are from energy limitations and stress conditions. Since
reproductive activity probably involves significant energetic expenditures, any stress
or diminution in energy resources affects sexuality or fecundity. This should be con-
sidered before formulating any general hypothesis on coral reproduction.
INTRODUCTION
Much information concerning reproductive biology of scleractinian corals has
recently become available. Fadlallah (1983) reviewed past studies and provided a list
of almost 90 species in which several known reproductive characteristics are pre-
sented. More recent studies (Harriott, 1983; Szmant-Froelich et ai. 1984; Shlesinger
and Loya, 1985; Wallace, 1985; Willis et ai., 1985; Babcock et al, 1986; Szmant,
1986) provide information on reproductive patterns of more than 100 additional
species of corals.
Although this list of studied species is impressive, data on scleractinian reproduc-
tion is still scanty, especially that dealing with their reproductive ecology. These stud-
ies evaluated sizes, shapes, and numbers of gonads, and attempted to establish repro-
ductive seasonality, lunar periodicity, mode of reproduction, planula characteriza-
tions, and behavior. However, most studies were based on observations and
Received 30 January 1987; accepted 31 July 1987.
* Present Address: Hopkins Marine Station of Stanford University, Pacific Grove, California 93950.
335
336 B. RINKEVICH AND Y. LOYA
experiments carried out within a period of a year or less. Only a few studies dealt
with longer periods ranging from two (Atoda, 1947a, b; Harriott, 1983; Jokiel, 1985;
Wallace. ' 985 ) to three years (Kojis and Quinn, 198 la; van Moorsel, 1983; Stoddard
and Black, 1985). Consequently, studies on sexual reproduction often fall short in
documenting many aspects of coral reproduction (Fadlallah, 1983). Detailed infor-
mation on coral reproduction could clarify many aspects of their life history patterns
and provide a better understanding of the coral reef as a whole.
Stylophora pistillata (Esper) is one of the most abundant coral species in the Gulf
of Eilat, Red Sea. Some aspects of the reproduction of this species have already been
studied in the field and the laboratory. Descriptions of planulae and gonads have
been made (Rinkevich and Loya, 1979a). In addition, synchronization in breeding,
colony size in relation to fecundity, onset of reproduction, reproduction within a
single colony, and seasonality of planulation were also reported (Rinkevich and Loya,
1979b). This paper summarizes results of a ten-year study on the reproduction of S.
pistillata which elucidate some general conclusions characterizing coral reproductive
activities.
MATERIALS AND METHODS
Reproductive activity of S. pistillata was studied from March 1974 to January
1984 (most intensively from 1976 to 1980). The study area was located in front of
the H. Steinitz, Marine Biological Laboratory at Eilat, Gulf of Eilat, Red Sea, and
was visited regularly once a month during the ten-year study period. Large colonies
(mean geometric radius, 7 > 20 cm) were sampled from both shallow (3-5 m) and
deep water (25-60 m) populations.
Reproduction was studied by two techniques: collections of shed planulae (see
below) and examinations of gonads in histological sections (Rinkevich and Loya,
1979a, b). A single branch was sampled from each colony. This branch represents the
reproductive state of the entire colony (Rinkevich and Loya, 1979b). The number of
female gonads was counted within serial sections for each tested polyp and quantita-
tive data were obtained on the average number of eggs per polyp in a given specimen
(6-18 polyps per sample). Male gonads were not counted because of the difficulty of
following them in serial histological sections as a result of the irregular shape of a
typical male gonad (Rinkevich and Loya, 1979a). Tissue samples were always taken
near the bases of branches since few polyps from the tips contain genital cells (Rinkev-
ich and Loya, 1979b; Kojis and Quinn, 198 la).
Early in the study, planulae were collected in situ by covering large colonies with
plankton nets in the late afternoon and removing the nets at midnight (Rinkevich
and Loya, 1979a). However, due to the difficulties with this technique during night
diving (especially with the deep-water colonies), planulae were collected from coral
branches that were brought into the laboratory. The branches were carefully removed
underwater using wire cutters, and transported to the laboratory within 30 min after
sampling in closed, separate plastic bags. The water in each bag was checked for the
appearance of planulae. Each sample was put separately in a 5 1 glass aquarium, con-
taining filtered seawater, and left overnight. Planulae were shed during the night (Rin-
kevich and Loya, 1 979a). Although handling stimulated planula release, it is assumed
that these planulae were prepared for shedding. This assumption was supported by
the finding that the released planulae were fully developed. Since conditions in all
treated samples were similar, we concluded that collection procedures did not affect
the results. Planulae were counted by sight and removed by pipettes. The seawater
was then filtered through a plankton net (100 ^m) and all remaining planulae were
VARIABILITY IN SEXUAL REPRODUCTION OF A CORAL
337
TABLE I
Some characterizations of recorded southern storms in Eilat
Waves
Winds
Date
No. of
storms
Max. height
(m)
Max. length
(m)
General
direction
Max. speed
(km/h)
Feb. 1979
3
2
12
SE
nd
Apr. 1979
3
1 +
nd
S
nd
Nov. 1979
1
1
6
SE
20
Dec. 1979
2
2
nd
SE
nd
Feb. 1980
2
2.5
10
SSE
35
Mar. 1980
1
nd
nd
nd
nd
Apr. 1980
1
nd
nd
nd
nd
Jan. 1981
2
1.5
20
S, SE, SW
25
Feb. 1981
3
1.2
27
SSE
18
Mar. 1981
1
0.5
nd
S.SE
nd
nd = no available data. (Personal communication, C. Porter, Israel Oceanographic and Limnological
Research Ltd., Eilat).
collected and counted. Sampled branches were placed on a filter paper for 15 min to
remove excess water and weighed (accuracy to the nearest 1 g). In most cases branch
weights ranged between 100-200 g. Results are presented as number of released plan-
ulae per 100 g of coral skeleton, during one night.
In addition, the release of planulae from mature colonies ( r > 20 cm) was checked
each month in situ where several branches were carefully broken from many colonies.
This procedures stimulated the release of planulae in colonies which were in a repro-
ductive state. The shed planulae were easily seen and traced by sight underwater. A
planular index was then formulated (see below), which took into account the relative
number of planulae shed and the percentage of reproducing colonies. Since variabil-
ity in the fecundity between different colonies within the population was high (Rin-
kevich and Loya, 1979b), up to 90 large colonies were sampled each month (in 2-3
replicates, at least 30 colonies in each) to assess the validity of the planular index. The
index sign (-) was given when none of the sampled colonies released any planulae;
(H — ) when very few planulae were released (total number of 1-5 planulae from the
30 tested colonies in each replicate); (+) when about one third of the colonies released
few planulae; (++) when up to two thirds of the colonies shed planulae (many or few;
many = any small fragment broken from the colony released about two planulae);
(+++) when most or all of the colonies shed planulae.
The most severe storms in the sea at Eilat are known as southern storms, which
occur during the winter and spring. Some physical parameters of these storms are
partly documented from February 1979 (Table I).
RESULTS
Long-term study on seasonally of planulae shedding
Plantation in S. pistillata was continuously studied between March 1974 to Jan-
uary 1 984 by sampling more than 9000 colonies (Table II). The two questions investi-
gated were whether plantation occurs in the same months from one year to the next
and how the planular index in the shallow water population fluctuated during the
338 B. RINKEVICH AND Y. LOYA
TABLE II
Monthly planular index in shallow water populations of Stylophora pistillata
during 119 months of observations
Planular index* in
Year Jan. Feb. Mar. Apr. May Jun. Jul. Aug. Sep. Oct. Nov. Dec.
1974
1975
1976
1977
1978
1979
1980
1981
1982
1983
1984
* (-), No planulae; (H — ), very few; ( + ), few; ( + + ), intermediate; (+++), large numbers.
119 months of observations. S. pistillata has a long reproductive season (planulae
release) lasting approximately 8 months, from December to July (Table II). However,
the reproductive season ranged 6 months (in 1976) to 9 months (in 1975). In the
three-month period from August to October, no planulation was ever recorded. Only
once during the ten-year investigation were very few planulae observed in November
(in 1975). A marked variation was noted in the December-January-February index
between different years. Although these months represent the beginning of the repro-
ductive season (Rinkevich and Loya, 1979b), this variability might also be the result
of the southern storms which are most severe during the winter (Table I). This phe-
nomenon is also demonstrated in another part of the present study: in April 1980 a
southern storm interrupted our field sampling. Branch samples were collected before
the storm from 10 mature colonies. Nine of them released high numbers of planulae
(average of 30 ± 28 planulae, per 100 g skeleton, per colony). One day after the storm
samples were collected for histological study from 1 3 other mature colonies inhabit-
ing the same area and depth. Only eight colonies contained low numbers of female
gonads while the others were either sterile or contained only male gonads. The aver-
age number of female gonads per polyp, per colony was very low (0.4 ± 0.6), much
lower than other April months (for more detail, see Tables VI and IV, respectively).
Long-term study on reproductive states
Two separate sets of experiments followed the long-term state of reproduction in
shallow water populations. In the first experiment 20 large colonies (7 > 20 cm) were
chosen (December 1 976) and sampled for histological study two to three times a year
e.g., in the beginning, the peak and the end of the reproductive season over four
successive reproductive periods, until the deaths of all of them were recorded (Febru-
ary 1980). Since synchronization in the reproduction activity exists between branches
(Rinkevich and Loya, 1979b), only one branch was sampled each time from each
colony. This sampling procedure did not affect survivorship or reproduction (un-
pub.). Colony mortality was high (Table III), although colonies were carefully chosen
on the basis of their healthy state (without dead branches or tissue damage). One and
VARIABILITY IN SEXUAL REPRODUCTION OF A CORAL 339
TABLE III
Reproductive state and average number of female gonads per polyp in Stylophora pistillata
colonies sampled during Dec. 1976 to Feb. 1980
Average numbers of female gonads per polyp in
Coral
no. Dec. 76 Apr. 77** Dec. 77 Apr. 78 Jun. 78 Dec. 78 Apr. 79 Dec. 79 Feb. 80
1
-(10)
0.7(10)*
D
2
0.2(10)
0.5(10)*
D
3
0.6(10)
2.9(10)*
1.2(6)
2.6(7)*
0.7(6)*
-(10) D
4
2.4(10)
1.4(10)*
0.2(6)
+(10)
D
5
0.4(10)
0.4(10)*
D
6
0.1 (10)
1.9(10)*
+(6)
-(10)
-(10)
-(8) D
7
1.9(10)
1.1 (10)*
0.5(6)
D
8
-(10)
D
9
0.6(10)
1.1 (10)*
0.7(7)
2.2(6)*
S
10
-(10)
0.4(10)*
+(6)
+(6)
-(8)
1.4(7) 0.4(7) -(9) D
11
-(10)
D
12
-(10)
D
13
-(10)
2.5(10)*
2.1(10)
2.5(10)*
-(6)
1.8(6) 1.0(8) D
14
-(10)
0.5(10)*
-(5)
1-9(11)
-(10)
-(10) -(10) D
15
-(10)
1.6(10)*
1.0(5)
S
16
1.1(10)
1.3(10)*
2.3(10)
S
17
-(10)
D
18
-(10)
0.4(10)*
D
19
-(10)
0.9(10)*
-(11)
3.3(7)
S
20
0.2(10)
0.9(10)*
+(7)
2.1(8)
-(10)
1.2(6) D
December and February months refer to the beginning of the reproductive season, April months to
the peak of reproduction, and June to the decline phase of the reproductive season (numbers in parentheses
refer to the number of polyps examined).
-, Sterile colony; +, Only male gonads present; D, The colony was found dead; *, Planulae detected
in histological sections; **, Planulae were found in all plankton nets put on marked colonies; S, Destroyed
by storm.
two years after the beginning of the study, 60% and 30%, respectively, of the colonies
were alive. Only one colony of the 20 samples (5%) remained alive after 3 years (Table
III). A decrease in fecundity was repeatedly observed several months prior to the
natural death of many of the colonies. In four out of the six dead colonies following
a period of high fecundity (colonies 9, 15, 16, 19; Table III), the death was attributed
to southern storms. None of the dead colonies during the first 4 months of the study
(colonies 8, 11, 12, 17; Table III) contained any genital cells when first sampled.
The results (Table III) also indicate variability in sexuality (reproductive states:
male, hermaphrodite, or sterile modes of reproduction) and fecundity of a specific
colony in different years. Hermaphroditic colonies which exhibit high fecundity in
one reproductive season may differ in the following reproductive season in which
they become sterile (colonies 6, 14; Table III), or male (colonies 4, 10; Table III) and
vice versa. The changes in colony reproductive patterns are further demonstrated in
colonies sampled in three to four consecutive December months (colonies 3, 6, 10,
13, 14, 20; Table III). Sexuality or fecundity of five out of these six colonies were
altered in each December month.
In the second set of experiments (Table IV), 1 55 large shallow water colonies were
sampled over 10 successive sampling periods at the beginning and during the peak of
5 reproductive seasons (April 1 976-April 1 980). Changes among the different seasons
340
B. RINKEVICH AND Y. LOYA
Stylophora
TABLE IV
pistillata: reproductive states of shallow water populations
Colony reproductive state (%)
A f 1 J
No. of
Average temale gonads
Date
colonies
Hermaphrodites
Males only
Sterile
(polyp"1 colony"')
Apr. 76
17
94
6
1.5 ±0.9
Dec. 76
20
45
—
55
0.4 ±0.7
Apr. 77
16
100
—
—
1.2 ±0.8
Dec. 77
12
67
25
8
0.7 ±0.8
Apr. 78
9
67
22
11
1.6± 1.3
Jun. 78
16
31
—
69
0.2 ±0.4
Dec. 78
14
50
—
50
0.5 + 0.6
Mar. 79
26
88
—
12
1.5± 1.1
Dec. 79
12
50
25
25
0.5 + 0.7
Apr. 80
13
62
—
38
0.4 ± 0.6
either in sexuality or fecundity were observed. For example, each one of the 4 differ-
ent December months (years 1976, 1977, 1978, 1979) represented different patterns
of reproductive states: 8-56% of the colonies were sterile, 0-25% males and 45-67%
were hermaphrodites among the different December months. The same pattern was
recorded for sexuality of March to April months: 0-38%, 0-22% and 62-100%, re-
spectively (Table IV). It is concluded that "one year of sampling" is not enough for
the characterization of reproductive states in this species.
Reproduction in .shallow versus deep water populations
Possible differences in reproduction between shallow and deep water populations
were tested in two sets of experiments. In the first, we analyzed serial histological
sections of 90 shallow water colonies (from Table IV). The results were compared to
those of 77 deep water colonies (25-45 m, Table V) sampled on the same days
during three successive reproductive seasons (April 1978-April 1980). Shallow water
colonies possessed up to 5 times more female gonads per polyp per colony than deep
water colonies (P < 0.01, Wilcoxon's signed rank test; Sokal and Rohlf, 1981). This
phenomenon was most clear during the peak of the reproductive season, March to
Stylophora pistillata:
TABLE V
reproductive states of deep water colonies
Colony reproductive state (%)
Depth
No. of
Average temale gonads
Date
(m)
colonies
Hermaphrodites
Males only
Sterile
(polyp"1 colony"1)
Mar. 78
60
1
100
0.7
Apr. 78
25-30
15
40
40
20
0.3 ±0.4
Jun. 78
25
15
7
—
93
0.0 ±0
Dec. 78
27-30
15
86
7
7
0.7 ±0.6
Mar. 79
40-45
11
36
36
28
0.4 ±0.8
Dec. 79
27-30
9
—
—
100
0
Apr. 80
25-30
12
8
50
42
0.0 ±0.1
VARIABILITY IN SEXUAL REPRODUCTION OF A CORAL 341
TABLE VI
Shedplanulae in sample branches of shallow and deep water Stylophora pistillata colonies
Shallow
Deep
Colonies
which
Average
no. of
Colonies
which
Average
no. of
shed
planulae
shed
planulae
Depth
No. of
planulae
(per 100 g
Depth
No. of
planulae
(per 100 g
Date
(m)
colonies
(%)
skeleton)
(m)
colonies
(%)
skeleton)
Jan. 79
3-6
5
80
32 ±49
27
6
0
0
Feb. 79
3-6
6
100
85 ±95
27-30
4
25
1 ± 1
Mar. 79
5
6
100
22 ±20
40-42
5
20
0.4 ± 1
Jun. 79
5
4
100
31 ±22
34
5
20
2 ± 5
Dec. 79
5
4
0
0
30
3
0
0
Jan. 80
3-5
9
89
14±31
39-42
9
33
4 ± 10
Feb. 80
5
6
83
4± 2
30
6
0
0
Apr. 80
3-6
10
90
30 ±28
25-27
9
44
3 ± 6
May 80
3-8
10
90
44 ±47
27
10
70
4 ± 7
Jun. 80
6-9
10
100
14 ± 10
27-30
10
30
3 ± 7
Jul. 80
4-6
10
70
3± 6
30
10
20
0.2+ 0.4
Jan. 81
3-5
5
60
4± 8
30-35
5
0
0
Feb. 81
3-5
6
67
5± 3
27-30
6
33
1 ± 1
April (the average female gonad per polyp in April 1980 is lower than other April
months because samples were taken immediately after a storm).
In the second set of experiments, planulae were collected in the laboratory from
branch samples of 91 shallow water and 88 deep water colonies, on 13 collecting
dates (Table VI). Significantly more planulae were shed by shallow water colonies
than by deep water colonies (P < 0.0 1 ; Wilcoxon's signed rank test; Sokal and Rohlf,
198 1 ). At the peak of the reproductive season about 20-80 planulae on average were
shed per 100 g skeleton from shallow water colonies during one night. In deep water
colonies the number did not exceed four planulae. An additional 55 deep water colo-
nies were sampled during summer and fall (July-November) to examine whether the
reproductive season there differs from that of shallow water populations. All histolog-
ical sections were free of eggs. Moreover, no planulae were shed during a parallel
study where branches were carefully broken in situ from an additional 80 colonies.
These results indicate that the reproductive season of deep water colonies is probably
two to three months shorter than that of shallow water populations.
DISCUSSION
The study of coral reproductive biology may be engaged with ambiguous defini-
tions which could lead to wrong interpretations. For example, Fadlallah (1983) indi-
cated that confusion arises from the applications of the term hermaphroditism, which
describe two different life history processes: ( 1 ) development of monoecy over the
lifetime of a specimen and, (2) sequential maturation of female and male products
within one breeding period. Thus, it was accepted that 5". pistillata (Rinkevich and
Loya, 1979a, b) and Goniastrea australensis (Kojis and Quinn, 198 la, b) were prot-
androus hermaphrodites over their lifetime, but protogynous hermaphrodites in each
single reproductive season. The present study indicates that either sexuality and/or
fecundity may be completely altered from one reproductive season to the next. Her-
342 B. RINKEVICH AND Y. LOYA
nfaphroditic colonies which exhibited high fecundity in one season became male or
even sterile thereafter, and vice versa. Small colonies (geometric mean radius 7 < 2
cm) which invest much energy in rapid growth (Loya, 1985), possess only male go-
nads ir their hrst year of reproduction. An increase in colony size correlated with an
increa:,., in percentage of hermaphroditic colonies within the population (Rinkevich
and Loya, 1979b). Reproduction of injured colonies of S. pistillata which invested
energy in growth and regeneration was significantly reduced for at least two successive
reproductive seasons after the fracturing event (Rinkevich, 1982). In addition, the
fecundity of dying colonies was reduced several months before their death (Table III
and Rinkevich and Loya, 1986), and dying colonies often changed their sexuality
before their mortality and became male. Field experiments also demonstrated that
the number of female gonads per polyp in S. pistillata was significantly reduced in
colonies competing intraspecifically and the typical synchrony in reproduction
among different branches of a given colony was changed and disynchronized (Rin-
kevich and Loya, 1985).
From the above results, we suggest that sexuality and fecundity in S. pistillata are
responsive to the general state of health of the colony and its energetic limitations.
Studies also addressed reproduction/energy allocation questions in other coral
reef species. Kojis and Quinn (1985) found lower fecundity in damaged Goniastrea
favulus colonies compared to unharmed controls and suggested that this resulted
from reallocation of resources to growth activities that would repair damaged tissue
and cover the broken skeletons. Richmond (1984) indicated that reef corals may
allocate energy into new tissue via budding for colony growth, or via planulation
for production of new colonies. He found that colonies of Pocillopora damicornis at
Enewetak atoll, Marshall Islands, allocated the majority of their reproductive energy
into larva production while in the eastern Pacific the same species channels energy
into colony growth. Thus, internal and/or external (see below) determinants may
play a significant role in the expression of sexuality or fecundity in hermatypic corals,
although the mechanisms are not yet understood.
Stimson (1978) proposed that coral species which release planulae are characteris-
tic of shallow water environments such as reef flats, and hypothesized that shallow
water species should planulate to facilitate early settlement in the parental habitat.
Conversely, deep water corals should release eggs and sperm into the water to facili-
tate dispersal. More recent studies, however, suggest that the mode of reproduction
is related to more complex factors than habitat alone (Harriott, 1 983; Szmant, 1 986).
Thus it is of interest to study the mode of reproduction of the same species in two
different depths. Karlson (1981) found a reduction in reproductive activity with in-
creasing depth in two Jamaican species ofZoanthus. Kojis and Quinn (1983) further
indicate that fecundity of Acropora palifera decreased with depth. Colonies at depths
greater than 1 2 m had approximately half the fecundity of surface colonies. These
studies support the results of the present study (Tables IV, V, VI) which indicate high
differences in fecundity between shallow and deep water 5". pistillata colonies.
The importance of available energy for reproduction is apparent from the de-
crease of fecundity in deep water populations. S. pistillata invests photosynthetically
derived energy in reproduction (Rinkevich, 1982; Rinkevich and Loya, 1983). Mc-
Closkey and Muscatine (1984) found that the daily CZAR (the percentage contribu-
tion of zooxanthellae-translocated carbon to animal maintenance respiration) in S.
pistillata in deep water was less than half of that in shallow water. Mean CZAR at 35
m was 78%, compared to 157% at 3 m. They also found that the decreased carbon
availability to the host animal at 35 m was the consequence of both decreased net
carbon fixation and decreased percentage of net fixed carbon translocated to the host.
VARIABILITY IN SEXUAL REPRODUCTION OF A CORAL 343
Therefore, we suggest, that the generous daily carbon supply in shallow water colonies
enables them to channel significantly more energy to reproduction than deep water
colonies.
It is hard to separate the two explanations for the differences in reproductive activ-
ity between shallow and deep water colonies e.g.. energy limitations versus selective
pressures. The present results point to energy limitation in deep water colonies rather
than to the suggestion of selection pressures which favor high fecundity of shallow
water colonies (Stimson, 1978).
This study provides for the first time results of long-term (1974-1984) experi-
ments and observations on reproductive activities in a hermatypic coral. These and
earlier (Rinkevich, 1982; Rinkevich and Loya, 1985; 1986) results indicate that sexu-
ality as well as fecundity are determined and regulated by a variety of internal pro-
cesses (such as the state of health of the colony, regeneration of broken branches,
energy limitation, senescence, and death) and external, physical parameters (such as
storm activities). The only other relevant papers on external parameters are the recent
works of Jokiel (1985) and Jokiel et al. (1985) who suggested that environmental
factors including temperature, salinity, tidal cycle, visible, and ultraviolet radiation
influence the number of planulae released by Pocillopora damicornis per spawning
cycle, as well as the synchronization of lunar release of planula larvae. Therefore it
is concluded that reproductive activity in S. pistillata involves significant energetic
expenditures that otherwise would be allocated into other physiological pathways
such as growth and maintenance. As a result, any significant stress or diminution in
energy resources affects at least one of the reproductive characteristics of this species.
Physical and biological parameters have often been directly related to the distribution
of a given species. However, the present study indicates that even reproduction can
be directly altered as a result of the influence of these parameters. The changes in the
reproductive activity of S. pistillata colonies in the field may not be a function of a
single parameter. The combined effect of several external and internal factors on
gravid colonies should be examined experimentally before and during the reproduc-
tive season. These should be considered before formulating any general hypothesis
on coral reproduction.
ACKNOWLEDGMENTS
We are grateful to A. Shafir and to Y. Shapira for field assistance; to C. Porter for
the data presented in Table I; and to Y. Benayahu, Z. Wolodarsky, and Y. Shlesinger
for their great help and encouragement throughout the study. We thank L. Fishelson
for his help and advice and N. D. Holland, J. Pearse, and K. Ishizuka for their com-
ments on the manuscript. Appreciation is extended to the staff members of the MBL
at Eilat for their hospitality and the use of facilities. This study was partly supported
by the United States-Israel Binational Science Foundation (BSF) and the Tel Aviv
University Fund for Basic Research.
LITERATURE CITED
ATODA, K. 1 947a. The larva and postlarval development of some reef building corals. I. Pocillopora dami-
cornis cespitosa (Dana). Sci. Rep. Tohoku Univ. (4th ser.) 18: 24-47.
ATODA, K. 1947b. The larva and post larval development of some reef building corals. II. Stylophora
pistillata (Esper). Sci. Rep. Tohoku Univ. (4th ser.) 18: 48-64.
BABCOCK, R. C., G. D. BULL, P. L. HARRISON, A. J. HEYWARD, J. K. OLIVER, C. C. WALLACE, AND
B. L. WILLIS. 1 986. Synchronous spawnings of 1 05 scleractinian coral species on the Great Barrier
Reef. Mar. Biol. 90: 379-394.
344 B. RINKEVICH AND Y. LOYA
FADLALLAH, Y. H. 1983. Sexual reproduction, development and larval biology in scleractinian corals. A
review. Coral Reefs 2: 129-150.
HARRIOTT, V. J. 1^83. Reproductive ecology of four scleractinian species at Lizard Island, Great Barrier
Reef. Coral Reefs 2: 9- 1 8.
HARRIS >•• P. L., R. C. BABCOCK, G. D. BULL, J. K. OLIVER, C. C. WALLACE, AND B. L. WILLIS. 1984.
tss spawning in tropical reef corals. Science 223: 1 186-1 189.
JOKJEL, P. L. 1985. Lunar periodicity of planula release in the reef coral Pocillopora damicornis in relation
to various environmental factors. Pp. 307-312 in Proc. 5th Int. Coral Reef Congr. Vol. 4, C.
Cabrie and B. Salvat, eds. Tahiti.
JOKIEL, P. L., R. Y. ITO, AND P. M. Liu. 1985. Night irradiance and synchronization of lunar release of
planula larvae in the reef coral Pocillopora damicornis. Mar. Biol. 88: 167-174.
KARLSON, R. H. 1981. Reproductive patterns in Zoanthus spp. from Discovery Bay, Jamaica. Pp. 699-
704 in Proc. 4th Int. Coral Reef Symp., Vol. 2, E. D. Gomez, C. E. Birkeland, R. W. Buddemeier,
R. E. Johannes, J. A. Marsh, Jr., and R. T. Tsuda, eds. Manilla.
KOJIS, B. L., AND N. J. QUINN. 198 la. Aspects of sexual reproduction and larval development in the
shallow water hermatypic coral, Goniastrea australensis (Edwards and Haime, 1 857). Bull. Mar.
Sci. 31:558-573.
KOJIS, B. L., AND N. J. QUINN. 1 98 1 b. Reproductive strategies in four species of Porites (Scleractinia). Pp.
145-15 1 in Proc. 4th Int. Coral Reef Symp., Vol. 2, E. D. Gomez, C. E. Birkeland, R. W. Budde-
meier, R. E. Johannes, J. A. Marsh, Jr., and R. T. Tsuda, eds. Manilla.
KOJIS, B. L., AND N. J. QUINN. 1983. Coral fecundity as a biological indicator of reef stress. Am. Zool. 23:
961.
KOJIS, B. L., AND N. J. QUINN. 1985. Puberty in Goniastrea favulus. Age or size limited? Pp. 289-293 in
Proc. 5th Int. Coral Reef Congr. Vol. 4, C. Cabrie and B. Salvat, eds. Tahiti.
LOYA, Y. 1985. Seasonal changes in growth rate of a Red Sea coral population. Pp. 187-191 in Proc. 5th
Int. Coral Reef Congr. vol. 6, C. Cabrie and M. Harmelin- Vivien, eds. Tahiti.
McCLOSKEY, L. R., AND L. MUSCATINE. 1 984. Production and respiration in the Red Sea coral Stylophora
pistillata as a function of depth. Proc. R. Sot: Lond. B 222: 2 1 5-230.
VAN MOORSEL, G. W. N. M. 1983. Reproductive strategies in two closely related stony corals (Agaricia,
Scleractinia). Mar. Ecol. Prog. Ser. 13: 273-283.
RICHMOND, R. H. 1984. An energetic approach relating fecundity, growth and reproduction in the reef
coral Pocillopora damicornis. Am. Zool. 24: 27A.
RINKEVICH, B. 1982. Stylophora pistillata. Ecophysiological aspects in the biology of a hermatypic coral.
Ph.D. thesis, Tel Aviv University, Israel. 300 pp. (in Hebrew with English summary).
RINKEVICH, B., AND Y. LOYA. 1979a. The reproduction of the Red Sea coral Stylophora pistillata. I.
Gonadsand planulae. Mar. Ecol. Prog. Ser. 1: 133-144.
RINKEVICH, B., AND Y. LOYA. 1979b. The reproduction of the Red Sea coral Stylophora pistillata. II.
Synchronization in breeding and seasonality of planulae shedding. Alar. Ecol. Prog. Ser. 1: 145-
152.
RINKEVICH, B., AND Y. LOYA. 1983. Short term fate of photosynthetic products in a hermatypic coral. J.
Exp. Mar. Biol. Ecol. 73: 175-184.
RINKEVICH, B., AND Y. LOYA. 1985. Intraspecific competition in a reef coral: effects on growth and repro-
duction. Oecologia 66: 100-105.
RINKEVICH, B., AND Y. LOYA. 1986. Senescence and dying signals in a reef building coral. Experientia
40: 320-322.
SHLESINGER, Y., AND Y. LOYA. 1985. Coral community reproductive patterns: Red Sea versus the Great
Barrier Reef. Science 228: 1333-1335.
SOKAL, R. R., AND F. J. ROHLF. 1981. Biometry. Freeman, NY. 859 pp.
STIMSON, J. S. 1978. Mode and timing of reproduction in some common hermatypic corals from Hawaii
and Enewetak. Mar. Biol. 48: 173-184.
STODDARD, J. A., AND R. BLACK. 1985. Cycle of gametogenesis and planulation in the coral Pocillopora
damicornis. Afar. Ecol. Prog. Ser. 23: 153-164.
SZMANT, A. M. 1986. Reproductive ecology of Caribbean reef corals. Coral Reefs 5: 43-53.
SZMANT-FROELICH, A., L. RIGGS, AND M. REEUTTER. 1983. Sexual reproduction in Caribbean reef corals.
Am. Zool. 23: 96 1.
WALLACE, C. C. 1985. Reproduction, recruitment and fragmentation in nine sympatric species of the coral
genus Acropora. Mar. Biol. 88: 217-233.
WILLIS, B. L., R. C. BABCOCK, P. L. HARRISON, J. K. OLIVER, AND C. C. WALLACE. 1 985. Patterns in the
mass spawning of corals on the Great Barrier Reef from 1981 to 1984. Pp. 343-348 in Proc. 5th
Inter. Coral Reef Congr. Vol. 4, C. Cabrie and B. Salvat, eds. Tahiti.
Reference: Biol. Bull. 173: 345-354. (October, 1987)
VITELLINS AND VITELLOGENINS OF THE TERRESTRIAL ISOPOD,
ARMADILLIDIUM VULGARE
SACHIKO SUZUKI
Laboratory of Biology, Kanagawa Prefect ural College, Nakaocho 50-1, Asaliiku. Yokohama 241, Japan
ABSTRACT
Four forms of vitellogenin (Vg- l-Vg-4) in the hemolymph and four forms of
vitellin (Vn- l-Vn-4) in the oocytes of reproductive females of Armadillidium vul-
gare were detected by polyacrylamide gel electrophoresis (PAGE) at stage D of the
molting cycle. All vitellogenins decreased and were not detected in the hemolymph
at stage E. At stage E, Vn • 1 - Vn • 3 disappeared; Vn • 4 was the major vitellin compo-
nent in mature oocytes. The electrophoretic patterns of vitellogenin and vitellin re-
vealed changes correlated with the molting cycle and oocyte growth.
Vitellogenins were electrophoretically identical to vitellins. Rabbit anti-Vn • 4 an-
tibody cross-reacted with vitellogenins. Using Slater's method, the four forms of vitel-
lin were glycolipoproteins, with molecular weights of Vn • 1 -700,000, Vn • 2-620,000,
Vn • 3-540,000, and Vn - 4-470,000. In SDS-PAGE, vitellin Vn - 4 yielded three main
polypeptide components with molecular weights of 80,000, 99,000, and 1 27,000. The
polypeptide compositions among vitellins ( Vn • 1 -Vn • 4) were similar.
INTRODUCTION
The correlation between the molting cycle and secondary vitellogenesis was de-
scribed in the amphipod Orchestia gammarellus (Meusy and Charniaux-Cotton,
1984). In the isopod Porcellio dilatatus, the fat body (subepidermal adipose tissue) is
the site of vitellogenin synthesis (Picaud and Souty, 1980). The cauterization of Y-
organs(Souty et al, 1982; Picaud, 1 983) and ovariectomy (Picaud and Souty, 1981)
of P. dilatatus lowered the rate of vitellogenin release into the hemolymph. However,
no study elucidates the hormonal mechanisms which regulate the vitellogenin syn-
thesis in Crustacea.
I am investigating the hormonal control of reproduction — especially hormonal
regulation of vitellogenin synthesis — in Armadillidium vulgare. Previous work on A.
vulgare indicated that rapid oocyte growth occurs at stage D of the molting cycle.
Oocytes did not continue to grow in Y-organ ablated females (Suzuki, 1986). Y-
organ (molting hormone) was needed for oocyte growth. However, it is not yet known
whether vitellogenin synthesis is induced by molting hormone in A. vulgare.
The present study identified female-specific proteins (vitellins and vitellogenins)
of A. vulgare by electrophoresis. The data will contribute to the understanding of
vitellogenin synthesis and its hormonal (molting hormone) regulation. This paper
identifies four forms of vitellogenins and vitellins, and describes their characteristics
in A. vulgare. Three of the forms were described by Picaud (1983).
Received 9 February 1987; accepted 30 July 1987.
345
346
S. SUZUKI
I
•
SCO -
,00
£ 4OO
O
TJ
a, 3OO
o
O
200
100
:x
i_
o
o
100 \
80
60
tf)
0)
o
o
o
t
Ov-i
10
t
RI
20
t
St
30
EC
40 0)
.a
E
3
20 Z
EAJ3] Molting stages
Days
Ov-2
FIGURE 1 . Oocyte growth during the molting cycle following first oviposition in Armadillidium vul-
gare. — O — , diameter of oocyte;-- • --, number of oocytes; Ov- 1 , first oviposition; Ov-2, second oviposi-
tion; RI, release of larva; St. sternoliths; EC, ecdysis. Ventrical lines around the points show standard errors.
MATERIALS AND METHODS
Animals
Armadillidium vulgarewas collected near Yokohama in early March 1986. Sev-
eral females (10-12 mm body length) were kept in a petri dish with moistened soil.
Two adult males were added to the dish for a normal reproductive cycle. They were
maintained at 25 ± 2°C in natural daylight, and fed decayed leaves and rat chows.
To observe oocyte growth, fresh ovaries were examined microscopically.
Preparation of hemolymph and ovarian homogenate to electrophoresis
Hemolymph samples were collected using a capillary tube placed in a hole in the
tergite of the seventh thoracic segment. It was diluted 1/10 with Tris-HCl buffer (20
mM, pH 7.6 containing 130 mM NaCl and 5 mM EDTA). Dissected ovaries were
rinsed and homogenated in 300 ^1 Tris-HCl buffer. Ovarian homogenate was passed
through prefilter (milipore, AP) to remove lipids, centrifuged at 20,000 X g for 15
min at 4°C, and the supernatant was collected as a sample of ovarian homogenate.
Polyacrylamide gel electrophoresis
Polyacrylamide gel electrophoresis (PAGE) was performed using 5% acrylamide
monomer (Davis, 1964). Polyacrylamide gel electrophoresis in sodium dodecyl sul-
ISOPOD VITELLIN AND VITELLOGENIN 347
HEMOLYMPH
34 567
Vg-l
Vg-2
» Vg-3
Vg-4
f
III II
OOCYTE- 150 25O 35O 450 55Opm -DIAMETER
III I I
Vn-l
Vn-2
Vn-3
Vn-4
I
8 9 10 II 12
OVARIAN HOMOGENATE
FIGURE 2. Electrophoretic pattern of the hemolymph and the ovarian homogenate on 5% PAGE. 1 ,
hibernating adult females; 2-6 and 8-12, vitellogenic females; 7, adult male; Vg- l-Vg-4, vitellogenin;
Vn-l-Vn-4, vitellin.
fate (SDS-PAGE) was also performed (Laemmli, 1970) using 10% acrylamide. Pore-
limited electrophoresis was carried out on 5-25% acrylamide gradient slab gels
(PAGGE) (Slater, 1969) using Davis's buffer system.
348
S. SUZUKI
FIGURE 3. PAGE of the ovarian homogenate (350-450 ^m in oocyte diameter) stained for protein
with Coomassie blue (1), for glycoprotein with periodic acid/Schiff reagent (2), and for lipoprotein with
Sudan black B (3).
-C
o>
0)
3
o
0)
8O
60
4O
20
10 15
mm from origin
20
FIGURE 4. Molecular weight determination of vitellins by pore-limited electrophoresis (5-25%
PAGGE) of ovarian homogenate (350-450 ^m). Molecular weights were calculated from mobilities rela-
tive to each standard proteins (A-C, Pharmacia). A, thyroglobulin (669,000 M. W.); B, ferritin (440,000);
C, catalase (232,000); Vn - 1 -700,000; Vn . 2-620,000; Vn • 3-540,000; Vn • 4-470,000.
ISOPOD VITELLIN AND VITELLOGENIN 349
M. W. x IO'3
127
94
80
67
43
30
20
14.4
B
FIGURE 5. Molecular weight determination of Vn • 4 polypeptide by SDS-PAGE of ovarian homoge-
nate. A, slab gel of standard proteins (phosphorylase b-94,000; bovine serum albumine-67,000; ovalbumin-
43,000; carbonic anhydrase-30,000; soybean trypsin inhibitor-20,000; «-lactalbumin- 14,400, Pharmacia).
B, slab gel of ovarian homogenate ( 1 50 /^m in oocyte diameter and stage C). C, disc gel of ovarian homoge-
nate (500 ^m and stage E).
After electrophoresis, proteins were stained with Coomassie blue and glycopro-
teins were visualized using the periodic acid Schiff(PAS) method (Zacharius et al,
1969). Lipoprotein samples were stained with Sudan black B (Sano, 1981) prior to
electrophoresis.
Preparation of antibody and immunodiffusion test
VN-4 was used as antigen. Mature oocytes (200, 550 ^m in diameter) were ho-
mogenized in Tris-HCl buffer and centrifuged. The resulting 1.5 ml containing about
1 mg/ml total proteins (Lowry et al, 195 1, with BSA as a standardj was applied to
the top of a slab gel (PAGE, 3 mm thick, 10.5 X 1 1.5 cm, 30 mA for 2 h). After
electrophoresis, both sides of the gel were stained with Coomassie blue to define the
vitellin band. The Vn • 4 band was then cut from the unstained portion of the slab gel
and thoroughly homogenized with 1.5 ml Tris-HCl buffer. The gel homogenate (3
ml) was stored frozen at -80°C until use.
One milliliter of the homogenate (350 Mg/ml proteins) and Freund's complete
350 s. SUZUKI
flMH *ijijfc'
^^^, ,dtfMlM|
8
FIGURE 6. Polypeptide analysis of vitellin Vn- l-Vn-4. Ovarian homogenote (350-450 /urn) was
prestained with Sudan black B. After PAGE of homogenate, each vitellin band was cut out of the disc gels
and eluted by homogenizing the gel bands in 20 mA/ Tris-HCl buffer. PAGE patterns of separated Vn • 1
(1), Vn-2 (2), Vn-3 (3), and Vn-4 (4). Polypeptide analysis of each separated Vn- 1 (5), Vn-2 (6), Vn-3
(7), and Vn-4 (8) on disc SDS-PAGE.
adjuvant ( 1 ml) were injected subcutaneously into the back of a male rabbit twice
every 10 days. A booster injection was given once, on the 10th day after the second
antigen injection. Five days later the rabbit was bled and an immunoglobulin (IgG)
fraction was precipitated by ammonium sulfate (40% saturation). After dialysis
against Tris-HCl buffer, this antiserum (anti-Vn • 4 IgG) was stored frozen at -80°C.
Immunodiffusion tests were performed (Ouchterlony, 1 949). Samples were tested
on 1% agarose gel plates with 0.5 M Tris-HCl buffer. Both antigen and antibody were
allowed to diffuse for one day at room temperature and examined for precipitin lines.
RESULTS
Changes of vitellin and vitellogenin during the molting cycle
Young oocytes were seen (Fig. 1 ) in the ovaries after oviposition. They gradually
increased in size during stage C of the molting cycle. Rapid oocyte growth began soon
after the appearance of the sternoliths, which were evident in stage D. Oocytes were
about 550 /j.m at stage E, and ecdysis occurred in the posterior and then anterior
region of the female's body before oviposition.
The electrophoretic pattern of vitellogenins and vitellins was investigated on disc
PAGE. Diluted hemolymph (20 ^1) and ovarian homogenate (30 /A) were subjected
to electrophoresis at 2 mA per tube for 3 h. Hemolymph analysis revealed four forms
of vitellogenin (Vg- l-Vg-4) present in the hemolymph of stage D females with sub-
ISOPOD VITELLIN AND VITELLOGENIN
351
FIGURE 7. Identification of vitellogenin by immunoelectrophoresis. Hemolymph from reproductive
female (1) and male (3) at stage D of the molting cycle were subjected to PAGE. Disc gels were then
embedded in a layer of 1% agarose gel on a glass plate. The trough (2) of the agarose gel was cut and filled
with anti-Vn-4 serum. After 24 h diffusion at room temperature, PAGE gels of (1 and 3) were removed
and the precipitate line of immunochemical reaction was stained with Coomassie blue. The other PAGE
gels of ( 1 and 3), stained with Coomassie blue, were placed on each original site of the agarose gel.
mature oocytes (350-450 yum) (Fig. 2-4, 5). These forms were not detected in females
with mature oocytes (550 /j.m) at stage E of the molting cycle (Fig. 2-6). Vitellogenin
was not detected either in stages A and B of the reproductive females or in the hemo-
lymph of males (Fig. 2-7) throughout the molting cycle.
Analysis of ovarian homogenate revealed four forms of vitellin (Vn- l-Vn-4) in
immature oocytes of stage D females (Fig. 2-10, 11). These forms of vitellin exhibited
the same electrophoretic pattern as vitellogenin. At stage E, Vn- l-Vn-3 were not
detected; Vn-4 was the major component of vitellin found in mature oocytes (Fig.
2- 12). Vn-4 was also the primary vitellin in young oocytes of stage C females (Fig.
2-8,9).
Characterization ofvitellogenins and vitellins
The four bands of vitellin and vitellogenin had low electrophoretic mobility and
were located closely together (Fig. 2-5, 11). Figure 3 shows that the vitellins were
352 s. SUZUKI
FIGURE 8. Ouchterlony agar diffusion analysis showing reaction between anti-Vn-4 serum (A)
against ovarian homogenate ( 1 and 2) or hemolymph of females (3-6). 1 and 4, stage C; 2 and 5, stage D;
3, hibernating; 6, stage E.
glycolipoproteins, as stained with periodic acid/Schiff reagent and Sudan black B.
The ovarian homogenate, prepared from immature oocytes (350-450 j/m), was ana-
lyzed by the pore-limited method (PAGGE) using standard proteins to determine the
molecular weight of the four vitellins (Fig. 4). They were estimated to be Vn- 1-
700,000 (700K), Vn - 2-620,000 (620K), Vn • 3-540,000 (540K), and Vn - 4-470,000
(470K), respectively.
Figures 5 and 6 show the polypeptide composition of vitellin separated in SDS-
PAGE. Vn-4, the major vitellin in stages C and E (Fig. 2-8, 12), yielded three main
polypeptides with molecular weights estimated to be 80,000 (80K), 99,000 (99K),
and 127,000 (127K) (Fig. 5). To compare the polypeptide compositions of Vn- 1-
Vn-4, each vitellin prestained with Sudan black B was isolated from immature oo-
cytes using PAGE. After electrophoresis, the gel band of each vitellin was cut out and
eluted in Tris-HCl buffer. Although the isolated vitellin overlapped with other vitel-
lins (Fig. 6- 1-4) each vitellin was analyzed on SDS-PAGE. Vn- l-Vn-4 were sepa-
rated into several polypeptides; their main polypeptide compositions were very sim-
ilar (Fig. 6 • 5-8). The lower polypeptides (MW 80K and 99K) were common through
Vn-1 -Vn-4 (Figs. 5,6).
Immunochemical reactions of and- Vn-4 serum against ovarian homogenate (vi-
tellin) and diluted hemolymph (vitellogenin) were examined by double diffusion
analysis in agarose gel. The four vitellogenins gave a single precipitate line when anti-
Vn • 4 serum was reacted with the hemolymph of stage D females. The precipitate
line was absent when the serum was reacted with hemolymph of males (Fig. 7). Figure
8 presents the reactions between anti-Vn-4 serum and various stage hemolymph or
ovarian homogenates. These results indicate that anti-Vn-4 antibody cross-reacted
with vitellins and vitellogenins.
DISCUSSION
Four forms of vitellin and vitellogenin were identified electrophoretically from
Armadillidium vulgare. Picaud (1983) found three forms from this species by electro-
phoresis. Vitellins are glycolipoproteins with higher molecular weights (700 K-470K).
ISOPOD VITELLIN AND VITELLOGENIN 353
Vn-4, the smallest vitellin, contains three main polypeptides at MW 80K, 99K, and
127K. The polypeptide compositions of Vn- l-Vn-4 are quite similar. Vitellins of
the isopods Porcellio d Hat at us (315K), Idotea balthica (290K), and Ligia oceanica
(320K) have similar molecular weights (Picaud, 1983); molecular weights of A. vul-
gare vitellins are higher. However, the main polypeptide compositions of Vn-4 from
A. vulgare (8QK, 99K, and 127K) have molecular weights similar to those of P. dilata-
tus (78K, 97K, and 180K) (Picaud, 1983).
The presence of a precursor (vitellogenin) of egg yolk protein (vitellin) shows that
A. vulgare vitellin appears to be synthesized at an extra ovarian site (possibly the fat
body) and then transported to the ovary through the hemolymph, as in Porcellio
dilatatus (Picaud and Souty, 1980). However, Souty (1983) reported that the ovary
of P. dilatatus can synthesize its proteinic yolk.
Changes of vitellin and vitellogenin were observed during the molting cycle and
oocyte growth. All forms of vitellin and vitellogenin were observed at stage D and,
except for Vn-4, they declined and disappeared during oocyte growth. The major
peak of vitellogenin synthesis was observed at stage D of the isopod in Idotea balthica
by Souty and Picaud ( 198 1 ) and in Porcellio dilatatus by Picaud and Souty ( 198 1 ).
The question remains whether there is a correlation between the changes in the ap-
pearance of vitellin and/or vitellogenin and the Y-organ (molting hormone) in A.
vulgare. In isopods, however, Charniaux-Cotton (1985) found that molting hormone
is necessary for vitellogenin synthesis and is regulated by a feedback mechanism.
Since vitellin Vn- l-Vn-4 have common polypeptides and a common antigenic
determinant, the smallest vitellin (Vn-4) may be accumulated throughout oocyte
growth. Vn • 1-Vn • 3 seem to undergo a proteolytic processing in the oocytes. Analo-
gous phenomena have been reported (Baert, 1985, 1986) for Pereneris cultrifera
(polychaete). During oocyte growth, the higher molecular weight vitellins disap-
peared leaving the single form (the lowest vitellin) at immaturity. These results sug-
gest that a progressive proteolytic cleavage of vitellin occurs in polychaetes. Vitello-
genin processing has also been reported in amphipods (Junera and Meusy, 1982) and
in the locust (Chen et ai, 1978). The processing of vitellin and vitellogenin is pres-
ently unclear in A. vulgare and awaits further study.
ACKNOWLEDGMENTS
The author thanks Prof. Y. Katakura of Keio University and Dr. K. Yamasaki of
Tokyo Metropolitan University for helpful suggestions during this study.
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occurrence and relation to vitellin maturation in the oocyte. Camp. Biochem. Physiol. 81 B: 851-
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BAERT, J. L. 1986. Evidence for vitellin maturation within oocytes of Perinereis cultrifera (polychaete
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CHEN, T. T., P. W. STRAHLENDORF, ANDG. R. WYATT. 1978. Vitellin and vitellogenin from locusts
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Acad.Sci. 121:404-427.
JUNERA, H., AND J.-J. MEUSY. 1982. Vitellogenin and lipovitellins in Orchestia gammarellus (Pallas)
(Crustacea, Amphipoda): labelling of subunits after in vivo administration of 3H-leucine. Expe-
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354 s. SUZUKI
LAEMMLI, U. K. 1970. Cleavage of structure proteins during the assembly of the head of bacteriophage
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LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with
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A SCANNING ELECTRON MICROSCOPE STUDY OF ASCIDIA
MALAGA EGG (TUNICATE). CHANGES IN THE CELL SURFACE
MORPHOLOGY AT FERTILIZATION
LUISANNA VILLA AND ELEONORA PATRICOLO
Institute of Zoology. University of Palermo, ViaArchira.fi 18, 90123 — Palermo, Italy
ABSTRACT
Ascidia malaca eggs with and without envelopes were studied using the scanning
electron microscope. Follicle cells, chorion, and test cells were examined and com-
pared with those of other ascidian species. No appreciable differences were found.
The surface topography of dechorionated eggs differed before and after fertilization.
The pole of the unfertilized egg was indicated by a small smooth region beneath
the polar pit. The remaining surface of the egg was undulated. Short microvilli were
scattered on the cell membrane except in the area nearest the polar pit. Surface dis-
placements occurred in the fertilized egg, changing its morphological features, at vari-
ous intervals after sperm penetration. The smooth region expanded shortly after fer-
tilization, and the inferior part of the vegetal hemisphere was corrugated by pro-
nounced undulations. Two new types of microvilli appeared. After the ejection of the
first polar body the appearance of the egg surface changed: the animal hemisphere
became corrugated, bearing numerous short microvilli, and the vegetal hemisphere
showed slight undulations. At the vegetal pole microvilli concentrated to form a pro-
tuberance. After the ejection of the second polar body a diffusion of elongated mi-
crovilli was observed. The present results indicate that during ooplasmic segregation
the movement of the cell membrane components produces changes in the surface
topography. These govern the rearrangement of the cytoplasm.
INTRODUCTION
Ascidian eggs show characteristic deformations at fertilization and in the interval
between the ejection of the first and second polar bodies. Cell surface movements
after sperm penetration were studied by Ortolani (1955). Colored chalk granules were
bound to the plasma membrane to mark the surface of the unfertilized egg. After
fertilization their displacement was observed using the light microscope; the polar pit
was used as a landmark. Her observations suggested that the movements of the sur-
face and the modifications in shape were caused by a cortical contraction.
The present SEM study examined the morphology of Ascidia malaca eggs with
and without envelopes. The dechorionated egg was examined both before and after
fertilization to observe the modifications which characterize the surface topography.
We followed the distribution and organization of the microvilli at different intervals
after sperm penetration.
Previous ultrastructural investigations of Ascidia malaca eggs have been per-
formed, but they were primarily concerned with oogenesis (Materazzi and Bondi,
1973; Gianguzza and Dolcemascolo, 1978; 1979), egg morphology (La Spina
Received 13 May 1987; accepted 22 July 1987.
Abbreviations: SEM = Scanning electron microscopy; TEM = Transmission electron microscopy.
355
356 L. VILLA AND E. PATRICOLO
D'Anna, 1974), sperm morphology (Villa, 1975; Villa and Tripepi, 1983), and egg-
sperm interaction (Villa, 1977). No information exists on the egg plasma membrane.
Transmission and scanning electron microscope studies of egg surface changes
and early • vents in ascidian development used only Ciona intestinalis (Sawada and
Osanai, 1981). The primary objective of this study was to elucidate the mechanism
of ooplasmic segregation. The present results not only extend our knowledge of the
ascidian egg envelope but also reveal differences in the egg surface between the animal
and vegetal hemispheres, which become more evident after fertilization. During
ooplasmic segregation significant changes and apparent transposition of the features
of the two halves take place. Modifications in the distribution and shape of the surface
microvilli continue to occur until the ejection of the second polar body.
MATERIALS AND METHODS
Adult specimens ofAscidia malaca were collected from the Gulf of Palermo. Male
and female gametes were obtained surgically from gonoducts of dissected animals.
Eggs removed from the oviducts were washed in Millipore-filtered seawater; some
were left intact with egg envelopes, others were transferred to agar-coated Syracuse
dishes and dechorionated by hand using steel needles.
The experiments were performed at 22°C by fixing unfertilized eggs and eggs 3,
7, and 30 minutes after insemination.
Transmission electron microscopy
For conventional TEM studies, unfertilized and fertilized (3 min after insemina-
tion) intact eggs were fixed with 3% glutaraldehide in 0. 1 Mcacodylate buffer in sea-
water (pH 7.2) containing 4% sucrose for 30 min at room temperature, and postfixed
in 1% osmium tetroxide in the same buffer for 1 h at 4°C. The specimens were dehy-
drated in an ethanol-propylene oxide series and embedded in Dow epoxy resins
(Lockwood, 1964). Sections were stained with saturated uranyl acetate and lead ci-
trate (Venable and Coggeshall, 1965), and examined with a Siemens Elmiskop Ib
TEM operating at 80 kV.
Scanning electron microscopy
Unfertilized and fertilized intact and hand-dechorionated eggs were fixed as de-
scribed for TEM, then ethanol-dehydrated, critical-point dried, sputter-coated with
gold, and observed with JEOL JSM 1 5 and ISI DS 1 30 SEMs. Other intact eggs were
fixed and critical-point dried as above, but before they were coated with gold the
follicle cell layer and chorion were partially dissected with a fine sharp needle. The
dissected specimens were coated and viewed under the SEM as described above.
Intact and dechorionated eggs show no evidence of distortion after specimen prep-
aration; however shrinkage is generally observed (about 20%). Shrinkage is greater in
intact eggs since the chorion is greatly reduced by SEM preparation.
RESULTS
Egg envelope morphology
The Ascidia malaca egg envelope consists of an acellular layer — the vitelline coat
(i.e. chorion) — lying between two cellular layers, the external follicle cells and inter-
nal test cells (Figs. 1-3, 5, 6). The follicle cells constitute a single layer of conical-
SURFACE CHANGES IN ASCIDIAN FERTILIZATION
357
FIGURE 1 . Phase contrast micrograph of a living unfertilized Ascidia malaca egg. fc = follicle cell; tc
= test cell; vc = vitelline coat. 270X.
FIGURE 2. Transmission electron micrograph (TEM) of envelopes of unfertilized egg. Arrows indi-
cate coating of the outer vitelline coat; pm = plasma membrane. 2500X.
FIGURE 3. Phase contrast micrograph of a living fertilized egg; test cells have migrated towards the
vegetal hemisphere (arrows), vp = vegetal pole. 290X.
FIGURE 4. TEM of a head of a fully differentiated spermatozoon from the sperm duct, m = mito-
chondrion; n = nucleus. lO.OOOX.
FIGURE 5. TEM of envelopes of a fertilized egg; spermatozoa are found in the vitelline coat (arrow-
heads). 5000X.
shaped, highly vacuolated hexagonally arranged cells; their convex basal region forms
indentations in the vitelline coat, which are clearly seen when the latter is cut away
(Fig. 1 1 ). The follicle cells touch one another only at their basal region, where the
plasma membranes of adjacent cells seem to interdigitate, separated by narrow clefts.
The clefts are the only means by which the sperm can reach the subjacent vitelline
coat, where the "sperm reaction" — involving swelling, migration, and loss of the mi-
tochondrion— occurs. In fact, many "remnants" of spermatozoa (i.e. mitochondrion
plus tail), and only a few intact ones, are found mainly in the cleft zone 3 minutes
FIGURE 6. Scanning electron micrograph (SEM) of a fertilized egg; follicle cells form a hexagonal
pattern (asterisks). 520X.
358
SURFACE CHANGES IN ASCIDIAN FERTILIZATION 359
after fertilization (Figs. 7-10). At high magnification the follicle cells show a reticu-
lated lace-like membrane (Fig. 7).
The chorion shows different morphological features and different thicknesses de-
pending on fixation techniques; however, a thin homogeneous electron-dense outer
layer with an organized coating (previously called "chorial membrane," Villa, 1977)
and a thicker fibrous inner layer are always observed (Figs. 2, 5). The thickness of the
chorion (ca. 20 nm in vivo) decreases during TEM preparation, and even further
during that for SEM.
The chorion encloses the test cells that, in unfertilized eggs, are close to the egg
plasma membrane. The test cells form a single but discontinuous layer of roundish or
oval, moderately vacuolated cells which move freely within the texture of the chorion.
In fertilized eggs the test cells migrate towards the vegetal hemisphere where they
accumulate in layers (Figs. 3, 5).
SEM examination of eggs in which the follicle cell and chorion layers had been
partially cut away prior to gold coating revealed that the test cell plasma membranes
have either pseudopodia-like extensions or invaginations of corresponding size (Fig.
1 1); holes can also be observed in the plasma membrane where the numerous micro-
villi would normally be located (Figs. 12, 13).
Surface morphology
Unfertilized egg. A well-defined polarity is observed: the plasma membrane at the
tip of the animal hemisphere is slightly undulated, while the remaining parts are
highly undulated with surface folds of random orientation (Fig. 14). The animal pole
is marked by a pit, from the bottom of which the first polar body will emerge (Fig.
1 5). Short microvilli (approximately 0.2 ^m in length) are fairly uniformly distributed
on the egg surface (Fig. 16) except in the region of the polar pit that is relatively devoid
of microvilli.
Eggs 3 min after insemination. The shape of the fertilized egg changes rapidly
(Fig. 17): it elongates, becoming pear-shaped with transient bulges, and then rapidly
regains its original shape. A marked polarity of the folded areas and two new types of
microvilli also characterize this stage. The animal hemisphere and the upper half
of the vegetal hemisphere are slightly undulated, while the lower half of the vegetal
hemisphere is corrugated (Fig. 1 9): short microvilli, similar to those of the unfertilized
eggs, are scattered over the entire surface except in the polar pit. Moreover, a few
FIGURE 7. High magnification of the follicle cell layer showing clefts between the cells and lace-like
reticular membrane. Arrow indicates an intact spermatozoon; arrowheads indicate clefts, st = sperm tail.
3000X.
FIGURE 8. SEM of the follicle cell layer of a fertilized egg. Arrows indicate numerous remnants of
spermatozoa (i.e., tail + mitochondrion) in the clefts between adjacent follicle cells. lOOOx.
FIGURE 9. SEM of the cleft zone showing intact sperm (arrowhead) and two spermatozoa beginning
the "sperm reaction" (arrows). 2000X.
FIGURE 10. High magnification of the cleft zone showing mitochondria left out and a still intact
spermatozoon. 4500X.
FIGURES 11-13. SEM of unfertilized eggs in which follicle cells and vitelline coat were partially cut
away. Figure 1 1: View of the underside of the vitelline coat showing indentations of the follicle cells (aster-
isks); numerous test cells with pseudopodia-like extensions or with invaginations (arrowheads) are left on
the vitelline coat during dissection. 2710X. Figures 12, 13: View of the egg plasma membrane, on which
some test cells rest, showing numerous pore-like openings corresponding to the base of the microvilli.
Figure 12, 2050X. Figure 13, 3150X.
FIGURES 14-16. SEM of unfertilized eggs showing polarity (Fig. 14), smooth and slightly microvil-
lated surface of the egg near the polar pit (Fig. 15), and high magnification of microvilli of highly undulated
vegetal area (Fig. 16). ap = animal pole; pp = polar pit. Figure 14, 700X. Figure 15, 5460X. Figure 16,
7600X.
360
SURFACE CHANGES IN ASCIDIAN FERTILIZATION 361
large stumpy microvilli (approximately 0.8 nm in length) are randomly scattered in
the equatorial area, and numerous slender microvilli (0.5 /j.m by 0. 1 nm) can be ob-
served very densely arranged in the lower half of the vegetal hemisphere (Fig. 18).
Eggs 7 min after insemination. After the ejection of the first polar body, striking
but transient modifications in shape — such as a lobe formation at the vegetal pole
(Fig. 21) — occur. An extensive reorganization of the egg surface results in the rear-
rangement of microvilli and in the apparent transposition of the folded areas from
the vegetal to the animal hemisphere. The plasma membrane is highly undulated by
pronounced folds in the animal hemisphere (Fig. 20), while in the vegetal hemisphere
it is only slightly wrinkled (Fig. 22). The stumpy microvilli of the previous stage are
still found in the equatorial area (Fig. 23); the slender microvilli also persist in the
vegetal half. At the vegetal pole a well-defined circular protuberance covered by a
dense clump of short microvilli appears (Figs. 22, 24); spermatozoa are occasionally
encountered on the pole or in the surrounding region.
Eggs 30 min after insemination. The egg regains its spherical shape after ejection
of the second polar body. The distribution of the folded areas is similar to that of the
previous stage (Fig. 25), although the microvilli reorganize; they are again uniformly
distributed over the whole surface and appear slightly longer when compared with
those of the previous stages (Fig. 27). Moreover the numerous spermatozoa left on
the plasma membrane after insemination are now concentrated at the vegetal pole in
which the protuberance of clumped microvilli has gradually faded (Figs. 26, 28).
DISCUSSION
This study provides further information on the morphology of the accessory cells,
and new data on nude surface topography.
To understand their role in development, egg envelopes of many ascidian species
have been subjects of extensive morphological and biochemical studies. However,
previous SEM studies of egg envelopes were performed only on a few species includ-
ing A scidiella aspersa (Mansueto and Villa, 1983), dona intestinalis (Bates, 1980;
De Santis et ai. 1980) and Phallusia mammillata (Honnegger, 1982, 1986). The
general morphological characteristics ofAscidia malaca egg envelopes are similar to
those of the aforementioned species.
However, our study has detected some peculiarities of the test and follicle cells.
The test cell membrane forms pseudopodia and invaginations, which probably reflect
dynamic cell-to-cell contact, while the follicle cell membrane shows a reticulated lace-
like structure and clefts in the basal region (previously observed only in Ciona intesti-
nalis, Bates 1980). The size of these clefts might be involved in the ascidian "sperm
reaction" (described by Lambert, 1982): loss of the mitochondrion would reduce
the diameter of the spermatozoon to a size allowing easy penetration. Moreover,
only intact spermatozoa have been detected on the plasma membrane of dechorion-
ated eggs.
The morphological features of the surface of the dechorionated egg differ greatly
before and after fertilization.
A smooth area around the polar pit marks a polarity in the unfertilized egg.
FIGURES 17-19. SEM of eggs fixed 3 min after fertilization showing shape modification (Fig. 17),
different distribution of microvilli in equatorial area: (A) upper vegetal hemisphere (B) and lower vegetal
hemisphere (C) (Fig. 18), and marked polarity of the folded areas (Fig. 19). Arrows indicate stumpy micro-
villi; arrowheads indicate slender microvilli. Figure 17, 700X. Figure 18, 4350X. Figure 19, 700X.
FIGURES 20-24. SEM of eggs fixed 7 min after fertilization showing changes in surface morphology.
Figure 20: Supraequatorial area, ah = animal hemisphere; pb = polar body. 620X . Figure 2 1 : Subequatorial
area with a lobe formation at the pole, vh = vegetal hemisphere. 620X. Figure 22: View of the vegetal
362
SURFACE CHANGES IN ASCIDIAN FERTILIZATION 363
After sperm penetration and between the ejection of the first polar body and the
second maturation division, egg morphology changes drastically. In addition to the
known shape deformations which may be caused by a cortical contraction during
ooplasmic segregation (Ortolani, 1955; Reverberi, 1971; Sawada and Osanai, 1981),
other fine modifications of the surface architecture occur.
In the newly fertilized egg (3 min), the mildly undulated animal area expands over
the whole hemisphere as far as the subequatorial zone; the remaining vegetal part
is corrugated. This rearrangement corresponds with the displacement of the chalk
granules observed with the light microscope: divergent at the animal pole and conver-
gent at the vegetal pole (Ortolani, 1955). These membrane modifications cause the
test cells to migrate downwards and accumulate in the lower part of the vegetal region,
as described by Conklin (1905).
Changes in the organization and distribution of the microvilli also occur at this
time. In addition to the short type found in the unfertilized egg, two new types of
microvilli appear: large stumpy microvilli in the equatorial area and slender ones
condensed in the vegetal hemisphere, probably where the myoplasm accumulates.
After the ejection of the first polar body (7 min after sperm penetration) an appar-
ent transposition of the features of the two halves is observed. The animal region
becomes more undulated and is now densely covered by short microvilli. This occurs
almost simultaneously with the disappearance of the polar pit. The vegetal region is
less undulated since the egg elongates again, forming a lobe at the pole. This is fol-
lowed by the ejection of the second polar body. According to the Jeffery and Meier
model (1983) the protrusion is formed by a tight contractile ring of actin filaments
that seems to push the endoplasm towards the animal pole creating a myoplasmic
lobe in the vegetal pole. In the egg which has regained the spherical shape, microvilli
concentrate to form a protuberance at the end of the vegetal hemisphere. Supernu-
merary spermatozoa begin to collect in this region. In fact, the spermatozoa follow
the surface movements towards the vegetal pole, as do test cells (Conklin, 1905),
chalk granules (Ortolani, 1955), and lectins (Monroy etal. 1973; O'Dell et ai, 1974;
Ortolani et al, 1977; Zalokar, 1980). The migration of these external components
appears to be coordinated with that of the myoplasm with which pigment granules,
mitochondria, ribosome-like granules, subcortical granules, and filamentous struc-
tures cosegregate (Conklin, 1905; Reverberi, 1956; Berg and Humphreys, 1960;Man-
cuso, 1964; Sawada and Osanai, 1981; Jeffery and Meier, 1983).
When the egg is mature it is again spherical. Its surface is undulated primarily in
the animal region and exhibits an almost homogeneous distribution of elongated
microvilli; a small clump of spermatozoa stays at the vegetal pole.
We did not observe development after the fusion of the pronuclei began. We sug-
gest that the elongation and diffusion of the microvilli might constitute a reserve
of plasma membrane for the two cell stage. In Halocynthia roretzi (Satoh and Deno,
hemisphere showing the vegetal pole covered by clumped microvilli (asterisk). 620X. Figure 23: Magnifi-
cation of the equatorial area showing stumpy microvilli. 3300X. Figure 24: Magnification of the clumped
microvilli at the vegetal pole. 2550X.
FIGURES 25-28. SEM of eggs fixed 30 min after fertilization. Figure 25: Distribution of folded areas
in animal and vegetal hemispheres: the vegetal pole is marked by clumping of supernumerary spermatozoa
(asterisk). 580x. Figure 26: View of the vegetal hemisphere showing the polar zone covered by spermatozoa
(asterisk). 580X. Figure 27: Magnification of elongated microvilli. 4730X. Figure 28: Magnification of the
vegetal polar zone showing intact spermatozoa. 4980X.
364 L. VILLA AND E. PATRICOLO
1984) the appearance and disappearance of microvilli is associated with cleavage
cycles.
Disappearance, concentration, and elongation of the microvilli in different re-
gions of the activated egg could be the expression of a dynamic condition of the
plasm:, membrane. Not only can the microvilli be considered a reserve of membrane,
but they could also reflect a reorganization of the cell surface on which cytoplasmic
events depend.
Changes in density, distribution, and organization of the microvilli have been
observed after fertilization in sea urchin (Eddy and Shapiro, 1976; Schroeder, 1979;
Longo, 1986), fish (Iwamatsu, and Keino, 1978), mouse (Nicosia et al, 1978), and
amphibian eggs (Monroy and Baccetti, 1975; Charbonneau and Picheral, 1983). Lo-
cal surface differentiation consisting of special microvilli occur on the polar lobes in
the egg of some gastropods (Dohmen and Van der Mey, 1977); it is therefore sug-
gested that a relationship exists between the surface structures and the localization or
expression of the morphogenetic factors in the polar lobes.
In ascidian eggs the cell surface elements seem to be connected to the cytoskele-
ton; the presence in the cortex of a contractile actin-network which produces the force
causing cytoplasmic movements has been demonstrated in Stye/a, Boltenia, and Ci-
ona eggs (Jeffery and Meier, 1983, 1984; Sawada and Osanai, 1984, 1985). According
to Jeffery (1984), ooplasmic segregation includes the movements of cell surface com-
ponents, cytoskeleton, cytoplasmic organelles, and localized maternal mRNA mole-
cules, which are associated in a cytoplasmic complex. These authors, therefore, pro-
posed that the cortical contraction is the main cause of the polarized ooplasmic move-
ments in the ascidian species.
Several other results from studies ofAscidia malaca and Phallusia mammillata
eggs suggest that a pattern of developmental information is localized in the plasma
membrane; an early surface specialization which reflects a cytoplasmic compartmen-
talization of morphogens may also exist (Monroy et al., 1973; CTDell et al, 1974;
Ortolani et al, 1977; Zolokar, 1980). The lack of external K+ ions affects cellular
activities by acting directly on the membrane of the unfertilized egg; the probable
rearrangement of the membrane structures provokes, among other things, a change
in the detectability of Con A binding sites (Di Pisa et al., 1 982). The role of the plasma
membrane during embryonic development has been demonstrated by the differenti-
ation of neural structures and of tissue-specific enzyme obtained through trypsin in-
duction (Ortolani et al., 1979).
Based on these considerations we suggest that in ascidian eggs the cell membrane
components and related topographic changes could be the first cause of the ooplamsic
movements, which are driven by the contractile actin-network connected to the
plasma membrane; therefore, ascidian ooplasmic segregation could depend on sur-
face reorganization.
ACKNOWLEDGMENTS
We thank the Directors of the Institutes of Pathological Anatomy and Human
Anatomy (Faculty of Medicine, University of Palermo) for the use of the scanning
electron microscopes.
We would particularly like to thank Drs. Maria Campione (Pathological Anat-
omy) and Vito Marciano (Human Anatomy) for their invaluable technical assistance.
This work was supported by grants from M.P.I. (40% 1986) and CNR (Ct.
86.00814.04).
SURFACE CHANGES IN ASCIDIAN FERTILIZATION 365
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THE GREEN HYDRA SYMBIOSIS: ANALYSIS OF
A FIELD POPULATION
G. MULLER-PARKER* AND R. L. PARDY
School of Biological Sciences. University of Nebraska, Lincoln. Nebraska 68588-0118
ABSTRACT
Green hydra were abundant on the alga I 'aucheria taylorii in a shallow woodland
stream near Lincoln, Nebraska, from March to June 1985. Green hydra were also
found in low numbers throughout the winter. The algal-animal biomass characteris-
tics of field populations of green hydra are compared to those of cultures established
from the field populations and maintained under defined laboratory conditions. Al-
though of similar protein biomass, freshly collected hydra contained greater numbers
of symbiotic algae than did cultured hydra. Algae in field hydra were larger and con-
tained more chlorophyll than algae in cultured hydra. Field populations of green
hydra were highly productive; 16 ;ug C-h~' -mg hydra protein'1 were fixed by the
endosymbiotic algae at an irradiance of 28 ^E • m~2 • s" ' .
INTRODUCTION
The green hydra-Chlorella association has been intensively studied for two de-
cades. The association involves C/ilore/la-\ikQ algae living within the digestive cells
of the freshwater polyp Hydra viridissima. It is one of a group of algal-invertebrate
associations called phycozoans (Pardy, 1983) to emphasize their algal-animal com-
posite nature. Much is known about the metabolic interdependency of the bionts,
the recognition processes whereby hydra acquire algae, and some of the regulatory
processes which stabilize the association. Comprehensive reviews concerning the as-
sociation may be found in Cook ( 1 980, 1981,1983).
The majority of experimental studies have used laboratory cultures of green hy-
dra. These hydra have been cultured under defined conditions with respect to temper-
ature, light intensity, and feeding and maintenance schedules. Such stringent culture
methods yield populations of green hydra of uniform size and age distributions, with
stable densities of symbiotic algae. Although these laboratory cultures of green hydra
are useful for certain experimental studies, they may bear little resemblance to green
hydra living in ponds and streams. While a variety of ecological studies (Welch and
Loomis, 1924; Miller, 1936; Bryden, 1952; Carrick, 1956; Cuker and Mozley, 1981;
Ribi el al, 1985) have attempted to describe the seasonal distribution and abundance
of non-symbiotic hydra, similar studies of green hydra are lacking. Although green
hydra have been collected from a variety of habitats [standing water (Whitney, 1907;
Lashley, 1915), a swamp (Carrick, 1956), a river (McAuley, 1984), and roadside
ditches (Forrest, 1959)], none of these studies have described the algal-animal bio-
mass characteristics of freshly collected hydra.
In this paper we analyze the biomass parameters of symbiotic algae in field popu-
lations of green hydra, and compare these parameters to those of cultures established
Received 13 April 1987; accepted 22 July 1987.
* Present address: University of Maryland, C.E.E.S., Chesapeake Biological Laboratory, Solomons,
Maryland 20688-0038.
367
368 G. MULLER-PARKER AND R. L. PARDY
from the field populations and maintained under defined laboratory conditions. We
also describe the seasonal variation in algal-animal biomass parameters of green hy-
dra, and provide some estimates of the productivity of field populations. The results
show that field populations of green hydra contain greater numbers of algae and are
far more productive than hitherto measured in laboratory-cultured green hydra.
MATERIALS AND METHODS
Location of study
A small stream near a large reservoir (Pawnee Reservoir) 10 miles northwest of
Lincoln, Nebraska (40°50'37"N, 96°5 1'37"W) was chosen as a study site, as green hy-
dra were known to occur in this stream (Pardy and Glider, 1984). The spring-fed
stream (Pawnee stream) extended 1 .5 km above the study site. There were two small
ponds upstream from the site, one 122 m long and the other 50 m long. At the study
site the water depth varied from 15 cm to 60 cm, depending on local rainfall. The
bottom was muddy and often filled with litter from overhanging trees.
Collection and maintenance of hydra
Leaf litter and attached submerged vegetation were collected from the bottom of
Pawnee stream; floating Lemna sp. plants were collected when present. As hydra
were initially found in great abundance on algal mats of the filamentous chrysophyte
Vaucheria taylorii, these mats were collected on a regular basis and examined for
green hydra. Hydra were routinely sampled by removing four to five mats, about 200
cm2 each, of Vaucheria taylorii and placing these into separate containers. In the
laboratory, green hydra detached from the algal filaments and were readily collected
with a Pasteur pipette. All analyses on hydra were performed within 24 h after col-
lection.
To compare the biomass parameters of freshly collected hydra (Afield hydra) with
those of hydra maintained under defined conditions in the laboratory, hydra collected
in March 1985 were brought into culture. These hydra were slowly acclimated to M
solution (Muscatine and Lenhoff, 1965) by gradually replacing stream water with M
solution. Cultures were then maintained under continuous light at two irradiances
(5 and 30 ;uE • irT2 • s~ ' ) at 21°C and fed three times each week with freshly hatched
Artemia nauplii. The culture medium was replaced daily; twice daily on feeding days.
After 1 5 months of laboratory culture, population growth rates of Pawnee hydra
maintained at 5 and 30 ME-m~2-s~' were measured as described by Muller-Parker
and Pardy (1987). Population growth rate constants and doubling times were calcu-
lated according to Loomis (1954).
Ultrastructure of symbiotic algae
Transmission electron microscopy was used to examine the ultrastructure of sym-
biotic algae in freshly collected hydra. Hydra collected on March 18, 1985 were cut
into pieces in phosphate buffered (0. 1 M, pH 6.8) 3% glutaraldehyde at room temper-
ature. The segments were fixed for 2 h in the glutaraldehyde fixative, then post-fixed
with 1% OsO4 in buffer for 1 h. Following dehydration in a graded series of ethanol,
specimens were embedded in Epon 812. Sections stained in 2% aqueous uranyl ace-
tate and lead citrate were viewed in a Philips 20 1 electron microscope operated at
60 kv.
GREEN HYDRA SYMBIOSIS 369
Hydra biomass parameters
Algal and animal biomass parameters (protein, number of algae per /^g hydra
protein, number of algae per hydra digestive cell, algal chlorophyll and algal cell vol-
umes) were measured on pooled samples of 100 hydra using previously described
procedures (Muller- Parker and Pardy, 1987), with the exception that from 5 to 25
hydra were pooled to measure the number of algae per digestive cell. Biomass param-
eters of laboratory cultures of Pawnee hydra were measured after 15 months of cul-
ture. These hydra were last fed 48 hours before analysis.
Productivity of field hydra
The productivity of Pawnee hydra collected on April 3 and June 3, 1985 was
measured in the laboratory. Carbon fixation at various irradiances was measured 24
h after collection. Groups of 25 hydra, each with one bud for uniformity, were incu-
bated with NaH14CO3 (0.8 ^Ci-ml ') in 5.0 ml of filtered (>0.45 /mi) streamwater
supplemented with 5 mM NaHCO3 in glass beakers covered with various layers of
screen. Replicate groups of hydra were incubated in the dark to correct for any dark
fixation of NaH14CO3 . Incubation media were sampled at the start of the experiments
for total 14C activity. At the end of the one-hour incubations hydra were thoroughly
rinsed in cold, filtered streamwater and then homogenized in distilled water. Organic
14C retained by hydra, protein biomass, and numbers of algae were determined as
previously described (Muller-Parker and Pardy, 1987). Total CO2 in filtered stream-
water was calculated from the total alkalinity-measured potentiometrically (Goiter-
man, 1969) — of samples of filtered streamwater collected on July 17, 1986. After
correction for dark fixation, the amounts of 14C retained by hydra tissues were con-
verted to rates of carbon fixation (Vollenweider, 1969) normalized to hydra protein
biomass and to numbers of algae.
RESULTS
Distribution of hydra in Pawnee stream
The distribution of green hydra in Pawnee stream from March 1985 to May 1986
was highly variable and appeared related to the presence of the filamentous chryso-
phyte alga, Vaucheria taylorii (Blum, 1971). Green hydra on V. taylorii were not
immediately obvious, as the slender polyps and color of hydra bore remarkable re-
semblance to the algal filaments. Other samples of submerged vegetation yielded few
hydra although individuals were occasionally found anchored to Lemna leaves as
found by Pardy and Glider ( 1 984).
Specimens of Vaucheria taylorii were identified by the characteristic structure of
the antheridia and oogonia (Blum, 1971). These reproductive structures developed
frequently in algae maintained in the laboratory at 2 1 °C under continuous light. Thus
it was possible to verify the taxonomic identity of this alga from various collections
made throughout the year. This alga (formerly named Vaucheria geminata var. ra-
cemosa) has been reported from creeks around Lincoln, Nebraska (Saunders, 1894).
V. taylorii was common in shallow, unshaded areas of the stream where there was
slow water flow. The depth of the water above algal mats varied from 1 to 12 cm.
Peak abundances of field hydra averaged about 500 hydra per 200 cm2 mat of V.
taylorii, and occurred from March to June 1985. From June to September 1985 the
stream occasionally dried up and did not contain aquatic vegetation or hydra. The
site was not visited until January 1 986, at which time V. taylorii was abundant and
370
G. MULLER-PARKER AND R. L. PARDY
FIGURE 1 . Algal symbiont in green hydra freshly collected from Pawnee stream. Scale bar = 1
hydra were found in low densities (1-10 individuals per 200 cm2 mat). Hydra per-
sisted on V. taylorii throughout the winter and spring of 1 986. At all times, algal mats
contained large numbers of zooplankton and the hydra were frequently observed to
feed on these. Large nonsymbiotic brown hydra were often found among the green
hydra, but never in great abundance.
All green hydra collected in March and April 1985 were asexual. Many had from
one to five buds per individual. By early May, half of the hydra were sexual, bearing
both ovaries and testes. Ninety-five percent of the collected hydra had gonads in late
May. In early June the number of hydra bearing gonads had decreased to 50%; most
of these bore testes only. Embryonic thecae resembled those described by McAuley
(1984) for Hydra viridissima. A two-chambered theca, characteristic of Chlorohydra
hadleyi (Forrest, 1959), was never seen. Released eggs did not develop under labora-
tory conditions.
Morphology, size, and chlorophyll content of algal symbionts from green hydra
The algal symbiont of hydra collected from Pawnee stream resembles the algae
found in the English strain (Pardy, 1976; Jolley and Smith, 1978) of green hydra in
that it possesses a pyrenoid traversed by a single thylakoid (Fig. 1 ).
The size of algae in field and cultured populations of green hydra is given in Table
GREEN HYDRA SYMBIOSIS
371
TABLE I
Si:e of symbiotic algae in green hydra either (a) freshly collected or (b) maintained
in culture for 15 months
Algal diameter
(urn)
Algal cell volume
(Mm3)
(a) March 17, 1986
May?, 1986
4.38(±0.93)a
3.44 (±0.85)
44
21
(b) Low light (5 ME -nT2^1)
High light (30 M£- m2-s-')
2.90 (±0.55)
2.59 (±0.41)
13
9
±SD;n = 100.
I. Algae varied greatly in size in hydra collected on two dates in 1986, but cell volumes
of algae in freshly collected hydra were at least twice those of algae in cultured green
hydra (Table I).
Algae in field hydra contained more than four times the amount of chlorophyll
measured in algae from cultured hydra (Table II). Although the chlorophyll content
of algae varied greatly between cultured and field hydra, the ratio of chlorophyll a to
chlorophyll b was about one in algae obtained from all hydra (Table II).
Algal-animal biomass parameters of green hydra
Algal numbers in hydra are readily obtained by counting the number of algae in
dissociated digestive cells. Figure 2 shows the variation in numbers of algae in diges-
tive cells in hydra collected in 1985 and 1986 from Pawnee stream. The average
number of algae per cell ranged from 14 (March 1986) to 34 (June 1985). Ambient
stream temperatures are also given in Figure 2. There appears to be no relationship
between algal densities in green hydra and water temperature. Hydra collected in
near-freezing waters (January and February 1 986) under substantial layers of ice and
TABLE II
Chlorophyll content of algae isolated from green hydra either (a) freshly collected in 1985
or (b) maintained in culture for 15 months
Total
Chi- cell' '
Chi a -cell"1
Chi b -cell'1
(Pg)
(Pg)
(Pg)
alb
(a) March 18
0.63
0.32
0.31
1.01
March 20
0.80
0.44
0.36
1.20
April 3
0.68
0.35
0.33
1.06
May 7
0.77
0.40
0.37
1.05
May 22
0.45
0.21
0.23
0.92
June 3
0.49
0.28
0.21
1.34
(b) Low light
0.15(±.02)a
0.08 (±.01)
0.07 (±.01)
1.14 (±.06)
High light
0.04 (±.01)
0.02 (±.004)
0.02 (±.006)
1.16(±.24)
±SD; n = 5.
372
G. MULLER-PARKER AND R. L. PARDY
35
S 25
I
to
0)
o
CT
20
15
14
17 15 13
I
3 6 14 19 19
I
MAR APR MAY JUNE
1985
JAN FEB MAR APR MAY ' LAB
1986
FIGURE 2. Number of algae per digestive cell (open circles, ± SE) in hydra collected from Pawnee
stream in 1985 and 1986. Ambient streamwater temperatures measured on the collection dates are in-
cluded near the origin of the y-axis. For comparison, the mean numbers of algae per digestive cell of
cultured Pawnee hydra maintained at 5 /uE • m 2-s ' (solid triangles) and at 30 /uE-m 2-s '
are included.
(open triangles)
snow contained a full complement of algae (Fig. 2). The numbers of algae per diges-
tive cell of cultured hydra are included in Figure 2 for comparison. Digestive cells of
cultured hydra contained under 20 algae, whereas 50 or more algae per digestive cell
were frequently counted in freshly collected green hydra. In general, digestive cells of
field populations of these green hydra contained greater numbers of symbiotic algae
than those of hydra maintained in laboratory culture.
The density of symbiotic algae in freshly collected hydra was also measured by
determining the number of algae per jug hydra protein in 1985 (Fig. 3a). Algal densi-
ties varied two-fold over a four month period; algal densities measured in June were
double those obtained in March. This change in algal density coincided with a shift
from a predominantly asexual population to a predominantly sexual population. Al-
gal densities in cultured hydra were within the range obtained for field hydra; hydra
maintained at a low irradiance contained more algae than those maintained at a high
irradiance (Fig. 3a).
The size of field hydra was estimated by measuring the weight of protein per hydra
individual. Figure 3b shows that the protein content of field hydra ranged from 2 to
8 jug protein, and that the increase in algal density (Fig. 3a) was accompanied by a
decrease in protein content. Thus, hydra collected in June 1985 were smaller and
contained higher densities of algae than those collected in March 1985.
Laboratory cultures of Pawnee hydra contained high protein biomass and low
algal densities in comparison to field populations (Fig. 3a, b). Hydra maintained at 5
• rrT2 • s"1 were larger and contained more algae than individuals maintained at 30
GREEN HYDRA SYMBIOSIS
373
o
*.4
Tc
3
0)
o 2
e
t4
0.2
. (a)
-(b) q
6— -O,
O
MARCH
APRIL
MAY
1985
JUNE
LAB
FIGURE 3. (a) Number of algae per ng hydra protein in hydra collected from Pawnee stream in
1985 (open triangles), (b) Protein biomass of hydra collected from Pawnee stream in 1985 (open circles).
Parameters obtained in cultured hydra (Lab) maintained at 5 ^E-m":-s ' (solid symbols) and at 30 nE-
m 2 • s ' (open symbols) are included to the right of the field data.
/uE-rn 2-s '. Population growth rate constants of Pawnee hydra maintained at both
irradiance levels were the same and averaged 0.28 day"1, which resulted in a doubling
time of 2. 5 days.
Productivity of field hydra
The productivity of green hydra collected from Pawnee stream on April 3, 1985
and on June 3, 1985 was measured in the laboratory. The amount of carbon fixed by
these hydra increased linearly with increase in irradiance from 0 to 30 ME-m~2-s~'
(Fig. 4). Photosynthetic efficiencies (the slopes of the lines in Fig. 4) were normalized
to protein biomass and number of algae. Photosynthetic efficiencies of April hydra
were 0.45 jug C • (^E • m~2 • s~ ' )" ' • h ' • mg ' protein and 0.022 ^g C •
h~ ' • 1 06 algae^ ' , whereas those of June hydra were 0.60 jug C • (yuE • m"2 • s~ ' )~ ' • h
m~ • s~ ' )
~ ' ~ '
mg ' protein and 0.0 16
-IT1 • 106 algae"1. Hydra collected on
these two dates differed greatly in algal density (Fig. 3a) and in protein biomass (Fig.
3b). The hydra collected on April 3 contained twice the amount of protein and half
the number of algae as hydra collected on June 3, 1985. Figure 4a shows that on a
protein basis the smaller hydra with greater numbers of algae (June hydra) were more
productive than the April hydra. When the data were normalized to numbers of algae,
hydra with low densities (April hydra) were more productive on a cell basis (Fig. 4b).
These results suggest that although high algal densities result in greater productivity
on a unit biomass basis, the amount of carbon fixed per alga is greatly reduced.
DISCUSSION
We have shown that field populations of symbiotic hydra are highly productive
and maintain high densities of algae throughout the year. There may be significant
374
G. MULLER-PARKER AND R. L. PARDY
18
I '4
"o
Q.
O
10
o
0>
T 0.6
0>
o
&•
O
VD
QQ.4
!c
6
o>
3-0.2
(a)
i i i i i
(b)
10 20 30
IRRADIANCE (/uE rrf2.SH)
FIGURE 4. Productivity of field hydra at different irradiances. Rates were normalized to (a) protein
biomass and (b) numbers of algae. Open circles represent production rates of hydra in April 1985 and
closed circles are those obtained for hydra in June 1 985.
differences in the algal and animal biomass parameters and productivity of field and
laboratory populations of green hydra. Symbiotic algae isolated from field popula-
tions of green hydra are larger and contain more chlorophyll than those isolated from
cultured hydra. When maintained under controlled laboratory conditions, the algal-
animal biomass parameters and growth rates of these hydra resemble those of the
Florida hydra strain (Mu Her- Parker and Pardy, 1987) kept under the same con-
ditions.
Algal-animal biomass parameters
The biomass parameters of green hydra collected in 1985 and 1986 from one site
were quite variable. Variation in protein biomass of hydra, in numbers of algae, and
size and chlorophyll content of the algae can result from several processes which are
not mutually exclusive. Changes in the physical environment (light, temperature,
water flow) and in prey availability may affect these parameters. Genetic differences
in hydra populations collected at different times may be important (McAuley, 1984).
The relative influence of these factors cannot be presently assessed.
GREEN HYDRA SYMBIOSIS 375
Hydra were found in Pawnee stream throughout the winter. Others have found
both symbiotic hydra (Whitney, 1907) and non-symbiotic hydra (Carrick, 1956) in
water of very low temperatures. However this is the first report which shows that
green hydra maintain high densities of symbiotic algae throughout the winter.
There was a great increase in algal density and decrease in protein biomass of field
hydra during the spring of 1985 (Fig. 3a, b). At the same time these hydra shifted
from a predominantly asexual population to a sexual population. This suggests that
sexual individuals may contain higher densities of algae than non-sexual hydra.
This needs to be confirmed in hydra maintained under controlled conditions in the
laboratory.
The most striking difference between the algal-animal biomass parameters of
freshly collected and cultured Pawnee hydra was the large difference in algal cell vol-
ume (Table I) and chlorophyll content of the algae (Table II). The large size of the
algae and the great number of algae per digestive cell (Fig. 2) in freshly collected
hydra show that these hydra contain a proportionately greater ratio of plant to animal
biomass than cultured hydra. This suggests that regulatory processes governing the
number of algae in hydra cells may be substantially different under field conditions.
Hydra collected from Pawnee stream and maintained in culture for 15 months
showed similar responses to light as the Florida strain of hydra maintained under the
same conditions (Muller-Parker and Pardy, 1987). In both Pawnee and Florida hy-
dra, protein biomass and algal densities decreased with increase in culture irradiance,
whereas population growth rates were unaffected by irradiance. An increase in algal
cell volume and decrease in chlorophyll per alga occurred in both Pawnee and Florida
hydra with increase in culture irradiance. These results suggest that green hydra from
different localities respond in a consistent manner to changes in culture irradiance.
Pawnee hydra were smaller than Florida hydra as the protein biomass of cultured
Pawnee hydra maintained at 5 and 30ME-m~2-s~' (7.6 and 6.3 /^g) was less than that
of Florida hydra maintained at the same irradiances ( 1 1 .0 and 9.5 yug; Muller-Parker
and Pardy, 1987). The population growth rates of Pawnee hydra were slightly higher
than those of Florida hydra; population doubling times for Pawnee hydra were 2.5
days whereas those of Florida hydra averaged about 3 days. Algal cell volume and
the number of algae per digestive cell were similar for both Pawnee and Florida hydra.
Productivity of field collected Pawnee hydra
The productivity of hydra collected from Pawnee stream on two dates was high;
rates of carbon fixation were over four times as great as those obtained with cultured
Florida hydra incubated under similar conditions (Muller-Parker and Pardy, 1987).
Although field Pawnee hydra and cultured Florida hydra are not strictly comparable,
the productivity of cultured Pawnee hydra was not measured in this study. The previ-
ous light history may affect photosynthetic rates, as photosynthetic efficiencies de-
rived for Pawnee hydra were greater than those obtained for cultured Florida hydra
(Muller-Parker and Pardy, 1987). Differences in productivity cannot be attributed to
differences in protein biomass and density of algae of field and laboratory hydra, since
Pawnee hydra collected in April and June 1985 were substantially different in these
two parameters. However, algal cell size and chlorophyll content of algae were much
greater in freshly collected Pawnee hydra than in cultured Florida hydra, which may
account for the high productivity of Pawnee hydra.
ACKNOWLEDGMENTS
We thank K. Lee for the transmission electron micrograph (Fig. 1 ) and J. Rosow-
ski for identification of Vaucheria taylorii. Research supported by grants from the
376 G. MULLER-PARKER AND R. L. PARDY
National Science Foundation (PCM 8314590) and the University of Nebraska-Lin-
coln Research Council to R. L. Pardy.
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Reference: Biol. Bull. 173: 377-386. (October, 1987)
ZOOPLANKTON FEEDING ECOLOGY: CONTENTS OF FECAL
PELLETS OF THE COPEPOD CENTROPAGES VELIFICATUS FROM
WATERS NEAR THE MOUTH OF THE MISSISSIPPI RIVER
JEFFERSON T. TURNER*
National Marine Fisheries Service, NOA4, Beaufort. North Carolina 285 16
ABSTRACT
The in situ diet of the copepod Centropages velificatus was investigated using
scanning electron microscopy. Contents of fecal pellets produced upon natural diets
were compared with assemblages of available phytoplankton. Samples came from
continental shelf waters near the mouth of the Mississippi River. A wide variety of
particle sizes and types were ingested. The dominant phytoplankton taxa in pellets
sometimes mirrored those dominant in the water, particularly if these were large soli-
tary cells or elongate chains of smaller cells. However, in other cases the dominant
phytoplankton remains in pellets were those of cells that were not abundant in the
water. Crustacean remains and fine particles of silt from the river plume were also
frequent dominant components. C. velificatus is clearly an omnivore.
The often poor correspondence between ingested and abundant phytoplankton
taxa and the frequency of crustacean remains suggests that this copepod spends much
of its time feeding as a raptorial carnivore upon other zooplankton.
INTRODUCTION
Omnivorous feeding is common in planktonic marine copepods. Numerous spe-
cies ingest both phytoplankton and zooplankton (reviewed by Turner, 1984a), and
some large copepods can eat larval fish (Turner et ai, 1985 and references therein).
However, most studies of copepod omnivory have used laboratory diets such as cul-
tured phytoplankton and zooplankton. Examinations of the diets of omnivorous co-
pepods feeding upon natural food assemblages have been less frequent.
As part of a study of planktonic food webs that support larval fish in northern
Gulf of Mexico continental shelf waters, an effort has been made to define diets of
copepod species that are prey of fish larvae (Turner, 1984b, c, d, 1985, 1986a, 1986b).
Contents of fecal pellets produced from food ingested prior to capture have been
compared with co-occurring phytoplankton species assemblages. A general pattern
that has emerged for many calanoid species is that of indiscriminant suspension feed-
ing; phytoplankton taxa in fecal pellets reflect the composition of the natural phyto-
plankton. However, certain copepods such as the large calanoid Labidocera aestiva
(Turner, 1 984c) or several cyclopoid species (Turner, 1 986b) did not exhibit this pat-
tern as strongly as other typically herbivorous species. The fecal pellets of L. aestiva
and the cyclopoids contained considerable crustacean remains, and disproportion-
ately high abundances of large phytoplankton cells that were not particularly abun-
dant in the water. These results suggest that these copepods are primarily raptorial
omnivores, feeding frequently on other animals and large phytoplankton cells.
Received 23 October 1986; accepted 22 July 1987.
* Present address: Biology Department, Southeastern Massachusetts University, North Dartmouth,
Massachusetts 02747.
377
378 J. T. TURNER
In addition to the largely qualitative fecal pellet studies, we have performed quan-
titative grazing studies on three species of calanoid copepods: Acartia tonsa, Eucala-
nus pileatus, and Centropages velificatus (Turner and Tester, 1986; Tester and
Turner. 1 986). Although A. tonsa and E. pileatus exhibited nonselective grazing, with
phytoplankton taxa being ingested in proportion to their abundance, this pattern
did not hold for C. velificatus. Regression coefficients between rates of ingestion of
individual phytoplankton taxa and abundance of those taxa, or between total phyto-
plankton ingestion rate and total phytoplankton abundance were low. This suggested
that C. velificatus was feeding primarily as a carnivore, a detritivore, or as a selective
herbivore upon less-abundant phytoplankton. Accordingly, the contents of C velifi-
catus fecal pellets produced at these and other northern Gulf of Mexico stations were
examined with the scanning electron microscope (SEM).
Centropages velificatus Oliveira is widely distributed throughout the tropical and
subtropical western Atlantic and Gulf of Mexico (Fleminger and Hulsemann, 1973).
This species is the Atlantic cognate of the Indo-Pacific species Centropages furcatus
Dana (Fleminger and Hulsemann, 1 973). Thus, numerous Atlantic and Gulf of Mex-
ico records for C. furcatus (Owre and Foyo, 1967 and references therein; Bowman,
197 1 ; Paffenhofer and Knowles, 1980) apply to C. velificatus. This copepod is one of
the numerically dominant mesozooplanktonic calanoids in continental shelf waters
of the northern Gulf of Mexico (Marum, 1979; Minello, 1980), and it is consumed
in these waters by several species offish larvae (Govoni et al., 1983).
Various Centropages species have long been known to be omnivores, ingesting
both cultured phytoplankton. Anemia nauplii, or cultured copepod nauplii (re-
viewed by Turner, 1984a). Paffenhofer and Knowles (1980) found that C. velificatus
from the southeastern United States continental shelf ingested a higher percentage of
its body nitrogen in the form of laboratory-reared copepod nauplii (Pseudodiaptomus
coronatus) than as large diatoms of the genus Rhizosolenia. However, the temperate
congener C. hamatus ingested a higher percentage of its body carbon as natural phy-
toplankton than as field-caught copepod nauplii (mostly Acartia spp.) (Conley and
Turner, 1985). In addition, C. typicus has been shown to ingest yolk-sac larvae of two
fish species (Turner et al., 1985).
Results presented here support previous Centropages feeding studies, as well as
the suggestion from our quantitative grazing data (Turner and Tester, 1986; Tester
and Turner, 1 986) that C. velificatus is a raptorial omnivore, feeding to a great extent
upon large phytoplankton cells and other crustaceans. This is, to my knowledge, the
first study of the in situ feeding habits of this copepod.
MATERIALS AND METHODS
Samples were collected from ten stations (Table I) on three cruises in 1982 and
1983. Surface water samples (500 ml) for phytoplankton analyses were preserved in
UtermohPs solution (Guillard, 1973) immediately prior to zooplankton tows. Sur-
face tows with 363 yum mesh nets were used for copepod collection. The mesh was
coarse enough to allow passage of all phytoplankton and microzooplankton, thereby
preventing possible net feeding. Copepods were immediately sorted by pipette and
isolated in surface seawater within 5- 1 5 min of collection. After pellets reflecting gut
contents upon capture had been produced (usually < 0.5 h), they were individually
removed by pipette, placed in a mixture of filtered seawater and 20 jum-mesh-
screened surface water (containing natural microbes), and left for 24-36 h at approxi-
mately 20°C for microbial stripping of pellet peritrophic membranes, which if left
intact, mask contents (Turner, 1978; 1979). Pellets were preserved in 5% formalin:
seawater solutions for analyses ashore.
ZOOPLANKTON FEEDING ECOLOGY
379
100
- 80
ro
O
- 60
CO
40
20
0
100
DIATOMS
DINOFLAGELLATES
3FEB
1982
(2000)
I5DCC
1982
0930)
I9NOV
1963
(1400)
22NOV
1983
(2035)
24NOV
1983
(0605)
24NOV
1983
(1340)
27NOV. 28NOV. 3ONOV I DEC.
1983 1983 1983 1983
(0900) (0905) (0920) (083O)
Z
O
t 50
o
a.
O
o
O
m
o
m
,
C - Coscinodiscus spp.
DB-Ditylum brightwellii
NC'Nitzschio dosterium
PC-Prorocentrum compressum
RA-Rtiizosolenia alata
RF-Rhizosolenia fragilissima
RS-Rhizosotenia stolterfothii
SC'Skeletonema costatum
TL'Thalassiosira spp.
TX-Thdassiothrix spp.
|^| OTHER DIATOMS
• DINOFLAGELLATES
FIGURE 1. Abundance (upper) and composition (lower) of natural phytoplankton assemblages in
surface waters at sampling stations.
Preserved pellets were individually sorted by pipette and drawn onto Whatman*
GFC glass fiber filters. Filters with attached pellets were washed in distilled water for
salt elimination, dehydrated in a graded ethanol series, critical point dried, coated
with gold:palladium, and examined with a ISI-30 SEM at 15 kV.
All fecal pellets were from Centropages velificatus adult females. One hundred
twenty-two (122) micrographs were taken from 38 fecal pellets. SEM examination
preceeded phytoplankton analyses to avoid possible bias in characterization of pellet
contents. Entire visible sides of each pellet were examined. The 26 micrographs pre-
sented here (21% of those taken) are representative of pellet contents. Phytoplank-
ton analyses were made with the Utermohl inverted microscope technique (Lund
etai, 1958).
RESULTS
Abundance and species composition of surface phytoplankton varied considera-
bly (Fig. 1). Abundance ranged from 5.4-105.1 X 103 cells/1. With the exception of
* Reference to trade names does not imply endorsement by the National Marine Fisheries Service,
NOAA.
380 J. T. TURNER
TABLE I
Locations, limes, and surface salinities of sampling stations
Date
Local
time
Latitude
(N)
Longitude
(W)
Surface salinity
(%•)
5 Feb. 1982
2000
28°53'
89°29'
21.5*
15 Dec. 1982
1930
28°54'
89°28'
18.1*
1 9 Nov. 1983
1400
28°52'
89°29'
*
22 Nov. 1983
2035
28°50'
98°30'
31.5
24 Nov. 1983
0605
28°53'
89°29'
31.8
24 Nov. 1983
1340
28°51'
89°25'
29.6*
27 Nov. 1983
0900
29°02'
89°30'
26.1
28 Nov. 1983
0905
28°48'
89°57'
26.6
30 Nov. 1983
0920
28°48'
89°58'
30.7
1 Dec. 1983
0830
28°52'
89°29'
21.0*
* Stations with a high silt load.
two stations, assemblages were dominated by diatoms. However, on 15 December
1982, the dinoflagellate Prorocentrum compression comprised 17% of total cells, and
on 1 December 1983 a combination ofthedinoflagellatesP. compression, P. micans,
and Gyrodinium sp. comprised 35% of cells present. Abundant diatoms included
large solitary cells such as Ditylum brightwellii (16.5 x 148.5 ^m), Coscinodiscus sp.
(33-53 /urn diameter), and Thalassiosira sp. (13-36 ^m diameter). Also abundant
were various chain-forming diatoms such as Rhizosolenia fragilissima, R. stolter-
fothii, and Skeletonema costatum.
In addition to phytoplankton, silt from the plume of the Mississippi River was
abundant in surface waters from half of the stations (Table I).
Contents of fecal pellets varied widely among stations. At three stations from the
river plume (5 February 1982, 19 November 1983, and 24 November 1983 at 1340
h) silt was the only component of fecal pellets (Figs. 2a, b, c), despite the presence of
abundant phytoplankton (Fig. 1 ). Water samples from the station on 15 December
1982 also contained a heavy silt load, but fecal pellets contained primarily cells of the
large (33 /um diameter) dinoflagellate Prorocentrum compression (Fig. 2d) in addition
to silt. It is interesting that at this station P. compression comprised only 11% of
cells counted, whereas the similarly large (33-53 yum diameter) diatom Coscinodiscus
radiatus comprised 41% of cells counted, but no C. radiatus remains were observed
in pellets. At other stations, however, large solitary Gentries of the genus Thalassiosira
(26-33 /um diameter) were present in pellets as either intact cells or cell fragments
(Figs. 2e, f, 3a, b, c, d). Thalassiosira abundance at these stations was high, with
values of 35.7, 23.4, and 6.5 X 103 cells/1 on 24 November, 28 November, and 1
December 1983, respectively, accounting for 74%, 19%, and 56% of cells on these
dates. In addition to Thalassiosira spp., pellets from 24 November 1983 (0605 h)
contained fragments of P. compression cells (Fig. 3e) and remains of crustaceans (Fig.
3f). Crustacean remains, probably of copepod nauplii, were present in pellets from
all remaining stations (Fig. 4c, d, 5d, 6f), along with remains of both small and large
diatoms (Figs. 4a, b, 5a, b, 6a, b, c, d) and dinoflagellates (Figs. 5c, 6e). These three
stations (22 November, 27 November, 30 November 1983) all had intermediate lev-
els of phytoplankton abundance (Fig. 1), intermediate to high salinity, and reduced
silt load (Table I). It is interesting that phytoplankton taxa of long linear dimension
such as solitary Thalassiothrix sp. cells (up to 1 54 jum long) or chains of Skeletonema
costatum (maximum chain length unknown) were the most common phytoplankton
ZOOPLANKTON FEEDING ECOLOGY
381
FIGURE 2. Contents of Centropages velificatus fecal pellets, a. 24 November 1983 ( 1 340), b. 19 No-
vember 1983, c. 5 February 1982 (note peritrophic membrane, PM), d. 15 December 1982 (note peritro-
phic membrane, PM, and Prorocentrum compressum cell, Pr), e. 1 December 1983 (note Thalassiosira sp.
cell at arrow), f. 28 November 1983 (note Thalassiosira sp. cells).
observed in pellets from 27 and 30 November (Figs. 5a, b, 6a, b), even though these
taxa comprised only 3% each, respectively, of the cells in these samples. Small solitary
diatom cells, though sporadically present in pellets (Fig. 4a, b, 6c), were never a domi-
nant component.
DISCUSSION
Centropages velificatus is clearly an omnivore. Fecal pellets representing in situ
feeding contained primarily the remains of other crustaceans and large or elongate
phytoplankton taxa. These field results support previous laboratory studies with the
same species (PafFenhofer and Knowles, 1980) or laboratory and field studies with
382
J. T. TURNER
««£*«*&,•?
FIGURE 3. Contents of Centropages velificatus fecal pellets from the station on 24 November 1983
(0605). a. and b. Thalassiosira sp. cells (T), c. Thalassiosira sp. (T) and small centric (SC) diatom cells, d.
Thalassiosira sp. fragments (T), e. Prorocentrum compression cell (arrow), f. crustacean fragment (arrow).
congeners (Turner 1984a and references therein; Conley and Turner, 1985; Turner
etai, 1985).
The apparently frequent use of carnivory by C. velificatus is also suggested by
quantitative grazing data from the northern Gulf of Mexico (Turner and Tester,
1986; Tester and Turner, 1986). Regression coefficients for grazing versus available
phytoplankton gave poor correlation. Such uncoupling of grazing effort and phyto-
plankton abundance would be expected if C. velificatus feeds primarily as a carnivore,
or selectively upon less-abundant phytoplankters.
Although the presence of primarily large phytoplankton cells in fecal pellets is
suggestive of selective grazing on large particles, such a conclusion is not without
ambiguities. For reasons detailed in Turner (1984c, p. 279) and (1984 d, p. 82), it is
impossible to realistically make SEM analyses of fecal pellets quantitative. Further,
it is not clear why Prorocentrum compression cells were common in pellets but un-
FIGURE 4. Contents of Cenlropages velificatus fecal pellets from the station on 22 November 1983.
a. Fragments of an unidentified diatom (UD) and a small centric (SC) diatom, b. pennate diatom (arrow),
c. and d. crustacean fragments (arrows).
FIGURE 5. Contents of Centropages velificatus fecal pellets from the station on 27 November 1983.
a. and b. Thalassiothrix sp. (T) and Skeletonema costatum (S) fragments, c. unidentified dinoflagellate
fragment (arrow), d. crustacean remains (arrow).
384
J. T. TURNER
FIGURE 6. Contents of Centropages velificatus fecal pellets from the station on 30 November 1983.
a. Skeletonema costatitm fragments (arrows), b. Skeletonema costatum (S) and Thalassiosira sp. (T) frag-
ments, c. intact centric diatom cell (arrow), d. Thalassiothrix sp. fragments (arrow), e. fragment of an
unidentified dinoflagellate (arrow), f. crustacean remains (arrows).
common in the water (11% of total cells) on 1 5 December 1 982, whereas the similarly
large (33 /urn diameter, or larger) diatom Coscinodiscus radiatus was not observed in
these pellets even though it comprised 41% of available cells. In other cases, large
Thalassiosira spp. cells were abundant in pellets, but since they were also abundant
in the water, this pattern is more indicative of nonselective than selective feeding.
Conversely, the disproportionately high abundances in pellets of elongate cells of
Thalassiothrix sp. or Skeletonema costatum cells from elongate chains, when these
taxa comprised only a small proportion of available cells, may indicate size selection
on 27 and 30 November 1983. In short, there was no consistent pattern of ingestion
of large versus small, solitary versus chain-forming, or diatom versus dinoflagellate
cells. The production of fecal pellets containing primarily fine-grained silt particles
reveals that small particles can also be ingested in large numbers. Centropages velifi-
catus apparently employs a variety of mechanisms to capture food from a broad range
ZOOPLANKTON FEEDING ECOLOGY 385
of particle sizes. Such varied feeding mechanisms have been demonstrated with high-
speed cinematography for Centropages typicus (Cowles and Strickler, 1983) and
other copepods (Price et at, 1983).
The raptorial omnivory of Centropages velificatus evidenced by fecal pellet con-
tents is atypical of many other copepod species studied with the same techniques.
Mainly nonselective suspension feeding characterized Eucalanus pileatus (Turner,
1984b; Tester and Turner, 1986), Paracalanus quasimodo (Turner, 1984b), Acartia
tonsa (Turner, 1984c; Tester and Turner, 1986), Temora lurbinata and T. stylifera
(Turner, 1984d), Anomalocera ornata (Turner, 1985), and Undinula vulgaris
(Turner, 1986a). However, the frequent feeding of C. velificatus upon other crusta-
ceans and larger, often less-abundant phytoplankters is similar to the patterns for
Labidocera aestiva (Turner, 1984c) and several cyclopoids (Turner, 1986b). These
results emphasize that when considering the trophic positions or impacts of "cope-
pods" it is important to specify which ones.
ACKNOWLEDGMENTS
I thank W. Conley, M. Dagg, D. Daley, D. Hoss, and J. White for shipboard
assistance, T. Charles for assistance with SEM specimen preparation, C. Bland for
access to the electron microscopy facilities at East Carolina University, H. Gordy for
access to his darkroom, and S. Freitas for typing. The officers and crew of the FRY
Oregon II, as always, did their best to facilitate work at sea. P. Tester provided helpful
criticism of the manuscript. This research was supported by a contract from the
Ocean Assessment Division, National Ocean Service, NOAA, to the Southeast Fish-
eries Center's Beaufort Laboratory, NMFS.
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Plankl. Res. 2: 355-365.
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Reference: Biol. Bull. 173: 387-397. (October, 1987)
AN ANATOMICAL STUDY OF THE RETINA OF
NAUTILUS POM PI LIU S
W. R. A. MUNTZ AND S. L. WENTWORTH
School of Biological Science, Stirling University, Scotland FK9 4L.4, and Faculty of Science, Monash
University, Clayton, Victoria, Australia 3168[
ABSTRACT
The receptor outer segments of Nautilus have several rhabdomeres of parallel
microvilli occurring more or less equally at all orientations. The microvilli of adjacent
receptors overlap substantially. The supporting cells of the retina possess both micro-
villous processes and small cilia. The receptor cells become disorganized towards
their tips, although supporting cell processes can still be distinguished. There is no
limiting membrane. Similarities to and differences from the retinas of adult and em-
bryonic coleoid cephalopods are discussed.
The myeloid body of Nautilus is more developed than in other cephalopods, ap-
pearing in electronmicrographs as a complex of loops or circles. A series of dimpled
plates stacked in register one above the other could generate most or all of these
apparently complicated structures depending on the plane of section. Myeloid bodies
occur both externally and internally to the nucleus, and occasionally within the outer
segment.
At its margin the retina merges smoothly with the columnar epithelium lining the
inside of the iris. The margin of the retina contains relatively more supporting cells
than more central regions. Myeloid bodies first appear about 200 jum central to the
point at which the retina starts to thicken.
INTRODUCTION
The Nautiloidea, a highly successful group of cephalopods which originated in
the Cambrian period, are today only represented by a few species of Nautilus which
are restricted to deep waters in tropical areas of the Pacific and possibly the Indian
oceans. Nautilus differs in many respects from the more recent coleoid cephalopods
and, although undoubtedly specialized in many ways for its deep-water habitat
(Landman and Saunders, 1987), shows many characteristics that appear to have re-
mained unchanged since Jurassic times. Therefore, its study is of special interest in
that it may provide some insight into how the organization of the modern cephalo-
pods arose.
The visual system is one of the features that may be primitive in Nautilus. The
eye is structurally simple, consisting of a cup formed by an invagination of skin,
communicating directly with the surrounding water by way of the open pupil. The
outer and inner surfaces of the iris are lined with columnar epithelium bearing mi-
crovilli, which on the inside merges through a transitional zone with the retina itself;
as in other cephalopods, the rhabdomeres of the outer segments of the retinal recep-
tors are formed from modified microvilli (Messenger, 1981). The inner segments of
the receptors contain nuclei and myeloid bodies [Merton's (1905) "phaosomes"].
The latter, which are probably concerned with visual pigment metabolism (Hara and
Received 12 January 1987; accepted 27 July 1987.
1 Address for reprints.
387
388 W. R. A. MUNTZ AND S. L. WENTWORTH
Hara, 1972; Breneman et al, 1986), are especially interesting because they are much
more structurally complex than those found in the more recent coleoid cephalopods.
The structure of the eye has been the subject of a number of histological studies
at both the light and the electron microscope levels (e.g., Hensen, 1 865; Griffin, 1900;
Owen., 1932;Mugglin, 1937; Young, 1965; Barber and Wright, 1969; Muntz and Raj,
1984). Behavioral studies have also shown, as would be expected in an eye that has
no lens but operates on the principle of a pin-hole camera, that both visual acuity
and sensitivity are poor in comparison to equivalent lens-bearing eyes (Muntz and
Raj, 1984; Muntz, 1987).
The present paper presents further information on various aspects of retinal struc-
ture: in particular on the interrelationship between the rhabdomeres of neighboring
receptors and the supporting cells, the transitional zone between the epithelium lining
the inside of the iris and the retina, and the structure of the supporting cell processes
and the myeloid bodies.
MATERIALS AND METHODS
Specimens of Nautilus were trapped off the main reef at Suva, at depths between
165 and 600 m, and kept in glass aquaria in an air conditioned room at water temper-
atures between 7 and 1 3°C. The water was changed regularly, and the animals fed
occasionally on prawns. Under these conditions they remained alive for several
weeks, and one animal showed reliable positive phototactic behavior after more than
1 1 months in the laboratory. However histological preparations show some degenera-
tion of the retina after a prolonged period in the laboratory, and the optomotor re-
sponse also becomes less reliable (Muntz and Raj, 1 984). Therefore the present paper
reports results on animals used within 3 or 4 days of capture.
Small pieces of iris and retina were fixed in 2% glutaraldehyde made up in either
Sorensen's phosphate buffer or filtered seawater. After 1.5 h fixation the tissue was
washed in several changes of buffer or seawater as appropriate. The tissue was then
post-fixed in 1% osmium tetroxide for 1 h. In some cases this post-fixation was done
in Fiji and the tissue taken through to 70% ethanol before being brought back for
further treatment. In most cases, however, the tissue was stored in fresh buffer or
seawater and the further treatment, including post-fixation, carried out at Stirling or
Monash University. Following post-fixation the tissue was either block stained in 2%
uranyl acetate for 1 h or immediately dehydrated. Either Emix (EMscope, Ashford,
England) or Spurr's resin (Spurr, 1969) were used to embed the tissue. Blocks were
sectioned on either a Reichert OM3 Ultratome or an LKB "Ultratome III." Thin
sections (2-4 /urn) were taken and stained with Toluidine Blue (pH 9.0) and examined
under the light microscope. Ultrathin sections were stained with a saturated solution
of uranyl acetate in 50% methanol and post-stained in lead citrate. The sections were
examined using a JEOL JM 100QX) or an AEI Corinth 275 electron microscope.
In this paper the words inner or internally, when applied to the retina, mean to-
wards the center of the animal, while outer or externally mean in the opposite direc-
tion, that is towards the light.
RESULTS
Outer segments and supporting cells
Figures la and b show electron micrographs of sagittal sections through the outer
segments and supporting cells of the retina, confirming the structure described by
Barber and Wright (1969). The tubules or microvilli that make up the rhabdomeres
RETINA OF NAUTILUS POMPILIUS 389
can be seen leaving the outer segments, and where the plane of section cuts across the
microvilli they appear, as in other cephalopods (e.g., Cohen, 1973), to be closely
packed. The length of the microvilli varies from 2 to 6 p.m between different rhab-
domeres, but within a given rhabdomere their length does not change appreciably
with position. The rhabdomeres are separated from each other by the supporting cells
which contain a number of parallel processes extending over the whole depth of the
outer segment layer of the retina (300-500 /um) and which, from their appearance in
both transverse and longitudinal section, are clearly microvillar in nature. Figure Ib
shows the origin of the supporting cell processes at the cell body. Small primary cilia
are also found in the supporting cells (Fig. 3). It is not clear if they also occur in the
receptor cells.
A tangential section through the outer segments is shown in Figure 2. The rhab-
domeres radiate between the outer segments of neighboring receptors, separated by
the processes of the supporting cells. The microvilli from neighboring receptors inter-
digitate, individual microvilli passing most, or perhaps all, of the way from one outer
segment to another. Supporting cells have on average 46 processes (SD = 9, n = 30):
on three occasions the processes from 2 or 3 supporting cells have apparently run
together resulting in groups of about 90 or 1 50 processes.
Figure 4 shows, at the light microscope level, a sagittal section through the edge
of the retina and the transitional zone between the retina and the iris. It can be seen
that over about the outer third of their length the structure of the receptors becomes
disorganized. Figure 5 shows the appearance of the outer region, near the receptor
tips, as seen under the electron microscope. Processes belonging to the supporting
cells can be seen, but the structure of the receptors themselves is no longer clearly
organized into receptor cell bodies and rhabdomeres containing parallel microvilli.
There is no sign of a limiting membrane overlying the outer ends of the receptors
such as occurs in thecoleoid cephalopods (Zonana, 1961; Yamamoto et ai, 1965).
Transitional zone between iris and retina
Figure 4 shows that at the junction of the iris and the retina there is a smooth
increase in the thickness of the outer layer of the retina due to the increasing length
of the receptor outer segments or supporting cell processes. The myeloid bodies do
not start to occur at the point at which the retina thickens, but considerably further
in: thus in preparations from four animals the distance from the first myeloid body
to the edge of the retina (as indicated by the arrows in Fig. 4) ranged from 130 ^m to
240 ^rn (mean 193 ^m).
The edge of the retina also contains relatively few receptor cells relative to support-
ing cells compared to more central regions (Fig. 6). It is likely that the outer layers
of the retina take on their final form at the same point as the myeloid bodies appear.
Structure of the myeloid bodies
The appearance of the myeloid bodies under the transmission electron micro-
scope varied widely from a simple appearance of a number of wavy membranous
structures, to a complex of loops, whorls, and circles (Figs. 7, 8, 9). The myeloid body
is usually located in the inner segment of the receptors, although displaced myeloid
bodies lying in the outer segment occasionally are found. In electron micrographs of
52 receptor inner segments in which both the myeloid body and the nucleus could
be seen, the myeloid body lay internally to the nucleus on 33 occasions and externally
on 20 occasions. The fine membranous structure of the myeloid body is clearly shown
in Figures 8 and 9.
390 W. R. A. MUNTZ AND S. L. WENTWORTH
DISCUSSION
In the coleoid cephalopods that have been studied to date the arrangement of the
retina as seen in tangential section is very precise. The basic plan, first described by
Schultze (1869), is that each receptor outer segment carries a rhabdomere on two
opposite faces, and that the rhabdomeres from four receptors combine to make a
square rhabdome (Young, 1 962) — originally thought by analogy with the anthropods
to be the retina's basic functional unit. As a result of this structure the microvilli of
the rhabdomeres form two orthogonal sets, oriented in the vertical and horizontal
planes, respectively. There are some exceptions, and individual receptors can on oc-
casion contribute to up to eight rhabdomeres (Zonana, 1961; Cohen, 1973), but the
underlying plan is clear. The rhabdomeres of adjacent receptors remain separate,
without any overlap in their microvilli. In contrast, each receptor outer segment in
Nautilus has several rhabdomeres occurring more or less equally at all angles, and
with substantial or complete overlap between the microvilli of neighboring receptors.
There is no obvious regular grouping of the rhabdomeres from neighboring receptors
to form a structure analogous to the rhabdome.
The orthogonal arrangement of the rhabdomeres of coleoid cephalopods, and the
parallel relationship of the microvilli within them, is commonly assumed to underlie
the ability of cephalopods to discriminate the plane of polarization of light (e.g.,
Saidel et al, 1983). The orthogonal arrangement does not occur in Nautilus, but the
microvilli remain parallel to each other within any given rhabdomere, and the angu-
lar separation between each rhabdomere is still considerable even though less than
90°. Plane polarized light should thus still be able to affect the receptors of Nautilus
differentially, but without any precise or predetermined pattern between the plane of
polarization and the set of receptors that would be affected.
The interdigitation of the microvilli of neighboring receptors shown in Figure
2 is also visible in the electron micrographs published by Barber and Wright (1969),
although not mentioned in that paper. Such interdigitation of microvilli is not unique
to Nautilus, but occurs, for example, in some arachnids [e.g., Lampona (Gnaphosi-
dae). Blest, 1 985] and is an arrangement that might be expected to affect visual acuity.
Thus the simplest hypothesis relating retinal structure to acuity is that two objects
will be discriminable when their retinal images are far enough apart that at least one
receptor remains unstimulated between them (Helmholtz, 1924). Since microvilli
overlapping will increase the separation between stimuli necessary to achieve this, a
loss of resolution at the retinal level will result. However, in Nautilus the pin-hole
arrangement for image formation appears to be the limiting factor for visual resolu-
tion with the retinal mosaic considerably finer than is needed for the quality of the
image that is formed (Muntz and Raj, 1984). The overlapping of the microvilli, even
if this halved the effective fineness of the retinal mosaic, should not affect the animal's
ability to resolve detail. However, the overlap could benefit sensitivity, important for
an animal living at depth in the sea, if sensitivity is limited to some degree by random
retinal events that are unrelated to the stimulus, such as the spontaneous breakdown
of visual pigment molecules or the spontaneous release of transmitters at synapses
(visual "noise"). Overlap will increase the number of receptors simultaneously acti-
vated by a given stimulus, whereas "noise" events will presumably be independent
in time between the different receptors. Provided the central nervous system is able
to detect the simultaneity of messages from the receptors, sensitivity should be im-
proved.
The retina of Nautilus also differs from that of the coleoid cephalopods in that the
supporting cells have small processes that are ciliary in nature, as well as the microvil-
lous processes extending between the receptors. Ciliary structures have not hitherto
RETINA OF NAUTILUS POMPILIUS
391
FIGURE 1 . a. TEM of a sagittal section through the outer segments and supporting cells of the retina
of Nautilus pompilius. os: outer segment, rh: rhabdomere, s: supporting cell. The arrow shows a microvillus
opening into the outer segment. Fixative: 2% glutaraldehyde in seawater. Scale bar = 1 ^m. b. TEM of a
sagittal section of the receptor and supporting cells showing the origin of the supporting cell processes and
receptor microvilli. Fixative: 2% glutaraldehyde in seawater. Scale bar = 1 ^m.
FIGURE 2. TEM of a tangential section through outer segments and supporting cells of the retina.
Labels as in Figure 1. Scale bar = 2 ^m. The insert shows the appearance at higher magnification. The
arrows show interdigitating microvilli from neighboring receptors. Fixative: 2% glutaraldehyde in seawater.
Scale bar = 1 //m.
392
3d
W. R. A. MUNTZ AND S. L. WENTWORTH
5
» -ttr
•
/;
,
,
'
.
• •••
-•
I a* >
• v
t • -—
••*-..
•
'
FIGURE 3. TEMs showing primary cilia in supporting cells and associated centriolar structures. Fixa-
tive: 2% glutaraldehyde in phosphate buffer, (a) Sagittal section of the retina. The cilium is indicated by
the arrow. Scale bar = 1 ^m. (b) Transverse section of the basal body of a cilium showing pinwheel struc-
ture. Scale bar = 0.2 ^m. (c) Sagittal section of the retina showing diplosomal centrioles cut both longitudi-
nally and obliquely. Scale bar = 0.2 ^m.
FIGURE 4. LM of a sagittal section through the edge of the retina. The arrows show ( 1 ) the point at
which the outer segments start to lengthen and (2) the first visible myeloid body. Fixative: 2% glutaralde-
hyde in seawater. Scale bar = 100 ^m.
FIGURE 5. TEM of a sagittal section through outermost tips of the receptors. Supporting cell pro-
cesses are still clearly visible. Fixative: 2% glutaraldehyde in phosphate buffer. Scale bar = 5
RETINA OF NAUTILUS POM PI LIU S
393
•• 7*-. * V
-,'-«"'. V J /) .4*
•.V'V- « - '*
-if: , "^;^r tS
f *, ;^^V^v' rife ;
' :i- «
FIGURE 6. TEM of a sagittal section through the outer segments at the transitional zone at the edge
of the retina. The iris is to the right. Fixative: 2% glutaraldehyde in seawater. Scale bar = 2 ^m.
FIGURE 7. TEM of sagittal section through the inner segments of retinal receptors showing the rela-
tive positions of nuclei and myeloid bodies. The bottom of the photograph is internal. Fixative: 2% glutaral-
dehyde in seawater. Scale bar = 10 ^m.
FIGURE 8. TEM of a sagittal section through the outer segments of the retina showing displaced
myeloid body. Microtubules and supporting cell processes are clearly visible. Fixative: 2% glutaraldehyde
in phosphate buffer. Scale bar = 1 ^m.
FIGURE 9. TEM of a sagittal section of a myeloid body showing membranous structure. Fixative: 2%
glutaraldehyde in phosphate buffer. Scale bar = 2
394 W. R. A. MUNTZ AND S. L. WENTWORTH
been reported in the retina of any adult cephalopod. The photosensitive organs of
many ani mals have receptors of ciliary origin, or cilia, presumed not to be photosensi-
tive, in' 'rrningled with the receptors (Vanfleteren, 1982). Many rhabdomeric recep-
tors possess cilia in their embryonic or larval stages but not in the adult stage (Van-
fleteren, 1982). Yamamoto (1985) reported that such structures are present in both
the receptors and supporting cells of the embryos of the cuttlefish, Sepiella japonica,
persisting longer in the supporting cells than in the receptors. The retina of the 5".
japonica embryo resembles that of Nautilus in a number of other respects. These
include the fact that in early stages the nuclei of both supporting and receptor cells
are external to the basal membrane, whereas in adults only the supporting cell nuclei
remain external; that the microvilli of the receptor cells occur initially more or less
equally in all directions, only achieving the regular rectilinear arrangement of adults
at a relatively late stage of development; and that the supporting cells in embryos
send long microvillous processes between the receptor cells over the whole length of
their outer segments. They also possess cilia.
The myeloid bodies of Nautilus, which are found in the inner segments of the
receptors, are much more complex than those found in the coleoid cephalopods.
Barber and Wright (1969) state that they are usually found externally to the nucleus,
while Merton's ( 1 905) figures show them equally on either side of the nucleus. In our
preparations they lay internally to the nucleus more often than externally, and they
were also on occasion found displaced into the outer segment.
Myeloid bodies did not occur at the retinal margin, but only some 100-200 /urn
inwards after the point at which the retina outer layer starts to lengthen. The edge of
the retina also contains relatively few rhabdomeres and many supporting cells. The
presence of the myeloids may thus correlate with the presence of rhabdomeres.
The structure of the myeloid bodies has been described from light microscope
studies as a stacked series of cubes, or a honeycomb structure (Merton, 1905), and
from electron micrographs as a complex tubular array (Barber and Wright, 1969).
Both Barber and Wright's electron microscope results and those shown in Figures 7,
8, and 9 of the present paper appear to confirm both the complexity of and on many
occasions an apparently tubular structure. However, the photographs often give the
impression of a series of stacked wavy plates, or of a structure where both forms of
organization alternate or merge.
Despite the apparent complexity, there is a comparatively simple structure that
can generate most or all of these different appearances depending on the plane of
section, namely a series of dimpled plates stacked in register one above the other.
Mathematically the surface of each individual plate can be approximated by the rela-
tionship z = cos x cos y (Fig. 10). A section in the plane of the plates will clearly
produce a pattern of circles, squared off to a greater or lesser extent depending on
how close the section is to the point where z == 0, which could be interpreted as a
tubular structure. Similarly, a section perpendicular to the plane of the plates will
produce an appearance similar to a stacked series of wavy plates, the amplitude of
the waves depending on how close the section is to the lines denned by cos x or cos y
= 0. Various intermediate patterns similar to those seen in the electron micrographs
are generated when the section cuts the plate at an angle (see examples in Fig. 1 1 ). Of
course these patterns will be repeated with a frequency which will depend on the
spacing between the plates constituting the stack. The individual plates, or the whole
stack, could also be curved, which would affect the appearance of the myeloid bodies.
The function of myeloid bodies remains uncertain. However the available evi-
dence shows that in other cephalopods retinochrome is found in association with the
myeloid bodies, and that the distribution of retinoids between the myeloid bodies
RETINA OF NAUTILUS POMPILWS
395
FIGURE 10A. Suggested structure of myeloid bodies. Appearance of a surface defined by
cos x cos y = z.
and the outer segments of the receptors can be affected by the light regime under
which the animals are kept (Hara and Hara, 1972; Breneman ct al, 1986). Thus the
myeloid bodies are implicated in the visual pigment metabolism of cephalopods,
most likely being concerned with pigment regeneration. Metabolic events often occur
at the surfaces of membranes and thus depend on the membrane surface area. A stack
of dimpled plates of the sort proposed represents a simple way of increasing the total
surface area of membrane present in the myeloid body without increasing its gross
dimensions. Why Nautilus should have a myeloid body that is so structurally compli-
cated compared to other cephalopods remains unresolved.
FIGURE 10B. Suggested structure of myeloid bodies. Any plane of section of the surface x, y can be
defined by the two angles a and /3 and by the value of 7.
396
W. R. A. MUNTZ AND S. L. WENTWORTH
37I--
\n
n
FIGURE 11. A to E, contour maps of the surface shown in Figure 10A, with the appearance of the
cut surfaces that would be produced by sections cut in planes denned by different values of a and 7, /3
having a fixed value of TT and z a maximum of 1 in all cases. The contour lines show heights and depths
above and below the plane of the surface for values of 0, 0.2, 0.4, 0.6, and 0.8 of the maximum. A; a
= 0.257T, 7 = 1 .Sir. B; a = 0.25*-, y = 0. C; a = 0.5*-, 7 = *-. D, a = 0.5*-, 7 = 0.257T. E; « = 0.5*-, 7 = 0.5*-.
F, appearance of sections perpendicular to the plate (i.e. ft = 90°), for values of a of O.STT and values of 7
from left to right of 0.5 TT, 0.25*-, and 1.757T respectively. Superimposed on a contour map such sections
would appear as straight lines: they are shown here as they would be seen from a position perpendicular to
the plane of section.
ACKNOWLEDGMENTS
We are very grateful to the Director, Dr. U. Raj, and staff of the Institute of Marine
Resources at the University of the South Pacific, Suva, Fiji, for facilities, assistance,
and much helpful discussion; to Mr. T. Forrest of Stirling University, Scotland, for
technical help with the early stages of the electron microscopy; to Dr. J. C. Stillwell,
RETINA OF NAUTILUS POMPILIUS 397
Monash University, for helping over the model of the myeloid bodies; and to
the Science & Engineering Research Council of the United Kingdom for financial
assistance.
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BRENEMAN, J. W., L. J. ROBLES. AND D. BOK.. 1986. Light-activated retinoid transport in cephalopod
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Reference: Biol. Bull. 173: 398-406. (October, 1987)
SPECTRA SENSITIVITY OF THE COMPOUND EYES IN THE PURPLE
LAND CRAB GECARCINUS LATERALIS (FREMINVILLE)
ABNER B. LALL' AND THOMAS W. CRONIN2
1 The Thomas Jenkins Department of Biophysics, The Johns Hopkins University, Baltimore, Maryland
21218 and 2Department of Biological Sciences, University of Maryland at
Baltimore County, Catonsville, Maryland 2 1228
ABSTRACT
The spectral sensitivities (S( A)) of dark-adapted compound eyes of the purple land
crab Gecarcinus lateral is possess a broad maximum in the blue-green, 420-530 nm,
when measured by electroretinographic (ERG) techniques. Selective adaptation ex-
periments showed large changes in sensitivity but did not isolate different receptor
types. A photopigment with maximal absorption at 487 nm was identified in the
rhabdoms by microspectrophotometry. Besides the presence of a dominant green
receptor system, the existence of a hump in the short wavelength region in S(X) sug-
gests the presence of a blue-sensitive system as well. It is hypothesized that two photo-
pigments (P487 and P440) in conjunction with screening pigment(s) mediate broad
visual maximum in the blue-green in the purple land crab.
INTRODUCTION
One or two receptor systems have been described in the intertidal and subtidal
crabs. Behavioral evidence for color discrimination exists for portunid crab Carcinus
(von Buddenbrock and Friedrich, 1961) and for two Uca species (Hyatt, 1975). This
can be accounted for by the presence of different receptor types produced either by
different visual pigments (Wald, 1968) or by a single pigment in conjunction with
screening pigments (Leggett, 1 979). To date, microspectrophotometric (MSP) studies
have revealed only a single visual pigment in crabs (e.g., spider crab Libinia emargi-
nata: X max 493 nm. Hays and Goldsmith, 1969; blue crab Callinectes spidicus: X
max 500 nm, Bruno and Goldsmith, 1974; green crab Carcinus maenas: X max 505
nm, Bruno et al, 1973, crab species, Cronin and Forward, 1987).
Electrophysiological studies have produced varying results regarding the types
and numbers of photoreceptors in eyes of intertidal and subtidal crabs, even in the
same species. Some workers using electroretinographic (ERG) or single unit record-
ings have located only a single receptor system. For example, Scott and Mote (1974)
observed a single maximum at 510 nm in crabs inhabiting diverse habitats (Calli-
nectes sapidus, Sesarma reticulatum, Uca pugilator, and U. pugnas). Wald (1968)
reported that the subtidal Libinia emarginata had a single receptor type (X max 490
nm), and Fernandez (1973) located only one system in the deep-water species Pleu-
roncodes planipes (X max 523 nm). Similarly, Bruno et al. (1973) described a single
493 nm maximum in Carcinus maenus. However other workers have uncovered an
additional, shorter wavelength receptor system in several of these species: C. maenas
(Wald, 1968; Martin and Mote, 1982), C. sapidus (Martin and Mote, 1982), and U.
pugilator (Hyatt, 1 975). Thus it seems likely that crabs in general are dichromats.
Received 15 June 1987; accepted 27 July 1987.
398
VISION IN THE LAND CRAB 399
The land crabs are decapod Crustracea which have made the transition from wa-
ter to land. During their evolutionary adaptation to a new niche, a selection of a new
set of physiological and behavioral mechanisms differing from their ancestors would
be expected which could produce alterations in their visual systems. The visual pig-
ments of several intertidal crab species have been described (Cronin and Forward,
1 987), and some semiterrestrial crab species have been investigated electrophysiologi-
cally as described in the previous paragraph. Nevertheless, the spectral characteristics
of vision are not known for the purple land crab. The objective here is to fill this
gap in our present knowledge. The land crabs live in burrows in the sand or in the
mangroves. Most of them forage at twilight or at night in a light limiting condition.
MATERIALS AND METHODS
Animals
The purple land crabs (4-6 cm carapace width) used in these experiments came
from the West Coast of Florida and were kept in containers in the laboratory at room
temperature (2 1 -23°C) with a daily light cycle of 1 2 h light and 1 2 h dark.
Experimental preparation
Electrical recordings were taken from the corneal surface in live, intact prepara-
tions. The crabs were immobilized by bandaging heavily with gauze and then further
secured with adhesive tape. The eye of the crab is retractable, so it was immobilized
with dental cement to ensure that it remained easily accessible for experimentation.
The animals were allowed to dark-adapt for one hour. All the experiments were con-
ducted during the photophase at room temperature (21-23°C).
Electrical recordings
ERGs elicited by illumination of the eye were recorded by a glass pipette (tip
diameter 5-10 ^m) filled with physiological saline and inserted underneath the cor-
nea. The reference electrode, also filled with saline, was inserted into a small hole
bored in the shell of the animal. An Ag-AgCl wire made the connection between the
electrodes and the grid of a high impedance preamplifier. The output of the preampli-
fier was fed to a DC coupled cathode ray oscilloscope (CRO) and a chart recorder.
Optical system
A two-channel optical system was utilized (details given in Lall el ai, 1982). One
beam was for testing and other for chromatic adaptation. The test beam was obtained
from a 150 watt xenon arc operated at 7.5 amps, with a regulated power supply. The
light beam passed a high intensity grating monochromator. Quartz lenses collimated
the test beam and focussed it on the entrance of a quartz light pipe (3 X 360 mm).
The adapting beam was obtained from a 500 watt tungsten quartz iodide lamp, and
was collimated with a quartz lens and superimposed on the test beam using a beam
splitter. The quantum flux in both light beams was controlled by calibrated neutral
density filters. Both beams were interrupted by Uniblitz photographic shutters which
controlled the duration of the test flash and adaptation times.
Intensity calibration
The quantum flux of the test and the adaptation beam were determined by using
a calibrated PIN-10UV Schottky barrier photodiode with a Model 210A amplifier
400 A. B. LALL AND T. W. CRONIN
(United Detector Technology, Inc.). The photodiode was positioned directly at the
end of the quartz light pipe in the same way the eye was positioned during experimen-
tation and its output read on the CRO.
Experimental procedures
Light flashes of varying duration (0. 1-0.4 s) at 25 nm or 20 nm steps from 340 to
680 nm were administered over 5 log units of intensity change. The ERGs elicited by
these flashes were recorded. To maintain a constant steady dark-adapted state, the
test flashes were delivered at least 30 s to 90 s apart depending upon the intensity of
the test flash.
Spectral sensitivity curves
The amplitude of the ERG was used as an index of the sensitivity of the eye to the
quantum flux and the wavelength composition of the photic stimulus. The spectral
sensitivity functions were obtained by first determining the number of photons
needed to elicit a criterion amplitude of the ERGs for different stimulus wavelength.
A plot of 1/Q as a function of wavelength gave the spectral sensitivity function. Two
variations of the criterion method were used for determining the spectral sensitivity
curves, (a) ERGs were recorded at different levels of intensity for a stimulus wave-
length and V/logI function was obtained. Similar functions were obtained for all the
selected stimulus wavelengths across the spectrum. The reciprocal of the quanta
needed to elicit a chosen criterion amplitude response across the stimulus wave-
lengths, as a function of wavelength, gave the spectral sensitivity curve. This is a
lengthy procedure, and in most cases a shorter one was adopted, (b) One person
observed the CRO screen while another adjusted the intensity of the test flash with
neutral density filters at each wavelength until the observer signaled that a criterion
response (50 n\ or 200 yuV) had been met.
Several chromatic adaptation experiments were conducted. Either Corning glass
filters: blue (Cs5-60) and orange (Cs2-73) for broad-band irradiation, or Baird Atomic
Interference filters for monochromatic (440 nm and 610 nm) intense light were
placed in the path of the adapting beam of the stimulator and the eye was allowed to
chromatically adapt continuously during experimentation. The test flashes for deter-
mining the spectral sensitivity were superimposed on the chromatic adaptation beam.
The spectral sensitivity curves under chromatic adaptation were obtained by using
the second criterion method described above.
Microspectrophotometry
A single-beam instrument described in Cronin (1984) was used. Animals were
dark-adapted for several days, following which eyes were removed and ground in
2.5% gluteraldehyde in pH 7.5 MBL crustacean Ringer's (Cavenaugh, 1956). After
15 minutes of fixation at 0°C, the mixture of eye debris and photoreceptors (rhab-
doms) was centrifuged, resuspended in pH 7.5 Ringer's, and maintained at 0°C. Indi-
vidual rhabdoms were scanned as described in Cronin (1984). The rhodopsin absorp-
tion spectrum was determined by taking the difference between the absorption of a
fully dark-adapted rhabdom and the absorption of the same rhabdom after a 5 minute
photobleach with bright white light. Data from 1 3 rhabdoms were averaged and fit
with a Dartnall (1953) nomogram as described in Cronin and Forward (1987).
VISION IN THE LAND CRAB
401
60Or
500
ji.4OO
UJ
Q
P 300
200
100
0
LOG INTENSITY (photons /cm2s)
FIGURE 1 . V/logI curves for a dark-adapted compound eyes in Gecarcinns lateralis. The number at
the bottom of the curve is the log photons for the lowest response. Note that the curves for different wave-
lengths are similar in slope.
RESULTS
Electroretinograms (ERGs)
The ERGs were recorded from the corneal surface of the compound eyes after an
initial latency period (20-60 ms) from the onset of illumination. The response was
an "on" negative potential consisting of an initial phasic component followed by a
maintained or plateau component which lasted for the total duration of the illumina-
tion. At low levels of illumination only the plateau component was recorded. The
phasic component appeared at the intermediate levels of the illumination and in-
creased sharply with bright illumination. The "on" negative ERGs in the land crab
were similar to the ones recorded from the compound eyes of many arthropods which
have scotopic eyes (e.g., horseshoe crab; Chapman and Lall, 1967; Crustacea; Wald,
1968). The response waveform of the ERGs elicited by the stimuli of different wave-
lengths tended to be similar.
Intensity-response (V/log I) functions
Figure 1 shows the amplitude of the phasic component of the ERG plotted as a
function of log intensity of the stimuli of different wavelengths and intensities. The
slopes of these V/logI functions for the phasic components did not vary with stimulus
wavelength. These V/logI curves were used for: (a) determining the spectral sensitivi-
ties in Figure 3 (only the DA curve) and (b) determining whether there were any
wavelength-dependent changes in the slope of the V/logI functions. Systematic
changes in the slopes of the response curves for different wavelengths have been taken
as evidence for the presence of different receptor types as in the median ocelli of
Limulus (Chapman and Lall, 1967) and in the compound eyes of whirligig beetle
(Bennett, 1967) and wolf spider (DeVoe et al, 1969). The V/logI curves extended
over 4 to 5 log units of intensity change, which indicated that the photoreceptor could
function over 10,000 to 100,000 fold change in stimulus intensity.
402
A. B. LALL AND T. W. CRONIN
Or*
Q
_l
O
ui
cr
i
t-
i
o
420nm
560nm
0 5 10 15 20 25 30
TIME IN THE DARK (minutes)
FIGURE 2. Dark-adaptation of the purple land crab compound eye measured at two wavelengths.
The curves tended to be hyperbolic initially and follow a similar time course.
Dark-adaptation
Figure 2 shows the time course of dark-adaptation measured at two wavelengths
(560 nm and 420 nm). The eye was light adapted for 1 minute with white light, and
then the responses during dark-adaptation were tested alternately at 560 nm and 420
nm. Initially the threshold decreased hyperbolically, and then after about 20 minutes
the threshold decrease was linear. These two curves are parallel, showing that the
eye maintains constant relative sensitivities to the two wavelengths throughout dark
adaptation.
Spectral sensitivity
Figure 3 shows the S(X) functions under dark- and chromatic adaptation condi-
tions in G. lateralis. The dark-adapted S(X) curves showed a very broad sensitivity in
the blue-green (440-520 nm) region of the spectrum. In two animals (A, Fig. 3),
the sensitivity in the blue (430-460 nm) region was pronounced. Under chromatic
adaptation conditions with both Corning glass niters and narrow band interference
niters, the S(X) curves exhibited a decrease in sensitivity of about 1 .5 to 2.5 log units,
but a distinctive and pronounced selective effect with differential suppression of
different parts of the spectrum was not observed. The chromatic adaptation curves
tended to be broad and rather flat across the spectrum (compare curves a, b, and d
for different chromatic adaptation conditions in Fig. 3), except for a small hump in
the blue under red selective adaptation light. However, it should be noted that the
ERG is a gross response from the whole eye, and chromatic adaptation experiments
may poorly separate different receptor types even when the receptors are as far apart
in the spectrum as near-UV and green (e.g., Dineutes: Bennett, 1978; Photuris versi-
color; Lall, 1981). This difficulty in receptor isolation is further compounded when
the receptors are as adjacent in the spectrum as blue versus green, and when the
number of blue receptors is only a very small fraction of the green as in the blue crab
Callinectes (Martin and Mote, 1982).
Microspectrophotometry(MSP)
The absorption spectrum of the visual pigment found in the rhabdoms ofGecarci-
nus lateralis is shown in Figure 4 and represents an average curve of the difference
VISION IN THE LAND CRAB
403
. Gecarcmus lateralis
i
in
CVJ
'EIO
u
CO
z
o
I'll
0.
12
CO
z
LJ
CO
13
Nomogram P440
G. late rails visual pigment
Dark-adapt
Red-adapt oCs2-73 *6lOnm
Blue-adapt • Cs5-€0 o440nm
350 400 450 500 550 600 650
WAVELENGTH (nm)
FIGURE 3. Spectral sensitivity of the purple land crab compound eyes under dark- and chromatic
adaptation conditions. Nomogram (Ebrey and Honig, 1977) for P440( • • • ) and G. lateralis visual pigment
( ) from Figure 4 are superimposed for the data.
spectra between bleaches and dark-adapted preparations of 1 3 individual photore-
ceptors. The curve possesses a peak in the blue-green. The Dartnall (1953) nomogram
curve for P487 nm closely matches this difference spectrum curve (Fig. 4).
DISCUSSION
The purple land crab Gecarcinus lateralis possesses a primary blue-green sensitive
receptor system. This is supported by the following observations: (a) similarity of the
ERG waveforms as a function of wavelength, (b) univariance of V/logI slopes as a
function of wavelength (Fig. 1), (c) broad dark-adaptation curves with maximum in
the blue-green (Fig. 3), (d) a lack of isolation of either blue or green receptor system
under conditions of chromatic adaptation (Fig. 3), and (e) the presence of a blue-
green absorbing (X max 487 nm) photopigment in the rhabdoms (Fig. 4). However
this does not rule out the possibility of a second photoreceptor system, since it is
possible that the contribution of a receptor type consisting of only few ommatidia
can be masked by the dominant receptor system. In the retina of the swimming blue
crab Callinectes sapidus, green-sensitive cells (X max 508 nm) were dominant, while
only a few cells restricted to the ventral border region were blue-sensitive (X max 440
404
A. B. LALL AND T. W. CRONIN
Gecarcinus lateralis
Nomogram P487
0
400
500 600
Wavelength (nm)
700
FIGURE 4. Average absorption spectrum of the rhodopsin in 1 3 rhabdoms determined microspectro-
photometrically by taking the difference between spectra obtained from rhobdoms when dark-adapted and
again when photobleached with white light. The solid line represents a Dartnall's nomogram curve for
P487.
nm, Martin and Mote, 1982). Consequently, earlier studies using both single cell
(Scott and Mote, 1974) and ERG (Goldsmith and Fernandez, 1968) techniques did
not uncover the blue-sensitive receptor system. In our data, the presence of high sensi-
tivity in the blue in a few recordings strongly suggests that a similar blue-sensitive
receptor system (P440) exists in the purple land crab (Fig. 3).
It should be noted that the peak of the S(X) function in G. lateralis is difficult to
establish in our ERG data (Fig. 3). The maximal sensitivity in the blue-green is much
broader than the absorption spectrum of the most prevalent visual pigment, P487
(Fig. 4), presumably responsible for the sensitivity in the green (Fig. 3). The presence
of screening pigments have been implicated in modifying the visual spectral sensitiv-
ity mediated by the visual pigment(s). This modification in some Crustacea has led
to a broadening of the S(X) functions in the green (i.e., Leptograpsus variegatus,
Stowe, 1980; Carcinus maenas, Wald, 1968, Bruno et ai. 1973). While in other spe-
cies a shifting of the lambda maximum of the S(X) functions to longer wavelengths
occurs. For instance, in the lobster the peak of the ERG S(X) function is at 525 nm
(Kennedy and Bruno, 196 1 ), whereas the peak of the difference spectrum for bleach-
ing of lobster visual pigment is at 500 nm (Wald and Hubbard, 1957; Bruno et ai,
1977). Similarly, in the crayfish the species visual pigment X max appears to be at
530-535 nm (Goldsmith, 1978a) whereas the peak of the ERG S(X) function is at
565-570 nm (Kennedy and Bruno, 1961; Goldsmith and Fernandez, 1968; Wald,
1968), a dramatic shift of about 35 nm towards the red. These bathochromic shifts
in the peaks of the S(X) functions from the peaks of the species rhodopsins have been
attributed to the presence of distal screening pigment granules in some Crustacea
(crayfish: Goldsmith, 1978b; crab L. variegatus: Stowe, 1980). It is quite conceivable
that P487 overlaid by screening pigment(s) could cause a broadening of the S(X) func-
tions in blue-green in the purple land crab.
VISION IN THE LAND CRAB 405
The purple land crab G. lateralis generally inhabit tropical coastal hammocks
(Bliss el al, 1978). For protection the land crab burrows into the sand; it never wan-
ders too far away from the safety of its burrow. The crab is active under environmen-
tal conditions of low ambient light, temperatures from about 18.5°C to 30°C, and
high humidity. Unlike the giant crab, Cardisoma guanhumi, G. lateralis is not
primarily nocturnal and has been observed to be active in the subdued illumination
under heavy vegetation during the day. For effective functioning in a such an environ-
ment, a medium sensitivity, low threshold receptor with maximal sensitivity in the
green would be ideal for the purple land crab. The presence of a blue receptor would
enable the crab to discriminate open space from closed space. Our data suggest that
indeed G. lateralis possesses its highest sensitivity in the blue-green region of the
spectrum.
ACKNOWLEDGMENTS
The technical support for this project was provided by undergraduate research
students: Sarah Weatherall, Kathryn Peper, and Keith Hunter. Supported by NSF
grants # BNS 83-1 1 127, BNS 83-1 1 157, BNS 85-18769, and NIH 5 R01 EY 00520.
LITERATURE CITED
BENNETT, R. R. 1967. Spectral sensitivity studies of the whirligig beetle Dineutes ciliatus. J. Insect Phvsiol.
13:621-633.
BLISS, D. E., J. VAN MONTFRANS, M. VAN MoNTFRANS, AND J. R. BovER. 1978. Behavior and growth of
the land crab Gecarcinus lateralis (Freminville) in southern Florida. Bull. Am. Mus. Nat. Hist.
160: 115-151.
BRUNO, M. S., AND T. H. GOLDSMITH. 1 974. Rhodopsin of the blue crab Callinectes: evidence for absorp-
tion differences in vitro and in vivo. Vision Res. 14: 653-658.
BRUNO, M. S., M. I. MOTE, AND T. H. GOLDSMITH. 1973. Spectral absorption and sensitivity measure-
ments in single ommatidia of the green crab, Carcinus. J. Comp. Phvsiol. 82: 151-163.
BRUNO, M. S., S. N. BARNES, AND T. H. GOLDSMITH. 1977. The visual pigment and visual cycle of the
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VON BUDDENBROCK, N., AND H. H. FRIEDRICH. 1961. Cited by Waterman, T. H., Light sensitivity and
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York.
CAVENAUGH, G. M. 1956. Formulae and methods of the Marine Biological Laboratory chemical room.
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CHAPMAN, R. M., AND A. B. LALL. 1967. Electroretinogram characteristics and the spectral mechanisms
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CRONIN, T. W. 1984. The visual pigment of a stomatopod Crustacea, Squilla etnpusa. J. Comp. Phvsiol.
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CRONIN, T. W., AND R. B. FORWARD JR. 1987. The visual pigments of crabs I. Spectral characteristics. /.
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DARTNALL, H. J. A. 1953. The interpretation of spectral sensitivity curves. Br. Med. Bull. 9: 24-30.
DEVOE, R. D., R. J. W. SMALL, AND J. E. ZVAZGULIS. 1969. Spectral sensitivities of wolf spider eyes. J.
Gen. Phvsiol. 54: 1-32.
EBREY, T. G., AND B. HONIG. 1977. New wavelength dependent visual pigment nomograms. Vision Res.
17: 147-151.
FERNANDEZ, H. R. 1973. Spectral sensitivity and visual pigment of the compound eye of the galatheid
crab Plewoncodes planipes. Mar. Biol. 20: 148-153.
GOLDSMITH, T. H. 1978a. The spectral absorption of crayfish rhabdoms: pigment, photoproduct and pH
sensitivity. Vision Res. 18:463-473.
GOLDSMITH, T. H. 1978b. The effects of screening of pigments on the spectral sensitivity of some Crustacea
with scotopic (superposition) eyes. Vision Res. 18:475-482.
GOLDSMITH, T. H., AND H. R. FERNANDEZ. 1968. Comparative studies of crustacean spectral sensitivity.
Z. Vergl. Phvsiol. 60: 156-175.
HAYS, D., AND T. H. GOLDSMITH. 1969. Microspectrophotometry of the visual pigment of the spider crab
Libinia emarginata. Z. Vgl. Phvsiol. 65: 218-232.
406 A. B. LALL AND T. W. CRONIN
HYATT, G. W. 1975. Physiological and behavioral evidence for color discrimination by fiddler crabs (Bra-
chyura, Ocypodidae, Genus Uca). Pp. 333-365 in Physiological Ecology ofEstuarine Organisms,
J. F. Vernberg, ed. University of South Carolina Press, Columbia, SC.
KENNEDY, D., AND M. S. BRUNO. 1961. The spectral sensitivity of crayfish and lobster vision. J. Gen.
Physiol. 44: 1089-1102.
LALL, A. B. 1981. Electroretinogram and the spectral sensitivity of the compound eyes in the firefly Pho-
turis versicolor (Coleoptera: Lampyridae): a correspondence between green sensitivity and species
bioluminescence emission. J. Insect Physiol. 27: 461-468.
LALL, A. B., E. T. LORD, AND C. O. TROUTH. 1982. Vision in firefly Photuris lucicrescens (Coleoptera:
Lampyridae): spectral sensitivity and selective adaptation in the compound eyes. J. Comp. Phvs-
iol. 147: 195-200.
LEGGETT, L. M. W. 1979. A retinal substrate for color discrimination in crabs. /. Comp. Phvsiol. 133:
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MARTIN, F. G., AND M. I. MOTE. 1982. Color receptors in marine crustaceans: a second spectral class of
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554.
SCOTT, S., AND M. I. MOTE. 1974. Spectral sensitivity in some marine Crustacea. Vision Res. 14: 659-
663.
STOWE, S. 1980. Spectral sensitivity and retinal pigment movement in the crab Leptograpsus variegatus
(Fabricius). J. Exp. Biol. 87: 73-98.
WALD, G. 1968. Single and multiple visual systems in arthopods. J. Gen. Physiol. 51: 125-156.
WALD, G., AND R. HUBBARD. 1957. Visual pigment of a decapod crustacean: the lobster. Nature 180:
278-280.
Reference: Biol. Bull. 173: 407-418. (October, 1987)
CELL VOLUME REGULATION BY MOLLUSCAN ERYTHROCYTES
DURING HYPOOSMOTIC STRESS: Ca2+ EFFECTS ON IONIC
AND ORGANIC OSMOLYTE EFFLUXES
LAURENS H. SMITH JR.1 AND SIDNEY K. PIERCE12
1 Department of Zoology, University of Maryland, College Park, Maryland 20742 and 2Center of Marine
Biotechnology, 600 East Lombard St.. Baltimore. Maryland 21202
ABSTRACT
The role of Ca2+ in volume regulation remains obscure. Before it can be investi-
gated, however, the time courses of osmolyte and cell volume regulation and the
effect of Ca2+ must be simultaneously specified in a suitable cell type. We have tested
the red blood cells of Noetia ponderosa in that context. Our results show that the
regulation of cell volume of the erythrocytes following hypoosmotic stress has two
components. The first is an efflux of intracellular K+ and Cl~ (but not Na+) that
begins immediately with the onset of hypoosmotic exposure. The second component,
an efflux of taurine, follows the first, but only after many minutes. In addition, clam
erythrocyte volume regulation is dependent on external [Ca2+]. Volume recovery is
potentiated in hypoosmotic media containing elevated Ca2+ levels. Taurine efflux
from clam erythrocytes in hypoosmotic conditions is reduced in Ca2+-free media and
potentiated in high Ca2+ media. In contrast, the effluxes of K+ and Cl are not sensi-
tive to extracellular Ca2+ levels in either isosmotic or hypoosmotic media. Thus, the
effluxes of ionic and organic osmolytes from these cells are controlled by mechanisms
that differ in response time and Ca2+ sensitivity. These results suggest that the clam
cells have an unexceptional volume regulatory mechanism and should therefore
make an excellent model with which to study the role of Ca2+ in that process.
INTRODUCTION
Cell volume in response to hypoosmotic stress is controlled by regulation of the
amount of intracellular osmolytes. Cell volume increases rapidly when water enters
in response to hypoosmotic exposure, but then decreases as an efflux of intracellular
osmolytes removes osmotically obligated water.
The cells of marine invertebrates use intracellular free amino acids (FAA, for re-
view see Pierce, 1 982) or other small nitrogenous compounds (proline betaine. Pierce
et al, 1984; glycine betaine. Warren and Pierce, 1982) as osmolytes. In some inverte-
brate cell types, an intracellular inorganic component to cell volume regulation has
been found in addition to the organic osmolytes (Limulus myocardium. Warren and
Pierce, 1982; Glycera coelomocytes, Costa and Pierce, 1983; Noetia red blood cells,
Smith and Pierce, 1983; Cancer leg muscle, Moran and Pierce, 1984). The ions (Na+,
or K+, and Cl") leave the cell as soon as the hypoosmotic stress begins, and the organic
efflux follows at a slower rate.
In addition to the above characteristics, cell volume regulation in response to a
hypoosmotic stress seems to be affected by external Ca2+ concentrations (for a review
see Pierce and Amende, 1981; Pierce, 1982). Generally, when Ca2+ is reduced or
Received 2 January 1987; accepted 27 July 1987.
Abbreviations: FAA, free amino acids; ASW, artificial seawater, EGTA, ethyleneglycol-bis-(|S-amino
ethyl ether) N,N'-tetraacetic acid.
407
408 L. H. SMITH JR. AND S. K. PIERCE
omitted from the medium, cell volume regulation is either blocked or reduced. Free
[Ca2+]i is too low to exert a substantial osmotic effect. Thus, the role played by Ca2+
in the volume recovery must be either to activate or to regulate the osmolyte efflux
mechanism. Since understanding that regulatory mechanism is a central issue in cell
volume regulation, we have begun to investigate the Ca2+ effects more closely.
Unfortunately, the characteristics of the volume regulatory mechanism have not
been examined in sufficient detail in any single cell type to serve as a point of depar-
ture. Therefore we have selected a single cell type, the red blood cell of the bivalve,
Noetia ponderosa, and have described the entire process of osmolyte regulation dur-
ing the volume response to a hypoosmotic stress on this system. In addition, we have
determined the effect of [Ca2+]0 on each step of the process. Our results provide the
first simultaneous chronology of all the events (ion, amino acid, and volume changes)
that constitute cell volume recovery from a hypoosmotic stress, including the effects
of Ca2+ on that chronology. In addition, the similarity of our results to those from
other cells indicates that the Noetia cells are a useful model with which to study the
effects of Ca2+ further. A preliminary report of this study has been published earlier
(Smith and Pierce, 1983).
MATERIALS AND METHODS
Preparation oferythrocytes
N. ponderosa were collected by commercial dredge and maintained as described
by Amende and Pierce ( 1 980a).
Blood was collected from the clams at room temperature by forcing the valves
open and slashing the mantle. The blood was diluted with artificial seawater (ASW,
935 mosm) made according to Amende and Pierce (1980) and buffered to pH 7.4
with 5 mAf MOPS. The blood was then filtered through polyester wool. The erythro-
cytes were centrifuged twice at 500 X g; the supernatants were discarded and the cells
were resuspended in ASW between the spins. After the second centrifugation, the
cells were resuspended in ASW and centrifuged at 3000 X g, and the top layer of
reproductive and amoebocytic cells was removed by aspiration. The erythrocyte pel-
let was washed twice more and the erythrocytes were then resuspended in approxi-
mately 1 ml of ASW for further use.
Measurement of cell volume regulation
Cell volumes of Noetia erythrocytes were measured in isosmotic (935 mosm) and
hypoosmotic (560 mosm) ASW, or in iso- or hypoosmotic media containing in-
creased Ca2+ or no Ca2+ (Table I). Hypoosmotic ASW was made by diluting isosmotic
ASW with glass distilled water. MOPS was always kept at 5 mM, and the pH at 7.4.
The osmotic concentrations of the solutions containing 150 mA/Ca2+ were matched
to the control solutions by reducing the NaCl content. The high Ca2+ isosmotic solu-
tion was made fresh just before use as follows: first, the chloride salts of Na+, K+, and
Mg2+ were dissolved in distilled water in the concentrations indicated in Table I.
CaCl2 was then added to the mixture to produce a final concentration of 1 50 mM.
Finally, predissolved solutions of Mg2SO4 and NaHCO3 were added very slowly with
stirring to produce the concentrations of the ions listed in Table I.
NaCl content was increased to account for the osmotic deficit produced by Ca2+
removal in the Ca2+-free ASW. This last solution also contained 1 mA/ EGTA. The
osmotic concentrations of all solutions were determined before use with a vapor pres-
sure osmometer (Wescor, model 5 100C).
Approximately 4.0 X 106 washed cells were suspended in each experimental me-
OSMOLYTE REGULATION BY CLAM BLOOD CELLS 409
TABLE I
Ionic composition (in mmoles/liter) ofisosmotic artificial seawaters used as experimental media
Ion Control Ca2+-Free HighCa2+
NaCl
389.0
400.1
249.6
MgCl2
24.4
24.4
24.4
CaCl2
9.7
0
1 50. 1
KC1
9.7
9.7
9.7
MgSO4
27.0
27.0
27.0
NaHCO,
2.3
2.3
2.3
EGTA
—
1.0
MOPS
5.0
5.0
5.0
dium and the distribution of cell volumes in a 10,000 cell aliquot was measured after
5, 10, 20, 60, and 120 min of exposure to the media using an electronic cell sizer
(Coulter Counter Model ZB) and Coulter Channelyzer (Coulter Electronics Inc., Hia-
leah, FL) interfaced with an Apple 11+ computer. The time course of erythrocyte
volume change was measured in both high Ca2+ hypoosmotic and Ca2+-free hypoos-
motic ASWs following a 30 min preincubation of the cells in 935 mosm ASW con-
taining high Ca2+ and no Ca2+, respectively.
Mean cell volumes from a given experiment were expressed as a percent of the
mean cell volume at zero time. The differences in the percent initial cell volume in
experimental media at all sampling times were tested using analysis of variance and
the Student-Newman Keuls multiple range test (Steele and Torrie, 1960). P < 0.05
was considered significant.
Taurine efflux measurements
The amino acid efflux from Noetia erythrocytes exposed to hypoosmotic media
consists primarily of taurine (Amende and Pierce, 1 980). In addition, our preliminary
experiments showed that when a treatment altered the amino acid efflux, the release
of each amino acid was changed by the same proportion; the effluxes of the amino
acids were not altered differentially. In particular, taurine always accounted for 60-
70% of the total FAA efflux regardless of the magnitude of the FAA efflux. Therefore,
we used taurine to represent amino acid efflux.
Clam erythrocytes were removed from the animal and prepared as described
above. The cells were then placed into one of the experimental media. At appropriate
intervals, aliquots containing 4-7 X 106 cells were removed from the experimental
cell suspension and centrifuged at 5000 X g for 10 min. The taurine content of the
supernatant was then determined as follows. An equal volume of 80% ethanol was
added to the supernatant and the solution was placed into a boiling water bath for 20
min. Precipitated protein was removed by centrifugation at 25,000 X g for 20 min.
The supernatant was then lyophilized, the residue taken up in 0.2 TV lithium citrate
buffer, pH 2.2, and the taurine content measured with an amino acid analyzer (JEOL
JLC-6AH). The cell number in three 1 5-20 ^1 aliquots from each cell suspension was
counted three times with the Coulter Counter and averaged to express the taurine
levels in terms of cell number.
Significant differences in taurine efflux were determined by analysis of variance
and the Student-Newman Kuels multiple range test.
410 L. H. SMITH JR. AND S. K. PIERCE
Intracellular K+, Na+, and Cl~
Erythrocytes were isolated as described above, and 10-20 X 106 cells were sus-
pended in isosmotic media. Six 0.2 ml aliquots (approximately 1-8 X 106 cells) were
then removed for measurement of intracellular K+, Na+, or Cl~ at zero time. The rest
of the isosmotic suspension was divided in half and both halves centrifuged at 3000
( g for 5 min. The supernatants were aspirated and the cells resuspended in either
isosmotic or hypoosmotic media and sampled at intervals. At each sampling time,
triplicate 0.2 ml aliquots (approximately 1-8 X 106 cells) were removed from the
experimental cell suspension for the measurement of intracellular K+, Na+, or CT.
Intracellular K+ was measured by the method of Costa and Pierce (1983). The 0.2
ml aliquot of cell suspension was layered onto 0. 1 ml of silicone oil (Wm. F. Nye,
Inc., New Bedford, MA 02742, USA) which had been layered over 0.1 ml of 25%
glycerol. This preparation was then centrifuged at 3000 X g for 3 min (Beckman
Microfuge II) which pelleted the cells through the oil into the glycerol where they
lysed. The experimental medium remained on top of the oil. After the medium and
oil layers were aspirated, the glycerol phase containing lysed erythrocytes was pre-
pared for K+ measurement using atomic absorption spectroscopy (Perkin-Elmer
model 560).
The amount of medium carried with the cells as they moved through the oil into
the glycerol was determined for parallel replicates of cell suspension by the method
of Freel et al. ( 1 973) using 14C-PEG as an extracellular space marker. The K+ content
of the trapped medium (usually about 1% of the total cell pellet K+) was subtracted
from the K+ content of the glycerinated cell pellet to yield intracellular K+.
Intracellular Na+ was measured using the same technique described above for K+,
except that the trapped volume was measured directly for each sample instead of for
parallel replicates. Since ASW Na+ concentrations are high, the Na+ level of the
trapped medium could account for up to 60% of total cell pellet Na+. After the cells
had been sampled as described above, the medium and oil layers were aspirated,
and an additional 0. 1 ml of 25% glycerol was added to the cell pellet. This glycerol
suspension was homogenized, and 0.05 ml was removed for trapped volume estima-
tion. The remaining 0.15 ml was used to measure cellular Na+ content by atomic
absorption spectroscopy.
The technique for determining intracellular Cl~ was identical to that used for Na+,
except that CT was measured by amperometric titration (Chloridometer, Buchler-
Cotlove).
Statistical differences in intracellular K+, Na+, and CT content were determined
using analysis of variance and the Student-Newman Kuels multiple range test.
RESULTS
Noetia erythrocytes swell rapidly in hypoosmotic ASW, but within 10 min (P
< 0.05) after hypoosmotic exposure they begin to recover towards their original vol-
ume (Fig. 1 ). Although volume recovery continues for at least two hours after hypoos-
motic exposure, very close to half of the volume decrease happens within 20 min of
hypoosmotic exposure.
Taurine efflux from Noetia erythrocytes in hypoosmotic media increases signifi-
cantly during the volume regulatory period. However, most of the taurine efflux oc-
curs between 10 and 60 min of hypoosmotic exposure (Fig. 2).
There is no significant difference between the Na+ content of isosmotic and hypo-
osmotic cells over the duration of the two hour time course (Fig. 3a). However, intra-
cellular K+ decreases by about 15%, from 98 to 85 nmoles/106 cells within 10 min
OSMOLYTE REGULATION BY CLAM BLOOD CELLS
411
UJ
o
_l
<
150
140
130
120
110
100 -
mosm
935 560
O • CONTROL
D • Co2* FREE
A A HIGH Co2'
0 5 10 20 60
120
TIME (min)
FIGURE 1. Volume changes of Noetia ponderosa erythrocytes exposed to isosmotic (935 mosm) or
hypoosmotic (560 mosm) ASW of varying Ca2+ concentration. Vertical bars are ± 1 standard error of the
mean (S.E.M.).
<4
_l
_l
Ul
O
« 3
-
0
\
Ul
Z 2
-
rr
ID
H
W 1
"o
T
E
c
[-}-
l-j-
/-v
935
560 935
560
935 560
935560
0
10
60
120
TIME (min)
FIGURE 2. Taurine efflux from Noetia ponderosa erythrocytes in isosmotic (935 mosm) and hypoos-
motic (560 mosm) ASW. Vertical bars are ± 1 S.E.M.
412
L. H. SMITH JR. AND S. K. PIERCE
A.
CO
o
<£>
o
o
z
</>
.SJ
o
E
B.
o
1C
O
s
o
CO
UJ
o
o
10
_«»
O
E
60
40
20
0
co 100
80
60
TIME (min)
FIGURE 3. Intracellular Na+ (A), K+ (B), and Cl~ (C) content of Noetia ponderosa erythrocytes in
isosmotic (closed circles) and hypoosmotic (open circles) ASW. Vertical bars are ± 1 S.E.M.
after hypoosmotic exposure, and little further decrease occurs subsequently (Fig. 3b).
In addition, intracellular Cl~ decreases by 50% within 10 min of hypoosmotic expo-
sure, from about 70 to 35 nmoles/106 cells (Fig. 3c). Thus, K+ and CT~, but not Na+,
leave Noetia erythrocytes after hypoosmotic exposure, and in contrast with the tau-
rine efflux, this movement of intracellular ions begins immediately. The CT efflux
from Noetia erythrocytes was only partially balanced by the K+ loss. Thus, other
cations besides K+ (but not Na+) must leave the clam erythrocyte during the early
phase of volume recovery.
In the absence of Ca2+, the volume regulation in response to hypoosmotic ASW
is partially inhibited (Fig. 1 ). Cells in the hypoosmotic Ca2+-free medium recovered
slightly less than half the volume of control cells in normal hypoosmotic medium.
Ca2+ lack did not affect the volume of erythrocytes in isosmotic ASW (Fig. 1 ).
Isosmotic Ca2+-free ASW had no significant effect on cellular K+ (Fig. 4a). More-
over, the usual hypoosmotic effect on cellular K+ content occurred whether or not
Ca2+ was present in the medium (Fig. 4a). The effect of Ca2+ lack on intracellular
Cl~ content was similar in pattern to that of K+. The Cl content of the cells was
OSMOLYTE REGULATION BY CLAM BLOOD CELLS
413
mosm
935 560
• • CONTROL
Ca2+-FREE
CO
LJ
O
100
80
60
o
E 40
4
I I
B.
CO
LJ
O
(O
O
o
o
E
c
80
60
40
20
0 10
60
120
TIME (min)
FIGURE 4. Intracellular K+ (A) and Cl~ (B) content ofNoetia pondcrosa erythrocytes in control and
Ca2+-free isosmotic (935 mosm) and hypoosmotic(560 mosm) ASW. Vertical bars are ±1 S.E.M.
altered only by the usual amounts in response to the osmotic stress, regardless of
[Ca2+]0 (Fig. 4b).
In contrast, taurine efflux from Noetia erythrocytes in Ca-+-free hypoosmotic
ASW was significantly less than that of cells in hypoosmotic ASW after 60 and 120
min (Fig. 5). A small yet significant efflux of taurine occurred from clam erythrocytes
in Ca2+-free isosmotic ASW (Fig. 5). Therefore, taurine efflux was inhibited in Ca2+-
free hypoosmotic ASW, while K+ and Cl~ effluxes were unchanged.
Cell volume regulation by clam erythrocytes in media containing elevated Ca2^
was potentiated (Fig. 1 ). Hypoosmotically stressed clam erythrocytes recovered to
1 19% of initial cell volume in the presence of high Ca2+, while the cells in hypoos-
motic medium containing normal Ca2+ recovered to only 1 29% of initial cell volume.
No significant change in erythrocyte volume occurred upon exposure of the cells to
isosmotic high Ca2+ ASW.
High Ca2+ had no effect on taurine efflux from clam erythrocytes in isosmotic
ASW (Fig. 7) or on erythrocyte K+ or CP content in either hypoosmotic or isosmotic
media (Fig. 6a, b). The usual decrease in intracellular K+ and Cl within 10 min
414
CO
UJ
O
°0
\
LJ
a:
L. H. SMITH JR. AND S. K. PIERCE
5 ,- 560 mosm
4
3
2
I
o
C CF
C CF
C CF
C CF
o
e
C
2r
935 mosm
r-t-rn
c CF
0
C CF
10
TIME (min)
FIGURE 5. Taurine efflux from Noetia ponderosa erythrocytes in normal (C) and Ca2+-free (CF)
isosmotic (935 mosm) and hypoosmotic (560 mosm) ASW. Vertical bars are ± 1 S.E.M.
of exposure to hypoosmotic media (about 10 and 30 nmoles/106 cells, respectively)
occurred in the presence of high Ca2+ (Fig. 6a, b). In contrast, the taurine efflux from
Noetia erythrocytes in high Ca2+ hypoosmotic media was significantly greater, almost
twice that of control cells (Fig. 7).
Thus, both Ca2+-free hypoosmotic ASW and high-Ca2+ hypoosmotic ASW affect
the volume regulation and taurine efflux, but not K+ or Cl~ efflux, from clam eryth-
rocytes.
DISCUSSION
Our results confirm our earlier report (Smith and Pierce, 1983) that cell volume
regulation by Noetia erythrocytes in response to hypoosmotic conditions results from
the efflux of both organic and inorganic osmolytes. Noetia erythrocytes use effluxes
of K+, Cl~, and amino acids to regulate cell volume during a hypoosmotic stress. The
ion efflux occurs immediately in response to the stress while the efflux of taurine is
delayed. Both cell volume regulation and taurine efflux are dependent on extracellu-
lar [Ca2+] but the ion efflux is not. Therefore, a mechanism is present which allows
the two independent membrane permeability systems to produce a coordinated efflux
of organic and inorganic osmolytes from Noetia erythrocytes. Both K+ and Cl leave
OSMOLYTE REGULATION BY CLAM BLOOD CELLS
415
A.
LJ
o
E
80
60
o
\
^ 40
20
mosm
935 560
CONTROL
HIGH Co2'
B.
Ld
O
»
O
O
tn
£
O
£
80
60
40
20
0 10
60
120
TIME (min)
FIGURE 6. Noetia ponderosa erythrocyte intracellular K+ (A) and Cl~ (B) content in control and
high Ca2+ isosmotic ( 1 50 mM, 935 mosm) and hypoosmotic (90 mAI. 560 mosm) ASW. Vertical bars are
±1 S.E.M.
the Noetia erythrocytes immediately after the salinity stress while taurine concentra-
tions do not change for many minutes. Thus, the two ions act alone as the osmolytes
during the initial period of volume recovery. The amount of volume recovery result-
ing from the ionic efflux may be substantial. We do not know the maximum volume
attained by the Noetia cells, but it is likely that volume recovery is well underway at
our first sampling interval (5 min) (see, for example. Costa et ai, 1980). Therefore,
the initial stage of ionic efflux accounts for most of the volume recovery.
The significance of a dual osmolyte efflux system is not yet clear, although it is
not uncommon (Vislie. 1980; Costa and Pierce, 1983; Warren and Pierce, 1983;
Moran and Pierce, 1984; Hoffman et al, 1984). The differences between the ionic
and organic osmolyte effluxes may provide a clue. Although the inorganic osmolyte
efflux from the Noetia cells occurred immediately in response to hypoosmotic expo-
sure, the concentrations of the ions may not remain reduced. In at least one case,
after the initial reduction, the intracellular ion concentration was partially restored
as the organic components were reduced (Warren, 1982). On the other hand, while
the efflux of organic osmolytes is initiated later in the response, no restoration of
pre-stress concentrations occurs. Although the data are limited, they suggest that the
organic osmolytes are not functioning as effectors in direct response to the osmotic
stress, but rather as osmotic replacements as the ions are regulated back to homeo-
static levels. The organic osmolytes are kept at levels which produce cellular osmotic
stabilization without the detrimental effects on cell functions that might be caused
416
L. H. SMITH JR. AND S. K. PIERCE
UJ
o
UJ
E
20 r
16
12
8
4
0
560 mosm
C HC
C HC
C HC
C HC
to
.2?
o
935 mosm
TIME (min)
FIGURE 7. Taurine efflux from Noetia jwndcwsa erythrocytes in control (C) and increased Ca2+
(HC) isosmotic ( 1 50 mAf, 935 mosm) and hypoosmotic (90 mM, 560 mosm) ASW. Vertical bars are ± 1
S.E.M.
by marked reduction in inorganic ion concentration. We are presently evaluating
these ideas experimentally.
Our results indicate that the Noetia erythrocyte displays the components of vol-
ume regulation found among many other cell types. Rapid decreases of intracellular
K+ following hypoosmotic treatment occur in other invertebrate (Kevers el a/.,
1979a, b, 1981; Costa and Pierce, 1983; Moran and Pierce, 1984) and vertebrate
(Kregenow, 1971;Cala, 1977; Cheung^/ ai, 1982) cells. A hypoosmotically induced
Cl efflux concomitant with K+ release occurs in crustacean axons (Kevers ct ai,
1979b) and teleost erythrocytes (Lauf, 1982). Cl" loss is also associated with Na+
efflux in Limulus myocardium (Warren and Pierce, 1982) and rat liver (van Rossum
and Russo, 1 984). Thus, the initial ionically based stage of hypoosmotic volume regu-
lation in Noetia cells is qualitatively similar to the responses of many other cell types.
The hypoosmotically induced Na+ and Cl effluxes from Limulus myocardium,
like the ionic effluxes from Noetia blood cells, are unaffected by the absence of Ca2+
(and Mg2+) (Warren, 1 982). However, the ionic components of the volume regulating
systems of some other cell types are sensitive to the ambient level of Ca2+. For in-
stance, the K+ efflux from Glycera coelomocytes exposed to either isosmotic or hypo-
osmotic Ca2+-free media is potentiated (Costa and Pierce, 1983). External Ca2+ is
OSMOLYTE REGULATION BY CLAM BLOOD CELLS 417
important in the regulation of K+ efflux from vertebrate cells as well. For example,
K+ efflux from amphibian red blood cells was inhibited in Ca2+-free hypoosmotic
media (Cala, 1983). Likewise, K+ efflux and volume regulatory ability were impaired
in Ehrlich ascites tumor cells (Hoffman et ai, 1984) and human lymphocytes
(Grinstein, 1983) by Ca2+-free conditions.
Both volume regulation and taurine efflux are inhibited in Noetia erythrocytes
exposed to Ca2+-free hypoosmotic ASW, and both are potentiated in high Ca2+ hypo-
osmotic ASW. Similarly, cell volume regulation is inhibited by Ca2+-free hypoos-
motic media in Callinectes axons (Gerard, 1975) and Glycera coelomocytes, although
the FAA efflux from Glycera coelomocytes in divalent cation-free hypoosmotic me-
dia is potentiated (Costa and Pierce, 1983). In summary, the Ca2+ effects on volume
recovery and the effluxes underlying it are not the same in every cell type. Ionic or
organic osmolytes may be effected by Ca2+ change, but in the Noetia cells only the
amino acid portion of the response is Ca2+ sensitive.
An additional aspect of Ca2+ regulation is suggested by the small amount of tau-
rine released from Noetia erythrocytes in the Ca2+-free isosmotic medium. A similar
leak of amino acids occurred from Glycera coelomocytes in divalent cation-free isos-
motic media (Costa and Pierce, 1983) and mussel ventricles (Pierce and Greenberg,
1973). The increase in FAA permeability in Ca2+-free isosmotic media, in contrast
to its decrease in hypoosmotic media, suggests that different Ca2+ sensitive mecha-
nisms exist for the control of membrane permeability at normal and lowered osmotic
concentrations. The increased membrane permeability to taurine caused by Ca2+-
free isosmotic media may be due to a lack of divalent cations that bind to and stabilize
membranes (Lin and Macey, 1978; Papahadjopolous, 1978; Swinehart et al., 1980).
But in any case, Ca2+ may have more than one action on osmolyte permeability
depending upon the osmotic environment.
In conclusion, the characteristics of the volume response of the Noetia cells and
their similarity to those of other cell types suggests that the clam red blood cell will
be an excellent model with which to examine further effects and the roles of Ca2+ in
the regulation of cell volume.
ACKNOWLEDGMENTS
This work was supported by N.I.H. grant #GM-23731, and by the Chesapeake
Bay Fund. We wish to thank the Terry family of Willis Wharf, VA, for providing
Noetia and Alex Politis for perfecting the oil partition technique used in this study to
measure Na+ and Cl". This paper is Contribution No. 270 from the Tallahassee,
Sopchoppy, and Gulf Coast Marine Biological Association, Inc.
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CALA, P. M. 1977. Volume regulation by flounder red blood cells in anisotonic media. J. Gen. Phvsiol. 69:
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CALA, P. M. 1983. Cell volume regulation by Amphiuma red blood cells. The role of Ca2+ as a modulator
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CHEUNG, R. K., S. GRINSTEIN, H. M. DOSCH. AND E. W. GELFAND. 1982. Volume regulation by human
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COSTA, C. }., S. K. PIERCE, AND M. K. WARREN. 1980. The intracellular mechanism of salinity tolerance
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in polychaetes: volume regulation by isolated Glycera dibranchiata red coelomocytes. Bio/. Bull.
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COSTA, C. J., AND S. K. PIERCE. 1 983. Volume regulation in the red coelomocytes of Glycera dibranchiata:
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FREEL, R. >. G. MEDLER, AND M. E. CLARK. 1973. Solute adjustments in the coelomic fluid and
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GERARD. J. F. 1975. Volume regulation and alanine transport. Response of isolated axons ofCallinectes
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GRINSTEIN, S., S. COHEN, B. SARKADI, AND A. ROTHSTEIN. 1983. Induction of 86Rb fluxes by Ca2+ and
volume changes in thymocytes and their isolated membranes. J. Cell Physiol. 1 16: 352-362.
HOFFMAN, E. K., L. O. SIMONSEN, AND I. H. LAMBERT. 1984. Volume-induced increase of K+ and Cl~
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KEVERS, C., A. PEQUEX, AND R. GILLES. 1979a. Effects of hypo- and hyperosmotic shocks on the volume
and ion contents ofCarcinus meanus isolated axons. Comp. Biochcm. Physiol. 64A: 427-43 1 .
KEVERS, C., A. PEQUEX, AND R. GILLES. 1979b. Effects of an hypo-osmotic shock on Na+, K+, and Cl
levels in isolated axons ofCarcinus meanus. J. Comp. Physio/. 1 29: 365-37 1 .
KEVERS, C., A. PEQUEX, AND R. GILLES. 1981. Role of K+ in the cell volume regulation response of
isolated axons of CY//r/>n« meanus submitted to hypo-osmotic conditions. Molec. Phvsiol. 1: 13-
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KREGENOW, F. M. 197 1 . The response of duck erythrocytes to non-hemolytic media. J. Gen. Phvsiol. 58:
372-412.
LAUF, P. K. 1982. Evidence for chloride dependent potassium and water transport induced by hypoos-
motic stress. J. Comp. Physiol. 146: 9-16.
LIN, G. S. B., AND R. I. MACEY. 1978. Shape and stability changes in human erythrocyte membranes
induced by metal cations. Biochim. Biophys. Ada 512: 270-283.
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PIERCE, S. K. 1982. Invertebrate cell volume control mechanisms: a coordinated use of intracellular amino
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PIERCE, S. K.. S. C. EDWARDS, P. M. MAZZOCCHI, L. J. KLINGER, AND M. K. WARREN. 1984. Proline
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PIERCE, S. K., AND M. J. GREENBERG. 1973. The initiation and control of free amino acid regulation of
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VAN ROSSUM, G. D. V., AND M. A. Russo. 1984. Requirement of Cr and Na+ for the ouabain-resistant
control of cell volume in slices of rat liver. J. Membrane Biol. 77: 63-76.
SMITH, L. H., AND S. K. PIERCE. 1983. Noetia ponderosa erythrocytes use intracellular ions for initial
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SWINEHART, J. H., J. H. CROWE, A. P. GIANNINI, AND D. A. ROSENBAUM. 1980. Effects of divalent ion
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Reference: Biol. Bull. 173: 419-449. (October, 1987)
ABSTRACTS OF PAPERS PRESENTED AT THE GENERAL SCIENTIFIC
MEETINGS OF THE MARINE BIOLOGICAL LABORATORY
17-19 AUGUST 1987
Abstracts are arranged alphabetically by first author within the fallowing categories:
cell motility ami cytoskeleton; comparative physiology; developmental biology and
fertilization; ecology; integrative neurohiology and behavior; neurobiology; and vi-
sion. Author and subject references will be found in the regular volume index in the
December 1987 issue.
CELL MOTILITY AND CYTOSKELETON
Ultrathin optical-sectioning-tomography achieved with the light microscope. SHINYA
INOUE (Marine Biological Laboratory).
We have achieved optical sections as thin as 0. 1 urn for rectified polarized light and phase contrast
microscopy and 0.2 /urn for DIG microscopy. Rapid, programmed, serial through-focusing is also possible.
The conditions used to achieve the ultrathin optical sections were: ( 1) a new generation of well-corrected,
high N. A. objective lenses (Nikon new series 100/ 1 .4 and 60/ 1 .4 and Zeiss Axiophot 63/ 1 .4, Plan Apochro-
mats); (2) a high N.A. condenser fully and uniformly illuminated with high intensity monochromatic (546
nm) light through a light scrambler (Ellis 1985, J. Cell Bio/. 101: 83a; Inoue 1986, Video Microscopy,
Figs. III-2 1 , 111-22); (3) optical contrast enhancement and image correction with a condenser rectifier for
polarization and DIG optics, together with improved lens coating; (4) analog enhancement with black level
and gain controls on the video camera (DAGE-MTI 65M Newvicon); and (5) further digital enhancement
(UIC lmage-I). Optical sectioning, through-focal sectioning, recording, and analysis were also aided by (6)
a solid, optical bench microscope with precision stage and drives (Inoue 1 986, ihid)\ (7) stepper motor and
timed controller for the fine focus; and (8) a 450 TV line-resolution laser disk recorder (Panasonic OMDR
T2-2021 FBC). Examples of through ultrathin optical sectioning shown were: 5 nm immuno-gold stained
mitotic microtubules, and prophase chromatin, in Haemanthus; Golgi-stained neuronal axon and den-
drites; and surface filaments on buccal epithelial cells.
Supported by grants NSF DCB-85 1 8672 and NIH 5 R37 GM-3 1617.
Dynamics of spindle microtubules visualized in vivo by high resolution video polar-
ization microscopy. SHINYA INOUE (Marine Biological Laboratory), EDWARD D.
SALMON, AND LYNNE CASSIMERIS (Biology, UNC, Chapel Hill, NC).
We can now visualize individual microtubules (MTLs) directly in living cells using ultrathin optical
sectioning in video polarized light microscopy. We report here the dynamic lateral association of MTLs
seen in the mitotic spindle of newt lung epithelial cells using this new method. Throughout mitosis in the
half spindle, two or more MTLs associate laterally over distances of 1 to 2 /urn for 1 to 10 second durations,
forming short-lived birefringent rods. In time-lapse recording, the 100 or so rods that stochastically appear
in the 0. 1 ^m-thick optical section appear to shimmer (the northern lights flickering seen at lower resolu-
tion). In prometaphase, MTLs attached to the kinetochore form discrete, birefringent bundles, one to a
kinetochore. The 0.2 /urn-diameter bundle (of 10-20 MTLs) can be over 10 ^m long or even reach the
spindle pole. While the bundle is considerably more stable than the rods, it also changes length with time.
Presumably as the extent of lateral association varies, the MTLs bundle or splay polewards. The MTLs
may also assemble and grow, or disassemble and shorten. Each chromosomal spindle fiber (CSF) contains
many more MTLs than those in the bundle; the dynamic lateral association of the kinetochore and non-
kinetochore MTLs together contribute to the mechanical integrity and paradoxical lability of the CSFs. In
anaphase the kinetochore MTLs are commonly not associated into bundles but fan out diffusely poleward.
In late anaphase and telophase, individual waving MTLs and those splaying off the tips of the forming stem
bodies are clearly seen between the daughter set of chromosomes. Birefringent actin filament (bundles) in
the cleavage contractile ring and microvilli are also clearly visible.
Supported by grants: NSF DCB-85 18672 and NIH 5 R37 GM-3 16 17 to S.I.; NIH GM-24364 and
NSF DCB-86 1 662 1 to E.D.S.
419
420 ABSTRACTS FROM MBL GENERAL MEETINGS
The heat-stability of squid axoplasm neurofilaments provides a rapid method for their
purification. KENNETH S. KOSIK (Harvard Medical School) AND J. METUZALS.
A number of procedures have been used to purify neurofilaments from the squid giant axon. The
polypeptides that are most consistently associated with the filamentous fraction are P60 (apparent Mr
60,000), P200 (apparent Mr 200,000), and a high molecular weight band (HMW) with an apparent Mr of
-400,000. Purification of these polypeptides includes cytochrome c precipitation, discontinuous sucrose-
gradient centrifugation and gel nitration, and Millipore filtration. We find that the neurofilament polypep-
tides from Loligopealei have heat-stability which permits their rapid purification.
The giant axon was dissected, the axoplasm extruded, and the cleaned sheath and axoplasm immedi-
ately frozen on dry ice. These tissues were homogenized in a microtubule assembly buffer ( 1 00 mAf PIPES
pH 6.6, 1.0 mAf EGTA, 1.0 mAf MgSO4) with 2 mAf phenylmethyl-sulfonyl fluoride and 5 mAf EDTA.
The homogenate was spun at 25,000 x g for 1 5 min. The supernatant was made 0.4 Af NaCl and immersed
in a boiling water bath for 4 min. After boiling the tube was plunged into ice and spun at 100,000 X g for
30 min. The supernatant was dialyzed and analyzed by SDS-PAGE.
Coomassie staining of the axoplasmic preparation revealed polypeptides of molecular weights consis-
tent with the squid neurofilament proteins. The only contaminating bands were a group of polypeptides
of Mr ~ 18,000 which may represent calmodulin. Trace amounts of identically migrating bands were
present in the axonal remnant and sheath preparation. The putative P200 and HMW bands were stained
by a squid neurofilament antibody directed against phosphorylated epitopes. Anti-IFA. which recognizes
an epitope shared by all classes of intermediate filaments (Prussc/ al. 1981, Cell '21: 419), immunoreacted
with the putative P60 and P200. RT97 (Anderton et al. 1982, Nature 298: 84), which is directed against a
phosphorylated epitope of mammalian high and mid-sized neurofilament, immunoreacts with squid P60,
P200, and HMW. When mice were immunized with the heat-stable squid axoplasmic fraction, the resul-
tant monoclonal antibodies were generally directed against both the P200 and HMW or principally against
P60. The biophysical property of heat-stability suggests that the molecular structure of the neurofilament
polypeptides is elongated with few hydrophobic domains relative to their mass.
Supported by grant MA-8605 from the Medical Research Council of Canada to J.M.
Dynamics ofactin, myosin, and membranes in living cells during cell division. JEAN
M. SANGER, JEFFREY S. DOME, BALRAJ MITTAL, AND JOSEPH W. SANGER (Uni-
versity of Pennsylvania).
The distribution ofactin and myosin during cell division was followed in living cells by microinjection
of fluorescent G-actin or fluorescent myosin light chains into PtK2 cells. Cytoplasmic membranes were
labeled by exposing the PtK: cells to the dye 3,3' dihexyloxacarbocyanine iodide. During prophase, the
first stress fibers to disassemble were located over the condensing chromosomes. Stress fiber shortening
accompanied the rounding up of the prophase cell. Fluorescent actin and myosin were concentrated in the
mitotic spindle like all other fluorescent proteins that were injected into cells. During mid-anaphase, a
dramatic accumulation of myosin and actin fluorescence occurred in a band beneath the membrane at the
location of the former metaphase plate. The fluorescent band grew wider and brighter as cytokinesis began
and persisted as a bright band until the terminal stage of cytokinesis when mid-body formation occurred.
At this time two small bands of fluorescence formed at either side of the mid-body, perhaps acting as mini-
cleavage rings that maintain the integrity of the mid-body during the spreading of the two daughter cells.
Unlike actin and myosin, which were present in the mitotic spindle and cleavage furrow, membranes, as
revealed by 3,3' dihexyloxacarbocyanine iodide, were absent from these structures. Observations of in-
jected cells exposed to the dye showed no obvious concentration of membranes in the contractile ring. The
striking exclusion of most membranous organelles from the mitotic spindle probably accounts for the
concentration of contractile and non-contractile proteins that can be seen in the spindle when fluorescent
analogues are microinjected into cells. We conclude that the actin-myosin contractile ring responsible for
cytokinesis is formed not by the rearrangement ofactin and myosin filaments already present in the cell
cortex, but by a recruitment of these proteins from throughout the cytoplasm at mid-anaphase into a
contractile band.
Calcium transients are required for mitosis. ROBERT B. SILVER (Laboratory of Mo-
lecular Biology, University of Wisconsin, Madison) AND SHINYA INOUE.
Mitosis is thought to be regulated by transient elevations of cytoplasmic Ca2+ concentration. This
study tested directly if: (a) such Ca2+ transients occur during the cell cycle, and (b) if such Ca:+ transients
are used by the cell to signal the initiation of Ca2+-dependent biochemical pathways requisite for specific
mitotic events [e.g., nuclear envelope breakdown (NEB), anaphase onset (AO)]. Such a model would re-
quire a source/sink for Ca2+ and a Ca:+ binding target within the cytoplasm. Therefore, inhibition of the
CELL MOTILITY AND CYTOSKELETON 42 1
function of any one component in such a system should result in an arrest of Ca2+-dependent steps in
mitosis. An affinity purified antibody to the Ca2+-pump of smooth muscle sarcoplasmic reticulum (SR)
was prepared. This antibody inhibits ATP-dependent Ca2+ uptake by SR and mitotic apparatus (MA), and
recognizes a single membrane protein of 105 kD in these membrane preparations. Microinjection of this
IgG into second cell cycle E. panna blastomeres inhibits mitosis within two minutes of injection. Injections
in mid-anaphase halt chromosome movement; there is a loss of MA birefringence. Injection ofCa2+ chan-
nel antagonists specific for endomembrane Ca2+ efflux channels 6-7 min prior to NEB or 2 min prior to
AO prevents these events in a dosage-dependent fashion. Complete inhibition is achieved with 1 mg/ml
TMB-8 or 7 ng/m\ ryanodine, consistent with their relative effectiveness upon isolated membranes. Thus,
there are both a Ca2+-pump and a Ca2+ channel required for NEB and AO. Existence of cytoplasmic sites
which required Ca2 + -binding for activation of NEB and AO was tested by increasing intracellular Ca2+
buffering capacity by injecting increasing doses of antipyrylazo III (ApIII) (KD Ca2+ of 2 X 10~8 M). Cells
injected with 10 pi 0-30 pM ApIII continued normal mitotic cycling. Injection of 50 nAI ApIII between 6
and 5 min prior to NEB, or 2 min prior to AO inhibited these mitotic steps. Injection of 40 ^M ApIII was
inhibitory in only 60% of the cases. The timing of the injections was critical — the sensitive period (and
apparent Ca2+ pulse) occurring 6 min before NEB with a 40-45 s duration. This arrest by ApIII was not
spontaneously reversible. This NEB arrest was reversible by a subsequent injection of CaCli ( 10 pi 100 nAf
CaCl:). The double injected cells did not resume cycling activity immediately, but underwent NEB and
mitosis one cell cycle behind the control sister blastomeres. Injection of isoaequorins A, D, or E was used
to detect directly these cytoplasmic Ca2+. Cytoplasmic Ca2^ concentration drops as the cell enters mitosis.
Transient elevations of cytoplasmic Ca24 were found 6 min prior to NEB (40 s duration), 2 min prior to
AO (10-20 s duration), and during cytokinesis. Rapid sampling methods demonstrated that these Ca2+
pulses were composed of rapid (8 s frequency) pulses. These results demonstrate that transient elevations
of cytoplasmic Ca2+ are required for NEB and AO. They also suggest that the Ca2+ transients are used to
synchronize parallel biochemical pathways required for NEB, AO, and mitosis.
Supported by N.S.F. PCM-8703969 to R.B.S., N.S.F. DCB 85-18672 and NIH 5 R37-GM 31617 to
S.I., and N.S.F. DMB 87-03463 and N.I.H. GM 31314 to Osamu Shimomura who generously provided
the isoaequorin preparations.
COMPARATIVE PHYSIOLOGY
A subarachnoid space in the elasmobranch brain — macro and microscopic evidence
using large molecular weight fluorescent markers. ARTHUR M. BuTT(Dept. Phys-
iology, School of Medicine, East Carolina University, Greenville, NC 27858).
The brain of elasmobranchs is classically considered to be enclosed within a single ill-defined meninx
primiliva with only intraventricular (IV) cerebrospinal fluid (CSF). In fact, the skate (Raja erinacea) and
the dogfish (Mustclus canis) appear to possess three meningeal layers analogous to the dura, arachnoid,
and pia mater of higher vertebrates, and there is direct communication between the ventricular CSF and a
subarachnoid space via a large pore in the posterior choroid plexus.
Fifteen minutes after injection of FITC-Dextran (MW 70 kD) intravascularly and Evans blue al-
bumin(EBA, MW 60 kD) IV or subdurally, the brain was fixed and sections studied under the fluorescent
microscope. EBA was observed to exit through the posterior pore and fill the subarachnoid space when
injected IV, darkly staining the brain surface and subarachnoid blood vessels. There was further restriction
to EBA movement between the subarachnoid CSF and brain tissue. There was no apparent loss of FITC
from intracerebral or subarachnoid blood vessels. Also, EBA evidently diffused from the subarachnoid
space down alongside larger vessels as they passed through the brain surface. EBA did not cross the arach-
noid mater when injected IV or subdurally.
Elasmobranchs are therefore similar to other vertebrates, and have both arachnoid and dura mater
with a subarachnoid space directly connected with the ventricular CSF and separated from the subdural
space by a meningeal barrier. The perivascular space of intracerebral blood vessels is continuous with the
subarachnoid CSF but is separated from the brain interstitial fluid by the blood-brain barrier. A barrier is
indicated in subarachnoid vessels although its site was not discernible, and at the brain surface presumably
at the level of the subpial glia.
This work was made possible by a Grass Fellowship at the Marine Biological Laboratory, Woods Hole.
Effects of age on the skin pigmentation ofthegnppy(Poeci\ia reticulata). M. C. DICK-
MAN (Department of Biological Sciences, University of California, Santa Barbara,
CA, 93106).
Development of color patterns and ultrastructural changes of the pigment cells that form these color
patterns were studied in maturing male guppies using light and electron microscopy. Immature males were
422 ABSTRACTS FROM MBL GENERAL MEETINGS
tatooed for future identification and photographed using a stereo microscope for 21/: months. These were
compared to photographs taken of fully mature males over the same time period.
Melanophores first appeared on the dorsal regions of the fish. As they matured, young fish exhibited a
rapid migration of melanophores to all regions of their bodies. These melanophores formed a crisscross
pattern or<- >r two cells wide. They also formed massive aggregations in front of the caudal and behind the
ventral fins. These aggregations developed into black spots. As the fish aged more spots formed on other
regions of the body. Scales were removed from the spots and examined using a light microscope. These
scales revealed large aggregations of melanophores lacking any distinct shape. In contrast, scales removed
from non-spotted regions revealed melanophores of a stellate shape typical of teleost melanophores. The
melanophores from scales of spotted areas did not respond when treated with epinephrine or atropine
sulfate to induce aggregation or dispersion, respectively. When the melanophores from scales of other areas
of the body were chemically stimulated, both aggregation and dispersion of the melanin granules were
induced.
Electron microscopic studies of scale tissues taken from non-spotted regions of the body displayed
morphological features typical of teleost pigment cells. In contrast, scale tissues taken from spotted regions
revealed morphological features not found in scales of non-spotted regions. One type consisted of highly
compressed aggregations of melanin granules embedded in an electron-dense cytoplasmic matrix. Another
characteristic was an apparent disorganization of the cytoskeletal framework. Both electron microscopic
and immunocytochemical studies are in progress to examine these characteristics.
Transection of the spinal cord near the obex abolishes cardiovascular compensation
for gravity in bluefish (Pomatomus saltatrix). S. HUNTER Fox, CHRISTOPHER S.
OGILVY, AND ARTHUR B. DuBois (John B. Pierce Foundation Laboratory, New
Haven, CT).
The object was to determine if gravitational tolerance of the circulation of bluefish is abolished by
transection of the spinal cord. Bluefish were lightly anesthetized with tricaine, placed on a V-board, and
their gills perfused with seawater. Blood pressure (BP, mm Hg) was measured in the ventral aorta. The V-
board was tilted head up in air at 10° or 20° for 2 min, and 30° for 5 min or 30 min. Before transection of
the spinal cord, mean blood pressure decreased only slightly during the 10, 20, and 30° tilts. For example,
in a group of 5 fish, a control BP of 52 mm Hg decreased to 43 SE 1 .4 and heart rate (HR) increased from
53 to 68 per min SE 1 1 .2 at one min of a 30° tilt. Transection of the cord at C- 1 , 7 mm caudal to the obex,
did not alter the BP and HR response to tilting. Their BP decreased from 53 to 43 (SE 6.0) at 1 min of a
30° tilt and HR changed from 40 to 47 during the tilt. However, in 6 other fish, cord transection near the
medulla (3 mm caudal to the obex) produced immediate tachycardia and a fall in BP during tilting to 10,
20, or 30°. After the cord section, BP fell from 48 to 38 SE 2.3 during a 10° tilt, from 43 to 33 SE 2.5 at
20°, and from 44 to 29 SE 3.0 at 30°. HR was 76 before and 78 during the 30° tilt. This region of the cord
supplies sympathetic fibers to the heart and blood vessels in codfish [Nilsson, S. 1970 Ada Zool. (Stock-
holm) 57: 69-77]. We conclude that gravitational tolerance of the circulatory system in bluefish depends
on integrity of the spinal cord and its adjacent nerves just caudal to the obex, but not on the integrity of
the cord below the first cervical vertebra.
Initial results of lead measurements of deciduous teeth. ANN LEWANDOWSKI, MI-
CHAEL RABINOWITZ, ALAN LEVITON, KIRSTEN IVERSEN, AND SUSAN ROSE
(Neuroepidemiology Unit, Children's Hospital, Boston).
We have completed an analysis of 817 teeth as part of a prospective study of child development and
environmental lead. These teeth are from children whose umbilical cord blood was measured for lead in
1979-80. These children represent a general population not especially at high risk for lead poisoning.
Lead tends to concentrate in calcifying tissues. In teeth, a portion of incisor dentine is mineralized
concurrently with critical events of cortical development such as neuron migration and synaptogenesis;
this portion may be sensitive to lead. From each tooth collected by mail, those portions of secondary
dentine were sampled in duplicate ( 10 mg each), dissolved in acid, and analyzed for lead by anodic stripping
voltammetry (<0.05 ^g) under filtered air and with purified reagents. Recoveries of 0 to 100 nonagrams of
added lead were 100 ± 5 (SE) %.
We found an average of 3.8 ^g/g (ppm) lead (SD = 2.5, range 0 to 45, 2% > 10 ppm). Blanks contribute
about 0.2 ppm to these values; the average difference between the two splits of the same tooth are 1.05
ppm, and the mean difference between different teeth from the same child are 1 . 1 ppm (n = 24). Correlation
of tooth lead and umbilical cord blood lead is 0.12 (Spearman r, P = 0.02). The tooth lead also correlates
with the age of the tooth at shedding (r = 0. 17, P = 0.004). As in cord lead, there are demographic trends
in tooth lead relative to race and financial status. Thus, the tooth lead concentrations in this population,
although low on average, are sufficiently variable to provide a retrospective marker of lead insult.
COMPARATIVE PHYSIOLOGY 423
Inhibition by heparin of endotoxin-dependent coagulation ofamebocyte lysatefrom
Limulus polyphemus. JAMES A. MARCUM (Harvard Medical School) AND JACK
LEVIN.
Heparin is a highly sulfated complex carbohydrate composed of repeating glucosamine and uronic
acid moieties. It exhibits potent anticoagulant activity in vertebrate clotting systems. Limulus amebocyte
lysate (LAL) contains a cascade of activators which generate a clotting enzyme that produces gelation of a
clottable protein. We have examined the effect of heparin on endotoxin-dependent activation of LAL.
Addition of commercially available (mammalian) heparin to LAL, prior to the addition of the bacterial
lipopolysaccharide, inhibited coagulation, as determined by a gel end-point assay. Using a chromogenic
substrate (S-2222) and diluted LAL (1/100), heparin inhibited activation of the lysate by endotoxin in a
dose-dependent manner, with 50% of enzymatic activity inhibited by 2-3 Mg uronic acid/ml. Addition of
heparin after addition of the lipopolysaccharide to LAL did not inhibit the amidolytic activity of the clot-
ting enzyme. The specificity of heparin inhibition was investigated by examining endotoxin-dependent
activation of cell lysate in the presence of chondroitin 4-sulfate, chondroitin 6-sulfate, heparan sulfate,
dermatan sulfate, and hyaluronic acid. Addition of each glycosaminoglycan ( 10 ng uronic acid/ml) sepa-
rately did not inhibit LAL activation. To examine if the unique heparin structure that binds vertebrate
antithrombin is responsible for the inhibition of LAL activation, the complex carbohydrate was fraction-
ated into anticoagulantly active and inactive species, using antithrombin-sepharose. Both heparin species
comparably inhibited lipopolysaccharide-dependent activation of cell lysate. The mechanism by which
heparin inhibits LAL activation was examined by fractionating cell lysate into proactivator(s) and proclot-
ting enzyme preparations with heparin-sepharose under endotoxin-free conditions. Studies conducted
with the proactivator(s), proclotting enzyme, lipopolysaccharide, and chromogenic substrate revealed that
heparin did not inhibit the activation of the proactivator(s) by endotoxin or its amidolytic activity, but
that the complex carbohydrate did inhibit the generation of the clotting enzyme via the activator(s). The
physiological relevance of the regulation of LAL coagulation by heparin has not been established.
This work was supported, in part, by a Summer Fellowship (Marine Biological Laboratory) awarded
toJ.A.M.
The antiquity oftransglutamina.se: an intracellular enzyme from marine sponge cells
enhances clotting of lobster plasma. REED BROZEN, PETER SANDS, WILLIAM RIE-
SEN, GERALD WEISSMANN, AND LASZLO LORAND (Marine Biological Labora-
tory).
The aggregation of dissociated cells ofMicrociona prolij'era is the most ancient example ( 109 years) of
stimulus/secretion coupling in multicellular animals. The cells secrete a species-specific aggregation factor
in response to activators of protein kinase C and Ca ionophores (Weissmann ft al. 1986 Proc. Nail. Acad.
Sci. 83: 2914-2918). The clumped cells in their matrix resemble a blood clot and we therefore determined
whether sponge cells contained transglutaminase activity (Lorand and Conrad 1984, Mol. Cell. Biochem.
58:9-35). Whereas unbroken cells showed no such activity, high-speed ( 150,000 X#. 60min)supernatants
of sonicated cells contained transglutaminase as judged by four criteria: ( 1 ) time-, concentration-, and Ca-
dependent incorporation of l4C-putrescine into N,N'-dimethylcasein; (2) incorporation of dansylcadaver-
ine into dimethylcasein (0.3%) in agarose gels subject to electrophoresis; (3) clotting of citrated lobster
plasma (gamma glutamyl-epsilon amino lysine crosslinking of fibrinogen); and (4) inhibition by primary
amines (e.g.. histamine, dansylcadaverine) but not tertiary amines (e.g., dimethyldansylcadaverine). The
enzyme was concentrated over 25-fold from sponge cell sonicates and resolved on gel filtration chromatog-
raphy (Sephadex G-100). Peak fractions were concordant for l4C-putrescine incorporation and clotting of
lobster plasma; both activities were inhibited by histamine and dansylcadaverine, but not dimethyldansyl-
cadaverine. The sponge cells also contained an endogenous acceptor of l4C-putrescine; incorporation was
Ca-dependent and inhibited by primary amines. The data show that transglutaminase activity is present
in species without blood, hemolymph, or closed coelom. The enzyme may play a role in the formation of
new sponges from cell aggregates.
DEVELOPMENTAL BIOLOGY AND FERTILIZATION
The totipotent development of myoplasm-enriched ascidian embryos. W. R. BATES
(Department of Zoology, University of Kyoto, Kyoto 606, Japan).
The rapid development of the ascidian larva (Urochordata) is thought to be controlled primarily by
factors localized in the egg cytoplasm (Conklin 1905, /. Acad. Nat. Sci. (Phila.) 13: 1-1 19). These factors
424 ABSTRACTS FROM MBL GENERAL MEETINGS
may be localized into distinct cytoplasmic regions of the fertilized egg by a series of precisely oriented
movements termed ooplasmic segregation. The yellow pigmented cytoplasm — the myoplasm — is moved
from its i-ntial position in the egg periphery into the vegetal hemisphere. It is subsequently partitioned
into the i <uscle and mesenchyme cell lineages by a determinant type of cleavage pattern. In the present
investigation fertilized eggs ofStyela plicata or S. clava were bisected approximately along the equatorial
plant: into animal hemisphere fragments that lacked myoplasm and vegetal hemisphere fragments that
contained all of the egg myoplasm. The egg fragments were cultured in pasteurized, millipore-filtered
ater until the controls reached the larval stage (18-20 h). Eighty-two percent (88/107) of the vegetal
(myoplasmic) fragments cleaved while none of the anucleate animal (nonmyoplasmic) fragments cleaved.
Twenty-two percent (24/107) of the vegetal fragments cleaved normally but arrested prior to gastrulation.
Sixty percent (64/107) of the vegetal fragments developed into larvae (yellowhead larvae) in which my-
oplasm was present in many of the endoderm cells situated in the head region as well as in the tail muscle
cells. In all of the yellowhead larvae tested, the pattern of expression of AchE activity (an enzyme marker
for muscle cells; 22 tested), alkaline phosphatase (an enzyme marker for endoderm cells; 1 5 tested), and a
muscle cell-specific antigen (detected by indirect immunofluorescence staining with a monoclonal anti-
body that was prepared by Mita-Miyazawa et al. 1987, Development 99: 155; 64 tested) was normal as
compared to the controls. These results suggest that: ( 1 ) altering the distribution and concentration of the
various kinds of egg cytoplasm into the embryonic progenitor cells before first cleavage does not affect the
specification of larval cell types, despite Conklin's suggestion (1905) that the fertilized egg is comprised of
distinct regions of tissue-specific morphogens and (2) a regulatory mechanism resides in the vegetal hemi-
sphere of the egg that can compensate for the removal of the animal hemisphere without disrupting either
the ascidian type of cleavage pattern or the specification of larval cell types.
Time dependent shift in fluorescence emission in gossypol treated Aibacia sperm. MA-
RIO H. BURGOS (IHEM, U.N. Cuyo, Mendoza, Argentina) AND ROBERT B. SIL-
VER.
Gossypol, the yellow pigment of the cotton seed, can produce sterility in mammals including human
males, as described by the Chinese and confirmed by many investigators. The mechanism of action is
not completely understood. However, gossypol is known to interfere with sperm motility and disturbs
spermiogenesis and sperm maturation. The nature of the interaction between gossypol and macromole-
cules appears to be by a Schiff base formation and hydrophobic and nucleophilic attraction.
Two years ago we observed by epifluorescence that gossypol-treated Arbacia sperm acquire a rapid
fluorescence which was difficult to analyze. With more adequate equipment we have analyzed this phe-
nomenon.
Spermatozoa shed by nine-volt stimulation were suspended in filtered seawater and placed in wedge-
sealed chambers. Spermatozoa attached to the cover glass were observed by an objective piano apo rectified
X 100 under immersion oil. After washing the chamber with filtered seawater (f.s.w.), pictures were taken
with DIC and by epifluorescence microscopy every five seconds for one minute. Other chambers similarly
prepared and washed with f.s.w. were filled with (±) gossypol, (-) gossypol, or ( + ) gossypol, each at a
concentration of 100 ^Mol. The same procedure was applied to the three types of gossypol; the ( + ) and
(-) are optical isomers of the racemic (±) gossypol.
During the first 5 seconds of exposure to the (±) gossypol and to the (-) gossypol, an orange-yellow
fluorescence appears. In less than 10 seconds this color fades and is replaced by a stronger yellow fluores-
cence and then by a yellow-green one. The ( + ) gossypol did not produce fluorescence. The three gossypols
stopped sperm motility and produced swelling of the midpiece.
A series of fluorescent pictures show a change in mitochondrial characteristics: a single fluorescent
body changes into a group of fluorescent clumps.
This study concludes that (-) gossypol is the only fluorescent isomer. As such it acts like a fluorescent
probe whose main target appears to be the mitochondrion. We postulate that the swelling of the midpiece
may result from the fusion of gossypol with the plasma membrane phospholipids, causing alteration of its
permeability.
Supported by Rockefeller Foundation.
Phosphatidylinositol hydrolysis after Spisula oocyte fertilization. WILLIAM R. ECK-
BERG (Howard University, Washington, DC) AND ETE Z. SZUTS.
We recently reported evidence that activation of C-kinase is involved in GVBD in Spisula oocytes.
C-kinase is normally activated in cells by a transient increase in the diacylglycerol content of membranes
as a result of phosphatidylinositol hydrolysis. To test whether C-kinase could be activated in this manner
in Spisula oocytes, we labelled oocyte lipids with [32P]orthophosphate and determined the radioactivity in
DEVELOPMENTAL BIOLOGY AND FERTILIZATION 425
various inositol lipids after fertilization or artificial activation by excess K+. Fertilization resulted in a rapid
loss of radioactivity from phosphatidylinositol(4,5)bisphosphate (PtdInsP:). Thereafter, the radioactivity
in PtdlnsP: increased and equalled the preinsemination value by 3 min after insemination. K+/activation
resulted in a comparable, but more transient loss of radioactivity from PtdInsP2 . Furthermore, the radioac-
tivity exceeded that in unactivated oocytes by 3-5 min after activation. Fertilization also resulted in a
rapid, transient loss of radioactivity from phosphatidylinositol(4)phosphate (PtdlnsP). However, K+ acti-
vation did not affect the specific radioactivity of PtdlnsP. These data indicate that ( 1 ) Spisula oocyte C-
kinase can be activated by pathways similar to those which activate it in other eukaryotic cells and (2) that
phosphatidylinositols are metabolized in both fertilized and K+ activated oocytes, but their metabolism
differs after fertilization or artificial activation.
Interaction ofavidin with Spisula oocyte proteins. TATSUJI HANEJI (The Population
Council) AND S. S. KOIDE.
Avidin is a protein present in avian egg white; it binds biotin. Biotin is a vitamin and a constituent of
decarboxylases, enzymes involved in CO2 production. This shows that Spisula oocytes contain avidin-
interacting protein ( AIP) and the reaction involves biotin.
Biotin-containing proteins were determined by a transblot method and identified by a cytochemical
staining reaction using avidin-peroxidase. Spisula oocytes were homogenized and fractionated by centrifu-
gation. The cytosolic proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis
and transferred to a nitrocellulose paper. The paper was treated with 4% BSA solution to block non-specific
binding sites and incubated with avidin-peroxidase. The staining reaction was performed by development
in 4-chloro-l-naphthol and hydrogen peroxide.
Intense staining occurred with two oocyte proteins with Mr of 70 and 1 18 kD. Proteins of other
Spisula tissues showed none to minimal staining. The staining activity of avidin-peroxidase was blocked on
incubating with biotin, demonstrating that avidin-protein interaction involves biotin.
To determine whether AIPs are related to oocyte maturation, Spisula oocytes were treated with 50
mM KC1 to induce germinal vesicle breakdown. The staining reaction of the 70 and 1 18 kD proteins in
the samples prepared from oocytes treated with KC1 was intensified. The location of AIPs in oocyte cyto-
plasm was confirmed by a cytofluorescence technique using fluorescein isothiocyanate-avidin conjugate.
Supported by grant No. INT 82 1 1 350 from NSF and GA PS 87 1 2 from The Rockefeller Foundation.
Injected calcium buffers block fucoid egg development. L. F. JAFFE, M. H. WEIS-
ENSEEL, AND J. E. SPEKSNUDER (Marine Biological Laboratory).
Formation of a local growth zone in the developing fucoid egg may require the establishment of a
zone of high cortical calcium in this region. To test and explore this hypothesis, we injected calcium buffers
of the BAPTA type into six-hour-old Pclvetia eggs and observed their development. Such buffers should
shuttle calcium from regions of high concentration to ones of lower concentration and thus flatten out and
delocalize incipient gradients. Such a shuttle mechanism requires a buffer weak enough to release calcium
at a sink as well as strong enough to pick it up at a source.
In fact, injection of critical intracellular buffer concentrations does reproducibly block the initiation
of tip growth: such "baptized" cells may live for up to two weeks but they do not visibly differentiate or
grow. The critical concentration varies with the calcium dissociation constant, or KD of the buffer. So far,
the higher the KD and thus the weaker the buffer, the more effective it is: our weakest buffer — the newly
synthesized 5,5' mononitromonomethylBAPTA with an intracellular KD of about 30 /J.A1 — inhibits devel-
opment down to a final intracellular concentration of only 30 nM; our strongest — dimethylBAPTA with
a KD of about 0.4 \iM — takes about 4 mAf to inhibit; while dibromoBAPTA, with a KD of about 4 pM,
takes about 1 mAf. This shows that the nitromethylBAPTA (KD about 30 ^Af) is about 100 times as
effective in inhibiting the initiation of tip growth than the stronger dimethylBAPTA (KD about 0.4 nM).
These results support the calcium gradient hypothesis and suggest that the incipient tip region may
contain free calcium at levels as high as 30 ^Af or more.
Supported by NIH grants to L.F.J. and a DFG grant to M.H.W.
Effect ofUV irradiation on axis and muscle cell specification in embryos of the ascid-
ian Styela. WILLIAM R. JEFFERY (University of Texas at Austin).
Eggs of Styela clava contain colored ooplasms which segregate into specific regions of the zygote
following fertilization but before first cleavage. A yellow ooplasm, the myoplasm, first accumulates as a
cap at the vegetal pole, then extends into a vegetal crescent, and eventually enters embryonic muscle cells.
426 ABSTRACTS FROM MBL GENERAL MEETINGS
The role of localized cytoplasmic region in axis and muscle cell determination has been examined by UV
irradiation. In initial experiments, the entire surface of yellow crescent-stage zygotes was exposed to UV
light. At a dose of approximately 330 ergs/urn2, 100% of the embryos cleaved normally, but failed to
complete g;ts; relation and arrested as radialized blastulae. Muscle cell development, as determined by a
cloned must k~ pecific actin probe, a muscle-specific monoclonal antibody, and acetylcholinesterase local-
ization, w.-.s normal in the radialized embryos. The effective dose for elimination of muscle cell develop-
ment was approximately 1000 ergs/^m2. To determine the UV sensitivity periods for axis and muscle cell
development, embryos were irradiated at various times between fertilization and gastrulation. Embryos
irradiated between fertilization and the yellow crescent stage became radialized, whereas those irradiated
after myoplasmic localization developed a normal axis. In contrast, muscle cell development was sensitive
to irradiation throughout early development until about the 32-cell stage. In a second series of experiments,
zygotes at the yellow cap stage were irradiated locally in either the animal or vegetal hemisphere. In general,
UV light was more effective in suppressing axis and muscle cell specification when focussed in the vegetal
hemisphere. The results suggest that the axis and muscle cells are specified by UV-sensitive factors which
are localized in the vegetal hemisphere region of the zygote and function during distinct intervals of early
embryonic development.
This research was supported by NIH Training Grant HDO709810 for support of the Embryology
Course at the Marine Biological Laboratory, Woods Hole, MA.
Oocyte maturation-inducing substance (OMIS) in Spisula. A. L. KADAM (Population
Council), S. J. SEGAL, AND S. S. KOIDE.
Full-grown Spisula oocytes are arrested at the dictyate stage of meiosis and undergo maturation follow-
ing fertilization. Hirai et al. (Biol Bull. 167: 5 1 8) demonstrated that serotonin induces spawning and oocyte
maturation in the surf clam, Spisula solidissima. Toraya et al. (in press, Experientia), reported that Spisula
body fluid contains a substance that potentiates serotonin action in inducing oocyte maturation. The pres-
ent study was undertaken to purify and characterize OMIS from Spisula body fluid and ganglion cells.
Spisula body fluid and nerve ganglion cells were collected. The ganglion cells were suspended in artifi-
cial seawater (ASW) and lysed by sonication. The body fluid and ganglion homogenate were filtered
through a PM-10 membrane filter. The filtrate was adsorbed with activated charcoal and extracted with
0.1 HC1. The extract was concentrated and purified by HPLC using a reverse-phase ODS column, eluted
with 0.1 A' acetic acid containing 10% methanol. Maturation-inducing activity was assayed with washed
oocytes and scored for germinal vesicle breakdown (GVBD) by light microscopy.
The HPLC purified substance obtained from the body fluid and ganglion cells induced GVBD in
Spisula oocytes in a dose-dependent manner. OMIS is stable to trypsin and heat treatment, adsorbed by
activated charcoal, and not retained on dialysis in a tubing with a mol. wt. cutoff of less than lOOOdaltons.
Its retention time on HPLC elution is coincident with that of reference serotonin creatinine sulfate. In
conclusion, OMIS is present in Spisula body fluid and in ganglion cells and is probably serotonin.
This study was supported by grant no. INT 82 1 1 350 from NSF and GA PS 87 1 2 from The Rockefeller
Foundation.
Heterospecific sperm motility enhancement by sea urchin oocyte pept ides. S. J. SEGAL
(Rockefeller Foundation), M. H. BURGOS, S. INOUE, AND H. UENO.
Two sperm-activating peptides have been isolated from the egg jelly coat ofHemicentrotuspulcherri-
mus (Suzuki et al. 1981, Biochem. Biophys. Res. Comm. 99: 1238-1244) and characterized as adecapep-
tide, Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly and a nonapeptide, Gly-Phe-Asp-Leu-Thr-Gly-Gly-
Gly-Val. Samples of the polypeptides have been synthesized by R. Lundin, Pharmacia Co., Uppsala, Swe-
den, by Merrifield's solid-phase peptide synthesis procedure. We tested these substances (designated 7003
and 7203, respectively) for their ability to stimulate oxygen uptake and the speed of forward motion of
sperm from the marine worm, Chaetopterus pergamentaceus. Oxygen uptake was measured with an oxy-
gen electrode. For motility measurements, sperm were introduced into a wedge chamber and recorded
with a 20 X 10.50 NA rectified objective lens in the presence of a A/10 compensator, in a high extinction
video polarizing microscope. The video signal captured on a Newvicon video camera was processed with
the motion tracking function displayed every 1/5 s by the Image- 1 digital processor (Universal Imaging
Co.). Washed Chaetopterus sperm moved at a speed of less than 100 ^m/s. The addition of either polypep-
tide at a concentration of 10 nM increases the speed of movement to 200 ^m/s after 2 min of treatment.
This elevated rate of movement was observed for 7 min. We conclude that sperm-activation polypeptides
can accelerate sperm motility in a different species.
DEVELOPMENTAL BIOLOGY AND FERTILIZATION 427
Evidence that a G-protein mediates 1-methyladenine induced maturation of starfish
oocytes. FRASER SHILLING (Univ. of Southern California) AND LAURINDA A.
JAFFE.
In response to 1-methyladenine (1-MA), starfish oocytes undergo germinal vesicle breakdown
(GVBD) and initiation of meiosis. To investigate the possible role of a guanine nucleotide binding protein
(G-protein) in transducing the hormonal signal across the plasma membrane, we microinjected oocytes of
Asterias forbesi and Asterias vulgaris with the G-protein inhibitors guanosine-5'-O-(2-thiodiphosphate)
(GDP-/J-S) and pertussis toxin (PTX). GDP-/3-S, a metabolically stable analog of GDP, binds to G-proteins
and inactivates them. Similarly, PTX inactivates certain G-proteins by catalyzing their ADP-ribosylation.
Thirty minutes after applying 1-MA (10 7-10 5 A/), 100% of oocytes injected with control buffer had
undergone GVBD (n = 35), compared to 4% of oocytes injected with GDP-/J-S (2-4 mA/ final concentra-
tion in the oocyte, n = 28). Fifty minutes after 1-MA addition, only 36% of the GDP-/3-S injected oocytes
had undergone GVBD. PTX (2-6 ng/m\ final concentration in the oocyte) was also inhibitory. Prior to
injection, the PTX was activated by heating with dithiothreitol at 35°C for 30 min; the control solution
contained unheated PTX and dithiothreitol. Activated PTX or the control solution was injected into the
oocytes, and 80 to 160 min later 1-MA ( 10 6 A/) was applied. One hour after applying 1-MA, only 12% of
the oocytes injected with activated PTX had undergone GVBD (n = 25), compared to 100% of the control
injected oocytes (n = 10). The PTX was most effective if injected >80 min before 1-MA treatment, presum-
ably because of the time needed to ADP-ribosylate the G-protein. Inhibition of oocyte maturation by
GDP-/i-Sand PTX indicates the involvement of a G-protein in the coupling of 1-MA binding to initiation
of meiosis.
This work was supported by NIH Training Grant 5-T35-HD07098 awarded to the Embryology
Course, Marine Biological Laboratory, Woods Hole, MA, and by NIH Grant HD 14939 to L. A. Jaffe.
Entry of sperm into the animal pole of the egg of the ascidian Phallusia mammillata.
J. E. SPEKSNIJDER, C. SARDET, AND L. F. JAFFE (Marine Biological Laboratory).
It has long been believed that sperm tend to enter near the vegetal pole of ascidian eggs (Conklin 1905,
J. Acad. Nat. Sci. (Phila.) 13: 1-1 19). However, several observations have indicated that both animal and
vegetal fragments of various ascidian eggs can be fertilized. We have reinvestigated this matter during a
study on calcium waves at fertilization in ascidian eggs.
Unfertilized Phallusia eggs were preloaded with the Hoechst 33342 DNA dye (10 ng/m\, 30 min),
inseminated with preactivated sperm to ensure synchronous fertilization, and fixed in 5.6% formaldehyde
in seawater as early as 6 s after insemination. Both the meiotic spindle at the animal pole as well as the
nucleus of the sperm entering the dye-loaded egg become stained, and the angle between the two was
determined. At 6 s after insemination, only 1-7% of the eggs are fertilized. Forty-two percent of these eggs
show the sperm at an angle of 0-45° from the meiotic site, 38% at 45-90°, 1 7% at 90-1 35°, and only 3% at
135-180° (n = 57). This means that 80% of the eggs show the fertilizing sperm in the animal hemisphere.
At 2 min after insemination, those percentages are 18, 25, 30, and 27, respectively (n = 133), and at 5 min
they are 0, 6, 18, and 76 (n = 59), meaning that at this time the sperm is located in the vegetal hemisphere
in 94% of the eggs.
We conclude that the sperm normally prefers entry into the animal hemisphere. The male pronucleus
then moves towards the vegetal pole during the first phase of ooplasmic segregation, which occurs within
the first few minutes after fertilization.
Supported by NIH (L.F.J.); CNRS and NATO (C.S.).
Differentiation of Arbacia punctulata is blocked by the protease inhibitor leupeptin
after fertilization. WALTER TROLL (NYU Medical Center) AND SARAH DAVID-
SON.
Protease inhibitors such as leupeptin (Leup) acetyl-leucyl-leucyl-argininal, were shown to suppress
transformation of NIH3T3 cells after transfection with the activated H-ras oncogene (Garte et al. 1987,
Cancer Res. 47: 3159-3162). A possible mechanism of protease inhibitor action is interference of activa-
tion of DNA polymerase a necessary for specific DNA expression. Protease inhibitors are unique in only
interfering in selective DNA expressions showing negligible general toxicity. We demonstrate that 50 ^g
of Leup present 30 s after fertilization totally blocks differentiation of Arbacia to skeletal plutei. Leup
partially interferes with plutei formation when added 7 or 12 h after fertilization but has no action, even at
much higher concentrations, when added 20 h after fertilization. Plutei formation was usually observed at
24 h. Thus, the protease inhibitor Leup interferes with early genetic expression leading to differentiation
and has no action after the genetic expression has occurred. Several proteases inhibited by Leup are candi-
428 ABSTRACTS FROM MBL GENERAL MEETINGS
dates for the genetic amplification mechanism. These include the trypsin type enzymes plasminogen acti-
vator, thrombin, and sulthydryl proteases such as calpain. Inhibitors of the trypsin class, tosyl-L-arginine
methyl ester and 6-aminocaproic acid, interfered with plutei formation added 30 s after fertilization in
millimolar quantities. In addition, the amino acid lysine in millimolar quantities interfered with plutei
differentiation , while the amino acids arginine and ornithine had no effect. The mechanism of lysine inter-
ference with only negligible antiprotease action requires further study. It is tempting to speculate that lysine
interferes with DN A expression by inhibiting ornithine decarboxylase-forming polyamines.
This investigation was supported in part by USPHS Grant CA 37858 awarded by the National Cancer
Institute. S.D. was supported by the honors program of NYU Medical School.
Effects ofgossypol analogs on Spisula sperm. H. UENO (Rockefeller University), C.
PORTER, R. KAWASHIMA, M. H. BURGOS, K. WATANABE, S. J. SEGAL, AND
S. S. KOIDE.
Gossypol, an anti-fertility agent for men, inhibits sperm motility. To evaluate its structure-function
relationship, we prepared various derivatives and analogs ofgossypol (16 compounds). Effects of these
compounds upon sperm fertilizability, motility, and oxygen uptake were examined. The results obtained
were compared with gossypol and the mechanism of action ofgossypol was investigated.
Sperm fertilizability was examined by scoring the % GVBD of Spisula oocytes after incubation with
sperm which was treated with the drugs. Four out of the 1 7 compounds (gossypol, gossypolone, 516, and
518) significantly inhibited sperm fertilizability. Sperm motility was examined by video-microscopy with
special diffusion chamber designed by S. Inoue. Three of the above 4 compounds (gossypol, gossypolone,
and 518) strongly inhibited sperm motility under the experimental conditions. Oxygen uptake of intact
sperm in ASW (MBL) was measured by using a Clark type oxygen electrode in the closed chamber with
constant temperature system. We found that gossypol increased the oxygen uptake with an optimum
around 10-20 pM. A similar observation was reported with isolated mitochondria. This phenomenom is
known to be due to the uncoupling of oxidative phosphorylation. Only gossypol and a derivative with
substitution at 6-OH groups act as an uncouples Neither gossypolone nor apo-gossypol show any uncou-
pling activity. The present findings suggest that there is no correlation between an increase in oxygen
uptake and inhibition of sperm motility. Furthermore, the formation of hemiacetal, or the presence of
aldehyde and 1-OH group is essential for the activity. Schiff base derivatives ofgossypol are inactive.
Uncoupling activity ofgossypol is due to the oxidation ofgossypol to gossypolone and this requires a free
1-OH group and a free 4-position to form quinone.
We now propose the following hypothesis as the mechanism of action ofgossypol. Gossypol acts in
two steps: ( 1 ) conversion of gossypol to gossypolone which consumes molecular oxygen leading to the
reduction of oxygen tension in the sperm. (2) Subsequent metabolism of gossypolone to form dihydrogos-
sypolone may be mediated with NADH. The net result is a lowering of the level of NADH in the mitochon-
dria, and an interfering with the electron transport system. Both steps would inhibit sperm motility.
This study was supported by the grant from the Rockefeller Foundation (RF86068 Allocation #48
toH.U.).
Neural induction in ascidian embryos redivivus. J. R. WHITTAKER (Marine Biological
Laboratory).
This study has reinvestigated neural induction (evocation) in the development of the ascidian larva as
first described extensively by Reverberi and Minganti (1946, Pubbl. Sta:. Zool. Napoli 20: 199-252; 1947,
Pubbl. Staz. Zool. Napoli 21: 1-35). These authors observed that some brain tissues (and the melanocytes,
found exclusively in the brain) originated in cell lineages derived from the animal-anterior (a4.2) cell pair
of the bilaterally symmetrical 8-cell stage; expression of histological neural features depended on cellular
interaction with endodermal-notochordal derivatives of the vegetal-anterior (A4. 1 ) cell pair. Visible differ-
entiation of melanized (black) melanocytes in the larva of dona infest inalis was used as the test system to
reinvestigate these conclusions. Results of microsurgically isolating the blastomere pairs from the 8-cell
stage and recombining them in various ways confirmed that one or more ectopically located melanocytes
develop only in the a4.2/A4. 1 combination of quarter embryos. No isolated quarter embryo by itself devel-
ops melanocytes, except very occasionally an a4.2 quarter embryo autonomously differentiates internally
located melanocytes. Embryos were observed in culture for 24 h; dona larvae begin to hatch and are fully
differentiated at 1 8 h (at 1 8°C). In addition, endodermal-notochordal enriched cells (the B5. 1 pair) isolated
from the vegetal-posterior lineage (B4.1) at the 16-cell stage and combined (at their "64-celI" stage) as a
partial embryo with a4.2 cells (at the "16-cell" stage) act as an effective inducer of melanocytes in the
combination. This finding illustrates that neural inductive properties are a general feature of endoderm-
DEVELOPMENTAL BIOLOGY AND FERTILIZATION 429
notochord cell lineages and not solely a positional feature of the vegetal-anterior cells. The results of such
a combination also confirm experimentally the conclusion of other investigators from various cell lineage
marking experiments: A4. 1-derived cells can not serve as precursors of brain melanocytes.
This work was supported by NIHHS Grant HD-2 1 823.
ECOLOGY
Does copper affect the mating behavior of Gammarus annulatus Smith, 1873 (Amphi-
poda: Gammaridae)? GUSTAVO BISBAL (School of Oceanography, University of
Rhode Island, Narragansett, RI 02882).
The effects of copper on the mating behavior of G. annulatus are examined. This species exhibits a
precopula stage in which the female is grasped and held dorsally by the male and carried in this position
for several weeks.
To investigate the way in which copper promotes pair separation thus diminishing the probability of
reproductive contact, established pairs were exposed to three treatments during 96 hours: ( 1) control (fil-
tered seawater); (2) 0.025 ppm of Cu and; (3) 0.1 ppm of Cu. In addition, the probable effect of Cu
on preventing the precopula amplexus was tested on artificially separated pairs exposed to the same
treatments.
Three 1.0 1 plastic chambers, each with ten pairs, were used for each treatment. The medium was
renewed every 12 h. At the same intervals the number of pairs in amplexus was recorded, dead animals
were removed and sexed, and a water sample was taken for Cu (AAS) analysis, oxygen content, salinity,
and pH measurements. Temperature was maintained at 20 ± PC. Chambers were aerated constantly and
a summer photoperiod was maintained. Animals were fed squid daily. In assessing behavior, mortality
effects were removed by expressing the number of joined pairs as a percentage of the maximum possible
number of pairs per treatment.
At 1 2 hours the number of observed pairs did not differ between initially joined and separated pairs,
and the observed trend under each treatment showed no significant differences throughout the whole exper-
iment. A significantly higher cumulative mortality was detected at 72 and 96 h for the 0. 1 ppm treatment.
However, mating behavior was unaffected by the copper concentrations evaluated under laboratory condi-
tions, suggesting that the instinct for reproduction is manifested even under the chemically stressed condi-
tions of this experiment.
Acknowledgments are given to MBL and the Edwin Grant Conklin Memorial Fund for financial
support, and to the 1987 Marine Ecology Course staff, Geoff Trager, and Claudia Olivieri for their valu-
able help.
Relationship between trace metal distribution and sulfate reduction in surface sedi-
ment. PATRICIA M. A. BYRNE (Dept. of Oceanography, University College Gal-
way, Ireland).
Fe and Mn hydrous oxide coatings on recently sedimented particles may be reduced beneath the
sediment surface, releasing solubilized metals into pore waters. Conversely, dissolved Fe and Mn may be
precipitated as hydrous oxides (oxidizing conditions) or very commonly as sulfides (reducing conditions).
To obtain estimates of trace metal distribution and rates of sulfate reduction with depth, two sediment
cores were taken from Buzzards Bay (15 m), sectioned at 2-cm intervals, divided into several fractions, and
anoxically incubated for 0, 1 .5, 4, 7, 10, and 1 3 days. After incubation, pore water was extracted from the
sediment for determination of sulfate, sulfide, Fe, and Mn concentrations.
Initial pore water sulfate concentrations were roughly constant to a depth of 1 8 cm; diagenetic model-
ling suggested that this feature must be due to infaunal irrigation of burrows. Sulfate reduction rates were
highest (0.83 mM/day) at the surface and lower at all other depths (x = 0.097 /iA//day). Observed sulfide
production was very low (x = 0.006 ^A//day) relative to the known sulfate reduction rate, implying sulfide
precipitation; 98.9% of sulfur was either reoxidized to sulfate or incorporated into sulfide precipitates.
The most significant changes in metal concentrations occurred at the 0-2 cm interval. Pore water Fe
and Mn increased after 1 .5 days, probably due to reduction of Fe and Mn oxides. As sulfate reduction was
initiated and sulfide produced, the metal concentrations decreased implying the formation of metallic
sulfides.
I thank Don Rice, Peter Frank, Patricia Dell'Arciprete, and the staff and students of the Marine Ecol-
ogy Course. I would like to acknowledge MBL, the Faith Miller Scholarships Fund, UCG (Ireland), the
John F. Kennedy Fund, Uadaras na Gaeltachta, Pfeizer Chemical Corporation, and Carroll Industries
PLC, for providing support.
430 ABSTRACTS FROM MBL GENERAL MEETINGS
Effect of segment loss on reproductive output in Capitella sp. I (Polychaeta). SUSAN
D. HILL (Michigan State University), JUDITH P. GRASSLE, AND MICHAEL J. FER-
KOWICZ.
La lii'tta sp. I and sp. II are considered the most opportunistic of the Capitella complex. They co-
occur in the Woods Hole vicinity, but may have differential seasonal success in colonizing new areas.
Regenerating worms are frequently found in collected samples. In experiments similar to those reported
previously for Capitella sp. II. we investigated the effects of repeated tail removal (at 4-week intervals) and
subsequent regeneration on reproductive output in Capitella sp. I.
At 20°C, Capitella sp. I females undergoing repeated amputation of posterior segments showed no
significant reduction in the number of eggs produced per female until at least 4 weeks after the first amputa-
tion when compared with a matched sample of intact females. In comparison, Capitella sp. II females
showed a significant decrease by the second week. In Capitella sp. I, the number of broods produced by
regenerating and intact animals remained very similar for 6 weeks following first amputation. The observed
reduction in total egg production resulted from a decrease in the number of eggs per brood.
In the reciprocal experiment, Capitella sp. I females at 20°C undergoing simultaneous reproduction
and regeneration produced significantly fewer posterior segments in 4 weeks than a matched cohort in
which reproduction was blocked by the absence of males.
Oosorption occurs in these species. We find that oosorption may occur in Capitella sp. I fed and
starved regenerating females, and in isolated females in which normal spawning is blocked by the absence
of a mate. The conditions leading to oosorption and the role of oosorption in providing resources for other
processes are being investigated.
Support for this research was provided by NSF, OCE-8509169 (S.D.H.).
A green algal (chlorophycophytal) infection of the dorsal surface of the exoskeleton,
and associated organ structures, in the horseshoe crab, Limulus polyphemus.
Louis LEIBOVITZ' (Laboratory for Marine Animal Health, Marine Biological
Laboratory) AND GREGORY A. LEWBART.
Known diseases of the horseshoe crab, including two blue-green (cyanobacterial) diseases, are re-
viewed. The results of a six-year (198 1-1987) study of a previously unreported green algal infection of the
exoskeleton in adult wild and captive horseshoe crabs are reported. Progressive chronic degenerative le-
sions in the dorsum of the exoskeleton, the eyes (ocelli, and large lateral eyes), the arthrodial membrane,
and the base of the telson were observed. The disease often produced loss of tissue structures and functions,
including shell deformities, abnormal molts, degeneration and loss of eye structures (including the cornea
and ommatidia), erosion and perforations of the arthrodial membrane, and hemorrhaging from the heart.
No loss of function could be attributed to telson lesions.
Direct microscopic studies of the green algae in the affected tissues and algal cultures revealed the
young germlings' (zygotes) ability to extend their rhizoidal processes into and between the microscopic
chitinous lamina that compose the horseshoe crab's exoskeletal surface structures and organs. This resulted
in progressive tissue degeneration, necrosis, sloughing, and loss of tissue structure. Secondary bacterial and
mycotic infections followed algal invasion into the deeper tissues.
Morphological and cultural studies of the green algal organism, at the light and electron microscopic
levels, indicated that the pathogen belongs in the family Ulvaceae. Work is underway to further character-
ize the agent and the disease it produces, and develop methods for the prevention and control of the disease.
The results of this study were considered from the standpoint of the evolutionary development of a
disease, comparative pathology, wildlife management, and management of laboratory colonies of horse-
shoe crabs employed for biomedical studies.
This study is supported in part by a grant from the Division of Research Resources, National Institutes
of Health (P-40-RR 1 333-07).
The effect of the arborescent bryozoan Bugula spp. on the settlement, growth, and
mortality of the colonial encrust ingtunicateBolryUoides leachii. PHILIP E. MYERS
(University of South Carolina).
Space is often seen as a primary limiting factor in fouling communities and I examined how Bugula
maintains itself in these communities when it is a poor space competitor. Three treatments were used to
1 Professor, Department of Avian and Aquatic Animal Medicine, New York State College of Veteri-
nary Medicine, Cornell University, Ithaca, New York 14853.
ECOLOGY 43 1
examine if Bugula affected Botrylloides: ( 1 ) clean fouling plates (Alone); (2) plates with Bugula alone; and,
(3) plates in which the fouling community was allowed to grow and interact. For each treatment four 12
X 9 cm black plexiglas plates were hung 0.5 m below a floating dock in Eel Pond, Woods Hole. On alternate
days the number of newly settled Botrylloides were counted and the area of the ten oldest colonies was
measured to estimate growth. Excluding the first ten colonies that settled, all newly settled Botrylloides
were removed. If a monitored colony was missing it was counted as a mortality. Bugula and Alone treat-
ments had no mortality, while the whole fouling community had high mortality (77.5%). The high mortal-
ity was attributed to large settlement of the solitary tunicate Mogula spp. which covered most of the moni-
tored Botrylloides colonies. Bugula seemed to have a strong positive settlement effect on Mogula, although
it was quantified. Both settlement and growth were significantly different in all three treatments with Alone
greater than Bugula. which was greater than the whole fouling community. The majority of the growth
differences between Alone and Bugula treatments probably resulted from food competition, because Bu-
gula cannot outcompete Botrylloides for space. Competition for food may be related to flow and differences
in flow over a plate may be the cause of lower settlement in the Bugula treatment. Flow changes and the
usurping of available space (95-100%) by Mogula, and overgrowth of existing colonies probably caused
the fouling community treatment differences in settlement, growth, and mortality.
Image enhancement of wet seals on rocks and sand as the sample in population ecol-
ogy of Phoca vitulina concolor and Halichoerus grypus, basic research for the
closed model. DAVID PATON (Marine Biological Laboratory, Woods Hole, MA).
Population estimates of seals require a series of single observations. A vertical aerial photographic
method has been standardized that allows investigators to classify the animals according to age and color
without disturbing them. This method avoids distortion of the animals' resting spine length and provides
resolution to 4 cm. The resolution is dictated by the altitude flown, film choice, and the focal length of the
camera. This method, using a Cesna 172 aircraft and 70 mm format, has been developed for modest
budget researchers, enabling them to participate in a western North Atlantic survey. Five days of coincident
sampling must be synoptically charted. The catch sample is then repeated to confirm the number of seals.
Interviews conducted with prospective participants in the population survey require film processing,
materials, cameras, training, and funds to cover costs of extra surveys. Color film (ASA 1 20) would be used
with an intervelometer of the type designed at the University of Vermont, and flown with a boxed Pentax
250 mm camera fitted with a bubble level. The aircraft must be modified for noise reduction and camera
port fitment. This fitting would require four shop hours. It would not require USA/FAA certification or
defeat the status of the aircraft.
Forty-three gray seals were photographed on Wasque Shoal during a two-day lull between April storms
last winter. Transparencies were back lit using an illuminated stage. Grain on the transparency was resolved
using a 600x photographic microscope. Thirty-five millimeter slides were made at just above grain resolu-
tion using tungsten film matched to the irradient temperature. Four yearling pups were detected. Enhance-
ment of contrast using color filters revealed moult patches on the larger seals.
Effects of aerobic versus anoxic conditions on glutamine synthetase activity in Zostera
marina roots: possibilities for regulation of ammonium assimilation. A. MAR-
SHALL PREGNALL ( Vassar College).
Eelgrass (Zostera marina L.) assimilates ammonium from sediment pore water via the enzyme gluta-
mine synthetase (GS) in the roots. During summer, plants growing at depth have greater GS activity than
plants growing in shallow water. Sediment-free plants were held in continuous light for several days, caus-
ing prolonged root aerobiosis; root GS activity decreased below that of control plants held under normal
light-dark cycles. In contrast, plants held in sediments for several days in continuous darkness, causing
prolonged root anoxia, showed much increased root GS activity. This increase may result from de novo
synthesis of GS or from feedback effects of different amino acids and adenylates that fluctuate with root
anoxic/aerobic conditions. One hour pretreatment of root extracts with alanine (1-25 mM), which accu-
mulates in anoxic roots, only slightly inhibited GS activity; 7-amino butyric acid, which also accumulates
during anoxia, enhanced GS activity. Pretreatment with glutamate or glutamine (1-25 mA/), which accu-
mulate during root aerobiosis, also increased GS activity. Pretreatment with ATP (1-10 mM) slightly
decreased subsequent GS activity, while pretreatment with AMP and ADP enhanced subsequent activity.
Excised roots were incubated with cycloheximide (1-20 ppm) while under nitrogen for two days; GS activ-
ity was lower than in roots without cycloheximide, suggesting that part of the enhancement of GS activity
during extended root anoxia results either from de novo synthesis of GS molecules or from some other
protein-synthesis-dependent process. Thus it appears that hypoxic/anoxic root conditions produce changes
432 ABSTRACTS FROM MBL GENERAL MEETINGS
in both metabolite levels and protein synthesis that enhance the ammonium assimilation potential of
Zostera marina roots following the return to aerobic conditions.
The chloroplast-retaining dilates as a component oj the primary productivity in Great
Harbor, Woods Hole, Massachusetts. MICHAEL S. ROBERTS (Department of Bi-
c4ogy, Wesleyan University, Middletown, CT 06457).
Certain marine planktonic ciliates have been known since the late nineteenth century to sequester
chloroplasts from microalgae, but functional photosynthesis by chloroplast-retaining ciliates has only re-
cently been demonstrated. As planktonic ciliates, primarily tintinnids (Order Choreotrichida) and oligo-
trichs (Order Oligotrichida), are an important component of the microplankton biomass in coastal waters,
it is possible that mixotrophic ciliates which contain functional chloroplasts comprise a significant compo-
nent of the neritic primary productivity. To determine the significance of ciliate-retained chloroplast pro-
ductivity, water samples were collected from Great Harbor at the surface, 1.5, and 3.0 meters depth in
early August. In surface waters, 43% of the planktonic ciliates possessed chloroplasts although the surface
water chlorophyll a content was only 1.2 mg-m 3. At 1.5 and 3.0 meter depths, chloroplast-retaining
ciliates comprised 37% and 58% of planktonic ciliate density, respectively. These deeper neritic waters
possessed an average chlorophyll a content of 1.3 mg-m 3. Measurements of primary productivity by
means of I4C uptake and Winkler titration indicated a net photosynthetic deficit in surface water and water
at 3.0 meters depth. Net photosynthesis exceeded respiration in water at 1 .5 meters depth, reaching a value
of 30 mg-C-m 3-h '. Although the chloroplast-retaining ciliates remain an important component of the
planktonic biomass in late summer, their contribution to community primary productivity is apparently
negated by the increased density of heterotrophic ciliates.
Acknowledgments: the author thanks the faculty, staff, and students of the Marine Ecology Course
for their tremendous patience and guidance in this project. A very special thanks to Diane Stoecker for her
invaluable help and insight. This work was generously supported by the Bio Club Scholarship Fund, the
Francis S. Claff Memorial Scholarship Fund, and the William Morton Wheeler Family Founders' Scholar-
ship. La vie dansante.
Nitrate reductase activity in Zostera marina. NINA C. ROTH AND A. MARSHALL
PREGNALL ( Vassar College).
Nitrate reductase (NR) activity in Zostera marina was investigated using an in vivo assay; the optimal
incubation medium contained 60mA/ nitrate, 100mA/ phosphate, and 0.5% 1-propanolatpH 7.0. Zostera
leaves showed significantly higher NR activity than roots [350 nmoles nitrite/(g FW*h) versus 50 nmoles
nitrite/(g FW*h)]. The effects of depth (deep, middle, shallow) and location within the eelgrass meadow
(patch edge versus middle) on NR activity were examined using plants collected from three locations in
Woods Hole, Massachusetts. Nitrate enrichment experiments (200 nM NO, for six days) were conducted
to determine if NR activity can be induced. Results indicate that neither depth nor location within the
patch significantly affect nitrate reductase activity. Certain plants (shallow, 1.2 m depth) did respond to
the enrichment treatment, showing a significant increase in nitrate reductase activity over six days [<100-
950 nmoles nitrite/(g FW*h)]. It appears that shallow edge Zostera plants growing near a shoreline may be
affected by run-off or ground water percolation, since plants from this area exhibited rates up to 1600 nmol
nitrite/(g FW*h). Water samples from this location revealed higher nitrate concentrations ( 1 .35 nM NO3)
than other collection sites (0.7 jiA/). Thus, it is possible that chronic run-off causes sufficient nitrate enrich-
ment in the water column to induce nitrate reductase activity in Zostera leaves.
An estimate of primary productivity in Waquoit Bay National Estuarine Sanctuary,
Falmouth, Massachusetts. EDWARD T. ROWAN (Science Department, Falmouth
High School).
Waquoit Bay National Estuarine Sanctuary for Research and Education is located in Falmouth, Cape
Cod, Massachusetts. The watershed of this restricted opening bay is prime real estate for development. A
survey of this area was initiated so that students at Falmouth High School might participate in monitoring
the bay.
Eight stations within the bay were compared to a station established in Vineyard Sound. Consideration
of freshwater inputs and exchange with Vineyard Sound led to the positioning of the stations. A bouy
capable of floating at least four 300 ml glass BOD bottles was anchored at each station by two cement
construction blocks. All stations were sampled for temperature, salinity, dissolved oxygen, and pH at 0.5
ECOLOGY 433
meter intervals through the water column during differing tidal phases to determine the degree of mixing.
A transect for salinity was taken along the channel to assess the exchange of water with the sound.
Primary productivity experiments were performed at each station. Two sets of light-dark bottles were
suspended in the mid-water column. All bottles were filled with mid-column water; one set was enriched
with nutrients. All samples were incubated for four hours. A sample was returned to the lab for the determi-
nation of concentration of chlorophyll a. Light attenuation coefficients were calculated for each site.
Net productivity was generally low, as was the concentration of chlorophyll a. The nutrient-enriched
samples were more productive than those without this treatment. Two stations showed increased net pro-
ductivity that may be linked to higher levels of chlorophyll a. These stations do not appear to be nutrient-
limited and are located closest to the major freshwater inputs. These data lack the fidelity that would come
with increased samples.
The support of the Monsanto Corporation, the Associates of The Marine Biological Laboratory, and
the Falmouth School Committee is gratefully acknowledged.
Don't cat if Neptune is angry. BERND SCHIERWATER AND GEOFF TRACER (Marine
Biological Laboratory, Woods Hole, MA 02543).
The medusa stage of the hydrozoan Eleutheria dichotoma is very unusual in its biology compared to
other hydromedusae. This tiny medusa (umbrella diameter less than 0.5 mm) lives half-sessile in shallow
coastal waters and is not capable of swimming. In respect to the generally high water velocities in this
environment, a half-sessile lifestyle poses the risk of being detached by currents. In this study, we investi-
gated this organism's ability to remain attached to the substrate in high speed currents.
A special laser optical path and videorecording were used to determine water velocities at the moment
of detachment. We found statistically significant differences in ability to resist detachment between fed
and nonfed medusae. At water velocities ranging from 24 to 70 cm/s we could detach 87% of the freshly
fed medusae. Whereas only 25% of nonfed medusae could be detached at water velocities from 33 to more
than 70 cm/s. The results indicate that because of drastically increased surface area after feeding, drag
forces necessary to remove medusae are achieved at lower velocities. The range of calculated drag forces
at detachment for fed medusae was 3.6 X 10 6 to 3 1 X 10~6 Newton. Videotaping also showed that under
conditions of high water velocities the medusae move their body center down to the bottom to come into
the slow-moving boundary layer.
E. dichotoma medusae seem highly adapted to a half-sessile lifestyle, being capable of holding on to
the substrate at very high velocities. Feeding also significantly increases the risk of being detached.
Acknowledgments: We thank Dr. R. Strickler for providing laboratory space, technical equipment,
and fruitful discussions. This work was supported by "Herbert W. Rand Fellowship," Woods Hole, and
by the "Deutscher Akademischer Austauschdienst," Bonn.
Protein synthesis and degradation rates in two ecophenotypes of the cord grass Spar-
tina alterniflora Loisel from Great Sippewissett Salt Marsh, New England.
KRISHNAN THANKAVEL (Centre of Advanced Study in Marine Biology, Anna-
malai University, Portonovo 608 502, Tamil Nadu, India), MARSHALL PREG-
NALL, AND KANNUPANDI THADAMA.
The salt marsh cord grass, Spartina alternijlora Loisel, is the most productive of the marsh grasses and
has two ecophenotypes, viz. tall form and short form. This study attempts to estimate the rates of protein
synthesis and protein degradation in S. alterniflora and their role in the nutrient cycle in a salt marsh.
Young leaves at the moment of their expansion and old leaves were detached and placed in the dark.
Protein and chlorophyll a contents and the rates of protein synthesis and degradation were measured at
different times from 0 to 4 days after detachment. Rates of protein synthesis were measured by the incorpo-
ration of [4,5-H3] leucine into proteins. Rates of protein degradation were determined from the disappear-
ance of radioactivity from proteins already labeled with [4,5-H3] leucine.
The short form of S. alterniflora had more protein than the tall form and concentrations of protein
increased as the leaves senesced. Chlorophyll a decreased with time. Old leaves of the tall form of S. alter-
niflora took up the maximum tritiated leucine on the third day after detachment. In the short form the
young leaf took maximum leucine one day after detachment. Measurement of degradation indicates that
unlike most other plants, young and old leaves of both ecophenotypes of S. alterniflora synthesize and
accumulate protein until three days after detachment. Thus, S. alternijlora leaves may act temporarily as
a nitrogen sink or trap which contributes to the nitrogen limitation in the salt marsh.
T. Krishnan is grateful to the Council of Scientific and Industrial Research, New Delhi, for the Senior
Research Fellowship award. This work is supported by Frank R. Lillie, Caswell Grave, and Lucretia
434 ABSTRACTS FROM MBL GENERAL MEETINGS
Crocker Fellowships to T. Krishnan at the Marine Biological Laboratory, Woods Hole, USA. A very special
thanks to the staff and students of the Marine Ecology Course.
Life rough when vou are small. GEOFF TRACER (Marine Biological Laboratory,
Woods Hole, MA).
Small sessile suspension feeders in aquatic environments are faced with certain physical problems
with respect to size. In low Reynold's number environments, water is viscous, and small organisms such
as bryozoans and newly settled barnacles must spend considerable energy to move water past their feeding
structures. In this study a special laser optical pathway and video recording were used to analyze the relation
between structure, behavior, and water velocity in both large and small barnacles.
Results indicate that barnacles feed actively in slow currents by raking the water with cirri but change
to passive suspension feeding, where cirri are held stationary in the current, at higher velocities. This switch
occurs in a specific flow speed range. The range is lower for big barnacles than for small ones. Because of
small-scale viscous and boundary layer effects, big barnacles appear to be able to take advantage of energy
in ambient currents more readily than small barnacles. Results show barnacles to be sensitive to changes
in water velocity, and to be well-adapted for suspension feeding in a fluctuating flow environment.
I thank Dr. J. R. Strickler for providing laboratory space, special optical equipment, and useful dis-
cussion.
Bacterial uptake of glutamic acid in oxic and anoxic waters in a coastal pond. DIANA
E. VARELA (Centre Nacional Patagonico, Puerto Madryn, Argentina) AND JOHN
HELFRICH.
Heterotrophic activity of bacteria in aquatic environments uses dissolved organic compounds to pro-
duce paniculate organic material which forms the basis of the food chain. The bacterial activity at different
depths can be compared by measuring the turnover time and the rate of uptake of a dissolved amino acid
such as glutamic acid.
Measurements were made on samples from Siders Pond, a brackish water coastal pond with no river
input and a stable stratification. As a result, the bottom layer is anoxic and contains high concentrations
of nutrients. Glutamic acid labeled with 14C was added to water samples to observe the amino acid incorpo-
ration and respiration rate. The results were analyzed by Michaelis-Menten kinetics equations.
The depth profile of glutamic acid uptake was determined in samples collected at 2-m intervals. Deeper
samples were maintained under an N2 atmosphere to preserve anoxic conditions. The profile showed
greater uptake in the layer above 10 m. Accordingly, samples for time series and kinetics measurements
were taken at 2 and 12m.
The time series experiment showed similar turnover times for the bacterial populations from both
depths (3.2 h for 2 m and 2.9 h for 12 m). The kinetics experiments confirmed these results and yielded a
ten-fold greater maximum uptake velocity at 2 m (67 versus 6 nmoles/1 • h). The sum of the uptake constant
and the concentration of naturally occurring substrate (K. + S) was higher at 2 m than at 12 m(155 nmoles/
1 versus 20 nmoles/1).
These results demonstrate that the bacterial population at 2 m turns over a greater quantity of dis-
solved amino acids than that at 12m.
D.E.V. thanks John Hobbie and Ivan Valiela for their patient guidance and the staff and students of
the Marine Ecology Course at the Marine Biological Laboratory for their constant help. Funds were
awarded for this project from the Frank R. Lillie Fellowship.
Co-selection for clumping and phosphorus accumulation by bacteria isolated from
waste-water treatment systems. W. S. VINCENT AND SARA PRATT (University of
Delaware).
Previous studies described the clumping behavior of strains of bacteria isolated from a waste-water
treatment system, and showed that the system acts as an artificial ecosystem which imposed rigid selective
restraints on the component bacterial populations. The ability to form clumps is one of the traits which is
selected. Other characteristics of the bacterial system which are obviously required are oligotrophy and
facultative anaerobiosis.
Sludges formed in the waste-water system studied (Air Products and Chemicals, Inc. patented A-O
system) also contained 5-10% dry weight concentrations of polyphosphate. The selective value of this trait
is not immediately obvious. This led us to examine the phosphorus accumulating properties of 34 different
strains (non-Acinetobacters) isolated from A-O sludges. All of the 20 different clumping strains in this set
ECOLOGY 435
accumulate phosphorus to some degree with 1 0-fold variations among the strains in both dilute and normal
culture media where phosphorus was not limiting. In media designed to stimulate maximum phosphorus
accumulation, 6 of the strains contained more than 10% dry weight of P. Overall, the mean P content of
the 20 strains in this medium was 7. 1%, approximately the average of the in situ system sludges.
We conclude that clumping and the ability to take up and store P are co-selected in many species in
thissvstem.
INTEGRATIVE NEUROBIOLOGY AND BEHAVIOR
Signalled avoidance learning of eye withdrawal in (he green crab is predominantly
Pavlovian in mechanism. CHARLES I. ABRAMSON, PHILIP M. ARMSTRONG,
ROBIN A. FEINMAN, AND RICHARD D. FEINMAN (SUNY Health Science Center
at Brooklyn, Brooklyn, NY 1 1203).
Signalled avoidance learning was readily demonstrated in the green crab, Carcinm maenas. A mild
vibration to the carapace served as a warning (conditioned) stimulus (CS). Eye withdrawal during the CS
led to the omission of an otherwise scheduled puff of air to the eye (US). Acquisition was rapid, reaching
about 75% avoidance after about 30 trials. Extinction occurred slowly over the course of 40 CS only trials.
The comparative analysis of learning has shown that at least two underlying mechanisms, one Pavlovian
and the other operant, can govern the performance of animals in signalled avoidance situations. To deter-
mine the mechanism involved in learning in the crab, additional groups of animals were subjected to ( 1) a
classical conditioning paradigm in which CS responses had no effect on US presentation, (2) avoidance (60
trials) followed by classical conditioning (40 trials), or (3) classical conditioning followed by avoidance.
The behavior of all groups was essentially identical indicating that the association between the CS and US
was sufficient to effect conditioning and that the animals were unaffected by the consequence of their
actions. In this they resemble some avoidance behaviors such as the nictitating membrane response of the
rabbit or shuttle box performance in teleost fish, which show predominantly Pavlovian behavior, rather
than the shuttle box avoidance in rats where true operant avoidance has been demonstrated.
Functional organization of the sonic motor system in sea robins. ANDREW BASS (Cor-
nell University, Ithaca, NY), MICHAEL WEISER, AND ROBERT BAKER.
Sea robins generate sounds by contracting a set of bilateral 'sonic' muscles that are intrinsic to the
walls of the swimbladder. Each muscle is innervated ipsilaterally by a sonic motor nucleus (SMN) that lies
along the ventrolateral margin of the caudal medulla and rostral spinal cord. Bipolar stimulating electrodes
were implanted in each of the sonic muscles to antidromically activate its respective SMN. Individual
motoneurons were identified on the basis of the timing of action potential generation relative to outputs
from the sonic nerves as monitored with bipolar silver ball electrodes placed intracranially upon ventral
motor roots that arise from the SMN. Intracellular injections of horseradish peroxidase (HRP) demon-
strated that motoneurons typically have a teardrop-shaped soma with a small basal dendritic tree and a
large apical dendrite which ascends dorsolaterally to end in a large dendritic tuft. A single, unbranched
axon exits via a ventral root. There was no evidence for any direct connections between the two sonic
motor nuclei.
Central stimulation in the midbrain, using brief trains of low amplitude pulses, evoked an asynchro-
nous response in the ventral roots similar in frequency and duration to natural vocalizations of 100-200
Hz. We conclude that the distinctly bilateral sonic motor system in sea robins is functionally correlated
with the asynchronous firing of the two sides of the swimbladder. By firing the two sonic muscles at the
same frequency but out of phase with one another, sea robins can effectively double the fundamental
frequency of their 'vocal' signals. The location and properties of the pacemaker neurons that determine
the degree of asynchrony in firing between each SMN remain to be shown.
A.B. was the recipient of an H. Burr Steinbach fellowship.
Extrinsic optical signals, evoked field potentials, and single unit recordings from the
olfactory bulb of the skate (Raja erinacea). A. R. CINELLI AND B. M. SALZBERG
(University of Pennsylvania).
We have used multiple site optical recording of transmembrane voltage (MSORTV), together with
conventional electrophysiological techniques to study electrical activity in various layers in the in vitro and
436 ABSTRACTS FROM MBL GENERAL MEETINGS
in vivo olfactory bulb of the Atlantic skate Raja erinacea. In the living animal, orthodromic stimulation
evoked fieid potentials characterized by an initial triphasic wave, reflecting the compound action potential
in the olfactory nerve, followed by negative waves (N 1 , N3, N4, and, somewhat variably, N2). The current
genr- : T ihese waves were studied by laminar and current-source density analyses. Nl corresponds
to th .'ion of the glomerulus, N3, depolarization of granule cells, and N4, probably reflects the
re-excitation of mitral cells.
Paired stimuli were used to study the properties of these waves. The N 1 wave first exhibits a period of
facilitation, followed by a period of partial suppression. N3 has a period of nonlinear suppression. N4
exhibits a long-lasting suppression with a duration greater than 2 s.
Single unit discharges, presumably from mitral cells, also exhibited a period of suppression of the test
response, following paired stimuli. Some neurons showed a labile period of re-excitation. Late evoked
responses with similar properties were also seen.
Optical recordings of electrical activity from 500 ^m slices of skate olfactory bulb were obtained using
the extrinsic absorption changes exhibited by the pyrazo-oxonal dye RH 155. Following orthodromic stim-
uli, two depolarizing responses were evoked. A fast signal, observed in the upper region of the glomerular
layer, represents the compound action potential in the olfactory nerve. A signal of longer duration was
observed in the zone between the glomerulus and the mitral somata, and in deeper layers. Both signals
exhibited a wavelength dependence characteristic of RH 1 55, and were abolished in the presence of 2 ^m
TTX. Cadmium (100 ^m) eliminated the slow component without affecting the fast signal, suggesting that
the former is synaptically mediated. Barium (5-10 mA/), which depolarizes glial cells, increased the size
of the slow component, suggesting that this optical signal does not reflect a glial response to [K+]0. Different
condition/test intervals produced partial or complete suppression of the test response, depending upon the
location of the recording site, and upon the stimulus intensity. The inhibition could last more than 5 s; at
brief intervals (50 ms), the test responses were shortened as a consequence of the enhanced inhibition
arising from re-excitation of the mitral cells.
We conclude that, following a volley in the olfactory nerve, extremely prolonged changes in excitabil-
ity occur. First, there appears to be a period of facilitation in the dendritic arborization of mitral cells.
This is followed by a long lasting period of inhibition. The underlying neural mechanisms are unknown.
However, the study of electrical phenomena at the level of the dendritic processes of neurons, particularly
the spatio-temporal distribution of graded potentials, could be extremely important in elucidating the
mechanisms of plasticity in this system as well as others.
Supported by USPHS grant NS 16824 and Grass Foundation Fogarty Fellowships to A.R.C.
Initial survey of the chemosensory response properties of lobster mouthparts: spectral
populations and tuning breadth. FRANK COROTTO AND JELLE ATEMA (Boston
University Marine Program, Marine Biological Laboratory).
This is the first investigation of the physiological response properties of primary chemoreceptor cells
from the third maxilliped of the lobster, Homanis americanus. Of interest were the presence of different
spectral populations, and their tuning breadth, particularly in comparison with known cell populations in
three other chemoreceptor organs in the same animal.
Chemoreceptor cells were located by injecting a one second search stimulus pulse (equimolar mixture
of L-aspartate, L-glutamate, ammonium chloride, L-proline, sucrose, L-lysine, L-glutamine, betaine, L-
arginine, hydroxy-L-proline, ethanol, L-alanine, glycine, taurine, and leucine), each at maximum applied
concentrations of 10 nM into a carrier flow of artificial seawater which bathed the excised appendage. So
far, 18 cells were located and tested with all 15 compounds injected separately as 1 s pulses with applied
concentrations of 10 nAl. Action potentials were recorded extracellularly.
Seven cells were narrowly tuned (responses to other compounds were minimal). Of these, three re-
sponded best to betaine, two to L-glutamate, one to ammonium, and one to taurine. Less narrowly tuned
cells were also found: four responded best to hydroxy-L-proline, two responded best to glycine and one to
L-arginine.
If receptor physiology reflects behavioral function, we speculate that taurine cells, common in anten-
nules and thus far rare in maxillipeds, take advantage of the naturally high signal to noise ratio of taurine —
due to its low background in seawater — to detect distant odor plumes. Maxillipeds guard food intake
(Derby and Atema 1982, J. E.\p. Biol. 98: 317-327) which sets different constraints on the receptor cells
of that taste organ. Our current survey indicates that maxillipeds are less specialized than antennules or
walking legs in terms of receptor populations.
Supported by NSFBNS8 5 12585.
INTEGRATIVE NEUROBIOLOGY AND BEHAVIOR 437
Suppression of fictive feeding in vitro by foot shock in Limax maximus: neural corre-
lates in withdrawal and feeding systems. K. DELANEY (Princeton University,
Princeton, NJ 08544) AND J. J. CHANG.
Strong electric shock delivered to the head region produces a vigorous withdrawal response and cessa-
tion of feeding in intact L. maximus. This stimulus can be used to condition food aversion (Delaney and
Gelperin 1984, Neurosci. Abstr. 10: 691 ). An //; vitro preparation consisting of the central nervous system
with lips attached to the cerebral ganglia generates a stereotyped bursting pattern in buccal ganglia nerves
(fictive feeding, FF) when taste stimuli, such as potato juice, are applied to the lips. During a bout of FF,
electrical stimulation of the anterior portion of the foot or the anterior pedal nerves (APN's) which inner-
vate this tissue immediately stops all buccal nerve activity and bursting in buccal motor neurons recorded
intracellularly. Thirty to sixty seconds of foot shock (0.5-5 Hz, 3-10 V) will block or reduce FF triggered
with potato extract for up to 6 minutes after the shock. Suppression of FF is determined by comparing the
number of bites in a feeding bout 2-10 minutes after foot shock with the mean number of bites in control
bouts before and after the shock. Fifty percent of preparations showed suppression at 5 or 6 minutes post-
shock (n = 8 preps showing suppression > 25% at 5 or 6 minutes, mean suppression 82%). No preps
showed suppression at 10 minutes. Suppression of FFat all intervals was strongly correlated with transient
suppression of spontaneous bursting in the salivary fast burster neuron suggesting that variability in sup-
pression may relate to variability in the effectiveness of the shock stimulus.
Foot shock or APN stimulation produces inhibition of buccal motor neurons as determined by intra-
cellular depolarizing current injection or decreases in spontaneous firing or bursting rates which persists
for 30 seconds to 2 minutes. Strong inhibition of cerebral to buccal interneurons (CB's) which participate
in triggering and modulating FF is reliably seen. CB, , CB3, CB4,andCBECare inhibited for up to 6 minutes
after foot shock and clearly show increased levels of spontaneous inhibitory synaptic inputs for up to 8-
10 minutes post-shock. Taste-evoked activity in these CB's is reduced for up to 5 minutes post-shock.
Spontaneous activity recorded in the nerve innervating the buccal mass retractor muscle, the muscle which
produces head withdrawal is elevated for up to 20 minutes following 30 seconds of foot shock.
Support by the Grass Foundation is gratefully acknowledged.
Electromyographic recording of classical conditioning of eye withdrawal in the green
crab. RICHARD D. FEINMAN (SUNY Health Science Center at Brooklyn, Brook-
lyn, NY 1 1203), CHARLES I. ABRAMSON, AND ROBIN R. FORMAN.
The eye withdrawal reflex of the green crab, Carcinm maenas, is an anatomically and physiologically
well characterized system with many simplifying features that make it attractive for the study of neuronal
correlates of learning. The main retractor muscle, 19a, is innervated by two neurons: a fast one that medi-
ates retraction and a slow one that maintains the retracted state. The retraction proceeds without proprio-
ceptive feedback and overrides more complex movements of the eyes. Myographic activity was recorded
from the eye, using 50 n silver wires inserted into muscle 19a during a classical (Pavlovian) conditioning
procedure. The unconditioned stimulus (US) was a 0.5-s puff of air to the eye that invariably caused retrac-
tion into the carapace. This was correlated, in the myograms, with a rapid burst of spikes due to the fast
retractor neuron followed by tonic firing by the slow retractor. The conditioned stimulus (CS) was a 5-s
mild vibration to the carapace which had no effect on observable behavior of naive animals. Pairing of the
CS with the US led to the appearance of retraction of the eye, and a characteristic electromyographic
(EMG) response during CS presentations. The major findings were that: (1) myograms can be used to
record the progress of classical conditioning of the eye withdrawal reflex. (2) The conditioned EMG re-
sponse is, in general, of the same nature as the unconditioned EMG response, but is frequently more
robust. (3) Acquisition of EMG response during CS presentation in animals with immobilized eyes, and
their subsequent behavior in extinction and re-acquisition, is similar to the pattern of behavioral changes
in unoperated animals. This indicates that the open loop nature of the reflex is preserved in this learning
situation.
Supported, in part, by grants from James C. Marias and the SUNY Research Capital Equip-
ment Fund.
Organization of the vestibulo-ocular and vestibulo-spinal reflex pathways in the toad-
fish, Opsanus tau: anatomy and electrophysiology. R. KITCH, T. C. TRICAS, AND
S. M. HIGHSTEIN (Marine Biological Laboratory).
Retrograde transport of horseradish peroxidase (HRP), electrophysiology, and the intraaxonal injec-
tion of HRP were used to study vestibular reflex pathways in anesthetized toadfish. Eighth nerve branches
438 ABSTRACTS FROM MBL GENERAL MEETINGS
were labeled with HRP and alternate sections reacted with TMB/DAB. Five contiguous vestibular nuclei
are distinguished after McCormick(l 982, J. Morph. Ill: 159): from rostral to caudal these are the nucleus
anterior octavus (AO), n. magnocellularis (Mg), n. tangentialis (T), n. descending octavus (DO), and n.
posterior octavus (PO). The central projections of the three semicircular canals (SCC) overlap extensively.
They cover J!K- entire AO, T, and PO, and the lateral and ventral aspects of both Mg and DO. The saccular
and lagcn-: projections to the AO and PO overlie those of the SCC; however they do not project to T, and
in contrasi to the SCC, project dorsally to DO and Mg. The utricular nerve projections cover the entire
AO, T. and PO nuclei, the medial and ventral regions of Mg, and are seen diffusely throughout DO,
overlying both saccular and SCC projections.
Four central pathways were identified by intraaxonal recordings in the brain during ipsilateral eighth
nerve stimulation. One tract projects anteriorly in the medial longitudinal fasciculus (MLF) to the oculo-
motor nuclei with a mean latency of 0.8 ms. Three descending pathways were identified: ( 1 ) an ipsilateral
MLF pathway (mean latency = 0.8 ms, range = 0.5-1.2 ms). Intraaxonal injection revealed that these
axons arose from cell bodies in Mg and descended to give a terminal collateral in the midline medullary
reticular formation and terminals in the cervical anterior horn. (2) Another group of axons were recorded
lateral and deep to group 1 and may correspond to the lateral vestibulo-spinal tract. (3) Finally, the contra-
lateral MLF also contained a group of axons monosynaptically activated.
Supported by NIH NS2 1055.
Detection of chemical contrast in hermit crabs. LESLIE SAMMON AND JELLE ATEMA
(Boston University Marine Program, Marine Biological Laboratory, Woods
Hole, MA 02543).
Contrast detection is an important feature of sensory perception, but poorly understood in chemore-
ception. Yet, recent results seem to indicate that chemoreceptor cells in lobsters are designed to enhance
contrast (Atema 1985, Soc. E.\p. Biol. Symp., 39: 387-423): they are narrowly tuned and adjust response
functions to background concentrations of their best compound (self-adaptation). They cross-adapt to
compounds that stimulate the cell.
We developed a behavioral assay to test chemical contrast detection in whole animals by measuring
hermit crab (Pagurus longicarpus) responses to two complex odors against four different backgrounds. In
a small flume, six animals were presented for 5 minutes with a stimulus concentration series offish extract
(FE) and mussel extract (ME) injected into a background of raw seawater (RSW). A response function
resulted (n = 24 animals for each dilution). FE was slightly more stimulatory than ME. When RSW back-
grounds were changed to 10 4 FE or ME backgrounds, the response functions shifted up to the level of the
background indicating self-adaptation (ME in ME or FE in FE), but were unaffected in cross-adaptation
(ME in FE or FE in ME). The responses to ME and FE dropped in artificial seawater (ASW) background.
This contradicts our expectation that lower organic backgrounds would result in greater contrast and en-
hanced responses.
We then tested effects of living in an altered organic background by keeping the crabs for 7 days in
ASW, or in 10 4 dilution of FE or ME. ASW-exposed animals responded as in RSW, apparently adjusting
to this background that had suppressed their responses in the short term; responses were still not enhanced
as predicted. FE and ME-exposed animals showed response functions similar to those in short term self-
and cross-adaptation. When tested subsequently in RSW, the response remained suppressed indicating
that changes in thresholds may persist after long term exposure to high background levels.
Supported in part by the Armstrong Fellowship of Mount Holyoke College (to L.S.) and the Whitehall
Foundation (to J.A.).
Migratory behavior of individual horseshoe crabs. MARY ANNE SYDLIK, ROBERT B.
BARLOW JR., ANDREW STULL, DONALD R. NELSON, AND CARRIE KAMMIRE
(Marine Biological Laboratory).
During the breeding season horseshoe crabs (Limulus polyphemus) migrate to shore for mating and
nesting. On Cape Cod these migrations are correlated with lunar cycle, tidal cycle, and time of day. During
the 1987 season we tracked the migrations of individual males and females with tags and a computerized
ultrasonic telemetry system.
We tagged 576 individuals (152 clasped pairs, 253 solitary males, and 19 unmated females) over a
four-week period. We found that animals were most likely to revisit the beach within two to three days of
tagging. This suggests that individuals within the population have different rhythms for when to move into
shore or that they use the same beach for a few days and then switch to another location. There was no
relationship between the time of day an animal was tagged and when it was next seen at the beach. A larger
percentage of unmated males (24%) than individuals tagged as part of a pair ( 1 2%) were found on the beach
INTEGRATIVE NEUROBIOLOGY AND BEHAVIOR 439
on subsequent tides. Fewer females (8%) than males ( 1 8%, solitary and clasped combined) were seen at the
beach on more than one occasion, indicating that males and females may use different rules for how often
to travel into shore and therefore may respond differently to environmental cues for migration.
An unmated male, an unmated female, and a clasped female were fitted with ultrasonic transmitters.
All three animals were subsequently located in the bay using hand held hydrophones. We used triangulated
hydrophones and a computer to track the path of the male as he moved within the mating area.
We conclude that tagging and computerized ultrasonic tracking, combined with measures of environ-
mental variables at the staging site, can provide a rich source of information about the factors influencing
movement patterns of individual horseshoe crabs.
Supported by M. E. Lewis Postdoctoral Fellowship for Vision Research, BNS 83203 1 5 and EY-00667.
NEUROBIOLOGY
Characterization of phospholipid enzymes in squid axoplasm and giant fiber lobe.
MARIO ALBERGHINA, SERAFINA SALVATI, AND ROBERT GOULD (Institute for
Basic Research in Developmental Disabilities, 1050 Forest Hill Rd., Staten Is-
land, NY).
Proteins and phospholipids synthesized in the neuronal perikarya are axonally transported at a rapid
rate as part of the mechanism for membrane growth and maintenance in the axon and synaptic endings.
In addition, phospholipid enzymes are transported to axons and synaptic endings where they can modify
the structure of membranes in these specialized neuronal regions. The squid giant axon is an ideal prepara-
tion to demonstrate lipid enzymes in axoplasm since pure axoplasm can be obtained by simple extrusion
and axoplasmic enzymes can be characterized biochemically. Here we report on the characteristics of three
axonally localized enzymes, ( 1 )CDP-diacylglycerol:myo-inositol phosphotransferase, (2)acyl-CoA: 1-acyl-
sn-glycero-3-phosphocholine acyltransferase, and (3) phospholipase A2. Activities in axoplasm were com-
pared with activities in the neuronal perikarya-rich giant fiber lobe (GFL), and other neural tissues of the
squid, Loligo pealei. Properties of phosphatidylinositol synthase (enzyme 1) were quite similar for axo-
plasm and giant fiber lobe extract. Km values were: 0.44 mA/ (axoplasm) and 0.8 mA/ (GFL) for myo-
inositol and 0.025 mA/ (axoplasm) and 0.030 mA/(GFL) forCDP-dicaproin. Both activities were activated
by Mg++, inhibited by low (0.01 mA/) concentrations of Ca+ + and Mn++, and were inactivated by heating
(70°C for 10 min). One striking difference was found: the axoplasmic activity was strongly inhibited by N-
ethylmaleimide whereas the GFL enzyme was rather insensitive to this sulfhydryl group reagent. Acyltrans-
ferase activity in axoplasm, GFL, optic nerve, optic lobe, and fin nerve had similar properties, including:
Km values for lysophosphatidylcholine [i.e.. 0.008 mA/ (axoplasm) and 0.01 mA/(GFL)] and for oleoyl-
CoA [i.e.. 0.003 mA/ (axoplasm) and 0.005 mA/(GFL)], pH optimum (7.4-8.0), inactivation by heat and
by sulfhydryl group reagents. Phospholipase A2 activity was characterized with l-palmitoyl-2-['4C]-oleoyl-
sn-glycero-3-phosphocholine as substrate. In all tissues examined, enzyme activity was linear with protein
concentration and time for up to two hours. In axoplasm and GFL, activity exhibited a pH optimum
between 7.4 and 8.0. The activity was sensitive to heat and p-bromo-phenylacylbromide. Calcium was not
required for full activity. These results further characterize the de novo synthesis of phosphatidylinositol
in axoplasm and demonstrate that there are enzyme activities in axoplasm that modify the acyl side chains
of phospholipids through the acylation-deacylation cycle.
Supported by a grant from the NIH (NS 13980, RMG).
Calcium block of K channels in squid giant fiber lobe neurons. C. M. ARMSTRONG
(University of Pennsylvania, Philadelphia, PA) AND Y. PALTI.
We studied the block of K channels by internal calcium and strontium ion in cells isolated from the
giant fiber lobe of the squid stellate ganglion. Freshly dispersed cells have large potassium currents, a small
calcium current, and essentially no sodium current. Cells were whole-cell patch-clamped, using pipettes
that contained 20 mA/ CaCl2 or SrCl2, plus KC1 and K glutamate. The external solution was 440 mA/
NaCl and 50 CaCl2 . The Ca or Sr effect developed with a lag, making it possible to record near-normal K
current immediately after breaking into the cell. Ca and Sr blocks were apparent only at membrane voltages
above about 50 mV. Below this level K current had an almost normal appearance. Above 50 mV, current
developed normally for a fraction of a millisecond, and then 'inactivated' as Ca or Sr entered the channel.
Block deepened with increasing voltage up to about 150 mV, but was never complete, with about 15% of
current remaining. Calcium entry rate could be measured by opening the channels at 30 mV, where block
is slight, then following the decline of the current following a step to higher voltage. On stepping from 30
to 1 30 mV, current jumped in magnitude by a factor of about two because of increased driving force, then
440 ABSTRACTS FROM MBL GENERAL MEETINGS
decayed to about one-third of this level with a time constant of about 0.5 ms. Analysis suggests that the
calcium entry rate increases with voltage. Calcium-blocked channels at 130 mV recover from block when
voltage is lowered to 30 mV, with a time course that approximates the final phase of channel opening on
stepping from -70 to 30 mV. The results in general are compatible with the idea that calcium block is an
element of normal gating, and that the final step in opening of a K channel involves the release of a calcium
ion bound in the channel. Driving calcium into the channel from inside appears to reverse this final step.
Adenosine depresses spontaneous transmitter release from frog motor nerve terminals
by acting at anAl receptor. SUSAN R. BARRY (University of Michigan).
During nerve stimulation adenosine triphosphate (ATP) may be released with acetylcholine from
vertebrate motoneurons and hydrolyzed to adenosine in the synaptic cleft (Silinsky 1975, J. Physiol. 247:
145-162). Since adenosine levels at the neuromuscular junction (NMJ) may increase with neuronal activ-
ity, adenosine may play a feedback role in neuromuscular transmission.
Adenosine depresses spontaneous transmitter release from frog motor nerve terminals. Spontaneous
miniature endplate potentials (mepps) were recorded with intracellular microelectrodes from motor end-
plates of the frog cutaneous pectoris muscle. Adenosine reversibly depressed mepp frequency by up to 50%
but had no effect on mepp size. This inhibitory action was mediated by an adenosine receptor since the
effect of 10 nM adenosine was blocked by 20 nM theophylline, an adenosine receptor antagonist.
Two types of adenosine receptors, Al and A2, have been described on several cell types. Adenosine
depresses adenylate cyclase activity at the Al receptor but enhances adenylate cyclase activity at the A2
receptor. Adenosine analogs act at nanomolar concentrations at the A 1 receptor but at micromolar concen-
trations at the A2 receptor. The analog, L-N6-phenylisopropyladenosine (L-PIA), is more potent than 5'-
N-ethylcarboxamidoadenosine (NECA) at the Al receptor, while the reverse is true at the A2 receptor.
Finally, L-PIA is at least 100 times more potent than its stereoisomer, D-PIA, at the Al receptor but only
three times more potent than D-PIA at the A2 receptor.
The effects of L-PIA, NECA, and D-PIA were tested on spontaneous transmitter release at the frog
NMJ. All three compounds depressed mepp frequency without affecting mepp size. The threshold concen-
tration for the effect of L-PIA was about 1 nA/. L-PIA was 12 times more potent than NECA and 300
times more potent than D-PIA at reducing mepp frequency. These data indicate that adenosine depresses
spontaneous transmitter release from frog motoneurons by acting at an A 1 receptor. Future experiments
will determine whether adenosine reduces spontaneous transmitter output by inhibition of adenylate
cyclase.
Supported by NSF grant #BNS 85-06778 and a Kufller Fellowship from M.B.L.
Do arachidonic acid metabolites mediate modulation of K and Ca currents by FMRF-
amide in Aplysia neurons? VLADIMIR BREZINA (Department of Biology, UCLA).
Piomelli el al. ( 1987, Nature 328: 38-43) recently suggested that lipoxygenase metabolites of arachi-
donic acid act as second messengers mediating the inhibitory response, due to increased activity of K
channels of the 'S' type, to the neuropeptide FMRFamide in Aplvsia sensory neurons. Work in our labora-
tory (Brezina el al. 1987, J. Physiol. 382: 267-290; 388: 565-595) showed that the inhibitory response to
FMRFamide in other identified Aplysia neurons, the abdominal ganglion cells L2-L6 and R2, also consists
of enhancement of the 'S' or a similar K current (as well as suppression of the Ca current). Therefore I
used the methods of Piomelli el al. to investigate if the FMRFamide response in these cells might also be
mediated by arachidonic acid metabolites.
In the sensory neurons, Piomelli el al. found that the effect of FMRFamide was blocked by 4-bromo-
phenacyl bromide, an inhibitor of receptor-mediated release of arachidonic acid from membrane phospho-
lipids, as well as by nordihydroguaiaretic acid, which inhibits lipoxygenase pathways of arachidonic acid
metabolism, but not by indomethacin, an inhibitor of the cyclooxygenase pathway. In voltage-clamped
L2-L6 and R2 cells, none of these inhibitors (up to 100 ^A/) had any effect on the FMRFamide response.
Piomelli el al. also found that direct application to sensory neurons of arachidonic acid (dissolved in tolu-
ene, then dried and redissolved in the bath medium) mimicked the effect of FMRFamide. In cells L2-L6
and R2, this procedure, like FMRFamide, also elicited large currents that were outward at -40 mV and
were probably carried by K+. However, further investigation showed that the active substance was not
arachidonic acid, but contaminant toluene. Low concentrations («0.1%) of toluene alone (as well as of
benzene, xylene, and hexane, another solvent used by Piomelli el al.} elicited currents with current- voltage
characteristics identical to those presented by Piomelli el al. It also reproduced the hyperpolarization of
sensory neurons that Piomelli el al. attributed to the action of arachidonic acid.
I conclude that there is no present evidence to suggest that the effect of FMRFamide on cells L2-L6
and R2 is mediated by arachidonic acid metabolites. Furthermore, many of the experiments of Piomelli
et al. demonstrate effects of the solvent, not of arachidonic acid.
NEUROBIOLOGY 441
Supported by a fellowship from the Grass Foundation, and by USPHS grant R01 NS08364 to the late
Dr. R. Eckert.
The effect of strontium, barium, and strychnine on the synapse made by barnacle
photoreceptors. JOSEPH C. CALLAWAY AND ANN E. STUART (University of North
Carolina).
The presynaptic terminals of barnacle (Balanus nubilus) photoreceptors (PRs) have a K conductance
activated by the entry of Ca (CaGK). This CaGK might affect the membrane potential locally at the release
sites, influencing the release of transmitter and thus the postsynaptic (I-cell's) response. We tested this idea
by attempting to eliminate the CaGK while recording the I-cell's response to a 2 s pulse of light. Normally
this response consists of an initial peak that sags to a plateau. We tested whether substitution of Ca with Sr
or Ba might eliminate the PR's CaGK and eliminate this sag.
Twenty millimolar Ba (0 Ca) permeates the cell but does not activate the CaGK. It causes an action
potential (AP) without the prolonged undershoot which is due to the CaGK. However, in 20 mM Ba the
I-cell's response is totally abolished. Two millimolar Ba added to a normal (20 Ca) saline does not eliminate
the AP undershoot in the PR and thus does not block CaGK from the outside. We suspect that no Ba
enters the cell due to the much stronger affinity of Ca for an intrapore binding site (Tsien el ai 1987, Ann.
Rev. Biophys. Chem. 16: 265-290): 2 mA/Ca added to 20 mM Ba decreases the duration of the AP, as if it
were preventing Ba entry.
Twenty millimolar Sr (0 Ca) supports release of transmitter but also activates the CaGK, since the AP
in the PR has an undershoot in the Sr saline. Thus neither Ba nor Sr substitution have proven useful in
testing the hypothesis.
Surprisingly, strychnine ( 10 4 A/) eliminates the sag in the I-cell's light response. Strychnine, however,
does not affect the presynaptic CaGK. It has no effect on the light response recorded in the PR terminals
or on the amplitude, duration, or undershoot of APs set up there (in 20 mA/Ca and 20 mA/ tetraethylam-
monium ion). This suggests that the sag is a postsynaptic phenomenon and is not due to the PR's CaGK.
Supported by NIH grant EY03347 to A.E.S.
Resting conductance of the squid axon membrane. D. C. CHANG, J. R. HUNT, AND
P. Q. GAO (Baylor College of Medicine).
The resting membrane of the nerve cell is semi-permeable to cations. Our previous studies suggest
that such permeability properties are not determined by the excitable Na and K channels which are respon-
sible for generating the action potential. To characterize the conductance pathways of the resting mem-
brane, we employed internal perfusion and voltage clamp techniques to study the current-voltage relation-
ship of squid giant axons near the resting membrane potential. Tetrodotoxin was used to block the excitable
Na channel, and tetraethylammonium (TEA) and/or 4-aminopyridine (4-AP) to block excitable K chan-
nels. In most experiments symmetrical internal and external K concentrations (100 mM) were used to
shift the K+ reversal potential away from the resting potential. Under these conditions, we found a small
component of K conductance (denoted here as gK:) which is activated at a potential far more negative than
the classical delayed rectifier K channel (gKi). With a long depolarizing pulse (100 ms) the I-V curve of
steady-state current shows two "hooks," one of which represents inactivation of gK2 and the other inactiva-
tion of gK1 . While gK: is not blocked by 20 mM TEA, it appears to be blocked by 2 mM 4-AP. Its ion
selectivity is K > Rb > NH4 > Na. At this point it is not clear whether g^ is completely unrelated to gK)
or is a component of gM induced by high [K]0. When both TEA and 4-AP are applied, both gK1 , and g^
are removed, but the resting membrane potential is reduced by only 10 mV, and the resting potential is
still strongly dependent on [K]0, but is not very sensitive to [Na]0, much like an untreated axon. Further-
more, the resting conductance under TEA + 4-AP still shows an ion selectivity of K > Rb > NH4 > Na.
These observations suggest an additional K conductance, gK3, contributes to the resting potential. We find
that gK3 is not measurably voltage dependent. In conclusion, our results suggest multiple pathways (e.g.,
gK-> and gK;0 for the resting current in the squid giant axon.
Supported by ONR contract N00014-85-K-0424 and NSF grant BNS-84-06932.
Classification of presynaptic calcium channels in the squid giant synapse. MILTON P.
CHARLTON (University of Toronto) AND GEORGE J. AUGUSTINE.
Recent studies demonstrate that pharmacological and other criteria may be used to classify Ca chan-
nels into at least three different types. We attempted to extend this classification scheme to the Ca channels
responsible for mediating transmitter release at the squid giant synapse.
Our experiments focused on two different types of ligands — omega toxin from the snail Conns geogra-
442 ABSTRACTS FROM MBL GENERAL MEETINGS
phus and dihydropyridines — that were used to discriminate between the two types of Ca channels proposed
to mediate transmitter release at other synapses. These agents were dissolved in saline containing 1/5 of
the normal Ca concentration and were perfused through the artery that normally supplies blood to the
squid stellate ganglion. In this saline, the giant synapse responds to presynaptic action potentials with
subthreshold synaptic responses (PS Ps) that we used as indirect, but sensitive, indicators of the presynaptic
Ca channel activity.
Omega Conns toxin had no effect on PSPs at concentrations as high as 500 nM. Addition of cyto-
chrome C ( 1 mg/ml) to the saline to reduce nonspecific toxin binding did not increase the effectiveness of
the toxin. Reduction of the divalent ion concentration of the saline to 0.75 mA/Ca and 15.5 mA/ Mg still
did not allow the toxin to decrease PSP amplitude. Further, the dihydropyridine Ca channel antagonist
nitrendipine (1-10 /uA/) and the Ca channel agonist Bay K 8644 (1-10 ^A/) had no effect on PSP amplitude.
In summary, neither Conns toxin nor dihydropyridines alter transmission at the squid giant synapse.
From this we conclude that there must be more than three types of Ca channels because the Ca channels
responsible for mediating transmitter release at this synapse do not correspond to any of the three types of
Ca channels previously classified.
Supported by a MRC (Canada) grant to M.P.C. and NIH grant NS-2 1624 to G.J.A.
C-kinase activation mediated by proteolysis modulates K+ conductances in Hermis-
senda B-photoreceptors. CHONG CHEN (Caltech 164-30, Pasadena, CA 91 125),
DANIEL L. ALKON, AND PAUL E. GALLANT.
C-kinase activation by phorbol esters together with Ca2+-loading conditions reduces Hermissenda K+
currents. IAand Ic and enhances ICa:+ (Alkon et al. 1986, Biochem. Biophys. Res. Comm. 134: 1245-1253;
Farley and Auerbach 1986, Nature 319: 220-223). The activation depends on translocation of C-kinase
from the cytosolic to the membrane compartment. It was initially reported that K-kinase could be activated
by limited proteolysis with trypsin or calpain, forming an enzymatically active M-kinaseflnouefM/. 1977,
J. Biol. Chem. 252: 7610-7616). The physiological significance of the activation of C-kinase by protease
remains obscure. The two-microelectrode voltage-clamp technique was used to study whether the proteoly-
sis of C-kinase could modulate membrane conductances of isolated B-photoreceptor somata. The cell was
held at -60 mV. A +60 mV voltage step elicited two major K+ currents, IA and Ic. The average currents
before addition of the drug were 50 ± 5.5 nA S.E. for IA and 22.0 ± 2.5 nA for Ic. The iontophoretic
injection of trypsin inhibitor, leupeptin, 25 ng/^\ in 0.9 A/ KAC reduced IA to 86.7 ± 2.5% S.E. (n = 18)
and Ic by 88.8 ± 2% S.E. (P < 0.005). Pre-exposing cells to 0.5 fiM phorbol ester DPBA reduced the effect
of leupeptin injection on the IA to 97.8 ± 2.7% S.E. and to a lesser extent Ic to 9 1 .7 ± 4%. Bath application
of C-kinase inhibitor H7 (60 ^M in ASW) reduced the effect of leupeptin injection on IA to 95.9 ± 3.0%
and Ic to 97.7 ± 4%. The iontophoresis of trypsin, 2.5 ng/n\ in 0.9 A/ KAC, increased IA to 106 ± 3% and
Ic to 107 ± 2%. The DPBA exposure reduced the leupeptin effect on IA to 99.7 ± 1 .5% and on Ic to 100. 1
± 1.6% of control. This study demonstrates an interaction between protease inhibitor/stimulator and C-
kinase inhibitor/stimulator. The study suggests that there may be another C-kinase activation pathway
which also modulates K+ channels. The C-kinase activation by proteolysis interacts with phorbol esters/
Ca2+-activated C-kinase in such a way that they modulate K+ channels in two opposite directions, i.e.,
enhancement and reduction.
Inactivation rate is not voltage dependent in pituitary sodium channels. GABRIEL
COTA AND CLAY M. ARMSTRONG (Dept. of Physiology, University of Pennsylva-
nia).
Papain inside GH3 cells removes the inactivation of the sodium channels. We recorded macroscopic
sodium currents at 15-20°C with patch pipettes, using supercharging to increase voltage-clamp speed
(Armstrong and Chow 1987, Biophys. J. 52: 133-136). Papain (1 mg/ml) was delivered through the patch
pipette. The sodium current usually changed little within the first three minutes of whole-cell recording,
and then gradually increased in size with a clear prolongation of its rising phase and loss of inactivation
(incomplete decay of the current during the 10-ms test pulse). The enzymatic removal of fast inactivation
was nearly complete 10 min later. As in neuroblastoma cells (Gonoi and Hille 1987, J. Gen. Physiol. 89:
253-274), the prolongation of the rising phase and the increase of current amplitude induced by papain
were more pronounced for a small depolarization than for a larger one.
The rate constant of the inactivation step can be determined by comparing current traces at several
voltages before and after removal of inactivation. The rate constants determined at -20, 0, +40, and +60
mVwere 1.53, 1.58, 1.44, and 1.50ms ^respectively (15°C, 1 mA/ external Ca). We conclude that sodium
channels inactivate with a voltage-independent rate constant.
NEUROBIOLOGY 443
Incorporation of tritiated inositol and choline into phospholipids in the squid stellate
ganglia with special reference to the giant synapse. ROBERT M. GOULD (Institute
for Basic Research in Developmental Disabilities, 1050 Forest Hill Rd., Staten
Island, NY), JOHN HOLSHEK, AND DAVID W. PUMPLIN.
Isolated squid stellate ganglia with attached pallial nerves and hindmost giant axons were incubated in
artificial seawater (MBL formulation) at room temperature (22°C) with either tritiated inositol or tritiated
choline (200 ^Ci/3 ml) for three hours. The preparations were washed in seawater and some were stimu-
lated for 10 minutes at 10 Hz. They were then fixed in 2.5% glutaraldehyde in 0.8 A/ sucrose and 0.1 M
cacodylate buffer, pH 7.4, for several hours. After fixation, the samples were washed for several days with
buffer to remove water soluble precursors, embedded in Spurrs embedding resin under conditions that
limit extraction of the lipid. Light and electron microscopic (EM) autoradiographs were then prepared
(Gould el al. 1987, J. Nenrochem. 48: 1121-1131). With light microscopic autoradiography, the major
sites of phosphatidylcholine (choline precursor) and phosphatidylinositol (inositol precursor) formation
were the cell body-rich giant fiber lobe and the neuropil adjacent to the giant fibers and their synaptic
specializations. Choline appeared to label the neuronal perikarya more strongly than inositol. Labeling at
the synapse was higher than in the pre- or postsynaptic axoplasm. Quantitative EM autoradiography was
used to characterize the relative incorporation by different pre- and postsynaptic elements. Both choline
and inositol were selectively incorporated into lipid within glial elements at the junction. One difference
between the precursors was that lipid formation from choline was more prominent in postsynaptic struc-
tures and in postsynaptic axoplasm, whereas lipid formation from inositol showed preference for presynap-
tic structures including the regions rich in synaptic vesicles. A further study compared inositol lipid distri-
bution between a resting preparation and one stimulated ( 10 min at 10 Hz) following labeling. We found
that the stimulated preparation showed a marked decline in the proportion of silver grains over postsynap-
tic structures compared with the non-stimulated preparation. Further preparations are being analyzed to
substantiate these findings.
Supported by grants from the NIH (NS 1 3980, R.M.G.) and from NSF (D.W.P.).
Two classes of miniature end-plate potentials are present in the isolated, innervated
electrocyte. M. E. KRIEBEL (Dept. Physiology, Health Sci. Ctr., Syracuse, NY),
G. D. PAPPAS, AND G. Q. Fox
The electric organs of the skate (Raja erinacea) were treated with 1% collagenase in elasmobranch
saline for two hours so that individual electrocytes could be separated with gentle shaking. These electro-
cytes were washed with saline and placed in a small bath attached to the stage of a compound microscope.
Some small electrocytes were spherical (50 ^m dia.) and innervated on about 30% of their surface. The
innervated surface invaginates during differentiation to form the "cup" shape of the mature cell. Intracellu-
lar recordings from electrocytes show resting potentials of -65 mV. Nerves were stimulated by drawing
the innervated surface against a suction electrode and threshold analyses indicate three to six efferent
nerves. End-plate potentials (50 mV) and spontaneous intracellular miniature end plate potentials
(MEPPs) were recorded. The two classes of MEPPs from small cells are based on amplitudes. The smaller
class (sub-MEPPs) is about l/10th the size of the larger class (1-2 mV). The smallest electrocytes show the
greatest percentage of sub-MEPPs. Both classes have the same time characteristics which indicate that both
classes are generated by the same postsynaptic mechanism. Both classes are recorded at the same site
showing that they are from the same presynaptic release area. If vesicle size alone determines quantal size
then there should be two classes of vesicles based on diameters (with volume ratios of 10: 1 ). Histograms of
synaptic vesicle diameters show a smooth, slightly log-normal distribution with a mean of 60 nm. No
vesicles with a mean of 30 nm were found to account for the sub-MEPP quantal class.
Autoradiography utilizing [3H] phorbol esters is potentially useful for cellular analysis
of protein kinase C activity in hippocampus slices. ALAN M. KUZIRIAN (Lab. of
Biophysics, NINCDS-NIH, Marine Biological Laboratory), BARRY BANK, JO-
SEPH LOTURCO, AND DANIEL L. ALKON.
Recent studies characterizing eyelid conditioning induced changes in ionic currents in the CA1 pyra-
midal cells of rabbit hippocampus implicate protein kinase C (PKC) (Disterhoft et al. 1986, PNAS 83:
2733-2737). The current changes resemble those previously reported for Hermissenda B photoreceptors
following conditioning and direct iontophoretic injection of purified PKC (Kubota et al. 1986, Soc. Neu-
rosci. Abstr. 12: 599). Bank et al. (manuscript in review) found evidence for classical conditioning (eyelid)
444 ABSTRACTS FROM MBL GENERAL MEETINGS
induced translocation of cytosolic PKC into the membranes of CA1 cells but with no change in total
activity.
Because these conditioning specific changes are similar in both animals and are mimicked by phorbol
esters which specifically bind to PKC, we attempted to determine the distribution of PKC at the cellular
leve! <••• 'jl'DBu in rabbit hippocampus (CA1 region).
. ocampus slices (300-700 /urn thick) from conditioned, pseudoconditioned, and naive animals
wc< •• teneraily prefixed (2.5% glutaraldehyde in 0.05 M cacodylate, pH 7.4; 10 min), incubated in either
2.5 oM [3H]PDBu alone or combined with 2.5 pM non-radioactive PDBu (non-specific labelling control)
for 60 min, buffer washed (30 min), further fixed (60 min), and embedded in 5% agarose. Vibratome
sections ( 10-20 ^m) were cut and mounted on subbed slides, coated with Ilford KD- 5 emulsion, exposed
for 4 to 6 weeks, and developed. Silver grains were counted over the strata oriens and radiatum and pyrami-
dal cell layer.
Scintillation data of treated sections indicated no binding activity differences between prefixed or fresh
slices while non-specific labelling represented approximately 50% of the total amount bound under both
conditions. Silver grains were equally distributed over the strata oriens and radiatum is well as CA1 soma
region (4.7 ± 0.2 grains/ 1 00 ^m2). Silver grain counts from slices incubated with [3H] and non-radioactive
PDBu decreased slightly over the CA 1 soma region and stratum oriens (4.2 ± 0. 1 grains/ 1 00 nm2). How-
ever, the number of silver grains decreased 49% over the stratum radiatum (2.3 grains/ 100 /urn2).
These results compliment those of Worley el al. (1986, J. Neurosci. 6: 199-207). However, data from
the non-specific binding experiment clearly show that PKC is concentrated in the stratum radiatum (the
area containing the CA 1 apical dendrites) with a minor amount in the stratum oriens adjacent to the CA 1
soma region. Although no statistically significant differences in silver grain densities over the areas sampled
could be directly related to training differences, this possibility will be pursued.
Calcium affects the birefringence response of the squid giant axon. DAVID LAN-
DOWNE (University of Miami School of Medicine).
Segments of squid axons were internally perfused and voltage-clamped with an axial internal electrode
assembly. A beam of plane polarized light from a tungsten-halogen bulb passed through the preparation
from below at 45° with respect to the axis. A second polarizer perpendicular to the first was mounted above
the nerve and a photodetector placed above this analyzer. The birefringence response is seen as a change
in light level at the detector associated with a change in nerve membrane potential. The control external
solution contained (in mA/) 88 Na, 352 TMA (tetramethylammonium), 2 HEPES buffer and 50 Ca.
During a depolarizing voltage pulse the birefringence decreases rapidly for the first few hundred micro-
seconds and then more slowly for the first few milliseconds. After the pulse the birefringence returns to the
resting level at first rapidly and then more slowly. The sodium permeability of the membrane increases
with a delay after the fast decrease in birefringence. At the end of a brief pulse the permeability decreases
with the same time course as the rapid birefringence change. These two changes in birefringence, the rapid
and the slower change, have been associated with molecular motion underlying activation and inactivation
of the sodium channels respectively (Landowne, 1985,7. Membr. Biol. 88: 173-185).
Elevating the external Ca concentration from 50 mA/ to 250 mA/at constant Na, while maintaining
osmolarity by replacing TMA, decreases the amplitude and rate of rise of the sodium permeability change
during the pulse and increases the rate of decline following the pulse. High Ca decreases the slope of the
slow birefringence change both during and after the pulse. The rapid change after the pulse is distinctly
more rapid in high Ca, the rapid change at the beginning of the pulse is unchanged or slightly faster.
Reducing Ca to 10 mA/ (TMA replacement) has the opposite effects, increasing the slope of the slow
changes in birefringence and slowing the fast recovery after the pulse. These results illuminate the molecu-
lar basis of calcium's effect on the sodium permeability change and thus on nerve excitability.
This work was supported by a grant from the Whitehall Foundation and NSF grant BNS-8514312.
Sectionless sectioning: a systematic method for scanning electron microscopic exami-
nation of embedded tissue. STEPHEN B. LEIGHTON AND ALAN M. KUZIRIAN
(NINCDS-NIH, Marine Biological Laboratory, Woods Hole, MA 02543).
We have developed a new method for obtaining serial images of epoxy embedded tissue, without much
of the tedium, distortion, and possible loss of sections associated with traditional sectioning. A miniature
microtome was built which operates inside a scanning electron microscope (SEM). As sections are cut,
they are discarded and the remaining block face is sequentially: etched with an oxygen plasma, coated by
a gold sputtering device, and imaged — all within the SEM. Because the block face is imaged while fixed
with respect to the electron optics (except to advance for the next cut), problems of section handling,
NEUROBIOLOGY 445
distortion, and alignment are avoided. The plasma etching differentially etches the epoxy faster than the
tissue, thus providing relief to the block face and improving image quality.
Serial images were obtained of a nerve trunk emanating from the pedal ganglion of Hermissenda
crassicornis, which was fixed in glutaraldehyde/osmium and embedded in epoxy resins. Section thickness
was approximately 0.75 micron and features as small as 100 nm were resolved. Use of a tungsten filament
in the SEM reduced the beam current to a low enough level (300 picoamps at 10 kV) to eliminate the
problem of beam damage to the block, previously observed with a Lanthanum Hexaboride filament.
We anticipate that this technique may significantly aid three-dimensional reconstructions of a broad
range of biological tissues including neural networks and whole embryos for ontogenetic studies. It may
also be useful for systematic sampling of tissues in the SEM, concurrent with x-ray analyses. The method
uses to its best advantage the large depth of field and full range of magnifications of the SEM to sample
large surface areas rapidly, thus making it ideal for bridging the gap between light microscopy and TEM.
The system also has the potential to be automated to a high degree.
Acknowledgment: We wish to thank Dr. Daniel Alkon for his generous support and encouragement
of this research.
Behavioral experiments suggest G protein modulation of calcium channels in Parame-
cium. ANITA D. MC!LVEEN (University of Connecticut Health Center, Farming-
ton, CT), SARAH GARBER, AND BARBARA E. EHRLICH.
Backward swimming of Paramecium is regulated by the opening of voltage-dependent calcium (Ca)
channels in the cilia. Previous work showed that the duration of backward swimming in potassium-con-
taining solutions correlates with the magnitude of the Ca current. Using this behavioral assay, we tested
compounds that may modulate Ca channels in Paramecium calkinsi, a marine ciliate. To incorporate
putative regulatory compounds intracellularly, cells were permeabilized with EDTA ( 1 mA/). After addi-
tion of the test compound(s), the cells were resealed with 1.2 mA/MgCl2. In all cases 6-carboxyfluorescein
was added as a permeabilization indicator. After washing the cells, the effect of the test compound(s) on
the behavior of fluorescent cells was measured. We found that intracellular concentrations of GTP7S, a
nonhydrolyzable analog of GTP which binds to and activates G proteins, of 1 ^Af and greater caused the
cells to swim backwards irreversibly (^900 seconds). Mean recovery time for control cells was 180 seconds.
Other compounds such as GDP/iS, ATP-yS, AMPPNP, and GNPPNP (each at 100 nM) had no effect on
the length of backward swimming suggesting that the action of GTP-yS is specific. When 1 j/A/GTP-ySand
10 juA/ GDP/iS were incorporated simultaneously, the effect of GT?7S alone was entirely eliminated.
These results strongly suggest that the backward swimming behavior of Paramecium is modulated by G
proteins. Since behavioral experiments cannot distinguish between an effect on the Ca channel and an
effect on the switch controlling the direction of ciliary motion, electrophysiological experiments will be
used to distinguish between these alternatives.
We thank Drs. K. Dunlap and J. Lechleiter for the use of their fluorescence microscope; without it
we could not have done this work. This work was supported in part by a Kuffler Fellowship. B.E.E. is a
PEW Scholar in the Biomedical Sciences.
Voltage-clamp reversal oj the sodium pump in dialyzed squid giant axons. R. F. RA-
KOWSKI, DAVID C. GADSBY, AND PAUL DE WEER (Marine Biological Labora-
tory).
We have used a low-noise voltage clamp to examine current-voltage (I-V) relationships of the electro-
genie sodium pump in internally dialyzed squid giant axons under various experimental conditions. Na
and K conductances were inhibited by 200 nA/ tetrodotoxin and 1 mA/ 3,4-diaminopyridine in the bathing
solution and 20 mA/ 3-phenylpropyltriethylammonium in the dialysate. The I-V relationship of the Na
pump was determined as the difference between steady-state I-V curves obtained in the presence and ab-
sence of 0.1 mA/ dihydrodigitoxigenin (H2DTG). Membrane currents in the presence of H2DTG were
insensitive to external [K]. Under conditions designed to support Na/K pumping in the forward direction
only (50 mA/ Na,, 0 Na0, 0 K,, 10 K0, 5 ATP), pump current declines slightly and linearly with hyperpolar-
ization (10-20% between 0 and -60 mV). Addition of 400 mA/ external Na renders the pump current
more steeply voltage dependent (30-50% decline between 0 and -60 mV), with a hint of nonlinearity at
very negative potentials (< - 100 mV). When the sodium pump is constrained to run backward only (0
Na,, 400 Na0, 140 K,, 0 K<,, 0 ATP, 5 ADP, 5 P,), its I-V relationship is sigmoid, saturating at voltages
more negative than - 100 mV and approaching zero at positive potentials ( 10 nM atractyloside and 2 mA/
diadenosine pentaphosphate were present to suppress endogenous ATP generation.) We also produced
conditions in which both forward and reverse pumping can occur within the accessible range of membrane
446 ABSTRACTS FROM MBL GENERAL MEETINGS
potentials, by lowering dialysate [ATP] to 0. 1 mM and raising [ADP] and [P,] to 10 mM each. Resulting
pump I-V relationships were approximately linear from -100 to +20 mV, and clearly crossed the zero
current axis. Our observations demonstrate that the direction of the sodium pump under constant ionic
and biochemical conditions can be reversed by varying membrane potential alone. The pump remains
electrogenic on either side of reversal without abrupt change in slope, allowing determination of pump
reversal potential by interpolation.
Supported by NIH grants NS22731, HL36783, and NS1 1223.
Calcium channels required for neuropeptide release in the intact nerve terminals of
vertebrate neurohypophyses are sensitive to u-conoto.xin and insensitive to dihy-
dropyridines: optical studies with and without voltage-sensitive dyes. B. M. SALZ-
BERG, A. L. OBAID, AND R. FLORES (University of Pennsylvania).
Optical techniques can facilitate the study of excitation-secretion coupling, especially in the small
nerve terminals of vertebrates. Extrinsic absorption changes exhibited by potentiometric dyes have estab-
lished the ionic basis of the action potential in synchronously activated populations of nerve terminals in
the intact amphibian and mammalian neurohypophyses (Salzberg et al. 1983, Nature 306: 36-40; Obaid
et al. 1985, J. Gen. Physiol. 85: 481-489.) Also, large and rapid changes in light scattering, measured as
transparency, follow membrane depolarization and are intimately associated with the release of neuropep-
tides from the nerve terminals of the mouse neurohypophysis (Salzberg el til. 1985, J. Gen. Physiol. 86:
395-4 1 1 ; Gainer et al. 1 986, Neuroendocrinology 43: 557-563).
We report some experiments that help to define the pharmacological profile of the calcium channels
present in intact neurosecretory terminals of vertebrates. We used the dihydropyridine compounds nifedi-
pine and Bay-K 8644, at concentrations ranging from 2 to 5 nM. and the peptide toxin w-conotoxin GVIA
(Peninsula) ( 1-5 //A/). In the frog neurohypophysis, neither dihydropyridine compound had any effect on
the calcium-dependent components of the action potential or on the regenerative calcium response elicited
in the presence of 1-2 nM TTX/5 mM TEA. These spikes remained absolutely unchanged for up to one
hour in the presence of nifedipine and Bay-K 8644. co-Conotoxin GVIA, however, abolished the after-
hyperpolarization of the normal action potential, and dramatically reduced the height of the upstroke and
the size of the undershoot in the regenerative calcium spike.
The effects of these calcium channel modifiers on the action potential recorded optically from the
terminals of the Xenopus neurohypophysis was faithfully reflected in the behavior of the light scattering
changes observed in the neurohypophysis of the CD- 1 mouse. Dihydropyridines left unaffected the intrin-
sic optical signal associated with secretion, while w-conotoxin GVIA (5 /xA/) reduced its size by 50%. These
observations demonstrate for the first time that the type of calcium channels that dominates the secretory
behavior of intact vertebrate nerve terminals is blocked by u>-conotoxin GVIA and is insensitive to dihydro-
pyridines.
Supported by USPHS grant NS 16824 and by NATO and Philippe Foundation Fellowships to R.F.
(INSERM).
Fura-2 imaging of calcium transients in squid giant presynaptic terminal. STEPHEN
]. SMITH (Yale University), Luis R. OSSES, MILTON P. CHARLTON, AND GEORGE
L AUGUSTINE.
We employed the fluorescent Ca-indicator dye fura-2 and digital video microscopy to obtain 2-dimen-
sional images of stimulus-induced changes in intracellular Ca in the giant presynaptic terminals of squid.
These terminals were microinjected with fura-2 to a final concentration of approximately 100 ^M. Binding
of Ca to fura-2 was monitored by collection of video images with fluorescence excitation at 350 nm and at
390 nm. Electrical stimuli eliciting 25-50 presynaptic action potentials were used to open presynaptic Ca
channels and allow Ca to accumulate in presynaptic cytoplasm. Laterally disposed terminals were used to
permit visualization of both longitudinal and lateral gradients of intracellular Ca. Abrupt longitudinal Ca
gradients were observed at the boundary between presynaptic axonal and terminal regions, with Ca signals
at least 10 times larger in the terminal than in the axon. This indicates that Ca channels opened by presyn-
aptic depolarization are more concentrated in terminals than in the axon. In addition, steep lateral gradi-
ents of Ca accumulation were observed, with the larger Ca signals being observed in cytoplasm immediately
adjacent to the synaptic cleft. These observations imply that the Ca channels are localized to the immediate
zone of contact between pre- and post-synaptic neurons. Thus, Ca channels appear to have a restricted
distribution in the presynaptic membrane, being most abundant at sites of transmitter release.
Supported by HHMI and Whitaker Fdn. funds to S.J.S., MDA Postdoctoral Fellowship to L.R.O., a
MRC grant to M.P.C., and NIH grant NS-2 1624 to G.J.A.
NEUROBIOLOGY 447
Morphological characterization of isolated, concentrated nerve endings of the skate
electric organ. H. STADLER (Max Planck Inst. F. Biophysik. Chemie, Goettingen,
FRG), G. Q. Fox, G. D. PAPPAS, AND M. E. KRIEBEL.
Skate (Raja crinacea) electric organ isolated from the tail by blunt dissection and incubated in 1%
collagenase produces homogeneous fractions of component nerve endings and electrocytes. Two hours of
enzyme treatment result in the separation of the nerve plexus from the cup-shaped electrocyte. However,
the electrocyte continues to elicit miniature end plate potentials (MEPPs) indicating that the presynaptic
terminals remain in functional contact with the cell. An additional two hours of treatment eliminates the
MEPPs and produces an electrocyte free of terminals as determined by electron microscopy. The presynap-
tic terminals are covered or capped by a thin Schwann cell layer and are released from the electrocyte by
the collagenase as "strips" of synaptosomes. The presynaptic surface of the synaptosomes remains uncov-
ered and represents approximately 20% of the synaptosomal plasma membrane. If cytochalasin is added
to the collagenase incubation medium, MEPPs of longer duration are observed. This may indicate that
cytochalasin promotes a more gradual separation of the entire efferent nerve. Attention is now focused on
developing means by which to completely remove the Schwann cell cap.
Kinetics of two calcium channel types in chick sensory neurons. D. SWANDULLA AND
C. M. ARMSTRONG (Univ. of Pennsylvania, Philadelphia, PA).
We studied whole cell Ca and Ba currents in chick dorsal root ganglion cells which were kept 6 to 10
h in culture. Voltage steps were imposed on the membrane in 1 5 /us using an improved patch clamp circuit
(Armstrong and Chow 1987, Biophys. J. 52: 133-136), and changes in membrane current were measured
30 ^s after the initiation of the step. Tail currents associated with Ca channel closing decayed in two clearly
distinct phases which could be well fitted with two exponentials. The time constants for the tail components
were ~ 160 /us and ~ 1.5 ms at -80 mV, 20°C. The slow tail component inactivated almost completely as
the test pulse duration increased up to 100ms. It was strongly reduced when changing the holding potential
from -80 to -40 mV or when adding nickel (100-200 nM) to the external medium. This behavior indi-
cates that the slow tail component is due to closing of the low voltage activated (l.v.a.) Ca channels (Carbone
and Lux 1984, Biophys. J., 46: 413-418). The fast tail component was fully activated with 10 ms test
pulses to +20 mV, 20°C, and inactivated to roughly 30% during 500 ms pulses. It was hardly affected by
micromolar concentrations of nickel, and only reduced in amplitude when holding at -40 mV. The time
course of tail current decay did not change as the fast deactivating channels activated with short test pulses
or inactivated with long ones. Neither were deactivation kinetics affected by changing the holding potential
nor by varying the test pulse amplitude. Lowering the temperature from 20 to 10°C decreased the fast time
constant by a factor of ~2.5. In all cases the fast tail current component was very well fit by a single
exponential. There was no indication for an additional exponential of significant size.
Our findings indicate that the fast tail current component in this preparation is due to closing of a
single class of Ca channels which differs both in its voltage range of activation as well as its pharmacology
from the well characterized l.v.a. channel type.
D.S. is supported by the Max Kade Foundation. C.M. A. is supported by NIH grant NS 1 2543.
VISION
Rapid desensitization terminates the response of Limulus photoreceptors to brief in-
jections ofinositoltrisphosphate. RICHARD PAYNE AND ALAN FEIN (Marine Bio-
logical Laboratory).
The rhabdomeral (R)-lobe of Limulus ventral photoreceptors contains stores of calcium that can be
released by inositol 1,4,5 triphosphate (InsP,). The released calcium activates a conductance in the plasma
membrane, depolarizing the cell. Photoreceptors were impaled in their R-lobes with pipettes containing
100 nM InsP3. A brief (< 100 ms) pressure injection of InsP3 delivering approx. 1 pi was followed by a
second injection 1-30 s later. The first injection caused a smooth, transient depolarization of 20-45 mV.
The depolarization had typically declined to 10% of its peak value within 1 s, at which time a second
injection of InsP} was ineffective. Thus the decline of the depolarization may be due to desensitization to
InsP3, in addition to its dilution and metabolism. The amplitude of the depolarization caused by the second
injection recovered fully given longer intervals between injections, 2-5 s being required for half maximal
recovery. The rapid desensitization to InsP3 could be caused by depletion of calcium stores. However,
replacing extracellular calcium by EGTA had no effect on the time course of recovery from desensitization.
Thus, if calcium stores are depleted following an injection, the rapid recovery of sensitivity is not due to
448 ABSTRACTS FROM MBL GENERAL MEETINGS
refilling using extracellular calcium. Alternatively, released calcium may feedback to locally desensitize
the response to InsP, (Payne et al. 1986, J. Gen. Physio/. 127: 107-126). Negative feedback is indicated by
oscillatory bursts of depolarization following prolonged injections of InsP3. The interval between bursts of
membrane depolarization, 2-20 s, is similar to that required for the recovery from desensitization.
Rermular cells in slices of the lateral eye of Limulus were also impaled with pipettes containing 100
nM f risPv Pressure injections produced oscillatory bursts of depolarization that were diminished by light-
adaplaiion or prior injection of InsP,. Thus InsP, acts similarly in lateral and ventral eyes.
supported by grant EY03793.
Modulation of retinal sensitivity by putative efferent neurotransmitters. MELISSA R.
SCHNEIDER AND ROBERT B. BARLOW JR. (Institute for Sensory Research, Syra-
cuse University).
In many animals the brain forms a neural link with the eye to modulate retinal function. In Limulus
the efferent input from a circadian clock in the brain terminates on retinal cells releasing one or more
neurotransmitters. The neurotransmitter(s) mediate multiple changes in retinal structure and function that
increase overall visual sensitivity. Three major changes in photoreceptor function include: ( 1 ) an increase
in response per photon or gain; (2) an increase in photon catch; and (3) a reduction in spontaneous optic
nerve activity. Are these changes produced by a single neurotransmitter or are multiple transmitters in-
volved?
To answer this question we assessed the effects of various pharmacological agents on afferent optic
nerve activity. In particular we assessed the effects of octopamine, octopamine antagonists, and forskolin
on retinal sensitivity. We also assessed the action of octopamine antagonists on the effects of efferent
neurotransmission.
We report that octopamine ( 10~6-10~3 M) and a potent octopamine antagonist NC-5 ( 10~5-10~3 A/),
when injected subcorneally into the lateral eye /// situ, increased gain and photon catch without reducing
spontaneous optic nerve activity. The increase in gain was complete within 10 minutes of the injection
whereas the increase in photon catch required at least 45 minutes to reach completion. Forskolin ( 10~5-
10 4 A/), an adenylate cyclase activator, also increased gain and photon catch without changing spontane-
ous activity. Metoclopramide (10 4 A/), an octopamine antagonist (type 2A), completely blocked the
effects of both octopamine and NC-5. Phentolamine ( 10 4 A/) partially blocked the effects of octopamine
and completely blocked those of NC-5. Yohimbine ( 10 3- 10 4 A/) did not block the effects of octopamine.
Our results indicate that the three retinal properties of gain, photon catch, and spontaneous activity
can change independently of one another. Gain and photon catch can increase without a simultaneous
reduction in spontaneous activity. More than one neurotransmitter may therefore mediate the circadian
changes in retinal structure and function.
Supported by the Senior Fellowship Program of Hamilton College, Clinton, NY, National Science
Foundation grant BNS 83203 1 5, and National Institutes of Health grant EY-00667.
Aluminum flouride and GTP increase inositol phosphate production in distal seg-
ments of squid photoreceptor s. SUSAN F. WOOD (Marine Biological Laboratory),
ETE Z. SZUTS, AND ALAN FEIN.
Light releases inositol triphosphate (IP}) in squid photoreceptors (Szuts et al. 1986, Biochem. J. 240:
929-932). This release may be mediated by a GTP binding (G-) protein which activates a phospholipase.
To test for the role of a G-protein, stimulation of inositol phosphate production in the dark by aluminum
fluoride was measured. Distal segments (labeled with 3H-inositol) were shaken off in an artificial cytosol
containing dithiothreitol, protease inhibitors, and 10 mA/ EGTA. This suspension was filtered, pelleted at
13,000 X g for 1 min and resuspended in the above solution plus 10 mA/ 2,3 diphosphoglycerate (2,3
DPG), an IP, phosphatase inhibitor. EGTA was lowered to . 1 mA/ for the aluminum fluoride experiments.
Distal segments were incubated in the dark in 100 nM A1C1, and 10 mA/KFat room temp, for 2-15 min.
Under these conditions, the A1F4 form will predominate (Goldstein 1964, Anal. Ctiem. 36: 243-244).
This form activates G-proteins (Bigay et al. 1985, FEBS 191: 181-185). After 5-15 min incubation with
A1F4 , all inositol phosphates (IP, , IP;, and IP,) increased. Of the lipid precursors, only phospatidylinositol
bisphosphate (PIP2) decreased in response to A1F4~, suggesting a possible source of the increased inositol
phosphates. The possibility that A1F4 could act as a phosphatase inhibitor was tested by incubating distal
segments with 3H-IP, in the presence or absence of A1F4 . No effect on the breakdown of IP, and IP2 was
seen even in the absence of 2,3 DPG.
Endogenous levels of GTP under these conditions are estimated to be ~ 1 ^A/ (as measured by HPLC).
This is sufficient for a 3-4 fold increase in IP, after a light flash. Added GTP ( 1 mA/) caused an increase in
the light response. IP, and IP2 increased by 28% + 6% and 14% + 7% (x + SE, n = 8), respectively over
VISION 449
that seen in the absence of GTP. These results suggest that a G-protein may be involved in the light-
stimulated production of IP3.
Near-UV light effects on the dogfish (Mustelus canis) lens. SEYMOUR ZIGMAN (Uni-
versity of Rochester School of Medicine, Rochester, NY 14642) AND KRIS LOWE.
Dogfish (Mustelus canis) were used to assess the effects of near-U V radiation on lenses with no prior
exposure history. Fresh lenses were incubated in elasmobranch Ringer's solutions with 95% air:5% CO2
for 18 h while being exposed through a pyrex beaker to near-U V radiation (365 nm max; 3 mW/cm2) from
a Woods lamp. Two known near-U V sensitizers, riboflavin or tetracycline (0.5 mAl), were added in some
experiments; dark controls were also studied. Lenses were blotted and weighed before and after incubation.
Epithelium (plus capsule) and the outer cortex were dissected and homogenized in Dounce glass homoge-
nizers. Insoluble fractions were sedimented at 10,000 rpm; high molecular weight (HMW) colloidal frac-
tion at 30.000 rpm; water-soluble lens crystallins remained in the supernatant (TSP). Protein contents were
estimated spectrophotometrically. Polyacrylamide gel electrophoresis (PAGE) on 10% gels with 1% SDS
+ 50 m.\f DTT buffer to dissolve proteins revealed the protein subunits in each fraction. No consistent
change in lens weight relative to U V-exposure with or without sensitizers was observed. U V-exposed lenses
with and without sensitizers developed a diffuse subcapsular opalescence. In the cortex, UV-radiation
caused a decrease in soluble, and an increase in HMW and insoluble proteins. This was exaggerated by
added sensitizers. Insoluble to soluble protein ratios increased in the epithelium, especially with added
sensitizer. Using PAGE, it was found that only the HMW peptide profile contained aggregated material,
indicating a covalent crosslinking. We conclude that near-U V light (especially in the presence of sensitizers)
leads to enhancement of crosslinked HMW proteins in the periphery. No specific crystallin was altered.
Support: Research to Prevent Blindness, Inc.; Mullie and Pledger Funds (University of Rochester);
NIH(EY 00459).
ABSTRACTS
ABSTRACTS OF PAPERS PRESENTED AT THE GENERAL SCIENTIFIC MEETINGS
OFTHE MARINE BIOLOGICAL LABORATORY 419
Cell motility and cytoskeleton J. rt^ .- 419
Comparative physiology 42 1
Developmental biology and fertilization ...^ 423
Ecology .."..-. , 429
Integrative neurobiology and behavior .^ . !=435
Neurobiology 439
Vision . 447
CONTENTS
BEHAVIOR
BOTTOM, MARK L., AND ROBERT E. LOVELAND
Orientation of the horseshoe crab, Limulus polyphemus, on a sandy
beach 289
KAHAN, DAVID, THEODORA BAR-EL, NORBERT WILBERT, SAMSON LEIKEH-
MACHER, AND SAMUEL OMAN
The feeding behavior of Paranophrys carnivora (Ciliata, Philasteridae) 299
DEVELOPMENT AND REPRODUCTION
GRIFFIN, FRED J., WALLIS H. CLARK JR., JOHN H. CROWE, AND Lois M.
CROWE
Intracellular pH decreases during the in vitro induction of the acrosome
reaction in the sperm ofSicyonia ingentis 311
MARTIN, VICKI J.
A morphological examination of gastrulation in a marine athecate hy-
drozoan 324
RlNKEVICH, B., AND Y. LOYA
Variability in the pattern of sexual reproduction of the coral Stylophora
pistil lata at Eilat, Red Sea: a long-term study 335
SUZUKI, SACHIKO
Vitellins and vitellogenins of the terrestrial isopod, Armadillidium vul-
gare . 345
VILLA, LUISANNA, AND ELEONORA PATRICOLO
A scanning electron microscope study ofAscidia malaca egg (Tunicate).
Changes in the cell surface morphology at fertilization 355
ECOLOGY AND EVOLUTION
MULLER-PARKER, G., AND R. L. PARDY
The green hydra symbiosis: analysis of a field population 367
TURNER, JEFFERSON T.
Zooplankton feeding ecology: contents of fecal pellets of the copepod
Centropages velificatus from waters near the mouth of the Mississippi
River ,.. 377
GENERAL BIOLOGY
MUNTZ, W. R. A., AND S. L. WENTWORTH
An anatomical study of the retina of Nautilus pompilius 387
PHYSIOLOGY
LALL, ABNER B., AND THOMAS W. CRONIN
Spectral sensitivity of the compound eyes in the purple land crab Gecar-
cinus lateralis (Freminville) 398
SMITH, LAURENS H., JR., AND SIDNEY K. PIERCE
Cell volume regulation by molluscan erythrocytes during hypoosmotic
stress: Ca2+ effects on ionic and organic osmolyte effluxes 407
Continued on Cover Three
Volume 173
Number 3
THE
BIOLOGICAL BULLETIN
PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
RUSSELL F. DOOLITTLE, University of California at
San Diego
WILLIAM R. ECKBERG, Howard University
ROBERT D. GOLDMAN, Northwestern University
C. K. GOVIND, Scarborough Campus, University
of Toronto
/ ' V ' v "
JUDITH P. GRASSLE, Marine Biological Laboratory
MICHAEL J. GREENBERG, C. V. Whitney Marine
Laboratory, University of Florida
MAUREEN R. HANSON, Cornell University
JOHN E. HOBBIE, Marine Biological Laboratory
LIONEL JAFFE, Marine Biological Laboratory
HOLGER W. JANNASCH, Woods Hole Oceanographic
Institution
WILLIAM R. JEFFERY, University of Texas at Austin
GEORGE M. LANGFORD, University of North
Carolina at Chapel Hill
GEORGE D. PAPPAS, University of Illinois at Chicago
SIDNEY K. PIERCE, University of Maryland
HERBERT SCHUEL, State University of New York at
Buffalo
VIRGINIA L. SCOFIELD, University of California at
Los Angeles School of Medicine
LAWRENCE B. SLOBODKIN, State University of New
York at Stony Brook
JOHN D. STRANDBERG, Johns Hopkins University
DONALD P. WOLF, Oregon Regional Primate Center
SEYMOUR ZIGMAN, University of Rochester
Editor: CHARLES B. METZ, University of Miami
DECEMBER, 1987
Printed and Issued by
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PRINCE & LEMON STS.
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- FEB 1 1 1988
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THE
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IV
CONTENTS
No. 1, AUGUST 1987
Annual Report of the Marine Biological Laboratory 1
INVITED REVIEW
STANLEY-SAMUELSON, DAVID W.
Physiological roles of prostaglandins and other eicosanoids in inverte-
brates 92
BEHAVIOR
CHADWICK, NANETTE E.
Interspecific aggressive behavior of the corallimorpharian Corynactis
californica (Cnidaria: Anthozoa): effects on sympatric corals and sea
anemones 110
DEVELOPMENT AND REPRODUCTION
BOSCH, ISIDRO, KATHERINE A. BEAUCHAMP, M. ELIZABETH STEELE, AND
JOHN S. PEARSE
Development, metamorphosis, and seasonal abundance of embryos and
larvae of the antarctic sea urchin Sterechinus neumayeri 126
ECOLOGY AND EVOLUTION
ALEXANDER, STEPHEN P., AND TED E. DELACA
Feeding adaptations of the foraminiferan Cibicides refulgens living epi-
zoically and parasitically on the antarctic scallop Adamussium colbecki 1 36
BORRERO, FRANCISCO J.
Tidal height and gametogenesis: reproductive variation among popula-
tions ofGeukensia demissa 160
MARCUS, NANCY H.
Differences in the duration of egg diapause of Labidocera aestiva (Co-
pepoda: Calanoida) from the Woods Hole, Massachusetts, region .... 169
GENERAL BIOLOGY
HOSE, Jo ELLEN, GARY G. MARTIN, VAN ANH NGUYEN, JOHN LUCAS, AND
TEDD ROSENSTEIN
Cytochemical features of shrimp hemocytes 178
MACKIE, G. O., AND C. L. SINGLA
Impulse propagation and contraction in the tunic of a compound
ascidian 188
MANGUM, C. P., K. I. MILLER, J. L. SCOTT, K. E. VAN HOLDE, AND M. P.
MORSE
Bivalve hemocyanin: structural, functional, and phylogenetic rela-
tionships 205
OKAMURA, BETH
Particle size and flow velocity induce an inferred switch in bryozoan
suspension-feeding behavior 222
vi CONTENTS
PHYSIOLOGY
DEATON. LEWIS E.
Epithelial water permeability in the euryhaline mussel Geukensia
demissa: decrease in response to hypoosmotic media and hormonal
modulation ............................................ 230
ENGE^, DAVID W., AND MARIUS BROUWER
Metal regulation and molting in the blue crab, Callinectes sapidus: met-
allothionein function in metal metabolism ..................... 239
FELBECK, HORST, AND SANDRA WILEY
Free D-amino acids in the tissues of marine bivalves .............. 252
HAND, STEVEN C.
Trophosome ultrastructure and the characterization of isolated bacte-
riocytes from invertebrate-sulfur bacteria symbioses .............. 260
WISEMAN, ROBERT W., AND W. Ross ELLINGTON
Energetics of contractile activity in isolated radula protractor muscles
of the whelk Busycon contrarium: anaerobic end product accumulation
and release ............................................. 277
No. 2, OCTOBER 1987
BEHAVIOR
BOTTON, MARK L., AND ROBERT E. LOVELAND
Orientation of the horseshoe crab, Limulus polyphemus, on a sandy
beach ................................................. 289
KAHAN, DAVID, THEODORA BAR-EL, NORBERT WILBERT, SAMSON LEIKEH-
MACHER, AND SAMUEL OMAN
The feeding behavior of Paranophrys carnivora (Ciliata, Philasleridae) 299
DEVELOPMENT AND REPRODUCTION
GRIFFIN, FRED J., WALLIS H. CLARK JR., JOHN H. CROWE, AND Lois M.
CROWE
Intracellular pH decreases during the in vitro induction of the acrosome
reaction in the sperm ofSicyonia ingentis ...................... 311
MARTIN, VICKI J.
A morphological examination of gastrulation in a marine athecate hy-
drozoan ............................................... 324
RlNKEVICH, B., AND Y. LOYA
Variability in the pattern of sexual reproduction of the coral Stylophora
pistillata at Eilat, Red Sea: a long-term study .................... 335
SUZUKI, SACHIKO
Vitellins and vitellogenins of the terrestrial isopod, Armadillidium vul-
gare .................................................. 345
VILLA, LUISANNA, AND ELEONORA PATRICOLO
A scanning electron microscope study ofAscidia malaca egg (Tunicate).
Changes in the cell morphology at fertilization .................. 355
ECOLOGY AND EVOLUTION
Mui LER-PARKER, G., AND R. L. PARDY
The green hydra symbiosis: analysis of a field population ........... 367
CONTENTS vii
TURNER, JEFFERSON T.
Zooplankton feeding ecology: contents of fecal pellets of the copepod
Centropages velificatus from waters near the mouth of the Mississippi
River 377
GENERAL BIOLOGY
MUNTZ, W. R. A., AND S. L. WENTWORTH
An anatomical study of the retina of Nautilus pompilius 387
PHYSIOLOGY
LALL, ABNER B., AND THOMAS W. CRONIN
Spectral sensitivity of the compound eyes in the purple land crab Gecar-
cinus lateralis (Freminville) 398
SMITH, LAURENS H., JR., AND SIDNEY K. PIERCE
Cell volume regulation by molluscan erythrocytes during hypoosmotic
stress: Ca2+ effects on ionic and organic osmolyte effluxes 407
ABSTRACTS
ABSTRACTS OF PAPERS PRESENTED AT THE GENERAL SCIENTIFIC MEETINGS
OFTHE MARINE BIOLOGICAL LABORATORY 419
Cell motility and cytoskeleton 419
Comparative physiology 42 1
Developmental biology and fertilization 423
Ecology 429
Integrative neurobiology and behavior 435
Neurobiology 439
Vision 447
No. 3, DECEMBER 1987
DEVELOPMENT AND REPRODUCTION
LYNN, JOHN W., AND WALLIS H. CLARK JR.
Physiological and biochemical investigations of the egg jelly release in
Penaeus aztecus 451
ECOLOGY AND EVOLUTION
HILBISH, THOMAS J., AND F. JOHN VERNBERG
Quantitative genetics of juvenile growth and shape in the mud crab
Eurypanopeus depressus 46 1
RINKEVICH, BARUCH, AND IRVING L. WEISSMAN
The fate of Botryllus (Ascidiacea) larvae cosettled with parental colo-
nies: beneficial or deleterious consequences? 474
GENERAL BIOLOGY
LATZ, MICHAEL I., TAMARA M. FRANK, MARK R. BOWLBY, EDITH A. WID-
DER, AND JAMES F. CASE
Variability in flash characteristics of a bioluminescent copepod 489
viii CONTENTS
MCAULEY, P. J.
Quantitative estimation of movement of an amino acid from host to
Cl: ' symbionts in green hydra 504
PHYSIOLOGY
AKKETT, S. A., G. O. MACKIE, AND C. L. SINGLA
Neuronal control of ciliary locomotion in a gastropod veliger (Callio-
stoma) 513
GROSVENOR, W., AND G. KASS-SIMON
Feeding behavior in Hydra. I. Effects of Anemia homogenate on nema-
tocyst discharge 527
MUTHIGA, NYAWIRA A., AND ALINA M. SZMANT
The effects of salinity stress on the rates of aerobic respiration and photo-
synthesis in the hermatypic coral Siderastrea siderea 539
SHORT REPORTS
HOLLAND, NICHOLAS D., ALEXANDER B. LEONARD, AND J. RUDI STRICKLER
Upstream and downstream capture during suspension feeding by Oligo-
metra serripinna (Echinodermata: Cridoidea) under surge conditions 552
SHORT, FREDERICK T., LISA K. MUEHLSTEIN, AND DAVID PORTER
Eelgrass wasting disease: cause and recurrence of a marine epidemic .. 557
ABSTRACTS
ABSTRACTS OF PAPERS PRESENTED AT THE MARINE BIOLOGICAL LABORA-
TORY: NORTHEASTERN REGIONAL CONFERENCE ON DEVELOPMENTAL
BIOLOGY 563
INDEX TO VOLUME 173 . 575
Reference: Biol. Bull. 173: 451-460. (December, 1987)
PHYSIOLOGICAL AND BIOCHEMICAL INVESTIGATIONS OF THE
EGG JELLY RELEASE IN PENAEUS AZTECUS
JOHN W. LYNN AND WALLIS H. CLARK JR.
Department of Zoology and Physiology. Louisiana State University, Baton Rouge, Louisiana 70803 and
Bodega Marine Laboratory, University of California, Bodega Bay, California 94923
ABSTRACT
Following contact with seawater, Penaeus aztecus ova undergo a massive release
of extracortical jelly precursor material which is transformed into a layer of jelly-
like material surrounding the ova. Release and dissipation of the precursors can be
irreversibly inhibited by the protease inhibitors N-a-p-tosyl-L-lysine chloromethyl
ketone and soybean trypsin inhibitor, implicating trypsin-like proteases in the pro-
cess. Treatment with the less-specific enzyme inhibitor phenylmethyl sulfonyl fluo-
ride also irreversibly inhibits the release of the cortical material. Jelly precursor in
whole mature ovaries stain positive with PAS. Staining with alcian blue reveals acid
mucopolysaccharides in the investment coat of the ova but not in the jelly precursors.
Precursors isolated from whole mature ovaries are approximately 25-30% carbohy-
drate (anthrone sulfuric acid reaction) and 70-75% protein (Lowry's and Bradford's
protein determinations). No sialic acids are detected in the isolates (thiobarbituric
acid assay). Trypsin is effective in dissipating the precursor isolates. Amino acid anal-
ysis reveals high ratios of cysteic acid. Significant biochemical differences between P.
aztecus egg jelly material and sea urchin egg jelly are discussed.
INTRODUCTION
Spawning of eggs from the ovary of the penaeid shrimp into the surrounding sea-
water results in a dramatic and massive release of a jelly precursor from extracellular
cortical crypts (Hudinaga, 1942; Clark et ai, 1980, 1984). This release is initiated by
contact with seawater. According to the morphological descriptions of Clark et al.
(1980, 1984) a membrane fusion event is not involved. Initially, the jelly precursor
components are stored in crypts in the surface of the mature egg separated from the
environment by only a thin egg investment coat (Clark et al, 1980, 1984). Transfor-
mation of the jelly precursors into the jelly coat surrounding the eggs of P. setiferus
and P. aztecus has been demonstrated to be a Mg+2-dependent event (Clark and
Lynn, 1977). The rod-shaped jelly precursors contained in ovarian eggs of P. setifer-
ous were originally described by King (1948) as peripheral bodies and recognized as
jelly precursors by Hudinaga (1942) and later investigators. The biochemical compo-
sition and physiology of the release of the precursors is still poorly understood. We
now report new data on the physiological parameters involved in the egg jelly release
and present biochemical data on the composition of the released material.
MATERIALS AND METHODS
Animal collection
Using a standard otter trawl, brown shrimp (P. aztecus) were collected 80-100
miles south of Galveston, Texas. Animals were transported to the laboratory in a 1 50-
Received 17 August 1987; accepted 22 September 1987.
Abbreviations: periodic acid-Schiff, PAS; isolation medium, IM; ethylene-diaminetetraacetic acid,
EDTA; soybean trypsin inhibitor, SBTI; N-a-p-tosyl-L-lysine chloromethyl ketone, TLCK; phenylmethyl
sulfonyl fluoride, PMSF.
451
452 J w LYNN AND W. H. CLARK JR.
gal tank at 1 'j >"C. In the laboratory, gravid female shrimp were placed in aerated,
,rboys, and the water temperature was slowly raised to 28°C to induce
btained from these animals were used for investigations on jelly pre-
dease.
Jelly precursor isolation
Mature ovaries suspended in an isolation medium (IM) (500 mM NaCl, 9 mM
CaCl2, 14mMKCl, 15 mMMgC!2,and 1 OmM Tris, pH 7.6) containing 30% sucrose
or 35% glycerol were homogenized with a Potter-Elvehjem tissue grinder. The ho-
mogenate was centrifuged (1000 X g for 5 min). The pellet was resuspended in IM,
layered over IM containing 60% sucrose or 70% glycerol, and centrifuged (8000 X g
for 60 min). The resulting pellet, consisting mainly of the jelly precursor, was washed
four times to remove contaminant sucrose or glycerol. These isolates were either used
immediately or freeze-dried and stored at -80°C. All isolates were assayed for purity
using light and electron microscopy.
Protein analysis
A Lowry's total protein reaction (Lowry et al, 195 1) was performed on the iso-
lated precursor (40 mg/ml) and spectrophotometrically measured on a Coleman 124
double beam spectrophotometer. Protein was quantitated using a coomassie blue
stain for total proteins (Bradford, 1976) and measured spectrophotometrically as
above. Serum albumin was used as a standard for both protein measurement tech-
niques. Amino acid analysis was performed on freeze-dried isolates. Samples were
acid hydrolyzed with HC1 or performic acid (Moore and Stein, 1954) and assayed on
a Durum D500 amino acid analyzer.
Carbohydrate analysis
To determine the presence of carbohydrates, a molisch alpha napthol or anthrone
sulfuric acid assay (Dische, 1955) was performed on isolates (40 mg/ml). An L-cyste-
ine sulfuric acid assay (Dische, 1955) was also performed to determine the presence of
hexoses, 6-deoxyhexoses, 2-deoxypentoses, pentoses, hexuronic acids, and heptoses.
Standards used for this assay were: fucose, glucose, glucuronic acid, sedoheptulose,
2-deoxy-D-ribose, and ribose (Sigma). Either the Ehrlich reaction (Werner and Odin,
1 952) or the thiobarbituric acid assay (Warren, 1 959) was used to test for the presence
of sialic acids. N-acetyl neuraminic acid (Sigma) was used as a standard (2 Mg/ml for
the thiobarbituric acid assay and 200 Mg/ml for the Ehrlich reaction).
Enzymatic digestion and inhibition
Sensitivity to enzymatic degradation was tested on fresh and freeze-dried isolates
at 24-26°C. Enzymes (Sigma) used were: 0. 1% trypsin (bovine pancreatic) in 0.46 M
Tris, pH 8.1, containing 0.012 MCaQ2; 0.1% alpha chymotrypsin (bovine pancre-
atic) in 8 mA/Tris, pH 7.8, containing 0. 1 MCaCl2; 0.2% aryl sulfatase (Aerobacter)
in 0.2 M sodium acetate, pH 5.0; 0.02% hyaluronidase (bovine testis) in 0. 1 M mono-
sodium phosphate, pH 5.3, containing 0. 1 5 M NaCl; 0. 1% collagenase (Clostridium)
in 0.05 MTris, pH 7.5, containing 0.35 MCaCl2; 0.1% lipase (Candida cylindracea)
in 1.0 M Tris, pH 8.1. All solutions were prepared according to the Worthington
manual (1972). Effects of these enzymes on precursor isolates were observed with
microscopy for periods up to 5 hours. As controls, precursor isolates were held
5 hours in the buffer systems used for each enzyme.
PENAEID EGG JELLY RELEASE 453
Spawned eggs were collected in artificial seawater (Cavanaugh, 1956) containing
either 0.1% soybean trypsin inhibitor (SBTI) (Sigma), 0.1% N-a-p-tosyl-L-lysine
chloromethyl ketone (TLCK) (Sigma), or 0.1% phenylmethylsulfonyl fluoride
(PMSF) (Sigma). Eggs were either held in these solutions for observation or returned
to normal seawater within 10 minutes after treatment in these solutions for observa-
tion. Control eggs were placed in normal seawater previously filtered through a milli-
pore (0.2 jum) filter or held in a solution of artificial seawater (Cavanaugh, 1956).
Microscopic techniques
Ovarian tissue was dissected from either wild mature animals or animals induced
to mature by bilateral eyestalk ablation (Duronslett et ai, 1975). These tissues were
fixed in phosphate-buffered (pH 7.8) 10% formalin or Bouin's fixative (Thompson,
1966) and embedded in paraffin. Sections (5 urn) were stained with alcian blue 8GX
at pH 2.0 (Thompson, 1966), periodic acid-Schiff reagent (PAS) (Thompson, 1966),
aldehyde fuchsin (Thompson, 1966), or mucicarmine (Thompson, 1966).
Spawned eggs and isolated jelly precursor were fixed for 1-2 hours in a 0.2 M
phosphate-buffered (pH 7.5) paraformaldehyde-glutaraldehyde solution (Karnovsky,
1965) for electron microscopy. Samples were post-fixed in 0.1 M phosphate-buffered
(pH 7.5) osmium tetroxide (1%) for 30 minutes, rapidly dehydrated in a graded ace-
tone series, and embedded in a low- viscosity epoxy resin (Spurr, 1969). Sections were
cut with glass or diamond knives on a Porter Blum MT2-B ultramicrotome. Thin
sections were stained with saturated methanolic uranyl acetate and aqueous lead ci-
trate (Venable and Coggeshall, 1965) and examined on an Hitachi HS-8 electron
microscope.
RESULTS
Mature P. aztecus oocytes are approximately 265 ^m in diameter and are isoleci-
thal (Clark et al. , 1 980). Extracellular club-shaped jelly precursors lie within membra-
nous invaginations of the oolemma (crypts) and are separated from the environment
by a thin vitelline envelope. The substructure of the jelly precursors consists of feath-
ery elements. Contact with seawater initiates expulsion of the precursor from invagin-
ations or crypts. As a result of the precursor expulsion, the vitelline envelope is lifted
from the oolemma and is lost. Once released, the precursor elements dissipate form-
ing a homogenous transparent jelly layer around the oocyte.
The jelly precursor elements within the crypts of an oocyte stain with PAS, but
not alcian blue; however, the vitelline envelope exhibits a positive reaction with both
stains. The vitelline envelope and the precursor material do not stain with either
aldehyde fuchsin or mucicarmine.
Purified precursor isolates are shown in Figure 1 at the light level and in Figure 2
as observed with electron microscopy. The jelly precursor from each crypt maintains
its structural integrity after isolation. Even the feathery substructural units of the
precursor, originally described by Clark et al. (1980), are still apparent in fresh and
freeze dried isolates (Fig. 3). Isolated precursor material was stable in several solvents
(Table I). Sulfuric acid and sodium hydroxide completely dissipated the isolates and
were compatible with the biochemical assays.
Table II shows assays for protein and carbohydrate components. The Lowry and
Bradford assays were positive. From these determinations and the amino acid pro-
files, lyophilized precursor isolates are approximately 70-75% protein by weight.
Both HCl-hydrolyzed and performic-acid-hydrolyzed isolates were analyzed for
amino acid content since HC1 hydrolysis partially or completely destroyed methio-
454
J. W. LYNN AND W. H. CLARK JR.
FIGURE 1. Light micrograph of a rehydrated sample of a purified jelly precursor isolate. Arrow:
individual precursor element. Bar = 100 nm.
FIGURE 2. Transmission electron micrograph of rehydrated purified precursor isolates used for the
biochemical assays. Only small amounts of contaminant were apparent and appeared to be primarily yolk
in nature. CR: precursor element; arrow: suspected contaminant. Bar = lO^m.
FIGURE 3. High magnification transmission electron micrograph of feathery substructural elements
of isolated jelly precursor. Bar = 0.5
PENAEID EGG JELLY RELEASE 455
TABLE I
Solvent extraction of jelly precursor isolates
Solvent
Precursor dissipation
Absorption peaks of solutes
SWNaOH
Complete
260 nm, 225 nm
2 ./VNaOH
Complete
270 nm, 220 nm
ISTVH.SO,
Complete
465 nm, 370 nm
320 nm, 250 nm
12JVHC1
Partial*
280 nm, 220 nm
2NHC\
Partial*
280 nm, 220 nm
PO4 buffer, pH 5.8
Partial*
265 nm, 250 nm
Acetone
None
None
Chloroform
None
None
Petroleum ether
None
None
Mercaptoethanol, pH 8.8
None
None
Methanol
None
None
Triton X- 100(1%)
None
None
* Swells and becomes flocculent.
nine and cysteine. Amino acid analysis showed a predominance of aspartic acid and
glycine with relatively high ratios of cysteine (Table III).
The molish alpha napthol and the anthrone sulfuric acid assays indicated carbo-
hydrates. Specific carbohydrate groups in the isolates were assayed using the L-
cysteine sulfuric acid assay. With this assay, an initial peak in the sample was observed
at 396 nm (Fig. 4), indicating a 6-deoxyhexose and possible overlapping absorption
due to hexoses or pentoses. Following the addition of water and a 6-h waiting period,
peaks were observed at 5 10 nm, 460 nm, and 410 nm. The peaks at 5 10 nm and 460
nm suggested heptoses. The peak at 410 nm probably resulted from a shift in the
absorption of light in the range indicating a 6-deoxyhexose, since the addition of
water destroys hexose absorption (Dische, 1955). Hexoses were suggested, however,
by the decrease in the absorption at 396 nm after the addition of water. The sample
probably did not contain pentose or 2-deoxyhexose, for no peaks comparable to the
standards were observed. Sucrose contamination resulting from the isolation proce-
TABLE II
Summary of biochemical assays on jelly precursor isolates
Assay Compounds reacting Assay results
Lowry's Protein +
Bradford's Protein +
Molisch alpha-napthol assay Carbohydrates +
Anthrone sulfuric Hexoses, pentoses, 6-deoxyhexoses, +
acid assay Hexuronic acid, heptoses +
L-cysteine sulfuric Pentoses
acid assay Hexuronic acid
Hexoses +
Heptoses +
6-Deoxyhexoses +
2-Deoxypentoses +
Ehrlich reaction Neuraminic acid
Thiobarbituric acid assay Neuraminic acid
456 J- w LYNN AND W. H. CLARK JR.
TABLE III
Am/no aci , •>« of isolated jelly precursor
Amino acid Composition, moles/ 100 mg
Asparticacid 126.1
Threonine 57.4
Serine 50.6
Glutamicacid 61.9
Proline 28.7
Glycine 72.4
Alanine 51.0
Valine 57.1
Methionine 13.3
Cysteine 30.8
Isoleucine 30.0
Leucine 61.2
Tyrosine 20.7
Phenylalanine 29.4
Histidine 13.9
Lysine 37.3
Arginine 18.9
dure was discounted as the source of carbohydrates because the results remained the
same for preparations isolated over glycerol. A glycerol standard gave no peaks with
the L-cysteine reaction. Carbohydrate content was estimated to be approximately
25-30% of the lyophilized material weight, using the anthrone sulfuric acid assay.
Assays for sialic acid were negative.
Table IV summarizes the results of enzyme treatments of isolated jelly precursor
elements and shows that trypsin effected complete element dispersal. Light micros-
copy revealed a period of swelling followed by a loss of element morphology. After a
5-h treatment with chymotrypsin, the isolates swelled but did not dissipate com-
pletely. Jelly precursor elements held in the same buffers used with the enzyme assays
maintained a normal morphology and did not swell or dissipate. In addition, jelly
formation was inhibited in eggs spawned into seawater containing either SBTI,
TLCK, or PMSF and the effect was not reversible on return to normal seawater. If
0.1% SBTI was added to seawater that contained eggs already in the process of jelly
precursor element expulsion or dissipation, jelly formation was inhibited.
DISCUSSION
The oocytes of penaeid shrimp contain a jelly-like precursor in crypts of the oo-
lemma prior to spawning (Clark el al. , 1 980, 1 984). This material is released at spawn-
ing and forms a jelly investment around the egg (Hudinaga, 1942; Clark et al., 1980,
1984). In contrast to the jelly layers that invest the ova of many animals, the penaeid
coat is a primary investment produced by the oocyte (unpub. data). The penaeid
material also differs in its biochemical properties from the jellies of other animal ova.
Thus, while the P. aztecus coat may be analogous to other egg jellies, it does not
appear to be homologous. While there are several biochemical differences between
the penaeid shrimp jelly and the jellies in other animal ova, it must be noted that the
biochemical characteristics reported in this paper deal with a precursor form of the
penaeid jelly. Subtle changes in the bonding and components may occur as the heter-
ogeneous form of the jelly is transformed into the translucent homogenous form.
Jellies of spawned eggs from sea urchins and amphibians are composed of gly-
PENAEID EGG JELLY RELEASE
457
w
u
g
l.O-i
0.8 —
0.6 —
0.2-
0.0
350
I
400
450
wavelength
500
550
600
FIGURE 4. Spectrophotometric tracing of wavelength scan for the L-cysteine sulfuric acid reaction.
Dotted line represents initial reaction; solid line represents reaction after a 6-hour waiting period and the
addition of water. Tracing is a computer digitized reconstruction of the original scan.
cosaminoglycans, sulfate esters, and sialic acids (Vasseur, 1948; Monne and Slautter-
back, 1950; Humphries, 1966; Lee, 1967; Freeman, 1968; Isaka et ai, 1970; Hotta
et al, 1970a, b, 1973, 1977; Ishihara et al, 1973; Katagiri, 1973; Lorenzi and He-
drick, 1973; SeGall and Lennarz, 1979). The jelly material released from teleost and
nereid eggs is also reported to contain acid mucopolysaccharides with sulfate esters
and, occasionally, a neutral mucopolysaccharide (Costello, 1949; Yamamoto, 1956;
Raven, 1961; Yamamoto, 1961).
Biochemical assays suggest that, unlike most other egg jellies, the penaeid jelly
precursor contains a substantial amount of protein compared to carbohydrate (70-
TABLE IV
Enzymic treatments of jelly precursor isolates
Enzyme
Concentration
Effect on isolated rods
Trypsin
alpha-Chymotrypsin
aryl-Sulfatase
Hyaluronidase
Collagenase
Neuramindase
Lipase
alpha-Amylase
0.1%
0.1%
0.2%
0.02%
0.1%
0.1%
0.1%
0.1%
+ + dissipated; + swelling, no dissipation; — no effect.
458 J- W. LYNN AND W. H. CLARK JR.
75% prot carbohydrate). Alcian blue staining indicated carboxylated
and/or & i i bohydrate groups in the vitelline envelope but not in the precursor
material. ^cid was conspicuously absent from the isolates when tested by the
Warren (1959), whereas it may compose over 25% of purified samples of
gg jelly (Hotta, 1977). In addition, enzymes capable of dispersing sea
in egg jellies (aryl sulfatase and neuraminidase) had no detectable effect on the
material released from P. aztecus eggs. The significance of these differences is unclear.
P. aztecus egg jelly also differs strikingly in protein content from sea urchin jelly.
Sea urchin egg jelly contains approximately 20-25% protein (for review see Hunt,
1970) whereas P. aztecus egg jelly contains approximately 70-75% protein. Amino
acid ratios, however, are similar in the two animals with the exception that the pen-
aeid jelly material has higher cysteic acid ratios. Although sulfhydryl linkages are
present in penaeid jelly material, they do not appear to be primarily responsible for
structural integrity, since sulfhydryl reducing agents do not dissipate the jelly
precursor.
Two lines of evidence indicate that trypsin-like protease enzymes are involved in
the in vivo release and dispersion of the jelly precursor. Isolates were effectively dissi-
pated by trypsin and to a much lesser degree by chymotrypsin. Secondly the specific
serine-protease inhibitor SBTI inhibited this release and dispersion in vivo. Although
TLCK and PMSF may also inhibit SH proteases in addition to serine proteases ( Whi-
taker and Perez-Villasenor, 1968), a serine protease is preferentially supported since
SBTI is not reported to inhibit SH proteases and it is capable of completely inhibiting
the jelly release. Regardless, a trypsin-like enzyme is suggested, but isolation and in
vitro characterization of the enzyme will be essential in identifying the specific class
of proteases represented by these enzymes.
Proteases are additionally implicated by the ability of the inhibitors to stop jelly
formation at two stages: when the precursors are released from their crypts, and later,
when the material is transformed into a translucent jelly layer. It is particularity inter-
esting that these two stages of jelly release and transformation are also completely
inhibited by a deficiency of Mg+2 in the seawater (Clark and Lynn, 1977) suggesting
that the proteases involved are also Mg+2-dependent. A similar protease-dependent
jelly precursor release and transition of the jelly precursor into a homogenous jelly
layer has also been observed in the eggs ofSicyonia ingentis (unpub. data). The initial
phase of release which appears to be less Mg+2-sensitive in the S. ingentis egg, seems
to be mediated by failure of the vitelline envelope to break down, preventing jelly
expulsion. A similar phenomenon may be responsible for preventing jelly release in
the P. aztecus egg. The location of the proposed protease involved in the penaeid egg
release is unknown. Therefore, specific activities of the enzymes involved must be
localized and described to understand the sequence of events of the egg jelly release.
Despite the differences in chemical composition between egg jellies reported in
other species and the jelly formed by P. aztecus, it is likely that the different types of
jelly layers in question may have very similar functions. For example, the jelly forma-
tion in P. aztecus may protect the early zygote from the environment. Although mor-
phologically the precursors are rapidly dissipated, their chemical constituents may
remain around the ova for a long time. In this form, these chemical constituents
could act as an antibacterial agent or as a repellent to other microorganisms. Prelimi-
nary studies reveal zones of inhibition around jelly precursor isolates placed on
freshly streaked plates of shrimp exoskeletal bacteria (unpub. data). This exciting
possibility should be further pursued in vivo.
An additional or alternative role for the formation of the jelly layer in P. aztecus
y involve the acrosome reaction of the sperm. Acrosomal inducing abilities of egg
jstment coats have been demonstrated in sea urchins (SeGall and Lennarz, 1979)
PENAEID EGG JELLY RELEASE 459
and sturgeon (Cherr and Clark, 1985). Indeed, the investment coats of many species
may contain components which trigger the acrosome reaction in sperm (Lopo, 1983,
for review). Although the role of the P. aztecusjefty as an acrosomal reaction inducer
has not been tested, a component of egg jelly from a related penaeid shrimp, S. in-
gentis has recently been demonstrated to induce the acrosome reaction (Clark et
ai, 1984).
This study shows that the jelly formation in P. aztecus differs markedly from the
cortical reactions of other animals, such as sea urchins. Instead, this reaction appears
to be a delayed jelly-coat formation. It remains to be seen whether the physiological
and biochemical differences of the shrimp jelly coat indicate, reflect, or match func-
tional similarities of jelly investments of other animals.
ACKNOWLEDGMENTS
We thank Kathyrn Kanagaki for performing the amino acid analysis, Dr. Ron
Sizemore for assistance in testing inhibition of microbial growth, and Ann McGuire
for editorial assistance. This research was funded in part by Texas Sea Grant #NOAA
04-3-158-18 and by California Sea Grant #NOAA 04-m01-189. We would also like
to thank the National Marine Fisheries Institute in Galveston, Texas, for use of ani-
mals and facilities.
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Reference: Biol. Bull. 173: 461-473. (December, 1987)
QUANTITATIVE GENETICS OF JUVENILE GROWTH AND SHAPE IN
THE MUD CRAB EURYPANOPEUS DEPRESSUS[
THOMAS J. HILBISH AND F. JOHN VERNBERG
Department of Biology and Belle W. Baruch Institute, University of South Carolina,
Columbia, South Carolina 29208
ABSTRACT
Rates of growth and development were measured for the first six molts following
the crab 1 stage in the mud crab Eurypanopeus depressus. The genetic contribution
to variation in growth rate, development rate, and shape was determined for each
molt interval. Genetic variation in growth rate, measured as increases in both width
and length, was evident at most molt intervals. There were also significant genetic
effects upon the intermolt interval. Growth rates for each molt interval, calculated
on a daily basis to remove the interaction between growth rate and development rate
also showed genetic variation. There was no evidence that genetic variation in these
parameters changed during early juvenile development; there were substantial levels
of genetic variation in growth rate at most ontological stages. Despite high levels of
genetic variation for growth rate in dimensions of the carapace, there was no evidence
of genetic variation in shape. This analysis does not provide a quantitative estimate
of the levels of genetic variance for these traits but does indicate that the magnitude
of this source of variance must be very significant.
INTRODUCTION
Variance in many traits may have a large genetic component. High heritabilities
have been demonstrated for many traits, including morphology (van Noordwijk et
al, 1980; Boag, 1983), behavior (Arnold, 1981a, b; Via, 1984a, b), physiology (Curt-
singer and Laurie-Ahlberg, 1981), and other traits that are ecologically important and
have a strong influence upon fitness. Current studies focus on understanding the role
of development in genetic variation for these traits. It is important to determine
whether quantitative genetic variation for a trait is stable throughout the develop-
ment of an organism. If the heritability of a trait changes during ontogeny, then natu-
ral selection can only influence the trait during intervals of high heritability. Con-
versely, if natural selection only occurs during certain periods of development, then
the trait will be more free to vary during other portions of ontogeny.
The mud crab Eurypanopeus depressus (Smith) has several features which will
make it suitable for a quantitative analysis of growth and development. Like other
arthropods, changes in size and shape in Eurypanopeus is restricted to a short interval
following a molt while the new exoskeleton is still flexible. In addition, molting of the
exoskeleton uniquely defines developmental events. In many nonarthropod species
the accurate assessment of developmental progress or developmental staging is either
difficult or is a largely arbitrary process. Accurate developmental assessment is essen-
Received 22 June 1987; accepted 26 August 1987.
1 Contribution number 688 from the Belle W. Baruch Institute for Marine Biology and Coastal Re-
search.
461
462 T. J. HILBISH AND F. J. VERNBERG
tial to an upderstanding of changes in heritability during ontogeny. Finally, in Eury-
pan- . enile development includes a rapid change in shape. During the
first Ihte 1C molts, these crabs transform from a megalops with a square cara-
. trapezoidal shape typical of the adult stage in this species. These character-
istics allow us to address several fundamental questions on the quantitative genetics
of juvenile growth and form. We will determine the relative importance of genetic
causes to the variance in early juvenile growth and shape in Eurypanopeus. We will
also determine whether genetic variation in these traits varies during development
and whether the genetic component of variance changes during the transformation
in shape that occurs early in juvenile development.
MATERIALS AND METHODS
Eurypanopeus depressus is the most common mud crab inhabiting oyster reefs
along the east coast of the United States (Williams, 1 984) and is abundant in intertidal
oyster reefs in South Carolina. Ovigerous females are found from April to November
in the North Inlet estuary (Georgetown, South Carolina). Costlow and Bookhout
( 196 1 ) described the larval development of this species reared under laboratory con-
ditions. They reported four zoeal stages and a megalops.
Gravid Eurypanopeus depressus females were collected at the Baruch Institute
Field Laboratory near Georgetown, South Carolina, during the summer of 1978. All
females used in this study were collected at one time. Each female was maintained in
the laboratory at 25°C and a 14:10 h light:dark cycle in an individual bowl of 30%o
seawater until an egg mass was expelled. Egg clutches from six females were hatched
and individually reared in beakers through the zoeal stages. Zoeae were maintained
under the same environmental conditions as their mothers and were fed freshly
hatched Anemia ad libitum.
Upon metamorphosis to the megalops stage, juveniles crabs were transferred to
individual cells in a compartmented box where they were maintained with seawater
(30%o and 25°C). To reduce the effects of a shared environment, the compartmental-
ized boxes were returned to the incubator in a random placement following a daily
change in seawater. Juvenile crabs were fed excess amounts of freshly hatched Ar-
temia daily. Individual crabs were checked daily for a molted exoskeleton. Upon
molting the exoskeleton was removed and the crab was measured for both carapace
length and width to the nearest 0.01 mm using an ocular micrometer on a dissecting
microscope. Carapace length was measured as the distance between the anterior and
posterior margins of the shell. Width was measured as the maximal distance across
the lateral margins of the shell. The number of days elapsed since metamorphosis to
megalops was recorded at each molting. During the experiment individual animals
died, so sample sizes vary throughout the analysis. Measurements continued for eight
successive molts but numbers declined significantly after the sixth molt. Therefore,
the analysis reported here is restricted to the first six molts.
Genetic and statistical analysis
Data were collected such that members of the same family could be distinguished
throughout the analysis. Quantitative genetics uses the similarity among relatives to
determine the proportion of the phenotypic variance in a trait that can be explained
by genetic variance (Falconer, 198 1). Therefore, if the mean value for a trait, such as
body size, varies significantly among families this implies that close relatives may
GENETICS OF GROWTH AND SHAPE IN EURYPANOPEUS 463
appear similar because of the genes they share in common. It is also possible that
relatives are similar in appearance for non-genetic reasons. These possibilities are
discussed below. In this analysis we know whether two individuals share the same
mother; we do not know whether they share the same father. Therefore it is unknown
whether offspring derived from the same clutch are full- or half-siblings. Without
knowing the exact genetic relationship among siblings it is impossible to estimate
heritability (genetic variance/total phenotypic variance) accurately. However, we can
estimate whether there is a significant genetic effect upon the phenotypic variance
observed in these samples of juvenile crabs. This analysis is similar to that used in
other studies of quantitative genetics using wild-caught pregnant females (Arnold,
1981a,b).
Relative growth rates were determined by regressing the change in size between
successive molts against initial size for each individual within a family. Variation
among families in relative growth was then analyzed using analysis of covariance.
Relative growth rates are reported as mm growth/mm initial size. Relative growth
rates were determined for both length and width and were calculated on a per molt
and per day basis (see below). Development rate was calculated as the number of
days required to proceed from one molt to the next. Variation among families in
development rate was analyzed using ANOVA. Eurypanopeus depressus varies dra-
matically in shape during juvenile development. Shape can be expressed as the ratio
of width to length. However, ratios have unusual sampling distributions and ANOVA
is not an appropriate methodology for their analysis (Atchley et ai, 1976). Therefore,
carapace length was regressed against width for each family and analysis of covariance
used to determine whether there was significant variation among families in either
slopes or adjusted means of the regressions.
Relative growth rates are expressed in two ways. First, the relative growth between
two successive molts was determined by regressing the change in size against initial
size for each family. Variation among families was then analyzed using ANCOVA.
The analysis of covariance reports the average change in size for each family adjusted
to the average initial size for all families. Relative growth rates were then calculated
by dividing each adjusted mean by the average initial size. Therefore, relative growth
rates are reported as mm growth/mm initial size/molt (mm/mm/molt). This method
of expressing relative growth indicates the change in size between molts but does
not account for the amount of time required to proceed from one molt to the next.
Therefore growth rates were also calculated by determining the change in size be-
tween successive molts for each individual and dividing by the intermolt interval for
that individual. This quantity was then regressed against initial size to determine
relative growth rates and the data analyzed by ANCOVA.
The family means for all growth and development rate measurements are pre-
sented in graphical form. It was not possible to simultaneously present the standard
errors of the means for these analyses without obscuring the graphical presentations.
Therefore the following convention was adopted to provide a representation of the
variance about each family mean. The error mean square is reported for each analysis
of variance in tabular form. Combined with the sample sizes for each family these
data may be used to calculate either standard errors or confidence limits for the family
means (Sokal and Rohlf, 1981). The six families used in this study varied in size. In
addition, some individuals died and occasionally individual measurements were lost.
Therefore sample sizes within a family vary slightly from one experiment to the next.
Average sample size and the range in sample size for each family are: family 1, 10.2
464
T. J. HILBISH AND F. J. VERNBERG
TABLE I
F-te , , nong families in growth and development rates. Error mean squares
foi each test
Molt interval
1-2
2-3
3-4
4-5
5-6
GROWTH RATE
Length/Molt
F-value
3.61(102)**
3.29(110)**
1.98(112)
2.14(107)
1.83(84)
error MS
0.0098
0.0085
0.0170
0.0079
0.0058
Width/Molt
F-value
3.21(112)**
1.65(117)
6.20(115)***
0.23(112)
1.65(89)
error MS
0.0137
0.0100
0.0075
0.0041
0.0044
Length/Day
F-value
4.00(94)***
0.54(112)
0.92(112)
2.64(107)*
4.09 (78)**
error MS
0.00105
0.00105
0.00047
0.00023
0.00005
Width/Day
F-value
4.75(102)***
0.74(117)
4.56(115)***
1.62(112)
3.33(88)*
error MS
0.00105
0.00104
0.00023
0.00018
0.00009
DEVELOPMENT RATE
F-value
error MS
4
5
.10(1
.99
10)***
1.25
2.02
(120)
3,
4.
.29(1
29
19)**
2.70(1
24.95
15)*
6.04(94)***
17.53
The denominator degrees of freedom are reported in parenthesis. The numerator degrees of freedom
are equal to 5 in all cases. The significance of the F-test is indicated by an asterisk (*P < 0.05; **P < 0.01;
***P < 0.00 1 ). F- values without an asterisk are not significant at the 5% level.
(7-12); family 2, 13.5 (1 1-15); family 3, 17.1 (10-20); family 4, 21.7 (18-24); family
5, 38.6 (3 1-42); and family 6, 12.1 (11-13).
RESULTS
Relative growth per molt
Relative increases in length varied with development and among families. Be-
tween the first and second molt the average relative growth rate was 0.25 mm/mm/
molt and there was significant variation among families in their average growth rate
(P < 0.01, Table I). Families 1, 3, 5, and 6 exhibited high growth rates of approxi-
mately 0.25 mm/mm/molt while families 2 and 4 exhibited much lower growth rates
of approximately 0.17 mm/mm/molt (Fig. 1). The error mean square for relative
growth rates over this first interval and all subsequent analyses are presented in Ta-
ble I.
Between the second and third molt families 1, 3, 5, and 6 continued to grow at
significantly higher rates than did families 2 and 4 (P < 0.01, Table I). The highest
relative growth rates were observed between the third and fourth molt, averaging 0.36
mm/mm/molt (Fig. 1). However the variation among families was not significant
during this interval. Average growth rates dropped to 0. 16 mm/mm/molt in the next
two molt intervals (molt 4-5 and 5-6), and there was no significant variation among
families (Fig. 1 ; Table I). In summary, there was significant variation among families
in relative growth rate for length in the first two molt intervals. There was no signifi-
GENETICS OF GROWTH AND SHAPE IN EURYPANOPEUS
465
0.4-,
1 0.3
UJ
| 02 -I
£
o
tr
o
^ O.I
<
UJ
5
0.0
XX
MOLT
FIGURE 1. Relative growth rates in length for Eurypanopeus depressus for each intermolt interval.
Growth rates are reported in mm/mm/molt. Each symbol indicates the mean growth rate for each of the
six families used in the analysis. Significant variation among families is indicated along the abcissa with an
asterisk (*P < 0.05, *V < 0.0 1 , **T < 0.00 1 ).
cant variation among families in relative growth rate as development progressed be-
yond the third molt.
Similar to growth in length, relative changes in width depended on both develop-
ment and family. Relative growth rates were initially high, averaging 0.30 mm/mm/
molt between molts 1 and 2. Between molts 5 and 6 these rates declined to an average
value of 0.17 mm/mm/molt (Fig. 2). There was significant variation among families
in relative growth rate between molts 1 and 2 (P < 0.01, Table I) and molts 3 and 4
(P < 0.001, Table I). Family 6 was the fastest growing group in virtually all molt
intervals. Families 2 and 4 were initially the slowest growing families, but by the final
molt interval they were among the most rapidly growing families. This change in
relative growth rates was similar to that observed for growth in length. Variation in
relative growth in width was not significant among families in any other molt interval
(Fig. 2; Table I).
Relative daily growth
Relative growth rates were also calculated by dividing the change in size between
molts by the interval between molts. This method of expressing relative growth pro-
vides a daily estimate of growth but also includes two potentially genetically variable
parameters; growth and development time. An alternate way of viewing this expres-
sion of growth is that it removes a potential artifact in the previous expression by
controlling for variation in intermolt intervals. In many species growth between molts
466
T. J. HILBISH AND F. J. VERNBERG
0.4 -
I03
E
LU
0.2 H
O.
yj
0.0
**
***
i
2
i
3
MOLT
FIGURE 2. Relative growth rates in width for Eurypanopeus depressm for each intermolt interval.
Growth rates are reported in mm/mm/molt. Each symbol indicates the mean growth rate for each of the
six families used in the analysis. Significant variation among families is indicated along the abcissa with an
asterisk (*P < 0.05, **P < 0.0 1 , ***P < 0.00 1 ).
is a function of the length of time between molts. Therefore the previous analysis
may confuse variation in the interval between molts with variation in growth rate.
This artifact is minimized by expressing relative growth on a daily rather than per
molt basis.
Relative daily growth in length varied during development and was strongly de-
pendent upon family. Relative increase in length was initially high, averaging 0.049
mm/mm/day, but declined steadily with each molt (Fig. 3). The average daily growth
rate was 0.0 1 5 mm/mm/day between the fifth and sixth molts. Daily growth rates in
length also exhibited a significant family effect. Between molts 1 and 2, families 1, 3,
5, and 6 grew at nearly double the rates of families 2 and 4 (P < 0.001 ; Table I; Fig.
3). In the interval between molts 2 and 3 and between molts 3 and 4 there were no
significant differences among families in daily growth rates (Table I). The variance in
daily growth rate among families was again significant (P < 0.05; Table I) between
molts 4 and 5. There was also significant variation among families between molts 5
and 6 (P < 0.01, Table I). There was a clear change in the rank order of relative
growth rates among the families with progressing development. Families 2 and 4 were
initially the slowest growing groups, but by the final molt interval they were the fastest
growing families (Fig. 3).
Relative daily growth rates for width decreased steadily with each successive molt.
Initially relative growth rates in width were 0.067 mm/mm/day and declined to 0.0 1 6
mm/mm/day by the final molt interval (Fig. 4). As with previous measures of growth,
relative daily growth in width also depended upon family. Between molts 1 and 2,
the variation among families was highly significant (P < 0.001, Table I). Between
GENETICS OF GROWTH AND SHAPE IN EURYPANOPEUS
467
0.07-
•8 0.06 -
0.05 -
0.04 -i
E
,E
J
ui
0.03 -
o
or
0.02 -
0.01 -
0.00
**
I
2
3 4
MOLT
I
5
i
6
FIGURE 3. Relative daily growth rates in length for Eurypanopeus depressus for each intermolt inter-
val. Growth rates are reported in mm/mm/day. Each symbol indicates the mean growth rate for each of
the six families used in the analysis. Significant variation among families is indicated along the abcissa with
an asterisk (*P < 0.05, **P < 0.0 1 , ***P < 0.00 1 ).
molts 2 and 3 there was no significant variation among families in relative daily
growth. Between molts 3 and 4 there was again significant variation among families
(P < 0.001, Table I). Between molts 4 and 5 there was no significant variance among
families while in the final molt interval the variation among family means was sig-
nificant (P < 0.05, Table I). As with previous analyses, families 2 and 4 initially exhib-
ited low relative growth rates and ultimately became the fastest growing families by
the final molt (Fig. 4).
Development rate
The intermolt interval increased with development. The interval between the first
and second molt averaged 5.2 days while it required about 12.8 days to proceed from
molt 5 to 6 (Fig. 5). The variation among families changed substantially over the
course of development. Early in development, between molts 1 and 2, variation
among families was highly significant (P < 0.001, Table I). This variation was due
primarily to the relatively long intermolt period of families 3 and 4 (Fig. 5). The time
required to proceed from molt 2 to 3 was virtually identical in all cases; the variation
among families was not significant. After the third molt, families 2 and 4 exhibited a
large decrease in the intermolt interval relative to the other families. By the final molt
interval these two families had an intermolt period 35% lower than the other four
families (Fig. 5). The variation among families in intermolt time was also significant
for the intervals between molts 3 and 4, molts 4 and 5, and molts 5 and 6 (Table I).
Shape
Carapace width initially increased at much higher rates than did length. Ulti-
mately the rates of increase in width and length converged by the final molt interval.
468
T. J. HILBISH AND F. J. VERNBERG
0.09 -
-S, 0.08 -
o
•p
§ 0.07
E
LU
I
0.06-
0.05 -
§ 0.04
e>
UJ
^ 0.03
5
a 0.02 -
0.01 -
0.00
***
***
I
2
3 4
MOLT
I
5
FIGURE 4. Relative daily growth rates in width for Eiirypanopens depressus for each intermolt inter-
val. Growth rates are reported in mm/mm/day. Each symbol indicates the mean growth rate for each of
the six families used in the analysis. Significant variation among families is indicated along the abcissa with
an asterisk (*P < 0.05, **P < 0.0 1 , ***P < 0.00 1 ).
This resulted in a major change in shape over the first few juvenile molts. After meta-
morphosis to the first crab stage the carapace of Eurypanopens depressus is approxi-
mately square, with an average width to length ratio of 0.98 (Fig. 6). During the
second molt the ratio of width to length increased to 1 .08. The ratio increased to 1.2
during the third molt. During molts 4, 5, and 6 there was a gradual but continual
increase in the width to length ratio up to an average of 1 .24 (Fig. 6). By the sixth molt
the crabs had adopted the trapezoidal shape characteristic of adult Ewypanopeus.
The analysis of shape was designed to test two questions: do families vary in shape
and where in ontogeny is this source of variance of greatest importance? Carpace
width was regressed against length for each family, and ANCOVA was used to deter-
mine if there was significant variation in shape among families at each molt. Signifi-
cant variation in adjusted means indicates that some families are consistently broader
or narrower than others. Significant variation among slopes of these regressions indi-
cates that the manner in which width scales onto length depends upon family. There-
fore, either variation in adjusted means or slopes would be indicative of shape varia-
tion among families.
Variation in adjusted means was not significant for any of the six molts (Fig. 6;
Table II). With the exception of the sixth molt there were also no significant differ-
GENETICS OF GROWTH AND SHAPE IN EURYPANOPEUS
469
14-
12-
10-
8-
oe
4-
0
WWW
r\'r\'r\
*X
***
MOLT
FIGURE 5. Mean development rates for six families of Eurypanopeus depressus for each intermolt
interval. Development rates are reported as the days required to proceed from one molt to the next. Signifi-
cant variation among families is indicated along the abcissa with an asterisk (*P < 0.05, **P < 0.01, ***P
< 0.001).
ences among families in the slope of the width on length regressions (Table II). In
the sixth molt there was significant variation among families in the slopes of these
regressions (0.01 < P < 0.05). However this one case of significance may be due to
random chance. There were 1 2 tests of significance performed in this analysis, of
which one was significant at the 0.05 level. Rejections at this significance level should
occur by chance 5% of the time (0.6 times in 1 2 tests). It should also be noted from
Figures 1 and 2 that there was no significant variation among families in increases in
either width or length which also suggests that the significance of variation in shape
among families during this interval was spurious. In summary, there is no convincing
evidence of significant variation in shape among the six families used in this analysis.
DISCUSSION
There was significant variation among families in relative growth rates. This was
true for rates of increase in both width and length and for growth rates calculated on
both a per molt and daily basis. Variation in growth rate was observed throughout
juvenile development; every molt interval included significant family variation in at
least one of the measures of relative growth rate. Natural selection must operate on
traits that retain significant genetic variation and there has been considerable interest
in the manner in which heritable variation may be distributed through development.
For Eurypanopeus depressus, genetic variation for growth rate persists for all juvenile
470
T. J. HILBISH AND F. J. VERNBERG
1.3-
I.2--
i i.i H
1
0.9 -
I
2
3
MOLT
i
5
FIGURE 6. Width to length ratios for six families of Eurypanopeus depressus after the first six juvenile
molts. Width to length ratios are mean values for each family.
developmental stages. Therefore, natural selection could potentially affect growth
rate in this species at any point during its juvenile development.
The development rate also varied among families. This variation must explain in
part the growth rate variation observed among families. In general, high growth rates
in any given interval were inversely correlated with the duration of an intermolt inter-
val. Rapid growth and rapid development appear to co-occur during development.
However, this correlation has little bearing on the variation in growth observed
TABLE II
Results ofANCOVA of carapace width regressed against length for each family at each molt
Molt
F (adjusted means)
F (slopes)
1 1.57(121)
1.32(119)
1.40(118)
4 1.26(118)
0.85(114)
6 0.89(90)
1.85 (116)
0.76 (114)
1.80 (113)
0.53 (113)
1.05 (109)
2.59* (85)
F values for tests of homogeneity of adjusted means and slopes are given. The numerator degree of
freedom was 5 in every test. The denominator degrees of freedom are given in parenthesis.
*P<0.05.
GENETICS OF GROWTH AND SHAPE IN EURYPANOPEUS 471
among families. We correlated the growth and development rates of each family
within a molt interval; there were no significant correlations between growth and
development rates within any molt interval. This lack of correlation is exemplified
by the interval between the second and third molt in which intermolt intervals were
identical for all six families yet there was considerable variation in growth. In addi-
tion, one would expect that expressing growth rate on a daily basis would reduce any
role of development rate in indirectly explaining among family variation in growth.
This was clearly not the case — the expression of growth on a daily basis appeared to
increase the differences among families. Therefore, it appears that families ofEurypa-
nopeus depressus differ in both growth and development rates and that these interact
in a complex manner.
While it is apparent that there is substantial variation among families in rates
of growth and development, the origin of this variation is not obvious. Significant
covariation among family members may be due to genetic causes or to a shared envi-
ronment (Falconer, 198 1 ). The most obvious source of a common environment that
may lead to covariation among siblings is a maternal effect. However it is also possible
that a shared environment during the larval culture phase could lead to significant
elevation in the similarity among relatives. With the experimental design used here
it is not possible to unequivocally reject the hypothesis of maternal effects leading to
the observed variation among families. However, the pattern of growth rate variation
among families indicates that maternal effects are an unlikely explanation for these
results. One consistent trend in the growth data was that families 2 and 4 initially
exhibited low growth rates which increased during development until in most cases
they became the most rapidly growing families. The other four families were typically
fast growing during the initial molts and then declined in their relative growth rates.
Maternal effects might have a reasonably consistent impact on the growth of each
family. For example, if a female produces high quality eggs, perhaps by the inclusion
of atypically high yolk levels, the offspring born to that female should consistently
grow faster than offspring produced from inferior eggs. What was observed here was
a relative growth advantage of some families that changes during ontogeny. This
switching of growth advantage is difficult to explain by maternal or other common
environmental effects. Therefore we conclude that a significant proportion of the
variation in growth and development observed among families is of genetic origin.
In a conventional quantitative genetic design, controlled matings are produced in
which paternal effects can be quantified and the degree of genetic relation among
offspring known. The study of genetic variation in natural populations has certain
liabilities that are exemplified in this study. The first has been discussed. By using
wild-caught ovigerous females the among family variation includes both genetic and
maternal effects. This design is similar to that of Arnold ( 1 98 1 a, b) who used pregnant
female garter snakes to generate families of full-siblings.
A second problem with using wild-caught pregnant females is that we are uncer-
tain as to the genetic relationship among siblings. Individuals born to a given female
may be either full- or half-siblings depending upon the mating system of the popula-
tion under investigation. Many species of brachyuran crabs store sperm from multi-
ple matings (Sastry, 1983). Therefore the offspring of a single Eurypanopeus female
are likely to be a combination of full and half-siblings. Without knowing the exact
genetic relationship among offspring, it is impossible to partition the variance in a
trait into genetic and environmental sources or to subpartition the genetic variance
into additive and nonadditive components. While we cannot accurately estimate the
magnitude of the genetic contribution to the variation in growth and development
472 T. J. HILBISH AND F. J. VERNBERG
rates, it is ck; - substantial fraction of this variation must be genetic in origin.
Familv e^ re very striking; in many incidences family explained greater than
20% variation observed in relative growth rates. Therefore genetic varia-
tio:: h in Eurypanopeus depressus is prevalent but a more controlled breed-
ing design would be required to produce an accurate estimate of its magnitude.
Genetic variation in growth has been observed in other marine invertebrates. Sin-
gle or multilocus genetical analysis of enzyme-coding genes has often revealed a sig-
nificant genetic component to growth (Zouros et al, 1 980; Carton, 1 984; Koehn and
Gaffney, 1984). Quantitative genetic analysis also revealed that a substantial fraction
of the observed variation in growth in marine bivalves is of genetic origin (Lannan,
1972; Newkirk et al., 1977, 1981). This study likewise found a large proportion of
the variation in growth is due to genetic variation.
There was no evidence that shape of the carapace in Eurypanopeus depressus was
genetically variable. This was surprising in light of the highly significant variation
among families in increase in both width and length which are the two parameters
used to determine carapace shape. There is increasing interest in the analysis of shape
and in the role ontogeny plays in altering genetic variances in morphology (Atchley
and Rutledge, 1980; Leamy and Cheverund, 1984). Recently, Riska et al. (1984)
demonstrated that morphological traits may have high levels of quantitative genetic
variation associated with them early in juvenile development, but following the at-
tainment of adult stature this genetic variation is sharply reduced. This study on mice
illustrates the commanding role ontogeny may have in the expression of genetic vari-
ation. In the present study, we expected to observe significant family effects on shape,
particularly between the second and third molt when shape changed rapidly. This
was not the case. There was only one incidence of significant variation among families
in shape and this case was probably not meaningful. Therefore we conclude that in
Eurypanopeus depressus growth and development rates are genetically variable while
shape is a highly conserved trait, exhibiting virtually no genetic variation during juve-
nile development.
ACKNOWLEDGMENTS
We thank Dr. David Lincoln for critically reading an earlier draft of this paper.
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ATCHLEY, W. R., C. T. GASKINS, AND D. ANDERSON. 1976. Statistical properties of ratios. I. Empirical
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Reference: Biol. Bull 173: 474-488. (December, 1987)
IE OF BOTRYLLUS (ASCIDIACEA) LARVAE COSETTLED
WITH PARENTAL COLONIES: BENEFICIAL OR
DELETERIOUS CONSEQUENCES?
BARUCH RINKEVICH12 AND IRVING L. WEISSMAN2
1 Hopkins Marine Station of Stanford University, Pacific Grove, California 93950, and2 Laboratory of
Experimental Oncology, Department of Pathology. Stanford University School
of Medicine, Stanford, California 94305
ABSTRACT
The consequences of settlement of Botryllus larvae close to or on parental colonies
were followed in two sets of experiments. In the first, 28 experimental progeny settled
adjacent to 6 parents; 207 other sibling progeny served as controls. Four different
types of interactions between parent colony and offspring were observed: fusion and
resorption of the offspring, fusion and separation, tunic-to-tunic contact and separa-
tion, tunic-to-tunic contact, and the death of offspring. Offspring interacting with
parents had significantly higher mortality than control offspring. Resorption was the
fastest process (one week on average); the two "separation" processes lasted approxi-
mately two months. Twenty of the 2 1 progeny that died after interacting with parents
did not grow at all (even after 75 days). All 7 offspring that separated from their
parents grew. In two cases of fusion between offspring and adults, large eggs were
found within the progeny zooids. Presumably the eggs translocated from the maternal
colony through the connecting blood vessels. Only five progeny survived in this set
of experiments, a phenomenon which coincided with the degeneration or the mortal-
ity of the parent. In the second set of experiments all 93 progeny which had settled
on old, dead tunics of 5 parental colonies died within 8 weeks.
These results indicate that cosettlement of offspring proximal to their parental
colony is usually deleterious in the long term to the progeny, both when they fuse
with or when they merely contact the parent. This phenomenon was also recorded
in field observations. We suggest that the phenomenon of gregarious settlement of
Botryllus larvae near their parents, although characterized by the loss of many prog-
eny, is nonetheless advantageous in response to biotic interactions such as interspe-
cific competition. In this view resorption may have evolved as a secondary process,
as a result of the nature of self/nonself recognition in Botryllus.
INTRODUCTION
Colonies of the tunicate Botryllus originate from a sexually produced plaktonic
larva. The larva attaches to a substrate and there metamorphoses into a "founder"
oozooid. Colonies arise by asexual multiplication (budding) from this "founder" oo-
zooid. The result is a colony of morphologically and genetically identical zooids. The
star-shaped groups of zooids in the colony all share a common blood-vascular system
embedded in a continuous gelatinous matrix, the tunic. Studies of colony specificity
show self/nonself discrimination in the genus Botryllus (reviewed by Taneda et al,
1985). When the growing parts of two adjacent colonies of the same species come
Received 27 January 1987; accepted 30 September 1987.
474
TUNICATE LARVAE SETTLED NEARBY ADULTS 475
into direct contact, they either reject one another or fuse. This histocompatibility (or
fusibility) discrimination is controlled by a single gene locus with multiple codomi-
nantly expressed alleles (Oka and Watanabe, 1960; Sabbadin, 1962; Scofield et al,
1982; Scofield and Nagashima, 1983). Consequently, two colonies sharing one or
both alleles at the fusibility locus can fuse through their vascular blood systems. Colo-
nies that do not share either allele at the fusibility determining locus reject each other.
Grosberg and Quinn (1986) suggested that larvae of B. schlosseri from Woods
Hole, MA, distinguish kin on the basis of shared fusibility locus alleles, a mechanism
which promotes cosettlement of histocompatible colonies. A gregarious settlement
of Botryllus sibling larvae was also recorded from Monterey, CA (Scofield et al.,
1982), and in populations from the Mediterranean Sea (Sabbadin, 1978). Sibling Co-
settlement in Botryllus may be intensified rapidly by the mechanism of larval meta-
morphosis after larval release into the water column (Grave and Woodbridge, 1924;
Sabbadin, 1978; Grosberg and Quinn, 1986). This promotes cosettlement of larvae
in proximity to their parental colonies (Sabbadin, 1978; Grosberg and Quinn, 1986;
our unpubl. data). Offspring share at least one allel with their parental colony at the
fusibility-histocompatibility locus. Therefore they have the capacity to fuse with the
parent colony.
Grosberg and Quinn ( 1 986) proposed that colony fusion among kin may be bene-
ficial to both members of the chimera in several ways. For example, colony fusion
immediately increases the chimeric-colony size. Since survivorship and onset of re-
production are known to be size dependent, fusion might reduce the likelihood of
mortality and lower the age of first reproduction. Here we describe a laboratory study
of the consequences of offspring cosettlement near their natal colony. Survivorship
and growth rates of the larvae were followed and compared to other, control offspring.
In another set of experiments we followed the survivorship of progeny settled on their
maternal-colony tunic.
MATERIALS AND METHODS
Eleven large sexually matured colonies of Monterey Botryllus schlosseri were used
in two sets of experiments. The sexual maturity of each colony was determined by
embryos developing inside the zooids. All the large colonies were born and raised in
the laboratory in a standing seawater system (Boyd et al., 1986) until their use in
experiments. Thereafter they were maintained in a running seawater system (Rinkev-
ich and Weissman, 1987a). Two sets of experiments were performed. In the first,
larvae were allowed to settle near their parental colonies. In the second, larvae were
allowed to settle on maternal-colony tunic.
First set of experiments
Six colonies (= cases) were placed separately, in six 4 1 glass tanks just before
larvae were hatched. The tanks were aerated by an airstone and maintained in 1 8°C
by a SOW aquarium heater. The glass slide on which each colony had been grown (5
X 7.5 cm) was placed vertically in a slot of a glass staining rack. A blank slide was
placed in the nearest slot, facing the sexually matured colony. Hatched larvae settled
immediately after their release on the maternal colony tunic, adjacent to the parental
colony, or on the blank slide. Larvae metamorphosed to oozooids rapidly thereafter.
The slides were transferred to 1 7 1 tanks in a running seawater system two days later.
Progeny that settled on the colony tunic were removed and discarded, as were dying
or poorly developed zooids that settled on the two glass slides. Several developed
476 B. RINKEVICH AND I. L. WEISSMAN
oozooids (3- :xperimental oozooids in each case) were left undisturbed near their
par ay. Under the dissecting microscope other well-developed oozooids
tfuily peeled from the blank slides with small pieces of razor blade attached
to a firm handle. They were translocated with a Pasteur pipette to other 5 X 7.5 cm
slides to serve as controls (20-44 controls per case, total of 207 control offspring).
Dbservations were taken at least once each week, when the mother-colonies and the
offspring were cleaned with a soft small paint brush. Experimental offspring that in-
teracted with the colonies were observed 4-5 times a week. All control offspring of a
given colony together with their natal colony and the experimental offspring were
kept within the same tank under comparable conditions.
Second set of experiments
Five large, older colonies (ages 1 5-20 months) were used. Each colony had grown
over most of its glass-slide surface area. However, at least 30% of the animal surface
area was characterized as "dead tunic." A dead tunic is designated as the tunic of the
resorbed, old part of a large colony that does not encompass ampullae and active
blood vessels. A dead tunic quite often also appears between systems which spread
apart, in a growth pattern distinct from most colonies found in the wild but which
often characterizes laboratory-cultured Botryllus colonies. Dead tunic frequently in-
cludes relics of resorbed zooids, buds and unhatched embryos, and is covered on its
upper side by fouling organisms, and on its lower side (facing the glass substrate) by
unicellular algae. In this set of experiments we followed the survivorship of offspring
settled on dead tunic of their natal colonies. All other offspring settled on the slide,
on the mother colony zooids, or near the big colony were removed by a small piece
of razor blade.
RESULTS
First set of experiments
Twenty-eight experimental progeny interacted with the six parental colonies
(cases I-VI) in the first set of the experiments. Four different types of interactions
were observed and designated as: A == fusion, leading to resorption of the offspring;
B =: fusion, followed later by separation of the progeny and the maternal colony
(disconnection); C = tunic-to-tunic contact between the offspring and the parental
colony accompanied by the death of the progeny; D =-- tunic-to-tunic contact, fol-
lowed by disconnection of the two interacting individuals (Table I). The colony size
of each observed experimental progeny and the mean body size of the control prog-
eny for each case are given in Figures 1 and 2. In this series of experiments (Figs. 1-
4, Tables I, II) zooids which remained in contact with the natal colony suffered growth
failure and poor survivorship, whereas those zooids which subsequently disconnected
from the natal colony both survived and grew. These results are detailed below in a
case-by-case analysis.
Case I (Table I, Figs. 1, 3a-d). Seven offspring directly contacted with the parental
colony. Five of them (nos. 1, 3, 5, 6, 7; Fig. 1) fused or interacted by tunic-to-tunic
contact only and thereafter died or were resorbed without growing. In one case (prog-
eny no. 5) the offspring survived for 76 days, whereas oozooid no. 1 resorbed 48 h
after fusion was recorded. When oozooid no. 1 's body was resorbed, the left ampullae
and blood vessels continued to operate for an additional one month while they grad-
ually disintegrated (Fig. 3a-d). Only two colonies (nos. 2, 4) survived: offspring no. 4
remained attached to the dead tunic of the mother colony for 7 1 days and then sepa-
TUNICATE LARVAE SETTLED NEARBY ADULTS 477
TABLE I
Summary of interactions between offspring and their parental colonies in the first set of experiments
Case
No. of
offspring
Interaction type3
A
B
C
D
Remarks"
I
7
3
1
2
1
B = Disconnected after 20 days as a result of subsequent
degeneration of the mother colony. D = The
offspring attached to "dead" tunic of the mother
colony.
II 5 14 B = Disconnected after 57 days as a result of subsequent
degeneration of the mother colony and the
offspring.
III 3 1 B = Disconnected after 8 1 days as a result of subsequent
degeneration of the mother colony and the
offspring.
IV 3 3
V 6213 B = Disconnected after 61 days as a result of subsequent
degeneration of the mother colony.
VI 4 1111 B = Disconnected after 63 days as a result of subsequent
degeneration of the mother colony. D = The
offspring attached to "dead" tunic of the mother
colony.
Total: 28 6 5 15 2
a The interaction type: A = Fusion and resorption of the offspring; B = Fusion and disconnection; C
= Tunic-to-tunic contact and death of the offspring; D = Tunic-to-tunic contact and disconnection.
b "Dead" tunic = A layer of tunic which attached to the colony but does not harbor zooids, blood
vessels, or ampulae.
rated, and no. 2 fused with the degenerating part of the parent for 20 days and then
separated. This last colony exhibited a significantly higher growth rate than the con-
trols, and on the final day of the experiment (age of 125 days) it possessed 25 zooids
(in 2 systems) compared to 6.8 ± 3.4 zooids of the control offspring (P < 0.001,
/-test).
Case II (Table I, Fig. 1). Five offspring attached to their maternal colony tunic.
Four of them (nos. 1, 3, 4, 5; Fig. 1) died. Offspring no. 2 remained fused with the
mother colony for 57 days and separated as a result of reciprocal degeneration of the
two members of the chimera. This progeny continued to degenerate and died 5 1
days thereafter. Twelve days after the fusion, when progeny no. 2 possessed only
four zooids, we observed two large eggs in its buds. We had followed the interactions
between this progeny and the parent every other day from the day of fusion and did
not observe any development of female or male gonads in the progeny. Five days
later, seven large eggs were recorded in progeny no. 2's buds. After 9 additional days
and up to the death of this progeny (at the age of 152 days) no more eggs were re-
corded. The mother colony contained many eggs during the first 14 days after fusion;
these disappeared thereafter concomitantly with its own degeneration.
Case III (Table I, Figs. 1, 3e-h). Three offspring attached to the parent tunic.
Two (nos. 2, 3; Fig. 1) died without fusion. Offspring no. 1 fused and disconnected
81 days later as a result of reciprocal degeneration (Figs. 1, 3e-h). This progeny con-
tinued to degenerate and died 2 1 days thereafter.
Case IV (Table I, Fig. 2). Three progeny attached to the mother colony tunic.
They died 12-47 days thereafter without growth.
478
B. RINKEVICH AND I. L. WEISSMAN
_
'.40! '.'•-" <J8> <25X25) (23) (22) [Te] < 1 5)
CONTROLS (N)
» » j ~~- ^ ' 1 1
20 40 60 80 100 120 140 160
0Fusion & resorption
Fusion & disconnection
T T T & death
0 T T T 4 disconnection
. Control colonies
|29J (28) <24) (22) (12) (ID
(29) <Z5) (23) (19X18X16) <I4> [jjz] (12) <IO> (9X9) CONTROLS (N)
20 40 60 80 100 120 140 160
Age (days)
(44) (37) 36 (32) (30)
(44) (41) (36) (36) (32) (27X27X23) (20) ( 14) IlOl (7X7) (7)(6) COKTROLS (N)
20 40 6O 80 100 120 140 160
FIGURE 1. Botryllus schlosseri. Cosettlement of offspring near their maternal colony. Cases I to III,
represented by 7, 5, and 3 experimental offspring, respectively (each experimental offspring is marked by a
number). The average body size of the controls is marked by a solid line and black circles. The experimental
offspring are marked by open circles. A small black circle within an open one represents a situation in which
the body sizes of the controls and the experimental offspring are the same. The numbers in parentheses in
the upper part of each case represent the number of surviving controls, while those in boxes provide the
number of controls on the day of first offspring-parental interaction and on the day of termination of the
last offspring-parental interaction. A black arrowhead = fusion; a black arrow with an open head
= disconnection; TTT = tumc-to-tunic contact.
TUNICATE LARVAE SETTLED NEARBY ADULTS
479
o
o
X
2) (12) (9><7XS)(3M3I3> m (3) CONTROLS (N>
o 20 <»o 60 so 100
Age (days)
0Fusion &. resorption
Fusion & disconnection
._<, T T T & death
_ ^o T T T & disconnection
Control colonies
(34) |33| (25) <20 (ii)
(38) <3<> <26> <2D (19X19X18) [j*] (I THIS) CONTROLS (K)
20 40 60 80 100 120
Age (days)
CONTROLS <N>
20 40 60 80 100 120 140 ISO 180 2OO
Age (days)
FIGURE 2 Botrvllus schlosseri. Cosettlement of offspring nearby their maternal colony. Cases IV to
VI, represented by 3, 6, and 4 experimental offspring, respectively (each experimental offspring is marked
by a number). See legend to Figure 1 for other details.
480
B. RINKEVICH AND I. L. WEISSMAN
FIGURE 3. Resorption and disconnection of progeny cosettled with their parent, a-d: Case I, off-
spring no. 1 (refer to Fig. 1). (a) Immediately after resorption, the relict of the resorbed zooid is still seen,
with five operating ampullae connected with the parent by one vessel, (b) Six days later, the zooid's body
is completely resorbed, while the ampullae are still functional, (c) After an additional eight days, the ampul-
lae are resorbed but the blood vessel still permits blood flow, (d) A month after the zooid's resorption, the
blood vessel and ampullae have disintegrated. The dead tunic, overgrown by bacteria, was loosely adherent
to the substrate and was detached from the substrate three days later, during a routine cleaning of the
TUNICATE LARVAE SETTLED NEARBY ADULTS 481
Case V (Table I, Figs. 2, 4a-e). Five of the six offspring that attached tunic-to-
tunic or fused with their maternal colony died without growth. A typical fusion-re-
sorption process is documented with offspring no. 4 (Fig. 4a-b). Progeny no. 3 repre-
sents another case of egg transfer from the mother colony (Fig. 4c-e). This progeny
grew to 2 and 3 zooids 6 and 14 days after fusion, respectively. In this last day (age
43 days) one big egg was observed in a bud (Fig. 4c), but no male gonads were found.
The maternal colony contained many similar-size eggs. Progeny no. 3 grew rapidly
and 6 1 days after fusion it disconnected from the parent as a result of degeneration
and the death of the mother colony. At the end of the observation period (day 118)
progeny no. 3 contained 1 3 zooids and was significantly larger than the controls (7.0
± 3.4 zooids; P < 0.01, Mest).
Case VI (Table I, Fig. 2). Two (nos. 2,3) of the four offspring which attached to
the parent colony died or were resorbed within 1 1 days. Offspring no. 1 attached to
the mother colony tunic for 63 days and disconnected because of degeneration of the
old part of the parent. This colony grew significantly slower than the controls, and
on the day of separation (age 129 days) it contained only 6 zooids compared to 21.9
± 9.4 zooids of controls (P < 0.01; Mest). Offspring no. 4 fused with the parent and
disconnected after 62 days, concomitant with the large colony's degeneration. On the
day of separation (age 1 38 days) this offspring contained 1 5 zooids (compared to 2 1 .7
± 9.5 zooids of controls, P > 0.05, Mest). Offspring no. 4 multiplied to a 30 zooid
colony after an additional period of 47 days.
We calculated the controls' survivorship-percentage in each case by subtracting
the number of surviving controls on the day of termination of the last offspring-
parental interaction from the number of controls on the day of first offspring-parental
interaction, and found 46.3% survivorship (68 out of 147 of the control offspring
were alive). By contrast, when we group the offspring which were eliminated by re-
sorption and mortality together, only 7 out of 28 experimental offspring survived
(25%). In fact, survivorship eventually was even less: two experimental offspring [case
112 and case III1] were included in the group of survivors although they were already
in the process of dying, since according to the pre-determined criterion, they were
still alive when reseparated from their parental colonies. The experimental offspring
have a significantly higher proportion of "exclusion" than the controls (P < 0.05;
testing equality of two percentages). However, there is the argument that the resorp-
tion of the fused oozoids by their parent-colonies cannot be equated with, and is not
equivalent to, the death of those offspring which attached but did not fuse with natal
colonies. According to this point of view only 2 out of 1 7 offspring ( 1 1 .8%) survived.
This mortality rate is significantly higher than that of control offspring (P < 0.0 1 ;
testing equality of two percentages).
animals, e-h: Case III, offspring no. 1 (refer to Fig. 1 ). (e) Fusion of the offspring with the parent involves
one vessel. The offspring possessed two zooids. (f) Seventeen days later, the offspring is a small colony with
two zooids and four buds, before changing asexual generations. The ampullae in the parent colony have
retreated, and there was no growth of the parent. The nearest two systems to the fused offspring contained
21 zooids. (g) Forty-one days later, degeneration and partial mortality of the parent has occurred, with the
two nearest systems to the offspring containing only eight zooids. All ampullae of the parent retreated,
exposing a bare, dead tunic. The offspring was still connected by one operating blood vessel to the mother
colony. No growth occurred in the offspring colony, (h) Nine days later, resorption and death of most
of the maternal colony continued with only four zooids remaining near the offspring. The offspring
had disconnected from the mother colony, was in fair condition, but with no growth, a = ampullae,
b = bud, d = dead tunic, rz = resorbed zooid, v = blood vessel, z = zooid. Scale bar: a-d = 0.2 mm,
e-h = 0.5 mm.
482
B. RINKEVICH AND I. L. WEISSMAN
FIGURE 4. Settlement of Botryllus larvae near or on the parental colony, a-e: Case V (refer to Fig.
2). a-b: Offspring no. 4, represents a typical resorption. (a) Immediately after fusion, (b) Eight days later,
resorption, only ampullae remained, c-e: Offspring no. 3, eggs translocated from the mother colony to the
progeny, (c) Fourteen days after fusion, the offspring had three zooids and one egg in a developed bud
(picture was taken from underneath the colony), (d) Twelve days later, the offspring colony with seven
zooids and seven large eggs (only 5 can easily be recognized) in the developed buds (picture was taken from
underneath the colony), (e) Twenty-three days later, shortly before disconnection, the dead tunic was
connected between the fused colonies. (0 Second set of experiments. Settlement of oozooid on dead tunic
of its maternal colony, a = ampulla, b = bud, d = dead tunic, e = egg, o = oozooid, v = blood vessel,
z = zooid. Scale bar = 0.5 mm.
Table II presents some characteristics of the fusion and the tunic-to-tunic interac-
tions between the offspring and parental colonies. The processes which resulted in
the elimination of the offspring (resorption and tunic-to-tunic contact followed by
a death) lasted a shorter period than the other two processes which resulted in the
reseparation of the offspring from the parents. Resorption is the fastest process (on
the average about one week, Table II) while the two disconnecting processes lasted
on average about 2 months. In addition, most of the offspring which ultimately were
resorbed and killed during tunic-to-tunic contact did not grow at all (20 out of 21).
TUNICATE LARVAE SETTLED NEARBY ADULTS 483
By contrast growth was recorded in all the 7 offspring in the other two types of interac-
tions that led to disconnection (P < 0.0 1 ; Fisher's exact test for independence).
Second set of experiments
The survivorship of 93 offspring settled on the dead tunic of 5 parental colonies
was recorded every 2 weeks for a period of 2 months (Table III). The experimental
colonies were carefully chosen for their size and the relative large surface area which
encompassed dead tunic only (Fig. 4f). Some of the offspring died (natural death, or
the effect of the mother colony?). However, most of them were killed by the lifting of
the degenerated parental colony tunic from the substrate. Since the offspring were
attached to the tunic and not to the substrate, they were swept out from the slide
and died. After one month only 18.3% of the offspring survived. Two months after
settlement no offspring survived (Table III).
DISCUSSION
Co-settlement ofBotnilus progeny adjacent to their maternal colony (first set of
experiments) or on their mother colony's dead tunic (second set of experiments)
clearly resulted in increased offspring mortality. Most of the results in this study were
obtained within 2 months of the first tunic-to-tunic contact. Resorption was the fast-
est interaction recorded (about 1 week) and is much faster than the resorption ob-
tained when two large colonies or two big subclones of different colonies fused (up to
8 months after fusion; Rinkevich and Weissman, 1987a). The fast resorption re-
corded in the present study could be the result of the huge body-size differences be-
tween the parent and the offspring. The colony body size was found to be an impor-
tant factor for determination as to which colony in a chimera will be resorbed (Rin-
kevich and Weissman, 1987a). Only 3 out of 11 (27.3%) progeny survived the
resorption and the separation from the parent (Figs. 1, 2; Table II). This number
is too low to support the proposal that fusion of closely related genotypes on the
histocompatibility locus is always beneficial. In fact, the results of the present study
indicate that the survivorship of progeny fused with a parent is possible only when
associated with the degeneration or mortality of the parental colony (Table I).
Another interesting result in the first set of experiments is that most of the off-
spring (60.7%) did not fuse with their maternal colonies, although they share alleles
in common with the parents in the fusibility locus. Part of this is due to offspring
settlement near an "old" part of the parent which did not possess active ampullae.
Active, healthy ampullae of growing parts are vital for a successful fusion between
compatible colonies (Taneda, 1985; Y. Saito, pers. comm.). However, in other cases,
although offspring and parents confronted active, good ampullae, they did not fuse
for a long time (up to 2 months); thereafter the offspring died. Out of the 17 tunic-
to-tunic interacting pairs only 2 progeny survived: those that reseparated from their
parental colony (Table II). In addition, these two offspring (cases 14, VII; Figs. 1, 2)
grew significantly slower than the controls. These results may indicate a possible role
of humoral factors and/or cellular elements present in the tunic of intraspecifically
interacting Botryllus colonies. Diffusing of allogeneic humoral factors through the
text matrix was shown to exist in at least two species of botrylloid ascidians, B. primi-
genus and Botrylloides simodensis (reviewed by Taneda et al., 1985).
In two cases mature eggs were observed within the offspring soon after fusion with
their parents. The parental colonies contained at the same time many similar eggs.
In contrast, none of the control progeny exhibited any sign of sexual development.
484
B. RINKEVICH AND I. L. WEISSMAN
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TUNICATE LARVAE SETTLED NEARBY ADULTS 485
TABLE III
Survivship of offspring settled on "dead tunic" of their maternal colonies
No.
of offspring after
Colony
No. of
no.
settled offspring
2 weeks
4 weeks 6 weeks
8 weeks
1
25
15
3
0
0
2
18
16
2
0
0
3
12
10
1
0
0
4
22
18
6
2
0
5
16
13
5
1
0
Total
93
72
17
3
0
Egg development in Botryllus differs from any other ordinary case of protogyny in
that the first developed young ova migrate in the blood stream to the second genera-
tion of buds developed from the existing generation of zooids. These ova continue to
develop, although they still do not reach maturity. Rather, they migrate again into
the next generation of blastozooids. It is not until the seventh or eighth generation
that ova are fertilized (Herdman, 1925). Since no such egg development was observed
in the fused progeny nor the typical simultaneous male gonad development (Mukai,
1977; Sabbadin and Zaniolo, 1979) the observed large eggs in the fused progeny likely
migrated from the mother colony through the connecting blood vessels. Germ cell
exchange between fused Botryllus colonies has been recorded before (Sabbadin and
Zaniolo, 1979). In some hatches of offspring, the progeny released from fused colo-
nies were mostly or totally heterochthonous (Sabbadin, 1 982). This suggests the possi-
bility of germ cell parasitism (discussed by Buss, 1982), in addition to the resorption
which is related to somatic cell parasitism (Rinkevich and Weissman, 1987a,b). An-
other pattern of parasitism, oriented translocations of materials from the inferior col-
ony towards the superior member within a chimera, was recorded in fused colonies
of the hermatypic coral Stylophora pistillata from the Red Sea (Rinkevich and Loya,
1983). This phenomenon was coupled with a significant reduction of growth rate
and reproduction of the inferior colony (Rinkevich and Loya, 1985). These findings
suggest a possible complex network of physiological interactions occurring after a
fusion between two compatible colonies is established. Unfortunately, in these exper-
iments the potential germline chimerism, or parasitism, could not be tested critically,
as fertilization of the eggs did not occur. Thus we can neither affirm that the eggs
were of parental origin nor that they might have enjoyed a selective reproductive
advantage.
Grosberg and Quinn (1986) postulated that Botryllus schlosseri larvae recognize
kin on the basis of shared alleles at the histocompatibility locus and that this recogni-
tion promotes cosettlement of histocompatible individuals. They suggested that fu-
sion which subsequently evolved from cosettlement of the closely related genotypes
is beneficial among kin in several ways, as initially proposed by Buss (1982). One of
these benefits is that fusion may increase the probability of survivorship and growth.
In contrast, the present paper provides evidence (Figs. 1, 2, 3, 4; Tables I, II, III) that
the settlement of offspring very near or on their parental colony may in fact reduce
survivorship and growth rates. Similar results of reduction in survivorship and growth
rates were obtained in other experiments at Hopkins Marine Station in which pairs
of large colonies (Rinkevich and Weissman, 1987a,b) or pairs of sibling oozooids
(unpub.) were cosettled.
486 B. RINKEVICH AND I. L. WEISSMAN
A number of theories have been proposed that the ability to recognize one's kin
is beneficial (reviewed by Hepper, 1986). It is a fact that sibling larvae of Botryllus
sel . in aggregations (Grosberg and Quinn, 1 986; this paper) and near their parental
•ony (Sabbadin, 1978; this paper). However, by keeping in mind the deleterious
effects of the fusion, how does one explain the existence of cosettlement of closely
related individuals such as in the case ofBotryllusI One possible explanation consid-
ers the selective pressure of other biotic interactions, such as interspecific com-
petition.
Botryllus colonies in the field compete for the substrate with other sessile organ-
isms. For example, B. schlosseri from Woods Hole is usually overgrown and killed by
Botrylloides (Grosberg, 1 982). In addition, this competition also mediates the relative
frequency of different types of life history tactics presented by Botryllus colonies
(Grosberg, 1982). In Monterey, California, even large Botryllus colonies are often
overgrown by Diplosoma, a fast-growing colonial tunicate (unpub.).
Buss (1981) has shown that the formation of aggregations in the colonial bryozoan
Bugula turrita is a response to interspecific competition. B. turrita also suffers signifi-
cant intraspecific competition. However, in high densities B. turrita is rarely over-
grown or killed by interspecific competition (Buss, 1981). The costs incurred by in-
creased intraspecific competition involve a loss of proliferative potential, whereas the
benefits accrued by reduced interspecific competition involve whole-colony mortality
(Buss, 198 1 ). Since mortality results in a greater loss in fitness than does a reduction
in growth rate, the evolutionary selection for the gregarious settlement is plausible. If
the persistence of dense assemblages is necessary in sedentary marine animals for
excluding possible interspecific competitors (Jackson, 1983), our results could reflect
a general pattern of habitat selection in sessile marine invertebrates which aggregate
in settlement versus those that do not (Knight-Jones and Moyse, 1961; Crisp, 1979;
Buss, 1981; Jackson, 1983; and literature therein).
Working on another colonial bryozoan, Keough (1984) demonstrated that larvae
of Bugula neritina settle preferentially near conspecific larvae. This observation, cou-
pled with the observation that isolated juveniles of this species did not reach adult
size, was attributed to the selective effects of predation by fishes. Fishes rarely took
more than five bites on any one feeding visit, so groups of colonies might only be
damaged at the periphery. A strong advantage to gregariousness resulted since central
colonies in a group were left untouched. There is no record effusion between Bugula
colonies, although self/nonself recognition has been described in bryozoans (Keough,
1984).
In Botryllus, on the other hand, one common allele at the fusibility locus is suffi-
cient for fusion between colonies (Scofield el al, 1982). Keeping in mind the two
studies on gregarious bryozoans (Buss, 1981; Keough, 1984), we suggest that fusion
of cosettled compatible Botryllus colonies may have long-term deleterious conse-
quences and may not be functionally beneficial to the chimera per se, as suggested by
Grosberg and Quinn (1986). We propose that the phenomena of resorption and/or
death of interacting conspecific colonies may have evolved as a necessary conse-
quence of the self-recognition phenomenon in Botryllus, which allows two different
genotypes sharing in common only one allele at the fusibility locus to fuse and form
a chimera. More specifically, we suggest that the gregarious settlement of Botryllus
larvae in the proximity of the parental colonies and the nature of self/nonself recogni-
tion of this species may have initially evolved as two independent phenomena (or
strategies) which are in the process of adaptation. As a result, the cost/benefit out-
comes depend on the view of the observer. The concept for the general costs effusion
was first presented by Buss ( 1 982) and further discussed in Rinkevich and Weissman
TUNICATE LARVAE SETTLED NEARBY ADULTS 487
(1987b). It is not the purpose of the present paper to discuss again the costs for the
chimera in details. However, following our previous discussion, in our view chimera
formation in Botryllus could develop only if the evolutionary benefits of cosettlement
exceeded the disadvantage of the harmful consequences of fusion. Interspecific com-
petition between Botryllus and other sessile organisms (Grosberg, 1982) may provide
this very selective pressure (Buss, 1981). Gregarious settlement of Botryllus larvae,
even with the loss of many progeny (as the results of resorption and/or death), would
be evolutionarily selected if the number of survivors in gregarious settlement would
exceed the number of survivors of settlement in a random distribution. Perhaps the
resorption and death of one of the two colonies in parabiotic union allows the unit
of reproductive selection to be the survivor of both intra- and interspecific competi-
tion. If so, it shall be most important to define the number and character of genes
involved in the self/nonself recognition events leading to death and/or resorption of
contacting Botryllus kin. Therefore the ecological advantages of gregarious settlement
in Botryllus and the role of interspecific competition must be addressed in further
field and laboratory experiments.
ACKNOWLEDGMENTS
We are grateful to K. Ishizuka and C. Patton for technical assistance. Our appreci-
ation to Y. Saito for his valuable advice during the study and to S. Gaines, J. Danska,
K. Ishizuka, and two anonymous referees for their comments on the manuscript. B.
Rinkevich is a Lucille P. Markey Fellow of the Life Sciences Research Foundation.
The study was supported by grants GM 25902 and CA 42551 from the National
Institutes of Health.
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488 B. RINKEVICH AND I. L. WEISSMAN
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Reference: Biol. Bull. 173: 489-503. (December, 1987)
VARIABILITY IN FLASH CHARACTERISTICS OF A
BIOLUMINESCENT COPEPOD
MICHAEL I. LATZ, TAMARA M. FRANK, MARK R. BOWLBY,
EDITH A. WIDDER, AND JAMES F. CASE
Department of Biological Sciences and Marine Science Institute, University of California,
Santa Barbara, California 93106
ABSTRACT
Bioluminescence of the copepod, Pleuromamma xiphias, was investigated with
an optical multichannel analyzer (OMA) to measure emission spectra, an integrating
sphere-photon counting detector system to determine flash kinetics and quantum
emission, and an ISIT video system to image spatial patterns of emission.
Light emission was in the blue spectral region, with maximum emission at ap-
proximately 492 nm. Spectral waveforms were unimodal, or bimodal with the sec-
ondary peak at 472 nm.
Flashes in response to a single stimulus consisted of two components: a fast com-
ponent attaining maximum intensity in under 100 ms, and a slow element which
peaked after 600 ms. The fast component originated from thoracic and abdominal
light organs while the slow component represented a large expulsion of luminescent
material from the abdominal organ only. Both components exhibited first order ex-
ponential decay although the decay rate of the fast component was approximately
one order of magnitude greater. The typical flash response to a single stimulus exhib-
ited a response latency of 30 ms, initial rise time of 87 ms, duration of 2.4 s, and
quantum emission of 1.4 X 10'° photons flash"1. Quantum emission increased with
increasing stimulus strength.
Both response waveform and total quantum emission were affected by the fre-
quency of electrical stimuli. Stimulation at 1 Hz generated the greatest luminescence,
averaging 1.1 X 10" photons response"1 for 11 s emissions. Higher rates of stimula-
tion decreased total quantum emission and response episode duration, and resulted
in greater temporal summation of the emission waveform.
Variability in flash characteristics due to electrical stimulation suggests a versatil-
ity of luminescent displays in situ.
INTRODUCTION
Recent bathyphotometer measurements in the Sargasso Sea suggest that most sti-
mulable bioluminescence in epipelagic waters originates from zooplankton, princi-
pally crustaceans such as euphausiids, ostracods, and copepods, as well as other or-
ganisms such as larvaceans and radiolaria (Swift et al, 1983, 1985). Copepods repre-
sent approximately 70% of the zooplankton specimens in the upper 200 m of the
Sargasso Sea. Calanoid copepods are more abundant than other copepods (Deevey,
1971) and include many luminescent species (Herring, 1978, 1985).
The secreted bioluminescence of copepods originates from multiple glands (Gies-
brecht, 1895; Clarke et al, 1962), and presumably functions as part of an escape
Received 6 February 1987; accepted 22 September 1987.
Abbreviations: OMA — optical multichannel analyzer, MCA — multichannel analyzer, S/N — signal to
noise ratio, FWHM — full width at half maximum amplitude, PMT — photomultiplier tube.
489
490 M. I. LATZ ET AL.
response from predators (David and Conover, 1961; Buck, 1978; Young, 1983).
Flashes can be induced by mechanical, electrical, photic, or vacuum (presumably
act as mechanical) stimulation, as well as by the presence of potential predators
(D <d Conover, 1961; Clarke et al, 1962; Barnes and Case, 1972; Lapota and
84; Herring, 1985; Yevstigneyev, 1985). After a brief latency following the
stimulus, copepods respond with a flash that rapidly rises to maximum intensity and
lasts from less than 1 s to more than 10 s.
A striking aspect of copepod bioluminescence is the variability in flash waveforms
and kinetics (David and Conover, 1961; Clarke et al., 1962; Barnes and Case, 1972).
This makes it difficult to identify trends in flash responses due to experimental manip-
ulations or other factors. Only in coastal ostracods (Morin and Bermingham, 1980;
Morin, 1 986) has the variability in flash responses been correlated with different lumi-
nescent behaviors.
Our study examines variability in the spectral and temporal characteristics of bio-
luminescence of the calanoid copepod, Pleuromamma xiphias. Members of the ge-
nus Pleuromamma are numerous throughout the year in the Sargasso Sea (Deevey,
1971) and at times it is one of the dominant genera (Fish, 1954). They are active
vertical migrators (Roehr and Moore, 1965), ascending from daytime depths of 350
m to epipelagic depths at night where they contribute to measured bioluminescence
(Swift et al., 1983, 1985). The flash of/*, xiphias is similar to that of other members
of the Metridiidae and is readily elicited by electrical pulses (Clarke et al., 1962; Yevs-
tigneyev, 1985). Our results indicate that the nature of the electrical stimulus dictates
the type of flash response observed, and suggest that complex neural or other factors
play significant roles in regulating the kinetics and quantum emission of the flash
response.
MATERIALS AND METHODS
Adult specimens of Pleuromamma xiphias (Giesbrecht, 1 889) were collected and
studied during the April, 1985, Biowatt cruise aboard the RV Knorr, and during a
subsequent cruise on the RV Endeavor in May, 1987, from stations between 28° and
35° N 70° W in the Sargasso Sea. Plankton nets with 333 ^m mesh and 0.5 or 1 m
mouth diameters were towed at night for 20 min at depths of 80-100 m. Seawater
temperatures at these depths ranged from 18-22°C (T. Dickey, pers. comm.). Speci-
mens were sorted and maintained in filtered seawater in darkness until use. All experi-
ments were performed at room temperature (22 ± 2°C) within 10 h of collection.
Subsequently, specimens were individually preserved in 4% formalin for later identi-
fication.
Spectral measurements
Bioluminescence emission spectra were measured with a Princeton Applied Re-
search optical multichannel analyzer (OMA) system. The OMA detector (EG&G
PARC Model 1420) consists of a linear array of 700 intensified photodiodes which
simultaneously collects the light signal across a 350 nm spectral window of a poly-
chromator. The OMA system has the requisite high sensitivity, high resolution, and
fast response time necessary for registering dim, brief bioluminescent emissions. De-
tails of OMA operation and calibrations have been previously described ( Widder et
al., 1983).
Specimens were suspended in a drop of filtered seawater between a pair of tung-
sten electrodes and stimulated at 20 Hz for approximately 4 s with 10 V, 5 ms dura-
tion monophasic pulses from a Grass model S44 stimulator. Bioluminescence was
COPEPOD BIOLUMINESCENCE 491
focused onto a 1 mm entrance slit to the polychromator by quartz optics and was
integrated over a period of 1-2 s by the OMA system. Spectra with signal to noise
ratios (S/N) less than 30 were not used ( Widder et at, 1 983).
Measurements of flash kinetics and quantal output
The temporal characteristics of bioluminescence were measured with an integrat-
ing sphere-photon counting detection apparatus. An integrating sphere is considered
critical to precise measurements of quantum emission from sources that may not
emit isotropically, e.g., most organisms with photophores. Single specimens were sus-
pended in a drop of filtered seawater between a pair of tungsten electrodes while
enclosed in a 10 inch diameter integrating sphere (Labsphere, Inc.). The inside sur-
face of the sphere is coated with white Polane polyethylene paint to ensure maximum
reflectance (97% reflectance at 500 nm) and minimize damage to the reflector surface
from contact with seawater. A baffle between the source and detector assured that
only light that had undergone multiple reflections within the sphere was measured.
Bioluminescence was detected by a photon counting RCA No. 8850 photomultiplier
tube, operating at - 1 700 V with a calibrated discriminator setting of -0.3 1 5 V, that
viewed the interior of the sphere through a 4 cm diameter port. The entire apparatus
was calibrated for photon emission both before and after the cruise with an Optronic
Laboratory model 310 calibration source referenced to an NBS standard. Quantum
calibration took into account not only the spectral responsivity of the integrating
sphere and photomultiplier tube but also the spectral emission of bioluminescence
of P. xiphias as measured by the OMA. The calibration of the system was frequently
checked at sea with a C14 phosphor (I-Lite, 0.05 mCi) referenced to the Optronics
source.
Bioluminescence was stimulated by single or repetitive electrical pulses at various
frequencies, while the photomultiplier signal was monitored for either 8 or 20 s with
an Ortec No. 776 counter/timer and a Norland No. 5400 multichannel analyzer
(MCA). Flash waveforms displayed on the MCA were either directly photographed
or videotaped. Printed copies of the flash waveforms were later obtained from the
video record after processing by a Megavision model 1024XM image analysis system.
Flash kinetics were derived from the printed waveform on a Summagraphics digitiz-
ing pad.
Flash characteristics are denned as follows: response latency = time from presen-
tation of the stimulus to beginning of the response; total rise time = time from begin-
ning of the response to maximum intensity of emission; 50% decay time - time from
maximum intensity to an intensity one-half that value; total flash or response dura-
tion = time from beginning to end of response; response episode = light emission
during repetitive stimulation; (total) quantum emission = total integrated photons
of response episode; maximum response = response with greatest total integrated
photons.
Image intensification
Individual specimens were placed between two metal electrodes in a clear leucite
chamber containing approximately 2 ml of filtered seawater. The chamber was en-
closed in a sealed box with white reflective internal surfaces. Bioluminescence was
viewed from above through a port in the box with an intensified SIT video camera
(Dage-MTI Model 66) fitted with a 105 mm Nikon F/4.0 lens. A photon counting
detection system (described above) obtained simultaneous measurements of flash
waveforms and kinetics. The photon counting tube viewed the interior of the box
492
M. I. LATZ ET AL.
UJ
1
0.8
0.6
0.4
0.2
350
450
550
650
350
450
550
650
WAVELENGTH (nm)
FIGURE 1. Mean emission spectra of the bioluminescence of Pie uromamma. (A) Pleiiromamma
xiphias spectra are displayed on the same intensity scale but are vertically displaced for clarity, (a) Uni-
modal spectral distribution (dashed line) representing emissions from three specimens; max = 493 nm,
FWHM = 83 nm, S/N = 103. (b) Bimodal spectral distribution (solid line) from four specimens; max
= 492, 472 nm, FWHM = 74 nm, S/N = 97. (B) Spectral distribution representing two specimens of P.
abdominalis; max = 486, 465 nm, FWHM = 75 nm, S/N = 70.
through a 4 cm diameter port, and measured only reflected bioluminescence. Quan-
tum calibration of this system was not performed.
Light production was stimulated electrically by single or repetitive pulses. The
COPEPOD BIOLUMINESCENCE
493
B
i
»—
0.1
0
1.00n
0.30
0.10-1
0.03
TIME (s)
FIGURE 2. Luminescent responses of two specimens of Pleurornamma xiphias to a single 1 0 V, 5 ms
duration electrical stimulus. (A) Typical flash response exhibiting fast and slow components. The relative
intensity of emission is displayed with time; time bar = 1 s. (B) Decay kinetics of both components of the
flash displayed in (A). Relative intensity (log scale) is shown as a function of time. For each component
the slope of the calculated linear regression (solid lines) reflects the rate of exponential decay (refer to text).
Decay rate of the fast component (a) was -8.3 while the decay rate of the second component (b) was -0.9
(R = 0.99 for each). (C) Flash lacking the second component. As in (A) except that the vertical scale is
magnified 10 times. (D) Decay of light emission of the flash displayed in (C). As in (B). The decay rate was
-2.3 (R = 0.99).
methods of collection and analysis of data were identical to those described in the
previous section.
RESULTS
Spectral characteristics
Bioluminescence emission spectra from 7 specimens of Pleurornamma xiphias
were centered in the blue region of the visible spectrum with maxima at approxi-
mately 492 nm. Two types of spectral distributions were measured; about half of the
specimens produced unimodal spectra while the others generated bimodal spectra
(Fig. 1). Regardless of the spectral shape, the dominant emission was at 492-493
nm while the short-wavelength 472 component was present either as a subpeak or
shoulder.
Bimodal emission spectra were also measured from the two specimens of P. ab-
dominalis tested. They differed only slightly from those of P. xiphias, having maximal
emission at 486 nm and a short-wavelength subpeak at 465 nm (Fig. 1). Neither
species gave evidence of sexual differences in spectral emissions.
494
M. I. LATZ ET AL.
B
COPEPOD BIOLUMINESCENCE 495
Temporal characteristics and quantum emission
Flashes from approximately 350 specimens of Pleuromamma xiphias were ana-
lyzed for kinetics and quantum emission. No spontaneous flashes were observed from
specimens in the apparatus prior to testing, although luminescence often was elicited
by handling during preparation. Even though there was much variability in the kinet-
ics and quantum emission of luminescent responses, it was possible to identify gen-
eral trends in responsiveness.
The waveform of a flash response to a single electrical pulse (Fig. 2A) was resolved
into two components. The first, characterized by fast rise and decay times, was fol-
lowed, after a slight decay in intensity, by a second element consisting of a slower rise
and decay (see Fig. 5B for expanded waveform). The maximum intensity of the sec-
ond peak was equal to or greater than the first peak. The majority (25 out of 46) of
flashes stimulated by single electrical pulses contained both components. The re-
maining flashes were composed of either the fast or slow component only (7 and 6
flashes, resp.), or had a waveform that was not possible to resolve (8 flashes). Quan-
tum emissions of flashes with only one component (Fig. 2C) were from one to two
orders of magnitude lower than those of two-component flashes.
Preliminary observations indicated that mechanical stimulation of light emission
also elicited flashes with fast and slow components, although the kinetics and quan-
tum emission of mechanically stimulated flashes were not investigated.
Image intensification of single specimens during the production of two-compo-
nent flashes revealed the spatial pattern of emission (Fig. 3). The fast component was
typically produced by a brief emission from a luminescent gland located laterally on
thoracic segment 3 or 4, and simultaneous light production from another light organ
located on the caudal rami (Fig. 3b). The luminescent material was not violently
expelled from the body during this fast component. The decrease in light intensity
following the fast component resulted from a cessation of thoracic light organ produc-
tion along with a slightly diminished emission from the abdominal light organ (Fig.
3c). The slow flash component was due to increased production of luminescent mate-
rial by the abdominal light organ and subsequent expulsion from the body (Fig. 3d).
No other light organs were observed to be active at this time. The decay of the slow
component was due to a gradual decrease in the amount of luminescent material
produced by the abdominal light organ (Fig. 3e, f).
Flashes in response to a single 10 V stimulus (Table I) had a mean stimulus-
response latency of 1 8 ms. The initial peak of the fast component occurred within
100 ms while the slower second component reached maximal intensity approxi-
mately 600 ms after the stimulus presentation. Total flash duration was 2.6 s, and the
average quantum emission of a single flash was 1 .4 X 10'° photons flash"1 (maximum
of 7.1 X 10'° photons).
Decay of light emission was measured for each component of two-component
flashes (n = 7). Bioluminescence decreased exponentially with time according to the
FIGURE 3. Luminescent response of a single specimen of Pleuromamma xiphias to a single 40 V, 5
ms electrical stimulus monitored by simultaneous image intensification and photomultiplier recording.
(A) Flash waveform from the MCA showing fast and slow components. Intensity is shown as a function of
time. Time bar = 2 s. (B) Simultaneous images of animal (lateral view) obtained from single frames of the
video record. Scale bar = 1 mm. Letters (a) through (f ) in both portions of the figure correspond to identical
time periods during the flash: (a) prior to stimulation; (b) maximum emission during fast component; (c)
decay in intensity of fast component; (d) maximum emission during slow component; (e) and (f) decay of
slow component.
496
M. I. LATZ ET AL.
TABLE I
Response A ; W quantum emission of flashes of Pleuromamma xiphias stimulated with single
electnad pulses (10 V, 5 ms duration)
Total rise time (ms)
Latency
(ms)
Fast
component
Slow
component
50% decay
time (s)
Total Hash
duration
(s)
Quantum emission
(photons flashr1)
18.4*
±4.2
(0.5-59)
87.1
± 15.8
(27-202)
603.0**
±254.7
(552-838)
0.9
±0.3
(0.02-2.8)
2.6
±0.7
(0.1-10)
1.4 x 10'°
±0.6X 10'°
(6.1 X 107-7.1 X 10'°)
* Values represent the mean values ± standard errors of the mean, with ranges in parenthesis, for 14
specimens.
** Precise measurements of second component kinetics were possible in only three specimens.
standard equation for exponential decay, Y = A * e(B*4), where A is the y-intercept and
B is the decay rate constant. Decay rates were calculated from the linear regressions of
the natural logarithm of intensity with time (Fig. 2B, 2D; R = 0.99 for all regressions).
The mean (±S.E.) rate of decay of the fast component of — 14.6 ± 4.4 was an order
of magnitude greater than the rate of decay of the second component (— 1 .25 ± 0.5).
A separate experiment demonstrated that flash quantum emission increased as a
function of stimulus strength when animals were tested with single stimuli of 5 ms
duration from 2-10 V in magnitude (Fig. 4). Minimum responses were at 2 and 4 V,
where the average emission was approximately 3.8 X 109 photons flash"1. Threshold
<
o
2 4 6 8 10
STIMULUS MAGNITUDE (volts)
12
FIGURE Quantum emission of Pleuromamma xiphias flashes as a function of the voltage of single
5 ms duration rtrical pulses. For each stimulus condition, 6-7 specimens were tested. Mean values
(± standard em re: 2 V, 3.9 ± 2.7 X 109 photons; 4 V, 3.7 ± 1.8 X 109 photons; 6 V, 7.3 ± 0.5 X 109
photons; 8 V, l.ti 9 x 10'° photons; 10 V, 1.3 ± 0.9 X 10'° photons. Due to the large standard errors,
mean values were no; 'nificantly different from one another (/-test, P > 0.05), although maximum flash
quantum emission at ei i stimulus voltage level increased.
COPEPOD BIOLUMINESCENCE
497
B
FIGURE 5. Luminescent responses of Pleuromamma xiphias as a function of repetitive stimulation
with 10 V, 5 ms duration electrical pulses. Intensity of output (same relative scale) is displayed as a function
of time. Time bars = 2 s except in (B). The maximum intensity of the responses was near the threshold of
non-linearity of the detection system and may underestimate true intensity levels. (A) Response episode
of one specimen to 0.5 Hz stimulation. (B) Detail of individual flashes comprising response episode for 0.5
Hz stimulation; fast and slow components of each flash are evident. Time bar = 1 s. (C) Response to 2 Hz
stimulation; temporal summation produces a single prolonged response episode. (D) Response to 10 Hz
stimulation. The response is similar in waveform to that for single pulse stimulation but of longer duration.
was apparently near 2 V, since at that level only 50% of the specimens tested produced
luminescence, while at higher voltages all stimuli elicited a response. Stimulus voltage
had no effect (Student's /-test, P > 0.05) on response latency (mean and standard
error = 36 ± 7 ms), initial rise time ( 123 ± 21 ms), 50% decay time (856 ± 201 ms),
or flash duration (2.8 ± 0.5 s). These values are not significantly different from those
of the previous experiment with a constant 10 V stimulus (Student's /-test, P > 0.05).
Stimulation with repetitive pulses had the most pronounced effect on response
waveform and quantum emission (Fig. 5). At the slowest stimulus rate of 0.5 Hz (Fig.
5A), the response episode consisted of individual flashes separated by partial decay
of light intensity. There was a 1 : 1 stimulus-response correlation and no temporal
summation or facilitation of single flashes. In many instances luminescence was not
exhausted during the 20 s data collection period, which was equivalent to 1 1 stimuli;
therefore the measured quantum emission underestimated the total stimulable emis-
sion. The waveform and kinetics of single flashes comprising the response episode
were similar to those for single pulse stimulation; in fact it was usually possible to
resolve the fast and slow flash components (Fig. 5B).
When stimulated at 1 and 2 Hz, components of individual flash responses to each
stimulus pulse were still evident, but were temporally summated to form a single
prolonged emission with an average duration of approximately 8 s (Fig. 5C). At 1 Hz
stimulation, the average total duration of the response episode was approximately 14
s and the mean total emission was 1.1 X 10" photons episode"1 (Table II). The largest
response episode measured had a total emission of 2.9 X 10" photons episode"1.
Higher rates of stimulation (10 and 20 Hz) actually resulted in decreased emis-
498 M. I. LATZ ET AL.
TABLE II
Resp-- •• and quantum emission of response episodes of Pleuromamma xiphias stimulated with
rept till ;/ stimuli (10 V, 5 ms duration) at frequencies ranging from 0.5 to 20 Hz
Latencyf
(Hz) (ms)
Response
duration (s)
Total quantum emission
(photons episode"1)
Maximum response
(photons episode"1) n
0.5
36.0* >17.1**(2.2)ft
±18.2 ±2. 3 (±0.4)
5.2 X 10'°**
± 1.8 X 10'°
1.6X10"** 9
1
12.2
±3.0
>13.8**
±2.5
11. OX 10'°**
±4.9x 10'°
2.9X10"** 6
2
17.1
±5.4
8.1
± 1.4
7.2 X 10'°
± 1.4 X 10'°
2.5X10" 11
5
21.7
±6.6
8.8
± 1.8
8.0 X 10'°
±2.1 X 10'°
2.0X10" 12
10
18.8
±4.3
7.5
± 1.3
5.2 x 10'°
± 1.1 X 10'°
1.0X10" 10
20
33.2
±8.4
5.4
± 1.3
4.8 x 10'°
± 1.3 X 10'°
1.0 x 10" 10
* Values represent mean ± standard error of the mean.
** The reported value underestimates the actual value since some responses persisted beyond the 20 s
data collection period.
f Stimulus-response latencies for different stimulus frequencies were not significantly different from
one another (Mest, P> 0.05).
ft Measurements from single flashes comprising the entire response episode.
sion; average duration of the response episode was less than 6 s with a quantum
emission averaging 5 X 10'° photons episode'1 (Table II). Summation was so com-
plete that individual flashes comprising the response episode were not recognizable
(Fig. 5D). In fact, the resultant waveform was similar to the flash response for single
pulse stimulation (Fig. 2A), although of longer duration and with greater quantum
emission.
Since stimulus-response latencies were unaffected by stimulus magnitude and fre-
quency, the values for all experiments were pooled (Fig. 6). The most common la-
tency values occurred between 5 and 20 ms with a median latency of 16 ms (n
: 152). This is similar to latencies of 7 to 9 ms reported for the Metridiidae (David
and Conover, 1961; Clarke et al, 1962). Minimum flash latencies of 4 to 18 ms have
been reported for other species of the Metridiidae (David and Conover, 196 1 ; Clarke
et al, 1962; Barnes and Case, 1972); in the present study 6% of all specimens tested
exhibited latencies less than 5 ms. The briefest flash latency measured was 2 ms,
probably reflecting a direct electrical effect on the light organ (Baguet, 1975; Baguet
et al., 1980) rather than one mediated through sensory or central pathways, since
synaptic delay alone accounts for approximately 1 ms (Katz and Miledi, 1965) in
chemically transmitting crustacean neuromuscular synapses.
Observations suggested that subsequent flashes from a specimen exhibited differ-
ent flash kinetics and quantum emission from those of first flashes. This was tested
by subjecting 5 specimens to a second 10 V, 5 ms duration pulse approximately 4
minutes following an original stimulus pulse of the same magnitude (Table III). While
statistically not significantly different (Mest, P > 0.05), all specimens exhibited a sec-
ond flash response that occurred after a longer latency, had a longer rise time, and
COPEPOD BIOLUMINESCENCE
499
o
uj
£
32 -i
28 -
24
20
16
12 -
•l.ll I .i ... .
1 — ' — i — '""I""' — i — ' — i — ' — i — ^i
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180190200
LATENCY (ms)
FIGURE 6. Frequency histogram of the distribution of stimulus-response latencies for 1 52 specimens
of Pleuromamma xiphias. The statistical mode of the distribution was 16 ms.
was of shorter duration than the first flash. In addition there was an average 35%
reduction in quantum emission for the second flash.
DISCUSSION
The most striking feature of the bioluminescence of freshly collected specimens
of the copepod, Pleuromamma xiphias, was the variability in spectral and temporal
characteristics and quantum emission of the flash response. Such variability has been
TABLE III
Comparison of response kinetics of flashes of Pleuromamma xiphias between initial single electrical pulse
(10 V, 5 ms duration) and an identical stimulus delivered 4 min later
Total rise time
Latency (ms) (ms)
50% decay time Total flash duration
(s) (s)
Total quantum emission
( photons flash ')
A. First stimulus
9.0*
103.1
1.4
3.0
1.6 X 10'°
±2.4
±24.2
±0.6
±0.6
±0.7X 10'°
B. Second stimulus**
21.8
212.5
0.3
2.0
0.6 X 10'°
±5.1
±75.6
±0.2
±0.6
±0.4X 10'°
* Values represent mean ± standard error of the mean for 5 specimens.
** While mean values for flash kinetics and total quantum emission were not significantly different
from those of the first flash response (Mest, P > 0.05), for each specimen the second response always had a
longer latency, slower rise time, faster decay time, shorter flash duration, and diminished flash quantum
emission (Sign test; Zar, 1974).
500 M I- LATZ ET AL
noted but not investigated in previous studies of copepod flashing (David and Con-
over, 1961; Clarke et al, 1962; Barnes and Case, 1972; Lapota and Losee, 1984).
Emission spectra
The spectral distributions of Pleuromamma xiphias bioluminescence were either
birnodal or unimodal. Herring (1983) measured bimodal spectra in P. borealis, but
the luminescence for other copepod genera which have been studied exhibits uni-
modal distributions (David and Conover, 1961; Herring, 1983; Widder et al., 1983).
P. xiphias contains 11 luminescent glands (Giesbrecht, 1895; Clarke et al., 1962)
of unknown variability in spectral emissions. Since variability in the recruitment of
luminescent glands is well known for copepods (David and Conover, 1961; Barnes
and Case, 1972), there may be a spatial origin to the variability in the emission spec-
tra. Observation of the sites of light emission during spectral measurements is neces-
sary to determine whether luminescence from different body regions has different
spectral properties.
Flash kinetics
Luminescence by calanoid copepods is believed to occur by the expulsion of the
contents of paired sacs comprising the luminescent gland through a pore and subse-
quent mixing outside the body (David and Conover, 196 1 ; Clarke et al, 1 962; Barnes
and Case, 1972; Herring, 1985). The activation/expulsion process presumably in-
volves at least three steps: ( 1 ) activation within the photocytes, (2) transport through
internal channels, culminating in (3) expulsion through pores to outside the body.
In the present study the flash of P. xiphias was resolved into two components.
The fast component of the flash appears to involve steps ( 1 ) and (2) above, with little
or no expulsion from the body. Two glands are responsible for light emission: the
caudal organ located on the posterior tip of the abdomen, and one or more of several
thoracic light organs. The slow flash component consists of all three steps, with con-
siderable spewing of luminescent material from the posterior light organ into the
external environment. Although both components exhibit first order exponential de-
cay, the different decay rates also suggest that the two components are separate events
involving different emission mechanisms.
The wide range of values measured for the rise time of P. xiphias flashes (mean
170 ms, range 15 to 535 ms) reflect the variability known for copepods. Previous
measurements for the Metridiidae range from 30 to 900 ms (Clarke et al., 1962;
Barnes and Case 1972; Lapota and Losee, 1984). The variability of these measure-
ments may result from the presence of a rapid, neurally triggered response coupled
with a mechanism with an inherently slower temporal element, such as glandular
mixing and expulsion.
Flash durations for Pleuromamma xiphias were stimulus-dependent and ex-
tended over 2 orders of magnitude, ranging from 1 00 ms to greater than 20 s. Previous
measurements of flash durations for Pleuromamma species range from 200 ms to
over 6 s (Clarke et al., 1962; Yevstigneyev, 1985), and 100 ms to 1 1 s for other cope-
pods (Barnes and Case, 1972; Lapota and Losee, 1984). In this study the maximum
period over which luminescence could be expressed was not investigated; however,
the copepod Gaussia is able to respond to single electrical pulses delivered at 0. 1 Hz
for more than 3 min (Barnes and Case, 1972).
Quantum emission
Considering the abundance of luminescent plankton, there are few measurements
available of the quantum emission of individual flashes, in part due to the difficulty
COPEPOD BIOLUMINESCENCE
501
TABLE IV
Total quantum emission of single flashes from planktonic organisms
Organism
Mode of
stimulation
Mean total quantum
emission (photons flash"')
Reference
Protozoa
Colonial radiolaria
Dinophyta
Nocliluca miliaris
Ceratium horridum
Ceratium breve
Pyrocystis noctiluca
Pyrocystisfusiformis
Gonyaulax polyedra
Crustacea
Euphausiacea
Euphausia eximia calyptopis I
Nyctiphanes simplex furcilia I
A', simplex furcilia III
Copepoda
Pleiiromamma \iphias
Corycaeus latus
Centropages furcatus
Corycaeus speciousus
Paracalanus indicus
Ostracoda
Conchoecia secernenda
Mechanical
Electrical
Vacuum*
Vacuum
Mechanical
Mechanical
Spontaneous
Vacuum
Vacuum
Vacuum
Single electrical
Repetitive elect.
Vacuum
Vacuum
Vacuum
Vacuum
Electrical
1 X 109
2X 109
4X 107
1 X 10"
7X 109
2X 10'° first flash
0.1 X 10'° subsequent flash
1 X 107
1 X 10'°
6X 10'°
1 X 10"
1 X 10'°
9X 10'°
1 X 108
8X 107
5X 107
3X 107
3X 10'°
Lalzetal.. 1987
Eckert, 1967
Lapota and Losee, 1 984
Lapota and Losee, 1 984
Latz and Case, unpub.
Latz and Case, unpub.
Latz and Case, unpub.
Lapota and Losee, 1 984
Lapota and Losee, 1984
Lapota and Losee, 1 984
Present study
Lapota and Losee, 1984
Lapota and Losee, 1984
Lapota and Losee, 1 984
Lapota and Losee, 1 984
Latz, Frank, and Case,
unpubl.
* Flashes were induced by removing water from chamber, stranding organisms on filter paper.
in making these measurements. Electrical stimulation allows for precise control of
stimulus parameters such as pulse strength, duration, and frequency, and the results
of the present study have demonstrated that changes in these parameters greatly in-
fluence the resulting quantum emission of the luminescent responses. Quiescent
specimens of P. xiphias were extremely sensitive to handling; it was not uncommon
for some bioluminescence to be triggered during handling. Therefore, the present
measurements of flash output are conservative estimates of luminescence capacity.
Light emission by planktonic organisms for which data are available (Table IV)
ranges from 1 X 107 photons flash"1 for the dinoflagellate Gonyaulax (Latz and Case,
unpub.) to approximately 1 X 10" photons flash"1 for larval euphausiids (Lapota
and Losee, 1984). The quantum emission of luminescence by P. xiphias is situated
at the upper portion of this range, and is 1.5-2.5 orders of magnitude greater than
that reported for smaller copepods by Lapota and Losee (1984), although these
differences may also reflect differing excitational and recording methods as well as
species differences.
The present data on the total quantum emission of Pleiiromamma xiphias lumi-
nescence reflect the variability in responses previously observed in the Metridiidae
(David and Conover, 1961; Clarke et a/., 1962; Barnes and Case, 1972). The maxi-
mum total quantum emission measured following electrical stimulation was equiva-
lent to the bioluminescence potential, or total luminescent capacity of P. xiphias,
which, based on measurements of total mechanically stimulated bioluminescence, is
estimated to be approximately 1 X 10" photons (Latz and Case, unpub.). Therefore
502 M. I. LATZ ET AL.
a single f;a^. represented approximately 4-15% of total possible light emission. Thus,
p X' us well as other copepods, is capable of emitting numerous flashes before
responi ,:• faiiure occurs (Clarke et al, 1962; Barnes and Case, 1972; present study).
In the present study, electrical stimulation at 2 Hz effectively evoked the total lumi-
nest :;-u! capacity, while other stimulus frequencies generated flash episodes which
utilized 50% or more of total luminescent capacity.
Implications of flash variability
Previous studies of copepod luminescence have noted the "irregular" shapes of
the flash responses (Clarke et al., 1962; Barnes and Case, 1972). Generally, response
episodes to repetitive stimulation exhibited more complex and variable waveforms
than flashes induced by single stimuli. This trend was also true for P. xiphias. For this
organism, stimulus frequency was a predictable source of variability of the response
waveform. However, the general pattern was the higher the stimulus frequency, the
simpler the flash waveform, due in part to different degrees of temporal summation of
individual flashes comprising the response episode. There was no standard response
waveform for all stimulus protocols.
Such a large capacity for luminescence, plus the variability in flash waveform and
quantum emission, suggest that copepods are not limited to a single type of flash but
instead may exhibit a versatility of luminescent behaviors. Behavioral versatility in
zooplankton-secreted luminescence is in fact known for coastal ostracods, for which
three types of behaviorally significant luminescent displays have been described
(Morin and Bermingham, 1980; Morin, 1986). These displays involve not only con-
trol of the interval between flashes but also of flash duration, the latter through mech-
anisms that may involve differences in the chemical composition of the luminescent
secretion. In some species individual ostrocods produce dozens of flashes during the
signaling sequence. Evidence for behavioral versatility of luminescent displays also
exists for squid (Young et al, 1982), fish (McFall-Ngai and Dunlap, 1983), and for
counter-illuminating organisms (reviewed by Young, 1983).
There is evidence for multiple modes of copepod luminescence. Metridia gener-
ates two types of flashes during feeding experiments with euphausiids as predators
(David and Conover, 1961). Single bright flashes may be associated with an escape
response, while multiple flashes over a 30-s interval may be associated with successful
predation.
In terms of the present measurements of the physical characteristics of the flash
of Pleuromamma xiphias, variability in the emission spectra, flash kinetics and flash
quantum emission most likely result from a combination of several factors: spatial
origin of emission from the body, temporal summation and complex neural processes
(as determined by stimulus strength and frequency), previous excitation history, col-
lection and handling artifacts, and general physiological state of the organism. Until
additional experiments can correlate flash patterns with specific behaviors, the vari-
ability in bioluminescence of P. .xiphias only suggests the physiological mechanisms
responsible for possible differences in luminescent displays.
ACKNOWLEDGMENTS
We thank E. Swift, E. Buskey, C. Mann, J. Dugas, and the captains and crews of
the RV Knorr and RV Endeavor for assistance with organism collection. We are
grateful to R. Bidigare for technical assistance at all hours, and S. Bernstein for com-
puter applications. This work was supported by the Office of Naval Research (con-
tract numbers N00014-84-K-0242 to J.F.C. and N00014-84-C-0132 II A to John
Marra). Biowatt contribution #06.
COPEPOD BIOLUMINESCENCE 503
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Reference: Biol. Bull. 173: 504-512. (December, 1987)
QUA 4TITATIVE ESTIMATION OF MOVEMENT OF AN AMINO ACID
3M HOST TO CHLORELLA SYMBIONTS IN GREEN HYDRA
P. J. McAULEY
Department of Plant Sciences, Oxford University, Agricultural Science Building, Parks Road,
Oxford OX 1 3PF. United Kingdom
ABSTRACT
Washing symbiotic Chlorella algae freshly isolated from green hydra with 0.05%
sodium dodecyl sulphate was shown to remove virtually all contaminating host mate-
rial, previously a severe constraint in quantifying movement of metabolites from host
to symbionts. When brine shrimp labelled with 3H-leucine were fed to hydra in sym-
biosis either with the native strain of Chlorella (E/E hydra) or two strains cultured
from Paramecium bursaria (E/3N and E/NC hydra), it was found that after 24 h 3-
4% of the total radioactivity retained by the symbiosis was present in the algae. Analy-
sis of the free amino acid pool of symbiotic algae from E/E hydra showed that over
70% of the radioactivity was associated with leucine, and significant amounts of ra-
dioactivity were retained by these algae for at least five days following a single feeding
with radioactive brine shrimp. In both E/E and E/NC hydra, the amount of radioac-
tivity per unit protein was considerably less in the symbionts than in the host, suggest-
ing that access to host amino acid pools were limited. These results are discussed in
terms of the possible role and regulatory significance of amino acids as a nitrogen
source to symbiotic Chlorella, and of the cost to the host in maintaining the sym-
biosis.
INTRODUCTION
While it is well known that Chlorella algae symbiotic with green hydra release
photosynthetically fixed carbon that is used by their hosts (Cernichiari et al, 1969;
Mews, 1980; Mews and Smith, 1982), it has become apparent only recently that
movement of metabolites in the opposite direction may also be important in main-
taining the symbiosis. In hydra grown in darkness, host supply of metabolites, which
from indirect evidence may include glucose (McAuley, 1986a), is necessary to main-
tain a reduced population of symbionts. In light, cell division of the algae appears to
be dependent upon a 'factor' present in host food, possibly one or more amino acids
(McAuley, 1985, 1986b).
Amino acids may also have an important role in nitrogen supply to the symbionts.
Although it was long believed that algae symbiotic with green hydra used ammonium
produced by host catabolic processes, as in symbioses between marine invertebrates
and zooxanthellae (Muscatine, 1980; Wilkerson and Muscatine, 1984; Summons et
al., 1986; Anderson and Bums, 1987), recent research has shown that high levels
of host glutamine synthetase and low perialgal vacuolar pH may prevent uptake of
ammonium by algae in green hydra (Rees, 1986). Since the symbiotic algae also lack
nitrate reductase and nitrite reductase (D. McKinney and H. LenhofT, University of
California, Irvine, pers. comm.), it has been suggested that host supply of amino acids
Received 17 July 1987; accepted 30 September 1987.
504
HETEROTROPHY BY CHLORELLA IN SYMBIOSIS 505
may form the primary source of nitrogen for algae symbiotic with green hydra
(McAuley, 1986b, 1987;Rees, 1986).
Direct evidence for movement of metabolites from host to symbiotic algae has
been difficult to obtain. Logically, it could be studied by supplying food containing
radioactively labelled metabolites, then isolating algae from the symbiosis and assess-
ing uptake of radioactivity. Although this approach has been used (Cook, 1 972; Thor-
ington and Margulis, 198 1 ), the interpretation of the results is now in doubt because
it was subsequently realized that algae isolated by centrifugation of homogenates of
green hydra are heavily contaminated with host material, despite washing the algal
pellet in several changes of distilled water or culture medium (Cook, 1983; Douglas
and Smith, 1983; McAuley, 1986c).
Recently, it was demonstrated that complete removal of contaminating host ma-
terial could be achieved by washing the algal pellet with a dilute solution (0.05%,
w:v) of the surfactant sodium dodecyl sulphate (SDS) (McAuley, 1986c). Meints and
Pardy (1980) earlier used SDS to isolate algae from green hydra, but at a higher con-
centration which was subsequently found to affect the viability of the algae. However,
neither the viability, nor the photosynthetic capacity, nor the ability to sequester and
retain amino acids were affected after washing with 0.05% SDS (McAuley, 1986c;
Douglas, in press). In this paper the SDS-washing technique was used to separate
algae from host material in homogenates of hydra which had been supplied with food
labelled with 3H-leucine. Evidence for the transfer of leucine from host food to the
symbiotic algae is described and discussed.
MATERIALS AND METHODS
Maintenance of organisms
Green and aposymbiotic hydra of the European strain of Hydra viridissima PAL-
LAS were cultured in M solution (Muscatine and Lenhoff, 1965) at 18°C in continu-
ous light (60 juEinsteins m~2 s"1). Cultures were fed each Monday, Wednesday, and
Friday with freshly hatched nauplii of the brine shrimp Anemia salina (Lenhoff and
Brown, 1970). Green hydra were symbiotic with either the native strain ofChlorella
algae (E/E hydra) or were derived from aposymbionts artificially reinfected with
NC64A (E/NC hydra) or 3N813A (E/3N hydra) strains ofChlorella originally iso-
lated from Paramecium bursaria (Muscatine et al, 1 967; Weis, 1 978). All experimen-
tal hydra had been fed 72 h prior to use and each bore a single bud. Cultured 3N8 1 3A
algae were grown as previously described (McAuley, 1986a).
Radioactive labelling of brine shrimp
Freshly hatched nauplii of Artemia salina were incubated at a density of 30
nauplii ml"1 in artificial seawater (Tropic Marin: Dr. Seiner Aquarientechnik, War-
tenberg, W. Germany) containing 0.5 ^Ci ml"1 L-[4,5-3H] leucine (Amersham Inter-
national pic, England). After 24 h in darkness at 25°C (normal hatching conditions),
Artemia were washed with M solution and added to dishes containing hydra (two
shrimp per hydra). Determination of radioactivity in homogenates of Artemia
showed that on average each contained 7260.7 ± 390.3 dpm (mean ± S.E. of five
separate determinations). Almost 80% of the label was associated with TCA-ethanol
insoluble material, suggesting that most of the leucine had been incorporated into
protein.
506 P- J- McAULEY
Separation of radioactivity in algal and animal fractions of hydra
Twenty-four hours after being fed with 3H-leucine labelled Anemia, hydra were
washed in M solution, homogenized, and the volume made up to 1.5 ml. Three 100-
H\ aiiquots of homogenate were immediately counted (total radioactivity), a further
three were taken to determine total protein content, and numbers of algae were deter-
mined by counting a diluted aliquot using a hemocytometer. The algae in the rest of
the homogenate were washed with 0.05% (w:v) SDS and distilled water (McAuley,
1986c), slightly modifying the technique to take account of the small volumes used.
The washed algal pellet was finally resuspended in 0.45 ml distilled water. Three 100-
fj\ aiiquots were counted for radioactivity and a further 100 /A was diluted and num-
bers of algae determined so that dpm per SDS-washed algal cell could be calculated.
Radioactivity was measured by making aiiquots up to 1 ml with distilled water in a
plastic scintillation vial, adding 8 ml scintillation fluid (1000 ml toluene, 500 ml Tri-
ton X-100, 6 g 2-5-diphenyloxazole), and counting using a Beckman LSI 801 scintil-
lation counter. Disintegrations per minute were calculated using the H number
method.
Protein content of homogenates and SDS washed algae
Samples were freeze-thawed, extracted with an equal volume of 0.4 A/NaOH for
1 h, and protein content determined as previously described (McAuley, 1986c).
Thin layer chromatography
Ethanolic extracts of SDS-washed algae were spotted on a TLC plate together with
a small volume containing unlabelled authentic amino acids. Chromatograms were
run as previously described (McAuley, 1987). Amino acids were identified by spray-
ing with ninhydrin; spots were scraped off and suspended in scintillation fluid (1000
ml toluene, 5 g 2-5-diphenyloxazole, 0.3 g (l,4-bis[5-phenyl-2-oxazolyl]-benzene;
2,2'-p-phenylene-bis[5-phenyloxazole), and radioactivity counted.
RESULTS
Effectiveness ofSDS-washing in removal of animal contamination
To test if SDS-washing removed significantly more animal contamination from
the algal pellet than simply washing with distilled water, E/E hydra were fed 3H-
leucine labelled Anemia, homogenized after 24 h, and the algae in the homogenate
washed either with distilled water (controls) or 0.05% SDS. The algal pellets washed
with distilled water contained almost nine times the radioactivity of those washed
with SDS, while comparison of the amount of label per algal cell (as recovery of algae
is lower after SDS-washing) showed that controls appeared to contain six times as
much radioactivity as algae washed with SDS (Table I). To test if SDS washing re-
moved only radioactivity associated with contaminating host material, and not radio-
activity already sequestered by the algae, cultured 3N8 1 3 A algae ( 107 cells per ml in
20 mM phosphate buffer, pH 6.3) were incubated in 0. 1 mM 3H-leucine (5 mCi per
mmol) for 1 h, diluted with ice cold buffer to stop uptake, and then washed either
with SDS and distilled water, or with distilled water only. Aiiquots were filtered onto
glass fiber disks and radioactivity was determined as previously described (McAuley,
1986a). Radioactivity per cell in algae washed with either SDS or distilled water was
compared to that in algae filtered immediately at the end of the incubation period
(controls). Paired /-tests of four replicate experiments showed no significant differ-
HETEROTROPHY BY CHLORELLA IN SYMBIOSIS 507
TABLE I
Comparison of apparent uptake of 3H-label by algae isolated from hydra fed with 3H-leucine labelled
shrimp by conventional or SDS-washing techniques
dpm/ 1 00 M! no algae x 1 05/
algal pellet 100 /zl pellet dpm/algal cell
Wash
Distilled water 9537.6 ± 1390.1 7.241+0.895 0.01318 ± 0.00142
SDS 830.3 ± 68.6 3.7 16 ±0.451 0.00223 ± 0.00032
Eighty hydra were homogenized 24 h after being fed with 3H-leucine labelled Anemia and aliquots of
the homogenate were washed with distilled water or SDS and numbers of algae and radioactivity deter-
mined as described in Materials and Methods. Figures are means ± S.E. of four independent experiments.
ence (P > 0.10) in radioactivity retained by controls (0.01050 ± 0.00099 dpm per
cell) compared to radioactivity retained after washing with either SDS (0.00993
± 0.00096 dpm per cell) or distilled water (0.0 1 0 1 4 ± 0.00 1172 dpm per cell). There-
fore, it was concluded that loss of radioactivity from algae during SDS washing was
insignificant.
Further experiments determined whether all label associated with contaminating
host material could be removed from the algal pellet by SDS-washing. Aposymbiotic
hydra were homogenized 24 h after having been fed with 3H-leucine labelled Anemia,
and appropriate numbers of unlabelled cultured 3N813A algae were suspended in
the homogenate and washed either with distilled water or with SDS. Counts showed
that over 10% of radioactivity added to the algae remained after washing with distilled
water, but less than 0.2% remained after washing with SDS (Table II). The results in
Tables I and II suggest that if algae are washed only with distilled water, 80% of the
radioactivity apparently associated with them is due to animal contamination. This
is almost entirely removed by washing with SDS. A similarly high level of contamina-
tion was measured in zooxanthellae isolated from tentacles of specimens of the sea
anemone Aiptasia pulchella which had been fed Artemia labelled with 35S-methio-
nine(Steen, 1986a).
TABLE II
Effect of SDS washing on removal of contaminating aposymbiotic hydra homogenate
from unlabelled cultured 3N813A cells
Experiment 1
Experiment 2
dpm %
dpm
%
Label added/ 100 n\ algae
19821.9 ± 125.8 100.00
22562.2 ±235.2
100.00
recovered
Label/ 1 00 ^1 algae washed
2127.8 ± 40.7 10.74
3019.6 ± 36.8
13.38
with distilled water
Label/ 100 ^1 algae washed
8.6 ± 0.5 0.04
33.7+ 2.6
0.15
with 0.05% SDS
Appropriate numbers of unlabelled cultured 3N8 1 3 A cells were suspended in homogenate of aposym-
biotic hydra which had been fed with 3H-leucine labelled Artemia 24 h previously. Radioactivity was
determined before and after washing algal cells with distilled water or 0.05% SDS. Figures are means
+ S.E. of four replicate aliquots.
508 P- J- MCAULEY
TABLE III
Partiti ' •" 1-labL-i in green hydra
Total dpm/ 100 M!
homogenate
dpm/algal cell
dpm/total
algae in 100/xl
% label
in algae
Symbiosis
E/E
34803.3
+
3575.9
0.003493
+ 0.00030
1191
.4 +
141
.5
3.4
E/3N
30574.3
+
2820.7
0.005592
± 0.000564
1304
,2±
178
.7
4.3
E/NC
24263.2
+
2694.9
0.002220
±0.000193
694
0 +
53
.7
2.9
Forty hydra were homogenized in 1.5 ml 24 h after having been fed 3H-leucine labelled Anemia, and
label in animal and algal fractions determined as described in Materials and Methods. Figures are means
± S.E. of four independent experiments.
Partition of 3H-label supplied to green hydra by holozoic feeding
To measure movement of amino acid from host food to symbiotic algae, green
hydra were fractionated (see procedure in Materials and Methods) 24 h after being
fed with 3H-leucine labelled A rtemia. Transfer of 3H-label to symbiotic algae occurred
both in the normal (E/E) symbiosis and in artificial (E/3N and E/NC) symbioses,
amounting to 3-4% of the total radioactivity retained by the hydra (Table III). In one
experiment, amino acids were extracted in 80% ethanol from algae isolated from
E/E hydra which had been fed 3H-leucine labelled shrimp 24 h previously. Extract
was co-chromatographed with authentic markers, and ninhydrin positive spots were
scraped off and radioactivity determined. About 10% of identified 3H-label in the
ethanol-soluble pool was arginine, about 7% alanine, 3-5% each of aspartate, gluta-
mine, and glutamate, and over 70% leucine. These results suggest that symbiotic algae
were able to take up amino acids supplied to green hydra via holozoic feeding.
Further comparisons were made on the basis of specific activities per unit animal
and algal protein in E/E and E/NC hydra. The total amount of protein in samples
was determined directly, while the amount in algal cells was calculated from separate
determinations of algal cell protein content and estimation of numbers of algae per
sample. Calculations showed that in both symbioses, algal cells contained less radio-
activity per unit protein than hydra tissue (Table IV). The amount of radioactivity
per unit protein in E/E algae was about 24%, and in E/NC algae 18%, that per unit
TABLE IV
Partition of 3H-label in green hydra according to protein content of animal and algal fractions
Protein/algal
cell (pg)
Protein in algae/
1 00 n\ homogenate dprn/^g Protein in animal/ dpm//xg
(fig) algal protein 1 00 n\ homogenate animal protein
Symbiosis
E/E 13.08 ±0.1 4
E/NC 10.85 ±0.43
4.44 + 0.33 268.6 ±23.0 3 1.61 ±2.66 1109.8 ±202.7
3.44 + 0.33 204.6±17.8 21.99±2.49 1145.7±229.5
Protein content of algal cells measured in three (E/NC) or four (E/E) separate determinations in 2-4
replicate samples containing 5 X 106 SDS-washed algae. Protein content of animal and algal fractions
determined from measurements of total protein/ 1 00 n\ homogenates (from experiments described in Table
HI), number of algae/ 1 00 ^1 and protein content of algal cells. Figures are means ± S.E. of four independent
exneriments.
experiments.
HETEROTROPHY BY CHLORELLA IN SYMBIOSIS
509
protein of hydra tissue. This suggested that availability to the algae of amino acid
derived from holozoic feeding was restricted, either because of some host mechanism
directly controlling supply, or because a large proportion was immediately seques-
tered by host cells upon entry.
Retention of 3H-label by symbiotic algae
The amount of radioactivity retained by symbiotic algae was measured for five
days subsequent to feeding green hydra with 3H-leucine labelled Anemia. At 24-h
intervals, 40 hydra were homogenized, total dpm in aliquots of homogenate and per
SDS-washed alga were determined, and the percentage of the total dpm found in the
algae was calculated as described in Materials and Methods. Net uptake by the algae
of radioactively labelled amino acid was found to continue up to 48 h after feeding
(Fig. 1), but thereafter dpm per algal cell slowly declined. In contrast, the percentage
of the total label retained by the symbiosis which was found in the algal population
continued to increase over the period of measurement. This difference may be due
to either of two reasons. First, uptake of amino acid by the algae could have been
diluted by algal cell division; although algal mitosis reaches a maximum about 12 h
after host feeding, it continues, although at a declining rate, for several days thereafter
(McAuley, 1 982). Second, increase in the proportion of label retained by algae despite
a fall in dpm per algal cell may be a result of differences in the ability of algal cells
and animal tissue to retain radioactively labelled amino acid. Possibly, the rate of
protein catabolism was faster in host cells than in symbiotic algae during host
starvation.
DISCUSSION
Contamination of the algal pellet with host material has previously been a severe
constraint on determining how radioactive metabolites are partitioned between the
o
5*4
o
tsi
O
I2
0
O
O»
4 O
4
o
2 o
0
Time after feeding (days)
FIGURE 1. Retention of 3H-label by symbiotic algae in E/E hydra fed 3H-leucine labelled Anemia.
Hydra were fed radioactive brine Anemia as described in Materials and Methods. At daily intervals 40
hydra were homogenized and dpm per algal cell (• — - •) and % total 3H-labeI retained by the symbiosis
found in algal cells (O O) were calculated. Each point is the mean ± S.E. of three independent experi-
ments.
510 P- J- MCAULEY
host and < nonts in algal/invertebrate symbioses. Although conventional wash-
ing by repealed centrifugation and resuspension in fresh medium will remove up to
90C cmtaminating host material from algae isolated from green hydra (Cook,
1972; " ouglas and Smith, 1983), the remaining 10% may contain several times the
radioactivity actually sequestered by the algae. Contamination represents at least the
same amount of protein as contained by algal cells (Douglas and Smith, 1983;
McAuley, 1986a), and results described here showed that host material contained
ihree to four times as much radioactivity per unit protein as did the algae. Therefore,
unless contamination is accurately quantified or completely removed, uptake of ra-
dioactivity by symbiotic algae in vivo is liable to be severely overestimated.
In our experiments, SDS-washing was shown to remove from the algal pellet all
but a fraction of 1% of radioactive label associated with host contamination. This
enabled accurate measurement of the partition of label between host and symbiotic
algae in hydra that had been fed with 3H-leucine labelled Anemia salina nauplii. In
both the natural symbiosis (E/E) and in artificial symbioses with Paramecium algae
(E/NC, E/3N), radioactivity moved into the algae after hydra were fed with 3H-leu-
cine labelled Anemia, and algae in E/E hydra were found to retain significant
amounts of radioactivity for at least five days after feeding.
Although the algae constitute about 1 5% of the total protein content of the symbi-
osis, only 3-4% of radioactivity retained by green hydra was found in the algae after
24 h, rising to just over 5% after 120 h. This suggests that access by the algae to amino
acids derived from host digestion of food may be restricted. Since amino acids must
pass through the host-derived vacuolar membrane which surrounds each alga, it is
possible that the host cell can directly control the rate of amino acid supply (McAuley,
1986b). Carroll and Blanquet (1984) found that a low molecular weight factor iso-
lated from host tissue inhibited uptake of alanine by zooxanthellae symbiotic with
the jellyfish Cassiopea xamachana. Alternatively, differences in specific labelling of
algal and animal protein may be due to competition between uptake by the algae and
host utilization of amino acid for protein synthesis. That algae contain less label than
expected also may be due to recycling to the host of a proportion of sequestered
amino acid.
Demonstration of 'reverse translocation' suggests that maintenance of the green
hydra symbiosis depends on flow of metabolites in both directions. Not only is mal-
tose exported from the algae and used by the host (Mews, 1980; Mews and Smith,
1982), but amino acids and possibly other metabolites (McAuley, 1986a) derived
from host feeding are transported into the perialgal vacuole and used by the symbiotic
algae. The significance of reverse translocation is two-fold.
First, as suggested by Douglas and Smith (1983), supply of metabolites to the algae
may impose a net 'cost' upon the host in certain circumstances. When hydra are
grown in continuous darkness, where maltose release is reduced and the algae must
depend upon host-derived metabolites for growth, those with a population of algae
grow more slowly than aposymbionts artificially rid of their algae (Douglas and
Smith, 1983). A similar net cost has been found in the sea anemone Aiptasia pul-
chella, whose symbiotic zooxanthellae sequester radioactivity when hosts are given
35S-methionine labelled food (Steen 1986a). Symbiotic anemones starved in darkness
showed a significant decline in the adenylate ratio of ATP: (ATP + ADP) compared
to aposymbionts, but in light the adenylate ratio declined at the same rate in starved
symbiotic and starved aposymbiotic anemones (Steen, 1986b).
Second, there is now persuasive evidence that uptake of ammonium by algae in
symbiosis with green hydra is prevented by low perialgal vacuolar pH and high levels
of host glutamine synthetase (Rees, 1986). Furthermore, measurements of amino
HETEROTROPHY BY CHLORELLA IN SYMBIOSIS 5 1 1
acid pool size and of uptake and metabolism of amino acids indicate that symbiotic
algae may be nitrogen-limited (McAuley, 1986b, 1987). Experiments described here,
demonstrating movement of 3H-leucine from host food to symbiotic algae, support
the suggestion that in green hydra amino acids may form the principal supply of
nitrogen in symbiosis (McAuley, 1986b, 1987; Rees, 1986). This differs from symbio-
ses between marine invertebrates and zooxanthellae, in which nitrogen is supplied to
the symbionts as ammonium (Muscatine, 1980; Wilkerson and Muscatine, 1984;
Summons el ai, 1 986; Anderson and Burris, 1 987). These experiments also suggested
that in vivo uptake of 3H-leucine by symbiotic algae may be restricted. Host control
of supply of nitrogen, or of one or more specific amino acids, may be the mechanism
whereby cell growth of symbiotic algae is reduced, and cell division is inhibited except
at host cell division (McAuley, 1981, 1985, 1986d).
ACKNOWLEDGMENTS
I thank Professor Sir David Smith, Biological Secretary, Royal Society for suggest-
ing improvements to a draft of this paper. This work was supported by the Science
and Engineering Research Council.
LITERATURE CITED
ANDERSON, S. L., AND J. E. BURRIS. 1987. Role of glutamine synthetase in ammonia assimilation by
symbiotic marine dinoflagellates (zooxanthellae). Mar. Biol. 94: 45 1-458.
CARROLL, S., AND R. S. BLANQUET. 1984. Alanine uptake by isolated zooxanthellae of the mangrove
jellyfish Cassiopea xamachana. II. Inhibition by host homogenate fraction. Biol. Bull. 166: 419-
426.
CERNICHIARI, E., L. MUSCATINE, AND D. C. SMITH. 1969. Maltose excretion by the symbiotic algae of
Hydra viridis. Proc. R. Soc. Land. B 173: 557-576.
COOK, C. B. 1972. Benefit to symbiotic zoochlorellae from feeding by green hydra. Biol. Bull. 142: 236-
242.
COOK, C. B. 1983. Metabolic interchange in algae-invertebrate symbiosis. Int. Rev. Cytol. 14: 177-209.
DOUGLAS, A. E. The influence of host contamination on maltose release by symbiotic Chlorella. Limnol.
Oceanogr. (in press).
DOUGLAS, A. E., AND D. C. SMITH. 1983. The cost of symbionts to their host in green hydra. Pp. 631-
648 in Endocytobiology II, H. E. A. Schenk and W. Schwemmler, eds. Walter de Gruyter, Berlin.
LENHOFF, H., AND R. BROWN. 1970. Mass culture of hydra: an improved method and its application to
other aquatic invertebrates. Lab. Anim. 4: 139-154.
MCAULEY, P. J. 1981. Control of cell division of the intracellular Chlorella symbionts in green hydra. J.
CellSci.41: 197-206.
MCAULEY, P. J. 1982. Temporal relationships of host cell and algal mitosis in the green hydra symbiosis.
J.CellSci. 58:423-431.
McAULEY, P. J. 1985. The cell cycle of symbiotic Chlorella. I. The relationship between host feeding and
algal cell growth and division. J. Cell Sci. 77: 225-239.
MCAULEY, P. J. 1986a. Glucose uptake by symbiotic Chlorella. Planta 168: 523-529.
McAULEY, P. J. 1986b. Uptake of amino acids by cultured and freshly isolated symbiotic Chlorella. New
Phytol. 104:415-427.
MCAULEY, P. J. 1 986c. Isolation of viable uncontaminated Chlorella from green hydra. Limnol. Oceanogr.
31:222-224.
McAULEY, P. J. 1986d. The cell cycle of symbiotic Chlorella. III. Numbers of algae in green hydra digestive
cells are regulated at digestive cell division. J. Cell Sci. 85: 63-7 1 .
McAULEY, P. J. 1987. Nitrogen limitation and amino acid metabolism of Chlorella symbiotic with green
hydra. Planta 171: 532-538.
MEINTS, R. H., AND R. L. PARDY. 1980. Quantitative demonstration of cell surface involvement in a
plant-animal symbiosis: lectin inhibition of reassociation. J. Cell Sci. 43: 239-25 1 .
MEWS, L. 1980. The green hydra symbiosis. III. The biotrophic transport of carbohydrate from alga to
animal. Proc. R. Soc. Lond. B 209: 377-401.
MEWS, L., AND D. C. SMITH. 1982. The green hydra symbiosis. VI. What is the role of maltose transfer
from alga to animal? Proc. R. Soc. Lond. B 216: 397-4 1 3.
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MUSCA ' LI 980. Uptake, retention, and release of dissolved inorganic nutrients by marine algal-
invertebrate associations. Pp 229-244 in Cellular Interactions in Symbiosis and Parasitism,
ook, P. W. Pappasand E. D. Rudolph, eds. Ohio State University Press, Columbus.
MUSOA :NfE, L., AND H. M. LENHOFF. 1 965. Symbiosis of hydra and algae. I. Effects of some environmen-
; tions on growth of symbiotic and aposymbiotic hydra. Biol. Bull. 128: 415-424.
E, L., S. J. KARAKASHIAN, AND M. W. KARAKASHIAN. 1967. Soluble intracellular products of
algae symbiotic with a ciliate, sponge and a mutant hydra. Comp. Biochem. Physiol. 20: 1-12.
REES, T. A. V. 1986. The green hydra symbiosis and ammonium. I. The role of the host in ammonium
assimilation and its possible regulatory significance. Proc. R. Soc. Lond. B 229: 299-3 14.
STEEN, R. G. 1986a. Evidence for heterotrophy by zooxanthellae in symbiosis with Aiptasia pulchella.
Biol. Bull. 170: 267-268.
STEEN, R. G. 1 986b. Impact of symbiotic algae on sea anemone metabolism: analysis by in vivo 3IP nuclear
magnetic resonance spectroscopy. J. Exp. Zool. 240: 3 1 5-325.
SUMMONS, R. E., T. S. BOAG, AND C. B. OSMOND. 1986. The effect of ammonium on photosynthesis
and the pathway of ammonium assimilation in Gymnodinium microadriaticum in vitro and in
symbiosis with tridacnid clams and corals. Proc. R. Soc. Lond. B 227: 1 47- 159.
THORINGTON, G., AND L. MARGULIS. 1981. Hydra viridis: transfer of metabolites between hydra and
symbiotic algae. Biol. Bull. 160: 175-188.
WEIS, D. S. 1978. Correlation of infectivity and concanavalin A agglutinability of algae exsymbiotic from
Paramecium bursaria. J. Protozool. 25: 366-370.
WILK.ERSON, F. P., AND L. MUSCATINE. 1984. Uptake and assimilation of dissolved inorganic nitrogen by
a symbiotic sea anemone. Proc. R. Soc. Lond. B 221: 7 1-86.
Reference: Biol. Bull. 173: 513-526. (December, 1987)
NEURONAL CONTROL OF CILIARY LOCOMOTION IN A
GASTROPOD VELIGER (CALLIOSTOMA)
S. A. ARKETT, G. O. MACKIE, AND C. L. SINGLA
Department of Biology, University of Victoria, Victoria, British Columbia V8W 2Y2
ABSTRACT
Intracellular recordings from pre-oral ciliated cells of competent Calliostoma liga-
tum veligers were used to demonstrate the mechanisms of neuronal control of ciliary
locomotion. During normal ciliary beating at 5-7 Hz, the membrane potential shows
no oscillations or spiking activity. It remains at a resting potential of about —60 mV.
Depolarization from resting potential is due to excitatory input from the CNS and,
depending upon the kind of input, veligers appear to show two types of locomotory
behavior. In one type, normal ciliary beating is periodically interrupted by rapid,
velum-wide ciliary arrests. These arrests are caused by a propagated, Ca++-dependent
action potential in the pre-oral ciliated cells. The second type is characterized by
either a velum-wide or local slowing of normal ciliary beating, and appears to result
from a slow depolarization of the ciliated cell membrane. Pre-oral ciliated cells are
electrically coupled to each other. This property may ensure the synchrony of velum-
wide ciliary arrests or differential velar slowing of ciliary beating. These findings dem-
onstrate some of the mechanisms of the fine control veligers possess over their loco-
motory and feeding behavior.
INTRODUCTION
Many metazoans that use cilia to move or to produce feeding currents can control
the frequency of ciliary beating. This control is often revealed as intermittent, rapid
arrests of ciliary beating. Ciliary arrests have been correlated with chemically medi-
ated, rapid, postsynaptic depolarizations of the ciliated cell membrane (Mackie et al,
1974; Murakami and Takahashi, 1975; Mackie et al., 1976; Moss and Tamm, 1986;
Arkett, 1987). These depolarizations are rapidly conducted through gap junctions,
which connect the ciliated cells (Gilula and Satir, 1971; Mackie et al., 1974). Addi-
tionally, some animals appear to have a finer degree of control of ciliary beating. For
example, the ciliary beating frequency of lateral cilia of Mytilus gill is variable and
dependent upon dopaminergic and serotonergic CNS input (Paparo and Aiello,
1970; Paparo et al., 1975; Aiello et al., 1986). Larvae of many marine invertebrates
are also capable of modulating the beat frequency of their locomotory cilia (see review
by Chia et al., 1984), yet the mechanisms by which gradual changes in ciliary beat
frequency are generated are not well understood.
Veliger larvae use the pre-oral (locomotory) cilia of the velum to move through
the water column. Upward swimming is periodically interrupted, either spontane-
ously or upon contact with "foreign" objects, by sudden ciliary arrests whereupon the
veliger rapidly sinks (Garstang, 1 929). These ciliary arrests have long been assumed to
be under neuronal control because neuronal processes, emerging from the cerebral
ganglion, ramify across the velum to the pre-oral ciliated cells (Carter, 1926; Carter,
Received 13 May 1987; accepted 30 September 1987.
513
514 S. A. ARKETT ET AL.
1928; Wt, . ' 955; Fretter, 1967) and form chemical synapses with them (Mackie
et a!. I .trical activity recorded extracellularly and intracellularly from pre-
ora ells shows no spiking activity during normal ciliary beating, but large
dep*. .ations occur one-for-one with velum-wide ciliary arrests (Mackie et al,
6). These arrests are absent when the velum is isolated from the rest of the veliger.
Pre-oral cell action potentials appear to be Ca++-dependent since elevated Ca++
causes longer duration ciliary arrests and addition of Co++ blocks arrests (Korobtsov
and Sakharov, 1971). Serotonin has a cilio-excitatory effect (Koshtoyants et al., 1961)
and an unknown "inhibitory substance," extracted from nudibranch veligers, causes
ciliary arrest (Buznikov and Manukhin, 1962).
Some veligers are also capable of finer control of ciliary beating. This control is
usually expressed as a gradual decrease in the frequency of the beating cilia. Veligers
are thus able to slowly descend with cilia beating at "a reduced level" (Cragg, 1980).
Furthermore, there is evidence that veligers are capable of integrating sensory infor-
mation to alter locomotory and feeding behavior since the beating "vigor" and "rate
of [food] collection" by pre-oral cilia varies with the degree of satiation (Fretter and
Montgomery, 1968). During this study, we developed a new preparation, exploiting
the large, pre-oral ciliated cells of a gastropod veliger larva, to examine the mecha-
nisms by which the larval nervous system modulates ciliary beating and hence con-
trols locomotion.
We demonstrate that excitatory input from the CNS modulates the inherent beat-
ing frequency of pre-oral cilia ofCalliostoma ligatum (Gould, 1 849) veligers. We also
show that the ciliated cells are electrically coupled to each other, a property which
may ensure the velum-wide synchronization of rapid ciliary arrest. These features
endow the veliger with two distinct types of locomotory behavior.
MATERIALS AND METHODS
Larvae were raised in the laboratory. Adult Calliostoma ligatum, collected from
San Juan Island, Washington, were spawned in separate bowls, containing 20°C,
coarse-filtered seawater. Animals were placed foot up and usually spawned between
30 min to 1 hour later. Gelatinous egg strands were dissociated by repeatedly drawing
the egg mass into a Pasteur pipette. Eggs were then transferred to additional bowls at
a concentration of several hundred eggs per bowl. Approximately 2 ml of sperm from
several different males were diluted in 500 ml of filtered seawater. Eggs were fertilized
by adding about 0.5 ml of diluted sperm to each bowl. Developing veligers were kept
on a seawater table at 10-12°C. Seawater in the bowls was changed twice daily and
unfertilized eggs and moribund veligers were removed. Veligers developed rapidly
within their egg capsules and usually hatched and began swimming around the bowl
after about 5 days. We found that the electrical properties of pre-oral ciliated cells
and ciliary activity of veligers as young as 72 hours post-fertilization were nearly iden-
tical to that of older veligers, but younger veligers did not retract into their shell as
readily. For this reason, we often used these younger veligers for intracellular record-
ings after excapsulating them with sharpened tungsten needles.
Hatched or excapsulated larvae were held in position and manipulated for record-
ing by attaching a small bore suction (polyethylene tubing) electrode to the shell or
foot, or by placing the shell into a small depression in the Sylgard- (Dow Corning)
lined recording dish. The frequency of ciliary beating was monitored by holding a
larva in the beam of a low power laser (Spectra Physics Model 155; 0.95 mW, 632.8
nm). The beam was aimed at a photodiode and the beating cilia on one velar lobe
alternately bisected the beam (Fig. 1 D). Voltage changes across the photodiode were
CONTROL OF VELIGER LOCOMOTION 5 1 5
amplified by an AC-coupled preamplifier (Grass Model P-15) with the low pass filter
set for 1 s (the longest r possible). Stroboscopic measurements of changes in ciliary
beat frequency were made with a Chadwick-Helmuth Strobex. Conventional intra-
cellular recording techniques were used to record from pre-oral ciliated cells (3 M
KCl-filled glass electrodes; 20-30 M12). For dye injection, electrodes were tip-filled
with 5% Lucifer Yellow CH (Sigma) in distilled water and back-filled with 1 MLiQ2
(90-100 MO). Dye was injected with pulsed hyperpolarizing current (0.5-1.0 nA)for
up to ten minutes. Lucifer Yellow-filled velar cells were viewed live with a fluores-
cence microscope and photographed. All recordings were made in 15-1 8°C seawater
unless otherwise stated. Artificial seawater was used for experiments requiring altered
ionic content. Normal artificial seawater was composed of 430 mM NaCI, 10 mM
CaCl2 , 1 0 mM KC1, 30 mM MgCl2 , 20 mM MgSO4 , 1 0 mM Tris HC1 pH - 7.8.
Larvae were processed for electron microscopy by first anesthetizing them in a
1:1 mixture of seawater and isotonic (0.33 M) MgCl2 and then fixing in a solution
containing 2.0% TEM grade formaldehyde, 1.5% glutaraldehyde, 0.2 M sodium
phosphate (monobasic), 0.2 M sodium phosphate (dibasic) buffer, pH = 7.2 on ice.
Larvae were rinsed, post-fixed in 2% osmium tetroxide in the same buffer, dehydrated
through graded alcohols and propylene oxide, and embedded in EPON 8 12. Sections
were stained with uranyl acetate and lead citrate.
RESULTS
Ciliary beating and arrest behavior
The pre-oral cilia of C. ligatum larvae beat in laeoplectic metachronal waves (Fig.
1A, B) with a frequency of 5-7 Hz (Fig. ID). This ciliary beating propels larvae up-
ward with the velum and foot leading and the shell trailing. Stoppages of the pre-oral
cilia (ciliary arrest) occur either spontaneously, or when larvae contact the air-water
interface or other obstacles. Ciliary arrest is characterized by a synchronized stoppage
of all pre-oral cilia and varying degrees of contraction of the velar lobes. Cilia are held
in a cone-shaped tuft over the disc of the velum (Fig. 1 c) during arrest, and thus reside
at the beginning of the effective stroke. After a few seconds, the pre-oral cilia begin
to beat and the metachronal wave is re-established. Although the arrest of all cilia is
synchronized, the resumption of ciliary beating is not always uniform around the
velum. Clearly the cilia of some portions of the velum start beating before others.
Strong or repetitive stimuli may also cause a withdrawal of the velum and foot into
the shell. Recovery from this contracted position usually takes longer than recovery
from a simple ciliary arrest.
Ciliary arrests, exhibited by encapsulated or free-swimming larvae, are often
rhythmical. A sampling often different larvae, all of which were about 96 hours old
and still in egg capsules, showed 13.9 to 22. 1 ciliary arrests per minute. The interval
between arrests is usually very regular. As an example, one larva showing an arrest
frequency of 17.6 arrests/min had a mean (±SE, n = 3 1 ) period of 3.46 (0.07) seconds.
Free-swimming veligers also showed rhythmical arrests, but the rhythmicity was of-
ten interrupted by external factors such as debris, air-water interface, or other veligers.
In addition to these coordinated velum-wide arrests, we have observed a general slow-
ing of ciliary beating. The slowing in the sinking rate or upward swimming that we
have observed in free-swimming veligers appears to be due to this general slowing of
ciliary beating.
516
S. A. ARKETT ET AL.
FIGURE 1 A-D. A. Free-swimming veliger of Calliostoma ligatum showing the laeoplectic metachro-
nal waves of the pre-oral cilia. Waves move clockwise in this view. Note the outlines of individual pre-oral
ciliated cells (arrow). Scale = 100 ^m. B. 96-hour-old veliger in egg capsule showing normal ciliary beating.
C. Same larva as in B, but with cilia in the "arrested" position. Cilia are held in the characteristic conical
tuft. Note that the foot and velum are still extended, but severe stimuli may cause their withdrawal into
the shell. Scale for B and C = 100 Mm. Light micrographs were made using a Zeiss strobe flash. D. Laser
arrangement used to monitor the frequency of metachronal waves and ciliary arrest. Laser beam is aimed at
a photodiode and the veliger is positioned so that the cilia alternately bisect the beam. The large downward
deflections in the voltage record (arrows) correspond to velum-wide ciliary arrests. The small oscillations
between arrests represent the metachronal waves. L — laser.
CONTROL OF VELIGER LOCOMOTION
517
J L
FIGURE 2 A-C. Intracellular electrode recordings from pre-oral ciliated cells ofCalliostoma ligatum
veligers. A. Single electrode recording from a ciliated cell (top) with a simultaneous photodiode voltage
record (bottom) of ciliary beating and arrests. Note that each spike corresponds 1:1 to a large downward
deflection of the diode voltage record, indicating a velum-wide ciliary arrest. The third spike also caused a
velum-wide arrest, but the veliger had moved slightly to a position less than optimal for detecting ciliary
arrest. Cilia gradually begin to beat within 1 -2 s after a spike. Recommencement of ciliary beating is shown
by the small oscillations which gradually increase in amplitude until the normal metachronal wave is re-
established. B. Single oscilloscope sweep of a spontaneous, ciliated cell action potential. Note the absence
of EPSP activity on the depolarizing phase. Horizontal scale = 50 ms, vertical scale = 10 mV. C. Dual
electrode recording from opposite velar lobes, showing synchrony of action potentials. Note the distinct
shoulder on the rising phase of the smaller amplitude second spike. Horizontal scale = 200 ms, vertical
scale = 20 mV.
Physiology of pre-oral ciliated cells
Intracellular recordings from pre-oral ciliated cells show a resting potential of -60
mV. These cells exhibit no spiking activity or membrane potential oscillations during
normal ciliary beating. However, coordinated, velum-wide arrests of pre-oral cilia
correlate 1 : 1 with action potentials (Fig. 2A). These spikes have a positive phase last-
ing 40 ms, a peak amplitude of 45-50 mV, and a 10 mV hyperpolarizing undershoot
lasting 700 ms (Fig. 2B). The hyperpolarizing phase is usually absent upon initial
penetration of a cell, although spikes, lacking this phase, still cause ciliary arrest. Re-
cordings from some ciliated cells show summing excitatory postsynaptic potentials
(EPSPs) leading to an action potential, while others show no such EPSPs (Fig. 2B,
C). The absence of EPSPs in some spikes may indicate that the synaptic site(s) is far
enough away from the recording site such that EPSPs have decayed. We do not know
if every pre-oral cell is innervated, but these results would suggest that they are not.
Spontaneous spikes recorded from opposite velar lobes are well synchronized (Fig.
2C), showing about a 1 ms peak-to-peak delay. A stimulated spike propagates at
about 20 cm/s. About 1-2 s after a spike, cilia slowly begin to beat again and the
metachronal wave is re-established. However, at spiking frequencies greater than ap-
proximately 0.4 Hz, cilia remain arrested or twitching in the arrested position. Spike
bursts rarely occurred at higher frequencies, but Figure 2C shows one such example.
Note that the amplitude of the second spike in the burst is slightly reduced. The
absolute refractory period for stimulated spikes is about 200 ms.
518
S. A. ARKETT ET AL.
J
FIGURE 3 A. Intracellular electrode recording from pre-oral ciliated cells of Calliostoma ligatum
veligers. Horizontal scale = 50 ms. (1, 2, 3) 200 ms (4, 5), vertical scale = 10 mV. 1. Single oscilloscope
sweep of a spontaneous action potential in normal seawater. Note the short duration positive phase and
long duration hyperpolarizing undershoot. 2. One minute after beginning replacement of normal CaCl2
seawater with 10 mA/CoCl2 seawater. Note the loss of the hyperpolarized undershoot and the broadening
of the spontaneous action potential. Spike still caused a velum wide ciliary arrest. 3. After two minutes,
depolarization no longer caused velum-wide ciliary arrest, although localized "stutters" in ciliary beating
were visible. 4. Three minutes in 10 mA/ CoCl2 seawater, small spontaneous EPSPs are still present. 5.
Five minutes in 10 mA/CoC!2 seawater. All synaptic activity is abolished. Spikes could not be initiated by
intracellular stimulation. Upon return to normal seawater, spikes return as in 1. Same electrode penetra-
tion for 1-5. B. Spontaneous action potential of pre-oral cells in normal seawater (1) and about 5 minutes
after the addition of 5 mA/ 4-aminopyridine (2). Horizontal scale = 20 ms, vertical scale = 10 mV. C.
Action potential of pre-oral cells in normal seawater (1) and two minutes after the addition of 10 mA/
tetraethylammonium chloride. Horizontal scale = 20 ms, vertical scale = 10 mV.
Pre-oral ciliated cell action potentials are reversibly reduced in amplitude and are
eventually abolished when CaCl2 is replaced by an equimolar concentration of CoCl2
(Fig. 3A). Cilia beat continuously and action potentials could not be elicited by intra-
cellular electrode stimulation in 10 rrLMCoCl2 seawater. Increasing concentrations
of Mg++ produced a similar effect, but even at 120 mAf Mg++, a 5-10 mV depolariza-
tion was still detectable. It appears that Ca++ alone is responsible for the depolariza-
tion since a reduction of Na+ to 25% of normal seawater (107.5 mA/ NaCl and 322.5
mA/choline chloride) had no effect on spike amplitude or duration (not shown). The
initial effect of the addition of Co++ on the spike is a broadening of the positive phase
and a loss of the distinct hyperpolarizing undershoot (Fig. 3A). These effects suggest
that Ca++-dependent potassium currents are responsible for the hyperpolarization
phase and these currents might be blocked by K+ channel blockers (Hille, 1984).
Addition of 5 mM 4-aminopyridine (4-AP) or 10 rrLMtetraethylammionium chloride
(TEA) (Sigma) mimics the initial Co++ effects by eliminating the hyperpolarizing
undershoot and broadening the spike (Fig. 3B, C).
Pre-orai ciliated cells are electrically coupled to each other. Depolarizing and hy-
perpolarizing current injected into one cell causes a proportional depolarization and
hyperpolarization in neighboring cells (Fig. 4A). A brief pulse of suprathreshold posi-
tive current can produce a spike that propagates throughout the velum and causes
CONTROL OF VELIGER LOCOMOTION
519
_J20mV
t»
B
J
FIGURE 4 A, B. Intracellular electrode recordings from pre-oral ciliated cells of Calliostoma ligatwn
veligers. A. Simultaneous dual electrode recording demonstrating electrical coupling between ciliated cells.
Hyper- and depolarizing current (±5 nA) injected into electrode #2 causes a corresponding hyper- and
depolarization of a neighboring ciliated cell (electrode #1 ). Similar results were observed when electrodes
were located on opposite velar lobes. Note the synchrony and similarity of the spontaneous spikes in
both recordings. Electrode bridge was not balanced. B. Successive sweeps of stimulated ciliated cell action
potentials, using the same electrode penetration. In the first sweep, a brief subthreshold pulse of depolariz-
ing current caused no spike. A spike was elicited in the second sweep when a suprathreshold current pulse
was injected into the cell. Horizontal scale = 50 ms, vertical scale = 10 mV.
ciliary arrest (Fig. 4B). Ciliated cells are dye coupled to each other as Lucifer Yellow
injected into one cell rapidly spreads throughout all pre-oral ciliated cells (Fig. 5).
We found that it was possible to modulate the inherent frequency of ciliary beat-
ing by injecting ramp depolarizing current into a ciliated cell. As the membrane po-
tential of a ciliated cell is depolarized from resting potential, cilia of the impaled cell
and those immediately adjacent to it begin to slow. As the cell is depolarized further,
a wave front of slowing cilia spreads symmetrically about the electrode until all pre-
oral cilia slow and eventually stop beating (Fig. 6a, c). If the stimulus current is turned
off rapidly, cilia of all cells begin to beat again. However, if the current is slowly
reduced back to zero, then the cilia far from the electrode begin to beat first, followed
by those close to the electrode. From these results, it appeared that the frequency of
pre-oral ciliary beating was dependent upon the level of depolarization of the ciliated
cell membrane, although the laser arrangement shown in Figure ID did not have the
resolution to detect small changes in ciliary beat frequency that accompanied the slow
depolarizations. We tried to document this phenomenon by making stroboscopic
measurements of changes in ciliary beat frequency while depolarizing the pre-oral
cells. This was done by increasing the concentration of KC1 in the bath to depolarize
the velum as a whole, rather than from a point source as in Figure 6A, C. Increases
in the concentration of KC1 to 0.5, 0.75, 1.0, 1.25, 1.75, 2.0 mM, above normal
seawater linearly depolarizes the resting potential by 4, 6, 7, 8, 10, 11 mV, respec-
tively. While depolarizing the membrane potential, however, the spiking frequency
520
S. A. ARKETT ET AL.
FIGURE 5 A, B. Lucifer Yellow dye coupling of pre-oral ciliated cells of live Calliostoma ligatum
veligers. A. Brightfield micrograph of the velum. Cilia extend toward the top of the photo. B. Fluorescence
micrograph of the same portion of the velum as A. A single cell was filled and dye spread throughout all
cells. Note the outlines of individual cells. Scale = 30 ^m.
B
J
J
FIGURE 6 A-C. Intracellular electrode recordings from pre-oral ciliated cells of Calliostoma ligatum
veligers. A. Ramp depolarizing current injected into a single ciliated cell causes an initial slowing of cilia
at position 1 . As the ramp continues, the wave front of slowing cilia follows the arrows and cilia at position
2 begin to slow when those around 1 have stopped. This wave front passes through 3 to 4, whereupon all
cilia have stopped. Laser record (top) shows that when the current is terminated, cilia begin to beat again.
The laser beam passed through position 3 and thus detected the arrest of cilia in this area alone. All cilia
were arrested before the current ramp reached its maximum. B. Simultaneous dual electrode recording
from ciliated cells on opposite velar lobes. Top trace shows a spontaneous slow depolarization with numer-
ous high frequency EPSPs while bottom trace remains at resting potential. Horizontal scale = 1 s, vertical
scale = 10 mV. C. Simultaneous dual electrode recording from ciliated cells on opposite velar lobes. Depo-
larizing current (up to 5 rt A) was gradually injected into electrode #2. Electrode # 1 shows the corresponding
depolarization. Cilia beg^ to slow during the depolarization and eventually arrested at the position of
electrode # 1 at the arrow. Electrode bridge was not balanced. Horizontal scale = 2 s, vertical scale = 20
mV.
CONTROL OF VELIGER LOCOMOTION 521
increases and consequently the cilia remain arrested, or the metachronal wave was
disrupted to the extent that visual synchronization was impossible. This fact ham-
pered the visual measurement of ciliary beat frequency for a range of membrane
potentials, and thus we were only able to document beat frequency reductions in
response to small depolarizations. In one veliger, ciliary beat frequency in normal
seawater was 7.2 Hz. This frequency was reduced by 13% to 6.3 Hz after increasing
the concentration of KC1 by 1.5 mM. This increase in KC1 depolarized the —60 mV
resting potential by 9 m V or 1 5%.
We have observed occasional spontaneous, high frequency EPSPs that are super-
imposed on long duration depolarizations (Fig. 6B). These slow depolarizations likely
correspond to the general slowing of ciliary beating as described above, although we
could not monitor ciliary beat frequency during dual intracellular electrode record-
ings. Slow depolarizations may be recorded on one velar lobe and not the other (Fig.
6B), whereas spikes are always observed velum-wide. These collective findings sug-
gest, although admittedly do not prove unequivocally, that the frequency of pre-oral
ciliary beating is dependent upon the depolarization level of the ciliated cell mem-
brane and this level is modulated by excitatory synaptic input.
Morphology of pre-oral ciliated cells
The morphology of pre-oral ciliated cells of C. ligatum are very similar to those
ofMangelia spp., which have been well described by Mackie et al. (1976). Thus, only
a brief description of these velar cells of C ligatum will be given here. Pre-oral ciliated
cells are characterized by the presence of numerous scythe-like compound cilia (Fig.
7A). There are usually about 25 of these cilia per cell and each one is composed of
approximately 50 individual cilia. Each individual cilium is anchored in the cell by a
distinct basal body and a long striated rootlet (Fig. 7B). The rootlets are robust and
extend well into the cell. Numerous mitochondria are concentrated just below the
basal bodies. Another distinct feature of these cells is the presence of numerous, large
lipid vacuoles, concentrated near the base of the cell (Fig. 7A, D). These vacuoles
appear to be similar to the "reserve food vacuoles" described by Carter (1928). Dis-
tinctively less dense sheath cells lie on the oral and aboral sides of ciliated cells as in
Mangelia spp. (Mackie et al., 1976). Sheath cells bear numerous microvilli and only
a few simple cilia. Lipid vacuoles are conspicuously absent from sheath cells. Pre-oral
ciliated cells are connected to each other by numerous gap junctions and a few septate
demosomes (Fig. 7B, C). Ciliated cells are richly innervated by axons containing elec-
tron-lucent vesicles (Fig. 7D). Most of these neuro-ciliary synapses are found at the
proximal end of the cells, although we have observed some distally.
DISCUSSION
We have provided evidence to suggest that the beating frequency of the locomo-
tory, pre-oral cilia of competent veligers of C ligatum is dependent upon the level of
depolarization of the ciliated cells' membrane and that this level is modulated by
excitatory neuronal input. Pre-oral cilia appear to have an inherent beating frequency
which is seen at resting potential, or when the ciliated cells are isolated from excitatory
inputs either physically by dissociation or chemically in high Mg++ solutions. The
inherent beating frequency is reduced by excitatory inputs, presumably originating
from the cerebral ganglion. This ganglion is highly developed in early veliger stages
ofAplysia californica (Kandel et al., 1980) and its axons have been shown to inner-
vate the velar ciliated cells in several veligers (Carter, 1 926, 1 928; Mackie et al., 1 976).
522
S. A. ARKETT ET AL.
• " v= '
4 •
-
. - »' -
FIGURE 7 A-D. A. Side view of a single, isolated pre-oral ciliated cell from Calliostoma ligatnm
Aboral end is to the right, oral end is to the left. Effective stroke is from left to right. Note the scythe-like
compound cilia which he in rows orthogonal to the direction of beat. Prominent spheres at the base of the
CONTROL OF VELIGER LOCOMOTION 523
We also find many neuro-ciliary synapses (Fig. 7D), but we have not determined the
origin of these axons, nor do we know if all pre-oral cells are innervated.
Control of locomotion
Veligers are capable of modulating the inherent frequency of ciliary beating to
yield two distinct types of locomotory behavior. In the first type, normal ciliary beat-
ing is periodically interrupted by rapid, coordinated, velum-wide ciliary arrest (Figs.
1 D, 2A). Coordinated velum-wide ciliary arrests are caused by a ciliated cell action
potential, which results from summing EPSPs. This spike propagates rapidly
throughout the electrically coupled pre-oral cells (Fig. 2C). Cilia remain arrested with
spiking frequencies of 0.4 Hz or greater. One to two seconds after the spike, cilia begin
to beat and during normal ciliary beating, the membrane potential remains at resting
potential and little synaptic activity is observed. This modulation of ciliary beating is
similar to that in other veligers (Mackie et ai, 1976) and other metazoan ciliated
systems (Mackieet al., 1974; Saimi etai, 1983a; Arkett, 1987) where a rapid stoppage
of ciliary beating is advantageous (see below).
We have provided several pieces of evidence to suggest that the action potential
of the pre-oral ciliated cells is dependent upon an influx of Ca++. Replacement of
Ca++ by Co++ reduces the amplitude and eventually blocks the spike and prevents
ciliary arrest. (Fig. 3A). This finding corroborates the study by Korobtsov and Sakha-
rov (1971) which showed that Co+ blocks ciliary arrest behavior. We have also
shown that the K+ channel blockers, 4-AP and TEA, eliminate the long hyperpolariz-
ing undershoot and broaden the spike, suggesting Ca++-dependent K+ channels are
present in the pre-oral cells. After the large influx of Ca++ during the depolarizing
phase of the spike, these K+ channels may be activated, thereby hyperpolarizing the
cell and preventing further Ca++ entry (Hille, 1984). As the internal Ca++ concentra-
tion is reduced, cilia may resume beating and a second spike may be initiated. These
large ciliated cells should prove to be a valuable preparation for more fully character-
izing the currents involved in ciliary arrest and the resumption of beating.
The second type of locomotion is produced by a gradual slowing of ciliary beating,
rather than by full velum-wide ciliary arrests. High frequency EPSPs generate long
duration, slow depolarizations of the ciliated cells (Fig. 6B, C). As the ciliated cells
gradually depolarize, the frequency of ciliary beating appears to drop. We have not
observed veligers of this species to use this method of modulating ciliary beating fre-
quency to stop ciliary beating, only to slow it. Localized excitatory input to pre-oral
cells can alter the ciliary beating frequency of one velar lobe and not the other (Fig.
6B). This differential ciliary beating may be used by the veliger in conjunction with
velar muscle contractions to make turns in the swimming path.
We have demonstrated that an action potential is actively propagated throughout
the ciliated pre-oral velar epithelium. The rapid depolarizations associated with cili-
ary arrests in ascidian branchial basket (Mackie et al., 1974; Arkett, 1987) and Myti-
lus gill (Murakami and Machemer, 1982; Saimi et al., 1983a) appear to be conducted
cell are lipid vacuoles. Scale = 25 nm. B. Electron micrograph of the distal portion of two ciliated cells
connected by gap junctions (single arrows) and a septate desmosome (double arrow). Note the robust
striated rootlets. Scale = 0.5 p.m. C. A single gap junction connecting two pre-oral ciliated cells. Scale
= 0.1 nm. D. An axon (AX) containing electron lucent vesicles at the base of a ciliated cell. LV — lipid
vacuole; Scale = 0.5
524 S. A. ARKETT ET AL.
passi < :»ugh gap junctions connecting ciliated cells. We have also provided evi-
denc t suggests a dependence of ciliary beat frequency on the level of depolariza-
tion ;e ciliated velar cells, although unequivocal demonstration of this point was
ssible using the intact veliger. The mechanisms for Mytilus gill appear to differ
Saimi et al. (1983b) stated that the ciliary beat frequency is independent of the
liated cell membrane potential. Yet, Aiello et al. (1986) showed that dopamine
caused a dose-dependent decrease in the endogenous ciliary beating frequency of
Mytilus lateral cilia, but they did not monitor ciliated cell membrane potential. In
Paramecium, forward swimming increases as the membrane potential becomes more
hyperpolarized (Machemer, 1974; Bonini et al., 1986). We found that hyperpolariza-
tion of veliger pre-oral ciliated cells has no apparent effect on beating frequency. Pre-
oral cilia never appear to beat faster than their inherent frequency.
Importance of electrically coupled ciliated cells
The pre-oral ciliated cells are electrically coupled to each other (Figs. 4 A, 5) by
numerous gap junctions between the cells (Fig. 7B, C). Strong electrical coupling
should ensure that EPSPs will summate spatially from velum-wide excitatory input to
ciliated cells and temporally due to a reduction in the junctional shunting of current
between the cells and a longer time constant (Getting, 1974; Berry and Pentreath,
1977). These features lead to a regenerative action potential in the pre-oral cells. Elec-
trical coupling of these cells should ensure that an action potential generated at one
point on the velum will propagate rapidly throughout the pre-oral cells, thereby syn-
chronizing the arrest of all pre-oral cilia (Figs. 2C, 4A, B). This property of rapid
junctional transmission is one of the most important features of electrically coupled
systems (Bennett, 1977). However, we cannot rule out an alternative explanation,
namely that ciliary arrest in the two velar lobes is synchronized by bilaterally symmet-
ric pairs of neurons in the cerebral ganglion.
The electrical coupling properties of pre-oral cells may also make possible small,
local changes in the membrane potential without spikes. Local CNS input may depo-
larize a portion of the velum, but because the input resistance of the velar cells is low,
current is shunted throughout the pre-oral cells and no spikes are generated. How-
ever, such local input can result in long duration, slow depolarizations (Fig. 6B, C)
and thus a differential slowing of ciliary beat frequency on different sides of the velum.
Functional significance of locomotory control
We have demonstrated that the veligers of C. ligatum are capable of controlling
locomotory behavior. This control appears to be expressed in two different types of
locomotion. The alternating upward swimming and sinking upon ciliary arrest is
probably the best known type of locomotion and it has been proposed as a mecha-
nism for regulating position in the water column (Richter, 1 973; Cragg and Gruffydd,
1975; Hidu and Haskin, 1978). Net upward movement could be accomplished by
decreasing the frequency or duration of ciliary arrests, consequently increasing the
amount of time spent swimming. Since veligers generally sink faster than their maxi-
mum ascent speed (Cragg, 1980; Chia et al., 1984), an increase in the frequency of
ciliary arrest should result in a net downward movement. Although the main use of
this type of locomotion may be in regulating diel vertical migration patterns (Richter,
1973), it may also be important as a defence mechanism. Contact with "foreign"
objects or potential predators might cause a rapid ciliary arrest and sometimes a re-
traction of the body into the shell, whereupon the larva sinks rapidly (Fretter, 1967).
CONTROL OF VELIGER LOCOMOTION 525
The second type of locomotion is characterized by more subtle changes in normal
ciliary beating. The gradual, velum-wide slowing of locomotory cilia without full ar-
rest would enable the veliger to either swim slowly upward or sink at a slower rate
than during full ciliary arrest (Richter, 1973; Cragg and Gruffydd, 1975). This behav-
ior appears to be closely linked to the veliger' s satiation level (Fretter and Montgom-
ery, 1 968) and may enable feeding veligers to remain within a zone of abundant food.
The differential ciliary beating of velar lobes observed in C. ligatum in conjunction
with muscle contraction of the velum may enable the veliger to vary its swimming
direction.
ACKNOWLEDGMENTS
We thank Drs. A. O. D. Willows, Director of Friday Harbor Laboratories, for
providing research facilities; R. W. Meech for helpful discussions on this work; D. H.
Paul for helpful criticisms of this manuscript; M. Strathmann for bringing these veli-
gers to our attention; and J. Voltzow for suggestions on fixatives. This study was
supported by NSERC grant #A1427 to G. O. Mackie.
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FEEDING BEHAVIOR IN HYDFL4. I. EFFECTS OF ARTEMIA
HOMOGENATE ON NEMATOCYST DISCHARGE*
W. GROSVENOR AND G. KASS-SIMON
Department of Zoology, University of Rhode Island, Kingston, Rhode Island 02881
ABSTRACT
Inhibition of desmoneme and stenotele nematocyst discharge occurs when Hydra
attenuata are fed to repletion. Inhibition can be induced by the application of prey
homogenates in the external medium. The onset of inhibition is relatively rapid (<30
s) while the release from inhibition is much slower (>20 min). Inhibition is concen-
tration-dependent. Gel chromatography separation of homogenate shows that the
inhibitory substance(s) have a molecular weight greater than 5000. These substances
cause the strongest stenotele inhibition and are least effective in activating the feeding
reflex (mouth opening and tentacle concerts) which is caused by smaller molecular
weight substances. The receptor sites for the inhibitory substance(s) are located on
the external surface of the hydra tentacle. Accumulation of prey substances may be
the mechanism by which stenotele discharge is inhibited when hydra are fed to
repletion.
INTRODUCTION
Nematocysts are highly specialized organelles that serve several functions in coel-
enterates. Nematocysts are used to help capture and kill prey. In some coelenterates,
nematocysts are also necessary for locomotion and defense (Picken and Skaer, 1966;
Mariscal, 1974).
In Hydra two types of nematocysts are involved with the feeding response. Des-
monemes function in prey capture and have a tightly wound thread which wraps
around the prey. Stenoteles have a penetrant shaft and function as killing nemato-
cysts by piercing the prey and releasing a lethal toxin (Ewer, 1947). When hydra are
fed to repletion, however, they lose their ability to discharge nematocysts. Several
causes for nematocyst inhibition have been proposed. ( 1 ) Hydra become less respon-
sive to stimulation and fewer stenoteles are brought to bear against the prey (Burnett
et al, 1961). (2) Either a metabolite from the prey or a product of digestion inhibits
nematocyst discharge (Smith et al., 1974). (3) A factor from the nematocysts them-
selves inhibits nematocyst discharge (Ruch and Cook, 1984).
We now present evidence that the discharge of stenotele nematocysts can be inhib-
ited by the external application of prey substances and that the receptor sites for these
substances are located on the tentacle.
MATERIALS AND METHODS
Specimens of Hydra attenuata from a single asexually reproducing clone were
used in all experiments (Kass-Simon and Potter, 1971). They were raised in BVC
Received 26 March 1987; accepted 23 September 1987.
* An abstract of this work was presented at The Symposium on the Biology of Nematocysts, Irvine,
California, August 1986.
527
528 W. GROSVENOR AND G. KASS-SIMON
solutio: omis and Lenhoff, 1956), and were fed daily from a culture of Anemia
naupl ; shrimp).
shrimp homogenates were prepared by concentrating large quantities of
brii -np nauplii in a filter (Whatman #4 paper) and washing them thoroughly
with distilled water. The nauplii were drawn off with a syringe and the concentrate
was repeatedly forced through the bore of an 18 gauge needle until its consistency
was smooth. This crude homogenate was centrifuged in a clinical centrifuge (~ 1000
< g, 5 min). The water-soluble middle layer was drawn off and recentrifuged to com-
plete the removal of insoluble material. The finished homogenate was then divided
into samples of 1 to 3 ml and frozen at — 5°C until use.
The relative concentrations of various homogenates were determined by collect-
ing 1 ml subsamples of brine shrimp from the shrimp collected from the mass cul-
tures. These were counted and the number of brine shrimp per ml computed.
Stenotele discharge was defined as being inhibited when a hydra rejected, in suc-
cession, five offered brine shrimp that made contact with the tentacles and that were
still swimming normally after 30 s (Smith et al., 1 974). Brine shrimp were introduced
into the medium surrounding the hydra with a pasteur pipet and were directed to-
wards the tentacles with forceps. To be classified as having made contact, brine
shrimp must have distinctly moved a tentacle. Slight brushes with the tentacle were
difficult to discern and were not counted. Hydra were transferred from dish to dish
using large bore dropping pipets.
Brine shrimp killing was grouped into two categories: ( 1 ) brine shrimp that were
captured and killed, and (2) those that made contact but were not captured or killed.
Brine shrimp which were initially captured, but then escaped were classified in
group 2.
Feeding hydra to repletion
Hydra were placed in 10 ml of BVC solution in a 60 X 15 mm glass petri dish,
and were offered 2 brine shrimp every 5 minutes for 150 min (30 trials). During the
experiment the number of brine shrimp captured, killed, and ingested was moni-
tored. If brine shrimp made contact with the tentacles and were not captured, brine
shrimp continued to be offered until either two brine shrimp were captured or a total
of five offerings were made.
Inhibition ofstenotele discharge
Hydra were placed in 5 ml of 1/25 dilutions of brine shrimp homogenates and
were tested for inhibition of stenotele discharge as above. Animals which displayed
significant inhibition ofstenotele discharge were then transferred to fresh BVC solu-
tion for 1 min. They were again transferred to fresh BVC solution for 45 min and
again tested with 5 brine shrimp. Control animals were placed in 5 ml of BVC solu-
tion and tested for stenotele discharge after 5 min.
Effects of homogenate on stenotele discharge
Two sets of experiments were performed to test the effects of homogenate on
nematocyst discharge. Hydra tentacles were ablated 24 h prior to testing. They were
then placed in 1/25 dilutions (0.74 mg/ml protein) of brine shrimp homogenates.
First, individual tentacles were placed on a glass slide in the diluted homogenate for
5 min and covered with a coverslip; the number of discharged nematocysts were
counted under 400X magnification. An equal number of control tentacles were
NEMATOCYST DISCHARGE IN HYDRA 529
placed in BVC solution. In addition, tentacles in diluted homogenate were placed in
a 0. 1 5 X 1 .0 cm cell and the number of discharged nematocysts were counted after 5
and 25 min while being observed under an inverted microscope (400X). Control ten-
tacles were placed in BVC solution.
Time course of inhibition and dose response
The onset of inhibition was determined by placing individual hydra in 1/25 dilu-
tions of brine shrimp homogenate. Hydra were transferred into the homogenate and
tested with five brine shrimp at given time intervals after transfer.
To test for recovery from inhibition, several hydra were placed in 1/25 dilutions
of homogenate for 5 min, rinsed with fresh BVC solution, and then rapidly trans-
ferred into separate dishes containing 5 ml of BVC solution. They were then tested
with 5 brine shrimp at regular time intervals ranging from 2.5 min to 22.5 min.
To determine the concentration of homogenate required for inhibition, the ho-
mogenate was diluted serially by factors of 10, resulting in dilutions of 10"' to 10~6.
Individual hydra were placed in 5 ml of diluted homogenate for 5 min and were
then tested with 5 brine shrimp. The protein concentration of the homogenate was
determined by measuring the protein concentrations of an homogenate from the
same batch (Lowry et al, 195 1 ).
Extraction of inhibitory substances
Gel filtration chromatography (Determan, 1969) was used to fractionate the ho-
mogenate. A 1 5.5 by 1.5 cm column with a polystyrene bed support was packed with
sephadex G-25-80 beads (Sigma Chemical Co.). Solvent (BVC solution) was placed
in a reservoir raised several feet above the column to create a slight positive pressure.
A 1-ml sample of homogenate was used for each experimental run. Sample collection
began 50 drops prior to the first appearance of homogenate fractions from the col-
umn. Fifteen 20-drop samples were collected. The first appearance of homogenate
fractions from the column could be clearly observed due to the cloudiness of the
fraction and the slight orange color. This correlated closely with calculations of pro-
tein elution from Determann (1969).
Each of the 15 fractions was diluted by 1/20 with BVC solution. One hydra was
placed in 5 ml of diluted fraction for 5 min. The level of stenotele discharge, mouth
opening, and tentacle activity was determined. Numerical values, as given in Table
I, were assigned to each of the responses for data analysis. In a separate set of experi-
ments, 5 X 10~4 M reduced glutathione (GSH, Sigma) was run through the column
to locate the fractions in which substances that activate the feeding response would
be found (Loomis, 1955).
Localization of receptors
To test for receptors in the gastrovascular cavity, injections of homogenate were
made through the basal pore. The injection apparatus was composed of drawn out
polyethylene tubing attached to a Hamilton 1 0 microliter syringe (Smith et al. , 1 974).
First, hydra were injected with 1 .0 /zl of whole homogenate, and tested with 2
brine shrimp every 5 min for 35 min. At the end of 35 min additional homogenate
was injected until it could be seen leaking from the hypostome. Two minutes later
the hydra were again tested for stenotele discharge using 5 brine shrimp. As a control,
1 .5 ^1 of air was injected. Air bubbles remain intact and can easily be seen in the gut.
After 5 min the hydra were tested for stenotele discharge with 5 brine shrimp.
530 W. GROSVENOR AND G. KASS-SIMON
TABLE I
Numc'-u s. assigned to specific aspects of the feeding behavior in hydra:
mouth opt '. >ng and tentacle activity
Mouth opening response: Numerical value:
Hypostome protruding from tentacle ring. Slight opening. 1
Moderate mouth opening. Mouth clearly open. 2
Large mouth opening, hydra may be inverting itself. 3
Tentacle activity:
Slight activity, periodic concerts. 1
Continuous concerts. 2
Tentacle writhing. 3
Tight writhing, tentacles may have entered mouth. 4
Partial values given for activities between the defined steps.
In another series of experiments, hypostomes with attached tentacles (ablated 24
h earlier) were placed in 0.25 ml of BVC solution containing 10 ^1 of homogenate.
The animals were allowed 5 min to relax and were tested with 5 brine shrimp. Control
animals were placed in BVC solution only. In addition, isolated tentacles were tested
by the same procedure.
Different regions of a given tentacle were also tested for inhibition of stenotele
discharge. A small quantity of homogenate was applied to the proximal half of a
tentacle using a 10 yul syringe. The extent of the cloud of homogenate could clearly
be seen. The proximal half of the tentacle was kept in the homogenate for 2 min and
then relocated to a clear region of the depression slide. Brine shrimp were added to
the culture medium and the number of random contacts or kills were recorded for 2
min for each half of the tentacle.
If the proximal half of the tentacle became inhibited, that portion was ablated
from the whole tentacle and placed in fresh BVC solution. It was allowed 1 h to
recover and was then retested for stenotele discharge as above.
Statistical analyses were done on the University of Rhode Island computer system
with software from Statistical Analysis Systems (Box 800, Cary, North Carolina).
Where applicable, Wilcoxon Rank Sums, Kruskal-Wallis K Samples, and Linear Re-
gression tests were used. Paired data were analyzed using the Wilcoxon Signed-Rank
Test (Lentner, 1975).
RESULTS
Our observations with respect to hydra's behavior when fed to repletion (Fig. 1 )
essentially confirm the results of Smith et al. (1974) and Ruch and Cook (1984).
The hydra readily killed the brine shrimp offered to them (trials 1-10). However, as
additional shrimp were added to the hydra, the number of brine shrimp killed de-
creased. When the killing of brine shrimp began to decrease (trial # 1 1 ) the hydra had
killed 169 out of 170 brine shrimp (>99%). From trial #1 1 through #30 killing was
reduced to <30% (89/320 brine shrimp). Ingestion was also reduced; 92% of the brine
shrimp killed in the first 10 trials were ingested, whereas in the latter trials <40% were
ingested.
During the latter trials the killing of brine shrimp by individual hydra were quite
variable. A given hydra could be completely inhibited for two or more periods and
NEMATOCYST DISCHARGE IN HYDIL4
531
NUMBER
OF
ARTEMIA
KILLED
2-
1 -
0-
i
4
i
8
10 12 14 16
i
18
20 22 24
i
26
i
28
i
30
TRIAL NUMBER
FIGURE 1. Number of brine shrimp killed/hydra when hydra were offered 2 brine shrimp at 5-min
intervals for 30 trials. Arrow indicates the mean trial in which column contractions began, n = 8.
then kill one or two brine shrimp. Sometimes, brine shrimp that were captured were
not killed. These would eventually pull free from the tentacles and continue swim-
ming normally.
Application of brine shrimp homogenate (1/25 dilution) to the medium sur-
rounding a hydra caused mouth opening and tentacle writhing responses. In these
cases, when brine shrimp are placed near the tentacles of animals in homogenate,
there is a significant decrease in the killing response compared to control animals in
BVC solution (P = .001, Wilcoxon Rank Sums, Fig. 2). After a washing out period
in which the hydra are placed in clean BVC solution, stenotele discharge returned
and was (T2, Fig. 2) significantly greater than that in the test animals (T, , Fig. 2) (P
< 0.01 Wilcoxon Signed-Rank).
Microscopic examination of hydra tentacles indicate that 1/25 dilutions of brine
shrimp homogenates do not stimulate nematocysts to discharge. In both sets of exper-
iments, test tentacles (dilute homogenate) were not significantly different from con-
trol tentacles (BVC solution). The five tentacles placed in homogenate for 5 min
and covered with a coverslip produced no discharged nematocysts. Among control
tentacles, one tentacle discharged a single stenotele. Among tentacles observed on an
inverted microscope with no coverslip for 5 min, 3 out of 10 in homogenate dis-
charged stenoteles (mean = 2.33 stenoteles/tentacle). Among 10 control tentacles, 4
discharged stenoteles (mean = 3.25 stenoteles/tentacle) and one discharged a desmo-
neme. No additional discharge was found at the 25 min interval. The results indicate
that homogenate alone does not induce nematocyst discharge.
Almost complete inhibition appears to occur within 30 s of placing a hydra in
diluted homogenate. No significant differences were found among the 5 tested time
periods (30 s, 1,2, 3, and 4 min.), indicating that the onset for inhibition was less
than 30 s. Release from inhibition occurred in 20 min, after which the killing was
restored to normal levels (Fig. 3).
In serial dilutions of homogenate, a linear relationship (r = 0.82) was found be-
tween the concentration of homogenate and the number of brine shrimp killed (Fig.
4). Although the number of brine shrimp killed by individual hydra varied, as a group
532
W. GROSVENOR AND G. KASS-SIMON
5-
NUMBER
OF 4.
ARTEMIA
KILLED
3-
2-
1 -
O-1-
T, T,
FIGURE 2. Number of brine shrimp killed (mean ± SD) during external application of 1/25 dilutions
of homogenate. Each hydra was tested with five brine shrimp. C = Controls, in BVC solution; TI = tests,
in dilute homogenate; T2 = retests, retested after 45 min in BVC solution, n = 6.
NUMBER
OF
ARTEMIA
KILLED
5-
4-
3-
2-
1-
0-
• • •
• •
i i
2.5 5
10
i
15
i
20
TIME IN MINUTES
FIGURE 3. Release of stenotele nematocysts from inhibition. Hydra were placed in 1/25 dilutions of
homogenate for 5 min and then transfered to fresh BVC solution. They were tested with five brine shrimp
at given time intervals, n = 8, r = 0.67.
NEMATOCYST DISCHARGE IN HYDR.4
533
5-
NUMBER 4-
OF
ARTEMIA
KILLED 3.
2-
1-
0-
icr
I0~5 I0"4 I0~3 I0"
HOMOGENATE DILUTIONS
10'
FIGURE 4. Serial dilutions of homogenate ( 1 8 mg/ml) that induce inhibition of stenotele discharge.
Hydra were placed in given dilutions of homogenates for 5 min and then tested with 5 brine shrimp, n
= 6. A linear regression was calculated between 10~5 and 10~2. r = 0.82.
there was a 50% reduction in killing at a 10 4 dilution of homogenate. The protein
concentration of the undiluted homogenate was 1 8 mg/ml. At 50% inhibition the
protein concentration of the diluted sample was 1.8 ng/m\.
Brine shrimp homogenates separated according to molecular weight were tested
for their effects on stenotele discharge and for various aspects of the feeding response
(Fig. 5). Significant differences were found between the killing of brine shrimp in
different fractions (P = .000 1 , Kruskal-Wallis). The differences were between the first
two fractions (control) and the largest molecular weight fractions, 3 and 4 (Multiple
Comparisons based on Kruskal-Wallis). The other samples did not show a significant
loss in stenotele discharge compared to the controls.
There were also significant differences in the mouth opening response between
the different fractions (P = .000 1 , Kruskal-Wallis). Fractions 8 through 1 1 had a
significantly higher mouth opening response. Fractions 3 and 4 (the large mol. wt.
fractions) caused little or no response. Significant differences were also found for ten-
tacle activity (P = .000 1 , Kruskal-Wallis). Fractions 8 through 1 2 caused significantly
more activity than the other samples.
GSH (5 X 10"4 M) was also run through the column. The results indicate that
GSH had no effect on brine shrimp killing. Samples 7 through 1 1 produced at least
'/2 maximal mouth opening response (Table I). Tentacle activity responses were at
least l/z maximal in samples 6 through 1 1 (Fig. 5). Little or no activity was found in
samples 3 and 4 for either mouth opening response or tentacle activity.
Injections of whole homogenate into the gastrovascular cavity had no effect on
stenotele discharge (Fig. 6). One hundred percent of the brine shrimp were killed
during the seven testing periods. There was no indication of mouth opening or tenta-
cle activity.
Only when homogenate leaked out of the hypostome (Fig. 6, T2) was there a sig-
nificant inhibition of stenotele discharge compared to control animals (P = .009,
Wilcoxon Rank Sums). Leakage of homogenate can be caused by injecting excess
amounts into the gastrovascular cavity and can be readily observed as a murky cloud
534
W. GROSVENOR AND G. KASS-SIMON
NUMBER OF ARTEMIA KILLED
5 --
4 -
3 -
2 -
I •
0 -L
MOUTH OPENING RESPONSE
3 -r
2 -
0 -L
•GSH
TENTACLE ACTIVITY
4 -
3-
2 -
I -
0-L
•GSH
i
9
i i i i i i
10 11 12 13 14 15
SAMPLE *
FIGURE 5. Homogenate separation using gel filtration chromatography. Values in each scale are
denned in the methods section. The GSH lines indicate the samples with at least 'A maximal response to 5
X 1(T4 reduced glutathione. Arrow indicates the beginning of the fractionated homogenate in the samples.
n = 7.
around the tentacles. When this occurred, mouth opening responses and tentacle
concerts or writhing were also observed.
Neither columns nor hypostomes are required for stenotele discharge to be inhib-
ited (Fig. 7). Both ablated hypostomes with tentacles and isolated tentacles displayed
significant inhibition of stenotele discharge compared to controls (P = .00 1 , both
experiments, Wilcoxon Rank Sums). Similar results were found by Smith et al
(1974) and Ruch and Cook (1984) using hypostomes and tentacles.
Treatment of the base of tentacles with homogenate (Fig. 8, T,) resulted in a sig-
nificant loss of stenotele discharge compared to the tentacle base of control animals
(P = .003, Wilcoxon Rank Sums). Brine shrimp killing for the tips of the tentacles,
which had not been treated, were not significantly different from their corresponding
controls. After placing the base of the tentacles in fresh B VC solution for 1 h, stenotele
NEMATOCYST DISCHARGE IN HYDH4
535
PERCENT
OF
ARTEMIA
KILLED
100%-
75%-
50%~
25%~
• 1
• 1
• •
* •
• »
• • 4
• •
• •
* *
• •
• •
* •
• •
• *
• •
.v
* •
• •
• •
* •
• •
• *
* •
• •
• •
• •
• *
* •
t • •
•J9
» « •
••
1 • •
• *
• •
* *
• •
T, T2
FIGURE 6. Injections of homogenate into the gastrovascular cavity through the basal pore. Control
animals were injected with air. Results expressed as % brine shrimp killed (mean ± SD) by the tentacles.
Percents were used since the total number of brine shrimp used varied in each test (T, = 14 brine shrimp,
T2 = 5). C = Controls, air injections; TI = test, homogenate injections; T2 = retests, homogenate leaking
out of the gastrovascular cavity, n = 5.
discharge in the base of the tentacles (Fig. 8, T2) largely returned and was significantly
different from the base of the test tentacles (P < 0.01 Wilcoxon Signed-Rank).
DISCUSSION
Stenotele discharge in hydra becomes inhibited when animals are fed to repletion.
After repletion and the onset of inhibition, the animals remain at least partially inhib-
ited until the end of the experiment (95 min). This compares favorably with results
by Smith et al. ( 1 974) which indicate that stenotele discharge remains partially inhib-
ited until regurgitation (over 4 h).
Externally applied brine shrimp homogenates produced a rapid onset of inhibi-
tion of stenotele discharge (<30 s). Since the methods used to measure the time of
onset were not sensitive enough to resolve times less than 30 s, a more exact time for
onset of inhibition could not be determined. The release from inhibition is relatively
slow. These findings also agree with work by Smith et al., (1974) in which the hypo-
stome and tentacles were removed from inhibited animals and placed in fresh culture
solution; the killing response returned in 30 min.
The inhibitory effects of the homogenates were dependent upon concentration.
At 50% inhibition of stenotele discharge, the concentration of homogenate was equal
to the water soluble layer of 0.043 brine shrimp/^1 ( 1 .8 vg/m\ protein). This is a very
small amount compared to the average number ingested by experimental animals
(mean = 32 brine shrimp/hydra). Although the number of shrimp necessary to in-
duce 50% inhibition of stenotele discharge is greater than the average number in-
gested by a hydra, it is probably incorrect to calculate the concentration of the inhibi-
tory substances, surrounding a repleted and inhibited hydra, as though they were
dissolved in the entire experimental dish. Loomis( 1964) and Lenhoff( 1965) showed
that a concentration gradient does exist in the microenvironment surrounding a hy-
536
W. GROSVENOR AND G. KASS-SIMON
5-
NUMBER
OF 4_
ARTEMIA
KILLED
3-
2-
1 -
o-1-
HYPOSTOMES
AND
TENTACLES
TENTACLES
FIGURE 7. Inhibition of stenotele discharge in hypostomes and tentacles of hydra. Ablated hypo-
stomes with tentacles (n = 8) and isolated tentacles (n = 9) were placed in 1/25 dilutions of homogenate
for 5 minutes and tested with 5 brine shrimp. C = controls, in BVC solution; T = tests, in dilute homoge-
nate. Bars represent standard deviations.
dra which can have behavioral effects. The effective concentration of metabolites,
surrounding a hydra, can not be extrapolated from our data.
Two responses are elicited when homogenates are applied to the medium sur-
UJ
LJ
I-
tE
u.
O
cr
UJ
CD
O
O
O
CC
UJ
n
i.oo -
75 -
.50 -
.25 -
0 -
C T, T2
BASE
OF
TENTACLE
TIP
OF
TENTACLE
FIGURE 8. Bases and tips of intact tentacles were tested for inhibition of stenotele discharge in 1/25
dilutions of homogenate. The base of the tentacle was covered with homogenate for 2 min and then the
entire tentacle was tested with excess brine shrimp for 2 min. Inhibited regions of the tentacle were ablated,
placed in fresh BVC solution, and retested after 1 h. The scale represents the ratio of the number of brine
shrimp killed over the total number of contacts (mean ± SD). C = controls, tentacle placed in BVC solution
only; T, = test, base of tentacle placed in homogenate; T2 = retest, base of tentacle after washing for 1 h in
BVC solution, n = 8.
NEMATOCYST DISCHARGE IN HYDRA 537
rounding a hydra: ( 1 ) an activation of the feeding reflex (mouth opening and tentacle
movement) and (2) an inhibition of stenotele discharge. The strong activation of the
feeding reflex does not cause the decrease in the killing of brine shrimp because there
is no decrease in killing when GSH is applied to the external medium (GSH causes a
strong activation of the feeding reflex).
Ruch and Cook ( 1 984) proposed that inhibition of stenotele discharge was due to
the previous discharge of nematocysts. They found that instead of inhibition, brine
shrimp homogenates (5.8 fj.g/1.5 ml) caused a 35% increase in stenotele discharge.
Only when they applied a "crude homogenate" (of an unknown concentration) were
they able to get a significant decrease in stenotele discharge. They attributed this inhi-
bition to the large number of stenotele nematocysts that were discharged by the ho-
mogenate itself. According to their data 75 to 100 nematocysts were discharged
per hydra.
We are unable to confirm these findings with our experiments. In our experi-
ments, homogenates diluted to a similar protein concentration (1.8 Mg/ml) induced
50% inhibition of stenotele discharge. The inhibition of stenotele discharge is not due
to previous nematocyst discharge, since at higher protein concentrations (0.74 mg/
ml), where there is complete inhibition of stenotele discharge, no significant amount
of nematocyst discharge occurred. Our work is consistant with that of Pantin (1942)
and Ewer (1947) who found that food extract alone does not elicit nematocyst dis-
charge, although it does lower the threshold to discharge when applied locally. Since
homogenate is the only variable altered in our experimental design, we believe that a
substance in the homogenate causes the inhibition of stenotele nematocysts.
Ruch and Cook (1984) showed that nematocyst-rich tissues will cause complete
inhibition of stenotele discharge. Furthermore, Clark and Cook (1986), in the colo-
nial hydroid Halocordyle disticha, were able to induce complete inhibition using the
discharge products of large numbers of purified stenotele nematocysts. It is possible
that more than one mechanism for inhibition of stenotele discharge exists or that the
various mechanisms may employ a similar substrate.
Gel filtration chromatography demonstrates that the substances activating the
feeding reflex and those inhibiting stenotele discharge do not have the same molecu-
lar weight. The largest molecular weight fraction (>5000), which caused the strongest
inhibition of stenotele discharge, displayed very little or no activation of the feeding
reflex, whereas the samples causing the strongest feeding reflex also produced the
highest level of killing. Since gel filtration chromatography separates substances based
upon molecular weight, the partial inhibition in later column samples indicates either
that inhibition of stenotele discharge may be caused by more than one substance or
that there was incomplete separation of the homogenate samples.
Injections of brine shrimp homogenates into the basal disc of hydra do not cause
any inhibition of stenotele discharge or activation of the feeding response until ho-
mogenate leaks into the external environment. Furthermore, different body regions
or even different regions of a tentacle behave independently from each other.
Whether inhibition occurs directly at the nematocyte or involves some other compo-
nent of the battery cell complex (Hufnagel et al, 1985) remains to be determined.
In summary, inhibition of stenotele discharge can be induced with external appli-
cation of prey homogenates. The onset of inhibition is rapid (<30 s), while the release
from inhibition is relatively slow (>20 min). Inhibition is dependent upon the con-
centration of homogenate. The inhibitory substances are different from those which
activate the feeding reflex. The receptors for these inhibitory substances are found on
the external surface of the hydra tentacle and may be associated with the nematocyte
directly or with some other component of the battery cell complex.
538 W. GROSVENOR AND G. KASS-SIMON
ACKNOWLEDGMENTS
We would like to thank L. M. Passano for critically reading the manuscript.
LITERATURE CITED
BURNETT, A. L., T. LENTZ, AND M. WARREN. 1960. The nematocysts of Hydra (Part 1 ): the question of
control of the nematocyst discharge reaction by fully fed Hydra. Ann. Soc. R. Zoo/. Belg. 90:
247-267.
CLARK, S. D., AND C. B. COOK. 1986. Inhibition of nematocyst discharge during feeding in the colonial
hydroid Halocordyle disticha (= Pennaria Tiarella). Biol. Bull. 171: 405-416.
DETERMANN, H. 1969. Gel Chromatography, second edition. Springer- Verlag, New York. 202 pp.
EWER, R. F. 1947. On the functions and mode of action of the nematocysts of Hydra. Proc. Zoo/. Soc.
Lond. 117:305-376.
HUFNAGEL, L. A., G. KASS-SIMON, AND M. K. LYON. 1985. Functional organization of the battery cell
complexes in tentacles of Hydra attenuata. J. Morphol. 176: 323-341.
KASS-SIMON, G., AND M. POTTER. 197 1 . Arrested regeneration in the budding region of Hydra as a result
of abundant feeding. Dev. Biol. 24: 363-378.
LENHOFF, H. M. 1965. Some physicochemical aspects of the macro- and microenvironments surrounding
Hydra during activation of their feeding behavior. Am. Zoo/. 5: 5 1 5-524.
LENTNER, M. 1975. Introduction to Applied Statistics. Prindle, Weber and Schmidt Inc., Boston, MA. 388
PP-
LOOMIS, W. F. 1955. Glutathione control of the specific feeding reactions of Hydra. Ann. N. Y. Acad. Sci.
62:209-228.
LOOMIS, W. F., AND H. M. LENHOFF. 1956. Growth and sexual differentiation of Hvdra in mass culture.
J. Exp. Zoo/. 132: 555-573.
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LOWRY, O. H., N. J. ROSBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the
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MARISCAL, R. N. 1974. Nematocysts. Pp. 129-178 in Coelenterate Biology: Reviews and New Perspectives,
L. Muscatine and H. M. Lenhoff, eds. Academic Press, New York.
PANTIN, C. A. F. 1942. The excitation of nematocysts. J. Exp. Biol. 19: 294-3 10.
PICKEN, L. E. R., AND R. J. SKAER. 1966. A review of researches on nematocysts. Pp. 19-50 in The
Cnidaria and Their Evolution. W. J. Rees, ed. Academic Press, New York.
RUCH, R. J., AND C. B. COOK. 1984. Nematocyst inactivation during feeding in Hvdra litoralis. J. Exp.
Biol. 111:31-42.
SMITH, S., J. OSHIDA, AND H. BODE. 1974. Inhibition of nematocyst discharge in Hvdra fed to repletion.
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Reference: Biol. Bull. 173: 539-551. (December, 1987)
THE EFFECTS OF SALINITY STRESS ON THE RATES OF AEROBIC
RESPIRATION AND PHOTOSYNTHESIS IN THE HERMATYPIC
CORAL SIDER.4STREA SIDEREA
NYAWIRA A. MUTHIGA1* AND ALINA M. SZMANT2**
^Department of Oceanography. Florida State University, Tallahassee, FL 32306 and2 Rosenstiel School of
Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway. Miami, FL 33149
ABSTRACT
Corals are reputed to have low tolerance to salinity fluctuations. Yet the sclerac-
tinian coral Siderastrea siderea commonly inhabits reef zones and nearshore areas
that experience salinity fluctuations of 5 to 10%o. Small colonies of this species were
subjected to both long-term and sudden decreases or increases in salinity. Their rates
of aerobic respiration and photosynthesis, measured as changes in oxygen concentra-
tion, were followed for up to 144 hours after the sudden changes.
Normal salinities of coastal waters near Panacea, Florida, are 28 to 30%o, but S.
siderea was able to acclimate to 42%o when salinity was increased slowly over a 30-
day period. Neither respiratory nor photosynthetic rates of S. siderea were affected by
changes in salinity of less than 10%o above or below the acclimation salinity. Greater
changes in salinity (either up or down) caused decreases in respiratory and photosyn-
thetic rates proportional to the magnitude of the salinity change. Decreases in chloro-
phyll per algal cell and in assimilation number were associated with and possibly
responsible for some of the decreases in photosynthetic rates.
These results show that S. siderea is able to withstand sudden and prolonged,
environmentally realistic changes in salinity without measurable whole-animal
effects. Further studies are needed to determine whether this species is remarkable in
its ability to tolerate salinity change, or whether reef corals are more tolerant to salin-
ity change than is generally believed.
INTRODUCTION
Observations on the distribution and vigor of coral reefs have suggested a relation-
ship between major environmental factors and the physiological well being of herma-
typic corals. In general, coral reefs only flourish within narrow ranges of salinity,
temperature, and turbidity (Wells, 1957; Yonge, 1963; Stoddart, 1969; Connell,
1973; Glynn, 1973). Although there have been a few experimental studies on the
effects of temperature and sedimentation on the physiology of corals, the effects of
salinity remain poorly studied.
Support for the generally accepted relationship between salinity and coral reef
distribution is indirect rather than experimental. Freshwater runoff or heavy rain on
shallow reef flats, or coincident with low spring tides, may lower local salinities and
lead to physiological damage to reef organisms. Excessive rain killed shallow water
biota on reefs in Tahiti (Crossland, 1928) and Jamaica (Goodbody, 1961). Runoff
following such storms may especially damage reefs close to river mouths (Squires,
Received 13 April 1987; accepted 30 September 1987.
* Present address: Kenya Marine Fisheries Research Institute, P. O. Box 81651, Mombasa, Kenya.
** To whom reprint requests should be sent.
539
540 N. A. MUTHIGA AND A. M. SZMANT
1962). The outflow of freshwater from a freshwater lens along atoll margins has been
suggested to limit coral growth on inner reef flats, as observed on Arno atoll
(Hiatt, 1957).
At the other extreme, high salinities resulting from prolonged drought have been
observed to occur in the lagoons of Turneffe Atoll, Belize (Smith, 1 94 1 ). Evaporation
in tide pools during low tides may also cause salinities to rise to stressful levels.
It is generally agreed that most scleractinian corals can survive only small varia-
tions in salinity, with death resulting when salinity drops below 25%o (Edmondson,
1928) or increases above 40%o (Jokiel et al, 1974). The few experimental studies of
the effects of salinity on coral mortality have reported tolerances ranging from lower
limits of 17.5 to 28%o (Vaughan, 1919; Edmondson, 1928) and higher limits of 38.5
to 52.5%o (Wells, 1957; Edmondson, 1928). Kinsman (1964), however, found reefs
growing in salinities of more than 42%o in the Persian Gulf with large heads ofPorites
spp. flourishing at 48%o. Observations of coral reef distribution will provide by infer-
ence information on the salinity tolerances of corals, however, there is a need for
systematic studies on the effects of salinity on corals.
Detrimental effects of salinity on hermatypic corals can occur due to physiological
stress on the coral animal or the corals1 algal symbionts. The photosynthetic products
of symbiotic zooxanthellae contribute to coral metabolism (Muscatine, 1967; von
Holt and von Holt, 1968). If the symbiotic relationship between coral and algae is
disrupted due to salinity stress, there may be a profound effect on coral metabolism.
Several studies have reported the effects of temperature and light on zooxanthellae
photosynthesis (Clausen and Roth, 1975; Coles and Jokiel, 1 977; Jacques and Pilson,
1980; Jacques et al., 1 983) but similar information on the effects of salinity is lacking.
Goreau ( 1 964) observed that flood waters during Hurricane Flora lowered the salinity
to less than 30%o for more than 5 weeks resulting in mass loss of zooxanthellae by
many reef flat corals.
The objective of this study was to determine the effects of changes in salinity on
coral respiration and photosynthesis. The experimental organism was the hermatypic
coral Siderastrea siderea, which has a wider geographic distribution than most west
Atlantic reef corals. It has been reported as far north as North Carolina (Maclntyre
and Pilkey, 1969) and is common wherever there is shallow hard substrate in the
northeastern Gulf of Mexico. It is abundant on Floridian and Caribbean coral reefs,
especially in shallow reef-flat and back-reef areas where salinity fluctuations are the
greatest (A. M.S., unpub. obs.). In the Florida panhandle area of the Gulf of Mexico,
this species occurs as small nodules 5 to 10 cm in diameter on rocky bottoms near
shore. Salinities in these nearshore areas are usually 28 to 30%o but can drop rapidly
5 to 10%o during periods of runoff(unpub. obs.).
MATERIALS AND METHODS
Colonies of Siderastrea siderea 4 to 8 cm in diameter were collected from hard-
bottom outcrops located 30 to 40 meters offshore of St. Teresa Beach, Florida, at a
depth of 2 to 3 meters. They were maintained in a recirculating filtered seawater
system in a constant temperature room. The volume of the system was approximately
400 liters of seawater. Water was changed each time a new batch of corals was col-
lected, so that the initial salinity was identical to that at the time of collection. Light-
ing of approximately 100 ^uEin m"2 s~', measured with a Licor quantum sensor, was
provided by banks of four 40 watt cool-white fluorescent bulbs suspended above the
aquaria. Timers controlled i 12-hour light:dark cycle.
All salinity experiments were conducted at temperatures between 22 and 26°C.
EFFECTS OF SALINITY ON SIDER.4STREA 541
During the winter months when the field temperatures were lower, the corals were
brought to laboratory temperature by slowly raising the temperature a few degrees
each day. Respiration and photosynthesis measurements were made daily until the
laboratory temperature was reached. After one to four days at constant temperature,
the salinity change was initiated. Tests with salinity changes of 30 to 25%o, 30 to 20%o
and 30 to 16%o were done this way. The remaining tests were conducted during the
summer at the temperature at which the corals were collected.
Each salinity test was begun by measuring the rates of aerobic respiration (oxygen
consumption) and photosynthesis (oxygen production) for each coral at the control
salinity (salinity at time of collection). Eight to ten corals and two control chambers
without corals were used in each test. Measurements were made daily for two to four
days before exposing some of the corals to the new salinity. Half of the corals (n - 4
or 5) were then exposed to the test salinity. A second set of incubations were begun
immediately after, with the experimental group in the test salinity and the control
group remaining at the environmental salinity. Thus, the first measurement of change
in respiration was measured over the first hour after exposure to a new salinity, and
photosynthesis over the second hour of exposure. The corals were later returned to
maintenance aquaria at their respective salinities. Incubations were repeated at 24-
hour intervals for up to a week.
During incubations, daytime respiration measurements were done first by cover-
ing the incubation system with several layers of black plastic sheeting to exclude light.
A second incubation in the light to measure photosynthesis by the zooxanthellae was
begun immediately following the dark incubation. Individual colonies were incu-
bated in plexiglas chambers in a water bath to maintain constant temperature. Incu-
bations lasted from 45 to 60 minutes. The dissolved oxygen concentration (DO) of
the filtered seawater used to fill the chambers was measured and the seawater bubbled
with air if it was less than 95% saturation. Water samples were taken from chambers
at the beginning and at the end of the incubation. DO measurements were made
using the Winkler method (Strickland and Parsons, 1 972). Gross photosynthetic rates
(hereafter referred to as photosynthetic rates) were estimated by summing oxygen
production measured in the light with oxygen consumption measured in darkness.
At the end of each experiment, coral tissues were removed from the skeletons
with a jet of filtered seawater from a Water-Pik (Johannes and Wiebe, 1970) and
homogenized. Aliquots of the homogenates were preserved with LugoFs iodine
(Throndsen, 1978) for microscopic determination of zooxanthellae density, or fil-
tered onto glass fiber filters for chlorophyll measurements using a fluorometric tech-
nique (Strickland and Parsons, 1972). Surface areas of each coral were measured by
the aluminum foil method of Marsh (1970), and were used to normalize the oxygen
flux rates of the various sized colonies (McCloskey el a/., 1978). Oxygen flux rates are
reported as the mean (n == 4 or 5) ±1 standard deviation. Statistical comparisons
between means for each treatment were done using a two-tailed /-test.
RESULTS
Daytime respiration
All of the respiratory rates reported below for the salinity tests were measured
during normal daylight hours. Before accepting this experimental protocol, we tested
to determine whether respiratory rates measured thus were similar to respiratory rates
measured during normal nighttime hours. The results (Table I) show that respiratory
rates of individual colonies varied by as much as a factor of two. However, for each
542 N. A. MUTHIGA AND A. M. SZMANT
TABLE I
Daytime versus nighttime respiratory rates of individual colonies of Siderastrea siderea
Daytime respiration rate Nighttime respiration rate
Colony number (nmolO2cm~2 h~') (nmol O2cm 2 h ')
1
411
411
2
824
828
3
573
552
4
810
755
Mean 655 ± 207 637 ± 256
Each rate represents a single one-hour long measurement.
colony, respiratory measurements made during the daytime were similar to those
measured at night. Therefore we accepted the procedure of measuring respiration
rates during daytime incubations.
Temperature acclimation
As salinity tests were conducted year-round at similar temperatures (22-26°C),
corals used in experiments conducted during the winter were necessarily exposed to
large temperature increases just before exposure to salinity changes.
Respiration rates (R) of corals collected at 16°C(R = 371 ±67 nmo!O2 cm"2 h"1;
n - 10) increased significantly when water temperatures were gradually increased to
22°C over a 72 hour period (R = 532 ± 90 nmol cm 2 h"1; P < 0.01) (Fig. 1 ). Photo-
synthetic rates also increased significantly (P < 0.005) from 890 ± 200 nmol O2 cm"2
h"1 at 16°C to 1462 ± 229 nmol O2 cm"2 h"1 at 22°C during the same time period.
Photosynthetic rates increased more (39%) than respiratory rates (30%). The Q)0s
calculated according to Schmidt-Nielsen (1979) were 1.82 for respiration and 2.29
for photosynthesis.
Responses to sudden changes in salinity
Eight tests were conducted: six on the effect of decreases and two on the effects of
increases in salinity on respiratory and photosynthetic rates. Corals in the first three
tests (30%o to 25, 20, and 16%o) were collected at 1 5°C and were brought to laboratory
temperatures, as described above, before beginning the salinity changes. Two tests
studied the responses of corals acclimated to 42%o when exposed to salinities of 35
and 22%0.
The mean respiratory and photosynthetic rates of control and test groups before
and at the end of the exposure periods are summarized in Table II. There was a large
amount of variability between individual colonies before exposure, and mean rates
of the control groups often increased or decreased during the exposure period. How-
ever, the mean rates of the controls were similar to those previously reported for reef
corals (Wethey and Porter, 1976; McCloskey et ai, 1978; Davies, 1978; Szmant-
Froelich et a!., 1981). The post-exposure values in Table II are the mean rates mea-
sured at the end of each salinity test, and tests varied in duration of exposure. How-
ever, whenever a significant effect was seen, it generally occurred within a few hours
of exposure (Fig. 2).
Overall, the mean change in respiration of the control corals and of the corals
EFFECTS OF SALINITY ON SIDER.4STREA
543
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I
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TEMPERATURE ( ° C )
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TEMPERATURE ( ° C )
FIGURE 1 . Mean (±1 S.D.) rates of respiration (A) and gross photosynthesis (B) of colonies ofSider-
astrea siderea exposed to 2°C increases in temperature every 24 hours, n = 10.
exposed to salinity changes of 5%o and 7%o was an increase of 61 ±116 nmol O2 cm 2
h~' (n = 40), and this mean was not significantly different from zero (t --•• 1.84, P
> 0.05). The mean change in photosynthesis for the same groups was an increase of
151 ± 181 nmol O2 cm"2 h~' (n = 40), and this mean change was significantly greater
than zero (/ = 2.48, P < 0.02). The gradual increase over time in photosynthesis by
the control corals could be explained as a gradual adaptation by their zooxanthellae
to higher light levels in the laboratory as compared to the low light levels in the muddy
coastal waters where the corals were collected. These changes (increases) in respira-
tory and photosynthetic rates exhibited by the control corals over the one to five day
experimental periods (Table II) are, in general, small and in the opposite direction to
the more dramatic changes (decreases) in respiratory and photosynthetic rates exhib-
ited by test corals exposed to the larger changes in salinity. In two cases for respiration
(42 to 35%o tests; Table II) and in three cases for photosynthesis (same two plus 42 to
544
N. A. MUTHIGA AND A. M. SZMANT
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EFFECTS OF SALINITY ON SIDEH4STREA
545
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FIGURE 2. Mean (± 1 S.D.) respiration (A) and photosynthesis (B) rates of Siderastrea siderea accli-
mated to 28%o salinity and abruptly exposed to 16%o salinity for a period of one week, n = 4.
22%o; Table II) pre-exposure rates of the control groups were significantly different
from those of the experimental groups. In these cases, post-exposure rates of the ex-
perimental groups were tested against their own pre-exposure rates.
Salinity decreases of up to 10%o (e.g., 30%o-20%o) caused no significant effect on
either respiratory or photosynthetic rates of test corals, but a decrease of 14%o (from
30%o to 16%o) caused a significant decrease in both rates (Mest, P < 0.025). Respira-
tion decreased by 48% after 19 hours of exposure and photosynthesis decreased by
67% after 20 hours of exposure to 16%o. This particular salinity change was repeated
during the summer when the ambient temperature at the collection site was the same
as that in the laboratory; ambient salinity, however, was slightly lower (28%o). The
corals in this later test group also showed a rapid and significant decrease in both
respiration and photosynthesis when exposed to 16%o (P < 0.025) (Fig. 2). Respira-
tion decreased by 36% and photosynthesis by 33% after 44 hours of exposure, similar
to the decreases observed in the first test. Further gradual lowering of salinity caused
the rates of both respiration and photosynthesis to continue to decrease to 53% and
56% (respectively) of the original rates. The corals eventually died after exposure
to 12%o.
Salinity increases from 32%o to 42%o (A10%o) caused no significant change in res-
546 N. A. MUTHIGA AND A. M. SZMANT
TABLE III
Zooxcmhellui' density and chlorophyll content of the zooxanthellae of Siderastrea siderea colonies used in
the s, Measurements were made shortly after the end of each experiment. Control corals are
>.' maintained at normal salinities throughout the test period. Test corals are those
exposed to lowered salinities for the durations listed in Table II.
Zoox.
density
Chlorophyll
a
Chi a/Zoox.
Salinity
106 cells/cm2
Mg/cm2
Mg/106 cells
change %«
Control
Test
Control
Test
Control
Test
30 to 25 (5)
1
.49 ± 0
,30
1.32±
0.20
9.6 ± 1.8
8.8
±3.
0
6.6 ±0.9
7.0 ±2.7
30 to 20 (5)
1
.46 + 0
.36
2.00 ±
0.34**
16.4 + 4.9
14.0
± 1,
3
12.1 ±5.5
7.1 ± 1.4
28 to 12* (4)
0
.72 ±0
,11
0.53 ±
0.15
7.2 ± 1.6
3.4
± 1,
2**
10.3 ±3.7
6.4+ 1.8
42 to 35 (4)
0
.96 ±0
.07
0.86 ±
0.07
9.1 ± 1.3
6.9
± 1,
,**
9.5 ±2.0
8.1 ± 1.8
42 to 22 (4)
—
9.8 ± 1.7
6.7
±2,
,7
—
—
Mean
1
,19±0
.41
10.8 ±4.5
9.6 ±3.9
* This is the same test that is listed in Table II as a change in salinity from 28 to 16%». Salinity was
further reduced to 1 2%o after the conclusion of the test and before the tissues were collected for the zoo-
xanthellae and chlorophyll measurements.
** Denotes test groups that were significantly different from their controls (Mest, P < 0.05). Values
are means ± 1 S.D. (n).
piration, but did cause a 25% decrease in photosynthesis (Table II). Corals exposed
to a 14%o increase (from 28%o to 42%o) had significant decreases in both respiration
and photosynthesis (P < 0.025). Photosynthesis decreased by 39% after 47 hours and
respiration decreased by 22% within 43 hours.
Several colonies of S. siderea were gradually acclimated to 42%o by allowing the
seawater in the recirculating system to slowly evaporate over a month-long period.
Respiratory and photosynthetic rates of these acclimated colonies were similar to
those collected at 28 to 30%o (Table II). When these acclimated corals were exposed
to a 7%o decrease in salinity (to 35%o) there were no measurable effects, but there were
large decreases in respiration and photosynthesis when corals acclimated to 42%o were
exposed to 22%o, a change in salinity of 20%o. Respiration decreased by 82% and
photosynthesis by 81% after 12 hours of exposure.
Zooxanthellae density and chlorophyll content
The control corals had variable zooxanthellae densities ranging from 0.82 to 1 .49
X 106 cells/cm2 (Table III) in measurements made after the completion of the salinity
tests. There were no significant differences between the zooxanthellae densities of
control and test groups, except for the 30 to 20%o test group which had a significantly
greater zooxanthellae density than its control. The chlorophyll content of the control
corals ranged from 5.8 to 17.4 /ug/cm2. In all cases, the experimental corals had lower
chlorophyll contents than their control groups, but the differences were only statisti-
cally significant (Mest, P < 0.05) for the 28 to 12%o, and 42 to 35%o salinity tests (Table
III). In the former tests the decrease in chl a coincided with significant reductions in
photosynthetic rates.
DISCUSSION
Temperature effects
Oxygen consumption is the standard method for measuring routine metabolism
of an animal and is generally equated to aerobic respiration (Prosser, 1973). Within
EFFECTS OF SALINITY ON SIDERASTREA
547
A Resp= 483 - 46 ( A
o
IN
10 15
SALINITY (A °/oo)
FIGURE 3. Plot of the mean change in respiration rate of colonies of Siderastrea siderea after 24
hours of exposure to reduced or increased salinity (Y axis) versus the change in salinity (X axis). Change
in respiration rate was calculated for each coral as the difference between its respiration rate measured 24
hours after exposure and the mean of several measurements made over a 24 to 48 hour period before the
exposure began. The changes in respiration of the control groups were plotted versus the changes in salinity
of their respective test groups rather than opposite a zero change in salinity. The regression line was calcu-
lated using a least squares method; the 5%o salinity change point (0) was omitted from the regression for
reasons explained in the text, n = 4 or 5.
the temperature range that an organism can tolerate, the rate of oxygen consumption
of heterothermic animals is often found to increase in a fairly regular manner with
increasing temperature. The average Qi0 over the temperature range of 16 to 22°C
for the respiration rate of Siderastrea siderea was 1.82, which is similar to the Q10s
for respiration reported for various coelenterates (Lenhoff and Loomis, 1957; Sass-
man and Mangum, 1970; Mangum et al., 1972). Photosynthesis is also influenced by
temperature in a similar manner. The average Qi0 for photosynthesis by the zooxan-
thellae of S. siderea was 2.29 for the same temperature range. This compares well
with those found for many plants ( 1 .0-2.7) (Salisbury and Ross, 1969).
Responses to changes in salinity
According to Vernberg and Vernberg (1972), there are several typical physiologi-
cal responses to salinity stress. These include (a) an increase in respiration when sub-
jected to salinity stress (regardless of the direction of salinity change); (b) a decrease
in respiration regardless of direction of salinity change; (c) an increase in respiration
if salinity is lowered but a decrease if salinity is increased; and (d) no change in respira-
tion. In addition, there is often an initial transitory change in respiration after which
a new steady state is achieved.
Siderastrea siderea shows a combination of responses (b) and (d) above, depend-
ing on the magnitude of the salinity change (Fig. 3, 4). Changes in salinity of less than
10%o had no significant effect on respiration, nor, in many cases, on photosynthesis
(response d). Thus, within the environmentally realistic salinity range for this species
5. siderea is able to tolerate sudden and prolonged exposures to fairly large (10%o)
changes in salinity. Changes in salinity greater than 10%o and 9%o caused significant
decreases in respiration and photosynthesis, respectively, regardless of whether salin-
548 N. A. MUTHIGA AND A. M. SZMANT
ity was increased or decreased (response b). There was no initial transitory burst of
respiratory activity within the first hour of exposure to the altered salinity (results in
Fig. representative). Rates remained lower for at least a week during the present
experiments.
ure 3 includes a least-squares linear regression of the change in respiration after
24 hours of exposure to altered salinity versus the change in salinity over that period.
The data from the A5%o test were omitted from the regression because we suspect
these animals of having been incompletely adjusted to the laboratory temperature at
the time the salinity change test began, and simultaneous changes in salinity and
temperature may have a synergistic detrimental effect (Coles and Jokiel, 1978). All
control groups (except for the A5%o one) and the A7%o experimental group had in-
creases in respiration over the 24-hour experimental period. The regression shows
that only salinity changes greater than 10.5%o resulted in respiration depression. If
this coral is an osmoconformer like many coelenterates (Ranklin and Davenport,
1981), its extracellular fluid osmotic pressure will fall or rise with changes in the envi-
ronment. This would lead eventually to cellular swelling or shrinking, and to cell
disruption. But even before such damage is done, the changes in cell size and alter-
ations in internal geometry might disturb cell functions and possibly cause decreases
in metabolic rates (Ranklin and Davenport, 1981). Alternatively, corals could con-
tract their polyps thus reducing their contact with the adverse salinity conditions.
Shumway (1978) has shown that the sea anemone Metridium senile contracts when
exposed to lower salinity. Contraction would (a) decrease gas exchange with the exte-
rior, and thus contribute to lower respiration rates, and (b) reduce the exposure of
the zooxanthellae to light, and contribute to a lower rate of photosynthesis. However,
contraction might have a similar effect on oxygen flux rates regardless of the magni-
tude of the change in salinity. Thus it is not a completely satisfactory explanation for
the linear decrease in respiratory and photosynthetic rates that occurred as the size
of the salinity change increased. No systematic observations on changes in coral be-
havior due to salinity change were carried out. However, casual observations revealed
that S. siderea exposed to high or low salinities sometimes retracted into their skele-
tons for extended periods. Further studies should include experiments on the effec-
tiveness of retraction as an avoidence mechanism in corals, and on the effect of retrac-
tion on the measurement of respiratory and photosynthetic rates.
The ability of this species to acclimate to abnormally high salinities (42%o) shows
that the animal can acclimate to a large net salinity change when the change occurs
slowly, whereas the same salinity change if experienced suddenly might be fatal. As
recovery experiments were not performed, we do not know at what point the depres-
sion in respiration is still reversible upon return to normal salinities, nor whether
acclimation after a sudden large salinity change will occur. In the single longer experi-
ment where salinity effects were found early on (Table II: 28 to 16%o), respiration was
still depressed after 5 days of exposure to the lower salinity.
Figure 4 shows a similar linear regression of change in photosynthesis after 24
hours of altered salinity versus change in salinity. The minimum salinity change that
caused depression in photosynthesis was 9%o which is slightly lower than that required
to cause a depression in respiration. There were also large decreases in the chlorophyll
content per algal cell in all of the test groups where it was measured, except for the
30 to 25%o salinity change group (Table III). The decreases in photosynthesis appear
to have been caused by the combined effect of small decreases in zooxanthellae den-
sity, chlorophyll content per algal cell and, in one case, by a decrease in the assimila-
tion number (oxygen produced per amount of chlorophyll. Table IV). Polyp retrac-
tion also could have played a role as discussed above.
EFFECTS OF SALINITY ON SIDERASTREA
549
to
600
IT 400
-
200
0
-200
o i
°- I -400
S --600
LJ
o -5 -800
^ i-1000
o
-1200
Control
-Test
O
D
*
APhoto=874-99.5 (A °/oo)
r2= 0.81
5 10 15
CHANGE IN SALINITY (A °/<x>)
20
FIGURE 4. Plot of the mean change in photosynthesis rate of colonies of Siderastrea siderea after 24
hours of exposure to altered salinity (Y axis) versus the change in salinity (X axis). See legend of Figure 3
for further explanation, n = 4 or 5.
In conclusion, respiratory and photosynthetic rates of the coral Siderastrea sid-
erea were not adversely affected by changes in salinity of less than 10%o, although their
chlorophyll content began to decline after changes of 7%o salinity. When exposed to
salinity changes greater than 10%o for 24 hours, respiratory and photosynthetic rates
both decreased regardless of whether salinity was increased or decreased. Linear re-
gressions showed a significant linear relationship between the degree of respiratory
and photosynthetic depression and the magnitude of the salinity change. Finally, it
is important to note that scleractinian corals are generally considered intolerant to
salinity change, yet S. siderea was able to experience rather large ( 10%o) changes in
salinity without any demonstrable effect on respiratory or photosynthetic rates. It will
be interesting to determine whether this species is remarkable in this regard, and thus
suited to live in reef-flat and coastal areas where salinity changes are more frequent,
or whether scleractinian corals are more tolerant to salinity change than is generally
believed.
TABLE IV
Photosynthetic performance by the zooxanthellae of Siderastrea siderea exposed to changes in salinity.
Duration of exposure for each test can be found in Table 11
Salinity
nmol O2 1
; 106 Cells)" 'h'1
nmol O2 (A
•gchlar'h-
Change %o
Control
Test
Control
Test
30 to 25 (5)
775 ± 135
825 ± 95
120 ±25
138+ 59
30 to 20 (5)
453 ± 118
393 ± 85
44 ± 19
57+ 15
28 to 12(4)
2173 ±342
1350 ±614**
222 ± 42
228 ± 133
42 to 35 (4)
1797 ±448
1968 ±566
188 + 21
260 ± 121
42 to 22 (4)
—
—
192 + 52
56 ± 35**
** Denotes test groups that were significantly different from their controls (/-test, P < 0.05). Values
are means + 1 S.D. (n).
550 N. A. MUTHIGA AND A. M. SZMANT
ACKNOWLEDGMENTS
This work is part of a thesis submitted by N.M. in partial fulfillment of an M.S.
degree. We thank Dr. R. Iverson for his advice, Drs. D. C. White and W. Herrnkind
for loan of laboratory equipment, T. McClanahan for help with field work and read-
ing the manuscript, and Drs. P. Walsh, P. Lutz, and two anonymous reviewers for
helpful comments on the manuscript.
This work was supported by an ITT HE Fellowship and a student grant from the
Department of Oceanography to N.M., and by NSF OCE-8208560 and OCE-
8315191toA.S.F.
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UPSTREAM AND DOWNSTREAM CAPTURE DURING SUSPENSION
FEEDING BY OLIGOMETRA SERRIPINNA (ECHINODERMATA:
CRINOIDEA) UNDER SURGE CONDITIONS
NICHOLAS D. HOLLAND, ALEXANDER B. LEONARD, AND J. RUDI STRICKLER1
Marine Biology Research Division, Scripps Institution of Oceanography, La Jolla, California 92093 and
' Boston University Marine Program, Marine Biological Laboratory, Woods Hole, Massachusetts 02543
ABSTRACT
The crinoid Oligometra serripinna is a suspension feeder that usually experiences
unidirectional tidal currents from which it extracts food particles by downstream
capture (i.e., while the food grooves face downcurrent). However, near slack tide,
wave surge may cause brief current reversals, each lasting about 2 s at roughly 10 s
intervals. To test if a crinoid can engage in upstream capture (i.e., while the food
grooves face upcurrent) during brief current reversals, we approximated these surge
conditions in a laboratory flume. In the laboratory, as in the field, the crinoid oriented
its food grooves downstream with respect to the predominant current, and the body
posture did not change during the brief intervals of reversed flow. Brine shrimp cysts
were added to the flume, and video recordings were made of the crinoid capturing
these particles. Under surge conditions, the crinoid (1) captured 16.2% of the ap-
proaching particles while its food grooves faced downstream and (2) captured 8.0%
of the approaching particles while its food grooves faced upstream. Thus O. serrip-
inna used its filter both for upstream capture and for downstream capture, although
the former was only about half as efficient as the latter.
INTRODUCTION
Many suspension feeding animals use a portion of their body as a filter to capture
particles from the passing water. The filter parts that capture and transport the parti-
cles may be oriented toward the current (i.e., upstream capture, as in bivalves) or
away from the current (i.e., downstream capture, as in entoprocts). Although these
definitions work well for many filter feeders, there is some question as to whether
crinoids, which are generally considered downstream capturers (Magnus, 1967;
Meyer, 1979, 1982; Holland et al., 1986), might sometimes function as upstream
capturers. In some crinoids, an animal may orient some parts of its filter upstream
and other parts downstream (e.g., in the cone posture and in the partial arm fan
described by La Touche, 1 978, and by Byrne and Fontaine, 1981). It has been implied
but not clearly shown that such crinoids can engage in upstream and downstream
capture simultaneously. Furthermore, in another crinoid (Oligometra serripinna),
the filter is oriented downstream in the unidirectional tidal current that predominates
in the field. However, at slack tide, wave surge causes periodic reversals in the current
direction (Leonard et al., 1987). During each reversal, O. serripinna does not change
its body posture. Thus the filter is oriented upstream. The present note (1) demon-
Received 6 August 1987; accepted 21 September 1987.
552
UPSTREAM AND DOWNSTREAM CAPTURE
553
AN
PIFG
PI
FIGURE 1 . (A) Diagram of a crinoid showing the oral (mouth-bearing) side of the body. The mouth
and anus open on the central disc from which the arms radiate. Each arm (usually more numerous than
the 5 shown here) is fringed on either side by short side branches called pinnules. (B) Enlargement of the
region opposite bracket x in Figure 1 A. The arm food groove and pinnular food grooves run along the oral
side of the arm and pinnules, respectively. Each pinnule is fringed by tube feet. (C) Cross section of three
adjacent pinnules cut through plane y-/ in Figure IB. The pinnular food grooves are on the oral side of
the body, which is depicted facing upward. Downstream capture is diagrammed for the triangular particle,
and upstream capture is diagrammed for the circular particle. Abbreviations: AN = anus, AR = arm,
ARFG = arm food groove, DSC = downstream capture, MO = mouth, PI = pinnule, PIFG = pinnular
food groove, TF = tube foot, USC = upstream capture.
strates that under surge conditions O. serripinna can alternate between upstream and
downstream capture and (2) compares the efficiencies of these two processes.
The crinoid filter is diagrammed in Figure 1 A,B. Approaching particles adhere to
the adhesive tube feet, which rapidly bend and transfer the particles into the food
grooves for transport to the mouth. The gut openings and food grooves are situated
on the oral side of the body (i.e., the side on which the mouth opens), which faces
downcurrent during downstream capture. This behavior has been studied in detail
for O. serripinna by Holland et al. (1986) and by Leonard et ai (1987). Downstream
capture and upstream capture are shown diagrammatically in Figure 1C.
MATERIALS AND METHODS
We studied a feather star, Oligometra serripinna, living on sea fans at a depth of
10 m at North Reef, Lizard Island ( 14°38'S; 145°28'E), Great Barrier Reef, Australia.
Current speed and direction in the microhabitat of O. serripinna were determined
from underwater video recordings of a dye trail released by the non-motorized dye
injector described by Colman et al. (1984). The study site and orientation of the
injector relative to the crinoid are described by Leonard et al. (1987). We analyzed
554
N. D. HOLLAND ET AL.
P
R
L
E
V
P
R
L
—
FS
—
(
A
u -
) ® =
FS
-^m
1!
M
a»*
2— ^>
-^— 1
FIGURE 2. Diagram of 1 1 -liter flume (45 cm long) for producing surging flow; the water level is
indicated by the dotted line. To obtain flow in the predominant direction (indicated by arrows labeled 1),
one propeller ( = PR) is turned clockwise and the other ( = PR' ) is turned counterclockwise for 6 s. To obtain
flow in the reverse direction (indicated by arrows labeled 2), the action of each propeller is reversed for 2 s.
At either end of the working section of the flume, there is a flow straightener (=FS and FS'). A bright beam
of light is focused into the flume from above with a lens (=LE). A first video system records the speed and
direction of brine shrimp cysts visualized in side view at the position marked by the circled A. An open
mesh perch (shown in side view, =PE) supports the crinoid (not illustrated), which is illuminated with fiber
optics (=FO). A second video system records the upstream and downstream captures in side view at the
position marked by the circled B.
100 s of a recording made near slack tide at about 8: 15 h on 16 August 1986. During
every third of a second (i.e., over 10 consecutive video frames), the speed and direc-
tion of the current was measured from the movement of irregularities in the dye
stream. The predominant current toward the east-northeast was recorded as positive,
and reversed current toward the west-southwest was recorded as negative.
The surge in the field was approximated in the laboratory in an 1 1 -liter flume
(Leonard etal, 1987) to which an additional propeller and flow straightener had been
added (Fig. 2). The two propellers were turned by a reversible, adjustable speed mo-
tor. One propeller turned clockwise and the other turned counterclockwise for 6 s to
produce a. predominant flow of approximately 5 cm/s. The action of the two propel-
lers was then quickly reversed to produce a reverse flow for 2 s before the predominant
flow was re-established and the cycle repeated.
The surge experiment in the flume was performed at 25°C, the approximate tem-
perature in the field at the time. A specimen ofOligometra serripinna was allowed to
attach to an open mesh perch with the food grooves facing downstream relative to
the predominant current direction. Brine shrimp cysts that had been soaked in seawa-
ter for 8 h were then added to the flume. The concentration and speed of the cysts
(and thereby the current speed) were measured as they passed through a segment of
a light beam 2 cm in diameter and 1.8 cm from top to bottom (at position A in Fig.
2). The passing cysts were recorded in side view with a video system (Leonard et ai,
1987). A 100-s interval on the tape was quantified. Current speed was determined
from the distance iravelled by cysts over three consecutive video frames (i.e., over an
UPSTREAM AND DOWNSTREAM CAPTURE
555
10
20
50
_60_
90
100
+6-
+ 3-
i
; O
'-3H
A.. A, A A
10
20 30
SECONDS
40
SO
60
70
80
90
100
FIGURE 3. (A) Fluctuations in current velocity and direction (positive values are toward the east-
northeast, and negative values are toward the west-southwest) in the microhabitat ofOligometra serripinna.
(B) Fluctuations in current velocity and direction in a laboratory flume with surging flow (positive values
are in the predominant direction, and negative values are for the relatively brief intervals of reversed flow).
Each filled circle marks a point in the flow cycle where a brine shrimp cyst was captured.
interval of 0. 1 s). Motion in the predominant direction was recorded as positive, and
motion in the reverse direction was recorded as negative. During the same 100-s
interval, one third of a single O. serripinna arm, with 20 pinnules (10 on each side),
was recorded with a second video system, a duplicate of the first. The arm was re-
corded (at position B in Fig. 2) in side view a few degrees off the perpendicular to
provide a foreshortened view of the pinnules on either side.
RESULTS AND DISCUSSION
Figure 3A plots fluctuations in current speed and direction near slack tide in the
microhabitat ofOligometra serripinna. During the 100-s period, there were 10 con-
spicuous reversals in current direction, most "negative flow" periods lasted about 2 s
and reached speeds of approximately 3 cm/s.
The results of the laboratory flume surge experiment are shown in Figure 3B,
which places cyst capture in the context of fluctuations in current speed and direction.
During the 100-s period, 41 cysts were captured during the cumulative 70 s that flow
was in the predominant direction (i.e., downstream captures), and 10 cysts were cap-
tured during the cumulative 30 s that flow was in the reverse direction (i.e., upstream
captures). The video recordings showed that all the food grooves faced directly up-
stream throughout each current reversal. Thus, upstream capture was unequivocal.
The capture efficiencies during downstream and upstream capture, respectively,
were calculated by counting the cysts crossing the light beam during the 70 s of pre-
dominant flow and during the 30 s of reverse flow. Each count was divided by the
recorded area of the light beam normal to the flow (3.8 cm2) and multiplied by the
recorded area of the arm normal to the flow ( 1 .2 cm2). This gave the number of cysts
approaching the recorded part of the crinoid's filter as 252 and 125 during predomi-
nant and reversed current flow, respectively. The capture efficiency (percentage of
captures normalized to approaches) was 16.2% during downstream feeding and 8.0%
during upstream feeding. We do not know why upstream capture was markedly less
efficient than downstream capture. An explanation of this difference would probably
556 N. D. HOLLAND ET AL.
require a detailed knowledge of flow through the gaps in the filter and the thickness
of the boundary layers around the tube feet and pinnules.
The distinction between upstream capturers and downstream capturers appears
to be useful for suspension feeders that actively pump water past their filters in a
single direction. However, this distinction may not apply for passive suspension feed-
ers, which depend on exogenous currents to bring particles to their filters. For exam-
ple, Patterson's (1984) work indicates that a given polyp of an octocoral can engage
in either upstream or downstream capture depending on the strength and direction
of the exogenous current. Moreover, the present study is the first clear demonstration
that at least some crinoids can augment their usual downstream capture with up-
stream capture.
In the field, Oligometra serripinna probably obtains only a small proportion of its
food by upstream capture because there are no flow reversals during most of the tidal
cycle (Leonard et al, 1987) and upstream capture is markedly less efficient than
downstream capture. Even so, it is possible that upstream capture may be relatively
important in some other crinoid species — especially those living within the infra-
structure of reefs where slow, meandering flows predominate.
ACKNOWLEDGMENTS
This work was supported by NSF grants to N. D. Holland (OCE84-0067 1 ) and to
J. R. Strickler (OCE84- 1 626 1 ). We are deeply indebted to the administration and staff
of the Lizard Island Research Station, Queensland, Australia, for their hospitality and
assistance. Our manuscript was constructively criticized by L. Z. Holland.
LITERATURE CITED
BYRNE, M., AND A. R. FONTAINE. 1 98 1 . The feeding behaviour ofFlorometra serripinna (Echinodermata:
Crinoidea). Can. J. Zool. 59: 1 1-18.
COLMAN, R. S., H. C. CRENSHAW, D. L. MEYER, AND J. R. STRICKLER. 1984. A non-motorized dye
injector for visualization of flow in situ and its use with coral reef crinoids. Mar. Biol. 83: 125-
128.
HOLLAND, N. D., J. R. STRICKLER, AND A. B. LEONARD. 1986. Particle interception, transport and rejec-
tion by the feather star Oligometra serripinna (Echinodermata: Crinoidea), studied by frame
analysis of videotapes. Mar. Biol. 93: 1 1 1-126.
LA TOUCHE, R. W. 1978. The feeding behaviour of the featherstar Antedon bifida (Echinodermata: Cri-
noidea). J. Mar. Biol. Ass. U. K. 58: 877-890.
LEONARD, A. B., J. R. STRICKLER, AND N. D. HOLLAND. 1987. Effects of current speed on filtration during
suspension feeding in Oligometra serripinna (Echinodermata: Crinoidea). Mar. Biol. (in press.)
MAGNUS, D. B. E. 1967. Ecological and ethological studies and experiments on echinoderms of the Red
Sea. Stud. Trap. Oceanogr. 5: 635-664.
MEYER, D. L. 1979. Length and spacing of the tube feet in crinoids (Echinodermata) and their role in
suspension-feeding. Mar. Biol. 51: 361-369.
MEYER, D. L. 1982. Food and feeding mechanisms: Crinozoa. Pp. 25-42 in Echinoderm Nutrition, M.
Jangoux and J. M. Lawrence, eds. Balkema, Rotterdam.
PATTERSON, M. R. 1984. Patterns of whole colony prey capture in the octocoral, Alcyonium siderium.
Biol. Bull. 167:613-629.
Reference: Biol. Bull. 173: 557-562. (December, 1987)
EELGRASS WASTING DISEASE: CAUSE AND RECURRENCE
OF A MARINE EPIDEMIC
FREDERICK T. SHORT1, LISA K. MUEHLSTEIN2, AND DAVID PORTER2
1 Jackson Estuarine Laboratory, University of New Hampshire, RFD 2. Adams Point, Durham, New
Hampshire 03824 and2 Department of Botany, University of Georgia, Athens, Georgia 30602
ABSTRACT
Eelgrass populations are currently infected with a disease that produces symptoms
and epidemiology reminiscent of the famous eelgrass wasting disease of the 1930s.
This disease virtually eliminated eelgrass (Zostera marina L.) in the North Atlantic
for three decades. For 50 years scientists have speculated about the cause of the 1930s
eelgrass decline. We have now proven that the causal organism of the present epi-
demic is a pathogenic strain ofLabyrinthula, which was suspected, but never conclu-
sively shown to cause the 1 930s wasting disease. We have isolated the infectious form
of Labyrinthula from eelgrass from Maine to North Carolina on the Atlantic coast,
and from Puget Sound on the Pacific coast; disease-related dieoffs of eelgrass beds are
confirmed in Maine, New Hampshire, and Massachusetts.
DISCUSSION
A recurrence of the wasting disease, which almost eliminated eelgrass (Zostera
marina L.) in the 1930s, was discovered on the border of New Hampshire and Maine
in the early 1980s (Short et ai, 1986). Since then, eelgrass populations exhibiting
symptoms and epidemiology comparable to the 1930s epidemic have been found
from Nova Scotia to North Carolina. The eelgrass wasting disease of the 1 930s consti-
tuted a marine epidemic which disrupted highly productive coastal ecosystems and
fisheries. The disease had run its course by the 1940s; healthy eelgrass populations
generally were reestablished by the 1 960s. Over the past 50 years, scientists have pro-
posed pathogenic organisms, temperature changes, reduced light, and combined en-
vironmental factors as causes of the 1930s disappearance of eelgrass. In this report,
we present proof that the causal organism of the current epidemic is a pathogenic
strain of Labyrinthula and describe our findings concerning the range of the disease.
The wasting disease that devastated eelgrass populations throughout the North
Atlantic between 1 930 and 1 933 dramatically disrupted coastal and nearshore ecosys-
tems. The most obvious impact was the reduction or loss of migratory waterfowl
populations (Addy and Aylward, 1944). Equally important, though not immediately
apparent, was the impact on commercial fisheries. The loss of the scallop fishery in
the mid- Atlantic coast of the United States is best documented (Thayer et al., 1984).
The 1 930s eelgrass loss altered coastal habitats and changed for decades the character-
istics of nearshore soft sediment environments (Rasmussen, 1973, 1977). In fact,
some locations were permanently altered, and eelgrass never returned.
The cause of the 1 930s wasting disease was never conclusively determined (John-
son and Sparrow, 1961; Den Hartog, 1987). However, two main alternative theories
Received 3 1 August 1987; accepted 29 September 1987.
557
558
F. T. SHORT ET AL.
Q WASTING DISEASE DISTRIBUTION - 1986
EELGRASS GEOGRAPHIC DISTRIBUTION
FIGURE 1 . Geographic distribution of eelgrass and of the current wasting disease on the east coast of
North America.
were promoted: first, that a microorganism was infecting and killing plants (Peterson,
1934; Renn, 1935), and second, that environmental stress from abnormally warm
temperatures increased the susceptibility of the plants to ever-present microorgan-
isms (Rasmussen, 1977). The microorganism most commonly implicated was a
slime-mold-like protist, Labyrinthula (Renn, 1934; Cottam and Addy, 1947), identi-
fied as L. macrocystis Cienk. (Young, 1943). In the decades since the first reports of
the wasting disease there has been significant organism-specific research on Labyrin-
thula (Pokorny, 1967; Olive, 1975; Porter, 1988). The early work during the disease
period was not conclusive because the necessary methods for axenic culture of Laby-
rinthula had not been developed (Renn, 1936; Johnson and Sparrow, 1961). Al-
though axenic cultures of Labyrinthula (Watson and Ordal, 1957) were developed in
the 1950s, tests of Koch's postulates were never attempted.
The current eelgrass wasting disease, first reported in the Great Bay Estuary, New
Hampshire (Short et ai, 1986), occurs in two stages: ( 1 ) the initial infection of eelgrass
leaves; and (2) the subsequent mass mortality of eelgrass. The infection is character-
ized by dark necrotic lesions on both young and old eelgrass leaves. The infection has
now spread throughout the Great Bay Estuary, but the complete dieoff of eelgrass
beds is restricted to local areas. Like the disease of the 1930s, the current epidemic is
limited in the estuary by salinity; eelgrass growing in low salinity waters is less suscep-
tible to infection. The decline is not universal; many areas showing infection symp-
toms as yet demonstrate no mass mortality.
The infection of eelgrass with the characteristic symptoms of the wasting disease
is now widespread along the Atlantic coast of North America. Eelgrass collected in
the summers of 1986 and 1987 from numerous sites between Nova Scotia, Canada,
EELGRASS WASTING DISEASE
559
autoclave
Labyrinthula
reisolated
FIGURE 2. Procedure for laboratory infection of eelgrass, Zostera marina L., by Labyrinthula. Steril-
ized 1 cm pieces of uninfected, green eelgrass leaves were invaded with an axenic culture of Labyrinthula
and then attached to a leaf of a healthy, green eelgrass shoot growing in an individual incubation flask.
When a pathogenic strain was thus tested, the necrotic, blackened patches symptomatic of the wasting
disease appeared on the eelgrass leaves within 14 h on some and within 24 h on all the plants. Labyrinthula
was reisolated from the diseased leaves, thus satisfying Koch's postulates.
and Connecticut, USA, showed the infection (Fig. 1). Additionally, in 1986 infected
eelgrass plants were found in Roscoff, France, and in Friday Harbor, Washington,
and Beaufort, North Carolina, USA. The simultaneous occurrence of the wasting
disease on both sides of the Atlantic is reminiscent of the reports of the 1930s disease
(Fisher-Piette et al, 1932; Huntsman, 1932; Cotton, 1933; Lewis and Taylor, 1933;
Peterson, 1933; Taylor, 1933). The appearance of the disease on the Pacific coast was
reported in the late 1930s (Young, 1938), with significant eelgrass decline evident in
1941 (Moffit and Cottam, 1941).
Our recent research has concentrated on determining the cause of the current
eelgrass disease. Following Koch's postulates, we have successfully identified the
causal agent to be a pathogenic strain of Labyrinthula (Fig. 2). We have regularly
isolated this strain of Labyrinthula from diseased eelgrass leaves from Great Bay,
New Hampshire and also from Beaufort, North Carolina and Friday Harbor, Wash-
ington. The pathogenic strain has never been isolated from healthy, green eelgrass
560 F. T. SHORT ET AL.
TABLE!
Labyrinthula infection experiments on eelgrass (Zostera marina) in laboratory culture
Labyrinthula source: Substrate, location Number of replicates Percent infection
Diseased eelgrass, Great Bay, NH 33 100
Diseased eelgrass, Puget Sound, WA 8 100
Diseased eelgrass, Beaufort, NC 4 100
Healthy eelgrass, Beaufort, NC 4 0
Spartina, Sapelo Is., GA 6 0
Codium drift, Weekapaug, RI 90
Mangrove leaf. Longboat Key, FL 7 0
leaves. Both pathogenic and non-pathogenic strains of Labyrinthula were isolated
and then grown in pure culture using previously described procedures (Porter, 1988).
The eelgrass infection experiments were performed in laboratories at both the Uni-
versity of Georgia and the University of New Hampshire (Table I). Every shoot ex-
posed to the pathogenic strain of Labyrinthula (45 shoots of a total of 45) exhibited
the disease symptoms. None of the nine control shoots, which were treated identi-
cally, but without Labyrinthula in the sterilized inoculum leaf piece, showed disease
symptoms; all remained healthy. During the first week, the infection spread quickly,
with the dark patches increasing to 3-4 cm lesions along the inoculated leaves. After
two weeks, inoculated leaves were completely black or brown and, on most shoots,
the infection had spread to other leaves. After three weeks, several of the infected
shoots were completely brown and dead, while on other shoots the spread of the
infection stopped; the growth of all the infected plants was greatly reduced relative to
the control plants. Labyrinthula was reisolated from the infected leaves, thus satisfy-
ing Koch's postulates.
For the present study, four other strains of Labyrinthula were isolated from green
eelgrass leaves and various other marine plants. When these strains were tested for
pathogenicity, as described above, none of the 26 inoculated plants produced any
signs of the disease symptoms (Table I).
It is significant that we found both pathogenic and non-pathogenic strains of Lab-
yrinthula widely distributed in estuarine environments. It is possible that these repre-
sent different species, but the present taxonomic understanding of the species Laby-
rinthula is poor at best (Olive, 1975; Porter, 1988). A critical monograph of the genus
is clearly needed.
Despite the widespread infection of eelgrass with the wasting disease, there is as
yet relatively little documented evidence of disease-related declines. As mentioned
above, the carefully monitored decline in the Great Bay Estuary has expanded (Short
et al., 1986). Since 1984, entire eelgrass beds have died and large portions of other
beds have disappeared. A nearly complete decline of eelgrass at Cape Ann, Massachu-
setts in 1984 (Dexter, 1985), has been linked to disease through subsequent sampling
of a few remnant eelgrass plants which proved to be infected.
Other declines in eelgrass have been reported. Loss of eelgrass from estuaries in
both North America and Europe has been shown to result from pollution of coastal
regions (Jones and Tippie, 1983; Kemp etal., 1983;Neinhuis, 1 983; Orth and Moore,
1983). Elsewhere in the world, other seagrasses have also experienced pollution-re-
lated declines (Cambridge and McComb, 1984). Loss of eelgrass was reported from
several other areas along the east coast of the U. S. in 1986, including Buzzards Bay
EELGRASS WASTING DISEASE 561
and Cape Cod, Massachusetts, where the cause was pollution and Great South Bay,
New York, where the cause was shading by a plankton bloom. Although the wasting
disease has also been discovered in some of these locations, there is no evidence that
it has contributed to any of these declines. However, we believe that the combined
effects of the wasting disease and pollution could devastate eelgrass populations.
Whether the current eelgrass wasting disease will produce a catastrophic eelgrass
decline is unknown. Although the current wasting disease has not yet caused a wide-
spread decline of eelgrass populations, the disease poses a major threat to coastal
fisheries, waterfowl populations, numerous marine habitats, and the health of estua-
rine environments. What has produced the recurrence of this widespread epidemic,
what circumstances might bring this disease to the stage that devastates eelgrass popu-
lations, and what role environmental factors may play in this transition remain mat-
ters of conjecture and further investigation.
ACKNOWLEDGMENTS
This research was supported by the New Hampshire Waterfowl Association,
the New Hampshire Department of Fish and Game, and the National Estuarine
Research Reserve program, NOAA. Jackson Estuarine Laboratory contribution
number 146.
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Manage. 8: 269-275.
CAMBRIDGE, M. L., AND A. J. McCOMB. 1984. The loss of seagrasses in Cockburn Sound, Western Aus-
tralia. I. The time course and magnitude of seagrass decline in relation to industrial development.
Aquat. Bot. 20: 229-243.
COTTAM, C., AND C. E. ADDY. 1947. Present eelgrass conditions and problems on the Atlantic coast of
North America. Twelfth North American Wildlife Conference, San Antonio, TX. Pp. 387-398.
COTTON, A. D. 1933. Disappearance of Zostera marina. Nature 132: 277.
DEN HARTOG. C. 1987. "Wasting disease" and other dynamic phenomena in eelgrass beds. Aquat. Bot.
27:3-14.
DEXTER, R. W. 1985. Changes in the standing crop of eelgrass, Zostera marina L., at Cape Ann, Mass.
since the epidemic of 1932. RhodoraSl: 357-366.
FISHER-PIETTE, E., R. HEIM, AND R. LAMI. 1932. Not preliminaire sur une maladie bacterienne des Zost-
eres. Comptes Rendus Acad. Sci. Paris 195: 1420-1422.
HUNTSMAN, A. G. 1932. Progress Reports. Atlantic Biol. Sta. and Fish. Exp. Sta. 5: 1 1-15.
JOHNSON, T. W., JR., AND F. K. SPARROW. 1961. Fungi in Oceans and Estuaries. J. Cramer, Weinheim.
Pp. 57-62 and 225-229.
JONES, G. A., AND V. K. TIPPIE. 1983. Chesapeake Bay Program: Findings and Recommendations. U. S.
Environmental Protection Agency, Philadelphia, 48 pp.
KEMP, W. M., W. R. BOYNTON, R. R. TWILLEY, J. C. STEVENSON, AND J. C. MEANS. 1983. The decline
of submerged vascular plants in Upper Chesapeake Bay: Summary of results concerning possible
causes. Afar. Tech. Soc. J. 17: 78-89.
LEWIS, H. F., AND W. R. TAYLOR. 1933. Disappearance of Zostera in \912.Rhodora35: 152-154.
MOFFIT, J., AND C. COTTAM. 1 94 1 . Eelgrass depletion on the Pacific coast and its effect upon black brant.
U. S. Fish and Wildl. Sen'., Wildl. Leaflet 204: 1-26.
NEINHUIS, P. H. 1983. Temporal and spatial patterns of eelgrass (Zostera marina L.) in a former estuary
in the Netherlands, dominated by human activities. Afar. Tech. Soc. J. 17: 69-77.
OLIVE, L. S. 1975. The Mycetozoans. Academic Press, New York. 293 pp.
ORTH, R. J., AND K. A. MOORE. 1983. Chesapeake Bay: an unprecedented decline in submerged aquatic
vegetation. Science 22: 5 1-52.
PETERSON, H. E. 1933. Wasting disease of eelgrass (Zostera marina). Nature 132: 1004.
PETERSON, H. E. 1934. Wasting disease of eelgrass. Nature 134: 143.
POKORNY, K. S. 1967. Labyrinthula. J. Protozoa. 14: 697-708.
562 F. T. SHORT ET AL.
PORTER. D. 1988. Labyrinthulomycota. In Handbook of Protoctista, L. Margulis et ai, eds. Jones and
Bartiett, Boston.
RASMUSSFN, E. 1973. Systematics and ecology of the Isefjord marine fauna (Denmark). (With a survey of
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(973.495pp.
RASMUSSEN, E. 1977. The wasting disease of eelgrass (Zostera marina) and its effects on environmental
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C. Helfferich, eds. Marcel Dekker, New York.
RENN, C. E. 1934. Wasting disease of Zostera in American waters. Nature 134: 416.
RENN, C. E. 1935. A mycetozoan parasite of Zostera marina. Nature 135: 544-545.
RENN, C. E. 1936. The wasting disease of Zostera marina. Biol. Bull. 70: 148-158.
SHORT, F. T., A. C. MATHIESON, AND J. I. NELSON. 1986. Recurrence of the eelgrass wasting disease at
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TAYLOR, W. R. 1933. Epidemic among Zostera colonies. Rhodora 35: 1 86.
THAYER, G. W., W. J. KENWORTHY, AND M. S. FONESCA. 1984. The ecology of eelgrass meadows of the
Atlantic coast: a community profile. U. S. Fish Wildl. Serv. FWS/OBS-84/02, 147 pp.
WATSON, S. W., AND E. J. ORDAL. 1957. Techniques for the isolation of Labyrinthula and Thraiistochy-
trium in pure culture. J. Bacterial. 73: 589-590.
YOUNG, E. L. 1938. Labyrinthula on Pacific coast eelgrass. Can. J. Res. 16: 115-117.
YOUNG, E. L. 1943. Studies on Labyrinthula. The etiologic agent of the wasting disease of eelgrass. Am. J.
Bot. 33: 586-593.
Reference: Biol. Bull. 173: 563-574. (December, 1987)
ABSTRACTS OF PAPERS PRESENTED AT THE MARINE BIOLOGICAL
LABORATORY: NORTHEASTERN REGIONAL CONFERENCE
ON DEVELOPMENTAL BIOLOGY
24-27 SEPTEMBER, 1987
The conference was organized by Lorraine Gudas (Dana-Farber Cancer Institute and
Harvard Medical School) and Greenfield Sluder (Worcester Foundation for Experi-
mental Biology). Support for graduate student participation was provided by the Na-
tional Science Foundation (DCB-8713713).
ABSTRACTS
In vivo properties of primary cilia in cultured kidney epithelial cells. S. S. BOWSER,
K. E. ROTH, AND C. L. RIEDER (Wadsworth Ctr. Labs. & Res., NY State Dept.
Health, Albany, NY).
A single primary (1°) cilium is generated from the parent centriole of the centrosome in most embry-
onic and terminally differentiated cell types. 1° cilia are oriented perpendicular to the dorsal cell surface,
which makes them difficult to observe by conventional LM preparative methods. As a result, their proper-
ties and function remain unknown. To overcome this restriction, we grow kidney epithelial cell monolayers
on plastic films. A side view of the monolayer is obtained by folding the film, cell-side out, and sandwiching
it in a simple coverslip chamber. Along the folded edge, 1° cilia are perpendicular to the optical axis and
readily imaged by real-time or time-lapse video-LM. 1° cilia appear as rigid, 0.25 /im-diameter rods and
their average length is characteristic for each kidney cell line examined. 1° cilia show no beating motions
but passively bend in response to flow. 1 ° cilia frequently possess one or more varicosities or swellings along
the ciliary shaft. Time-lapse video LM reveals that these varicosities move bidirectionally between the
ciliary base and tip. Same-cell correlative LM and high-voltage EM indicates that these varicosities are
delimited by the ciliary membrane and contain a granular cytoplasmic matrix devoid of vesicles or other
organelles. Our finding that cytoplasmic transport occurs along 1 ° cilia suggests they may serve as a conduit
for exchange between the extracellular milieu and the nucleus.
Time-lapse photographic study of neural tube closure defects in the chick. K. T. BUSH,
R. G. NAGELE, AND H. LEE (Department of Biology, Rutgers University, Cam-
den, NJ and Department of Pediatrics, University of Medicine & Dentistry of
New Jersey-School of Osteopathic Medicine, Camden, NJ).
A new method was described whereby early chick embryos were explanted using a modified New's
technique [in which (1) avian Ringer's solution replaced Pannett-Compton saline and (2) the vitelline
membrane with an adherent blastoderm was placed over the glass ring] and photographed with a Nikon
Diaphot inverted microscope equipped with both phase-contrast optics and photomicrographic accessories
maintained in an incubator. This method permits for the first time recording of sequential changes in the
morphology of early chick embryos. Specifically, 4-somite stage embryos were explanted, grown in nutrient
medium with or without a teratologic dose of xylocaine (lidocaine HC1, Elkins-Sinn), and were photo-
graphed at 2-3 hour intervals. We found, among other things, that a characteristic neural tube closure
defect often seen in the brain region of xylocaine (200 ^g/ml)-treated chick embryos was consequence of
failure of the neural tube to withstand the tension generated by the rapidly expanding cephalic region
which occurred when the corresponding controls had advanced to 1 1 -somite stage.
Interspecific comparisons of the period gene. H. V. COLOT, G. PETERSEN, Q. Yu,
D. WHEELER, J. C. HALL, AND M. ROSBASH (Department of Biology, Brandeis
University, Waltham, MA).
We have cloned and sequenced substantial portions of the period (per) gene from Drosophila pseudo-
obscura, D. virilis, and D. simulans as part of our continuing investigation into the function of the per gene
563
564 DEVELOPMENTAL BIOLOGY CONFERENCE ABSTRACTS
in circadian and ultradian rhythms. The D. pseudoobscura gene has been examined in the most detail and
compared with L'IC well-characterized per gene of D. melanogaster. The locomotor activity of D. pseudoob-
scura d; lantially in period length and strength, as well as overall pattern, from that of D. melano-
gaster. DNA sequence comparison between these two species shows blocks of diverged coding sequence
inte; ;ed with blocks of conserved coding sequence. Surprisingly, the TG-repeat region of D. melano-
gaster is replaced in D. pseudoobscura by a longer, totally different repeat that is preceded by only a few
TG and SG pairs. (Note that deletion of most of the TG region of D. melanogaster affects the species-
specific courtship song rhythm but not the circadian rhythm.) Therefore, the TG repeat is probably not a
series of (obligatory) glycosaminoglycan attachment sites but may confer species-specific properties on the
protein. Experiments involving cross-species transformation of the per gene have been initiated.
Pattern formation with Fusarium illustrates a principle for generation of cell pattern. J.
DAS AND H. BUSSE (Biochem. Inst. Med. Fak., Univers. Kiel, GFR and Harvard
Medical School, Dept. Physiology & Biophys., Boston, MA).
The fungus Fusarium usually grows by extending its mycelium outwards. If hyphae try to grow into a
region already occupied by the mycelium of a neighboring fungus, then either of the two following pro-
cesses can occur: (1) the hyphae do not recognize each other and intermingle; or (2) the hyphae recognize
that the neighboring region is already occupied and stop growing.
In the second case, the pattern formed in the plane of growth can be understood in terms of a specific
distance measure. A distance measure is a mathematical function which allows an object (a hypha in our
case) to determine its distance relative to other objects in the neighborhood. If the distance measure reaches
a preset limit a decision is made. In our example: hyphae stop to grow. That distance measures can produce
spatial pattern is illustrated for Fusarium. It is likely that patterns in differentiating animal tissue can also
be described by distance measures.
Integrin structure, function, and developmental expression. D. W. DESIMONE, M. A.
STEPP, R. PATEL, E. MARCANTONIO, AND R. O. HYNES (Center for Cancer Re-
search, M.I.T., Cambridge, MA).
The integrins are a family of cell-surface receptor complexes that participate in a variety of cell-cell
and cell-extracellular matrix interactions, which are involved in morphogenesis, cell migration, hemostasis,
oncogenesis, and immune function. Integrins exist as heterodimers made up of distinct a and 0 subunits.
We have isolated cDNA clones of integrin subunits from several species. Comparisons among the cDNA
sequences of p subunits from Xenopus laevis, chicken, mouse, and human reveal that the integrin family
can be organized into at least three classes. We estimate that the three classes of /3 subunits diverged from
a common ancestral gene at an early stage of metazoan evolution. Functional heterogeneity within integrin
classes is probably conferred by a large number of distinct, yet structurally related, a subunits. The struc-
tural relatedness of the integrin receptors will be discussed in view of results obtained by Northern and
Southern blot hybridization analyses, cDNA sequencing, and immunologic cross-reactivity. Similar ap-
proaches are being used to determine the developmental expression and functional significance of the
integrins during amphibian embryogenesis.
Identification of a calcium-calmodulin dependent protein kinase associated with the
sea urchin mitotic apparatus. J. H. DINSMORE AND R. D. SLOBODA (Dept. of
Biological Sciences, Dartmouth College, Hanover, NH 03755).
Mitotic apparatuses (MAs) isolated from the sea urchin, Lytechinus pictus, were assayed for protein
kinase activity; phosphorylation of endogenous MA substrates was determined by SDS-polyacrylamide gel
electrophoresis and autoradiography. MAs were isolated by the Salmon (Methods Cell Biol. 25: 71-102,
[1982]) detergent lysis method with the following modifications: embryos were treated with 3% hexylene
glycol for 3 min prior to lysis; 50 nM GTP and protease inhibitors were included in the lysis buffer; and
MAs were washed two times in 50 mM PIPES, pH 6.9 plus 10 jzA/taxol to obtain the final MA preparation.
Isolated MAs were incubated with [732P]ATP, Mg2+, and one of the following; cAMP; cGMP; Ca2+; cal-
modulin; Ca2+ and calmodulin; Ca2+, calmodulin and calmidizolium. Specific phosphorylation of only a
single protein of Mr 62 kD occurred when both Ca2+ and calmodulin were present. Incubation of intact,
birefringent MAs with 40 fiM ATP plus 0.3 nM calmodulin resulted in an increased rate and extent of
depolymerization of MA microtubules in response to micromolar Ca2+. In the absence of either ATP or
calmodulin this effect of Ca2+ on microtubule stability was not observed. Finally, in vivo phosphorylation
experiments showed that protein phosphorylation in the embryo rose and fell with the cell cycle, with peak
DEVELOPMENTAL BIOLOGY CONFERENCE ABSTRACTS 565
levels of phosphorylation occurring at the metaphase-anaphase transition. The results suggest that specific
Ca2+-calmodulin dependent protein phosphorylation affects MA stability, and may be important in con-
trolling the metaphase-anaphase transition in vivo. [Supported by NSF BNS85-03597 to RDS and a R. M.
Cramer Graduate Fellowship and Sigma Xi Grant-in-Aid to JHD]
Inducible c-myc overexpression and F9 teratocarcinoma stem cell differentiation.
C. A. DIONNE AND L. J. GUDAS (Division of Cancer Genetics, Dana-Farber Can-
cer Institute and Pharmacology Department, Harvard Medical School, Bos-
ton, MA).
The retinoic acid-induced terminal differentiation of F9 teratocarcinoma stem cells is accompanied
by a very early and rapid decrease in c-myc proto-oncogene expression. We have isolated several F9 cell
lines which have stably integrated a tightly regulated, highly inducible c-myc expression vector. The mor-
phological and biochemical differentiation of these transfected cell lines was similar to that of F9 wild type
cells despite continuous induced c-myc overexpression throughout the differentiation period. In addition,
c-myc overexpression did not prevent the decreased rate of cellular proliferation in response to retinoic
acid. Our results argue that the previously reported dominant effects of c-myc on differentiation and cellu-
lar proliferation are not observed in embryonic stem cells such as F9 teratocarcinoma stem cells.
Nucleoprotein complexes that regulate gene expression in adipocyte differentiation:
direct participation ofc-fos. ROBERT J. DISTEL, HYO-SUNG Ro, BARRY S. RO-
SEN, DOUGLAS L. GROVES, AND BRUCE M. SPIEGELMAN (Dana-Farber Cancer
Institute and Department of Pharmacology, Harvard Medical School, Bos-
ton, MA).
Adipocyte differentiation is accompanied by the transcriptional activation of many new genes, includ-
ing a putative lipid binding protein termed adipocyte P2 (aP2). The aP2 gene contains a regulatory element
(FSE2) 124 bases 5' to its start of transcription. This DNA sequence binds nuclear factors in a sequence-
specific fashion as determined by its altered mobility in gel retardation assays. Deletion analysis of promo-
tor-linked transfection assays and competition of these constructions in cells with synthetic FSE2 elements
suggests that trans-acting factors bind in this region and act as negative regulators of aP2 gene activity in
preadipocytes. c-fos appears to participate directly in this nucleoprotein complex, as demonstrated by the
ability of antibodies to c-fos to disrupt specific binding of factors and the FSE2 sequence but not to factor-
binding sequences from several other genes. Antibodies to c-fos specifically immunoprecipitate protein
complexes covalently bound to FSE2 DNA via UV cross-linking.
Intracellular patterning and the problem of assembly. GARY W. GRIMES (Department
of Biology, Hofstra University, Hempstead, NY 1 1550).
Ciliated protozoa typically are described on the basis of their highly asymmetric pattern of the ciliature.
However, we have microsurgically induced a ciliate possessing bilateral symmetry which is stably inherited
during asexual (and presumably sexual) reproduction. Because these bilaterally symmetrical cells are essen-
tially "Siamese Twins" fused side-by-side, with one half the mirror image of the other, one would predict
that the asymmetry of the individual ciliary structures would correspondingly be mirror-imaged. However,
they are not. Rather, they are assembled typically (i.e., not mirror-imaged) and patterned in a mirror-
imaged manner, or they are assembled in an inverted fashion but outwardly organized in a mirror-imaged
pattern in order to correspond to the global asymmetry of the mirror-imaged half of the cell. These data
suggest the presence of a global patterning mechanism imposed on the cortex of these ciliates to which the
individual elements of the ciliature must respond during their assembly. Constraints on the way in which
the ciliature can be assembled thus determines whether or not they are assembled in a "typical" or inverted
manner. The general principles of "directed assembly" and "directed patterning" are emphasized by these
studies. Supported by Research Grants from the NSF and Hofstra University.
Cell surface reorganization in the fertilized egg of the zebrqfish. N. H. HART AND
J. S. WOLENSKI (Department of Biological Sciences, Rutgers University, New
Brunswick, NJ).
The time-course of surface reorganizational events and their sensitivity to microfilament inhibitors in
the fertilized egg of the zebrafish (Brachydanio) were studied with light and electron microscopy. A single
566 DEVELOPMENTAL BIOLOGY CONFERENCE ABSTRACTS
sperm binds to a predetermined site on the egg oolemma within 5 s of insemination. A distinct fertilization
cone formed at this site between 45-60 s. Complete incorporation of sperm head, midpiece, and a portion
of the ftageilum occurred between 60 and 120 s. The second polar body formed by 4 min near the site of
gamete fusion. Cortical granule exocytosis, initiated beyond the site of gamete union by 30 s, was completed
by 4 I he reaction in the vicinity of the fertilizing sperm was visible by 60s. Freeze fracture analysis
showed higher IMP density on the P leaflet of the egg plasma membrane than on the P face of the cortical
granule membrane. These differences persisted after cortical granule breakdown, suggesting that the two
membrane domains do not mix rapidly. Cortical granule exocytosis and fertilization cone formation were
unaffected by either cytochalasin B or D ( 1 0 /ug/ml) treatment. Sperm incorporation was inhibited by either
10 ng/ml CB or 50 fig/ml CD. Sperm entry into the egg appears to require actin polymerization.
Molecular studies of biological rhythms in Drosophila. F. R. JACKSON AND K. J.
ELLIOTT (Worcester Foundation for Experimental Biology, Shrewsbury, MA).
The fruitfly Drosophila is an excellent model system for genetic studies of biological clocks. As a
consequence, several Drosophila genes have been identified that determine the properties of biological
oscillators. One of these genes, period (per), encodes a proteoglycan-like glycoprotein whose function is
currently being investigated in several laboratories. We have recently employed per-gene probes to identify
and isolate segments of Drosophila genomic DNA containing related sequences. One of these '/w-homolo-
gous' clones appears to encode a proteoglycan which contains Thr-Gly repeats similar to those seen in the
per protein. Experiments are underway to determine the relevance of this '/^"-homologous' gene for the
development and/or maintenance of circadian rhythms.
Another Drosophila clock gene, Andante, maps to chromosomal region 10E and lengthens periods of
circadian and ultradian rhythms. We have cloned DNA sequences from the 10E region, and initiated a
chromosomal walk towards the And locus. Chromosomal breakpoints which flank the And gene are cur-
rently being localized in cloned sequences to define the physical limits of the locus.
Isolation and characterization of an mRNA sequence (ERA- 1) exhibiting a rapid and
protein synthesis independent induction during the retinoic acid- induced differen-
tiation ofteratocarcinoma stem cells. G. J. LAR.OSA AND L. J. GUDAS (Program
on Cell and Devel. Biology, Harvard Medical School and Dana-Farber Cancer
Institute, Boston, MA).
Vitamin A and its derivatives (retinoids) exhibit profound effects on the proliferation and differentia-
tion of many cell types. F9 teratocarcinoma stem cells, which differentiate into non-tumorigenic primative
endoderm cells in response to retinoic acid (RA), serve as an excellent in vitro model for molecular studies
of cellular differentiation and the mechanism by which RA can set this complex process into motion. In
order to begin to analyze events which occur rapidly after RA treatment of F9 cells, we have constructed a
cDNA library from RNA of F9 cells treated for 8 h with RA. By screening with a cDNA probe enriched
for RA-induced sequences by subtractive solution hybridization, we have been able to isolate a cDNA
clone for an RNA which exhibits the properties of a primary target of RA action. This gene, £arly /?etinoic
/lcid-1 (ERA-1), encodes a 2.2 kb polyadenylated RNA which is rapidly induced by RA, in a dose-depen-
dent manner, both in the absence or presence of protein synthesis inhibitors. The isolation of this gene
sequence demonstrates that RA can influence gene expression very rapidly and also provides us with an
important tool to further analyze the initial intracellular action of RA in this F9 differentiation model
system.
Cellular interactions during amphibian gastrulation. J. LEBLANC (The College of
Staten Island, CUNY, NY), M. YODER AND I. BRICK (New York Univer-
sity, NY).
Epithelial movement during amphibian gastrulation is responsible for much of the reorganization
that establishes the primitive body plan. If cells are to form an epithelium and then exhibit epithelial
translations, sufficient intercellular cohesion, adhesions along their adjacent edges, would be required to
maintain integrity. Cellular interactions at the dorsal and lateral lip areas of the blastopore in Ranapipiens
embryos at various stages during gastrulation have been investigated by TEM.
Areas of unspecialized apposition and desmosomes were observed between adjoining cell surfaces ( 1 )
of lip cells lining the blastoporal groove, (2) of lip cells at the surface of the groove and underlying lip cells,
(3) of cells of the yolk plug lining the blastoporal groove, (4) of yolk plug cells at the surface of the groove
and underlying yolk plug cells, and (5) of lip and yolk plug cells. The cellular arrangements and intercellular
DEVELOPMENTAL BIOLOGY CONFERENCE ABSTRACTS 567
junctions suggest that the layer of lip cells and the layer of yolk plug cells that line the blastoporal groove
are both an epithelium.
Primordial germ cells of day 5 chick embryos. B. Y. LEE, K. T. BUSH, R. G. NAGELE,
AND H. LEE (Department of Pediatrics, University of Medicine & Dentistry of
New Jersey-School of Osteopathic Medicine, Camden, NJ and Department of
Biology, Rutgers University, Camden, NJ).
Primordial germ cells (PGCs) of the chick are initially identifiable with certainty in the germinal cres-
cent area of the definitive streak stage embryo. As embryonic development proceeds, most PGCs migrate
to the area vasculosa, enter blood vessels, and then reach the gonads. By day 5 of development, nearly all
PGCs, which will eventually settle in the gonads, are already in the gonads. These PGCs are distinguishable
from somatic (germinal epithelial) cells in that they have well-defined cell boundaries, numerous periodic
acid Schiff-positive granules, prominent lipid droplets, ring-shaped Golgi complex, and fragmented
nucleoli. They appear to relocate themselves in an ameboid fashion within the gonads. Of the several
methods used for isolating PGCs from the gonads, treatment with 0.2% collagenase-0. 1% trypsin inhibitor
in calcium- and magnesium-free Hanks' balanced salt solution followed by the discontinuous Percoll densi-
ty-gradient centrifugation was found to be most effective. Isolated PGCs, when grown in vitro, retained
both their distinguishing features and the ability to migrate actively.
Leukemic viral expression with induced changes in hematopoietic microenvironment.
G. P. LEONARDI, M. MANTHOS, J. LoBuE, D. ORLIC, AND J. MITRA (Dep't of
Biology, New York Univ., New York, NY and Dep't of Anatomy, N. Y. Medical
College, Valhalla, NY).
A transplantable, granulocytic leukemia has been established from BALB/c mice inoculated with a
variant of Rauscher Leukemia Virus that normally induces severe derangement of erythropoiesis (RLV-
A; LoBue et al, 1972). Six weeks before, and following inoculation of this erythroblastic virus, these mice
were kept at elevated red cell volumes by injection of 75% packed red cells (i.e.. hypertransfusion) every 7
days. Such hypertransfusion in mice eliminates red cell formation and alters the microenvironment mak-
ing it more suitable for granulopoiesis. Development of the "typical" RLV-A pathogenesis resulting in
erythroblastosis and fatal anemia was not observed in these animals. Instead, the appearance of massive
numbers of leukocytic elements including transplantable monomyelocytic leukemic cells was seen in four
of the six animals by 1 8-40 weeks post-viral inoculation. Hypertransfused control animals did not exhibit
these reactions. Hypertransfusion eliminates production of red cells and their precursors, and modifies
bone marrow stromal cells thus changing the microenvironment (Brookoff and Weiss, 1982). Sustained
hypertransfusion by ( 1 ) eliminating virally susceptable erythroid cells or (2) modifying the hematopoietic
microenvironment or (3) as a consequence of both resulted in the expression of a different viral oncogenic
expression. The authors acknowledge the generous financial support of Dr. and Mrs. P. C. Chan for these
studies.
Interleukin-2 (IL-2) distribution in adult newts (Notophthalmus viridescens) during
regeneration and following non-amputational wounding. M. F. LOMBARD' AND
R. E. SiCARD2 ('Department of Biology, Regis College, Weston, MA; and De-
partment of Pediatrics, Rhode Island Hospital, Providence, RI).
A role for the immune system in epimorphic regeneration has been proposed, but not proved. Accord-
ingly, [3H]-IL-2 (a potent lymphokine that modulates immune response through its actions on macro-
phages and lymphocytes) was used to explore changes in distribution and availability of IL-2 responsive
cells during regeneration and after non-amputational wounding. IL-2 content was reduced in major hemo-
poietic organs 2-8 days after trauma. Progressive increases, to control levels, occurred in livers of both
groups; however, IL-2 content remained depressed in spleens of regenerating, but not "wounded," animals
between 15-21 days post-trauma. During this same period, IL-2 content was reduced peripherally, as
reflected by decreased content in non-traumatized forearms. Initially, IL-2 content of non-amputational
wound sites was low, but progressively increased from 8-2 1 days post-trauma. In contrast, IL-2 content of
stumps and blastemas decreased between 2-21 days postamputation. The apparent pattern of availability
and distribution of IL-2 responsive cells during regeneration differed from that seen during repair of non-
amputational wounds. These data are consistent with immunological influence on regeneration and sug-
gest that further exploitation of IL-2 as a probe might help elucidate that role.
568 DEVELOPMENTAL BIOLOGY CONFERENCE ABSTRACTS
Sea urchin H2A.F/Z: an unusually conserved H2A variant gene. ROBERT MclSAAC,
HEIDI MILLER, CAROL A. BRENNER, CATHERINE NOCENTE-MCGRATH, SUSAN
FRANCIS, AND SUSAN G. ERNST (Department of Biology, Tufts University, Med-
forcL MA).
Sea urchin development is distinct in its sequential and overlapping use of multiple histone classes.
In addition, variants have been isolated which are differentiation specific. We have identified a cDNA clone
that encodes an H2A variant histone gene which is distinct from any class of sea urchin histones. The
coding region is 74% homologous to the chicken H2A.F gene and a remarkable 97% conservation exists
when comparing the putative amino acid sequences. Developmental expression does not coincide with
any embryonic histone class and the message has been found in all adult tissues examined. The 1.7 kb
transcript contains an unusually long 3' untranslated region and binds to oligo dt cellulose. In contrast to
the early gene set, this sequence is present at only a few copies per haploid genome. Both the sea urchin
and the chicken gene encode a protein related to the replication-independent protein H2A.Z found in
vertebrates. This protein is present at a level of 1 - 1 0% of the total H2A. Its low abundance, highly conserved
nature, and broad phylogenetic distribution all suggest a unique role for this protein in chromatin organi-
zation.
This work was supported by a Biomedical Research Grant and Tufts University Faculty Research
Award to Susan G. Ernst.
Luminescence proteins exhibit circadian rhythms but their mRNAs do not. D.
MORSE, P. MILOS, E. Roux, AND J. W. HASTINGS (Biological Labs, Harvard
University, Cambridge, MA 02 1 38).
At least three components of the bioluminescent system in Gonyaulax polyedra are under circadian
regulation: the substrate (luciferin), the luciferin binding protein (LPB), and the enzyme luciferase. LPB
cDNA was isolated by immunological screening from a cDNA library subcloned into an expression vector.
Its identity was confirmed by in vitro translation of hybrid selected mRNA. Northern hybridization to
mRNAs isolated at 8 different time points spanning a 24-hour period showed that the amounts of the LBP
mRNA were invariant with time. A putative luciferase cDNA has also been isolated and experiments
similarly indicate constant levels of the corresponding mRNAs. In a complementary approach, in vitro
translation of the mRNAs used in the Northern experiments showed equal synthesis of LBP at all time
points. These results indicate that circadian control of the amount of binding protein is exerted at the
translational rather than the transcriptional level.
The guinea pig sperm surface protein PH-20 is anchored in the membrane by a phos-
phatidylinositol lipid. B. PHELPS, P. PRIMAKOFF, D. E. KOPPEL, AND D. G.
MYLES. (Dept. of Physiology, University of Connecticut Health Center, Farming-
ton, CT).
PH-20 is an integral protein of the guinea pig sperm plasma membrane, for which there is evidence
for a role in sperm-zona pellucida binding [Primakoff et ai, J. Cell Biol. 101: 2239 (1985)]. PH-20 is
uniformly distributed over the entire cell surface of testicular sperm, but is localized to the posterior head
region of distal cauda epididymal sperm. Following the exocytotic acrosome reaction the plasma mem-
brane becomes contiguous with the inner acrosomal membrane and PH-20 migrates to the anterior head
region [Myles and Primakoff, J. Cell Biol. 99: 1634 (1984)]. Measurement of the diffusion coefficient of
PH-20 using the technique of fluorescence recovery after photobleaching (FRAP), show that PH-20 diffu-
sion on testicular sperm is highly restricted within the plane of the bilayer, while PH-20 on acrosome-
reacted sperm is freely diffusing. At the intermediate stage, acrosome-intact cauda epididymal sperm, the
protein diffuses at a rate between these two extremes; it is mobile but not freely diffusing. Exposure of
testicular sperm, acrosome-intact, and acrosome-reacted epididymal sperm to a phosphatidylinositol-spe-
cific phospholipase C results in the release of PH-20 from the cell surface. Thus, a cell that is no longer
capable of protein synthesis is able to modulate, during its differentiation, both the surface distribution and
the rate of diffusion of an integral membrane protein that is anchored in the bilayer by a lipid. Supported by
GM23585 to D.E.K. and NIH HD 16580 to D.G.M.
DEVELOPMENTAL BIOLOGY CONFERENCE ABSTRACTS 569
Molecular analyses of early neural pattern decisions in Xenopus. CAREY PHILLIPS
(Department of Biology, Bowdoin College, Brunswick, ME 0401 1).
Orientation of the Xenopus nervous system results from an interplay between the animal-vegetal axis,
established during oogenesis, and intracellular rearrangements, apparently cued by the point of sperm
entry. Many subsequent cellular pathway choices are necessarily involved in construction of a nervous
system and these choices may be reflected by changes in molecular synthetic patterns. Several molecular
probes have been constructed to use in assaying for early determinative events leading to neural differentia-
tion. Epi 1 , a monoclonal antibody, is being used as a molecular probe to distinguish between cells proceed-
ing along a neural pathway from cells destined to become epidermis. Using the Epi 1 probe, we have
determined that the position of the presumptive neural tissue is approximately, although reversibly, estab-
lished by at least the third cleavage division. We have also determined that the information necessary for
expression of the Epi 1 antigen is associated with the cortex of the uncleaved embryo. The molecular nature
of this information is being addressed experimentally. We are now in a position to use Epi 1 and other
molecular probes to study how a series of pathway choices might interact to produce the final position of
neural structures.
Protein-DNA interactions on the 5' non-transcribed spacer of Tetrahymena ther-
mophila rDNA. K. RIEKKI AND R. E. PEARLMAN (Department of Biology, York
University, Toronto, Ontario, Canada, M3J 1P3).
The 5' non-transcribed spacer (NTS) of the rDNA of T. thermophila contains sequences specifying a
number of important functions including bidirectional origin of replication, promotion of RNA polymer-
ase I catalyzed transcription, nucleosome phasing, rDNA copy number control, and topoisomerase I cleav-
age. We have initiated studies to correlate structure with function and to look at sequence specific protein-
DNA interaction in this region.
Various restriction fragments in the 5' NTS have extremely anomalous mobility when electrophoresed
through polyacrylamide gels suggesting sequence directed bending. In the center proximal (650 bp) Taq\-
Xba\ fragment, the bending locus appears to be in the 3' portion of this fragment. Using crude nuclear
protein extracts and an electrophoretic mobility shift assay to assess protein-DNA interaction, we have
demonstrated protein-DNA interaction in this Taq\-Xba\ fragment. Greater interaction and specificity
appears to occur with the 5' region of this sequence. Binding of protein from the same nuclear extracts also
occurs to the 420 bp Xba\-Xba\ fragment. Competition for this binding with unlabelled DNA from wild
type C3 and from the rmm 1 mutation (Larson el ai, Cell 47: 229-240 (1986)) suggests interesting and
possibly functionally significant specificity of this interaction. DNase I footprint experiments and protein
fractionation are in progress in attempts to further assess the specificity of these protein-DNA interactions.
Effect of photoperiod and melatonin in glucosaminidase activity during newt limb
regeneration. M. RIVERA, R. APONTE, F. CALIMANO, AND F. VALLES (Cayey
University College, Department of Biology, Cayey, P.R. 00633).
The effect of continuous light, continuous darkness, and melatonin on the activity of the lysosomal
enzyme N-acetyl glucosaminidase during limb regeneration in the newt Notophthalmus viridescens was
studied. Animals exposed to continuous light showed more enzyme activity than the controls during the
first 1 2 days after amputation. In these animals the highest peak of activity was exhibited on day 7, whereas
the controls showed the highest peak at day 5. The pattern of activity is the same in both groups. In animals
exposed to continuous darkness, the highest peak is observed on day 10 after amputation; the activity
pattern is delayed as compared with controls. Animals whose forelimbs were amputated and were injected
with melatonin showed a decrease in enzyme activity. The highest peak was detected on day 12 after
amputation. The relationship between darkness and melatonin is discussed. Part of this work is supported
by NIH Grant 5 S14 RR02640.
570 DEVELOPMENTAL BIOLOGY CONFERENCE ABSTRACTS
Studies :>n a transplantable monomyelocytic leukemia and in the offspring from
es between transplanted and normal BALB/c mice. E. RODRIGUEZ, J. Mi-
TRA, AND J. LoBUE. (Department of Biology, New York University, New York
City, New York).
In vivo cytogenetical studies on a transplantable monomyelocytic leukemia (MML) initially induced
in female BALB/c mice (Fredrickson et al., J. Nat I. Cancer Inst. 48: 1597-1605, 1972) by the Rauscher
leukemia virus (RLV) has revealed: (a) The presence of a marker deleted chromosome 18 in all somatic
tissues examined (bone marrow, peripheral blood, and spleen) restricted to female transplanted cells; (b) a
high degree of centromeric associations mainly in peripheral blood; and (c) in MML transplanted mice,
chromosome #19 shows presence of NOR in addition to chromosomes 12, 15, 16, and 18, whereas control
animals have NORs on chromosomes 12, 15, 16, and 18.
The disease has been shown to cause abnormalities in meiosis (Segenreich et al., Genetics 113: s20,
1986). Male MML transplanted animals have been mated at mid stage of the disease (third day) with
normal females. Analysis of the offspring is presently being performed. Litter size and phenotypical charac-
teristics of the offspring were normal. To date no significant cytogenetical abnormalities have been found
in the somatic tissues of the offspring and hematological parameters on peripheral blood seem to be normal.
Localization oflaminin and collagen IV transcription in mouse teratocarcinomas and
embryos by in situ hybridization. M. B. ROGERS AND L. J. GUDAS (Dana-Farber
Cancer Institute and Department of Biological Chemistry and Molecular Phar-
macology, Harvard Medical School, Boston, MA).
Mouse F9 teratocarcinoma cells can be induced in culture to form two distinct extra-embryonic cell
types: parietal and visceral endoderm. F9 cells grown in monolayer and treated with retinoic acid (RA)
differentiate into parietal endoderm while cells allowed to form aggregates in the presence of RA form
embryoid bodies with an outer layer of visceral endoderm. Visceral endoderm is characterized by the
synthesis of alpha-fetoprotein and the basement membrane components laminin and collagen IV. The
transcription of these genes has been characterized by in situ hybridization of radioactive recombinant
probes to frozen sections of embryoid bodies. In contrast to previous observations from other laboratories
on basement membrane protein expression, laminin and collagen IV mRNAs were found in the outer cell
layer of embryoid bodies whether or not RA was present. Additional experiments are extending the tech-
nique to sections of preimplantion mouse embryos.
Meiotic chromosome studies in BALB/c mice bearing a transplantable mono-myelo-
cytic leukemia (MML) and in the offspring of normal females crossed with trans-
planted males. E. J. SEGENREICH, J. MITRA, AND J. LoBuE (Department of Biol-
ogy, New York University, New York, NY).
Meiotic chromosome studies have been performed on BALB/c mice transplanted with mono-myelo-
cytic leukemia (MML), an acute disease induced by a virally (RLV) transformed cell (Fredrickson et al.,
J. Natl. Cancer Inst. 48: 1597-1605, 1972). Results indicate that MML transplantation is associated with
increases in meiotic chromosomal aberrations. Levels of aneuploidy, polyploidy, structural alterations,
and precocious separations rise throughout the six day disease. Spermatogonial chromosomes also show
increases in aneuploidy, polyploidy, and structural alterations. These mitotic chromosomes exhibit a
higher degree of anomalies than those in other tissues of the leukemic animal (E. Rodriguez, unpub. data).
Successful mating of normal females and mid-stage (day 3) MML transplanted males has produced pheno-
typically and, as of one year of age, hematologically normal litters. Preliminary studies of the offspring
have revealed mostly insignificant changes in the frequencies of meiotic aberrations compared to the con-
trol litters. Only a slight increase in polyploidy is observed. Spermatogonial mitosis also shows slight rises
in the level of polyploidy along with centromeric affinity. The nature of susceptability to the pathological
parameters of the disease as well as to meiotic disturbances, will be determined by MML transplantation
into the offspring.
DEVELOPMENTAL BIOLOGY CONFERENCE ABSTRACTS 571
Spatial patterning of neuronal differentiation in the leech. M. SHANKLAND AND M. Q.
MARTINDALE (Department of Anatomy and Cellular Biology, Harvard Medical
School, Boston, MA).
We have examined the spatial patterning of neuronal differentiation in the metameric nerve cord of
the leech using an antibody to molluscan small cardioactive peptide B (SCP). In the adult, anti-SCP stains
an unpaired interneuron that is present only on the right or left side of the segmental ganglion in abdominal
segments 1-3 and 18-21. Both antibody staining and intracellular injection of Lucifer Yellow reveal that
this neuron extends an axon through the connective nerve into adjacent ganglia. The unpaired neuron is
equally likely to lie on either side of a given segment, but there is greater than 95% likelihood that it will be
on alternate sides (e.g., right:left) in successive segments. In the embryo, every abdominal ganglion initially
has a bilateral pair of these neurons and both express SCP-like immunoreactivity. As development pro-
ceeds one member of the pair ceases to stain in segments 1-3 and 18-21, while both members of the pair
cease to stain in segments 4-17. The right:left alternation observed in the mature pattern could arise be-
cause the unpaired neuron in one ganglion influences the rightileft decision in adjacent ganglia through its
interganglionic axon. We have obtained support for this idea by showing that the pattern of alternation is
locally disrupted by transecting the embryonic nerve cord prior to the onset of asymmetry.
Atrial natriuretic peptide (ANP) levels during rat ontogeny. R. E. SICARD AND J. C.
WERNER (Division of Pediatric Cardiology, Department of Pediatrics, Rhode Is-
land Hospital, & Program in Medicine, Brown University, Providence, RI).
ANP is an important modulator of fluid/electrolyte and pressure homeostasis. Its role and mecha-
nisms of action have been extensively explored in adults; however, adequate appreciation of the develop-
mental physiology of ANP is limited. Accordingly, we have begun exploring ANP metabolism during rat
development. Levels of immunoreactive ANP (ir-ANP) were determined by radioimmunoassay (reagents
from Amersham) in amniotic fluids or plasma during the last trimester of pregnancy and the first 2 months
of life. During the last trimester, amniotic fluids displayed a biphasic change in ir-ANP levels: rising from
<10 fmol/ml (13 days gestation) to 31.4 ± 7.3 fmol/ml (n = 29; 18-19 days gestation), then falling to < 10
fmol/ml (n = 5). Plasma concentrations of ir-ANP remained <10 fmol/ml during this time, then rose
during the first month [25.9 ± 7.1 fmol/ml, n = 21] to approximately adult levels [22.3 ± 5.4 fmol/ml, n
= 29]. These data suggest that significant changes in ANP metabolism and activity might be occurring
during the perinatal period in rats.
Localization of a gene product in Drosophila that influences biological rhythms.
K. K. SIWICKI, C. EASTMAN, M. ROSBASH, AND J. C. HALL (Biology Department,
Brandeis University Waltham, MA 02254).
The period (per) gene of Drosophila rnelanogaster influences the period of circadian rhythms as well
as an ultradian rhythm (period ca. 1 min) in the fly's courtship song. To begin to investigate how the gene
regulates these complex behaviors, we used an antibody specific for the per protein to examine its anatomi-
cal distribution with immunocytochemical methods. Staining was detected in the eyes, optic lobes, and
brain of wild type flies, but was not present in per~ mutants. Rhythmic fluctuations in the intensity of the
staining were apparent: the pattern was much more prominent at night than during the day. The day/night
cycling was observed during entrainment ( 12 h light/ 12 h dark) and continued to cycle in constant dark-
ness, suggesting that an endogenous circadian oscillator may regulate the expression of the per gene, which
itself influences circadian rhythms. Supported by NS-07873 to K.K.S. and GM-33205 to M.R. and J.C.H.
Centrosomes in echinoderm development. G. SLUDER, F. J. MILLER, K. LEWIS, AND
C. L. REIDER (Worcester Foundation for Experimental Biology, Shrewsbury,
MA 01545).
In animal cells, centrosomes are an ensemble of poorly understood organelles found at the ends of the
mitotic or meiotic spindles. Centrosomes are required for the establishment of the bipolar spindle axis,
chromosome orientation/alignment, chromosome movement, and the establishment of the cleavage fur-
row. We will briefly introduce the centrosome and then discuss the origin of the centrosomes used in the
development of the echinoderm zygote. We demonstrate for sea urchin eggs that the centrosomes used in
development come only from the sperm; the egg centrosome is apparently lost. We then describe our
572 DEVELOPMENTAL BIOLOGY CONFERENCE ABSTRACTS
characterization of centrosome inheritance in starfish eggs. This system is interesting because the egg is
fertilized at miosis I when it contains two functional centrosomes. However, only the male centrosome is
used in .."^velopment; the female centrosomes are functionally lost prior to first mitosis. We will describe
ongoi • <s xperiments which seek to: (a) provide an understanding of what functional and structural aspects
of the female centrosomes are lost; (b) show that male and female centrosomes have intrinsically different
stabilities; (c) determine when, during the meiotic sequence, the female centrosomes are destabilized.
Mouse cellular retinoic acid binding protein: cloning, cDNA sequence and mRNA
expression during the retinoic acid-induced differentiation ofF9 wild type andRA-
3-10 mutant teratocarcinoma cells. CAROL M. STONER AND LORRAINE J. GUDAS
(Dana-Farber Cancer Institute and Dept. of Pharmacology, Harvard Medical
School, Boston, MA).
Retinoic acid, a natural derivative of vitamin A (retinol), induces mouse F9 teratocarcinoma stem
cells to differentiate into nontumorigenic parietal endoderm cells. The mouse cellular retinoic acid binding
protein (CRABP) has been implicated in the mechanism of action of retinoic acid (RA). A mutant F9
teratocarcinoma cell line, RA-3- 10, which possesses less than 5% of the wild type level of CRABP activity,
fails to differentiate in response to RA. To study the role that CRABP plays in the RA-induced differentia-
tion process, we cloned the mouse CRABP cDNA and determined its full-length sequence. Southern analy-
sis of F9 genomic DNA hybridized to CRABP cDNA suggests that the CRABP gene is present as a single
copy gene in the haploid genome and that the gene contains two introns. Northern analysis shows that the
CRABP mRNA is a single, low abundant mRNA approximately 800 bases in length. The steady state
CRABP mRNA level remains relatively constant during the RA-induced differentiation of F9 wild type
cells. The CRABP mRNA level is approximately 20-fold lower in the RA-3- 10 mutant stem cells than in
F9 wild type stem cells.
Cyclins and the cell cycle of early clam embryos. K. I. SWENSON' AND J. V. RUDER-
MAN2 ('Dept. of Anatomy and Cellular Biology, Harvard Medical School, Bos-
ton, MA; 2Dept. of Zoology, Duke University, Durham, NC).
Fertilized oocytes of the surf clam require new protein synthesis in order to complete meiosis. New
protein synthesis is also required during each cell cycle of the early embryo for the completion of the
mitotic divisions as well (Hunt and Ruderman, unpub.). Possible candidates for these M-phase inducing
proteins are the cyclins A and B, the levels of which periodically accumulate and disappear as a function
of the mitotic cell cycle (Evans el ai, 1983. Cell 31: 389; Swenson el ai. 1986. Cell 41: 861; Westendorf,
unpub.). The suspected M-phase inducing role of cyclin A has been confirmed by showing that Xenopus
oocytes, when microinjected with in vitro synthesized SP6 cyclin A mRNA, enter meiosis. The activity of
cyclin B in this assay system is unknown. Recently we found that cyclin A was tightly associated with a
kinase activity. We are interested in the functional properties and specificities of this kinase activity and
its involvement, if any, in the ability of cyclin A to induce M-phase.
Molecular genetics of early embryogenesis in C. elegans. A. TELFER, D. LEVITAN, U.
GIAMBARELLA, AND D. T. STINCHCOMB (Harvard University, Cambridge, MA).
The first division of embryogenesis in the nematode, Caenorhabditis elegans, produces blastomeres
that display different cell division patterns and are determined to express different developmental poten-
tials. Mutations in one class of maternal effect genes disrupt early development: the first division produces
blastomeres that divide synchronously and fail to express certain differentiated cell types. These mutants
are termed par for they are defective in the partitioning of germ line granules, they fail to segregate factors
that control cell cycle rates, and they possibly fail to partition determinants of some cell types. We have
identified and isolated restriction fragment polymorphisms (RFLPs) on either side of ihepar-1 locus. DNA
between the two markers has been cloned. We will delimit the par- 1 gene by identifying maternal tran-
scripts and by microinjecting cloned DNA to assess its function. Similarly, a RFLP in or near the par-2
gene has been identified and adjacent DNA has been isolated. Characterization of the par gene products
may help us understand how early blastomeres become determined during development.
DEVELOPMENTAL BIOLOGY CONFERENCE ABSTRACTS 573
Retinoids and pattern formation in vertebrate limbs. CHRISTINA THALLER AND
GREGOR EICHELE (Dept. of Physiology, Harvard Medical School, Boston, MA).
All-/ra«5-retinoic acid induces striking digit pattern duplications when locally applied to the develop-
ing chick limb bud. Instead of the normal digit pattern (234) a mirror-symmetrical 432234 pattern can be
specified. Hence, retinoic acid closely mimics posterior limb bud tissue (known as the zone of polarizing
activity, ZPA) that causes very similar duplications when grafted to an anterior site of a host limb bud.
This raises an intriguing possibility: that retinoic acid is a natural signalling substance involved in limb
pattern formation. We discovered that chick limb buds contain endogenous retinoic acid and found that
retinoic acid but not its biologically inactive precursor retinol, forms a concentration gradient across the
limb anlage with a highpoint in the posterior domain of the limb bud, the part that contains the ZPA.
Moreover, the amount of endogenous retinoic acid detected in the limb bud is the same as required to
induce duplications when retinoic acid is provided exogenously. To examine whether retinoic acid is pro-
duced in situ, we locally applied its precursors, all-/ra«s-retinol and all-/ram-retinal, in radioactive form.
Both compounds are metabolized: retinol to retinal and retinal to retinoic acid. These observations
strengthen the hypothesis that retinoic acid is a local chemical mediator involved in the specification of
the anteroposterior limb pattern.
Region specific expression of mouse homeo box genes. M. F. UTSET' A. AWGULE-
wiTSCH2, W. McGiNNis5, AND F. H. RUDDLE' 2 (Departments of1 Human Genet-
ics, 2Biology, and 3Molecular Biophysics and Biochemistry, Yale University, P.O.
Box 6666, New Haven, CT 065 10).
Mammalian homeo box genes show extensive homology to Drosophila homeotic and segmentation
genes. As a step toward determining their functions, we are studying the patterns of homeo box gene
expression during mouse development. Results from a number of laboratories indicate that several mouse
homeo box genes display region specific patterns of expression within the developing central nervous sys-
tem and mesoderm. For example, Hox-2.1 expression within the central nervous system of the newborn
mouse and the 13.5 day mouse embryo appears restricted to the medulla and spinal cord, whereas at the
same stages Hox-3.1 expression is found within the spinal cord posterior to the third cervical vertebra
(Science 235: 1379-1382). At earlier embryonic stages Hox-3.1 expression is also detected in a spatially
restricted pattern in mesodermal tissues. These patterns of expression are consistent with the hypothesis
that mouse homeo box genes perform region specific developmental functions akin to those of Drosophila
homeotic genes.
Visualization ofactin with rhodamine phalloidin in thezebraftsh egg. J. S. WOLENSKI
AND N. H. HART (Department of Biological Sciences, Rutgers University, New
Brunswick, NJ).
The distribution of polymerized actin in zebrafish (Brachydanio) eggs was determined using rhoda-
mine phalloidin and fluorescence microscopy. Unactivated eggs exhibited a prominent and continuous
band of fluorescence just beneath the plasma membrane. Whole eggs showed little evidence of staining in
the remaining cortical cytoplasm. However, staining was visible around individual cortical granules in
cortical fragments prepared from formaldehyde-fixed cells. A prominent, narrow band of staining was also
detected at the periphery of activated eggs. Continuity of this fluorescent layer was interrupted at sites of
fusion between the egg plasma membrane and exocytosing cortical granules. Gaps in the fluorescence were
also evident in eggs treated with cytochalasin B. Our results suggest that polymerized actin is present in
cortices of eggs including the site of sperm entry.
Developmental regulation of three testis-specific genes during mouse spermatogenesis.
P. C. YELICK, Y. KWON, P. A. BOWER, K. C. KLEENE, AND N. B. HECHT (Depart-
ment of Biology, Tufts University, Medford, MA 02155).
Mouse spermatogenesis is the continuous process of development in which a progenitor germ cell
differentiates into mature spermatozoa. We have identified three testis-specific genes, mouse protamine 1
(mPl), mouse protamine 2 (mP2), and mouse transition protein 1 (mTPl), all of which are expressed in
the haploid phases of spermatogenesis. All three gene products are involved in the nuclear condensation
events which occur during the sequential replacement of the nucleosomal histones by mouse protamines.
574 DEVELOPMENTAL BIOLOGY CONFERENCE ABSTRACTS
All three genes are very similarly regulated. mPl, mP2, and mTPl genes are first transcribed in the round
spermatid stage of spermiogenesis. All three mRNAs are translationally regulated in that they are first
transcribed and then stored for up to eight days before they are translated. The translated forms of all three
mRNAs, as present on polysomes, differ from the RNP-complexed forms in that the poly (A)+ tails are
shortened by about 140 nucleotides. We also demonstrate the presence in rat and hamster of gene se-
quences and testis-specific mRNAs homologous to mPl and mP2 cDNAs. Comparison of protamine 1
and 2 gene expression in rat, hamster, and mouse demonstrates variable expression of the protamine 1 and
2 genes that is also reflected in the protamine 1 and 2 content of hamster, rat, and mouse sperm.
INDEX
A pH decrease at sperm activation, 3 1 1
ABRAMSON, CHARLES I., PHILIP M. ARMSTRONG,
ROBIN A. FEINMAN, AND RICHARD D. FEIN-
MAN, Signalled avoidance learning of eye with-
drawal in the green crab is predominantly Pav-
lovian in mechanism, 435
ABRAMSON, CHARLES I., see Richard D. Feinman,
437
Acrosome reaction, 3 1 1
Actin, 188,420
Actin filaments, 573
Adenosine depresses spontaneous transmitter re-
lease from frog motor nerve terminals by act-
ing at an A 1 receptor, 440
Aequorin, 420
Aging, 421
Aggression, 1 10
ALBERGHINA, MARIO, SERAFINA SALVATI, AND
ROBERT GOULD, Characterization of phos-
pholipid enzymes in squid axoplasm and giant
fiber lobe, 439
ALEXANDER, STEPHEN P., AND TED E. DELACA,
Feeding adaptations of the foraminiferan Cibi-
cides refulgens living epizoically and parasiti-
cally on the antarctic scallop Adamusstum col-
becki, 136
ALKON, DANIEL L., see Chong Chen, 442; and Alan
M. Kuzirian, 443
Aluminum flouride and GTP increase inositol
phosphate production in distal segments of
squid photoreceptors, 448
Ambient flow velocity, 222
Amino acid uptake, 504
Ammonium assimilation, 43 1
Amphibian gastrulation, 566
Amphipods, 429
Anaerobic metabolism of radula muscles, 277
Anatomical study of the retina of Nautilus pompil-
ius. An, 387
Antarctic echinoid reproduction, 126
Antarctica, 126, 136
Anthopleura elegantissima, 1 10
Anticoagulation, 423
Antiquity of transglutaminase: an intracellular en-
zyme from marine sponge cells enhances clot-
ting of lobster plasma, The, 423
Aplysia, 440
APONTE, R., see M. Rivera, 569
Arachidonic acid, 440
ARKETT, S. A., G. O. MACKIE, AND C. L. SINGLA,
Neuronal control of ciliary locomotion in a
gastropod veliger (Calliostoma), 5 1 3
ARMSTRONG, C. M., AND Y. PALTI, Calcium block
of K channels in squid giant fiber lobe neuron,
439
ARMSTRONG, CLAY M., see Gabriel Cota, 442; and
D. Swandulla, 447
ARMSTRONG, PHILIP M., see Charles I. Abramson,
435
Ascidia malaca egg, 355
Ascidian, 423, 428
Ascidian egg, 427
Assembly constraint, 565
Asynchrony, 435
ATEMA, JELLE, see Frank Corotto, 436; and Leslie
Sammon, 438
Atrial hormone, 57 1
Atrial natriuretic peptide (ANP) levels during rat
ontogeny, 571
AUGUSTINE, GEORGE J., see Milton P. Charlton,
44 1 ; and Stephen P. Smith, 446
Autoradiography utilizing [3H] phorbol esters is po-
tentially useful for cellular analysis of protein
kinase C activity in hippocampus slices, 443
Avidin, 425
Avoidance conditioning, 435
AWGULEWITSCH, A., see M. F. Utset, 573
Axis and muscle cell determination, 425
Axon, 443
B
Bacteria, 434
Bacterial selection, 434
Bacterial uptake of glutamic acid in oxic and anoxic
waters in a coastal pond, 434
Bacteriocytes, 260
BAKER, ROBERT, see Andrew Bass, 435
BANK, BARRY, see Alan M. Kuzirian, 443
BAR-EL, THEODORA, see David Kahan, 299
BARLOW, ROBERT B., JR., see Mary Anne Sydlik,
438; and Melissa R. Schneider, 448
Barnacle, 44 1
BARRY, SUSAN, R., Adenosine depresses spontane-
ous transmitter release from frog motor nerve
terminals by acting at an A 1 receptor, 440
BASS, ANDREW, MICHAEL WEISER, AND ROBERT
BAKER, Functional organization of the sonic
motor system in sea robins, 435
BATES, W. R., The totipotent development of my-
oplasm-enriched ascidian embryos, 423
BEAUCHAMP, KATHERINE A., see Isidro Bosch, 1 26
Behavior, 110,438
feeding, 437
withdrawal, 437
Behavioral experiments suggest G protein modula-
tion of calcium channels in Parameciurn, 445
Betaine, 299
Biological rhythms, 566
Bioluminescence, 489
Biomechanics, 433, 434
575
576
INDEX TO VOLUME 173
Biotin,42.'
Birefringence, 443
BISBAL, G!_ STAVO, Does copper affect the mating
behavior ofGammarus annulatus Smith, 1 873
(Amphipoda: Gammaridae)? 429
Bivalve, 205, 230
Bivalve hemocyanin: structural, functional and
phylogenetic relationships, 205
Blood clotting, 423
Blood-brain barrier, 42 1
Blue crab metal metabolism, 239
BORRERO, FRANCISCO, J., Tidal height and game-
togenesis: reproductive variation among popu-
lations ofGeukensia demissa, 160
BOSCH, ISIDRO, KATHERINE A. BEAUCHAMP, M.
ELIZABETH STEELE, AND JOHN S. PEARSE, De-
velopment, metamorphosis, and seasonal
abundance of embryos and larvae of the ant-
arctic sea urchin Sterechinus newnayeri, 1 26
Botryllus, 474
BOTTON, MARK L., AND ROBERT E. LOVELAND,
Orientation of the horseshoe crab, Limuluspo-
Ivphemus, on a sandy beach, 289
BOWER, P. A., see P. C. Yelick, 573
BOWLBY, MARK. R., see Michael I. Latz, 489
BOWSER, S. S., K. E. ROTH, AND C. L. RIEDER, In
vivo properties of primary cilia in cultured kid-
ney epithelial cells, 563
BRENNER, CAROL A., see Robert Mclsaac, 568
BREZINA, VLADIMIR, Do arachidonic acid metabo-
lites mediate modulation of K and Ca currents
by FMRF-amide in Aplysia neurons? 440
BRICK, I., see J. LeBlanc, 566
BROUWER, MARIUS, see David W. Engel, 239
BROZEN, REED, PETER SANDS, WILLIAM RIESEN,
GERALD WEISSMAN, AND LASZLO LORAND,
The antiquity of transglutaminase: an intracel-
lular enzyme from marine sponge cells en-
hances clotting of lobster plasma, 423
Bryozoan suspension-feeding, 222
Bugula, 430
BURGOS, M. H., see S. J. Segal, 426; and H. Ueno,
428
BURGOS, MARIO H., AND ROBERT B. SILVER,
Time dependent shift in fluorescence in gossy-
pol treated Arbacia sperm, 424
BUSH, K. T., R. G. NAGELE, AND H. LEE, Time-
lapse photographic study of neural tube clo-
sure defects in the chick, 563
BUSH, K. T., see B. Y. Lee, 567
BUSSE, H., see J. Das, 564
Busycon contrarium, 211
BUTT, ARTHUR M., A subarachnoid space in the
elasmobranch brain — macro and microscopic
evidence using large molecular weight fluo-
rescent markers, 42 1
BYRNE, PATRICIA M. A., Relationship between
trace metal distribution and sulfate reduction
in surface sediment, 429
C-Kinase activation mediated by proteolysis modu-
lates K+ conductances in Hermissenda B-pho-
toreceptors, 442
Ca channels, 44 1 , 445, 446, 447
Calcium, 420, 441,446
Calcium affects the birefringence response of the
squid giant axon, 444
Calcium block of K channels in squid giant fiber
lobe neurons, 439
Calcium channels required for neuropeptide release
in the intact nerve terminals of vertebrate neu-
rohypophyses are sensitive to w-conotoxin and
insensitive to dihydropyridines: optical studies
with and without voltage-sensitive dyes, 446
Calcium gradient, 425
Calcium ion, 439
Calcium transients are required for mitosis, 420
CALIMANO, F., see M. Rivera, 569
CALLAWAY, JOSEPH C., AND ANN E. STUART, The
effect of strontium, barium, and strychnine on
the synapse made by barnacle photoreceptors,
441
Calyptogena magnified, 260
Capitella, 430
Carnivorous ciliates, 299
CASE, JAMES F., see Michael I. Latz, 489
CASSIMERIS, LYNNE, see Shinya Inoue, 419
Cell
cycle, 572
division, 420
migration, 566, 567
permeabilization, 445
surface morphology, 355
Cell surface reorganization in the fertilized egg of
the zebrafish, 565
Cell volume regulation by molluscan erythrocytes
during hypoosmotic stress: Ca2+ effects on
ionic and organic osmolyte effluxes, 407
Cell volume regulation, 407
Cell-adhesion, 564
Cellular interactions during amphibian gastrula-
tion, 566
Cellular retinoic acid binding protein, 572
Centropages velificatus, 377
Centrosome, 571
Centrosomes in echinoderm development, 57 1
Cerebrospinal fluid, 42 1
CHADWICK, NANETTE E., Interspecific aggressive
behavior of the corallimorpharian Corynactis
California (Chidaria: Anthozoa): effects on
sympatric corals and sea anemones, 1 10
Chaetopterus sperm, 426
CHANG, D. C., HUNT, J. R., AND P. Q. GAO, Rest-
ing conductance of the squid axon membrane,
441
CHANG, J. J., see K. Delaney, 437
Channel, 441
Characterization of phospholipid enzymes in squid
axoplasm and giant fiber lobe, 439
INDEX TO VOLUME 173
577
CHARLTON, MILTON P., AND GEORGE J. AUGUS-
TINE, Classification of presynaptic calcium
channels in the squid giant axon, 44 1
CHARLTON, MILTON P., see Stephen J. Smith, 446
Chemoautotrophy, 260
Chemoreception, 436, 438
Chemosensory response, 299
CHEN, CHONG, DANIEL L. ALKON, AND PAUL E.
GALLANT, C-kinase activation mediated by
proteolysis modulates K+ conductances in
Hermissenda B-photoreceptors, 442
Chick embryos, 563, 567
Chick sensory neurons, 447
Chlorella, 367, 504
Chlorophycophytal, 430
Chloroplast-retaining ciliates as a component of
the primary productivity in Great Harbor,
Woods Hole, Massachusetts, 432
Choline, 299
Chromatin organization, 568
Cibides refulgens, 1 36
Cilia, 513
CINELLI, A. R., AND B. M. SALZBERG, Extrinsic op-
tical signals, evoked field potentials, and single
unit recordings from the olfactory bulb of the
skate (Raja erinacea), 435
Circadian rhythms, 448, 563, 568, 57 1
Circulation, 422
CLARK, WALLIS H., JR., see Fred J. Griffin, 311;
and John W. Lynn, 45 1
Classical conditioning, 437
Classification of presynaptic calcium channels in
the squid giant synapse, 441
Clonal organisms, 1 10
Closed capture recapture, 43 1
Clumping and phosphorus accumulation in bac-
teria, 434
Cnidaria, 110,324
Co-selection for clumping and phosphorus accu-
mulation by bacteria isolated from waste-water
treatment systems, 434
COLOT, H. V., G. PETERSON, Q. Yu, D. WHEELER,
J. C. HALL, AND M. ROSBASH, Interspecific
comparisons of the period gene, 563
Community ecology, 1 10
Competition, 110,430
Control of veliger locomotion, 5 1 3
Copepod, 169,489
Copper toxicity, 429
Coral, 110
Coral reef, 335
Coral reproduction, 335
Corallimorpharian behavior, 1 10
COROTTO, FRANK, AND JELLE ATEMA, Initial sur-
vey of the chemosensory response properties of
lobster mouthparts: spectral populations and
tuning breadth, 436
Corynactis californica, 1 1 0
Cosettlement, 474
COTA, GABRIEL, AND CLAY M. ARMSTRONG, In-
activation rate is not voltage dependent in pitu-
itary sodium channels, 442
CRONIN, THOMAS W., see Abner B. Lall, 398
CROWE, JOHN H., see Fred J. Griffin, 3 1 1
CROWE, Lois M., see Fred J. Griffin, 3 1 1
Crustacea, 345, 435, 437
Crustacean hemocytes, 1 78
Cyclins and the cell cycle of early clam embryos,
572
Cytochemical features of shrimp hemocytes, 178
Cytogenetics, 570
Cytoplasmic
determinants, 423
localization, 425
transport, 563
D
rDNA, 569
D-alanine, 252
D-amino acids in bivalves, 252
D-aspartate, 252
DAS, J., AND H. BUSSE, Pattern formation with Fu-
sarium illustrates a principle for generation of
cell pattern, 564
DAVIDSON, SARAH, see Walter Troll, 427
DE WEER, PAUL, see R. F. Rakowski, 445
DESIMONE, D. W., M. A. STEPP, R. PATEL, E.
MARCANTONIO, AND R. O. HYNES, Integrin
structure, function, and developmental ex-
pression, 564
DEATON, LEWIS E., Epithelial water permeability
in the euryhaline mussel Geukensia dernissa:
decrease in response to hypoosmotic media
and hormonal modulation, 230
DE LACA, TED E., see Stephen P. Alexander, 136
DELANEY, K., AND J. J. CHANG, Suppression of
fictive feeding in vitro by foot shock in Limax
maximus: neural correlates in withdrawal and
feeding systems, 437
Dendrites, 435
Detection of chemical contrast in hermit crabs, 438
Development, 126, 425, 427
Development, metamorphosis, and seasonal abun-
dance of embryos and larvae of the antarctic
sea urchin Sterechinns neumayeri. 126
Developmental regulation of three testis-specific
genes during mouse spermatogenesis, 573
Diapause, 169
DICKMAN, M. C., Effects of age on the skin pigmen-
tation of the guppy, 42 1
Didemnidae, 188
Differences in the duration of egg diapause ofLabi-
docera aestiva (Copepoda: Calanoida) from
the Woods Hole, Massachusetts, region, 169
Differentiation of Arbacia punctulata is blocked by
the protease inhibitor leupeptin after fertiliza-
tion, 427
DINSMORE, J. H., AND R. D. SLOBODA, Identifica-
tion of a calcium-calmodulin dependent pro-
tein kinase associated with the sea urchin mi-
totic apparatus, 564
DIONNE, C. A., AND L. J. GUDAS, Inducible c-myc
overexpression and F9 teratocarcinoma stem
cell differentiation, 565
578
INDEX TO VOLUME 173
Distance, 564
DISTEL, ROBERT!., HYO-SUNG Ro, BARRY S. RO-
SEN, DOUGLAS L. GROVES, AND BRUCE M.
SPIEGELMAN, Nucleoprotein complexes that
regulate gene expression in adipocyte differen-
!;on: direct participation of c-fos, 565
Do arachidonic acid metabolites mediate modula-
tion of K and Ca currents by FMRF-amide in
Aplysia neurons? 440
Does copper affect the mating behavior of Gam-
mams annulatus Smith, 1873 (Amphipoda:
Gammaridae)? 429
Dogfish lens, 449
DOME, JEFFREY S., see Jean M. Sanger, 420
Don't eat if Neptune is angry, 433
Drosophila, 566,571
DuBois, ARTHUR B., see S. Hunter Fox, 422
Duration of egg diapause, 169
Dynamics of actin, myosin, and membranes in liv-
ing cells during division, 420
Dynamics of spindle microtubules visualized in
vivo by high resolution video polarization mi-
croscopy, 419
E
EASTMAN, C., see K. K. Siwicki, 57 1
ECK.BERG, WILLIAM R., AND ETE Z. SZUTS, Phos-
phatidylinositol hydrolysis after Spisula oo-
cyte fertilization, 424
Ecology, 136
Eelgrass wasting disease: cause and recurrence of a
marine epidemic, 557
Effect of UV irradiation on axis and muscle cell
specification in embryos of the ascidian Stvela,
425
Effect of photoperiod and melatonin in glucosami-
nidase activity during newt limb regeneration,
569
Effect of segment loss on reproduction output in
Capitella sp. I (Polychaeta), 430
Effect of strontium, barium, and strychnine on the
synapse made by barnacle photoreceptors,
The, 441
Effect of the arborescent bryozoan Bugula spp. on
the settlement, growth, and mortality of the co-
lonial encrusting tunicate Botrylloides leachii,
The, 430
Effects of aerobic versus anoxic conditions on gluta-
mine synthetase activity in Zostera marina
roots, 43 1
Effects of age on the skin pigmentation of the
guppy, 42 1
Effects of gossypol analogs on Spisula sperm, 428
Effects of salinity stress on the rate of aerobic respi-
ration and photosynthesis in the hermatypic
coral Sideraslrea siderea. The, 539
EHRLICH, BARBARA E., see Anita D. Mcllveen, 445
EICHELE, GREGOR, see Christina Thaller, 573
Eicosanoids, 92
Elasmobranch, 42 1
Electric organ, 447
Electrical coupling, 513
Electromyogram, 437
Electromyographic recording of classical condi-
tioning of eye withdrawal in the green crab,
437
Electron microscopy, 42 1
Electrocyte, 443
Eleutheria dichotoma, 433
ELLINGTON, W. Ross, see Robert W. Wiseman,
277
ELLIOT, K. J., see F. R. Jackson, 566
Embryogenesis, 572
Embryonic induction, 428
Endocrinology, 230
Endosymbiosis, 432
Energetics of contractile activity in isolated radula
protractor muscles of the whelk Busycon con-
trarium: anaerobic end product accumulation
and release, 277
ENGEL, DAVID W., AND MARIUS BROUWER, Metal
regulation and molting in the blue crab, Calli-
nectus sapidus: metallothionein function in
metal metabolism, 239
Entry of sperm into the animal pole of the egg of
the ascidian Phallusia mammillata, 427
Epi 1,569
Epidermal specific antigen, 569
Epithelial water permeability in the euryhaline
mussel Geukensia demissa: decrease in re-
sponse to hypoosmotic media and hormonal
modulation, 230
Epithelial water permeability, 230
ERNST, SUSAN G., see Robert Mclsaac, 568
Estimate of primary productivity in Waquoit Bay
National Estuarine Sanctuary, Falmouth,
Massachusetts, An, 432
Euroepidemiology, 422
Eurypanopens, 46 1
Evidence that a G-protein mediates 1-methylade-
nine induced maturation of starfish oocytes,
427
Extracellular matrix, 564
Extrinsic optical signals, evoked field potentials,
and single unit recordings from the olfactory
bulb of the skate (Raja erinacea), 435
FMRF-amide, 440
Fate of Botryllus (Ascidiacea) larvae cosettled with
parental colonies: beneficial or deleterious
consequences, 474
Fecal pellets, 377
Feeding adaptations of the foraminiferan Cibicides
refulgens living epizoically and parasitically on
the antarctic scallop Adamussium colbecki,
136
Feeding behavior, 299, 437, 527
Feeding behavior in Hydra I. Effects of Anemia ho-
mogenate on nematocyst discharge, 527
Feeding behavior of Paranophrys carinovora (Cili-
ata, Philasteridae), The, 299
INDEX TO VOLUME 173
579
Feeding strategies, 222
FEIN, ALAN, see Richard Payne, 447; and Susan F.
Wood, 448
FEINMAN, RICHARD D., CHARLES I. ABRAMSON,
AND ROBIN R. FORMAN, Electromyographic
recording of classical conditioning of eye with-
drawal in the green crab, 437
FEINMAN, RICHARD D., see Charles I. Abramson,
435
FEINMAN, ROBIN A., see Charles I. Abramson, 435
FELBECK, HORST, AND SANDRA WILEY, Free D-
amino acids in the tissues of marine bivalves,
252
FERKOWICZ, MICHAEL J., see Susan D. Hill, 430
Fertilization, 427
Fertilized egg, 565
Fish, 422, 435
Flash kinetics, 489
FLORES, R., see B. M. Salzberg, 446
Fluorescence, 424
Forelimb regeneration, 567
FORMAN, ROBIN R., see Richard D. Feinman, 437
Fox, G. Q., see M. E. Kriebel, 443, and H. Stadler,
447
Fox, S. HUNTER, CHRISTOPHER S. OGILVY, AND
ARTHUR B. DuBois, Transection of the spinal
cord near the obex abolishes cardiovascular
compensation for gravity in bluefish, 422
FRANCIS, SUSAN, see Robert Mclsaac, 568
FRANK, TAMARA M., see Michael I. Latz, 489
Free D-amino acids in the tissues of marine bi-
valves, 252
Fucoid egg, 425
Functional organization of the sonic motor system
in sea robins, 435
Fura-2 imaging of calcium transients in squid giant
presynaptic terminal, 446
Fusarium, 564
G-protein, 427, 445
GADSBY, DAVID C, see R. F. Rakowski, 445
GALLANT, PAUL E., see Chong Chen, 442
Gametogenesis, 160
GAO, P. Q., see D. C. Chang, 441
GARBER, SARAH, see Anita D. Mcllveen, 445
Gastrulation in hydrozoan, 324
Gecarcinus lateralis, 398
Gene expression, 427, 565
Genes, 566
Genetics of growth and shape in Eurypanopeus, 46 1
Geotaxis, 289
Germinal vesicle, 426
Geitke nsia demissa, 160
GIAMBARELLA, U., see A. Telfer, 572
Glucosaminidase, 569
Glutamic acid uptake, 434
Glutamine synthetase regulation, 43 1
Gossypol, 424, 428
GOULD, ROBERT M., JOHN HOLSHEK, AND DAVID
W. PUMPLIN, Incorporation of tritiated inosi-
tol and choline into phospholipids in the squid
stellate ganglia with special reference to the gi-
ant synapse, 443
GOULD, ROBERT, see Mario Alberghina, 439
GRASSLE, JUDITH, P., see Susan D. Hill, 430
Gravity, 422
Green algae infection, 430
Green algal (chlorophycophytal) infection of the
dorsal surface of the exoskeleton, and associ-
ated organ structure, in the horseshoe crab,
Limulus polyphemus. A, 430
Green hydra, 367, 504
Green hydra symbiosis: analysis of a field popula-
tion, The, 367
GRIFFIN, FRED J., WALLIS H. CLARK JR., JOHN H.
CROWE, AND Lois M. CROWE, Intracellular
pH decreases during in vitro induction of the
acrosome reaction in the sperm ofSicyonia in-
gentis, 3 1 1
GRIMES, GARY W., Intracellular patterning and the
problem of assembly, 565
GROSVENOR, W., AND G. KASS-SIMON, Feeding
behavior in Hydra. I. Effects of Anemia ho-
mogenate on nematocyst discharge, 527
GROVES, DOUGLAS L., see Robert J. Distel, 565
Growth, 46 1
GUDAS, L. J., see C. A. Dionne, 565; G. J. LaRosa,
566; M. B. Rogers, 570; and Carol M. Stoner,
572
Guinea pig sperm surface protein, 568
Guinea pig sperm surface protein PH-20 is an-
chored in the membrane by a phosphatidylino-
sitol lipid. The, 568
H
Halichoerus grvpus, 431
HALL, J. C., see H. V. Colot, 563; and K. K. Siwicki,
571
HAND, STEVEN C., Trophosome ultrastructure and
the characterization of isolated bacteriocytes
from invertebrate-sulfur bacteria symbioses,
260
HANEJI, TATSUJI, AND S. S. K.OIDE, Interaction of
avidin with Spisula oocyte proteins, 425
HART, N. H., AND J. S. WOLENSKI, Cell surface re-
organization in the fertilized egg of the zebra-
fish, 565
HART, N. H., see J. S. Wolenski, 573
HASTINGS, J. W., see D. Morse, 568
Heat-stability of squid axoplasm neurofilaments
provides a rapid method for their purification.
The, 420
HECHT, N. B., see P. C. Yelick, 573
HELFRICH, JOHN, see Diana E. Varela, 434
Hemocyanin, 205
Hemoglobin, 205
Heparin, 423
Hermit crab, 438
580
INDEX TO VOLUME 173
Hett :•: ;ic sperm motility enhancement by sea
urchin oocyte peptides, 426
Heterotrophy by Chlorella in symbiosis, 504
HIGHSTEIN, S. M., see R. Kitch, 437
HILBISH, THOMAS J., AND F. JOHN VERNBERG,
Quantitative genetics of juvenile growth and
shape in the mud crab Eurypanopeus depres-
sus, 46 1
HILL, SUSAN D., JUDITH P. GRASSLE, AND MI-
CHAEL J. FERKOWICZ, Effect of segment loss
on reproductive output in Capitella sp. I (Poly-
chaeta), 430
Hippocampus, 443
Histidine, 299
Histone variant— H2A. F/Z, 568
HOLLAND, NICHOLAS D., ALEXANDER B. LEON-
ARD, AND J. RUDI STRICKLER, Upstream and
downstream capture during suspension feed-
ing by Oligometra serripina (Echinodermata:
Cridoidea) under surge conditions, 552
HOLSHEK, JOHN see Robert M. Gould, 443
Homarus americanus, 436
Homeo box, 566
Homeo box genes, 573
Horseshoe crabs, 289, 438
HOSE, Jo ELLEN, GARY G. MARTIN, VAN ANH
NGUYEN, JOHN LUCAS, AND TEDD Ro-
SENSTEIN, Cytochemical features of shrimp
hemocytes, 178
HUNT, J. R., see D. C. Chang, 441
Hydra, 527
Hydrothermal vents, 260
Hydrozoan, 324
HYNES, R. O., see D. W. DeSimone, 564
Hypoosmotic stress, 407
Identification of a calcium-calmodulin dependent
protein kinase associated with the sea urchin
mitotic apparatus, 564
Image enhancement of wet seals on rocks and sand
as the sample in population ecology of Phoca
vitulina concolor and Halichoerus grypus, 43 1
Image intensification, 489
Impulse propagation and contraction in the tunic
of a compound ascidian, 1 88
In situ hybridization, 570
In vivo properties of primary cilia in cultured kid-
ney epithelial cells, 563
Inactivation rate is not voltage dependent in pitu-
itary sodium channels, 442
Incorporation of tritiated inositol and choline into
phospholipids in the squid stellate ganglia with
special reference to the giant synapse, 443
Inducible c-myc overexpression and F9 teratocarci-
noma stem cell differentiation, 565
Inhibition by heparin of endotoxin-dependent co-
agulation of amebocyte lysate from Limulus
polyphemus. 423
Initial results of lead measurements of deciduous
teeth, 422
Initial survey of the chemosensory response proper-
ties of lobster mouthparts: spectral popula-
tions, and tuning breadth, 436
Injected calcium buffers block fucoid egg develop-
ment, 425
Inositol lipid, 424
Inositol phosphates, 448
Inositol triphosphate, 447
INOUE, S., see Robert B. Silver, 420; and S. J. Segal,
426
INOUE, SHINYA, EDWARD D. SALMON, AND
LYNNE CASSIMERIS, Dynamics of spindle mi-
crotubules visualized in vivo by high resolution
video polarization microscopy, 419
INOUE, SHINYA, Ultrathin optical-sectioning-to-
mography achieved with the light microscope,
419
Integrin structure, function, and developmental ex-
pression, 564
Interaction of avidin with Spisula oocyte proteins,
425
Interleukin-2 (IL-2) distribution in adult newts (No-
tophthahnus viridescens) during regeneration
and following non-amputational wounding,
567
Interspecific aggressive behavior of the coralli-
morpharian Corynactis californica (Cnidaria:
Anthozoa): effects on sympatric corals and sea
anemones, 1 10
Interspecific comparisons of the period gene, 563
Intracellular pH decreases during the in vitro induc-
tion of the acrosome reaction in the sperm of
Sicyonia ingentis, 3 1 1
Intracellular patterning and the problem of assem-
bly, 565
Invertebrate-bacteria symbioses, 260
Ion channel gating, 439
Isolation and characterization of an mRNA se-
quence (ERA- 1 ) exhibiting a rapid and protein
synthesis independent induction during the re-
tinoic acid-induced differentiation of terato-
carcinoma stem cells, 566
Isopod vitellin and vitellogenin, 345
IVERSEN, KJRSTEN, see Ann Lewandowski, 422
JACKSON, F. R., AND K. J. ELLIOT, Molecular stud-
ies of biological rhythms in Drosophila, 566
JAFFE, L. F., M. H. WEISENSEEL, AND J. E. SPEK-
SNIJDER, Injected calcium buffers block fucoid
egg development, 425
JAFFE, L. F., see J. E. Speksnijder, 427
JAFFE, LAURINDA A., see Fraser Shilling, 427
JEFFERY, WILLIAM R., Effect of UV irradiation on
axis and muscle cell specification in embryos
of the ascidian Styela, 425
INDEX TO VOLUME 173
581
K channels, 439
K+ currents, 442
KADAM, A. L., S. J. SEGAL, AND S. S. KOIDE, Oo-
cyte maturation-inducing substance (OMIS)
in Spisula, 426
KAHAN, DAVID, THEODORA BAR-EL, NORBERT
WILBERT, SAMSON LEIKEHMACHER, AND
SAMUEL OMAN, The feeding behavior ofPara-
nophrys carnivora (Ciliata, Philasteridae), 299
KAMMIRE, CARRIE, see Mary Anne Sydlik, 438
KASS-SIMON, G. see W. Grosvenor, 527
KAWASHIMA, R., see H. Ueno, 428
Kinetics of two calcium channel types in chick sen-
sory neurons. 447
KITCH, R., T. C. TRICAS, AND S. M. HIGHSTEIN,
Organization of the vestibulo-ocular and vesti-
bulo-spinal reflex pathways in the toadfish, Op-
sanus (an, 437
KLEENE, K. C., see P. C. YELICK, 573
KOIDE, S. S., see Tatsuji Haneji, 425; A. L. Kadam,
426; S. J. Segal, 426; and H. Ueno, 428
KOPPEL, D. E., see B. Phelps, 568
KOSIK, KENNETH S., AND J. METUZALS, The heat-
stability of squid axoplasm neurofilaments
provides a rapid method for their purification,
420
KRIEBEL, M. E., see H. Stadler, 447
KRIEBEL, M. E., G. D. PAPPAS, AND G. Q. Fox,
Two classes of miniature end-plate potentials
are present in the isolated, innervated electro-
cyte, 443
KUZIRIAN, ALAN M., BARRY BANK, JOSEPH Lo-
TURCO, AND DANIEL L. ALKON, Autoradiog-
raphy utilizing [3]phorbol esters is potentially
useful for cellular analysis of protein kinase C
activity in hippocampus slices, 443
KUZIRIAN, ALAN M., see Stephen B. Leighton, 444
KWON, Y., see P. C. Yelick, 573
LAROSA, AND L. J. GUDAS, Isolation and charac-
terization of an mRNA sequence (ERA-1) ex-
hibiting a rapid and protein synthesis indepen-
dent induction during the retinol acid-induced
differentiation of teratocarcinoma stem cells,
566
Labyrinthula, 557
LALL, ABNER, B., AND THOMAS W. CRONIN, Spec-
tral sensitivity of the compound eyes in the
purple land crab Gecarcinus lateralis (Fremin-
ville), 398
Laminin collagen IV. 570
LANDOWNE, DAVID, Calcium affects the birefrin-
gence response of the squid giant axon, 444
Larvae, 5 1 3
LATZ, MICHAEL L, TAMARA M. FRANK, MARK R.
BOWLBY, EDITH A. WIDDER, AND JAMES F.
CASE, Variability in flash characteristics of a
bioluminescent copepod, 489
LEBLANC, J., M. YODER, AND I. BRICK, Cellular
interactions during amphibian gastrulation,
566
Lead, 422
Learning, 435
LEE, B. Y., K. T. BUSH, R. G. NAGELE, AND H.
LEE, Primordial germ cells of day 5 chick em-
bryos, 567
LEE, H., see K. T. Bush, 563, and B. Y. Lee, 567
Leech embryo, 571
LEIBOVITZ, Louis, AND GREGORY A. LEWBART,
A green algal (chlorophycophytal) infection of
the dorsal surface of the exoskeleton, and asso-
ciated organ structures, in the horseshoe crab,
Limulus polyphemus, 430
LEIGHTON, STEPHEN B., AND ALAN M. KUZIRIAN,
Sectionless sectioning: a systematic method for
scanning electron microscopic examination of
embedded tissue, 444
LEIKEHMACHER, SAMSON, see David Kahan, 299
Lens proteins, 449
LEONARD, ALEXANDER B., see Nicholas D. Hol-
land, 552
LEONARDI, G. P., M. MANTHOS, J. LoBuE, D. OR-
Lic, AND J. MITRA, Leukemic viral expression
with induced changes in hematopoietic micro-
environment, 567
Leukemic viral expression with induced changes in
hematopoietic microenvironment, 567
LEVIN, JACK, see James A. Marcum, 423
LEVITAN, D., see A. Telfer, 572
LEVITON, ALAN, see Ann Lewandowski, 422
LEWANDOWSKI, ANN, MICHAEL RABINOWITZ,
ALAN LEVITON, KIRSTEN IVERSEN, AND SU-
SAN ROSE, Initial results of lead measurements
of deciduous teeth, 422
LEWBART, GREGORY A., see Louis Leibovitz, 430
LEWIS, K., see G. Sluder, 57 1
Life history, 169
Life is rough when you are small, 434
Light microscope, 4 1 9
Limb regeneration, 569
Limulus algal infection, 430
Limulus polyphemus, 289, 423, 447
Lipid enzymes, 439
Lipid metabolism, 443
LoBUE, J., see G. P. Leonardi, 567; E. J. Segen-
reich, 570; and E. Rodriguez, 570
LoTURCO, JOSEPH, see Alan M. Kuzirian, 443
Localization of a gene product in Drosophila that
influences biological rhythms, 571
Localization of laminin and collagen IV transcrip-
tion in mouse teratocarcinomas and embryos
by in situ hybridization, 570
LOMBARD, M. F., AND R. E. SICARD, Interleukin-
2 (IL-2) distribution in adult newts (Notoph-
thalmus viridescens) during regeneration and
following non-amputational wounding, 567
Lophora pistillata, 335
LORAND, LASZLO, see Reed Brozen, 423
LOVELAND, ROBERT E., see Mark L. Botton, 289
582
INDEX TO VOLUME 173
LOWE, CHRIS, see Seymour Zigman, 449
LOYA, V '., see B. Rinkevich, 335
LUCAS, JOHN, see Jo Ellen Hose, 178
Lucinafloridana, 260
Luminescence proteins exhibit circadian rhythms
but their mRNAs do not, 568
LYNN, JOHN W., and WALLIS H. CLARK JR. Physi-
ological and biochemical investigations of egg
jelly release in Penaeus aztecus, 45 1
Lysosomes, 178
M
M-phase, 572
iMEPPS, 443
MACKIE, G. O., AND C. L. SINGLA, Impulse propa-
gation and contraction in the tunic of a com-
pound ascidian, 188
MACKIE, G. O., see S. A. Arkett, 5 1 3
MANGUM, C. P., K. I. MILLER, J. L. SCOTT, K. E.
VAN HOLDE, AND M. P. MORSE, Bivalve he-
mocyanin: structural, functional, and phyloge-
netic relationships, 205
MANTHOS, M., see G. P. Leonardi, 567
MARCANTONIO, M., see D. W. De Simone, 564
MARCUM, JAMES A., AND JACK LEVIN, Inhibition
by heparin of endotoxin-dependent coagula-
tion of amebocyte lysate from Limulus poly-
phemus, 423
MARCUS, NANCY H., Differences in the duration of
egg diapause ofLabidocera aestiva (Copepoda:
Calanoida) from the Woods Hole, Massachu-
setts, region, 169
MARTIN, GARY G., see Jo Ellen Hose, 1 78
MARTIN, VICKI J., A morphological examination
of gastrulation in a marine athecate hydro-
zoan, 324
MARTINDALE, M. Q., see M. Shankland, 571
Mating behavior, 429
Maturation, 424, 427
Maxilliped, 436
McAULEY, P. J., Quantitative estimation of move-
ment of an amino acid from host to Chlorella
symbionts in green hydra, 504
McGlNNlS, W., see M. F. Utset, 573
MclLVEEN, ANITA D., SARAH GARBER, AND BAR-
BARA E. EHRLICH, Behavioral experiments
suggest G protein modulation of calcium chan-
nels in Paramecium, 445
MCISAAC, ROBERT, HEIDI MILLER, CAROL A.
BRENNER, CATHERINE NOCENTE-MCGRATH,
SUSAN FRANCIS, AND SUSAN G. ERNST, Sea
urchin H24. F/Z: an unusually conserved H2A
variant gene, 568
Meiosis, 570, 571
Meiotic chromosome studies in BALB/c mice bear-
ing a transplantable mono-myelocytic leuke-
mia (MML) and in the offspring of normal fe-
males crossed with transplanted males, 570
Melanocyte, 428
Melanophore, 421
Melatonin, 569
Membrane receptors, 564
Metal metabolism, 239
Metal regulation and molting in the blue crab, Cal-
linectes sapidus: matallothionein function in
metal metabolism, 239
Metridium senile, 110
METUZALS, J., see Kenneth S. Kosik, 420
Microciona prolifera, 423
Microtubule depolymerization, 564
Microtubules, 419
Migratory behavior of individual horseshoe crabs,
438
MILLER, F. J. see G. Sluder, 57 1
MILLER, HEIDI, see Robert Mclsaac, 568
MILLER, K. I., see C. P. Mangum, 205
MILOS, P., see D. Morse, 568
Mirror imagery, 565
Mitosis, 4 19, 420, 564, 571
MITRA, J., see G. P. Leonardi, 567; E. J. Segenreich,
570; and E. Rodriguez, 570
MITTAL, BALRAJ, see Jean M. Sanger, 420
Modulation of retinal sensitivity by putative effer-
ent neurotransmitters, 448
Molecular analyses of early neural pattern decisions
in Xenopus, 569
Molecular genetics of early embryogenesis in C.
elegans, 572
Molecular studies of biological rhythms in Dro-
sophila, 566
Mollusc, 437
Molting hormone, 345
Mono-myelocytic leukemia, 570
Morphological characterization of isolated, con-
centrated nerve endings of the skate electric or-
gan, 447
Morphological examination of gastrulation in a
marine athecate hydrozoan, A, 324
MORSE, D., P. MILOS, E. Roux, AND J. W. HAS-
TINGS, Luminescence proteins exhibit circa-
dian rhythms but their mRNAs do not, 568
MORSE, M. P., see C. P. Mangum, 205
Moult, 431
Mouse cellular retinoic binding protein: cloning,
cDNA sequence and mRNA expression dur-
ing the retinoic-acid induced differentiation of
F9 wild type and RA-3-10 mutant teratocarci-
noma cells, 572
Mouse development, 573
Mouse teratocarcinomas, 570
MUEHLSTEIN, LISA K., see Frederick T. Short, 557
MULLER-PARKER, G. AND R. L. PARDY, The green
hydra symbiosis: analysis of a field population,
367
MUNTZ, W. R. A., AND S. L. WENTWORTH, An an-
atomical study of the retina of Nautilus pom-
pilius, 387
Murine
erythroleukemia, 567
leukemia, 570
myeloid leukemia, 567
MUTHIGA, NYAWIRA A., AND ALINA M. SZMANT,
INDEX TO VOLUME 173
583
The effects of salinity stress on the rates of aero-
bic respiration and photosynthesis in the her-
matypic coral Siderastrea siderea, 539
Myeloid body in Nautilus, 387
MYERS, PHILIP E., The effect of the arborescent
bryozoan Bugula spp. on the settlement,
growth, and mortality of the colonial encrust-
ing tunicate Botrylloides leachii, 430
MYLES, D. G., see B. Phelps, 568
Myocyte, 188
Myosin, 420
N
NAGELE, R. G., see K. T. Bush, 563, and B. Y. Lee,
567
Nautilus, 387
Near-UV light effects on the dogfish (Mustelus
canis) lens, 449
NELSON, DONALD R., see Mary Anne Sydlik, 438
Nematocyst discharge in Hydra. 521
Nerve endings, 447
Nerve terminals, 446
Neural differentiation, 57 1
Neural induction in ascidian embryos redivivus,
428
Neural pattern, 569
Neuroepidemiology, 422
Neurofilament, 420
Neuroid conduction, 188
Neuromuscular junction, 440
Neuronal control of ciliary locomotion in a gastro-
pod veliger (Calliosloma), 5 1 3
Neurotransmitters, 448
Neurulation, 563
NGUYEN, VAN ANH, see Jo Ellen Hose, 178
Nitrate reductase activity in Zostera marina, 432
NocENTE-McGRATH, CATHERINE, see Robert
Mclsaac, 568
Non-muscle contractility, 188
Notophlhalmus viridescens. 567
Nucleoprotein complexes that regulate gene expres-
sion in adipocyte differentiation: direct partici-
pation of c-fos, 565
Nutrient enrichment, 432
o
OBAID, A. L., see B. M. Salzberg, 446
Offspring studies, 570
OGILVY, CHRISTOPHER S., see S. Hunter Fox, 422
OKAMURA, BETH, Particle size and flow velocity in-
duce an inferred switch in bryozoan suspen-
sion-feeding behavior, 222
Olfactory bulb, 435
Oligometra serripinna, 552
Oligotrichida, 432
OMAN, SAMUEL, see David Kahan, 299
Oocyte, 424
Oocyte maturation-inducing substance (OMIS) in
Spisula, 426
Optical recording, 435, 446
Optimal foraging, 222
Organization of the vestibulo-ocular and vestibulo-
spinal reflex pathways in the toadfish Opsanus
tan: anatomy and electrophysiology, 437
Orientation of a horseshoe crab, Limulus polyphe-
mus, on a sandy beach, 289
Orientation of intertidal Limulus, 289
ORLIC, D., see G. P. Leonardi, 567
Osmolyte regulation by clam blood cells, 407
Osmoregulation, 230, 252
OSSES, Luis R., see Stephen J. Smith, 466
PAGE, 345
PALTI, Y., see C. M. Armstrong, 439
PAPPAS, G. D., see M. E. Kriebel, 443; and H.
Stadler, 447
Paranophrys carinvora, 299
PARDY, R. L., see G. Muller-Parker, 367
Parental colonies, 474
Particle size and flow velocity induce an inferred
switch in bryozoan suspension-feeding behav-
ior, 222
PATEL, R., see D. W. DeSimone, 564
PATEL, DAVID, Image enhancement of wet seals on
rocks and sand as the sample in population
ecology of Phoca vitulina concolor and Hali-
choerus grypus, basic research for the closed
model, 43 1
PATRICOLO, ELEONORA, see Luisanna Villa, 355
Pattern formation, 423, 564, 573
Pattern formation with Fusariwn illustrates a prin-
ciple for generation of cell pattern, 564
PAYNE, RICHARD, AND ALAN FEIN, Rapid desensi-
tization terminates the response of Limulus
photoreceptors to brief injections of inositol
triphosphate, 447
PEARLMAN, R. E., see K. Riekki, 569
PEARSE, JOHN S., see Isidro Bosch, 126
Pecten, 136
Penaeid egg jelly release, 45 1
Penaeus aitecas, 45 1
Period gene, 563, 571
PETERSON, G., see H. V. Colot, 563
PHELPS, B., P. PRIMAKOFF, D. E. KOPPEL, AND
D. G. MYLES, The guinea pig sperm surface
protein PH-20 is anchored in the membrane
by a phosphatidylinositol lipid, 568
PHILLIPS, CAREY, Molecular analyses of early neu-
ral pattern decisions in Xenopus, 569
Phoca vitulina concolor, 43 1
Phorbol ester autoradiography, 443
Phosphatidylinositol hydrolysis after Spisula oo-
cyte fertilization, 424
Phospholipase, A2, 439
Photoreceptor, 441, 447, 448
Photosynthesis, 539
Physiological roles of prostaglandins and other ei-
cosanoids in invertebrates, 92
584
INDEX TO VOLUME 173
Physiological and biochemical investigations of the
egg jelly release in Penaeus aztecas, 45 1
PIERCE, SIDNEY, K., see Laurens H. Smith Jr., 407
Pituitary cells, 442
Polarization microscopy, 4 1 9
PORTER, C, see H. Ueno, 428
PORTER, DAVID, see Frederick T. Short, 557
PRATT, SARA, see W. S. Vincent, 434
PREGNALL, A. MARSHALL, Effects of aerobic versus
anoxic conditions of glutamine synthetase ac-
tivity in Zostera marina roots: possibilities for
regulation of ammonium assimilation, 431
PREGNALL, A. MARSHALL, see Nina C. Roth, 432,
and Krishnan Thankavel, 433
PRIMAKOFF, P., see B. Phelps, 568
Primary cilia, 563
Primary productivity, 432
Primordial germ cells of day 5 chick embryos, 567
Productivity, 432
Prostaglandins, 92
Protamines, 573
Protease inhibitor leupeptin, 427
Protein kinase C, 443
Protein phosphorylation, 564
Protein synthesis and degradation rates in two eco-
phenotypes of the cord grass Spartina alter-
niflora Loisel from Great Sippewissett Salt
Marsh, New England, 433
Protein-DNA interactions on the 5' non-tran-
scribed spacer of Tetrahvmena thermophila
rDNA, 569
Proteolysis, 442
PUMPLIN, DAVID W., see Robert M. Gould, 443
Q
Quantitative estimation of movement of an amino
acid from host to Chlorella symbionts in green
hydra, 504
Quantitative genetics of juvenile growth and shape
in the mud crab Eurypanopeus depressus, 46 1
Quantum emission, 489
R
RABINOWITZ, MICHAEL, see Ann Lewandowski,
422
Radula protractor muscles, 277
RAKOWSKJ. R. F., DAVID C. GADSBY, AND PAUL
DE WEER, Voltage-clamp reversal of the so-
dium pump in dialyzed squid giant axon, 445
Rapid desensitization terminates the response of
Limulus photoreceptors to brief injections of
inositol trisphosphate, 447
Rat development, 57 1
Red blood cell, 205
Red Sea, 335
Regeneration, 430
Region specific expression of mouse homeo box
genes, 573
Regulatory physiology, 92
REIDER, C. L., see G. Sluder, 57 1
Relationship between trace metal distribution and
sulfate reduction in surface sediment, 429
Reproduction, 1 26, 430, 45 1
Respiration, 539
Resting conductance of the squid axon membrane,
441
Resting potential, 441
Retina of Nautilus pompilius, 387
Retinal anatomy, 387
Retinoic acid, 572
Retinoic acid induced gene, 566
Retinoids and pattern formation in vertebrate
limbs, 573
Retroviruses, 567
Rhodamine phalloidin, 573
REIDER, C. L., see S. S. Bowser, 563
RIEKKI, K., AND R. E. PEARLMAN, Protein-DNA
interactions on the 5' non-transcribed spacer of
Tetrahymena thermophila rDNA, 569
RIESEN, WILLIAM, see Reed Brozen, 423
Riftia pachyptila, 260
RINKEVICH, B., AND Y. LOYA, Variability in the
pattern of sexual reproduction of the coral Sty-
lophorapistillata at Eilat, Red-Sea: a long-term
study, 335
RINKEVICH, BARUCH, AND IRVING L. WEISSMAN,
The fate of Botryllus (Ascidiacea) larvae coset-
tled with parental colonies: beneficial or delete-
rious consequences? 474
Risks of feeding, 433
RIVERA, M., R. APONTE, F. CALIMANO, AND F.
VALLES, Effect of photoperiod and melatonin
in glucosaminidase activity during newt limb
regeneration, 569
Ro, HYO-SUNG, see Robert J. Distel, 565
ROBERTS, MICHAEL S., The chloroplast-retaining
ciliates as a component of the primary produc-
tivity in great Harbor, Woods Hole, Massachu-
setts, 432
RODRIGUEZ, E., J. MITRA, AND J. LoBuE, Studies
on a transplantable monomyelocytic leukemia
and in the offspring from crosses between
transplanted and normal BALB/c mice, 570
ROGERS, M. B., AND L. J. GUDAS, Localization of
laminin and collagen IV transcription in
mouse teratocarcinomas and embryos by in
situ hybridization, 570
ROSBASH, M., see H. V. Colot, 563; and K. K. Si-
wicki, 571
ROSE, SUSAN, see Ann Lewandowski, 422
ROSEN, BARRY S., see Robert J. Distel, 565
ROSENSTEIN, TEDD, see Jo Ellen Hose, 1 78
ROTH, K. E., see S. S. Bowser, 563
ROTH, NINA C., AND A. MARSHALL PREGNALL,
Nitrate reductase activity in Zostera marina,
432
Roux, E., see D. Morse, 568
ROWAN, EDWARD T., An estimate of primary pro-
ductivity in Waquoit Bay National Estuarine
Sanctuary, Falmouth, Massachusetts, 432
INDEX TO VOLUME 173
585
RUDDLE, F. H., see M. F. Utset, 573
RUDERMAN, J. V., see K. I. Swenson, 572
SALMON, EDWARD D., see Shinya Inoue, 419
Salt marsh, 433
SALVATI, SERAFINA, see Mario Alberghina, 439
SALZBERG, B. M., A. L. OBAID, AND R. FLORES,
Calcium channels required for neuropeptide
release in the intact nerve terminals of verte-
brate neurohypophyses are sensitive to w-con-
otoxin and insensitive to dihydro-pyridines
optical studies with and without voltage dyes,
446
SALZBERG, B. M., see A. R. Cinelli, 435
SAMMON, LESLIE, AND JELLE ATEMA, Detection of
chemical contrast in hemit crabs, 438
SANDS. PETER, see Reed Brozen, 423
SANGER, JEAN M., JEFFREY S. DOME, BALRAJ
MITTAL, AND JOSEPH W. SANGER, Dynamics
of actin, myosin, and membranes in living cells
during cell division, 420
SANGER, JOSEPH, W., see Jean M. Sanger, 420
SARDET, C, see J. E. Speksnijder, 427
Scaling, 434
Scanning electron microscope study ofAscidia ma-
laca egg (Tunicate). Changes in the cell surface
morphology at fertilization. A, 355
Scanning electron microscopy, 444
SCHIERWATER, BERND, AND GEOFF TRACER,
Don't eat if Neptune is angry, 433
SCHNEIDER, MELISSA R., AND ROBERT B. BAR-
LOW JR., Modulation of retinal sensitivity by
putative efferent neurotransmitters, 448
SCOTT, K. E., see C. P. Mangum, 205
Sea anemones, 1 10
Sea urchin H2A.F/Z: an unusually conserved H2A
variant gene, 568
Sea urchin development, 427, 568
Sea urchin egg peptides, 426
Seals, grey and common, 43 1
Sectionless sectioning: a systematic method for
scanning electron microscopic examination of
embedded tissue, 444
SEGAL, S. J., M. H. BURGOS, S. INOUE, AND H.
UENO, Heterospecific sperm motility enhance-
ment by sea urchin oocyte peptides, 426
SEGAL, S. J., see A. L. Kadam, 426; and H. Ueno,
428
SEGENREICH, E. J., J. MITRA, AND J. LoBuE, Mei-
otic chromosomes studies in BALB/c mice
bearing a transplantable mono-myelocytic leu-
kemia (MML) and in the offspring of normal
females crossed with transplanted males, 570
Segmentation, 571
Serial sectioning, 444
Serotonin, 426
Sessile organisms, 110
Settlement, 430
SHANKLAND, M., AND M. Q. MARTINDALE, Spa-
tial patterning of neuronal differentiation in
the leech, 571
SHILLING, FRASER, AND LAURINDA A. JAFFE, Evi-
dence that a G-protein mediates 1-methylade-
nine induced maturation of starfish oocytes,
427
SHORT, FREDERICK T., LISA K. MUEHLSTEIN, AND
DAVID PORTER, Eelgrass wasting disease:
cause and recurrence of a marine epidemic,
557
SICARD, R. E., AND J. C. WERNER, Atrial natri-
uretic peptide (ANP) levels during rat ontog-
eny, 571
SICARD, R. E., see M. F. Lombard, 567
Siderastrea siderea, 539
Signalled avoidance learning of eye withdrawal in
the green crab is predominantly Pavlovian in
mechanism, 435
SILVER, ROBERT B., AND SHINYA INOUE, Calcium
transients are required for mitosis, 420
SILVER, ROBERT B., see Mario H. Burgos, 424
SINGLA, C. L., see G. O. Mackie, 188; and S. A.
Arkett, 513
SIWICKI, K. K., C. EASTMAN, M. ROSBASH, AND
J. C. HALL, Localization of a gene product in
Drosophila that influences biological rhythms,
571
Skate, 443, 447
SLOBODA, R. D., see J. H. Dinsmore, 564
SLUDER, G., F. J. MILLER, K. LEWIS, AND C. L.
REIDER, Centrosomes in echinoderm develop-
ment, 571
SMITH, LAURENS H., JR., AND SIDNEY K. PIERCE,
Cell volume regulation by molluscan erythro-
cytes during hypoosmotic stress: Ca2+ effects
on ionic and organic osmolyte effluxes, 407
SMITH, STEPHEN J., Luis R. OSSES, MILTON P.
CHARLTON, AND GEORGE J. AUGUSTINE,
Fura-2 imaging of calcium transients in squid
giant presynaptic terminal, 446
Sodium
channel gating, 442
current, 442
pump, 445
Sonic motorneurons, 435
Spartina alterniflora, 433
Spatial patterning of neuronal differentiation in the
leech, 571
Spectral sensitivity of the compound eyes in the
purple land crab Gecarcinus laterally (Fremin-
ville), 398
SPEKSNIJDER, J. E., C. SARDET, AND L. F. JAFFE,
Entry of sperm into the animal pole of the egg
ascidian Phallusia mammillata, 427
SPEKSNIJDER, J. E., see L. F. Jaffe, 425
Sperm, 311,428
Sperm motility, 426
Spermatogenesis, 573
Spermatozoa, 424
SPIEGELMAN, BRUCE M., see Robert J. Distel, 565
Spisula, 428
586
INDEX TO VOLUME 173
Spisula oocyte maturation, 425
Squid, 420
Squid axon, 439, 44 1,445
Squid sieilate ganglion, 443
STADLER, H., G. Q. Fox, G. D. PAPPAS, AND
M. E. KRIEBEL, Morphological characteriza-
tion of isolated, concentrated nerve endings of
the skate electric organ, 447
STANLEY-SAMUELSON, DAVID W., Physiological
roles of prostaglandins and oher eicosanoids in
invertebrates, 92
Starfish, 427
STEELE, M. ELIZABETH, see Isidro Bosch, 1 26
STEPP, M. A., see D. W. DeSimone, 564
STINCHCOMBER, D. T., see A. Telfer, 572
STONER, CAROL M., AND LORRAINE J. GUDAS,
Mouse cellular retinoic acid binding protein:
cloning, cDNA sequence and mRNA expres-
sion during the retinoic acid-induced differen-
tiation F9 wild type and RA-3-10 mutant tera-
tocarcinoma cells, 572
STRICKLER, J. RUDI, see Nicholas D. Holland, 552
STUART E., ANNE, see Joseph C. Callaway, 44 1
Studies on a transplantable monomyelocytic leuke-
mia and in the offspring from crosses between
transplanted and normal BALB/c mice, 570
STULL, ANDREW, see Mary Ann Sydlik, 438
Subarachnoid space in the elasmobranch brain —
macro and microscopic evidence using large
molecular weight fluorescent markers, 42 1
Sulfate reduction, 429
Suppression of fictive feeding in vitro by foot shock
in Limax maximus: neural correlates in with-
drawal and feeding systems, 437
Surface changes in Ascidian fertilization, 355
Surface reorganization, 565
Suspension feeding, 434, 552
SUZUKI, SACHIKO, Vitellins and vitellogenins of
the terrestrial isopod, Armadillidium vulgare,
345
SWANDULLA, D., AND C. M. ARMSTRONG, Kinet-
ics of two calcium channel types in chick sen-
sory neurons, 447
SWENSON, K. I., AND J. V. RUDERMAN, Cyclins
and the cell cycle of early clam embryos, 572
Switching in bryozoan feeding modes, 222
SYDLIK, MARY ANNE, ROBERT B. BARLOW JR.,
ANDREW STULL, DONALD R. NELSON, AND
CARRIE KAMMIRE, Migratory behavior of in-
dividual horseshoe crabs, 438
Symbiosis, 504
Symbiotic algae, 367
Synapse, 44 1 , 446
SZMANT, ALINA M., see Nyawira A. Muthiga, 539
SZUTS, ETE Z., see Susan F. Wood, 448; and Wil-
liam R. Eckberg, 424
TELFER, A., D. LEVITAN, U. GIAMBARELLA, AND
D. T. STINCHCOMB, Molecular genetics of
early embryogenesis in C. elegans, 572
Teratocarcinoma cell differentiation, 566
Teratocarcinoma cells, 572
Territoriality, 1 10
Tetrahymena thermophila, 569
THADAMA, KANNUPANDI, see Krishnan Thanka-
vel, 433
THALLER, CHRISTINA, ANDGREGOR EICHELE, Re-
tinoids and pattern formation in vertebrate
limbs, 573
THANKAVEL, KRISHNAN, MARSHALL PREGNALL,
AND KANNUPANDI THADAMA, Protein syn-
thesis and degradation rates in two ecopheno-
types of the cord grass Spartina alterniflora
Loisel from great Sippewissett Salt March,
New England, 433
Three-dimensional reconstruction, 444
Tidal height and gametogenesis: reproductive vari-
ation among populations of Geukensia de-
missa, 160
Time dependent shift in fluorescence emission in
gossypol treated Arbacia sperm, 424
Time-lapse photographic study of neural tube clo-
sure defects in the chick, 563
Tomography, 419
Totipotent development of myoplasm-enriched as-
cidian embryos. The, 423
Trace metals, 429
TRACER, GEOFF, Life is rough when you are small,
434
TRACER, GEOFF, see Bernd Schierwater, 433
Transection of the spinal cord near the obex abol-
ishes cardiovascular compensation for gravity
in bluefish, 422
Transglutaminases, 423
Transition protein 1, 573
Transmitter release, 440
TRICAS, T. C., see R. Kitch, 437
Trimethylamine oxide, 299
TROLL, WALTER, AND SARAH DAVIDSON, Differ-
entiation of Arbacia punctulata is blocked by
the protease inhibitor leupeptin after fertiliza-
tion, 427
Trophosome ultrastructure and the characteriza-
tion of isolated bacteriocytes from inverte-
brate-sulfur bacteria symbioses, 260
Tunic response system, 188
Tunicate larvae settled nearby adults, 474
TURNER, JEFFERSON T., Zooplankton feeding
ecology: contents of fecal pellets of the cope-
pod. Centropages velificatus from waters near
the mouth of the Mississippi, 377
Two classes of miniature end-plate potentials are
present in the isolated, innervated electrocyte,
443
Tail currents, 447
Teeth, 422
Telemetry, 438
U
UV irridation, 425
UENO, H., C. PORTER, R. KAWASHIMA, M. H.
INDEX TO VOLUME 173
587
BURGOS, K. WATANBE, S. J. SEGAL, AND S. S.
KOIDE, Effects of gossypol analogs on Spisitla
sperm, 428
UENO, H., see S.J.Segal, 426
Ultrathin optical-sectioning-tomography achieved
with the light microscope, 419
Ultraviolet light, 449
Upstream and downstream capture during suspen-
sion feeding by Oligometra serripinna (Echi-
nodermata: Cridoidea) under surge condi-
tions, 562
UTSET, M. F., A. AWGULEWITSCH, W. MC&NNIS,
AND F. H. RUDDLE, Region specific expression
of mouse homeo box genes, 573
VALLES, F., see M. Rivera, 569
VAN HOLDE, K. E., see C. P. MANGUM, 205
VARELA, DIANA E., AND JOHN HELFRICH, Bacte-
rial uptake of glutamic acid in oxic and anoxic
waters in a coastal pond, 434
Variability in flash characteristics of a biolumines-
cent copepod, 489
Variability in sexual reproduction of a coral, 335
Variability in the pattern of sexual reproduction of
the coral Stylophorapistillata at Eilat, Red Sea:
a long term study, 335
Vaucheria taylorii, 367
VERBERG, F. JOHN, see Thomas J. Hilbish, 46 1
Vertebrate limbs, 573
Vestibular, 437
Vestibulo-ocular, 437
Vesitbulo-spinal, 437
Videomicroscopy, 419, 563
VILLA, LUISANNA, AND ELEONORA PATRICOLO, A
scanning electron microscope study ofAscidia
malaca egg (Tunicate). Changes in cell mor-
phology at fertilization, 355
VINCENT, W. S., AND SARA PRATT, Co-selection
for clumping and phosphorus accumulation
by bacteria isolated from waste-water treat-
ment systems, 434
Vision in the land crab, 398
Vision, 448
Visualization of actin with rhodamine phalloidin in
the zebrafish egg, 573
Vitellins and vitellogenins of the terrestrial isopod,
Armadillidium vulgare, 345
Voltage-clamp reversal of the sodium pump in dia-
lyzed squid giant axons, 445
W
Waquoit Bay National Estuarine Sanctuary, 432
Waste-water treatment, 434
Wasting disease, 557
WATANABE, K., see H. Ueno, 428
Water column, 434
Water flow control, 188
Water permeability, 230
WEISENSEE, M. H., see L. F. Jaffe, 425
WEISER, MICHAEL, see Andrew Bass, 435
WEISSMAN, IRVING L., see Baruch Rinkevich, 474
WEISSMANN, GERALD, see Reed Brozen, 423
WENTWORTH, S. L., see W. R. A. Muntz, 387
WERNER, J. C., see R. E. Sicard, 571
WHEELER, D., see H. V. Colot, 563
WHITTAK.ER, J. R., Neural induction in ascidian
embryos redivivus, 428
WIDDER, EDITH, A., see Michael I. Latz, 489
WILBERT, NORBERT, see David Kahan, 299
WILEY, SANDRA, see Horst Felbeck, 252
WISEMAN, ROBERT W., AND W. Ross ELLINGTON,
Energetics of contractile activity in isolated
radula protractor muscles of the whelk Busy-
Ion contrarium: anaerobic end product accu-
mulation and release, 277
WOLENSKI, J. S., see N. H. Hart, 565
WOLENSKJ, J. S., AND N. H. HART, Visualization
of actin with rhodamine phalloidin in the ze-
brafish egg, 573
WOOD, SUSAN F., ETE SZUTUS, AND ALAN FEIN,
Aluminun flouride and GTP increase inositol
phosphate production in distal segments of
squid photoreceptors, 448
YELICK, P. C., Y. KWON, P. A. BOWER, K. C.
KLEENE, AND N. B. HECHT, Developmental
regulation of three testis-specific genes during
mouse spermatogenesis, 573
YODER, M., see J. LeBlanc, 566
Yu, Q., see H. V. Peterson, 563
Zebrafish, 565, 573
ZIGMAN, SEYMOUR, AND CHRIS LOWE, Near-UV
light effects on the dogfish (Mustelus canis)
lens, 449
Zoochlorellae, 367
Zooplankton feeding ecology: contents of fecal pel-
lets of the copepod Centropages velificatus
from waters near the mouth of the Mississippi
River, 377
Zostera marina, 43 1 , 432, 557
057
CONTENTS
• ~\/
^ T DEVELOPMENT AND REPRODUCTION
LYNN, JOHN W., AND WALLIS H. CLARK JR.
Physiological and biochemical investigations of the egg jelly release in
Penaeus aztecus . . . /y; >, -/. .\.-'.^. ............. ............. 45 1
/ ECOLOGY AND EVOLUTION
HILBISH, THOMAS J., AND F. JOHN VERNBERG
and shape in the mud crab
461
ahiit&fiv^ and shape in the mud crab
-"-
RlNKEVICFJ, BARUCH,¥;a3R^I^G L. WEtfSMAN
The| fate °f ^{n:7ii/5 (Ascidiacea) larvae cosettled with parental colo-
nies benencial or del^&gipp&corjseqiiences? . .V. ^ .............. 474
'' ''• '- '-'•'"' '•' -^^f- /Sr'«J>-~? ' . '»;v« - "~
GENERALJBIOLOGY
•• - • jf
LATZ, Ml(?HAEE^/f ^A^R?f M^K-A^K, MARK R. BOWLBY, EDITH A. WlD-
DER,'AND ijA-MES F-.-GA&E, ~. :-.-^ ~ - -^
Variability in flash characteristics of a bioluminescent copepod ...... 489
McAuLEY, P. J..
Quantitative estimation of movement of an amino acid from host to
Chlorella symbionts in green hydra . p.^-j^v. . '-^ ....... W^, • • v • • 504
~ '*: ^^t- PHYSIOLOGY J ' ^"
ARKETT, S. A., G. O. MACKIE, AND C. L. SINGLA
Neuronal control of ciliary locomotion in a gastropod veliger (Callio-
stomd) . ,\. . .x. . . , .--; ...... %jj<) ,, ^ .^. -. *J£-\*) ............... . . 513
GROSVENOR, W., AND G. KASS-SIMON
Feeding behavior in Hydra. I. Effects of Anemia homogenate on nema-
tocyst discharge ^. rr^ .)(. . .............. ^^-'. - -'-^ •^'-^.^ • • '.- 527
MUTHIGA, NYAWIRA A., AND ALINA M. SZMANT
The effects of salinity stress on the rates of aerobic respiration and photo-
synthesis in the hermatypic coral Siderastrea siderea . y. J.\ ., . /-.- . . .\ . 539
/ ^4i" ^T I\ SHORTREPORTS^ ^ ^, /" - ^
HOLLAND, NICHOLAS D., ALEXANDER B. LEONARD, AND J. RUDI STRICKLER
Upstream and downstream capture during suspension feeding by Oligo-
metra serripinna (Echinodermata: Cridoidea) under surge conditions 552
SHORT, FREDERICK T., LISA K. MUEHLSTEIN, AND DAVID PORTER
Eelgrass wasting disease: cause and recurrence of a marine epidemic .. 557
-:U '^ ^ ^r'\^. \~',/:-.\ ABSTRACTS ^^ s 0^
ABSTRACTS OF PAPERS PRESENTED AT THE MARINE BIOLOGICAL LABORA-
TORY: NORTHEASTERN REGIONAL CONFERENCE ON DEVELOPMENTAL
BIOLOGY . ^ .\. .^.,. . :", .--^. ^ ,;.} ^^ 85 ...^ ................... 56^
INDEX TO VOLUME 173 . 575
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