i
THE
BIOLOGICAL BULLETIN
PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
JOHN M. ANDERSON, Cornell University MEREDITH L. JONES, Smithsonian Institution
EDWARD M. BERGER, Dartmouth College GEORGE O. MACKIE, University of Victoria
„ _ HOWARD A. SCHNEIDERMAN, Monsanto Research
STEVEN C. BROWN, State University of New York Corporation
at Albany
RALPH I. SMITH, University of California, PHILIP B. DUNHAM, Syracuse University Berkeley
CATHERINE HENLEY, National Institutes of Health F' J°HN VERNBERG' University of
J. B. JENNINGS, University of Leeds EDWARD O. WILSON, Harvard University
W. D. RUSSELL-HUNTER, Syracuse University Managing Editor
VOLUME 157
TO DECEMBER, 1979
(/
Printed and Issued by
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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. Agent for Great Britain : Wheldon and Wesley, Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London, W. C. 2. Single numbers, $10.00. Subscription per volume (three issues). $27.00.
Communications relative to manuscripts should be sent to Dr. diaries B. Metz, Marine Biological Laboratory, Woods Hole, Massachusetts 02543 between June 1 and September 1, and to Dr. Charles B. Metz, Institute For Molecular and Cellular Evolution, University of Miami, 521 Anastasia, Coral Gables. Florida 33134 during the remainder of the year.
THE BIOLOGICAL BULLETIN (ISSN 0006-3185) Second-class-postage paid at Woods Hole, Mass., and additional mailing offices.
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CONTENTS
No. 1 , AUGUST, 1^7()
ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 1
BIRD, DENNIS J., AND ALBERT F. EBLE
Cytology and polysaccharide cytochemistry of the gill of the American
eel, A nguilla rostrata 104
BRIGGS, R. P.
Fine structure of musculature in the copepod Paranthessius anemoniae Claus 112
FELDER, DARRYL L.
Respiratory adaptations of the estuarine mud shrimp, Callianassa jamaicense (Schmitt, 1935) (Crustacea, Decapoda, Thalassinidea) . . 125
HARRIS, LARRY G., AND NATHAN R. HOWE
An analysis of the defensive mechanisms observed in the anemone Anthopleura elegantissima in response to its nudibranch predator Aeolidia papillosa 138
KUHL, DEIRDRE L., AND LARRY C. OGLESBY
Reproduction and survival of the pileworm Nereis succinea in higher Salton Sea salinities 153
LANG, WILLIAM H., RICHARD B. FORWARD, JR., AND DON C. MILLER
Behavioral responses of Balanus improvisus natiplii to light intensity
and spectrum _._ 1 66
NAKATANI, ISAMU, AND TAKASHI OTSU
The effects of eyestalk, leg, and uropod removal on the molting
and growth of young crayfish, Procambarus clarkii 182
STOKES, DARRELL R., AND NORMAN B. RUSHFORTH
Evoked responses to electrical stimulation in the colonial hydroid Clava squamata: A contraction pulse system 189
WENNER, ADRIAN M., AND CRAIG FUSARO
An analysis of population structure in Pacific mole crabs (Ilippa pacifica Dana) 205
No. 2, OCTOBER, 1979
CORNELL, JOHN C.
Salt and water balance in two marine spider crabs, Libinia emarginata
and Pugettia producta. I. Urine production and magnesium regulation. 221
D'ABRAMO, Louis R.
Dietary fatty acid and temperature effects on the productivity ot the cladoceran, Moina macrocopa 234
FUJIMORI, TAKUMI, AND SETSURO HIRAI
Differences in starfish oocyte susceptibility to polyspermy during
the course of maturation 249
KOMATSU, MlEKO, YASUO T. KANO, HlDEKI YOSHIZAWA, SHOJI
AKABANE, AND CHITARU OGURO
Reproduction and development of the hermaphroditic sea-star, Asterina minor Havashi . .. 258
iv CONTENTS
LANE, JACQUELINE Moss, AND JOHN M. LAWRENCE
The effect of size, temperature, oxygen level and nutritional condition
on oxygen uptake in the sand dollar, Mellita quinquiesperforata (Leske) 275
LEE, HSUEH-TZE, AND RICHARD D. CAMPBELL
Development and behavior of an intergeneric chimera of hydra (Pelmatohydm oligactis interstitial cells : Hydra attennata epithelial cells) . . . . ' 288
MARCUS, NANCY H.
On the population biology and nature of diapause of Labidocera aestiva (Copepoda : Calanoida) 297
MOFFETT, STACIA
Locomotion in the primitive pulmonate snail Melampus bidentatus: foot structure and function 306
ROBERTSON, ROBERT, AND TERRY MAU-LASTOVICKA
The ectoparasitism of Boonea and Fargoa (Gastropoda: Pyramid- ellidae) 320
SCARBOROUGH, ANN, AND EARL WEIDNER
Field and laboratory studies of Glugea hertwigi (Microsporida) in the rainbow smelt Osmerus mordax 334
WHITTAKER, J. R.
Development of tail muscle acetylcholinesterase in ascidian embryos lacking mitochondrial localization and segregation 344
ABSTRACTS OF PAPERS PRESENTED AT THE MARINE BIOLOGICAL
LABORATORY. 356
No. 3, DECEMBER,
AUDESIRK, TERESA E.
A field study of growth and reproduction in Aplysia calijornica 407
CORNELL, JOHN C.
Salt and water balance in two marine spider crabs, Libinia emarginata
and Pugettia producta. II. Apparent water permeability 422
COSTOPULOS, JAMES J., GROVER C. STEPHENS, AND STEPHEN H. WRIGHT
Uptake of amino acids by marine polychaetes under anoxic conditions 434
CUKIER, MARTA, GRACIELA ALICIA GUERRERO, AND MARIA CRISTINA
MAGGESE
Parthenogenesis in Coptopteryx viridis, Giglio Tos (1915) (Dyctiop- tera, Mantidae) 445
GALLARDO, C. S.
Developmental pattern and adaptations for reproduction in Nucella crassilabrum and other muricacean gastropods 453
LAMBERT, GRETCHEN
Early post-metamorphic growth, budding and spicule formation in
the compound ascidian Cystodytes Icbitiis 464
MINASIAN, LEO L., JR., AND RICHARD N. MARISCAL
Characteristics and regulation of fission activity in clonal cultures
of the cosmopolitan sea anemone, Plaliplandla luciae (Verrill) 478
CONTENTS v
RUDLOE, ANNE
Locomotor and light responses of larvae of the horseshoe crab, Limulus polyphemus (L.) 494
SATTERLIE, RICHARD A., AND JAMES F. CASE
Development of bioluminescence and other effector responses in the pennatulid coelenterate Ren ill a kollikeri 506
STRATHMANN, R. R., AND E. LEISE
On feeding mechanisms and clearance rates of molluscan veligers 524
ZAMER, WILLIAM E., AND CHARLOTTE P. MANGUM
Irreversible nongenetic temperature adaptation of oxygen uptake in clones of the sea anemone Haliplanella luciae (Verrill) 536
INDEX TO VOLUME 157. 548
*
Volume 157 Number 1
THE
BIOLOGICAfcBULLETIN
AUG 2 2 1979
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
VvOOuo fi*-'! v ;>,
Editorial Board EDWARD M. BERGER, Dartmouth College
JOHN M. ANDERSON, Cornell University MEREDITH L. JONES, Smithsonian Institution
GEORGE O. MACKIE, University of Victoria
HOWARD A. SCHNEIDERMAN, University of STEPHEN C. BROWN, State University of New York California, Irvine
at Albany
RALPH I. SMITH, University of California,
PHILIP B. DUNHAM, Syracuse University Berkeley
„ F. JOHN VERNBBRG, University of
CATHERINE HENLEY, National Institutes of Health South Carolina
J. B. JENNINGS, University of Leeds £. O. WILSON, Harvard University
W. D. RUSSELL-HUNTER, Syracuse University Managing Editor
AUGUST, 1979
Printed and Issued by LANCASTER PRESS, Inc.
PRINCE & LEMON STS. LANCASTER, PA.
THE BIOLOGICAL BULLETIN
THE BIOLOGICAL BULLETIN is published six times a year by the Marine Biological Laboratory, MBL Street, Woods Hole, Massachusetts 02543.
Subscriptions and similar matter should be addressed to THE BIOLOGICAL BULLETIN, Marine Biological Laboratory, Woods Hole, Massachusetts. Agent for Great Britain : Wheldon and Wesley, Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London, W. C. 2. Single numbers, $8.00. Subscription per volume (three issues), $22.00, (this is $44.00 per year for six issues).
Communications relative to manuscripts should be sent to Dr. W. D. Russell-Hunter, Marine Biological Laboratory, Woods Hole, Massachusetts 02543 between June 1 and September 1, and to Dr. W. D. Russell-Hunter, P.O. Box 103, University Station, Syracuse, New York 13210, during the remainder of the year.
Copyright © 1979, by the Marine Biological Laboratory
Second-class postage paid at Woods Hole, Mass., and additional mailing offices.
ISSN 0006-3185
INSTRUCTIONS TO AUTHORS
THE BIOLOGICAL BULLETIN accepts original research reports of intermediate length on a variety of subjects of biological interest. In general, these papers are either of particular interest to workers at the Marine Biological Laboratory, or of outstanding general significance to a large number of biologists throughout the world. Normally, review papers (except those written at the specific invitation of the Editorial Board), very short papers (less than five printed pages), preliminary notes, and papers which describe only a new technique or method without presenting substantial quantities of data resulting from the use of the new method cannot be accepted for publication. A paper will usually appear within four months of the date of its acceptance.
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3. A condensed title or running head of no more than 35 letters and spaces should be included.
4. Literature Cited. The list of references should be headed LITERATURE CITED, should conform in punctuation and arrangement to the style of recent issues of THE BIOLOGICAL BULLETIN, and must be typed double-spaced on separate pages. Note that citations should include complete titles and inclusive pagination. Journal abbreviations should normally follow those of the U. S. A. Standards Institute (USASI), as adopted by BIOLOGICAL ABSTRACTS and CHEMICAL ABSTRACTS, with the minor differences set out below. The most generally useful list
Continued on Cover Three
NOTICE TO SUBSCRIBERS
Effective with the first issue of Volume 158 (February. 1980), the subscription price for THE BIOLOGICAL BULLETIN will be raised. Subscription per volume (three issues) will be $27.00 (this is $54.00 per year for six issues) ; single num- bers will be $10.00 each. Continuing 1980 subscriptions for which payment has actually been received before November 1, 1979 will be honored at the old rate.
Vol. 157, No. 1 Auguust, 1979
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
THK MAR1NK BIOLOGICAL LABORATORY
ElGHTY-FIRST REPORT, FOR THK YKAK 1 978 X IN Ei\ -FIRST YKAR
I. TRUSTEES A.\D EXECUTIVE COMMITTEE (AS OF AUGUST, 1978) . 1 II. CERTIFICATE OF ORGANIZATION. ....
III. ARTICLES OF AMENDMENT. ... 6
IV. BYLAWS OF THE CORPORATION 7 Y. REPORT OF THE DIRECTOR 12
Addenda :
1. The Staff. 19
2. Investigators, Fellowships, and Students 41
3. Scholarships 56
4. Tabular View of Attendance, 1974 1978. 57
5. Institutions Represented 57
6. Friday Evening Lectures 59
7. Members of the Corporation. . . 61 VI. REPORT OF THE LIBRARIAN. 91
VII. REPORT OF THE TREASURER. 92
I. TRUSTEES
Including Action of 1978 Annual Meeting
PROSSER GIFFORD, Chairman of the Board of Trustees, Dean of Faculty, Amherst College, Amherst, Massachusetts 01002
GERARD SWOPE, JR., Honorary Chairman of the Board of Trustees, Croton-oji- Hudson New York, New York 10520
DENIS M. ROBINSON, Honorary Chairman of the Board of Trustees, High Yoltage Engi- neering Corporation, Burlington, Massachusetts 01803
1
Copyright © 1979, by the Marine Biological Laboratory
Library of Congress Card No. A38-518
(ISSN 0006-31 85 i
1 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
JONATHAN O'HERRON, Treasurer, Lazard Freres and Company, 1 Rockefeller Plaza, New York, New York 10020
PAUL R. GROSS, President of the Corporation, and Director of the Laboratory, Marine Biological Laboratory, Woods Hole, Massachusetts 02543
GERALD FISCHBACH, Clerk of the Corporation, Harvard Medical School, Boston, Massa- chusetts 02215
EMERITI
PHILIP B. ARMSTRONG, State University of New York, Upstate Medical Center
ERIC G. BALL, Marine Biological Laboratory
DUGALD E. S. BROWN, Placida, Florida
FRANK A. BROWN, JR., Northwestern University
ANTHONY C. CLEMENT, Emory University
KENNETH S. COLE, National Institutes of Health
ARTHUR L. COLWIN, University of Miami
LAURA H. COLWIN, University of Miami
D. EUGENE COPELAND, Marine Biological Laboratory
PAUL S. GALTSOFF, Woods Hole, Massachusetts
HARRY GRUNDFEST, College of Physicians and Surgeons
DOUGLAS MARSLAND, Woods Hole, Massachusetts
HAROLD H. PLOUGH, Amherst, Massachusetts
C. LADD PROSSER, University of Illinois
JOHN S. RANKIN, JR., Ash ford, Connecticut
A. C. REDFIELD, Woods Hole, Massachusetts
MARY SEARS, Woods Hole, Massachusetts
CARL C. SPEIDEL, University of Virginia
H. BURR STEINBACH, Woods Hole, Massachusetts
ALBERT SZENT-GYORGYI, Marine Biological Laboratory
W. RANDOLPH TAYLOR, University of Michigan
GEORGE WALD, Harvard University
CLASS OF 1982
EVERETT ANDERSON, Harvard Medical School GEORGE H. A. CLOWES, JR., Boston City Hospital ELLEN R. GRASS, The Grass Foundation JOHN P. KENDALL, Boston, Massachusetts EDWARD A. KRAVITZ, Harvard Medical School HANS LAUFER, University of Connecticut MARJORIE R. STETTEN, National Institutes of Health WALTER S. VINCENT, University of Delaware J. RICHARD WHITTAKER, Wistar Institute
CLASS OF 1981
JOHN M. ARNOLD, University of Hawaii JANE FESSENDEN, Marine Biological Laboratory HELEN HOMANS GILBERT, Dover, Massachusetts STEPHEN W. KUFFLER, Harvard Medical School MAURICE LAZARUS, Federated Department Stores, Boston ROBERT MAINER, Boston Company, Incorporated GEORGE PAPPAS, University of Illinois Medical Center W. D. RUSSELL-HUNTER, Syracuse University RAYMOND E. STEPHENS, Marine Biological Laboratory
TRUSTEES
CLASS OF 1980
PHILIP B. DUNHAM, Syracuse University TIMOTHY H. GOLDSMITH, Yale University BENJAMIN KAMINER, Boston University KEITH R. PORTER, University of Colorado LIONEL I. REBHUN, University of Virginia DOROTHY RO\VE, Boston, Massachusetts \V. NICHOLAS THORNDIKE, Boston, Massachusetts EDWARD O. WILSON, Harvard University
CLASS OF 1979
ROBERT D. ALLEN, Dartmouth College
FRANCIS D. CARLSON, The Johns Hopkins University
HAYS CLARK, Avon Products, Incorporated
DENNIS FLANAGAN, Scientific American
PHILIP GRANT, University of Oregon
CATHERINE HENLEY, National Institutes of Health
E. SWIFT LAWRENCE, Pawtucket Institution for Savings
JOHN W. MOORE, Duke University Medical Center
MELVIN SPIEGEL, Dartmouth College
STANDING COMMITTEES
EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES
PROSSER GIFFORD, ex officio FRANCIS CARLSON, 1980
PAUL R. GROSS, ex officio PHILIP GRANT, 1980
JONATHAN O'HERRON, ex officio BENJAMIN KAMINER, 1979
JOHN YV. MOORE, 1981 KEITH R. PORTER, 1979 MELVIN SPIEGEL, 1981
BUDGET COMMITTEE
JOHN \V. MOORE, Chairman JONATHAN O'HERRON, ex officio
JOHN M. ARNOLD JEROME SCHIFF
GEORGE H. A. CLOWES, JR. HOMER P. SMITH, ex officio
PAUL R. GROSS, ex officio WALTER S. VINCENT WILLIAM T. GOLDEN
BUILDINGS AND GROUNDS COMMITTEE
FRANCIS HOSKIN, Chairman AUDREY E. V. HASCHEMEYER
FRANK CHILD FRANK LONGO
LAWRENCE B. COHEN ROBERT D. PRUSCH
ALAN FEIN RONALD PRZYBYLSKI
DANIEL GILBERT ALLEN SCHUETZ
ROBERT GUNNING, ex officio EVELYN SPIEGEL
COMPUTER SERVICE COMMITTEE
JOHN HOBBIE, Chairman E. F. MAcNiCHOL, JR.
WILLIAM J. ADELMAN MELVIN ROSENFELD, JR.
FRANCIS P. BOWLES CONSTANTINE TOLLIOS ALLAHVERDI FARMANFARMAIAN
4 ANNUAL i'l I'ORT OF THE MARINE BIOLOGICAL LABORATORY
EMPLOYEE RELATIONS COMMITTEE
JOAN HOWARD, Chairman LEWIS LAWDAY
CAROL EBERHARD DONALD LEHY
CHARLOTTE FRANK RAYMOND STEPHENS JUDITH GRASSLE
HOUSING, FOOD SERVICE AND DAY CARE COMMITTEE
J i Di in GKASSLK, Chairman BRIAN M. SALZBERG
DANIEL ALKON HOMER P. SMITH, ex officio
EARLENE ARMSTRONG ANN STUART
SAMUEL I. BEALE LEON P. WEISS
. \IMLEE LADERMAN JANE ZAKEVICIUS
INSTRUCTION COMMITTEE
BENJAMIN KAMINER, Chairman R. K. JOSEPHSON
DANIEL ALKON MORTON MASER, ex officio
ROBERT ALLEN MERLE MIZELL
CLAY M. ARMSTRONG JOEL ROSENBAUM
ESTHER GOUDSMIT SHELDON J. SEGAL
ARTHUR HUMES GEORGE WOODWELL
INVESTMENT COMMITTEE
YV. NICHOLAS THORNDIKE, Chairman MAURICE LAZARUS
PROSSER GIFFORD, ex officio JOHN W. MOORE
WILLIAM T. GOLDEN JONATHAN O'HERRON, ex officio
LIBRARY COMMITTEE
KEITH R. PORTER, Co-chairman STEPHEN W. KUFFLER EDWARD A. ADELBERG, Co-chairman LUIGI MASTROIANNI, JR.
FRANK M. CHILD ROBERT MORSE
BERNARD DAVIS ARTHUR PARDEE
JOHN DOWLING SHELDON J. SEGAL
J. W7. HASTINGS MARJORIE R. STETTEN
SHINYA INOUE STANLEY WATSON
M\( N SCHOLARSHIP COMMITM-!!'
JOEL E. BROWN, Chairman WILLIAM W. SUTTON
ALLEN COUNTER JAMES TOWNSEL
LOWELL DAVIS CHARLES W^ALKEK MORTON MASER, ex officio
MARINE RESOURCES COMMITTEE
SEARS CROWELL, Chairman ROBERT D. PRUSCH
FRANCIS P. BOWLES JOHN S. RANKIN
TOM HUMPHREYS CARL P. SWANSON
JACK LEVIN JONATHAN WITTENBERG
CYRUS LEVINTHAL JOHN VALOIS, ex officio ROBERT PRENDERGAST
CERTIFICATE OF ORGANIZATION RADIATION ( OMMITTEE
\\.\LTER S. YlMKVI, Clinirnniii JOHN lloMBIE
EUGENE BELL I I MAcNicnoi., JR.
FRANCIS BOWLES MORTON MASER, e\ officio
RICHARD L. CHAPPELL HARRIS RIPPS PAUL J. DE\VEER
RESEARCH SERVICES COMMITTEE
ARTHUR DuBois, Chairman T. NARAHASHI
NINA S. ALLEN GEORGE PAPPAS
FRANCIS P. BOWLES JEROME SCHIFF
S. S. KOIDE BRUCE SZAMIER
E. F. MAcNiCHOL, JR. JAY WELLS
MORTON MASER, ex officio SEYMOUR ZIGMAN
RESEARCH SPACE COMMITTEE
TIMOTHY GOLDSMITH. Chairman MORTON MASER, ex officio
JOHN ARNOLD BRIAN SALZBERI.
PHILIP GRANT ANN STUART
FREDERICK GRASSLE BETTY WALL
GEORGE LANGFORD GEORGE WOODWELL
SAFETY COMMITTEE
ROBERT GUNNING, Chairman E. F. MACNICHOL, JR.
DANIEL ALKON MORTON MASER, ex officio
FRANCIS P. BOWLES TOSHIO NARAHASHI
LEWIS LAWDAY RAYMOND E. STEPHENS
DONALD LEHY FREDERICK THRASHER
II. CERTIFICATE OF ORGANIZATION
(On File in the Office of the Secretary of the Commonwealth) No. 3170
We, Alpheus Hyatt, President, William Stanford Stevens, Treasurer, and William T. Sedgwick, Edward G. Gardiner, Susan Minis and Charles Sedgwick Minot being a majority of the Trustees of the Marine Biological Laboratory in compliance with the requirements of the fourth section of chapter one hundred and fifteen of the Public- Statutes do hereby certify that the following is a true copy of the agreement of associa- tion to constitute said Corporation, with the names of the subscribers thereto :-
\\ e, 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 Massa- chusetts, and the Acts in amendment thereof and in addition thereto.
The name by which the Corporation shall be known is THE MARINE BIOLOGICAL LABORATORY
6 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
Tht 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 Minis, B. H. Van Meek.
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 Minis, Charles Sedgwick Minot.
(Approved on March 20, 1888 as follows:
1 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 require- ments 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}
III. 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 meetings of the corporation:
VOTF.I): That the Certificate of Organization of this corporation be and it hereby is amended by the addition of the following provisions:
'Xo Officer, Trustee or Corporate Member of the corporation shall be personally liable for the payment or satisfaction of any obligation or liabilities incurred as a result of, or otherwise in connection with, any commitments, agreements, activities or affairs of the corporation.
BYLAWS OF THE CORPORATION 7
"Except as otherwise specifically provided by the Bylaws of the corpora- tion, meetings of the Corporate Members of the corporation may be held anywhere in the United States.
"The Trustees of the corporation may make, amend or repeal the Bylaws of the corporation in whole or in part, except with respect to any pro- visions thereof which shall by law, this Certificate or the Bylaws of the corporation, require action by the Corporate Members."
The foregoing amendment will become effective when these articles of amendment are filed in accordance with Chapter 180, Section 7 of the General Laws unless these articles specify, in accordance with the vote adopting the amendment, a later effective date not more than thirty days after such filing, in which event the amendment will become effective on such later date.
In Witness whereof and Under the Penalties of Perjury, we have hereto signed our names this 2nd day of September, in the year 1975, James D. Ebert, President; David Shepro, Clerk.
(Approved on October 24, 1975, as follows:
I hereby approve the within articles of amendment and, the filing fee in the amount of $10 having been paid, said articles are deemed to have been filed with me this 24th day of October, 1975.
PAUL GUZZI
Secretary of the Commonwealth)
IV. BYLAWS OF THE CORPORATION OF THE MARINE BIOLOGICAL LABORATORY
(Revised August 11, 1978)
I. (A) The name of the Corporation shall be The Marine Biological Laboratory. The Corporation's purpose shall be to establish and maintain a laboratory or station for scientific study and investigation, and a school for instruction in biology and natural history.
(B) Marine Biological Laboratory admits students without regard to race, color, sex, national and ethnic origin to all the rights, privileges, programs and activities generally accorded or made available to students in its courses. It does not discriminate on the basis of race, color, sex, national and ethnic origin in employment, administration of its educational policies, admissions policies, scholarship and other programs.
II. (A) The members of the Corporation ("Members") shall consist of persons elected by the Board of Trustees, upon such terms and conditions and in accordance with such procedures, not inconsistent with law or these Bylaws, as may be determined by said Board of Trustees. Except as provided below, any Member may vote at any meeting, either in person or by proxy executed no more than six months prior to the date of such meeting. Members shall serve until their death or resignation unless earlier removed, with or without cause, by the affirmative vote of two-thirds of the Trustees then in office. Any Member who has attained the age of seventy years or has retired from his home institution shall automatically be designated a Life Member provided he signifies his wish to retain his membership. Life Members shall not have the right to vote and shall not be assessed for dues.
8 ANNUAI KH'ORT OF THE MARINE BIOLOGICAL LABORATORY
(B) The Associates of the Marine Biological Laboratory shall he 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 ^rcond Tuesday in August in each year at the Laboratory in Woods Hole, Massachu- setts, at 9:30 a.m. Subject to the provisions of Article VI 1 1 (2), at such meeting the Members shall choose by ballot six Trustees to serve four years, and shall transact such other business as may properly come before the meeting. Special meetings of the Members may be called by the Chairman or Trustees to be held at such time and place as may be designated.
V. Twenty rive Members shall constitute a quorum at any meeting. Except as otherwise required by law or these Bylaws, the affirmative vote of a majority of the Members voting in person or by proxy at a meeting attended by a quorum (present in person or by proxy) shall constitute action on behalf of the Members.
VI. (A) Inasmuch as the time and place of the Annual Meeting of Members are fixed by these Bylaws, no notice of the Annual Meeting need be given. Notice of any special meeting of Members, however, shall be given by the Clerk by mailing notice of the time and place and purpose of such meeting, at least 15 days before such meeting, to each Member at his or her address as shown on the records of the Corporation.
(B) Any meeting of the Members may be adjourned to any other time and place by the vote of a majority of those Members present or represented at the meeting, whether or not such Members constitute a quorum. It shall not be necessary to notify any Member of any 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 tiled with the records of the meeting, or if he shall attend the meeting without pro- testing 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 procedures, not inconsistent with these Bylaws, as the Trustees shall have deter- mined. 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,
BYLAWS HI- THK CORPORATION <)
(2) Trustees ("Board Trustees") elected by the Trustees then in office according to such procedures, not inconsistent with these Bylaws, as the Trustees shall have determined. Except as provided below, such Board Trustees shall be divided into four classes of three, one class to be elected each year to serve for a term of four years. Such classes shall be designated by the year of expiration of their respective terms. It is contemplated that, unless otherwise determined by the Trustees for good reason, Board Trustees shall be individuals who have not been considered for election as Corporate Trustees.
(3) Trustees ex officio, who shall be the Chairman, the President, the Director, the Treasurer, and the Clerk.
(4) Trustees emeriti who shall include any Member who has attained the age of seventy years (or the age of sixty five and has retired from his home institution) and who has served a full elected term as a regular Trustee, provided he signifies his wish to serve the Laboratory in that capacity. Any Trustee who qualifies for emeritus status shall continue to serve as a regular Trustee until the next Annual Meeting whereupon his office as regular Trustee shall become vacant and be filled by election by the Members or by the Board, as the case may be. The Trustees ex officio and emeriti shall have all the rights of the Trustees, except that Trustees emeriti shall not have the right to vote.
(B) The aggregate number of Corporate Trustees and Board Trustees elected in any year (excluding Trustees elected to fill vacancies which do not result from expira- tion 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 suc- cessors are chosen in their stead.
(D) Any Trustee may be removed from office at any time with or without cause, by vote of a majority of the Members entitled to vote in the election of Trustees; or for cause, by vote of two-thirds of the Trustees then in office. A Trustee may be removed for cause only if notice of such action shall have been given to all of the Trus- tees or Members entitled to vote, as the case may be, prior to the meeting at which such action is to be taken and if the Trustee so to be removed shall have been given reasonable notice and opportunity to be heard before the body proposing to remove him.
(E) Any vacancy in the number of Corporate Trustees, however arising, may be filled by the Trustees then in office unless and until filled by the Members at the next Annual Meeting. Any vacancy in the number of Board Trustees may be filled by the Trustees.
(F) A Corporate Trustee or a Board Trustee who has served an initial term of at least 2 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 a Trustee.
IX. (A) The Trustees shall have the control and management of the affairs of the Corporation. 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 Corpora- tion 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 elect a Treasurer and Clerk to serve one year, and 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
10 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
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 ex- pedient in addition to those powers conferred by Article X. They shall from time to time elect Members to the Corporation upon such terms and conditions as they shall have determined, not inconsistent with law or these Bylaws.
(B) The Board of Trustees shall also have the power, by vote of a majority of the Trustees then in Office, to elect an Investment Committee and any other committee and, by like vote, to delegate thereto some or all of their powers except those which by law, the Articles of Organization or these Bylaws they are prohibited from delegating. The members of any such committee shall have such tenure and duties as the Trustees shall determine; provided that the Investment Committee, which shall oversee the management of the Corporation's endowment funds and marketable securities, shall include the Chairman of the Board of Trustees, the 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 Trus- tees, 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 Committee, and the President as Vice Chairman. A majority of the members of the Executive 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 Com- mittee shall determine.
(C) The Executive Committee shall have and may exercise all the powers of the Board during the intervals between meetings of the Board of Trustees except those powers specifically withheld from time to time by vote of the Board or by law. The Executive Committee may also appoint such committees, including persons who are not Trustees, as it may from time to time approve to make recommendations with respect to matters to be acted upon by the Executive Committee or the Board of Trustees.
(D) The Executive Committee shall keep appropriate minutes of its meetings and its action shall be reported to the Board of Trustees.
(E) The elected Members of the Executive Committee shall constitute as a standing "Committee 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, Presi- dent, 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,
BYLAWS OF THE CORPORATION' 11
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 Ex- ecutive Committee or any other committee appointed by the Trustees may also parti- cipate 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 Trustee shall be necessary to dissolution of the Marine Biological Laboratory. In case of dissolution, the property shall be disposed of in such manner and upon such terms as shall be determined by the affirmative vote of two-thirds of the Board of Trustees then in office.
XIV. These Bylaws may be amended by the affirmative vote of the Members at any meeting, provided that notice of the substance of the proposed amendment is stated in the notice of such meeting. As authorized by the Articles of Organization, the Trustees, by a majority of their number then in office, may also make, amend, or repeal these Bylaws, in whole or in part, except with respect to (a) the provisions of these Bylaws governing (i) the removal of Trustees and (ii) the amendment of these Bylaws and (b) any provisions of these Bylaws which by law, the Articles of Organiza- tion or these Bylaws, requires action by the Members.
No later than the time of giving notice of the meeting of Members next following the making, amending or repealing by the Trustees of any Bylaw, notice thereof stating the substance of such change shall be given to all Corporation Members entitled to vote on amending the Bylaws.
Any Bylaw adopted by the Trustees may be amended or repealed by the Members entitled to vote on amending the Bylaws.
XV*. The account of the Treasurer shall be audited annually by a certified public accountant.
XVI. The Corporation will indemnify every person who is or was a trustee, officer or employee of the Corporation or a person who provides services without compensa- tion to an Employee Benefit Plan maintained by the Corporation, for any liability (including reasonable costs of defense and settlement) arising by reason of any act or omission affecting an Employee Benefit Plan maintained by the Corporation or affect- ing the participants or beneficiaries of such Plan, including without limitation any damages, civil penalty or excise tax imposed pursuant to the Employee Retirement Income Security Act of 1974; provided, (1) that the Act or omission shall have occurred in the course of the person's service as trustee or officer of the Corporation or within the scope of the employment of an employee of the Corporation or in connection with a service provided without compensation to an Employee Benefit Plan maintained by the Corporation, (2) that the Act or omission be in good faith as determined by the Corporation (whose determination made in good faith and not arbitrarily or capriciously shall be conclusive), and (3) that the Corporation's obligation hereunder shall be offset to the extent of any otherwise applicable insurance coverage, under a policy maintained by the Corporation or any other person, or other source of indemnification.
12 ANXl'AI. KKI'ORT OF THE MARINE BIOLOGICAL LABORATORY
V. RKPORT 01 THK DIRECTOR
I o: THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY
"We should be careful to get out of an experience only the wisdom that is in it and stop there; lest we be like the cat that sits down on a hot stove-lid. She will never sit down on a hot stove-lid again — and that is well ; but also she will never sit down on a cold one anymore."
—Mark Twain
The calendar year to which this report refers saw many changes. Not the least of these was the departure of James Ebert for the Presidency of the Carnegie Institution of Washington. Ebert's tenure extended through about half of the year, although he was present in person, and by way of generous advice and guidance from Washington in spirit, for a far larger part of it. Other changes have taken place; some in manage- ment, some in programs and courses, and some in the Laboratory's rules of operation. A most significant event of the latter kind was adoption by the Trustees, in February of 1979, of a comprehensive seven-year plan for programs, space utilization, and campus improvement.
Many of the transitional changes, like the Trustees' plan and its related actions, came in 1979, and will therefore not be covered in this report. Their occurrence is felt, however, at the time of writing, and their impact upon the style of life and work at the Laboratory will doubtless increase in the year to come.
James D. Ebert
Although a State of the Institution report is not the appropriate place for a tribute to its retired chief executive officer, there being better occasions and places, I cannot let pass the opportunity to mark the debt we owe to Dr. Ebert. His long association with the MBL, which we trust will continue, reached a peak of intensity during an influential tenure as the Laboratory's first full-time Director and President. Always forceful and direct in his views, more analytical and better-prepared than most who disagreed with him, Ebert brought about a series of major alterations in the way the MBL functions, meeting successfully, thereby, a number of crises in finance and management. He was able to accomplish this without any infringement of program quality nor of the tradi- tional rights and responsibilities of the Corporation.
I have a more detailed view of his efforts than is generally available, owing to the records of his work, upon which we are daily dependent, and to my now frequent con- tacts with friends of the Laboratory to whom Ebert first took the MBL's case. There can be no denial of the fact that his efforts were as fruitful as they were tireless and self- sacrificing. I believe that his contribution to the Laboratory will stand permanently as having been central to its survival in strength.
Research
To separate research from education is a process even more artificial for the MBL than it is for universities. The intertwining of the two is here less an accident and more a design than in any other scientific institution, ft has been so ever since Director Whitman wrote in the first Annual Report:
"Other things being equal, the investigator is always the best instructor. The highest grade of instruction in any science can only be furnished by one who is thoroughly imbued with the scientific spirit, and who is actually engaged in original work. Whence the propriety — 'and, I may say, the necessity — of linking the function of instruction with that of investigation."
REPORT OF THE 1)1 HECTOR 13
It is nevertheless iiM-tul to report separately upon re-search activity at the MBL, to the extent that is characterized by numbers of persons so engaged, by the sums awarded and spent for the purpose, and by the facilities given up to it, as opposed, for example, to those employed solely for classroom activities.
\Ye can be proud that in a time of declining real support for research nationwide, and of exponentially-growing costs and demands upon physical facilities, the MBL has been able, upon a comparatively uncertain financial base, to enlarge the scope and im- pact of its research, and to do so without denying access to its facilities by any properly qualified person or group.
There are four broad areas of research that together encompass most, although not all, investigative activities at the Laboratory. These are (1) Ecology, (2) Cellular, Developmental, and Reproductive Biology, (3) Xeuroscience, Physiology, and Bio- physics, and (4) Marine Biology and Biomedicine. In each of these areas the Labora- tory provides for permanent (or year-round) as well as transient (mainly, but not exclusively summer) programs.
In 1978 there was growth in all four, as measured by people, funds, and — since no decline of standards was, or will ever be permitted — by impact upon biology as a whole, i.e., by quality. The simple argument underlying this last conclusion is from informa- tion theory, in which one obtains first the information content per individual in an assemblage (the units being "bits" or "nats," or some other, depending upon the base of logarithms chosen), and then the information content of the entire assemblage by multiplying the result by the number of individuals.
Lest this be interpreted as an argument for unlimited growth, I make haste to demur. It is not. It is, rather, a way of saying that, within limits, growth accompanied by maintained standards is ipso facto an increase in total quality.
The Ecosystems Center, the Boston L^niversity Marine Program, the Laboratory of Sensory Physiology, and Laboratory of Biophysics, and a group of some twenty individual research programs operated at the MBL have all performed as well or better in 1978 than they did in 1977. By performance I mean the sum of those measures employed in our system of program evaluations: important publications; grants awarded; honors and awards to staff; presentations of important lectures; quality of students and other participants attracted ; and the like.
These activities, which comprise the currently permanent part of the MBL's re- search effort, have already reached a size and level of achievement such that to single out one or two for special mention here would be impolitic. It may well be that we shall soon need a separate Annual Report on Research, in order to do justice to the accomplishments of our permanent programs. Suffice it to say that the health and quality of year-round research at MBL are no longer at issue. The issue will instead be, for the future, to control growth while still retaining a decent flexibility of operation, so that life at MBL does not decay to the level of that lived in many universities, where changes in space assignment are accomplished only at the expense of internecine warfare.
In the summer of 1978 research space at the Laboratory was fully occupied, except for a few rooms that fell vacant at a late hour, owing to changes of plans. For the summer of 1979 there were far more applications even than in the prior year. \Ye have been able, at least at the time of writing, to accommodate all those whose proposed research was judged appropriate to our standards by the Research Space Committee. This was accomplished by several devices, including the use for research of rooms not formerly rented for that purpose, and a vigorous effort to encourage the sharing of space, when that would not interfere with research quality. I may say that applicants so approached by us, including Corporation members who have enjoyed a certain luxury in past summers, responded magnificently. No such suggestion for sharing failed to be received courteously and to be considered seriously.
U ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
The above is a quantitative statement, of course, about the summer research pro- gram, but the argument made earlier about population and information content ap- plies: there is a significant and continuing increase in the demand for research space and facilities here assignable for such use, and the overall quality is rising. All indicators suggest that the trend will continue.
It is evident from the foregoing that we shall have a great deal of decision-making to do in the future as regards deployment of space and facilities. This is a challenge that we ought not to fear: far better to be in a position of making fine distinctions— however fallible peer judgments may sometimes be, and however painful rejection is— than to be unable to assign all the available space for want of good contenders.
A few sample numbers: overhead recovery from non-MBL research grants (via space charges) was $424,631 in 1977, $503,576 in 1978, and we have budgeted $562,699 for 1979. The corresponding figures for MBL-administered grants (government and private) are $251,916; $297,136; $391,766. These number do, of course, have an inflation component, and hence do not imply a proportionate increase in the absolute size of the research effort, but they do reflect a real and impressive size increment nevertheless. Were it not that the inflationary rise of operating costs easily matches them, I might be moved to call such figures "healthy."
Some losses and gains of research persons should be mentioned, although of course space limitation prohibits anything like a comprehensive listing. There was the very tragic loss of Dr. Fred Lang, of the B.U.M.P., in an automobile accident, and that must be mentioned. Two other honored and well-known Corporation members died in the course of the year: Jean Clark Dan and Lester G. Barth.
It is a personal pleasure for me and splendid news for the Laboratory that Professor Shinya Inoue, of Pennsylvania, a distinguished cell biologist and long-time Corporation member, will henceforth be in permanent residence at the MBL, and will play an im- portant role in the planning and growth of our cell biology programs. Several addi- tional appointments of importance have been made or are in the making. They will be announced in due course.
Education
As indicated earlier, every research activity at the MBL has a closely-related educational component; hence a mere catalogue of the formal courses does not convey the pervasiveness of learning and instruction that characterizes work here. As is true for research, however, a simple listing can indicate something of the scope of the pro- gram, and of changes taking place within it, for Corporation members already familiar with it.
Each of the five January courses offered in 1978 was continued in 1979: Behavior (J. Atema) ; Comparative Pathology of Marine Invertebrates (F. Bang) ; Develop- mental Biology (W. Vincent) ; Ecology (G. \Yoodwell) ; and Neurobiology (Alan Fein, together with E. F. MacNichol, Jr. and others, who carried on following the death of Fred Lang). One hundred fourteen students completed courses in the now well- established January semester, to which contributions were made by a group of dis- tinguished teachers from around the nation, as well as from the Woods Hole resident community. The January semester is an obvious success, but its growth in future years will be small, limited by the space and facilities available for it.
Since it is clear that the January semester is to be a recurrent MBL activity, we shall be engaged during months to come in a number of management and quality control initiatives. These will bring the entire program under the same sort of continuous review, eventually, as is applied to the summer courses, possibly by the same committee of the Corporation. We will also attempt to regularize the financial arrangements, i.e.,
REPORT OF THE DIRECTOR 15
those governing tuition charges, reimbursements and compensation for faculty and guest speakers, and the assignment of operating costs among various cost centers of the budget. It is even now clear, however, that the January semester places no great financial burden upon the Laboratory, while yielding large benefits to the community intellectual life during what was formerly a slow month. Of course it provides invalu- able educational benefits to the students, whose many letters attesting thereto are a gratifying chapter of our files.
All seven of the distinguished summer courses continued under the same directorates as in 1977, with one hundred forty-eight participants. During the year a number of searches for directorships due to become vacant in 1980 were initiated. Thomas Reese and John Hildebrand have been appointed co-directors of the Xeurobiology Course to succeed Edward Kravitz. Outcomes of other searches will be announced in the summer of 1979. The excellent Microbial Ecology program has been elevated, under stimulus of the recommendations of an ad hoc committee and with approval of the Standing Committee on Instruction, to the status of a regular MBL summer course. Ably directed in 1978 and in prior years by Holger Jannasch, it will for 1979 and 1980 have Jannasch as co-director with Harlyn Halvorson, who will then continue as Instructor- in-Chief for an additional three years. A major effort is underway to provide this new course with suitable quarters in the Loeb Building by the summer of 1980.
Because of the tragic death of Mrs. Gelperin in May, 1979, Alan Gelperin will be unable to serve, at least for 1979, as director of the course on Neural Systems and Be- havior. Faculty member Ronald Hoy agreed promptly and generously to step in as interim director. For this reason we anticipate a smooth operation of the relatively new offering despite the absence, hopefully a temporary one, of its energetic first director.
Active planning was begun in April, 1979, for a 1980 summer course in the Biology of Parasitism. The planning group includes, in addition to a number of MBL ad- ministrators and our own Frederik Bang, director of the January course on comparative pathology and a scholar of international reputation in the field, several accomplished members of the Harvard and Rockefeller University faculties, together with scientific officers of the Rockefeller and Edna McConnell Clark Foundations. The MBL Com- mittee on Instruction, also represented among the planners (by Chairman Kaminer), will be concerned with detailed proposals during the summer of 1979.
The spring course in Aquatic Veterinary Medicine, a combined offering of the LTniversity of Pennsylvania, Cornell University, and three of the four Woods Hole institutions, directed energetically by Donald Abt, was offered for the second time, and with pronounced success. Interest in the program is sufficiently high to justify not only its continuation, but also expansion of the effort to include post-course research and, possibly, a number of more permanent activities centered at the MBL.
Morton Maser's program of continuing education, in the form of Short Courses, continues to merit the praise accorded it in Dr. Ebert's 1977 report. In 1978 there were eleven such courses, most of them concerned with advanced instrumentation and re- search technology, and featuring faculties of an exceptional expertise. Among the Instructors-in-Chief were Robert Allen, Blair Bowers, Lee Peachey, Bruce Wetzel, Russell Steere, Eduardo Macagno, Yukata Kobayashi, Patrick O'Farrell, Robert Ivarie, and Maser. A total of 157 participants came to Woods Hole in the months of March, April, May, October, November and December for these high-intensity courses. Their quality has been the subject of uniformly favorable comment, from students and faculty alike. A most enthusiastic article about them has been published in the Norelco Reporter.
Funding of all the formal educational offerings is at a high level, although as uni- versity science administrators well know, it is impossible to recover the current real
16 ANNUAL REPOR1 OF Till''. MARINE UK )!.()( ,K.\I. LABORATORY
costs uf advanced laboratory teaching- Nearly every summer course receives direct sii|)port from government or private grants, or from a combination of the t\vo. Those few courses that \\ere temporarily without directed support in 1978 have it or are very likely to have it in 1979. With reorganization of the financing and accounting for the January Semester, it will become practical to initiate an energetic program of fund- raising for that venture. Short Courses come very close to paying for themselves; indeed, they are upon a simple cash basis truly self-supporting.
This section cannot close without mention of the Boston University Marine Pro- gram, which continues as a leading opportunity, nationally, for graduate students seeking to specialize in marine biology. The MBL's pride in the Program and pleasure in its fine students and faculty is matched by opinion of the faculty and central ad- ministration at Boston University. An outstanding new appointment to the B.U.M.P. faculty has been made this year (Dr. Sidney Tamm, a cell biologist who will work with the already prominent MBL motility group). Dr. Christopher H. Price, an active in- vestigator in the field of neurobiology, has been appointed to replace Dr. Lang.
.^i/t'iitifii Meetings and Conferences
Among the blessings that some of us take for granted at the MBL is the skill and efficiency with which housing and other maintenance needs of transient participants in our programs are handled. It is a task of the greatest complexity, involving not only the work of a specialized support staff, but also attention to detail, patience, and tact, coupled with a willingness to deal under pressure with many demands for exceptions to rules. All this Homer Smith does, as he has done for many years, quietly and effectively.
The more remarkable, therefore, is the rapidly increasing exploitation of our Swope Center, comprising as it does a significant additional burden of work. This is done, again, with minimum fuss, and by a support staff that would be viewed as skeletal by the management of a small motel.
In 1978, there were only two months (June and December) — and those with good reason — during which there were not significant numbers of working visitors to the MBL, residing in the Swope Center and making use of the Laboratory's facilities for one or another scientific or educational purpose. The total number of such persons reached a remarkable 2,300. Activities and institutions represented included : Massachusetts Marine Educators, St. Mary's College, Purdue University, National Marine Fisheries Service, Yale University, East Coast Nerve Net, International Symposium on the Spermatozoon, M.I.T., the University of Pennsylvania, Harvard University, Drew University, Upsala College, the Society of General Physiologists, the Marine Science Librarians, University of Maryland, Maine Audubon Society, a conference on Bio- medical Applications of Limiilits, Brown University, the New England Estuarine Society, Simmons College, and many others.
The value of such utilization of our facilities, quite aside from the implied financial benefits, is incalculable. All the Woods Hole institutions are beneficiaries (as is the village as a whole), and those perceived values attest to the wisdom of MBL people whose labors and gifts made the Swope Center a reality.
Once again, our problem is now one of choice among worthy competing alternatives, rather than of recruitment of users, — one of growth control, rather than of its simple encouragement. Again, I should be inclined to describe the whole phenomenon as unqualifiedly healthy, were there not the ghost of concern about inexorable inflation of operating costs, which, however carefully they are held down by means of economies, tend to alter what should be a surplus-producing function to one that may just pay for itself. The matter of costs and charges in this sphere will receive serious review in the year to come.
KKI'ORT OF TIIK DIKF.CTOR 1 7
Management
Significant changes in management siruciure and uvlmique will certainly take place in the two or three years ahead, but \ve have been very cautious about changes during this year of transition. Caution is demanded for more than the usual reasons of morale: most changes in management that ought to be made will cost more than current systems, but we are determined that administration shall set an example of cost-consciousness for the institution as a whole.
Francis Bowles, who has served effectively as head of the Department of Research Services, has relinquished that position in favor of an association with the Ecosystems Center, which provides him a deserved increase of opportunity to pursue his own re- search and technical interests. He has been replaced by Morton Maser, who adds this responsibility to his already major commitment in continuing education and in the operations of service laboratories. In recognition thereof, Maser has been named Assistant Director for Educational and Research Services, with a broad range of de- fined duties and responsibilities concerned with research support services, equipment space deployment, admissions, and the day-to-day operations of our formal courses. He will work closely with the Director, with the Instructors-in-Chief, and with the Committee on Instruction in an effort to bring an increase of order and predictability into what is an unnecessarily variable system of management.
All activities enumerated in earlier sections need and deserve published notice at a number of levels of specificity — from in-house posting of events schedules to journal articles and press releases. These are the purview of the Public Relations Department which, with minimal staff under the direction of Anne Camille Maher, has met the demand with honor. Among particularly useful ventures of the past year (quite aside from such public events as MBL Day) have been the appearance of a regular and timely calendar of scientific activities, and a new and greatly improved directory of personnel and programs.
Our able Controller, Edward Casey, and his competent staff continue to get things done accurately and on time, and to accomplish that miracle with a staff, space, and machinery that would be more appropriate to an institution half the size of the MBL. Some relief from the burden of work and responsibility will have to be provided for that department soon. In the meantime it is to be commended for its management of accounting and financial control functions. Among those are the duties of Joan Howard, MBL Grants and Contracts Administrator, who deals competently with a volume of government support for research and teaching that would, in better-endowed circumstances, call for an office full of people.
The Library, too, deserves commendation, for the self-effacing but highly profes- sional style with which its services are managed and rendered to the entire community, again under circumstances of fiscal restraint. \Ye will be announcing during the summer of 1979 a number of encouraging initiatives and gifts which, all together, will make easier the work of our Library staff and improve significantly the facilities and services offered to users.
Support
Details of Foundation, Corporate, and other support received during 1978 appear below in a separate listing. For the present it is sufficient to note that Foundation sup- port, specifically, came to $719,142 in 1978, as compared with $763,699 in 1977. This is a good showing, considering that it was a year of management transition, requiring a certain temporary slowing of fund-raising activity. In fact gifts received early in calendar year 1979, and others in prospect, make it certain that the two-year totals for 1978 and 1979 will exceed by a large factor those obtained in any prior period ot similar
IS ANNUAL REPORT OF THE MARINE BIOLOCICAI. LABORATORY
length. This will, it is hoped, he the subject lor considerable discussion and action beginning in the late summer of 1979. It is to be noted that the Annual Campaign yielded twice as much support in 1978 as in 1977.
Atlantic Richfield Foundation
Charles Ulrick & Josephine Bay Foundation
Charles E. Culpeper Foundation
Fred Harris Daniels Foundation, Inc.
Arthur Vining Davis Foundations
Henry L. and Grace Doherty Charitable Foundation
Eastern Associated Foundation
Exxon Corporation
Foundation for Microbiology
Walter Henry Freygang Foundation
Friendship Fund
General Electric Foundation
Gillette Charitable & Educational Foundation
Grace Foundation, Inc.
Grass Foundation
Lillia Babbitt Hyde Foundation
IBM Corporation
Edward Bangs Kelley & Elza Kelley Foundation
Henry P. Kendall Foundation
Charles A. King Trust
Josiah Macy, Jr. Foundation
NL Industries
Jessie Smith Noyes Foundation
Pfizer, Inc.
Rockefeller Foundation
Rowland Foundation, Inc.
Sandoz Foundation
Alfred P. Sloan Foundation
Seth Sprague Educational & Charitable Foundation
Surdna Foundation
UPS Foundation
Conclusion
I take the liberty, in closing, of making a personal comment. I hope to be forgiven for it, because the comment is in effect a message to Trustees, but one that ought to be proffered in print.
I have now had a sufficient opportunity to study the MBL and its operations to have some confidence in a judgment of its health. I find the Laboratory in a state of flourishing health; this by comparison with other academic and research institutions with which I have been, and in some cases remain, associated. By health I refer to the entire organism; its history, its personality, its prospects, and its physical body. Parts of the body need attention, to be sure, and some growth of the parts is urgent'; but that can and will be done. There is every reason to believe that the unique and influential history of the Laboratory is predecessor to a future just as unique and of just as great an influence.
To play a role in fashioning that future, and in solving the important problems that must be solved, is a privilege. To be sure the privilege has to be paid for; the work is as taxing, physically and psychologically, as any I have ever done, and despite every
REPORT OF THE DIRECTOR 19
effort to the contrary I have had to slow somewhat, for a year or so, my efforts in re- search that I care about a great deal. I am certain now, however, of what I could not have been certain in July of 1978, when I arrived for full-time residence in Woods Hole: the job can be done. It is as important as any job it has ever been my good fortune to work at .
1. THE STAFF MARINE ECOLOGY
I. INSTRUCTORS
JOHN M. TEAL, Woods Hole Oceanographic Institution, co-director of course IVAN VALIELA, Boston University Marine Program, co-director of course CHARLENE VAN RAALTE, Hampshire College
II. ASSISTANTS
MIKE PETROVSKI, Boston University Marine Program CHRISTOPHER VAN RAALTE, Dalhousie University
III. SPECIAL LECTURERS
ARNE JENSEN, University of Aarhus
THOMAS FENCHEL, University of Aarhus
BRUCE J. PETERSON, Ecosystems Center
JOHN E. HOBBIE, Ecosystems Center
JAMES W. PORTER, University of Georgia
KAREN G. PORTER, University of Georgia
LAUREN HAURY, Woods Hole Oceanographic Institution
ROBERT PAINE, University of Washington
JOY GEISELMAN, Woods Hole Oceanographic Institution
WENDY WILTSE, Woods Hole Oceanographic Institution
DONALD RHOADS, Yale University
THOMAS JORDAN, Boston University Marine Program
RICHARD HAEDRICH, Woods Hole Oceanographic Institution
JOHN STEELE, Woods Hole Oceanographic Institution
SUSAN PETERSON, Woods Hole Oceanographic Institution
JOHN MASON, Woods Hole Oceanographic Institution
WILLIAM ODUM, University of Virginia
HOWARD SANDERS, Woods Hole Oceanographic Institution
C. A. S. HALL, Cornell University
IV. LECTURES
I. VALIELA Introduction to course
J. TEAL Introduction to the oceans
C. VAN RAALTE Marine ecology and Woods Hole
A. JENSEN Salt marsh vegetation in Denmark
I. VALIELA Structure and function of salt marsh ecosystems
A. JENSEN Phenology and gro\vth of Halimione
B. PETERSON Techniques and results of phytoplankton production
studies
B. PETERSON The role of plankton in the global carbon cycle
I. VALIELA Nutrient budget of a salt marsh
20
\\\r\l K'KI'ORT OF THE MARINE Bl( >l .< >< ,l( AL LABORATORY
I". FICN< in i
J. Ho 15 DIE J. HoBBIE
J. PORTER J. PORTER
J. PORTER K. PORTER I.. HAURY R. PAINE R. PAINE J. GEISELMAN R. PAINE
\V. WlLTSE
T. FENCHEL
C. VAN RAALTE
D. RHOADS D. RHOADS T. JORDAN R. HAEDRICH J. STEELE
S. PETERSON I. VALIELA J. MASON W. ODUM \V. ODUM \Y. ODUM H. SANDERS C. A. S. HALL C. A. S. HALL
Microbial processes in esluarinr sediments I
Kaclcria in aquatic systems 1
Bacteria in aquatic systems II
Life in three dimensions: open ocean vs. terrestrial
ecosystems Life in t\vo dimensions: substrate-hounded marine
ecosystems
The ecology of marine invertebrate-algal symbioses Grazing and planktonic community structure Patchiness in Cape Cod Bay Plant-herbivore relationship in marine systems Disturbance and diversity in the intertidal Chemical defenses in marine algae Higher order carnivores Effects of the predatory snail, Polinices, on community
structure
Microbial processes in estuarine sediments II Nitrogen fixation in salt marshes Infaunal deposit-feeding: climax mud communities Bioturbation and sediment binding Nitrogen budget of mussels The nature of deep sea fishes
Problems with the management of marine resources Changing values in uses of salt marshes Experimental studies of salt marsh pollution Distribution and behavior of Atlantic blue fin tuna The ecology of mangroves Detritus feeding in coastal ecosystems Venice : a case study The West Falmouth oil spill
Environmental impact of coastal zone energy facilities Models and decision making: Hudson River
EMBRYOLOGY
I. INSTRUCTORS
JOAN V. RUDERMAN, Harvard University, co-director TOM HUMPHREYS, University of Hawaii, co-director SUSAN V. BRYANT, University of California, Irvine GARY L. FREEMAN, University of Texas CYRUS LEVINTHAL, Columbia University EDUARDO R. MACAGNO, Columbia University
II. CONSULTANTS
R. K. HUNT, Johns Hopkins University J. P. TRINKAUS, Yale University J. R. \YHITTAKER, Wistar Institute
III. ASSISTANTS
CLARISSA CHENEY, University of Pennsylvania NIGEL HOLDER, University of California, Irvine MARC LAUFER, University of Pennsylvania
REPORT OF THE DIRECTOR
21
G. L. FREEMAN* G. L. FREEMAN
N. H. VERDOUK M. R. DOHMEN J. R. WHITTAKER
L. WOLPERT J. P. TRINKAUS
J. P. TRINKAUS
J. V. RUDERMAN
T. HUMPHREYS T. HUMPHREYS
LAN Bo CHEN R. K. HUNT
R. K. HUNT C. LEVINTHAL
E. R. MACAGNO C. LEVINTHAL S. BRYANT L. JAFFEE S. INOUE
N. DETERRA
N. B. GILULA N. B. GILULA P. BRYANT J. SAUNDERS X. HOLDER L. ITEN S. KAUFMAN
K. KALTHOFF X. LEDOUARIN
J. ROSENBAUM
R. SAGER R. SAGER
G. STENT
IV. LECTURES
Localization and the specification of form in development Localization of embryonic determinates in Cerebratulus
eggs and early embryos
The origin of spatial organization in Molluscan develop- ment The relationship between local surface differentiation and
cytoplasmic localization Localization and development in Ascidians eggs and
embryos : I and II
Pattern formation in chick limb development Meroblastic development and gastrulation in Fundulus
embryos
Mechanisms of cell movement in vivo during gastrulation Maternal mRNA and localization The marine sponge, Microciona prolifera and other
aggregating cell systems The molecular organization of M. prolifera aggregation
factor complex and its active fragments Studies on intercellular fibronectin matrices Specification of positional information in amphibian eye
development
Positional information in neural development Three dimensional computer reconstruction of neural
anatomy Development of synaptic connections in invertebrate
visual systems Development and characterization of identified cells in
isogenic fish Pattern regulation in amphibian limb regeneration: I and
II Activation and patterning of animal eggs and oocytes by
steady ion currents
Mitosis: Cell structural organization and mechanisms Control of cell division and morphogenesis in Stentor Gap junctions and communication between cells Communication in development and differentiation Pattern formation in the imaginal disks of Drosophila Positional signaling in chick limb bud development Programmed development of the chick wing Pattern regulation in chick limbs
Control of sequential commitment in Drosophila develop- ment Analysis of an anterior morphogenetic determinant in the
egg of an insect, Smitta sp., Chironimidae Migration and differentiation of neural crest cells studied
in avian embryos with interspecific chimeras Control of flagellar protein synthesis in Chlamydomonas Somatic cell genetics: Methodology Somatic cell genetics: Application to some fundamental
problems in biology Neuroembryology of the leech
22 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
Y. PENG LOH Corticotropin and endorphin peptides. Biosynthesis and
their role as intercellular messengers N. MARCUS Normal and aberrant phenotypes in the developing sea
urchin
P. GRANT, S. SHARMA, C. LEVINTHAL, AND R. K. HUNT New looks at retinal-tectal connections, a microsymposium
NEURAL SYSTEMS AND BEHAVIOR
I. INSTRUCTORS
ALAN GELPERIN, Princeton University, director of course
JAMES L. GOULD, Princeton University
ADRIANUS KALMIJN, Woods Hole Oceanographic Institution
RONALD HOY, Cornell University
DAVID J. PRIOR, University of Kentucky
WILLIAM KRISTAN, JR., University of California, San Diego
RANDOLF MENZEL, Free University, Berlin
FERNANDO NOTTEBOHM, Rockefeller University
II. ASSISTANTS
CHRISTINA MYLES, Manchester Community College S. REINGOLD, Princeton University
III. VISITING LECTURER J. NICHOLLS, Stanford University
IV. SPECIAL LECTURERS
GEORGE GERSTEIN, University of Pennsylvania
ROBERT CAPRANICA, Cornell University
WILLIAM G. QUINN, Princeton University
ERIC R. KANDEL, Columbia University
LARRY COHEN, Yale University
TOM EISNER, Cornell University
DONALD GRIFFIN, Rockefeller University
MICHAEL V. L. BENNETT, Albert Einstein University
EDUARDO MACAGNO, Columbia University
GUNTHER STENT, University of California, Berkeley
V7. LECTURES
J. GOULD Introduction to classical ethology
J. GOULD Neuroethology of E. coli
J. GOULD Communication in honey bees
J. GOULD Orientation and navigation in honey bees
A. KALMIJN Electroreception in fish
A. KALMIJN Active and passive electro-orientation
A. KALMIJN Physics and physiology of electroreception
A. KALMIJN Magnetic orientation
R. HOY Genetics and neurobiology of cricket song
REPORT OF THE DIRECTOR
23
Acoustic interneurons
Specificity and mechanisms of neural regeneration
Temperature acclimation in neural systems
Behavioral switching
Environmental modulation of reflex pathways
The leech : Sensory and motor neurons
The leech : After-effects of activity
The leech : Chemical and electrical transmission and
quantal analysis Pattern generation Leech heartbeat control system Leech swimming I Leech swimming II Neuroethology of molluscan learning Serotonin and synaptic modulation Comparative physiology of feeding Color vision in invertebrates Wavelength selective behavior Physiology of insect learning Herbivore-plant interactions II Selective learning Evolution of vocal learning
Origins and mechanisms of hemispheric dominance Birdsong, a neuroethological model
EXPERIMENTAL MARINE BOTANY
(COMPARATIVE BIOLOGY AND BIOCHEMISTRY OF ALGAE)
I. INSTRUCTORS
JEROME A. SCHIFF, Brandeis University, director of course
JAMES FIORE, Suffolk University, laboratory instructor
HARVARD LYMAN, State University of New York at Stony Brook, laboratory instructor
DAVID MAUZERALL, Rockefeller University
ROBERT TROXLER, Boston University
II. CONSULTANTS
ROBERT L. GUILLARD, Woods Hole Oceanographic Institution FRANK A. LOEWUS, Washington State University RALPH S. QUATRANO, Oregon State University
III. STAFF ASSOCIATES
SEYMOUR COHEN, State University of New York at Stony Brook CATHERINE FUSSEL, Pennsylvania State University HANS GAFFRON, Woods Hole, Massachusetts and Sanibel, Florida ANDREW HOLOWINSKY, Brown Universitv
R. HOY R. HOY D. PRIOR D. PRIOR D. PRIOR J. NICHOLLS J. NICHOLLS J. NICHOLLS
B. KRISTAN B. KRISTAN B. KRISTAN B. KRISTAN A. GELPERIN A. GELPERIN A. GELPERIN R. MENZEL R. MENZEL R. MENZEL T. EISNER
F. NOTTEBOHM
F. NOTTEBOHM F. NOTTEBOHM F. NOTTEBOHM
IV. ASSISTANTS
JOANNE LUEBBERT, State University of New York at Stony Brook SCOTT SCHATZ, University of Rhode Island SHARON SUBYAK, Suffolk University
' I ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
V. SPE< i VL LECTURERS
\ KKNMN AIIMADJIAN, Clark 1 1 nivcrsi t \
MARY M. ALLEN, \\VlU-slcy College
MARTHA BERLINER, Simmons College
ANNETTE COLEMAN, Brown I'm versify
FRANKLIN FONG, Texas A & M University
JANE GIBSON, Cornell University
ROBERT L. GUILLARD, Woods Hole Oceanographic Insti union
PETER HEPLER, University of Massachusett.--
PETER HEYWOOD, Brown University
LIONEL JAFFE, Purdue University
ALFRED LOEBLICH, Harvard Universit\
LYNN MARGULIS, Boston University
EYTANA PADAN, Hadassah Medical School
CARL PRICE, Rutgers University
JOEL ROSENBAUM, Yale University
WOLFHARDT RiJDiGER, University of Munich
GREGORY SCHMIDT, Rockefeller University
JAMES R. SEARS, Southeastern Massachusetts University
RAYMOND E. STEPHENS, Marine Biological Laboratory
ARTHUR I. STERN, University of Massachusetts
STAN WATSON, Woods Hole Oceanographic Institution
ROBERT WILCE, University of Massachusetts
C. L. F. WOODCOCK, University of Massachusetts
VI. LECTURES
J. A. SCHIFF Chemical Phase of Evolution; biogeochemistry
J. A. SCHIFF Appearance of Oxygen
J. A. SCHIFF Evolution of Procaryotes
J. A. SCHIFF Evolution of Eucaryotes & Organelles
J. A. SCHIFF Evolution of Life Cycles
J. A. SCHIFF Nutritional Cycles
J. A. SCHIFF Metabolism of Nitrogen and Sulfur
L. MARGULIS Microbial Evolution
C. L. F. WOODCOCK Biology of Acetabularia
R. TROXLER Biosynthesis of Heme and Chlorophyll, Early Stages
R. TROXLER Biosynthesis of Heme and Chlorophyll, Later Stages
R. TROXLER Open Chain Tetrapyrroles
D. MAUZERALL Photochemical Principles
D. MAUZERALL Photochemistry of Photosynthesis
J. GIBSON Photosynthetic Electron Transport
A. STERN Photophosphorylation
L. JAFFE Development of Polarity
J. ROSENBAUM Control of Flagellar Development in Chlamydomonas
R. E. STEPHENS Microtubules in Plant Development
A. HOLOWINSKY Wall Synthesis, Deformation and Morphogenesis in Algae
F. FONG Phototaxis in Eugleua
J. SEARS Vertical Distribution of Algae
R. WILCE Phytogeographic Studies in the North Atlantic
J. FIORE Life Histories of the Brown Algae
P. HEPLER Stomatal Mechanisms
M. M. ALLEN Biology of Blue Green Algae (Cyanobacteria) I
REPORT OF THE DIRECTOR
25
Polyamines
Chloroplast Development I
Chloroplast Development II and 111
Circadian Rhythms
Ouantitative Measurement of Cell Constituents
Pigment Methodology
Xanthophyta, Chrysophyta, Bacillariophyta
Unique Cytology and Molecular Biology of Dinoflagellates
Euglenophyta and Cryptophyla
Chloromonads
Chlorophyta I : Unicells
Development of Single Cells from IJrasiolti
Chlorophyta II: Biology of the Colonial Green Flagellates
Field and Preservation Methods
Phytoplankton Ecology I and Ecology 1 1
Chlorophyta III, Chlorophyta IV and Chlorophyta V
Principles Affecting the Isolation of Cells and Subcellular
Structures
Pheophyta I, II, and III Rhodophyta I. II and III Distribution of Eastern Macroalgae Phytochrome-Con trolled Responses Procaryotic Membranes
Biology of Blue Green Algae (Cyanobacteria) 1 1 Cyanophages
In Vitro Synthesis and Assembly of Chloroplast Proteins Experimental Studies of the Lichen Symbiosis Protoplasts of Unicellular Green Algae Algal Symbioses
Facultative anoxygenic photosynthesis in blue-green algae Studies of phytochrome and chlorophyll synthesis
NEUROBIOLOGV
I. INSTRUCTORS
EDWARD A. KRAVITZ, Harvard Medical School, director of course
JONATHAN B. COHEN, Harvard Medical School
EDWARD J. FURSHPAN, Harvard Medical School
DAVID D. POTTER, Harvard Medical School
IOHN G. HILDEBRAND, Harvard Medical School
GERALD D. FISCHBACH, Harvard Medical School
PETER R. MACLEISH, Harvard Medical School
STORY C. LANDIS, Harvard Medical School
JOHN HEUSER, University of California, San Francisco Medical School
PAUL O'LAGUE, University of California, Los Angeles
THOMAS S. REESE, National Institutes of Health
1 1. STAFF ASSOCIATES
S. MATSUMOTO, Harvard Medical School B. BATTELLE, National Institutes of Health R. HARRIS-WARRICK, Harvard Medical School M. NELSON, Harvard Medical School
S. COHEN
J. A. SCHIFF
H. LYMAN
A. HOLOWINSKV
H. LYMAN
H. LYMAN
H. LYMAN
A. LOEBLICH
J. A. SCHIFF H. LYMAN
P. HEY WOOD
H. LYMAN
J. A. SCHIFF
A. COLEMAN
J. FlORE
R. GUILLAHU
J. FlORE
C. PRICE
J. FlORE J. FlORE J. FlORE
A. HOLOWINSKY STAN WATSON M. M. ALLEN S. COHEN G. SCHMIDT V. AHMADJIAN M. BERLINER H. LYMAN E. PADAN \Y. RUDIGER
26 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
S. GLUSMAN, Harvard Medical School
D. LANDIS, Harvard Medical School M. RHEUBEN, Perm State University
III. ASSISTANTS
SUSAN HUTTNER, University of California, Los Angeles J. LApRATTA, Harvard Medical School RUTH SIEGEL, Harvard Medical School R. NEUBIG, Harvard Medical School B. REESE, National Institutes of Health
IV. SYMPOSIA AND SEMINARS Symposium I Slow Physiological Events
S. W. KUFFLER, Harvard Medical School R. W. TSIEN, Yale University
E. R. KANDEL, Columbia University
Symposium II Selectivity
D. PURVES, Washington LTniversity
B. G. WALLACE, Stanford University U. J. McMAHAN, Stanford University J. DIAMOND, McMaster University
Special Saturday Seminars
C. ARMSTRONG, University of Pennsylvania T. N. WIESEL, Harvard Medical School
R. RAHAMIMOFF, Hebrew University, Israel
Regular Seminar Series
D. D. POTTER, Harvard Medical School
J. BROWN, State University of New York at Stony Brook A. E. STUART, Harvard Medical School T. S. REESE, National Institutes of Health R. LLINAS, New York University
D. GOODENOUGH, Harvard Medical School L. JAN, Y. N. JAN, Harvard Medical School
E. MACAGNO, Columbia University
M. GUTHRIE, Commission to Combat Huntington's Disease
R. MURPHY, State University of New York Albany
J. CARTAUD, Paris
H. POLLARD, National Institutes of Health
R. HOY, Cornell University
T. SEELEY, Harvard University
J. KIRZ, State University of New York at Stony Brook
V. LECTURES
J. HEUSER Structural features of nervous systems
S. LANDIS Presynaptic structure
REPORT OF THE DIRECTOR
27
S. LANDIS T. REESE
G. FlSCHBACH G. FlSCHBACH
N. LEDOURAIN P. MACLEISH G. FlSCHBACH
J. COHEN R. NEUBIG
J. COHEN J. CARTAUD J. COHEN
J. HlLDEBRAND J. HlLDEBRAND
E. KRAVITZ M. NELSON E. KRAVITZ B. BATTELLE E. KRAVITZ
Postsynaptic structure
Growth cones and synaptic development
Myogenesis and development of chemosensitivity
Neurogenesis (neural tube) and development of electrical excitability
The neural crest
Synapse formation: I. Early stages of transmitter release
Synapse formation: II. Organization of the postsynaptic membrane
Subcellular fractionation of Torpedo electric organ
Identification of neurotransmitter receptors by ligand binding
Biochemical characterization of the nicotinic cholinergic receptor
Morphological studies of nicotinic cholinergic receptors and acetylcholinesterase
Neurotransmitter receptors and their responses, an over- view
Introduction. Acetylcholine
Catecholamines
GABA
Neuroethology
Amines and Modulation
Cyclic AMP
Peptides
PHYSIOLOGY I. INSTRUCTORS
K. E. VAN HOLDE, Oregon State University, director of course
MARK MOOSEKER, Yale University
RAY STEPHENS, Marine Biological Laboratory
JAY BROWN, University of Virginia
JOHN WOOLEY, Princeton University
TOM BALDWIN, University of Illinois
F. DAHLQUIST, University of Oregon
SUSAN ASTRIN, Institute of Cancer Research
II. CONSULTANTS
RUTH SAGER, Sidney Farber Institute for Cancer Research ALEX RICH, Massachusetts Institute of Technology
III. STAFF ASSOCIATES
AL CHRISTOPHER, University of Chicago MIRIAM BALDWIN, University of Illinois \V. DENTLER, University of Kansas JERRE SUMMERS, Cancer Research Institute K. TATCHELL, Oregon State University
IV. ASSISTANTS
PAT GALVIN, University of Denver DAVID VONHIPPEL, University of Oregon
28
\\Nl\l. REPORT OK THE MA K' INK BIOLOGICAL LABORATORY
V. SPECIAL LECTI RKKS
PHIL BASSFORD, Harvard Medical School
Su CHUNG, Harvard University
\OKL DK'TERRA, Haneman Hospital
H. EISENBERG, Weizmann Institute, Israel
SARAH ELGIN, Harvard University
R. GENNIS, University of Illinois
N. B. GILULA, The Rockefeller Universiu
ALFRED GOLDBERG, Harvard Medical School
STEVE HARRISON, Harvard University
Knu CHI HUANG, John Hopkins University
S. INOUE, University of Pennsylvania
I. ISENBERG, Oregon State University
ROBERT JACKSON, The Rockefeller University
NICOLE LE DOUARIN, Marine Biological Laboratory
ALLAN MAXAM. Harvard University
T. POLLARD, Johns Hopkins University
HANS Ris, University of Wisconsin
JOEL ROSENBAUM, Yale University
PETER SETLOW, University of Connecticut Health Center
ALBERT SZENT-GYORGII, Marine Biological Laboratory
ANDREW SZENT-GYORGII, Brandeis University
L. TILNEY, University of Pennsylvania
ANNA MARIE WEBER, University of Pennsylvania
K. E. VAN HOLDE [. WOOLEY
T. BALDWIN- ANDREW SzENT-GYORt.II
A. M. WEBER M. MOOSEKER
L. TILNEY
M. MOOSEKER
R. STEPHENS
A. MAXAM
\Y. DENTLER
K. E. VAN HOLDE
S. CHUNG
J. WOOLEV
S. ELGIN
I. ISENBERG
H. Ris
J. BROWN
R. GENNIS
T. WEGMANN
VI. LECTURES
Introduction to cromatin structure: Evidence from nu- clease digestion and hydrodynamic studies
Introduction to chromatin structure: evidence from elec- tron microscopy and scattering studies
Bioluminescence and bacterial luciferase
Introduction to contractile proteins
Properties of the actin filament
The control of actomyosin-mediated motility in non- muscle cells
The role of actin in non-muscle motility
Actin-membrane association
Microtubules, I and II
Nucleic acid chemistry and DNA sequences
Microtubule/membrane interaction in cilia and rlagella
DNA-protein interactions in the nucleosome
Characterization of the histone core complex
Molecular architecture of the nucleosone
Chromosomal structure and function in Drosophila
Histone-histone interaction
Higher order structure in chromosomes
The fluid mosaic model of membrane structures
E. coli pyruvate oxidase — studies on lipid-protein inter- actions
Structure and function of histocompatability antigen on a membrane protein
REPORT OF THE DIRECTOR
P. BASSFORD R. DAHLQUIST R. DAHLQUIST S. INOUE
X. DETER R A X. B. GlLULA X. B. GlLULA H. ElSENBERG S. ASTRIN S. ASTRIN
J. BROWN A. GOLDBERG
T. BALDWIN' R. JACKSON
P. SETLOW R. C. HUANG
X. I.EDOUARIN J. ROSENBAUM
R. SAGER R. SAGER
S. HARRISON
A. RICH
ALBERT SZENT-GYORGII
Transport and secretion in gi;im nruame b.i< tcria
Lipid-protein interactions
Bacterial chemolaxi^, a model M-n^ing >\>tem
Mitosis cell structural nrgani/at ion and mechanisms
Control of cell division and morphogenesis in Stenlo.'-
dap junctions and communication between cells
Communication in development and differentiation
Protein-DX'A interactions
Control of transcription in eukaryots
Control of endogenous viral gene expression in chick
embryos
Protein components of the L-cell plasma membrane Studies on the mechanism of protein breakdown in animal
and bacterial cells
Role of proteases in controlling enzyme levels Signal peptidase and its role in the transfer of poly-
peptides across the RER membranes Protein regulation during bacterial spore germination Initiation of RNA synthesis in vitro Migration and differentiation of neural crest cells studied
in avian embryos with interspecific chimeras Control of flagellar protein synthesis in chimeras Somatic cell genetics I. methodology Somatic cell genetics II. Application to some fundamental
problems in biology Protein flexibility and macromolecular assembly: virus
structures at high resolution
Molecular structure and biological function of t-RXA The living state
RESEARCH PROGRAM IX MICROBIAL ECOLOGY
I. INSTRUCTORS
JANE A. GIBSON, Cornell University
ROBERT E. HUNGATE, University of California, Davis
HOLGER \Y. JANNASCH, Woods Hole Oceanographic Institution
ALEX KEYNAN, The Hebrew University, Jerusalem
EDWARD R. LEADBETTER, University of Connecticut, Storrs
JEANNE S. POINDEXTER, Public Health Research Institute, New York
1 1. CONSULTANTS
HARLYN O. HALVORSON, Brandeis University J. WOODLAND HASTINGS, Harvard Universit\ ROGER Y. STANIER. Institute Pasteur, Paris EDWARD O. WILSON, Harvard University
III. STAFF ASSOCIATES
RUSSEL L. CUHEL. Woods Hole Oceanographic Institution
MARK A. SCHNEIDER, Amherst College
CRAIG D. TAYLOR. Wood- Hole Oceanographic Institution
30 \\NUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
IV7. ASSISTANT STEPHEN J. GIOVANNONI, Boston University
\". SPECIAL LECTURERS
RICHARD P. BLAKEMORE, University of New Hampshire, Durham
ALASDAIR COOK, Cornell University
BERNARD D. DAVIS, Harvard University
ARNOLD L. DEMAIN, Massachusetts Institute of Technology
IAN DUNDAS, University of Bergen, Norway
JOHN W. FARRINGTON, Woods Hole Oceanographic Institution
TOM FENCHEL, University of Aarhus, Denmark
JAMES G. FERRY, Virginia Polytechnical Institute, Blacksburg
JOEL C. GOLDMAN, Woods Hole Oceanographic Institution
EVERETT P. GREENBERG, Harvard University
HARLYN O. HALVORSON, Brandeis University
J. WOODLAND HASTINGS, Harvard University
ETANA PADAN, The Hebrew University, Jerusalem
ANTONIO H. ROMANO, University of Connecticut, Storrs
E. G. RUBY, Harvard University
IVAN VALIELA, Marine Biological Laboratory
JOHN B. WATERBURY, Woods Hole Oceanographic Institution
VI. LECTURES
H. W. JANNASCH Introduction to microbial ecology I and II
H. W. JANNASCH Continuous culture of microorganisms
H. W. JANNASCH Continuous culture in microbial ecology
H. W. JANNASCH Practice of the chemostat
H. W. JANNASCH Experiments in deep sea microbiology
J. A. GIBSON The photosynthetic bacteria I and II
J. A. GIBSON Uptake measurements in microbial ecology
J. A. GIBSON Nutrient uptake in cyanobacteria
J. A. GIBSON Survival of prokaryotes: envelope changes
E. R. LEADBETTER Ways of making a living: anaerobes
E. R. LEADBETTER Ways of making a living : aerobes
E. R. LEADBETTER Microbial attack on hydrocarbons
E. R. LEADBETTER Survival of procaryotes: ecological aspects
E. R. LEADBETTER Microbiology of the tooth surface
R. E. HUNGATE Analysis of a microbial ecosystem
R. E. HUNGATE Cultivation of anaerobes
R. E. HUNGATE The rumen as an ecosystem I and II
J. S. POINDEXTER The bacterial prostheca: occurrence, structure and
possible function
J. S. POINDEXTER The bacterial prostheca: developmental studies
J. S. POINDEXTER Survival of prokaryotes: the vegetative cell
A. KEYNAN Introduction to the light emitting bacteria II
A. KEYNAN Experimentation with bacterial bioluminescence
A. KEYNAN Survival of prokaryotes : the endospore
C. D. TAYLOR The biology of methane formation
C. D. TAYLOR The ecology of methane formation
C. D. TAYLOR The effect of pressure on bacterial growth
R. L. CUHEL Psychrophilic bacteria
REPORT OF THE DIRECTOR 31
R. P. BLAKEMOKK Magnetotactic bacteria
B. D. DAVIS Mechanism of protein secretion across membranes A. COOK Microbial degradation of pesticides
A. L. DEMAIN Do antibiotics have a function in natural populations of
microorganisms?
I. DUNDAS Halophilic bacteria
J. \\'. FARRINGTON Biochemistry of methane in oceanic environments
T. FENCHEL Microbial transformations in sediment I and II
J. G. FERRV The biology of sulfate reduction
f. G. FERRY Sulfate reduction in marshes and estuaries
J. C. GOLDMAN Continuous culture of algae
E. P. GREENBERG The Spirochaetes
H. O. HALVORSON Survival of prokaryotes: molecular aspects
J. \V. HASTINGS Introduction to the light emitting bacteria I
E. PADAN Anoxic photosynthesis in cyanobacteria
A. H. ROMANO The biology of Sphaerotilus
E. G. RUBY Taxonomy and distribution of luminous bacteria
I. YALIELA The Sippewisset marsh
J. B. WATERBURY Marine cyanobacteria
YEAR-ROUND PROGRAMS 1978
BOSTON UNIVERSITY MARINE PROGRAM
THE STAFF
ARTHUR HUMES, Boston University Marine Program, director JELLE ATEMA, Boston University Marine Program BJORN CANNING, University of Stockholm, Sweden STJEPKO GOLUBIC, Boston University
C. K. GOVIND, University of Toronto
FREDERICK LANG, Boston University Marine Program CHARLES LENT, Brown University IVAN YALIELA, Boston University
THE ECOSYSTEMS CENTER
GEORGE M. WOODWELL, director
JOHN E. HOBBIE, senior scientist; DANIEL B. BOTKIN, associate scientist; FRANCIS P. BOWLES, JERRY M. MELILLO, BRUCE J. PETERSON, assistant scientists; RICHARD H. BURROUGHS, senior fellow; JOHN T. FINN, postdoctoral fellow; ERENE Y. PECAN, assistant to the director and research associate; MARY LOUISE MONTGOMERY, assistant to the director; RICHARD A. HOUGHTON, PAUL A. STEUDLER, research associates; WILLIAM J. BEHRENS, JOANNE CLARK, COLLEEN M. CAVANAUGH, KEITH X. ESHLEMAN. JOHN Y. HELFRICH, VOYTEK KIJOWSKI, FREDERIC LIP- SCHULTZ, KATHERINE C. PARSONS, DAVID S. SCHIMEL, JEFFREY B. SHELKEY, HEDY SLADOVICH, research assistants; PAUL DETWILER, graduate research fellow; JAMES T. MORRIS, WILLIAM B. BOWDEN, JAMES P. REED, graduate research assistants; MICHELLE DIONNE, graduate assistant; GREGG DIONNE, LAWRENCE HOBBIE, ANN LEWANDOWSKI, ANDREA R. TURNER, laboratory assistants; MARK WTHITE, technician; JOAN M. UPTON, secretary; NANCY L. CAMPBELL, JEANNE FERRARI, FRANCES A. SEYMORE, typists.
; • MUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
JANUARY COURSES 1978 BEHAVIOR
(Offered Jointly by Boston University Al urine Program and the Marine Biological Laboratory)
I. INSTRUCTORS
JELLE ATEMA, Boston University, director of course
ADRIANUS KALMIJN, Woods Hole Oceanographic Institute
TERRY CROW, Marine Biological Laboratory
TONY SWAIN, Boston University
VINCENT DETHIER, University of Massachusetts
ROBERT BARLOW, Syracuse LIniversity
MARGARET NELSON, Harvard University
BEHRUS JAHAN-PARWAR, The Worcester Institute
IZJA LEDERHENDLER, Marine Biological Laboratory
DANIEL STENZLER, Boston University
LEHR BRISBIN, University of Georgia
JOHN PALMER, University of Massachusetts
MEL KREITHEN, Cornell University
ELIZABETH RUSSELL, The Jackson Laboratory
TIMOTHY WILLIAMS, Swarthmore College
DOUGLAS S. RIGGS, Hampshire College
WILLIAM W ATKINS, Woods Hole Oceanographic Institute
KATHY PAYNE, The Rockefeller University
BERNT WURSIG, State University of New York at Stony Brook
JANE FRICK, Woods Hole, Massachusetts
DIETLAND MiJLLER-ScHWARZE, State University of New York College of Environmental
Science and Forestry
ARTHUR SILVERSTEIN, Johns Hopkins School of Medicine EMIL MENZEL, State University of New York at Stony Brook BORI OLLA, National Marine Fisheries Service ALASTAIR STUART, University of Massachusetts at Amherst GEORGE MICHEL, Boston University FRANCIS BOWLES, Marine Biological Laboratory STUART MACKAY, Boston University DANIEL ALKON, Marine Biological Laboratory
II. LECTURES
J. ATEMA Introduction
J. ATEMA Sensory physiology and behavior
J. ATEMA Chemo- and mechanoreceptors
A. KALMIJN Electroreception with and without electric organs
J. ATEMA Evolution of chemical senses
A. KALMIJN Electroreception: the detection of inanimate electric fields
A. KALMIJN The physics and physiology of electroreception
T. CROW Statistical considerations in the design of behavioral
experiments
A. KALMIJN Electroreception: more questions with or without answers
V. DETHIER Insect taste: Food discrimination in plant-feeding insects
T. SWAIN Chemical compounds in plants affecting insect herbivores
V. DETHIER Whal chemosensory neurons tell rhe brain
REPORT OF THE DIRECTOR
33
R. BARLOW
R. BARLOW
M. NELSON
M. NELSON
B. JAHAN-PARWAR
I. LEDERHENDLER
D. STENZLER
L. BRISBIN
L. BRISBIX J. ATEMA J. PALMER J. PALMER J. PALMER M. KREITHEN M. KREITHEN T. WILLIAMS D. RIGGS \Y. WATKINS K. PAYNE
B. Wt'RSIG
D. SMITH D. SMITH J. FRICK
D. MtiLLER-SCHWARZE D. MULLER-SCHWARZE D. MULLER-SCHWARZE
D. MULLER-SCHWARZE
A. SlLVERSTEIN
E. MENZEL E. MENZEL
E. MENZEL
B. OLLA
A. STUART A. STUART J. ATEMA G. MICHEL
J. ATEMA
F. BOWLES S. MAC KAY D. ALKON D. ALKON
The eye and the brain
Vision and Limulus: a multidisciplinary analysis
Introduction to neural control of beha\Tior
Hissing and social behavior in cockroaches
Chemosensory behaYior in the seahare, Aplysia
Social behaYior in Aplysia
The burial alarm response and olfaction in the mud snail
Ecology, domestication and the behavior of dogs: some
principles and their application to present-day problems
of men, wolves, and dogs
The domestic dog today: Training and tracking Catfish social behavior data analysis Introduction to biological rhythms Biological rhythms in shore dwelling animals Human rhythm- Introduction to bird migration Orientation and infra-sound
Pleiotropism and the nature of the YY-Locus in the mouse Selected topics in biomathematics Sperm whale acoustic behavior Annual change in songs of Humpback whales Group composition and stability of coastal bottlenose
porpoises
Introduction to social behavior in birds Acoustic and visual signals in redwing blackbirds Migration of green turtles Introduction to pheromones Mammalian pheromones
Predator-prey relations in Antarctic bird communities Ecology of the reindeer culture The generation of immunologic diversity: Phylogenists vs.
Ontogenists
Social organization in Chimpanzees Social organization in Macaques Primate intelligence Social behavior and rhythms in marine fishes as related to
environmental factors Social behavior of insects Termite communication and social behavior Social behavior and pheromones in lobsters The European Ethologists as seen by an American
Psychologist
Behavior assays in pollution research Territories and social behavior of lobster fishermen Bio-medical telemetry in behavioral studies Associative training in a nudibranch mollusc Tier mis senda Neural substrates of associative training in Hermissenda
DEVELOPMENTAL BIOLOGY
I. INSTRUCTORS
YYALTER S. VINCENT, University of Delaware, director of course NANCY H. MARCUS. Woods Hole Oceanographic Institution
34
ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
JAMES D. EBERT, Marine Biological Laboratory
KENNETH T. EDDS, Marine Biological Laboratory
SUSAN GERBI, Brown University
SHINYA INOUE, University of Pennsylvania
WILLIAM MASSOVER, Brown University
RICHARD MILLER, Temple University
RAYMOND E. STEPHENS, Marine Biological Laboratory
II. ASSISTANT DAVID Moss, University of Delaware
III. SPECIAL LECTURERS
EUGENE BELL, Massachusetts Institute of Technology
DAVID FRANCIS, University of Delaware
HARLYN HALVORSON, Brandeis University
ARTHUR HUMES, Boston LIniversity Marine Program
HANS LAUFFER, University of Connecticut
DOLORES SCHENDEL, Sloan-Kettering Institute
RICHARD TASCA, University of Delaware
W. S. VINCENT W. S. VINCENT N. H. MARCUS
W. MASSOVER W. MASSOVER W. M \SSOVER S. INOUE S. INOUE S. INOUE S. INOUE J. D. EBERT
R. MILLER W. S. VINCENT
R. MILLER M. D. MASER K. T. EDDS W. S. VINCENT E. RUSSELL K. T. EDDS W. S. VINCENT W. S. VINCENT S. GERBI
H. LAUFFER H. LAUFFER J. COLLIER
IV. LECTURES
Introduction to course
Gametogenesis I, II, and III
Differentiation and development of sea urchins in labora- tory culture I and II
Comparative oogenesis I
Maturation and ovulation I and 1 1
Vitellogenesis
Spicule development in sea urchins I and II
Techniques of phase and polarizing microscopy
Experimental analysis of spindle structure I
Experimental analysis of mitosis
Ionic regulation of embryonic induction, differentiation in growth
Gamete activation I and II
Principles and practices of cell fractionation and gradient analysis I
Fertilization I and II
Techniques and demonstration of electron microscopy
Cleavage and control of cell divisions I and II
Principals and practice of gradient analysis II
Plieotropism and the nature of the W-locus in mice
The role of microfilaments in cell division
Processing of eucaryotic transcripts
Sequence conservation in ribosomal RNA
DNA sequence organization on eucaryotic genomes I and II
Analysis of gene activity in Chironomus development
Specific message isolation and identification in Chironomus
Normal development of Spiralia
REPORT OF THE DIRECTOR 35
J. COLLIER Experimental analysis of the development of spiralians
J. COLLIER Molecular biology of spiralian development I and II
E. ANDERSON Early embryogenesis in mammals
A. SILVERSTEIN The generation of immunologic diversity: phylogenists
vs. ontogenists
R. E. STEPHENS Chemistry and structure of microtubules
R. E. STEPHENS Morphogenesis of cilia
H. O. HALVORSOX New techniques in approaching developmental problems
J. D. EBERT Birth defects: Biological and ethical considerations
\V. S. YIXCENT The politics of the recombinant DXA controversy
X. H. MARCUS Phenotypic plasticity in marine invertebrates
ALBERT SzENT-GYORGYl Protein, ascorbic acid and cancer
ECOLOGY
I. INSTRUCTORS
GEORGE M. WOODWELL, The Ecosystems Center, director ol course DANIEL B. BOTKIN, The Ecosystems Center JOHN E. HOBBIE, The Ecosystems Center JERRY M. MELILLO, The Ecosystems Center
II. SPECIAL LECTURERS
S. H. BERWICK, Yale
KENNETH O. EMERY, Woods Hole Oceanographic Institution
T. FENCHEL, University of Aarhus, Denmark
C. A. S. HALL, Cornell University
HOLGER \Y. JANNASCH, Woods Hole Oceanographic Institution
MARILYN J. JORDAN, The Ecosystems Center
THOMAS E. LOVEJOY, World Wildlife Fund
BRUCE J. PETERSON, The Ecosystems Center
G. ROWE, Woods Hole Oceanographic Institution
J. H. RYTHER, Woods Hole Oceanographic Institution
HOWARD L. SANDERS, Woods Hole Oceanographic Institution
LAWRENCE B. SLOBODKIN, State University of New York at Stony Brook
FREDERICK E. SMITH, Harvard University
J. STEELE, Woods Hole Oceanographic Institution
IVAN VALIELA, Boston University
III. LECTURES
G. M. WOODWELL Structure of the biosphere I and II
H. SANDERS Natural communities and evolutionary strategies I and II
L. SLOBODKIN Evolution: The development of species
L. SLOBODKIN Group selection: The development of communities
K. O. EMERY Geological factors: Continental drift
G. M. WOODWELL Climatic factors: The vegetation of the earth
G. ROWE The oceans: Distribution of primary production
G. ROWE The oceans: Secondary production
G. M. WOODWELL Primary production and the metabolism of the earth
G. M. WOODWELL The world carbon budget: The predominance of forests
J. E. HOBBIE The world carbon budget: The role of oceans
J. E. HOBBIE Primary productivity in aquatic systems I and II
36 . \XNUA1. REPORT OF THE MARINE BIOLOGICAL LABORATORY
C. A. S. HALL Secondary productivity I and 1 1 S. H. BERWICK Biogeography I and II
D. B. BOTKIN Succession and stability I and II
G. M. WOODWELL Nutrients and communities I and II
H. W. JANNASCH The microbiology of S-transformations I and 1 1
B. J. PETERSON Nutrient limitation in aquatic ecosystems
B. J. PETERSON Phosphorus cycle of the ocean
J. M. MELILLO Nutrient cycling in forested basins I and II
I. VALIELA Nutrient cycling in a salt marsh
G. M. WOODWELL Ecosystems, energy and world politics
T. E. LOVEJOY Endangered species
J. H. RYTHER Oceanic productivity
J. STEELE The North Sea fishery I and II
S. B. PETERSON Northwest Atlantic fishery I and II
K. O. EMERY Mineral resources of the deep sea and international
politics
F. E. SMITH Urban ecology I and 1 1 M. J. JORDAN Ecological effects of toxins
G. M. WOODWELL Biotic impoverishment and the threshold dilemma: A
major challenge for science and government
NEUROBIOLOGY
(Offered jointly by Boston University Marine Program and the Marine Biological Laboratory)
I. INSTRUCTORS
ALAN FEIN, Marine Biological Laboratory, director of course FREDERICK LANG, Boston University Marine Program, director of course EDWARD F. MACNICHOL, JR., Marine Biological Laboratory, laboratory director
II. SPECIAL LECTURERS
WILLIAM J. ADELMAN, NIH-NINCDS, Woods Hole
DANIEL L. ALKON, NIH-NINCDS, Woods Hole
JELLE ATEMA, Boston University Marine Program
ROBERT BARLOW, Syracuse University
THOMAS CAREW, Columbia University
MELVIN COHEN, Yale University
VINCENT G. DETHIER, University of Massachusetts
FREDERICK DODGE, Rockefeller University and IBM Watson Labs
ERIC FRANK, Harvard Medical School
DONALD FRAZIER, University of Kentucky
ALFREDO GORIO, The Rockefeller University
RAMI GROSSMAN, NIH-NINCDS
ADRIANUS KALMIJN, Woods Hole Oceanographic Institution
EHUD KAPLAN, Rockefeller University
CHARLES LENT, State University of New York at Stony Brook
SIMON LEVAY, Harvard Medical School
DOUGLAS S. RIGGS, Hampshire College
ELIZABETH S. RUSSELL, The Jackson Laboratory
ARTHUR SILVERSTEIN, Johns Hopkins University
oi I Ml' DIRECTOR
A. FEIN' S. LEVAY J. AT EM A M. COHEN A. KALMIJX A. FEIN A. FEIN A. FEIN A. FEIN W. ADELMAN V. DETHIER R. BARLOW R. BARLOW F. DODGE
E. KAPLAN
F. LANG F. LANG F. LAXG A. FEIN
F. LAN*, F. LANG
E. RUSSELL
F. LANG F. LANG A. Go RIO D. RIGGS A. GORIO A. GORIO
L. MARSHALL
D. RIGGS
E. FRANK D. RIGGS
F. LANG
D. FRAZIER
A. SlLVERSTEIN
E. KRAVITZ
C. LENT
R. GROSSMAN
T. CARE\V
C. LENT
D. ALKON D. ALKON
[IT. LECTURES
Cell permeability and the plasma membrane
Microscopic anatomy of the nervous system
Evolution of chemical senses
Involution of neuron structure and function
Physics and physiology of electroreception
The resting membrane potential
Cable properties of neurons
Action potentials
Saltatory conduction in myelinated axons
Neuronal currents, channels, spaces and clefts
What chemosensory neurons tell the brain
The eye and the brain
Vision in Li mid us: a multidisciplinary analysis
A horseshoe crab-eye view of Great Harbor
Receptor properties of Limnlus lateral eye in situ
Synaptic transmission — The soups vs. the sparks
Mechanisms of transmitter release
Ionic basis of synaptic potentials
Role of intracellular Ca++ and Na+" in adaptation of
Limulus photoreceptors Quantal release Excitation-secretion coupling
Pleiotropism and the nature of the W-locus in the mouse Origin and fate of synaptic vesicles Neurotrophic influences
Intracellular recording of synaptic potentials Pleasures and pitfalls of biological modeling Acetylcholine compartments in the mouse diaphragm The mode of action of black widow spider venom Factors influencing reinnervation of skeletal muscle The steady-state behavior of biological feedback systems Formation of nerve-muscle synapses in tissue culture Fitting straight lines when both X and Y are subject to
error Developmental neuroethology : Physiological basis for
changes in fight and flight behavior during growth of
the lobster
Putative transmitters in vertebrate respiratory neurons The generation of immunologic diversity: Phylogenists vs.
ontogenists Three neurohormones in the lobster : Studies on the cellular
localization, release and physiological actions of octo-
pamine, serotonin, and dopamine Organizational and neural properties of leech CNS Conduction of action potentials along nonhomogenous
axons The utility of the marine mollusc Aplysia for the cellular
analysis of behavior
Cellular studies of amine neurons in leech Associative training in a nudibranch mollusc, Hermissenda Xcural substrates of associative training in Hermissenda
38 \\NUAL REPORT OF THK MAKIXK IJK >!.<)< HCAL LABORATORY
COMPAKATIVK PATI K )!.( )( \\ OF MARINK INVKRTHBRATHS
I. INSTRUCTORS
FREDERICK B. BANG, Johns Hopkins l'ni\ rrsily
BETSY BANG, Johns Hopkins University
JACK LEVIN, Johns Hopkins University
ROBERT PRENDERGAST, Johns Hopkins University
C. AUSTIN FARLEY, National Marine Fisheries Service at Oxford, Maryland
KENNETH EDDS, Marine Biological Laboratory
JACK MARCHALONIS, Frederick Cancer Research Center
CAROL REINISCH, Sydney Farher Cancer Research Institute in Boston
II. LECTURES
F. B. BAN<; Cellular clumping in seastars, hermit crabs, and clotting
factors in Carcinus
J. LEVIN Extracellular clotting
J. LEVIN Limulus and endotoxin
J. LEVIN Comparison of invertebrates and vertebrates
B. G. BANG Evolution of mucociliary system
B. G. BANG Sipunculus and urn cells
F. B. BANG Comparative aspects of mucociliary systems
K. EDDS Inflammations of Invertebrates
C. A. FARLEY Normal Anatomy of the Oyster C. A. FARLEY Pathology of the Oyster
C. A. FARLEY Infectious Diseases in the Oyster
C. A. FARLEY Tumors in Oysters
C. A. FARLEY Virus Diseases in Oysters
F. B. BANG Specific Bacteriological and Virus Diseases of crabs and
lobsters
J. PEARCE Pathological pollution aspects of ecology
C. REINISCH Tumors and regeneration
C. REINISCH Recognition in invertebrates
R. PRENDERGAST Starfish factor — effects in vertebrates
F. B. BANG Elements of immune response — Sipunculus, stars
R. PRENDERGAST Introduction to the vertebrate immune response
J. MARCHALONIS Agglutinins and antibodies
J. MARCHALONIS The evolution of the immune response
LABORATORY OF BIOPHYSICS, NINCDS-NIH
WILLIAM J. ADELMAN, JR., chief of the laboratory and head, section on neural membranes
DANIEL L. ALKON, head, section on neural systems
ALAN J. HODGE senior scientist; JAY WELLS, research physiologist ; DAVID E. GOLDMAN, guest worker; TERRY CROW, staff fellow; IZIA LEDERHENDLER, ROBERT J. FRENCH, MITSUO TABATA, visiting fellows; JONATHAN SHOUKIMAS, THOMAS JERUSSI, LEON SHIMAN, I PA fellows; JOSEPH NEARY, biochemist; RICHARD WALTZ, mathe- matician/programmer; CLYDE TYNDALE, electronics specialist; RUTHANNE MUELLER, research technician; JUNE HARRIGAN, mariculturist
LABORATORY OF SENSORY PHYSIOLOGY
EDWARD F. MACNICHOL, JR., director
ALAN FEIN, associate scientist and deputy director; FERENC I. HAROSI, associate
REPORT OF THE DIRECTOR 39
scientist; ETE Z. SZUTS, assistant scientist; BARBARA ANN COLLINS, senior research associate; D. WESLEY CORSON, postdoctoral fellow; JOSEPH LEVINE, graduate student, Harvard University; KATHLEEN FRENCH, graduate student, Boston University Marine Program; STEVEN L. GOODMAN, research assistant; MENACHEM HANANI, National Institutes of Health; THEODORE P. WILLIAMS, Florida State University; YVETTE KUNZ RAMSAY, University of Dublin; LEO V.. LIPETZ, Ohio State University; PAUL WITKOVSKY, GUIDO HASSIN, State University of New York at Stony Brook, visiting scientist
NATIONAL FOUNDATION FOR CANCER RESEARCH
ALBERT SZENT-GYORGYI, Director
JANE A. MCLAUGHLIN; PETER R. C. GASCOYNE; RICHARD MEANY; T. JOHN LEWIS, Professor, University College of North Wales; RONALD PETHIG, Lecturer, Uni- versity College of North Wales; PAUL ELVIN, Graduate Student, Brunei University, Uxbridge, England ; JOSHUA SELIG, part-time glassware cleaner.
INDEPENDENT YEAR-ROUND PROGRAMS
RAYMOND STEPHENS, principal investigator MELANIE PRATT; MARY E. PORTER
SHINYA INOUE, principal investigator ANDREW EISEN; MARK B. FIENBERG
KENNETH EDDS, principal investigator NORMAN R. JARVIS
ERIC BALL, principal investigator
D. EUGENE COPELAND, principal investigator
JUDITH GRASSLE, principal investigator
FERN BIRTWISTLE; CYNTHIA LANYON-DUNCAN; LINDA PHILBIN-MUNSON
RUTH D. TURNER, principal investigator CARL J. BERG; GREGORY A. TRACEY
ROBERT RICE, principal investigator
PRISCILLA ROSLANSKY; SUSAN M. HOUGHTON; REBECCA LASH
ERIC KANDEL, principal investigator
CARL J. BERG; THOMAS CAPO; SUSAN PERRITT
KEITH R. PORTER, principal investigator RANDOLPH H. BYERS; MARK McNivEN
LEWIS TILNEY, principal investigator LAURINDA A. JAFFE
YEAR-IN-SCIENCE 1977-1978
The Year-in-Science program at the MBL is for advanced undergraduates and beginning graduate students. For undergraduates the program is equivalent to a university honors program ; for beginning graduate students it is designed to accommodate those who require the staff and special facilities of the laboratory in support of their re- search. Students join staff of the MBL in a variety of studies, participate in courses at
40 \\NUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
the Woods Hole Oceanographic Institution, the Boston University Marine Program, and in regularly scheduled seminars with staff of The Ecosystems Center and others at the MBL.
THE STAFF
GEORGE M. WOODWELL, director, The Ecosystems Center
DANIEL B. BOTKIN, associate scientist
FRANCIS P. BOWLES, assistant scientist
JOHN E. HOBBIE, senior scientist
JERRY M. MELILLO, assistant scientist
BRUCE J. PETERSON, assistant scientist
THE LABORATORY STAFF
(Including all whose services began or ended during the year)
HOMER P. SMITH, General Manager
LUCENA J. EARTH, Director of Admissions
FRANCIS P. BOWLES, Coordinator of Research Services
EDWARD G. CASEY, Controller
JANE FESSENDEN, Librarian
A. ROBERT GUNNING, Superintendent, Buildings and Grounds
LEWIS M. LAWDAY, Assistant Manager, Department of Marine Resources
KARLENE LUKOVITZ, Assistant Editor, The Biological Bulletin
ANNE C. MAKER, Public Relations Officer
MORTON D. MASER, Coordinator of Continuing Education
LAURIE A. MORSE, Assistant Editor, The Biological Bulletin
WESLEY N. TIFFNEY, Curator, Gray Museum
JOHN J. VALOIS, Manager, Department of Marine Resources
GEORGE M. WOODWELL, Director, The Ecosystems Center
EDUCATION OFFICE ESTHER M. BROWNE PAMELA V. SAWDO
DIRECTOR'S OFFICE MAUREEN M. MORRIS
PUBLIC RELATIONS OFFICE
FERN P. CALLIS DENICE WYE
R. ANN PERRY
GENERAL MANAGER'S OFFICE
FLORENCE S. BUTZ AGNES L. GEGGATT
ELAINE C. CROCKER FRANCES N. JOHNSON
CONTROLLER'S OFFICE
RUTH B. CAMPBELL SUZANNE J. SEMINO
DORIS C. DAVIS LAUREL SWAIN
NANCY L. ELLIS ANNE S. WILLIAMS JOAN E. HOWARD
KKl'ORT OF THE DIRECTOR 41
LIBRARY
JUDITH A. ASHMORE JOAN H. GRICE
REBECCA J. BAILEY E. LENORA JOSEPH
NANCY M. CAPUANO HOLLY E. KARALEKAS
LYNNE A. DOELLING LAUREL SWAIN
DAVID J. FITZGERALD M. Axx WHITE
CHARLOTTE F. FRANK DENICE WYE
BUILDINGS AND GROUNDS
LEE E. BOURGOIX RICHARD A. LOVERING
MADELINE H. BRODERICK ALAN G. LUNN
TIMOTHY CLINTON JOHN B. MACLEOD
FRANCIS J. COPPOLA JOHN E. MAURER
JOSEPH E. DONOHOE STEPHEN A. MILLS
GLENN R. ENDS MORGAN MOORE
CHARLES K. FUGLISTER SUSAN NICKERSON
ELIZABETH J. GEGGATT SIMONE ST. JEAN
RICHARD E. GEGGATT, JR. CLAYTON SEARS
ROBERTO G. GIBBONS GLENN I. SHEAR
ROGER W. HOBBS, JR. GILBERT F. SILVIA
THOMAS N. KLEINDIXST MERILYN A. SMART
ELISABETH KUIL CHRISTOPHER STONE
DONALD B. LEHY JANE E. SYLVIA
RALPH H. LEWIS FREDERICK THRASHER
SOFIEA LEWIS FREDERICK WARD
WILLIAM M. LOCHHEAD RALPH WHITMAN
DANIEL LOEWUS WILLIAM WHITTAKER
DEPARTMENT OF MARINE RESOURCES
EDWARD G. ENDS, JR. JOHN RYTHER, JR.
JOYCE ENDS EUGENE TASSINARI
ROBERT M. HEBDEN BRUNO TRAPASSO
HOWARD LANE JOHN VARAO MARK Muxsox
RESEARCH SERVICES
JULIE A. ANDRADE ROBERT J. COLDER THOMAS R. ANTHOXY ANDREW HODGDOX FRAXKLIX D. BARNES DAVID JUERS JOHN S. BARNES LOWELL V. MARTIN- CATHY A. CARRINGTON JOAN PETERS-GILMARTIN CAROL A. EBERHARD FRANK E. SYLVIA LINDA M. GOLDER
2. INVESTIGATORS; RESEARCH FELLOWSHIPS; STUDENTS Independent Investigators, 1978
ADEJUWON, CHRISTOPHER A., Research Fellow, The Population Council, The Rockefeller Uni- versity ARMSTRONG, CLAY M., Professor of Physiology, University of Pennsylvania
42 ANNUAL REPORT OI-' 'I' UK MARINE BIOLOGICAL LABORATORY
AKMSTKOM., I'KTHK B., Associate Professor of Zoology, University of California
ARNOLD, JOHN M., Professor, University of Hawaii, Kewalo Marine Laboratory
ASTRIN, SUSAN M., Assistant Member, The Institute for Cancer Research
BARB, RICHARD S., Research Fellow, Albert Einstein College of Medicine
BALDWIN, THOMAS O., Assistant Professor of Biochemistry, University of Illinois, Urbana
BANG, FRF.DKRIK B., Professor of Pathobiology, The Johns Hopkins School of Hygiene and
Public Health BANG, BETSY G.
BARLOW, ROBERT B., Professor, Syracuse University BAUER, G. ERIC, University of Minnesota
BAUMGOLD, JESSE, Staff Fellow, NIMH, National Institutes of Health
BEAUGE, Luis A., Associate Professor of Biophysics, University of Maryland, School of Medicine BEGENISICH, TED, Assistant Professor of Physiology, LIniversity of Rochester BELL, EUGENE, Professor, Massachusetts Institute of Technology BENNETT, MICHAEL V. L., Director, Division of Cellular Neurobiology, Albert Einstein College
of Medicine
BISHOP, STEPHEN H., Associate Professor, Iowa State University BORGESE, THOMAS A., Associate Professor of Biology, Herbert H. Lehman College, The City
University of New York BRINLEY, FLOYD J., JR., Professor, Department of Physiology, University of Maryland, School of
Medicine BRODWICK, MALCOLM S., Assistant Professor of Physiology and Biophysics, University of Texas,
Medical Branch
BROWN, JAY C., Associate Professor, University of Virginia, School of Medicine BROWN, JOEL E., Professor of Physiology and Biophysics, State University of New York at
Stony Brook
BRYANT, SUSAN V., Associate Professor, University of California, Irvine BURDICK, CAROLYN J., Associate Professor of Biology, Brooklyn College, The City University of
New York
BURGER, MAX M., Chairman of the Biocenter, University of Basel, Switzerland BURKART, Werner R., Postdoctoral Research Fellow, Biocenter, University of Basel, Switzerland BURNS, ROY GORDON, Lecturer, Imperial College of Science and Technology, London, England CASADAY, GEORGE, B., Postdoctoral Fellow, Cornell University CHANG, DONALD C., Assistant Professor, Baylor College of Medicine
CHAPPELL, RICHARD L., Associate Professor, Hunter College, The City University of New York CHUNG, SU-YUN, Research Fellow, Harvard University
COHEN, JONATHAN B., Assistant Professor of Pharmacology, Harvard Medical School COHEN, LAWRENCE B., Associate Professor, Yale University, School of Medicine COHEN, SEYMOUR S., Professor, State University of New York at Stony Brook COHEN, WILLIAM D., Associate Professor of Biological Sciences, Hunter College, The City Uni- versity of New York
COOPERSTEIN, SHERWIN J., Professor of Anatomy, University of Connecticut CRANDALL, EDWARD D., Assistant Professor of Physiology and Medicine, University of Pennsyl- vania, School of Medicine
DAHLQUIST, FREDERICK W., Associate Professor, University of Oregon DAVIS, EDWARD M., Postdoctoral Fellow, Department of Biology, Yale University DENTLER, WILLIAM L., Assistant Professor, University of Kansas
DETERRA, NOEL, Research Associate Professor of Anatomy, Hahnemann Medical College DE WEER, PAUL, Associate Professor of Physiology and Biophysics, Washington LIniversity,
School of Medicine DiPoLO, REINALDO, Associate Investigator, Institute Venezolano de Investigaciones Cientificas,
Venezuela
DOWLING, JOHN E., Professor of Biology, Harvard University DuBois, ARTHUR B., Director, The John B. Pierce Foundation Laboratory DUNHAM, PHILIP B., Professor of Biology, Syracuse University EATON, DOUGLAS C., Assistant Professor of Physiology and Biophysics, University of Texas,
Medical Branch ECKBERG, WILLIAM R., Graduate Assistant Professor, Howard University
REPORT OK THE DIRECTOR 43
EDWARDS, DONALD H., JR., Postdoctoral Fellow, Stanford University
EHRENSTEIN, GERALD, Research Physicist, National Institutes of Health
EISNER, THOMAS, Jacob Gould Schurman Professor of Biology, Cornell University
ELLISON REBECCA P., Postdoctoral Fellow, The Population Council, The Rockefeller University
ERBER, JOACHIM, Assistant Professor, Institut fur Tierphysiologie, Freien Universitat, Berlin,
Germany
ESUMI, HIROYASU, Research Associate, National Cancer Center Research Institute, Japan FAIN, GORDON L., Assistant Professor of Ophthalmology, University of California at Los Angeles FARMANFAMAIAN, A., Professor of Physiology, Rutgers University FENCHEL, Tom M., Professor of Ecology, University of Aarhus, Denmark FIORE, JAMES, Associate Professor of Biology, Suffolk University FISCHBACH, GERALD, Professor, Harvard Medical School FISHMAN, HARVEY M., Professor of Physiology and Biophysics, University of Texas, Medical
Branch at Galveston
FLAVIN, MARTIN, National Institutes of Health FLETCHER, DONALD J., Postdoctoral Fellow, Emory University FOHLMEISTER, JURGEN, Lecturer, University of Minnesota FREEMAN, GARY, Associate Professor of Zoology, University of Texas, Austin FURSHPAN, EDWIN J., Professor of Neurobiology, Harvard Medical School FUSSELL, CATHERINE P., Associate Professor, Pennsylvania State University GAINER, HAROLD, Head, Section of Functional Neurochemistry, National Institutes of Health GELPERIN, ALAN, Associate Professor of Biology, Princeton University
GIBSON, JANE, Associate Professor of Biochemistry, Molecular and Cell Biology, Cornell Uni- versity
GILBERT, DANIEL L., Research Physiologist, National Institutes of Health GLANTZ, RAYMON M., Associate Professor of Biology, Rice University GLUSMAN, SILVIO, Research Associate, Harvard Medical School GOLDSMITH, PAUL K., Biologist, National Institutes of Health GOULD, JAMES L., Assistant Professor, Princeton University
GOULD, ROBERT M., Senior Research Scientist, Institute for Basic Research in Mental Retarda- tion in New York
GREENBAUM, ELIAS, Research Scientist, Corporate Research Laboratory, Union Carbide Corpora- tion
GROSCH, DANIEL S., Professor of Genetics, North Carolina State University GROSSMAN, ALBERT, Professor, New York University Medical School
GUTTMAN, RITA, Professor of Biology, Brooklyn College, The City University of New York HALVORSON, HARLYN O., Director, Rosenstiel Center, and Professor of Biology, Brandeis Uni- versity HARDING, CLIFFORD V'., Professor and Director of Research, Kresge Eye Institute of Wayne
State University
HARRIS-WARRICK, RONALD, Postdoctoral Fellow, Harvard Medical School HASCHEMEYER, AUDREY E. V., Professor of Biology, Hunter College, The City University of New
York
HEPLER, PETER K., Associate Professor of Botany, University of Massachusetts, Amherst HILDEBRAND, JOHN G., Associate Professor of Neurobiology, Harvard Medical School HOLDER, NIGEL, Postdoctoral Research Associate, University of California, Irvine HOLOWINSKY, ANDREW W., Associate Professor of Biology, Brown University HOSKINS, FRANCIS, Illinois Institute of Technology
HOY, RONALD R., Associate Professor of Neurobiology and Behavior, Cornell University HUMPHREYS, SUSIE, Assistant Researcher, University of Hawaii HUMPHREYS, TOM, Professor of Biochemistry, University of Hawaii
HUNGATE, ROBERT E., Emeritus Professor of Bacteriology, University of California, Davis HUNT, R. KEVIN, Assistant Professor of Biophysics, The Johns Hopkins University li, ICHIO, Research Associate, Department of Biology, University of Virginia JACOB, MICHELE, Postdoctoral Fellow, Columbia University JAFFE, LIONEL, Professor of Biology, Purdue University JAN, LILY YEH, Instructor in Neurobiology, Harvard Medical School JAN, YUH NUNG, Research Fellow in Neurobiology, Harvard Medical School
44 ANNUAL RKI'OKT OK TIIK .\I.\K1.\K I'.H >l .< >< ,ICAI. I.AIU )R. \TORY
| \NN \M ii. I IIH (.1 i; \\ ., Course Director, Woods Hole Oceanographic Institution
Ji HI K\. \\II.I.I\M I-., Assistant Professor of Zoology, University of Texas, Austin
JOHNSTON, DANIEL, Assistant Professor, Baylor College of Medicine
Ji INNER, RONALD W., Assistant Professor, Department of Physiology, University of Iowa
KALMIJN, AD. J., Associate Scientist, Woods Hole Oceanographic Institution
K \\IINER, BENJAMIN, Professor and Chairman, Boston University School of Medicine
KAMMER, ANN E., Associate Professor, Kansas State University
KAPLAN, EHUD, Assistant Professor, The Rockefeller University
KENNEDY, BRIAN, Postdoctoral Fellow, Washington University Medical School
KEYNAN, A., Professor of Biology, Hebrew University of Jerusalem, Israel
KIRSCH, GLENN E., Postdoctoral Fellow, Northwestern University
KLINE, RICHARD P., Research Fellow, Harvard University
KOIDE, SAMUEL S., Senior Scientist, Biomedical Division, The Population Council
KRAVITZ, EDWARD A., Professor of Neurobiology, Harvard Medical School
KRISTAN, WILLIAM B., JR., Assistant Professor of Biology, University of California, San Diego
KUFFLER, STEPHEN W., John Franklin Enders University Professor, Harvard Medical School
KUHNS, WILLIAM J., Professor, University of North Carolina
KUNZ-RAMSAY, Yvette, University College, Ireland
KUSANO, KIYOSHI, Professor, Illinois Institute of Technology
LANDIS, STORY, Instructor, Harvard Medical School
LANDOWNE, DAVID, Associate Professor, University of Miami
LASEK, RAYMOND J., Associate Professor, Case Western Reserve University
LASH, JAMES W., Professor of Anatomy, University of Pennsylvania, School of Medicine
LAUFER, HANS, Professor of Biology, University of Connecticut
LEADBETTER, E. R., Executive Officer, University of Connecticut
LIPETZ, LEO E., Professor, Ohio State University
LIPICKY, RAYMOND J., Professor of Pharmacology and Medicine, Director of Clinical Pharmacy,
University of Cincinnati
LISMAN, JOHN E., Assistant Professor of Biology, Brandeis University LLINAS, R., Professor and Chairman, Department of Physiology and Biophysics, New York
University Medical Center LOEWENSTEIN, W. R., Professor and Chairman of the Department of Physiology and Biophysics,
University of Miami, School of Medicine LOFTFIELD, ROBERT B., Professor of Biochemistry, Chairman, University of New Mexico, School
of Medicine
LOH, Y.-PENG, Senior Staff Fellow, National Institutes of Health LONGO, FRANK J., Associate Professor of Anatomy, University of Iowa LORAND, L., Professor of Biochemistry and Molecular Biology, Northwestern University LORAND, JOYCE BRUNER Lux, HANS DIETER, Head, Neurophysiological Laboratory, Max Planck Institute of Psychiatry,
Germany LYMAN, HARVARD, Associate Professor, Biology Department, State University of New York at
Stony Brook
MACAGNO, E. R., Assistant Professor, Columbia University MACLEISH, PETER R., Postdoctoral Fellow, Harvard Medical School
MARUO, TAKESHI, Postdoctoral Fellow, The Population Council, The Rockefeller University MASTROIANNI, LUIGI, JR., Professor and Chairman, Department of Obstetrics and Gynecology,
University of Pennsylvania, School of Medicine
MATHEWS, RITA W., Senior Research Associate, Hunter College, The City University of New York MATSUMOTO, STEVEN G., Postdoctoral Research Fellow, Harvard University Medical School MAUZERALL, D., Professor, The Rockefeller University MEEDEL, THOMAS H., Postdoctoral Trainee, The Wistar Institute MEISS, DENNIS, Postdoctoral Research Associate, Scarborough College, University of Toronto.
Canada
MENZEL, RANDOLF, Professor, Freien Universitat, Berlin, Germany METUZALS, J., Professor and Director of the Electron Microscopy Unit, University of Ottawa,
Faculty of Medicine, Canada METZ, CHARLES B., Professor, University of Miami
REPORT OF THE DIRECTOR 45
MILLER, RICHARD S., Professor, Yale University
MITCHELL, RALPH, Gordon McKay Professor of Applied Biology, Harvard University
MOORE, JOHN W., Professor of Physiology, Duke University
MOORE, L. E., Professor, University of Texas, Medical Branch
MOOSEKER, MARK S., Research Fellow, Harvard Medical School
MULLINS, L. J., Professor of Biophysics and Chairman, University of Maryland, School of Medicine
MYLES, CHRISTINA Jo, Assistant Professor of Life Science, Manchester Community College
NAGANO, TOSHIO, Professor and Chairman of the Department of Anatomy, Chiba University,
School of Medicine, Japan
NAGEL, RONALD L., Associate Professor of Medicine, Albert Einstein College of Medicine NAKAJIMA, SHIGEHIRO, Professor, Purdue University
NAKATSUJI, NORIO, Research Associate, Massachusetts Institute of Technology NARAHASHI, TOSHIO, Professor and Chairman, Northwestern University Ni'.ARY, JOSEPH T., Staff Scientist, Laboratory of Biophysics
NELSON, MARGARET C., Research Fellow in Xeurobiology, Harvard Medical School NICHOLLS, JOHN, Professor of Neurobiology, Stanford University, School of Medicine NICOLI, MIRIAM Z., National Institutes of Health Postdoctoral Fellow, University of Illinois NICOSIA, SANTO V., Assistant Professor of Obstetrics, Gynecology, and Pathology, University of
Pennsylvania
NOE, BRYAN D., Assistant Professor of Anatomy, Emory University NOTTEBOHM, FERNANDO, Professor, The Rockefeller LIniversity
OBAID, ANA LIA, Postdoctoral Research Associate, University of Pennsylvania, Medical School OERTEL, DONATA, Postdoctoral Research Fellow, Harvard Medical School OHKI, SHINPEI, Associate Professor, State University of New York at Buffalo O'LAGUE, PAUL H., Assistant Professor, University of California, Los Angeles ORNBERG, RICHARD L., Postdoctoral Research Associate, National Institutes of Health OVADIA, MICHAEL, Postdoctoral Fellow, University of Pennsylvania, School of Medicine OXFORD, GERRY S., Assistant Professor of Physiology, University of North Carolina PANT, HARISH C., Senior Staff Fellow, National Institutes of Health
PAPPAS, GEORGE D., Professor and Head of Anatomy, University of Illinois, College of Medicine PARMENTIER, JAMES L., Medical Research Assistant Professor, Duke University PERSON, PHILIP, Medical Investigator, Veteran's Administration Hospital PETHIG, RONALD, Lecturer, University of Wales, Bangor, United Kingdom
PICHON, YVES, Maitre de Recherche, Centre National de la Recherche Scientifique, Paris, France PIERCE, SIDNEY K., Associate Professor of Zoology, University of Maryland POINDEXTER, JEANNE S., Associate, Public Health Research Institute of New Y'ork POLLARD, HARVEY B., Senior Investigator and Medical Officer, National Institutes of Health POTTER, DAVID D., Harvard Medical School POUSSART, DENIS, Professor, Universite Laval, Quebec, Canada
POZNANSKY, MARK, Associate Professor of Physiology, University of Alberta, Canada PRENDERGAST, ROBERT A., Associate Professor, The Johns Hopkins LIniversity, School of Medicine PRICE PHILIP, Postdoctoral Fellow, The Rockefeller University PRIOR, DAVID J., Assistant Professor, University of Kentucky PRUSCH, ROBERT D., Assistant Professor of Biology, Brown University QUIGLEY, JAMES P., Associate Professor of Microbiology and Immunology, State University of
New York, Downstate Medical Center, Brooklyn RAMON, FIDEL, Assistant Professor, Duke University REBHI N, LIONEL L, Professor of Biology, University of Virginia
REESE, THOMAS S., Head, Section on Functional Neuroanatomy, National Institutes of Health REINGOLD, STEPHEN C., Postdoctoral Research Fellow, Princeton University REQUENA, JAIME, Assistant Investigator, Centre de Biofisica Y Bioquimica, Caracas, Venezuela REYNOLDS, GEORGE T., Professor of Physics, Princeton LIniversity RHEUBEN, MARY B., Assistant Professor, Pennsylvania State University RICKLES, FREDERICK R., Veteran's Administration Hospital and University of Connecticut Health
Center RIPPS, HARRIS, Professor of Ophthalmology and Physiology, New York University, School ot
Medicine ROSE, BIRGIT, Research Associate Professor, LIniversity of Miami, School of Medicine
46 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
ROSENKRANZ, HERBERT S., Professor and Chairman, Department of Microbiology, New York Medical College
RUDERMAN, JOAN V., Assistant Professor, Department of Anatomy, Harvard Medical School
RUSHFORTH, NORMAN B., Professor and Chairman, Department of Biology, Case Western Reserve University
RUSSELL, JOHN M., Assistant Professor, University of Texas, Medical Branch
RUSSELL-HUNTER, W. D., Professor of Zoology, Syracuse University
SAGER, RUTH, Professor, Harvard Medical School
SCARPA, ANTONIO, Associate Professor of Biochemistry and Biophysics, University of Pennsyl- vania, School of Medicine
SCHACHER, SAMUEL, Research Associate, Columbia University College of Physicians and Surgeons
SCHIFF, JEROME A., Professor of Biology and Director of the Institute for Photobiology of Cells and Organelles, Brandeis University
SCHUEL, HERBERT, Associate Professor of Anatomy, State University of New York at Buffalo
SCHUETZ, ALLEN W., Professor, Department of Population Dynamics, The Johns Hopkins Uni- versity
SEALOCK, ROBERT W., Assistant Professor, University of North Carolina at Chapel Hill
SEGAL, SHELDON J., Vice President and Director, Biomedical Division, The Population Council
SEJNOWSKI, TERRANCE J., Research Fellow, Princeton University
SEYAMA, ISSEI, Associate Professor, Hiroshima University, Japan
SHARNOFF, MARK, Professor of Physics, University of Delaware
SHIELDS, DENNIS, Assistant Professor of Anatomy, Albert Einstein College of Medicine
SHRIVASTAV, BRIJ B., Medical Research Assistant Professor, Duke University
SIMMONS, ROBERT M., Lecturer, University College, London, England
SIMPSON, TRACY L., Professor, Department of Biology, University of Hartford
SMITH, JANIE E., Biologist, National Institutes of Health
SPECK, WILLIAM T., Associate Professor of Pediatrics, Case W7estern Reserve University
SPIEGEL, EVELYN, Research Associate Professor, Dartmouth College
SPIEGEL, MELVIN, Professor of Biology, Dartmouth College
SPRAY, DAVID, Assistant Professor, Albert Einstein College of Medicine
SPIRA, MICHA E., Professor, Albert Einstein College of Medicine
STETTEN, MARJORIE R., Biochemist, NIAMDD, National Institutes of Health
STOKES, DARREL R., Assistant Professor of Biology, Emory University
STRACHER, ALFRED, Professor and Chairman, Department of Biochemistry, State University of New York, Downstate Medical Center
STUART, ANN E., Assistant Professor, Harvard Medical School
STUNKARD HORACE W., Research Associate, American Museum of Natural History
SUGIMORI, MUTSUYUKI, Research Assistant Professor, New York University Medical Center
SUMMERS, JESSE W., Member, Institute for Cancer Research
SUMMERS, ROBERT G., Associate Professor of Anatomy, State University of New York at Buffalo
SUSSWEIN, ABRAHAM J., Research Fellow, Albert Einstein College of Medicine, Yeshiva University
SZENT-GYORGYI, ANDREW G., Chairman, Department of Biology, Brandeis University
SzENTKiRALYi-SzENT-GYORGYi, EVA M., Research Associate, Brandeis University
TABATA, MITSUO, Research Fellow, Laboratory of Biophysics
TAMM, SIDNEY, Associate Scientist, University of Wisconsin
TAMM, SIGNHELD, Research Associate, University of Wisconsin
TASAKI, ICHIJI, Chief, Laboratory of Neurobiology, NIMH, National Institutes of Health
TEN£ICK, ROBERT E., Associate Professor of Pharmacology, Northwestern University
TERAKAWA, SUSUMU, Visiting Associate, National Institutes of Health
TIFFERT, TERESA, Assistant Professor, Department of Physiology, University of Maryland, School of Medicine
TRINKAUS, J. P., Professor of Biology, Yale University
TROLL, WALTER, Professor of Environmental Medicine, New York University Medical Center
TROXLER, ROBERT F., Associate Professor of Biochemistry, Boston University, School of Medicine
TWEEDELL, KENYON S., Professor, University of Notre Dame
TYTELL, MICHAEL, Postdoctoral Fellow, Case Western Reserve University, School of Medicine
VAN HOLDE, K. E., Professor, Oregon State University
VAN RAALTE, CHARLENE, Instructor, Hampshire College
REPORT OF THE DIRECTOR 47
VINCENT, WALTER S., Professor of Cell and Molecular Biology, University of Delaware, School of
Life and Health Sciences
WAGNER, RICHARD W., Postdoctoral Fellow, University of Miami, School of Medicine WATERS ROBERT S., Postdoctoral Fellow, The Rockefeller University WAXMAN, STEPHEN G., Associate Professor of Neurology, Harvard Medical School WEBER, ANNMARIE, University of Pennsylvania, School of Medicine WEISSMANN, GERALD, Professor of Medicine, New York University Medical Center WHITTAKER, J. RICHARD, Associate Professor, The Wistar Institute
WIERCIXSKI, FLOYD J., Professor, Department of Biology, Northeastern Illinois University WILLIAMS, T. P., Professor of Biological Sciences, Florida State University WILSON, DARCY B., Professor of Pathology, University of Pennsylvania, School of Medicine WOLF, DON P., Associate Research Professor, University of Pennsylvania, School of Medicine WOOLEY, JOHN C., Research Fellow in Biochemistry, Harvard University WORGUL, Basil V., Research Associate, Department of Ophthalmology, Columbia University WTu, CHAU H., Assistant Professor of Pharmacology, Northwestern University YEH, JAY Z., Assistant Professor, Northwestern University ZIGMAN, SEYMOUR, Professor of Ophthalmology and Biochemistry, University of Rochester,
School of Medicine and Dentistry ZIRKIN, BARRY R., Associate Professor, Division of Reproductive Biology, The Johns Hopkins
University
Lillie Fellow, 1978
LE DOUARIN, NICOLE, Centre National de la Recherche Scientifique, Institut D'Embryologie, France
Alexander Forbes Lecturer, 1978
KRNJEVIC, KRESIMIR, Department of Research in Anaesthesia, Mclntyre Medical Sciences Building, Canada
Rand Fellow, 1978 MKN/EL, RANDOLF, Institut fur Tierphysiologie, Freien Universitat, Berlin, West Germany
Grass Fellows, 1978
ASHCROFT, FRANCES M., Grass Foundation Fellow, Department of Zoology, Cambridge Uni- versity, United Kingdom
DAY, JOHN W., Albert Einstein College of Medicine EDWARDS, DONALD H., Postdoctoral Fellow, Stanford University FRAZIER, DONALD, Professor, University of Kentucky, School of Medicine GOLDBERG, MARK T., Grass Fellow, Memorial University of Newfoundland, Canada LUND, ALBERT E., Postdoctoral Fellow, Northwestern University Medical School MEYER, DAVID J., Grass Fellow, Health Sciences Center, State University of New York at Stony
Brook
NASS, MENASCHE M., Research Fellow in Biology, California Institute of Technology PELLMAR, TERRY, Research Fellow, National Naval Medical Center, Bethesda QUANDT, FREDERICK N., Postdoctoral Fellow, University of California, Los Angeles SATTERLIE, RICHARD A., Grass Fellow, University of California, Santa Barbara SCOTT, SHERYL A., Postdoctoral Fellow, Carnegie Institution of Washington SULLIVAN, ROBERT E., Postdoctoral Fellow, University of Hawaii at Manoa WATSON, WINSOR H., Ill, Research Associate, University of Massachusetts ZOTTOLI, STEVEN J., Research Scientist II, Research Institute on Alcoholism
Macy Scholars, 1978
ARMSTRONG, EARLENE, Assistant Professor, University of Maryland
HILL, ANITA V., Assistant Professor of Biology, Grambling State University
4S ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
JAMES C., JR., Department of Anatomy, Howard University College of Medicine HOWZE, GWENDOLYN B., Assistant Professor, Texas Southern University IVENS, M. SUE, Associate Professor, Natural Sciences, Dillard University PORTER, CHARLES W., Associate Professor of Biology, San Jose State University WALKER, DOROTHY G., Associate Professor, Howard University
Summer Research Scholarships, 1978 (Steps Toward Independence)
ARMETT-KIBEL, CHRISTINE, Assistant Professor of Biology, University of Massachusetts
BOURNE, GEORGE B., Assistant Professor, The University of Calgary, Canada
CHUNG, SU-YUN, Research Fellow in Chemistry, Harvard University
FUJIWARA, KEIGI, Assistant Professor of Anatomy, Harvard Medical School
HOFFMAN, RICHARD J., Assistant Professor of Biological Sciences, University of Pittsburgh
HUFNAGEL, L. Assistant Professor of Microbiology, University of Rhode Island
KASS-SIMON, M. Assistant Professor of Zoology, University of Rhode Island
LANGFORD, GEORGE M., Assistant Professor of Anatomy, Howard University, College of Medicine
LEWIS, LARRY M., Assistant Professor of Biology, Millersville State College
O'MELIA, ANNE F., Assistant Professor of Biochemistry, Louisiana State University Medical
Center
RUDY, BERNARDO, Assistant Professor of Physiology, Eastern Pennsylvania Psychiatric Institute SALZBERG, BRIAN, Assistant Professor of Physiology, University of Pennsylvania TREISTMAN, STEVEN N., Assistant Professor of Biology, Bryn Mawr College YOUNG, LILY Y., Assistant Professor of Environmental Microbiology, Stanford University
Research Assistants, 1978
ANDERSON, DAVID J., The Rockefeller University
ANSTROM, JOHN, State University of New York at Buffalo
ANTONELLIS, BLENDA, University of Rochester School of Medicine
ARVAN, PETER, Yale University
AUGUSTINE, GEORGE J., JR., University of Maryland
BARNES, EDWARD S., Columbia University
BATRA, RANJAN, Institute for Sensory Research, Syracuse University
BEHRMAN, AMY, Swarthmore College
BENNETT, HOLLY V. L., The Albert Einstein College of Medicine
BODICK, NEIL, Columbia University
BOSLER, ROBERT B., Harvard University Medical School
BOYLE, MARY B., Yale University
BRANDON, CHRISTOPHER, Baylor College of Medicine
BUSH, JAMES, Rutgers College
BYERS, H. RANDOLPH, Harvard Medical School
CALLE, PAUL, University of Pennsylvania
CAPCO, DAVID GEORGE, University of Texas
CHAN, YUE-WAH STEVEN, Baylor College of Medicine
CHELSKY, DANIEL, University of Oregon
CHENEY, CLARISSA M., University of Toronto
COHEN, RICHARD, Hunter College
COLUCCI, BARBARA ANN, Herbert H. Lehman College, The City University of New York
CRAWFORD, WILLIAM, University of Hawaii
CRITZ, ANNE MC£LROY, University of Pennsylvania
CROOP, ROBERT, University of Pennsylvania
DAHL, ROBERT F., Princeton University
DANDEKAR, PRAMILA, State University of New York at Buffalo
DAVIDSON, DOROTHY E., University of Delaware
DRAKE, PETER F., Bryn Mawr College
EAGLE, JANE S., University of Connecticut Health Center
EIGNER, E. ANN, University of New Mexico School of Medicine
FALKOW, STEPHEN, New York University, School of Medicine
REPORT OF THE DIRECTOR 4()
FIELD, NANCY, \\Vsleyan University
FINE, ALAN, University of Pennsylvania
FINKEL, TOKEN, University of Pennsylvania School of Medicine
FITT, WILLIAM K., University of California, Santa Barbara
FLEMING, JUDY, University of Colorado
FRIZZELL, KAREN L., University of Massachusetts
GALVIN, PATRIC N., University of Colorado at Denver
GILLY, WILLIAM F., University of Pennsylvania
GLICKSMAN, MARGIE, Brown University
GONCALVES, MARK A., University of Colorado
GORDON, DORIA, Oberlin College
GREEN, JEFFREY D., State University of New York at Buffalo
( "IREENWALT, DALE, Iowa State University
GREGA, DEBRA S., University of Kentucky
GRUPP, STEPHEN, University of Cincinnati
HARRIS, ANDREW L., The Albert Einstein College of Medicine
HARRIS, VICKI KAY, Emory University
HEINRICHS, STEFAN
HENRY, JONATHAN J., University of Hawaii
HERLANDS, Louis, Yale University
HERSCH, STEVEN M., Boston University
Ho, SIMON MING HUNG, University of Toronto, Canada
HOLMES, DON J., Washington University Medical School
HOWARD, LOUISA, Dartmouth College
HURST, TERRY W., University of Texas, Medical Branch
HUSE, WILLIAM, Albert Einstein College of Medicine
HUTTNER, SUSANNE, University of California, Los Angeles
HYLANDER, BONNIE, State University of New York at Buffalo
LMHOF, RUTH, Biozentrum University of Basel, Switzerland
JAEGER, RICHARD R., Hunter College, The City University of New York
JANUS, TODD, J., Northwestern University
JASLOVE, STEWART, Duke University Medical Center
JIMENEZ, RAMON J., University of Guadalajara, Mexico
JUMBLATT, JAMES E., University of Basel, Switzerland
KATAYAMA, ROBERT N., University of Pennsylvania
KENDALL, DEBRA A., Northwestern University
KIEHART, DANIEL P., University of Pennsylvania
KIRK, MARK D., Rice University
KOUMJIAN, LAUREN, Mount Holyoke College
LAUFER, MARC R., University of Pennsylvania
LEIBOWITZ, DAVID H., Columbia University
LESHER, SARAH, Yale University
LEVINTHAL, ADAM
LEWIS, STEPHEN, University of Cincinnati
LITTLEFIELD, PETER, University of Maryland
LIVINGSTONE, M. S., Harvard Medical School
LUEBBERT, Jo ANN, State University of New York at Stony Brook
LUTTINGER, KARL M., Columbia University
MARSDEN, J. ELLEN
MEEDEL, THOMAS H., The Wistar Institute
MEYER, MICHAEL A., University of North Carolina at Chapel Hill
MIR, FRANCOISE, University of Basel, Switzerland
MONTANARO, GEORGE D., University of Iowa
MORELLO, ROBERT S., University of Rochester
MORGAN, CHARLES R., Emory University
MORGAN, GINA, Howard University
MORRIS, JAMES R., Case Western Reserve University
NARAHASHI, TARO, Northwestern University
50 \\XTAL REPORT OK THE MARINE BIOLOGICAL LABORATORY
NECLES, NICHOLAS, Columbia University
NEMHAUSER, IRIS, Hunter College, The City University of New York
NEUBIG, RICHARD R., Harvard Medical School
PELLEY, CHIQUITA, University of Oregon
PORTER, MARY E., University of Pennsylvania
RAMOS, TALIA, The Johns Hopkins University School of Hygiene and Public Health
REDMANN, GREG A., University of Texas Medical Branch
REISS, PAUL, University of Maryland
RENDER, Jo ANN, University of Texas, Austin
RHO, JAI-HYON, The Johns Hopkins University
ROBERTSON, LOLA E., American Museum of Natural History
ROLLINS, SHARON, Cornell University
ROSENKRANZ, PNiNA, Princeton University
ROSMAN, GARY, New York University
RUMER, NINA, University of Delaware
SAMSON, DOUGLAS A., The Johns Hopkins School of Hygiene and Public Health
SAUNDERS, MARY J., University of Massachusetts
SCARBOROUGH, ANN, Louisiana State University
SCHENCK, KATHLEEN, Princeton University
SCHMIDT, JEFF, University of California, Los Angeles
SCHMIDT, MADELYN R., Harvard Medical School
SCHNEIDER, MARK ALAN, Amherst College
SCRUGGS, VIRGINIA
SHURE, MICHAEL S., Yale University
SIEGEL, RUTH E., Harvard Medical School
SIMON, SANFORD, New York University Medical Center
Socci, ROBIN R., Rutgers, The State University of New Jersey
SOLOMON, DENNIS J., Massachusetts Institute of Technology
SOLWAY, ALAN, Wayne State University, School of Medicine
STAMLER, JOHN F., University of Iowa
STEELE, JOY ANN, University of Alberta, Canada
STICH, THOMAS J., University of Maryland
SUBYAK, SHARON E., Suffolk University
SUH, KYUNGSUN, Columbia University
SWAN, MICHAEL C., University of California, Los Angeles
SWENSON, RANDOLPHE P., University of Pennsylvania
TALIAN, JOHN, Carnegie-Mellon University
TAYLOR, CHRISTOPHER E., The Johns Hopkins School of Hygiene and Public Health
TIMPE, LESLIE C., Harvard Medical School
TRAVIS, MARK A., Greenville College
TYNER, EMILY M., Vassar College
VON HIPPEL, DAVID F.
WALDROP, BRIAN, Rice University
WALTON, KERRY, New York University Medical Center
WARREN, MARY K., University of Maryland
WEEKS, JANIS C., University of California, San Diego
WEISS, KEVIN, University of Pittsburgh
WESTERFIELD, MONTE, Max Planck Institute fur Psychiatric, Germany
WHEATLEY, RICHARD, Columbia University
WICK, SUSAN M., University of Massachusetts
WILLIAMS, CAROLYN H., Emory University
WILLIAMS-ARNOLD, Lois D., University of Hawaii
WODLINGER, HAROLD M., University of Toronto, Canada
YONEMOTO, WES, University of Hawaii
YULO, TERESA, University of Rochester School of Medicine and Dentistry
ZAKEVICIUS, JANE, New York University School of Medicine
ZUKOWSKI, ANTHONY J., University of Hawaii
REPORT OK THE DIRECTOR 51
Library Readers 1Q78
ADELHERG, EDWARD A., Professor of Human Genetics, Vale University
ALLEN, NINA STROMGREN, Assistant Professor, Dartmouth College
ALLEN, ROBERT DAY, Professor and Chairman, Dartmouth College
ANDERSON, EVERETT, Professor of Anatomy and Associate Director, Laboratory of Human
Reproduction and Reproductive Biology, Harvard University Medical School BEAN, CHARLES P., Biophysicist, General Electric Company BOURNE, DONALD, Marine Research Inc.
CANDELAS, GRACIELA C., Professor, University of Puerto Rico
CARRIERE, RITA, Assistant Professor, State University of New York, Downstate Medical Center CHILD, FRANK M., Professor of Biology, Trinity College
CLIFFORD, SISTER ADELE, Professor of Biology, College of Mount St. Joseph on the Ohio COLE, KENNETH S., Research Biophysicist Emeritus, National Institutes of Health CONDOURIS, GEORGE A., Professor and Chairman, New Jersey Medical School COPELAND, DONALD EUGENE, Professor of Biology, Tulane University CORNWELL, ANNE CHRISTAKE, Assistant Professor of Ophthalmology, Montefiore Hospital and
Medical Center
COUCH, ERNEST F., Associate Professor of Biology, Texas Christian University DAVIS, BERNARD D., Professor, Harvard Medical School DEHN, PAULA F., Graduate Assistant, University of Southern Florida
DETTBARN, WoLF-D., Professor of Pharmacology, Vanderbilt University, School of Medicine DUDLEY, PATRICIA L., Professor of Biological Sciences, Barnard College, Columbia University DUNDAS, IAN, Associate Professor, University of Bergen, Norway EBERT, JAMES D., President, Carnegie Institute of Washington EDDS, LOUISE L., Associate Professor, Zoology and Microbiology, Ohio University ELDER, HOWARD A., Professor of Medicine, Albert Einstein College of Medicine EISEN, HERMAN N., Professor of Immunology, Massachusetts Institute of Technology ENGLANDER, SOL WALTER, University of Pennsylvania FISCHMAN, DONALD A., Professor and Chairman, State University of New York, Downstate
Medical Center
FISHER, SAUL H., Clinical Professor of Psychiatry, New York University, School of Medicine FRANZINI- ARM STRONG, CLARA, Associate Professor of Anatomy, University of Pennsylvania GABRIEL, MORDECAI L., Professor of Biology, Brooklyn College, City University of New York GAGNE, GERARD D., Doctoral Candidate, University of Maine, Orono GALATZER-LEVY, ROBERT M., Lecturer, Associate Attending Psychiatrist, University of Chicago
and Michael Reese Hospital
GOLDMAN, ROBERT D., Professor of Biological Sciences, Carnegie-Mellon University GOLDSTEIN, MOISE H., JR., Professor, The Johns Hopkins University GOUDSMIT, ESTHER M., Associate Professor, Oakland University, Rochester GOULD, STEPHEN JAY, Professor of Geology, Harvard University GRANT, PHILIP, Professor of Biology, University of Oregon HAUBRICH, ROBERT, Professor of Biology, Denison University HENLEY, CATHERINE, National Eye Institute, National Institutes of Health HILLMAN, PETER, Associate Professor, The Hebrew University of Jerusalem, Israel HINSCH, GERTRUDE W., Associate Professor, University of Florida HOCHSTEIN, SHAUL, Lecturer, The Hebrew University of Jerusalem, Israel
HUBERMAN, MICHAEL H., Intern — Internal Medicine and Pre-doctoral Fellow, New York Uni- versity
HUETTNER, ROBERT J., Assistant Professor, Columbia University, School of Dentistry HUNTER, R. DOUGLAS, Assistant Professor of Biological Sciences, Oakland University, Rochester ILAN, JOSEPH, Professor, Case \Vestern Reserve University, School of Medicine INOUE, SADAYUKI, Assistant Professor, McGill University, Canada IRELAND, LEONARD, Associate, Bermuda Biological Station ISENBERG, IRVIN, Professor of Biophysics, Oregon State University ISSELBACHER, KURT J., Mallinckrodt Professor of Medicine, Chief of the Gastrointestinal Unit,
Harvard Medical School and Massachusetts General Hospital ISSIDORIDES, MARIETTA R., Research Professor in the Department of Psychiatry, University of
Athens, Medical School, Greece
52 ANNUAL REPORT OF TI1K MARINE BIOLOGICAL LABORATORY
I \\ITT, NORMAN B., Professor of Medicine, Head, Division of Gastroenterology, Cornell Uni- versity Medical College
JOHNSON, WILLIAM H., Professor of Biology, Rensselaer Polytechnic Institute KALTENBACH, JANE C., Professor of Biological Sciences, Mount Holyoke College KARUSH, FRED, Professor of Microbiology, University of Pennsylvania, School of Medicine KEAN, EDWARD L., Associate Professor, Case Western Reserve University KEOSIAN, JOHN P., Author KIRSCHENBAUM, DONALD M., Associate Professor, State University of New York, Downstate
Medical Center
KLEIN, MORTON, Professor of Microbiology, Temple University Medical School KOBAYASHI, MAKOTO, Professor of Physiology, Hiroshima University, Japan KOGUT, M ARGOT, Lecturer, King's College, London, England KRNJEVIC, K., Professor and Director, Department of Anaesthesia Research, McGill University,
Montreal, Canada
LADERMAN, AIMLEE, Doctoral Candidate, State University of New York at Binghamton LAZAROW, JANE K., Senior Research Analyst, Minnesota Department of Health LE DOUARIN, NICOLE, Professeur, Directeur de 1'Institut d'Embryologie du C. N. R. S. et du
College de France, Centre National de la Recherche Scientifique
LEFEVRE, MARIAN E., Associate Scientist, Medical Department, Brookhaven National Laboratory LEIGHTON, JOSEPH, Professor and Chairman, Department of Pathology, Medical College of
Pennsylvania
LEVITAN, HERBERT, Associate Professor, University of Maryland, College Park LIEBERMAN, MICHAEL W., Associate Professor of Pathology, Washington University, School of
Medicine, St. Louis LINCK, RICHARD W., Assistant Professor, Harvard Medical School
LlNEAWEAVER, THOMAS H., Ill, Author
Lux, HANS DEITER, Max Planck Institute of Psychiatry, Germany
MARSLAND, DOUGLAS, Research Professor Emeritus, New York University
MASLAND, RICHARD H., Assistant Professor of Physiology
MITCHELL, JAMES B., Associate Professor of Biology, Moravian College
MIZELL, MERLE, Professor of Biology, Tulane University
MORRELL, FRANK, Professor of Neurology, Rush Medical College
NEWBURY, THOMAS, Assistant Professor, University of Hawaii
OJAKIAN, GEORGE, Assistant Professor, State University of New York, Downstate Medical Center
PALMER, JOHN D., Chairman and Professor of the Zoology Department, University of Massa- chusetts, Amherst
PEARLMEN, ALAN L., Associate Professor of Physiology and Neurology, Washington University, School of Medicine
PLOCKE, DONALD J., Associate Professor and Chairman of Biology, Boston College
ROSENBAUM, JOEL L., Professor of Biology, Yale University
ROSENBERG, EVELYN, Professor, Jersey City State College
SALMON, EDWARD D., Assistant Professor, Zoology Department, University of North Carolina, Chapel Hill
SANGER, JOSEPH W., Associate Professor, University of Pennsylvania
SAUNDERS, JOHN W., Professor, State University of New York at Albany
SCHLESINGER, WALTHER R., Professor and Chairman, Department of Microbiology, Rutgers Medical School, College of Medicine and Dentistry of New Jersey
SCOTT, GEORGE T., Professor of Biology, Oberlin College
SHEMIN, DAVID, Professor and Chairman, Department of Biochemistry and Molecular Biology, Northwestern University
SHEPRO, DAVID, Professor, Boston University
SMITH, MICHAEL A., Research Associate, Hunter College, The City University of New York
SONNENBLICK, B. P., Professor Emeritus, Rutgers, The State University of New Jersey
SPECTOR, ABRAHAM, Professor of Ophthalmic Biochemistry, Department of Ophthalmology, Columbia University
SUGDEN, BILL, Assistant Professor, University of Wisconsin
SZAMIER, R. BRUCE, Assistant Professor, Harvard Medical School
TRACER, WILLIAM, Professor, The Rockefeller University
REPORT OF THE DIRECTOR
53
WAINIO, WALTER, Professor and Chairman, Department of Biochemistry, Rutgers College,
Rutgers, The State University of New Jersey WAKSMAN, BYRON H., Professor of Pathology, Yale University WEBB, H. MARGUERITE, Professor of Biological Sciences, Goucher College WEISS, LEON, Chairman, Department of Animal Biology; Professor of Cell Biology, University
of Pennsylvania, School of Veterinary Medicine
WHEELER, GEORGE E., Professor of Biology, Brooklyn College, Biology Department WILSON, THOMAS H., Professor of Physiology, Harvard Medical School WITTENBERG, JONATHAN B., Professor of Physiology, Albert Einstein College of Medicine WOLF, JASON, Associate Professor, Wesleyan University
YNTEMA, C. L., Professor Emeritus, Upstate Medical Center, State University of New York ZACKS, SUMNER I., Professor and Chairman, Section of Pathology, Brown University; Pathologist -
in-Chief, Laboratory Director, The Miriam Hospital
Students, 1978
All students listed completed the formal course programs. pleting post-course research programs.
Summer Programs 1978 MARINE ECOLOGY
Asterisk indicates those com-
*BAXTER, MOLLY
BRONSON, BECKY
CHEN, CELIA Y. *COPLEY, NANCY JEANETTE *DAY, MARGARET ELISABETH
DEMUTH, ROBERT EMERSON
DONN, THEODORE E., JR. *FAGAN, JOANNE
FREDERICQ, SUZANNE *JENSEN, CYNTHIA LUND
KATZ, LAWRENCE C. *KIRCHMAN, DAVID Louis
LECHLEITNER, RICHARD A. *MOGAN, ARTHUR *RIETSMA, CAROL S.
ROSLANSKY, LOUISE
SlLBERHORN, ERIC MARTIN
SKINNER, CAROL ANN *TALBERT, JEAN E.
EMBRYOLOGY
ANDERSON, KATHRYN VIRGINIA BROWN, KENNETH MICHAEL CHILTON, BEVERLY SUE EDWARDS, MARY KAYE PLASTER, MURRAY S. GIBSON, BARBARA LOUISE GUIDICE, GEORGE J., JR. JOHNSON, THOMAS EUGENE KORNHER, JOHN STEPHEN KOSOFSKY, BARRY EVAN LAXDZBERG, JOEL SERGE MARUSICH, MICHAEL F. MILLER, CAROL LYNN
MUNEOKA, KEN RICE, DOUGLAS SALIK, JANE ANNE SANDRIDGE, PAUL TIMOTHY SCHULZ, STEPHANIE SZARO, BEN GREGORY TANSEY, TERESE R. WANEK, NANCY LYNN WATANABE, MICHIKO WEHRMAKER, ALFRED WISE, LAWRENCE DAVID YOUNG, NEVIN DALE YOUNG
*BLEISCH, WILLIAM VIRGIL CONDON, TIMOTHY COQUELIN, ARTHUR DAVID, WILLIAM S. EPEL, DEBRA LYNN FURBISH, DEAN RUSSELL HOLZ, GEORGE G., Ill
NEURAL SYSTEMS AND BEHAVIOR
HURLBERT, ANYA HURWITZ, JODIE LINDA JAVITT, DANIEL C. KUWADA, JOHN *LEVY, DANIEL JONATHAN MARCOTTE, RONALD R. MCMANUS, MARY ELLEN
54
ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
MEHLER, BRUCE LEO * MILLER, MARK W.
MOISEFF, ANDREW
OLSON, LEAH M.
ORR, DOMINIC PING-VIM *READY, NEAL EDWARD
ROSE, BARRY *ROSE, ROBERT D.
ROSELLI, CHARLES EUGENE
SCHESSEL, DAVID SCHILDBERGER, KLAUS
*SCHMITT, BRIAN C.
*SOLON, MICHAEL H. TANELIAN, DARRELL LEE TOMPKINS, LAURIE
*TREVARROW, WILLIAM W. TURNER, JEFFREY SCOTT WEI, AGUAN DANIEL
EXPERIMENTAL MARINE BOTANY
ASHENDORF, DfiBRA S.
BERENBERG, CATHY Jo BOCZAR, BARBARA A. *DOLYAK, BARBARA LORRAINE DUNLAP, JULIE J. FAVREAU, MITCHELL R. FITTER, MINDY SUE
"GRAVES, JOSEPH L., JR. JOHNSON, PATRICIA A. KAUFMAN, LON MONAR, KENNETH PEARCE, ROBERT ELLIOTT SHELINE, JONATHAN LEE WHITTAKER, CARLA J.
NEUROBIOLOGY
DODD, JANE FUKADA, KEIKO GARCIA-ARRARAS, JOSE E. LEMOS, JOSE RAMON MARCEY, DAVID JAMES PINE, JEROME
ROOF, DOROTHY J.
SCHNEIDERMAN, ANNE M.
SEJNOWSKI, TERRENCE J. STOCKBRIDGE, NORMAN LANDER THOMAS, WILLIAM ERIC YODLOWSKI, MARILYN L.
PHYSIOLOGY
BARR, ALAN HOWARD *BECKWITH, KIRK D.
BEHER, MICHAEL GARY
CARMAN, ALICE BLANCHE
CHANG, JANE
COLLINS, ELIZABETH T. *COLLINS, MARY
COOPER, JOHN A. *CUTLER, MIRIAM RUTH *DAYTON, ANDREW IMBRIE *GERSHON, NAHUM DAVID *GRIFO, JAMES ANTHONY
HINTZE, T.
JAFFE, LAURINDA ANN
LIBBER, MICHAEL R.
MARQUEZ-STERLING, NUMA R.
MATTSON, JOAN CRAWFORD *McCARTHY, ROBERT ALAN
*MULHOLLAND, JOYCE L *OTTER, TIMOTHY
*PlCKETT, CECIL
*PINE, RICHARD IRA *PROFFITT, JOHN HOUSTON *RAUSCH, STEVEN *REBERS, JOHN ERIC *ROBINSON, MARGARET S. *SELICK, HAROLD E. *SHAHIN, ROBERTA D. SULLIVAN, CHARLES HENRY, II *SULLIVAN, SUSAN JEAN *SUPRENANT, KATHY ANN *TRAVIS, JEFFREY L. *Wooo, STEVEN CHENAULT *Wu, CHAO-TING *WYBAN, JAMES ALLEN
*AMY, PENNY S. *BELLAS, CHRISTINE MARIE *GOLDFARB, DAVID SCOTT GRAHAM, JULIA BELL *KASTER, ALLAN GERARD
MICROBIAL ECOLOGY
PASTER, BRUCE JAY POTRIKUS, CATHERINE J. REED, WILLIAM MICHAEL SISCOE, PEGGY JEAN WARD, BESS B.
REPORT OF THE DIRECTOR
55
January Programs 1978 BEHAVIOR
ASHKENAS, LINDA R. BENI, JANINE E. BROWN, ADOLF MAXIMILIAN BRYANT, BRUCE P. CLARK, STEPHEN J. DERBY, CHARLES DORSETT DUWALDT, SARAH ELGIN, RANDALL HULL HERNANDEZ, TERESA
JONES, MAURICE, JK. MILLER, DEBORAH LYNN OBIN, MARTIN S. POETHKE, DAVID JOHN RAGLAND, HARRY CRAIG STRATTON, ANDREA WOOD WILLIAMS, ISABELLE P. WOODBURY, PATRICK B.
DEVELOPMENTAL BIOLOGY
BARBACCI, JOSEPH J., JR. BRODELL, GEORGE KLINE BRYAR, BETSY A. BURKE, DANIEL J. EVARTS, JONATHAN HAROLD FORD, WILLIAM C. GILLESPIE, LAURA LEE GOLD, ALBERT MARK GUSTAFSON, KIRK ROBERT HOLCOMB, CHERIE HOWELL, MARGARET A. MCGIMSEY, WILLIAM CLAYWELL
ABOYA, ELLEN GWEN BEHL, ANN BERGANTZ, JAMES M. BIXLER, ROBERT P.
COUPARD, MlCHELE
DEPELTEAU, AUDREY MARIE DETWILER, RALPH PAUL FARRELL, THOMAS A. FELDMAN, IRA ROBERT GREENE, GEORGE D. GROFF, JOSEPH MATTHEW HOCHBERG, ANN PATRICIA KELLEY, JULIA CROCKER
AABY, TRYGVE GENE APATAOFF, BRIAN R. BAGGOTT, BRIAN BURKE BODENSTEIN, LAWRENCE E. CARR, CATHERINE EMILY CHU, KA Hou DILLSAVER, MARGARET DOLBER, PAUL CHRISTIAN GARBER, NANCY D. HABER, MICHELE A. HEINEKE, ERIC WAYNE HIGGINS, GERALD ARTHUR JAMES, DEMETRIA ELIZABETH KENT, KARLA S.
MCMANUS, GERALDINE A. MANNING, MICHAEL SCOTT MILANI, SUSAN PATTERSON, NANCY PERRY, GAIL E. RHINEHART, MARY ELLEN RIDDELL, DEBORAH CHRISTIE SMILEY, LAURA ELLEN TOTH, LESLIE ELLEN TURNBULL, SARAH W. VOWELL, JOANNA LYNN
ECOLOGY
KELLOGG, DEAN LUNDT, JR. LARSON, WENDELIN MAP. LAWRENCE, VIKI ANN LEAMAN, DANNA Jo MACFARLANE, LINDA LOUISE MACWrHORTER, SUSAN E. McGowAN, MICHAEL F. NAUGHTON, DARLENE A. PERFETTI, PATRICIA ANN STEELE, PAMELA CURTIS THEBY, JANET ELAINE WALKER, SHARON LESLIE
NEUROBIOLOGY
KRASNOW, MARK ALAN LOPEZ, JOSE R. MACHLIS, LEE ELLEN MORNAY, SHARON MARIE MOULTON, JOHN FREEMAN, 1 1 1 OGONOWSKI, MICHAEL M. STEINBERG, ALAN B. VALENTICH, JOHN D. VERRETT, JOYCE M. WEINBERG, BRADLEY ADAM RIND, JEFF SLADOVICH, HEDY E. FEIGENBAUM, DAVID L.
56 ANNUAL RH'ok'l OF THE MARINE BIOLOGICAL LABORATORY
COMPARATIVE PATHOLOGY OF MARINE INVERTEBRATES
Bt RNS, CARY UEWrn CARDENOSA, GILDA CARLSON, GEORGE A. CHORNEY, MICHAEL J. COOPER, KEITH RAYMOND COUCH, DAVID ROBERT DEAKINS, LYNN WHEELER GOLDBERG, HARRY R. GULKA, GARY J. HEATFIELD, BARRY MARK KELLER, THOMAS EARL
KEYT, BRUCE ALAN LOGUE, MAUREEN UENISK LOMBARDI, ROSEMARIE MILLER, TIMOTHY K. MULVEY, MARGARET E. NEWMAN, MICHAEL C. SEIFERT, RONALD A.
SONSTEGARD, RONALD A.
SYPEK, JOSEPH P. WALLACE, JAMES L. YEATON, ROBIN L. W.
BOSTON UNIVERSITY MARINE PROGRAM
AARONSON REBECCA ASHKENAS, LINDA BESSE, SHEILA BOTERO, LEONOR BRYANT, BRUCE BUCHSBAUM, ROBERT COLE, JAMES COLE, TIMOTHY CUSHMAN, MARY DAVIS, CABELL DERBY, CHARLES DOJIRI, MASAHIRO DOURDEVILLE, THEODORE DUNCAN, THOMAS EDER, SUSAN EISEN, JUDITH FABRIZIO, MARY FOREMAN, KENNETH FRENCH, KATHLEEN GIBLIN, ANNE
GIBSON, DANIEL HILL, RUSSELL HOWES, BRIAN JORDAN, THOMAS KENT, KARLA KIPP KATRINA KOLBA, CLIFFORD KOUMJIAN, LAUREN LANGBAUER, WILLIAM MACIOLEK, NANCY OGONOWSKI, MARK PASCOE, NATALIE PIOTROWSKI, MICHAEL POOLE, ALAN REID, ROBERT WALTHALL, WALTER WERME, CHRISTINE WIER, SUSAN WILLIAMS, ISABELLE WILSON, JOHN
Spring 1978
WILLIAM B. BOWDEN SARAH M. MILLS JAMES T. MORRIS JAMES P. REED MARK N. WHITE
YEAR-IN-SCIENCE
Summer 1978
WILLIAM B. BOWDEN AMY FRIEDLANDER RICHARD GRANT JAMES T. MORRIS JAMES P. REED MARK N. WHITE
Winter 1978
WILLIAM B. BOWDEN MICHAEL S. MANNING CHARLES P. MCCLAUGHERTY JAMES T. MORRIS JAMES L. OLDS JAMES P. REED
Bio Club:
Gary H. Calkins:
Lucretia Crocker : Art hur Klorfcin :
3. SCHOLARSHIPS, 1978 MARY ELLEN McMANus
DOMINIC ORR ROBERT ROSE
BARBARA BOCZAR DAVID KIRCHMAN
WILLIAM BLEISCH DANIEL LEVY
REPORT OF THE DIRECTOR 57
TOSHIO NAGANO NEAL READY ROBERT ROSE CHRISTOPHER TAYLOR
Josiah Macy, Jr. : January Summer
SHARON MORNAY CKCIL PICKETT
JOYCE \"ERRETT PATRICIA CHASE
WILLIAM FORD CAROL BAGNELL
MARGARET HOWELL PATRICIA JOHNSON-
WILLIAM THOMAS JOSEPH GRAVES, JR. PAUL SANDRIDGE CARLA WHITTAKER Society of General
Physiologists: JOHN COOPER
MIRIAM CUTLER ANDREW DAYTON
4. TABULAR VIEW OF ATTENDANCE, 1974-1978
1974 1975 1970 1977 1978
|
\\\ \' STIGATORS — TOT-\L |
508 |
511 |
535 |
501 |
522 |
|
Independent • . . . |
302 |
301 |
312 |
280 |
268 |
|
Library Readers |
75 |
81 |
93 |
82 |
106 |
|
Research Assistants |
131 |
129 |
130 |
139 |
148 |
|
STUDENTS — TOTAL |
158 |
212 |
249 |
212 |
250 |
Summer Courses
Ecology.. 14 18 18
Embryology. 21 24
Experimental Invertebrate Zoology. . 30 34 36 33
Experimental Marine Botany. 11 14 16 16 14
Marine Ecology. ... 19
Neural Systems and Behavior. 31
Neurobiology. 12 12 12 12 12
Physiology. 40 33 41 34 34
January Courses
Behavior. 17 20 17
Biosphere 17
Comparative Pathology of Marine Invertebrates. . .
Developmental Biology. . 30 20 32 14 21
Ecology. 29 17
Neurobiology. . 23 24 27
TRAINEES— TOTAL. . 41 31 9 10 11
TOTAL ATTENDANCE.. 707 754 793 723 783
Less persons represented in two categories. . 00 1 00
707 754 792 723 783
INSTITUTIONS REPRESENTED — TOTAL. . 222 237 234 226 196
FOREIGN INSTITUTIONS REPRESENTED. . 31 26 33 33 30
5. INSTITUTIONS REPRESENTED, 1978
Albert Einstein College of Medicine Boston College
American Museum of Natural History Boston University
Amherst College Boston LIniversity School of Medicine
Baylor College Brandeis University
58
ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
Brookhaven National Laboratory
Brooklyn College, The City University of New
York
Brown University Bryn Mawr College California Institute of Technology California, University of, Davis California, University of, Irvine California, University of, Los Angeles California, University of, San Diego California, University of, Santa Barbara Carnegie Institution of Washington Carnegie-Mellon University Case Western Reserve University Case Western Reserve University, Medical
School
Chicago, University ot Cincinnati, University of College of Mount St. Joseph on the Ohio Colorado, University of Columbia University Columbia University, College of Physicians
and Surgeons
Columbia University, School of Dentistry Connecticut, University of Connecticut, University of, Health Center Cornell University Cornell University Medical College Dartmouth College Delaware, University of Denison University Dillard University Duke University Duke University Medical Center Eastern Pennsylvania Psychiatric Institute Emory University Florida, University of Florida State University General Electric Company Goucher College Grambling State University Grass Foundation Greenville College Hahnemann Medical College Hampshire College Hartford, University of Harvard Medical School Harvard University Hawaii, University of Herbert Lehman College, The City University
of New York Howard University
Howard University, College of Medicine Hunter College, The City University of New
York
Illinois, University of
Illinois, University of, College of Medicine Illinois Institute of Technology
Institute for Basic Re-search in Mental Re- tardation
Institute for Cancer Research, The
Iowa, University of
Iowa State University
Jersey City State College
John B. Pierce Foundation Laboratory
Johns Hopkins University, The
Johns Hopkins University, The School of Hygiene
Johns Hopkins University, The School of Medicine
Kansas, University of
Kent State University
Kentucky, University of
Kresge Eye Institute
Laboratory of Biophysics, NINCDS NIH
Louisiana State University
Louisiana State University Medical Center
Maine, University of, Orono
Manchester Community College
Maryland, University of
Maryland, University of, School of Medicine
Massachusetts, University of
Massachusetts General Hospital
Massachusetts Institute of Technology
Miami, University of
Miami, University of, School of Medicine
Millersville State College
Minnesota Department of Health
Minnesota, University of
Miriam Hospital, The
Mount Holyoke College
Montefiore Hospital & Medical Center
Moravian College
National Institute of Mental Health, NIH
National Naval Medical Center
New Jersey Medical School
New Mexico, University of, School of Medicine
New York University
New York University Medical Center
New York University School of Medicine
North Carolina, University of, at Chapel Hill
North Carolina State University, Raleigh
Northeastern Illinois University
Northwestern University
Notre Dame, University ot
Oakland University
Oberlin College
Ohio State University
Ohio University
Oregon, University of
Oregon State University
Pennsylvania, University of, School of Medicine
Pennsylvania, University of, School of Vet- erinary Medicine
Pennsylvania State University
Pittsburgh, University of
Population Council, The
REPORT OF THE DIRECTOR
59
Princeton University
Public Health Research Institute of The City
of New York, Inc. Purdue University Michael Reese Hospital Rensselaer Polytechnic Institute Research Institute on Alcoholism Rhode Island, University of Rice University Rochester, University of Rochester, University ot, School of Medicine
& Dentistry
Rockefeller University, The Rush Medical College
Rutgers — The State University of New Jersey Rutgers University Medical School San Jose State University South Florida, University of Stanford University
Stanford University School of Medicine State University of New York, Downstate
Medical Center State University of New York, Upstate Medical
Center
State University of New York at Albany State University of New York at Binghamton State Universitv of New York at Buffalo
State University of New York at Stony Brook
Suffolk University
Swarthmore College
Syracuse University
Temple University Medical School
Texas Christian University
Texas Southern University
Texas, University of, Austin
Texas, University of. Medical Branch
Trinity College
Tulane University
Union Carbide Corporation
Vanderbilt University
Vassar College
Veterans' Administration Hospital, Brooklyn
Virginia, University of
Virginia, University of, School of Medicine
Washington University School of Medicine
Wayne State University
Wesleyan University
Wisconsin, University of
Wistar Institute
Woods Hole Oceanographic Institution
Yale University
Yale University School of Medicine
Yeshiva Universitv
FOREIGN INSTITUTIONS REPRESENTED, 1978
Aarhus, University of, Denmark
Alberta, University of, Canada
Athens, University of, Medical School, Greece
Basel, The University of, Biocenter, Switzer- land
Bergen, University of, Norway
Bermuda Biological Station
Calgary, University of, Canada
Cambridge, University of, England, U.K.
Centre National de la Recherche Scientifique, France
Centre de Biofisica Y Bioquimica, Caracas, Venezuela
Chiba University, Japan
College de France, Paris, France
Free University, Berlin, Germany
Guadalajara, University of, Mexico
Hebrew University, The, of Jerusalem, Israel
Hiroshima University, Japan
Imperial College of Science & Technology,
London, England, U.K. Instituto Venezolano de Investigaciones,
Venezuela
King's College, London, England, U.K. Laval, Universite, Canada Max Planck Institute for Psychiatry, Munich,
West Germany
Memorial University of Newfoundland, Canada National Cancer Center Research Institute,
Japan
North Wales, University of, Wales, U.K. Ottawa, University of, Canada Puerto Rico, LTniversity of Toronto, University of, Canada LIniversity College, London, England, U.K. University College, University of Dublin,
Ireland Wales, LTniversity of, Bangor, U.K.
ft. FRIDAY EVENING LECTURES, 1978
June 30
J. RICHARD YV'HITTAKER. The Wistar Institute
Egg cytoplasmic determinants of tissue differ- entiation
60 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
July 7
BEATRICE MINT/ Putting mutant genes into mice
Institute for Cancer Research Ninetieth Anniversary Lecture
July 13
Thursday
KRESIMIR KRNJEVIC Intraneuronal factors that influence excitability
McGill University 1 ) Regulation of neuronal tiring by internal Ca2+
Alexander Forbes Lecturer at the MBL
July 14
KRESIMIR KRNJEVIC 2) Intracellular actions of synaptic transmitters
July 21
DANIEL MAZIA Mitotic center^
University of California, Berkeley
July 2«
ROBERT H. BURRIS Omnipresent \2 — how organisms convert it to
University of Wisconsin NHs
August 4
STEPHEN JAY GOULD Ontogeny and phylogeny
Harvard University
August 11
HARRIS RIPPS A ray of light on night blindness
New York University Medical Center
August 18
ROBERT H. \\ HITTAKER Theory of species diversity
Cornell University
August 25
DANIEL ALKON A neural basis for associative learning
Laboratory of Biophysics, NINCDS, at the MBL
REPORT OF THE DIRECTOR 61
7. MEMBERS OF THE CORPORATION, 1978
Including Ad ion of 1978 Annual Meeting
Life Members
ADOLPH, DR. EDWARD F., University of Rochester, School of Medicine and
Dentistry, Rochester, New York 14627 BEAM, DR. HAROLD W., Department of Zoology, State University of Iowa,
Iowa City, Iowa 52240
BEHRE, DR. ELLINOR H., Black Mountain, North Carolina 28711 BERTHOLF, DR. LLOYD M., 307 Phoenix Ave., Bloomington, Illinois 61701 BOLD, DR. HAROLD C., Department of Botany, University of Texas, Austin,
Texas 78712
BRIDGMAN, DR. A. JOSEPHINE, 715 Kirk Rd., Decatur, Georgia 30030 BROWN, DR. DUGALD E. S., Cape Haze, Box 426, Placida, Florida 33946 BURDICK, DR. C. LALOR, The Lalor Foundation, 4400 Lancaster Pike, Wilming- ton, Delaware 19805
CARPENTER, DR. RUSSELL L., 60 Lake St., Winchester, Massachusetts 01890 CHASE, DR. AURIN, Professor of Biology, Emeritus, Princeton University.
Princeton, New Jersey 08540
CLARKE, DR. GEORGE L., 44 Juniper Road, Belmont, Massachusetts 02178 CLEMENT, DR. ANTHONY C., Department of Biology, Emory University, Atlanta,
Georgia 30322
COLE, DR. KENNETH S., 2404 Loring Street, San Diego, California 92109 COLWIN, DR. ARTHUR L., 320 Woodcrest Rd., Key Biscayne, Florida 33149 COLWIN, DR. LAURA H., 320 W^oodcrest Rd., Key Biscayne, Florida 33149 COSTELLO, DR. HELEN M., Morgan Rd., Wroods Hole, Massachusetts 02543 CROUSE, DR. HELEN V., Institute of Molecular Biophysics, Florida State Uni- versity, Tallahassee, Florida 32306
DILLER, DR. IRENE C., 2417 Fairhill Avenue, Glenside, Pennsylvania 19038 DILLER, DR. WILLIAM F., 2417 Fairhill Avenue, Glenside, Pennsylvania 19038 ELLIOTT, DR. ALFRED M., P.O. Box 564, Woods Hole, Massachusetts 02543 FERGUSON, DR. JAMES K. W., 56 Clarkson St., Thornhill, Ontario, Canada FISCHER, DR. ERNST, 3110 Manor Drive, Richmond, Virginia 23230 FRIES, DR. ERIK F. B., 3870 Leafy Way, Miami, Florida 33133 GAFFRON, DR. HANS, P.O. Box 308, Sanibel, Florida 33959
GALTSOFF, DR. PAUL S., National Marine Fisheries Service, Woods Hole, Massa- chusetts 02543 GRAY, DR. IRVING E., Department of Zoology, Duke University, Durham, North
Carolina 27701 GRUNDFEST, DR. HARRY, Department of Neurology, Columbia University,
College of Physicians and Surgeons, New York, New York 10032 HAMBURGER, DR. VIKTOR, Department of Zoology, Washington University, St.
Louis, Missouri 63130 HAMILTON, DR. HOWARD L., Department of Biology, University of Virginia,
Charlottesville, Virginia 22901 HARTLINE, DR. H. KEFFER, The Rockefeller University, New York, New York,
10021 HiHHARt), T)K. HOFM-. 143 1C. College Si.. Apt. 309. Oberlin. Ohio 44074
62 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
HISAW, DR. F. L., 5925 S. W. Plymouth Drive, Corvallis, Oregon 97330 HOLLAENDER, DR. ALEXANDER, Associated University, Inc., 1717 Massachusetts
Ave., N. W., Washington, D. C. 20036 HuGHES-ScHRADER, DR. SALLY, Department of Zoology, Duke University,
Durham, North Carolina 27706
IRVING, DR. LAURENCE, University of Alaska, College, Alaska 99701 JOHNSON, DR. FRANK H., Department of Biology, Princeton University, Prince- ton, New Jersey 08540
KAAN, DR. HELEN, 62 Locust St., Apt. 244, Falmouth, Massachusetts 02540 KAHLER, ROBERT, P.O. Box 423, Woods Hole, Massachusetts 02543 KILLE, DR. FRANK R., 500 Osceola Ave., Winter Park, Florida 32789 KLEINHOLZ, DR. LEWIS, Department of Biology, Reed College, Portland, Oregon
97202
LOCHHEAD, DR. JOHN H., 49 Woodlawn Rd., London, SW6 6PS, England U.K. LYNN, DR. W. GARDNER, Department of Biology, Catholic University of America,
Washington, D. C. 20017
MAGRUDER, DR. SAMUEL R., 270 Ceder Lane, Paducah, Kentucky 42001 MALONE, DR. E. P., 6610 North llth Street, Philadelphia, Pennsylvania 19126 MANWELL, DR. REGINALD D., Department of Biology, Syracuse University,
Syracuse, New York 13210
MARSLAND, DR. DOUGLAS, 3523 Loquat Ave., Miami, Florida 33133 MILLER, DR. JAMES A., Department of Anatomy, Tulane University, New
Orleans, Louisiana 70112 MILNE, DR. LORUS J., Department of Zoology, University of New Hampshire,
Durham, New Hampshire 03824
MOUL, DR. E. T., 42 F. R. Lillie Rd., Woods Hole, Massachusetts 02543 NICOLL, DR. PAUL A., 6636 E. Street Rd. 46, Bloomington, Indiana 47401 PAGE, DR. IRVING H., Cleveland Clinic, Euclid at E. 93rd Street, Cleveland, Ohio
44106
PLOUGH, DR. HAROLD H., 31 Middle Street, Amherst, Massachusetts 01002 POLLISTER, DR. A. W., Box 23, Dixfield, Maine 04224 POND, SAMUEL E., P.O. Box 63, E. Winthrop, Maine 04343 PORTER, DR. H. C., University of Pennsylvania, Philadelphia, Pennsylvania 19174 PRYTZ, DR. MARGARET McD., 21 McCouns Lane, Oyster Bay, New York 11771 REZNIKOFF, Dr. PAUL, 11 Brooks Rd., Wroods Hole, Massachusetts 02543 RICHARDS, DR. A. GLENN, Department of Entomology, UJniversity of Minnesota,
St. Paul, Minnesota 55101
RICHARDS, DR. OSCAR W., Pacific University, Forest Grove, Oregon 97116 SCHARRER, DR. BERTA, Department of Anatomy, Albert Einstein College of
Medicine, 1300 Morris Pkwy., New York, New York 10461 SCHMITT, DR. F. O., 165 Allen Dale St., Jamaica Plain, New York 02130 SEVERINGHAUS, DR. AURA E., 375 West 250th Street, New York, New York 10071 SHEMIN, DR. DAVID, Department of Biochemistry and Molecular Biology,
Northwestern University, Evanston, Illinois 60201
SICHEL, DR. ELSA K., 4 \Vhitman Rd., Woods Hole, Massachusetts 02543 SMITH, DR. DIETRICH C., 216 Oak Forest Ave., Catonsville, Maryland 21228 SONNEBORN, DR. T. M., Department of Zoology, Indiana University, Blooming- ton, Indiana 47401
REPORT OF THE DIRECTOR 63
SPEIDEL DR. CARL ('., 1873 Field Road, Chariottesville, Virginia 22903 STEINBACH, DR. H. BURR, one Bell Tower Lane, Woods Hole, Massachusetts
02543 STRAUS, DR. W. L., JR., Department of Anatomy, The Johns Hopkins University
Medical School, Baltimore, Maryland 21205 STUNKARD, DR. HORACE W., American Museum of Natural History, Central
Park West at 79th Street, New York, New York 10024 TAYLOR, DR. W. RANDOLPH, Department of Botany, University of Michigan,
Ann Arbor, Michigan 48109
TEW'INKEL, DR. Lois E., 4 Sanderson Ave., Northampton, Massachusetts 01060 WALD, DR. GEORGE, Higgins Professor of Biology, Emeritus, Harvard University,
Cambridge, Massachusetts 02138
WALLACE, DR. LOUISE B., 359 Lytton Ave., Palo Alto, California 94301 WARREN, DR. HERBERT S., % Leland C. Warren, 721 Conshohocken State Road,
Penn Valley, Pennsylvania 19072 WEISS, DR. PAUL, The Rockefeller University, 66th St. and York Ave., New
York, New York 10016
WHITING, DR. ANNA R., Woods Hole, Massachusetts 02543 WICHTERMAN, DR. RALPH, 31 Buzzards Bay Ave., \Voods Hole, Massachusetts
02543
YOUNG, DR. D. B., Main Street, North Hanover, Massachusetts 02357 ZINN, DR. DONALD J., P.O. Box 589, Falmouth, Massachusetts 02541 ZORZOLI, DR. ANITA, Department of Biology, Vassar College, Pou^hkeepsie,
New York 12601
Regular Members
ABBOTT, DR. MARIE B., High Street, Coventry, Connecticut 06238
ACHE, DR. BARRY W7., Department of Biological Sciences, Florida Atlantic
UJniversity, Boca Raton, Florida 33432
ACHESON, DR. GEORGE H., 25 Quissett Ave., Woods Hole, Massachusetts 02543 ADELBERG, DR. EDWARD A., Department of Microbiology, Yale University
Medical School, New Haven, Connecticut 06510
AFZELIUS, DR. BJORN, Wenner-Gren Institute, University of Stockholm, Stock- holm, Sweden ALKON, DR. DANIEL, Laboratory of Biophysics, Marine Biological Laboratory,
Woods Hole, Massachusetts 02543 ALLEN, DR. GARLAND E., Biology Department, Washington University,
St. Louis, Missouri 63130 ALLEN, DR. NINA S., Department of Biology, Dartmouth College, Hanover,
New Hampshire 03755 ALLEN, DR. ROBERT D., Chairman, Department of Biology, Dartmouth College,
Hanover, New Hampshire 03755 ALSCHER, DR. RUTH, Department of Biology, Manhattanville College, Purchase,
New York 10577
AMATNIEK, ERNEST, 4797 Boston Post Rd., Pelham Manor, New York 10803 ANDERSON, DR. EVERETT, Department of Anatomy and Laboratories of Human
Reproductive Biology, Harvard Medical School, Boston, Massachusetts 02115
64 \\\T.\I k'KI'OKT OK THE MARINE i:i< >[.<>< ilCAL LABORATORY
ANDERSON, DK. J. lM., Division of Biological Sciences, Emerson Hall, Cornell I'liiversity, Ithaca, New York 14X5.*
ARMSTRONG, Du. CLAY M., Department of Physiology, University of Pennsyl- vania, School of Medicine, Philadelphia, Pennsylvania 10174
ARMSTRONG, DR. PETER B., Department of Zoology, University of California, Davis, California 95616
ARMSTRONG, DR. PHILLIP B., 51 Elliot Place, Rutherford, New Jersey 07070
ARNOLD, DR. JOHN MILLKR, Kewolo Marine Lab., Pacific Biomedical Research Center, 41 Ahui St., Honolulu, Hawaii 96813
ARNOLD, DR. WILLIAM A., Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830
ATEMA, DR. JELLE, 9 Millfield St., Woods Hole, Massachusetts 02543
ATWOOD, DR. KIMBALL C., 100 Haven Ave., Apt. 21 -E, New York, New York 10032
AUSTIN, DR. MARY L., 506^ North Indiana Avenue, Bloomington, Indiana 47401
BACON, ROBERT, Church Street, Woods Hole, Massachusetts 02543
BALDWIN, DR. THOMAS O., Department of Biochemistry, University of Illinois, Urbana, Illinois 61801
BALL, DR. ERIC G., P.O. Box 406, Falmouth, Massachusetts 02541
BANG, DR. BETSY, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21205
BANG, DR. F. B., Department of Pathobiology, The Johns Hopkins University School of Hygiene, Baltimore, Maryland 21205
BARKER, DR. JEFFERY L., Bldg. 36, Room 2002, National Institutes of Health, Bethesda, Maryland 20014^
BARLOW, DR. ROBERT B., JR., Institute for Sensory Research, .Syracuse Uni- versity, Merrill Lane, Syracuse, New York 13210
BARTELL, DR. CLELMER K., Department of Biological Sciences, Louisiana State University, New Orleans, Louisiana 70113
BARTH, DR. LUCENA J., Marine Biological Laboratory, Woods Hole, Massa- chusetts 02543
BARTLETT, DR. JAMES H., Department of Physics, University of Alabama, P.O. Box 1921, University, Alabama 35486
BAUER, DR. G. ERIC, Department of Anatomy, University of Minnesota, Minne- apolis, Minnesota 55414
BEAUGE, DR. Luis ALBERTO, Department of Biophysics, University of Maryland School of Medicine, 660 W. Redwood St., Baltimore, Maryland 21201
BECK, DR. L. V., DEPARTMENT of Pharmacology, Indiana University, School of Experimental Medicine, Bloomington, Indiana 47401
BELL, DR. EUGENE, Department of Biology, Massachusetts Institute of Tech- nology, Cambridge, Massachusetts 02139
BENNETT, DR. MICHAEL V. L., Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Ave., New York, New York 10461
BENNETT, DR. MIRIAM F., Department of Biology, Colby College, Waterville, Maine 04901
BERG, DR. CARL J., JR., Marine Biological Laboratory, Woods Hole, Massa- chusetts 02543
REPORT OF THE DIRECTOR 65
HERMAN, DR. MONES, National Institutes of Health, Theoretical Biology NCI, Bldg. 10, 4B56, Bethesda, Maryland 20014
BERNARD, GARY D., Department of Ophthalmology and Visual Science, Yale University, 333 Cedar St., New Haven, Connecticut 06510
BERNE, DR. ROBERT \V., University of Virginia School of Medicine, Charlottes- ville, Virginia 22903
BERNHEIMER, DR. ALAN \Y., New York University College of Medicine, New York, New York 10016
BIGGERS, DR. JOHN DENNIS, Department of Physiology, Harvard Medical School, 25 Shattuck St., Boston, Massachusetts 02115
BISHOP, DR. DAVID \\'., Department of Physiology, Medical College (A Ohio, P.O. Box 6190, Toledo, Ohio 43614
BISHOP, DR. STEPHEX H., Department of Zoology, Iowa State University, Ames, Iowa 50010
BLAUSTEIN, MORDECAI P., Department of Physiology and Biophysics, Washing- ton University School of Medicine, St. Louis, Missouri 63110
BLUM, DR. HAROLD F., 612 E. Durham St., Philadelphia, Pennsylvania 19119
BODIAN, DR. DAVID, Department of Otolaryngology, The Johns Hopkins Uni- versity, Traylor Building, Room 424, 1721 Madison St., Baltimore, Maryland 21205
BOETTIGER, DR. EDWARD G., Department of Zoology, University of Connecticut, Storrs, Connecticut 06268
BOOLOOTIAN, DR. RICHARD A., President, Science Software System, Inc., 11899 West Pico Blvd., Los Angeles, California 90064
BOREI, DR. HAXS G., Department of Zoology, University of Pennsylvania, Philadelphia, Pennsylvania 19174
BORGESE, DR. THOMAS A., Department of Biology, Lehman College, City LTni- versity of New York, Bronx, New York 10468
BORISV, DR. GARY G., Laboratory of Molecular Biology, University of Wis- consin, Madison, Wisconsin 53715
BOSCH, DR. HERMAN F., % F. Taylor, Box 147, South Berwick, Maine 03908
BOTKIN, DR. DANIEL B., Wilson Center, Smithsonian Institution, Washington, D. C. 20560
BOWEN, DR. VAUGHN T., Woods Hole Oceanographir Institution, Redfield Bldg. 3-32, \Voods Hole, Massachusetts 02543
BOWLES, DR. FRANCIS P., Marine Biological Laboratory, \Voods Hole, Massa- chusetts 02543
BRANDT, DR. PHILIP WILLIAMS, Department of Anatomy, Columbia University, College of Physicians and Surgeons, New York, New York 10032
BRINLEY, DR. F. J., JR., Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201
BRODERICK, DR. MALCOLM S., Department of Physiology and Biophysics, Uni- versity of Texas Medical Branch, Galveston, Texas 77550
BROOKS, DR. MATILDA M., Department of Physiology, Lhiiversity of California, Berkeley, California 94720
BROWX, DR. FRANK A., JR., Department of Biological Sciences, Northwestern University, Evanston, Illinois 60201
66 AXNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
BROWN, DR. JOEL E., Department of Physiology and Biophysics, Bldg. E, State
University of New York at Stony Brook, Stony Brook, New York 11794 BUCK, DR. JOHN B., Laboratory of Physical Biology, National Institutes of
Health, Bethesda, Maryland 20014
BURBANCK, DR. MADELINE PALMER, Box 15134, Atlanta, Georgia 30333 BURBANCK, DR. WILLIAM D., Box 15134, Atlanta, Georgia 30333 BURDICK, DR. CAROLYN J., Department of Biology, Brooklyn College, Brooklyn,
New York 11210 BURGER, DR. MAX M., Department of Biochemistry, University of Basel, CH.
4056-Klingelbergstrasse 70, Basel, Switzerland BURKY, DR. ALBERT J., Department of Biology, University of Dayton, Dayton,
Ohio 45469
BURR, DR. ARTHUR H., Department of Biological Sciences, Simon Eraser Uni- versity, Burnaby, British Columbia, Canada V5A 1S6 CANDELAS, DR. GRACIELA C., Department of Biology, University of Puerto Rico,
Rio Piedras, Puerto Rico 00931 CARLSON, DR. ERANCIS D., Department of Biophysics, The Johns Hopkins
University, Baltimore, Maryland 21218 CASE, DR. JAMES F., Department of Biological Sciences, University of California,
Santa Barbara, California 93106 CASSIDY, REV. JOSEPH, O.P., Department of Biological Sciences, Northwestern
University, Evanston, Illinois 60201 CEBRA, DR. JOHN J., Department of Biology, The Johns Hopkins University,
Baltimore, Maryland 21218
CHAET, DR. ALFRED B., University of West Florida, Pensacola, Florida 32504 CHAMBERS, DR. EDWARD L., Department of Physiology and Biophysics, Uni- versity of Miami School of Medicine, P.O. Box 52087, Biscayne Annex,
Miami, Florida 33152 CHAPPELL, DR. RICHARD L., Department of Biological Sciences, Hunter College,
The City University of New York, New York, New York 10021 CHAUNCEY, DR. HOWTARD H., 30 Falmouth St., \Vellesley Hills, Massachusetts
02181
CHENEY, DR. RALPH H., 11 Park Street, Woods Hole, Massachusetts 02543 CHILD, DR. FRANK M., Department of Biology, Trinity College, Hartford,
Connecticut 06106
CITKOWITZ, DR. ELENA, 410 Livingston St., New Haven, Connecticut 06511 CLARK, DR. A. M., Department of Biological Sciences, University of Delaware,
Newark, Delaware 19711 CLARK, DR. ELOISE E., National Science Foundation, 1800 G Street, Washington,
D. C. 20550 CLARK, HAYS, Executive Vice-President, Avon Products, Inc., 9 W7est 57th
Street, New York, New York 10019 CLARK, DR. WALLIS H., Aquaculture Program, College of Agricultural and
Environmental Sciences, 228 Mark Hall, University of California, Davis,
California 95616 CLAUDE, DR. PHILIPPA, Primate Center, Capital Court, Madison, Wisconsin,
53706
REPORT OF THE DIRECTOR 67
CLAYTON, DR. RODERICK K., Section of Genetics, Development and Physiology,
Cornell University, Ithaca, New York 14850 CLOWES, DR. GEORGE H. A., JR., The Cancer Research Institute, 194 Pilgrim
Road, Boston, Massachusetts 02215 CLUTTER, DR. MARY, Developmental Biology Program, National Science
Foundation, Washington, D. C. 20550
COBB, DR. JEWEL P., Dean, Douglass College, New Brunswick, New Jersey 08903 COHEN, DR. ADOLPH I., Department of Ophthalmology, Washington University,
School of Medicine, 660 S. Euclid Ave., St. Louis, Missouri 63110 COHEN, DR. LAWRENCE B., Department of Physiology, Yale University, 333
Cedar St., New Haven, Connecticut 06510
COHEN, DR. SEYMOUR S., Department of Pharmacological Science, State Uni- versity of New York at Stony Brook, Stony Brook, New York 11790 COHEN, DR. WILLIAM D., Hunter College, New York, New York 10021 COLLIER, DR. JACK R., Department of Biology, Brooklyn College, Brooklyn,
New York 11210 COOPERSTEIN, DR. SHERWIN J., University of Connecticut, School of Medicine,
Farmington Ave., Farmington, Connecticut 06032 CORLISS, DR. JOHN O., Department of Zoology, University of Maryland, College
Park, Maryland 20742
CORNELL, DR. NEAL W., 1914 Marthas Rd., Alexandria, Virginia 22307 CORNMAN, DR. IVOR, Townfield, Port Royal, Virginia 22535 COUCH, DR. ERNEST F., Department of Biology, Texas Christian University,
Fort Worth, Texas 76110
CRANE, JOHN O., 315 West 106th Street, New York, New York 10025 CREMER-BARTELS, DR. GERTRUD, Universitats Augenklinik, 44 Munster, West
Germany CRIPPA, DR. MARCO, Department de Biologic animale, Embryologie Moleculaire,
154 route de Malagnou, CH-1224, Chene-Bougeries, Geneve, Switzerland CROWELL, DR. SEARS, Department of Zoology, Indiana University, Bloomington,
Indiana 47401 DAIGNAULT, ALEXANDER T., W. R. Grace Co., 1114 Avenue of the Americas,
New York, New York 10036 DAN, DR. KATSUMA, Professor Emeritus, Tokyo Metropolitan University,
Meguro-ku, Tokyo, Japan DANIELLI, DR. JAMES F., Life Sciences Department, Worcester Polytechnic
Institute, Worcester, Massachusetts 01609 DAVIS, DR. BERNARD D., Bacterial Physiology Unit, Harvard Medical School,
25 Shattuck Street, Boston, Massachusetts 02115 DAW, DR. NIGEL W7., 78 Aberdeen PL, Clayton, Missouri 63105 DEGROOF, DR. ROBERT C., Department of Pharmacology, Thomas Jefferson
University, 1020 Locust St., Philadelphia, Pennsylvania 19174 DEHAAN, DR. ROBERT L., Department of Anatomy, Emory University, Atlanta,
Georgia 30322
DELANNEY, DR. Louis E., The Jackson Laboratory, Bar Harbor, Maine 04609 DEPHILLIPS, DR. HENRY A., JR., Department of Chemistry, Trinity College,
Hartford, Connecticut 06106
68 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
DETTBARN, DR. WOLF-DIETRICH, Department of Pharmacology, Vanderbilt
University, School of Medicine, Nashville, Tennessee 37217 DEWEER, DR. PAUL J., Department of Physiology, Washington University,
School of Medicine, St. Louis, Missouri 63110 DISCHE, DR. ZACHARIAS, Columbia University, Eye Institute, 630 W. 165th
Street, New York, New York 10032 DIXON, DR. KEITH E., School of Biological Sciences, Flinders University, Bedford
Park, South Australia DOWDALL, DR. MICHAEL J., Max Planck-Institut fur Biophysikalische Chemie,
D-3400 Gottingen, West Germany DOWLING, DR. JOHN E., Biological Laboratories, Harvard University, 16 Divinity
Avenue, Cambridge, Massachusetts 02138
DRESDEN, DR. MARC H., Department of Biochemistry, Baylor College of Medi- cine, Houston, Texas 77025 DUDLEY, DR. PATRICIA L., Department of Biological Sciences, Barnard College,
Columbia University, New York, New York 10027 DUNHAM, DR. PHILIP B., Department of Biology, Syracuse University, Syracuse,
New York 13210 EBERT, DR. JAMES DAVID, Carnegie Institution of Washington, 1530 P Street,
Northwest, Washington, D. C. 20008
ECKBERG, DR. WILLIAM R., Department of Zoology, Howard University, Wash- ington, D. C. 20059 ECKERT, DR. ROGER O., Department of Zoology, University of California, Los
Angeles, California 90024
EDDS, DR. KENNETH T., Box 348, Woods Hole, Massachusetts 02543 EDDS, DR. LOUISE, College of Osteopathic Medicine, Grosvenor Hall, Ohio
University, Athens, Ohio 45701 EDER, DR. HOWARD A., Albert Einstein College of Medicine, New York, New
York 10461 EDWARDS, DR. CHARLES, Department of Biological Sciences, State University
of New York at Albany, Albany, New York 12203 EGYUD, DR. LASZLO G., The Sonntag Institute For Cancer Research, Boston
College, Chestnut Hill, Massachusetts 02167 EHRENSTEIN, DR. GERALD, National Institutes of Health, Bethesda, Maryland
20014 EICHEL, DR. HERBERT J., Department of Biological Chemistry, Hahnemann
Medical College, Philadelphia, Pennsylvania 19174 EISEN, DR. ARTHUR Z., Division of Dermatology, Washington University,
School of Medicine, St. Louis, Missouri 63110 ELDER, DR. HUGH Y., Institute of Physiology, University of Glasgow, Glasgow,
Scotland, U. K. ELLIOTT, DR. GERALD F., The Open University Research Unit, Foxcombe Hall,
Berkeley Road, Boar Hill, Oxford, England, U. K.
EPEL, DR. DAVID, Hopkins Marine Station, Pacific Grove, California 93950 EPSTEIN, DR. HERMAN T., Department of Biology, Brandeis University, Wal-
tham, Massachusetts 02154
ERULKAR, DR. SOLOMON D., Department of Pharmacology, University of Penn- sylvania Medical School, Philadelphia, Pennsylvania 19174
REPORT OF THE DIRECTOR 69
ESSNER, DR. EDWARD S., Kresge Eye Institute, Wayne State University, School
of Medicine, 540 E. Canfield Ave, Detroit, Michigan 48201 ETIENNE, DR. EARL M., Department of Anatomy, Harvard Medical School,
Boston, Massachusetts 02115 FAILLA, DR. PATRICIA M., Office of the Director, Argonne National Laboratory,
Argonne, Illinois 60439
FARMAXFARMAIAN, DR. ALLAHVERDI, Department of Physiology and Biochem- istry, Rutgers University, New Brunswick, New Jersey 08903 FAUST, DR. ROBERT GILBERT, Department of Physiology, University of North
Carolina, Medical School, Chapel Hill, North Carolina 27514 FAWCETT, DR. D. W., Department of Anatomy, Harvard Medical School,
Boston, Massachusetts 02115 FEIN, DR. ALAN, Marine Biological Laboratory, Woods Hole, Massachusetts
02543 FERGUSON, DR. F. P., National Institute of General Medical Sciences, National
Institutes of Health, Bethesda, Maryland 20014 FESSENDEN, JANE, Librarian, Marine Biological Laboratory, Woods Hole,
Massachusetts 02543 FISCHBACH, DR. GERALD, Department of Pharmacology, Harvard Medical
School, 25 Shattuck St., Boston, Massachusetts 02115 FISHER, DR. J. M., Department of Biochemistry, University of Toronto, Toronto,
Ontario, Canada FISHMAX, DR. HARVEY M., Department of Physiology, University of Texas,
Medical Branch, Galveston, Texas 77550
FISHMAN, DR. Louis, 143 North Grove Street, Valley Stream, New York 11580 FLANAGAN, DENNIS, Editor, Scientific American, 415 Madison St., New York,
New York 10017 Fox, DR. MAURICE S., Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139 FRAENKEL, DR. GOTTFRIED S., Department of Entomology, University of Illinois,
Urbana, Illinois 61801 FRANZINI, DR. CLARA, Department of Biology G-5, University of Pennsylvania,
School of Medicine, Philadelphia, Pennsylvania 19174 FRAZIER, DR. DONALD T., Department of Physiology and Biophysics, University
of Kentucky, School of Medicine, Lexington, Kentucky 40507 FREEMAN, DR. ALLAN R., Professor and Chairman, Department of Physiology,
Temple University School of Medicine, 3420 N. Broad St., Philadelphia.
Pennsylvania 19140 FREEMAN, DR. GARY L., Department of Zoology, University of Texas, Austin,
Texas 78710
FRENCH, DR. ROBERT J., 17 Quissett Ave., Woods Hole, Massachusetts 02543 FREYGANG, DR. WALTER J., JR., 6247 29th Street, N. W., Washington, D. C.
20015 FULTON, DR. CHANDLER M., Department of Biology, Brandeis University,
Waltham, Massachusetts 02154 FURSHPAN, DR. EDWIN J., Department of Neurophysiology, Harvard Medical
School, Boston, Massachusetts 02115
70 ANNUAL REPORT OF THK MARINE BIOLOGICAL LABORATORY
I ISELER, DR. JOHN W., Department of Cell Biology, 5323 Harry Mines Blvd., University of Texas Medical Branch, Dallas, Texas 75235
FYE, DR. PAUL M., Woods Hole Oceanographic Institution, Woods Hole, Massa- chusetts 02543
GABRIEL, DR. MORDECAI L., Department of Biology, Brooklyn College, Brooklyn, New York 11210
GAINER, DR. HAROLD, Head, Section of Functional Neurochemistry, National Institutes of Health, Bldg. 36, Rm. 2A21, Bethesda, Maryland 20014
GALL, DR., JOSEPH G., Department of Biology, Yale University, New Haven, Connecticut 06520
GELFANT, DR. SEYMOUR, Department of Dermatology, Medical College of Georgia, Augusta, Georgia 30904
GELPERIN, DR. ALAN, Department of Biology, Princeton University, Princeton, New Jersey 08540
GERMAN, DR. JAMES L.. III. The New York Blood Center, 310 East 67th Street, New York, New York 10021
GIBBS, DR. MARTIN, Institute for Photobiology of Cells and Organelles, Brandeis University, Waltham, Massachusetts 02154
GIBSON, DR. A. JANE, Wing Hall, Cornell University, Ithaca, New York 14850
GIFFORD, DR. PROSSER, Dean, Amherst College. Amherst, Massachusetts 01002
GILBERT, DR. DANIEL L., Laboratory of Biophysics, NINCDS, National Institutes of Health, Building 36, Room 2A29, Bethesda, Maryland 20014
GILMAN, DR. LAUREN C., Department of Biology, Box 249118, LTniversity of Miami, Coral Gables, Florida 33124
GIUDICE, DR. GIOVANNI, University of Palermo, Via Archirafi 22, Palermo, Italy
GLUSMAN, DR. MURRAY, Department of Clinical Psychiatry, Columbia Uni- versity, 722 \V. 168th St., New York, New York 10032
GOLDEN, WILLIAM T., 40 Wall Street, New York, New York 10005
GOLDMAN, DAVID E., 63 Loop Rd., Falmouth, Massachusetts 02540
GOLDSMITH, DR. MARY H. M., Department of Biology, Kline Biology Tower, Yale University, New Haven, Connecticut 06520
GOLDSMITH, DR. TIMOTHY H., Department of Biology, Yale University, New Haven, Connecticut 06520
GOLDSTEIN, DR. MOISE H., JR., 506 Tray lor Bldg., The Johns Hopkins Uni- versity, School of Medicine, 720 Rutland Ave., Baltimore, Maryland 21205
GOOCH, DR. JAMES L., Department of Biology, Juniata College, Huntington, Pennsylvania 16652
GOODCHILD, DR. CHAUNCEY G., Department of Biology, Emory University, Atlanta, Georgia 30322
GOODMAN, DR. LESLEY JEAN, Department of Zoology and Comparative Physi- ology, Queen Mary College, Mile End Rd., London, El 4 NS, England, U. K.
GOOTSCHALL, DR. GERTRUDE Y., 315 East 68th Street, Apartment 9M, New York, New York 10021
GOUDSMIT, DR. ESTHER M., Department of Biology, Oakland University, Rochester, Michigan 48063
GOULD, DR. STEPHEN J., Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts 02138
REPORT OF THE DIRECTOR 71
GRAHAM, UK. HERBERT, 36 Wilson Road, Woods Hole, Massachusetts 02543
GRANT, DR. PHILLIP, Department of Biology, University of Oregon, Eugene, Oregon 97403
GRASS, ALBERT, The Grass Foundation, 77 Reservoir Road, Quincy, Massa- chusetts 02170
GRASS, ELLEN R., The Grass Foundation, 77 Reservoir Road, Quincy, Massa- chusetts 02170
GRASSLE, DR. JUDITH P., Marine Biological Laboratory, Woods Hole, Massa- chusetts 02543
GREEN, DR. JAMES W., Department of Physiology, Rutgers University, New Brunswick, New Jersey 08903
GREEN, DR. JONATHAN P., Department of Biology, Reed College, Portland, Oregon 97202
GREENBERG, DR. MICHAEL J., Department of Biological Sciences, Florida State University, Tallahassee, Florida 32306
GREGG, DR. JAMES H., Department of Zoology, University of Florida, Gainesville, Florida 32601
GRIEF, DR. ROGER L., Department of Physiology, Cornell University Medical College, New York, New York 10021
GRIFFIN, DR. DONALD R., The Rockefeller University, 66th Street and York Avenue, New York, New York 10021
GROSCH, DR. DANIEL S., Department of Genetics, Gardner Hall, North Carolina State University, Raleigh, North Carolina 27607
GROSS, DR. PAUL R., President and Director, Marine Biological Laboratory, Woods Hole, Massachusetts, 02543
GROSSMAN, DR. ALBERT, New York University Medical School, New York, New York 10016
GUNNING, MR. A. ROBERT, 377 Hatchville Road, Hatchville, Massachusetts 02536
GUTTMAN, DR. RITA, Department of Biology, Brooklyn College, Brooklyn, New York 11210
GWILLIAM, DR. G. F., Department of Biology, Reed College, Portland, Oregon 97202
HALL, DR. ZACK W., Department of Physiology, University of California, San Francisco, California 94143
HALVORSON, DR. HARLYN O., Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02154
HANXA, DR. ROBERT B., State University of New York, College of Environ- mental Science and Forestry, Syracuse, New York 13210
HARDING, DR. CLIFFORD V., JR., Professor and Director of Research, Kresge Eye Institute, Wayne State University, School of Medicine, 540 E. Canfield, Detroit, Michigan 48201
HAROSI, DR. FERENC I., Marine Biological Laboratory, Woods Hole, Massa- chusetts 02543
HARRIGAN, DR. JUNE F., Marine Biological Laboratory, Woods Hole, Massa- chusetts 02543
HARRINGTON, DR. GLENN W7., Department of Microbiology, University of
72 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
Missouri, School of Dentistry, 650 E. 25th Street, Kansas City, Missouri
64108 HASCHEMEYER, DR. AUDREY E. V., Department of Biological Sciences, Hunter
College, 965 Park Avenue, New York, New York 10021 HASTINGS, DR. J. WOODLAND, The Biological Laboratories, Harvard University,
Cambridge, Massachusetts 02138 HAXO, DR. FRANCIS T., Department of Marine Biology, Scripps Institution of
Oceanography, University of California, La Jolla, California 92038 HAYASHI, DR. TERU, 3100 S. Michigan, Chicago, Illinois 60616 HAYES, DR. RAYMOND L., JR., Department of Anatomy, School of Medicine,
Morehouse College, Atlanta, Georgia 30314 HENLEY, DR. CATHERINE, 5225 Pooks Hill Road, Apt. 1120 North, Bethesda,
Maryland 20014 HERNDON, DR. WALTER R., 506 Andy Holt Tower, University of Tennessee,
Knoxville, Tennessee 37916
HERVEY, JOHN P., Box 85, Penzance Road, Woods Hole, Massachusetts 02543 HESSLER, DR. ANITA Y., 5795 Waverly Avenue, La Jolla, California 92037 HEUSER, DR. JOHN, Department of Physiology, School of Medicine, University
of California, San Francisco, California 94143 HIATT, DR. HOWARD H., Office of the Dean, Harvard School of Public Health,
677 Huntington Ave., Boston, Massachusetts 02115 HIGHSTEIN, DR. STEPHEN M., Division of Cellular Neurobiology, Albert Einstein
College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 HILL, DR. ROBERT H., Department of Zoology, University of Rhode Island,
Kingston, Rhode Island 02881 HILLMAN, DR. PETER, Department of Biology, Hebrew University, Jerusalem,
Israel HINEGARDNER, DR. RALPH T., Division of Natural Sciences, University of
California, Santa Cruz, California 95060 HINSCH, DR. GERTRUDE W., Department of Biology, University of South Florida,
Tampa, Florida 33620 HOBBIE, DR. JOHN E., The Ecosystems Center, Marine Biological Laboratory,
Woods Hole, Massachusetts 02543 HODGE, DR. ALAN J., Marine Biological Laboratory, Woods Hole, Massachusetts
02543
HODGE, DR. CHARLES, IV, P. O. Box 4095, Philadelphia, Pennsylvania 19118 HOFFMAN, DR. JOSEPH, Department of Physiology, Yale University School of
Medicine, New Haven, Connecticut 06515 HOFFMANN, DR. RICHARD J., Department of Biological Sciences, University of
Pittsburgh, Pittsburgh, Pennsylvania 15260 HOLLYFIELD, DR. JOE C., Baylor School of Medicine, Texas Medical Center,
Houston, Texas 77030 HOLTZMAN, DR. ERIC, Department of Biological Sciences, Columbia University,
New York, New York 10027 HOLZ, DR. GEORGE G., JR., Department of Microbiology, State University of
New York, Upstate Medical Center, Syracuse, New York 13210 HOSKIN, DR. FRANCIS C. G., Department of Biology, Illinois Institute of Tech- nology, Chicago, Illinois 60616
REPORT OF THE DIRECTOR 73
HOUSTON, HOWARD, Preston Avenue, Mericlen, Connecticut 06450 HUBBARD, DR. RUTH, The Biological Laboratories, Harvard University, Cam- bridge, Massachusetts 02138 HUMES, DR. ARTHUR G., Boston University Marine Program, Marine Biological
Laboratory, Woods Hole, Massachusetts 02543 HUMMON, DR. WILLIAM D., Department of Biology, Ohio University, Athens,
Ohio 45701 HUMPHREYS, DR. SUSIE HUNT, University of Hawaii, Pacific Biomedical Research
Center, 41 Ahui St., Honolulu, Hawaii 96813 HUMPHREYS, DR. TOM D., University of Hawaii, Pacific Biomedical Research
Center, 41 Ahui St., Honolulu, Hawaii 96813 HUNTER, DR. BRUCE, Administrative Office, Tulane University, New Orleans,
Louisiana 70118
HUNTER, DR. R. DOUGLAS, Department of Biological Sciences, Oakland Uni- versity, Rochester, Michigan 48063
HUNZIKER, H. E., Main St., Falmouth, Massachusetts 02540 HURWITZ, DR. CHARLES, Basic Science Research Laboratory, VA Hospital,
Albany, New York 12208 HURWITZ, DR. JERARD, Department of Molecular Biology, Albert Einstein
College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 HUXLEY, DR. HUGH E., Medical Research Council, Laboratory of Molecular
Biology, Cambridge, England, U. K. HYDE, DR. BEAL B., Department of Botany, University of Vermont, Burlington,
Vermont 05401 ILAN, DR. JOSEPH, Department of Anatomy, Case Western Reserve University,
School of Medicine, Cleveland, Ohio 44106 INOUE, DR. SADAYUKI, Department of Pathology, Pathology Institute, McGill
University, 3775 University Street, Montreal 112, Quebec, Canada INOUE, DR. SHINYA, 217 Leidy Lab Building, Department of Biology, University
of Pennsylvania, 38 and Hamilton Walk, Philadelphia, Pennsylvania 19174 ISENBERG, DR. IRVING, Department of Biochemistry and Biophysics, Oregon
State University, Corvallis, Oregon 97331
ISSELBACKER, DR. KURT J., Massachusetts General Hospital, Boston, Massa- chusetts 02714 IZZARD, DR. COLIN S., Department of Biological Sciences, State University of
New York at Albany, Albany, New York 12207 JACOBSON, DR. ANTONE G., Department of Zoology, University of Texas, Austin,
Texas 78712 JAFFEE, DR. LIONEL, Department of Biology, Purdue University, Lafayette,
Indiana 47907 JAHAN-PARWAR, Dr. BEHAUS, Worcester Foundation for Experimental Biology,
222 Maple Avenue, Shrewsbury, Massachusetts 01545 JANNASCH, DR. HOLGER W., Woods Hole Oceanographic Institution, Woods
Hole, Massachusetts 02543 JEFFERY, DR. WILLIAM R., Department of Zoology, University of Texas, Austin,
Texas 78712 JENNER, DR. CHARLES E., Department of Zoology, University of North Carolina,
Chapel Hill, North Carolina 27514
74 ANNUAL RKl'ORT OF T1IK MAK1NK BIOLOGICAL LABORATORY
JENNINGS, DR. JOSKPII B., Department <>i /oology, University of Leeds, Leeds
LS2 9JT, England, U. K. JONES, DR. MEREDITH L., Division of Worms, Museum of Natural History,
Smithsonian Institution, Washington, D. C. 20550 JONES, DR. RAYMOND F., Department of Biology, State University of New York
at Stony Brook, Stony Brook, New York 11753 JOSEPHSON, DR. R. K., School of Biological Sciences, University of California,
Irvine, California 92717 JOYNER, DR. RONALD W., Department of Physiology, University of Iowa,
Iowa City, Iowa 52242 JUNQUEIRA, DR. Luiz CARLOS, Department Histologia, Institute Ciencias Bio-
medicas, C.P. 4365, Sao Paulo, Brazil KABAT, DR. E. A., Department of Microbiology, Columbia University, College
of Physicians and Surgeons, 630 W. 168th St., New York, New York 10032 KAFATOS, DR. FOTIS C., The Biological Laboratories, Harvard University, 16
Divinity Avenue, Cambridge, Massachusetts 02138 KAJI, DR. AKIRA, Department of Microbiology, University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania 19174 KALEY, DR. GABOR, Department of Physiology, Basic Sciences Building, New
York Medical College, Valhalla, New York 10595 KALTENBACH, DR. JANE, 139 Cold Hill, Granby, Massachusetts 01033 KAMINER, DR. BENJAMIN, Department of Physiology, Boston University School
of Medicine, 80 E. Concord St., Boston, Massachusetts 02118 KAMMER, DR. ANN E., Division of Biology, Kansas State University, Manhattan,
Kansas 66502 KANE, DR. ROBERT E., Pacific Biomedical Research Center, 41 Ahui Street,
University of Hawaii, Honolulu, Hawaii 96813 KANESHIRO, DR. EDNA S., Department of Biological Sciences, University of
Cincinnati, Cincinnati, Ohio 45221 KARAKASHIAN, DR. STEPHEN J., 165 West 91st Street, Apt. 16-F, New York,
New York 10024 KARUSH, DR. FRED, Department of Microbiology, University of Pennsylvania,
School of Medicine, Philadelphia, Pennsylvania 19174 KATZ, DR. GEORGE M., Department of Neurology, Columbia University, College
of Physicians and Surgeons, 630 West 168th Street, New York, New York
10032 KEAN, DR. EDWARD L., Departments of Biochemistry and Ophthalmology, Case
Western Reserve University, Cleveland, Ohio 44101 KEMP, DR. NORMAN E., Department of Zoology, University of Michigan, Ann
Arbor, Michigan 48104
KENDALL, MR. JOHN P., One Boston Place, Boston, Massachusetts 02108 KEOSIAN, DR. JOHN, P. O. Box 193, W7oods Hole, Massachusetts 02543 KETCHUM, DR. BOST\VICK H., P. O. Box 32, Woods Hole, Massachusetts 02543 KEYNAN, DR. ALEXANDER, Vice President, Hebrew University, Jerusalem, Israel KING, DR. THOMAS J., Program Director, Division of Cancer Research, Resources
and Center, National Institutes of Health, Bldg. 31, Room 10A03, Bethesda,
Maryland 20014
REPORT OF THE DIRECTOR 75
KINGSBURY, DR. JOHN M., Department of Botany, Cornell University, Ithaca,
New York 14850
KIRSCHENBAUM, DR. DONALD, Department of Biochemistry, College of Medi- cine, State University of New York, 450 Clarkson Avenue, Brooklyn, New York 11203
KLEIN, DR. MORTON, Department of Microbiology, Temple University, Phila- delphia, Pennsylvania 19122
KLOTZ, DR. I. M., Department of Chemistry, Northwestern University, Evans- ton, Illinois 60201
KOHLER, DR. KURT, Biologische Institut der Universitat Stuttgart, D-7, Stuttgart 60, Ulmer Str. 227, West Germany
KOIDE, DR. SAMUEL S., Population Council, The Rockefeller University, 66th St. and York Ave., New York, New York 10021
KONINGSBERG, DR. iRWiN R., Department of Biology, Gilmer Hall, University of Virginia, Charlottesville, Virginia 22903
KOSOWER, DR. EDWARD M., Department of Chemistry, University of California, San Diego, La Jolla, California 92093
KRAHL, DR. M. E., 2783 West Casas Circle, Tucson, Arizona 85704
KRANE, DR. STEPHEN M., Massachusetts General Hospital, Boston, Massa- chusetts 02114
KRASSNER, DR. STUART MITCHELL, Department of Developmental and Cell Biology, University of California, Irvine, California 92717
KRAUSS, DR. ROBERT, Dean, School of Science, Oregon State University, Cor- vallis, Oregon 97331
KRAVITZ, DR. EDWARD A., Department of Neurobiology, Harvard Medical School, 25 Shattuck St., Boston, Massachusetts 02115
KRIEBEL, DR. MAHLON E., Department of Physiology, State University of New- York, Upstate Medical Center, Syracuse, New York 13210
KRIEG, DR. WENDELL J. S., 1236 Hinman, Evanston, Illinois 60602
KRUPA, DR. PAUL L., Department of Biology, The City College of New York, 139th St. and Convent Avenue, New York, New York 10031
KUFFLER, DR. STEPHEN W., Department of Neurophysiology, Harvard Medical School, Boston, Massachusetts 02115
KUSANO, DR. KIYOSHI, Department of Biology, Illinois Institute of Technology, 3300 South Eederal Street, Chicago, Illinois 60616
LAMARCHE, DR. PAUL H., 593 Eddy St., Providence, Rhode Island 02903
LANCEFIELD, DR. REBECCA C., The Rockefeller University, 1230 York Ave., New York, New York 10021
LANDIS, DR. DENNIS, Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts 02114
LANDOWNE, DR. DAVID, Department of Physiology, University of Miami, Miami, Florida 33124
LANGFORD, DR. GEORGE M., Department of Anatomy, Howard University, College of Medicine, Washington, D. C. 20059
LASH, DR. JAMES W., Department of Anatomy, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19174
LASTER, DR. LEONARD, President, Health Services Center, University of Oregon, Portland, Oregon 97201
76 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
LAUFER, DR. HANS, Biological Sciences Group 1-42, I'niversity of Connecticut, Storrs, Connecticut 06268
LAUFFER, DR. MAX A., Department of Biophysics, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
LAWRENCE, E. SWIFT, Box 228, West Falmouth, Massachusetts 02574
LAZAROW, JANE, 221 Woodlawn Avenue, St. Paul, Minnesota 55105
LEADBETTER, DR. EDWARD R., Biological Sciences Group U-42 University of Connecticut, Storrs, Connecticut 06268
LEAK, DR. LEE VIRN, Department of Anatomy, Howard University, College of Medicine, Washington, D. C. 20059
LECAR, DR. HAROLD, Laboratory of Biophysics, National Institute of Neuro- logical Diseases and Stroke, National Institutes of Health, Bethesda, Mary- land 20014
LEDERBERG, DR. JOSHUA, President, The Rockefeller University, New York, New York 10021
LEE, DR. JOHN J., Department of Biology, City College of the City University of New York, Convent Avenue and 138th Street, New York, New York 10031
LEFEVRE, DR. PAUL G., Department of Physiology, Health Sciences Center, East Campus, State University of New York at Stony Brook, Stony Brook, New York 11794
LEIGHTON, DR. JOSEPH, Department of Pathology, Medical College of Penn- sylvania, 3300 Henry Ave., Philadelphia, Pennsylvania 19129
LENHER, DR. SAMUEL, Box 1573, Vineyard Haven, Massachusetts 02568
LERMAN, DR. SIDNEY, Laboratory for Ophthalmic Research, Emory University, Atlanta, Georgia 30322
LERNER, DR. AARON B., Yale Medical School, New Haven, Connecticut 06510
LEVIN, DR. JACK, Hematology Division, The Johns Hopkins Hospital, Baltimore, Maryland 21205
LEVINE, DR. RACHMIEL, 2024 Canyon Road, Arcadia, California 91006
LEVINTHAL, DR. CYRUS, Department of Biological Sciences, 908 Schermerhorn Hall, Columbia University, New York, New York 10027
LEVITAN, DR. HERBERT, Department of Zoology, University of Maryland. College Park, Maryland 20742
LEWIN, DR. RALPH A., Scripps Institution of Oceanography, La Jolla, California 92037
LING, DR. GILBERT, 307 Berkeley Road, Merion, Pennsylvania 19066
LINSKENS, DR. H. P., Department of Botany, University of Driehuizerweg 200, Nijmegen, The Netherlands
LIPICKY, DR. RAYMOND J., Department of Pharmacology, College of Medicine, University of Cincinnati, 231 Bethesda Avenue, Cincinnati, Ohio 45267
LITTLE, DR. E. P., 216 Highland Street, West Newton, Massachusetts 02158
LIUZZI, DR. ANTHONY, Department of Radiological Sciences, University of Lowell, Lowell, Massachusetts 01854
LLINAS, DR. RODOLFO R., Department of Physiology and Biophysics, New York University Medical Center, 550 First Ave., New York, New York 10016
LOEWENSTEIN, DR. WERNER R., Department of Physiology and Biophysics, Uni- versity of Miami, School of Medicine, P. O. Box 520875, Miami, Florida 33152
REPORT OF THE DIRECTOR 77
LOEWUS, DR. FRANK A., Department of Agricultural Chemistry, Washington
State University, Pullman, Washington 99164 LOFTFIELD, DR. ROBERT B., Department of Biochemistry, University of New
Mexico Medical School, 900 Stanford N. E., Albuquerque, New Mexico 87106 LONDON, DR. IRVING M., 16-512, Massachusetts Institute of Technology, Cam- bridge, Massachusetts 02139 LONGO, DR. FRANK f., Department of Anatomy, University of Iowa, Iowa City,
Iowa 52442 LORAND, DR. LASZLO, Ann: Mrs. P. Velasco, Department of Biochemistry and
Molecular Biology, Northwestern University, Evanston, Illinois 60201 LURIA, DR. SALVADOR E., Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139
LYNCH, DR. CLARA J., 4800 Filmore Avenue, Alexandria, Virginia 22311 MAcNiCHOL, DR. EDWARD F., JR., Marine Biological Laboratory, Woods Hole,
Massachusetts 02543 MAHLER, DR. HENRY R., Department of Biochemistry, Indiana University,
Bloomington, Indiana 47401 MALKIEL, DR. SAUL, Sidney Farber Cancer Center, 35 Binney Street, Boston,
Massachusetts 02115 MANALIS, DR. RICHARD S., Department of Physiology, University of Cincinnati,
College of Medicine, Eden and Bethesda Aves., Cincinnati, Ohio 45267 MAGNUM, DR. CHARLOTTE P., Department of Biology, College of William and
Mary, Williamsburg, Virginia 23185 MARKS, DR. PAUL A., Columbia UJniversity College of Physicians and Surgeons,
630 West 168th Street, New York, New York 10032 MARSH, DR. JULIAN B., Department of Biochemistry and Physiology, Medical
College of Pennsylvania, 3300 Henry Ave., Philadelphia, Pennsylvania 19129 MARUO, DR. TAKESHI, The Population Council, The Rockefeller University,
1230 York Avenue, New York, New York 10021
MASER, DR. MORTON, The Marine Biological Laboratory, Woods Hole, Massa- chusetts, 02543 MASTROIANNI, DR. LUIGI, JR., Department of Obstetrics and Gynecology,
Hospital of the University of Pennsylvania, 3400 Spruce St., Philadelphia,
Pennsylvania 19174 MATHE\YS, DR. RITA W., Hunter College, Box 1075, 695 Park Ave, New York,
New York 10021 MAUTNER, DR. HENRY G., Department of Biochemistry and Pharmacology,
Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massa- chusetts 02111 MAUZERALL, DR. DAVID, The Rockefeller University, 66th Street and York
Avenue, New York, New York 10021 MAXWELL, DR. ARTHUR, Provost, Woods Hole Oceanographic Institution, Woods
Hole, Massachusetts 02543 MAZIA, DR. DANIEL, Department of Zoology, University of California, Berkeley,
California 94720 McCANN, DR. FRANCES, Department of Physiology, Dartmouth Medical School,
Hanover, New Hampshire 03755
78 ANNUAL REPORT OK TIIK MARINE HlOU H.ilCAL LABORATORY
McCLOSKEY, DR. LAWRENCE R., Department of Biology, Walla Walla College,
College Place, Washington 99324
MCLAUGHLIN, JANE A., P. O. Box 187, Woods Hole, Massachusetts 02543 McMAHON, DR. ROBERT F., Department of Biology, University of Texas,
Arlington, Texas 7601^ McREYNOLDS, DR. JOHN S., Department of Physiology, University of Michigan,
Ann Arbor, Michigan 48104 MEINKOTH, DR. NORMAN A., Department of Biology, Swarthmore College,
Swarthmore, Pennsylvania 19081 MELLON, DR. DEFOREST, JR., Department of Biology, University of Virginia,
Charlottesville, Virginia 22903 METZ, DR. C. B., Institute of Molecular Evolution, University of Miami, 521
Anastasia St., Coral Gables, Florida 33134 MIDDLEBROOK, DR. ROBERT, 86 Station Road, Btirley-In-Wharfdale, West
Yorks, England, U. K. MILKMAN, DR. ROGER D., Department of Zoology, University of Iowa, Iowa
City, Iowa 52242 MILLS, DR. ERIC LEONARD, Institute of Oceanography, Dalhousie University,
Halifax, Nova Scotia, Canada
MILLS, ROBERT, 56 Worcester Ct., Falmouth, Massachusetts 02540 MITCHELL, DR. RALPH, Pierce Hall, Harvard University, Cambridge, Massa- chusetts 02138 MIZELL, DR. MERLE, Department of Biology, Tulane LIniversity, New Orleans,
Louisiana 70118
MONROY, DR. ALBERTO, Stazione Zoologica, Villa Communale, Napoli, Italy MONTROLL, DR. ELIOTT W., Institute for Fundamental Studies, Department of
Physics, University of Rochester, Rochester, New York 14627 MOORE, DR. JOHN A., Department of Biology, University of California, River- side, California 92502 MOORE, DR. JOHN W., Department of Physiology, Duke University Medical
Center, Durham, North Carolina 27706 MOORE, DR. LEE E., Department of Physiology and Biophysics, University of
Texas Medical Branch, Galveston, Texas 77550 MORAN, DR. JOSEPH F., JR., 23 Foxwood Drive, RR#1, Eastham, Massachusetts
02642 MORIN, DR. JAMES G., Department of Biology, University of California, Los
Angeles, California 90024 MORRELL, DR. FRANK, Department of Neurological Sciences, Rush Medical
Center, 1753 W. Congress Pkwy., Chicago, Illinois 60612 MORRILL, DR. JOHN B., JR., Division of Natural Sciences, New College, Sarasota,
Florida 33580
MORSE, DR. RICHARD STETSON, 193 Winding River Road, Wellesley, Massa- chusetts 02181 MORSE, ROBERT W., Associate Director, Woods Hole Oceanographic Institution,
Woods Hole, Massachusetts 02543 MOSCONA, DR. A. A., Department of Zoology, University of Chicago, Chicago,
Illinois 60627
REPORT OF THE DIRECTOR 79
MOTE, DR. MICHAEL I., Department of Biology, Temple University, Philadelphia, Pennsylvania 19122
MOUNTAIN, DR. ISABEL M., Brookfield PI., Pleasantville, New York 10570
MULLEN, GEORGE, Belknap and McLain, 650 Pleasant Street, Watertown, Massachusetts, 02172
MUSACCHIA, DR. XAVIER J., (Graduate School, University of Louisville, Louisville, Kentucky 40208
NABRIT, DR. S. M., 686 Beckwith Street S. W., Atlanta, Georgia 30314
NACE, DR. PAUL FOLEY, 5 Bowditch Rd., Woods Hole, Massachusetts 02543
XACHMANSOHX, DR. DAVID, Department of Xeurology, Columbia University, College of Physicians and Surgeons, Xew York, Xew York 10032
XAKAJIMA, DR. SHIGEHIRO, Department of Biological Sciences, Purdue Uni- versity, West Lafayette, Indiana 47907
NAKAJIMA, DR. YOSUKO, Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907
XARAHASHI, DR. TOSHIO, Department of Pharmacology, Xorthwestern Uni- versity Medical Center, 303 E. Chicago Ave., Chicago, Illinois 60611
XASATIR, DR. MAIMON, Department of Biology, University of Toledo, Toledo, Ohio 43606
XELSOX, DR. LEONARD, Department of Physiology, Medical College of Ohio at Toledo, Toledo, Ohio 43699
NICHOLLS, DR. JOHN GRAHAM, Department of Xeurobiology, Stanford Uni- versity, Stanford, California 94305
XICOSIA, DR. SANTO Y., School of Medicine, University of Pennsylvania, Phila- delphia, Pennsylvania 19174
XIELSEN, DR. JENNIFER B. K., Waksman Institute for Microbiology, Rutgers University, Piscataway, Xew Jersey 08854
XOE, DR. BRYAN D., Department of Anatomy, Emory University, Atlanta, Georgia 30345
XOVIKOFF, DR. ALEX B., Department of Pathology, Albert Einstein College of Medicine, Bronx, Xew York 10461
OCHOA, DR. SEVERO, 530 East 72nd Street, Xew York, Xew York 10021
ODUM, DR. EUGENE, Department of Zoology, University of Georgia, Athens, Georgia 30601
OERTEL, DR. DONATA, Department of Xeurophysiology, University of Wisconsin, Madison, Wisconsin 53706
O'HERRON, JONATHAN, Lazard Freres and Company, 1 Rockefeller Plaza, Xew York, Xew York 10020
OLSON, DR. JOHN M., Department of Biology, Brookhaven Xational Laboratory. Upton, Xew York 11973
OSCHMAN, DR. JAMES L., Marine Biological Laboratory, Woods Hole, Massa- chusetts 02543
OXFORD, DR. GERRY S., Department of Physiology, University of Xorth Carolina, Chapel Hill, Xorth Carolina 27514
PALMER, DR. JOHN D., Department of Zoology, University of Massachusetts, Amherst, Massachusetts 01002
PALTI, DR. YORAM, Head, Department of Biophysics, University of Maryland, Baltimore, Maryland 21201
80 \\.\TUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
PAPPAS, DR. GEORGE D., Department of Anatomy, University of Chicago,
College of Medicine, 1853 W. Polk St., P. (). Box 6998, Chicago, Illinois 60612 PARDEE, DR. ARTHUR B., Department of Pharmacology, Harvard Medical
School, Boston, Massachusetts 02115 PARDY, DR. ROSEVELT L., School of Life Sciences, University of Nebraska,
Lincoln, Nebraska 68588 PASSANO, DR. LEONARD M., Department of Zoology, University of Wisconsin,
Madison, Wisconsin 53706 PEARLMAN, DR. ALAN L., Department of Physiology, Washington University
School of Medicine, St. Louis, Missouri 63110 PERKINS, DR. C. D., National Academy of Engineering, 2101 Constitution Ave.,
N. W7., Washington, D. C. 20418 PERSON, DR. PHILIP, Special Dental Research Program, Veteran's Administration
Hospital, Brooklyn, New York 11219 PETTIBONE, DR. MARIAN H., Division of Worms, \V-213, Smithsonian Institution,
Washington, D. C. 20560 PFOHL, DR. RONALD J., Department of Zoology, Miami University, Oxford.
Ohio 45056
PHILPOTT, DR. DELBERT E., MASA Ames Research Center, Moffett Field, Cali- fornia 94035 PIERCE, DR. SIDNEY K., JR., Department of Zoology, University of Maryland,
College Park, Maryland 20740 PINTO, DR. LAWRENCE, Department of Biological Sciences, Purdue University,
West Lafayette, Indiana 47907 POLLARD, DR. HARVEY B., National Institutes of Health, F. Bldg. 10, Rm. 10B17,
Bethesda, Maryland 20014 POLLARD, DR. THOMAS D., Department of Cell Biology and Anatomy, School of
Medicine, Johns Hopkins University, Baltimore, Maryland 21205 POLLOCK, DR. LELAND W7., Department of Zoology, Drew University, Madison,
New Jersey 07940
PORTER, DR. BEVERLY H., 14433 Taos Court, Wheaton, Maryland 20906 PORTER, DR. KEITH R., 748 llth Street, Boulder, Colorado 80302 POTTER, DR. DAVID, Department of Neurobiology, Harvard Medical School,
Boston, Massachusetts 02115 POTTER, DR. H. DAVID, Center for Neural Sciences, Indiana University,
Bloomington, Indiana 47401 POTTS, DR. WILLIAM T., Department of Biology, LTniversity of Lancaster,
Lancaster, England, U. K. POUSSART, DR. DENIS, Department of Electrical Engineering, Universite Laval,
Quebec, Canada PRENDERGAST, DR. ROBERT A., Department of Pathology and Ophthalmology,
The Johns Hopkins University School of Medicine, Baltimore, Maryland
21205 PRICE, DR. CARL A., Waksman Institute of Microbiology, Rutgers University,
P.O. Box 759, Piscataway, New Jersey 08854 PRICE, DR. CHRISTOPHER H., Marine Biomedical Institute, LIniversity of Texas,
Galveston, Texas 77550
REPORT OF THE DIRECTOR S1
PRIOR, DR. DAVID JA.MKS, Department of Biological Sciences, University of
Kentucky, Lexington, Kentucky 40506 PROSSER, DR. C. LADD, Department of Physiology and Biophysics, Burrill Hall
524, University of Illinois, Urbana, Illinois 61801 PROVASOLI, DR. LUIGI, Haskins Laboratories, 165 Prospect Street, New Haven,
Connecticut 06520 PRUSCH, DR. ROBERT D., Division of Biomedical Sciences, Brown University,
Providence, Rhode Island 02904 PRZYBYLSKI, DR. RONALD J., Department of Anatomy, Case Western Reserve
University, Cleveland, Ohio 44101
RABIN, DR. HARVEY, Box 239, Braddock Heights, Maryland 21714 RAMOX, DR. FIDEL, Duke University Medical Center, Durham, North Carolina
27706
RANKIN, DR. JOHN S., Box 97, Ashford, Connecticut 06278 RANZI, DR. SILVIO, Department of Zoology, University of Milan, Via Celonia 10,
Milan, Italy RATNER, DR. SARAH, Department of Biochemistry, The Public Health Research
Institute, 455 First Avenue, New York, New York 10016 REBHUN, DR. LIONEL I., Department of Biology, Gilmer Hall, University of
Virginia, Charlottesville, Virginia 22901 REDDAN, DR. JOHN R., Department of Biological Sciences, Oakland University,
Rochester, Michigan 48063
REDFIELD, DR. ALFRED C., Maury Lane, Woods Hole, Massachusetts 02543 REESE, DR. THOMAS S., Head, Section on Functional Neuroanatomy, National
Institutes of Health, Bethesda, Maryland 20014 REINER, DR. JOHN M., Department of Biochemistry, Albany Medical College
of Union University, Albany, New York 12208 REINISCH, DR. CAROL L., Department of Tumor Immunology, Sidney Farber
Cancer Institute, 44 Binney St., Boston, Massachusetts 02511 RENN, DR. CHARLES E., Route 2, Hampstead, Maryland 21074 REUBEN, DR. JOHN P., Department of Neurology, Columbia University, College
of Physicians and Surgeons, New York, New York 10032 REYNOLD, DR. GEORGE THOMAS, Department of Physics, Princeton University,
Princeton, New Jersey 08540
RICE, DR. ROBERT VERNON, Marine Biological Laboratory, Woods Hole, Massa- chusetts 02543 RICKLES, DR. FREDERICK R.., University of Connecticut, School of Medicine,
Newington, Connecticut 06111 RIPPS, DR. HARRIS, Department of Opthalmology, New York University, School
of Medicine, 550 First Ave., New York, New York 10016 ROBERTS, DR. JOHN L., Department of Zoology, University of Massachusetts,
Amherst, Massachusetts 01002
ROBINSON, DR. DENIS M., 19 Orlando Avenue, Arlington, Massachusetts 02174 ROCKSTEIN, DR. MORRIS, Department of Physiology, University of Miami
School of Medicine, P. O. Box 975, Biscayne Annex, Miami, Florida 33152 RONKIN, DR. RAPHAEL E., 3212 McKinley St., N. W., Washington, D. C. 20015 ROSE, DR. BIRGIT, Department of Physiology, University of Miami School of
Medicine, Miami, Florida 33152
82 A \\UAL RErORT OF THE MARINE BIOLOGICAL LABORATORY
ROSE, DR. S. MERYL, 34 High St., Woods Hole, Massachusetts 02543 ROSENBAUM, DR. JOEL L., Kline Biology Tower, Yale University, New Haven,
Connecticut 06510 ROSENBERG, DR. EVELYN K., Jersey City State College, Jersey City, New
Jersey 07305 ROSENBERG, DR. PHILLIP, Division of Pharmacology, University of Connecticut,
School of Pharmacy, Storrs, Connecticut 06268 ROSENBLUTH, DR. JACK, Department of Physiology, New York University,
School of Medicine, 550 First Avenue, New York, New York 10016 ROSENBLUTH, RAJA, 3380 West 5th Avenue, Vancouver, British Columbia,
Canada V6R 1R7 ROSENKRANZ, DR. HERBERT S., Department of Microbiology, New York Medical
College, Valhalla, New York 10595
ROSLANSKY, DR. JOHN, 26 Albatross, Woods Hole, Massachusetts 02543 ROSLANSKY, DR. PRISCILLA F., Box 208, Woods Hole, Massachusetts 02543 Ross, DR. WILLIAM N., Department of Neurobiology, Harvard Medical School,
Boston, Massachusetts 02115 ROTH, DR. JAY S., Division of Biological Sciences, Section of Biochemistry and
Biophysics, University of Connecticut, Storrs, Connecticut 06268 ROWE, Miss DOROTHY, 88 Chesnut St. Boston, Massachusetts 02165 ROWLAND, DR. LEWIS P., Department of Neurology, Columbia University,
College of Physicians and Surgeons, 630 W. 168th St., New York, New
York 10032 RUBINOW, DR. SOL I., Department of Biomathematics, Cornell University,
Medical College, New York, New York 10012 RUDERMAN, DR. JOAN V., Department of Anatomy, Harvard Medical School,
Boston, Massachusetts 02115 RUSHFORTH, DR. NORMAN B., Department of Biology, Case Western Reserve
University, Cleveland, Ohio 44106 RUSSELL, DR. JOHN M., Department of Biophysics, University of Texas, Medical
Branch, Galveston, Texas 77550 RUSSELL-HUNTER, DR. W. D., Department of Biology, Lyman Hall, Syracuse
University, Syracuse, New York 13210 RUSTAD, DR. RONALD C., Department of Radiology, Case Western Reserve
University, Cleveland, Ohio 44106 RYTHER, DR. JOHN H., Woods Hole Oceanographic Institution, Woods Hole,
Massachusetts 02543
SAGER, DR. RUTH, Sidney Farber Cancer Center, 44 Binney St., Boston, Massa- chusetts 02115 SALMON, DR. EDWARD D., Department of Zoology, University of North Carolina,
Chapel Hill, North Carolina 27514 SALZBERG, DR. BRIAN M., Department of Physiology, University of Pennsylvania,
4010 Locust St., Philadelphia, Pennsylvania 19174 SANDERS, DR. HOWARD L., Woods Hole Oceanographic Institution, Woods Hole,
Massachusetts 02543 SATO, DR. HIDEMI, Sugashima Marine Biological Laboratory, Nagoya University,
Sugashima-cho, Toba-shi, Mie-ken, 517, Japan
RKl'OKT OF THE DIRECTOR S3
SAUNDERS, DR. JOHN \\'., JR.. Department of Biological Sciences, State University of New York at Albany, Albany, New York 12222
SAZ, DR. ARTHUR KENNETH, Department of Microbiology, Georgetown Uni- versity Medical and Dental Schools, 3900 Reservoir Road, N. W., Washing- ton, D. C. 20051
SCHACHMAN, DR. HOWARD K., Department of Molecular Biology, University of California, Berkeley, California 94720
SCHIFF, DR. JEROME A., Institute for Photobiology of Cells and Organelles, Brandeis University, Waltham, Massachusetts 02154
SCHLESINGER, DR. R. WALTER, Department of Microbiology, Rutgers Medical School, P. O. Box 101, Piscataway, New Jersey 08854
SCHMEER, SISTER, ARLINE C., American Cancer Research Center and Hospital, 6401 W. Colfax Ave., Denver, Colorado 80214
SCHNEIDERMAX, DR. HOWARD A., Center for Pathobiology, School of Biological Sciences, University of California, Irvine, California 92717
SCHOLANDER, DR. P. F., Scripps Institution of Oceanography, La Jolla, Cali- fornia 92037
SCHOPF, DR. THOMAS J. M., Department of the Geophysical Sciences, University of Chicago, 5734 S. Ellis Avenue, Chicago, Illinois 60637
SCHOTTE, DR. OSCAR E., Department of Biology, Amherst College, Amherst, Massachusetts 01002
SCHUEL, DR. HERBERT, Department of Anatomical Sciences, State University of New York, Buffalo, New York 14214
SCHUETZ, DR. ALLEN WALTER, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205
SCHWARTZ, DR. TOBIAS L., Biological Sciences Group, University of Connecticut, Storrs, Connecticut 06268
SCOTT, DR. ALLAN C., 1 Nudd St., Waterville, Maine 04901
SCOTT, DR. GEORGE T., Department of Biology, Oberlin College, Oberlin, Ohio 44074
SEARS, DR. MARY, Box 152, Woods Hole, Massachusetts 02543
SEGAL, DR. SHELDON J., Director, Population Division, The Rockefeller Founda- tion, 1133 Avenue of the Americas, New York, New York 10036
SELIGER, DR. HOWARD H., McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218
SELMAN, DR. KELLY, Department of Anatomy, College of Medicine, University of Florida, Gainesville, Florida 32601
SENFT, DR. JOSEPH P., Organic Gardening and Farming Research Center, Box 323, Kutztown, Pennsylvania 19530
SHANKLIN, DR. DOUGLAS R., P. O. Box 1267, Gainesville, Florida 32602
SHAPIRO, DR. HERBERT, 6025 North 13th Street, Philadelphia, Pennsylvania 19141
SHAVER, DR. JOHN R., Department of Zoology, Michigan State University, East Lansing, Michigan 48823
SHEPHARD, DR. DAVID C., P. O. Box 44, \Voods Hole, Massachusetts 02543
SHEPRO, DR. DAVID, Department of Biology, Boston University, 2 Cummington Street, Boston, Massachusetts 02215
S4 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
SHERMAN, DK. l.\\'., Division of Lift- Sciences, University of California, Riverside,
California 92502 SHILO, DR. MOSHE, Heiid, Department of Microbiological Chemistry, Hebrew
University, Jerusalem, Israel SIEGEL, DR. IR\YIN M., Department of Ophthalmology, New York University
Medical Center, 550 First Avenue, New York, New York 10016 SIEGELMAN, DR. HAROLD W., Department of Biology, Brookhaven National
Laboratory, Upton, New York 11973 SIMON, DR. ERIC J., New York University Medical School, 550 First Avenue,
New York, New York 10016 SJODIN, DR. RAYMOND A., Department of Biophysics, University of Maryland
School of Medicine, Baltimore, Maryland 21201 SKINNER, DR. DOROTHY M., Biology Division, Oak Ridge National Laboratory,
Oak Ridge, Tennessee 37830 SLOBODKIN, DR. LAWRENCE B., Department of Biology, State University of
New York at Stony Brook, Stony Brook, New York 11790 SMITH, HOMER P., General Manager, Marine Biological Laboratory, Woods Hole,
Massachusetts 02543
SMITH, DR. MICHAEL A., Marine Biological Laboratory, Woods Hole, Massa- chusetts 02543
SMITH, PAUL FERRIS, Church Street, Woods Hole, Massachusetts 02543 SMITH, DR. RALPH I., Department of Zoology, University of California, Berkeley,
California 94720
SONNENBLICK, DR. B. P., Department of Zoology and Physiology, Rutgers Uni- versity, 195 University Avenue, Newark, New Jersey 07102 SORENSON, DR. ALBERT L., Department of Physiology, Albert Einstein College
of Medicine, 1300 Morris Park Ave., Bronx, New York 10461 SORENSON, DR. MARTHA M., Department of Neurology, Columbia University,
College of Physicians and Surgeons, New York, New York 10032 SPECK, DR. WILLIAM T., Department of Pediatrics, Case Western Reserve
University, Cleveland, Ohio 44106 SPECTOR, DR. A., Black Bldg., Rm. 1516, Columbia University, College of
Physicians and Surgeons, New York, New York 10032 SPIEGEL, DR. EVELYN, Department of Biological Sciences, Dartmouth College,
Hanover, New Hampshire 03755 SPIEGEL, DR. MELVIN, Department of Biological Sciences, Dartmouth College,
Hanover, New Hampshire 03755 SPIRTES, DR. MORRIS ALBERT, Veterans' Administration Hospital, 1061 Perdido
Street, New Orleans, Louisiana 70112 SPRAY, DR. DAVID C., Department of Neuroscience, Albert Einstein College of
Medicine, Bronx, New York 10461 STARZAK, DR. MICHAEL E., Department of Chemistry, State University of
New York, Binghamton, New York 13901 STEINBERG, DR. MALCOLM S., Department of Biology, Princeton University,
Princeton, New Jersey 08540 STEINHARDT, DR. JACINTO, 306 Reiss Bldg., Georgetown University, Washington,
D. C. 20007
STEPHENS, DR. GROVER C., School of Biological Sciences, University of Cali- fornia, Irvine, California 92717
REPORT OF THE DIRECTOR 85
STEPHENS, DK. RAYMOND E., Marine Biological Laboratory, Woods Hole, Massa- chusetts 02543
STETTEN, DR. MARJORIE R., National Institutes of Health, Bldg. 10, 9B-02, Bethesda, Maryland 20014
STOKES, DR. DARRELL R., Department of Biology, Emory University, Atlanta, Georgia 30322
STRACHER, DR. ALFRED, State University of New York, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, New York 11203
STREHLER, DR. BERNARD L., 2310 N. Laguna Circle Dr., Agoura, California 91301
STRETTON, DR. ANTHONY O. W., Department of Zoology, University of Wisconsin, Madison, Wisconsin 53706
STUART, DR. ANN E., Department of Neurobiology, Harvard Medical School, 25 Shattuck St., Boston, Massachusetts 02115
STMMERS, DR. WILLIAM C., Huxley College, Western Washington State College, Bellingham, Washington 98225
SUSSMAN, DR. MAURICE, Department of Life Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
SWOPE, GERARD, JR., Blinn Road, Box 345, Croton-on-Hudson, New York 10520
SZABO, DR. GEORGE, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, Massachusetts 02115
SZAMIER, DR. ROBERT BRUCE, Harvard Medical School, Berman-Gund Labora- tory, Eye and Ear Infirmary, 243 Charles Street, Boston, Massachusetts 021 14
SZENT-GYORGYI, DR. ALBERT, Institute for Muscle Research, Marine Biological Laboratory, WToods, Hole, Massachusetts 02543
SZENT-GYORGYI, DR. ANDREW G., Department of Biology, Brandeis University, Waltham, Massachusetts 02154
TAKASHIMA, DR. SHIRO, Department of Bioengineering, University of Pennsyl- vania, Philadelphia, Pennsylvania 19174
TANZER, DR. MARVIN L., Department of Biochemistry, Box G, University of Connecticut, School of Medicine, Earmington, Connecticut 06032
TASAKI, DR. ICHIJI, Laboratory of Neurobiology, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20014
TAYLOR, DR. DOUGLAS L., The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138
TAYLOR, DR. ROBERT E., Laboratory of Biophysics, National Institute of Neu- rological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland 20014
TAYLOR, DR. W. RO\YLAND, 1540 Northbourne Rd., Baltimore, Maryland 21239
TELFER, DR. WILLIAM H., Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19174
DETERRA, DR. NOEL, Department of Anatomy, Hahnemann Medical College, 230 N. Broad St., Philadelphia, Pennsylvania 19102
THORNDIKE, W. NICHOLAS, Wellington Management Company, 28 State St., Boston, Massachusetts 02109
TIFFNEY, DK. WESLEY N., 226 Edge Hill Rd., Sharon, Massachusetts 02067
TRAGER, DR. WILLIAM, The Rockefeller University, 66th Street and York Avenue, New York, New York 10021
TRAVIS, DR. D. M., Department of Pharmacology, University of Elorida, Gaines- ville, Elorida 32601
86 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
TRAVIS, DR. DOROTHY F., 733 Sligo Ave., Apt. 503, Silver Spring, Maryland 20910
TRINKAUS, DR. J. PHILIP, Osborn Zoological Laboratories, Department of Zoology, Yale University, New Haven, Connecticut 06510
TROLL, DR. WALTER, Department of Environmental Medicine, New York University, College of Medicine, New York, New York 10016
TROXLER, DR. ROBERT F., Department of Biochemistry, Boston University School of Medicine, 80 E. Concord St., Boston, Massachusetts 02118
TURNER, DR. RUTH I)., Mollusk Department, Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts 02138
TWEEDELL, DR. KENYON S., Department of Biology, University of Notre Dame, Notre Dame, Indiana 46656
URETZ, DR. ROBERT B., Division of Biological Sciences, University of Chicago, 950 E. 59th St., Box 417, Chicago, Illinois 60637
VALIELA, DR. IVAN, Boston University Marine Program, Marine Biological Laboratory, Woods Hole, Massachusetts 02543
VALOIS, JOHN, Marine Biological Laboratory, Woods Hole, Massachusetts 02543
VAN HOLDE, DR. KENSAL EDWARD, Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331
VILLEE, DR. CLAUDE A., Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115
VINCENT, DR. WALTER S., Chairman, Department of Biological Sciences, Uni- versity of Delaware, Newark, Delaware 19711
WAINIO, DR. W. W., Bureau of Biological Research, Rutgers University, New Brunswick, New Jersey 08901
WAKSMAN, DR. BRYON, Department of Pathology, Yale University, New Haven, Connecticut 06510
WALKER, DR. CHARLES A., 3113 Shamrock South, Tallahasee, Florida 32303
WALL, DR. BETTY' J., Marine Biological Laboratory, Woods Hole, Massachusetts 02543
WALLACE, DR. ROBIN A., P. O. Box Y., Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830
WANG, DR. A., Bedford Road, Lincoln, Massachusetts 01773
WARNER, DR. ROBERT C., Department of Molecular and Cell Biology, Uni- versity of California, Irvine, California 92717
WARREN, DR. LEONARD, Department of Therapeutic Research, Anatomy- Chemistry Bldg., Rm. 337, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19174
WATERMAN, DR. T. H., 610 Kline Biology Tower, Yale University, New Haven, Connecticut 06520
WATSON, DR. STANLEY WAYNE, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543
WEBB, DR. H. MARGUERITE, Department of Biological Sciences, Goucher College, Towson, Maryland 21204
WEBER, DR. ANNEMARIE, Department of Biochemistry, University of Pennsyl- vania School of Medicine, Philadelphia, Pennsylvania 19174
WEBSTER, DR. FERRIS, Associate Director for Research, Woods Hole Oceano- graphic Institution, Woods Hole, Massachusetts 02543
REPORT OF THE DIRECTOR 87
WEIDNER, DR. EARL, Department of Zoology and Physiology, Louisiana State
University, Baton Rouge, Louisiana 70803 WEISS, DR. LEON P., Department of Animal Biology, University of Pennsylvania,
School of Veterinary Medicine, Philadelphia, Pennsylvania 19174 WEISSMANN, DR. GERALD, Professor of Medicine, New York University, 550
First Avenue, New York, New York 10016 WERMAN, DR. ROBERT, Department of Zoology, Hebrew University, Jerusalem,
Israel WHITTAKER, DR. J. RICHARD, Wistar Institute for Anatomy and Biology, 36th
Street at Spruce, Philadelphia, Pennsylvania 19174 \VIERCINSKI, DR. FLOYD J., Department of Biology, Northeastern Illinois
University, Chicago, Illinois 60625 WIGLEY, DR. ROLAND L., National Marine Fisheries Service, Woods Hole,
Massachusetts 02543 WILBER, DR. C. G., Chairman, Department of Zoology, Colorado State U^ni-
versity, Fort Collins, Colorado 80521 WILSON, DR. DARCY B., Department of Pathology, University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania 19174
WILSON, DR. EDWARD O., Department of Zoology, Harvard University, Cam- bridge, Massachusetts 02138 WILSON, DR. T. HASTINGS, Department of Physiology, Harvard Medical School,
Boston, Massachusetts 02115 WILSON, DR. WALTER L., Department of Biology, Oakland University, Rochester,
Michigan 48063 WITKOYSKY, DR. PAUL, Department of Anatomical Sciences, Health Science
Center, State University of New York at Stony Brook, Stony Brook, New
York 11794 WITTENBERG, DR. JONATHAN B., Department of Physiology and Biochemistry,
Albert Einstein College of Medicine, New York, New York 10461 WOELKERLING, DR. WILLIAM J., Department of Botany, Latrobe University,
Bundoora, Victoria, Australia 3083 WOLF, DR. DON P., University of Pennsylvania, School of Medicine, 314 Medical
Labs 6/3, Philadelphia, Pennsylvania 19174
WOODWELL, DR. GEORGE M., Director, The Ecosystems Center, Marine Bio- logical Laboratory, Woods Hole, Massachusetts 02543 WYTTENBACH, DR. CHARLES R., Department of Physiology and Cell Biology,
University of Kansas, Lawrence, Kansas 66045 YNTEMA, DR. C. L., Department of Anatomy, State University of Xew York,
UJpstate Medical Center, Syracuse, New York 13210 YOUNG, DR. DAVID K., Department of the Navy, NORDA, Code 334, NSTL Station,
Missouri 35929 YPHANTIS, DR. DAVID A., Department of Biochemistry and Biophysics, UJni-
versity of Connecticut, Storrs, Connecticut 06268
ZIGMAN, DR. SEYMOUR, University of Rochester School of Medicine and Den- tistry, 260 Crittenden Boulevard, Rochester, New York 14620 ZIMMERMAN, DR. A. M., Department of Zoology, University of Toronto, Toronto
5, Ontario, Canada Z^YEIFACH, DR. BENJAMIN YY., rp Ames, University of California, San Diego,
Lajolla, California 92037
ANNUAL REPORT OF THE MARINE IUOl.(H,ir.\l. LABORATORY
Associate Members
ABELSON, UK. AND MRS. PHILIP II.
ACHESON, DR. AND MRS. GEORGE ACKROYD, DR. AXD MRS. FREDERICK
\Y.
ADELBERG, L)R. AND MRS. EDWARD A. ADELMAN, DR. AND MRS. WILLIAM J. AHEARN, MR. AND MRS. DAVID C. ALLEN, Miss CAMILLA K. ALLEN, MRS. A. D. ALLEN, MRS. ROBERT D. AMBERSON, MRS. WILLIAM R. ARMSTRONG, DR. PHILIP ARMSTRONG, DR. AND MRS. SAMUEL C. ARNOLD, DR. AND MRS. JOHN ATWOOD, DR. AND MRS. KIMBALL C. BACON, MR. ROBERT BALL, DR. AND MRS. ERIC G. BALLANTINE, DR. AND MRS. H. T., JR. BANG, DR. AND MRS. FREDERICK B. BANKS, MRS. AND MRS. W. L. BARROWS, MRS. ALBERT W. BENNETT, MRS. GEORGE F. BENNETT, DR. AND MRS. MICHAEL
V. L.
BERNSTEIN, MR. AND MRS. HERMAN BERNHEIMER, DR. ALAN W. BIGELOW, MRS. ROBERT BLACKBURN, DR. AND MRS. GEORGE L.
BODEEN, MR. AND MRS. GEORGE H. BOETTIGER, DR. AND MRS. EDWARD G. BOLTON, MR. AND MRS. THOMAS C.
BORGESE, MRS. THOMAS A. BOTKIN, DR. DANIEL B. BOWLES, MR. AND MRS. FRANCIS P. BRADLEY, DR. AND MRS. CHARLES C. BRONSON, MR. AND MRS. SAMUEL C. BROWN, DR. AND MRS. DUGALD E. S. BROWN, DR. AND MRS. F. A., JR. BROWN, DR. AND MRS. THORNTON BUCK, MRS. JOHN B.
BUFFINGTON, MRS. ALICE H. BUFFINGTON, MRS. GEORGE
BURGER, DR. AND MRS. MAX M. BURROUGH, MRS. ARNOLD H. BURT, MR. AND MRS. CHARLES E. BUTLER, MR. AND MRS. RHETT W. BUTLER, MRS. E. G. CALKINS, MR. AND MRS. G. N., JR.
CAMPBELL, MR. AND MRS. WORTHING-
TON, JR.
CARLSON, DR. AND MRS. FRANCIS CARLTON, MR. AND MRS. WINSLOW G. CASHMAN, MR. AND MRS. EUGENE R. CHAMBERS, DR. AND MRS. ED\VARD I,. CHENEY, DR. RALPH H. CLAFF, MR. AND MRS. MARK CLARK, DR. AND MRS. ARNOLD M. CLARK, MR. AND MRS. HAYS CLARK, MRS. JAMES McC. CLARK, DR. AND MRS. LEONARD B. CLARK, MR. AND MRS. W. VAN ALAN CLEMENT, DR. AND MRS. A. C. CLOWES FUND, INC. CLOWES, MR. ALLEN W. CLOWES, DR. AND MRS. G. H. A., JR. COHEN, DR. AND MRS. SEYMOUR CONNELL, MR. AND MRS. W. J. COOPER, MR. AND A/IRS. JOHN H., JR. COPELAND, MRS. D. EUGENE
COPELAND, MR. AND MRS. PRESTON S.
COSTELLO, MRS. DONALD P.
CRAMER, MR. AND MRS. IAN D. W.
CRANE, MR. JOHN
CRANE, JOSEPHINE FOUNDATION
CRANE, MRS. W. CAREY
CROSS, MR. AND MRS. NORMAN C.
CROSSLEY, MR. AND MRS. ARCHIBALD
M.
CROWELL, DR. AND MRS. SEARS CURTIS, DR. AND MRS. W. D. DAIGNAULT, MR. AND MRS. A. T. DANIELS, MR. AND MRS. BRUCE G. DANIELS, MRS. F. HAROLD DAY, MR. AND MRS. POMEROY DuBois, DR. AND MRS. A. B.
DUNKERLEY, MR. AND MRS. GORDON H.
DuPoNT, MR. A. FELIX, JR. DYER, MR. AND MRS. ARNOLD W. EASTMAN, MR. AND MRS. CHARLES E. EBERT, DR. AND MRS. JAMES D. EGLOFF, DR. AND MRS. F. R. L. ELLIOTT, MRS. ALFRED M. ELSMITH, MRS. DOROTHY O. EPEL, MRS. DAVID EVANS, MR. AND MRS. DUDLEY
REPORT OF THE DIRECTOR
89
EWING, DR. AND MRS. GIFFORD C. FENNO, MRS. EDWARD N. FERGUSON, DR. AND MRS. J. ]., JR. FINE, DR. AND MRS. JACOB FIRESTONE, MR. AND MRS. EDWIN FISHER, MRS. B. C. FISHER, MR. FREDERICK S., Ill FISHER, DR. AND MRS. SAUL H. FRANCIS, MR. AND MRS. LEWIS W., JR. FRIES, DR. AND MRS. E. F. B. FULLER, MR. AND MRS. BENNETT B. FYE, DR. AND MRS. PAUL M. GABRIEL, DR. AND MRS. MORDECAI L. GAISER, DR. AND MRS. DAVID W. GARFIELD, Miss ELEANOR GARREY, DR. AND MRS. WALTER GELLIS, DR. AND MRS. SYDNEY GERMAN, DR. AND MRS. JAMES L., Ill GIFFORD, MR. AND MRS. JOHN A. GIFFORD, DR. AND MRS. PROSSER GILBERT, DR. AND MRS. DANIEL L. GILDEA, DR. MARGARET C. L. GILLETTE, MR. AND MRS. ROBERT S. GLASS, DR. AND MRS. H. BENTLEY GLAZEBROOK, MRS. JAMES R. GLUSMAN, DR. AND MRS. MURRAY GOLDMAN, DR. AND MRS. ALLEN S. COLORING, DR. IRENE P. GOLDSTEIN, MRS. MOISE H., JR. GRANT, DR. AND MRS. PHILIP GRASSLE, MR. AND MRS. J. K. GREEN, Miss GLADYS M. GREENE, MR. AND MRS. WILLIAM C. GREER, MR. AND MRS. W. H., JR. GREIF, DR. AND MRS. ROGER L. GROSCH, DR. AND MRS. DANIEL S. GROSS, MRS. PAUL R. GRUSON, MRS. MARTHA GUNNING, MR. AND MRS. ROBERT HALVORSON, DR. AND MRS. HARLYN O. HANDLER, DR. AND MRS. PHILIP HARVEY, DR. AND MRS. EDMUND N.,
JR.
HARVEY, DR. AND MRS. RICHARD B. HASKINS, MRS. CARYL P. HASSETT, DR. AND MRS. CHARLIE HASTINGS, MRS. J. WOODLAND HAWKINS, MR. RICHARD H. Ill HEFFRON, DR. AND MRS. RODERICK
HENLEY, DR. CATHERINE HIAM, MR. AND MRS. E. W. HIATT, DR. AND MRS. HOWARD HIBBARD, Miss HOPE HILL, MRS. SAMUEL E. HlLSINGER, MR. AND MRS. ARTHUR HlRSCHFELD, MRS. NATHAN B. HOBBIE, DR. AND MRS. JOHN HOCKER, MR. AND MRS. LON HOPKINS, MRS. HOYT S. HORWITZ, DR. AND MRS. NORMAN H. HOUGH, MRS. GEORGE A., JR. HOUSTON, MR. AND MRS. HOWARD PI.
HUETTNER, DR. AND MRS. ROBERT HUNZIKER, MR. AND MRS. HERBERT E. HUTCHINSON, MR. AND MRS. JOHN
HYNES, MRS. NICOLE E. INOUE, MRS. SHINYA IRELAND, MRS. HERBERT A. ISSOKSON, MR. AND MRS. ISRAEL IVENS, DR. SUE JANNEY, MRS. WISTAR JEWETT, G. F., FOUNDATION JEWETT, MR. AND MRS. G. F., JR. JONES, MR. AND MRS. DEWITT, III JORDAN, DR. AND MRS. EDWIN P. KAAN, DR. HELEN W. KAHLER, MR. AND MRS. GEORGE A. KAHLER, MRS. ROBERT W. KAIGHN, DR. MORRIS E. KAMINER, MRS. BENJAMIN KARUSH, DR. AND MRS. FRED KEITH, MR. JEAN R. KEOSIAN, MRS. JESSIE
KlEN, MR. AND MRS. PlETER
KINGWELL, THE REV. AND MRS. WIL- BUR J.
KlNNARD, MR. AND MRS. L. R. KIVY, DR. AND MRS. PETER KOELSCH, MR. AND MRS. HERBERT KOHN, DR. AND MRS. HENRY I. ROLLER, DR. AND MRS. LEWIS R. KRIS, DR. AND MRS. ANTON O. KUFFLER, MRS. STEPHEN W. LADERMAN, MR. AND MRS. EZRA LASH, DR. AND MRS. JAMES LASTER, DR. AND .MRS. LEONARD LAUFER, DR. AND MRS. HANS LAWRENCE, MR. FREDERICK V.
90
ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
LAWRENCE, MRS.. WILLIAM
LAZAROW, MRS. ARNOLD
LEMANN, MRS. LUCY B.
LENHER, MR. AND MRS. SAMUKI.
LEVINE, DR. AND MRS. RACHMIEL
LEVENTHAL, Ms. MONIKA MEYER
LEWIS, MR. T. HOHN
LILLIE, MRS. KARL C.
LILLY, MR. AND MRS. JOSIAH K., Ill
LOBB, PROF. JOHN
LOEH, MRS. ROBERT F.
LONG, MRS. G. C.
LORAND, MRS. LASZLO
LOWENGARD, MRS. JOSEPH
LOWE, DR. AND MRS. CHARLES W.
LURIA, DR. AND MRS. S. E.
MACKEY, MR. AND MRS. WILLIAM K.
MACLEISH, MRS. WILLIAM
MACNARY, MR. B. GLENN
MAcNicHOL, DR. AND MRS. EDWARD
F.,jR.
MAKER, Miss ANNE CAMILLE MARKS, DR. AND MRS. PAUL A. MARSLAND, DR. AND MRS. DOUGLAS MARTYNA, MR. AND MRS. JOSEPH MARVIN, DR. DOROTHY H. MASER, DR. AND MRS. MORTON MASTROIANNI, DR. AND MRS. L., JR. MATHER, MR. AND MRS. FRANK J., Ill MATTHIESSEN, MR. AND MRS. G. C. MAYOR, MRS. JAMES W., SR.
McCuSKER, MR. AND MRS. PAUL T.
MCELROY, MRS. NELLA W.
McGlLLICUDDY, DR. AND MRS. J. J.
McLANE, MRS. T. THORNE MEIGS, MR. AND MRS. ARTHUR MEIGS, DR. AND MRS. J. WISTER MELILLO, DR. AND MRS. JERRY THE MELLON FOUNDATION METZ, MRS. CHARLES B. MEYERS, MR. AND MRS. RICHARD MILLER, DR. DANIEL A. MIXTER, MR. AND MRS. W. J., JR. MONTGOMERY, DR. AND MRS. CHARLES
H.
MOORE, MR. JOHN W. MORSE, MR. AND MRS. CHARLES L., J R. MORSE, MR. AND MRS. RICHARD S. MOSES, MR. AND MRS. GEORGE L.
Mori., MRS. EDWIN T. NEUBERGER, MRS. HARRY H. NEWTON, C. H., BUILDERS, INC. NEWTON, Miss HELEN K. NICHOLS, MRS. GEORGE NlCKERSON, MR. AND MRS. FRANK L. NORMAN, MR. AND MRS. ANDREW E. NORMANDIE FOUNDATION O'HERRON, MR. AND MRS. JONATHAN OLMSTED, MR. AND MRS. CHRISTOPHER ORTINS, MR. ARMAND PAPPAS, DR. AND MRS. GEORGE D. PARK, MR. AND MRS. FRANKLIN A. PARK, MR. AND MRS. MALCOLM S. PARMENTER, Miss CAROLYN L. PARMENTIER, MR. GEORGE L. PATTEN, MRS. BRADLEY M. PECAN, Ms. ERENE V. PENDERGAST, MRS. CLAUDIA PENDELTON, 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 PHILIPPE, MR. AND MRS. PIERRE PORTER, DR. AND MRS. KEITH R. PROSSER, MRS. C. LADD PUTNAM, MR. ALLAN RAY PUTNAM, MR. AND MRS. W. A., Ill RATCLIFFE, MR. THOMAS G., JR. RAYMOND, DR. AND MRS. SAMUEL READ, Ms. LEE
REDFIELD, DR. AND MRS. ALFRED C. RENEK, MR. AND MRS. MORRIS REYNOLDS, DR. AND MRS. GEORGE REYNOLDS, MR. AND MRS. JAMES T. REZNIKOFF, DR. AND MRS. PAUL RIGGS, MR. AND MRS. LAWRASON, HI RIINA, MR. AND A/IRS. JOHN R. ROBB, Ms. ALISON A. ROBERTSON, MRS. C. STUART ROBERTSON, DR. AND MRS. C. W. ROBINSON, DR. AND MRS. DENIS M. ROGERS, MRS. JULIAN ROOT, MRS. WALTER S. Ross, MRS. JOHN ROWE, MRS. WILLIAM S.
REPORT OF THE TREASURER
91
RUBIN, DR. JOSEPH
RUGH, MRS. ROBERTS
RUSSELL, MR. AND MRS. HENRY D.
RYDER, MR. AND MRS. FRANCIS C.
SAUNDERS, DR. AND MRS. JOHN \Y.
SAUNDERS, MRS. LAWRENCE
SAVERY, MR. ROBER
SAWYER, MR. AND MRS. JOHN E.
SCHLESINGER, MRS. R. WALTER
SCOTT, MRS. GEORGE T. SCOTT, MRS. XORMAN SEARS, MR. AND MRS. HAROLD B. SEGAL, DR. AND MRS. SHELDON SHAPIRO, MRS. HARRIET S. SHEMIN, DR. AND MRS. DAVID SHEPROW, DR. AND MRS. DAVID SHERMAN, DR. AND MRS. IRWIN
SlMKINS, MRS. WlLLARD S.
SLATER, MR. DAVID
SMITH, MR. AND MRS. DIETRICH C.
SMITH, MRS. HOMER P.
SMITH, MR. AND MRS. ROBERT I.
SMITH, MR. YANDORN C.
SNIDER, MR. ELIOT
SONNEBEND, MR. AND MRS. PAUL
STRACHER, DR. AND MRS. ALFRED STEINBACH, DR. AND MRS. H. B. STETTEN, DR. AND MRS. DEWITT, JR. STONE, DR. AND MRS. WILLIAM STUART, ANN STUNKARD, DR. HORACE SWANSON, DR. AND MRS. CARL P. SWOPE, MR. AND MRS. GERARD L. SWOPE, MR. AND MRS. GERARD, JR. SWOPE, Miss HENRIETTA H. TANNER, DR. AND MRS. HARVEY A. TARTAKOFF, DR. HELEN
TAYLOR, DR. AND MRS. W. RANDOLPH TIETJE, MR. AND MRS. EMIL D., JR. TITTLER, MRS. SYLVIA TODD, MR. AND MRS. GORDON F.
TOLKAN, MR. AND MRS. NORMAN X.
TOMPKINS, MRS. B. A. TRACER, MRS. WILLIAM TROLL, DR. AND MRS. WALTER TULLY, MR. AND MRS. GORDON F. VALOIS, MR. AND MRS. JOHN VEEDER, MRS. RONALD A. VINCENT, MRS. WALTER S. WAKSMAN, DR. AND MRS. BRYON H. WARE, MR. AND MRS. J. LINDSAY WARREN, DR. AND MRS. SHIELDS WATT, MR. AND MRS. JOHN B. WEISBERG, MR. AND MRS. ALFRED M, WENGREN, MR. RICHARD WEXLER, ROBERT H. FOUNDATION WHEATLEY, DR. MARJORIE A. WHEELER, DR. AND MRS. PAUL S. WHEELER, DR. AND MRS. RALPH E. WHITNEY, MR. AND MRS. GEOFFREY G., JR.
WlCHTERMAN, DR. AND MRS. RALPH WlCKERSHAM, MR. AND MRS. A. A.
TlLNEY WlCKERSHAM, MRS. JAMES H., JR.
WILHELM, DR. HAZEL S. WILSON, MR. AND MRS. ROBERT E., J K. WlTMER, DR. AND MRS. ENOS E. WOLFINSOHN, MR. AND MRS. WOLFE WOODWELL, MRS. GEORGE YNTEMA, DR. AND MRS. CHESTER L. ZINN, DR. AND MRS. DONALD J. ZWILLING, MRS. EDGAR
VI. REPORT OF THE LIBRARIAN
This year we received some relief in the crowded stack area. Dr. Gross ap- proved the purchase of 300 additional metal shelves to be added to the top stack in the wing. This stack was not filled to capacity when originally built, so we were able to fit nine banks of shelving along each side and five banks at the back without crowding the Reserve Desks on the top floor. This new shelf space will hold approximately three years' growth in journal acquisitions. Before the end of that period we expect to have additional space in the Lillie Building made available to the Library.
Relief for Library salaries was also achieved this year. Specific MBL em- ployee salaries were presented to the Members of the Board and Executive Com-
U2 ANNUAL KKI'OKT OK THE MAR1XE BIOLOGICAL LABORATORY
mittee. The result was increased salaries for many, somewhat comparable to those at the Woods Hole Oceanographic Institution.
A thorough inventory of the book section revealed a total of 25,000 volumes. When added to the journal collection, the complete volume count is now 180,000.
For this annual printed record, I would like to include a part of the preface of Dr. Stephen J. Gould's new book, "Ontogeny and Phylogeny," Harvard Uni- versity Press, 1977:
''. . . and, above all, to an institution that has its own humanity and seems to me more an organism than a place — the Library of the Marine Biological Laboratory at Woods Hole. XVhere else would an idiosyncratic worker like me find a library open all the time, free from the rules and bureaucracy that stifle scholarship and "protect" books only by guarding them from use. It is an anomaly in a suspicious and anonymous age. May it survive as it is, despite all the improbabilities."
VII. REPORT OF THE TREASURER
I am pleased to report that the Marine Biological Laboratory made progress in 1978 in reducing its operating deficit. In 1978, the operating loss was $35,454, a reduction from $85,414 in 1977. Operating results excluded approximately $282,000 of depreciation charges recorded in each year.
Total revenues in 1978 amounted to $2,502,077, an increase of $420,583 over the prior year. The principal sources of the increased revenues were higher tuition as the result of expanded programs and increased overhead recovery due to a 5% increase in the overhead rate, coupled with increased year-round occupancy. In addition, unrestricted gifts increased by almost $100,000.
Total 1978 expenses amounted to $2,537,531, an increase of $370,623 over 1977. This increase was principally due to performing critical plant repairs which had been deferred in prior years for budgetary reasons, expansion of the Continuing Education programs begun in late 1977, the addition of two new course offerings, and a significant increase in the volume of the Chemical Room. Of this $2,537,531, recurring operating expenses represented $2,418,697, an in- crease of $251,789, or 12%, over 1977. In our report last year, we mentioned that our 1978 budget anticipated that such expenses would increase only 5% from the 1977 level. This unanticipated and unfavorable variation in the expense budget is mainly attributable to the additional Continuing Education course offerings and greater volume in the Chemical Room — both of which generated additional offsetting revenues.
Investment income amounted to $341,846 in 1978, an increase of 6% over 1977. Total investments at December 31, 1978 were valued at $5,605,947 at market and $5,739,745 at cost. The return, as a percentage of average market value, was 6.2%.
As was mentioned to you last year, methods ot improving the rate ot return on investments were to be examined during 1978. Accordingly, based on the recommendation of the Investment Committee, the Executive Committee, at its February 1979 meeting, appointed Standish, Ayer & Wood, Inc. of Boston to be the investment advisor for all of the Laboratory's investment portfolios.
We have budgeted in 1979 for a further reduction in the operating deficit and our objective for 1980 is to record a modest surplus.
REPORT OF THE TRKASTRER 93
The following is a statement of the auditors: To the Trustees of Marine Riologicul Laboratory, Woods Hole, Massachusetts:
\Ye have examined the balance sheets of Marine Biological Laboratory as of December 31, 1978 and 1977, and the related statements of current funds revenues, expenditures, and other changes and changes in fund balances for the years then ended. Our examinations were made in accordance with gen- erally accepted auditing standards and, accordingly, included confirmation from the custodians of securities owned at December 31, 1978 and 1977, and such tests of the accounting records and such other auditing procedures as we considered necessary in the circumstances.
As more fully described in Note B to the financial statements, the Labora- tory excludes certain costs of buildings and equipment from the balance sheet. In our opinion, generally accepted accounting principles require that such costs be included as investment in plant in the financial statements.
In our opinion, except for the effects on the financial statements of the matter discussed in the preceding paragraph, the aforementioned financial statements present fairly the financial position of Marine Biological Labora- tory at December 31, 1978 and 1977, and its current funds revenues, expen- ditures and other changes and the changes in fund balances for the years then ended, in conformity with generally accepted accounting principles applied on a consistent basis.
Our examinations were made primarily for the purpose of rendering an opinion on the basic financial statements (pages 94 to 99, inclusive), taken as a whole. The summary of investments (page 102) although not considered necessary for a fair presentation of the financial position at December 31, 1978 and 1977, current fund revenues, expenditures and other changes, and the changes in fund balances for the years then ended in accordance with generally accepted accounting principles, is presented primarily for purposes of supplementary analysis. This additional information has been subjected to the audit procedures applied in the examination of the basic financial statements and, in our opinion, is fairly stated in all material respects in rela- tion to the basic financial statements taken as a whole.
Boston, Massachusetts
March 30, 1979 COOPERS & LYHRAND
94 ANNUAL REPORT OF TIIH MARINK BIOLOGICAL LABORATORY
MARINK BIOLOGICAL LABORATORY BALANCE SHEETS
December 31, 1978 and 1977
Assets 1078 1977
Current Funds:
Unrestricted:
Cash, including deposits at interest $ 382,396 $ 517,513
Accounts receivable, net of allowance for
uncollectible accounts of $4,500 in 1978
and $12,955 in 1977 699,708 540,743
Other assets 15,367 15,996
Due to restricted current funds. .. (278,075) (294,485)
Due from invested funds. .. 103,248 105,133
Total unrestricted .. 922,644 884,900
Restricted :
Cash 18,973 87,013
Investments, at cost; market value: 1978 —
$1,297,551; 1977— $1,118,917 (Note A,
Schedule I) 1,297,530 1,112,683
Due from unrestricted current fund 278,075 294,485
Due from invested funds 260,967 173,191
Total restricted .. 1,855,545 1,667,372
Total current funds. . $ 2,778,189 $ 2,552,272
Invested Funds:
Cash.... 6.982 102,031 Investments, at cost; market value: 1978 —
$3,950,184; 1977— $3,799,434 (Note A,
Schedule I).. . 4,084,003 3,822,798
Due to unrestricted current fund (103,248) (105,133)
Due to restricted current funds. . (260,967) (173,191)
Total invested funds. . $ 3,726,770 $ 3,646,505
Plant Fund:
Land, buildings and equipment at cost (Note B) . . . . 12,421,929 12,374,587
Less accumulated depreciation 4,060,407 3,778,545
Total plant fund $8,361,522 $ 8,596,042
The accompanying notes are an integral part of the financial statements.
REPORT OF THE TREASURER 95
MARINE BIOLOGICAL LABORATORY BALANCE SHEETS
December 31, 1978 and 1977
Liabilities and Fund Balances 1Q78 1V77
Current Funds: Unrestricted :
Accounts payable and accrued expenses $ 316,657 $ 195,418
Deferred income 61,432 62,131
Fund balance 544,555 627,351
Total unrestricted 922,644 884,900
Restricted :
Fund balances:
Unexpended gifts and grants 1,777,677 1,597,841
Unexpended income of endowment funds. . . 77,868 69,531
Total restricted 1,855,545 1,667,372
Total current funds. .. $ 2,778,189 $ 2,552,272
Invested Funds:
Endowment funds 2,174,027 2.172,160
Quasi-endowment funds 934,143 934,143
Retirement fund (Note C). . 618,600 540,202
Total invested funds $ 3,726,770 $ 3,646,505
Plant Fund:
Invested in plant 8,361,522 8,596,042
Total plant fund. . $ 8,361,522 $ 8,596,042
The accompanying notes are an integral part of the financial statements.
ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
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100 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
MARINE BIOLOGICAL LABORATORY
NOTES TO FINANCIAL STATEMENTS i
A. Significant Accounting Policies:
Basis of Presentation — Fund Accounting
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 activities or objectives specified. In the accompanying financial statements, funds that have similar characteristics have been combined.
Externally restricted funds may only be utilized in accordance with the purposes established by the source of such funds. However, the Laboratory retains full control over the utiliza- tion 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 expended for current operating purposes. Unrestricted revenue is re- ported as revenue in the unrestricted current fund when received.
Endowment funds are subject to restrictions requiring that the principal be invested and only the income utilized. Quasi-endowment funds have been established by the Laboratory for the same purposes as endowment funds, however, any portion of these funds may be expended.
Investments
Investments purchased by the Laboratory are carried at cost. Investments donated to the Laboratory are carried at fair market value at date received. For determination of gain or loss upon disposal, cost is determined based on the specific identification method.
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 the market value at the beginning of the quarter, adjusted for the cost of any additions or disposals during the quarter.
Indirect Cost Recovery
The Laboratory recovers indirect costs by charging occupants of laboratory space fees based on a negotiated fixed indirect cost rate for the period the space was occupied. When actual rates are subsequently determined, the difference is reflected in the next negotiated fixed rate.
REPORT OF THE TREASURER 101
B. Land, Buildings and Equipment:
Following is a summary of the plant fund assets:
Classification 1978 1977
Land $ 639,693 $ 639,693
Buildings 10,190,430 10,143,088
Equipment 1,591,806 1,591,806
12,421,929 12,374,587
Less accumulated depreciation 4,060,407 3,778,545
$ 8,361,522 $ 8,596,042
The original cost of land, buildings and related initial furnishing equipment is capitalized when the assets are acquired. The cost of subsequent additions and purchases, repairs and remodeling is expensed when incurred. Equipment and remodeling expenditures amounted to approximately $125,000 and $110,000 in 1978 and 1977, respectively.
Depreciation is computed using the straight-line method over estimated useful lives of 40 years for buildings and 20 years for equipment.
C. Retirement Fund:
The Laboratory has a noncontributory pension plan for substantially all full-time em- ployees which complies with the requirements of the Employee Retirement Income Security Act of 1974. The actuarially determined pension expenses charged to operations in 1978 and 1977 were $76,374 and $64.277, respectively. The Laboratory's policy is to fund pension costs accrued.
D. Pledges and Grants:
As of December 31, 1978 and 1977, the following amounts remain to be received from previous gifts and grants for specific research and instruction programs, and are expected to be received as follows:
1978 1977
1978 $1,697,486
1979 $2,196,201 115,000
1980 28,000
$2,224,201 $1,812,486
102 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
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CYTOLOGY AND POLYSACCHARIDE CYTOCHEMISTRY OF THE GILL OF THE AMERICAN EEL, ANGV1LLA RO STRATA
DENNIS J. BIRD1 AND ALBERT F. EBLK
Department of Bioloi/y. Trenton State College. Trenton, Nczv Jerscv, 08625
Much work has been published on the gills of teleosts, (Vickers, 1961 ; Munshi, 1964; Steen and Kruysse, 1964; Hughes and Grimstone. 1965; Newstead, 1967; Hughes and Wright, 1970). Morgan and Tovell (1973) and Morgan (1974) described the structure and development of secondary lamellae in gills of trout. Work on eel gills has included cytological studies, ( Ogawa, 1962; Yamada and Yokote, 1975), electron microscopic investigations of gill fine structure (Mizuhira, Amakawa, Yamashina, Shirai, and Utida, 1969), and studies of osmotic adapta- tion of eels to freshwater and sea water for the Japanese eel, Anguilla japonica. Keys and Willmer (1932) described chloride-secreting cells in the common eel, Anguilla vulgaris ; Getman (1950) and Doyle and Epstein (1972) reported on osmotic effects and adaptive changes of chloride cells in the American eel, Anguilla ro strata.
The present study describes cytological details of gill filaments and secondary lamellae of freshwater-adapted early juvenile and adult American eels. Arrange- ment and morphology of epithelium, gill rays, mucous-secreting cells, pillar cells, and blood spaces within secondary lamellae are described and illustrated. Poly- saccharide cytochemistry as revealed by periodic-acid-Schiff (PAS) staining as well as Alcian blue reactions at various pH values and with increasing concentra- tions of MgClL> of all cell types and connective tissue are described ; results are compared with similar tissues in the Anguilla species, and in teleosts in general.
MATERIALS AND METHODS
Fresh water-adapted juvenile and adult eels were obtained from the Aquaculture Laboratories, Mercer Generating Station, Trenton, New Jersey. Animals were killed by decapitation and gills were excised and fixed in Davidson's solution at room temperature for 24 hr. Tissues were processed and embedded in paraffin (Humanson, 1972) ; sections were cut at 5 /xm.
Stains used were hematoxylin and eosin, periodic-acid-Schiff (PAS) (McManus, 1948), Feulgen Picro-Amido-black (modified from Farley, 1969), Alcian blue 8GX (Gurr, London) at pH 0.5 (Lev and Spicer, 1964). Alcian blue pH 2.6, and Alcian blue pH 5.7 with post-treatment in ascending concentrations of MgCL (Scott and Dorling, 1965; Mowry, 1970).
Slides were examined and photographed with the Zeiss Photomicroscope II using Kodak SO-410 Monochrome Photomicrography film with a Kodak #66 Wratten gelatin filter.
1 Present address : Department of Biology, Northeastern University, Boston, Massachusetts 02115.
104
CYTOLOGY OF GILLS OF AMERICAN EEL
105
FIGURE 1. Cartilaginous gill rays (CT) support filaments of the eel gill. Mucus-secret- ing cells (MC) appear at bases of filaments. Hematoxylin and eosin stained; scale bar is 25 /on.
FIGURE 2. Basement membrane (BM) in gill filament stained by PAS. Scale bar is 25 ,um.
FIGURE 3. Two-celled layered epithelium of secondary lamella of adult eel. Nuclei of inner cell layer (IEP) shown over pillar cell body (PC) ; pillar cells delimit blood spaces (BS) in lamellae. Note nucleus of outer layer of epithelium (OEP) ; scale bar is 10 /on.
FIGURE 4. Secondary lamellae of early juvenile eel, showing single epithelial layer (EP) with nuclei over pillar cells (PG). Blood cells crowd blood spaces between pillar cells. Hematoxylin and eosin stained ; scale bar is 10 fj.ni.
FIGURE 5. Chloride cells (CC) at the base of lamellae near afferent lamellar artery (AA). Note the orominent nucleolus. visible with Feuleen Picro-Amido-Black stain: scale bar is 10 Mm.
106
D. J. BIRD AND A. F. EBLE
OEP
IEP
FIGURE 6. Diagram of secondary lamella of an adult eel. Pillar cells (PC) and their cytoplasmic flanges (PCF) delimit blood spaces (BS) within lamella. Two-cell layered epithelium supported by basement membrane (BM) covers secondary lamella. Nuclei (NU) of the inner epithelium (IEP) are more flattened than those of the outer epithelial cells (OEP) and lie over pillar-cell bodies. Scale bar is 5 /mi.
RESULTS
General morphology
Gills of the eel consist of four bony gill arches on either side of the pharynx. Gill arches bear two rows of flattened filaments supported for approximately two- thirds of their length by cartilagenous gill rays. Filaments each bear two rows of secondary lamellae.
Cytology and cytochemistry
Gill filaments contain a wide, centrally-located gill ray (Fig. 1) which stains intensely in PAS, Alcian bine pH 0.5, and Alcian blue pH 5.7 with high concentra- tions of MgClo (Table I). Large mucus-secreting cells are found at both base and tip of filaments ; cell secretions stain prominently with PAS reactions as well as with Alcian blue pH 2.6. They appear unstained in Alcian blue pH 0.5. A well-defined basement membrane supports the filament epithelium (Fig. 2) ; this connective tissue encircles afferent lamellar arteries and extends into secondary lamellae as the basement membrane for lamellar epithelia. It is stained intensely by PAS, Alcian blue pH 0.5, and Alcian blue pH 5.7 with concentrations of MgClo up to 1.30 M (Table I).
CYTOLOGY OF GILLS OF AMERICAN EEL 107
Blood supplied by afferent lamellar arteries flows through blood spaces delimited by pillar cells and their cytoplasmic flanges (Figs. 3, 6). Pillar cell nuclei typically contain two small areas of chromatin material with fine granular material distributed around the margins of the nuclear envelope. Nuclei occupy nearly the entire volume of the cell, with a width of approximately 4 p.m and a height of 8 ^m.
A two-layered epithelium covers secondary lamellae in adult eels, supported by a basement membrane continuous along pillar-cell flanges. Nuclei of inner epithelial layers frequently lie directly over pillar-cell bodies (Figs. 3, 6), with nuclei flattened against the basement membrane. Several dense areas of chromatin appear near the center of the nucleus, with smaller areas of chromatin adhering to the nuclear envelope. Nuclei of the outer epithelial layer appear more spherical than inner epithelial nuclei ; one dark nucleolus is usually visible sur- rounded by smaller chromatin masses (Fig. 6). Cytoplasm of outer epithelial cells is much less dense than that of inner epithelial cells, with distal portions of cytoplasm staining lightly with Alcian blue pH 2.6. A single-layer epithelium covers secondary lamellae in early juvenile eels (Fig. 4) ; epithelial nuclei are located over pillar-cell bodies.
Chloride-secreting cells surround afferent lamellar arteries, crowded in the "v" between adjacent secondary lamellae (Fig. 5). Cells are large, with a granular, eosinophilic cytoplasm and a large nucleus with a prominent nucleolus surrounded by small regions of chromatin material (Fig. 5). Crowding of cells in this area of filaments causes them to be elongated, with nuclei typically located at the base of cells. Chloride-cell cytoplasm stains heavily in PAS reactions and exhibits some reaction to Alcian blue pH 2.6, especially at distal portions of cells. In Alcian blue pH 0.5 there is little reaction in chloride cells; further, in Alcian blue pH 5.7, cells reach their extinction point at a critical electrolyte concentration of 0.030 M MgClo (Table I).
DISCUSSION
Cartilage supporting gill filaments shows heavy concentrations of sulfated muco- polysaccharides, staining with Alcian blue pH 0.5 (Lev and Spicer, 1964). Poly- sulfate groups stain selectively as pH is lowered to a point below the pH of carboxyl groups ; sulfate groups still dissociated are free to bind the cationic dye. Mowry (1963) reported that, at low pH, hyaluronic acid, heparin, and chondroitin readily stain in Alcian blue ; the color reactions are identical but chondroitin com- prises only a minor portion of extracellular material. However, the sulfate ester derivatives, chondroitin sulfate A and chondroitin sulfate C, are major structural components of vertebrate cartilage (Lehninger, 1970). In Alcian blue pH 5.7 cartilage reacts intensely when treated with high concentrations of MgClo (Table I). Mowry (1970) noted that binding of the cationic dye in 0.30 M or higher concentrations of MgClo indicated the presence of sulfated polvanions ; the higher the ionic strength of Mg2+ the higher the degree of sulfation. Addition of MgCU dissolves the stained complexes formed by the polyanion's reaction with Alcian blue (Scott and Dorling, 1965). The critical electrolyte concentration (lowest concentration of Mg2+ at which a given polyanion is no longer stainable) is 1.20M MgClo for gill-ray cartilage. The presence of highly sulfated chondroitin
10S
D. J. BIRD AND A. F. EBLE
TABLE I
Results of cytochemical tests .
|
Stain |
Mucous cells |
Basement membrane |
Cartilage |
Chloride cells |
|
Hematoxvlin and eosin |
+ |
+ + |
+ + + |
+ + + |
|
PAS |
+ + + + + |
+ + + + |
+ + + |
+ + + |
|
Feulgen-Picro Amido-black |
+ + |
+ + + |
+ + + |
+ + + + |
|
Alcian blue pH 0.5 |
|
+ + + + + |
+ + + + |
+ + |
|
Alcian blue pH 2.6 |
+ + + + |
+ + |
+ + + |
+ + |
|
Alcian blue pH 5.7 with |
||||
|
0.00 M MgCl2 |
+ + + |
+ + + |
+ + + |
+ + |
|
0.05 M MgCl2 |
+ + + |
+ + + |
+ + + |
+ + |
|
0.10 M MgCl, |
+ + + |
+ + + |
+ + + |
+ |
|
0.20 M MgCl, |
|
+ + + |
+ + + |
+ |
|
0.30 M MgCl2 |
|
+ + + |
+ + + |
— |
|
0.40 M MgCl, |
|
+ + + |
+ + + |
|
|
0.50 M MgCl2 |
|
+ + + |
+ + + |
|
|
0.60M MgCl2 |
|
+ + + |
+ + + |
|
|
0.70M MgCl2 |
|
+ + + + |
+ + + |
|
|
0.80 M MgCI2 |
|
+ + + + |
+ + + |
|
|
0.90 M MgCl2 |
|
+ + + + |
+ + |
|
|
LOOM MgCl2 |
|
+ + + + |
+ + |
|
|
1.10M MgCl2 |
|
+ + + + |
+ + |
|
|
1.20M MgCl2 |
|
+ + + + + |
+ + |
|
|
1.30M MgCl2 |
— — — |
+ + + + + |
|
• — • — — |
— , No reaction; ++, weak reaction;
strong reaction.
derivatives in cartilage accounts for the intensity of reactions to Alcian blue pH 0.5 and Alcian blue pH 5.7 with high concentrations of MgClo-
Mucous cells found on filaments at both base and tip have secretions which exhibit properties of acid mucopolysaccharides with vicinal hydroxyl as well as carboxyl groups. Cell secretions stain heavily in Alcian blue pH 2.6, with no staining in Alcian blue pH 0.5. Further, mucous cell secretions reach their extinction point at a critical electrolyte concentration of 0.20 M MgCl2 in Alcian blue pH 5.7 series (Table I). Mowry (1963) stated that basophilia in a 0.30 M or lower concentrations of MgCb indicated the presence of polycarboxylates. In addition, Alcian blue is a reliable and sensitive test for carbohydrate polycar- boxylates.
Some variation in the exact chemical structure of mucus secretions apparently exists within the Anguilla species. Yamada and Yokote (1975) reported the presence of sulfated mucopolysaccharides in mucous cells of the Japanese eel, Anguilla japonica, and described staining of cells in a range from 0.10 to 0.60 M MgCl2 ; those secretions show a higher degree of sulfation than those of Anguilla rostrata using similar procedures (Table I). Mucous cells always appear at the base and tip of gill filaments in the eel, as described for trout (Morgan and Tovell, 1973) and many other teleosts (Newstead, 1967; Hughes and Wright, 1970).
Electron microscope studies on epithelial basement membranes in gills from rainbow trout (Morgan and Tovell, 1973) and other teleosts (Hughes and Grim-
CYTOLOGY OF GILLS OF AMERICAN EEL 109
stone, 1965; Newstead, 1967) showed a close association of basement membrane and pillar-cell flanges. In the present study, epithelial basement membrane is also in contact with cytoplasmic flanges of pillar cells (Fig. 6) ; it also surrounds afferent lamellar arteries at bases of secondary lamellae (Fig. 2) and supports gill-filament epithelia. The basement membrane shows heavy concentrations of sulfated mucopolysaccharides, staining intensely in Alcian blue pH 0.5 ; in Alcian blue pH 5.7 the basement membrane can also be distinguished when treated with concentrations of MgClo up to 1.30 M (Table I). Magnesium chloride provides better discrimination of polyanions than other salts (Scott and Dorling, 1965) and the critical electrolyte concentration is a reflection of the type of polyanions pres- ent, as well as the concentration. In Anguilla ro strata, basement membranes exhibit an even greater degree of sulfation than gill-ray cartilage (Table I).
Blood supplied to secondary lamellae is channeled through blood spaces formed by overlapping pillar cell flanges (Fig. 6). These blood spaces appear to be somewhat similar to capillaries ; however, no endothelium could be dis- cerned. Studies on trout (Morgan and Tovell, 1973) and other teleosts (Hughes and Grimstone, 1965 ; Newstead, 1967 ; Hughes and Wright, 1970) indicated that blood spaces in secondary lamellae were entirely delimited by extensions of pillar- cell cytoplasm. Morgan (1974) further stated that, developmentally, pillar cells originate directly from mesenchymal cells, and not from cells having affinities to endothelial cells.
Above the pillar cells and basement membrane a two-cell, layered epithelium covers secondary lamellae in adult eels (Fig. 3). Nuclei of inner epithelial cells lie directly over pillar cell bodies (Figs. 3, 6) ; similar arrangements have been reported to exist in trout (Morgan and Tovell, 1973) and many other teleosts (Newstead, 1967; Hughes and Wright, 1970). Hughes and Grimstone (1965) suggested that location of epithelial nuclei over pillar cell bodies could be adaptive, as little gas exchange would be expected at those points. In the present study, the average water-to-blood distance for adult eels is 5 to 8 /xm, but at points where epithelial nuclei are located, the distance is nearly doubled from the free edge to the blood spaces. In early juvenile eels (2-3 g body weight) there is only one epithelial layer in secondary lamellae; nuclei of these cells invariably lie over pillar cell bodies (Fig. 4). Here, the water-to-blood distance is only 3 to 4 jam.
Keys and Willmer (1932) reported only a single layer of epithelium in second- ary lamellae of the common eel, Anguilla vulgaris, but they gave no information concerning the size of animals used in their study. From the evidence of the twro cell layers found in Anguilla rostrata and other teleosts, it is apparent that, as the animals mature, a second cell layer appears and the water-to-blood distance increases slightly.
Chloride-secreting cells appear in clusters at bases of secondary lamellae, in close proximity to afferent lamellar arteries (Fig. 5). Cytoplasm of chloride cells exhibits high concentrations of carbohydrate polycarboxylates staining intensely with PAS reactions (Table I) and Alcian blue pH 2.6. In the latter, it is interest- ing to note that staining is limited to the distal portions of chloride cells; basal portions of cells show no reaction to the stain.
Appearance of chloride cells near afferent lamellar arteries is well known for the Japanese eel, Anguilla japonica (Ogawa, 1962; Shirai and Utida, 1970; Utida,
110 D. J. BIRD AND A. F. EBLE
Kamiya, and Shirai, 1971) as well as other teleosts (Vickers, 1961 ; Munshi, 1964; Newstead, 1967). Getman (1950) suggested that in Anguilla rostrata, the location of chloride cells in interlamellar epithelium allows access to a good blood supply and insures exposure to the environment for salt secretion. Shirai and Utida (1970) studied the development and degeneration of chloride cells when eels were adapted to fresh water and to sea water; the secretory mechanism was examined by Utida, ct al. (1971) to determine the relationship between Na+-K+-ATPase and numbers of chloride cells in seawater-adapted animals. In the present study, animals were freshwater adapted, and chloride cells were similar in appearance, location, and stainability to those of freshwater teleosts (Munshi, 1964).
In conclusion, we would like to thank Dr. Joseph A. Vena for his advice and assistance with staining procedures, and Mr. Gerald Nicholls for his suggestions and cooperation on photographic techniques.
SUMMARY
Gills of the American eel were found to be morphologically similar to those of other members of the Anguilla species, and to teleosts in general. Gill filaments contain cartilagenous gill rays rich in polysulfates, and stain intensely in PAS and Alcian blue pH 0.5.
Pillar cells delimit blood spaces within secondary lamellae ; they were found to be covered by a thin connective tissue supporting a single-layered epithelium in early juvenile animals and a two cell, layered epithelium in adult eels. In the latter, nuclei of the outer layer were much larger and not as densely stained as those of the inner epithelial cells, whose nuclei appeared flattened over pillar-cell bodies.
Basement membranes supporting epithelia of secondary lamellae and gill fila- ments exhibited heavy concentrations of sulfate groups shown by reactions in Alcian blue pH 0.5 and Alcian blue pH 5.7 with high concentrations of MgClo.
Chloride cells were found in the interlamellar epithelium, especially surrounding afferent lamellar arteries. They had a granular, eosinophilic cytoplasm with carbo- hydrate polycarboxylates concentrated in distal portions of cells; nuclei had a prominent, centrally-situated nucleolus surrounded by small chromatin masses.
Results of cytochemical tests for all cell types were reported, and information correlated to previous findings on eel gills in particular, and teleost gills in general.
LITERATURE CITED
DOYLE, W. L., AND F. H. EPSTEIN, 1972. Effects of cortisol treatment and osmotic adaptation
on the chloride cells in the eel, Anguilla rostrata. Cytobiologie, 6 : 58-73. FARLEY, C. A., 1969. Probable neoplastic disease of the hematopoietic system in oysters,
Crassostrca virginica and Crassostrea gigas. Natl. Cancer hist. Monogr., 31 : 541-555. GETMAN, H. C., 1950. Adaptive changes in the chloride cells of Anguilla rostrata. Biol. Bull.,
99 : 439-445. HUGHES, G. M., AND A. V. GRIMSTONE, 1965. The fine structure of the secondary lamellae of
gills of Gadus pollachius. Q. J. Microsc. Sci., Pt. 4, 106: 343-353. HUGHES, G. M., AND D. E. WRIGHT, 1970. A comparative study of the ultrastructure of the
CYTOLOGY OF GILLS OF AMERICAN EEL 1 1 1
water-blood pathway in the secondary lamellae ot teleost and elasmobranch fishes—
benthic forms. Z. Zcllforsch. Mikrosk. Anat., 104: 478-493. HUMANSON, G. L., 1972. Animal Tissue Techniques. W. H. Freeman and Company, San
Francisco, 641 pp. KEYS, A. B., AND E. N. WILLMER, 1932. "Chloride secreting" cells in the gills of fishes, with
special reference to the common eel. /. Physiol., 76 : 368-378.
LEHNINGER, A. L., 1970. Biochemistry. Worth Publishing Incorporated, New York, 838 pp. LEV, R., AND S. S. SPICER, 1964. Specific staining of sulphate groups with Alcian blue at low
pH. /. Histochem. Cytochcw., 12: 309. McMANUs, J. F. A., 1948. Histological and histochemical uses of Periodic acid. Stain
TechnoL, 23 : 99-108.
MIZUHIRA, V., T. AMAKAWA, S. YAMASHINA, N. SHIRAI, AND S. UTIDA, 1969. Electron micro- scopic studies on the localization of sodium ions and sodium-potassium-activated adeno-
sinetriphosphatase in chloride cells of eel gills. Exp. Cell. Res., 59 : 346-348. MORGAN, M., AND P. W. A. TOVELL, 1973. The structure of the gill of the trout, Salmo
gairdncri (Richardson). Z. Zcllforsch. Mikrosk. Anat., 142: 147-162. MORGAN, M., 1974. Development of secondary lamellae of the gills of trout, Salmo gairdncri
(Richardson). Cell Tissue Res., 151: 509-523. MOWRY, R. W., 1963. The special value of methods that color both acidic and vicinal hydroxyl
groups in the histochemical study of mucins. With revised directions for the colloidal
iron stain, the use of alcian blue 8GX, and their combination with the periodic
acid-Schiff reaction. Ann. N.Y. Acad. Sci., 106: 402-423. MOWRY, R. W., 1970. Analytical methods for carbohydrates. IV. Histochemical methods.
Pages 777-807 in W. Pigman and D. Horton, Eds., The carbohydrates; chemistry and
biochemistry, Vol. 3B, Academic Press, London. MUNSHI, J. S. D., 1964. "Chloride cells" in the gill of freshwater teleosts. Q. J. Microsc. Sci.,
Pt. 1, 105 : 79-89. NEWSTEAD, J. D., 1967. Fine structure of the respiratory lamellae of teleostean gills. Z.
Zcllforsch. Mikrosk. Anat., 79 : 396-428. OGAWA, M., 1962. Chloride cells in Japanese common eel, Anguilla japonica. Sei. Rep.
Saitama Univ. Ser. B. Biol. Earth. Sci., 4: 131-137. SCOTT, J. E., AND J. DORLING, 1965. Differential staining of acid glycosaminoglycans
(Mucopolysaccharides) by alcian blue in salt solutions. Histochemie, 5: 221-233. SHIRAI, N., AND S. UTIDA, 1970. Development and degeneration of the chloride cell during
seawater and freshwater adaptations of the Japanese eel, Anguilla japonica. Z. Zcll-
forsch. Mikrosk. Anat., 103: 247-264. STEEN, J. B., AND A. KRUYSSE, 1964. The respiratory function of teleostean gills. Comp.
Biochem. Physiol, 12 : 127-142. UTIDA, S., M. KAMIYA, AND N. SHIRAI, 1971. Relationship between the activity of Na+-K'~
activated adenosinetriphosphatase and the number of chloride cells in eel gills with
special reference to seawater adaptation. Comp. Biochem. Physiol., Pt. A, 38 : 443-447. VICKERS, T., 1961. A study of the so-called "chloride-secretory" cells of the gills of teleosts.
Q. J. Microsc. Sci., Pt. 4, 104: 507-518. YAMADA, K., AND M. YOKOTE, 1975. Morphochemical analysis of mucosubstances in some
epithelial tissues of the eel, Anguilla japonica. Histochemistry, 43: 161-172.
Reference: 7?iW. />'»//. 157 : 112-124. (August 1979)
FINE STRUCTURE OF MUSCULATURE IN THE COPEPOD PARANTHESSIUS ANEMONIAE CLAUS
R. P. BRIGGS i Department of Zoology, The Queen's University of Belfast, Northern Ireland
Paranthcssiiis ancmoniae Claus is a cyclopoid associate of the snakelocks anemone Anemonia snlcata (Pennant). First described by Claus (1889) in the Adriatic Sea and later by Bocquet and Stock (1959) from Mediterranean waters, Paranthcssiiis has only recently been recorded from British waters (Gotto and Briggs, 1972; Briggs and Gotto, 1973; Briggs, 1973). Other recent studies of Paranthessiiis have described this copepod's general ecology (Briggs, 1976), alimentary canal (Briggs, 1977a), larval development (Briggs, 1977b) and inte- gument (Briggs, 1978). Copepod muscle has been investigated by Hartog (1888), Scott (1901), Lowe (1935), Changeux (1960), Fahrenback (1962) and Park (1966). Ultrastructural studies of Cyclops by Bouligand (1962, 1963 and 1964) and of Macrocyclops albidits by Fahrenbach (1963) are among the few detailed studies of copepod muscle.
MATERIALS AND METHODS
Copepods were fixed for 12 hr at 4° C in 5% gluteraldhyde in 0.12 M Millonig buffer (pH 7.4) containing 3% NaCl and 0.1 mM CaCl. Fixed speci- mens were processed for light and electron microscopy.
Light microscopy
Copepods fixed in gluteraldehyde were dehydrated though ethylene glycol and •embedded in glycol methacrylate (G.M.A.) which was polymerized in gelatin cap- sules at 60° C for 48 hrs. Sections 1 to 2 //, thick were cut on a Reichert OMU2 ultratome and stained on glass slides with mercuric bromo phenol blue (method vl Maiza, Brewer, and Alfert, 1953).
Electron microscopy
Fixed copepods were washed in Millonig buffer-wash (2-8 hrs), post-fixed for 2 hrs in \% osmium tetroxide and the dehydrated through ethanol to propylene oxide and embedded in araldite. Sections were cut with a Reichert OMU2 ultra- tome, mounted on copper grids, stained with uranyl acetate and lead citrate and examined in an AE1 EM801 electron microscope operating at 60 kV.
i Present Address: Department of Agriculture (NI), Fisheries Research Laboratory, Castleroe Road, Coleraine, Northern Ireland.
112
THE MUSCULATURE OF PARANTHESSIUS
113
z
A
1u
I Li. ,
t
I-
- : • f-,<: , " •. -r*
' • • ••'
FIGURE 1. Electron micrograph of striated muscle in Paranthcssiiis anemoniae. (a) Longi- tudinal section through muscle. Z line, A and I band are arrowed, (b) Transverse section of muscle fibrils at both A band and I band levels, (c) Transverse section of muscle show- ing longitudinal sarcoplasmic reticulum element (SL) actin filaments (AC) and myosin. (d) actin (AC) and myosin filaments (MY).
114 R. P. BRIGGS
RESULTS
The general body muscle
Light microscopy has shown the longitudinal muscles in Paranthessius to be composed of bundles of muscle fibers which pass along either side of the mid-dorsal line. A similar pair of longitudinal muscle bundles are situated in a ventro-lateral position. Treatment with periodic-acid-Schiff (method of McManus, 1946) demon- strated the presence of large amounts of glycogen within the muscle.
Examination of ultra thin sections with the electron microscope shows the muscle of Paranthcsssius to be striated (Fig. la). A and I bands are clearly visible, the former having a well-defined central H zone as is usual for striated muscle (Hanson and Huxley, 1953). The functional units of muscle (sarcomeres) are separated from one another by an electron-dense Z line. Transverse section shows the muscle to be composed of polygonal-shaped myofibrils measuring between 1 and 4 /JL in diameter (Fig. Ib). Both thick (myosin) and thin (actin) filaments are present. The myosin filaments, which appear to be hollow, measure 12 nm in thickness with an average length of 1100 nm and are spaced about 25 nm apart. The actin filaments, on the other hand, are on average 4 nm in thickness and are each placed equidistant between two myosin filaments. The sarcomere has an aver- age length of 1200 nm, though this varies with the state of muscle contraction.
Superficial examination of Paranthessius muscle reveals an apparent similarity to vertebrate muscle with each myosin filament surrounded by six similar filaments and six actin filaments in hexagonal array (Figs. Ic, d). More detailed examina- tion, however, shows that the actual position of the actin filaments in relation to the myosin filaments is different from vertebrate muscle in that each actin fila- ment does not lie equidistant from three myosin filaments (Fig. 2). If the myosin filaments in Paranthessius are imagined to be the apices of an equilateral triangle, then the actin filaments occur in the center of each side (Fig. 2). This arrangement of myofilaments is similar to that described for other copepods, for example Bouligand (1962), Fahrenbach (1963) and Raymont, Krishnaswamy, Woodhouse, and Griffin (1974).
Sections through the muscle show the myofibrils to be surrounded by men- branous material which constitutes the sarcoplasmic reticulum. This is also seen to be regularly distributed within the myofibrils. In transverse section, the interfibrillar sarcoplasmic reticulum appears as circular membranous zones, often paired, measuring 50 nm in diameter. The single or paired units are regularly spaced between 200 nm and 400 nm apart (Fig 3a). Longitudinal sections show the sarcoplasmic reticulum to be in the form of canals measuring an average of 50 nm across and of varying length (Fig. 3b), and appearing elliptical in slightly oblique sections (Fig. 3c).
Examination of a large number of muscle sections has revealed that the sar- coplasmic reticulum is a branching tubular system that runs longitudinally through and around the myofibrils. The intrafibrillar elements link up with those surround- ing the fibril in the region of the Z line by means of transverse tubules of sarco- plasmic reticulum. This feature is evident in both transverse and longitudinal sections (Fig. 3d, 4a). The canals surrounding the myofibrils are continuous with the membrane of the sarcolemma surrounding the muscles. This implies the
THE MUSCULATURE OF PARANTHESSIUS
115
T.S. THICK & THIN FILAMENTS
T.S.'H' ZONE
T.S. 'I' ZONE
T.S. VERTEBRATE MUSCLE
FIGURE 2. Diagrammatic representation of the myofilament arrangement in copepod muscle. Vertebrate muscle is also represented for comparison. If the myosin filaments in copepod muscle are imagined to be the apices of an equilateral triangle, then the actin filaments occur in the center of each side. In vertebrate muscle each actin filament lies equidistant from three myosin filaments.
existence of a continuous system of sarcoplasmic reticulum, leading from the muscle surface and running between the muscle fibers, myofibrils and myofilaments.
The paired nature of some of the longitudinal tubules wthin the myofibrils seen in transverse section is attributed to elongate membranous vesicles that lie along
116
R. P. BRIGGS
"' >
v. • . . th.1* "• . • ' ." I -
tM
^i-SL
^ ,-.,••;'
% - •:^tV\CtV*-X2^^^^^H
-.••>^J-S;v.-- •• ^i-f
\+ > :2*-'^v " '--, * ' - . - '•-^c-icA?*
• ii
't.S »'-*^ •V*'''- 'f:' ^\ %'?''•" •"***
^)sSS8^^8gSSi:
8^^-AvJ' H'JJSigg^
FIGURE 3. (a) Longitudinal sarcoplasmic re iculum elements (SL) and blind ending vesicles (V) in transverse section. The blind ending vesicles have a characteristic granular appearance to their lumen, (b) Longitudinal (SL) and transverse elements (ST) of sarco- plasmic reticulum as seen in longitudinal section, (c) Slightly oblique section through muscle showing longitudinal sarcoplasmic reticulum (SL) elements as oval vesicles, (d) Transverse section of Paranthcssius muscle showing both longitudinal (SL) and transverse elements (ST) of sarcoplasmic reticulum in the I band (I). No transverse elements were observed in the A band (A).
THE MUSCULATURE OF PARANTHESSIUS
117
*' w - '•'
w
£?* V>.-
FIGURE 4. (a) Longitudinal section of Paranthessius muscle fibril showing junction of transverse sarcoplasmic reticulum (ST) and longitudinal sarcoplasmic reticulum (SL). (b) Mitochondria (M) and glycogen granules (G) on surface of muscle fibril.
longitudinal elements. The lumina of these vesicles have a granular appearance and their length rarely exceeds that of a sarcomere (Fig. 3a, 5). The general irregular nature and the occasional folding and branching of the longitudinal tubules
118
R. P. BRIGGS
SAL
RSI-
FIGURE 5. Three-dimensional diagram of the sarcoplasmic reticulurn system in Paran- thessins ancmoniae muscle showing longitudinal sarcoplasmic reticulum (SL), vesicles (V), peripheral sarcoplasmic reticulum (PSL), transverse sarcoplasmic reticulum (ST), Z line (Z), actin filament (AC), myosin filament (MY), sarcolemma (SAL) and its imaginations (IS).
is probably accounted for by the occurrence of more than two units in some sec- tions examined.
Scattered on the muscle surface under the sarcolemma are numerous mito- chondria that may measure up to 2 ^ in length, and 0.6 /j. in width. The mito- chondria of the muscle (or sarcosomes) are characterised by the possession of numerous cristae (Fig. 4b). Granules of glycogen (Fig. 4b) measuring about 25 nm in diameter occur commonly between the myofibrils and are thought to be responsible for the strong positive reaction to the P.A.S. test for carbohydrates
THE MUSCULATURE OF PARANTHESSIUS
119
? * ri:."
f**,3BLv *•' *• *
•4» 'nk CL -« -* 5 - ' . -
-MY
•--
*
'"'
K.XK*. *
«r
0-1
•-.
.
p
- '
I— •
-
.
.;*•
jfl 'v :* rfflP - s * tv]
• i|- , • *3
r-.^/, »Vv ' S Sfef»?IXl^
rro^^i-- }fe
<*• v ••Jfeft-- -.U-1 -• J^JKS
''//*•
.•- ,i C -' fill)*.
"^v^T^-iyA A$ ' J^*!
FIGURE 6. (a) Transverse section through muscle of alimentary canal showing both actin (AC) and myosin (MY) filaments, (b) Details of muscle junction with cuticle (CU) showing attachment fibrils (T) and dense terminal region (DTK) of muscle, (c) Further details of attachment of muscle (MU) to cuticle (CU) by tonofibrils (T). (d) Transverse section of the tonofibrils which attach muscle to cuticle in Paranthessius.
120
R. P. BRIGGS
TABLE I
Dimensions of muscle fibrils in Paranthessius, Cyclops, Macrocyclops and, a vertebrate.
|
Myosin |
|||||
|
Species |
Actin thickness |
Reference |
|||
|
length |
thickness |
separation |
|||
|
Paranthessins |
1100 nm |
12 nm |
25 nm |
4 mn |
|
|
Cyclops |
1500 nm |
12 nm |
25 nm |
4 nm |
Bouligand |
|
(1964) |
|||||
|
Macrocyclops |
— |
15 nm |
48 nm |
— |
Farenbach |
|
(1963) |
|||||
|
Vertebrate |
1500 nm |
10 nm |
45 nm |
6 mn |
Threadgold |
|
(1967) |
observed in light microscope studies. The muscle nuclei are a flattened ovoid shape and have a single nucleolus. They are found in close contact with the muscle surface and were not commonly encountered during these investigations. The longitudinal muscle bundles and those supplying the appendages are constructed as described.
The muscle of the alimentary canal
The alimentary canal was found to be surrounded by muscle of a slightly different structure. There are several isolated bundles or strands of longitudinal muscle measuring 0.5 to 1.0 /A thick lying beneath the basement membrane of the digestive tract epithelium (Fig. 6a). Surrounding these longitudinal muscles is a layer of circular muscle of 1 /j. average thickness. The myosin filaments of the gut muscle were seen in transverse section to be surrounded by 10 to 12 actin filaments instead of the six noted in the general body muscle. Although the myosin filaments are about the same distance apart as the general muscle (25 nm) they are somewhat thicker (15 nm). No internal ramification of sarcoplasmic reticulum is seen in this muscle.
The attachment of the muscle to the cuticle
Near the site of attachment to the cuticle the muscle fibrils terminate at an irregular electron-dense line that can be seen in longitudinal sections to traverse the fibril (Fig. 6b). More detailed examination, however, reveals that this electron- dense line is composed of the sarcolemma of the muscle, near to which, at a distance of 30 to 40 nm, is another plasma membrane that gives rise to fine tubules or tonofibrils (Fig. 6c, d). The tonofibrils measure about 20 nm in diameter and seem to pass, in groups, through thickened electron-dense zones, from each of which emerges an electron-dense fiber of roughly 400 nm thickness. These fibers ramify through the cuticle forming a firm attachment. The length of the tonofibrils ranges from 5 to 0.5 p. in different parts of the animal. Attachment is sometimes made to normal cuticle, while in other places the endocuticle invaginates to form an apophysis for muscle attachment (Briggs, 1978).
THE MUSCULATURE OF PARANTHESSIUS \1\
DISCUSSION
The observations made on Paranthessius muscle agree in many respects with those made by Bouligand (1962, 1963, 1964) on Cyclops and Acanthocvclops. by Fahrenbach (1963) on Macrocyclops and by Raymont ct ol. (1974) on Calanus. In Cyclops the myosin filaments measure 1.5 ^ in length compared to 1.1 //. in Paranthessius. Macrocyclops has myosin filaments 15 mm thick, which are further apart than those of both Cyclops and Paranthessius. No detailed measurements are available for Calanns muscle. Dimensions of muscle structures given in Table T includes details of typical vertebrate muscle for comparison.
In a study of Acanthocyclops, Bouligand (1964) describes zones of double overlapping between the two sets of actin filaments of the sarcomeres when the muscle is contracted. A transverse section through this zone shows twice the number of actin filaments found in other regions of the muscle. In longitudinal sections of contracted muscle these overlapping zones or CM bands are characterised by appearing as a dark band across the muscle in the center of the H zone Examination of many sections of Paranthessius muscle did not show this feature to be present. It is possible that the muscle examined here was fixed in the relaxed state. This is considered unlikely, however, since copepods always ex- hibited strong locomotory movements on encountering fixatives. The probability that all sections cut were of relaxed muscle is, therefore, very low. The general arrangement of the myofibrils is the same as that described for other copepods.
The sarcoplasmic reticulum in Paranthessius is very similar to that described by Bouligand (1962, 1963) for Cyclops, though it tends to be somewhat less elaborate than that studied by Fahrenback (1962) in Macroc\clops. In this species, the blind-ending membranous vesicles found in Paranthessius and also in Cyclops are more expanded, forming a well developed system of cisternae. As was found with the vesicles of Paranthessius muscle, the cisternae do not join with the elements of the sarcoplasmic reticulum, but come to within 10 nm in most regions of the muscle. The term dyad, used by Smith (1961). to describe the association between sarcoplasmic reticulum tubules and the cisternae in the beetle Tcnebrio is used here to describe similar structures in copepod muscle. In Paranthessius a dyad represents the paired membranous tubules seen in a transverse section of muscle myofibrils. This is an association between a sarcoplasmic reticulum tubule and a blind ending vesicle.
The sarcoplasmic reticulum system of copepods may be contrasted with that of vertebrate muscle. In most vertebrates the sarcoplasmic reticulum forms a sleeve around the muscle fibril ending in a number of finger-like projections in the region of the Z line, where it comes into close contact with the invagi- nated membrane of the sarcolemma. This association forms a triad, com- posed of two sarcoplasmic reticulum elements (one from each side of the Z line of the fibril) and the membrane of the sarcolemma. The sarcolemma membrane is, therefore, not a continuous tubular system ramifying longitudinally through the myofibrils as is found in copepod muscle.
Fahrenbach (1963) stressed the importance of efficient diffusion of "transmitter substances" (Ca++) in fast acting muscles. Slower contracting vertebrate muscles have the discontinuous triad structure described, in which the sarcolemma membrane
\22 K. I'. HKI<;<;S
is not connected to the sarcoplasmic reticulum system. The myofilaments in the center of the myofibrils are not brought into such close proximity with a potential impulse-conducting element as is found in the arthropodan continuous dyad system of fast contracting muscle. In most fast muscle studied the distance that calcium ions have to diffuse in order to reach the center of the myofibrils to trigger con- struction is maintained at a minimum distance of less than 1 /A. Examples include 0.3 to 0.35 /j. in the fast muscle of the dragon fly Acshna (Smith, 1961). 0.18 to 0.2 p. in the toadfish Opsanus (Fawcett and Revel. 1961). 0.15 to 0.25 p. in the bat Eptesicus (Revel. 1962) and 0.2 /A in the copepod Macrocyclops (Fahrenbach 1963). The value for Paranthessius was found to be on average 0.25 /A. Fahren- bach (1963) proposes that this is the reason why the longitudinal tubules are arranged in a regular hexagonal manner in fast copepod muscle.
Bouligand (1962) suggests that the longitudinal sarcoplasmic reticulum ele- ments of Cyclops are regularly arranged, so that their tendency to expand when the muscle contracts, (due to the hydrostatic pressure of their contained fluid) will not disorientate the myofilaments. Bouligand proposes that evidence for this may be gained from observation of true transverse sections of hexagonal array of the tubules. It is visualised that expansion force lines from these tubules would pass through the myosin filaments towards another expanding tubule, which would be exerting a similar force. This implies that the position of the myosin filaments would be undisturbed during muscle contraction. If the expansion forces tended to act between the myosin filaments the latter would be displaced to either side. Both Fahrenbach's and Bouligand's interpretation of the regular arrangement of sarcoplasmic reticulum are applicable to Paranthessius muscle, which has a similar structure to that of the species studied by these authors. Although the sarcoplasmic reticulum is not so complex as that of Macrocyclops, all other structural features indicate that the muscle of Paranthessius is of a fast contracting type. The muscles of the alimentary canal are probably slower acting, since they have nearly twice as many actin filaments as the general body muscles which is a characteristic of "slower" muscle (Fahrenbach 1967).
Parasitic forms usually have smooth or "slow contracting muscle" (Capart, 1948). It is noteworthy that Paranthessius has muscle characteristics of free-living forms, i.e., "fast acting muscle". This is not surprising when it is considered that Paranthessius is quite a mobile associated form (Briggs, 1974). It is of survival value for Paranthcssuis to lie capable of rapid swimming in order to regain a position on its host if dislodged. Since the larval instars of Paranthessius live freely in the plankton, the infective stage must possess efficient locomotion for host location. The elaborate musculature of the adult may, therefore, represent a legacy from the free living phase of the life cycle.
Apart from the gripping claws of the second antenna and spinal reduction in the mouth parts, Paranthessius is relatively unmodified morphologically and bears a strong resemblance to free-living cyclopoids. It is, moreover, associated with the external surface of the host, never being found in the gastrovascular cavity, and is very mobile both on and off the anemone. These features together with the elaborate musculature described here suggest that in an evolutionary context Paranthessius is a recent invader of Aneinonia siilcata. Comparative studies of the musculature in other associated species which exhibit varying degrees of host
THE MUSCULATURE OF PARANTHESSWS 123
dependence and morphological modification might add support to these specula- tions on the evolution of parasitism in copepods.
I wish to thank Professor Gareth Owen and Dr. R. V. Gotto of the Queen's University of Belfast for their invaluable encouragement and advice during this study, undertaken during the tenure of a postgraduate studentship from the Depart- ment of Education for Northern Ireland.
SUMMARY
1. Paranthessius ancinoniac has striated muscle composed of actin and myosin myofilaments arranged hexagonally, as in free living copepods.
2. The sarcoplasmic reticulum is continuous with the membrane of the sarco- lemma in the region of the Z line and forms a continuous system of tubules which ramify through the muscle.
3. Blind-ending vesicles form dyads with the longitudinal sarcoplasmic reticulum tubules.
4. Attachment of the muscle to the cuticle is by tonofibrils.
5. A relatively short sarcomere length, complex sarcoplasmic reticulum and high proportion of myosin to actin filaments indicate the "phasic" nature of the general body muscle in Paranthessius.
6. The muscle of the alimentary canal is characteristic of "tonic" muscle.
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12: 571-574. SCOTT, A., 1901. Lepeophtheirus nnd Lcniaca. Liverpool Marine Biology Committee Memoirs,
VI 62 pp. SMITH, D. S., 1961. Innervation of the fibrillar flight muscles of an insect : Tcncbrio niolitar
(Coleoptera). /. Biophys. Biochein. Cytol. (2)8: 447-466. THREADGOLD, L. T., 1967. The Infrastructure of the Animal Cell. Pergamon Press, Oxford,
313 P1).
Reference: Hiol. Hull. 157 : 125-137. ( August 1979 )
RESPIRATORY ADAPTATIONS OF THE ESTUAR1NE MUD SHRIMP,
CALLIANASSA JAMA/CENSE (SCHMITT, 1935) (CRUSTACEA.
DECAPODA. THALASSIXIDEA) '
DARRYL L. FELDER
Department 0} Bioloyy. L'uh'ersity of Southwestern Louisiana, Lafayette. Louisiana 7i>?«1
Thalassinid mud shrimps connnonly burrow in hypoxic marine sediments, and their success in these habitats seems, at least in part, predicated upon metabolic adaptations. Recent studies of thalassinids from the Pacific coast of North America have identified a number of behavioral and physiological respiratory adaptations to the hypoxic habitats of intertidal species (Farley and Case, 1968; Thompson and Pritchard, 1969; Roxby, Miller, Blair, and Van Holde. 1974; Miller and Van Holde, 1974; Miller, Pritchard. and Rutledge. 1976; Torres, Gluck. and Childress, 1977; Hawkins, 1971, unpublished M.S. thesis. Oregon State University). A rich thalassinid fauna occurs in intertidal and sublittoral habitats along coasts of the western Atlantic, but metabolic regulation among these species has been investigated only in Upogebia affinis by Mangum and van Winkle (1973).
The present study concerns Callianassa jainaiccnsc (Schmitt) (Callichinis jaiiiaiccnsc according to the generic scheme of de Saint Laurent, 1973), a common inhabitant of estuarine mud flats in the northern Gulf of Mexico (Felder, 1978) and other areas of the western Atlantic (Rodrigues, 1971). On the Louisiana coast, dense populations of C. jamaiccnsc are found in muddy substrates where low- salinity interstitial water is markedly hypoxic. Tidal exposure of these substrates frequently subjects such populations to extended periods of anoxia. Studies were undertaken to identify respiratory adaptations of C. jamaiccnsc to such hypoxic habitats. Specifically, this paper reports (i) survival under aquactic and aerial anoxia, (ii) aerial respiration, (iii) effects of oxygen tension on metabolic rate, and (iv) post-anoxia metabolic rates.
MATERIALS AND METHODS
Animals were collected from a tidally influenced pond on Grand Terre Island, Louisiana. Methods of collecting, transporting, maintaining and salinity-acclimating animals were the same as previously described (Felder, 1978). Animals were accli- mated to a salinity of 2Q%c in dark, 25° C incubators; all were maintained at this salinity for nine days before experiments were initiated. Animals were not fed. and aeration was provided during all phases of salinity-acclimation. Only inter- molt, uninjured adult males were used in respiration studies. Wet weights were determined by thoroughly blotting animals with tissue and then weighing to the nearest milligram. All sea water used in experimental studies was carefully main- tained at a salinity of 20 ± 0.3/{c and temperature of 25 ± 0.2° C.
Anoxic sea water was prepared by gassing sea water with nitrogen. In one
1 In part adapted from a doctoral dissertation submitted to the Department of Zoology and Physiology, Louisiana State University, Baton Rouge.
125
126
DARRYL L. FELDER
AERATED WATER
DEOXYGENATED WATER
FIGURE 1. Diagrammatic cut-away view of water bath showing components of flow- through respirometer : A, influent oxygen electrode ; B, effluent oxygen electrode ; C, bypass shunt; D, respiration chamber; E, magnetic stirrers ; F, differential oxygen me'.er ; G, integrat- ing chart recorder; H, peristaltic pump. A small bubble trap (not shown) was installed between components H and A.
experiment anoxic sea water was siphoned into BOD (300-ml biochemical oxygen demand) bottles containing one animal each, and bottles were sealed until death occurred. Other animals in individual, perforated vials were placed as a group into 5 liters of anoxic sea water which was replaced daily ; whenever the 5-liter jar was opened for removal of dead animals, it was regassed with nitrogen. Control animals were maintained in continuously aerated sea water. Tolerance of aerial anoxia was determined by supporting animals on the rack of a desiccator over water and continuously gassing the water with nitrogen. Control animals in an aerial environment were likewise maintained, but underlying water was gassed with air.
Aerial Vo2 was measured in a Gilson respirometer with 130-ml respirometry flasks and equivalent ballast. Filter paper wicks and 30% KOH were added to each flask side-arm to absorb COo. Each flask contained one animal and one milliliter of sea water to maintain water saturation of air. One hour was allowed for equilibration ; thereafter oxygen consumption was read at 30-min intervals.
Oxygen consumption at decreasing oxygen tensions was measured by placing the animal into a 13.5-mm ID (inner diameter) plastic tube with openings at both ends. The tube was wedged vertically against the wall of a BOD bottle, and a small stirring bar was placed at the center of the bottle. An oxygen electrode was fitted snugly into the bottle opening, and depletion of oxygen was recorded with a Beck- man oxygen analyzer. The analyzer was calibrated in air-saturated sea water and checked by Winkler titration (Strickland and Parsons, 1972) before each run. Temperature was maintained by a water bath supported over the magnetic stirrer. The stirring rate was set at the lowest speed, which produced maximum deflection of the oxygen meter. The displacement volume of the animal, tube, and stirring bar
CALLIANASSA RESPIRATORY ADAPTATIONS
127
was subtracted from the bottle volume. Pleopod ventilatory strokes were counted during 5-min intervals and expressed as mean number/min. To minimize effects of handling, each animal was transferred to a plastic tube and placed (anterior end up) into a BOD bottle 30 min before it was sealed ; as an additional precaution, the first 30 min of recorded oxygen depletion were discarded.
A flow-through respirometer was assembled from a dual-probe International Biophysics differential oxygen analyzer, a Houston Instrument integrating chart recorder, a peristaltic pump, two magnetic stirrers, a constant temperature water bath, and a 16-mm ID glass respiration chamber (Fig. 1). Flow rate was maintained at ca. 10 ml/min and was precisely determined by measuring the volume of effluent ; injection of a dye at this flow rate indicated thorough mixing of water as it passed through the respiration chamber. Each animal was placed into the chamber with its anterior toward the influent opening ; aerated water was provided for 1 hr before oxygen consumption was read. Prior to each run, the oxygen analyzer was calibrated by Winkler titration and the differential between the elec- trodes was set to zero. Altered oxygen tensions were achieved by controlled mix- ing of fully aerated and nitrogen-saturated water. Whenever oxygen content of influent wrater was altered, 20 min were allowed for the flow-through system to flush before oxygen consumption was read. Anoxic conditions were provided by pumping deoxygenated water into the respiration chamber and then closing valves at either end (Fig. 1). The flow-through respiration chamber was lined with fine-mesh plastic gauze to provide traction for thoracic legs during pleopod beating. Pleopod strokes were counted as previously described.
Field measurements of oxygen in exposed and submerged burrows were made during an afternoon low tide in July 1972. Oxygen concentration of water over-
100
80-
60-
40-
20-
100
300
Exposure time, hours
FIGURE 2. Survival among specimens of Callianassa jainaicciisc under aerial and aquatic anoxia compared to survival of controls under normoxia. Experimental conditions include anoxic water with accumulation of metabolic wastes (crosses) ( N = 35 ) , anoxic water changed daily (solid circles) ( N — 30), aerial anoxia (open circles) (N = 30), aerial normoxia (squares) (N = 15), and aquatic normoxia (triangles) (N = 50); N is the number of animals initially exposed to each condition. Temperature was maintained at 25 ± 0.2° C; salinity was 20 ± 0.3',,
128 DARRYI. I.. FKLDER
lying C. jamaiccnse l)urrovvs was measured in situ with an air-calibrated Yellow Springs Instrument oxygen meter. Water from burrows of C. janiaiccnsc was sampled and analyzed as described by Thompson and Pritchard (1969).
RESULTS Survival under ano.via
Survival of specimens of C. janiaiccnsc under aquatic and aerial anoxia is plotted in Figure 2. The LD-)0 (mean lethal dose) was lowest, ca. 3.2 days, when anoxic water was not changed and metabolic wastes accumulated for the duration of survival. Under such conditions, with individual animals sealed into 300-ml BOD bottles of anoxic water, ambient pH dropped from an intial level of 8.7 ± 0.3 to 6.9 ± 0.5 at the time of death. When anoxic water was replaced daily, the LD50 increased to ca. 4 days. The LD-,,, for animals held in aerial anoxia, ca. 5 days, exceeded that for animals subjected to aquatic anoxia. Among those animals subjected to aerial anoxia or daily changes of anoxic water, a few survived more than two times the exposures producing LD50's.
Under normoxic conditions, losses of control animals in a water-saturated environment approached the LD50 on the 16th day of exposure. Mortalities among control animals in normoxic water did not exceed 2% within the same 16-day time period.
Aerial o.\'\gen consumption
After 60 min of equilibration in a 25° C Gilson respirometer, oxygen con- sumption (Vo2) in water-saturated air was read at 30-min intervals over an additional 2-hr period. Wet weights of the 25 animals used in the aerial respira- tion experiments ranged from 3.51 to 5.25 g. Mean VOL, rates and standard errors over the four successive 30-min time periods were 18.9 ± 1.53, 16.2 ± 1.29, 19.0 ±1.39, and 18.9 ±1.12 ^/(g wet wt-hr). respectively. Activity in the respirometer flasks was not quantitatively monitored, but animals were for the most part quiescent during the Vo., determinations.
Effects of low o.rygen tension on aquatic oxygen consumption
As oxygen was depleted from sealed BOD bottles, specimens of C. janiaicense regulated V0., until oxygen tension (P0,) decreased to ca. 20 mmHg (Fig. 3). The critical oxygen tension (P(.) ranged from 10 to 25 mmHg among the 10 animals studied. The slightly higher V,,, at 120 mmHg is of questionable significance as it may relate to disturbance of animals when placing them into the BOD bottles at the beginning of the experiment. Mean pleopod ventilatory rates ranged from 20 to 33 strokes/min at oxygen tensions above the P,.. As PO2 fell from 20 to 10 mmHg, pleopod activity increased to near 60 strokes/min; concurrent increases in V0o occurred in some animals and accounted for the large range of V02 at oxygen tensions between 12 and 15 mmHg (Fig. 3). Pleopod activity decreased as P02 dropped below 9 mmHg and was again near 39 strokes/
CALLIANASSA RESPIRATORY ADAPTATIONS
129
100-
OJ
_,- o
-a o a. o
_a;
Q_
50-
0 J
T
100
40
60 80
Po2 mm Hg
100
120
FIGURE 3. Mean oxygen consumption ( open circles ) and mean ventilatory rate ( solid circles) among specimens of Calliainissa jainaiccnsc as oxygen is depleted from a sealed bottle. Each open circle is mean value for 10 animals and each solid circle is mean value for eight animals. Vertical lines indicate ranges ; rectangles indicate standard errors ; horizontal lines indicate span of oxygen tension over which means are taken. Wet weights range from 1.45 to 3.82 g. Temperature was maintained at 25 ± 0.2° C; salinity was 20 ± Q.3f/ff.
min at 0 minHg. Animals held at complete anoxia continued to decrease pleopod activity and, after 2 to 4 hr, stopped ventilating unless disturbed.
\Yhen P,,, was abruptly decreased from normoxia ( 150 mmHg) to hypoxia (37 inmHg) in a flow-through respirometer, specimens of C. jainaiccnsc reduced \"(), by more than SO'/f for 2 to 3 hr (Fig. 4). Oxygen consumption gradually increased after 5 hr of hypoxia and after 9 hr was near 75% of V(), in normoxia. In normoxia, pleopod activity ranged from 14 to 18 strokes min. As Po2 decreased to 37 mmHg, pleopod activity at first increased slightly but soon subsided to rates less than those in normoxia.
\Yhen introduction of hypoxic water (37 mmHg) followed 12 hr of anoxia, the Vo2 of C. jainaiccnsc was initially just above that observed under normoxia (Fig. 5). The V02 in hypoxia decreased slowly from the rates measured shortly after termination of anoxia. Pleopod activity, which was negligible during anoxia,
130
DARRYL L. FELDER
100-
30-
CD
3
40-
20-
-150
-125
100 1
-75 •§
-50
-25
i.o
2.0 4.0 6.0
Exposure time, hours
8.0
10.0
FIGURE 4. Temporal variations in aquatic oxygen consumption (open circles) and pleopod ventilatory rate ( solid circles ) among specimens of Callianassa jamaicense when ambient oxygen tension ( crosses on heavy line ) is abruptly reduced. Each value is mean rate for five animals. Vertical lines indicate ranges ; rectangles indicate standard errors ; horizontal lines indicate time spans over which means are taken. Temperature was maintained at 25 ± 0.2° C ; salinity was 20 ± 0.3'A.
dramatically increased with introduction of hypoxic water. As hypoxic water entered the respiration chamber following 12 hr of anoxia, animals invariably moved to the influent opening of the chamber (Fig. 1) and began rapid ventilation with their pleopocls. The accelerated pleopod activity was maintained near 50 strokes/ min for ca. 30 min after hypoxic water was introduced into the chamber, and animals spent almost all of this time near the influent opening of the respiration chamber. A gradual decrease of pleopod activity paralleled the slowly decreasing V0o which began near the middle of hour 14 and continued through hour 15.
When anoxia was terminated by introducing normoxic (150 mmHg) water, the Vo2 increased to two times the rates preceding anoxia (Fig. 6). Five hours after anoxia was terminated, Vo2 approached that observed before anoxia. With the reintroduction of normoxic water, animals moved to the influent opening of the respiration chamber and rapidly ventilated with their pleopocls as when hypoxia (37 mmHg) followed anoxia (Fig. 5). However, animals neither remained at the incurrent opening nor maintained accelerated pleopod activity for as long as when hypoxic water followed anoxia.
Field measurements of dissolz'ed oxygen
Burrows of C. jamaicense contained very low concentrations of dissolved oxygen when located above the waterline. On Grand Terre Island fluctuating tides ex- posed numerous burrows along pond margins for periods varying from a few hours to several days. Oxygen tension in water from five active burrows located from 1 to 3 m outside the pond ranged from 0 to 5 mmHg (x == 2.2 mmHg),
CALLIANASSA RESPIRATORY ADAPTATIONS
131
100
- 150
- 125 a
- 100 e
75 1
TC
01
n:
-50
- 25
13.0 Exposure time, hours
14.0
15.0
FIGURE 5. Temporal variations in aquatic consumption (open circles) and pleopod ventila- tory rates (solid circles) among specimens of Calliauassa jauiaiccnsc when ambient oxygen tension (crosses on heavy line) is dropped to anoxia and then raised to hypoxia. Each value is mean rate for five animals. Vertical lines indicate ranges ; rectangles indicate standard errors; horizontal lines indicate time span over which means are taken. Temperature was maintained at 25 ± 0.2° C ; salinity was 20 ± 0.3&-.
100-
80-
60-
CNJ
O
40-
20-
12 HOURS ANOXIA
-150 -
- 125
-100 I
-75 -50 -25
1.0 12.0 15.0 1-4.0
E , L F ire tiniL . . ur?
16.0
FIGURE 6. Temporal variations in aquatic consumption (open circles) and ventilatory rates (solid circles) among specimens of Callianassa jamaicense when ambient oxygen tension (crosses on heavy line) is dropped to anxoia and returned to nonnoxia. Each value is mean rate for five animals. Vertical lines indicate ranges; rectangles indicate standard errors; horizontal lines indicate time spans over which means are taken. Temperature was maintained at 25 ±0.2° C; salinity was 20 ±0.3',,.
UJ DAkkYI. L. I'KI.DKR
which was well below critical oxvgen tension* ( I',.) established fur specimens of C. jamaicense in the laboratory. Longer periods of isolation from estuarine pond waters were caused by storm-effected movements of sand which elevated large areas of the pond above water level, sometimes for periods of several months. Exca- vation of one such area which had been isolated from the pond for 2 months produced numerous living specimens of C. jatnaicense, although most were mori- bund ; some of these moribund specimens appeared to be occupying portions of burrows above the water table.
Higher and more variable oxygen tensions occurred in burrows at the immediate edge of the pond and just inside the pond. Oxygen tensions in 10 of these burrows, located from 0.2 to 2.0 m inside the pond, ranged from 11 to 119 mmHg (x — 73.4 mmHg). However, at the time of sampling, oxygen tensions in surface waters were from 151 to 161 mmHg, or well above concentrations in burrows. Oxygen tensions in water of the estuarine pond probably approached these levels only during the periods of photosynthetic activity. Diel cycles of oxygen in water overlying C. famaicense burrows on Grand Terre Island were characterized by decreasing tensions after dark as photosynthesis was replaced by net community oxygen consumption. Diel variations were monitored in October, 1974 (J. Day, personal communication), and P(), of water overlying C. jainaiccnsc burrows re- mained below the P(. of C. famaicense during a 4 to 5 hr period just before to just after dawn.
DISCUSSION
The limited studies available to von Brand ( 1946) led him to conclude that decapod crustaceans show little tolerance of anoxia. Extended tolerance of anoxia is, however, one adaptation exhibited by a number of decapods which burrow in potentially hypoxic substrates. For instance, mud-burrowing crayfish survive anoxia four times longer than those inhabiting swift streams (Bovbjerg, 1952). Among the thalassinid decapods, Upogebia pugettensis and Callianassa californiensis survive anoxia for at least three days (Thompson and Pritchard, 1969) and Callianassa jainaiccnsc survives anoxia for three to four days (Fig. 2). These and other thalassinids, such as Callianassa affinis in Southern California (Congleton, 1974) and Callicliints forcsti from west Africa ( LeLoeuff and Intes, 1974), are highly specialized for a burrowing existence in shallow, hypoxic, marine substrates, and their tolerance of anoxia is clearly an adaptation to habitat.
Under aquatic anoxia, the longer survival of specimens of C. jainaiccnsc when anoxic water is changed daily, compared to survival of specimens when anoxic water is not changed, may reflect the effects of accumulated metabolic wastes. Products of anaerobic metabolism could account for the decrease in pH observed when individuals of C. jainaiccnsc are sealed into IK )l) bottles of anoxic water and left until death occurs. The buffering characteristics of burrow water, particularly in the lower-salinity extremes of C. jainaiccnsc habitat, or the ability to exchange anoxic burrow water might thus affect survival of C. jainaiccnsc when it is sub- jected to periods of anoxia in nature. Short-term survival of C. janiaiccnsc does not, however, appear to be affected by the physical presence or absence of the burrow, although MacGinitie (1934) reports that a specimen of Callianassa
CALLIANASSA RESPIRATORY ADAPTATIONS 133
californiensis will soon flic if not maintained with its body contacting the wall of a tube. In either anoxia or normoxia, survival of specimens of C. jauiaicense does not seem to be influenced by whether animals are maintained in plastic tubes or individually in larger bottles and open dishes. The possibility remains, however, that the tube facilitates efficient respiration in hypoxic water.
Survival and oxygen consumption by specimens of C. jauiaicense in air were investigated despite the lack of direct evidence that this species resorts to aerial respiration in nature. However, on several occasions living animals were collected from substrates which had been exposed for up to 2 months, and on one occasion they were collected from a mud bank more than 1 m above the water table. As negligible concentrations of oxygen are usually found in water of exposed burrows, and as C. jauiaicense has the ability to survive (Fig. 2) and respire in water- saturated air, occupancy of exposed upper portions of the burrows seems at least a plausible alternative to longterm anoxia. Aerial respiration is best documented among terrestrial and semiterrestrial decapods but is also used on an "emergency" basis by a number of aquatic species (Wolvekamp and Waterman, 1960). For ex- ample, Carcinits niaenas may raise its body and aerially ventilate when stranded in hypoxic ponds (Taylor and Butler, 1973). The humid environment of Cal- lianassa burrows fulfills an important requirement for aerial respiration as oxygen diffuses most rapidly across a wet cuticle (Lockwood, 1967).
The lower V02 of C. jauiaicense in saturated air than in water could be attributed in part to decreased activity under aerial conditions as high P,,., is main- tained at the respiratory surface without the need for extensive ventilatory move- ments. The aerial \*()J was less than 40 9r of aquatic \T0- measured in BOD bottles or the flow-through system. This difference in aerial versus aquatic Vo-_. is much greater than that reported for well-adapted semi-terrestrial decapods and suggests that the ability for aerial uptake of oxygen is not particularly well developed. It is not known to what degree C. jainaicense depends upon anaerobic pathways under such conditions as at least a partial source of energy. As suggested by Miller ct al. ( 1976), it would lie interesting to investigate the possible use of anaerobic pathways even at high oxygen tensions.
Within the P(>., range of respiratory independence, V,,., of C. jauiaicense is ca 68 fjd/(g wet wt-hr ) in a stirred BOD bottle from which oxygen is being depleted and 50 to 55 //,!/( g wet wt-hr) in a flow-through ( 10 nil min ) respirometer. Both of these methods involve placement of the animal into a small diameter tube which simulates a burrow and allows the animal to brace itself while ventilating. Measurement of V0.. in tubes seems to provide the better index of "routine" (sensu Fry, 1975 ) metabolic rates in burrowing thalassinids as this situation most closely approximates the natural mode of respiration. The reported respiration rates for intermolt specimens of Callianassa californiensis over the P,»., range of respiratory independence are for animals not in tubes (Thompson and Pritchard, 1969; Miller ct al., 1976; Torres ct al., 1977), and these rates vary from ca. 18 to ca. 34 p.1 (g wet wt-hr). Farley and Case (1968) have shown that pleopod activity is clearly greater when a specimen of Callianassa californiensis is placed into a small diameter tube than when it is placed into a tube too large for it to brace against tube walls while countering pleopod strokes. In the present study smaller tubes were used in BOD bottles and this necessitated the
KU DARK VI. I.. FEEDER
use of smaller animals (1.45-3.82 g) than in the flow-through respirometer (4.55-7.13 g) ; this size difference, as previously suggested by Torres et al. (1974), could account for observed differences in Vo2. Differences in the stirring or flowing of water during measurements of V<>2 in BOD bottles and the flow- through system may additionally contribute to a difference in Vo2 measured by those methods, as flow characteristics can engender adaptive respiratory responses (Mangum and van Winkle, 1973).
Regardless of the method of measurement, the metabolic rates here reported for C. jainaiccnsc rank among the lower known for crustaceans at similar tempera- tures (Wolvekamp and Waterman, 1960) and reflect metabolic adaptation to a hypoxic habitat. Low metabolic rates in Callianassa californiensis and Upogcbia pugcttcnsis at 10° C are also considered adaptations to a similarly hypoxic habitat (Thompson and Pritchard, 1969). Montuori (1913) reports a much higher Vo2 of 132 /xl/(g wet wt-hr) in Callianassa subterranea and 368 |ul/(g wet wt-hr) in Gebia litoralis (Upogcbia litoralis) at 25° C, but experimental conditions of his study differ too greatly to permit detailed comparisons of data.
The low critical oxygen tension (Pc) for Callianassa jainaiccnsc likewise sug- gests a metabolic adaptation. Metabolic regulation is common to a large number of aquatic crustaceans, and van Winkle and Mangum (1975 ) note that such regulation is expected where the path of oxygen permeation is restricted to an indirect route by way of circulating body fluids. The Pc between 10 and 25 mmHg for C. jainaiccnsc (Fig. 3), like that reported for other thalassinids (Thompson and Pritchard, 1969; Miller ct al., 1976; Torres ct al., 1977) is well below the Pc for most crustaceans (Wolvekamp and Waterman, 1960). Hypothetical curves of oxygen consumption over decreasing P02, as predicted by a polynomial model (Mangum and van Winkle, 1973), suggest a P(. for Upogcbia affinis similar to that reported for U. piigcttcnsis but not as low as those in Callianassa californiensis or C. jainaicensc. It has been suggested that the Pc of crustaceans represents the Po2 at which blood pigment fails to become saturated at the gills (Redmond, 1955) or, more recently, that it reflects the initiation of anaerobiosis (Young, 1973). Regardless, maintenance of aerobic respiration until a very low Po2 is reached would seem a conservative adaptation for burrowers in hypoxic substrates.
Present data do not explain the mechanics involved in metabolic regulation by Callianassa jainaiccnsc at low P(>2. Neither pleopod activity (Fig. 3) nor heart rate (Thompson and Pritchard, 1969) shows a linear increase with decreasing ambient Po2- The findings of Torres ct al. (1977) additionally show that complete immobilization of the pleopods causes no appreciable change in the Pc. However, scaphognathite ventilation rates were not monitored, and it remains to be seen whether scaphognathite ventilation rate, cardiac output, and circulation patterns undergo proportional increases at lowered PO-... Studies with totally bled speci- mens of Callianassa suggest that at least part of the ability to regulate V02 is due to respiratory properties of the blood itself (Miller ct al., 1976).
The increase in pleopod activity at and just below the Pc may reflect an "escape" reaction ; escape from low P02 in nature could be achieved by rapid pleopod ventilation which would replace low P02 burrow water with higher P02 water from overhead. Periodic ventilatory pulses, such as those reported for Callianassa filholi when specimens are confined to a glass tube ( Devine, 1966), may likewise
CALLIANASSA RESPIRATORY ADAPTATIONS 135
be such reactions triggered by depletion of oxygen to a concentration near the Pc. Such reactions and taxic responses exhibited during flow-through respirometry suggest the presence of an internal or external oxygen receptor in C. jautaicensc; Farley and Case (1968) have previously postulated the existence of such a receptor in Callianassa calijornicnsis and C. affinis, but direct evidence for an oxygen recep- tor is still lacking for any thalassinid species.
The drop in V,,., following abrupt exposure of specimens of Callianassa jainai- censc to hypoxic water (Fig. 4) indicates that regulation of metabolic rates in low Po2 is dependent upon how fast hypoxia is approached. The decrease in \ro2 suggests a partial shutdown of aerobic respiration or loss of metabolic regulatory ability unless hypoxia is approached slowly. Mangum (1970) reports aerobic shut- down in bloodworms rapidly introduced into hypoxic water, and Kushins and Mangum (1971) note that metabolic response of the snail, Nassanus, depends upon how rapidly hypoxia is approached. Similarly, Hiestand (1931) reports that a crayfish which normally responds as a metabolic regulator will metabolically con- form if placed into a small volume of water where Po2 is reduced rapidly or if depletion of oxygen in a large jar commences at less than air saturation. Because V0o of C. jainaiccnsi i'owly increases after several hours in hypoxic water (Fig. 4 ) , it appears that a time-dependent internal change, such as descrease in pH or re-establishment of diffusion gradients, is linked to ability to regulate Vo2. This suggests that some degree of Iow-Po2 acclimation is induced in C. jauiaiccnse after six or more hours of hypoxia.
The lowest ambient oxygen tensions in the Callianassa jaiiiaicense habitat occur on occasions when burrows are exposed and animals cannot ventilate by pumping water from overhead. An increase in Y,>2 after termination of anoxia (Fig. 6) suggests the development of an oxygen debt under such conditions; a similar compensatory increase occurs in Callianassa calijornicnsis and Upogebia pugcttcnsis after exposure to anoxia (Thompson and Pritchard, 1969). Although evidence of oxygen debts among crustaceans is meager ( Lockwood, 1967), clear evidence of anaerobic glycolysis in Callianassa calijorniensis tends to support this hypothesis ( Ha\vkins, 1971, unpublished M. S. thesis, Oregon State University). Published field observations of several Pacific coast thalassinids (MacGinitie, 1935) and present observations of Callianassa jainaiccnsc, C. major, and C. islagrande on the Louisiana coast indicate these animals move to the upper portions of the burrows as high tides flood burrows exposed earlier by low tides. Such behavior would facilitate the most rapid exchange of burrow water and payment of an oxygen debt developed during anaerobiosis. The magnitude and duration of elevated V02 rates in C. jauiaicensc following anoxia are determined by the ambient P02 provided at the termination of anoxia, as evident in comparing Figures 5 and 6. The rate of oxygen uptake under such circumstances is at least passively affected by the blood-to-water gradient of Po2, and a pattern for metabolic regulation is temporarily supplanted by metabolic conformation and higher respira- tory rates in normoxia. It seems very unlikely that the observed differences in post-anoxia V,,2 rates can be attributed to activity, because activity is greater and maintained at elevated rates for a longer period during slightly elevated V02 in hypoxic water following anoxia (Fig. 5) than during the greatly elevated VO2 in normoxic water following anoxia (Fig. 6).
136 DAKKVL I.. FKLDEK
Oxygen uptake via areas of the integument other than gills has not heen investigated in C. jainaiccnsc, hut as these animals have a thin exoskeleton, such extrabranchial uptake of oxygen seems a strong possibility and could prove advantageous in a hypoxic habitat. Although there is no conspicuous morpho- logical evidence of specialized, extrabranchial respiratory surfaces in C. jainaiccnse, accessory pleopodal gill filaments occur on some thalassinids (de Saint Laurent, 1973 ). The experiments of Torres ct al. ( 1977 ) show no evidence of extrabranchial uptake in pleopods of Callianassa califoniiensis, but further investigations of accessory uptake are warranted ; such studies could possibly explain the low oxygen gradients between prebranchial and postbranchial blood reported by Miller ct al. (1976).
SUMMARY
Callianassa jainaiccnsc survives exposure to aquatic and aerial anoxia for more than 3 days. In normoxic water-saturated air it survives for ca. 16 days. The rate of oxygen consumption (Vo2) in air is less than 40% of Vo2 in water. Aquatic V,,., is regulated above critical oxygen tensions (Pc.) of 10 to 25 mmHg when animals are allowed to slowly deplete oxygen from a sealed bottle. Mean aquatic Vo2 of animals in a flow-through respirometer or in tubes placed into sealed BOD bottles ranges from 50 to 68 />d/(g wet wt-hr) over oxygen tensions (P0,) above the P,.
After a 12-hr exposure to anoxic water, VO2 is not regulated; post-anoxia Vo2 in hypoxic water (37 mmHg) is initially less than VQ., measured in normoxic water (150 mmHg) before exposure to anoxia; post-anoxia V0l> in normoxic water is initially two times the pre-anoxia Vo2 and suggests the development of an oxygen debt during anoxia. When P02 of ambient water is abruptly dropped from 150 to 37 mmHg, specimens of C. joinaiccnsc exhibit a partial shutdown of aerobic metabolism, but the Vo2 begins to recover after 6 hr in hypoxia.
When oxygen tension is slowly decreased, pleopod ventilation rate varies little as POL- changes from 120 to 20 mmHg. The pleopod ventilation rate increases as POL, falls 20 to 10 mmHg, but decreases below 10 mmHg and stops after several hours under anoxia. The rapid response of taxis and pleopod activity when C. jainaicense is exposed to altered P02 suggests rapid perception of external oxygen levels and provides further circumstantial evidence of an oxygen receptor in thalassinids.
Tolerance of anoxia, metabolic regulation to a low Pc, low metabolic rates, metabolic responses following anoxia, and taxic response to altered Po2 constitute adaptations to the hypoxic habitat of C. jainaiccnsc.
LITERATURE CITED
BOVBJERG, R. V., 1952. Comparative ecology and physiology of the crayfish Orconcctcs
propinquns and Cainbanis fodicus. Physiol. Zoo/., 25 : 34-56. VON BRAND, T., 1946. Anacrobiosis in Invertebrates. Biodynamica Monographs, Normandy.
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CALLIANASSA RESPIRATORY ADAPTATIONS 137
FARLEY, R. D., AND J. F. CASE, 1968. Perception of external oxygen by the burrowing shrimp,
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Reference : liiol. Bull. 157 : 138-152. (August 1979)
AN ANALYSIS OF THE DEFENSIVE MECHANISMS OBSERVED IN
THE ANEMONE ANTHOPLEURA ELEGANTISSIMA IN
RESPONSE TO ITS NUDIBRANCH PREDATOR
AEOLIDIA PAPILLOSA
LARRY G. HARRIS AND NATHAN R. HOWE 1 /iu'lin/y Department, University of Nczv Hampshire, Durham, Nciv Hampshire 03S24
The sea anemone Anthopleura clegantissiina (Brandt, 1835) is a conspicuous member of mid-intertidal communities along the exposed rocky west coast of the United States (Hand, 1955; Ricketts and Calvin, 1962; Sebens, 1977). Several recent studies (Waters, 1973; Harris, 1973; Edmunds, Potts, Swinfin and Walters, 1975, 1976) have reported that A. clegantissiina is a preferred prey of the anemone-eating aeolicl nudibranch, ^-leolidia papillosa (Linnaeus, 1767). Ed- munds ct a!., (1976) described behavioral reactions of A. elegantissima to attack, including bulging of the column at the site of attack, crawling, and releasing from the substrate. Howe and Sheikh ( 1975) characterized an alarm pheromone, anthopleurine. from A. elegantissima and described the behavioral response it elicited in the anemone. Howe and Harris (1978) demonstrated that A. papillosa acquires anthopleurine when feeding on A. elegantissima and that leakage of the pheromone caused the alarm response in other individuals. Waters (1973) speculated that A. papillosa is evolving to specialize on A. elegantissima.
Anthopleura elegantissima has well-developed behavioral responses to attack by A. papillosa, but none of these behaviors provides an effective defense in the laboratory. In addition, A. papillosa is consistently found associated with one of its least preferred prey, the subtidal anemone Mctridiiim senile (Linnaeus, 1767) (Harris, 1973; Brewer, 1977). The ineffectiveness of the defenses of A. elegantis- sima against one of its chief predators under laboratory conditions and the fact that the predator is primarily associated with a less preferred prey suggests that the defenses may be more effective under natural conditions. The purpose of this study was to investigate a series of potential defensive mechanisms which may pro- vide at least partial protection for A. elegantissima against A. papillosa. The effec- tiveness of these mechanisms were then evaluated in the context of the environment in which this predator-prey association is found.
MATERIALS AND METHODS
This study was conducted during the period of January to June 1976, though L.G.H. had been making observations on A. papillosa since 1964. The laboratory experiments and observations were conducted at the facilities of Hopkins Marine Laboratory, Pacific Grove, California. Field studies were clone at the laboratory and at two nearby locations in Monterey Bay (36° 37' N, 121° 53' W).
Some of the specimens of Anthropleura elegantissima were collected from a rock
1 Present address : Department of Biology, University of Houston, Houston, Texas 77004.
138
ANEMONE DEFENSIVE MECHANISMS 139
outcropping on the east side of the laboratory and adjacent to a ramp for an old boat works (Site 1). These rocks face east and are exposed to direct sunlight from sunrise to late afternoon (1500-1600 hr). Most of the specimens of Antho- pleura clcgantissima and some of the specimens of Aeolidia papillosa were collected from the rocky intertidal zone under abandoned sardine canneries (Site II) located adjacent to the laboratory ; this site is shaded by the buildings and only receives direct sunlight from sunrise until about 1200 hr. Most of the specimens of A. papillosa were obtained from a subtidal site under Wharf No. 2 in Monterey (Site III). At this site, specimens of A. papillosa of various sizes can be obtained throughout the year, feeding on the large concentrations of Metridium senile (Yarnall, 1972; Brewer, 1977). Animals were maintained in running sea water at ambient temperatures (14° C).
It is important to distinguish between two forms of A ntlio pleura elegantissima present in Monterey Bay and farther south. The clonal form of A. elegantissima is restricted to the midtide zone, forms clones by binary fission on open rock surfaces, and seldom grows to a column diameter greater than 50 mm. A solitary form of A. elegantissima is found in the lower intertidal zone and to depths of 15 m; it attaches in cracks in the rock substrate and has not been observed to reproduce asexually. Individuals of this anemone may attain a column diameter of greater than 80 mm. In this study, we will refer only to the clonal form.
Intensive surveys of two rocky intertidal sites (Sites 1 and II) were undertaken in March and again in May, 1976, to determine densities of A. papillosa associated with A. elegantissima. Site II under the canneries faced northeast and received direct sunlight only during the morning hours. Small specimens of A. papillosa were common among clones of A. clcgantissima at this site from January through March, 1976. Sampling was done using a 1/64-m quadrat to determine the relative density of A. papillosa compared to the density of A. elegantissima. No specimens of A. papillosa were ever found associated with clones of A. clcgantissima at Site I, so no quadrat sampling was done there nor at Site II in June.
Three experiments were conducted to assess the importance of desiccation as an environmental stress on A. papillosa. In the first, six specimens of A. papil- losa and six specimens of A. clcgantissima were placed in stacking dishes without water for 6 hr. The experiment was done in the laboratory to approximate the time of exposure at neap low tide on a sunless day. In the second and third experiments, groups of A. papillosa were placed on rocks among clones of A. elegantissima immediately after the water had receded below the clones and were retrieved just prior to reflooding by the incoming tide. At the end of each experiment, animals were placed in dishes containing fresh sea water and held for 24 hr. Those animals which were moribund or unable to hold onto the glass dish after 24 hr were considered lost, while those which were attached and crawling normally were rated as surviving the low tide. The justification was that animals which were too weak to remain attached to the rock would be washed free by wave action. A nudibranch washed off a rock in the midtide may ultimately survive, but it is at least removed from the anemones on which it had been feeding. Controls were left in running sea water during the experiments.
Observations were made on feeding encounters between A. elegantissima and A. papillosa to determine the sequence of attack, the behavioral responses of A.
140 L. G. HARRIS AND N. R. HOWE
elegantissima and the preference of A. papillosa for particular regions of the anemone. In the first experiment, a number of anemones were removed from rocks and placed in a dish coated with silicone grease which prohibited their attach- ment. These anemones were left in running sea water for 24 hr and at the time of the experiment a majority of them were open. Fifty nudibranchs (body length 2-3 cm) were released into the dish and allowed to feed for 5 hours. To determine sites of attack by A. papillosa, the anemones were then relaxed in 7% MgCl (in fresh water) and surveyed for tissue damage which was obvious by direct observation. In a second experiment, approximately 100 specimens of A. elegantissima from a single clone were placed in a running sea-water table and groups of four to five nudibranchs were placed among the anemones ; attacks were described and tabulated.
Observations showed that contact of any part of the nudibranch with the column of an anemone caused local swelling of the column at the site of contact. Preliminary experiments showed that the anemones were responding to A. papillosa mucus. A series of experiments were conducted to determine the site of receptivity of A. elegantissima to A. papillosa mucus, the duration of the response and the specificity of this response relative to other nudibranch species.
The tentacles or the column of the anemones wrere touched with mucus-covered or blank cotton swabs in the first experiment. In experiment two, mucus was obtained from the back or the foot of A. papillosa, from the aeolid Hcnuissenda crassicornis (Eschscholtz, 1831) and from the dorid nudibranch Anisodoris nobilis (Odhner, 1907). A. nobilis, which eats only sponges, served as a control in the second set of experiments. In all tests, nudibranchs were rubbed writh a wet cotton swab which was then applied to a part of the body of the anemone. A separate swab was used for each anemone. The behavior of the anemones and duration of any response was described from observations made several times an hour.
RESULTS AND OBSERVATIONS
Qualitative sampling of a number of exposed rocky intertidal habitats along the California coast by both authors over several years showed that A. papillosa is found in association with clones of A. elegantissiiiia throughout the year. The density of nudibranchs tends to be higher in the winter than in the summer, and a majority of the animals seen and/or collected were large — between 30 and 80 mm in length.
At certain times of the year, A. papillosa can be very common in association with A. elegantissiiiia (Table I). Small specimens of A. papillosa (mean length 12 mm; maximum 22 mm) were prevalent at the canneries site (Site II) from January through March. However, the mean length of animals observed and/or collected did not increase over this 3-month period, and they were not present a few hundred meters away at Site I. In late April, a storm pounded the intertidal zone with approximately 2.5-m waves. From late April through July no speci- mens of A. papillosa were observed at either of the intertidal stations, although at Site III all sizes were common in subtidal fouling communities (Table I). A. papillosa grows from 1 mm to over 30 mm in about 2 months when feeding on A. elegantissima in the laboratory (Harris, in preparation) ; this suggests that
ANEMONE DEFENSIVE MECHANISMS
141
TABLE I
Comparison of Aeolidia papillosa density in association with anemone clones at two intertidal and one subtidal (8 m] sites in Monterey Bay in winter and summer. Site I is n rocky outcropping, on the beach on the NE side of Hopkins ^f urine Slut ion. Site II is a rocky ledge und'T the canneries about 300 m E of Site I. Site III is i/n/l :r commercial \\~harf No. 2 in Monterey. Site I and II contain clones of Anthopleura elegantissiina and Site III is dominated by clones «f Metriclium senile. Where no quadrat numbers are given, a minimum of 5 hr of observations were made at each site during that month.
Number A. papillosa/m'1 of anemone clone
|
Site |
February 1976 |
April 1976 |
June 1976 |
Time of direct exposure to sunlight |
|
I |
0/m2 |
0 |
0 |
sunrise to ^-1600. |
|
II |
24.3. in'-' |
0 |
0 |
sunrise to ^1200. |
|
(104 1 64 m2) |
||||
|
III |
86 m2 |
present |
''.5 ni- |
none |
|
(10 1, 10 m2) |
(20 1/10 in-'i |
nudibranchs were continuously recruiting to the site under the canneries but that they were not surviving long enough to reach sexual maturity.
Small specimens of A. papillosa (< 15 mm length) were typically found within 2 cm of the anemones. Often this was at the edge of a clone or within a scattered aggregate of anemones. A nudibranch might be in a small depression or crack in the rock surface, but the majority of individuals were exposed on flat surfaces, apparently having made no attempt to hide when the tide receded. Large specimens of A. papillosa (> 15 mm) may be found farther from their prey and typically seek out cracks in which to hide when not feeding. However, even with intensive
TABLE II
A summary of quadrat analyses done to assess the relative densitv of Aeolidia papillosa in relation to the percentage of free space among Anthopleura elegantissiina clones. The sampling was done at Station II under the canneries on 12 February 1976. The quadrat size was 1 f>4 m-, and 104 quadrats were sampled.
|
% Free space |
0% |
25% |
50% |
75% |
Total |
|
Number of quadrats |
22 |
39 |
31 |
12 |
104 |
|
Number of quadrats with |
|||||
|
A. papillosa |
2 |
17 |
11 |
4 |
34 |
|
Number of A. papillosa |
2 |
18 |
18 |
5 |
44 |
|
Mean |
|||||
|
Number of .4. papillosa |
|||||
|
per quadrat |
0.091 |
0.46 |
0.56 |
0.41 |
0.42 |
|
Density of A. papillosa |
|||||
|
per m2 of A . |
|||||
|
elegantissima clone |
5.8 |
29.4 |
35.8 |
26.2 |
24.3 |
|
% Quadrats with A. |
|||||
|
papillosa |
9% |
43% |
35% |
33% |
33% |
1 Significant at <0.01 (/-test).
142
L. G. HARRIS AND N. R. HOWE
TABLE III
Results of desiccation experiments in which Aeolidia papillosa was exposed to air for 6 hr and then returned to fresh sea water. Survival was determined after 24 hr. Experiment 1 was done in the laboratory while experiments 2 and 3 were conducted in the field by placing nudibranchs adjacent to clones of Anthopleura elegantissima on the receding tide and retrieving them just prior to submergence on the incoming tide. The results were significant at less than 0.01% using chi square.
|
Experiment |
1 |
2 |
3A |
3B |
|
Number of A. papillosa |
6 |
10 |
32 |
33 |
|
Number surviving after |
||||
|
24 hr (%) |
4 (66%) |
4 (40%) |
16 (50%) |
13 (39%)' |
|
Control survival |
100% |
100% |
100% |
100% |
1 Eighteen nudibranchs disappeared when the tide washed over the rock containing the animals before they could be retrieved; only 15 animals remained and the 18 missing A. papillosa were considered killed.
searching, no nudibranchs larger than 22 mm were found at Site II during the period from January through June 1976.
Results of the quadrat sampling at Site II illustrate the tendency of small nudibranchs to be found in open areas adjacent to groups of A. elegantissima (Table II). The quadrats were only placed over areas containing A. elegantissima, because numerous observations indicated that A. papillosa remains close to its prey. Only two nudibranchs were found within what appeared to be solid masses of anemones while the vast majority occurred at the periphery of clones. Close examination revealed that even in the most tightly packed clones, there was usually bare rock between pedal disks. The sampling data suggest that anemones within tightly packed clones are essentially free from predation by A. papillosa. While A. papillosa tended to be found at the periphery of clones where there is more free space, two other predators or parasites, the prosobranch Epitonium tine turn (Carpenter, 1864) and the pycnogonid Pycnogonnm stcarnsi (Ives, 1892) were common only within these tightly packed aggregations.
While no individuals of A. papillosa were collected at Sites I and II during the late spring and summer of 1976, one of us (L.G.H.) had previously collected specimens of A. papillosa and egg masses at several locations (Eagle Point, San Juan Islands, Washington; Dillon Beach and Bodega Bay, California) during the months of June and July. These sites are more exposed open coastal locations than the protected environment of Monterey Bay. In each case, the density of A. papillosa was well below 1/nr of A. elegantissima clone. The majority of the nudibranchs were large (> 40 mm), sexually mature, and were located in tidepools or at the lower end of the anemone's distribution in the intertidal.
Results of tests on the effects of desiccation on A. papillosa are recorded in Table III. In both the laboratory and the field experiments, survival of test animals exposed to air for 6 hr was approximately 50%, while control animals maintained in running sea water had 100% survival. In the initial laboratory test, two animals died even though they were sitting in small amounts of residual water and mucus. Most of the nudibranchs were still reactive after 6 hr out of water, but those that ultimately died were unable to hold onto the dish after water was added.
ANEMONE DEFENSIVE MECHANISMS
143
Two smaller nudibranchs (about 20 mm) set out at Site I were so desiccated that they had to be scraped from the rock surface.
In Experiment 3B, (see Table III), 33 specimens of A. [>a[>illosa were placed among a clone of A. clcgantisshiia at Site II. Due to a miscalculation, the nudi- branchs were not retrieved until after a few small swells had already washed over the area. Onlv 15 out of the 33 animals were still attached to the rocks; the other
FIGURE 1. Photographic illustration of the initial contact between specimens of A. papil- losa and A. clcfiantissiinn and two resulting feeding behaviors and anemone responses: (a) the initial contact involving the nudibranch's rhinophores and the anemone's tentacles; (b) the mutual retraction that typically occurs following first contact; (c) this nudibranch fed on the column for about 3 hr and made numerous attempts to reach t'-e tentacles which were out of reach due to bulging of the column; (d) this nudibranch reached the tentacles before bulging of the column began and was lifted free of the substrate. The nudibranch fed in this position for about 3 hr; note line of mucus and detritus on the g'ass left by the anemone as it crawled during the attack.
144
L. G. HARRIS AND N. R. HOWE
TABLE IV
Feeding / \l»'i •/ incuts to <l ••termini' u'lietlier Aeolidia papillcsa shows a preference for specific body regions when attacking Anthopleiira de^antissima. In Experiment A the tinemnnes were nut attached and were lying on their sides so an approaching nndihrancli had one chance to encounter the oral area first, two chances for lite column and one chance for the ped:il disk. In Experiment B, the anemones were altucheil and a nitdihranch would typically make contact with a tentacle first, but then it would touch the lolumn i\rn if it attacked the tentacles. The results were significant at less than 0.01',, using chi square.
|
A |
B |
|||
|
Experiment |
Predicted |
Actual |
Predicted |
Actual |
|
Column |
16.5 (50%) |
4(12%) |
30.5 (50' , i |
19 (31%) |
|
Tentacles |
8.25 (25%) |
18 (54' , ) |
30.5 (50' , ) |
42 (69%) |
|
Pedal disk |
8.25 (25%) |
11 (33%) |
Ol |
0 |
1 Anemones were attached so pedal disk was not available.
18 animals had disappeared, presumably washed away by slight swells (about 30 cm). Of the 15 animals remaining, 13 survived.
The results of a separate experiment also suggest the unsuitability of the intertidal for A. papillosa in the late spring and summer. On 2 May, 1976, 18 small specimens of A. papillosa (mean size 15 mm) were placed among marked clones in protected habitats under the canneries. The animals were placed among clones where there were cracks and algae to provide refuges from desiccation. Three days later, only one nudibranch could still be found in the area where it was placed. By the eighth day, no specimens of A. papillosa remained in the vicinity. During the late spring and summer this protected habitat, which is shaded after 1200 hr is apparently too stressful for A. papillosa.
The behavior of the nudibranch attack and anemone responses have been described previously (Russell, 1942; Waters, 1973; Harris, 1973; Edmunds ct al., 1976), but they will be reviewed because it is relevant to understanding the mechanism behind the bulging behavior described by Edmunds ct al., (1976). Figure 1 illustrates the sequence of attack. Initial contact is typically between the nudibranch's rhinophores and the anemone's tentacles (Fig. la). The tentacles and nudibranch both retract (Fig. Ib). A. papillosa then moves into attack with buccal mass extended, rhinophores retracted and cerata bristled. The oral tentacles touch the column causing it to bulge in the area touched (Figs. Ic, d). The bulging behavior consists of an exaggerated relaxation of the column wall where contact has been made. Waves of contraction run up and down the column and changes in height and shape are frequent during bulging. This behavior continues for up to 6 hr after a single contact with A. papillosa mucus. A result of this behavior is to raise the tentacles out of reach of the nudibranch (Fig. Ic). If the A. papillosa does reach the tentacles, before bulging is initiated, it may be lifted free of the substrate (Fig. Id).
Experiments testing for a preferred site of feeding by A. papillosa on A. elegantissiuia showed that the tentacles are selected over the column (Table IV). Xudibranchs also select the pedal disc over the column when anemones are not
ANEMONE DEFENSIVE MECHANISMS
145
attached. This preference for the tentacles is even more obvious when the attack sequence is observed; as in Figure Ic, nudibranchs often finally fed on the column, because the anemone's behavioral responses prevented easy access to the tentacles.
The bulging behavior of A. elegantissima is elicited by mucus from A. papillosa and also from the aeolid Hcniiisscnda crassicornis which occasionally attacks anemones (Table V). That mucus from A. papillosa caused bulging was first indicated during attempts to observe the alarm response in a tidepool containing several anemones. An A. papillosa was placed in the pool to determine if feeding on one anemone would cause the alarm response in other individuals. The nudi- branch crawled between two anemones, but did not attack either. Within minutes the contacted anemones had elongated their columns ; they remained in this expanded and distinctive posture for over 2 hr. This indicated to us that mucus from the nudibranch may be initiating this behavior. Subsequent experiments (Table V) verified this hypothesis. Mucus from A. papillosa caused the column to bulge, and this behavior pattern lasted for 3 to 6 or more hours even without further stimuli from the nudibranch. In addition to inflation of the column on the side touched, the verrucae dropped their attached sand grains and shell bits.
Tests conducted to determine sites of sensitivity in A. elegantissima to A. papillosa mucus revealed that only the column was responsive. The tentacles contracted when touched by mucus, but did not elicit any inflation behavior by any part of the column and returned to their normal relaxed position within a minute or two. Howe and Sheikh (1975) showed that the tentacles were the site of greatest sensitivity to the alarm pheromone anthopleurine. Mucus from any part of A. papillosa and from Hcniiisscnda crassicornis elicited column bulging, while mucus from Anisodoris nobilis had no observable effect on anemone be- havior. Some anemones continued to show bulging reactions for 6 or more hours after a single contact with mucus from A. papillosa even in running sea water. Since 6 hr is the approximate time of submergence during a tidal cycle and A. papillosa only feeds during high tide, these results suggest a two-part mecha- nism in which anthopleurine causes a quick and short-term contraction of the tentacles which increases the likelihood that A. papillosa will contact the column first. Contact with the column will result in mucus from the nudibranch being
TABLE Y
ry of two experiments t» test the effects of Aeolidia papillosa mucus on column bulging in Anthopleura elegantissima. Mucus secreted from glands on the foot and on the ccruta was compared. Mucus from two other nudibranchs was also used; Hermissenda crassicornis occasionally attacks anemones, while Anisodoris nobilis cats only sponges and served as a control.
|
% Anemones bulging over time in minutes |
|||||||
|
0 |
min 60 |
120 |
180 |
240 |
300 |
360 |
|
|
A. papillosa foot (34) 1 |
0% |
76 |
62 |
53 |
29 |
18 |
6 |
|
A. papillosa cerata (37) |
<)', |
81 |
84 |
81 |
62 |
32 |
22 |
|
H. crassicornis (37) |
<)', |
65 |
84 |
78 |
51 |
24 |
22 |
|
A. nobilis (40) |
o% |
2.5 |
5 |
7.5 |
7.5 |
2.5 |
0 |
1 Total number of anemones used in two replicate experiments.
146 L. G. HARRIS AND N. R. HOWE
sensed by the column receptors ; this will initiate a slow but long-lasting inflation of the column that will raise the tentacles far above the substrate, often out of reach of the nudibranch.
Verification that A. papillosa mucus elicits bulging under field conditions occurred during desiccation experiment 3 (Table III). Nudibranchs were placed in clear areas within scattered clones. Water and mucus from the nudibranchs drained down the rock surfaces and around anemones below the nudibranchs. Thirty minutes after the experiment began, anemones which contacted the fluid draining from A. papillosa were bulging; many dropped their sand grain cover and several had released from the rock. The unattached anemones were lying on their sides with the column bulged, but the slope was too gentle for them to fall or roll from their original position without water movement. The next day the anemones were reattached, contracted and recovered with sand grains. How- ever, comparison of photographs taken during the experiment and the following day showed that a number of anemones had moved 2 to 6 cm and four anemones had disappeared.
When attacked by A. papillosa, individuals of A. elcgantissima crawl in the opposite direction. In 3 hr animals may have crawled as far as 4 cm. In the next 24 hr animals continued to crawl for another 3 to 4 cm, although limited observa- tions suggest that in the field they may not crawl as far as they do in the laboratory.
In the laboratory, approximately 10% of the anemones attacked by A. papillosa released from the substrate during the attack. We do not know whether the percentage of release is as high in the field where there is active water movement, though both authors have encountered A. papillosa in tide pools in the process of attacking unattached anemones. Verrucae of detached anemones are extremely ad- hesive and attach quickly to any object that they contact. Unless there is active water movement at the time anemone releases, it may ultimately reattach in the same area.
DISCUSSION
The clonal form of Anthopleura elcgantissima occurs primarily in the midtide zone on the exposed coastline of the west coast of North America (Hand, 1955 ; Ricketts and Calvin, 1962; Dayton, 1971; Francis, 1973a, b; Sebens, 1977). In this habitat individuals are exposed to wave action and six or more hours of exposure to air twice every 24 hr. Connell (1972) has suggested that the upper limit of a species in the intertidal is due to physical factors, especially physiological tolerance to exposure, and that the lower limits are due primarily to biological factors such as competition and predation. Dayton (1971) showed that A. clegantissima cannot survive in more protected areas of the San Juan Islands, because it is incapable of withstanding the long mid-day low tides in the summer. The exposure experiments (Table III) and collecting data suggest that specimens of A. papillosa are less able to withstand desiccation than their prey and that affects their ability to hold on to the substrate when the tide returns. The effect of desiccation appears to be greatest in the warmer months of the year and in protected areas such as Monterey Bay, since A. papillosa can be collected with A. elegantissima in the summer at exposed, open coastal sites (Waters, 1973).
ANEMONE DEFENSIVE MECHANISMS 147
A. papillosa presumably survives better in exposed habitats because spray from waves decreases the threat of desiccation.
The majority of large, reproductive A. papillosa found associated with clones of A. elegantissima have been at the lower end of the anemone's range in the intertidal. The mean size of A. elegantissima is greatest in these habitats (Sebens, 1977). Sebens (1977) has suggested that the longer submersion time increases feeding time for the anemones. A similar phenomenon has been described for the gastropod Tegula funebralis (Paine, 1969). The largest specimens of T. funebralis were found in the lower intertidal where the increased food supply and time for feeding would be translated into greater reproductive output. However, this was offset by increased threat of predation by the starfish Pisastcr ochraceus. It is likely that the intertidal distribution of A. elegantissima represents at least a partial or seasonal refuge from predation by A. papillosa and that the lower limit of the anemone's range is influenced by where the balance between the potential for increased reproductive output and the threat of predation comes out in favor of predation.
Clone formation in A. elegantissima has been described as a competitive strategy that allows for rapid space utilization (Sebens, 1977). The fact that A. elegantis- sima has a well developed aggressive response to encroachment by other anemones similar to that reported for corals by Lang (1973) reinforces the likelihood that clone formation is an adaptation for space competition (Francis, 1975b, 1976; Purcell, 1977). Clone formation may also have adaptive significance as a defense against predation. Veligers of A. papillosa settle on the rock surface adjacent to their anemone prey (Harris, unpublished observations) ; therefore, only the periphery of a clone will be available for recruitment of the predator. The circum- ference of a clone of anemones is less than the sum of the circumferences of the same number of individual anemones were they dispersed, so there is less surface area for settlement. Also, the anemones at the center of the clone will be relatively free from predation (Table II) while those at the periphery of the clone will be exposed to the greatest damage from nudibranch attacks.
Francis (1976) has shown that anemones at the periphery of a clone serve as soliders which expend most of their energy in the production of nematocysts and regenerating wounded areas after aggressive encounters. These soldiers do not contribute directly to asexual or sexual reproductive efforts of the clone. Assum- ing that clone formation is primarily a competitive strategy similar to encrusting colonial growth as proposed by Jackson (1977), the additional adaptive value of minimizing the threat of predation for a majority of clone-mates should have a synergistic effect in reinforcing selection for cloning.
A. papillosa shows a clear preference for the tentacles of A. elegantissima (Table IV). The final feeding site is dependent on several factors including relative sizes of the predator and prey, the initial position of the tentacles, and the reaction of the anemone. If the length of the nudibranch is equal to or greater than the column diameter of the anemone, then the aeolid is large enough to readily attack the tentacles of an anemone in the normal open position (see Fig. la). Should the tentacles be raised in an alarm response or chance behavior, then the likelihood of the nudibranch reaching the tentacles is reduced, particularly if the anemone responds to contact with the column by bulging (Figs. Ic, d).
14S L. G. HARRIS AND N. R. HOWE
A. papillosa that have previously eaten A. clegantissiiita leak anthopleurine for up to 7 days and anthopleurine leaked from a nudihranch will initiate the alarm response (Howe and Harris, 1978). The alarm response, which involves a quick general contraction of the tentacles, may be of some advantage to an anemone in that it does increase the possibility that A. papillosa will not contact the tentacles first. A number of encounters have been observed in which the nudibranch gave up when it failed to reach the tentacles of an anemone. A speci- men of A. papillosa feeds daily and primarily at high tide. The response to anthopleurine leaking from a nearby nudibranch should be greatest at the beginning of a high tide, since the anemones will have been free of the water-carried signal during low tide ; continued exposure to anthopleurine causes fatiguing of the response over time (Howe and Sheikh, 1975).
The alarm response is of short duration and therefore not likely to deter a large, persistent nudibranch. However, raising the tentacles does increase the likelihood that the predator will touch the column. The column responds to A. papillosa mucus by swelling and this behavior pattern lasts for several hours (Table V).
There are two possible advantages to bulging behavior. The first is protection of the tentacles. It should be selectively advantageous to keep the feeding structures intact and instead to lose tissue from the column. Attacks to the column typically involve removal of epidermal tissue which will be regenerated, and very seldom result in complete penetration of the body wall. The second advantage is that when a specimen of A. papillosa attempts to reach or succeeds in reaching the tentacles, it must release at least partially from the rock surface (Figs. Ic, d) and will be vulnerable to being dislodged by wave action. Being washed off the rock by surge may not kill the nudibranch, but it removes it from further predation on the same clone.
The site of reception for the water-transmitted pheromone, anthopleurine, is the tentacles of A. elegantissima, and the alarm response involves short duration, generalized contraction of the tentacles (Howe and Sheikh, 1975). Acolidia papillosa mucus is detected by contact, the receptors are in the column and the response is a localized inflation of the column. It is predictable that the receptors for a water-transmitted pheromone would be in the tentacles, for they extend farthest from the central axis of the animal. The generalized alarm response should be most effective if it occurs before contact is made with the predator since no directionality of response is required. It also seems likely that the receptors for a localized response to a slow-moving predator would be at the site of the response as is the case with the receptors for the bulging behavior.
Crawling by an anemone after an attack will potentially decrease the likelihood of a second attack because of the presence of the other members of the clone. Nudibranchs attack the first individual they encounter when foraging. This would spread the damage produced by a nudibranch to several members of a clone and decrease the chances for the loss of an individual and shrinkage of the clone. Another effect of crawling behavior would be to isolate the nudibranch on the rock substrate, increasing the chances of desiccation. This is most likely to occur in young nudibranchs that do not show a strong tendency to hide at low tide.
ANEMONE DEFENSIVE MECHANISMS 149
Approximately 10% of the encounters observed in the laboratory between A. papillosa and A. clegantissima resulted in the anemone releasing from the sub- strate. Rosin (1969) reported a similar escape response in Anthopleura nigresccns (Verrill) to its predator, the aeolid nudibranch Hcrviella sp. In the field, detaching from the rock substrate will potentially result in the anemone being carried to new habitats which may be viewed as a means of dispersal of the clone, and therefore, a positive side result of this association.
A specimen of A. papillosa consumes 50 to 100% of its wet body weight each time it feeds, which is at least once a day (Howe and Harris, 1978). Therefore, the nudibranch becomes an increasing threat as it grows to a point where it is capable of killing an anemone in a single meal ; this suggests that the selective Value of the defensive mechanisms discussed would be most effective against young nudibranchs. A. papillosa is about 0.5 mm in length when it first meta- morphoses. Nudibranchs about 1 mm in length grow to about 35 mm and become sexually mature in a little over two months in the laboratory (Harris, in prep- aration). This suggests that the time and size must be considered in evaluating the selective value of defensive mechanisms since it will be at least 2 months after metamorphosis before a nudibranch is able to cause serious damage during an encounter or before it begins to reproduce.
None of the defensive adaptations described in this paper stop A. papillosa veligers from metamorphosing in a clone, nor are they effective in detering predation in any given encounter between a nudibranch and an anemone either in the field or in the laboratory. We suggest that these mechanisms interact in such a way that the predator is killed before it reaches sexual maturity. The overall defensive strategy seems to be to minimize damage to clone members and to increase the likelihood that the predator will be killed or removed by desiccation and/or wave action.
The best evidence for this proposed defensive strategy is the fact that A. papillosa is primarily associated with the subtidal anemone Mctridinui senile (Harris, 1973, 1976. and in preparation). A similar pattern occurs in the Atlantic where A. papillosa is associated with Metridiitm senile (Stehouwer, 1952), Sagartia elegans (C. Todd, University of North Wales, personal communication) and Cereus pcduncnlatiis (J. Tardy, University of Poitiers, personal communication) ; these three anemones were found to be among the least preferred in laboratory studies by Edmunds ct al. (1975). Swennen (1961) reported large numbers of specimens of A. papillosa feeding on Actinia eqnina, a preferred species found in a similar habitat to that of Anthopleura elegantissiuia. Swennen's observations were made during the winter when the threat of desiccation would be least.
To influence the prey preference hierarchy of A. papillosa, the defensive strategies of anemones should focus on preventing the nudibranch from reaching sexual maturity and reproducing. Young nudibranchs seldom leave the prey where they have metamorphosed unless the prey is consumed or the nudibranch attains sexual maturity and searches for a mate (Harris, 1973) ; therefore, the choice of initial prey species takes place at the veliger stage prior to metamorphosis. The primary criterion for the choice of prey species must be survival to sexual maturity and reproduction. The prey preferences reported in the literature (Waters, 1973; Harris, 1973; Edmunds, et al., 1975) are derived from laboratory
150 L. G. HARRIS AND N. R. HOWE
choice experiments with adult nudibranchs. Harris (1976) proposed that the prey preference hierarchy for the veliger stage of A. papillosa should be limited to relatively few species of anemone in an area and the principle criterion for selection should be survival to reproduction. In contrast, adult nudibranchs should be much less selective in their choice of prey since continued survival and reproduction should be the primary consideration at this stage. A. papillosa attains sexual maturity 2 to 3 months after metamorphosis and at a length of about 35 mm ; an individual nudibranch is capable of continuing to reproduce for 6 or more months while growing to about 120 mm (Swennen, 1961; Harris, 1973; Clark, 1975). Adult specimens of A. papillosa show ingestive conditioning to even non-preferred anemones and will seek out prey they are conditioned to, unless they make contact with a more preferred species, and then they will switch (Harris, 1973; Wood, 1968; Murdoch, 1969). A hungry specimen of A. papillosa will attempt to feed on virtually any anemone species and even the corallamorpharian Corynactis cali- jornica Carlgren, 1936 (Waters, 1973; Harris, in preparation; Edmunds ct al., 1975).
In conclusion, the clonal form of Anthoplcura elegantissiuia has evolved a series of defensive mechanisms including intertidal position, cloning, the alarm response to the pheromone, anthopleurine, column bulging initiated by nudibranch mucus, crawling and releasing from the substrate. None of these adaptations prevent veligers of A. papillosa from metamorphosing in association with A. elegantissiuia nor do they prevent an attack. However, they do combine in the context of the natural environment to form a very effective defensive strategy which increases the chances that the predator will be removed and/or killed by desiccation and/or wave action before it grows to sexual maturity. By preventing reproduction in the majority of nudibranchs which do metamorphose on this species, A. elegantissiuia exerts negative selective pressure on the prey preference hierarchy of the veliger stage. The success of this defensive strategy is illustrated by the fact that A. papillosa is primarily associated with one of the least preferred anemones of adult A. papillosa.
Mechanisms like cloning and the release of anthopleurine may also serve other functions such as competition or communication between clonemates. The fact that these mechanisms have adaptive value for more than one aspect of the anemone's biology should increase the selection for these traits.
We wish to thank the University of New Hampshire for a Summer Faculty Fellowship to Larry G. Harris and the National Science Foundation and the ARCS Foundation for graduate fellowships to Nathan R. Howe. The research was done while both of us were in residence at Hopkins Marine Station. Luther Black, Terrence Gosliner, Alan Hulbert, Alan Kuzirian, James Taylor, Charles Walker and John Sasner read drafts of the manuscript and made numerous con- structive comments.
SUMMARY
1. The defensive mechanisms shown by the west coast, intertidal sea anemone, Anthoplcitra elegantissiuia, in response to its nudibranch predator Acolidia
ANEMONE DEFENSIVE MECHANISMS 151
papillosa are identified and evaluated in the context of the environment where A. elegantisshna occurs. The defensive mechanisms include intertidal distribution, clone formation, alarm response, bulging of the column, crawling and releasing from the substrate.
2. A. papillosa are primarily located at the periphery of clones so that anemones in the interior of the clone have a refuge from predation. Assuming that cloning is an adaptation for space competition in A. clcgantissinia, then the additional advantage derived as a defensive mechanism should increase selection for clone formation.
3. A. papillosa was less able than A. clcganiiss'nna to withstand desiccation from exposure at low tide. This suggests that the intertidal distribution of A. ele- gantissiina is a defensive adaptation which reduces the threat of predation by A. papillosa at least during the warmer months of the year.
4. The bulging of the column at the site of contact was found to be a localized response of several hours duration. The mucus of A. papillosa stimulated the response and the receptors were found to be situated in the column. Mucus from the coelenterate-eating aeolid nudibranch, Hcnnissenda crassicornis, also initiated the response while neither the mucus from the sponge eating dorid, Anisodoris nob His, nor control swabs dipped in sea water caused bulging.
5. None of the defensive mechanism directly protects an anemone from attack by A. papillosa. The defensive mechanisms all interact to minimize damage to the clone until the predator is removed by desiccation and or wave action. This strategy is most effective during the 2- to 3-month period between when the veliger metamorphoses and when the nudibranch reaches sexual maturity.
6. This defensive strategy of killing the young nudibranch before it reproduces may negatively influence prey selection by the veliger stage. The evolution of the prey preference hierarchy of the veliger stage should be based on the criterion of survival to sexual maturity. Evidence for the effectiveness of this defensive strategy is that Aeolidia papillosa is primarily associated with the subtidal anemone, Metridinni senile, one of the least preferred prey of adult nudibranchs.
LITERATURE CITED
BREWER, B. A., 1977. The association between an endoparasitic rhabdocoel, Family Fecampidae, and the nudibranch, Aeolidia papillosa (Linnaeus, 1761). Masters Thesis, San Jose State University. 43 pp.
CLARK, K. B., 1975. Nudibranch life cycles in the Northwest Atlantic and their relationship to the ecology of fouling communities. Hclgol. iviss. Mccrcsunters. 27 : 28-69.
CONNELL, J. H., 1972. Community interactions on the marine rocky intertidal shores. Ann. Rev. Ecol. Syst., 3 : 169-192.
DAYTON, P. K., 1971. Competition, disturbance, and community organization : the provision and subsequent utilization of space in a rocky intertidal community. Ecol. Monogr., 41: 351-389.
EDMUNDS, M., G. W. POTTS, R. C. SWINFEN, AND V. L. WATERS, 1975. The feeding prefer- ences of Aeolidia papillosa (L.). (Mollusca, Nudibranchia). /. Mar. Biol. Assoc. U.K., 54 : 939-947.
EDMUNDS, M., G. W. POTTS, R. C. SWINFEN, AND V. L. WATERS, 1976. Defensive behavior of sea anemones in response to predation by the opisthobranch mollusc Aeolidia papil- losa (L.) /. Mar. Biol. Assoc. U.K., 56 : 65-83.
FRANCIS, L., 1973a. Clone specific segregation in the sea anemone Antho pleura elegantisshna. Biol. Bull, 144 : 64-72.
152 L. G. HARRIS AND N. R. HOWE
FRANCIS, L., 1973b. Intraspecific aggression and its effect on the distribution of Anthoplcura
elcgantissima and some related sea anemones. Biol. Bull., 144: 73-92. FRANCIS, L., 1976. Social organization within clones of the sea anemone Anthoplcura clc-
yantissima. Biol. Bull., 150 : 361-376. HAND, C., 1955. The sea anemones of central California. Part II. The endomyarian and
mesomyarian anemones. Wasmann J. Biol., 13 : 37-99. HARRIS, L. G., 1973. Nudibranch associations. Pages 213-314 in T. C. Cheng, Ed., Current
topics in comparative patJwbiology. Academic Press, New York. HARRIS, L. G., 1976. Comparative ecological studies of the nudibranch Acolidia papillosa and
its anemone prey Mctridhtm scnsile along the Atlantic and Pacific coasts of the
United States. /. Moll Studies., 42 : 301. HOWE, N. R., AND L. G. HARRIS, 1978. Transfer of the sea anemone pheromone, anthopleurine,
by the nudibranch Acolidia papillosa. Chem. Ecol., 4 : 551-561. HOWE, N. R., AND Y. M. SHEIKH, 1975. Anthopleurine : a sea anemone alarm pheromone.
Science, 189 : 386-388. JACKSON, J. B. C., 1977. Competition on marine hard substrata : the adaptive significance
of solitary and colonial strategies. Amcr. Nat., Ill : 743-767. LANG, J., 1973. Interspecific aggression by scleractinian corals. 2. Why the race is not only
to the swift. Bull. Mar. Sci.. 23 : 260-279.
LENHOFF, H. M., 1968. Behavior, hormones, and hydra. Science, 161 : 434-442. MURDOCH, W. W., 1969. Switching in general predators: experiments on predator specificity
and stability of prey populations. Ecol. Monogr., 39 : 335-354. PAINE, R. T., 1969. The Pisaster-Tegula interaction: prey patches, predator food preference,
and intertidal community structure. Ecology, 50 : 950-961. PURCELL, J. E., 1977. Aggressive function and induced development of catch tentacles in
the sea anemone Mctridhtm senile (Coelenterata, Actiniaria). Biol. Bull. 153 : 355-368. RICKETTS, E. F., AND J. CALVIN, 1962. Bctivcen Pacific Tides, 3rd. Ed. Stanford University
Press, Stanford, California, 516 pp. ROSIN, R., 1969. Escape responses of the anemone Antlwpleura nigrescens (Verrill) to its
predatory aeolid nudibranch Hcri'iella (Baba). Vcligcr, 12: 74-77. RUSSELL, H. D., 1942. Observations on the feeding of Acolidia papillosa (L.) with notes on
the hatching of the veligers of Cuthona ainocna (A. and H.) Nautilus, 55: 80-82. SEBENS, K. P., 1977. Habitat suitability, reproductive ecology and the plasticity of body size
in two sea anemone populations (Anthoplcura elegantissiina and A. xanthogramrnicd):
Ph.D. Thesis, University of Washington. 331 pp. STEHOWER, H., 1952. The preference of the slug Aeolidia papillosa (L.) for the sea anemone
Metridium senile (L.). Arch. Nccrl. Zoo/., 10 : 161-170. SWENNEN, C., 1961. Data on distribution, reproduction and ecology of the nudibranchiate
molluscs occurring in the Netherlands. Ncth. J. Sea. Res., 1 : 191-240. WATERS, V. L., 1973. Food-preference of the nudibranch Acolidia papillosa, and the effect
of the defenses of the prey on predation. Vcligcr, 15 : 174-192. WOOD, L., 1968. Physiological and ecological aspects of prey selection by the marine gastropod,
Urosalpinx cinerea (Prosobranchia, Muricidae). Malacologia, 6: 267-320. YARNALL, J. L., 1972. The feeding behavior and functional anatomy of the gut in the eolid
nudibranchs Hcrmisscnda crassicornis (Eschscholtz, 1831) and Acolidia papillosa
(Linnaeus, 1761). Ph.D. Thesis, Stanford University. 126 pp.
Reference: Biol Bull. 157: 153-165. (August 1979)
REPRODUCTION AND SURVIVAL OF THE PILEWORM
NEREIS SUCC1NEA IN HIGHER
SALTON SEA SALINITIES
DEIRDRE L. KUHL AND LARRY C. OGLESBY * Department of Bioluc/y, Pomona College, Claremont, California 91711
The Salton Sea is a large (200 square miles) closed salt lake in a below-sealevel depression in the Colorado Desert of southeastern California. The Sea was formed accidentally from 1905 to 1907 when flood waters of the Colorado River broke through poorly constructed headgates of an irrigation canal. Following repair of the break, the new Salton Sea evaporated to a low of 250 feet below sea level by 1925. Since then the Sea has gradually increased in volume and surface elevation because agricultural waste water inputs exceed the annual evaporation of about 6 feet. In 1977, surface elevation was —228 feet, highest since 1914, and salinities averaged about 37/£o in summer. Early history of the Salton Sea is given by MacDougal (1914), and later history by Walker (1961).
The original biota of the Salton Sea, freshwater organisms from the Colorado River and a few species native to desert springs, died out by the mid-1920's. Attempts to introduce sportfishes did not succeed until the early 1950's, with the establishment of the croaker (Bairdiclla icistia), sargo (Anisotremus davidsoni), and orange-mouth corvina (Cynoscion .ranthitliis), all from the Gulf of California. Several successful introductions of invertebrates have also occurred. The Salton Sea is eutrophic and highly productive (Walker, 1961), but as there are no planktivorous fishes, most plankton die and are converted to benthic detritus (Arnal, 1961). Nereis (Neanthcs) snccinca Leuckart, a polychaete annelid intro- duced in 1930, is the most important benthic detritivore and the most important food source for the sportfishes. The quasi-marine ecosystem of the Salton Sea is discussed by Walker (1961) and Young (1970). Ecology and reproductive biology of N. succinea in the Salton Sea have been discussed by Carpelan and Linsley (1961a, b) and Walker (1961).
With the salinity of the Salton Sea approaching 4Qf/co in the early 1970's, much concern has been expressed over the future of the sportfishery. one of the most important in California (Hanson, 1972). Increasing development of local geo- thermal energy resources may result in spills of highly saline waste brines into the Sea, which may also affect the sportfishes or their food supply (Shinn. 1976). Reproduction by the three sportfishes is limited to salinities below 40'.; f ( Brocksen and Cole ,1972 ;'Lasker, Tenaza, and Chamberlain, 1972 ; May, 1975, 1976). There is little published information on high salinity adaptations in polychaetes (Bayly, 1972; Oglesby, 1978), and only one published study on the effects of higher salinities on N. snccinca in the Salton Sea. Hanson (1972) observed that pile- worms survived 96-hr exposures to salinities as high as 67.5'vV, but speculated
1 Address for reprint requests: L. C. Oglesby, Department of Biology. Pomona College, Claremont, California 91711.
153
154 1). 1- KUHL AND L. C. OGLESBY
that "reproduction of the pileworm would probably be adversely affected at lower salinities."
It is the purpose of the present study to establish upper salinity limits for reproduction and survival of N. siiccinca in Salton Sea waters, in order to assess the possibility that pileworm failure due to increased salinity from either evapora- tion or brine spills may adversely affect the sportfishery.
MATERIALS AND METHODS
Pileworms were collected from the northeastern Salton Sea, at Mecca Beach in the Salton Sea State Recreational Area. Worms were picked by hand from shallow sediments, particularly from barnacle shell rubble cemented with gypsum crystals. While a few heteronereids could be collected from the benthos in this fashion, it was necessary to collect by night-lighting in order to secure adequate numbers for experiments on fertilization and development.
In the laboratory, worms were maintained in glass tubes in finger bowls, covered to reduce evaporation. For most experiments worms were maintained at 21 to 23° C; for some experiments, worms were kept at 34° C, somewhat warmer than summer water temperatures in 1976-77, but not as warm as maximum water temperatures of 36° C recorded by Walker (1961).
Salton Sea water was collected from Mecca Beach. Higher salinities were made by evaporating 36/fo Salton Sea water at about 50° C until it reached a concentration of about 90/Yf. This was then mixed with 36%c Salton Sea water to provide a full range of salinities. During the concentration process, calcium salts precipitated. Thus, in the final mixtures, calcium may have been some- what undersaturated. For some experiments media were made up from Instant Ocean, an artificial salt mixture resembling- ocean sea wrater in ionic ratios. All media were routinely filtered through activated charcoal in Whatman Grade 3 filter paper before use. Media were changed in the culture dishes daily. No anti- biotics were used.
Laboratory fertilizations were carried out according to the procedures detailed in Costello, Davidson, Eggers, Fox, and Henley (1957) and Smith (1964). Water was changed daily, involving some loss of developmental stages by decanting. In some cultures, bacterial or protozoan contamination was present ; however, this did not pose a severe problem experimentally. Since no supplemental food was supplied to the developing larvae, cultures died at a late trochophore or early 3-setiger stage.
Salinities in the field and during ordinary laboratory operations were measured with an American Optical total solids refractometer, calibrated against known solutions measured with a Hewlett-Packard vapor pressure osmometer and with a Buchler-Cotlove chloridometer. Chlorinities were converted to %0 Salton Sea water using the ion ratios given in Carpelan (1958), Walker (1961), and Young (1970).
RESULTS
Effects oj hiyhcr Salton Sea salinities on sitn'h'al of atokotis worms
Experiments on survival of large atokes (immature worms) involved either direct transfers to the full range of salinities, or gradual increases of salinity in
NEREIS SUCCINEA IN HIGH SALINITIES
155
Nereis succlnea Average ST5Q
Salton Sea water • T= 21 - 23 C O T = ~ 34°C
"Instant Ocean" T= 21 - 23 C
CS
50
30
40
50
60 70
Medium: °oo,salinity
80
FIGURE 1. Survival time of Nereis snccinca in increasing concentrations of Salton Sea water, expressed as average ST.™. Points are averages of all trays (initially 10 worms each) in each salinity, with the standard error indicated. Arrows indicate the Critical Salinity (CSso) where survival is reduced to 50% of that in 36C/CC Salton Sea water.
increments of lO/tc until the final test salinity was reached. Calculation of the time for 50% survival (ST50) of a test group of worms (usually four trays with 10 worms each) began with the day the worms first were placed in the final test salinity. Graphs of ST50 as a function of the final salinity were used to determine the Critical Salinity (CS5o), the salinity where survival is reduced to SQ% of the survival in 36/{< Salton Sea water. This calculation of CS50 is possibly biased in those experiments involving graduated salinity increases, since it ignores the mortality of worms in lower salinities during the adaptation period. However, otherwise comparable experiments in which worms were transferred directly to the final salinities gave similar results. Results of both types of experiments are combined for the following analyses.
Results for all experiments on survival of atokes are summarized in Figures 1 and 2. These are averages and calculations based on over 280 worms in 36/tV. over 140 worms in each salinity between 45 and 60/£c, and between 50 and 100 worms in all higher salinities. Figure 1 shows the average survival time (ST50) for all worms in all salinities, with the standard error indicated. Survival was high at salinities of 50/^ and lower, but declined in higher salinities. The critical salinity CS50 is estimated to be slightly lower than 65%c. Figure 1 also provides the results of two related experiments: the effects of elevated temperature (34° C rather than the usual 21-23° C), and the effects of transfer to Instant Ocean. In both cases, overall survival seems to be diminished as compared with worms in
156 I'. I- KUHL AND I.. C. OGLKSBY
Salton Sea water in 21 to 23° C, though the general pattern is similar. For worms in Instant Ocean, CS50 is estimated to be somewhat higher than 65%c. It was not possible to provide an estimate of CS50 for the worms at 34° C, for it was higher than the highest salinity used in this experiment, 65/{0. It appears that the Critical Salinity is not lowered for Salton Sea pileworms exposed either to ocean sea water or to temperatures only slightly lower than maximum summer water temperatures in the Salton Sea.
Much of the variability in the estimates of average SToo (Fig. 1) is caused by the fact that in all salinities, all the worms in a given tray often would die soon after the first worm died, presumably because of the accumulation of toxic materials before the medium was changed. Not only is variability increased by this phenomenon, which may not be salinity-related, but also the average ST5o is decreased. Die-off of entire trays of 10 worms was particularly a problem at 34° C, due to increased bacterial activity. In order to provide an estimate of survival that avoids this problem, Figure 2 shows the survival time for the longest surviving worm in each salinity. At least some pileworms survived longer than the length of the experiments in nearly all salinities of 65C/CC and lower, over 4 weeks for the worms in Salton Sea water at 21 to 23° C, and over 2.5 weeks for the worms in Instant Ocean and in 34° C. At higher salinities maximum survival time declined rapidly. No worms survived even a day at salinities higher than 85%o at 21 to 23° C, nor higher than 75%v in Instant Ocean. In both these experi- ments, CS5o was 70%e. No salinities higher than 65%o were used in the experiments at 34° C, and since at least one individual lived as long as 8 days in this salinity, it is not possible to estimate CS5o, which must be higher than 6S%C.
Most worms in the laboratory did not survive more than a week or two, even when maintained in 36%o Salton Sea water. Carpelan and Linsley (1961a) indi- cated that N. succinca takes over a month to reach sexual maturity in the Salton Sea, so preumably pileworms must live well over a month in their natural habitat waters. Diminished survival in the laboratory may be a consequence of accumula- tion of metabolites, to lack of adequate food, or to other artificial causes. For these reasons, the survival times of pileworms in these laboratory experiments should not be taken as an exact counterpart of survival in the Salton Sea as salinity there increases. Rather, these laboratory results should be taken as an index of survival.
Both indices of survival (Figs. 1, 2) indicate that atokous pileworms survive increased Salton Sea salinities up to 60 to 65%c. Survival is considerably diminished at salinities in excess of 70%c, with no survival above 8Sc/(.c, a concentration which is well over twice the present concentration of the Salton Sea. Even high summer water temperatures of 34° C do not decrease the survival limits of the pileworms. There is no indication that pileworms live longer in ocean water than in Salton Sea water.
Effects o] higher Salton Sea salinities un heteronereid development
Studies of sexual development and spawning indicate that while N. succinca breeds year-round in the Salton Sea, reproductive activity is somewhat depressed during the summer months of high water temperature (Carpelan and Linsley,
SVCCINEA IN HIGH SALINITIES
157
Nereis succinea
Maximum survival Salton Sea water • T= 21 - 23 C O T=~34°C "Instant Ocean" D T= 21 - 23 C
50 60 70
Medium: %6 salinity
90
FIGURE 2. Maximum survival time of Nereis succinea in increasing concentrations of Salton Sea water. Circled points are those where worms were still alive hy the end of the experiment.
1961a, b). Nevertheless, heteronereids (sexually mature adult worms) were collected throughout the summers of 1976 and 1977. The sex ratio of worms col- lected from the benthos at Mecca Beach was about one-third males and two-thirds females. As noted by Costello, ct al. (1957) and Carpelan and Linsley (1961a), the sex ratio of swarming heteronereids is strongly reversed, males outnumbering females by about 10 to 1. This was also the case in our own collections. Hetero- nereids regularly developed in laboratory cultures, with the sex ratio comparable to that encountered in benthic worms during collection, about 2 : 1 females to males. Heteronereid development in the laboratory was strongly influenced by salinity
158
I). L Kflll. AM) L C. (H.I.KSI'.Y
(Fig. 3). About lSf/c of the pileworms maintained in lo\\- salinities (36 and 40/co) matured as lieteronereids during the summer of 1(>77. A somewhat lower per- centage (just under \2'/< ) matured in 50';,. but in higher salinities maturation of lieteronereids was greatly depressed, and there was no maturation in salinities of 70(/t c or higher. There may be a differential effect of higher salinities on matura- tion of the two sexes, development of males being more strongly depressed than that of females. This observation mav be an artifact of very small numbers of hetero-
o>
CO
5 CD g?
Heteronereid development in higher Salton Sea salinities
Medium: %o salinity
FIGURE 3. Effect of increasing Salton Sea salinities on heteronereid production by Nereis succinea. Actual numbers of lieteronereids produced are indicated. Arrow indicates CSsa where heteronereid production is reduced to 50% of that in 2>6r/c( Salton Sea water.
NEREIS SUL'CIMiA IX HIGH SALINITIES 159
nereids in the higher salinities. The estimated CS.-.n for heteronereid development is about 5Sc/co (Fig. 3).
Effects of higher Sol ton Sea salinities on fertilization and Uirn.il development
Laboratory fertilizations, using heteronereicls either collected directly from the Salton Sea or produced in laboratory cultures, were readily accomplished. Development of larvae in 36/^c Salton Sea water appeared normal, and had the same time course as in previous studies on Salton Sea worms (Carpelan and Linsley, 1961a, b) and on this species at \Yoods Hole ( Costello, et al., 1957). In all these studies, temperatures were similar, about 20 to 25° C.
Fertilization in various salinities. Fertilization of eggs of N. succinea could be accomplished at salinities as high as 50/w, although the percentage of successful fertilizations was much reduced at 50/<V. In one experiment, there was less than 5% successful fertilization at 55C/CC, and in a second experiment, no success at this salinity. In these experiments, eggs from females maintained in 36^V were initially transferred to culture dishes containing 100 ml of the experimental salinity, and sperm from males opened in 36c/ro added. Eggs transferred to salinities of 45',, and higher shrank from osmotic water loss immediately after the transfer. Shrink- age was particularly noticeable in 50%c and higher, many eggs becoming greatly distorted. It is estimated that the CS-,o for fertilization is 4S(/((. While there was a small proportion of apparently successful fertilizations in 55(/(C in one experi- ment, there was no further development even to the first cleavage. The highest salinity in which any development takes place following fertilization is 50/£<-. These results are summarized in Figure 4.
Fertilization in 36%c Salton Sea water, jolloived by transfers to I'arions salinities. Smith (1964) demonstrated that later developmental stages of N. diversicolor were less sensitive both to lower and to higher salinities than early stages. To determine if this is the case with N. sitecinea, several experiments were conducted in which eggs were fertilized in 36'^ Salton Sea water .and embryos transferred to higher salinities at several different times during development.
In the first group of experiments, eggs were transferred as soon as possible after fertilization into a full range of salinities. There was excellent survival and subsequent development in salinities up through 45/^c, although in 45', f there was some evidence of shrinkage, and a few zygotes cleaved abnormally. These abnormally cleaving embryos (such as "dumbells" which cleaved no further, and irregular cell clusters) did not develop successfully. There was poor survival of embryos transferred to 5Qc/cc, with abnormal cleavages and much shrinkage, but a few normal trochophores developed at this salinity. At 55 and 60/-rr there was temporary survival, a high proportion of abnormal cleavage patterns, and no successful development. Embryos transferred to salinities higher than 60r,, shrank excessively and showed no type of cleavage. These early cleavage stages seem as sensitive to higher salinities as fertilization itself ; it is estimated that the CSso for early cleavage is 4SC/(C, with no successful development higher than 50/^r. These results are summarized in Figure 4.
This same experiment was conducted at 34° C to determine if summer water temperatures were more stressful on development than 21 to 23° C. There were
160 D. L. KUHL AND L. C. OGLESBY
no observed differences in survival and developmental success between 34° C and 21 to 23° C, otlier than a considerable acceleration of cleavage at the higher temperature. There seems to be no additional stress on development in tempera- tures as high as summer water temperatures in the Salton Sa. On the other hand, in either temperature regime, development in salinities of 45/£c was markedly delayed as compared with development at lower salinities.
In a second group of experiments, fertilized eggs were allowed to develop in 36%0 Salton Sea water until the early trochophore stage, about 8 to 10 hr. These early trochophore stages were then transferred to salinities up to 75c/cc. There was excellent survival and development at all salinities up through 50/£o. No shrinkage of trochophores was noticed at 45% c. In 50/£o there was some shrinkage, and trochophore cilia ceased beating temporarily. At 55%c these shock effects were more severe, but there was considerable survival and subsequent development. At 60%c there was no recovery of ciliary motion following transfer, and while it was estimated that perhaps 5% of the early trochophores survived the transfer, there was no subsequent development. There was no survival at higher salinities. Thus, early trochophores are less sensitive to high salinities than earlier cleavage stages. The estimated CSr,o is 50%c, but there was no development above 55%c. These results are summarized in Figure 4.
In the final experiment, fertilized eggs were allowed to develop in 36%c Salton Sea water until the swimming trochophore stage, about 24 hr. Swimming trocho- phores were then transferred to salinities up to 75C/(C, and survival and subsequent development monitored to the beginning of segmentation about 18 hr later. Survival was not as good as in the experiments involving transfers at earlier stages, but there was some survival and development up through 55%c. Swimming trochophores transferred to 50 and 55%o showed some shrinkage, and ciliary activity temporarily stopped. In 60/{<? these shock effects were more severe, and survival was low; however, some trochophores were still alive after 18 hours, though development had not progressed. In higher salinities there was little or no survival following transfer, shock effects were very severe, and there was no subsequent development. It appears that swimming trochophores have about the same sensi- tivity to higher Salton Sea salinities as do early trochophores. These results are summarized in Figure 4.
DISCUSSION
The present experiments supplement and extend the results to Hanson (1972) in showing that atokous N. succinea can survive for extended periods of time in very high Salton Sea salinities, at least as high as 65%c, with perhaps some reduc- tion of survival in 7Q'/cc. There is short term survival in salinities as high as 80/£P, more than twice the present salinity of the Salton Sea. There are reports of nereid and other polychaetes in high salinities in lagoons and estuaries, but few reports are for salinities in excess of 5Q%0 (Bayly, 1972; Oglesby, 1978). There have been no experiments on any of these other polychaetes to determine actual upper salinity tolerances, or if they actually breed in such high salinities.
Hanson's (1972) prediction that reproduction of the pileworm in the Salton Sea would be adversely affected at salinities lower than the limit for adult sur-
NEREIS SUCCINEA IN HIGH SALINITIES
161
Atokous Stage
1 ) Maximum survival -
2) Maximum ST^g —
3) Average STVn
Heteronereid development
Fertilization-
1st cleavage - 2nd cleavage - 3rd cleavage -
Early trochophore
Pigmented trochophore
Segmentation
-10
20
30
40
50
Atokous Stage •
50%
1 Maximum
Nereis succinea
Salinity tolerance profile of development and survival in higher Salton Sea salinities
M Habitat * I
Hours after fertilization at - 23 C
30 40 50 60 70
Medium: %osalinity
80
FIGURE 4. Salinity tolerance profile of development and survival of N. succinea in higher Salton Sea salinities. Stages and times are approximate for 21 to 23° C. The solid line indi- cates the Critical Salinity (CSw), where survival and development are reduced to 50% of that in 36#0 Salton Sea water. The dashed line indicates the maximum salinity at which any survival or development occurred.
vival is confirmed by these experiments, fur there was no successful fertilization or cleavage in salinities higher than SQf/((, and reasonable success was limited to salin- ities of 45%c and lower. Early and late trochophores could tolerate somewhat higher salinities, up to 50%c. Assumption of the high salinity tolerance of the atokes must come later in development than the appearance of segmentation, the termination of the present experiments.
Table I summarizes the literature on the effects of low salinities on development of N. succinea. Taken together, these reports suggest that N. succinea, like many other estuarine and marine polychaetes, cannot reproduce at salinities below the horohalinicum of 5 to 8>c/fr (Oglesby, 1978). even though atokous N. succinea can
162
I). I.. KUHL AND L. C. OGLES BY
survive at considerably lower salinities (Oglesby, 1965; Hogue and Oglesby, unpublished results). The report by Foster (1972) that N. succinea can survive in fresh water is not supported by published data (Oglesby, 1965, 1978). The studies summarized in Table I, combined with the present results, provide a picture that is comparable to N. divcrsicolor (Smith, 1964), showing a "bottleneck" of salinities above and below which cleavage is blocked (Fig. 4). That is, while adult and atoke survival limits range from about \7rr to as high as 8Q'/(c, develop- ment is successful only between 10 and 45/{r.
It is premature to conclude that when the Salton Sea finally exceeds 45 to 50%p, reproduction of N. succinea will be blocked. As Smith (1964, 1977) has discussed, at least some of the reason for lack of reproductive success at extreme salinities may be mechanical, due to osmotic swelling in low salinities and osmotic shrinkage in high salinities. Shrinkage was very apparent in the present experi- ments at 50f/((l and higher in eggs and embryos transferred from 36(/((. It would have been desirable to attempt fertilizations with eggs and sperm taken from adults adapted to much higher salinities, to avoid the initial osmotic problem. However, even though heteronereids were produced in the laboratory in salinities as high as 65%c (Fig. 3), never were a male and a female mature at the same time in a salinity higher than 36%c. Smith (1964) reported that some populations of N. diversiclor in northern Europe reproduce in salinities greater than the upper salinity limit for reproduction of other populations of the same species. It may
TABLE I Effects of lower salinities on development of Nereis succinea*
Stage and treatment
Observed effects on development
Reference
Unfertilized eggs Fertilized eggs
Development in 91% SW after 1 hr exposure to dilute SW
Development in varied SW after fertilization in 91% SW
Development in varied SW after fertilization in 100% SW
Development in varied SW and temperatures after fertilization at 20%fl
Survival down to 9.6%0.
Very low resistance to dilute sea water.
Normal development above 16%o; diminished success at 11-14%0, though some eggs develop normally.
Normal development above 16%0; diminished success at 14%0, but some normal development; cleavage but not trochophores in 11, 13%0.
Normal development of nectochaeta larva in 14-35%0; normal develop- ment of trochophore in 8%0 ; no cleavage in 2-6%0.
Development at 10, 15, 20%c, except none at 10%0 at 10° C. Develop- ment accelerated at higher salinity and higher temperature.
Just (1928) Just (1928)
Just (1930a)
Just (1930b)
Kinne (1954)
Dean and
Mazurkiewicz
(1975)
* Nereis limbata Ehlers, the name used by Just (1928, 1930a, b) is synonymous with N. succinea Leuckart. Just (1928, 1930a, b) did not provide the actual concentration of his "100% SW." Salinities presented in this table are based on data of Cole (1940) for summer water salinities at Woods Hole. Since there are no significant fresh-water inflows in the area, it is reasonable to assume that \Voods Hole summer sea water averages about 32%r, or 91 % of 35%c salinity.
NEREIS SUCCINEA IX HK.H SAI.IMTIKS 163
be that as the salinity of the Salton Sea gradually rises, there will be genetic selection for N. siiccinea with higher limits for reproductive success.
Developmental events were somewhat slowed at higher salinities, but at salinities below 45 to 50/£c development was otherwise normal. Smith (1964) observed that low salinities also delayed development, particularly cleavage, in N. divcrsicolor.
The present experiments indicate that temperatures as high as 34° C do not have any marked effect on adult survival and reproduction in N. succinca. At the present time there is year-round reproduction in the Salton Sea, and this pattern should continue as long as the overall salinity itself does not become too high.
Interestingly, Salton Sea pileworms do not seem to have reduced survival in Salton Sea water as compared with ocean water (Figs. 1, 2). May (1976) found that eggs and larvae of the croaker Bairdiclla survived well in sea water, but had very poor survival in Salton Sea water of the same salinity. He sug- gested that this poor survival of the fish was related to the unusual ionic com- position of the Salton Sea. This seems not to be a problem with N. succinca.
Spills of geothermal waste brines of 5 to 10 times the salinity of the Salton Sea (California Department of Water Resources, 1970; Shinn, 1976) would be expected to eliminate pileworms from the affected area, as well as other benthic and pelagic organisms. These adverse effects could be caused not only by the excessively high salinity f^er sc, but also by concomitant elevated temperatures or reduced oxygen concentrations. Pileworm larvae are in the Salton Sea plankton all year (Carpelan and Linsley. 1961a, b), and so there would be a constantly available source for recolonization. Only if there were contamination by heavy metals would there be a long-term problem ( Reish and Carr, 1978). Thus, it is unlikely that a spill of even highly saline geothermal waste brines would have any more than a temporary and localized effect on the population of N. succinea in the Salton Sea.
The present experiments indicate that reproduction of N. succinea in the Salton Sea will continue with undiminished success at salinities at least as high as 4S(/fp, and probably as high as SQ(/CC. This means that gradually increasing salinities of the Salton Sea will not adversely affect the pileworm until some years after the collapse of the sportfishery, which seems sensitive to salinities no higher than
It is a pleasure to thank Pomona College students Charles Anderson, William R. Hargreaves, E. Wayne Hogue, and Jan Low, who have assisted in various aspects of this study, as we'll as many other students and faculty who helped with the col- lections. We appreciate the helpful discussions and assistance of C. A. Engle, Area Manager, and R. C. Johnson, of the Salton Sea State Recreational Area ; J. E. Fitch, Research Director of the California Department of Fish and Game; R. R. Ireland of the Imperial Valley Environmental Project, Lawrence Livermore Laboratory; Dr. Ralph I. Smith, University of California at Berkley: and Dr. Alice Shoemaker Oglesby. This research was supported in part by Subcontract #345003 from the Lawrence Livermore Laboratory, University of California, Imperial Valley Environmental Project, supported by the U. S. Department of Energy under Contract No. W-7405-ENG-48.
164 l>. L. KUHL AND L. C. OGLESBY
SUMMARY
The polychaete annelid Nereis (Ncanthcs) succinca is the major benthic detritivore in the Salton Sea, an inland salt lake in southeastern California, and is critical in the trophic chain leading to the sportfishery. In view of the increasing salinity of the Salton Sea, laboratory experiments were conducted to determine critical upper salinity limits for reproduction and survival of pileworms. Atokous (immature) pileworms can survive for extended periods in Salton Sea salinities at least as high as 65% o, with some reduction of survival in 70%c, and with only short term survival in 80% c, more than twice the present salinity of the Salton Sea (36%c). Heteronereid production is depressed by salinities higher than 50% r. Reproduction of N. succinca is successful at salinities at least as high as 45%c, and probably as high as 50% c. Fertilization and early cleavage stages are less tolerant of elevated salinities than are later development stages such as trochophores.
LITERATURE CITED
ARNAL, R. E., 1961. Limnology, sedimentation, and microorganisms of the Salton Sea, Cali- fornia. Gcol. Soc. Am, Bull., 72 : 427-478. BAYLY, I. A. E., 1972. Salinity tolerance and osmotic behavior of animals in athalassic
saline and marine hypersaline waters. Annu. Rcz>. Ecol. Syst., 3 : 233-268. BROCKSEN, R. W., AND R. E. COLE, 1972. Physiological responses of three species of fish to
various salinities. /. Fish. Res. Board Can., 29 : 399-405. CALIFORNIA STATE DEPARTMENT OF WATER RESOURCES, 1970. Geothermal wastes and the
water resources of the Salton Sea area. D]]'R Bulletin No. 143-7. 123 pp. CARPELAN, L. H., 1958. The Salton Sea. Physical and chemical characteristics. Limnol.
Occanogr., 3 : 373-386. CARPELAN, L. H., AND R. H. LINSLEY, 1961a. The pileworm, Ncanthcs succinca (Frey and
Leuckart). Calif. DC p. Fish Game Bull, 113 : 63-76.
CARPELAN, L. H., AND R. H. LINSLEY, 1961b. The spawning of the pileworm Ncanthcs suc- cinca in the Salton Sea. Ecology. 42 : 189-190. COLE, W. H., 1940. The composition of fluids and sera of some marine animals and of the
water in which they live. /. Gen. Physio!,. 23 : 575-584. COSTELLO, D. P., M. E. DAVIDSON, A. EGGERS, M. H. Fox, AND C. HENLEY, 1957. Methods
for Obtaining and Handling Marine Eggs and Embryos. Marine Biological Laboratory,
Woods Hole, Massachusetts. 246 pp. DEAN, D., AND M. MAZURKIEWICZ, 1975. Methods of culturing polychaetes. Pages 171-197
in W. L. Smith and M. H. Chanley, Eds. Culture of Marine Invertebrate Animals.
Plenum Publ. Co., New York.
FOSTER, N., 1972. Freshicater Polychaetes (Annelida) of North America. Biota of Fresh- water Ecosystems Identification Manual No. 4, U. S. Environmental Protection
Agency, Stock Number 5501-0368. 15 pp. HANSON, J. A., 1972. Tolerance of high salinity by the pileworm, Ncanthcs succinca. from
Salton Sea, California. Calif. Fish Game, 58: 152-154. JUST, E. E., 1928. Hydration and dehydration in the living cell. I. The effect of extreme
hypotony on the egg of Nereis. Physiol. Zoo/., 1 : 122-135. JUST, E. E., 1930a. Hydration and dehydration in the living cell. III. The fertilization of
Nereis eggs after exposure to hypotonic sea-water. Protoplasma, 10 : 24-32. JUST, E. E., 1930b. Hydration and dehydration in the living cell. IV. Fertilization and
development of Nereis eggs in dilute sea-water. Protoplasma, 10 : 33-40. KINNE, O., 1954. Uber das Schwarmen und die Larvalentwichklung von Nereis succinca
Leuckart ( Polychaeta) . Zoo/. Ans., 153: 114-126. LASKER, R., R. H. TENAZA, AND L. L. CHAMBERLAIN, 1972. The response of Salton Sea
fish eggs and larvae to salinity stress. Calif. Fish. Game, 58 : 58-66. MAcDouGAL, D. T., Ed., 1914. The Salton Sea. A Study of the Geography, the Geology,
NEREIS SUCCINEA IN HIGH SALINITIES 165
the Floristics, and the Ecology of a Desert Basin. Carnegie lust, ll'ash. PubL, 193L : 1-182.
MAY, R. C., 1975. Effects of temperature and salinity on fertilization, embryonic development, and hatching in Bairdiclla icistia (Pisces: Sciaenidae), and the effect of parental salinity acclimation on embryonic and larval salinity tolerance. Fish. Bull., 73 : 1-22.
MAY, R. C., 1976. Effects of Salton Sea water on the eggs and larvae of Bairdiclla icistia (Pisces : Sciaenidae). Calif. Fish Game. 62 : 119-131.
OGLESBY, L. C., 1965. Steady-state parameters of water and chloride regulation in estuarine nereid polychaetes. Cotnp. Biochcm. Physio!., 14: 621-640.
OGLESBY, L. C., 1978. Salt and Water Balance. Pages 555-668 in P. J. Mill, Ed. Physiology of the Annelida. Academic Press, London and New York.
REISH, D. J., AND R. S. CARR, 1978. The effect of heavy metals on the survival, reproduction, development, and life cycles for two species of polychaetous annelids. Mar. Pollut. Bull, 9(1) : 24-27.
SHINN, J. H., Ed., 1976. Potential Effects of Geothcrmal Energy Conversion on Imperial Valley Ecosystems. Lawrence Livermore Laboratory, University of California, Livermore. UCRL-52196. 77 pp.
SMITH, R. L, 1964. On the early development of Nereis dircrsicolor in different salinities. /. Morfihol., 114: 437-464.'
SMITH, R. L, 1977. Physiological and reproductive adaptations of Nereis diversicolor to life in the Baltic Sea and adjacent waters. Pages 373-390 in D. J. Reish and K. Fauchald. Eds. Essays on Polychaetous Annelids in Memory of Dr. Olga Hartman. Uni- versity of Southern California, Allan Hancock Foundation Special Publication.
WALKER, B. W., Ed., 1961. The Ecology of the Salton Sea, California, in Relation to the Sportfishery. Calif. Def>. Fish Game Fish Bull., 113: 1-204.
YOUNG, D. R., 1970. The Distribution of Cesium, Rubidium, and Potassium in the Quasi- Marine Ecosystem of the Salton Sea. Ph.D. Dissertation, University of California, San Diego. 213 pp.
Reference: Hiol. Hull. 157: K.6-1.S1. (August 1979)
BEHAVIORAL RESPONSES OF BALANUS IMPROVISUS NAUPLII TO LIGHT INTENSITY AND SPECTRUM
WILLIAM H. LANG, RICHARD B. FORWARD, JR. 1 AND DON C. MILLER
Environmental Protection Agency, Environmental Research Laboratory, South Ferry Road,
Narragansctt, Rhode Island 02882
Although barnacle larvae have been used in numerous classical studies on photo- taxis and spectral response, results have been predominantly qualitative in nature. Groom and Loeb (1890), Ewald (1912), and Rose (1925) studied phototaxis in stage I-II nauplii of Balanus pcrforatits. Vischer and Luce (1928) attempted to define the spectral sensitivity of cyprid Balanus aniphitritc and "B. improinsus" . Barnes, Crisp, and Powell (1951) demonstrated orientation to light during settle- ment of cyprid Scniibalaints balanoides and Balanus crcnatns. Based on these and other studies, Thorson (1964) characterized barnacles as maintaining positive phototaxis throughout their larval life.
More recent studies have begun to quantify light responses of barnacle larvae. Barnes and Klepal ( 1972) determined the spectral sensitivity and threshold of photo-response at 522 nm for stage I nauplii of Eltniniits niodcstits and -5\ balanoides. Crisp and Ritz (1973) studied the effects of dark-adaptation on relative light sensi- tivity for stage II E. nwdestus and 5". balanoides, and limiting intensities of white light for photo responsiveness of stage II ^. balanoides and B. crcnatns and cyprid 5\ balanoides.
With the exception of a series of studies on the zoea of the estuarine decapod crustaceans (Forward, 1974; Forward and Costlow, 1974; Forward, 1976b, 1977; Forward and Cronin, 1978), quantitative phototactic and spectral studies of larval crustacean groups are sparse (see Forward, 1976a; Aiken and Hailman, 1978).
In (this study, short-term phototaxis (direction) and orthophotokinensis (velocity) of Balanus improvisus nauplii are investigated using a new method of video-computer quantification which greatly facilitates studies of movement in small organisms (Davenport, Culler, Greaves, Forward, and Hand, 1970). The ability to rapidly quantify and compare movement paramenters makes this system a potentially powerful tool for behavioral biassays (Anderson, 1971; Olla, 1974). Thus this study demonstrates the capabilities of this system and provides a rigorous analysis of the photobiology of barnacle larvae.
MATERIALS AND METHODS Experimental animal
Balanus improvisus nauplii were sorted from surface plankton tows taken at Pettaquamscutt River near Narragansett, Rhode Island. During the collection period (24 Oct-28 Nov., 1977) B. improvisus nauplii were abundant and easily
1 Duke University Marine Laboratory, Beaufort, North Carolina 28516; and Zoology Department, Duke University, Durham, North Carolina 27706.
166
I.KiHT RESPONSES OF BAKXACI.K X.UTU! 167
sorted from plankton samples (Lang, 1979). Water temperature and salinity at collection times ranged from 7 to 14° C. 13 to 20% c. Nauplii were immediately transferred to filtered sea water at 15° C, 15% o and placed in either 15° or 20° C temperature boxes with constant illumination. For experiments conducted at 30%c, the temperature of the lS%c water was first allowed to equilibrate at 20° C and nauplii were then transferred to 5%c salinity increments at one hr intervals.
All larvae were maintained overnight (8-12 hr) at specified temperature/ salinity before being used experimentally. Isochrysis galbana and Tetrasehnis snecia were added as food. The following morning, nauplii of the desired stage were sorted from initial cultures and groups of 20 transferred to 5-ml beakers. Light-adapted nauplii were exposed to room lights supplemented with a 60-W incandescent bulb for at least 1 hr prior to experimentation ; dark-adapted nauplii were kept in dark temperature-controlled boxes for at least 1 hr before experimenta- tion. Studies with other arthropods indicate that these times are adequate for light and dark adaptations (e.g., Hamdorf and Schwemer, 1975; Barnes and Goldsmith, 1977). To minimize effects of possible larval diurnal cycles (Singara- jah, Moyse, and Knight-Jones, 1967), all experiments were conducted between 13:00 and 16:30 hr. All nauplii were tested within 26 hr of capture.
Light stimulus and video system
A microscope and closed circuit television system were used to monitor and record swimming behavior of nauplii. Dark field substage illumination, interference filtered to 830 nm (about 15 nm half width), provided light for a Colin 4400 tele- vision camera mounted on a Wild M-5 microscope body (Lang, Lawrence, and Miller, 1979). Larvae were placed in a 1.2 X 1.2 X 1.0 cm Incite cuvette filled to 0.5 cm depth. Movement was monitored in the horizontal plane.
A light stimulus presented horizontally and perpendicular to the cuvette wall was provided by two sources. For initial studies on light response of nauplii, a grating monochromater with 150-W xenon short arc lamp (Oriel Corporation) was used. For spectral and intensity studies a slide projector with 300-W tungsten bulb and thin film absorption filters (Ditric Optics) ranging from 440 to 640 nm in 20-nm intervals (about 7 nm half-width) was used (Latz and Forward, 1977). In both cases light intensity of quantum levels were regulated by neutral density filters. Intensity was measured by a YSI model 64 A radiometer.
Experimental procedure
Experiments were conducted in a temperature-controlled, darkened room. A preparation of 20 nauplii was transferred from a 5-ml beaker to the test cuvette ; the cuvette was then aligned on the dark field stage. Light-adapted nauplii were transferred under room lights ; dark-adapted nauplii were transferred under dim light (interference filtered at 700 nm). All lights were extinguished and nauplii were allowed at least 30 sec prior to experimentation to recover from movement of the test cuvette during placement on stage.
For initial studies on naupliar response to light stimulus, five intensities of 480-nm light ranging from 27 to 0.0027 W/m2 were used. Replicate preparations of nauplii were tested at each intensity in ascending order with 45-sec intervals
168 LANG, FORWARD, JR. AND MILLER
between 2.5 sec stimuli. The response of nauplii 2.5 sec prior to stimulation, during stimulation, and 3.0 sec following stimulation were analyzed by computer.
For spectral response, the 830-nm substage light was turned on 5 to 15 sec prior to light stimulus, a 2.5-sec stimulus applied, and the substage light ex- tinguished. After 1 minute, a second wavelength stimulus (in ascending order) was applied. After four exposures the preparation of dark-adapted nauplii was changed. For both spectral and intensity studies, computer analysis was limited to naupliar response during the latter 2.0 sec of the 2.5-sec stimulus.
The general procedure for testing response to light intensities was similar. One preparation of nauplii was exposed to a complete sequence of seven intensities of 480-nm light starting with an intensity estimated as subthreshold. All stimuli were 2.5 sec in duration with 30- to 40-sec intervals between each stimulus.
At least three replicates were run with each plankton sample. With the excep- tion of stage III nauplii, at least two different plankton samples were represented.
Computer analysis
Video recordings of naupliar response to light stimuli were played back through a video-to-digital processor, the "Bugwatcher" (Greaves, 1975). Outlines of each nauplii within the camera field of view are delimited as X-Y coordinates. For this study, video tapes were analyzed by computer at 10 frames per second; every sixth frame of the normal 60 f/sec recording was fed into a Data General Eclipse S/200 computer. Time series of X-Y coordinates (video files) indicating displace- ment of nauplii at 0.1 -sec intervals were generated for each replicate sample. A tone generator synchronized with the light stimulus shutter control marked periods of stimulation; a tone detector in the Bugwatcher determined light stimulus duration to the nearest 0.1 sec on video files.
Video files were analyzed using second generation programs developed by Wilson and Greaves (1979). Processed video files (see Greaves, 1975) yielded a time-scaled computer track of naupliar movement (Fig. 1A). About 10 to 15 nauplii were tracked simultaneously. Linear velocity (Fig. IB) and direction of travel (Fig. 1C) were calculated for each 0.1-sec interval of the tracks; mean values for individual nauplii during a given time interval were determined (Fig. ID). Mean direction of travel (DOT) and mean linear velocity (MLV) for all nauplii of an experimental group were then pooled to calculate sample means. Sample DOT distributions were tested using a Chi-square test (Batschelet, 1965). Standard SPSS (Nie, Hull, Jenkins, Steinbrenner, and Bent, 1975) and SAS (Bar, Goodnight, Sail, and Helwig. 1976) analysis of variance programs w^ere used to test sample MLV data.
RESULTS
Initial studies on stage II naupliar light response
Preliminary observations indicated that stage II B. iinproi'isns nauplii were strongly positively phototactic to lower light intensities (about 10 W/nr2), par- ticularly near 500 nm. The following experiments were devised to further characterize the responses of light-adapted nauplii.
LIGHT RESPONSES OF BARXACLE NAUPLII
169
1 mm
480nm
|
D |
MLV mm/sec |
MEAN VECTOR 6 | r |
|
|
DARK 0-2.6 sec |
1 01 |
1 70 |
0.2 |
|
LIGHT Z6- 5.1 sec |
1 84 |
329 |
0.8 |
B
o
LU
DARK
LIGHT
65%
0 -2.6
SEC
2.6-5.1 SEC
01 2345
SECONDS
FIGURE 1. An example of computer analysis of a single Balanus itufrovisns nauplius. Digital processing of a video tape produces a computer tracking of the nauplius (A). A light stimulus (480 nm, 0.06 W/nr) was applied from 2.6 to 5.1 sec. Linear velocity (B) and direction of travel (C) are calculated at 0.1-sec intervals (stimulus at 0°) and mean values for desired time intervals (D) determined.
Light-adapted stage II nauplii (15° C, 20/^) were exposed to 480 nm light at five intensities ranging from 27 to 0.0027 W/nr. The initial MLV of nauplii exposed only to 830-nm darkfield illumination does not vary significantly (P = 0.05) between sample group (Table I). During light stimulation, the MLV for groups at the upper three light intensities increases significantly ; recovery to MLV statistically equal to control levels does not occur within the three sec following stimulation. No significant change in MLV" occurs at the lower two light intensities
17(1
I.ANG, FORWARD, JR. AND MILI.KR
(Table I ). Plotting the running mean velocities of each sample group at 0.1 -sec intervals shows the time sequence of naupliar photokinetic response (Fig. 2). The change in MLV is delayed in onset and return to normal, relative to light stimulus duration.
A change in MLV (orthophotokinesis) is observed only above 0.027 W/nr. However a directional response to light (phototaxis) occurs at all intensities (Table I, Fig. 3). DOT distributions during the initial "dark" interval do not vary significantly (P -- 0.05) from a theoretical uniform distribution. During the "light" interval, nauplii at all light intensities exhibit nonrandom distributions and a significant difference from initial distributions. Computer plotted histograms (Fig. 3) illustrate a directed response toward the light source.
The individual nauplii paths (e.g. Fig. 1A) and time analysis of MLV responses (Fig. 2) indicate that a delay of about 0.5 sec often occurs between stimulation and naupliar response. The DOT distribution for the entire 2.5-sec light stimulus interval clearly indicates a general positive phototactic movement (Fig. 4A). If the initial 0.5-sec "orientation period" at light stimulation is omitted from DOT determinations, a strong directed response becomes evident (Fig. 4B). For the following experiments, mean naupliar direction and MLV during the latter two sec of a 2.5-sec light stimulus are presented. Positive phototaxis is considered a mean direction of travel ± 15° of the light source ; negative phototaxis is a DOT ± 15° in the opposite direction. Comparative results using a ±45° "windows" in respective directions are also included.
To correlate with other laboratory studies (Lang, ct <//., 1979; Lang, Miller, Lawrence, Marcy, and Clem, in progress), light intensity and spectral experiments were conducted at 20° C, using a tungsten light source and filters. Essentially the same positive phototaxis to 480-nm light was found using the new light source and at the higher temperature.
TABLE I
Mean linear velocity and \2 directional response comparison of light-adapted stage II Balanus ini- provisus nauplii (15° C 20 %c) during initial "dark" interval (830-nm substage light) at 0-2.5 sec, 480-nm light stimulation at 2.5 to 5.0 sec, recovery "dark" interval at 5.0 to 8.0 sec. A null hypothesis that the distribution of direction of travel means (60° intervals) for each sample group of 30 to 38 nauplii is equal to a theoretical uniform distribution was tested using the x2 test, x2 values for P < 0.05 are indicated by an asterisk where P represents the probability of rejecting the null hypothesis when actually true.
|
Intensity W/ms at 480 nm |
Mean linear velocity mm /sec |
Direction of travel Chi-square: experimental vs. uniform distribution |
||||
|
Initial "dark" |
Light stimulation |
Recovery "dark" |
Initial "dark" |
Light stimulation |
Recovery "dark" |
|
|
27 |
1.35 ±0.35 |
2.13 ± 0.65 |
1.97 ±0.63 |
7.14 |
33.67* |
8.80 |
|
2.7 |
1.54 ±0.55 |
2.29 ± 0.66 |
1.91 ± 0.61 |
4.43 |
64.14* |
5.00 |
|
0.27 |
1.25 ±0.39 |
1.82 ±0.52 |
1.66 ± 0.55 |
7.86 |
29.81* |
15.29* |
|
0.027 |
1.46 ±0.36 |
1.60 ±0.45 |
1.55 ±0.48 |
5.00 |
61.50* |
8.74 |
|
0.027 |
1.46 ±0.31 |
1.51 ± 0.38 |
1.50 ±0.36 |
4.42 |
19.60* |
8.47 |
I.UiHT RESPONSES OF BARXACLE NAUPLII
171
O 0
LU CO
.i 2
1
2
1
1
I NITIAL
4n8°STIMULUS
RECOVERY
B
N-34
N-38
N-35
N-30
345
SECONDS
8
FIGURE 2. The running mean linear velocity ± variance of stage II Balanus improvisus nauplii in response to a 480-nm light stimulus applied at 2.6 to 5.1 sec: (A) 27 W/nr; (B) 0.27 W/nr; (C) 0.027 W/m2; (D) 0.0027 W/m2; (E) 0.00027 W/m2. The number of naupliar paths analyzed (N) is indicated. Initial and recovery represent "dark" periods of 830-nm sub- 3tage illumination only.
172
LANG, FORWARD, JR. AND MILLER
INITIAL
DARK
0°-480nm
LIGHT
FINAL
DARK
90°
FIGURE 3. Distribution of mean direction of travel of stage II Bulaints iinprurisus during initial 2.5-sec "dark" interval, 2.5 sec 480-nm light stimulus at 0°, and final 3.0-sec "dark" interval: (A) 27 W/m2; (B) 2.7 W/m2; (C) 0.27 W/m2; (D) 0.027 W/m2; (E) 0.0027 W/m". Sample numbers and "dark" condition are as in Figure 2. Significantly (P <. 0.05) nonrandom distributions are indicated in Table I.
LIGHT RESPONSES OF BARXACLE NAUPLII
173
'45%
45?;
FIGURE 4. Distribution of mean direction of travel for 38 stage II Balanus improvisus exposed to a 480 nm, 2.7 W/nr light stimulus at 0° : (A) directional response during full 2.5-sec stimulus; (B) directional response during the latter 2.0 sec of the same stimulus.
Stage II response spectrum
Dark-adapted stage II nauplii (20° C, l5(/cc) were exposed to filtered light from 440 to 640 nm in 20-nm increments. Quantal intensity was balanced to approxi- mately 0.07 X 1016 quanta iir sec at each wavelength (calculated values ranged
100-
li! CO
z O a.
CO
LU
QC
50-
A
CONTROLS
A*
440
480 520 560
WAVELENGTH (nm)
600
640
FIGURE 5. Response spectrum for positive phototaxis (% response). Open circle, stage II Balanus improvisus; response ± 45° of light source; closed circle; ± 15° of light source. Open triangle, stage VI B. improvisus; ±45° of light source; closed triangle, ±15° of light source. See Table II for sample numbers.
174
LANG, FORWARD, JR. AND MILLER
100
C/}
z
o
W50
LU DC
AO
CONTROLS
10
-7
10'
r6
10
,-5
10
-4
10
-3
10
-2
i
10"
1.0
INTENSITY ( W/m2)
FIGURE 6. Intensity response for positive phototaxis of stage II Bulaiius improvisns at 480 nm. Closed triangle, dark-adapted at 20° C, lS(/fc (N = 26-47) ; open triangle, light-adapted at 20° C, \5%c (N = 53-60); open circle, light-adapted at 20° C, 30#c (N = 22-29).
from 0.062-0.088 X 101"). This quantal intensity was chosen because at 480-nm light it produced a clear positive phototactic response, but did not evoke a maximal response.
Analysis of DOT for nauplii indicates a broad response spectrum (Fig. 5). Increasing the ±15° "window" for positive response to ± 45° produces a similar curve at response levels 30 to 40% higher. The peak percentage of positive photo- taxis occurs at 480 nm (60% ± 15° ; 97% ±45°), however similar strong responses are evident at 500 to 520 nm. A significant negative response did not occur at any wavelength.
Although peak swimming speeds were found between 460 and 520 nm, distinct differences in MLV relative to wavelength are absent. One way analysis of variance followed by the Duncan test (P -- 0.05) yields three broadly overlapping homo- genous subsets, where the difference in the means of any two groups within a subset is not significant (Table II).
Intensity response
Light- and dark-adapted stage II nauplii (20° C, 15%C) were exposed to seven intensities of 480-nm light. In both groups, response to light was either positive or not evident; negative phototaxis did not occur significantly above random pre- dictions (Fig. 6).
In close agreement with initial results, light-adapted nauplii exhibit a signif- icant MLV increase at light intensities above 10~3 W/m2 (Table III). Dark- adapted nauplii significantly increase MLV above 10~* W/m2 and, in contrast to
LIGHT RESPONSES OF BARNACLE NAITL1I
175
100
LU CO
z O
LU
tr
50
^CONTROLS
10
-7
10
-6
,-5
10
-4
10
-3
10
-2
10"
1.0
I N
TENSITY ( W/m2)
FIGURE 7. Intensity response for positive phototaxis of stage III and stage VI Balanns improrisns at 480 nin. Open triangle, light-adapted stage III at 20° C, 30(/<e (N = 22-29); open circle, light-adapted stage VI at 20° C, 30#f (N = 25-42); closed circle, dark-adapted stage VI at 20° C, 30#f (N = 46-56).
light-adapted larvae, dark-adapted larvae show no increase in MLV at full intensity (Table III).
Directional response results further demonstrate the effects of dark adapta- tion. Light-adapted nauplii show a plateau of strong positive phototaxis from 10~3 to 10'1 W/m2; a similar pattern occurs from 10 4 to 10~2 W/m2 for dark-adapted nauplii (Fig. 6).
A significant positive phototactic response is present at all intensities tested for dark-adapted larvae. For light-adapted larvae, no significant response occurs below 10~4 W/m2. In respect to percent response and MLV, dark-adapted nauplii appear to have a somewhat "stronger" response to favorable light intensities relative to light-adapted nauplii (Fig. 6).
Some light-adapted stage II nauplii were also investigated at 30/£c, 20° C, to permit comparison with later stage nauplii tested at higher salinity. A similar, but perhaps enhanced response, relative to results at 15#0 was observed (Fig. 6). The change in salinity does not alter the basic pattern of response in stage II nauplii.
Light response in later stage nauplii
Smaller numbers of stage III and stage VI nauplii were tested for response to light, in addition to stage II nauplii. The later stage B. iinprovisus larvae are likely to be carried into increased salinity water ('Bousfield. 1955). Hence, these initial studies were conducted at 30#r.
Phototactic response of light-adapted stage III naupli (30/rr, 20° C) is similar to stage II nauplii (Fig. 7). The control MLV of stage III nauplii tends
176
LAN(i, FORWARD, JR. AND MILLER
TABLE II
Mean linear velocities of stage II and stage IV Balanus improvisus nauplii in response to 2.5-sec stimuli of light at different -wavelengths. Homogeneous subsets as determined by the Duncan s multiple range test (P = 0.05) are indicated (see Nie, et al., 1975).
|
Wavelength 7.0-9.0 X 1011 quanta/m3 |
Stage II |
Stage VI |
||||
|
N |
MLV ± s.d. mm /sec |
subset |
N |
MLV ± s.d. mm /sec |
subset |
|
|
440 |
26 |
1.75 ±0.66 |
BC |
35 |
3.72 ± 1.35 |
A |
|
460 |
46 |
1.97 ± 0.77 |
AB |
33 |
3.21 ± 1.19 |
AB |
|
480 |
40 |
1.85 ± 0.83 |
ABC |
35 |
3.36 ± 1.38 |
AB |
|
500 |
45 |
2.02 ± 0.99 |
AB |
35 |
3.26 ± 1.49 |
AB |
|
520 |
45 |
2.13 ± 0.79 |
A |
40 |
2.94 ± 1.07 |
AB |
|
540 |
40 |
1.92 ± 0.62 |
ABC |
37 |
2.83 ± 1.51 |
B |
|
560 |
34 |
1.69 ± 0.75 |
BC |
34 |
2.80 ± 1.37 |
B |
|
600 |
50 |
1.56 ± 0.60 |
C |
36 |
3.05 ± 1.37 |
AB |
|
620 |
34 |
1.81 ± 0.60 |
ABC |
37 |
3.16 ± 1.18 |
AB |
|
640 |
55 |
1.78 ±0.66 |
BC |
48 |
2.89 ± 1.18 |
B |
to be greater than stage II (Table III). Stage III nauplii show no significant increase in MLV during any light stimulus tested (Table III) therefore the photo- kinetic response seen in stage II nauplii is absent.
Both the response spectrum and responses to different intensities were tested for stage VI nauplii. Photo-responsiveness of stage VI larvae is generally less than that observed for stage II and III larvae; individual variation in nauplii is noticeably increased.
The spectral response function of stage VI nauplii using a ± 15° "window" for positive photo-response yields a generally low response of about 25 to 35% at all wavelengths, except 520 and 600 nm (Fig. 5). Increasing the window to ±45° produces a curve similar to stage II with peak response at 480 nm, however, respon- siveness is 20*/6 or more below stage II results (Fig. 5). Decreased responsiveness of stage VI nauplii at 520 nm and increased responsiveness above 600 nm relative to stage II larvae is evident (Fig. 5). Unlike stage II results, increasing the window to ± 45° changes the basic shape of the response curve for stage VI nauplii and is perhaps indicative of less precise orientation to the light stimulus during the 2.5-sec test interval.
The response of stage VI nauplii to different intensities of 480-nm light is most interesting in respect to dark adaptation. Light-adapted stage VI nauplii first show a significant phototactic response above 10~4 W/m2 (Fig. 8), which is in agreement with stage II results (Fig. 7). However, unlike stage II nauplii, the positive photo- taxis of stage VI nauplii rapidly diminishes above 10^2 W/m2. Dark adaptation of stage VI nauplii, although enhancing phototaxis between 10~5 and 10'* W/m2 (Fig. 7), does not appreciably alter the threshold of response. Finally, in marked contrast to stage II (Fig. 7), dark adaptation does not decrease responsiveness of stage VI nauplii to higher light intensities (Fig. 7).
The MLV of stage VI nauplii did not vary significantly as a function of light intensity or wavelength (Table II, III). The MLV for all samples (3.05/ mm/sec) was significantly above stage II and III results.
LIGHT RESPONSES OF BARXACLE NAUIM.II
177
TABLE III
Mean linear velocities of Balanus iniprovisus naupliar stages II, III, and VI in response to 2.5 sec 480-nm light stimuli of different intensities. Control represents swimming speeds with 830-nm sub- stage illumination. Homogeneous subsets as determined by the Duncan's multiple range test (P = 0.05) are indicated for each naupliar test group (see Nie, et al., 1975).
|
Dark-adapted II |
Light-adapted II |
|||||
|
Intensity |
||||||
|
N |
MLV ± s.d. |
subset |
N |
MLV ± s.d. |
subset |
|
|
6.2 X 10-' |
32 |
1.63 0.84 |
BC |
60 |
1.91 0.81 |
A |
|
x io-2 |
33 |
2.22 0.96 |
A |
59 |
1.92 0.88 |
A |
|
x 10-' |
26 |
2.11 1.10 |
A |
58 |
1.77 0.65 |
AB |
|
x io-1 |
37 |
1.94 0.73 |
AB |
59 |
1.58 0.60 |
BC |
|
X IO-3 |
36 |
1.55 0.588 |
C |
60 |
1.44 0.51 |
C |
|
x io-« |
47 |
1.54 0.51 |
C |
54 |
1.63 0.66 |
BC |
|
x io-7 |
39 |
1.41 0.57 |
C |
53 |
1.54 0.53 |
BC |
|
Control |
40 |
1.52 0.55 |
C |
55 |
1.48 0.49 |
C |
|
Light-adapted III |
Dark-adapted VI |
|||||
|
Intensity |
||||||
|
N |
MLV ± s.d. |
subset |
N |
MLV ± s.d. |
subset |
|
|
4.5 X IO-1 |
24 |
2.14 0.86 |
A |
50 |
2.94 1.52 |
A |
|
x io-2 |
29 |
2.40 0.70 |
A |
50 |
3.00 1.61 |
A |
|
x io-3 |
25 |
2.32 0.81 |
A |
49 |
3.03 1.74 |
A |
|
x io-4 |
22 |
2.24 0.87 |
A |
47 |
3.15 1.84 |
A |
|
X 10-s |
24 |
2.18 0.90 |
A |
46 |
2.60 1.29 |
A |
|
X IO-6 |
24 |
2.23 0.88 |
A |
56 |
3.00 1.50 |
A |
|
x 10-- |
23 |
2.08 0.96 |
A |
49 |
3.08 1.79 |
A |
|
Control |
23 |
2.20 0.73 |
A |
50 |
2.98 1.25 |
A |
DISCUSSION
The photophysiology of zooplankton has been recently reviewed by Forward (1976b). In general, zooplankton living in coastal and fresh water tend to have their primary spectral maximum in the 500- to 600-nm region, the wavelengths best transmitted in these waters. Visscher and Luce ( 1928) noted maxi- mal response of B. amphitrite and "B. improvisus" cyprids at 530 to 545 nm (although reported as B improvisus cyprids, the time of collection and simi- larity in size to B. amphitrite cyprids suggest Balanus cburcns as the more probable species tested (McDougall, 1943; Lang, 1979)). In more precisely controlled experiments using stage I E. inodcstus and S. balanoidcs nauplii, Barnes and Klepal (1972) found maximal spectral sensitivity between 520 and 530 nm, with a marked shoulder of strong responses at 450 to 530 nm.
Stage II B. improvisus nauplii have a maximal spectral sensitivity at 480 to 520 nm, somewhat shorter wavelengths than expected considering their estuarine habitat and other previous findings for barnacle larvae. Strong photo response occurs within this spectral range with maximal positive phototaxis at 480 nm (Fig. 6) and maximal MLV response to light stimuli at 520 nm (Table II).
Light response studies of stage VI nauplii are hindered by high individual vari- ability of responsiveness; some nauplii appear highly sensitive to light stimuli while
17S LANG, KORWAKD. JR. AND MII.I.KR
others are unresponsive. Those stage VI nauplii which do respond show strong- sensitivity to 440 to 500 nm, are curiously less sensitive to 520-nni light, and appear to be more sensitive to wavelengths at and above 540 nm relative to stage II (Fig. 6). Considerable structural and, presumably, physiological changes occur in stage VI nauplii in preparation for metamorphosis to cyprid (Walley, 1969). This includes development of compound eyes under the dorsal cephalic shield. Stage VI nauplii may have, depending on their age, only a single median naupliar eye, or two well pigmented compound eyes ( Kaufmann, 1965 ; Lang, 1979). Since the larval samples tested in this study included all phases of stage VI development, variability of responsiveness is not unexpected. Increased sensitivity to higher wavelengths is perhaps related to development of compound eyes ; studies on specific phases of stage VI development and, more importantly, the cyprid stage, are needed to verify this hypothesis.
Contrasting qualitative observation of photokinetic responses in barnacle larvae have been reported in the literature. Ewald (1912) noted a charatceristic sinking reaction in stage II specimens of Balanus perjoratus following a sudden dark-to- light transition. Similarly Crisp and Ritz (1973) saw decreased activity in S. balaiwides cyprids exposed to sudden light increase and conversely, increased activity following light intensity reduction. Essentially opposite reactions were observed in our laboratory for stage II Balanus 1'cniistns, B. improvisus, and B. amphitrite; MLV sharply increased following sudden exposure to bright white light and MLV sharply decreased following removal of the light stimulus (Lang, ct al., 1979). Photokinetic responses were found absent in stage II nauplii of S. balanoidcs and E. modcstits (Crisp & Ritz, 1973) and in Chthaiualus fragilis (Lang, ct al., 1979).
The present study shows that stage II nauplii of B. iiiiproi'isus consistently increases MLV following exposure to a specific range of light intensities at 480 nm, the range being determined, in part, by the initial state of naupliar light adaptation. Dark-adapted nauplii show photokinetic response at reduced light intensities relative to light-adapted nauplii. The upper intensity, if any, which inhibits increased MLV in light-adapted nauplii was above the maximum tested (27 W/m2). An increase in MLV appears to represent a second aspect in stage II light response. At detectable, but suboptimum intensities, stage II nauplii exhibit positive photo- taxis but show no significant change in MLV. Only within a narrower range of light intensities inducing maximal response does MLV increase. A similar change in MLV does not occur in light-adapted stage III nauplii or light- and dark-adapted stage VI nauplii. Results indicate at least two possibilities; either stage II nauplii exhibit a "stronger" response to light incorporating both directional and kinetic reactions or, later stages normally swim at or near their potential MLV and are incapable of further MLV increase in response to light stimulii.
Earlier studies (Lang, ct al., 1979) showed that B. iuiproi'isns nauplii will briefly stop or reduce MLV when a strong white light source was removed. Re- moval of the 480-nm light stimulii in present studies did not induce this response. We assume this response occurs only with more intense and/or longer light stimula- tion.
The level of previous adaptation to light has been shown to significantly effect the photoresponse of zooplanktons (Forward. 1976b). Crisp and Ritz (1973)
LIGHT RESPONSES OF BARNACLE NAUPLII 179
demonstrated relative reductions in barnacle naupliar light responsiveness following exposure to strong light. Continual exposure to light of sufficient intensity will induce a photonegative response in harnacle nauplii (Groom and Loeh, 1890).
A change in responsiveness to light following dark-adaptation is clearly seen here in B. iniproiisns stage II nauplii. The threshold for positive phototaxis in light-adapted nauplii is at least an order of magnitude greater relative to dark- adapted naupliar responses. Conversely, light-adapted phototaxis is significantly less suppressed at higher light intensities (Fig. 6). Positive phototaxis for at least I0(/c (above control) of dark-adapted stage II nauplii tested was seen at 6.2 X 10~7 W/nr at 480 nm. A consistent, strong response (i.e., 30% or more of the test population) occurred above 10~5 W/nr. These values bracket the same order of magnitude for light responsiveness reported by Barnes and Klepal (1972) and Crisp and Ritz (1973) for three barnacle species of naupliar stages I-II.
A similar clear shift in responsiveness of light- and dark-adapted stage VI nauplii was not seen. In particular, dark-adapted nauplii remained equally or more responsive to higher light intensities as compared to light-adapted nauplii (Fig. 7). Cypricl larvae of some barnacles are known to exhibit at least two fundamental light responses; orientation at fixation and shade-seeking during exploration (Crisp and Ritz. 1973). As with spectral results, we would eventually like to demonstrate whether idiosyncrasies in stage VI light behavior relate to light responses in B. improvisus cyprids.
The degree of light-adaptation, although it affects the intensity sensitivity of B. iuiproi'isus nauplii, does not alter the basic types of response. Under the stable and presumably favorable salinity-temperature conditions tested, the imme- diate reaction of B. improvisus to light stimulation is either positive phototaxis or no response. A characteristic "shadow response'' with negative phototaxis seen in light-adapted brachyuran zoea (Forward, 1976a, 1977) tested under similar light stimuli is absent.
In his classic field study, Bousfield (1955) showed that stage II B. improvisus nauplii maintained an average water column position near the surface, while later naupliar stages were found at progressively lower average depths. The strong posi- tive photo response of stage II nauplii and marked decline of photo responsiveness in stage VI nauplii seen here correlates well with these field observations. How- ever, a discussion of ecological implications of larval photobehavior is best deferred until further studies are complete.
This study most importantly demonstrates a new research technique. Video- computer systems decidedly enhance the ability to convert visual (video) records into quantified data. "With this ability comes a new potential to initiate studies on locomotory behavior involving various parameters and to measure responses with a resolution previously not readily obtainable.
Additional software development for analysis of phototactic responses was provided by Dr. Robert Wilson. Technical assistance for Bugwatcher operations was provided by Dr. John Greaves. Martha Marcy and Sally Lawrence con- tributed significant assistance in larval maintenance and data analyses.
180 LANG, FORWARD, JR. AND MILLER
SUMMARY
A video-computer behavioral analysis system — the "Bugwatcher"-— was found to be capable of rapidly and accurately analyzing the phototactic movements of stage II, III, and VI nauplii of the barnacle Balaints iinprorisits. Under the test conditions these larvae only display a positive phototactic response ; a negative response was not observed. The response spectrum of dark-adapted stage
II shows a plateau of strongest positive phototaxis between 480 and 520 nm with about 60/£> of test larvae swimming ±15° toward the light stimulus. In contrast, stage VI nauplii are generally less phototactic. The response spectrum changes to have a depression at 520 nm and enhanced responsiveness to longer wavelengths of 540 to 580 nm.
Responsiveness to different light intensities changed upon light adaptation. Upon stimulation with 480 nm lights, dark-adapted stage II nauplii show a significant positive phototaxis at 6.2 X 10~7 W/m2. Peak response occurs between 10 * and 10~2 W/nr. A signficant increase in mean linear velocity (MLV) accompanies the maximal phototactic response. In contrast, upon light adaptation, stage II nauplii show a significant positive phototaxis only to intensities above 10~4 W/m2, 480 nm. Peak response occurs between 10~3 and 10'1 W/m2. A significant increase in MLV occurs from about 10~3 to at least 27 W/m2. Light-adapted stage
III nauplii show intensity sensitivity similar to stage II nauplii ; however, no in- crease in MLV occurs during light stimulation. Stage VI nauplii show a reduced percent phototactic response at all intensities as compared to stage II-III. Light- er dark-adaptation does not result in clear shifts in intensity sensitivity as evident in stage II responses. Differences in stage VI naupliar light responses may be related to impending metamorphisis to cyprid. A study of cyprid light response is needed to clarify this.
LITERATURE CITED
AIKEN, R. B., AND J. P. HAILMAN, 1978. Positive phototaxis of the brine shrimp Arlcmia
salina to monochromatic light. Can. J. Zoo!., 56: 708-711. ANDERSON, J. M., 1971. II. Sublethal effects and changes in ecosystems. Assessment of the
effects of pollutants on physiology and behaviour. Proc. R. Soc. Loud., B. Biol. Sci.,
177: 307-320. BARR, A. J., J. H. GOODNIGHT, J. P. SALL, AND J. T. HELWIG, 1976. A User's Guide to SAS.
SAS Institute Inc., Raleigh, North Carolina, 329 pp. BARNES, H., D. J. CRISP, AND H. T. POWELL, 1951. Observations on the orientation of some
species of barnacles. /. Anim. Ecol., 20 : 227-241. BARNES, H., AND W. KLEPAL, 1972. Phototaxis in stage I nauplius larvae of two cirripedes.
/. Exp. Mar. Biol. Ecol. 10 : 267-273. BARNES, S. N., AND T. H. GOLDSMITH, 1977. Dark adaptation, sensitivity and rhodopsin level
in the eye of the lobster, Homarus. J. Coin p. Physio!., 120: 143-159.
BATSCHELET, E., 1965. Statistical Methods for the Analysis of Problems in Animal Orienta- tion and Certain Biological Rhythms. American Institute of Biological Sciences,
Washington, D. C., 57 pp. BOUSFIELD, E. L., 1955. Ecological control of the occurrence of barnacles in the Miramichi
Estuary. Natl. Mus. Can. Bull., No. 137 : 1-69. CRISP, D. J., AND D. A. RITZ, 1973. Responses of Cirripedc- larvae to light. I. Experiments
with white light. Alar. Biol. (Berlin), 23 : 327-335. DAVENPORT, D., G. J. CULLER, J. O. B. GREAVES, R. B. FOREWARD, AND W. G. HAND, 1970.
The investigation of the behavior of microorganisms by computerized television.
IEEE Trans. Bioined. Eng., BME-17 : 230-237,
f.IGHT RESPONSES OF BARNACLE NAUPLII 181
EWALD, W. F., 1912. On artificial modification of light reactions and the influence of electro- lytes on phototaxis. /. Exp. Zool., 13 : 591-612.
FORWARD, R. B., 1974. Negative phototaxis in crusteacean larvae : possible functional sig- nificance. /. Exp. Mar. Biol. Ecol., 16 : 11-17. FORWARD, R. B., JR., 1976a. A shadow response in a larval crustacean. Biol. Bull. 151 : 126-
140.
FORWARD, R. B., JR., 1976h. Light and diurnal vertical migration : photobehavior and photo- physiology of plankton. Pages 157-209 in K. Smith, Ed., Photochemical and photo- biological rci'icn's. Volume 1. Plenum Press, New York. FORWARD, R. B., JR., 1977. Occurrence of a shadow response among brachyuran larvae. Mar.
Biol., 39: 331-341. FORWARD, R. B., JR., AND J. D. COSTLOW, 1974. The ontogeny of phototaxis by larvae of
the crab Rhithropanopcus harrisii. Mar. Biol., 26 : 27-33. FORWARD, R. B., JR., AND T. W. CRONIN, 1978. Crustacean larval phototaxis: Possible
functional significance. Pages 253-261 in D. S. McLusky and A. J. Berry, Eds., Proc.
12th European Symp. Mar. Biol., Pergamon Press, New York. GREAVES, J. O. B., 1975. The bugstystem : the software structure for the reduction of quantized
video data of moving organisms. Proc. lust. Electrical Electronics Engr., 63: 1415-
1425. GROOM, T. T., AND J. LOEB, 1890. Der Heliotropismus der Nauplien von Balaiius perforate
und die periodischen Tiefenwanderungen pelagischer Tierre. Biol. ZentralbL, 10 :
160-177. HAMDORF, K., AND J. SCHWEMER, 1975. Photoregeneration and adaptation process in insect
photoreceptors. Pages 263-289 in A. W. Snyder and R. Menzel, Eds., Photorcception
Optics, Springer, Berlin. KAUFMAN, R., 1965. Zur Embryonal- and Larvalentwicklung von Scalpcllum scalpcllum L.
(Crust. Cirr.) mit einen Beitrag zur Autokologie dieser Art. Z. Morpholo. Oekol.
Ticre, 55 : 161-232. LANG, W. H., 1979. Larval development of shallow water barnacles of the Carolinas (Cirri-
pedia : Thoracica) with keys to naupliar stages. NOAA Tech. Rep., NMFS Circ.,
421: 1-39. LANG, W. H., S. LAWRENCE, AND D. C. MILLER, 1979. The effects of temperature, light, and
exposure to sublethal levels of copper on the swimming behavior of barnacle nauplii.
In F. Jacoff, Ed., Proc. Symp. The State of Marine Environmental Research, U. S.
Govt. Print. Office, Washington, D. C. (in press). LATZ, M. L, AND R. B. FORWARD, JR., 1977. The effect of salinity upon phototaxis and
geotaxis in a larval crustacean. Biol. Bull., 153: 163-179. McDoucALL, K. D., 1943. Sessile marine invertebrates at Beaufort, North Carolina. Ecol.
Mongr., 13 : 321-374.
NIE, N. H., C. H. HULL, J. G. JENKINS, K. STEINBRENNER, AND D. H. BENT, 1975. Sta- tistical Package for the Social Sciences, McGraw-Hill, New York. 675 pp. OLLA, B. L., (Ed.), 1974. Behavioral bioassays. Pages 1-31 in G. V. Cox, Chairman, Marine
Bioassays Workshop Proceedings, Marine Technology Society, Washington, D. C.,
308 pp. ROSE, M., 1925. Contribution a letude de la biologic du plancton ; le probleme des migrations
journalieres. Arch. Zool. Exp. Gen., 64: 387-542. SINGARAJAH, K. V., J MOYSE, AND E. W. KNIGHT-JONES, 1967. The effect of feeding
upon the phototactic behaviour of cirripede nauplii. /. Exp. Mar. Biol. Ecol., 1 :
144-153. THORSON, G., 1964. Light as an ecological factor in the dispersal and settlement of larvae of
marine bottom invertebrates. Ophelia, 1 : 167-208. VISSCHER, J. P., AND R. H. LUCE, 1928. Reactions of the cyprid larvae of barnacles to light
with special reference to spectral colors. Biol. Bull., 54 : 336-350. WALLEY, L. J., 1969. Studies on the larval structure and metamorphosis of Balanus balanoidcs
(L.). Phihs. Trans. R. Soc. Land., B. Biol. Sci., 256: 237-279. WILSON, R. S., AND J. O. B GREAVES, 1979. The evolution of the bugsystem : recent progress
in the analysis of bio-behavioral data. In F. Jacoff, Ed., Proc. Symp. The State of
Marine Environmental Research, U. S. Govt. Print. Office, Washington, D. C., (in
press).
: />'/„/. />'?,//. 157: 182-188. I August ]<)7<) >
THE EFFECTS OF EYESTALK, LEG, AND UROPOD REMOVAL ON THE MOLTING AND GROWTH OF YOUNG CRAYFISH,
PR 0 CA MBA R US CLA RKII
IS AMU NAKATANI AND TAKASHI OTSU Department of Bioloi/y, Faculty of Science, Yaniai/ata I'liii'ersity. Yainaiiata. 9Q0 Japan
The effects of eyestalk removal on molt have been studied in decapod crustaceans (Brown and Cunningham, 1939; Ahramowitz and Abramowitz, 1940; Smith, 1940). but few crustaceans have been studied during several consecutive molts, probably because of a high mortality. According to Abramowitz and Abramowitz (1940), however, the removal of the eyestalk itself is unlikely to be the reason for the mortality, because some of their blinded crabs survived for 11 weeks after the removal of eyestalks.
In our preliminary experiments (unpublished), 14 of 90 eyestalkless young crayfish survived after more than 10 molts, though the rest died within 4 months. In most cases, the eyestalkless crayfish died at the time of molt or within 1 or 2 days after the molt, probably because of bacterial infection at the time of molt. If this is so, a high mortality might be avoidable to some extent by keeping the container water clean, without disturbing the molting animals.
The molt cycle in Crustacea is thought to be regulated by two hormones, a molt hormone and a molt-inhibiting hormone. It is believed that the X-organ in the eyestalk produces the molt-inhibiting hormone, which is then stored in the sinus gland. Eyestalk removal, therefore, shortens the following intermolt cycle (Brown and Cunningham, 1939; Smith, 1940). The molt-inhibiting hormone inhibits the activity of the Y-organ, which secretes the molt hormone (Passano, 1960). When the eyestalk is removed the Y-organ is no longer inhibited. If the larvae of the prawn Palacinonctcs kadiakensis are destalked at metamorphosis, they grow larger in size than the untreated ones, without causing any alteration on the duration of the larval instars (Hubschman, 1963).
It is known that leg removal causes precocious molt in land crabs, Gecarcinus latcralis (Skinner and Graham, 1970), freshwater shrimp, Palaeinonctcs kadiakctuis (Stoffel and Hubschman, 1974), and crayfish, Procauibarns clarkii ( Bittner and Kopanda, 1973). Skinner and Graham (1972) hypothesized that severing a critical number of leg nerves stimulates the precocious molt.
In the present study, the molt interval and the growth rate of the crayfish were studied during five consecutive molts when the eyestalks. the legs or the uropods were removed.
MATERIALS AND METHODS
Specimens of the crayfish, Procambarus clarkii, used in this work were collected from ponds in the suburbs of Yamagata. Only crayfish of 8 to 12 mm in length (the length was measured from the tip of rostrum to the hind margin of cephalothorax carapace) that molted once in the laboratory (initial molt) were
182
CRAYFISH MOLTI.M,
183
chosen as experimental material. They were kept separately, first in polypropylene containers ( Lustro ware of Boclen Co., 70 X 85 X 45 mm), then in large con- tainers (105 X 120 X 53 mm) containing dechlorinated tap water at 22 to 23° C when they grew to more than 15 mm in carapace length. They were kept under a photoperiodic light condition of 14-hr light and 10-hr dark. The animals were fed fish food pellets and fallen dead leaves of persimmon. The dead leaves were effective in keeping the animals healthy. \Yhen water in the containers was renewed each day, the crayfish were immersed in 1.3% NaCl solution for 20 to 30 sec to prevent infection by bacteria or protozoa.
The crayfish were classified into four experimental groups : ( 1 ) untreated intact crayfish as control ; (2) crayfish from which a pair of eyestalks was removed ; (3) crayfish from which three pairs of the second, third and fourth walking legs were removed, and (4) crayfish from which a pair of uropods was removed. Each group consisted of 23 individuals that had just finished the initial molt in the laboratory, and every operation \vas performed on the day following this initial molt. The organs were cut off at their bases with scissors. After the third molt following the operation, the regenerated legs or uropods were removed again. Carapace length was measured two days after each molt.
RESULTS Molting
Molting rate was recorded daily for 230 days after the initial molt. The results are illustrated in Figure 1A-D. The crayfish which died during the course of the experiments were excluded from the data. Four crayfish from the control group, one from the legless group, and one from the uropodless group failed to complete the fifth molt even after 230 days. Since these animals had a reduced fecal output, their failure to molt may have been the result of factors other than hormonal or nervous controls.
Untreated crayfish (controls). Fourteen individuals completed the fifth molt, but three molted four times and one completed only the third molt. The average time required for 50% of the individuals to reach each consecutive molt (T50) was 17 days for the first molt, 50 days for the second, 87 days for the third, 128 days for the fourth, and 164 days for the fifth, after the initial laboratory molt.
TABLE I
The days of the intenuolt cycles (molt to molt) in crayfish, Procambarus clarkii.
|
Molt |
Control (14) |
Eyestalkless (ID |
Legless (20) |
L'ropodless (20) |
|
Initial to 1st |
19.6 ± 2.7 |
6.5 ± 0.2 |
14.1 ± 1.0 |
19.7 ± 2.3 |
|
1st to 2nd |
23.8 ± 2.9 |
7.1 ± 0.3 |
12.6 ± 0.9 26.9 ± 2.9 |
|
|
2nd to 3rd |
33.6 ± 4.2 |
7.5 ± 0.2 |
31.5 ± 3.8 |
33.6 ± 4.2 |
|
3rd to 4th |
31.3 ± 3.1 |
8.8 ± 0.2 |
21.1 ± 1.6 |
29.9 ± 2.6 |
|
4th to 5th |
43.2 ± 6.0 |
9.2 ±0.4 |
35.2 ± 4.6 |
39.0 ± 3.4 |
The number of individuals is in parentheses. Data were obtained from crayfish which had completed five consecutive molts. The standard error of the mean is shown.
184
I. NAKATANI AND T. OTSU
100 80 60
40 20
0
100 80 60
*7 40 ~ 20 o 0
80 60 40 20 0
100 80 60 40 20 0
A
B
IV
c
0 20 40 60 80 100 120 140 160 180 200 220
DAYS
FIGURE 1. Molting percentage of surgically treated crayfish, specimens of Procambarus clarkii, at five consecutive molts after the initial laboratory molt. A, untreated crayfish (control) ; B, both eyestalks removed; C, three pairs of walking legs removed; D, both uropods removed. In the groups where legs or uropods were removed, the regenerated legs or uropods were removed again one day after the third molt. Roman numerals I, II, III, IV and V, show the 1st, 2nd, 3rd, 4th, and 5th molt, respectively.
Large variation, however, was observed among the individuals. For example, in the first molt the earliest crayfish molted on the seventh day but the last crayfish molted on the fifty-third day. As clearly shown in Figure 1 , this variation became more pronounced in the later molts.
Eyestalkless crayfish. Twelve of 23 treated crayfish died between the third and fifth molts. The remaining 11 completed all five consecutive molts within 43 clays. T50 from the first to the fifth molt was 6 (35.3% compared to the value of
CRAYFISH MOLTING
control animals), 13 (26.0%), 20.5 (23.6%), 29 (22.7%), and 39 (23.8%) days, respectively. The first molt occurred on the fifth clay and all the treated individuals molted by the eighth day. All 23 crayfish completed the second molt between 6 and 8 days after the first molt and finished the third molt between 7 and 8 days after the second molt. Two animals, however, died on the first and fifth day respectively, after the third molt.
The surviving crayfish completed the fourth molt by the intermolt cycle of 7 to 12 days. Seven animals died within two days after the fourth molt. The 14 survivors completed the fifth molt by the intermolt cycle of 8 to 12 days. Three died at the time of the fifth molt.
Legless crayfish. Twenty of 21 animals completed five consecutive molts (one animal completed only four). Tr,0 from the first to the fifth molt was 13 (76.5% of the control value)', 24 (48.0%), 55 (63.2%), 77 (60.2%), and 99 (60.4%) days, respectively.
A few animals had longer molt cycles than the others. Roughly speaking, the operated animals could complete five molts during the time in which the untreated crayfish completed four molts. The walking legs regenerated to normal size after the second molt. Two crayfish died ; one at the time of its third molt and another at its fourth molt.
2140
o~~
' ' 120 100
CO <
LU CT CJ
o
LU
_l
LU
<
CJ
80
60
20
0
O Control
£ Uropods removed A Legs removed Eyestalkless
0 20 40 60 80 100120140160 DAYS
FIGURE 2. The average percentage of increase in carapace length of specimens of the crayfish, Procambarus clarkii, to the original length after each of the five consecutive molts. Each point represents the mean days required for each molt following the initial laboratory molt.
I. XAKATAM AXI) T. OTSl'
TAHLI. 1 1
/'//c average carapace length (mm) after each molt in crayfish, 1'rocambarus clarkii.
|
Initial |
1st |
2nd |
3rd |
4th |
5th |
Mean |
|
|
Control Eyestalkli-s-i Legless Uropodless |
11.1 ±0.2 11.5 ± 0.3 11.2 ± 0.3 1 1 .6 ± 0.3 |
12.3 ±0.3 (10.5) 13.9 ± 0.3 (20.7) 12.1 ± 0.3 (8.1) 12.7 ± 0.3 (8.9) |
13.5 ±0.4 (10.4) 16.3 ± 0.4 (17.5) 13.3 ± 0.3 (10.0) 14.1 ± 0.5 (11.3) |
14.9 ± 0.5 (10.2) 19.5 ± 0.5 (19.8) 14.6 ± 0.4 (10.2) 15.5 ± 0.6 (9.7) |
16.1 ± 0.5 (8.2) 22.8 ± 0.6 (16.8) 15.9 ± 0.4 (8.6) 16.X ± 0.7 (8.6) |
17.5 ±0.6 (8.5) 26.2 ± 0.7 (14.7) 17.1 ± 0.5 (7.8) 18.3 ± 0.9 (8.6) |
(9.6) (17.9) (8.9) (9.4) |
The numerals in parentheses are the average percentage of increased carapace length for each molt.
Uropodless crayfish. Twenty of 21 animals completed five consecutive molts, one animal staying at the fourth molt during the experimental period. Tr>0 from the first to the fifth molt was 12 (70.0% of the control value), 39 (78.0%). 73 (83.9%>) 96 (75.0%), and 136 (82.9%} days, respectively. Regeneration of the uropods was observed on all crayfish after the second molt. Two crayfish died ; one at the first molt and another at the third molt.
Gro^(.<th rates
The mean values of growth rate for crayfish which completed five molts wrere measured after each of five consecutive molts with special reference to the mean value of the initial carapace length, which was measured after the initial laboratory molt (Fig. 2). As is clear from Figure 2, the rate of increase of carapace length of the eyestalkless group is more than six times greater than that of the control group.
The percentage increases in carapace length from between each premolt and corresponding postmolt stage are shown in Table II. The average percentage of increase in carapace length per molt was 9.6% for untreated group, 17.9% for eyestalkless group, 8.9% for legless group, and 9.4% for uropodless group.
DISCUSSION
The results suggest that the removal of eyestalks, legs, or uropods stimulates molt in the crayfish, Procauibants clarkii. The results support the findings of other authors studying eyestalkless crayfish, Cainbarus clarkii (Smith, 1940), fiddler crabs, Uca pugilator ( Abramowitz and Abramowitz, 1940) and land crabs, Gecarciuns lateralis (Skinner and Graham, 1972) ; and walking legless crayfish, Procauibants clarkii ( Bittner and Kopanda. 1973), freshwater shrimp, Palacinonctcs kadiakcHsis (Stoffel and Hubschman, 1974) and land crabs, Gecarciuns lateralis (Skinner and Graham, 1970). Brown and Cunningham (1939) and Smith (1940) discussed the possible mechanisms concerning the effects of eyestalk removal on the molt, suggesting that the eyestalks contain the inhibiting hormone which delays molting.
Abramowitz and Abramowitz (1940) found that in fiddler crabs both molt and growth were stimulated by eyestalk removal. In our experiments, we found that the average growth rate of carapace length in the eyestalkless crayfish after every molt was about twice that of the intact animals, and that the duration of the molt
CR. \VKISII MOLTING 18?
cycles of the former was about one fourth of that of the latter. From these data, the mean weight increase of eyestalkless animals was about 15 times larger than the corresponding weight of the control animals 40 days after the operation. On the other hand, the molts of both the leg- or uropod-removed crayfish were stimulated, but the carapace length growth rate after every molt was less than that of the controls. Thus, it is clear that growth was accelerated by eyestalk removal, and that the secretion of molting hormone was induced by the growth of the body.
Weis (1976) found that the removal of seven legs from a fiddler crab did not result in a significant increase in carapace width after molt and regeneration. Krishnakumaran and Schneiderman (1970) found that ecdysterone did not increase DNA synthesis in the epidermis, muscles, nerve cells, and connective tissue of cray- fish, Procambarus clarkii, although it induced molting. In the present experiments, the carapace length increased after molt in crayfish whose legs or uropods were removed, but the rate of increase was less than that of the untreated control.
It may be safely concluded that the removal of legs or uropods has the same effects on molt and growth in crayfish. This would support the hypothesis of Skinner and Graham ( 1972) that molt inhibitory factors do not exist in the limbs of Crustacea and that precocious molt is stimulated by the severing of a critical number of nerves. Stoffel and Hubschman (1974) pointed out that the loss of several walking legs stimulates the neurosecretory cells of the X-organ via nervous impulses to stop releasing the molt-inhibiting hormone. According to Holland and Skinner (1976), the removal of one or more limb buds of Gecarcinus lateralis inhibited growth of the remaining limb buds until re-regenerates reached an appro- priate size. In the present experiment, both legless or uropodless crayfish molted before they grew in body size sufficient for molt. Therefore, if the crayfish is missing legs or uropods. it may be possible that the regenerating buds grow faster than all the other parts of the body, and the regenerating buds stimulate the release of molt hormone. The intensity of stimulus may depend, to some extent, on the number of buds, or on the surface of the cut ends or organs. It is known that land crabs missing many legs prepare for molt sooner than those which are missing one or two legs (Skinner and Graham. 1970).
It has been reported that the mortality is high in destalked crustaceans (Abramowitz and Abramowitz, 1940; Smith, 1940; Skinner and Graham, 1972). According to Abramowitz and Abramowitz (1940), the viability is concerned in some way with the eyestalks. In general, death during molt by intact Crustacea is common. In the present experiments, five crayfish of the control group, two of the legless group, and two of the uropodless group died at the time of molt or within a day after their molt. In the eyestalkless group, twelve individuals died at the time of molt or within a few days after their molt. The mortality of the eye- stalkless group, therefore, is higher than the other groups. It may be possible that the eyestalkless crayfish grow too rapidly to prepare properly for molt and this leads to failure at molt.
Our sincerest thanks go to Dr. H. Bernard Hartman, Associate Professor of Biology, Texas Tech University, for his courteous reading and exact revising of the manuscript.
188 I. NAKATANI AND T. OTM
SUMMARY
1. Removal of a pair of eyestalks induces precocious molt and accelerates the growth of the crayfish, Procarnbanis clarkii.
2. Removal of three pairs of walking legs or a pair of uropods induces pre- cocious molt without any effects on the growth of the body.
3. The average time required for 50% of the individuals to reach the fifth molt after the intial molt is 39 days for eyestalkless crayfish, 99 days for legless, and 136 days for uropodless crayfish. The untreated crayfish (controls) required 164 days to attain the fifth molt.
4. The average percentage of increase in carapace length at the time of the fifth molt is 9.6% for the untreated crayfish, 17 '.6% for eyestalkless, 8.9% for legless, and 9.4% for the uropodless group.
5. The mortality during the approximately eight-month experimental period was 5:23 for the untreated group, 12:23 for the eyestalkless group, 2:23 for the legless group and 2 : 23 for the uropodless group. The eyestalkless crayfish were healthy until the third molt, but experienced great mortality at the time of the fourth and fifth molt. The failure of molt in eyestalkless crayfish may be due to too rapid increase in the body size, imparing preparations for molt.
LITERATURE CITED
ABRAMOWITZ, R. K., AND A. A. ABRAMOWITZ, 1940. Moulting, growth, and survival after
eyestalk removal in Uca pugilator. Biol, Bull., 78: 179-188. BITTNER, G. D., AND R. KoPANDA, 1973. Factors influencing molting in the crayfish Pro-
cainbarus clarkii. J. Exp. Zool., 186 : 7-16. BROWN, F. A. JR., AND O. CUNNINGHAM, 1939. Influence of the sinus gland of crustaceans on
normal viability and ecdysis. Biol. Bull.. 77 : 104-114. HOLLAND, C. A., AND D. M. SKINNER, 1976. Interactions between molting and regeneration
in the land crab. Biol. Bull., 150 : 222-240. HUBSCHMAN, J. H., 1963. Development and function of neurosecretory sites in the eyestalks of
larval Palacmonetcs (Decapoda: Natantia). Biol. Bull.. 125: 96-113. KRISHNAKUMARAN, A., AND H. A. SCHNEIDERMAN, 1970. Control of molting in mandibulate
and chelicerate arthropods by ecdysones. Biol. Bull., 139: 520-538. PASSANO, L. M., 1960. Molting and its control. Pages 473-536 in T. H. Waterman, Ed.,
The Physiology of Crustacea. I. Metabolism and Groi^'th. Academic Press, New York
and London. SKINNER, D. M., AND D. E. GRAHAM, 1970. Molting in the land crab: Stimulation by leg
removal. Science, 169 : 383-384. SKINNER, D. M., AND D. E. GRAHAM, 1972. Loss of limbs as a stimulus to ecdysis in Brachyura
(true crabs). Biol. Bull., 143 : 222-233. SMITH, R. I., 1940. Studies on the effects of eyestalk removal upon young crayfish (Cambarus
clarkii GIRARD). Biol, Bull,, 79 : 145-152. STOFFEL, L. A., AND J. H. HUBSCHMAN, 1974. Limb loss and the molt cycle in the freshwater
shrimp, Palacinonctcs kadiakensis. Biol, Bull., 147 : 203-212. WEIS, J.. 1976. Effects of environmental factors on regeneration and molting in fiddler crabs.
Biol. Bull., 150: 152-162.
Reference : Biol. Bull. 157 : 189-204. (August 1979)
KVOKED RESPONSES TO ELECTRICAL STIMULATION IN THE
COLONIAL HYDROID CLAVA SQUAMATA:
A CONTRACTION PULSE SYSTEM 1
DARRELL. R. STOKES AND NORMAN B. RUSHFORTH
Department of Biology, Emory University, Atlanta, Georgia 30322; Department of Biology,
Case Western Reserve University, Cleveland, Ohio 44106; and The Marine Biological
Laboratory, Woods Hole, Massachusetts 02543
Electrical activity has been recorded from several hydrozoans (phylum Cnidaria) representing two distinct classes of polyps — those which produce recurring behavioral events of spontaneous origin, for example, Corymorpha (Ball, 1973; Ball and Case, 1973); Hydra (Josephson, 1967; Josephson and Macklin, 1969; Passano and McCullough/ 1962, 1963, 1964, 1965; Rushforth, 1966, 1971; Rushforth and Burke, 1971); Millepora (deKruijf, 1976); Obelia (Morin and Cooke, 1971a, b, c) ; Tnbnlaria (deKruijf, 1977; Josephson, 1962, 1965, 1974; Josephson and Mackie, 1965 ; Josephson and Rushforth, 1973 ; Josephson and Uhrich, 1969) ; and those which are behaviorally quiescent, e.g. Cordylophora (Josephson, 1961b; Mackie, 1968) ; Hydractinia (Stokes, 1974a, b) and Probosci- dactyla (Spencer, 1974). Despite these obvious behavioral differences, a review of these studies (Rushforth and Stokes, 1978) showed that there is at least one element of electrically evoked activity which is common to all species of both classes. These common elements are the large (0.5-15 mV), slow conducting (2-20 cm/sec), long duration (20-500 msec) potentials which are correlated with contraction of whole or isolated parts of a polyp. Examples include the Josephson Pulses (IPs) of Cord\lophora, Hydranth Pulses (HPs) and Neck Pulses (NPs) of Tiibularia, Stalk Pulses (SPs) and Hydranth Pulses (HPs) of Corymorpha, Contraction Pulses (CPs) or Contraction Bursts (CBs) of Hydra, Symmetrical Contraction Pulses (SCPs) of Hydractinia, Contraction Pulses (CPs) of Millepora, Contraction Pulses (KPs) of Obelia and the Tentacle Contraction Pulses (TCPs) and Colonial Pulses (CPs) of Proboscidactyla,
It has been proposed (Rushforth and Stokes, 1978) that these pulses are homol- ogous representations of a fundamental conducting system termed the 'Contraction Pulse System' (CPS). This system functions to activate muscles of widespread distribution, or isolated blocks of muscles. In an effort to strengthen this proposal, we have investigated the evoked responses of the colonial gymnoblastic hydroid, Clara squaniata. The purpose of the present study was to examine the electrical activity of this behaviorally quiescent species.
MATERIALS AND METHODS Collection and maintenance of animals
The Supply Department of the MBL, Woods Hole, provided all animals for this study. Only colonies growing on Ascophyllum were used. Colonies in which the
1 Supported in part by PHS grant number MH 10734-10 to N. B. Rushforth. and a grant from the McCandless Foundation, Atlanta, Georgia, to D. R. Stokes.
189
I). K. STOKES AND N. B. RUSHFORTM
polyps appeared unhealthy or damaged, or in which the alga had begun to decay, were discarded. All specimens were placed in an aerated, refrigerated (14° C) Instant Ocean System containing sea water, and were fed daily on newly hatched Artemla nauplii. Collected colonies were studied within 2 to 3 days. Glass micro- scope slide cultures of Clai'a were also made by dissecting two to three inter- connected polyps from a colony and attaching them to the slide by fine surgical thread. Such cultures were maintained under conditions similar to those of the freshly collected colonies. New stolons grow out from the transplant, adhere to the glass slide and give rise to new polyps. After about 1 week the original transplant and the surgical thread can be removed, leaving only new growth attached to the slide.
Most experiments used whole colonies on their algal substrate. The alga was trimmed to about 2 to 3 cm length and pinned to the Sylgard bottom of a dish containing 500 ml of sea water. The temperature of the sea water in the dish was also maintained at 14° C by an outer jacket of circulating, refrigerated water. The attached hydroids did not appear to be damaged by this process and survived for several days under such conditions, when appropriately fed. In certain experiments single polyps were used. These polyps were excised from the colony by means of iridectomy scissors at the junction between polyp and stolon.
Stimulation and recording techniques
Suction electrodes were used for recording and stimulation. Plastic Tygon or polyethylene tubing was flame heated and drawn to diameters appropriate for attachment to a specific region of the polyp or stolon, usually 50 to 100 jam. The tubing was then squarely and evenly cut to insure minimal damage to the soft tissue. Glass electrodes modeled after those described by Josephson (1967) were used in experiments on isolated polyps. These electrodes have a bell-shaped tip in which the polyp can be held without damaging the tissues. Both kinds of electrodes can be attached by means of mild suction obtained by manipulation of a hypodermic syringe at the opposite end of the tubing or glass. Micromanipulators served to facilitate movement and placement of the electrodes. Both stimulation or recording could be achieved through these suction electrodes, and in either case, the indif- ferent electrode was a coil of chloridecl silver wire placed in the experimental dish. Electrical stimuli were single or repetitive pulses delivered through a stimulus isolation unit. Conventional capacitor coupled amplifiers and display devices were used.
RESULTS
General organization of the colony
Clara may be found at Woods Hole, Massachusetts in association with an intertidal brown alga, Ascophyllmn. It is generally located at the branch points of the alga or within damaged flotation sacs, and only rarely along the lengthy exposed parts of the stem. Occasionally one can find Clara on rocks or wharf pilings. Colonies growing on Ascophyllitin are comprised of upwards of 50 to 100 monomorphic polyps ; the hydranth of each bears a naked hypostome and terminal
HYDROZOAN BEHAVIORAL PHYSIOLOGY 1M1
mouth, and some 20 to 30 filiform tentacles. The tentacles are uniformly distributed over the hypostomal region and do not form a distinct ring or rings. The lengthy stalk region below the hypostome is naked in immature polyps. Gono- phores bud off from this stalk region 1 to 2 mm below the most proximal tentacles in mature polyps. The polyps vary in length, from about 0.5 to 1.5 cm, and are joined basally by a coenosarc of interconnecting stolons. The sexes are separate and a colony is comprised of all male or all female polyps, probably originating from a single planula. Occasionally, as reported by Fb'yn (1927), male and female colonies will occur together in the same clump.
General behavior and responses to mechanical stimuli
Spontaneous and rhythmically recurring behavior of individual polyps or groups of polyps does not occur under uniform conditions of illumination in Clava. Polyps may bend slowly or reorient individual tentacles, but the general observation is prevailing quiescence. Endogenous pacemaker systems like those responsible for the rhythmic behavior of Tubularia (Josephson, 1962; Josephson and Mackie, 1965) or Hydr-a (Passano and McCullough, 1962, 1965) do not appear to be present in Clara.
Mechanical stimulation of a polyp may induce weak or vigorous polyp activity depending on the intensity of the stimulus. Pinching an individual tentacle can result in contraction of that tentacle alone ; contraction of the tentacle and movement of the hydranth towards the stimulated side ; or contraction of the entire polyp. Contraction of the polyp may be graded, but in most cases, par- ticularly colonies growing on Ascophyllum, it was not observed to spread to neighboring polyps. In rare cases, vigorous pinching of the hydranth resulted in a vigorous contraction of the polyp, and that contraction spread in a graded fashion to the neighboring polyps. Pinching a stolon also can elicit the same response; however, in no case were more than three polyps involved in this coordinated response.
Long-term electrical recordings from individual polyps show that electrical activity does not occur in Clava in the absence of behavioral events. Furthermore, many complex behaviors, particularly those associated with feeding, occur in the complete absence of detectable electrical events. Other complex responses induced by certain amino acids are associated with the generation of electrical potentials ; however their role in feeding, if any, is not known.
Following mechanical stimulation of a Clava polyp, either single or multiple pulses can be recorded with a short latency (less than 400 msec) which correlate with the visual observation of overall polyp contraction. \Yeak pinching of any part of a polyp produces single electrical pulses with each contraction, whereas vigorous stimulation produces multiple electrical pulses and a more vigorous polyp contraction. These same results can be obtained by mechanical stimulation of the interconnecting stolons.
Electrical stimulation-correlated behavior and electrical activity
The electrical responses in this study have been recorded extracellularly from rather large blocks of tissue comprised of ectoderm, niesoglea and endoderm.
192
I). R. STOKES AND N. B. RUSHFORTH
V
Multiple CPs
FIGURE 1. Clava polyp schematic showing representative kinds of evoked electrical pulses. A, delayed burst potentials similar to those shown in record B, but recorded at faster time base. Stimulus not indicated. B, a sequence of CPs and two bursts of DBFs evoked by four stimuli (dots beneath recording) applied to the burst generator region (hatched region). C, a typical CP evoked by stimulation of any part of the body column or tentacles. D, typical record of multiple CPs recorded from the polyp stalk. E, multiple CPs similar to those recorded in D, but at a faster time base. Dot indicates stimulus artifact. G, gonophores, S, stolon; Vertical scale A applies also to B, C, D, E, =0.5 mV; Horizontal Scale A applies also to C, E, =0.6, 0.2, 0.3 sees, respectively; Horizontal Scale B applies also to D =5.0 sec.
Positive identification of the underlying morphological source of electrical signals generated by these tissues is difficult and hence, for the moment, we cannot identify these events as neural, muscular, or epithelial. Instead the neutral term "conduct- ing system" (Josephson and Mackie, 1965) will be used to define the substrates of these electrical events.
Two distinct kinds of electrical activity can be recorded from Clava depending on the stimulus site. Evidence presented here and in a subsequent report (Rush- forth and Stokes, in preparation) indicates that these two events represent activity in separate conducting systems. One of these conducting systems is activated only by direct stimulation of the hyclranth distal to the gonophore stalks (Fig. 1, hatched region). The electrical events (Figs. 1A, B) occur as a burst or a
HYDROZOAX BEHAVIORAL PHYSIOLOGY
193
TABLE I Characteristics of electrically stimulated contraction pulses in Clava : mean ± s.e.
|
Pulse type |
Latency (msec) |
Interpulse interval (msec) |
Pulse duration |
Pulse amplitude mV |
Conduction velocity cm /sec |
||
|
Rise time (msec) |
+ve to — ve Peak (msec) |
Total (msec) |
|||||
|
A. Single contraction pulses B. Multiple contraction pulse bursts |
331 ± 109 (3)* 330 ± 70 (2) |
371 ± 19 (9) |
74 ±26 (2) 73 ± 6 (2) |
48 ± 11 (4) 50 ± 7 (9) |
144 ± 20 (4) 140 ± 8 (9) |
0.53 ±0.03 (4) 0.61 ± 0.10 (9) |
2.6 ±0.2 (11) 2.1 ± 0.3 (5) |
* Values in parentheses = X .
program of multiple bursts of pulses alter a long latency (x=: 19.2 ± 1.4 sec, Ar = 18) and are referred to as Delayed Burst Potentials (DBFs). A burst of DBFs on an expanded time scale is shown in Figure 1A. In no case have DBFs been initiated by stimulation of stolons or the stalk region proximal to the gonophores. DBFs correlate with a synchronized depression of the tentacles and, at least to the first few bursts of a program, with a symmetrical shortening of the body stalk. The stalk contraction element is not apparent after the first few bursts of a program. Also, the pulses within a burst appear much more homogeneous following loss of an observable stalk contraction. The conducting system under- lying these bursts of pulses, called the Delayed Burst System (DBS) is the subject of a subsequent report (Rushforth and Stokes, in preparation).
A second type of electrical event can be evoked by stimulation of the polyp at any location — tentacles, hypostome, body column or base. Characteristically, these potentials are single (Figs. IB, C) or multiple pulses (Figs. ID, E), which are similar in all respects to those evoked by mechanical stimulation. These potentials
B
\ \
O.SmV
5 Sec
FIGURE 2. Facilitation of CP potentials within a "burst", recorded at two sites (A and B) on a single polyp stalk. Two stimuli were applied (dots) adjacent to site B.
194 1). K. STOKKS AND N. B. RUSHFORTH
are correlated with a symmetrical, often localized contraction of the polyp and are thus referred to as Contraction Pulses (CPs). The conducting system under- lying the CPs is referred to as the Contraction Pulse System (CPS). The threshold for activation of the CPS is generally below that for the DBS. Both single and multiple CPs appear to originate within the tissues beneath the stimulat- ing electrode and have a short latency (about 300 msec) in comparison to the DBPs. Characteristics of the electrical pulses associated with single and multiple CPs are presented in Table I. In summary, both single CP events and individual CPs of a multiple sequence are nearly identical with respect to pulse rise time (75 msec), positive to negative peak (50 msec), total pulse duration (140 msec) and pulse amplitude (0.5-0.6 mV). The refractory period for single CPs was determined to be about 200 to 250 msec, and the interpulse intervals for multiple CPs are on the average about 370 msec. As shown in Figure IE, the interpulse intervals tend to elongate towards the end of a CP burst. CPs are generally biphasic, although triphasic CPs have also been recorded, usually following the first stimulus of a regime of stimuli given to a fully expanded polyp.
Both single and multiple CPs are nonpolarized conducted events (Table I). The conduction velocity was determined by recording from two Tygon suction electrodes attached to the same side of the poylp while stimulating through a glass holding electrode. From these records, the time delay between negative peaks of the electrical event recorded at the two sites and the measured distance between the recording electrodes were used to compute the conduction velocity. The mean conduction velocity for single CPs is 2.6 cm/sec (N — 11) and for multiple CPs 2.1 cm/sec (N = 3). For three animals, conduction velocities for single CPs were determined for both the proximal and distal directions. No sig- nificant differences were observed (x = 2.3 ±0.1 distally and x = 2.2 ± 0.3 proxi- mally). The mean conduction velocity for DBPs is 9.8 cm sec (N = 7).
The responses of a single polyp to electrical stimulation appear to be graded with stimulus intensity. A single threshold shock to a tentacle may result only in contraction of the stimulated tentacle. A small CP is associated with the tentacle contraction. Similarly, threshold stimulation of the hydranth may result only in contraction of the hydranth region, with associated CPs, and in no apparent contraction of the stalk region. Responses of the polyp are more extensive and also more vigorous with increasing numbers and intensity of stimuli. The ampli- tude and number of recorded CPs decrement with distance from the stimulus site. Multiple CPs of large amplitude occur in regions of vigorous contraction, the number and amplitude diminishing in regions where contraction intensity is also reduced. During repetitive stimulation, contraction spreads to and involves a greater part of the polyp, an observation which appears to correlate with facilitation of CPs to each shock. Single threshold shocks generally give a localized polyp contraction and a single CP. Multiple shocks of threshold intensity or single suprathreshold stimulation of either proximal or distal regions of the polyp stalk can evoke multiple firing of the CP system. Some 30 or more CPs have been recorded to a single suprathreshold stimulus. Multiple firing of the CP system such as that shown in Figures ID, E is correlated with a prolonged, continuous shortening of the polyp stalk, which often reduces the polyp to a stubby ball. In
HYDROZOAN BEHAVIORAL PHYSIOLOGY
195
B
.5mV
5 Sec
.5mV 5Sec;Ts"ec.
FIGURE 3. Interpolyp communication. A, simultaneous recordings from two interconnected polyps (Pi and P-) following application of four stimuli to one of them (asterisk). Largest events (stimulus artifacts) correlated to stimulus record (lower channel). Note that a burst (DBPs) occurs only in the stimulated polyp. B, same as A, but note that a CP occurs in Pi shortly after a burst of DBPs in P2. C, simultaneous recordings from a polyp ( P) and an attached stolon (S) while stimulating the same stolon (asterisk). Note the small potentials in S correlate with repetitive CPs (dots) in P which facilitate to each stimulus (marked in lower trace).
this condition, the polyp is refractory to further stimulation, and it may take 30 min or more before the polyp fully expands once again.
Dual recordings from a single polyp during multiple CP firing ( Fig. 2} also show more vigorous contraction adjacent to the stimulus site and uniformly large CPs (Fig. 2B), while at a distant recording site the contraction may not be apparent initially but becomes increasing more vigorous throughout a CP burst. The CPs in such a burst show a marked facilitation (Fig. 2A). As a wave of contraction passes a recording site, subsequent stimulation can resttlt in a marked reduction and even a defacilitation of CPs within a burst.
Stimulation of the hydranth region of a polyp, as shown in Figure IB, can evoke both single and multiple CPs in addition to DBPs. In this record, the first of four stimuli initiated some four CPs and the third stimulus a single CP of smaller amplitude. And finally, some 12 sec after the fourth stimulus, the DBS fired a burst of potentials.
D. R. STOKES AM) X. I',. RUSH FORTH
Sea Water
B
MgCI2-10Min.
C
MgCI2-50Min.
D
Sea Water- 30 Min.
.5mV
5 Sec.
FIGURE 4. Effects of MgCU. A, recording from an isolated polyp in sea water. Stimuli in this and following records are marked in lower trace. B, same as A, after 10 min in MgCU. C, same as A, after 50 min in MgCU. D, same as C, following return of polyp to normal sea water for 30 min.
Interpolyp communication
Communication from polyp to polyp by means of the interconnecting network of stolons has been difficult to demonstrate in wild colonies. In such colonies, the polyps arise from a tangled mass of stolons which makes it difficult to determine
HYDROZOAN BEHAVIORAL PHYSIOLOGY 1<)7
which of the polyps are directly connected. In addition there are numerous symbionts (crustaceans, platyhelniinths, protozoans) which live in association with the tangled stolons, many of which have been observed to feed on the soft tissues of the colony. It seems likely that these symbionts disrupt the structural organiza- tion of the colony, leaving whole portions of the colony or even individuals func- tionally isolated from each other. In only a few colonies did mechanical stimulation of one polyp affect the behavior of adjacent polyps. These were young colonies with new growth and no apparent damage to the stolons joining the two polyps. In all cases, very intense mechanical stimulation was necessary to show spread of excitation to an adjoining polyp, and in all cases only two polyps were involved.
Slide culture colonies provide a better preparation for the study of interpolyp communication. The stolons grow out in rather straight formations and give rise to polyps at intervals of about 3 to 5 mm. One can maintain cultures free of symbionts and also easily observe the integrity of the stolon network. Recording from and stimulating stolons and polyps which arise from them is greatly facilitated in culture colonies, and consequently it is easier to demonstrate interpolyp com- munication. Three examples of such are shown in Figure 3. In record A, record- ing electrodes were placed on two polyps in such a culture colony. One polyp, P2, was stimulated with four shocks in the region on the burst generator. CPs were recorded first in P-2 (the polyp stimulated), which eventually, after the fourth stimulus, were conducted through the stolon to PI where two small CPs were recorded. Po was observed to contract following the first stimulus, PI not until after the fourth stimulus. The DBS was activated in P2 as can be seen by the delayed burst in this record, but the DBFs did not conduct through the stolons to the distant polyp (Pi). In Figure 3B, the polyp stimulated (Po) gave rise to CPs and DBFs, the second burst of DBFs appears to activate the CP system of the distant polyp. While this is not conclusive evidence for an interaction between the DBS and CPS, it is supportive of additional evidence for such an interaction presented subsequently (Rushforth and Stokes, in preparation). There is no evidence that CPs initiate activity in the DBS.
In Figure 3C, recording electrodes were placed on a stolon (S) and a polyp (P) arising from that same stolon. The stolon was stimulated with three stimuli all of which initiated a small (150 //V), fast spike in the stolon which correlated with CPs in the distant polyp. These CPs facilitated to each stimulus. In one case the small spike fired independently of an electrical stimulus, and it too cor- related with a CP in the polyp and contraction of the polyp. DBFs were never initiated by stimulation of the stolon. Repetitive stimulation of a stolon results in a graded spread of excitation involving only the CP system. Spread of excita- tion to some 8 to 10 interconnected polyps in a colony has been observed, polyps closer to the stimulus site showing more CP activity than those more distant.
Effects of MgCl,
The effects of isosmotic MgClo on the mechanical and electrical activity of attached individual polyps are shown in Figure 4, Electrical activity and the visually monitored behavioral responses were first studied in normal sea water and when typically consistent responses were observed, isosmotic MgClo was
D. R. STOKES AND N. B. RUSHFORTH
4 on
t
on
.5mV
5Sec
FIGURE 5. Light induced CP activity (A) and multiple CP activity (B) of Clava following a 15 min period of dark-adaptation. Onset of illumination is indicated by the arrows.
added to the experimental dish until a final concentration of 40% was obtained. The polyps were stimulated in the burst generator region with four shocks at 2-second intervals; the stimulus burst being repeated at 10-minute intervals. Only polyps giving both CPs and DBFs to two consecutive stimulus tests 10 minutes apart in normal sea water were used. A typical record in normal sea water is shown in Figure 4A. Both visual and electrical records were made follow- ing each stimulus regime. The results were similar for five polyps, one from each of five colonies.
Isosmotic MgClo abolishes nearly all CP activity as well as the correlated behavioral responses after ten minutes exposure (Fig. 4B). The DBFs, however, persist in somewhat altered form and increased latency, despite the absence of a behavioral response. The bursts appear to consist of uniformly facilitating pulses of somewhat diminished amplitude from that in normal sea water. Delayed bursts have been recorded for up to 3 hours in MgQ2 without further change. Both CPs and typical DBF bursts are restored following exposure to normal sea water for a brief period (10-30 min ; Fig. 4D).
Effects of light
Whole colonies of Clava respond to sudden sharp increases in illumination after an apparent delay of some several seconds. Polyps contract symmetrically and often in distinct steps in much the same manner as occurs following CP activity. There does not appear to be a systematic co-ordination of polyp contraction throughout the colony ; polyps nearest the source of illumination generally con- tract first, but there is no set order of responses. Synchronized tentacle depres- sion characteristic of DBS activity has not been observed. The latency of the contraction responses coupled with the absence of tentacle depression led to a series of experiments designed to determine whether electrical events result in CP activ- ity, DBS activity, or perhaps an as yet undescribed conducting system.
HYDROZOAN BEHAVIORAL PHYSIOLOGY 199
TABLE 1 1 Responses of dirk-adapted Clava polyps to the onset of illumination, means ± s.e. (N = 5).
|
Number of pulses |
Latency* to first pulse |
|||
|
Before |
During |
After |
||
|
a) Intact polyp Midstalk |
0.3 ± 0.2 |
10.9 ± 2.7 |
0.6 ± 0.3 |
35.4 ± 4.8 |
|
h) Intact polyp Hydranth |
0.8 ± 0.8 |
13.5 ± 2.4 |
1.8 ± 1.8 |
23.4 ± 5.8 |
|
Prox. stalk |
1.0 ±0.7 |
12.1 ± 2.0 |
3.1 ± 2.7 |
24.9 ± 7.0 |
|
c) Transected |
||||
|
polyp Hvdranth |
0.5 ± 0.5 |
6.3 ± 4.0 |
0.6 ± 0.6 |
10.2 ± 1.2 |
|
Prox. stalk |
0.6 ± 0.2 |
11.8 ± 0.7 |
0.8 ± 0.4 |
30.4 ± 6.4 |
* Defined as the time interval in seconds from the onset of illumination to the intitation of electrical activity.
Recordings were made from single Clara polyps of a whole colony which had been dark-adapted for 15 min. The results of these experiments showred that polyps are quite sensitive to light, producing both single (Fig. 5A) and multiple (Fig. 5B) electrical pulses during the initial stages of stimulation. The numbers of pulses recorded from single (midstalk) and dual sites (hydranth and proximal stalk) were determined for a control period of 2 min before light stimulation, and a 2-min period following light stimulation. The light source for all experiments was a 6-V bulb from a microscope lamp set about 15 cm from the preparation. Light intensity was similar throughout. The experimental regime was repeated for a mimimum of five trials with a 15-min period of dark-adaptation between succes- sive light exposures, for five polyps. The results for single recordings for all experiments are shown in Table II. The mean number of pulses recorded during the 2-minute light exposures for all experiments were on the order of 10 to 40 times greater than either before or after the light stimulation period. The mean latencies (defined as the time interval from the onset of illumination to the onset of the electrical response) varied from about 10 sec in the isolated hydranth preparation to 35 sec in stalk recordings. Although most of the electrical activity appears in the form of single or doublets of pulses, they are often produced in distinct groups. The mean number of pulses per group is significantly different for the hydranth and proximal stalk regions (5.1 ±0.8 and 2.0 ± 0.1, respectively). An intermediate value of 3.4 ±0.1 was obtained from midstalk recordings.
Electrical responses to the onset of illumination have been recorded also from isolated tentacles (three of five preparations) isolated hypostomes (three of five preparations) and hydranth preparations from which all tentacles and the hypostome were removed (six of six preparations). We have not examined isolated pieces of stolon.
Addition of MgClo abolishes the characteristic responses to the onset of illumination within a period of five minutes. The only exception occurred in a single isolated hypostomal preparation which produced DBFs that persisted in somewhat modified form for three hr. with no observable behavioral correlate.
200 I). K. STOKES AND N. B. RUSHFORTH
I >ISCUSSION
Evidence from this shulv indicates that the CP system of Claz'a is another example of a fundamental muscle activating conducting system found in hydrozoan polyps. Electrical potentials from this system are correlated with symmetrical contractions of whole, or regions of whole, polyps, and are similar in pulse characteristics and conduction velocity to the electrical potentials correlated with contractions in other hydroids. Such pulses have the common features of long duration (up to 500 msec), relatively large amplitude (up to 15 mV), and slow conduction velocity (2—21 cm/sec) when recorded externally (Rushforth and Stokes, 1978).
Activation of CPs in Clara results always in some degree of symmetrical shortening of the polyp in the direction of the interconnecting network of stolons. The polyp may shorten in one or more steps depending upon the intensity and frequency of stimulation. Following successive contractions, the polyp is reduced to a stubby ball. Furthermore, when contracted, the polyps are shielded between the branches or within the damaged flotation sacs of the alga upon which they naturally occur. These responses are adaptive in that they provide a limited degree of protection for the exposed, softbodied polyp from potentially hazardous environ- mental stimuli. Similar protective functions of polyp contraction are apparent also in Hydractinia where the polyps ultimately contract below a layer of chitinous spines (Stokes, 1974b) ; in Millcpora. where the polyp contracts into a calcified skeleton (deKruijf, 1976 ; and in Obdia where the polyp withdraws into a hydrotheca (Morin and Cooke. 1971a). Protective withdrawal, like the escape responses of insects and crustaceans with giant fiber systems, may be the major role of the CP system in Clara. Behavioral activities such as those associated with prey capture, feeding, and defecation are more complex and are probably integrated by other conducting systems within the polyp. Multiple conducting systems have been physiologically identified in all hydroid polyps thus far examined, though in no case is the behavior totally attributable to known conducting systems. Clara has at least two distinct conducting systems within the polyp. In addition to the CPS, a non-polarized Delayed Burst System (DBS) has been identified which produces programs consisting of bursts of pulses. Such programs are initiated after a- long delay (about 20 sec) following stimulation. The DBS can be dis- tinguished from the CPS by its somewhat higher threshold of activation; resistance to Mg2+ ; restricted location within the hydranth of the polyp ; and different be- havioral correlates (tentacle depression vs. polyp contraction). The potentials produced by the DBS also have different conduction velocity and pulse character- istics. DBPs have shorter duration (50-60 msec), shorter interpulse intervals (170-230 msec), and faster conduction (8-12 cm sec) than multiple contraction pulses ((-/• Table I ).
Though the functional significance of the DBS is not known, there is evidence to suggest that it interacts with the CPS. Tentacle depression together with polyp contraction is observed during the initial delayed bursts following DBS activation. These initial delayed bursts often appear to contain CPs interspersed with the DBPs (Stokes and Rushforth, personal observations). The CP elements in the delaved bursts are absent in the latter phase of a long program of bursts,
HVDROZOAX BEHAVIORAL PHYSIOLOGY 201
when the polyp is reduced to a stubby ball and contractions are no longer apparent. In this contracted state the CP system is not evoked by electrical stimulation. In addition, exposure of a polyp to Mg-+ eliminates contractions, and results in delayed bursts which appear to consist of a single pulse type, presumably DBFs. These observations suggest that the CP system is excited initially by the DBS and polyp contraction is caused in the early phase of a burst program. However, the system becomes increasingly refractory and CPs drop out in the final stages of the program.
Simultaneous recordings from two polyps connected by a stolon support the hypothesis that the DBS excites the CPS. Activation of the DBS by stimulation of the hydranth of one polyp can trigger CPs in the same polyp which then give rise to a CP in a neighboring second polyp. Very small potentials recorded in the muscle-free stolons interconnecting the two polyps correlate with the observed polyp contractions. These pulses may represent activity in nerve cells of an interconnecting nerve net. Nerve cells have been identified in the stolons of Hydractinia (Stokes, 1974a) but have not yet been looked for in Clara stolons. Single or multiple CPs of one polyp can also serve to initiate CPS activity in a second interconnected polyp. However, we have observed no case of CP triggering of DBPs. Interactions of the DBS and CPS in individual polyps and interactions of the CPS from polyp to polyp provide for a means of colonial co-ordination. Foyn (1927) was able to identify members of individual, interspersed colonies of Clava by pinching one polyp and observing which additional polyps contracted. Co-ordinated responses of polyps comprising a colony have been observed also in Cordylophora (Josephson, 1961b) and Hydractinia (Josephson, 1961a; Stokes, 1974b) where they are presumed to be protective and co-ordinated by conducting systems underlying muscle contraction. Clearly all members of the colony would be served by advanced notice of a predator attempting to feed on one member of the colony.
The electrical activity evoked by dark-adapted Clara polyps which occurs after the onset of illumination has certain features of both DBS and CPS activation. The recorded latencies following the onset of illumination to the initiation of elec- trical activity are usually quite long, sometimes on the order of 35 sec. The latencies of DBPs evoked by electrical stimulation are often equally as long. However, despite such long latencies, the following evidence suggests that light activates the CPS ; removal of the burst generator region by transection of a polyp well below the hydranth does not affect the generation of pulses in the remaining proximal stalk region, clearly demonstrating that the burst generator region is not necessary for the light induced responses. Though patterns of light-induced potentials some- times consist of programs of burst, such bursts are closer in pulse characteristics to multiple CPs than DBPs. Frequently the light response consists of sets of widely spaced single or double pulses ; Mg2+ abolishes the light-induced activity and the behavioral responses in the same time course as CPs.
The long latency of the photic responses in Clava is similar to that recorded for Hydractinia (21-70 sec; Stokes, 1972). Such long latencies may result from a similar mechanism of pulse generation and spread of excitation. In fact, it is a contraction pulse system (the SCP) which is activated by light in Hydractinia. The latency of the response may reflect the levels of photosensitive pigments which
J()_> D. K. STOKES AND N. B. RUSHFORTH
have accumulated during the period of dark adaptation (Ballard, 1942). In Clava we have preliminary data showing that there is an inverse relationship between the length of the dark adaptation period and the latency to the response. The light receptors, be they photosensitive pigments or some as yet unidentified photo- receptor, would appear to be widespread throughout a polyp. However, since more pulses are induced in the hypostomal region, than in the body column and base of the polyp, there may be relatively more pigment or more photoreceptors in this region. In Hydractinia electrical activity occurs only by direct photic stimulation of the basal mat (Stokes, 1972). We have not examined the light sensitivity of Clava stolons.
Very little is known of the morphological substrates of the CPS or DBS in Clara. Preliminary histological studies utilizing reduced methylene blue show the presence of nerve cells in the stalk region. The burst generator region and the stolons remain to be examined. However, electrical recordings suggest that CPs are not solely a result of neuronal activity. The electrical potentials are too large and of too long in duration to originate from the small nerve cells. Further- more, they are conducted much more slowly than one would expect for purely neuronal pathways. It has been suggested that similar large potentials from other hydroids are propagated in epithelial sheets via low resistance junctional specializa- tions (Josephson, 1967). Septate junctions have been found connecting epithelio- muscular cells of Hydra (Wood, 1959) and Hydractinia- (Stokes, 1974a) where they have been implicated in contraction responses. Josephson and Macklin (1967) have shown that the CP of Hydra is a transepithelial event. On the other hand, as Mackie (1970) suggests, conduction of such large potentials may combine both neuronal and epithelial elements.
In this study we have shown that the colonial hydroid, Clava squauiata possesses a Contraction Pulse System whose properties are similar to the CP systems of other hydroids. It provides further evidence that the CP system is a common conducting system in hydroid polyps. Studies with other hydrozoans should indicate whether it is a universal feature.
The authors wish to thank Drs. R. Josephson, University of California, Irvine, California, W. Schwab, Virginia Polytechnic Institute, Blacksburg, Virginia, and R. Ritzman, Case Western Reserve University, Cleveland, Ohio, for comments on an early draft of this manuscript.
SUMMARY
1 . At least two conducting systems are present in the colonial hydroid, Clara squamata, the contraction pulse system (CP system) which initiates symmetrical polyp contraction, and a delayed burst system (DBS) which is correlated with tentacle depression and polyp contraction.
2. The CP system has properties similar to contraction pulse systems of other hydroids; its electrical pulses are of large amplitude (greater than 0.5 mV) and long duration (150 msec), and slow conduction velocity (2-3 cm/sec).
3. The CP system courses through the polyps and their interconnecting stolons.
HYDROZOAX BEHAVIORAL PHYSIOLOGY
Electrical stimulation of a single polyp gives rise to CPs associated with contraction of that polyp, which sometimes can be recorded also in adjacent polyps.
4. Isosmotic MgCU abolishes CPs and associated column contractions, but does not suppress delayed burst pulses.
5. Light initiates contractions of the polyp and correlated CPs.
6. It is postulated that the CP system of Clara is similar to contraction pulse systems previously described for other hydroids.
LITERATURE CITED
BALL, E. E., 1973. Electrical activity and behaviour in the solitary hydroid Corymorpha pahna.
I. Spontaneous activity in whole animals and in isolated parts. Biol. Bull., 145 :
223-242. BALL, E. E., AND J. F. CASE, 1973. Electrical activity and behaviour in the solitary hydroid,
Corymorpha pahna. II. Conducting systems. Biol. Bull. 145: 243-264. BALLARD, W. W. 1942. The mechanism of synchronous spawning in Hydractinia and Pennaria.
Biol. Bull.. 82: 329-339. DEKRUIJF, H. A. M., 1976. Spontaneous electrical activity and colonial organization in the
hydrocoral Millcpora (Milleporina, Coelenterata ). Mar. Behar. Physiol., 4: 137-159. DEKRUIJF, H. A. M., 1977. Bursting pacemaker activity in the solitary hydroid Titbularia
solitaria. J. Exp. Biol., 68 : 19-34. FOYN, B., 1927. Studien Uber Geschlecht und Geschlechtszellen bei Hydroiden. I. 1st Clara
sqiiamata (Miiller) eine gonochoristische oder hermaphrodite Art? Arch. Entivick-
hingsmcch Org. (Wilhelm Roux), 109: 513-534.
JOSEPHSON, R. K., 1961a. Colonial responses of hydroid polyps. /. Exp. Biol.. 38: 559-577. JOSEPHSON, R. K., 1961b. Repetitive potentials following brief electrical stimuli in a hydroid.
/. Exp. Biol., 38 : 579-593. JOSEPHSON, R. K., 1962. Spontaneous electrical activity in a hydroid polyp. Coinp. Biochcin.
Physio I.. 5: 45-58. JOSEPHSON, R. K., 1965. Three parallel conducting systems in the stalk of a hydroid. J.
Exp. Biol. ,42: 139-152. JOSEPHSON, R. K., 1967. Conduction and contraction in the column of hydra. /. Exp. Biol.,
47: 179-190. JOSEPHSON, R. K., 1974. Factors affecting muscle activation in the hydroid Tiibularia. Biol.
Bull.. 147 : 594-607. JOSEPHSON, R. K., AND G. O. MACKIE, 1965. Multiple pacemakers and the behaviour of the
hydroid Tiibularia. J. Exp. Biol., 43 : 293-332. JOSEPHSON, R. K., AXD M. MACKLIN, 1967. Transepithelial potentials in hydra. Science,
156: 1629. JOSEPHSON, R. K., AND M. MACKLIN, 1969. Electrical properties of the body wall of Hydra.
J. Gen. Physiol., 53: 638-665. JOSEPHSON, R. K., AND X. B. RUSHFORTH, 1973. The time course of pacemaker inhibition
in the hydroid Titbularia. J. Exp. Biol.. 59 : 305-314. JOSEPHSON, R. K., AXD J. UHRICH, 1969. Inhibition of pacemaker systems in the hydroid
rnbnlaria. J. Exp. Biol.. 50 : 1-14.
MACKIE, G. O., 1968. Electrical activity in the hydroid Cordylophora. J. Exp. Biol.. 4 : 387-400. MACKIE, G. O., 1970. Neuroid conduction and the evolution of conducting tissues. Q. tier.
Biol., 45 : 319-332. MORIN, J. G., AND I. M. COOK, 1971a. Behavioural physiology of the colonial hydroid Obelia.
I. Spontaneous movements and correlated electrical activity. /. Exp. Biol., 54 : 689-706.
MORIX, J. G., AND I. M. COOK, 1971b. Behavioural physiology of the colonial hydroid Obelia.
II. Stimulus-initiated electrical activity and bio-luminescence. /. Exp. Biol., 54: 707-721.
MORIN, J. G., AND I. M. COOK, 1971c. Behavioural physiology of the colonial hydroid Obelia.
III. Characteristics of the bioluminescent system. /. Exp. Biol., 54 : 723-735.
204 D. R- STOKES AND N. B. KUSHFORTH
PASSANO, L. M., AND C. B. McCuLLOUGH, 1962. The light response and the rhythmic potentials
of Hydra. Proc. Natl. Acad. Sci. U.S.A., 48 : 1376-1382. PASSANO, L. M., AND C. B. McCuLLOUGH, 1963. Pacemaker hierarchies controlling the
behaviour of Hydra. Nature, 199 : 1174-1175. PASSANO, L. M., AND C. B. McCuLLOUGH, 1964. Coordinating systems of behaviour in
H\dra. I. Pacemaker systems of the periodic contractions. /. R.rp. Biol., 41 : 643-
664. PASSANO, L. M., AND C. B. AlcCuLLOUGH, 1965. Coordinating systems and behaviour in
Hydra. II. The rhythmic potential system. /. E.rp. Biol., 42: 205-231. RUSHFORTH, N. B.. 1966. An analysis of spontaneous contraction pulse patterns in Hydra.
Am. Zool., 6 : 524. RUSHFORTH, N. B., 1971. Behavioral and electrophysiological studies of Hydra. I. Analysis
of contraction pulse patterns. Biol. Bull., 140 : 255-273. RUSHFORTH, N. B.,, AND D. S. BURKE, 1971. Behavioral and electrophysiological studies of
Hydra. II. Pacemaker activity of isolated tentacles. Biol. Bull.. 140: 502-519. RUSHFORTH, N. B., AND D. R. STOKES, 1978. Contraction pulse systems in hydroids. Am. Zool.
18: 605. SPENCER, A. M., 1974. Behaviour and electrical activity in the hydrozoan Proboscidactyla
flavicirrata (Brandt). I. The hydroid colony. Biol. Bull., 146: 100-115. STOKES, D. R., 1972. Functional organization of conducting systems in the colonial hydroid
Hydractinia echinata Fleming. PhD thesis, University of Hawaii, Honolulu, Hawaii,
319 pp.
STOKES, D. R., 1974a. Morphological substrates of conduction in the colonial hydroid, Hydrac- tinia echinata. I. An ectodermal nerve-net. /. E.rp. Zool., 190 : 19-46. STOKES, D. R., 1974b. Physiological studies of conducting systems in the colonial hydroid,
Hydractinia echinata. I. Polyp specialization. /. Exf- Zool., 190: 1-18. WOOD, R. L., 1959. Intercellular attachment in the epithelium of Hydra as revealed by electron
microscopy. /. Biophys. Biochcm. CytoL, 6 : 343-352.
Reference: Biol. Bull. 157: 205-220. (August 1979)
AN ANALYSIS OF POPULATION STRUCTURE IN PACIFIC MOLE CRABS (HIPPA PACIFICA DANA)
ADRIAN M. WENNER AND CRAIG FUSARO
Marine Science Institute. University of California. Santa Barbara. California 931t>t>
Hippa pacifica Dana, a hippid mule crab, inhabits the intertidal zone of tropical and subtropical Pacific island beaches. At Oahu, Hawaii, it primarily scavenges Physalia, the Portuguese man-of-war which washes onto beaches from the open ocean (Matthews, 1955; Wenner, 1977). At Enewetak Atoll. Marshall Islands, although it sometimes receives an abundance of Physalia (S. Smith, personal communication), the mole crab apparently depends more upon lagoon-produced mysids and other zooplankton which wash ashore at night (Wenner, 1977).
A comparison of population samples in this species, both in Hawaii and at Enewetak, provided some unexpected observations. In Hawaii, samples of Hippa pacifica suggested a remarkably consistent population structure. During a 5- nionth period, samples were essentially identical to one another, whether obtained from different beaches or from different parts of the same beach on Oahu. How- ever, the consistent results obtained in Hawaii did not hold for extensive sampling at Enewetak Atoll. Samples from populations on different beaches at that atoll showed wide differences when sex ratio was analyzed as a function of crab size.
Wenner (1972) earlier had examined the question of sex ratio and size for many marine crustaceans. At that time he conjectured that the several distinct patterns found might be species-specific, on the basis of data available. However, this hypothesis failed with the initial comparison of data from Hawaiian and Enewetak mole crab samples. Hawaiian population samples fell into what had been termed an "intermediate" pattern, whereas the first year's samples at Enewetak formed the "reversal" pattern found in protandrous species such as the pandalid shrimps studied by Butler (see Wenner, 1972; Fig. 8).
The second year of sampling at Enewetak yielded conflciting data among various islets and led to a reconsideration of the premise that the sampling method provided representative data, even though this discrepancy among samples was not initially obviously related to sampling method (the same procedure had been used in all cases). To test the efficacy of sampling technique, a beach of limited extent was sampled at Enewetak in the usual manner, except that animals were not returned as had been done earlier. Instead, crabs were removed during a 3-week period until few or no crabs came to the individual bait stakes (sampling with removal). Initial samples, which represented the normal sampling procedure, could then be compared with the larger segment of population removed from the beach. The question became : In what ways did the initial samples represent the larger beach population ?
MATERIALS AND METHODS
The study site at Enewetak Islet was a 250-m long beach which terminated on its eastern end at a solid-walled cargo pier and tapered in the other direction to
205
206 V M. WENNKH AND C. FUSARO
a narrow strip of sand among concrete blocks and limestone rubble. Much of the western half of the beach fronted a limestone reef flat. At low tides the sand on that portion of the beach bordered on exposed coral reef.
During a 3-week period in 18 separate sessions (1-2 hr each), a total of 4011 animals was removed from the beach with the use of a baiting procedure described fully by Wenner (1977). Stakes baited with shark meat and placed approximately 10 m apart along the entire stretch of beach during each sampling session attracted the crabs. Thirty to forty stakes were kept in the sand for approximately 20 min and were reset two or three times more during each session.
Mole crabs, apparently reacting to chemical stimuli (Matthews, 1955), scurried toward the bait as it was repeatedly covered by wave action. Successive sets of bait during each sampling session usually yielded progressively fewer animals per bait stake. By the end of each session, even fresh bait placed between the sampling stakes did not generate an increase in catch rate.
Animals were usually collected during the mid-point of an outgoing tide, because that appeared to be the optimal time (on the basis of earlier experience). They were occasionally collected during other parts of the tidal cycle in order to sample sand patches covered by water only at such times. Such attempts, how- ever, never yielded as many animals per unit effort as did those run during out- going tides. Accordingly, in some comparisons samples were grouped by twos in order to offset those small numbers and to better reveal trends.
After animals were sieved from the sand, they were measured with the aid of an automatic sizing device (Wenner, Fusaro, and Oaten, 1974). Tallies included size, sex, and percentage of females carrying eggs within each size class. Females were returned alive to beaches at the opposite end of the islet. Males were kept in a sea water table and evenutally returned alive to the original beach as part of another experiment.
RESULTS Changes in catch during removal
Animals, when considered by sex and size, did not come uniformly to the bait during the 3-week period. The overall catch of males fluctuated until mid-way through the program and then began tapering off (Fig. 1A). By contrast, female catch was greatest at first, with a rather consistent decline thereafter (Fig. IB). Small females (those equivalent in size to the male size range — solid bars in Fig. IB) showed much the same catch pattern as males, however, indicating that size rather than sex was the factor responsible for the differences in catch pattern between males and females.
One prominent feature of the change which occurred during the removal pro- gram was the selective catch of large females during the early part of that period (Figs. IB, C). More than 80% of the 378 females caught in the first two samples were greater than 12.4-mm carapace length (the mean minimum size of egg-carrying females). In the last six samples, however, fewer than 18% of the females caught were at least that large (N -- 319).
The percentage of those females larger than 12.4-mm carapace length which carried eggs also varied at the removal beach during the 3-week period (Fig. ID).
POPULATION STKIATURK IX MOLE CRAMS 207
In the first two samples combined, 64.2%) of the 307 larger females carried eggs, whereas 85. 0% of the remaining number of larger females had eggs (N == 860). A 9 by 2 x- test for homogeneity failed (x2 == 77.00, P < 0.001), indicating that the percentage of egg-carrying females changed significantly during the 3-week period. Since egg development time is approximately 20 days (unpublished results), such a change with time would not be unexpected.
Results from the other two islets which had been repeatedly sampled yielded somewhat more consistent data. At Jedrol (David) Islet, where the removal pro- gram was started later and run only 10 days, the percentages of large females in berry for the five samples were 87.2, 85.7, 86.0, and 83.6%, respectively, with data for the last two samples combined to eliminate small sample error (N -- 376, 434, 150, and 116, respectively). Those percentages did not show a significant change with time (x2 -- 1.05, P < 0.05). At Boken Islet, two samples taken 6 days apart had 89.3 and 84.2% of the larger females in berry (N -- 337 and 505, respec- tively), a marginal significant difference (x2 ; 4.53, P < 0.05).
Sex ratio fluctuated more markedly than any other variable measured during the removal program. The first three of the 18 samples yielded a total of 923 animals, with each of those three having a low percentage of males (27.7, 28.2, and 30.1%, respectively). In later samples the percentage of males varied widely, from a low of 40.6% males (fifth sample) to a high of 78.8% males (last sample). However, a persistent upward trend in that percentage became evident when the percentage of males was considered as a function of cumulative number of animals caught (Fig. IE). Beginning with the fourth sample, the overall percentage of males increased uniformly. After 4011 animals had been collected, 48.4% of them were males. When those data were grouped into four blocks of 923, 1127, 896, and 1065 animals, a 4 by 2 x2 test yielded a value of 234.36 (P < 0.001). When the same test was run with the first group omitted, the data remained heterogeneous (x2 = 113.00, P < 0.001). This level of significance, coupled with the close fit of the line to the points, indicates that males were under-represented in the first group of three samples and over-abundant in the last group of 1065 animals (last seven samples combined).
It was after the trend shown in Figure IE began to emerge that a total of 1679 animals was removed from the beach on Jedrol (Rex) Islet, where a parallel set of results emerged (Fig. IF). Unfortunately, time did not permit additional sampling at that islet.
Two samples from Boken (Irwin) Islet were also obtained. The male per- centage there rose from 1.7 to 6.4% during removal of a total of 346 and 456 animals, respectively. Boken Islet differs from the other two in being downwind from the lagoon waters during trade wind conditions and is, presumably, in a more food-rich location (Fusaro, 1978b; Wenner, 1977).
Estimates of population size
Since catch per unit effort generally declined during removal trapping, and since rate of decline is directly related to size of total population and to number removed, the total population size can be estimated by various methods (South- wood, 1966, pp. 181-186). However, not all of the four conditions as listed by
208
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FIGURE 1. Changes in population samples during the three-week removal program. The bars depict sequential male (A) and female (B) catches, with the 18 samples grouped in pairs. Hours spent collecting each pair of samples were : 2, 2, 2, 2.25, 4, 2.5, 2.5, 3 and 3, respectively. The dashed line in Figure 1A indicates a consistent decline in catch per unit effort starting midway through the removal program; the solid portion of each bar in Figure IB represents small females (females <[12.5-mm carapace length, the mean maximum size of males). The line in Figure 1C shows the decline in percentage of females which were large (females ;>12.4-mm carapace length, the mean minimum size at onset of egg-bearing), while Figure ID illustrates the differences obtained in egg-bearing percentages for those larger females during
POPULATION STRUCTURE IX MOI.K CRABS
TABI.F I
Various estimates of the number of animals inhabiting the study beach on Eneivetak Islet, determined by different methods based upon changes in catch pattern during removal sampling.
|
Estimates of total population size |
||||
|
males |
Total females |
Small females |
Total animals |
|
|
Method |
||||
|
Max. likeli- |
||||
|
hood |
||||
|
(±s.e.) Regression "Corrected |
9575 (±2996) 8661 |
2374 (±351 2532 |
6091 (±6182) 4859 |
6279 (±208) 6539 |
|
Regression" Time-Unit |
2661 2967 |
2389 2223 |
1271 1518 |
4658 4752 |
|
1 : 1 Sex ratio |
2250 |
2250 |
4500 |
|
|
Total caught |
1940 |
2071 |
1008 |
4011 |
Southwood (1966) were met in this present study. One condition in particular, "The chance of being caught must be equal for all animals" was clearly violated ; males and small females were not caught as readily in the first eight sessions as were large females (first four bars in Figs. 1A, B). Nevertheless, a use of the various methods did provide a set of estimates (Table I), from which inferences can be drawn. In each case, estimates were made independently from the data for males, females, small females, and total number of animals, on the basis that the catch patterns differed markedly from one another in those different categories.
The method based on maximum likelihood, which Southwood (1966) called "the most accurate method", yielded the least consistent set of results. The male estimate (9575 ± 2995) exceeded the estimate for total number of animals (6279 ± 208). Likewise, the calculated number for small females exceeded the number estimated for all females.
Use of the regression method initially provided the same lack of consistency as that yelded by the maximum likelihood method ; estimates for the number of males and small females were far too high to correspond with the estimates for total numbers of all animals or of all females. By inspection of Figures 1A and IB, on the other hand, such a result would be expected, since males and small females clearly began a consistent decline in numbers only after the first eight samples (first four bars in those figures) had been removed from the beach.
Alternatively, one can adapt the regression method by applying it to the data
sequential removal. In both Figures 1C and ID a weighted mean (x) for the last three paired points compensates for small sample variation. The line in Figure IE represents the percentage of males as a function of cumulative number of animals caught in the 18 samples at the main removal beach, while the line in Figure IF shows the same relationship for animals taken from Jedrol Islet.
210 A. M. VVENNEK AND C. FUSARO
for the last 10 samples (last five bars). The estimates derived in each case can l)e added to the numbers caught in the first eight samples. Serious inconsistencies then disappear ("corrected regression" in Table I).
The "time-unit" method for estimating population size (Kono, in Southwood, 1966) relies heavily on data obtained at only three times (as well as on the cumulative catch at those times) : the first sample, the mid-point sample, and the last sample. An application of that method to the data yielded estimates relatively close to those produced by the "corrected regression" method, with males and small females again being somewhat over-estimated because of their under-representation in the first sample.
Finally, if one assumes a 1 : 1 sex ratio for the population (the megalopa stage of Emerita analog a arrives on the beach in a 1:1 sex ratio, Wenner, 1972), an extrapolation of the line in Figure IE to the 50% mark would yield an estimate of about 4500 animals. The 2250 females so estimated (assuming a 1 : 1 sex ratio) closely matches each of the other estimates for the total number of females.
The various estimates shown in Table I, qualified by the nature of the violation of conditions outlined by Southwood (1966), would indicate that most of the animals were removed from the study beach (between 87% and 93 %, particularly if one relies on the quite consistent set of results for the total number of females).
The foregoing analysis now permits an assessment of how well the initial samples represented the larger beach population.
Modal size classes
The data for all samples combined fell into discrete modal size classes (Fig. 2), when separated by the method outlined by Cassie (1954). Data for males separated cleanly into only three modes, with the third mode having a slight inflection above the 95% level. The steep slope of Mode 3 suggests a maximum size for that sex. Female data actually fell into five modes. However, since the third and fourth modes did not differ appreciably, those data were combined and were thereafter treated as Mode 3 (Figs. 2, 3). The data thus reveal that the beach likely experienced four or five periods of recruitment during the year or two prior to sampling. (It should be noted, however, that the mode of largest females is based upon only seven animals caught during the first three sampling periods.)
To illustrate some of the differences in modal size class structure which arose during the removal process, data from the first three samples (N = 923) are herein compared to data obtained from the last six samples (N = 876). In all cases, modes were again separated by the same method.
Females in the first three samples (N — 657) fell primarily into the four or five modes mentioned earlier (Fig. 3A), but females in the last three samples (N = 319) provided data for only three distinct modes (Fig. 3B). The large displace- ment in the two sets of lines in Figure 3 does not represent an appreciable change in the size of animals within each mode. Rather, the displacement reflects a change in relative percentage of females which fell into each of the three modal size classes when one compares the beginning and end of the 3-week sampling period. For example, as can be seen in Figure 3, most females in the first three samples were in the third mode (79% of 657 females), whereas the second mode contained
POPULATION STRUCTURE IN MOLE CRABS
211
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FIGURE 2. Male and female modal size classes for all data (N = 4011). Males (broken lines) fell into three distinct size classes when modes were separated by the method of Cassie (1954). Females (solid lines) continued growth beyond the maximum male size. Approxi- mate numbers within each mode are indicated below each line representaing male modes and above each line representing female modes.
most of the females in the last six samples (72% of 319 females). Thus, t\vn facets of modal size class structure should be distinguished : first, the estimated mean size of animals within each mode and second, the relative percentage of animals within each mode.
The data derived from the captured males (Fig. 4) differed from that for females in some important respects. Only two male modes occurred in the first three samples combined (Fig. 4A), while four or five females modes were evident in those combined samples (Fig. 3A). The smallest and largest females had no counterpart among the males in the first three samples. However, the last six
212 A. M. WENNER AND C. EUSARO
99.9
CO
UJ
99
S 95
LU U_
h-
LU 70
01 50
LU
°" 30
LU
> 15
h-
3 5
r>
0 I
X = 15.6 ± 0.78
(20}
22.8 ± 1.18
_J I I ' -
4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
SIZE CLASS (mm)
FIGURE 3. Changes in cumulative percentage of females as a function of size during removal. Data for the first three samples combined (open circles) fell into at least four modes (A). Data from the last six samples (closed circles) formed only three discrete modes, displaced upward on the graph due to a later catch of a higher percentage of smaller females (B). The x symbols indicate the relationship for all female size data (N = 2071). The sets of numbers below the lines indicate means, standard deviations, and approximate number (in parentheses) of animals within each mode in Figure 3A, and the numbers above the lines represent the same characteristics for Figure 3B. (See Fig. 2 for separation of modes for the total female data.)
samples yielded three modes for the males (Fig. 4B). modes which could be matched quite readily with the three modes representing smaller females in Figures 2 and 3B.
On the other hand, data for males obtained in the first three and in the last six samples (Fig. 4) did not show that same parallel relationship found in the female data (Fig. 3). The lines representing the upper mode for males in first and last samples converged. S. R. Haley (personal communication) and M. Page (personal communication) have concluded from laboratory results that males reach a maximum size and cease growth, though they continue molting. Females in the laboratory, according to Haley and Page, showed no such cessation of growth under similar circumstances.
The relative percentage of males which fell into each mode also differed markedly in the first and last samples (Fig. 4). The upper (third) mode in the first three samples contained approximately 75% of the 266 males, while 6\c/( of the 557 males caught in the last six samples were in the second mode.
POPULATION STRUCTURE IN MOLE CRABS
213
CO
999 -
99 -
95
85 70 50
LJ O
LJ >
30
i= 15
=> 5
o
I
O.I
X = 12.2 ± 0.47 (139)
9.2 ± 1.39
12.4 ± 0.48
B
3 45 6 7 8 9 10 II 12 13 14 SIZE CLASS (mm)
FIGURE 4. Changes which occurred in cumulative percentages of males, as in Figure 3 for females. Initial samples (A) yielded only two modes, as against the four to five modes for females. Final samples (B), however, resulted in three discrete modes for males. The convergence of the two lines representing the largest males agrees well with the concept of of a maximum size reached by that sex. Means, standard deviations, and sample sizes are as shown in Figure 3.
Sex ratio curves
An earlier analysis (Wenner, 1972) revealed that sex ratio may vary with size in marine Crustacea. At the time, such variation was believed to form a pattern characteristic of a species or population. It is clear from more recent results obtained from mole crabs both in Hawaii and Enewetak that the "characteristic pattern" hypothesis is now untenable. The data published in 1972 indicated an "intermediate" sex ratio pattern for Hippa pacific a in Hawaii, but data gathered later at Enewetak in 1972 yielded a "reversal" sex ratio pattern for the same species (see Fig. 5 A).
'14
A. M. WENNER AND C. FUSARO
100
(J4ff)
80
CD UJ
60
UJ
o
UJ CL
40
20
(/V--/9) o
o o
\
l° A
4 56 78 9 10 II 12 13 14 15 16 17 18 SIZE CLASS (mm)
FIGURE 5. A discrepancy between sex ratio curves (sex ratio as a function of size— Wenner, 1972). A sigmoid curve ("reversal pattern") was obtained in 1972 when approxi- mately 2000 animals were collected from six beaches on five different islets (A). A new pat- tern ("oscillation pattern") emerged when 4011 animals were removed from a single beach in the present study (B). Out of several hundred small animals in 1972, only one was female (the first point in Fig. 1A).
Data for initial samples in the current study yielded neither the Hawaii nor the earlier Enewetak sex ratio patterns ; a sex ratio pattern existed which had not been found earlier (Fig. 5B). Instead, the percentage of males oscillated with increase in crab size. It is further evident that the number of oscillations cor- responded well with the basic number of modal size classes found for males and females in the comparable size ranges (i.e.. Fig. 2).
The "oscillation" sex ratio pattern is what one might expect if the following conditions apply : first, the population consists of different cohorts which have arrived at different times from the plankton (three recent cohorts in this case), second, one sex grows faster than the other (females in this case — see Haley, 1979), and third, one sex reaches a maximum size, while the other sex continues growth beyond that size (females in this case).
For the first three samples of the present study, the sex ratio curve formed by the data for all animals greater than 10-mm carapace length nearly exactly matched that same portion of the curve for the total number of animals collected, as seen in Figure 5B. For the 86 animals in the first three samples which were 10 mm and smaller, however, only 28% were males, a point which falls far below
POPULATION STRUCTURE IN MOLE CRABS J15
any of the points on the curve for all animals. This result corresponds with the under-representation of males as seen in Figure 4.
Despite the large discrepancy between sex ratio curves at the two localities and in different years, it should be noted that the sigmoid portions (for animals larger than 12 mm carapace length) of the curves in Figures 5 A and 5B differ from one another by only about f-mm carapace length at the 50% level in the graph. The mean maximum size of males (12.5-mm carapace length) also falls close to that same level. It is also noteworthy that an analysis of Hippo, cubcnsis data (from Hanson, 1969) places that portion of the curve for his species between the two curves shown in Figure 5.
Size at onset of egg production
The mean minimum size of egg production can be a useful measure in popula- tion studies if one wishes to compare the success of animals which live in one habitat with the success of animals which live elsewhere (Wenner, Fusaro, and Oaten, 1974). It is essential, however, that initial sample data accurately repre- sent the entire population before one makes that comparison. (In decapods the mean minimum size at which females can extrude eggs is one convenient measure of sexual maturity, provided conditions are optimum for egg production, since eggs are usually retained on the pleopods until they hatch.)
Unfortunately, samples of Hippa pacifica populations in both Hawaii and at Enewetak taken in earlier years did not permit a determination of the mean minimum size at which egg-bearing occurred because small females were seldom caught. The previous sections document one possible reason for such a failure- samples taken early in the sampling period did not include an accurate representa- tion of small females present in that population.
The removal of a larger percentage of the population in the present study pro- duced a sizeable number of smaller females and permitted the derivation of a curve which represented the mean minimum size of egg production for this particular beach (x= 12.4 ± 1.02 mm; N = ca. 1000). The curve had a striking simi- larity to the shape of a comparable curve published earlier for Enierita analoga (Wenner, Fusaro, and Oaten, 1974).
A question remained as to whether initial samples would indicate a different size at onset of egg production than would data for the population as a whole. Consequently, data for the first three samples combined were compared to the data for the total 3-week catch. Although the available sample size for the early data was quite small (and although more scatter existed among the points), it was evident that a marked difference did not exist between early samples (x = 12.7 ±4.64 mm; N -= ca. 150) and total data in this comparison. Only 0.3-mm carapace length difference existed between means for best-fit lines from all data compared to data for only the first three samples.
DISCUSSION
Pacific mole crabs (Hippa pacifica) apparently live only in the intertidal zone (Wenner, 1977) ; the same habit was reported for Hippa cubensis by Hanson
216 A. M. WENNER AND C. FUSARO
(1969). (However, see Borraclaile, 1906.) This restricted habitat, together with a behavior of readily coming to bait, makes these animals particularly suitable for studies of crustacean biology. Large numbers of these carnivores can be collected in a relatively short time. In addition, the animals can be measured quite rapidly while still alive by means of a graded sieve (Wenner, Fusaro, and Oaten, 1974). In the present study, these combined attributes permitted measurement and removal of a large percentage of Pacific mole crabs from a beach limited in length. From population estimates (Table I), based on changes which occurred during sampling (Southwood, 1966), it would appear that approximately 9090 of the animals which inhabited that short stretch of beach on Enewetak Islet, Enewetak Atoll had been removed.
Some of the contrasting results between first and last samples were unexpected, in the sense that earlier (1970-71) samples had yielded repeatable data. At that time samples from different beaches or islets were very similar, and repeated samples (sampling with replacement) from the same beach matched one another. In retrospect, one might conclude that the sampling bias could have been antici- pated, since animals were caught by "trapping" (e.g., Gilbert, Gutierrez, Frazer, and Jones, 1976).
Trawling for animals can apparently lead to problems similar to those posed by trapping with bait. Gotshall (1972), while sampling shrimp during a 4-year period, experienced a bias problem similar to that encountered in the present study.
Small animals were caught less frequently at first and were primarily males ; this discrepancy led to an imbalance in sex ratios throughout the sampling pro- gram. Hanson (1969; p. 15) earlier found such a discrepancy in his sampling program with Hipf>a ciibcnsis. He wrote: "Length-frequency distributions of the (trapped animal) samples showed close conformation to those obtained by the more exact procedure of sieving sand samples from different levels of the beach, except for the smallest crabs (4-8-mm carapace length). Only 17.8</o of the Bellair (trapped) samples were in this size range while 39.2% of the crabs from the Paynes Bay sieved samples were in this range." By contrast, Hanson derived an overall figure of 48.3% males for the animals from the several Paynes Bay beaches from initial samples, identical to the percentage we finally obtained only after removing more than 4000 animals from one beach. In addition, the first few samples in this present study were remarkably similar to one another, and by then 23% of the total catch had been removed. It was not until the fourth sample that a marked change in catch pattern occurred.
Although some changes during sampling were appreciable, the overall effort provided a rare opportunity : comparing all of the data from the removal beach with data obtained from the first few samples.
In studies of crustacean populations, a number of measures can assess just how discrete populations differ from one another in response to differences in environmental influences, but one must first have some confidence that initial samples have provided an accurate estimate of population characteristics before comparing populations with one another. Among the measures one can use are : first, modal size classes, including number of each sex in each size class and per- centage of animals within each mode ; second, size at onset of egg production (Wenner, Fusaro, and Oaten, 1974) ; third, extent of egg production, including
POPULATION STRUCTURE IX MOLE CRABS 217
percentage of females carrying eggs and number of eggs per given size of female ; fourth, "instantaneous" growth rate (Fusaro, 1978a), including field molt rate and size increment at molt; and fifth, sex ratio patterns (Wenner, 1972), including shape variations and pattern displacement. Age is generally not used, since that measure is very difficult or impossible to determine directly for crustaceans in nature (Wilder, 1953).
This current study has shown that initial samples provided reliable data for most of the above measures, but not all aspects, as discussed below.
Overall, the percentage of males and females within the different modes in the first three samples did not accurately represent the population structure obtained during the entire sampling program (Figs. 3, 4). Neither small males nor small females were properly represented in those first samples. Also, the seven large females caught at first were apparently the only very large animals on the beach.
However, although the percentage of males and females within each mode changed drastically during removal, the mean size of animals within each mode did not change appreciably (Figs. 3, 4). This means that population samples for this species can be compared to one another through time, if one takes into account the fact that the absolute percentages can vary greatly within each mode.
The first three samples yielded a reliable estimate for mean minimum size of egg production (Wenner, Fusaro, and Oaten, 1974), when compared to that estimate obtained in later samples or to that obtained from the total data (12.4-mm carapace length). Hanson (1969) found a 15-mm carapace length for the same characteristic in Hip pa citbcnsis.
It is not yet certain whether the percentage of mature females which bears eggs is reliably determined from initial samples, but this seems to be the case. At the primary removal beach (Fig. ID), an estimate derived from the first two samples (64.2%) was significantly lower than that obtained from all remaining samples (85.0%). However, data obtained from Jedrol Islet were quite consistent; the percentage of larger females (females greater than 12.4-mm carapace length) bearing eggs did not change during removal. It is therefore possible that an influx of food had occurred just prior to the beginning of sampling at the removal beach on Enewretak Islet (see Wenner, 1977, Table I). If so, it may be that the rising percentage of ovigerous females reflected that particular energy input.
At the removal beach, the sex ratio pattern (sex ratio as a function of size) obtained from initial samples quite clearly did not characterize the species or population as suggested earlier (Wenner, 1972, p. 344) for at least two reasons: first, small animals did not come to the bait in proportion to their numbers in the population when removal was begun ; and second, it is now apparent from other work that both shape and position of the sex ratio curve can differ between popula- tions and at different times of the year (see Fusaro, 1977).
A striking contrast between various Hawaii results (Wenner, 1972, Fig. 10; and Haley. 1979, Fig. 2) and Enewetak data (Figs. 5 A, B, this study) can now be reconciled. The "intermediate" (Wenner, 1972, Fig. 10), "reversal" (Fig. 5A, this study), and "anomalous" (Haley, 1979, Fig. 2) sex ratio curves obtained from initial samples at various times likely did not accurately represent that aspect of population structure for the small animals. Rather, the differences be- tween these patterns in the lower size classes probably reflect the degree to which
218 A. M. WENNER AND C. FUSAKO
small animals had not been captured or the degree to which they might not have been seen during hand catching (Haley, 1979).
Three out of four of the previously described types of sex ratio patterns (Wenner, 1972) have thus now been obtained for a single species, apparently reflecting both sampling bias and changes in population structure. It is further apparent that each of those curves probably represented part of yet another sex ratio pattern (an "oscillation" pattern, Fig. 5B), a pattern which has now also been found for a confamilial mole crab, Emcrita analoga (Fusaro, 1977). Further- more, the number of oscillations in that pattern matches the number of major modal size classes found for males and small females (see Fig. 2), oscillations which may represent the number of major influxes of young in the recent months or years, modified by a differential growth rate between sexes (Haley, 1979).
In retrospect, it would appear that points which deviate from the sex ratio curve for other crustaceans (see Fig. 11 for Calcinus latciis in an earlier analysis— Wenner, 1972) could well represent real deviations.
The results of this study indicate that males reach a maximum size in this species, a parameter which could be quite valuable for comparing populations one with another. In such comparisons, however, the largest male which can be found in each population is not the best estimate of that parameter (the "largest male size" normally increases with sample size). Rather, one can use the 50, 95, or 99% level in the mode representing the largest males. In Figure 2 the cor- responding values would be: 12.5 ± 0.59, 13.8, and 14.3, respectively. Statistical comparisons between populations are feasible, of course, only if one uses the first of these estimates (because one then has an estimate of standard deviation).
The initial three samples provided an accurate estimate of mean maximum size of males when compared to the data for all males. The first three samples combined yielded a mean and standard deviation of 12.4 ± 0.48 mm carapace length (N = 200) ; the remaining large males had a comparable mean of 12.5 ± 0.52 mm (N = 479).
The fact that males reach a maximum size while females continue growing be- yond that size provides a related measure for comparing populations : an arbitrary point on the sigmoid portion of the sex ratio curve, where females first become dominant in the larger size classes (13.7 ± 0.47 mm at the 50% level in Fig. 5B). Since these sigmoid portions of the sex ratio curve are straight lines on probability paper, one can derive an estimate of the mean and standard deviation for that 50% transition point. This transition size was also quite accurately determined from intial samples. The first three samples yielded values of 14.0 ± 0.73 mm carapace length (N = 393), compared to the overall value shown above of 13.7 ± 0.47mm (N == 745).
Southwood (1966) outlined methods by which some systems might be sampled so as to reduce the amount of bias inherent in estimates of population structure. Gilbert, Gutierrez, Frazer, and Jones (1976, p. 57) updated the approaches outlined and admonished : "The sample must represent the defined 'population' of animals or plants we wish to investigate." The concern of these workers has been supported only in part by the current study. Initial samples did accurately represent some aspects of population structure. It should also be stressed that researchers who would like to understand crustacean biology better are not merely "choosing suit-
POPULATION STRUCTURE IN MOLE CRABS 219
able species for ecological work" (Gilbert, Gutierrez, Frazer, and Jones, 1976, pp. 58, 59). Marine forms can often be readily caught only by some form of trapping (i.e., bait, nets, etc.). Although there is perhaps no way to know if initial samples contain a bias without going through a removal process, it is also obvious that a bias can be tolerated once one knows its nature.
Other researchers have reported similar discrepancies. In an extreme example, Bolin (1961 ) found that plankton hauls caught only female lantern fish (Tarleton- bcania crennlaris}, while albacore tuna stomachs contained males but no females. The discrepancy became partially resolved when an unexplained event caused a mas- sive death, resulting in millions of these fish being washed onto the beaches in central California. Of 521 specimens examined, 43.25% were males. Bolin speculated that males, being faster than females, could escape plankton nets. Conversely, he felt that females, having no photophores, would not be seen by tuna at that depth and could escape that mode of predation.
In a less extreme but still significant example, Gotshall (1972), who found marked discrepancies in catches of Pandahis jordaui with respect to some year classes (size classes), suggested possibilities for those anomalous results, and re- viewed some similar problems encountered by other researchers.
In the present study, although initial samples did not accurately portray a few of the conventional measures used by population biologists, reasonable esti- mates were gained of several parameters, as outlined above. The present study thus demonstrates the value of comparing data obtained from initial samples with actual population structure, especially when a biased sampling technique is the only feasible way to capture a sample from a population.
We thank S. Smith and E. S. Reese of the Mid-Pacific Marine Laboratory for providing space, facilities, and financial support. Some of the work was supported by a faculty research grant from the University of California. We also thank P. and J. Lambertson and P. Allen for their assistance at the laboratory, as well as P. Lewis and C. Akers for typing the final draft of the manuscript. T. Ebert, B. Fitt, S. R. Haley, P. Leviten, E. Noble, A. Oaten, M. Page. H. Wells, and P. Wells provided helpful comments on the manuscript.
SUMMARY
1. Essentially all of a population of mole crabs (Hip pa pacifica Dana) was removed from an isolated beach at Enewetak Atoll, Marshall Islands. Estimates of poplation size, based on catch pattern, indicate that 87 to 93 9e of the animals were bait-trapped out of their restricted habitat. The sequential trapping permitted a number of comparisons between initial sample data and actual population structure.
2. The first three samples were large and yielded highly consistent data when compared to one another, but some aspects of population structure were nonethe- less non-representative. For example, the percentage of crabs within each modal size class in the total population differed markedly from that estimated by initial samples. The location of those modal size classes did not change during the removal process.
J20 V M. WENNER AND C. FUSARO
3. After the first three samples (where male percentages were about 29%), sex ratio fluctuated wildly in individual samples, apparently as a consequence of size discrepancy in arrival at the bait. Eventually, males comprised about 48.4% of the 401 1 animals removed.
4. Several other aspects of population structure were accurately estimated in early samples, including : mean size at onset of egg production, percentage of mature females carrying eggs, the sex ratio-size class relationship (for larger crabs), and the mean maximum size of males.
LITERATURE CITED
BOLIN, R. L., 1961. The function of the luminous organs of deep-sea fishes. Proceedings of
the Ninth Pacific Science Congress (1957), 10: 37-39. BORRADAILE, L. A., 1906. The Hippidea, Thalassinidea and Scyllaridea. Pages 750-754 in
J. S. Gardiner, Ed., The Fauna and Geography of the Maldivc and Laccadirc Archi- pelagoes. Vol. 2. Cambridge University Press, London.
CASSIE, R. M., 1954. Some uses of probability paper in the analysis of size frequency distribu- tion. Aitst. J. of Mar. Freshwater Res., 5 : 513-522. FUSARO, C., 1977. Population structure, growth rate, and egg production of the sand crab,
Emcrita analoya in two different environments : a comparative analysis. Ph.D.
Disseration, University of California at Santa Barbara, Santa Barbara, California. FUSARO, C., 1978a. Growth of the sand crab, Emerita analoga (Hippidae), in two different
environments. U. S. Fish ll'ildl. Serv. Fish Bull., 76 (2) : in press. FUSARO, C., 1978b. Food availability and egg production: A field experiment with Jlippn
pacifica Dana (Decapoda, Hippidae). Pac. Sci., 32: 17-23. GILBERT, N., A. P. GUTIERREZ, B. D. FRAZER, AND R. E. JONES, 1976. Ecological Relationships.
W. H. Freeman & Co., San Francisco, 157 pages. GOTSHALL, D. W., 1972. Population size, mortality rates, and growth rates of Northern
California ocean shrimp, Patidahis jordani, 1965 through 1968. Calif. Dep. Fish
Game Fish Bull., 155. 47 pages. HALEY, S. R., 1979. Sex ratio as a function of size in Hippo, pacifica Dana (Crustacea,
Anomura, Hippidae) : A test of the sex reversal and differential growth hypotheses.
Am. Nat., 113: 391-397. HANSON, A. J., 1969. The life-history of the sand crab, Hippa cnbensis Saussure living on a
small island. M. S. Thesis. University of British Columbia, Vancouver. MATTHEWS, D. C., 1955. Feeding habits of the sand crab Hippa pacifica (Dana). Pac. Sci.,
9: 382-386.
SOUTHWOOD, T. R. E., 1966. Ecological Methods. Methuen, London. 391 pages. WENNER, A. M., 1972. Sex ratio as a function of size in marine Crustacea. Am. Nat.. 106 :
321-350. WENNER, A. M., 1977. Food supply, feeding habits, and egg production in Pacific mole crabs
(Hippa pacifica Dana). Pac. Sci., 31 : 39-47. WENNER, A. M., C. FUSARO, AND A. OATEN, 1974. Size at onset of sexual maturity and
growth rate in crustacean populations. Can. J. Zoo!., 52: 1095-1106. WILDER, D. G., 1953. The growth rate of the American lobster (Honuirus nmcricaints} . J.
Fish. Res. Board Can., 10 : 371-512.
Continued from Cover Two
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CONTENTS
\
ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY
BIRD, DENNIS J., AND ALBERT F. EBLE
Cytology and polysaccharide cytochemistry of the gill of the American
eel, Anguilla rostrata ......................................... 104
BRIGGS, R. P.
Fine structure of musculature in the copepod Paranthessius anemoniae Claus ............................................ 112
FELDER, DARRYL L.
Respiratory adaptations of the estuarine mud shrimp, Callianassa jamaicense (Schmitt, 1935) (Crustacea, Decapoda, Thalassinidea) . . 125
HARRIS, LARRY G., AND NATHAN R. HOWE
An analysis of the defensive mechanisms observed in the anemone Anthopleura elegantissima in response to its nudibranch predator Aeolidia papillosa ..... * ...................................... 138
KUHL, DEIRDRE L., AND LARRY C. OGLESBY
Reproduction and survival of the pileworm Nereis succinea in higher Salton Sea salinities ................................... 153
LANG, WILLIAM H., RICHARD B. FORWARD, JR., AND DON C. MILLER
Behavioral responses of Balanus improvisus nauplii to light intensity and spectrum ............................................... 166
NAKATANI, ISAMU, AND TAKASHI OTSU
The effects of eyestalk, leg, and uropod removal on the molting and growth of young crayfish, Procambarus clarkii ............... 182
STOKES, DARRELL R., AND NORMAN B. RUSHFORTH
Evoked responses to electrical stimulation in the colonial hydroid Clava squamata: A contraction pulse system .................... 189
WENNER, ADRIAN M., AND CRAIG FUSARO
An analysis of population structure in Pacific mole crabs (Hippa pacifica Dana) ............................................... 205
Volume 157
\.M Biological
I 'RRARY
OCT 291979
-J^oods Hole, Mass.
Number 2
BIOLOGICAL BULLETIN
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Vol. 157, No. 2 October, 1979
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
Reference: Biol. Hull., 157: 221-233. (October, 1979)
SALT AND WATER BALANCE IN TWO MARINE SPIDER CRABS,
LIBINIA EMARG1NATA AND PUGETTIA PRODUCTA. I. URINE
PRODUCTION AND MAGNESIUM REGULATION
JOHN C. CORNELL 1
Department of Zoology, University of California, Berkeley, California 94720, U.S.A., and the Bodega Marine Laboratory, Bodega Bay, California 94923, U.S.A.
The excretory organs of decapod crustaceans function in magnesium regulation (Robertson, 1957; Lockwood, 1962; Potts and Parry, 1964) and to a lesser extent, in nitrogen excretion (Delaunay, 1931; Binns and Peterson, 1969). Magnesium regulation has been observed in decapods from a variety of habitats (Robertson, 1939, 1949, 1953; W7ebb, 1940), the usual pattern being a lowered blood concen- tration and an elevated urine concentration, with respect to the medium. This phenomenon has been investigated extensively in two osmoregulating crabs, PachygrapSHS crassipes (Prosser, Green and Chow, 1955; Gross and Marshall, 1960; Gross and Capen, 1966) and Carcinus tnaenas (Riegel and Lockwood, 1961 ; Lockwood and Riegel, 1969), but little is known about magnesium regulation in osmoconforming crabs.
A problem in the study of magnesium regulation, and other aspects of salt and water balance, is that of making accurate estimates of urine production rates. Cannulation is usually not feasible because of the geometry and delicacy of the excretory duct. Many methods have been used, perhaps the best known being nephropore-occlusion, but only a few estimates have been made for an extended period of time by the continuous collection of urine: Procambarus clarkii (Kame- moto and Ono, 1968; Ono and Kamemoto, 1969); Paranephrops zcalandicus (Wong and Freeman, 1976) ; Cancer tnagister (Holliday, 1977) ; Callinectes sapid us (Cameron and Batterton, 1978). A technique for the continuous collec- tion of urine has been used in the present study.
MATERIALS AND METHODS
Specimens of the osmoconforming crab, Pugettia prodncta, were collected near the Bodega Marine Laboratory, Bodega Bay, California, and maintained at 10 to
1 Present address : Department of Zoology, Washington State University, Pullman, Washington 99164.
221
Copyright © 1979, by the Marine Biological Laboratory
Library of Congress Card No. A38-518
(ISSN 0006-3185)
222 JOHN C. CORNELL
12° C, either in running sea water (SW) at the Bodega Marine Laboratory, or in filtered SW (about one crab per 4 liters) at the University of California, Berkeley, California. Intermolt crabs were used except where otherwise noted.
Estimates of urine production were made by continuous collection of urine. A polyester resin cast of the region surrounding the two nephropores was made in order to position two polyethelene tubes. In making the cast, the crab was clamped in a supine position and the periopods secured with rubber bands. The ventral surface was dried with compressed air and those portions of the third maxillipeds distal to the ischia were cut off. The region surrounding the nephro- pores was lightly swabbed with vaseline which acted as a mold releasing agent. A transverse dam of plasticine, placed distal to the third maxillipeds, prevented the casting material from flowing into the mouth parts. A freshly mixed polyester resin, such as "G-R-R-R-I-P" (Idaho Chemical Industries, Inc.) was applied so that it extended posteriad to the plasticine dam, anteriad to the antennules, and laterad to about 5 mm beyond the nephropores. After 15 min, the cast was removed and the crab returned to SW. This procedure was carried out at least 24 hr before the start of an experiment.
The impressions of the opercula that cover the openings of the nephropores could clearly be seen in the cast. Each impression was used to center a hole of 0.063 inch (1.60 mm) diameter. A drill, larger by about 20% than the diameter of the opercula, was used to countersink the first pair of holes to a depth slightly greater than the diameter of the opercula. This allowed sufficient room for the opercula to open. Polyethelene tubing (P.E.Intramedic 190, Clay Adams) was pressed into the holes in the cast. Silicone grease was carefully applied around the periphery of the cast and between the two holes on its inner face. The crab was replaced in the clamp, the periopods secured with rubber bands, and the region surrounding the nephropores dried with compressed air. The cast, with the attached tubing, \vas positioned and pressed onto the crab. In most cases it snapped into position and was firmly held in place by a rubber band.
The crab was then suspended in an aquarium so that the top of the carapace was just submerged. The seal between the cast and the animal was checked by blowing into the tubes and watching for air bubbles. The tubes were led over the lip of the aquarium, about 3 to 5 cm above the nephropores, and fed into two vials, positioned at about the same level as the nephropores. The urine was collected under mineral oil.
Urine production was also estimated by blocking the nephropores with Eastman 910 cement (Armstrong Cork Co.) and measuring the change in weight. The Eastman 910 was allowed to set for 10 min before the crabs were returned to SW. During this period, excess water was removed from the branchial chambers, the animals were dried for one minute with compressed air, and weighed.
The concentrations of sodium, magnesium, calcium, potassium, chloride, and ninhydrin positive substances (NFS), and osmotic pressure, in blood, urine and SW were measured. Blood samples were withdrawn by puncturing an arthrodial membrane at a leg base with a drawn-out Pasteur pipet. Samples were centrifuged under mineral oil for 10 min in either an International Clinical Centrifuge at about 6000 rpm at room temperature or a Sorval model RC2-B, at 10,000 rpm at 1° C, the latter being used in the preparation of samples for osmotic pressure
SALT AND WATER BALANCE IN CRABS 223
determinations. Urine samples were also collected at the nephropore. A small hook, guided with the aid of a dissecting scope, was used to lift an operculum and the urine was collected in a drawn-out Pasteur pipet. Samples of plasma, urine and medium were stored under mineral oil in polystyrene vials.
Concentrations of cations were measured with a Perkin-Elmer model 290 atomic absorption spectrophotometer. Concentrations of chloride were measured on a Buchler-Cotlove chloridometer. NFS were determined by the method of Fowden (1951). Plasma was deproteinized by the addition of an equal volume of 10% trichloroacetic acid. For uniformity, urine received the same treatment. Samples were read on a Klett colorimeter against glycine standards. Measure- ments of osmotic pressure were made with an Advanced Instruments "Osmette" osmometer. Measurements of electrical potential difference across the body wall were made with an Analog Devices model 40 J operational amplifier (input impedance, 1011 ohms) as a preamplifier and voltages were read on a Tektronix oscilloscope. Chlorided silver wires in 3 M KC1 were used to make the electrical connections to 3 M KCl-agar bridges. One bridge served as the reference electrode while the other was placed in a hole in the top of the carapace, which remained out of water.
RESULTS
Urine production rates estimated by continuous collection
Figure 1 shows the results of an experiment where urine was collected over a 24-hr period. The sum of the cumulative volume of urine released from both nephropores has been arbitrarily fitted with a fifth order polynomial. The urine production rate, the first derivative of this polynomial, is also included. The rate of urine production was usually greater during the first 12-hr period than during subsequent collecting periods. The rates which will be reported were obtained after a 12-hr lapse from the start of collection. Urine release is intermittent, the interval between successive releases can vary greatly, and urine is usually released simultaneously from both nephropores.
Intermolt specimens of Piigettia producta (average weight, 101 g) in 100% SW produced urine at 6.40 ± 3.08 (22) % body weight (bw)/day, mean ± SD (N). Postmolt crabs (73 g) produced urine at 2.89 ± 3.68 (5) % bw/day and premolt crabs (65 g) produced urine at 29.5 ± 3.98 (3) % bw/day. A single classification analysis of variance indicates that there are significant differences among these means (P < 0.01) and a priori tests indicate that there are differences between the rates for intermolt and premolt crabs (P < 0.01), and intermolt and postmolt crabs (P < 0.05). The cause for these differences is not known.
Urine production rates for 10 crabs (114 g) were compared on two successive days. The average rate on the first day was 5.06 ± 3.68% bw/day ; on the second day it was 5.48 ± 3.27 % bw/day. These means are not significantly different (paired f-test). For one crab, urine production was measured for a 2-week period. During this time, the rate varied from 2 to 6% bw/day, the average being 3.02% bw/day. There was no apparent pattern to the daily fluctuations, and it is possible that the changes could be accounted for by changes in the volume of urine held in the bladders, which in Pitgcttia can exceed 5% bw (Cornell, 1976).
224
JOHN C. CORNELL
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FIGURE 1. Urine production in a 46 g specimen of Pmjcttia product a from the time of placement of the polyester resin cast until the 24th hr. The cumulative volume of urine pro- duced from the left and right nephropores is indicated by the square boxes and open circles, respectively, and can be read on the left ordinate. The sum of the cumulative volume of urine from both nephropores, indicated by solid circles, has been arbitrarily fitted with a fifth order polynomial, indicated by the solid curve. The first derivative of this polynomial, or urine production rate, is indicated by the dashed line and can be read on the right ordinate.
Urine production estimated from weight gain
Intermolt specimens of nephropore-occluded Pugettia (43.54 g) gained 3.38 ± 2.10 (12) % bw after 24 hr, while a control group (46.10 g) gained 0.43 ± 0.88 (9) % bw in the same period. Four crabs with blocked nephropores gained an additional 1.0% between 24 and 48 hr. These animals were markedly swollen, the posterior region of the carapace being lifted away from the abdomen. The urine production rate for intermolt crabs determined by weight gain is significantly less than that determined by continuous collection (P < 0.005, f-test), suggesting that the rates estimated by weight gain are under-estimates of the true rate, and that significant back pressures can occur.
Some major constituents of blood and urine
Measurements of some major constituents of blood, urine and medium for crabs in 100% SW are presented in Table I. An approximate statistical test (Sokal and Rohlf, 1969, p. 372), analogous to a single classification analysis of variance, was used to test for equality among means since these data are heteroscedastic.
The concentrations of sodium and chloride in the blood were both 98% of
SALT AND WATEK BALANCE ix CKAJJS
225
their respective concentrations in the medium. In the urine, the concentration of chloride was about equal to, while sodium was 96% of, the concentration in the medium. The concentrations of magnesium, calcium and potassium in the blood were 88, 118, and 105% of their respective concentrations in the medium. In the urine, these same ions were 135, 124, and 114% of the medium, respectively. NFS were about 10 times more concentrated in the blood than in the urine. The osmotic pressure of the blood was about 2 mosM greater than that of the medium and the urine was isosmotic to the blood, but not statistically different from the medium.
The concentrations of inorganic ions were more variable in the urine than in the blood; in particular, magnesium was the most variable, as judged by the ratio of the standard deviation to the mean. Magnesium concentrations ranging from 48 to 128 HIM were observed in the urine. Table II shows two correlation matrices, one each for the blood and the urine. In each matrix, a rank correlation coefficient has been computed for each inorganic ion writh every other inorganic ion. There is a significant negative correlation between sodium and magnesium in the urine. There are also significant positive correlations between magnesium and calcium and between magnesium and potassium in the urine.
In the blood, magnesium concentrations were also relatively more variable than the other ions which were measured. A significant negative correlation be- tween magnesium and calcium was found, the reverse of the condition in the urine. Significant positive correlations between magnesium and chloride, and between sodium and potassium, were also found.
TABLE I
Concentrations of some major constituents of the blood and urine of specimens of Pugettia producta in sea water. Relative concentrations are expressed as a percentage of the medium. Absolute concen- trations of inorganic ions, ninhydrin positive substances (NFS) and osmotic pressure (OP) are expressed in mM, mM glycine, and mosM, respectively. F(df) indicates the F ratio of an approximate test with the calculated degrees of freedom. One and two asterisks denote P < 0.05 and P < 0.01, respectively.
|
Fluid |
|||||
|
Relative concentration |
|||||
|
C 1 4. |
Absolute concentration, mean ± sd~(N) |
F |
|||
|
Solute |
(df) |
||||
|
Blood |
Urine |
Medium |
|||
|
98.0% |
99.4% |
100% |
4.24* |
||
|
Gl- |
524 |
± 12.9 (13) |
532 ± 27.9 (14) |
535 ± 2.73 (6) |
(2, 18) |
|
97.8% |
96.1', |
100' , |
9.60** |
||
|
Na* |
450 |
± 16.0 (23) |
442 ± 22.1 (37) |
460 ±5.74 (6) |
(2, 30) |
|
88.3', |
135', |
100% |
75.7** |
||
|
Mg2+ |
46 |
1 ± 2.48 (23) |
70.5 ± 19.2 (37) |
52.2 ± 0.65 (6) |
(2, 24) |
|
1189; |
124% |
100', |
95.6** |
||
|
Ca2+ |
11 |
8 ± 0.52 (13) |
12.4 ± 1.11 (22) |
10.0 ± 0.17 (6) |
(2, 43) |
|
105% |
114', |
100% |
11.5** |
||
|
K+ |
10 |
2 ± 0.38 (13) |
11.1 ± 1.13 (22) |
9.74 ±0.19 (6) |
(2, 21) |
|
— |
— |
— |
38.4** |
||
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NPS |
3 |
72 ± 2.00 (14) |
0.39 ± 0.12 (5) |
— |
(1, 13) |
|
100.2% |
100.2' , |
100% |
3.69* |
||
|
OP |
1016 |
± 2.47 (10) |
1016 ± 3.77 (10) |
1014 ± 1.19 (10) |
(2, 15) |
226
JOHN C. CORNELL
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SALT AND WATER BALANCE IN CRABS
227
TABLE III
Magnesium and sodium concentrations in the blood of normal and nephropore-occluded specimens of Pugettia producta in sea water. See text for statistical tests.
|
Ion |
Time |
|||||
|
0 hr |
24 hr |
48 hr |
||||
|
Normal |
Occluded |
Normal |
Occluded |
Normal |
Occluded |
|
|
Mg2+ (HIM) sd, N |
47.1 2.05, 7 |
47.5 2.48, 10 |
46.8 1.01, 7 |
48.5 4.33, 10 |
46.1 2.44, 7 |
51.4 3.87, 10 |
|
Na+ (mM) sd, N |
448 14.8, 7 |
454 20.1, 10 |
450 4.5, 7 |
462 28.9, 10 |
446 16.8, 7 |
473 23.9, 10 |
The effects of nephrop ore-occlusion on magnesium regulation
Of the inorganic ions studied, magnesium represents the clearest example of an ion which is regulated by the excretory system. Its concentration in the blood, urine, and medium suggest that it is continuously diffusing down the concentra- tion gradient from medium to blood, and that this gradient is maintained by its active removal by the excretory system. Estimates of the electrical potential difference across the body wall of three crabs indicate a difference of less than 0.1 mV. Thus, the driving force for the diffusion of magnesium from the medium to the blood must be the difference in chemical potential.
Blocking the nephropores should stop the removal of magnesium from the blood, and thus the magnesium level in the blood should rise, reaching equilibrium with the magnesium in the medium. The results of such an experiment appear in Table III. The magnesium concentration in the blood of the experimental animals with blocked nephropores increased ; after 48 hr the ratio of the magnesium concentration in experimental crabs to that in control crabs was 1.12. There was also an increase in sodium concentration in the blood of experimental crabs, the above ratio being 1.06. This may indicate that sodium is also normally regulated below its equilibrium concentration. The sodium and magnesium data were separately analyzed by two-way analysis of variance after randomly removing the data for three crabs in the experimental group in order to facilitate the calcula- tions. These analyses indicate that there are significant increases in sodium and magnesium concentrations in the blood of nephropore-occluded crabs (P < 0.01 for both ions in the sub-groups Normal and Occluded ; for both ions, the F ratios were not significant in the sub-group Time, and for the interaction between sub- groups Normal and Occluded X Time).
In Pach\grapsits crassipes, Gross and Capen (1966) demonstrated that the magnesium concentration in the urine is a direct function of the time the urine is held in the bladder. This does not seem to be the case in Pngcttia. When, after 86 hr, the magnesium concentration in the urine of nephropore-occluded and con- trol crabs were compared, the magnesium concentration in the control crabs, 81.9 ± 26.8 (7) HIM, was greater than that in the experimental crabs, 58.4 ± 9.93 (9) mM. This unexpected result seems to have been caused by chance, since the
228 JOHN C. CORNELL
concentration in the control group was greater than expected. Also, since one of the crabs in the experimental group had died, the remaining animals may not have been in good condition and the transport of magnesium could have been reduced. The experiment was repeated using a paired design and a shorter period of time to minimize the effects of individual variation and nephropore-occlusion. No differences were found in 10 crabs between the magnesium concentrations at 0 hr, 65.3 ± 20.7 HIM, and 24 hr, 64.2 ± 18.3 HIM, after nephropore-occlusion. These experiments suggest that the bladders of Pngcttia do not secrete magnesium into the urine.
DISCUSSION
Decapod crustaceans have considerable ability to regulate their internal ionic compositions (Robertson, 1949, 1953). This appears to be independent of the ability to osmoregulate and has been defined by Robertson (1949, p. 182) as the "maintenance in a body fluid of concentrations of ions differing from those of a passive equilibrium with the external medium." Since the blood of decapods con- tains considerable amounts of non-diffusible, negatively charged protein, the internal/external concentration ratio of an ion can differ passively from unity. This ratio is expected to be less than 1.0 for the anions and greater than 1.0 for the cations. Robertson (1953) found, using a dialysis technique, that it was often 1.03 for the cations, except for calcium for which it was estimated that up to 20% was complexed with proteins. Greenaway (1976) has confirmed these results for calcium.
The present data suggest that Pugettia prodncta hypo-regulates magnesium and sodium ; potassium is probably slightly hyper-regulated, while chloride is prob- ably very close to its equilibrium concentration. From these data it is not possible to determine if calcium is regulated, since an unknown amount is com- plexed with proteins. The comparison of the concentrations of various ions in the blood of Pngcttia with those of other decapod crustaceans, tabulated by Prosser (1973), indicates that the pattern of regulation in Pngcttia is similar to that in other marine decapods.
Expressed as HIM glycine, the blood and urine of Pngcttia contain 3.7 and 0.39 HIM NFS, respectively. It was the usual policy to use crabs which had not been fed for a week. Thus, NFS in the blood of Pngcttia fresh from the field could be different. A comparison of these values with those reported for Carcinus maenas (Binns, 1969b ; Evans, 1972) indicates that the blood concentrations are similar in both crabs when differences in technique are accounted for (see Evans, 1972). The concentration of NFS in the urine of Pngcttia appears to be about half of that in Carcinns, but this can be quite variable and may not represent a true difference. The antennal gland of crustaceans is not a major route for nitrogen loss. Binns and Peterson (1969) estimate that in the spiny lobster Jasns edzvardsi, 90% of all the soluble nitrogen is excreted extra-renally.
The urine production rate for intermolt specimens of Pngcttia, determined by continuous collection, was 6.40% bw/day. However, the rate determined by weight gain was less than half of this, 2.95% bw/day after correction for the control value. There is good reason to believe that the former estimate is the more correct, the latter being reduced by back pressure. Although few data exist, there appears
SALT AND WATER BALANCE IN CRABS
229
TAKLK IV
Magnesium excretion rates and magnesium permeabilities for some decapod crustaceans in sea water. One dagger indicates that Cm and Cb are magnesium concentrations (mM) in medium and blood, respectively. Two daggers: see text for discussion of the effects of the electrical potential on these values. Three daggers denote the references: (1) Gross and Marshall, 1960; (2) urine and blood [ATg2+] — Riegel and Lockwood, 1961; urine production rate — Binns, 1969a; (3} this report.
|
Mg'+ |
Me2"1" |
|||||
|
Animal |
Blood |
Urine |
Medium |
Urine flow |
excretion rate |
-*-*•>•& permeability |
|
(reference) ftt |
mM |
mM |
mM |
rate % bw/day |
jumol |
^mol |
|
g • day |
(Cm-Cb)g-dayt |
|||||
|
Pachygrapsus |
20 |
305 |
52.0 |
3.9 |
11.9 |
0.37ft |
|
crassipes (1) |
||||||
|
Carcinus |
31 |
250 |
52 (?) |
4.4 |
11.0 |
0.52ft |
|
maenas (2) |
||||||
|
Libinia |
44 |
66 |
49.0 |
5.1 |
3.4 |
0.68 |
|
emarginata (3) |
||||||
|
Pugettia |
46 |
70 |
52.2 |
6.4 |
4.5 |
0.72 |
|
producta (3) |
to be a relationship between urine production and molt stage since premolt crabs produced urine at greater rates, and postmolt crabs produced urine at lesser rates, than intermolt crabs. The cause of this relationship is a matter for speculation.
The urine production rate was also determined by continuous collection for the osmoconforming spider crab Libinia emarginata (5.1% bw/day) and the fresh- water crayfish Pacifastacus Icniiiscnlns (6.0% bw/day). At present, it is dif- ficult to explain, considering water permeabilities and osmotic pressures of blood and media, why Libinia, Pacifostaeiis and Pugettia produce urine at comparable rates. Regardless, it is clear from many studies that most moderate-sized decapods produce urine at 2 to 10% bw/day when tested at salinities representative of normal habitat salinity, the exception being the freshwater brachyurans which produce no more than about 1% bw/day (Shaw, 1959; Thompson, 1970; Harris, 1975).
The present data suggest that the excretory system of Pugettia is responsible for the lowered magnesium concentration in the blood since magnesium is concen- trated in the urine, and since blocking the nephropores results in elevated levels of magnesium in the blood. Calcium and potassium are also concentrated in the urine, although not to so great a degree as magnesium. There appears to be some relationship among magnesium, calcium, and potassium in the urine, the concentration of the latter two ions being positively correlated with that of magnesium. By contrast, the calcium concentration in the urine of Carcinus is independent of the magnesium concentration (Lockwood and Riegel, 1969).
The concentration of magnesium in the urine of Pugettia is negatively correlated with that of sodium. Similar relationships between sodium and magnesium have been reported for Carcinus (Webb, 1940; Riegel and Lockwood, 1961), Pachy- grapsus crassipes (Prosser, Green and Chow, 1955; Gross and Marshall, 1960; Gross and Capen, 1966) and Cancer magister (Hunter and Rudy, 1975). Thus, some from of Na+/Mg2+ exchange mechanism may exist. Little is known about the mechanisms of magnesium transport. However, Holliday (1978) has found
230 JOHN C. CORN HI. I.
that the net magnesium flux in the isolated bladder of Cancer is inhibited by ouabain and that only a small part of the opposing net sodium flux is associated with the magnesium flux. In the isolated gut of the insect Hyalophora cecropia, magnesium transport is independent of sodium and related, in a complex way, to potassium transport (Wood, Jungreis and Harvey, 1975).
The reabsorption of fluid from the urine, as suggested by the urine/blood (U/B) ratio of filtration markers, is insufficient to account for the U/B ratios of magnesium in a number of decapods (Gross and Capen, 1966; Lockwood and Riegel, 1969; Franklin, Teinsongrusmee and Lockwood, 1978). In Pugettia, the U/B ratio of magnesium is about 1.5 and the U/B ratio of inulin may approach this value (Cornell, 1976). However, there is some difficulty in the interpretation of inulin U/B ratios in animals with large bladders (see Riegel, Lockwood, Norfolk, Bulleid and Taylor, 1974; and Cornell, 1976). Thus, it seems unwarranted to conclude that fluid reabsorption accounts for the concentration of magnesium in the urine of Pugettia.
Gross and Capen (1966) have shown that the magnesium concentration in the urine of Pachygrapsus is a function of the time the urine is held in the bladder. However, during nephropore-occlusion the magnesium concentration in the urine did not increase in Pugettia, suggesting that magnesium is concentrated in the antennal gland. A similar experiment on Pachygrapsus produced an in- crease in urine magnesium. In Figure 3 of their report, Gross and Capen (1966) presented results which suggest that the fluid entering the bladders contains 38 mM magnesium, about 20 mM higher than the blood. The urine of Pugettia is about 24 mM higher in magnesium than the blood. The implication is that the antennal glands of both animals perform equivalent tasks, and that it is the bladders of Pachygrapsus which concentrate most of the magnesium, a function which the bladders of Pugettia do not seem to perform.
The concentration of magnesium in the blood must be a function of at least three parameters : the permeability of the body wall, the excretion rate, and the electrochemical potential across the body wall. Neglecting for the moment the electrical potential, and dividing the weight-specific magnesium excretion rate by the concentration difference between the blood and the medium, an estimate of the permeability of the body wall can be obtained. In Table IV these quantities have been computed for four decapods. Taking into account the electrical po- tential does little to change these results. In Pugettia and Libinia, the potential is negligible, 0 ± 0.1 mV. Potentials of -2.0 mV (inside negative) for Pachy- grapsus (Rudy, 1966) and Carcinus (Greenaway, 1976) have been reported for animals in SW. This is a small potential and for present purposes may be neglected, however, the permeability of Pachygrapsus and Carcinus may be slightly less than indicated. The data in Table IV suggest that those crabs which have much reduced the magnesium concentration in their blood have done so by lowering the magnesium permeability and by raising the rate of magnesium ex- cretion.
The significance of magnesium regulation in crustaceans, first noted by Robertson (1949, 1953), is that a strong negative correlation exists between the activity of an animal and the magnesium concentration in its blood. Thus, animals with low magnesium levels tend to be highly active, while those with high levels
SALT AND WATER BALANCE IN CRABS 231
tend to be lethargic. Robertson (1953) has pointed out that high extracellular levels of magnesium have been found to block neuromuscular transmission in Carcinus (Katz, 1936) and to reduce the mechanical response of isolated crayfish legs to electrical stimulation (Waterman, 1941).
This paper constitutes part of a doctoral dissertation submitted to the Depart- ment of Zoology, University of California, Berkeley. It is a pleasure to thank Dr. Ralph Smith for his encouragement and guidance during the course of this work. I am grateful to Dr. Cadet Hand and the laboratory staff for the use of the facilities of the Bodega Marine Laboratory, Bodega Bay, California. I thank Dr. Robert Josephson for the opportunity to pursue my work while acting as a course assistant in the Experimental Invertebrate Zoology Course at the Marine Biological Laboratory, Woods Hole, Massachusetts. Financial assistance in the form of a one-year University Fellowship was greatly appreciated. My wife, Mary, deserves special thanks.
SUMMARY
1. A new technique for the continuous collection of crab urine is described. Estimates of urine production, based on this technique, indicate that specimens of Pugettia producta in sea water produce urine at 6.4% body weight (bw)/day. Premolt and postmolt crabs produce 30 and 3.0% bw/day, respectively. Inter- molt specimens of Libinia cmarginata produce 5% bw/day.
2. The urine production rate for specimens of Pugettia, estimated by weight gain following 24 hr of nephropore-occlusion, is 3.0% bw/day. This is significantly less than that determined by the continuous collection of urine, suggesting that back pressure can interfere with urine production.
3. Ion regulation was examined in specimens of Pugettia. When expressed as a percentage of their concentrations in sea water, the values in blood plasma of chloride, sodium, magnesium, calcium, and potassium are 98, 98, 88, 118, and 105%, respectively, for crabs in sea water. Likewise in the urine, the values for these same ions are 99, 96, 135, 124, and 114%, respectively. Ninhydrin positive substances, measured with glycine standards, are 3.7 and 0.39 mM in blood plasma, respectively. The electrical potential across the body wall of both species of crab is zero.
4. In Pugettia, blocking the nephropores causes an increase in the magnesium concentration in the blood, suggesting that the excretory system is mainly respon- sible for regulating this ion. However, blocking the nephropores causes no change in the magnesium concentration of urine stored in the bladder, which suggests that the antennal gland is mainly responsible for concentrating magnesium in the urine. Libinia and Pugettia excrete magnesium at a lower rate, and are more permeable to magnesium, than brachyurans which are strong magnesium regulators.
LITERATURE CITED
BINNS, R., 1969a. The physiology of the antennal gland of Carcinus utacims (L.). II. Urine production rates. /. Exp. Biol., 51 : 11-16.
232 JOHN C. CORNELL
BINNS, R., 1969b. The physiology of the antennal gland of Carcinus maenas (L.)- IV. The reabsorption of amino acids. /. Exp. Biol., 51 : 29-39.
BINNS, R., AND A. J. PETERSON, 1959. Nitrogen excretion by the spiny lobster Jasus cdwardsi (Hutton) : the role of the antennal gland. Biol. Bull., 136: 147-153.
CAMERON, J. N., AND C. V. BATTERTON, 1978. Antennal gland function in the freshwater blue crab, Callincctcs sapid us: water, electrolyte, acid-base and ammonia excretion. /. Comp. P/iysiol,. 123: 143-148.
CORNELL, J. C., 1976. Aspects of salt and water balance in two osmoconforming crabs Libinia emarginata and Pugcttia prodncta (Brachyura: Majidae). Ph.D. thesis, University o\ California, Berkeley, 243 pp. (University Microfilms/Dissertation Ab- stracts International No. 77-15,646.)
DELAUNAY, H., 1931. L'excretion azotee des invertebres. Biol. Rev., 6: 265-301.
EVANS, P. D., 1972. The free amino acid pool of the haemocytes of Carcinus macnas (L.). /. Exp. Biol, 56: 501-507.
FOWDEN, L., 1951. The quantitative recovery and colorimetric estimation of amino-acids separated by paper chromatography. Biochcm. J. 48 : 327-333.
FRANKLIN, S. E., B. TEINSONGRUSMEE, AND A. P. M. LOCKWOOD, 1978. Inhibition of mag- nesium secretion in the prawn Palaemon scrratus by ethacrynic acid and by ligature of the eyestalks. Pages 173-193 in K. Schmidt-Nielsen, L. Bolis, and S. H. P. Mad- drell, Ed., Comparative physiology: water, ions and fluid mechanics. Cambridge Uni- versity Press, New York and London.
GREENAWAY, P., 1976. The regulation of haemolymph calcium concentration of the crab Carcinus maenas (L.). /. Exp. Biol., 64: 149-157.
GROSS, W. J., AND L. A. MARSHALL, 1960. The influences of salinity on the magnesium and water fluxes of a crab. Biol. Bull., 119: 440-453.
GROSS, W. J., AND R. L. CAPEN, 1966. Some functions of the urinary bladder in a crab. Biol. Bull., 131 : 272-291.
HARRIS, R. R., 1975. Urine production rate and urinary sodium loss in the freshwater crab Potamon cdulis. J. Comp. Physiol., 96 : 143-153.
HOLLIDAY, C. W., 1977. A new method for measuring the urinary rate of a brachyuran crab. Comp. Biochcm. Physiol., 58A : 119-120.
HOLLIDAY, C. W., 1978. Magnesium transport by the bladder of the crab, Cancer magister. Am. Zool., 18 : 577.
HUNTER, K. C., AND P. P. RUDY, 1975. Osmotic and ionic regulation in the Dungeness crab Cancer magister Dana. Comp. Biochem. Physiol., 51A: 439-447.
KAMEMOTO, F. L, AND J. K. ONO, 1968. Urine flow determinations by continuous collection in the crayfish Procambants clarkii. Comp. Biochcm. Physiol., 27 : 851-857.
KATZ, B., 1936. Neuro-muscular transmission in crabs. /. Physiol., 87 : 199-221.
LOCKWOOD, A. P. M., 1962. The osmoregulation of Crustacea. Biol. Rev., 37 : 257-305.
LOCKWOOD, A. P. M., AND J. A. RIEGEL, 1969. The excretion of magnesium by Carcinus maenas. J. Exp. Biol., 51 : 575-589.
ONO, J. K., AND F. I. KAMEMOTO, 1969. Annual and proecdysial variations in urine produc- tion in crayfish. Pacific Sci., 23 : 305-310.
POTTS, W. T. W., AND G. PARRY, 1964. Osmotic and ionic regulation in animals. Pergamon Press, London, 423 pp.
PROSSER, C. L., 1973. Inorganic ions. Pages 79-110 in C. L. Prosser, Ed., Comparative animal physiology, 3rd ed. Saunders, Philadelphia.
PROSSER, C. L., J. W. GREEN, AND T. J. CHOW, 1955. Ionic and osmotic concentrations in the blood and urine of Pachygrapsus crassipes acclimated to different salinities. Biol. Bull., 109: 99-107.
RIEGEL, J. A., AND A. P. M. LOCKWOOD, 1961. The role of the antennal gland in the osmotic and ionic regulation of Carcinus maenas. J. Exp. Biol., 38 : 491-499.
RIEGEL, J. A., A. P. M. LOCKWOOD, J. R. W. NORFOLK, N. C. BULLEID, AND D. A. TAYLOR, 1974. Urinary bladder volume and the reabsorption of water from the urine of crabs. /. Exp. Biol., 60 : 167-181.
ROBERTSON, J. D., 1939. The inorganic composition of the body fluids of three marine inverte- brates. J. Exp. Biol, 16: 387-397.
SALT AND WATER BALANCE IN CRABS 233
ROBERTSON, J. D., 1949. Ionic regulation in some marine invertebrates. /. Exp. Biol., 26: 182-200.
ROBERTSON, J. D., 1953. Further studies on the ionic regulation of marine invertebrates. /. Exp. Biol., 30: 277-296.
ROBERTSON, J. D., 1957. Osmotic and ionic regulation in aquatic invertebrates. Pages 229- 246 in B. T. Scheer, T. H. Bullock, L. H. Kleinholz, and A. W. Martin, Ed., Recent advances in invertebrate physiology. University of Oregon Publications, Eugene, Oregon.
RUDY, P. P., 1966. Sodium balance in Pachygrapsus crassipes. Comp. Biochem. Physiol., 18: 881-907.
SHAW, J., 1959. Salt and water balance in the east african fresh-water crab Potainon niloticus (M. Edw.). /. Exp. Biol., 36: 157-176.
SOKAL, R. R., AND F. J. ROHLF, 1969. Biometry. W. H. Freeman and Co., San Francisco, 776 pp.
THOMPSON, L. C, 1970. Osmoregulation of the fresh water crabs Metopaulias depressus (Grapsidae) and Pscitdothelp/nisa jouyi (Pseudothelphusidae). Ph.D. thesis, Uni- versity of California, Berkeley, 101 pp. (University Microfilms/Dissertation Abstracts International No. 71-9938.)
WATERMAN, T., 1941. A comparative study of the effects of ions on whole nerve and isolated single nerve fiber preparations of crustacean neuromuscular systems. /. Cell Comp. Physio!., 18: 109-126.
WEBB, D. A., 1940. Ionic regulation in Carcinus macnas. Proc. R. Soc. London, B. Biol. Sci., 129: 107-136.
WONG, J. M., AND R. F. H. FREEMAN, 1976. Osmotic and ionic regulation in different popula- tions of the New Zealand freshwater crayfish Parancphrops zcalandicus. J. Exp. Biol., 64: 645-663.
WOOD, J. L., A. M. JUNGREIS, AND W. R. HARVEY, 1975. Active transport of mangesium across the isolated midgut of Hyalophora cccropia. J. Exp. Biol., 63 : 313-320.
Reference: Bwl. Bull., 157: 234-248. (October, 1979)
DIETARY FATTY ACID AND TEMPERATURE EFFECTS
ON THE PRODUCTIVITY OF THE CLADOCERAN,
MOINA MAC ROC OP A
LOUIS R. D'ABRAMO * Department of Biology, Yale University, Neiv Haven, Connecticut 06520
Analyses of the relationships between physical parameters and the quantity and quality of food in relation to zooplankton physiological processes such as ingestion, assimilation (growth) and reproduction have been scarce. These studies are dif- ficult since experimental techniques have lacked means to keep biotic factors con- stant while modifying a physical factor, or vice versa. As a result, the nutritional quality of a particular food under certain physical conditions has not been evaluated.
The study of interactions between the quantity and quality of food available for a zooplankter and existing physical parameters is very important. The struc- ture of zooplankton communities may be partially determined by the relative abilities of fauna to efficiently utilize and process the available food and thereby satisfy their individual nutritional requirements.
Investigators who have classified the nutritional suitability of a particular food for a predator have observed effects on ingestion, assimilation and reproduction. No study has adequately controlled experimental conditions so that the inter- relationships among a physical factor, the chemical composition of diet, and the population dynamics of a zooplankton species could be identified.
Stuart, McPherson, and Cooper (1931) studied the relative value of a variety of bacterial species as food for the aseptic cladoceran, Moina macrocopa. They found differences in growth, fertility, and fecundity. Lefevre (1942) demonstrated that the normal growth and reproduction of various cladoceran species were depend- ent upon the suitability (physical or physiological) of species of fresh water algae. Monoxenic culture of two species of Crustacea, Arteinia salinct, and Tigriopus japonicus, an harpacticoid copepod, by Provasoli, Shiraishi and Lance (1959) demonstrated that many unialgal diets either failed to permit growth to adulthood or allowed only a few consecutive generations. Interestingly, a phytoplankton species that was nutritionally good for one species was not always good for the other species. The apparent nutritional deficiencies of some of the unialgal diets could often be rectified by the addition of specific vitamins or other organic com- pounds (Shiraishi and Provasoli, 1959). Lee, Tietjen, and Garrison (1976) observed a seasonal "switching" of nutritional requirements for Nitocra typica, an harpacticoid copepod from salt marsh aufwuchs communities. In these studies the comparative nutritional value of unialgal diets of various species and strains was determined by growth rate and fecundity measurements. In some cases the nutritional adequacy of a particular algal diet was temperature dependent. Guerin and Gaudy (1977) and Gaudy and Guerin (1977) grew the harpacticoid copepod, Tisbe holothuriae, on a variety of artificial chemically undefined participate diets. The dry weight, elementary chemical composition, fecundity, sex ratio, and result-
1 Present address ; Bodega Marine Laboratory, Box 247, Bodega Bay, CA 94923.
234
PRODUCTIVITY OF MOIX. / 235
ing population dynamics of this species were significantly affected by the quality of food. Schindler (1971) showed that the assimilation efficiencies of the copepods Diaptomus gracilis, Cyclops strennus, and the cladoceran, Daphnia longispina are dependent upon the type of food eaten. Roman (1978) speculated that the successful association of the blue-green alga Trichodesmium and the harpacticoid copepod Macrosetella gracilis may be due to the copepod's efficient conversion of the carbon and nitrogen fixed by the alga into secondary production.
A precise understanding of how zooplankton population dynamics are influenced by the chemical composition of the diet can only be realized when proper conditions will permit the experimenter to independently control qualitative and quantitative changes. The axenic culture of a zooplankter on a chemically defined artificial diet would satisfy such controlled conditions. Such a diet must permit not only adequate ingestion, digestion, and assimilation but also continuous reproduction so that large populations and successive generations may be studied.
The biphasic medium for the axenic culture and continuous reproduction of Moina •inacrocopa formulated by Conklin and Provasoli (1978) provides for the precise study of food/zooplankton relationships. The medium consists of a soluble phase and a participate phase. Water soluble vitamins, nucleic acids, amino acids, and inorganic salts comprise the soluble phase, while coagulated protein-starch and protein-lipid particles comprise the participate phase. The medium has permitted the first analysis of the nutritional requirements of a crustacean (Conklin and Provasoli, 1977). By employing this medium the experimenter can control the chemical composition of the diet as well as the mode of presentation to the herbivore (i.e., number of particles/ml, amount of chemical compound/particle). Since the particles, unlike natural phytoplankton and bacteria, are inert to the chemical or quantitative changes associated with physical environmental factors, precise relationships between diet composition and some physical variable can be determined.
This study is directed toward analysis of dietary fatty acid and temperature interrelationships and the effects upon the productivity of Moina inacrocopa. Undoubtedly there are other chemical compounds which affect the normal growth and reproduction of Moina (Conklin and Provasoli, 1977). Experimentation with fatty acids was chosen because, for this class of compounds, there are unique compositional differences amongst the orders of algae (Wood, 1974) and because past research demonstrates the essentiality of lipid factors for sustained fertility in Moina inacrocopa and Daphnia magna (Provasoli, Conklin, and D'Agostino, 1970; Viehoever and Cohen, 1938).
The importance of dietary lipids for the Crustacea was also inferred from the research on the phylogenetically related insects; lipids affect their metamorphosis, diapause, fertility, and fecundity (Beck, Lilly, and Stauffer, 1949; Vanderzant, Kerue, and Reiser, 1957; Tamaki, 1961 ; Dadd, 1960; Nayar, 1964; Adkisson, Bell and Wells, 1963; Bull and Adkisson, 1960; Foster and Crowder, 1976; Ikan, Stanic, Cohen and Shulov, 1970; Chumakova, 1962).
MATERIALS AND METHODS
The organism employed in this research was Moina inacrocopa americana, a member of the order Qadocera. Descriptions and illustrations may be found in
236 LOUIS R. D'ABRAMO
Goulden's monograph of the Moinidae (Goulden, 1968). Reproduction occurs primarily by parthenogenesis. At times sexual females and males will appear in populations and sexual reproduction will occur (D'Abramo, in preparation). Par- thenogenetic eggs are released from a pair of ovaries into a brood pouch. The neonates are born viviparously and are miniature images of the adults. Neonates pass through four stages of growth (instars) before becoming sexually mature. During the fourth or adult instar a female lays her first brood of eggs. Generation time is temperature-dependent but is rapid compared to many other Crustacea (4-5 days at 26° C).
Axenization
A serial dilution method as described by Conklin and Provasoli (1978) was employed to free the experimental organisms from bacteria. Recently born neonates were washed for 15 min in 15 separate baths composed of 10 ml of DM? medium (Provasoli and D'Agostino, 1970) in 6-cm diameter Petri dishes. All baths contained two drops of an antibiotic mix (D'Agostino and Provasoli, 1970). The eighth bath of the series contained a suspension of bacteria-free Chlamydo- monas reinhardii (GMS~), to allow for feeding and clearing the gut of bacterial flora. After the entire washing procedure, individual animals were transferred to 20 X 125 mm screwcap test tubes containing 10 ml of DM7 and a suspension of C. reinhardii. After two days growth at room temperature under a bank of cool white fluorescent bulbs, 0.5 ml of the culture medium was dispensed into semisolid DA medium (D'Agostino and Provasoli, 1970) for detection of any bacterial or fungal contaminants. This sterility test was incubated at room tem- perature for 15 to 20 days.
Once monoxenic cultures were established and found aseptic, young were aseptically transferred to 20 X 125 mm screwcap test tubes that contained 10 ml of a sterile artificial medium, K733, which is a modification of the artificial medium developed for Moina by Conklin and Provasoli (1977) (Table I). Acclimation to the artificial medium occurred within three generations and was determined by visual observation of the number of progeny per brood and of a normal swimming behavior. Culturing of animals fed artificial food was done in the darkness at room temperature. Growth in the dark discourages algal reproduction and all traces of algal cells generally disappeared by the third transfer in artificial media.
Populations of Moina were maintained on artificial and natural diets. Main- tenance populations on artificial medium K733 were incubated at 19° C in dark- ness to prevent photodeterioration of riboflavin. Growth at this temperature slowed population growth rates, thereby increasing the time between transfers. Popula- tions fed Scenedcsinits nacyalii (Chodat) and C. reinhardii (CMS") were grown at 19° C with a 16:8 L/D photoperiod. New cultures of natural and artificial diets were usually started every three weeks.
Diet I annul at ion
The artificial media used in the experimentation with Moina macrocopa were modifications of the Fl medium of Conklin and Provasoli (1977). Modifications may be found in Table I. The media contained two types of particles, starch-
PRODUCTIVITY OF Mal\'.l
237
TABLE I Modifications of Conklin-Provasoli Fl Medium.
Common basal medium (per cent w or v/v)
Changes: (a) Metal mix P II to metal mix L; Metal mix L, 1 ml == Na2EDTA-2H2O, 3.81 g;
Zn(asSO4"), 0.30 mg; B, 0.12 mg; Mn(asCl-), 0.087 mg; Fe(as NH4SOr),
0.06 mg; Co(as Cl"), 0.024 mg; Cu(as SO4=), 0.024 mg; Mo(as NH4+),
0.036 mg.
(b) Liver infusion L25 at 70 mg to defatted liver infusion L25 (lipid extraction by
chloroform/methanol (2/1) (v/v) for three hr under N2) at 40 mg. Additions: (a) glycogen 10 mg
(b) globulin + 2X crystalline egg albumin, 1 ml, 1 ml == bovine a globulin (Frac- tion IV) 2.25 mg + 2X crystalline egg albumin (ICN), 0.75 mg. Mixture is formed by dissolving components in water ; coagulating this mix by autoclaving; homogenizing (2600 rpm for 5 min) the coagulum to produce particles; autoclaving the particles and rehomogenizing. Deletions: (a) DF2
Particles Changes:
(a) SA gel particles:
(b) FV particles:
Artificial medium
Basal medium 96 ml = 540 X 103 ml
1 ml == 10 mg rice starch + 4.10 mg 2X crystalline egg albumin.
1 nil == 8 mg 2X crystalline egg albumin + 0.75 egg lecithin + 1 mg BHT (butylated hydroxytoluene) + 0.66 ergo- calciferol + 0.25 retinopalmitate + 2 mg dl-atocopherol + fatty acids in variable quantities and qualities, depend- ing upon experiment. Fatty acids for K733 medium : 1 mg palmitic acid + 0.3 mg oleic acid + 0.7 mg linoleic acid + 1 mg a-linolenic acid.
3 ml SA gel + 1 ml FV particles, pH == 8.0, Particle concentration
protein and lipid-protein. All inorganic and organic additions were made from prepared stock solutions. Generally inorganic stock solutions were stored at room temperature while organic solutions or suspensions were stored either at 8° C or frozen. To prevent bacterial contamination, 0.5 ml of a volatile preservative solution (Hutner and Bjerknes. 1948) was added to all stock solutions. The volatile preservative vaporizes during autoclaving. Stock solutions were usually renewed within a four-month period. All fatty acids used in the diets were 99+% pure and were stored frozen under a N2 atmosphere. Prepared media varied in the qualitative and quantitative composition of fatty acids absorbed onto the protein particles. Qualitative fatty acid additions were made to simulate the unique proportional differences found amongst four orders of algae, Cyanophyceae, Cryptophyceae, Chlorophyceae, and Bacillarophyceae (Table II).
Since the work involved the effect of quantitative and qualitative changes in dietary fatty acids upon the productivity of Moina, concern developed regarding the possible differential uptake of particular fatty acids by the albumin. To insure that results could be genuinely attributed to particular diets, particles prepared 2 months previously were subjected to a lipid compositional analysis by thin layer and gas chromatographic techniques. These analyses were kindly performed by Dr. David H. Beach of the Department of Microbiology, State University New York, Upstate Medical Center at Syracuse. Lipid analysis of the particles revealed
238
LOUIS R. D'ABRAMO
TABLE II
/•'</// v acid mixtures simulating average percent composition of representative algal classes (total fatty acids = 3 mg).
|
Mixtures |
||||
|
Patty acids (ing) |
Cyanophyceae1 |
Chlorophyceae |
Cryptophyceae |
Bacillariophyceae |
|
14:0 (myristic) |
— |
. — - |
0.30 |
— |
|
16:0 (palmitic) |
1.25 |
1.00 |
0.45 |
0.75 |
|
16:1 (palmitoleic) |
0.80 |
0.10 |
— |
1.20 |
|
18:0 (stearic) |
0.12 |
— |
— |
— |
|
18:lco9 (oleic) |
0.30 |
0.25 |
0.30 |
0.15 |
|
18:2co6 (linoleic) |
0.40 |
0.35 |
0.30 |
— |
|
18:3co3 (a-linolenic) |
0.13 |
1.30 |
0.90 |
— |
|
20:5o;3 (eicosapentaenoic) |
— |
— |
0.45 |
0.90 |
|
22:6w3 (docosahexaenoic) |
— |
— |
0.30 |
— — |
1 The 18:4 (octadecatetraenoic acid) which comprises from 15 to 30% of the cryptophyceae fatty acids was not incorporated into the mixture because of the lack of a conveniently available and highly pure (99 -+- %) source.
that the experimentally intended dietary differences, both qualitative and quantita- tive, were genuine. The lipophilic albumin had no tendencies toward differential absorption of the fats and vitamins. Uptake was complete.
Determination of particle sise and particle concentration — optical transmission relationships
The number of particles in media of different particle concentrations was deter- mined through the use of a Coulter Counter (Model ZB, Counter Electronics, Hialeah, Florida). The counter was calibrated with the use of 10.2-ju, diameter pollen. Counts of artificial particles were performed using a ICO-/* aperture, an amplification of four and an aperture current equal to one half. The lower and upper threshold settings were 1 and 40, respectively. The threshold factor mea- sured as the average volume of the known system divided by the lower threshold dial setting at half count equalled 16.8186. Particle size frequency distribution for a sample was determined using a Coulter Channelyzer (base channel threshold — 1, window width = 100) and an X-Y Recorder II for automatic plotting. The size of the particles was determined by employing the formula. Channel number X window width/100 + Base Channel Threshold X Threshold Factor = cubic microns.
Aliquots of variable volumes from samples of percent transmissions ranging from 45 to 85% were diluted to 20 ml by means of a special electrolyte solution, Isoton II (Curtin Matheson Scientific, Inc.). From these diluted suspensions ten separate 500-ju.l samples were counted, from which an average was computed. Counts for these samples ranged from 10,000 to 30,000 per 500 /*!. Background counts did not exceed 100 per 500 jul. The derived average particle concentration (number per 500 ju.1) was then multiplied by the dilution factors to obtain the number of particles per ml. A standard curve relating particle concentration (number/ml) to per cent transmission was then constructed. Particle concentration within the media was altered by additions from the stock mixtures. All prepared media were
PRODUCTIVITY OF MOINA 239
adjusted to pH 8.0 with a pH meter and were then dispensed with a macro- pipette (Macroset-Oxford Laboratories) as 10 ml aliquots into 20 X 125 -mm screw cap culture tubes (Pyrex #9825). The media in the culture tubes were then autoclaved, and, after cooling to room temperature, were stored at 8° C until use. Experiments involving the comparative quality of diets were all performed with media containing the same initial number of particles. Normal concentration was 540 X 103 particles ''nil.
Preparation of inoculum and productivity determinations
To determine the relative nutritive quality of diets with each particular fatty acid composition, 10 to 12 first instar females were inoculated into separate culture tubes containing the same variable. These inocula were n collates of the first and second broods of single females that had been previously isolated from maintenance cultures and grown at 19° C in 20 X 125-mm screw cap culture tubes containing 10 ml of K733 media. From these, replicate inocula populations were allowed to develop by parthenogenesis at various temperatures and defined times. The particles of the artificial media were kept in suspension by daily mixing with a Vortex- genie mixer (Scientific Industries, Inc.). Particle sedimentation rates were slow and most of the particles (ca 70%) would remain in suspension over a 24-hr period. From the 10 to 12 growing populations a set of three or four culture tubes were harvested at three different predetermined particle concentrations (60— 70%, 70-80%, 80%+ optical transmission at 650 iim). Optical transmission was measured by an instrument similar to a Spectronic 20 (Bausch and Lomb) but modified to accept culture tubes.
The sequential harvesting at different particle concentrations permitted an analysis of the changing structure of the Moina populations through time. Animals were killed by the addition of 0.5 ml of ethyl alcohol and then were transferred by pipette to a modified Bogorov counting tray (Wickstead, 1965). With the aid of a binocular stereoscope (36x) each individual comprising a population was counted, sized, and categorized. Categories included female instar I-IV, male instar I-IV, adult females, adult males, ephippial females, and ephippia. Measurements of the animals were made with the use of an ocular micrometer. Measurements were made anteroposteriorly, from a point just distal to the eye to the caudal tip of the carapace.
Average dry weights of the four female instars were determined by selecting a sufficient number of animals of a particular instar and placing them on a pre- tared aluminum dish. These samples were then dried at 60° C for 24 hr in a laboratory oven (Model 10-200C, Grieve-Hendry, Co., Chicago, 111.), cooled for one hr in a desiccator, and weighed immediately on a Calm gram electrobalance (Model G-Cahn Instrument Co., Paramount, California). Additional weighings of a sample were performed until no change in weight could be detected. Average dry weight of a particular instar was determined by dividing the biomass in the dish by the number of instars which comprised the sample. Three samples of well-fed animals were taken for the average weight determination of each instar. Each sample was derived from a population growing on a different diet. A logio- logio plot of average length of an instar vs. average dry weight indicated that
240
LOUIS R. D'ABRAMO
1600-
30°C
FAT MIXTURE TYPE A CRYPTOMONAD • DIATOM
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500
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FIGURE 1. Fatty acid composition dependent qualitative effect. 30° cryptomonad, diatom, and blue green fatty acids at 3 mg%.
the dry weight of Molna increases as the cube of the body length. The equation is: dry weight =1.1 body length3; corr. = 0.9992. The total biomass of a population of Molna was then determined by converting lengths of each individual comprising the population into dry weight and summing the total.
Productivity was determined by dividing the total biomass of an harvested population by the total number of days since the appearance of the first brood of the original female inoculum. The day when the inoculum's first brood appeared was considered day one in order to eliminate some of the observed individual variability associated with time to maturity and thereby provide comparable data among samples of the same medium.
Many of the populations analyzed contained males and ephippial females but their presence did not affect productivity calculations since they invariably had not attained sexual maturity when the populations were harvested.
PRODUCTIVITY OF MOINA
241
1200^
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800-
400-
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FAT MIXTURE TYPE
• CHLOROPHYTE
• DIATOM
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— I 1 1 ' 1~ ~~T~
6.0 7.0 8.0 9.0
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11.0
FIGURE 2. Fatty acid composition dependent quantitative effect. 26° C; diets containing chlorophyte and blue green fatty acids at 3 mg% and diatom fatty acids at 1.5 mg%.
All experimental populations were incubated in darkness. This procedure was used to eliminate the photoperiod factor which has been associated with the transition to sexuality in some species of Cladocera and to prevent the photodegrada- tion of riboflavin, a required vitamin present in all media. Exposure to light wras minimal and occurred only when cultures were shaken or when optical transmission measurements were taken. The source of light was indirect, consisting of a 10 watt light bulb fitted into a photographic safelight stand which was covered with red acetate film. Within the optical transmission apparatus red acetate material covered the slit (passage) between the light source and the area where the culture tube was positioned for a measurement. Light transmitted by the red acetate material was 600 to 700 nanometers. Past research indicates that Cladocera are minimally respon- sive to wavelengths in this region in both visual and nonvisual sensitivity (Scheffer, Robert, and Medioni, 1958).
242
LOUIS R. D'ABRAMO
RESULTS
Particles
Two types of particles were used as food in all media. It was important, there- fore, to determine their size distribution. Although formulated separately, starch- protein and lipid-protein particles did not differ in size distribution. Particle size ranged from 2 to 20 ^3 with the majority of particles (80%) in the 2 to 10 |U,3 range. Because of the similar size distributions it was assumed that there was no different sedimentation rate which could affect the probabilities of ingestion of the two types of particles.
Effects of dietary fatty acids and temperature on productivity
A series of experiments was done to define the effect of varying the quantity and quality of dietary fatty acids at different temperatures. Results of the experi- ments are shown in Figures 1, 2, and 3. Points of graphs represent either a single determination or an arithmetic average of multiple (2-4) determinations of biomass of populations developed on particular diets at selected days of harvest. Points derived from multiple observations include the range of values (vertical bars). Computed regression lines are included. Mortality within populations did not affect productivity determinations since, in most cases, the maximum length of an experimental period never exceeds the life span of Moina. If mortality was ob- served the population was discarded.
o
03
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r i i i i ' i
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FIGURE 3. Fatty acid composition dependent temperature effect. Diets containing chloro- phyte and diatom fatty acids at 3 mg%, 30° C and 22° C.
PRODUCTIVITY OF MOINA
243
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244 LOUIS R. D'ABRAMO
Figure 1 illustrates the qualitative effect of dietary fatty acid composition. At 30° C and 3 mg% total fatty acids, a diet containing cryptomonad-type fatty acids is more productive than those of diatom and blue green type fatty acids. Figure 2 depicts the quantitative effect of dietary fatty acid composition. In this experiment the three diets were supplied at one-half the normal total particle concentration and the content of total fatty acids per particle was varied. At 26° C the diatom type fatty acid diet containing 1.5 mg% of fatty acids is as productive as the blue green type diet which contains 3.0 mg% fatty acid. The chlorophyte fatty acid diet at 3.0 mg% fatty acid concentration is more productive than both. Hence at 26° C the qualitatively poor blue green diet barely equalled the produc- tivity of the qualitatively better diatom diet even with twice the total calorific content.
Figure 3 exemplifies the effect of temperature in relation to the quality of dietary fatty acids. At 30° C and 3 mg% fatty acid concentration, the diet containing diatom fatty acids is slightly more productive than the diet containing the chlorophyte fatty acids. This relationship is inversed at 22° C.
One way ANOVA indicates that productivity differences between chlorophyte (3 mg%) and diatom (1.5 mg%) fatty acid diets at 26° C, the chlorophyte (3 mg%) and blue green (3 mg%) fatty acid diets at 26° C and the chlorophyte (3 mg%) and diatom (3 mg^o) fatty acid diets at 22° C are significant (P < 0.05).
The productivity of every diet decreased as the incubation temperature de- creased. This relationship is illustrated in Table II where productivity values of a cryptomonad fatty acid diet at 3 mg% fatty acid and a blue green fatty acid diet at 3 mg% fatty acids are listed at five different temperatures, 30°, 26°, 22°, 18°, and 14° C. One way ANOVA indicates that differences between these diets at 22° and 18° C are significant (P < 0.05).
Increasing the total amount of fatty acids per particle in diets while keeping the starch-protein ratio constant at 1.5 : 1 had an effect on productivity at the three different temperatures studied. Three diets were made to contain cryptomonad-type fatty acids in concentrations of 1.5, 3.0 and 6.0 mg% respectively. Table III shows that at those temperatures investigated, 22°, 18°, and 14° C, productivity increased from 1.5 to 3.0 mg%. However, an increase to 6.0 mg% reduced productivity, indicating that a too high dietary fat content is inhibitory. An increase in total fatty acids from 3.0 to 6.0 mg% in the chlorophyte type fatty acid diet was also inhibitory at 22° C.
DISCUSSION
Axenic culture of Moina tnacrocopa in almost chemically defined biphasic media has allowed a precise analysis of some dietary factors affecting its population dynamics, and particularly a better understanding of the combined effects of abiotic and biotic factors. It has been established that the chemical composition of food, particularly the type and content of fatty acids, can directly affect productivity. Qualitative fatty acid differences in the diets were formulated in an attempt to simulate the general composition that is unique to each of four orders of algae, Cyanophyceae, Chlorophyceae, Bacillarophyceae, and Cryptophyceae. Variations
PRODUCTIVITY OF MOIX.I 245
in productivity amongst the diets can most probably be attributed to the combined effects of differences in generation time, brood size, and time between broods. The comparative quality of a particular diet can also be temperature dependent.
The poor quality, in general, of those diets containing Cyanophyceae-type fatty acids may partially explain the observed poor nutritional value of some species of blue-green algae (Arnold, 1971). A partial explanation of the different nutri- tiousness of various species of algae for zooplankton species may in fact be the qualitative and quantitative content of dietary fatty acid. Though no fatty acid analyses were performed on Moina organisms grown on particular diets, the notable differences in productivity of the various diets suggest that body fatty acids are derived entirely from dietary sources. The qualitative fatty acid com- position of the lipids of Moina has been found to be significantly affected by diet (Watanabe, Arakawa, Kitajima, Fukusho, and Fujita, 1978). It appears that the zooplankter's capacity for efficient lipid biosynthesis or inter-conversion is poor. Fatty acids were found to be essential to fertility in Moina (Conklin and Provasoli, 1977).
The results of the research conducted with increased fatty acid quantities (1.5 mg%-3 mg%-6 mg%) suggest that subtle interrelationships can exist amongst macronutrient dietary components. Little or no increase in the productivity of Moina was attained by increasing the total cryptomonad fatty acids concentration from 3 to 6 mg% at the different temperatures. The same results occurred for a chlorophyte type fatty acid diet at 22° C. Protein and carbohydrate concentrations remained constant in these experiments. Such observations add to the convincing evidence that crustaceans cannot tolerate high levels of dietary lipid. Andrews, Sick, and Baptist (1972) demonstrated that a dietary lipid supplement (1/3 beef tallow, 1/3 corn oil, and 1/3 menhaden oil) at levels >10% adversely affected growth and survival in the shrimp Pcnaeus setijenis. Forster and Beard (1973) supplemented a shrimp meal based diet for Palaemon serratus with 7.5 and 15% levels of cod liver and corn oil. At the 15% level significant growth inhibition was observed for both lipid sources. The inhibitory effects of increased fatty acids in the diet may also partially explain the nutritional inadequacy of senescent algal cells. Twenty-five percent of the total dry weight of these cells is known to be fat (Fogg, 1965).
Optimal productivity of Moina macrocopa requires the presence of macro- nutrients in the proper proportions. Provasoli and D'Agostino (1969) showed that optimal starch-protein ratios for the growth of Artemia salina were 5: 1 and 10: 1. A 1 : 1 ratio was inhibitory. Hence, the potential quality of food sources cannot be entirely evaluated from considerations of calorific content.
The interesting physiological responses of Moina to varied nutrition permit some speculation. It seems plausible to assume that given the same food, the growth and reproductive capacities of species of zooplankton can be entirely different. Zooplankton community structure in an aquatic system may in part be determined by competitive processes whose outcome is based upon the satisfaction of nutritional requirements and/or the most efficient systems of biochemical assimilation and conversion.
The subtle nutritional interrelationships between predator and prey must be
246 LOUIS R. D'ABRAMO
identified if successful continuous culture of other phagotrophic invertebrates like Moina is to be realized. With a proper understanding of nutritional requirements and efficient energy budgets, the potential husbandry of marine particulate feeders such as lobsters, oysters, scallops, and shrimp can be greatly enhanced. Knowledge of crustacean nutrition may permit the use of cladoceran cultures as an alternative secondary sewage treatment. Filter feeding of participates by the Cladocera would eliminate much of the potential biological oxygen demand (BOD) of primary treat- ment effluent and resulting populations could be harvested and used as fish food.
This work represents part of a dissertation submitted to the Graduate School of Yale University in partial fulfillment of the requirements for the Ph.D. degree, granted in May, 1979. I am deeply grateful to Dr. Luigi Provasoli for his advice and encouragement. This research was supported by a Sigma Xi grant and NSF grant DEB77-07226 to the author and by NSF grant DEB77-05433 to L. Provasoli.
SUMMARY
1. The cladoceran, Moina inacrocopa americana was cultured axenically on an artificial diet consisting of a particulate and soluble phase. The effect of changes in the quantitative and qualitative dietary fatty acid composition was investigated.
2. Qualitative fatty acid differences were made to simulate the unique pro- portional differences found among four orders of algae, Cyanophyceae, Chloro- phyceae, Cryptophyceae, Bacillarophyceae.
3. The quality of dietary fatty acids available to Moina exerts an effect upon productivity and the nutritional value of a particular diet in relation to fatty acid composition can be temperature dependent.
4. Increased levels of fatty acids in the diet of Moina reduces productivity.
5. The cladoceran, Moina macrocof>a americana, may be entirely dependent upon diet for its source of fatty acids.
6. A partial explanation for the differential nutritiousness of particular species of algae may be their qualitative and quantitative fatty acid content.
LITERATURE CITED
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induction of diapause in the pink bollworm, Pcctinophora gossypiella (Sanders).
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energy levels on growth and survival of Penaeid shrimp. AquacnUure 1 : 341-347. ARNOLD, D. E., 1971. Ingestion, assimilation, survival, and reproduction by Daphnia pulex
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PRODUCTIVITY OF MOINA 247
CHUMAKOVA, B. M., 1962. Significance of individual food components for the vital activity
of mature predatory and parasitic insects. Voprosy Ekologii i Biotsenologii, 8 :
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DADD, R. H., 1960. The nutritional requirements of locusts. /. Insect Physiol., 4: 319-347. D'AGOSTINO, A. S., AND L. PROVASOLI, 1970. Dixenic culture of Daphnia magna, Straus.
Biol. Bull., 139: 485-494. FOGG, G. E., 1965. Algal cultures and phytoplankton ecology. University of Wisconsin Press,
Madison, Wis. 126 pgs. FORSTER, J. R. M., AND T. W. BEARD, 1973. Growth and experiments with the prawn Palacinon
serratus Pennant fed with fresh and compounded diets. Fish Invest., Ser. II., 27(7) :
16 pp.
FOSTER, D. R., AND L. A. CROWDER, 1976. Fatty acids of diapause and nondiapause pink boll- worm larvae, Pectinophora gossvpiella (Saunders). Contp. Biochcni. Ph\siol., 55B :
519-521.
GAUDY, R., AND J. P. GUERIN, 1977. Dynamique des populations de Tisbc holothuriae (Crusta- cea: Copepoda) en evelage sur trois regimes artificiels differents. Mar. Biol. 39:
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chimique elementaire de Tisbc holothuriae (Copepoda: Harpacticoida) eleve sur dif- ferents regimes artificiels. Mar. Biol., 44 : 65-70. HUTNER, S. H., AND C. A. BJERKNES, 1948. Volatile preservatives for culture media. Proc.
Soc. Exp. Biol. Med., 67: 393-397. IKAN, R., V. STANIC, E. COHEN, AND A. SHULOV, 1970. The function of fatty acids in the
diapause of the Khapra beetle Trogoderma granarium Everts. Cowp. Biochcni.
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requirements of Nitocra typica, an harpacticoid copepod from salt marsh aufwuchs
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NAYAR, J. K., 1964. The nutritional requirements of grasshoppers. Can. J. Zool, 42: 11-22. PROVASOLI, L., D. E. CONKLIN, AND A. S. D'AGOSTINO, 1970. Factors inducing fertility in
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and Tigriopus in monoxenic culture. Ann. N.Y. Acad. Set., 11 : 250-261. ROMAN, M. R., 1978. Ingestion of the blue green alga Trichodesmium by the harpacticoid
copepod, Macrosctclla gracilis. Limnol. Occanogr. 23(6) : 1245-1247. SCHEFFER, D., P. ROBERT, AND J. MEDIONI, 1958. Reactions oculomotrices de la Daphnie
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SCHINDLER, J. R., 1971. Food quality and zooplankton nutrition. /. Aniin. Ecol., 40 : 589-595. SHIRAISHI, K., AND L. PROVASOLI, 1959. Growth factors as supplement to inadequate algal food
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248 LOUIS R. D'ABRAMO
VANDERZANT, E. S., D. KERUE, AND R. REISER, 1957. The role of dietary fatty acids in the
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quality of living feed from the viewpoint of essential fatty acids for fish. Bull. Jap.
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Reference: B'wl. Bull, 157: 249-257. (October, 1979)
DIFFERENCES IN STARFISH OOCYTE SUSCEPTIBILITY TO POLYSPERMY DURING THE COURSE OF MATURATION 1
TAKUMI FUJIMORI2 AND SETSURO HIRAI 3
Marine Biological Station of Asamushi, Tohoku University. Asannishi, Aoinori-city 039-34 Japan
It is known that the eggs of marine invertebrates such as the sea urchin and starfish lose the capability to fertilize or to develop normally when they are main- tained in sea water for too long a period after being shed (Goldforb, 1935 ; Clark, 1936). In starfish oocytes, the optimum period for insemination is between germinal vesicle breakdown and the formation of the first polar body (Fol, 1879; Delage, 1901) ; insemination after the formation of the first polar body results in a decreased rate of subsequent normal development (Lillie, 1915).
Some earlier investigators reported that starfish oocytes, when inseminated in sea water for several hours, lose their capacity to resist polyspermy (Chambers, 1923; Clark, 1936). Thus, the decreased rate of normal development after the formation of the first polar body may be related to polyspermy. On the other hand, Chambers and Chambers (1949) have suggested that during the course of oocyte maturation, there is a specific period of ripening of cytoplasm when insemination would result in normal fertilization and development; after this period there is a decline in proper functional interrelation between the sperm and cytoplasm.
The present paper reports findings in support of the work of Chambers (1923) and Clark (1936).
MATERIALS AND METHODS
Oocytes from Astcrina pcctinifera and Astcrias aiintrcnsis were used. The starfish were collected at the seashore near the Marine Biological Station of Tohoku University, Asamushi. Specimens of Asterina pcctinijcra were collected in September and kept in an aquarium supplied with circulating cold sea water at 10 to 13° C for two months. Specimens of Asterias amnrcnsis were collected in April and maintained in running sea water tanks at 7 to 9° C for a month in the laboratory.
Asterlna pectinifera. All but a few of these oocytes showed no conspicuous change when isolated from the ovary in sea water. The isolated full-grown oocytes with germinal vesicles were prepared by tearing the ovaries with forceps in sea water and washed three times with sea water. The oocytes were kept for at least 1 hr to make sure that they did not undergo spontaneous maturation.
Asterias amnrcnsis. These oocytes usually undergo spontaneous maturation when
1 Contribution No. 438 from Marine Biological Station of Asamushi, Tohoku University. Asamushi.
2 Present address : Shogakukan, 1 Hitotsubashi, Chiyoda Ward, Tokyo.
3 To whom reprint requests should be sent.
249
250 T. FUJIMORI AND S. I Ilk, VI
Asterias amurensis 13°C
Asterina pectinifera 20° C
0 40 80 120 160 200
t A
FIGURE 1. The time course of oocyte maturation in Asterina pectinifera and Asterias amurensis. This shows the time (min) when germinal vesicle breakdown occurs, and the first and second polar body form. 1-Methyladenine was added at A.
isolated in sea water. To obtain oocytes, ovaries were directly immersed in 1-methyladenine sea water. Full-grown oocytes which began germinal vesicle breakdown were spawned and these oocytes were used.
1-Methyladenine. 1-Methyladenine (Sigma) dissolved in deionized water at a concentration of 10~3 M was served as stock solution, and diluted with sea water to a concentration of 10~G M before use.
Schedule of oocyte maturation. As shown in Figure 1, in an oocyte of Asterina pectinifera, the germinal vesicle disappears completely 40 min after being placed in 1-methyladenine sea water (1O6M) and the first polar body forms at 65 to 75 min and the second polar body at 105 to 115 min, respectively at 20° C. In Asterias amurensis, oocytes which began germinal vesicle breakdown were released from ovary 40 min after ovary was immersed in 1-methyladenine sea water (1Q-6 M). The first polar body forms at 105 to 115 min and the second polar body at 170 to 180 min at 12° C.
Spermatozoa. To obtain a sperm suspension, an isolated testis was torn with forceps in an empty Petri dish. Before use, 10 //,! of semen was sucked up into a micropipette and diluted into 10 ml of 10"4 M histidine sea water to increase the sperms' motility. These sperm suspensions were diluted from 10-fold to 100,000- fold with fresh sea water and the number of spermatozoa was counted with haematometer. Inseminations were carried out by mixing 9 ml of oocyte suspension with 1 ml of sperm suspension.
Cytological procedures. Examined oocytes were fixed with Carnoy's solution and were sectioned by the usual paraffin method. The sections, 10 p in thickness, were stained with Feulgen's reaction and counterstained with fast green.
POLYSPERMY AND OOCYTE MATURATION 251
Experimental procedures. The capacity of oocytes for normal development was investigated as follows ; oocytes of Asterina pcctinifcra were used. Isolated immature oocytes were immersed in 1-methyladenine sea water (1O6 M) at 20° C and then, one part of these oocytes was transferred to another Petri dish containing fresh sea water and inseminated at one period, the other part was transferred to another Petri dish and inseminated at another period, respectivly, so on. Oocytes were 2000 to 2500 per 10 ml sea water and concentration of sperm was finally 2 X 106 per ml. The number of the oocytes which formed fertilization membranes was counted 5 min after each insemination and then these oocytes were kept for 20 hr at 20° C. The oocytes were allowed to develop to early gastrula and the number was counted under a light microscope.
The occurrence of normal first cleavage was checked as follows : the ovaries of specimens of Asterias auntrcnsis were directly immersed in 1-methyladenine sea water (10~6 M) and one part of released oocytes (2000-2500) was transferred to Petri dishes containing 10 ml fresh sea water at 40, 110, 150, and 180 min after the immersion in 1-methyladenine sea water and inseminated respectively. The concentration of sperm used was finally 8 X 105 per ml. Formation of fertiliza- tion membrane and first cleavage were observed 2.5 hr after each insemination. This experiment was performed at 12° C.
The relation between occurrence of abnormal cleavage and sperm concentration was examined as follows: oocytes of Asterias amurensis (2000-2500) were trans- ferred to Petri dishes containing 10 ml fresh sea water at 40 and 180 min after addition of 1-methyladenine sea water (10~6 M) and inseminated with various sperm concentrations which were from 8 X 101 to 8 X 106 per ml, respectively. Formation of fertilization membrane and first cleavage were checked at 2.5 hr after each insemination. This experiment was done at 12° C.
RESULTS
Oocytes of Asterina pcctinifcra were immersed in 1-methyladenine sea water for various periods ranging from 40 to 180 min. The oocytes were inseminated in fresh sea water, at various intervals, after being placed in 1-methyladenine sea water. The formation of fertilization membrane was checked 5 min after insemina- tion, and then the oocytes were continuously kept in sea water for 20 hr ; at the end of the experiment the number of oocytes which developed to early gastrula was counted under a light microscope.
When inseminations were performed between 40 and 80 min after being placed in 1-methyladenine sea water, 73 % of the oocytes developed to early gastrula. Forty-five per cent developed to gastrula, when inseminated at 120 min (period of the formation of second polar body) and only 2% at 180 min, respectively (Fig. 2).
Usually, germinal vesicle breakdown occurs spontaneously in a few per cent of the oocytes after isolation in sea water without 1-methyladenine application in Asterina pec tin if era oocytes. In order to synchronize the course of oocyte maturation, isolated oocytes were kept in sea water for 1 hr and then spontaneously matured oocytes were checked. In this experiment, the mean rate of spontaneous maturation was 19%. For these spontaneously matured oocytes, about 100 min had
252
T. FUJIMORI AND S. HIRAI
100
80
60
AO
20
0
AO
80
120
160
200
FIGURE 2. The percentage of fertilization membrane formation and normal early gastrula in Astcrina pcctinifcra oocytes. Abscissa: time (min) after 1-methyladenine application; ordinate : the rate (%) of fertilization membrane and normal early gastrula formation. Open circles, fertilization membrane formation; closed circles, normal early gastrula. Each point shows mean ± of five experiments.
lOOr
80
60
AO
20
0
AO
80
120 160
200
FIGURE 3. The percentage of fertilization membrane formation and normal first cleavage in Astcrias amurensis oocytes. Abscissa: time (min) after 1-methyladenine application; ordinate: the rate (%) of fertilization membrane formation and normal first cleavage. Open circles, fertilization membrane formation; closed circles, normal first cleavage. Each point shows mean ± of four experiments.
POLYSPERMY AND OOCYTE MATURATION
253
already elapsed after isolation in sea water, before insemination, and these spon- taneously matured oocytes developed abnormally upon insemination. Subsequently, the maximum rate of oocytes which developed to early gastrula did not exceed 73 % even in case of the insemination at 40 min.
Next, the occurrence of first cleavage was checked 2.5 hr after insemination. In this experiment, oocytes of Astenas amurensis were used. After being placed in 1-methyladenine sea water for various times, oocytes were transferred to fresh sea water and inseminated. Although the formation of fertilization membrane occurred in all oocytes, the rate of normal first cleavage decreased with increased intervals after being placed in 1-methyladenine sea water; 82%, when inseminated at 40 min, 69% at 110 min (period of first polar body formation) and only 4% at 180 min (period of second polar body formation) (Figs. 3, 4).
In addition, these abnormal cleavages occurred even if 1-methyladenine was completely removed by washing with fresh sea water after occurrence of germinal
B
FIGURE 4. Normal first cleavage and abnormal cleavage in Asterias amurcnsis oocytes when inseminated at 40 min (A) and 180 min (B) after 1-methyladenine application.
254
T. FUJIMORI AND S. HIRAI
100
80
60
40
20
0
8x10 8x!02 8xl03
8xi05 8x1 06
FIGURE 5. Relation between sperm concentration and normal first cleavage. Abscissa : sperm concentration (per ml); ordinate : the rate (%) of fertilization membrane formation and normal first cleavage. Solid squares, fertilization membrane formation upon insemina- tion at 40 min ; open squares, fertilization membrane formation upon insemination at 180 min ; closed circles, normal first cleavage upon insemination at 40 min; open circles, normal first cleavage upon insemination at 180 min. Each point shows mean ± of five experiments.
vesicle breakdown. Therefore, this abnormality was not due to the excess of 1-methyladenine.
Next, oocytes of Asterias ainnrensis were inseminated at 40 and 180 min after being placed in 1-methyladenine sea water, with various sperm concentrations ranging from 8 X 103 to 8 X 106 per ml. When inseminations were performed
POLYSPERMY AND OOCYTE MATURATION 255
FIGURE 6. Section through an Astcrina pcctiiiifcra oocyte which was inseminated at 180 min after 1-methyladenine application. Arrows show sperm nuclei.
40 min after being placed in l-rnethyladenine sea water, the rate of fertilization mem- brane formation increased with the increased sperm concentrations. The rate of normal first cleavage showed only a slight decrease with higher sperm concen- trations (Fig. 5) ; 87% of these oocytes cleaved normally when inseminated with heavy sperm concentration (8 X 106 per ml). On the other hand, when insemina- tions were performed at 180 min (period of second polar body formation), the rate of normal first cleavage showed a sharp decrease accompanying the in- creased sperm concentrations, although the rate of fertilization membrane formation showed a similar curve seen at 40 min ; the rate of normal first cleavage was 53%, with moderate sperm concentration (8 X 104 per ml) which brought normal cleavage for almost all oocytes that were inseminated at 40 min, 8% with 8 X 105 per ml and only 1% with heavy sperm concentration (8 X 106 per ml). These results showed that the increase of abnormality in the oocytes inseminated at 180 min was due to the increased sperm concentration. Therefore, it seemed that polyspermy occurred in the oocytes inseminated at 180 min. This probability was confirmed cytologically (Fig. 6). Oocytes of Asterina pcctinifcra were inseminated 120 min after being placed in 1-methyladenine sea water with a moderate sperm concentration (5 : 105 per ml), and 5 min after insemination they were fixed with Carney's solution. Microscopic examination showed that most oocytes contained more than two sperm nuclei.
In addition, when insemination was performed with moderate sperm con- centration (8 X 104 per ml) at the period of second polar body formation, the rate of fertilization membrane formation did not attain one hundred per cent (Fig. 5), but about 50% of the fertilized oocytes showed normal first cleavage and most of them developed to early normal bipinnaria.
DISCUSSION
During the course of starfish oocyte maturation, the optimum period for insemination is between germinal vesicle breakdown and first polar body formation
256 T. FUJIMORI AND S. HIRAI
(Fol, 1879; Delage, 1901). Insemination after first polar body formation results in a tendency toward a decline of normal development ; abnormality becomes more pronounced when oocytes are inseminated 30 to 60 inin after second polar body formation (Lillie, 1915). But in these studies, the cause for occurrence of abnormality was not clarified. In our results, when inseminations were per- formed between germinal vesicle breakdown and first polar body formation, ferti- lization membrane formed normally in all cases and the first cleavage occurred normally in most oocytes. However, when inseminations were performed after first polar body formation, the rate of first cleavages and subsequent development apparently decreased. Only a small percentage of oocytes underwent normal cleavage when inseminated after second polar body formation, although fertiliza- tion membrane was formed normally in all oocytes. Figure 5 shows the increased rate of abnormal cleavage accompanying increased sperm concentrations in oocytes with second polar body: with moderate sperm concentration (8 X 104 per ml) the rate of normal cleavage was 53%, but with high sperm concentration (8 X 106 per ml), the rate of normal cleavage was only one per cent. However, even at the same high sperm concentration, normal cleavage occurred in 87% of the oocytes which completed germinal vesicle breakdown. Thus, these results sug- gested that the increased rate of abnormal cleavage was due to the occurrence of polyspermy. The occurrence of polyspermy was confirmed by cytological observa- tion.
Chambers and Chambers (1949) have suggested that there is a decline in proper functional interactions between sperm and egg cytoplasm when insemination is performed after the optimum ripening period. In the present study, however, some fertilized oocytes, which were inseminated after second polar body forma- tion, showed normal cleavage and developed to early bipinnaria if the sperm concentration was light. It is suggested that as long as polyspermy was prevented, the oocyte remained viable, even after second polar body formation.
Earlier investigators reported that starfish oocytes become susceptible to poly- spermic fertilization after standing in sea water less than two hrs (Chambers, 1923; Clark, 1936). In their studies, the occurrence of polyspermy and abnormal development were not considered in relation to various periods of oocyte maturation. Our study confirms that the number of starfish oocytes showing polyspermy gradually increases with increasing time intervals between first polar body formation and sperm addition, and that almost all the oocytes show poly- spermy when the insemination is performed after second polar body formation. Even in the latter case, however, fertilization membrane is normally formed. It is postulated that polyspermy occurs before fertilization membrane formation and that there is some mechanism (s) of protection against polyspermy between the first sperm entry and fertilization membrane formation. The mechanism (s) may be gradually lost after first polar body formation and disappear completely after second polar body formation.
On the other hand, polyspermy occurs regardless of the fertilization membrane formation in immature oocytes which have intact germinal vesicles (Cayer, Kishi- moto and Kanatani, 1975; Hirai, 1976). Thus, the protective mechanism(s) against polyspermy may be acquired during the maturation process.
In sea urchin eggs, it was recently demonstrated by Jaffe (1976) that poly- spermy was quickly inhibited as a result of electrical depolarization brought about
POLYSPERMY AND OOCYTE MATURATION 257
by the entry of the first spermatozoon. In starfish oocytes, it was reported that potassium conductance of oocyte membrane changed during maturation, thus altering the form of action potential subsequently generated (Miyazaki, Ohmori and Sasaki, 1975). Potassium conductance was small before first polar body forma- tion, and larger after it (Miyazaki, Ohmori and Sasaki, 1975). It is suggested that the change of potassium conductance during oocyte maturation may affect electrical depolarization brought about by the entry of the first spermatozoon. Further examination would be desirable.
We thank Dr. K. Osanai for critical advice and Dr. H. Kanatani, Dr. S. Shirai and Dr. T. Kishimoto for helpful discussion. We also wish to thank Dr. Y. Nagahama for his critical reading of the manuscript. This work has been supported in part by a grant in aid from the Ministry of Education.
SUMMARY
During the course of starfish oocyte maturation, the optimum period for insemi- nation is between germinal vesicle breakdown and the first polar body formation. The rate of polyspermy increases with increasing time intervals between the first polar body formation and sperm addition, although the fertilization membrane is formed normally in all oocytes. As long as polyspermy is prevented, however, the oocytes remain viable even after the formation of second polar bodies. Thus, it is postulated that there is some mechanism (s) of protection against polyspermy between the first sperm entry and the fertilization membrane formation. The mechanism (s) may be gradually lost after the first polar body formation and disappear completely after the second polar body formation.
LITERATURE CITED
CAYER, M. L., T. KISHIMOTO, AND H. KANATANI, 1975. Formation of the fertilization
membrane by insemination of immature starfish oocytes pretreated with calcium-free
sea water. Dcv., Groivth Differ., 17: 119-125. CHAMBERS, R., 1923. The mechanism of the entrance of sperm into the starfish egg. /. Gen.
Physiol., 5: 821-829. CHAMBERS, R., AND E. L. CHAMBERS, 1949. Nuclear and cytoplasmic interrelations in the
fertilization of the Astcrias egg. Biol. Bull., 96 : 270-282.
CLARK, J. M., 1936. An experimental study of polyspermy. Biol. Bull., 70 : 361-384. FOL, H., 1879. Recherches sur la fecondation et le commencement de I'henogenie chez divers
animaux. Geneve Soc. Phys. Mem., 26 : 89-397. DELAGE, Y., 1901. Etudes experimentales sur la maturation cytologigue et sur la partheno-
genese artificielle ches les echinodermes. Arch. Zoo/. E.rp. 3 scr., 9 : 285-326. GOLDFORB, A. J., 1935. Viscosity changes in aging unfertilized eggs of Arhacia punctuhitn.
Biol. Bull, 68: 191-206. HIRAI, S., 1976. The role of germinal vesicle material on the swelling of sperm nuclei
penetrated in immature oocytes of the starfish. Bull. Mar. Biol. Stn.. Asainushi, 15 :
165-171. JAFFE, L. A., 1976. Fast block to polyspermy in sea urchin eggs is electrically mediated.
Nature, 261: 68-71. LILLIE, R. S., 1915. Momentary elevation of temperature as a means of producing artificial
parthenogenesis and the conditions of its action. /. E.rp. Zoo/., 5 : 375-428. MIYAZAKI, S., H. OHMORI, AND S. SASAKI, 1975. Potassium rectifications of the starfish
oocyte membrane and tleir changes during oocyte maturation. /. Physiol., 246 : 55-78. MIYAZAKI, S., H. OHMORI, AND S. SASAKI, 1975. Action potential and non-linear current- voltage relation in starfish oocytes. /. Physiol., 246 : 37-54.
Reference: Diol. Unll., 157: 258-274. (October, 1979)
REPRODUCTION AND DEVELOPMENT OF THE HERMAPHRODITIC SEA-STAR, ASTERINA MINOR HAYASHI
MIfiKO KOMATSU, * YASUO T. KANO, HIDEKI YOSHIZAWA, SHOJI AKABANE AND CHITARU OGURO
Department of Biology, Faculty of Science, Toyama University, Toyama 930;
Uozu Aquarium, Uozu, Toyama 937; Matsumoto Dental College,
Shiojiri, Nagano 399-07, Japan
A number of papers have been published on the development of sea-stars belonging to the genus Asterina, but the entire process of the development from eggs to juveniles is known only in four species: A. gibbosa (Ludwig, 1882; Goto, 1898; MacBride, 1896), A. burtoni (James, 1972), A. coronata japonica (Komatsu, 1975) and A. bathcri (Kano and Komatsu, 1978). Among these, description of A. burtoni is quite brief and information on the development of other asterinids is fragmental.
Sea-stars generally are gonochoric, but a few hermaphroditic asterinid species, namely A. bathcri, A. gibbosa, A. scobinata and A. panccrii (Ohshima, 1929; Delavault, 1966; Dartnall, 1970) have been known. The details of breeding in these hermaphroditic species, however, are not well documented, although the gonadal structure and development of some species like A. gibbosa (MacBride, 1896) and A. bathcri (Ohshima, 1929; Kano and Komatsu, 1978) have been reported in detail.
The present study was initiated to determine the breeding and development of A. minor, which was recently described as a new species (Hayashi, 1974). The preliminary observations revealed that the present species had a distinct breeding behavior. Moreover, it is found that A. minor is hermaphroditic and is able to self-fertilize. This feature being unique among sea-stars, it was felt worthwhile to have a thorough understanding of the breeding and development of this species.
The present paper describes the structure of the gonad, breeding behavior, and development throughout metamorphosis in A. minor. It also incorporates the observations on isolated cultures studied during the breeding season.
MATERIALS AND METHODS
Specimens of A. minor were collected from the undersurface of stones at the intertidal zone of Kushimoto, Wakayama Pref., Japan, on several occasions during May of 1972 to June of 1977. They were brought to the laboratory of Toyama University and kept either in groups or individually.
The development of the species from fertilization to the completion of metamorphosis was observed with the help of a dissecting microscope and an inverted microscope. Measurements of living embryos were executed with an ocular micrometer. Examination of the skeletal system wras performed on alcohol- fixed specimens treated with potassium hydroxide solution.
For microscopic observation of the reproductive organs, fresh specimens were measured and weighed, and then fixed in Bouin's solution. The fixed material was
1 Address for reprints : Dr. Mieko Komatsu, Department of Biology, Faculty of Science, Toyama University, Toyama 930, Japan.
258
ASTERINA BREEDING AND DEVELOPMENT
259
A
am
FIGURE 1. A) Aboral view of the arrangement of mature gonads in the specimen, col- lected on April 25, 1975, just before spawning. Aboral body wall and viscera were removed. am, ampulla; g, gonad ; is, interradial septum; vr, vertebral ridge; Scale = 1 mm. B) Rem- nants of germinal substance, mostly egg fragments, attached to the gonopores in the specimen, collected on June 13, 1974, which was after spawning, ad, adambulacral plate ; mt, remnants of germinal substance; o, oral plate. Scale = 500 pm. C) Aboral view of the gonads in the specimen collected on June 13, 1974, which was after spawning, am, ampulla; g, gonad; is, interradial septum ; vr, vertebral ridge. Scale = 500
serially sectioned at 7 hematoxylin and eosin.
by routine paraffin method and stained with Delafield's
OBSERVATIONS AND RESULTS Breeding season
In the present study, no observations were made to confirm spawning in the field. However, it is felt that a possible breeding season of A. minor is during May at Kushimoto. This prediction is based on the following circumstantial evidence. In the specimens examined, the gonads were largest in size and nearly mature in late April (Figs. 1A, 2A-F). In adults collected in June, some particles, possibly fragments of ova, were often observed near the gonopores (Fig. IB). Similar particles were usually observed in the specimens spawned under laboratory con- ditions. The gonads of the individuals collected in June showed an atrophy and contained degenerating ova (Figs. 1C, 3A, 3B). Spawning in the laboratory as observed for 3 years occurs in the month of May (Table I). The juveniles of this species, each bearing two pairs of tube-feet in each arm, were collected in the month of June. They were estimated to be 20 to 30 days old since their fertilization. In the later months (July onward), the juveniles found in the field were larger than those collected in June (Table II).
Structure of the gonad
A pair of gonads lies at each interradial portion and each gonad is composed of cluster of tubules (Figs. 1A, 1C). Histological observations on the specimens
260
KOMATSU ET AL.
TABLE I
Occurrence of spawning of specimens of Asterina minor in the laboratory.
|
Number of animals kept together |
Date of collection in the field |
Time and date of the commencement of spawning in the laboratory |
|
40 |
April 25, 26, 1974 |
21:20 May 8, 1974 |
|
2 |
April 25, 1975 |
17:05 Mav 2, 1975 |
|
to |
April 25, 1975 |
13:00 Mav 11, 1975 |
|
4 |
April 25, 1975 |
9:20 May 24, 1975 |
|
2 |
Feb. 16, 1976 |
20:25 Mav 12, 1976 |
|
2 |
April 28, 1976 |
17:30 Mav 19, 1976 |
|
2 |
April 28, 1976 |
20:55 Mav 22, 1976 |
|
1 |
April 28, 1976 |
0:00 May 25, 1976 |
|
2 |
April 28, 1976 |
20:00 Mav 25, 1976 |
measuring 2.5 to 6.0 mm in R show that this species is a spatial hermaphrodite. Each gonad consists of both the ovarian tubules and the testicular ones (Figs. 2A, 2B) and has a common gonoduct which opens on the oral side of the disk (Figs. 3 A, 3B). In the breeding season, the ovarian tubules show pale yellow color and the testicular tubules are whitish and semi-transparent. In general, the testicular tubules lie near the gonoduct and are smaller than the ovarian tubules located in the peripheral region (Fig. 2C). The distribution of the ovarian and testicular tubules, however, varies in different gonads and also in different individuals. In some cases, both sex elements exist in a single tubule (Figs. 2D, 2F). After spawning, all gonads become shrunken and transparent and show a green tint (Figs. 1C, 3A, 3B).
Breeding behavior
Breeding assemblage was observed in the laboratory every year. Although spawning was not observed in the field, assemblage of this species was often found
TABLE II
Number and size distribution of juveniles (under 17 pairs of tube-feet) collected in the field.
|
Number of tube-feet in the longest arm (in pairs) |
June 10-12, 1975 |
Julv 3-6, 1974 |
Sept. 11-13, 1973 |
Feb. 16, 1976 |
|
2 |
10 |
4 |
||
|
3 |
31 |
1 |
||
|
4 |
27 |
5 |
||
|
5 |
6 |
26 |
||
|
6 |
32 |
|||
|
7 |
42 |
|||
|
8 |
29 |
|||
|
9 |
1 |
7 |
1 |
|
|
10 |
1 |
3 |
||
|
11 |
4 |
5 |
||
|
12 |
22 |
19 |
||
|
13 |
36 |
9 |
||
|
14 |
18 |
3 |
||
|
15 |
6 |
2 |
||
|
16 |
3 |
1 |
||
|
17 |
2 |
1 |
ASTERINA BREEDING AND DEVELOPMENT
261
A' ".*>". .. ^^fc-— - '
FIGURE 2. Section of the gonad of a specimen of Astcrina minor, which was fixed just before spawning. Hematoxylin-eosin stain. A) A testicular and an ovarian tubule, containing mature sperms and ova, respectively. B) Magnified picture of the testicular tubule in Figure 2A.
C) Section of gonad showing that testicular tubules (te) are situated close to the gonoduct (d).
D) A hermaphroditic gonad, in which ovarian part is dominating. E) A hermaphroditic gonad, in which testicular part is dominating. F) Magnified picture of a part of Figure 2E. Scale = 100 //m in A, C, E. Scale = 50 Aim in B, D, F.
in the field in May. The following is a description of the typical process of breed- ing assemblage.
Forty animals which were collected on April 25 and 26, 1974, began to organize into two groups in a large glass container in the laboratory several days after their collection. These groups were not very stable and animals moved frequently from one group to the other. Generally after about 10 days the assem- blages became stable, and no animals seemed to move from their respective group. At this time animals in either group clung to one another along the margins of their bodies, or they were imbricated with others to some extent.
262
KOMATSU ET AL.
B
oo
FIGURE 3. Sagittal section of the gonad of a specimen of Astcrina minor, showing gonoduct and gonopore. A) This specimen was fixed during spawning. Note a spawned egg (ov) close to the body surface of the adult and fertilization membrane (f). d, gonoduct; oo, ovarian ova; v, ventral body wall of the adult. Scale = 50 ,um. B) This specimen was fixed just after spawning was finished. Note a remnant of sperms (s) in the gonoduct (d) and degenerating ova (do), oo, ovarian ova; te, testis. Scale = 25 /j.m.
At 21 : 20 May 8, one animal in an assemblage comprised of 25 individuals began to spawn (a, in Fig. 4). As will be described later the eggs got attached to the substratum after their release. About 15 min thereafter, about 80 eggs were laid by this animal. At 22: 50, three other animals (b, c, d, in Fig. 4) began to spawn and several minutes later each animal had delivered about 20 eggs. About 30 min thereafter, four more animals (e, f, g, h, in Fig. 4) began to spawn. Two
TABLE III Spawning of specimens of Asterina minor in isolated culture.
|
Size of animal (mm) R r |
Fresh weight of newly collected animal (mg) |
Fresh weight o' animal after spawning (mg) |
Decrease of weight by spawning (%) |
Number of eggs spawned |
Term in isolated culture (day) |
|
7.0 4.2 |
188.3 |
106.0 |
44 |
297 |
27 |
|
6.5 4.5 |
149.4 |
100.4 |
33 |
233 |
87 |
|
5.6 3.3 |
148.3 |
— |
— |
180 |
85 |
|
5.5 4.1 |
94.1 |
77.7 |
17 |
243 |
21 |
|
5.4 4.0 |
196.4 |
92.8 |
53 |
261 |
21 |
|
5.1 3.7 |
79.0 |
69.7 |
12 |
127 |
19 |
|
5.1 3.3 |
62.3 |
45.2 |
27 |
132 |
29 |
|
5.0 3.8 |
89.3 |
69.8 |
22 |
137 |
39 |
|
5.0 3.2 |
67.6 |
56.3 |
17 |
116 |
28 |
|
4.7 3.5 |
37.8 |
29.5 |
22 |
87 |
21 |
|
4.2 3.8 |
45.8 |
34.9 |
24 |
44 |
100 |
|
4.1 2.9 |
16.4 |
10.5 |
36 |
6 |
33 |
ASTERINA BREEDING AND DEVELOPMENT
263
9.20 p.m., V-8
l.20p.m.,V-8
6.30<J.m.,V-9
2.30 a.m., V-9
FIGURE 4. Sequential change of the distribution of individuals in a breeding assemblage. Dotted areas show the deposition of eggs. See text for detail.
hours later, three more animals (i, j, k, in Fig. 4) were found to be spawning, and soon four other animals (1, m, n, o, in Fig. 4) followed. After that, the majority of animals in this assemblage were found to be spawning. About 6 hr after the first spawning, all animals seemed to have completed spawning. At 6:30 the following morning (May 9), the assemblage was almost disordered. In the other assemblage, spawning commenced 3 hr after the onset of the spawning in the first one. The process of spawning in the other assemblage was very similar to that in the first. In the evening of May 9, the assemblages were completely disorganized and constituent individuals in either assemblage got mixed. After the disorganization of the assemblages, two egg masses were found, and these were not protected by the adults.
Isolated culture, a proof of self-fertilisation
From the characteristic breeding assemblage observed, it seems that this species engages in cross-fertilization. However, there is a possibility that the species also indulges in self-fertilization, since histological observations show
264
KOMATSU ET AL.
FIGURE 5. Development of specimens of Astcrina minor. All pictures show living specimens. A) Egg just after spawning. Fertilization membrane was not yet formed. Sperms are crowding around the jelly layer. B) A mass of sperms (arrow) is seen near the spawning
ASTERINA BREEDING AND DEVELOPMENT 265
both male and female elements simutlaneously maturing in one individual. In order to know whether the species was able to self-fertilize, observations were made on individuals in isolated culture. Specimens, immediately after their col- lection in the field, were kept individually in small glass jars. Temperature during the culturing was maintained similar to that of the natural habitat, 15 to 25° C. The majority of animals in the isolated culture deposited eggs and these eggs were fertilized with the sperms ejected from the same animal in the absence of any artificial treatment. The details in respect of isolated culture and self-fertilization are given in Table III.
The eggs possibly became mature during the travel through the gonoduct or else as soon as they were released. The number of eggs spawned by one adult is related to the size of the adult. The self-fertilized eggs developed into normal embryos and became normal juveniles through metamorphosis (Fig. 5K). No differences were observed in the fertilization rates or developmental processes in the specimens held in mass culture and those kept in an isolated culture. Adults were collected in different months of the year and kept separately, but they spawned within a limited span of time in the laboratory.
The above observations demonstrate that A. minor has an ability to self- fertilize when kept singly. However, it is not clear whether or not self-fertilization occurs when the animal is spawning in the breeding assemblage.
Development
In this species no fertilization membrane was observed in the freshly laid eggs (Fig. 5 A).
The egg is spherical with a diameter of 437 ± 5 /xm (mean ± s.e., n = 37). They are transparent yellow and have a jelly layer of about 20 /xm thick. A -few minutes after spawning, sperm masses are released from the same adult (Fig. 5B) and the sperms are dispersed. The head of the sperm is spherical, about 3 /xm in diameter, and the tail is about 30 /xm in length. The eggs a few minutes after spawning, attach to the substratum and the neighbouring eggs by their sticky jelly layer. Ten minutes after spawning, elevation of the fertilization membrane is recognized. Figure 5C shows fertilization membrane in an egg 30 min after spawn- ing. The spawned eggs are laid on the bottom in one layer (Figs. 5D, 5E). About 2 hr after spawning, polar bodies are seen in the perivitelline space, which is about 50 /xm in height (Figs. 5F, 5G). Three hours after spawning at 20 to 23° C, the first cleavage occurs through the animal-vegetal axis (Figs. 5H, 6A), and 40 min thereafter it is followed by the second cleavage which is perpendicular
adult (at). Fertilization membrane is being formed in an egg (o), about 10 min after spawning. C) Egg about 30 min after spawning. D) Adult (at) and eggs spawned from it, 1 hr after the commencement of spawning. E) Magnified picture of a part of Figure 5D. Two eggs are attached to each other by their jelly layers (arrows), which lie outside of the fertiliza- tion membrane. F) Eggs 2 hr after spawning. G) Magnified view of F. Note polar bodies (arrow). H) First cleavage, 3 hr after spawning. I) Hatching brachiolaria, view from the ventro-lateral (left) side. Long and short arrows indicate fertilization membrane and brachiolar arms, respectively. J) Developed brachiolariae, creeping on the substratum. K) Metamorphosed juveniles, 15 days after spawning. Scale = 50 /mi in D, G. Scale = 100 /mi in A, B, C, E, F, H, I. Scale = 200 /mi in J. Scale = 100 /mi in K.
266
KOMATSU ET AL.
FIGURE 6. Development of specimens of Astcrina minor. All drawings are from a living specimen, jelly layer is not shown except in A. All scales show 100 /j.m. A) Two-cell stage, f, fertilization membrane; j, jelly layer. B) Eight-cell stage, 4.5 hr after spawning, f, fertiliza- tion membrane. C) 64-cell stage, 6.5 hr after spawning, bm, blastomere. D) Early wrinkled blastula, 10 hr after spawning, cm, cell mass; et, egression tract. E) The most wrinkled blastula, 12.5 hr after spawning. F) Early gastrula, 18 hr after spawning. Egression tracts (et) are still recognized on the surface of the embryo, view from the vegetal pole, bp, blasto- pore. G) Early gastrula, 18 hr after spawning, view from the animal pole, et, egression tract. H) Gastrula, 36 hr after spawning. I) Gastrula, 40 hr after spawning, bp, blastopore. J) Brachiolaria, 2 and a half days after spawning, view from the ventro-lateral (right) side. K) Brachiolaria, 3 days after spawning, ventral view, ba, brachiolar arm ; su, sucker. L) Brachiolaria at the commencement of hatching, 4 days after spawning, view from the ventro- lateral (left) side, f, fertilization membrane. M) Brachiolaria, just after hatching, ventral view, ba, brachiolar arm; su, sucker. D) Same as M, dorsal view, ba, brachiolar arm; hp, hydropore. O) Same as M, ventro-lateral (right) view, hp, hydropore.
ASTERINA BREEDING AND DEVELOPMENT 267
to the first. The cleavage is total, equal, and radial. The embryo reaches the eight-cell stage (Fig. 6B) and the 64-cell stage (Fig. 6C), 4.5 and 6.5 hr respec- tively after spawning. Eight hours after spawning, the embryos enter into the wrinkled blastula stage. The process of wrinkling has been earlier reported in detail (Komatsu, 1976). Figures 6D and 6E illustrate an early wrinkled blastula and most wrinkled blastula, respectively. Twenty-four hours after spawning, the wrinkled blastula stage is completed.
At the end of the wrinkled blastula stage, gastrulation by invagination takes place (Figs. 6F, 6G). Thirty-six hours after spawning, the gastrula begins to rotate within the fertilization membrane (Fig. 6H) and becomes enlarged along the archenteric axis. Figure 61 shows a gastrula 40 hr after spawning. It measures about 500 p,m in length and 350 ^m in width. Two days after spawning, the ventral side of the embryo becomes flattened and the rudiments of the brachiolar arms (lateral arms) appear at the A'entro-lateral side of the embryo. These arms grow gradually and the third arm (anterior arm) emerges from the ventral side of the anterior portion of the embryo. Two and a half days after spawning, the embryo becomes pear shaped, with three distinct brachiolar arms and a rudimentary sucker which appears in the area surrounded by the brachiolar arms (Fig. 6J). Three days after spawning, the brachiolaria measures 550 //m in total length (Fig. 6K). The anterior half of the embryo, which bears three brachiolar arms and a sucker, corresponds to the stalk of the larva and the posterior half is the disk of the larva. Lateral arms are longer, 150 /xtn in length, than the anterior arm, which is about 100 /mi. A blastopore and a hydropore are observed at the posterior tip and at the dorsolateral (right) side of the larva, respectively. Three and a half days after spawning, the blastopore is closed.
About four days after spawning, the brachiolaria is hatched from the fertilization membrane (Figs. 51, 5L), usually at the anterior portion. Figures 6M, 6N and 60 show a freshly hatched larva. The present species has no pelagic life and the brachiolariae creep on the substratum with their developed brachiolar arms through- out the brachiolaria stage (Fig. 5J). One day after hatching, the brachiolariae attach to the substratum with their brachiolar arms and not with the sucker (Figs. 7A, 7B). This attachment is a sign of the commencement of meta- morphosis. At this stage, the brachiolar arms are markedly long and appear semi- transparent excepting at the tip ends. The anterior arm measures 250 /u.m, and the lateral arms are 350 ^m in length.
About 12 hr later, the disk begins to transform into a subpentagonal form (Fig. 7C), and the stalk begins to shrink rapidly. The rudiments of the tube-feet are seen on the hydrolobes, these are clearly visible in the future oral side of the adult disk. Figures 7D and 7E show a larva one day after the one shown in Fig. 7C. This larva is about 500 /j.m in diameter and has a hydropore in an inter- radius of the future aboral side of the disk. A degeneration of the stalk, including the brachiolar arms and sucker, progresses gradually. About 7 days after spawning, the larvae are freed from the substratum due to the extreme degeneration of the brachiolar arms. They are able to move by means of their tube-feet (Figs. 7F, 7G). On the aboral side of the larva at this stage, one central and five inter- radial plates are recognized (Fig. 7H). Metamorphosis is completed with the opening of the mouth about 10 days after spawning (Figs. 71, 7J). At this stage,
268
KOMATSU ET AL.
K
r\
ASTERINA BREEDING AND DEVELOPMENT 269
the juvenile is 700 //.m in diameter, and bears a red eye-spot at the basal portion of each terminal tentacle. On the oral side, a rudiment of the stalk is still observed. In each interradius, the formation of a pair of oral plates is noticed. Skeletal plates on the aboral side are shown in Fig. 7K. It is of interest to note that a slit is seen at the midline of each terminal plate, making it appear as if one terminal plate is composed of two pieces. Fifteen days after spawning, that is about 5 days after the completion of metamorphosis, the juveniles start moving from the place where they were laid (Figs. 5K, 8A). At this stage, each oral plate bears a spine which is pointed at the center of the mouth (Figs. SB, SC). On the aboral side of disk, small radial plates are being formed (Fig. 8D). After the spawning the juveniles grow to the size of 1000 and 1200 /mi in diameter after 20 and 30 days, respectively. In specimens 30 days after spawning, each radial plate bears one spine at the center. In each interradius of the aboral side, one pair of the secondary plates is formed. On the oral side, a pair of adambulacral plates is formed in each arm and each adambulacral plate is furnished with one spine. The juveniles were kept in the laboratory several months after the completion of metamorphosis, but no further development was observed beyond the stage 30 days after spawning (20 days after the completion of metamorphosis). Field surveys were then undertaken to obtain supplementary material for understanding post-metamorphic development. As shown in Table II, a number of juveniles were collected. Among these, two animals are described here in detail. One animal collected on July 3, 1974, was 1500 /*.m in diameter and had three pairs of the tube-feet in each arm (Figs. 9, 10, 11 A). At each interradius, one pair of superomarginal plates was present, each plate having two prominent spines. On the oral side, five pairs of oral plates encircle the mouth. In each arm, two pairs of adambulacral plates and one pair of inferomarginal plates were present. The first adambulacral plate had two small spines. The second adambulacral plate was smaller than the first, and each of them had one spine. The other speci- men collected on Sept. 7, 1973, was 3000 //.m in diameter and possessed seven pairs of tube-feet in each arm (Figs. 11B, 11C). This specimen had well-developed aboral skeletal plates, some of which were imbricated with each other. Between the neighboring interradial plates, there was papular area, each of which had one papula. In one of the interradial plates there was a concave portion and this might be a rudiment of the madreporite. Each arm had several pairs of supero-
FIGURE 7. Development of specimens of Astcrina minor. All drawings are made from living specimens, except H and K, which were treated with KOH solution before examination. All scales show 100 ^m. A) Brachiolaria, 5 days after spawning, strongly attached to substratum with brachiolar arms (ba), dorsal view. B) Same as A, ventral view, ba, bra- chiolar arm; su, sucker. C) Metamorphosing larva, 12 hr after that shown in Figures 7 A and B, view from the future aboral side of the juvenile, ba, brachiolar arm D) Metamorphosing larva, 1 day after that shown in Figure 7C, future oral view, ba, brachiolar arm ; h, hydrolobe ; su, sucker. E) Same as D, future aboral view, ba, brachiolar arm. F) Metamorphosing larva, 7 days after spawning, future oral view, st, stalk ; tf, tube-foot ; tt, terminal tentacle. G) Same as F, future aboral view. H) Aboral skeletal plates, in the same stage shown in Figures 7F and G. c, central plate ; i, interradial plate ; ts, spines on the terminal plate. I) Juvenile just after the completion of metamorphosis, 10 days after spawning, aboral view. J) Same as I, oral view, e, eye-spot; mo, mouth; st, markedly degenerated stalk. K) Aboral skeletal plates, in the same stage shown in Figures 71 and J. c, central plate ; i, interradial plate ; t, terminal plate ; ts, spines on the terminal plate.
270
KOMATSU ET AL.
A
t
C
B
D
FIGURE 8. Juvenile of specimens of Asterina minor, five days after the completion of metamorphosis, having two pairs of tube-feet. A and B are drawn on living specimens and C and D are on KOH treated specimens. All scales show 100 /jfn. A) Aboral view, c, central plate; i, interradial plate; r, radial plate; t, terminal plate; tf, tube-foot. B) Oral view, os, oral spine; tf, tube-foot; tt, terminal tentacle. C) Skeletal plates of an arm, oral view, a, ambulacral plate ; o, oral plate ; os, oral spine ; t, terminal plate ; ts, spine on the terminal plate. D) Aboral skeletal plates, c, central plate; i, interradial plate; r, radial plate ; t, terminal plate.
marginal plates and inferomarginal plates, the latter both in size and number were larger than the former. Six pairs of adambulacral plates were present in each arm, and each of the three proximal plates (1st, 2nd, and 3rd) possessed three spines. In the interradial area of the oral side, there were several ventro-lateral plates in addition to adambulacral plates and inferomarginal plates. One slit was always recognizable along the midline of the terminal plate.
ASTERINA BREEDING AND DEVELOPMENT
271
sm
FIGURE 9. Aboral skeletal plates of a juvenile specimen of Asicrina minor, with three pairs of tube-feet in each arm, KOH treated, c, central plate; i, interradial plate; r, radial plate ; sm, supermarginal plate. Scale = 250 /mi.
FIGURE 10. Skeletal plates of a juvenile specimen of Aster ina minor, in the same stage shown in Figure 9, KOH treated. A) Shadow picture showing spines on terminal plates and contour of the body. Scale = 500 /am. B) Picture of plates in one arm and disk, in the same specimen shown in Figure A. Shaded area corresponds to plates in the oral side. Scale = 150 /mi. C) Magnified picture of the central portion of Figure B. Scale = 150 /mi.
272
KOMATSU ET AL.
A
-
B
FIGURE 11. Skeletal plates of juvenile specimens of Astcrina minor, treated with KOH. A) Oral skeletal plates in one arm in the specimen shown in Figure 9. a, ambulacral plate ; ad, adambulacral plate ; im, inferomarginal plate ; o, oral plate ; os, oral spine ; vl, ventro-lateral plate. Scale = 200 /itn. B) Aboral skeletal plates of a specimen having seven pairs of tube- feet in each arm, hatched portions show papular area, m, interradial plate with a rudiment of madreporite. Scale = 450 /uni. C) Skeletal plates in the oral side of one arm in the specimen shown in Figure B. ad, adambulacral plate ; im, inferomarginal plate ; o, oral plate ; vl, ventro-lateral plate. Scale 500 = /um.
DISCUSSION
A. minor lacks the bipinnaria stage and develops without pelagic life as known in A. gibbosa and A. cxiyna ( Ludwig, 1882; MacBride, 1896; Mortensen, 1921). In general, asteroids whose development is direct have pear-shaped brachiolaria with three brachiolar arms. In species belonging to Asterina, the brachiolar arms of A. batheri and A. coronata japonica are short and blunt (Komatsu, 1975; Kano and Komatsu, 1978). The brachiolariae of these species have pelagic life. On the other hand, the brachiolar arms of A. minor are well developed and resemble those of A. gibbosa and A. c.vigna, and yet these brachiolariae spend benthonic life creeping on the substratum before metamorphosis. It is likely, therefore, that well-developed brachiolar arms are one of the adaptive characters of the benthonic life.
ASTERINA BREEDING AND DEVELOPMENT 273
As described before, asteroids, barring a few hermaphroditic species, are gono- choric (Delavault, 1966). Sexuality of asterinids has been fairly well studied since Cuenot (1887) reported the occurrence of protandric hermaphroditism in A. gibbosa. Among asterinid species, in addition to A. gibbosa, hermaphroditism is known in A. panccrii, A. batheri and A. scobinata (Ohshima, 1929; Cognetti, 1954; Dartnall, 1970; Kano and Komatsu, 1978). In A. batheri a few hermaphroditic individuals occur among gonochoric individuals (Ohshima, 1929). No difference seems to exist in the sexual status of this species from different geographical regions (Kano and Komatsu, 1978). In this study, it was found that A. minor is a spatial hermaphroditic and all individuals bear hermaphroditic gonads comprising both functional testes and ovaries. In the breeding season, both elements mature simultaneously and the eggs can be fertilized with the sperm released from the same individual. This study has presented for the first time evidence of definite self-fertilization in asteroids.
Chia (1968) reported that Leptasterias Iie.vactis aggregates under rocks during breeding season. Such a gathering is usual behavior in brooding species (Kubo, 1951). It is interesting to note that A. minor shows a distinct breeding assemblage, although it is not a brooding species and is capable of self-fertilizing. These facts may imply a complex historical background through which A. minor has been speciated.
The data accumulated so far indicate that genus Asterina, or its closely related groups, despite intimate similarity in adult morphology in different species, shows a remarkable variety in the mode of development and the method of breeding (Ludwig, 1882; MacBride, 1896; Goto, 1898; Mortensen, 1921; James, 1972; Komatsu, 1975; Kano and Komatsu, 1978). This observation may bring out the importance of studies on the development of Asterina group which may not only clarify the ontogeny of a given species but may also help towards understanding the evolution of asteroid development as a typical group having divergent breed- ing methods and development.
The authors are indebted to Professor Emeritus Katsuma Dan, Tokyo Metro- politan University, and Professor Emeritus Ryoji Hayashi, Toyama University, for their valuable advice. Thanks are also extended to Drs. Hiro'omi Uchida, Takeshi Tatsuki, and members of Sabiura Marine Laboratory of the Marine Parks Center of Japan, for their kind cooperation in the collection of the specimens. The present study was supported in part by Grants-in-Aid from the Ministry of Educa- tion of Japan (Nos. 054012, 074102/174234).
SUMMARY
1. The breeding season of Asterina minor is estimated to be during the month of May in Kushimoto, Japan. A. minor shows a characteristic breeding assemblage and the eggs are laid on the substratum in a mass spawning. The eggs are not protected by the adults.
2. A. minor is a spatial hermaphrodite, where ovaries and testes in an individual become mature simultaneously. Isolated individuals are capable of self-fertilizing and the self-fertilized eggs develop normally.
274 KOMATSU ET AL.
3. The spawned eggs are spherical, yellow, and 437 /mi in average diameter. They attach to the substratum with a sticky jelly layer. Cleavage is total and radial.
4. Eggs through the wrinkled blastula stage develop into a pear-shaped brachiolaria bearing three brachiolar arms within the fertilization membrane.
5. About four days after spawning, the brachiolariae hatch from the fertiliza- tion membrane and creep on the substratum with well-developed brachiolar arms. There is no evidence of pelagic life in the present species.
6. One day after hatching, brachiolariae attach firmly to the substratum with the brachiolar arm and undergo a rapid transformation of the body (metamorphic climax). Metamorphosis is completed with the opening of the mouth about 10 days after spawning. The newly metamorphosed juvenile is about 700 //,m in diameter and each arm bears two pairs of the tube-feet and one red eye-spot at the base of the terminal tentacle.
7. The reproduction and larval development of A. minor are unique, and the study may prove a good guideline for understanding the evolution of reproduction and development in Asteroidea.
LITERATURE CITED
CHIA, F-S., 1968. The embryology of a brooding starfish, Leptastcrias hexactis (Stimpson).
Acta Zool, 49: 321-364. " COGNETTI, G., 1954. La proteroginia in una popolazione di Astcrina panccrii Gasco del Golfo
di Napoli. Boll. Zool, 21: 77-80. CUENOT, L., 1887. Contributions a 1'etude anatomique des Asterides. Arch. Zool. Exp. Gen.,
2(5) : 1-144. DARTNALL, A., 1970. Some species of Asterina from Flinders, Victoria. Victorian Nat., 87 :
1-4. DELAVAULT, R., 1966. Determinism of sex. Pages 615-638 in R. A. Boolootian, Ed., Physiology
of Echinodcnnata. Interscience, N. Y. GOTO, S., 1898. Some points in the metamorphosis of Asterina gibbosa. J. Sci. Coll. Imp.
Univ. Tokyo, 12: 227-242. HAYASHI, R., 1974. A new sea-star from Japan, Astcrina minor sp. nov. Proc. Jap. Soc. Syst.
Zool., 10: 41-44. JAMES, D. B., 1972. Note on the development of the asteroid Asterina burtoni Gray. /. Mar.
Biol. Assoc. India, 14 : 1-2. KANO, Y. T., AND M. KOMATSU, 1978. Development of the sea-star, Astcrina batheri Goto.
Dcv. Growth Differ., 20 : 107-114. KOMATSU, M., 1975. Development of the sea-star, Asterina coronata japonica Hayashi. Proc.
Jap. Soc. Syst. Zool., 11 : 42-48. KOMATSU, M., 1976. Wrinkled blastula of the sea-star, Astcrina minor Hayashi. Dev. Grozvth
Differ., 18: 437-440. KUBO, K., 1951. Some observations on the development of the sea-star, Lcf>fasterias ochotensis
siinilispinis (Clark). /. Fac. Sci., Hokkaido Univ. Ser. VI, Zool., 10: 97-105. LUDWIG, H., 1882. Entwicklungsgeschichte der Astcrina gibbosa Forbes. Z. Wiss. Zool.,
37: 1-98.
MACBRIDE, E. W., 1896. The development of Astcrina gibbosa. Q. J. Microsc. Sci., 38 : 339-411. MORTENSEN, TH., 1921. Studies of the development and larval forms of cclnnoderms. G. E. C.
Gad, Copenhagen, 216 pp. OHSHIMA, H., 1929. Hermafrodita marsetelo Asterina batheri Goto. Annot. Zool. Jap., 12:
333-349.
Reference: Biol Bull, 157: 275-287. (October, 1979)
THE EFFECT OF SIZE, TEMPERATURE, OXYGEN LEVEL AND
NUTRITIONAL CONDITION ON OXYGEN UPTAKE IN THE
SAND DOLLAR, MELLITA QUINQUIESPERFORATA
(LESKE)
JACQUELINE MOSS LANE AND JOHN M. LAWRENCE
Department of Biology, University of South Florida, Tampa, Florida 33620
Because metabolism often represents a large source of expended energy, accurate measurement of metabolism is essential for construction of energy budgets. Metabolic processes, as measured indirectly by oxygen uptake, however, may vary with physical condition and physiological state. It is often difficult to assess the results of all these variables on metabolism, and some effects are either dis- counted or estimated.
Such estimations may be misleading, especially in echinoids, where relatively few studies have been made and where different species of echinoids may respond differently to certain variables. The purpose of this study is to obtain a relatively accurate estimation of metabolic energy expenditure in the sand dollar, Mellita quinquiesperforata, by measuring oxygen consumption seasonally at ambient water temperatures, under various degrees of starvation, during the day and night, and at different oxygen concentrations on various size animals.
MATERIALS AND METHODS
For monthly measurement of oxygen consumption, five or six animals of various sizes were collected from Mullet Key, a sub-tidal, semi-protected sandy beach area along the Gulf of Mexico, adjacent to the mouth of Tampa Bay, Florida (27° 38' N; 82° 44' W). After allowing the guts to clear overnight, animals were placed in individual dishes containing either 200 ml (for small animals), 500 ml (for medium-sized animals) or 1000 ml (for large animals) of freshly aerated sea water. After 0.5 hr, a 1-cm layer of mineral oil (which blocks oxygen diffusion) was poured over the surface of the water. At this time, an initial water sample was taken from a dish with no animal (control). A known volume of water (approx. 30 ml) \vas removed from each dish at 1 and 2 hr after sealing; the water was gently stirred just before sampling. Oxygen in wrater samples was measured using the micro-Winkler method (Hoar and Hickman, 1967). Sea water used for all experiments was collected from the field and filtered through 0.22 //, Mille- pore filters before use. A constant temperature chamber (dimly lit) stimulated monthly environmental water temperatures and fluctuated less than 0.2° C during each experiment. Salinity varied little between experiments (33.0-34.9%o). After 2 hr, each animal was removed, weighed, and placed in a drying oven at 80° C. Dried animals were weighed and ground to powder in a \Yylie mill. A small sample of this material was used for measurement of total nitrogen by the micro-Kjeldahl (Holland, 1964). Monthly rates of oxygen consumption are expressed on a wet, dry and total body nitrogen weight basis. Analysis of the
275
276 J. M. LANE AND J. M. LAWRENCE
data was done on values obtained after 1 hour as tests showed M. qninqiiiespcrforata to be an oxygen conformer.
To ascertain how rate of oxygen consumption is affected by reduced oxygen tensions in sea water, animals of similar weight (7 g dry weight) were placed in dishes containing a known volume of sea water (100 ml) in ten experiments. The dishes were sealed with a lid through which an oxygen electrode was mounted. The rate of respiration in these closed dishes was measured every 10 minutes. The water was stirred during each reading by a magnetic stirbar located just beneath the oxygen electrode. The water temperature in all these experiments was 25° C.
In addition to monthly measurements using a closed system, the rate of oxygen consumption was measured in June in an "open" or "flow-through" system, similar to the one described in Hoar and Hickman (1967). Six animals were placed in individual dishes which were closed to general water circulation except for entrance and exit holes. Water entered the dishes and was continuously siphoned into stoppered Erlenmeyer flasks. The rate of flow through the dishes was measured each day and adjusted so that oxygen tension never fell below 80% saturation. Animals used in this experiment were acclimatized to summer water temperatures (29-33° C). Experiments were run at 15, 20, 25, 30, and 35° C. For each experimental temperature, the water in the flow-through apparatus was changed and different animals were used. Animals were not placed into the dishes until all feces had been expelled (usually overnight) ; oxygen readings were begun after animals had been held at experimental temperatures in the dishes for one day. Oxygen concentration was measured in the Erlenmeyer flasks using a Beckmann Field-Lab oxygen meter fitted with a platinum electrode. Readings were taken at 4-hr intervals for 48 continuous hours. The experimental tempera- ture was maintained by placing the whole flow-through apparatus in a constant temperature chamber. A dim light switched on at 0700 hr and off at 2200 hr simulating day-night conditions. After each experiment, animals were removed from the dishes, weighed and dried as described above. Results from the flow- through system were compared with results from a closed respiratory system. One-hour closed system measurements were run at the same temperature at the beginning of each flow-through experiment.
The effect of starvation upon the rate of oxygen consumption was measured at different times of the year by starving animals in filtered sea water for periods up to 1 month. Water in the holding tanks was renewed every 2 days with freshly collected and filtered sea water. During starvation, water temperature in tanks simulated ambient temperatures. The oxygen consumption of animals which were starved for various times (5-30 days), was measured in a closed system.
Results of all oxygen uptake experiments are expressed as log-log linear regression equations with logio rate of oxygen uptake and logio body weight (wet, dry or body nitrogen weight) being the two variables. Slopes and intercepts from different seasons or experiments were compared using analysis of covariance (Snedecor and Cochran, 1971). Variances were checked with Bartlett's Chi- square (Snedecor and Cochran, 1971). For ease in comparing rates of oxygen consumption at different temperatures, pooled slopes were used when statistically valid and intercepts were adjusted as follows; new intercept equals average of Y values minus pooled slope times average of X values.
OXYGEN UPTAKE IN THE SAND DOLLAR
277
4-r
t--*-^v w-*
432
OXYGEN CONCENTRATION-ML
FIGURE 1. Rate of oxygen uptake (//,! O2/hr/g dry weight) and percent utilization of avail- able oxygen by Mcllita quinquiesperforata in decreasing oxygen concentrations in three repre- sentative experiments. A closed system was used for measurement.
RESULTS
Rates of respiration of Mcllita quinquiesperforata conform to oxygen concentra- tions in water (Fig. 1). Oxygen consumption decreases with decreasing oxygen pressure in a nearly linear manner. At very low levels of external oxygen (< 1 ml Oo/liter), a low level of respiration is maintained.
In monthly oxygen measurements at ambient temperatures, rates of respira- tion are dependent on both body weight (wet, dry or body nitrogen) and water
278
J. M. LANE AND J. M. LAWRENCE
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OXYGEN UPTAKE IN THE SAND DOLLAR
279
TABLE II
Summary of analysis of covariance for testing significance of differences between slopes and intercepts for regression equations of oxygen uptake in Mellita quinquiesperforata at different temperatures, in different nutritional states, on a dry (D]V) and nitrogen (XIV) weight basis, and in closed and open respirometers. The form of the linear regression equation is logio ml Ot respired /hr per animal (F) equals loglo g dry bodv weight or nitrogen weight (A') times slope plus intercept. For comparing slopes, summer months are considered as VI, VII, and VIII; fall months are IX, X, and XI; winter months are XII, I, and II; and spring months are III, IV, and V.
|
Dates or experiments being tested |
Pooled slope |
X- variance |
"F" for slope |
df |
"F" for intercept |
df |
Tests for heterogeneity among experimental dates
|
All non-starved dates (D\V) |
0.5485 |
13.61 |
0.3027 |
9, 36 |
||
|
Summer non-starved |
||||||
|
dates (D\V) |
0.5374 |
5.10 |
0.1323 |
2, 11 |
21.7575** |
2, 27 |
|
Fall non-starved dates (DW) |
0.5614 |
4.20 |
2.2056 |
2, 9 |
0.3768 |
2, 23 |
|
Winter non-starved |
||||||
|
dates (DW) |
0.5327 |
0.90 |
0.0827 |
2, 12 |
7.8714** |
2, 29 |
|
All non-starved dates (XW) |
0.5670 |
14.55 |
1.1036 |
8, 32 |
1154.6722** |
8, 327 |
|
All starved dates (DW) |
0.8216 |
2.37 |
1.5747 |
4, 14 |
||
|
All starved dates (XW) |
0.8132 |
7.77 |
2.5699 |
4, 14 |
15.9071** |
4, 75 |
Tests for heterogeneity among grouped regressions
|
Starved vs non-starved |
||||||
|
summer dates (DW) |
0.5789 |
0.83 |
7.7766** |
1, 24 |
2.3639 |
1, 25 |
|
Starved vs non-starved |
||||||
|
fall dates (DW) |
0.6611 |
15.43** |
6.2446** |
1, 21 |
1.8405 |
1. 27 |
|
Starved vs non-starved |
||||||
|
summer dates (XW) |
0.6359 |
0.01 |
2.1658 |
1, 21 |
0.0364 |
1, 22 |
|
Starved vs non-starved |
||||||
|
fall dates (XW) |
0.6572 |
10.42** |
2.9027 |
1. 21 |
0.2687 |
1, 22 |
|
Closed vs open at 25° C |
0.6772 |
1.09 |
0.0750 |
1, 8 |
62.4133** |
1, 9 |
|
Closed vs open at 20° C |
0.5496 |
0.90 |
0.0436 |
1, 26 |
23.2153** |
1, 27 |
|
Closed vs open at 30° C |
0.4115 |
12.52** |
0.0456 |
1, 20 |
11.8181** |
1, 21 |
* Significant at 95% level. ** Significant at 99% level.
temperature (Table I; Fig. 2). Small animals have proportionally higher rates of respiration than do large animals when values are expressed on a dry weight (pooled yearly slope of 0.5485; Table II) or a nitrogen weight (pooled yearly slope of 0.5670; Table II) basis. Differences between slopes are not sig- nificant when slopes are compared together by year or by season (Table II). Degree of gonadal development had no effect on respiratory rates.
Although there was no significant difference between slopes, rates of respira- tion did change significantly with water temperature, as indicated by significant "F" values for intercepts (Table II). To simplify comparison at different tempera- tures, intercepts were adjusted using a pooled slope of 0.5485 (monthly slopes were not significantly different) for all experiments. Using these adjusted intercepts, a 5-g animal (dry weight) would have highest rates of respiration on VII/4/72 at 31° C (0.4259 ml O2/hr) and lowest rates on 11/27/73 at 15° C (0.1845 ml O2/hr) (Fig. 2). Rates of respiration slowly decrease between 30 and 22° C (fall months) but decline greatly between 31 and 30° C (VII/4/72 and VII/25/72, respectively). The respiratory rate is lower at 33° C than at 31° C (Fig. 2). Q10 values for field acclimatized animals are 1.38 from the temperature range 15 to 33° C, 1.69 from 15 to 31° C, 1.93 from 22 to 31° C, 1.13 from 22 to 30° C and 1.42 from 15 to 22° C.
280
J. M. LANE AND J. M. LAWRENCE
450-,
400- •
350- -
cr 300
I
Q
LJJ
250+
LLJ
cr
c?
200- -
150- -
100
35
on-starved starved
30
25
20
15
TEMPERATURE- C
FIGURE 2. Relationship between oxygen consumption (/A O2 respired/hr per animal) and water temperature for a starved and a non-starved 5-g (dry) specimen of Mcllita qitinqnics- pcrforata. Rates were calculated from regression equations given in Table I.
Respiratory rates decline with starvation ; the degree to which the rate declined depended on length of time animals were starved, water temperatures during starva- tion, and weight of the animal. Because animals were starved for different lengths of time at different water temperatures, it is difficult to assess the effect of these variables independently. Respiratory rates became more depressed as the period of starvation increased (Table I). A 1-g dry animal which was starved for 18 days at 31° C (VII/21/72) would use 53.63 fA Oo/hr, whereas a 1-g animal starved for 5 days at 30° C (VI 1/30/72) would use 76.24 /J O2/hr (calculated using a pooled slope of 0.8670 and adjusted intercepts, Table I).
Effects of starvation depend on weight of the animals. On the basis of body dry weight, starvation has a proportionally greater effect on the respiratory rate of small starved animals as indicated by higher values for "starved" slopes (Table I). A 20-g (dry weight) starved animal has slightly higher rates and a
OXYGEN UPTAKE IN THE SAND DOLLAR
281
5-g (dry weight) starved animal has lower rates when compared with non-starved animals of similar size (Table I; Fig. 2). On a seasonal basis, the slopes for starved and non-starved animals are significantly different (Table II).
\Yhen respiratory rates are expressed on a nitrogen basis, Oo uptake of small, starved animals is not as depressed when compared to non-starved animals (Fig. 3) and the regression slopes and intercepts of the two groups are not significantly different (Table II). Starved animals of all sizes have significantly less nitrogen/g dry weight than non-starved animals (Lane, 1977; Fig. 4) and small animals lose (metabolize) slightly more nitrogen during starvation than do larger animals (Lane, 1977). Small animals have lower rates of respiration when starved, but they have less body nitrogen as well when compared with larger animals.
Rates of respiration depend on the type of system used for measuring respira- tion. Comparison of respiration in closed and open systems at the same tempera- ture shows intercepts, hence rates, of Oo consumption are significantly higher in the open system (Table II). Using pooled slopes and adjusted intercepts for each
non-starved starved
10 T5
DRY WEIGHT
20
25
FIGURE 3. Relationship between oxygen consumption (ml O? respired/hr/g nitrogen) and dry body weight for starved and non-starved specimens of Mellita quinquiespcrforata at fall water temperatures. Rates were calculated as follows ; non-starved animals-logio ml Os/hr/g nitrogen equals 1.1414 minus 0.3823 times logio g dry body weight; starved animals-logio ml O2/hr/g nitrogen equals 0.9740 minus 0.1835 times logio g dry body weight.
282
J. M. LANE AND J. M. LAWRENCE
9-r
8--
z <
LLJ
o
o
cr
7--
6--
5--
4-1 VI
non-starved
starved
\ --•
VII VIII IX
X XI XII MONTH
I
II
III
FIGURE 4. Seasonal changes in the amounts of total nitrogen in a starved and non-starved 1-g specimen of Mcllita quinquicspcrforata. Values were calculated using pooled seasonal slopes and adjusted intercepts. Slopes used for non-starved animals are: summer, 0.8989; fall, 0.9462; winter, 1.0301. Slopes used for starved animals are: summer, 0.9561; fall, 1.0499.
temperature, a 10-g animal (dry weight) would respire 0.3003 ml O2/hr per animal and 1.0598 ml O2/hr per animal at 25° C; 0.2252 ml O2/hr per animal and 0.5564 ml O2/hr per animal at 20° C ; and 0.4990 and 0.8158 ml O2/hr per animal at 30° C in closed and open systems respectively. Comparison of respiration during the day and night in an open system showed no trends.
DISCUSSION
As found with many other echinoderms and invertebrates in general, the respira- tory rates of Mcllita quinquiesperforata are modified by a variety of factors. Body weight or age is one such factor. Smaller (younger) animals have proportionally higher rates of respiration than larger (older) animals when rates are based on both body weight or body nitrogen. Similarly, this effect of size (age) on rate of O2 uptake has been found in all other echinoids studied and regression slopes (logio ml O2 respired/hr per animal versus logio body weight) for echinoids generally fall" between 0.500 and 0.700 (Lewis, 1967, 1968; McPherson, 1968; Percy, 1971; Webster, 1972; Miller and Mann, 1973). An adequate or complete explanation for this phenomenon is lacking. The decreasing surface area with increasing weight explanation does not appear to apply in the case of M. quinquies-
OXYGEN UPTAKE IN THE SAND DOLLAR
283
pcrforata. Surface area (from shape) is approximately body weight 0.00. while respiration is body weight 0.5485. In addition, the body wall of echinoids has been shown to be the principal consumer (90%) of oxygen in whole respiring animals (Giese, Farmanfarmaian, Hilclen, and Doezema, 1966; Webster, 1972; Belman and Giese, 1974; "Webster and Giese. 1975). Stephans, Volk, Wright, and Backlund (1978) similarly concluded that the epidermis of the test of the sand dollar is responsible for a large portion of the total oxygen consumption by the sand dollar. Dendrastcr c.vcentricus. Since the outer surface of the body wall is directly exposed to oxygenated water, the surface to volume argument should hardly apply to these echinoids.
In M. quinquiesperforata, it would appear that smaller (younger) animals may simply have 'faster' metabolic systems, i.e., shorter half-lives of enzymes, faster turnover of proteins. Evidence for this comes from similar decreases in rate with increasing animal weight which were found for the processes of feeding, carbon excretion, ammonia excretion and radioactive uptake in this sand dollar (Lane, 1977). With starvation, smaller sand dollars metabolize proportionally greater amounts of nitrogen than larger animals. Fuji (1962, 1967) and Miller and Mann (1973) found similar allometry in different functions of other echinoids. Stephans (•/ al. (1978), however, found that differences in rates of amino acid uptake between small and large sand dollars disappeared when surface area was used as the basis of expression.
As with M. quinquiesperforata, other echinoids have depressed respiratory rates with starvation. Farmanfarmaian (1966) observed that Y,,,'s were reduced by as much as 50% after a month of starvation. Giese (1967) noted a general
TABLE III
Summer and fall comparisons for calories expended in respiration month of starved and non-starved Mellita quinquiesperforata. For starved animals, values were calculated from VIII 14 72 (summer) u'ith a slope of 0.867 and an adjusted intercept of -1.3353 and XI 15 73 (fall) with a slope of 0.7720 and an adjusted intercept of — 1.3<)9>>. For non-starved animals, a slope of 0.5485 and an adjusted intercept of —0.7541 (VII 4 72) was used for calculating summer values and a slope of n.54\5 and an adjusted intercept of —1.0119 (XI / 15/72) was used for fall values. 4.8 cal ml 02 respired was used to convert ml 0-j into calories.
|
Dry weight of animals |
Cal respired/ month /starved animal |
Cal respired/ month /non-starved animal |
Starved values/ nun-starved values X 100 |
Summer comparison
|
1 g |
159.67 |
608.80 |
26.22% |
|
10 g |
1175.71 |
2152.65 |
54.61% |
|
20 g |
2144.11 |
3148.39 |
68.10% |
|
-tOg |
3910.80 |
4604.72 |
83.93% |
Fall comparison
|
1 g |
169.31 |
336.26 |
50.35% |
|
10 g |
1001.55 |
1188.98 |
84.23% |
|
20 g |
1710.05 |
1738.95 |
98.33% |
|
40 g |
2919.97 |
2543.33 |
114.80% |
284 J. M. LANE AND J. M. LAWRENCE
decline in rate of oxygen uptake over a month of starvation. Webster (1972) found that the greatest decrease in Vo2 occurred in the first week of starvation with little change thereafter. Boolootian and Cantor (1965) reported that the respira- tory rate fell to a low level after three weeks of starvation and remained constant thereafter. Differences in response of respiratory rate among echinoids with starvation may perhaps be explained by differences in nutritional his- tory at the beginning of the experiment and consequent differences in sub- strate being metabolized during starvation. Wallace (1973) found that the V02 of starving crabs fell in steps corresponding to the type of nutrient reserve being metabolized. Carbohydrate reserves were first utilized, with lipid next and protein last. Differences in response of small and large specimens of M. quinquiesperjorata to starvation may also be due to differences in substrate being metabolized. If so, large animals may have had proportionally greater re- serves of either carbohydrate or lipid (in the gonad). Consequently, small animals may have been metabolizing protein while larger animals were metabolizing carbohydrate or lipid, resulting in more depressed rates of respiration in small animals. The proportionally greater decrease in amount of body nitrogen in small, starved animals seems to support this conclusion.
Higher rates of respiration in small, non-starved specimens of Mellita may also have been due to effects of SDA or greater activity in non-starved sand dollars. Effects of both factors, however, appear to be minimal. Activity of starved and non-starved animals was similar in closed containers. As reported by Lilly (1979) for two species of sea urchins, the effect of SDA was most pronounced just after feeding and declined to pre-f ceding levels after 3 hr. Rates of C>2 uptake in the sand dollar were measured approximately 24 hr after feeding. Hence SDA should be of slight consequence.
Differences between respiratory rates in starved and non-starved animals may
represent energy used in growing (Table III). If so, 26% of respiration energy
is used for summer maintenance and the remainder (74%) is energy expended for
growth in a 1-g, non-starved specimen of M. qninquiesperforata. In an actively
growing 20-g animal (dry weight), 68% of the respiration energy is used for
summer maintenance and 32% respresents energy for growth (Table III). In
fall, more non-starved respiration energy is used in maintenance and less in
growth for all size sand dollars. Both the greater amounts of energy expended
in growth by small sand dollars and the seasonal difference in amounts of energy
for growth by all size animals are consistent with information from growth
studies on this animal (Lane, 1977). From growth studies, a 1-g animal would
expend 136 calories for growth/month in the summer (Lane, 1977). Difference
between respiratory rates of starved and non-starved 1-g animals indicate 449
calories/month would be spent in growth. Likewise in fall, a 1-g animal would
add 91 calories/month in growth (Lane, 1977) and expend 167 calories/month
in growing (from difference between starved and non- starved respiration). For
larger animals in fall, starved respiration was greater than non-starved. This
anomaly may have resulted from differences in the nutritional condition of "freshly
collected" animals. Negative growth reported for large animals in fall (Lane, 1977)
could indicate that "freshly collected" specimens of M. qninquiesperforata were
OXYGEN UPTAKE IN THE SAND DOLLAR 285
already starved when taken from the field, and starvation in the laboratory produced no further change in respiratory rate.
Although laboratory experiments were not specifically designed to characterize type of acclimation response in M. quinquiesperforata, monthly oxygen measure- ments made at ambient water temperatures did demonstrate partial acclimatization in sand dollars. Vo2's were higher in January at 20° C than in December at the same temperature. Likewise, acclimatization to summer water conditions may have resulted in lower rates of respiration on VI 1/25/72 at 30° C as compared to the much higher rates three weeks earlier on VI 1/4/72 at 31° C. As with many other echinoids (Farmanfarmaian and Giese, 1963; Moore and McPherson, 1965; McPherson, 1968; Percy, 1971; Webster, 1972), this compensatory response was only partial in Mellita since respiration was still higher in summer than in winter.
The sensitivity of the respiratory rate in M. quinquiesperforata (Qio) was relatively low between 22 and 30° C. In the Tampa Bay area, the change in water temperature over the 22 to 30° C range is rapid during the spring warming and fall cooling. The respiratory insensitivity over this range would, therefore, appear to have some adaptive value. The decline in Vo2 at 33° C may indicate metabolic malfunction as this temperature is close to the lethal limits (^ 38° C) of this sand dollar.
Oxygen consumption may be modified by type of system used for measuring respiration. Miller and Mann (1973) for 5\ droebachiensis, Webster and Giese (1975) for Strongylocentrotns purpiiratus and this study found that rates of respiration were higher in flow-through systems when compared with closed sys- tems. Although this increased rate may be caused by increased availability of oxygen, activity of animals may also be increased in flowing water. Activity of M. quinquiesperforata was much greater in the flow-through system than inter- mittent activity of sand dollars in closed systems. Although sand dollars are exposed to a continual exchange of water in their environment, much of the time they are partially buried and do not or can not continually wave their spines back and forth as was the case in the open system. Therefore, the monthly oxygen measurements made in closed dishes are considered more representative of routine metabolism.
Due to various factors which modify metabolic rate, and the different units used to express respiratory rate, it is difficult to compare absolute rates of respiration among various echinoids. When compared to the respiratory rates of tropical and temperate echinoids as given by Webster (1975), Vo.2's of M. quinquies- perforata (as found in this study) are slightly higher than rates for tropical echin- oids of similar weight.
SUMMARY
1. Rates of respiration in a closed vessel conformed to oxygen concentrations in surrounding water until a low level (1 ml Oo/liter) of oxygen was attained.
2. Respiratory rates of small animals were proportionally higher on both a dry weight and nitrogen weight basis than rates of large animals (slope for ml O2 respired/hr per animal was 0.5485 for dry weight and 0.5670 for nitrogen) in a closed vessel.
286 J. M. LANE AND J. M. LAWRENCE
3. Respiratory rates of field acclimatized animals were slightly higher at suininer temperatures (30-33° C) than at winter water temperatures (15-20° C) with evidence for partial acclimatization presented.
4. Respiratory rates of starved animals were lower than rates of non-starved animals with starvation depressing the rates of smaller animals more than rates of larger animals.
5. Respiratory rates in an open system were approximately twice as high as rates in a closed system due to greater activity of animals in the open system.
6. Comparison of day versus night respiration showed no obvious trends.
7. Respiratory rates of M . quinquiesperforata are slightly higher than rates of a similar weight, tropical, regular urchin as reported in the literature.
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OXYGEN UPTAKE IX THE SAXD DOLLAR 287
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WALLACE, J. C., 1973. Feeding, starvation and metabolic rate in the shorecrab, Carcuius inaenas. M\,r. Biol.. 20: 277-281.
\\"i -.i:> 1 1 K, S. l\., \()~2. The respiratory physiology of Strongylocentrotus pitrpiiratits Stimpson with particular reference to the reproductive cycle. Ph.D. dissertation, Stanford I'm- •:'ersity, 120 pp.
WEBSTER, S. K., 1975. Oxygen consumption in echinoderms from several geographical loca- tions, with particular reference to the Echinoidea. Biol. Bull., 148: 157-164.
\\ i ISSTER, S. K., AND A. C. GIESE, 1975. Oxygen consumption of the purple sea urchin with special reference to the reproductive cycle. Biol. Bull.. 148 : 165-180.
kHn-ence: Biol. Bull., 157: 288-296. (October, 1979)
DEVELOPMENT AND BEHAVIOR OF AN INTERGENERIC CHIMERA
OF HYDRA (PELMATOHYDRA OLIGACTIS INTERSTITIAL
CELLS: HYDRA ATTENUATA EPITHELIAL CELLS)
HSUEH-TZE LEEi AND RICHARD D. CAMPBELL
Department of Developmental and Cell Biology, and Center fur Patlwbioloc/y, University of California, Irvine, California 92717
Hydra has so few cell types that it should be possible to map out the develop- mental and behavioral functions of each. By combining cell types of different species, one might trace roles by identifying species characters in the resulting chimeric animals.
Many of hydra's highly specialized cell types (nerve cells, nematocytes, gametes) are part of a single lineage of cells that is continually being renewed by proliferation and differentiation of a stem cell called the interstitial cell or 'T cell." The entire interstitial cell lineage can be removed from a hydra by various means (Marcum and Campbell, 1978a; Campbell, 1979). The resulting animal is termed an "epithelial hydra," and is composed only of ectodermal and endodermal epithelial cells. This viable epithelial shell can then be repopulated by I cells, since they will migrate throughout the depleted animal from a small, temporary graft of normal tissue. A number of chimeric strains in hydra have been made in this fashion (Saffitz, Burnett and Lesh, 1972; Sugiyama and Fujisawa, 1978; Marcum and Campbell, 1978b). In order to assign roles to the different cell lineages, one would use the most dissimilar species as parents. However, grafting success (and hence presumably tissue compatibility) decreases as species diversity increases (Campbell and Bibb, 1970), so that most hydra chimeras have been constructed of cells from the same or closely related species.
One pair of dissimilar species, Hydra attenuata and Pdmatohydra oligactis. will partially tolerate intergrafting and a considerable literature suggests that it may be possible to make a stable chimera between their cells (Evlakowa, 1946; Brien and Reniers-Decoen, 1955; Kolenkine and Bonnefoy, 1976). Since it is possible to remove the interstitial cell lineage from H. attcniiata, we repopulated epithelial H. attenuata with P. oligactis interstitial cells. The reciprocal graft is not possible since a technique for removing I cells from P. oligactis has not been found. This report describes some developmental and behavioral similarities and differences between the chimeras and the two parental species.
MATERIAL AND METHODS
Specimens of Hydra attenuata from Lake Zurich, specimens of Pelmatohydra oligactis collected in Grant Lake, Mono County, California, and the chimeras were all grown in "M" solution lacking bicarbonate (Muscatine and Lenhoff, 1965) by standard methods (Lenhoff and Brown, 1970). Epithelial specimens of H. attenuata were produced by a double colchicine treatment (Marcum and Campbell,
1 Present address : Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543.
288
HYDRA CHIMERA
289
1978a) to eliminate I cells. Distal halves of epithelial specimens of H. attcnnata were repopulated with I cells by axially grafting them to proximal halves of normal P. oligactis polyps. The graft junction wras marked by a permanent constriction in the ectoderm (Kolenkine and Bonnefoy, 1976) and by different coloration of the endoderm in these two species. Grafts were left intact for 3 to 5 days and then the P. oligactis epithelial tissue was cut away. The resulting repopulated hydra were maintained for up to 6 months by methods appropriate for epithelial hydra (Marcum and Campbell, 1978a).
Cell compositions were determined using David's (1973) maceration procedure. Heat shocks were applied by immersing 10-ml test tubes containing individual hydra in 2 ml of medium into a preheated waterbath for 30 min. Afterwards the tubes were left at room temperature for 12 hr, and then the hydra were cultured normally. Time-lapse films were made using a 16-mm Bolex camera outfitted with extension tubes behind the lens, with illumination through heat filters.
Feeding response was measured using the methods of Lenhoff (1969). Inhibi- tion of nematocyst discharge (Smith, Oshida and Bode, 1974) was carried out by feeding hydra to repletion and then releasing single shrimp, successively, onto tentacles at measured times later. The number of shrimp contacts which occurred before two became trapped was recorded. Only 20 trials were offered each polyp ; a score of 20 indicated that the hydra did not catch the Artcinia.
RESULTS
Genetic composition oj chimeras
Although the methods used for producing chimeras seem straightforward, we considered it important to demonstrate that the chimeras were, in fact, composed of H. attcnnata epithelial and P. oligactis interstitial cells.
The genetic origin of interstitial cells was verified by analysis of chimera nematocysts. Table I shows the dimensions and Figure 1 shows the morphologies
TABLE I
Nematocyst sizes. Length and width (standard deviations in parenthesis) are all in ^m. Each measurement represents about 20 nematocysts. Chimera polyps a, b, c, and d are progeny from different grafts.
|
Hydra strain |
Stenotele |
Atrichous isorhiza |
Desmoneme |
|
H. attenuata |
14.8 X 11.4 |
9.7 X 4.0 |
8.1 X 5.6 |
|
(2.2) (1.9) |
(1.0) (0.2) |
(0.6) (0.4) |
|
|
P. oligactis |
12.9 X 9.7 |
7.8 X 4.3 |
6.7 X 4.8 |
|
(0.5) (0.5) |
(0.5) (0.5) |
(0.4) (0.3) |
|
|
Chimera polyp a |
12.1 X 9.5 |
8.2 X 4.0 |
5.7 X 4.2 |
|
(0.7) (0.7) |
(0.7) (0.2) |
(0.5) (0.4) |
|
|
polyp b |
11.9 X 9.1 |
8.8 X 4.4 |
6.2 X 4.6 |
|
(0.6) (0.8) |
(0.5) (0.6) |
(0.4) (0.4) |
|
|
polyp c |
11.0 X 8.3 |
8.8 X 3.S |
6.1 X 4.0 j |
|
(1.0) (0.7) |
(0.6) (0.3) |
(0.5) (0.1) |
|
|
polyp d |
11.7 X 8.9 |
9.1 X 4.0 |
6.1 X 4.2 | |
|
(0.6) (0.5) |
(0.8) (0) |
(0.2) (0.2) |
H. LEE AND R. D. CAMPBELL
FIGURE 1. Nematocyst structures of //. attcmiata (left), P. oHgactis (middle), and chimera (right). Nematocyst types, bottom to top, are: stenotele, holotrichous isorhiza, atrichous isorhiza, and desmoneme. All figures except the chimera holotrichous isorhiza represent mature, mounted nematocysts. Chimeras rarely have mounted holotrichous isorhizas, but the species specific tubule pattern is visible in complete but immature capsules in gastric region nests, as shown here. Scale indicates 10 /urn.
of nematocysts of the chimeras and both parents. These data are taken from animals more than 4 months after chimeras were made. The small sizes of the chimera nematocysts are similar to those of P. oligactis and not H. attcnnata. The morphologies of the chimera nematocysts (Fig. 1) are also unambiguously those of P. oligactis. The absence of transverse coils in the holotrichous isorhiza is the
TABLE II
Relative proportions of nematocyst types in tentacles. Numbers express percentages of total nemato- cysts. (Between 500 and 1000 nematocysts were counted in the distal parts of tentacles, with the number of polyps indicated at left}.
Nematocyst type
|
Stenotele |
Isorhizas |
Desmoneme |
|
|
H. attenuata (n = 2) P. oligactis (n = 3) Chimera (n = 6) |
3.5 (±0.7) 34 (±9) 37 (±8) |
12.5 (±4.9) 5.7 (±1.5) 6.7 (±1.0) |
84 (±4) 60 (±9) 57 (±7) |
HYDRA CHIMERA 291
most notable character of P. oligactis stingers (Ewer, 1948), and the chimera also lacks these. In addition, the slender shape of the stenotele, the bluntly oval shape of the holotrichous isorhiza, and the reniform atrichous isorhiza are all characteristically P. oligactis
The two parental species differ in the relative abundances of the different nematocyst types. Table II shows that in this character the chimeras closely resemble P. oligactis and not H. attcniiata. However, one abnormality of the chimeras was the nearly complete absence of mature holotrichous isorhizas. None were found on animals whose nematocytes were measured (Table I), and few were seen in this study. The photograph in Figure 1 is of an immature holo- trichous isorhiza.
The genetic origin of epithelial cells was ascertained in two ways. First, in color both the ectoderm and endoderm were found to resemble H. attcniiata rather than P. oligactis. The chimeric ectoderm was colorless, and the endoderm pink, as in H. attcniiata. P. oligactis has yellow granules in the ectoderm and an orange endoderm. Second, it is known that epithelial tissue controls graft tolerance (Campbell and Bibb, 1970). Therefore, two chimeras that had been established for 6 months were bisected and halves were grafted back to the two parental strains. In the chimera///, attcniiata grafts, the graft junctions became imperceptible within a day and no incompatibility was detected during the next 8 days of culture. In the chimera/P. oligactis grafts, the graft junctions were still constricted after 1 day and by the sixth day the two halves had separated.
We conclude that the chimeras had H. attcniiata epithelial cells and P. oligactis interstitial cell lineage, and that this composition remained stable throughout the study.
Morphology of chimeras
Chimeras were always smaller than either parent (Fig. 2). Measurements of ten chimeras and parents grown on a regime of six shrimps day averaged 2.0 mm (chimera), 7.0 mm (H. attcnitata) and 9.0 mm (P. oligactis} in extended length. Tentacle number of budding individuals averaged 5.8 (chimeras), 6.5 (//. attcniiata) and 6.4 (P. oligactis) per polyp. The body column and tentacles of chimeras never seemed to elongate as much as those of either parental species, and this was a contributing factor to their short lengths. The chimeras were slightly more stalked than the H. attcnnata parent, but not as pronouncedly so as the P. oligactis parent.
Budding
The most clear-cut difference between buds of the parental species is the arrangement of tentacle rudiments. Buds of P. oligactis first acquire two lateral tentacles, and after these have grown long, two more intercalated rudiments arise (Fig. 3a). In //. attcniiata the tentacle rudiments arise nearly synchronously and are all about the same length (Fig. 3c). The chimeric pattern (Fig. 3b) is clearly of the H. attcniiata type.
The budding rates of chimeras were about normal. In one experiment three polyps each of //. attcniiata. P. oligactis and chimera were fed six shrimps/day.
292
H. LEE AND R. D. CAMPBELL
FIGURE 2. Morphology of hydra, (a) H. atfcnuata (left), chimera (middle) and P. oligactis (right). All three hydras are of the same age, growing under the same conditions, (b)-(d) different chimera individuals showing typical poses. Scale indicates 1 mm.
HYDRA CHIMERA
293
FIGURE 3. Pattern of tentacle development on buds, (c) H. attenuate!.
(a) P. oligactis (b) chimera,
long- enough for each to produce five buds. Budding rates for these three strains were, respectively, 0.26, 0.26, and 0.20 buds/day. Early morphogenesis, until basal disk formation, of the developing buds occurred at normal rates. However, chimera buds remained attached to their parents for an average of 8 days, while both parental buds detached after 3 days. Mature chimera buds were very small, averaging 1.2 mm in length (extended), whereas H. attenuate, buds average 5.1 mm and P. oligactis buds averaging 5.4 mm in length. Chimera buds did not them- selves begin to bud for 19 days, while parental buds budded after 10 (H. attenuata} or 11 (P. oligactis) days.
Regeneration
Polyps that had been fed six shrimps/day for 6 days were cut in half trans- versely and both parts allowed to regenerate, without feeding. Tentacle regenera- tion was assayed by the presence of tentacle rudiments. Basal disk regeneration was assayed by adherence to the dish.
The chimeras (n = 3) and both parental species (H. attenuata, n = 8; P. oligactis, n == 7) regenerated tentacles from the proximal half in 2 days. The distal halves of chimeras and of H. attenuata regenerated basal disks in 2 days. How- ever, the P. oligactis proximal halves had not regenerated basal disks in 26 days. Thus, in basal disk regeneration the chimera resembled the epithelial cell parent.
Feeding behavior
The chimeras (as well as the two parental strains) showed typical (Lenhoff, 1969) feeding responses to Artemia. When shrimp touched the tentacles they adhered, indicating desmoneme nematocyte discharge, and were paralyzed, indicat- ing stenotele nematocyte discharge. Tentacles holding shrimp underwent consider- able writhing, and shrimp were brought to the mouth repeatedly. However, in the chimera the shrimp were never swallowed. The chimeras were thus unable to feed themselves.
To determine if the swallowing behavior itself wras deficient in the chimeras,
2<)4
H. LEE AND R. D. CAMPBELL
TABLE 1 1 1
IiilnhitinH of ncmatocyst discharge following satiation. Numbers represent average number of trial* before hydra caught and paralyzed two Artemia. Twenty was the maximum nnniher of trials al- lowed for any polyp. These data represent about 100 sets of trials.
|
Hydra strain |
|||
|
//. at/t'tuiata |
/'. oliyactis |
Chimera |
|
|
10-30 |
5.8 |
7.5 |
5.4 |
|
30-60 |
12 |
16 |
8.4 |
|
60-120 |
10 |
20 |
6.7 |
we strung isolated hyclranths on nylon fishing line in "M" solution and measured the creeping movement along the line. In normal hydra the hydranth will move rapidly along the line in the direction of the mouth, as the hydranth attempts to swallow the line. After the first 20 min, H. attcnnata (n -- 1) had moved 2.4 mm and P. oligactis (n-- 1) had moved 0.9 mm. Two chimera hyclranths failed to move during 3.5 hr of observation.
Normal hydra exhibit a mouth opening response to glutathione. The dura- tion of opening reflects the intensity of the response (Lenhoff, 1969). The durations of the feeding responses to 10~5 M glutathione (26° C, pH 7.0) were measured with the following results: H. attcnnata (n~10), 35.2 ± 18.6 min; P. oligactis (n =: 11), 41.9 ± 13.5 min ; chimera (n == 26), 21.7 ± 6.8 min.
We also tested the satiation response, as manifested by the failure of nemato- cysts to discharge, and consequently the failure to trap Artcn/ia, after the hydra is fed (Smith, Oshida and Bode, 1974 ). Table III shows data from these experiments. The chimeras showed a reduced satiation response up to two hours after feeding.
Body niotility
Chimeras were much less active than were the two parents. Chimeras never somersaulted nor extended fully, while parental polyps frequently did. We analyzed body contractions and pulsations using time-lapse motion pictures show- ing all three polyp types in the same dish. By "contractions" we refer to a marked shortening of the body column followed by a reextension. "Pulsation" refers to peristaltic waves traveling proximally down the column.
In contraction frequency, chimeras (0.25/min) were intermediate between H. attcnnata (0.08/min) and P. oligactis (0.34 /''min). In pulsation frequency chimeras (1.07/min) resembled H. attcnnata (1.03/min). P. oligactis did not exhibit pulsations.
DISCUSSION
The major objective of this research was to produce a chimeric strain of hydra in order to distinguish developmental and behavioral contributions of the epithelial and interstitial cell lineages. Other studies of this nature (Sugiyama and Fujisawa, 1978; Marcum and Campbell, 1978b) showed the feasibility of this approach using closely related strains or species. In the present study we sought to examine the
HYDRA CHIMERA 295
feasibility of using distantly related species, in this case species of separate genera.
In several traits it was possible to attribute chimeric development and behavior to particular cell types. The pattern of tentacle origin on buds, the rate of basal disk regeneration, heterospecific grafting specificity, color, and prominence of columnar peristaltic waves were all clearly characteristic of H. attciutata. Thus, these traits are determined by epithelial cells. On the other hand, nematocyst mor- phology, nematocyst concentration, and interstitial cell temperature sensitivity (Fradkin, Lee, and Campbell, unpublished) were distinctively those of the P. olhjactis, indicating that these characteristics are due to the interstitial cell lineage. These results fit the pattern so far uncovered (Campbell, 1979) that morphological and morphogenetic traits derive principally from the epithelial cell genotype.
In several respects the evolutionary divergence between H. attenuate, and P. olhjactis was apparently too great to allow normal chimera functioning. These chimeras were very delicate, and had such altered behavior that they could neither eat nor sommersault by themselves. Repopulation of epithelial hydra by interstitial cells of the same species yields hydra of normal behavior ( Marcum and Campbell, 1978b) suggesting that the behavioral defects observed here are due to cell incompatibilities rather than due to the repopulation procedure. Therefore, in constructing chimeras for purposes of deducing cellular roles one must work with more closely related species. However, chimeras containing cells as divergent as H. attcnnitta and P. oliyactis certainly may be useful in unravelling cellular mechanisms. It would be interesting, for example, to see if abnormal neuromuscular contacts might be responsible for the inadequacy of the feeding behavior or the absence of column elongation.
\Ye thank Margaret Chow, Beverly Marcum, and Nancy \Yanek for help in these studies. Supported by research grants PHS NS 12446 and XSF PCM-02276.
SUMMARY
An intergeneric chimera was produced by repopulating epithelial Hydra attcnnata (lacking the interstitial cell lineage) with interstitial cells of Pelmatohydra oligactis. The chimera's morphology and morphogenesis generally resembled that of H. attcnnata, for example in the pattern of bud tentacles, in basal disk regeneration rate, and in heterografting specificity. Nematocyst characters of the chimera were the P. oligactis type. In behavior the chimeras were inter- mediate in some respects but deficient in others. For example, chimeras were unable to feed by themselves or to extend the column. This study illustrates the value of chimeras in deducing which cell types control the various aspects of develop- ment and behavior.
NOTE ADDED IN PROOF
Data from cell type composition studies show that the percentage of nerves among the total cells counted for H. attcnnata is 5.5^, for P. oligactis, ?.7c/c, and for the Chimera, 6.4%. A total of 5000 to 9000 cells were counted for 6 to 9 indi- viduals (6 individuals of H. attcnnata, 6 of P. olit/actis. and 9 of Chimera).
296 H. LEE AND R. D. CAMPBELL
LITERATURE CITED
BRIEN, P., AND M. RENIERS-DECOEN, 1955. La signification des cellules interstitielles des
hydres d'eau douce et le probleme de la reserve embryonnaire. Bull. Biol. Fr. Bclg.,
89: 285-325. CAMPBELL, R. D., 1979. Development of hydra lacking interstitial and nerve cells ("epithelial
hydra") Symp. Soc. Dev. Biol., 37: 267-293. CAMPBELL, R. D., AND C. BIBB, 1970. Transplantation in coelenterates. Transplant. Proc.,
2: 201-211. DAVID, C. N., 1973. A quantitative method of maceration of hydra tissue. Wilhelm Roux'
Arch. Etitu'ickhtin/sineeli. Org., 171 : 259-268. EVLAKOWA, V. F., 1946. Form-building migration of regenerative material in the hydra.
C.R. Academy of Science. Moscozv, 53 : 373-376. EWER, R. F., 1948. A review of the Hydridae and two new species of Hydra from Natal.
Proc. Zoo/. Soc. Land. 118: 226-244. KOLENKINE, X., AND A. M. BoNNEFOY, 1976. Etude structurele des jonctions et "contacts"
myoepitheliaux dans 1'affrontment heterospecifique Pchnatohydra oligactis et Hydra
attemtata. J. Microscop. Biol. Cell, 27 : 59-68. LENHOFF, H. M., 1969. pH profile of a peptide receptor. Comp. Biochcm. Ph\siol., 28:
571-586. LENHOFF, H. M., AND R. D. BROWN, 1970. Mass culture of Hydra: an improved method and
its application to other aquatic invertebrates. Lab. A trim., 4: 139-154. MARCUM, B. A., AND R. D. CAMPBELL, 1978a. Development of Hydra lacking nerve and
interstitial cells. /. Cell. ScL, 29: 17-33.
MARCUM, B. A., AND R. D. CAMPBELL, 1978b. Developmental roles of epithelial and inter- stitial cell lineages in hydra : analysis of chimeras. /. Cell Sci., 32 : 233-247. MUSCATINE, L., AND H. M. LENHOFF, 1965. Symbiosis of Hydra and algae. I. Effects of
some environmental cations on growth of symbiotic and aposymbiotic Hydra. Biol.
Bull.. 128: 415-425. SAFFITZ, J. E., A. L. BURNETT, AND G. E. LESH, 1972. Nervous system transplantation
in hydra. /. Ex p. Zoo/., 179: 215-223. SMITH, S., J. OSHIDA, AND H. BODE, 1974. Inhibition of nematocyst discharge in hydra fed
to repletion. Biol. Bull, 147 : 186-202. SUGIYAMA, T., AND T. FUJISAWA, 1978. Genetic analysis of developmental mechanisms in
hydra. V. Cell lineage and development of chimera hydra. /. Cell Sci., 32 : 215-232.
Reference: Biol. Bull., 157: 297-305. (October, 1979.)
ON THE POPULATION BIOLOGY AND NATURE OF DIAPAUSE OF LABIDOCERA AESTIVA (COPEPODA: CALANOIDA) 1
NANCY H. MARCUS
Woods Hole Occanograpliic Institution, Woods Hole, Massachusetts 02543
Knowledge of the seasonal distribution patterns of many marine copepods led Fish and Johnson (1937) to postulate that these species might produce resting eggs as an adaptive mechanism to insure survival at times of the year unfavorable to a planktonic existence. It was not until 1972 (Zillioux and Gonzalez, 1972) that conclusive results demonstrating the production of resting eggs by a marine copepod were obtained. During the last decade, it has become increasingly evident that the production of resting eggs by temperate, inshore marine copepods is a wide- spread event in this group (Zillioux and Gonzalez, 1972; Kasahara, Onbe and Kamigaki, 1975; Landry, 1975a; Grice and Gibson, 1975, 1977; Grice and Law- son, 1976; Uye and Fleminger, 1976). These studies have documented the existence of resting eggs in bottom sediments (Kasahara, Uye and Onbe, 1974, 1975 ; Kasahara, Onbe, and Kamigaki, 1975 ; Grice and Gibson, 1975, 1977 ; Uye and Fleminger, 1976) and demonstrated their production by females collected from the plankton (Grice and Gibson, 1975, 1977; Grice and Lawson, 1976; Uye and Fleminger, 1976; Zillioux and Gonzalez, 1972). These investigations have shown that physical factors such as temperature, light, salinity, and oxygen con- centration affect the maintenance and termination of the resting egg. The events which actually trigger the induction of resting egg production are still poorly understood. The elucidation of factors which influence the induction, maintenance, and termination of copepod resting eggs is necessary to achieve a better under- standing of the biology, evolution and distribution patterns, both in time and space, of neretic temperate-boreal copepods which are key elements in the food web.
Labidoccra aestiva is a large calanoid copepod reported to occur in coastal waters from the Gulf of St. Lawrence to the Gulf of Mexico (Wheeler, 1901 ; Grice, 1956; Fleminger, 1957; Cronin, Daiber and Hulbert, 1962; Anraku, 1964; Van Engel and Tan, 1965; Bowman, 1971; Fleminger, 1975). The populations occurring north of Cape Hatteras are seasonally abundant in the plankton, with maximum numbers of nauplii, copepodites, and adults occurring in the summer and fall, and disappearing by mid-December. Grice and Gibson (1975) and Grice and Lawson (1976) have demonstrated the production of resting eggs by females of L. aestiva collected in the field and their presence in bottom sediments.
During the summer-fall period in 1977, a preliminary investigation of subitane- ous and resting egg production by L. aestiva from Vineyard Sound, Massachusetts was conducted. This work indicated that the production of resting eggs commenced in early September. The surface water temperature was 19.5° C. The data presented in this paper document the seasonal variation in subitaneous and resting
1 Contribution Number 4345 from the Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543.
297
298
NANCY H. MARCUS
UJ M
in
O
CD
U
100
I
U
50 < I
D
FIGTRK 1. Trends in body size (cephalothorax, solid square, total length solid circlet, percent hatch (open square), and surface water temperature (open circle) for the sampling period July 7, 1978 to December 6, 1978.
egg production during the summer-fall period in 1978. It is shown that the proportion of subitaneous eggs produced is greater during the summer, decreases markedly in September, and persists as a small percent throughout the fall. More- over, the major change from subitaneous to resting egg production precedes the initiation of increasing adult body size and decreasing surface water temperatures by 2 weeks. It is demonstrated that resting eggs which are chilled at 5° C for a minimum period of 30 days, will hatch synchronously within 2 days when warmed to 21° to 23° C. Comparable eggs kept continuously at 19° to 23° C will hatch over a longer period of time and not synchronously. This paper discusses the significance of these findings as they relate to diapause induction, maintenance, and termination, as well as the population dynamics of L. acstiva.
MATERIALS AND METHODS
Specimens of Labidoccra acstlva were collected from July through December at weekly intervals from a 1 to 2 m depth in Vineyard Sound, Massachusetts by tow- ing a 1-m, 243-/x mesh net for a 10 min period. Adult males and females were removed from these two samples by pipette. Twenty individuals of each sex were preserved in 5% buffered formalin each week, except for collections in late fall when very few adults were present. Two measurements of body size were obtained for each
lUAPATSK. OK /.. AESTIVA 299
preserved specimen (i.e., cephalothorax, and total length) using a stemnnicruscope and ocular micrometer. During the summer months, eggs were collected by placing 10 females together in separate 100-ml dishes. Gynmodiniitni nelson! was added as food, and the dishes were placed overnight in an incubator at 19° C, with a 12 to 12-hr photoperiod. The following morning eggs were removed by pipette and counted. The percent hatch was determined for an aliquot (100-120) of these eggs. During the summer, eggs usually hatched within 2 days. The same procedure of collection, counting, and determination of percent hatch was followed for fall eggs. Fall eggs which did not hatch within 4 days were placed in filtered sea water (glass fiber) in 75-ml screw-top glass jars. The jars were incubated at 19° C or refrigerated at 5° C. At the appropriate time, the refrigerated jars were warmed to 21° to 23° C and percent hatch determined. At the same time, the percent hatch in the jars kept at 19° C was determined.
RESULTS
The trends in body size, percent immediate hatch, and surface water tempera- ture for the six-month period between July and December 1978 are shown in Figure 1. The standard deviations for body size ranged between 0.01 and 0.17. For each sex, changes in ceplialothorax length paralleled total length. At each census throughout the sampling period, female body size was larger than male body size. An inverse relationship was observed for body size and surface water temperature for both females and males, and is depicted for adut females in Figure 2a. The smallest adults were collected during July and August when water temperatures ranged between 20° to 22° C. A marked increase in size began in September reaching a maximum in late November.
The percent immediate hatch (i.e., within 2-4 days) of eggs collected from grouped females was greatest from July to mid-August, ranging between 74 and 93%. Subsequently the percent immediate hatch began to decrease and was observed to vary between 6 and 27% from mid-September to early December. The marked decrease in the portion of eggs hatching immediately began in late August, and preceded the initial drop of surface water temperatures to below 20° C (Figs. 1, 2b) and the time at which body size began to increase. No obvious dif- ference in total egg production was observed, although differences may exist.
The percent hatch of eggs incubated at 19° or 5° C and then later warmed to room temperature (21°-23° C) is shown in Table I. The data presented are for eggs laid within 1 day of collection for five samplings between early September and late November. In general the percent hatch of eggs kept at 5° C and then warmed to 21° to 23° C was higher than those incubated at 19° C. Moreover, the hatch of the 5 ° C eggs occurred synchronously within two days of being warmed to 21° to 23° C. It was determined from periodic examination of the jars held at 19° C that non-synchronous hatching occurred throughout the incubation interval. Some of the unhatched eggs at 19° C appeared dead (brown, granulated interior), whereas others appeared viable (greenish hue).
DISCUSSION
An inverse relationship between body size and temperature has been demon- strated for a number of marine and freshwater copepods (Coker, 1933; Marshall,
300
NANCY H. MARCUS
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3.0
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TEMPERATURE °C
25
FIGURE 2. Scatter diagrams of female total body length (a), and percent hatch (b) versus surface water temperature at the time of collection.
Nicholls, and Orr, 1934; Aycock, 1942; Deevey, 1960a, b; McLaren, 1965 ; Deevey, 1966; see review in Hutchinson, 1967; Durbin and Durbin, 1978). The seasonal pattern of size variation reported herein for Labidocera aestiva from Vineyard Sound is similar to that reported by Deevey (1960a) for another population of the species from Delaware Bay studied in 1930. However, the patterns are shifted in time. The smallest individuals are found during August in Vineyard Sound and September in Delaware Bay. Deevey (1960a) reports that the average surface water temperature in Delaware Bay was 22° C during September. This value
DIAPAUSE OF L. AESTIVA
301
TABLE I
Percent hatch of incubated (19°-23° C) and refrigerated Clutch 1 eggs after -warming to 21°-23° C, for five samplings between early September and late November.
|
Date incubated |
Period of incubation |
|||
|
Date collected |
or refrigeration |
', Hatch |
||
|
0° |
19-23° |
(days) |
||
|
Sep 27 |
Oct 2 |
75 |
96 |
|
|
Sep 27 |
Oct 2 |
75 |
61 |
|
|
Oct 5 |
Oct 10 |
111 |
95 |
|
|
Oct 5 |
Oct 10 |
111 |
42 |
|
|
Sep 7 |
S.-p 1 1 |
58 |
83 |
|
|
Sep 7 |
Sep 1 1 |
58 |
2 |
|
|
Oct 19 |
Oct 23 |
126 |
95 |
|
|
Oct 19 |
Oct 23 |
126 |
55 |
|
|
Nov 21 |
Nov 27 |
87 |
95 |
|
|
Nov 21 |
Nov 27 |
87 |
95 |
corresponds to temperatures recorded for Vineyard Sound in August (Fig. 1). The pattern, however, varies from year to year within an area, inasmuch as the smallest individuals during 1932 in Delaware Bay were collected in early August, when temperatures were then highest. It is obvious that body size is correlated more with the surface water temperature than with the time of collection.
In the present study, there was no evidence of discrete generations for L. acsth'a in Vineyard Sound. Breeding appeared to be continuous throughout the summer and fall. Deevey (1960a) suggested that spawning was continuous during the summer for the Delaware Bay populaton, but that a break occurred between early August and September, resulting in a burst of larger individuals late in October. Moreover, it was suggested that the offspring of the large-sized fall individuals would not mature until the following year. More recent studies of L. acstiva from Vineyard Sound (Grice and Gibson, 1975; Grice and Lawson, 1976) have demon- strated that the offspring of the fall individuals survive the winter as resting eggs, and hatch the following spring. It is probable that the same life history pattern is common to the Delaware Bay population.
In Vineyard Sound, the population continues to breed at temperatures as low as 8.5° C (Fig. 1), producing both subitaneous and resting eggs. On the other hand, Deevey (1960a) reports that 12° C is the lower limit of breeding in Delaware Bay. This difference could reflect acclimatization or genetic adaptation of individuals to the slightly differing thermal regimes of Vineyard Sound and Delaware Bay (see Bumpus, 1957). It has been shown for a wide variety of marine invertebrates that the tolerance limits for a number of biological functions differ between populations exposed to different environmental regimes (see Battaglia and Beardmore, 1978).
Whatever the factor (s) inducing the production of resting eggs by L. acstira from Vineyard Sound, it is shown that the response (i.e. initiation of dormancy) is first expressed in late August or early September. The intensity of the response increases as time progresses and the surface water temperatures decrease. This intensification is expressed as an increase in the percent production of resting
302 NANCY H. MARCUS
eggs and a decrease in subitaneous eggs. There is a strong correlation between temperature and the type of egg produced (Fig. 2b). However, temperature alone is not sufficient to account for the observed switch in egg production in early September (Fig. 1). The same temperatures (i.e. 18° -20° C) which are co- incident with the production of resting eggs in September are observed in early summer when production is restricted to subitaneous eggs. Temperature may influence the type of egg produced, but it would appear that other factors are also involved.
The data show that subitaneous eggs are produced throughout the summer-fall period, and that resting eggs are produced only during the fall. There are clearly two egg types being produced in the fall. Subitaneous eggs produced at this time take longer to hatch (2-4 days) at 21° to 23° C than those produced in the summer (1-2 days).
The rate of embryonic development of copepod eggs is influenced by egg size, temperature, and environmental conditions experienced by the parents (McLaren, 1965; 1966; McLaren, Corkett, and Zillioux, 1969; Landry, 1975b; Hart and McLaren, 1978). For L. acstiva the observed difference in rate of development most probably reflects a maternal effect relating to the physiological condition of the female at the time of collection, as was shown for Pseudocalanns by Hart and McLaren (1978). No obvious size difference was observed between summer and fall subitaneous eggs of L. acstiva in the present study.
The fall subitaneous eggs of L. acstiva were detected by placing females collected at that time in an incubator at 19° C. If the subitaneous eggs produced by fall females were collected and held at temperatures equivalent to the ambient values in Vineyard Sound, their development would be retarded and they could be mistaken for resting eggs. On the other hand, fall eggs which do not hatch within 2 to 4 days at 21° to 23° C are to be regarded as resting eggs. As first demon- strated by Grice and Gibson (1975) resting eggs of L. acstiva remain viable for as long as 120 days at 5° C. It was suggested that a minimum chilling period of of 2 to 4 weeks at 5° C was required to break the dormant condition. The results of the present study show that if resting eggs are placed at 19° C without chilling, hatching will take place, but it takes longer and occurs sporadically. Chill- ing of resting eggs results in the reduction of time in actual diapause and synchroniza- tion of hatching. The diapause and hatching response of resting eggs of L. aestiva to different temperatures corresponds to several examples of insect and fresh- water copepod diapause (Church and Salt, 1952; see Hutchinson, 1967; Stress, 1969b ; Dean and Hartley, 1977a, b). The dormant state in these animals consists of a period of diapause followed by post-diapause development and then hatching (see Mansingh, 1971). During the diapause period, development is arrested and cannot resume even if conditions are favorable. Once the diapause is broken the individual is competent to resume development as soon as adverse conditions are terminated. For L. acstiva the actual breaking of diapause (re- activation) can take place at 19° C, but the process is faster at colder temperatures. Post-diapause development, however, is slower at reduced temperatures than at high temperatures. Therefore, individual chilled eggs terminate diapause at different times, and further development is retarded as long as the eggs remain chilled. The chilled eggs accumulate at a stage of readiness, and when exposed to higher tempera-
DIAPAUSE OF L. AESTIVA 303
tures are competent to proceed with development, resulting in synchronous hatching. On the other hand, individual resting eggs held continuously at 19° C terminate the diapause condition at different times (as the chilled eggs), but since the higher temperature is favorable for post-diapause development to proceed there is no accumulation of individuals at the boundary between diapause and post-diapause so that hatching is asynchronous.
Two attributes expressed by resting eggs are cold-hardiness and synchronous hatching. Resting eggs remain viable at 2° C for as long as 6 months (Grice and Gibson, 1975) whereas summer subitaneous eggs remain viable for no longer than 15 days (Grice, unpublished). The tolerance of fall subitaneous eggs has not been investigated. The cold resistance of resting eggs enables survival during the winter. Synchronization of development promotes the reproductive success of the first generation appearing in the early summer by ensuring that individuals will attain reproductive maturity at the same time. If hatching was sporadic, the number of mature individuals in the population at any one time might not attain a size sufficient for successful mating encounters to occur.
The effects of temperature on the maintenance and termination of resting eggs of L. acsti-ra are similar to those observed for other plants and animals which overwinter. In the majority of cases tor which diapause induction has also been investigated, photoperiod and temperature have been shown to be the two most important factors affecting the initiation of dormancy (Harvey, 1957; Stross and Hill, 1965; see Hutchinson, 1967; Stross, 1969a, b; Watson and Smallman, 1971; see Mansingh, 1971; de March, 1977; see Clutter, 1978). The pattern of egg production observed in this study wrould occur if a developmental stage perceived a cue which then triggered an irreversible sequence of events leading to one type of egg or another. The fact that resting egg production precedes the decline of surface water temperatures suggests that such a stimulus may exist. The success- ful species will evolve a dormancy response to a factor which closely parallels the stress (in this case temperature) but which itself is extremely stable (such as photoperiod). That two egg types are produced during the fall could be due to the extended survival of summer females which lay subitaneous eggs and the newly developed fall females programmed to produce resting eggs. Moreover, because no two individuals are alike, some females may respond to a weak stimulus, whereas others require a more intense exposure. In the spruce budworm (Harvey, 1957), diapause-free development occurs under a long day regime (i.e., greater than 15 hr of light) in a portion of the population. As day length is increased the number of non-diapausing insects increases, reaching 100% in continuous light. Universal diapause occurs when day length is less than 15 hr. Current progress in our laboratory indicates that short photoperiods (less than 12 hr light) are effective in inducing the production of resting eggs by laboratory-reared L. acstira. This will be reported on at length in a subsequent paper.
I thank George Grice for introducing me to the phenomenon of resting egg production by marine copepods and for the many hours of discussion of the prob- lem. I also thank G. Grice and T. Cowles for their helpful criticism of the manuscript. This work was supported by NSF grant OCE-7808857 and the Ocean Industry Program at the Woods Hole Oceanographic Institution.
304 NANCY H. MARCUS
SUMMARY
The calanoid copepod, Labidocera acstwa was collected from Vineyard Sound, Massachusetts between July and December 1978. Adult size (cephalothorax and total body length) was inversely proportional to surface water temperature at the time of collection. The major switch from subitaneous to resting egg production occurred during late August to early September, but a small percent of subitaneous eggs continued to be produced throughout the fall. Resting eggs were cold-resistant and when chilled at 5° C hatched synchronously when warmed to 21° to 23° C. Individual resting eggs kept continuously at 19° C took longer to hatch and emer- gence was asynchronous. The resting eggs of L. aestiva appear to be in a state of diapause similar to many insects, and it is suggested that photoperiod is the primary cue inducing the production of resting eggs.
LITERATURE CITED
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in eggs of Melanoplus bivattatus (Say) (Orthoptera: Acridae). Can. J. Zool., 30:
90-171. CLUTTER, M., 1978 (ed.). Dormancy and Developmental Arrest. Academic Press: New
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DIAPAUSE OF /.. AESTIVA 305
FLEMINGER, A., 1957. New calanoid copt-puds of Pontclla Dana and t.abidocera Lubbock
with notes on the distribution of genera in the Gulf of Mexico. Tulane Stud. Zool.,
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/. Fish. Res. Bd. G/H.. 34: 410-412. GRICE, G. AND T. LAWSON, 1976. Resting eggs in the marine calanoid copepod, Labidoccra
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budworm, Choristoncura funifcrana (Clem.) (Lepidoptera : Tortrichidae). Can. J.
Zool., 35: 549-572. HUTCHINSON, G. E., 1967. A Treatise on Limnology. Vol. II. John Wiley and Sons, Inc. :
New York, 1115 pp. KASAHARA, S., T. ONBE, AND M. KAMIYAKI, 1975. Calanoid copepod eggs in sea-bottom
buds. III. Effects of temperature, salinity, and other factors on the hatching of
resting eggs of Tortanus forcipatus. Mar. Biol., 31 : 31-35. KASAHARA, S., S. UYE, AND T. ONBE, 1974. Calanoid copepod eggs in sea-bottom muds.
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Seasonal cycles of abundance in the populations of several species of copepod and
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528-538.
MCLAREN, I., 1966. Predicting development rate of copepod eggs. Biol. Bull.. 131 : 457-469. McLAREN, I., C. J. CORKETT, AND E. J. ZiLLious, 1969. Temperature adaptations of copepod
eggs from the Arctic to the tropics. Biol. Bull., 137 : 486-493.
STROSS, R., 1969a. Photoperiod control of diapause in Daphnia. II. Induction of winter dia- pause in the Arctic. Biol. Bull., 136: 264-273. STROSS, R., 1969b. Photoperiod control of diapause in Daphnia. III. Two stimulus control
of long-day, short-day induction. Biol. Bull., 137 : 359-374.
UYE, S., AND A. FLEMINGER, 1976. Effects of various environmental factors on egg develop- ment of several species of Acartia in southern California. Mar. Biol., 38 : 253-262. VAN EXCEL, W., AXD E. TAN, 1965. Investigations of inner continental shelf waters of lower
Chesapeake Bay. Part VI. The copepods. Chesapeake Sci., 6: 183-189. WATSOX, N., AXD B. SMALLMAX, 1971. The role of photoperiod and temperature in the
induction and termination of an arrested development in two species of freshwater
cyclopoid copepods. Can. J. Zool., 49 : 855-862. WHEELER, W., 1901. The free-swimming copepods of the Woods Hole region. Bull. U. S.
Fish Comm. (1899) : 157-192. ZILLIOUX, E., AND J. GONZALEZ, 1972. Egg dormancy in the neretic calanoid copepod and its
implications to overwintering in boreal waters. Arch. Oceanogr. Limnol., European
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Ki-tVmicc- : Kiol. Hull., 157: 306-319. (October, 1979)
LOCOMOTION IN THE PRIMITIVE PULMONATE SNAIL MELAMPUS BIDENTATUS: FOOT STRUCTURE
AND FUNCTION
STACIA MOFFETT
Department of Zoology, Washington State University, Pullman, Washington 99164, and Marine Biological Laboratory, Woods Hole, Massachusetts 1)25-13
Most land pulmonates crawl by means of muscular waves that travel along the foot from posterior to anterior. These waves, classified as direct waves by Vies (1907), have been analyzed by Jones (1973) for the land slug Agriolimax. A great variety of wave types has been described for prosobranch gastropods ; the most primitive type is thought to be the retrograde type, in which the waves travel in a direction opposite to the gastropod's progress (Miller, 1974; Trueman, 1975). Jones and Trueman (1970) have analyzed the mechanism of retrograde waves in the limpet Patella and have reviewed gastropod locomotory waves (Jones, 1975; Trueman, 1975).
The purpose of the work reported here has been to analyze the unusual locomo- tion of the snail Melainpits bidcntatus. This basomtnatophoran snail is a member of one of the most primitive families of pulmonates, the Ellobiidae. Information on its mode of locomotion may cast light on the origin of the locomotory behavior exhibited by the more advanced land pulmonates.
Although Morton (1955) indicated that land-inhabiting ellobiids, including M clam pus, crawl in a way that others have classified as retrograde waves (Jones, 1975), my examination of events in M clam pus locomotion has not supported that classification. Rather, Melampus exhibits a form of locomotion that fits neither the direct nor retrograde wave category nor any of the other locomotor categories of Miller (1974). Mclain[>ns locomotion consists of a repeated sequence of events, the crawl-step, in which the posterior of the foot slides along but the anterior is lifted and placed. Hydraulic forces, described by Chapman (1958; 1975) for a variety of soft-bodied animals, are an important component in the locomotion of M clam pus. The blood is acted on by the columellar muscles as well as the intrinsic pedal musculature, and the transverse subdivision of the foot allows the posterior-to-anterior transfer of blood to be coordinated with the muscular events.
MATERIALS AND METHODS
Annual. Adult specimens of Melampus bidcntatus Say with shell lengths of 7 to 11 mm were collected in salt marshes of Woods Hole, Massachusetts. They were maintained in large, covered fingerbowls lined with filter paper moistened with 75% sea water (Instant Ocean) and provided with napa (Chinese) cabbage and crushed eggshells. A photoperiod of 8L: 16D and constant temperature of 16° C suppressed reproductive activity (Apley, 1968).
Photographic Techniques. Crawling snails were filmed with a Bolex Macro-
306
LOCOMOTION IN MELAMPUS 307
zoom 160 super 8 mm camera at 18 frames per second. Side and bottom views were obtained simultaneously by the use of mirrors.
Marking the Foot. Snails were anaesthetized according to the method of Price (1977). Three or more spots of India ink were injected just beneath the surface of the sole. The animals were allowed to recover overnight in fresh 75% sea water.
Anatomy. Pedal and columellar muscles were studied in live and narcotized snails and specimens fixed in alcoholic Bouins. Serial sections of snails quick- frozen in liquid nitrogen while crawling (Jones, 1973) provided information on the muscle contraction patterns during locomotion. The 10 /xm sections were stained with Mallory's triple stain (Pantin, 1948).
Force Recordings. Snails were allowed to crawl over a plexiglass platform having a 2-mm gap into which a 1-mm bar was inserted. The bar was attached to a force transducer (Statham Micro-Scale Accessory Model UL5) connected to an amplifier (Gilson IC-MP module). The resulting forces were recorded on a chart recorder (Gilson ICT-SH). The transducer was connected so as to measure either upward and downward forces or backward and forward forces as the snail's foot passed across it. The translucent platform on which the snail crawled was positioned just above the writing surface of the recorder so that the crawling snail was filmed with the chart recorder's activity in the background. The absolute magnitude of the forces was not relevant to this analysis.
RESULTS
Stages in the crawl-step
As illustrated in Figure 1, the foot of Mclanipiis bidentatns is divided anatomically into an anterior region (the propodium) and a posterior region (the metapodium) by a permanent transverse groove. The mouth region sur- rounded by the oral veil or lappets forms an important part of the locomotory surface.
In Melampus locomotion a single series of muscular events propagates along the foot at one time. I have called this mode of locomotion a crawl-step because the anterior part of the foot is lifted free of the substratum. Characteristic postures assumed by the foot during the crawl-step have been identified in frame-by-frame analysis of motion picture films of crawling snails. Postures identified with three easily recognized stages of the crawl-step are shown in Figure 1. Each stage is characterized by the following events :
Stage I. Metapodial shortening /propodial extension. The posterior third of the metapodium shortens as a single movement and the propodium simultaneously extends to its longest dimension. The head and oral veil are lifted off the sub- stratum.
Stage II. Metapodial extension. A series of local muscular contractions within the metapodium produce one or more waves that ripple forward along the sides of the metapodium from the medial region of the metapodium toward the transverse groove. The region of the metapodium ahead of the elevations bulges outward and when the bulge reaches the transverse groove the posterior edge of the pro- podium is displaced upward and forward by the expanded metapodium. Mean-
308
STACIA MOFFETT
T=0
Stage I ov
T=1.6
Stage Ilia
T=2
Stage Illb
5 mm
FIGURE 1. The crawl-step of AIclniiipus bulciitntus. Side and bottom views traced from motion picture frames for Stage I : metapodial shortening/propodial extension, Stage II : metapodial extension, and Stage III : propodial elevation, posterior (a) and then anterior (h) phase. Cumulative elapsed time in seconds is given to the left. Dotted lines (side views) give the snail's posture at onset of the movement characteristic of each stage and the unbroken lines represent the posture upon completion of the movement. Stippled regions (bottom view) indicate portions that were lifted during each stage. Subdivisions of the locomotory surface are labeled in the bottom view of Stage I: OV, oral veil-mouth region; P, propodium of the foot; M, metapodium of the foot.
while the posterior region of the metapodium narrows and flattens dorsoventrally and the shell tilts forward as the oral veil is lowered to the substratum.
Stage III. Propodial elevation. The posterior region of the propodium is elevated first (Ilia, Fig. 1) and then the anterior region is elevated (Illb, Fig. 1). The oral veil-mouth region forms an area of contact with the substratum throughout this stage. The shell tilts backwards.
In the motion picture from which the tracings shown in Figure 1 were made, the snail progressed 2.2 mm in 2.3 sec. Snails with shell lengths of 10 to 1 1 mm typically crawled 2 to 4 mm/step at a rate of 10 to 20 steps/min at 20° C.
Foot morphology
Figure 2 shows the relationship of the columellar muscles to the foot. In Mchunpus the columella itself is largely resorbed during development, leaving
LOCOMOTION IN MEL. IMP US
309
RAPM
B
LACM
RAPM
B LACM
FIGURE 2. Relationship between columellar muscles and the foot in Melampus. A, view of animal's left side; B, right side. Left anterior cephalic muscle (LACM) is unshaded, the right anterior and right and left posterior muscle (RAPM) stippled, and buccal muscle (B) crosshatched.
the columellar muscles attached to the inner wall of the .shell (Morton, 1955). The muscles follow the inner surface of the shell within the body whorl in approxi- mately a 360° turn from origin to insertion in the extended foot. In addition to the buccal retractor muscle, two major subdivisions of the columellar muscle are apparent in Melainpiis. The left anterior and cephalic muscle (LACM, Fig. 2) originates in several adjacent bundles and further subdivides into muscle bands that insert around the head, into the left tentacle and oral veil, the left side of the propodium and approximately the anterior third of the left side of the metapodium. The right anterior and right and left posterior muscle (RAPM, Fig. 2) has a single origin on the shell and divides into muscle bands that insert on the right tentacle, the right oral veil, the right propodium, the anterior portion of the metapodium and bilaterally in the posterior region of the metapodium. Thus the muscles enter the foot in a quite asymmetrical pattern with those muscles on the left side that support the shell being the more massive. The columellar muscle system of Mela in pits probably includes both the longitudinal and columellar muscle groups described for Lyninaea by Plesch, Janse and Boer (1975).
In serial sections of snails quick-frozen in the act of crawling, there is no evidence that intrinsic bands of muscle fibers form discrete layers above the sole of the foot. Rather, the fibers that could be traced appear to be derived from columel- lar muscles. This is apparent in the parasagittal section which cuts through the left anterior and cephalic columellar muscle (Fig. 3C). The muscle fibers form a mesh- work enclosing small, spherical blood spaces, shown in a region of the metapodium (Fig. 3E). These are similar to those in the foot of the limpet, Patella (Jones and Trueman, 1970 ) . These small spaces are contrasted with the large blood sinuses that are present in the anterior region of the foot (Fig. 3 A, D).
\Yhen sections of snails frozen in different stages of the step cycle are com- pared, one of the most obvious differences is in the orientation of the transverse groove. In Stage I, the metapodial shortening/propodial extension results in a propodium that is long and a transverse groove that is shallow and anteriorly- slanting (Fig. 3A). In Stage Ilia elevation of the transverse goove and posterior region of the propodium pulls the groove into a deep backward-slanting indentation (Fig. 3C). As the propodial elevation moves into the anterior propodium (Stage
310
STACIA MOFFETT
FIGURE 3. Sections prepared from specimens frozen in liquid nitrogen while crawling. In each case, anterior is to the left. A. Stage I posture, mid-sagittal section showing propodial region of foot and tranverse groove (arrow). Scale bar 250 /JL. B. Stage II posture, mid- sagittal section showing metapodial region with two metapodial waves (arrows). Scale bar 250 (JL. C. Stage Ilia posture, left para-sagittal section through left anterior and cephalic muscle bands in the propodial region of the foot. Arrow indicates transverse groove. Scale bar 250 /j.. D. Stage Illb posture, mid-sagittal section showing propodial region of the foot and transverse groove (arrow). Scale bar 250 /j.. E. Detail of metapodial tissue showing small blood spaces. Scale bar 25 ^. F. Detail of muscle contraction pattern in the metapodial waves in Figure 3B. Scale bar 75 p. Abbreviations used: BS, large blood sinus; MG, suprapedal mucus gland; PG, pedal ganglion.
I lib) the transverse groove region is relaxed and assumes a condition intermediate between that of Stage Ilia and Stage I (Fig. 3D).
Metapodial contractile waves are shown in Figure 3B and the muscle pattern
LOCOMOTION IN MELAMPUS
311
contributing to them is apparent at higher magnification in Figure 3F. The sole of the foot is lifted straight up rather than pulled in a slanting angle forward. It appears that the elevations are caused by a local tightening of the mesh-work of fibers within the foot. Anterior to the elevations, the blood spaces are larger than in the contracted regions behind the waves. A consequence of anterior move- ment of such a pattern of waves would be the forward displacement of blood.
Marked-foot experiments
In this and the following sections an attempt was made to characterize more precisely the sequence of events and the forces underlying forward progression during the crawl-step. The aim of marked-foot experiments was to analyze temporally the changes in the relative size of the propodium and metapodium and the movement of points within those regions. This was achieved by filming the foot of snails that had spots of ink injected into the sole. In every fourth frame of the film the position of the ink spots was determined, relative to a fixed point behind the snail's foot. Sample data from five crawl-steps are illustrated in Figure 4. The beginning and end of one crawl-step is included between the vertical dashed lines.
The following features are apparent from such an analysis : First, shortening of the posterior metapodium, measured as advancement of the end of the foot, is mirrored in time and magnitude by advancement of the anterior edge of the propodium. Thus during posterior shortening the length of the snail's foot does not change. The remaining events in the crawl-step are all forward shifts of intermediate points along the foot, relative to the fixed posterior and anterior
14- 12- 10-
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FIGURE 4. Progress of the anterior (A) and posterior (E) edges of the foot and three spots of injected ink (B-D) was charted for every fourth frame of an 18 frames/sec motion picture film. The events of one complete crawl step occur between the vertical dashed lines.
312 STACIA MOFFETT
Stage I
Stage II
Stage Ilia
Stage Illb
FIGURE 5. Diagrammatic illustration of the relationship of an ink spot to the transverse groove. Motion picture films indicated that during locomotion the spot of ink disappeared within the transverse groove from Stage Illb through Stage I and was visible during Stages II and Ilia.
edges of the foot. These forward shifts proceed from posterior to anterior : the metapodial waves carry point D and then point C forward. Finally, point B moves forward as the anterior propodium is lifted oft" the substratum.
Films of a snail with a differently placed ink spot shed light on events occurring in the region of the transverse groove during locomotion. The ink spot was in the anterior metapodium almost within the transverse groove. During locomotion, the spot appeared and disappeared with each crawl-step. The position of the spot during the crawl-step is illustrated diagrammatically in Figure 5. It became visible during metapodial extension in Stage II and was eclipsed by expansion of the posterior propodium as the anterior propodium was elevated (Stage Illb). Both this and the histological evidence indicate that although the transverse groove is a constant feature of the foot, a greater or lesser region of the pedal sole anterior or posterior to this region can be drawn up into the groove. Both changes in blood volume in the propodium and metapodium adjacent to the groove, and contraction of columellar muscles which insert close to the groove, alter its configuration.
Up^vard and doumzvard forces
Direct observations and motion picture films indicate that portions of the Melampus foot are elevated during locomotion. Forward sliding movements along the mucus-covered substratum might also be expected to exhibit an upward com- ponent. Transducer recordings of upward and downward forces were obtained
LOCOMOTION IN MHLAMl'US
313
to determine which portions of the sole are experiencing upward or downward (weight-bearing) forces during each stage in the crawl-step cycle.
The forces exerted during locomotion were measured as snails crawled across a moveable bar positioned in a slit in a plexiglass platform. Five to six step cycles were required for snails to crawl across the bar and therefore at each successive step the transducer measured forces from a more posterior region of the foot. The pattern of upward and downward deflections recorded in this manner was fairly constant from crossing to crossing and animal to animal. Figure 6A is typical of the recordings that were produced. These data were interpreted as described in the methods. Postures characteristic of Stages I-III for each step were identified with particular points on the force recording as illustrated in Figure 6A.
Upward
* *
Downward 1 III 211
B
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Stage I
765432
Stage II
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Stage III
FIGURE 6. A is the force transducer recording of upward and downward forces exerted on a bar as a snail crawled across it. A portion of the snail's foot was in contact with the bar for six complete steps and the end and beginning of two other steps, for a total of 24 sec. Each point at which the simultaneously-recorded motion picture film indicated that the snail assumed one of the postures characteristic of Stage I, II or III of the crawl step cycle was marked on the transducer recording. The dotted line represents a baseline adjustment that was inserted to compensate for the overwhelming effect of passage of the shell mass onto the bar. In B the information obtained from the force recording is represented as a pattern of upward and downward-pointing triangles showing the distribution of vertical forces exerted by the foot on the substratum at Stages I, II and III of the step cycle. Regions exerting no vertical force are indicated by squares. The position of the triangles or squares under the foot marks the position of the recording bar each time the posture characteristics of a particular stage was assumed.
314 STACIA
Note that in the force recording in Figure 6 A there is a dramatic downward registration following stage II of the fourth step. At that time the shell weight shifts forward onto the har. From then until the foot is pulled off the bar, all the upward deflections fail to rise above the original baseline. The dotted line in Figure 6A was inserted as a relative baseline in an attempt to correct for the overwhelming effect of shell weight on the force recordings. Using that correction, the results of these experiments were summarized in Figure 6B. The following conclusions can be drawn : In metapodial shortening/propodial extension the pro- podium is weight-bearing while the posterior region of the metapodium exerts an upward force on the substratum. In metapodial lengthening the anterior region of the propodium and the posterior region of the metapodium are weight- bearing while the waves moving forward in the anterior region of the metapodium exert an upward force. In propodial elevation the oral veil and the entire metapodium are weight-bearing during elevation of the posterior and anterior propodium.
Forzi'ard and backward jurccs
Lissmann (1946) defined the sliding progression of a snail's foot along a substratum as being the product of forces acting parallel to the ground, including internal and external forces acting to change the shape of the snail's body and the reactions from the ground. The concept of the internal forces has been advanced and direct measurements of fluid pressure made for a variety of animals since that time (see Chapman, 1975). The reactions of the ground to the passage of the snail's foot were measured in this study to indicate what was happening along the length of the foot at each stage in the crawl-step cycle. Lissmann's (1946) reactions from the ground were defined as the static reaction, which is the force exerted on the substratum in a backward direction by stationary areas while other regions of the foot are advanced, and sliding friction, which is the forward force that moving portions of the foot exert. These backward and forward forces were recorded and interpreted in a manner similar to that described for upward and downward forces, but with the transducer measuring forces in the horizontal plane.
In general the sole of the foot did not noticeably protrude into the space in the plexiglass slit adjacent to the recording bar, but rather moved smoothly onto the bar. There were two notable exceptions to this generalization : if the spacing of the animal's approach to the bar was such that anterior metapodial advancement or propodial advancement began at the edge of the slit, then the foot region bulged downward into the gap and thereby displaced the bar forward by pushing it from behind. A sample recording of the forward and backward forces is shown in Figure 7A. The labels on the recording show the points at wrhich the snail's posture matched the characteristic postures for stages I, II, and III in each of the six crawl steps. The distribution of forces beneath the foot at each stage is illustrated in Figure 7B. During metapodial shortening/propodial extension, the posterior metapodium and the anterior propodium register forward sliding friction while the anterior metapodium and posterior propodium show the static reaction. In metapodial extension the entire metapodium registers a forward force and the propodium registers a backward force. In propodial elevation the propodium is the only region showing forward sliding friction.
LOCOMOTION IN MELAMPUS
315
Backward
Forward
B
65432
Stage I
r — x
5432
Stage II
FIGURE 7. A is the force transducer recording of forward and backward forces exerted by a snail's foot as it crawled across a bar. The snail required four complete crawl-step cycles and portions of two other cycles to complete the crossing. Information was obtained in a manner similar to that described for Figure 6. B is the pattern of forward and back- ward forces exerted by the foot on the substratum in Stages I, II and III of the crawl-step.
DISCUSSION
The crawl-step of Melainpits is a complex sequence of events compared with the direct pedal waves of most land pulmonates. Figure 8 gives a model for Melampus locomotion that was constructed from the histology, cinematography and force transducer recordings presented in the results.
According to this model the Stage I metapodial shortening/propodial extension result from a single muscular event : contraction of columellar muscles in the posterior half of the metapodium. This action not only draws the end of the foot upward and forward, as indicated by the transducer recordings, but it causes some blood to leave the posterior region of the foot. This blood could flow across the low transverse groove into the propodium and into the anterior region of the body. The pattern of forces recorded from the foot at this stage indicates that the weight is primarily borne by the posterior propodium and that it and the anterior metapodium experience a backward drag (the static reaction). The combination of downward and forward forces recorded from the anterior propodium are what would be expected in a region experiencing invasion of blood from the posterior. The observation that the propodium bulged downward into the crack
316
STACIA MOFFETT
Stage I
Stage
Stage Ilia
Stage 1Mb
FIGURE 8. Model of mechanical events producing locomotion in Mclampiis. Arrows below the foot show forces exerted on the substratum ; arrows within the body show fluid dynamics ; dark regions in the body indicate muscle contraction patterns. See text for discussion.
adjacent to the recording bar indicates that relaxed tonus in the propodium may contribute to the hydraulic expansion.
In Stage II, metapodial extension is produced by low-amplitude waves travelling from the middle toward the anterior metapbdium. Their action is depicted as squeezing blood forward within the ventral portion of the foot until it accumulates behind the transverse groove. The resulting change in orientation of the groove causes blood in the posterior propodium to move up into the large blood sinuses in the dorsal propodium and anterior metapodium. The snail's weight rests on the anterior propodium and the posterior metapodium while the middle of the foot shifts forward. The observed narrowing of the metapodium that begins in this stage and continues through Stage III probably exerts a tonic force on the blood and favors forward movement of the foot. This tonic force may be produced
LOCOMOTION IN MELAMPUS 317
by body wall musculature rather than by pedal muscle fibers derived from the columellar muscles.
In Stage III, propodial elevation results from contraction of columellar muscle bands, chiefly those of the left anterior and cephalic columellar muscle. The transverse groove region is elevated in Stage Ilia. This allows the anterior edge of the metapodium to expand into the space formerly occupied by the posterior propodium. Again, the combination of downward and forward forces indicates that metapodial extension results from an hydraulic event. By Stage Illb the more anterior region of the propodium is elevated by columellar muscles and the posterior region returns to the substratum in advance of its former position. This forward displacement is due, at least in part, to the extension of the metapodium in Stage Ilia. Throughout Stage III the oral veil and the posterior metapodium are static, weight-bearing regions that support the forward progression of the anterior region of the foot.
The hydraulic expansion of the anterior region of the foot of Melampus is similar to hydraulic events described for burrowing in the naticid snail Polinices josephinus (Trueman, 1968). The work of Schiemenz (1884) and, more recently, Russell-Hunter and Russell-Hunter (1968) and Russell-Hunter and Apley (1968) revealed that in naticids water is taken up into special channels to aid in the expansion of the foot. Both the burrowing Polinices and the crawling Melampus advance in a similar stepwise fashion.
The deeply cleft transverse groove and elevation of the propodium in a stepping locomotion are features that have arisen independently in several lines of Ellobiidae that are adapted to a hard substratum (Morton, 1955). Morton (1955) suggests that it may be especially adaptive in progression over broken or irregular surfaces. The model presented above indicates that the transverse groove has a valve-like function, rendering the metapodium relative!}- independent of the propodium during metapodial extension. This enhanced control over hydraulic events may have been important in the transition from the marine habitat to land in the Ellobiidae.
Morton (1955) describes the locomotion of ellobiids with a divided foot as ". . . fixing down the anterior third of the foot well in advance of the animal and drawing the remainder forwards upon it" (p. 151). This brief description neglects the hydraulic expansion of the propodium and thereby exaggerates the superficial similarity between Melampus locomotion and the "loping" locomotion of another primitive pulmonate, Ot'ma oils. According to Vies' (1913) account of Otina locomotion, the transversely subdivided foot is used in a stepping fashion in which the anterior is lifted and placed forward, rendering the foot long, and then the posterior is pulled forward, rendering the foot short. The hydraulic component that is responsible for maintaining the Melampus foot at a relatively constant length is apparently absent in Otina locomotion. Furthermore, no matter in wrhat order the events of Melampits locomotion are considered, the contraction pattern within the propodium clearly passes from posterior to anterior and therefore the crawl-step locomotion exhibited by this snail cannot be considered a retrograde wave. Thus the locomotion of Melampus cannot be cited to support the supposition that the retrograde wave locomotion seen in stylommatophoran gastropods during escape behavior is a primitive type of locomotor behavior within the pulmonates. The onlv feature of Melampus locomotion that is similar to the locomotion of
318 STACIA MOFFETT
higher land snails and slugs is the anteriorly-travelling ripple that produces metapodial lengthening. This wave of contraction is apparently produced by intrinsic pedal musculature and, although the Melampus foot appears to lack the specialized musculature that produces the waves in Agriolimax (Jones 1973), observation of the side view clearly shows that the elevated region of the wave is compressed and its forward movement forces a bolus of fluid forward. The fact that these metapodial waves do not originate at the end of the foot is very significant in an attempt to relate them to the direct waves of stylommatophorans. This is the case because, as Jones (1975) has pointed out, the multiple direct wave pattern of higher pulmonates could not be derived by adding waves to the single direct wave type of locomotion such as that exhibited by Onchidclla, in which the wave begins by shortening of the posterior region of the foot (Vies, 1907). Such an action renders the foot shorter during locomotion than when it is at rest and the propagation of many waves at the posterior of the foot before the first wave passes off the anterior would cause the foot to become prohibitively short. The wave pattern in higher pulmonates, in contrast, is initiated at the onset of locomotion at a site near the anterior part of the foot, where constriction of the elevated region results in a compensatory stretching of a more posterior part of the foot and the foot length remains constant (Lissmann, 1945; Jones, 1975). Thus the metapodial waves of Melampus could represent the survival of the motor pattern that gave rise to multiple direct wave locomotion in stylommatophorans. Morton's (1955) descrip- tion of locomotion in ellobiids that lack a transverse groove suggests that this wave pattern may be more pronounced in less specialized members of the family.
I would like to thank Dr. Christopher H. Price and Dr. Kenneth V. Kardong for reading and commenting on the manuscript and Gail McDole for help on the histological work. This work was supported by a fellowship from the Grass Foundation, National Science Foundation grant BNS 76-09706 and Public Health Service grant NS 14333-02 (NEUB).
SUMMARY
The foot of Melauipns is subdivided into an anterior propodium and a posterior metapodium by a permanent transverse groove. Locomotion in Melampus consists of repetition of a cycle of events that pass from posterior to anterior; this cycle has been named a crawl-step Three stages in the crawl-step have been identified: Metapodial shortening is produced by the action of columellar muscles and this action forces blood anteriorly to extend the propodium. Metapodial lengthening is produced by muscle action within the metapodium and extends the metapodial region forward at the expense of the propodium. Propodial elevation is pro- duced by columellar muscles and prepares the propodium to "step" forward while fluid invasion occurs in the first stage.
LITERATURE CITED
APLEY, M. L., 1968. Field and experimental studies on pattern and control of reproduction in Melampus bidentatus (Say). Ph.D. dissertation, Syracuse University, 154 pp. Diss. abstr. V29, p. 1527B (#68-13,810).
LOCOMOTION IN MELAMPUS 319
CHAPMAN, G., 1958. The hydrostatic skeleton in the invertebrates. Biol. Re:: 33: 338-71.
CHAPMAN, G., 1975. Versatility of hydraulic systems. /. Ex p. Zool, 194: 249-270.
JONES, H. D., 1973. The mechanism of locomotion of Agriolimax rcticulatus ( Mollusca :
Gastropoda). /. Zool., (Loud.}, 171 : 489-98. JONES, H. D., 1975. Locomotion. Pages 1-32. in V. Fretter & J. Peake, Eds., Pulmonatcs,
Academic Press, London. JONES, H. D., AND E. R. TRUEMAN, 1970. Locomotion of the limpet, Patella ruluata L.
/. Ex p. Biol.. 52: 201-16. LISSMAN, H. W., 1946. The mechanism of locomotion of gastropod molluscs. II. Kinetics.
/. Exfi. Biol., 22: 37-50. MILLER, S. L., 1974. The classification, taxonomic distribution, and evolution of locomotor types
among prosobranch gastropods. Proc. Malacol. Soc. Land.. 41 : 233-72. MORTON, J. E., 1955. The evolution of the Ellobiidae with a discussion on the origin of tin-
Pulmonata. Proc. Zool. Soc. Loud.. 125 : 127-68. PANTIN, C. F. A., 1948. Notes on Microscopical Technique for Zoologists. Cambridge,
Cambridge University, 79 pp. PLESCH, B., C. JANSE, AND H. H. BOER, 1975. Gross morphology and histology of the
musculature of the freshwater pulmonate L\mnaca stagnalis ( L. ) . Neth. J. Zool.,
25: 332-52. PRICE, C. H., 1977. Regeneration in the central nervous sys'.em of a pulmonate mollusc,
M clam pus. Cell Tissue Res., 180: 529-36. RUSSELL-HUNTER, W. D. AND M. RUSSELL-HUNTER, 1968. Pedal expansion in the naticid
snails. I. Introduction and weighing experiments. Biol. Bull.. 135 : 548-62. RUSSELL-HUNTER, W. D. AND M. L. ApLEY, 1968. Pedal expansion in the naticid snails.
II. Labelling experiments using inulin. Biol. Bull., 135 : 563-73. SCHIEMENZ, P., 1884. Uber die Wasseraufnahme bei Lamellibranchiaten und Gastropoden.
Mitt. Zoul. Stn. Neapcl, 5: 509-43. TRUEMAN, E. R., 1968. The mechanism of burrowing in some naticid gastropods in comparison
with that of other molluscs. /. Exp. Biol., 48 : 663-78. TRUEMAN, E. R., 1975. The Locomotion of Soft-Bodied Animals. American Elsevier,
New York, 200 pp. VLES, F., 1907. Sur les ondes pedieuses des mollusques reptateurs. Comptes Rendu Acad.
Sci, 145: 276-8.
VLES, F., 1913. Observations sur la locomotion d'Otina otis Turt. Remarques sur la progres- sion des gasteropodes. Bull. Soc. Zool. Fr., 38: 242-50.
Reference: Biol. Hull., 157: 320-333. (October, 1979)
THE ECTOPARASITISM OF BO ONE A AND FARGO A (GASTROPODA: PYRAMIDELLIDAE) *
ROBERT ROBERTSON AND TERRY MAU-LASTOVICKA
Department of Malacology, The Academy of Natural Sciences of Philadelphia, Pennsylvania 19103
Pyramidellids are now well known to be ectoparasites feeding on the body fluids of invertebrates (Ankel, 1938, 1949a, 1949b, 1959; Fretter and Graham, 1949, 1962; Fretter, 1951). Most of the verified hosts are polychaetes, gastropods and bivalves, but there also appear to be various minor host groups such as poly- placophorans and echinoderms (Robertson and Orr, 1961). A defect of many of the reports of hosts and even of "feeding" experiments is that no definite obser- vations on feeding were made. It is unsatisfactory to report a "host" when a pyramidellid is merely found "on" or "with" an invertebrate, whether it be in the field or in the laboratory. Pyramidellids, being foraging animals, sometimes assume positions on living substrates other than their hosts. Also, there can be behavioral preludes to feeding that do not culminate in feeding. A dilemma is that feeding cannot be verified in situ in the field, while in the laboratory some pyramidellids will feed on "hosts" they would never naturally encounter; the feeding of other pyramidellids is observed with great difficulty in the laboratory, even when the probable natural host is offered. Under the circumstances, what should be done is to observe for consistent associations with "hosts" in the field and then to deter- mine whether these "hosts" are fed on in the laboratory.
The degree to which pyramidellids are host-specific remains unresolved. Fretter and Graham (1949, 1962), even while recording the European species Odostoniia ambigiia (0. "eulimoides") on Pcctcn maximus, Chlamys opercularis and Ostrca cdiilis, emphasized that pyramidellids are host-specific. Berry (1955) went so far as to suggest that host-specificity accounts for the large number of pyramidellid species. There may be pyramidellids that are specific to one host species, but there are a few suggestive data that others are not. Ankel and Christensen (1963) observed Odostoniia rissoides (0. "scalaris") in Denmark feeding on five species in the laboratory: Lacuna vincta (L. "divaricata"), Littorina "sa.vatilis", Hydrobia nlvae, Rissoa incmbranacca and Chlamys opercularis. All but the last species live in the same habitat. There are, too, literature records of 0. rissoides with My til us cditlis. Ankel and Christensen (1963) also assembled literature data showing that 0. ambigiia had been found with four pectinid species, Mytilus editlis, Ostrca cdiilis, Hiatclla ("Sa.vicava") rugosa and Turrit ella coiuiuttnis. Minichev (1971) found Odostomia fiijitanii on various mollusks in the Sea of Japan, and under experimental conditions it fed readily on Littorina brcvicula, Tegula rustica, Uniboniiiin costatum, Turritclla jortilirata, Area boitcardi, etc. According to LaFollette (1979), the southern California species ChrysaUida clncta has at least six hosts: Haliotis corrugata, H. julgcns, Tegula ciscni, Norrisia •norrisi, Astraca undosa and A. gibbcrosa (all archaeogastropods).
1 Woods Hole Oceanograpbic Institution Contribution No. 4313.
320
BOONEA AND FARGOA ECTOPARASITISM 321
The purposes of the present paper are : to tabulate literature records of the "hosts" of all eastern North American odostomioids (Boonca and Fargoa species), to present (with emphasis on Boonca seiuinuda and B. bisiituralis) original field data on their occurrences and frequencies with "hosts", to present laboratory data on the "hosts" that were fed upon, and to present laboratory data on the preferences that B. scniimtda and B. bisittitralis had for two hosts. Of prime interest was the sympatric occurrence and abundance of these two species, and how they divide resources. Two species in the related genus Fargoa were also of interest by way of comparison, but fewer data are available about them.
Justification for separating the genera Boonea and Fargoa from Odostoinia has recently been presented elsewhere (Robertson, 1978). The systematics of the species reported on here was also treated in the same paper. Boonea imprcssa is con- sidered one species, although the populations in North Carolina and northwest Florida have planktotrophic larval development while a Texas population is lecithotrophic. The nomenclature and systematic sequences of the non-pyramidellid mollusks in the tables follow Abbott (1974) except that Bitthiin is used instead of Diastoma (Houbrick, 1977).
MATERIALS AND METHODS
Odostomioids were collected in the vicinities of Plymouth and Woods Hole, Massachusetts, and Beaufort and Wilmington, North Carolina. Correspondents sent living specimens and habitat data from Sapelo Island, Georgia, and Galveston, Texas. In all, seven species were collected alive, and careful note was taken on whether they were consistently on a particular living substrate — the presumptive host. "Hosts" were obtained near Woods Hole, on the New Jersey coast, near Beaufort, and from Texas.
Quadrats were used to determine the abundance of Boonca relative to its hosts and to other mollusks in the same habitats. The quadrats were placed at random in habitats suitable for two Boonea species (for B. scuiinuda, the muddy sand shallow subtidal zone where Crepidula jornicata is common at the N end of Ouissett Harbor, 3 km NE of Woods Hole, mid-August 1978; for B. bisiituralis, the stony low intertidal zone, underlain by sand and peat, where Littprina littorea is common at the shore opposite Flume Pond, S of Gunning Point, 4 km NNE of Woods Hole, early and late August, 1978). There was time only for two quadrats for each species. Three of the quadrats were 1.0 X 1.0 in; the fourth, because of a rising tide, had to be 0.5 X 0.5 m. All mollusks (except Bittinin alternatuin in the Boonca seinhutda habitat) in each quadrat were collected, identi- fied, counted and weighed (the weights include the shells). The five species of Boonca and Fargoa whose presumptive hosts were determined in the field were kept with these hosts in bowls of sea water in the laboratory. After it was learned that Boonca would feed on mollusks other than the presumptive hosts, all mollusks found in Boonea habitats plus a selection of species from other habitats were offered. Two polychaete species found in the same habitat as B. bisiituralis were also offered, as was the ascidian Molgula. The two Fargoa species were offered the serpulid Hydroides diaitthiis, a few other (unidentified) polychaetes, and various mollusks (Littorina littorea, Crepidula fornicata and Anachis avara} .
322
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324 R. K015KRTSOX AND T. MAU-LAST< )\MCKA
To be considered in a jccd'uuj position odostomioids had to be stationary on a "host" for about two or more minutes, and less than a proboscis length away from an accessible portion of the potential "host's" soft tissues. Usually after assuming a feeding position, odostomioids probing their "hosts" partially everted and inverted their proboscises one or more times, near or onto the "host's" soft tissues (probing rarely occurs away from any host). Feeding was recorded only when the proboscis was fully everted, when the "host" was pierced, and when the buccal pump within the proboscis "vibrated" to and fro with peristaltic waves.
The Boonca and Fargoa animals and the "hosts" were kept together until feed- ing, death, or termination of the experiment occurred. The interactions were monitored with a binocular dissecting microscope. Differences in the number of "hosts" available and the time available for observations account for the differences in the "hosts" that were offered each species in Table V.
The movements of two Boonca species towards two hosts at a distance were quantified. Round, plastic washbasins (diameter 30 cm) were filled with sea water to a depth of 5 cm. Four Crcpidula jornicata chains were placed at equal distances around the perimeter and four groups of four large specimens of Littorina littorea were placed between them. Sixty individuals of Boonea seininuda or B. bisuturalis (all about the same size) were placed in the center of each basin and allowed to move freely. The littorinas also moved freely and occasionally one had to be placed back in the water after it crawled up the side of the basin. Otherwise the basins were undisturbed for 3 hrs, after which time the littorinas and crepidulas were removed and the number of Boonea individuals on each was counted. All Boonea animals were then removed from their hosts and the basin. The basin was washed and new sea water was added. Each experiment was done twice each day. Boonea was not allowed to feed except when in the basin (6 hrs per day). There were ten trials for B. seininuda, and eight trials for B. bisuturalis. The data for each species were tested for homogeneity to determine whether the two sets of data could be lumped and treated as two experiments (Sokal and Rohlf, 1969). Chi-squares were then computed.
RESULTS
There are literature records of three species of Boonca with 22 species of mollusks, one species of polychaete and one species of ascidian (Table I). Only 18 of the 44 records were based on actual feeding observations. Table I also shows that two species of Fargoa are recorded with four species of Hydroides. Only two of the seven records were based on feeding observations.
In the present study, three species of Boonea were found alive and with hosts (all of them molluscan ; Table II). Four species of Fargoa were found alive, but only two of them were with hosts (both Hydroides diantJius; Table II).
Four Boonca seininuda animals were found in the winter with Littorina littorea in the habitat occupied at other seasons by B. bisuturalis (shore opposite Flume Pond, near Woods Hole). B. bisuturalis was occasionally found in the summer with B. seininuda on Crcpidula fornicata (N end of Quissett Harbor, near Woods Hole).
The quadrat data (Tables III and IV show that Boonea seininuda and B.
BOONEA AND PARGOA ECTOPARAS1T1SM
325
TABU-; II
Field observations on Boonea and Kargoa species found alive in the present study: localities and presumptive hosts.
|
Species |
Area |
In feeding position on |
Association |
|
B. semi nuda |
\Yoods Hole |
C rep id ul a fornicata |
common |
|
B. seminuda |
Woods Hole |
Littorina littorea |
rare |
|
B. seminuda |
Woods Hole |
Argopecten irradians |
rare |
|
B. seminuda |
Beaufort |
Crepidula fornicata |
common |
|
B. seminuda |
Beaufort |
Argopecten irradians |
common |
|
B. bisuturalis |
Plymouth |
Ilyanassa obsoleta |
common |
|
B. bisuturalis |
Woods Hole |
Littorina littorea |
common |
|
B. bisuturalis |
Woods Hole |
Crepidula fornicata |
fairly rare |
|
B. bisuturalis |
Woods Hole |
Argopecten irradians |
fairly rare |
|
B. impressa* |
Beaufort |
Crassostrea virginica |
common |
|
B. impressa* |
Wilmington |
Crassostrea virginica |
common |
|
B. impressa* |
Sapelo I. |
Crassostrea virginica |
common |
|
B. "impressa"^ |
Galveston |
Crassostrea virginica |
common |
|
B. "impressa "f |
Galveston |
Ischadium recurvum |
rare |
|
B. "impressa "f |
Galveston |
Geukensia demissa |
rare |
|
F. dianthophila |
Woods Hole |
Hydro ides d fan thus |
common |
|
F. dianthophila |
Beaufort |
Hydroides dianthus |
common |
|
F. bushiana |
Beaufort |
not found |
|
|
F. bartschi |
Woods Hole |
Hydroides dianthus |
common |
|
F. bartschi |
Beaufort |
not found |
|
|
F. bartschi |
Wilmington |
Hydroides dianthus |
common |
|
F. bartschi |
Texas |
not found |
|
|
F. gibbosa |
Beaufort |
not found! |
* Planktotrophic. t Lecithotrophic. t Muddy sand Zostera substrate.
bisuturalis occur at low densities relative to the biomass and numbers of their hosts. The percentages derived from the weights in Tables III and IV are all similar, ranging from 0.035 to 0.165 with only the predominant hosts considered and 0.029 to 0.079 when all potential hosts are pooled.
When starved to some degree, Boonea species fed on many of the gastropods and bivalves that were offered them (Table V), including not only their probable predominant hosts and other species in the same habitats (Tables Ill-TV) but even species from other habitats. B. bisuturalis fed on a record 37 mollusk species (Table V). It did not feed on the polychaetes Nereis (NeantJies] sncciiica and Hydroides diantliiis, the chiton Chaetoplcura apiculata or the ascidian Molgula manhattensis.
Aside from a few interactions that probably were not monitored long enough, the only proffered gastropod and bivalve "hosts" that were not observed even to elicit feeding positions or probing were a large gastropod with a thick integument (Busycon), and small species with tight closure (Caecum, Mysella and Gemma}. The bivalves Anadara, Anomia, Tcllina and Cumingia were fed on reluctantly or not at all.
Starved Texas Boonea "impressa" was frequently observed to feed cannibalisti-
,•526
R. ROBERTSON AND T. MAU-LASTOVICKA
TABLI-: 11!
Weights and numbers of individual mollusks from two 1.0 X 1.0 m quadrats in a habitat where Boonea seminuda is relatively abundant on Crepiclula fornicata, showing the abundance of the Boonea.
|
Species |
Quadrat 1 Weight (g)/ [no. individuals] |
Quadrat 2 Weight (g)/ [no. individuals] |
|
1. Crepidula fornicata (adults) |
256.484 [74] |
159.472 [49] |
|
2. Littorina littorea (mainly large adults) |
29.874 [15] |
6.526 [4] |
|
3. Crassostrea virginica (juvenile and adults) |
25.143 [5] |
0.531 [1] |
|
4. Bittium alter naium (juveniles) |
not weighed or |
counted* |
|
5. Anomia simplex (juvenile to medium-sized) |
1.221 [21] |
3.972 [42] |
|
6. Crepidula convexa (mainlv juveniles) |
1.187t[78J] |
3.228f [140J] |
|
7. Urosalpinx cinerea (adults) |
3.707 [2] |
0 [0] |
|
8. Crepidula plana (juvenile and adults) |
0.477 [2] |
0.006 [1] |
|
9. Boonea seminuda (mainly juveniles) |
0.090 [24] |
0.063 [25] |
|
Totals |
318.183 [221] |
173.798 [262] |
|
(excluding Bittium) |
||
|
Weight of Boonea: |
||
|
percent of weight of Crepidula fornicata |
0.035 |
0.040 |
|
percent of weight of all "hosts" |
0.029 |
0.037 |
* Very numerous mainly above the substratum on the red alga Polysiphonia; rank estimated, f Including egg sacs. J Excluding egg sacs.
cally (Table V), unlike the other species. B. bisuturalis was once seen to feed on another odostomioid, Fargoa bartschi (Table V).
The degree of unselectiveness in feeding differs among the Boonea species. B. "impressa" (from Texas) fed on 36 out of the 37 gastropod and bivalve species offered to it (97%} (Table V). B. bisntiira-Iis fed on 37 out of the 46 gastropod and bivalve species offered to it (80%) (Table V). B. seinln-nda fed on 22 out of the 36 gastropod and bivalve species offered to it (61%) (Table V). B. seminuda is more selective than either of the other species, feeding reluctantly or not at all on most neogastropods (Urosalpinx through Ilyanossa in Table V) and many bivalves.
The "hosts" were pierced in all accessible places (Table V), including tentacles which might be thought too sensitive for this (Ankel and Christensen, 1963). Mclainpus and Littorina "sa.ratilis", kept submerged with difficulty, were fed on only under water.
Boonea was never seen to feed on individual hosts much smaller than itself. (In the case of Odostotma, Ankel and Christensen, 1963, often observed that the parasite was larger than the host.) Only tiny juvenile B. Insiituralls were seen to feed on tiny juvenile Littorina littorea. The many juveniles of the smaller mollusks encountered in the quadrats (Tables III-IV) no doubt would be immune from parasitism by adult Boonea.
In the laboratory, Fargoa dlanthophila and F. bartschi probed and fed only on Hydroldes dianthns. F. dianthophlla fed readily both on the collar and on the branchiae. F. bartschi was seen to feed (on the collar) only once.
In the experiments on the presumably chemosensory responses of Boonea to
BOONEA AND FARGOA ECTOPARASITISM
327
two hosts at a distance, Boonca scininuda had a total of 234 individuals (77%) on Crepidula fornicata and 72 individuals (23%) on Littorina littorea in ten trials. The data for B. scminitda were found to be sufficiently homogeneous (0.70 < P < 0.80) to be treated as a single sample. A Chi-square with one degree of freedom, corrected for continuity, was then performed, resulting in a probability much less than 5 in 10,000 (P « 0.0005 ) that B. scininuda was randomly distributed between C. fornicata and L. littorea. B. scininuda showed a clear preference for C. fornicata. Boss and Merrill (1965) also demonstrated the same preference.
In the 8 trials of Boonea bisittitralis a total of 102 individuals (29%) were on Crepidula fornicata, while 246 individuals (71%) were on Littorina littorea after 3 hr. The data for B. bisittitralis were also tested for homogeneity. These data were found not to be uniform in magnitude, although they all differed in the same direction. The pooled Chi-square was highly significant, but the heterogeneity Chi-square was significant as well. Chi-squares were then computed for each of the eight trials. The Chi-squares for the first three trials are not significant. In the final five trials, after acclimating to laboratory conditions, B. bisiititralis showed a clear preference for L. littorea (P « 0.0005 in trials 4-6; 0.005 > P > 0.001 in trials 7-8).
DISCUSSION
Boonca scininuda and B. bisiititralis occupy different (but slightly overlapping) habitats. Judging by the Woods Hole field data (Tables II-IV), the feeding
TABLE IV
Weights and numbers of individual mollusks (juveniles as well as adults) from one 1.0 X 1.0 m quadrat and one 0.5 X 0.5 m quadrat in a habitat where Boonea bisuturalis is relatively abundant near and on Littorina littorea, showing the abundance of the Boonea.
|
Species |
Quadrat 1 (1.0 X 1.0 m) Weight (g)/ [no. individuals] |
Quadrat 2 (0.5 X 0.5 m) Weight (g)/ Cno. individuals] |
|||||
|
1. |
Littorina littorea |
132 |
.005 |
[2,156] |
68 |
.647 |
[708] |
|
2. |
Alercenaria mercenaria |
0 |
CO] |
33. |
997 |
[2] |
|
|
3. |
My a ar en aria |
0 |
.219 |
[7] |
25, |
,828 |
[14] |
|
4. |
Petricola pholadiformis |
0 |
.901 |
[3] |
11 |
,051 |
[13] |
|
5. |
Urosalpinx cinerea |
20 |
.418 |
[48] |
2 |
,288 |
[5] |
|
6. |
Littorina "saxatilis" |
1 |
.511 |
[63] |
0. |
530 |
[17] |
|
7. |
Boonea bisuturalis |
0 |
.069 |
[10] |
0 |
,113 |
[24] |
|
8. |
Crepidula convexa |
0 |
.147* |
[18t] |
0 |
[0] |
|
|
9. |
Mitrella lunata |
0 |
.021 |
[3] |
0 |
.029 |
[7] |
|
10. |
Bittium alternatum |
0 |
.046 |
[5] |
0 |
.013 |
[5] |
|
11. |
Littorina obtusata |
0 |
.001 |
[1] |
0 |
.019 |
[1] |
|
12. |
Argopecten irradians |
0 |
.017 |
[2] |
0 |
[0] |
|
|
13. |
Anomia simplex |
0 |
.006 |
[1] |
0 |
.001 |
[1] |
Totals
Weight of Boonea:
percent of weight of Littorina littorea percent of weight of all "hosts"
155.361 [2,317]
0.052 0.044
142.516 (797]
0.165 0.079
* Including one cluster of egg sacs, t Excluding one cluster of egg sacs.
328
R. ROBERTSON AND T. MAU-LASTOVICKA
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BOONEA AND FARGOA ECTOPARASITISM
329
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330 R. ROBERTSON AND T. MAU-LASTOV1CKA
experiments (Table V) and the experiments on the attractiveness of different hosts, B. scm'muda clearly feeds primarily on Crcpidnla fornicata and B. bisnturalis primarily on Littorina littorca. Thus the two Boonca niches differ in respect to preferred habitat and predominant host, and competition for space and food is largely avoided.
Assuming that what happens in the laboratory also happens in nature and that preferences for the predominant hosts are not overriding, it seems probable that some of the other mollusk species living in the same habitats serve as natural secondary hosts for Boonca. Leaving aside Crepidula jornicata, B. scminuda occurs with five other common mollusk species ; four of these were fed on in the laboratory (Table V). Four rare species co-occur, and three of these were also fed on in the laboratory (Table V). Leaving aside Littorina littorca, B. bisnturalis occurs with seven other common species ; all of these were fed on in the laboratory (Table V). Fifteen rare species co-occur, and all but two of these were also fed on in the laboratory (Table V). Rarely, B. scminuda is associated with Littorina littorca in the field (Table II) and hence rather definitely feeds on it in nature. The same holds for B. bisnturalis with Crepidula jornicata (Table II). Pre- dominant hosts are thus reversed occasionally. Leaving aside Crassostrea virginica, B. "impressa" occurs with six other common species; all of these were fed on in the laboratory (Table V). Of these, Gcnkensia dcmissa and the Ischadinm appear in Table II and hence are particularly likely natural secondary hosts.
Molgnla and an unidentified polychaete are the only non-molluscan hosts reported to be fed on by a Boonca (Allen, 1958; B. impressa'). Molgnla was not fed on by B. bisutnralis despite intermittent monitoring for three weeks. B. bisuturalis sometimes assumed a feeding position on either of the siphons, just as it does on bivalves. Probing into a siphon was seen once. Allen (1958) may also have seen probing. The tunic of Molgnla is probably too thick and tough for penetration by a Boonca proboscis. The cuticle of Nereis is probably also impenetrable.
Boonca clearly is a generalist molluscivore, by no means being host-specific. If some selective catastrophe befell any of the predominant hosts, the Boonea populations no doubt would be greatly reduced but would survive on other molluscan hosts.
Boonea bisutnralis is an indigenous species at Woods Hole, having been described (as Turritclla bisutnralis) by Say (1822) with "Boston harbour" as the type-locality. Littorina littorca, on the other hand, is introduced and has been at Woods Hole only since 1875 (Wells, 1965). (This in itself indicates that B. bisnturalis is not host-specific.) It would be interesting to know the host or hosts of B. bisnturalis before this introduction. Assuming that there has been no habitat change, Mya arcnaria (see Medcof, 1948), Pctricola pholadijormis, Uro- salpinx cincrea and Merccnaria mcrccnaria are possibilities. Note also that at Plymouth, Ilvanassa obsolcta was a common substrate (Table II) in a mud habitat. Crassostrea virginica, the predominant host of B. impressa (which hybridizes or intergrades clinally with B. bisuturalis in the New York-New Jersey area), was never found to be a living substrate for B. bisnturalis at Woods Hole. Crassostrea was, however, reported as a natural host elsewhere in New England by Loosanoff (1956) and Boss and Merrill (1965).
BOON E A AND FARGO A ECTOPARASITISM 331
In terms of biomass, Crcpidula fornicata and Littorina littorea, the pre- dominant hosts of Boonea scinimtda and B. bisntnralis, are dominant in their respective habitats (Tables III and IV). L. littorea is also dominant numerically. The low densities of the two Boonea species relative to the biomass and numbers of their hosts (Tables III and IV) make it likely that these odostomioids do little harm to their hosts — injuries such as reported by Cole and Hancock (1955). Loosanoff (1956), and Allen (1958) were not observed. Judging by the four quadrats and the two species, a given biomass of host may support a relatively stable percentage biomass of Boonea. Note that the percentages derived from the weights in Tables III and IV are all similar.
The anomalous results of the experiments with B. bisittiiralis are probably due to the individuals having just been collected. The trials with B. seniiniida started after these animals had been in the laboratory for two weeks feeding on Crepidula fornicata and Littorina littorea. Something caused the B. bisntnralis to move randomly in their first 48 hr in the laboratory. Possibly they were disoriented or hungry.
In contrast to the pair of Boonea species at Woods Hole, the pair of Fargoa species is in direct competition with each other for space and food (Hydroides dianthiis). They occasionally exist even on the same individual w7orm. F. diantlio- phila is the commoner and smaller of the two species. Sometimes it rides in and out of the worm's tube while sitting on the worm's operculum. At other times it sits on the tube. F. bartschi never enters the tube since (at least when adult) it is too long to turn around.
In the Woods Hole area Hydroides dianthiis is sparsely and patchily dispersed, and spatfalls vary in success at each locality from year to year (personal observa- tions). Not surprisingly, the Fargoa populations are commensurately sparse, patchy and sporadic. Hydroides dianthiis is sympatric with other species of Hydroides (Eupomatus is a synonym) off North Carolina, northeast and north- west Florida. Fargoa dianthophila occurs with three of these other Hydroides species (Wells and Wells, 1969). The same is probably true of F. bartsehi, which ranges from Massachusetts to Texas. Thus the two Fargoa species seem both to be specialists host-specific to the genus Hydroides. This occurrence of more than one odostomioid on the same serpulid is reminiscent of Odostoiuia plicata, 0. Inkisi, 0. unidcntata and "Chrysallida" (Partnlida) spiralis of Europe, all of which parasitize Pornatoceros triqiicter ( Ankel, 1959; Fretter and Graham, 1962).
Boonea is a generalist and Fargoa a specialist. It seems likely that the former condition is the more primitive, and that pyramidellids were not originally host- specific.
This work was supported by National Science Foundation Grant DEB 76-18835. Constance (Mrs. Hollis Q.) Boone kindly sent live specimens from Texas; Dr. John N. Kraeuter kindly sent specimens from Georgia. Dr. Rudolf S. Scheltema generously provided space in his laboratory at Woods Hole, and I spent one summer at the Duke University Marine Laboratory. Isabelle Williams identified the Nereis. The following kindly read and criticized various drafts of the manuscript : Dr. George M. Davis, Robert Hershler and Virginia Orr Maes.
332 R. ROBERTSON AND T. MAU-LASTOVICKA
SUMMARY
1. Three Boonca species (occurring in sympatric species pairs) occupy dif- ferent habitats and have different molluscan host preferences. In the field, B. seniinnda is preferentially with Crepidula fornicata or Argopccten irradians, B. bisiititralis with Littorina littorea (introduced), Ilyanassa obsoleta or Crassostrea virginica, and B. iinpressa with C. rirtjinica. Weights of the first two species are about 0.03 to 0.17% those of their hosts. In the laboratory, B. sciiiinnda was attracted much more to Crepidula fornicata than to Littprina littorea. With B. bisutiiralis it was vice versa.
2. In the laboratory, B. seininiida fed on 22 out of the 36 gastropod and bivalve "hosts" offered; B. bisittnra/is fed on 37 out of 45, and B. iinprcssa fed on 36 out of 37. Some of these mollusks probably serve as secondary hosts in nature. Boonca definitely is not host-specific. Polychaetes, Chaetopleura and Molgula were not fed on.
3. Fargoa dianthophila and F. bartschi, two much rarer species, compete writh each other for space and food by both obligately parasitizing species in the genus Hydroides, sometimes co-occurring on the same individual. Fargoa species do seem to be host-specific.
LITERATURE CITED
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ALLEN, J. F., 1958. Feeding habits of two species of Odostouiia. Nautilus, 72: 11-15. ANKEL, F., AND A. M. CHRISTENSEN, 1963. Non-specificity in host selection by Odostomia
scalaris Macgillivray. Vidcnsk. Mcdd. Dan. Naturhist. Forcn., 125: 321-325. ANKEL, W. E., 1938. Beobachtungen an Prosobranchiern der schwedischen Westktiste. Arkiv
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77: 79-82. ANKEL, W. E., 1949b. Die Nahrungsaufnahme der Pyramidelliden. Vcrhandlungen der
Dcutschen Zoologcn, Kiel, 1948: 478-484. ANKEL, W. E., 1959. Beobachtungen an Pyramidelliden des Gullmar-Fjordes. Zoo/. Anz.,
162: 1-21. BERRY, S. S., 1955. Importance of the large pyramidellid elements in the West American
fauna. Am. Malacol. Union Annn. Kept., 1954: 25. Boss, K. J., AND A. S. MERRILL, 1965. Degree of host specificity in two species of Odostomia
(Pyramidellidae : Gastropoda). Proc. Malacol. Soc. Loud., 36: 349-355, pi. 15. BULLOCK, R. C., AND K. J. Boss, 1971. Non-specificity of host-selection in the ectoparasitic
snail Odostomia (Mcnestlw) bisnturalis (Say) (Gastropoda: Pyramidellidae).
Brcviora, 363 : 1-7. COLE, H. A., AND D. A. HANCOCK, 1955. Odostomia as a pest of oysters and mussels. /. Mar.
Biol. Assoc. U.K., 34: 25-31, 2 pis. DALL, W. H., 1883. On a collection of shells sent from Florida by Mr. Henry Hemphill.
Proc. U. S. Natl. Mus., 6: 318-342, pi. 10. FRETTER, V., 1951. Turbonilla clegantissima (Montagu), a parasitic opisthobranch. /. Alar.
Biol. Assoc. U.K., 30: 37-47. FRETTER, V., AND A. GRAHAM, 1949. The structure and mode of life of the Pyramidellidae,
parasitic opisthobranchs. /. Mar. Biol. Assoc. U.K., 28 : 493-532. FRETTER, V., AND A. GRAHAM, 1962. British prosobranch molluscs; their functional anatomy
and ecology. Ray Society, London, xvi + 755 pp.
BOON E A AND FARGO A ECTO PARASITISM 333
HACKNEY, A. G., 1944. List of Mollusca from around Beaufort, X. Carolina, with notes
on Tcthys. Nautilus, 58 : 56-64. HOPKINS, S. H., 1956. Odostomia impressa parasitizing southern oysters. Science. 124
628-629. HOUBRICK, R. S., 1977. Reevaluation and new description of the genus Biltinm ( Cerithiidae) .
Vcligcr, 20: 101-106, 1 pi. LAFOLLETTE, P. I., 1979. Observations on the larval development and behavior of Chrysallida
cincta Carpenter, 1864 (Gastropoda: Pyramidellidae ). }\'cst. Soc. Malacol. Anini.
Kept., 11: 31-34. LOOSANOFF, V. L., 1956. Two obscure oyster enemies in New England waters. Science, 123 :
1119-1120. MEDCOF, J. C, 1948. A snail commensal with the soft-shell clam. /. Fish. Res. Board Can..
7: 219-220. MERRILL, A. S., AND K. J. Boss, 1964. Reactions of hosts to proboscis penetration by Odostomia
scminuda (Pyramidellidae). Nautilus, 78: 42-45, pis. 4-5. MINICHEV, Y. S., 1971. Contribution to the biology of some Pyramidellidae (Gastropoda,
Pyramidellidae) of the Pos'yet Bay of the Sea of Japan (in Russian). Acad. Sci.
U.S.S.R., Zool. I nst. Explorations of the fauna of the seas, 8(16) : 221-229. MOORE, D. R., 1961. The marine and brackish water Mollusca of the state of Mississippi.
Gulf. Res. Rep. 1(1) : 1-58. PELSENEER, P., 1928. Les parasites des mollusques et les mollusques parasites. Bull. Soc. Zool.
Fr., 53: 158-189.
PORTER, H. J., 1974. The North Carolina marine and estuarine Mollusca — an atlas of occur- rence. University of North Carolina Institute of Marine Sciences, Morehead City,
vi + 351 pp. PORTER, H. J., 1976. Spiral cord variation of Odostomia inipressa (Say) and O. seininuda
( C. B. Adams); family Pyramidellidae. Bull. American Malacol. Union Inc. 1976:
38-41. ROBERGE, A. G., 1968. Odostomia dianthophila (Gastropoda, Pyramidellidae) from Buzzard's
Bay, Mass., a northern range extension. Nautilus, 81(4) : iii. ROBERTSON, R., 1957. Gastropod host of an Odostomia. Nautilus, 70 : 96-97. ROBERTSON, R., 1967. The life history of Odostomia bisutnralis, and Odostomia spermatophores
(Gastropoda: Pyramidellidae). YearB. Am. Philos. Soc., 1966: 368-370. ROBERTSON, R., 1978. Spermatophores of six eastern North American pyramidellid gastropods
and their systematic significance (with the new genus Boonea). Biol. Bull., 155:
360-382. ROBERTSON, R., AND V. ORR, 1961. Review of pyramidellid hosts, with notes on an Odostomia
parasitic on a chiton. Nautilus, 74: 85-91. SAY, T., 1822. An account of some of the marine shells of the United States. /. Acad. Nat.
Sci. Phila., 2 : 221-248, 257-276, 302-325. SCHELTEMA, A. H., 1965. Two gastropod hosts of the pyramidellid gastropod Odostomia
bisutnralis. Nautilus, 79 : 7-10. SOKAL, R. R., AND F. J. ROHLF, 1969. Biometry; the principles and practice of statistics in
biological research. Freeman, San Francisco, xxi + 776 pp.
WELLS, H. W., 1959. Notes on Odostomia imprcssa (Say). Nautilus, 72: 140-144. WELLS, H. W., 1965. Maryland records of the gastropod, Littorina littorea, with a dis- cussion of factors controlling its southern distribution. Chesapeake Sci., 6 : 38-42. WELLS, H. W., AND M. J. WELLS, 1961. Three species of Odostomia from North Carolina,
with description of new species. Nautilus, 74: 149-157.
WELLS, H. W., AND M. J. WELLS, 1969. New host and distribution records of Odostomia dian- thophila. Nautilus, 82: 109-110. WELLS, H. W., M. J. WELLS AND I. E. GRAY, 1964. The calico scallop community in North
Carolina. Bull. Mar. Sci. Gulf Carihh., 14: 561-593.
Reference-: liwl. Hull.. 157: 334-343. (October, 1979.)
FIELD AND LABORATORY STUDIES OF GLUGEA HERTWIGI
(MICROSPORIDA) IN THE RAINBOW SMELT
OSMERUS MORDAX *
ANN SCARBOROUGH AND EARL WEIDNER
flic Marine Biological Laboratory, ]]'oods Hole, Massachusetts 02543, and the Department of Zoology and Physiology, Louisiana State University,
Rouc/e. Louisiana 70893
Glitgca hertwigi-induced microsporidosis is a disease of the smelt, Osinerus inorda.r. 0. iiiorda.v is an anadromous species that has been successfully introduced to temperate freshwater areas. Haley (1957) has provided some evidence which indicates G. hcrtwigl was in part responsible for the decline of the smelt fishery in the Atlantic. The incidence of Glitgca infection reaches a seasonal peak of nearly 9Qr/c in juvenile Lake Erie smelt each summer and fall ; Nepszy, Budd, and Dechtiar (1978) estimate vast economic losses in the smelt fishery in the Great Lakes due to mortality of infected juveniles.
G. heriu'iyi infections typically localize as parasite colonies in the suhmucosal layer of the intestine. Mortaility of the smelt host is believed to occur by starva- tion ; in addition, infected fish have navigation problems, are more susceptible to predation, and less able to recover from environmental stress (Legault and Delisle, 1967; Nepszy and Dechtiar, 1972). Spawning female smelt in Lake Erie characteristically have numerous G. hcrtu'igi cysts in the ovaries as well as along the intestine. Chen and Power (1972) reported a 42% decrease in fecundity of Glitgca infected females. The microsporidan Plistophora ovariac infecting the golden shiner Notcinigoticus crysolcitcas is an example of transovarian parasite transmission (Summerfelt and Warner, 1970).
G. hertwigi is an obligate intracellular parasite completing its life cycle within a single host cell. After ingestion by a smelt, the infective spore is believed to discharge a polar filament with a velocity suitable for penetrating the intestinal mucosal cell layer (Ishihara, 1968; Weidner, 1972, 1976). The vegetative stages of the parasite do not cause host cell degeneration but stimulate hypertrophy and abnormal development into a "xenoma" ( Weissenberg, 1968, 1976; Weidner, 1974). "Xenoma" refers to the unique association between an hypertrophied host cell and developing intracellular parasites (Weissenberg, 1968, 1976) . The host-cell component is induced to undergo extensive growth during vegetative de- velopment (schizogony) by the parasite. Eventually the parasite differentiates into mature spores (sporogony) which fill the central region of the xenoma. By this stage, a combination of host animal response and parasite growth transform the xenoma into a thick-walled "Glugea-cyst" filled with innumerable spores (Sprague and Vernick, 1968; Weissenberg, 1968, 1976; Weidner. 1973, 1976). G. hertivigi cysts range from 0.4 to 5.0 mm in diameter; heavy smelt infections
1 This investigation was supported by NSF Doctoral Dissertation Grant #DEB 77-08413.
334
GLUGEA HERTWIGI IN OSMERUS MORD.iX 335
number over 200 xenomas per host (Legault and Delisle, 1967; Nepszy and Dechtiar, 1972).
McVicar (1975), Olson (1976) and Weissenberg (1968) transmitted Glitgea species to fish held in the laboratory. Weidner (1973) and Stunkard and Lux (1965) suggested that invertebrate filter-feeders may serve as natural vectors or transport hosts for fish microsporidans. Olson (1976) determined a low level of Ghtgca stephani infection occurred by ingestion of spores directly. McVicar (1975) transmitted G. stephani through spore-carrying vectors and by injection of spores into the peritoneal cavity of adult fish. Weissenberg (1968) did not determine whether G. a no mala was militated by a spore-carrying vector or by direct ingestion of the spores.
We thought it would be of interest to examine G. hertwiyi growth and multiplication in ovaries of spawning female smelt ; follow transmission of infections ; and examine the microstructure of early xenoma growth.
MATERIALS AND METHODS
Adult smelt
Spawning female smelt were collected from Wheatley, Ontario, on Lake Erie and from the Jones River, near Plymouth, Cape Cod, Massachusetts. A total of 150 fish from each location were examined internally for the presence of Ghtgca hcrtwigi cysts. The intestine and ovaries from infected and non-infected fish were excised, cut into small pieces and fixed in pH 7.4 phosphate buffered glutaraldehyde overnight at 4° C. After several buffer rinses, the material was post-fixed in phosphate buffered 2°fo osmium tetroxide for 2 hr at 4° C, dehydrated in ethanol and embedded in Epon. One micron sections were cut on a Dupont-Sorvall MT-2B microtome and stained with \% toluidine blue. Parasite cysts were removed from adult smelt, homogenized, pelleted in pH 7.4 phosphate buffer, stored at 4° C and used for the transmission experi- ments.
Transmission experiments
Eggs and milt were stripped from spawning smelt, mixed 1 : 5 respectively and kept in a well-aerated nylon mesh cone. Naturally fertilized eggs were also collected from the river-lied. Anticipating large mortalities, a non-infected smelt population was located in Long Pond, Cape Cod, Massachusetts. Several years' examination of smelt from this pond had indicated they were completely free of G. hertu'igi infection. Young smelt (20-25 mm) in Long Pond were attracted at night to a strong light at the surface and collected by hand-net. All fish were maintained at the National Marine Fisheries Service Aquarium, Woods Hole, in 20° C filtered fresh water taken from the Jones River well above the spawning sites. All fish were maintained on a diet of phyto- and zooplankton seined from Long Pond. The following methods of parasite transmission were attempted.
Experiment 1. Laboratory reared and collected smelt were exposed to a suspension of G. hcrtwiyi spores placed in the tanks. The water was well aerated but not filtered for the following 48 hr.
336
A. SCARBOROUGH AND E. WEIDNER
FIGURE 1. Young specimen of the smelt, Osincnts mordax, experimentally infected with Glugca liertivigi. The yearly incidence of natural infections nears 90% in juveniles with consequent vast mortalities. Seven days post infection, xenomas (white arrows) develop proximate to larger 4-\veek-old "Glugea cysts" (black arrows). Bar represents 2.5 mm.
Experiment 2. Laboratory reared smelt (10 mm, 6 weeks post spawning) were fed spore-carrying zooplankton ( cladocerans and copepods) on 2 consecutive days. The plankton was first exposed to a spore suspension for 30 min, washed once with water, examined to ensure the presence of spores in their digestive tracts and then fed to the smelt.
Experiment 3. Collected smelt (15 mm, approximately 8 weeks post spawning) were fed spore-carrying plankton as above.
Experiment 4. Smelt from Experiment 1, 4 weeks after exposure to a spore suspension, were fed spore-carrying plankton as above. As a control, a number of laboratory reared and collected smelt were maintained unexposed to spores.
Development of xenomas
Young smelt from all tanks were observed and photographed with a Wild M-4 Makroskope 5 clays, 1, 2 and 3 weeks after spore feeding. Intestinal tissue from experimental and control smelt were prepared for microscopy as outlined above.
TABLE I
|
Number of smelt infected |
||||||
|
Experiment number |
Source of smelt |
Sample size |
Method of exposure |
Period of incubation |
||
|
Light < 5 |
Heavy > 50 |
|||||
|
(per host) |
(per host) |
|||||
|
1 |
mixture of |
20 |
spore suspension |
28 days |
8 |
0 |
|
lab reared |
||||||
|
and |
||||||
|
collected |
||||||
|
2 |
lab rearc-d |
5 |
spore-carrying |
7 days |
0 |
5 |
|
plankton |
||||||
|
3 |
collected |
14 |
spore-carrying |
7 days |
0 |
14 |
|
plankton |
||||||
|
4* |
mixture |
8 |
spore-carrying |
7 days |
0 |
8 |
|
plankton |
* Smelt from Experiment 1, carrying light infections 28 days after exposure to the spore sus- pension.
GLUGEA HERTWIGl IN OSMERUS MORD.IX
RESULTS
337
Adult smelt
In spawning females Gliiga Jicrtzvigi cysts were found in the ovaries of more than 50% of the infected fish in Lake Erie and in 25% of infected fish in the Jones River. The ovaries of Lake Erie fish were heavily infected whereas those of the Jones River \vere lightly infected. Sections of infected ovaries showed the parasite was isolated from the scattered ova by the cyst wall. No free spores or developing stages of G. hcrtza'igi were observed outside the cysts, in ova or in ovarian tissue.
Parasite transmission
The results of the experiments are summarized in Table I. Positive results were obtained from both methods of spore transmission ; however, the intensity of infection and its effect differed.
sb
FIGURE 2. Young smelt, 12 days after feeding on spore-carrying plankton. Numerous xenomas (arrows) begin just behind the stomach and continue along the gut to the vent. S, stomach ; sb, swimbladder. Bar represents 180 yum.
FIGURE 3. Young smelt previously infected after exposure to a spore suspension developed numerous new xenomas (arrows) along the intestine when fed spore-carrying plankton. X, xenomas from exposure to spores directly. Bar represents 750 /j.m.
338 A. SCARBOROUGH AND E. WEIDNER
Experiment 1. Laboratory-reared and collected fish were exposed to a suspension of G. hcrtiviyi spores. Four weeks after exposure, 40% of the smelt exhibited one or two Glugca cysts in the posterior region of the intestine. These cysts were uniformly dense and protruded from the submucosa distending the peritoneal cavity. The fish fed continually throughout the experimental period. Several fish died during the period, were examined and one was found infected with a single Glugea cyst.
Experiment 2. Laboratory-reared fish were exposed to spore-carrying plankton. One week after exposure all fish exhibited numerous small xenomas beginning just behind the stomach and continuing along the entire length of the intestine to the vent (Fig. 2). Gradually the fish stopped feeding, had difficulty swimming and all died by 16 days after exposure to the spores.
Experiment 3. Collected smelt were exposed to spore-carrying plankton. The results were similar to those in Experiment 2 ; however, these fish stopped feeding and died at about 25 days after exposure to the parasite.
Experiment 4. Smelt from Experiment 1, 4 weeks after being exposed to a spore suspension, were fed spore-carrying plankton. One week after feeding, all fish exhibited numerous small xenomas along the intestine as well as the large posterior xenomas (Fig. 3). All fish died within 2 weeks after exposure to the spore-carrying vectors. Controls were examined periodically and found free of infection.
Parasite development
Parasite growth was rapid at 20° C. Examination of the intestine from a heavy infection (Experiments 2, 3, and 4) showed the extensive tissue involvement. In- fections protruded to the serosa and were easily dissected away intact (Fig. 4). Extensive host cell hypertrophy was the obvious feature of sectioned material (Fig. 5). Xenomas ranged from 20 to 50 /AUI in diameter. Smaller xenomas contained one or two greatly enlarged host cell nuclei ; whereas the larger xenomas
\
FIGURE 4. Whole intestine 1 week after exposure to spore-carrying plankton. Xenomas (arrows) protrude to the serosa and are easily dissected away intact. Bar represents 80 /mi.
GLUGEA HERTiriGI IX OSMERUS MORDAX
339
• '. *r ••• • * j
* A
V
^ •»
-
-
FIGURE 5. Cross section of intestine similar to that shown in Figure 4. At least 40 xenomas (arrows) 20 to 50 /j.m in diameter develop within the suhmncosa and protrude from the mucosa. Infected host cells hypertrophy and contain early schizont stages of the parasite, e, epithelium. One micron Epon section; \% toluidine blue stain; bar represents 50 /j.m.
FIGURE 6. Cross section of intestine from lightly infected smelt 2 weeks after feeding on a spore suspension. Progressive hypertrophy increased the xenomas to 100 to 125 ^um in diameter. Xenomas develop in the submucosal layer causing mechanical distension of the epithelium (e). Sporogony stages and free dense spores fill the central region of the xenoma. One micron Epon section; \% toluidine blue stain; bar represents 50 ^m.
340 A. SCARBOROUGH AND E. WEIDNER
were multinucleated with many nuclei lobed or branched, indicating nuclear division. At 1 week the parasite formed schizont colonies peripheral to the host cell nuclei. After 2 weeks of growth there was considerable increase in host cell hypertrophy and Glugca maturation to spores. Xenomas ranged from 100 to 125 /iin in diameter with host cell nuclei, cytoplasmic components and G. hertzvigi schizonts particularly obvious in the peripheral region of the xenomas ; sporo- blastic stages of the parasite were common in the central region (Fig. 5).
DISCUSSION
Transovarian parasite transmission is known from a number of microsporidan species (Kudo, 1966). Recently, Summerfelt and Warner (1970) demonstrated a Plistophora ovariae infection in viable eggs of the golden shiner, Noteinigonciis crysolcucas. Although the ovaries of spawning female smelt were often loaded with G. Jicrtwigi parasites, our thorough examinations indicate G. hcrtivigi are not present in germinative or egg cells. Previously, there has been limited success in experimental peroral transmission of fish microsporidans (Delisle, 1969; McVicar, 1975; Summerfelt and Warner, 1970; Stunkard and Lux, 1965). How- ever, Olson (1976) successfully transmitted G. stcphani to the English sole, Para- phyrys vet nl its in water temperatures above 15° C. Several authors are convinced that transport vectors are necessary to concentrate Glugca spores for natural trans- mission of certain fish microsporidosis (Haley, 1957; Putz and McLaughlin, 1970; Stunkard and Lux, 1965). Small filter-feeding animals may serve as transport hosts and in addition, may stimulate the spores to hatch and infect the fish. Weidner (unpublished observations) has observed such a phenomenon with G. stcphani in the winter flounder, Pseitdopleiircncctcs amcricanns. In this study, transmission of G. hertwigi to both laboratory-reared and collected smelt was successful at 20° C, either by direct spore consumption or ingestion of spore- carrying vectors; however, a major magnitude of difference exists in the intensity between direct spore and vector transmitted infections. Vector transmission produced massive infections along the entire intestine in all test subjects.
Smelt are selective predators, taking cyclopoid and calanoid copepods and several species of cladocerans as their first food (Reif and Tappa, 1966; Siefert, 1972). Presumably, the natural G. Jicrtzvigi infection occurs through the ingestion of spore-carrying filter feeders by very young smelt. Release of spores from in- fected adult smelt occurs via two routes. Scarborough (unpublished observations) has observed the expulsion of parasite xenomas from ovaries during spawning. In this manner, female adult smelt may concentrate G. hertzvigi spores in the immediate vicinity of developing young. Further, infected smelt carrion were seen being preyed upon by small crustaceans in the nursery areas after spawning. Nepszy and Dechtiar (1972) found that heavily infected adult smelt were unable to recover from spawning stress ; mass mortalities in the spawning grounds con- sisted of infected adults.
Massive infections of G. hertwigi consequent to ingestion of spore vectors are fatal to both collected and laboratory reared smelt. Mechanical distention of the intestinal tissue and starvation are thought to be the cause of death. Osmcrus mordax is a difficult species to raise in the laboratory and the minimal condition
GLUGEA HERTWIG1 IN OSMERUS MORDAX 341
of the reared fish probably precluded a greater susceptibility to the effects of multiple infections. Young smelt tolerate light infections for at least several weeks and likely carry them into adulthood. Nepszy and Dechtiar (1972) found that G. hcrtivigi colonies can remain in smelt for much of the host's life, and stress will significantly increase the mortality rate in these fish over uninfected smelt.
It is well documented that the life cycle of microsporidans begins with injection of the sporoplasm through a spore tube into the host (Vavra, 1976; Weidner, 1972). It is assumed that the discharging tube of G. hcrtivigi spores penetrates through the gut basal lamina delivering the parasite into submucosa cells.
Whether transmitted directly by spores or via spore-carrying vectors, de- velopment of G. hcrtivigi was identical, and paralleled that described for other Glngca species (Sprague and Vernick, 1968; Weissenberg, 1968, 1976). Changes in macroscopic appearance of xenomas correlated with microscopic examinations of the sectioned material. While the host cell component of the xenoma remains viable, the parasite develops numerous schizonts within the peripheral cytoplasm. The host cytoplasm and nucleus hypertrophy in apparent response to the parasite's pres- ence ; subsequently, the nucleus undergoes an amitotic budding. As the xenoma size increases, a cellular capsule delimits the xenoma from the surrounding tissue ; this capsule becomes enveloped by host connective tissue layers. While maturation of the parasite progresses through sporogony, host cell components begin to degenerate and spores fill the interior. The cyst stage consists of spores and scattered vestiges of host cell components surrounded by a wall.
Although Weissenberg (1976) believed the host cell to be a presumptive macro- phage, the cell type which can support G. hcrtivigi remains undetermined. Our observations indicate massive infections will produce xenomas easily separated from the intestine. Xenomas were observed associated with various visceral organs, including cells below the peritoneal lining. It is not known how G. hcrtivigi enters ovarian tissue; presumably, initially infected host cells enter the blood stream and are delivered to favorable environs for growth, such as the highly vascularized ovaries. Noscnia inicJiaelis, a microsporidan infecting the blue crab, Callencctcs sapidns, undergoes vegetative growth in the gut wall and these cells subsequently circulate to muscle tissue for continued development (Weidner, 1972).
This particular host-parasite association likely will lie of some use in the study of xenomas. The transparency of young smelt lends itself to in I'ii'o study of drug effects on parasite infection and xenoma development. Tissue culture techniques may now be carried out on isolated xenomas since these larger tumor cells are readily detectable in young smelt and easily removed aseptically from the gut serosa.
Thanks are recorded to Mr. Charlie Wheeler of the National Marine Fisheries Service Aquarium. Woods Hole, Massachusetts ; to Mr. Stephen Xepszy of the Canadian Ministry of Xatural Resources, Lake Erie Fisheries Research Station, Wheatley, Ontario, and special appreciation to Mr. James Kennedy of the Massa- chusetts Fish and Wildlife Service, Buzzards Bay, Massachusetts.
SUMMARY
Glugca hertwigi-induced microsporidosis is a disease of the smelt Osincnts mordax. The yearly incidence of infection reaches over 50rr in adult smelt and as
342 A. SCARBOROUGH AND E. WEIDNER
high as 90r/( in juveniles. Primary infections localize as large intracellular colonies in submucosal cells of the digestive tract. Field observations indicate the ovaries of spawning females are the secondary site of infection. G. hcrtwigi was success- fully transmitted to both laboratory-reared and collected young smelt at 20° C by small filter-feeding vectors and by direct ingestion of spores. Infections transmitted by spore-carrying vectors numbered hundreds per animal, and were visible along the intestine one week after feeding. Large parasitized host-cells (xenomas) extended from the intestinal serosa and were easily recovered. G. her twig i infec- tions acquired by direct spore feeding numbered one or two per animal ; these fish have the capacity to develop many new infections by feeding on spore- carrying vectors. Microscopic study revealed that G. hcrtwigi development was indeed within a single greatly hypertrophied host cell. After 1 week of growth, 20 to 50-/Aiii xenomas contained a few enlarged host nuclei and vegetative G. Jicrtwigi ; after 2 weeks, the xenomas measured 100 to 125 yu.ni, exhibited multiple host nuclei and numerous G. hertwigi sporoblasts and spores.
LITERATURE CITED
CHEN, M., AND G. POWKK, 1972. Infection of American Smelt in Lake Ontario and Lake Erie
with the microsporidian parasite Glugca hcrtiviqi (Weissenberg). Can. J. Zool.,
50: 1183-1188. DELISLE, C., 1969. Bimonthly progress of a non-lethal infection of Glugca hcrt-a'igi in young-
of-the-year smelt. Osincrns c per Inn us inorda.v. Can. J. Zoo!., 47 : 871-876. HALEY, A. J., 1957. Microsporidian parasite, Glugca hertwigi in American Smelt from Great
Bay region. New Hampshire. Trans. Am. Fish. Sac., 83 : 84-90. ISHIHARA, R., 1968. Some observations on the fine structure of sporoplasm discharged from
spores of a microsporidian, Noscina boinbycis. J . Ini'crtcbr. Pathol., 12 : 245-257. KUDO, R. R., 1966. Protozoology. 5th ed. Charles C Thomas, Publ., Springfield. 1135 pp. LEGAULT, R. O., AND C. DELISLE, 1967. Acute infection by Glugca hertu'igi (Weissenberg)
in young-of -the -year rainbow smelt, Osmcrus cpcrlanus morda.r (Mitchill). Can. J.
Zoo/., 45: 12912. McViCAR, A. H., 1975. Infection of plaice Plcitrnncctcs platcssa L. with Glugca (Noscina)
stcpliani ( Hagenmuller, 1899) (Protozoa: Microsporidia) in a fish farm and under
experimental conditions. /. Fish. Bio!., 7: 611-619. NEPSZV, S. J., J. BUDD, AND A. O. DECHTIAR, 1978. Mortality of young-of-the-year rainbow
smelt ( Osmcrus morda.r ) in Lake Erie associated with the occurrence of Glugca licrt-
u'igi. J. ll'ildl. Dis., 14(2) : 233-239. NEPSZY, S. J., AND A. O. DECHTIAK, 1972. Occurrence of Glugca hertwigi in Lake Erie
ranbow smelt (Osincrns morda.r) and associated mortality of adult smelt. /. Fish.
Res. Bd. Can., 29 : 1639-1641.
OLSON, R., 1976. Laboratory and Field Studies on Glugca stcpliani (Hagenmuller), a micro- sporidian parasite of pleuronectid flat-fishes. /. Protozool., 23(1) : 158-164. PUTZ, R. E., AND J. A. MCLAUGHLIN, 1970. Biology of Nosematidae (Microsporidia) from
freshwater and euryhaline fishes. Pages 124-132 in S. F. Snieszko, Ed., A Sym- posium on Diseases of Fislics and Shellfishes.. Special Publication No. 5, Am. Fish.
Soc., Washington, D. C. RIKF, C. B., AND D. W. TAPPA, 1966. Selective predation; smelts and cladocerans in
Harveys Lake. Limnol. Occanog., 11 : 437. SIEFERT, R. E., 1972. First food of Larval Yellow Perch, White Sucker, Bluegill, Emerald
Shiner, and Rainbow Smelt. Trans. Am. Fish. Soc., 101(2): 219-225. SPRAGUE, B., AND S. H. VERNICK, 1968. Light and electron microscope study of a new species
of Glugca (Microsporidia, Nosematidae) in the 4-spined stickleback Apcltcs quadraus.
J. Protocol, 15(3) : 547-571. STUNKARD, H., AND F. E. Lux, 1965. A microsporidian infection of the digestive tract of the
winter flounder, Psciidoplcuronectes amcricanus. Biol. Bull.. 129: 371-385.
GLUGEA HERTU'IGI IN OSMERUS MORD.IX 343
SUMMERFELT, R. C., AND M. C. WARNER, 1970. Incidence and intensity of Plistophora ovariae,
a microsporidian parasite of the Golden Shiner, Notemigoneus crysolcucus. Pages
142-160 in S. F. Snieszko, Ed., A Symopsiiiin on Diseases of Fishes and Shellfishes.
Special Publication No. 5, Am. Fish. Soc., Washington, D. C. VAVRA, J., 1976. Development of Microsporidia. Pages 87-110 in T. C. Cheng and L. A. Bula,
Tr., Eds., Comparative Pathologv Vol. 1. Biology of the Microsporidia. Plenum
Publ., New York. WEIDNER, E., 1972. Ultrastructural study of microsporidian invasion into cells. Z. Parasitenk.,
40: 227-242. WEIDNER, E., 1973. Studies of microsporidian disease transmission in winter flounder and
smelt. Biol. Bull., 145(2) : 459. WEIDNER, E., 1974. Microsporidiosis in aquatic animals. Pages 77-82 in R. L. Amborski, Ed.,
Proceedings of Gulf Coast Regional Symopsium on Diseases of Aquatic Animals.
Center Wetland Resources, LSU Press, Baton Rouge. WEIDNER, E., 1976. Some aspects of microsporidian physiology. Pages 110-126 in T. C.
Cheng and L. A. Bulla, Jr. Eds., Comparative Pathobiology Vol. 1, Biology of the
Microsporidia. Plenum Publ., New York. WEISSENBERG, R., 1968. Intracellular development of the microsporidia Glugea anomala
( Moniez) in hypertrophying migratory cells of the fish Gastcrotcits acitlentiis L.,
an example of the formation of "Xenoma" tumors. /. Protozool., 14 : 44-56. WEISSENBERG, R., 1976. Microsporidian interactions with host cells. Pages 203-207 in T. C.
Cheng and L. A. Bulla, Jr. Eds., Comparative Pathobiology, Vol. 1, Biology of the
Microsporidia. Plenum Publ., New York.
Reference: Biol. Bull., 157: 344-355. (October, 1979)
DEVELOPMENT OF TAIL MUSCLE ACETYLCHOLINESTERASE
IN ASCIDIAN EMBRYOS LACKING MITOCHONDRIAL
LOCALIZATION AND SEGREGATION
J. R. WHITTAKER
The U'istar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104 and Marine Biological Laboratory, Woods Hole, Massachusetts 02543
Ascidians (subphylum Urochordata ; class Ascidiacea) develop a large number of mitochondria during oogenesis. At fertilization many of these become localized into a myoplasmic crescent and are subsequently segregated by the determinate cleavage mechanism into the muscle lineage cells of the developing larva. In some species colored yolk granules are associated with the mitochondria ; they serve as visible markers of the mitochondria! localization and its segregation (Conklin, 1905; Berrill, 1929; Berg and Humphreys, 1960). Many different staining techniques have been used to establish that oviparous species have mitochondrial segregation even in the absence of a visible crescent {e.g., Meves, 1913; Duesberg, 1915; Mancuso, 1952; Reverberi, 1956).
This obvious association of large numbers of mitochondria with the ascidian larval muscle cell lineage has raised questions about whether the crescent mito- chondria are (a) permissive, (b) selective, or even (c) instructive of muscle differentiation. An equally important question is whether segregation of the cytoplasmic determinants responsible for muscle differentiation (Whittaker, 1973) is linked directly to the mechanisms that localize and segregate mitochondria into the muscle cells. The results of various observations and experiments that have attempted to answer these questions prove to be contradictory. The suggestion has therefore persisted in some of the review literature that mitochondrial localizations are causally related to muscle cell determination and development (Brachet, 1960, 1974; Minganti, 1961; Reverberi, 1961, 1971).
Conklin (1931) displaced mitochondria by centrifugation of unfertilized ascidian eggs and concluded from his results that localizations of mitochondria were not the cause of muscle determination. Mitochondria could be driven out of the finely granular plasm in which they were found without preventing the formation of muscle during subsequent development. When the plasm itself was displaced, the larval muscles were also displaced. Tung, Ku, and Tung (1941) noted that mitochondrial masses moved by centrifugation to neural and ectodermal regions did not cause these cells to develop myofibrillae.
The first contradictory evidence came from centrifugation experiments by Ries (1939) who found that displacement of the indophenol oxidase-containing plasm of the ascidian egg resulted in a change in muscle development; he did not know at the time that this enzyme was a mitochondrial oxidase. La Spina (1958) also showed that mitochondrial displacement resulted in some abnormalities of muscle development. Direct interference with mitochondrial function, using inhibi- tors of the mitochondrial enzymes, resulted in the development of embryos markedly
344
ASCIDIAN MUSCLE DEVELOPMENT 345
deficient in muscle structures (de Vincentiis, 1956; Reverberi, 1957). Recently, Bell and Holland (1974) have found by microsurgically dividing centrifugally strati- fied eggs in various orientations and fertilizing the fragments that a certain limited number of mitochondria appear to be necessary for muscle differentiation. Their data also confirm the possibility that mitochondrial localizations might be causally related to muscle differentiation.
One of the difficulties with these various traumatic and disruptive experimental interventions is that they cause severe abnormalities in the embryos. Results are subject to considerable selection and interpretation by the investigator. In nature, however, a situation occurs in which the question can be clearly resolved in a quali- tative way. Certain ascidian species have secondarily evolved anural larvae which no longer develop the larval tail (Berrill, 1931). One of these species, Molgula arenata, differentiates larval tail muscle up to the point of producing histospecific muscle acetylcholinesterase (Whittaker, 1979). This species does not localize mito- chondria or segregate them into the muscle lineage cells.
MATERIALS AND METHODS
Materials
The specimens of Molgnla arenata Stimpson used in this study were dredged from sand flats near Senator Shoal in northern Nantucket Sound at Cape Cod, June through November. Control observations were made on embryos of two urodele species: Ciona intestinalis (L. ), obtained in the vicinity of Woods Hole, Massachusetts, and Molgnla occidcntalis Traustedt of the Florida Gulf, purchased from the Gulf Specimen Company, Panacea, Florida. These three species do not have a visibly colored mitochondrial crescent.
Gametes wrere obtained by techniques described previously (Whittaker, 1979). Embryos were cultured in filtered sea water at 18 ±0.1° C using a refrigerated constant temperature water bath. Since the time of first division is variable in M. arenata (Whittaker, 1979), development times are expressed as time after the 2-cell stage.
Histo chemistry
Acetylcholinesterase wjas localized in embryos by the Karnovsky and Roots (1964) procedure after 2 to 3 min fixation in cold (5° C) 80% ethanol (Durante, 1956). Incubation was for 12 hr at 18° C. Various substrate and inhibitor controls for the identity of the enzyme are presented elsewhere (W'hittaker, 1979).
Succinic dehydrogenase activity was detected by the standard technique for bound mitochondrial enzymes described by Pearse (1972) using sodium succinate as substrate and nitro blue tetrazolium as the electron acceptor. Cytochrome oxidase was localized by the diaminobenzidine reaction of Seligman. Karnovsky, Wasserkrug, and Hanker (1968) including 20 /xg/ml Sigma C-40 catalase in the reaction medium to prevent peroxidase activity. The fixative used for both enzymes was Karnovsky's (1965) fixative, but with formaldehyde and glutaralde- hyde each reduced to 1.5^-, half of the originally recommended strength. Fixation was for 5 to 15 min at 5° C. Incubation time for each of the mitochondrial enzvmes
346 J- R- WHITTAKER
acetylcholinesterase synthesis
puromycm
sensitivity
actinomycin D
sensitivity
2-cell gastrula neurula hatch
02 46 8 10 12
HOURS AFTER 2-CELL
FIGURE 1. Time of various developmental stages and other events in specimens of Molyula arcnata at 18° C.
was 90 to 120 min at 22° C. Succinic dehydrogenase staining did not occur when sodium succinate was omitted from the incubation medium ; the enzyme activity was not affected significantly by 10 HIM sodium azide. Cytochrome oxidase activity did not occur in the presence of either 10 HIM sodium azide or 10 HIM potassium cyanide. Cytochrome c greatly enhances the sensitivity of the reaction but cytochrome oxidase gives a modest reaction with 3.3'-diamino- benzidine in the absence of substrate. Concentrations of the reaction products in both of these histochemical reactions appear to follow the Beer- Lambert law ; color density is proportional to enzyme activity and therefore proportional to the numbers of mitochondria (Cabrini, Vinuales, and Itoiz, 1969; Marines, 1978). After the respective histochemical reactions the embryos were dehydrated in ethanol, cleared in xylene, and mounted in damar resin. The three enzyme pro- cedures produced essentially permanent color reactions.
RESULTS
Development of acetylcholinesterase
Development of larval tail muscle acetylcholinesterase in Molgnla arcnata initially followed the same pattern found in urodele ascidian species : the enzyme became histochemically detectable at neurulation. Color first appeared at 6 hr after the 2-cell stage (Fig. 1) and enzyme activity accumulated gradually over the next few hours until a modest level of activity was attained (Fig. 2). In some larvae this activity reached a level as high as 20% of that found in the larvae of comparable urodele species of Molgula, but the mean activity found was 5 to 6% of the urodele level (Whittaker, 1979). Tail development was completely suppressed in M. arenata and except for acetylcholinesterase development presump- tive muscle tissues did not otherwise develop beyond the early neurula stage ; there was no obvious myofibrillar synthesis.
ASCIDIAN MUSCLE DEVELOPMENT
347
B5.2
FIGURES 2-5. Embryos of Molgula arcnata stained histochemically for acetylcholinesterase.
FIGURE 2. Hatched larva.
FIGURE 3. Embryo cleavage-arrested in cytochalasin B at the 4-cell stage and reacted for enzyme at 8 hr after the time of the 2-cell stage.
FIGURE 4. Embryo cleavage-arrested at the 8-cell stage and reacted for enzyme at 8 hr after the 2-cell stage.
FIGURE 5. Embryo cleavage -arrested at the 16-cell stage and reacted for enzyme at 8 hr after the 2-cell stage.
The muscle cell lineage designations are according to Ortolani (1955). All figures are tlie same magnification ; the bar in Figure 2 is 40 jum long.
Segregation of a muscle determinant
Previous studies with cleavage-arrested embryos of C. intcsthialis have shown that blastomeres of the muscle lineage in arrested embryos will eventually develop acetylcholinesterase (Whittaker, 1973). Similar experiments with M. arcnata.
348
J. R. WHITTAKER
B4.1
ASCIDIAN MUSCLE DEVELOPMENT 349
produced the same results, at least in the earlier stages. Such observations support the theory that an autonomously acting cytoplasmic determinant of acetylcholin- estrase development is being segregated in the muscle lineage cells.
Embryos of M. arcnata were placed in 2 /Ag/ml cytochalasin B (Sigma) at various cleavage stages after fertilization. These were then reacted for acetyl- cholinesterase 8 to 9 hr after the 2-cell stage. The maximum cell numbers producing acetylcholinesterase and the relative positions of these cells in the embryo matched those of the known ascidian muscle cell lineage : one blastomere at 1-cell, two at 2-cell, two at 4-cell, two at 8-cell, and four at 16-cell. Figures 3-5 depict the last three of these stages.
A majority of the embryos at all of the various cleavage-arrested stages developed acetylcholinesterase in one or more of the muscle lineage blastomeres and a modest number of embryos at 2-, 4- and 8-cell stages produced the maximum lineage numbers of reacting cells (two). Interestingly, few embryos at the cleavage- arrested 16-cell stage produced enzyme in more than two cells, one on each bilateral side. Figure 5 illustrates one of the embryos in which all four of the muscle lineage cells produced acetylcholinesterase. Usually only the B5.1 cells synthesize acetyl- cholinesterase. This restriction to two rather than four cells is conceivably related to the lesion that results in a limited expression of acetylcholinesterase in M. arcnata.
A limitation of expression seemed also to occur in later stages as well, but there the results were less certain. The chorion of M. arcnata adheres closely to the embryo and appears to exert tension during development. Consequently, the acetylcholinesterase-producing muscle lineage cells in cleavage-arrested 32-cell and 64-cell stages tend to remain aggregated together in two bilateral groups. Aggrega- tion combined with high yolk content of the cells and the large number of nuclei which accumulate make it essentially impossible to discern cell boundaries within groups of older myoblasts.
Acetylcholinesterase dependence on protein and RNA synthesis
The time at which enzyme was first detected histochemically (Fig. 1) is apparently the time of first acetylcholinesterase synthesis. When 200 j".g/ml puromycin di-HCl (Sigma) was added to embryos of M. arcnata 30 min before the time (at 6 hr) of first acetylcholinesterase staining no enzyme was detected histochemically at 8 to 9 hr time. Similarly, embryos placed in puromycin at
FIGURES 6-13. Embryos of Ciona intcstinalis stained histochemically for succinic dehydro- genase.
FIGURE 6. Unfertilized egg.
FIGURE 7. 2-cell stage.
FIGURE 8. 4-cell stage.
FIGURE 9. 8-cell stage. Side view of the bilaterally symmetrical embryo.
FIGURE 10. 16-cell stage.
FIGURE 11. 32-cell stage.
FIGURE 12. 64-cell stage.
FIGURE 13. Middle tailbud stage at about the time of first melanocyte differentiation (12 hr development at 18° C).
The muscle lineage desginations are according to Ortolani (1955). All figures are the same magnification; the bar in Figure 13 is 40 /im long.
350 J. R. WHITTAKER
6 hr produced, even after many hours, only the slight amount of enzyme activity that would ordinarily he detected at 6 hr time. Puromycin at 200 jug/ml causes 95 to 99% inhibition of protein synthesis in ascidian embryos (Whittaker, 1973, 1977). Presumably the cytoplasmic factor being segregated is not a preformed inactive acetylcholinesterase since the occurrence of activity seems to depend directly on protein synthesis.
There was also an actinomycin D sensitivity period for acetylcholinesterase synthesis in this species. This occurred between 2 and 3 hr before the time of enzyme synthesis (Fig. 1). When embryos were treated continuously with 20 Mg/ml actinomycin D (Sigma, Grade III) beginning at 3 hr after the 2-cell stage, no acetylcholinesterase developed subsequently. If treatment was started at 4 hr some slight amount of enzyme activity was found at hatching time. Progressively more enzyme activity was found the later after 4 hr that actinomycin D treat- ment was started. Since actinomycin D at this concentration produces a maximal inhibition of RNA synthesis in ascidian embryos (Smith, 1967; Mansueto- Bonaccorso, 1971), occurrence of acetylcholinesterase probably requires a specific embryonic period of RNA synthesis. Enzyme messenger RNA (mRNA) synthesis most likely occurs during this time. On the basis of other studies (Whittaker, 1977), one can assume that enzyme synthesis would be resistant to actinomycin D if a performed mRNA for the enzyme were present.
Mitochondrial segregation in iirodelc embryos
Succinic dehydrogenase reactions in early embryonic stages of C. intestinalis showed clearly the pattern of mitochondrial distribution in the embryos (Figs. 6-13). Before germinal vesicle breakdown, which occurs in the oviduct, mito- chondria are distributed uniformly in the subcortical cytoplasm of the egg, as seen by the distribution of succinic dehydrogenase in cryostat sections (Patricolo, 1964). After germinal vesicle breakdown and before fertilization there is a migra- tion of mitochondria to the vegetal half of the egg (Fig. 6). After fertilization they migrate further into the vegetal half of the egg and many of them eventually form a mitochondrial crescent, which is seen most clearly at the 2-cell stage (Fig. 7).
Although mitochondria were distributed elsewhere in the egg and embryos, a major concentration occurred in the muscle lineage cells, according to the lineage patterns established by Conklin (1905) and Ortolani (1955). One should note particularly the distribution of activity at the 4-cell stage (Fig. 8) where there is obviously segregation of much more activity into the B3 pair of blastomeres. At the tailbud stages one sees the strong localization of enzyme in the differentiating muscle cells of the tail (Fig. 13). Identical results were obtained with embryos of another species, M. occidcntalis.
Similar enzyme distributions can be shown with a second mitochondrial enzyme, cytochrome oxidase. Histochemical localizations of this enzyme were identical in both C. intestinalis and M. occidcntalis to those shown in Figs. 6-13 for succinic dehydrogenase. This duplication lends additional confidence to the likelihood that these staining reactions reveal the mitochondrial distribution rather than simply reactive peculiarities of a particular enzyme.
. \SCIDI.\X Ml'SCI.K DKVKI.OI'MKXT
351
Mitochondrial enzymes in aniircil embryos
There was no evidence at any embryonic stage that mitochondria were differentially segregated into nuiscle lineage hlastomers of M. arenata. Embryos were examined at cleavage stages and at various later developmental stages. At second cleavage the two posterior (B3) blastomeres are slightly larger than the
FIGURES 14-17. Embryos of Molgula arenata stained histochemically for the mitochondrial enzymes succinic dehydrogenase and cytoclirome oxidase.
FIGURE 14. 4-cell stage embryo stained for succinic dehydrogenase.
FIGURE 15. Embryo 6 hr after the 2-cell stage stained for succinic dehydrogenase.
FIGURE 16. 4-cell stage embryo stained for cytochrome oxidase.
FIGURE 17. Hatching larva stained for cytochrome oxidase. The arrow indicates the position of one arm of the muscle cell crescent.
All figures are the same magnification; the bar in Figure 17 i::. 40 //m long-.
35 _! J. R. WHITTAKER
anterior pair. Figure 14 shows that distribution of succinic dehydrogenase occurs in proportion to the size of the hlastomeres. There is no obvious differential segregation of activity into the posterior pair. At the neurula stage (Fig. 15) the developing larva had no enzyme localization in those presumptive muscle rudi- ments that so clearly showed the presence of acetylcholinesterase in other experi- ments (Fig. 2). Cytochrome oxidase staining revealed the same result: no segregation of enzyme at either the 4-cell stage (Fig. 16) or in the hatching larva (Fig. 17). As described in detail elsewhere (Whittaker, 1979), the hatching larva emerges "head" end first from the chorion; in Fig. 17 the smaller bulge at the left is the emerging anterior end of the larva. The crescent of myoblasts is located at the position of the arrow. There is no differential accumulation of cytochrome oxidase staining in this region of the embryo, or elsewhere.
DISCUSSION
Preformation of mitochondria in the ascidian oocyte is an apparent adaptation to the needs of rapid development in oviparous embryos. Presumably the embryo is unable to synthesize sufficient mitochondria during a brief development time to meet the high energy requirements of larval tail muscle, and must prepare some of these in advance of embryonic development. Ascidian embryos synthesize addi- tional mitochondria during development. Mancuso (1962) noted bilobed mito- chonclrial structures especially in myoplasmic regions of the Ciona embryo, and concluded that muscle mitochondria multiply during embryogenesis. Measure- ments of mitochondria! enzyme activity indicate that only about half of the number of mitochondria found at larval hatching occur preformed in the fertilized egg (D'Anna and Metafora. 1965; D'Anna. 1966).
According to enzyme activity measurements by Berg ( 1956, 1957) on separated blastomeres at the 4-cell stage roughly two-thirds of the original mitochondria are segregated (in Ciunu) into the two B3 muscle lineage blastomeres. A large pro- portion of these become actively segregated into the later cells of the muscle lineage. While mitochondria] preformation and segregation may be necessary for optimal physiological function of the larva at hatching, the important question is whether this initial concentration directly provides information in the developing muscle system.
Larvae of most ascidian species have a histospecific acetylcholinesterase that occurs in the tail muscle cells ( Durante, 1956, 1959). One anural species, M. arcnata, also developed enzyme in the presumptive muscle cells of the aborted tail (Whittaker, 1979), which indicates that these cells are unquestionably pro- grammed to become muscle. Although myofibrils were not obvious, and probably did not occur, the cells nevertheless differentiated part of their histospecific acetyl- cholinesterase.
Evidence from experiments with cleavage-arrested embryos of Ciona (Whit- taker, 1973) and microsurgically isolated partial embryos (Whittaker, Ortolani, and Farinella-Ferruzza, 1977) suggests that ascidian embryos localize and segregate a cytoplasmic determinant concerned with synthesis of acetylcholinesterase by the developing muscle cells. This factor appears to be neither the enzyme itself nor a preformed mRKA for acetylcholinesterase. and is probably an agent responsible
ASCIDIAN MUSCLE DEVELOPMENT 353
for activating particular genetic programs in the embryo (Whittaker, 1973). A similar determinant apparently occurs in M. arcnaia. Segregation of a determinant was demonstrated by cleavage-arresting embryos of M. arcnata with cytochalasin B, and finding the eventual development of acetylcholinesterase in blastomeres of the ascidian muscle lineage pattern. Also, normal occurrence of acetylcholine- sterase activity was blocked by puromycin and actinomycin D, suggesting thereby that there is neither preformed enzyme nor preformed mRNA for acetylcholin- esterase.
Histochemical staining of the mitochondria! enzymes succinic dehydrogenase and cytochrome oxidase in embryos and larvae of M. arcnata showed unequivocally that no "crescent" localization of mitochondria occurred after fertilization and no subsequent differential segregation of mitochondria took place. The conclusion that anural embryos lack a differential localization and segregation of mito- chondria is thought to be valid for two reasons. Results with the same techniques in a normal urodele embryo series (Figs. 6-13) unambiguously show differential mitochondria! distribution. Secondly, concentrations of the histochemical reaction products follow the Beer-Lambert law. and the human eye is sufficiently sensitive to distinguish even minor concentration differences in the products.
In M. arcnata there is an uncoupling of mitochondria! behavior and muscle cell differentiation. Mitochondria are not localized and not differentially segregated ; yet there is determination of larval muscle and its partial differentiation. This shows without question that the mitochondria! accumulation in muscle cells of ascidian embryos has no direct informational relationship to the cellular dif- ferentiation. At the same time it is likely that the cytoplasmic determinants of muscle are not physically associated with the myoplasmic mitochondria. One cannot, however, rule out the possibility that the same physical mechanism is involved in both mitochondria! segregation and segregation of the determinants responsible for muscle differentiation. If so. the processes operate independently of one another.
The demonstration of disjunction between mitochondrial segregation and muscle differentiation is a potent example of the intrinsic superiority of nature's experiment. Theodore Boveri urged us long ago to seek the natural experiment where possible: "the investigator of living processes will make it his special concern to find out abnormalities, in which lie has not intervened with his crude methods, where he can penetrate into the nature of the alteration" (Boveri, 1908, p. 216).
I thank Dr. Arthur Humes, Director of the Boston University Marine Program at the Marine Biological Laboratory, for the use of dredging facilities, and Mr. Charles H. Henry Jr. of the Case Western Reserve University Dental School for his patient preliminary study of succinic dehydrogenase staining in embryos of dona. The work was supported by Grant HD 09201, awarded by the National Institute of Child Health and Human Development, DHE\Y.
SUMMARY
The ascidian Molgula arcnata produces an anural larva in which development of the tail and other urodele features has been suppressed. The embryos never-
354 J. R. WHITTAKER
theless developed part of the- histospecific tail muscle acetylcholinesterase; the presumptive myoblasts have obviously acquired the muscle differentiation program. \\ hen cleavage-stage embryos were prevented from undergoing further division by treatment with cytochalasin R, acetylcholinesterase evenutally developed in blasto- meres of the muscle lineage. These anural embryos apparently segregate a cyto- plasmic determinant concerned with acetylcholinesterase development into cells of the muscle lineage. In this species there was no localization and segregation of mitochondria! succinic dehydrogenase and cytochrome oxidase in the muscle lineage, as found in embryos of two urodele ascidians, dona intestinalis and Molgula occidentals. The causal determinant of histospecific acetylcholinesterase expression is not, therefore, a differential localization of mitochondria nor is segrega- tion of the muscle determinants linked directly to mitochondrial segregation.
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BERG, W. E., 1956. Cytochrome oxidase in anterior and posterior blastomeres of dona intesti- nalis. Hiol. Bull, 110: 1-7. BERG, W. E., 1957. Chemical analyses of anterior and posterior blastomeres of Cionu intestinalis.
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BKACHET, J., I960. The Biochemistry <>f Development, Pergamon Press, London, 320 pp. BRACHET, J., 1974. Introduction to Molecular Embryology, Springer-Verlag, New York, 176 pp. CABRINI, R. L., E. J. VINUALES, AND M. E. ITOIZ, 1969. A microspectrophotometric method
for liistochemical quantification of succinic dehydrogenase. Acta Histochcni., 34:
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development of ascidian eggs. Actn Embryul. Morphol. /:.r/>., 8: 267-277. DE VINCENTIIS, M., 1956. Sullo sviluppo delle uovo di 1'lialli/sia mamillata in condizioni di
anaerobiosi. E.rperientia. 12: 381-382. DUESBERG, J., 1915. Recherches cytologiques sur la fecondation des Ascidiens et sur leur
developpement. Cunici/ic lust, ll'asli. Contril'. Einbryol.. 3: 33-70. DURANTE, M., 1956. Cholinesterase in the development of dona intestinalis (Ascidia).
Experientia. 12: 307-308. DURANTE, M., 1959. Sulla localizzazione istochimica della acetilcolinesterasi lungo lo sviluppo
di alcune ascidie e in appendicularie. Acta Embryol. Morphol. E.vp. 2 : 234-243. KARNOVSKY, M. J., 1965. A formaldehyde-gkitaraldehyde fixative of high osmolality for use in
electron microscopy. J. Cell Biol.. 27: 137A-138A. KARNOVSKY, M. ]., AND L. ROOTS, 1964. A "direct-coloring" thiocholine method for cholin-
esterases. /. Histochcni. Cvtocliem., 12: 219-221.
ASCIDIAN MUSCLE DEVELOPMENT 355
LA SPIXA, R., 1958. Lo spostamento dei plasmi mediante centrifugazione nell'uovo vergine di
ascidie e il conseguente sviluppo. Acta Embryol. Morphol. E.rp.. 2: 66-78. MAXCVSO, V., 1952. Ricerche istochimiche nell'uovo di Ascidie. II. Distribuzione delle
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265-269. MAXCUSO, V., 1962. L'uovo di Ciona intestinalis ( Ascidia) allo stadio di 8 blastomeri osservato
al microscopic elettronico. Acta Embryol. Morphol. E.rp., 5 : 32-50. MANSUETO-BONACCORSO, C., 1971. Incorporazione di precursor! marcati in presenza di actino-
micina nello sviluppo delle nova di Ascidie. Penetrazione dell'actinomicina D-H:i nei
diversi stadi. Atti Accad. Xaz. Lined Rend., 50: 164-166. MARIXOS, E., 1978. Cytochrome oxidase activity of mitochondria in Xenopus laevis previtello-
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der Befruchtung des Eies von Phallusia inamillata. Arch. Mikrosk. Anat. Ent-tAck-
lidii/smecli.. 82 : 215-260. MINGANTI, A., 1961. Recent investigations on tbe development of ascidians. Pages 255-276
in S. Ranzi, Ed., Symposium on the Genii Cells and Earliest Stat/cs of Development,
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(II-B) : 71-76. PEARSE, A. G. E., 1972. Histocliemistry. Theoretical and Applied. Third Edition. Vol. 2
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340 in A. Tyler. R. C. Borstel, and C. B. Metz, Eds., The Bei/innini/s of Embryonic
Development, American Association for the Advancement of Science, Washington, D. C. REVERBERI, G., 1961. The embryology of ascidians. A dr. Morplioi/.. 1 : 55-101. REVERBERI, G., 1971. Ascidians. Pages 507-550 in G. Reverberi, Ed., Experimental Embryoloi/y
of Marine and Fresh-loafer Invertebrates. North-Holland/American Elsevier, New
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bedifferenzierung bei Ascidien. Arcliiv fiir E.rperimentclle Zellforschun//. 23 : 95-121. SELIGMAX, A. M., M. T. KARXOVSKY, H. L. WASSERKRUG, AXI> J. S. HANKER. 1968. Nondroplet
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osmiophilic reagent, diaminobenzidine (DAB). /. Cell Biol.. 38: 1-14. SMITH, K. D., 1967. Genetic control of macromolecular synthesis during development of an
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WHITTAKER, J. R., 1973. Segregation during ascidian embryogenesis of egg cytoplasmic in- formation for tissue-specific enzyme development. Proc. Xatl. A cad. Sci. L .S.A.. 70 :
2096-2100. WHITTAKER, J. R., 1977. Segregation during cleavage of a factor determining endodermal
alkaline phosphatase development in ascidian embryos. /. E.rp. Zoo/., 202 : 139-153. WHITTAKER, J. R., 1979. Development of vestigial tail muscle acetylcholinesterase in embryos
of an anural ascidian species. Biol. Bull., 156: 393-407. WHITTAKER, J. R., G. ORTOLAXI, AXD X. FARIXELLA-FERRTZZA, 1977. Autonomy of
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ABSTRACTS OF PAPERS PRESENTED AT THE MARINE BIOLOGICAL LABORATORY
.Ihslracts arc arranged alphabetically by first author. Author and subject rejer- I-HCCS id II I'C jonnd in the regular i-olntuc inde.r, appearing in flic I>cccnibcr issue.
GENERAL SCIENTIFIC MEETINGS AUGUST 20-22, 197<>
Morphogenesis in (/raited Hydra attenuata : positive dominance, negative dominance, and pattern regulation. JAMES ALFRED ADAMS.
Positive dominance (induction) and negative dominance (inhibition) have been studied in grafted Hydra attenuata. Grafted animals are prepared having three gastric regions, tandemly arranged, with original disto-proximal orientation of the graft pieces being maintained. Grafted animals prepared in this fashion ( 3g hydras ) have the ability to regenerate heads and feet in the vicinities of the graft borders. These regenerates are referred to as a secondary head (2°h) and secondary feet (2°f). Gradients in the frequencies of both 2°h and 2°f regeneration are observed in these animals. Using Chi-square methods and a 95% level of confidence, one finds a significantly greater frequency of 2°h regeneration at the graft border farthest from the terminal head ( 0.050 > P > 0.010). Likwise, a higher frequency of 2°f regeneration occurs at the graft border farthest from the terminal foot (0.005>P). These data suggest that the terminal head or foot, by its presence, either inhibits or causes inhibition of a like structure occurring (negative dominance). This inhibitory effect decreases in a linear fashion with increasing distance from the terminal head or foot as the case may be.
However, when a tentacle and a small piece of attached hypostomal tissue is left on the middle and proximal graft pieces there is no inhibition of head formation at either graft border. This result shows that the inductive capacity (positive dominance) of the tentacle- hypostome region can overcome the negative dominance of the terminal head. This technique also affects 2°f formation on the 3g animal. The frequency of 2°f regeneration is enhanced significantly at the proximal graft border and decreased significantly at the distal border, with the effect that the gradient in foot formation observed in control 3g animals is abolished. Thus, a direct relationship, and perhaps an interdependency between the gradients of 2°h and 2°f regeneration in 3g H. attenuata is demonstrated.
Alternate pathi^a\s in the biosynthesis of testosterone bv Rana pipiens. CHRISTOPHER A. ADEJUWON, SHELDON J. SEGAL, AND S. S. KOIDE.
The absence of immunoreactive progesterone, despite the presence of appreciable amounts nf immunoreactive testosterone in unstimulated testes taken from specimens of Rana f'ipicns, led us to investigate the precursor role of progesterone in testicular testosterone biosynthesis in this amphibian species. Three sets of experiments were performed. The first was chromatographic analysis of testicular steroids. Testis slices (60-80 mg) were placed in 2.0 ml ringer's solu- tion and were agitated with 10.0 ml petroleum ether and then with choloroform. After evaporat- ing the solvent, the residue was dissolved in chloroform : methanol and applied to rtourescent- coated silica gel thin-layer chromatography (TLC) plates. The chromatogram was developed in ethyl acetate : water : n-hexane : ethanol. The resulting spot was visualized with ultraviolet light using testosterone as a marker. The steroid present in the testicular extract had an Rf value of 0.63 compared to the Rf values of 0.67 and 0.72 for testosterone and progesterone, respectively. Radiolabeled pregnenolone co-migrated with progesterone in this system.
In the second experiment, in vitro stimulation of testosterone production by human chorionic gonadotropin (hCG) was demonstrated by incubating Rana testis slices with ringer's solution containing varying concentrations of hCG. Aliquots (100 /JL\) of the incubation medium were removed between 0.5 and 2.0 hr of incubation for testosterone determination
356
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 357
by radioimmunossay (RIA). Testosterone production was stimulated by hCG in a dose- related manner ; the lower limit of sensitivity was 2 ng. Thirdly, in vitro conversion of precursor to 3H-testosterone was demonstrated by incubations of 3H-progesterone and :!H-pregnenolone with testicular slices in ringer's solution containing a nicotinamide adenine dinucleotide phosphate- generating system. Steroids were extracted and analyzed on TLC. About 8.8% of ;<H-pro- gesterone and 8.7% of 3H-pregnenolone were transformed to 3H-testosterone in 4 hr of incubation The present results, based on RIA, TLC, and hCG-stimulation show that testosterone is produced by testes taken from mature male frogs of the R. pipicns species. The A" and A4 biosynthetic pathways appear to be equally important for in vitro testosterone formation. HCG concentra- tions as low as 2 ng stimulate testosterone production.
Supported by a grant from the George Hecht Fund. C. A. Adej union is a recipient of a Rockefeller Foundation Fellowship.
Characterization of Spisula sperm protamhic. JUAN Ausio AND K. E. VAN HOLDE.
The major protein component of mature Spisula sperm nuclei appears to be a protamine. This material has been purified from ripe sperm by the following procedure : sperm were washed gently from excised gonads and homogenized in 5% acetic acid. After low-speed centrifugation, the nuclei were re-extracted with 25% acetic acid. These extractions remove most of the contaminating histories. The nuclei were next extracted with 0.25 x HC1, and the protamine so released was purified by chromatography on a CMC-25 column, using 1.2- 1.8 M NaCl gradients.
The protamine so prepared migrated as a single band on urea gel electrophoresis, with the reservation that a small amount of material always remained at the top of the gel. Sedimenta- tion equilibrium studies at pH 9.2, and treatment with 0.4 M NaCl revealed a nearly homogeneous material of molecular weight 29,400. The sedimentation coefficient was determined at a number of concentrations in 0.4 M NaCl, and the extrapolated value of S3j° ,„• = 2.0 X 10~13 was obtained. This value, small for a protein of this mass, corresponds to a frictional ratio f/f0 = 1.55, indicat- ing asymmetry or unfolding of the molecule. Upon increasing the ionic strength above 1 M, the sedimentation coefficient increases about 25%, probably from a condensation of this highly charged molecule.
This research was supported in part by grant T32-GM-077S4 from the PHS.
Phosphorylation of the 40s ribosomal subunit after fertilization of Arbacia punc- tulata eggs. DENNIS BALLINGER, SCOTT PETERSON, AND TIM HUNT.
A particularly prominent alteration in the pattern of protein phosphorylation after fertiliza- tion of Arbacia punctithita eggs occurs in a 31,000 MW protein whose labeling increases rela- tive to other proteins. This increase can be detected 5 min after fertilization in eggs which have been labeled for 2 hr with ^POj.
The following criteria identity this protein as a component of the 40s ribosomal subunit : first, it co-sediments with 80s ribosomes on a low salt sucrose gradient ; secondly, it co-sediments with the 40s ribosomal subunit on a high salt sucrose gradient; thirdly, its mobility in a two dimensional gel system designed to separate basic ribosomal proteins (acid urea/SDS) suggests that it may be the sea urchin analogue of protein S6 of yeast and mammalian ribosomes, but this identification is tentative at present. We hope to use this analytical system to determine whether the increased labeling represents dc n/>vt> phosphorylation, or simply an increased rate of turnover of preexisting phosphate residues. Our data indicate, however, that at least one of these parameters must change after fertilization.
We have made one test of the hypothesis that the phosphorylation of this ribosomal protein may be connected in some way with the 20- to 30-fold increase in protein synthesis which occurs in the sea urchin after fertilization by asking whether the labeled subunits were preferentially associated with polysomes. We labeled embryos with 32PO4 for 4 hr following fertilization, and analyzed extracts on sucrose gradients. The labeled 31,000 MW protein was found associated to the same extent with 80s ribosomes and polysomes in terms of cpm/Aaio. These results are
35«S PAPERS PRESENTED AT MAR1XE BIOLOGICAL LABORATORY
indicative of little or no preferential association of phosphorylated suhunits with mRXA in 4-hr embryos. Thus, the significance of this phosphorylation, while suggestive, remains uncertain.
Difference between SDS-PAGE patterns oj Labyrinthula slimeways and vegetative cells. EUGENE BELL, NORIO NAKATSUJI, AND STEPHANIE SHER.
The slimeways of Labyrinthula are synthesized by the cells and contain contractile proteins organized, in part, into filaments. To examine the relative protein complexities of cells and slimeways, colonies were labeled with HC reconstituted protein hydrolysate, homogenized, fractionated into cells and slimeways and then subjected to SDS PAGE. Labeled homogenates of whole colonies gave a minimum of 55 radioactive bands. Bands comigrating with chick skeletal muscle myosin and chicken gizzard were identified. The slime-way fraction showed five times fewer bands than the whole colony homogenate or the cell fraction homogenate, but the film densities of actin bands of the cell and slimeway fractions was the same, while the myosin band of the slimeway fraction was even more dense.
To try to understand the segregation of cell and slimeway proteins in the three-dimensional colony, we have reviewed the fine structure of Labyrinthula and propose the following scheme : In the extrusion of "extracj~'u1ar" memVrnes and slimeway proteins from a cell bounded only by its plasma membr: ne (cell membrane I), the first event important for slimeway formation may be the ballooning of membrane vesicles from bothrosome apertures. The vesicles with asso- ciated proteins flatten into two layers and open where the layers make hairpin turns. The layer adjacent to the cell, cell membrane II, which is attached to the cell through the bothrosome, fuses with comparable layers from other bothrosomes of the same cell to form a second mem- brane around the cell. The cell is now invested with a second membrane of reverse polarity with respect to the plasma membrane. Thus, through vesicle formation and rupture, selected proteins and formed membrane pieces are brought to the exterior for slimeway construction.
Supported by grant no. 04-7-158-44079 from NOAA to E. Bell.
Conduction and dielectric studies of protein-methylglyoxal coinple.ves. STEPHEN BONE.
Dielectric and conduction studies of bovine serum albumin (BSA), casein, collagen, and lysozyme treated with methylglyoxal have been made in an attempt to provide evidence for the existence of the charge-transfer mechanism proposed by Albert Szent-Gyorgyi (1979, Biol. Bull., 157: 398). The dry treated protein samples show a dielectric dispersion at sub- Hertz frequencies whereas no such trend is observed for the pure proteins. A similar low fre- quency dispersion has been shown to exist in the dielectric studies of the conventional charge- transfer complex (perylene + chloranil). These dispersions have been found to be associated with a bulk rather than an electrode interfacial effect. The observation of an increased DC conductivity and lowered conductivity activation energy for the protein-methylglyoxal complexes as compared with the untreated proteins is consistent with the formation of a charge -transfer complex. Analysis of the dielectric dispersions indicate that this dielectric loss cannot be interpreted using conventional dipolar relaxation theories, but they may be under- stood in terms of the hupping of mobile charge carriers. These carriers are thought to hop over potential energy barriers associated with intra- and inter-protein boundaries, and their mean free path length has been determined to be of the order of 1.5-1.8 nm for the BSA, casein, and lysozyme complexes and 16 nm for the collagen complex. These studies provide strong evidence for the existence of a charge-transfer interaction between proteins and methylglyoxal as originally proposed by Szent-Gyorgyi.
Pressure-flow relations in the perfused systemic circulation of squid. GEORGE B. BOURNE.
An artificially perfused preparation of the posterior systemic circulation of squid was developed to permit examination of some physical and cholinergic factors affecting pressure-
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 359
flow relations. The posterior aorta was cannulated via the systemic heart, after which pres- sure was monitored by a strain-gauge manometer connected to the cannula at a T-junction by polyethylene tubing. Flow was measured by an electromagnetic flmvmeter and also was monitored by sensing loss of fluid from the perfusion reservoir via an isometric muscle lever. The signals from the transducers were read out on a Brush recorder.
The preparation took approximately 15 min to stabilize when perfused by vigorously oxygenated, filtered sea water. Stability was maintained for 1 to 2 hr, after which the prepara- tion decayed rapidly. The decay was characterized by increasing vascular resistance and severe oedema.
When flow was plotted against pressure, a curve which obeyed a power function was obtained. Acetylcholine caused a marked vasodilation. The threshold of response to acetyl- choline was lO'11^! with 75% of maximal response occurring at 10~s M. The vasodilatory action of acetylcholine was blocked by d-tubocurarine.
This work was supported by a Steps Toward Independence Program Fellowship and by an operating grant from the National Scientific and Engineering Research Council of Canada.
The numbers and sizes of cells in inollitscan ganglia; simultaneous optical recording of activity from many neurons. [Demonstration.] M. B. BOYLE, L. B. COHEN, AND E. R. MACAGNO.
We have compared ganglia from the central nervous systems of a number of gastropod molluscs with the object of finding an animal having relatively large and relatively feu nerve cells. We hope to use optical methods for monitoring activity to help us unravel the neuronal basis of behaviors. Freshly dissected ganglia were left in a 0.1 to 0.3 mg/ml solution of methylene blue in saline for 12 to 18 hr at room temperature, fixed in 4c/r formalde- hyde in saline for about 30 min, dehydrated in 30, 60, 90, and 100r/<- acetone, and cleared in methyl salicylate. The ganglia were then whole-mounted between cover slips using Permount. This procedure is designed to selectively stain cell bodies. We showed examples of buccal ganglia from Aplysia etiliforiiiea. Dendronatns iris, and Tritouiu diomedia. and of circumesophageal ganglia from Hermissenda crassicornis, Dendnn/otiis iris, and Tritoiiin dioiucdia. As viewed under the dissecting scope, the buccal ganglia of Tritnnw and Aplysia appear to contain a relatively large number of small cells as compared with J >ciidroiiottis. The circumesophageal ganglia of Hermissenda seem to consist of cells whose average size is significantly smaller than that in either Tritonia or Dcndronotits. Dendrouotits appears to have relatively few small cells in either the buccal or the circumesophageal ganglia.
We also showed an example of a pre-recorded experiment done at Friday Harbor Laboratories on Dendroronotus iris. We detected spontaneous activity and activity in response to suction electrode stimulation of nerves from a circumesophageal ganglion using the merocyanine-rhodanine dye WW433.
Supported by NIH grants NS08437. XS 14946. and RR00442.
Optimal prey selection of Littorina littorea bv green crahs. JOHN E. BOYNTON.
Optimum foraging theory predicts that predators will be adapted to select prey of a size that returns maximal food energy for time invested. Ciirciinis nnicnas (green crab) has a varied diet which includes Littoriini littorea (periwinkle). Crabs attack snails by several methods : crushing, shell edge chipping, and shell boring. Predictions of optimal foraging theory and correlations of attack armature and defensive armor were tested using this predator-prey system. Snails of all sizes were fed to a size-spectrum of individual crabs, and prey size selected was determined. Snail shell-length : dry-weight relations were measured. Parameters of crab claws were examined as well as snail shell thickness and length relations. Only snails in the size range of 5 to 17 mm were selected by crabs. Dry weight results indicate that snail soft tissue increases exponentially. Crabs would benefit more from going after larger snails rather than smaller snails. Crabs mostly crushed their prey. Crabs chose snails that fell within the uooer ranee of that permitted by the individuals' claw gape, and
360 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
ignored smaller snails. This corresponds with data on snail populations in the salt marsh. Claw size seems to determine the prey size that can he attacked.
Chemoreception in aitfisli: effects of diet on behni'ior and body odor. BRUCE BRYANT AND JELLE ATEMA.
Whole hody-odor complexes are important in maintaining social structure, hut this maintenance is not based on a rigid stimulus-response relationship as found in typical pheromone-mediated social behavior. In aquaria, the brown bullhead, Ictuliirns ncbitlosus, forms territories and dominance hierarchies. The importance of body odor in maintaining these territories and hierarchies was investigated by observing the effects of a changing body odor of one member of a territorial pair. Body-odor change was induced by a be- haviorally non-invasive manipulation — a change in diet.
Pairs of fish, fed identically for at least three weeks (Purina trout chow), were observed and both social and solitary behavioral units were recorded. A handling control preceded the diet manipulation and consisted of isolating and feeding the subdominant C/3) fish on chow in a separate tank for 1 day. Two days after reintroduction into the main tank, (/3) was isolated again but this time fed an identical volume of beef liver. In three of four tanks, a change from display behavior to aggressive nips and bites by a occurred when comparing control and experimental reintroductions of p. The fourth tank showed no change in aggres- sive behavior but a change in territorial status, p's territory suffered significantly more incur- sions following manipulation than during the control periods ( P < 0.05, Friedman 2-way ANOVA). Inspection of p's solitary behavior units did not reveal effects of the diet change. The changes in behavior associated with a change in diet suggest that the maintenance of territorial hierarchy is dependent on chemical recognition of conspecifics which have become familiar ; diet manipulation disrupts this recognition. The degree and time course of this disruption seems to suggest that it is not complete and that familiarity is regained within days.
New developments hi the mariculture of Aplysia californica. THOMAS R. CAPO, SUSAN E. PERRITT, AND CARL J. BERG, JR.
It recently has become possible to study the developmental neurobiology of Aplysia culi- fornica through the use of laboratory cultured animals. In order to optimize culture condi- tions we have emphasized three approaches. The first involved the evaluation of various antibiotics. The combination of Penicillin-G sodium salt ( 100.5 units/ml ) and Streptomycin- sulfate ( 50 /ug/nil ) was routinely found to improve the survival of veliger cultures. The second approach was to find substrates other than Laiircncia pacifica, for triggering the metamorphosis of A. californica. Two red alga available in the Woods Hole region, Neoagard- hcilla bailcyi and Gracilaria sp., have been successful. Neoagardheilla bailcyi. Strain A,, has been cultured in the laboratory for six months ( T. Capo, in preparation ) . With present tech- niques, more than adequate amounts of epiphyte-free N. bailcyi have been grown for meta- morphic and early juvenile stages. Finally, the technique for handling metamorphic stages has been simplified with the use of disposable 50-ml conical centrifuge tubes. By using suf- ficient algae to cover the base of the tube, sinking animals are brought into increased contact with the substrate on which metamorphosis takes place. The combined effects of these three approaches have simplified and improved the mass culture of Aplysia californica.
This work was funded by a grant from the Klingenstein Foundation.
Motor fields of pharvnycal inotonenrons in Navanax, an opisthobranch mollusc. MITCHELL S. CAPPELL, DAVID C. SPRAY, DAVID H. HALL, ABRAHAM J.
SUSSWEIN, AND MlCIIAEL V. L. BENNETT.
Three orthogonal muscle groups comprise the Xtrraua.r pharynx : radials which run through the pharyngeal thickness and mediate expansion, and superficial circumferentials and longi-
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 361
tudinals which cause constriction and shortening. Stimulation of identified neurons in the huccal ganglion cause these pharyngeal movements. This work provides evidence that these neurons are true motoneurons, with specific and reproducible motor fields. Evidence indicat- ing that these cells directly innervate muscle includes: constant latency EMGs are evoked by intrasomatic stimulation at moderately high frequencies, antidromic impulses are evoked by electrically stimulating the appropriate muscle area, and a peripheral nerve net is apparently absent.
Circumferential and longitudinal muscles consist of a superficial gridwork of a very con- sistent number of discrete bands. Localizability and reproducibility of motor fields was quantitatively determined with respect to these pharyngeal landmarks.
Motoneuron fields were identified by : movements following intrasomatic stimulation, area producing 1-1 EMGs with intrasomatic impulses, areas yielding antidromic impulses at low threshold, and axonal course through nerves innervating specific areas. Generally, these methods gave mutually consistent results.
To date fields of 10 motoneurons have been characterized. These fields show little vari- ability. Four circumferential motoneurons, two per side, innervate fused circumferential bands located at the anterior of the pharynx. Of the two per side, one innervates ipsilaterally, the other bilaterally. Another left circumferential motoneuron bilaterally innervates the fused bands located posteriorly. Both giant ( G) expansion motoneurons innervate radial muscles of the entire pharynx. The ventral left medium-sized (M) cell innervates the anterior dorsal pharynx bilaterally from about circumferential band 25 to 31. An adjacent left expansion motoneuron innervates the more posterior ventral and dorsal pharynx ipsilaterally. A small ( S ) left expansion motoneuron has a bilateral field comprising about the anterior half of the M cell. About another dozen observed fields are not yet adequately established. These include longitudinal motoneurons, none of which have been discovered previously. These data provide a further step towards understanding the neural control of feeding in Narana.i-.
Mitchell Cappell is supported in part by NIH training grant no. 5T 32 GM 7288.
Ionophore induced sodium loading of nerve terminals: a model for long term facili- tation of transmitter release. MILTON P. CHARLTON, CHARLIE S. THOMPSON, AND HAROLD L. ATWOOD.
Accumulation of sodium ions caused by sodium pump inhibitors, metabolic poisons, repeti- tive stimulation or direct injection increases spontaneous and evoked transmitter release at synapses. We induced sodium loading in nerve terminals by application of the sodium iono- phore, Monensin. In the stretcher muscle of the spider crab (Lihinia), application of 6 MM Monensin caused a 2- to 8-fold increase in the amplitude of postsynaptic potentials ( psp ) evoked when the excitor motorneuron was stimulated. Recovery following washout of Monensin took up to 50 min. A second dose gave a smaller response or none. The rate of increase of psp's following application of Monensin was greater in saline containing the normal concentra- tion of sodium than in saline in which two-thirds of the sodium had been replaced by sucrose. This is consistent with a sodium ionophore role for Monensin in this system. Fol- lowing application of Monensin in saline containing no calcium, normal saline (no Monensin) was reapplied and there was an immediate increase in psp amplitude as if Monensin had been working during its application in zero calcium saline. This and its miniscule calcium transporting ability militate against a calcium ionophore role of Monensin in our experiments. Extracellular focal recordings at synaptic sites showed that quantal content was increased by Monensin. Spontaneous transmitter release was increased by Monensin in crab, lobster and frog ( cutaneus pectoralis ) muscles ; in the latter there was a 20-fold increase in spontaneous release. No changes in membrane potential or input resistance of muscle fibers were seen with Monensin treatment. The amount and duration of long lasting potentiation produced by tetanic stimulation were similar to the effects produced by Monensin. The data support a role for sodium in long-term facilitation and demonstrate the usefulness of Monensin for loading sodium in small cells.
Supported by NRC, MDAC, Banting Research Foundation, NIH.
362 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Some biochemical properties oj an l\N A-inslructed DNA polymenise in developing sea urchin ( Lyiecliinus pictus) embryos. BEVERLY S. CHILTON, MARC R. LA r PER, AND SANTO V. NICOSIA.
The rate of protein synthesis in unfertilized (UF) sea urchin eggs increases dramatically during early cleavage. This has been attributed in increased oogenic mRNA availability and translational efficiency. The presence of an RNA-instructed DNA polymerase in UF eggs and early cleavage stage embryos suggests that the sea urchin may inherit a mechanism for the amplification of existing maternal mRNA's.
An RNA-instructed DNA polymerase was partially purified according to Slater and Slater (1972, Nature Nnv Biolotiy 237: 81-87) from UF eggs and early (2-16 cell) embryos. Using the synthetic template-primer (rA)n-(dT),,, incorporation of (3H)-dTTP (305 cpm/pM) by the polymerase from UF eggs and 16-cell embryos was maximal at 15° C. Incorporation kinetics were linear for 150 min. The effects of polymerase concentration on the rate of DNA synthesis resulted in similar sigmoidal curves for the enzyme from both UF eggs and 16-cell embryos. This suggests that the polymerase has a similar subunit structure and relation- ship to template-primer prior to and during embryogenesis.
Polymerase activity, expressed as pM ( :'H ) -dTTP/^g protein, was 0.17 and 0.53 for UF eggs ( n = 3 animals/determination ) , and averaged 0.64 for two- and four-cell embryos. Specific activity (mean±s.e. ) was not significantly different for UF eggs (0.45 ±0.08) compared to 16-cell embryos (0.55 ±0.14). Thus RNA-dependent DNA polymerase is detectable throughout early development. However, when expressed on a per cell basis, specific activity decreases during this time. The partially purified polymerase from 16-cell embryos banded at a sucrose buoyant density of 1.15 to 1.16 g/ml, the same density as the particulate polymerase isolated from human tumors. This implies a viral association for the sea urchin RNA-dependent DNA polymerase which may play a role in early development.
Supported by USPHS HD-06274-Sub 4.
Hydrogen peroxide release from sea urchin e<j<js during fertilisation: importance in the block to pol\'spenn\<. MICHAEL COBURN, HERBERT SCHUEL, AND WALTER TROLL.
Polyspermy results in abnormal cleavage and halting of development by the blastula stage in sea urchins. For successful fertilization polyspermy must be avoided. We noted a novel block to polyspermy employed by Arbucin pitiictitl<it<i: the release of hydrogen peroxide after fertilization. We suggest that this hydrogen peroxide inactivated other sperm after entry of the first, ensuring proper development.
We have demonstrated the crucial role of hydrogen peroxide release in preventing poly- spermy in Arhacia by causing 100';; polyspermy through the addition of catalase to the sea water in which eggs were fertilized. Catalase is an enzyme of high specificity for the decom- position of H-Ou to water and oxygen. An early finding was confirmed by showing that sperm are inactivated by minute concentrations of H^O-, and that this action was blocked by catalase. We have further shown that catalase produces polyspermy within the first minute after fertilization, when present at the time of the addition of sperm to eggs.
Protease inhibitors such as soybean trypsin inhibitor (SBTI) have been shown to cause polyspermy in Arbuciu. This and other protease inhibitors also act to prevent oxygen uptake and H2Oa production in human white blood cells. We now note that SBTI prevented the release of H2Oa from fertilized Arbacia eggs, similar to its action in white cells. Thus, the cause of polyspermy produced by protease inhibitors could be the inhibition of H«O2 release, rather than the prevention of normal raising of the protective fertilization envelope as was suggested previously. These observations reinforce the relationship between proteases and H^O- produc- tion in biological systems.
We acknowledge grant support from NIH-CA-16060 to Walter Troll and from the New York University School of Medicine Honors Program to Michael Coburn.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 363
Association oj centriole-like structures with the marginal band oj a inollitscan red cell. \YILLI.\M D. COHEN AND IRIS NEMHAUSER.
Marginal bands (MBs) are continuous circumferential bundles of microtubules found classically in all non-mammalian vertebrate erythrocytes and in platelets. MBs are believed to function in cellular morphogenesis, but relatively little is known about their formation.
We have been engaged in a continuing survey of blood cells of local (MBL) invertebrates for possible presence of AIBs, with the objectives of establishing the phylogenetic distribution of the MB system identifying useful cell types for experimental work on MB structure and physiology. Previously we have established that MBs are present in sipunculan red cells, Limulus amebocytes, and lobster and crab coelomycetes. We report here the presence of MBs in the hemoglobin-containing red cells of the arks Auudara ovalis and Anadara transrcrsa. A striking feature of these molluscan red cell MBs is the obvious presence, on or adjacent to each MB, of a pair of centriole-like structures which we have tentatively named "perioles". The living red cells (A. transvcrsn) are highly flattened and generally slightly elliptical (10-15 ^m long axis). In cells lysed with Triton X-100 under microtubule-stabilizing conditions, the nucleus, cytoplasmic particles, and MB with associated perioles are directly observable in phase contrast (oil immersion). The MBs are usually elliptical, with the periole pair appearing at various points on the ellipse but most frequently near one end. The two perioles are normally adjacent; however, in some cases a gap (0.25-1.5 /urn ) occurs between them. In fixed, uranyl acetate-stained whole mounts of lysed cells ( TEM ) MB microtubules and the densely-staining perioles are readily visible. A trans-MB network of fine filaments is also present. The perioles appear to be within or on the MB, with extra-MB microtubules often 'focussing' upon them in pole-like fashion. Their centriole-like features as observed in whole mounts include (a) close pairing, sometimes with approximately right angle positioning, (b) cylindrical appearance with one closed end, and (c) size, approximately 0.25X0.17 ^m. Wre suggest that the perioles may be MB organizing centers, functionally analogous to centrioles and basal bodies.
Supported by CUNY PSC-BHE grants 12260 and 13051, and by NIH grant HL 20902 from the National Heart, Lung, and Blood Institute.
Dependence of discrete wai'c frequency on pH in Limulus ventral photoreceptors. D. \YESLEV CORSON AND ALAN FEIN.
Discrete waves in the ventral photoreceptors of Limulus are thought to result from either direct or spontaneous activation of visual pigment molecules by the absorption of photon energy or thermal kinetic energy. We have found that the average frequency of occur- rence of spontaneous discrete waves in darkness rises 15-fold over a 2 pH unit range (6.0-8.0) of artificial sea water that bathes the receptors. Over this same range, the incre- ment of discrete wave frequency added by a steady dim light remains essentially constant.
Artificial sea water was buffered with 10 HIM each of Pipes (pK = 6.8), Hepes ( pK = 7.55) and Tris (pK = 8.30) and titrated to pH values in the range from 6.0 to 8.0 in half pH unit increments. The frequency of discrete waves was determined by counting the number of discrete waves in five 40-sec intervals at each experimental pH after a 10 min exposure. A dim steady light was adjusted to give an increment of approximately 1 discrete wave per second ( 0.99 ± 0.66 dw/sec ; x ± s.d. ; n = 9 ) over the spontaneous frequency ( 0.89 ± 0.42 dw/sec, n = 9 ) at pH 7.0. Dim 20-msec test flashes showed that changes in the average discrete wave frequency were not the result of changes in the average discrete wave amplitude, and data were taken only from cells in which the spontaneous discrete wave frequency returned to its control level following each exposure.
In seven of nine cells, the spontaneous discrete wave frequency rose within the pH range of 6.0 (0.16 ±0.1 7 dw/sec, 11 = 4) to 8.0 (2.41 ±0.60 dw/sec, n = 4), while in the remaining two cells there was no appreciable rise in frequency from pH 7.0 (0.17 ±0.06 dw/sec) to pH 8.0 (0.17 ±0.10 dw/sec). No appreciable rise in the light-induced frequency increment occurred in either the seven responsive cells (1.30 ±0.33 dw/sec at pH 6.0 rs. 0.79 ± 0.76 dw/ sec at pH 8.0) or the two unresponsive ones (0.43 ±0.14 dw/sec at pH 7.0 vs. 0.48 ± 0.26 dw/sec. at pH 8.0).
Supported by a grant from N.E.I., N.I.H., and the Rowland Foundation.
364 PAl'KRS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Production and metabolism of steroids in Homarus americanus. ERNEST F. COUCH, C. A. ADEJUWON, AND S. S. KOIDE.
We have reported that the niandihular gland of the lobster contains progesterone as determined by radioiinmunoassay (Couch r/ <//. J-iiol. null. 155: 433, 1978). To learn the fate i)f the progesterone, tritium-labeled progesterone ( 1 < 10" cpm ) was injected into the abdominal muscle of an adult female lobster. After 1.5 hr various tissues (blood, urine, heart, green gland, hind gut, gills, mandibular gland, hepatopancreas, ovary, nerve cord, and muscle) were excised, homogenized, and extracted with petroleum ether and ethyl ether. The radioactivity in tin- extracts was measured. The highest incorporation of progesterone occured in the mandibular gland (7500 cpm/mg wet wt.) and in the green gland (2750 cpm/mg wet wt.).
To determine the capability of lobster tissues to transform steroids, the mandibular gland. hepatopancreas, and ovary were incubated with :'H-pregnenolone for 6 hr ;';; 1'ilro at room temperature. The reaction mixture was extracted with petroleum ether and ethyl ether. The extracts were pooled and dried. The residue was dissolved in chloroform : methanol (1:1) and spotted on silica gel TLC plates. The plates were developed in a mixture of petroleum ether : ethanol : water ( 100 : 80 : 20 by volume ) . The spots were visualized under UV light. By this method pregnenolone and progesterone were not separated. However, in the extract of the hepatopancreas, a unique radiolabeled metabolite (Rt = 0.71) was detected which was distinctly different from progesterone, testosterone, estrone, estradiol, and estriol. This UV-absorbing compound was also found in untreated hepatopancreas, but it was absent in ovary and mandibular gland. These results indicate that pregnenolone is transformed into a metabolite in the hepatopancreas under physiological conditions.
To establish a relationship between mandibular gland function and reproduction, ovaries from lobsters at varying stages of oocyte development were excised and fixed for morphological studies. Previous studies had shown the presence of small amounts of progesterone (about 0.2 ng/g of wet wt.) in immature ovaries but not in mature ovaries. In the present study. we observed that the size of the mandibular gland appears to vary during ovarian develop- ment ; i.e.. the gland was small (about 12 mg/100 g of body wt. ) during the dormant stage of egg production. It increased in size (about 25 mg/100 g of body wt. ) during the time of early oocyte growth and development and receded to about 9 to 11 mg/100 g of body weight as maturation of oocytes occurred. These results suggest that mandibular gland function might be associated with ovarian development.
Supported by a grant from the George Hecht Fund.
Response specificity folloiciin/ behavioral training in the nudibranch inollnsk Hermissenda crassicornis. TERRY CROW AND NANCY OFFENBACH.
Photopositive responses of the nudibranch mollusk Hermissenda crassicornis exhibit long- term behavioral plasticity that has been examined at the cellular level. The behavior is modified by an automated training procedure that stimulates the eyes and statocysts using light paired with rotation while the animals are confined to the ends of glass tubes filled with sea water. Random control procedures have demonstrated that the long-term behavioral modification is dependent on pairing light and rotation. Response latencies to move into an illuminated area are significantly increased for animals that received light paired with rotation. The increased response latencies following training are not the result of a decrease in general activity.. When the same animals were tested in both light and dark after training we found that only response latencies to enter the illuminated area were increased (P 0.01). Latencies to move to the end of the tubes in the dark wre not significantly changed. Significant light-dark differences were not found for random control or normal untrained animals. We next examined the response latencies of animals to initiate movement towards the illuminated area at the opposite end of the tube. We found a linear relationship between the start latencies (initiation of movement toward the light) and the latencies to enter light for normal untrained animals ( r = 0.93, P<0.01). Start latencies for trained animals were correlated with the latencies to enter light before (r = 0.97) and after training (r = 0.99). We found significant increases in start latencies following training for the light paired with rotation group (P < 0.005) while the start latencies did not change for random control groups.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 365
The increase in the response latencies to enter light after training can he accounted for by the significant increase in start latencies for trained animals. This specific modified behavioral response to light following training will be examined at the cellular level in intact and semi- intact preparations.
In vitro transcription in nuclei fro/n . \rhacia punctulata. LISA M. DAVIS.
A technique for isolating nuclei which are capable of incorporating 3H-UTP into TCA precipitatable counts has been devised. This incorporation is inhibited by the presence of a-amanitin, it is dependent upon the presence of all four ribonucleotide triphosphates, and the reaction yields a product which is Ribonuclease A sensitive. The isolation procedure yields nuclei which are not aggregated and which are free of cytoplasmic contamination. Conditions for maximizing the reaction, with regards to the divalent metal ions and the salt concentrations, were also investigated. The procedure for isolating transcriptionally active nuclei called for the presence of MgCk NP-40, and EGTA, in the homogenization buffer. The former two ingredients were important for obtaining clean, non-aggregated nuclei, and the latter for inhibiting nucleases. Nuclei from embryonic stages other than gastrula were not assayed for this transcriptional activity. However, the observation was made that nuclei isolated from embryos grown at 25° C were only 25% as active as those from embryos grown at 18° C. Following isolation, the nuclei were either assayed immediately, or stored at —20° C, with no loss of activity for at least 2 weeks.
The a-amanitin profile of this activity was determined ; the reaction was inhibited 50% by both high and low levels of the toxin ( 100 and 5 /-eg/ml, respectively). Whether this result is due to the absence of any polymerase III activity, or to the possibility that the enzyme is sensitive to higher levels of amanitin, was not determined at the time of this writing. However. attempts were made to optimize for activity by polymerase III, by altering the Mg2* concen- trations and using Mir* and KC1 concentrations which typically stimulate this activity. It was determined that the time course of the reaction, as well as the amanitin profiles, were identical with either 1 HIM, 5 IBM, or 10 HIM MgLV. (The assay mix also contained 1 HIM Mir* and 150 nut KC1).
This research was supported in part by training grant no. 32-GM-07784 from P.H.S.
Chemical search image: prey e.rposiire improves selective chemical detection b\ a predator (Homarus americanus). CHARLES DERBY AND JELLE ATE MA.
Considered as a species, lobsters (Honmnis n/ncricauiis ) are omnivores ; individuals, how- ever, can be selective in prey preferences. Experience with prey can alter prey preferences by various methods : learning to search for prey in particular areas ; learning to handle prey more efficiently ; and learning to detect prey. The latter case, where encounters with prey improve the predator's detection of that prey, is called search image formation. This report describes chemical search image formation in lobsters.
Behavioral responses of ten lobsters to prey body odors were observed in 2.5-m-long troughs. Body-odor stimuli of two prey species, Mytilus cditlis ( blue mussel ) and Modiolus modiolus (horse mussel), were prepared by placing five mussels in sea water for a fixed amount of time. For each stimulus, lobsters were observed for 10 min before and 10 min after stimulus introduction. Movements of appendages associated with alerting and searching ( antennules, antennae, maxillipeds, claws, walking legs) were quantified, and differences between pre- and post-introduction periods were determined by Chi-square analyses. Detection thresholds for the two stimuli were determined by randomly presenting seawater controls and log-dilutions of both odors. Effects of feeding experience on thresholds were examined by initially feeding starfish and fish to all ten lobsters. After determining thresholds, lobsters were placed in a tank with either live Mytilus or live Modiolus. After 30 days of this exposure, thresholds were again determined. Changes in responses to experienced prey odors were compared relative to changes in response to nonexperienced prey odors. Seven lobsters became relatively more sensitive to odors of experienced prey ; two showed no change ; one became relatively less sensitive. Wilcoxon matched-pairs signed-rank test demonstrates significant at P < 0.05. This
366 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
experiment therefore demonstrates that chemical detection of prey improves as a function of experience with that prey ; this supports the idea that lobsters can form chemical search images.
Effects oj unilateral antennule ablutions on food odor orientation in Homarus americanus. DANA DEVINE AND JELLE ATEMA.
Recent electrophysiological investigations of the lobster antennule have suggested that the lobster can orient to odors by gradient discrimination through simultaneous comparison of bilateral receptor input from the two lateral flagella of the biramous antennule. Behavioral evidence supporting this hypothesis is anecdotal and scant, prompting the present study.
Lobsters were placed individually in 600-liter aquaria, where they occupied a shelter in the middle of the back wall. Two air life water flows were directed toward the center of the tank from the side walls, running continuously. For testing, a standard food odor was injected in either flow. The lobsters' behavior was observed and timed in response to this chemical stimulus. Three sets of data were obtained per lobster in the following order : untreated, after ablation of the right lateral flagellum, and after subsequent ablation of the ipsilateral medial flagellum. The following results were obtained. First, the initial direction choice during searching changed dramatically from 100% correct to roughly random upon ablation of one lateral flagellum. This illustrates the importance of lateral flagellar input for direction choice at the start of a search. Secondly, the paths followed by ablated animals when searching for the source of the stimulus were significantly more random than those of normal lobsters. Thus, lobsters missing one lateral flagellum could no longer efficiently follow the changing stimulus concentrations within the odor cloud. However, circus movements were never seen in ablated animals. Thirdly, ablation did not impair the lobsters' ability to detect the presence of a stimulus : the alert time did not increase. After ablation of the ipsilateral medial flagellum, there was a significant decrease in alert time which has no obvious explanation. These data give behavioral support to the hypothesis that the receptor input from both lateral flagella (perhaps from the aesthetasc hairs themselves) are used for efficient orientation to a food odor.
./// the poly(A) ( + ~)mRNA sequence complexity also occurs in poly(A)(—}- ni l\ X.I in sea urchin embryos. ROGER DUNCAN AND TOM HUMPHREYS.
Polysomal RNA was purified from Lytcchiints pictns hatched blastulae, and fractionated into poly (A) ( + ) and poly (A) ( — )RNA by multiple passages through an oligo d( T ) -cellulose column. A cDNA probe was synthesized using the poly(A) (+)mRNA as the template and AMV reverse transcriptase. The extent to which the poly (A) ( + )mRNA sequence com- plexity also occurs in poly ( A ) ( — )niRNA was determined by hybridizing the cDNA to both RNA populations. The complete hybridization reaction of the cDNA to its template poly- (A)( + )mRNA spans about 3 decades on a log R0t axis, indicating that a range of mRNA abundancies exists, and terminates with greater than 80% of the cDNA in hybrid form. The poly(A) ( — )RNA also hybridizes greater than 80% of the cDNA, indicating that all the poly(A) (+)mRNA sequence complexity is also present in mRNA molecules containing little or no poly (A). The cDNA-poly(A) ( — )RNA hydrization kinetics show that the poly- (A)( — )mRNA molecules which are driving the reaction are represented in the polysomes at about the same frequency as the poly(A) ( + )mRNAs. The poly(A) and oligo(A) length and content of both RNA preparations was examined by digestion with RNase followed by gel electrophoresis. Comparison of the relative amount of poly(A) in the poly(A)( + ) and poly- (A) ( — )RNA preparations showed that greater than 90% of the mRNA molecules driving the cDNA-poly(A) ( — )RNA hybridization reaction lack a poly (A) segment longer than 20 nucleo- tides. We conclude that a specific set of mRNA sequences which are exclusively polyadenylated does not exist in sea urchin embryo mRNA, confirming a recent in vitro translation comparison.
Supported by NIH training grant #TG HD 07098 and a grant from the National Institute of Child Health and Human Development.
PAPERS PKKSKXTED AT AIARIXK BIOLOGICAL LABORATORY 367
Studies on actin mRNA-covuplimentary genomic DXA sequences in the sea urchin, S. purpuratus. DAVID S. DURICA, JEFFERY A. SCHLOSS, AND \YILLIAM R. GRAIN.
An actin gene-containing plaMiiid recently isolated from Drosophila melanogaster ( K. Fyrberg and N. Davidson, personal communication) was tested for interspecific sequence homol- ogy to sea urchin (5". purpuratus} DXA. The actin coding region of the cloned Drosophila DNA cross-reacted significantly in Southern transfer experiments using several restriction enzymes, hybridizing to a limited number of discrete bands (4-8 depending on the enzyme). Solu- tion hybridization followed by HAP chromatography also indicated significant cross-reaction, with reassociation kinetics suggesting a homogeneous component repeated less than 10 copies per genome. Sequences which specifically cross-reacted with the Drosophila probe were cloned into the bacterial plasmid vector pBR322. These clones fall into two size classes which cor- respond to genomic Hind III fragments of 3.6 and 7.0 kb. Both clones tested positive when assayed for the ability to selectively capture actin mRNA from a total mRNA population. Both plasmids are presently being characterized by restriction and heteroduplex mapping to determine regions of homology between the two sea urchin plasmids and the Drosophila clone.
D.S.D. is a fellow- of the Muscular Dystrophy Association. This research was also supported by NIH Training Grant #TG-HD07098. '
Studies of the tmnslational regulation oj histone synthesis in Arbacia punctulata. CHRISTOPHER EARL AND TIM HUNT.
Histone synthesis in the somatic and embryonic cells of a variety of organisms is coupled to DNA replication. Control is exerted at the levels of transcription and translation, but the molecular basis of the regulatory mechanisms is unknown. We decided to test the hypothesis that histones bind specifically to histone mRNAs and block their translation. We prepared histones and histone mRNA from hatching blastulae of A. punctulata and added both to reticulocyte lysates. We found that histones did indeed inhibit protein synthesis, but that this inhibition was not specific for histone mRNA.
Incubation of A. punctulata embryos in 1 HIM hydroxyurea inhibits both DNA synthesis and histone synthesis within 30 min, as measured by incorporation of radiolabeled thymidine and methionine or lysine, respectively. We therefore examined the stability of translatable histone mRNA in vitro. We prepared cytoplasmic extracts from four hour blastulae, untreated or treated for 90 min with 1 mM hydroxyurea. After homogenization and cenrifugation, the postmitochondrial supernatants were equilibrated with reticulocyte salts on a Sephadex G-50 column. We mixed 3 volumes extract with 1 volume reticulocyte lysate to boost in vitro translation. Histone synthesis was significantly reduced in the hydroxyurea extracts after treatments longer than 1 hr, but it is not clear whether this result is due to active degradation of the histone messages or to a halt in their transcription and an unaltered rate of nonspecific turnover.
Supported by NIH training grant TG-HD07098.
Temperature-dependent Timing of mitosis and cleavage In Lytechinus variegatus. ANDREW EISEN AND SHINYA INOUE.
At a constant temperature individual embryos of the sea urchin Lytechinus vancijatus divide at intervals with precision far greater than apparent in a batch of sibling embryos. A series of events recur with the exact same interval for the first four divisions. Taking the completion of cleavage as unity, the events occur at the following fractions of the cleavage interval: nuclear envelope breakdown (0.45), spindle formation (0.50), metaphase (0.06), anaphase-A onset (0.71), anaphase-B onset (0.79), cleavage onset (0.85), cleavage 50% com- pleted (0.93). The fractions remain constant over the full range of temperatures which permit development (17-33° C). Thus a plot of division events vs. time yields a straight line for a single embryo at a given temperature. All lines intercept the abscissa just as the "nuclear streak" appears. Between 17° C and 27° C the slopes increase with temperature with a Qio
368 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
of r<;. 2. While the division schedule suggests a continuously operating temperature-dependent "clock", such is not the case. When the temperature is raised from 17 to 27° C, the schedule immediately shifts to the new rate. However, when the temperature is dropped during the second division from 27 to 17° C, the rate does not immediately shift. Rather, the shift is variously delayed. The delay can be as great as 23 min when the embryo is chilled very early after cleavage. Before metaphase the delay progressively decreases as though cells enter the new schedule at around metaphase regardless of the stage at chilling. Thus portions of the cell-division cycle (ca. 0.0-0.65) appear insensitive to temperature drops (from 27-17° C) and their rates are pre-determined by the temperature at an earlier time.
Supported by NIH GM 23475-14, NSF PCM 76-81451, and NIH MSTP GM 07170-05.
Population density, sl.cc distribution and home range of tlie American Eel (Anguilla rostrata) in the Great Sippewissett salt niarsli. TIM FORD AND EVAN MERCER.
The American Eel is increasing in commercial importance, yet little demographic or be- havorial information is available for eels in coastal and inland habitats. Local eel populations were studied by trap-recapture methods along 600 m of tidal creek in Great Sippewissett marsh, Falmouth, Massachusetts, during summer 1979. The creek system varied in bottom topography and sediment type and encompassed both high and low marsh. Conical minnow traps (Gee-Gee Co.) were used which captured eels larger than 15 cm (standard length) ; the largest eel trapped was 62.5 cm. Eels were fin-clipped, using a site specific code. Additionally, animals were measured, and body blemishes noted. Intersection of body length, blemishes or marks, and fin notches allowed individual eels to be distinguished. A total of 300 eels were marked and 67 individuals recaptured ; some of these several times. Capture-recapture data was analysed with Jolly's model, which estimated a population of 350 eels in the 600-m creek system. Of this population small eels (less than 30 cm) dominated captures in the narrow, soft-bottomed creeks of the high marsh, and larger eels (greater than 30 cm) in the broader, soft mud to sandy bottomed creeks of the low marsh. Distances travelled by recaptured eels gave an indication of home range. Two eels dispersed over 200 m; the majority dispersed over much less distance (average 38 ± 54 m), which suggests a small home range. Little movement was observed for eels greater than 40 cm and the largest eels were commonly recaptured several times at specific sites. The results indicate large eels may establish territories in low marsh, restricting small eels to high marsh creeks.
Studies by fluorescence of protochlorophyll(ide') and its photo trans formation in dark-grown Euglena gracilis rar. bacillaris. MARCIA A. FREY, RANDALL S. ALBERTE, AND JEROME A. SCHIFF.
Intact cells show a fluorescence emission peak at about 635 to 638 nm from excitation of protochlorophyll(ide) (Pchl(ide)) 634; no emission peak is seen in the 650 to 660 nm region. Emission peaks at 712 and 730 nm, also due to Pchl(ide) excitation, may represent aggregates. Emission at 642 nm appears to be due to protophenophytin/phorbide (Ppheo) based on excitation spectra. On exposure to light, the 638 nm peak decreases and chlorophyll (Chi) and, per- haps, pheophytin a ( Pheo a ) emission appears, hut the 712 and 730 nm emissions do not change. Acetone extracts transferred to diethyl ether show emission bands at : 630 nm (Pchl(ide) : excitation maxima at 435, 535, and 571 nm) ; 590 nm (Mg protoporphyrin IX (monomethyl ester?) : excitation maxima at 417 and 551 nm) ; 512 nm (phytofluene: excitation maxima at 333, 348, and 368 nm) ; and in the 642 nm region (Ppheo: excitation maxima at 417, 525, 565, 585, and 638 nm). Acidification and washing of the ether yields more Ppheo and less Pchl(ide) and phytofluene. Extracts of light exposed cells show similar spectra but Pchl(ide) is diminished and an emission at 673 nm appears due to a mixture of Chi and Pheo a based on excitation spectra. On acidification, this emission decreases and appears to be due solely to Pheo a. As found previously by absorption, Pchl(ide) 634 is present and phototransformable to Chi but Pchl(ide) 650 is undetectable. Ppheo is detected in intact dark-grown cells and is probably not an extraction artifact; Ppheo to Pchl(ide) ratios of about 10:1 were found previously by absorption spectroscopy of extracts. Since Pheo a is
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 369
found after light exposure, one possibility of many is a photoconversion of Ppheo to Pheo a. The detection of Mg protoporphyrin IX suggests a control point just after this compound in the biosynthetic pathway of Chi. Fluorescence has permitted the detection of phytofluene, a precursor of carotenoids, in these cells.
Supported by the Experimental Marine Botany Program and grants from the National Science Foundation (PCM 78-10535; BMS 7300987A01 ; PCM 79-06638) from the National Institutes of Health (GM 2394-01 A2; GM 14595).
Electron spin resonance studies oj protein-methylglyoxal completes and model systems. PETER R. C. GASCOYNE.
The interaction of methylglyoxal with bovine serum albumin, collagen, lysozyme and casein has been studied using electron spin resonance spectroscopy. A radical signal centered at g = 2.005 accompanied the reaction of methylglyoxal with the proteins studied and this radical was found to be stable to drying. If the lysine residues in the protein were first blocked by dimethylation, however, no such radicals could be induced by methylglyoxal treatment, indicating that the lysine sidechains of the protein were involved in the radical formation. This observation of the primary importance of lysine is consistent with the spectroscopic findings of Pethig and McLaughlin (1979, Biol. Bull., 157: 388).
In order to further understand the interaction between methylglyoxal and the protein lysine sidechains, a simple model, methylamine, has been investigated using a computer con- trolled stop-flow technique. In this way the development of the various radical species accom- panying the mixing of molar methylglyoxal and methylamine solutions has been followed from 0.01-100 sec. Under a nitrogen atmosphere a singlet centered at g = 2.005 appeared in about 2 seconds followed by the development in a few tens of seconds of a septet with 8.5 Gauss splitting, and later by other radicals. The addition of sodium ascorbate greatly enhanced and accelerated the formation of the observed radicals and the presence of at least one additional radical species was detected after 20 sec. This observation of the importance of ascorbate is in agreement with the suggestion of Szent-Gyorgyi (1979, Biol. Bull.. 157: 398) that the ascor- bate molecule tends to facilitate the process of molecular charge transfer in methylglyoxal-amine systems.
Is there titbitlin in spirochaetesf SHARON GREENBERG.
The theory of the exogenous origin of microtubules in eucaryotic cells is based on the hypothesis that an early symbiotic relationship between primitive procaryotes, specifically spirochaetes, and eucaryotic cells, led to the evolution of motile organelles of existing eucaryotic organisms. This theory predicts the existence of tubulin-like proteins in spirochaetes.
The presence of tubulin in spirochaetes was investigated using the free-living, facultatively aerobic, halophilic spirochaete, Spirochacta halophila. Microtubule-like cytoplasmic fibrils have previously been demonstrated in this bacterium by electron microscopy. Cultures of S. halophila were analysed for tubulin-like proteins by comparison of migration patterns on two polyacrylamide gel systems of whole-cell spirochaete protein fractions with an axoneme- derived tubulin standard prepared from sea urchin sperm. On 7.5% Laemmli slab and tube gels, there were protein bands in the spirochaete sample which apparently comigrated with a- and /3-tubulin in the axoneme standard. On Fairbanks gels, the axoneme-derived tubulin standard produced the predicted single band at 55,000 MW. The putative spirochaete tubulin, however, ran as multiple bands ; in some cases lower than the axoneme tubulin standard.
It would seem that on the basis of the Laemmli gel system there are proteins in the spirochaete sample with mobilities similar to that of authentic tubulins. When the same sample is run on Fairbanks gels, however, the apparent homology between the proteins breaks down. A conclusive statement cannot be made at this time, based on the results obtained from polyacrylamide gel electrophoresis, concerning the presence of tubulin-like proteins in the spirochaete 5\ halophila.
This research supported in part by training grant T23-GM-07784 from P.H.S.
370 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Structural changes associated with increased coupling resistance in the septate ax on of the crayfish. ROBERT B. HANNA, GEORGE D. PAPPAS, AND MICHAEL V. L. BENNETT.
Increased coupling resistance was brought about by mechanical injury to identified segments of the lateral septate axon in the ventral nerve cord of the crayfish. The axons were fixed at specific time intervals following injury and prepared for electron microscopy. Both freeze- fracture replicas and thin sections were utilized. The intramembranous particles of the control electrotonic junctions are 12.5 nm in width, have a center-to-center spacing of 20 nm, and are organized in a loose hexagonal array. Following injury, the intramembranous particles of the identified high resistance electrotonic junctions were found to lose their organization and become more widely spaced. In some experiments the particle spacing and organization remained unchanged for as long as 45 min following injury. Thin sections cor- roborated the findings from freeze-fracture replicas.
Following injury there is an increased density of the axoplasm and an increased number of cellular organelles in the uninjured axon proximal to the junction. Concomitant with the increased cytoplasmic density, there is a mobilization of the Schwann cells which become interposed between the injured and uninjured segments (Pappas, Asada, and Bennett, 1971, /. Cell Bio!., 49: 173-188). The mobilized Schwann cells develop increased extent of anastomosing tubular networks, which is continuous with their plasma membrane. Eventually the junctional complex is apparently internalized in the axoplasm of the uninjured axon.
Absorption properties of Platymonas sp. Rev 2 containing a high proportion of chlorophyll b. B. A. HAYHOME, JEROME A. SCHIFF, ROBERT R. L. GUILLARD, AND RANDALL S. ALBERTE.
Platymonas sp. Rey 2, a marine green prasinophyte alga, recently obtained in axenic culture (from Venezuela), has 55% of its total chlorophyll (a + b) in the form of chlorophyll b as determined by absorption spectra in i'ii'0 and in vitro, by chromatographic separation of the pigments followed by spectrophotometric determination and by fluorescence excitation spectra of the whole cells. Absorption spectra of the intact cells show a greatly increased absorption in the chlorophyll b region (653 nm) and a sharper drop on the long wavelength side of the chlorophyll a peak compared with organisms having the usual levels of chlorophyll b (e.g., Chlorclla pyrcnoidosa Emerson strain) or low levels (Euglcna gracilis var. bacillaris). The total absorption cross section in the red region of the spectrum, however, is very similar for all three types of organism. This suggests that some antenna chlorophyll a of system 2 of photosynthesis is replaced by increased chlorophyll b which maintains the effective cross section for absorption but moves the absorption region to somewhat shorter wavelengths in the red in these organisms ; this may be advantageous in competition for light with organisms having normal chlorophyll b levels. Plating efficiencies of 100% have been achieved for this organism and the conditions for treatment with chemical mutagens have been worked out ; chlorophyll-b-deficient mutants would be of help in learning more about the biosynthesis of chlorophyll b and its role in photosynthesis.
Supported by the Experimental Marine Botany Program and grants from the National Science Foundation (PCM78-10535 ; BMS73-00987 A01; OCE-7808858; PCM 7906638) and from the National Institutes of Health (GM 2394-01 A2; GM 14595); B.A.H. was supported by the Steps Towards Independence Program of M.B.L.
The major protein in Spisula sperm nuclei is a protainine. Louis HERLANDS AND JUAN Ausio.
When SDS gels are used to analyze the 0.25 x HC1 extractable proteins isolated from Spisula solidissiina sperm nuclei, a histone pattern similar to that of somatic cells is found. But in comparison with Spisula embryos there are few other proteins present, presumably because of the lack of contaminating cytoplasm and ribosomes in the sperm system. This
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observation has led others to believe that Spisitla is a typical histone-type sperm that would be a good model system for studying both nucleosome structure and the fate of sperm nuclear proteins after fertilization. However, when the same proteins are run on HOAc/urea gels, we see a new band which corresponds to an unusual protamine-like protein which is insoluble in SDS. More than 80% of its amino acid residues are Lys, Arg, Ser, or Ala. This new band is the major component; only small amounts of histones are present. These histones most likely come from immature sperm since first, there are several acetylated forms of H4; and secondly, gel scans indicate that the amount of histone in relation to protamine is variable. Digestion of Spisnla sperm nuclei with micrococcal nuclease indicates that Spisula sperm chromatin is primarily nut in the nucleosome configuration. Only small amounts of 140 BP protected DNA fragments are generated at early digestion times. The amount of this particle does not increase as digestion proceeds. The origin of this particle may either be immature sperm or perhaps a subset of chromatin in the mature sperm that is still organized with histones. Even though Spisula sperm contains predominantly protamine, an extremely stable PCA soluble limit, similar to somatic chromatins, is reached. But unlike chromatin, the DNA fragments are heterogeneous in size.
L. H. is a John Courtney Murray fellow, supported also by a grant in research from Sigma Xi. The research has been supported in part by grant #T32-GM-07784 from the PHS.
Light-harvesting pigment-protein complexes from brown algae and diatoms: implications jor the organization of the photosynthetic unit. D. L. GUSTAFSON, A. L. FRIEDMAN, M. S. RUDNICK, H. LYMAN, AND R. S. ALBERTE.
The pigment-protein complexes of three macrophytic brown algae, Lannnaria saccharina, Chorda fihtin and Pylaiella littoralis and the diatom Skclctoncina costatitin \vere examined with respect to the organization of the photosynthetic unit (PSU) by fluorescence emission and excitation analysis and polyacrylamide gel electrophoresis of membrane detergent extracts. The fluorescence characterization of whole thalli, chloroplast membrane fragments, detergent solubilized thylakoids and gel electrophoretic zones were used to determine the paths of energy transfer among the light-harvesting pigment of the PSU. Pigment composition, pigment- protein biochemistry and physiological data support two paths of energy transfer. One path involves the coupled transfer of excitation energy from chlorophyll (Chi) c to Chi a, while the other involves energy transfer from the carotenoid fucoxanthin to Chi a and not to Chi c. Polyacrylamide gel electrophoresis of SDS solubilized thylakoid membranes of these algae results in two major pigment-protein zones. The slowest migrating zone shows fluor- escence and absorption characteristics of a pigment-protein containing Chi a and Chi c with energy transfer from Chi c to Chi a. The other major zone shows spectral characteristics of a pigment-protein complex containing Chi a and fucoxanthin with energy transfer from fucoxanthin to Chi a. Physiological data obtained during nitrogen starvation of the diatom Skclctoncina demonstrates a specific loss in fucoxanthin and Chi a. Accompanying these pig- ment losses are decreases in PSU size and a reduction in photosynthetic rates. In contrast, supraoptimal nitrogen levels lead to a cellular increase in these pigments and the pigment- protein complexes. The increase in pigment-protein may represent a nitrogen storage mecha- nism for these cells, and as such implies an opportunistic strategy for their adaptation to fluctuating nutrient levels.
Research supported by the Experimental Marine Botany Program, National Science Foundation grants PCM 78-10535 and PCM 79-06638, and National Institutes of Health grant GM 2394-0 1A2.
Water relations and photosynthetic characteristics oj the tall and short ecoplienes oj Spartina alterniflora. ARTHUR M. B. HOGAN, DAVID MAUZERALL, AND RANDALL S. ALBERTE.
The tall ecophene of Spartinn ultcrniflora in Sippuvissett Marsh is inundated for the greatest part of the tidal cycle while the short ecophene is infrequently inundated. The present
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investigation sought to account for the morphological differences by examining the water relations, morphological features, protein content and photosynthetic characteristics of the two ecophenes. All parameters were replicated at least ten times and data was significant to at least the 95% level. It was found that the short form was more succulent (41% greater) and heavier (18% greater) than the tall form. Both forms had similar leaf widths. The relative water content of the short form was lowest at midday during a high tide cycle and hence the short form was more water stressed. At pre-dawn the short form recovered to hydration levels similar to the tall form. Protein content per unit leaf area was 34% higher and 20% higher on a dry weight basis in the short form, strongly suggesting that nitrogen is not limiting growth of this ecophene. Leaf chlorophyll (Chi) content is similar (2.9-3.2 X 10~4 mg/mm2) in the two forms when expressed per unit leaf area but 15% greater in the tall form when expressed on a dry weight basis. The Chi a : b ratios for both forms were 3.1 ±0.1. Photosynthetic rates of the short form measured by oxygen evolution were 26% greater when expressed on either a Chi or an area basis than the tall form. Furthermore the photosynthetic unit (PSU) size in the short form was about 40% smaller than in the tall form. Based on the differences in leaf thickness, the short form possessed about 30% more PSUs per unit area as a result of the greater amount of tissue per unit surface area. The greater numbers of PSUs per unit area fully explains the higher photosyntlietic rates per area in this form. The alterations in the packaging of the photosynthetic machinery probably represents an adaptive response to a more stressful environment of higher salinity and lower water availability. In addition, photosynthetically competent protoplasts were isolated in good yield from both ecophenes which will allow for further detailed examinations of these ecophenes. Research was supported by the Experimental Marine Botany Program and National Science Foundation grants PCM 79-06638 and PCM 78-10535.
Brush border calmodulin: A structural protein of the uiicrorillns core. CHRISTINE L. HOWE AND MARK S. MOOSEKER.
We are investigating the molecular basis for calcium regulation of microvillar motility in the brush border of intestinal epithelial cells. Results indicate that calmodulin is present in brush borders isolated from chicken, and that it is a major constituent of the microvillar filament bundle. Calmodulin is prepared by boiling suspensions of either Triton-treated brush borders or demembranated microvilli. No calmodulin is detected in the solubilized brush-border membrane fraction. The supernate derived from boiled microvilli contains one major polypeptide identified as calmodulin by first, comigration with brain calmodulin on SDS gels and on alka- line urea gels, where both proteins undergo a shift in mobility with Ca"+ present ; and secondly, 4-fold activation of phosphodiesterase in the presence but not absence of calcium. The molar ratio of calmodulin to actin is approximately 1 : 10 in the intact brush border and increases to 1 : 3 in the isolated microvillus, indicating that calmodulin is predominantly localized in this structure. Calmodulin remains tightly associated with the microvillus core in the presence or absence of calcium. Furthermore, calmodulin does not bind to muscle F-actin in vitro, indicating that the binding of calmodulin to the microvillus core may involve protein (s) other than actin. These results suggest an intimate association between the microvillar actin filament bundle and the regulatory functions of this multifaceted protein in the brush border. Examples may include regulation of actomyosin interactions responsible for microvillar move- ment and calcium transport.
Supported by NIH AM-25387 and T32-GM-07784.
Fairy rings: membrane particle arrays present during early stages of de novo cilio genesis in Tetrahymena. L. A. HUFNAGEL.
During a study of membrane-cytoskeletal interactions related to morphogenesis in Tetra- hymena, freeze-fracture EM was used to characterize membrane structural changes preceding formation of somatic cilia in log phase cells and oral cilia in cells synchronized for oral replace- ment. During early neogenesis of somatic cilia, a ring of irregularly spaced 100 to 130 A particles was observed in the E-face of the plasma membrane. The diameter of the ring was
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similar to that of cross-fractured ciliary axonuncs, and a central group of 2-9 100 to 130 A particles usually was seen within the ring. Ring particles numbered from 20 to 90, with an inverse relationship to the number of central particles. The rings resembled mushroom rings found in lawns after rainstorms; hence the name "fairy rings." Unlike the E-face, the P-face of the plasma membrane was evenly studded with small particles, with no apparent arrangement. Fairy rings were associated with specializations in membrane topography. Based on the observations, a tentative sequence for ring formation, origin of the ciliary neck- lace (a membrane specialization of the mature cilium) and concurrent topographical changes has been developed. According to this sequence, the central particles and parasomal sac (a basal body-associated plasma membrane imagination) appear earliest and may be intimately involved in positioning of basal bodies, while the fairy ring develops later, perhaps under the influence of the basal body. The fairy ring then migrates into the basal region of the nascent cilium, to become the ciliary necklace. These ideas are currently under further investigation by means of TEM. In contrast with these observations, fairy rings have not been observed during neogenesis of oral cilia. This supports the idea that the rings do not assist in the positioning of basal bodies and also suggests that they are not necessary for growth of cilia. Supported by a Marine Biological Laboratory Steps Toward Independence Fellowship.
Structure of aggregatoin factor on sponge cells and in gels. SUSIE HUMPHREYS AND T. S. REESE.
Aggregation factor has been visualized on aggregating Microciona frolijcra cells and in factor gels by electronmicroscopy of specimens rapidly frozen and then either freeze-substituted and thin-sectioned, for freeze-fractured, deep-etched, and replicated without chemical fixation or cryoprotectant. Each factor macromolecule, a glycoprotein (MW 18x10" daltons), is a ring from which radiate several side chains which give it a "sunburst" shape which is well preserved by our freezing methods. Thus, the relationships of the factor molecules with each other and the cell surfaces of aggregating cells can be directly visualized. Preliminary examination of factor alone gelled in 10 mM CaCU showed many images suggestive that factor retains its sunburst configuration in gels and that contact between side arms is the rule. Chemical dissociation of sponge tissue by divalent cation depletion extracts aggregation factor but cells aggregate rapidly when divalent cations and aggregation factor are restored (T. Humphreys). Replicas of cells aggregated in factor showed factor molecules over much of their surfaces, suggesting that factor "receptors" may be evenly distributed over the whole cell surface. In sections of reaggregating cells we could sometimes see the circular backbone sandwiched between two cells ; some side chains contacted one cell and some from the same molecule contacted an adjacent cell. Our results suggest that cell-recognition sites are localized in the factor side chains and that the entire connection between cells could be made by single macromolecule bridges. Most significantly, our results demonstrate that factor could function by actually forming bridges between cells.
This work was supported by NSF grant PCM 78-09309 and by IRP, NINCDS. Purified factor was provided by Tom Humphreys.
Tubulin antibody induces inicrotiibule depolymerization in vivo and in vitro. SHINYA INOUE, KEIGI FUJIWARA, AND ELAINE D. PAPAFRANGOS.
Antibodies can be used for cytochemically localizing antigens or for functionally blocking reactions involving the antigen. We now find that a tubulin antibody (Anti-TL) can also depolymerize labile microtubules. The antibody was prepared against tubulin paracrystals induced in sea urchin Strongyrocentrotus purpuratus eggs by vinblastine. Upon micro- injection of ca. 0.4 nanoliters of Anti-TL solution (containing ca. 150 mg/ml of immunoglobulin), the meiotic metaphase arrested spindle in the oocyte of Chactoptcrus pcrgamentaceous, and the mitotic cleavage spindles and asters in Lytcchinus varicgatus, substantially lose their birefringence and grow smaller in minutes. The reaction resembles cold or colchicine treat- ment. Spindle birefringence and size are not appreciably reduced by injection of 100 mM KC1, buffer solution alone (100 mM KG, 10 m\i phosphate buffer pH 6.9, 0.02% Na-azide),
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or pre-immune IgG in buffer; if the birefringence decreases it recovers, although some cells injected with large volumes of IgG (with or without Anti-TL) blebbed extensively and lysed after approximately half an hour. Mouse and embryonic chick brain microtubules assembling in vitro are also depolymerized by Anti-TL, but again not by pre-immune IgG or buffer alone. The rise of specific viscosity ceases and decays exponentially as soon as Anti-TL is added to the polymerizing solution (2.5-3 mg/ml tubulin, 0.1 M PIPES, 1 mM DTT, 1 mM EGTA, 0.5 mM MgCU, 0.1 mM EDTA, 4 M glycerol). We do not yet know whether the Anti-TL depolymerizes microtubules by shifting the microtubule-subunit equilibrium, or by directly attacking the microtubules themselves.
Supported by NIH GM23475-14 and NSF PCM 76-81451 to S.I. and NIH-GM 25637 to K.F.
Field and laboratory observations of lobster inatiin/ behavior. ELISA KARNOFSKY AND JELLE ATEMA.
Courtships and mating behavior of the lobster (Homanis aincricanns) have been observed in our laboratory in large naturalistic aquaria. Field observations are now being made to com- pare laboratory observations to behavior in the natural environment. In the laboratory, dominant males establish stable residences and regularly displace other individuals from their burrows. There is female mate selection : females approach the dominant male burrow 1 to 5 days premolt, exhibiting ritualized behavior as do males during these entrance ceremonies. The female may leave the burrow periodically, chasing other lobsters from the area. This may be part of a generally observed premolt aggression peak. The male often lunges at the opening, preventing the female from leaving. Approximately 20 min after the female molts, the male mates with her. He then feeds on and later rejects the molt shell. Cohabitation without leaving continues for 1 to 5 days. Females may leave for short excursions before leaving permanently. Mating in the field was observed by direct and indirect clues. Two lobsters were discovered in a large burrow. The male had previously been tagged and the female had no crusher claw. This couple was observed for 8 days during which the female molted as indicated by a regenerated crusher ; also, a molted seizer claw was found outside the shelter. Male entrance ceremonies were performed, as was blocking of the entrance. On the 8th day the female disappeared. The same day a tagged female appeared and cohabited for 7 days. She molted between the 1st and 3rd day. During this time the male was seen evicting another male from a nearby burrow.
This study shows that observations in naturalistic seetings contribute continuity to scant and difficult-to-obtain field observations, while field studies modify and add reality to still rather artificial laboratory observations in large aquaria.
Properties of polymcrizablc tnbitlin from isolated Spisula spindles. THOMAS C. S. KELLER in AND LIONEL I. REBHUN.
Spindles were isolated from activated eggs of the surf clam Spisula solidissiina in a buffer containing 3.4 M glycerol, 100 HIM PIPES, 1 tmi MgCU, 5 HIM EGTA, 1% NP-40 pH 6.8. Tubulin was recovered from the isolated spindles by washing them free of the glycerol into a buffer containing 100 imr PIPES, 1 HIM MgCU, 5 mM EGTA pH 6.8 and placing them on ice until birefringence disappeared. The nonbirefringent spindle remnants were pelleted leaving the cold depolymerized spindle tubulin in the supernatant. Upon addition of GTP (to 1 run) and warming to 24 or 37° C, the spindle tubulin polymerized into microtubules detectable by an increase in light scattering and by the appearance of birefringent strands. The reassembled spindle tubulin was further purified through two additional cycles of tempera- ture dependent assembly-disassembly at either 24 or 37° C. After three cycles, the spindle tubulin cycled at 37° C contained predominantly tubulin, small amounts of a high molecular weight component (which differed in mobility from Spisula axoneme dyneins and bovine brain HMW's) and a doublet approximately 33,000 daltons. Tubulin cycled at 24° C con- tained at least 3 additional proteins normally lost at 37° C. Polymerization of the cycle- purified spindle tubulin was both concentration- and temperature-dependent with critical
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concentrations of 0.09 mg/ml at 37° C and 0.24 mg/ml at 24° C. To determine whether the associated proteins were necessary for polymerization, they were removed by DEAE-cellulose and phosphocellulose chromatography. The resulting purified tubulin polymerized with critical concentrations of 0.11 mg/ml at 37° C and 0.28 mg/ml at 24° C, not significantly different from those obtained with cycle-purified tubulin containing associated proteins. Thus Spisiila spindle tubulin polymerizes at physiological temperatures (24° C) and the extent of this polymeriza- tion is independent of associated proteins.
Preparation and characterization of phagosome membranes from Arbacia punctulata embryo cells. DAVID KE\V, CHRISTINE KAZNOWSKI, AND JAY BROWN.
Phagosomes from dissociated Arbacia embryos at eight-cell and hatched blastula stage were characterized by scanning and transmission electron microscopy, by enzyme assays, and polyacrylamide gel electrophoresis. Phagosomes were isolated after allowing the single cells to phagocytize latex beads.
Cysteine/papain treated eggs were grown to eight-cell stage for dissociation in Ca'+, Mg2*- free sea water. Untreated eggs were grown to hatched blastula for dissociation — Ca"^-free sea water washes, then shearing in sucrose/citrate/EDTA. Washed cells were incubated with 0.7 micron latex beads — 3 to 4 hr for eight-cell stage, and 6 to 8 hr for blastula. Washed cells were lysed in hypotonic buffer and lightly homogenized. Phagosomes containing beads were isolated on a sucrose step gradient spun at 100,000 X g for 90 min, at the 15 to 20% interface. Washed phagosomes contained an average of 0.6% of the homogenate protein. SEM series show progressive development of bead-size lumps in the blastula cells, and TEM sections show beads deep in the cells. Lactoperoxidase-catalyzed 125I-labeling showed some major different proteins labeled on living cells compared to washed phagosomes, when the proteins were separated by PAGE on Matsudara gels. Membrane enzymes assayed were : cytochrome c oxidase for mitochondria, NADH diaphorase for smooth ER, acid phosphatase for lysosomes, 5'nucleotidase and phosphodiesterase for plasma membrane. The non-plasma membrane markers were greatly reduced in the phagosomes relative to the homogenate, except acid phosphatase was slightly increased in the blastula cells. This increase is probably due to phagosome-lysosome fusion after the long incubation. 5'nucleotidase was assayed for eight- cell stage, at 7-fold phagosome enrichment. Phosphodiesterase was assayed at blastula stage, at 10-fold phagosome enrichment. The method shows a good yield and purification of mem- brane recently phagocytosed. It offers an opportunity to investigate properties and changes in a developing system, and as a non-developmental system to investigate transport, turnover, or pharmacological effects.
This research was supported in part by P.H.S. training grant T32-GM-07784.
Adaptation of bacteria to rapidly changing environmental conditions. DAVID
KlRCHMAN AND RALPH MlTCHELL.
We used tidal flow in the Great Sippewissett salt marsh, Massachusetts, as a model system to determine the response of bacteria to rapidly changing environmental conditions. The number of bacteria was measured over two tidal cycles using the acridine orange direct count method (AODC). The total number of free bacteria was approximately 2.0 X 10'Vml at low tide. As the tide came in, total numbers decreased to a minimum at high tide of 1.0 X 10B/ml. The number of free bacteria increased again to approximately 2.0 X 10(i/ml as the tide left the marsh. We measured growth rates of bacteria from Buzzards Bay and from the marsh at low tide, in filtered sterilized (0.22 /ttm Millipore) water from Buzzards Bay and from the marsh. Growth rates were determined by measuring changes in bacterial numbers with AODC over 10 hr in 300-ml BOD bottles incubated in the marsh. Marsh bacteria grew in Buzzards Bay water at lower growth rates than in marsh water. Minimal growth of Buzzards Bay bacteria in Buzzards Bay water was detected in the 10 hr experiment. Bacteria from Buzzards Bay did grow in marsh water, but this growth does not appear to be fast enough to account for the observed changes in bacterial numbers over a tidal cycle at Great Sippewissett. While bacterial numbers doubled in 6 hr from high to low tide,
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significant growth by bacteria from Buzzards Bay in marsh water was not evident until the eighth hour. The fate of marsh bacteria carried into Buzzards Bay is unclear over a longer time scale. Bacteria from Buzzards Bay do not appear to adapt to the changing environ- mental conditions in the marsh rapidly enough to account for the pattern in bacterial num- bers over a tidal cycle. A fresh inoculum of bacteria from the sediment at each tidal cycle may explain the large population at low tide.
This work was supported in part from NOAA sea grant to Harvard University NA 79-AA-00091.
How photoisomerizable asobcnzenc compounds affect acctylcholinc receptors of skate muscle. M. KROUSE, H. LESTER, AND M. WEINSTOCK.
Previously, we have studied the effects of azobenzene compounds on Elcctroplionis electroplaques. This work extends those investigations to the skate, Raja crlnacca. Photo- isomerizable drugs were applied to the depressor rostri muscle. Effects of these drugs on endplate potentials and endplate currents (recorded under voltage clamp) were examined. Trans-Bis-Q is a potent agonist in the eel electroplaque (Kr) = 150 nM at —150 mV). A 30 /UM solution of Bis-Q containing predominantly the inactive cis isomer was added to the voltage clamped muscle. Then, a light flash increased the concentration of the trans isomer from about 5 to 11 /AM and thus induced a net inward current of 15 nA. This increased inward current was, however, transient even though both isomers are stable in the dark. Ten /aM 2BQ, a competitive antagonist (in its cis isomer) on the eel electroplaques (Ki = 150 nM) reduced the amplitude of the endplate current to about 25% of its control value. This effect was seen with both the cis- and trans-configurations of 2BQ. Ten /XM benzyl Bis-Q inhibits the response to carbachol in the eel but in the skate muscle the effect is to increase the ampli- tude of the endplate potential and to prolong its decay, suggesting that it is a cholinesterase inhibitor. A small antagonist effect would not have been seen. EW-1, a local anesthetic (in its cis isomer) on the eel electroplaque ( Ki = 25 /AM) has no effect on endplate currents with concentrations as high as 100 /AM. While there are differences between eel electroplaque and skate muscle in regard to drug action and potency, the observations reported here serve to demonstrate that photoisomerizable compounds may be profitably used on preparations other than Elcctrophorns.
Supported by M.D.A. (grant in aid), X.T.H. (fellowship to M.W., RCDA NS-11756 to H.L.), and N.S.F. (grant PCM-74-02140).
Characteristics of the phofosynthetic unit in macrophytic red algae. T. A. KURSAR, D. MAUZERALL, AND R. S. ALBERTE.
Phycobilisomes were isolated from field-collected Griffithsia sp. and Champia f>arvula. Excitation of intact phycobilisomes with 500 nm light results in a small fluorescence emission at 576 nm from phycoerythrin and a large peak at 672 nm. Griffithsia sp. phycobilisomes in 0.65 M Sorensen's buffer have an uncorrected sedimentation coefficient of 83 S.
The in vivo fluorescence of wild type Gracilaria tikvahiae and an orange mutant ( P-7-1, obtained from J. van der Meer, NRC, Halifax, Nova Scotia) was also measured. Excitation of chlorophyll a with 430 nm light resulted in emission at 696 nm in both wild type and the mutant. Excitation of phycoerythrin at 550 nm in the wild type resulted in phycoerythrin emission at 577 nm, an unidentified emission at 644 nm, and a prominent shoulder at 683 nm, whereas the mutant ( P-7-1 ) showed a decreased 644 nm emission, almost no 683 nm emission and an emission band at 700 nm. The excitation spectrum of the 690 nm emission includes phycoerythrin and a large contribution from phycocyanin and allophycocyanin in the wild type, whereas for P-7-1 the 690 nm fluorescence is principally excited by phycoerythrin with a small contribution from phycocyanin and no apparent contribution from allophycocyanin. The chlorophyll content of the wild type and P-7-1 are the same, but P-7-1 is low in carotenoids. The growth rate of P-7-1 is about half that of the wild type. Our preliminary conclusion is that the light harvesting system of photosystem I is normal in P-7-1 and that the lesion
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has altered the phycobilisome or the light harvesting chlorophylls and carotenoids associated with photosystem II (or both). These results also suggest that in P-7-1 excitation energy transfer may go directly from phycocyanin to chlorophyll a and bypass allophycocyanin.
Research supported by the Experimental Marine Botany Program, National Science Foundation grants PCM 78-10535 and PCM 79-06638, and National Institutes of Health grant GM 2394-0 1A2.
Slow changes in the magnitude of the potassium current associated ivith changes in the internal perfusion solution in squid a.rons. DAVID LANDOWNE AND VIRGINIA SCRUGGS.
Experimentally \ve find it difficult to alter the internal potassium concentration in internally perfused squid giant axons. Apparently there is no inert replacement monovalent cation which does not iself reduce the potassium current. When we reduced the internal potassium, replacing it with sucrose, we find the magnitude of the potassium current declines in two phases. There is a rapid decline during the first 1 to 2 min which corresponds with the time required to change the internal solution. This is followed by a slow decline over tens of minutes. This is reversible, on switching back to a high potassium solution the current increases in two phases. The relative proportion of the two phases varies. We think this is asociated with damage to the interior of the axon, with less damage favoring more slow phase. Prolonged treatment with pronase removes the slow phase.
From these current measurements it seems that the residual axoplasmic lattice retains some of the potassium and only slowly equilibrates with the perfusion fluid when no cation is provided to exchange for potassium. It will similarly retain sodium or TMA. When the internal solution was changed from one containing sodium and potassium to one containing potassium only, and with sugar replacing sodium, the current-voltage curve retained the negative conductance region associated with the presence of sodium. A 1-min rinse with high potas- sium followed by return to low potassium removed the negative conductance.
Phosphocellulose purification of dogfish and skate brain tiibulin. GEORGE M. LANG- FORD, LASCELLES E. LYN-COOK, AND DANIEL ROBBINS.
Dogfish shark and skate brain tubulins were purified first by two cycles of a temperature dependent assembly-disassembly procedure and then by phosphocellulose (PC) column chroma- tography. The cycled tubulins were capable of spontaneous assembly but the phospho- cellulose purified tubulins were not. The cycled tubulins, nevertheless, contained no high molecular weight, microtubule associated proteins (MAPs). The high molecular weight pro- teins present in the supernatant and pellet fractions obtained after each of the assembly-dis- assembly steps were determined by sodium dodecyl sulfate, polyacrylamide gel electrophoresis (SDS-PAGE). The gels of the initial extract show two major high molecular weight protein bands of appoximately 280,000 and 320,000 daltons. At least two minor bands in the molecular weight range of 300,000 daltons could also be seen. One of the two major proteins, the 280,000 daltons protein, is lost in the first cold pellet. The 320,000 dalton protein which co-migrates with ciliary dynein 1, co-purifies with tubulin through the largest number of steps. Sometimes it is retained after completion of two cycles of purification but most often it too is lost in an earlier step. The twice cycled tubulins free of high molecular weight proteins were put on a phosphocellulose column. Because phosphocellulose sequesters Mg2+, this ion was immediately added to each of the column fractions. The fractions containing tubulin were combined and concentrated by a Millipore immersible ultrafilter. Phosphocellulose tubulin, in the range of 0.5 to 1.0 mg/ml, did not spontaneously assemble. When mixed with cycled tubulin, PC tubulin was incorporated into microtubules after a lag time of 1 min. Electron microscopy revealed that the tubules formed from a mixture of PC and cycled tubulins were often defective. Incomplete closure of tubules resulting in regions that appear as flat ribbons of protofilaments were often seen. These data show that high molecular weight MAPs do not cycle with the fraction of tubulins obtained by temperature cycling. Since PC
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tubulins do not spontaneously initiate assembly, there may be MAP fragments or lower molecular weigh proteins required for initiation of polymerization.
Tidal rhythm and tissue organization in the neural gland of Ciona intestinalis : correlates with cellulose and fibronectin. JAMES W. LASH, MICHAEL OVADIA, CHARLES H. PARKER, AND CLIVE N. SVENDSEN.
Rarely have tidal rhythms been associated with specific tissue changes. It is more common that such rhythms are expressed as behavioral changes. In the ascidian Ciona intestinalis tidal rhythms have been found to be correlated with specific alterations in the neural ganglion and gland. At high tide the ganglion secretes PAS positive granules, but not at low tide. At low tide the gland is organized as a parenchyma and secretes an Alcian Blue positive substance. At high tide the neural gland undergoes a marked reorganization, and the gland cells now adhere to new:ly appearing PAS and Alcian Blue positive fibers. The function of the gland is poorly understood and the nature of the secretory products is not known. Ganglion/gland complexes isolated from the animal undergo complete transition from low tide phase to that of high tide, but not the reverse. In an attempt to mimic the gravitational forces thought responsible for these changes, intact animals were centrifuged for one tidal interval at 1.5 Xa. This permitted the change from low tide phase to that of high tide, but maintained the high tide phase. Various enzymes were used to obtain evidence of the con- stituents of the ganglion/gland complex. Hyaluronidase digested the ganglion secretory granules. Cellulase removed most of the fibers in the gland at high tide phase. Using human and hamster fibronectin antiserum and fluorescein conjugated IgG, fluorescence was seen primarily in the neural gland, coating the fibers at high tide and generally distributed around the gland cells at low tide. Thus the extra-cellular matrix of the gland appears to contain cellulose and fibronectin.
Supported by NIH research grant HD-00380 to JWL.
The growth and reproduction of selected species of ineiofaiina in selected natural inicrofloral assemblages. JOHN J. LEE AND MONICA J. LEE.
Previous research has shown that various species of meiofauna are highly selective in their feeding habits and that when incubated in gnotobiotic cultures with different species of algae and bacteria their growth and fecundity vary greatly. Sometimes two or more species are needed to satisfy the nutritional requirements of an animal. Enigmatically most of the meiofaunal species studied gnotobiotically are most fecund on species with low natural abundances (<.3%). This raises the question of whether littoral benthic microbial assemblages are consumer con- trolled or whether they selectively control meiofaunal abundances and fecundity. Experiments carried out this summer in both the field and the laboratory provided more data on this important question.
Natural microfloral assemblages were gently sieved ( 38 /urn ) to remove all larger organisms and meiofauna. After the sieved mixtures were examined and the juveniles, nauplii, larvae, ciliates, etc. were picked out with the aid of small capillary pipettes, the mixtures were used as inocula for growth experiments. Selected meiofaunal species were inoculated into tissue culture flasks with the "natural mixtures" and incubated in situ. The tissue culture flasks had nylon filter (3.0 fj.m ) covered windows cut into their surfaces which allowed free passage of sea water while retaining the algal assemblages. Vessels in assemblages without meiofauna and vessels incubated in the laboratory under constant environmental conditions served as controls.
Allogroinia laticollaris, a foraminiferan, steadily increased in all the natural assemblages into which it was introduced. Populations of Chrounidorina c/cniiunica, a nematode, reproduced vigorously and then crashed without recovery in one set of experiments and failed to reproduce in a mixture incubated later in the summer. Rhabditis marina, another nematode, failed to reproduce in cither experiment. Nitocra typica, an harpacticoid copepod, reproduced after a lag of 3 weeks and maintained a high population for 7 weeks. Permanent preparations were made
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 379
of the diatom assemblages of the control and experimental vessels. Studies of these preparations are now in progress.
Supported by NSF grant OCE 7825798.
The photosensitizing action of psoralcns in dogfish ocular tissues. S. LERMAN, J. M. MEGA\V, AND Y. TAKEI.
The photosensitizing action of 8-methoxypsoralen (8-MOP) has led to increasing concern regarding potential ocular complications in patients treated \vith this drug. Free 8-MOP can be demonstrated in the dogfish ocular lens and retina for 24 hr following an intraperitoneal injection of 1 mg of this drug. The 8-MOP can be detected in the lens by phosphorescence spectroscopy and labelled 8-MOP (25-50 /^Ci 3H or 15 ^Ci 14C) by autoradiography. When the fish is kept in the dark, the free 8-MOP diffuses out of these tissues after 24 hr. However, photic stimulation by exposure to G.E. BLB fluorescent light (mainly 365-nm radiation) for 1 to 15 days, results in the formation of photoproducts involving lens proteins and 8-MOP. The extracted and purified soluble protein fractions (after repeated TCA washing) had a significant activity (both for SH and 14C) compared with background levels in proteins from controls kept in the dark. Autoradiographic studies were also positive in the tissues obtained from UV-exposed fish. These data correlate well with phosphorescence, EPR, and NMR experiments which demonstrate at least one photoproduct in the soluble lens protein fraction (in vitro and in vivo) following exposure to UV radiation longer than 320 nm. We have shown that the tryptophan 8-MOP photoreaction requires O°, in contrast with the cyclobutane photoproducts formed between 8-MOP and thymine, which are not Q» dependent. Singlet O2 plays a role in the 8-MOP-tryptophan photoreaction. Because the ocular lens is a completely encapsulated organ which grows throughout life and never sheds any of its cells, the photo- binding of 8-MOP and lens proteins (as well as with DNA) will result in the permanent retention of new photosensitizers within this organ. This could enhance the UV-induced changes already occurring in the normal aging lens and might even have a cataractogenic potential. Furthermore, the occular lens normally acts a a UV filter, preventing UV radiation (longer than 300 nm) from damaging the retina. Thus the presence of 8-MOP in the retinas of aphakic patients could also prove hazardous.
Supported by NIH grant EY-01575, EY-01967 and AGO-1309.
Possible mechanisms for inhibition of cellular actii'ity by dyes with highly negative reduction potentials. DEBORAH LIPMAN AND SEYMOUR ZIGMAN.
Cyanine (methine) dyes with reduction potentials ( EK ) more negative than —1.0 V have been found by us to inhibit mitosis in sea urchin embryos at 10~5 to 10~" M. Chemically similar dyes wih ER values less than —1.0 V were ineffective. The motility of the dynoflagellate Gymnodinium brcvc was also eliminated by dyes with ER values more negative than —1.0 V, but not with ER values more positive (calomel electrode). Consideration of the possible mechanisms behind these varied effects and also the inhibitory effects on the synthesis of DNA and protein in sea urchin eggs by these dyes led to a hypothesis that the dyes with highly negative ER's interfere with cellular electron transport in oxidative metabolism and with energy production. The highly negative potentials (i.e.. greater than —1.22 V using the hydrogen electrode) of these dyes compared to those of NAD, FAD, and the cytochrome c half cells (all considerably more positive) would predict an action due to electronic saturation. Observations of the ability of sea urchin eggs (Arbacia punctiilata) to divide, and measurements of their ATP content (measured by the firefly tail assay) after treatment with dyes having highly negative ER'S indicated that no major reduction in ATP content resulted. These dyes may alter electron transport reactions involved in other phases of cellular oxidative metabolism, for example cytochrome c. Thus the inhibition of growth and reduction of activity of cells due to their exposure to dyes with highly negative ER'S may not result from interference with energy metabolism, but could result from electronic saturation of the components of cellular respira- ion with other consequences.
(Dyes supplied by Eastman-Kodak Co.)
380 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Presynaptic calcium current and postsynaptic response generated by a presynaptic action potential ; a voltage clamp study in flic squid giant synapse. R. LLINAS,
M. SUGIMORI, AND S. SlMON.
The present study was designed to characterize the presynaptic calcium current ( Pre Ica) and its relation to postsynaptic potential (PSP) and current (PSI) during a presynaptic action potential. This was accomplished by driving the command amplifier of the presynaptic voltage clamp circuit with a prerecorded spike. In addition, the experiments would test whether the properties of the Pre Ira predicted for a presynaptic spike by our model (Llinas ct al., 1976, Proc. Nat. Acad. Sci., 73: 2918) could be reproduced experimentally.
In the present study the waveform of a presynaptic action potential was recorded prior to the application of tetrodotoxin tetraethylammonium and 3-aminopyridine. Following addi- tion of these agents, voltage clamp conditions at —70 mV holding potential were simultaneously set (via two independent circuits) to both pre- and postsynaptic elements in the junction in order to measure ICa and PSI (Llinas and Sugimori, 1978, Biol. Bull, 155: 454). The Pre Ica measured during the spike-like presynaptic voltage clamp depolarization started near the peak of the presynaptic "spike", and its maximum amplitude had a sigmoidal dependence on the amplitude of the presynaptic voltage transient. Both peak and total postsynaptic current and peak PSP were linearly related to peak or total presynaptic Ica. This study confirmed the linear relationship between presynaptic calcium and postsynaptic response. Furthermore, the time course and magnitude for Ica coincided with those predicted by the model and with the conclusion that synaptic transmission is mainly produced by an "off" calcium current (i.e., generated at the falling phase of the action potential).
Research was supported by USPHS research grant NS14014.
Stimulation of testosterone production by mouse Lcydig cells with factors isolated from a microorganism and Ovalipes ocellatus. TAKESHI MAURO, AMY R. SEGAL, AND S. S. KOIDE.
During the course of determining choriogonadotropin(CG)-like substance in tissues of marine organisms, we have demonstrated that a trypsin-like protease present in the crab (Ovalipes ocellatus) stomach mimics CG in the radiommunossay (RIA) and the radioreceptor- assay (RRA) systems and is capable of stimulating rat ovarian adenylate cyclase activity in vitro.
In this communication evidence will be presented to show that a CG-like factor is pro- duced by a micoorganism. Extract of an acetone-dried preparation of a culture content of a microorganism tentatively named as "Progenitor crytocidcs" was prepared. The extract con- tained a CG-like factor as determined by RIA with the antiserum to hCG/3-COOH-terminal peptide and RRA using bovine corpus luteum membranes. The CG-like factor was purified by chromatography on Sephadex G-100, Concanavalin A-Sepharose, and DEAE-Sephadex A-50. Interference by proteases in the extract was excluded. The bacterial factor was adsorbed on ConA-Sepharose, suggesting that it contains glucose or mannose moieties. Gel filtration on Sephadex G-100 demonstrated that the purified factor was eluted at the same position as standard hCG.
When the in ^nvo biological activity of the purified factor was determined by the uterine weight and the ovarian weight assays using immature female rats, the potency of the factor was 380 lU/mg and 900 lU/mg, respectively. Moreover, it stimulated testosterone production by mouse Leydig cells. Its biological potency was estimated to be equivalent to 3400 lU/mg. The CG-like factor purified from crab stomach induced a slight stimulation of testosterone production by mouse Leydig cells. The relative biological potency was equivalent to 0.3 mlU/ mg. On the other hand, trypsin and chymotrypsin did not stimulate steroid formation.
The present findings suggest two alternative hypotheses for the presence and expression of mammalian gene(s) in bacteria and invertebrates. One is that the CG gene has its origin early in evolution, possibly as a protease. The other is that it is a consequence of a natural process of recombination.
Supported by a grant from the George Hecht fund. T. Maruo is a recipient of a Rocke- feller Foundation Fellowship.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Studies on urn cell complexes of Sipunculus nuclus (Linnaeus) : influence of phys- iologic and pathologic mammalian sera on mucus secretion. LUIGI MASTROI- ANNI, JR., SANTO V. NICOSIA, ELLEN STREIBEL, AND GILBERT HAAS.
Serum may play an important role in both vertebrate and invertebrate mucus secretion. This possibility is intriguing since many factors which induce serum transudation, such as estrogen and immune complexes, are also associated with enhanced mucus release. In order to study the influence of different hormonal milieux and of altered immunologic states on the mucus-stimulating activity (MSA) of serum, urn cell complexes (UCC) of Sipunculus uudus, 50 to 100/exp., were incubated with unheated physiologic and pathologic sera. Sera were diluted 1 : 1 with filtered sea water (FSW). Mucous tails (mean /urn tail length ± s.e.) were produced in response to male and female rabbit, mouse, and human sera obtained during endocrinologically different states. A significant (P < 0.001) enhancement of mucus release was induced by estrous mouse sera (67.22 ± 4.45 vs. 43.90 ± 3.06 at diestrus). Mucous tails of significantly greater length (P< 0.001) were also observed with sera obtained from estrous rabbits 12 to 15 hr after treatment with 5.0 mg of conjugated estrogen (45.97 ± 4.11 vs. 17.88 ± 1.87, before) and 20 tnin after a single intravenous injection of 25 fig of estradiol-17/i (37.91 ± 3.75 vs. 14.00 ± 2.01, before). The estrogen-enhanced MSA of serum was not altered by extraction with activated charcoal and, as indicated by dialysis, the active factor had a molecular weight greater than 13,000. Mucus release did not occur with FSW alone and with FSW containing 10 >j.g/fj.\ of estradiol-170 or 10 to 20 mg/ml of bovine serum albumin. UCC also released mucous tails of significantly greater length (P < 0.001) when exposed to pathologic sera obtained from infertile or vasectomized men exhibiting circulating antisperm agglutinins (64.12 ±1.57 vs. 16.82 ± 1.83, normal serum). This secretory response was commensurate with agglutinin titers. These results indicate that mammalian sera may contain more than one fatcor capable of inducing augmented mucus secretion.
Supported by USPHS HD-06274 Sub-4.
Effects of extreme temperatures on protein synthesis in the toadfish. RITA W. MATHEWS AND AUDREY E. V. HASCHEMEYER.
The toadfish (Of>sanns tan), a temperate species occurring along the eastern Atlantic from the Gulf of Maine to Cuba, tolerates a broad range of temperatures both in nature and in the laboratory. Studies of protein synthesis in liver in vivo have shown a moderate temperature dependency (Cji,i = 2.5) from 15° to 30° C. As temperature is decreased, Qio increases dramatically ; however, even at 4° C all steps of protein synthesis continue to operate slowly without apparent differential breakdown in function. Rates return to normal when fish are warmed.
At temperatures above 30° C polypeptide chain elongation showed a normal rate increase with temperature ( Qio about 2.5), reaching 6 amino acid residues per second at 37° C, as in mammalian liver. However, total incorporation of radioactive amino acids into protein, reflecting all steps in protein synthesis, declined precipitously above 32° C. Results were obtained as fractional incorporation rate (incorporation into protein divided by total radio- activity in liver and by incubation time). At 30° C the fractional incorporation rate for 14C-leucine is 0.10 min'1 representing 10% turnover of the intracellular leucine pool per minute. In 1-min pulse experiments carried out in vivo immediately after the fish was warmed to the experimental temperature fractional incorporation was found to follow an Arrhenius relationship downward with Qm about 1/40 up to the lethal limit of 39° C. Fish returned to 30° C after 5-min exposure to higher tempera' ures showed nearly complete recovery of synthetic rate up to an exposure temperature of 38° C. The results suggest a partially reversible lesion in the system involving initiation or recycling of ribosomal subunits.
Supported by National Science Foundation grant DPP 77-20461.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
The size of the photosynthetic mill and its turnover time in various seaweeds. D. MAUZERALL AND D. WITTENBERC,.
The size of the photosynthetic unit (PSU), defined as the moles of chlorophyll (Chi) per mole of Oa emitted in a single flash was determined following the classical experiment of Emerson and Arnold. Our previous measurements have shown that green seaweeds have about the same PSU size (2000 Chl/Oi;) as freshwater green algae and higher plants, but that the red seaweeds have a smaller size based on Chi a. We have extended these measurements to grasses and to other red and brown seaweeds. Spartina altcrniflora. short : 2100 ± 300, tall : 3600 ±900 (A. Hogan) ; Zostera marina, 2200 (L. Mazzella); Champia parvula, 1500; Ceramiion ruhnttn. 1100; Cracilaria vcrrucosa, high light 1600, low light 2100 ( T. Kursar ) ; Fucus z'csicjilosis. 1800; Laminaria sacchariua 3300 (S. Schatz) ; Sart/assum filipcudula, 2000; Sphaerotrichia divaricata, 1900. Light absorption of the accessory pigments makes up for the missing Chi in the red seaweeds, but add to the absorption of the brown algae. A quantitative measure of pigment efficiency is the fraction of solar photons absorbed by the pigment. This is roughly equal for all the pigments over the full solar spectrum. This selection weakness may allow a more random play of evolution and account for the wide diversity in amount and kind of pigments observed. The turnover time is remarkably constant, near 0.5 msec, for all the species studied. This result shows a constancy in the evolution of photosynthesis that transcends the variable color and morphology of the benthic algae.
We thank Dr. J. Fiore for help in collecting and identifying the seaweeds, the named stu- dents in the program for data, and the Experimental Marine Botany Program and the NSF (grants PCM 74-11747 and 79-06638) for support.
Photosynthetic characteristics of Zostera marina L. (Eel grass). L. MAZZELLA, D. MAUZERALL, H. LYMAN, AND R. S. ALBERTE.
The photosynthetic activity of Zoster, i marina, from a tidal bed in the Woods Hole area, was studied. It was found that the chlorophyll (Chi) content (mg Chl/g fresh weight and mg Chl/cnr), oxygen evolution rates [/xmol O;>/( cnr -min) and fj.mo\ (O-2/(mg Chl-min)] and numbers of photosynthetic units (PSU) per unit leaf area varied in response to leaf age, light exposure and degree of epiphytization. These parameters were measured for different leaves from the youngest innermost leaf to the oldest outermost epiphytized leaves, and in different portions of the leaves from the youngest lowest portion to the oldest upper more epiphytized portion. The Chi content was lowest in the base of the youngest innermost leaves (0.002 mg Chl/cnr), while the highest concentration was found in the middle portion of old epiphytized leaves (1.56 mg Chl/cnr). The lowest photosynthetic activity [2.5 X 10"1 /umol O-/ (mg Chl-min)] was found in the base of the youngest leaves while the highest activity was observed in the tip of old epiphytized leaves [(10.5 X 10"1 ,u,mol Os/dng Chl-min) ]. In general a gradient in Chi content and photosynthetic activity from the tip to the base of all leaves was found except in epiphytized old leaves from which the epiphytes were removed. The disappearance of this gradient from the latter leaves and the highest activity seen in the tip of old epiphytized leaves suggests that a significant portion of photosynthetic activity of older leaves is due to the presence of epiphytes. A gradient in Chi content and photosynthetic activity in the younger portion of all leaves from the outermost to the innermost leaves was also observed. The different maturity of the tissue and the different exposures to light intensity could explain this gradient in the young portion of different leaves. In conclusion, the photosynthetic activity of Zostera leaves is regulated by three factors: first, age of tissue; secondly, light intensity exposure ; and thirdly, presence of epiphytes with all other parameters constant (i.e. temperature, depth). For the young part of all leaves the light intensity and the maturity of tissue play an important role. For the oldest epiphytized leaves, the presence of epiphytes is more important than the other factors in the dynamics of the photosynthetic activity and biomass production.
Research supported by the Experimental Marine Botany Program and National Science Foundation grants PCM 79-06638 and PCM 78-10535.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 383
Interaction of lipsosomes with dogfish lens capsules. J. M. MEGAW, S. LERMAN, AND Y. TAKEI.
Liposomes are microscopic vesicles composed of lipid bilayers. They have been used to deliver drugs and enzymes into tissues and cultured cells. Techniques employed to enhance the specificity of delivery have included antibody and lectin-mediated binding. We have investigated the possibility of binding concanavalin A (Con A) containing liposomes to the lens capsule of the dogfish both in ntro and in rii'o. Liposomes were prepared by injection of ethanol solutions of phosphatidyl serine and phosphatidyl choline into PBS alone, or PBS containing either unlabeled or fluorescein labeled Con A. The vesicle size was increased by incubation in the presence of CaCU followed by addition of EDTA. The liposomes were then washed by centrifugation and suspended in PBS. Whole dogfish lenses, some pre-incubated with unlabeled Con A, were incubated in the various liposome preparations and then washed in PBS. The capsules were examined by scanning electron microscopy (SEM) or by fluorescence micros- copy (FM). For the in 1'ii'o studies, small volumes of aqueous humor were withdrawn from the eyes and were replaced with fluorescein-labeled liposomes in PBS. After 6, 23, or 29 hr, the eyes were removed and portions of the cornea, angle, iris, retina and lens were fixed for SEM or were examined by FM. SEM and FM demonstrated that liposomes containing Con A were bound to the lens capsule in vitro. Pre-incubation of lenses with unlabeled Con A resulted in decreased binding of Con A containing liposomes to the lens capsules. FM of eye tissues from animals injected with fluorescein-labeled Con A liposomes indicated binding of fluorescent material to the anterior lens capsule and to a lesser extent, to corneal endothelium. No fluorescence was noted in vitreous, retina or posterior lens capsule.
This work was supported by NIH (NEI) grants EY-01575, AGO-1309 and EYO-1967.
Nicotinamide deamidase actii'ity in oocytes of Spisula solidissima. AKIRA MOMII AND S. S. KOIDE.
We have demonstrated previously that 5 HIM nicotinamide inhibits germinal vesicle break- down (GVBD) in Spisula oocytes and that nicotinamide is rapidly converted to a metabolite by Spisitla oocytes. These findings suggest that the active inhibitor of GVBD may not be nicotinamide itself, but a metabolite. The present study was performed to identify the metabolite, to determine the enzymatic system mediating the transformation, and to investigate the mechanism of GVBD inhibition induced by incubation with nicotinamide.
Packed oocytes (2.5 ml) were suspended in 9.5 ml artificial sea water .(ASW) and incubated with 0.5 ml of 14C-nicotinamide (25 /uCi) at 22 to 23° C for 3 min. The final concentration of nicotinamide was 5 mM. The oocytes were washed with ASW three times and extracted with 2% HC1O4. The extract was neutralized with 1 N KOH and centrifuged. The metabolite in the supernatent was identified as nicotinic acid by PEI-cellulose and DEAE-cellulose thin-layer chromatography (TLC). No nicotinamide was detected. Since it has been demonstrated previously that nicotinic acid has no influence on GVBD in Spisula oocytes, it is unlikely that this metabolite is the active agent. Since ammonia is a product of the transformation, the ability of ammonium bicarbonate to influence GVBD was tested. At 15 HIM, ammonium bicarbonate blocked GVBD induced by insemination (45% GVBD com- pared to 95% for the control). Nicotinamide deamidase activity (EC 3. 5. 1.19) of Spisula oocytes was determined. Oocytes homogenate was incubated with 11C-nicotinamide at 37° C for 15 min. The resulting nicotinic acid was quantified after separation on PEI-cellulose TLC developed with H2O for 5 cm and with 1 N HCOOH for 10 cm. The deamidase activity is greater than 700 nmoles/(mg protein -hr) and does not change after fertilization. This value is far greater than that reported for mammalian tissues or the level found in Arbacia eggs. Spisula hepatopancreas has deamidase activity equivalent to that found in Spisula oocytes.
It can be concluded that Spisula oocytes possess an active deamidase system that rapidly transforms nicotinamide to nicotinic acid. The liberated ammonia may be reponsible for the inhibition of GVBD in Spisula oocytes induced by nicotinamide.
Supported by a grant from the George Hecht Fund.
384 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Differential solubilities oj cytoskeletal proteins in squid a.roplasin. JAMES R. MORRIS AND RAYMOND J. LASER.
We have studied the forms which cytoskeletal proteins take under physiologic conditions using squid giant axon axoplasm. The axoplasm consists principally of cytoskeletal proteins (tubulin, neurofilament proteins (NFP), and actin). A cyclinder of fresh axoplasm was extruded from the giant axon directly into a physiologic axoplasmic buffer (buffer P). Using SDS-PAGE, proteins diffusing into buffer P were compared to those remaining in axoplasm. After 24 hr at 20° C in buffer P, essentially all of the NFP (95%) remain in the axoplasm while 83% of the tubulin and 75% of the actin diffused into buffer P. Most of the other axoplasmic proteins appear only in buffer P. The axoplasm maintains its cylindrical morphol- ogy throughout the experiment. The mitochondria are retained in the axoplasm. Electron microscopy shows the presence of a network primarily composed of neurofilaments (NF). Microtubules are absent. This structure is referred to as the axoplasmic ghost.
Because all the NFP remained in the ghost and electron microscopy shows the ghost to be principally NF, we conclude that essentially all axonal NFP are normally polymerized in NF. A fraction of the tubulin and actin also remain attached to the ghost. This fraction must also exist as stable polymer. Most of the tubulin and actin diffused into buffer P. This diffusable component exists either as monomers or as a polymer which is soluble under physiologic conditions. We distinguish between monomeric and polymeric forms by analyzing the kinetics of pro'.ein diffusion into buffer P. This analysis shows that the diffusion of tubulin and actin includes a fraction which is slowed when compared to physico-chemical predictions. Thus, we have quantitatively analyzed three forms of cytoskeletal proteins in axoplasm : stable polymer, soluble polymer, and diffusable monomer. NFP differ from tubulin and actin in that NFP exist solely as stable polymer while tubulin and actin exist in all three forms in the axon.
Cytoskeletons in Labyrinthula slimeways. NORIO NAKATSUJI AND EUGENE BELL.
Electron microscopic observations have shown the existence of three kinds of filaments in Labyrinthula slimeways ; many actin-like 6-nm filaments, rare short filaments that are thicker than 6-nm, and numerous short 2.3-nm filaments. The latter two kinds of filaments may be myosin aggregates and individual myosin molecules respectively. Decoration of the 6 nm filaments with myosin S-l fragments was done after mild glycerination. They made clear arrow-head structures, showing that actin is contained in the filaments. Controls with 4 rmi ATP showed no decoration. Polarity of parallel filaments was not always in the same direction. Decorated filaments were frequently observed to terminate in dense plaques associated with the slimeway membrane. Immune-fluorescent staining, SDS-polyacrylamide gel electrophoresis and arrow-head decoration of filaments support the probability that actin and myosin occur in the slimeways.
Mg2+ and Ca2+-free sea water cr.uses the slimeway to break into many beads. Thin sections of these beads show the attachment of 6-nm filaments to the slimeway membrane plaques clearly. Some thin sections of glycerinated slimeways suggest that 6-nm filaments are also attached to the outer surface of the unit membrane of intra-slimeway vesicles. Thus, for a model mecha- nism of cell translocation, we propose that actin filaments are attached to the inner surface of the slimeway membrane and to the outer surface of the membrane that is investing the spindle cells, to permit myosin-mediated sliding between these filaments.
Supported by NOAA sea grant 04-7-158-44079.
Marginal band systems in blood cells oj marine species: visualisation by indirect immunofluoresence. IRIS NEMHAUSER, WILLIAM D. COHEN, AMY MILSTED, AND ROBERT D. GOLDMAN.
Marginal bands (MBs) and associated paired centriole-like structures ("perioles"; pos- sible MB organizing centers) were observed in Triton-lysed red cells of the mollusc Anadara
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 385
transvcrsa, using indirect FITC immunofluorescence with anti-tubulin. Only MBs and the associated perioles fluoresced, with the disposition of the latter matching that observed in the same lysed cells in phase contrast. In some cases MB fluorescence was so great as to obscure that of the perioles, though they were clearly visible in phase contract. Similar results were obtained with preparations of lysed dogfish erythrocytes (Mustclus canis), except that MB- associated, paired fluorescing structures were observed in a minority of cells. It is possible that they are present only in immature cells or obscured in most cells by the MBs or other structures. The results suggest that centriole-like structures may be associated with the MBs of diverse species, and that a careful search for them is warranted.
Anti-tubulin binding was also employed to follow structural alterations occurring in MBs of lobster coelomocytes (Hoinarus aincricanus) as they undergo changes in morphology while spreading on glass substrata (at room temperature, approximately 21° C). At t = 0 (no spread- ing) the cells are flattened and somewhat elliptical, with virtually all of their microtubules contained within intact circular or elliptical MBs. After spreading for 5 min (t = 5 min), most MBs are still recognizable but show signs of disorganization, with figure-8 forms prevalent. At t = 10 min MBs are no longer recognizable as such, with bundles of microtubules splayed in various directions. At t = 20 min the microtubule network has disappeared, leaving only scattered points of fluorescence visible in some cells. This is in contrast to other cell types such as those of cultured cell lines, which reorganize microtubule networks as they spread. Perhaps, in this instance, the cells are engaged in a process of self-destruction related to a clotting role in vivo.
Supported by CUNY PSC-BHE grants 12260 and 13051, and by NIH grant HL 20902 to W.D.C.
Ultrastructure of urn cell complexes of Sipunculus nudus (Linnaeus) before and after serum-induced mucus release. SANTO V. NICOSIA AND JANICE SEWINSKI.
This report analyzes the cytology of urn cell complexes (UCC), a currently used inverte- brate model of humorally-regulated mucus secretion. UCC are 60-80 /urn in size and are composed of one vesicle cell (VC) and one basal cell (BC) marginally joined by prominent desmosomes. VC's are thin-walled structures whose cytoplasm con'.ains few mitochondria and scattered, sudanophilic and osmiophilic, lipid droplets. These cells surround a vesicular cavity which contains mucus-like, microfibrillar material and is continuous with the extra- cellular milieu through the BC-delimited opening of UCC. There is a distinct polarity in cilia distribution and secretory activity in the BC. Rows of 5 to 10 fj.m long cilia are present, amidst microvilli, exclusively in the outer lining of BC. These cilia are anchored by 3 to 5 /urn long, curving rootlets which exhibit a periodicity of 65 nm and may have, together with desmosomes, a role in UCC plasticity during the mechanical stresses of mucus release, debris sweeping, and forward propulsion. In addition to numerous ribosomes, scattered mitochondria, and few lipid droplets, BC contain approximately 8 to 10 X 103 secre- tory-like granules which are uniformly electron-dense and lack a distinct limiting membrane in glutaraldehyde-osmium fixed UCC. However, they display cytochemical characteristics of mucous granules (diastase resistance, periodic acid-Schiff positivity, metachromasia, and immunoperoxidase-detectable lysozyme activity). The number of these granules is also drastically reduced after serum-induced mucus release. Typical exocytotic discharge of secretory material into the vesicular cavity of UCC is rare. More often these granules are found around or within intracellular canaliculi or sacs which extend up to the inner lining of BC where mucus release takes place. BC also contain 4 to 5 circular bundles of 14 to 17- nm wide microfilaments whose integrity, along with the UCC ability to release mucus, is dis- rupted by cytochalasin B. This study suggests that mucous tails are formed on the inner aspect of BC by a microfilament-mediated extrusion of mucus from storage reservoirs (intra- cellular canaliculi and/or the vesicular cavity) of UCC.
Supported by USPHS HD-06274 Sub-4.
386 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Particle size selectivity in Mocliolus clemissus and Balanus balanoides. DEANNA H. OLSON.
In great Sippivvissett Salt Marsh, Fahnouth, Massachusetts, Balanus balannidcs settles on Modiolus dcmissus, as M. dcmissus shells comprise most of the available hard substrate in the marsh system. Particle-size selectivity of these filter feeders was studied and related to posible food competition which may limit growth in areas of the marsh. Each species was allowed to filter natural sea water in a standing culture system. The water contained a de- creasing logarithmic progression of particles with increasing size. Particle concentration was determined (Coulter counter: 5-49 ^m size range) before introduction of the species to the system, and after 2 and 4 hr of nitration (7-11 replications).
Mean percent removal of seven particle size classes, within the range studied, was cal- culated for M. dcmissus (valve length range: 71-74 mm) and for a number of individuals of B. balanoides which corresponded to the average observed on marsh mussels. For both species, percent removal of particles increased positively with increasing particle size. Within 2 hr, the removal of particles in each size class by M. dcmissus ranged from 68 to 94% and by B. balanoides from 10 to 90%. After four hr, M. dcmissus and B. balanoides filter 93 to 99% and 19 to 95% of available particles, respectively. Particle-removal spectra for M. dcmissus suggested generalized filtration while B. balanoides selectively removed larger particles. Equal biomasses of these species (obtained by adjusting barnacle numbers) yielded particle removals similar to the above. Therefore competition between these filter feeders occurs only for the larger-sized particles studied.
Ammonification and nitrification potentials of soils from a northern hardwood forest and a pine plantation. K. C. PARSONS AND J. M. MELILLO.
In a laboratory study, rates of ammonification and nitrification were measured for organic and mineral soils from a northern mixed hardwood forest and a pine plantation. Two-normal KC1 extractions of organic soil samples from both stands yielded no nitrite-nitrate after nine weeks of incubation, although more than 250 fj.g of ammonium-nitrogen per gram soil (dry weight) was extracted from the same samples. Only mineral soil samples from the hard- woods produced significant, albeit low, levels of nitrite-nitrate ( 8.0 fig NOa + NOa-N/g soil from the upper 15 cm mineral soil, 2.5 /tg NO2 + NOs-N/g soil from 15 to 30 cm mineral soil horizon). Levels of ammonium extracted from mineral soils from the hardwood stand ranged from 2.5 to 8.0 fig NH4-N/g soil. Mineral soils from the pine plantation produced 1.5 to 5.0 Mg NH4-N/g soil.
Other soil samples were leached with distilled water periodically for thirteen weeks to give rates of nitrification. Levels of nitrite-nitrate were significantly greater than ammonium in samples from the upper 15 cm of mineral soil from the hardwoods. No nitrite-nitrate was leached from the organic soils from either stand, and none was produced by the mineral soils from the pine plantation. Ammonification was evident in all soil samples, but especially prominent in the organic horizons. When soils were fertilized with phosphorus, nitrification rates remained essentially zero. Ammonification in amended samples was not significantly different from controls in any soil type from either stand. Both systems are concluded to be ammonium-dominated. These experiments show nitrifiers not to be phosphorus-limited. Directions for further research are suggested.
The histocheinistry of muscle fiber types in the regenerating clau's of the lobster, Homarus americanus. NATALIE G. PASCOE.
Lobsters regrow autotomized claws initially as soft limb buds, which upon the subsequent molt become hard-shelled, functional claws. Histochemical techniques developed by Mark Ogonowski and Fred Lang for the characterization of lobster muscle in respect to fast and slow properties were used to examine the state of differentiation of regenerating opener and closer muscles in claw limb buds from adult animals. Early in the development of a bud, the muscles stain darkly for NADH diaphorase, an indicator of oxidative capacity (fatigue
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 387
resistance). However, initially, niyofibrillar ATPase, an enzyme that regulates speed of con- traction, stains faintly. The staining patterns in mature buds are similar to those of the adult cutter and crusher claws. In the closer muscle of the mature bud alternate staining of sequential sections reveals that fibers that stain intensely for myofibrillar ATPase stain lightly for NADH diaphorase. Conversely, fibers that stain darkly for NADH diaphorase show little myofibrillar ATPase activity. Some single fibers of intermediate staining intensity are found, and they stain in an intermediate extent for both enzymes. High specific activity myofibrillar ATPase seems to exclude the property of high oxidative capacity, or vice versa.
Opener muscles show regional differentiation in respect to NADH diaphorase; however, they do not have high specific activity myofibrillar ATPase in areas of lo\v NADH diaphorase. They may lack the potential for this.
The diurnal response of the gray seal, Halichoerus grypus, to tide and insolation during the month oj April at Aluskcgct Shoals, Nantuckct Sound, Massachusetts, USA. DAVID PATON.
The integument of the gray seal undergoes a once-yearly molt during April in these latitudes. This metabolic activity increases the nutritional requirements of the animal because it remains on land. Heating of the skin by insolation augments this metabolic stress to the advantage of the animal. If the seal can spend the time of the molt cycle in air (a fluid of a lower rate of thermal conductance than water), it can sustain the molt in less time, thence sav- ing energy that would normally be used in finding and processing food at sea. The gray seal has been observed to respond directly to the period of the diurnal tide cycle outside of the breeding and molting periods of the year. This behavior is supposed to be associated with feeding at sea.
During 5 days in April, a pod of gray seals was observed at Nantucket Sound. White light reaching the area was measured with a hand held Vivitar light meter, M90, and compared with a pyronometer, Epply 6-90, located at Woods Hole. Cloud cover, wind, sea and air temperature were also noted. Tide movement was observed on the study site and compared with published tide tables for the effects of wind fetch and current. Two gales swept the area during this time. The animals were not observed at night. A control animal, being offered its usual diet, was reported to have molted in an aquarium on Cape Cod isolated from tides. Climate diagrams were plotted for four size-color classes of model gray seals. The gray seals were observed to respond to insolation of white light rather than the period of the tide during the days of April 12th to 17th, 1979. Thermal regulatory behavior was observed. Further investiga- tions into the bioenergetic bounding effect of this habitat on the gray seal will be continued.
Research supported by members of the Yarmouth Conservation Trust for 1978.
Distribution of (jamctophytcs and sporophytes oj Chondrus crispus /;/ the vicinity oj Woods Hole. NANCY PENNCAVAGE, SCOTT SCHATZ. ESTHER MCCANDLESS, AND JAMES FIORE.
The red alga Chondrus crispus of the North Atlantic coast derives economic importance from its high content of kappa and lambda carrageenans, sulfated galactans which differ in degree and position of sulfation and in the presence of an anhydro-ring in the former. Kappa carrageenan gels in the presence of K+ ; lambda carrageenan solutions are viscous but do not gel. The variability in proportions of the two polysaccharides in carrageenan preparations was ex- plained recently by the discovery by one of us that kappa carrageenan is produced only by gametophytes, lambda carrageenan only by sporophytes of C. crispus and certain other red algae. In these species, qualitative analysis for anhydrogalactose can therefore be used to determine the phase of nonsporulating plants. Northern collections of C. crispus have demonstrated that the percentage of sporophytes in populations increased with depth, but no information is available on relative proportions or distribution of the phases here near the southern limits of the species.
TAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Between June 21 and August 18 we made 11 collections from 8 different Massachusetts sites: Black Rock, Cuttyhunk jetty, NMFS jetty, MBL beach, Menemsha jetty, Nahant, Nobska, and West Falmouth jetty. At NMFS jetty, Menemsha and Nobska, collections were made at several depths. A number of randomly selected plants from each collection (30 to 40) were analyzed for anhydrogalactose. Usually the proportion of sporophytes at mean low water (MLW) level was very low, approximately 5% of plants. At Menemsha this increased to 40% at —12 feet (below MLW), a distribution similar to that seen farther north. However, on both Nobska jetty and NMFS jetty the proportion of sporophytes was higher at MLW (22-28%) and reduced with greater depth (5% at — 7 ft and 11% at —9 ft, respectively.) Explanation of these observations is not yet apparent. (Support of the Experimental Marine Botany program and NSF grant PCM -7906638 is acknowledged.)
Spectroscopic and chemical studies oj protein-methylglyoxal complexes. RONALD PETHIG AND JANE A. MCLAUGHLIN.
Szent-Gyorgyi has drawn attention (1979, Biol. Bull., 157: 398) to the role of methyl- glyoxal (MG) in forming charge-transfer reactions with protein molecules. Such a concept has received strong experimental support from the studies of dielectric and electron spin resonance properties of protein — MG complexes (see Bone, 1979, Biol. Bull., 157: 358; Gascoyne, 1979, 157: 369). Our purpose has been to investigate the basic chemical reaction responsible for such a charge-transfer interaction. A neutral aqueous mixture of bovine serum albumin (BSA), casein or lysozyme with twice distilled MG assumes a yellow color having an absorption peak at 330 nm and a "shoulder" at around 350 nm. Addition of NaBH4 removes these absorption peaks. If the reaction is performed at pH4 the initial solution remains colorless. Adjustment of a neutral protein-MG aqueous mixture to pH3 modifies the absorption to give a new peak at 340 nm and no "shoulder" at 350 nm, and this effect is reversible on readjustment to pH7. The e-amino groups of the lysine side-chains of BSA and casein have been reductively dimethylated and after this treatment the proteins no longer react with MG to form a yellow color. The blocking of the arginine side-chains of BSA with cyclohexanedione has produced no observable effect on the color reaction with MG. Studies have also been made using methanol-water mixtures.
We consider that these studies show that the relevant reaction for our studies involves the formation of a Schiff base between tnethylglyoxal and the protein lysine side-chains. Theoretical calculations by others show that such a Schiff base provides a good electron acceptor for charge-transfer interactions with neighboring peptide units. This forms a strong theoretical basis for the electronic desaturation concepts described by Szent-Gyorgyi as being relevant to the "living-state."
L-lcuchic transport b\> toad fish Ih'cr studied by the Oldendorj method in vivo. ROGER PERSELL AND AUDREY E. V. HASCHEMEYER.
A technique originally developed by Olendorf for the study of transport in mammalian brain was extended by Pardridge (1977, Am. 3. Pliysiol.. 232: E492-E496) to amino acid transport in rat liver. A pulse injection of 14C -amino acid and 'HOH is given via the hepatic portal vein, and retention of amino acid relative to water is evaluated as the Liver Uptake Index (LUI).
We have examined L-leucine uptake by toadfish liver in t'iz'o at two temperatures by this technique. Hepatic clearance of 3HOH was followed as a function of time and the LUI for L-leucine determined. Maximal 3HOH extraction was about 80% of dose, similar to the value found in rat, but washout was much slower. In the time range up to 1.5 min, tj was 2.5 min at 20° C and 2.8 min at 10° C, compared to 0.8 min for rat. This represents a three-fold difference in rate of portal blood flow between the two species. LUI for leucine in toadfish was 0.7 to 0.8 for the first 4 min after injection and then increased as ;)HOH continued its efflux from the liver. At t = 10 min, LUI based on free (acid-soluble) radioactivity was 2.0 at 20° C. If protein-bound radioactivity is included in the LUI, following the method of Pardridge,
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 389
LUI is as high as 5. The latter value, however, reflects protein synthesis more than transport activity.
Previous studies in toadfish liver have shown concentration of L-leucine relative to an extracellular space marker, "H-inulin. This procedure has been used to study saturation and competition for the carrier-mediated transport process. The Oldendorf technique appears to be less useful in the fish because of the slowness of blood flow. Thus, the kinetics of liver amino acid transport when analyzed relative to 3HOH at the critical early times after injection are obscured by the slow water efflux.
Supported by National Science Foundation grant PCM77-07164.
The relative importance of bacterial and fungal bioinass and Spartina organic matter in the nutrition of tu'o species of salt marsh amphipods. NEAL W. PHILLIPS.
Several recent studies question the generalization that microbes are the major food source for detritus-feeders. I examined the relative importance of microbial and non-microbial biomass in the nutrition of two species of marsh amphipods, Orchcstia grillus and O. sfartino- phila. Assimilation efficiencies for total organic matter and for bacteria and fungi were esti- mated by an ash-tracer method which requires measurements of the ash fraction and of bacterial and fungal densities in food (dead leaves of Spartina alterniflora) and feces.
Specimens of O. spartinophila were fed submersed in chambers designed to minimize micro- bial growth on fecal pellets. Specimens of O. c/rillus were fed in moist fingerbowls from which fecal pellets were rinsed every 4 hr. After 12 hr of feeding, fecal pellets and uneaten food were homogenized and preserved for bacterial and fungal counts. Bacteria were enumerated by the acridine-orange direct count method ; fungal hyphae were measured microscopically on filtered subsamples incubated with water-soluble aniline blue, a fluorescent stain. Fecal pellets from food of known ash content were dried, weighed, combusted (500° C, for 3 hr) and reweighed to obtain ash fractions.
Orchcstia spartinophita assimilated 64 and 23% of total organics in two experiments. Assimilation efficiencies (5 replicates) for bacteria ranged from 23 to 75(/c and for fungi from 0 to 60%. The animals did not scrape fungi from leaf surfaces. Orchcstia grillus apparently assimilated 39% of total organics and 60 to 70% of bacteria, but fungal assimilation efficiencies were highly negative. This suggests that the animals selectively ingest fungi, and that the calculated efficiency for total organics is incorrect. Bacteria averaged 0.11% of litter dry weight, and fungi 0.24%. Microbial biomass may represent a small fraction of total assimilable organic matter.
fictcction of a sync.vin-likc soluble factor in anglerfish islet tissue that aggregates islet secretory granules in the presence of small amounts of calcium. HARVEY B. POLLARD, BRYAN D. NOE, AND G. ERIC BAUER.
Exocytosis is a common mechanism for the secretion of hormones and transmitters that are transiently sequestered in secretory granules, and depends in many cell types on an initial rise in the intracellular calcium concentration. Elevation in calcium appears to cause secretory granules to become closely associated with either plasma membranes or, frequently, with the membranes of other secretory granules that have already secreted but are still attached to the plasma membrane. This calcium-dependent membrane interaction may be mediated by a new protein called synexin. Synexin was recently discovered in chromaffin cells where it caused chromamn granules to aggregate with each other and with plasma membranes only in the presence of calcium. Synexin activity can be quantitated simply by following the increase in turbidity of a granule suspension at A 420 nm that accompanies aggregation, and we have utilized this approach to search for synexin activity in crude extracts of Anglerfish (Lophius amcricdints) islet tissue. We found that the post-microsomal supernatant fraction contained a potent islet granule aggregating activity which could also be observed directly by phase micros- copy. Further analysis showed that the reaction was strictly dependent upon calcium,
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since neither barium, strontium nor magnesium (all 2 HIM) could either substitute for or inhibit calcium action (1.2 /AM). A careful titration of the calcium concentration dependence with an EGTA buffer revealed that the K ( 1/2) for granule aggregation was ca. 0.04 /AM, with a Hill coefficient of ca. 3.5. Furthermore, the activity was stable to both dialysis and boiling, the latter observation clearly distinguishing Anglerfish islet factor from bovine synexin. Nonetheless, the general similarities of the reactions catalyzed by both factors lead us to conclude that secretion of some hormones found in islet tissue may prove to be mediated by a synexin-like activity similar in some ways to the protein previously characterized in chromafnn tissue.
Succession of fiz'e common salt marsh detritivores on Spartina alterniflora detritus of decreasing particle size and increasing age. CATHERINE N. POURREAU.
Detritus from the common cord grass Spartina alterniflora in little Sippewissett salt marsh, Falmouth, Massachusetts, is the primary source of food for five invertebrate detritivores (high marsh: Philoscia rittata (isopod), Orchcstia t/rillns (amphipod) ; low marsh: Gammarus palnstris (amphipod), Mclampns bidcntatus and Littorina littorca (gastropods)). Feeding preferences of these detritivores for different physical and chemical states of decomposition of 6". alterniflora leaves were studied by observing feeding activity in a quartered petri dish. Experiments (replicated three times) consisted of observations of animal positions in the dish every 10 min. Orchcstia grillus and P. rittata proved photosensitive, hence were tested in the dark. M clam pus bidcntatus fed only in the dark.
Selection for specific detrital sizes and age classes were detected in all species. Coarse particles (0.6 cm leaf fragments) were selected by M. bidcntatus and L. littorca. fine particles (0.6 mm) by (/. palnstris. and very fine (0.2 mm) by P. rittata and O. grillus. Conversely, 0- to 1-month-old detritus was preferred by P. rittata, 2- to 5-month-old detritus by M. bi- dcntatus, 6- to 9-month-old detritus by G. palnstris. and 9 months or older detritus by L. littorca and O. griUus. Relative importance of detrital particle size versus age for detriti- vore was tested by offering detritus of preferred age of an unpreferred particle size, and rice versa. Philoscia rittata and O. grillus responded more strongly to detrital age ; G. palnstris, M. bidcntatus. and L. littorca to particle size. Consideration of these results and habitats of these detritivores suggests that the detrital resource is partitioned and does not overlap.
Transnieinbrane movements o] suljur compounds in the squid giant a.ro)i. R. D. PRUSCH AND F. C. G. HOSKIN.
The intracellular ionic content, including anions, is closely regulated by a variety of diffusional and active processes. Isethionate, 2-hydroxyethanesulfonate, accounts for nearly half of the total anion balance in the squid giant axon. Both sulfide and cysteine (specifically the sulfur of cysteine) are involved in the biosynthesis of isethionate and hence in the maintenance of the anion balance. Thus measurements of transmembrane movements of both cysteine and sulfide are important to an understanding of the anion balance in the squid axon.
When isolated squid axons were equilibrated with ^S-cysteine (10~5M cysteine externally) dissolved in sea water ( 1 HIM HEPES, 10"4 M DTT, pH 7) a steady state intracellular concen- tration of 18 /AM was reached in 2 to 3 hr. Cyanide in the bathing medium (1 HIM) reduced the internal cysteine concentration to 8 /AM in the same time. When isolated axons were injected with ;15S-cysteine, an efflux rate constant of about 1.15 X 10~3 min'1 was attained after a 1 to H hr equilibration period. A reversible increase in cysteine efflux was brought about by 1 niM CN or 1 mM cysteine in the external medium. When the experiment was performed with 10~5 M NaaS, the internal concentration of sulfide after 5 hr was 10 /IM. Cyanide (1 mM) reduced this to 6 /AM. The rate constant for ^S-sulfide efflux was found to be 2 X 10~3 min"1 2 hr after injection of the isotope. This was reversibly increased to 3 X 10~3 min"1 by 1 mM sulfide in the external medium and to 2.7 X 10"3 min"1 in the presence of 1 mM CN.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 391
In summary both cysteine and sulfide uptake are reduced by CN, and the efflux of both are increased by CN. More importantly, cysteine efflux is stimulated by external cysteine, and sulfide efflux is stimulated by external sulfide. These observations indicate that both cysteine and sulfide are transported by carrier-mediated processes.
Supported by NIH grant ES-02116.
Chevnoreception in Homarus americanus: responses of primary receptors to second- ary plant compounds. PAMELA REILLY, CHARLES DERBY, AND JELLE ATEMA.
Secondary plant compounds are known feeding inhibitors in terrestrial systems ; their role in the marine environment, however, has only recently been examined. The major secondary compounds of terrestrial plants have generally not been found in marine plants ; other com- pounds, such as phenolics, seem to be more important. The chemosensory basis of detection of these compounds by marine consumers is unknown. This study is a neurophysiological analysis of the sensitivity of lobster chemoreceptors to terrestrial and marine secondary plant compounds, including a comparison of responses of two chemoreceptive appendages : antennules and walking legs.
Extracellular responses from nerves of excised appendages were recorded w'hile chemical stimuli were injected into sea water flowing over the chemoreceptive region of the appendage. Secondary plant compounds tested include those known in terrestrial environments (atropine sulfate, sinigrin, caffeine, salicin, amygdalin, morin, quinine sulfate, heliotropine ) and marine environments (ferulic acid, />-coumaric acid, gallotannin, phloroglucinol, bromoform, diiodo- methane). Mean responses (average number of spikes per trial) for these compounds were compared to those of two known excitatory stimuli : L-glutamate of equimolar concentration (10~5 M ) and a standard mussel extract. Mean responses to L-glutamate were identical for leg and antennular chemoreceptors : 22% of the mussel extract response. The mean responses to secondary plant compounds were generally low, ranging from 0 to 60% of the L-glutamate mean response. The mean responses to these compounds were almost always higher for leg than for antennular chemoreceptors ; this is due, at least in part, to the larger number of responding neurons in the legs. This implies that there is a larger population of receptors sensitive to secondary plant compounds in legs. This greater sensitivity of legs to secondary plant compounds indicates a functional difference between antennular and leg chemoreceptors, the latter being more important in the ultimate acceptance or rejection of food. This difference parallels that of smell and taste and is another argument in the functional separation of two chemoreceptor systems in aquatic vertebrates and arthropods.
Image intensification as a tool in low level fluorescence studies o] living cells. GEO. T. REYNOLDS.
Wolniak ct al. (1979, Biol. Bull.. 157: 402) describe results obtained using a high gain image intensification system for fluorescence studies of Hacinaiithus cells in a situation where the excitation intensity level required by conventional recording techniques destroys the cell development under study. The high gain system is an area detector consisting of a 4-stage intensifier capable of photon gains up to 10", and the output phosphor is viewed with a plumbicon vidicon, SIT vidicon, or conventional film camera. In this work the excitation level was reduced by several orders of magnitude by means of filters. This, and even further reduction made possible by the high gain system, is also an advantage in studies in which : first, the high level excitation normally required "bleaches" the fluorescent probe ; secondly, fluorescence tagging is limited in order to avoid interfering with normal cell processes ; or thirdly, receptors for the probe are sparse. In observations in which background fluorescence is a problem, it is possible to utilize a very narrow band width excitation to enhance the signal to background ratio. With the low excitation levels possible using high gain image intensification, the required bandwidths can be achieved using a suitable monochrometer rather than with the relatively complex and expensive tunable dye lasers required for high intensity excitation.
Helpful discussion with Elliot Elson is gratefully acknowledged. This work is supported by DOE contract EY-76-S-02-3120.
392 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
The use of Limulus amebocyte lysate (LAL) for the removal of lip o poly sac charide from biological reagents. FREDERICK R. RICKLES AND JACK LEVIN.
We have previously reported that many reagents are contaminated by lipopolysaccharide (LPS) (or endotoxin). Contamination of materials by LPS may interfere with the inter- pretation of results in various biological systems since LPS can mimic the action of many reagents and inhibit the action of o'Jiers. For example, concanavalin A (con A), which is a potent mitogen for mammalian mononuclear cells, is frequently contaminated by LPS, thus limiting its usefulness as a specific B-cell lectin. We report here the results of separation experiments designed to remove LPS from con A by differential absorption with a lysate made from the amebocytes of Limulus polyphemus. Con A was radiolabeled with 125I and then mixed 1 : 1 ( V/V) with LAL. After gelation and high speed centrifugation only 33% — 9.8 (s.e. ) of the radioactivity could be recovered from the supernatant. If, however, the lectin was pre-incubated with the disaccharide alpha-methyl-D-mannoside, 80.5% ± 10.6 of the radio- activity \vas in the supernatant. Four different preparations of con A have undergone purifica- tion by this method with complete removal of LPS. Each preparation has remained fully mitogenic when tested with human mononuclear cells (mean maximum DNA synthetic response = 7259 ± 2355 cpm before LAL absorption: 7477 ± 3410 cpm after LAL absorption). Similar results have been obtained with other protein antigens and lectins. Therefore, LAL can be used to remove LPS from a variety of proteins without loss of activity. In this way reagents can be prepared which have greater specificity and the pitfalls of LPS contamination can be avoided.
Supported by the Medical Research Service of the Veterans Administration and by Research grant HL-01601 from the National Heart, Lung and Blood Institute.
Intracellular pH regulation in squid giant a.vons. J. M. RUSSELL AND W. F. BORON.
Squid axons respond to internal acid loads by returning intracellular pH (pHi) toward normal ( — 7.3), in a process ("acid extrusion") which requires internal Cl~ and external Na+ and HCO:f. Thomas (1977, /. Pliysiol., 273: 317-338) has proposed that external Na+ and HCO:T exchange for internal H+ and Cl~. Alternatively, Na+ may combine with CO3= to form the NaCOs" ion pair which enters in exchange for Cl~. Both models predict a stoichiometry of 1 Na+ : 1 Cr : 2 H+ neutralized intracellularly. We have now used "Na, 38C1, and pH microelectrodes to measure the stoichiometry on internally dialyzed squid giant axons ( inside : pH = 6.5, [Cl'l = 150 HIM ; outside : pH = 8.0, [Na+] = 425, [HCCXf ] = 12, TTX = 10'7, ouabain 10'5). The HCOr-dependent Na+ influx was 3.1 ±0.5 (s.e.) (n = 5) pmol/( cnr-sec). Na+ efflux and Cl~ influx were —0.1 ±0.3 (3) and —0.2 ±0.3 (3), respectively. Cl~ efflux, taken from 1976 experiments (similar conditions), was 3.9. The acid extrusion rate, calculated from the pHi recovery rate after halting dialysis, was 8.8 ± 0.8 (TO). These data are thus consistent with predicted 1:1:2 stoichiometry. In addition, we studied the dependence of acid extrusion rate on [HCOa"]0 and on [Na+J0. Values for apparent Km and Vmax were 2.3 ± 0.2 mM and 1.1 ± 0.05 pmol/(cnr-sec), respectively, for HCO:r, and 77 ± 13 mM and 1.1 ± 0.6 for Na+. When these acid extrusion rates were plotted as a function of [NaCO3~]0, the data from the HCO3~ and the Na+ experiments fell on the same curve, with best fit values of 74 ± 3 V.M for Km, and 1.1 ± 0.2 for Vmax. This is consistent with the ion pair model, but does not rule out the Thomas model. Acid extrusion was reversibly inhibited 80 to 85% by 1 mM furosemide and by 1 mM 4,4'-dinitro-2,2'-stibenedisulfonic acid.
Supported by NIH grants NS-11946 and GM -06499.
A comparative study on Laminaria saccharina (Phaeophyta) infected by Phyco- melaina laminariae (Ascomycotina ). SCOTT SCHATZ, DAVID MAUZERALL, AND JAMES FIORE.
A comparative study on the brown alga, Laminaria saccharina infected by the ascomycete, Phycomclaina laminariae is presented. Light microscopy reveals that fungal hyphae grow
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intercellularly in the epidermis and outer cortex of host stipe tissue. Affected host cells become elongated and distended prior to cell-wall breakdown. Host plant susceptibility appears to be in part a function of the aging process with sporophytes younger than one year old displaying complete resistance to infection. The percentage of the population infected increased from 14.8 in May to 42 in August. Growth rates of infected plants was much less than that measured in healthy plants. Examination of the photosynthetic capabilities of blade tissue from healthy and infected plants shows that the photosynthetic unit sizes ( psu ) are similar. The psu in healthy plants is 3400 ± 100 while in infected plants the psu is 3000. However, the rate of oxygen evolution in saturating light was much greater in healthy plants than in infected plants. Significant differences were also found when transmitted light was reduced by 56, 74, and 90%. These data suggest that the dark reactions of photosynthesis are inhibited and not the light reactions. The following hypotheses are presented as possible mechanisms by which growth and photosynthesis are inhibited in infected host plants : first, the fungus serves as a sink, drawing away metabolites essential for the dark reactions of photosynthesis; secondly, the transport of photosynthetic products into the stipe is reduced resulting in a feedback inhibition of the dark reactions in the blade ; thirdly, a mycotoxin produced by the fungus is transported from the stipe into the blade ; and fourthly, these phenomena are simply a result of natural senescence processes typified by reduced metabolic activity thereby predisposing the plant to fungal infestation.
Support of the Experimental Marine Botany Program and NSF (grant 79-06638) is gratefully acknowledged.
Direct effect of LHRH on tcsticitlar steroidogenesis in Rana pipiens. S. J. SEGAL AND C. A. ADEJUVVON.
The LHRH (luteinizing-hormone-releasing-hormone) decapeptide has been identified in hypothalami of mammals, birds, and amphibia. For all vertebrate species studied, the role of releasing hormone in controlling LH secretion by the pituitary can be demonstrated. Recently, attention has been directed toward possible extra-pituitary effects of the decapeptide in order to explain paradoxical inhibitory action of LHRH or its analogues on gonadal function in mammals.
The direct effect of synthetic LHRH and one of its analogues (d-ser(bu* )"desgly10 LHRH ethylamide) on steroidogenesis by the testis of Rana pipiens has been investigated. The study includes the influence of the polypeptides on the well-known stimulatory action of hCG on the frog testis. Individual testes of specimens of R. pipiens ( in breeding season and producing abundant spermatozoa) were incubated in amphibian ringer's solution at room tem- perature. One hundred /j.\ aliquots of incubation medium were taken at 2 or 4 hr and assayed for testosterone ( T ) by radioimmunoassay. Two-hour control value of T production per individual testis is 22 ng. Addition of 16 ng, 32 ng, or 64 ng hCG to the incubation medium stimulates T production to levels of 38 to 95 ng. Adding 100 ng of LHRH (without hCG) results in 2-hr T production of 103 ng. When 64 ng hCG and 100 ng LHRH are included in the 2-hr incubation together, an intermediate production level of 80 ng is obtained. Similarly, the values obtained when a dose of 100 ng LHRH is included along with 32 ng or 16 ng hCG are intermediate between the values obtained with LHRH or hCG individually. The 4-hr T production level of R. pipiens testis in vitro is 20 ng in this study. Adding hCG (16, 32, or 64 ng) raises the production to 60 to 115 ng. The T produced by incubating testis slices with 100 ng LHRH for 4-hr is 124 ng. When both hormones are added, intermediate levels are produced. Parallel results are obtained with the LHRH analogue. A dose of 100 ng stimulates T production at 2 hr to 63 ng and at 4 hr to 83 ng. When the analogue (100 ng) is added along with 16 ng, 32 ng, 64 ng hCG the stimulatory effects of the two hormones are not additive.
These results demonstrate that : first, LHRH and one of its analogues directly stimulate steroidogenesis by the K. pipiens testis in vitro; secondly, the stimulatory effect of the deca- peptide or its analogue is not additive to the stimulatory action of hCG; thirdly, at the time and doses employed, the LHRH analogue is approximately equipotent to LHRH in causing T production by the frog testis in vitro.
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The gift of the LHRH analogue from Roussel UCLAF of Paris, France is gratefully acknowledged. C. A. Adejuwon is the recipient of a Rockefeller Foundation Fellowship. Sup- ported by a grant from the George Hecht fund.
./ i/cneral method, employing arsenazo III in liposomes, for the study of calcium ionophores: Results with A23187 and prostaglandins. CHARLES SERHAN, PAUL ANDERSON, ELIZABETH GOODMAN. ELISABET SAMUELSSON, AND GERALD WEISSMANN.
Multilamellar (MLV) and large unilamellar (LUV) lipid vesicles (liposomes) trap the metallochromic dye arsenazo III (2,7-bis(arsonophenylazo)-l,8-dihydroxynaphthalene-3,6-di- sulfonic acid) in their aqueous compartments. When ionophore A23187 was preincorporated into lipid bilayers of either MLV's or LUV's above 0.001 molar %, addition of Ca to the outside of liposomes produced spectral shifts (max. at 656 nm) characteristic of the AIII-Ca2 complex. The method permitted detection of two molecules of A23187 per liposome. Liposomes with A23187 were permselective : Mg or other divalent cations were not translocated. Integrity of liposomes was monotered by addition of excess EGTA which dissociated extra- liposomal AHI-Caa complexes. Incorporation of A23187 did not enhance permeability of liposomes to glucose, nor did valinomycin or gramicidin provoke Ca uptake. Ca uptake was not influenced by omission of cholesterol from the usual molar lipid composition of MLV's or LUV's (phosphatidyl choline 7: dicetylphosphate 2: cholesterol 1). Since prostaglandins may act as calcium ionophores, we have incorporated into MLV's and LUV's stable prostag- landins (PGE2, PGIs, PGBi), endoperoxides ( PGH^-analogues), and a wrater-soluble, poly- meric derivative of PGB, : PGBX. None acted as ionophores. In contrast, when added to the outside of preformed MLV's or LUV's PGBx, at concentrations above one micromolar, provoked permselective Ca uptake equivalent to that induced by 10~8 M A23187. These studies demonstrate not only that liposomes containing arsenazo III may be employed in a sensitive assay method to study agents which form channels for divalent cations, but that a water-soluble derivative of a naturally occuring fatty acid, PGBx, is a potent calcium ionophore.
Suljatc-depletion profiles and snlfatc-rcditction rates for a salt marsh. SUSAN SHEN.
Sulfate reduction is a major process in salt marsh decomposition, and the resulting reduced inorganic sulfur compounds are a potential source of energy to the coastal food web. As a step toward a better understanding of the process of sulfate reduction in salt marshes, the extent of sulfate depletion and the rate of sulfate reduction were measured in several sites in the Great Sippewissett Salt Marsh. Profiles of sulfate depletion in peat over depths up to 23 cm were constructed for various types of marsh sediments : dwarf Spartina altcnriflora, tall S. latcrniftora, S. patens, and creek bottom. Profiles are highly variable for the sites sampled, with the exception of a creek bottom site where distinctly less sulfate is found at greater depths. Variations were also found among cores taken at the same site. There are two possible explanations for such variability in sulfate depletion profiles: first, the hydrology of these marsh sediments is very complex, affecting the time during which sulfate reduction has occurred, and secondly, the extent of sulfate reduction and re-oxidation varies with depth and vegetation type.
Sulfate reduction rates were measured in the salt marsh at sites containing homogeneous stands of dwarf Spartina altcrniflora. A method was used to determine the rate of dis- appearance of sulfate in replicate cores incubated for periods of up to 2 weeks. Rates were consistently high for the top 5 cm of the peat and tended to be much lower at other depths. Although reduction rates varied greatly between similar sites and among replicate cores, they are well within the range of rates of reduction previously determined for dwarf .9. altcrniflora sediments during the summer by a radio-tracer technique. Much of this variation may be due to variation in the initial sulfate concentrations among the replicate cores.
Supported by the Jessie Noyes Smith Foundation and the SURDNA Foundation.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 395
Repeated genomic sequences cloned from the sea urchin Lytechinus pictus. JONATHAN SMITH, LESLIE SERUNIAN, WILLIAM PHILLIPS, ANDREW MURK.U , SHARON HOROWITCH, AND GERALD RUBIN.
The sea urchin genome is composed of approximately 25% repeated DNA sequences, most of which are short (average length 300 bases) and well characterized. However, 6 to 8% of the genome consists of repeated elements greater than 2 kilobases in length. Although much attention has been directed toward the tandemly repeated sequences (histone and ribosomal RNA genes), the dispersed repetitive sequences have been virtually ignored, except for studies showing that they are more highly conserved than the short interspersed repeats.
We have studied the long repeated sequences in the genome of Lytechinus pictus by isolat- ing and characterizing genomic DNA fragments using recombinant DXA technology. Re- combinant plasmids were constructed by ligating Hind III digested L. pictus genomic DNA into the Hind III site of bacterial plasmid pBR322 and were used to transform E. coli. Eight hundred and fifty transformants were isolated by resistance to ampicillin and screened with a 3L'P-labeled probe enriched in long repeated sequences. This probe was prepared by reassociating DNA to low Cot values, digesting with SI nuclease, size-selecting on sucrose gradients, and nick translating. When the SI -resistant DNA was electrophoresed on agarose gels, discrete bands, 500 and 800 nucleotides in length, were observed. Approximately 100 of the original clones hydridized to the repeated DNA probe ; 48 of these were hybridized to 3"P-complementary DNA synthesized from polyadenylated RNA isolated from hatching blastulae and pluteus embryos. One single clone hybridized to both of these probes indicating that the genomic fragment contains both repeated and transcribed sequences. DNA was also prepared from two clones containing long repeats ( 10 and 13 kilobases in length). The genomic inserts represented by repeated sequences were localized by hybridization of the repetitive DNA probe to restriction fragments of clones transferred to nitrocellulose according to the method of Southern.
Supported by NIH training grant no. TG-HD07098.
A comparative study of the marginal bands in newt ( Xotophthalmus viridescens) and cluck (Callus domesticus) crythrocytes. DENICE SMITH.
Although the erythrocytes of newt and chick are like the red cells of other non- mammalian vertebrates in that they possess a microtubule-containing structure called a mar- ginal band, these cells differ considerably in cell size and numbers of microtubules present in the marginal bands and they additionally differ in the degree to which the marginal band can be isolated from the nucleus, suggesting that the marginal bands in the two systems might be organized quite differently.
While marginal bands from chick erythrocytes are bound tenaciously to the nucleus by a fibrous matrix, newt marginal bands can be released from the nucleus (and possibly the matrix) by treatment of the erythrocytes with 0.5% Triton X-100 followed by a separation procedure in a sucrose step gradient. Analysis of these preparations by SDS PAGE reveal proteins which migrate in regions corresponding to MAPs, spectrin, tubulin, and actin when com- pared to standards known to contain these proteins, suggesting a possible association of mar- ginal bands with the cytoskeleton.
Treatment of marginal bands with 0.5 M KC1, which is known to disrupt the trans-band material, permits the individual microtubules of chick marginal bands to be visualized in negative stained preparations. When newt marginal bands are treated with 0.5 M KC1 for 1 hr, the marginal bands appear under phase contrast microscopy to be released from the cell, with microtubules splaying apart. Preliminary analysis by SDS PAGE suggests a selective removal of high molecular weight proteins by this procedure. Treatment of chick and newt erythrocytes with DNAase ( Img/ml ) for 1 hr appears to release the marginal band from the nucleus. Although the fibrous matrix appears to be disrupted, the marginal bands retain their characteristic curvature. Preliminary analysis of the preparations by SDS PAGE sug- gests a selective removal of high molecular weight proteins as well as spectrin.
This research was supported in part by grant T32-07784 from the PHS.
396 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Heavy metal effects on intestinal absorption oj nutrients in the toad fish, Opsanus tau. ROBIN Socci, JOHN MERCURO, AND A. FARMANFARMAIAN.
Heavy metals (Cd, Hg, Cr, Co) have been shown to inhibit the activity of various enzymes in different animal and plant tissues. Less is known of the inhibitory effect of these heavy metals on membrane transport mechanisms and, in particular, intestinal epithelium which acts as the first and most important barrier to the entry of these heavy metals in higher inverte- brates and all vertebrates. We have examined the effect of CdCb on the absorption of amino acids in the intestine of the toadfish in riro.
The test solution consisted of Forster Taggart fish ringer, a 14C-labeled amino acid (L-alanine, L-leucine), with or without a chosen level of the heavy metal inhibitor (CdCU). A closed loop of the midgut was cannulated under anesthesia and the test solution inserted for a 10 min incubation period. This solution was mechanically mixed by an oscillating pump stirrer. Two concentrations of substrate were usually used. Tritiated inulin was included as a water marker and radioisotopes were measured by liquid scintillation spectrometry. At 40 mM L-leucine and 5 X 10~4 M CdCl2, there was a reduction in the absorption rate of 36% (P^O.Ol), compared to that of the control without CdCU. However, at the 6.6 mM level, no significant inhibition was observed. When the CdCl2 level was reduced to 1 X 10~4 M, uptake of L-leucine was reduced by 52% (P < 0.001). At 1 X 10"5 M CdCU, there was no significant (P^O.05) inhibition of leucine transport. Two concentrations of CdCl2 (1 X 10~4 and 5 X 10~4 M) were tested with 6.6 HIM L-alanine. In both cases there was no significant (P <, 0.05 ) cadmium inhibition.
Supported by Center for Coastal and Environmental Studies, Rutgers University.
Behavioral evidence for two populations of amino acid receptors in catfish taste. ANN STEWART, BRUCE BRYANT, AND JELLE ATEMA.
From electrophysiological studies on the maxillary barbel of the catfish, it appears that there are two populations of receptors for amino acids : a generalist and a specialist ( Caprio, J. and D. Tucker, 1976, Soc. Ncurosci., 2: 152). The generalists respond best to alanine, but also to all other amino acids, while the specialists respond only to L-arginine, its methyl-ester, and slightly to alanine. In cross-adaptation experiments, a 10~4 M solution of alanine raised the threshold of all amino acid responses except arginine to about 10~3 M ; conversely an adapting arginine background had a minimal effect on the thresholds for other amino acids.
We tested behaviorally by touching the maxillary barbels of eight resting catfish (Ictalunis )icbulosus) with cotton swabs dipped in stimulus solutions. The fish were first conditioned to not respond to the tactile stimulus of a blank swab. Then thresholds for alanine and arginine were determined. Both ranged from 10~° to 10~NM. When the aquaria were flooded with a 10~4M background of arginine, the alanine threshold was raised by 0.7 or more log units in four fish and unchanged in four. When 10~4 M alanine was used as the adapting background, increases of 0.7 log units or greater were found for six fish (P C 0.05, sign test). Thus the alanine effect on arginine thresholds was slightly larger than the converse effect. When arginine and alanine stimuli were presented in their own background, the stimuli had to be 5 to 25% above background to elicit responses. This behavioral study confirms earlier electro- physiological work and demonstrates that catfish have an alanine and an arginine taste system which operate rather independently. Such a system would make it possible to detect arginine in high backgrounds of other amino acids. The significance for the catfish's natural environmet has yet to be determined.
Evidence for three conducting systems in the Jiydroid Clava. DARRELL R. STOKES AND NORMAN B. RUSHFORTH.
Three nonpolarized conducting systems can be activated by electrical stimulation of the colonial hydroid Clai'a squaiiitita. Two of these systems have been described within individual polyps: a Contraction Pulse System (CPS) and a Delayed Burst System (DBS). Stimula- tion of the CPS produces Contraction Pulses (CPs) which may occur as single events or as
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 397
bursts of potentials of facilitated amplitude (about 0.5 mV), and are correlated with polyp contraction. Stimulation of the DBS produces a program of bursts of pulses (DBPs) beginning about 20 sec after a shock. DBPs facilitate within a burst to amplitudes of about 0.5 mV and correlate with symmetrical depression of the tentacles.
Electrical stimulation of the stolons which interconnect polyps of the colony activates a third system: a Fast Pulse System (EPS). Fast Pulses (FPs), with no behavioral correlate, are of smaller amplitude (ca. 0.05 mV) and shorter duration (ca. 20 msec) than pulses from the CPS or DBS. Such pulses are conducted at a velocity of about 10 cm/sec, similar to that of the DBPs but three times faster than CPs. Production of FPs by electrical stimulation of the stolons induces CPs in adjacent polyps. In contrast, stimulation of a polyp to produce CPs does not evoke FPs in the stolons. This suggests that polarized trans- mission junctions occur between the stolons and the polyps.
Supported in part by NIH grant MH-10734 to N. B. Rushforth.
Stotocyst cilia transmit rather than transduce mechanical stimuli. E. W. STOMMEL, R. E. STEPHENS, AND D. L. ALKON.
We have investigated the role of motile cilia (hairs) in mechanotransduction by the stato- cyst of the nudibranch mollusc Heriuisscnda crassiconiis. Movement of hairs which experience the weight of statoconia causes variance of voltage noise and membrane depolarization. Two complementary approaches were used to immobilize the cilia. Vanadate anion (V+5) was iontophoretically injected into hair cells, using 0.1 nA currents through an electrode filled with 0.01 M NaVO3 and 0.1 M Na-acetate. This reversible inhibitor of dynein cross-bridge cycling initially caused the cilia to lose their rigid, vibratile motility and assume a more classic, pliable beat pattern. When the concentration of vanadate approached 10~5 M, the cilia were arrested against the cell membrane. Voltage noise decreased as the cilia slowed and bent more extremely, nearly disappearing as motility was lost. The cell no longer depolarized upon gravitational or mechanical stimulation. Rapid reversal with norepinephrine or slow reversal with time restored both the noise and depolarization response. Cilia were also rendered rigid by covalent cross-linkage of the membrane "sleeve" to the 9 + 2 axoneme, using the photoactivated, lipophilic, bifunctional agent 4,4'-dithiobisphenyl azide. In initial stages of cross-linkage, the cilia remained vibratile but slowed and moved through wider excursions. Voltage noise decreased in frequency but increased in amplitude. When the cilia were fully arrested, voltage noise was minimized while the resting potential remained essentially constant. Since cilia which are partially arrested by vanadate undergo considerable bending but show decreased noise, neither the axoneme nor the ciliary membrane proper appear to be sites of transduction. In full vanadate arrest, the exposed plasma membrane itself shows no response to stimulation. However, in beating, stiffened cilia, voltage noise becomes amplified, implying an increased efficiency of transduction. Therefore, the basal region is the most likely transduction site, being the leverage point to which force is applied via the ciliary shaft.
The larval stages of Lepocreadium areolatum (Linton. 1VOO) Stunkard. 1969, ( Trematoda: Digenea ). HORACE W. STUNKARD.
At the General Scientific Meetings a year ago, it \vas reported that certain hydrozoan and scyphozoan medusae and ctenophores in the Woods Hole area harbor unencysted metacercarial stages of digenetic trematodes. Five species have been recognized and the life-cycles of three of them have been worked out and published: that of Neopechona pvrifonnc Linton, 1900) Stunkard, 1969 by Stunkard (1969, Biol. Bull., 136: 96-113), of Lepocreadium setifcroidcs (Miller and Northup, 1926) Martin, 1938 by Stunkard (1972, Biol. Bull., 142: 326-334); and of Lintonium vibc.r (Linton, 1900) Stunkard and Nigrelli, 1930 by Stunkard (1978, Biol. Bull., 155: 383-394). The discovery on 20 July 1978 of an ophthalmotrichocercous cercaria from Nassarius tririttatus. whose structure agreed with an unidentified metacercaria, first noted in 1973, led to the elucidation of the life-cycle. The incidence of infection by this species is very light ; the cercaria had not been found before in the examination of thousands of
398 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
A . trivittatus. Only OIK- infected snail was found in 1978 and only one in the examination of hundreds of snails in 1979. The adult worm produces very few, very large eggs. A happy ecological situation facilitated experimental procedure. Crowell (1945) reported that the hydroid, Podocoryne carnca. lives on the shells of N. trivittatus. This snail harbors the trematode infection and liberates cercariae ; the hydroid liberates medusae, which are attacked and invaded by the cercariae. Medusae, infected in the laboratory and embedded in bits of squid and clam, were fed to cunners, Tautogolabrus adspcrsiis. This method yielded juvenile and gravid specimens of Lcpocrcadiuin arcolatitin, reported initially from white perch and cunners by Linton (1900, Bull. U. S. Fish Commission (1898) : 267-304; 1901, Bull. U. S. Fish Coin- mission (1899) : 405-492) and known also from puffers and other fishes since that time. Investigation supported by NSF DEB-74-145340 AO1.
I'ancrcatic secretion f/rannles arc associated icitli niicrotiibiiles. K. A. SUPRENANT AND \Y. L. DENTLER.
Secretion granules (SG) isolated from goosefish pancreatic islets specifically associate with microtubules assembled in intro from chick brains. Microtubules and SG's were mixed and associations were monitored in solution by darkfield light microscopy. Associations were confirmed by electron microscopy. SG's only bound to microtubules with high molecular weight associated proteins (MAP-MTs). In the presence of 0.2 HIM ATP, SG's bound to 43% of the MAP-MT's. Upon addition of lOO-^M cAMP, 57% of the MAP-MT's bound SG's. Microtubules also aggregated in the presence of cAMP to form large bundles associated with granules. The addition of 5-mM ATP to the cAMP treated aggregates, released the SG's from the MAP-MT's and dissociated the MT aggregates. Only 12% of these MAP-MT's had granules bound. The release of the SG's and the dispersion of the MT's are nucleotide specific, since pyrophosphate, AMP, ADP, GTP, and ITP have no effect on the release or dissociation. Polystyrene beads did not associate with the MT's under any assay conditions. The association between SG's and MAP-MT's was strong enough to be maintained during ultracentrifugation. SG's and MAP-MT's were mixed in 2M sucrose and centrifuged through a step gradient. The granules moved upward in the gradient with MAP-MT's associated to the SG membrane. In thin section, the association appeared to be mediated by the filamentous MAP proteins. Intact MT's from goosefish islets were isolated in the presence of hexelene glycol. SG's were attached along the length of the microtubules as assayed by negative staining.
This research was supported in part by a training grant T 32-GM -07784 from P.H.S. and by NIH AM-21672.
The living state. ALBERT SZENT-(JYORGYI.
"What is life" is the main problem of biology. We can ask the same question in a more meaningful way : what is the difference between animate and inanimate. The difference is so great that we are justified to speak of the "living state" as a special physical state. Material systems can be decomposed into molecules, molecules into atoms, atoms into electrons and nuclei. Electrons and nuclei can be decomposed still further but the energies needed are far beyond biological dimensions. The great sensitivity and reactivity of living systems indicate that the smallest independently moving particles are very small. We have to look for them among electrons and nuclei.
The number of electrons of the electron cloud, is given by the atomic numbers. With this number of electrons the electronic cloud is closed, the atom is a "closed shell" atom. Closed shell atoms build closed shell molecules. In them the electrons have to be immobile. To make them mobile electrons have to be taken out. Electrons cannot be taken out without disturbing the electroneutrality. Electrons can be transferred to another atom by charge transfer, and form then a charge transfer complex which is electroneutral. In the one of the two atoms or molecules the cloud will be desaturated and electrons will have a mobility. Charge transfer has been studied on various models having methylglyoxal as electron acceptor and methylamine, or symmetrical dimethylethylenediamine. The properties of these models are
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 399
discussed. Charge transfer introduces an electronic mobility and reactivity, as shown by the optical and electron spin resonance spectra. The living state is an electronically desaturated state.
Migration rate of mud bacteria as a junction of magnetic field strength. BARBARA D. TEAGUE, MICHAEL GILSON, AND AD. J. KALMIJN.
Certain marine and freshwater mud bacteria are endowed with a permanent magnetic dipole moment. This moment is attributed to an endogenous chain of tightly coupled, single- domain magnetite crystals. When separated from the mud, these magnetic bacteria swim north, following the earth's magnetic field lines. As at Woods Hole, Massachusetts, the field lines are steeply vertically inclined, the bacteria rapidly return to the bottom sub- strate where they seem to thrive best. To quantify this migration, we measure the time to traverse the distance between two lines, 1 mm apart, as a function of the ambient magnetic field strength. Using dark-field illumination, we observe single organisms as they migrate in a low-oxygen hemocytometer chamber. We control the ambient magnetic field by regulating the current through a Helmholtz-coil system. At high magnetic field strengths, the bacteria follow a virtually straight path, swimming at rates around 150 /mi/sec. At lower field strengths, they take a more random path which reduces their migration rate. Although they swerve moderately at the earth's magnetic field strength (0.5 gauss), the bacteria still achieve about 80% of their maximum migration rate observed at higher-gauss fields. This suggests that the bacterial dipole moments are well adapted to orientation in the earth's magnetic field. Since the strength of their magnet determines the degree to which the organisms overcome random motion, we can estimate the magnitude of their dipole moment.
This research is conducted under the auspices of the Office of Naval Research, as part of Kalmijn's WHOI-MBL project N00014-79-C-0071 NR 083-004, in collaboration with Drs. R. P. Blakemore and R. B. Frankel.
Excitation of squid giant a.von membrane exposed to an identical solution intra- ccllularly and cxtracelliilarly. SUSUMU TERAKAWA.
An electrical excitability was demonstrated in a squid giant axon membrane exposed to an identical solution intracellularly and extracellularly. The solution used for the intra- cellular and extracellular perfusion contained glycerol ( 12% in volume ) and one of cobalt-, manganese-, barium-, or nickel-salts at the concentration of 2 HIM. Usually, nothing else was added to the solution. The membrane potential measured by a KCl-containing glass pipette electrode stayed in the range of —10 to +10 mV during the intracellular and extra- cellular perfusions with an identical solution. When a constant inward current was passed through the membrane under such perfusions, oscillatory changes in the membrane potential and the membrane conductance were observed. The shape and the time course of these changes were very similar to those of repetitively fired action potentials. When calcium-, magnesium-, or strontium-salt was used as a sole electrolyte species, the oscillatory response did not appear. The oscillatory response was reversibly suppressed by 4-aminopyridine, but could not be sup- pressed either by tetrodotoxin or by D-600. A voltage clamp study revealed the N-shaped I-V characteristic of the membrane. Interpretations proposed by Teorell (1959, /. Gen. Physiol.. 42: 847) and by Katchalsky (1968. Q. Rev. Biophys.. 1: 127) for oscillatory phenomena in inanimate membranes may be helpful in understanding the results obtained.
Comparison of long-lasting hyper polarization produced synaptically z^ith that induced b\ cyclic AMP in Aplysia pacemaker neurons. STEVEN N. TREISTMAN.
Cell R15 of Aplysia possesses an endogenous rhythm in which bursts of action potentials alternate with interburst hyperpolarizations. A few stimuli to the branchial nerve can silence the cell for periods of minutes to hours. We had previously shown that phos- phodiesterase inhibitors could augment the hyperpolarization evoked by branchial nerve
400 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
stimulation, suggesting that cyclic nucleotides may play a role in the generation of these long-lasting potentials. Intracellular injection of cAMP derivatives, as well as GMP-PNP, an adenylate cyclase activator, caused sustained hyperpolarization of R15, strengthening this hypothesis. In this presentation, a voltage clamp paradigm is used to generate the pseudo- steadystate current-voltage curve, and compare the conductance changes produced by nerve stimulation with those induced by cAMP manipulation. Branchial nerve stimulation at high stimulus strengths caused an increased slope conductance in the R15 plot. Moreover, the negative slope region in the plot thought to be basic to generation of bursting activity was obliterated. Similar results were obtained after intracellular injec'.ion of GMP-PNP, cAMP derivatives, or cAMP in cells pretreated with the phosphodiesterase inhibitor, Ro 20-1724. These results are compatible with a cAMP mediation of the long-lasting synaptic event seen in R15 after branchial nerve stimulation.
This work was supported by a grant from the National Institutes of Health, and a Steps-Toward-Independence Fellowship from the Marine Biological Laboratory.
Mechanism of red algal bile pigment formation. ROBERT F. TROXLER, STANLEY B. BROWN, AND KEVIN M. SMITH.
The photosynthetic accessory pigment phycocyanobilin is structurally closely related to bilirubin, the end product of heme turnover in mammals. The latter process involves insertion of two oxygen atoms and lsO-labeling experiments have shown that these atoms are derived from two oxygen molecules (Two-Molecule Mechanism) rather than from one oxygen molecule ( One-Molecule Mechanism ) . The similarity in structure between bilirubin and algal bile pigments suggests that heme may be an intermediate in phycocyanobilin biosynthesis. However, since plants manufac;ure substantial quantities of magnesium protoporphyrin IX, it has been suggested that phycocyanobilin might arise from the magnesium branch of the porphyrin pathway.
We have performed preliminary 18O-labeling experiments with the unicellular rhodophyte, Cyanidinm calduriniii. which suggested phycocyanobilin synthesis by the Two-Molecule Mecha- nism. However, photosynthetic production of I0> "Oa prevented a precise quantitative interpreta- tion of the results. In the present work, 18O-labeling experiments were performed using cells in which photosynthesis was inhibited with the inhibitor, 3-( 3, 4-dichlorophenyl )-!,!-( di- methylurea) . Quantitative measurements on the mass spectra of phycocyanobilin isolated from these cells, revealed high 1SO incorporation and clearly demonstrated synthesis by the Two- Molecule Mechanism. This process probably involves independent attack by two oxygen molecules followed by intramolecular rearrangement and release of an oxygen molecule con- taining atoms from each of the attacking oxygen molecules.
To evaluate the possible origin of phycocyanobilin via the magnesium branch of the porphyrin pathway, a chemical model system was studied in which a magnesium chlorin was converted to a dihydrobiliverdin derivative, chemically analagous to phycocyanobilin. A thin-layer chromatography system was developed by which the product could be purified and isolated for mass spectrometry. ISO-labeling experiments on this model system are in progress. If the reaction occurs by the One-Molecule Mechanism, then phycocyanobilin syn- thesis via the magnesium branch is very unlikely. If, however, a Two-Molecule Mechanism is found, further experiments to distinguish between the magnesium and iron branches of the porphyrin pathway will be necessary.
Supported by NATO research grant no. 1721, NSF grant PCM79 24139 and CHE78 25557, and NIH grant GM 22822.
Glial-axonal protein transfer: its functional significance. MICHAEL TYTELL AND RAYMOND J. LASEK.
In the squid giant axon, many proteins synthesized by the adaxonal glial sheath cells are actively transferred into the axon. We examined these transferred proteins and compared them with the proteins of axoplasm (AXM) and the stellate ganglion of the giant axon. Proteins synthesized by the sheath and ganglion were labeled bv incubating the giant
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 401
axon and ganglion in 1 mCi/ml 3H-leucine in artificial sea water for 60 to 360 min at 19 to 21° C. Then the AXM, containing labeled transferred proteins, was extruded. The empty sheath, AXM, and ganglion were each homogenized in either a physiological buffer or electro- phoresis solubilization buffer. The homogenates were analyzed by ultracentrifugation, Sephadex column chromatography, and 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE).
The centrifugation and column chromatography experiments revealed that the proteins transferred into the AXM were contained in a particle which was unaffected by hypotonic shock or 400 niM KI, but was partially disrupted by 1% Triton and completely disrupted by 0.1% SDS. The 2D-PAGE analysis of the transfer particle in AXM showed that it consisted of a select group of about 50 sheath proteins, one of which has been tentatively identified as actin.
Eleven of the transfer particle proteins in the 2D-PAGE analysis were coincident with and therefore, identical to major axonal proteins. Since axonal proteins are made in the soma and supplied to the axon via axonal transport, we compared the 2D-PAGE pattern of proteins synthesized in the ganglion with these 11 transfer particle proteins. All but one were also made in the ganglion. Thus, some of the major proteins of axoplasm, presumably supplied by the soma, are also selectively transferred to the axon in particulate form from the adaxonal glial cells. We propose that glial-axonal protein transfer may serve to supple- ment a specific fraction of axonally transported proteins.
RNA synthesis in isolated chloroplasts. ELIZABETH VIERLING.
Chloroplasts were isolated from Hordcum vulgarc cv. Himalaya (barley) using 10 to 80% preformed Percoll silica sol gradients. These gradients separate intact plastids from broken plastids, mitochondria and other cellular debris. As judged by phase contrast micros- copy the preparations were free of nuclear and bacterial contamination. Chloroplasts were prepared for electron microscopy using the Miller spreading technique after a brief lysis in 0.005% Triton X-100. DNA fibrils observed at the periphery of lysed Chloroplasts were morphologically distinct from both eukaryotic chromatin and prokaryotic DNA. DNA strands had a diameter several times that of double-stranded DNA and were irregularly beaded. Structures which could be identified as transcriptionally active were not observed. For in vitro transcription, chloroplasts were incubated in the presence of 3H-UTP at a chlorophyll concentration of 100 /ug/ml. Incorporation of "H-UTP into an RNAase-A-sensitive product proceeded linearly for 30-45 min. Incorporation was a-amanitin insensitive at a concentration of 10 Mg/ml and was not light dependent. The time course and level of RNA synthesis were identical with or without the addition of 1 niM MnCU. This observation is consistent with chloroplast but not with nuclear RNA polymerase activity. Initiation of transcription was assayed for using Hg-agarose affinity chromatography of 7-thiol ATP or GTP labelled RNA. Results indicated that 0.4% of the 3H-UTP incorporated represented new transcript initiation.
This work was done in the MBL Physiology course, 1979 with funding by PHS training grant no. T 32-GM -07784.
Plasma fibroncctin (CIG) of the dogfish plasma mediates attachment of phagocytes to collagen substrates. GERALD WEISSMANN, JAMES W. LASH, G. E. SIEFRING, J. IBERS, AND LASZLO LORAND.
Plasma fibronectin (cold-insoluble globulin CIG) mediates the attachment of mammalian cells to collagenous substrates : we sought evidence for a similar protein in plasma of Muslelus canis. Blood phagocytes of the dogfish (10") were exposed either to uncoated Sepharose 4B beads ( 104 ) or to beads coated with gelatin by means of cyanogen bromide. In heparinized, complement-sufficient plasma, 58 and 90%, of control and gelatin beads, respectively bound three or more phagocytes (30 min, 23° C) ; evidence that complement activation (as in human plasma) by Sepharose 4B mediates cell adhesion to polysaccharide particles. In decomplemented (30 min, 56° C) plasma, only 4% of control, but 75% of gelatin beads had phagocytes attached. When plasma was also depleted of CIG (by preincubation with gelatin beads), cells attached to 5% of control, and to only 28% of gelatin beads. Small petri dishes were prepared with
402 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
purified mammalian collagens (Types I, II, and III) with and without proteoglycan aggregates. Dogfish phagocytes in decomplemented plasma avidly adhered to each type of collagen (but not to uncoated areas of the dish) and proteoglycans diminished attachment. Dogfish CIG was isolated by affinity chromatography on Sepharose 4B -gelatin and eluted in 2 M arginine ; yield : 0.38 mg/nil plasma. After dialysis, the isolated protein migrated identically to human CIG on 3.8% polyacrylamide gel electrophoresis (MW = 220,000) and dissociated to a single chain in the presence of dithiotheitol. Gelatin — but not control — beads exposed to either decomplemented dogfish plasma or purified CIG showed specific fluorescence when first reacted with goat anti-human CIG or rabbit anti-hamster fibroblast fibronectin and then with rabbit anti-goat IgG. Data suggest that CIG is conservative both in structure and function: attachment of dogfish and mammalian phagocytes is mediated by the same recognition protein.
Protein synthesis in cell free extracts of Lytechinus pictus eggs. MATT WINKLER, ELLEN BAKER, AND TIM HUNT.
The rate of protein synthesis increases 20- to 30-fold upon fertilization of the sea urchin egg as maternal mRNA is mobilized into polysomes. The increase in intracellular pH accompanying fertilization appears to play a major regulatory role in this activation. A cell- free protein synthesizing system prepared from Lytccliiims pictus eggs was used to study this regulatory process. These cell-free systems show excellent incorporation of amino acids into protein after an initial, somewhat variable lag. Raising the pH shortens this lag and appears to mimic the activation of protein synthesis ;'» vii'o.
These systems will translate added globin, histone and TMV mRNAs at both pH's, as measured by the appearance of new labeled bands on SDS gels. The added mRNAs, like the endogenous mRNAs, are translated to a much lesser extent at pH 6.9 than at 7.4. Addi- tion of even large amounts (up to 320 /ug/ml ) of histone mRNA results in at most a 2-fold stimulation of protein synthesis at pH 6.9 and some degree of inhibition at pH 7.4. Analysis of products suggests that the added mRNA competes with the endogenous mRNA at pH 7.4, while it augments existing protein synthesis at pH 6.9. Previous analysis of the protein synthetic machinery in the intact egg has been interpreted to indicate that the maternal mRNA is unavailable for translation before fertilization; but the very modest stimulation of protein synthesis by added mRNA at pH 6.9 seems inconsistent with this idea, and suggests that other components of the translational machinery may be in a dormant state before fertilization. The transit time for a specific mRNA was measured in this system by the addition of TMV mRNA and assaying for the first appearance of completed product on SDS gels. The maximum transit time for the synthesis of the 130,000 MW product is 20 min at pH 7.4, or approximately one amino acid per second.
Supported by NIH training grant no. TG-HD07098.
Low level excitation of cJilorotetracycline fluorescence in Haemanthus endosperm cells using image intensification. STEPHEN M. WOLNIAK, PETER K. HEPLER, WILLIAM T. JACKSON, AND GEO. T. REYNOLDS.
Cell plate formation in dividing endosperm cells of Hacnuintliiis involves the migration of calcium-rich vesicles to the equatorial region of the cell, followed by their coalescence at the site of incipient wall deposition. The calcium-membrane probe, chlorotetracycline (CTC, 10-50 /AM ) permits visualization of the plate-forming vesicles in living cells with fluorescence microscopy. CTC fluorescence is punctate and is localized at the spindle poles from prophase through late anaphase. Previous studies have shown that the poles are enriched in endoplasmic reticulum, mitochondria and plastids. By the onset of telophase, there is a dispersion of fluorescence at the poles and a concomitant increase in the region of the forming plate. Since visualization of fluorescence with intense near-UV light (390-410 nm) often disrupts normal divisions, we reduced incident excitation light by a factor of approximately 250 with a neutral density filter. We then employed image intensification of the fluorescence emission to study plate deposition as a dynamic process in living sells. Fluorescence micrographs were
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 403
taken at 15 min intervals during anaphase and telophase. In agreement with the high intensity fluorescence images, we first observed an increase in spindle midzone fluorescence when the poleward migration of the kinetochores was only 70 to 90% complete. At this stage of division, trailing chromatids extend through the equatorial region of the spindle. CTC-fluorescent material appears to aggregate at numerous sites in the phragmoplast, and as chromatid migration progresses, the highly fluorescent plate thickens. This technique demonstrates the feasibility of image intensification fluorescence studies on living cells that are normally adversely affected by intense short wavelength light (Reynolds, 1979, Biol. Bull.. 157: 391).
Supported by NIH grant RO1-GM -25120 to P. K. H. ; NSF grant PCM-7805172 to W. T. J.; and DOE contract EY-76-S-02-3120 to G. T. R.
Characterization of the hemoglobin of the elani, Astarte castanea. THOMAS D. YAGER AND K. E. VAN HOLDE.
The hemoglobin of the clam Astarte castanea is a large ( 10T dalton) protein which binds oxygen non-cooperatively. At pH > 9 in the absence of divalent cations, this protein dis- sociates into a single subunit.
Clams were placed on ice 10 min and then easily opened by severing the adductor muscles. At 4° C, hemolymph was collected from the pallial cavities by pipette, centrifuged 7 min at 13,000 X g, scrubbed with CO and dialyzed against tris or bicarbonate containing 10 mM EDTA. The dialysate is a virtually pure solution of the subunit. Polyacrylamide gel-electrophoresis in SDS revealed one band at 340,000 daltons, with a trace higher-MW contaminant (the sample having been boiled in \% SDS//3ME for 2 min and run upon 7.7 X 2.6 and 3.1 X 2.6 Laemmli slabs). Analysis in the ultracentrifuge gave Sai. »" = 11.3 S and, for the concentration-dependence of S, ks = 45 cm3/g (with v assumed to equal 0.7425 cm'Vg).
Purification of the subunit was achieved by molecular sieve chromatography of the dialysate (through Bio-Rad A-1.5 m beads, mesh 200, on an 80 X 1.6 cm Pharmacia column). A single eluent peak was observed, for which the ratio of 414/280 nm absorbance was constant at 2.3. The trailing edge of the peak was homogeneous in sedimentation velocity and in SDS/PAGE (with the sample heated to 60° in 1% SDS/ySME for 2 min, and run upon a 5.2 X 2.6 Laemmli slab). Sedimentation equilibrium analysis also showed a single component, with MW = 342,000. Using the Sao, •»•" and MW values from ultracentrifugation, the frictional coefficient was cal- culated to be 1.50. This relatively high value indicates that the subunit has some asymmetry.
This work was done in the MBL Physiology course, with funding from PHS training- grant no. T32-GM-07784.
In vitro reassembly oj squid brain nenrofilainents and their purification by assembly- disassembly. ROBERT V. ZACKROFF, ANNE E. GOLDMAN, AND ROBERT D. GOLDMAN.
Neurofilaments from squid brain tissue were disassembled at high ionic strength and reassembled upon lowering the ionic strength to physiological levels. Brains were homogenized in and extracted with disassembly buffer ( 1 M KC1, 0.25 M MES, 5 mM EGTA, 1 mM PMSF, pH 6.6) and centrifuged at 250,000 X </ for 90 min at 4° C. The resulting supernatant contained soluble subunits which rapidly reassembled into neurofilaments upon dilution with 9 volumes of reassembly buffer ( 0.25 M MES, 5 mM EGTA, 1 HIM PMSF, pH 6.6). Reassembled neurofilaments harvested by centrifugation consisted of one major polypeptide of approximately 60,000 daltons, and three minor polypeptides of higher molecular weight which were barely detectable on normally loaded (15 /ug) slab gels. Further purification by a second cycle of disassembly, clarification, and reassembly resulted in retention of all four poly- peptides. Negative staining of 1 -cycle purified and subsequently depolymerized neurofilaments revealed the presence of ca. 3 nm diameter, 5 to 25 nm long subunits. Upon dilution into reassembly buffer, the turbidity increased abruptly and 10-nm diameter filaments appeared. This abrupt turbidity increase was followed by a slower turbidity rise to plateau which was
404 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
correlated with neurofilament elongation. Reassembled neurofilaments exhibited the light- scattering properties of long rods, indicating that turbidity may be used as a measure of poly- mer weight concentration. In addition to neurofilaments, negative staining revealed the presence of dense structures from which filaments often appeared to radiate. The dense structures were also present after salt-induced disassembly of neurofilaments, and were retained through at least two assembly-disassembly cycles. These results constitute the first successful purification of neurofilaments by assembly-disassembly. Analysis of possible homologies of neurofilament polypeptides and polypeptides of intermediate filaments from non-neural cells is in progress.
This work was supported by NSF and the Runyon-Winchell Cancer Fund.
Bacterial denitrification: a gas chroinatograpliic stitdv using acetylene inhibition of N-20 rcditctase in two hour incubations. C. M. ZACKS, P. A. STEUDLER, AND J. M. MELILLO.
Current techniques for measuring denitrification potential in soil by the acetylene block method require incubation times of many hours or days. We have determined that denitri- fication characteristics of soil change rapidly after sampling, suggesting that misleading rates will be obtained unless the incubation is initiated within a day of collection, and is completed as soon as possible. A system was developed to measure denitrification in unaltered field samples in a 2-hr period. This was made possible by detection of N-O in the 0.02-0.2 ppm range by electron capture, and use of N2 instead of He as the balance gas in incubation vessels. In this range, short-term incubations required greater reproducibility of N^O concentration values than could be obtained with a N^O-He mixture. Improvement was noted when N» was used in place of He, which was attributed to improved homogeneity of the N2O-N2 system over N-O-He.
In preliminary studies of a forest ecosystem, soil from clearcut and undisturbed areas was assayed without further laboratory alteration. It was found that activity in clearcut areas was up to 30 times greater than in the undisturbed control (650 pg N-O-N/hr vs. 20 pg N2O-N/(hr-g organic wt). This large difference was confined to surface layers alone. Correlations were found between denitrification potential and the concentration of mineral nitrogen ions, as determined by extraction with 2 N KC1. Nitrate-nitrite maintained a constant low level over a range of denitrification rates, suggesting rapid nitrate uptake, while ammonium concentrations were found to be high in high potential areas, and low in inactive sites. Rapid uptake of nitrate is supported by the finding that addition of this ion induced dramatic increase in activity, but further investigation is required to assess the relative contributions of nitrate and ammonium precursors to the denitrification pathway.
Fetaturcs of elasinobranch eye lenses relative to those of humans. SEYMOUR ZIG- MAN AND TERESA PAXHIA.
Comparisons of elasmobranch lenses with those of humans reveal differences in size, visual accommodation and pigmentation and similarities in growth processes and structural protein distribution. Most similar is the age-related buildup of highly aggregated proteins that derive from lower molecular weight precursors. These aggregates are firmly bound to the fiber cell membranes, which are structurally similar in both species. Dogfish (Mustclus canis ) lenses remain transparent in spite of the presence of these aggregated complexes which would cause light scattering in human lenses. Visibly fluorescent pigment is present at very low levels in dogfish lenses, at intermediate levels in those of surface-swimming sharks, and at very high levels in human lenses, especially in the membrane complexes. Lens fiber cells of elasmobranchs have similar shapes and knoblike interdigitations to those of man, but are several times larger. Their membranes accumulate soluble crystallins which cannot be removed by extensive washing with 8u urea. The major SDS-solubilized membrane protein
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 405
of dogfish lenses has a molecular weight of 25,000 daltons, but this may include a certain amount of soluble crystallins. Heating in SDS does not aggregate this protein fully. Near- UV light exposure of lens homogenates in the presence of 1 tmi tryptophan increased the binding of protein and non-tryptophan fluorescence to dogfish fiber cell membranes substantially, and electron microscopic examination revealed much greater electron density. The data suggests that additional binding of non-membrane protein to lens membranes could result from exposure of lenses to near-UV light, with fluorescent and pigmented tryptophan photo- products serving as crosslinking agents.
Supported by University of Rochester (Pledger and Mullie Funds), National Eye Institute of the PHS (EY 00459), and Research to Prevent Blindness.
Continued from Cover Two
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CONTENTS
CORNELL, JOHN C.
Salt and water balance in two marine spider crabs, Libinia emarginata and Pugettia producta. I. Urine production and magnesium regulation 221
D'ABRAMO, LOUIS R.
Dietary fatty acid and temperature effects on the productivity of the cladoceran, Moina macrocopa 234
FUJIMORI, TAKUMI, AND SETSURO HIRAI
Differences in starfish oocyte susceptibility to polyspermy during
the course of maturation 249
KOMATSU, MI£KO, YASUO T. KANO, HIDEKI YOSHIZAWA, SHOJI AKABANE, AND CHITARU OGURO
Reproduction and development of the hermaphroditic sea-star, Asterina minor Hayashi 258
LANE, JACQUELINE Moss, AND JOHN M. LAWRENCE
The effect of size, temperature, oxygen level and nutritional condition on oxygen uptake in the sand dollar, Mellita quinquiesperforata (Leske) 275
LEE, HSUEH-TZE, AND RICHARD D. CAMPBELL
Development and behavior of an intergeneric chimera of hydra (Pelmatohydra oligactis interstitial cells : Hydra attenuate epithelial cells) 288
MARCUS, NANCY H.
On the population biology and nature of diapause of Libidocera aestiva (Copepoda : Calanoida) 297
MOFFETT, STACIA
Locomotion in the primitive pulmonate snail Melampus bidentatus: foot structure and function 306
ROBERTSON, ROBERT, AND TERRY MAU-LASTOVICKA
The ectoparasitism of Boonea and Fargoa (Gastropoda : Pyramidellidae) 320
SCARBOROUGH, ANN, AND EARL WEIDNER
Field and laboratory studies of Glugea hertwigi (Microsporida)
in the rainbow smelt Osmerus mordax 334
WHITTAKER, J. R.
Development of tail muscle acetylcholinesterase in ascidian embryos lacking mitochondrial localization and segregation 344
ABSTRACTS OF PAPERS PRESENTED AT THE MARINE BIOLOGICAL
LABORATORY. , ... 356
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Continued on Cover Three
Vol. 157, No. 3 December, 1Q79
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
Reference: Biol. Bull. 157: 407-421. (December, 1979)
A FIELD STUDY OF GROWTH AND REPRODUCTION IN APLYSIA CALIFORNICA1
TERESA E. AUDESIRK-
Catalina Marine Science Center, University of Southern California.
Aralon, California 90704
Some aspects of reproduction in the marine opistobranch Aplysia californica have been the subject of considerable research over the past ten years. Descrip- tion of a cluster of neurosecretory cells in the central nervous system was followed by extensive work directed toward characterization of their secretory product, which was found to induce egg-laying (Arch, 1972; Arch, Earley, and Smock, 1976; Kupfermann, 1967, 1970; Pinsker and Dudek, 1977). Strummwasser, Jacklet, and Alvarez (1969) noted a yearly cycle both in egg-laying hormone production and in receptivity to the substance. Thus initial investigation of the reproductive cycle in A. californica was from the standpoint of its neurohormonal control.
To date, no long-term studies of natural populations of this species have been published. Aplysia piinctata, found in the eastern Atlantic, has been more extensively investigated. A 16-month study of this species from Trearddur Bay, Anglesey (Carefoot, 1967a) led to the conclusions that A. piinctata is an annual, breeding between May and October, with major settling in the autumn. The red alga Plocamium coccintitui supplies a substantial part of the diet of the Trearddur Bay population (Carefoot, 1967a), in addition to supporting more rapid growth than several other algal species being consumed (Carefoot, 1967b, c).
A major difference between A. piinctata and A. californica is size. While A. piinctata rarely exceeds 80 g (Carefoot, 1967a), A. californica often exceeds 3000 g with a species record of 6800 g reported by MacGinitie and MacGinitie (1968). The growth rate and life span necessary to achieve such size have not been documented, nor have seasonal changes in abundance, weight, or reproductive
1 Contribution Xo. 36 from Catalina Marine Science Center, University of Southern California.
2 Present address : Division of Biological Sciences, University of Missouri, Columbia.
Missouri 65211.
407
Copyright © 1979, by the Marine Biological Laboratory
Library of Congress Card No. A38-518
(ISSN 0006-3185)
408 TERESA F, AUDES1RK
activities of a natural population been reported. This paper describes the results of such ati investigation.
MATERIALS AND METHODS
This study was conducted between July 1972 and August 1975 at the University of California's Catalina Marine Science Center on Santa Catalina Island, 25 km off the California coast south of Los Angeles. Two field study areas were selected which were in close proximity to the marine laboratory and known to support subtidal populations of A. calif arnica (subsequently referred to as Aplysia}. Site 1 covered part of Bird Rock, a small island approximately 500 m from shore. The area investigated covered about 35 m of shoreline and extended to a depth of 8 m. The total area of ocean floor studied was roughly 5600 m2. The sea bed sloped gently within the study area, but dropped more precipitously around the rest of the rock island.
Site 2 consisted of approximately 140 m of coastline starting inside Big Fisher- man's Cove on Catalina Island, extending a short distance east along the coast and to a depth of about 8 m for a total sea bed area of approximately 2800 m".
Except for the collections described below, the Aplysia populations in these areas were protected from human disturbance.
Collections were made once each month at each site, beginning in July of 1972 at Site 1, and in June of 1973 at Site 2. A monthly collection at each site was continued through August of 1975. Each month, weather permitting, from two to five divers using SCUBA covered the bottom in a zigzag pattern between the depths of 1 and 8 m along the length of the study area (depths were approximate as indicated by divers' depth gauges). Because most specimens of Aplysia were found in water shallower than 5 m, on some dives the area below this depth was not searched. Each specimen of Aplysia encountered was collected. Divers rarely found animals weighing less than 20 g due to the searching techniques used, and the tendency of smaller animals to be camouflaged amidst algal fronds. The data on abundance and weight presented in this paper therefore exclude all newly metamorphosed specimens and most juveniles of less than 20 g.
From July of 1972 through March of 1973, the presence or absence of copulat- ing individuals was noted during the monthly collections. From April 1973 through the end of the study period, additional data were collected on underwater slates during the dives. These included the number of animals paired or aggregated, and in many cases the number of animals in individual aggregations. Paired animals were usually copulating and were always in physical contact with one another. An aggregation was defined as a group of three or more speci- mens of Aplysia in physical contact with one another. Such groups generally included individuals engaged in egg-laying, copulation, or both. At any given time the aggregation could include (or on rare occasions be entirely composed of) animals not engaged in either of these activities (see also Kupfermann and Carew, 1974).
After collection, the animals were brought into the laboratory where each was weighed (after being placed on a paper towel for a few seconds to drain off
APLYSIA GROWTH AND REPRODUCTION 409
excess water). Beginning in August 1973, each animal was tagged with a one-half inch Floy Tag (Floy Tag and Manufacturing, Seattle, WA), colored with waterproof marker and stamped with an identifying number or letter. A tagging gun was used to insert the tag through one of the parapodia. While in the laboratory, the animals were housed in aquaria supplied with a continuous flow of fresh sea water within 1 ° C of ambient ocean temperature. After being tagged and weighed, the animals were returned to the study site, usually within 24 hr of the time of capture.
Histological examination of gonads from Aplysia was performed each month from October 1973 through September of 1975. To assure a supply of gonads throughout the year and to conserve local populations, gonads were obtained from specimens of Aplysia sacrificed for neurophysiological research at the California Institute of Technology in Pasadena, California. These animals were obtained (through Pacific Biomarine Supply Co.) from Palos Verdes on the California coast about 25 km north of Catalina Island. These were maintained in a recirculating sea water system without feeding on a regular schedule of 12 hr of darkness alternating with 12 hr of light. The temperature was maintained at a constant 14° C.
The animals whose gonads were examined had been maintained under these conditions for an average of 11 days. Each animal was weighed after removal of excess water as described above. A piece of gonad (3 to 5 mm2) was removed (preliminary measurements had shown that average oocyte diameter is uniform throughout the gonad) and fixed in Bouin's solution made with sea water. The tissue was then dehydrated, cleared, and embedded in Paraplast. Non-serial (usually every tenth section was retained) 5-mm sections were stained using Cason's modification of the Mallory-Heidenhain stain (Humason, 1967). The stained sections wrere examined microscopically, and oocyte diameter measured with an ocular micrometer. To establish a standard point of reference for measure- ment, only oocytes in which nucleoli were visible were measured. According to Thompson and Bebbington (1969), the nucleolus is prominent until the Aplysia oocyte has completely matured, at which time it disappears. Thus a slight bias against very large oocytes may be introduced by this method. The average diameter of the first 50 oocytes in which the nucleolus was visible was deter- mined. Fixation seemed to have little effect on oocyte size, as the diameter of the largest oocytes measured (80 /u,m) wyas approximately the same as a newly laid egg. In extremely immature gonads, oocytes were small and difficult to measure with certainty. Based on initial measurements, an average value of 7 /j.m was assigned to the oocytes of such immature gonads. The number of animals sampled each month ranged from 5 to 20, but in 19 of the 24 months, ten or more animals were examined.
RESULTS
Seasonal variation in weight
Figure 1 shows size-frequency distributions of all collections at Site 1 in which more than six animals were found. Presentation of the data in this form
410
TERESA E. AUDESIRIC
reveals some trends as well as the considerable variability in population structure from one year to the next. The tendency of small animals to appear in the winter and spring can be seen in 1973 and 1975. An increase in overall size during late spring and summer is evident in 1974 and 1975. Unpredictable fluctuations in size and numbers also occurred. In late winter and early spring of 1973, juveniles appeared as expected, and had begun to increase in size by May. In June, however, the population inexplicably disappeared and remained absent for the remainder of the summer and fall. A few animals appeared in December, and specimens of Aplysia in a range of sizes were present through the spring of 1974. The sizable component of small animals present in the spring of the preceding and succeeding years was absent.
Figure 2, which shows the trends apparent over the three-year sampling period, was generated by combining the data for each month throughout the sampling- period. Each site is graphed separately, with each point representing the weighted average of the collections made during that month. Seasonal differences in the size of the animals are apparent. Highest weights were recorded during the summer months of June through August, while weights were generally lowest between February and May. There is also a significant difference in the weights of the populations found at the two study sites. These were compared by performing a
12 1 73 2
nc nc 117
10
0
I
I
I
10
n c
12 1 75 2
3 35 69
LLLL
FIGURE 1. Size-frequency distribution of all collections at Site 1 in which more than six animals were found. The width of the figure corresponds to the percentage of animals found in each size class. The uppermost number is the date, while the underlined number imme- diately below it shows the number of specimens of A. calif arnica collected that month. N.C. : no collection.
•IPLYSIA GROWTH AND REPRODUCTION
411
1200-
wt
S.2.
g
800
400-
0 N D
FIGURE 2. Average weights of specimens of A. californica collected at each study site, plotted by month. S. 1 : Site 1 ; S. 2 : Site 2. The upper of the two numbers associated with each data point is the total number of animals collected during that particular month throughout the study period. The lower number is the number of years in which samples were collected during that month. Only months in which six or more specimens of A. cali- jornica were collected are graphed.
paired /-test on the average weights for the ten individual months in which more than six animals were collected at each site (two-sided paired f-test ; t = 3.6, P < 0.01). The two study sites differed in the availability of food algae readily consumed by Aplysla. Site 1 was dominated by beds of the surfgrass Phyllospadix to a depth of about 3 m. This plant is not normally consumed by Aplysla (Winkler and Dawson, 1963; personal observation). Below 3 m, the giant kelp Macrocystls predominated. Numerous other algal species were present on the rocky substrate including several which have been reported to serve as food for Aplysla (Leighton, 1966). Of these, only two were readily consumed under laboratory conditions. Laurencia and Plocanrium. Neither of these algae was abundant at Site 1. Site 2 supported (among many other algal species) large beds of Plocamiwn between the depths of 1 and 4 m. Below7 this depth, Macrocystls predominated.
412
TERESA E. AUDES1KK
100
80-
AWt
60
40-
20^
0 --
-20
J FMAMJ JASOND
FIGURE 3. Graph of data from Table I. Changes in weight are expressed as percentages of initial body weight and plotted according to month. Data from both study sites collected throughout the tagging program are presented. Only months in which five or more animals were recaptured are graphed.
The average weights of animals used at Caltech also show seasonal variation, in spite of attempts by Pacific Biomarine Supply Co. to provide animals of roughly uniform size. Figure 6 shows that animals were smallest during late winter and early spring, while maximum weights were reached during the summer and fall. This parallels the trend seen in the populations studied on Catalina Island.
Tagging and recapture
A total of 154 specimens of Aplysia were recaptured between June of 1973, when the tagging program was initiated, and August of 1975, when the study was completed. Altogether, 728 animals were tagged during this period. Of those recaptured, 101 had been tagged the previous month. Only these were used to generate Figure 3, to best illustrate the growth trends typical of individual months. In Figure 3, field weight changes have been expressed as percentages of beginning body weight. Data have been combined for animals collected at both study sites, and for the same month throughout the tagging period. Only months in which five or more individuals were recaptured are graphed. Since few animals were found in winter (Fig. 4) there were few recaptures between October
APLYSIA GROWTH AND REPRODUCTION
413
and January. Again, strong seasonal trends are evident, with the maximum per- cent increase occurring in late winter and early spring when the animals are rela- tively small, and decreasing as the average weight of the population increases. A net loss of weight occurred during August and September. Table I shows the number of recaptured animals which had gained or lost weight during each month of the year. Weight loss was almost never observed between January and June, but after June, the majority of animals recaptured had lost weight.
In some instances, the same individual was recaptured more than once, pro- viding information on the weight changes which individuals experience with age and season. These cases reveal that this species is capable of considerable growth during a single month. One animal from Site 2 gained 910 g between February and March of 1975, and an additional 1178 g between March and April of that year. Two animals which were captured first in the spring and later in the summer each showed an initial \veight gain, followed by summer weight loss.
In this study, specimens of Aplysia were tagged with considerable success. Nine animals were recaptured after four or five months, indicating that some individuals spend a considerable time within a limited area. This species is capable of traveling relatively long distances, however. Kupfermann and Carew (1974) found that A. calijornica could easily cover a distance of 75 m (net) in a two-day period. Because many animals could have moved out of the study areas from one month to the next, recapture data from this study were not dependable for population size estimates.
Seasonal changes in abundance
It was noted during collections that the abundance of Aplysia seemed to vary seasonally, with a maximum during the spring, and a nearly total disappearance during the months of October and November. Abundance was estimated by determining the number of animals captured per unit effort, with one unit of effort defined as one diver-hour. The trend that emerges when data taken through- out the study are graphed by month is shown in Figure 4A. While few or no animals were found during regular monthly dives between October and Decem- ber, juveniles ranging from 1 mm to 2 cm in length were found on Plocamium
TABLE I Weight changes under field conditions.
|
Month |
N |
Initial Wt. (mean, g) |
Wt. Change (mean, %) |
No. gaining |
No. losing |
|
Jan |
2 |
380 |
42 |
2 |
0 |
|
Feb |
5 |
475 |
85 |
4 |
1 |
|
Mar |
16 |
386 |
54 |
16 |
0 |
|
Apr |
11 |
695 |
98 |
11 |
0 |
|
May |
7 |
545 |
68 |
7 |
0 |
|
Jun |
12 |
865 |
30 |
10 |
2 |
|
Jul |
15 |
1060 |
35 |
7 |
8 |
|
Aug |
31 |
644 |
-13 |
3 |
27 |
|
Sep |
5 |
906 |
-11 |
2 |
3 |
414
TERESA E. AUDESIRK
25
201
15
10
5
0
30 25 20 15 10 5 0 40 35 30 25 20 15
io
5^ 0
15
A
B
C
1972-1975
S.2.
JASONDJFMAMJJASONDOFMAMJJASONDJFMAMJJA
1972 1973 1974 1975
FIGURE 4. Abuandance of animals expressed as Aplysia per diver-hour, plotted against the month. A : data from both study sites over the entire collection period are combined in a single "composite year". B : abundance at Site 2 plotted against actual time. C : abundance at Site 1 plotted against actual time. D : average abundances for both sites graphed for that period during which both sites were sampled. Dotted lines indicate that one or more collections were missed during the period covered.
APLYSIA GROWTH AND REPRODUCTION 415
and Lanrencia brought into the laboratory. Juveniles very similar in color to the red algae were found hidden among the fronds, and these small animals seemed to be especially abundant during fall, winter and early spring.
Although distinct trends emerged from the total data collected (Figure 4A), Figures 4B, C and D show the considerable variability of the two sites from year to year. For example, collections at Site 1 during June, July and August 1973 produced a total of five individuals, while collections during the same months of 1975 produced 183 animals (including 25 recaptures).
A predictable occurrence at both sites was the nearly total disappearance of animals in the fall (October through December). Many individuals collected during this period exhibited symptoms of physical deterioration. These included erosion of the free edges of the parapodia and loss of pigment resulting in pale patches on the body. Such animals frequently died in captivity, an unusual occur- rence at other times of the year. Few dead animals were found in the field at any time of the year.
Reproductive activity
When collections were initiated at Site 1 in July of 1972, copulation was already in progress. The following year, no copulation or egg-laying was observed there due to the disappearance of the population in June (May was the last successful collection in 1973 at Site 1, and no mating was observed at that time.) Collec- tions at Site 2 began in June 1973, at which time copulation was occurring. In 1974, a few large animals were first seen mating in April at Site 1, and in May at Site 2. In 1975, again a few large animals were seen mating at Site 2 as early as April, but at Site 1, copulation and egglaying were not observed until July, at which time many animals were participating.
Data on the percentage of animals aggregated, and the number of animals making up aggregations, are plotted in Figure 5. In April and May, the per- centage of animals observed in pairs and aggregations was small, but increased to 80% or more during the months of July and August (Fig. 5A). Fifty-three aggregations are plotted according to the number of animals participating (Fig. 5B), which ranged from 2 to 19. Pairs of animals were observed more frequently than any other size grouping. Since Aplysia mates in chains of indefinite length, a pair of animals may be considered the shortest possible mating chain.
Seasonal changes in oocyte diameter
The data graphed in Figure 6 show the changes in oocyte diameter which under- lie seasonal breeding in A. calif ornica. Most animals collected in winter (January through March) have extremely small gonads containing few recognizable oocytes, these averaging about 7 p.m in diameter. In April and May, the gonad is charac- terized by a mixture of oocytes of all sizes, ranging up to 80 ,u.m. the approximate diameter of a newly laid ovum. The proportion of large oocytes increases with time, until, by the months of September, October, and November, mature animals show few of the small oocytes which predominate in the spring. The low standard error in average oocyte diameter in animals collected between January and October inidicates that oocyte maturation is relatively synchronized. In November and
416
TERESA E. AUDES1RK
December, the standard error increases due to the inclusion of some sexually immature animals. It should be noted that, during the winter months, size is not an accurate indicator of sexual maturity. Sexually immature specimens of up to 600 g have been found at this time.
22-
20-
18
•J
O 14
o-
W>
<T> 12
^
W 10
ons
A
B
8 10 12 14
- in Aggregation
16
18
n
20
FIGURE 5. A : percentage of the total number of specimens of Aplysia captured each month that were paired or aggregated. Data from both study sites throughout the collection period are combined. B : frequency of observation of aggregation plotted against aggregation size.
APLYSIA GROWTH AND REPRODUCTION
417
800-
Wt
g
400 H
60-
OD gm
40-
20-
— i 1 1 1 1 1 1 1 1 r~
0 H D J F M A M J J
19X3 1974
SONDJFMAMJJAS
1975
FIGURE 6. Mean weights (upper line) and oocyte diameters (lower line) of specimens of Aplysia plotted against time. These animals were collected at Palos Verdes, California, by Pacific Biomarine Supply Co. Most data points represent at least 10 individuals (see text). These were sacrificed throughout the month indicated, and collected an average of 11 days prior to being killed. Bars indicated standard error of the mean.
DISCUSSION
Data presented in this paper support the hypothesis that Aplysia have a life expectancy of approximately one year, and a single extended period of copulation and egglaying which begins late in spring and ends in the fall with the death of the sexually mature population. Although migration into deeper water and reappearance in the spring cannot be entirely ruled out, there is presently no evidence in support of this hypothesis. Animals found in the spring tend to be smaller and more abundant than those found later in the year. They appear healthy and live for long periods under laboratory conditions, in contrast to animals obtained in the fall whose skin is often eroded and who usually die after a short period of captivity.
The extended period of egg production might be expected to result in asynchrony with respect to size and sexual maturity within the population. However, there
41 S TERESA E. AUDESIRK
is evidence that increasing water temperature in the spring provides a synchronizing cue for the initiation of gonadal development (Audesirk, 1976; Smith and Care- foot, 1967). Differences in survival and growth rate during different seasons might also result in greater uniformity in size than would be predicted from the long period of egglaying. Further study is required to test these hypotheses. The disappearance of nearly all animals larger than 20 g at Catalina Island as early as October is in contrast to the situation at Palos Verdes where sexually mature specimens are found until December. A comparison of environmental factors including water temperature, food availability, and wave exposure between i he two locations might provide clues to this discrepancy.
The mean weights of the populations at Palos Verdes and Catalina show pro- nounced seasonal differences consistent with an annual species (Figures 1, 2, and 6). On Catalina, small specimens of Aplysia, presumably offspring of the previous reproductive season, make their appearance between January and May. Maximum size is reached in June or July, and shows a decrease in August and September. In October, November and December, few animals are found (Fig. 4). Although trends in mean weight were roughly parallel for the two Catalina Island popula- tions sampled, a consistent and significant size difference was observed (Fig. 2). The apparent explanation for this is the relative availability of food algae at the two sites. At Site 1, no algal species known to be readily consumed by Aplysia under laboratory conditions was found in abundance. In contrast, Site 2 included large beds of the red alga Plocainiiini which A. californica will eagerly consume. In his study of A. pnnctata, Carefoot (1967a) found a strong correlation between the size of the animals and the abundance of Plocaiiiiuin. In a comparison of eight algal species consumed by A. pinicfata, Plocaiiiiuin was preferred over six other species, and supported most rapid growth (Carefoot, 1967b).
Data from tagging and recapture (Fig. 3; Table I) indicate that, at Catalina Island, A. californica experiences fastest growth between February and April, prior to the onset of breeding. The animal's capacity for rapid growth explains how some individuals may achieve sizes in excess of 3000 g in a single year. Growth slows as breeding intensity increases, and the average size reaches a peak in June or July. In August, when breeding is at its most intense (as indicated by the percentage of animals in pairs or aggregations in Fig. 5), a net loss of weight occurs. This loss is possibly attributable to two major causes. The first is loss of foraging time due to time spent in reproductive activities. Although the per- centage of time spent in breeding has never been quantified, field observations indi- cate that individuals could remain in aggregations for a day or longer. Kupfermann and Carew (1974) tagged the five animals at one aggregation site and, during four visits during the next five days, always observed one or more tagged animals at the site. The animals were not individually identified, and some had certainly departed and returned, but others may have remained. Aggregations and pairs of individuals appear almost exclusively during months when copulation is observed. It is hypothesized that the animals in aggregations which are not engaged in egglaying or copulation at any given time may have recently completed one or more of these activities, or may be about to engage in them. Further study is needed to test this hypothesis. During the present study, at Site 1 nearly all the mating aggregations were discovered in dense beds of the surfgrass Phyllo-
.IPLYSIA (ikOVYTH AXI) REPR( )I >tVTU )X 419
spadix, whose closely spaced blades seem to present an ideal anchoring site for the tangled egg masses. Since they do not consume this plant, the animals at Site 1 probably did not eat during the time spent in aggregations. Kupfermann and Carew (1974) also never observed feeding by animals in breeding aggregations.
The second major factor contributing to weight loss is the massive egg produc- tion by these hermaphrodites (MacGinitie, 1934) which requires large energy expenditures.
Although seasonal trends in abundance are evident, these are superimposed upon dramatic fluctuations from one year to the next. Beeman (1977) noted that opisthobranch populations "tend to be erratic and sporadically explosive," a gen- eralization with some applicability to Aplysia. Eales (1921) and Carefoot (1967a) reported similar fluctuations in populations of A. punctata.
Like A. pnnctata, which is found dead or dying in the field between September and December (Carefoot, 1967a), A. califoniica shows physical deterioration manifested externally as tissue erosion during the same months. A related phe- nomenon was reported by Lickey, Wozniac, Block, Hudson, and Augter (1977). who noted that specimens of A. califoniica in the laboratory seldom die between December and July, but rarely survive more than a month between August and November. For unknown reasons, dead animals were rarely found in the field at Catalina Island. In the laboratory, however, death was common during these months.
The brief life cycle of A. califoniica and the dramatic changes in its physiology and behavior as it progresses from sexual immaturity through the reproductive state to senescence have important implications for investigations of Apl\sia neurobiology. Changes in neurohormones with respect to season have already been noted (Strummwasser, Jacklet, and Alvarez, 1969). The formation of breeding- aggregations, copulation, and egglaying are dramatic behavioral changes correlated with sexual maturity. The imminent death of mature animals obtained in the fall must also be taken into account during investigations of Apl\<sia behavior and nervous system function.
SUMMARY
1. Observations of field growth rates, reproductive activities, and abundance of Aplysia calfiornica were made over a three-year period on Santa Catalina Island off southern California.
2. The mean weight of the population was found to vary with the location in which the animals were collected, presumably as a result of differing availability of food.
3. Seasonal weight differences were also apparent. In general, small specimens of A. calif arnica appear between February and May. Mean weight reached a maximum between June and August. Considerable variability was encountered from year to year.
4. Tagging and recapture showed that growth rates reached a maximum in the spring just prior to breeding. The rate decreased thereafter until weight loss was experienced in August and September.
420 TERESA K. AUDKSIRK
5. A. calif arnica was usually most abundant in the spring, with numbers de- creasing during the summer. The animals almost completely disappeared during the months of October, November, and December with the exception of extremely small specimens found on algae.
6. Breeding activity was occasionally observed as early as April and reached its greatest intensity during July and August when at least 80% of the animals collected were in breeding aggregations.
7. Histological examination of gonads showed maximum oocyte diameter between June and October, and minimum between January and March.
8. Data are consistent with an annual species whose extended summer breeding period is terminated by the death of mature individuals during the fall.
LITERATURE CITED
ARCH, S., 1972. Polypeptide secretion from the isolated parietovisceral ganglion of Aplysia
californica. J. Gen. Pliysiol., 59 : 47-59.
ARCH, S., P. EARLEY, AND T. SMOCK, 1976. Biochemical isolation and physiological identifica- tion of the egg-laying hormone in Aplysia californica. J. Gen. Pliysiol., 68 : 197-210. AUDESIRK, T., 1976. The Role of Seasonal Periodicity, Chemical Communication, and Chemo-
receptive Organs in Reproduction in Aplysia californica Cooper; Ph.D. thesis, Uni- versity of Southern California, Los Angeles, California, 201 pp. BEEMAN, R. D., 1977. Gastropoda: O pisthobranchia. Pages 115-180 in A. C. Giese and J. S.
Pearse, Eds., Reproduction of Marine Invertebrates, Vol. IV. Academic Press,
New York. CAREFOOT, T. H., 1967a. Studies on a sublittoral population of Aplvsia pnnctata. J. Mar. Biol.
Assoc. U.K., 47: 335-350. CAREFOOT, T. H., 1967b. Growth and nutrition of Aplysia pnnctata feeding on a variety of
marine algae. /. Mar. Biol. Assoc. U.K., 47 : 565-589. CAREFOOT, T. H., 1967c. Growth and nutrition of three species of opisthobranch molluscs.
Comp. Biochcm. Pliysiol., 21 : 627-652. EALES, N. B., 1921. Aplysia. Liverpool Marine Biological Memoirs No. 24. Proc. Trans.
Liverpool Biol. Soc.. 35 : 183-266. HUMASON, G. L., 1967. Animal Tissue Techniques. W. H. Freeman and Co., San Francisco,
569 pp. KUPFERMANN, L, 1967. Stimulation of egg-laying; possible neuroendocrine function of bag
cells of abdominal ganglion of Aplysia California. Nature, 216: 814-815. KUPFERMANN, L, 1970. Stimulation of egg-laying by extracts of neuroendocrine cells (bag
cells) of abdominal ganglion of Aplysia. J. Ncurophysiol., 33 : 877-881. KUPFERMANN, L, AND T. J. CAREW, 1974. Behavior patterns of Aplysia californica in its natural
environment. Bchav. Biol., 12: 317-337. LEIGHTON, D. L., 1966. Studies on food preferences in algivorous invertebrates of southern
California kelp beds. Pac. Sci., 20 : 104-113. LICKEY, M. E., J. A. WOZNIAC, G. D. BLOCK, D. J. HUDSON, AND G. K. AUGTER, 1977. The
consequences of eye removal for the circadian rhythm of behavioral activity in Aplysia.
J. Comp. PhysioL, 118: 121-143. MAC&NITIE, G., 1934. The egg-laying activities of the sea hare Tctliys calijornicus (Cooper).
Biol. Bull, 67: 300-303. MACGINITIE, G., AND N. MAC&NITIE, 1968. Natural History of Marine Animals (Second
Edition) McGraw-Hill, San Francisco, 523 pp. PINSKER, H. M., AND F. E. DUDEK, 1977. Bag cell control of egg-laying in freely behaving
Aplysia. Science, 197 : 490-493. SMITH, S. T., AND T. H. CAREFOOT, 1967. Induced maturation of gonads in Aplysia punctata
Cuvier. Nature, 215 : 652-653.
APLYSIA GROWTH AND REPRODUCTION 421
STRUM WASSER, F., J. W. JACKLET, AND R. B. ALVAREZ, 1969. A seasonal rhythm in neural
extract induction of behavioral egg laying in Aplysia. Comp. Biochem. Physiol., 29 :
197-206. THOMPSON, T. E., AND A. BEBBINGTON, 1969. Structure and function of the reproductive
organs of three species of Aplvsia (Gastropoda: Opisthobranchia) . Malacologia, 7:
347-380. WINKLER, L. R., AND E. Y. DAMSON, 1963. Observations and experiments on the food habits
of California sea hares of the genus Aplysia. Par. Set., 17 : 102-105.
Reference: Biol. Bull. 157: 422-433. (December, 1979)
SALT AND WATER BALANCE IN TWO MARINE SPIDER CRABS,
LIBINIA EMARGINATA AND PUGETTIA PRODUCTA. IT.
APPARENT WATER PERMEABILITY
JOHN C. CORNELL i
Department of Zoology. Uiiit'crsity of California, Berkeley, California 94/20, U. S. A., and the Bodcita Marine Laboratory, Bodega Bay, California 94923, U. S. A.
Iii studies of salt and water balance, permeability is an important parameter since knowledge of it, together with a knowledge of the appropriate driving force, permits predictive statements concerning net fluxes. A complication in this matter is that permeability may vary. A reduction in tracer water exchange rate has been found in a number of osmoregulating decapod crustaceans transferred to a dilute medium (Rithropanopeus harrisn, Smith, 1967; Capen, 1972: Carcinus maenas, Smith, 1970; Berlind and Kamemoto, 1977: Hemigrapsus nndus, Smith and Rudy, 1972; Palaemonetes png'w, Roseijadi, Anderson, Petrocelli and Giam, 1976). However, not all osmoregulating decapods respond to a dilute salinity in this fashion (Palaemonetes various, Parry, 1955: Carcinus maenas, Rudy, 1967: Penaeus duorarum and Uca spp., Hannan and Evans, 1973), nor does the same species necessarily respond in the same way under similar conditions. Also, there are alternative explanations for changes in water exchange other than a true change in permeability (Smith, 1970, 1976). However, the adaptive value of a reduction in water permeability when the osmotic gradient is increased is quite clear and, on the basis of the available evidence, a strong argument can be made in favor of changes in water permeability.
There is, perhaps, little reason to predict that an osmoconforming crab would respond to dilute salinity with a reduction in permeability ; however, when speci- mens of Libinia emarginata were transferred from 100 to 80% sea water, there was a reduction of 30% in water exchange after the first hour (Cornell, 1973). It can be argued that a reduction in water permeability is also an adaptive response to dilute salinity in an osmoconformer, but the argument is open to question. In the present study, some aspects of apparent water permeability have been examined in the osmoconforming crabs Libinia emarginata and Pugettia producta and in the osmoregulating crab Carcinus maenas. An attempt is made to identify some factors which may be important in changes in water exchange rates.
MATERIALS AND METHODS
General procedures
Specimens of Pugettia producta were maintained at 10-12° C in filtered sea water (SW), about one crab per 4 liters, at the University of California, Berkeley, California. Specimens of Carcinus maenas and Libinia emarginata were main- tained at 19 to 21° C in running SW at the Marine Biological Laboratory, Woods
1 Present address : Department of Zoology, Washington State University, Pullman, Washington 99164, U. S. A.
422
SALT AND WATER BALANCE IN CRABS 423
Hole, Massachusetts. The SW used at Berkeley was obtained from the Bodega Marine Laboratory, Bodega Bay, California and had an osmotic pressure of about 1015 milliosmols, while that at Woods Hole was about 945 milliosmols.
Exchange rates of tracer water were measured using D2O according to the technique described by Welsh, Smith and Kammer (1968). Specimens of Carcinits and Libinia were immersed for 11 and 15 min, respectively, in aerated 3-liter solutions of sea water (SW) plus 2.0 to 2.5% DoO maintained at constant temperature (21 ± 0.5° C) in a bath of running SW. Samples of blood wrere with- drawn by puncturing an arthrodial membrane with a drawn-out Pasteur pipet.
Heart rate was determined by implanting chronic platinum electrodes which just penetrated the hypodermis over the region of the heart. The electrodes were cemented to the carapace with quick-setting ("five-minute") epoxy glue, after scraping away the epicuticle from the region where the bond was to be made. Electrodes were implanted at least 24 hr prior to an experiment.
Measurements of pressure changes caused by the beat of the scaphognathite were made with a Statham model P23BC pressure transducer connected by polyethylene tubing (P.E. 190, 0.047 inch =1.2 mm inside diameter) to the cut-oft" tip of an 18-gauge hypodermic needle. A hole, positioned by external landmarks, was drilled through the carapace, and the needle tip was inserted through the hole and into the branchial chamber, just posterior to the scaphognathite. The needle tip was cemented in place with quick-setting epoxy. Measurements of heart and scaphognathite rates were recorded on a Grass Instrument Co. model 7a polygraph.
Isolated gills from Libinia were first flushed and then perfused with a solution containing inorganic ions in ratios given for Cancer perfusion fluid (Welsh, Smith and Kammer, 1968) and dextrose (1.5 HIM). The solution was buffered to pH 7.2 with "Tris" (about 25 HIM) and the final solution was adjusted to 945 milliosmols. A peristaltic pump, either a Buchler Instruments Polystatic Pump or a Sage model 375A, controlled the rate of perfusion and recirculated fluid between the gill and a small reservoir from which samples were taken. The total fluid volume of the system was about 1.5 to 2.0 ml. Polyethelene tubing was fitted to the afferent and efferent vessels and secured with silk thread. A small clamp, placed about 5 mm from where the tubing entered the gill, main- tained the proper spatial relationship between the two tubes. A rod, attached to the clamp, suspended the preparation in a beaker containing 300 ml of aerated SW plus y/o D2O maintained at 20 ±0.1° C. Constant stirring was provided by a magnetic stirrer.
A practical approach to estimates o\ apparent i^atcr permeability
Two different measurements of water permeability can be made, osmotic perme- ability and diffusive permeability.
Osmotic water permeability (Posm) - - LPRT/VW, where Lp is the hydraulic conductivity, R is the gas constant, T is the absolute temperature, and Vw is the partial molar volume of water. Lp is a measurement of permeability and, simply stated, is a flux divided by a driving force. The advantage of converting L,, into Posm is that by doing so, it is possible to compare P,,sm with estimates of diffusive water permeability, since both can be expressed in the same units.
424 JOHN C. CORNELL
Unfortunately, the conditions under which Lp may be determined are incompatible with most whole-animal experiments. However, the concept of expressing a net flux divided by a driving force is useful and has been adopted here. The symbol Lp* will be used to indicate a net flux divided by osmotic pressure and values will be expressed in ^l/(g-hr-osmol). Posm* will be expressed in cm3/ (g-sec) in keeping with the more conventional units [cm3/ (cm2 -sec) == cm/sec]. When expressed in cm3/(g-sec), Lp* is in the units of cm3/(g-sec-atm), R = 82.04 cm3/ (atm- mole -degree), T is absolute temperature in degrees K, Vw = 18 cm3/mole. Since permeable surface area is unknown in a whole animal, weight has been substituted and allowances must be made for this factor in comparisons where large weight differences occur.
In a decapod LP* wrill reflect not only water permeability, but other factors as well. Lp* only provides an adequate description of water movements across an ideal semi-permeable membrane which separates two solutions containing non- electrolytes. The characterization of a membrane which is permeable to solutes is more complex. When the solute is composed of univalent ions, the phenomeno- logical approach based on irreversible thermodynamics requires the determination of six membrane coefficients (see review by House, 1974). Since water move- ment is influenced by the presence of permeant ions, Lp must be measured in the absence of an osmotic gradient and current flow, and in the presence of a driving force of hydrostatic pressure. Clearly, the measurement of net water fluxes under these conditions would be difficult in an intact decapod crustacean. It is believed, despite some objections, that the present approach provides a meaningful way of dealing with the problem of making measurements of "water permeability" in a whole animal.
Diffusive water permeability (Pd) " kv/A, where v is the volume of the water pool, A is surface area and k is a rate constant. In a well-stirred, two-compart- ment system in a steady state, the tracer concentration, f(t), in the very small compartment which initially contains no tracer is given by f(t) = C(l -- exp — kt), where t is time, C is the concentration in the very large compartment, and k is a rate constant. The value of k is proportional to the diffusive water permeability, and in similar animals the constant is similar, so that k should provide a reasonable basis on which to make comparisons of permeability. In the present study, the results of the experiments with tracer water will be expressed in terms of the rate constant k in (1/hr), where k== (1/t) loge[C/(C -f(t))]. The symbol P,,* in cm3/ (g-sec) will indicate the analog to the more familiar P(1 in cm/sec.
RESULTS
Estimates of Lp*
For an animal in a steady state, Lp* may be estimated from the urine production rate and the osmotic difference between the blood and the medium. This approach is well suited to osmoregulators, but in an osmoconformer, the osmotic difference can be small (about 2 milliosmols in Pugettia} and thus subject to large errors in measurement. An alternative approach is to determine the rate of weight gain at time 0, when an animal is transferred to a dilute test medium.
SALT AND WATER BALANCE IN CRABS
425
TABLE I
Estimates of Lf in normal and nephrc pore-occluded specimens of Libinia emarginata and Pugettia producta. Analysis of covariance of the regressions of the transformed weights (see text) vs time indicates that there are significant differences between the initial rates of weight gain for normal speci- mens of Libinia (N == 19) and Pugettia (N ---- 10) (P <0.001 ) and for nephropore-occluded speci- mens of Libinia (N = 7) and Pugettia (N = 7) (P < 0.05).
|
Animal |
Libinia |
Pugettia |
||
|
State of nephropores |
Normal |
Occluded |
Normal |
Occluded |
|
Average weight (g) |
152 |
144 |
121 |
117 |
|
Rate of weight gain at / = 0, |
||||
|
expressed in volume |
||||
|
[Ml/(g-hr)] |
30 |
33 |
70 |
67 |
|
Osmotic gradient at t = 0 |
||||
|
(osmols) |
0.189 |
0.189 |
0.203 |
0.203 |
|
LP* CA^/Cg-hr-osmol)] |
159 |
174 |
345 |
330 |
At this moment, urine production is equal to that in the normal medium, the osmotic gradient is known, and the rate of weight change can be assumed to equal the net flux of water.
Empirically, it has been found that the equation f (t) = Ct exp — jt (where f (t) is the weight at time t, C is a constant and j is a rate constant) provides a good description of weight changes in a number of decapods transferred to a dilute medium. At time 0, the rate of change of f(t) can be determined from the first derivative and is simply C. Thus, by plotting the transformed weight data (transformed by loge[ ( (Wt/W0) •• l)(l/t)], where t is time and Wt and W0 are the weights at times t and 0, respectively) against time, the y intercept can be determined, and the value of C is equal to antiloge of the y intercept. The resulting plots are seldom strictly linear, but they are sufficiently so that it is an "easy task to estimate the intercept.
Using the above technique, the rates of weight gain were obtained from data on normal and nephropore-occluded animals (Cornell, 1976). Values for Lp* were calculated from these rates and the changes in osmotic gradients. The results, presented in Table I, suggest that Pugettia is about twice as permeable to water as Libinia. The average for normal and nephropore-occluded animals is 166 and 338 /Ml/(g-hr-osmol) for Libinia and Pugettia, respectively. From these estimates, it is possible to calculate the expected osmotic difference necessary to produce an influx of water equal to the rate of normal urine production (2.1 and 2.5 ju.l/(g-hr) for Libinia and Pugettia, respectively, Cornell, 1979) ; thus, 2.1/166 = 13 milliosmols and 2.5/388 == 7.4 milliosmols for Libinia and Pugettia, respectively. The estimate of 7.4 is greater than the observed value of 2 milliosmols in Pugettia. The osmotic difference between the blood and the medium is not known for Libinia; however, in both cases, these estimates are greater by about an order of magnitude than the colloid osmotic pressure (Mangum and Johansen, 1975). These discrepancies raise questions concerning the mechanism for the entry of water in an osmoconforming crab. One interpretation of the present results is that, whatever the mechanism, the driving force is equivalent to an osmotic pressure of between 7 and 13 milliosmols.
I !6
JOHN C. ( OKNKI.I
Libinia
IN = 16 JN=10 ]±2 SE
0
5 10
HOURS IN 80% SW
20
FIGURE 1. Changes in D»O water exchange rates in specimens of Libinia cmarginata and Carcinus macnas transferred from 100 to 80% SW, expressed as k in (1/hr), the hourly water exchange fraction. For specimens of Libinia, the results from two experiments are shown: the first, is indicated by the solid circles (average weight of crabs, 168 g) ; the second, is indicated by the open circles (220 g). For specimens of Carchnis, the results are indicated by the open squares (22 g).
I have calculated Lp* for specimens of Puycttia during the first several hours after transfer to 80% SW. The calculations suggest that there is a reduction in Lp* of about 60% during the first hour; however, this may be an artifact. The calculations, based on measurements which have relatively large variances (weight gain, urine production, and the osmotic gradient, Cornell, 1976), become increasingly sensitive with time to errors in measurement. Since a plot of the logarithm of the osmotic gradient between the blood and the medium versus time is not significantly curvilinear, a large reduction seems unlikely. Perhaps the safest conclusion is that the data are inadequate to indicate whether or not a short-term change in Lp* occurs.
Etitnatcs of k
The results of two experiments in which specimens of Libinia were transferred from 100 to 80% SW are shown in Figure 1. For practical reasons, the points at time 0 were actually determined 24 hr prior to the transfer of animals to 80% SW. In the first experiment, k in (1/hr) was 5.68 for animals in SW and 3.99 for the same group after one hour in 80% SW, a reduction of 30% (P < 0.001, paired i-test). The second experiment is similar to the first, but was carried out for a longer period of time. In this experiment, k, measured after one hour of
SALT AND WATER BALANCE IN CRABS
427
60 f
| 50
(ft •*->
no <U
-Q
LU40
\—
< DC.
CC
30
20
0
2 3
HOURS IN 80% SW
FIGURE 2. Changes in heart rate of specimens of Libinia einaryinata transferred from 1UO to 80% SW. Rates were determined from 2-min intervals (average weight of crabs, 150 g).
exposure to 80% SW, was not significantly different from k in 100% SW, but after five hours there was a significant decrease (P < 0.005, paired Mest). The reduction in k appears to be a transient response, since k returned to normal after ten hours. The cause of the difference in k between the two groups of animals is unknown.
While the reduction in k in Libinia, an osmoconforming crab, appears to be a transient response, long-term reductions have been reported for a number of osmoregulating crabs transferred to dilute salinities. The time course for this change has been reported for Rithropanopeus (Capen, 1972). In this crab, there is a reduction in k within the first hour of transfer from 75 to 10% SW. The initial phase of this reduction is not unlike that in Libinia, both in the initial rate of change and in the final extent of the change. It is of some interest to know if the time course in Rithropanopeus is similar to that in other osmoregulating crabs that have a reduced k in dilute media. To determine this, k was measured in specimens of Carcinus mac mis transferred from 100 to 80% SW. The results of this experiment appear in Figure 1. For specimens of Carcinus in 100% SW, k was 1.94. After one hour of exposure to 80% SW, there was little change in k, but after five hours of exposure, the value had decreased by about 30% (k -- 1.41, P < 0.005, paired Mest).
In the present study, k was determined in animals which were in a steady state, and in animals which were adapting to 80% SW and were not in a steady state. Therefore, it seems appropriate to ask if the reduction in k can be attributed solely to a change in state. The answer is probably "no." Two variables will be considered, surface area and volume of the water pool. The
428
JOHN C. CORNELL
0
2 3
HOURS IN 80% SW
FIGURE 3. Changes in the amplitude of scaphognathite beat in specimens of Libinia emarginata transferred from 100 to 80% SW. Results are expressed as a percentage of the pressure developed by beats at time 0. The average amplitude of beat at time 0 was 0.7 cm H2O and the average rate, 180/min (average weight of crabs, 163 g).
first, although difficult to evaluate, may be dismissed at the gross level on the grounds that whatever tendency for change exists is for an increase in surface area — a direction opposite to that necessary to account for the reduced k. The second may be dismissed on the grounds that, although an increase in volume occurs, and this would tend to reduce the value of k, the extent of the volume change (0.56% body weight in Libinia and much less in Carcinus} is insufficient to change the volume of the water pool (about 70% body weight) by an amount necessary to account for the reduction. The presence of more than one water compartment can also be considered ; although this was not found in preliminary studies in Libinia, it has been reported for Carcinus (Rudy, 1967). Rudy found that the fast compartment was 60 to 80% of the total volume of exchangeable water ; if all of the increase in volume occurred in this compartment, it would not account for the decrease in k.
Two possible mechanisms for changes in k
There is some similarity in the initial change in k in specimens of Carcinus, Libinia and Rithropanopeus transferred to a dilute salinity. Although this similar- ity may be superficial, there could be a common mechanism. There are, of course, many possible changes which could account for a reduction in k, but for present purposes, we will consider changes which occur in the unstirred layers at a liquid- solid interface. The concentration of a substance within an unstirred layer may be quite different from its concentration in the free medium, and under some condi-
SALT AND WATER BALANCE IN CRABS
42V
TABLE II
Comparison of Posm* and P,t* for some crabs. References are indicated by superscripts: a, this report; b, Born (1970); c, urine production from Shaw (1961), osmotic gradient from Smith (1970); d, urine production from Binns (1969), osmotic gradient from Smith (1970); e, Rudy (1967); f, Smith (1970).
|
RT |
kv |
||
|
Animal |
Medium (% SW) |
Posm* = Lp* — X 10' Vw [cmV(g-sec)] |
pd* = -- X 10' [cmV(g-sec)] |
|
Libinia emarginata |
100 |
28a |
lla |
|
Pugettia producta |
100 |
56a |
36b |
|
Carcinus maenas |
100-94 |
1.5a, 4.6f, 3.7a |
|
|
80-70 |
5.3«, 5.4d |
1.5°, 5.3f, 2> |
|
|
50-40 |
4.3C, 4.4d |
1.4e, 3.9[ |
|
|
25-35 |
5.1% 7.5d |
3.4{ |
tions, the thickness of this zone is an inverse function of the free stream velocity. Unstirred layers have a large influence on the rate of tracer water movement across very permeable membranes, since as permeability increases, diffusion through the unstirred layers becomes rate limiting.
For several crabs (Rithropanopeus, Capen, 1972; Uca, Hannan and Evans, 1973), it has been shown that 86 to 90% of tracer water exchange occurs in the gill chambers. Thus a change in blood circulation, or external irrigation of the gills could have a major influence on total water exchange. When specimens of Libinia were transferred to 80% SW there was a reduction in heart rate (Fig. 2). This could indicate a reduction in the blood circulation in the gills, and might account for the reduction in k. This idea received some support when it was found that water exchange was a function of the perfusion rate in isolated gills of Libinia maintained in SW : after 10 min of perfusion at 25, 47, and 90 ml/hr, the concentrations in the perfusion fluid, expressed as a percentage of the external medium, were 6.3 ± 2.84 (6), 14.2 ± 4.56 (6)*, and 18.2 ± 10.5 (5) % D2O (mean ± SD (N), * = P < 0.001, paired f-test), respectively.
These is also evidence for a reduction in the rate of irrigation of the gills. In a typical 150-g specimen of Libinia in SW, the scaphognathite, or gill bailer, beats 150 to 200 times per minute. When this crab was transferred from 100 to 80% SW, there was a somewhat erratic, but definite reduction in the amplitude of the pressure pulse (Fig. 3), and a small tendency for a reduction in the rate of beat. Furthermore, in some animals, the scaphognathite appeared to stop for periods of several minutes. Most recordings were carried out for a 5-hour period, at which time the scaphognathite beat had not returned to normal. In one 15-hr recording, the scaphognathite beat returned to normal at about 9 hr. Considering the time courses, the change in the amplitude of the scaphognathite beat is better correlated with the change in k than the change in heart rate.
DISCUSSION
Comparative studies of decapod crustaceans, using tracer water (Rudy, 1967; Thompson, 1970; Hannan and Evans, 1973) and other techniques (Nagel, 1934; Gross, 1957; Herreid, 1969), have shown that there is a relationship between
430 JOHN C. CORNELL
"permeability" and habitat. In general, permeability decreases sequentially in animals from marine, brackish-water, and fresh-water habitats. Among closely related osmoconformers, permeability, as indicated by weight gain, seems to decrease with increasing euryhalinity (Davenport, 1972). Libinia etnarginata and Pugettia producta provide an additional example of this type of relationship, since the lower apparent permeability of Libinia (Table II), can be correlated with its great salinity tolerance; Libinia can be adapted to 40% SW (Gilles, 1970) while Pitgettla, to only 50 to 55% SW. These differences between Libinia and Pugettia are possibly related to the fact that environmental salinity fluctuations are greater on the east coast of the United States, where Libinia is found, than on the west coast of the United States, where Pugettia is found.
The relationship between tracer water exchange rate (k) and habitat reinforces the belief that k is a meaningful measure of permeability ; however, some reserva- tions about the meaning of small changes may be necessary. Generally among decapod crustaceans, changes in salinity have been found to affect k by about 20 to 40%. This change in k is small enough that factors other than permeability could be responsible. One such factor is a change in unstirred layers. In speci- mens of Libinia exposed to a dilute medium, the transient reduction in k, which initially resembles the long-term reduction in Rithropanopeus karris ii (Capen, 1972) and Carcinns maenas (this report), can be correlated with a reduction in the amplitude of the beat of the scaphognathite. In other studies, there are also indications of reductions in gill irrigation of crabs exposed to a dilute medium (Libinia and Maja verrucosa, King, 1965; Carcinns madias, Hume and Berlind, 1976). A temporary reduction in gill irrigation could provide a simple mecha- nism for short-term reductions in k and might confuse the issue of permeability changes since the scaphognathite is not only affected by salinity changes, but oxygen tension (McMahon and Wilkens, 1975) and tidal cycle (Arudpragasam and Naylor, 1964) as well.
Changes in blood flow could also affect k, but the evidence for this is not strong. Reductions in the heart rates of Libinia and Carcinns were thought by Cornell (1973, 1974) to provide a possible explanation for reductions in k; however, further data make this possibility less likely. In addition, Hume and Berlind (1976) found that the heart rate in Carcinns increases in dilute media. The cause of this discrepancy between the results of Hume and Berlind and my own is not known.
It is generally believed that the effects of unstirred layers can be much greater on estimates of diffusive water permeability (Pd) than on osmotic water perme- ability (Posm) (House, 1974). However, much of the discrepancy between these estimates may be eliminated by adequate stirring (Dainty and House, 1966). Thus, one possible way of evaluating the effects of unstirred layers comes from the comparison of POSm and Pa.
It should be pointed out that it is difficult to justify the necessary assumptions in comparing Posm and Pd in something as "simple" as an isolated sheet of epithe- lium, let alone an intact animal. But, we are guided by the words of House (1974, p. 341) : "In view of the dubieties about errors in Pd and Lp values one has no proper right to compare them. Nevertheless, I shall do so." Table II shows Posm* and P,,* for Carcinns, Libinia and Pugettia. In Libinia and Pugettia. P,,sm* was determined from the rate of weight gain in 80% SW at time 0 and
SALT AND VYATKR BALANCE IX CJKAI'.S -Ml
the osmotic gradient caused by the transfer from 100% SW. In Carcinus, Posm* was determined from published values of urine production rates, and osmotic- gradients, for animals in a steady state in dilute SW. For estimates of Pd*, it was assumed that the exchangeable water in all crabs was 70% body weight. P0sm*/Pa* is greater than unity in all cases. Interpreting these results is a matter for speculation, but I suggest that these data are consistent with the possibility that changes in unstirred layers could have a major influence on water exchange rates in all of these crabs since the ratio of P0sm*/Pd* does not differ greatly among Carcinus, Libinia and Pugettia.
Thus, a transient increase in the thickness of unstirred layers surrounding the gill might explain the transient reduction in k in a crab such as Libinia. Such a mechanism could also affect the initial phases of a long-term reduction in k in a crab such as Carcinus, but the evidence suggests that the maintenance of long- term reductions in k are the result of some other mechanism, which may be under hormonal control (see Tullis and Kamemoto, 1974, and Berlind and Kamemoto, 1977). The existence of this other mechanism is demonstrated by the fact that the isolated gills of Callinectcs spadius and Cancer irroratns from crabs adapted to 40% SW have a lower water exchange rate than do those from crabs adapted to 100% SW (Cantelmo, 1977).
In the strictest sense, it is possible to state that a change in water permeability has not been demonstrated beyond doubt in any decapod crustacean. But regard- less, a process that can reduce the effect of an osmotic gradient is potentially of adaptive value. Thus, we should ask if a reduction in k reflects the action of such a process. A definitive answer to this question is not yet available for Libinia, Pugettia. and many other decapod crustaceans.
This paper constitutes part of a doctoral dissertation submitted to the Depart- ment of Zoology, University of California, Berkeley. It is a pleasure to thank Dr. Ralph Smith for his encouragement and guidance during the course of this work. I am grateful to Dr. Cadet Hand and the laboratory staff for their assistance and for the use of the facilities of the Bodega Marine Laboratory, Bodega Bay. California. I thank Dr. Robert Josephson for the opportunity to pursue my work while acting as a course assistant in the Experimental Invertebrate Zool«^\ Course at the Marine Biological Laboratory, Woods Hole, Massachusetts. Financial assistance in the form of a one-year University Fellowship was greatly appre- ciated. My wife, Mary, deserves special thanks.
SUMMARY
1. Estimates of osmotic water permeability suggest that Libinia anarginata [166 ^/(g-hr-osmol)] is less permeable to water than Pugettia prodncta [338 fjl/ (g-hr-osmol)]. Data on tracer water exchange supports this conclusion and the differences in apparent permeability can be correlated with differences in habitat.
2. Short-term changes in D2O water exchange were examined in specimens of Carcinus viaenas and Libinia cinarginata. When these crabs were transferred from 100 to 80% sea water, there was an initial reduction in k in (1/hr), the
432 JOHN C. CORNELL
hourly water exchange fraction, of from 1.9 to 1.4 and from 5.8 to 4.3 in Card nns and Libinia, respectively. In both crabs, the initial response to the dilute medium is similar; however, in Libinia. the reduction in k is transient while in (.'arcinus it is a long-term response.
3. Estimates of osmotic (POSm*) and diffusive (IV:) water permeabilities for Carcinns, Libinia and Pugcttia indicate that the ratio of P,,sra*/Pd* is about 2, which suggests that unstirred layers could have a major influence on tracer water movement in all of these crabs. It is proposed that the initial changes in k, which occur during adaptation to a dilute medium, are at least partly the result of an increase in thickness of the unstirred layers surrounding the gills, caused by a reduction in the flow of medium through the gill chamber.
LITERATURE CITED
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euryhaline crabs and in isolated, perfused gills. Comp. Biochetn. Physiol., 58A : 383-385. BINNS, R., 1969. The physiology of the antennal gland of Carcinns maenas (L.). II. Urine
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SALT AND WATER BALANCE IN CRABS 433
KING, E. N., 1965. The oxygen consumption of intact crabs and excised gills as a function
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UPTAKE OF AMINO ACIDS BY MARINE POLYCHAETES UNDER ANOXIC CONDITIONS
JAMES J. COSTOPULOS, GROVER C. STEPHENS, AND STEPHEN H. WRIGHT
Department of Developmental and Cell Biology, University of California, Irvine,
Irvine, California 92/17
Representative species of at least twelve different families of the class Poly- chaeta have served as experimental organisms in studies of transepidermal trans- port of small organic solutes in several laboratories. These studies include many published reports dealing with specific aspects of this phenomenon (reviewed by Stephens, 1972; Jdrgensen, 1976) as well as unpublished work in this laboratory. Since marine annelids are often conspicuous members of the infauna, are easy to collect and maintain for brief periods in the laboratory, and live in habitats relatively rich in dissolved organic resources, they continue to be used for such work (Ahearn and Gomme, 1975 ; Rice and Chien, 1978; Stephens, 1975). Despite this attention, basic characteristics of this influx remain unexamined. We under- take to explore two aspects of influx of amino acids in two representative polychaetes.
First, the relation between influx of labeled substrates and net movement of substrate into or out of the animal is still in question. Stephens (1975) showed net influx of known amino acids in Nereis diversicolor and Capitella sp. using a fluorometric procedure to follow disappearance of added substrate. However, labeled substrates were not employed in this work. Details of the relation between influx of substrate followed radiochemically and net movement of substrate followed chemically have been described and analyzed for molluscs (Wright and Stephens, 1977, 1978 ; Crowe, Dickson, Otto, Colon and Farley, 1977) and for enchinoderms (Stephens, Volk, Wright and Backlund, 1978). The only report concerning annelids is that of Johannes and Webb (1970) in which they describe influx of 14C-labeled glycine accompanied by net efflux of amino acids in Clymcnclla. Since many of the reports concerning influx of 14C-labeled substrates overtly or tacitly assume that such influx represents net removal of substrate from the medium by the animal and draw conclusions on this basis, the subject merits investigation.
Second, infaunal polychaetes undergo periods of relative oxygen deprivation or anaerobiosis. If net influx of small organic molecules is to be invoked as a potential supplementary form of nutrition for these animals, the response of the process to relative or complete anoxia is important.
Stephens (1963) reported that influx of 14C-labeled amino acids in Clyinenella was unimpaired after long exposure to sea water through which gaseous N2 was bubbled. However, no direct measurements of Po2 were made. Several authors have used inhibitors of aerobic metabolism and reported partial inhibition of influx rates in some cases, and no clear response in other cases. None of this work includes independent criteria to assess inhibitor effects, and in most cases does not demonstrate reversibility of effects reported.
The present work reports the relation between influx and net flux of glycine in two genera of polychaetes. This relation is reported over a range of Po2 including
434
AMINO ACID TRANSPORT IN WORMS 435
anoxic conditions. The effects of anoxia are compared to effects of cyanide inhibi- tion of aerobic metabolism.
MATERIALS AND METHODS
Specimens of Marphysa sanguined (Eunicidae) were collected intertidally from Upper Newport Bay, Newport Beach, California. Animals were found in muddy sediment under and between intertidal rocks. Specimens of Pareurythoe calijornica (Amphinomidae) were collected subtidally from a shallow lagoon at Point Mugu Naval Base near Port Hueneme, California. The animals occurred in sandy sedi- ment just below mean low tide. Animals were maintained in the laboratory in aerated containers at 16° C. Specimens of both genera were selected in the wet weight range of 0.7 to 1.4 g for observations. Experiments were carried out at room temperature (23° C) ; animals were acclimated to the temperature change for several hours before use.
Influx of 14C-glycine into animals was followed by periodic sampling of the medium in which animals were incubated. Experimental media were prepared from 14C-gly (UL) at 20 to 40 //.Ci/liter plus sufficient 12C-gly to achieve the desired chemical concentration. All media were prepared in artificial sea water (MBL) prepared according to Cavanaugh (1956) filtered through a Millipore filter (0.45-/xm pore size). Duplicate 0.2-ml samples of medium were taken periodically, acidified to drive off CO2, and added to a toluene-based scintillation cocktail containing a detergent. Radioactivity was determined using a Beckman CPM-100 scintillation counter. Details of sampling protocol vary according to the experi- mental procedure.
Net change in ambient glycine concentration wras followed using fluorescamine to determine changes in primary amines in solutions in which animals were incu- bated. The procedure has been described (Stephens, 1975; Stephens et al, 1978). The reagent, fluorescamine, reacts with primary amines to produce a fluorescent product wTith an absorption maximum at 390 nm and emission peak at 480 nm. Fluorescence was measured using a Perkin-Elmer spectrophotofluorom- eter. Initially, fluorescence reflects glycine concentration since glycine is the only primary amine present in the medium. After incubation, fluorescence represents remaining glycine plus any primary amines which may be present as a result of efflux from the animal. Fluorescence is expressed in units of equivalent glycine concentration.
Influx and net flux of glycine under anaerobic conditions was measured as follows : Several hundred ml of MBL sea water was placed in a large culture flask with a port at the base in which an oxygen electrode (YSI) was mounted. Nitrogen gas was passed through acid pyrogallol and bubbled through the culture flask using a breaker stone. Oxygen content was monitored with the oxygen meter until anoxic conditions were achieved ; approximately 5 min were required to reach the same oxygen reading as that obtained for sea water chemically de- oxygenated with dithionite. Four samples of 50 ml each were then siphoned into four flasks, each containing one worm. Two of these flasks were placed in the N2 gas train and maintained anoxic. The other two were reoxygenated using an air pump and served as controls. Worms were allowed to adapt to these conditions
436
COSTOPULOS, STEPHENS AND WRIGHT
TABLE I
A'i//c.s of uptake of glycine in specimens of Pareurythoe and Marphysa under aerobic and anaerobic i mi/lit ituis. In all cases, initial concentration of glycine was 20 /uM. Influx was calculated from the rate of depletion of uC-glycine from the medium; net influx refers to the rate of disappearance of total primary amines from the medium, and is expressed in terms of gly cine-equivalents.
|
Experimental condition |
Influx [moles X 10-'/(g-hr)] |
Net influx [moles X lO-V(g-hr)] |
|
Pareurythoe Anaerobic |
2.9 |
3.2 |
|
3.0 |
3.2 |
|
|
2.0 |
1.3 |
|
|
2.5 |
2.3 |
|
|
Aerobic |
Average 2.6 ± 0.3 (s.d.) 5.4 |
2.5 ± 0.8 5.7 |
|
4.6 |
5.2 |
|
|
8.9 |
8.2 |
|
|
7.8 |
8.0 |
|
|
Marphysa Anaerobic |
Average 6.7 ± 1.7 2.2 |
6.8 ± 1.3 |
|
1.9 |
— |
|
|
1.2 |
1.2 |
|
|
3.1 |
3.3 |
|
|
Aerobic |
Average 2.1 ± 0.7 7.4 |
2.3 |
|
8.5 |
. — |
|
|
4.9 |
4.9 |
|
|
5.2 |
4.9 |
|
|
Average 6.5 ± 1.5 |
4.9 |
for 15 to 30 min. Substrate (0.5 ml) was then added using a hypodermic syringe to initiate the experimental period. Samples were withdrawn periodically using a hypodermic syringe and were used for duplicate determinations of radioactivity and duplicate determinations of fluorescence at each time point.
Observations on influx and net flux of glycine in Pareurythoe were also carried out using a flow system. Filtered MBL sea water containing "C-labeled glycine (20 /mi) was placed in a flask to serve as a medium reservoir. A metering pump was used to produce a flow of medium through a chamber with sintered glass discs at either end (internal diameter =: 16 mm, length — 75 mm, approxi- mate volume ~ 15 ml). Medium from the reservoir was led to the chamber via a sampling port which permitted monitoring Po2 as it passed into the chamber. The medium reservoir was initially deoxygenated and then reoxygenated to the desired level. By using a relatively large volume of medium in the reservoir with a small free surface, negligible drift in Po2 was encountered during the course of observations. Flow rate was calculated to be sufficiently rapid that oxygen consumed by the worm would represent a depletion of 10% or less of the oxygen content of sea water in equilibrium with air. This represents a necessary compro- mise which allowed for a minimal flow rate sufficient to permit reliable deter- minations of the difference between inflow and outflow concentrations. Actual flow rates ranged from 3 to 6 ml/min depending on the weight of the worm. At
AMINO ACID TRANSPORT IN WORMS
437
each level of P02 tested, medium was pumped through the chamber for 15 min prior to sampling. Samples were then taken each 5 min for the following 30 min and analyzed in duplicate for radioactivity as described. In some experiments the fluorescence at inflow and outflow ports was also determined.
In all cases where medium depletion was followed with time, a straight line was obtained when log radioactivity or log fluorescence was plotted against time, indicating that depletion followed first order exponential kinetics. Uptake rates are presented as moles glycine/(g wet weight -hour) from an ambient concentration of 20 /AM. Data from the first hour of sampling was used to establish the rate constant. Uptake rates obtained using the flow system were calculated from the average difference between inflow and outflow samples as described and are ex- pressed in the same units.
RESULTS
Table I presents rates of influx and net rates of influx for Pareurythoe and Marphysa under anaerobic and aerobic conditions. There is no significant difference
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FIGURE 1. Effect of cyanide on the influx and net influx of glycine into A) Marphysa and B) Pareurythoe. Influx (shaded bars) was calculated from the rate of depletion of wC-glycine from the experimental medium; net influx (open bars) was determined from the rate of depletion of total primary amines. In all cases the initial concentration of glycine was 20 MM ; the concentration of KCN was 2 mM. Bars represent + 1 standard deviation.
438
COSTOPULOS, STEPHENS AND WRIGHT
Aerobic
Anoxic
TIME (hrs)
1 2
TIME (hrs)
FIGURE 2. Effect of anoxia on the time course of influx and net influx of glycine into Parcurythoe. Solid circles represent measured levels of 14C-glycine ; open circles are concen- trations of total primary amines expressed as equivalent glycine concentration. Anoxic con- ditions were produced by bubbling N2 through experimental media containing 2 mM KCN. Each point is the mean of three separate determinations with individual worms ; bars represent ± 1 s.d. .
between rates of influx (14C depletion) and net influx (decrease in primary amines). Hence there is a net transfer of substrate from the medium to the animal at a rate accurately estimated by either procedure. Rates of uptake under anaerobic conditions are reduced to levels 32 to 46% of aerobic rates.
Figure 1 presents data for influx and net influx of glycine in the presence and absence of 2 mM KCN. Oxygen consumption for three individuals of Marphysa was measured using an oxygen electrode. Exposure to 2 mM KCN for 30 min inhibited oxygen consumption to 13% of control values. Normal rates of oxygen consumption were restored when animals were permitted to recover for 24 hr. Influx and net flux of glycine into three individuals of each species before, during and after exposure to 2 mM KCN was measured on two separate occasions. Influx and net flux were reduced in the presence of cyanide to levels essentially the same as those observed under anaerobiosis (40 and 46% of control values). Recovery was complete.
Figure 2 presents data for Pareurythoe showing the time course of influx of 14C-glycine and net flux of total primary amines under aerobic and anoxic conditions. The data for each experimental condition are mean values of separate studies on three worms. Anoxic conditions were produced by bubbling N2 through experimental media also containing 2 mM KCN. Similar results were obtained using Marphysa. Note the close correspondence between influx as determined by depletion of radioactivity and net disappearance of substrate as indicated by determination of fluorescence. This is the case under both sets of conditions. In
AMINO ACID TRANSPORT IN WORMS
439
both cases, determinations of fluorescence begin to diverge from determinations of radioactivity after two hours. In both cases, radioactivity does not continue to decline indefinitely. This does not represent a limitation of the transport system since 14C-glycine at concentrations of 2 to 4 X 1O7 M is removed exponentially by both species.
Figure 3 presents data for two individuals of Parcitrythoc in which influx and net influx of glycine was measured in the flow system. Error bars represent tl standard deviation of the average difference of seven determinations of radio- activity or fluorescence at inflow and outflow ports. Actual differences range from 0.6 to 7c/o of inflow. Since P<>_. w7as measured at the inflow, the values are systematic overestimates of the average PO2 experienced by the animal during the observation period. Six such experiments \vere performed. In all cases there was a sustained high rate of influx, essentially comparable to that observed in aerobic depletion experiments, until ambient Po2 was reduced to approximately 10% to 2Q% of air saturation values. In some cases, there was some decrease with decreasing P02 prior to the more conspicuous decrease observed at very low Po2- The data are too variable to justify mathematical treatment but suffice to demon- strate that influx and net influx are not linearly related to P02 over this range of oxygen concentrations. In these observations, influx and net influx of glycine were inhibited to a greater extent under anoxic conditions than the inhibition
t
20
40
60
80
100
FIGURE 3. Effect of P02 on influx and net influx of glycine into Parcurythoc. Circles and squares represent separate experiments on two individual worms. Closed symbols represent influx of 14C-glycine ; open symbols represent net influx of total primary amines. Bars are ± 1 s.d. ; those cases in which open symbols have no error bars indicate that the variability was too large to include effectively in the figure.
440 COSTOPULOS, STEPHENS AND WRIGHT
TABLE II
Nutritional role of amino acid uptake in Pareurythoe and Marphysa: examination of critical param- eters. See text for the basis of the calculations.
Pareurythoe Marphysa
|
Concentration of free amino acids in environment GUM) |
123 |
131 |
|
Oxygen consumption [ml/(g-hr)] |
0.13 |
0.15 |
|
Influx [moles X 10-7/(g-hr)] (cf. Table I, for 20 /*M) |
6.7 |
6.9 |
|
Contribution for 20 ^uM gly Gug) |
50.5 |
44.6 |
|
% oxygen requirement |
17.4 |
13.8 |
|
Glycine concentration for 100% oxygen requirement GUM) |
115 |
145 |
|
Mixed amino acid concentration for 100% oxygen requirement GUM) |
52 |
65 |
observed in depletion experiments. Anaerobic influx rates were approximately to 20% of those observed at higher Po2-
DISCUSSION
There is no significant difference between rates of 14C-glycine influx in Marphysa and Pareurythoe estimated from radiochemical measurements and rates of net influx of glycine estimated from fluorometric determinations from an ambient gly- cine concentration greater than 10 /AM (Figure 2, Table I). There is a slow efflux of primary amines (fluorescamine-positive material) with time in both aerobic and anoxic conditions (Figure 2). These findings agree with similar studies of molluscs (Wright and Stephens, 1977, 1978) and echinoderms (Stephens ct or/., 1978). In all of the soft-bodied marine invertebrates examined adequately, influx of 14C-labeled amino acids reflects net influx quite accurately at concentrations greater than 10 JU.M ; however, this process is accompanied by a slow efflux of primary amines.
These results agree with the reports of Johannes, Coward and Webb (1969) and Johannes and Webb (1970) using the flatworm, Bdclloura Candida and the polychaete, Clyuicndla. They supplied labeled substrates at low concentrations (0.6 /AM and 1.0 /AM in the studies cited) and observed a slow net efflux of amino acids with time at rates broadly comparable to those reported here and those reported by Stephens (1968). However, they interpreted their data as evidence for exchange diffusion and questioned the occurrence of net influx of substrate into marine invertebrates, suggesting that earlier literature might represent a misinterpretation of such exchange diffusion. However, exchange diffusion is clearly excluded as a mechanism of efflux by data showing that efflux is essentially independent of ambient concentrations, as appears to be the case in the examples previously cited. Beyond this, there is currently no evidence concerning the route or mechanism of efflux.
Table II presents measurements of naturally occurring primary amines in the immediate habitat of Pareurythoe. Data were obtained by expressing interstitial water from freshly collected sediment cores in the sandy subtidal habitat of Pareurythoe. Total free amino acids were estimated by the fluorescamine technique. Some of these data are reported in Stephens et al. (1978) where methodological details are presented. The samples ranged from 54 to 244 /AM (average 123 ± 54
AMINO ACID TRANSPORT IN WORMS 441
, n=: 13). Free amino acids in the habitat of Marphysa were estimated in a similar fashion. However, it was not possible to obtain cores in the immediate area of the rocks where the animals were found. Therefore, cores were taken from otherwise comparable muddy sediment in immediately adjacent areas of Newport Bay. Average free amino acids ranged from 88 to 180 //.M (average 131 ± 35 /AM, n = 7). Table II also presents data for oxygen consumption of Pareurythoe in the size range employed in these experiments [0.13 ml/(g-hr)± 0.02, n == 6]. For Marphysa, oxygen consumption was 0.145 ± 0.25 (n = 8). Table II also includes average rates of glycine influx (average of influx and net influx from Table I) for the two worms. Given these data, it is possible to calculate numbers to estimate the significance of amino acid influx as compared to metabolic require- ments estimated from oxygen consumption. Complete oxidation of 1.0 nig of glycine requires 2.23 ml O2 (STP). One can calculate the percentage contribution of glycine influx from an ambient concentration of 20 /AM (where measurements were made) for the two cases. By making the assumption that influx at higher concentrations is linearly related to concentration, the glycine concentration in the medium which would provide sufficient reduced carbon to account for oxidative metabolism can be calculated. In fact, interstitial amino acids from the immediate habitat of Pareurythoe have been identified chromatographically (Stephens ct al., 1978). Glycine is present as well as seven other identifiable amino acids accounting for approximately 85% of total primary amines as estimated by fluorescamine. Thus it is more realistic to use a conversion factor of 1 ml of Oo required for complete oxidation of a mixture of amino acids provided one assumes that rates of glycine influx are comparable to influx rates for other amino acid substrates. The table also includes the ambient free amino acid concentration (based on these assumptions) required to provide reduced carbon equivalent to oxygen consumption. These data and calculations indicate that influx of free amino acids from ambient solution occurs at rates that are of the same order of magnitude as the requirement for reduced carbon to sustain oxidative metabolism ; i.e. the process of transepi- dermal influx represents a potentially important source of supplementary nutrition for these animals. This conclusion is based on the assumption that bulk con- centrations of free amino acids measured in interstitial water of the sediment habitat are a reasonable estimate of concentrations available to the animals. There is no direct information on this point, though Stephens (1975) has provided evidence that irrigation activity of infaunal annelids may increase local free amino acid concentration in interstitial water.
These data and calculations are not relevant to conditions during periods of anaerobiosis which infaunal worms may undergo periodically. Under such cir- cumstances, influx rates for glycine in Pareurythoe and Marphysa decrease to 32 to 46% of aerobic rates. Requirements for reduced carbon will be considerably increased during periods of anaerobiosis. However, net influx continues from ambient concentrations in the range found in the habitat under anoxic conditions for up to 4 hr. Thus the process continues though its rate and overall contribution to metabolic requirements are both considerably reduced.
The presence of 2 HIM KCN in the medium almost completely inhibits aerobic metabolism (oxygen consumption was reduced by 83%). This effect would be predicted based on the binding of CN~ to heme groups and the resulting interdiction
442 COSTOPULOS, STEPHENS AND WRIGHT
of electron transport. Thus the concentration used and the time of incubation employed were sufficient to force dependence on anaerobic metabolism. The result- ing decrease in influx (40 to 45% of control values) agrees well with the decrease observed under anoxic conditions. Cyanide inhibition, both of influx and of oxygen consumption, were reversible ; no mortality \vas encountered in these experiments. Thus this is additional evidence for the ability of these animals to tolerate periods of anaerobiosis. There is no evidence for a specific inhibitory effect of cyanide on the transepidermal transport system.
Mangum (1976) reviews respiratory adaptations of annelids. In her view irrigation of the burrow is one of the major adaptations to the sediment habitat in this group. Although data for Marphysa and Parenrythoe are not reported, it is reasonable to assume that they also irrigate their burrows. Mangum tabulates data comparing average PO^ in the microhabitat (i.e. in the burrow) with P0o in the water column. The former ranges from 73 to 104 mm Hg when the overlying water column is essentially saturated. Thus the ability of the annelids examined in the present work to sustain normal rates of net influx at partial pres- sures of oxygen below saturation (Fig. 3) is of interest. There is little decline in influx rate until Po2 drops below 20% of saturation. Since these estimates of the Po2 actually encountered by the worms in the flow chamber are systematic overestimates (measured at the inflow end of the chamber), this suggests that the worms are functioning as aerobes at quite low Po2's and hence at realistic levels of oxygen availability under normal circumstances. The depression of influx under anoxic conditions was greater in these experiments than was the case in the medium depletion experiments (15 to 20% rather than 32 to 46% of control values). The flow chamber was large in diameter compared to the worms. Under conditions of very low Po2. the worms typically coiled tightly. In contrast, medium depletion experiments were done with continuous gentle agita- tion by the gas stream (N2 or air). It is possible that the greater depression of influx in the flow system is simply the result of this behavior and the reduction in surface exposed to the medium by the worms in the two situations.
The limitations of the use of oxygen consumption as a measure of metabolic requirements should be reemphasized. These limitations are based on the neces- sary assumption that metabolism is aerobic (clearly not always the case for annelids) and on the fact that requirements for organic material which must support growth and balance losses by other pathways do not enter the estimation process. It would be far more appropriate to undertake measurement of heat production (Pamatmat, 1978) and estimate minimum requirements on that basis; however, this has yet to be done for annelids. The calculations presented in Table II indicate that influx of naturally occurring free amino acids may provide a major input under aerobic circumstances. However, the significance of influx under anaerobic conditions will certainly be less, both by virtue of the reduction in rate and because of the increased requirements for substrate. In any case, the conclusion drawn from this work is that uptake of amino acids is a supplement. Both worms certainly have other feeding methods at their disposal.
The metabolism of facultatative anaerobic invertebrates is complex and involves a variety of end products (de Zwaan, Kluytmans, and Zandee, 1976). Some of the pathways demonstrated produce amino acids such as alanine and succinate
AMINO ACID TRANSPORT IN WORMS 443
and these compounds might be expected to accumulate under anoxic conditions (Hochachka, Fields, and Mustafa, 1973). Though such reactions may well be involved in facultative anaerobiosis in annelids, the resulting amino acids are not perceptively lost to the medium in Marphysa and Pareurythoe; rates of efflux of primary amines are not increased under anoxic conditions (Figure 2).
The extent to which the two species examined in this work actually experience anoxic periods is probable very different. Marphysa was collected from a popula- tion in the intertidal from an area characterized by fine-grained sediment of high organic content. At low tide, animals almost certainly experience anoxia. Pareurythoe was collected from a shallow sub-tidal population in an area of coarser sediment. It is not clear why Pareurythoe would ever experience anoxia so long as burrow irrigation continues. Both species tolerate anoxic periods well and behave similarly in the present observations. Mangum (1976) suggests that the circulatory system in annelids may play a more important role in acquisition of oxygen from the environment than in distribution to deeper tissues ; in many species deeper tissues are virtually avascular. Possibly the widespread tolerance of anoxia among annelids may reflect the fact that deeper structures may be poorly supplied with oxygen even in aerobic conditions.
Finally, the relation of this work to earlier reports of influx of 14C-labeled sub- strates into marine polychaetes should be mentioned. Since it is now quite simple to examine influx and net flux of amino acids, further work in this area should include such examination. However, the coupling of influx as estimated radio- chemically and net influx examined chemically has now been reported for several major groups of marine invertebrates and in each case examined, influx has been shown to be a close estimate of net flux provided concentrations are realistic and initial rates are compared. It seems increasingly likely that the early reports can be accepted provisionally as providing evidence for the existence of transport systems capable of net transport of substrate from the medium into the animal. The potential contributions of such transport will depend then on environmental availability of substrate and will no doubt vary among species. The wide distribu- tion of transepidermal transport systems among marine invertebrates indicates that further work is desirable to describe the extent to which energy flow by this pathway may contribute to the trophic organization of marine communities.
This work was supported by grant OCE-09017 from the National Science Foundation.
SUMMARY
1. The effect of anoxia on influx and net flux of amino acids from dilute solu- tions into two species of marine polychaetes was studied.
2. Rates of influx and net flux correspond quite closely at ambient concen- trations greater than 10 //,M. Anoxic conditions, produced by incubating speci- mens of Marphysa and Pareurythoe in solutions containing 2 niM KCN or through which N2 was bubbled, did not affect the tight correspondence between influx and net flux, though rates were reduced by approximately 50%.
3. The effect of P02 on influx and net flux was examined using a continuous
444 COSTOPULOS, STEPHENS AND WRIGHT
flow system. Influx and net influx remained at control rates down to PO-'S 10 to 20% of air saturation values.
4. Comparisons of rates of net flux to measured values of O* consumption indicate that these animals can acquire sufficient reduced carbon to account for their oxidative needs if their surfaces are exposed to amino acid levels on the order of 50 to 65 /XM.
5. Primary amines in the interstitial water of sediments in the immediate vicinity of populations of these worms averaged between 123 and 131 JU.M.
6. Marphysa and Pareurythoe live in habitats that are relatively rich in amino acids, and they possess transport systems capable of the net accumulation of these compounds at rates sufficient to provide a significant supplement to other forms of feeding. The uptake process continues during periods of anoxia, though its rate and overall contribution to metabolic requirements are reduced.
LITERATURE CITED
AHEARN, G. A., AND J. GOMME, 1975. Transport of exogenous D-glucose by the integument of a polychaete worm (Nereis divcrsicolor Muller). /. Exp. Biol., 62: 243-264.
CAVANAUGH, G. M. (Ed.), 1956. Formulae and Methods, IV, of the Marine Biological Labora- tory Chemical Room. Marine Biological Laboratory, Woods Hole, Massachusetts, 61 pp.
CROWE, J. H., K. A. DICKSON, J. L. OTTO, R. D. COLON, AND K. K. FARLEY, 1977. L^ptake of amino acids by the mussel Modiolus demissus. J. Exp. Zoo!., 202 : 323-332.
HOCHACHKA, P. W., J. FIELDS, AND T. MUSTAFA, 1973. Animal life without oxygen: basic biochemical mechanisms. Am. Zool. 13 : 543-555.
JOHANNES, R. E., S. J. COWARD, AND K. L. WEBB, 1969. Are dissolved amino acids an energy source for marine invertebrates ? Comp. Biochcm. Physiol., 29 : 283-288.
JOHANNES, R. E., AND K. L. WEBB, 1970. Release of dissolved organic compounds by marine and freshwater invertebrates. Pages 257-273 in D. Hood, Ed., Organic Matter in Natural Waters, Univ. Alaska Inst. Mar. Sci. Occasional Pub. #\.
J^RGENSEN, C. B., 1976. August Putter, August Krogh, and modern ideas on the use of dis- solved organic matter in aquatic environments. Biological Revicivs, 51 : 291-328.
MANGUM, C. P., 1976. Primitive respiratory adaptations. In: R. Newell Ed., Adaptation to Environment : Essays on the Physiology of Marine Animals. Butterworths, London, pp. 191-278.
RICE, M. A., AND P. K. CHIEN, 1978. The effects of divalent cadmium on the uptake kinetics of glycine by the polychaete, Neanthcs vircns. ll'asinann J. Biol., 35 : 137-143.
STEPHENS, G. C., 1963. Uptake of organic material by aquatic invertebrates. II. Accumula- tion of amino acids by the bamboo worm, Clymenella torquata. Comp. Biochem. Physiol. 10: 191-202.
STEPHENS, G. C., 1968. Dissolved organic matter as a potential source of nutrition for marine organisms. Am. Zool., 8 : 95-106.
STEPHENS, G. C., 1972. Amino acid accumulation and assimilation in marine organisms. Pages 155-184 in J. W. Campbell, L. Goldstein, Eds., Nitrogen Metabolism and the Environment. Academic Press, N.Y.
STEPHENS, G. C., 1975. LTptake of naturally occurring primary amines by marine annelids. Biol. Bull., 149 : 397-407.
STEPHENS, G. C., M. J. VOLK, S. H. WRIGHT, AND P. S. BACKLUND, 1978. Transepidermal accumulation of naturally occurring amino acids in the sand dollar, Dcndraster cxcentricus. Biol. Bull., 154: 335-347.
WRIGHT, S. H., AND G. C. STEPHENS, 1977. Characteristics of influx and net flux of amino acids in Mytilus californianus. Biol. Bull., 152 : 295-310.
WRIGHT, S. H., AND G. C. STEPHENS, 1978. Removal of amino acid during a single passage of water across the gill of marine mussels. /. Exp. Zool., 205: 337-351.
ZWAAN, A. DE, J. H. F. M. KLUYTMANS, AND D. L. ZANDEE, 1976. Facultative anaerobiosis in molluscs. Biochcm. Soc. Symp. 41 : 133-168.
Reference: Biol Bull. 157: 445-452. (December, 1979)
PARTHENOGENESIS IN COPTOPTERYX VIRIDIS, GIGLIO TOS (1915) (DYCTIOPTERA, MANTIDAE)
MARTA CUKIER, GRACIELA ALICIA GUERRERO AND MARIA CRISTINA MAGGESE
Laboratorio dc Embriologia Animal, Departamcnto de Cicncias Biologicas, Facultad de Cicncias Exactas y Naturalcs, Univcrsidad de Buenos Aires,
Buenos Aires (Argentina)
The mantid Coptotcry.v z'iridis has been studied for several years in our laboratory. As its behavior is similar to that of other species of the sub-order Mantodea, we considered the possible existence of a parthenogenetic reproduction mechanism. These animals are solitary and sedentary, and the female often kills the male before copulation takes place. The average adult life of the female is twice as long as that of the male (Guerrero, Maggese and Cukier, 1977).
Observations made by other workers indicate that Bninncria borcalis reproduces exclusively by parthenogenesis (White, 1948a) and Mioniantis savignii (Adair, 1925) reproduces both by parthenogenesis and by being fertilized. The present investigation was designed to determine the existence and type of parthenogenesis in C. z'iridis.
MATERIALS AND METHODS
Oothecas of Coptoptcrv.v riridis were gathered in the vicinity of the slaughter- house of Lisandro de la Torre in Buenos Aires city, and were kept in separate flasks. The nymphs which emerged from these oothecas were kept in individual cages for their whole life, at room light and temperature. The cages and the method of feeding were as described by Guerrero and De Carlo (1976).
Male nymphs of the 5th and 6th nymphal stages were employed for determina- tion of the karyotype, and females of the 7th nymphal stage were used to corroborate the results. The testes and ovaries were immersed in an hypotonic solution of 0.77o Na citrate, fixed in Carnoy and stained with Giemsa.
Levan's classification was used to determine the karyogram where the centro- meric index is Ic = 100 s/c where s is short arm and c is whole length. Chromosomes with values of the index (Ic) of 50 are termed metacentric (type M) ; of from 50 to 37.5, metacentric (type m) ; 37.5 to 25, submetacentric (type Sm) ; 25 to 12.5, subelocentric (type st) ; 12.5 to 0, acrocentric (type t) ; and of 0 are termed telocentric (type T).
Study of the embryos involved fixation in Bouin, followed by manual elimination of the chorion, embedding in parafin, sectioning at 7 ^ thickness, and staining with hematoxylin-eosin.
RESULTS
Seven of the 13 females which arrived at the adult stage laid oothecas without fertilization. The number of oothecas laid by a female varied from 1 to 7, the variation depending on the time they lived as adults and on the time of the year in which they attained adulthood.
445
446
CUKIER, GUERRERO AND MAGGESE
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03
PARTHENOGENESIS IN MANTIDAE 447
A total of 22 oothecas was laid by the 7 females (Table I). The time that elapsed between the achievement of the adult stage and the laying of the first ootheca varied from 30 to 80 days. The female that had laid her first ootheca on the 30th day had become an adult towards the end of the summer (2-Mar-76), whereas the one that delayed 80 days in laying her first ootheca had attained adulthood at the beginning of the summer (26-Dec-75). The rest of the females laid their first ootheca towards the end of the summer, having attained the adult stage at the beginning of that season. In general, the sooner they became adults the longer they delayed in laying their first ootheca.
The first ootheca was laid on 26-Feb-76 and the last one on 4-May-76, a span of two and a half months. The intervals of time between one laying and the next for all the females was from 6 to 19 days. The average intervals between successive layings per female varied between 9 and 13 days and the general average of intervals for all the oothecas laid by the 7 females was 11 days.
As a rule the females died immediately after their last laying, or a few days after it. The exception was one female (XII-82) which had arrived at the adult stage at the end of the summer, and lived 106 days after her last laying. As this laying took place at the beginning of May, it might be asked whether cessation of laying was due to low temperature, photoperiod or any other reason. Her ovaries were full of mature eggs.
The female IX-I died 3 days after laying her last ootheca, with her ovaries full of mature eggs. They were not vestigial eggs since the last ootheca laid was of an enormous size. The female X-22 died 7 days after her last laying, and she had her ovaries full of mature eggs. The female X-30 laid her last ootheca on the day of her death, and her ovaries had no mature eggs. The female X-37 lived 15 days of adult life and her ovaries were full of mature eggs. She did not lay oothecas.
According to the present observations (Table I) it seems that the female would need a stimulus to start laying oothecas. This stimulus could be the presence of a male, if there was one; otherwise the female would wait until the last moment to lay her oothecas. Other probable stimuli could be environ- mental factors such as temperature and photoperiod. Once the process of laying started, it would not cease until the female died, or the environmental conditions turned unfavorable. It seems that the laying would depend on a certain period of the year more than on the chronotropic age of the individual. These data also indicate that about 30 days are needed to begin laying and that the yolk replacement is significant.
The next step was to wait until the oothecas hatched, since we were not sure that they contained viable eggs. The first nymph hatched 6 months and 18 days after laying, on ll-Oct-76 (Table II) and the last one on the 15-Jan-77.
According to our data for non-parthenogenetic populations, hatching never occurs after this date (Guerrero, Maggese, Cukier, 1977). The average interval between the laying of the ootheca and the first hatching was 7.5 months. From the 22 oothecas laid, 9 hatched nymphs, and 8 of the remaining 13 oothecas exhibited exposed cells that caused the eggs to dry. The number of nymphs ecloded per ootheca was very low, between 1 and 5. A total of 23 nymphs were hatched.
448
CUKIER, GUERRERO AND MAGGESE
TABLE II
|
Individual N |
Ootlieca N |
Number of hatched nymphs |
Sex |
Date of laying |
Date of the first hatching |
Time between laying and hatching |
Date of the last hatched nymphs |
|
X-22 |
1 |
2 |
female |
15-Mar-76 |
3-Nov-76 |
7 months and |
13-Dec-76 |
|
19 days |
|||||||
|
X-3 |
1 |
4 |
female |
!7-Mar-76 |
22-Nov-76 |
8 months and |
2 5- No v- 7 6 |
|
8 days |
|||||||
|
X-22 |
2 |
3 |
female |
23-Mar-76 |
ll-Oct-76 |
6 months and |
lS-Jan-77 |
|
18 days |
|||||||
|
1-17 |
1 |
5 |
female |
24-Mar-76 |
l7-Nov-76 |
7 months and |
13-Dec-76 |
|
24 days |
|||||||
|
X-3 |
2 |
1 |
female |
31-Mar-76 |
7-Dec-76 |
8 months and |
7-Dec-76 |
|
7 days |
|||||||
|
X-19 |
1 |
2 |
female |
31-Mar-76 |
29-Nov-76 |
8 months |
7-Dec-76 |
|
XII-82 |
1 |
1 |
female |
3-May-76 |
29-Dec-76 |
7 months and |
29-Dec-76 |
|
24 days |
|||||||
|
X-19 |
2 |
1 |
female |
7- Apr- 7 6 |
6-Dec-76 |
8 months |
6-Dec-76 |
|
X-22 |
3 |
4 |
female |
15- Apr- 7 6 |
22-Oct-76 |
6 months and |
13-Dec-76 |
|
3 days |
The nymphs were all females, with a highly restricted viability since none of them passed the second nymphal stage (they lived between 0 and 14 days). Due to these results, the oothecas were kept for observation.
At the beginning of May, some of the parthenogenetic oothecas were opened and eggs in perfect state were found inside them. These eggs were fixed, embedded in paraffin and stained, revealing a very early stage of development. They presented a blastoderm along the surface with a ventral thickening that would represent the beginning of the development of the germinal band.
Oothecas collected from nature after the hatching period (3 months) also con- tained fresh eggs in the same stage of development. In these last oothecas it was found that several of those that had yielded a low progeny had a great number of empty chorions.
1 234 56 7 8 9 10 11 12 13 X
A
10 u
FIGURE 1. Diploid karyogram of Coptopteryx viridis. This was made from two meta- phases of the second meiotic division.
PARTHENOGENESIS IN MANTIDAE
449
B
v
*c»
IQu
FIGURE 2. Chromosomes of Cof>toptcry.r viridis. A) Diakinesis. B) two metaphases of the second meiotic division (with 13 and 13-X chromosomes). C). A metaphase of the second meiotic division with 13 chromosomes and an anaphase with 28 chromosomes. D) Telophase with 13 and 13 chromosomes. E) Telophase with 13 and 13-X chromosomes.
This evidence gave rise to two possibilities: either the existence of an annual- biennial cycle gave rise to two types of eggs, some with a slower development than others, or the development of the eggs after a certain stage had ceased for some reason (the structure of the ootheca would have allowed them to remain fresh). Kume and Dan (1968) observed in Paratcnodcra aridifolia that in the diapause
450 CUKIER, GUERRERO AND MAGGESE
coincident with hibernation the embryos were in the same stage of development as ours.
It was decided to wait until the next spring to see which of the two alternatives was correct. The first proved to be valid, since on 22-Nov-77 nymphs started hatching from the parthenogenetic oothecas laid during the Summer-Autumn period of 1976 and which had already hatched nymphs in the Spring-Summer of 1976-1977 ; nevertheless the birth frequency was very low.
The next step was to determine the karyotype of the non-parthenogenetic individuals of this species. It was found that the male has 13 autosomes and a sexual chromosome as haploid number. Figure 1 shows the diploid karyogram of the species. It can be seen that it has acrocentric autosomes of the "t" type according to the classification of Levan, Fredga and Sandberg (1964), and the X chromosomes is subtelocentric of the "st" type according to the same classification.
This karyogram was built from two metaphases of the second meiotic division, with 13 and 13-X chromosomes respectively. As can be seen in the diakinesis of Figure 2a and in many others observed in this study, no trivalent chromosomes were found, which led us to the conclusion that this species belongs to the XX system for the female and XO for the male. Figure 2b shows two metaphases of the second meiotic division, with 13 and 13-X chromosomes respectively.
Figure 2c shows a metaphase of the second meiotic division with 13 chromo- somes and an anaphase with 28 chromosomes which is the result of the separation of chromatids from the metaphase of the previous figure (2b). Figure 2d and 2e show two telophases, one with 13 and 13 chromosomes and the other with 13 and 13-X chromosomes respectively, indicating the end of the meiosis seen in figure 2c. All of these, and other figures studied, made us think that this species belongs to the type XX-XO with a diploid number of 27 chromosomes for the male and 28 for the female.
DISCUSSION
The only species studied with this type of reproduction has been Brunneria borealis, from Central Texas and North Carolina, which reproduces exclusively by parthenogenesis (White, 1948) and Miomantis savignii (Adair, 1925) from Egypt, which reproduces both by parthenogenesis and after fertilization.
It has been seen in Coptpptcryx viridis, that the beginning of oviposition varies within a very wide range and is related to the moment in which the animals attain the adult stage. This laying would be related to a certain period of the year more than to the age of the individual. This might explain why the female that reached the adult stage at the beginning of summer delayed 80 days in laying her first ootheca, whereas the one attaining maturity at the end of the same season took 30 days in doing so (Table I). From the analysis of the ovaries studied it is not clear whether or not the growth of the oocytes is synchronous with the process of nuclear maturation (meiosis).
The study of the oogenesis of this species will help to clarify this point. It was observed that the chromosome morphology of Coptopteryx viridis differs from that of other species of mantids described by White (1941) and Hughes-
PARTHENOGENESIS IN MANTIDAE 451
Schrader (1943). These authors studied species from Europe, Africa and Central and North America and found that in most of them the autosomes are metacentric or "M" type, according to the classification of Levan (1964), with some of the submetacentric or "sm" type, according to the same classification. Nevertheless, the sexual chromosomes have the same configuration. This shows a marked difference with the chromosomes of Coptopteryx viridis where the autosomes are acrocentric or of the "t" type and the X chromosome is subtelocentric or of the "st" type according to Levan (1964). These data suggest that this is a diploid parthenogenesis, since haploid parthenogenesis never yields females. From this point, two possibilities arise : that the parthenogenesis is automictic or apomictic. In the first case the parthenogenesis would be exclusively thelytoky because of the sexual configuration of the species. In the second case, and due to the low number of hatched nymphs, it could be a thelytoky or amphitoky parthenogenesis. This last type of parthenogenesis would yield males by abnormal meiosis, as occurs with the Aphides (Homoptera) which have a XX-XO sexual system (Lees, 1961). This did not occur in Coptopteryx viridis, therefore we are inclined to think that this is a case of diploid parthenogenesis, automictic or apomictic, but exclusively thelytoky. Nevertheless, due to the low viability of the indviduals, it is possibly an automictic parthenogenesis, since homocygosis favors the expres- sion of deletereous genes.
The authors express thanks to Dr. C. Naranjo for photographic assistance.
SUMMARY
Several years of observations of the behavior of the mantid Coptoteryx viridis suggested evidence of parthenogenesis in this species. C. viridis is a solitary, sedentary animal, where the female often kills the male before copulation takes place, and the average male adult life is half that of the female.
Virgin females were reared in our laboratory from their hatching to the end of their lives; these laid oothecas. From these oothecas, parthenogenetic nymphs were born, all of the female sex and with a very low viability. The karyo- type of the non-parthenogenetic individuals of this species was found to be XO-XX with a diploid number of 27 chromosomes for the male and 28 for the female. The autosomes were acrocentric or "t" type while the X chromosome was sub- telocentric or "st" type, according to Levan's classification.
LITERATURE CITED
ADAIR, E. W., 1925. On parthenogenesis in Miomantis saingnii Saussure (Orth). Bull. Soc.
R. Entouwl. Egyptc, 8 : 104-148. GUERRERO, G. A., AND J. M. DE CARLO, 1976. Contribution al conocimiento del aparato genital
femenino y fases del desarrollo de Coptopteryx viridis (Insecta, Mantodea). Ph\sis
(B. Aires), 35 (#90 C) 125-137. GUERRERO, G. A., M. C. MAGGESE, AND M. CUKIER, 1977. Estudio de una poblacion de
laboratorio de Coptopteryx viridis Giglio Tos (1915) (Insecta, Mantodea). Physis
(B. Aires), 36 (#92 C) 295-303. HUGHES-SCHRADER, S., 1943. Meiosis without chiasmata in diploid and tetraploid spermatocytes
of the mantid Callimantis antillanun Saussure. /. MorphoL, 73: 111-141.
452 CUKIER, GUERRERO AND MAGGESE
KUME, M., AND K. DAN, 1968. Invertebrate Embryology. Printed in Yugoslavia by Prosveta,
Belgrade, 467 pages.
LEES, A. D., 1961. Clonal polymorphism in Aphids. Symp. R. Entomol. Soc. Loud., 1 : 68-79. LEVAN, A., K. FREDGA, AND A. A. SANDBERG, 1964. Nomenclature for centromeric position on
chromosomes. Hcrcditns, 52(2) : 201-220. WHITE, M. J. D., 1941a. The evolution of the sex chromosomes. I. The XO and XXY
mechanism in praying mantids. /. Genet., India, 42 : 143-172. WHITE, M. J. D., 1948a. The chromosomes of the parthenogenetic mantid Brunncria Borcalis.
Evolution, 2: 90-93.
Reference: Biol. Bull. 157: 453-463. (December, 1979)
DEVELOPMENTAL PATTERN AND ADAPTATIONS FOR
REPRODUCTION IN NUCELLA CRASSILABRUM
AND OTHER MURICACEAN GASTROPODS x
C. S. GALLARDO Institute dc Zoologia, Univcrsidad Austral de Chile, Casilla 567, Valdivia, Chile
An extensive literature about development in muricacean gastropods from the northern hemisphere and tropical coasts has been developed ; however, many of these works are strongly descriptive and do not always present an adaptive inter- pretation of features observed. In recent years a valuable advance has been achieved by Spight, based on his extensive field experience with thaidids. Some muricid developmental rules proposed by Spight refer to factors influencing pre-hatching time, ecology of hatching size, hatching type in relation to latitude and habitat conditions as well as factors conditioning selection of spawning sites (Spight, 1975, 1976b, 1977a, c).
To determine how far these rules are applicable in controlling the evolution of developmental patterns in muricaceans requires more extensive comparative knowledge of species from geographical areas not yet explored. In this sense, the coast line of Chile is very promising, as it comprises an extensive latitudinal range accompanied by great diversity in habitat conditions. Some years ago, a comparative study of reproduction in Chilean muricids was begun. Preliminary interest was focused on the commercially important species Concholepas concho- lepas (Gallardo, 1973; Gallardo, in press). Later, egg masses, embryo feeding and hatching type of Chorus gigaiitcus were also analyzed ; these results have been discussed in relation to habitat conditions (Gallardo, in press). In the present paper, studies on egg masses and embryos of the intertidal snail Nncclla crassilabntni from the locality of Mehuin, a small bay near Valdivia, are reported ; this informa- tion is complemented with field observations on habitat, spawning sites, embryo mortality and pre-hatching time at two different seasons. A discussion follows in order to interpret some of these features ; hatching type and hatching size are analyzed in relation to the rules earlier set out by Spight. Emphasis is given to embryo feeding patterns and their possible adaptive significance within the holo- benthic muricaceans, a question still not completely answered.
MATERIALS AND METHODS
Egg capsules of Nucella crassilabnnii were collected on the intertidal rocky shore of Mehuin (39° 25' S, 73° 10' W) from March, 1976 until April, 1977. Capsules of one cluster were separated and monitored to record capsule size com- position as well as synchronism of development between different capsules in each group. Development of embryos was followed by opening capsules in different
1 This research was supported by DI-UACH S78-8 Research Project.
453
454
C. S. GALLARDO
FIGURE 1. N. crassilabrum. Egg masses attached on vertical rocky substrate at the inter- tidal of Mehuin.
embryonic phases. Measurements and drawings were made from living material observed under a stereo-microscope provided with a micrometer eyepiece. Number of nurse eggs ingested per embryo was estimated from capsules in which all nurse eggs had been eaten ; careful dissections of embryos in early trochophore phase made it possible to determine the number ingested and its variation.
Pre-hatching time was recorded in clusters that were spawned at different seasons of the year. For this purpose, two egg masses containing eggs in early cleavage stage were selected and tagged. One of these was spawned during late autumn and the other during late spring. Each spawning site was periodically visited on tidal exposure periods until the snails had hatched ; on each visit, 3 or 4 capsules were collected for further examination at the laboratory. Water temperature of the sea is recorded daily at the Laboratory of Mehuin ; this is the information used for the present paper. Spawning sites used by N. crassilabrum were inspected to record habitat preferences. Embryos killed by physical stresses were identified by their pink color.
RESULTS
Egg capsules and masses
Shape of the egg capsule of Nucella crassilabrum resembles that of its congener, N. lapillus (Ankel, 1937). Each flattened capsule has concave and convex sides; when seen from either the convex or concave side, the structure above the peduncle appears nearly oval in shape, with a gradual increase in breadth toward the top. At the top of the capsule there is a circular exit hole which is closed by a
DEVELOPMENT IN A MARINE SNAIL 455
prominent plug. It is possible that, as observed in other neogastropods (Hyman, 1967), this plug weakens and dissolves as the embryos reach the hatching stage. The exit hole diameter is approximately 875 to 1200 /mi. Inside the capsule, eggs and mucus-like fluid are contained in a thin transparent sac. Each capsule possesses a short stalk; stalks of various capsules are cemented to the substrate in a con- tinuous band. The capsule wall is fairly transparent, showing the embryos inside. Clusters are yellowish, due to the yellowish eggs when freshly laid, and fade to dull grey with the development of the larvae. Size of egg capsules depends on size of the female producing them. The length of the capsules we have observed, exclud- ing the stalk, varies between 5.0 to 12.8 mm.
The capsules are laid very close to each other with a distance of approxi- mately 2.0 to 2.5 mm between stalks. Field observations suggest that, as typical in certain muricids, communal spawning is also the rule in this species. The clusters (Fig. 1) are laid close to each other, making it difficult to ascertain the number of egg capsules laid by a single female. Nevertheless, in certain cases, the orientation and capsule size allow one to distinguish egg masses laid by dif- ferent females; such clusters may contain up to 60 egg capsules. Capsules of the same cluster are arranged in a definite pattern, all of them facing the same direction ; capsules arranged in a given row alternate in position with respect to those from a contiguous row as seen in Figure 1. Direction in which the different clusters are oriented on the substrate appears random.
Spazvning sites
Egg masses are attached to rocky substrates, most frequently in crevices and on vertical surfaces and least frequently in tidepools and on horizontal surfaces. In general, permanently wet and shaded inter tidal sites, from extreme low water of spring tides to about mean low w^ater of neap tides, are preferred. Capsules at this last intertidal level were found in a group of rocks partially buried in the soft sandy beach. In this area, extreme seasonal sand fluctuations of about 80 or 100 cm are observed and a great quantity of sand is deposited during the summer, greatly reducing the rocky surface merging above the soft bottom. In that case, manv tss masses of Nucella crassilabntm are covered by the sand. Furthermore,
*> Oc5 •*
the retreating tide regularly exposes them to dry air and wind, especially throughout late spring and summer ; snails become exposed to air longer and more frequently than lower on the shore and many egg masses do not complete develop- ment, probably owing to the effect of these environmental stresses. Capsules writh embryos killed by physical stresses change from the normal yellow color to pink or purple. The most favorable situation for subsequent hatching of capsules was at those sites where they were permanently submerged in the sea water.
Eggs and embryos
The eggs are creamy white, their diameter varying between 204 and 293 /tin with a mean of 240 /*m. The number of eggs per capsule varies from 134 to 1116. A significant correlation observed between capsule length and number of eggs per capsule (Fig. 2) accounts for this variation. Besides the normally viable eggs there are many others that undergo atypical development and serve as nurse eggs.
456
C. S. GALLARDO
1200-1
1000-
LJ
ID800-
Q_ O
QC LU
0-.
3 600-
400H
200-
y=-347.5+93.68x
n=109 r= 0.818 P<0.01
|
1 5 |
i b |
i 7 |
i 8 |
i 9 |
i 10 |
11 |
i 12 |
13 |
14 |
CAPSULE SIZE (mm) FIGURE 2. N. crassilabrum. Relationship between number of eggs per capsule and capsule size.
Counts in three egg capsules of different sizes (Table I) reveal that only 6.6 to 7.9% of the total eggs in a capsule are viable. The number of embryos also increases in proportion to capsule size (Fig. 3) ; it varies between 10 and 122.
The intracapsular development of N. crassilabrum is of direct type. In viable embryos it follows the normal spiral pattern of cleavage. On the other hand, atypical cleavage is observed in non-viable nurse eggs which appear very variable in shape. The atypical development of these nurse eggs is finally arrested. The fertile larvae begin ingesting nurse eggs when they attain an early trochophore stage. By then, the mouth, esophagus and body expand to enclose entire nurse eggs as they are pushed down the digestive tract by the cilia lining it. The
DEVELOPMENT IN A MARINE SNAIL
457
TABLE I. Xucella crassilabrum. Percentage of viable embryos and nurse-eggs in three different capsides.
|
Capsule size |
Total nurse |
Viable embryos and |
|
(mm) |
eggs |
percentage |
|
7.9 |
406 |
32 (7.3%) |
|
6.8 |
342 |
24 (6.6%) |
|
8.6 |
397 |
34 (7.9%) |
ingested eggs are visible as distinct bulges in the body wall (Fig. 4b) and will spill out intact from an embryo opened during the feeding period. In the succeeding stages (Figs. 4c, d) and intracapsular veliger is gradually developed; by then, the embryos show the appearance of the shell and foot and the velar lobes are partially expanded. The embryo is now clearly divided into head (anterior to the shell), foot (ventrally), and visceral hump (covered by the shell) ; nurse eggs ingested still obscure details of internal structure (Fig. 4e). The posterior
120-
110-
100-
90-
S 80 o
LiJ O.
cn 60 o
2 50-
LU
40 30 20 10
y=-35.87i-9.3x
Ti=108 r=0.678 P<0.01
— i 1 1 1— — r~ ~T— — r~ — r~ ~~r~
56 8 9 10 11 12 13 14
CAPSULE SIZE (mm)
FIGURE 3. N. crassilabrum. Relationship between number of viable embryos per capsule and capsule size.
458
C. S. GALLARDO
£f
FIGURE 4. N. crassilabrum. Different stages of intracapsular development, a) a fertile egg (left side) next to a nurse-egg, in cleavage stage, b) trochophore stage with the outlines of whole nurse-eggs seen through the body wall, c-d) early veliger stage, e-f) mean and advanced veliger stage, g) pre-hatching stage with the velum in reabsorption process, h) hatching juvenile stage. The lines equal 500
face of the foot gradually differentiates a small, thin operculum. On either side of the foot a spherical statocyst is visible. Within the velar rim and around the stomodaeum, rudiments of the adult head are now beginning to become organized and a pair of black eyespots is visible on the base of recently developed tentacles
DEVELOPMENT IN A MARINE SNAIL
459
(Fig. 4f). Ingestion and feeding activity may continue in some capsules up to the veliger stage illustrated in Figure 4e.
The next stages of the intracapsular veliger (Figs. 4g, h) show a gradual and extensive increase in size, especially of the shell, foot and tentacles. The yolk gradually disappears in the visceral hump with accompanying differentiation of the viscera. A columella muscle faintly visible on the left side of the visceral hump is able to effect withdrawal of both head and foot into the shell. By the end of intra- capsular development, the lateral lobes of the velum have been resorbed and this pre-hatching juvenile shows an active crawling foot and large tentacles.
Embryonic feeding and rate of development
During intracapsular development, some embryos acquire more nurse eggs than their capsulemates and this is reflected in the size distribution of embryos and hatchlings. Embryos in trochophore phase as small as 450 /mi were found together with others as large as 775 /mi after all nurse eggs had been eaten. By this time, the number of eggs eaten by each larva varies from 3 to 20 as shown in Figure 5 (left side) for three capsules with different numbers of capsulemates. This figure also shows the frequency at which embryos ingest different numbers
• n
<s>
•'.
LU
I
n
n
UJ
•
e m
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: LLl
L
X= 11.67n.e.per embryo Ti- 34 embryos ; 392n.e.
5-
>= 31embryos
|
X=12.68-n.e. |
X=1157ijm |
|||||||||
|
- |
n=32 ; 406Tve. |
71=18 |
||||||||
|
5- |
5- |
|||||||||
|
- |
1 i |
|||||||||
X=14.25n.e.
10-1 o
; 330n.e.
2' V 6' 8 10 12 14 16 18 20 NURSE EGGS INGESTED
|
X=1230/im •n=20 n rfh |
i— |
— , |
— i |
|
//b=i i i U ' ' '-' ' ' Ci I i fc |
~ |
B
m
X
•
HATCHING SIZE (jjm)
FIGURE 5. N. crassilabrum. Left, frequencies of embryos ingesting different numbers of nurse-eggs in three different capsules. Right, hatching size frequencies between embryos of three different capsules, n.e. = nurse-eggs.
460 C. S. GALLARDO
of nurse eggs. Hatching size distributions for juveniles from three different cap- sules are shown in Figure 5 (right side). While some juveniles attain 825-/xm shell length at the moment of hatching, others are as large as 1300 jam. Mean number of nurse eggs ingested per embryo per capsule as well as mean hatching size, seem to change regularly as the number of embryos per capsule decreases. The effect of haphazard distribution of embryos among capsules on hatching size, has been previously studied by Spight (1976a) in the muricids Thais cinarghiata and Acanthina spirata. While embryo counts are less predictable in these species, nurse eggs per capsule are distributed more regularly. Some embryos share their yolk supplies with many more capsulemates than others and this factor is most important in determining a variable hatching size between nurse egg feeders' embryos (Spight, 1976a).
Time required to complete intracapsular development was estimated from two egg masses spawned at different seasons of the year. At the time the observations began, the egg capsules were in an early cleavage stage. An egg mass controlled during autumn and winter months took between 70 and 80 days to fully develop and hatch ; mean of monthly water temperature from May to August, 1976 at Mehuin, varied between 9.67 and 10.60° C. In other masses that developed during late spring and early summer, the time was significantly reduced to approximately 55 to 65 days ; in this case, mean of monthly water temperature from November, 1976 to January, 1977 varied between 11.40° and 14.10° C. Doubtlessly, dif- ferences in developmental rates in nature are well accounted for by seasonal changes in sea water temperatures at Mehuin.
DISCUSSION
Nucclla crassilabniiii showys the most evolved pattern of development known for Chilean muricids if we compare it with observations in C. concholcpas (Gallardo, 1973) and in Ch. gigantcus (Gallardo, in press). The type of development of N. crassilabniiii agrees with that reported for its congener N. la pill us (Pelseneer, 1910). Such pattern of development is expected in high latitude muricaceans inhabiting interidal rocky shores (Spight, 1977a). Spight establishes that, among the rocky intertidal muricaceans, the hatching type evolves markedly according to a latitudinal gradient ; while in this habitat all tropical species maintain planktonic larvae, all high latitude species metamorphose before hatching. In this sense, N. crassilabniiii , whose geographic distribution extends along the Chilean coast to the Magellan Strait, may be included within the muricid group showing such reproductive tendency. In a paper about hatching type of Chorus giganteus (Gallardo, in press), a case of coincidence with Spight's predictions for muricaceans from another type of habitat has also been reported. According to Spight (1977a), muricaceans with an intermediate hatching type (a non-feeding veliger near to metamorphosis) had been found to inhabit a shallow water sand habitat; the findings in Ch. gigantcus agree, as this sand bottom muricid also possesses such hatching type. Causes for these tendencies in muricid developmental patterns when related to latitude and habitat conditions are unknown.
All reproductive patterns should involve adaptations to maximize survival of embryos and, consequently, their reproductive fitness. Some of these adaptations
DEVELOPMENT IN A MARINE SNAIL 461
should include ability of adults to discriminate and choose good spawning sites, as well as an appropriate developmental time and hatching size. Our field observations in N. crassilabruin reveal that mortality of embryos is of common occurrence at least at the upper shore spawning sites. Probably some sites offer more protection than others ; for instance, one would expect physical stresses to be minimal in tidepools, but they are used less frequently than vertical surfaces exposed to repeated dehydration during low tides. Accord- ing to observations of the intertidal muricid Thais lamcllosa (Spight, 1977c), spawning sites selected would reflect the conflicting demands of different life history stages. According to Spight, vertical surfaces are least accessible to predators and often are covered with food (barnacles), but provide little protec- tion from physical stresses. Crevices offer some protection from physical stresses and usually are covered with barnacles but are accessible to predators. Understone surfaces and tidepools offer protection from stresses but are accessible to predators and are often distant from food sources. With these considerations in mind, Spight concludes that T. laindlosa females deposit capsules most frequently on the surfaces that are most suitable when the demands of all life history stages are considered. Studying the congeneric species N. lapillus, Feare (1970) found that physical stresses operating during exposure to air are responsible for hatching success of G/-O at mean tide level and 57'yo at mean low water of neap tides. Our field observations in N. crassilabnnn suggest that this species also uses poor spawn- ing sites regularly and it probably does so for the same reasons that T. lamellosa does. For instance, different environmental stresses seem to be influencing sur- vival of N. crassilabruin adults when closely located sites are compared (Gallardo, in preparation) ; demands of this life history stage could be in part conditioning the selection of spawning sites that are most suitable.
Hatching size of intertidal muricaceans is shorter (0.6 to 1.3 mm; X = 1.01 mm) than that of species living in subtidal habitats (0.6 to 2.5 mm ; X = - 1.54 mm) or on coral reef flats (1.1 to 1.9 mm; X== 1.40 mm) [Spight, 1976b; (X) = mean values calculated from a few species where data are available]. According to this, the hatching size of Ar. crassilabnnn (0.82 to 1.30 mm) corresponds with the habitat this muricid occupies. To attain the appropriate hatching size, holo- benthic muricaceans have followed two evolutionary paths : provision of nurse eggs or increase in size of a self-sufficient fertile egg. Now the question arises : which selective forces favor one or the other of these developmental options? It has been hypothesized that providing much of the yolk as nurse eggs may allow a snail to have a large hatching size and at the same time a relatively brief developmental time (Spight, 1975). In order to prove the validity of this hypothesis between muricaceans, we may use information available about developmental time, hatching size and embryonic feeding source both in N. crassilabnnn (this paper) as in other species (Spight, 1975, 1976b) ; this information is compared in relation to habitat. We may see that among intertidal muricaceans adaptation for nurse egg feeding is of common occurrence ; in a few species of this habitat, energy for developing embryos is totally obtained from larger fertile eggs (Thais lamcllosa 590 fj-m ; T. canaliculate,, 620 /xm). T. lamcllosa reveals a developmental time of 67 to 91 days at 9.6° to 11° C, more or less similar to that of N. crassilabruin, although the upper limit of its range, 91 days, slightly exceeds that of N. eras-
462 C. S. GALLARDO
sihibrum; SO days at 9.6° to 10.6° C. On the other hand, subtidal species usually require a large hatching size. To attain it, they usually have evolved a large fertile egg (Ceratostotna joliatum, 720 /xin ; Torvamurex territus, 67 S /xm) ; numerous nurse eggs per embryo are consumed in species of this habitat showing such feeding mechanism (Mure.v senegalensis, 35 n.e. per embryo ; M. qiiadrifrons, 91 n.e. per embryo). Subtidal species whose egg exceeds 600 /mi in diameter, reveal a markedly slow developmental rate; for instance, T. territus takes 90 days to hatch at 20° C and C. foliatum takes 120 days to hatch at 10° to 12° C. Our observations in N. crassilabrum and those of Spight (1977c) and Feare (1970) reveal that one of the most important sources of embryo mortality in muricaceans laying in the intertidal, are the physical stresses operating at this level ; an embryo of T. lamellosa cannot tolerate even one two-hour exposure (Spight, 1977a). Similar observations have been made by Pechenik (1978) in prosobranchs of the family Nassidae. Pechenik found that egg capsules of Nassarius obsoletus did not afford substantial protection against desiccation; 0.5-hr exposures to 75% relative humidity killed 17.5% of the embryos. It is reasonable to assume that such mortality would be reduced by laying the egg capsules in protected micro- environments, as N. obsoletus clearly does (Pechenik, 1978), or by shortening developmental time of clusters spawned at more exposed sites of the intertidal. When, for reasons considered above, an intertidal muricid does not always use good spawning sites for embryos, the possibility that it is shortening developmental time should be preferred. An evolutionary option in this sense may be to change to another form of embryonic feeding, that is the provision of nurse-eggs. But, how to explain nurse-egg feeding by some subtidal muricaceans? In this case, this embryonic feeding adaptation seems to have been specially favored to attain an extremely large hatching size. Relatively large muricid eggs range from 675 to 920 /Am in diameter (T. territus, Thais lima) ; nevertheless the hatchlings developed from such eggs do not exceed from 1.3 to 1.5 mm in length. In subtidal species, usually requiring a larger hatchling (1.6 to 2.0 mm), each embryo is provided with numerous nurse eggs (M. quadrifrons, M. senegalensis}. These preliminary considerations suggest that a delicate compromise between at least the optimal hatching size and developmental time, could be orienting evolution of embryonic feeding adaptations among holobenthic muricaceans. However, further work is necessary to reinforce this hypothesis. The results for the small number of species considered in this discussion cannot, by themselves, support it, but they can add to the data for future comparative studies. Information about developmental time, hatching size, habitat, and mortality sources is still lacking in various muricids. An optimal material to evaluate the advantages of evolving nurse eggs is offered by T. canaliculata ; this intertidal snail appears to be in the course of evolving from one reproductive mode (self-sufficient large egg) to the provision of nurse- eggs for embryos (Spight, 1977b).
SUMMARY
1. Eggs of Nitcella crassilabrum range from 204 to 293 /-an in diameter (mean = 240 jum). Only 6.6 to 7.9% are fertile; the remaining are ingested as nurse eggs.
2. Embryos metamorphose before hatching. Pre-hatching time ranges from 55 to 80 days according to seasonal temperature fluctuations.
DEVELOPMENT IN A MARINE SNAIL 463
3. Hatching size varies from 0.82 to 1.3 mm, depending on number of nurse- eggs ingested per embryo (from 3 to 20). Number of fertile embryos per capsule (10 to 122) depends on capsule size.
4. Hatching type and hatching size shown by N. crassilabntm agree with those of other muricaceans living in similar habitat conditions.
5. Pre-hatching time and hatching size data of various muricaceans are analyzed to determine to what extent they influence embryonic mode of nutrition, namely the presence of nurse-eggs or alternatively large and fertile self-sufficient eggs. Provision of nurse-eggs for embryos is of common occurrence among intertidal muricaceans and this mode of nutrition seems to have been favored in such habitats to reduce developmental time. Providing the yolk as nurse-eggs seems also to contribute to a larger hatching size, as suggested by some subtidal murica- ceans with such embryo support patterns.
LITERATURE CITED
ANKEL, W. E., 1937. Der feinere Bau des Kokons der Purpurschnecke Nucella la pill us (L.)
und seine Bedeutung fur das Laichleben. Vcrh. Dtsch. Zool. Gcs., 1937 : 77-86. FEARE, C. J., 1970. Aspects of the ecology of an exposed shore population of dogwelks Nucella
lapillus (L.). Oecologia, 5 : 1-18. GALLARDO, C. S., 1973. Desarrollo intracapsular de Concholcpas concholcpas (Brugiere)
(Gastropoda Muricidae). Musco National dc Historia Natural, Santiago, Chile.
Pub!. Ocas., 16 : 1-16. GALLARDO, C. S., 1979. El ciclo vital del muricido Concholcpas concholcpas y consideraciones
sobre sus primeras fases de vida en el bentos. Biol. Pcsq. Chile (In press). GALLARDO, C. S., 1979. Postura y estadio de eclosion del gastropodo Muricidae Chorus
giganteus (Lesson, 1829). Studies on Neotropical Fauna and Environment (In press). HYMAN, L. H., 1967. The Invertebrates. Vol. VI. Mollusca I. McGraw-Hill Book Com- pany, New York, 792 pp. PECHENIK, J. A., 1978. Adaptations to intertidal development : studies on Nassarius obsoletus.
Biol. Bull., 154:282-291. PELSENEER, P., 1910. Recherches sur I'embryologie des Gastropodes. Mem. Acad. R. Belg.,
Ser. 2, Vol. 3 : 1-167. SPIGHT, T. M., 1975. Factors extending gastropod embryonic development and their selective
cost. Oecologia, 21 : 1-16. SPIGHT, T. M., 1976a. Hatching size and the distribution of nurse eggs among prosobranch
embryos. Biol Bull, 150 : 491-499.
SPIGHT, T. M., 1976b. Ecology of hatching size for marine snails. Oceologia, 24 : 283-294. SPIGHT, T. M., 1977a. Latitude, habitat and hatching type for muricacean gastropods. The
Nautilus, 91(2) : 67-71. SPIGHT, T. M., 1977b. Is Thais canaliculata (Gastropoda: Muricidae) evolving nurse eggs?
The Nautilus, 91(2) : 74-76. SPIGHT, T. M., 1977c. Do intertidal snails spawn in the right places? Evolution, 31: 682-691.
Reference: Biol. Bull. 157: 464-477. (December, 1979)
EARLY POST-METAMORPHIC GROWTH, BUDDING AND SPICULE
FORMATION IN THE COMPOUND ASCIDIAN
CYSTODYTES LOBATUS
GRETCHEN LAMBERT
Department of Biological Science, California State University, Fullcrton, California 92634 and Hopkins Marine Station of Stanford University, Pacific Grove, California 93950
Compound ascidian colonies are comprised of many individuals wholly embedded in a common tunic. Budding is accomplished by active epidermal synthesis and constriction, with the regenerating buds moving through this common tunic to form a new system (see Berrill, 1951, 1961 for reviews of early papers; Sebastian, 1957; Levine, 1960; Freeman, 1971 ; Nakauchi, 1966a, b, c, 1970, 1977; Nakauchi and Kawamura, 1974a, b, 1978). Cystodytes, unlike other genera in the suborder Aplousobranchia, family Polycitoridae, is distinguished by large numbers of cal- careous spicules surrounding the abdomen of each zooid, forming a spicular sac separating each individual from neighboring zooids (Ritter, 1900; Van Name, 1945). During budding this spicular sac must be reorganized and reformed around the abdomen of each bud, but the way in which this is accomplished is completely unknown. The common tunic overlying the spicular layer contains numerous large, closely packed, acid-filled bladder cells (Abbott and Newberry, 1980) ; any rupture of these cells results in immediate dissolution of nearby spicules and evolution of CO2. The spicules might be dissolved and reformed during budding or just reallocated in some way among the buds; there appear to be no life history studies on any species of the genus Cystodytes, even though the genus is common and widespread (Van Name, 1945; Millar, 1975). Therefore, the present study is an examination of the general ecology and method of budding in Cystodytes lobatus (Ritter, 1900), a species that occurs abundantly in the low intertidal and subtidal zones along the central California coast. The time course of tadpole release, spicule formation in oozooids (the individuals developing from newly-settled tadpoles before the first budding), and the method of budding with each zooid isolated in its own spicular sac are discussed here. Included is the discovery that the spicules are contained within a discrete extra-cellular membrane. This membrane has been named the tunic spicular lamina and is concluded to form an organic matrix for spicule formation.
MATERIALS AND METHODS
This work was carried out at the Hopkins Marine Station, Pacific Grove, California, between February and August, 1978. Animals were collected from rocks in front of the marine station and at Pt. Pinos, about two miles away. Three color variants exist: white, pink and orange. Since the taxonomy of these variants has not been studied, and since Ritter (1900) based his original description of the species on the white form, only white colonies were used throughout this study.
464
ASCIDIAN BUD AND SPICULE FORMATION 465
Colonies were maintained in unfiltered running sea-water aquaria in the laboratory ; between February and August the temperature varied only from 13° to 16° C. Colonies usually lived at least a month in the laboratory, and if they reattached to the bottom of the aquarium they survived even longer.
Colonies with tadpoles were easily recognized by the presence of the bright pink yolky embryos. Since it is known that some colonial ascidians release their tadpoles in response to light (e.g., Watanabe and Lambert, 1973), the colonies were kept in the dark at night. Tadpole collection was accomplished by removing the colonies in the morning to a clear plastic aquarium supplied with running sea water. Drainage was through a hose penetrating the aquarium at one end, with water flow adjusted so that the water level was at the hose exit. In this way, as tadpoles were released from the colonies and swam upwards they were skimmed off and carried through the hose to a tadpole collector, consisting of a short piece of polyvinyl chloride pipe with 300-ju.m Nytex glued to the bottom of it and resting in a small dish. Tadpoles were released throughout the day. Periodically they were removed from the collector to a petri dish with a glass slide in the bottom of it centered over a piece of black plastic under the dish to induce settling of the tadpoles in the darkest area. These slides were then placed in a plexiglass slide holder and submerged in a large cement tank in the laboratory filled with running unfiltered sea water where they were maintained for several months. Slides were removed from the holder and placed in a sea water-filled petri dish for examination of the living zooids, using an American Optical dissecting microscope with phototube. All photographs were taken with an Olympus OM-2 35-mm SLR camera with microscope adapter.
A Beckman Expandomatic IV pH meter equipped with a rapid-response M 1-410 combination pH probe (Microelectrodes, Inc., Londonderry, N. H.) was used for all pH determinations. Bladder cell pH was determined in two ways. Adult colonies were washed several times in distilled water, blotted dry with Kim wipes, then either the pH electrode was carefully inserted about 2 to 4 mm into the superficial layer of the tunic or bladder cells in the upper tunic layer were broken by agitation with a fine probe and then the pH electrode was immersed directly into the resulting pool of (mostly) bladder cell fluid. Results were the same for both methods. Bladder cell contents were analyzed for the presence of chloride and sulfate ions by probing them directly with hand-made finely drawn out glass micropipettes under a dissecting microscope. The fluid collected was expelled into a watch glass, and a drop of either silver nitrate or barium chloride was added. (AgNO3 forms a precipitate of AgClo in the presence of chloride ions, BaClo forms a precipitate of BaSO4 in the presence of sulfate ions.)
Because alcohol dehydration and even fixation in buffered formalin did not prevent rupture of bladder cells and dissolution of spicules, razor blade sections of living colonies were made and stained supravitally with PAS, aldehyde fuchsin, alcian blue pH 2.5, alcian blue pH 1.0, sudan black B, 0.1% toluidine blue, or neutral red, using the methods in Pearse (1968), in order to analyze the tunic for acid mucopolysaccharides and other structural materials.
A few 4-wk-old oozooids settled on slides were relaxed in 100 ml sea water to which had been added 1 drop of menthol-saturated 95% ethanol, according to the method of Abdel-Malek (1951), When total relaxation was achieved in 3 to
466 GRETCHEN LAMBERT
6 lir, 10' ^ formalin was added drop by drop while stirring, until the animals were dead. The zooids were transferred to \()% formalin for 24 hr, then removed in tin their slides, washed, dehydrated in alcohols and embedded in the Polysciences 1 154 plastic embedding medium. After 24 hr the plastic blocks were trimmed and sectioned at 1 to 2 /tin. Sections were removed one at a time, placed in a drop of 1'y ammonia on a slide to flatten out. then flame-dried and stained with methylene blue or O.I'/ toluidine blue.
RESULTS
General biology
Cystodytes lobutns grows in large mats, up to a half meter across or more; whether each mat is one colon\' or many that have fused or abutted wras not determined. Colonies bv the Hopkins Marine Station averaged 5 mm in thickness. However, material collected elsewhere and in D. P. Abbott's private collection may be up to 1 cm or more in thickness (Abbott and Xewberry, 1980).
Colonies always occurred in the low intertidal or subtidal : a —0.6 ft tide or lower was necessary to collect intertidally. The best intertidal sites were under overhanging rocks and to a lesser extent on the north side of vertical rocks, away from direct sunlight and (possibly) from competition with plants.
Large colonies were commonly observed to have overgrown many barnacles and polychaete worm tubes, resulting in a mat with many superficial ridges and knobs. Cross sections indicated colonies to be of variable thickness ; some ridges covered overgrown barnacles while others were present without any underlying irregularity of the substratum. Other compound ascidians, common in the same area as Cystodytes, were Aplidunn calijornicuin and Archidistoina psammion; colonies of both these species also attained large size, and a dynamic situation appeared to be present of overgrowth of one ascidian by another — either overgrowth of Cystodytes by Aplidinni or Archidistoina or vice versa.
Very few predators were observed feeding on C. lobatns; those animals found on or near colonies in the field were the starfish Patiria ininiata and the gastropods CaUiostoina ligatnin, Tegula fitncbralis, Mcgathitra crcnitlata, and Lamcllaria dicgoensis. However, these observations were made intertidally at low tide when Cystodytes was out of water ; high tide observations might be different. When Cystodytes is out of water it forms a slimy coating of an (apparently) mucus-like material that can be seen hanging in long strands and dripping off the colonies. This material is not present in submerged colonies until the surface is rubbed. The significance of this is as yet unknown.
Laboratory feeding observations in the above-mentioned animals found on or near colonies showed that all but Tegula junebralis would eat Cystodytes in the laboratory. Stomach contents of subtidal Calliostoiua ligatnui and Mcgatliura creimlata collected on or near Cystodytes yielded chunks of colony with the spicules mostly intact (Sellers, 1977 and personal communication). Lamellaria dicgoensis was maintained in the laboratory from March 10 until August 31 on a diet of nothing but Cystodytes lobatus; during this period its weight increased from 2.2 grams (May 3) to 6.0 grams (August 21). A study of feeding of
ASrmiAN P.IM) AND SPICULE F< )KM ATK >N
467
FIGTKK 1. Two Cystodytes lnhatus systems comprised of 5 and 3 xooicls. Scale l)ar 1 mm.
Lamellaria on Cystodytes, including a calorific analysis of C \stod\tes colonies utilizing a semimicro bomb calorimeter, will IK- published separately (Lambert, 1979).
Other animals associated with Cystodytes as well as other colonial ascidians are the clam Mytilimeria nitttalli and the amphipocl Polyclicria osborni, both of which live embedded in the ascidian test (Abbott and Newberry, 1980). Very little is known of the relationship of these species to their ascidian hosts (Skogsberg and Vansell, 1928; Yonge, 1952).
Colony organization and tadpole release
The zooids of Cystodytes (Fig. 1) are arranged in systems (Van Name, 1945) with a mean of 4 or 5 zooids per system (Fig. 2). The atrial siphons open separately at the surface (Fig. 1). The abdomens are surrounded by a layer of overlapping calcareous spicules (Figs. 3, 4) and the test matrix is filled with bladder cells (Van Name, 1945) tightly packed and ranging in size roughly from 35 to 80 /mi. These cells are filled with sulfuric and possibly hydrochloric acids; tests were positive for the presence of both sulfate (Abbott and Newberry, 1980)
14
i a • 10
8 6-
12345671
Number of zooid s/s y s t e m FIGURE 2. Number of zooids per system, n = 55 systems, x — 4.36 zooids/sys'.em, s.d. = 1.69.
k.s
GRETCHEN LAMBERT
FIGURE 3. Underside of a C. lobaius system. Scale bar 1 mm. FIGURE 4. C. lobatns oozooid 32 days old. Scale bar 0.5 mm.
and chloride ions. The pH of bladder cell contents was determined to he l.o.
Kmhryos develop within the atrial chamber of the adult zooids (Van Name, 1945). Because of their bright pink color they could be observed easily and were recorded in nearly all colonies collected between March 7 and August 18.
Tadpoles were collected in the tadpole collector and settled on glass in order to observe the growth of zooids in the laboratory, the time course of spicule and bladder cell formation, and the method of budding. The time course of tadpole release (Fig. 5) reveals that more tadpoles were released after 3 to 4 hr in the light following overnight dark adaption under black plastic than at any other time, and tadpoles were released more or less continuously all day. Colonies left in an uncovered aquarium overnight in order to observe their reaction to natural dawn also released larvae sporadically throughout the day.
Cystodytes tadpoles are large, with a mean body length of 1.27 mm (n = 25, s.d. "0.113), tail length of 2.59 mm (n == 25, s.d. == 0.141), and tail width of
80- 70 • 60 SO 40 30 20 10-
3456
Hours in light
FIGURE 5. Tadpole release as related to duration of light period after darkness. Numbers are totals for 10 days of observations.
ASCIDIAN BUD AND SPICULE FORMATION
469
0.76 mm (n -- 25, s.d. — 0.049). The heart beats somewhat erratically but does reverse, as in the tadpoles of Distaf>lia and Diplosoma (Cloney, personal com- munication) and Pycnoclavclla stanleyi (Trason, 1963). Bladder cells are already densely packed in the test matrix of the tadpole. The pH of six individuals homogenized in a few drops of distilled water was 2.85. A few tadpoles meta- morphosed within 15 min of being released, though most had a fres larval life of 1 to 3 hr. Settlement was greatly enhanced by using slides that had been soaked in sea water for several days.
Post-inctainorphic groi^th and budding
Young oozooids begin to feed 3 to 4 days after settlement (as determined by the presence of food pellets in the gut) and by one week of age the gut has dif- ferentiated into five well-defined regions similar to those in many aplousobranchs : esophagus, stomach, post-stomach, mid-intestine, and intestine or rectum. The stomach and intestine are orange-brown; the rest of the zooid is colorless or nearly so. By 24 hr or so after settlement, oozooids have four rows of stigmata, the same as the adult blastozooids. This is similar to the closely related poly- citorid Archidistoma ritteri ( Levine, 1960) in which both oozooids and blasto- zooids have three rows. In the polyclinid Amaroucium multiplicatum (Nakauchi, 1966a), in contrast, the oozooids have four rows but after budding the blastozooids have six or seven rows of stigmata. In (". lobatns there are usually four stigmata per side in the anteriormost row at metamorphosis ; at budding this row has 17 or 18 stigmata per side. \Yith successive buddings the blastozooids orient vertically with the abdomen directly beneath the thorax rather than curved around it is in the oozooid, but otherwise the oozooids and blastozooids appear to be the same morphologically. \Yhether oozooids ever from gonads, however, was not determined.
There is great variation among oozoids in the quantity of spicules produced, even those arising from tadpoles from a single colony reared on the same slide (compare Fig. 4 with Figs. 6 and 8). A number of tadpoles were collected on May 25 and allowed to settle on three slides which were maintained in the
TABLE I.
Relationship between spicule density and time to budding, number of abdominal buds and time until buds begin feeding in C. lobatus.
|
n |
X |
s.d. |
|
|
Days from settlement to budding for zooids with few |
|||
|
spicules |
32 |
29.72 |
3.0 |
|
Days to budding for zooids with many spicules |
18 |
37.33 |
6.28 |
|
Number of buds, few-spiculed zooids |
32 |
3.59 |
0.61 |
|
Number of buds, many-spiculed zooids |
18 |
2.89 |
0.47 |
|
Days from budding until buds begin feeding in few- |
|||
|
spiculed zooids |
24 |
5.58 |
0.50 |
|
Days from budding until buds begin feeding, many- |
|||
|
spiculed zooids |
15 |
5.67 |
0.62 |
470
CKKIVHKN I.AMI'.RRT
FIGURE 6. T \venty-five-day-old oozooid. Arrow indicates position of new stomach. Scale bar 0.5 mm.
FIGURE 7. Thirty-one-day-old oozooid. Arrow indicates new stomach at posterior end of mother bud. Scale bar 0.5 mm.
FIGURE 8. Thirty-three-day-old oozooid; budding is nearly complete. Scale bar 1 mm.
FIGURE 9. Four-week-old oozooids, 1 /uni sections, (a) entire zooid ; scale bar 250 /*m. (b) Enlargement of tunic region; scale bar 100 /urn. (c) TSL region of tunic; scale bar 20 fj.m. b = bladder cell, o = oozooid, s = spicule, TSL = tunic spicular lamina, zc = zooid cavity.
FIGURE 10. Thirty-six-day-old oozooid forming 3 abdominal buds ; scale bar 0.5 mm. B = bud, TSL =• disrupted tunic spicular lamina.
FIGURE 11. Spicule from an adult C. lobatus colony. Scale bar 50
ASCIDIAN BUD AND SPICULE FORMATION 471
laboratory until the end of August. Table I compares oozooids that developed few spicules with those that developed many. Zooids with many spicules took longer to bud (37 days as compared with 30 days) and produced fewer buds. (These are lab times; colonies occurring naturally in the field grew more rapidly than those raised in the lab, and achieved larger size). Without exception, buds always produced the same (subjective) quantity of spicules as the mother had: all buds of a many-spiculed oozooid developed a heavy coating of spicules, and all buds of a few-spiculed oozooid developed only a sparse coating of spicules. This may explain why in some colonies in the field all the zooids have few spicules, while in other colonies all zooids have many spicules.
An attempt was made to study growth rate in a group of zooids by removing slides from the slide holders every other day for measurement of the zooids settled on them. However, this had an obviously detrimental effect on the zooids: the time to budding was longer, only two buds were produced per oozooid, and nearly all the animals ultimately degenerated, so these data were discarded. Other workers contemplating using this technique should be aware of these possible effects.
Although the process of budding has not previously been described for Cysto- dytcs, it is in fact similar to budding in other polycitorids (Oka, 1942; Oka and Usui, 1944; Berrill, 1947, 1948; Levine, 1960; Nakauchi, 1966b, c) and in the polyclinids (Nakauchi, 1966a, 1970, 1974, 19/7). Cystodytes exhibits Xakauchi's (1966a) Type I budding (abdominal ; all buds receive some digestive and epicardial tissue). As in Aniarouchtni ycunazii (Nakauchi, 1970), budding in Cystodytes is preceded by an elongation and enlargement of the posterior end of the esophagus which begins at least 10 days prior to budding (Fig. 6). This esophageal enlarge- ment will become the new stomach of the mother bud (Figs. 7,8). A few hours before budding begins, the orange-brown stomach and intestine elongate greatly, as does the epicardium. Feeding ceases, the thorax contracts, and all fecal pellets in the digestive tube collect at the end of the rectum. Budding proceeds posteriorly, with the first constriction occurring at the anterior end of the old stomach. Sometimes the heart of this first bud (called the mother or thoracic bud) can be seen beating at this time. Three or four abdominal buds form, with the terminal bud receiving the mother's old heart. All buds receive some of the orange-brown stomach and intestinal tissue as well as some epicardial tissue. After budding is completed and the mother bud's digestive tube has fully regenerated, the thorax relaxes, the old fecal pellets are ejected, and feeding resumes. Buds begin feeding after 5 to 6 days, and the next budding occurs in about 3i weeks (lab time). Buds usually orient to the mother but one or two might join a neighboring system; this is probably the reason for the large range in number of zooids per system in Figure 2.
Spiculc formation and rcallocation at budding
As mentioned earlier, a major difference from the Polyclinidae and from other genera in the Polycitoridae is the formation of calcareous spicules in Cystodytes. Using a dissecting microscope at 40 or 80 X one can distinguish the first spicules as early as 5 days after settlement. At first, the spicules are very tiny and cannot be individually discerned ; only a white spot is noticeable constituting the
472 GRETCHEN LAMBERT
entire amount of spicular material. Spicule formation always begins on the inner side of the abdomen at the junction between abdomen and thorax. Gradually the spicules appear to migrate as they increase in number and size until at the end of 4 weeks (laboratory growth time) a zooid's abdomen is completely covered with somewhat overlapping non-birefringent spicules (Fig. 4). These spicules are not attached to the abdomen, though ; they are embedded in the innermost layer of tunic that lines the zooid cavity. This layer has been named the "tunic spicular lamina" (TSL). Figure 9 shows the newly forming spicules embedded in the middle of the TSL. Above it are the closely packed bladder cells extending all the way to the outer tunic surface, with the common tunic consisting of the material between the bladder cells. The TSL appears to be completely extracellular, because when a razor blade cross section is made through an adult colony, the zooids can be removed easily from the tunic leaving all the spicules behind. The TSL thus forms a spicular sac in the tunic surrounding but separated from the abdomen of each zooid.
The fate of the spicules at the time of budding was next studied, to determine whether they are dissolved and reformed or simply divided up among the buds in some way. A careful examination at 2 to 3 hr intervals of a few budding zooids showed that first the abdominal epidermal constrictions separated the buds within the zooid cavity. When this process was at least partially completed, the spicular sac began to constrict, resulting ultimately in the abdominal portion of each bud being surrounded by a small spicule sac (Fig. 10). At no time was any dissolution of spicules observed ; some of the larger spicules could be followed as they migrated to the buds. During the first stages of spicule "reallocation" many of the spicules were disoriented from their previously regular overlapping pattern. Instead of lying parallel to the abdominal epidermis, they might now7 be perpendicular to it. This would be expected if the spicules were embedded in a membrane and the entire membrane were constricting. A few spicules were some- times "left behind" in the test, where they remained isolated. However, active and rapid synthesis of new spicules was also occurring during this time, and approximately 3 to 4 days after the onset of budding all buds were close together but separated from one another by their own spicular sacs, with the spicules regularly aligned parallel to the inner edge of the tunic lining the zooid cavity.
New spicules continued to lie added as the buds grew, and the old spicules increased in diameter. Indeed, in adult colonies some spicules may be 1 mm or more in diameter; these might have been carried through a number of generations. They exhibit a complex configuration of knobs not found on small, newly formed spicules (Fig. 11).
Histochcmistry of the tunic spicular lamina
Hunt (1970) indicated that in molluscs, the formation of calcium carbonate may depend upon induction by an organic matrix with the presence of sulfated acid mucopolysaccharides being important to this process. Recent papers on tunic composition listed these and other substances found in ascidian tunic and the stains appropriate for their detection (Deck, Hay and Revel, 1966; Smith,
ASCIDIAN BUD AND SPICULE FORMATION
473
TABLE II. Histochemistry of C. lobatus tunic.
|
Tunic region |
||||
|
Stain |
Specific for |
Time (min.) |
||
|
TSL* |
Common tunic |
|||
|
0.1% toluidine blue in |
Sulfated acid mucopoly- |
10-30 |
+ + + + |
+ + |
|
30% ethanol |
saccharide |
B-metachromatic |
||
|
Aldehyde fuchsin |
Sulfated acid muco- |
5-15 |
+ + |
+ + |
|
substances |
||||
|
Alcian blue pH 2.5 |
Sulfated-acid muco- |
14-40 |
+'+ + + |
+ + |
|
polysaccharide |
||||
|
Alcian blue pH 1.0 |
Sulfated acid muco- |
15-30 |
+ + + |
+ + |
|
polysaccharide |
||||
|
Periodic acid-Schiff |
Cellulose-like compounds |
10 |
+ + |
+ + |
|
Sudan black B |
Lipid |
40 |
+ + + |
+ + |
*TSL = tunic spicular lamina.
1970; Stievenart, 1970, 1971). The techniques described by Pearse (1968) for making and using these stains were applied to fresh thin razor blade slices of adult Cystodytcs lobatus colonies ; the results are listed in Table II. Alcian blue and toluidine blue, two stains specific for sulfated acid mucopolysaccharides, stained more heavily around the spicules and inner edge of tunic lining the zooid cavities than elsewhere in the tunic, thus definitely delineating the tunic spicular lamina (TSL). Even those spicules isolated in the tunic during budding and no longer associated with any particular zooids retained this darkly staining membrane around them. (The fact that aldehyde fuchsin did not stain this region in a similar fashion supports Pearse's (1968) statement that it is not the specific stain it has been considered to be).
DISCUSSION
Cystodytcs is one of the few genera of ascidians containing mineral concretions that persist in the sediments after the animal's death (Herdman, 1884). In several cases new fossil species of Cystodytcs have been described solely on the basis of the spicules (Bonet and Benveniste- Velasquez, 1971; Monniot, 1970a; Monniot and Buge, 1971). The taxonomic significance of these spicules depends on the determination of whether or not the spicules form in a species-specific fashion. Monniot (1970b) concluded that polycitorid and didemnid spicules form as a "physico-chemical precipitation of aragonite in the tunic independent of cellular action" and could be used taxonomically only in a general way, to indicate the ascidian group. However, she did intimate that the tunic must play some part in spicule formation due to its fibrous or lamellar structure, but she did not elaborate. The present study proves the existence of an organic matrix for spicule formation (the TSL), differing from the surrounding common tunic in the concentration of sulfated acid mucopolysacharides. Also, the newly forming spicules have a lumen and are incompletely mineralized (Fig. 9c), while the spicules of adult C. lobatus are completely mineralized ; this may indicate
474 (iRETCHEX LAMBERT
another organic matrix for mineralization within each spicule (Lowenstam. personal communication). Cellular action is implicated by the fact that the spicules form in a particular region of the animal, at the junction between abdomen and thorax, and migrate out from that point to the TSL. In addition, a genetic component is implied by the great variation among colonies in the extent of spicule formation. Lafargue and Kniprath's (1978) paper proves the cellular origin of spicules in the Didemnidae. They identified the organ of spicular origin and also found that the spicules are surrounded by a discrete double- layered membrane. Thus their study and this study contradict the findings of Prenant (1925), Peres (1948) and Monniot (1970b). Peres was obviously troubled by his inability to determine the reasons for tunic stratification in the Polyclinidae, which he stated was especially noticeable around the periphery of zooids in addition to the thin outer cuticle.
Examination of the spicules in Cystodytcs lobatus was difficult because nearly any treatment (fixation, sectioning, staining, even relaxation of live zooids) usually resulted in some disruption of the bladder cells and partial dissolution of the spicules. This is why all of the staining was done supravitally, as suggested by Pearse (1968). Nevertheless, some stains (aldehyde fuchsin and alcian blue pH 1.0 especially) changed the spicules into needle-like clusters within a few minutes. Fixation in alcohol or buffered formalin also disrupted bladder cell membranes, and after being left for a few weeks in fixative the surface irregularities disappeared from many spicules in adult colonies. Indeed, some Cystodytcs colonies after several years on a museum shelf have few if any recognizable spicules left, a fact which undoubtedly has led to erroneous calculations of the abundance and type of spicules when the colonies were alive (Ritter, 1900; Van Name, 1945; Millar, 1962). Because of these difficulties, taxonomic descriptions of Cystpdytes and didemnid species should include photographs or at least descriptions of spicules from live colonies if possible.
The question remains of howr the tunic spicular lamina can "bud" when the zooid buds, a problem especially puzzling since the lamina appears to be com- pletely extracellular. At a magnification of 675 X, it is possible that a mem- brane was indistinctly seen in the l-//.m sections between the outer edge of the tunic spicular lamina and the beginning of the bladder cell region. If this is so, perhaps this membrane somehow separates from the rest of the tunic during budding. It will be necessary to embed and section zooids in the process of budding in order to examine this further.
My most sincere thanks go to Dr. Donald P. Abbott of the Hopkins Marine Station for suggesting this topic, sharing his laboratory with me, and offering me continued help and encouragement. I also thank P. Adams, F. Chapman, J. Cooper, Y. Fadallah, C. Harrold, A. Hines, C. Patten, V. Scofield, and R. Sellers for their assistance. Discussions with Drs. Todd Newberry and Richard Cloney greatly aided this work, and I especially want to thank Dr. Newberry for his excellent translation of a number of pages of French. Dave Behrens identified the Lamellar ia; Drs. Donald Abbott, Roger Seapy and Heinz Lowenstam con- siderably improved the manuscript by their critical comments and valuable sug-
ASCIDIAN BUD AND SPICULE FORMATION 475
gestions. My husband Charles spent much time in helping me with all aspects of the photography, microscopy, pH determinations and reading of the manu- script; I greatly appreciate his constant support throughout this study.
SUMMARY
1. The colonial ascidian Cystodytcs lobatus has a long breeding season (at least 6 months) and releases tadpoles sporadically throughout the day, indicating a long period of recruitment.
2. Tadpoles of C. lobatus were settled and reared in the laboratory in order to observe early growth, budding and spicule formation.
3. Budding is preceded by the formation of a new stomach at the posterior end of the esophagus and fits Nakauchi's Type I budding pattern.
4. Spicule formation begins within 5 days after settlement. The spicules appear to form in a particular region at the anterior end of the abdomen and migrate over the abdomen to form a single or slightly overlapping layer embedded in a "tunic spicular lamina." This lamina lies between the common tunic and the zooid cavity and forms a spicular sac in the tunic surrounding but separated from the abdomen of each zooid. It stains especially heavily for sulfated acid mucopoly- saccharide ; the spicules are concluded to form by cellular action in this organic matrix.
5. There is great variation among zooids in the quantity of spicules formed. These differences are maintained in the buds, resulting in colonies in which all zooids either have few or many spicules, and are therefore probably genetic in origin.
6. During budding the spicular sac becomes disrupted and appears to bud, resulting in a reallocation of the spicules to the buds and formation of separate spicular sacs around the abdomen of each bud. At budding there is apparently no disruption of bladder cell membranes in the tunic and no dissolution of spicules by the acids contained in the bladder cells.
LITERATURE CITED
ABBOTT, D. P., AND A. T. NEWBERRY, 1980. Urochordata : The Tunicates. In Morris, R.,
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MINT, S., 19/0. P olysaccharide-Protein Complexes in Invertebrates. Academic Press, New
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mutabilis (Ascidiae Compositae). Sci. Rep. Tokyo Bunrika Daigaku, Sec. B, 7: 23-53. PEARSE, A. G. E., 1968. Histochcmistry Theoretical and Applied. 3rd ed., vol. 1, Little,
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SKOGSBERG, T., AND G. H. VANSELL, 1928. Structure and behavior of the amphipod, Poly-
chcria osborni. Proc. Calif. Acad. Sci., 4th series, 17(10) : 267-295. SMITH, M. T., 1970. The blood cells and tunic of the ascidian Halocynthia aurantiuin (Pallas).
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Reference: Biol. Bull. 157: 478-493. (December, 1979)
CHARACTERISTICS AND REGULATION OE FISSION ACTIVITY IN
CLONAL CULTURES OF THE COSMOPOLITAN SEA ANEMONE,
MALI PLAN ELLA LUC I All (VERRILL) 1
LEO L. MINASIAN, JR. AND RICHARD N. MARISCAL
Department of Biological Science, Florida State University. Tallahassee, Florida 32306
The diversity of reproductive modes within the Cnidaria is greater than that of most other metazoan groups. This is particularly evident among the sea anemones, for which Chia (1976) has compiled a list of reproductive modes, and has speculated on their evolution and adaptive significance. Reproduction in Hali- planclla (-- Diaduiucnc ) Inciac Verrill is of special interest because H. liiciae has greatly expanded its geographical range since the turn of the century (Uchida, 1932; Stephenson, 1935; Hand., 1955; Shick and Lamb, 1977). This species now occurs intertidally on boreal (Uchida, 1936; Williams, 1973), temperate (Stephenson, 1935; Hand, 1955) and tropical (Dunn, personal communication; Belem and Monteiro, 1977) coasts.
The ability of H. luciac to establish new populations is extraordinary since, unlike other intertidal invertebrates, H. litciae has never been observed to produce larvae as agents of dispersal (Davis, 1919 ; Shick, 1976). All observed reproduction has been asexual, through longitudinal fission (Hargitt, 1912; Davis, 1919), and less commonly by pedal laceration (Atoda, 1954; Johnson and Shick, 1977). Although a single fission event infrequently produces multiple individuals, most fission events are binary, and analogous to cytokinesis (Atoda, 1976; Minasian, 1976).
Longitudinal fission permits the rapid establishment of intertidal clones (Chia, 1976; Francis, 1976). The strategic advantages of fission have been recently discussed by Francis (1976) and Shick and Lamb (1977). Hoffmann (1976) and Shick and Lamb (1977) described the genetic composition of different con- specific clones of anemones, and have made valuable inferences concerning advan- tages of asexual reproduction in contrast to sexual reproduction.
Understanding the contribution of asexual reproduction to the success of H. luciac makes necessary the analysis of asexual reproductive rates and their regulation. However, few studies have evaluated asexual reproductive rates of sea anemones, partly due to a lack of standardized methods.
Researchers of hydroid development have calculated exponential rates of increase (symbolized "A'") in numbers of polyps in laboratory cultures (Loomis, 1954), and have related these rates to environmental variables (Fulton, 1962; Davis, 1971). Minasian (1976) determined k (fission rate) in laboratory populations of H. liiciae, and quantified the effect of feeding frequency on k. The occurrence of fission in H. Inciac is affected by temperature (Miyawaki, 1952) and fluctuating
1 Contribution No. 51 of the Tallahassee, Sopclioppy & GuU' Coast Marine Biological Association.
478
REGULATION OF FISSION IN H. LUCIAE 479
temperature-emersion treatments (Johnson and Shick, 1977) ; but the effect of temperature on k was not quantified in those studies.
The present study quantifies the combined effects of temperature and feeding frequency on A' in H . luciac, and provides a comprehensive analysis of longitudinal- fission activity in a sea anemone. This includes previously undescribed char- acteristics of fission activity, essential to the understanding of the regulation of longitudinal fission. In addition, we describe a method for establishing and main- taining permanent in ritro clonal cultures of H. luciac. This has enabled us to obtain a range of possible values of k in a clone of H. luciac from northwestern Florida.
MATERIALS AND METHODS
Establishment of clonal cultures
Specimens of H. Inciae were collected on 2 October from a sand-flat intertidal region adjacent to the Florida State University Marine Lab near Turkey Pt, on the N. W. Florida Gulf coast. These anemones occur on the undersides of sedimentary stones, accompanied by barnacles (Chthamalus fragilis} in the upper littoral region, and oysters (Crassostrca virginica) in the mid-littoral region. //. luciac cloned from this location (29° 54.8' N, 84° 30' \Y ) have a brownish-green column with 12 to 24 yellowish-orange longitudinal stripes in large individuals. Shick and Lamb (1977) found a sample of striped H. luciac from this population to consist of two different genotypes (i.e. clones). The specimens of H. luciae cloned in this study are presumed to belong to the same genotype, since they are phenotypically (sex and color) identical and phenotypically distinguishable from the second striped clone present at the collection site.
To establish clonal cultures, three individual anemones of average diameter (about 5 mm) were isolated in three covered glass storage dishes (Corning #3250), each containing approximately 200 ml natural sea wrater (28 to 30/£<r). These cultures were maintained at room temperature (22 to 25° C) under a 14/10 hr light-dark cycle provided by a fluorescent (cool-white) light source, and fed to repletion (30 to 60 min ) with newly hatched Artcinia nauplii every second day. All culture dishes were rinsed briefly with distilled water and refilled with fresh sea water daily. The number of anemones in each culture increased rapidly through longitudinal fission.
After 3 months each clonal culture was divided into two cultures, producing a total of six clonal cultures. These served as stock cultures for the experiments, and w^ere kept under the same photoperiod at a temperature of 16 to 18° C ; feeding- frequency was reduced to twice per week. Every 4 to 6 weeks each clonal culture was transferred to clean glassware. Clonal cultures were maintained under this standardized regimen for at least 4 months before being used in experiments. Anemones from stock cultures were less than 7 mm in diameter ; histological examination showed that none of these anemones bore gonads.
Culture experiments
For each experiment, six duplicate cultures were set up by placing 10 anemones from each stock culture in glass bowls (glass stacking dish, Wheaton #350134)
480 L. L. MINASIAN, JR. AND R. N. MARISCAL
which were 11.5 cm in diameter, and contained approximately 150 nil of sea water. Sets of six cultures were then placed under one of nine different experimental regimens. These regimens, evaluating combined temperature and feeding effects upon fission activity, were as follows: fed once every 2 days at 26, 21 or 16° C; fed once every 4 days at 26, 21 or 16° C; starved at 26, 21 or 16° C. Thus the entire experiment involved a total of 54 clonal cultures, each initially containing 10 anemones. In addition, fission activity in stock cultures was evaluated by setting up six experimental cultures as above, and observing fission activity for 3 months under maintenance conditions (fed twice per week at 16 to 18° C). Experi- mental cultures were exposed to the new temperature for 2 days before beginning the experiment, providing time for attachment of anemones to culture bowls. The second day after transfer to culture bowls (day 0) marked the start of the experi- ment ; for experimental groups receiving food, day 0 was the day of the first feeding. These cultures were also under a 14/10 hr light-dark cycle. Anemones were counted daily, at which time they were fed, if necessary, and the sea water changed.
Sta fist ical analysis
Fission rates were calculated from the least-squares regression equation of the natural log number of anemones as a function of time in culture : InA^ = kti + \nN0. Here the rate of increase in log number of anemones (InAfj) on a given day (£1) is defined by the fisson rate (k), which is the slope of the regression line. The number of polyps present at the start (day 0) of the culture interval, and Y- intercept of the regression line, is \nN(l. This equation was employed by Loomis (1954) and Fulton (1962) to calculate the exponential rate of increase (k) for asexual production of hydroid polyps, in which Ni = N0e.k<i.
Since exogenous factors can impose a variable delay period prior to the initiation of fission, it was necessary to recognize two distinct parameters for fission rate, defined as follows. The overall fission rate (k} is the rate of increase during the entire culture interval, including the initial delay before the onset of fission. The second parameter, /c.,,,j, gives the rate of increase subsequent to the initial delay period. Adjusted fission rate (A'a,ij) is calculated by the same method as k, except that the first data entry, day 0, is the day prior to the first occurrence of fission. Hence, &a(]j designates the rate of increase only after the initiation of fission, rather than during the entire culture period, and better estimates the rate of active, sustained fission. Where the delay before onset of fission is absent or very short, k — ka<\j. Both k and k.^ were calculated for each culture, except where fission activity was not sustained and considered to be nonexponential. The delay before the onset of fission also was calculated for each culture.
The effects of temperature and feeding on k and &adj were evaluated by means of a two-way analysis of variance (ANOVA). Paired comparisons between sta- tistical means were performed using /-tests.
The percentage of anemones undergoing fission per day was calculated as follows OVi — AVi) -100/Arj, where N{ is the number of anemones on a given day, and Afj-i is the number present on the previous day. An angular transformation was performed on these percentage data, and means and standard deviations
REGULATION OF FISSION IN H. LUCIAE
481
10
8
FIGURE 1. Semilogarithmic plot of numerical increase through longitudinal fission in cultures of H. luciac, reared under conditions of routine maintenance (17±1° C, fed twice per week). Day 0 is 2 days after transfer to culture bowls from stock cultures. Each point is a mean of six cultures, each intially containing 10 anemones; error bars are standard deviations. The slope of the regression equation, InNi = kti + \nN0, is the fission rate, k (see Materials and Methods for further explanation). The slope of the solid line (£) is 0.0149 ±0.0017 (mean ± s.d.) ; the slope of the broken line, A-adJ (0.0162 ± 0.0010), is corrected for the initial delay period prior to the start of fission activity.
calculated for each regimen. On plots of the temporal pattern of fission pulses, the scale of the ordinate conforms to the transformation, whereas the units on the ordinate are actual, untransformed percentages. This transformation is necessary to assume normality (Sokal and Rohlf, 1969). Means and standard devia- tions were calculated for relative maxima of all fission pulses (pulse maxima), and for relative minimal values between fission pulses (pulse minima) ; these were evaluated using a multiple-range (Student-Neuman-Keuls) test. Lengths of intervals between fission-pulse maxima were also determined. A G-test for independence (Sokal and Rohlf, 1969) was performed on frequency data for pulse intervals. Test statistics corresponding to probabilities of less than 0.05 were regarded as significant.
RESULTS
Characteristics and exogenous regulation of fission activity
Figure 1 illustrates prominent characteristics of fission activity in clonal cultures reared under the standard maintenance regimen. These characteristics
482
L L \II.\ASI.\.\, JR. AND K. X. MAR1SCAL
-a- 26°C
-b- 21°C
-c- 16°C
20
24
28
Days
FIGURE 2. Semilogarithmic plot of numerical increase through longitudinal fission in cultures of H. luciac fed once every 2 days. Each point represents a mean of six cultures, each initially containing 10 anemones ; error bars are standard deviations. Three experimental culture temperatures were used: (a), 26° C; (b). 21° C ; (c), 16° C.
included : first, an initial delay period before the onset of fission ; second, an exponential fission rate followed by a temporary cessation of fission ; and third, resumption of the exponential fission rate. The variable delay lasted for 21.33 ± 4.46 (mean ± s.d.) days, although one culture initiated fission as early as day 18 (Fig. 1). This delay was initiated by the mechanical disturbance imparted in transferring anemones to new culture bowls. For the entire culture interval of 92 days, k -- 0.0149 ± 0.0017 (mean ± s.d.) ; with the delay period omitted from calculation, A'a,ij = 0.0162 ± 0.0010. The calculation for fcadj usually increased the estimate of fission rate in addition to shifting the regression line to the right.
Figures 2 and 3 demonstrate the numerical increase in cultures of H. htciac
REGULATION OF FISSION IN H. LUCIAE
483
which were fed Artemia. At 26° C little or no delay occurred prior to the onset of fission ; hence k and k.lAj were essentially identical. Only when anemones were fed once every two days at 16° C was AVIJ significantly larger than k (Mest, P<0.05).
Values of fcadj in experimental cultures ranged from 0.0278 (doubling time = 24.9 days) at 16° C to 0.0728 (doubling- time == 9.5 days) at 26° C, in cultures fed every second day. Analyses of temperature and feeding effects showed both factors to significantly affect k and £ailj, based upon comparisons between 21° and 26° C regimens (Table I). Temperature coefficients (Qio) for A'adj over the 21 to 26° C temperature range were 1.6042 and 1.6759 for feeding frequencies of 2 and 4 days, respectively (Table II). Thus, the difference in feeding frequency did not influence the effect of temperature upon &a(lj in this range. Since the Qm for the 16 to 21° C range was much larger (Table II), the greatest effect of temperature on £!Klj occurred below 21° C.
-a- 26°C
-b- 21 °C
-c- 16°C
Days
FIGURE 3. Semilogarithmic plot of numerical increase through longitudinal fission in cultures of H. liiciac fed once every 4 days. Each point represents a mean of six cultures, each initially containing 10 anemones; error bars are standard deviations. Three experi- mental culture temperatures were used: a), 26° C; (h) 21° C; (c), 16° C.
484
L. L. MINASIAN, JR. AND R. N. MARISCAL
TABLE I
Means ± standard deviations for values of k and kadj for H. luciae reared at different temperatures and feeding frequencies. Each experimental mean consisted of six bowls, each initially containing 10 ane- mones. The resulting two-way ANOVA was performed on four means (21° vs. 26° C, fed every second or fourth day). The ANOVA results were the same for both k and kadj, and indicate if an effect upon k was statistically significant ( + ) or not significant ( — ) at the 5% level; n.c. = fission activity not sufficient to calculate k.
Feeding frequency
Temperature
16 ± 1° C
21 ± 1° C
26 ± 1° C
Values of k
|
2 days 4 days |
0.0207 ± 0.0036 n.c. |
0.0569 ± 0.0040 0.0518 ± 0.0031 |
0.0728 ± 0.0035 0.0695 ± 0.0032 |
Values of
|
2 days 4 days |
0.0278 ± 0.0047 n.c. |
0.0574 ± 0.0035 0.0533 ± 0.0045 |
0.0727 ± 0.0036 0.0690 ± 0.0025 |
ANOVA: Effects for 21° vs. 26° C
|
Temperature |
Feeding |
Interaction |
|
( + ) |
( + ) |
(-) |
Both temperature and feeding significantly affect length of the delay period prior to the initiation of fission (Table III). The duration of delay periods varied from 1 day or less at 26° C, to over 21 days at 16° C. Table III shows that decreasing the feeding frequency from 2 to 4 days further lengthened this delay. Tempera- ture and feeding frequency acted synergistically upon the pre-fission delay, as indicated by the significant ANOVA interaction term (Table III). For example, at 26° C halving the feeding frequency (from every second to every fourth day) increased the mean delay period by 1.3 days ; at 16° C, halving the feeding frequency increased the delay by over 10 days (Table III).
Fission activity in starved cultures at 26° C and 21° C was limited to two major pulses of fission activity (Fig. 4) ; starved anemones at 16° C did not
TABLE II
Temperature coefficients (Qio) for fission rate (k0rf>) in H. luciae at two different feeding frequencies; n.c. = insufficient data for calculation.
Temperature Range
|
r eeuiug irequtfiiuy |
16°-21° C |
21°-26° C |
16°-26° C |
|
2 days 4 days |
4.3567 n.c. |
1.6042 1.6759 |
2.6436 n.c. |
REGULATION OF FISSION IN //. LUC f 'AK 485
TABLE III
Means ± standard deviations for delay to onset of fission (days) in H. luciaea/ different temperatures and feeding frequencies. Each experimental mean consisted of six bowls, each initially containing 10 individual anemones. The two-way A NOVA result indicates if an effect on fission delay is statistically significant ( + ) or not significant ( — ) at the 5% level.
|
Feeding frequency |
Temperature |
||
|
16 ± 1° C |
21 ± 1° C |
26 ± 1° C |
|
|
2 days 4 days |
11.6667 ± 2.3381 >21.0 |
1.8333 ± 0.9832 5.5000 ± 1.3784 |
0.0000 ± 1.0954 1.3333 ± 1.5055 |
ANOVA: Effects for 21° vs. 26° C
|
Temperature |
Feeding |
Interaction |
|
(+) |
( + ) |
( + ) |
undergo fission. Nonetheless, these cultures remained healthy throughout the experiment.
Temporal pattern of fission activity
After long delay periods a major pulse of synchronous fission occurred, fol- lowed by a temporary cessation of fission (Figs. 1, 2c). The resumption of fission after this initial pulse was either less synchronous and without additional major plateaus (Fig. 1), or continued to show additional, brief cessations of fission (Fig.2c).
H. luciae exhibited distinct pulses of increased fission activity, with relative maxima (peaks) and minima (troughs), shown in Figures 5 and 6. These occurred
28
Days
-a- 26°C
-b- 21 °C
-c-
16°C
FIGURE 4. Semilogarithmic plot of numerical increase through longitudinal fission in starved cultures of H. luciae. Each point represents a mean of six cultures, each initially containing 10 anemones ; error bars are standard deviations. Three experimental culture temperatures were used: (a), 26° C; (b), 21° C; (c), 16° C.
486
L. L. MINASIAN, JR. AND R. N. MARISCAL
Days
FIGURE 5. Percentages of H. Indue undergoing fission per culture per day in cultures fed once every 2 days. Each percentage is a mean of six cultures ; arrowheads indicate feeding days. Data \vas subjected to angular transformation prior to statistical computations; plotted means are transformed data. The ordinate indicates actual, untransformed percentages. Three culture temperatures are represented: (a) 26° C; (b) 21° C; (c) 16° C.
REGULATION OF FISSION IN //. LUCIAE
487
TABLE IV"
Different lengths of time periods between peak values of fission activity (pulse maxima), observed in II. luciae culture populations. Cultures initially consisted of 10 anemones each and were reared under different temperature and feeding regimens for 1 month. Six clonal cultures were reared under each different culture regimen. The percentage of fissions occurring each day were then averaged, and the peaks of fission activity determined. Occurrence of the various lengths of pulse intervals depend upon culture conditions (G-test for independence, P < 0.05).
|
Length of period between pulse maxima (days) |
||||
|
2 |
3 |
4 |
>4 |
|
|
26° C, fed every 2 days |
7 |
0 |
3 |
1) |
|
26° C, fed every 4 days |
0 |
5 |
1 |
1 |
|
2 1 ° C, fed every 2 days |
5 |
0 |
4 |
0 |
|
2 1 ° C, fed every 4 days |
1 |
3 |
2 |
0 |
|
16° C, fed every 2 days |
4 |
0 |
1 |
1 |
in cycles, with intervals between pulse maxima usually lasting for 2 to 4 days. These pulses of fission had a phasic dependence upon the feeding regimen. In cultures which were fed every second day, pulse maxima occurred only on feeding days, although not on all feeding days (Fig. 5). Therefore, periods between pulse maxima were multiples of 2 days, in cultures fed every second day. At 26° and 21° C most pulse maxima had a 2-day periodicity, with fewer pulse maxima being 4 days apart (Fig. 5a, b). In the 16° C cultures, a 6-day period between pulse maxima occurred at one point, although 2-day periods were still most frequent (Fig.Sc).
In cultures fed at 4-day intervals pulse maxima often occurred on days other than feeding days, and were usually 3 or 4 days apart (Fig. 6). Thus, the periodicity of pulse maxima was longer than that in cultures receiving food at
TABLE V
Means and standard deviations for fission-pulse maxima and minima observed in cultures ofH. luciae reared under different culture regimens. Each determination was taken from angular-transformed, averaged data from six clonal cultures. Means are for pulse maxima or minima occurring over a one- month period. Sample sizes are in parentheses. Asterisks denote means which are significantly different from all other means in the same column (Student- Neuman-Keuls test, P < 0.05).
Culture regimen
Pulse maximumt
Pulse minimumf
26° C, fed every 2 days
£adj = 0.0727 26° C, fed every 4 days
&adj = 0.0690 21° C, fed every 2 days
£adj =0.0574 21° C, fed every 4 days
&adj = 0.0533 16° C, fed every 2 days
/fead = 0.0278
18.359 ± 4.601 (11) 16.967 ± 5.039 (8) 13.393 ± 5.014 (10) 15.602 ± 7.650 (.8) 7.920 db 5.390 (7)*
8.823 ± 1.893 (11)* 6.486 ± 3.790 (8) 6.828 ± 3.467 (10) 3.658 ± 3.226 (8)* 0.417 ± 1.021 (6)*
f angular transformation (arcsin-V ' r fissions/day) is necessary to assume normality.
488
L. L. MINASIAN, JR. AND R. N. MARISCAL
18 14-
10-
6.0- 4.0-
2.0-
1.O- O5-
-g 0.1
C
o
(0 (0
22-
18-
(a)
(b)
Days
FIGURE 6. Percentages of H. luciac undergoing fission per culture per day in cultures fed once every 4 days. Each percentage is a mean of six cultures, each containing 10 or more anemones; arrowheads indicate feeding days. Data was subjected to angular transforma- tion prior to statistical computations ; plotted means are transformed data. The ordinate indicates actual, untransformed percentages. Two culture temperatures are represented : (a) 26° C; (b) 21° C.
REGULATION OF FISSION IN H. LVCIAE 489
2-day intervals. Only one period of 2 days was observed at the 4-day feeding frequency (Fig. 6b). Table IV summarizes the occurrence of pulse periodicity, which is dependent upon culture conditions (G-test, P < 0.05).
Pulse maxima often attained 10 to 20% of the culture population undergoing fission per day (untransformed percentages are given on the ordinate in Figs. 5 and 6). Pulse minima usually dropped below 4% fissions per day in cultures exhibiting lower values of k (Table V). Mean values of pulse maxima were generally indicative of values of £adj. For example, a mean pulse maximum of 13.393 (arcsin-V%fissions/day) corresponded to a mean k.,it]j of 0.0574, while a significantly lower (Student-Neuman-Keuls, P < 0.05) mean pulse maximum of 7.920 (arcsin-V% fissions/day) accompanied a A'a(,j of 0.0278 (Table V). Like- wise, significantly lower values of A'!1(ij accompanied lower values for pulse minima (Table V).
DISCUSSION
The distinction between k and fca(lj is important for the analysis of fission activity in H. hiciae. Fluctuations in population size and density are best considered in terms of k rather than fcadj, since periods of fission delay, as opposed to active periods of fission, are not easily distinguished in field studies. Moreover, fission-related morphological variability (Minasian, 1979) is best understood in relation to the total number of fissions over an entire interval (k}, rather than the sustained or maximal rate of fission during part of that interval (7ca,ij). The parameter &afii is a more accurate indication of active fission than is k. Hence, A'adj should be used to examine experimental effects upon fission rate. The calculation of k^ requires that data be collected at frequent, preferably daily intervals.
The present study reveals three different effects of temperature upon fission activity. The first effect is upon fission rate. The Qm value for £a(11 in the 16 to 21° C range was three times greater than the Qio value for the 21 to 26° C range. Sassaman and Mangum (1970) similarly found that oxygen consumption in H. Iiiciac showed disproportionately large Qio values over a range of 10 to 17.5° C. The second effect of temperature, a lengthening of the delay period, was also greatest below 21° C. Temperature also affected fission-pulse patterns: in cultures fed every second day, a 10° C decrease in culture temperature caused a 40% drop in the frequency of fission pulses, and a signficant decrease in values of pulse maxima and minima.
The great response of fission activity to small changes in temperature indicates that temperature is the foremost exogenous regulator of k. Miyawaki (1952) believed that a temperature threshold of approximately 15° C, below which no fission occurred, existed for H. luciae. He observed no longitudinal fission at temperatures below 20° C. The present study shows that values of k for this Florida clone decrease sharply as temperatures descend to 15° C, and in this respect agrees with Miyawaki's (1952) observations on Japanese H. luciae. However, a temperature threshold which limits fission in H. luciae is dependent upon other parameters which also affect k, such as food availability. Thus, k is best regarded as having a graded response to temperature, rather than an absolute threshold of 15° C.
4()() I. L. MINASIAN, JR. AND R. N. MARISCAL
Tlie effect of feeding frequency upon k appears to be temperature-dependent. Although halving the feeding frequency resulted in reductions of k by only 4.5% at 26° C, and 8.9% at 21° C, a relatively large effect of decreased feeding frequency upon k occurred at 16° C, where the delay period was greatly lengthened. A temperature-dependent effect of feeding on the morphology of H. luciac reflects such interaction of effects on k (Minasian, 1979).
Exogenous factors determine sensitivity to mechanical stimuli which interrupt fission activity. Transferring anemones to new culture vessels (when setting up experimental cultures) imparted a mechanical disturbance, which caused little or no fission delay at high temperature and feeding frequency ; but at low temperatures and feeding frequencies this delay may last for several weeks. Johnson and Shick (1977) demonstrated that fluctuating immersion-emersion cycles decrease fission activity in H. luciae. The mechanism underlying this effect may involve mecha- nically induced delays.
The synchrony of fission cessation following initial fission activity in low-tem- perature cultures (Fig. 1) is reminiscent of a synchronized cell culture (e.g., Zeuthen and Scherbaum, 1954; Zeuthen, 1964), and implies the existence of relatively constant periods between fissions among individuals within the culture population. This long-term fission synchrony is most evident at low temperatures because periods between fission events are lengthened. When exogenous factors permit only short periods between fissions, such long-term synchronization is not observed. Studies on the lengths of inter-fission periods must be made on individual anemones to ascertain if inter-fission periods are relatively constant in duration, and how they change due to exogenous influences. The inter-fission period may simply be a delay in response to the previous fission event, which itself constitutes a mechanical disturbance.
The coincidence of fission pulses with feeding days, in cultures fed every second day, points to a regulatory role for other exogenous, and possibly endogenous factors. Photoperiodic and feeding-digestion cycles appear to be involved. For example, cultures of H. luciae are more active during dark portions of the photo- periodic cycle, and usually exhibit greater fission activity in the dark (Minasian, unpublished data). Similarly, Batham and Pantin (1950) observed pedal loco- motory activity of the sea anemone, Metridium senile, to occur only at night. Although feeding causes long-term enhancement of fission, Torrey and Mery (1904) suggested that fission in PI. luciac is inhibited by feeding. Thus, interaction of photoperiodic rhythms and short-term inhibition by feeding produces a 2-day pulse pattern in cultures fed at 2-day intervals: fission is inhibited during the first dark period subsequent to feeding, followed by a release from inhibition and increased fission activity during the second dark period after feeding. This inhibition of fission during hours subsequent to feeding explains the even-day duration of periods between fission-pulse maxima in cultures fed at 2-day intervals.
In cultures fed at 4-day intervals, periods between fission-pulse maxima are longer, with pulses of fission activity often beginning during the 24 hr subsequent to feeding. Minasian (1976) similarly observed that H. luciae undergo more synchronous fission activity in response to renewed feeding when starved for longer periods. Thus, cultures fed at different frequencies have inherent differences in the response of fission activity to feeding. If there exists an endogenous influence
REGULATION OF FISSION IN //. LUCIAE 491
on the fission-pulse pattern, it may permit synchronization of fission pulses with 2-day feeding intervals, hut not with 4-day intervals.
The extreme sensitivity of fission activity to temperature indicates that the clone of H. hiciac examined in these culture experiments must achieve recruitment primarily when temperatures exceed 20° C. Below 20° C, low-temperature inhi- hition is reinforced by tidal cycles, which can limit food availability and impart mechanical disturbance through immersion-emersion effects. Hence, below 20° C k will be small, and delays between major pulses of fission activity will be long. Previous studies (Shick and Lamb, 1977; Minasian 1979) have stated that the absence of gametogenesis (i.e., absence of sexual reproduction) in H. luciae is associated with small size (and hence high k}. Thus, the 15° to 20° C temperature range probably marks a crucial transition from small, sterile and strictly asexual anemones exhibiting high k, to larger, sexually reproductive anemones which exhibit only infrequent fission. Under present culture conditions, periodic, pulsed increments of fission activity are best interpreted in relation to two periodic, exogenous stimuli : photoperiod and feeding. In natural populations of H. luciae, periodic stimuli include photoperiod and tidal fluctuation, of which the latter involves both feeding and a mechanical (immersion-emersion) effect (Johnson and Shick, 1977). Therefore, it is possible that such pulses of fission activity have a phasic dependence upon tidal and photoperiodic cycies in intertidal populations of H. luciae. An investigation of fission activity under field conditions may elucidate this relationship.
The authors thank Dr. D. A. Meeter for consultation regarding the statistical analyses, and Dr. W. H. Heard and R. W. Seaton for comments on the manuscript.
SUMMARY
1. Permanent cultures of a clone of H. luciae from N. W. Florida were reared under different temperature and feeding regimes in order to identifiy and quantify parameters of asexual reproduction.
2. The principle components of fission activity include fission rate, a delay period following a mechanical disturbance, and periodic pulses of increased fission activity ; all components are regulated by temperature and feeding frequency.
3. A distinction is made between fission rate including the delay period (k), and fission rate following the delay period (&arij)-
4. Fission rates (&a<jj) ranged from 0.0162 (doubling time =: 42.8 days) at 17° C to 0.0727 (doubling time == 9.5 days) at 26° C.
5. Temperature is the foremost regulator of k ; the greatest influence of feeding frequency was upon periodic pulses of fission activity.
6. Culture data indicate that recruitment in natural populations of this clone is restricted by seasonal temperature; below 20° C there is a sharp reduction in k. It is suggested that inhibition of k by temperatures below 20° C favors a transition from asexual to sexual reproduction.
7. The pulsatile, periodic character of fission activity is prominent in laboratory cultures, and suggests that such activity in natural habitats may have a phasic dependence upon tidal and photoperiodic cycles.
492 L. L. MINASIAN, JR. AND R. X. MARISCAL
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New York.
K.-ierence: BioL Bull. 157: 494-505. (December, 1979)
LOCOMOTOR AND LIGHT RESPONSES OF LARVAE OF THE HORSESHOE CRAB, LIMULUS POLYPHEMUS (L.)
ANNE RUDLOE
Department of Biological Sciences, Florida State University, Tallahassee, Florida 32301
The American horseshoe crab, Llinnhis polyphemus is an abundant and con- spicuous member of the estuarine fauna of the Atlantic coast of the United States and the Gulf of Mexico. Its importance as a laboratory animal for neurophysio- logical, biochemical and medical research is well established (Wolbarsht and Yeandle, 1967; Cohen, 1979). Despite these facts, remarkably little has been recorded on the ecology and natural history of the species. The limited informa- tion available (Sinister, 1958; Sokoloff, 1978; Rudloe, 1979; Rudloe, 1978; Rudloe and Herrnkind, 1976) has primarily been concerned with the adult phase of the life cycle, especially the emergence of breeding crabs onto sandy beaches.
The juvenile and larval phases of the life cycle are of equal interest, however. Juveniles are important predators of the sandy intertidal community in areas adjacent to adult breeding beaches (Green and Hobson, 1970; Rudloe, 1978), while the resemblance of the larval instar to certain trilobites has long been a source of comment and is sometimes cited as an indication of the phylogenetic antiquity of the species.
Larvae hatch approximately 5 weeks after the female lays her eggs near the high tide mark. The time may vary depending on ambient temperature, and other environmental factors (Jegla and Costlow, 1979). The embryological develop- ment of Limulus eggs is well described by Kingsley (1892). However, field studies of this phase of the life cycle are limited to observations by Shuster (1958) on nematode, oligochaete and maggot activity in the nests, and Hummon, Fleeger and Hummon (1976) who described the interaction of beach meiofauna with developing eggs.
Newly laid eggs are sticky and occur as tightly clumped balls. \Yhen the eggs hatch, the larvae remain in distinct aggregations at depths comparable to those of newly laid eggs. The larvae eventually reach the surface of the sand and emerge into the water column. When larvae emerge from the nest or when they are removed and exposed to water, they exhibit a "swimming frenzy" reminiscent of neonate sea turtles, swimming vigorously and continuously for hours. They are also, like the larvae of many other marine invertebrates, strongly positively phototactic, orienting immediately to any available light source (Thorson, 1964; Rudloe, 1978). Loeb (1893) first mentioned the response of Lhnnlns larvae to light, while the development of the swimming behavior has been described by Pearl (1904) as a continuum from embryo to larvae. More recently, French (1977) and French and Doliner (1978) have also described aspects of larval light responses in the laboratory.
While juvenile and adult horseshoe crabs are very similar in morphology, the larval (post hatching) instar is morphologically specialized and unlike all sub-
494
EMERGENCE OF LARVAL I.IMULUS 495
sequent stages. This larval instar is confronted with the necessity of moving successfully from a nest buried several centimeters beneath the sand surface at a high level of the beach out into the marine environment. Once this transition is achieved, the animal will not re-enter the upper beach environment until it is a sexually active adult. At that time it will be vastly changed, both behaviorally and morphologically, from the larval stage. The timing and mechanisms of release of larval specimens of Limiilns, as well as the characteristic behavioral patterns of the larvae that contribute to achieving this movement away from the nest and which differentiate it from other phases of the life cycle were the objectives of this study.
MATERIALS AND METHODS
Field studies
Activity of larvae was monitored at Mashes Sands Beach, Wakulla County, Florida from May through November, 1977, June and July, 1978, and in June, 1979. Nests in this area occurred in a distinct zone at the level of extreme high tide and could be easily located by digging at that height on the beach. A series of nests was dug open and the depth from the center of the nest to the surface was measured at periodic intervals ranging from 3 to 7 days between May and November, 1977, for a total of 21 samples. The number of nests measu-red on each date varied from 10 to 25. Digging was always done within three hours of the daylight high tide.
In addition, the depths of six nests chosen haphazardly each hour were checked over an 11-hr period on the night of full moon in June, 1978. Different nests were measured at each hour, for a total of 66 nests. The nests were dug open by hand and the depth from the center of the larval aggregation to the sur- face was measured. Nests remained tightly packed after hatching so that accurately locating the center was not difficult. Sampling was also done at the hour of high tide at seven-day intervals thereafter to ascertain nest depths at high tides on the following new moon spring tide and at the following two neap tides. Hourly sampling of six nests was conducted on the night of new moon in June, 1979, using the method described above. Nests were checked from low tide until the hour of high tide, for a total of 48 nests.
Surface plankton tows, each of 4-min duration, were made parallel to the beach at a depth of 1 m for day and night high tides on 128 tides between May and November, 1977, using a coarse mesh net constructed of wire window screening. This mesh retained the macroscopic Linntlus larvae while passing smaller zoo- and phyto-plankters. The captured larvae were counted and released.
Laboratory analysis of locomotor activity rhythms
Larvae were examined in the laboratory for locomotor activity rhythms using time-lapse photography. An automatically triggered motordrive 35-mm camera using black and white negative film was used, as was an 8-mm movie camera set for timed exposures. The still camera recorded a frame once every 20 min, whereas the 8-mm camera recorded every 2 min. In all cases the data were grouped into 1-hr intervals.
496 ANNE RUDLOE
Activity was recorded under various light regimes. Ambient light (with care taken to insure that no artificial light source was present) with an approximate light regime of fifteen hours of light and nine hours of darkness (LD) and water temperatures ranging between 28° C and 34° C was used to approximate conditions of light and temperature encountered in the upper intertidal zone. Constant light and temperature (LL) and constant darkness and temperature (DD) were also used.
Five hundred larvae were used in each of three DD trials, and 1000 larvae were used in all other trials. These densities did not result in any significant mortality, but, assessed visually, did approximate concentrations observed in the field. Larvae were collected from the field prior to each trial on the nights of full moon, new moon, and at 7-day intervals before and after (the lunar quarters), by digging nests of hatched but unreleased animals. All trials were run until the larvae molted into the morphologically distinct first juvenile instar, usually for 6 to 8 days. Animals held under DD did not molt this rapidly and were tested for 12 days.
An additional four sets of 1000 larvae were tested for 1 week each over 1 lunar month, from full moon to full moon in August, 1978. Trials were initiated on the nights of full moon, full moon plus 7 days, new moon, and new moon plus 7 days, and continued until molting of the larvae as above. Larvae were collected from the field 8 hr prior to the start of each trial, by digging nests of hatched but unreleased animals. An ambient light and temperature regime was employed, and activity was recorded as described above, using time-lapse photography. Lunar variability in the positive response of larvae to a light source was also tested during these trials by exposing the larvae to a constant light gradient during the hours of darkness. This light was provided by an overhead source (Tensor Hi Intensity Lamp #C3812), at a distance of 1 m from the water surface. This illuminated 25% of the test aquarium with white light. The remainder of the aquarium was shaded from the light with an opaque cover, creating a steep gradient of light intensity between the lighted quadrant and the rest of the aquarium.
All trials were conducted in 20-gal glass aquaria with a sand substrate into which animals buried when they were inactive. A closed system of circulation was employed with subsand filtration. Temperatures ranged from 28° to 34° C during the ambient light regime trial and were held at 24° C under constant light and darkness. Salinity was held at 2Q(/rc in all trials.
RESULTS Field studies
Nest depths in the field showed no consistent change from the beginning to the end of the 8-month breeding season. The mean depth for larvae (13.2 ± 2.9 cm) was not significantly different than that for newly laid and immobile eggs (14.6 ± 1.9 cm) although the standard deviation was somewhat larger (t = 1.25, n.s.). However, there was a pronounced short term movement of larval nests upward from a mean depth of 17 cm at low tide to the surface 6 hr later at high tide on the night of full moon (Fig. 1). This was followed by movements of the larval nests back down to depths of approximately 14 cm by the time of the next low tide. Nests could be observed at the surface for 30 min prior to and 30
EMERGENCE OF LARVAL LIMULUS
497
0
E o
Q_
LJ Q
8
12
16
20
2130 0030 0330
TIME
0630 0815
FIGURE 1. Movement of larval nests to and away from the surface of the beach during nights of full moon (solid line) and new moon (dotted line). The arrow indicates the time of high tide in each case. Bars indicate 1 s.d. Movement of nests to the surface on the night of full moon was not repeated on the night of new moon.
min after the high tide. Usually, some 15 to 20 individual larvae become partially exposed at the surface, with the rest of the nest remaining immediately below the surface. Each wave washed away the surface animals and carried them down the beach into the water. Many were cast back up onto the beach and stranded as the tide receded, where they formed drift lines of larvae on the beach. Most of these animals remained at the surface until daylight, after which they disappeared, falling prey to shore birds at dawn. The willet, Catoptrophorus semipalmatus, was a major predator. Reburial was probably not of great signficance since scat- tered individuals were only occasionally found beneath the surface. Thus, larvae were seen to move in the field from their original burial depths to the surface and back down again over a 12 hr tidal cycle on the night of full moon. Such pro- nounced movements to the surface did not occur on the night of new moon. There was no significant difference between mean nest depth at low tide and mean nest depths at high tide on the night of new moon in 1979.
The depths of nests at the hour of high tide on the subsequent neap tide, new moon spring tide and second neap tide of the lunar month in 1978 are presented in Figure 2. Only on the night of the full moon spring tide did larvae come to the surface. The water did not reach the level of the nests at any time other than during spring tides at full and new moon.
The results of the plankton sampling adjacent to the breeding beach on day and night tides throughout the breeding season are provided in Figure 3. The major releases occurred on the night of full moon with two exceptions. And, as with breeding adults (Rudloe 1978), a strong nocturnal activity rhythm is evident.
498
ANNE RUDLOE
01
~ 5
o 10
E u
CL UJ O
15
20
l
I
I
FULL F + lwk NEW LUNAR PHASE
F-Hwk
FIGURE 2. Mean depth of larval nests at the hour of high tide at each lunar phase for one month. Bars indicate 1 s.d.
There was also a major release of larvae whenever localized storms with strong onshore winds coincided with high tide, producing an unusually heavy surf. Under these circumstances, larvae were released by day as well as by night. Following a massive storm release in July, the following full moon did not show the usual peak of emergence. The larvae that would have emerged then may have been washed out in the preceding storm. Thus, the occurrence of storm conditions that coincide with a sufficiently high tide are an additional release mechanism.
Laboratory studies
When larvae were maintained under ambient light and temperature regimes, they displayed strong nocturnal activity peaks for the duration of the larval instar (Fig. 4). Larvae remained buried below the surface of the sand substrate during daylight hours. The abruptness with which activity appeared at the same hour each evening and terminated at the same hour each morning suggests that activity might be triggered very precisely by some as yet undescribed factor such as light intensity.
That the nocturnal rhythmicity of behavior might be endogenous in nature is suggested by the results of the trials in which activity was recorded under constant conditions of darkness and temperature. In Figure 5 larval activity is seen to occur during the hours corresponding to normal darkness almost exclusively. It was also noted that none of the larvae used in these trials molted as rapidly to the first juvenile instar as did those maintained under ambient light and tem- perature conditions.
When maintained under LL conditions and constant temperature, the larvae substained slight nocturnal activity peaks for the first 3 days (Fig. 6) of the trial, although the amplitude of the variation was considerably reduced, and substantial numbers of larvae remained active during daylight hours. Overall activity was well
EMERGENCE OF LARVAL LIMULUS
499
800.-
784
600-
LU
cr
_i
u_ o
*
400-
LIMULUS LARVAL RELEASE " NIGHT " DAY " STORM
322
200-
7/16 7/30 8/14 N F N
9/14
N
9/27 F
TIDE
FIGURE 3. Appearance of free-swimming larvae in plankton samples adjacent to a breeding beach throughout a breeding season. F = full moon ; N = new moon. Open peaks represent night high tides, closed peaks represent day high tides, and shaded peaks represent storm release. The asterisk denotes the July full moon, when larval release was not significant. This may be due to the extreme release of larvae on the preceding full moon when a massive storm release coincided with the lunar release.
below that under DD and ambient light regimes, however, and reached a level of zero by the fifth day of the trial. When light was removed for 30 min on the fifth night, swimming activity resumed within 5 min of the onset of darkness. After 30 min of darkness, light was restored and activity showed a steady decline throughout the rest of the night. This experiment was terminated thereafter due to the molting of the larvae.
A comparison of levels of activity and light responses of the larvae tested for 7 days each for each week of a lunar month are presented in Table I. Larvae were more active during weeks of spring tides (new and full moon) than on the intervening weeks of neap tides (£-=5.49, P<0.01). Differences in levels of activity for the nights of new and full moon in the laboratory were not significant, however.
Although there was no significant difference in over-all first night activity be- tween new and full moon, there was a marked difference in the pattern of activity
300
ANNE RUDLOE
u_ O
UJ
UJ
40-
20-
TIME
7/10
8
FIGURE 4. Activity patterns of 1000 Liinulus larvae maintained in the laboratory under conditions of ambient light and temperature. Swimming activity began at 2200 hr eacli night and terminated at 0800 hr each morning. Larvae remained buried in the sand substrate when not actively swimming. Data is present as number of larvae visible in 1 frame of film at each hour.
200-
o
UJ
1 300
a: u.
u a.
UJ
200
100
300
200
100
NTGHT
7/5
7/9
DAY
NIGHT
7/2
7/6
^7/10
DAY
NIGHT
7/3
7/7
7/11
DAY
NIGHT
7/4
7/8
7/12
DAY
2100 0600 2100
0600 2100 0600 2100 TIME (hrs.)
0600
FIGURE 5. Swimming activity of 500 Limit I us larvae maintained in the laboratory under conditions of constant temperature and darkness. One trial is presented \vith a duration of 12 days, after which the larvae molted.
EMERGENCE OF LARVAL LIMULUS
501
600
LLJ
o 400 in
L- O
#
200
D
6/4
6/5
6/6
6/7 TIME
6/8
6/9
FIGURE 6. Swimming activity of 1000 Liinulus larvae maintained in the laboratory under conditions of constant temperature and light. Activity terminated on the fifth night but was resumed immediately when light was removed for 5 min at 0100 hr. With restoration of the light, activity again declined. Animals molted during the next 24-hr period and the experiment was terminated. D is daylight at 1200 hr, X is night at 2400 hr. Number of animals swimming was plotted hourly.
during these nights. During the course of the full moon night, activity increased by a factor of 114% between 10 PM and 7 AM while it declined by 29% during the same period during the course of the new moon night. It declined even more steeply, by factors of 79% and 81% on the nights of neap or quarter moon tides. Activity also declined steeply from first to last nights of the session for the full moon (82%) and neap tide (78, 89%) weeks, but declined less sharply from first to last night during the new moon week (30% decline in activity >.
Larvae were significantly attracted to light in all lunar weeks (Table I) except that of the new moon. Although larvae \vere active and swimming on the night of new moon, they showed no significant response to light as measured by concen- tration of individuals in the lighted quadrant of the aquarium. This lack of respon- siveness was maintained throughout the entire week of the new moon trial.
DISCUSSION
The use by Liinuhts of the intertidal sandy beach as a breeding site and the burial of nests well below the surface affords a great deal of protection for develop- ing eggs, and most survive to hatching. The occurrence of larval nests in a dis- tinct zone in the upper intertidal in the northeastern Gulf of Mexico may be related
502
ANNE RUDLOE
TABLE I
Lunar variation in larval Sicilian ing activity and light responses under laboratory conditions. Four sets of 1,000 larvae, one for each lunar week, iverc tested. The larvae responded to light at all times other than the week of new moon.
|
Lunar phase |
||||
|
Full |
New |
Full - 1 week |
Fuii ^ 1 week |
|
|
Mean activity, first night |
580 |
485 |
329 |
393 |
|
% decline in activity, first night |
(increased) |
29 |
79 |
81 |
|
% decline in activity, first to sixth |
||||
|
night |
82 |
30 |
78 |
89 |
|
% responding to light, first night |
63 |
31 |
78 |
89 |
|
% decline in light response, first night |
31 |
• — • |
38 |
57 |
|
% decline in light response, first to sixth |
||||
|
night |
32 |
— |
(increased) |
38 |
|
113 |
||||
|
X2, light response, first to sixth night |
49.8 |
9.44 |
49.8 |
64.6 |
|
P < 0.01 |
N.S. |
P < 0.01 |
P < 0.01 |
|
|
X2, light response, first night |
246 |
6.52 |
69.5 |
131.7 |
|
P < 0.01 |
N.S. |
P < 0.01 |
P < 0.01 |
n = 1000 in each trial.
to the small tidal amplitude of approximately 1 m. According to Shuster (per- sonal communication), nests are found in Delaware Bay, where the spring tide range is approximately 2 m, over a much wider band — from the high tide line through 60% of the exposed intertidal. In Cape Cod Bay, where the range is 3 m or more, nests are also widely distributed but tend to be most numerous in the mid tidal area.
In addition to protection, this nesting pattern also creates substantial prob- lems of emergence and dispersal of the young into the aquatic environment. How- ever, Li 111 ul us larvae are a behaviorally and morphologically specialized phase of the life cycle, that cope well with the problems of nest release. With an initial swim- ming frenzy and positive phototaxis, and with lunar and circadian activity rhythms that synchronize activity with water levels on the beach, the larvae are well adapted for emergence from the nests and dispersal. After this transition is achieved, larvae molt into the substantially different morphology and behavior patterns characteristic of the benthic portion of the life cycle.
Although further work is required to be conclusive, the nocturnal swimming of Limulus larvae under DD conditions suggests an endogenous rhythm. The peaks of activity appear to occur progressively later during the night as the experiment progresses, and these peaks are approximately associated with the time of low tide in the field. Whether this is in fact reflective of an advancing endogenous tidal cycle or is a shifting of the circadian rhythm is not established at this time. The observed suppression of activity under LL is characteristic of both aquatic and terrestrial nocturnal species (Aschoff, 1960).
Semi-lunar reproductive rhythms are \vell known among intertidal crustaceans, snails, and bivalves (Gifford, 1962; Warner, 1967; Russell-Hunter, Apley and
EMERGENCE OF LARVAL LIMULUS 503
Hunter, 1972; Wheeler, 1979). The field emergence of larvae on the full moon and the absence of larval release on the new moon therefore poses an intriguing question. Is it reflective of an edogenous lunar rhythm, or is moonlight a key stimulus in orienting the movement of larvae to the surface and into the water column ?
Based on the activity of larvae in the laboratory, there appears to be a lunar rhythm of responsiveness to light that is somewhat different from the lunar loco- motory rhythms observed. While larvae were active at both full and new moons (i.e. on spring tides), they were responsive to light only at full moon. This is consistent with the lack of field larval release during the spring tides of the new moon.
Furthermore, neurophvsiological sensitivity to light several hundred times dim- mer than moonlight has been recorded for the Llnnilns median ocellus by Lall and Chapman (1973) who obtained responses to levels of illumination as low as 1.4 : : 1O10 /A¥/(cnr-nm) at 360 NM. Moonlight intensity of 1O3 ^W/(cm2-nm) has been reported by Kampa (1970). Therefore moonlight is well within the sensitivity range of adult specimens of Liinulus.
A model to explain the observed pattern of larval release in the field is presented. It is based on the comparison of field release with the activity and light response patterns seen in the laboratory. The following factors are apparently of significance : an endogenous readiness to respond to a dim light source that peaks at the time of full moon and is minimal at the time of new moon ; the availability of an appropriate light source in the form of a full moon ; a water level reaching the nests only during periods of spring tides ; and the suppression of activity by the bright light of day that overrides afternoon spring high tides. This pattern of larval release is supplemented by occasional mass releases caused by storms that coincide with high tides at times other than full moon.
This model seems to be most consistent with the data presently available. In particular, the full/new moon differences in response to light in the laboratory cor- respond to the lack of field release at new moon. However, whether moonlight or the lack of it is detectable to larvae buried several centimeters in the sand prior to high tide has not been determined. If it is not detectable at the depth of the nests, then some other factor may be instrumental in initiating movement toward the surface.
If inactive larvae are removed from a nest and placed in water, swimming activity is immediately initiated. Increasing interstitial moisture associated with the rising tide might conceivably initiate activity, for instance, with moonlight becom- ing the dominant stimulus only as the animals approach the surface. The apparent partial movement of larvae toward the surface on the night of new moon in June, 1979, suggests the existence of some stimulus in addition to moonlight. The identification of that stimulus, should it exist, as well as testing of the other components of the proposed model, awaits additional research.
I gratefully acknowledge the field assistance of Mr. Richard Shebler and Mr. Doug Gleeson. Mr. Jack Rudloe and Dr. W. F. Herrnkind also provided field assistance as well as valuable suggestions and comments throughout the course of
504 ANNE KUDLOE
this work. This research was funded by a grant from the GrifTis Foundation and the American Littoral Society.
SUM MARY
The horseshoe crab Liniitliis [>olypltciiiits lays its eggs on sandy beaches at the level of the highest high tide in the northeastern Gulf of Mexico, buried approxi- mately 18 cm below the surface. When they hatch, the larvae must move from the buried nest site into the marine environment.
In the field, nests of larvae move to the sand surface and emerge at the spring high tide on the night of full moon. They may also be released by heavy surf asso- ciated with storms. No release occurs on the spring high tides associated with new moon.
In the laboratory, larvae are seen to be noctnrnally active, both under ambient and DD photoperiods. Activity peaks at times of full and new moons, and larvae are positively phototatic at all lunar phases except newr moon.
A model to account for observed field behavior in light of laboratory activity and light responses is presented.
LITERATURE CITED
ASCHOFF, J., 1960. Exogenous and endogenous components in circadian rhythms. Cold Sprin/i
Harbor Symp. Quant. Biol, 25 : 11-28. COHEN, E., Ed., 1979. ftiomedical applications of the Horseshoe Crab (Liinitlidae). Alan R.
Liss, Inc., New York, 688 pp. FRENCH, K., 1977. An effect of light on the hatching pattern of Limit/us polvplieuitts. Biol.
Bull., 153 : 425. FRENCH, K., AND S. DOLINER, 1978. Photospositive behavior of young Linntlns. Biol. Bull.,
155: 437-438. GIFFORD, C. A., 1962. Some observations on the general biology of the land crab Cardiosoma
guanhumi ( Latreille) in South Florida. Biol. Bull., 123 : 207-223.
GREEN, R. H., AND K. D. HOBSOX, 1970. Spatial and temporal structure in a temperate inter- tidal community, with special emphasis on Gemma gemma. (Pelecypoda: Mollusca).
Ecology, 51: 999-1011.
HUMMON, W. D., J. W. FLEECER, AND M. R. HUMMON, 1976. Meiofauna-macrofauna inter- actions. I. Sand beach meiofauna affected by maturing Limulus eggs. Chesapeake
Sci.. 17 : 292-298. JEGLA, T. C., AND J. D. COSTLOW, 1979. The Linntlns bioassay for ecdysteroids. Biol. Bull.,
156: 103-104. KAMPA, E. M., 1970. Underwater daylight and moonlight measurement in the eastern North
Atlantic. /. Mar. Biol. Assoc'lLK., 50 : 397-420.
KINGSLEY, J. S., 1892. The embryology of Limulus. J . Morphol., 7 : 35-68. LALL, A., AND R. CHAPMAN, 1973. Phototaxis in Limulus under natural conditions : evidence
for reception of near ultraviolet light in the median dorsal ocellus. /. E.rp. Biol., 58:
213-224. LOEB, J., 1893. Uber kunstliche Unwandlung positiv heliotropischer Tiere in negativ helio-
tropische und umgekchit. /. Arch. yes. Physiology, 54: 81-107. PEARL, R., 1904. On the behavior and reactions of Limulus in early stages of development.
/. Coinp. Neurol. Psycho!.. 14: 138-164. RUDLOE, A., 1978. Some ecologically significant aspects of the behavior of the horseshoe crab,
Limulus pol\phcmus. Ph.D. dissertation, Florida State University, Tallahassee, Florida.
246 pp. RUDLOE, A., 1979. Limulus polyphemns: a review of the ecologically significant literature.
Pages 27-35 in E. Cohen, Ed., Biomedical Application of the Horseshoe Crab
(Liinnli(ltie). Alan R. Liss, Inc., New York.
EMERGENCE OF LARVAL LIMULUS 505
RUDLOE, A., AND W. F. HERRNKiND, 1976. Orientation of Linudns polyphemus in the vicinity
of breeding beaches. Mar. Behav. PhysioL, 4 : 75-89. RUSSELL-HUNTER, W. D., M. L. APLEY, AND R. D. HUNTER, 1972. Early life history of
Mclampns and the significance of semi-lunar synchrony. Biol. Bull.. 143 : 623-656. SHUSTER, C. N., JR., 1958. On morphometric and serological relationships within the
Limulidae, with particular reference to Liunilns polyphemus (L.). Ph.D. dissertation,
Nezv York University, Neiv York. Diss. Abstr., 18 : 371-372, 287 pages. SOKOLOFF, A., 1978. Observations on populations of the horseshoe crab. Limit/its ( = Xipho-
sura) polyphemus. Res. Popul. Ecol. (Kyoto), 19: 222-236. THORSON, G., 1964. Light as an ecological factor in the dispersal and settlement of larvae
of marine botton invertebrates. Ophelia, 1 : 167-208. WARNER, G. F., 1967. The life history of the mangrove tree crab, .trains pismii. J. ZooL,
153 : 321-335. WHEELER, D. E., 1979. Semi-lunar hatching periodicity in the mud fiddler crab I'ca ptti/im.r
(Smith). Estuaries, 1 : 268-269. WOLBARSHT, M., AND S. YEANDLE, 1967. Visual processes in the I.iiiinliis eye. .{nun. Rev.
Phvsiol.. 29: 513-542.
KdVrence: B'wl. null. 157: 506-523. (December, 1979)
DEVELOPMENT OF BIOLUMINESCENCE AND OTHER EFFECTOR RESPONSES IN THE PENNATULID COELENTERATE
RENILLA KOLLIKERI
RICHARD A. SATTERLIE 1 AND JAMES F. CASE
Department of Biological Sciences. University of California, Santa Barbara, California 93106
In benthic marine invertebrates with planktonic larval forms, the conversion from larva to adult usually involves significant morphological and physiological changes. The morphological changes can involve destruction and resorption as well as elaboration and reorganization of larval structures, the production of new tissues and structures, and renewed differentiation of adult organ rudiments (see, for example, Bonar, 1978; Cameron and Hinegardner, 1974, 1978). Despite a wealth of information concerning morphological changes which occur at meta- morphosis of marine invertebrate larvae, little is known about the physiological changes at this critical time. In particular, little is known about functional changes in the nervous and effector systems during larval maturation and trans- formation to the adult form.
The ideal preparation for studying changes in larval neuroeffector function is one in which the organization of nervous and effector elements is simple, the systems in question can be easily monitored, the adult neuroeffector systems are well understood, and the larval stages are relatively large and easy to raise through metamorphosis. These conditions are approached in the octocoral Renilla kollikcri. The nervous elements of Renilla are arranged in a diffuse neural network (Satterlie, Anderson and Case, 1976). This nerve net is through-conducting, that is, an impulse initiated in one area of the net is transmitted throughout the remaining portion without decrement (Anderson and Case, 1975 ; Satterlie, Anderson and Case, 1976). Effector function in Renilla, and other anthozoans, is based on per- haps the simplest integrative mechanism, peripheral frequency dependent facilitation (Pantin, 1935; Parker, 1920; Nicol, 1955a, b) and is therefore relatively predict- able. Although larvae of Renilla are not large enough for electrophysiological recording, they possess an effector system, the bioluminescent system, which is easily monitored photometrically. Furthermore, in live preparations, the lumi- nescent cells (photocytes) fluoresce when illuminated with the proper exciting wavelength of light. The luminescent system can therefore be monitored both morphologically and physiologically in live, intact specimens. The neuroeffector systems of adult Renilla colonies, including the luminescent system, have been examined in some detail (Anderson and Case, 1975). Electrical impulses were recorded from a colonial conduction system which controls bioluminescence, polyp withdrawal and colonial contraction. Semi-autonomous polyp conduction systems are also active (Anderson and Case, 1975).
1 Present address : Department of Zoology, University of Alberta, Edmonton, Alberta, Canada T6G 2E9.
506
EFFECTOR CONTROL IN RENILLA LARVAE 507
Our studies indicate that the development of Rcnilla kollikcri is similar to that of Rcnilla reniformis (Wilson, 1883) and other octocorals (Matthews, 1916; Gohar, 1940a, b; Chia and Crawford, 1973). The eggs of Renilla are large and yolky, and are usually spawned in large numbers. A swimming planula settles and metamorphoses into a primary polyp. Secondary polyps bud from the primary polyp to form the colony (see Wilson, 1883).
We have followed the development of Rcnilla from the fertilized egg to the mature primary polyp during three reproductive seasons in order to describe the onset of effector form and function, with particular reference to three activities ; ciliary swimming, muscular activities, and bioluminescence. The observations of effector function have been utilized to describe the functional development of the nervous system (s) that underlie effector activation during the various stages of larval life. In another report, these observations will be supplemented by an ultrastructural investigation of the developmental stages.
MATERIALS AND METHODS
Colonies of Renilla kollikcri Pfeffer were collected in shallow water off Zuma Beach, California and in the Santa Barbara Channel by divers. Healthy specimens were kept in large aerated aquaria at 18 to 20° C and checked several times daily for spontaneous spawning. Colonies that had been dark conditioned for 1 to 2 days in running seawater aquaria frequently spawned following transfer to lighted battery jars of still sea water. Larvae were reared in 3-liter flasks or beakers of aerated sea water, changed twice daily. Late swimmers (see results for descrip- tion) were transferred to 250-ml beakers with or without sand. The lecithotrophic eggs permitted development to the primary polyp stage without feeding.
Fluorescence
Living larvae were observed in one of three types of squash preparations. Light and medium squashes involved bridging a coverslip across two other cover- slips (No. 1-J- thickness) on a slide. For light squashes, the slide was flooded with sea water and for medium squashes, the animal was placed in a small drop. In heavy squashes, which frequently resulted in tissue damage, a coverslip was placed directly on a small drop of sea water containing the specimen. A Zeiss Universal Research Microscope was used with mercury lamp illumination, BG 12 and BG 38 exciting filters, and a 500 nm cut-off barrier filter. Larvae were anesthetized in a 1 : 1 solution of 0.37 M MgCU: sea \vater for photograph}-.
Bioluminescence
Bioluminescence was detected with an EMI 9601 B end-window photomultiplier tube operated at —950 V, giving a high signal-to-noise ratio. A Uniblitz 100-2 electric shutter was threaded onto the window end of the tube and operated from outside a dark experimental box. Signals from the photomultiplier tube were amplified with a Keithley 427 current amplifier leading to a 7P1F DC amplifier of a Grass 79D polygraph.
The larvae were tested in laminated plexiglass blocks with 2.5 cm diameter and
508
R. A. SATTERLIE AND J. F. CASE
FIGURE 1. Fertilized egg approximately 15 min after release. The ciliary coating is made up of thousands of sperm adhering to the egg surface (verified by E.M.). X76, light squash.
FIGURE 2. About 8-cell stage. The initial cleavages are irregular and apparently incom- plete. At this stage, most of the sperm coating is gone. One hour embryo. X76, light squash.
FIGURE 3. About 64-cell stage. Three hour embryo. X76, light squash.
FIGURE 4. Pre-swimmer planula. At this stage, the endoderm is fully formed and the gastric cavity begins to appear. Ciliary activity is weak so the pre-s\vimmer merely spins in circles on the bottom. X76, light squash.
FIGURE 5. Early swimmer planula. Ciliary activity is sufficient to lift the planula off the bottom. No muscular activity is evident. X76, light squash.
EFFECTOR CONTROL IX REX ILL. I LARVAE
1 -cm-deep center wells (same diameter as shutter opening) which were positioned to allow a 3-cm specimen-to-pm tube window distance. Chemicals could he intro- duced via PE 50 intramedic polyethylene tubing penetrating the wall of the chamber. Single larvae were placed in 1 ml of sea water in the center well, which could be centered under the shutter pm tube by touch. One milliliter of test solution was slowly injected. The test solutions were 0.53 M KC1, 0.1 M CaClo, 3% HoO2, and sea water controls. The electrical test chamber consisted of a similar plexiglass block with a pair of horizontal 180 mesh silver screens (Unique Wire Weaving Co., Hillside, N. J. ), leading to a Grass S9 stimulator. Test animals were placed on the bottom screen with enough sea water to cover them but prevent swimming. The upper screen wras then pressed down to contact the larva or sea water. Large stimulating voltages were frequently required due to the shunting effect of the sea water. All luminescence experiments were carried out in a dark box within a darkroom at ambient air temperature (20° to 25° C).
RESULTS
Pennatulid colonies have three distinct parts : the peduncle, rachis, and polyps. The basal peduncle is an anchoring device which is inserted into the substratum by peristaltic movements. The rachis, or main tissue mass, supports an array of two types of secondary polyps, the siphonozooids and the autozooids. The rachis and peduncle are derived from the primary polyp, and the colony is formed by budding of the secondary polyps (Wilson, 1883). The rachis of Rcnllla kdllikcri is flattened and leaf-shaped. The ventral surface is devoid of polyps and normallv lies in the substratum. The dorsal surface bears scattered autozooids.
j
inhalent siphonozooid clusters, and a single exhalent siphonozooid.
Ren ilia kdllikcri colonies are dioecious. Gametes are borne on the autozooid septal filaments. When mature, the eggs are tan in color and approximately 0.3 to 0.4 mm in diameter. In male colonies, sperm are packed in follicles which are the same size as the eggs, but white in color. Both eggs and sperm follicles are covered by a single layer of ciliated endodermal cells until spawning takes place. During the reproductive season, the septal filaments of each autozooid contain more than 20 sperm follicles or eggs at different stages of development.
Spawning
The reproductive season for Ren ilia kdllikcri in the Santa Barbara area extends from May to late July or early August, a period centered around the summer solstice. During this period colonies may spawn many times, in small groups or en masse. In female colonies, spawning begins with an extreme inflation of the
FIGURE 6. Swimmer planula. The planula is now hollow and the septa begin to form by outgrowth of the endoderm. Swimming is active. Local muscular contractions can be elicited. X76, light squash.
FIGURES 7-8. Swimmer planula, approximately 10 to 15 hr older than in Figure 6. The same animal was used for both bright field (7) and fluorescence (8, traced from original to show position of dimly fluorescent photocytes) micrographs. The first sign of fluorescence is evident at this time. Note that the anthocodial septa are well formed (Fig. 7, arrows). The mouth ( M, Fig. 7) is not yet open. X56, heavy squash.
510
R. A. SATTERLIE AND J. F. CASE
rachis, in conjunction with accelerated and pronounced rachidial peristalsis. In a 9.5 cm colony, each peristaltic wave passed across the rachis in 45 to 60 sec, a conduction velocity of 0.16 to 0.21 cm/s (21° C). This is compared to a rate of 0.11 to 0.13 cm/s (23° C) in non-spawning Rcnilla. Two and sometimes three waves are present on the rachis of spawning colonies at all times. During spawning, the autozooids remain extended with the tentacles bent slightly in an aboral direction. In female colonies, the shedding of eggs appears to result from the peristaltic movements. In one instance, three waves were required to squeeze an egg out of the autozooid mouth.
When released, the eggs are oblong, but become sperical in 15 to 30 min (Fig. 1). They are neutrally buoyant, and float at all levels in an aquarium of still \vater. All released eggs, as well as some dissected from a spawning colony, were already fertilized, indicating that fertilization occurs before release. Sperm follicles are not released intact during spawning. The follicles rupture within the autozooids, and the sperm are released through the autozooid mouths and the exhalent siphonozooid.
TABLE I
Summary of early development of Renilla kollikeri with observations on effector function (25° C). The numbers in parentheses indicate the corresponding text figures.
|
Hours from spawning |
Stage |
Ciliary swimming |
Muscle activity |
Fluorescence |
Bioluminescence |
|
0 |
Fertilized |
— . |
— |
— |
— |
|
i-3 |
egg (1) First |
||||
|
1-3 |
cleavages (2) About |
_ |
_ |
||
|
7 |
64-cell (3) Stereoblastula |
||||
|
32 38 60 |
Pre- swimmer (4) Early swimmer (5) Swimmer (6) |
Spinning on bottom Swimming Swimming |
Local |
• — • |
• — |
|
contractions |
|||||
|
84 |
Swimmer (7) |
Swimming |
Conducted |
First sign (8) |
— |
|
contractions |
|||||
|
131 |
Late swimmer (9) Settled (12) |
Swimming, sinking Ceases |
Conducted contractions Conducted |
+ (Id) + + (11) |
- (20B) |
|
contractions |
|||||
|
136| 191 274 |
Settled (13) Tentacle buds (14) Pinnules (15) |
— |
Separate responses Separate responses Separate |
+ + + (13) as in adult + 4-4- (15, 16) |
First Sign (20C) + (20E) + + (20F) |
|
323 |
Primary polyp |
— |
responses Separate |
+ + + (17) |
+ + + (20G) |
|
responses |
EFFECTOR CONTROL IN RENILLA LARVAE
511
Larval development
The early cleavages and larval development of Rcnilla kollikcri are similar to that described by Wilson (1883) for Rcnilla rcnifonnis. The intial cleavages are extremely irregular and variable. Seldom can 2-, 4-, or 8-cell stages be recognized. The first detectable cleavages (Rcnilla kollikcri') occur 20 min to 3 hr after spawn- ing, at which time embryos of 8 to approximately 64 cells are observed (Fig. 3). Prior to this stage the embryos are very irregular in shape (Fig. 2).
Early development progressed more rapidly at 25° C than at lower tempera- tures tested. Cooler temperatures, down to 6° C, retarded development without apparent harmful effects. A timetable for the development of Renilla kollikeri (25° C) is shown in Table I, and all further mention of age will refer to this table and temperature.
Just prior to cleavage, neutral buoyancy is lost, and the embryos settle to the bottom of the aquarium. No ciliary activity is apparent. Subsequent cell divisions give rise to stereoblastulae. Endoderm formation is followed by gradual disappear- ance of the central, yolky cells. Swimming planulae are formed 38 hr after spawn- ing (Figs. 4, 5). The planulae are barrel-shaped and swim at the air-water inter-
FIGURE 9. Late swimmer planula. The eight anthocodial septa Cone indicated, large arrow) are visible, two of which extend to form the peduncular septum (small arrow). XlOl, light squash.
FIGURE 10. Late swimmer planula. Tracing of a fluorescence micrograph indicating the position of the dimly fluorescent photocytes, which are arranged in rows along the septa. X47, medium squash.
512
EFFECTOR CONTROL IN RENILLA LARVAE 5U
face (early swimmers, Table I). During the swimming period (4 to 6 days) the planulae gradually elongate (swimmers. Figs. 6, 7) and undergo considerable cellular differentiation. During this time the peduncular septum, as well as the eight anthocodial septa are formed (Fig. 7). The differentiation of muscle and nerve cells begins early in the planula stage (early swimmers ; Satterlie and Case, in preparation ) .
When 5 days old, the planulae (late swimmers. Fig. 9) begin to sink to the bottom of the aquarium. The larvae settle oral end first on clean glass or in sand (Fig. 12). Settlement is achieved more rapidly when a sand substratum is provided, as opposed to clean glass, the difference being as much as 8 hr. Once attached to the substratum by the oral end, the larvae "roll" to the side and eventually attach by the aboral peduncle. The oral end is then raised. When settled in sand, the larvae move between sand grains and in some cases burrow in approximately a half-centimeter.
Tentacle buds first appear as conical projections on the anthocodium at 8 days (Fig. 14). The tentacles gradually elongate and sprout lateral pinnules (Fig. 15). At 13 days, the juveniles represent structurally complete primary polyps (Figs. 18, 19). The juveniles survived as single polyps for up to 3 weeks in the laboratory, but did not bud secondary autozooids. See Wilson (1883) for an account of colony formation in Rcnilla renifonnis.
Development of effector systems
Ciliary swimming. The ectoderm of adult specimens of Ken ilia is not heavily ciliated except for the invaginated pharynx of each polyp. However, for a short time during development, the planulae rely on a dense ectodermal ciliary coat for locomotion. Ciliary activity is continuous, and no ciliary reversals or arrests could be demonstrated. When a planula encountered a solid object, for instance the side of a container, it would continue to swim in a forward direction (aboral end first) until it eventually slid awray. The swimming motion produces a rotation about the oral-aboral axis, and in the late swimmer, which maintains a bend in the aboral end, produces a cork-screw-like motion. All ectodermal ciliary activity ceases upon settlement. If larvae- are immediately dislodged, however, ciliary activity reappears. This ability is not apparent after the larvae have remained
FIGURE 11. Freshly settled planula. Fluorescence micrograph showing the photocytes arranged along the septa. X56, heavy squash.
FIGURE 12. Freshly settled planula, longitudinally contracted. X78, light squash.
FIGURE 13. Settled juvenile. This animal produced a luminescent response similar to that in Figure 20C. The fluorescent cells are as bright as those in the la'er stages. Note the constriction in the "neck" region. The anthocodium is inflated, and the peduncle deflated, demonstrating the separation in responses in the two areas. X58, medium squash.
FIGURE 14. Tentacle bud stage. Again note the inflated anthocodium and contracted peduncle. X80, light squash.
FIGURE 15. Tentacled polyp at stage of pinnule development. Note the photocyte processes. X66, medium squash.
FIGURE 16. Higher magnification fluorescence micrograph of the photocyte cluster marked by the arrow in Figure 15. X214, medium squash.
FIGURES 17-18. Anthocodial region of a primary polyp. The photocytes are found in the lateral sides of the tentacle bases. X43, medium squash.
514
R. A. SATTERLIE AND J. F. CASE
FIGURE 19. Full view of a primary polyp. Two siphonozooids are visible (arrows). X14, medium squash.
settled for an hour or more. If the larvae are not allowed to settle, the late swimmer stage is prolonged and further morphological changes, such as tentacle budding, are delayed.
Muscular activity. Obvious muscular activity is lacking until the swimmer stage (60 hr), after elongation is underway. At this point a mechanical or electrical stimulus to any part of the planula produces a local contraction which pulls the stimulated tissue away from the probe. This muscular contraction is not conducted circularly or longitudinally. Within 2 to 5 hr, similar stimuli produce a more widespread circular constriction, and the planulae contract longitudinally to about two thirds of the relaxed length. At this time, the septa are well formed (Fig. 7). By the late swimmer stage, the larvae are capable of bending movements as well as "protective" longitudinal contractions. These conducted muscular events are not observed when the planulae are placed in excess Mg++ for 5 min. In the anesthetized state, stimulation only produces local twitches as in the earlier planulae.
At the time of planula attachment, a division between polyp and peduncle re- actions is evident. A stimulus to the peduncle produces a conducted contraction, but not always with an accompanying polyp contraction. Similarly, polyp stimula- tion does not always produce peduncular contractions. As soon as tentacle buds
EFFECTOR CONTROL IN RENILLA LARVAE
515
are apparent, the primary polyp behaves much like the autozooids of a mature colony. An electrical or mechanical stimulus to the polyp causes an inversion of the antho- coclium and an overall contraction of the polyp.
Peristaltic movements are not apparent until settlement, when the peduncle begins rapid, strong peristaltic contractions. If an animal is dislodged, the peristaltic contractions cease until resettlement occurs. As with muscular reactions, a definite separation is evident between the anthocodium and the peduncle, and each is capable of separate peristaltic and bending movements.
Development of the biolitminescent system
Fluorescence. The first sign of fluorescence occurs in the swimmer stage approximately 80 hr after spawning. Extremely dim fluorescent cells are evident on the eight anthocodial septa (Figs. 7, 8). The cells are oval to round, 5 to 10 fjLin in maximum length, and without noticeable processes. The size of the fluo- rescent cell bodies does not appear to change much as the larvae grow. Cell processes are first observed as small, stubby projections after settlement has taken place. By the time of tentacle pinnule development, the cells appear morphologically similar to photocytes of mature autozooids (Figs. 15, 16).
The intensity of fluorescence increases until around 140 hr. when it is approxi- mately as intense as in mature autozooid photocytes. No subsequent increase in fluorescence intensity is apparent. If larvae are prevented from settling, fluorescence increases in intensity at the normal rate, but the appearance of the photocyte cyto- plasmic processes is delayed. The increase in fluorescence is not affected by the type of settling substratum.
TABLE II
Summary of chemical bioliiminescence tests expressed as number of successful trials/number of trials. The data on substrate effects are from a separate experiment using KCl as the stimulant.
|
Larval stage |
KCl |
CaCh |
H2O2 |
|
64 Cell stage |
0/30 |
— |
0/5 |
|
Early swimmer |
0/33 |
— |
0/5 |
|
Swimmer |
0/26 |
0/1 |
0/5 |
|
Late swimmer |
0/34 |
0/2 |
0/5 |
|
Settled (oral attachment) |
1/24 |
0/2 |
0/5 |
|
Settled (peduncle) |
13/16 |
1/2 |
3/5 |
|
Swimmers (same age as peduncle settled) |
0/14 |
— |
— |
|
Tentacle buds |
23/23 |
2/2 |
4/5 |
|
Swimmers (same age as tentacle buds) |
8/12 |
— |
— |
|
Mature tentacles |
11/11 |
5/5 |
5/5 |
Substratum effect on KCl response — peduncle settled larvae approximately
160 hr from spawn
|
Glass settled |
(no sand) |
5/32 |
||
|
Sand settled |
19/20 |
|||
|
Glass settled |
(sand in container) |
5/8 |
516 k. A. S. \TTKKI. IK A.ND J. I-'. CASK
As the tentacles begin to form, the fluorescent cell groups appear more pe- ripherally in the septa (compare Figs. S, 10. 11. 13, 15, and 17). When the tentacles are fully formed, the fluorescent cells are found between the tentacle bases (Fig. 17) as in mature autozooids.
Bioluminescence. The bioluminescent capability of Rcnilla was tested with three chemicals known to induce luminescence in adult animals. Potassium chloride, isotonic with sea water, produces transient flashing presumably by randomly de- polarizing cells. H^Oo produces similar results. Hypotonic CaCU was used by Anderson and Cormier ( 1973 ) to induce luminescence from lumisomes, isolated subcellular particles which contain all components of the luminescent reaction. In severed autozooids from adult colonies, 0.1 M Cadi- produces steady glowing for up to 45 min.
No bioluminescence was measurable up to the time of planula settlement (Table II and Fig. 13). Even when 12 late swimmer larvae, ready to settle, were stimulated together with KC1, luminescence was still not recordable. For com- parison, Obclia sp. hydroids were tested with KC1. In Obelia the photocytes, which are fluorescent, are scattered in the stolons and uprights. Thus a piece of tissue can be dissected which contains only one photocyte. The record from a test of such a piece of Obclia is shown in Figure 20A, and represents a base of com- parison for the Rcnilla luminescence tests.
Freshly settled planulae do not luminesce (Fig. 20B ). One to three hours after settlement, the first light is recorded (Fig. 20C). At this stage, the juveniles are attached by the peduncle with the anthocodium raised, and the fluorescence intensity of the photocytes is equal to that of mature autozooid photocytes. In general, no bioluminescence is recorded from animals in which the fluorescence is less intense than in adult autozooids. If larvae are detached prior to rolling over to the peduncular attachment, and prevented from settling, luminescent competence is delayed by up to 24 hr ( Table II ).
The substratum is important to the development of luminescent ability (Table II). Late swimmers will settle on the sides and bottom of glass beakers if no other substratum is available. Appearance of bioluminescence in glass settled juveniles is delayed just as if settlement is prevented. Occasionally, planulae settled on the glass sides of a sand-substratum container, in which case lumi- nescence developed normally.
The waveform of chemically-induced luminescence is initially irregular (Figs. 20C, D). When approximately 190 hr old, the juveniles exhibit a luminescent waveform similar to that of the adult, namely a rapid rise to peak followed by a slower decay (Figs. 20E-G). At the tentacle bud stage, light emission takes the form of several individual peaks (Fig. 20E) possibly indicating the sequential stimulation of photocytes or photocyte groups. By the tentacled stage, the emis- sion consists of broad flashes up to 4 sec in duration (Fig. 20G). Multiple flashes are frequently encountered, with the initial flash normally being the brightest (Fig. 20F). In general, the intensity of bioluminescence increases with age of the juvenile. The above descriptions of the stages of bioluminescent competence represent the norm of multiple tests. The developmental rates of individuals vary slightly.
EFFECTOR CONTROL J.\ K1LN1U..I LARVAE
517
557
FIGURE 20. Chemically induced bioluminescence in Rcnilla larvae (B-G, see Table II). (A) — KC1 induced bioluminescence from a piece of Obclia sf>. upright containing only one photocyte. The gain is the same for all records. An event marker, superimposed on the time trace (1 -second intervals), was used to indicate the beginning (downward mark) and end (upward mark) of the KC1 injection. (B)— Newly settled planula ; (O— Settled planula, attached by peduncle ; (D)— Juvenile settled for 12 hr ; ( E)— Tentacle-bud stage ; (F)— Pinnule development stage; (G) — Primary polyp. Bioluminescence could not be induced prior to planula settlement.
Settled juveniles luminesce in response to electrical stimulation. Up to the tentacle bud stage (Figs. 21 A, B), the voltages required (60 to 80 V/5 ms) result in tissue damage. The responses, however, are similar to those of the KC1 tests (Figs. 21A — 20C, D; 21 B, C -< 20E ; 21D-20F; 21E, F-20G). At the tentacle bud stage and later, the luminescent flashes occur in a 1 : 1 ratio with stimuli, although in some cases several stimuli are required to initiate the response (Fig. 21C). In subsequent stages, the responses begin with the first or second stimulus (Figs. 21D, E). Stimulus thresholds are on the order of 20 to 40 V/5 ms. Facilitating responses (Fig. 21F), characteristic of adult specimens of Rcnilla. are obtained when the tentacles are fully formed, 320 to 370 hr after spawning
-SIS
k A. SATTERL1K AND J. F. CASK
B
JUUL-LjUUL
l I I I I I I I I
vMirr^W^-^-^W^^U^H^
i i i i
V
i i i i i i i i l i i
Vvj^U..
t L L L,
FIGURE 21. Electrically induced bioluminescence in Rcnilla larvae. The top trace represents light output; the center trace, time (1-second intervals); and the bottom trace, stimulus markers (1/s). The scale bar represents the amplitude of the single Obclia photo- cyte luminescent response (Fig. 20A). The gain for traces (A-D) are the same as in Figure 20. The gain for (E) is reduced by 2.5 times, and that for (F) by 5 times. (A) — Se tied planula : (B, C) — Tentacle bud stage; (D) — Pinnule development stage; (E, F) — Primary polyp. Bioluminescence responses are initially irregular, eventually following stimuli 1:1. The final stage of maturation of the bioluminescent system is the appearance of facilitating responses (F) similar to those of adult colonies.
(Figs. 18, 19). The stimulus threshold for bioluminescent responses of the mature primary polyp is as low as 5 V/l ms.
DISCUSSION
The initiating stimuli for spawning in Rcnilla kollikcri are unknown although light seems to be important. Wilson (1883) found that Rcnilla rcniformis spawned between 5 AM and 7 AM in the laboratory. Although Rcnilla kollikcri spawned at all times of the day, most spontaneous spawning occurred between 12 noon and 3 PM. By altering the light regime, small spawns could be induced. Released sperm are swept into the female colonies and circulated by the water vascular system, into which the ripe gonads extend. Fertilization is probably internal and perhaps
EFFECTOR CONTROL IN REN ILL A LARVAE 519
stimulates female spawning. This behavior could serve to increase the probability of egg fertilization, which would otherwise be jeopardized by the surging currents in which Rcnilla colonies are found (Kastendiek, 1976).
Dispersal of Rcnilla larvae has two phases. The neutral buoyancy of the fertilized eggs allows them to be swept away from the parent colonies. Following- loss of neutral bouyancy in the early cleavage stages, the swimming planulae re-enter the water column for 4 to 6 days.
The ciliary swimming behavior of Rcnilla planulae is noteworthy in that a coordinated ciliary reversal or arrest response is lacking. The forward propulsive beat continues regardless of obstacles. A reversal or arrest would require some form of cell-to-cell communication, be it mechanical or bioelectrical. In larvae of many organisms (see Spencer, 1974), as well as in many adult coelenterates (Mackie, 1965, 1975, 1976; Mackie and Passano, 1968; Spencer, 1971, 1975. 1978) impulses are conducted in excitable epithelia. Such a conduction system seems not to be involved in the swimming behavior of Rcnilla planulae unless in the normal swimming metachronism. Intercellular gap junctions could not be found in any of the larval stages of Rcnilla (Satterlie and Case, in preparation).
Muscular activity is first apparent in the swimmer stage, mostly as local responses. The coordinated contractions of late swimmers suggest the presence of a functional conduction system. Both nervous and muscular elements are present by the swimmer stage, and may represent components of this conduction system (Satterlie and Case, in preparation). It is also possible, however, that coordina- tion is due to electrically or mechanically coupled muscle or epithelial cells. Mechanical coupling is counterindicated by the magnesium sensitivity of the muscular responses, which suggests neural control of musculature at this early stage. At least one neural conduction system is apparently present and functional prior to settlement and metamorphosis.
A proper substratum promotes planula settlement. Chia and Crawford (1973) found that sand grain size was not important to the settlement of the pen- natulid Ptilosarcns yitrneyi, but that an organic film on the sand grains was essential to induce settlement. Miiller (1973) found that in the hydrozoan Hydractinia settlement and metamorphosis is triggered by a lipid produced by marine bacteria. Our findings suggest that a similar situation exists in Rcnilla since settlement of planulae is delayed if clean glass is the only available substratum. As in other octocorals, prevention of attachment prevents or delays metamorphosis. In several xeniid and gorgonian octocorals. delayed settlement increased the num- ber of abnormalities, such as retarded tentacle development (Gohar, 1940b) and axial skeleton formation (von Koch, cited in Gohar, 1940b). Chia and Craw- ford (1973, 1977) found that planulae of Ptilosarcns would continue to swim indefinitely if the proper substratum was not available. Comparing planulae in which settlement was prevented to primary polyps of the same age, they noted sig- nificant differences in ultrastructure despite the identical ages of the two groups. Of 9 cell types in the planulae, and 12 cell types in the polyps, only 7 were common (Chia and Crawford, 1977). This reorganization in cellular composition can be attributed to settlement-metamorphosis. Similar settlement-induced changes may parallel the physiological changes which are apparent in the three effector systems
520 K. A. SATTEKUE AND .1. F. CASE
of Rcnilla at the time of peduncle attachment and metamorphosis. The first of these changes is that the ectodermal ciliation becomes inactive and the cilia are apparently resorbed. A second change is that peristaltic movements of the peduncle become intense and a separation can be seen between the muscular activities of peduncle and anthocodium. A separation of conduction systems, into peduncular (future colonial) and anthocodial (future polyp), is therefore suggested. This separation was shown electrophysiologically in adult Rcnilla by Anderson and Case (1975). The metamorphosis-induced changes in neuromuscular organi- zation of Rcnilla larvae probably involve regional differentiation of the planula conduction system since the reactions of the anthocodium and peduncle do not change. Stimuli to either area still produce conducted muscular activity in the stimulated region. During the third change, the juveniles gain the ability to bioluminesce (when about 5i clays old). The green fluorescent protein (see Morin, 1974; Cormier, Hori and Anderson, 1974; Cormier. Lee and Wampler, 1975 for reviews of Rcnilla bioluminescence biochemistry) is produced prior to settlement, and its manufacture is not drastically altered during metamorphosis. The photocytes of the primary polyp are already differentiating prior to settlement, although some morphological changes do occur at this time, such as growth of photocyte processes. The cause of the immediate appearance of bioluminescent com- petence is not known. The possibility exists that the bioluminescent system was functional prior to settlement, and we were unable to detect the light. However, the multiple animal tests tend to refute this claim. Also, it would be difficult to explain why delays in settlement would also delay the onset of measurable light output.
Colonial bioluminescent responses in adult Rcnil'.a colonies are controlled by a colonial nerve net (Anderson and Case, 1975; Satterlie, Anderson and Case, 1976). Light emission generally occurs in a 1:1 ratio with stimuli, after the first, and exhibits a facilitation in intensity which is dependent upon interpulse interval (Nicol, 1955a, b). The primary polyps do not exhibit similar responses until tentacle maturation. The sequence of luminescent responses represented in Figures 21C-F could reflect maturation of the neuro-effector hook-ups (within the polyp system), of the conduction systems (colonial and polyp), of the con- nection between the two conduction systems, or a combination of such develop- ments. Maturation of the polyp neuro-effector system appears to occur between the tentacle bud stage and the tentacled stage, as indicated by the shift from short multiple flashes to "coordinated" broad flashes in the KG tests. In the latter stage, the presence of a conduction system common to the eight septa, and thus linking the photocyte groups, can be inferred.
The responses of the tentacle-bud stage juveniles are reminiscent of recordings obtained by Nicol (1955a) during bridge experiments on adult Rcnilla. With a small bridge of tissue separating two lobes of the rachis, several stimuli were required before luminous waves were observed in the distal lobe. Interneural facilitation (Pantin, 1935) within the colonial conduction system was proposed as a vehicle for the response. Similar experiments on two other pennatulids, Stylatula and Virgularia, using electrophysiological recordings of colonial nerve net activity, support tin's interpretation ( Satterlie, Anderson and Case, in prepara-
EFFECTOR CONTROL IN A'1-XILLA LARVAE 521
tion). Immature juvenile conduction systems, or incomplete connections between systems could give rise to local responses. If so, the maturation of the conduction systems or connections, as well as that of the neuro-effector junctions could be represented by a gradual shift to a 1 : 1 stimulus-to-response ratio with a facilitating intensity output (Figs. 21C-F).
There are two colonial conduction systems in adult specimens of Rcnilla, in addition to semi-autonomous polyp conduction systems (Anderson and Case, 1975). We have demonstrated that at least one conduction system is present in the swimming planula prior to settlement. This conduction system mediates muscle activity of the entire larva (future polyp and colony tissue), and may represent the future colonial nerve net. At the time of settlement and metamorphosis the separation between the polyp and peduncular systems that becomes evident prob- ably involves separation and elaboration of the polyp conduction system(s) and formation of colony-polyp connections. In light of the morphological reorganiza- tion that occurs in other pennatulids (Ptilosarciis, Chia and Crawford, 1977) at metamorphosis, reorganization of conduction systems in .Rcnilla is certainly pos- sible. The final stage in development of the neuro-effector systems, the appearance of facilitating responses, probably involves maturation of the neuro-effector junc- tions themselves. The role of the second colonial conduction systems of adult Renilla colonies is uncertain, and this report does not contribute to its elucidation.
This work was supported by the U. S. Office of Naval Research through Contract N00014-75-C-0242. \Ye thank Mark Lowenstine for technical assistance, Shane Anderson and Gary Robinson for collecting the animals, and F. S. Chia, A. N. Spencer, L. R. Bickell, R. A. Koss, and C. M. Young for commenting on the manuscript.
S KM MARY
1. The development of the pennatulid coelenterate Rcnilla kollikcri was followed from fertilized egg to primary polyp stage, including observations on the develop- ment of effector responses such as ciliary swimming, muscular reactions and bioluminescence.
2. Ciliary swimming is first apparent in the early planula, 32 hr after the .spawn. Ciliary activity persists until settlement, at approximately 130 hr.
3. Muscular reactions are first evident as local contractions in the swimmer stage (about 60 hr). Conducted contractions can be elicited at 80 hr, suggesting that a functional conduction system is present in the planula larva. At settlement, separate peduncular and anthocodial muscular responses and peristaltic contractions are evident.
4. The future photocytes first fluoresce at about 80 hr, and thereafter fluorescence intensity increases until the time of settlement. The first sign of bioluminescence follows settlement, and is delayed if settlement is prevented. Bioluminescent responses do not exhibit normal facilitation until the primary polyp stage, and responses of the preceding stages may reflect maturation of the
522 R- A. SATTERLIE AND J. F. CASE
colonial and polyp conduction systems as well as of connections between the two systems.
5. Settlement and metamorphosis are delayed when planulae are reared in con- tainers without sand.
LITERATURE CITED
ANDERSON, J. M., AND M. J. CORMIER, 1973. Lumisomes, the cellular site of bioluminescence"
in Coelenterates. /. Biol. Chcin., 243 : 2937-2943. ANDERSON, P. A. V., AND J. F. CASE, 1975. Electrical activity associated with luminescence
and other colonial be'-avior in the pennatulid Rcnilla kollikcri. Biol. Bull., 149: 80-95. BONAR, D. B., 1978. Morphogenesis at metamorphosis in opisthobranch molluscs. Pages
177-196 in F. S. Chia and M. E. Rice, Eds., Settlement and Metamorphosis of Marine
Invertebrate Larvae. Elsevier, New York. CAMERON, R. A., AND R. T. HINEGARDNER, 1974. Initiation of metamorphosis in laboratory
cultured sea urchins. Biol. Bull., 146 : 335-342. CAMERON, R. A., AND R. T. HINEGARDNER, 1978. Early events in sea urchin metamorphosis,
description and analysis. /. Morphol., 157 : 21-32.
CHIA, F. S., AND B. J. CRAWFORD, 1973. Some observations on gametogenesis, larval develop- ment and substrate selec ion of the sea pen Ptilosarcus gurneyi. Mar. Biol.. 23 : 73-82. CHIA, F. S., AND B. J. CRAWFORD, 1977. Comparative fine structural studies of planulae and
primary polyps of identical age of the sea pen Ptilosarcus gurne\i. J . Morplwl., 151 :
131-157. CORMIER, M. J., K. HORI, AND J. M. ANDERSON, 1974. Bioluminescence in coelenterates.
Biochiiu. Biophys. Acta, 346 : 137-164. CORMIER, M. J., J. LEE, AND J. E. WAMPLER, 1975. Bioluminescence: Recent advances. Annu.
Rev. Biochem., 44 : 255-272. GOHAR, H. A. F., 1940a. Studies on the Xeniidae of the Red Sea. Publ. Mar. Biol. Stn. a!
Gharadqa, 2: 25-118. GOHAR, H. A. F., 1940b. The development of some Xeniidae ( Alcyonaria). Publ. Alar. Biol.
Stn. al Ghardaqa, 3 : 27-78. KASTENDIEK, J., 1976. Behavior of the sea pansy Rcnilla kollikcri Pfeffer (Coelenterata :
Pennatulacea) and its influence on the distribution and biological .interactions of the
species. Biol. Bull, 151 : 518-537. MACKIE, G. O., 1965. Conduction in the nerve-free epithelia of siphonophores. Am. ZooL,
5: 439-453.
MACKIE, G. O., 1975. Neurobiology of Stomotoca. II. Pacemakers and conduction path- ways. /. Neurobiol., 6: 357-378. MACKIE, G. O., 1976. Propagated spikes and secretion in a coelenterate glandular epithelium.
/. Gen. Physio!., 68: 313-325. MACKIE, G. O., L. M. PASSANO, 1968. Epithelial conduction in hydromedusae. J. Gen. Physio!.,
52: 600-621. MATTHEWS, A., 1916. The development of Alcyonhtm digitatum with some notes on the early
colony formation. Q. J. Microsc. Sci., 62 : 43-94. MORIN, J. G., 1974. Coelenterate bioluminescence. Pages 397-438 in L. Muscatine and H. M.
Lenhoff, Eds., Coelenterate Biology. Academic Press, New York. MULLER, W. A., 1973. Metomorphose-Induktion bei Planulalarven. I. Der bakterielle Induk-
tor. Wihclin Ron.r' Arch. Entivicklungsmcch Org., 173 : 107-121. NICOL, J. A. C, 1955a. Observations on luminescence in Rcnilla (Pennatulacea). /. Ex p. Biol.,.
32: 299-320. NICOL, J. A. C., 1955b. Nervous regulation of luminescence in the sea pansy Renilla kollikeri.
J. Exp. Biol., 32 : 619-635. PANTIN, C. F. A., 1935. The nerve net of the Actinozoa. I. Facilitation. /. Exp. Biol., 12 :
119-138. PARKER, G. H., 1920. Activities of colonial animals. II. Neuromuscular movements and
phosphorescence in Renilla. J. Exp. ZooL, 31 : 475-513. SATTERLIE, R. A., P. A. V, ANDERSON, AND T. F. CASE, 1976. Morphology and electrophysiology
EFFECTOR CONTROL IN RENILLA LARVAE 523
of the through-conducting systems in pennatulid coelenterates. Pages 619-627 in G. O.
Mackie, Ed., Coclcnterate Ecology and Behavior. Plenum Press, New York. SPENCER, A. N., 1971. Myoid conduction in the siphonophore Nanomia bijuga. Nature, 233 :
490-491. SPENCER, A. N., 1974. Non-nervous conduction in invertebrates and embryos. Am. Zool.,
14: 917-929 SPENCER, A. N., 1975. Behavior and electrical activity in the hydrozoan Proboscidactyla
flavicirrata (Brandt). II. The medusa. Biol. Bull.. 149: 236-250. SPENCER, A. N., 1978. Neurobiology of Polyorchis. I. Function of effector sys'ems. /.
Ncurobiol, 9: 143-157. WILSON, E. B., 1883. The development of Rcnilla. Philos. Trans. R. Soc. Loud. B Biol. Sci..
174: 723-815.
Reference: liiol. null. 157: 524-535. (December, 1979)
ON FEEDING MECHANISMS AND CLEARANCE RATES OF MOLLUSCAN VELIGERS
R. R. STRATHMANN AND E. LEISE
Department of Zoology and Friday Harbor Laboratories, University ol Washington,
Friday Harbor, Washington 98250
The teeth of a mammal or the mouthparts of a copepod can tell a knowledgeable biologist much about that animal's feeding habits. Deductions based on the structure of ciliary bands can be at least as useful. This study is part of a larger effort to relate quantitative aspects of ciliary feeding to the morphology of ciliary feeders. Much more extensive biogeographic or taxonomic comparisons of developmental adaptations can be made when feeding capacities of larvae can be predicted from the length of cilia and the lengths of their ciliated bands.
Molluscan veliger larvae possess a lobed velum which produces both feeding and locomotory currents. Those veligers which feed on suspended particles con- centrate these particles between two opposed bands of cilia which line the velar edge (Fretter, 1967; Strathmann, Jahn, and Fonseca, 1972; Thompson, 1959; Werner, 1955). The preoral band consists of long compound cilia which produce the swimming and feeding currents. The postoral band consists of shorter cilia which beat towards the preoral band. The combination of the two bands captures and retains particles. Between these bands is a food groove with small cilia which transport particles towards the mouth.
Veligers are tiny feeding machines which convert small eggs into larger juveniles. Parents which produce small eggs can produce large numbers of offspring, but there are costs that limit larval success as larval size is reduced. A reduced feeding capacity is one cost of a decreased larval size. This can take the form of a reduction in the clearance rate (volume of water cleared of particles per unit time) or restriction to a smaller range of particle sizes. In molluscan veliger larvae the clearance rate is likely to be limited by both the length of the velar edge and the length of the preoral cilia (Strathmann, ct al., 1972). The size of the particles captured is probably limited by the length of the preoral cilia.
Here we are testing the hypothesis that longer preoral cilia contribute to higher clearance rates. The test consists of comparative observations with high speed microcinephotography of movements of the preoral cilia, the postoral cilia and particles captured at the velar edge. Clearance rates are calculated from these measurements. We used three species whose veligers have different lengths of preoral cilia : the oyster Crassostrea giyas, the nudibranch Tritonia diomedea and the mud snail Nassarins ubsolctus. Movements of cilia and particles, but no particle captures, were also observed for a veliger of an unidentified species of prosobranch gastropod. Our cinefilms of feeding larvae also have extended pre- vious interpretations of the veliger feeding mechanism.
524
CILIARY FEEDING OF VELIGEKS
525
METHODS
Oyster (Crassostrea yiyus) gametes were obtained by placing adults in sea water at 30° C until spawning occurred ( 3 to 4 brs). The eggs \vere fertilized at ambient sea water temperatures (12 to 16° C). Larvae of T. diomcdca and .V. obsoletns were obtained from eggs laid in laboratory aquaria. The unidentified prosobranch veliger was taken from the plankton in Friday Harbor. All larvae were reared in culture dishes in the laboratory at 12 to 16° C and were fed the green flagellate Dnnaliclla tertiolccta.
Test subjects were starved for 24 hr before being filmed to promote maximum feeding rates during filming. Coverglasses were supported by plasticene feet just low7 enough to impede larval swimming and high enough not to impede velar cilia in the plane of focus. The larvae were usually filmed with plane of focus per- pendicular to the velar edge, as in Figure 4, for velocities of cilia and particles. In some cases larvae were filmed with plane of focus parallel to the velar edge for metachronal wavelength.
Larvae were filmed with Nomarski differential interference optics with 16 or 40 X objectives, a high speed cinecamera at 100 or 200 frames per second, and continuous light. A timing light exposed the margin of the film every 0.01 seconds and is accurate to ±\% according to its manufacturer, the Redlake Corpora- tion. Temperatures were maintained at 12° to 13° C with a Cloney cooling stage (Cloney, Schaadt, and Durdeen. 1970) except for some sequences of C. gigas filmed at 20° to 22° C.
Particles used for feeding observations were 2-/xm plastic spheres and the flagellates Diinaliella tertiolccta and Monochrysis Inthcri (5 to 10-/xm). We observed only one capture of a plastic sphere so the data on captures apply to the flagellates. High concentrations of particles were used to insure some captures in the plane of focus during high speed filming. Since particles are rapidly removed from suspension by the larvae and also settle in the confined space on a slide, particle concentrations could not be accurately determined. Therefore no cell counts were made.
Tracings of the films were made frame by frame for cilium and particle paths. All velocities were calculated from the tracings. The fraction of particles captured \vas calculated using all particles passing within reach of the preoral cilia from the beginning to the end of several filmed sequences.
TABLE I
|
Species |
Hgg Diameter (Aim) |
Shell length at start of feeding (Aim) |
Preoral cilium length (/tin) |
|
Crassostrea gigas |
45-50 |
90* |
30 |
|
Tritonia diomedea |
90 |
145* |
40 |
|
Nassarius obsoletns |
165* |
270* |
70 |
* Data from Costello, Davidson, Eggers, Fox, and Henley (1957), Kempf & Willows (1977), Ouayle (1969), Scheltema (1967).
526
R. R. STRATHMANN AND E. LE1SE
RESULTS
We assume that the veligers are actively feeding whenever numerous particles are passing around the food groove to the mouth. When this occurs both the preoral and postoral bands are beating. Occasionally both bands would cease beating but we did not observe the preoral band beating while the postoral band stopped. Crassostrea gigas, Tritonia diomedea, and Nassarius obsoletus are consistent with our impression that the size of veligers and the length of preoral cilia increase with egg size (Table I).
Velocities o\ cilia and particles
Among species, the velocities of the tips of the preoral cilia in the effective stroke increase with the length of cilium (Fig. 1). It is difficult to see the bases of cilia in many filmed sequences, so radius of arc was estimated by project- ing the straight section of a cilium in successive frames back to a point of inter- section. For each species the mean estimated radius of arc is greater than the lengths of those cilia which could be measured accurately. The velocities are taken from the maximum movement observed between two successive frames during the effective stroke.
Angular velocities are more convenient for comparisons among species and calculation of clearance rates. The angular velocities plotted in Figure 2 are calculated by dividing the tip velocity by the radius of arc. For cilia which are held
o
$12 \
E E
8
Q_ .— 4
C. gigas = °
T. diomedea * A
N. obsoletus • o
Unid. *
* **»*»*,
*.' •
V- cB
20
60
100
140
RADIUS of ARC (jjm)
FIGURE 1. Maximum observed tip velocities of preoral cilia during their effective strokes versus radius of arc (estimate of cilium length) for veligers of Crassostrea gigas, Tritonia diomedea, Nassarins obsoletus and an unidentified prosobranch. Open circles are effective strokes associated with particle captures ; solid circles with no capture.
CILIARY FEEDING OF VELIGERS
527
140
o o>
T3
ro
> 60
20
C. gigas T. diomedea N. obsoletus Unid
.
«*•
t . 8
40 80
RADIUS of ARC (jum)
120
FIGURE 2. Maximum observed angular velocities of preoral cilia during their effective strokes (same larvae as in Figure 1). Open circles are effective strokes associated with particle captures ; solid circles with no capture.
straight during the effective stroke, the angular velocity is constant over almost the entire length of the cilium. Angular velocity may increase with cilium length (see below) but the trend is less striking than the increase of tip velocity with the length of the cilium (Fig. 1).
One-way analysis of variance of the angular velocities of the four species indicates significant differences among the species (Table II). A multiple com- parison among species indicates that the species fall into three significantly dif- ferent groups : C. gigas with the lowest mean angular velocity, T. diomedea in
TABLE II Comparisons among means for species. Ho: Means equal; reject if P < 0.001
|
Species |
Mean angular velocity (radians/ sec) |
(n) |
Mean ratio of velocities of cilium and particle (C/P) |
Range of (C/P) |
(n) |
|
Crassostrea gigas Tritonia diomedea Nassarius obsoletus Unidentified |
32 54 70 63 |
(19) (51) (25) (24) |
1.6 2.0 1.5 1.5 |
1.1-2.1 1.1-3.0 1.0-2.4 1.1-1.9 |
( 6) (36) (20) (21) |
|
Reject Ho (one way Anova) |
Reject Ho (Kruskal-Wallis test) |
||||
|
P < o.om |
P < 0.001 |
Groups by Student-Newman Keuls multiple comparison (reject Ho if P < 0.01): significantly different groups are (1) C. gigas, (2) T. diomedea, and (3) N. obsoletus and unidentified veliger.
528
R. K. STRATHMANN AND F, LEISK
TABLE III
Two sample rank tests for C/P (ratio velocities of cilium and particle) and W (angular velocity of cilium) Ho: Groups same; reject Ho if P < 0.05. Ho accepted for all texts (Mann-Whitney U, two /ailed).
|
Species |
Comparison |
(ni, ns) |
p |
|
Crassrstrea g/g«s |
Wat 12° Cand 20° C W for captures and misses C/P for captures and misses |
(24, 23) ( 3, 3) ( 3, 3) |
>0.20 >0.20 >0.20 |
|
Tritonia diomedea |
W for captures and misses C/P for captures and misses |
(19, 17) (19, 17) |
>0.20 >0.20 |
|
Nassarius obsoletus |
W for captures and misses C/P for captures and misses C/P for algae and 2 /xm spheres |
( 5, 16) ( 5, 16) (12, 9) |
>0.20 >0.05 >0.20 |
|
Unidentified |
C/P for algae and 2 jum spheres |
(16, 5) |
>0.20 |
the middle, and N. obsoletus and the unidentified veliger with the highest mean angular velocities (Table II). Preoral cilium length increases along with the angular velocity in these three groups. Thus among species angular velocities of preoral cilia may increase with the lengths of preoral cilia, but more species must be examined to confirm this trend. The observed differences in angular velocities may be associated with the species or the taxonomic order rather than the lengths of the preoral cilia. Also, the mean angular velocity of N. obsoletus is greater than for the unidentified prosobranch veliger which has much longer preoral cilia. The implications of the anomalously lower angular velocity in the unidenti- fied veliger are not obvious because this veliger was not observed to catch particles. It is nevertheless clear from these data that the small veligers are not compensating for the shorter length of preoral cilia with increased angular velocities.
Temperature could affect the rate of beat of cilia but we did not observe this. The mean angular velocity of the preoral cilia of C. giyas at about 20° C is only slightly greater than at 12 to 13° C, and the difference is not significant (Table III).
The preoral cilia in their effective strokes move one to three times faster than the nearby particles. We could not demonstrate that the ratio of cilium velocity to particle velocity (C/P) nries with the angular velocity of the cilium or the type of particle (Tables III, IV). In T. diomedea particle velocities increase and then decrease with the distance from the base of preoral cilium (Fig. 3). The velocities were measured for particles near the middle of the effective stroke of the preoral cilia. Since velocity of the cilium increases from the base to the tip, the ratio of cilium to particle velocity should increase near the tip, but we cannot demonstrate this (Table IV, C/P versus D). Because we cannot show the existence of these possible confounding factors, we have lumped all observations on C/P for each species in a test for differences among species. Differences in C/P among species are highly significant (Table II), but the cause of the difference is not clear. The species with a substantially different ratio of cilium velocity to particle velocity is T. diomedea, with a mean ratio of 2 as opposed to about 1.5 for
CILIARY FEEDING OF VELIGERS
529
u
0>
E E
co Q.
20 40 60
Distance from Cilium Base
80
FIGURE 3. Particle velocity versus distance from base of preoral ciliurn for veliger of Tritonia diomcdca. L — length of preoral cilium. R = portion blocked by recovery stroke.
the others. The preoral cilia of T. dioin-edea are of intermediate length. We can- not show that the ratio of cilium velocity to particle velocity changes in a regular way with cilium length, cilium speed, or food particle. Therefore, longer preoral cilia, which have angular velocities greater than or equal to those of shorter cilia, move more particles and presumably more water past the preoral band of cilia.
Clearance rate
The volume of water moving through the preoral band can be estimated from the above data. Our estimate combines the angular velocity (W), the ratio of cilium velocity to particle velocity (C/P), the length of the preoral cilium (L). and a correction for the part of the current blocked by the recovery stroke (R). Our reasoning is as follows. The pie-shaped area within an arc of one radian with
TABLE IV
Regression lines for C/P (ratio of velocities of cilium and particles) against W (angular velocity) or D (distance toward base from tip of cilium) Ho: slope equals 0; reject Ho if P < 0.05 (t-test, two tailed).
|
Species |
Comparison |
(n) |
P |
|
Crassostrea gigas |
C/P versus W C/P versus D |
(6) (6) |
> 0.50 o.K) > P > 0.05 |
|
Tritonia diomeden |
C/P versus W C/P versus D |
(36) (36) |
> 0.50 > 0.50 |
|
Nassarius obsoletus |
C/P versus W C/P versus D |
(21) (21) |
0.50 > P > 0.20 0.10 > P > 0.05 |
|
Unidentified |
C/P versus W C/P versus D |
(21) (21) |
0.50 > P > 0.20 0.50 > P > 0.20 |
530
k. k. STRATHMANN AND E. LEISE
TABLE V
|
L preoral |
R recovery |
V = (L2 - R2)WP/ |
F fraction |
FV clear- |
||
|
Species |
cilium length |
stroke cor- rection |
2C volume through preoral band |
caught out of n |
(n) |
ance rates (jim'/sec |
|
(Mm) |
(^m) |
(Mm3/sec Mm) |
particles |
Mm) |
||
|
Crassostrea gigas |
30 |
9 |
8,200 |
0.44 |
(13) |
3,600 |
|
Tritonia diomedea |
40 |
9 |
21,000 |
0.24 |
(36) |
4,900 |
|
Nassarius obsoletus |
70 |
9 |
110,000 |
0.15 |
(23) |
17,000 |
|
Unidentified |
100 |
9 |
210,000 |
|
1 |
|
radius equal to L is L2/2. Subtracting the smaller pie-shaped area blocked by the recovery stroke gives (L2-R2)/2. We assume that particle velocity equals water velocity, so this pie-shaped area times the angular velocity times a unit length of the velar edge divided by the ratio of cilium to particle velocities gives the volume of water passing a unit length of the preoral band per unit time as (L2-R2)WP/2C. This volume increases with the length of the preoral cilia (Table V).
This estimate includes only the water out to the tips of the cilia and so does not include all the water moved past the velar edge. Three sources of bias may enter this estimate of water flow. First, our measured radius of arc exceeds the length of the cilia, so estimates of angular velocity may be low. Second, we assume a constant ratio of cilium velocity to particle velocity along the cilium, whereas this ratio may increase near the tip. Third, some cilia may have been pushing particles faster than the water. The first underestimates water flow ; the second and third overestimate it. For the unidentified prosobranch veliger there is a fourth source of bias. Tips of its preoral cilia are often still bent in the recovery stroke as the base begins the effective stroke, so the effective length of the preoral cilium is less than indicated. The value of R is about the same for all species, as diagrammed in Figure 4, but could not be measured to the nearest ju.m. Also, R varies around the arc of the effective stroke. Despite these qualifications, the calculation gives an indication of the effectiveness of the cilia in moving water.
To convert this volume per time to an estimate of clearance rate, it is neces- sary to multiply by the fraction of passing particles which are actually captured. Particles further from the base of the cilia are less likely to be captured, but there is no sharply defined point beyond which no particles are captured and within which all are captured. In Figure 4 velar edges of three species are diagrammed to the same scale. The dashed lines indicate approximate paths of some particles, although the paths vary considerably even at the same distance from the base of the cilia. The films show no captures beyond the outer dashed line. Almost all the particles within the inner dashed line are captured. Particles also tend to travel across the arc described by a cilium. An effective length of cilium is there- fore not readily defined.
To obtain the fraction of passing particles which are captured, we recorded the total number of particles passing the preoral band in a filmed sequence of an actively feeding veliger for each species and noted the fraction captured (F, Table V). The sample of captures is small but the sample confirms our impression that the longer preoral cilia are less efficient at removing particles of this size from suspen-
CILIARY FKKDIM, OF VKI.K,KK>
531
C gigas
T.diomedea
N.obsoletus
', \ /
50 |Jm
FIGURE 4. Optical section across the velar edge diagrammed for three species to same scale. Preoral and postoral cilia indicated but food groove cilia omitted. Preoral cilium shown in several positions. Dashed lines mark two particle paths (see text).
sion. The clearance rate is estimated by multiplying the volume passing the cilia by the fraction of particles cleared. Though longer cilia are less efficient, there is a definite gain in rate of clearance per unit length of ciliated band with longer preoral cilia.
Mechanisms of concentrating particles
If particles are to be concentrated, they must be moved relative to the water. \Ye have no means of observing water movement in this study, but the observed motion of cilia and particle gives some information on the mechanism by which particles are concentrated. Several combinations of events could occur during concentration : the particles could move faster or slower than the water by either adhering to the cilia or being sieved by two adjacent cilia in their effective strokes. Our observations indicate that more than one mechanism of concentration could be operating.
The simplest hypothesis for concentration is that preoral cilia push adhering particles faster than the water during the effective stroke. In many captures, a preoral cilium overtakes a particle and then moves along next to it. This motion is consistent with the hypothesis that the cilium is pushing an adhering particle faster than the water. Captures occur in which no cilia are observed to touch the particles, but a capturing cilium may have been out of focus.
532 K. k. STRATHMANN AND I-;. I.KISE
Particles reverse their direction and move from posterior to anterior into the food groove beneath the preoral cilia in the recovery stroke (Fig. 4). It seems unlikely that the particles are concentrated when they are held or moved against a posterior current here, but we cannot rule out the possibility.
Particles might also be concentrated by a sieve formed by adjacent cilia in their effective strokes. In four captures by the Tritonia diomedea veligers two cilia in their effective strokes were separated by an angle of less than 11°. The pair of cilia move next to a particle during part of the effective stroke and could be pushing it faster than the water. Since the gap between the cilia is less than the diameter of the particle, the pair of cilia may be forming a sieve. Because the depth of focus is small, these pairs of cilia may be immediate neighbors. Captures of this sort were not observed with veligers of Crassostrea gigas and Nassarius obsoletits.
Pairs of cilia separated by a small angle are not visible in most capture sequences. Adjacent preoral cilia are often separated by a large angle in their effective strokes. In such cases the metachronal wavelength rather than the dis- tance between neighboring cilia must set the pore size of the sieve. The preoral cilia of Nassarius obsoletits were filmed in a plane perpendicular to the plane of the effective stroke as well as parallel to it. The gap between preoral cilia in the same position of the effective stroke exceeded 20 /mi in most instances, whereas these veligers capture particles of a diameter less than 10 /mi. For veligers of this size the sieve formed by the metachronal wave is only effective for rather large particles, if it is effective at all.
Another hypothesis is that particles are retained by a sieve composed of both the preoral and postoral cilia in the latter part of their effective strokes. Under this hypothesis water in the current generated by the effective strokes would be squeezed out past the cilia of both bands at then end of the effective strokes when the tips of the opposing cilia are close together. We observed a postoral cilium tip meeting a preoral cilium tip in only one of the frames examined. Observed closest approach between preoral and postoral cilium tips in N. obsoletus is about 15 /mi, which is about twice the diameter of the captured particles. The actual distances may be somewhat less, however, because the closest approach can fall in the interval between frames of the film.
The films therefore indicate that the spacing of cilia does not set the minimum size for captured particles and that other mechanisms must be operating either in addition to such sieving or instead of it. The hypothesis of weak adhesion of cilia and particles is consistent with observed movements during captures ; the lower frequency of capture of particles nearer cilium tips where differences in velocities are greatest ; and the lower efficiency of capture with longer preoral cilia of equal or higher angular velocities. However, direct evidence for adhesion is still lacking.
The role of the postoral cilia in capturing particles is not clear. Occasionally particles enter the food groove past the postoral band but most captured particles do not follow this route. Yet Strathmann ct al. (1972) found that captures by a serpulid trochophore ceased when the postoral band ceased beating but the preoral band continued beating. Possibly the postoral band is necessary for retaining particles but does not aid in concentrating them. Captured particles reverse their direction of motion where the preoral cilia end their effective stroke. The captured
CILIARY FEEDING OF VELIGERS 533
particles then move anteriorly into the food groove beneath the preoral cilia in their recovery stroke. The recovery strokes of the preoral cilia may be insufficient by themselves to carry the particles into the food groove and keep them there. The current from the postoral cilia may help retain particles during capture and subsequent transport toward the mouth.
DISCUSSION
Our studies of three species of veliger larvae suggest that shorter preoral cilia clear particles from the water current more efficiently than longer preoral cilia but produce lower clearance rates per unit length of velar edge. This lower clear- ance rate is the result of both shorter cilia and lower angular velocities. However, larvae with longer preoral cilia may capture small particles less efficiently than large particles. Our observations with small flagellates may therefore underestimate the advantages associated with longer preoral cilia. We are not aware of any studies comparing length of preoral cilia to egg size in species of veligers, but our impression, from past casual observations and the three species reared from eggs in this study, is that veligers from larger eggs tend to have longer preoral cilia when they begin feeding. Our tentative conclusion is that veligers from smaller eggs have both a shorter velar edge and a lower clearance per unit length of velar edge, so their maximum clearance rates are lower.
The techniques of this study could be extended to more species and a greater range of sizes of food particles. A broader comparison could establish quantitative relationships between easily measured traits (cilium length and length of velar edge) and feeding capabilities (maximum clearance rate and efficiency of clearance of particles of different sizes). This would permit comparisons of feeding capabilities of larvae in numerous species which would test hypotheses on costs associated with small egg size and geographic shifts in developmental strategies.
Strathmann et al. (1972) , using Harris' (1961) argument, speculate that with preoral cilia, which are each composed of a bundle of numerous individual cilia, cilium length could vary independently of cilium velocity. There seems to be no physical necessity for higher angular velocities with longer preoral cilia. Observa- tions on more species are therefore needed to establish the trend found with the species studied here.
Rubenstein and Koehl (1977) have categorized the mechanisms by which sus- pension feeders concentrate food particles. Our observations of veligers indicate direct interception of a particle by a fiber, with the preoral cilium as the fiber. Our best guess from these limited observations is that preoral cilia overtake the particles in the latter part of the effective stroke and weakly adhere to them, pushing them faster than the water. Possibly two preoral cilia sometimes act together as a sieve. The postoral cilia probably help retain particles but do not necessarily play an important role in concentrating particles.
The veligers in this study were maintained at comfortable temperatures during filming, but they were confined on a microscope slide, exposed to bright light, and fed unnaturally high concentrations of particles. They could have reduced their rate of clearance by some means which was not detected. Bayne (1976) calculates a clearance rate of 10.000 to 15.000 ^mVsec per /j.m of velar edge for the veliconcha
534 K- K. STRATHMANN AND E. LEISE
of Mytilus edit/is. This value is based on measurement of the velum and on clearance rates calculated from rates of ingestion by unconfined veligers in a known concentration of Isochrysis yalbana. Bayne's values fall between those determined here for T. diomcdea and N. obsoletus. The shell length of 250-ju.m for the M. ednlis veliconcha falls between those of early stage T. diomcdca and N. obsoletus, so Bayne's values appear to be in close argeement with our estimates, although the length of preoral cilia of M. cdulis veligers at this stage is not given. In the worst case, if the preoral cilia of the M. ednlis veliconcha are about the same as in the early stage C. gigas veligers, then our estimates of clearance rates could be one third to one half the maximum rate found by Bayne. In either case, Bayne's result indicates that our values are not far from maximum clearance rates for unconfined veligers.
Strathmann (1971) calculated clearance rates of 5000 to 10,000 /xnr/sec per ^m of ciliated band for echinoderm larvae feeding on Amphidinium carleri. This flagellate is somewhat larger than Monochrysis luthcri. The cilia of these larvae are shorter than the preoral cilia of C. gigas veligers, and the feeding mechanism is different, but the rates are in rough agreement with those determined here. As in Bayne's study, the estimate for the echinoderm larvae is based on ingestion rates of unconfined larvae.
Ratios of preoral cilium velocity to particle velocity in veligers are similar to ratios reported by Sleigh and Aiello (1972), who found ratios of about 1.6, 3.3, and 4.0 near the cilium tips of Pleurobrachia pilens comb plates, Mytilus edulis gill lateral cilia, and Stentor polymorphus membranelles. The plot of particle velocity against distance from cilium base for the veliger of T. diomedea is similar to the plots of Sleigh and Aiello for Stentor and Pleurobrachia, except that the velocities decrease more abruptly beyond the tips of the T. diomedea preoral cilia.
This study was supported by NSF grant OCE-7818608. R. L. Fernald, J. Lewin, B. Masinovsky, and T. E. Schroeder provided some of the organisms and equipment used. Space was provided at the Friday Harbor Laboratories.
SUMMARY
1. Beat of preoral cilia and particle paths were filmed for veligers of Crassostrea gigas, Tritonia diomcdca, Nassarius obsoletus and an unidentified prosobranch. Particle captures were filmed for the three identified species.
2. Clearance rates per unit length of velar edge are estimated from the equation (L2-R2)WPF/2C, where L is cilium length, R a correction for recovery stroke, W angular velocity, C/P the ratio of velocities of cilium and particle, and F the fraction of particles captured. The clearance rates are in rough agreement with Rayne's values for veligers of Mytilus cdulis.
3. In the three identified species, longer preoral cilia clear particles at a higher rate but with less efficiency. Since veligers from larger eggs generally have both longer preoral cilia and a longer velar edge, a larger egg generally produces a veliger with a higher maximum clearance rate when the veliger begins to feed.
CILIARY FEEDING OF VELIGERS 535
4. Angular velocities increase with ciliuni length in the three identified species of veligers but the larger unidentified species did not continue this trend.
5. Preoral cilia in their effective strokes move 1 to 3 times faster than particles travelling in about the same arc with a mean of about 1.5 times the speed of the particles. In mid effective stroke, the ratio of velocities of cilia and particles is not significantly different for captured and non-captured particles, nor does the ratio vary significantly with angular velocity of ciliuni. The ratio does vary significantly among species.
6. Particles passing closer to the base of the preoral cilia are more likely to be captured.
7. We hypothesize that suspended particles are concentrated when they are overtaken by preoral cilia in their effective stroke, weakly adhere to the preoral cilia, and are pushed faster than the water. Capture is completed when particles are drawn into the food groove, probably by the action of the recovery stroke of preoral cilia, the current from postoral cilia, or both.
LITERATURE CITED
BAYNE, B. L., 1976. The biology of mussel larvae. Pages 81-120 in B. L. Bayne, Ed., Marine
Mussels: their Ecology and Physiology, Cambridge University Press, Cambridge,
London. CLONEY, R. A., J. SCHAADT, AND J. V. DURDEEN, 1970. Thermoelectric cooling stage for the
compound microscope. Ada Zool (Stockh.), 51: 95-98. COSTELLO, D. P., M. E. DAVIDSON, A. EGGERS, M. H. Fox, AND C. HENLEY, 1957. Methods
for Obtaining and Handling Marine Eggs and Embryos. Marine Biological Laboratory,
Woods Hole, Massachusetts, 247 pp.
FRETTER, V., 1967. The prosobranch veliger. Proc. Malacol. Soc. Land., 37 : 357-366. HARRIS, J. E., 1961. The mechanics of ciliary movement. Pages 22-36 in J. A. Ramsey, V. B.
Wigglesworth, Eds., The Cell and the Organism. Cambridge University Press,
Cambridge, England. KEMPF, S. C., AND A. O. D. WILLOWS, 1977. Laboratory culture of the nudibranch Tritonia
diomedca Bergh (Tritoniidae : Opisthobranchia) and some aspects of its behavioral
development. /. Ex p. Mar. Biol. Ecol, 30 : 261-276. QUAYLE, D. B., 1969. Pacific oyster culture in British Columbia. Bull. Fish. Res. Bd. Can.
169: 1-192. RUBENSTEIN, D. L, AND M. A. R. KOEHL, 1977. The mechanisms of filter feeding : some
theoretical considerations. Am. Nat., Ill : 981-994. SCHELTEMA, R. S., 1967. The relationship of temperature to larval development of Nassarius
obsoletus (Gastropoda). Biol. Bull.. 132: 253-265. SLEIGH, M. A., AND E. AIELLO, 1972. The movement of water by cilia. Acta Protosool., 11 :
265-278.
STRATHMANN, R. R., 1971. The feeding behavior of planktotrophic echinoderm larvae : mecha- nisms, regulation, and rates of suspension feeding. /. 7:.r/>. Mar. Biol. Ecol., 6 :
109-160. STRATHMANN, R. R., T. L. JAHX, AND J. R. C. FONSECA, 1972. Suspension feeding by marine
invertebrate larvae : clearance of particles by ciliated bands of a rotifer, pluteus, and
trochophore. Biol. Bull.. 142 : 508-519.
THOMPSON, T. E., 1959. Feeding in nudibranch larvae. /. Mar. Biol. Assoe. U.K.. 38: 239-248. WERNER, B., 1955. Uber die Anatomic, die Entwicklung und Biologic des Veligers und der
Veliconcha von Crepidula fornienta L. (Gastropoda Prosobranchia) . Helgoland.
Uf'iss. Meeresunters., 5: 169-21 7.
Reference: Biol. Bull. 157: 536-547. (December, 1979)
IRREVERSIBLE NONGENETIC TEMPERATURE ADAPTATION OF
OXYGEN UPTAKE IN CLONES OF THE SEA ANEMONE
HALIPLANELLA LUCIAE (VERRILL)
WILLIAM E. ZAMER AND CHARLOTTE P. MANGUM Department of Biology, College of William and Mary, Williamsburg, Virginia 23185
For many years physiological differences have been observed among latitudinally separated populations of the same species (Bullock, 1955 ; Prosser, 1955 ; Vernberg, 1962). These differences have been attributed to a variety of phenotypic adapta- tions and also to genetic variation.
One of the earliest examples of physiological variation was the difference in acutely measured bell pulsation rates of the jellyfish Aurelia aurita collected from Nova Scotia and Florida (Mayer, 1914), the direction of which agreed with Krogh's (1916) prediction of increased rates in cold-adapted animals compared to warm-adapted animals, when the rates are measured at a common temperature. More recent studies of oxygen uptake as well as motor activity in cnidarians con- firmed the existence of distinct responses in latitudinally separated populations within a species (Sassaman and Mangum, 1970; Mangum, Oakes, and Shick, 1972;Shick, 1976).
In many examples of intraspecific variation, latitudinal differences disappear with acclimation to common conditions, indicating their phenotypic origin. In several studies of latitudinally separated populations however, the differences persist despite prolonged exposure to common conditions, which has been interpreted alternatively as genetic divergence and "irreversible nongenetic adaption" (Kinne, 1962). There are numerous examples of variation which is highly likely to have a genetic basis, e.g. interspecific variation (Vernberg, 1962). The possibility of nongenetic but irreversible adaptation within species is less certain.
Evidence of this phenomenon in Drosophila subobscura was presented by Smith (1956), who induced variation in heat tolerance among groups of flies raised at different temperatures. The differences, which did not disappear completely upon acclimation to a common temperature, were adaptive in character ; flies developing at the higher temperature survived longer at an upper lethal limit. However, selec- tion, operating during development, could conceivably explain these results. Kinne (1962) reported that fish developed from eggs which had been transferred to a different salinity 3 to 6 hr after spawning exhibit lower food conversion efficiencies relative to controls developed from eggs which remained in the spawning salinity. He does not, however, compare food conversion efficiencies of fish acclimated to a common salinity which had developed at different salinities. It is not clear, therefore, whether the observed differences in food conversion efficiencies are reversible upon acclimation to common conditions. Bradley (1978) showed that copepods raised at 20° C are more tolerant of high temperatures than those raised at 10° C. However, these results could have arisen from selection during rearing or to a thermal acclimation that requires more than two days' exposure to a common
536
IRREVERSIBLE TEMPERATURE ADAPTATION 537
temperature to disappear. Schneider (1968) concluded that differences in accli- mated oxygen uptake rates among populations of the crab Rhithropanopeus harrisi can be reversed only in part by breeding the crabs at a common temperature, but the full report of these data has not appeared.
The mode of reproduction in the actinian Haliplanella luciae (Verrill) offers an interesting approach to the question of nongenetic adaptation which is irreversible within a period of prolonged acclimation to common conditions. This species re- produces largely by asexual mechanisms, longitudinal fission being the most common (Shick and Lamb, 1977). The fission process in Haliplanella luciae and the effects of temperature and other environmental factors on it have been studied in detail by several authors (Torey and Mery, 1904; Davis, 1919; Uchida, 1932; Miyawaki, 1952; Minasian, 1976). Longitudinal fission can be induced in the laboratory by storing the animal at 10° C or below for 6 to 8 weeks, followed by raising the temperature above 10° C (Sassaman, personal communication). This treatment apparently simulates increasing water temperatures during spring. After fission the progeny regenerate tissues to close the body wall torn in the process. Thus isogenic clones of animals can be raised under different environmental condi- tions and later compared, virtually eliminating the possibility of genetic variability among the experimental animals.
In the present study, three generations of isogenic anemones were reared at two temperatures, and their rates of oxygen uptake compared after acclimation to common thermal conditions. A few observations were also made on the electro- phoretic banding patterns of five enzymes and on the dimensions of the gas exchange surface.
MATERIALS AND METHODS
Collection, maintenance of animals and experimental design
Specimens of Haliplanella luciae (Verrill) were collected from the York River estuary (15 to 21#c) at the mouth of Indian Field Creek, Virginia in January 1977 HO0 C). Included in this sample were animals of Uchida's (1932) color types 1 and 3, indicating genetic heterogeneity in the population. Assuming all animals to be genetically unique, single individuals were placed in 30-ml plastic beakers filled with York River estuary water. One half of the animals were placed at 28° C and the remainder stored at 18° C. After approximately 2 weeks the animals used at both temperatures had undergone fission, each original individual producing two or more progeny ('clonemates). When regeneration was complete, the animals were transferred to 5° C for 6 to 8 weeks. At the end of this period, the clonemates were separated and allowed to undergo fission and subsequent forma- tion of new tissue at different developmental temperatures: one member of each clone was randomly chosen and placed at 18° C and its clonemate was placed at 28° C. After fission and regeneration, the second generation clonemates were separated, stored at 5 ° C for 6 to 8 weeks and then returned to their developmental temperature, where a third generation was produced. Thus there are four groups of animals according to developmental temperature and the number of generations produced at a given temperature : two consist of animals taken from the field that
538
\Y. K. /AM KM AN'D C. I'. M AN GUM
Tempe ratur e (°C)
IS
Collection Site IO°C
Temperature (C)
28
18
28
18
M LJ U
I I I I
A A B B
I I I
till
B > »>
28
^
A
/ \ / \
/ \
B B
\ / H
\
FIGURE 1. Cloning of H. luciac at 18° and 28° C. The example shows two animals (A and B) assumed to be genetically unique. Solid arrows indicate transfer from one temperature to another ; dotted arrows indicate fission and regeneration. Roman numerals indicate generation.
divided and formed new tissue three times at either 18° or 28° C; one consists of animals that divided first at 18° C and a second and third time at 28° C, and one of the animals that divided a second and third time at 18° C. Because of the difference in the number of fissions at each developmental temperature (Fig. 1), a control was necessary to permit pooling the data for all clones at a particular developmental temperature. The oxygen uptake rates for animals which divided twice at a developmental temperature were compared with those for animals which had divided three times at that developmental temperature. The important aspect of the experimental design is, however, that each individual has at least one clonemate treated in the same way but given the alternative thermal regime (Fig. 1), and that each of the four experimental groups contains members of the same clones, in the same proportions. Thus the rearing procedures cannot have permitted selection.
Development and growth occurred at the specified temperature in York River estuary water (15 to 2l(/ce, with members of all groups given the same salinity regime). At least 4 days prior to the measurement of oxygen uptake, the salinity was brought to 18#c. Animals were fed an excess of freshly hatched Artcmia nauplii every other day.
Oxygen uptake measurements
Oxygen uptake (Vo2) °f the third generation clonemates was measured at the developmental temperature (TD), the same as the acclimation temperature
IRREVERSIBLE TEMPERATURE ADAPTATION 539
(TA). These data are designated TD = 18° C TA and TD = 28° C == TA. Other, but isogenic, individuals were transferred to the alternative temperature, held there for 2 to 4 weeks and oxygen uptake measured. These data are designated TD == 18° C TA == 28° C and TD == 28° C, TA == 18° C. All measurements were made in millipore filtered water at \Sf/(c salinity by adjusting the concentration of natural York River estuary water (15 to 2\%c) with dis- tilled water or commercial sea salt (Dayno Corp). Animals were starved for 60 to 84 hr before an experiment, and transferred to the respirometry chambers the night before the measurement to permit attachment to the wall of the chamber. Oxygen depletion was measured with a Yellow Springs Instrument Co. Model 53 Biological Oxygen Monitor, using a high sensitivity (0.0005 in) teflon membrane and a 1 X 10 mm stirring bar. The animals were permitted to adjust to stirring for 1 hr before sealing the chamber and the data were discarded when the tentacles were retracted before completion of the measurement. All data in the Po2 interval 110 to 159 mm Hg were analyzed. For each experiment a measure- ment of the oxygen uptake of the electrode was made using an empty chamber, and this value was subtracted from the experimental rate.
At the end of an experiment animals were stimulated to expel fluid from the gastrovascular cavity, removed from the chambers, lightly blotted and weighed to the nearest tenth of a milligram (Calm Electrobalance, Model G).
Electro phoresis
Animals were starved for one week and then frozen whole, either singly or in groups from the same clone and treatment group, in two volumes of buffer: 0.05 M tris (titrated to pH 8 with concentrated HC1) and 0.001 M EDTA to which NADP (4 mg/liter) was added. Freezing took place quickly in a bath of dry ice and acetone, and the samples were then stored at —70° C until homogenized. Samples were thawed, homogenized and centrifuged. When necessary the super- natants were diluted as much as 50% with distilled water to obtain 25 /j.1 which were loaded into slots of horizontal starch gels ( 13% w/v ; Sigma starch).
Gel and electrode buffers for glucose-6-phosphate dehydrogenase (G-6-PDH, E.C. 1.1.1.49) and isocitrate dehydrogenase ( IDH, E.C. 1.1.1.42) were modified from Markert and Faulhaber (1965) : 0.9 M tris, 0.5 M boric acid, 0.02 M EDTA diluted 1 : 20 for the gel buffer and 1 : 6 for the electrodes ; in addition, 30 mg and 10 mg NADP were added to the gel and to the cathodal buffer tray, respectively. The gel and electrode buffers for malate dehydrogenase (MDH, E.C. 1.1.1.37) were those described by Nichols and Ruddle (1973). The discontinuous LiOH buffer system of Selander, Hunt, and Yang (1969) was used for phosphoglucose isomerase (PGI, E.C. 5.3.1.9) and hexokinase (HK, E.C. 2.7.1.1.). The period of electro- phoresis was 3 to 4 hr. After electrophoresis at 350 volts, gels were sliced and stained with slight modifications of the procedures described by Brewer (1970) and Shaw and Prasad (1970). The gels were preserved overnight in 50% ethyl alcohol, blotted and stored in plastic wrap until photographed.
Gas exchange surface area
Animals from the TD =: 18° C TA and the TD == 28° C = TA treatment were anesthetized with MgCK Tentacles were counted and the external
540 W. E. ZAMER AND C. P. MANGUM
surface areas of the body wall and the tentacles were approximated from measure- ments made at 10 to 25 X, using formulas for the frustum of a cone and for a cylinder, respectively.
Statistical analyses
Mean Vo2 values were computed by determining the rate during each time interval of the continuous record of the change in oxygen concentration. Thus, unless specifically stated otherwise, the N value given in the results represents the sum of all data obtained from the members of an experimental group, which are treated as a homogenous population.
The comparison was complicated, however, by the different body sizes of the experimental groups (see below). Two alternative procedures were used to eliminate differences due to body size from the conclusions. First, a regression coefficient of —0.43 was used to correct rates to a common weight of 1.6 mg, a representative value, by covariance analysis. This coefficient was determined by performing a regression analysis on data from the TD = 18° C = TA treatment group (n = 14 animals, r — 0.75, P < 0.001). This relationship is homogenous, showing no evidence of the discontinuity observed by Shick, Brown, Dolliver, and Kayar (1978). Second, the data were grouped by weight classes established from an analysis of frequency distributions, and the comparison made within the classes.
Although the experimental design permits analysis of the data as paired observa- tions, the available procedures for computing Student's t require an equal number of observations in each member of the pair, a condition which was not met. The element of genetic homogeneity was maintained in the experimental design by keep- ing virtually constant the percent of the total number of observations made on each clone under each combination of developmental and experimental temperatures.
RESULTS
Effect of developmental temperature on body size
At 28° C fission occurred more rapidly and more frequently than at 18° C, resulting in more numerous but smaller progeny (see below). As suggested earlier by a number of investigators (Ray, 1960; Mangum, 1963; Shick, 1972) this finding suggests that the often observed phenomenon of a larger body size in the colder regions of a species range results from latitudinal differences in develop- mental temperature.
The frequency distribution of weight in each experimental group (Fig. 2) indicates, in general, that body size of the animals is more widely distributed in the 18° C developmental group than in the 28° C developmental group. Almost 73% of the animals in the TD = 28° C = TA treatment group weigh 1.1 mg or less, and 91% of the animals in the TD == 28° C, TA -- 18° C experimental group weigh 2.0 mg or less. In contrast, only 29% of the animals in the TD =: 18° C = TA group and 17% of those in the TD =: 18° C, TA == 28° C treatment group weigh 1.1 mg or less. In these groups, reared at low temperature, greater fre- quencies of animals in the higher weight classes are also evident (Fig. 2). A
IRREVERSIBLE TEMPERATURE ADAPTATION
541
2
TD= I8"C TA- I8°C
0.7 1.0 1.3 1.6 1.92.22.52.8 3.7 I 2~ 3 4 5
TD = I8°C TA =28°C
0.7 1.0 1.3 1.6 1.9 2.22.52.8 3.7 1234 5
o
E
TO = 28°C TA = 28°C
0.7 1.0 1.3 1.6 1.9 2.2 2.52.8 3.7 2
I
34 5
Body We i g ht (m g) Weight Gloss No
TD - 28"C TA - I8°C
0.7 1.0 1.31.6 1.92.22.52.8 3.7 4
I
234 5
Body Weig ht (mg) Wei g h t Class No.
FIGURE 2. Frequency distribution of body weight in treatment groups, designated by developmental (TD) and acclimation (TA) temperature.
comparison of the frequency distributions by weight class of the two developmental temperature groups emphasizes the trends (Fig. 3). The mean body weight of the TD == 28° C TA and TD == 28° C, TA == 18° C animals is 1.4 mg; the mean weight of the two TD =: 18° C groups is 2.0 mg. The median weight class of the two TD = 28° C groups is 0.6 to 0.8 mg, and the corresponding figure for the TD = 18° C groups is 2.1 to 2.3 mg.
Oxygen uptake
Three clones of animals which first divided at 18° C and one clone of animals which first divided at 28° C were used to measure Oo uptake; all clones produced third generation animals at both developmental temperatures. No significant dif- ferences were found between the control groups (P > 0.85) indicating that VO2 in clones that have produced two generations at a particular developmental tem- perature is the same as in clones that have produced three generations at that developmental temperature.
542
W. E. ZAMRR AND C. P. MANGUM
9r I — l
o
E
j/> 5 o
E c
< 3
6
z
0.7 1.0 1.3 1.6 l_9 2.22.52.8 3.7 1234 5
Body Weight (mg)
Weight Class No. 7r
B
o
E
< 3
o
z
0.7 1.0 1.3 1.6 1.92.22.52.8 Body Weight (mg)
3.7
0.7 1.0 1.3 1.6 1.92.22.52.8 Body We ight (mg)
37
FIGURE 3. Frequency distribution of body weight in (A) all animals used in the oxygen uptake experiment, (B) TD = 28° C developmental groups only, and (C) TD = 18° C develop- mental groups only.
The mean weight corrected oxygen uptake rates of the two TD = 18° C groups are significantly higher (P < 0.01) than those of the two TD = 28° C groups at both acclimation temperatures (Table I). At the test temperature of 18° C the rates in the TD =: 18° C groups are 18% higher than those of the TD == 28° C groups, and at 28° C the rates are 19% higher. However, using the raw data for the smaller animals produced at 28° C and the larger ones produced at 18° C. the difference diminished to 4% at the test temperature of 18° C and it is actually reversed at 28° C (—8%). Thus a confounding influence of developmental temperature, its effect on body size, may obscure the direct effect on the acclimated rate of oxygen uptake.
The frequency distribution by weight of all animals used in the oxygen uptake experiments is shown in Figure 3. When the rates are compared by weight class, significant differences between the TD -- 18° C and the TD = 28° C groups are
IRREVERSIBLE TEMPERATURE ADAPTATION
543
TABLE I
A. Oxygen uptake (nl/g-hr) in isogenic specimens of Haliplanella luciae reared for two to three gener- ations at different temperatures. Mean ± s.e. (N). Probability values from one-way analyses of vari- ance comparing developmental temperature groups at each test (= acclimation) temperature.
|
Test temperature (°C) |
Body wt. (mg) |
Developmental temperature (°C) |
P |
|
|
18 |
28 |
|||
|
18 |
1.9 ± 0.2 |
243.7 ± 12.0 |
235.0 ± 16.2 |
n.s. |
|
28 |
1.3 ± 0.1 |
(HI) 487.1 ± 24.1 |
(72) 524.7 ± 21.6 |
U.S. |
|
(36) |
(54) |
B. Oxygen uptake (pl/g*hr) corrected to a common body weight (1.6 mg) by covariance. Data analysis as above.
|
Developmental temperature (°C) |
|||||
|
Test temperature (°C) |
P |
||||
|
18 |
Qio |
28 |
Qio |
||
|
18 |
257.0 ± 9.4 |
218.7 ± 13.9 |
P < 0.001 |
||
|
(HI) |
(72) |
||||
|
1.99 |
1.96 |
||||
|
28 |
510.3 ± 22.6 |
427.8 ± 14.5 |
0.0018 |
||
|
(36) |
(54) |
found in one of the four possible comparisons at 18° C and in two of three at 28° C (Table II). The trend of higher rates in the TD -- 18° C groups is uniformly consistent, but the probability levels rise due to the smaller number of observa- tions (N).
TABLE 1 1
Weight corrected oxygen uptake rates (^l/g-hr), Mean ± s.e. (N). Probability (P) values from one- way analyses of variance comparing the developmental temperature group rates at each experimental (= acclimation) temperature for each weight class.
|
Experimental |
Weight class |
Developmental temperature (°C) |
|||
|
temperature |
p |
||||
|
(°C) |
No. |
Range (mg) |
18 |
28 |
|
|
18 |
1 |
(0.6-0.8) |
270.5 ± 46.2 (10) |
194.5 ± 33.2 (19) |
n.s. |
|
2 |
(0.9-1.1) |
269.8 ± 38.0 (11) |
146.6 ±41.4 (7) |
0.0499 |
|
|
3 |
(1.2-1.7) |
218.6 ±22.7 (23) |
159.1 ± 21.8 (16) |
n.s. |
|
|
4 |
(1.8-2.0) |
— |
288.5 ± 18.7 (24) |
— |
|
|
5 |
(2.1-3.7) |
254.7 ± 11.2 (67) |
258.8 ± 4.1 (6) |
n.s. |
|
|
28 |
1 |
(0.6-0.8) |
— |
444.0 ± 22.8 (20) |
— |
|
2 |
(0.9-1.1) |
413.3 ± 38.1 (6) |
417.4 ± 27.3 (20) |
n.s. |
|
|
3 |
(1.2-1.7) |
568.9 ± 13.8 (5) |
408.3 ± 37.7 (9) |
0.003* |
|
|
4 |
(1.8-2.0) |
608.9 ± 34.3 (13) |
439.9 ± 24.1 (5) |
0.01 |
|
|
5 |
(2.1-3.7) |
427.4 ± 31.6 (12) |
|
|
* Bartlett's Test indicates non-homogenous variances for the two developmental groups of this weight class. A Separate Variance Estimate /-test was used to make this comparison.
544
W. E. ZAMER AND C. I'. MANGUM
TABLE III
Gas exchange surface area (cm-/g wet wt.) -in each developmental temperature group of Haliplanella luciae. N = Number of animals. Weight classes as in Table II.
|
Tentacle surface area |
|||||
|
Developmental temperature (°C) |
Weight class |
X |
No. tentacles |
||
|
cmVg |
% Total body- surface area |
||||
|
18 |
1 |
2 |
13 |
5.6 |
33.9 |
|
2 |
2 |
16 |
5.0 |
57.5 |
|
|
3 |
3 |
20 |
7.6 |
42.7 |
|
|
28 |
1 |
2 |
18 |
19.2 |
73.5 |
|
2 |
4 |
30 |
14.7 |
72.9 |
|
|
3 |
3 |
29 |
18.6 |
78.7 |
Qio values for both developmental temperature groups (Table II) are similar, and they also approximate the values reported previously over the same tempera- ture range (Sassaman and Mangum, 1970). Thus, no evidence of a change in temperature sensitivity with developmental temperature is seen. No overall pat- tern of temperature sensitivity is apparent in the data grouped by weight class, thus supporting previous conclusions that it is not correlated with body size (Sassaman and Mangum, 1970). Nor is there a trend in Qw with developmental temperature.
Gas exchange surface
A possible explanation of the irreversible difference between cold and warm- reared anemones is a change in gas exchange surface area. This hypothesis would be supported by an increase in tentacular surface, the primary site of oxygen uptake in epifaunal anemones (Sassaman and Mangum, 1972; Shick ct al., 1978) in the TD =: 18° C groups. In fact, measurements of gas exchange surface (Table III) indicate the opposite relationship, suggesting that the difference would be even larger if the gas exchange surface had remained the same.
Surface area is not correlated with body size. Pooling data for all weight classes at each developmental temperature, anemones in the TD = 28° C groups have more tentacles and a significantly larger (Mann- Whitney U-Test) total surface area available for gas exchange than anemones in the TD = 18° C groups. Not only are the tentacle numbers greater in the TD = 28° C groups, but the average surface area per tentacle is greater as well. Moreover, the percent of the total body surface area contributed by the tentacles always exceeds 73 in the TD -- 28° C groups, compared to a maximum of 57.5% in the TD = 18° C animals (weight class 2).
Electrophoresis
The enzymes examined showed no qualitative differences in banding pattern either among the separate clones of one treatment group or among clonemates of different treatment groups. The results for all of the enzymes tested are similar
IRREVERSIBLE TEMPERATURE ADAPTATION 545
and they suggest that the population of H. luciae at Indian Field Creek exhibits no variation at these loci. Of the five enzyme systems examined, only IDH was clearly polymorphic, the banding pattern suggesting fixed homozygosity at two loci. However, no qualitative differences in banding pattern were detected, either between clones or within clones. Since these loci differ in latitudinally separated populations and within populations of H. luciae (Shick and Lamb, 1977), the uniform pattern could reflect conservative selection pressures on these loci for this particular population. Regardless, there is no evidence that the different alleles were either induced or repressed by the formation of tissues at different temperatures.
DISCUSSION
Differences in aerobic metabolism that cannot be reversed by 2 to 4 weeks acclimation to common conditions clearly result from reproduction, regeneration and the formation of new tissues at different temperatures, when the effects of other variables are eliminated from the data. The direction of the change is generally compensatory, resulting in higher metabolic rates in the animals pro- duced at low temperatures. The magnitude of the change, however, is not great enough to override opposing effects of developmental temperature on body size and gas exchange surface area. Oxygen uptake rates go up at low developmental temperature but down with increasing body size and decreasing tentacular surface, and body size increases and tentacular surface area decreases at low developmental temperature. The net outcome of the concomitant and counteracting trends is little or no difference between the two groups, when body size and gas exchange surface are retained as variables.
The question remains of the origin of the metabolic difference in animals of the same size. One alternative is a direct but irreversible effect of developmental temperature on the metabolic machinery within the cell. The five enzyme systems examined were selected in part for their variability in the species (and in other anemones; Manwell and Baker, 1970), and in part for their relation to aerobic metabolism. Hexokinase, phosphoglucose isomerase and isocitrate dehydrogenase vary in different populations of H. luciae (Shick and Lamb, 1977) ; isocitrate dehydrogenase and malate dehydrogenase catalyze reactions in the tricarboxylic acid cycle and glucose-6-phosphate dehydrogenase regulates a branchpoint of the pentose phosphate pathway. No qualitative changes with developmental tem- perature or acclimation temperature were found for any of the five enzymes tested. Quantitative data, on various enzyme systems, however, are not available. Robert and Gray (1972) have shown an increase in the specific activity of glucose-6-phos- phate dehydrogenase and 6-phosphogluconate dehydrogenase of the blue crab Callinectes sapid iis during cold exposure, accompanied by no qualitative electro- phoretic changes. A thorough test of this possibility, which we regard as a promising hypothesis, would entail an exhaustive investigation of polymorphic enzyme systems that influence the rate of oxidative reactions, directly or indirectly.
The only obvious alternative explanation would be an irreversible effect of developmental temperature on the gas exchange system. Since fluid movements on both sides of the tentacles are generated by cilia, an irreversible difference in oxygen convection would seem to be highly unlikely. An irreversible change in gas
546 W. E. ZAMER AND C. P. MAN GUM
exchange surface was detected, but its effect on oxygen uptake should he the opposite of that observed. Thus a systemic explanation seems highly unlikely.
Regardless of its basis, the present findings clearly demonstrate the reality of irreversible physiological adaptation, and they raise the possibility that the compensatory responses found earlier in geographically separate populations of the species may result from developmental as well as acclimation temperature.
We are grateful to R. J. Hoffmann for the use of his facilities and expertise in performing the electrophoreses, and to D. Reed for his help with data analysis.
SUMMARY
1. Isogenic clones of //. Inciac were raised at each of two developmental tem- peratures, 18° and 28° C. Despite prolonged acclimation to common thermal con- ditions, oxygen uptake rates differ according to the temperature of reproduction, regeneration and development.
2. The effects of developmental temperature, however, are masked by body size differences. Only when this variable is eliminated can the underlying effect of developmental temperature be detected.
3. The irreversible change is not due to an increase in the gas exchange surface area at the primary site of Oo uptake, the tentacles.
4. No qualitative changes in banding patterns for five enzymes ( HK, PGI, IDH. MDH, G-6-PDH) were found.
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INDEX
Ablation of antennules in lobster, effect > on
food odor orientation in, 366 Absorption properties of Platymonas contain- ing high levels of chlorophyll b, 370 Acetylcholine receptors, effects of azobenzene
on, 356
Acetylcholinesterase, ascidian larval, develop- ment in absence of mitochondrial localiza- tion, 344
Acid cells, sulfuric, in tunic of colonial ascid- ian, 464 Actin gene-containing plasmid from Droso-
pltila uiclanogastcr, 367
Actin myofilaments, in copepod muscle, 112 ADAMS, J. A. Morphogenesis in grafted Hydra attcnuata : positive dominance, negative dominance, and pattern regulation, 356 Adaptation, of saltmarsh bacteria, 375 for reproduction, in Nucella crassUabrum,
453 temperature, of oxygen uptake in Hali-
planclla Inciac, 536
ADEJUWON, C. A. See E. F. Couch, 364 ADEJUWON, C. A., S. J. SEGAL, AND S. S. KOIDE. Alternate pathways in the bio- synthesis of testosterone by Rana pipiens, 356
ADEJUWON, C. A. See S. J. Segal, 393 Aeolidia papillosa, response of Anthopleura to
predator attacks by, 138 Aggregation factor, effect of, on sponge cells,
373
AKABANE, S. See M. Komatsu, 258 ALBERTE, R. S. See A. M. B. Hogan, 371 ; B. A. Hayhome, 370; D. L. Gustafson, 371 ; L. Mazzella, 382 ; M. A. Frey, 368 ; and T. A. Kursar, 376 ALKON, D. L. See E. W. Stommel, 397 Amino acid receptors in catfish taste, 396 transport by marine polychaetes, 434 transport in toadfish liver, 388 Ammonincation of soil in hardwood and pine
forests, 386
Amphipod nutrition, importance of bacterial hiomass, fungal biomass, and Spartina detritus in, 389
ANDERSON, P. See C. Serhan, 394 Anemone, defensive mechanisms of, 138 Anglerfish islet tissue, synexin-like factor in,
Aiuiiiillci, population studies on, in the Great Sippewissett salt marsh, 368
Ant/uilla rostrata, cytology of the gill of, 104
Anoxia, tolerance of, in Callianassa, 125
Anoxic conditions, effects on amino acid trans- port in marine polychaetes, 434
Antibody, tubulin, induces microtubule de- polymerization, 373
Anthopleura elegantissima, defensive mech- anisms against nudibranch attacks, 138
Apl\sia calijornica, growth and reproduction,
"407 mariculture of, 360
ApJysia pacemaker neuron, synaptic hyper- polarization in, 399
Appendage removal, effect on molting and growth in crayfish, 182
Arbacia eggs, phosphorylation of protein after fertilization in, 357
Arbacia embryos, phagosomes from, 375
Arbacia punctulata, transcription in nuclei of, 365
Arbacia, translational regulation of histone synthesis in, 367
Arsenazo III in liposomes, for study of cal- cium ionophores, 394
Ascidian, budding, colony and spicule forma- tion in, 464
larval tail muscle determination and dif- ferentiation, 344
Ascomycete, Laminaria infected by an, 392
Asexual reproduction in clonal cultures of HaliphmcUa luciae, 478
Astarte, characterization of hemoglobin in, 403
Asterina minor, breeding and development of, 258
Asterias aniurcnsis, oocyte susceptibility to polyspermy, 249
Asterina pectinifcra, oocyte susceptibility to polyspermy, 249
ATEMA, J. See A. Stewart, 396; B. Bryant, 360; C. Derby, 365; D. Devine, 366; E. Karnofsky, 374 ; and P. Reilly, 391
ATWOOD, H. L. See M. P. Charlton, 361
AUDESIRK, T. E. A field study of growth and reproduction in Aplysia californica, 407
Ausio, J. AND K. E. VAN HOLDE. Character- ization of Spisula sperm protamine, 357 Ausio, J. See L. Herlands, 370 Axenic culture, of Moina, 234 Azobenzene compounds, effects on acetylcho- line receptors of skate, 376
548
INDEX
549
Bacterial biomass, importance in amphipod
nutrition, 389
BAKER, E. See M. Winkler, 402 Balanus improvises, photoresponse of nauplii,
166
Balanus, particle size selectivity in, 386 BALLINGER, D., S. PETERSON, AND T. HUNT. Phosphorylation of the 40s ribosomal sub- unit after fertilization of Arbacia punctu- lata eggs, 357
BAUER, G. E. See H. B. Pollard, 389 Behavior, in hydrozoan colonies, 189
of Limuhts larvae, in reference to lunar and
tidal rhythms, 494
BELL, E., N. NAKATSUJI, AND S. SHER. Dif- ference between SDS-PAGE patterns of Labyrinthula slimeways and vegetative cells, 358
BELL, E. See N. Nakatsuji, 384 BENNETT, M. V. L. See M. S. Cappell, 360;
and R. B. Hanna, 370 BERG, C. J., Jr. See T. R. Capo, 360 Bioluminescence, development of, in Rcnilla,
506
Biomechanics of crawling, in RIcIanipus, 306 Biosynthesis of testosterone, by Rana pipiens,
356
BIRD, D. J. AND A. F. EBLE. Cytology and
polysaccharide cytochemistry of the gill of
the American eel, Anguilla rostrata, 104
Blood of spider crabs, major constituents of,
221 BONE, S. Conduction and dielectric studies of
protein-methylglyoxal complexes, 358 Boonca, ectoparasitism of, 320 BORON, W. F. See J. M. Russell, 392 BOURNE, G. B. Pressure-flow relations in the perfused systemic circulation of squid, 358 BOYLE, M. B., L. B. COHEN, AND E. R. MA- CAGNO. The numbers and sizes of cells in molluscan ganglia ; simultaneous optical recording of activity from many neurons, 359 BOYNTON, J. E. Optimal prey selection of
Littorina littorea by green crabs, 359 Brachiolaria sea star lacking pelagic life, 258 Brain tubulin, purification of, from dogfish
and skate, 377
Breeding assemblage of Astcrina, 258 BRIGGS, R. P. Fine structure of musculature in the copepod Paranthessius anemoninc Claus, 112 Brown algae and diatoms, pigment-protein
complexes from, 371 BROWN, J. See D. Kew, 375 BROWN, S. B. See R. F. Troxler, 400
BRYANT, B. AND J. ATEMA. Chemoreception in catfish : effects of diet on behavior and body odor, 360
BRYANT, B. See A. Stewart, 396 Budding in a compound ascidian, 464
Calcium ionophores, arsenazo III in liposomes, for the study of, 394
Callianassa jamaiccnsc, respiration in, 125
Callichirus jamaiccnsc, respiration in, 125
Calmodulin, a protein in microvilli, 372
CAMPBELL, R. D. See H.-T. Lee, 288
CAPPELL, M. S., D. C. SPRAY, D. H. HALL, A. J. SUSSWEIN, AND M. V. L BENNETT. Motor fields of pharyngeal motoneurons in Naranax, an opisthobranch mollusc, 360
CAPO, T. R., S. E. PERRITT, AND C. J. BERG, JR. New developments in the mariculture of Aplysia californica, 360
Carcimts maenas, salt and water balance in, 422
Catfish, chemoreception and social behavior,
360 amino acid receptors in, 396
Centriole-like structures, association with the marginal band of a molluscan red cell, 363
CHARLTON, M. P., C. S. THOMPSON, AND H. L. ATWOOD. lonophore induced sodium loading of nerve terminals : a model for long term facilitation of transmitter re- lease, 361
Chemical search image in lobsters, 365
Chemoreception, in catfish social behavior, 360 in lobster, responses to secondary plant com- pounds, 391
CHILTON, B. S., M. R. LAUFER, AND S. V. NICOSIA. Some biochemical properties of an RNA-instructed DNA polymerase in developing sea urchin (Lytcchhnis pictus) embryos, 362
Chimeras, hydra, development and behavior of, 288
Chlorophyll b, absorption properties of Platy- monas containing it, 370
Chloroplasts, RNA synthesis in, 401
Chlorotetracycline fluorescence in Hacinantlius cell division using image intensification, 402
Chondrus, gametophytes and sporophytes of, 387
Choriogonadotropin-like substances from a microorganism and a crab, 380
Cilia feeding mechanisms, clearance rate of, in molluscan veligers, 524
Ciliary swimming in planula larvae, 478
550
INDEX
Ciliogenesis, membrane particle arrays in Tet-
niliyincini during, 372 ('uniti, tidal rhythm in tin- neural gland of,
378
Cirripedia, photoresponses in, 166 Cladocerans, effects of dietary fatty acid and
temperature on productivity of, 234 Clara sqnainata. evoked responses to electrical
stimulation of, 189
Clava, three conducting systems in, 396 Clearance rates, of molluscan veligers, 524
COBURN, M., S. SCHUEL, AND W. TROLL. Hy-
clrogen peroxide release from sea urchin eggs during fertilization : importance in the block to polyspermy, 362
COHEN, L. B. See M. B. Boyle, 359
COHEN, W. D. AND I. NEMHAUSER. Associa- tion of centriole-like structures with the marginal band of molluscan red cell, 363
COHEN, W. D. See I. Nemhauser, 384
Colonial reorganization and spicule formation in a tunicate colony, 464
Conducting systems, electrical, in colonial hy-
droids, 189 three, in Clava, 396
Conduction studies of protein-methylglyoxal complexes, 358
Contractile proteins, in Labyrinthula slime- ways, 358
Copepods, diapausing eggs of, 297 fine structure of musculature in, 112
Coptopteryx viridis, parthenogenesis in, 445
CORNELL, J. C. Salt and water balance in two marine spider crabs, Libinia emargin- ata and Pugettia producta. I. Urine pro- duction and magnesium regulation, 221
CORNELL, J. C. Salt and water balance in two marine spider crabs, Libinia emargin- ata and Pugettia producta. II. Apparent water permeability, 422
CORSON, D. W. AND A. FEIN. Dependence of discrete wave frequency on pH in Limulus ventral photoreceptors, 363
COSTOPULOS, J. J., G. C. STEPHENS, AND S. H. WRIGHT. Uptake of amino acids by ma- rine polychaetes under anoxic conditions, 434
COUCH, E. F., C. A. ADEJUWON, AND S. S. KOIDE. Production and metabolism of steroids in Hoinanis aincricanus, 364
Coupling resistance associated with structural changes in the septate axon of crayfish, 370
Crabs, spider, apparent water permeability in, 422
GRAIN, W. R. See D. S. Durica, 367
( 'rassfistrca (fig as, feeding mechanisms of, 524
Crawl-step, stage of locomotion in Mclampus, 306
Crayfish, septate axon of, coupling resistance associated with structural change in, 370
CROW, T. AND N. OFFENBACH. Response specificity following behavioral training in the nudibranch mollusk, Hermissenda crassicornis, 364
CUKIER, M., G. A. GUERRERO, AND M. C. MAGGESE. Parthenogenesis in Copto- pteryx I'iridis, Giglio Tos (1915) (Dyc- tioptera, Mantidae), 445
Cyclic nucleotides, effects on hyperpolarization of Aplysia pacemaker neuron, 399
Cystodytcs lobatus, growth, budding, and spicule formation in, 464
Cytochemistry, of eel gill, 104
Cytology, of eel gill, 104
Cytoskeleton, in Labyrinthula slimeways, 384 proteins in squid axoplasm, 384
D
D'ABRAMO, L. R. Dietary fatty acid and tem- perature effects on the productivity of the cladoceran, Moina macrocopa, 234
DAVIS, L. M. /;; vitro transcription in nuclei from Arbacia punctulata, 365
Defensive strategy of Anthoplcura against nudibranch predators, 138
Denitrification, soil, using the acetylene block method, 404
DENTLER, W. L. See K. A. Suprenant, 398
DERBY, C. AND J. ATEMA. Chemical search image : prey exposure improves selective chemical detection by a predator (Hoina- rus aincricanus) , 365
DERBY, C. See P. Reilly, 391
Development of Asterina minor, 258 of neuroeffector function in RcniHa, 506 pattern, of Nucclla, 453
temperature, effects on size and oxygen up- take in a sea anemone, 536
DEVINE, D. AND J. ATEMA. Effects of uni- lateral antennule ablations on food odor orientation in Hoinanis aincricanus, 366
Dielectric studies of protein-methylglyoxal complexes, 358
Dietary fatty acid and temperature effects on Moina, 234
Differentiation, ascidian larval muscle, in ab- sence of mitochondria! localization, 344
Diapause, in a copepod, 297
Disassembly of squid brain tissue neurofila- ments, 403
Discrete waves, in Lhnulus ventral photore- ceptors, 363
INDEX
551
Diurnal response of the gray seal, Halichoenis, to tide and insolation, 387
DNA polytnerase in embryos of Lytechinus,
362 repetition in sea urchin genome, recombinant
studies in, 395
sequence homologies, between sea urchin DNA and a Drosophila- actin gene-con- taining plasmid, 367
Dogfish eye lenses, compared to human eye lenses, 404
Drosophila, actin gene-containing plasmid from, 367
DUNCAN, R. AND T. HUMPHREYS. All the poly (A) ( + )mRNA sequence complex- ity also occurs in poly (A) ( — )mRNA in sea urchin embryos, 366
DURICA, D. S., J. A. SCHLOSS, AND W. R. GRAIN. Studies on actin mRNA-compli- mentary genomic DNA sequences in the sea urchin, 5". purpuratus, 367
Dyes with negative reduction potentials, effect on mitosis, 379
Dynetic trematodes, life cycles of, 397
E
EARL, C. AND T. HUNT. Studies of the trans-
lational regulation of histone synthesis in
Arbacia punctulata, 367 EBLE, A. F. See D. J. Bird, 104 Ecosystem, forest, bacterial denitrification in,
404
Ectoparasitism of Boonca and Fanjint, 32(1 Eel gill cytology, 104 Egg production, by Hippa pacifica, 205 EISEN, A. AND S. INDUE. Temperature-de- pendent timing of mitosis and cleavage in
Lytechinus raricgatus, 367 Electrical pulses in Clara squainata, 189 Electron charge transfer and the living state,
398 spin resonance studies of protein-methyl-
glyoxal complexes, 369 Embryos, Lvtcchinus, DNA polymerase in,
362 Xucclla, feeding and rate of development in,
453 Endotoxin, interaction with Li in ill us amebocyte
lysate, 392 Erythrocytes, newt and chick, marginal bands
in, 395 Euglena, fluorescence studies of protochloro-
phyll in, 368
Eye lenses, human and dogfish, 404 Eyestalk removal, effect on molting and
growth in crayfish, 182
Eye tissues of dogfish, photosensitizing action of psoralens in, 379
Facilitation of transmitter release by sodium, 361
Fargoa, ectoparasitism of, 320
FARMANFARMAIAN, A. See R. Socci, 396
Feeding behavior, of hydra chimeras, 288 mechanisms and clearance rates of molluscan veligers, 524
FEIN, A. See D. \V. Corson, 363
FELDER, D. L. Respiratory adaptations of the estuarine mud shrimp, Callianassa jamai- ccnsc (Schmitt, 1935) (Crustacea, Deca- poda, Thalassinidea), 125
Fertilization, hydrogen peroxide as block to
polyspermy in sea urchin eggs, 362 self, in a sea-star, 258
Fibronectin, plasma, and dogfish phagocytes, 401
FIORE, J. See N. Penncavage, 387; and S. Schatz, 392
Fission, regulation of, in the sea anemone, Haliplanclla luciae, 478
Fluorescence studies of living cells, image in- tensification in, 391 of photochlorophyll(ide) in Euglena, 368
FORD, T. AND E. MERCER. Population density, size distribution, and home range of the American Eel (Anyuilla rostrata) in the Great Sippewissett salt marsh, 368
FORWARD, R. B. JR. See W. H. Lang, 166
Food odor orientation, effects of antennule ablation in lobster on, 366
Foot structure and function, of Mclitnipits. 306
FREY, M. A., R. S. ALBERTE, AND J. A. SCHIFF. Studies by fluorescence of proto- chlorophyll(ide) and its phototransforma- tion in dark-grown Euglena gracills var. bacillaris, 368
FRIEDMAN, A. L. See D. L. Gustafson, 371
FUJIMORI, T. AND S. HIRAI. Differences in starfish oocyte susceptibility to polyspermy during the course of maturation, 249
FUJIWARA, K. See S. Inoue, 373
Fungal biomass, importance in amphipod nu- trition, 389
FUSARO, C. See A. M. Wenner, 205
(IALLARDO, C. S. Developmental pattern and adaptations for reproduction in Nucella erassihihruni and other muricacean hosts,
453
552
IXDKX
Gametophytes of Chondrus, 387
GASCOYNE, P. R. C. Electron spin resonance
studies of protein-methylglyoxal com- plexes and model systems, 369 Giant axon, squid, transmembrane movements
of sulfur compounds in, 390 Gill structure, of the American eel, 104 GILSON, M. See B. D. Teague, 399 Glial-axonal protein transfer in squid giant
axon, 400 Glugca hcrtwigi, intracellular parasite of
smelt, 334
GOLDMAN, A. E. See R. V. Zackroff, 403 GOLDMAN, R. D. See R. V. Zackroff, 403;
and I. Nemhatiser, 384 GOODMAN, E. See C. Serhan, 394 Grafted H\dra attcnuata, morphogenesis in,
356 Great Sippewissett salt marsh, population
studies of Auguilla in the, 368 GREENBERG, S. Is there tubulin in spiro-
chaetes, 369 Green crabs, selection of Littorina littorea as
prey, 359 Growth, and reproduction of meiofauna in
selected natural microfloral assemblages,
378
in a compound ascidian, 464 in Procambarus clarkii, 182 of Aplysia in the field, 407 GUERRERO, G. A. See M. Cukier, 445 GUILLARD, R. R. L. See B. A. Hayhome, 370 GUSTAFSON, D. L., A. L. FRIEDMAN, M. S.
RUDNICK, H. LVMAN, AND R. S. ALBERTE.
Light-harvesting pigment-protein com- plexes from brown algae and diatoms : im- plications for the organization of the photosynthetic unit, 371
H
HAAS, G. See L. Mastroianni, 381
Hacmanthus cell plate formation, chlorotetra- cycline fluorescence in, using image in- tensification, 402
Halichoerus, diurnal response of, to tide and insolation, 387
Haliplanella luclae, regulation of fission activ- ities in, 478
Haliplanella luciae, temperature adaptation of oxygen uptake in, 536
HALL, D. H. See M. S. Cappell, 360
HANNA, R. B., G. D. PAPPAS, AND M. V. L. BENNETT. Structural changes associated with increased coupling resistance in the septate axon of the crayfish, 370
HARRIS, L. G. AND N. R. HOWE. An analysis of the defensive mechanisms observed in
the anemone Anthoplcura clcgantissima in response to its nudibranch predator Aeo- lidia papillosa, 138 HASCHEMEYER, A. E. V. See R. W. Math-
ews; and R. Persell, 388 HAYHOME, B. A., J. A., SCHIFF, R. R. L. GUILLARD, AND R. S. ALBERTE. Absorp- tion properties of Platymonas sp. Rey 2 containing a high proportion of chloro- phyll b, 370 Hemoglobin, characterization of, in Astartc,
403
HEPLER, R. K. See S. M. Wolniak, 402 HERLANDS, L. AND J. Ausio. The major pro- tein in Spisula sperm nuclei is a prota- mine, 370
Hermaphroditic sea-star, Asterina minor, 258 Hermissenda, training response in, 364 Hippa pacifica, sampling bias and population
structure, 205
HIRAI, S. See T. Fujimori, 249 Histochemistry, acetylcholinesterase localiza- tion in ascidian larvae, 344 of regeneration, in lobster claw muscle, 386 Histology, of Aplysia gonad, 407 Histone synthesis in Arbacia, translational
regulation of, 367
HOGAN, A. M. B., D. MAUZERALL, AND R. S. ALBERTE. Water relations and photosyn- thetic characteristics of the tall and short ecophenes of Spartina alt erni flora, 371 Homarus americanns, mating behavior in, 374 Horohalinicum, Nereis biology in the, 153 HOROWITCH, S. See J. Smith, 395 HOSKIN, F. C. G. See R. D. Prusch, 390 Host preferences of Boonea and Fargoa, 320 HOWE, C. L. AND M. S. MOOSEKER. Brush border calmodulin : A structural protein of the microvillus core, 372 HOWE, N. R. See L. G. Harris, 138 HUFNAGEL, L. A. Fairy rings : membrane particle arrays present during early stages of dc noi'o ciliogenesis in Tetrahymena, 372 Human eye lenses, compared to dogfish lenses,
404
HUMPHREYS, S. AND T. S. REESE. Structure of aggregation factor on sponge cells and in gels, 373
HUMPHREYS, T. See R. Duncan, 366 HUNT, T. See D. Ballinger, 357; C. Earl,
367; and M. Winkler, 402 Hydra attcnuata, development and behavior of
an intergeneric chimera of, 288 morphogenesis in grafted, 356 Hydrogen peroxide, block to polyspermy dur- ing fertilization of sea urchin eggs, 362
INDEX
553
Hydroids, contraction pulses in, 189 Hyperpolarization, synaptic, in Aflysia pace- maker neurons, 399
IBERS, J. See G. Weissmann, 401
Image intensification in fluorescence studies of
living cells, 391
using chlorotetracycline fluorescence in Haemanthus, 402
Induction, in grafted Hydra attenuate,, 356
Inhibition of mitosis by dyes with highly nega- tive reduction potentials, 379
INOUE, S., K. FUJIWARA, AND E. D. PAPA- FRANGOS. Tubulin antibody induces micro- tubule depolymerization in vivo and in vitro, 373
INOUE, S. See A. Eisen, 367
Ion regulation, in spider crabs, 221
Islet tissue, anglerfish, synexin-like factor in, 389
JACKSON, W. T. See S. M. Wolniak, 402
K
KALMIJN, A. J. See B. D. Teague, 399
KANO, Y. T. See M. Komatsu, 258
KARNOFSKY, E., AND J. ATEMA. Field and laboratory observations of lobster mating behavior, 374
Karyograms, of mantid chromosomes, 445
KAZNOWSKI, C. See D. Kew, 375
KELLER, T. C. Ill, AND L. I. REBHUN. Prop- erties of polymerizable tubulin from iso- lated Spisula spindles, 374
KEW, D., C. KAZNOWSKI, AND J. BROWN. Preparation and characterization of phago- some membranes from Arbacia punctulata embryo cells, 375
KIRCHMAN, D. AND R. MITCHELL. Adapta- tion of bacteria to rapidly changing en- vironmental conditions, 375
KOIDE, S. S. See C. A. Adejuwon, 356; E. F. Couch, 364; T. Mauro, 380; and A. Momii, 383
KOMATSU, M., Y. T. KANO, H. YOSHIZAWA, S. AKABANE, AND C. OGURO. Reproduc- tion and development of the hermaphro- ditic sea-star, Asterina minor Hayashi, 258
KROUSE, M., H. LESTER, AND M. WEINSTOCK. How photoisomerizable azobenzene com- pounds affect acetylcholine receptors of skate muscle, 376
KUHL, D. L. tion and succinea 153
KURSAR, T.
ALBERTE. synthetic 376
AND L. C. OGLESBY. Reproduc- survival of the pilewonn Nereis in higher Salton Sea salinities,
A., D. MAUZERALL, AND R. S. Characteristics of the photo- unit in macrophytic red algae,
Labidocera aestiva, population biology of, 297
Labyrinthula slimeways, 358, 384
LAMBERT, G. Early post-metamorphic growth, budding and spicule formation in the compound ascidian Cystodytcs lobatus, 464
Lainiiiaria infected by an ascomycete, 392
LANDOWNE, D. AND V. SCRUGGS. Slow changes in the magnitude of the potassium current associated with changes in the in- ternal perfusion solution in squid axons, 377
LANE, J. M. AND J. M. LAWRENCE. The effect of size, temperature, oxygen level, and nutritional condition on oxygen uptake in the sand dollar Mellita quinqmesper- forata (Leske), 275
LANG, W. H., R. B. FORWARD, JR., AND D. C. MILLER. Behavioral responses of Balanus iinproristis nauplii to light intensity and spectrum, 166
LANGFORD, G. M., L. E. LYN-COOK, AND D. ROBBINS. Phosphocellulose purification of dogfish and skate brain tubulin, 377
Larval development, in a pennatulid coelenter- ate, 478
Limiilus behavior, 494
LASER, R. J. See J. R. MORRIS, 384
LASER, R. J. See M. Tytell, 400
LASH, J. W., M. OVADIA, C. H. PARKER, AND C. N. SVENDSEN. Tidal rhythm and tis- sue organization in the neural gland of Ciona intestinalis: correlates with cellu- lose and fibronectin, 378
LASH, J. W. See G. Weissmann, 401
LAUFER, M. R. See B. S. Chilton, 362
LAWRENCE, J. M. See J. M. Lane, 275
LEE, H.-T. AND R. D. CAMPBELL. Develop- ment and behavior of an intergeneric chimera of hydra (Pehnatohydra oligactis interstitial cells : Hydra attcnuata epi- thelia cells), 288
LEE, J. J. AND M. J. LEE. The growth and reproduction of selected species of meio- fauna in selected natural microfloral as- semblages, 378
LEISE, E. See R. R. Strathmann, 524
554
INDEX
l.en> cap.sule*. dogii.sh, interaction of liposomes \\itli, 383
Lepocreadium areolatum, lite-cycle of, 397
LKRMAN, S., J. M. MEGAW, AND Y. TAKEI. The photosensitizing action of psoralens in dogfish ocular tissues, 379
LEHMAN, S. See J. M. MEGAW, 383
LESTER, H. See M. Krouse, 376
LEVIN, J. See F. R. Rickles, 392
Leydig cells in mice, testosterone production of, 380
LHRH decapeptide, effect on steroidogenesis in Rana pipicns, 393
Lil'inia cntarc/inata, salt and water balance in, 221, 422
Life-cycle of Lepocreadium urcolatuin, 397
Light, effects on electrical activity in Clara,
189 responses of larval Limit! us, 494
Liniitlus amebocyte lysate, interaction with _ endotoxin, 392 " larval behavior, 494
ventral photoreceptors, discrete waves in, 363
LIPMAN, D. AND S. ZIGMAN. Possible mecha- nisms for inhibition of cellular activity by dyes with highly negative reduction po- tentials, 379
Liposomes, arsenazo III in, for study of cal- cium ionophores, 394 interaction of, with dogfish lens capsules, 383
Littorina littorca, prey of green crabs, 359
Living state and electron charge transfer, 398
[.UNAS, R., M. SUGIMORI, AND S. SlMON.
Presynaptic calcium current and post- synaptic response generated by a pre- synaptic action potential, a voltage clamp study, in the giant squid synapse, 380 Lobster, claw muscle, histochemistry of re- generation of, 386 chemoreception in, response to secondary
plant compounds, 391 effects of antennule ablations in, on food
odor orientation, 366 steroid production in, 364 chemical search images in, 365 Locomotion, in a pulmonate snail, 306 Locomotion, in Limuliis larvae, effects of
lunar and tidal periodicities in, 494 LORAND, L. See G. Weissmann, 401 Lunar and tidal periodicity of Limnlits larvae
emergence, 494
LYMAN, H. See D. L. Gustafson, 371 LYMAN, H. See L. Mazzella, 382 LYN-COOK, L. E. See G. M. Langford, 377 iiiHs, DNA polymerase in embryos of,
complexity of polysomal RNA in, 366
effects, of temperature on timing of mitosis in, 367
M
MACAGNU, E. R. See M. B. Boyle, 359 Macrophytic red algae, phycobilisomes in, 376 MAGGESE, M. C. See M. Cukier, 445 Magnesium regulation in spider crabs, 221 Magnetic field, effects on migration rate of
mud bacteria, 399
MANGUM, C. P. See W. E. Zamer, 536 Mantids, asexual reproduction in, 445 MARCUS, N. H. On the population biology and nature of diapause of Labidocera acstiva ( Copepoda : Calanoida), 297 Marginal bands, and perioles in marine ani- mal blood cells, 384 centriole-like structures in, in molluscan red
cells, 363
in newt and chick erythrocytes, 395 Mariculture of Aplysia califoniica, 360 MARISCAL, R. N. See L. L. Minasian, Jr.,
478 Marplivsa siiin/iiinin. uptake of amino acids in,
434
MASTROIANNI, L., JR., S. V. NICOSIA, E. STREIBEL, AND G. HAAS. Studies on urn cell complexes of Sipunculus mid us (Lin- naeus) : influence of physiologic and pathologic mammalian sera on mucus secretion, 381
MATHEWS, R. W. AND A. E. V. HASCHE- MEYER. Effects of extreme temperatures on protein synthesis in the toadfish, 381 Mating behavior in lobster, 374 MAURO, T., A. R. SEGAL, AND S. S. KOIDE. Stimulation of testosterone production by mouse Leydig cells with factors isolated from a microorganism and Ovalipes occl- hitus, 380
MAUZERALL, D. AND D. WITTENBERG. The size of the photosynthetic unit and its turnover time in various seaweeds, 382 MAUZERALL, D. See A. M. B. Hogan, 371 ; L. Mazzella, 382; S. Schatz, 392; and T. A. Kursar, 376
MAU-LASTOVICKA, T. See R. Robertson, 320 MAZZELLA, L., D. MAUZERALL, H. LYMAN, AND R. S. ALBERTE. Photosynthetic char- acteristics of Zostcra marina L. (Eel grass), 382
MCCANDLESS, E. See N. Penncavage, 387 MCLAUGHLIN, J. A. See R. Pethig, 388
INDEX
Mechanical stimuli, transmission by statocyst cilia, 397
MEGAW, J. M., S. LERMAN, AND Y. TAKEI. Interaction of liposomes with dogfish lens capsules, 383
MEGAW, J. M. See S. Lerman, 379
Meiofauna, growth and reproduction of, in selected natural microfloral assemblages, 378
IfcIatupHs bidentatus, locomotion in, 306
MELILLO, J. M. See C. M. Zacks, 404; and K. C. Parsons, 386
Mcllita quinquiesperforata, effect of size, tem- perature, oxygen level, and nutritional condition on oxygen uptake, 275
Membrane particle arrays during ciliogenesis in Tctraliymcna, 372
MERCER, E. See T. Ford, 368
MERCURO, J. See R. Socci, 396
Metabolic inhibitors, effects on amino acid
transport in marine polychaetes, 434 rates, in Callianassa, 125
Metal, heavy, effects on gut function in toad- fish, 396
Metamorphosis, in a pennatulid coelenterate,
478 of Asterina minor, 258
Methylglyoxal, protein complexes of, 358
MgCla, effects on electrical activity in Clava, 189
Microfloral assemblages, growth and repro- duction of meiofauna in, 378
Microscopy, electron, of isolated chloroplasts,
401
electron, of Labyrinthula slimeways, 384 light, of eel gill, 104
SEM and fluorescence, of dogfish lens cap- sules, 383
TEM of copepod muscle, 112 TEM and SEM of Arbacia phagosomes,
375 TEM of sponge aggregation factor, 373
Microsporida, infecting Osincrus niorda.r, 334
Microtubules, association with pancreatic se- cretion granules, 398
Microtubule depolymerization, induced by tubulin antibodies, 373
Microvillar protein, calmodulin, 372
MILLER, D. C. See W. H. Lang, 166
MILSTED, A. See I. Nemhauser, 384
MTNASIAN, L. L., JR., AND R. N. MARISCAL. Characteristics and regulation of fission activity in clonal cultures of the cosmo- politan sea anemone, Haliplanella liiciac (Verrill), 478
MITCHELL, R. See D. Kirchman, 375
Mitochondria, localization and segregation in
ascidian embryos, 344 Mitosis, effects of temperature on timing of,
in Lytechinus, 367 inhibition of, by dyes with highly negative
reduction potentials, 379 Modiolus, particle size selectivity in, 386 MOFFETT, S. Locomotion in the primitive
pulmonate snail Mclanifits bidentatus:
foot structure and function, 306 Moina uuicrocopa, productivity of, 234 Mole crabs, sex ratio and modal size classes of,
205 Molgula arenata, development of tail muscle
acetylcholinesterase in, 344 Molluscan ganglia, comparison of buccal and
circumesophageal ganglia, 359 optical recording in, 359 Molluscan parasites, host preferences, 320 Molluscan red cell, centfiole-like structures
in association with marginal bands in, »
363 Molting and growth of the crayfish, Procam-
banis clarkii, 182 MOMII, A. AND S. S. KOIDE. Nicotinamide
deamidase activity in oocytes of Spisuhi
solidissima, 383 Monensin induced sodium loading, of nerve
terminals, 361
MOOSEKER, M. S. See C. L. Howe, 372 Morphogenesis, in grafted specimens of Hydra
attcnuata, 356 determinant of ascidian larval tail muscle
acetylcholinesterase, 344 Morphology, of hydra chimeras, 288 MORRIS, J. R. AND R. J. LASEK. Differential
solubilities of cytoskeletal proteins in squid
axoplasm, 384
Motor fields, of pharynx, in Xai'ana.v, 360 Mud bacteria, migration rate as a function of
magnetic field, 399 Muricaceans, developmental patterns and
adaptations for reproduction in, 453 MURRAY, A. See J. Smith, 395 Musculature, fine structure of, in Paranthes-
sius anemoniae, 112
Muscular development, in a pennatulid coe- lenterate, 506 Myosin myofilaments, in copepod muscle, 112
N
XAKATANI, I. AND T. Oxsu. The effects of eyestalk, leg, and uropod removal on the molting and growth of young crayfish, Procambartts clarkii, 182
NAKATSUJI, N. AND E. BELL. Cytoskeletons in Labyriiithula slimeways, 384
556
INDEX
\.\KATSUJI, N. See E. Bell, 358
Nassarius chsolctus, feeding mechanisms of, 524
A"<u'<;;;(7.r, motor fields in pharynx of, 360
NEMHAUSER, I., W. D. COHEN, A. MILSTED, AND R. D. GOLDMAN. Marginal band systems in blood cells of marine species : visualization by indirect immunofluores- ence, 384
NEMHAUSER, I. See W. D. Cohen, 363
Nereis succinct], survival in, in Salton Sea salinities, 153
Nerve terminals, Monensin induced sodium loading of, 361
Neural gland of dona, tidal rhythm in, 378
Neuroeffector development in a pennatulid coelenterate, 506
Neurofilaments, squid brain, in vitro reas- sembly of, 403
NICOSIA, S. V. AND J. SEWINSKI. Ultra- structure of urn cell complexes of Sipuncu- Ins n ud us (Linnaeus) before and after serum-induced mucus release, 385
NICOSIA, S. V. See B. S. Chilton, 362; and L. Mastroianni, 381
Nicotinamide deamidase activity in oocytes of S pi sul a, 383
Nitrification of soils in hardwood and pine forests, 386
NOE, B. D. See H. B. POLLARD, 389
Nuclear protein, in Sfisula, 357
Nuclei, from Arbacia punctnlata, transcription in, 365
NuccUa crassilabrum, reproduction in, 453
Nudibranch, prey-preference hierarchy, in- fluence of anemone defenses on, 138 training response in Hermlssenda, 364
Nutrition, of Moina, 234
of saltmarsh invertebrates on Spartinn detritus, 390
Nutritional condition, effect on oxygen uptake, in a sea star, 275
o
OFFENBACH, N. See T. Crow, 364
OGLESBY, L. C. See D. L. Kuhl, 153
OGURO, C. See M. Komatsu, 258
OLSON, D. H. Particle size selectivity in Modiolus demissus and Balanus bala- noidcs, 386
Oocyte, maturation in starfish, 249
susceptibility to polyspermy, in starfish, 249 of Spisula, nicotinamide deamidase activity in, 383
Opsanus, temperature effects on protein syn- thesis in, 381
Optical recording in molluscan ganglia, 359
Osincrus mordu.v, intracellular parasite of, 334 Osmoregulation of Nereis in Salton Sea
salinities, 153
OTSU, T. See I. Nakatani, 182 OVADIA, M. See J. W. Lash, 378 Oxygen uptake, effect of environmental con- ditions on, in a sea star, 275 temperature adaptation of, in Haliplanella luciac, 536
Pancreatic secretion granules, association with microtubules, 398
PAPAFRANGOS, E. D. See S. Inoue, 373
PAPPAS, G. D. See R. B. Hanna, 370
Paranthcssius anemoniae Claus, fine structure of musculature in, 112
Parasite transmission, infection of smelt by a microsporid, 334
Parcur\thoc californica, uptake of amino acids in/ 434
PARKER, C. H. See J. W. Lash, 378
PARSONS, K. C. AND J. M. MELILLO. Am- monification and nitrification potentials of soils from a northern hardwood forest and a pine plantation, 386
Parthenogenesis, in Coptopteryx viridis, 445
PASCOE, N. G. The histochemistry of muscle fiber types in the regenerating claws of the lobster, Homarus ainericanus, 386
PATON, D. The diurnal response of the gray seal, Halichocrus grypus, to tide and in- solation during the month of April at Muskeget Shoals, Nantucket Sound, Massachusetts, U. S. A., 387
PAXHIA, T. See S. Zigman, 404
Pelmatohydra oligactis, development and be- havior of an intergeneric chimera of, 288
PENNCAVAGE, N., S. SCHATZ, E. McCAND- LESS, AND J. FIORE. Distribution of gametophytes and sporophytes of Chon- drus crispus in the vicinity of Woods Hole, 387
Perfusion experiments, in squid giant axons, 399
Perioles and marginal bands in marine animal blood cells, 384
Permeability, apparent water, in spider crabs, 422
PERRITT, S. E. See T. R. Capo, 360
PERSELL, R. AND A. E. V. HASCHEMEYER. L-leucine transport by toadfish liver studied by the Oldendorf method in vivo, 388
PETERSON, S. See D. Ballinger, 357
INDEX
557
PETHIG, R. AND J. A. MCLAUGHLIN. Spec-
troscopic and chemical studies of protein-
methylglyoxal complexes, 388 Phagocytes, dogfish, and plasma fibronectin,
401
Phagosomes from Arbacia embryos, 375 Pharynx, of Navana.r, motor fields of, 360 PHILLIPS, N. W. The relative importance of
bacterial and fungal biomass and Spartina
organic matter in the nutrition of two
species of salt marsh amphipods, 389 PHILLIPS, W. See J. Smith, 395 Phosphorylation, of protein after fertilization
in Arbacia eggs, 357 Photocytes, development of, in Rein ilia larvae,
506 Photoperiod, effects on resting egg production,
in a copepod, 297 Photopositive responses, in a nudibranch mol-
lusk, 346 Photoreceptors, discrete waves in, in Liinulus,
363
Photoresponse of Balanus nauplii, 166 Photosensitizing action of psoralens in eye
tissues of dogfish, 379 Photosynthetic unit, organization of, in brown
algae and diatoms, 371 the, turnover time in seaweeds, 382 Photosynthesis and water relations in Spar-
tiiia, 371
in macrophytic red algae, 376 in Zostcra, 382
Phycobilisomes in macrophytic red algae, 376 Phycocyanobilin, mechanism of formation, in
red algae, 400 Pigment-protein complexes from brown algae
and diatoms, 371 Plant compounds, secondary, chemoreceptor
responses to, in lobster, 391 Planula larvae, neuroeffector development in
a pennatulid, 506
Platymonas containing high levels of chloro- phyll b, absorption properties of, 370 POLLARD, H. B., B. D. NOE, AND C. E. BAUER,
Detection of a synexin-like soluble factor
in anglerfish islet tissue that aggregates
islet secretory granules in the presence of
small amounts of calcium, 389 Polychaete nutrition, 434 Polyp contraction, in response to electrical
impulses, in Clava, 189 Polysomal RNA, sequence complexity of, in
Lyt echinus eggs, 366 Polyspermy, starfish oocyte susceptibility to,
249 Population dynamics, of Aplysia, 407
size estimate, effect of sampling bias, 205
studies on Anguilla in the Great Sippewissett
salt marsh, 368 Potassium current changes in squid giant
axons, 377 Postsynaptic response to presynaptic calcium
current, 380 POURREAU, C. N. Succession of five common
salt marsh detritivores on Spartina alter-
niflora detritus of decreasing particle size
and increasing age, 390 Predator-prey relationships, crabs and Lit-
torina, 359 use of chemical search images in, in lobster,
365
Pressure-flow relations in squid, 358 Presynaptic calcium current, postsynaptic re- sponse to, 380
Procambarus clarkii, molting and growth, 182 Production of steroids in lobster, 364 Productivity, fatty acid and temperature
effects on, in Moina, 234 Protamine in Spisula sperm nuclei, 370 sperm, characterization of Spisula, 357 Protein-methylglyoxal complexes, dielectric
studies of, 358
electron spin resonance studies of, 369 spectroscopic and chemical studies of, 388 Protein synthesis in sea urchin eggs, 402
in toadfish, temperature effect on, 381 Protein transfer, glial-axonal, in squid giant
axon, 400 Protochlorophyll, fluorescence studies of, in
Euglcna, 368 Prey selection of Littorina Httorea by green
crabs, 359
PRUSCH, R. D. AND F. C. G. HOSKIN. Trans- membrane movements of sulfur compounds
in the squid giant axon, 390 Psoralens, photosensitizing action of, in eye
tissue of dogfish, 379 Pugcttia producta, salt and water balance in,
221, 422
Pulmonate snail, locomotion in Mcliiinpus, 306 Pyramidellidae, ectoparasitism of, 320
R
Rana pipiens, biosynthesis of testosterone by,
356 effect of LHRH decapeptide on steroido-
genesis in, 393
REBHUX, L. I. See T. C. Keller, III., 374 Recombinant studies on DXA repetition in
sea urchin genome, 395 Red algae, mechanism of phycocyanobilin
formation, 400 REESE, T. S. See S. Humphreys. 373
INDEX
Regeneration <>f claw muscle in lobsk-r, histo- cliemisty of, 386
Regulation of iiitracellular pH in squid giant axons, 392
REILLY, P., C. DERBY, AND J. ATEMA. Chemo- reception in Homarus amcricanus: re- sponses of primary receptors to second- ary plant compounds, 391
Rcnilla, development of neuroeffector function in, 506
Reproduction, asexual, in mantids, 445 in Nucclla crassilabrum, 453 in Aplysia, seasonal cycle of, 407 success of Nereis siiccinca at high salinities and temperatures, 153
Respiration, in a sea star, 275 in Calliciiuissa janiaicense, 125
Resting eggs, copepod, 297
REYNOLDS, G. T. Image intensification as a tool in low level fluorescence studies of living cells, 391
REYNOLDS, G. T. See S. M. Wolniak, 402
Ribosomal subunits, in Arbacia eggs, 357
RlCKLES, F. R. AND J. LEVIN. The USC of
Limulus amebocyte lysate (LAL) for the removal of lipopolysaccharide from bio- logical reagents, 392
RNA synthesis in isolated chloroplasts, 401 ROBBINS, D. See G. M. Langford, 377 ROBERTSON, R. AND T. MAU-LASTOVICKA. The ectoparasitism of Boonca and Fargoa (Gastropoda: Pyramidellidae), 320 RUBIN, G. See J. Smith, 395 RUDLOE, A. Locomotor and light responses of larvae of the horseshoe crab Limulus pulyphcnius (L.), 494 RUDNICK, M. S. See D. L. Gustafson, 371 RUSHFORTH, N. B. See D. R. Stokes, 189,
396
RUSSELL, J. M. AND \V. F. BORON. Intra- cellular pH regulation in squid giant axons, 392
Salt marsh, sulfate depletion profiles for a, 394
Saltmarsh bacteria, adaptation of, 375
Salinity tolerance in Nereis. 153
Salton Sea salinities, effects on survival of atokous worms, 153
Sampling bias, effect of. on population size estimates, 205
SAMUELSON, E. See C. Serhan, 394
SATTERLIE, R. A. AND J. F. CASE. Develop- ment of bioluminescence and other effector responses in the pennatulid coelenterate Kcnilla knllikeri. 506
SCARBOROUGH, A. AND E. WEIDNER. Field and laboratory studies of Glugea hcrtivigi (microsporida) in the rainbow smelt Os- uict'HS ini'i'du.r, 334
SriiATZ, S., D. MAUZERALL, AND J. FIORE. A comparative study on Latninaria saccha- riini (Phaeophyta) infected by Phycomc- laina laniinanac ( Ascomycotina), 392
SCHATZ, S. See N. Penncavage, 387
SCHIFF, J. A. See B. A. Hayhome, 370; and M. A. Frey, 368
SCHLOSS, J. A. See D. S. Durica, 367
SCHUEL, H. See M. Coburn, 362
SCRUGGS, V. See D. Landowne, 377
Sea anemones, effects of temperature and feeding on asexual reproduction in, 536
Sea urchin DNA, sequence homologies with an actin gene-containing plasmid from Dro- sopliila in, 367 eggs, hydrogen peroxide as block to poly-
spermy, 362
eggs, protein synthesis in, 402 genome, recombinant studies on DNA rep- etition in, 395
Seasonal variation of body length, in copepods, 297
Seaweeds, turnover time of photosynthetic unit in, 382
SEGAL, A. R. See T. Mauro, 380
SEGAL, S. J. See C. A. Adejuwon, 356
SEGAL, S. J. AND C. A. ADEJUWON. Direct effect of LHRH on testicular steroidoge- nesis in Rana fipieiis, 393
Selectivity of particle size in Modiolus and ttalanns, 386
Septate axon of crayfish, coupling resistance associated with structural changes in, 370
Sequence complexity of polysomal RNA in Lytcchiinis eggs, 366
SERHAN, C., P. ANDERSON, E. GOODMAN, E. SAMUELSSON, AND G. WEISSMANN". A general method, employing arsenazo III in liposomes, for the study of calcium iono- phores : results with A23187 and prosta- glandins, 394
SERUNIAN, L. See J. Smith, 395
SEWIN.SKI, J. See S. V. Nicosia, 385
Sex ratio and modal size classes in Pacific mole crabs, 205
SIIEN, S. Sulfate-depletion profiles and sul- fate-reduction rates for a salt marsh, 394
SHKK, S. See E. Bell, 358
SIEFKING, G. E. See G. Weissmann, 401
SIMON, S. See R. Llinas, 380
Sipuncnlns, urn cell complexes and mucus secretion in, 381
INDEX
559
Sipunculus, urn cell complexes, ultrastructure
of, 385
Size, effect on oxygen uptake in sand dollars, 275
Slimeways, Lab\ritil1iuhi, contractile proteins in, 358
Smelt, laboratory transmission of parasites of, 334
SMITH, D. A comparative study of the mar- ginal bands in newt (Notophthalmus n'n- dcsccns) and chick (Callus doincsticus) erythrocytes, 395
SMITH, J., L. SERUNIAN, W. PHILLIPS, A. MURRAY, S. HOROWITCH, AND G. RUBIN. Repeated genomic sequences cloned from the sea urchin Lytcchinus pictus, 395
SMITH, K. M. See R. F. Troxler, 400
Soccr, R., J. MERCURO, AND A. FARMANFAR- MAIAX. Heavy metal effects on intestinal absorption of nutrients in the toadfish, Opsamis tan, 396
Social behavior, pheromone-mediated, in cat- fish, 360
Spartitia detritus, importance in amphipod
nutrition, 389 nutrition of saltmarsh invertebrates on, 390
Spawning of Asterina minor, 258
Specificity of Boonca and Fargoa, 320
Spectroscopic and chemical studies, of protdn- methylglyoxal complexes, 388
Sperm nuclei, Spisula, protamine in, 370
Spicule formation, in Cystodytcs lobatus, 464
Spider crabs, urine production and magnesium regulation in, 221
Spindles, Spisula, tubulin from, 374
Spirochaetes, tubulin in, 369
Spisula. nicotinamide deamidase activity in
oocytes of, 383
sperm nuclei, protamine in, 370 sperm protamine, characterization of, 357 tubulin from isolated spindles of, 374
Sponge cells, effect of aggregation factor on, 373
Sporophytes of L'hondrus, 387
SPRAY, D. C. See M. S. Cappell, 360
Squid axoplasm, cytoskeletal proteins in, 384 regulation of intracellular pH in, 392
Squid brain, reassembly of neurofilaments from, 403
Squid giant axon, glial-axonal protein transfer
in, 400
perfusion experiments, 399 transmembrane movements of .sulfur com- pounds in, 390 potassium current changes in, 377
Squid, pressure-flow relations in systemic cir- culation of, 35S
Starfish, oucyte maturation, 249
Starvation, effect on oxygen uptake, in a sea star, 275
Statocyst cilia transmit mechanical stimuli, 397
STEPHENS, G. C. See J. J. Costopulos, 434
STEPHENS, R. F. See E. W. Stommel, 397
Steroidogenesis, in Ratio effect of LHRH dec- apeptides on, 393
Steroid production in lobster, 364
STEUDLER, P. A. See C. M. Zacks, 404
STEWART, A., B. BRYANT, AND J. ATEMA. Be- havioral evidence for two populations of amino acid receptors in catfish taste, 396
STOKES, D. R. AND N. B. RUSHFORTH. Evi- dence for three conducting systems in the hydroid, Clara, 396
STOKES, R. R. AND N. B. RUSHFORTH. Evoked responses to electrical stimulation in the colonial hydroid Clara squatnata: a contraction pulse system, 189
STOMMEL, E. W., R. E. STEPHENS, AND D. L. ALKON. Statocyst cilia transmit rather than transduce mechanical stimuli, 397
STRATHMANN, R. R. AND E. LEISE. On feed- ing mechanisms and clearance rates of molluscan veligers, 524
STREIBEL, E. See L. Mastroianni, 381
STUNKARD, H. \V. The larval stages of Lcpocrcadium arcolatitni ( Linton, 1900) Stunkard, 1969, ( Trematoda : Digenea), 397
Subitaneous eggs, copepod, 297
SUGIMORI, M. See R. Llinas, 380
Sulfate-depletion profiles for a salt marsh, 394
Sulfates, in eel gill filaments, 104
Sulfur compounds in the squid giant axon, 390
SUPRENANT, K. A. AND W. L. DENTLER. Patl-
creatic secretion granules are associated with microtubules, 398 SUSSWEIN, A. J. See M. S. Cappell, 360 SVENDSEN, C. N. See J. W. Lash, 378 Synexin-like factor in anglerfish islet tissue,
389
Systemic circulation, squid, pressure-flow rela- tions of, 358 S/KXT-GvoRovr. A. The living state, 398
Tadpoles, ascidian, settling and early growth.
budding, and spicule formation, 464 TAKEI, Y. See S. Lerman, 379; and J. M.
Megaw, 383
Tl-AliUE, B. D., M. GlLSON, AND A. J. KAL-
MI.TN. Aligration rate of mud bacteria as a function of magnetic field strength, 39Q
560
INDEX
Teleosts, gill structure of, 104 Temperature, and feeding effects on reproduc- tion in sea anemones, 536 -dependent timing of mitosis, in L\techinins,
367 developmental, effects on size and oxygen
uptake in a sea anemone, 536 effect on oxygen uptake in sand dollars, 275 effects on productivity of Moina, 234 effects on protein synthesis in toadfish, 381, TERAKAWA, S. Excitation of squid giant axon membrane exposed to an identical solu- tion intracellularly and extracellularly, 399 Testosterone, biosynthesis of, by Rana pipicns,
356
production by Leydig cells in mice, 380 Tetrahymena, membrane particle arrays in,
during ciliogenesis, 372 Thalassinids, respiration in, 125 THOMPSON, C. S. See M. P. Charlton, 361 Tidal (and lunar) periodicity of Liinulus
larvae emergence, 494
rhythm in the neural gland of dona, 378 Timing of mitosis in Lytcchinus, effects of
temperature, 367 Toadfish, heavy metal effects on gut function
in, 396
liver, amino acid transport in, 388 temperature effects on protein synthesis in,
381 Training response in the nudibranch, Hermis-
senda, 364 Transcription in Arbacia punctulata nuclei,
365 Translational regulation of histone synthesis
in Arbacia, 367
Transmembrane movements of sulfur com- pounds in squid giant axon, 390 Transport, amino acid, in toadfish liver, 388 TREISTMAN, S. N. Comparison of long-lasting hyperpolarization produced synaptically with that induced by cyclic AMP in Aplysia pacemaker neurons, 399 Trltonia diomedea, feeding mechanisms of, 524 TROLL, W. See M. Coburn, 362 TROXLER, R. F., S. B. BROWN, AND K. M. SMITH. Mechanism of red algal bile pig- ment formation, 400 Tubulin, dogfish and skate brain, purification
of, 377
from isolated Spisula spindles, 374 in spirochaetes, 369 Tunicate, larval tail muscle determination and
differentiation, 344
TYTELL, M. AND R. J. LASER. Glial-axonal protein transfer : its functional signif- icance, 400
u
Ultrastructure of musculature in copepods, 112
of urn cell complexes of Sipunculus, 385 Urine production in spider crabs, 221 Urn cell complexes and mucus secretion in Sipunculus, 381, 385
V
VAN HOLDE, K. E. See T. D. Yager, 403 VAN HOLDE, K. E. See J. Ausio, 357 Veligers, molluscan clearance rates and feed- ing mechanisms, of, 524
Video-computer analysis of phototactic move- ments of barnacle nauplii, 166 VIERLING, E. RNA synthesis in isolated
chloroplasts, 401
Voltage clamp study, of postsynaptic response to presynaptic calcium current, 380
W
Water balance, in osmoregulation and osmo-
conforming crabs, 422 Water relations and photosynthesis in Spar-
tina, 371
WEIDNER, E. See A. Scarborough, 334 WEINSTOCK, M. See M. Krouse, 376 W'EISSMANN, G., J. W. LASH, G. E. SIEFRING, J. IBERS, AND L. LORAND. Plasma fibro- nectin (CIG) of the dogfish plasma medi- ates attachment of phagocytes of collagen substrates, 401
WEISSMANN, G. See C. Serhan, 394 WENNER, A. M. AND C. FUSARO. An analysis of population structure in Pacific mole crabs (Hip pa pacifica Dana), 205 WHITTAKER, J. R. Development of tail muscle acetylcholinesterase in ascidian embryos lacking mitochondrial localization and seg- regation, 344
WINKLER, M., E. BAKER, AND T. HUNT. Pro- tein synthesis in cell free extracts of Lytechinus pictus eggs, 402 WITTENBERG, D. See D. Mauzerall, 382 WOLNIAK, S. M., P. K. HEPLER, W. T. JACK- SON, AND G. T. REYNOLDS. Low level excitation of chlorotetracycline fluores- cence in Haemanthus endosperm cells us- ing image intensification, 402 WRIGHT, S. H. See J. J. Costopulos, 434
YAGER, T. D. AND K. E. VAN HOLDE. Char- acterization of the hemoglobin of the clam, Astarte castanca, 403
YOSHIZAWA, H. See M. Komatsu, 258
I X I )EX
561
ZACKKOFF, R. Y., A. E. GOLDMAN, AND R. D. GOLDMAN. /;; vitro reassembly of squid brain neurofilaments and their purification by assembly-disassembly, 403
ZACKS, C. M., P. A. STEUDLER, AND J. M. MELILLO. Bacterial denitrification : a gas chromatographic study using acetylene in- hibition of N2O reductase in two hour in- cubations, 404
XAMER, W. E. AND C. P. MANGUM. Irre- versible nongenetic temperature adaptation of oxygen uptake in clones of the sea anemone, Haliplanella luciae (Verrill), 536
ZIGMAN, S. AND T. PAXHiA. Features of elasmobranch eye lenses relative to those of humans, 404
ZIGMAN, S. See D. Lipman, 379
Zostcra, photosynthesis in, 382
Continued from Cover Two
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CONTENTS
AUDESIRK, TERESA E.
A field study of growth and reproduction in Aplysia calif ornica .... 407
j '' - *•• '
CORNELL, JOHN C.
Salt and water balance in two marine spider crabs, Libinia emarginata and Pugettia producta. II. Apparent water permeability . . 422
COSTOPULOS, JAMES J., GROVER C. STEPHENS, AND STEPHEN H. WRIGHT
Uptake of amino acids by marine polychaetes under anoxic conditions 434
CUKIER, MARTA, GRACIELA ALICIA GUERRERO, AND MARIA CRISTINA MAGGESE
Parthenogenesis in Coptopteryx viridis, Giglio Tos (1915) (Dyctioptera, Mantidae) 445
GALLARDO, C. S.
Developmental pattern and adaptations for reproduction in Nucella crassilabrum and other muricacean gastropods 453
LAMBERT, GRETCHEN
Early post-metamorphic growth, budding and spicule formation in
the compound ascidian Cystodytes lobatus 464
MlNASIAN, LEO L., JR., AND RICHARD N. MARISCAL
Characteristics and regulation of fission activity in clonal cultures
of the cosmopolitan sea anemone, Haliplanella luciae (Verrill) 478
RUDLOE, ANNE
Locomotor and light responses of larvae of the horseshoe crab, Limulus polyphemus (L.) >•;/? . , 494
SATTERLIE, RICHARD A., AND JAMES F. CASE
Development of bioluminescence and other effector responses in
the pennatulid coelenterate Renilla kollikeri 506
STRATHMANN, R. R., AND E. LEISE
On feeding mechanisms and clearance rates of molluscan veligers . . 524
ZAMER, WILLIAM E., AND CHARLOTTE P. MANGUM
Irreversible nongenetic temperature adaptation of oxygen uptake
in clones of the sea anemone Haliplanella luciae (Verrill) 536
INDEX TO VOLUME 157 . 548
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