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THE

BIOLOGICAL BULLETIN

PUBLISHED BY

THE MARINE BIOLOGICAL LABORATORY

Editorial Board

JOHN M. ANDERSON, Cornell University ARTHUR L. COLWIN, Queens College, New York

DONALD P. COSTELLO, University of

North Carolina

FRANK M. FISHER, JR., Rice University

CATHERINE HENLEY, University of

North Carolina

MEREDITH L. JONES, Smithsonian Institution

ROBERT K. JOSEPHSON, Case Western

Reserve University

CHARLES B. METZ, University of Miami

LEONARD NELSON, Medical College of Ohio

at Toledo

HOWARD A. SCHNEIDERMAN, University of

California, Irvine

STEPHEN A. WAINWRIGHT, Duke University CARROLL M. WILLIAMS, Harvard University

W. D. RUSSELL-HUNTER, Syracuse University Managing Editor

VOLUME 139

JULY TO DECEMBER, 1970

Printed and Issued by

LANCASTER PRESS, Inc.

PRINCE & LEMON STS.

LANCASTER, PA.

11

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, $5.00. Subscription per volume (three issues), $14.00.

Communications relative to manuscripts should be sent to Dr. W. D. Russell-Hunter, Marine Biological Laboratory, Woods Hole, Massachusetts 02543 between May 23 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.

Second-class postage paid at Lancaster, Pa.

LANCASTER PRESS, INC., tANCASTEK, PA

CONTENTS

No. 1. AUGUST, 1970

PAGE Annual report of the Marine Biological Laboratory 1

ROE, PAMELA

The nutrition of Paranemertes peregrina (Rhynchocoela : Hoplonemertea).

I. Studies on food and feeding behavior 80

GIBSON, RAY

The nutrition of Paranemertes peregrina (Rhynchocoela : Hoplonemertea) .

II. Observations on the structure of the gut and proboscis, site and se- quence of digestion, and food reserves 92

CHIU, K. W., W. G. LYNN AND J. P. LEICHNER

Environmental temperature and thyroid activity in the lizard, Sceloporus occidentalis 107

REELING, ALFRED W., PATRICK) BERNAL AND ALEJANDRO ZULETA

Emersion of the amphibious Chilean clingnsh, Sicyases sanguineus 115

FRAENKEL, G. AND JAN ZDAREK

The evaluation of the "Calliphora Test" as an assay for ecdysone 138

HEATFIELD, BARRY M.

Calcification in echinoderms : effects of temperature and diamox on incor- poration of calcium-45 in vitro by regenerating spines of Strongylocentrotus purpuratus 151

LACOMBE, DYRCE

A comparative study of the cement glands in some balanid barnacles (Cirripedia, Balanidae) 164

RICE, NOLAN E. AND W. ALLAN POWELL

Observations on three species of jellyfishes from Chesapeake Bay with special reference to their toxins. I. Chrysaora (Dactylometra} qiiinquc- cirrha 180

ROBERTS, MORRIS H., JR.

Larval development of Pagunts longicarpus Say reared in the laboratory.

I. Description of larval instars 188

ROOSEN-RUNGE, EDWARD C

Life cycle of the hydromedusa Phialidinni gregarium (A. Agassiz, 1862)

in the laboratory 203

SLAM A, K., K. HEJNO, V. JAROLIM AND F. SORM

Natural and synthetic materials with insect hormone activity. 5. Specific juvenile hormone effects in aliphatic sesquiterpenes 222

iv CONTENTS

WEATIIERLEY, A. H. PAGE

Effects of superabundant oxygen on thermal tolerance of goldfish 229

No. 2. OCTOBER, 1970

FROMSON, DAVID AND J. R. WHITTAKER

Acetylcholinesterase activity in eserine-treated ascidian embryos 239

HAVEN, DEXTER S. AND REINALDO MORALES-ALAMO

Filtration of particles from suspension by the American oyster Crassostrea virginica 248

HENLEY, CATHERINE

Changes in microtubules of cilia and flagella following negative staining with phosphotungstic acid 265

LENSKY, Y., C. COHEN AND H. A. SCHNEIDERMAN

The origin, distribution and fate of the molting fluid proteins of the ce- cropia silkworm 277

MACKINTOSH, F. ROY AND EUGENE BELL

Reversible response to puromycin and some characteristics of the uptake and use of amino acids by unfertilized sea urchin eggs 296

MADERSON, P. F. A., K. W. CHIU AND J. G. PHILLIPS

Changes in the epidermal histology during the sloughing cycle in the rat snake Ptyas korros Schlegel, with correlated observations on the thyroid gland 304

MOWBRAY, RODNEY C., GEORGE GORDON BROWN AND CHARLES B. METZ

Cytological and immunological investigation of sperm-egg interactions in selected decapods (Crustacea) and Limnlus polyphcinns L. (Merosto- mata) 313

MURPHY, JAMES S.

A general method for the monoxenic cultivation of the Daphnidae 321

SAROYAN, J. R., E. LINDNER AND C. A. DOOLEY

Repair and reattachment in the Balanidae as related to their cementing mechanism 333

SMITH, RALPH I.

The apparent water-permeability of Carcinus maenas (Crustacea, Brachy- ura, Portunidae) as a function of salinity 351

SQUIRE, RICHARD D. AND DANIEL S. GROSCH

The effects of acute gamma irradiation on the brine shrimp, Arteniia. I. Life spans and male reproductive performance 363

SQUIRE, RICHARD D.

The effects of acute gamma irradiation on the brine shrimp, Artemia. IT. Female reproductive performance 375

CONTENTS v

TSUJI, FREDERICK I., RICHARD V. LYNCH, III AND YATA HANEDA PAGE Studies on the bioluminescence of the marine ostracod crustacean Cypri- dina serrata 386

Abstracts of papers presented at the Marine Biological Laboratory 402

X<>. 3. DECEMBER, 1970

EARTH, R. H., JR AND WILLIAM J. BELL

Physiology of the reproductive cycle in the cockroach Byrsotria fumigata (Guerin) ". 447

BASKIN, DENIS G. AND DAVID W. GOLDING

Experimental studies on the endocrinology and reproductive biology of the viviparous polychaete annelid, Nereis limnicola Johnson 461

BRADSHAW, WILLIAM E.

Interaction of food and photoperiod in the termination of larval diapause

in Chaobonis americanus (Diptera : Culicidae) 476

D'AGOSTINO, ANTHONY S. AND LUIGI PROVASOLI

Dixenic culture of Daphnia magna, Straus 485

GRIFFIN, DONALD R. AND RODERICK A. SUTHERS

Sensitivity of echolocation in cave swiftlets 495

KAPUR, S. P. AND A. SEN GUPTA

The role of amoebocytes in the regeneration of shell in the land pulmo- nate, Enplecta indica (Pfieffer) 502

KEETON, WILLIAM T.

"Distance effect" in pigeon orientation : An evaluation 510

KRISHNAKUMARAN, A. AND H. A. SCHNEIDERMAN

Control of molting in mandibulate and chelicerate arthropods by ecdysones 520

NEWTON, W. DONALD

Gastrulation in the turbellarian H \droHuia.\- c/risca (Platyhelminthes ; Plagiostomidae) : Formation of the epidermal cavity, inversion and epiboly 539

NOBLE, P. B.

Coelomocyte aggregation in Cucumaria jrondosa: Effect of ethylenedia- minetetraacetate, adenosine, and adenosine nucleotides 549

NYBLADE, CARL F.

Larval development of Pag urns annulipes (Stimpson, 1862) and Pagurus poUicaris Say, 1817 reared in the laboratory 557

UMMINGER, BRUCE L.

Effects of subzero temperatures and trawling stress on serum osmolality

in the winter flounder, Pseudopleuronectes americamis 574

Vol. 139, No. 1 August, 1970

THE

BIOLOGICAL BULLETIN

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY

THE MARINE BIOLOGICAL LABORATORY SEVENTY-SECOND REPORT, FOR THE YEAR 1969 — EIGHTY-SECOND YEAR

1. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST, 1969) 1

1 1 . ACT OF INCORPORATION 4

III. BYLAWS OF THE CORPORATION 5

IV. REPORT OF THE DIRECTOR 7

Addenda :

1. Memorial 9

2. The Staff 11

3. Investigators, Lalor and Grass Fellows, and Students. . . 22

4. Fellowships and Scholarships 36

5. Training Program 36

6. Tabular View of Attendance, 1965-1969 39

7. Institutions Represented 39

8. Friday Evening Lectures 41

9. Tuesday Evening Seminars 42

10. Members of the Corporation 43

V. REPORT OF THE LIBRARIAN 70

VI. REPORT OF THE TREASURER. 71

I. TRUSTEES Including Action of 1969 Annual Meeting

GERARD SWOPE, Jr., Chairman of the Board of Trustees, Croton-on-Hudson, New York,

New York 10520

ALEXANDER T. DAIGNAULT, Treasurer, 7 Hanover Square, New York, New York 10005 H. BURR STEINBACH, Director, President of the Corporation, and Professor of Zoology,

University of Chicago EUGENE BELL, Clerk of the Corporation, Massachusetts Institute of Technology

1

Copyright © 1970, by the Marine Biological Laboratory Library of Congress Card No. A38-518

2 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

EMERITI

WILLIAM R. ARMSTROXG, Falmouth, Massachusetts

PHILIP B. ARMSTRONG, State University of New York, College of Medicine, Syracuse

DETLEV W. BRONK, The Rockefeller University

C. LALOR BURDICK, The Lalor Foundation

E. G. BUTLER, Princeton University

C. LLOYD CLAFF, Brockton, Massachusetts

KENNETH S. COLE, National Institutes of Health

PAUL S. GALTSOFF, Woods Hole, Massachusetts

M. H. JACOBS, University of Pennsylvania

DOUGLAS MARSLAND, Marine Biological Laboratory

CHARLES W. METZ, Woods Hole, Massachusetts

CHARLES PACKARD, Woods Hole, Massachusetts

HAROLD H. PLOUGH, Amherst, Massachusetts

A. C. REDFIELD, Woods Hole Massachusetts

CARL C. SPEIDEL, University of Virginia

A. H. STURTEVANT, California Institute of Technology ALBERT SZENT-GYORGYI, Marine Biological Laboratory W. RANDOLPH TAYLOR, University of Michigan

B. H. WILLIER, The Johns Hopkins University

CLASS OF 1973

JAMES CASE, University of California, Santa Barbara

ARTHUR L. COLWIN, Queens College

WILLIAM T. GOLDEN, New York, New York

GEORGE G. HOLZ, JR., State University of New York, Upstate Medical Center, Syracuse

SHINYA INOUE, University of Pennsylvania

CHARLES B. METZ, University of Miami

GEORGE T. SCOTT, Oberlin College

MALCOLM S. STEINBERG, Princeton University

EDGAR ZWILLING, BRANDEIS University

CLASS OF 1972

JOHN B. BUCK, National Institutes of Health

ANTHONY C. CLEMENT, Emory University

DONALD P. COSTELLO, University of North Carolina

GEORGE H. A. CLOWES, JR., Harvard Medical School

TERU HAYASHI, Illinois Institute of Technology

ALBERTO MONROY, University of Palermo, Italy

JOHN W. SAUNDERS, JR., State University of New York at Albany

HOWARD A. SCHNEIDERMAN, University of California, Irvine

ANDREW SZENT-GYORGYI, Brandeis University

«

CLASS OF 1971

FRANK A. BROWN, JR., Northwestern University

D. EUGENE COPELAND, Tulane University

SEARS CROWELL, Indiana University

HARRY GRUNDFEST, Columbia University, College of Physicians and Surgeons

LEWIS H. KLEINHOLZ, Reed College

TRUSTEES

SAMUEL LENHER, Wilmington, Delaware

C. LADD PROSSER, University of Illinois

S. MERYL ROSE, Tulane University

\V. D. RUSSELL-HUNTER, Syracuse University

CLASS OF 1970

ROBERT D. ALLEX, State University of New York at Albany

ERIC G. BALL, Harvard Medical School

FRANCIS D. CARLSON, The Johns Hopkins University

JAMES D. EBERT, Carnegie Institution of Washington

J. WOODLAXD HASTINGS, Harvard University

RUDOLF T. KEMPTOX, Vassar College

RICHARD S. MORSE, Boston, Massachusetts

CLARK P. READ, Rice University

WALTER S. VINCENT, University of Pittsburgh

STANDING COMMITTEES

EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES

GERARD SWOPE, JR., ex officio TERU HAYASHI, 1971

ALEXAXDER T. DAIGXAULT, ex officio SEARS CROWELL, 1970

H. BURR STEINBACH, ex officio FRAXCIS D. CARLSOX, 1970

CLARK P. READ, 1972 LEWIS H. KLEINHOLZ, 1970 J. WOODLAND HASTINGS, 1971

LIBRARY COMMITTEE

JOHN B. BUCK, Chairman ARNOLD LAZAROW

MELVIN ROSEXFELD, JR. DAVID A. Ross

NORMAN B. RUSHFORTH T. H. WILSON

RESEARCH SERVICES COMMITTEE

MARTIN MENDELSON, Chairman WILLIAM J. ADELMAN, JR.

EUGENE BELL HOWARD A. SCHNEIDERMAN

ANDREW SZENT-GYORGYI DAVID A. YPHANTIS

SUPPLY DEPARTMENT COMMITTEE

FRAXK M. FISHER, JR., Chairman SEARS CROWELL

MILTON FINGERMAN LIONEL I. REBHUN

W. D. RUSSELL-HUNTER ROBERT T. WlLCE

INSTRUCTION COMMITTEE

FRANCIS D. CARLSON, Chairman MICHAEL V. M. BENNETT

PHILIP B. DUNHAM PHILIP GRANT

J. WOODLAND HASTINGS ALBERTO MONROY JOHN M. TEAL

BUILDINGS AND GROUNDS COMMITTEE

RUDOLF T. KEMPTON, Chairman D. EUGENE COPELAND

JAMES W. GREEN TERU HAYASHI

PHILIPP STRITTMATTER LEON P. WEISS

ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

RADIATION COMMITTEE

S. J. COOPERSTEIN, Chairman DANIEL S. GROSCH

LASZLO LORAND GEORGE T. REYNOLDS

RONALD C. RUSTAD

RESEARCH SPACE COMMITTEE

J. WOODLAND HASTINGS, Chairman ARTHUR L. COLWIN

JAMES W. LASH WALTER S. VINCENT

COMMITTEE FOR THE NOMINATION OF OFFICERS

FRANCIS D. CARLSON SEARS CROWELL

J. WOODLAND HASTINGS TERU HAYASHI

LEWIS H. KLEINHOLZ CLARK P. READ

FOOD SERVICE COMMITTEE

GEORGE G. HOLZ, JR., Chairman JOHN M. ARNOLD

FR. J. D. CASSIDY S. J. COOPERSTEIN

GlLLES H. COUSINEAU RlTA GUTTMAN

LEWIS H. KLEINHOLZ

II. ACT OF INCORPORATION

No. 3170

COMMONWEALTH OF MASSACHUSETTS

Be It Known, That whereas Alpheus Hyatt, William Sanford Stevens, William T. Sedgwick, Edward G. Gardiner, Susan Minns, Charles Sedgwick Minot, Samuel Wells, William G. Farlow, Anna D. Phillips, and B. H. Van Vleck have associated themselves with the intention of forming a Corporation under the name of the Marine Biological Laboratory, for the purpose of establishing and maintaining a laboratory or station for scientific study and investigation, and a school for instruction in biology and natural his- tory, and have complied with the provisions of the statutes of this Commonwealth in such case made and provided, as appears from the certificate of the President, Treasurer, and Trustees of said Corporation, duly approved by the Commissioner of Corporations, and recorded in this office;

Now, therefore, I, HENRY B. PIERCE, Secretary of the Commonwealth of Massachu- setts, do hereby certify that said A. Hyatt, W. S. Stevens, W. T. Sedgwick, E. G. Gardi- ner, S. Minns, C. S. Minot, S. Wells, W. G. Farlow, A. D. Phillips, and B. H. Van Vleck, their associates and successors, are legally organized and established as, and are hereby made, an existing Corporation, under the name of the MARINE BIOLOGICAL LAB- ORATORY, with the powers, rights, and privileges, and subject to the limitations, duties, and restrictions, which by law appertain thereto.

Witness my official signature hereunto subscribed, and the seal of the Commonwealth of Massachusetts hereunto affixed, this twentieth day of March, in the year of our Lord One Thousand Eight Hundred and Eighty-Eight.

[SEAL] HENRY B. PIERCE,

Secretary of the Commonwealth

BYLAWS OF THE CORPORATION 5

III. BYLAWS OF THE CORPORATION OF THE MARINE BIOLOGICAL LABORATORY

(Revised August 12, 1966)

I. The members of the Corporation shall consist of persons elected by the Board of Trustees.

II. The officers of the Corporation shall consist, of a Chairman of the Board of Trustees, President, Director, Treasurer and Clerk.

III. The Annual Meeting of the members shall be held on the Friday following the second Tuesday in August in each year at the Laboratory in Woods Hole, Massachusetts, at 9 :30 A.M., and at such meeting the members shall choose by ballot a Treasurer and a Clerk to serve one year, and nine 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 Trustees to be held at such time and place as may be designated.

IV. Twenty-five members shall constitute a quorum at any meeting.

V. Any member in good standing may vote at any meeting, either in person or by proxy duly executed.

VI. 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.

VII. The Annual Meeting of the Trustees shall be held promptly after 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, and the Secretary shall give notice thereof by written or printed notice, mailed to each Trustee at his address as shown on the records of the Corporation, at least one (1) week before the meeting. At such special meeting only matters stated in the notice shall be considered. Seven Trustees of those eligible to vote shall constitute a quorum for the transaction of business at any meeting.

VIII. There shall be three groups of Trustees:

(A) Thirty-six Trustees chosen by the Corporation, divided into four classes, each to serve four years. After having served two consecutive terms of four years each, Trustees are ineligible for re-election until a year has elapsed.

(B) Trustees ex officio, who shall be the Chairman, the Director, the Treasurer, and the Clerk.

(C) Trustees Emeriti, who shall be elected from present or former Trustees by the Corporation. Any regular Trustee who has attained the age of seventy years shall con- tinue to serve as Trustee until the next Annual Meeting of the Corporation, whereupon his office as regular Trustee shall become vacant and be filled by election by the Corpora- tion and he shall become eligible for election as Trustee Emeritus for life. 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.

The Trustees and officers shall hold their respective offices until their successors are chosen and have qualified in their stead.

IX. The Trustees shall have the control and management of the affairs of the Cor- ration. They shall elect a Chairman of the Board of Trustees who shall be elected an- nually and shall serve until his successor is selected and qualified and who shall also pre- side at meetings of the Corporation. They shall elect a President of the Corporation

6 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

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 appoint a Director of the Laboratory for a term not to exceed five years, provided the term shall not exceed one year if the can- didate 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 com- pensation and define the duties of all the officers and agents; and may remove them, or any of them except those chosen by the members, at any time. They may fill vacancies occurring in any manner in their own number or in any of the officers. The Board of Trustees shall have the power to choose an Executive Committee from their own number, and to delegate to such Committee such of their own powers as they may deem expedient. They shall from time to time elect members to the Corporation upon such terms and conditions as they may think best.

X. The Associates of the Marine Biological Laboratory shall be an unincorporated group of persons (including associations and corporations) interested in the Laboratory and shall be organized and operated under the general supervision and authority of the Trustees.

XI. 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.

XII. The account of the Treasurer shall be audited annually by a certified public accountant.

XIII. These bylaws may be altered at any meeting of theTrustees, provided that the notice of such meeting shall state that an alteration of the bylaws will be acted upon.

RESOLUTIONS ADOPTED AT TRUSTEES' MEETINGS

EXECUTIVE COMMITTEE

I. RESOLVED:

(A) The Executive Committee is hereby designated to consist of not more than ten members including the ex officio members who shall be the Chairman of the Board of Trustees, President, Director and Treasurer; six additional Trustees, two of whom shall be elected by the Board of Trustees each year, to serve for a three-year term. (August 11, 1967).

(B) The Chairman of the Board of Trustees shall act as Chairman of the Executive Committee, and the President as Vice President. A majority of the members of the Executive Committee shall constitute a quorum and a majority of those present at any properly held meeting shall determine its action. It shall meet at such times and places and upon such notice and appoint such sub-committees as the Committee shall deter- mine. (August 12, 1966).

(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 the Board or law. (August 16, 1963)

(D) The Executive Committee shall keep appropriate minutes of its meetings, and its action shall be reported to the Board of Trustees. (August 16, 1963).

II. RESOLVED

The elected members of the Executive Committee shall be constituted as a standing "Committee for the Nomination of Officers," responsible for making nominations at the Annual Meeting of the Corporation and of the Board of Trustees, for candidates to fill each office as the respective terms of office expire (Chairman of the Board, President, Director, Treasurer, and Clerk).

REPORT OF THE DIRECTOR 7

IV. REPORT OF THE DIRECTOR

To: THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY Gentlemen :

In the important aspects of the life of the Marine Biological Laboratory this has been a good year. The scientific work has been maintained at a high level. Indeed, the activities of the scientists working at the Laboratory are a magnificent testimonial to the ability of good people to carry on good work regardless of financial stringency and budget cuts.

In terms of our physical plant, this has been the year in which the long-envisioned plans to house our instructional program in modern, fireproof quarters have been realized. As Conklin wrote in 1900, the wooden buildings all were of a temporary character and could be used only in summer. The then stated need for "at least one substantial, fire-proof building . . . which can be used the year around" increased with the years. Now three such year around laboratory buildings have replaced the wooden laboratory buildings to make possible the sustained service to scientists envisioned by Conklin. Although the passing of Old Main may be regretted by some, there can be no doubt that the Whitman and Loeb Buildings fulfill a long-standing need for con- temporary structures. True, the quality of instruction depends in the first instance on the quality of the instructors and the students — of the senior and junior scientists, if you will. However, adequate modern fireproof quarters will certainly enhance the ability of our people to achieve the standards consistent with the changing requirements of modern science.

It is fitting here to recall the devotion with which Dr. Arthur Parpart as President of the Corporation of the Marine Biological Laboratory worked for the fulfillment of our building program. His untimely death in 1965 was a blow to us all; and the new buildings, completed and under construction, are a fine testament to the dedicated efforts of Arthur Parpart. We are fortunate indeed that the development of this pro- gram was carried out by Dr. Philip B. Armstrong initially during his tenure as Director and subsequently as Chairman of the Building Committee.

The MBL and the expanding world of marine sciences

The Marine Biological Laboratory was founded by a group of imaginative and dedi- cated scientists who visualized a facility serving the interests of all biology. Dr. Conklin, in a report written in 1900, speaks as follows:

"In his address at the opening of the Laboratory and in subsequent publications, Professor Whitman took the position that there was great need for a laboratory which should represent, (1) the whole of biology; (2) both teaching and research; (3) the widest possible cooperation of educational and scientific institutions. Such a labora- tory should not be merely a collecting station, nor a summer school, nor a scientific work shop, nor a congress of biologists, but all of these; an institution combining in itself the functions and features of the best biological institutes of the world, having the cooperation of the biologists of this country, and thus forming 'a national center of instruction and research in every department of Biology.' The history of the Labora- tory has shown that this ambitious project is not only highly desirable, but that it is entirely feasible and has justified the claim of the Director that such an institution is the greatest need of Americal biology."

The aims and aspirations set forth by C. O. Whitman and expounded by Conklin seventy years ago still have a scope and validity that is undeniable. The Marine Biological Laboratory has become indeed a national resource and all associated with the

ANNi:.\L KKI'ORT OK THK MAKINK BIOLOGICAL LABORATORY

Laboratory must share the responsibility of maintaining the viability of this intellectual asset of the world.

The Marine Biological Laboratory was founded by scientists with the major objective of getting on with science. The Laboratory is designed to provide services to biologists with facilitities open to all within the limits of physical space. The early evolution of the Laboratory depended not only on the efforts of scientists, but the growth for the first half century or more was also accomplished with the assistance of funds from individuals and private foundations. Now the pattern has changed. Federal funds are needed in larger proportion, and the research facilities of our sister institutions, the universities and colleges of the world, have expanded enormously over what they were many years ago. Clearly there is a continued and enhanced need to look to the future — to insure that the MBL continues to serve its fundamental purposes.

The new facilities, the Loeb Building and the new dining-dormitory complex, allow us to stretch our active calendar in a way that was not possible before. Particularly, the MBL can now truly cooperate with neighboring institutions of higher learning by making available superb seaside facilities for year around courses of instruction and advanced training. The fine effort by Boston University (Boston University Marine Program) has provided a valuable model for one such type of service we can render.

During the forthcoming season, plans will be developed to enhance the scope of activity of the MBL and to seek stable financing. We are indeed fortunate that Dr. James D. Ebert, Director-Designate, has assumed major responsibility for develop- ing these forward-looking plans.

During the 1969 season, Dr. Case undertook the direction of the zoology course (Experimental Invertebrate Zoology), and Dr. Siegelman, the botany course (Experi- mental Botany). Both courses, under the new directors, achieved exemplary levels of innovation. Also, Dr. Adelman managed a most successful training program in Excitable Membrane Physiology and Biophysics. During the 1969 season, plans were made by Drs. Bennett and Dowling for the new course in neurobiology, and that pro- gram is demonstrating the imaginative approaches which we cherish at the MBL.

It is, of course, impossible to make any proper institutional assessment of the scientific advances made by independent investigators during their stay at MBL. The benefits of work here include not only research done in residence, but also ideas exchanged and new viewpoints generated. What can be said with great assurance is that the level of intellectual activity was clearly visible and was good.

A fine illustration of the potentialities of the MBL for extended season work occurred during this past year. Dr. Summers, in charge of our squid survey program, was able to provide squid to Dr. Adelman well on into the late fall. The squid were taken as a part of the ecological survey, and proved to be fine, healthy animals. It is now definitely established that squid users could be provided with animals throughout the year except during a few of the worse winter months. Thus squid join the ranks of other familiar organisms which are available to investigators on a year around basis.

The Systematics-Ecology Program

This program has celebrated its eighth anniversary. Well over a hundred major publications have appeared from work within or sponsored by the Program. Of special interest to the MBL in general has been the "Keys to Marine Invertebrates," of which nearly five thousand copies have been distributed through the Supply Department.

During the eight years of growth, 33 visiting investigators have been on deck, with 24 postdoctoral fellows and 16 graduate trainees. Field sampling for the biotic census of Cape Cod Bay has been completed, and the analysis of the results is proceeding. Staff members have contributed assistance to the Boston University Marine Program

REPORT OF THE DIRECTOR 9

and to other visiting groups. The record of accomplishment of this innovative venture is very refreshing indeed and the program may well serve as one model for future year around development of our activities.

The future of the MBL is bright. The problems looming ahead are challenges, not obstacles. We welcome Dr. James Ebert as incoming Director and wish him well.

1. MEMORIAL ALBERT TYLER BY CHARLES B. METZ

Albert Tyler's sudden and untimely death on November 9, 1968 ended a distin- guished career in developmental biology and a long association with the Marine Bio- logical Laboratory. Albert Tyler was born in Brooklyn, New York on June 26, 1906. He began his higher education as a chemistry major at Columbia University, where he soon came under the influence of T. H. Morgan. Upon completion of the bachelor's degree in 1927, he became Morgan's graduate student. He obtained the master's degree at Columbia in 1928 and then moved to the California Institute of Technology with Morgan where he received a combined biology and chemistry Ph.D. in 1929. He was then appointed to the Institute's faculty and rose through the ranks to the pro- fessorship of embryology which he held the rest of his life. Tyler and Morgan were close friends and scientific colleagues for the remainder of Morgan's life. Both men were highly imaginative, novel and wide-ranging in their thinking and interests. Their special concerns were problems of developmental and marine biology. This last interest almost certainly originated from Morgan's experiences as a student at the Annisquam Marine Laboratory the summer of 1886 and led to his long association with its immediate successor, the Marine Biological Laboratory. In due course Morgan transmitted his interest in marine organisms and the Marine Biological Laboratory to Albert Tyler.

Albert Tyler's direct association with the Marine Biological Laboratory began the summer of 1926, following his junior year at Columbia. He was then listed as a Be- ginning Investigator at the Laboratory, a status he held each summer until 1929 when he was promoted to Independent Investigator. He was elected to Corporation Member- ship in 1931, served as an Instructor in the Embryology Course from 1946 through 1950, a Trustee of the Laboratory from 1948 to 1962 and a member of the Editorial Board of the Biological Bulletin from 1951 through 1954. His concern for the Labora- tory and his influence on many of its activities were very great.

Albert Tyler was one of the leaders in the transition from classical experimental embryology to the molecular biology of development of the present decade. He was almost uniquely equipped for this role writh his strong training in the physico-chemical sciences coupled with the very best possible instruction in classical genetics, cytology and developmental biology obtained from T. H. Morgan, E. B. Wilson and others at the time when these subjects were at their peak. Albert Tyler's first research was per- formed here at the Marine Biological Laboratory and followed the classical tradition. It concerned the production of double embryos in annelids and molluscs. Two papers and his Ph.D. thesis resulted from this work. His first officially recorded seminar at the Marine Biological Laboratory was delivered in 1931 on the subject of artificial parthenogenesis in the eggs of the Pacific Coast echiuroid, Urechis caupo. Shortly, however, his interests turned to more dynamic aspects of development. This was reinforced during the year 1932-33 while a National Research Council Postdoctoral Fellow in Warburg's Laboratory and at the Zoological Station at Naples. This new line of investigation concerned the energetics of development and particularly the energy

10 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

requirements for form changes in the embryo. The first paper in this series appeared in 1933 and the work is now regarded as a classical application of physico-chemical principles to the study of developmental processes. This research led to studies on metabolic changes and particularly the increase in respiration that follows fertilization in sea urchin eggs. In the late 1930's Albert Tyler and several of his colleagues includ- ing S. H. Emerson, L. C. Pauling and A. H. Sturtevant recognized that the modern immune-chemistry, then emerging, had significance for several biological disciplines and could also provide tools for study of other fundamental problems. Albert Tyler took this as an opportunity to explore the macro-molecular events of fertilization and develop- ment in a more modern and rigorous fashion. These studies and their offshoots held his interest for the next 20 years.

His investigations on fertilizin and antifertilizin modernized F. R. Lillie's antigen- antibody analogy, and extended our knowledge of fertilizin and antifertilizin chemistry, specificity, mechanism of interaction and role in fertilization. He investigated specific egg membrane lysins from spermatozoa of molluscs. These substances clearly can contribute to fertilization specificity and certainly are essential to provide a passage for the spermatozoan to the egg surface. In collaboration with Harry Grundfest and Alberto Monroy here at the Marine Biological Laboratory he demonstrated a resting potential and a change in potential at fertilization in the echinoderm egg. Later, electron microscope studies on sperm-egg interaction resulted in provocative theories to relate the ultrastructural observations to the role of fertilizin and antifertilizin in fertilization.

Characteristically, he introduced many conceptual and experimental novelties into his work. F<jr example, to study the role of antigens in fertilization he developed a method for preparing non-agglutinating and non-precipitating antibodies by photo- oxidation some fourteen years before the discovery of enzymatic digestion methods for preparing univalent antibodies. His studies on sea urchins led him to formulate an autoantibody theory of cell structure which he proceeded to test by examining for specific complementary substances in pneumococci and for antivenom production in Gila monsters. Later he published a theory of cancer and, as usual, undertook extensive laboratory tests, in this case with mice. But problems of fertilization and reproductive physiology commanded his major interest during this period. His precise thinking and laboratory skill set the standard for all who worked in this field.

During the last ten years Albert Tyler concentrated on the molecular biology of fertilization and development. He was among those who first provided evidence that messenger RNA must be present in an inactive or "masked" form in the unfertilized sea urchin egg and he early appreciated that such stored informational RNA might constitute the classical "formative substances" responsible for determination in the egg. This began an intensive study of the nucleic acids of the egg including cytoplasmic DNA and the mechanism of activation of protein synthesis at fertilization. These investiga- tions were cut off when, at age 62, Albert Tyler was at the height of his extraordinarily productive career.

The impact of Albert Tyler's published contributions will long endure. Likewise his influence will continue to be significant through the many students, postdoctorals and friends who received training and inspiration from him in his own laboratory, here at the Marine Biological Laboratory, and at other institutions during visits. Even brief visits had a lasting effect on students. This resulted from an extraordinary combination of energy, personality, imagination and immediate transfer of ideas to laboratory tests.

Albert is survived by his wife, Betty, and two sons: James, a successful engineer, and Steven, a graduate student at Santa Barbara. The family ties were very strong, and all shared Albert's enthusiasm for science and participated in it. Betty and James are co-authors on papers with Albert.

REPORT OF THE DIRECTOR 11

Albert Tyler was sought after to organize and chair many symposia and to function in advisory and consultant capacities to government agencies and private foundations. He served as President of the American Society of Naturalists and the Society of Gen- eral Physiologists. Nevertheless, Albert was alway an informal man who abhored pretense and display. He especially enjoyed small informal gatherings. Here he shared his zest for science through discussion with friends. His many friends here at Woods Hole, as elsewhere, will long remember such discussions and miss the excitement and challenge of Albert's subtle wit and penetrating mind.

2. THE STAFF EMBRYOLOGY

I. CONSULTANT EVERETT ANDERSON, Professor of Biology, University of Massachusetts

II. INSTRUCTORS

MALCOLM S. STEINBERG, Professor of Biology, Princeton University, in charge of course JOHN M. ARNOLD, Assistant Professor of Cytology, Pacific Biomedical Research Center,

University of Hawaii

MAX BURGER, Associate Professor of Biology, Princeton University GARY FREEMAN, Assistant Professor of Biology, University of California at San Diego RALPH T. HINEGARDNER, Associate Professor of Biology, University of California at

Santa Cruz

ANTONE JACOBSON, Professor of Biology, University of Texas HANS LAUFER, Associate Professor of Zoology, University of Connecticut

III. LECTURERS

LIONEL JAFFE, Professor of Biology, Purdue University RAYMOND RAPPAPORT, PROFESSOR OF BIOLOGY, UNION COLLEGE PAUL B. WEISZ, Professor of Biology, Brown University

IV. LABORATORY ASSISTANTS

NORMAN A. GRANHOLM, University of Oregon ROBERT S. TURNER, University of Oregon

V. LECTURES

M. S. STEINBERG Introduction to the course

ANTONE JACOBSON Introduction to the embryology of teleost fishes

\V. \V. BALLARD Gastrulation in teleosts

ANNA RUTH BRUMMETT Experiments on the dynamics of embryo formation in

teleosts

ANTONE JACOBSON Experiments on the control of organ determination

M. S. STEINBERG Self-assembly of multicellular complexes

HERBERT M. PHILLIPS Equilibrium measurements of embryonic cell adhesiveness

M. S. STEINBERG Morphogenetic phenomena in sponges

M . S. STEINBERG Developmental control processes in coelenterate ontogeny

STANLEY SHOSTAK Form-determining mechanisms in hydra

12

ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

EVERETT ANDERSON JEAN C. DAN RALPH T. HINEGARDNKK RAYMOND RAPPAPORT RAYMOND RAPPAPORT RALPH T. HINEGARDNKK LEWIS TILNEY

PAUL GROSS PAUL GROSS A. B. PARDEE LIONEL JAFFE

ERIC DAVIDSON

JOHN M. ARNOLD JOHN M. ARNOLD JOHN M. ARNOLD JACK COLLIER HANS LAUFER HANS LAUFER HERBERT OBERLAXDER GARY FREEMAN IRWIN KOXIGSBERG GARY FREEMAN J. RICHARD WHITTAKER ROGER D. MILKMAN BEATRICE MINTZ

PAUL B. WEISZ

The fine structure of eggs

Fertilization

Echinoderm development: egg to pluteus

Cytokinesis : establishment of the mechanism

Cytokinesis : nature and operation of the mechanism

Echinoderms: life cycle and experimental embryology

Microtubules in mesenchyme formation and differentiation

in Arbacia

Regulation of macromolecule synthesis I Regulation of macromolecule synthesis II Division of bacterial cells Localization in the developing fucus egg, and the general

role of localizing currents Localization of "morphogenetic factors" in the cytoplasm

of animal eggs, and its possible significance Normal development of the spiralian embryo An analysis of spiralian development Development of the cephalopod embryo Gene transcription during Ilyanassa embryogenesis Embryonic development in Crustacea Post embryonic development in Crustacea Effects of insect hormones on imaginal discs in vitro The organization of the ascidian egg Clonal development of embryonic myoblasts in vitro Metamorphosis and asexual reproduction in ascidians Pigment cell differentiation in ascidian embryo Development and genetics of Botryllus schlosseri Four-parent mice offer clues about gene control of mam- malian differentiation The significance of larvae

PHYSIOLOGY

I. CONSULTANTS

MERKEL K. JACOBS, Professor of Physiology, University of Pennsylvania ALBERT SZENT-GYORGYI, Director, The Institute for Muscle Research, Marine Bio- logical Laboratory

W. D. McELROY, Director, McCollum-Pratt Institute, The Johns Hopkins University J. WOODLAND HASTINGS, Professor of Biology, Harvard University

II. INSTRUCTORS

ANDREW G. SZENT-GYORGYI, Professor of Biology, Brandeis University, in charge of

course SYDNEY BRENNER, Medical Research Council, Laboratory of Molecular Biology,

Cambridge, England

RODERICK K. CLAYTON, Professor of Biophysics, Cornell University SEYMOUR S. COHEN, Professor of Biochemistry, University of Pennsylvania HUGH E. HUXLEY, Medical Research Council, Laboratory of Molecular Biology,

Cambridge, England

MAURICE SUSSMAN, Professor of Biology, Brandeis University DAVID A. YPHANTIS, Professor of Biology, University of Connecticut

REPORT OF THE DIRECTOR 13

III. SPECIAL LECTURERS

HARLYN HALVORSON, Professor of Bacteriology, University of Wisconsin

SHINYA INOUE, Professor of Biology, University of Pennsylvania

K. E. VAN HOLDE, Professor of Physical Chemistry, University of Oregon

IV. STAFF ASSOCIATES

RAYMOND E. STEPHENS, Department of Biology, Brandeis University

ANNEMARIE WEBER, Department of Biochemistry, St. Louis University

RAQUEL SUSSMAN, Department of Biology, Brandeis University

PETER NEWELL, Department of Biology, Brandeis University

EUGENE KATZ, Department of Biology, Brandeis University

MICHAEL JOHNSON, Department of Biophysics, University of Connecticut

DENNIS ROARK, Department of Biophysics, University of Connecticut

WALTER F. STAFFORD, III, Department of Biophysics, University of Connecticut

PETER B. MOORE, Department of Molecular Biology, Yale University

DARRELL FLEISCHMAN, Charles F. Kettering Research Laboratory

CELIA E. FREDA, Department of Therapeutic Research, University of Pennsylvania

V. RESEARCH ASSISTANTS

RICHARD WAYNE LINCK, Department of Biology, Brandeis University RITA BURKI, Department of Biochemistry, St. Louis University ELLEN RAYNER, Department of Biology, Brandeis University

B. J. CLAYTON, Department of Genetics, Development and Physiology, Cornell Uni- versity ELLEN STREIBEL, Department of Therapeutic Research, University of Pennsylvania

VI. LABORATORY ASSISTANTS

MARGARET KETCHUM, College of Liberal Arts, Boston University DAVID SLOANE WILSON, University of Rochester

VII. LECTURES

ANDREW G. Aspects of chemistry of muscle contraction

SZENT-GYORGYI Assembly and organization of the filaments of molluscan

muscles

ANNEMARIE WEBER Control of contraction and relaxation

RAYMOND E. STEPHENS "All microtubules are equal but some are more equal than

others"

DAVID A. YPHANTIS Physical approaches in biochemistry I

Physical approaches in biochemistry II Physical approaches in biochemistry III

RODERICK K. CLAYTON Photosynthesis: ancient history: biochemical and evolu- tionary speculations Photosynthesis: breathes there a man with soul so tough,

he thinks two systems aren't enough? Photosynthesis: the value of absorbtion and emission

spectroscopy MAURICE SUSSMAN DNA and RNA in eucaryotic cells: an embarrassment of

riches

The cellular slime molds XLVI The cellular slime molds XLVII

14

ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

HARLYN HALVORSON SEYMOUR S. COHEN

HAROLD S. GINSBERG HENRY MAHLER RACHMIEL LEVINE HUGH E. HUXLEY

IAN R. GIBBONS SHINYA INOUE RUTH SAGER BERNARD D. DAVIS M. V. L. BENNETT

LAWRENCE B. COHEN EDWARD A. KRAVITZ

RUTH HUBBARD RICHARD CONE JOHN E. DOWLING ALBERT SZENT-GYORGYI ARTHUR PARDEE J. WOODLAND HASTINGS DARRELL FLEISCHMAN

HENRY LINSCHITZ

ALVIN NASON KEITH R. PORTER L. C. JUNQUIRA RUTH E. BENESCH REINHOLDT BENES( 11 SYDNEY BRENNER

ALEX KEYNAN

JOHN C. ECCLES K. E. VAN HOLDE GUIDO GUIDATTI KLAUS WEBER EUGENE KENNEDY RICHARD L. SIDMAN EDWARD A. ADELBERG DAVID F. WAUGH

Developmental control in XLVIII

Synthesis of phage proteins I

Synthesis of phage proteins II

Functions of polyamines

Synthesis of adenoviruses

Biogenetic autonomy of yeast mitochondria

Action of insulin

X-ray diffraction results on striated muscle

Functional implications of muscle structure

Aspects of ciliary movement

Inscrutable fibers of the spindle

What does chloroplast DNA do?

Ribosome polysome cycle

Central dogma of neurophysiology I

Central dogma of neurophysiology II

Changes in optical properties of nerves during activity

Biochemical studies on single neurons and preliminary observations on lobster ganglia in long-term culture

Chemistry of visual photoreception

Photoreceptor excitation mechanisms

The vertebrate retina: an approachable piece of the brain

Biology of Donor-Acceptor interactions

Biochemical studies in active transport

The chemistry and biology of excited singlet oxygen

Luminescence and membrane potential in photo-syn- thetic bacteria

Kinetic studies of photoreaction in phytochrome, chloro- phyl and rhodopsin

Assimilatory nitrate reductase from Nenrospora

Microtubules in fish chromatophores

Control mechanism for oxygen release by hemoglobin I

Control mechanism for oxygen release by hemoglobin II

Control mechanisms I

Control mechanisms 1 1

Control mechanisms III

The outgrowing bacti-rial endospore as a system for the

study of unicellular differentiation

Integration of information by cerebellar Purkinje cells Pulsed-field electric dichroism of macromolecules Structure and function of hemoglobin Structure of aspartic transcarbamylase Membrane structure and function Cell interaction in developing mouse brain DNA transfer and bacterial conjugation Clotting mechanisms, implants and thrombosis

ALAN WEEDS JOSEPH ILAN

VIII. SPECIAL SEMINARS

The light chains of the myosin molecule Translational control of specific mRNA during insect metamorphosis

REPORT OF THE DIRECTOR 15

EXPERIMENTAL MARINE BOTANY

I. CONSULTANTS

STERLING B. HENDRICKS, U. S. Department of Agriculture WILLIAM S. HILLMAN, Brookhaven National Laboratory BESSELL KOK, Research Institute for Advanced Studies JOHAN A. HELLEBUST, Harvard University

II. INSTRUCTORS

HAROLD W. SIEGELMAN, Plant Biochemist, Brookhaven National Laboratory, in charge

of course

TREVOR W. GOODWIN, Professor of Biochemistry, University of Liverpool, England ROBERT R. L. GUILLARD, Associate Scientist, Woods Hole Oceanographic Institution FRANK A. LOEWUS, Professor of Biology, State University of New York at Buffalo JOHN M. OLSON, Biophysicist, Brookhaven National Laboratory ROBERT T. WILCE, Associate Professor of Botany, University of Massachusetts

III. SPECIAL LECTURERS

LAWRENCE BOGORAD, Professor of Biology, Harvard University

MARTIN GIBBS, Professor of Biology, Brandeis University

SARAH GIBBS, Associate Professor of Botany, McGill University

Louis A. HANNIC, Assistant Professor of Botany, Dalhousie University

JOHN A. HELLEBUST, Associate Professor of Biology, Harvard University

HARVARD LYMAN, Associate Professor of Biology, State University of New York at

Stony Brook

GORDON MACLACHLAN, Associate Professor of Botany, McGill University CARL A. PRICE, Professor of Botany, Rutgers University JOSEPH RAMUS, Assistant Professor of Biology, Yale University JEROME SCHIFF, Professor of Biology, Brandeis Universitv

IV. ASSISTANT AND COLLECTOR WILLIAM A. McDANiEL, Botany Department, University of Massachusetts

V. LECTURES

H. W. SIEGELMAN Photochemically-active chromoproteins of plants

C. A. Price Zonal centrifugation

JOHN M. OLSON Photosynthesis — evolution, structure and function

J. A. HELLEBUST Excretion of organics by phytoplankton

FRANK A. LOEWUS Structure and biosynthesis of plant cell walls and poly-

saccharides

SARAH GIBBS Autoradiographic studies on RNA synthesis in chloroplasts

G. MACLACHLAN Cellulose metabolism in relation to growth

T. W. GOODWIN] Introduction to the carotenoids, _steroids, and related

substances

HARVARD LYMAN Euglena I

R. R. L. GUILLARD Nutritional requirements and productivity of phyto-

plankton

MARTIN GIBBS Path of carbon in photosynthesis

L. A. HANIC Plant cell wall with special reference to the algae

16 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

MAX BLOOMER The role of hydrocarbons in the sea

L. BOGORAD Chloroplast development

ROBERT T. WILCE Introduction to the major groups of benthic marine algae

J. RAMUS Aspects of the fine structure and developmental mor-

phology of Pseudogloiophloea in culture J. SCHIFF Euglena II

EXPERIMENTAL INVERTEBRATE ZOOLOGY

I. CONSULTANTS

FRANK A. BROWN, JR., Morrison Professor of Zoology, Northwestern University

C. LADD PROSSER, Professor of Physiology, University of Illinois

CLARK P. READ, Professor of Biology, Rice University

ALFRED C. REDFIELD, Woods Hole Oceanographic Institution

W. D. RUSSELL-HUNTER, Professor of Zoology, Syracuse University

II. INSTRUCTORS

JAMES F. CASE, Professor of Biology, University of California, Santa Barbara, in charge

of course

FRANK M. FISHER, JR., Associate Professor of Biology, Rice University DAVID C. GRANT, Assistant Professor of Biology, Davidson College JONATHAN P. GREEN, Assistant Professor of Biology, Brown University MICHAEL J. GREENBERG, Associate Professor of Biology, Florida State University ROBERT K. JOSEPHSON, Associate Professor of Biology, Case Western Reserve Uni- versity

CHARLOTTE P. MANGUM, Associate Professor of Biology, College of William and Mary ALAN GELPERIN, Assistant Professor of Biology, Princeton University

III. SPECIAL LECTURERS

EDWARD R. BAYLOR, Professor of Biology, State University of New York at Stony

Brook

W. D. RUSSELL-HUNTER, Professor of Zoology, Syracuse University MAHLON E. KRIEBEL, State University of New York, Upstate Medical Center JAMES G. MORIN, Harvard University C. LADD PROSSER, Professor of Physiology, University of Illinois

IV. LABORATORY ASSISTANTS

ELDON E. BALL, University of California, Santa Barbara ALAN C. EGGLESTON, University of California, Santa Barbara ROGER C. HALVERSON, University of California, Santa Barbara

V. LECTURES

H. BURR STEINBACH Introduction to the Marine Biological Laboratory

ROBERT K. JOSEPHSON Coelenterata and Ctenophora

JAMES F. CASE The study of invertebrates

FRANK M. FISHER, JR., Platyhelminthes, Aschelminthes, Nemertea, I and II

DAVID C. GRANT The Cape Cod environment

JAMES F. CASE Porifera

REPORT OF THE DIRECTOR

17

CHARLOTTE P. MANGUM JONATHAN P. GREEN MICHAEL J. GREENBERG W. D. RUSSELL-HUNTER ALAN GELPERIN JAMES F. CASE DAVID C. GRANT FRANK M. FISHER, JR.,

CHARLOTTE P. MANGUM C. LADD PROSSER CHARLOTTE P. MANGUM

DAVID C. GRANT MICHAEL J. GREEXBERG

MAHLON E. KRIEBEL MICHAEL J. GREENBERG

JONATHAN P. GREEN

JAMES F. CASE EDWARD R. BAYLOR

JAMES F. CASE JAMES G. MORIN JAMES F. CASE ROBERT K. JOSEPHSON

ALAN GELPERIN

Annelids

Arthropoda, I and II

Mollusca

Ancestors and archetypes with reference to Neopilina

Echinodermata

Protochordata

Plankton

Cellular mechanisms of ingestion

Ingestion mechanisms of multicellular animals

Digestive physiology

Respiration : principles

Thermal adaptation

Respiration : exchange mechanisms

Respiration : transport mechanisms

Community structure and diversity

Circulation: some aspects of hemodynamics including

flow7 in ciliated tubes Physiology of the tunicate heart Circulation: comparative physiology of the heart, ABRM

and other muscles

Xeuroendocrinology of annelids, arthropods and molluscs Physiology of the arthropod exoskeleton Osmoregulation Luminescence Stimuli associated with feeding behavior and aggregation

of zooplankton in the sea Sensory physiology : I Obelia luminescence Sensory physiology: II

Behavior without brains: I. plants, protozoa, and sponges Behavior without brains: II, cnidarians Increasing neural complexity in lower metazoa Strategies of behavioral physiology Executive neurons Complex behavior in simple systems

MARINE ECOLOGY

I. CONSULTANTS

MELBOURNE R. CARRIKER, Director, Systematics-Ecology Program BOSTWICK H. KETCHUM, Woods Hole Oceanographic Institution EDWIN T. MOUL, Rutgers University

JOHN H. RYTHER, Woods Hole Oceanographic Institution HOWARD L. SANDERS, Woods Hole Oceanographic Institution

II. INSTRUCTORS

LAWRENCE B. SLOBODKIN, Department of Biology, State University of New York at

Stony Brook, in charge of course EDWARD R. BAYLOR, Department of Biology, State University of New York at Stony

Brook

18

ANN I Al. k I- I 'OK I OK Till; MAKIXK BIOLOGICAL LABORATORY

SUMNER RICHMAN, Department of Biology, Lawrence University

JOHN STEELE, Department of Agriculture and Fisheries, Aberdeen, Scotland

W. ROWLAND TAYLOR, Department of Earth and Planetary Sciences, The Johns

Hopkins University and the Chesapeake Bay Institute EDWARD O. WILSON, Department of Biology, Harvard University

III. SPECIAL LECTURER LEV FISHELSON, University of Tel Aviv, Israel

IV. LABORATORY ASSISTANTS

HERMAN F. BOSCH, Department of Earth and Planetary Sciences, The Johns Hopkins

University WAYNE H. BELL, Department of Biology, Middlebury College

LAWRENCE SLOBODKIN

\Y. ROWLAND TAYLOR

S. KATONA SUMNER RICHMAN

SUMNER RICHMAN

LAWRENCE SLOBODKIN EDWARD O. WILSON

JOHN STEELE

R. MITCHELL JOHN STEELE

V. LECTURES

Introduction to the course

The politics of ecology

Approaches to ecology

The strategy of evolution

The marine environment I. Chemistry of seawater

The marine environment II. Solar radiation through sea- water

Phytoplankton I

Phytoplankton II

Primary productivity

Zooplankton

Introduction to ecological energetics

Measurement of zooplankton feeding

Factors affecting zooplankton feeding

Feeding of Calanus on synchronously growing cultures of diatoms

Bomb calorimetry and respiration

Predation theory

Classical models of ecological systems

Animal communication I

Animal communication II

Colonization and species equilibrium I

Colonization and species equilibrium II

Speciation I

Speciation II

North Sea food chains

Chlorophyll and C14 uptake in sand

Antagonistic interactions between micro-organisms

Pelagic food chains

Oxygen uptake by sand

Benthic food chains

Energy flow in sand

Theoretical models of food chains

An experimental study of a marine food chain.

REPORT OF THE DIRECTOR 19

EDWARD R. BAYLOR Light-stimulated animal behavior

Analysis of behavior by experiment and computer Cladoceran behavior — stimuli Cladoceran behavior— hunting and feeding Plankton patches and sea surface circulation I Plankton patches and sea surface circulation II

HOWARD L. SANDERS Physical and biological control of communities

EDWARD R. BAYLOR Plankton patches and sea surface circulation III

LEV FISHELSON The ecology of coral reefs in the Gulf of Aquaba

SYSTEM ATI CS-ECOLOGY PROGRAM

THE STAFF

Director: MELBOURNE R. CARRIKER

Resident Systematist (Zoology) : ROBERT P. HIGGINS

Acting Resident Systematist (Botany) : ROBERT T. WILCE

Resident Ecologist : DAVID K. YOUNG

Assistant Ecologist: KATHARINE D. HOBSON

Postdoctoral Fellows and Research Associates: DAVID G. COOK, CELIA R. HAIGH,

LAWRENCE R. MCCLOSKEY, NORMAN R. SINCLAIR, WILLIAM C. SUMMERS GraduateResearch Trainees : WILLIAM R. COBB, JOAN R. CONWAY, MARY ANN GILBERT,

\VILLIAM H. GILBERT, WILLIAM D. HUMMON, JEFFREY S. LEVINTON, ALLAN D.

MICHAEL, LELAND W. POLLOCK, JAMES R. SEARS Visiting Investigators in Residence: EDWARD L. BOUSFIELD, LOUISE BUSH, MARTIN

A. BUZAS, HOWARD H. CHAUNCEY, PATRICIA L. DUDLEY, MARIE U. NYLEN, JOEL

S. O'CONNOR, PHILIP PERSON, HAROLD H. PLOUGH, DONALD C. RHOADS, WESLEY

N. TIFFNEY, RUTH D. TURNER, VICTOR Z. ZULLO

Consultants : WILLIAM RANDOLPH TAYLOR, RUTH D. TURNER, ROBERT T. WILCE Curator: JOHANNA M. REINHART

Assistant Curator (Gray Museum Herbarium) : JOAN R. CONWAY Technical Field Assistant: PETER J. OLDHAM Field Assistant: FRANCIS DOOHAN

Scientific Illustrators: RUTH von ARX, SUSAN P. HELLER Captain, R/V A. E. YERRILL: JAMES P. OSTERGARD Mate, R/V A. E. YERRILL: PETER GRAHAM

Administrative Assistants: HAZEL VV. FERMINO, CONSTANCE A. BRACKETT Program Secretary : EVA S. MONTIERO Research Assistants: DONALD F. ALLEN, ANNE C. COLLINS, MARY ANN GILBERT,

WILLIAM H. GILBERT, GAIL GINGRASS, THEODORE J. GRANT, DAVID J. HARTZBAND,

MARLYN J. HUMMEL, RICHARD A. MCGRATH, CHARLES R. McKAY, JOHN J. Mc-

MAHON, ELLEN P. Me REYNOLDS, LELAND W. POLLOCK, ALFRED POOR, CAROL Q.

SCHWAMB, ANNE SMARSH, MICHAEL A. SOUKUP, MARTHA SPIERS, WILLIAM STAMM,

RICHARD TRAVERSE, LINDA VACCA, DIRK VAN ZANDT Visitors: MOHAMED HYDER, PIERRE LASSERRE, BARRY A. WADE

SEMINARS (WINTER INCLUDED) KARL BANSE Oxygen consumption by the subtidal sea bed and the

importance of the macrofauna

JOHN S. RANKIN, JR. Antarctic benthic communities

CELIA R. HAIGH Polygamy in the redwinged blackbird

NORMAN R. SINCLAIR Studies on the heterophyid trematodes Apophallus brevis

and A. imperator ROBERT R. HESSLER Deep sea isopods

20

ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

WILLIAM D. HUMMON SANFORD A. Moss WILLIAM GOSLINE

TOM FENCHEL JAMES A. SLATER JAMES R. SEARS

ROBERT A. MURCHELANO DAVID K. YOUNG JOANNA Z. PAGE WESLEY N. TIFFNEY, JR.

WILLIAM C. LEGGETT

ROBERT MARAK BARRY A. WADE

LAWRENCE R. MCCLOSKEY PHILIP B. ROBERTSON R. JACK SCHULTZ MELBOURNE R. CARRIKER

PIERRE LASSERE JOEL O'CONNOR

NORMAN HOLME ROBERT L. VADAS EDWARD L. BOUSFIELD

VICTOR A. ZULLO

DONALD C. RHOADS DAVID W. FOLGER

LEWIS A. HANIC IVAN VALIELA LELAND W. POLLOCK MARTIN A. BUZAS

JOHN C. Russ CLAY SASSAMAN M. PATRICIA MORSE LOUISE F. BUSH

Interspecific competition between gastrotrichs on several

Woods Hole beaches Heated effluents and the behavior of young shad exposed

to thermal gradients Some relationships between functional morphology and

bony iish classification Aspects of research in interstitial ecology Aspects of biogeography in South Africa Aspects of life history and habitat ecology of sub-littoral benthic marine algae

Bivalve larval culture — bacteriological considerations Biogenic reworking of marine sediments Control of reproduction in Derbesia Relationship between tree-line and snow cover in the White

Mountains, N. H. Observations on the migratory behavior of American shad

in the Connecticut River utilizing ultrasonic tracking Determination of peak spawning times of haddock Kingston Harbor — a case study in conservation and

development

Ecology of an echinospira larvae Larval development of scyllarid lobsters Evolution of unisexuality and polyploidy in fish Excavation of boreholes by the gastropod Urosalpinx :

an analysis by means of light and scanning electron

microscopy Distribution of meiobenthic oligochaetes as explained by

physiological ecology Distribution of fishes over natural and sand-filled bottoms

in a Long Island estuary Benthos study techniques Agarum and the kelp bed community

Adaptive radiation in sand-burrowing amphipod crusta- ceans Temperate marine faunas: a paleontologist's view of the

present

The use of bivalves as environmental data recorders Land-derived biogenic participates recovered from surface

water and air during two Atlantic crossings Seaweed farming — problems and solutions Invasion and structuring of dung arthropod communities Aspects of the ecology of interstitial tardigrades Foraminiferal densities and environmental variables: the

Linear model in marine ecology Characteristics and applications of the scanning electron

microscope Aspects of temperature and low oxygen adaptations in

coelenterates Direct development in the nudibranch mollusc, Coryphella

salmonacea Couthouy Small marine turbellaria of Cape Cod: some history,

comments and questions

REPORT OF THE DIRECTOR 21

THE LABORATORY STAFF HOMER P. SMITH, GENERAL MANAGER

Miss JANE FESSENDEN, Librarian ROBERT KAHLER, Superintendent Build-

CARL O. SCHWEIDENBACK, Manager, Sup- ings and Grounds

ply Department ROBERT GUNNING, Assistant Superin-

JOHN J. VALOIS, Assistant Manager, Sup- tendent, Buildings and Grounds

ply Department ROBERT B. MILLS, Manager, Department

FRANK A. WILDES, Controller of Research Service

GENERAL OFFICE

MRS. VIRGINIA R. ARTHUR MRS. VIVIAN I. MANSON

EDWARD J. BENDER Miss ELAINE C. PERRY

MRS. FLORENCE S. BUTZ MRS. CYNTHIA S. REGAN

MRS. COLLEEN P. HALL Miss MARY TAVARES MRS. ANN W. LOOMIS

LIBRARY

MRS. VIRGINIA BRANDENBURG MRS. LENORA JOSEPH

Miss ELIZABETH A. BROWN MRS. DORIS RICKER

DAVID J. FITZGERALD

MAINTENANCE OF BUILDINGS AND GROUNDS

ELDON P. ALLEN RALPH H. LEWIS

JOHN T. BRADY RUSSELL F. LEWIS

BERNARD F. CAVANAUGH KENNETH J. MARKS

CECIL COSTA STEPHEN A. MILLS

JOHN V. DAY WILLY M. NEILSON

MANUEL P. DUTRA ROBERT A. SERRANO

CHARLES FUGLISTER FREDERICK E. THRASHER

RICHARD E. GEGGATT, JR. FREDERICK E. WARD

DONALD B. LEHY EDWARD J. WHITE

DEPARTMENT OF RESEARCH SERVICES

GAIL M. CAVANAUGH MRS. JULIETTE T. LAUZON

LOWELL V. MARTIN FRANK E. SYLVIA

SUPPLY DEPARTMENT

COURTNEY BARBER, III Miss JOYCE B. LIMA

DAVID H. GRAHAM EUGENE TASSINARI

ROBERT HAMPTON BRUNO F. TRAPASSO

LEWIS M. LAWDAY JOHN VARAO ROBERT O. LEHY

22 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

DINNING HALL AND HOUSING

ROBERT T. MARTIN, Manager, Food Service NORMAN MICHAUD, Head Chef MRS. ELIZABETH KUIL, Supervisor, Dining Room MRS. ELLEN T. NICKELSON, Supervisor, Dormitories ALAN G. LUNN, Supervisor, Cottage Colony

3. INVESTIGATORS: LILLIE, GRASS AND RAND FELLOWS; STUDENTS

Independent Investigators, 1969

ABRAHAMSON, EDWIN \V., Professor of Chemistry, Case Western Reserve University ADELMAN, WILLIAM J., JR., Professor of Physiology, University of Maryland, School of Medicine AGARWAL, RAM, A., Research Associate, Florida State University AKIYAMA, TOYOHIRO, Research Associate, Columbia University

ALLEN, ROBERT DAY, Chairman and Professor, Department of Biological Sciences, State Uni- versity of New York at Albany

ANDERSON, EVERETT, Professor of Zoology, University of Massachusetts, Amherst APRIL, ERNEST W., Postdoctoral Fellow, Columbia University ARMSTRONG, PHILLIP B., Professor of Anatomy, State University of New York, Upstate Medical

Center

ARNOLD, JOHN M., Assistant Professor, University of Hawaii ATKINSON, BURR G., Postdoctoral Research Associate, Florida State University ATWOOD, K. C., Professor of Microbiology, University of Illinois, Urbana AUCLAIR, WALTER, Associate Professor, Rensselaer Polytechnic Institute BAL, ARYA K., Assistant Professor, University of Montreal BARTELL, CLELMER K., Research Associate, Tulane University BAUER, G. ERIC, Assistant Professor, University of Minnesota BAYLOR, E. R., Professor of Biology and Marine Sciences, State University of New York at

Stony Brook

BAYLOR, MARTHA B., State University of New York at Stony Brook BELAMARICH, FRANK A., Associate Professor, Boston University BELL, ALLEN L., Assistant Professor, University of Colorado Medical Center BENAMY, DANIEL A., Research Associate, Columbia University BENNETT, M. V. L., Professor of Anatomy, Albert Einstein College of Medicine BIGGERS, JOHN D., Professor of Biology, The Johns Hopkins School of Hygiene and Public

Health BRANDT, PHILIP WILLIAMS, Associate Professor, College of Physicians and Surgeons, Columbia

University BRENNER, SYDNEY, Head of the Division of Molecular Genetics, Medical Research Council,

Laboratory of Molecular Biology, Cambridge England

BROWN, FRANK A., JR., Morrison Professor of Biology, Northwestern University BROWN, JOEL E., Associate Professor of Physiology, Massachusetts Institute of Technology BORTOFF, ALEXANDER, Associate Professor, State University of New York, Upstate Medical

Center

BUCK, JOHN B., Chief, Laboratory of Physical Biology, National Institutes of Health BURGER, MAX M., Associate Professor of Biology, Princeton University BUSH, LOUISE, Visiting Investigator in Residence, Systematics-Ecology Program BUZAS, MARTIN A., Visiting Investigator in Residence, Systematics-Ecology Program CARNAY, LAURENCE D., Research Associate, National Institutes of Health CARRIKER, MELBOURNE R., Director, Systematics-Ecology Program CASE, James F., Professor of Zoology, University of California, Santa Barbara CASS, ALBERT, Fellow of the Interdepartmental Institute for Neurological Sciences, Albert

Einstein College of Medicine CASSIDY, FR. JOSEPH D., O. P., Assistant Professor of Biology and Honors Professor of Modern

Genetics, Providence College and Northwestern University

REPORT OF THE DIRECTOR

CHAMBERS, EDWARD L., Professor of Physiology and Biochemistry, University of Miami CHAUNCEY, HOWARD, Chief, Research in Oral Diseases, Veterans Administration Central Office,

Washington, D. C.

CHIARANDINI, DANTE T., Research Associate, Columbia University CHILD, FRANK M., Associate Professor of Biology, Trinity College CLAYTON, RODERICK K., Professor of Biology and Biophysics, Cornell University CLEMENT, A. C., Professor of Biology, Emory University COGXETTI, GOFFREDO, University of Palermo, Italy

COHEN, LAWRENCE B., Assistant Professor of Physiology, Yale University School of Medicine COHEN, SEYMOUR S., Chairman and Professor, Department of Therapeutic Research, University

of Pennsylvania Medical School

COLE, KENNETH S., Staff Member, Laboratory of Biophysics, National Institutes of Health COLWIX, ARTHUR L., Professor of Biology, Queens College, The City University of New York COLWIN, LAURA HUNTER, Professor of Biology, Queens College, The City University of New York COOHILL, THOMAS P., Assistant Professor, Veterans Administration Hospital, Leech Farm and

University of Pittsburgh Medical School

COOK, DAVID G., Research Associate, Systematics-Ecology Program COOPERSTEIN, SHERWIN J., Professor of Anatomy, University of Connecticut COPELAND, DONALD EUGENE, Professor of Biology, Tulane University COSTELLO, DONALD P., Kenan Professor of Zoology, University of North Carolina COUSINEAU, GILLES H., Associate Professor, University of Montreal CRIPPA MARCO, Assistant Professor, University of Palermo, Italy CROWELL, SEARS, Professor of Zoology, Indiana University DAN, JEAN CLARK, Professor, Ochanomizu University, Tokyo

DAVIS, ROBERT F., Trainee Fellow, College of Physicians and Surgeons, Columbia University DAW, NIGEL \V., Assistant Professor, Washington University School of Medicine and Harvard

Medical School

DAWE, CLYDE J., Investigator, Laboratory of Pathology, National Cancer Institute DEGUCHI, TAKEHIKO, Research Associate, Duke University Medical Center DEHAAN, ROBERT L., Research Staff Member, Carnegie Institution of Washington DE LORENZO, A. J. DARIN, Director and Professor of Laboratory of Neurobiology, The Johns

Hopkins University School of Medicine

DEPHILLIPS, HENRY A., JR., Associate Professor of Chemistry, Trinity College DETTBARN, WOLF-DIETRICH, Professor of Pharmacology, Vanderbilt University School of

Medicine

DE WEER, PAUL J., Instructor, University of Maryland School of Medicine DIKE, SUE CARLISLE, Postdoctoral Fellow, Rice University DODGE, FREDERICK A., JR., Associate Professor, The Rockefeller University DOWLING, JOHN E., Associate Professor of Ophthalmology and Biophysics, The Johns Hopkins

University

DRESDEN MARC H., Assistant Professor, Baylor University College of Medicine DUDLEY, PATRICIA L., Visiting Investigators in Residence, Systematics-Ecology Program DUNHAM, PHILIP B., Associate Professor of Zoology, Syracuse University ECCLES, JOHN C., Distinguished Professor of Biophysics and Physiology, State University of

New York at Buffalo ECCLES, HELENA T., Assistant Research Professor of Physiology, State University of New York

at Buffalo

ECKMAN, PETER, Graduate Student, New York University School of Medicine EGYUD, LASZLO G., The Institute for Muscle Research

EHRENSTEIN, GERALD, Physicist, Laboratory of Biophysics, National Institutes of Health EISEN, ARTHUR Z., Associate Professor of Medicine, Washington University School of Medicine ELKINS, WILLIAM L., Assistant Professor, University of Pennsylvania School of Medicine ELLIOTT, GERALD F., Scientific Staff, Medical Research Council, London ELLIS, KEITH, Posdoctoral Fellow, University of Cincinnati EPSTEIN, HERMAN T., Professor of Biophysics, Brandeis University ERULKAR, SOLOMON DAVID, Professor of Pharmacology, University of Pennsylvania FABER, DONALD S., Postdoctoral Fellow, State University of New York at Buffalo FARMANFARMAIAN, A., Associate Professor of Physiology, Rutgers University

24 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

FEDER, NED, Chief, Section on Biophysical Histology, National Institutes of Health

FERNANDEZ, HUGO L., DSR Staff Member, Massachusetts Institute of Technology

FINGERMAN, MILTON, Chairman and Professor of Biology, Tulane University

FERTZIGER, ALLEN P., Postdoctoral Fellow, Albert Einstein College of Medicine

FISHER, F. M., JR., Associate Professor of Biology, Rice University

FISHMAN, HARVEY M., National Institutes of Health

FLEISCHMAN, DARRELL E., Staff Scientist, Charles F. Kettering Research Laboratory

FRAZIER, DONALD T., Associate Professor of Physiology, University of Kentucky

FREDA, CELIA E., Postdoctoral Fellow, University of Pennsylvania

FREEMAN, ALAN RICHARD, Associate Professor of Physiology, Rutgers Medical School

FREEMAN, GARY, Assistant Professor of Biology, University of California, San Diego

FUORTES, M. G. F., Chief, Laboratory of Neurophysiology, National Institutes of Health

GIBBONS, IAN R., Professor of Biophysics, University of Hawaii

GELPERIN, ALAN, Assistant Professor of Biology, Princeton University

GILBERT, DANIEL L., Head, Section on Cellular Biophysics, National Institutes of Health

GOLDIZEN VERNON C., Director of Research, Aquarium Systems, Inc.,

GOLDSMITH, TIMOTHY H., Associate Professor of Biology, Yale University

GOOCH, JAMES L., Assistant Professor of Biology, Juniata College

GORMAN, ANTHONY L. F., Research Physiologist, National Institutes of Health

GRAHAM, DONALD, NIH Special Fellow, Case Western Reserve University

GRAHAM, LEWIS T., JR., Assistant Professor, Institute Psychiatric Research, Indiana University

Medical Center

GRANT, DAVID C., Assistant Professor of Biology, Davidson College GRANT, PHILIP, Professor of Biology, University of Oregon GREEN, JONATHAN P., Assistant Professor, Brown University GREENBERG, MICHAEL J., Associate Professor, Florida State University GROSCH, DANIEL S., Professor of Genetics, North Carolina State University, Raleigh GROSS, PAUL R., Professor of Biology, Massachusetts Institute of Technology GROSSMAN, ALBERT, Assistant Professor, New York University Medical School GRUNDFEST, HARRY, Professor of Neurology, Columbia University GUILLARD, ROBERT R. L., Associate Scientist, Woods Hole Oceanographic Institution GUTTMAN, RITA, Associate Professor of Biology, Brooklyn College, The City University of New

York

HAIGH, CELIA R., Research Associate, Systematics-Ecology Program HALVORSON, HARLYN O., Professor of Molecular Biology, University of Wisconsin HARRINGTON, GLENN W., Research Associate, State University of New York, Upstate Medical

Center

HARRISON, STEPHEN C., Junior Fellow in the Society of Fellows, Harvard University and Child- ren's Cancer Research Foundation

HARTSHORNE, DAVID J., Assistant Professor, Mellon Institute, Carnegie-Mellon University HASCHEMEYER, AUDREY E. V., Associate in Biological Chemistry, Massachusetts General Hospital

and Harvard Medical School

HASTINGS, J. WOODLAND, Professor of Biology, Harvard University HAYASHI, HIDEO, Research Associate, Columbia University

HAYASHI, TERU, Chairman, Department of Biology, Illinois Institute of Technology HEILBRONN, EDITH, Associate Professor, Research Institute of National Defence, Sweden and

Biochemistry Institute, University of Uppsala, Sweden

HENKIN, ROBERT L, Chief, Section of Neuroendocrinology, National Institutes of Health HENLEY, CATHERINE, Visiting Associate Professor of Zoology, University of North Carolina HERVEY, JOHN P., Senior Electronics Engineer, The Rockefeller University HIGGINS, DON C., Associate Professor of Neurology, Yale University School of Medicine HINEGARDNER, RALPH T., Associate Professor, University of California, Santa Cruz HINSCH, GERTRUDE W., Institute of Molecular Evolution, University of Miami HOLT, CHARLES E., Associate Professor of Biology, Massachusetts Institute of Technology HOLTZMAN, ERIC, Assistant Professor of Biological Sciences, Columbia University HOLZ, GEORGE G., JR., Chairman and Professor of Microbiology, State University of New York,

Upstate Medical Center

REPORT OF THE DIRECTOR 25

HOSKIN, FRANCIS C. G., Assistant Professor of Biochemistry, College of Physicians and Surgeons,

Columbia University

HUBBARD, RUTH, Research Associate and Lecturer, Harvard University HUGHES, GEORGE M., Head and Professor of Zoology, University of Bristol, England HUXLEY, HUGH E., Laboratory of Molecular Biology, Cambridge, England ILAN, JOSEPH, Assistant Professor of Biology, Temple University ILAN, JUDITH, Research Associate, Temple University

INOUE, SADAYUKI, Assistant Professor of Biology, University of Montreal, Canada INOUE, SHINYA, Professor of Biology, University of Pennsylvania ITO, SUSUMU, Associate Professor of Anatomy, Harvard Medical School JACOBSON, ANTONE G., Professor of Zoology, University of Texas at Austin JANISZEWSKI, LESZEK, Chairman of Department of Neurophysiology, N. Copernicus University,

Poland

JONES, MEREDITH L., Curator, Division of Worms, Smithsonian Institution JOSEPHSON, ROBERT K., Associate Professor of Zoology, Case Western Reserve University KAMINER, BENJAMIN, Institute for Muscle Research

KANATANI, HARUO, Associate Professor, Ocean Research Institute, University of Tokyo KANEKO, AKIMICHI, Research Fellow in Neurobiology, Harvard Medical School KATZ, GEORGE M., Assistant Professor, Columbia University KEM, WILLIAM READE, Postdoctoral Fellow, Duke University

KEYNAN, ALEXANDER, Head and Professor of Biological Sciences, Hebrew University, Jerusalem KITAI, S. T., Associate Professor, Wayne State University School of Medicine KOBATAKE, YONOSUKE, Visiting Scientist, National Institutes of Health KOHLER KURT, Professor, CNRS, Montpellier, France KREMZNER, LEON T., Assistant Professor of Biology, College of Physicians and Surgeons, Columbia

University KRIEBEL, MAHLON E., Assistant Professor of Physiology, State University of New York, Upstate

Medical Center

KRUPA, PAUL L., Assistant Professor of Biology, The City College of New York KUSANO, KIYOSHI, Associate Professor, Indiana University Medical School LAKI, KOLOMAN, Chief, Laboratory of Biophysical Chemistry, National Institutes of Health LAMARCHE, PAUL H., Research Associate and Associate Physician-in-Chief, Departments of

Pathology and Pediatrics, Rhode Island Hospital

LANDOWNE, DAVID, Postdoctoral Research Fellow, Yale University School of Medicine LASTER, LEONARD, Chief, Section on Gastroenterology, National Institutes of Health LAUFER, HANS, Associate Professor of Biology, University of Connecticut LAZAROW, ARNOLD, Head and Professor of Anatomy, University of Minnesota LERMAN SIDNEY, Professor of Ophthalmology and Biochemistry, McGill University, Canada LEVY, MILTON, Chairman and Professor of Biochemistry, New York University College of

Dentistry

LINDAUER, MARTIN, Professor, Biological Institute, University of Frankfort, Germany LIUZZI, ANTHONY, Assistant Professor, School of Public Health, Yale University School of Medicine LOEWENSTEIN, WERNER R., Professor of Physiology, Columbia University LOEWUS, FRANK, Professor of Biology, State University of New York at Buffalo LORAND, JOYCE BRUNER, Research Associate, Northwestern University LORAND, L., Professor of Chemistry, Northwestern University MAcNicHOL, EDWARD F., JR., Director, National Institute of Neurological Diseases and Stroke,

National Institutes of Health

McCLOSKEY, LAWRENCE R., Research Associate, Systematics-Ecology Program MANGUM, CHARLOTTE P., Associate Professor of Biology, College of William and Mary MARSLAND, DOUGLAS, Research Professor Emeritus, New York University MARUYAMA, KOSCAK, Associate Professor, University of Tokyo MATSUMURA, FUMIO, Associate Professor of Entomology, University of Wisconsin MAUTNER, HENRY G., Professor of Pharmacology Yale University School of Medicine McCuTCHAN, MARY CAROLINE, Postdoctoral Fellow, University of Pennsylvania McREYNOLDS, JOHN S., Staff Associate, National Institutes of Health

MEHTA, PANKAJ, Lecturer in Ophthalmology and Biochemistry, McGill University, Canada MENDELSON, MARTIN, Assistant Professor of Physiology, New York University School of Medicine

26 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

METUZALS, J., Professor of Histology, University of Ottawa, Canada

METZ, CHARLES B., Professor of Zoology, Institute of Molecular Evolution, University of Miami

MILKMAN, ROGER D., Professor of Zoology, University of Iowa

MILLER, RICHARD L., Assistant Professor of Biology, Temple University

Mi ROW, SUSAN, Teaching Assistant, New York Medical College

MISRA, BRAJ M., Research Fellow, Harvard Medical School

MONKOY, AI.HKRTO, Professor of Anatomy, University of Palermo, Italy

MOORK, John W., Professor of Physiology and Pharmacology, Duke University

MOORK, PETER B., Assistant Professor of Biophysics, Yale University

MORGADES, PILAR P., Visiting Scholar, Columbia University College of Physicians and Surgeons

MORIN, James G., Graduate Student, Harvard University

MORLOCK, NOEL, Assistant Professor, Columbia University

MOTE, MICHAEL I., Postdoctoral Research Fellow, Yale University

MULLINS, LORIN J., Chairman and Professor of Biophysics, University of Maryland School of

Medicine

MURAYAMA, KoiCHi, Research Associate, Duke University

NADELHAFT, I., Research Physicist, Veterans Administration Hospital, Pittsburgh NARAHASHI, TOSHIO, Associate Professor of Physiology, Duke University NELSON, LEONARD, Chairman, Department of Physiology, Medical College of Ohio at Toledo NEWELL, PETER C., Postdoctoral Fellow, Brandeis University NYSTROM, RICHARD A., Associate Professor, University of Delaware OBERPRILLER, JEAN C., Assistant Professor of Anatomy, LTniversity of North Dakota OBERPRILLER, JOHN, Assistant Professor of Anatomy, University of North Dakota OLIVEIRO-CASTRO, GILBERTO, Research Associate, College of Physicians and Surgeons, Columbia

University

OLSON, JOHN M., Biophysicist, Brookhaven National Laboratory PALMER, JOHN D., Chairman, Department of Biology, New York University PALTI, YORAM, Lecturer in Physiology, Hebrew University Medical School, Jerusalem PAPPAS, GEORGE D., Professor of Anatomy, Albert Einstein College of Medicine PEARLMAN, ALAN L., Assistant Professor of Physiology and Neurology, Washington University

School of Medicine and Harvard Medical School

PERSON, PHILIP, Chief, Special Research Laboratory for Oral Tissue Metabolism, Veterans Ad- ministration Hospital, Pittsburgh

PORTER, KEITH R., Professor of Biology, Harvard University PROSSER, C. LADD, Head and Professor of Physiology and Biophysics, University of Illinois,

Urbana

READ CLARK P., Professor of Biology, Rice University

REEDY, MICHAEL K., Professor of Physiology, University of California, Los Angles REUBEN JOHN P., Associate Professor, Columbia University REYNOLDS, GEORGE T., Professor of Physics, Princeton University RHOADS, DONALD C., Systematic-Ecology Program, Marine Biological Laboratory RICE, ROBERT V., Professor of Biochemistry, Mellon Institute of Carnegie-Mellon University RICHMAN, SUMNER, Associate Professor of Biology, Lawrence University RIPPS, HARRIS, Professor of Experimental Ophthalmology, New York University School of

Medicine

ROBERTS, JOHN L., Associate Professor of Zoology, University of Massachusetts, Amherst ROSE, FLORENCE C., Research Associate, Tulane University

ROSE, S. MERYL, Professor, Laboratory of Developmental Biology, Tulane University ROSENBERG, PHILIP, Head and Professor, Division of Pharmacology, University of Connecticut

School of Pharmacy

ROTH JAY S., Professor of Biochemistry, University of Connecticut RUIZ-MANRESA, FRANCISCO, Research Fellow, Columbia University

RUSHFORTH, NORMAN B., Associate Professor of Biology, Case Western Reserve University RUSSELL-HUNTER, W. D., Professor of Zoology, Syracuse University RUSTAD, RONALD C., Associate Professor of Radiology and Biology, Case Western Reserve

University SAUNDERS, JOHN W., JR., Professor of Biological Sciences, State University of New York at

Albany

REPORT OF THE DIRECTOR 27

SCHMEER, ARLINE C., O. P., Director, Life Science Research and Professor of Biology, Ohio Dominican College

SCHMIDT, MICHAEL J., Graduate Student, Vanderbilt Medical School

SCHOPF, THOMAS J. M., Assistant Professor, Center for Marine and Environmental Sciences, Lehigh University

SCHROEDER, THOMAS E., Research Fellow, Harvard University

SCHWARTZ, TOBIAS L., Assistant Professor of Regulatory Biology, University of Connecticut

SCOTT, GEORGE T., Professor of Biology, Oberlin College

SHAW, STEPHEN R., Visiting Scientist, National Institutes of Health

SHEMIN, CHARLOTTE N., Research Associate, Northwestern University

SHEMIN, DAVID, Professor of Biochemistry, Northwestern University

SHE PRO, DAVID, Professor of Biology, Boston University

SHERMAN, IRWIN W., Associate Professor of Zoology, University of California, Riverside

SHIMONO, TOSHIO, Research Associate, Wayne State University School of Medicine

SIEGEL, IRWIN M., Associate Professor of Experimental Ophthalmology, New York University Medical Center

SIEGELMAN, H. W., Biochemist, Brookhaven National Laboratory

SIMON, ERIC J., Associate Professor of Experimental Medicine, New York University Medical School

SINCLAIR, NORMAN R., Research Associate, Systematic-Ecology Program, Marine Biological Lab- oratory

SLOBODKIN, LAWRENCE B., Professor of Biology, State University of New York at Stony Brook

SOKOLOVE, PHILLIP G., Graduate Student, Harvard University

SORENSON, ALBERT LEE, Postdoctoral Trainee, College of Physicians and Surgeons, Colubmia University

SPIEGEL, MELVIN, Professor of Biology, Dartmouth College

SPIRTES, M. A , Clinical Associate Professor of Pharmacology, University of Pittsburgh and Associate Chief of Staff, Veterans Administration Hospital, Pittsburgh

STEELE, JOHN H., Senior Principal Scientific Officer, Marine Laboratory, Aberdeen, Scotland .

STEINBACH, ALAN BURR, Research Associate, Albert Einstein College of Medicine

STEINBACH, H. BURR, Director, Marine Biological Laboratory and Professor of Zoology, Uni- versity of Chicago

STEINBERG, MALCOLM S., Professor of Biology, Princeton University

STELL, WILLIAM K., Staff Associate, National Institutes of Health

STEPHENS, RAYMOND E., Assistant Professor of Biology, Brandeis University

STEVENSON, NANCY R., Postdoctoral Fellow, Rutgers Medical School

STILLMAN, IRVING M., Research Associate, National Institutes of Health

STRAUSS, ELLIOTT W., Associate Professor, Division of Biomedical Sciences, Brown University

STRITTMATTER, PHILIPP, Professor of Biochemistry, University of Connecticut

STUNKARD, HORACE W., Research Associate, American Museum of Natural History

SUMMERS, WILLIAM C., Research Associate, Systematic-Ecology Program, Marine Biological Laboratory

SUSSMAN, MAURICE, Professor of Biology, Brandeis University

SZENT-GYORGYI, ALBERT, Director, The Institute for Muscle Research

SZENT-GYORGYI, ANDREW, Professor of Biology, Brandeis University

TANZER, MARVIN L., Assistant Professor of Biochemistry, University of Connecticut Medical School

TASAKI, ICHIJI, Chief, Laboratory of Neurobiology, National Institutes of Health

TAYLOR, W. ROWLAND, Associate Professor of Oceanography, The Johns Hopkins University

TAYLOR, WM. RANDOLPH, Emeritus Professor of Botany and Curator of Algae, University of Michigan

TERWILLIGER, ROBERT C., Research Associate, Boston University

TILNEY, LEWIS G., Assistant Professor of Biology, University of Pennsylvania

TRINKAUS, JOHN P., Professor of Biology, Yale University

TROLL, WALTER, Associate Professor of Environmental Medicine, New York University Medical Center

TUPPER, JOSEPH T., Graduate Student, State University of New York at Albany

TWEEDELL, KENYON S., Professor of Biology, University of Notre Dame

ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

VAN BREEMEN, C., Assistant Professor, Department of Pharmacology, University of Florida and

Department of Medicine, University of Medicine

VAN HARN, GORDON L., Associate Professor of Biology, Oberlin College VAN HOLDE, K. E., Professor of Biophysics, Oregon State University VILLEE, CLAUDE A., Andelot Professor of Biochemistry, Harvard University VINCENT, W. S., Associate Professor of Anatomy and Cell Biology, University of Pittsburgh,

School of Medicine

VRABEC, FRANTISEK, Visiting Professor, Columbia University

WAGNER, HENRY G., Director of Intramural Research, National Institutes of Health WALD, GEORGE, Higgins Professor of Biology, Harvard University WARREN, LEONARD, Professor of Therapeutic Research, University of Pennsylvania School of

Medicine WATANABE, AKIRA, Consultant, National Institutes of Health and Tokyo Medical and Dental

University, Tokyo

WATKINS, DUDLEY T., Assistant Professor of Anatomy, University of Connecticut Health Center WEBB, H. A^ARGUERITE, Chairman and Professor of Biological Sciences, Goucher College WEBER, ANNEMARIE, Professor of Biochemistry, St. Louis University WELSCH, FRANK, Graduate Student, Vanderbilt University School of Medicine WERBLIN, FRANK S., Professor of Electrical Engineering, University of California, Berkeley WHITTAKER, J. RICHARD, Associate Member, Wistar Institute of Anatomy and Biology,

Philadelphia

WHITTAKER, NOEL DE TERRA, Assistant Member, The Institute for Cancer Research WIERCINSKI, FLOYD J., Professor of Biology, Northwestern Illinois State College WILCE, ROBERT T., Associate Professor of Botany, University of Massachusetts WILSON, DARCY B., Assistant Professor of Medical Genetics, University of Pennsylvania School

of Medicine

WILSON, EDWARD O., Professor of Zoology, Harvard University WITKOVSKY, PAUL, Assistant Professor of Physiology, Columbia University WITTMAN, KARL S., Instructor, Hudson Valley Community College WOESE, CARL R., Associate Professor of Microbiology, University of Illinois WYTTENBACH, CHARLES R., Assistant Professor of Zoology, University of Kansas YAMAGISHI, SHUNICHI, Research Associate, Columbia University YONATH, ADA E., Fellow, Mellon Institute of Carnegie-Mellon University YOUNG, DAVID K., Resident Ecologist, Systematics-Ecology Program YPHANTIS, DAVID A., Professor of Biology, University of Connecticut YUYAMA, SHUHEI, Postdoctoral Fellow, University of Toronto, Canada ZIGMAN, SEYMOUR, Associate Professor of Ophthalmology and Biochemistry, University of

Rochester Medical School ZIMMERMAN, ARTHUR M., Professor of Zoology, University of Toronto, Canada

Lillie Fellow, 1969 JEAN CLARK DAN, Ochanomizu University, Tokyo

Grass Fellows, 1969

EDUARDO DE ROBERTS, Senior Fellow, Director and Professor of Institute, University of Buenos

Aires

PETER ECKMAN, New York University Medical Center KEITH O. ELLIS, University of Cincinnati College of Medicine DONALD S. FABER, State University of New York at Buffalo DONALD T. FRAZIER, University of Kentucky L. T. GRAHAM, JR., Indiana University Medical School MICHAEL J. SCHMIDT, Vanderbilt University PHILIP G. SOKOLOVE, Harvard University ALAN BURR STEINBACH, Albert Einstein College of Medicine JOSEPH T. TUPPER, State University of New York at Albany GORDON L. VAN HARN, Oberlin College

REPORT OF THE DIRECTOR 29

Rand Fellow, 1969 MARTIN LINDAUER, Professor, University of Frankfurt, Germany

Research Assistants, 1969

ALTMAN, MORRIS D., New York University

ANDERSON, KAREN LEE, University of Colorado Medical Center

ANN, SONGZA, Washington University School of Medicine

ANTONELLIS, BLENDA, Case Western Reserve University

APRIL, STEPHANIE P., Columbia University

AFTER, MATTHEW N., George Washington University School of Medicine

ARISPE, NELSON, Duke University

AZARNIA, ROOBIK, University of Miami

BAGLEY, SUSAN JEAN, University of Rochester

BAKER, NORMA, Brown University

BALL, ELDON E., University of California, Santa Barbara

BARCHI, ROBERT L., University of Pennsylvania

BARNES, STEPHEN N., University of Colorado Medical Center

BEAULIEU, RENEE, L. University of Connecticut

BECKER, JUDITH S., University of Connecticut Health Center

BELANGER, ANN M., Case Western Reserve University

BELANGER, SANDRA E., The Biological Bulletin

BELL, WAYNE, Middlebury College

BELLEMARE, GUY, University of Montreal

BERGER, EDWARD M., Syracuse University

BERKLEY, PATRICIA, Massachusetts Institute of Technology

BIGELOW, ELAINE L., University of Connecticut

BOSCH, HERMAN F., The Johns Hopkins University

BOTOS, PAUL, JR., Princeton University

BOYER, STANLEY, Northwestern University

BRINK, JOHN M., National Institutes of Health

BRODSKY, DIANE, Northwestern University

BROOME, MARIANNE Florida State University

BUDAY, AHILA, University of Montreal

BUIKE, R. BRUCE, Albany Medical School

CAMPBELL, LAURIE K., Northwestern University

CAYER, MARILYN, University of Miami

CHEVALIER, ROBERT L., University of Chicago

CLARK, ANDREA, State University of New York at Stony Brook

COBB, WILLIAM R., Systematics-Ecology Program

COLGAN, JAMES A., Columbia University

CONWAY, JOAN, Systematics-Ecology Program

COOLEY, REBECCA A., State University of New York at Albany

Cox, EDWIN B., Duke University School of Medicine

DAIGNEAULT, REJEAN, University of Montreal

DAVIDSON, ALICE, Case Western Reserve University

DAVIDSON, JAMES A., Case Western Reserve University

DOLE, W'ILLIAM P., New York University School of Medicine

DOMANIK, RICHARD A., Northwestern University

DON, IRL J., Washington University School of Medicine

DOOHAN, FRANCIS, Systematics-Ecology Program

DREXLER, ANDREW JAY, New York University School of Medicine

DUNHAM, JOYCE E., Smithsonian Institution

EDDS, KENNETH, State University of New York at Albany

EGGLESTON, ALAN C., University of California, Santa Barbara

ELLSWORTH, JANET P., State University of New York, Upstate Medical Center

ELSTER, JAMES R., New York University Medical Center

ETTIENNE, EARL M., State University of New York at Albany

30 ANNUM REPORT OF THE MARINE BIOLOGICAL LABORATORY

FISHER, LINDA, Rutgers University

FOUCAR, ELLIOTT, Oberlin College

Fox, DlANNE H., University of Pennsylvania

FUSELER, JOHN WM., JR., University of Pennsylvania

GILBERT, MARY ANN, Systema tics-Ecology Program

GILBERT, WILLIAM H., Systematics-Ecology Program

GILL, LORRAINE ANN, Massachusetts General Hospital

GODDARD, JAN, University of Pennsylvania

GOFF, ROBERT W., University of Massachusetts

GOLDMAN, JAMES E., New York University

GOLDSTEIN, MARTIN, Washington University

GONZALEZ, NICHOLAS J., Brown University

GOSPODNETIC, MARIJAN, University of Ottawa, Canada

GRANHOLM, NORM A., University of Oregon

GREENBERG, MICHAEL A., Case Western Reserve University

CREEP, NANCY, Yale University

GRZYB, STANLEY EDWARD, University of Connecticut School of Medicine

HACHMEISTER, LON E., University of Washington

HALVERSON, ROGER C., University of California, Santa Barabra

HANSON, MUSETTA, Ohio Dominican College

HARRIS, EDWARD M., Duke University

HARTZBAND, DAVID J., Systematics-Ecology Program

HAYASHI, SALLY, Yale University Medical School

HECKER, BARBARA, Columbia University

HEYMANN, PETER W., John Carroll University

HILLMAN, GILBERT R., Yale University

HINEGARDNER, ELENA C., Columbia University

HIRSHFIELD, MICHAEL F., Princeton University

HOBSON, KATHARINE D., Systematics-Ecology Program

HOFFMAN, RICHARD J., College of William and Mary

HOLZ, RONALD, Albert Einstein College of Medicine

HUBBARD, RUSSELL BRUCE, Vanderbilt University School of Medicine

HUEBNER, ERWIN, University of Massachusetts

HUMMON, WILLIAM D., Systematics-Ecology Program

HUNTER, R. DOUGLAS, Syracuse University

JOHNSON, MICHAEL L., Oregon State University

JOHNSON, ROYCE O., II, Princeton University

JONES, WESLEY, Tougaloo College

KAUFMANN, KARL W., JR., Lehigh University

KELLEHER, SARAH, Case Western Reserve University

KENNEY, DIANNE M., Boston University

KIMURA, JOHN E., Stanford University

KING, JOHN M., Aquarium Systems, Inc.

KIRSCHNER, DANIEL A., Harvard Medical School

KOEPPE, JOHN E., Tulane University

KOTLER, DONALD P., Rutgers University

LAMPERT, SCOTT, University of Pennsylvania

LAMPING, JOHN A., Northwestern University

LANG, FRED, University of Illinois

LAZAROW, PAUL B., The Rockefeller University

LESTER, HENRY A., The Rockefeller University

LEVY, RICHARD A., University of Delaware

LINCK, RICHARD W., Brandeis University

LIPSON, ROBERT, Columbia University

MCCAFFREY, FRANCIS, University of Massachusetts

McELWEE, DENNIS, Tulane University

McGRATH, RICHARD A., Systematics-Ecology Program

McGRATH, STEVEN, Systematics-Ecology Program

REPORT OF THE DIRECTOR 31

McKAY, CHARLES R., Systematics-Ecology Program

McMAHON, JOHN, Systematics-Ecology Program

McMAHON, ROBERT F., Syracuse University

MILLER, JAY E., Temple University

NAGEL, SIDNEY R., Princeton University

NEFF, MARY ROSE, Brown University

NICKERSON, KENNETH W., Oregon State University

NICKERSON, RICHARD P., Syracuse University

NOE, BRYAN D., University of Minnesota

NOLTE, JOHN, Massachusetts Institure of Technology

OAKS, JOHN A., Tulane University

OLDHAM, PETER J., Systematics-Ecology Program

PASBY, TERRY L., University of Illinois

PAUL, IZHAK, University of Toronto, Canada

PHELPS, PATRICIA, University of Iowa

PILLSBURY, STEPHEN, University of Connecticut School of Medicine

PIERCE, SIDNEY K., JR., Florida State University

PIPER, JUDITH ANN, University of Connecticut

POOR, ALFRED, Systematics-Ecology Program

POSTON, ROBIN, Duke University

PRUSCH, ROBERT D., Syracuse University

RAVITZ, MELVYN JAY, Albert Einstein College of Medicine

RAYNER, ELLEN P., Brandeis University

REINHART, JOHANNA M., Systematics-Ecology Program

RIGGIO, BONNIE L., University of Massachusetts

ROARK, DENNIS E., University of Connecticut

ROBERTSON, LOLA E., American Museum of Natural History

RODEWALD, RICHARD D., University of Pennsylvania

ROESSNER, KAREN D., Case Western Reserve University

RORKE, CHARLES, Wistar Institute of Anatomy and Biology

ROSE, BIRGIT, College of Physicians and Surgeons, Columbia University

ROWEN, ADAM J., Yale University

RUBINSTEIN, NEAL A., Dartmouth College

SACHS, MARTIN, University of Massachusetts

SAGE, JEAN A., Indiana University Medical Center

SALMON, EDWARD, University of Pennsylvania

SANDLIN, RONALD A., National Institutes of Health

SASSAMAN, CLAY, College of William and Mary

SAUL, RICHARD, University of Maryland

SCHMIDLIN, ANNELISE, Princeton University

SCHWARTZMAN, JOSEPH D., Dartmouth College

SEARS, JAMES R., Systematics-Ecology Program

SHEPARD, CHARLES A., Oberlin College

SHIROKY, DOROTHY V., The Johns Hopkins University

SINGER, IRWIN I., New York University

SLAUGHTER, MARGARET ANN, Yale University School of Medicine

SLAVIN, WILLIAM R., University of Connecticut

SMARSH, ANNE, Systematics-Ecology Program

SOGIN, STEPHEN J., University of Illinois

SOUKUP, MICHAEL A., Systematics-Ecology Program

STAFFORD, WALTER, University of Connecticut

STAMM, WILLIAM C., JR., Drew University

STARLING, JANE A., Rice University

STEWART, JAMES R., Brown University

STRAUS, ANDREA, Kirkland College

STREIBEL, ELLEN, University of Pennsylvania

SUDDITH, ROBERT L., Indiana University

SUSSMAN, JOEL L., Columbia University

32 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

SZAMIER, ROBERT B., Albert Einstein College of Medicine

SZONYI, ESZTER, Institute for Muscle Research

TANG, CECILIA, University of Connecticut

TANN, EDWARD M., Ohio Dominican College

TATUM, SUSAN M., University of Pennsylvania

TEREBEY, NICHOLAS, State University of New York, Upstate Medical Center

THEILE, JUDITH A., Rensselaer Polytechnic Institute

THIEME, ELISABETH, Rutgers University

TRUCHAN, SISTER LEONA, Northwestern University

TUCKER, GAIL S., University of Kansas

TURNER, ROBERT S., JR., University of Oregon

VAN WIE, DONALD G., Harvard University

VAN ZANDT, DIRK, Systematics-Ecology Program

WEXLER, ANDREW M., Phillips Academy

WIDEMAN, CYRILLA, Illinois Institute of Technology

WILLIAMS, Lois D., University of Hawaii

WILSON, DAVID S., Brandeis University

WOLLEY, ROBERT C., Tulane University

WOLSEY, PENNY, McGill University, Canada

WOODCOCK, FRANCES J., University of Miami

WOZENSKI, JANET, Syracuse University

YEE, ANN G., Harvard University

YOUNG, JANICE E., Northwestern University

ZAKEVICIUS, JANE M., New York University Medical Center

ZAVITKOVSKI, JERRY, Oregon State LTniversity

ZIPSER, BIRGIT, Albert Einstein College of Medicine

Library Readers, 1969

ALLEN, GARLAND E., Assistant Professor of Biology, Washington University

ANDERSON, RUBERT S., Woods Hole, Massachusetts

APPEL, ANTOINETTE R., Queens College

BALL, ERIC G., Professor of Biological Chemistry, Harvard Medical School

BERLIN, RICHARD D., Assistant Professor of Physiology, Harvard Medical School

BERNE, ROBERT M., Chairman and Professor of Physiology, University of Virginia School of

Medicine

BODANSKY, OSCAR, Vice President, Sloan- Kettering Institute for Cancer Research BOETTIGER, EDWARD G., Professor of Physiology, University of Connecticut CARLSON, FRANCIS D., Professor of Biophysics, The Johns Hopkins University CHASE, AURIN M., Emeritus and Senior Research Biologist, Princeton University CRANE, ROBERT K., Chairman and Professor of Physiology, Rutgers Medical School DAVIS, JOHN D., Assistant Professor of Biological Sciences, Smith College DE ROBERTIS, EDUARDO, Professor and Director of Institute, University of Buenos Aires DUBOFF, GREGORY S., Professor of Experimental Medicine, University of Michigan School of

Medicine

EBERT, JAMES, Director and Professor of Embryology, Carnegie Institution of Washington EDDS, M. V., JR., PROFESSOR of Biology, Brown University

FLAVIN, REV. JOHN W., Chairman, Department of Biology, College of the Holy Cross GABRIEL, MORDECAI L., Chairman and Professor of Biology, Brooklyn College GERMAN, JAMES, Investigator and Director of Laboratory of Human Genetics, The New York

Blood Center

GIBBS, MARTIN, Professor of Biology, Brandeis University

GITLIN, DAVID, Professor of Pediatrics, University of Pittsburgh School of Medicine GOUDSMIT, ESTHER M., Research Associate, The Johns Hopkins University GRANT, ROBERT J., Assistant Professor of Biological Sciences, Hunter College GREEN, JAMES W., Professor of Physiology, Rutgers University HANDLER, PHILIP, Professor of Biochemistry, Duke University

REPORT OF THE DIRECTOR 33

HUBBARD, SUSAN BAUR, Marine Biological Laboratory

ISSELBACHER, KURT J., Professor of Medicine, Massachusetts General Hospital KALTENBACH, JANE C., Associate Professor of Biological Sciences, Mount Holyoke College KANE, ROBERT E., Assistant Director and Associate Professor Pacific Biomedical Research

Center, University of Hawaii

KEMPTON, RUDOLF T., Professor Emeritus of Biology, Vassar College KEOSIAN, JOHN, Professor of Biochemistry, Rutgers University KRAVITZ, EDWARD A., Professor of Neurobiology, Harvard Medical School LEVINTHAL, CYRUS, Chairman, Department of Biological Sciences, Columbia University LOCHHEAD, JOHN H., Professor of Zoology, University of Vermont MAHLER, HENRY R., Professor of Chemistry, Indiana University MARSHAK, ALRFED, Professor of Experimental Pathology, Tulane University NACHMANSOHN, DAVID, Professor of Biochemistry, Columbia University, College of Physician-

and Surgeons

NASATIR, MAIMON, Chairman and Professor of Biology, University of Toledo OSTERHOUT, MARION L., Marine Biological Laboratory RAVIN, HERBERT A., Director and Professor of Medicine, Sinai Hospital of Detroit and Wayne

State University ROSENBLUTH, JACK, Associate Professor of Physiology and Biophysics, New York University

College of Medicine

ROWLAND, LEWIS P., Chairman and Professor of Neurology, University of Pennsylvania RUBINOW, SOL I., Professor of Biomathematics, Cornell University Medical College SAGER, RUTH, Professor of Biological Sciences, Hunter College

SCHLESINGER, R. WALTER, Chairman, Department of Microbiology, Rutgers University SCOTT, ALAN, Professor of Biology, Colby College

SIRLIN, J. L., Associate Professor of Anatomy, Cornell University Medical College SMELSER, GEORGE K., Director of Eye Research, College of Physicians and Surgeons, Columbia

University

SONNENBLICK, B. P., Professor of Zoology and Radiation Science, Rutgers University SPECTOR, ABRAHAM, Professor of Ophthalmology, College of Physicians and Surgeons, Columbia

University

SPERELAKIS, NICK, Professor of Physiology, University of Virginia School of Medicine STETTEN, DEWITT, JR., Dean and Professor of Experimental Medicine, Rutgers Medical School STETTEN, MARJORIE R., Research Professor of Experimental Medicine, Rutgers Medical School STRACHER, ALFRED, Professor of Biochemistry, State University of New York, Downstate Medical

Center

TROTTER, PHILIP J., Interdisciplinary Postdoctoral Fellow, New England Institute, Inc. VAGELOS, P. ROY, Chairman and Professor of Biochemistry, Washington University School of

Medicine

WAINIO, WALTER, Chairman and Professor of Biochemistry, Rutgers University WAKSMAN, BYRON H., Chairman and Professor of Microbiology, Yale University WEISS, LEON, Professor of Anatomy, The Johns Hopkins University Medical School WHEELER, GEORGE E., Associate Professor of Biology, Brooklyn College WHITING, ANNA R., Consultant, Biology Division, Oak Ridge National Laboratory WICHTERMAN, RALPH, Professor of Biology, Temple University

WILSON, T. HASTINGS, Chairman and Professor of Physiology, Harvard Medical School WITTENBERG, JONATHAN B., Professor of Physiology, Albert Einstein College of Medicine Yow, FRANK \V., Professor of Biology, Kenyon College YNTEMA, CHESTER L., Professor of Anatomy, State University of New York, Upstate Medical

Center

ZACKS, SUMNER L, Associate Professor of Pathology, University of Pennsylvania ZEIDENBERG, PHILLIP, Senior Research Psychiatrist, New York State Psychiatric Institute ZIPSER, DAVID, Assistant Professor of Molecular Biology, Columbia University

Students, 1969

All students listed completed the formal course program, June 16-July 26. Asterisk indicates students completing post-course reaserch program, July 27-August 30.

34 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

ECOLOGY

ARNETT, PATRICIA M., Indiana State University *BLOOM, STEPHEN A., University of South Florida

BROTHERS, EDWARD B., Scripps Institution of Oceanography

FLESSA, KARL W., Brown University *GLEYE, LINDA G., Sarah Lawrence College *PERMESLY, LESTER S., Tufts University School of Medicine

QUICK, FRANK W., JR., University of Louisville *REX, MICHAEL A., Harvard University

SCHULTZ, JOHN C., University of Chicago *SHIMEK, RONALD L., Montana State University *SLOZAK, FRANCES, University of Massachusetts *SMITH, SHARON L., College of St. Teresa *WEINSTEIN, JUDITH D., University of Massachusetts *\VILLIAMS, ERNEST H., JR., Princeton University

WILLIAMSON, JANET L., Oherlin College

WING, DANIEL C., Oberlin College

EMBRYOLOGY

*BACHELER, LEE T., The Johns Hopkins University *BROYLES, ROBERT H., The Bowman Gray School of Medicine

BUCKLEY, PATRICIA A., University of Virginia

CALLAWAY, EDWARD E., University of California, Davis *CAMERON, ROBERT A., University of California, Santa Cruz *CONWAY, ARTHUR F., University of Miami *DESMOND, MARY E., University of Colorado

GORDON, RICHARD, Columbia University

HENDRICKSON, SCOTT A., Washington University *HOFMAN, FLORENCE C., University of Wisconsin

KLAYMAN, MICHAEL B., Union College

LIPTON, BRUCE H., University of Virginia *PACKARD, DAVID S., JR., Louisiana State University *SENTKOWSKI, ALAN M., The Johns Hopkins University *SHERMOEN, ANTONY W., Wesleyan University *THISTLETHWAITE, J. RICHARD, Amherst College

TISCHFIELD, JAY A., Yale University *TOOLE, BRYAN P., Massachusetts General Hospital *WIESCHAUS, ERIC F., University of Notre Dame *WOLIN, EDWARD M., Reed College

EXPERIMENTAL BOTANY

*BURR, JUDITH E., Drew University

CAMPBELL, ROBERT M., University of St. Andrews, Scotland *DINER, BRIAN A., The Rockefeller University *GROVES, SAMUEL T., Yale University *HUGHES, GLORIA J., University of Rhode Island

KAMYKOWSKI, DANIEL L., Scripps Institution of Oceanography *KENT, GRAHAM RONALD, University of Guelph, Canada *LEVI, CAROLYN A., University of Massachusetts

McCoRMiCK, JON M., Millersville State College *McDANiEL, WILLIAM A., University of Massachusetts *MOSHIER, SUZANNE E., University of Chicago *MOZGALA, WALTER J., University of Massachusetts

NIELSON, ANDREA L., University of Massachusetts *OWENS, OLGA V., University of Maryland

REPORT OF THE DIRECTOR 35

*SPERLING, PATRICIA G., Kent State College WILLIAMS, NINA S., University of Maryland

PHYSIOLOGY

*ANDREWS, THOMAS G., JR., College of Physicians and Surgeons, Columbia University

*BLEICH, HERMANN E., Columbia University

*BRACKENBURY, ROBERT W., California Institute of Technology

*BRONK, BURT V., Queens College

*CHAMBLISS, GLENN H., University of Chicago

*COLLINS, TERRYL R., Simon Eraser University, Canada

*DANIEL, JAMES L., Carnegie-Mellon University

*EPSTEIN, PAUL M., University of Massachusetts

"FLEMING, HONOREE, University of Chicago

*GOLDSMITH, MELVIN, University of Connecticut

*Hsi, EDWARD S., University of Minnesota

KOVACS, MARK A., Massachusetts Institute of Technology "LEHMAN, WILLIAM J., Princeton University *Li, JADE, Bryn Mawr College

*LucAS, ROGER C., Illinois Institute of Technology *MANSON, MICHAEL, The Johns Hopkins University *MAO, CHI-CHIANG, University of Oklahoma "MESSER, ANNE, University of Oregon

NIST, CYNTHIA R., The Rockefeller University

PASPY, TERRY L., University of Illinois, Urbana *RUDD, JENNIFER N., \Vesleyan University *SPOLSKY, CHRISTINA M., Yale University

STRALEY, SUSAN C., Cornell University *SzuTS, ETE Z., The Johns Hopkins University *TEICHBERG, SAUL, Columbia University *\\'ATERS, COREEN A., Brandeis University *\\"ATERSTON, ROBERT H., University of Chicago *\\~EISS, HOWARD D., Northwestern University Medical School *Wooo, DONALD S., Washington State University *\Yooo, KATHLEEN A., Washington State University

YONATH, JACOB, Weizmann Institution, Rehovoth

INVERTEBRATE ZOOLOGY

BILLINGSLEY, BkYNDER C., Tougaloo College

CALDAROLA, PATRICIA, Cornell University

CASADAY, GEORGE B., Cornell University

CHAIKEN, MICHAEL B., State University of New York at Stony Brook

CHIN, KENNETH W., Columbia University *CowAN, PHILIP E., Glasgow University, Scotalnd * DONALDSON, SVEN, New College, Sarasota *EGGLESTON, LINDA L., Rice University

ELLSWORTH, JANET P., State University of New York Upstate Medical Center

FANO, VIRGINIA, University of Wisconsin, Madison

FOELIX, RANIER F., National Science Foundation Department of Mental Health, Raleigh

GOODMAN, MARK L., University of Massachusetts, Amherst *HABERFIELD, EVE C., University of Rhode Island *HACKETT, JOHN T., University of Illinois

INHABER, FRANCINE, City College of New York *!RWIN, GEORGE H., Ill, Emory University

JUNGREIS, ARTHUR M., University of Minnesota *KAHLER, GEORGE A., Ill, Rice University *McARDLE, FLORENCE E., Columbia University

36 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

*MlYAMOTO, DAVID M., University of California, San Diego

NORELLI, LINDA K., Bryn Mawr College

ODESSBY, RICHARD, Harvard University

PEZALLA, PAUL D., University of Minnesota *PRIOR, DAVID J., University of Virginia *RAM, JEFFREY L., California Institute of Technology *REHM, JANET N., Scripps Institution of Oceanography

ROHRLICH, SUSANNAH T., Harvard University *SEGUIN, LANA R., Indiana University *SKARF, BARRY, The Johns Hopkins University

STAMM, WILLIAM C., JR., Drew University

TEREBEY, NICHOLAS, State University of New York, Upstate Medical Center

TESTERMAN, JOHN K., University of California, Irvine

VACCA, LINDA LEE, University of Southern California *\VESTON, JOANNE, Cornell University

WILSON, SAMUEL, Scripps Institution of Oceanography

4. FELLOWSHIPS AND SCHOLARSHIPS, 1969

The Lucretia Crocker Scholarship :

NINA S. WILLIAMS, Experimental Botany Course The James Watt Mavor Scholarship :

ROBERT M. CAMPBELL, Experimental Botany Course

PHILIP E. COWAN, Invertebrate Zoology Course The Edwin Grant Conklin Scholarship :

BRYON P. TOOLE, Embryology Course

5. TRAINING PROGRAMS

FERTILIZATION AND GAMETE PHYSIOLOGY RESEARCH TRAINING PROGRAM

I. INSTRUCTORS

CHARLES B. METZ, University of Miami, Program Chairman JOHN D. BIGGERS, The Johns Hopkins University MARCO CRIPPA, University of Palmero, Italy GERTRUDE W. HINSCH, University of Miami KURT KOHLER, University of Montpellier, France LEONARD NELSON, MEDICAL College of Ohio at Toledo

II. CONSULTANTS

JEAN CLARK DAN, Ochanomizu University, Tokyo PAUL GROSS, Massachusetts Institute of Technology CLAUDE VILLEE, Harvard Medical School

III. LABORATORY ASSISTANTS

MARILYN L. CAYER, Electron Microscope Assistant

LAURA HOSKIN, Secretary

FRANCES WOODCOCK, Photographic Assistant

IV. TRAINEES

BUTZEK, JAMES C., Illinois Institute of Technology CLARK, WALLIS H., University of California, Riverside CONWAY, CAROLYN M., University of Miami COOMES, ROGER K., Monkato State College ELINSON, RICHARD P., Yale University FENWICK, RAYMOND G., JR., Miami University FORTUNE, JOANNE E., Cornell University HOPPER, JAMES E., University of Wisconsin

REPORT OF THE DIRECTOR 37

KOSER, RICHARD B., Brooklyn College

LAVIN, LORA H., Rosemont College

LEE, HAROLD H.( University of Toledo

O'MELIA, ANNE F., Fordham University

SACCO, ANTHONY G., University of Tennessee

SCHROEDER, PAUL C., Washington State University

SHELLEY, THOMAS F., State University of New York, Upstate Medical Center

WEINBERG, ERIC S., The Rockefeller University

V. LECTURES

KURT KOHLER The passage of mRNA from the nucleus to cytoplasm

R. M. IVERSON RNA synthesis during oogenesis and development

W. J. HUMPHREYS Structure and function of cortical granules in invertebrate eggs

C. J. BROKOW Recent studies on sperm mobility

L. E. FRANKLIN The acrosome reaction and sperm penetration in the golden hamster egg

M. CRIPPA Mechanisms of control of ribosomal RNA synthesis during Xenopus oogenesis

MILDRED GORDON Cytochemical analyses of mammalian sperm flagella

J. GALL Differential gene synthesis during oogenesis

H. A. BERN Vertebrate prolactin: a reproductive hormone with non-reproductive be- ginnings

EXCITABLE MEMBRANE PHYSIOLOGY AND BIOPHYSICS TRAINING PROGRAM

I. INSTRUCTORS

WILLIAM J. ADELMAN, JR., University of Maryland School of Medicine, Program Chairman JOHN W. MOORE, Duke University School of Medicine TOSHIO NARAHASHI, Duke University School of Medicine YORAM PALTI, Hebrew University, Hadassah Medical School TOBIAS SCHWARTZ, University of Connecticut

II. CONSULTANTS

KENNETH S. COLE, National Institutes of Health

HARRY GRUNDFEST, College of Physicians and Surgeons, Columbia University

LORIN J. MULLINS, University of Maryland School of Medicine

ICHIJI TASAKI, National Institutes of Health

III. TRAINEES

CALLAHAN, LEO X., University of Maryland CLEAVES, CAROL ANN, Duke University DAVIS, DONALD G., University of Pittsburgh DAWSON, DAVID C., University of Pittsburgh DE HEMPTINNE, ALEX, Duke University Di POLO, REINALDO, Harvard University Medical School ERNAU, MILDRED C., State University of New York at Albany JENDRASIAK, GORDON L., University of Notre Dame JONES, CEDRON, Massachusetts Institute of Technology KORENBROT, JUAN I., The Johns Hopkins University MCCREERY, DOUGLAS B., University of Connecticut WAGGONER, ALAN S., University of Oregon

IV. LECTURES

H. BURR STEINBACH Introduction TOBIAS SCHWARTZ The flux equation

The Ussing-Teorell "unidirectional flux ratio" — osmotic equilibrium — the Staverman reflexion coefficient

The Gibbs-Donnan equilibrium

The Goldman equation

Simple diffusion regimes- — electrical equivalent circuits

38

ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

RAYMOND A. SJODIN

LORIN J. MULLINS

KENNETH S. COLE

C. R. WORTHINGTON

I. STILLMAN

KENNETH S. COLE JOHN W. MOORE

DANIEL L. GILBERT YORAM PALTI

R. FiTzHucH

DANIEL L. GILBERT WILLIAM J. ADELMAN

HARVEY FISHMAN RITA GUTTMAN TOSHIO NARAHASHI

ROBERT L. DE HAAN B. HILLE

LAWRENCE COHEN ICHIJI TASAKI

HARRY GRUNDFEST

D. E. GOLDMAN

V. WORKSHOPS PAUL DE WEER YORAM PALTI

Struction of cell membranes

Diffusion and permeation in cell membranes

Radioactive tracers and analysis of kinetics

Results: nerve, muscle and erythrocytes

Active ion transport

Active transport techniques in squid axons

Active transport: stoichiometry, substrate requirements and electrical

consequences

Models for active and passive ion transport Models for nerve excitation Passive membrane properties I Passive membrane properties II X-ray analysis of nerve myelin Myelin structure

Preparation of synthetic lipid bilayers K-selective macrocyclic polypeptides Voltage clamp strategy Voltage clamp technique: normal currents Ionic conductance kinetics: Hodgkin-Huxley equations Specifications for nerve membrane models Fixed surface charges Varying potential control voltage clamp Reconstruction of the membrane action potential Reconstruction of the propagated action potential and excitation

phenomena

Phase-space analysis of nerve models Dimensional analysis of nerve models Impulse propagation in a nerve fiber (film) Internal perfusion of squid axons: techniques Internal perfusion of squid axons Internal perfusion: voltage clamp results Membrane asymmetries Voltage clamp studies of frog skin Temperature and nerve membrane function Polyvalent cations in nerve excitation Membrane conductance and drug action I — tetrodotoxin Membrane conductance and drug action II — insecticides and tetraethyl-

ammonium

Electrical recording from single heart cells isolated in tissue culture Voltage clamp studies of frog myelinated axons Specifications for an ionic channel

Light scattering and birefringence changes during nerve activity Internal perfusion studies of the squid axon Electrophysiological properties of excitable membranes Optical properties of excitable membranes Varieties of excitable membranes General ionic requirements for electrogenesis Electric organs

Model making : general principles Basic physical mechanisms in excitability Survey of current model systems A detailed model of membrane excitability

Isotopic techniques

Digital computer analysis and reconstruction of nerve behavior

REPORT OF THE DIRECTOR

39

6. TABULAR VIEW OF ATTENDANCE, 1965-1969

INVESTIGATORS — TOTAL

Independent 284

1965 1966

572 555 287

Library Reader. . . . Research Assistants .

STUDENTS — TOTAL

I nvertebrate Zoology .

Embryology

Physiology

Experimental Botany Ecology

TRAINEES — TOTAL ,

TOTAL ATTENDANCE

Less persons represented in two categories.

INSTITUTIONS REPRESENTED — TOTAL. FOREIGN INSTITUTIONS REPRESENTED.

62

227

128 41 20 30 20 17

16

734 4

730 218

27

77 191

126

37 22 29 18 20

16

710 0

710 198

28

1967 590 313 78 199

132 41 20 31 20 20

16

738 4

734 177

29

1968

528

281

76

171

122 39 20 30 15 18

17

667

7

660 169

23

1969

566

310

68

188

118 35 20 30 16 17

29

708

5

703 187

24

7. INSTITUTIONS REPRESENTED, 1969

Albany Medical School

Albert Einstein College of Medicine

American Museum of Natural History

Aquarium Systems, Inc.

Barnard College

Baylor University College of Medicine

Boston University

Brandeis University

Brookhaven National Laboratory

Brooklyn College, The City University of New

York

Brown University Bryn Mawr College California Institute of Technology California, University of, Berkeley California, University of, Davis California, University of, Irvine California, University of, Los Angeles California, University of, Riverside California, University of, San Diego California, University of, San Francisco California, University of, Santa Barbara California, University of, Santa Cruz Carnegie Institution of Washington Case Western Reserve University Case Western Reserve University Medical

School

Chicago, University of Children's Cancer Research Foundation Cincinnati, University of

City College of New York, The

Colby College

College of the Holy Cross

College of Physicians and Surgeons, Columbia

University

College of William and Mary Colorado, University of Colorado, University of, Medical Center Columbia University Columbia University, College of Physicians and

Surgeons

Connecticut, University of Connecticut, University of, Health Center Connecticut, University of, Medical School Connecticut, University of, School of Pharmacy Cornell University Cornell University Medical College Dartmouth College Davidson College Delaware, University of Drew University Duke University- Duke University Medical Center Emory University Florida, University of Florida State University Fordham University George Washington University School of

Medicine Goucher College

40

ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

Harvard Medical School

Harvard University

Hawaii, University of

Hudson Valley Community College

Hunter College

Illinois, University of

Illinois Institute of Technology

Indiana State University

Indiana University

Institute for Cancer Research, The

Institute for Muscle Research, The

Institute of Molecular Evolution, University of Miami

Institute of Psychiatric Research, Indiana Uni- versity Medical Center

Iowa, University of

John Carroll University

Johns Hopkins University, The

Johns Hopkins University, The, School of Hygiene

Johns Hopkins University, The, School of Medicine

Juniata College

Kansas, University of

Kent State College

Kentucky, University of

Kenyon College

Kettering, Charles F., Research Laboratory

Kirkland College

Lawrence University

Lehigh University

Louisiana State University

Louisville, University of

Maryland, University of

Maryland, University of, School of Medicine

Massachusetts, University of

Massachusetts General Hospital

Massachusetts Institute of Technology

Medical College of Ohio at Toledo

Mellon Institute of the Carnegie-Mellon University

Miami, University of

Miami University

Michigan, University of, School of Medicine

Michigan, University of

Michigan State University

Millersville State College

Minnesota, University of

Monkato State College

Montana State University

Movint Holyoke College

National Cancer Institute

National Institutes of Health

National Science Foundation

Naval Undersea Warfare Center

New College, Sarasota

New England Institute, Inc.

New York Blood Center, The New York State Psychiatric Institute New York University

New York University College of Dentistry- New York University Medical College New York University School of Medicine North Carolina, University of North Carolina State University, Raleigh North Dakota, University of Northeastern Illinois State College Northwestern University Northwestern University Medical School Notre Dame, University of Oak Ridge National Laboratory Oberlin College Ohio Dominican College Oklahoma, University of Oregon, University of Oregon State University Pennsylvania, University of Pennsylvania, University of, School of Medicine Pennsylvania Hospital Pittsburgh, University of

Pittsburgh, University of, School of Medicine Princeton University Providence College Queens College, The City University of New

York

Reed College

Rensselaer Polytechnic Institute Rhode Island, University of Rhode Island Hospital Rice University Rochester, University of Rochester, University of, Medical School Rockefeller University, The Rosemont College Rutgers University Rutgers University Medical School St. Louis University St. Teresa, College of Scripps Institution of Oceanography Simon Fraser University Sinai Hospital of Detroit

Sloan-Kettering Institute for Cancer Research Smith College

Smithsonian Institution of Washington South Florida, University of Stanford University State University of Iowa 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 Buffalo State University of New York at Stony Brook

REPORT OF THE DIRECTOR

41

Syracuse University Temple University Tennessee, University of Texas, University of, Austin Toledo, University of Tougaloo College Trinity College

Tufts University School of Medicine Tulane University

Vanderbilt University School of Medicine Vassar College Vermont, University of

Veterans Administration Central Office, Wash- ington, D. C.

Veterans Administration Hospital, Brooklyn Veterans Administration Hospital, Pittsburgh Virginia, University of Virginia, University of, School of Medicine Washington State University Washington University Washington University School of Medicine Wayne State University Wesleyan University Wisconsin, University of Wistar Institute of Anatomy and Biology Woods Hole Oceanographic Institution Yale University Yale University School of Medicine

FOREIGN INSTITUTIONS REPRESENTED, 1969

Bristol, University of, England Buenos Aires, University of, Argentina Centre National de la Recherche Scientifique,

France

Frankfurt, University of, Germany Glasgow, University of, Scotland Guelph, University of, Canada Hebrew University Medical School, Jerusalem Institute Venezolano de Investigaciones Cien-

tificas, Venezuela

Laboratory of Molecular Biology, England Marine Laboratory, Aberdeen, Scotland McGill University, Canada Medical Research Council, England Montreal, University of, Canada N. Copernicus University, Poland Ocean Research Institute, University of Tokyo,

Japan

Ochanomizu University, Japan Ottawa, University of, Canada Palermo, University of, Italy Research Institute of National Defence, Sweden Simon Fraser University, Canada St. Andrews, University of, Scotland Tokyo, University of, Japan Toronto, University of, Canada Weizmann Institution, Israel

8. FRIDAY EVENING "LECTURES, 1969

July 4

TREVOR W. GOODWIN Stereospecific studies on biosynthesis of caro-

University of Liverpool tenoids and sterols in plants

July 11

JEAN CLARK DAN . .The acrosomal process membrane

Ochanomizu University, Tokyo Lillie Fellow at the MBL

July 17

EDUARDO DE ROBERTIS Synthesis, storage and release of transmitter

University of Buenos Aires substances

Alexander Forbes Lecturer at MBL Senior Grass Fellow at the MBL

fuly 18 EDUARDO DE ROBERTIS Isolation and nature of chemical receptors

July 25

JOHN H. STEELE Experimental studies of marine food chains

Marine Laboratory Aberdeen, Scotland

42

ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

August 1

HUGH E. HUXLEY The design requirements of striated muscle

Medical Research Council Cambridge, England

August 8

MARTIN LINDAUER Learning and forgetting in honeybees

University of Frankfurt, Germany Rand Fellow at the MBL

August 15

J. WOODLAND HASTINGS Living light: biochemical mechanisms and bio- Harvard University logical function of bioluminescence

August 22

SYDNEY BRENNER Prospects for the complete analysis of the ner-

Medical Research Council vous system of nematodes

Cambridge, England

9. TUESDAY EVENING SEMINARS, 1969 July 15

S. YUYAMA Temperature — pressure effects on RNA syn-

ARTHUR M. ZIMMERMAN thesis in synchronized Tetrahymena

HARUO KANATANI Effects of adenine derivatives on oocyte matura- tion and spawning in starfish

ARLINE C. SCHMEER, O.P Mercenaries (clam) extracts: effect on hela

cytokinetics

CATHERINE HENLEY The 9+1 pattern of microtubules in sperma-

D. P. COSTELLO tozoa of certain Turbellaria

M. B. THOMAS W. D. NEWTON

July 22

ROGER MILKMAN An exact study of polygenic variation in natural

populations

A. M. JUNGREIS Changes in metabolite regulation as functions of

acclimation temperature, starvation and sea- son in the frog Rana pipiens

L. T. GRAHAM, JR Dark adaptation: its influence on GABA in

frog retina, in vivo July 29

SEYMOUR ZIGMAN Variations in lens insoluble protein

R. RODEWALD Selective antibody transport in the proximal

small intestine of the neonatal rat

P. G. SOKOLOVE Role of sodium pump in stretch receptor adapta- tions

JOHN D. PALMER Preliminary findings on the effect of D2O on the

H. B. DOWSE period of circadian activity rhythms

REPORT OF THE DIRECTOR 43

August 5

W. J. DEAL Photoregulation of bioelectricity by photochromic

acetylcholine receptor inhibitors HENRY G. MAUTNER Studies of the conformation of small molecules

E. SHEFTER acting on acetylcholine receptors

M. J GREENBERG ... .Some properties of junctional cilia in molluscan

gills August 12

F. C. G. HOSKIX. . . ..Possible significance of "DFP-ASE" in squid

nerve TOBIAS L. SCHWARTZ The Goldman equation: its constraints and its

applicability in the face of electrogenic and

non-electrogenic pumps

A. L. PEARLMAN Color opponent cells in cat lateral geniculate

N. W. DAW nucleus

C. K. BARTELL Application of continuous particle electrophoresis

INI. FINGERMAN for the separation of lipoproteins having

melanin-dispersing activity from eyestalks of

fiddler crabs (Uca pugilator}

10. MEMBERS OF THE CORPORATION, 1969 Including Action of 1969 Annual Meeting

Life Members

ADOLPH, DR. EDWARD F., University of Rochester School of Medicine and Den- tistry, Rochester, New York 14620

BAITSELL, DR. GEORGE A., Winter Park Tower, Winter Park, Florida 32789 BERTHOLF, LLOYD M., Central State College Association, 2530 Crawford Avenue,

Evanston, Illinois 60201

BRODIE, MR. DONALD, 522 Fifth Avenue, New York, New York 10018 COLE, DR. ELBERT C., 2 Chipman Park, Middlebury, Vermont 05753 COWDRY, DR. E. V., 4580 Scott Avenue, St Louis, Missouri 63110 CRANE, MRS. W. MURRAY, 820 Fifth Avenue, New York, New York 10021 CURTIS, DR. MAYNIE R., Cancer Research Laboratory, School of Medicine,

University of Miami, Coral Gables, Florida 33146 DAWSON, DR. A. B., 12 Scott Street, Cambridge, Massachusetts 02138 DAWSON, DR. J. A., 129 Violet Avenue, Floral Park, Long Island, New York 11001 Hess, DR. WALTER, 787 Maple Street, Spartanburg, South Carolina 29302 HISAW, DR. F. L., Biological Laboratories, Harvard University, Cambridge,

Massachusetts 02138

IRVING, DR. LAURENCE, University of Alaska, College, Alaska 99735 JACOBS, DR. M. H., Department of Physiology, University of Pennsylvania,

Philadelphia, Pennsylvania 19104

LOWTHER, DR. FLORENCE, Barnard College, New York, New York 10027 MACDOUGALL, DR. MARY STUART, Mt. Yernon Apartments, 423 Clairmont

Avenue, Decatur, Georgia 30030 MALONE, DR. E. F., 6610 North llth Street, Philadelphia, Pennsylvania 19126

44 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

MEANS, DR. J. H., 15 Chestnut Street, Boston, Massachusetts 02108 MEDES, DR. GRACE, 303 Abington Avenue, Philadelphia, Pennsylvania 19111 PAGE, DR. I. H., Cleveland Clinic, Euclid at E. 93rd Street, Cleveland, Ohio

44106

PAYNE, DR. FERNANDUS, Indiana University, Bloomington, Indiana 47405 PLOUGH, DR. H. H., 15 Middle Street, Rt. 1, Amherst, Massachusetts 01002 POND, SAMUEL E., 53 Alexander Street, Manchester, Connecticut 06040 PORTER, DR. H. C., University of Pennsylvania, Philadelphia, Pennsylvania

19104

SCHRADER, DR. SALLY, Duke University, Durham, North Carolina 27706 SMITH, DR. DIETRICH C., 218 Oak Street, Catonsville, Maryland 12128 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. WM. RANDOLPH, Department of Botany, University of Michigan,

Ann Arbor, Michigan 48104

TURNER, DR. C. L., Northwestern University, Evanston, Illinois 60201 WAITE, DR. F. G., 144 Locust Street, Dover, New Hampshire 03820 WALLACE, DR. LOUISE B., 359 Lytton Avenue, Palo Alto, California 94301 WARREN, DR. HERBERT S., 2768 Egypt Road, Audubon, Pennsylvania 19407 WILLIER, DR. B. H., Department of Biology, The Johns Hopkins University

Baltimore, Maryland 21218

Regular Members

ABBOTT, DR. BERNARD C., Department of Biological Sciences, University of

Southern California, University Park, Los Angeles, California 90007 ADELBERG, DR. EDWARD A., Department of Microbiology, Yale University, New

Haven, Connecticut 06510 ADELMAN, DR. WM. J., JR., Department of Physiology, University of Maryland,

Medical School, Baltimore, Maryland 21201 ALLEN, GARLAND E., Biology Department, Washington University, St. Louis

Missouri 63103 ALLEN, DR. ROBERT D., Department of Biological Sciences, State University of

New York at Albany, Albany, New York 12203 ALSCHER, DR. RUTH, Department of Biology, Manhattanville College, Purchase,

New York 10577 AMATNIEK, MR. ERNEST, 34 Horner Avenue, Hasting-on-the-Hudson, New York

10706

AMBERSON, Dr. WILLIAM R., Katy Hatch Road, Falmouth, Massachusetts 02540 ANDERSON, DR. EVERETT, Department of Zoology, University of Massachusetts,

Amherst, Massachusetts 01003 ANDERSON, DR. J. M., Division of Biological Sciences, Emerson Hall, Cornell

University, Ithaca, New York 14850

ANDERSON, DR. RUBERT S., Box 113, Woods Hole, Massachusetts 02543 ARMSTRONG, DR. PHILIP B., Department of Anatomy, State University of New

York, College of Medicine, Syracuse, New York 13210

REPORT OF THE DIRECTOR 45

ARNOLD, DR. JOHN MILLER, P. B.R.C., 2538 The Mall, University of Hawaii,

Honolulu, Hawaii 96822 ARNOLD, DR. WILLIAM A., Division of Biology, Oak Ridge National Laboratory,

Oak Ridge, Tennessee 37830

ASHWORTH, DR. JOHN MICHAEL, Department of Biochemistry, Leicester Uni- versity, Leicester, England ATWOOD, DR. KIMBALL C., Department of Microbiology, University of Illinois,

Urbana, Illinois 61801 AUCLAIR, DR. WALTER, Department of Biology, Rensselaer Polytechnic Institute,

Troy, New York 12181 AUSTIN, DR. COLIN RUSSELL, Physiological Laboratory, Cambridge University,

Downing Street, Cambridge, England

AUSTIN, DR. MARY L., 506^ North Indiana Avenue, Bloomington, Indiana 47401 BACON, MR. ROBERT, Church Street, Woods Hole, Massachusetts 02543 BAKALAR, MR. DAVID, 330 Beacon Street, Boston, Massachusetts 02167 BALL, DR. ERIC G., Department of Biological Chemistry, Harvard Medical

School, Boston, Massachusetts 02115 BALLARD, DR. WTILLIAM W., Department of Biological Sciences, Dartmouth

College, Hanover, New Hampshire 03755 BANG, DR. F. B., Department of Pathobiology, The Johns Hopkins University

School of Hygiene, Baltimore, Maryland 21205 BARD, DR. PHILLIP, The Johns Hopkins Medical School, Baltimore, Maryland

21205 BARTH, DR. L. G., Marine Biological Laboratory, Woods Hole, Massachusetts

02543 BARTH, DR. LUCENA, Marine Biological Laboratory, Woods Hole, Massachusetts

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 BAYLOR, DR. E. R., State University of New York at Stony Brook, Long Island,

New York 11790 BAYLOR, DR. MARTHA A., State University of New York at Stony Brook, Long

Island, New York 11790 BEAMS, DR. HAROLD W7., Department of Zoology, State University of Iowa, Iowa

City, Iowa 52240 BECK, DR. L. V., Department of Pharmacology, Indiana University, School of

Experimental Medicine, Bloomington, Indiana 47401 BEHRE, DR. ELINOR M., Black Mountain, North Carolina 28711 BELAMARICH, DR. FRANK A., Department of Biology, Boston University, Boston,

Massachusetts 02215 BELL, DR. ALLEN, Department of Anatomy, University of Colorado, Medical

Center, Denver, Colorado 80220

BELL, DR. EUGENE, Department of Biology, Massachusetts Institute of Tech- nology, Cambridge, Massachusetts 02139 BENNETT, DR. MICHAEL V. L., Department of Anatomy, Albert Einstein College

of Medicine, Bronx, New York 10461

46 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

BENNETT, DR. MIRIAM F., Department of Biology, Sweet Briar College, Sweet

Briar, Virginia 24595 BERG, DR. WILLIAM E., Department of Zoology, University of California,

Berkeley, California 94720 BERMAN, DR. MONES, National Institutes of Health, Institute for Arthritis and

Metabolic Diseases, Bethesda, Maryland 20014 BERNE, DR. ROBERT M., University of Virginia School of Medicine, Charlottes-

ville, Virginia 22903 BERNHEIMER, DR. ALAN W., New York University College of Medicine, New

York, New York 10016 BERNSTEIN, DR. MAURICE, Department of Anatomy, Wayne State University

College of Medicine, Detroit, Michigan 48237 BERSOHN, DR. RICHARD, Department of Chemistry, Columbia University, 959

Havemeyer Hall, New York, New York 10027 BEVELANDER, DR. GERRIT, Dental Branch, Medical Center, University of Texas,

Houston, Texas 77025 BIGGERS, DR. JOHN DENNIS, The Johns Hopkins University School of Hygiene

and Public Health, Division of Population Dynamics, Baltimore, Maryland

21205 BISHOP, DR. DAVID W., Medical College of Ohio at Toledo, P.O. Box 6190,

Toledo, Ohio 43614

BLANCHARD, DR. K. C., The Johns Hopkins University Medical School, Balti- more, Maryland 21205

BLOCK, DR. ROBERT, Adalbertstr. 70, 8 Munich, 13 Germany BLUM, DR. HAROLD F., Department of Biological Sciences, State University of

New York at Albany, Albany, New York 12203 BODANSKY, DR. OSCAR, Department of Biochemistry, Memorial Cancer Center,

444 East 68th Street, New York, New York 10021 BODIAN, DR. DAVID, Department of Anatomy, The Johns Hopkins University,

709 North Wolfe Street, Baltimore, Maryland 21205 BOELL, DR. EDGAR J., Department of Biology, Kline Biology Tower, Yale

University, New Haven, Connecticut 06520 BOETTIGER, DR. EDWARD G., Department of Zoology, University of Connecticut,

Storrs, Connecticut 06268 BOLD, DR. HAROLD C., Department of Botany, University of Texas, Austin,

Texas 78712

BOOLOOTIAN, DR. RICHARD A., Box 24787, Los Angeles, California 90024 BOREI, DR. HANS G., Department of Zoology, University of Pennsylvania,

Philadelphia, Pennsylvania 19104 BORSELLINO, DR. ANTONIO, Institute di P'iscia, Viale Benedetto XV, 5 Genova,

Italy BOWEN, DR. VAUGHN T., Woods Hole Oceanographic Institution, Woods Hole,

Massachusetts 02543

BRADLEY, DR. HAROLD C., 2639 Durant Avenue, Berkeley, California 94704 BRANDT, DR. PHILIP WILLIAMS, Department of Anatomy, Columbia University,

College of Physicians and Surgeons, New York, New York 10032 BRIDGMAN, DR. ANNA J., Department of Biology, Agnes Scott College, Decatur,

Georgia 30030

REPORT OF THE DIRECTOR 47

BRINLEY, DR. F. J., JR., Department of Physiology, The Johns Hopkins Uni- versity Medical School, Baltimore, Maryland 21205 BRONK, DR. DETLEV W., The Rockefeller University, 66th Street and York

Avenue, New York New York 10021 BROOKS, DR. MATILDA M., Department of Physiology, University of California,

Berkeley, California 94720

BROWN, DR. DUGALD E., 34 Hyatt Road, Woods Hole, Massachusetts 02543 BROWN, DR. FRANK A., JR., Department of Biological Sciences, Northwestern

University, Evanston, Illinois 60201 BUCK, DR. JOHN B., Laboratory of Physical Biology, National Institutes of

Health, Bethesda, Maryland 20014 BULLOCK, DR. T. H., Department of Neurosciences, University of California,

San Diego, La Jolla, California 92038 BURBANCK, DR. MADELINE PALMER, Box 15134, Emory University, Atlanta,

Georgia 30322 BURBANCK, DR. WILLIAM D., Box 15134, Emory University, Atlanta, Georgia

30322

BURDICK, DR. C. LALOR, The Lalor Foundation, 4400 Lancaster Pike, Wilming- ton, Delaware 19805 BURGER, DR. MAX M., Department of Biology, Princeton University, Princeton,

New Jersey 08140 BURNETT, DR. ALLISON LEE, Department of Biology, Case Western Reserve

University, Cleveland, Ohio 44106 BUSSER, DR. JOHN H., American Institution of Biological Sciences, 3900 Wisconsin

Avenue NW, Washington, D. C. 20016 BUTLER, DR. E. G., Department of Biology, Princeton University, Princeton,

New Jersey 08540 CANTONI, DR. GIULLIO, National Institutes of Health, Mental Health, Bethesda

Maryland 20014 CARLSON, DR. FRANCIS D., Department of Biophysics, The Johns Hopkins

University, Baltimore, Maryland 21218

CARPENTER, DR. RUSSELL L., Tufts University, Medford, Massachusetts 02155 CARRIKER, DR. MELBOURNE R., Systematics-Ecology Program, Marine Biological

Laboratory, Woods Hole, Massachusetts 02543 CASE, DR. JAMES, Department of Biology, University of California, Santa

Barbara, California 93106 CASSIDY, REV. JOSEPH D., O.P., Department of Biological Sciences, Northwestern

University, Evanston, Illinois 60201 CATTELL, DR. McKEEN, Cornell University Medical College, 1300 York Avenue,

New York, New York 10021

CHAET, DR. ALFRED B., University of West Florida, Pensacola, Florida 32505 CHAMBERS, EDWARD L., University of Miami, School of Medicine Miami, Florida CHASE, DR. AURIN M., Department of Biology, Princeton University, Princeton,

New Jersey 08540

CHAUNCEY, DR. HOWARD H., Veterans Administration Central Office, Washing- ton, D. C. 20420 CHENEY, DR. RALPH H., Biological Laboratory, Brooklyn College, Brooklyn,

New York 11210

48 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

CHILD, DR. FRANK M., Department of Biology, Trinity College, Hartford, Con- necticut 06106

CLAFF, DR. C. LLOYD, P. O. Box 144, Woods Hole, Massachusetts 02543 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, DR. LEONARD B., 149 Sippewissett Road, Falmouth, Massachusetts

02540 CLARKE, DR. GEORGE L., Biological Laboratories, Harvard University, Cambridge,

Massachusetts 02138 CLAYTON, DR. RODERICK K., Division of Biological Sciences, Cornell University,

Ithaca, New York 14850

CLELAND, DR. RALPH E., Department of Botany, Indiana University, Blooming- ton, Indiana 47401 CLEMENT, DR. A. C., Department of Biology, Emory University, Atlanta, Georgia

30322 CLOWES, DR. GEORGE H. A., JR., Havard Medical School, Boston, Massachusetts

02115 COHEN, DR. SEYMOUR S., Department of Therapeutic Research, University of

Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 COLE, DR. KENNETH S., Laboratory of Biophysics, NINDS, National Institutes

of Health, Bethesda, Maryland 20014 COLLIER, DR. JACK R., Department of Biology, Brooklyn College, Brooklyn,

New York 11210

COLTON, DR. H. S., P. O. Box 699, Flagstaff, Arizona 86001 COLWIN, DR. ARTHUR L., Department of Biology, Queens College, Flushing, New

York 11367 COLWIN, DR. LAURA H., Department of Biology, Queens College, Flushing, New

York 11367

COOPERSTEIN, DR. SHERWIN J., School of Dental Medicine, University of Con- necticut, Hartford, Connecticut 06105 COPELAND, DR. D. EUGENE, Department of Biology, Tulane University, New

Orleans, Louisiana 70118

COPELAND, DR. MANTON, 88 Federal Street, Brunswick, Main 04011 CORNMAN, DR. IVOR, 10A Orchard Street, Woods Hole, Massachusetts 02543 COSTELLO, DR. DONALD P., Department of Zoology, University of North Carolina,

Chapel Hill, North Carolina 27514 COSTELLO, DR. HELEN MILLER, Department of Zoology, University of North

Carolina, Chapel Hill, North Carolina 27514 COUSINEAU, DR. GILLES H., Department of Biology, Montreal University, P. O.

Box 6128, Montreal, P. Q., Canada CRANE, MR. JOHN O., Woods Hole, Massachusetts 02543 CRANE, DR. ROBERT K., Department of Physiology, Rutgers Medical School,

New Brunswick, New Jersey 08903 CROASDALE, DR. HANNAH T., Dartmouth College, Hanover, New Hampshire

03755

REPORT OF THE DIRECTOR 49

GROUSE, DR. HELEN V., Institute for Molecular Biophysics, Florida State Uni- versity, Tallahassee, Florida 32306 CROWELL, DR. SEARS, Department of Zoology, Indiana University, Bloomington,

Indiana 47401 CSAPO, DR. ARPAD I., Washington University School of Medicine, 4911 Barnes

Hospital Plaza, St. Louis, Missouri 63110 DAIGNAULT, MR. ALEXANDER, T., W. R. Grace and Company, 7 Hanover Square

New York, New York 10005 DAN, DR. JEAN CLARK, Department of Biology, Ochanomizu University, Otsukaj

Bunkyo-Ku, Tokyo, Japan DAN, DR. KATSUMA, President, Tokyo Metropolitan University, Meguro-Ku,

Tokyo, Japan DANIELLI, DR. JAMES F., Department of Medicinal Chemistry, University of

Buffalo School of Pharmacy, Buffalo, New York 14214 DAVIS, DR. BERNARD D., Havard Medical School, 25 Shattuck Street, Boston,

Massachusetts 02115 DEHAAN, DR. ROBERT L., Department of Embryology, Carnegie Institution of

Washington, Baltimore, Maryland 21210 DELORENZO, DR. ANTHONY, Anatomical and Pathological Research Laboratory,

The Johns Hopkins Hospital, Baltimore, Maryland 21205 DEPHILLIPS, DR. HENRY A., JR., Department of Chemistry, Trinity College,

Hartford, Connecticut 06106 DETTBARN, DR. WOLF-DEITRICH, Department of Pharmacology, Vanderbilt

University, School of Medicine, Nashville, Tennessee 37217

DEViLLAFRANCA, DR. GEORGE W., Department of Zoology, Smith College, North- ampton, Massachusetts 01060 DIEHL, DR. FRED ALSON, Department of Biology, University of Virginia, Char-

lottesville, Virginia 22903

DILLER, DR. IRENE C., 2417 Fairhill Avenue, Glenside, Pennsylvania 19038 DILLER, DR. WILLIAM F., 2417 Fairhill Avenue, Glenside, Pennsylvania 19038 DODDS, DR. G. S. 829 Price Street, Morgantown, W^est Virginia 26505 DOOLITTLE, DR. R. F., Department of Biology, University of California, La Jolla,

California 92037 DOWBEN, DR. ROBERT, Division of Biomedical Sciences, Brown University,

Providence, Rhode Island 02912 DOWLING, DR. JOHN E., Department of Ophthalmology and Biophysics, Wilmer

Institute, Johns Hopkins University, Baltimore, Maryland 21205 DRESDEN, DR. MARCH., Department of Biochemistry, Baylor College of Medicine,

Houston, Texas 77025 DUNHAM, DR. PHILIP B., Department of Zoology, Syracuse University, Syracuse,

New York 13210 DURYEE, DR. WILLIAM R., Department of Pathology, George Washington

University, 2300 K Street, N. W., Washington, D. C. 20037 EBERT, DR. JAMES DAVID, Department of Embryology, Carnegie Institution of

Washington, Baltimore, Maryland 21210 ECCLES, DR. JOHN C., Department of Biophysics and Physiology, State University

of New York at Buffalo, Buffalo, New York 14214 '

50 ANM Al. REPORT OF THE MARINE BIOLOGICAL LABORATORY

ECKERT, DR. ROGER O., Department of Zoology, University of California,

Los Angeles, California 90024

EDDS, DR. MAC V., JR., Department of Medical Science, Box G, Brown Uni- versity, Providence, Rhode Island 02912 EDER, DR. HOWARD A., Albert Einstein College of Medicine, Bronx, 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 Institute for Muscle Research, Marine Biological

Laboratory, Woods Hole, Massachusetts 02543 EICHEL, DR. HERBERT J., Department of Biochemistry, Hahnemann Medical

College, Philadelphia Pennsylvania 19102 EISEN, DR. ARTHUR Z., Division of Dermatology, Washington University, School

of Medicine, St. Louis, Missouri 63130 EISEN, DR. HERMAN, Department of Medicine, Washington University, St. Louis,

Missouri 63110 ELDER, DR. HUGH YOUNG, Institute of Physiology, University of Glasgow

Glasgow, Scotland, U. K. ELLIOTT, DR. ALFRED M., Department of Zoology, University of Michigan, Ann

Arbor, Michigan 48104 ELLIOTT, DR. GERALD F., Walton Hall, Bletchley, Bucks, The Open University,

London, England, U. K. ESSNER, DR. EDWARD S., Sloan-Kettering Institute for Cancer Research, Rye,

New York 10580 EVANS, DR. TITUS C., State University of Iowa College of Medicine, Iowa City,

Iowa 52240 FAILLA, DR, P. M., Radiological Physics Division, Argonne National Laboratory,

Argonne, Illinois 60439

FARMANFARMAIAN, DR. ALLAHVERDI, Department of Physiology and Biochem- istry, Rutgers University, New Brunswick, New Jersey 08903 FAURE-FREMIET, DR. EMMANUEL, College de France, Paris, France 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, Havard Medical School, Boston,

Massachusetts 02115 FERGUSON, DR. F. P., National Institute of General Medical Sciences, National

Institutes of Health, Bethesda, Maryland 20014 FERGUSON, DR. JAMES K. W., Connought Laboratories, University of Toronto,

Toronto 5, Ontario, Canada FIGGE, DR. F. H. J., University of Maryland Medical School, Lombard and Green

Streets, Baltimore, Maryland 21201 FINGERMAN, DR. MILTON, Department of Biology, Tulane University, New

Orleans, Lousiana 70118 FISCHER, DR. ERNST, Department of Physical Medicine and Rehabilitation,

Albany Medical College, Albany, New York 12208 FISHER, DR. FRANK M., JR., Department of Biology, Rice University, Houston,

Texas 77001

REPORT OF THE DIRECTOR 51

FISHER, DR. JEANNE M., Department of Biochemistry, University of Toronto,

Toronto 5, Ontario, Canada FISHER, DR. KENNETH C., Department of Zoology, University of Toronto,

Toronto 5, Ontario, Canada

FISHMAN, DR. Louis, 143 North Grove Street, Valley Stream, New York 11580 FRAENKEL, DR. GOTTFRIED S., Department of Entomology, University of Illinois,

Urbana, Illinois 61801 FREEMAN, DR. ALAN RICHARD, Department of Physiology, Rutgers Medical

School, New Brunswick, New Jersey 08903 FREYGANG, DR. WALTER H., JR., 6247 29th Street, N. \V., Washington, D. C.

20015

FRIES, DR. ERIK F. B.. P. O. Box 605, WToods Hole, Massachusetts 02543 FULTON, DR. CHANDLER M., Department of Biology, Brandeis University

W^altham, Massachusetts 02154 FUORTES, DR. MICHAEL G. F., NINDS, National Institutes of Health, Bethesda,

Maryland 20014 FURSHPAN, DR. EDWIN J., Department of Neurophysiology, Harvard Medical

School, Boston, Massachusetts 02115

FURTH, DR. JACOB, 99 Fort Washington Avenue, New York, New York 10032 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 GAFFRON, DR. HANS, Department of Biology, Institute of Molecular Biophysics,

Florida State University, Tallahasee, Florida 32306 GALL, DR. JOSEPH G., Department of Biology, Yale University, New Haven,

Connecticut 06520

GALTSOFF, DR. PAUL S., Bureau of Commercial Fisheries, Woods Hole, Massa- chusetts 02543 GELFANT, DR. SEYMOUR, Department of Zoology, Syracuse University, Syracuse,

New York 13210 GERMAN, DR. JAMES L., Ill, The New York Blood Center, 310 East 67th Street,

New York, New York 10021 GIBBS, DR. MARTIN, Department of Biology, Brandeis University, Waltham,

Massachusetts 02154 GILBERT, DR. DANIEL L., Laboratory of Biophysics, NINDS, National Institutes

of Health, Building 36, Room 2A-31, Bethesda, Maryland 20014 GILMAN, DR. LAUREN C., Department of Zoology, University of Miami, Coral

Gables, Florida 33146

GINSBERG, DR. HAROLD S., Department of Microbiology, University of Pennsyl- vania School of Medicine, Philadelphia, Pennsylvania 19104 GIUDICE, DR. GIOVANNI, University of Palermo, Via Archirafi 22, Palermo, Italy GOLDEN, MR. WILLIAM T., 40 Wall Street, New York, New York 10050 GOLDSMITH, DR. TIMOTHY H., Department of Biology, Yale University, New

Haven, Connecticut 06520 GOODCHILD, DR. CHAUNCEY G., Department of Biology, Emory University,

Atlanta, Georgia 30322

52 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

GOTTSCHALL, DR. GERTRUDE Y., 315 East 68th Street, Apartment 9M, New York,

New York 10021

GRAHAM, DR. HERBERT, Bureau of Commercial Fisheries, Woods Hole, Massa- chusetts 02543

GRANT, DR. DAVID C., Box 2316, Davidson, North Carolina 28036 GRANT, DR. PHILIP, Department of Biology, University of Oregon, Eugene,

Oregon 97403 GRASS, MR. ALBERT, The Grass Foundation, 77 Reservoir Road, Quincy,

Massachusetts 02170 GRASS, MRS. ELLEN R., The Grass Foundation, 77 Reservoir Road, Quincy,

Massachusetts 02170 GRAY, DR. IRVINE E., Department of Zoology, Duke University, Durham, North

Carolina 27706 GREEN, DR. JAMES W., Department of Physiology, Rutgers University, New

Brunswick, New Jersey 08903 GREEN, DR. JONATHAN P., Department of Biology, Brown University, Providence,

Rhode Island 02912 GREEN, DR. MAURICE, Department of Microbiology, St Louis University Medical

School, St. Louis, Missouri 63103 GREENBERG, DR. MICHAEL J., Department of Biological Sciences, Florida State

University, Tallashassee, Florida 32306 GREGG, DR. JAMES H., Department of Zoology, University of Florida, Gainesville,

Florida 32601 GREGG, DR. JOHN R., Department of Zoology, Duke University, Durham, North

Carolina 27706 GREIF, DR. ROGER L., Department of Physiology, Cornell University Medical

College, New York, New York 10021 GRIFFIN, DR. DONALD R., The Rockefeller University, New York, New York

10021 GROSCH, DR. DANIEL S., Department of Genetics, Gardner Hall, North Carolina

State University, Raleigh, North Carolina 27607 GROSS, DR. JEROME, Developmental Biology Laboratory, Massachusetts General

Hospital, Boston, Masaschusetts 02114

GROSS, DR. PAUL, Department of Biology, Massachusetts Institute of Tech- nology, Cambridge, Massachusetts 02139 GROSSMAN, DR. ALBERT, New York University Medical School, New York, New

York 10016 GRUNDFEST, DR. HARRY, Department of Neurology, Columbia University,

College of Physicians and Surgeons, New York, New York 10032 GUTTMAN, DR. RITA, Department of Biology, Brooklyn College, Brooklyn

New York 11210 GWILLIAM, DR. G. F., Department of Biology, Reed College, Portland, Oregon

97202

HAJDU, DR. STEPHEN, National Institutes of Health, Bethesda, Maryland 20014 HALVORSON, DR. HARLYN O., Department of Bacteriology, University of Wis- consin, Madison, Wisconsin 53706 HAMBURGER, DR. VIKTOR, Department of Zoology, Washington University, St.

Louis, Missouri 63110

REPORT OF THE DIRECTOR 53

HAMILTON, DR. HOWARD L., Department of Biology University of Virginia,

Charlottesville, Virginia 22903

HARDING, DR. CLIFFORD V., JR., Oakland University, Rochester, Michigan 84063 HARRINGTON, DR. GLENN W., Upstate Medical Center, State University of New

York, Syracuse, New York 13210 HARTLINE, DR. H. KEFFER, The Rockefeller University, New York, New York

10021 HARTMAN, DR. FRANK A., Ohio State University, Hamilton Hall, Columbus,

Ohio 43210 HARTMAN, DR. H. BERNARD, Department of Zoology, University of Iowa, Iowa

City, Iowa 52240 HARTMAN, DR. KATHERINE A., Department of Physiology, Ohio State University,

Columbus, Ohio 43210 HARTMAN, DR. P. E., Department of Biology, The Johns Hopkins University,

Baltimore, Maryland 21218 HASCHEMEYER, DR. AUDREY E. V., Department of Biological Sciences, Hunter

College, 695 Park Avenue, New York, New York 10021 HASTINGS, DR. J. WOODLAND, Biological Laboratories, Havard University,

Cambridge, Massachusetts 02138 HAUSCHKA, DR. T. S., Roswell Park Memorial Institute, 666 Elm Street, Buffalo,

New York 14203 HAXO, DR. FRANCIS T., Department of Marine Botany, Scripps Institution of

Oceanography, University of California, La Jolla, California 92038 HAYASHI, DR. TERU, Department of Biology, Illinois Institute of Technology,

Chicago, Illinois 60616

HAYWOOD, DR. CHARLOTTE, Box 14, South Hadley, Massachusetts 01075 HEGYELI, DR. ANDREW F., 8018 Aberdeen Road, Bethesda, Maryland 20014 HENLEY, DR. CATHERINE, Department of Zoology, University of North Carolina,

Chapel Hill, North Carolina 27514 HERNDON, DR. WALTER R., Office of the Dean, College of Liberal Arts, University

of Tennessee, Knoxville, Tennessee 37916

HERVEY, MR. JOHN P., Box 735, Woods Hole, Massachusetts 02543 HESSLER, DR. ANITA Y., 5795 Waverly Avenue, La Jolla, California 92037 HIATT, DR. HOWARD H., Beth Israel Hospital, 330 Brookline Avenue, Boston,

Massachusetts 02215

HIBBARD, DR. HOPE, 366 Reamer Place, Oberlin, Ohio 44074 HILL, DR. ROBERT BENJAMIN, Department of Zoology, University of Rhode

Island, Kingston, Rhode Island 02881

HINEGARDNER, DR. RALPH T., Division of Natural Sciences, University of Cali- fornia, Santa Cruz, California 95060 HINSCH, DR. GERTRUDE W., Institute of Molecular Evolution, 521 Anastasia,

University of Miami, Coral Gables, Florida 33134 HIRSHFIELD, DR. HENRY I., Department of Biology, Washington Square Center,

New York University, New York, New York 10003 HOADLEY, DR. LEIGH, Biological Laboratories, Harvard University, Cambridge,

Massachusetts 02138

HODGE, DR. CHARLES, IV, Department of Biology, Temple University, Philadel- phia, Pennsylvania 19122

54 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

HOFFMAN, DR. JOSEPH, Department of'Physiology, Yale University School of Medicine, New Haven, Connecticut 06515

HOLLAENDER, DR. ALEXANDER, Biology Division, Oak Ridge National Labora- tory, Oak Ridge, Tennessee 37830

HOLTZMAN, DR. ERIC, Department of Biological Sciences, Columbia University, New York, New York 10032

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., Biology Department, Illinois Institute of Tech- nology, Chicago, Illinois 60616

HOSTETLER, DR. KARL Y., Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106

HOUSTON, MR. HOWARD, Preston Avenue, Meriden, Connecticut 06450

HUMPHREYS, DR. TOM DANIEL, Department of Biology, University of California, San Diego, La Jolla, California 92037

HUNTER, DR. FRANCIS R., Department of Biology, Centre Experimental de Estu- dios Superiores, Barquisimeto, Venezuela

HURWITZ, DR. CHARLES, Basic Science Research Laboratory, VA Hospital, Albany, New York 12208

HURWITZ, DR. J., Department of Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461

HUTCHENS, DR. JOHN E., Department of Physiology, University of Chicago, Chicago, Illinois 60637

HUXLEY, DR. HUGH E., A-Iedical Research Council, Laboratory of Molecular Biology, Cambridge, England, U. K.

HYDE, DR. BEAL B., Department of Botany, University of Vermont, Burlington, Vermont 05401

HYDE, ROBINSON, Princeton University, Princeton, New Jersey 08540

INOUE, DR. SHINYA, 217 Leidy Building, Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104

ISENBERG, DR. IRVIN, Science Research Institute, Oregon State University, Corvallis, Oregon 97330

ISSELBACHER, DR. KURT J., Massachusetts General Hospital, Boston, Massa- chusetts 02114

JAFFE, LIONEL, Department of Biology, Purdue University, Lafayette, Indiana 46207

JANOFF, DR. AARON, Department of Pathology, New York University School of Medicine, 550 First Avenue, New York, New York 10016

JENNER, DR. CHARLES E., Department of Zoology, University of North Carolina, Chapel Hill, North Carolina 27514

JOHNSON, DR. FRANK H., Department of Biology, Princeton University, Prince- ton, New Jersey 08540

JONES, DR. E. RUFFIN, JR., Department of Biological Sciences, University of Florida, Gainesville, Florida 32601

JONES, DR. MEREDITH L., Division of Worms, Museum of Natural History, Smithsonian Institution, Washington, D. C. 20650

JONES, DR. RAYMOND F., Department of Biology, State University of New York at Stony Brook, Long Island, New York 11753

REPORT OF THE DIRECTOR

JOSEPHSON, DR. R. K., Department of Biology, Case Western Reserve University,

Cleveland, Ohio 44106

KAAN, DR. HELEN W., Box 665, Woods Hole, Massachusetts 02543 KABAT, DR. E. A., Neurological Institute, Columbia University, College of

Physicians and Surgeons, New York, New York 10032

KALEY, DR. GABOR, New York Medical College, Flower and Fifth Avenue Hos- pitals, 5th Avenue at 106th Street, New York, New York 10029 KAMINER, DR. BENJAMIN, The Institute for Muscle Research, Marine Biological

Laboratory, Woods Hole, Massachusetts 02543 KANE, DR. ROBERT F., Pacific Biomedical Research Center, 2538 The Mall,

University of Hawaii, Honolulu, Hawaii 96822 KARAKASHIAN, DR. STEPHEN J., Department of Biology, State University of New

York College at Old Westbury, Oyster Bay, New York 11771 KARUSH, DR. FRED, Department of Microbiology, University of Pennsylvania

School of Medicine, Philadelphia, Pennsylvania 19104 KATZ, DR. GEORGE M., Department of Neurology, Columbia University, College

of Physicians and Surgeons, 630 W7est 168th Street, New York, New York

10032 KAUFMAN, DR. B. P., Department of Zoology, University of Michigan, Ann Arbor,

Michigan 48104 KELLY, ROBERT E., Department of Anatomy and Psychology, Dartmouth

Medical School, Hanover, New Hampshire 03755 KEMP, DR. NORMAN E., Department of Zoology, University of Michigan, Ann

Arbor, Michigan 48104

KEMPTON, DR. RUDOLF T., RR No. 1, Box 351, St Augustine, Florida 32084 KEOSIAN, DR. JOHN, Department of Biology, Rutgers University, Newark, New

Jersey 07102 KETCHUM, DR. BOSTWICK H., Woods Hole Oceanographic Institution, Woods

Hole, Massachusetts 02543

KEYNAN, DR. ALEXANDER, Institute for Biological Research, Ness-Ziona, Israel KILLE, DR. FRANK R., State Department of Education, Albany, New York 12201 KIND, DR. C. ALBERT, Department of Zoology, University of Connecticut, Storrs,

Connecticut 06268

KINDRED, DR. JAMES E., 2010 Hessian Road, Charlottesville, Virginia 22903 KING, DR. THOMAS J., Georgetown University, Department of Biology, Washing- ton, D. C. 20007 KINGSBURY, DR. JOHN M., Department of Botany, Cornell University, Ithaca,

New York 14850 KINNE, DR. OTTO, Biologische Anstalt Helgoland, 2 Hamburg-Altona, Palmaille

9, Germany

KLEIN, DR. MORTON, Department of Microbiology, Temple University, Phila- delphia, Pennsylvania 19122 KLEINHOLZ, DR. LEWIS H., Department of Biology, Reed College, Portland,

Oregon 97202 KLOTZ, DR. I. M., Department of Chemistry, Northwestern University, Evanston,

Illinois 60201 KOHLER, KURT, Department of Biochimie Macromoleculair, C.N.R.S., Universite

de Montpellier, Montpellier, France

56 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

KOLIN, DR. ALEXANDER, Department of Biophysics, California Medical School, Los Angeles, California 90024

KONIGSBERG, DR. IRWIN R., Department of Biology, Gilmer Hall, University of Virginia, Charlottesville, Virginia 22903

KORNBERG, DR. HANS LEO, Department of Biochemistry, University of Leicester, Leicester, England

KORR, DR. I. M., Department of Physiology, Kirksville College of Osteopathy, Kirksville, Missouri 63501

KRAHL, DR. M. E., Department of Physiology, University of Chicago, Chicago, Illinois 60637

KRANE, DR. STEPHEN M., Massachusetts General Hospital, Boston, Massa- chusetts 02114

KRASSNER, DR. STUART MITCHELL, Department of Organismic Biology, Uni- versity of California, Irvine, California 92650

KRAUSS, DR. ROBERT, Department of Botany, University of Maryland, Balti- more, Maryland 21201

KREIG, DR. WENDELL J. S., 303 East Chicago Avenue, Chicago, Illinois 60611

KRIEBEL, DR. MAHLON E., Department of Anatomy, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461

KUFFLER, DR. STEPHEN W., Department of Neurophysiology, Havard Medical School, Boston, Massachusetts 02115

KUNITZ, DR. MOSES, The Rockefeller University, 66th Street and York Avenue, New York, New York 10021

KUSANO, DR. KIYOSHI, Department of Psychiatry, Indiana University, Blooming- ton, Indiana 47401

LAMY, DR. FRANCOIS, Department of Biochemistry, University of Sherbrooke, School of Medicine, Sherbrooke, Quebec, Canada

LANCEFILLD, DR. D. E., 203 Arleigh Road, Douglaston, Long Island, New York 11363

LANCEFIELD, DR. REBECCA C, The Rockefeller University, 66th Street and York Avenue, New York, New York 10021

LANDIS, DR. E. M., Department of Biology, Lehigh University, Bethlehem, Pennsylvania 18015

LANSING, DR. ALBERT I., Department of Anatomy, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213

LASH, DR. JAMES W., Department of Anatomy, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

LASTER, DR. LEONARD, National Institutes of Health, Bethesda, Maryland 20014

LAUFER, DR. HANS, Department of Zoology and Entomology, University of Connecticut, Storrs, Connecticut 06268

LAUFER, DR. MAX A., Department of Biophysics, University of Pittsburgh, Pittsburgh, Pennsylvania 15213

LAVIN, DR. GEORGE I., 6200 Norvo Road, Baltimore, Maryland 21207

LAWLER, DR. H. Claire, 336 West 246th Street, Riverdale, New York 10471

LAZAROW, DR. ARNOLD, Department of Anatomy, University of Minnesota Medical School, Minneapolis, Minnesota 55455

LEDERBERG, DR. JOSHUA, Department of Genetics, Stanford Medical School, Palo Alto, California 94304

REPORT OF THE DIRECTOR 57

LEE, DR. Richard E., Cornell University College of Medicine, New York, New

York 10021 LEFEVRE, DR. PAUL G., University of Louisville School of Medicine, Louisville,

Kentucky 40208

LEHMANN, DR. FRITZ, Zoologische Inst., University of Berne, Berne, Switzerland LENHER, DR. SAMUEL, 1900 Woodlawn Avenue, Wilmington, Delaware 19806 LERMAN, DR. SIDNEY, Mclntyre Medical Science Center, McGill University,

Room 12H, Montreal, Canada

LERNER, DR. ARRON B., Yale Medical School, New Haven, Connecticut 06515 LEVIN, DR. JACK, Department of Medicine, The Johns Hopkins Hospital,

Baltimore, Maryland 21205 LEVINE, DR. RACHMIEL, Department of Medicine, New York University Medical

College, 5th Avenue at 106th Street, New York, New York 10029 LEVINTHAL, DR. CYRUS, Department of Biological Sciences, Columbia University,

908 Schermerhorn Hill, New York, New York 10027 LEVY, DR. MILTON, Department of Biochemistry, New York University School of

Dentistry, New York, New York 10010 LEWIN, DR. RALPH A., Scripps Institution of Oceanography, La Jolla, California

92037 LEWIS, DR. HERMAN W., Genetic Biology Program, National Science Foundation,

Washington, D. C. 20025

LING, DR. GILBERT, 307 Berkeley Road, Merion, Pennsylvania 19066 LINSKENS, DR. H. P., Department of Botany, University of Driehuizerweg 200,

Nijmegen, The Netherlands

LITTLE, DR. E. P., 216 Highland Street, West Newton, Massachusetts 02158 LLOYD, DR. DAVID P. C., The Rockefeller University, New York, New York 10021 LOCHHEAD, DR. JOHN H., Department of Zoology, Life Sciences Building, Uni- versity of Vermont, Burlington, Vermont 05401 LOEB, DR. R. F., 950 Park Avenue, New York, New York 10028 LOEWENSTEIN, DR. WERNER R., Department of Physiology, ColumbiaUniversity,

College of Physicians and Surgeons, New York, New York 10032 LOFTFIELD, DR. ROBERT S., Department of Biochemistry, University of New

Mexico Medical School, Albuquerque, New Mexico 87106 LONDON, DR. IRVING M., Department of Medicine, Albert Einstein College of

Medicine, New York, New York 10461 LORAND, DR. LASZLO, Department of Chemistry, Northwestern University,

Evanston, Illinois 60201 LOVE, DR. WARNER E., Department of Biophysics, Johns Hopkins University,

Baltimore, Maryland 21218 LUBIN, DR. MARTIN, Department of Microbiology, Dartmouth Medical School,

Hanover, New Hampshire 03755

LYNCH, DR. CLARA J., The Rockefeller University, New York, New York 10021 LYNN, DR. W. GARDNER, Department of Biology, Catholic University of America,

Washington, D. C. 20017 MAcNiCHOL, EDWARD F., JR., NIH, Bldg. 31 Room SA-52, Bethesda, Maryland

20014 MAGRUDER, DR. SAMUEL R., Department of Anatomy, Tufts University School

of Medicine, 135 Harrison Avenue, Boston, Massachusetts 02111

ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

MAHLER, DR. HENRY R., Department of Biochemistry, Indiana University,

Bloomington, Indiana 47401 MALKIEL, DR. SAUL, Children's Cancer Research Foundation, Inc., 35 Binney

Street, Boston, Massachusetts 02115 MANGUM, CHARLOTTE P., Department of Biology, College of William and Mary,

Williamsburg, Virginia 23185 MANWELL, DR. REGINALD D., Department of Zoology, Syracuse University,

Syracuse, New York 13210 MARKS, DR. PAUL A., Columbia University, College of Physicians and Surgeons,

New" York, New York 10032 MARSH, DR. JULIAN B. Department of Biochemistry, University of Pennsylvania

School of Dental Medicine, 4001 Spruce St., Philadelphia, Pennsylvania 19104 MARSHAK, DR. ALFRED, Tulane University Medical School, New Orleans,

Louisiana 70112 MARSLAND, DR. DOUGLAS, A., 48 Church Street, Woods Hole, Massachusetts

02543

MARTIN, DR. EARL A., 682 Rudder Road, Naples, Florida 33940 MATHEWS, DR. SAMUEL A., Thompson Biological Laboratory, Williams College,

Williamstown, Massachusetts 02167 MAUTNER, DR. HENRY G., Department of Pharmacology, Yale University, School

of Medicine, New Haven, Connecticut 06510 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 McDANiEL, DR. JAMES SCOTT, Department of Biology, East Carolina College,

Greenville, North Carolina 28734 MCDONALD, SISTER ELIZABETH SETON, Department of Biology, College of Mt. St.

Joseph on the Ohio, Mt. St. Joseph, Ohio 45051

MCELROY, DR. WILLIAM D., Department of Biology, The Johns Hopkins Uni- versity, Baltimore, Maryland 21218 MEINKOTH, DR. NORMAN, Department of Biology, Swarthmore College, Swarth-

more Pennsylvania 19081 MELLON, DR. DEFOREST, JR., Department of Biology, University of Virginia,

Charolottesville, Virginia 22903 MENDELSON, DR. MARTIN, Department of Physiology, New York University

Medical School, New York, New York 10016 METZ, DR. C. B., Institute of Molecular Evolution, University of Miami, Coral

Gables, Florida 33146

METZ, DR. CHARLES W., Box 174, Woods Hole, Massachusetts 02543 MIDDLEBROOK, DR. ROBERT, Dorunsway, School Lane, Kirk Ella, Hull, England,

U. K. HU107NR MILKMAN, DR. ROGER D., Department of Zoology, University of Iowa, Iowa

City, Iowa 52240 MILLER, DR. FAITH STONE, Department of Anatomy, Tulane University, New

Orleans, Louisiana 70112 MILLER, DR. J. A., JR., Department of Anatomy, Tulane, University, New

Orleans, Lousiana 70112

REPORT OF THE DIRECTOR 59

MILLOTT, DR. NORMAN, Department of Zoology, Bedford College, University of

London, Regents Park, London N.W.I., England MILLS, DR. ERIC LEONARD, Institute of Oceanography, Dalhousie University,

Halifax, Nova Scotia, Canada MILNE, DR. LORUS J., Department of Zoology, University of New Hampshire,

Durham, New Hampshire 03824 MONROY, DR. ALBERTO, CNR Laboratory of Molecular Embroyology, 80072

Arco Felice, Napoli, Italy MOORE, DR. JOHN A., Department of Life Sciences, University of California,

Riverside, California 92502 MOORE, DR. JOHN \V., Department of Physiology, Duke University Medical

Center, Durham, North Carolina 27706

MOORE, DR. R. O., Department of Biochemistry, Ohio State University, Colum- bus, Ohio 43210 MORAN, DR. JOSEPH F., JR., Department of Biology, Sacred Heart LTniversity,

Bridgeport, Connecticut 06604 MORLOCK, DR. NOEL, Department of Neurology, Columbia University, College

of Physicians and Surgeons, New York, New York 10032 MORRILL, DR. JOHN B., JR., Division of Natural Sciences, New College, Sarasota,

Florida 33478

MORSE, DR. RICHARD STETSON, 193 Winding River Road, Wellesley, Massa- chusetts 02184 MOSCONA, DR. A. A., Department of Zoology, University of Chicago, Chicago,

Illinois 60637 MOUL, DR. E. T., Department of Biology, Rutgers University, New Brunswick,

New Jersey 08903

MOUNTAIN, MRS, J. D., Charles Road, Alt. Kisco, New York 10549 MULLINS, DR. LORIN J., Department of Biophysics, University of Maryland

School of Medicine, Baltimore, Maryland 21201

MUSACCHIA, DR. XAVIER J., Department of Physiology, Medical Center, Uni- versity of Missouri, Columbia, Missouri 65201 XABRIT, DR. S. M., Texas Southern University, 3201 Wheeler Avenue, Houston,

Texas 77004 NACE, DR. PAUL FOLEY, Department of Biology, University of West Florida,

Pensacola, Florida 32504 NACHMANSOHN, DR. DAVID, Department of Neurology, Colubmia University,

College of Physicians and Surgeons, New York, New York 10032 NARAHASHI, DR. TOSHIO, Department of Physiology, Duke University Medical

Center, Durham, North Carolina 27706 NASATIR, DR. MAIMON, Department of Biology, University of Toledo, Toledo,

Ohio 43606 NASON, DR. ALVIN, McCollum-Pratt Institute, The Johns Hopkins University,

Baltimore, Maryland 21218 NELSON, DR. LEONARD, Department of Physiology, Medical College of Ohio at

Toledo, Toledo, Ohio 43614 NEURATH, DR. H., Department of Biochemistry, University of Washington,

Seattle, Washington 98105

60 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

NICHOLLS, DR. JOHN GRAHAM, Department of Neurobiology, Harvard Medical

School, 25 Shattuck Street, Boston, Massachusetts 02115 NICOLL, DR. PAUL A., R.R. 12, Box 286, Bloomington, Indiana 47401 Niu, DR. MAN-CHIANG, Department of Biology, Temple University, Phila- delphia, Pennsylvania 19122 NOVIKOFF, DR. ALEX B., Department of Pathology, Albert Einstein College of

Medicine, Bronx, New York 10461 OCHOA, DR. SEVERO, New York University College of Medicine, New York, New

York 10016 ODUM, DR. EUGENE, Department of Zoology, University of Georgia, Athens,

Georgia 30601

OLSON, DR. JOHN M., Brookhaven National Laboratory, Upton, New York 11973 OPPENHEIMER, DR. JANE M., Department of Biology, Bryn Mawr College, Bryn

Mawr, Pennsylvania 19010

OSTERHOUT, DR. MARION IRWIN, 160 E. 65th Street, New York, New York 10021 PACKARD, DR. CHARLES, 13 North Street, Woods Hole, Massachusetts 02543 PALMER, DR. JOHN D., Department of Biology, New York University, University

Heights, New York, New York 10053 PALTI, DR. YORAM, Hebrew University School of Medicine, Department of

Physiology, Box 1172, Jerusalem, Israel PAPPAS, DR. GEORGE D., Department of Anatomy, Albert Einstein College of

Medicine, Bronx, New York 10461 PARNAS, DR. ITZCHAK, Department of Zoology, Hebrew University, Jerusalem,

Israel PASSANO, DR. LEONARD M., Department of Zoology, University of Wisconsin,

Madison, Wisconsin 53706 PATTEN, DR. BRADLEY M., University of Michigan, 2500 East Medical Building,

Ann Arbor, Michigan 48104 PERSON, DR. PHILIP, Special Dental Research Program, Veterans Administration

Hospital, Brooklyn, New York 11219 PETTIBONE, DR. MARIAN H., Division of Marine Invertebrates, U. S. National

Museum, Washington, D. C. 20025 PHILPOTT, DR. DELBERT E., MASA Ames Research Center, Moffett Field,

California 94035 PICK, DR. JOSEPH, Department of Anatomy, New York University, Bellevue

Medical Center, New York, New York 10016 PIERCE, DR. MADELENE E., Department of Zoology, Vassar College, Pough-

keepsie, New York 12601 POLLISTER, DR. A. W., Department of Zoology, Columbia University, New York,

New York 10027

POND, DR. Samuel E., 53 Alexander Street, Manchester, Connecticut 06040 PORTER, DR. KEITH R., Biological Laboratories, Harvard University, Cambridge,

Massachusetts 02138 POTTER, DR. DAVID, Department of Neurophysiology, Harvard Medical School,

Boston, Massachusetts 02115 POTTS, DR. WILLIAM T. W., Department of Biology, University of Lancaster,

Lancaster, England, U. K.

REPORT OF THE DIRECTOR 61

PRICE, DR. CARL A., Department of Biochemistry and Microbiology, Rutgers

University, New Brunswick, \c\v Jersey 08803 PROCTOR, DR. NATHANIEL, Department of Biology, Morgan State College,

Baltimore, Maryland 21212 PROSSER, DR. C. LADD, Department of Physiology and Biophysics, Burrill Hall,

University of Illinois, Urbana, Illinois 61803 PROVASOLI, DR. LUIGI, Raskins Laboratories, 165 Prospect Street, New Haven,

Connecticut 06520

PRYTZ, DR. MARGARET R., 21 McCoune Lane, Oyster Bay, New York 11771 RABIN, DR. HARVEY, Institute for Comparative Biology, Zoological Society of

San Diego, Box 551, San Diego, California 92112 RAMSEY, DR. ROBERT W., Department of Physiology, Medical College of Virginia,

Richmond, Virginia 23150 RANKIN, DR. JOHN S., Department of Zoology, University of Connecticut, Storrs,

Connecticut 06268 RANZI, DR. SILVIO, Department of Zoology, University of Milan, Via Celonia 10,

Milan, Italy RAPPORT, DR. M., Department of Pharmacology, Columbia University, College

of Physicians and Surgeons, New York, New York 10032 RATNER, DR. SARAH, Department of Biochemistry, The Public Health Research

Institute of the City of New York, Inc., 455 First Avenue, Newr York, New

York 10016 RAY, DR. CHARLES, JR., Department of Biology, Emory University, Atlanta,

Georgia 30322 READ, DR. CLARK P., Department of Biology, Rice University, Houston, Texas

77001 REBHUN, DR. LIONEL I., Department of Biology, Princeton University, Princeton,

New Jersey 08540

RECKNAGEL, DR. R. O., Department of Physiology, Case Western Reserve Uni- versity, Cleveland, Ohio 44106

REDFIELD, DR. ALFRED C., Woods Hole, Massachusetts 02543 RENN, DR. CHARLES E., 509 Ames Hall, The Johns Hopkins University, Balti- more, Maryland 21218 REUBEN, DR. JOHN P., Department of Neurology, Columbia University, College

of Physicians and Surgeons, New York, Newr York 10032 REYNOLDS, DR. GEORGE THOMAS, Palmer Laboratory, Princeton University,

Princeton, New Jersey 08540

REZNIKOFF, DR. PAUL, 151 Sparks Ave., Pelham, New York 10803 RICE, DR. ROBERT VERNON, Mellon Institute, Carnegie-Mellon University, 4400

Fifth Avenue, Pittsburgh, Pennsylvania 15213 RICH, DR. ALEXANDER, Department of Biology, Massachusetts Institute of

Technology, Cambridge, Massachusetts 02139 RICHARDS DR. A., 2950 East Marble Street, Tucson, Arizona 85716 RICHARDS, DR. A. GLENN, Department of Entomology, University of Minnesota,

St. Paul, Minnesota 55101 RICHARDS, DR. OSCAR W., Pacific University, College of Optometry, Forrest

Grove, Oregon 97116

62 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

ROCKSTEIN, DR. MORRIS, Department of Physiology, University of Miami School

of Medicine, P.O. Box 875 Biscay ne Annex, Miami, Florida 33152 ROMER, DR. ALFRED S., Museum of Comparative Zoology, Harvard University,

Cambridge, Massachusetts 02138 RONKIN, DR. RAPHAEL R., National Science Foundation, O.I.S.A., Washington,

D. C. 20550 ROOT, DR. W. S., Department of Physiology, Columbia University, College of

Physicians and Surgeons, New York, New York 10032 ROSE, DR. S. MERYL, Laboratory of Developmental Biology, Tulane University,

F. Edward Hebert Center, Belle Chasse, Louisiana 70037 ROSENBERG, DR. EVELYN K., Armed Forces Radiobiology Research Institute,

National Naval Medical Center, Bethesda, Maryland 20014 ROSENBERG, DR. PHILIP, Division of Pharmacology, University of Connecticut,

School of Pharmacy, Storrs, Connecticut 06268

ROSENBLUTH, Miss RAJA, Kinsmen Laboratory for Neurological Research, Uni- versity of British Columbia, Vancouver 8, British Columbia, Canada ROSENKRANZ, DR. HERBERT S., Department of Microbiology, Columbia Uni- versity, College of Physicians and Surgeons, New York, New York 10032 ROSENTHAL, DR. THEODORE B., Department of Anatomy, University of Pitts-

gurgh Medical School, Pittsburgh, Pennsylvania 15213 ROSLANSKY, DR. JOHN, 26 Albatross, Woods Hole, Massachusetts 02543 ROTH, DR. JAY S., Division of Biological Sciences, Section of Biochemistry and

Biophysics, University of Connecticut, Storrs, Connecticut 06268 ROTHENBERG, DR. M. A., Dorset Test Center, Ft. Douglas, Salt Lake City, Utah

84113 ROWLAND, DR. LEWIS P., Department of Neurology, Hospital of the University

of Pennsylvania, 3400 Spruce Street, Philadelphia, Pennsylvania 19104 RUGH, DR. ROBERTS, Radiological Research Laboratory, Columbia University,

College of Physicians and Surgeons, New York, New York 10032 RUNNSTROM, DR. JOHN, Wenner-Grens Institute, Stockholm, Sweden RUSHFORTH, DR. NORMAN B., Department of Biology, Case Western Reserve

University, Cleveland, Ohio 44106 RUSSELL-HUNTER, DR. W. D., Department of Zoology, Lyman Hall, Syracuse

University, Syracuse, New York 13210 RUSTAD, DR. RONALD C., Department of Radiology, Case Western Reserve

University, Cleveland, Ohio 44106 RUTMAN, DR. ROBERT J., University of Pennsylvania, School of Veterinary

Medicine, Department of Animal Biology, 3800 Spruce Street, Philadelphia,

Pennsylvania 19104 RYTHER, DR. JOHN H., Woods Hole Oceanographic Institution, Woods Hole,

Massachusetts 02543 SAGER, DR. RUTH, Department of Biological Sciences, Hunter College, 695 Park

Avenue, New York, New York 10021 SANBORN, DR. RICHARD C., Dean, Purdue University Regional Campus, 1125

East 38th Street, Indianapolis, Indiana 46205 SANDERS, DR. HOWARD L., Woods Hole Oceanographic Institution, Woods Hole,

Massachusetts 02543

REPORT OF THE DIRECTOR 63

SATO, DR. HIDEMI, 217 Leidy Building, Department of Biology, University of

Pennsylvania, Philadelphia, Pennsylvania 19104 SAUNDERS, DR. JOHN W., JR., Department of Biological Sciences, State University

of New York at Albany, Albany, New York 12203 SAZ, DR. ARTHUR KENNETH, Department of Microbiology, Georgetown University

Medical and Dental Schools, 3900 Reservoir Road, Washington, D. C. 20007 SCHACHMAN, DR. HOWARD K., Department of Biochemistry, University of

California, Berkeley, California 94720 SCHARRER, DR. BERTA V., Department of Anatomy, Albert Einstein College of

Medicine, New York, New York 10461 SCHLESINGER, DR. R. WALTER, Department of Microbiology, Rutgers Medical

School, New Brunswick, New Jersey 08903 SCHMEER, SISTER ARLINE CATHERINE, O.P., Department of Biology, College of

St. Mary of the Springs, Columbus, Ohio 43219 SCHMIDT, DR. L. H., Southern Research Institute, 2000 Ninth Avenue South,

Birmingham, Alabama 35205 SCHMITT, DR. FRANCIS O., Neurosciences Research Program, Massachusetts

Institute of Technology, 280 Newton Street, Brookline, Massachusetts 02146 SCHMITT, DR. O. H., University of Minnesota, 200 T.N.C.E. Minneapolis,

Minnesota 55455 SCHNEIDERMAN, DR. HOWARD A., Department of Organismic Biology, School of

Biological Sciences, University of California, Irvine, California 92664 SCHOLANDER, DR. P. F., Scripps Institution of Oceanography, La Jolla, California

92037 SCHOPF, DR. THOMAS J. M., Department of the Geophysical Sciences, University

of Chicago, Chicago, Illinois 60637 SCHOTTE, DR. OSCAR E., Department of Biology, Amherst College, Amherst,

Massachusetts 01002 SCHRAMM, DR. J. R., Department of Botany, Indiana University, Bloomington,

Indiana 47401 SCHUEL, DR. HERBERT, Anatomy Department, Mount Sinai, School of Medicine,

New York, New York 10029 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., Colby College, Waterville, Maine 02901 SCOTT, DR. D. B. McNAiR, Lippincott Building, 25 Locust Street, Philadelphia,

Pennsylvania 19013 SCOTT, DR. GEORGE, T., Department of Biology, Oberlin College, Oberlin, Ohio

44074

SEARS, DR. MARY, Box 152, \Voods Hole, Massachusetts 02543 SELIGER, DR. HOWARD H., McCoLLUM-PRATT Institute, The Johns Hopkins

University, Baltimore, Maryland 21218 SENFT, DR. ALFRED \V., Department of Medical Sciences, Brown University,

Providence, Rhode Island, 02912 SEVERINGHAUS, DR. AURA E., 375 West 250th Street, New York, New York 10071

64 ANNUAL KKI'OKT OF THE MAU1NK BIOLOGICAL LABORATORY

SHAPIRO, DR. HERBERT, 6025 North 13th Street, Philadelphia, Pennsylvania 19141

SHAVER, DR. JOHN R., Department of Zoology, Michigan State University, East Lansing, Michigan 48823

SHEDLOVSKY, DR. THEODORE, The Rockefeller University, New York, New York 10021

SHEMIN, DR. DAVID, Department of Chemistry and Biological Sciences, North- western University, Evanston Illinois 60201

SHEPRO, DR. DAVID, Department of Biology, Boston University, 2 Cummington Street, Boston, Massachusetts 02215

SHERMAN, DR. I. W., Division of Life Sciences, University of California, Riverside, California 92507

SICHEL, MRS. F. J. M., Department of Biology, Trinity College, Burlington, Vermont 05401

SIEGELMAN, DR. HAROLD W., Department of Biology, Brookhaven National Laboratory, Upton, New York 11973

SILVER, DR. PAUL, Department of Botany, University of California, Berkeley, California 94720

SIMMONS, DR. JOHN E., JR., Department of Biology, University of California, Berkeley, California 94720

SJODIN, DR. RAYMOND A., Department of Biophysics, University of A/laryland School of Medicine, Baltimore, Maryland 21201

SLIFER, DR. ELEANOR H., 308 Lismore Avenue, Glenside, Pennsylvania 19038

SLOBODKIN, DR. LAWRENCE Basil, Department of Biology, State University of New York at Stony Brook, Stony Brook, New York 11790

SMELSER, DR. GEORGE K., Department of Anatomy, Columbia University, New York, New York 10032

SMITH, MR. HOMER P., Marine Biological Laboratory, Woods Hole, Massa- chusetts 02543

SMITH, MR. PAUL FERRIS, Clark Street, Woods Hole, Massachusetts 02543

SMITH, DR. RALPH I., Department of Zoology, University of California, Berkeley, California 94720

SONNENBLICK, DR. B. P., Rutgers University, 195 University Avenue, Newark, New Jersey 07102

SONNEBORN, DR. T. M., Department of Zoology, Indiana University, Blooming- ton, Indiana 47401

SPECTOR, DR. A., Department of Ophthalmology, Columbia University, College of Physicians and Surgeons, New York, New York 10032

SPEIDEL, DR. CARL C., 1873 Field Road, Charlottesville, Virginia 22903

SPIEGEL, DR. MELVIN, Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755

SPINDEL, DR. WILLIAM, Belfer Graduate School of Science, Yeshiva University, Amsterdam Avenue and 186th Street, Bronx, New York 10461

SPRATT, DR. NELSON T., Department of Zoology, University of Minnesota, Minneapolis, Minnesota 55414

SPRITES, DR. MORRIS ALBERT, Veterans Adminsitration Hospital, Leech Farm Road, Pittsburgh, Pennsylvania 15206

STARR, DR. RICHARD C., Department of Botany, Indiana University, Blooming- ton, Indiana 47401

REPORT OF THE DIRECTOR 65

STEINBACH, DR. H. BURR, Director, Marine Biological Laboratory, Woods Hole, Massachusetts 02543

STEINBERG, DR. MALCOLM S., Department of Biology, Princeton University, Princeton, New Jersey 08540

STEINHARDT, DR. JACINTO, Georgetown University, Washington, D. C. 20007

STEPHENS, DR. GROVER C., Division of Biological Sciences, University of Cali- fornia, Irvine, California 92650

STEPHENS, DR. RAYMOND E. Department of Biology, Brandeis University, Waltham, Massachusetts 02154

STETTEN, DR. DfiWiTT, Rutgers University Medical School, New Brunswick, New Jersey 08903

STETTEN, DR. MAJORIER., Rutgers University Medical School, New Brunswick, New Jersey 08803

STEWART, DR. DOROTHY, 317 Ardmore Circle, High Point, North Carolina 27261

STRACHER, ALFRED, Downstate Medical Center, State University of New York at Brooklyn, 450 Clarkson Avenue, Brooklyn, New York 11203

STREHLER, DR. BERNARD L., 5184 Willow Wood Road, Rolling Hills Estate, California 90274

STRITTMATTER, DR. PHILIPP, Department of Biochemistry, University of Con- necticut, School of Medicine, Health Center, Hartford Plaza, Hartford, Connecticut 06105

STURTEVANT, DR. ALFRED H., California Institute of Technology, Pasadena, California 91 109

SULKIN, DR. S. EDWARD, Department of Bacteriology, University of Texas, Southwestern Medical School, Dallas, Texas 75221

SUMMERS, WILLIAM C., Systematics-Ecology Program, Marine Biological Lab- oratory, Woods Hole, Massachusetts 02543

SUSSMAN, DR. MAURICE, Department of Biology, Brandeis University, Waltham, Massachusetts 02154

SWANSON, DR. CARL PONTIUS, Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218

SWOPE, MR. GERARD, JR., Croton-on-Hudson, New York, New York 10520

SZABO, DR. GEORGE, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, Massachusetts 02115

SZENT-GYORGYI, DR. ALBERT, Institute for Muscle Research, Marine Biological Laboratory, Woods Hole, Massachusetts 02543

SZENT-GYORGYI, DR. ANDREW G., Department of Biology, Brandeis University, Waltham, Massachusetts 02154

TANZER, DR. MARVIN L., Department of Biochemistry, University of Con- necticut, School of Medicine, Health Center, Hartford Plaza, Hartford, Connecticut 06105

TASAKI, DR. ICHIJI, Laboratory of Neurobiology, National Institutes of Health, Bethesda, Maryland 20014

TAYLOR, DR. ROBERT E., Laboratory of Biophysics, National Institutes of Health, Bethesda, Maryland 20014

TAYLOR, DR. W. ROWLAND, Department of Oceanography, Chesapeake Bay Institute, The Johns Hopkins University, Baltimore, Maryland 21218

TfiWiNKEL, DR. Lois E., Department of Zoology, Smith College, Northampton, Massachusetts 01060

66 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

THALER, DR. M. MICHAEL, University of California, San Francisco, California

94102

TRACER, DR. WILLIAM, The Rockefeller University, New York, New York 10021 TRAVIS, DR. D. M., Department of Pharmacology, University of Florida, Gaines- ville, Florida 32601 TRAVIS, DR. DOROTHY FRANCES, 1918 Northern Parkway, Greenberry Woods,

Baltimore, Maryland 21210 TRINKAUS, DR. J. PHILIP, Department of Biology, Yale University, New Haven,

Connecticut 06520 TROLL, DR. WALTER, Department of Industrial Medicine, New York University,

College of Medicine, New York, New York 10016 TWEEDELL, DR. KEN YON S., Department of Biology, University of Notre Dame,

Notre Dame, Indiana 46556 URETZ, DR. ROBERT B., Department of Biophysics, University of Chicago,

Chicago, Illinois 60637 VAN HOLDE, DR. KENSAL EDWARD, Oregon State University, Department of

Biochemistry and Biophysics, Corvallis, Oregon 97331 VILLEE, DR. CLAUDE A., Department of Biochemistry, Harvard Medical School,

Boston, Massachusetts 02115 VINCENT, DR. WALTER S., Department of Anatomy, University of Pittsburgh,

Pittsburgh, Pennsylvania 15213 WAINIO, DR. W. W., Bureau of Biological Research, Rutgers University, New

Brunswick, New Jersey 08903 WALD, DR. GEORGE, Biological Laboratories, Harvard University, Cambridge,

Massachusetts 02138 WALLACE, DR. ROBIN A., P. O. Box Y, Oak Ridge National Laboratory, Oak

Ridge, Tennessee 37890 WARNER, DR. ROBERT C., Department of Chemistry, New York University

College of Medicine, New York, New York 10016 WARREN, DR. LEONARD, Department of Therapeutic Research, University of

Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 WATERMAN, DR. T. H., 610 Klein Biology Tower, Yale University, New Haven,

Connecticut 06520

WATKINS, DR. DUDLEY TAYLOR, Department of Anatomy, University of Con- necticut, Storrs, Connecticut 06268 WATSON, DR. STANLEY WAYNE, Woods Hole Oceanographic Institution, Wroods

Hole, Massachusetts 02543 WEBB, DR. H. MARGUERITE, Department of Biological Sciences, Goucher

College, Towson, Maryland 21204 WEISS, DR. LEON P., Department of Anatomy, The Johns Hopkins University,

School of Medicine, Baltimore, Maryland 21205

WEISS, DR. PAUL A., The Rockefeller University, New York, New York 10021 WERMAN, DR. ROBERT, Department of Zoology, Hebrew University, Jerusalem,

Israel WHITAKER, DR. DOUGLAS M., Las Palmas Apartment 204, 1800 Palm Boulevard,

Brownsville, Texas 78520

WHITE, DR. E. GRACE, 1312 Edgar Avenue, Chambersburg, Pennsylvania 17201 WHITING, DR. ANNA R., 535 West Vanderbilt Drive, Oak Ridge, Tennessee 37830 WHITING, DR. PHINEAS, 535 West Vanderbilt Drive, Oak Ridge, Tennessee 37830

REPORT OF THE DIRECTOR 67

WICHTERMAN, DR. RALPH, Department of Biology, Temple University, Phila- delphia, Pennsylvania 19122 WIERCINSKI, DR. FLOYD J., Department of Biology, Northeastern Illinois State

College, 5500 North St. Louis Avenue, Chicago, Illinois 60625 WIGLEY, DR. ROLAND L., U. S. Fish and Wildlife Service, Bureau of Commercial

Fisheries, Woods Hole, Massachusetts 02543 WILBER, DR. C. G., Department of Zoology, Colorado State University, Fort

Collins, Colorado 80521

WTILCE, DR. ROBERT THAYER, Department of Botany, University of Massa- chusetts, Amherst, Massachusetts 01003 WILSON, DR. J. WALTER, Department of Biology, Brown University, Providence,

Rhode Island 02912 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 WINTERS, DR. ROBERT WAYNE, Department of Pediatrics, Columbia University,

College of Physicians and Surgeons, New York, New York 10032 W^ITSCHI, DR. EMIL, The Rockefeller University, New York, New York 10021 WITTENBERG, DR. JONATHAN B., Department of Physiology and Biochemistry,

Albert Einstein College of Medicine, New York, New York 10461 WRINCH, DR. DOROTHY, Department of Physics, Smith College, Northampton,

Massachusetts 01060 WYTTENBACH, DR. CHARLES R., Department of Zoology, University of Kansas,

Lawrence, Kansas 66044 YNTEMA, DR. C. L., Department of Anatomy, State University of New York,

Upstate Medical Center, Syracuse, New York 13210 YOUNG, DR. DAVID KENNETH, Systematics-Ecology Program, Marine Biological

Laboratory, Woods Hole, Massachusetts 02543

YOUNG, DR. D. B., Main Street, North Hanover, Massachusetts 02357 ZACK, DR. SUMNER IRWIN, The Pennsylvania Hospital, University of Pennsyl- vania School of Medicine, Philadelphia, Pennsylvania 19104 ZIGMAN, DR. SEYMOUR, University of Rochester School of Medicine and Dentistry,

260 Crittenden Boulevard, Rochester, New York 14620 ZIMMERMAN, DR. A. M., Department of Zoology, University of Toronto, Toronto

5, Ontario, Canada ZINN, DR. DONALD J., Department of Zoology, University of Rhode Island,

Kingston, Rhode Island 02881 ZIRKLE, DR. RAYMOND E., Department of Biophysics, University of Chicago,

Chicago, Illinois 60637 ZORZOLI, DR. ANITA, Department of Physiology, Vassar College, Poughkeepsie,

New York 12601 ZULLO, DR. VICTOR A., Department of Geology, California Academy of Sciences,

Golden Gate Park, San Francisco, California 94118 ZWEIFACH, DR. BENJAMIN, c/o Department of AMES, University of California,

San Diego, La Jolla, California 92073 ZWILLING, DR. EDGAR, Department of Biology, Brandeis University, Waltham,

Massachusetts 02154

68

ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

ASSOCIATE MEMBERS

ALLEN, Miss CAMILLA K. ALTON, MRS. BENJAMIN ANGUS, DR. AND MRS. RALHI C,. ANTHONY, MR. AND MRS. RICHARD A. ARMSTRONG, MRS. PHILIP B.

AUCLAIR, DR. AND MRS. WALTER

HACON, DR. CATHERINE L. BACON, MR. AND MRS. ROBERT BAKALAR, MR. AND MRS. DAVID BALL, MRS. ERIC G. BALLANTINE, DR. AND MRS. H.

THOMAS, JR.

BARBOUR, MRS. Lucius H. BARROWS, MRS. ALBERT W. BARTOW, MR. AND MRS. CLARENCE W. BARTOW, MRS. FRANCIS D. BEALE, MR. AND MRS. E. F. BIDDLE, DR. VIRGINIA BIGELOW, MRS. ROBERT P. BOETTIGER, MRS. EDWARD G. BRADLEY, DR. AND MRS. CHARLES BRONSON, MR. AND MRS. SAMUEL C. BROWN, DR. AND MRS. F. A., JR. BROWN, MRS. THORNTON BUCK, MRS. JOHN B.

BUFFINGTON, MRS. GEORGE

BURDICK, DR. C. LALOR BURT, MR. AND MRS. CHARLES E. BUTLER, DR. AND MRS. E. G. CALKINS, MR. AND MRS. G. NATHAN,

JR.

CAREY, Miss CORNELIA CARLTON, MR. AND MRS. WINSLOW G. CARPENTER, MR. DONALD F. CASHMAN, MR. EUGENE R. CHALMERS, MR. AND MRS. BRUCE CLAFF, MRS. C. LLOYD CLAFF, MR. AND MRS. MARK M. CLARK, MR. AND MRS. HAY'S CLARK, MRS. JAMES McC. CLARK, DR. AND MRS. LEONARD B. CLARK, MRS. LEROY CLARK, MR. AND MRS. W. VAN ALAN CLOWES, MR. ALLEN \Y. CLOWES, DR. AND MRS. GEORGE H. A.,

JR.

COPELAND, MRS. I). EUGENE COSTELLO, MRS. DONALD P. CRAMER, MR. AND MRS. IAN D. \\ . CRANE, MR. JOHN (Friendship Fund) CRANE, JOSEPHINE B., FOUNDATION CRANE, Miss LOUISE CRANE, MR. STEPHEN CRANE, MRS. W. CAREY CRANE, MRS. W. MURRAY CROCKER, MR. AND MRS. PETER J. CROSSLEY, MR. AND MRS. ARCHIBALD

M.

CROWELL, MR. AND MRS. PRINCE S. ( i RTIS, DR. AND MRS. WILLIAM D. DAIGNAULT, MR. AND MRS. A. T. DANIELS, MR. AND MRS. BRUCE G. DANIELS, MRS. F. HAROLD DAY, MR. POMEROY DRAPER, MRS. MARY C. Du Bois, DR. AND MRS. A. B. Du PONT, MR. A. FELIX, JR. DYER, MR. AND MRS. ARNOLD W. ELSMITH, MRS. DOROTHY O. EWING, DR. AND MRS. GIFFORD C. FAXON, DR. NATHANIEL W. FAY, MRS. HENRY H., JR. FENNO, MRS. EDWARD N. FERGUSON, DR. AND MRS. JAMES J., JR. FINE, DR. AND MRS. JACOB FIRESTONE, MR. AND MRS. EDWIN FISHER, MRS. B. C. FISHER, MR. FREDERICKS., III. FORBES, MRS. ALEXANDER FRANCIS, MR. LEWIS H., JR. GABRIEL, DR. AND MRS. MORDECAI L. GAISER, DR. AND MRS. DAVID W. GALTSOFF, DR. AND MRS. PAUL S. GAMBLE, MR. AND MRS. RICHARD B. GARFIELD, Miss ELEANOR GELLIS, DR. AND MRS. SYDNEY S. GERMAN, DR. AND MRS. JAMES L., Ill GIFFORD, MR. AND MRS. JOHN A. GIFFORD, DR. AND MRS. PROSSER GlLCHRIST, MR. AND MRS. JOHN M. GILDEA, DR. MARGARET C. L. GILLETTE, MR. AND MRS. ROBERT S.

REPORT OF THE DIRECTOR

69

GLAZEBROOK, MRS. JAMES R. GOLDMAN, DR. AND MRS. ALLEN S. COLORING, DR. IRENE GRASSLE, MR. AND MRS. J. K. GREEN, Miss GLADYS M. GREENE, MRS. WILLIAM C. GREER, MR. AND MRS. WILLIAM H., JR. GREIF, DR. AND iAlRs. ROGER GRUSON, MR. AND MRS. EDWARD GULESIAN, MRS. PAUL J.

GUREWICH, DR. AND MRS. VLADIMIR

HAMLEN, MRS. J. MONROE HANDLER, DR. AND MRS. PHILIP HANNA, MR. AND MRS. THOMAS C. HARE, DR. AND MRS. GERARD HARRINGTON, MR. AND MRS. R. D. HARVEY, DR. AND MRS. EDMUND N.,

JR.

HARVEY, DR. AND MRS. RICHARD HERVEY, MRS. JOHN P. HIAM, MR. AND MRS. EDWIN W. HlRSCHFELD, DR. AND MRS. NATHAN B. HOCKER, MR. AND MRS. LON HOPKINS, MRS. HOYTS HOUGH, MR. AND MRS. GEORGE A., JR. HOUSTON, MR. AND MRS. HOWARD E.

HUNZIKER, MR. AND MRS. HERBERT E.

JANNEY, MR. AND MRS. F. W/ISTAR JEWETT, MR. AND MRS. G. F., JR. JOHNSON, MR. AND MRS. CRAWFORD JORDAN, DR. AND MRS. EDWIN P. KEITH, MRS. HAROLD C. KEITH, MR. AND MRS. JEAN REID KENEFICK, MR. AND MRS. THEODORE

G. KEOSIAN, MRS. JOHN

KlNNARD, MR. AND MRS. L. RlCHARD

KOHN, DR. AND MRS. HENRY I.

KOLLER, DR. AND MRS. LEWIS R.

LASSALLE, MRS. NANCY N. LA\VRENCE, MR. AND MRS. MILFORD R. LAZAROW, DR. AND MRS. ARNOLD LEMANN, MRS. LUCY BENJAMIN LEVINE, DR. AND MRS. RACHMIEL LILLIE, MRS. KARL C. LOBB, PROFESSOR AND MRS. JOHN LOEB, DR. AND MRS. ROBERT F.

LOVELL, MR. AND MRS. HOLLIS R.

LOWENGARD, MRS. JOSEPH

MACKEY, MR. AND MRS. WILLIAM K. MAcNicHOL, MRS. EDWARD F., JR. MARSLAND, DR. AND MRS. DOUGLAS MARVIN, DR. DOROTHY MAST, MRS. S. O. MATHER, MR. FRANK J., Ill MAVOR, MRS. JAMES W., SR.

McCuSKER, MR. AND MRS. PAUL T.

MCELROY, MRS. NELLA W.

McGlLLICUDDY, DR. AND MRS. JOHN J.

McLANE, MRS. HUNTINGTON MEIGS, MR. AND MRS. ARTHUR MEIGS, DR. AND J. WISTER METZ, MRS. CHARLES B. MEYERS, MR. AND MRS. RICHARD MITCHELL, MRS. PHILIP MIXTER, MRS. W. I. MONTGOMERY, DR. AND MRS. CHARLES

H.

MORSE, MR. AND MRS. RICHARD S. NATERMAN, DR. AND MRS. HYMAN L. NEWTON, Miss HELEN K. NICHOLS, MRS. GEORGE NlCKERSON, MR. AND MRS. FRANK L. NORMAN, MR. AND MRS. ANDREW NORMANDIE FOUNDATION INC. PACKARD, MRS. CHARLES PARK, MR. AND MRS. FRANKLIN A. PARK, MR. MALCOLM S. PARPART, MRS. ARTHUR K. PATTEN, MRS. BRADLEY PENDLETON, DR. MURRAY E. PENNINGTON, Miss ANNE H. PERKINS, MR. AND MRS. COURTLAND

D.

PHILIPPE, MR. AND MRS. PIERRE PUTNAM, MR. AND MRS. WILLIAM A.,

Ill

RATCLIFFE, MR. THOMAS, JR. RAYMOND, DR. AND MRS. SAMUEL REDFIELD, DR. AND MRS. ALFRED REZNIKOFF, DR. AND MRS. PAUL RIGGS, MR. AND MRS. LAWRASON, III ROBERTSON, DR. AND MRS. C. W. ROBINSON, MR. DENIS M. ROGERS, MRS. CHARLES E. ROOT, DR. AND MRS. WALTER S.

70

ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

RUGH, DR. AND MRS. ROBERTS RYDER, MR. AND MRS. FRANCIS C. SAUNDERS, MR. AND MRS. LAWRENCE SCHWARTZ, MRS. VICTOR A. SHEPROW, DR. AND MRS. DAVID SHIVERICK, MRS. ARTHUR SMITH, MRS. HOMER P. SPEIDEL, MRS. CARL C. STEINBACH, MRS. H. BURR STETTEN, DR. AND MRS. DE\VITT, JR. STONE, MR. AND MRS. LEO STONE, MRS. SAMUEL M. STUNKARD, DR. HORACE W. SWANSON, MRS. CARL P. SWOPE, MR. AND MRS. GERALD, JR. SWOPE, MR. AND MRS. GERALD L. SWOPE, Miss HENRIETTA H.

TOLKAN, MR. AND MRS. NORMAN

TOMPKINS, MR. AND MRS. B. A. WAKSMAN, DR. AND MRS. BYRON H. WAKSMAN, DR. AND MRS. SELMAN A. WALLACE, DR. AND MRS. STANLEY L. WANG, DR. AND MRS. AN

WARE, MR. AND MRS. J. LINDSAY WARREN, DR. AND MRS. SHIELDS WATT, MR. AND MRS. JOHN B. WEBSTER, MRS. EDWIN S. WEISBERG, MR. AND MRS. ALFRED M. WHITELEY, MR. AND MRS. GEORGE C.,

JR.

WHITING, DR. AND MRS. PHINEAS W. WHITNEY, MR. G. G., JR.

WlCHTERMAN, MRS. RALPH WlCKERSHAM, MR. AND MRS. A. A.

TlLNEY

WlCKERSHAM, MRS. JAMES II. WlLHELM, MR. AND MRS. HlLMAR J.

WILSON, MRS. EDMUND B. WILSON, DR. MAY G. WlTMER, DR. AND MRS. ENOS WOLFE, DR. CHARLES WOLFINSOHN, MRS. WOLFE WRINCH, DR. DOROTHY YNTEMA, MRS. CHESTER L. ZWILLING, MRS. EDGAR

V. REPORT OF THE LIBRARIAN

The space problem in the stack area of the library has been solved temporarily. The reprints have been compressed and will be placed in the basement stack leaving no room for expansion. One entire floor of stacks is now available to the serials section, relieving the growth situation for the next few years. Unfor- tunately this move means that we will be unable to accept any reprints until more floor area is added to the library.

During 1969 we received 4563 requests for articles contained in the library. We made 370 requests to other libraries for use by investigators here. 2,076 volumes were sent to the bindery and holdings now total 140,111. This figure does not include the reprint collection.

Total Number of Serial Titles 4,054

Number received currently 2,384

(On subscription 958

On exchange 981

On gift basis 365)

Number of text books added 302

(Received from book exhibit 160)

Reprints added in 1969 1,106

REPORT OF THE TREASURER 71

VI. REPORT OF THE TREASURER

The market value of the General Endowment Fund and the Library Fund at December 31, 1969, amounted to $2,197,603 and the corresponding securities are entered in the books at a v^llue of $1,574,735. This compares with values of $2,403,484 and $1,575,096, respectively, at the end of the preceding year. The average yield on the securities was 4.37% of the market value and 6.09% of the book value. Uninvested principal cash was $1,104. Classification of the se- curities held in the Endowment Fund appears in the Auditor's Summary of Investments.

The market value of the Pooled Securities at December 31, 1969, amounted to $763,700 as compared to book values of $634,152. These figures compare with values of $824,502 and $666,202, respectively, at the close of the preceding year. The average yield on the securities was 3.57% of the market value and 4.30% oi the book value. Uninvested principal cash was in the amount of $447.

The proportionate interest in the Pool Fund Account of the various funds, as of December 31, 1969, is as follows:

Pension Funds . . 23.048%

General Laboratory Investment 20.659%

F. R. Lillie Memorial Fund .... 2.252%

Anonymous Gift 772%

Other:

Bio Club Scholarship Fund . . . .587%

Rev. Arsenius Boyer Scholarship Fund 709%

Gary N. Calkins Fund 671%

Allen R. Memhard Fund .129%

Lucretia Crocker Fund 2.438%

E. G. Conklin Fund 411%

Jewett Memorial Fund 217%

M. H. Jacobs Scholarship Fund 294%

Herbert W. Rand Fellowship 20.834%

Mellon Foundation 9.835%

Mary Rogick Fund 2.157%

Swope Foundation 5.412%

Clowes Fund 9.575%

Donations from MBL Associates for 1969 amounted to $9,246 as compared with $8,360 for 1968. Unrestricted gifts from foundations, societies and com- panies amounted to $18,065.

During the year we administered the following grants and contracts:

Investigators Training MBL Institutional

10 XIH 3 XIH 2 NIH

3 NSF 1 NSF 2 NSF

1 ONR 1 AEC

1 Whitehall 1 Ford

15 46

72 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

An overhead rate of 20% of allowable direct costs was still in effect for most federally funded research grants at the beginning of the year, but this rate was generally superseded by the new rate of 25% which had been negotiated. How- ever, Federal grants now require a cost sharing contribution by the Laboratory to research projects, usually at a rate of about 5%. An indirect cost rate based on the laboratory space allocated for a research project has now been negotiated with the federal agencies, for grants initiated after August 1, 1969.

The following is a statement of the auditors:

To the Trustees of the Marine Biological Laboratory, Woods Hole, Massachusetts

We have examined the balance sheet of Marine Biological Laboratory as at December 31, 1969, the related statement of operating expenditures and income and statement of funds for the year then ended. Our examination was made in accordance with generally accepted auditing standards, and accordingly included such tests of the accounting records and such other auditing procedures as we considered necessary in the circumstances. We examined and have reported on financial statements of theLaboratory for the year ended December 31, 1968.

In our opinion, the accompanying financial statements present fairly the assets, liabilities and funds of Marine Biological Laboratory at December 31, 1969 and 1968 and the results of its operations for the years then ended on a consistent basis.

The supplementary schedules included in this report were obtained from the Laboratory's records in the course of our examination and, in our opinion, are fairly stated in all material respects in relation to the financial statements, taken as a whole.

Boston, Massachusetts

March 20, 1970 LYBRAND, Ross BROS. AND MONTGOMERY

It will be noted from the operating statement that the Laboratory activities for the year under review, amounted to a figure of a little over 1.5 million dollars, which amount is comparable to last years results.

ALEXANDER T. DAIGNAULT,

Treasurer

REPORT OF THE TREASURER 73

MARINE BIOLOGICAL LABORATORY

BALANCE SHEETS December 31, 1969 and 1968

Investments

1969 1968

Investments held by Trustee:

Securities, at cost (approximate market quotation 1969 —

$2,197,603) $1,574,735 $1,575,096

Cash 1,104 624

1,575,839 1,575,720

Investments of other endowment and unrestricted funds:

Pooled investments, at cost (approximate market quotation 1969 —

$763,700) less $5,728 temporary investment of current fund

cash 628,424 660,474

Other investments 1,474,150 2,282,150

Cash 447 7,653

Accounts receivable 33 33

Due from current fund 61,622 53,276

$3,740,515 $4,579,306

Plant Assets

Land, buildings, library and equipment 6,072,007 5,732,782

Less allowance for depreciation (note 1) 1,802,581 1,711,311

4,269,426 4,021,471

Construction in progress \ 758, 341 1,316,468

Investments at cost (approximate market quotation 1969 — $527,106) 707,327 842,629

Due from current funds 13,401 38,531

$8,748,495 $6,219,099

Current Assets

Cash.. 163,471 251,061

Temporary investment in pooled securities 5,728 5,728

Accounts receivable (U. S. Government, 1969— $52,391 ; 1968— $33,824) 137,757 130,503

Inventories of supplies and bulletins 45,593 47,036

Other assets 6,619 7,146

Due to plant funds (13,401) (38,531)

Due to endowment funds (61,622) (53,276)

$ 284,145 $ 349,667

74 ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

MARINE BIOLOGICAL LABORATORY

BALANCE SHEETS December 31, 1969 and 1968

Invested Funds

1969 l<>68

Endowment funds given in trust for benefit of the Marine Biological

Laboratory $1,575,839 $1,575,720

Endowment funds for awards and scholarships:

Principal.... 427,663 427,663

Unexpended income 44,630 39,394

472,293 467,057

Unrestricted funds functioning as endowment 1,528,190 2,336,190

Retirement fund 217,433 192,543

Pooled investments — accumulated (loss) gain (53,240) 7,796

$3,740,515 $4,579,306 Plant Funds

Funds expended for plant, less retirements 9,598,348 6,886,046

Less allowance for depreciation charged thereto 1,802,581 1,711,311

7,795,767 5,174,735

Accounts payable 240,001 163,204

Unexpended plant funds 712,727 881,160

$8,748,495 $6,219,099 Current Liabilities and Funds

Accounts payable and accrued expenses 56,419 44,737

Advance subscriptions 30,141 23,712

Unexpended grants— research 33,918 25,727

Unexpended balances of gifts for designated purposes 23,278 23,764

Current fund 140,389 231,727

$ 284,145 $ 349,667

Note 1 — During the current year the Laboratory reduced the estimated useful lives of certain plant assets. This change had the effect of increasing depreciation expense for 1969 by approxi- mately $43,000. Depreciation charges remain, however, from 1% to 5% of the original cost of the assets.

Note 2 — The Laboratory has commitments of approximately $2,600,000 for the construction of a teaching laboratory and a dormitory dining-hall.

REPORT OF THE TREASURER

75

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76

ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

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REPORT OK THE TREASURER

77

MARINE BIOLOGICAL LABORATORY

STATEMENT OF FUNDS

Year Ended December 31, 1969

Balance Invest- Used or Other Balance

December Gifts and ment Ciin-cut Expendi- December

31, 1968 Other Receipts Incom,e Expenses lures 31, 1969

Invested funds $4,579,306 $ 31,635 $250,688 $ 229,997 $ 83,117 $3,740,515

(808,000) (3)

Unexpended plant funds $ 881,160 223,475 24,688 416,596 $ 712,727

Unexpended research

grants $ 25,727 2,254,014 781,586 1,464,237 $ 33,918

Unexpended gifts for

designated purposes $ 23,764 7,469 7,068 887 $ 23,278

(7,500)(2)

(25,122)(1) Current fund $ 231,727 808,000(3) 866,716 $ 140,389

$2,483,971 $275,376 $1,018,651 $2,831,553

Gifts 230,944

Grant for facilities

construction 1 ,464,237

Grants for research,

training and

support 789,777

Appropriated from

current income

and other 24,135

Net gain on sale of

securities

(1) Excess of current

expenditures over

income (25,122)

(2) Gift of boat

transferred to plant funds expended. . . .

(3) Transfer from

invested funds

$2,483,971

Expended for IH-\\ laboratory and dormitory- dining hall.. 2,747,548

Scholarship awards 8,930

Payments to

pensioners. ... 13,152

Loss on sale of

securities 61,036

Other. 887

$2,831,553

78

ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY

MARINE BIOLOGICAL LABORATORY

SUMMARY OF INVESTMENTS December 31, 1969

Securities held by Trustee: General endowment fund: jj ^ Government securities $	Cost 25,065 711,938 84,771 419,247	Per Cent of Total 2.0 57.4 6.8 33.8	Market Quotations $ 24,325 500,123 69,986 1,175,447	Per Cent Investment of Income Total 1969 1.3 $ 1,250 28.3 31,158 4.0 3,991 66.4 42,471
Preferred stocks
Common stocks
1	,241,021	100.0	1,769,881	100.0	78,870
General educational broad endowment fund: U S Government securities	53,113 184,745 15,476 80,380	15.9 55.4 4.6 24.1	51,569 134,487 11,753 229,913	12.1 31.4 2.7 53.8	2,650 8,107 1,244 5,065
Other bonds
Preferred stocks
Common stocks
	333,714	100.0	427,722	100.0	17,066
Total securities held by Trustee $1	,574,735		$2,197,603		95,936

Investments of other endowment and

unrestricted funds: Pooled investments : U. S. Governnu: Corporate bonds Preferred stocks Common stocks

Less temporary investment of current fund cash . .

5,728

lit securities	33,338	5.2	34,000	4.5	1,354
	134,850	21.3	114,244	15.0	8,274
	67,040	10.6	56,625	7.4	1,236
	398,924	62.9	558,831	73.1	16,400
	634,152	100.0 i	I 763,700	100.0	27,264

228

628,424

27,036

Other investments:

U. S. Government securities.

Other bonds

Common stocks

Real estate. .

27,938 15,029 49,634 17,549

Short-term commercial notes 1,364,000

1,474,150

Total investments of other endow- ment and unrestricted funds $2,102,574

1,133

750 2,697

134,134 138,714

165,750

Total .

261,686

REPORT OF THE TREASURER 79

MARINE BIOLOGICAL LABORATORY

SUMMARY OF INVESTMENTS (CONTINUED)

December 31, 1969

Investment

Income Cost 1969

Custodian's fees charged thereto 10,998

Investment income distributed to

invested funds 250,688

Plant investments:

Federal agency and corporate bonds 140,000 7,242

Common stock 564,456 17,394

Preferred stock. 2,871 52

707,327 24,688

Current investments:

Temporary investment in pooled

securities $ 5,728 228

Total investment income $275,604

Reference: liwl. Hull., 139: NO «M. (August, 1970)

THE NUTRITION OF PARANEMERTES PEREGRINA

(RHYNCHOCOELA : HOPLONEMERTEA) . I. STUDIES

ON FOOD AND FEEDING BEHAVIOR *

PAMELA ROE

Department of Zoology, University of IVashwijInn. Seattle, U'lishington 98105

Nemerteans are common organisms in the intertidal zones of temperate regions of the world (Coe, 1943). They live in a wide variety of habitats and can occur in large numbers. Most nemerteans are carnivorous (Coe, 1943), and as abundant predators they may well be important components of the communities in which they live. Since efficient exploitation of available food is a necessary requirement of all animals, studies of feeding and food preference should be of great importance to the field of ecology (Emlen, 1966). Both because of their predatory nature and because they have an interesting means of capturing food, nemerteans are well suited to the study of feeding and food preference.

This paper reports aspects of feeding behavior of Paranemertes peregrina Coe, 1901, hereafter referred to as Paranemertes, a predatory nemertean which occurs along the entire Pacific coast of North America from the Aleutian Islands in Alaska to Ensenada, Mexico (Coe, 1940) in both rocky and muddy bay intertidal • habitats. The feeding habits of Paranemertes have previously received little atten- tion, being only briefly mentioned by Coe (1905), MacGinitie and MacGinitie (1949), and Correa (1964). The MacGinities (1949, and personal communica- tion) reported that Paranemertes feeds at low tides on nereid polychaetes.

Specifically, this report concerns (1) a description of the feeding process of Paranemertes, including steps in prey capture; (2) a comparison of Paranemertes from exposed and sheltered rocky intertidal and muddy bay areas for food in nature and food preferences in laboratory experiments; and (3) a description of an escape response of nereid prey to Paranemertes. This study is the beginning of an ecological study now in progress on the role of Paranemertes as a predator in these types of communities.

MATERIALS AND METHODS

Four study areas were selected where Paranemertes is fairly abundant. These were: (1) English Camp at Garrison Bay, San Juan Island, Washington, a protected muddy bay area; (2) the ferry landing at Edmonds, Washington, a concrete and metal structure with large boulders piled against it; (3) boulders on the sandy beach to the north of the ferry landing at Edmonds; and (4) the rocky outcropping at Mukkaw Bay, south of Cape Flattery, open Pacific coast,

1 Part I of this paper is based on a thesis submitted by P. Roe in partial fulfillment of the requirements for a Master of Science degree, University of Washington, 1967.

80

FEEDING IN PARANEMERTES 81

Washington. Garrison Bay and the two rocky areas at Edmonds are in protected waters ; Mukkaw Bay is exposed to strong wave action. Paranemertes was found on the mud at Garrison Bay, and among and bclmv the barnacles and mussels on the rocks in the other areas.

Field observations and collections of over 500 Parancmerles were made at the four study areas during low tides from October 1966 through April 1967, monthly at Garrison and Mukkaw Bays and nearly weekly at Edmonds. Specimens of Paranemertes were put singly into 2-oz jars partly filled with clean cooled sea water. All specimens were kept near 10° C.

Polychaetes from each study area were collected to determine what species lived in association with Paranemertes and to be used in food preference experi- ments. Polychaetes were identified from Hartman, 1968 and Berkeley and Berkeley 1948, 1952. Potential prey from other phyla which occurred in these habitats were not used for food preference experiments since initial examina- tions of Paranemertes faeces and initial preference experiments gave positive results only for polychaetes.

Since each nemertean was kept in a separate container, its faeces from food eaten in nature could be collected from the water in which it was kept. Para- nemertes faeces consist of bundles of polvchaete setae, facilitating identification of the prey. Faeces and worms that had fed in nature were preserved in 70% alcohol.

In food preference experiments a potential prev was put into a container with a nemertean which had been kept without food for one week, and the reactions of the nemertean and prey were observed. Reactions of the nemerteans were graded in three categories, after contact was made with the prey. Step 1, absence of reaction or jerking back of the head was considered a negative response. Step 2, eversion of the proboscis, and Step 3, ingestion of prey, were considered positive responses. If a negative response occurred following presentation of a polvchaete other than a nereid, a nereid was offered subsequently, since preliminary work indicated that Paranemertes has a strong preference for nereids. The experi- ment was excluded from analysis of results if there was a negative response to the nereid control. Preliminarv data suggested that Paranemertes has a slight "memory," i.e., a nemertean offered a nereid followed soon bv a different food responds positively at first to the second food. This "memory" apparently does not last more than a few minutes. Therefore, if a given Paranemertes was used for more than one feeding experiment, it was returned to its container after each experiment and not used again for at least one hour. When possible, each prey type was presented in ten trials. The length of time before defecation was recorded in several instances. Fisher's exact test CSiegel, 1956) was used except where otherwise indicated to examine the significance of food preference results.

An escape response of nereid prey to Paranemertes was observed in nature and in Paranemertes food preference tests. Experiments were run to determine (1) if nereids responded more vigorously to Paranemertes than to simple tactile stimulus and (2) what parts of the nereid body are most sensitive to Paranemertes.

PAMELA ROE

OBSERVATIONS AND RESULTS General observations in nature

Paranemertes is an active predator during low tide. Worms were observed to emerge from crevices and from the mud as the tide receded and they were observed feeding out of water at low tide. They are not merely stranded on the rocks as the tide goes out.

Few Paranemertes come out when it is raining, even during low tides. During inclement weather aggregations of Paranemertes can be found in cracks or sheltered crevices in rocky areas. Paranemertes crawls out into the open more often at night or on cloudy days than it does on sunny days.

Feeding process

Feeding of Paranemertes has been analyzed as a 3-step process. Step 1, prey contact : Contact of the anterior edge of Paranemertes' head with a prey organism and recoil of Paranemertes' anterior end is the first step of the feeding process. This recoil almost always occurs, even if the prey proves an unacceptable species. Recoil is often accompanied by a 90° or a 180° turn of the head away from the prey and a crawling away if the prey is not acceptable. The initial head with- drawal is more vigorous if the prey is a nereid.

Preliminary tests and observations suggested that Paranemertes has little or no distance chemoreception for use in finding prey. Nereids had to come into contact with the anterior rim of a Paranemertes' head before the nemertean responded. Water from jars in which nereids lived caused no response when pipetted into a jar with a Paranemertes. In two of eleven tries, Paranemertes responded by slight eversion of the proboscis to the tube of a recently-removed nereid. Once a Paranemertes followed the fresh mucus trail of a nereid. Contact with nereids or their immediate and fresh products appears to be a requirement for recognition and elicitation of the feeding response by Paranemertes.

Step 2, prey capture : The second step in the feeding process is eversion of the proboscis, often rapidly and with great force. Prey much stronger than the nemertean may be paralyzed within a few seconds by this action. In the labora- tory, eversion of the proboscis occurs almost every time a nereid is presented, but only rarely in response to other prey organisms (Tables I-IV). For this reason Step 2 is considered a positive feeding response. In the laboratory Paranemertes often paralyzes a nereid, then does not eat it.

The proboscis everts to a given point, stops, then moves again, either inverting or everting more, then stops again. These stops are sometimes accom- panied by a visible jabbing motion. The stylet of Paranemertes is small (Coe, 1905) and difficult to follow during rapid proboscis movements. However, it was observed moving forward to the point where the end was in contact with the prey. It is believed that the stylet venom is pumped in the prey during these stops. Paralysis is complete only in the part of the prey around which the proboscis has been wound, and often a nemertean must envelop the prey 3 or 4 times in different places before the effect is complete. The proboscis can be everted a short or long distance and the stylet can be terminal in either case. When the nemertean is through puncturing the prey in one place, it can kwith-

FEEDING IN PARANEMERTES

draw the stylet, evert the end of the proboscis still more, and stab in a new and more distant place on the prey. The initial force for eversion is hydrostatic (Bohmig, cited by Clark, 1964), but further, more deliberate eversion appears to be controlled by the proboscis musculature. Either the central stylet is replaced with great rapidity or Paranemertes does not lose its stylet readily. Proboscides cut off while wrapped around a prey or shortly thereafter always showed the central stylet intact and always showed accessory stylets in the accessory sacs. The proboscis often winds tightly around the prey, and combined effects of prey movement, squeezing, and stylet action frequently cause the prey to tear apart. However, if the prey is not badly damaged, or if it is fairly large, the effect of the venom wears off in about 20 minutes. The venom paralyzes but does not kill the prey.

The proboscis is lined with conical gland cells that secrete a sticky mucus-like substance upon contact with a prey, enveloping the prey where the proboscis was wrapped around it. This sticky substance functions to hold the prey and may have some paralytic function as well.

Step 3, ingestion : After a prey is paralyzed Paranemertes withdraws the proboscis, losing contact with the prey for a few seconds. It then moves over the prey, feeling for a place to start the sucking-ingestion process, the third of the feeding stages. Paranemertes will start sucking any place on the prey which it can surround with its distended mouth. Prey can be ingested tail or head first, or be bent into a v- or j -shape and ingested from the side. Muscular peristalses from the posterior tip anteriorad often accompany ingestion. Most of ingestion is accomplished by the musculature around the mouth. There are sometimes posterior-to-anterior peristaltic waves of the region just posterior to the mouth. The lips also expand lengthwise, then close over a new portion of the prey, then contract, drawing in the prey. Sometimes small prey seem to be glided in as if by ciliary action. If a prey begins to move during ingestion, or if the prey is large and the Paranemertes is having difficulty ingesting it, the proboscis is often everted even while the anterior end is highly distended. When prey diameter is less than the distended mouth, ingestion only takes a few minutes ; the entire feeding process can take place in 2 to 3 minutes. The limiting factor in ingestion is prey diameter. Paranemertes cannot ingest a prey whose diameter is far greater than its own. Prey length makes little difference. One Paranemertes was observed eating a nereid longer than itself.

Slight variations in the feeding process occur when syllids or spionids are eaten. The greatest differences were observed during feeding on Syllis. Even though the syllid hardly moved, the nemertean everted its proboscis more than 10 times, yet the syllid was hardly affected, suggesting that syllids might be partially refractory to the venom. A posterior piece of the syllid was broken off, and the proboscis continued to entwine the piece. Ingestion took several minutes even though the syllid was thin and only about an inch long, and the piece being ingested even smaller. Eating consisted of tearing a piece off, wrapping the proboscis around that piece, ingestion, then repeating the process on another piece further anterior.

On a few occasions, when a Paranemertes was offered a nereid that had been previously paralyzed, step two was skipped.

84 PAMELA ROE

Length of time before defecation

Observations of 12 specimens of Paranemertes every 2 hours after laboratory feedings showed that defecation occurs from 12 to more than 33 hours after feeding. Observations at longer intervals from field collections and laboratory feedings gave the same results.

Observations of feedings in nature

At Garrison Bay 44 of 246 specimens of Paranemertes collected (18%) were observed feeding or defecated shortly after being taken to the laboratory. Of those that had fed in nature, 36 (81.8%) had eaten Platynereis bicanaliciilata, 7 (15.9%) had eaten other nereids (setae of nereids other than Platynereis were not identifiable to species), and only 1 (2.3%) had fed on a non-nereid polychaete. A total of 97.7% had eaten nereid prey.

At Mukkaw Bay 8 of 42 worms collected (19%) had recently fed, all on nereid prey, and probably all on Nereis vexillosa as that was the only nereid species found in that habitat.

Twenty of 56 worms (35%) collected at Edmonds beach had recently eaten. Seven of these were observed feeding. Food at this habitat consisted of 11 nereids (probably N. vexillosa) (55%), 8 Polydora sp. (40%), and 1 Syllis sp. (5%). Although the majority of worms from Edmonds beach had fed in nature on nereids, the nemerteans from this location had a more varied diet than did those at Garrison or Mukkaw Bays.

At the Edmonds ferry landing 33 of 140 worms collected (23.5%) had recently fed. Six of these were observed while feeding. The diet at the ferry landing consisted of 22 (67.7%) Syllis sp., 8 (24.2%) Polydora sp. and 3 (9.1%) Lepidonotus squamata. No nereid faeces were collected from Paranemertes from the ferry landing. Nereids were quite rare in numerous polychaete samples from the ferry landing rocks, and were probably too scarce to constitute much of the diet oi Paranemertes.

Food preference experiments

Tables I-IV show possible prey species tested with Paranemertes from each study area. In most cases prey species found in a given study area were tested with Paranemertes from the same area. However, Platynereis was tested with Paranemertes from all study areas even though it was found only at Garrison Bay. Members of all macroscopic polychaete families collected from each area were tested. One Neanthes brandti (Nereidae) was found at Garrison Bay, and indi- viduals of Paranemertes have been observed attacking Neanthes. However, adults are far too large for the nemerteans to be able to ingest ; they were there- fore not considered in laboratory experiments.

In the tables, steps 1, 2 and 3 refer to feeding reactions of Paranemertes to the prey. Step 1 is considered a negative response, steps 2 and 3 are considered positive responses. The column "total positive trials" sums all trials in which a Paranemertes responded positively either to a test prey or to a control nereid. The column "negative responses to controls" refers to the number of trials in

FEEDING IN PARANEMERTES

TABLE I Feeding experiments with Garrison Bay Paranemertes

85

	Steps in feeding response
Food		Final	Total positive	Negative responses to
	Initial		trials	nereid controls
	Step 1
		Step 2	Step 3
Lumbrineris inflata	5			5	4
Ophiodromus pugettensis	10			10	0
Nainereis laevigata	11			11	2
Glycinde sp.	6			6	3
Dorvillea sp.	8			8	1
Cirratulid	8	1		9	1
Terebellid	5			5	5
Phyllodocid	8			8	2
Polydora sp.	7	1		8	2
Lepidonotus sqiiamatiis	6		1	7	0
Arrnandia brevis	9		1	10	1
Nereis vexillosa	1	5	5	11	—
Platynereis bicanaliculata	10*		17	27	—
Totals	94	7	24	125	21

* These worms were fed by invertebrate zoology students. I do not know if the worms re- sponded at Step 1 or Step 2. These data are conservatively listed as negative responses.

which a Paranemertes did not respond to either test or control prey. There were no controls when nereids were the test prey.

In the laboratory specimens of Paranemertes from Garrison Bay show a very strong preference for nereids (Table I). In only one case was any other polychaete ingested, and in only 4 of 87 tries did a Paranemertes react positively to other possible prey organisms. The nemerteans from Garrison Bay did not prefer one nereid species to the other (x2 test with Yates' correction: P > 0.5).

Although there are few experiments with specimens of Paranemertes from Mukkaw Bay, results show (Table II) a significant difference in response by

TABLE 1 1 Feeding experiments with Mukkaw Bay Paranemertes

	Steps in feeding response
Food	Initial	Final	Total number of trials
	Step 1
		Step 2	Step .5
Phyllodocid	5			5
Syllis sp.	8			8
Nereis vexillosa		10	1	11
Platynereis bicanaliculata	1	10	1	12
Totals	14	20	2	36

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TABLE III

Feeding experiments with Edmonds Beach Paranemertes

	Steps in feeding response
			Total	Negative
Food		Final	positive	responses to
	Initial		trials	controls
	Step 1
		Step 2	Step 3
Hemipodus boreal is	7			7	0
Polydora sp.	9			9	2
Syllis sp.	8	1		9	2
Eteone sp.	10			10	2
Lepidonotus squamatus	7	1		8	1
Nereis vexillosa	1	5	1	7	—
Pla ty nereis b ica na licula ta	2	3	4	9	—
Totals	44	10	5	59	7

Pairs with Significant Difference at the 5% Level (Fisher's Exact Test)

Nereis— Svllis P = 0.01

Nereis— Polydora P < 0.005

Nereis— Lepidonotus 0.025 > P > 0.01

Nereis — Eteone P < 0.005

Nereis— Hemipodus 0.01 > P > 0.005

Platynereis — Lepidonotus P = 0.025

Platynereis— Polydora P = 0.005

TABLE IV

Feeding experiments with Paranemertes from Edmonds Landing

	Steps in feeding response
			Total	Negative
Food		Final	positive	responses to
	Initial		trials	controls
	Step 1
		Step 2	Step 3
Syllis sp.	9	4	2	15	2
Polydora sp.	7	3	3	13	0
Lepidonotus squamatus	10	1		11	1
Hemipodus borealis	8	1		9	0
Eteone sp.	8			8	0
Nereis vexillosa		6	4	10	—
Platynereis bicanalicula ta		3	5	8	—
Totals	42	18	14	74	3

Pairs with Significant Difference at the 5% Level (Fisher's Exact Test) Platynereis— Syllis 0.025 > P > 0.01

Platynereis — Polydora Platynereis — Lepidonotus Platynereis — Hem ipodus Platynereis — Eteone Syllis — Eteone Polydora — Eteone

0.05 > P > 0.025

P < 0.005

P < 0.005

P < 0.005

P = 0.05

P = 0.05

FEEDING IN PARANEMERTES

87

Paranemertes to Nereis or Platynereis and syllids (P < 0.005) and to Nereis (0.01 > P > 0.005) or Platynereis (P = 0.005) and phyllodocids. There was no significant difference in response to the nereid species offered. It can be concluded that Mukkaw Bay individuals also prefer nereid over non-nereid food.

There was a significant difference in the response of specimens of Paranemertes from Edmonds beach to Nereis or Platynereis and to non-nereid foods. There was no significant difference in responses among the non-nereid species, and there was no significant difference in response between the nereid species offered (Table III). It can be concluded that Edmonds beach individuals also prefer nereid over non-nereid food and have no preference among the nereids.

Specimens of Paranemertes from Edmonds ferry landing again showed preference of nereids to all other prey, and there was no significant difference in the response to the two nereid species offered. The nemerteans reacted positively to Syllis and Polydora, and even ingested them, and there was a significant differ- ence at the 5% level in the response of Paranemertes to these two species and Eteone, to which Paranemertes showed total lack of response.

Differences between nereids

Although specimens of Paranemertes showed no significant preference of one nereid over another, there was a significantly greater number of successful feedings during the food preference tests when Platynereis was the prey than when Nereis vexillosa was the prey. Several times Paranemertes could not find a place on Nereis from which to start the sucking ingestion process (step 3 of the feeding process). If trials in which step 3 was started, but in which Paranemertes derived no nourishment from the prey, are combined with step 2 as unsuccessful positive trials (Table V), Paranemertes was significantly more successful in feeding on Platynereis than on Nereis ve.villosa (x2 -- 5.957, 0.02 > P > 0.01).

Nereis vexillosa appears to be a more aggressive animal than Platynereis. On several occasions during feeding experiments a Nereis bit a Paranemertes' proboscis when the proboscis was wrapping around it. The Nereis sometimes

TABLE V

Unsuccessful positive responses versus completed feedings by Paranemertes to Platynereis and Nereis

Source of experimental animals	Food	Unsuccessful positive responses	Complete feedings
Garrison Bay	Nereis rexillosa	6	4
	Platynereis bica naliculata	10	17
Mukkaw Bay	Nereis vexillosa	10	1
	Platynereis bicanaliculata	11	0
Edmonds Beach	Nereis vexillosa	5	1
	Platynereis bicanaliciilata	4	3
Edmonds Landing	Nereis vexillosa	9	1
	Platynereis bicanaliculata	4	4
Totals	Nereis vexillosa	30	7
	Platynereis bicanaliculata	29	24

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PAMELA ROE

did not let go for more than an hour, making the feeding process very drawn out. On one occasion a Nereis bit the tail off a Paranemertes. These actions were never observed when Platynereis was the prey. Small Nereis were used for feeding experiments ; however, Nereis as small as some Platynereis used were difficult to find. Size difference might account for part of the increased difficulty Paranemertes had with Nereis.

Nereid escape response

It was observed, both in nature and in the laboratory food preference tests for Paranemertes, that nereid polychaetes have an escape response from Paranemertes. Nereids swim rapidly away upon contact with Paranemertes if water is present. If they cannot swim, as is often the case during low tides in nature when Paranemertes is feeding, they jerk away and crawl off rapidly. Responses of nereids in tests to determine if nereids responded to Paranemertes more strongly than to simple tactile stimulus and to determine their more sensitive parts were categorized as five levels (Table VI). Data from Nereis vexillosa and Platynereis were similar

TABLE VI

Responses of nereids to Paranemertes and to tactile stimulus

Part of nereid touched	Response to Paranemertes
Degree of response
0	1	2	3	4
Number of times response reached
Palps or anterior end Para podia Dorsum or venter Posterior cirri	2 0 0 0	0 0 0 0	4 1 1 6	0 0 0 3	1 2 4 5
Posterior cirri	Response to tactile stimulus
4	2	1	0	0

Degree of response by nereids to Paranemertes or to tactile stimulus

0 = neutral

1 == slight increase in crawling rate

= jerk and rapid crawling = flip of tail and rapid crawling 4 == swimming

and are lumped. Response to tactile stimulus was determined only for a sensitive part of the nereids, the posterior cirri.

If response levels 0 and 1 are considered negative and levels 2, 3, and 4 are considered positive responses, there is a significant difference (x2 : 15.90, P < 0.001) between nereid response to Paranemertes and to tactile stimulus from a pencil or probe. Fisher's exact test shows no significant difference in response

FEEDING IN PARANEMERTES 89

from any one of the four body parts tested. The difference between response to tactile stimulus and response to Paranernertes of nereid palps was not significant at the 5% level, but the difference was significant for the other three areas tested (parapodia- — -tactile stimulus, P - 0.05 ; dorsum— tactile stimulus, 0.025 > P 0.01 ; posterior cirri — tactile stimulus, P < 0.005).

Although very few trials were made, nereids did not respond with the swimming escape response when they came into contact with Amphiporus jormiddbilis, Emplectonema gracilc, or Zygonemertes virescens, other nemerteans which occur in the same habitats with Paranemertes and the nereids, but which to my knowledge, do not eat nereids.

DISCUSSION

The feeding process of Paranernertes differs in only minor details from feeding reported for other vermivorous nemerteans. For example, heteronemerteans as Linens do not lose proboscis contact with the prey while searching for a place to start ingestion (Beklemishev, 1955; Jennings, 1960) ; Paranemertes can afford such behavior since it paralyzes its prey. Paranemertes, like Cephalothrix species (Jennings and Gibson, 1969), cannot ingest polychaetes that are much greater in diameter than it is. Linens can ingest prey several times its diameter (Gontcha- roff, 1948; Beklemishev, 1955). Paranemcrtes has no distance chemoreception for finding food; Prostoma (Reisinger, 1926), Cerebratulus (Wilson, 1900; Coe, 1943), and two species of Linens (Jennings and Gibson, 1969) all find prey by long range chemoreception.

A major difference between feeding of Paranemertes and most other nemerteans is the high degree of specificity in food preference in Paranemertes. Amphiporus lactifloreus was reported to have narrow preference for Gammarus locusta in laboratory feedings (Jennings and Gibson, 1969), but most nemerteans studied so far eat a wide variety of prey (Roe, 1967; Gibson, 1968, 1970). The specialized preference of Paranemcrtes for nereids in the laboratory is reflected in nature by the high percentage of nereid food in the diet in areas where this preferred food is available. Although Paranemertes at the Edmonds ferry landing did not eat nereids because they were unavailable, these same nemerteans showed strong preference for nereid food in the laboratory experiments. These Paranemertes are quite small, even as mature adults, compard to the sizes of Garrison and Mukkaw Bay Paranemertes. Their small size is possibly an effect of the lack of preferred food in their diet. Paranemertes may be able to capture nereids with much less energy expended per unit energy gained than they can with other prey, as evidenced by the difficulty of feeding by Paranemertes on syllids. Paranemertes seems to have an optimal feeding strategy by specializing on nereids when they are available and taking a wider range of prey when nereids are uncommon. A maximum of selectivity in food types when food is common, and a decrease in selectivity when food is scarce is the optimal feeding strategy for predators (Emlen, 1966).

The absence of distance chemoreception in Paranemertes, combined with its highly specialized predation behavior and diet, plus the well-developed escape response of nereids to it suggest a pattern of evolution in environments where preferred food is so common that distance chemoreception is unnecessary.

90 PAMELA ROE

I wish to thank Dr. Alan J. Kohn for his assistance throughout this work. I would also like to thank Dr. Robert T. Paine for letting me go with him to Mukkaw Bay every month, and Dr. Robert Fernald for providing facilities at Friday Harbor Laboratories for niy use.

SUMMARY

1. Paranemertes percyrina, an abundant hoplonemertean in rocky and muddy intertidal habitats of Washington, feeds on polychaetes during low tide periods.

2. Feeding by Paranemertes involves three steps. The first stage is a recoil of the Paranemertes' head upon contact with a prey. Contact must be made with the prey; Paranemertes does not find prey by distance chemoreception. The proboscis is everted and wraps around the prey in step two. The prey is tem- porarily paralyzed or is killed. In step three the prey is ingested by means of sucking motions from muscles around the mouth. Defecation occurs from 12 to 33 hours after feeding.

3. Specimens of Paranemertes ate mainly nereid polychaetes at the three study areas where nereids were available. At the fourth study area the nemerteans ate a wider variety of polychaetes. Experiments to test food preference showed that specimens of Paranemertes from all study areas strongly preferred nereids to members of other polychaete families.

4. Nereid polychaetes have a swimming escape response from Paranemertes.

LITERATURE CITED

BEKLEMISHEV, K. V., 1955. Predatory nemertines. Priroda, 9: 108-109.

BERKELEY, E. AND C. BERKELEY, 1948. Annelida, Polychaeta errantia. In: Canadian Pacific

Fcnma, No. 9b(l). Fisheries Research Board of Canada, Toronto, 100 pp. BERKELEY, E. AND C. BERKELEY, 1952. Annelida, Polychaeta sedentaria. In: Canadian Pacific

Fauna, No. 9b(2). Fisheries Research Board of Canada, Toronto, 139 pp. CLARK, R. B., 1964. Dynamics in Metazoan Evolution. Clarendon Press, Oxford, 313 pp. COE, W. R., 1901. Papers from the Harriman Alaska Expedition, 20. The Nemerteans.

Proc. Wash. Acad. 5V/., 3 : 1-110. COE, W. R., 1905. Nemerteans of the west and northwest coasts of North America. Bull.

Mus. Comp. Zool, 47: 1-318. COE, W. R., 1940. Revision" of the nemertean fauna of the Pacific coasts of North, Central,

and northern South America. Allan Hancock Pac. Expcd., 2: 247-323. COE, W. R., 1943. Biology of the nemerteans of the Atlantic coast of North America. Trans.

Conn. Acad. Arts ScL, 35 : 129-328. CORREA, D. D., 1964. Nemerteans from California and Oregon. Proc. Calif. Acad. Sci., 31:

515-558. EMLEN, J. M., 1966. The role of time and energy in food preference. Amer. Natur., 100:

611-617. GIBSON, R., 1968. Studies on nutrition in the phylum Rhynchocoela, with observations on the

ecology of one entocommensal species. Ph.D. thesis, University of Leeds, Leeds,

England. GIBSON, R., 1970. The nutrition of Paranemertes peregrina (Rhynchocoela: Hoplonemertea).

II. Observations on the structure of the gut and proboscis, site and sequence of

digestion, and food reserves. Biol. Bull, 139 : 92-106. GONTCHAROFF, M., 1948. Note sur 1'alimentation de quelques nemertes. Ann. Sci. Natur.

Paris, Series 11, W: 75-78. HARTMAN, O., 1968. Atlas of the Errantiate Polychaetons Annelids front California. Allan

Hancock Foundation, Los Angeles, 828 pp.

FEEDING IN PARANEMERTES 91

JENNINGS, J. B., 1960. Observations on the nutrition of the rhynchocoelan Linens rnber

(O. F. Miiller). Biol Bull, 119: 189-196. JENNINGS, J. B., AND R. GIBSON, 1969. Observations on the nutrition of seven species of

rhynchocoelan worms. Biol. Bull., 136: 405-433. MACGINITIE, G. E., AND N. MAcGiNixiE, 1949. Natural History of Marine Animals.

McGraw-Hill, New York, 473 pp. REISINGER, E., 1926. Nemertini, pp. 7.1-7.24. In: P. Schulze, Ed., Biologic dcr Tiere

Deutschlands. Gebriider Bornsraeger, Berlin. Lief 17. ROE, P., 1967. Studies on the food and feeding behavior of the nemertean Parancmertcs

peregrina. M.S. thesis, University of Washington, Seattle, Washington. SIEGEL, S., 1956. N on parametric Statistics for the Behavioral Sciences. McGraw-Hill, New

York, 312 pp. WILSON, C. B., 1900. The habits and early development of Cerebratulus lacteus (Verrill).

Quart. J. Micro scop. Sci., 43: 97-198.

Reference : liiol. Bull, 139: 92-106. (August, 1970)

THE NUTRITION OF PARANEMERTES PEREGRINA (RHYNCHO-

COELA: HOPLONEMERTEA). II. OBSERVATIONS ON THE

STRUCTURE OF THE GUT AND PROBOSCIS, SITE AND

SEQUENCE OF DIGESTION, AND FOOD RESERVES1

RAY GIBSON 2 University of U'asliin/iton, Friday Harbor Laboratories, Washington 98250

The division of the Rhynchocoela into Anopla and Enopla is partly based upon the morphology of the gut and proboscis (Hyman, 1951). In anoplan nemerteans the mouth and proboscis pore are quite separate and the proboscis lacks stylet armature, whereas enoplans have the gut and proboscis opening anteriorly via a common rhynchodaeal aperture. In addition, the hoplonemertean enoplans have their proboscis armed by a characteristic stylet apparatus.

Despite these major differences, the basic digestive physiology of both groups is similar. It consists of an acidic extracellular proteolytic phase, followed by the phagocytosis of food particles and their subsequent intracellular digestion by means of proteases, carbohydrases and lipases acting in harmony (Jennings and Gibson, 1969). Intracellular digestion occurs in two stages, first acidic and secondly alkaline, with acid and alkaline phosphatases being associated with the respective phases.

An exception to this general rule is found in the atypical microphagous bdellonemerteans, where the loss of the carnivorous habit has resulted in a reduction of the emphasis placed upon proteolysis concurrent with an increase in the amount of carbohydrase activity at both the extra- and intracellular sites (Gibson and Jennings, 1969).

In all the hoplonemertean species so far investigated the endopeptidases responsible for extracellular proteolysis are produced and secreted by the gastro- dermal columnar cells. Functional gland cells are present in the intestinal epithelium, but their precise role in the digestive processes has not yet been determined.

Greater variation in hoplonemerteans is, however, found in the acid-secreting mechanisms of the foregut, and two distinct types can be recognized (Jennings and Gibson, 1969). Prostoma rubrum, in common with anoplan species, possesses acidophilic gland cells which at all times exhibit demonstrable carbonic anhydrase activity, but amphiporids and tetrastemrnids lack this enzyme completely al- though still producing acidic secretions. These variations in physiology may be attributed to differences in feeding, as Amphiporus, and probably Tetrastemma also, possesses a specialized feeding mechanism, in contrast to Prostoma which

1 Supported by National Science Foundation research grant number GB-5531.

2 Present address : Department of Biology, Edge Hill College of Education, Ormskirk, Lancashire, England, U. K.

92

NUTRITION IN PARANEMERTES PEREGRINA 93

feeds in the manner more typical of the phylum as a whole (various authors, summarized by Jennings and Gibson, 1969) .

The food reserves of nemertcans consist principally of fat globules stored in the gastrodermis, although some deposits on occasion can also be found in the general body parenchyma. No protein reserves have been recorded for the group, and the storage of glycogen appears to be of secondary importance. Gly- cogen occurs as tiny granules scattered throughout the gastrodermis, parenchyma and musculature, with occasional aggregations around the gonads (Reisinger, 1926 ; Jennings, 1960 ; Gibson and Jennings, 1969 ; Jennings and Gibson, 1969).

Until recently the common Pacific hoplonemertean Paranemertes peregrina Coe had been little investigated with respect to its nutrition. Roe (1967, 1970) has reported on the food and feeding behavior of this species, and the present study forms a logical sequence to this investigation as well as adding to our knowledge of the digestive physiology in nemerteans.

MATERIALS AND METHODS

Specimens of Paranenicrtes peregrina were obtained from intertidal muddy shores at Garrison Bay and Snug Harbor, San Juan Island, Washington, during July and August 1969.

Histological studies on the structure of the gut and proboscis were made on specimens fixed in marine Bourn, Susa, or 10% neutral formalin containing 3% sodium chloride. Paraffin wax (56° C m.p.) sections cut at 6-8 /x, were subse- quently stained by routine methods, including hematoxylin and eosin, Feulgen, Mayer's hemalum, periodic acid-Schiff (PAS), Mallory's trichrome, \% aqueous Alcian blue (for mucopolysaccharides), or by the bromphenol blue technique of Johri and Smith (1956) for proteins.

The site and sequence of digestion was determined from the examination of specimens fixed at progressive time intervals following an observed meal. In all cases the readily accepted nereid polychaete Platyncrcis bicanalicitlata, collected from the same areas as Paranemertes. was used as the food.

Specimens were fixed for 2-4 h at 4° C in 10% formalin containing 3% sodium chloride, phosphate-buffered to pH 7.0. Following fixation they \vere either washed in ice-cold distilled water and sectioned directly on an International Harris Model CTD cryostat, or dehydrated through graded acetones at 4° C, cleared in xylene at 18-22° C, and infiltrated in vacno in paraffin wax of melting point 45° C. Paraffin sections were mounted on albumenized slides, air dried at room temperature, and dewaxed before rinsing in absolute acetone prior to incuba- tion for enzyme visualization. Cryostat sections were similarly subjected to acetone treatment in order to remove fats before being incubated. All dehydration, clearing and infiltration times were kept to a minimum suitable to the size of the specimens being processed.

The following methods were used in the investigation of enzymes present : the Hausler (1958) method for carbonic anhydrase; the Hess and Pearse (1958) indoxyl acetate method for cathepsin-C type endopeptidases, as used by Jennings (1962a, 1962b), Rosenbaum and Ditzion (1963), Jennings and Mettrick (1968), Jennings and Gibson (1969), and Jennings and Gelcler (1969) ; the Burstone and Folk (1956) L-leucyl-/2-naphthylamide technique for exopeptidases of the leucine-

94 RAY GIBSON

aminopeptidase type; the indoxyl acetate (Holt, 1958) and a-naphthyl acetate (Gomori, 1952) methods for non-specific esterases; the Gomori (1952) Tween 80 method for lipase; the Burstone (1958) azo-dye technique for acid phosphatase; and calcium salt method (Gomori, 1939) for alkaline phosphatase.

Controls used for these histochemical methods included heat-inactivated sections and media from which the specific suhstrate had been omitted.

The distribution of the food reserves was studied in paraffin sections of specimens fixed either in Flemming's osmium tetroxide fluid (for fats), or 90% alcohol containing \% picric acid and subsequently stained by the PAS or Bauer methods (for glycogen).

OBSERVATIONS Structure of the gut and proboscis

As in other monostyliferous hoplonemerteans, the gut and proboscis of Paranemertes lack separate external openings. A single anterior pore, the rhynchodaeal aperture, opens from the anterior tip into a somewhat cone-shaped chamber, the rhynchodaeum, and it is from the back of this that the proboscis and gut open dorsally and ventrally respectively (Fig. 1).

The rhynchodaeal epithelium possesses no gland cells and consists only of ciliated cuboidal cells 6-9 /*, tall overlying a thin basophilic basement membrane. In common with the remainder of the gut, the rhynchodeum has no specific muscu- lature associated with it, although numerous obliquely arranged fibers are embedded in the surrounding parenchyma. In most specimens local aggregation of muscle fibers at the junction of proboscis and rhynchodeum is suggestive of a possible sphincter.

Around the rhynchodaeum can also be found the lobular frontal glands, which extend posteriorly to just behind the cerebral ganglia, and the paired cerebral organs, which open anteriorly into the cephalic slits.

The gut is divisible histologically into two distinct regions, the foregut and the intestine. Unlike the situation reported for many other hoplonemertean species, Paranemertes does not possess either a distinct esophagus or a pyloric tube, and the foregut opens directly into the rhynchodeum and intestine at the appro- priate points. The intestine extends ventrally and anteriorly as a blind-ending cecum, both intestine and cecum bearing numerous, often long, multilobed diverticula.

The foregut epithelium is folded and glandular, and consists of two distinct cell types. The principal components are columnar cells 45-60 ju, tall and 6-8 /* wide bearing dense distal cilia 4-5 p, long. These cells are filled with a coarsely granular basophilic cytoplasm that fails to react to histochemical stains for either mucus or protein. Their single oval or spherical nucleus, 3^.5 p. in diameter, is situated proximally. Evidence of cytoplasmic vacuolation can frequently be found, particularly in recently fed specimens.

Between the basophilic cells are non-ciliated pyriform gland cells of similar height but only 4-6 ^ width, packed with acidophilic proteinaceous spheres of 1 p. or less diameter. Gland cell nuclei, 4-5 //. long and 2-2.5 //. wide, are positioned proximally with their long axes approximately at right angles to the epithelial basement membrane.

NUTRITION IN PARANEMERTES PEREGRINA

95

Both cell types are secretory when fully developed. The basophils extrude their contents to the foregut lumen between their distal cilia, but the acidophils open either directly to the lumen or discharge their spheres via secretory tracts extending between the columnar cells. Shortly after ingestion is commenced, evidence of secretion can be found in the foregut lumen, the basophilic component appearing as finely participate and irregular "strings," the acidophilic spheres complete and isolated. There is some evidence to suggest that the acidophilic secretions initially expand after discharge, and then rupture to release their contents into the lumen.

c.d

FIGURE 1. Parancmcrtes peregrina. Stereogrammatic representation to show the relative positions of the proboscis and alimentary canal, characteristic of this type of hoplonemertean. The appearance of the body in transverse section is shown for two points in the lower diagrams ; c.d., cecal diverticulum; e.g., cerebral ganglion; f., foregut; i., intestine; i.e., intestinal cecum; p., proboscis ; r., rhynchocoel ; rd., rhynchodeum ; rd.p., rhynchodeal pore.

The distribution of the adipophilic glands in the foregut is such that they are concentrated in the anterior half where they and the columnar cells occur in approximately equal numbers. A short region immediately adjoining the rhynchodeum tends to lack these glands, however, and it is probable that this portion of the foregut is equivalent in the esophagus described for other hoplo- nemertean species. The posterior foregut epithelium shows a progressive decrease in the density of the acidophils as the intestine is approached, and in the region just anterior to the hindgut the ratio of gland to columnar cells is 1 : 20 or more.

A distinct pyloric tube is absent from Paranemertes and there is no decrease in epithelial height although the foregut tends to be less folded. At the junction of foregut and intestine a loose aggregation of circular and oblique muscle fibers is found in the surrounding parenchyma. These may serve as sphincter muscles to

96

RAY GIBSON

this part of the gut, although such an arrangement was not seen in all specimens examined.

The intestinal wall, or gastrodermis, is in its structure very similar to that described for other species. It consists of acidophilic pyriform gland cells inter- spersed between ciliated columnar cells. In starved specimens the columnar cells are 60-80 ^ tall and 6-8 ft wide, their sparsely distributed distal cilia extending 12-14 /x. into the intestinal lumen. Subspherical nuclei 2-2.5 /j. in diameter are embedded proximally in the cytoplasm, the latter being finely participate and possessing no particular staining affinities. The proximal regions of the columnar cells also contain variable numbers of acidophilic proteinaceous inclusions up to 3-4 /A diameter, and these react positively to the Hess and Pearse technique for endopeptidases.

II I I I I I I I I I I I I I I I I I 1 I

r.

r.m.

FIGURE 2. Paranemcrtcs peregrina. Diagram to show the relationships between the parts of the proboscis in the retracted (a) and protruded (b) positions. Note how in (b) the central stylet is terminal ; a.p., anterior proboscis epithelium ; c.s., central stylet positioned in muscular bulb ; p.p., posterior proboscis epithelium ; r., rhynchocoel ; r.e., rhynchocoel endo- thelium ; r.m., proboscis retractor muscle.

In contrast, the narrower gland cells, filled with acidophilic proteinaceous spheres of maximum diameter 1.5 /A, fail to react to histochemical methods for either endopeptidases or non-specific esterases, although their contents are dis- charged into the intestinal lumen and clearly play some part in the extracellular digestive processes.

Gland cells are most numerous in the anterior intestine and cecum, their numbers decreasing posteriorly so that they are almost absent from the region near the anus. The ratios of gland to columnar cells are about 1:1 and 1:30, respectively.

The proboscis of Parancmcrtcs is armed by a single, needle-shaped, central stylet, and lies coiled in a rhynchocoel which extends for only about one-quarter of this body length. The rhynchocoel is lined by a thin endothelium overlying muscle layers comprised of inner longitudinal and outer circular fibers.

NUTRITION IN PARANEMERTES PEREGRINA

97

Three distinct regions of the proboscis can be recognized. In the retracted position (Fig. 2a) these are an anterior thick-walled tube, a short central muscu- lar bulb housing the stylet apparatus, and a posterior acidophilic portion whose rearmost extremity is connected to the rhynchocoel by the proboscis retractor muscle.

The anterior proboscis epithelium is composed of two cell types arranged in a distinctive manner (Fig. 3). Elongate pyriform gland cells packed with pro- teinaceous acidophilic spheres of less than 1 /* diameter are interspersed with irregular-shaped columnar cells whose distal regions are filled with finely partial - late, nonproteinaceous basophilic cytoplasm. In the retracted position the cellular arrangement is somewhat obscured (Fig. 4), but when the proboscis is protruded the epithelium forms definite papillae, each papilla consisting of a series of

cm.-

•l.m.l

c.t-

l.m.2

l.p.n.

i

end.

FIGURE 3. Paranemertes pcregrina. Diagrammatic representation of the structure of the anterior proboscis when in the protruded position ; a., acidophilic gland cell ; b., basophilic columnar cell; c.m., circular muscle layer; c.t., connective tissue; end., endothelium; l.m.l, inner longitudinal muscle layer ; l.m.2, outer longitudinal muscle layer ; l.p.n., longitudinal proboscis nerve; pa., parenchyma.

groups of acidophilic cells flanked by columnal cells. The overall epithelial height at this time is approximately 40-50 p., with papillae extending outward for 0.1-0.12 mm (Fig. 3). Both cell types discharge their contents when the proboscis is in use, their secretions showing a similar appearance to those described for the foregut.

Beneath the epithelium parenchymatous tissue forms a layer that extends into the papillae to form a semi-rigid core. The parenchyma overlies three muscle zones consisting of a single layer of circular fibers and two outer layers of longi- tudinal fibers. The longitudinal muscles are separated by a narrow zone of con- nective tissue. Enclosing the anterior proboscis a thin endothelium lies next to the outer longitudinal muscle layer.

98 RAY GIBSON

The proboscis of Paranciucrtcs is furnished with fourteen longitudinal nerves that extend the full length of the anterior region in the outermost muscle layer and its adjoining connective tissue.

At its posterior end the anterior proboscis narrows into the central muscular bulb, which is divided into two parts. The anterior region contains the stylet apparatus. The single central stylet, SO /* long, is carried on a cylindrical waisted base of about 100 /j. length and 50 p, maximum diameter. On either side of, and slightly anterior to, the central stylet are the paired accessory stylet pouches, each containing from two to eight accessory stylets of similar size to the central one. Both central and accessory stylets show the braided or fluted effect described by Coe (1905) for the more southerly Californian variety of Parancmertcs peregrina.

The posterior half of the stylet bulb consists entirely of muscle fibers arranged around a sac-like central lumen, and it is probably through the contractions of these muscles that the proboscis secretions are forced into the body of the prey via the stylet wounds. The bulb lumen connects the anterior and posterior proboscis chambers by narrow canals (Fig. 2).

The third, posterior, region of the proboscis consists of a thin endothelium enclosing a single muscle layer of longitudinal and oblique fibers. Bordering the musculature a parenchymatous layer lies below the inner epithelium consist- ing of interstitial and gland cells that are not arranged into papillae. Glands, 15-20 /* long and 5-8 /A wide, are filled with irregular-shaped proteinaceous acidophilic granules of maximum dimension about 1.5 ju. There are no basophilic components in this proboscis region, and the interstitial cells possess no particular staining affinities. The proboscis lumen is often partly filled with a coarsely granular matrix that is secreted by the gland cells, and soon after proboscis eversion has taken place the lumenar contents show an increase in density at the same time as the glands can be seen discharging their contents.

The functions oj the proboscis

It seems evident that the two major proboscis regions possess distinctive roles in the function of the organ. When the proboscis is protruded the central stylet is terminal (Roe, 1967), and any secretions poured into the wound inflicted by the stylet, forced from the proboscis by the contractions of the muscular bulb, can only arise from the posterior gland cells (Fig. 2b ) . Immobilization of nereid prey does not occur in the absence of stylet penetration, and it must therefore be presumed that the paralytic toxins are produced and secreted only by the posterior proboscis epithelium. Any comparable substances discharged from the anterior proboscis papillae would be both distant to the stylet wound, and subject to dilu- tion from the surrounding sea water. However, these theoretical considerations are at variance with the findings of W. R. Kem, University of Illinois, unpublished results, who reports that the anabaseine toxin comprises some 7% of the wet tissue weight of the anterior proboscis, as well as being secreted by the general epidermis. This author fails to comment on the distribution of the toxin in the posterior proboscis and, in the light of this conflicting evidence, it seems inad- visable to draw further conclusions until additional investigations have been conducted.

NUTRITION IN PARANEMERTES PEREGRINA 99

The papilliary secretions, as noted earlier, closely resemble in appearance those of the foregut. The structure of the anterior proboscis suggests that the papillae assist in the organ gripping its catch, and it is likely that some of the secretions at least are viscous and play a supplementary role. Both non-specific esterase and acid phosphatase activity have been recorded in the acidophilic gland cells, with the former appearing additionally in the papilliary secretions. Since the basophilic secretions are distinctly "stringy" in appearance, it may be suggested that it is these that comprise the viscous component of the papilliary products, and that the gland cell esterases possess other roles. The precise function of the enzymes has not been established, but they may be involved in either initiating the disrup- tion of the polychaete epidermis or in enhancing the viscous properties of the basophilic secretions. There is no evidence to suggest that Parancincrtcs possesses an extracorporeal digestive phase, as reported for other hoplonemertean species by Jennings and Gibson ( 1969) .

The site and sequence of dit/cstion

The food and feeding mechanism of Paranemertes have been fully reported by Roe (1967). Xereid polychaetes are caught by the proboscis, immobilized by its secretions, and ingested whole by means of a sucking action. At this time the foregut actively discharges its secretions, which have an acidic pH value of 5.5-6.0, as determined from Platynereis specimens examined during ingestion after having been previously stained with indicator dyes. No carbonic anhydrase activity was ever observed in any part of the gut, so the acid secreting mechanisms must involve other enzymes, presumably associated with the proteinaceous acidophilic glands. The role of the foregut basophilic secretions is not clearly understood, but two pos- sibilities may be suggested. First, they may possess a lubricative function to facilitate ingestion or. second, they may activate the acidophilic secretions within the gut lumen.

Food material thus enters the intestine in an acidic medium, the acid secretions serving both to kill the prey and provide the appropriate pH level for subsequent extracellular proteolysis. As the food enters the intestine the gastrodermal gland cells discharge their contents. At the same time there is an increase in the number of the acidophilic inclusions within the columnar cells and they are discharged into the gut lumen between the cilia. As noted earlier, these inclusions contain cathepsin-C like endopeptidases (Fig. 5) and the amount of intralumenar activity of these enzymes increases as more of the inclusions are discharged.

Within two hours of a meal the food is sufficiently broken up to allow the phagocytosis of food particles by the columnar cells, food vacuoles formed at this time showing strong endopeptidase activity. There is no evidence to suggest that endopeptidases are secreted cytoplasmically, so that the intracellular activity observed presumably originated from the gut lumen, being taken into the cells during the phagocytotic processes. During this digestive phase strong acid phosphatase activity can be demonstrated in and around the food vacuoles (Fig. 6), where it is presumed to be concerned in some way with the intravacuolar maintenance of the acidic pH necessary for the efficient functioning of the proteases.

As time progresses the number of food vacuoles increases and six hours after a meal the columnar cells are packed with them. The early phase of intracellular

100

RAY GIBSON

••

6 c u

8

'

, ,JT

9

1O

FIGURE 4. Paranemertes pcrcynna. '1'ransverse section tlirough the anterior proboscis in retracted position, showing how the cellular arrangement of the papillae is obscured ; compare with Figure 3 ; Mallory ; scale : 20 fi.

NUTRITION L\ P AR AN EM ERTES PEREGRIN A 101

digestion lasts for up to thirty-six hours or more, by which time the residues of the meal have been evacuated from the gut lumen. Following this there is a decline in the amount of demonstrable acid phosphatase and endopeptidase activity, these enzymes becoming replaced by exopeptidases and alkaline phosphatase at the same sites. The activity of these enzymes persists within the columnar cells until digestion is completed, when the exopeptidases disappear and cannot be further demonstrated until the appropriate stage of a subsequent meal. This is the usual pattern of exopeptidase activity as reported for other nemertean species. Alkaline phosphatase activity, which is intense during peak exopeptidase visualization (Fig. 7), is at all other times present only as a faint zone of activity at the distal margins of the columnar cells. The enzymes responsible for the second, alkaline stage of intracellular digestion can be demonstrated within food vacuoles and their surrounding cytoplasm for as long as ninety-six hours after a meal.

Carbohydrases and lipases were not demonstrated at any stage in digestion, but the failure to visualize their activity could be explained by the relatively low amounts present. Lipases, for example, have only been successfully demonstrated in specimens fed on a high fat diet (Jennings, 1962a), and carbohydrases have been reported only by inference (Jennings and Gibson, 1969). There is no reason to suppose that Paranemertes in any way differs with respect to its carbohydrate- and fat-digesting enzymes.

The food reserves

Fat forms the principal food reserve in Paranemertes, being stored mainly in the gastrodermal columnar cells as globules of 2 /x or less diameter (Fig. 8). Occasional deposits up to 7 ^ across can be found, but these are irregularly distributed. Fat is deposited more or less uniformly throughout the cell cytoplasm, but tends to be absent from a distal zone 5-8 //, deep.

No fat deposits were found in the parenchyma, epidermis, foregut, blood system or body musculature, but occasional globules of 1.5-2 ^ diameter were observed

FIGURE 5. Paranemertes peregrina. Section through the gastrodermis of a starved specimen showing columnar cells with the acidophilic spheres which are the sites of endopeptidase activity (arrowed) ; Hess and Pearse method ; scale : 60 /*.

FIGURE 6. Paranemertes peregrina. Part of the gastrodermis six hours after a meal showing the distribution of acid phosphatase activity (black) in and around food vacuoles; Burstone's azo dye method ; scale : 30 p.

FIGURE 7. Paranemertes peregrina. Oblique section through the gastrodermis during the later stages of intracellular digestion to show the intense alkaline phosphatase activity (black) distributed throughout the columnar cell cytoplasm and in food vacuoles ; Gomori's calcium salt method ; scale : 60 /*.

FIGURE 8. Paranemertes peregrina. Longitudinal section through part of the gastro- dermis to show the distribution of fat globules; Flemming; scale: 12 /t.

FIGURE 9. Paranemertes peregrina. Transverse section of a part of the gastrodermis showing the restriction of glycogen storage to the distal regions of the columnar cells (arrowed) ; Bauer ; scale : 20 //..

FIGURE 10. Paranemertes peregrina. Section through two blood vessels (b.v.) to show the intense "leucine aminopeptidase" activity consistently present in their lining walls ; Burstone and Folk method; scale: 10 p.

FIGURE 11. Paranemertes peregrina. Longitudinal section through the body wall showing the narrow distal zone of non-specific esterase activity (arrowed) ; Gomori's -naphthyl acetate method ; scale : 20 /j..

102 RAY GIBSON

in the anterior proboscis, where they were restricted to the connective tissue separating the two layers of longitudinal muscle fibers.

At the time of collection mature specimens were not available, so no observations on fat deposition within the ova and ovarian endothelia can be made. These sites usually contain large amounts of fat in other species.

Small amounts of glycogen, occurring as tiny scattered granules, are stored in the distal regions of the gastrodermal columnar cells (Fig. 9), the body wall muscles (particularly the longitudinal layers), and in the parenchyma adjoining the gastrodermis.

Other sites of ensymic activity

Strong exopeptidase activity is consistently present in association with the blood vascular system (Fig. 10), a regular site for this enzyme previously reported in other species by Gibson and Jennings (1967).

Variable amounts of non-specific esterase activity were found in several other sites in the body, demonstrable by both the indoxyl acetate and a-naphthyl acetate techniques. At each site the intensity of activity is independent of the nutritive state.

Weak esterase activity was observed in the rhynchocoel endothelium, longi- tudinal body musculature, and endothelium and plasma of parts of the blood vascular system. Irregular, but stronger amounts were visualized in the connective tissue separating the two longitudinal muscle layers of the anterior proboscis, and in the outer sheath of parts of the main lateral nerve rords. The strongest esterase activity appeared in the tracts of the cerebral glands, and as a 2.5-4 /J. thick distal border to the epidermis (Fig. 11).

The only other enzymic activity demonstrated was acid phosphatase, parallelling the non-specific esterase distribution in the epidermis.

DISCUSSION

The digestive physiology of Paranemertes peregrina closely resembles that described for other nemertean species by Jennings and Gibson (1969). These authors showed that amongst hoplonemerteans interspecific differences occur principally with respect to the acid-secreting mechanisms of the foregut, specifically in the presence or absence of demonstrable carbonic anhydrase activity in the acido- philic gland cells. The occurrence of this enzyme appeared to be restricted to those species in which direct ingestion of the food was not preceded by an extra- corporeal digestive phase, this being true for palaeo- and heteronemerteans also. Paranemertes thus differs from most other nemerteans in lacking both extra- corporeal digestion and demonstrable carbonic anhydrase activity, although its foregut secretions are clearly acidic in nature. A similar situation is found in the bdellonemertean Malacobdella grossa, but physiological and morphological differ- ences in this species can be related entirely to its atypical commensal habits (Gibson and Jennings, 1969).

Paranemertes is less closely related to Amphiporus and Tetrastemma, which lack carbonic anhydrase, than is Prostoma, which possesses the enzyme. This suggests that amongst these species variations in foregut physiology cannot

NUTRITION IN PARANEMERTES PEREGRINA 103

simply be related either to systematic position or feeding mechanism, and it can be concluded only that this aspect of hoplonemertean digestive physiology is subject to interspecific alteration whose controlling factors are not yet understood.

The absence of a distinct esophagus and pyloric tube from Paranemertes is much more likely to be related to the manner of feeding. A similar absence of foregut differentiation is found in anoplan species where the food is ingested directly in a manner like that described for Paranemertes by Roe (1967, 1970). In contrast, hoplonemertean species such as Amphiporus, in which the foregut is partially protruded for feeding, possess not only a distinct oesophagus and an ex- tended pyloric tube, but also show much more folding of the foregut epithelium than is found in species feeding in the more conventional nemertean manner.

As in other carnivorous nemerteans, the emphasis in Paranemertes is placed upon the production of proteolytic enzymes by the gut, those acting extracellularlv being secreted by the gastrodermal columnar cells in the usual hoplonemertean fashion. The gland cells also discharge their products into the intestinal lumen, but the nature of their secretions remains undetermined. It is possible that they repre- sent additional proteolytic enzymes not demonstrable by the techniques employed in the present work.

The persistent zone of alkaline phosphatase activity in the gastrodermal border may be involved in the phagocytosis and absorption of food material from the gut lumen. These enzymes have been linked with the phosphorylative transfer of extracellular substances by Danielli (1952) and Erasmus (1957), and Halton (1967) has reported that in polyopisthocotylean Monogenea alkaline phosphatases are found distally in the gastrodermis during the absorption of food materials. Similar roles have been attributed to these enzymes in other nemerteans (Jennings, 1962a ; Jennings and Gibson, 1969 ; Gibson and Jennings, 1969), in archiannelids (Jennings and Gelder, 1969), and in rodents (Hugon and Borgers, 1968). The enzymes may further be concerned with the uptake of lipids, since Noma (1964) and Raghavan and Ganguly (1967) have demonstrated that shortly after a meal and during the active absorption of materials there is an increase in the phospholipid content of the intestinal mucosa.

Of the enzymes found at sites other than in the gut, exopeptidases consistently present in the blood vascular system are believed to be involved in the circulation of amino acids and simple peptides, as discussed by Gibson and Jennings (1967).

The role of the epidermal enzymes has not been established, but they may be concerned in one or both of two distinct mechanisms. W. R. Kem (un- published) recorded that 70% of the species' toxin is localized within the epi- dermal tissues, and the enzymes may thus play a part in the secretion of this substance as a defensive mechanism against predation. Distasteful epidermal secretions at least are apparently produced by other species, since Gibson (1968) recorded that the extremely voracious littoral fish Parenophrvs bnbalis consist-

j * -

ently refused to feed on lineids, even when starved and readily prepared to accept a wide variety of other natural and artificial foods.

A somewhat more plausible explanation of the role of these enzymes concerns the uptake of simple nutrient materials from the environment. Fisher and Cramer (1967) showed that glucose and amino acids were absorbed across the epidermis of Linens ruber, and concluded that the epidermal microvilli were involved in this process. Jennings and Gibson (1969), in reporting the occur-

104 RAY GIBSON

rence of epidermal enzymic activity in a number of nemertean species, suggested that these enzymes may be concerned with the extracorporeal digestion of simple proteins or polypeptides, which could then be absorbed across the microvilli. This postulated link between epidermal enzymes, microvilli and absorption is further discussed by Jennings (1969), who comments on the similarity between gastrodermal and epidermal microvilli, although noting that the former are not apparently concerned in the normal phagocytotic processes of the gut. The epidermal absorption of nutrient materials may thus be a general feature of nemerteans, in which case the blood system exopeptidases may well be additionally involved in this mechanism.

The roles of other non-digestive enzymes are far from being understood. In the anterior proboscis connective tissue an association between the non-specific esterases and fat reserves may tentatively be drawn, as certain esterases are involved in the hydrolysis of triglycerides (Reid and Dunnill, 1969), and are believed to be partly responsible for intracellular lipid metabolism in bdello- nemerteans (Gibson and Jennings, 1969). The absence of demonstrable lipases from these sites is, however, at variance with this suggestion.

\Yhether a similar role can be envisaged for esterases in other tissues is in doubt, particularly since fat deposits are otherwise restricted to the gastrodermis. Certainly in the cerebral glands, where strong esterase activity is persistently recorded, the enzymes must function in other metabolic processes. It is not known for certain whether these glands are involved with chemotactic (Reisinger, 1926), endocrine (Scharrer, 1941) or other functions.

The author wishes to extend his sincere appreciation to Dr. R. L. Fernald for providing facilities at the Friday Harbor Laboratories.

SUMMARY

1. Digestion in the hoplonemertean Parancuicrtcs peregrina is achieved by a combination of extra- and intracellular processes. The extracellular phase, effected in an acidic medium, involves endopeptidases secreted by the gastro- dermal columnar cells, and other, as yet unidentified, substances discharged from the intestinal gland cells. The semi-digested food is then phagocytosed and digestion completed intracellularly by peptidases, carbohydrases and lipases acting in harmony. Intracellular digestion is initially acid and then alkaline, with acid and alkaline phosphatases associated with the appropriate phases.

2. Nereids used as food are caught by the proboscis, and immobilized by secretions produced by the posterior proboscis gland cells. These secretions are pumped into the body of the prey via wounds caused by the central stylet. The nature of these secretions has not definitely been established, but they may contain the toxin anabaseine.

3. The anterior proboscis secretions are concerned with aiding the grip of the proboscis papillae and possibly with initiating the denaturation of the prey epidermis.

4. Acid secretions are produced by the foregut via a mechanism that does not involve carbonic anhydrase.

NUTRITION IN I'. IK. \\I-.MKRTES PEREGRIN A 105

5. Other sites of enzymic activity have heen reported, and where possible sug- gestions made as to their probable roles.

6. Fat forms the principal food reserve, with major deposits being stored in the gastrodermal columnar cells, but some glycogen is stored in a variety of body tissues.

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percgrina. M.Sc. thesis, University of Washington, 44 pp. ROE, P., 1970. The nutrition of Paranemertes peregrina (Rhynchocoela : Hoplonemertea).

I. Studies on food and feeding behavior. Biol. Bull, 139 : 80-91. ROSENBAUM, R. M., AND B. DiTziON, 1963. Enzymatic histochemistry of granular components

in digestive gland cells of the Roman Snail Helix pomatia. Biol. Bull, 124: 211-224. SCHARRER, B., 1941. Neurosecretion. III. The cerebral organ of the nemerteans. /. Comp.

N enrol, 74: 109-130.

Reference: Biol Bull, 139: 107-114. (August, 1970)

ENVIRONMENTAL TEMPERATURE AND THYROID ACTIVITY IN THE LIZARD, SCELOPORUS OCCIDENTALS

K. W. CHIU, W. G. LYNN AND J. P. LEICHNER Department of Biology, The Catholic University of America, Washington, D. C. 20317

A relation between environmental temperature and the secretory activity of the thyroid gland has been reported for a number of species of lizards on the basis of both field and laboratory studies (see reviews, Lynn, 1960, 1970). It has been suggested that the influence of temperature change upon the thyroid is an indirect one, mediated by the hypophysis (Eakin, Stebbins and Wilhoff, 1959). Increase in temperature is assumed to result in a rise in the release of TSH from the hypo- physis and decrease in temperature in a lowering of TSH release. The present expriments were designed to further investigate the relationship between tempera- ture and the hypophyseal-thyroidal axis by a study of radioiodine incorporation by the thyroid in intact, hypophysectomized and sham-hypophysectomized animals maintained at various temperatures ranging from 15° to 38° C and by examining the effects of TSH administration to hypophysectomized animals at the highest temperature, 38° C.

MATERIALS AND METHODS

A total of 200 specimens of Sceloporus occidentalis (Baird and Girard) were purchased from a supply company in California. Before being used for experi- mentation, the animals were kept for at least one week at room temperature (26 to 33° C) with an 8-hour daily period of illumination. They were fed with larval and adult specimens of Tenebrio and water was available to them at all times.

At the end of this period, fully adult lizards of fairly uniform weight were selected and these were divided into groups placed in constant temperature chambers kept at (a) 21 ± 2° C and 30 ± 1° C (experiments performed in October, 1968) and (b) 15 ± 1° C, 30 ± 1° C and 38 ± 1° C (experiments per- formed in September, 1969). This choice of temperatures was guided by the fact that they cover the range from minimum to maximum voluntary temperatures reported for the species (Brattstrom, 1965; Cunningham, 1966; McGinnis, 1966). After one week in the constant temperature chambers, each group was subdivided into three sets of animals as follows: (A) intact, untreated animals; (B) animals with the pars distalis of the hypophysis removed; (C) sham operated animals. The number of specimens in each group varied and will be indicated in the results (Table I).

At the end of the second week at the designated temperature (i.e., one week after operation), each animal was given an intraperitoneal injection of 5 /ic of carrier-free I131. The level of radioactivity in the thyroid region was then measured at 24-hour intervals for a period of seven days.

107

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TEMPERATURE AND THYROID IN LIZARDS 109

Measurements of radioactivity were made with a scintillation counter consisting of a Ijf X 2-inch Nal crystal, an RCA 6342A photomultiplier tube with a con- ventional amplifier, and a binary sealer. The crystal and photomultiplier were mounted in a lead cylinder 5.2 mm thick with a collimating slit measuring 4.0 X 120 mm. This was large enough to cover the area of the thyroid when the ventrum of the animal was properly placed over the slit. The animals remained in the constant temperature chambers throughout the week-long counting period during which they were not fed because it had previously been found that these animals show variations in thyroid activity levels during digestion.

To make a statistical comparison of the percentage I15 1 uptake by the thyroid in the various groups, we have used the mean value of the counts ± the standard error of the mean for each group on the sixth day after radioiodine administration ( "t'' test). This seems justifiable because we have found in previous experiments that the maximum uptake is usually reached by the fifth day and then remains at approximately the same level for about one week.

The results of these experiments indicated the desirability of a further study to test the effects of TSH administration to hypophysectomized animals. Specimens remaining in stock were used for this experiment. They were kept at 38 ± 1° C for one week, removal of the pars distalis was then carried out, and beginning 24 hours after the operation, all animals were given daily injections of 0.025 USP units of TSH per gram body weight in 0.015 ml of 0.7c/f saline, or just the saline without TSH, for 7 days. Three hours after the last TSH injection, each animal was given 5 p.c of carrier-free I131 and the radioactivity in the thyroid region was determined at 24-hour intervals for one week as described above. It is difficult to say whether the TSH dosage used is a "physiological" one for lizards since nothing is known of the secretion rate of the hormone in reptiles. It was chosen because our preliminary studies indicated that it resulted in I131 uptakes in hypophysec- tomized animals that were comparable to those found in normal, unoperated lizards.

Surgical removals of the pars distalis and sham-operation were performed under anesthesia with sodium pentabarbital (Nembutal, Abbott). The TSH was purchased from Nutritional Biochemical s Corporation and was reconstituted with 0.7% saline to a concentration of 10 USP units per ml.

RESULTS General observations

Lizards kept at 15° C became sluggish within a short time and remained inactive throughout the experiment. Those at 21° C were quiescent but, unlike those at 15° C, would move about for short periods when disturbed. At 30° and 38° C the lizards were highly active and very excitable, especially at the latter temperature. Only five specimens of the 73 used in this experiment died during the course of the work. One of these was a sham-operated animal kept at 15° C. The other four were lizards kept at 38° C, two of the four untreated controls and two of the five sham-operated animals.

110

K. W. CHILJ, W. G. LYNN AND J. I'. LE1CHNER

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DAYS AFTER INJECTION

FIGURE 1. Radioiodine uptake by the thyroid, in per cent of injected dose, at various temperatures in unoperated Sceloporus and in those with the pars distalis of the hypophysis removed.

TEMPERATURE AND THYROID IN LIZARDS

111

/131 accumulation by the thyroid

The percentage of I131 incorporated by the thyroid gland on successive days after injection is given in Table I for all groups except those given TSH treatment and these data are shown graphically in Figure 1. The data recorded for 30° C represent combined figures for an experiment carried out in two different years (Oct. 1968 and Sept. 1969). Statistical analysis showed no significant difference

1234

DAYS AFTER INJECTION

FIGURE 2. The effect of TSH administration upon radioiodine uptake by the thyroid, in per cent of injected dose, in Sceloporus with pars distalis removed and in sham-operated animals.

for these two groups of animals and it has seemed justifiable to combine the figures in the interest of brevity. It will be noted that intact, untreated controls kept at any of the high temperatures (21°, 30° or 38° C) showed a gradual rise in radio- activity in the thyroid region during the first 5 days after administration of the isotope and then a levelling off during the sixth and seventh days. However it is noteworthy that the maximum levels reached did not differ significantly at these three temperatures. Although the maximum figure for 38° C appears to be lower than those for 21° C and 30° C, statistical analysis shows that this difference is not significant (P > 0.8). At the lowest temperature (15° C), the untreated

112 K. W. CHIU, W. G. LYNN AND J. P. LEICHNER

controls showed a much lower rate of accumulation and no tendency to level off during the 7-day counting period. This was also true for sham-operated animals and operated animals maintained at this temperature. Comparison of the figures for untreated controls with those for sham-operated animals shows that the sham- operation had no significant effect upon I1;il uptake hy the thyroid. Surgical removal of the pars distalis of the hypophysis resulted in a significant decrease in thyroidal accumulation hy the thyroid at all of the high tempertures (21°, 30° and 38° C) but hypophysectomized animals kept at 15° C showed no significant difference in radioiodine uptake as compared with intact or sham-operated animals at that temperature.

The percentage uptake of I131 by the thyroids of hypophysectomized lizards injected with TSH is shown graphically in Figure 2. It may be seen that TSH administration resulted in a great increase in radioiodine accumulation especially during the first two days after injection of the isotope, which reached a maximum by the third or fourth day and then showed a slight decline.

DISCUSSION

The present results indicate a general pattern of I131 accumulation in the thyroid of Sceloporus occidentalis which is similar to that found for other squamate reptiles such as Gekko (Chiu et al., 1967) and Thamnophis (Chin and Lynn, unpublished) and for the turtles Pseudemys and Terrapcnc ( Shellabarger et al., 1956). How- ever, it should be noted that other experiments on Sceloporus occidentalis and Anolis carolinensis (Kobayashi and Gorbman, 1959) and on Xantusia henshaivi (Buckingham, 1970) have shown a more rapid rise in thyroidal radioiodine to reach a maximum value by the second day after I131 injection with a retention at this value for at least six more days, and still another study on Anolis caro- linensis (Lynn et al., 1965) indicates that, after reaching maximum levels at about two days, the levels fall rapidly over the next four days, especially in lizards kept at high temperature (35° C).

Comparison of data on radioiodine uptake by the thyroid in intact, untreated animals at the various temperatures used in the present study shows, rather sur- prisingly, that over the range of 21° C to 38° C, there is no evidence of any significant temperature-related change in thyroid activity. Only at 15° C is the thyroid functioning significantly inhibited. This would seem to indicate that there is a certain critical temperature, somewhere between 15° C and 21° C below which the thyroid gland is relatively inactive but that above this critical tem- perature the gland reaches a level of activity that is not modified by further temperature increase, at least over the 21° C to 38° C range. It appears that this finding by itself would oppose the theory of a direct effect of temperature change upon the thyroid in the sense of a regular increase in physiological activity with every temperature rise.

Moreover, it is well known that the functioning of the thyroid is under the control of a hypophyseal thyrotropic hormone. This has been demonstrated for the species used in these experiments (Gorbman, 1946; Eakin et al., 1959), as well as for other lizards such as Anolis (Nussbaum, 1963; Lynn et al., 1965) and Xantusia (Buckingham, 1970). In the present study, as shown by the graphs for the animals with the pars distalis of the hypophysis excised, absence of the hypo-

TEMPERATURE AND THYROID IN LIZARDS 113

physeal hormones results in a sharp decrease in thyroid activity in all animals except those kept at 15° C. This supports all previous studies on the importance of TSH in normal thyroid functioning in lizands, and also indicates that at 15° C the thyroids of intact, untreated animals are functioning at no higher level than are those of animals that completely lack TSH.

Wilhoft (1958) and Eakin et al. (1959) found that fence lizards kept at 35° C are much more active than those kept at room temperature and advanced the hypothesis that this is related to increased thyroid activity at higher temperature which is, in turn, due to a heightened release of TSH from the hypophysis. In view of the present results, it seems more likely that the role of temperature in thyroid activation is simply a permissive one. Below a certain temperature level there is no response to temperature change; ahove that level the thyroid is active regardless of the precise temperature, at least over the 21° to 38° C range. This is supported also by work done in this laboratory on Phrynosoma (Leichner, tin- published). This would mean either that TSH release from the hypophysis is subject to an "all or none" relation to temperature or, alternatively, that the responsiveness of the thyroid to TSH stimulation is so related.

Despite the small number of animals available, it seems clear that the dosage of TSH used (0.025 USP units per gram body weight daily for seven days) was sufficient to increase the uptake of radioiodine by the thyroids of hypophysecto- mized specimens to approximately the normal level for intact animals maintained at the same temperature (38° C). However, it must be emphasized that, although the level of uptake reaches similar values in these groups, the precise pattern of radioiodine accumulation in the hypophysectomized animals given exogeneous TSH at a single dosage level is quite different from the normal pattern. In the latter the accumulation is gradual over the seven-day counting period, whereas in the former the highest level is reached by the third day and then declines. This raises the interesting possibility that there is normally a continuous and constant rate of release of TSH from the hypophysis in many squamate and turtle species which accounts for the pattern of thyroidal accumulation of I131 in Gckko, Thainnophis. Tcrrapcnc and Pscndcmys, and Sceloporus. When a cyclic fluctuation in endo- geneous TSH production and release occurs, a different pattern of thyroidal I131 accumulation comparable to those reported for Anolis (Lynn et al., 1965) and Xantiisia (Buckingham, 1970) would result. In the present study, the pattern found for hypophysectomized Sceloporus receiving TSH injections would also be explicable on this basis.

SUMMARY

The relationship between temperature and the hypophyseal-thyroidal axis in Sceloporus occidentals has been investigated by a study of radioiodine incorpora- tion by the thyroid in intact animals, and animals with the pars distalis removed, kept at temperatures of 15°, 21°, 30° and 38° C. Over the range of 21 to 38° C there was no evidence of any significant temperature-related change in the normal and experimental animals, although the thyroid activity was reduced after the operation. At 15° C the thyroid functioning was significantly inhibited and there was no difference between the normal and experimental animals. It is suggested that the role of temperature in thyroid activation is a permissive one. Data for

114 K. W. CHIU, W. G. LYNN AND J. P. LEICHNER

I131 incorporation in hypophysectomized animals receiving TSH therapy offers a possible explanation for the different patterns of thyroidal I131 accumulation ob- served in different forms of lizards.

LITERATURE CITED

BRATTSTROM, B. H., 1965. Body temperature of reptiles. Amer. Midland Natur., 73 : 376-422. BUCKINGHAM, M. B., 1970. Effects of TSH and thiourea upon thyroid function in the

lizard Xantusia henshawi. Gen. Comp. Endocrinol., 14: 178-183. CHIU, K. W., J. G. PHILLIPS AND P. F. A. MADERSON, 1967. The role of the thyroid in the

control of the sloughing cycle in the Tokay (Gekko gecko, Lacertilia). /. Endo- crinol., 39 : 463-472. CUNNINGHAM, J. D., 1966. Additional observations on the body temperatures of reptiles.

Herptologica, 22 : 184-189. EAKIN, R. M., R. C, STEBBINS AND D. C. WILHOFT, 1959. Effects of parietalectomy and

sustained temperatures on thyroid of lizard, Sceloporus occidentalis. Proc. Soc. Exp.

Biol. Med., 101 : 162-164. GORBMAN, A., 1946. Qualitative variation of the hypophyseal thyrotropic hormone in the

vertebrates. Univ. Calif. Publ. Zool, 51 : 229-244. KOBAYASHI, H., AND A. GORBMAN, 1959. Thyroidal utilization of radioiodide in normal and

dehydrated lizards. Annot. Zool. Jap., 32 : 179-184.

LYNN, W. G., 1960. Structure and functions of the thyroid gland in reptiles. Amer. Mid- land Natur., 64 : 309-326. LYNN, W. G., 1970. The thyroid. In: A. d'A Bellair, C. Cans and E. Williams, Eds.,

The Biology of the Reptilia. Academic Press, in press. LYNN, W. G., J. J. McCoRMiCK AND J. C. GREGOREK, 1965. Environmental temperature and

thyroid function in the lizard, Anolis carolinensis. Gen. Comp. Endocrinol., 5: 587-595. McGiNNis, S. M., 1966. Sceloporus occidentalis: preferred body temperature of the western

fence lizard. Science, 152 : 1090-1091. NUSSBAUM, N., 1963. Hypophysectomy and thyroid function in Anolis (Iguanidae). Anat.

Rec., 145: 340. SHELLABARGER, C. J., A. GORBMAN, F. C. SCHATZLEIN AND D. McGiLL, 1956. Some quantitative

and qualitative aspects of I131 metabolism in turtles. Endocrinology, 59 : 331-339. WILHOFT, D. C., 1958. The effect of temperature on thyroid histology and survival in the

lizard, Sceloporus occidentalis. Copeia, 1958 : 265-276.

Reference: Biol. Bull., 139: 115-137. (August, 1970)

EMERSION OF THE AMPHIBIOUS CHILEAN CLINGFISH,

SICYASES SANGUINEUS

ALFRED W. EBELING, PATRICIO BERNAL, AND ALEJANDRO ZULETA

Department of Biological Sciences, University of California, Santa Barbara, California 93106 and I:stncit'»i de Bioloyia Marina, I'ina del Mar, Chile

Amphibious fishes ". . . spend periods of time out of water, on or above the ground surface, as normal parts of their life histories" (Gordon, Boetius, Evans, McCarthy, and Oglesby, 1969, page 141). Many inhabit marginal zones like the marine intertidal zone, which fluctuates between aquatic and terrestrial condi- tions. Some gobies and blennies briefly expose themselves as they scurry from one tidal pool to another; others may remain out of water for many hours while the tide is out, keeping moist in damp seaweed or under rocks (Gordon, 1966). \Yilliam H. Eger (in preparation) found several small species of clingfishes (Gobiesocidae) of the Gulf of California in moist areas under dry rocks or in clumps of snails far from the water's edge.

Sicyases sangidneus of coastal Chile and southern Peru is an exceptionally large clingfish which can live both below the cool and well-aerated surf and above water on exposed rocks (Buen, de, 1960). This Chilean clingfish attaches by means of a large disc formed of the fused and highly modified pelvic fins, bearing small and flattened dermal papillae (Briggs, 1955). The complementary forces of suction by the disc and adhesion by the papillae secure this clingfish to the rock, so that it resembles a large chiton or limpet blending with the dark sub- strate. Although young fish are relatively active as they occasionally scamper in and out of the water, adults are quiescent as they cluster on exposed rocks often well above water level (Vargas and Concha, 1957ab).

To survive above the surf, clingfish must (1) either tolerate large fluctuations of the environment or remain in the splash zone and (2) breathe air. If they leave the splash zone, they risk variable oxygen concentration, temperature, and salinity in isolated tidal pools, and desiccation and overheating on drying rocks (cf. Carter, 1931). If emerged fish were continuously wetted and cooled in the splash zone, however, they could survive with minimum adjustments, other than those necessary for breathing air. With this in mind, we set out: (1) to determine the optimal circumstances of the Chilean clingfish's life above water; (2) then, to find out why these circumstances generally prevail and if they can be largely compromised ; which (3) led to experimental studies of the mechanism of the fish's aerial respira- tion. Our investigations supplemented more intensive physiological studies by Gordon, Fischer, and Tarifeno (in preparation) of the survival of the fish out of water.

115

116 ALFRED W. EBELING, PATRIC1O BERNAL AND ALHJANDRO ZULETA

MATERIALS AND METHODS 1'ield observations

\Ye observed the behavior and distribution of fish at three localities along the coast of central Chile, north of Valparaiso in a subtropical region of light to moderate rainfall. At each locality fish were watched through binoculars as they clung to the vertical surfaces of three large rocks on progressively higher ground : rock 1 with base awash at low tide, to rock 3 with base awash only at high tide. These surfaces faced mostly southwest, so that they were shaded in the morning and sunlit in the afternoon. They either paralleled the surge or formed a lee, protecting the fish from the full force of the surf. Locality I was in a relatively urban area just north of the Estacion de Biologia Marina at Montemar, while localities II and III were in sparsely populated rural areas about 60 km north of I.

The observed surfaces of rocks 1 measured about four square meters at localities I and III, and about six square meters at locality II ; rocks 2 and 3 together measured barely three square meters at all localities. The surface of rock 1 was subjectively divided into two habitats : a lower half generally wet at low tide and an upper half generally dry at low tide. A fringe of brown algae about one meter above tidal low underlaid the surfaces of rocks 1 and 2. With approaching high tide, larger swells broke over the rocks to form characteristic rivulets down the sheltered surfaces. Clingfish often congregated in these relatively wet areas.

The three localities were visited a total of 38 times between October 7 and November 20, 1967. A set of 24 observations was made during each visit. The diel distribution of visits was: 5 visits between 0720 and 0913 hrs, 11 visits between 1019 and 1308 hrs, 3 visits between 1340 and 1515 hrs, 8 visits between 1526 and 1723 hrs, 8 visits between 1700 and 1825 hrs, 2 visits between 1820 and 1905 hrs, and one visit between 2020 and 2100 hrs by spotlight. Localities II and III were visited only five times each.

Fifteen physical and nine biological variables were measured to relate emergent clingfish with their environment (Table I) : locality, scored 1-3 : date, in days from the first visit; time of day (hrs), 0700-2100; wave height (m), from trough to crest of swells about 25 m offshore ; water level, scored 1-8, from minimum low tide in calm sea to maximum high in rough sea ; dryness, upper rocks, scored 1-3, from the condition when all surfaces of the three rocks were wet to that when the upper halves of rocks 1 and 2 and all of 3 appeared dry ; relative humidity (%} measured with a sling psychrometer ; air and water temperatures (°C), water measured in the surf with bucket thermometer ; wind direction, scored 1-4 — NW, WNW, W. SW — and velocity (mph), recorded as the average of three measure- ments by hand-held anemometer; overcast, scored 1-10, from clear and sunny to complete overcast ; si in on rocks, scored 1-3, from total shade, through hazy sun, to bright sun; irradiance (langlies/hr), measured in shade or sun, depending on the condition of light on the rocks, with an integrating photometer (Haley, 1967) ; barometric pressure (millibars), recorded off station from a barometer in the laboratory.

We observed fish from a distance of about 20-30 m ; closer approaches dis- turbed them. Recorded abundances were the averages of three counts, which included all visible fish along or above the upper margin of the seaweed fringe on the rock surfaces. Numbers of fish on rocks 1-3, recorded separately, were also

EMERSION OF AMPHIBIOUS CLINGFISH

117

summed to obtain total fish. Other observations were recorded for rock 1 only : the number of fish along the seaweed fringe, the per cent of fish on the lower half of the rock surface, the per cent of fish with heads pointed down, and the per cent of juvenile fish (young and halfgrown). The density of the fish in crevices and seaweed patches was scored 1-3 from scarce to abundant.

TABLE I

Variables possibly affecting clingfish emerged in the rocky intertidal

of central Chile*

Variable	Range	Mean	Standard deviation	Commun- ality
Locality	1-3	1.29	0.61	0.56
Date	1-51	28.1	13.6	0.23
Time of day (hrs)	815-2100	1396.	354.	0.53
Ware height (m)	0.6-2.75	1.33	0.67	0.60
Water level (score)	1-8	4.82	1.89	0.71
Dryness, upper rocks (score)	1-3	1.84	0.73	0.83
Relative humidity (%)	52-96	73.3	12.0	0.61
Air temperature (°C)	11.5-18.3	15.7	2.30	0.78
Water temperature (°C)	11.3-14.7	12.6	0.91	0.42
Wind direction (score)	1-4	2.68	1.27	0.50
Wind velocity (mph)	1.2-17.3	5.93	3.96	0.47
Overcast (score)	1-10	4.39	3.65	0.48
Sun on rocks (score)	1-3	1.87	0.85	0.64
I r radiance (langlies)	0-3.3	1.55	0.83	0.64
Barometric pressure (mb)	29.79-30.11	29.93	0.10	0.48
Fish, total	0-135.0	35.1	31.6	0.99
Fish, rock 1	0-131.3	26.0	30.0	0.99
Fish, rock 2	0-23.7	4.92	5.59	0.78
Fish, rock 3	0-18.0	4.19	4.56	0.66
Fish, seaweed fringe	0-12.1	2.26	2.56	0.50
Fish on lower half of rock (%)	0-100	80.4	28.1	0.48
Fish with heads down (%)	0-80	30.4	22.8	0.57
Juvenile fish (' ,	41-100	93.9	14.6	0.67
Fish in crevices, etc. (score)	1-3	1.97	0.71	0.11

* Each variable, expressed by 38 observations, in defined in the text. The communality (0-1) measures interactions with the other variables.

Factors were computed to assemble groups of interacting variables into a few causal arrays, each defining a system of interactions either within the environ- ment or between clingfish and environment (cf., Sokal and Daly, 1961). First a 24 by 24 matrix of correlation coefficients, one coefficient for each pair of variables, was "factored" to extract latent roots and orthogonal vectors of 24 principal factors. Then a much smaller number of factors was obliquely rotated to "simple structure," so that fewrer factors defined more natural groups of variables because these factors were no longer necessarily orthogonal to each other; i.e., they could be inter- correlated (e.g., Cattell, 1965; Harman, 1967). In this way, "factor analysis represents covariation by finding fewer dimensions of variation than the number of variables in a correlation matrix" (Thomas, 1968: 849). Because we had no a priori way of estimating the number of natural groups in the system of 24 variables, we compared three different representations composed of three, four, and

118 ALFRED W. EBELING, PATRICK) BERNAL AND ALEJANDRO ZULETA

five rotated factors, respectively. The four-factor representation of the system appeared to be the most meaningful, because (1) two of the five factors were strongly intercorrelated (rf., Thomas, 1968) and (2) three factors did not distinguish two important groups.

Each group was ordered into an array by decreasing magnitudes of the "loadings" of its several constituent variables on its factor (Table II). The loading of a variable was a measure of the variable's relative "importance" to, or correlation with, the factor, and was somewhat arbitrarily adjudged "significant" if it equaled or exceeded an absolute value of about 0.50 (Sokal and Daly, 1961).

Communalities estimated the proportion of the variation of each variable attributable to its covariation with the others (Cattell, 1965; Harman, 1967). Variables with high communalities interacted strongly with other variables. Vari- ables with low communalities, however, explained relatively little of the system, in that a relatively large part of their variation was not attributable to their covaria- tion with the others. This "error variance" was partitioned out of the analysis, so that it did not influence the factor loadings. The program BMDX72 for computa- tion of the correlation matrix and communalities, extraction of 24 principal factors, and the subsequent rotation of a few factors to simple structure (Dixon, 1967) was modified for the IBM 360-75 computer at the University of California, Santa Barbara Computer Center.

Laboratory observations

Caught near localities I and II, experimental fish lived unfed in the laboratory in 40-liter plastic aquaria half-filled with aerated seawater of salinity about 34%o at 12-17° C.

Responses of clingfish to aeration, stagnation, temperature, enforced emersion, and fresh water were noted of fish placed 1-3 at a time in covered 12-liter plastic aquaria. Seven of the fish were classified as adult (130-190 mm long), nine as halfgrown (100-120 mm), and two as young (60-90 mm). Submerged fish were observed in aerated seawater sun-warmed from 13 to 19° C or in seawater first aerated for 30 minutes, then allowed to stagnate in the shade. Emerged fish were observed on moist paper towels in an empty aquarium. In the two stagnating aquaria, body movements and opercular rates were recorded at 30-minute intervals during eight hours for three each of the halfgrown and adult fish as dissolved oxygen content, measured by galvanic-cell oxygen electrode, decreased from saturation. Six halfgrown and adult fish were subjected to serial dilutions of seawater.

Percentage concentrations of oxygen and carbon dioxide, by volume, of gas held in the gill cavities were measured after three fish, emerged for varying periods, had expelled it: an adult, 184 mm total length weighing 92.9 g: a half- grown, 101 mm, 11.9 g: and a young, 67 mm, 3.3 g. Each fish voided bubbles through its gill slits while being gently submerged on a mechanical platform in a 12-liter aquarium half filled with a \% solution of polyethylene oxide in seawater (Fig. 1). "Polyox" is a resin, dissolving slowly in seawater to form a non-toxic and viscous solution, which prevents the fish's ventilation and preserves any bubbles trapped in it (Todd, 1970). Expelled gas was caught in an inverted funnel filled with polyox and capped with a rubber diaphragm. Fish and funnel rested on a

EMERSION OF AMPHIBIOUS CLINGFISH

119

platform formed of a wire frame bent to two levels about one centimeter apart and surfaced with plastic screen. The platform was smoothly raised and lowered on a rack and pinion elevator from a photographic enlarger without disturbing the fish, which rested quietly on the upper level above the liquid while the inverted funnel waited on the lower level just below the surface.

FIGURE 1. Experimental aquarium about half filled with a 1% solution of polyethylene oxide in seawater. An emerged Chilean clingfish rests above the surface on a split-level mechanical platform, which also supports an inverted funnel filled with the same "polyox" solution. Ending a specified period of emersion, the fish was slowly lowered into the liquid by cranking down the ratchet bar supporting the platform, so that the fish expelled bubbles out its gill slits into the polyox. The surfaced bubbles were preserved by the viscous polyox and captured in the funnel for later analysis of their gas content.

The fish remained quiet until slowly submerged at the end of each trial. Bubbles expelled from the gill slits were captured on the surface in the funnel, where they collected in the capped spout and were drawn into a one-milliliter syringe with dead space filled with saturated acid citrate solution. The gasping fish was quickly returned to the holding aquarium of aerated seawater, where it

120 ALFRED W. EBELING, PATRICIO BERNAL AND ALEJANDRO ZULETA

soon recovered for tin- next trial. For the adult and halfgrown, the volume of expelled i;as was estimated in the syringe; for the young, in the capillary of a Scl inlander microgasometer, where the gas content was analyzed (Scholander, van Dam, ClarT. and Kanwisher, 1955). Because gas was usually expelled in several buhhles from both gill slits, some was probably lost and most volumetric measurements were probably minimal. Little, if any, oxygen diffused through the polyox film. The known oxygen content (15-16%) of artificially reduced gas bubbled through polyox and collected after about 30 seconds was unchanged.

FIGURE 2. Clingfish emerged on a damp surface ; A, flattened on the substrate to protect its delicate "frontal skin" on chin, leading edge of sucker, and pectoral fins; B, with head raised to expose this vasculose skin for cutaneous respiration.

Minimal rates of oxygen uptake were derived from oxygen depletion in the estimated volumes of branchial gas captured in the funnel, calculated as functions of presumed initial oxygen content (21%), content in the expelled gas, and the estimated total gas volumes (after Todd and Ebeling, 1966, wrhere the brackets in the expression I [(0.21 -- P') -- P] were inadvertently omitted). The initial volume (I) was taken as equal to or greater than the modal volume recorded for each fish. Volumes from the adult fish ranged from 1.4-2.5 ml, with the mode

EMERSION OF AMPHIBIOUS CLINGFISH

121

2.2 ml. Therefore, 2.2 nil \vas substituted for all smaller measurements, which were probably of incomplete bubbles or hubbies emitted from one side only. All volumes exceeding 2.2 ml (2.3-2.5) were used because overestimations were un- likely. The "I" values for the halfgrown fish (0.09-0.85 ml, mode 0.20 ml) and young fish (0.01-0.04 ml, mode 0.03 ml) were estimated in the same way. Even though one value for the halfgrown fish was four times larger than the mode, it was retained as a possible measure of maximum gulping capacity.

Rates of oxygen uptake (milliliters per kilogram of wet body weight per hour) were calculated as the oxygen volumes multiplied by 1000 -=- weight of fish (g), and by 60-=- period of emersion (min). Rates after the branchial gas was apparently

TABLE II

Factors that group the variables in Table I into causal arrays

Factor I : tranquillity	Factor III: abundance
* Wind velocity Fish, rock 2 Wave height * Fish, total Fish with heads down	-0.75 0.75 -0.68 0.58 0.55	* Fish, rock 1 *Fish, total Juvenile fish Fish, seaweed fringe Local i 'I v	0.99 0.97 0.77 0.68 0.54
*Fish, rock 1	0.46
Factor II: water-level	Factor IV: insolation
Dryness, upper rocks Water level Fish, rock 3 Overcast	-0.84 0.78 0.78 0.53	.1 //• temperature Relative humidity Water temperature * Irradiance	0.91 0.79 0.72 0.67
Fish on lower half of rock *Irradiance	-0.53 -0.48	Wind direction *Wind velocity	0.48 0.48

* Variables are ordered by their relative "importance" as indicated by their loadings (numbers at right). Those marked by asterisk load "significantly" or nearly so on two factors, thereby indicating the factors' mutual interaction. Derivation of the factors is explained in the text.

renewed during a single trial were estimated as functions of the total duration of the trial, minus the time to renewal as indicated by a sharp rise in oxygen content in previous measurements made after certain intervals : adult fish, 57 min ; halfgrown fish, 35 min; young fish, 12.5 min (Fig. 3).

RESULTS

Effects of the natural environment

Communalities of the variables ordered the whole system into a hierarchy of links (Table I). The biological variables averaged higher communalities (0.71) than the physical (0.57) because they responded to several physical causes as well as interacting strongly among themselves. (The variable fish in crevices was excluded from the averaging because it seemed to vary randomly.) The variables of fish abundance had high communalities because emergent fish responded to short- term changes in water level, dryness of upper rocks, and wave height, and tended

122 ALFRED W. HEELING, PATRICK) BERNAL AND ALEJANDRO ZULETA

to congregate in patterns. Physical variables like date and water temperature, on the other hand, had relatively low communalities because they probably involved long-term changes and, consequently, had little effect on the fish during the short study period. Water temperature, which varied but 3° C, may respond more to long-term fluctuations of the offshore current than to local heating during the Chilean spring. Even though they did not seem to affect fish abundances directly, the effects of insolation like air temperature and irradiance had fairly high communalities because they interacted strongly among themselves.

The factors divided the system into its component arrays of variables (Table II). The variable loadings identified the factors and revealed important environ- mental effects on the clingfish. The four factors were dominated by decreas- ing wind velocity and wave height ("tranquillity"), increasing water level with wetting of upper rocks ("water-level"), generally increasing fish abundance ("abundance"), and warming with decreasing humidity ("insolation"). For a given factor, physical variables should have the higher loadings if they evoke responses in the biological, but if biological loadings exceed physical, they may respond to other causes (Thomas, 1968). Physical variables of the tranquillity and water-level factors averaged higher loadings (0.72) than did the biological variables (0.64). Therefore, most fish came out onto wet rocks during calm periods when they often turned heads down and moved higher as the tide rose. The abundance factor, having no predominating physical loadings, simply indi- cated that more fish, especially adults, emerged in the more remote localities. The insolation factor, having no biological loadings, simply grouped the obvious effects of solar warming, which generated the land-sea breezes.

Several simple correlations apparently indicated indirect effects of the environ- ment. Six variables did not load "significantly" on any factor ; i.e., they fell out- side all four causal nexuses. Fish in crevices, date, and barometric pressure were apparently unimportant links within the system defined by the sampling regime. Time of day, wind direction, and sun on rocks, however, correlated significantly (P < 0.05) with several other variables in the insolation and abundance factors, which, in turn, interacted with the tranquillity factor (Table II). These simple correlations and the interactions between factors would seem to indicate that fish came out in the late afternoon when the surfaces of the observed rocks were in the sun, were it not for the multitude of fish counted during the single night station at Montemar. Perhaps more fish emerged at populous Montemar (where almost 75% of the stations were occupied) as people left the beaches in the late afternoon and, coincidentally, as sea breezes subsided and the sea calmed.

/''inersion in aquaria

When half grown and adult clingfish emerged head first from aerated seawater, they immediately gulped air and perhaps water, stopped all opercular movement, closed their gill slits, and occasionally after several minutes turned heads down. Usually, however, they remained submerged or only partly emerged. Fish com- pletely emerged and clinging to the smooth aquarium side gradually slipped back into the water until partly submerged. Adults placed on a horizontal platform relaxed immediately and soon appeared oblivious to laboratory activity, although emergent young moved their opercles and shifted position from time to time.

EMERSION OF AMPHIBIOUS CLINGFISH 123

When submerged horizontally on a mechanical platform, all fish first fluttered their pectoral fins to break the suction through a groove in their ventral sucker, then moved their opercles to expel the bubbles through their gill slits.

Effects of temperature

Water warmer than 15° C disturbed submerged fish, which often clustered near the aquarium aerator and "panted" with strong and rapid opercular beats. When the seawater had warmed to an afternoon high of 17.8° C (room temperature, 20.1° C), most fish had pushed either their heads or tails above water. Those with heads submerged continued opercular beats, while those with heads out of the water had stopped all such movement.

Warm air disturbed emerged fish and seemed to elicit compensatory behavior because these fish stopped all stress reactions when subsequently cooled. An adult and halfgrown fish transferred from aerated seawater at 16.8° C to an empty aquarium in air 18.5° C closed their gill covers and were initially quiet. After a few minutes, however, the halfgrown fish began to "pant," apparently trying to pump moisture from the aquarium bottom through its gill cavities. The adult interspersed longer quiescent periods with shorter panting episodes for about an hour, then rested with gill covers closed while the halfgrown fish continued panting. At first, bubbles surrounded the opercles of both fish, as though the fish were trying to use their pharyngeal pumps. But when obviously stressed and drying fish were lightly sprinkled with seawater, they quickly relaxed and stopped panting. The halfgrown fish died within five hours with its opercles spread. The adult lived for nine hours, during which its skin became tacky. (In another trial, an adult survived 20 hours, although a halfgrown fish lived but 5 hours.)

Morphological changes accompanied the behavioral reactions to this tempera- ture-induced respiratory stress. The pharyngeal epithelium of the emerged adult fish was reddish with vascularization and its dark red gills were engorged with blood. At one time the anterior holobranch adhered to the front of the gill chamber, while the others were clumped and pressed against the back. A thin membrane behind the chin closed the then cup-like chamber from beneath. In contrast with the pharynx, the mouth was pale to white. During active periods as the air temperature approached 18° C, the adult raised its head to expose a broad area of relatively delicate, vasculose, and unpigmented "frontal skin," extending from its chin over the broad front of its sucking disc to its pectoral fins. This area was previously hidden when the fish pressed flat to the substrate and exposed only its relatively thick, mucus-laden, and dark dorsal skin (Fig. 2A). When the fish reared its head, we saw that the frontal skin had been transformed from a white surface showing few capillaries to a reddish surface beset with conspicuous networks of engorged capillaries (Fig. 2B) .

Effects of dissolved oxygen

Submerged clingfish showed no particular resistance to critically low con- centrations of dissolved oxygen, although the fish usually emerged before it suffocated. In the two stagnating aquaria, adult and halfgrown fish behaved normally until the concentration fell below 2 ml/1, when all four fish had partly

124 ALFRED W. EBELING, PATRIC1O BERNAL AND ALEJANDRO ZULETA

emerged and had increased their opercular rates by almost 70% (Table III). While oxygen measured more than 1 ml/1, fish usually clung to the aquarium wall tails up with their heads submerged and opercles moving. Then, as oxygen continued to fall, more and more partly emerged fish clung heads up and stopped their opercular movement. While emerging, they appeared to gasp as their noses broke the water's surface. (One halfgrown fish paused with nose barely pro- truding the surface, while it repeatedly gulped air possibly mixed with water.) Submerged fish became restless, then breathed rapidly and laboriously for varying periods before emerging.

TABLE III Responses of initially submerged clingfish to decreasing dissolved oxygen*

T~\I **1 A		Position of fish	Opercular rate (beats/min)
Dissolved oxygen concentration	Water temperature (O f~*\	Adult	Halfgrown
(ml/1)	C)
								Adult	Halfgrown
		E/T	Pe	H/E	E/T	Pe	H/E
5.00-7.00	12.5-13.8	1/16	0.1	0/1	0/10	—	—	47-120	36-160
	(13.0)							(82)	(105)
2.00-3.51	12.3-14.6	4/13	0.2	0/4	0/13	• — •	—	46-113	55-105
	(13.3)							(82)	(80)
1.00-1.71	14.0-15.3	6/15	0.1-0.6	0/6	2/7	0.3-0.5	0/2	102-205	93-203
	(14.6)		(0.35)			(0.38)		(144)	(148)
0.45-0.89	12.2-15.8	11 19	0.1-0.5	6/11	9/14	0.1-1.0	3/9	121-198	105-206
	(14.5)		(0.38)			(0.44)		(140)	(152)

" Three adult and three halfgrown fish were observed in two aquaria that were first aerated, then allowed to stagnate. For columns under "position of fish," total observations (T) included the number of sightings of fish entirely or partly emerged (E) and the number entirely sub- merged (T-E). The observations of emerged fish (E) included the estimated proportions of their bodies above water (Pe) and the number of fish with heads uppermost (H), as compared with the number either lying parallel with the seawater surface or with tails uppermost (E-H). Ranges of observations precede averages in parentheses.

Survival of fish in deoxygenated seawater of less than 2 ml/1 varied con- siderably. Although one adult fish died in 4 hours, the other survived 36 hours, including 24 hours in water containing less than 1 ml/1 oxygen, as it mostly rested with parts of its body above water. One halfgrown fish survived three days in water aerated but once every 24 hours.

Effects of salinity

Clingfish showed a surprising tolerance of fresh water. Four adult and half- grown fish lived submerged without apparent distress through a series of dilutions : 24 hours in 50% seawater, then 5 hours in 25% seawater, 5 hours in 12% seawater and 24 hours in pure tapwater. Another halfgrown fish transferred directly from 100% to the 12% seawater, however, turned pale and panted for about ten minutes before returning to normal. Only this halfgrown fish and

EMERSION OF AMPHIBIOUS CLINGFISH

125

another adult that was placed directly into tapwater died before the end of the experiment, some 58 hours after the first dilution.

Gas exchange in gulped air

Of the three clingfish emerged above the polyox solution, the adult appeared most quiescent and seemed to replenish the gas held in its gill cavities after the longest intervals. Soon after gulping air upon its removal from aerated sea-

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FIGURE 3. Temporal decrease in percentage oxygen in new or renewed gas held in the gill cavities of emerged clingfish; top (solid circles), expelled from a 93 g adult fish; bottom (open circles), from a 12 g halfgrown fish; inset (crossed circles) from a 3.3 g young fish. Each circle represents one measurement of the percentage oxygen in expelled gas after the fish had remained emerged on a mechanical platform for the time indicated on the horizontal axis.

126 ALFRED W. REELING, PATRICIO BERNAL AND ALEJANDRO ZULETA

water, it stopped all opercular and body movements and appeared undisturbed by activities in the laboratory. Percentage oxygen in its branchial gas expelled into the polyox solution decreased up to almost one hour, when the final obser- vation indicated a sharp increase (Fig. 3, solid circles). This increase was unconfirmed because long experimental trials were difficult to complete. The struggling fish entering the polyox occasionally destroyed its expelled bubbles before they could be secured in the funnel, and the chance of disturbing the fish increased with time, so that reruns took up to four hours for one measurement.

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FIGURE 4. Temporal decrease in the estimated rate of oxygen uptake from gas held in the gill cavities of clingfish emerged in the experimental aquarium described in Figure 3 ; solid circles, adult fish; open circles, half grown fish, crossed circles, young fish. Parentheses enclose points depicting rates estimated from volumetric oxygen depletion after the gas had been once depleted and then renewed (see text). Circles at the upper left depict off-scale approxi- mations of rates (numbers at right) measured after the fish had been emerged for relatively short periods of time. Problematical values are queried. Arrows indicate the average of two or three observations.

The quicker response of the halfgrown fish supported the tenuous hypothesis of gas renewal as indicated by the adult trials. Nineteen trials showed a similar sharp increase in oxygen content after slightly more than 30 minutes (Fig. 3,

EMERSION OF AMPHIBIOUS CLINGFISH

127

open circles). Because individual trials were shorter, a complete breathing cycle was observed, showing that oxygen decreased after renewal as before: within 35 minutes, the fish had gulped, depleted, expelled, and replenished its branchial air. Like the adult, the halfgrown fish quickly relaxed on the platform, where the oxygen content of its branchial gas reached a minimum of 4-6%.

The young fish was easily disturbed, occasionally moving its opercles and frequently expelling its branchial gas. The general pattern of oxygen depletion in this gas, however, resembled the others (Fig. 3, crossed circles). Trials

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lasting up to 12 minutes showed a steady decrease of oxygen to 13.4%, more than twice the minimum of the adult and halfgrown fish.

All fish held about the same relative amount of oxygen in their branchial bubbles. To compare potential oxygen supplies, ratios of modal volumes of expelled gas to body weight were divided by hours to renewal and multiplied by 1000 to rid decimals. At first glance, the adult index (25) indicated that the adult fish carried about the same supply as the halfgrown fish (29), although both indices were considerably less than that for the young fish (45). Recall,

128 ALFRED W. REELING, PATRICIO BERNAL AND ALEJANDRO ZULETA

however, that the young fish used but 36% of its oxygen, compared with 76% for the others. A corrected index of 23 (36/76 > 45) showed that the young fish actually held only slightly less oxygen for its size than the others.

Rates of oxygen uptake from the branchial gas decreased precipitiously during the first ten minutes of emersion, from an almost inestimable high to only 5-10 ml/kg/hr (Fig. 4). After the initial decrease, the adult rate averaged 5.3 ml/kg/hr, the halfgrown 6.8, and the young 6.2. (At about eight minutes, however, the rate of the young fish was only half that of the others.) During a 17-minute trial, the halfgrown rate was 27 ml/kg/hr (queried in Fig. 4), as calculated from an exceptionally large volume of expelled gas. Rates estimated for periods after gas renewal (points in parentheses) approximated the others.

The respiratory quotient of clingfish, as estimated from branchial gas exchange, was not unusual. Plots of carbon dioxide increase as a function of oxygen decrease, however, were scattered because the small observed percentages of carbon dioxide approached the experimental error and because several trials of less than five minutes yielded bubbles with unusually high percentages of carbon dioxide (1.5— 3.8%), as though the gas were accrued near the onset of the trials (Fig. 5). The regression coefficient, which probably underestimated the respiratory quotient (ff., Carter, 1957), was 0.23, compared with 0.28 for airbreathing mudsuckers (Gillichthys -inirabilis), as determined by Todd and Ebeling (1966). The young clingfish expelled gas containing proportionately more carbon dioxide (avg. 1.8%) than either the halfgrown (1.5%) or adult fish (1.3%).

DISCUSSION Adaptations to optimal conditions

Factor analysis indicates that when the Chilean clingfish Sicyascs sangiimcns is in the field it avoids stress by its distribution and behavior. It positions itself so that it stays wet and relatively cool. It clings to exposed rocks continuously sprayed by the rough surf, which remains cool even on warm days. Fish even congregate on sunny surfaces so long as they are in a splash zone. The rise and fall of the water level apparently controls the vertical distribution of terrestrial fish, which cling to the upper rocks only when these rocks are splashed by high water. Beach crowds, however, may inhibit emersion of adults, even onto the preferred wet rocks with broad vertical surfaces.

Direct observations of clingfish both in the field and in the laboratory sub- stantiate these conclusions drawn from factor analysis. In the field, fish were either restricted to the spray zone or to narrow cascades left by waves breaking over the rocks. During especially low tides, fish left the drying upper rocks. In general, they were either splashed or submerged by waves about once every five minutes and were never seen to wait longer than 15 minutes between wettings. Laboratory fish, which were not continuously splashed, often remained submerged or assumed a half-in and half-out position. Perhaps this is a compromise between total emersion without splashing, which would cause water loss and overheating, and continuous submersion, which could seem unnatural to the fish. Disturbed fish in relatively warm laboratory air appeared noticeably relieved when sprinkled with cool seawater. Like the mudskipper Pcriophthahnits sobrinus of eastern

EMERSION OF AMPHIBIOUS CLINGFISH 129

Africa (Harms, 1935; Stebbins and Kalk, 1961), a terrestrial clingfish may nor- mally not lose much water because it keeps its skin moist. Rao and Hora (1938) observed that a blenny, Andamia hcteroptcra, of southeastern Asia always lives above water in the intertidal spray zone, following the rising and falling tides. It follows that the "insolation" factor does not directly affect clingfish because they avoid overheating as well as dehydration. They can markedly lighten or darken the mottled color pattern of their exposed skin, thereby controlling heat absorption. Fish held for several days in the relatively warm laboratory were generally lighter than recentlv captured fish, which, however, may have altered their color to blend with dark rocks in the intertidal. Stebbins and Kalk (1961) suggested that mudskippers may control their body temperature by chang- ing skin color and avoiding midday heat. Gordon, Boetius, Evans, McCarthy, and Oglesby (1969) presumed that for Nosy Be mudskippers, which are protected by scales, overheating may be more hazardous than dehydration.

Survival during stress

A Chilean clingfish perhaps stranded during a violent winter storm could apparently survive substantial water loss on a drying rock, or substantial warming, stagnation, and dilution in an isolated tidal pool. Gordon, Fischer, and Tarifeiio (in preparation) showed that fish survive substantial dehydration in the labora- tory and Eger (in preparation) showed that other, smaller species of clingfishes tolerate surprising amounts of evaporative water loss. Even though a Chilean clingfish can survive several hours of aquatic anoxia by partial emersion, it is no more tolerant of low dissolved oxygen when it is submerged than most fishes (rf., Jones, 1964). But like the Nosy Be mudskipper Periophthalmus sobrinns (Gordon, Boetius, Boetius, Evans, McCarthy, and Oglesby, 1965) and the estuarine mudsucker GiUicIitlivs mirabilis (Todd and Ebeling, 1966) it survives in very dilute seawater. In three months of searching during the relatively dry Chilean spring, however, we found only one fish isolated in an upper tidal pool, a tiny young individual. \\'e saw none on dry rocks. Also, we counted rela- tively few fish on exposed rocks during the only rainy day of the field study. Al- though we found no fish in bays, sheltered inlets, or river mouths, Dr. Hugo Campos of the Universidad Austral (personal communication) reportedly collected a small young individual in the Rio Valdivia, several kilometers from the sea.

Adaptation to airbreathing

Airbreathing in the Chilean clingfish may have originated as a means to survive stagnation in isolated tidal pools. In general, the ability of fish to live in deoxygenated water by breathing air at the surface may constitute a preadapta- tion to living on land (Carter, 1957; Saxena, 1963; Johansen, 1968). Although airbreathing is rare in most groups of marine fishes, it is relatively common among species of gobies, blennies, and clingfishes that inhabit estuaries or the intertidal zone (Schottle. 1931; Oglialoro. 1947; Berlin. 1958; Saxena, 1963; Gordon, 1966; Eger, in preparation). And many of these species can live out of the water, escaping the intense competition and predation from the more abundant and diverse communities of subtidal predators.

130 ALFRED W. REELING, PATRICK) BERNAL AND ALEJANDRO ZULETA

The Chilean clingfish seems to have adapted behaviorally, morphologically, and physiologically to breathe air through its gills. In general, respiratory organs of amphibious teleosts include the skin, gills, mouth, pharynx, gut, and swim- bladder (e.g., Carter and Beadle, 1931; Carter, 1957; Krogh, 1959; Saxena, 1963; Johansen, Lenfant, Schmidt-Nielsen, and Petersen, 1968). The clingfish lacks a swimbladder and does not swallow air. Therefore, gills, skin, and buccopharyngeal epithelium are potential respiratory surfaces for emerged fish, which may consume oxygen more rapidly than submerged fish (Gordon, Fischer, and Tarifeno, in preparation). Although all three organs may contribute sub- stantially to aerial respiration, the branchial organs seemed to be the most specialized. The tightly shut gill cavities of emerged fish may hold a mixture of air and water and serve as a kind of lung, protected from drying, sealed with liquid, and provided with a large lamellar surface covered with water for oxygen absorp- tion. The branchial gas is not used for flotation because the fish, which lacks a swimbladder, expels the gas as it submerges and quickly dives to the bottom. The gas is not used as an oxygen store in stagnant water because it is always expelled. Vargas and Concha (1957a) observed that emerged fish with gills blocked by algin paste survive only about one-sixth as long as control fish.

Clingfish always expelled the spent gas through their small gill slits. Under optimal conditions, furthermore, the fish's head-down position on a vertical surface would facilitate release of gas upward through their watery branchial cavities. Most other airbreathing teleosts expel such gas through their gill slits rather than out their mouth (Todd and Ebeling, 1966; Johansen, 1966; Johansen, Lenfant, Schmidt-Nielsen, and Petersen, 1968).

Relatively few fishes breathe air through their gills, because gill lamellae tend to clump in air and because oxygen is usually absorbed through some epithelium aside from the gill membrane, where carbon dioxide is most easily eliminated (Carter, 1957; Krogh, 1959; Johansen, 1966). Of those that do, a freshwater knife fish of South America, Hypopotnus brevirostris, gulps air at the surface (Carter and Beadle, 1931), while an eel, Symbranchus marmoratus, often emerges from stagnant swamp waters and even hides in terrestrial burrows (Johansen, 1966). Among estuarine fishes, the mud skipper of Australia Pcriophthahnodon anstraHs reporterly has its gills modified for aerial respiration (cf. Berg and Steen, 1965) and Periophthalmus sobrinus in eastern Africa carries mixtures of air and water in its pharyngeal and gill cavities for aerial respiration and forcefully expels the spent gas in a spray of water from its gill slits (Stebbins and Kalk, 1961). Across the Mozambique Channel, however, the conspecific Nosy Be mud- skipper apparently does not have to do this in order to breathe on land (Gordon, Boetius, McCarthy, and Oglesby, 1969). Eger (in preparation) observed that a small amphibious clingfish of the Gulf of California, Tomacodon hinneralis, holds bubbles of air in its moist gill cavities when it comes out of the water. Rao and Hora (1938) concluded that the terrestrial blenny Andamia heteroptera breathes air held in its tightly shut gill cavities.

The gills of Chilean clingfish are structurally adapted for airbreathing, more so in adult than in young fish (Vargas and Concha, 1957a). Adult fish have fewer lamellae per millimeter of gill filament and have more widely spaced lamellae on the exposed distal half of the filament than do young fish, which seem generally

EMERSION OF AMPHIBIOUS CLINGFISH

less well-adapted to terrestriality. Gordon, Fischer, and Tarifefio (in preparation) noted that young fish do not survive enforced emersion as well as do adults, and the present study indicated that emergent young are much less composed than adult fish. In general, teleostean fishes that have proportionately smaller gill surfaces may survive better in air because their lamellae are more dispersed {e.g., Schottle, 1931; Gray, 1954). Indeed, all amphibious fishes that reportedly breathe air through their gills have lamellae that are widely spaced or otherwise structurally modified to prevent clumping and collapsing in air (Carter and Beadle, 1931; Schottle, 1931 ; Johansen, 1966).

Clingfish out of water in laboratory aquaria waited surprisingly long times between breaths, while most of the oxygen was being depleted from the air held in their closed gill cavities. The adult fish waited for almost one hour, about twice as long as the halfgrown fish and four times as long as the young fish. Al- though the two larger fish had used up almost 80% of their oxygen, the young fish, occasionally moving its opercles between breaths, had used only about 40%. Johansen (1966) observed that Symbranchus marmoratus gulps air at the surface of deoxygenated water at varying intervals of time, averaging about 15-20 minutes while it uses up about 50% of its branchial oxygen, but occasionally ex- tending to 30 or 40 minutes while it uses some 80% of its oxygen. The obligatory airbreathing electric eel Electrophorus electricus ascends at least once every two minutes (Johansen, Lenfant, Schmidt-Nielsen, and Petersen, 1968). Out of the water, the European eel Angidlla vulgaris inflates its gill cavities with air, which it regularly renews about once a minute at room temperature (Berg and Steen, 1966). Precht (1939) showed that cycles of airbreathing vary consider- ably in freshwater pulmonate snails, which may use up as much as 99% of their oxygen between breaths.

Emerged clingfish slow their heart rate and breathe at varying rates (Gordon, Fischer, and Tarifeiio, in preparation). Submerged birds and mammals show a general diving syndrome including bradycardia, which prevents asphyxia and shunts oxygenated blood under pressure to the vital organs (Scholander, 1940; Andersen, 1966). Obligatory water-breathing teleosts respond in a similar way when they are emerged (Leivestad, Andersen, and Scholander, 1957; Garey, 1962). The Australian mudskipper Periophthahnodon australis, on the other hand, is so well adapted to life out of water that its heart beat actually slows when it is submerged, as though aquatic life were completely foreign to this fish (Garey, 1962). Like the Chilean clingfish, however, a few other amphibious fishes show some tendency toward a "diving syndrome" in air, although usually less so than typical water-dwelling fishes (Berg and Steen, 1965; Todd, 1970). In the African mudskipper Periophthahnus sobrinns, the response is not detect- able (Gordon, Boetius, Evans, McCarthy, and Oglesby, 1969) and in the eel Symbranchns, the heart rate increases after an initial period of bradycardia. Perhaps handling of the experimental clingfish intensified their bradycardia and thereby slowed their oxygen consumption (cf., Kisch, 1950; Leivestad, Andersen, and Scholander, 1957), although emerged mudsuckers (Gillichthys} slow their heart rate markedly even with their brain removed (Todd, 1970), and mud- skippers actually increase their heart rate when disturbed (Gordon, Boetius, Evans, McCarthy, and Oglesby, 1969).

132 ALFRED W. EBELING, PATRICK) EERNAL AND ALEJANDRO ZULETA

The metabolic requirements of clingfish in air may differ from those of fish in water. They breathe sporadically in air, sometimes at a faster rate than they do in water, but apparently slow down or stop breathing altogether at respiratory plateaus (Gordon, Fischer, and Tariferio, in preparation) : young fish out of water in moist respiratory chambers at room temperature consume oxygen at rates of 50-110 ml/kg/hr after 1-3 hours out of water and at 120-180 ml/kg/hr after 11-13 hours out of water; 30-80 gram halfgrown and adult fish at 2-93 (avg. 32) ml/kg/hr during 0-12 hours out of water and 4-44 (avg. 18) ml/kg/hr during 15-23 hours out of water ; and aquatic young and adult fish more regularly at 32-50 (avg. 40) ml/kg/hr. Vargas and Concha (1957a) noted that the average rate of oxygen uptake at room temperature for a 120 g airbreathing adult fish, 46 ml/kg/hr, is low, compared with rates of many aquatic teleosts. It approxi- mates the standard rate of the European eel breathing air for four hours (Berg and Steen, 1965) and of the adult mudsucker (Barlow, 1961), but approaches only half the rate of the Nosy Be mudskipper (Gordon, Boetius, Evans, and Oglesby, 1968). Although the European eel decreases its rate of oxygen uptake after several hours in air (Berg and Steen, 1965), Symbranchus actually in- creases its oxygen uptake in air (Johansen, 1966). Todd (1970) inferred that the mudsucker Gillichthys in an anoxic atmosphere can decrease its metabolism to almost zero before it finally suffocates in comparative peace.

The branchial oxygen of clingfish may sustain their lowest rates of aerial oxygen consumption, but only 12-30% of their average rate, assuming no replace- ment of the gas between breaths. Gordon, Fischer, and Tarifeno (in prepara- tion), furthermore, questioned the need of fish to close their gill cavities when out of the water in their natural habitat, where they are regularly splashed by cold water. Emerged Symbranchus mannoratits keeps its single gill slit tightly closed for about 30 minutes, but then becomes agitated and opens its mouth to facilitate gas exchange (Johansen, 1966). When GillicJitliys inirabilis comes out of the water, it moves its slightly opened mouth as it holds a bubble of air in its buccopharynx (Todd, 1968). Perhaps clingfish are more active and aware in the field than they are in the laboratory, and so replenish their branchial air more frequently in the intertidal splash zone. But they may have difficulty exposing their small mouth, which is inferior in position and, therefore, is usually pressed against the rock surface.

Terrestrial clingfish may also breathe through their skin. Krogh (1904) demonstrated that European eels with gills blocked can consume about 60% of their normal aquatic oxygen requirement through their skin. Berg and Steen (1965) concluded that eels consume only about one third of their total oxygen through inflated gill cavities and the rest through their skin (augmented briefly by swimbladder oxygen). Terrestrial mudskippers breathe cutaneously as well as branchially, perhaps balancing pathways without changing overall rates (Gordon, Boetius, Evans, McCarthy, and Oglesby, 1969), and occasionally consuming some 60% of their total oxygen through their skin (Teal and Carey, 1967).

Clingfish probably breathe through their relatively delicate and vasculose "frontal skin," because the tough, thick, dark, and mucus-laden dorsal skin pro- tecting most of their body and fins appears unsuitable for oxygen absorption (at least in adults). Fish often raise their head to expose their frontal skin, which

EMERSION OF AMPHIBIOUS CLINGFISH 133

is shaded when they cling with head pointed downward. Other amphibious teleosts have potential respiratory epithelium located near the front of their body. Mahajan (1964) observed that the lips, barbels, and ventral adhesive pad of the Indian catfish Glyptothorax telchitta redden conspicuously as the emerged fish gasps air, which is forced out through its gill cavities. Vessels in the pectoral fins of airbreathing mudsuckers become noticeably engorged with blood (Todd, 1970).

Contact with air apparently stimulates clingfish to gulp. When they emerged snout first, they gulped air immediately, then closed their opercles, but when they emerged tail first, they continued their opercular movements so long as their head remained under water. Also, fish in stagnating water did not necessarily emerge and occasionally remained "panting" under water until they suffocated. Stebbins and Kalk (1961) observed that emerging mudskippers of eastern Africa quickly expand their gill cavities, then tightly close their small gill slits to secure air and water for branchial respiration on land, although Gordon, Boetius, Evans, and Oglesby (1968) found no water in the buccopharynx and gill cavities of emerging Nosy Be mudskippers, even though they made similar gulping move- ments. Johansen (1966) concluded that the most effective way to stimulate air- breathing in Synibranchus is simply to drain its aquarium, a much more effective stimulant than either enforced hypoxia or hypercarbia.

Unlike the Chilean clingfish, airbreathing fishes that inhabit stagnant waters of tropical swamps and coastal estuaries gulp air when they encounter varying degrees of aquatic hypoxia (e.g., Todd and Ebeling, 1966; Johansen, Lenfant, and Grigg, 1967; Johansen, 1968). Both aquatic and atmospheric hypoxia stimu- late the European eel to breathe air (Berg and Steen, 1965, 1966), although only atmospheric hypoxia so stimulates the obligatory airbreathing electric eel, which does not normally come out of the water (Johansen, Lenfant, Schmidt-Nielsen, and Petersen, 1968). Willmer (1934), however, inferred that environmental concentrations of both dissolved oxygen and carbon dioxide interact to control respiration in a freshwater characid of South America, Erythrinus erythrinus, which breathes air through its vasculose and physostomous swimbladder. Also, Precht (1939) showed that dual effects of hypoxia and hypercarbia are additive as they stimulate and control ventilation of freshwater pulmonate snails. Various vasomotor and pressure responses may control blood flow through the respiratory organs of airbreathing fishes (cf., Steen and Kruysse, 1964; Berg and Steen, 1966 ; Johansen, 1968; Todd, 1970).

Relatively large proportions of carbon dioxide in the gill cavities of airbreath- ing clingfish did not noticeably affect their ventilation or general behavior. Concentrations as large as 4 vol. % were measured in bubbles of gas expelled from fish forcefully submerged in laboratory aquaria. Nevertheless, young fish, whose expelled gas contained proportionately more carbon dioxide than that of adult fish, were more active and easily disturbed when out of the water. Also, as emerged adults dried off, they became distressed and finally opened their gill cavities, raised their head, and panted. They seldom opened their small mouth, however, when they raised their head to expose their frontal skin.

Terrestrial fishes that are subject to drying may not be able to eliminate access carbon dioxide from closed respiratory organs. Johansen (1966) pointed out that such fishes risk severe hypercarbia, because the normal pathway for eliminating

134 AT.KKKI) W. EBELING, PATRICIO BERNAL AND ALEJANDRO ZULETA

carbon dioxide through their gills is blocked. During enforced periods of emersion, in fact, Symbranchus marmorattts becomes agitated and opens its mouth after about 30 minutes. Airbreathing European eels, on the other hand, may eliminate most of their excess carbon dioxide through their moist and vasculose skin (Krogh, 1904). Gas containing more than 5 vol. % carbon dioxide passed directly over the gills of emerged eels inhibits the depth and frequency of their ventilation, although similar gas mixtures passed over their skin have no such effect (Berg and Steen, 1965). Constantly wetted by ocean spray, therefore, Chilean clingfish in the field should easily eliminate excess carbon dioxide through their skin, even while their gill cavities are tightly closed. The branchial organs of fish moistened in the laboratory showed an expectedly low respiratory quotient.

We thank Dr. Malcolm Gordon for stimulating and encouraging our investiga- tions and for critically reviewing the manuscript. The Convenio offices at the University of California at Los Angeles and at the Universidad de Chile at Santiago, supported by the Ford Foundation, assisted in many ways and defrayed all expenses. At the Estacion de Biologia Marina and the U. de Chile Laboratorio de Fisiologia Celular at Montemar, Professors Aguayo, W. Fischer, and S. Fischer were most kind and helpful. Sr. E. Tarifeno directed our use of specialized equip- ment. We especially thank Sr. J. Soto, majordomo of the laboratory, for main- taining our facilities, providing all experimental fish, and many other services. In southern Chile, Ebeling profited greatly from the advice and direction of Profes- sors V. Gallardo of the Universidad de Concepcion and H. Campos of the Universidad Austral in Valdivia. Sr. Castro Segundo of the Estacinn de Biologia Marina at Mehuin provided important natural history observations.

Mr. R. Ibara kindly assisted Ebeling with all computations and completed the factor analysis at the U.C.S.B. Computer Center during Ebeling's sabbatical leave provided by the University of California.

SUMMARY

The amphibious clingfish Sicyases sanguincus attaches by means of its ventral sucker to vertical surfaces of large exposed rocks splashed by the cool and heavy surf of Chile and southern Peru. Although adult and halfgrown fish tolerate diluted seawater and can survive at least a few hours in warm and stagnant water, they seldom, if ever, occur in isolated tidal pools, bays, or estuaries.

Factor analysis indicates that clingfish come out of the water more abundantly during periods of calm and often turn head-down. They avoid drying rocks outside the spray zone and emerge onto higher rocks as the water level rises. Adult fish come out of the water more abundantly in remote areas relatively undisturbed by civilization. Insolation apparently does not directly alter the abundance of clingfish, which act so as to minimize evaporative water loss and overheating.

Terrestrial fish breathe air held in their gill cavities, probably through their gills. As they come out of the water, fish gulp air, then stop all opercular move- ments to seal their cavities, and often turn head-down. This positioning may facilitate airbreathing by easing the expulsion of spent gas upward through the watery gill cavities and by shading an area of delicate respiratory epithelium under

EMERSION OF AMPHIBIOUS CLINGFISH 135

the chin. The volume percentage oxygen in gas expelled into a viscous solution of resin in sea water decreased regularly for about 12 minutes in a young fish, about 30 minutes in a half grown fish, and about one hour in an adult before these fish renewed their branchial gas. Although a concomitant increase in percentage carbon dioxide indicated that the branchial gas contributed to respiration, rates of oxygen uptake calculated from modal volumes of expelled gas were only about 12-30% of the fish's total long-term rate in air as determined by other investiga- tors. As in other airbreathing fishes, however, cutaneous respiration may supple- ment branchial respiration, which fills the total need only when the metabolic rate falls.

Exposure of their head apparently stimulates clingfish to gulp, so that atmo- spheric air, rather than aquatic hypoxia, is their primary stimulation to air- breathing. Fish in stagnating water do not necessarily emerge and occasionally remain under water until they suffocate. Like other airbreathing fishes, clingfish appear insensitive to relatively large proportions of carbon dioxide in their branchial gas. And even with their opercles closed and their ventral mouths pressed against the substrate, clingfish in the field should easily eliminate excess carbon dioxide through their wet skin.

Vargas and Concha (1957a) emphasized the ontogenesis of terrestriality in clingfish from erratic young to well-regulated adult : modification of gill surface to minimize clumping, control of aerial oxygen uptake, and greater composure on land. The present study indicates that exposure of an anteroventral respiratory mem- brane, assumption of a head-down position, improvement of gulping technique, control and slowing of ventilation, and greater efficiency of exchange between branchial gas and blood also contribute critically to the maturing fish's increasing independence of aquatic life.

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THE EVALUATION OF THE "CALLIPHORA TEST" AS AN ASSAY FOR ECDYSONE1

G. FRAENKEL AND JAN ZDAREK 2

Department of Entomology, University of Illinois, Urbana, Illinois 61801

Thirty-five years ago one of us (Fraenkel, 1935) showed that abdomens of a fly larva, Calliphora crythrocephala, which had been isolated by ligation before a critical period and normally would never pupariate, could be induced to tan by injection of hemolymph from pupariating larvae. This experiment was con- firmed on Calliphora (Becker and Plagge, 1939) and Didacus ciliatus (Narayanan and Lai, 1954). It subsequently became the method, now commonly known as the "Calliphora test," by which ecdysone was isolated, and with little modification is still the only practical method for identifying and assaying ecdysone. The validity of the original experiment was recently cast in doubt by Ohtaki, Milkman and Williams (1968) who failed to obtain pupariation by the injection of hemolymph in a member of a different family, Sarcophaga peregrina. They also used for their assays animals prepared in an entirely different manner, and on the basis of their data considered the 1935 experiment an erroneous observation, based on the ap- pearance of "false positives." They stated that the entire larva at the critical period contained only 7% of a Sarcophaga unit of ecdysone, and quoted estimates of the ecdysone titre in C. erythroccphala by Shaaya and Karlson (1965) in support of this contention. A more recent analysis of the distribution of ecdysone in different tissues of C. erythroccphala by Shaaya (1969) was also in agreement with these views.

We have now reproduced the original results in C. erythroccphala and a related species, Phormia regina, as well as in Sarcophaga argyrostoma. Further probing into the conditions under which tanning occurs during puparium formation of flies led to the discovery of a neurohormonal effect which accelerates the onset of tanning (Zdarek and Fraenkel, 1969). This observation suggested that under natural conditions, the amount of ecdysone required for tanning might be smaller than what had hitherto been determined by the standard test as a Calliphora (or Musca or Sarcophaga} unit.

These considerations further led to an analysis of the various factors which affect the outcome of the traditional Calliphora test in four different species of flies : Calliphora erythrocephala, Phormia regina, Sarcophaga biillata, and S. argyrostoma.

Some preliminary remarks about the terminology concerning the Calliphora test for ecdysone are in place. Not only has the test been, and will be increasingly used with other fly species, such as S. peregrina (Oktaki, Milkman and Williams, 1967), 5". bullata, S. argyrostoma, and P. regina (this paper), Musca domestica

study was supported by NSF grant GB 5441X and NIH grants AI-00533 and S-K06-GM-18,49S.

2 On leave from the Institute of Entomology, Czechoslovak Academy of Science, Prague.

138

"CALLIPHORA TEST" FOR ECDYSONE 139

(Kaplanis, Tabor, Thompson, Robbins and Shortino, 1966; Adelung and Karlson, 1969), but several steroids of the general structure of ecdysone have been em- ployed. We shall refer to the test as the "pupariation test for ecdysone," speak of ecdysone (s) when referring to the whole group of compounds, and use the terms a-ecdysone and /3-ecdysone (rather than the synonyma ecdysterone, crustecdysone, 20-hydroxyecdysone) when referring to a particular compound. Instead of using "Calliphora unit" as a general term we shall refer to the particular fly species to which a particular determination applies, or use the general term "pupariation unit of ecdysone."

MATERIALS AND METHODS Species of flics used

Specimens of Calliphora erythrocephala Meigen, and Phormia regina L. (both Calliphoridae), Sarcophaga bullata Parker and 5". argyrostoma Robineau-Desvoidy (Sarcophagidae) were used.

Breeding techniques

In much of the work reported below we used a modification of the culture tech- nique which was suggested to us by Dr. W. L. Downes, Michigan State University. The feeding fly larvae with their food (pork liver) are loosely wrapped in a sheet of aluminum foil and placed on top of a layer of sawdust. Larvae which have finished feeding leave the food and descend into the sawdust. By removing larvae from the sawdust at regular intervals, groups of larvae are obtained of much more homogeneous physiological age than when keeping the whole batch together.

Ligation

In the standard tests larvae were ligated at a time when about 10-20% had already pupariated. Ligatures, using cotton thread, were placed well behind the ganglionic mass, approximately behind the 5th visible segment, at a distance of about one-third of the length of the larva counting from the front.

Injection

Hind sections of ligated larvae were injected by means of finely drawn glass pipettes with a diameter of about 40 p. at the tip, manufactured with the aid of a Micropipette Puller Ml (Industrial Science Associates, Inc., Ridgewood, New York). Pressure was applied by mouth via a piece of rubber tubing fixed to the pipette. Amounts injected were estimated by comparison with a calibrated length of the same tubing. Since the hind parts of larvae ligated in this position are paralyzed, no further immobilization is required. Solutions of ecdysone in distilled water were injected in volumes of 5 /*! or less per test abdomen.

Scoring

The degree of tanning produced in response to a particular injection was esti- mated by a method similar to that developed by the Karlson group (Karlson and

140 G. FRAENKEL AND JAN ZDAREK

Shaaya, 1964; Adelung and Karlson, 1969) and others. These authors score complete, marked, half, and no pupariation as 100%, 75%, 50% and 0%, with a 25% score added in the latest paper. We use the same principle, with the differ- ence, that all partial pupariation was scored as 50%. Our procedure yields essen- tially the same percentage figures of pupariation as that obtained by other authors. The ecdysone used throughout was /J-ecdysone (obtained from Syntex Corp., Palo Alto, California). The reason for using ft- rather than a-ecdysone were: (1) Its availability. (2) Demonstration that this form is the one present in fly larvae (Galbraith, Horn, Thompson, Neufeld and Hackney, 1969). (3) Demonstration that /3-ecdysone is more active in Sarcophaga peregrina than a-ecdysone (Ohtaki, Milkman and Williams, 1967).

EXPERIMENTS

I. The effect of Hgation on pupariation in the front and hind parts of the larvae of four different fly species

When a mature fly larva, with the crop empty, is ligated into two parts behind its ganglionic mass the anterior or posterior parts may or may not pupariate. It is generally known that if the ligature is made before a "critical period" only the anterior parts pupariate whereas the posterior parts remain untanned unless sup- plied with molting hormone (ecdysone). If ligated after the critical period, both sections tan, though the anterior part does so about two hours before the posterior part (Zdarek and Fraenkel, 1969). One would expect the anterior part to pupariate under all circumstances, since it contains the ring gland, the source of ecdysone. This is. however, not the case. The ligature seems to affect pupariation in the front part in various ways. Even when ligated after the critical period (when the hind part tans) the anterior part may fail to pupariate (for reasons never satisfactorily explained). This we consider an inhibition. We must expect similar incidents of inhibition also to occur in precritically ligated larvae. Ligation can also cause a delay in pupariation of the front part.

The foregoing remarks apply to all cyclorrhaphous flies, there are however, as will be presently shown, important specific differences in detail which have to be considered when drawing conclusions from one species to another. To this end we have timed the occurrence of pupariation in the front and hind parts of C. erythrocephala, P. regina, Sarcophaga bullata and *$". argyrostoma after Hgation and in relation to pupariation of unligated controls. The larvae were ligated when about 10% had already pupariated.

The reason why we have selected the 48 hours mark for discussing the results, and why the observations were not continued for much longer was the growing incidence of mortality after this period, and the increasing difficulty of discerning between anterior parts which are either dead, dying, or else no longer able to pupariate. The results are recorded in Figure 1, A-D.

In all four species the hind parts which pupariate do so about two hours after the front parts. (Cases where the posterior parts alone pupariated are excluded). No pupariation occurred in the hind parts later than 15 hours after ligaturing in Calliphora and Phormia, and later than 20 hours in the Sarcophaga species. The occurrence of "false positives" after a waiting period of 24 hours has not been

"CALLIPHORA TEST" FOR ECDYSONE

141

100

50

100

A

Calliphora erythrocephala

10 20 30 40 50

Hours

i

••

-5 so -

20 30 40 50

100

50

0 '

• _-•

Sarcophago argyrostoma

10

20 30

Hours

40

50

100

50

Sarcophogo bulloto

10

20 30

Hours

40

50

FIGURE 1. These graphs depict the course of pupariation in four species of flies in

unligated controls A - - A, the front parts O O and hind parts • - - • of ligated

mature larvae. The top line D D represents mortality. A. Calliphora erythrocephala.

B. Phormia regina. C. Sarcophaga argyrostoma. D. Sarcophaga bitllata.

recorded. This eliminates the need for a further waiting period. Pupariation in the anterior parts in relation to that in the non-ligated controls does not seem to be delayed to any significant degree during the first 5 to 10 hours after ligating, but such a delay becomes very considerable in precritically ligated specimens, and is more marked in P. regina and S. bullata, than C. erythrocephala and 5*. argyrostoma.

In addition to this delay there is also an inhibition whereby many anterior parts fail to pupariate altogether. The incidence of this inhibition was very different in the different species. The proportion of precritically ligated larvae which by

142

G. FRAENKEL AND JAN ZDAREK

48 hours after ligating had pupariated in the anterior part was 12% in S. bullata, 23% in P. regina, 59% in C. erythrocephala and 71% in Sarcophaga argyrostoma. The total percentage of larvae which had not pupariated in the front part after 48 hours was 65% for 6\ bullata, 57% for P. regina, 23% for C. erythrocephala, and 22% for 5*. argyrostoma. These figures are not the final results because more pupariation occurred at a later date. The count was terminated at this date for reasons stated above. Despite this uncertainty, the different trend in the different species is very clear.

100

50

I

0

Call/ phora erythrocephala

0005 001

005 O.I

/3-ecdysone

FIGURE 2. Calliphora erythrocephala. The effect of the injection of graded doses of /3-ecdysone on pupariation in test abdomens from larvae where the anterior part alone had pupariated within 24 hours, between 24 and 48 hours, and between 48 and 72 hours after ligation.

It is interesting that in 5. bullata and P. regina, where the delaying and in- hibitory effect on pupariation in the precritically ligated larvae is stronger than in C. erythrocephala and .S. argyrostoma, ligation after the critical period gave rise to many more specimens where the hind part alone pupariated (S. bullata 16%, P. regina, 45%, S. argyrostoma 4%, and C. erythrocephala 3%).

Since the first prerequisite for a successful ecdysone test is as high a yield as possible of specimens which pupariate in the anterior part alone within the first 24 hours after ligating, S. bullata and P. regina are far less suitable test subjects than C. erythrocephala and 6". argyrostoma.

II. Variability of the pupariation unit of ecdysone

Karlson and Hanser (1953), Karlson and Shaaya (1964), and Adelung and Karlson (1969) have previously shown that the amount of ecdysone necessary to induce pupariation in the ligatured hind part is very much dependent on the

"CALLIPHORA TEST" FOR ECDYSONE

143

physiological age of the preparation at the time of injection. This age effect should be considered from two different perspectives. One is the presumed age of the larva in relation to pupariation which, in view of the large individual variation in any one batch can only be guessed from the time of pupariation in the controls, and from the period between ligation and pupariation in the front part. Since, as seen in Figure 1, pupariation in the normal controls was about completed after 24 hours, we may assume that all anterior parts which are tanned by that time had done so at more or less their appropriate time. All pupariation subsequently is in- creasingly delayed. The second consideration concerns the time interval between pupariation in the front part and the actual injection.

TABLE I

Calliphora erythnxvphala, effect of delay of injection of ecdysone on pupariation of lest abdomens . fi-Ecdysone -was injected at different periods into test abdomens of larvae in which the anterior part had pupariated within 24 hours after ligaturing. Each horizontal line represents tests performed simultaneously on larvae of the same batch, n == number of test abdomens per test, % '•= score of pupariation.

	Score of pupariation in test abdomens injected at the following periods after ligaturing
^-Ecdysone per test abdomen (/ig)	24 hours	48 hours	72 hours
	n	%	n	%	n	%
0.006	12	42	11	32
	12	41	10	30
0.012	11	82	11	64
	11	45	10	40
0.025	11	86	11	64
	12	54	10	50
0.05	9	94	8	87
0.1	11	82	12	63	9	50
0.2	11	95	12	87	10	60

1. Injection into test abdomens of different physiological ages. Mature larvae of Calliphora ery throe ephala were ligated, and the larvae which subsequently pupariated in only the front section divided into three groups according to the interval between ligation and pupariation: (1) \Yithin 24 hours; (2) Between 24 and 48 hours; (3) Between 48 and 72 hours after ligation. Group 1 was injected 24 hours, group 2, 48 hours, group 3, 72 hours after ligation. Responses to injection of each particular dose of ecdysone were estimated on the basis of three assays in group 1 and 2, and 2 assays in group 3, comprising 10 to 15 specimens at each assay. It was necessary to make several replications for each dose to draw the straight lines of Figure 2, since response to a particular dose varied consider- ably when larvae from different batches were used. This, by itself, shows that the bioassay is not very well reproducible.

The results (Fig. 2) show that test abdomens of group 1 were greatly more sensitive than of group 2, with those of group 3 still less sensitive. The value of the Calliphora unit, as defined above, was 0.008, 0.035, and 0.07 /^g /?-ecdysone per test abdomen for groups 1. 2, and 3, respectively.

144

G. 'FRAENKEL AND JAN ZDAREK

2. Injection into test abdomens of the same physiological age after different "waiting periods. The posterior parts of larvae whose anterior part alone had pupariated up to 24 hours after ligating were injected with graded doses of /3- ecdysone at 24, 48, and 72 hours after ligating. The result (Table I) shows a lower sensitivity of test abdomens to injected /3-ecdysone when the test abdomens had been further "aged." The differences between the 24 and 48 hours injections are significant at the 0.004 level (two tailed), by the Wilcoxon paired-sample rank test.

3. The dilution effect. In the pupariation test for ecdysone various authors injected the hormone solutions in portions of 10 (Calliphora, Sarcophaga) or 5 /xl (Musca) (see Discussion). This amount of solvent represents a far from neg- ligible addition to the blood volume of the test abdomens. In order to examine the effect of such a dilution on pupariation /3-ecdysone was injected into test abdomens of C. crytJirocephala in volumes of 1 and 10 jA water. Table II shows that the pupariation score was consistently reduced with the higher volumes of injection.

TABLE II

Calliphora erythrocephala, effect of the volume of injection on the pupariation score of test ab- domens injected with ecdysone. Injections were made 24 hours or 48 hours after ligation (the anterior parts had pupariated before 24 hours or between 24 and 48 hours after ligation, respectively).

"Age" of test abdomens hrs	/9-Ecdysone (jig) per test abdomen	Score of pupariation
Injected in 1 /il	Injected in 10 ^1
n	%	n	%
24 24 48	0.01 0.01 0.02	15 12 12	60 58 37	15 12 12	40 33 18

4. The effect of a brain hormone on flic pupariation unit of ecdysone. In a preceding paper (Zdarek and Fraenkel, 1969) we had shown that the addition of an extract from neurosecretory cells in the brain not only accelerated the onset of pupariation but also increased the sensitivity of the test abdomens to injected ecdy- sone in Phormia rcgina. We have now investigated this phenomenon in greater detail in C. erythrocephala.

Precritically ligated larvae were divided into three groups according to the time when the anterior parts alone had pupariated : A. within 24 hours, B. between 24 and 48 hours, C. between 48 and 72 hours after ligation. The hind parts were injected with graded doses of /3-ecdysone dissolved in water, and with a homogenate of CNS of .V. bullata larvae from which the ring glands had previously been removed (Table III). Test abdomens of group A required less ecdysone for a given pupariation effect when also injected with the CNS extract. In applying the Wilcoxon paired-sample rank test, the percentages given in Table 3A were recalculated to the first place of decimals to resolve the ties between the first and fifth experiment. The differences in pupariation score in the presence or absence of CNS extracts were significant at the 0.04 level, two-tailed. Pupariation

"CALLIPHORA TEST" FOR ECDYSONE

145

TABLE III

Calliphora erythrocephala, effect of graded dose of 0-ecdysone on pupariation of lest abdomens ligated at different physiological ages, in the presence or absence of homogenates from brains of Sarco- phaga bullata.

Injected per test abdomen	Pupariation score in absence of brain homogenate	Pupariation score in presence of brain hormone	Difference of percentage
vol. Ml	(3-ecdysone Mg	n	%	brain equivalent*	n	%

A. Anterior parts pupariated within 24 hours after ligation; injected 24 hours after ligation.

5	0.004	10	35	1.5	13	27	-8
5	0.008	17	32	1.5	16	47	+ 15
10	0.01	9	17	2.0	10	55	+ 38
5	0.016	16	59	1.5	16	66	+ 7
10	0.02	14	46	2.0	14	54	+8
5	0.032	16	81	1.5	16	91	+ 10
10	0.04	14	61	2.0	14	79	+ 18

B. Anterior parts pupariated 24 to 48 hours after ligation; injected 48 hours after ligation.

5	0.004	11	27	1.5	17	6	-21
5	0.008	12	29	1.5	17	3	-26
5	0.016	11	54	1.5	17	9	-45
5	0.032	16	84	1.5	15	57	-27
10	0.05	12	21	2.0	11	45	+ 24
10	0.1	12	58	2.0	11	72	+ 14
7	0.05	12	58	1.5	12	42	-16
7	0.1	12	67	1.5	12	71	+4
7	0.2	12	100	1.5	12	75	-25

C. Anterior parts pupariated 48 to 72 hours after ligation; injected 72 hours after ligation.

10 10

0.1 0.2

11 11

36 68

2.0 2.0

11 11

18 32

-18 -36

* X umber of brains per abdomen.

was also considerably accelerated in the presence of the CNS extracts. The larvae of group B and C which had been ligated at a younger age did not show either of these effects. Those of group C seemed to show an opposite effect of decrease of ecdysone action in the presence of the neurohormorie.

III. Pupariation induced in test abdomens oj different fly species by the injection of hemolymph

In order to test abdomens at their most sensitive state (see above) most injec- tions were made 24 hours or earlier after ligaturing. Twenty-four hours after the injection the number and degree of pupariation, as well as the number of dead specimens, were recorded. The surviving untanned specimens which ap-

146

G. "FRAENKEL AND JAN ZDAREK

peared in a sufficiently healthy state were then injected a second time and again scored after 24 hours.

The results are shown in Table IV. The final column, giving the total ratio, sums up the results of the first and second injection with the percentages calculated from the number of survivors after the first injection.

TABLE IV

Puparium formation in test abdomens of larvae of Callipliora erythrocephala, Phormia regina, Sarcophaga bullata and S. argyrostoma as a result of injecting them with hemolymph from different donors at different times after the critical period.

Test abdomens, species, time of injection after ligation	Hemolymph injected	Pupariation 24 hrs after	Total pupariation score
amount test abdomen	Donor	1st injection	2nd injection*
n	%	n	%	n	%
C. erythrocephala
24hrs	10 Ml	C. erytlir., white puparia	26	35	14	11	26	40
24hrs	10 p\	C. erythr., feeding larvae	38	3	32	6	38	8
24 hrs	10 MI	.S'. bullata, white puparia	17	0	8	0	17	0
24 hrs	10 jul	S. argyr., white puparia	15	37	5	20	15	43
48 hrs	10 Ml	C. erythr., white puparia	35	0	27	20	35	16
48 hrs	10 n\	C. erythr., feeding larvae	27	0	22	0	27	0
48 hrs	10 rf	S. bullata, white puparia	25	0	22	0	25	0
P. regina
16 hrs	5 n\	P. regina, white puparia	24	12	16	16	24	23
16 hrs	5 n\	P. regina, feeding larvae	15	0	14	0	15	0
12 hrs	8-10 Ml	P. regina, white puparia	23	22	16	25	23	39
12 hrs	8-10 Ml	Ringer sol.	10	10	9	0	10	10
S. bullata
24 hrs	10-15 /il	.V. bullata, white puparia	30	2	26	2	30	3
24 hrs	10-15 /il	.S'. bullata, feeding larvae	24	6	19	0	24	6
24 hrs	15 n\	C. erythr., white puparia	23	4	22	2	23	7
24 hrs	15 rf	C. erythr., feeding larvae	22	0	22	0	22	0
5. argystoma
24 hrs	15 Ml	.S'. argyr., white puparia	32	2	31	2	32	3
24 hrs	25 /xl	.V. argyr., white puparia	16	28	11	14	16	37
24 hrs	15 n\	C. erythr., white puparia	15	0	• — •	—	—	—
24 hrs	25 Ml	C. erythr., white puparia	20	40	9	22	20	45
24 hrs	10 /xl	.V. argyr., concentrate**	15	17	—	• —	• —	• —
24 hrs	25 Ml	.V. argyr., concentrate**	12	67	—	• —	—	—
24 hrs	25 M]	Ringer sol.	18	6	—	—	• —	- —
24 hrs	—	nothing injected	26	4	•			~~

*Second injection into surviving larvae, 24 hours after first. ** Filtrate from heat-coagulated blood from white puparia concentrated 5 times.

There were conspicuous differences between the four species. Calliphora, Phormia, and S. argyrostoma yielded pupariation scores around 40%, figures similar to those previously reported, but only when tested under the most favorable circumstances. It was essential to control the time and dosage of injection. Calliphora responded when injected 24 hours, but not 48 hours after ligation. S. argyrostoma responded well to injection of 25 /xl of its own hemolymph, but not

"CALLIPHORA TEST" FOR ECDYSONE 147

at all to that of 15 /A. It responded equally well to 25 /xl but not at all to 15 //I of Calliphora hemolymph. Reversely, Calliphora responded to 10 /tl of hemolymph from S. argyrostonia, but not to that from S. brtllata. This would suggest that the activity of the hemolymph of 5\ argyrostonia is higher than that of S. bullata. S. bullata showed no response to 10-15 /*! of its own hemolymph or that of Calliphora. No further tests were undertaken with this species in view of the difficulty of obtaining suitable test abdomens (see above) .

Active hemolymph was taken throughout from white puparia which, according to Shaaya and Karlson (1965) and Shaaya (1969), are at the peak of ecdysone activity. Hemolymph from younger, still feeding larvae (i.e., at least a day before the critical period) was in all cases inactive, thus proving the specificity of the test.

In view of the larger size of Sarcophaga it is not surprising that a larger volume of injected hemolymph is required for pupariation. An attempt was made of preparing concentrates of hemolymph for the purpose of increasing its ecdysone content. Hemolymph was diluted with 3 parts of water, heated at 90° for 10 minutes, centrifuged, and the supernatent concentrated on the vacuum evaporator at 70° C to one-fifth of the original concentration. Ten microliters of this concen- trate evoked a weak but clear tanning response, while 25 p\ led to 67% pupariation. A comparison of the effects of 25 //.I of the original blood and 10 /xl of the con- centrates shows that some activity was lost in the course of preparation of the concentrate.

DISCUSSION

It has recently been several times reported that the ecdysone titre in the hemolymph of larvae of cyclorrhaphous flies prior and during pupariation is far lower than the amount of exogenous hormone necessary to cause pupariation in the pupariation test for ecdysone (Shaaya and Karlson, 1965; Ohtaki, Milkman and Williams, 1968; Shaaya, 1969). Yet, we (this paper) and others (Fraenkel, 1935; Becker and Plagge, 1939; Narayanan and Lai, 1954) have shown that the hemo- lymph of pupariating larvae is capable to induce tanning in the pre-critically ligated abdomens. In order to solve this paradoxical situation we analyzed the pupariation test in great detail and revealed a number of factors which can affect the outcome of the traditional test.

1. Age of the larvae at the time of ligation

We (Fig. 2), as similarly before Karlson and Hanser (1953), Karlson and Shaaya (1964), and Adelung and Karlson (1969). have shown that test abdomens become greatly more sensitive to ecdysone nearer the time of pupariation.

2. Waiting period between ligation and infection

The test abdomen loses in responsiveness if injection of ecdysone is delayed (Table I). In Ohtaki, Milkman and Williams (1968) standard test abdomens a waiting period of 40 hours increased the Sarcophaga unit from slightly over 0.01 to about 0.035 /xg. Loss of sensitivity by post-ligation aging was also recorded by Karlson and Hanser (1953). Waiting periods of 24 hours or longer were routinely employed by all previous authors.

148 G. 'FRAENKEL AND JAN ZUAREK

3. A dilution effect

When ecdysone is injected into test abdomens of Calliphora (Adelung and Karlson, 1969) or Sarcophaga (Ohtaki, Milkman and Williams, 1967) in portions of 10 /xl of an aqueous solution, or dissolved in 10% isopropanol, respectively, or of 5 ju,l in Alnsca (Adelung and Karlson, 1969), the hemolymph of the receivers becomes appreciably diluted (the blood volume of a Calliphora test abdomen is 20 to 25 /xl). The effect which this overall change in concentration (not only of ecdysone) may have on tanning does not seem to have been considered. It clearly leads to an overestimate of the pupariation unit of ecdysone (Table II). Such effects are also noticeable in the data of Table III.

4. The effect of a neurohormone

Our previous data (Zdarek and Fraenkel, 1969) from P. regina had suggested that a humoral factor from the brain which accelerated the onset of pupariation also increased the sensitivity of the test abdomens to injected ecdysone. The same effect has now been shown in C. erythrocephala, but only in relatively mature test abdomens (Table III). A similar "synergistic" effect of a combined action of a brain hormone and ecdysone, both prepared from Bomby.v, in the pupariation of C. erytJirocephaJa had been discovered by Kobayashi and Burdette (1961). Thus, the absence of the neurohormone, as is the case in the standard test for ecdysone, may increase the requirements for ecdysone.

In all our experiments where injection of hemolymph induced pupariation, test abdomens of the highest possible sensitivity were employed, i.e. ligated as closely as possible before the critical period, and injected without a further waiting period with the highest possible amount of hemolymph. Thus, in the light of the various factors enumerated above — age, dilution effect, presence of neuro- hormone— the true Calliphora unit of ecdysone under the most favorable conditions may be substantially lower than the figure now given for the synthetic hormone (0.01 /xg/test abdomen). Furthermore, the hemolymph of pupariating larvae con- tains, besides the humoral factors, a potent substrate-enzyme system, which may not be without a significance for the result of the test. Our results with inter- specific transfusion of hemolymph confirm not only the non-specificity of its activity (Becker and Plagge, 1939), but also suggest that this activity can vary in different species.

Ohtaki, Milkman and Williams (1968) in explanation of their data with Sarcophaga peregrina suggest a mechanism by which ecdysone acted by a gradual accumulation of covert effects in response to subthreshold doses, rather than by massive accumulation in the hemolymph. In a current investigation on S. argwos- totna, to be published shortly, we have obtained direct experimental evidence in support of this contention, namely, that the effects of both exogenous and endo- genous ecdysone can and do summate over a period of time. In the light of these findings it may well be that the amount of hormone contained in injected hemolymph which induces tanning in test abdomens is only the last quantity neces- sary to bring about the qualitative change — tanning — in test abdomens already pre- conditioned (sensitized) by subthreshold doses of the hormone. Then the final effect is the results of two factors, sensitivity of the test abdomen and activity of the hemolymph.

"CALLIPHORA TEST" FOR ECDYSONE 149

In considering the significance of the pupariation test for ecdysone we must realize that the Calliphora (Sarcophaga, Musca} unit of ecdysone can only refer to the particular and peculiar conditions of the test abdomens, and may bear little relation to the real hormone titre in the normal larva at the time of pupariation which is also affected by the rates of inactivation (Ohtaki, Milkman and Williams, 1968; Karlson and Bode, 1969; Shaaya, 1969) and continuous release of ecdysone.

We thank our colleagues, Drs. J. Willis and G. Bhaskaran for reading critically the manuscript. Dr. A. Ghent for the statistical treatment, and Dr. J. B. Siddall of Syntex Research, Palo Alto, for the supply of ^-ecdysone.

SUMMARY

1. The effect of ligation on pupariation in the front or hind parts of larvae of four species of flies, Calliphora ery throe ephala, PJwrmia regina, Sarcophaga bitllata, and S. argyrostoma was investigated. Ligation causes effects of delay or inhibition of pupariation which are very differently expressed in the four species. A large proportion of pre- or postcritically ligated specimens of P. regina and S. buJlata altogether fail to pnpariate in the anterior part. This makes these species unsuitable test subjects for the pupariation test for ecdysone.

2. Test abdomens of C. erythrocephala required significantly less ecdysone for a given pupariation effect when also injected with a CNS-extract. Tanning was also considerably accelerated in this case.

3. The value of the pupariation unit of ecdysone is influenced by a number of factors, such as age at the time of ligation, the waiting period between ligation and injection, the dilution effect of the solvent, and the simultaneous action of a neurohormone. The requirements for natural ecdysone in normal larvae at the time of pupariation are probablv substantially lower than the values which have been determined by others with test abdomens and the use of synthetic ecdysones.

4. In confirmation of older data, and contrary to recent claims, tanning was induced in test abdomens of the larvae of C. erythrocephala, P. regina, and 5". argyrostoma by the injection of hemolymph from pupariating larvae. Calliphora blood induced tanning in specimens of S. argyrostoma, and vice versa. The con- clusions are drawn that differences between the different species in the action of ecdysone are of a quantitative rather than qualitative nature.

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biologischen Auswertung des Insektenhormones Ecdyson. 7. Insect Physlol., 15:

1301-1307. BECKER, E., AND E. PLAGGE, 1939. tJber das die Pupariumbildung auslosende Hormon der

Fliegen. Biol. Zbl, 59 : 326-341. FRAENKEL, G., 1935. A hormone causing pupation in the blowfly, Calliphora erythrocephala.

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150 G. 'FRAENKEL AND JAN ZDAREK

KAPLANIS, J. N., L. A. TABOR, M. I. THOMPSON, W. E. ROBBINS AND T. I. SHORTINO, 1966.

Assay for ecdysone (moulting hormone) activity using the house fly, Musca domestica

L. Steroids, 8: 625-631. KARLSON, P., AND C. BODE, 1969. Die Inaktivierung des Ecdysons bei der Schmeissfliege

Calliphora erythrocephala Meigen. /. Insect Physiol., 15 : 111-118. KARLSON, P., AND G. HANSER, 1953. Bildungsort und Erfolgorgan des Puparisierungs-

hormones der Fliegen. Z. Naturforsch., 86 : 91-96. KARLSON, P., AND S. SHAAYA, 1964. Der Ecdysontiter wahrend der Insektenentwicklung.

1. Eine Methode zur Bestimmung des Ecdysonsgehaltes. /. Insect Physiol., 110:

797-804. KOBAYASHI, M., AND W. J. BuRDETTE, 1961. Effect of brain hormone from Bombyx mori

on metamorphosis of Calliphora erythrocephala. Proc. Soc. E.vp. Biol. Mcd., 107 :

240-242. NARAYANAN, E. S., AND R. LAI, 1954. Controlling mechanism of pupation in Didacus

ciliatus (Loew.) (Diptera). Indian J. Entorn., 16 : 176-188. OHTAKI, T., R. D. MILKMAN AND C. M. WILLIAMS, 1967. Ecdysone and ecdysone

analogues : Their assay on the flesh fly Sarcophaga percgrina. Proc. Nat. Acad. Sci.,

58 : 981-984. OHTAKI, T., R. D. MILKMAN AND C. M. WILLIAMS, 1968. Dynamics of ecdysone secretion

and action in the flesh fly Sarcophaga peregrina. Biol. Bull., 135 : 322-334. SHAAYA, E., 1969. Der Ecdysontiter wahrend der Insektenentwicklung, VI. Untersuchungen

iiber die Verteilung in verschiedenen Geweben von Calliphora erythrocephala und

uber seine biologische Halbwertszeit. Z. Naturforsch., 24b : 718-721. SHAAYA, E., AND P. KARLSON, 1965. Der Ecdysontiter wahrend der Insektenentwicklung. II.

Die postembryonale Entwicklung der Schmeissfliege Calliphora erythrocephala Meig.

/. Insect Physiol., 11 : 65-69. ZDAREK, J., AND G. FRAENKEL, 1969. Correlated effects of ecdysone and neurosecretion in

puparium formation (pupariation) of flies. Proc. Nat. Acad. Sci., 64: 565-572.

Reference : Biol. Bull., 139: 151-163. (August, 1970)

CALCIFICATION IN ECHINODERMS: EFFECTS OF TEMPERATURE

AND DIAMOX ON INCORPORATION OF CALCIUM-45

IN VITRO BY REGENERATING SPINES OF

STRONGYLOCENTROTUS PURPURATUS ^

BARRY M. HEATFIELD 2 Department of Zoology, University of California, Los Angeles, California 90024

Studies of the calcareous endoskeleton of echinoderms have been reviewed by Hyman (1955), Raup (1966), Swan (1966), Nicol (1967), and Nichols and Currey (1968). Recent work on the echinoderm endoskeleton has been carried out by numerous investigators including Ebert (1967, 1968), Towe (1967), Travis, Francois, Bonar, and Glimcher (1967), Donnay and Pawson (1969), Kobayashi and Taki (1969), Markel and Titschack (1969), Nissen (1969), Pilkington (1969), Weber (1969), and Weber, Greer, Voight, White, and Roy (1969). To date, emphasis has been placed on various aspects of skeleton composition, morphology, crystallography, growth and regeneration. However, no quantitative studies have been reported on the physiology of skeleton formation in echinoderms (see Robertson, 1941 ; Nichols, 1964; and Nicol, 1967).

Due to the absence of quantitative studies on calcification in echinoderms, calcified tissues of the sea urchin, Strongylocentrotiis purpuratus (Stimpson), were investigated with the objective of developing a method to study calcification rates under different conditions. Gradually, attention was focused on the spines. Preliminary experiments showed that intact spines incorporate variable and rela- tively low quantities of calcium-45. But, when spines were broken, rapid uptake of the label occurred as part of the process of skeleton regeneration. This obser- vation suggested that regenerating spines might be useful as a calcifying system, and subsequent experiments confirmed this possibility.

Reported here are the results of experiments establishing regenerating spines of 5\ purpuratus as a useful tool to study calcification rates in vitro under differ- ent conditions with calcium-45 as a tracer. Using this calcifying system, the effects of temperature and a carbonic anhydrase inhibitor, Diamox (acetazolamide), on calcification rates are measured quantitatively.

MATERIALS AND METHODS Animals

Adult specimens of 6\ purpuratus, ranging in wret weight from 40 to 70 grams, were collected subtidally at Flat Rock Point, Los Angeles County, California,

1 This work was supported by a National Aeronautics and Space Administration Pre- doctoral Traineeship and represents a portion of a doctoral dissertation submitted to the Department of Zoology, University of California, Los Angeles, California.

2 Present address : Department of Zoology, Duke University, Durham, North Carolina 27706.

151

152 BARRY M. HEATFIELD

and maintained in the laboratory under constant room lighting in filtered, recircu- lating sea water at 14° C. Several species of brown algae were supplied as food.

Experimental procedures

Aboral, primary interambulacral spines of similar size were fractured with scissors 2 to 3 millimeters above the milled ring. As a result of this procedure, the stubs were protected from damage during regeneration by the surrounding forest of intact spines about 10 millimeters longer. Usually, 5 to 8 spines were fractured on each of the 10 plate rows of the interambulacra, which yielded from 50 to 80 stubs per urchin depending on animal size.

Fractured spines were incubated in vivo or in vitro. In the latter case, the stubs were removed from the urchin by severing the tissue attaching the base to the underlying tubercle. Incubations were carried out under constant room lighting at 15° C in covered, plastic dishes with a capacity of 500 or 1000 milliliters (Stoway utility dish, Southern California Plastic Co., Glendale). Calcium-45 with a specfic activity of 19.9 mc/mg was obtained from New England Nuclear, Boston, Massachusetts. In all experiments, aeration of the incubation medium was achieved by means of water-saturated air bubbled through an air stone. In some of the experiments, stubs were incubated in vitro simultaneously with stubs in vivo, but in separate compartments within the incubation chamber to prevent damage to them by the activities of the animal. The compartments were constructed from halves of plastic, histological coverslip boxes fastened to the inside of the chamber with methylene chloride just below the water level. To estimate 45Ca incorporated into the skeleton by exchange, control spines pre- viously bleached for several hours in 5.25% NaOCl (commercial strength Purex or Clorox) were fractured as above and added to the incubation medium in some of the experiments. Further details of each experiment are given in the Results section.

After incubation, stubs were sampled, bleached in Purex for 2 to 3 hours or longer to remove soft tissue and unincorporated 45Ca, rinsed several times in distilled water adjusted to pH 7.0, and oven dried at 110° C on filter paper. Be- tween rinsings, stubs were drained briefly on filtered paper. Care was taken to protect the delicate regenerated mineral from damage during handling. Dried stubs were mounted in modeling clay and stored until the activity of 45Ca was determined.

Assay of 45Ca activity

Individual stubs were fixed vertically with modeling clay on planchets. The tip of the regenerated portion of the stub was then positioned 1 mm below the center of a thin end-window Geiger-Miiller tube (LND Corp., Oceanside, New York, #733 T) with the aid of an adjustable platform which permitted reproducible counting geometry. Activity was determined with a transistorized sealer (Nuclear Supplies Inc., Encino, California, Model SA-250). After incubation in ^'Ca for several days, some radioactivity was detectable on the periphery of the spine shaft in addition to that of the regenerated tip. Radioactivity on the shaft was reduced to background levels during counting by slipping a rubber disc with a small hole in the middle over the spine tip to a position just below the level of fracture,

CALCIFICATION IN SEA URCHIN SPINES

153

care being taken not to touch the regenerated skeleton. The disc, of the same diameter as the planchet, was cut from the cuff of a rubber autopsy glove and pierced with a hot pin. Spines of different diameter were shielded by discs with holes of appropriate size. Each spine was counted for three minutes and the activity expressed as counts per minute per spine. In some of the experiments,

7

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I 4

O

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O

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0

A B C

0

Time After Fracture (days)

FIGURE 1. Kinetics of 45Ca incorporation by regenerating spines of five specimens of S. pitrpnratits (A-E) incubated in vivo for five days following experimental fracture. Each point represents the mean value of 3 or 5 spines, with vertical bars indicating the range of all values. Control values are means of 3 bleached spines.

activity of 45Ca in the incubation medium and coelomic fluid was determined in triplicate, 50 microliter aliquots which were dried on planchets and counted for three minutes. This activity was expressed as counts per minute per milliliter. All counts were corrected for background.

In experiments on the effects of temperature and Diamox on calcification rates, activity was determined independently by liquid scintillation techniques using the sample preparation method of Carr and Parsons (1962), adapted here to the assay

154

BARRY M. HEATFIELD

of spines labeled with 4r'Ca. Scintillation counting eliminated error due to self absorption. Each stub was inverted over a liquid scintillation vial and the tip snipped off with scissors about 1 millimeter below the level of experimental frac- ture. This procedure insured complete recovery of all labeled mineral deposited on the fractured surface during regeneration. In some of the stubs employed in experiments on the effect of temperature on calcification rates, the second milli- meter of the shaft was also removed and placed in a separate vial to estimate the activity of 40Ca in the first millimeter which was included with the regenerated mineral as a consequence of the sampling procedure. Two milliliters of 0.5 TV HC1 were added to each vial to dissolve the calcareous fragments. The vials were then gradually heated to about 130° C to remove water and HC1. After cooling, 6 milliliters of a mixture of toluene and ethanol (5:1), and 5 g/L of 2,5-diphenyl- oxazole (PPO), were added to each vial. Activity of 45Ca was determined at room temperature in a Beckman liquid scintillation counter, Model LS-233. Counts

Time After Fracture (days)

FIGURE 2. Kinetics of 15Ca incorporation by regenerating spines of a single 5". fnrpit incubated in vivo or in vitro for five days following experimental fracture. Each point represents the mean value of 5 spines, with vertical bars (left, in vivo; right, in vitro} indicating ± S.D.

CALCIFICATION IN SEA URCHIN SPINES

155

were taken for five minutes, corrected for background, and expressed as counts per minute per spine. Error due to measurement of radioactivity was usually less than ±5%.

RESULTS

Comparison of calcification rates in vivo and in vitro

To determine when calcification was initiated during regeneration in vivo, five urchins with fractured spines were incubated individually in about 350 milliliters of sea water containing 1.0 /xc/ml of 45Ca. At daily intervals for five days, 3 or 5 stubs were removed from each animal and assayed for radioactivity.

Figure 1 shows that there was a lag period of about two days following fracture before 45Ca was incorporated. During this time, the amount of label incorporated by regenerating spines was not significantly different from that incorporated in

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• In vivo o In vitro

24

Time (hours)

FIGURE 3. Kinetics of ^Ca incorporation by spines of three specimens of S. purpuratus (A-C) incubated for 24 hours in vivo or in vitro after regeneration for four days in vivo in plain sea water following fracture. Each point represents the mean value of 10 spines, with vertical bars (left, in vivo ; right, in vitro) indicating ± S.D.

156 BARRY M. IIKATl'IKU)

bleached controls by exchange. Thereafter, uptake of 45Ca by regenerating spines increased rapidly, with exchange accounting for only about \% of the total activity incorporated four to five days after fracture.

During the first 24 hours' incubation in vivo there was a relatively rapid dis- appearance of 45Ca activity from the incubation medium followed by a more gradual, nearly linear rate of removal. At the end of the incubation period, nearly 20% of the initial radioactivity was removed. In another experiment, the dis- appearance of radioactivity from the medium during the first 24 hours' incuba- tion was concomitant with a rapid appearance of radioactivity in the coelomic fluid, which reached equilibrium with the medium after 30 to 36 hours' incubation.

To determine whether calcification of regenerating spines is initiated in vitro, fifty spines were fractured on a single urchin. Twenty-five stubs were then removed (explanted) and incubated in vitro simultaneously with the remainder /';/ vivo in about 700 milliliters of sea water containing 1.0 /Ac/ml of 45Ca. Five stubs were sampled daily from each group for five days and assayed for radio- activity.

Figure 2 shows that explanted stubs incorporated 45Ca during regeneration in vitro in a manner similar to that of stubs in vivo. These explants also showed a two-day lag period during which relatively little 45Ca was incorporated. There- after, a rapid increase in radioactivity was observed similar to that shown in Figure 1. A two-day lag period before rapid incorporation of 45Ca was also observed in additional experiments carried out for up to five days with stubs incubated only in vitro.

To avoid the two-day lag period so that short term experiments could be conducted while calcification was in progress, spines were fractured on three urchins and allowed to regenerate in vivo for four days in plain sea water. One half of the number of stubs was then explanted from each urchin and incubated in vitro simultaneously with the remainder in vivo in 300 to 350 milliliters of sea water containing 1.0 //.c/ml of 45Ca. Ten stubs were sampled from both groups from each urchin at intervals up to 24 hours and assaved for radioactivity.

Figure 3 shows that 45Ca incorporation by calcifying stubs over 24 hours was directly proportional to the length of incubation with no statistically significant difference between stubs incubated in vivo and those in vitro. Under conditions of the experiment, 45Ca activity was detected in both groups after incubation for as little as one hour.

The results of these experiments indicate that, after regeneration for four days in vivo following fracture, explanted stubs could be used alone in further studies on calcification with 45Ca as a tracer. The use of explants is advantageous since removal of 45Ca from the medium due to uptake by the urchin is avoided. In addi- tion, each urchin provides numerous regenerating stubs which can be presumed to be similar genetically and physiologically. Thus, it should be possible to conduct controlled experiments in vitro to evaluate quantitatively the effects of various parameters on calcification rates. In the present investigation, two parameters, (1) temperature, and (2) Diamox (acetazolamide), an inhibitor of the enzyme, carbonic anhydrase, were selected for study.

CALCIFICATION IN SEA URCHIN SPINES 157

Effect of temperature on calcification rates in vitro

To determine the influence of temperature on calcification rates of regenerating spines in vitro, five dishes containing 200 milliliters of sea water and 1.5 /zc/ml of 45Ca were allowed to equilibrate for 24 hours at temperatures of 4.7°, 9.7°, 15°, 20°, and 26° C. Fifty stubs were explanted from each of four urchins after regeneration for four days in vivo in plain sea water following fracture. Ten stubs from each group of fifty were placed in each of the five dishes giving forty stubs per dish. The four groups of ten stubs in each dish were isolated from

20

16

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E

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o I c-

;> 8

a 4

o

r>

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0

A

B

C

15

25

15

25

25

Temperature (°C)

FIGURE 4. Incorporation of 45Ca by spines of three specimens of 5". purpuratus (A-C) incubated in vitro for 24 hours at various temperatures after regeneration for four days in vivo in plain sea water following fracture. Each point represents the mean value of 10 spines, with vertical bars indicating ± S.D. Data for a 4th urchin are similar to those shown, except that only a slight increase in ^Ca incorporation occurred between 15° and 20° C.

one another by pieces of glass rod of -J inch diameter placed on the bottom of each dish. Temperatures varied by 1° C or less during the experiment. After incu- bation for 24 hours, all stubs from each animal were assayed for radioactivity by liquid scintillation.

Figure 4 shows that incorporation of 45Ca by explants was directly proportional to temperature between 4.7° and 20° C, at which a maximum occurred. At 26° C, little incorporation of the label took place, and tissue on all stubs appeared to be partially decomposed at the end of the experiment. Mean values for the tem- perature coefficient (Qio), and energy of activation (E) calculated over the intervals 4.7° to 9.7°, 9.7° to 15°, and 15° to 20° C, are 5.50, 2.08, 1.73; and 26,052, 11,491, and 8662 calories per mole, respectively, with overall means be- tween 4.7° and 20° C, of 2.72, and 15,504 calories per mole, respectively, for the four urchins studied. Activity of 45Ca measured in the second millimeter of the shaft of those stubs assayed was considered negligible.

158

HARRY VI. HEATFIELD

Effect of Diamox on calcification rates in vitro

In living systems, carbonate ion (CO3=) can be obtained via bicarbonate ion (HCO3~), which is formed by the hydration or hydroxylation of CC>2 (see Wilbur, 1964), although it is not known with certainty which of these two mechanisms is operative at physiological pH (Maren, 1967). The enzyme, carbonic anhydrase, has been shown to catalyze the conversion of COa to HCOs~ (Meldrum and Roughton, 1933). If the rate of deposition of CaCO3 is dependent upon the enzyme-catalyzed conversion of COo to HCOs", then the application of an appropriate inhibitor of carbonic anhydrase should depress the rate of calcification.

To test this hypothesis in calcifying sea urchin spines in vitro, a specific inhibitor of carbonic anhydrase, 2-acetylamino- 1,3,4, thiadiazole-5-sulfonamide (Miller, Des- sert, and Roblin, 1950), or acetazolamide, was obtained as the sodium salt, Diamox, from the Lederle Laboratories of the American Cyanamid Company,

TABLE I

The effect of various concentrations of a carbonic anhydrase inhibitor, Diamox (acetazolamide), on the incorporation- of calcium-45 by spines of S. purpuratus incubated in vitro for 24 hours after regeneration for four days in vivo in plain sea water following fracture. Shown are mean values in counts per minute ± S.D., for the number of spines in brackets. Probability values equal 0.01 or less (see text for statistical procedures).

Animal	Control	Counts per minute
10-6 u Diamox	10-5 I/ Diamox	10-i .!/ Diamox	10-3 ,i/ Diamox
1 2 3	9596 ± 2404 (10) 9000 ± 3102 (9) 9952 ± 1036 (10)	3316 ± 968 (10) 3928 ± 1008 (10) 3764 ± 920 (10)	4364 ± 2372 (10) 3544 db 1392 (10) 3784 ± 880 (10)	5080 ±1604 (10) 5060 ± 1472 (10) 3968 ± 540 (10)	4908 ± 2052 (10) 4280 ± 1656 (7) 5112 ± 1344 (10)
Mean % inhibition ± S.E.	61 ± 5	58 ±4	50 ±9	53 ± 8

Pearl River, New York. A sea water solution of 4 X 10"" ! M Diamox was ad- justed to pH 8.0 and added to plain sea water in four plastic dishes giving final concentrations of 10~3, 10-*, 10~5, and 10~6 M. A fifth dish without added inhibitor served as the control. The final volume in all dishes was 250 milliliters, with 1.0 /xc/ml of 45Ca. Fifty stubs were explanted from each of three urchins after regeneration for four days in vivo in plain sea water following fracture. Ten stubs from each group of fifty were placed in each of the five dishes giving thirty stubs per dish. The three groups of ten stubs in each dish were isolated from one another by pieces of glass rod as described earlier. Incubations were carried out at 15° C for 24 hours. All stubs from each animal were then assayed for radioactivity by liquid scintillation.

Table I shows that there was a statistically significant reduction in 45Ca incorporation by explants treated with the inhibitor compared to the untreated controls. Probability values of 0.01 or less were obtained using the "t" test (Simpson, Roe, and Lewontin, 1960, page 176). The data show a fairly consistent effect of Diamox over a wide range of concentrations with a maximum mean inhibition of 61% at a concentration of 10~6 M.

CALCIFICATION IN SEA URCHIN SPINES 159

DISCUSSION

The results of kinetic studies reported here show that regenerating spines of the sea urchn, 6\ purpurahts, incorporate 45Ca in vitro in a manner similar to that of spines from the same animal incubated simultaneously in vivo. The data demonstrate a lag period of about two days following experimental fracture before calcification is initiated. The lag period cannot be attributed to the time required for uptake of 45Ca by the urchin, since a similar lag was also shown to occur during regeneration of fractured spines in vitro. The results of histological and histochemical studies of fractured spines during regeneration in vivo and in vitro indicate that the lag more likely reflects the time required for wound healing and reorganization of tissue at the site of fracture (Heatfield, unpublished). Following the lag period, a rapid incorporation of 45Ca takes place in vivo and in vitro. The incorporation of 45Ca following the lag period parallels the appearance and growth of new mineral in the form of "micro-spines" on the fractured surface of the spine shaft as observed with the light and scanning elec- tron microscopes (Heatfield, 1969). In calcifying stubs which were prepared by allowing fractured spines to regenerate for four days in vivo in plain sea water, incorporation of 45Ca in vitro was shown to be equivalent to that in vivo during incubation for 24 hours. The nearly linear rate of incorporation of the label during this period indicates that equilibrium between the tissues of regenerating spines and the incubation medium takes place rapidly (less than one hour at the spine tip).

Using explanted, fractured spines as a calcifying system after regeneration for four days in vivo, temperature was shown to have a marked effect on calcification rates in vitro. Incorporation of 45Ca was directly proportional to temperature between 4.7° and 20° C, at which a maximum occurred. Little incorporation of the label took place at 26° C, which appears to be a lethal temperature. The temperature of the ocean in the vicinity of the collecting site ranges from about 13° to 20° C (Boolootian, 1961). This range is within that in which incorporation of 45Ca was found to occur in the present work and indicates that temperature is not a limiting environmental factor in the regeneration process in spines of S. purpitratiis. The temperature coefficient CQio) and the energy of activation (E) varied inversely with increasing temperature betwen 4.7° and 20° C. Valus of E obtained in the present study fall within the general range (5000 to 25,000 calories per mole, and higher) listed by Sizer (1943) for a large number of enzyme-catalyzed reactions, and by Crozier (1924) for a variety of physiological phenomena. In the present work, the variation of E (/* of Crozier) with tem- perature suggests that several rate-limiting reactions may be involved in the calcification process with the one in effect at any particular moment dependent upon the ambient temperature (see Crozier, 1924).

Few quantitative studies have been carried out on the effect of temperature on mineral deposition in other calcifying systems. Malone and Dodd (1967) found that incorporation of 45Ca into whole shells of the bivalve mollusc, Mytilus eduHs, varied directly with temperature over the range, 5° to 23.5° C. Incorpora- tion due to exchange was not estimated and it was concluded that activity of 45Ca in shells incubated at the lower temperatures might not be due to skeleto- genesis. In the present study, incorporation of 45Ca at 4.7° C was about \ of the

160 liARKV M. HKATFIKLU

maximum observed at 20° C, indicating that significant deposition of mineral takes place in regenerating spines of >S". purpuralus even at relatively low tempera- tures. Porcella, Rixford, and Slater (1969) found that uptake of 45Ca by the fresli water crustacean, Daphnia magna, was temperature dependent, and obtained mean values for E of 15,000 calories per mole between temperatures of 10° and 25° C. In the study reported here, a similar mean value of E of 15,504 calories per mole was obtained between 4.7° and 20° C. In the reef coral, Pocillopora damicornis, Clausen (unpublished, cited in Lenhoff, Muscatine, and Davis, 1968) measured the effect of temperature on the incorporation of 45Ca, and found an exponential increase in radioactivity between 12° and 25° C, with a Qio of 12.7 or 6.7 depending on the length of incubation. Incorporation at 12° was about 1/13 that at 25° C. Above 25° C, a decline in calcification rate was observed. Values for E of 43,000 and 33,000 calories per mole were obtained by Clausen depending on the length of incubation. These values are about twice as high as those obtained in the present study and by Porcella ct al. (1969), and indicate that the rate of calcification in this species of corai is very sensitive to changes in temperature.

The enzyme, carbonic anhydrase, is generally believed to be important in the biological deposition of calcium carbonate as inferred from the results of experi- ments on the effect of carbonic anhydrase inhibitors on the rate of mineral deposition. The data reported here show a statistically significant reduction in the incorporation of 45Ca by regenerating spines of .S\ purpuratus incubated in vitro in the presence of a carbonic anhydrase inhibitor, Diamox (acetazolamide) . In- hibition of 50% to 61% was obtained over a concentration range of 10~3 to 10~6 M. From these results it is inferred that carbonic anhydrase plays a role in calcification of regenerating spines of 6". purpuratus though no attempt has yet been made to determine the presence of this enzyme in spine tissues. Inhibition of calcification has been observed in other organisms in which carbonic anhydrase activity was detected. Wilbur and Jodrey (1955) obtained up to 48% inhibition of 45Ca incorporation by the oyster, Crassostrea virg'nnca, in the presence of 4 ) 10~5 M Diamox. Costlow (1959) observed an inhibition of shell growth in the barnacle, Balamis improvises, after treatment with Diamox at concentrations ranging from 3.7 X 10~4 to 2.99 X 10~5 M. In four species of reef coral, Goreau (1959) found an inhibition of 45Ca incorporation of 51% to 80% in experiments conducted in the light with Diamox at a concentration of 10~ M. Isenberg, Lavine, and Weissfellner (1963) obtained essentially complete inhibition of min- eralization in the coccolithophorid, H \menoinonas sp. after exposure to 10~5 M Diamox, though the demonstration of carbonic anhydrase was not attempted in this organism. After injection of Diamox into the hen, Callus domesticus, Bern- stein, Nevalainen, Schraer, and Schraer (1968) measured a reduction in egg-shell weight of approximately 59%.

The results of these studies with Diamox, with the possible exception of Costlow (1959) and Isenberg et al. (1963), indicate that carbonic anhydrase is generally not indispensable to skeletogenesis, since deposition of mineral occurs, though at a reduced rate. Concentrations of Diamox which have a marked effect on calcification rates do not appear to be toxic (see Wilbur and Jodrey, 1955; Costlow, 1959; Goreau, 1959; Freeman, 1960; Isenberg et al. ' (1963); and Maren, 1967). In the presence of another inhibitor, 2-benzothiazolesulfonamide,

CALCIFICATION IN SEA URCHIN SPINES 161

Stolkowski (1948) observed an inhibition of growth in the larval skeleton of the sea urchins, Panic entrotus lividus and Arbacia aequituberculata. However, com- plete inhibition of mineral deposition was obtained with this drug in C. viryinica by Wilbur and Jodrey (1955), and in the fresh water snail, Physa heterostropha, by Freeman (1960), which led these workers to conclude that the action of 2-benzo- thiazolesulfonamide was not limited to carbonic anhydrase.

In calcifying systems where deposition of mineral proceeds at a slow rate and the enzyme-catalyzed hydration or hydroxylation of CO:, is not rate-limiting, then the application of inhibitors of carbonic anhydrase may not show an effect. Such a result was obtained by Freeman (1960) in studies of shell growth of P. heterostropha grown at different rates in the presence of Diamox.

It is a pleasure to acknowledge the enthusiasm, support, and guidance shown throughout this work by Dr. Leonard Muscatine of the Department of Zoology, University of California, Los Angeles. I would also like to thank Dr. Karl M. Wilbur of the Department of Zoology, Duke University, for critically reading the manuscript.

SUMMARY

1. Calcification during regeneration of experimentally fractured spines of the sea urchin, Strongyloc entrotus purpuratus (Stimpson), was studied quantitatively under different conditions with calcium-45 as a tracer.

2. Fractured spines rapidly incorporated 45Ca in vivo or in vitro after a lag period of about two days. The lag period is attributed to wound healing and reorganization of tissue at the site of fracture.

3. Additional experiments were conducted while calcification was in progress by allowing fractured spines to regenerate for four days in vivo followed by incubation in 45Ca in vivo or in vitro up to 24 hours. In these experiments in- corporation of the label was nearly linear with time and no significant difference was observed in the rate of uptake of 45Ca between regenerating spines incubated in vivo and those from the same urchin incubated simultaneously in vitro.

4. Incorporation of 45Ca in vitro was directly proportional to temperature between 4.7° and 20° C, at which a maximum occurred. A temperature of 26° C appeared to be lethal and little incorporation of 45Ca took place. Values of QJO and the energy of activation varied inversely with temperature, with overall means of 2.72 and 15,504 calories per mole, respectively, between 4.7° and 20° C.

5. Diamox (acetazolamide) at concentrations from 10~5 to 10~6 M, inhibited incorporation of 45Ca in vitro by 50% to 61%. It is inferred from these results that carbonic anhydrase is involved in calcification of regenerating spines of S. purpuratus.

LITERATURE CITED

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162 BARRY M. HEATFIELD

BOOLOOTIAN, R. A., 1961. Shoreline surface water-temperature data between Clallam Bay,

Washington, and Punta Banda, Baja California, for the year 1960. University of

California, Scripps Institution of Oceanography, data report: Surface ivatcr tempera- tures at shore stations, U. S. West Coast and Baja California, 1960, SIO reference

61-14, 28 April, 1961 : pp. 44-47. CARR, T. E. F., AND B. J. PARSONS, 1962. A method for the assay of calcium-45 by liquid

scintillation counting. Int. /. . I/1/1/. Radiut. Isotop., 13 : 57-62. CLAUSEN, C., unpublished data. Cited in LENHOFF, H. M., L. MUSCATINE AND L. V. DAVIS,

1968. Coelenterate biology: experimental research. Science, 160: 1141-1146. COSTLOW.JR., J. D., 1959. Effect of carbonic anhydrase inhibitors on shell development and

growth of Balanus improvisns Darwin. Physiol. Zool., 32 : 177-184. CROZIER, W. J., 1924. On biological oxidations as a function of temperature. /. Gen. Physiol.,

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to food availability and spine abrasion. Ecology, 49 : 1075-1091. FREEMAN, J. A., 1960. Influence of carbonic anhydrase inhibitors on shell growth of a

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CALCIFICATION IN SEA URCHIN SPINES 163

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Reference: Biol Bull., 139: 164-179. (August, WO)

A COMPARATIVE STUDY OF THE CEMENT GLANDS IN SOME BALANID BARNACLES (CIRRIPEDIA, BALANIDAE)1

DYRCE LACOMBE

Institnto de Pcsqitisas de Marinha, Rio de Janeiro, Brasil

The structure of the cement glands in Cirripedia was only briefly mentioned by Darwin (1854), Krohn (1859), Koehler (1889), Gravel (1905a, b) and a few others. Recently, beginning with Thomas (1944), several authors studied various types of epidermal glands in this group of animals (Boucquet-Vedrine and Ovechko, 1960; Utinomi, 1960; Walley, 1967), no one, however, dealt with the specialized glands responsible for the secretion of cement. A detailed histo- logical study by Lacombe (1966) on adult specimens of Balanus tintinnabulum called attention to the ectodermal origin of the cement glands. Subsequent histochemical investigations (Lacombe, 1967a) suggested that the intra- and extracellular secretion of the cement glands may be a type of acid mucopolysac- charide. Arvy and Lacombe (1968) and Arvy, Lacombe and Shimony (1968) utilizing histoenzymological techniques showed that the secretion spread in the cell and the extruded secretion within the canal system give a positive reaction for alkaline phosphatase, but the rest of the cement gland cell remains negative. A comparative study of the cement gland in B. tintinnabulum and Lepas anatifera (Lacombe and Liguori, 1969) suggested that the structure of the cement glands may be less complex in the primitive barnacles, such as the Lepalidae.

METHODS

The ovaria with the cement glands of adult specimens of B. nubilis and B. psittacns were dissected for immediate fixation; in the case of B. eburneus, B. amphitrite and B. balanoides the entire animal was fixed.

The following fixatives were employed : Flemming, Bouin, Susa, Carnoy, Helly, and Altmann's fluid, as well as 10% formaldehyde. The material was embedded in paraffin and sections were cut at 5, 7 and 10 /x.

The sections were stained with Delafield's hematoxylin and Chromotrop 2R, Ehrlich's hematoxylin and Orange G. G., Weigert's hematoxylin and Alcian blue at pH 2.3, Mallory's Azan method with Congo red, Trypan blue with Chromotrop 2R, Aniline blue and Nuclear fast red with Naphthol green. For fine details Heidenhain's iron alum hematoxylin was used, following fixation in Flemming's fluid.

OBSERVATIONS

Typically, as in B. tintinnabulum (Lacombe, 1966), the secretory cells which elaborate the cement occur in groups (B. psittacns, B. eburneus and B.

1 Translated from the Portuguese by Dr. Sophie Jakowska, Universidad Autonoma de Santo Domingo, Dominican Republic.

164

CEMENT GLANDS IN SOME BARNACLKS

amphrite) or in rosettes (B. nnbilis) intermingled with the ovarian follicles. In B. balanoidcs, on the other hand, they are scattered individually among the con- nective tissues in the basal portion of the animal. The number of cement gland cells appears relatively smaller in B. balanoidcs than in the other species examined.

transversal muscle barnacle body

muscle nauplius larvae

blastula morula

buccal plate cirrus

external mantle internal mantle

collector canal

connective tissue

ovariole

muscle fiber

circular canal

secondary canal

principal canal

cement gland

cement

FIGURE 1. Schematic drawing of a longitudinal section in Balanns sp., showing the internal anatomy and cement glands with canal system.

Figure 1 is a schematic drawing of the anatomy of the typical balanid barnacle in longitudinal section. The structures include the cement glands among the ovarian follicles, the canal system, through which the cement secretion is conveyed to the basal portion of the animal, and the flat epithelium of the mantle, shown in lateral view.

A. Secretory elements

In B. nitbilis the cement glands are found in the connective tissue near the ovarian region. In the external zone of the mantle the glands appear to be in the early stages of development (Fig. 2), and consist of small cells differentiating in the walls of the principal and secondary canals (Fig. 3). The young cement glands form groups of 15 to 30 small cells which have dense homogeneous cytoplasm and a central nucleus rich in chromatin.

During their development the gland cells become embedded deeper in the interior of the mantle-chamber and located in the connective tissue near the

166

DYRCE LACOMBE

group Of cement gland

group of secondary canals

-

als

- '

collector canal

&•••'

•

' J| sec ret or pole secretion

« • • . • >

young cement gland

»'- 5

connective tissue <*"-

old cement gland

FIGURE 2. Balanns nubilis. Longitudinal section through secondary canal with young cement glands in development, Mallory-Azan.

FIGURE 3. Transverse section through principal canals near the hilum region, Mallory- Azan.

FIGURES 4 to 7. Transverse section through the cement glands in different stages ; nuclear fast red and naphthol green.

CEMENT GLANDS IN SOME BARNACLES

167

ovarian follicles (Figs. 4 to 7). At this stage the glands begin to elaborate the cement secretion (Fig. 4). The secretion originates in one zone of the cell, the secretory pole, and accumulates in another zone, the storage pole ; the latter connects with the collector canal (Fig. 5) ; that means, from the point of view of genesis of this type of cells, that the apical part of the gland cell stores the secretion and the basal part produces it. Occasionally the cells of the canal walls develop clusters of secretory cells which appear as partial or complete rosettes in histological sections (Fig. 6).

nucleus

old cement gland

secretion

cytoplasm

young cement gland

v

mature cement gland

secondary canals

FIGURE 8. Cement glands in Balanns psittacus showing different stages of development.

Within such clusters the cement secretion is clearly visible. Young cells form- ing the clusters have regular, centrally located nuclei, each with two to four nucleoli. In mature cells, the nuclei are polymorphic, with many nucleoli in varied positions. Older cement gland cells have large and more irregular nuclei, and an even larger number of nucleoli ; the nuclei are very poor in chromatin indicating an increase of nucleoplasm.

168

DVRCE LACOMHK

cement gland

secretory pole

•:.* .•:*•"«>-' i

secondary canal'

- 4

'

.

^mmmm4 S^THrV

clntnicns. Intercellular cement secretion; nuclear fast red and Azan

FIGURE 9. B method.

FIGURE 10. Same gland in higher magnification.

FIGURE 11. Longitudinal section through the secondary and collector canals.

CEA1FNT GLANDS IN SOME BARNACLES 169

In B. psittat'iis the cement glands are much easier to locate than in B. nnbilis. In both species the epithelium nf the mantle occupies a large part of the animal's shell. In the walls of the principal and secondary canals the gland cells grow out- ward from the lumen. All stages of cell development and maturation may be easily seen (Fig. 8). The cytoplasm of the gland cells exhibits very small vacuoles which increase in size as the cells mature. These vacuoles, full of cement, are distributed throughout the cytoplasm. When stained with the Mai- lory's Azan method. or \\ith nuclear fast red stain and Aldan blue, the cement gland cells take on a uniform coloration. In B. psiHuciis. an accumulation of the cement at the storage pole, such as in B. nnbilis. was not observed. The nuclei oi the gland cells are large and more polymorphic, with 10-14 nucleoli irregularly distributed through the nucleoplasm. The nuclear chromatin is poor and dispersed. Each group of cement glands is composed of 8 to 1-1- secretory units or cells in different stages of development.

The gland in B. chunicns consists of a few. well-defined cells (Fig. 9). The young gland cells are located very close to the basal plate or the point at which the gland cells begin to differentiate from the canal walls. The mature gland cells are located in the connective tissue among the ovarian follicles. In this species the gland cells do not change in shape and form during the development. The cyto- plasm is dense, homogeneous and devoid of vacuoles. The nuclei are constant in form and exhibit very little polymorphism; chromatin is mostly concentrated around the nucleoli, which number two to four. The cement secretion is visible as granular particles scattered in the cytoplasm at the secretory pole of the cell (Fig. 10). At the storage pole the secretion granules are very dense and they leave the cell through a fine membrane, passing into the lumen of the collecting duct (Fig. 11). A similar condition was observed in the cement gland of B. fintiiiiialutliiiii (Lacombe, 19(>(H. At the storage pole of each gland cell of B. cbuniens. it is seen with the light microscope that the cytoplasm projects into the nucleus as shown in Fig. 10: this condition results in an increase of surface of contact between the secretion and the nuclear elements. In B. tintinnabulum, the electron microscope revealed that the endoplasmic reticulum has a different arrangement in this region of contact (Lacombe, 1967b). and suggested a rela- tionship between the nuclear elements and the cement secretion.

The cement glands of B. balauoides differ distinctly from those of all the other species described in this paper. In this species the glands (Fig. 12) are located in the basal portion of the animal near the region where the shell increases in size. They never appear as a distinct group of cells or as rosette shaped clusters. The glands consist of single isolated cells and the mature elements are particularly conspicuous in the connective tissue. Young cement glands are rarely seen in adult B. balanoidcs, perhaps due to the fact that the growth of the glands closely parallels that of the animal. Another distinctive feature of these gland cells is the abundance of cytoplasmic vacuoles containing cement secretion (Fig. 13), which gradually increase in size. When many vacuoles containing secretion accumulate, they move toward the cell membrane nearest to the collector canal. As it was observed in Lepas anatijcra (Lacombe and Liguori. 1969), B. balanoides exhibits more than one way of discharging cement secretion into the lumen of the collector canals, and this corresponds to the primitive condition. Figure 12 clearly shows two typical storage and discharge points in a cement gland cell.

170

DYRCE LACOMBE

. cytoplasm - jfR, mature cement gland

*«••'' /Set ^-|P$-"' -Y-Y -

*

• '"•

siEgu:

vacuoli

FIGURES 12 and 13. Biilanus balanoides. Cement ylands showing the vacuoles and the paths of cement extrusion; Erlich's liematoxylin and chromotrope 2K.

FIGURE 14. Cement glands, with typical nucleoli in the polymorphic nuclei ; Heidenhain's Iron liematoxylin.

CEMENT GLANDS IN SOME BARNACLES

171

The cement glands of B. balanoidcs exhibit considerable nuclear polymorphism such as seen in B. cluimcits. The number of nucleoli varies from four to four- teen and they are clearly defined (Fig. 14). The schematic drawing (Fig. 15) shows different parts of this type of cell.

In B. anipliitritc, the cement glands closely resemble those of B. eburneus in size, localization and form. The glands of B. amphitritc are located among the ovarian follicles (Fig. 16) and most frequently near the white muscles. The glands are composed of groups of 6 to 10 cells and are seldom represented by single isolated cells (Fig. 17). During all stages of development the gland cells

lumen of secondary canal

vacuoli with cement secretion

chromatin

nucleus

nucleolus

collector canal secretion

vacuoli

FIGURE 15. Cement gland of Balatnts balanoidcs, showing vacuoles rich in cement secretion, nucleoli of different sizes in the polymorphic nuclei.

are regular in form ; their cytoplasm is dense and contains small vacuoles near the cell membrane. Usually different stages of developing cells are found growing among the old gland cells (Fig. 18). This condition differs from that observed in B. balanoides. The occurrence of differences of cell development simplifies the observation of the cytology of the various cell phases. In young gland cells, the nuclei are round, poor in chromatin and have centrally placed nucleoli. In mature glands, however, the nuclei tend to become polymorphic. The cement secretion of B. cnupliitrite is similar to that of B. chiiniens, and both species show very distinct storage and secretory poles in the mature gland cells.

172

DYKTK LACOMHK

secondary canal , "

•'

16

-

.*."

; •

cerhent gland

s,

young cement gland

-

<

18

mature cement gland

FIGURES 16 and 17. Balanus amplntrite. Groups of cement gland cells with secondary canals, Delafield's hematoxylin and chromatrope 2R.

FIGURE 18. Different stages of development of cement glands; Erlich's hematoxylin with Congo red and orange G.G.

CEMENT GLANDS IX SOME BARNACLES

L73

B. Accessory canal system

The accessory canal svsu-m. which distributes the cement, \va.s previously studied in B. tintinnabulum (Lacomhe, 196<>> . It originates by progressive imagination of the hypodermal cells of the exterior mantle wall after the cypris larva becomes attached. Thus begins the formation of a complex system consisting of circular, radial and principal canals. The principal canals grow inward and ramifv ex- tensively, giving rise to numerous secondary canals. Subsequently, the chitin of the mantle epithelium >preads as a lining over the entire lumen of this canal -\stem. Some cells in the walls of the principal canals »r the secondary canals begin to differentiate into the specialized cement glands.

19

secretion

wall of principal canal

connective tissue

principal canal

ovariole

egg

muscle

cement gland

'

{t?/ secondary canal

young glands

FIGURE 19. Ovarian region in Balaiins intbilis showing the elastic fibers around the principal and secondary canals, with young, mature and old cement glands and oocytes in development.

The configuration of the canal system varies to some extent in the five species of balanids studied in this paper, but in general the pattern resembles that of B. tintinnabulum or that in the schematic drawing of the internal anatomy of a typical barnacle (Fig. 1 |. The secretion passes through the cell membrane from the vacuoles or from the storage pole into the lumen of the collecting canals, from where it moves on to the secondary and to the principal canals. The principal canals conduct the secretion to the radial and circular canals, from where it spreads out beneath the basal plate by typical outlets.

In B. •nubilis the secondary canals are narrow and many developmental stages of gland cells may be seen in their epithelium (Fig. 19). The principal canals adjoining the ovarian region have a large diameter and their epithelial cells tend to be larger. The connective tissue fibers are concentrated around the principal

174

DYRCE LACOMBE

20'

connective tissue

•.* ~ *

- principal canal j

Ba^lMW^* ~ •» <r.v 1

principal canal

connective tissue

hypoderm cell

cement gland

connective tissue * •>• •

V.

secondary canal

FIGURE 20. Baltiiius f>sitttii-ns. Principal canals, showing connective tissue fibers, hypo- dermal cells and chitin ; Mallory's Azan method with Congo red.

FIGURE 21. Same section as in Figure 20 showing the cement secretion in the lumen of the principal canal, same stain.

CEMENT GLANDS IN SOME BARNACLES 175

canals and apparently function as a protective covering (Fig. 19). The chitinous layer within the lumen of the principal canals is very difficult to observe, but it becomes evident and is clearly seen with polarized light. In this species, as well as in B. psittacns, a hilum appears in regions where the secondary canals join the principal canals.

The hilum is absent in B. cbnniens, B. ainpliitrite and B. balanoides. The principal canals are composed of very simple, flat epithelial cells (Fig. 11) with elongated nuclei : these are rich in chromatin, a fact which indicates that these nuclei are out of function, their nucleoplasm was reduced and their chromatin appears now more concentrated. There are no connective tissue fibers around the principal or secondary canals. The arrangement of the canal system is similar to that of B. tintinndbulum.

In B. psittacns the hilum is greatly accentuated and in this region all the cement gland cells show discrete secretion. The structure of the principal canals, indicated in Figures 20 to 23, resembles that of B. nnbilis in which the principal canals have a tortuous course (Fig. 20) and the secondary and the principal canals (Fig. 22 and 24) are surrounded by a large number of connective tissue fibers. The latter feature appears to be typical of the larger species of balanids, such as B. psittacns and B. nnbilis, and it is not observed in the others. In B. psittacns, the chitinous layer in the principal canals (Fig. 23) is clearly shown by simple staining with Congo red. The hypodermal cells in this species possess dense cytoplasm and a large, basally situated nucleus, poor in chromatin. Figure 25 shows a general histological view of the hilum region when the cement gland cells and canals are filled with secretion. The secretion in this species is clearly visualized along the entire canal system with simple histological stains. This is not as easily observed with such methods in the other balanids studied where only small granulations appear (Fig. 20-21), but histoenzymological methods permit to demonstrate the cement in those species.

C. Extrusion of the cement secretion

In the serial sections of B. ebnrnens. B. balanoides and B. amphitrite, stained with Heidenhain's iron alum hematoxylin, the basal muscle of the mantle is easily seen; Gutmann (1960) called attention to these muscles, but did not associate them with the cement glands. These striated muscle fibers extend from the chitinous covering of the external mantle epithelium on the base plate (Fig. 26), to the thin chitin of the inner mantle layer, which covers the body of the barnacle.

The muscle fibers pass through the connective tissue between the ovarian follicles and the cement glands (Fig. 27). These fibers are very distinct, with long tonofibrils in the base plate (Fig. 26) and very short tonofibrils at the mantle side. The striated muscles are closely associated with the cement glands and ovarian follicles as may be seen in cross section (Figs. 27 and 28). Figure 28

FIGURE 22. Principal canal with cement secretion, same stain.

FIGURE 23. Principal canals showing chitin and hypodermal layer and connective tissue transverse section, same stain.

FIGURE 24. Secondary canal near the hilum region, showing distinct connective tissue fibers, transverse section, same stain.

FIGURE 25. General view of the hilum region with cement glands in different stages and the arrangement of the canal system, same stain.

176

DYKCK r.ACOMBE

muscle fi±>ers

V * * a Jf&&

• " collector canal I

^ muscle fibers

30

FIGURE 26. Longitudinal section through the base plate of Balanus clnirncns showing the long tonofibrils and the muscles of the mantle, Heidenhain's iron liematoxylin.

FIGURE 27. The cement gland in Balanus bul<iii<_n<l,'s and muscular system of the mantle, same stain.

CEMENT GLANDS IX SOME BARNACLES 177

shows the cement glands of B. amphitritc. with the collector canal in cross section next to the storage pole. It might be supposed that this musculature (Figs. 29 and 30) is involved in some way in the movement of the cement secretion from the secondary and principal canals to the basal system. The contraction of these muscle fibers probably compresses the entire ovarian region, which includes the cement glands and the connective tissue, and may cause the cement to move towards the basal canal. Perhaps this contraction helps extrude the cement secretion from the canal system to the substrate in the absence of additional musculature in the walls of the canals. The basic anatomical relationships, seen in Figure 1, suggest the above interpretations.

DISCUSSION

The pattern that seems to emerge from the observations on the Balanids selected for study suggest that the degree of development and differentiation in the cement gland system may be related to the phylogenetic position of the species.

B. balanoides is the species that exhibits a relatively simple pattern: (1) single large secretory cells situated at the base of the animal, away from the ovarian follicles; (2) the secretion scattered throughout the cytoplasm and collected in vacuoles which extend into the collector canal : ( 3 ) the absence of elastic fibers around the canals.

B. anipJiitrite, B. cbiinicits and B. tintinnabnliun I the last was studied previously by Lacombe, 1966), represent what may be considered as a typical pattern for balanids : 1 ) large secretory cells, arranged in irregular groups, inter- mixed with the ovarian follicles; 2) the secretion formed at the secretory pole of the cells and extruded at the opposite pole into the collector conduct without the formation of vacuoles; 3) the absence of elastic fibers in the walls of the canal system.

In contrast to the above specie>. /!. psittacits and B. nul'ilis both possess elastic fibers around the canals, and in both, the cement glands are intermixed with the ovarian follicles. In B. psittacits, however, the secretory elements consist of irregular groups of small and large cells and in B. nnbilis. all the cells are small and are arranged in form of rosettes. The secretion is not confined to the vacuoles or to the storage pole, but appears scattered throughout the cytoplasm.

It may be too early to speculate on the significance of these differences observed in the balanids under study. It is generally recognized, however, that B. balanoides may represent a primitive form, while the large barnacles, B. psittacus and B. nnbilis may be considered as the more advanced forms of balanids. The morphological differences observed in the cement gland system in these species point to interpretations involving phylogenetic concepts. In addition, one is impressed by the fact that adult specimens of B. balanoidcs exhibit only fully formed cement gland elements, while in the other species one may observe within the same adult specimens the progressive stages of differentiation of these elements.

FIGURE 28. Cement gland and muscle fibers in Balaints aniphitritc, showing the striated muscles, the storage pole of the gland cell and collector canal in transverse section, same strain.

FIGURE 29. Longitudinal section through the mantle of B. chitnicns, showing the muscle fibers, same stain.

FIGURE 30. General view of the muscle system of the mantle of B. cl'iirucits, same stain.

178 DYRCE LACOMBE

This may suggest that the maturation of the animal and of the cement gland elements may he synchronized with the molting cycle in the more primitive, but not in the more advanced balanids.

B. clutnicits and B. tintinnabulum appear to be highly suitable for further demonstration, histochemicallv and ultrastructurally, of the relationship between the secretion granules and the nuclear and cytoplasmic constituents of the cement gland cell.

The role of the basal muscles, such as those seen in B. ehnrnciis, B. balanoides and B. ainphitrite. should be considered as an added mechanism for the distribu- tion of cement to the basal plate region.

This project was supported by a grant from the Office of Naval Research (ONR. N-00014) and conducted at the Osborn Laboratories of Marine Sciences of the New York Zoological Society. The author expresses her thanks to Dr. Ross F. Nigrelli and to Dr. Vincent Liguori for the use of laboratory facilities.

SUMMARY

1. The histological characteristics of the cement gland cells of barnacles have been compared in B. iiululis, B. psittacus, B. chnrnciis, B. balanoides and B. ainphitrite.

2. In B. balanoides. the cement gland cells show a very simple composition ; they are situated at the base of the animal and the secretion appears scattered throughout the cytoplasm. The cement apparatus of B. ainphitrite and B. ebnrnens looks like that of B. tintinnabulum, but in B. psittacus and B. nnbilis the cement gland cells appear more complex.

3. The extrusion of the cement secretion is brought about by muscle fibers that pass through the connective tissue, and in B. psittacns and B. nnbilis by elastic fibers around the secondary and principal canal systems.

LITERATURE CITED

ARVY, L., AND LACOMBE, D., 1968. Activites enzimatiques traceuses dans 1'appareil cementaire

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phosphatase activity histochemicallv detectable in the cement apparatus of the

Balanidae (Crustacea-Cirripedia). Atner. Zoo/., 8: 817. BOUCQUET-VEDRINE, J., AND N. OVECHKO, 1960. Contribution a 1'etude des glandes salivaires

de Chthamalus stcllatns. Bull. Soc. Linn. Normandie, 10: 106-118. DARWIN, C., 1854. A Monograph of the Sub-class Cirripcdia. The Balanidae. J'olitme 2.

Ray Society, London, 684 pp. GRUVEL, A., 1905a. fitudes anatomiques sur quelques Cirrhipedes Operculaires du Chile.

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KOEHLER, R., 1889. Recherches sur 1'organization des Cirripedes. Arch. Biol. 9: 311-402. KROHN, A., 1859. Beobachtungen iiber den Cementapparat und die weiblichen Zeugungsorgane

einiger Cirripedien. Arch. Xatitr</esc!i., 25: 355-364.

CEMENT GLANDS IN SOME BARNACLES 179

LACOMBE, D., 1966. Glandulas de cirnento e seus canais em Balanus tintinnabulum. In:

Publica^ao Institute de Pesquisas da Marinha, Nota Tecnica n° 32, pp. 1-39. Rio de

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tintinnabulum. In: Publicaqao n° 11 do Institute de Pesquisas da Marinha, pp. 1-29.

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Navais. In: Publicac,ao n" 017 do Institute de Pesquisas da Marinha, pp. 1-22.

Rio de Janeiro, Brasil. LACOMBE, D., AND V. LIGUORI, 1969. Comparative histological studies of the cement apparatus of

Lcpas anatifera and Balanus tintinnabuliis. Biol. Bull.. 137: 170-180. THOMAS, H., 1(>44. Tegumental glands in the Cirripedia Thoracica. Quart. J. Microscop.

Sci.,84: 257-281. UTINOMI, H., 1960. Studies on the Cirripedia Acrothoracica. 2. Internal anatomy of the

female of Berndtia purf>urca Vtinomi. Publ. Setor Mar. Biol. Lab., 8: 223-279. WALLEY, L. J., 1967. The citral glands : a new type of epidermal gland in Cirripedes.

Crustaceana, 12: 151-158.

Reference: Bint. Hull., 139 180-187. (August, 1('70)

OBSERVATIONS ON THREE SPECIES OF JELLYFISHES FROM

CHESAPEAKE BAY WITH SPECIAL REFERENCE TO THEIR

TOXINS. I. CHRYSAORA (DACTYLOMETRA)

QUINQUECIRRHA x

NOLAN E. RICE AND W. ALLAN POWELL I >< f-artment of Biology and Department of Chemistry, University of Richmond, Virginia 23173

Chrysaora (Dactylometra) quinquecirrha, the stinging nettle, has attracted the attention of vacationers in the Chesapeake Bay and other areas for many years because of the irritating and sometimes serious nature of its stings. At the time the present work was initiated there was no published information on the nature of the toxic agent of this species. Various terms had been applied earlier to the toxins of different Cnidaria but it is not certain that the extracted substances, e.g., hypnotoxin, congestin, thallasin, etc. were isolated from the nematocyst capsules or that they were pure substances. Halstead (1965) has reviewed most of the important literature on the poisonous Cnidaria up to 1965.

Lane and Dodge (1958) and Lane (1960) isolated the contents of the nematocyst capsules of Physalia physalis (Portuguese man-of-war) and deter- mined that these appeared to be protein in nature. Welsh (1961), page 180, states, "Much evidence indicates that the paralyzing edema-producing action of coelenterate toxins is due in large measure to a protein component (s)." Subsequently other investigators (Burnett, Stone, Pierce, Cargo, Layne and Sutton, 1968; Shapiro, 1968; Endean, Duchemin, McColm and Eraser, 1968; Crone and Keen, 1969) reach the same conclusion.

Burnett et al. (1968) isolated four types of nematocyst s from C. quinquecirrha and state, page 336, "Almost all the toxic activity was localized in sediments of chemically or physically ruptured nematocyst suspensions," and conclude "'that the toxin is membrane bound." However, Barnes (1967) collected the toxins of Chironex fleckcri and Chiropsalmus quadrigatus after discharge of nematocysts through a membrane (isolated human amnion) and found them to be a fluid. Endean ct al. ( 1968) obtained evidence that the toxin of C. fleckcri is intracapsular and indicate that the capsules themselves are non-toxic.

Many of the observations reported in the present paper have been made over the past six years but the work on toxins was begun in July, 1967. This project involved (1) the extraction and isolation of the nematocyst toxins of the stinging nettle (Chrysaora quinquecirrha), the clover leaf jelly (Anrelia aurita}, and the pink or lion's mane jellyfish (Cyanca capillata), all of which are common in the Chesapeake Bay and parts of its tributaries at certain seasons of the year; (2) toxicity experiments; and (3) the determination of the chemical nature of the toxins.

1 This investigation was supported by several Faculty Research Grants of the University of Richmond and a Virginia Academy of Science Research Grant.

180

TOXIN OF CHRYSAORA QUINQUECIRRHA 181

This paper reports on the observations and experiments on the summer nettle (C. quinquecirrha) . Research in progress with A. aurita and C. capillata will be published later.

MATERIALS AND METHODS

Nettles were collected during the summer months in the Rappahannock River and Chesapeake Bay (salinity approximately 1.5%) near Deltaville, Virginia. Only the tentacles and oral lobes were used. Usually six to eight gallons of this material were processed at one time.

The following procedure for the extraction of toxin is a modification of that used by T.ane and Dodge (1958) for Physalia. The Chrysaora material was allowed to autolyze in the refrigerator at 5° C fur 24 to 48 hours after which it was diluted with an equal volume of 1.5% NaCl solution. This was strained through Marquisette Nylon Netting (20 openings/inch) and then through Swiss silk bolting cloth (No. 12, 125 mesh). The screened suspension was permitted to settle at 5° C in the refrigerator for 24 hours, the supernatant decanted, and discarded. The residue, consisting of nematocysts and cellular debris, was centrifuged at 7000 rpm ("6000 g} for 15 minutes. The supernatant was discarded and an equal volume of 1.5% NaCl solution was added to the residue. The centrifuge tubes were gently shaken to loosen the cellular debris overlaying the nematocysts which firmly adhered to the bottoms of the tubes. This process was repeated several times. Each time the loosened debris was poured off. The residue was then thoroughly mixed with 1.5% NaCl solution, recentrifuged at 7000 rpm for 15 minutes, the supernatant decanted, and the above described process of washing repeated until a mass consisting almost entirely of undischarged nematocysts was secured. The whole process was continued until the supernatant was Biuret negative.

Initially 5 ml of nematocysts was mixed with 35 ml of 1.5% NaCl solution and homogenized in a Potter-El vej hem homogenizer to disrupt the nematocysts and release their contents. However, since this was laborious and time consuming, several other methods of rupturing the capsules were tried : osmotic pressure change through the use of distilled water and ultrasonic rupture. The procedure finally adopted was that of repeated homogenization of a frozen suspension of nematocysts in a Potter-El vej hem homogenizer. In this way 75% or more of the nematocyst capsules were ruptured or discharged, liberating the contents.

The homogenate was centrifuged in early experiments at 9000 rpm (10,000 g), in later ones at 18.500 rpm (40,000 g}, for 1 hour. Most of the strongly Biuret positive supernatant was carefully pipetted off and frozen until used in toxicity and electrophoresis experiments. The residue, consisting of undischarged nemato- cysts, large amounts of capsular debris, and tubes was repeatedly washed in 1.5% XaCl solution and repeatedly centrifuged until the wash solution was Biuret negative. This residue was frozen for later use.

Some of the supernatant after centrifugation at 9000 rpm was pipetted off and recentrifuged at 18,500 rpm for 1 hour. The small amount of residue, consisting of microscopically fine participate matter, was used in one of the toxicity experiments. The supernatant was treated with ammonium sulfate, centrifuged at 7000 rpm for 15 minutes, and the resulting pellet was dialyzed in standard dialysis tubing. The dialysate was used in one of the experiments.

182 NOLAN E. RICE AND W. ALLAN POWELL

In all toxicity experiments materials for injection were made up in 0.9% NaCl solution and 0.9% NaCl solutions were also used as controls. In all cases 1 ml doses were introduced intraperitoneally into white Swiss mice (Wistar strain).

The following standard chemical tests were used: Biuret, ninhydrin, Molisch, and Benedict's. Van Gieson's picrofuchsin and Mallory's aniline blue stains were applied to nematocysts.

Polyacrylamide gels were used to obtain the electrophoresis pattern of the toxic supernatant. The procedure for preparation of the polyacrylamide gel was sug- gested by the Canal Instrument Corporation in their instructions for the Model 12 Electrophoresis Apparatus. The sample and stacking gels were prepared using a buffer solution of pH 6.8-7.0; the separating gel was prepared using a buffer of pH 8.8-9.0. At the anode and cathode the pH of the buffer was 8.2-8.4.

RESULTS

Over the past seven years it has been observed that contacts of different indi- viduals with the summer nettle produce effects varying from a very faint burning sensation, barely detectable, and erythema to a severe development of angry red wheals accompanied by systemic symptoms such as nausea, respiratory distress, etc. On numerous occasions live nettles were deliberately permitted to come in contact with the skin of the biceps area of the arm. In some cases this produced only the faintest stinging sensation with little or no accompanying or subsequent erythema. Many individuals, however, generally showed a somewhat more severe allergic reaction.

The present study revealed four types of nematocysts. Batteries of holotrichous isorhizas (several sizes) and larger atrichous isorhizas are distributed fairly evenly over the exumbrella, oral lobes, and tentacles. The capsules of the holotrichous isorhizas range in length from 8 to 21 //. and in width from 3 to 18 p.. The tubes of these when discharged measured from 200 to 500 /M in length, 0.5 /* in diameter. The capsules of the atrichous isorhizas range from 20 to 25 /JL in length and are about 15 /j, in width. Most of the tubes when discharged measured from 500 to 2000 /A in length and from 0.5 to 1.5 //, in diameter. The euryteles have capsules which range from 10 to 12 /j. in length and 5 to 6 /j. in width. The tubes measure 50 to 150 p. in length and approximately 0.5 p. in width. The butt is 10 to 12 /j. in length and approximately 1.5 //, in width. A fourth type has a round capsule 15 to 18 p, in diameter with tube length of 150 to 250 p. and diameter of 2 p.. Additional round nematocysts were observed with a diameter of 4 to 8 ^ which may be smaller forms of the fourth type.

Various methods were tried in order to effect discharge of nematocysts from both living nettles and also isolated undischarged nematocysts. Immersion in distilled water and 1 M sucrose were ineffective in either case. Discharge of large numbers of nematocysts from the tentacles of living nettles occurred when formalin-acetic-alcohol fixative (FA A) or faradic electric shocks were applied, but neither of these agents caused the discharge of isolated nematocysts.

Since experiments on human skin in situ seemed out of the question, in order to get some idea of the penetration power of nematocysts the following experiment was set up : Two per cent pure agar containing enough Grenadier's borax-carmine

TOXIN OF CHRVS.IORA QUINQUECIRRIIA 183

to impart a red color was poured t(» a depth of about one-fourth inch in Petri dishes. Several slots, each about one-half inch long and one-eighth inch wide were cut out of each of these plates. A short piece of live tentacle was placed in each slot on the bottom against one side wall. A drop of FAA was applied to effect discharge of the nematocysts. The plates were then inverted under the microscope. The colorless nematocyst tube> could easily be seen in the red agar and their lengths measured from agar wall to tip of the tubes. \Yell over a hundred such measure- ments showed that the majority of nematocysts penetrated the agar from 200 to 400 p.. Most of the tubes took an almost straight course through the agar.

0

FIGURE 1. Electrophoretogram of the toxin of Chrysaora nematocysts obtained using polyacrylamide gels with subsequent staining with Coomassie blue stain for protein.

A large number of experiments dealing with the toxicity of nematocyst con- stituents to mice were performed as described under Materials and Methods. The results of these experiments show that the supernatant, after centrifugation of homogenized nematocyst suspensions at 9000 and 18,500 rpm, contained the toxin. The toxin is non-dialyzable, retaining its toxicity after salting out and sub- sequent dialysis. The LD 50, based on air dried residues corrected for salt content, was determined in three separate experiments to be 19 pg/g, 15 ng/g, and 16 pg/g of body weight, respectively. The ages of the doses of supernatant were 47, 85, and 193 days, respectively. It appears from a comparison of these figures that storage in a frozen state has little effect on toxicity. The residue obtained after removal of salt and after freeze-drying the supernatant was found to kill mice when administered at a level of 7.5 /^g/g of body weight. Washed undis- charged capsules, capsular debris, and tubes had no effect on mice ; even the fine sediment after centrifugation at 18,500 rpm was non-toxic.

Various chemical tests were applied to undischarged nematocvst>. capsular and tube debris, supernatant, and residue following centrifugation. The tests indicate

184 NOLAN E. RICE AND W. ALLAN POWELL

that: (1) thoroughly washed capsular and tube debris are negative to ninhydrin, Biuret, Molisch, and Benedict's tests before and after boiling; (2) the capsular contents (supernatant) are ninhydrin and Biuret positive but give negative results with Molisch and Benedict's reagents; (3) the residue following centrifugation at 18,500 rpm is negative to all of the tests; (4) the fluid contents of undischarged nematocysts are Biuret positive; (5) the capsules, capsular debris, and tubes do not stain appreciably, if at all, with aniline blue or picrofuchsin.

Supernatant containing toxin was thawed from the frozen state and subjected to electrophoresis using polyacrylamide gel and subsequently stained with Coomassie blue stain for protein. The resulting electrophoretogram is shown in Figure 1. One main band can be seen indicating one major protein fraction. Three faint bands may also be observed. Repetition using the supernatant from nematocysts obtained from another collection of nettles confirmed this picture.

DISCUSSION

Burnett et al. (1968) observed four types of nematocysts from Chrysaora: Type T, oval atrichous isorhizas; Type IT, round structures; Type TTT, euryteles ; Type IV, small round structures. Halstead (1965) in a table adapted from several authors records three types of nematocysts from Chrysaora: atrichous isorhizas, holotrichous isorhizas, and heterotrichous microbasic euryteles. The last type is followed by a question mark indicating uncertainty. In general our observations agree with those of Burnett ct a!. (1968) except that they do not record holo- trichous isorhizas. The eurvteles observed by us do not appear to be hetero- trichous microbasic euryteles but probably fit better in the eurytele class than any other.

Several methods for causing nematocyst discharge from living nettles have been used. Kline and Waravdekar (1960) used electric shock to effectively pro- duce discharge of nematocysts of Hydra Httoralis. More recently Barnes (1967) employed electrical shock with success on C. fleckeri, C. quadrigatus and Cyanca capillata. We have found that faradic shocks are quite effective over small areas of the tentacles of Chrysaora. FAA fixative gave good results but distilled water and 1 M sucrose were of little value.

Phillips and Abbott (1957) effected discharge of isolated nematocysts of Metridium senile fimbriatum by subjecting this anemone to various chemical agents : distilled water ; methylene blue ; weak acids ; weak bases ; sucrose ; and glycerine. Burnett et a!. (1968) compared the effectiveness of a number of physical and chemical agents in producing nematocyst rupture (not discharge") in Chrysaora. among them grinding ; freeze thaw ; distilled water ; salts, acids ; bases, cholinergic and adrenergic drugs ; heat ; and sonication. In contrast with the above results we were able to observe little or no discharge or rupture with distilled water, 1 M sucrose, FAA fixative, or faradic shock. Sonication of suspensions of nematocysts resulted in somewhat better than 50% rupture. The homogenization of frozen nematocyst suspensions, the procedure finally adopted, was found to be much more efficient than the method of Lane and Dodge (1958"), yielding as high as 75% rupture.

Our studies of the penetration of nematocyst tubes into agar are the first to be made with Chrysaora. However, Cleland and Southcott (1965) record some

TOXIN OF CHRYSAORA (}ij I XQUECIRRHA 185

work on the discharge of nematocysts of Australian species of jellyfishes into human skin and the pathological effects. Barnes (1967) made observations on the penetration of nematocysts of Chironcx, Chiropsalmus and Cyanea into various materials : human amnion ; sheep intestine ; hog stomach ; fish swim bladders ; synthetic sausage casings; latex; cured rubber; cellulose and various polyethylene and polyvinyl films. Further investigations in addition to those noted here should give much useful information for different species of cnidarians relative to the force of discharge of nematocysts, depth of penetration of tubes and the paths taken in test materials of various kinds.

As stated earlier in this paper a number of investigators (Lane and Dodge, 1958; Lane, 1960; Welsh, 1961; Burnett ct al. 1968; Shapiro, 1968; Endean et al. 1968; Crone and Keen, 1969) have found the toxins of various cnidarians to be protein in nature. Burnett ct al. (1968) believe (p. 335) that "the toxin factor of Chrysaora is a protein complex or is associated with a protein." The present work leaves little doubt that the toxin of Chrysaora is a protein or several proteins.

Burnett ct al. (1968) state further that after nematocyst rupture (p. 335) "the toxin can be recovered in significant amounts in the sediments," ... (p. 336) "that the toxin is membrane bound," . . . and (p. 335) "that the toxin is not released as a free fluid after nematocyst rupture." In contrast, the results of the present experiments show that the toxin is (1) contained in the undischarged nematocysts and is released as a free fluid after rupture which agrees with the findings of Barnes (1967) and Endean ct al. (1968) for other species of jellyfishes; (2) the toxin is not membrane bound (that is, to the capsules) ; and (3) the toxin is not present in thoroughly washed empty capsules, capsular fragments, tubes, nor in sediments after centrifugation at 9000-18,500 rpm which Endean et al. (1968) also indicate is true of the nematocysts of C. fleckeri. The differences in the findings of Burnett et al. (1968) and the results obtained in the present wrork might be due to differences in procedures followed in the isolation of the toxin.

Our LD 50 determinations in general agree with those of Burnett et al. (1968). They conclude, however, that toxicity loss resulted from freezing refrigeration of nematocysts. The LD 50 values reported in the present work for three separate experiments indicate that storage in the frozen state for better than six months had little effect on the toxicity of nematocyst contents.

It has been suggested by several investigators, Lenhoff, Kline and Hurley (1957), Phillips (1956), Kline (1961), Goldher, Burnett, Stone and Dilaimy (1969), that the capsules of various Cnidaria are composed of, or contain, a protein of the collagenous group. If this is so, Chrysaora material would appear to be different from vertebrate collagen since the capsules do not stain with aniline blue nor picrofuchsin which are accepted stains for vertebrate collagen.

We acknowledge with thanks the assistance of the following persons : Mr. William A. Dorsey, Chief of Public Laboratories, Richmond City Health Depart- ment for supplying us with laboratory mice ; Mr. James Rose, Research Chemist, Department of Surgery, Health Sciences Division, Virginia Commonwealth Uni- versity, Richmond, for electrophoresis studies ; Dr. Wilton R. Tenney, Department of Biology, University of Richmond for the photograph of the electrophoretogram ;

186 NOLAN E. RICE AND W. ALLAN POWELL

and Dr. Francis B. Leftwich, Department of Biology, University of Richmond for technical assistance.

SUMMARY

1. The response of the human body to the toxin of the summer nettle, Chrysaora quinquecirrha, is allergic in nature.

2. Four types of nematocysts were identified : atrichous isorhizas ; holotrichous isorhizas ; euryteles ; and a round type.

3. Application of faradic electrical shocks and the fixative FAA were effective in producing discharge of nematocysts in living nettles but not of isolated nematocysts.

4. The tubes of freely discharged nematocysts measured from 500—2000 /x in length. When discharged into 2% agar, the majority of tubes penetrated a distance of only 200-400 p.

5. An efficient method of isolation and rupture of the nematocysts with subse- quent isolation of the toxin is described.

6. The toxin is non-dialyzable. It gives positive ninhydrin and Biuret tests but negative Molisch and Benedict's tests.

7. The toxin is a protein or several proteins and is contained in the free fluid discharged from the nematocyst capsules. It is not membrane bound.

8. When inoculated intraperitoneally into white mice, the toxin gave an LD 50 of 19 fj-g/g, 15 fjig/g, and 16 p-g/g in three experiments.

9. There is little if any loss in toxic activity of nematocyst contents after storage in the frozen state over a six-month period.

10. The toxicity of the supernatant is retained after freeze-drying.

11. Thoroughly washed empty capsules, capsular fragments, and tubes do not appear to have any toxic effect when injected intraperitoneally into white mice.

12. Thoroughly washed empty nematocyst capsules, capsular fragments and tubes, and residues from high speed centrifugation gave negative results when tested with ninhydrin, Biuret, Molisch, and Benedict's reagents.

13. Empty washed capsules, capsular debris, and tubes do not stain with aniline blue nor picrofuchsin which are accepted stains for vertebrate collagen.

LITERATURE CITED

BARNES, J. H., 1967. Extraction of cnidarian venom from living tentacle, pp. 115-129. In:

Russell, F. E., and P. R. Saunders, Eds., Animal To.rius. Pergamon Press, Oxford. BURNETT, J. W., J. H. STONE, L. H. PIERCE, D. G. CARGO, E. C. LAYNE AND J. S. SUTTON,

1968. A physical and chemical study of sea nettle nematocysts and their toxin. /.

Invest. Dcrmatol., 51 : 330-336. CLELAND, J. B., AND R. V. SOUTHCOTT, 1965. Injuries to man from marine invertebrates in

the Australian region. Australia. National Health and Medical Research Council

Special Report, Series No. 12: 1-282. CRONE, H. D., AND T. E. B. KEEN, 1969. Chromatographic properties of the hemolysin from

the cnidarian Chironex fleckcri. Toxic on, 7 : 79-87. ENDEAN, R., CLAUDINE DUCHEMIN, D. McCoLM AND E. HOPE ERASER, 1969. A study of

the biological activity of toxic material derived from nematocysts of the cubomedusan

Chironex fleckcri. Toxicon,6: 179-204. GOLDHER, M. D., J. W. BURNETT, J. H. STONE AND M. S. DILAIMY, 1969. A comparison of the

composition of sea nettle (Chrysaora quinquecirrha) nematocysts and tentacles. /.

Invest. DermatoL, 52 : 393.

TOXIN OF CHRYSAORA QUINQUECIRRHA 187

HALSTEAD, B. W., 1965. Poisonous and Venemous Marine Animals of the World. U.

Government Printing Office, Washington, 994 pp. KLINE, E. S., AND V. S. WARAVDEKAR, 1969. Inhibitor of succinoxidase activity from Hydra

littoralis. J. Biol. Chcm., 235: 1803-1808. LANE, C. E., AND E. DODGE, 1958. The toxicity of Physalia nematocysts. Biol. Bull., 115:

219-226.

LANE, C. E., 1960. The Portuguese man-of-war. Sci. Amcr., 202: 158-168. LENHOFF, H. M., E. S. KLINE AND R. HURLEY, 1957. A hydroxyproline-rich, intracellular,

collagen-like protein of Hydra nematocysts. Biochem. Hii>f>liys. Acta, 26: 204-205. PHILLIPS, J. H., 1956. Isolation of active nematocysts of Afetridiinn senile and their chemical

composition. Nature, 178 : 932. PHILLIPS, J. H., AND D. P. ABBOTT, 1957. Isolation and assay of the nematocyst toxin of

Metridium senile fimbriahtm. Biol. Bull.. 113 : 296-301. SHAPIRO, BERT I., 1968. Purification of a toxin from tentacles of the anemone Condylueti*

gigantea. Toxicon, 5 : 253-259. WELSH, J. H., 1961. Compounds of pharmacological interest in coelenterates, pp. 179-186.

/;;: Lenhoff, H. M., and W. F. Loomis, Eds., The Biology of Hydra and Some Other

Coelenterates. University of Miami Press, Coral Gables, Florida.

Reference: Biol. Bull, 139: 188-202. (August, 1970)

LARVAL DEVELOPMENT OF PAGURUS LONGICARPUS SAY

REARED IN THE LABORATORY, I. DESCRIPTION

OF LARVAL IN STARS 1

MORRIS H. ROBERTS, JR.2 Virginia Institute of Marine Science, Gloucester Point, Virginia 23062

Thompson (1903) described from the plankton four zoeae and a megalopa which he ascribed to Pagurus longicarpus. Although his verbal description of the external anatomy is incomplete, his figures are adequate to identify accurately the species with which he was working. His description of the internal anatomy is the most complete study available for any decapod larva. He indicated that some larvae of P. annulipes were included in his study material but that he was unable to differentiate the two species.

MacDonald, Pike, and Williamson (1957) and Pike and Williamson (1959, 1960) described the larvae of several British and Mediterannean Pagurus species from carefully constructed plankton series. They were able to distinguish three types of larvae in this genus, based on 12 larval characteristics (several of which were composites of two characteristics), suggesting that the genus Pagurus may be polyphyletic, but no division of the genus was attempted for lack of adult charac- teristics supporting the larval data.

Larvae of several additional Pagurus species have been described from culture experiments by Coffin (1958, 1960), Hart (1937), Provenzano and Rice (1964) and Shenoy (1967). In addition, P. bernhardus has been cultured by Bookhout (1964) and A. Le Roux (personal communication) and shown to agree in all essentials with the description given by MacDonald et al. (1957).

This paper deals with the external anatomy of P. longicarpus larvae reared in the laboratory. It is clearly demonstrated that Thompson (1903) attributed his larvae to the wrong species and a suggestion is made as to the correct identity of his larvae.

MATERIALS AND METHODS

Ovigerous P. longicarpus were collected from the beach at Gloucester Point, Virginia. Some crabs were maintained at room temperature in finger bowls with filtered water (salinity: 19-22%c). The water was replenished once or twice daily until hatching occurred. This method had two disadvantages : first, there was a danger of hatching occurring without molting the embryonic or prezoeal

1 Contribution Number 342 from the Virginia Institute of Marine Science, Gloucester Point, Virginia.

2 Present address : Department of Biology, Providence College, Providence, Rhode Island 02918. This paper is part of a dissertation submitted to the School of Marine Science of the College of William and Mary in partial fulfillment of the requirements for the Doctor of Philosophy Degree.

188

LARVAL STAGES, PAGUkUS LONGICARPUS 189

cuticle ; second, adult hermit crabs kept in small containers eat many larvae before they can be removed. These problems were overcome in some experiments by removing the eggs from the pleopods of the crabs and culturing the eggs arti- ficially (Costlowand Bookhout, 1960).

Larvae were cultured individually in compartmented plastic boxes with nauplii of Artemia salina as food. No bactericide was used and water was changed daily. Living specimens of each instar were preserved in 70% ethanol with 10% glycerol as were exuvia and dead specimens. Live specimens proved most satisfactory for study as they were less likely to be damaged and easier to handle and dissect.

Several larvae in each instar were dissected under 85 % lactic acid and mounted in a modified Hoyer's medium for study and figuring. Figures of entire specimens and various appendages were drawn with the aid of an ocular grid and graph paper. Verbal descriptions, prepared from figures and notes taken during study, were subsequently checked against additional specimens. Notes on color were made while observing living larvae immobilized mechanically as suggested by Dean and Hatfield (1963). Larvae handled in this manner survived when returned to the culture dish and showed no obvious deleterious effects.

The following abbreviations are used throughout the description : A 1 = anten-

nule, A 2 : : antenna, Mn = : mandible, MX 1 ; maxillule, MX 2 = maxilla, Mxp 1

: first maxilliped, Mxp 2 = : second maxilliped, Mxp 3 : : third maxilliped, P 1 to

= pereiopods 1 to 5, PI 2 to 5 — pleopods 2 to 5, U — uropods.

RESULTS

Four zoeal stages and a megalopa were obtained. No prezoea was observed except when culture conditions for the eggs were known to be suboptimal. Examination of egg membranes revealed that the embryonic or prezoeal cuticle normally is shed at the time of hatching and remains attached to the egg membrane. It is concluded that the prezoea is not a true planktonic stage and therefore no description is given.

Zoeal (Figure 1 )

Rostrum unornamented, curved slightly ventrad, approximately equal to A 1 and A 2. Carapace with small posterolateral spines pointed ventrally. Eyes un- stalked. Abdomen with 5 somites plus fused 6th somite-telson. Somites 2 through 5 with 2 pairs of posterodorsal spines and one pair of ventrolateral spines, those of somite 5 longest, reaching fusion of 6th somite-telson. Pleopod buds and uropods absent. Telson formula 7 + 7; process 1, lateral spur; process 2, short ventral hair ; processes 3 to 7, long articulated plumose setae, process 4 longest. Fine hairs between processes. Small median notch. Anal spine present.

A 1 (Fig. 1C) — Uniramous, unsegmented, with 4 terminal aesthetascs and a ' long subterminal plumose seta.

A 2 (Fig. ID) — Scale with 8 (7-9) plumose setae on inner margin and terminal spur. Endopod about \ length of scale, fused to peduncle, with 2 terminal plumose setae. Short strong seta with cuneate setules on peduncle near endopod.

Mn (Fig. IE) — Incisor and molar processes present, palp absent.

190

\M IKKIS II. K015KKTS. JR.

0 mm

D

B

0.5 mm

FIGURE 1. Zoea I of Pagitnts longlcarpus; A. dorsal view, B. lateral view, C. antennule, D. antenna, E. mandibles, F. maxillule, G. maxilla, H. 1st maxilliped, I. 2nd maxilliped, J. 3rd maxilliped.

MX 1 (Fig. IF) — Coxal endite with 6 setae, basal endite produced into 2 strong spines. Endopod 3-segmented with 3 terminal plumose setae and an additional seta distomedially on second segment.

MX 2 (Fig. 1G) — Coxal and basal endites bilobed, both with 5 setae on proximal lobe, 3 on distal lobe. Endopod unsegmented with 4 terminal and 2 subterminal setae. Scaphognathite with distal lobe bearing 5 marginal plumose setae.

Mxp 1 (Fig. 1H) — Coxa nude, basis with 7 setae on inner margin. Endopod 5-segmented : long plumose seta on outer margin of segments 1, 2, and 5; fringe

LARVAL STAGES, PAGURUS LONGICARPUS 191

of fine hairs on outer margin of segment 3; 3, 2, 1, 2, 4 setae on inner margin of segments 1 to 5. Exopod 2-segmented, with 4 terminal plumose setae.

Mxp2 (Fig. II) — Coxa nude, basis with 3 setae on inner margin. Endopod 4-segmented : long plumose seta on outer margin of segment 2 ; 3, 2, 2, 4 setae on inner margin of segments 1 to 4. Exopod 2-segmented, with 4 terminal plumose setae.

Mxp 3 (Fig. 1J) — Uniramous unsegmented rudiment, flexed anteriorly and medially between bases of Mxp 2.

P 1 to 5 (Fig. IB) — Barely distinguishable rudiments initially but noticeably larger by end of stage.

Pigmentation— Red chromatophore midway along rostrum, few if any chromatophores on rest of carapace at hatching, several pairs developing during this stage near posterior margin. Two red chromatophores in each eyestalk region. Red chromatophores on base of A 2, on all inner mouth parts, encircling labrum, and 2 red and yellow chromatophores on basis of Mxp 1, 1 on Mxp 2. Chromato- phores sometimes in proximal segment of exopod of Mxp 1 and 2. Chro- matophores in intestine in somites 2 and 4; 6th somite-telson with pair of red and 3 pairs of red-yellow chromatophores. In most specimens, a pair of red or yellow chromatophores located at posterolateral corners of telson. Pigmentation variable.

Zoea II (Figure 2)

Rostrum slightly longer than A 1 and A 2. Eyes stalked. Abdomen with 5 somites and fused 6th somite-telson. Pleopod buds and uropods absent, but uropod anlagen visible within telson late in stage. Telson formula 8 + 8; process 8 added medially to process 7 ; process 4 still articulated and longest. Anal spine present.

A 1 (Fig. 2C) — No change from Zoea I.

A 2 (Fig. 2D) — Slight increase in size; endopod articulated to peduncle.

Mn (Fig. 2E) — No change from Zoea I.

MX 1 (Fig. 2F) — Coxal endite with 7 setae, basal endite with 4 spines and a small seta, spines articulated. Endopod with 3 terminal plumose setae and 1 plumose seta on inner margin of second segment.

MX 2 (Fig. 2G) — Coxal and basal endites bilobed. Coxal endite with 7 setae on proximal lobe, 4 on distal lobe. Basal endite with 5 setae on proximal lobe, 4 on distal lobe. Endopod with 3 terminal, 1 subterminal, and 2 (3) medial plumose setae. Scaphognathite with distal lobe bearing 8 plumose setae.

Mxp 1 (Fig. 2H) — Coxa with 1 seta, basis with 10 setae on inner margin. Endopod : long plumose seta on outer margin of segments 1, 2, 3, and 5 ; 3. 2, 1, 2, 4 setae on inner margin of segments 1 to 5. Exopod with 7 terminal plumose setae.

Mxp 2 (Fig. 21) — Coxa nude, basis with 3 setae on inner margin. Endopod: long plumose seta on outer margin of segments 2. 3, and 4 ; 3, 2, 2, 4 setae on inner margin of segments 1 to 4. Exopod with 7 terminal plumose setae.

Mxp 3 (Fig. 2J) — Biramous rudiment. Endopod bud fused to basis ; 2 terminal setae. Exopod 2-segmented, with 6 terminal plumose setae, functional for swimming.

P 1 to 5 (Fig. 2B) — Small uniramous buds increasing in length somewhat during stage. Occasionally pseudosegmented at end of stage.

192

MORRIS 11. ROBERTS, JR.

FIGURE 2. Zoea II of Pagurus longicarpus; A. dorsal view, B. lateral view, C. antennule, D. antenna, E. mandibles, F. maxillule, G. maxilla, H. 1st maxilliped, I. 2nd maxilliped, J. 3rd maxilliped.

Pigmentation — Generally as in Zoea I but more extensive. Eyestalks with red and yellow chromatophores. Carapace with additional chromatophores on lateral and dorsal regions. Mouth parts pigmented as in Zoea I. Red and yellow chromatophores developing dorsally in abdominal somite 2. Pattern on carapace extremely variable.

Zoea III (Figure 5)

Rostrum and carapace unchanged. Abdominal somite 6 free from telson, with smooth posterior margin. Ventrolateral spines on somite 5 projecting posteriorly to about middle of somite 6. Telson formula 8 + 8 as in Zoea II ; process 4 fused to telson, greater than | maximum telson width.

LARVAL STAGES, PAGURUS LONGICARPUS

193

FIGURE 3. Zoea III of Pagurus longicarpux ; A. dorsal view, B. lateral view, C. antennule, D. antenna, E. mandibles, F. maxillule, G. maxilla, H. 1st maxilliped, I. 2nd maxilliped, J. 3rd maxilliped.

A 1 (Fig. 3C) — Biramous, with both rami unsegmented and articulated to peduncle. Outer ramus with 4 terminal aesthetascs plus several short setae ; inner ramus nude. Three long plumose setae on distal end of peduncle, 1 near articula- tion of outer ramus and 1 on each side of inner ramus.

194 MORRIS II. ROBERTS, JR.

A 2 (Fig. 3D) — Scale with 9 (8) plumose setae. Endopod equal to scale minus terminal spur, with 1 (2) terminal setae. Ventral seta present as in Zoea I and II plus a short spine added ventral to scale.

Mn (Fig. 3E) — No change from Zoea I.

MX 1 (Fig. 3F) — Coxal enclite with 7 setae, basal endite with 4 spines and a small seta, spines articulated. Endopod with 3 terminal plumose setae and 1 plumose seta on inner margin of second segment.

MX 2 (Fig. 3G) — Coxal and basal endites bilobed. Coxal endite with 7 setae on proximal lobe, 4 (3) on distal lobe. Basal endite with 6 setae on proximal lobe, 5 on distal lobe. Endopod with 4 terminal and 2 subterminal plumose setae. Distal lobe of scaphognathite with 1 1 plumose setae.

Mxp 1 (Fig. 3H) — Coxa with 1 seta, basis with 10 setae on inner margin. Endopod : long plumose seta on outer margin of segments 1, 2, 3 and 5 ; 3, 2, 1, 2, 4 setae on inner margin of segments 1 to 5. Exopod with 8 terminal plumose setae.

Mxp 2 (Fig. 31) — Coxa nude, basis with 3 setae on inner margin. Endopod: long plumose seta on outer margin of segments 2, 3 and 4 ; 3, 2, 2, 4 setae on inner margin of segments 1 to 4. Exopod with 8 terminal plumose setae.

Mxp 3 (Fig. 3J) Coxa and basis both nude. Endopod unsegmented, articu- lated with basis, with 1 terminal and 1 subterminal seta. Exopod with 8 terminal plumose setae.

P 1 to 5 (Fig. 3B) — Increase considerably in size during stage, pseudoseg- mented at end of stage. P 5 hidden behind P 1 to 4, not readily visible.

U (Fig. 3A, B) — Freely articulated, biramous ; exopod £ telson length with 6 plumose setae on inner margin plus terminal spur ; endopod rudimentary.

Pigmentation — Generally as in Zoea II. Chromatophores of abdominal somite 6 spilling over into somite 5 and telson. Small red and yellow chromatophores beginning to develop in pereiopod buds.

Zoea IV (Figure 4}

Carapace, abdominal somites, and telson as in Zoea III.

A 1 (Fig. 4C) — Peduncle with 2 short rami. Outer ramus unsegmented with 4 terminal aesthetascs and 5 (6) aesthetascs on inner margin, plus several fine terminal setae. Inner ramus unsegmented, nude. Three long plumose setae on distal end of peduncle as in Zoea III.

A 2 (Fig. 4D) — Scale with 9 (7-9) plumose setae and terminal spur. Endopod longer than scale, reaching tip of rostrum. Initially unsegmented, but segments evident beneath cuticle just prior to molt ; appears folded like an accordion.

Mn (Fig. 4E) — Unsegmented palp rudiment present. Molar and incisor processes unchanged.

MX 1 (Fig. 4F) — Coxal endite with 8 (7-9) setae, basal endite with 7 (6-8) spines. Endopod with 3 terminal plumose setae plus 1 plumose seta at distal end of second segment.

MX 2 (Fig. 4G) — Coxal and basal endites bilobed. Coxal endite with 7 (7-8) setae on proximal lobe, 4 (4-5) on distal lobe. Basal endite with 5 (4-6) setae on proximal lobe, 6 (4-6) on distal lobe. Endopod with 4 long terminal plumose setae and 3 (4) plumose setae midway along inner margin. Scaphognathite with

LARVAL STAGES, PAGURUS LONGICARPUS

195

FIGURE 4. Zoea IV of Pagimis longicarpus ; A. dorsal view, B. lateral view, C. antennule, D. antenna, E. mandibles, F. maxillule, G. maxilla, H. 1st maxilliped, I. 2nd maxilliped, J. 3rd maxilliped.

196 MORRIS H. ROBERTS, JR.

distal and proximal lobes present, with 18 (11-20) plumose setae. Lobes of equal length ; proximal only .1 width of distal.

Mxp 1 (Fig. 4H) — Coxa with 1 seta, basis with 10 (10-12) setae on inner margin. Endopod: long plumose seta on outer margin of segments 1, 2, 3 and 5; 3, 2, 1, 2, 4 setae on inner margin of segments 1 to 5. Exopod with 8 terminal plumose setae.

Mxp 2 (Fig. 41) — Coxa nude, basis with 3 setae on inner margin. Endopod 5-segmented, unlike previous stages ; long plumose seta on outer margin of seg- ments 2, 3 and 5 ; 3, 2, 0, 2, 4 setae on segments 1 to 5. Exopod with 8 terminal plumose setae.

Mxp 3 (Fig. 4J) — Coxa and basis nude. Endopod short, pseudosegmented, with a few scattered setae distally. Exopod with 8 terminal plumose setae.

P 1 to 5 (Fig. 4B) — Segmented; PI cheliform, others styliform. Increase considerably during stage until space beneath carapace filled, but remain flexed against cephalothorax.

PI 2 to 5 (Fig. 4B) — Pleopod buds paired on somites 2 to 5. Biramotis, with endopod minute, nude, inconspicuous. Exopod usually nude, rarely with 5 terminal setae. Buds increase in length by about twice during stage.

U (Fig. 4A, B) — Exopod with 6 plumose setae on inner margin and 1 (2) terminal spurs, approximately equal to telson (exclusive of telson processes). Endopod still rudimentary but longer, with 2 terminal setae.

Pigmentation — As in Zoea III, although more conspicuous on carapace. Abdominal somite 5 and telson increasingly pigmented. Mxp 3 with a red-yellow chromatophore in basis. Pereiopod buds with large chromatophores. As in previous stages, pattern extremely variable even among larvae from a single female.

Megalopa (Figure 5)

Carapace (Fig. 5 A) with no posterolateral spines, rostrum greatly reduced and broadly rounded as in adult. Eyes stalked, peduncle slightly swollen in middle; eye scales present. Abdomen with 6 discrete segments and telson, slightly asym- metric in many specimens. Telson (Fig. 5A, P) with 4 + 4 terminal plumose setae and a minute lateral spine.

A 1 (Fig. 5C) — Outer ramus 4-segmented with 3 aesthetascs on second seg- ment, 4 on third and proximally on last segment, terminated with 1 long and 3 short setae. Inner ramus 2-segmented with a few short setae.

A 2 (Fig. 5B) — Flagellum with 10 to 14 segments, all with short setae distally. Scale reduced, unidentate, with a few scattered setae.

Mn (Fig. 5D) — Of adult shape. Palp 3-segmented with a few setae on last segment.

MX 1 (Fig. 5E) — Coxal endite with short setae, basal endite with 3 rows of 5 teeth each. Endopod unsegmented, no palp.

MX 2 (Fig. 5F) — Coxal and basal endites bilobed with numerous short setae. Endopod unsegmented with 3 terminal and 1 subterminal setae. Scaphognathite with v32-35 setae.

Mxp 1 (Fig. 5G) — Coxal endite small, not completely formed, with 4 setae. Basal endite considerably larger, but also not of adult shape, with many short setae. Endopod slightly reduced, unsegmented, with few setae. Exopod 2-seg-

LARVAL STAGES, PAGURUS LONGICARPUS

197

FIGURE 5. Megalopa of Pagurus longicarpus; A. dorsal view, B. antenna, C. antennule, D. mandibles, E. maxillule, F. maxilla, G. 1st maxilliped, H. 2nd maxilliped, I. 3rd maxilliped, J. 4th pereiopod, K. 5th pereiopod, L. 2nd pleopod, M. 3rd pleopod, N. 4th pleopod, O. 5th pleopod, P. telson and uropods.

198

MORRIS H. ROBERTS, JR.

mented with proximal segment still narrow and lacking marginal setae. Distal segment with 6 plumose setae.

Mxp2 (Fig. 5H) — Coxa nude. Basis with 2 setae on inner margin. Endopod 5-segmented, with fewer setae than adult. Exopod 3-segmented, with 8 terminal plumose setae.

Mxp 3 (Fig. 51)— Coxa nude. Endopod 5-segmented; basis fused with ischium ; dentate ridge with 3 (4) teeth plus accessory tooth and 4 setae; all seg- ments with fewer setae than adult. Exopod 3-segmented, with 8 terminal plumose setae.

P 1 to 5 (Fig. 5 A, J, K) — P 1 cheliform, slightly asymmetric, right larger than left, reaching A 2. P 2 and P 3 of adult proportions, functional as ambulatory legs. P4 (Fig. 5J) subchelate, propodus with a single row of 5 tubercles repre- senting adult rasp, dactylus with 3 tubercles. P5 (Fig. 5K) with 10 tubercles arranged in several rows and several hooked setae on propodus, 3 tubercles and several hooked setae on dactylus.

TABLE I Carapace and total length (in mm) of Pagurus longicarpus and Pagurus annulipes larvae

	Zoea
	I	II	III	IV
Pagurus longicarpus
Carapace X	1.05	1.23	1.47	1.66	1.13
Length range	0.99-1.10	1.12-1.40	1.35-1.64	1.44-1.80	0.90-1.26
N	20	27	35	35	40
Total X	1.74	2.40	2.90	3.38	2.39
Length range	1.67-1.80	2.16-2.61	2.70-3.19	3.06-3.60	2.05--2.70
N	20	26	35	32	36
Pagurus annulipes*
Total range	1.9-2.7	2.7-3.0	3.5-4.0	4.0	2.8-3.3
Length

* after Thompson (1903) as P. longicarpus.

PI 2 to 5 (Fig. 5L, M, N, O) — Pleopods still paired on somites 2 to 5 ; biramous, endopod small ; 8, 8, 8, 5 (6) terminal plumose setae on exopods of PI 2 to 5, natatory.

U (Fig. 5 A, P) — Left larger, with 12 (14) plumose setae and 11 (13) tubercles on exopod, 3 plumose setae and 4 (6) tubercles on endopod. Right with 11 (13) plumose setae and 9 (11) tubercles on exopod, 3 plumose setae and 3 (4) tubercles on endopod. Basis with a small spine near origin of endopod.

Pigmentation — Many more chromatophores present, but pattern essentially same as in zoeae. As next molt approaches, chelipeds become pale yellow or white as in juvenile.

Growth — The larval stages increased relatively uniformly from a total length of 1.74 mm in Zoea I to 3.38 mm in Zoea IV (Table I). With the molt to the megalopa, the total length dropped about 1 mm or 30% to approximately the size of Zoea II. Comparison of carapace lengths revealed the same result. It would be

LARVAL STAGES, PAGURUS LONGICARPUS

199

TABLE II Summary of anatomical differences between Pagurus longicarpus and Pagurus annulipes larvae

Stage

Pagurus longicarpus

Pagurus annulipes

Zoea I Zoea II Zoea III

Zoea IV

All Zoeae

Megalopa

A 2 scale with 8 setae

A 2 endopod == \ scale

A 2 scale with 8 setae

MX 2 scaphognathite with 8 setae

A 2 scale with 9 setae

MX 2 scaphognathite with 11 setae

Abdominal somite 6 without median dorsal spine

Telson process 4 long

A 2 scale with 9 setae

Mn palp present

MX 2 scaphognathite with 18 setae; proximal lobe present

Mxp 2 endopod 5-segmented

U with 6 plumose setae

Abdominal somite 6 without median dorsal spine

Telson process 4 long

Red chromatophores absent ventro- laterally on carapace and on ab- dominal somites 2 and 5

MX 1 endopod 0, 1, 3 setae

Mxp 1 and 2 exopod 2-segmented Mxp 3 exopod 2-segmented

(Zoea II-IV) Lateral abdominal spines long

Mxp 1,2,3 well developed P 1 == A 2 < P 2 and P3 PI 2 to 5 with 8,8,8,5 setae

A 2 scale with 10 setae

A 2 endopod <\ scale

A 2 scale with 10 setae

MX 2 scaphognathite with 6 setae

A 2 scale with 10 setae

MX 2 scaphognathite with 10 setae

Abdominal somite 6 with median dorsal spine

Telson process 4 minute

A 2 scale with 8 setae*

Mn palp absent

MX 2 scaphognathite with 11 setae; proximal lobe absent

Mxp 2 endopod 4-segmented

U with 7 plumose setae

Abdominal somite 6 with median dorsal spine

Telson process 4 minute

Red chromatophores present ventro- laterally on carapace and on ab- dominal somites 2 and 5

MX 1 endopod 1, 1, 2 setae

Zoea I-III; 1,1,3 setae, Zoea IV

Mxp 1 and 2 exopod unsegmented

Mxp 3 exopod unsegmented

Lateral abdominal spines short

Mxp 1,2,3 poorly developed PI = = P 2 and P 3 > A 2 PI 2 to 5 with 7,7,9,7 setae**

* A 2 scale with 10 setae according to C. Nyblade. ** PI 2 to 5 with 9(10),9(10),9(10),8 setae according to C. Nyblade.

interesting to follow changes in weight through the larval sequence but this has not been done. The change in length probably reflects the radical change in shape rather than a true change in biomass or volume.

The larva described by Thompson (1903) was considerably larger than P. longicarpus, 0.5 mm or more at each instar (Table I). Again there was an in- crease in total length to Zoea IV and then a reduction to about the length of Zoea II. Thompson used the wide range of lengths within each zoeal stage to justify partially his contention that he was working with two species, but, with the exception of Zoea I, the range of total length which he observed was equal to or smaller than that observed in the present study.

DISCUSSION

The larvae described by Thompson (1903) differ from P. longicarpus as de- scribed above in many features, some quite conspicuous. Table II summarizes these

200

MORRIS II. K( (HERTS, JR.

TABLE III

Summary of characteristics defining the four groups of Pagurus larvae recognized •with a list of the species in each group (After MacDonald et al., 1957)

Characteristics	Group
A	B	C	D
Zoeal length and	elongate	not elongate	not elongate	not elongate
telson breadth	narrow	broad	broad	broad
Lateral spines somite 5	long	long	medium	long
Median dorsal spine somite 6	absent	present	absent	absent
Telson process 4,	long	reduced	medium	long
Zoea III and IV	>£ telson	<i telson	= J telson	>5 telson
	width	width	width	width
	fused	fused	articulated	fused
Number setae on endopod	0	2	2	2
of A 2
A 2 scale	straight	curved	curved	curved
	L > 6 X W	L <: 4 X W	L < 4 X \V	L <4 X W
	setae < 8	9 setae SI	9 setae SI	8setaeSI-II
		lOsetaeSII-IV	lOsetaeSII-IV	9setaeSIII-IV
Mandibular palp, Zoea IV	present	absent	absent	present
Number setae on U endopod	0	2	1	2
Number pleopods, Zoea IV,	4	4	4	4
Megalopa
A 2 of Megalopa	long	short	short	medium
	A 2 > P 1	A 2 < P 1	A2 < P 1	A 2 == P 1
Yellow chromatophore	present	absent	present	absent
over stomach
List of species	bernhardus1	pridc/iiixii1	anachoretusz	longicarpus
	pubescens1	cuanensis1	kulkarnii3
	samuelis* ( ?)	alatus2
	beringanus1	forbesii1,2
		annulipes6

1 MacDonald et al., 1957.

2 Pike and Williamson, 1960. 3Shenoy, 1967.

4 Coffin, 1958, 1960.

5 Provenzano and Rice, 1964.

6 Thompson, 1903.

7 Hart, 1937.

differences in detail with some additional data from Mr. Carl Nyblade, University of Washington, Seattle (personal communication).

Many differences, such as setation, might readily result from differing rates of morphogenesis in cultured versus planktonic specimens. This has been reported previously for Pisidia longicornis (LeRoux, 1966) and Euceramus praelongus (Roberts, 1968). This type of explanation is less likely for such differences as telson process 4, mandibular palp, and pigmentation. Unfortunately, no specimens of P. longicarpus from the field have been obtained for comparison with laboratory- reared specimens. There is little doubt, however, that Thompson was not working with P. longicarpus. Carl Nyblade (personal communication) has identified

LARVAL STAGES, PAGURUS LONGICARPUS 201

Thompson's larvae as P. annulipes by comparison with cultured larvae of that species.

MacDonald ct al. (1957) distinguished two groups of Pagnrns larvae which they designated Group A (Type: P. bcrnJiardns} and Group B (Type: P. pridean.rii ) . Later, Pike and Williamson (1960) recognized a third group, Group C (Type: P. anachorctus} which is similar to Group B but differs by the absence of a median dorsal spine on somite 6, length of telson process 4 (both like Group A), and number of setae on uropod endopod (like Anapagurus as described by MacDonald ct al.. 1957). Detailed comparisons of these groups and a list of the species assigned to each group to date appear in Table III. Until the present study, all described larvae of the genus Pagiints have fit into a pre- viously defined group. Larvae of P. beringanus, described by Hart (1937), belong to Group A. Larvae of P. soiiinclis, described by Coffin (1958, 1960), appear to belong to Group A but cannot be definitely categorized because his description is not complete. Provenzano and Rice (1964) placed the larvae of P. uiarslii in Group B. Larvae of P. knlkarnii, described by Shenoy (1967), probably belong to Group C, accepting Shenoy's conclusion that the "normal" number of instars is 4 and not 3 as he observed. The larvae are comparable in pigmentation of the 5th and 6th abdominal somites and telson, absence of median dorsal spine on somite 6, telson process 4 long, articulated in last zoeal stage observed, and other pertinent characters except size of A 2 in the megalopa.

The larvae of Thompson (1903), herein ascribed to P. annitlipes, were cor- rectly placed in Group B. P. longicarpus , however, does not belong to this group, nor does it belong to either of the other previously defined groups. It differs from Group A by being not elongate, telson broad, having two setae on the endopod of A 2 in Zoea I and II, a curved broad antennal scale, 2 setae on the uropod endopod in Zoea IV, A 2 short and equal to right cheliped in the megalopa, no yellow chromatophore dorsal to the zoeal stomach, from Group B by possessing a long telson process 4 in Zoea III and IV and a mandibular palp in Zoea IV, and from Group C by having 2 setae on the uropod endopod and a rudimentary mandibular palp in Zoea IV. Thus P. longicarpus constitutes the type of a fourth group, Group D, of Pagurns larvae.

Additional groups of similar larvae will probably be found as further Pagurns species are studied. Larvae of P. pollicaris, based on a preliminary examination of larvae reared in this laboratory and a description of cultured larvae by another investigator (Carl Nyblade, personal communication), do not belong to any group as defined above. These larvae resemble Group B except for the lateral spines on abdominal somite 5 and the length of telson process 4 which are com- parable to Group C and the A 2 scale which is comparable to Group D.

To establish whether this division of the genus based on larval characteristics is of phyletic significance, as suggested by MacDonald ct al. (1957), will require much further study of larvae and adults with consideration of all characteristics of possible systematic value. Regardless, it has value in identification of plankton specimens.

No attempt will be made to compare larvae of the genus Pagnrus with larvae or other genera in the family Paguridae or with larvae of the other "hermit crab" families, Diogenidae and Coenobitidae. The subject has recently been reviewed in

202 MORRIS H. ROBERTS, JR.

some detail by Provcnzano ( l(">X;i, 1>). althotigh the emphasis was on the latter two families.

I am indebted to Dr. Langley Wood, who served as chairman of my graduate committee, for his patience and helpful criticisms. 1 also appreciate the many fruitful discussions with Dr. Marvin L. \Yass and Mr. Willard A. Van Engel and their critical reading of the manuscript. Dr. Morris L. Brehmer provided space in his laboratory and a critical review of the manuscript.

I owe special thanks to my wife, Beverly Ann, for her constant encouragement, and aid in the laboratory.

During the course of this study I was the recipient of a National Science Foundation Graduate Fellowship.

LITERATURE CITED

BOOKHOUT, C. G., 1964. Salinity effects on the larval development of Pagurus bernhardus (L.)

reared in the laboratory. Ophelia, 1 : 275-294. COFFIN, H. G., 1958. The laboratory culture of Pagurus sainuells Stimpson. Walla Walla

Coll. PnbL, 22 : 1-5. COFFIN, H. G., 1960. The ovulation, embryology, and developmental stages of the hermit crab,

Pagurus sainuclis Stimpson. U'ulla U'alla Coll. Publ., 25: 1-30. COSTLOW, J. D., JR., AND C. G. BOOKHOUT, 1960. A method for developing Brachyuran eggs

in vitro. Liiniiol. Oceanogr., 5: 212-215. DEAN, D., AND P. A. HATFIELD, 1963. Holding small aquatic invertebrates for observation.

Turto.v News, 41 : 43. HART, J. F. L., 1937. Larval and adult stages of British Columbia Anomura. Canadian J.

Res.,15D: 179-220. LERoux, A., 1966. Le developpement larvaire de Porcellana longicornis Pennant (Crustace

Decapode Anomoure Galatheide). Cah. Biol. Mar., 7 : 69-78. MACDONALD, J. D., R. B. PIKE AND D. I. WILLIAMSON, 1957. Larvae of the British species

of Diogenes, Pagurus, Anapagitnts and Lithodes (Crustacea, Decapoda). Proc. Zool.

Soc. London, 128 : 209-257. PIKE, R. B., AND D. I. WILLIAMSON, 1959. Crustacea, Decapoda : Larvae. XI. Paguridae,

Coenobitidae. Dromiidea, and Homolidea. Cons. Int. Explor. Mer., Zooplankton

Identification Sheet 81. PIKE, R. B., AND D. I. WILLIAMSON, 1960. Larvae of Decapod Crustacea of the families

Diogenidae and Paguridae from the Bay of Naples. Pubbl. Sta. Zool. Napoli, 31 :

493-552. PROVENZANO, A. J., JR., 1968a. The complete larval development of the West Indian hermit

crab Pctrochirus dioqencs (L.) (Decapoda, Diogenidae) reared in the laboratory.

Bull. Mar. Sci., 18 : 143-181. PROVENZANO, A. J., JR., 1968b. Biological investigations of the deep sea. 37. Lithopagunis

yucatanicus, a new genus and species of hermit crab with a distinctive larva. Bull.

Mar. Sci., 18 : 627-644. PROVENZANO, A. J., JR., AND A. L. RICE, 1964. Larval stages of Pagurus marshi Benedict

(Decapoda; Anomura) reared in the laboratory. Crustaceana, 7: 217-235. ROBERTS, M. H., JR., 1968. Larval development of the decapod Euccramus praclongus in

laboratory culture. Chesapeake Sci., 9 : 121-130. SHENOY, S., 1967. Studies on larval development in Anomura (Crustacea, Decapoda) — II.,

pp. 777-804. In: Symposium on Crustacea, Pt. II. Marine Biological Association,

Mandapam Camp, India. THOMPSON, M. T., 1903. The metamorphosis of the hermit crab. Proc. Boston Soc. Natur.

Hist., 31: 147-209.

Reference: Biol Bull, 139: 203-221. i August, 1«>70)

LIFE CYCLE OF THE HYDROMEDUSA PHIALIDIUM GREGARIUM (A. AGASSIZ, 1862) IX THE LABORATORY

EDWARD C. ROOSEN-RUNGE

of Biological Structure, School of ^fedicinr. mnl f'nh'srsity <>i U'tixliiiii/lon Friday Harbor Laboratories, Seattle, Washington 98105

It has been known for more than a hundred years that it is not difficult to raise hydroids from the eggs of Phial idium hemisphericum ('\Yright, 1858). There is no report of the raising of hydroids of the medusa known as Phialidium gregarium (A. Agassiz, in L. Agassiz, 1862) and its hydroid has not been identified until now. This would seem to be an excellent reason to undertake the observa- tion of the whole life cycle of P. gregarium in the laboratory. There are, however, several equally cogent reasons. In the first place, the elaboration of a culture method which permits reliably raising hydroid colonies from single planulae, and medusae from single hydroid colonies, also enables us to study individual life cycles. Colonies found in nature and even colonies raised from batches of eggs are composites of many individual beings and the dynamics of their behavior are difficult to explain because the extent, the developmental stage and other specific conditions of the individual components are usually unknown. On the other hand, a hydroid "colony" derived from a single planula may be considered a single animal. By observing and comparing many of these animals, conclusions can be reached about age changes, responses to environmental conditions and about differences between animals under identical conditions which must be ascribed to genetic variability. Hydroids have been notorious for the taxonomic difficulties which they represent, and many descriptions of what are assumed to be single species show a wide and poorly delineated range of anatomical features. Detailed comparison of colonies arising from single planulae under controlled conditions can be expected to clarify some of the taxonomic issues. Finally, the breeding of medusae in clones derived from single eggs will permit more precise experimenta- tion, for instance, on sex determination and on physiological parameters such as light sensitivity (Roosen-Runge, 1962) where results have been ambiguous until now because of genetic heterogeneity.

MATERIALS AND METHODS

All observations and experiments on living animals were made between the end of July and the end of Xovember. Medusae were caught from the dock of the Friday Harbor Laboratories. They were brought up with a small, white enamelled saucepan on a 3^ foot handle from a float approximately 2 feet above the water. Therefore, the animals came from the surface layer down to little more than 3 feet. Freshly caught medusae, 30-40, with a preponderance of males, were left in large bowls (1000 cc) overnight and the eggs collected around 10 A.M. Special care was taken to make sure that every parent animal belonged to the species P. gregarium as defined by Kramp (1962).

20.?

204

EDWARD C. ROOSEN-RUNGE

Several settling experiments were undertaken (see Results). The simplest method proved to be the most efficient. A dozen 3 X 1 inch microscopic slides were arranged on the bottom of a large bowl containing many hundreds of planulae. After 6-12 days, 50-66% of the slides were found to have at least one primary hydroid growing on them. Such slides were hung in plastic frames, each holding 5 slides into the tanks in which most of the hydroid colonies and many medusae were raised. More than 30 cultures were observed in detail for nearly 2 months and many more were used occasionally for short-term observations.

The tanks were modified after the "Plankton-Kreisel" described by Greve (1968). The principle of the "Kreisel" is a rotary circulation in which inflow into an outer compartment and outflow from an inner compartment are separated from each other by the sand on the bottom. The "airlift" of the inner compart- ment was omitted as unnecessary in an open seaw-ater system. The Kreisels (Fig. 1) were made out of carboys by removing the tops. They were 31-32 cm

Inflow

Outflow

Inner compartment

FIGURE 1. Sketch of the modified "Kreisel" used in raising hydroid colonies and medusae.

high and 26i cm in diameter. A self-syphoning outflow was arranged so that the volume of water in the tank oscillated between approximately 15 and 12| liters. Flow was kept between 5 and 20 liters per hour. Occasional plugging of the water lines caused irregularities and standstills which did not noticeably influence the experiments. The tanks were standing in 5-8 cm of seawater with continuous flow which assisted in maintaining a low temperature even when the circulation through the Kreisels stopped. At the inflow into the water tables, the temperature of the seawater varied from 10-13.5° C depending on the outside temperature. At an average temperature of the inflow of approximately 12.5° C, the water in the tanks had a temperature of 14° C at a flow of 14 liters per hour, 15° C at a flow of 9 liters per hour. In general, temperatures declined toward the fall.

For examination and counts, the individual cultures on their slides were re-

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205

Culture 3H, 2 • =gonangium

Side ! ° ' hydranth

-&-- branched, (2), hydranths

A = empty hydrotheca

® = immature hydranth or sprout

K) - empty gonangium

ml ni

Side 2

FIGURE 2a and h. Semidiagrammatic representation of a clonal hydroid culture of Phialidium f/rcyariii>n on a 3 X 1 inch slide, after a direct tracing. The culture was drawn 34 days after the planula settled and 21 days after the first side branches appeared on the stolon. Figure 2a shows the side of the primary hydroid.

moved from the tank and inspected submersed in a Petri dish. Cultures which grew on both sides of the slides were supported by a plastic ring (diameter f inch). Counts of hydroids and gonangia were made with the help of a 1 X 3 inch glass slide on which 14 areas 1.5 X 1.1 cm were drawn. This was placed directly under the culture slide and under the plastic ring.

Microscopic observations, photographs and measurements were made of living hydroids, gonangia and small medusae on a cold stage using an electric module (Cloney and Schaadt, 1970). On this stage the animals and their parts were kept very conveniently in depression slides at 10-13° C.

206

EDWARD C. ROOSEN-RUXGE

RESULTS

Results will be presented by first recounting observations on phases of tbe life cycle: settling, primary hydroids, growth of a stolon and trophosome, formation of gonosome, and growth of planktonic medusae. Secondly, the morphological features of the hydroid will be reported in detail, and the hydroid be denned summarily. This definition will then be compared with descriptions of related species in the literature, and an attempt will be made to identify the whole animal, inclusive of both its hydroid and medusal phases, by suggesting a single proper name.

^40-

-Cl

Culture 1,14

O— o hydranths •--• gonongio

I first medusoe * liberated

oo — o ,/

120-

V.

-d

40-

10 15 30 35 40 Days

50 55 60

Culture H, 30

O— O hy d ranlh s •--• gonangia

I first medusae f liberated

20 25 30 35 40

50

160-1

120

V.

«J

c 80- t

I

40 i

Culture 1,11

O— O hydronths •--• gonangia

first medusoe t liberated

Cr-°

Culture 1,10

hydranths gonangia

30 35

40 45 Days

50 55 60

80n	\	\	first medusae liberated
|40-	^
X3			"•"Q>s-.
^ -	0^°" '' x	^	^O
0-	•-•' cxJJ		.--•••
1 i i	' 1 1
30 35 40 45	50	55 60
Days

FIGURES 3-6. Graph of numbers of hydroids and gonangia during development of clonal hydroid cultures. The lines between points were drawn only when frequent observations justified the assumption of continuity of slope, even if no counts were made in the interval.

The hydroid culture — settling

The planulae of P. gregarium settle on many substrates with great ease ; for instance, on glass, plastic, wood and algae. (The non-glycogen polysaccharide glue and the glandular cells which produce it have been discussed by Bonner, 1955.) They will settle on vertical as well as on horizontal surfaces. In one of the Kreisels in which large numbers of medusae were kept, several blank microscopic slides hanging vertically in the current began to show growth of hydroid colonies after a few weeks. In other settling experiments it was found that planulae often accumulated at the walls of the vessels near the waterline, and attempts were made to utilize this tendency by putting many slides in "staining jars" with the waterline running lengthwise along the middle of the slide. These experiments failed, perhaps, because the amount of water in these jars was very small.

In repeated experiments with large numbers of eggs the first planulae to become sluggish and settle were observed in the second half of the third day after

HYDROMEDUSA LIFE CYCLE

207

fertilization. Approximately half of the planulae settled on the fourth and fifth days, but even on the twelfth day a few planulae were still swimming. In this species which shows little selectivity in the substrate for its hydroid phase, the time of settlement appears to be in large part determined by an endogenous readiness of the planula.

O— O hydronths qononqio

25 30 35 40 45 Days

Culture 1,6
200-	O— — O hydronth s •--• gonangia
160-	first f medusae liberated
120-
80-
40- o- 3	c \- __^
0 35 40

45 Days

50

I60-,

120-

Culture 1,23

O— O hydronths gonangia f irst medusa

40 45 50 Days

55 60

Number -fc 00 ) 0 0	O — O hydranths Culture 11,5 • - -• gonangia 1 first medusae liberated 1 »-Q O ••-• «^	S
UT^ 20 25 30	35 40 45 Days	50

FIGURES 7-10. Graph of numbers of hydroids and gonangia during development of clonal hydroid cultures. The lines between points were drawn only when frequent observations justified the assumption of continuity of slope, even if no counts were made in the interval.

Primary hydroids; first growth oj stolon

Once a planula has settled it flattens, rounds off and spreads into a pedal disc with 4 to 6 lobes within 2 days. It then forms a stalk on which a hydranth develops. Hydranths capable of feeding usually are observed on the third or fourth day after settlement. The primary hydroid will be described in some detail below (Figs. 11-15). There appears to be a period of several days during which there is little visible change in the pedal disc. If a hydroid is fed abundantly during this period it may regress and the disc develop one or more new hydroids which are usually somewhat larger than the first.

JOS

EDWARD C. ROOSEN-RUNGE

11

12

19

FIGURES 11-13. Photomicrographs from living clonal cultures of Phialidium gregariwtn. (Figures 11-15 show primary hydroids.) The bar represents 500 /j. (also for Figures 12 and 13.) Compare hydroids in Figures 11-13 with respect to number of tentacles, size, proportions of hvdrotheca and features of annulations.

HYDROMEDUSA LIFE CYCLE 209

A stolon is visible usually a week after settlement. It grows frequently from one pole of the pedal disc, but sometimes from two opposite poles. Initially, the stolon advances in an almost straight line, even when it progresses in two opposite directions.

Secondary hydroids; brandling of stolon

As the stolon grows, secondary hydroids are sprouting from it at rather regular intervals ( 1-3 mm in young cultures) which tend to become larger as the colony expands. Hydroids tend to become taller, develop more tentacles and branch more frequently (details are described below) but the degree to which this happens depends largely on the amount of food available. As long as colonies received only occasional feedings by pipette in addition to a small amount of planktonic organisms from the water supply, growth was slow and the hydroids which developed were only slightly more differentiated than primary ones. The first month of culture I, 14 (Fig. 3) serves an example. A primary hydroid was present on day 7 after fertilization. Three hydroids were counted on day 13, 14 and 17. \Yhen the culture was next examined in detail, on day 31, it had 10 hydroids. The culture had only subsisted and had, in fact, been somewhat damaged, so that the stolon was interrupted in several places. From that day on it was fed with very large numbers of brine shrimp several times daily and after 10 days there were 34 feeding hydranths. many of which were large and had 20 tentacles or more. Despite continued abundant supply of food (on day 41 the culture was completely saturated with brine shrimp under the microscope, each hydranth receiving many shrimp), within the next 4 days the number of feeding hydranths fell to 3, but in the following 4 days the number of feeding hydranths went up to 54. The eight examples in Figures 3-10 and all other cultures observed showed the same phenomenon. Boosting the food supply did always initially stimulate the growth of hydranths and, to a lesser extent, of stolon and hydroids. On the other hand, the hydranths \vere short-lived, if they fed heavily. A single hydranth which had ingested 6, 8 or even 10 freshly hatched brine shrimp usually regressed within 2-1 — 18 hours and only the empty hydrotheca was found in its place. Hydranths which were fed poorly often lived for many days. A lifespan of a week was observed several times. The stolon did not appear to regress after any amount of feeding. Xeither did it easily respond to starvation. In cultures neglected for several weeks the stolon appeared to contain a living cenosarc

FIGURE 14. The bar represents 200 /JL. Phase optics permit identification of teeth at rim of hydrotheca. At left a "rib" may be discerned running downwards from one of the teeth.

FIGURE 15. The bar represents 50 /u. Phase picture of the distal annulations on the stem of a hydroid. Cytoplasmic processes are shown extending from cenosarc to hydrotheca in the region of a constriction.

FIGURES 16 and 17. Secondary hydroids. Bar represents 500 p. in Figure 16, 1 mm in Figure 17. Compare the hydranths in Figure 11 and 12 with that in Figure 16 (same mag- nification) and 17 (less than half the magnification). The hydroid in Figure 17 was 7 mm tall in life.

FIGURE 18. The bar represents 500 /*. Gonangium with 4 gonophores, arising from a stem.

FIGURE 19. The bar represents 200 /*. An immature gonangium (left), approximately 1 day old, and the base of a hydrocaulus (right) arising from a stolon. Note annulations and the difference in thickness of hydrotheca and gonotheca.

210 EDWARD C. ROOSEN-RUNGE

throughout, even when only a few small hydroids were present. These results are, in general, similar to those of Crowell (1953) who studied growth and regression in hydroids of Campanularia kept at various levels of nutrition (brine shrimp).

During the initial slow growth of the colony the stolon did not branch. The earliest branching was seen 2 weeks after fertilization in a culture which had settled 8 days previously. At first branching there were as few as 3 hydroids and as many as 10 or more. Branches tended to occur at right angles (Figs. 22-25). When branching began the culture expanded rapidly. It reached the edge of the slide somewhere before another week had passed. The edge presented no obstacle (Fig. 23). Figure 2, for example, demonstrates that 3 weeks after the first branching of the stolon a colony may have grown around the edge of the slide at 17 different points. A culture of approximately the same age not confined to a slide, but growing on the wall of the tank, extended over a circular area with a diameter of approximately 7\ cm. It appears that for the first 2 months of a colony's life the area presented by the two sides of a 3 X 1 inch slide does not limit expansion to any appreciable degree.

Growth of gonosome

The earliest formation of gonangia occurred 19 days after fertilization. This was observed several times in mass cultures from many eggs of a single spawning. In the cultures raised from one egg the earliest gonangia were seen after 21 days. In the 8 cultures represented in Figures 3-10 the first gonangia were observed on days 36-45. When a gonangium was first unmistakably recognized, it was a small, dense, elevated structure of mushroom shape (Figs. 19, 22, at arrows) very different from the thin stalks of budding hydroids. Such early stages of gonangia appeared in locations where 12 hours before there had been no indications.

Figures 3-10 demonstrate that the first appearance of gonangia is regularly preceded by an increase in the number of hydroids which begins 7-4 clays earlier. For instance, culture II, 30 (Fig. 4) showed an appreciable increase in the number of hydroids on day 34, culture I, 11 (Fig. 5) on day 41 or earlier; the first gonangia were observed on day 40 and 45, respectively. Culture I, 10 (Fig. 6) demonstrates two periods of gonangial growth, the first beginning on day 36, the second on day 52. The first was preceded by a burst of hydroid formation beginning on day 32, the second by one beginning on day 48. There was no clear correlation between the peaks of hydroid development and the beginning of gonangial growth. (Compare, for instance, Fig. 7 where a peak almost coincides with the appearance of gonangia with Fig. 8 where it comes much later, or Fig. 3 where it precedes.) Nor is every rise in the number of hydroids followed by the appearance of gonangia (Figs. 3, 7).

Gonangia develop either from the stolon directly (Fig. 22) or. more rarely, from the stems of hydroids (Fig. 18). When they spring from the stolon they do so almost invariably close to a hydroid stem and characteristically equi- distant on both sides of it (Fig. 22). Their location within the culture is predict- able in certain respects. They occur on parts of the stolon which have reached a certain minimal age (1-2 weeks?). They never are formed by new side branches of the trophosome but are most frequently found on the middle portions of long stretches of stolon which are by their very positions identified as relatively

HYDROMEDUSA LIFE CYCLE 211

mature. This is shown, for example, in Figure 2 which also demonstrates that the side of the slide on which the primary hydroid grew (Fig. 2a) has far more gonangia than the other side which is on the average younger. Because of the relatively short duration of tin- observations it cannot be stated whether there is also an upper age limit beyond which any part of the stolon becomes incapable of producing gonangia. In any case, this hydroid presents a pattern very different from one like Podocor\nc ( Braverman and Schrandt, 1969) in which a dense center is seen populated by sexual and nutritive polyps.

Xo hydroid culture was observed continuously for longer than 7 weeks. A few cultures were seen to have two phases of formation on gonangia during this period (Figs. 3, 6). The amounts of proliferation of stolon, hydroids and gonangia varied greatly from culture to culture, but appeared to be largely dependent on food supply in every case. There was no evidence that fluctuations in temperature or the lessening of daylight with approaching fall had any influence. At the end of September, 15 cultures, 2-2\ months old. were suspended from the dock at 15 foot depth for a wintering experiment. They were contained in a plastic cage which permitted open circulation. At the end of November, 6 of these cultures had been destroyed by some predator (nudibranch?). At this time, the cage was wrapped tightly in nylon screening to prevent further predation. Nine cul- tures were alive but much reduced from their state in September and showed only 1-6 hydranths of primary type, i.e., very small with relatively few tentacles. At the end of March the cultures were once again examined. Six of them were alive and showed vigorous growth which apparently was of recent occurrence because detritus on the hydranths was minimal and empty hydrothecae were rare. The largest cultures had more than 30 hydranths with up to 20 tentacles. Three cultures showed gonangia in small numbers, in stages of development which indicated that their development had begun from 2-7 days previously.

Liberation, growth and maturation of medusae

The first medusae were liberated from hydroid cultures 26 days after fertiliza- tion. This occurred usually after 35-45 days and once after 52 days. Many cultures had not begun the formation of gonangia when observation ceased in the fall, but none of these were more than 6 weeks old. and there is no reason to assume that they would not have produced medusae eventually. All cultures observed for 52 days finally did liberate medusae. There is no indication that productivity became less with the approach of fall.

The interval between the first appearance of gonangia and the first free medusae was always between 4 and 5 days (for instance, Figs. 3-10). After medusae first appeared the cultures continued to produce for 6-10 days, which reflects the duration of a single period of formation of gonangia. During such a period 70 to more than 250 medusae were produced.

Newly hatched medusae (Fig. 21) measured 1.2-1.4 mm in largest diameter. They had 4 tentacles and 8 lithocysts, conforming in this to the defining character- istics of the genus Clytia ( Hincks, 1868). In addition, they displayed 4 immature tentacular buds. The gonadal Anlagen were very inconspicuous but were clearly defined under higher magnifications and phase optics. The tiny medusae were able to feed on single brine shrimp while they were still connected to the blastostyle,

212

EDWARD C. ROOSEN-RUNGE

21

22

24

25

FIGURES 20-25.

HYDROMEDUSA LIFE CYCLE 213

but even when unfed they expanded rapidly to a diameter of 2.5-3 mm during the first 2 days and after that time they had 8 tentacles. Freshly hatched medusae tended to remain "hung up" within the hydroid culture, even when it was vertically suspended, and were often seen somewhat entangled in the tentacles of hydroids. There was no evidence that they were ever ingested or harmed.

A few medusae were raised to near sexual maturity in 4 weeks. These were kept in fmgerbowls, were handled and transferred into fresh seawater daily. After 3 weeks they had 23 tentacles in the average with 8 tentacular buds, and their diameter was approximately 6 mm. After 4 weeks they measured approximately 1 cm and immature oocytes were discernible in the female gonads under the micro- scope. It was evident that the rate of their development depended greatly on feeding, water circulation and temperature. Certainly, the conditions for their development were not optimal in the present investigation. As long as extensive attempts have not been made to establish a better method of raising medusae, possibly in the Kreisel, the fastest time of their development to sexual maturity remains unknown, but one can predict with confidence that it will be less than 4 weeks.

It is of some interest that small medusae with tentacle numbers and diameters very similar to those raised in 3—1 weeks in the laboratory were frequently found in the Friday Harbor Bay in the first 10 days of September.

Morphology of hydroids

In the following paragraphs the range of variability of features conventionally used in taxonomic identification is presented point by point for the hydroid of P. gregarhtm.

Trophosome — size and branching

A great number of primary hydroids were observed and measured. They branched only very rarely. Variations in size were relatively small. The distance from pedal disc to rim of hydrotheca was in the average 1.4 mm. the range 1.25—

FIGURE 20. Photomicrographs from living clonal cultures of Phialidium grcc/urittin. The bar represents 200 /j.. Phase picture of the terminal part of a 3-4 day old gonangium (same magnification as in Figure 19) showing the lid of the blastostyle and one gonophore. The upper two oval black spots are tentacular bulbs, the larger irregular black spot below is the manubrium.

FIGURE 21. The bar represents 500 /a. Medusa of Phialidium i/rcf/uriiiin just after hatch- ing. Four tentacular bulbs (2 out of focus), 4 buds of tentacles (2 out of focus) and 4 of 8 lithocysts are clearly seen. The medusa is slightly flattened in a depression slide and conse- quently appears wider and lower than normal.

FIGURE 22. The bar represents 1 mm in Figures 22-25. Gonangia are shown (arrows), the lower two originating on both sides of a hydroid stem departing from the stolon to the left; the upper gonangium originates near another hydroid stem seen as a hook off the stolon slightly to the left below.

FIGURE 23. A damaged stolon (arrow) winding around the edge of its slide. The cenosarc is discontinuous; the theca extends intact around the edge and the stolon is continued out of focus, above and parallel with the one on the upper side of the slide.

FIGURE 24. A crossing of one part of the stolon over another one.

FIGURE 25. The focus is on two hydranths. A characteristic pattern of hydroids and stolon branching is shown.

214 EDWARD C. ROOSEN-RUNGE

1.55 mm. Subsequent hydroids in an adequately fed colony arc. gradually in- creasing in size. In the beginning of colony formation, each new hydroid formed along a stolon may be larger than the last, but with the beginning of branching of the stolon new hydroids are not always bigger, but often much smaller than the last ones funned, particularly on new branches of the stolon. Branching becomes more frequent with age in most colonies, but not in all. After a few weeks some cultures show as many hydroids branching as non-branching; others may have hardly any branches but only relatively tall single hydroids. The largest single hvdroid measured was 7.52 mm tall from stolon to rim of hydrotheca with tentacles 1.6-1.8 mm long. Branched hydroids occasionally exceeded a centimeter in height. As no experiments were made with a continuous maximal food supply or with different types of food, it can only be surmised that under some conditions the size of the colonies may be greater than the largest observed in the present investigation.

The variability of the hydroids of P. gregarium with regard to size and branching, but also with regard to number of tentacles and annulations (see below) is, perhaps, characteristic for the genus Clytia. Berrill (1950) has pointed out that hydroids of johnstoni are similarly variable in contrast to hydroids of, for instance, Campanularia or Obelia.

Tentacles. Primary hydroids (Figs. 11-14) were observed to have 8-12 tentacles, 11 in the average. The tentacles were 400 /j. or less in length. Large secondary hydranths (Figs. 16, 17) had up to 25 tentacles, frequently 20-22, and the tentacles \vere 1.6-1.8 mm long. It should be emphasized, however, that the same colonies which possessed many large hydroids usually had some small ones with low numbers of relatively short tentacles.

Hydrotheca. The size of the hydrotheca varied greatly. The length from diaphragm to rim measured in 10 primary hydroids was in the average 440 ju, the range 385^470 /*.. The diameter at the rim, usually the largest diameter, was in the average 150 /*, range 110-185 p. In 10 large hydroids from mature colonies the average of the same measurements was 1100 ^ (range 1000-1185 /A) and 410 p. (range 390-430 ju). In general, secondary hydrothecae tended to be slightly less elongate in shape than the primary ones. Hydrothecae of primary hydroids had 8-11 teeth (Fig. 14) which were sometimes pointed, but often blunt or partially broken. Empty hydrothecae often showed no teeth at all. This variability was attributed to the fragility of the thin teeth. The tentacles usually are draped over the edge of the hydrotheca within the cusps between teeth. Their movements appear to wear the rim easily, particularly when brine shrimp are fed which are large in relation to the hydranth and cause great wear and tear of the tenuous perisarc. It was difficult to find intact hydrothecae on large secondary hydroids. Usually 10 or 11 teeth were counted, but one hydrotheca possessed 13 teeth. With phase optics 4 and occasionally 8 ridges could be seen to extend downward about ^ of the length of the hydrotheca (one is visible in Fig. 14).

Annulations. As a rule annulations were found distally, on the stem im- mediately under the hydrotheca (Figs. 11-14, 16) and basally where the stem sprang from the stolon (Figs. 11, 12). In primary hydroids the distal annuli varied from 3-7 (rarely more) and were in the average 33 /j, high (range 28-41 //.) ; basal annuli varied from 12-16 and were 31 ^ high (range 27—43 /A). The zones of annulation often ended sharply and the part of the stem between them was

HYDROMEDUSA LIFE CYCLE 215

smooth (Fig. 13), but not infrequently the annuli were seen to become shallower and higher ;md give way to undulations throughout the center part of the stem (Fig. 11). Large secondary hydroids had a different pattern. Distal annuli were greater in number, 6-10, and much higher, 66 ju, in the average (range 48-80 p.} (Fig. 16); basal annuli (Fig. 19) were less numerous, 9-14, and in the average, 48 /*, high (range 35-53 /*,). Cytoplasmic processes were seen which ran from cenosarc to perisarc, usually at the constrictions (Fig. 15, arrows). It was also observed that contractions of the cenosarc increased the curvature of the annuli. A thorough study of the way in which the hydrotheca is laid down and maintained may in the future bring a better understanding of the nature of the annuli, and clarify their value as a taxonomic feature. This topic has been discussed pertinently by Berrill (1950, 1961).

Gonosom c

Ori</in, size and shape. The size and shape of a gonangium depends to a large extent on the state of its development. Small gonangia are funnel-shaped and the gonotheca is closely applied to the early gonophores. At this stage, it may appear that the pedicel is quite long and has from 5-12 annuli (Fig. 19). The pedicel is relatively shorter and has less annuli in more mature gonangia and in gonangia originating from a stem (Fig. 18). The envelope of the gonangium about the pedicel is smooth, an important distinction in comparison with the corrugated gonangium of C. johnstoni. A gonangium containing 5 medusae may grow taller than 3 mm and exceed 4- mm in largest diameter which is at the equator of the largest gonophore and not at the upper rim (Fig. 20). The number of gonophores varies, however, from 2-7 and gonangia are consequently smaller or larger. Usually the majority arises directly from the stolon in the vicinity of a hydroid stem. Others arise on stems which often have 2 hydroids and may finally have also 2 gonangia. The frequency of these sites appears to vary distinctly between different colonies.

Summary definition of the hydroid of P. gregarium

Trophosome. Colonies of hydroids predominantly single but branched increas- ingly as a function of age and food supply. Height of primary hydroid less than 1.6 mm. Second hydroids up to 10 mm or more, depending on conditions. Hydrocaulus long, annulated at base and at top ; intermediate portion generally smooth or undulating. Hydrotheca deeply campanulate, length to width 2.5-3.0:1 expanding very slightly above, with 8-13 teeth. 4 or rarely 8 fine ridges from rim downwards through upper -J of hydrotheca. Stolon branching predominantly at right angles.

Gonosome. Gonothecae ovate, outline smooth or somewhat undulating with a distinct collar at the opening, 3 mm or more in height when mature, arising from stolon with relatively long pedicel (up to 14 annuli) or from stem with shorter pedicel (4-8 annuli) ; 2-7 gonophores in each gonangium.

Taxonomic consideration

Now that the life cycle of the leptomedusa authoritatively identified (Kramp, 1962) as Phialidiiim (/ret/ariitm (A. Agassiz) in L. Agassiz, 1862, page 353,

216 EDWARD C. ROOSEN-RUNGE

Oceania grcgaria. has been observed in the laboratory, the question of the proper scientific name of the animal must be briefly considered. This question contains two problems: (1) the identity of the hydroid which has been shown to develop from the eggs of P. gregarium; and (2) the selection of one name for both the planktonic and the sessile phase of the species.

Identification of hydroid. Certainly the hydroid belongs properly to the genus Clytia established by Lamouroux (1816) as used by Nutting (1915), Fraser (1937) and others. For convenience sake the hydroid is called Clytia X in the following paragraphs. Hincks (1868) in defining the genus stated as its main characteristic the production of medusae with 4 radial canals, 4 marginal tentacles and 8 lithocysts. Nutting (1915, page 53) expanded the definition by describing the trophosome : "Colony often simple but always consisting of a creep- ing rootstock from which spring pedicels which are not regularly branched as a rule. Hydrothecae companulate, hydranths with trumpet-shaped proboscis." All these characteristics are observed in Cl\tia X.

It is to be expected that the species of Clytia which liberates medusae con- forming as adults to the description of P. gregarium, will be found among the hydroid species described for the Pacific Coast and preferably for the Northwest Pacific Coast of North America. No more than two species of Phialidium are ordinarily found in Puget Sound and around the San Juan Archipelago, P. gregarium and P. Jicmisphcriciiin. The last is not discussed here, but I can state on good, if not conclusive, evidence, that it occurs. Fraser (1937) listed not 2 but 12 species of Clytia from the Pacific Coast. Five of these, namely C. attcnuata (Calkins, 1899), C. bakeri (Torrey, 1904), C. hendersoni (Torrey, 1904), C. minitta (Nutting. 1901) and C. universitatis (Torrey, 1904), may be excluded because of their large size and completely different growth habits. C. incoiispicua (Forbes, 1848) may be eliminated from consideration because it is in all probability synonymous with C. johnstoni (Alder, 1856). C. johnstoni has distinctive corrugations on the gonotheca. It is a well described species (Hincks, 1868; Russell, 1953) and has been clearly established as the hydroid of Phialidium hemisphericum (Wright, 1858). Calkins (1899) found C. inconspicua near Port Townsend in Puget Sound, but stated that Alder (1856) and Hincks (1868) had found it in England. Hincks reported that Wright ( 1858) had raised this hydroid from jellyfish, which appears to establish the fact that it is the sessile phase of P. hemisphericum (Wright, 1858) and not of P. gregarium (A. Agassiz, 1862) which has not been found in Atlantic waters.

The question remains whether any one of the three species, C. cylindrica (Agassiz, 1862), C. kinkaidi (Nutting, 1915) and C. longithcca (Fraser, 1914), can be singled out as uniquely fitting the description of Clytia X. The answer is somewhat ambiguous. C. cylindrica was well described by L. Agassiz (1862), page 306. In his illustration the gonangium appears very similar to that of Clytia -V, but is described as distinctly flattened while that of Clytia X is round. Agassiz found this hydroid on the New England Coast where P. gregarium has never been observed. Fraser (1937) stated that it was found at Friday Harbor, but his description while less clear than that of Agassiz adds another discrepancy, a short pedicel with only one or two annulations. C. kinkaidi is found in Puget Sound (Nutting, 1915) and is similar to colonies of Clytia X in its early phases;

HYDROMEDUSA LIFE CYCLK 217

it is described as not branching and as depicted by Fraser appears to be of nnicb smaller size than Clytia X. It is especially similar in the features of ribs on the hydrotheca and of long gonangial pedicels. Finally, C. longitheca, reported for California and Vancouver Island, is also generally similar to Clytia X and uniquely so with regards to its long hydrotheca. Branching is not one of its features, however, and the gonangia apparently were never observed to spring from the stems but only from the stolon.

In summary, although the above three species show many general similarities. none can be unequivocally identified with Clytia X. Jt is probable1 that the descrip- tions in the literature partly pertain to Clytki X in at least one of its growth phases but they remain ambiguous and confusing. It is, therefore, proposed that the hydroid of P. gregarium be named Clytia gregaria. The specific gregarium or -ia. is. indeed, the senior name of the species, if it is granted that "Clytia X" is excluded from any species previously recognized. This designation has the inherent advantage of being immediately associated with the planktonic phase of the hydroid. C. cylindrica, kinkaidi and longitheca may come to be considered synonyms of this species once the full range of variability is demonstrated.

Identification o\ the animal. Once an animal has been observed continuously throughout its life cycle, there is every reason to bestow a single scientific name on it. Vet, in the case of hydrozoa with two life forms, this has become a difficult matter because the traditional pattern of the nomenclature appears to present almost insuperable obstacles which can be resolved only through an inordinate amount of scholarly work. For instance, Clytia johnstoni and Phia'idiitin hemi- sphcricum unquestionably refer to the two phases of the same animal, but authori- ties, such as Russell (1953), have not proposed one proper name for the animal, and the case of C. gregaria and P. gregarium may be very similar. On first sight, the genus Clytia appears appropriate for reasons of priority, but there are probably difficulties in establishing the genus Clytia (and Campanularia for that matter ) beyond all doubt, a task which the present author considers beyond his competence. On the other hand, one might suggest that the species discussed in the present paper be named in both its forms Phialidium gregarium (A. Agassiz) in L. . \gassiz, 1862, p. 353. This circumvents the possible difficulty of having to revise the nomenclature with respect to the genera Clytia and Campanularia which have been thoroughly entrenched for more than a century.

DISCUSSION

It was demonstrated in the first part of this paper that the "Plankton- Kreisel" (Greve, 1968) facilitates the raising of a great number of hydroid cul- tures from single eggs. There is no claim that the Kreisel is the only or even the best method to do this. Rees and Russell (1937), for instance, have raised hydroids of Amphinema, Rathkea and Mitocomella in plunger jars and did not even find the renewal of water very necessary, although it proved useful for reviving unhealthy colonies. The hydroids of Phialidium grow quite easily in small vessels, particularly in mass cultures, if there is a continuous flow of water. Clonal cultures, however, appeared to be more visible, accessible and experi- mentally controllable in the Kreisels. For optimal culture conditions of small

218 EDWARD C. ROOSEN-RUNGE

medusae the Kreisel may well be uniquely suited, but the recent investigation offered only very limited experience for medusal culture.

The availability of many individual hydroid cultures created opportunities for observation and experiment which have only been explored tentatively. Only those of most immediate interest in connection with problems of the general biol- ogy of Phialidium will be discussed here. There has been much speculation as to what causes the waxing and waning of swarms of jellyfish. The medusae of P. gregarium appear each year around the middle of April at Friday Harbor and dis- appear almost completely by the middle of September. Medusae of the species tentatively identified as Phialidium hcmisphericum (Wright, 1858) behave in approximately the same way, which is of interest because this species in contrast to P. gregarium is circumpolar and its behavior in different localities has been well described. At Plymouth, England, large medusae are found in spring and summer (Russell, 1938, 1953), in Danish waters in winter and in spring (Kramp, 1929). On the other hand, small medusae are seen at Plymouth and in the Atlantic in midwinter and, indeed, throughout the year (Lebour, 1922). The behavior of hydroid cultures in the laboratory suggests a reasonable hypothesis on the cause of the appearance and disappearance of medusal swarms, which can be tested in the future. The hypothesis states that hydroid colonies of the genus Phialidium grow and produce medusae in direct response to the food supply. They react with increased formation of hydroids to major increases in the supply of brine shrimp, and a major increase in hydroid formation is followed by liberation of medusae within 2 weeks. It is evident in the laboratory that small amounts of food are not effective as stimulus; only a sustained, heavy supply of suitable food did induce medusae in the numbers necessary for the remarkably dense swarms often seen in the summer. According to the hypothesis, there should be a considerable increase in the density of planktonic organisms on which the hydroids feed 5 to 6 weeks before swarms of mature medusae are found. At Friday Harbor this appears to be the case. Johnson (1932) showed that swarms of copepods, which presumably constitute the bulk of hydroid prey showed a first noticeable rise in March and continued to increase in bursts through May. March and April also saw a great increase in barnacle nauplii. Particularly relevant may be the behavior of the copepod Calanus finmarchicns which appeared on limited occasions in March, April and May, very suddenly and for only a few days in tremendous numbers in the 5 years investigated. The average plankton density in surface waters usually rose in March, reached its peak in June or early July and fell precipitously in August. It is obvious that these data which take no cognizance of vertical migra- tions or of the preference of the hydroids for certain food organisms are not satis- factory for detailed support of the hypothesis, but they fit in a general way. A rise in plankton density in March should bring a great increase in mature medusae by the second half of April. The increase should continue until August, partly because further waves of food supply may repeatedly stimulate old hydroid colonies, and partly because the eggs of medusae spawning from April on must give rise to new hydroids and these to new medusae which will mature first in July (6-8 weeks after fertilization). It appears that the life span of individual medusae does not exceed 3 months (all findings on medusae raised in the laboratory either from hydroids or from young marine specimens support this view) so that medusae

HYDROMEDUSA LIFE CYCLE 219

liberated at the probable peak of gonangial production in June, should disappear in September. Small growing medusae with 16-32 tentacles are, however, found until early September, but they occur infrequently and at best in small swarms of low density which must have a very low fertility.

It is obviously not a new and original finding that hydroid colonies are dependent on food supply for their growth. In fact, the results in the present paper appear to confirm, in general and in many details, the more extensive investigations of Crowell (1957) who explored the responses of growth zones in the hydroids of Catnpannlaria to various nutritional levels, and who also used brine shrimp for food. He found that of all growth zones the tip of the main stolon is least affected by nutrition. Hydranth growth reacted strongly and within a very few days to restriction or increase in food. Gonangia, however, were produced only by the two best fed of 8 graded experimental groups.

From laboratory findings one would expect small numbers of medusae to be liberated even in times of relative scarcity of food because of short-term local abundance of one or the other food organism. There has been no thorough search around Friday Harbor for single medusae during the months from September to March, but occasional single specimens have indeed been found. It is important, however, to realize that in order to produce swarms of fertile density the rate of hydroid and medusal production must be extremely high, and that there is probably a critical limit below which such accumulations cannot occur. Swarms must originate in localities where production occurs with great temporal and spatial density. In laboratory cultures a few of the factors which further this can be observed. (I) The stimulus of food, provided it is of sufficient magnitude, appears to act on gonangial formation rather precisely and cause a great number of gonangia to arise within a relatively short time. (2) The liberation of medusae occurs most frequently between the hours of dusk and dawn, probably most usually in the early morning, which would tend to concentrate the release of medusae. (3) Small medusae tend to stay on the bottom and to be kept there in between the hydroids and gonangia, sharing the food supply with the hydranths. Water movement will flush large numbers of medusae out of the colonies within very short intervals of time. In addition, the medusae show a tendency to swim away at the end of the first week.

Finally, it is now possible to make an estimate within an order or two of magnitude of the productivity of a small hydromedusa such as P. gregarinm. A female may produce an average of 50 eggs per day for 60 days (Roosen-Runge, 1962) or 3000 eggs in its lifetime. Each egg may produce minimally 300 medusae in a season. The maximal number is as yet unknown and may be many times as large. Potential productivity of each fertile couple is, therefore, at least 1 million medusae or more, which are, in the average, reduced by predators, tides and winds to another couple upholding the race.

This research was supported by Grant GB 8094 from the National Science Foundation. I wish to thank Dr. Paul L. Illg for his invaluable, critical assistance with the taxonomic part of this paper. Thanks are also due to Mr. J. Rankin for his skillful construction of the Kreisels and much other equipment, to Miss U. L.

220 KDVVAKI) C. ROOSKN-RUNGE

Roosen-Runge for technical assistance and drawings, and to Mrs. I). Ringer and Mrs. J. Tada for secretarial help.

SUMMARY

1. A method was described by which hydroid colonies, each on a microscopic slide, were raised from individual planulae of the leptomedusa known as Phialidium gregarium.

2. Growth and behavior of approximately 30 cultures were observed and ([iiantitated through nearly 2 months. Cultures produced gonangia and medusae 3-7 weeks after fertilization of the egg.

3. Gonosome development was always preceded by a burst of hydroid develop- ment 5-9 days previously. This burst appeared to be initiated entirely by an abundant food supply (brine shrimp).

4. Medusae were liberated 4-5 days after gonangia first appeared. From the growth rate of medusae under the less than optimal laboratory conditions, it was estimated that sexual maturity may be reached in approximately 3 weeks. The lifespan of the medusae probably does not exceed 3 months.

5. A small number of cultures wintered in an open cage in the sea. At the end of November they were found in a greatly reduced state and possessed only a very few, very small hydranths. At the end of Marcli they were in the early phases of vigorous trophosomal growth and the gonosome had just begun to flourish.

6. From the behavior of clonal colonies in the laboratory, the hypothesis was derived that the swarms of mature medusae in nature are the direct result of a "bloom" in zooplankton which occurred 5-7 weeks previously and stimulated first hydroid development and in consequence the formation of gonangia.

7. Morphological characteristics of the hydroid colonies were described and their variability under laboratory conditions recorded and discussed. It was shown that many taxonornic features such as size, branching, number of tentacles and annulations depend quantitatively on the age of the individual colony, its state of nutrition and on genetic factors.

8. The hydroid belongs to the genus Clyti-a (Lamouroux, 1816). A careful comparison was made with species of the genus as described for the Puget Sound region and California. It was concluded that none of these delineates unambigu- ously the morphological features of this hydroid. A tentative name for the hydroid was assigned according to priority principles as Clytia grcgaria. It was suggested that I'liialidiuin c/rec/ariinn may be the most appropriate name for the species in both its phases.

LITERATURE CITKD

AGASSI/, L., 1862. The genera of the Campanularians. Contrih. Nat. Hist. U. -V.. 4 (Chapter

VII) : 353. ALDER, J., 1856. A notice of some new genera and species of British hydroid zoophytes.

Ans. Maf/az. Natur. Hist.. Scries 2, 18: 353. BERRILL, N. J., 1950. Growth and form in calyptoblastic hydroids. II. Polymorphism within

the Campanularidae. /. Mnrphol., 87: 1-26. BERRIU., N. J., 1961. Growth, Development mid Ptiltcrn. Freeman and Company, San

Francisco and London, 555 pp.

HYDROMEDUSA LIFE CYCLE 22\

BONNER, J. T., 1955. A note concerning the distribution <>f polysaccharides in the early

development of Phialidium gregarium. Biol. Bull.. 108 : 18-20. CALKINS, G. N., 1899. Some hydroids from Puget Sound. Proc. Boston Soc. Natitr. Hist.,

28 : 333-367.

CLONEY, R., AND J. SCHAADT, 1970. Thermoelectric cooling stage for the compound micro- scope. A eta Zool., in press. CROWELL, S., 1953. The regression-replacement cycle of hydranths of Obelia and Campanularia.

Physiol. Zool., 26 : 319-327.

("kowELL, S., 1957. Differential responses of growth zones to nutritive level, age and tem- perature in the colonial hydroid Campanularia. J. E.vp. Zool., 134: 63-90. FORBES, E., 1848. A monograph of the British naked-eye medusae. Ray Society, London,

104 pp. FRASER, C. M., 1937. Hydroids of the Pacific Coast of Canada and the United States.

University of Toronto Press, Toronto, Canada, 207 pp. FRASER, C. M., 1914. Some hydroids from the Vancouver Island region. Trans. Ro\. Sue..

Can.,*: 99-216. GREVE, W., 1968. The "Plankton-Kreisel," a device for culturing zooplankton. Marine

Biology, 1 : 201-203. HI.YCKS, T., 1868. A History of the British Hydroid Zoophytes, I'oliune 1. John van Voorst,

London. JOHNSON, M. W., 1932. Seasonal distribution of plankton at 'Friday Harbor, Washington.

Univ. of Wash. Publ. Oceanography, 1 : 1-38. KRAMP, P. L., 1929. The hydromedusae of the Danish waters. Mem. Aead. Sci. Lettr.

Denmark, Series 8, 12 : 1-291. KRAMP, P. L., 1962. Notes on some Eastern Pacific species of Phialidium ( Leptomedusae).

Pacif. Sci., 16 : 25-29. LAMOUROUX, J. V. F., 1816. Histoire des Polypicrs Cortillic/enes I'le.vihles, vitli/airenicnt

nommes zoophytes. F. Poisson, Caen, France, 599 pp.

LEKOUR, M. V., 1922. The food of plankton organisms. /. Mar. Biol. Assoc., 12 : 644-677. XrrriNG, C. C., 1901. The hydroids of the Woods Hole region. U. S. Commission Fish and

Fisheries Doc.. 455: ,325-386. [Washington, Government Printing Office] NUTTING, C. C., 1915. American hydroids, Part III. The Campanularidae and the

Bonneviellidae. Spec. Bull. 4, Smithsonian Institution, Washington. REES, W. J., AND F. S. RUSSELL, 1937. On rearing the hydroids of certain medusae, with an

account of the methods used. /. Mar. Assoc., U. K., 22 : 61-82. RoosEN-RuNGE, E. C., 1962. On the biology of sexual reproduction of Hydromedusae, genus

Phialidium Leuckhart. Pacif. Sci., 16: 15-24.

RUSSELL, F. S., 1938. The Plymouth off-shore fauna. /. Mar. Biol. Assoc. U. K., 22: 411-439. RUSSELL, F. S., 1953. The Medusae of the British Isles. Cambridge Univ. Press, 529 pp. TORREY, H. B., 1904. The hydroids of the San Diego region. Univ. Calif. Publ. Zool., 2 : 1-43. WRIGHT, T. S., 1858. Description of new protozoa and observations on British zoophytes :

1. Coryne gravata, 2. Stauridia producta, 3. Campanularia johnstoni. Edinburgh.

Neiv Philosophical Journal (AVr>' Series), 7 : 276-287.

Reference: Biol. Hull.. 139: 222-228. (August, 1970)

NATURAL AND SYNTHETIC MATERIALS WITH INSECT HORMONE

ACTIVITY. 5. SPECIFIC JUVENILE HORMONE EFFECTS

IN ALIPHATIC SESQUITERPENES

K. SLAMA, K. HEJNO, V. JAROLIM, AND F. SORM

Institute of Entomology, and Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences,

Insects belonging to different taxonomical groups often show great differences in their sensitivities to juvenile hormone analogues. For example, some com- pounds, such as the esters of monocyclic sesquiterpenes, have substantial activity on the hemipteran insects of the family Pyrrhocoridae, but little or no activity for most other insects including other families of the Hemiptera (Slama, Suchy and Sorm, 1968). By contrast, there are many compounds, such as methylenedioxy- phenylethers of geraniol (Bowers, 1969), which show great activity on pupae of Coleoptera but only slight activity on hemipterans.

In previous studies of juvenile hormone activity of farnesenic acid esters ( Slama, Romanuk and Sorm, 1969) we have noticed that minor changes in chemical structure, such as the introduction of hydrogen chloride across the 6,7 and 10,11 double bonds, may lead to as much as 10,000-fold increase in activity for the bug Pyrrhocoris aptcrus with a simultaneous 100-fold loss of activity for the beetle Tenebrio tiiolitor. Other studies on hemipteran insects ( Suchy, Slama and Sorm, 1968) have documented the considerable differences between the families or higher taxonomic groups in their response to individual juvenile hormone analogues.

In the present investigation we have explored the phenomenon in further detail. To this end we have determined the juvenile hormone activity of each of a series of fifteen synthetic esters of aliphatic sesquiterpenic acids when tested on eight genera of insects including one or more representatives of three families of Hemiptera and two families of Coleoptera. Special attention was centered on those features of molecular structure which enhance or detract from hormonal activity for individual families and genera of insects.

MATERIALS AND METHODS

Juvenile hormone activity was assayed on the following Hemiptera: Pyrrhocoris dptcrits L. and Dysdcrcns (Paradysdercus} cingitlatits (Fabr.) (family Pyrrhocori- dae) ; Lyyacns cyncstris L. (family Lygaeidae) ; Graphosoma italicum Mull., Aelia acuminata L., and Eurygaster integriceps Put. (family Pentatomidae). Each analogue was also assayed on the following Coleoptera: Tenebrio molitor L. (family Tenebrionidae) and Dermestes vulpinns Fabr. (family Dermestidae).

For topical assays the compounds were applied in a standard 1 fjii drop of acetone on uninjured cuticle of freshly molted last instar larvae (Hemiptera) or freshly molted pupae (0-20 hrs) (Coleoptera). For injection assays on Tenebrio

222

JUVENILE HORMONE ACTIVITY

111

COOCH-

IV V

R = R = R = R =

C2H5 CH3 CoHc

I//

• COOR

COOR

COOCH,

COOCH:

VIII IX

R =

R - C2H5

COOR

COOCH-

COOCH.

XII R = CH3

XIII R = C2H5

XIV

XV

COOCHo

COOCH-

FIGURE 1. List of the compounds used for assays of juvenile hormone activity.

and Dermestes the compounds were injected in a 1 (A drop of olive oil into the body cavity of freshly molted pupae. The activity was determined according to the degree of retention of the larval (Hemiptera) or pupal (Coleoptera) characters after the next ecdysis.

The dose-response experiments performed on Pyrrhocoris and Graphosoma revealed that the whole range of activity from zero (formation of perfect normal adults) to maximum (formation of morphologically perfect supernumerary larval

224 K. SLAMA, K. HEJNO, V. JAROLl'M AND K. SORM

instars) was realized with a 10-fold change in concentration. The reciprocal in- crease of larval epidermal patterns and decrease of adult patterns was linear when plotted against the logarithmic scale of concentrations. This allowed determi- nation of a standard intermediate effect with minimum error. In the pupae of Coleoptera the range of activity from zero to maximum (extra-pupal instars) was realized with 100 to 1000- fold concentration change. The activity is given in "ID-50 Morph." units. This unit indicates the amount of substance in micrograms per specimen which produces under the above described conditions of application half-larval (Hemiptera) or half-pupal (Coleoptera) adultoids. The unit concen- tration occurs in the middle of the concentration range necessary for zero to maxi- mum effect. The concentrations provoking the first signs of activity can easily be derived from the ID-50 values. For example, if the ID-50 for a hemipteran larva is 0.05, the first signs of activity occur at 0.01 ; so also, maximum activity would be attained by the application of approximately 0.1 /Ag. Each value in Table I represents a result of 4 to 5 tests at different concentrations, each concentration being assayed on 5 to 10 individuals.

The list of the compounds is presented in Figure 1. The compounds II to V, VIII, XII, and XIII were prepared by means of \Yittig's reaction in which the aliphatic methyl ketones were treated with carbomethoxymethylene-triphenylphos- phorane (for preparation of methyl esters) or carboethoxymethylene-triphenylphos- phorane (for preparation of ethyl esters). This method yielded rather pure products.

The starting material for preparation of compounds II and III was hexahydro- pseudoionone, for IV and V citronellylacetone, for VIII geranylacetone, and for XII and XIII pseudoionone. Compound I was prepared by hydrogenation of VIII in presence of Pd/C catalyst. Compound VII was obtained from IV by addition of hydrogen chloride. The epoxides VI, X, and XIV were obtained from IV, VIII and XII after treatment with perphthalic acid. Similar technique was used to prepare the diepoxyderivative XV (two equivalents of perphthalic acid were used). Compounds IX and XI were kindly provided by Dr. M. Romafiuk.

All the synthesized compounds were purified by thin-layer or column chroma- tography on silica gel and their purity was checked by gas-liquid chromatographv (with the exception of halogen-containing compounds). In some cases infra-red and mass spectrometry was used for further characterization.

RESULTS

All the compounds studied were methyl or ethyl esters of aliphatic Cm terpenoid acids which differed in the amounts and positions of the double bonds. For easier orientation we have divided the compounds into the following groups :

Group A — Methyl ester of 3,7,1 1-trimethyl-dodecanoic acid ( I ) with fully saturated

molecule. B — Esters of 3,7,ll-trimethyl-2-dodecenic acid (11, 111 > with one double

bond.

C — Esters of 3,7, 11 -trimethyl-2,10-dodecadienoic acid (IV. V) with 10,11- epoxy (VI) and 1 1 -chloro (VII) derivatives.

JUVENILE HORMONE ACTIVITY

225

D — Esters of 3,7,ll-trimethyl-2,6,10-dodecatrienoic ac{d (VIII, IX) with

three double bonds including also 10,11-epoxy (X) and 7,11-dichloro

(XI) derivatives. E— Esters of 3,7,1 l-trimethyl-2,4,6,10-dodecatetraenoic acid (XII, XIII j

with four double bonds including 10,11-epoxy (XIV) and 4,5,10,11-

diepoxy derivatives (XV).

The juvenile hormone actk'itv of simple esters

As summarized in Table I, the saturated ester (I) showed no detectable activity except when high concentrations were administered to pyrrhocorid bugs. By contrast, substantial activity was recorded for the esters (II, III) of group B

TABLE I

Juvenile hormone activity of the compounds listed in Figure 1. The values indicate ID-50 (Morph) units in fig per specimen

Family	Pyrrhocoridae	Lygaeidae	Pentatomidae	Tenebrionidae	Dermestidae
Species	Pyrrho- coris apterus	Dys- dercus cingu- latus	Lygaeus equestris	Graplio- soma itali- cum	Aelia acumi- nata	Eury- gaster integri- ceps	Tenebrio molitor	Dermestes vulpinus
Application	topical	topical	topical	topical	topical	topical	injection	topical	injection	topical
Compound No.
x	100	100	>500	>1000	>1000	>1000	>1000	>1000	>1000	>1000
II	5	10	7	100	70	>100	1	10	1	10
III	5	10	5	50	50	—	1	7	0.5	5
IV	5	5	3	50	50	100	5	20	5	—
V	5	5	3	8	—	—	5	10	5	10
VI	0.5	0.07	0.5	1	4	—	3	10	3	9
VII	0.08	0.05	0.9	5	5	—	1	1	5	5
VIII	50	50	50	50	50	100	10	100	8	—
IX	50	30	40	50	30	100	5	100	5	—
X	3	1	—	10	5	20	10	—	—	—
XI	0.0008	0.005	0.009	30	1	30	1000	>1000	25	50
XII	3	1	5	3	5	20	500	>1000	100	—
XIII	3	1	—	1	5	20	500	>1000	100	—
XIV	0.8	0.7	1	1	7	5	100	>1000	—	—
XV	0.8	0.7	1	5	7	8	>1000	>1000	>500	—

The tested compounds contained approximately I trans methyl-3 isomors.

which contain the conjugated double bond at €2- These compounds showed a 10- to 20-fold increase in activity for the pyrrhocorids, and the appearance of low but definite activity for the pentatomids. In the case of the two species of Coleoptera, the increase in activity was at least a thousandfold.

With further increase in the number of double bonds the activity undergoes definite changes distinctive of each taxonomic group. The results may be sum- marized as follows: (i) In the Pyrrhocoridae and Lygaeidae the activity does not show any considerable variations with increasing degree of unsaturation, i.e., critical doses are between 1 and 10 /xg with the exception of farnesenic acid esters (VIII, IX) which are less active. (ii) The Pentatomidae show a continuous increase of juvenile activity with increasing degree of unsaturation ; the difference is approximately 10 to 100-fold, (iii) Just the reverse is true for the coleopterans, Tenebrio and Dermestes, where the esters of group B (II, III) are the most active

226

K. SLAMA, K. HEJNO, V. JAROLIM AND F. SORM

and the highly unsaturated compounds of group E (XII, XIII) are 100 to 1000 times less active or completely inactive.

The effect of epo.ridation

When the 10,11 double bond of the compounds IV and V is saturated by an oxirane ring (VI), hormonal activity is substantially increased in hemipteran insects and slightly increased also in the beetles. The increase is smaller in XIV where the oxirane ring is placed across the 10,11 double bond of the highly un- saturated compound XII. The diepoxy-ester (XV) with both 10,11 and 4,5 double bonds epoxidated has approximately the same activity as the monoepoxy- ester (XIV), suggesting that the oxirane ring in the position 4,5 does not increase the activity as that in the position 10,11.

The effect of hydrochlorination

Substitution of the 10,11 double bond in compounds IV and V by hydrogen chloride has similar effects on hormonal activity as described above for the corre- sponding epoxy-derivative. When both the 10,11 and 6,7 double bonds of compounds VIII and IX are saturated by hydrogen chloride (XI) there occurs an

TABLE II

Summary of the relationships between the double bonds, their substitutions by hydrogen chloride and epoxide, and juvenile hormone activity

	Pyrrhocoridae Lygaeidae	Prntatomidae	Tenebrionidae Dermestidae
Increasing degree of unsaturation 10, 11 substitution 6,7 substitution 6,7 and 10,11 substitution	little or no change large increase large increase enormous increase	increase increase slight decrease slight increase	decrease slight increase large decrease large decrease

enormous increase in activity for pyrrhocorid and lygaeid bugs, whereas little change in activity is observed for pentatomids. Equally impressive is the great decline in activity for the beetles. Since the monohydrochlorinated compound VII is highly active on beetles, the 6,7 double bond and especially the status of C- seem crucial. Thus, when the CT is attached to a halogen atom or is bound to an oxygen atom of an epoxide, the compounds are very active on pyrrhocorids and very inactive on the beetles. By contrast, in the pentatomid bugs the activity is little influenced by changes at C7.

The effect of stereo chemical isomers

The cis and trans isomers of compounds II and VII were isolated and tested on Pyrrhocoris, Dysdercus, and Graphosoma by topical assays and on Tcnebrio by both topical and inject assays. The trans isomers were found approximately 10 times more active than the cis isomers in all the hemipterans. In Tcnebrio there were large variations in activity but, in general, the trans isomers appeared to be 5 to 100 times more active than the cis isomers.

JUVENILE HORMONE ACTIVITY

The effect of methyl mid ethyl ester radicals

In the present study we found rather small difference in the activity of methyl and ethyl esters on pyrrhocorids, and relatively small activity increase of ethyl esters over the corresponding methyl esters in other insects studied. Present indications are that the ethyl esters cause a greater increase in activity in com- pounds where the 10,11 double bond is saturated by hydrogen chloride or epoxide.

DISCUSSION

Jarolim, Hejno, Sehnal and Sorm (1969) have described the juvenile hormone activity of the compounds listed in Figure 1 on larvae and pupae of Galleria mellonclla (Lepidoptera, family Pyralidae). It appears that the action of these compounds on Galleria is in many respects similar to that which \ve have found on pupae of the beetles, Tenebrio and Dermestes.

It is known from the literature that there are certain structural features of juvenile hormone analogues which produce a general increase or decrease of activity in all species. Our results have confirmed these generalizations which can be summarized as follows: (i) higher activity of stereochemical trans isomers (Yamamoto and Jacobson, 1962; Roller and Dahm, 1969; Wigglesworth, 1969a; Slama ct al., 1969; (ii) necessity of the 2,3 double bond in aliphatic terpenes ; (iii) loss of activity after introduction of very polar groups such as hydroxyls (Suchy ct al., 1968) ; (iv) increased activity after addition of 10,11-epoxy group (Bowers, Thompson and Uebel, 1965; Roller and Dahm, 1969; Ratusky, Slama and Sorm, 1969; Wigglesworth, 1969a, 1969b ; Jarolim et al, 1969); (v) in- creased activity after additions of hydrogen chloride at the 10,11 double bond (Law, Yuan and Williams, 1966; Romanuk, Slama and Sorm, 1967; Slama ct al., 1969; Wigglesworth, 1969a) ; (vi) higher activity of ethyl esters (Law ct al., 1966 ; Roller and Dahm, 1969 ; Slama ct al., 1969 ; Wigglesworth, 1969a) .

As indicated in the present study, there are certain chemical changes which lead to predictable increase or decrease of the juvenile activity for individual families and orders of insects. These have been summarized in Table II. The structure-activity relationships of this type are not common in the literature for they require large biological screenings on representatives of several insect groups. We have already mentioned that the degree of unsaturation and the chemical configuration at C- are among the factors determining selective action in esters of aliphatic sesquiterpenes. Both these factors seem to be associated with one and the same biological mechanism since each group of insects which shows specific re- sponses to the degree of unsaturation is also sensitive to substitutions at C-.

We suspect that this type of information will be helpful in the preparation of new synthetic juvenile hormone analogues with more or less selective pesticide effects.

Acknowledgment is gratefully given to Professor C. M. Williams who pro- vided helpful comments on the typescript and to Mrs. Pichlova, Mrs. Piscakova and Mrs. Zdenkova for their technical assistance.

K. SLAMA. K. HE.) NO, V. JAROLIM AND F. SORM

SUMMARY

The juvenile hormone activity of ethyl or methyl esters of aliphatic sesquiterpenic acids with 0 to 4 double bonds was tested on 8 species of insects belonging to 5 families of Hemiptera and Coleoptera. Special attention was paid to the addition of hydrogen chloride or epoxide groups on or across the double bonds.

Certain chemical changes in the molecule appear to cause a general increase of the activity in all species studied. These are : the presence of 2,3 unsaturation conjugated with the carboxyl group ; the trans stereochemical position of the C-3 methyl; an introduction of 10,11 epoxide or hydrochloride ; and esterification with ethyl rather than with methyl.

There are also chemical changes which lead to genus- or family-specific varia- tions in juvenile hormone activity. With increasing amount of unsaturation the activity either remains almost unaffected (pyrrhocorid bugs) or increases (pen- tatomid bugs) or decreases considerably (tenebrionid and dermestid beetles). The addition of hydrogen chloride or epoxide to the 6,7 double bond causes enormous increase in the activity in the Pyrrhocoridae and Lygaeidae, no con- siderable change in the Pentatomidae, and great decreases in the beetles and Lepidoptera.

LITERATURE CITED

BOWERS, W. S., 1969. Juvenile hormone: activity of aromatic terpenoid ethers. Science,

154: 323-325.

BOWERS, W. S., M. J. THOMPSON AND E. C. UEBEL, 1965. Juvenile and gonadotropic hor- mone activity of 10,11 epoxyfarnesenic acid methylester. Life Sciences, 4: 2323-2331. JAROLIM, V., K. HEJNO, F. SEHNAL AND F. SORM, 1969. Natural and synthetic materials with

insect hormone activity. 8. Juvenile activity of the farnesane-type compounds on

GaUcria mcllonclla. Life Sciences, 8 : 831-841. LAW, J. H., C. YUAN AND C. M. WILLIAMS, 1966. Synthesis of a material with high juvenile

hormone activity. Proc. Nat. Acad. Sci., 55 : 576-578. RATUSKY, J., K. SLAMA AND F. SORM, 1969. Natural and synthetic materials with insect hor-

more activity. 6. Juvenile hormone effects of some alkyl-ethers derived from aliphatic

/3-hydroxy acids. /. Stored Prod. Res., 5 : 111-117. ROLLER, H., AND K. H. DAHM, 1968. The chemistry and biology of juvenile hormone. Recent

Prog. Hormone Res., 24 : 651-680.

ROMANUK, M., K. SLAMA AND F. SORM, 1967. Constitution of a compound with a pro- nounced juvenile hormone activity. Proc. Nat. Acad. Sci., 57 : 349-352. SLAMA, K., M. SUCHY AND F. SORM, 1968. Natural and synthetic materials with insect hormone

activity. 3. Juvenile hormone activity of derivatives of p-(l,5-dimethylhexyl) benzoic

acid. Biol Bull, 134: 154-159. SLAMA, K., M. ROMANUK AND F. SORM, 1969. Natural and synthetic materials with insect

hormone activity. 2. Juvenile hormone activity of some derivatives of farnesenic acid.

Biol. Bull, 136: 91-95. SUCHY, K., K. SLAMA AND F. SORM, 1968. Insect hormone activity of p-(l,5-dimethylhexyl)

benzoic acid derivatives in Dysdercus species. Science, 162 : 582-583. WIGGLESWORTH, V. B., 1969a. Chemical structure and juvenile hormone activity: comparative

tests on Rhodnius prolixus. J. Insect Physiol., 15 : 73-94. WIGGLESWORTH, V. B., 1969b. Chemical structure and juvenile hormone activity. Nature, 221 :

190-191. YAMAMOTO, R. T., AND M. JACOBSON, 1962. Juvenile hormone activity of isomers of farnesol.

Nature, 196: 908-909.

Reference : Biol. Bull., 139: 229-238. (August, 1970)

EFFECTS OF SUPERABUNDANT OXYGEN ON THERMAL TOLERANCE OF GOLDFISH

A. H. WEATHERLEY

Zoology Department, Australian National University, Canberra, A.C.T. Australia

The physiology and ecology of thermal tolerance in teleosts has long been the subject of research, and several extensive articles and reviews on this topic have appeared in recent years (Brett, 1956; Fry, 1958, 1967; Fisher, 1958; Weatherley, 1963a, b). Temperature tolerance is greatly affected by physiological pro- cesses, which are governed by the normally prevailing temperatures of the environment, and there has therefore been much study of the relationship between lethal temperature and acclimation temperature (e.g., Brett, 1956; Fry, 1967). The fundamental biological characteristics of impairment of animal function through high temperature have, however, remained rather obscure (see Fry, 1967). N< ti- ls there yet much understanding of the effects of environmental factors, other than acclimation temperature, on thermal tolerance. The importance of oxygen tension, salinity level, pH, etc.. are, for instance, largely unknown.

HYPOTHESIS OF OXYGEN-DEPENDENT THERMAL TOLERANCE

Despite the lack of real understanding mentioned above, it has, at least, become evident that simplistic ideas of the overriding importance of some single cause of heat death are unlikely to be satisfactory (Rose, 1967). On the other hand, it does appear likely that certain aspects of the physiology of the whole animal will prove more directly sensitive to high temperature than others (Weatherley, 1963a). Thus, it has been thought for many years that functional failure of the nervous system may be of key importance (Brett, 1956; Fisher, 1958; Fry, 1958, 1967). This suggestion has considerable logical appeal in view of the long-known vulnerability of vertebrate nervous systems to even short-term deprivations of oxygen. We might, for instance, advance an argument along the following lines to account for impairment of the animal through damage to the nervous system when exposed to high temperature.

If ambient temperature rises substantially, then oxygen requirements of all tissues may be expected to show a corresponding increase. However, the circula- tory system would be able to meet the initial need for an increase in blood flow, and so supply the additional oxygen required. Should their demand begin to exceed the maximum ability of the respiratory and circulatory systems to supply oxygen, some tissues may be expected to begin to suffer from an oxygen shortage. The severity and permanence of their resulting malfunction will then depend on their particular sensitivity to that shortage. Nervous tissue will be among those tissues— with a high inherent metabolic rate and marked sensitivity to oxygen lack — most rapidly and seriously affected. As the vertebrate respiratory /circulatory system is, at least partly, under the control of the nervous system, we can expect the estab-

229

230 A. H. WEATHERLEY

li.shment of a 'vicious cycle' of worsening failure of both systems, if the oxygen shortage is maintained. In addition, the systems for oxygen transport and for oxygen and carbon dioxide exchange in vertebrates may, of course, be directly affected by temperature.

An experimental finding for trout and roach by Alabaster and Welcomme (1962) lends support to these ideas. They found that if the oxygen tension in the water is significantly below air-saturation value, thermal tolerance is appreciably lowered.

The present paper describes experiments performed to test the hypothesis that the lethal temperature of goldfish may be affected by failure of the oxygen supply system. The method used was to augment the normal oxygen supply, in the expectation that a superabundance of oxygen might ensure ready entry of oxygen into the fish through the entire body surface. Thus, a functional failure of the respiratory, circulatory, or oxygen transport systems would be compensated, at least to some extent, by saturation of blood serum, lymph, tissue fluids, etc., with oxygen.

MATERIALS AND METHODS Plan of experiments

A first testing of the hypothesis was to compare mean survival time at a fixed upper lethal temperature among fish in oxygen-enriched water ('experimentals') with those in water at air-saturation ('controls'). The oxygen available to the 'controls' was limited to the quantity that dissolves in water at the altitude of Canberra (approx. 600 m). In this first group of experiments all goldfish were removed directly from an aquarium stock tank and placed immediately either into the oxygen-rich water or ordinary 'air-saturated' water in the experimental vessels.

In the second group of experiments batches of fish were placed in oxygen-rich water for periods of 2, 6\ and 27i h before testing them against 'controls', or normal 'experimentals' (those placed in oxygen-rich water immediately before testing) .

The results of these two groups of experiments (Tables I and II), indicated the desirability of determining the ability of very much higher concentrations of oxygen to ameliorate thermal stress. A third series of experiments was therefore carried out in a specially constructed compression chamber (Fig. 1), and the results are given in Figure 2.

The fi-sh

The goldfish Carassins an rat us used in these experiments, which were ob- tained from a commercial supplier, were mostly of the type known as 'comets', except for a few experiments (1 and 2, Table I) in which 'calicoes' were used. In any one series of experiments the fish were of relatively uniform size and were sexually immature. The fish were maintained in aerated, filtered water in glass aquarium tanks and received 12 hours of light and 12 of darkness per day. They received a standard fish food every two days, similar to that described by Weather- ley (1963a), and for which they showed g';od appetite.

OXYGEN AND THERMAL TOLERANCES IN GOLDFISH

231

FIGURE 1. Compressor unit (4.4 1 capacity) for experiments on upper lethal temperature at high tensions of oxygen. Chamber is constructed of stainless steel, with perspex end plate. Thermometer, pressure gauge, bleeder valve and high pressure oxygen hose are shown.

TABLE I

Survival times of goldfish in superoxygenated water compared with controls in air-saturated water when exposed to lethal temperature of 40°C. (approximate acclimation temperature 28° C).

					Standard
Experiment Vn	Treatment	Xo. of individuals	Mean survival	Sums of squares	deviation of diff.	V	Signifi- cance
*-> U.		n	time (sec)	2d"	of means		level (%)
			X		<rd
1	Control	8	192	64397	50.5	19.6	0.1
	High oxygen	8	1181	1245688
2	Control	8	264	206988	67.3	10.8	0.1
	High oxygen	8	989	2117888
3	Control	8	528	957552	80.1	8.0	0.1
	High oxygen	8	1170	2330400
4	Control	6	587	323334	75.7	7.8	0.1
	High oxygen	6	1175	913350
5	Control	8	203	152152	45.1	12.0	0.1
	High oxygen	8	746	890588

In general, acclimation temperatures in the stock tanks varied by ± 0.5° C daily about the means as indicated below and in Tables I and II ; Fig. 2. In any one series of experiments the fish were either drawn randomly from one large stock tank in which all had spent at least a fortnight prior to experiments, or from several

232

A. H. WEATHERLEY

smaller adjacent tanks with identical temperature records. Sample bias is believed to have been kept to a minimum by these procedures.

Oxygen-supersaturated water at normal atmospheric pressure.

For the first series of experiments the test containers consisted of two large beakers, each containing approximately 2 liters of aquarium water (Table I). In the second series of experiments (Table II) the somewhat larger size of the fish required the use of even larger beakers of 5 liters capacity. In both series of tests a temperature was selected as 'lethal' on the basis of simple preliminary pilot tests combined with consideration of the published records of lethal temperatures

TABLE II

Analysis of survival of goldfish held in oxygen-saturated water for various times before exposure to a lethal temperature of 35° C, compared with controls in air-saturated water (approximate ac- climation temperature 18° C).

				Sums of E
				scores pre-
Experi- ment No.	Treatment	No. of in- dividuals	Range of survival times (sec)	ceding each C score (Mann-	Significance level (%)
				Whitney
				•U' test)
6	Control	8	70-555
7	Placed in O2-sat. water immediately
	before test	8	195- > 7200	4	0.1
8	2 hr in Oo-sat. water before test	8	1 60- > 7200	14	3.2
9	65 hr in C>2-sat. water before test	8	295- > 7200	3	0.1
10	21 \ hr in Oa-sat. water before test	8	1 80- > 7200	12	1.9

C = 'control' values for survival times; E == 'experimental' values for survival times.

for goldfish at various acclimation temperatures (Brett, 1956). With these guides as a basis, 40° C proved convenient in experiments 1-5 (Table I), whereas in experiments 6—10 (Table (II), because of differences in the size and age of the fish and their acclimation temperature, 35° C was used. Temperature was main- tained in each beaker at the selected lethal value by placing them in a glass tank in which the water was kept at a constant temperature by a Braun 'Thermomix'. Temperatures in the beakers varied from the selected lethal temperature by no more than ±0.05° C; in most instances no changes at all were detected.

In the 'control' beakers the water was aerated, with air at 3 Ib per sq inch pressure passing through an aquarium airstone, for at least 10 min before each experiment. In the 'experimental' beakers water was supersaturated with oxygen by vigorous passage of pure oxygen for about 10 minutes.

Determinations of oxygen concentration in both containers at the atmospheric pressure of Canberra and the acclimation tempertures of these experiments ( 17- 28° C) showed the saturation value to be 92-93% of the sea level value, and 4-5 times this as a result of superoxygenation. The oxygen tension in the 'experi- mental' beakers was thus always beyond the range of an oxygen electrode, and was therefore determined by the Winkler method, using 100 ml samples.

OXYGEN AND THERMAL TOLERANCES IN GOLDFISH

233

41r

40

39

u

o

a:

Lt

LU

a.

38

37

36-

35

B

27°C

t

ACCLIMATION

TEMPERATURE

I

17°C

10 20 30

ATMOSPHERES OF OXYGEN

FIGURE 2. Effects of increasing concentrations of dissolved oxygen on mean upper lethal temperature (black circles), based on samples of five fish per point. Lower curve (curve A in text) was for fish acclimated at 17° C, upper curve (B in text) for fish acclimated at 27° C. Vertical bars represent twice the standard deviation of each mean. See text for further explanation.

In the beakers, the water was continuously aerated ('controls') or oxy- genated ('experimentals') during the actual exposures of fish to lethal temperatures.

The method of testing was to expose samples, usually of 8, occasionally of 6 or 7, fish to the test temperatures by placing all fish simultaneously into the

234 A. H. WEATHERLEY

beakers. Times to death were recorded for each individual. The criterion of death was cessation of movement, which was sometimes rather difficult to observe, though judgment rapidly improved during a few pilot tests. Cessation of opercular movement appeared to be closely correlated with that of other movements of the l)odv, so it was frequently employed as a convenient and sensitive index of death. Many fish judged 'dead' by the criterion of 'cessation of movement' could he readily revived if removed immediately to a cold surface or cool water, but, by the same token, fish in which movement had ceased failed to move again if maintained at the same temperature or if the temperature was raised still higher.

On removal from the lethal temperature test beakers fish were weighed and measured. pH in the beakers was measured before and after each lethal test.

Higher concentrations of oxygen

The results of exposing goldfish to concentrations of oxygen higher than those obtainable by vigorous aeration alone (Tables I and II) made it desirable to examine the effects of much higher tensions of oxygen. The construction of a compression chamber was needed for this investigation (Fig. 1). The chamber was built of | inch seamwelded stainless steel, in the form of a cylinder. A 1 -inch- thick perspex end wall may be bolted to a heavy steel collar welded to one end; an airtight seal is effected by means of a rubber O ring. The unit carries a pres- sure gauge, a tap from which rubber pressure hose leads to an oxygen cylinder, and a steel-sheathed thermometer which dips into the water of the chamber when in use.

Before use, the compressor was placed on end and filled with water to a volume of 3.1 1 (total capacity 4.4 1). Then oxygen was bubbled through this for 10 min to allow as much as possible to enter solution under normal atmospheric pressure. A randomly draw7ii sample of 5 goldfish was then placed in the water and the perspex end plate bolted on. With the chamber now set on its side, the air above the water was flushed out, with a powerful stream of oxygen, for one minute. With the outlet valve closed the pressure of the oxygen atmosphere above the water was then raised to whatever level was required.

Henry's Law states that the amount of gas which dissolves in a liquid is directly proportional to the partial pressure of the gas in the atmosphere above the liquid. The law was assumed to hold under the above experimental conditions, so that the tension of dissolved oxygen was computed from the recorded atmospheric pressure in the chamber (Fig. 2).

Apart from the higher oxygen concentrations obtained with this compression chamber it was also used to examine the influence on lethal temperature of various other concentrations of oxygen from a few per cent up to 400-500% of air saturation (Fig. 2). As in the first two series of experiments (Tables I and II) oxygen tensions of about 2 and about 4-5 atmospheres were obtained, without com- pression, merely by passing pure oxygen through the water. Tensions of oxygen less than air saturation were easily got by bubbling pure nitrogen through the water.

The aim in covering this very wide range of oxygen tensions (from near zero to about 30 atmospheres) was to test the influence of oxygen tension on thermal tolerance comprehensively. All oxygen tensions from 10-200% of air

OXYGEN AND THERMAL TOLERANCES IN GOLDFISH

235

saturation were determined by means of a temperature-compensated oxygen electrode.

Lethal temperatures were determined by placing the chamber containing each batch of fish, immediately after the end plate was bolted on (and after compression if needed), into a previously prepared glass tank. For experimental series A ( Fig. 2 ) the water in this tank was held at 41.5° C, and for series B (Fig. 2) at 43.1° C. These temperatures had been carefully predetermined as a result of pilot tests to discover the temperatures at which fish, acclimated at 17 and 27° C, would die. These tank temperatures were known to be sufficient to heat the water of the compression chamber past the lethal level, but to do it slowly enough for the accurate estimation of the temperature at death (movement cessation ) for each fish. The fish could be easily seen through the perspex wall of the compression chamber. The chamber was not constantly lighted, but a lamp was positioned so that it could be used to examine the rear end of the chamber if fish drifted away from the perspex.

u

o

Qi

LU

Q.

01 23456789 10 11

TIME (MINUTES)

FIGURE 3. Rate of increase in temperature in compressor (Fig. 1) with external water temperature of 43.1° C (upper curve) and 41.5° C (lower curve). Hatching indicates range of temperature over which fish died in the experiment represented by each curve.

Figure 3 gives two curves depicting temperature rise against time in the chamber. The curves start at different values, each adjusted to the acclimation temperature of the animals. The curves pass through the actual temperatures recorded at one minute intervals : there has been no artificial smoothing.

It is noteworthy that rate of heating is not particularly significant in itself in influencing the temperature at which fish die. In several tests not reported here, the rate of heating to the lethal temperature was considerably more rapid than usual, though other conditions (i.e. oxygen tensions) were comparable. In these tests, mean lethal temperatures showed little difference as a result of the more rapid heating.

236 A. H. WEATHERLEY

RESULTS Oxygen-saturated water at normal atmospheric pressure

In experiments 1-5 plus several not reported here, the mean survival times of goldfish exposed to a lethal temperature in oxygen-saturated water, as compared to those in aerated water, were ahout 2-5 times greater (Table I). The difference between these means was significant at the 0.1% level (V test; Fisher, 1954).

While they remained alive, the gills of the fish exposed in oxygen-rich water were also visibly redder than those in ordinary water. Suggestive evidence only, this observation nevertheless accords with the idea that respiratory /circulatory failure accompanies progressive physiological impairment in the thermally stressed fish, i.e. that blood flow through the gills becomes reduced more rapidly in re- sponse to a relative oxygen shortage.

The results of experiments 6-10 (Table II), dealing with fish held at high oxygen tensions for various periods before exposure to the lethal temperature again showed a strong tendency for survival to be much longer than in ordinary aerated water. However, though the 'control' goldfish all died within a few minutes, the fish in oxygen-rich water showed a pronounced tendency to survive so much longer in all 'experimental' instances that each test was terminated with some fish still alive after 2 h. In testing the significance of the difference in survival times between 'experimental' and 'control' groups the Mann-Whitney non-parametric 'U' test was used instead of the Y test (Siegel, 1956).

Results show that survivals in oxygen-rich water were all significantly better than in air-saturated water, but that fish placed directly in a high oxygen tension at the beginning of their 'lethal' test survived at least as well as those given longer periods in high oxygen tensions before testing. This disposes of the possibility that giving fish prolonged exposures to high oxygen before exposure to lethal temperature tests might cause them to survive even longer than those placed directly in high oxygen only at the beginning of the test.

In experiments 1-5 of Table I the pH in the control beakers was 7.5, and of the oxygen-rich ones 7.9 ; pH measured immediately before and after lethal tests showed no changes. Weights ranged from 0.4-1.3 g (mean 0.7 g) for 'calicoes' in experiments 1 and 2; from 0.3-1.1 (mean 0.7 g) for 'comets' in experiments 3 and 4 ; for experiment 5 the mean weight was 0.2 g.

In experiments 6-10 (Table II) pH of controls was 7.4 and in the oxygenated water was 7.6, whether after 2, 6] or 27] hr passage of oxygen. Weights of fish in this series ranged from 2.2-3.9 g (mean 3.0 g).

Higher concentrations of oxygen

Upper lethal temperature was obviously related closely to oxygen tension over a considerable range of values (Fig. 2). Mean lethal temperature increased in both series of experiments (i.e. with fish acclimated to 17 and 27° C, respectively) by about 2° C, from a low corresponding to oxygen tensions of about 10% of air saturation up to a maximum at approximately 5 atmospheres. This tension of oxygen is also about the same as that which produced highly significant increases in survival time in the first two series of experiments. Thus, increased ability to survive a high temperature because of oxygen abundance can be manifested either by

OXYGEN AND THERMAL TOLERANCES IN GOLDFISH 237

increased survival time at a lethal temperature or by a higher mean lethal tem- perature achieved. There is nothing remarkahle about these parallel associations ; Fry (1967) has pointed out that they are only to be expected. It is, however, reassuring to find them demonstrated within one experimental study.

There was no additional enhancement of thermal tolerance in response to exposure to oxygen tensions in excess of 5 atmospheres — even up to approxi- mately 30 atmospheres. The limit was reached quite abruptly. On the other hand, there was no evidence of oxygen toxicity effects at higher tensions.

In these experiments pH varied only over the range 7.4—7.5 in the com- pression chamber at the beginning of the tests. Weights in series A ranged from 2.9-4.4 g and in series B from 3.0-4.8 g.

DISCUSSION

The results do not appear to be out of harmony with the hypothesis on which the experiments were based, so perhaps it may be seriously contended that some important malfunction of mechanisms that supply oxygen to the tissues begins at or near the lethal temperature level. Two arguments may be advanced in support of this. The first is that temperature tolerance is lowered when oxygen tensions are appreciably reduced below normal air saturation values ; the second is that goldfish can survive for long periods in concentrations as low as 8% of air saturation at a temperature of 20° C (own unpublished observations). Blazka (1958) has shown that the closely related crucian carp C. carassius can live under conditions of almost if not complete anoxia for several months at low temperatures. So it seems that oxygen insufficiency is a relative problem for fish, exacerbated particu- larly when temperatures are dangerously high. This reasoning accords with the hypothesis of damage to respiratory, circulatory, and oxygen transport, systems. The fact that there is an increase in thermal tolerance in response to increased oxygen, of an order similar to the decrease in tolerance seen in oxygen-poor condi- tions, also supports the hypothesis — or certainly does it no violence.

On the other hand the suddenly achieved limit of this enhancement of toler- ance suggests the operation of a new effect at an apparently very definite 'breakpoint', and here we may be witnessing a physiological failure of a quite critical sort. The enhancement of thermal tolerance up to 5 atmospheres of oxygen ends so abruptly that one must suppose not that more oxygen cannot be accepted by the tissues and body fluids of the fish, but simply that it is unable to utilize this extra oxygen. There might be several reasons for this and in concluding I list these.

(i) Heat denaturation of proteins including respiratory enzyme systems may make it impossible for the fish, as a whole organism, to make full use of the oxygen available to it.

(ii) Heat destruction of the lipoprotein of the cell membrane may make oxygen import into the cell, and/or carbon dioxide export out of it, impossible. At one time lipid was certaintly envisaged as important in thermal tolerance of goldfish (Hoar and Cottle, 1952a, b ; Hoar and Dorchester, 1949).

(iii) Direct thermal death or irreversible damage of cells of the central nervous system may be quite unpreventable above a certain temperature maximum despite the presence of a superabundance of oxygen.

238 A. H. WEATHERLEY

\n\ or all of the above effects may be at work; but perhaps some system critically linked to temperature may be responsible. If the latter proved correct it would be very interesting in several ways. One of these is a major point of this paper, which is to suggest that another system (or systems) — apparently failing at a critical level of temperature or after a critical exposure time — can have its failure alleviated merely by experimental amelioration of some accompanying environmental conditions. In that case it might be possible to postulate even more significant alleviations (if we could determine other ameliorating conditions for the new limiting factors).

SUMMARY

A hypothesis may be made out that the respiratory /circulatory, and possibly the oxygen transport, systems are importantly involved in thermal death of gold- fish. Experiments showed that at two distinct levels of temperature acclimation, superabundant oxygen in the water could produce a definite improvement either in time of survival at a fixed lethal temperature or in lethal temperature reached as a result of heating.

Despite this positive effect of high oxygen a clear cut failure of oxygen, in excess of about 5 atmospheres (partial pressure), to produce further improvement in thermal tolerance suggests the failure of some critical system directly affected by temperature.

The experiments, while revealing nothing of the detailed mechanisms involved, certainly do not invalidate the hypothesis proposed, and open a way to further investigation.

LITERATURE CITED

ALABASTER, J. S., AND R. L. WELCOMME, 1962. Effect of concentration of dissolved oxygen on

survival of trout and roach in lethal temperatures. Nature, 194 : 107. BLAZKA, P., 1958. The anaerobic metabolism of fish. Physiol. Zoo]., 31 : 117-128. BRETT, R. J., 1956. Some principles in the thermal requirements of fishes. Quart. Rev. Biol.,

31 : 75-87. FISHER, K. C., 1958. An approach to the organ and cellular physiology of adaptation to

temperature in fish and small mammals, pp. 3-48. In: C. L. Prosser, Ed., Physiological

Adaptation. American Physiological Society.

FISTIER, R. A., 1954. Statistical Methods for Research Works. Oliver and Boyd, Edinburgh. FRY, F. E. J., 1958. Temperature compensation. A mm. Rev. Physiol, 20 : 207-224. FRY, F. E. J., 1967. Responses of vertebrate poikilotherms to temperature, pp. 375-409. In:

A. H. Rose, Ed., Thermobiology. Academic Press, London. HOAR, W. S., AND M. K. COTTLE, 1952a. Dietary fat and temperature tolerance of goldfish.

Can. J. Zool, 30 : 41-48. HOAR, W. S., AND M. K. COTTLE, 1952b. Some effects of temperature acclimatization on the

chemical conposition of goldfish tissues. Can. J. Zool., 30 : 49-54. HOAR, W. S., AND J. E. C. DORCHESTER, 1949. The effect of dietary fat on the heat

tolerance of the goldfish (Carassius anratus}. Can. J. Res., 27 : 85-91. ROSE, A. H., 1967 (Ed.), Thermobiology. Academic Press, London, 653 pp.

SEIGEL, S., 1956. Nonparametric Statistics for the Behavioural Sciences. McGraw-Hill, N. Y. WEATHERLEY, A. H., 1963a. Thermal stress and interrenal tissue in the perch (Perca

fluviatilis (Linnaeus). Proc. Zool. Soc. London, 141: 527-555. WEATHERLEY, A. H., 1963b. Zoogeography of Perca fluviatilis (Linnaeus) and Perca

flavescens (Mitchell) with special reference to the effects of high temperature. Proc.

Zool. Soc. London, 141 : 557-576.

Vol. 139, No. 2 October, 1970

THE

BIOLOGICAL BULLETIN

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY

ACETYLCHOLINESTERASE ACTIVITY IX ESERINE-TREATED

ASCIDIAN EMBRYOS

DAVID FROMSON1 AND J. R. WHITTAKER- Dcptirtjnciit of Zoology, Uiih-crsity of California, Los Aiu/clcs, California

Durante (1956, 1957) has shown histochemically that a cholinesterase is localized in the two lateral bands of muscle cells extending posteriorly in the larval tail of the ascidian. dona intcstinalis. Since there was inhibition of normal muscle function in Ciuno embryos and larvae exposed to eserine sulfate and other inhibitors of cholinesterase activity, the enzyme undoubtedly has a function in tail muscle contraction (Durante, 1958).

When Ciona embryos were treated with eserine during early embryonic stages, the inhibition of muscle activity which it caused was not reversible when the embryos were removed from the eserine solution, whereas, the inhibitory effect of eserine on fully developed tadpoles was reversible (Durante, 1958). Durante concluded that eserine may stop the synthesis of cholinesterase during development. The most interesting of the experiments she reports is a prefertilization effect of eserine. Ciona eggs placed in eserine for several hours prior to fertilization and subsequently fertilized and permitted to develop in eserine-free sea water, produced morphologically normal larvae which were quiescent, and which lacked sustained movements of the tail (Durante, 1958). This work supports the intriguing possi- bility that a specific enzyme inhibitor might prevent synthesis of the enzyme it inhibits.

The most plausible alternative to the explanation suggested by Durante is that eserine remains trapped in the egg or embryo, and that this residual eserine inhibits the muscular movement of the larva. However, it was the prefertilization experiment which led Reverberi (1961 ) to accept the conclusion that cholinesterase synthesis had been interfered with, because he considered it unlikely that the Ciona embryos could have retained eserine for such a relatively long time in pure sea water.

1 Present address: Department of Biology, McGill University, Montreal 110, P.Q. Canada. - Present address : The \Yistar Institute of Anatomy and Biology, Philadelphia, Penn- sylvania 19104.

239

Copyright © 1970, by the Marine Biological Laboratory Library of Congress Card No. A38-518

240 DAVID FKOMSnX" AND J. K. \\HITT.\KKk

In view of the potential importance of the Durante results, we have under- taken to repeat the prefertilization experiment and to examine the possibility of eserine retention. Our work unequivocally shows that eserine is trapped in the egg in sufficient quantity to inhibit most of the acetylcholinesterase activity in the developing embryo, and that acetylcholinesterase synthesis is not affected by the drug.

MATERIALS AND METHODS . Ininuils

Adult specimens of Ciona intcstinalis L. were collected at Marina del Rev, California, and gametes were obtained surgically as described by Costello, Davidson, Eggers, Fox and Henley (1957). The embryos were reared in filtered sea water and at a constant temperature of 18° C ± 0.5° C.

Chemicals

Reagent grade chemicals were used throughout these studies. Eserine sulfate (H2SO4) USP was purchased from Calbiochem, BW62C47 ( 1 :5-bis-(4-tri- methyl-ammoniumphenyl) pentan-3-one diiodide] was a gift from the Burroughs- \Yellcome Company, Tuckahoe, New York, and iso-OMPA (tetraisopropylpyro- phosphoramide ) was purchased from L. Light and Company Ltd., Colnbrook, England. Dithiobisnitrobenzoic acid was purchased from Aldrich Chemical Com- pany ; acetylthiocholine iodide and butyrylthiocholine iodide were obtained from Nutritional Biochemical s Corporation.

I'rcpanition o] lionioi/oiatcs for cuzyinc assay

Embryos of the desired stage were washed with filtered sea water, and collected by low speed centrifugation. One to five thousand embryos were homogenized in a variable speed motorized Potter-Elvehjem homogenizer with a teflon pestle in a medium consisting of 0.1 A I sodium phosphate buffer (pH 8.0) with 0.1 9^ Triton X-100 (Rohm and Haas, Philadelphia, Pennsylvania). Homogenates were centrifuged in an International Centrifuge Model V at 750 X g for 10 minutes, and the resulting supernatants assayed for acetylcholinesterase activity. Different homogenate supernatants contained 0.1-0.5 nig protein/ml.

Assa\ for acetylcholinestera-se activity

A slightly modified method of Ellman, Courtney, Andres and Featherstone, ( 1961 ) was used to measure the acetylcholinesterase activity of homogenate super- natants (Fromson, 1968). Reaction mixtures contained 2.6 ml of 0.1 M sodium phosphate buffer (pH 8.0), 100 /d 0.1 M dithiobisnitrobenzoic acid in 0.1 M sodium phosphate buffer ( pH 7.0), 100 /A 0.75 M acetylthiocholine iodide and 100 /A enzyme homogenate supernatant. The final substrate concentration in the reaction mixture was 2.5 X 10~2 M. This concentration was assumed to saturate the enzyme reaction based on the findings of Ellman ct al. (1961). The reaction mixtures were incubated at 20° C in a water bath. Under these assay conditions,

ASCI I )l A X AC KT YLCHOLIN EST 1-: RAS I-: -Ml

Ciona enzyme activity was directly proportional to incubation time and directly proportional to the amount of homogenate protein in the reaction mixture.

The enzyme reaction velocity studied was the change in optical density (A O.I).) at 412 nm in a Zeiss PMQII spectrophotometer. In Table I, where per cent activities of the enzyme were being compared, enzyme activity was ex- pressed as A O.D. 412 nm >: 10::/min. Otherwise, In Table II and Figure 1. enzyme activity was expressed as a specific activity, millimicromoles substrate hydrolyzed/min/mg protein, since results with different homogenates were being- compared directly. Four activity measurements were made on each homogenate used and the results expressed as a mean ± the standard error ( S.F. ) of the mean.

Protein aua/vsis

Protein was precipitated from samples of enzyme homogenate by the addition of trichloroacetic acid to a concentration of Sc/r . An acid-insoluble fraction was pre- pared according to \Yhittaker (1966). The Lowry method was used to measure total protein in these acid-insoluble fractions ( Lowry. Rosebrough, Farr and Randall, 1951) with crystallized bovine serum albumin (Armour Pharmaceutical Company ) as a quantitative standard.

Eserine treatment

Unfertilized eggs were removed surgically from the oviducts of adult specimens of Ciotw and washed in several large volumes of filtered sea water. Eggs from each adult organism were kept separately at 18° C for 90-120 minutes (more time than required for the first cleavage to occur). The eggs were examined micro- scopically and only batches of non-dividing eggs were used. These unfertilized eggs were pooled and divided into two groups. One group was held as a control. The other group was treated with 0.002% (3 >: 10"5 M) eserine sulfate for one hour, and washed free of drug with an excess of filtered sea water. Control and eserine-treated eggs were then fertilized by addition of a dilute sperm suspension. and the resulting embryos reared in eserine-free sea water to the tadpole larva stage (18-19 hours at 18° C). There was no evidence that fertilization occurred in the control and eserine-treated eggs prior to the addition of sperm suspension.

RESULTS Xatitre of the cholinesterase enrj

Since the measured cholinesterase activity could be caused by a pseudocholines- terase as well as a true acetylcholinesterase, two series of experiments were performed which would distinguish between these two enzymes. Activities w^ere measured after treatment of the enzyme preparation with three specific enzyme inhibitors, and in the presence of the cholinesterase substrates acetyl- and hutyryl- thiocholine. The results of these experiments are presented in Table I.

The cholinesterase inhibitor eserine sulfate (Chadwick and Hill, 1947) \vas added to larva homogenates (1.5 X 10 ; M final concentration). After a 20-30 minute incubation at room temperature, no enzyme activity was detected in this

242

DAVID KRO.V1SOX AND I. R. WHITTAKER

mixture. This rules out the possibilitv that a non-specific esterase is contributing to the cholinesterase reaction.

Larval cholinesterase was further characterized by the effect of specific enzyme inhibitors. Knzyme activity was measured in control homogenates, homogenates treated for 20 minutes with the pseudocholinesterase inhibitor iso-OMPA (tetra- isopropylpyrophosphoramide, 10 :: .17; Aldridge, 1953), and homogenates treated 20 minutes with the acetylcholinesterase inhibitor B\Y 62C47 I 1 :5-bis-(4-trimethvl-

* i

ammoniumphenyl )pentan-3-one diiodide; Burgen, 1949]. The cholinesterase activity was inhibited 95.8% by BW 62C47 and only 5.5% by iso-OMPA. These data are consistent with the proposition that most of the enzyme activity is acetvl- cholinesterase activity.

TABLK 1

Effect of substrates and specific enzyme -inhibitors on the chalhtcstemse activity of embryo homogenates

Addition to reaction mixture	Function of additive	Enzyme activity (AO.D412 nm X H>3 min) mean ± S.E.	Per cent activity
Homogenate 1 Acetvlthiocholine iodide (2.5 X Ur- .17) Butyrylthiocholine iodide	Cholinesterase substrate Pseudocholinesterase substrate	34. 10 ±0.36 1.53 ±0.1 2	100 4.5
Homogenate 2* None (control) Iso-OMPA (lO-3 AD BW62C47 (10-3 ,17) Eserine sultate (1.5 X 10-' M]	Pseudocholinesterase inhibitor Acetylcholinesterase inhibitor Cholinesterase inhibitor	16. 58 ±0.1 5 15.68±0.12 0.70±0.01 0	100 94.5 4.2 0

* Acetvlthiocholine iodide (2 X 10 2 M) used as substrate.

Acetylcholinesterase and pseudocholinesterase are also distinguished by their respective substrate specificities. Since acetylcholinesterase is highly specific for acetate containing substrates (Aldridge, 1953), enzyme activities were measured using the substrates acetyl- and butyrylthiocholine iodide. The measured enzyme activity with the butyrylthiocholine was only 4.5% of the activity measured using the acetylthiocholine iodide substrate. Therefore, the enzyme activity measured in tadpole homogenates is attributable to the presence of acetylcholinesterase.

Effects o\ eserine treatment

Unfertilized eggs were treated with 3 X 10~5 M eserine sulfate and washed free of the drug after one hour. These eggs were then fertilized and reared in eserine- free sea water. The resulting larvae exhibited a vastly reduced capacity for move- ment. Despite this striking physiological impairment, no morphological irregulari- ties were observed upon microscopic examination of these larvae. These findings are similar to those reported by Dnrante ( 1958 ) .

Acetylcholinesterase activity, measured in homogenates of larvae hatched from eserine-treated eggs (as described above), was inhibited 80-87% (Table II and

ASCIDIAN ACETYLCHOLINESTERASE 243

Kig. 1). These quantitative measurements of enzyme activity confirm l)urant< '-> (1('5S) hypothesis that emhryos treated with eserine prior to fertilization arc defective in acetylcholinesterase activity.

Eserine retention

Eserine accumulation and retention during the one hour prefertilization incuba- tion is indicated by the following results. Acetylcholinesterase activity assayed in homogenates of larvae hatched from eserine-treated eggs was inhibited 80% (Table II). Homogenate mixtures containing equal amounts of control and eserine-treated embryo homogenates should have a predictable enzyme activity calculated by averaging the mean activity values for each of the two component homogenates.

TABLE II Acetylcholinesterase activity of embryos treated prior to fertilization with eserine

Embryo homogenates

Ari-t ylcholinesterase activity

(m/imoles substrate hydrolyzed niin

mg acid-insoluble protein)

mean ± S.E.

Control

Eserine-treated

Calculated activity of a 1 :1 mixture of control and eserine- treated embryo homogenates

Actual activity in a 1:1 mixture of control and eserine- treated embryo homogenates

32.63 ± 0.57 6.60 ± 0.32 19.61

6.23 ± 0.09

In the experiment reported in Table II, this calculated level was 56% of the control enzyme activity level. However, the actual acetylcholinesterase activity detected in the homogenate mixture was only 17% of the control level. Apparently, unfertilized dona eggs treated with eserine accumulate and retain this drug in sufficient quantities to inhibit the majority of the acetylcholinesterase activity in the homogenate mixture. In fact, the acetylcholinesterase activity in the mixed homog- enate was not significantly different from the activity measured in the eserine- treated embryo homogenate alone.

Restoration of enzyme activity

If the sole action of the accumulated eserine is to inhibit enzyme activity, then the presence of this inhibitor during development should not interfere with acetylcholinesterase synthesis. Removal of the inhibitor from homogenates of prefertilization-treated embryos should restore acetylcholinesterase activity to the level of control homogenates. Eserine is a reversible inhibitor of acetylcholinester- ase activity: it can easily be removed from the enzyme by dialysis (Cohen, Kals- beek, and \Yarringa, 1948).

Homogenates were prepared from control and experimental larval stage embryos and acetylcholinesterase activity measured. The experimental embryos were treated with 3 X 10"5 Jl/ eserine sulfate for one hour prior to fertilization. In the experiment shown in Figure 1, eserine treatment resulted in an

244

DAVID FKOMSON AXI) I.

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CONTROL

ESERINE- TREATED

FIGURE 1. Unfertilized Ciona eggs incubated in 3 X 10 5 M eserine sulfate for 1 hour prior to fertilization. The eggs were washed free of eserine, fertilized, and allowed to develop into larvae. Acetylcholinesterase was measured in control and eserine-treated larva homog- enates. These two homogenates were then dialyzed against distilled water, and the post- dialysis enzyme activities of control and eserine-treated embryo homogenates measured. The bars represent the mean enzyme activity of 4 activity measurements on each homogenate ; the vertical lines indicate the standard error of the mean.

inhibition of enzyme activity. These control and eserine-treated embryo homogen- ates were dialyzed against distilled water for 55 hours at room temperature. After dialysis, the enzyme activities in the homogenates were determined once again. Eserine inhibition was completely reversed by dialysis ; acetylcholinesterase activi- ties in the dialyzed control and the dialyzed experimental embryo homogenates were equal (Fig. 1 ). Since dialysis restores the level of enzyme activity in the experimental homogenate to that found in the control homogenate, the presence of eserine has obviously not prevented the synthesis of a full complement of acetyl- cholinesterase in these embryos.

ASCIDIAN ACETYLCHOLINESTERASE 245

An interesting point concerning the stability of Ciona acetylcholinesterase is illustrated by the dialysis of control homogenate in Figure 1. Since there is no loss of activity following a dialysis of two days duration at room temperature, the enzyme is shown to be remarkably resistant to denaturation. This is also true for acetylcholinesterase from other animal sources ( Xachmansohn and \Yilson, 1955).

DISCUSSION

Sawyer (1943) has shown clearly that eserine does not prevent synthesis of cholinesterase in developing Amblystouia embryos. Therefore, there is little reason to accept the conclusion that Durante ( 1958) reaches from her experiments with Ciona embryos, namely, that eserine has interfered with the synthesis of cholinesterase. Unlike Sawyer, Durante has no supporting evidence from enzyme studies ; her conclusion is based solely on the irreversible inhibition of larval motility caused by eserine treatments. The prefertilization experiment with eserine provides the best circumstantial evidence that enzyme synthesis may be deficient, because, as Reverberi (1961) points out, eserine would have to be retained in the egg for an unusually long time if trapped eserine is the real cause of the inhibited motility.

We know of no evidence from any biological system that a specific enzyme inhibitor which is not also a substrate analog could possibly prevent synthesis of an enzyme. On further investigation we have discovered that the Durante work is no exception. There is enough eserine trapped in Ciona embryos following prefertilization treatment to inhibit the acetylcholinesterase of control embryos when homogenates of both are mixed together. Full acetylcholinesterase activity can be restored in homogenates of experimental embryos by simple dialysis. All of the Durante results can be explained by retention of eserine in the egg or embryo following even a brief exposure to the drug.

Based on the Durante (1958) experiments, eserine seems to be freely diffusible into the egg at any stage of development and retained by all the pre-hatching stages. Since reversal of the reduced mobility caused by eserine occurred only in larvae which were first treated with the drug after hatching, it seems likely that the egg membranes are a permeability barrier which restricts loss of eserine in eserine-free sea \vater. This question could be studied further using enzyme preparations which de-chorionate ascidian eggs (Berrill, 1937).

Our investigation also confirms an important observation made by both Sawyer (1943) and Durante (1958). Differentiation of the morphological structure as well as the potential physiological function of an acetylcholinesterase-containing tissue is independent of the functional activity of the enzyme during development. Likewise, the function of tyrosinase in ascidian pigment cells is not necessary for normal differentiation of the cells (Minganti, 1957; Whittaker. 1960, 1966). This principle probably applies to the specialized enzyme systems of most differentiating cells and tissues.

246 DAVID FROMSON AND J. R. \YHITTAKER

Tliis investigation was supported by a predoctoral fellowship of the National Institutes of Health to David Fromson, and by faculty research grants from the Committee on Research of the University of California to J. R. Whittaker.

SUMMARY

1. Fhe use of specific cholinesterase inhibitors and substrates demonstrated that the enzyme activity in dona hitcsthialis larvae is an acetylcholinesterase.

2. Eggs treated with eserine sulfate (an acetylcholinesterase inhibitor) for one hour prior to fertilization developed into larvae with defective muscular movements and greatly reduced levels of acetylcholinesterase activity.

3. Two kinds of experiments show that this reduced enzyme activity \vas caused by the retention of eserine and not by inhibition of acetylcholinesterase synthesis. Homogenates of embryos from eserine-treated eggs inhibit acetyl- cholinesterase activity when mixed with homogenates of control embryos. Full enzyme activity in homogenates of the experimental embryos could be recovered by dialysis.

LITERATURE CITED

ALDRIDGE, W. N., 1953. The differentiation of true and pseudocholinesterase by organo-

phosphorus compounds. Biochcin. J.. 53 : 62-67. BERRILL, N. J., 1937. Culture methods for ascidions. Pages 564-571 /'».; P. S. Galtsoff, F. E.

Lutz, P. S. Welch and J. G. Needham, Eds., Culture Methods for Invertebrate Ani- mals. Comstock, Ithaca. BURGEN, A. S. V., 1949. The mechanism of action of anticholinesterase drugs. Brit. J.

Pharmacol., 4: 219-228. CHADWICK, L. E., AND D. HILL, 1947. Inhibition of cholinesterase by diisopropylfluorophos-

l>hate, physostigmine, and hexaethyl tetraphosphate in the roach. /. Neurophysiol., 10:

235-246. COHEN, J. A., F. KALSBEEK AND M. G. P. J. WAKRINGA, 1948. Reversibility of the inhibition

of true cholinesterase by physostigmine. Biochim. Biophys. Acta. 2 : 549-560. COSTELLO, D. P., M. E. DAVIDSON, A. EGGE.RS, M. H. Fox AND C. HENLEY, 1957. Methods

for Obtaining and Handling Marine Eggs and Embryos. Marine Biological Laboratory,

Woods Hole, Massachusetts, 247 pp. DURANTE, M., 1956. Cholinesterase in the development of the ascidian, dona intestinalis.

Expcricntia, 12: 307-308. DURANTE, M., 1957. Cholinesterase in the anterior and posterior hemiembryos of dona

intestinalis. Acta Embryol. Morphol. E.vp., 1 : 131-133. DURANTE, M., 1958. Action of cholinesterase inhibitors on ascidian embryos. Acta Embrvol.

Morphol. E.\-p., 1 : 273-279. ELLMAN, G. L., K. D. COURTNEY, V. ANDRES AND R. M. FEATHERSTOXE, 1961. A new and

rapid colorimetric determination of acetylcholinesterase activity. Biochcin. Pharmacol.,

7 : 88-95. FROMSON, D. R., 1968. Synthesis of acetylcholinesterase during embryogenesis of C-iona

intestinalis. Ph.D. thesis, University of California at Los Angeles, 101 pp. LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, 1951. Protein measure- ment with the Folin phenol reagent. /. Biol. Chem., 193: 265-275. MINGANTI, A., 1957. Inhibition of melanogenesis in Phallnsia embryos (Ascidians). Acta

Embryol. Morphol E.vp.. 1 : 37-47. NACHMANSOHN, D., AND I. B. WILSON, 1955. Acetylcholinesterase. Pages 642-651 in S. P.

Colowick and N. O. Kaplan, Eds., Methods in Ensymology, J'ol. 1. Academic Press,

Inc., New York.

ASCIDIAN ACKTYLCHOLINESTKRASE 247

REVEKBEKI, G., 1961. The embryology of ascidians. Pages 55-101 in M. Abercrombk- and J.

Brachet, Eds., Advances in Morphogenesis, J'olume 1. Academic Press, Inc., New

York. SAWYER, C. H., 1943. Cholinesterase and the behavior problem in Amblystoma. I. The

relationship between the development of the enzyme and early motility. II. The

effects of inhibiting cholinesterase. /. E.rp. Zool., 92 : 1-30. WHITTAKER, J. R., 1960. An in vivo analysis of tyrosinase function and melanin formation in

ascidian embryos. Aunt. Rec., 138: 388-389. WHITTAKER, J. R., 1966. An analysis of melanogenesis in differentiating pigment cells of

ascidian embryos. Develop. Bio!., 14: 1-39.

Reference : />'/"/. Hull.. 139: 248-264. (October,

FILTRATION OF PARTICLES FROM SUSPENSION BY THE AMERICAN OYSTER CR.ISSOSTKR.-l I'IRGINICA1

DEXTER S. HAVEN AND REINALDO MORALES-ALAMO I'iri/inia Institute of M urine Science. Gloucester I'oinl. I'iry'mia 23062

The oyster Crassostrea i'i>-</inica is a suspension feeder which retains suspended participate material on its gills. These are lamellar and composed of rows of fila- ments in groups that constitute folds (plicae) in the lamellar surface (Nelson, 1960). Water is pumped through small interfilamentary openings (ostia) by the action of cilia on the filaments. The actual manner in which particles are retained on the gills is not completely understood (J0rgensen, 1966). Suggested mecha- nisms include entrapment in mucus, straining action of large latero-frontal cilia, and limitation of passage through the gills by the size of the interfilamentary ostia.

Two methods have been commonly used to study particle filtration by bivalves. One involves channeling water into and from shell cavities with devices which collect all water flowing through the gills (Galtsoff, 1928; Loosanoff and Engle, 1(>47; Tammes and Dral. 1955). These studies have been criticized because oysters may not behave normally when subjected to the stress of the collecting device. The second involves measurements of rates at which undisturbed lamelli- branchs clear particles in standing water. This latter method has been used extensively by many workers in recent years ( J0rgensen, 1949, 1960; Ballantine and Morton, 1956; Chipman and Hopkins, 1954; J0rgensen and Goldberg, 1953; Willemsen, 1952). Objections to studies in standing water are that previously filtered material may be resuspended and refiltered (recycled) and also that particle concentrations will change with time. With only several exceptions, as exemplified by the work of Loosanoff and Engle ( 1947), particle density was mea- sured indirectly by techniques such as isotope labeling, per cent light transmission, etc. Results of these studies differ widely in respect to the efficiency at which various size particles are filtered from suspension. No one has correlated the results of these investigations with the structures and mechanisms which determine them (J0rgensen, 1966).

The purpose of this investigation was to establish more precisely the size of the smaller particles removed from suspension by the American oyster Crassostrea I'irc/inica and the efficiency of removal and to relate our results to the most probable mechanism involved.

MATERIALS AND METHODS .-Ipparatus and materials

The basic design of the study was to hold oysters in troughs of flowing water and to measure differences in numbers of particles entering and leaving the system. Differences were attributed to filtration by the animals. The experimental ap-

1 Contribution No. 356 from the Virginia Institute of Marine Science. Tin's research \va* supported by LI. S. Atomic Energy Commission Grant No. AT- (40^1) -2789.

248

I'ARTICLK FILTRATION IJY OYSTERS

250 DEXTER S. HAVEN AND REINALDO MORALES-ALAMO

proach of iiur studv differed from previous studies in several respects. Particle filtration was studied simultaneously over a continuous spectrum of sixes ranging from about 1.0 /j. to 12.0 /*, and direct measurements and counts of the particles involved were made. Water flowed continuously over the oysters under conditions approaching those of the natural environment. This also served to avoid recycling. Filtration activity was not measured instantaneously but covered a period of approximately 12min.

The experiments were conducted at the Virginia Institute of Marine Science, Gloucester Point, Virginia. Preliminary studies began in 1964 and 1965, but most data were obtained during 1966 and 1967.

Water for all studies was pumped through plastic pipes from a source 0.5 m above the bottom, 90 m offshore in the York River, to a constantly overflowing overhead trough in the laboratory. Water flow in 1966 experiments \vas 1.01 1/min ; in 1967, flows were regulated to range from 0.19 to 1.01 1/min.

Data were collected using a system having continuous water flow over the oysters and controls (single oyster shells). Particle removal was studied in respect to 1 ) the natural particle complement of York River water ( in one series of experiments) and 2) kaolinite clay particles added to filtered York River water. Kaolinite (American Petroleum Institute standard no. 7, Bath, South Carolina) with a density of 2.6 was similar to the mineral components of suspended matter in the York River (Nelson, 1963 ) .

The apparatus used is shown in Figure 1. Water was pumped intermittently from an overhead trough (A) through two spool-wound yarn depth niters (porosity of 20 fj. and 1 p., respectively ) in series (B) into a 13-gal polyethylene carboy (C). An alternate nitration system (B Alternate) was used in series with river water without added materials. In these experiments a standard soil analysis sieve with mesh size of 44 /j. was substituted for the depth filters. Passage through the soil analysis sieve did not appreciably change quantity of suspended solids since it had been found previously that 97c/r by weight of the suspended material in the York River passes through a 44 ^ sieve (Haven and Morales- Alamo, 1968). The carboy served to maintain a relatively constant water head and to enhance dissipation of air bubbles.

Kaolinite particles added to the flowing water were maintained in suspension in an acrylic tank (D) with an electric stirrer. From this source they were metered with a peristaltic pump (Harvard Apparatus Co., Dover, Massachusetts) into a 4.000-cc mixing flask (F). A magnetic stirrer mixed the suspensions with the filtered water and a second 4.000-ml flask (G) in series with the first enhanced homogeneity.

The mixture passed through flow meters (H) into two identical acrylic plastic experimental troughs (I) holding live oysters and oyster shells, respectively; troughs were 47 cm long by 13 cm wide and 6 cm high. A small magnetic mixing bar at the head of each trough under the inflowing hose further mixed the inflowing suspension. Three oysters (or shells) were placed side by side in each of four successive compartments. Twelve oysters were used in all studies except several in 1967 which used 24. Water depth above oysters and shells was approximately 2 cm.

Oysters came from an area free of known oyster diseases (Horsehead Rock) in the James River, Virginia. They were about 2 years old and averaged 50 mm in

PARTiCLK FILTRATION BY OYSTERS ^1

height with a mean wet meat weight of 4.8 g. To minimize possibility of particle recycling, flows and oyster numbers were chosen so that flow exceeded the theo- retical pumping capacity of the oysters. Calculations showed that each oyster received a flow of 1.05 1/hr per gram of wet meat weight. J^rgensen (1966) summarized data showing that adult oysters may pump through their gills from 0.5 to 0.8 1/hr per gram of wet meat weight.

Oysters and shells were scrubbed, rinsed, and immediately placed in experi- mental troughs enclosed in a large, darkened box.

Total solids were measured by nitration through 0.45 ^ membrane niters. Salinities and temperatures were monitored daily during the experimental periods with a stem hydrometer and a Foxboro recording thermometer, respectively. During 1966, studies extended from 24 June to 14 October, during which period salinities ranged from 18.0 to 24.1 %o and temperatures from 17.9 to 28.0° C. In 1967, experiments were conducted from 10 May to 12 June, with temperatures ranging from 14.2 to 23.4° C and salinities from 19.0 to 20.2 %0.

Collection of samples

Samples of water for particle size analysis were collected simultaneously from two pairs of 3-mm I.D. glass tubes which siphoned water continuously from the troughs halfway between the water surface and the top of the compartmental baffles (Fig. 1). One pair of tubes was located over the compartment im- mediately preceding the oysters (or shells), a second pair over an empty com- partment at the outlet. Samples were not collected until oysters were actively pumping and producing faeces or pseudofaeces, a period ranging from ^ to 2 hr. Faeces or pseudofaeces produced by the oysters settled to the bottom of the troughs without contributing to the particle load flowing from the system. Each sample to be analyzed for particle size was obtained by combining five subsamples, each of which took about 30 sec to collect and was obtained at 3-min intervals over a 12-min period. Consequently, each sample represented oyster activity over a 12-min interval.

In many experiments it was necessary to dilute samples to retard flocculation and reduce inaccurate counts resulting from coincident passage of particles through the tube orifice of the Coulter Counter. York River water, filtered twice through 0.45 /j. membrane filters, was used as dilutant. Subsamples collected from the troughs were diluted immediately by pouring into a flask containing a predetermined volume of dilutant. Dilution factors ranged between 1.9 and 3.6, depending on particle concentrations flowing through the troughs. After collection, samples were held at 26° C in a constant temperature water bath until counted since it was earlier found that changes in sample temperature affected the counts recorded by the. Coulter Counter.

Coulter Counter and analysis oj samples

Particles in the water samples were measured and counted using a Model B Coulter Electronic Particle Counter (Coulter Electronics, Inc., Franklin Park. Illinois). The counter principle is based on particles suspended in an electrolyte being forced through a small orifice between two electrodes through which an electric current path has been established; each particle displaces electrolyte in

252

DKXTKK S. HAVEN AND REINALDO MORALES-ALAMO

the orifice essentially equal to its volume and creates a change in current resistance proportional to this volume ( Kubitschek, 1960; Sheldon and Parsons, 1967). The counter does not measure linear dimension, and particle diameter is calculated from the displaced volume and represents the diameter of a sphere having the same volume as the particle. Particle counts are recorded on a decade counter.

Cumulative particle counts made at increasing consecutive particle size settings were processed through an IBM 360 computer. Counts were corrected for coincident passage through the orifice, for background count in the dilutant water,

TAHLF. I

Mean and range of number of particles of different size fractions per liter ofu'itler entering troughs with oysters in 1966 series of experiments

Size fraction GO	No. replication-	No, of particles (millions)
Mean	Range

Natural particles

1.00-1.99	3	1001.4	790.4-1230.6
2.00-2.99	3	103.2	77.3-131.0
3.00-3.99	6	33.3	20.4-48.3
4.00-4.99	6	10.9	6.4-14.0
5.00-5.99	6	5.5	4.2-7.6
6.00-6.99	5	2.3	1.6-2.8
7.00-7.99	4	1.1	1.1-1.3
9.00-9.99	3	0.3	0.3-0.4
11.00-11.99	3	0.1	0.1-0.1

Kaolinite particles

1.00-1.99	34	921.2	391.7-1756.0
2.00-2.99	34	. 25.8	65.1-492.1
3.00-4.99	34	59.2	16.2-118.0
5.00-6.99	12	8.9	4.5-16.7

and for dilution, and were then converted to differential counts. The latter were subsequently converted to total volume of all particles in the size interval. Size intervals were 1.0 p in most instances; however, on several occasions intervals ranging from 0.1 to 0.5 p. were used for sizes near 1.0 p.. In several instances intervals of 2.0 p. were used for sizes larger than 5.0 ^. Efficiency of removal of particles is expressed as per cent difference in numbers of particles of a size interval between the inflowing and outflowing water samples.

Data collected in preliminary studies conducted in 1964 and 1965 were subject to excessive variations in the control data and are presented only as a supplement to data obtained in 1966 and 1967. Later modifications eliminated this problem (see Methods).

The data on per cent removed were compared by analysis of variance after being- transformed into degrees using the arcsin percentage transformation. Further

I'ARTICLH FILTRATION BY OVSTKRS

253

analysis was made using Scheffe's multiple mean comparison method (Guenther, 1964). Data are presented graphically in terms of the point and 95% interval estimate of the mean, .r ± .S' •?/<,. u.-,, where .r -= mean, Sx -- sample standard error, and /„,).-, = the 59r level of student's t distribution ( Snedecor, 195(> I.

RESULTS

Individual experiments with kaolinite and natural particles showed a consistent pattern. There was a sharp increase in per cent of particles removed as particle size increased from 1.0 to between 2.0 and 4.0 p.. At larger sizes the per cent removed leveled off with no indication of further increase in efficiency.

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FIGURE 2. Mean per cent difference between particle numbers in water entering and water leaving experimental troughs for separate size fractions in 1966 natural particles experiments, plotted at mid-point of size interval ; open bars = oysters, solid bars == shells ; horizontal line = mean, vertical line -- range, bar •- interval estimate ( .r ± Sjtfo.os) . Figures over bars indicate number of replications.

Variations were present between individual experiments, especially in those using kaolinite particles, but the general pattern described above appeared con- differences in the filtration activity of the oysters at the time a particular experiment was conducted. Insofar as temperature is concerned, Loosanofr" (1958) observed no significant change in pumping rate of oysters between 16 and 28° C. In the absence of any observed relation for these three parameters, data for particle -sistently in every experiment. These variations could not be correlated to differences in temperature, salinity or particle concentration and probably resulted from removal were combined for similar size intervals. Results of studies made at size intervals different from those included above are reported separately. During most studies, oysters produced faecal strings and pseudofaeces, indicating filtration of particles from suspension and ingestion of some.

254

DKXTF.R S. IIAYKN AN'D RKl.XAl.DO MORALES-ALAMO

I'iltration under flow o\ J.01 l/inin using 12 oysters

Filtration of natural particles was studied in 1966 using 12 oysters and a flow of 1.01 l/inin. A total of six studies was combined into a composite curve at intervals of 1.0 /* (Fig. 2).

Particles in the undiluted water entering the trough were by far the most abundant in the 1.0 to 2.0 /JL size range where an average of over 1 billion per liter was measured. Numbers decreased rapidly with size and only 70,000 per liter were

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FIGURE 3. Mean per cent difference between particle numbers in water entering and water leaving experimental troughs for separate size fractions in 1964-1965 kaolinite particles experi- ments; open bars — oysters, solid bars == shells; horizontal line =- mean, vertical line = range, bar = interval estimate ( .f ± S^to.os) . Figures over bars indicate number of replications.

measured in the 12.0 to 13.0 p size range (Table I). Total solids were not consistently measured ; however, during July and August 1966 in the York River they ranged from 4.7 to 29.0 nig/1, with a mean of about 10.0 nig/1 (Haven and Morales- Alamo, 1966a).

Oysters removed a mean of 18% of the particles between 1 and 2 /JL. Average removal increased to 53% between 3.0 and 4.0 /J-. Above this size, there was no evidence of further differences in efficiency of removal up to 12.0 p. Mean per cent differences between inflow and outflow in the controls were close to zero with a small variance for most size intervals. There was no evidence of a difference among these means.

PARTICLE FILTRATION BY OYSTERS

255

Two separate studies conducted in August 1966 using natural particles with 12 oysters and a flow of 1.01 1/niin included size intervals under 1 /x. In the first, oysters removed 34.8% of the particles between 0.8 and 0.9 /* and 37.8% of the particles between 0.9 and 1.0 p. The second experiment showed a removal of 28.5% for particles between 0.9 and 1.0 //,.

Data on filtration of kaolinite particles obtained during preliminary studies conducted in 1964 and 1965 showed a similar pattern of removal with increase in

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FIGURE 4. Mean per cent difference between particle numbers in water entering and water leaving experimental troughs for separate size fractions in 1966 kaolinite particles experiments ; open bars = oysters, solid bars - : shells ; horizontal line : : mean, vertical line : : range, bar = interval estimate (.v ± Sxto.o5). Figures over bars indicate number of replications.

particle size (Fig. 3). Removal efficiency increased from 21% between 1.0 and 2.0 //. to 38% between 2.0 to 3.0 p.\ there was no evidence of further change in efficiency at larger sizes. The values recorded in these data are not considered reliable because of the excessive variation observed in the data but are included here only to illustrate the recurrence of a plateau in removal efficiency at particle sizes larger than 3 /A.

Data on filtration of kaolinite particles in 1966 were obtained by combining 34 separate studies using a flow of 1.01 1/min and 12 oysters. Particles over 7.0 /JL in the diluted samples were too few fof precise counts and were not included in the analysis.

Particles in the undiluted inflowing water were most numerous in the 1.0 to 2.0 /A size range where they averaged 921 million per liter. Particle number decreased to 8.9 million between 5.0 and 6.0 ^ (Table I ) .

256

DEXTER S. HAVEN AXD REINALDO MORALES-ALAMO

Removal of particles between 1.0 and 2.0 /*. averaged 24% ('Fig. 4). This increased to 37% for 2.0-3.0 /j. particles and reached 42% between 5.0 and 7.0 p.. There was no evidence of a significant difference in mean per cent removed among the three particle size fractions included between 2.0 and 7.0 p. In the controls, mean differences in particle counts between inflow and outflow were close to zero, with only small variances for all si/e intervals. As in studies with natural

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particles, there was variation between rates of removal at various particle sizes. However, most of the studies fell within relatively narrow limits.

Two of the experiments with kaolinite particles conducted in August 1966 included counts made at intervals smaller than 1.0 //.. These showed an average per cent removal of 32.7% for particles between 1.1 and 1.5 /j., 43.9% for particles between 1.5 and 2.0 p., and 54.2% for particles between 2.0 and 2.5 /j,.

Filtration under variable flow and oyster number

It was possible that the basic pattern of particle removal for all preceding data was a direct result of the particular experimental conditions used, i.e., a flow of 1.01 1/min and 12 oysters. To test this possibility, experiments with natural particles were conducted in May 1967 at various flow rates and with 12 or 24 oysters.

PARTICLE FILTRATION BY OYSTERS 257

Per cent removal using the standard 1.01 1/min flow with 12 oysters increased from 2Z% for particles between 1.0 and 2.0 // to 56^ between 3.0 and 4.0 /*. Xo change in per cent removed was evident for sizes above 4.0 /;, (Fig. 5, solid circles I. These data were almost identical to data collected in the 1^66 experiments. Seven experiments were conducted at flows of 1.01 1/min but with 24 oysters. Doubling oyster number displaced the entire curve upward. The basic pattern, however, remained the same (Fig. 5, half-filled circles), with efficiency increasing

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in the 1.0 to 4.0 fi range and then leveling off at about 88//r. In effect, doubling oyster number nearly doubled rates of removal. A reduction in flow but with 12 oysters had nearly the same effect as doubling oyster number and the basic pattern was still evident (Fig. 5, open circles ).

Particle removal in terms of volume

It was shown previously that natural particles were numerically most abundant between 1.0 and 2.0 p. and decreased to much lower levels between 11.0 and 12.0 p.. A similar relationship was found when particle numbers were converted to volumes (Fig. 6). Mean total volume for particles between 1.0 and 2.0 // entering

258

DKXTKK S. HAVKN AM) RHIXALDO MORALES-ALAMO

the svsleni was about 2.5 limes greater than in either of the next two larger size fractions and decreased significantly with particle size in the size range included. A test on the slope of the regression of mean total volume removed on particle size showed that it was significantly different from 0 ( /•* < 0.05). Therefore, volume removed decreased with increase in particle size (r-- 0.971). The mean volume removed by oysters between 1.0 and 2.0 p, was 0.425 mm"', but only 0.066 mm3

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between 11.0 and 12.0 p.. The combined volume removed between 1.0 and 4.0 p. represented 52.0% of the total removed between 1.0 and 12.0 p.; that removed between 7.0 and 12.0 p. represented much less, being 13.5% of the total. The 4.0 to 7.0 p. fraction represented 34.5% of the total.

Conversion of kaolinite particle counts to volume also showed a greater mean total volume of particles between 1.0 and 2.0 p. than of any of the other size fractions entering the system ( Fig. C> ) . Volumes plotted at the mid-points for the 3.0-5.0 and 5.0-7.0 p, intervals are one-half of the total volumes in the 2.0 p. intervals. That is, the value plotted represents mean removal rates for a 1 .0 p. interval.

The data for volumes of kaolinite particles removed were distributed differently than those for natural particles. Although a relatively high correlation coefficient

PARTICLE FILTRATION BY OYSTKK'S

of r := 0.751 was obtained, further analysis showed no evidence that the regression slope of mean total volume on particle size was significantly different from 0 (P > 0.05). Therefore, the data do not show that volume removed decreased with increase of particle size. They do show, however, that as much volume between 1.0 and 2.0 //. was removed as of any other size fraction.

The preceding results show that volumes of suspended material removed by oysters in the vicinity of 1.0-3.0 p. are at least as great as those in larger size intervals measured when considered in terms of the total volume rather than in terms of the efficiency with which oysters remove them.

Volume data from individual experiments with kaolinite particles, conducted in 1966, were used to illustrate the absence of a relation between concentration of particles entering the system, expressed as mg/1, and per cent of the particles removed. For this purpose, the data were converted into mg/1 by multiplying total volume of all particles in the size spectrum included in each experiment by 2.6, the approximate density value for kaolinite. These data were then grouped into three intervals representing low, moderate and high concentrations. These were, respectively, 5.91-10.10, 11.40-19.60 and 20.34-37.66 mg/1 ( Fig. 7).

Results showed that, within the size and volume limits studied, the quantity of material present in the inflowing water did not affect the per cent of particles removed at any of the size intervals measured or the shape of the curve for per cent removed vs. particle size.

DISCUSSION

JoYgensen (1960; states that results obtained by different investigators often vary or even disagree and that quantitative data on feeding in lamellibranchs would be of greater value if they can be shown to have been derived from experiments on undisturbed animals. We consider that our experimental technique approached the undisturbed conditions of the natural environment.

Other workers have found a relation similar to that shown by our data between particle size and per cent removal for C. rinjinico while the data of others disagree. JoYgensen and Goldberg (1953) found that graphite particles of sizes down to a few microns were effectively retained by C. rirginica and that 80% of the 1.0 to 2.0 /j. particles passed through the gills. Loosanoff and Engle (1947) showed that cells of the flagellate Euglena 60 ^ in length easily passed through the gills of C. virginica and sometimes only 15% was removed from suspension. In the 5.0 //. size they found oysters retained 0 to 85% of the Chlorclla sp. cells. They concluded that little correlation existed between size of the microorganisms and per cent removed by the gills. It is possible that differences in experimental techniques and in materials being filtered may adequately account for differing results.

Our experiments show a well defined pattern of particle removal when results are expressed in terms of per cent removal. Consistently and regardless of experi- mental conditions, oysters removed particles in the size range of 1.0 to 2.0 /j. with less than half the efficiency of the larger particles. Efficiency increased with particle size up to 3.0-4.0 //, for natural particles and to 2.0-3.0 ^ for kaolinite and then leveled off, with no evidence of a further change in efficiency. Minimum size of natural particles removed was between 0.8 and 0.9 //,, when 34.8% of the avail- able particles were removed.

260 DEXTER S. HAVEN AND KKI . \ALIX > MORALES-ALAMO

The increased efficiency of particle- removal with an increase in particle size was not unique to flows of 1.01 l/niin and 12 oysters. Doubling oyster number at J.01 1/inin had the effect of displacing the entire curve upward. 24f/r in the 1.0- 2.0 I.L range and from 34 to 42',; in the larger si/.e range. The inflection point on all curves, however, was still in the 3.0—4.0 /*, range. A similar inflection point was also demonstrated when (lows were reduced.

The percentage value at which the curves level oft" in our experiments is interpreted as representing the relation between the quantity of water filtered through the oysters' gills and the total volume of water flowing through the trough. Where the curve levels off at a removal of 60r/r, it is suggested that oysters were only pumping through their gills about 60c/o of the water flowing over them. This interpretation is supported by the fact that the curve was displaced upward when flows were decreased or oyster number increased without any changes in the nature of the curve itself. Furthermore, it was outlined earlier that oysters in our study were receiving 1.05 1/br per gram of tissue. This is in excess of the 0.5 to 0.8 1/hr per gram of tissue pumping rates of adult oysters summarized by Jp'rgensen (1966). Leveling off of the curves, therefore, indicates that all particles larger than the size of which the inflection point occurs are being completely filtered out of the water pumped through the gills.

The constancy in location of the inflection point in the curves suggests that it represents the smallest particle size that oysters can retain with complete efficiency. JpYgensen (1966) stated that such a sharp size limit between retainable and non- retainable particles is an indication that it is particle size and therefore the porosity of a filter that is responsible for retention. He also stated that the critical size for particle retention in Crassostrca 'I'irtjinica corresponds to the distance between adjacent laterofrontal cilia on a filament, which according to Atkins (1938) varies between 1.5 and 3.7 ^. This range coincides with the range of particle sizes at which the inflection point appears on our curves. This correspondence reinforces the suggestion that the distance between adjacent latero-frontal cilia is the factor that determines the smallest particle size that can he completely retained by the oyster gill.

It is difficult to reconcile the preceding mechanism of removal with the observed quantity of material removed in the 0.8 to 3.0 p. range in our experiments. Several possibilities exist: ( 1) The Coulter Counter essentially measures volume of particles and results are expressed in terms of the diameter of spheres of equal volume. It would be illogical to assume that the smaller particles would be round since particles in the 0.8 to 3.0 /A size range might include lenticular clay particles, ovoid dinoflagellates, rod-shaped bacteria, etc. It is possible that the orientation of the long axis of these irregularly shaped particles as they strike the filtering mechanism results in retention of a fraction of their numbers. (2) Smaller par- ticles may be trapped in with the larger ones, as suggested by Smith (1958), or adhere in some way to mucus on the gills. (3) Passage of particles through the interplicar spaces may stimulate blood to lie pumped into the vascular tissue sur- rounding the ostia resulting in partial closure of these openings (Elsey, 1935). (4) The gill musculature may exert some regulation on the porosity of the gill filter by adjusting the width of the ostia (JoYgensen, 1(>(>6).

PARTICLE FILTRATION' BY OYSTKKS 261

The mucous sheet theory of MacGinitie (1941) cannot be considered as a possible mechanism to explain the removal of particles in the 0.8 to 3.0 ^ range in our studies. According to this theory, when the animal is feeding, a mucous sheet that covers the entire gill surface intercepts all particles from the water passing through the gills. In all individual experiments \ve conducted, efficiency ot removal was lower in the 1.0 to 3.0 p. size range than at larger sizes. Existence of a mucous sheet should have resulted in complete retention of the smaller particles rather than in the partial retention observed. The plot for per cent removal vs. size would have then been a straight line showing equal efficiency of removal of particles of all sizes instead of the curve we obtained (Figs. 2 through 5).

Much controversy has evolved around the mucous sheet theory of MacGinitie ( 1941 ) as opposed to the straining function of the latero-frontal cilia. MacGinitie claimed that when the mucous sheet was absent from the gills of the four pelecypocl species he studied, no feeding was taking place, in effect disregarding the involve- ment of ciliary mechanisms in particle retention.

Nelson (1960) objected to the mucous sheet theory on the basis that if a mucous net fine enough to trap bacteria were spread across the gills, it would be drawn down against the lateral cilia and into the ostia by the strong water currents pumped through the gills interfering with the function of both. JpYgensen (1966) stated that it is unlikely that the complicated sorting mechanisms of the bivalve gill should not play a part in their feeding process as would be the case if a mucous sheet was required. Owen (1966) expressed the same objection and added that such a continuous mucous sheet would be nearly impossible to maintain in the presence of the adjacent tracts of frontal cilia beating in opposite directions. Dral (1967) did not find any supporting evidence for existence of a mucous sheet in Myfilits cdnlis in heavy or dilute particle concentrations and pointed out that all observations indicated that the latero-frontal cilia constitute the filtration mechanism.

JoYgensen (1955, 1966) tried to conciliate the conflicting interpretations by suggesting that filtration by means of mucous sheets occurs in water that contains only small concentrations of suspended material and that in water with large particle concentrations, filtration is accomplished by ciliary mechanisms.

Our experiments covered a wide range of concentrations (Fig. 7). The curve for efficiency removal obtained in every experiment showed the same pattern described in the results. The presence of an inflection point in every instance, which has been given as an indication of the absence of a mucous sheet, suggests that particle retention was accomplished by a mechanism other than the mucous sheet at low as well as at high concentrations. Particle retention on the gills by latero-frontal cilia, however, is very probably enhanced by other responses such as muscular contraction of the gills and ostia, vascular enlargement of tissues around the ostia and secret'ion of mucus in a manner other than as a continuous sheet (Verwey, 1952; Owen, 1966).

The preceding discussion has considered particle filtration in terms of per cent removal since much of the previous work has been presented in this manner. From an ecological aspect, the data become more significant when presented in terms of volume. Although per cent removal was lower between 1.0 and 3.0 p. for natural particles and 1.0 and 2.0 //. for kaolinite than at larger sizes, volumes

262 DEXTER S. HAVEN AND REINALDO MORALES-ALAMO

removed in these size ranges constituted the largest single fraction within the total size range investigated. This resulted from the smaller particles hetween 1.0 and 3.0 p. heing many times more numerous than those of larger size (Tahle I).

The oysters filtered relatively large quantities of solids in the 0.8 to 3.0 //. size range. No effort was made in this study to differentiate hetween the frac- tion of the particles filtered out and ingested and that of particles filtered out hut rejected as pseudofaeces. However, data collected in concurrent studies suggested that particles in this size range are the major component of solids ingested by oysters. Approximately 95% of the particles counted microscopically in dispersed samples of oyster faeces were smaller than 3 /x (Haven and Morales- Alamo, 1966a). Eighty to ninety per cent by volume of the particles in similar samples were smaller than 4 /j. as determined with the Coulter Counter (Haven and Morales- Alamo, 1968).

These findings suggest that particles in this size range suspended in the wrater may play an important role in the nutrition of oysters and other lamellibranchs. A review of much of the existing information on size of particles suspended in coastal waters is given by JpYgensen (1966). He concludes that in respect to the phytoplankton which are of importance in the nutrition of lamellibranchs, the nanno- and ultraplankton (less than 5.0 /A) mostly constitute the larger and often even the dominant part of the phytoplankton. Included in the size class are the marine bacteria which may range in size from less than 1 /j. up to about 2.5 i^.

Organic detritus has also been suggested as being an important food source for filter-feeding lamellibranchs (Verwey, 1952). Mullin (1965a) found that the 1-10 p, fraction of the participate matter from 20 stations in the western Indian Ocean constituted on the average 58% of the total participate organic carbon measured. Subsequently, Mullin (1965b) stated that phytoplankton carbon ac- counted for less than 20% of the total participate carbon, making carbon in the form of detritus or heterotrophic organisms considerably more important than was suggested in his earlier report. Newell (1965) suggested that much of the food supply of filter feeders may be provided by microorganisms attached to fine size suspended organic and mineral participate matter.

Removal of fine particles in the 1 to 10 p. range by oysters and possibly by other filter feeders may also be an important factor in sedimentary processes affecting the physical and chemical characteristics of bottom substrates and the benthic communities they support (Haven and Morales-Alamo, 1966a, 1966b, 1968). In a similar manner, radionuclides adsorbed onto clay mineral particles or incorporated into bacterial and algal cells as a result of accidental contamination of an estuary may be removed from the water by the filtration activity of filter feeders and deposited on the bottom where their accumulation may become a hazard.

Appreciation is expressed to Mr. John Xorcross, Dr. Joseph Loesch, and Mr. Frank Wojcik of the Virginia Institute of Marine Science for assistance in statistical analysis and in preparation of computer programs.

SUMMARY

1. Particle filtration by the oyster Crassostrca 1'irr/inica was studied in the 1.0 to 12.0 p. size range in relation to naturally occurring particles and to kaolinite suspensions in filtered river water.

PARTICLE FILTRATION BY OYSTKRS 263

2. Oysters were held in troughs of flowing water under conditions similar to their natural environment. Particle number and volumes entering and leaving the troughs were enumerated using a Coulter electronic particle counter. Par- ticle diameter was expressed as that of a sphere having a volume equal to the particle.

3. Results of the study were expressed as per cent of total particles removed in various size increments or in volumes removed over the same size range.

4. Oysters filtered naturally occurring particles in the 1.0 to 3.0 //, range with about one-third the efficiency as larger particles. Above 3.0 //. there was no change in efficiency with increasing particle size. For kaolinite particles, oysters removed particles in the 1.0 to 2.0 p, range with about half the efficiency as larger particles. Above this size there was no change.

5. When results are expressed in terms of per cent removal, the importance of the small sized particles is minimized. In terms of volume, particles in the 1.0 to 3.0 //, range constitute the largest single size fraction over the 1.0 to 12.0 p. range.

6. The consistent presence of an inflection point at a particle size around 2.0- 3.0 p, in the curve for per cent removed rs. particle size is an indication that the distance between adjacent latero-frontal cilia is the factor that determines the smallest particle size that can be completely retained by the oyster gill.

7. Presence of the inflection point is also interpreted as negating the existence of a mucous sheet over the gills, as suggested by MacGinitie (1941).

8. Particles in the 1.0 to 3.0 ^ range may play an important role in the nutrition of oysters and other lamellibranchs. Their removal by these organisms may also be an important factor in sedimentary processes.

LITERATURE CITED

ATKINS, D., 1938. On the ciliary mechanisms and interrelationships of lamellibranchs. VII.

Latero-frontal cilia of the gill filaments and their phylogenetic value. Quart. J.

Microscof. Sci., 80 : 346-430. BALLAXTIXE, D., AXD T. E. MORTOX, 1956. Filtering, feeding, and digestion in the lamellibranch

Lasaca nibra. J. Mar. Biol. Ass. U. K., 35 : 241-274. CHIPMAX, W. A., AXD J. G. HOPKIXS, 1954. Water nitration by the bay scallop, Pecten

irradians, as observed with the use of radioactive plankton. Biol. Bull.. 107 : 80-91. DRAL, A. D. G., 1967. The movements of the latero-frontal cilia and the mechanism of particle

retention in the mussel. Xetlicrlaiids J. Sea Res., 3 : 391-422. ELSEY, C. R., 1935. On the structure and function of the mantle and gill of Ostrca

(Crassostrea) gigas and Ostrea lurida. Trans. Roy. Soc. Canada, 29: 131-160. GALTSOFF, P. S., 1928. Experimental study of the function of the oyster gills and its bearing

on the problems of oyster culture and sanitary control of the oyster industry. Bull.

Bur. Fish. U. S., 44 : 1-39. GUEXTHER, W. C., 1964. Analysis of Variance. Prentice-Hall, Englewood Cliffs, New Jersey,

199 pp. HAVEX, D. S., AXD R. MORALES-ALAMO, 1966a. Aspects of biodeposition by oysters and

other invertebrate filter feeders. Limnol. Oceanogr., 11 : 487-498. HAVEX, D. S., AXD R. MORALES-ALAMO, 1966b. Use of fluorescent particles to trace oyster

biodeposits in marine sediments. /. Cons. Perma. Int. Explor. Mcr, 30 : 267-269. HAVEX, D. S., AXD R. MORALES-ALAMO, 1968. Occurrence and transport of faecal pellets in

suspension in a tidal estuary. Sediment. Geol., 2: 141-151. J0RGEXSEN, C. B., 1949. The rate of feeding by Mytilus in different kinds of suspension. /.

Mar. Biol. Ass. U. K., 28 : 333-344. J0RGEXSEX, C. B., 1955. Quantitative aspects of filter feeding in invertebrates. Biol. Rcr., 30:

391-454.

264 DEXTER S. HAVEN AND REINALDO MORALES-ALAMO

J0RGENSEN, C. B., 1960. Efficiency of particle retention and rate of water transport in

undisturbed lamellibranchs. ./. Cons. Perma. Int. Explor. Mer, 26: 94-116. J0RGENSEN, C. B., 1966. Biology of Suspension Feeding. Pergamon Press, New York,

357 pp. J0RGENSEN, C. B., AND E. D. Goi.nHKkG, 1953. Particle filtration in some ascidians and

lamellibranchs. Biol. Bull., 105 : 477-489.

KUBITSCHEK, H. E., 1960. Electronic measurement of particle size. Research, 13: 128-135. LOOSANOFF, V. L., 1958. Some aspects of bebavior of oysters at different temperatures.

Biol. Bull., 144: 57-70.

LOOSANOFF, V. L., AND J. B. ENGLE, 1947. Effects of different concentrations of micro- organisms on feeding of oysters (O. virginica). Bull. Bur. Fish. U. S., 51 : 31-57. MACGINITIE, G. E., 1941. On the method of feeding of four pelecypods. Biol. Bull., 80: 18-25. MULLIN, M. M., 1965a. Size fractionation of particulate organic carbon in the surface waters

of the western Indian Ocean. Limuol. Occanogr., 10: 459-462. MULLIN, M. M., 1965b. Size fractionation of particulate organic carbon in the surface waters

of the western Indian Ocean. Addendum. Lininol. Oceanogr., 10: 610-611. NELSON, B. W., 1963. Removal of kraft effluent from river water by suspended sediment.

Tappi, J. Tech. Ass. Pulp. Pap. Ind., 46 : 277-284. NELSON, T. C., 1960. The feeding mechanism of the oyster. II. On the gills and palps of

Ostrca, edulis, Crassostrea virginica, and C. angulata. J. Morphol., 107: 163-191. NEWELL, R., 1965. The role of detritus in the nutrition of two marine deposit feeders, the

prosobranch Hydrobia ulvae and the bivalve Macoma baltliica. Proc. Zool. Soc.

London, 144 : 25-45. OWEN, G., 1966. Feeding. Pages 1-51 in K. M. Wilbur and C. M. Yonge, Eds., Physiology

of Mollusca. Academic Press, New York. SHELDON, R. W., AND T. R. PARSONS, 1967. A Practical Manual on the Use of the Coulter

Counter in Marine Science. Coulter Electronics, Ontario, 66 pp. SMITH, R. J., 1958. Filtering efficiency of hard clams in mixed suspensions of radioactive

phytoplankton. Proc. Nat. Shellfish. Ass., 48 : 115-124.

SNEDECOR, G. W., 1956. Statistical Methods. Iowa State College Press, Ames, Iowa, 534 pp. TAMMES, P. M. L., AND A. D. G. DRAL, 1955. Observations on the straining of suspensions by

mussels. Arch. Neerl. Zool, 1 1 : 87-112. VERWEY, J., 1952. On the ecology of distribution of cockle and mussel in the Dutch Waddensea,

their role in sedimentation and the source of their food supply. Arch. Neerl. Zool., 10 :

172-239. WILLEMSEN, J., 1952. Quantities of water pumped by mussels (Mytilus edulis) and cockles

(Cardium cdule). Arch. Neerl. Zool., 10: 153-160.

Reference : Biol Bull,, 139: 265-276. (October, 1970)

CHANGES IN MICROTUBULES OF CILIA AND FLAGELLA

FOLLOWING NEGATIVE STAINING WITH

PHOSPHOTUNGSTIC ACID 1

CATHERINE HENLEY

Department of Zoology and Laboratories for Reproductive Biology, University of North

Carolina, Chapel Hill, North Carolina 27514, and the Marine

Biological Laboratory, Woods Hole, Mass. 02543

Phosphotungstic acid (PTA) is a reagent commonly used for the negative staining of cilia, flagella and certain other structures (see, for example, Burton, 1970). In addition to its more or less passive role in outlining and filling such structures, however, there are also macerating and digestive effects on microtubules, and very little direct attention has been paid in the literature to these. The protein- precipitating action of PTA at pH 5 is well known, but to my knowledge its lysing action at pH 6.8 has never adequately been documented. The present report is an attempt to do so, and to give some data on the selective nature of such effects among various types of microtubules.

MATERIALS AND METHODS

Both freshwater and marine turbellarian flatworms were used, the former collected locally from University Lake and from Stone Mountain, Georgia, and the latter furnished by the Supply Department of the Marine Biological Labora- tory at Woods Hole, Massachusetts. They included the following : Mesostoma georgianum, MicrodalyeUia sp. and Macrostomum sp., all freshwater rhabdocoels; the alloecoel Monoophorum sp. (marine) ; and a marine polyclad, Stylochus zebra. Prostoma rubrnni, a freshwater rhynchocoel. was also studied.

Maceration and negative staining were done with a \% aqueous solution of phosphotungstic acid brought to a pH of 6.8 by the addition of 1 ./V KOH or NaOH. A trace of bovine serum albumin was added just before use. The larger animals were cut up with sharp needles and the pieces immediately put into PTA ; smaller forms, such as Macrostomum and MicrodalyeUia, were dropped intact into the reagent. At appropriate intervals, samples were removed to Formvar-carbon- coated 200-mesh copper grids and treatment for all was continued at room tem- perature for periods varying from 2 to 10 min, after which the PTA was rapidly withdrawn with filter paper and the preparation allowed to dry. Micrographs were made with the Zeiss 9 A and 9S electron microscopes.

Microtubules both of cilia (Prostoma, MicrodalyeUia, Monoophorum, Meso- stoma and Stylochus) and of flagella (spermatozoa of Mesostoma) were studied, as well as the ciliary rootlets of Macrostoiniim.

1 Aided by a grant from tbe National Institutes of Health, GM 1531 1.

265

266

CAT 1 1 K R 1 N K HE N LEY

FIGURES 1-4.

MACERATION OF MICROTUBUI.KS 15 V PTA 267

OBSERVATIONS

Disappearance of one or both of the t\v<» central .singlets in PTA-treated cilia and flagella, in the absence of noticeable effects on the doublets, is one of the very few predictable findings. This is well illustrated in Figure 1, showing 3 cilia, in varying stages of maceration but in close physical juxtaposition to one another on the same grid square. In cilium A ( right i all 9 doublets and the 2 central singlets are present; the boundary of the binding matrix (arrow) is considerably farther distal along the axoneme than it is in cilium B. and the microtubules are longer. Furthermore, one of the 2 central singlets in B is entirely absent and the other is present for only a short distance. There is some evidence in higher-magnification micrographs that at least one of the doublets in B has likewise begun to disintegrate. In cilium C (lower left corner I the binding matrix has almost entirely disappeared and only the 9 doublets remain.

This variability in macerating action is also apparent among different grid squares of the same preparation, as shown by the cilia in Figures 2-5, all of which were on one grid. An entire cilium is shown in the montage of adjacent micro- graphs in Figure 2 ; here, very little maceration has occurred at the distal end of the cilium (upper i. while at the basal region the binding matrix is absent and the 9 doublets and 2 singlets are well separated (Fig. 3), with very few, if any, signs of degeneration. The star-shaped basal plate in Figure 4 shows evidence of a considerable degree of degeneration ; the 9 doublets are all present but most are broken off from the plate. The connections of the doublets to the basal plate appear to be effected by tapering processes.

The microtubules and basal plate of the cilium in Figure 5 are in a rather advanced stage of deterioration. Only portions of the 2 singlets (s) are still present and their protofibrils are conspicuous. There is considerable variation along the lengths of the 9 doublets. Both subtubules are mostly intact in the two doublets marked with white arrows; all the others have at least portions of one member absent. There appears to be a general tendency for the degrada- tion process to begin proximally and proceed to completion distally, but sometimes this progression is interrupted, as at the breaks indicated by b. Behnke and Forer (1967) and Stephens (1970) have demonstrated that the B-member of the doublet is more thermolabile than the A-. Since the surviving members of

FIGURE 1. Three cilia in varying stages of maceration, from the same grid square. In cilium A, all 9 doublets, the 2 central singlets, the matrix and membrane (the position of which is designated by the arrow) still are present. In cilium B, all 9 doublets are present but only 1 of the 2 singlets, and that for only a short distance. Nearly all the binding material has disappeared from cilium C, as well as both central singlets, leaving just the 9 doublets; Prostoma rubrttin; 4-min treatment.

FIGURE 2. A montage of adjacent micrographs of a cilium; the basal plate end (bp) is well macerated and the 9 doublets and 2 central singlets are well denned here, but the membrane and matrix, and possibly some coagulated mucus, are still present along the distal two-thirds of the axoneme; Microdalyellia; 10-min treatment.

FIGURE 3. Enlargement of the area indicated on Figure 2. The two singlets lie on either side of the letter s. Scale designation should be 0.4 micron instead of 0.5.

FIGURE 4. End-on view of a ciliary basal plate (b) ; only the 9 doublets are present, and of these, only 4 have even a trace of remaining attachment to the plate; Microdalyellia; 10-min treatment.

268

CATHERINE HENLEY

FIGURE S.

MACERATION OF MICROTUBULES BY PTA 269

•each doublet in Figure 5 have complete walls, and since the A-member is known to be the complete one (Stephens, 1970), it seems safe to surmise that we have another example of the greater sensitivity of the B-tubule to chemical and physical agents (in this case, PTA). Besides bringing about a complete disappearance of the B-subtubule, the macerating action of PTA may result in localized loss or separation of some of the component protofibrils for short distances (black arrow), so that a longitudinal split is present.

Figures 6-9 are micrographs of successive levels along a cilium ca. 14 microns long; Figures 6 and 7 are immediately adjacent to one another, as are Figures 8 and 9. Here the macerating action of PTA after a 10-min treatment differs in another way from the effects discussed above. The two central singlets (arrows in Figure 6) have not yet disappeared, but have a very frayed appearance, with conspicuous protofibrils. This is also the case for the doublets along their entire lengths, although it is less striking than in the singlets. There is little evidence in the doublets of the white line which, in optimally "stained" PTA-treated mate- rial, marks the common wall between the A- and B-subtubules of a doublet. Quite frequent breaks also occur along the doublets, as well as bends (Fig. 7).

An even more drastic macerating action of PTA is seen in the ciliary micro- tubules shown in Figure 10. Degradation here is so advanced that one cannot state with certainty which are doublet and which singlet microtubules ; only 8 are present of the expected total of 11 and there was no evidence nearby on the grid square of the presence of the others. The existence of a helical arrangement of subunits is suggested at the point marked by the arrow, and elsewhere as well.

A difference in the effects of PTA on flagellar axonemes and on the cortical singlets of the spermatozoon of Mesostoma (Henley, Costello, Thomas and New- ton, 1969) is shown in Figure 11. Here, after a 3-min treatment, the flagellar axoneme (fa) is still almost unaffected by PTA, while the cortical singlets (cs) are thrown into a striking helical configuration. It is not apparent in this low power micrograph, but in addition to the gross PTA-stimulated spiralling, the cortical singlets have a marked helical pattern in their walls, when viewed at higher mag- nifications. This is very similar to the configuration demonstrated in cortical singlets of the spermatozoon of Stylechus by Thomas (1970). We have thus far found no evidence of comparable helical configurations in cilia on the same grids as the coiled cortical singlets of spermatozoa, suggesting that here is another differ- ential effect of PTA on different types of microtubules. The spiralling of the entire complement of cortical singlets shown here can readily be related to an abrupt coiling of certain spermatozoa (including that of Mesostoma) observed by phase contrast microscopy during treatment of living spermatozoa with PTA. This coiling may persist for a period of 20 min or more of continued treatment, after which it disappears and the spermatozoa again assume a more straightened form. There is thus the possibility that at a certain stage of its action, PTA stimu- lates the microtubule subunits to undergo some of the changes normally associated

"FIGURE 5. Considerable maceration of the microtubules has occurred in this cilium. The 9 doublets and fragments of the singlets (s) are present, but only the 2 doublets designated with white arrows are intact or nearly so. One member of the doublets (probably the B-subtubule) has completely disappeared at the regions marked b, and a longitudinal separation between the two is apparent at the black arrow, bp, basal plate; Microdalyellia; 10-min treatment.

270

CATHERINE HENLEY

MACERATION OF MICROTUBULES BY FT A 271

with the production of motility. Continued action of the PTA macerates the microtubules more, and their "contractility" is lost. There is as yet no way of knowing whether PTA induces spiralling of the singlets, or whether the coiling results from release of an inherent tendency towards spiralling which is facilitated by PTA's dissolution of the spermatozoon's plasma membrane.

The pH of the PTA solution appears to be of rather critical importance, for even very short treatments at pH 8.3 (Figs. 12 and 13) result in changes in the 2 central singlets of cilia which are quite unlike those observed after any other treatment thus far tested. The singlets become semi-fused together along much of their lengths, and their component protofibrils have a beaded appearance, similar to that described by Behnke and Forer (1967) for central singlets after negative staining at pH 7. Some breaks are also apparent in the doublets, but both members reappear beyond the breaks. The common walls between the subtubules can be seen to persist in the absence of members of the doublet at the point marked by an arrow.

One or more striated ciliary rootlets per cilium are present in sections of all the forms studied here, but are rarely encountered in PTA-treated material. This suggests that they are very sensitive to the digesting action of the reagent at the concentration and pH used, and that they therefore disappear rapidly. Figure 14 shows two rootlets which did survive the treatment; there is a conspicuous major periodicity of ca. 790 A and evidence of a longitudinal fibrous substructure as well. The montage of adjacent micrographs in Figure 15 shows a rootlet in which the process of degeneration is quite far advanced at one end, while the other end (at the top of the picture) still retains the clear 790 A repeating pattern. The longi- tudinal fibrous substructure is clear in the lower region, but the periodicity here is obscured. Dorey (1965 ) observed a 650-700 A repeating pattern in negatively stained ciliary rootlets from a number of acoels; he used 0.05% PTA at pH 6.2 (as opposed to 1% at pH 6.8 used in the present work) and treated for periods of 5-10 min. The differences in technique may account for the disparity in periodicity, or there may be species differences. Further work to elucidate this point is in progress.

DISCUSSION

The impressive variety of degenerative changes in microtubules and associated structures, and the general unpredictability of effects exerted by PTA lead one to the belief that perhaps a great deal of caution should be exercised in the inter- pretation of electron micrographs of negatively stained material. It is not clear, for example, exactly which of the changes described by such workers as Behnke and Forer (1967) and Burton (1968) were due to experimental manipulations (using such agents as colchicine, temperature, pepsin, etc.} and which to the action of PTA itself.

"FIGURES 6-9. Micrographs at successive levels along one cilium, showing conspicuous protofibrils and many breaks. The complete series of 7 micrographs comprising this group reveals that every doublet has at least one break along its length, and several have more than one. The arrows in Figure 6 designate the disintegrating central singlets. Note the rather sharp bends in the microtubules of Figures 7 and 9. Figures 6 and 7 are immediately adjacent to one another, as are Figures 8 and 9; Monoophorum; 10-min treatment.

272

CAT I IKK L\IC J1KXI.KY

FIGURES 10-13.

MACERATION OF MICROTUBULES BY PTA 273

Behnke and Forer (1967) found that the central singlets of the 9 + 2 con- figuration were more likely to be absent in PTA-treated material, confirming the results reported here and by many other workers (see their Table 3). Grim- stone and King ( 1966) showed that the central singlets in flagella of a number of protozoa from Cryptocercus and Zootermopsis only rarely survived even very short treatments (15 sec) with 1% PTA, pH 7.0. If the central singlets were present, however, these were observed to be uncollapsed, in contrast to the doublets which readily collapsed or frayed.

Another type of central element in axonemes has been studied by Burton (1968) and Thomas (1970), among others, in spermatozoa of the lungfluke Haemal oloechus and the polyclad turbellarian Stylochus, respectively. They both found the complex central core of the "9 +1" pattern to be more resistant than either the doublets or the cortical singlets to a variety of experimental treatments, followed by negative staining with PTA. These data of Burton and of Thomas for spermatozoa are in interesting contrast to those presented here for cilia, which implicate the central elements as the least resistant to the action of PTA. However, the relationship of central singlets in the 9 + 2 pattern to the comparatively massive core in the "9 +1" pattern is obscure and probably com- plex. It may be, also, that there is an inherent difference in the properties of microtubules in cilia and in spermatozoa of the same form. Paired "9+1" axonemes, as well as cortical singlets, are found in spermatozoa of 3 (Mesostoma, Micr odaly ellia and Stylochus} of the 6 forms included in the present report. The spermatozoon of Macrostomum has cortical singlets only, with no axonemes (Henley, unpublished data). In the other two (Monoophorum and Prostoma} the axonemal pattern of spermatozoa has not yet been studied, but on the basis of information we have gathered from 8 other free-living flatworms, 1 other rhynchocoel and 3 annelids, it is possible to state that there is no set relationship between the pattern of microtubules in spermatozoa and in cilia of the same form. In addition, the fact that the cortical singlets of the spermatozoon of Mesostoma (and of MicrodalycUia and Stylochus as well) react differently to PTA than do the microtubules of the flagella suggests yet another source of variability. Differences in susceptibility of microtubules to PTA (and to other treatments as well) such as those discussed above may very well involve subtle differences in the biochemical composition of these structures in the various groups, despite their

FIGURE 10. Ciliary microtubules so completely macerated that one cannot determine which (if any) are singlets and which douhlets. Arrow designates helical particulate sub- structure. Three of the normal total complement of microtubules are present; Mesostoma; 6-min treatment.

FIGURE 11. Cortical singlet microtubules (cs) of a spermatozoon (at right) are thrown into a conspicuous helical arrangement by PTA treatment, while the microtubules of the flagellar axoneme (fa) are not. The thick solid black structure in the center is probably an unmacerated spermatozoon; Mesostoma; 4-min treatment.

FIGURE 12. This ciliary axoneme was treated with PTA at pH 8.3, rather than the usual 6.8. All 9 doublets are present, as well as the 2 central singlets (cs), which appear to be almost fused together and which have a striking beaded appearance. The breaks in the doublets are somewhat sharper than those usually seen after treatment at the lower pH ; Stylochus; 2-min treatment.

FIGURE 13. Enlargement of the area indicated by the rectangle in Figure 12. (Micrograph by Mary Beth Thomas).

274

C \TIIKKI.\I- HKXI.I- Y

FIGURE 14. Two ciliary rootlets, negatively stained with PTA, showing a major repeating pattern of ca. 790 A. Note the longitudinal fibrous substructure; Macrostomum; 4-min treatment.

MACERATION OF MICROTL'BULES BY PTA -275

apparent morphological resemblances. Evidence for this is offered by the recent work of Behnke (1970), who studied the comparative sensitivity of microtubules in disk-shaped blood cells of the frog, chick, rat and man, to cold, Colcemid, N- ethylmaleimide and alkaloids of J'inca. He found very marked species differences in such susceptibility. In our material, there are undoubtedly great variations in such features as the toughness of the pellicle, the presence or absence of mucus, etc., which could affect the action of PTA and the required duration of treatment for adequate negative staining. Also, it is our experience that marine forms are considerably less sensitive to the macerating action of PTA than freshwater ones, perhaps because of the presence in seawater of divalent ions, which appear to inhibit the action of PTA. In this connection. Roth and Shigenaka (1970) have recently shown that microtubules of the heliozoan axopod respond to treatment with cupric ion very much as they do to colchicine. They were also found to be very susceptible to degradation in the presence of nickel ion. The concentrations of both these cations in sea water are low (copper, 0.001-0.09 mg/1, nickel. 0.0001 mg/1, according to the Handbook of Chemistry and Physics, 1970, page F-145), but they could well be involved in the observed variability in the action of PTA.

It may be pertinent to point out here that while the precise formula of phospho- tungstic acid is not entirely clear (due to the amount of included water of crystalliza- tion ), its molecular weight is high. Values of 3312.5 and 3132.4 are given (Hand- book of Chcinistrv and Physics, 1970), for molecules containing 24 and 14 units of water, respectively. Therefore, a 1% solution is only about 0.003 M. Since the spermatozoa of many marine forms are osmotically adjusted to survive for a considerable period of time in sea water of molarity equal to that of a 0.55 M NaCl solution, the \% PTA is decidedly hypotonic. Some of the gross changes in living sperm subjected to PTA treatment may be due to this factor.

One of the most unpredictable aspects of negative staining is the correct duration of treatment with PTA, to achieve optimal "staining" of the microtubules without overmaceration. This seems to be correlated, to some extent at least, with the species differences already alluded to; for each new form we study, the correct duration of treatment is determined empirically and has been found to range from a few seconds to 30 min or more.

Burton (1966) briefly discusses the variability in action of PTA, in material on consecutive grids treated in the "same" way, and points out (page 404) that studying "negatively stained material forces one to make value judgments." These value judgments involve such factors as taking into account the varying degrees of susceptibility of the different types of microtubules and other components of cilia and flagella, to the degenerative effects of PTA. That this is indeed the case is abundantly borne out by the results reported here, in negatively stained microtubules on the same grid and. in fact, on the same grid square.

FIGURE 15. Montage of three adjacent micrographs, showing progressive maceration of a negatively stained ciliary rootlet. The basal body end (top) is intact, but there is progressive disintegration, ending in a completely disorganized mass at the distalmost end; Macrosotomum; 10-min treatment.

276 CATHERINE HENLEY

I am indebted to Miss Mary Beth Thomas for permission to use her unpub- lished micrographs ('Figs. 12 and 13), and to Miss Thomas and Dr. Donald P. Costello for valuable assistance in preparation of the illustrations.

SUMMARY

1. Variability in the macerating action of 1% aqueous phosphotungstic acid, pH 6.8, is exemplified in microtubules of flatworm and rhynchocoel cilia, from one grid square to another of the same preparation and within a single grid square. The central singlets appear to be the most susceptible and are often completely absent, even in cases where the binding matrix is still present around the doublets. Maceration usually, but not always, begins at the distal tip of a cilium and proceeds towards the basal plate ; it is evident along the lengths of doublets as partial or complete loss of one subtubule, as breaks and bends, and as fraying into the compo- nent protofibrils, with disappearance of the white line marking the wall between the subtubules.

2. Cortical singlet microtubules of a spermatozoon were thrown into a helical configuration by the action of PTA, while the flagellar microtubules of the same spermatozoon were unaffected.

3. After treatment with 1% PTA at pH 8.3, the central singlets of cilia (but not the doublets) were semi-fused along most of their lengths, and had a beaded appearance; there were breaks in the doublets but otherwise they appeared to be unaffected.

4. Ciliary rootlets were rarely seen, but when present had a clear 790 A major repeating pattern and a longitudinal fibrous substructure.

LITERATURE CITED

BEHNKE, O., 1970. A comparative study of microtubules of disk-shaped blood cells. /.

Ultrastruct. Res., 31 : 61-75. BEHNKE, O., AND A. FORER, 1967. Evidence for four classes of microtubules in individual

cells. /. Cell Sci., 2 : 169-192. BURTON, P. R., 1966. A comparative electron microscopic study of cytoplasmic microtubules

and axial unit tubules in a spermatozoon and a protozoan. /. MorphoL, 120 : 397-423. BURTON, P. R., 1968. Effects of various treatments on microtubules and axial units of lung- fluke spermatozoa. Z. Zclljorsch. Mikrosk. Anat., 87 : 226-248. BURTON, P. R., 1970. Fine structure of mitochondria of Spirostomum ambiguum as seen in

sectioned and negatively stained preparations. /. ProtozooL, 17 : 295-299. DOREY, A. E., 1965. The organisation and replacement of the epidermis in acoelous

turbellarians. Quart. J. Microscop. Sci., 106 : 147-172. GRIMSTONE, A. V., AND A. KLUG, 1966. Observations on the substructure of flagellar fibres.

/. Cell Sci, 1 : 351-362. Handbook of Chemistry and Physics, 1970. [Fifth edition] Cleveland, The Chemical Rubber

Co., 2033 pp. HENLEY, C, D. P. COSTELLO, M. B. THOMAS AND W. D. NEWTON, 1969. The 9 + 1 pattern of

microtubules in spermatozoa of Mesostoma (Platyhelminthes, Turbellaria). Proc.

Nat. Acad. Sci. U. S. A., 64 : 849-856. ROTH, L. E., AND Y. SHIGENAKA, 1970. Microtubules in the heliozoan axopodium. II.

Rapid degradation by cupric and nickelous ions. /. Ultrastruct. Res., 31 : 356-374. STEPHENS, R. E,. 1970. Thermal fractionation of outer fiber doublet microtubules into A- and

B-subfiber components : A- and B-tubulin. /. Mol. Biol., 47 : 353-363. THOMAS, M. B., 1970. Transitions between helical and protofibrillar configurations in

doublet and singlet microtubules in spermatozoa of Stylochns zebra (Turbellaria,

Polycladida) . Biol. Bull, 138 : 219-234.

Reference: Biol. Bull., 139: 277-295. (October, 1970)

THE ORIGIN. DISTRIBUTION AND FATE OF THE MOLTING FLUID PROTEINS OF THE CECROPIA SILKWORM

Y. LEXSKY,1 C. COHEN AND H. A. SCHNEIDERMAN 2 Developmental Biology Center, Case Western Reserve University, Cleveland, Ohio 44106

Molting in insects is accompanied by the production of a molting fluid which fills the exuvial space between the new and old cuticles and digests the inner layers of the old cuticle. Most of the products of digestion are resorbed. This process has been studied in detail in the Cecropia silkworm by Passonneau and Williams (1953). During the pupal-adult transformation of the Cecropia silkworm, molt- ing fluid appears in the exuvial space as soon as the epidermis retracts from the old pupal cuticle. This fluid persists for the first 19 days of the 21 days of pharate adult life. For the first two-thirds of this period the molting fluid is gel-like and has no obvious effect on the overlying pupal cuticle. However, on about the fourteenth day of pharate adult development, the molting gel of the pharate adult liquifies and begins to hydrolyze the proteins and chitin of the pupal endocuticle. By the 20th day the endocuticle has disappeared, leaving a thin crisp exocuticle and epicuticle. On the 19th day, molting fluid begins to be absorbed and, by the 20th day, most of it is gone from the exuvial space. On the 21st day, the insect emerges.

It is generally believed that the epidermal cells secrete the molting fluid. Whether they synthesize all of it, or secrete into it some components that were synthesized elsewhere, is not known. The literature contains only scanty references to the protein components of molting fluid. Passonneau and Williams (1953) showed that the early molting gel of Cecropia contains about 5 per cent protein. They also demonstrated proteolytic and chitinolytic activity in the molting fluid. Chen and Levenbook (1966) examined the molting fluid and blood of the blowfly, Phormia regina, by disc electrophoresis and reported that both fluids contained several proteins with similar Rf values. Their electrophoretic techniques did not permit them to determine whether any proteins of the molting fluid were related to those of the blood, or were products of cuticular digestion.

The mechanism of absorption of molting fluid prior to ecdysis is also unknown. It is generally believed that the ability to absorb molting fluid is a generalized property of the integument and that absorption takes place through the general surface of the new cuticle (Passonneau and Williams, 1953; Wigglesworth, 1933, 1948, 1965). However, it is not known whether specific regions of the integument are specialized as sites of absorption. Also, although it has been shown that small

1 Visiting scientist. Present address : Faculty of Agriculture, Hebrew University, Rehovoth, Israel.

2 Present address : Developmental Biology Laboratory, University of California, Irvine, California 92664.

Ill

278 Y. LENSKY. C. COHHN AND ]|. A. SCU . \KIDKUMAN

molecules are absorbed (cf. review bv Noble-Nesbitt, 1()67), notbing is known of tbe possible absorption of macromolecules.

Tbis report examines tbe proteins of molting fluid, epidermis and blood during tbe pupal-adult transformation of tbe Cecropia silkworm, by immunological and electropboretic techniques. It considers tbe following questions :

1. Wbich proteins (if any) are found in botb molting fluid and blood, and where are these proteins made ?

2. To what degree do native proteins (from blood and molting fluid) and introduced foreign proteins (from rabbit serum) move from molting fluid to blood and vice versa during adult development. How much macromolecular "traffic" is there between the blood and tbe fluid in tbe exuvial space?

3. Are macromolecules absorbed when molting fluid is absorbed just prior to ecdysis ?

4. Is molting fluid absorbed by the general surface of the newly formed integu- ment or does absorption occur more readily at certain sites?

MATERIALS AND METHODS Experimental annuals

Larvae, pupae and pharate adults of the Cecropia silkworm, Hyalophora cecropia, were used in most experiments. Males and females were used inter- changeably since there appeared to be no sex-limited differences in the phenomena we examined. In a few experimnts larvae of Samia cyntliia ricini and Galleria mellonella were used. Methods of rearing, storing, handling and staging these insects have been described elsewhere (Krishnakumaran, Berry, Oberlander and Schneiderman, 1967; Schneiderman and Williams, 1954; Telfer and Rutberg, 1960) . All animals were maintained at 25° C.

Reagents

Chemicals used for disc electrophoresis and immunoelectrophoresis were routine commercial preparations. Serum proteins came from laboratory rabbits. Purified rabbit gamma globulins (7S and 19S) were obtained by fractionation on DEAE cellulose columns and were lyophilized. All of tbe insect tissues were washed in 0.85% NaCl.

Collection of fluid in tissue samples from developing adults

Molting fluid, blood and tissue samples were taken from Cecropia silkworms of specific ages. Phenylthiourea (PTU) was added to all wounds and to all samples of tissue and fluids. In most experiments the fluids or tissue homogenates were centrifuged at 15,000 rpm in a Beckman "microfuge" for four minutes and stored at — 20° C until used.

Molting fluid: A small piece of cuticle was excised from the tip of the abdomen, and approximately 200 //I of molting fluid was collected in a capillary. During this process it was necessary to avoid touching the delicate epidermis with the capillary. Otherwise, tissue damage occurred and caused blood and molting fluid

MOLTING FLUID PROTEINS OF CECROPIA 279

to mix. Molting fluid collected in the way described was not contaminated by blood (see Results). Occasionally, molting fluid was collected from antennae or wings for special purposes, but this fluid was often contaminated by blood.

Blood: Blood was collected from larvae, diapausing pupae and adults by means of fine glass needles inserted into the heart. To avoid contaminating the blood of pharate adults with molting fluid, special procedures were employed. In early pharate adults, an incision was made through the pupal cuticle along the dorsal midline of the abdomen. At this time, the epidermis is still closely pressed against the cuticle above the heart, and blood was collected from the heart without contami- nation by molting fluid. In late pharate adults, the pupal cuticle was removed, the animal washed in saline and blotted to avoid contamination by molting fluid. A sharp capillary was then inserted through the cuticle of the pharate adult into the heart to collect blood.

Epidermis: The developing wings and antennae are a convenient source of epidermis. However, the blood present in these appendages made it impossible for us to obtain epidermis free of blood, even though the tissues were cut into small fragments and washed repeatedly. In contrast, the abdominal epidermis could be isolated and freed of blood by careful dissection, fragmentation and repeated washing in ice cold saline. The fragments were blotted to remove excess saline, homogenized, centrifuged and the clear supernatant used for further analysis. Abdominal epidermis prepared in this way had no detectable amounts of major blood proteins.

Disc electrophoresis

For disc electrophoresis the procedure of Davis (1964) was followed, using £m-glycine buffer (pH 8.6) and applying 3 MA per gel for 30 minutes at 25° C. The procedure was modified slightly to meet our special needs (cf. Patel and Schneiderman, 1969 for details). Gels of several pore sizes were tested. A 4.3% gel provided the best resolution for blood, molting fluid and epidermal proteins and was employed in most experiments. Gels were stained after electrophoresis with Buffalo Black ( = Naphthol Blue Black, Allied Chemicals) and were destained and preserved in 10% acetic acid.

Immunological methods

Antisera to blood proteins of diapausing pupae and adults of male and female Cecropia silkworms were prepared in rabbits. The insect blood was emulsified in Freund's adjuvant and injected subcutaneously into rabbits in four to six sites along the vertebral column. Two weeks later boosters were injected into the same sites. The antisera were deepfrozen at —20° C until used. These antisera could detect up to twelve different Cecropia blood antigens in immunoelectrophoresis experiments. Sheep antisera against total rabbit serum proteins and against rabbit gamma globulins were prepared by conventional methods.

The immunoelectrophoretic procedures of Grabar and Williams (1955) and the modified technique of Scheidegger (1955) were used to detect blood antigens in various fluids and tissues. Standard microscope slides were covered with 2 ml of

280

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MULTIXG FI.l'IU PROTFIXS OF CKCKOI'IA 281

1', agar ( Difco. Special "Noble") in barbital acetate buffer at pH 8.6, p -- 0.025. Troughs and holes were cut in agar as follows : 3 holes ( 1 mm diameter, 5 mm apart) and two troughs (6 mm apart i (Figs. 3 and 5). Electrophoresis was performed at 200 volts, applying 7 to 8 MA per slide for 60 minutes at 3° C. After electro- phoresis, the separated proteins were reacted with rabbit sera against the blood of Cecropia pupae and adults. Precipitation arcs appeared after 12 to 48 hours at 25°, slides were washed in saline for three days, dried and stained with Buffalo Black.

Ouchterlony's double-diffusion analysis (Ouchterlony, 1958) was carried out in 5 cm Petri dishes covered with 4 ml of \% agar in barbital acetate buffer. One central and 6 circumferential wells were cut out with a 13 gauge syringe needle and spaced 5 mm apart from each other (Fig. 6). Diffusion took place for 12 to 4S hours at 25° C.

Injection of dyes and proteins info the e.vnz'ial space and body cavity

Rabbit serum and purified rabbit gamma globulins were used as foreign proteins to help us detect macromolecular "traffic" between the exuvial space and the body cavity. Their presence in fluid compartments and in tissues was tested for by double diffusion analysis using sheep antiserum against rabbit gamma globulin. The distribution and penetration of macromolecules from one fluid compartment into another were also studied, using both rabbit serum proteins and molting fluid proteins stained with Buffalo Black. A series of dialysis studies with rabbit serum proteins stained with Buffalo Black demonstrated that, at concentrations of 0.1%, the dye remained firmly bound to the protein for at least 24 hours. This staining technique provided us with an easy way of tagging various proteins and studying their movements between fluid compartments. It was always confirmed by immunological methods.

Injection of foreign proteins, dye-"tagged" proteins or dye alone was made either into the exuvial space or into the body cavity in the dorsal side of the second or third abdominal segment.

Injections into the exuvial space posed no problem. However, special tech- niques were required to prevent blood and molting fluid from mixing during injections into the body cavity. In pharate adults younger than 14 days, the injection was made in the second or third abdominal segment through the pupal cuticle and directly into the heart. The epidermis in this region is closely pressed against the cuticle and no detectable mixing of blood and molting fluid occurred. In older pharate adults, the following procedure was carried out to prevent the mixing of blood and molting fluid during injection or immediately thereafter. A 6 mm square of pupal cuticle was excised between the second and third abdominal segment above the heart and the exposed edges of the pupal cuticle were sealed to the adult cuticle with melted paraffin. The small depression formed by this procedure was washed with saline and a volume of blood com- parable to the amount of solution to be injected was removed from the heart. The experimental solution was introduced into the heart, and the exposed area sealed with paraffin.

Y. LENSKY, C. COHEN AND H. A. SCHNE IDERMAN

RESULTS Cotnpartvnentalization of the e.rui'ia! space

The first experiments were designed to determine whether compartmentaliza- tion exists within the exnvial space. About half of the molting fluid of the left antennal case of a 16-day old pharate adult was removed through an opening in the pupal cuticle and replaced by a mixture of molting fluid tagged with Buffalo Black. One day later, examination revealed that the stained protein had not diffused from the antennal case. In a second experiment, a similar injection was made into the left antennal case of a 20-day old pharate adult. Five hours after the injection, the Buffalo Black-tagged protein had spread from the left antennal case to the fore and hind wings, head and thorax.

In another experiment on a 20-day old pharate adult 1 ml of \% aqueous solution of Buffalo Black was injected into the exuvial space at the tip of the abdomen. Ten hours after the injection, the Buffalo Black-tagged protein had diffused into the wings, thorax and head.

These experiments were repeated several times with the same results. Clearly, the exuvial space is compartmentalized and the fluids in the compartments do not mix with one another until several days before emergence. For this reason, all samples of molting fluid used in this study were withdrawn from the same place, namely the tip of the abdomen. Apparently, molting fluid is formed separately in several regions between which parts of the pupal cuticle remain attached to the epidermis. These areas of persistent attachment effectively divide the exuvial space into compartments. Just prior to ecdysis, the pupal cuticle detaches in these areas, and the exuvial space becomes continuous.

The protein composition of blood and molting fluid as repealed by disc electrophoresis

Samples of blood and of molting fluid were taken from pharate adults at various stages of the pupal-adult transformation and from diapausing pupae. The samples were subjected to disc electrophoresis and some typical results are recorded in Figure 1. From an analysis of the gels illustrated in this Figure and more than 50 similar gels, the following facts emerged :

(1) The molting fluid was not contaminated by blood, because it was colorless. The slightest contamination could be detected by the occurrence of yellow pigments. Also, as Figure 1 shows, several of the densest blood protein bands (Rf 34 and 45) were absent from molting fluid.

(2 ) None of the major protein bands in the blood was a major component of the molting fluid and vice versa. Band Rf 66, a major blood protein component, was detectable in low concentrations in the molting fluid. All 5 major blood protein bands had Rf's between 0 and 66 while major molting fluid protein bands had R/s greater than 66.

(3) Molting fluid contained at least 10 proteins which were not detected in the blood.

(4) Profound changes occurred in the protein pattern of the blood and molting fluid during development. Only changes that occurred in the molting fluid will

MOLTING FLUID PROTEINS OF CECROPIA

283

be discussed here. The molting fluid un day 7 is of special interest because it is still inactive enzymatically and does not contain products of cuticular digestion. It contained 8 to 10 distinct protein bands, but none had Rf's less than 45. However, between day 13 and 17, a number of new bands appeared with Rf's ranging from 10 to 38, some of which persisted throughout development. Whether these proteins have anything to do with the enzymatic activity of the molting fluid, or include products of cuticular digestion, will be considered in the discussion.

(5) A number of bands in blood and molting fluid have similar Rfs. Which of these bands represented identical proteins is considered in the next section.

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Proteins common to molting fluid and blood

The following electrophoretic and immunological methods were used to determine which protein bands with similar Rfs in molting fluid and blood are identical.

Separation by disc electrophoresis and precipitation by antisera against blood proteins: Thirty jA of molting fluid from 17-day old pharate adult males were applied to each acrylamide gel and the proteins separated as previously described. Following electrophoresis, the gels were cut longitudinally into two halves ; one was stained with Buffalo Black to locate the protein bands and the other half was placed on a microscope slide coated with 2% agar. Rabbit antisera against male pupal blood and against male adult blood were added into two parallel troughs on each side of the split gel. Diffusion was allowed to occur until no new lines of

284

Y. LENSKV, C. COHEN AND H. A. SCI LNEIDERMAN

precipitation appeared. The results are recorded in Figure 2. Four lines of precipitation were formed with antiserum against adult blood serum and two with antiserum against pupal blood. The Rr values of the molting fluid protein hands which omld correspond to each of these lines are recorded in Figure 2. It is impossible to decide precisely which of these hands are the blood antigens. From this result it is clear that the molting fluid of 17-day old pharate adults contains 4 antigens found in adult blood. Two of these antigens are absent from pupal blood or present in much lower concentrations. Further support for these conclusions comes irom immunoelectrophoretic data.

FIGURE 3. Immunoelectrophoretic analysis of blood and molting fluid of 16-day old developing adult male Cecropia moth. The wells contained (1) 2 /il of blood, (2) 10 fj.1 of molting fluid, and (3) 2 /ul of blood. The longitudinal troughs had (a) rabbit antiserum against blood of male pupae, and (b) rabbit antiserum against blood of male adults. The numbered arrows indicate the lines of precipitation formed by blood antigens present in the Cecropia blood and molting fluid.

Imiiumoelectrophorcsis: Molting fluid and blood from pharate adults 13, 16, 19 and 20 days old were analyzed by precipitation with rabbit antisera against the blood of male pupae and against the blood of female pupae. Figure 3 shows a typical result obtained with a 16-day old pharate adult. The many lines formed by the blood need not concern us. The key result is the demonstration that molting fluid formed two lines of precipitation with antiserum against pupal blood and five lines with antiserum against adult blood. These lines corresponded to those formed by blood antigens as follows :

fl) Lines 2 and 3 formed with antiserum against male pupal blood. A diffuse line. No. 1, also formed but is not visible in the photograph.

(2) Lines 1 to 5 formed with antiserum against male adult blood. There was

MOLTING FLUID PROTEIN'S OF CECROPIA

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no basic change in the number or identity of the blood antigens present in molting fluid during the final seven days of pharate adult development. These results confirm and extend the combined disc electrophoresis-double diffusion experi- ments described above.

Ouchterlony double diffusion analysis confirmed the identity between several blood antigens and molting fluid antigens (cf. also results presented in section 4(c) below and Figure 6) .

The origin of proteins common to blood and molting fluid

It is generally believed that the epidermis is "the source of at least the principle constituents of the molting fluid" (Passonneau and Williams, 1953). To deter-

286

Y. LENSKY, C. COHEN AND R A. SCHNEIDERMAN

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FIGURE 5. Immunoelectrophoretic analysis of blood, molting fluid and epidermis of 11 -day old, pharate, adult male Cecropia moth. The wells contained (1) 2 iA of blood, (2) 10 fj.1 of epidermal homogenate, and (3) 10 /j,\ of molting fluid. In (A) rabbit antiserum against blood of male pupae was in both troughs, and in (B) rabbit antiserum against blood of male adults in both troughs. The numbered arrows indicate the lines of precipitation formed by Cecropia blood antigens. Lines 3 and 5 did not show up in the photograph.

mine whether the epidermis was the source of the five antigens found in both the molting fluid and blood, epidermal proteins were examined by disc electrophoresis, immunoelectrophoresis and double diffusion.

Figure 4 records the results of disc electrophoresis of epidermis, blood and molting fluid from 11-day old pharate adults and demonstrates that the epidermis and molting fluid have a number of proteins with the same Rf values. They both lack the same major blood proteins. In short, in terms of disc electro- phoresis patterns, epidermis resembles molting fluid more than blood.

The identity between certain epidermal, molting fluid and blood proteins was established by the following immunoelectrophoretic analyses. Blood, molting fluid and epidermis from 11 -day old pharate adults were subjected to immunoelectro- phoresis using antisera against male pupal blood and male adult blood. It is evident from the results in Figure 5 that blood antigens 1, 2 and 3 were present both in the epidermis and the molting fluid when reacted with antiserum against pupal blood. Lines of precipitation 1, 2, 4 and 5 were formed by the epidermis

MOLTING FLUID PROTEINS OF CECROPIA

2S7

and lines 1 to 5 by the molting fluid following their reaction with antiserum against adult blood. The yellow pigments characteristic of both pupal and adult blood, which are bound to certain blood proteins, were absent from the abdominal epidermis as well as from the molting fluid of pharate adults.

These observations show that epidermis contains at least 4 of the 5 blood antigens found in the molting fluid. Proof of the identity among the antigens in the epidermis, blood and molting fluid was established by Ouchterlony double-diffusion analysis as follows: Molting fluid, blood and abdominal epidermis of 11-day old pharate male adults were examined by the Ouchterlony double-diffusion technique

A B

FIGURE 6. Double diffusion analysis of blood, epidermis and molting fluid of 11-day old, pharate, adult moth absorbed with two different antisera. Surrounding wells were prepared with (1, 4) blood, (2, 5) abdominal epidermis, and (3, 6) molting fluid. In (A) the center well contains rabbit antiserum against male pupal blood, and in (B) the center well contains rabbit antiserum against male adult blood. Blood antigens present in these three protein mixtures which fuse and form lines of identity are indicated by arrows. Another line of identity in (A) between wells 2 and 3 and between wells 5 and 6 did not show up in the photograph.

for the presence of lines of identity between these three protein mixtures. Rabbit antisera against male pupal blood and male adult blood were used. The results in Figure 6 reveal that two identical lines of precipitation were formed by the molting fluid, epidermis and blood when tested with antiserum against pupal blood. Figure 6 also shows that at least two identical lines were formed by blood, molting fluid and epidermis when reacted with antiserum against adult blood. In both cases heavy lines of precipitation were formed only by the blood, and were absent from the epidermis and molting fluid.

Although the resolution of the double-diffusion technique is much less than that of immunoelectrophoresis, these observations are consistent with the results of immunoelectrophoresis and establish that several of the blood antigens present in molting fluid are identical with those present in the epidermis.

The route of entry of blood antigens into moltiny fluid

The occurrence of blood antigens in molting fluid raises the question of how they get there. Are they secreted by the epidermal cells into the exuvial space and also

Y. I.KXSKY, C. COIIKX AND H. A. SCHNKI DI-.RMAN

into the blood? Or, do they originate in some other tissue, to be transported by epidermal cells into the exuvial space? Indeed, this raises the more general question of what sort of commerce there is between the molting fluid and blood compartments of the insect.

To answer this question, rabbit whole serum and rabbit whole serum tagged with Buffalo Black was injected into the abdominal exuvial space or into the body cavity of pharate adult females of different ages. Double diffusion analysis with sheep antiserum against rabbit gamma globulin was used to detect the presence of the gamma globulin fraction of the injected serum in various fluid compartments and tissues. The distribution of Buffalo Black-tagged proteins was visually determined. Unless otherwise indicated each experiment was repeated on three animals.

Injection of rabbit serum proteins into the c.vnvial space: In the first experi- ment 200 /j.\ of molting fluid was removed from 7-day old pharate adults. Follow- ing this, 150 /xl of rabbit serum was injected into the exuvial space and the open- ing sealed with paraffin. Four or ten days after the injection, the animals were sacrificed and blood and molting fluid were analyzed by double diffusion. The results revealed that rabbit gamma globulins were detected in the molting fluid but not in the blood. Apparently, in 11- to 17-day old pharate adults, the rabbit serum proteins did not move from the exuvial space into the blood. Also, the rabbit serum proteins retained their antigenic activity for at least ten days in the molting fluid, notwithstanding the presence of proteolytic enzymes in this fluid (Passonneau and Williams, 1953).

In another experiment, between 20 and 100 /jl of whole rabbit serum protein were injected into the exuvial space of 19-day old pharate adults at the peak of molting fluid absorption. Twenty-four hours later, blood and molting fluid were tested by double diffusion analysis for the presence of rabbit serum proteins using sheep antiserum against whole rabbit proteins and sheep antiserum against rabbit gamma globulin. No rabbit serum proteins were detected in the blood, but they were found in the molting fluid.

In a third experiment 250 /A of whole rabbit serum was injected into the exuvial space of a 20-day old pharate adult. The insect resorbed its molting fluid four days later, whereupon blood, fat body and ovaries were examined by double diffusion analysis. No lines of precipitation were observed. Apparently, immunologically active rabbit serum proteins are not resorbed into the blood even when active molting fluid absorption takes place.

To summarize, foreign proteins injected into the exuvial space of pharate adults of different ages could not be detected in the blood of these animals by immunological methods. This finding indicates that macromolecules similar to rabbit gamma globulins do not penetrate the new adult cuticle and epidermis to enter the blood, prior to or during active molting fluid absorption.

Injection of rabbit serum proteins into the body cavity: The next question asked was whether macromolecules could cross the epidermis from the body cavity into the exuvial space. To answer this question, rabbit serum proteins were injected into the heart of pharate adults of different ages.

In the first experiment, 7-day old pharate adults were injected with 100 p\ of rabbit serum and killed 4, 10 or 15 days thereafter. Double diffusion analysis

MOLTING FLUID PROTEINS OF CECROPIA 289

of molting fluid, fat body and blood revealed rabbit gamma globulins only in the blood. Thus, although rabbit globulins were detected in the blood for 15 days after injection, these proteins failed to pass from the blood into the exuvial space at any stage.

A similar experiment was performed on 14-day old pharnte adults in which 350 fj.\ of rabbit serum protein tagged with Buffalo Black were injected. The pharate adults were killed five days after the injection, and blood, molting fluid and various tissue were examined visually for the presence of blue dye, and immunologically by double diffusion, for the presence of rabbit serum proteins. The results revealed that the oocytes were the only tissue, besides blood, which contained gamma globulins. The oldest oocytes did not stain blue, but younger ones did, and the oocytes as a whole contained gamma globulins. However, when gamma globulins were injected on days 19 or 20, none of the oocytes took up gamma globulins. Although the Malpighian tubules were stained blue, no antigenically active gamma globulins were detected. Apparently the blue color is due to dye molecules which dissociated from rabbit serum proteins that might have been degraded.

Taken together, these experiments indicate that foreign proteins do not penetrate from the exuvial space to the blood or vice versa. The presence of certain native proteins in the blood and their absence from the molting fluid, coupled with the presence of certain native proteins in the molting fluid and their absence from the blood, also demonstrates an effective separation between the two fluid compartments. The results suggest that the blood antigens found in the molting fluid are not transported from the blood into the molting fluid, but are secreted by the epidermis directly into both molting fluid and blood.

Sites of molting fluid absorption

To identify the sites of molting fluid absorption, the fate of Buffalo Black-tagged molting fluid was investigated. In pharate adults, molting fluid is absorbed on days 19 to 21. A series of developing adults 17 to 18 days old had the pupal cuticle on the tip of the abdomen removed, 0.1 ml of molting fluid was collected and mixed with 0.9 ml of 2% Buffalo Black in saline. The resulting mixture was reinjected into the exuvial space. Four days later, after emergence, the adult integument was examined. Only one part of the abdominal integument showed conspicuous blue staining. This was a narrow band located on each interseg- mental membrane close to the sclerotized ridge at the posterior margin of each segment (Fig. 7). This blue-staining band was made up of many small pits or depressions about 15 microns in diameter. These pits were the only part of the cuticular surface that stained blue (Fig. 8) and from which diffusion of blue dye down and laterally into the integument took place.

In another experiment pupal cuticle was removed from 19- and 20-day old pharate adults and a drop of Buffalo Black-tagged molting fluid was applied to various parts of the adult integument. The penetration of the blue dye through the pits could be followed under the microscope. Within 15 minutes the dye was absorbed through the pits and its centrifugal diffusion into the integument could be followed. It was absorbed only through the pits.

290 Y. LENSKY, C. COHEN AND H. A. SCHNEIDERMAN

These pits absorbed Buffalo Black-tagged molting fluid only prior to ecdysis. If Buffalo Black-tagged molting fluid was applied to the integument after ecdysis, the dye-protein mixture accumulated on the pits, but could be washed away with saline. It failed to enter the integument, presumably, because of a waterproof coating on the pits.

Similar pits are also found in corresponding parts of larvae and pupae. They are permeable to Buffalo Black-tagged molting fluid only before ecdysis. In fifth-stage larvae of Sitniia cynthia ricini, for example, five to six hours after ecdysis, the pits failed to absorb Buffalo Black-tagged molting fluid. In pre-

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FIGURE 7. Portion of dorsum of abdomen of newly emerged female Cecropia moth showing tonofibrillar insertions in the intersegmental membrane (arrows). Buffalo Black dye was applied to the surface of the cuticle to emphasize the bands of pits for photographic purposes (X 16).

pupae of Galleria mellonella Buffalo Black-tagged molting fluid was also absorbed via pits, but after pupation there was no absorption.

These pits appear to be principal sites of molting fluid absorption in the ab- domen. In the thorax, molting fluid is resorbed on the patagia, parapatagia, membranes at the bases of the legs and also in the membranes lying between epimeron 2 and 3 and subalare 2 and 3. In the head, some Buffalo Black-tagged molting fluid appeared to be sucked into the mouth and some was also absorbed by the integument beneath the mouth parts.

These pits mark the points at which tonofibrils occur. In the abdomen, the longitudinal muscles attaching adjacent segments and the lateral muscles within a segment make tonofibrillar insertions in these pits. In pharate adult Cecropia, the small portion of each tonofibril which attaches the pupal cuticle to the adult cuticle (and due to which the pharate adult is capable of moving the pupal cuticle) breaks down after about day 18. After this time, the absorption of molting fluid takes place through these pits in the adult integument.

.MOLTING FLUID PROTEINS OF CECROPIA

291

• y

FIGURE 8. Tonofibrillar insertions showing absorption of stained molting fluid proteins in a portion of the intersegmental membrane of a 21-day old, pharate, male Cecropia moth. Approximately 250 /ul of molting fluid tagged with Buffalo Black had been injected on day 18 into the exuvial space. Three days later, the animal was killed and a portion of the abdominal dorsum was free of adhering tissues and fixed. An overall view of a portion of a segment (X 320) is shown above (A), and a high power view of above (X 800) below (B). Labels indicate: (a) intersegmental membrane, (b) tonofibrillar insertions or "pits," (c) the sclerotized ridge, and (d) a segment showing scale sockets.

These experiments with Buffalo Black-tagged protein suggest that some macro- molecules are absorbed from the molting fluid. Proof that this occurs was provided by the following immunological experiment. An injection of 275 /*! of rabbit serum was made into the ecdysial space of an 18-day old developing adult. When it had emerged five days later the integument (adult cuticle plus epidermis) was freed of surrounding tissues and scales and washed thoroughly in saline. It was then homogenized and tested for the presence of rabbit serum proteins by double diffusion analysis. The results revealed rabbit serum proteins in the integument, while longitudinal muscles which are attached to the tonofibrils showed no reaction. Identical results were obtained with rabbit serum proteins tagged with Buffalo Black. These observations prove that the integument absorbs macromolecules along with molting fluid and these remain there for several days in an immunologically active state. The results indicate that in the normal process of absorption of molting fluid some macromolecules may be absorbed intact.

Y. LENSKY, C. COHEN AND II. A. SCHNEIDERMAN

Principal sites of absorption in tin- abdomen are the tonofibrillar insertions. Thr absorbed proteins do not appear to enter the blood but some of them enter the integument.

DISCUSSION

The results indicate that molting fluid is a separate fluid compartment and has at least 10 protein components which were not detected in the blood. These 10 components are not products of cnticular digestion since they are present in inactive molting fluid prior to cuticular digestion. The blood and molting fluid do share five antigens. One of these, a major blood protein band, is a minor component of molting fluid. The other four common protein components are minor compart- ments of both blood and molting fluid.

This different protein composition in the two fluid compartments depends on the epidermal cells which act as a barrier to most macromolecules in the molting fluid and the blood. The existence of this barrier is evident from the fact that numerous native blood proteins were never detected in the molting fluid, and many native molting fluid proteins were never detected in the blood. Moreover, Buffalo Black-tagged molting fluid proteins also failed to appear in the blood. Similar results were obtained with various rabbit serum proteins of different molecular weights which failed to cross from one fluid compartment to another at any stage of adult development.

The fact that the epidermis acts as a barrier to many macromolecules suggests that most molting fluid proteins are synthesized by the epidermis itself and are not synthesized elsewhere and transported to the epidermis. This being the case, the presence of five common antigens in both molting fluid and blood suggests that, either (a) the epidermis secretes certain proteins in only one direction, but can secrete other proteins (the five blood antigens) in both directions; or (b) the epidermis secretes the five antigens into the molting fluid and other tissues secrete the same proteins into the blood; (c) the common antigens are secreted by some other tissues into the blood and are transported by the epidermis into the molting fluid. We favor the first suggestion.

These observations lead us to question earlier conclusions that molting fluid contains blood (e.g., Jeuniaux, 1958). It seems likely that, in those cases where molting fluid contains blood, some damage to the delicate new cuticle and epidermis occurred which caused mixing. Indeed, the only bona fide cases where blood is to be expected as a normal component of the molting fluid are in insects like Sialis in which the larval gills with enclosed blood are trapped in the exuvial space at the time of pupation (Selman, 1960) .

It is also worth noting, that, unless great precautions are taken, it is difficult to obtain blood from pharate adult insects without contaminating it with molting fluid. There have been several reports of proteins that appear in the blood only at the time of molting (e.g., McCormick and Scott, 1966). One wonders whether some of these molt-connected blood proteins might be molting fluid pro- teins that contaminated the blood in the course of collecting blood samples.

As far as the exchange of small molecules between blood and molting fluid is concerned, we have no new data. \Yigglesworth (1933) and Jeuniaux (195Si

MOLTINr, FLUID PROTEINS OF CECKOI'IA 293

indicate that dyes of molecular weights up to at least 500 can move- from the molting fluid to the blood and Passonneau and Williams (1953) have shown that glycine -C14 readily moves from the exuvial space into tissues. Locke (1966) has pre- sented evidence that small molecules are absorbed through small 30 A pores in the cuticulin of Calpodes.

The change in protein composition of molting fluid during adult development requires comment. Recent electron micrographic studies of molting in Apterygota indicate that molting fluid initially consists of granules with inactive enzymes which later become active (Noble-Nesbitt, 1963a, 1963b). The mechanism of this activation is not understood. In the present experiments, there was a great increase in the number of protein bands in molting fluid during the last eight days of adult development, at which time the molting fluid becomes activated. Whether these new bands represent newly-secreted proteins or enzymes, a re- arrangement (activation?) of previously secreted proteins, or some cuticular breakdown products is not yet known.

The mechanism for absorption of the molting fluid during the final two days of adult development is unknown. Contrary to the generally held view, the absorp- tion does not take place through the integument as a whole, but through particular regions. In the head and thorax, these regions are principally various flexible membranes at the bases of the appendages. In the abdomen, the principal sites of resorption are pits which represent the points through which tonofibrils make attachment to the old cuticle. In all cases in which it has been studied carefully, the tonofibrils remain attached to the old cuticle until just before ecdysis (Lai-Fook, 1966; Noble-Nesbitt, 1963a, 1963b; Wachter, 1930; Wolfe, 1954).' This appears to be true also in developing adult Cecropia. Until the tonofibrils break, the old and new cuticles are closely bound together at the points of attachment. On about day 19, the attachments between the tonofibrils and the pupal cuticle ruptures, and the adult can now move within the old pupal cuticle. The cause of this rupturing is not clear, however. As a result of the rupturing, the part of the tonofibrils connecting the two cuticles detaches from the new adult cuticle and leaves the point of attachment on the new cuticle exposed. It is through this exposed surface of adult cuticle — the pit — that molting fluid is absorbed.

The particular ultrastructural features of the tonofibrils that make them suitable sites for resorption requires further study (cf. e.g., Auber, 1963; Bouligand, 1962). Perhaps it is simply that by day 19 most of the cuticle is covered with substances that render it impermeable to molting fluid, whereas the newly exposed cuticular surfaces of the pits are permeable to macromolecules. In any event, although the epidermis on the general surface of the abdomen may be able to absorb molting fluid, in practice, the principal sites of absorption are the pits.

It has been demonstrated previously that, after molting fluid absorption, some molting fluid proteins remain on the surface of the adult cuticle after ecdysis (Wolfe, 1954; Jeuniaux, 1957). However, so far as we are aware no one has demonstrated that macromolecules in the molting fluid get absorbed. The present experiments with rabbit serum proteins demonstrate absorption of foreign proteins from the molting fluid and also demonstrate that these proteins end up in rather than on the integument. Whether they are in endocuticle, epidermis or both was not determined.

2()4 Y. LENSKY, C. COHEN AND II. A. SCHNEIDERMAN

We wish to thank Dr. Narayan Patel and Mrs. Ilona Polony for performing a number of disc electrophoretic separations and Mr. Gordon Marsh for his helpful comments on the typescript. This research was supported in part by research grants, HE 06320 and HE 10082 from the United States Public Health Service.

SUMMARY

1. Molting in insects is always accompanied by the production of a molting fluid which tills the exuvial space between the new and the old cuticle and digests the inner layers of the old cuticle. In Hyalophora cecropia, molting fluid is secreted at the outset of adult development and persists until two days before eclosion, whereupon it is absorbed.

2. The present report examines the protein composition of the molting fluid of Cecropia, the origin of the molting fluid proteins, the relation of these proteins to blood proteins and the exchange of macromolecules between the molting fluid and the blood. It also examines the sites of absorption of molting fluid.

3. Disc electrophoresis on acrylamide gels reveals that the molting fluid of Cecropia contains about fifteen protein bands which can be resolved at pH 8.6. Some of these protein bands are detected in the molting fluid at all stages, whereas others appear only at specific times. About ten of the bands are peculiar to molting fluid and are not detected in the blood. About five bands are detectable in both blood and molting fluid, but none of these common bands appears to be a major component of the molting fluid, and only one is a major blood protein. In contrast, the epidermis contains most of the major protein bands found in molting fluid but lacks all but one of the major protein bands present in the blood.

4. Immunological analysis reveals that blood and molting fluid share five antigens. At least four of these common antigens also occur in the epidermis which appears to secrete these antigens into both the molting fluid and the blood.

5. Native and foreign proteins do not penetrate from the exuvial space into the blood or vice versa. Apparently the epidermis and cuticle act as a barrier to the exchange of most macromolecules between the blood and molting fluid. The exuvial space is clearly a separate fluid compartment.

6. In addition the exuvial space itself is compartmentalized and the fluids in the compartments do not admix several days before eclosion.

7. Absorption of molting fluid during the final two days of adult development occurs most readily through particular regions of the integument. In the abdomen the principal sites of absorption are pits which represent the points through which tonofibrils make attachment to the old cuticle. Two days before ecdysis, the attachments between the tonofibrils and the pupal cuticle rupture, exposing the points of attachment on the new cuticle. It is through these exposed surfaces that much of the molting fluid is absorbed. Molting fluid is also absorbed in the head and thorax through various flexible membranes at the bases of the appendages.

LITERATURE CITED

AUBER, J., 1963. Ultrastructure de la jonction myo-epidermique des muscles de vol de

Dipteres. /. Microscopic, 2 : 325-336. BOULIGAND, Y., 1962. Les ultrastructures du muscle strie et de ses attaches au squelette chez

les cyclops (Crustaces copepodes). /. Microscopic, 1 : 377-394.

MOLTING FLUID PROTEINS OF CECROPIA 295

CHEN, P. S., AND L. LEVENBOOK, 1966. Studies on the haemolymph proteins of the blowfly,

Phonnia rcqina. I. Changes in ontogenic patterns. /. Insect Physiol., 12: 1595-

1609. DAVIS, B. G., 1964. Disc clectruplioresis. II. Method and application to human serum

proteins. Ann. Xc-^- York .lead. Sci.. 121: 404-427. GRABAR, P., AXD C. A. WILLIAMS, 1955. Methode immuno-electrophoretique d'analyse de

melanges de substances antigeniques. Biochint. Biophys. Acta, 17: 67-74. JEUXIAUX, C., 1957. Les enzymes d'origine epidermique au cours du phenomene de la niiu-

chez les insects. . Iclcx Societe Linneene, Bordeaux, 97 : 77-84. JEUXIAUX, C., 1958. Resorption du liquide exuvial chez le ver a soie ( Boml^v.r inori L.).

Arch. Int. Physiol. Bi^chem., 66: 121-122. KRISHNAKUMARAX, A. K., S. J. BERRY, H. OBERLAXDER AXD H. A. SCHXEIDERMAX, 1967.

Nucleic acid synthesis during insect development — II. Control of DNA synthesis in

the Cecropia silkworm and other saturniid moths. /. Insect Physiol., 13 : 1-57. LAI-FOOK, J., 1966. The ultrastructure of muscle insertions in insects. Amcr. Zool., 6: 530. LOCKE, M., 1966. The structure and formation of the cuticulin layer in the epicuticle of an

insect. Calpodcs cthlins (Lepidoptera, Hesperiidae). /. Morphol., 118: 461-494. McCoRMiCK, F. W., AXD A. SCOTT, 1966. A protein fraction in locust hemolymph associated

with the moulting cycle. E.rpcrientia, 22 : 228-229. NOBLE-NESBITT, J., 1963a. The fully-formed intermoult cuticle and associated structures of

Podnra aquatiea ( Collembola). Quart. J. Microscop. Sci., 104: 253-277. NOBLE-XESBITT, J., 1963h. The cuticle and associated structures of Podnra aquatiea at the

moult. Quart. J. Microscop. Sci., 104 : 369-391. NOBLE-NESBITT, J., 1967. Aspects of the structure, formation and, function of some insect

cuticles. Pages 3-16 in : J. W. Beament and J. E. Treherne, Eds. Insects and

Physiology, Oliver and Boyd, London. OUCHTERLONY, O., 1958. Diffusion-in-gel methods for immunological analysis. Progr.

Allergy, 5 : 1-78. PASSOXNEAU, J. \'., AXD C. M. WILLIAMS, 1953. The moulting fluid of the Cecropia silkworm.

/. E.\-p. Bio!.. 30 : 545-560. PATEL, N. G., AXD H. A. SCHXEIDERMAX, 1969. The effects of perfusion on the synthesis and

release of blood proteins by diapausing pupae of the Cecropia silkworm. /. Insect

Physiol., 15: 643-660. SCHEIDEGGER, J. J., 1955. Une micro-methode de 1'immuno-electrophoreses. Int. Arch. Allergy

Appl. Immunol, 7 : 103-110. SCHNEIDERMAX, H. A., AXD C. M. WILLIAMS, 1954. Physiology of insect diapause. IX. The

cytochrome oxidase system in relation to the diapause and development of the Cecropia

silkworm. Biol. Bull'., 118: 338-351. SELMAN, B. J., 1960. On the tissue isolated in some of the larval appendages of Stalls lutaria L.

at the larval-pupal moult. /. Insect Physiol., 4 : 235-257. TELFER, W. H., AXD D. RUTBERG, 1960. The effects of blood protein depletion on the growth

of the oocytes in the Cecropia moth. Biol. Bull., 118: 352-366. WACHTER, S., 1930. The moulting of the silkworm and a histological study of the moulting

gland. Ann. Entonwl. Soc. Amcr., 23 : 381-389. WIGGLESWORTH, V. B., 1933. The physiology of the cuticle and of ecdysis in Rhodnhis proli.rus

( Triatomidae, Hemiptera) with special reference to the function of the oenocytes and

of the dermal glands. Quart. J. Microscop. Sci, 76 : 269-318. WIGGLESWORTH, V. B., 1948. The insect cuticle. Biol. Rev.. 23 : 408-451.

WIGGLESWORTH, V. B., 1965. The Principles of Insect Physiology. Methuen, London, 741 pp. WOLFE, L. S., 1954. Studies of the development of the imaginal cuticle of Calliphura crythro-

ccphala. Quart. J. Miernscop. Sci., 95 : 67-78.

Reference: Hiul. Hull., 139: 290-303. (October, 1970)

REVERSIBLE RESPONSE TO PUROMYCIN AND SOME

CHARACTERISTICS OF THE UPTAKE AND USE

OF AMI NO ACIDS BY UNFERTILIZED

SEA URCHIN EGGS ^

F. ROY MACKINTOSH 2 AND EUGENE BELL

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 and Marine Biological Laboratory, ]Voods Hole, Massachusetts 02543

Several groups have reported that all unfertilized sea urchin eggs incorporate labeled amino acids into protein, as judged by autoradiography (Bell and Mac- Kintosh, 1967; MacKintosh and Bell, 1967; Epel, 1967; Tyler, Tyler and Piatigorsky, 1968). However, when incorporation is measured biochemically (i.e. by homogenization and acid precipitation of a radioactively labeled sample contain- ing 1000 to 10,000 eggs) there is a strong theoretical possibility that a few "im- mature" eggs or ovarian fragments in the preparation could be contributing to it heavily. Thus experiments relying on measurement of the rate of incorporation in these preparations as a measure of the rate of protein synthesis in mature un- fertilized eggs (MacKintosh and Bell, 1967; Epel, 1967) could be in substantial error, as suggested by Stavy and Gross (1967 ) .

That it is not in error emerges from some basic facts presented in this work about the uptake and incorporation into protein of amino acids by unfertilized eggs. The basal level of incorporation in unfertilized egg preparations is, in fact, the rate of incorporation by mature unfertilized eggs, and is not attributable to contamination with immature cells. Support for this conclusion is based heavily on the differential response of unfertilized eggs as compared with oocytes to puromycin ; the effect of the antibiotic on the former is reversible, while on the latter it is not. It is also based on other data which is presented below.

MATERIALS AND METHODS . Inhiials and gametes

Specimens of Strongylocentrotits f>itrpin-atns were obtained from Pacific Bio- Marine, Venice, California. Specimens of Arbacia pnnctitlata were obtained from Mr. Norris Hill, Beaufort, North Carolina. Animals were maintained at about 12° C in aerated tanks with subsand filters, and were used or discarded within two weeks after arrival.

Gametes were obtained by injection of isotonic (0.53 M] KC1, a method which yielded egg preparations that were superior (in terms of freedom from contaminating ovarian material) to those obtained by excision of gonads.

Eggs were prepared for use by passing them through four layers of cheesecloth, washing twice by settling in Millipore filtered sea water, and suspending them in

1 Supported in part by Grant No. GB-7312 — from the National Science Foundation.

2 Present Address : Mt. Herman School, Mt. Herman, Massachusetts 01354.

296

PUROMYCIN EFFECT ON ECHINOID EGGS

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FIGURE 1. (a) Mature eggs of Arbacia were collected as described in Materials and Methods. Half were treated with puromycin at 235 /*g/ml for four hours, while controls remained in untreated sea water. Both groups were rinsed and placed in fresh sea water, and maintained for an additional hour. 14C amino acids were then added to both groups (1 /xc/ml) and samples were withdrawn from both at hourly intervals for analyses as usual. Data are presented as acid insoluble cpm/mg protein for controls ( •— — • ) and puromycin treated

(O ---- O). Samples were counted at 11% efficiency, (b) An egg suspension containing oocytes (eggs with a germinal vessel) was prepared by mincing the gonads of a female of Arhacia after it had been induced by KC1 injection to shed most of its mature eggs. The preparation was divided in half and treated exactly as above, except that puromycin treatment was for only 1.5 hours, and radioactivity was counted at 90% efficiency. The data of 'Figure la and Ib is summarized in the inset, recalculated as rate of incorporation relative to controls, and plotted together for comparison of mature eggs (O -- O) and oocytes ( A -- A).

Millipore filtered sea water containing penicillin (160 ^g/ml ) and streptomycin (100 jug/ml). Eggs were maintained at 20° C (Strongylocentrotns} or at room temperature (Arbacia), which varied from day to day hetween 21° and 24° C. in Erlenmeyer flasks on a rotary shaker at 60 to 70 rpm.

To determine their maturity and condition, we examined egg preparations microscopically before use, and discarded any with an excessive number of oocytes, fragments of ovaries or fertilized eggs. Oocytes are easily detected by the presence of a large germinal vessicle. Typical levels of contamination with oocytes were of the order of 0.1 to 0.3 per cent. Bacterial contamination was occasionally

298

F. ROY MACKINTOSH AND EUGENE BELL

monitored bv plating samples of the egg suspension on agar made up in 80 ])er cent sea water and 20 per cent Charitv Waymonth medium. Tvpical levels of con- tamination within live or six hours of the start of an experiment were of the order of 10—20 viable hacteria per ml. The maximum lonnd was 12X0 hacteria per ml, after over 24 hours of incubation of a culture. On the most gratuitous possible assumption a single bacterial cell could not yield more than 0.2 DPM (if uniformly labeled with 14C at 50 mc/mMole). Thus in no case could contami- nation represent more than 3% of the radioactivity of a sample, and in most cases the amount would be far smaller than this. This level of contamination is not significant. Tyler, Tyler, and Piatigorsky (1%X) also report negligible bacterial contamination.

TABLIC I Uptake of "C' amino acids by eggs after short exposure and after rinsing

Exposure time	No rinse	1 rinse	4 rinse
.-! niin	—	3,900	—
,3 niin	— •	4,050	— •
1 hr	106,000	94,100	94,700
1 hr	102,000	97,000	95,600

Eggs of Strongylocentrotus were labeled for times indicated with 1 yuc/ml 14C amino acids. "No rinse" eggs were pipetted into 10 volumes of sea water (20° C) in a conical centrifuge tube, sedimented, resuspended and transferred to a clean tube to be dissolved in 8 M urea withuot further rinses. "1 rinse" consisted of suspending "no rinse" eggs in 2 ml cold homogenization medium and resedimenting and dissolving in 8 M urea. "4 rinses" consisted of three sea water washes prior to the wash with homogenization medium. Data are cpm/mg protein in the ho- mogenates. Each figure represents a single determination on a single homogenate. All samples came from the same egg preparation.

Assay of incorporation of radioactivity, even at the iiia.viiiniin observed levels of contamination

14C labeled reconstituted protein hydrolysate (a mixture of L amino acids with an average specific activity of about 140 mc/mMole, obtained from Schwarz Bioresearch, Orangeburg, New York ) was used as a protein precursor. To measure incorporation in eggs which had been exposed to labeled precursor, a 0.5 or 1 .0 ml sample of egg suspension was withdrawn from the incubation vessel and pipetted into a 12 ml conical centrifuge tube containing 10 nil of ice cold sea water. The eggs were sedimented by a brief centrifugation (about 5 seconds at full power in a clinical centrifuge) and the supernatant withdrawn. The eggs were then rinsed once with 2 ml cold homogenization medium (0.25 M sucrose, 0.24 M NH4C1, 0.01 M MgCl., 0.01 717 Tris, pH 7.4; described by Spirin and Nemer (1965)] and dissolved overnight in 8 M urea. The urea treatment was found to render soluble the radioactivity which was hot acid labile, i.e.. such non protein incorporation as amino acyl t-RNA labeling. The urea homogenate was routinely analyzed as follows : a portion was precipitated by the addition of bovine serum albumin as carrier if needed and 30 per cent ( w/vv ) TCA to give a final concentration of 15 per cent TCA and the precipitate collected on Millipore filters

PUROMYCIN EFFECT ON KCHIXOID EGGS

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FIGURE 2. (a) Unfertilized eggs of Strongylocentrotus were exposed to 1 //c/ml "C amino acids for three hours, washed, and placed in sea water ( « ) or sea water containing 0.01 M ethionine (O). Both groups incorporated labeled amino acids from their endogenous pools into acid insoluble material (lower curves). The level of uptake (upper curves) remained essen- tially constant for controls, i.e., the labeled amino acids accumulated during the three hour exposure do not wash off or leak out. Eggs exposed to ethionine lost a substantial portion of their endogenous label during this time. Note that ethionine does not significantly affect the rate of incorporation from the endogenous pool into protein until after four hours of treatment, (b) Eggs were maintained in sea water (•) or sea water containing unlabeled amino acids (methionine HI, ethionine O, or leucine A) at 0.01 M for five hours, washed, and given one hour pulse labels with 14C amino acids at intervals thereafter. The eggs which were exposed to unlabeled amino acids incorporate at a rate 3.0 times higher than controls (overall average). The overall loss of amino acids from pools caused by the amino acid pretreatment, as judged from Figure 2a, is a factor of 2.8. Thus the effect of the amino acid pretreatment seems to be the exchange of the internal pools of many amino acids for the single amino acid present in the medium, lowering the size of pools of all amino acids except that supplied by approximately a factor of three and thereby inflating subsequent incorporation rates by a factor of three. That the value of cpm in the homogenate (uptake) accurately reflects these changes is evidence for its validity as a measure of radioactivity in endogenous amino acid pools.

in the usual way ; these were counted with a low background gasflow counter at 11 per cent efficiency or in a Packard Tri-carb scintillation counter at 90 per cent efficiency ; a second portion of the homogenate was used for estimation of proteins by the method of Lowry (Lowry, Rosebrough, Farr and Randall, 1951) ; a third portion of the homogenate was pipetted directly into a scintillation vial and counted in an appropriate medium [either Bray's fluid (Bray, 1960) or a mixture of four parts ethylene glycol monoethyl ether and six parts of the usual toluene based scintillation fluid). The value obtained in this manner, normalized to amount of protein in the sample, is referred to as "uptake."

300 F. ROY MACKINTOSH AND Kl'f.KNE BKI.L

The value obtained by the Lowrv reaction lor protein content of the samples was used to normalize uptake and incorporation value to equal amounts of protein, and therefore presumably to equal numbers of eggs.

RESULTS

Differential response of mature and immature ct/t/s to puromycin

When mature unfertilized eggs are exposed to puromycin for four hours or less (at physiological temperatures), and then placed in puromycin-free water containing 14C amino acids, within three hours they attain a rate of incorporation similar to that observed in untreated controls (Fig. 1). Slightly longer treatment with puromycin is followed by a stimulation of protein synthesis as reported previously (MacKintosh and Bell, 1967). Irreversibility begins to appear only after 6 hours

TAHLK 1 1

Incorporation of 14C amino acids by eggs into acid soluble and insoluble material in the presence of unlabeled amino acid in the medium

	Uptake	Acid insoluble	Ratio
control ethioniiiL-	210,000 24,000	2133 219	0.0100 0.0091

Eggs of Strongylocentrotus were exposed for one hour to 1 juc/ml 14C amino acids in the presence or absence of 0.01 M ethionine. Samples were assayed as usual for radioactivity in the homogenate and for acid insoluble incorporation. Values are per mg protein.

of treatment of unfertilized eggs of Arbacia, and after more than 8 hours of treat- ment of unfertilized eggs of Strongylocentrotus.

On the other hand, when fertilized eggs are treated with puromycin, the effects become irreversible within one hour of treatment (Ellis, 1966). Similarly, when preparations of unfertilized eggs containing 30 per cent immature eggs (oocytes) are exposed to puromycin for 1.5 hours, protein synthesis in preparations washed free of the antibiotic is greatly depressed. After 3 hours in puromycin-free medium, incorporation in a population of mixed mature and immature eggs is reduced to 35 per cent of controls (Fig. lb). The capacity of mature unfertilized eggs to recover from puromycin treatment is seen in Figure la. The rate of incorporation of precursor into acid insoluble material has exceeded that of controls by 3 hours after washing with puromycin-free sea water.

The effect of concentration and competition on uptake and use of radioactive amino acids

The value obtained by counting radioactivity in the total homogenate is believed to represent uptake of amino acids from the medium as opposed to some type of nonspecific absorption on the following basis :

(1) Eggs which are exposed to 14C amino acids briefly have very low uptake values (Table I).

PUROMYCIN EFFECT ON ECHINOID EGGS

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FIGURE 3. Arbacia eggs were pulse labeled for one hour with the following concentrations of 14C amino acids: 0.1, 0.2, 1.0, 2.0, 5.0 /tc/ml. Eggs were processed as usual and the acid insoluble radioactivity plotted against the total radioactivity in the homogenates. The linearity of the resulting curve shows that incorporation into acid insoluble material is a constant fraction of uptake independent of the actual level of uptake.

(2) Rinsing the eggs does not substantially change the value obtained (Table I).

(3) When eggs are labeled with 14C amino acids, washed and returned to sea water, their uptake (counts in whole homogenate) does not change substantially for several hours (Fig. 2a). (This is also true of eggs exposed to puromycin after labeling.)

(4) If eggs are labeled and washed and placed in sea water containing a single nonradioactive amino acid at 0.01 M, their content of radioactivity (uptake) decreases threefold over a period of five hours. Conversely, if the eggs are first exposed to the unlabeled amino acid for five hours and then exposed to labeled amino acids, their rate of uptake is unchanged but their rate of incorporation into acid insoluble material is trebled. See legend. Figure 2b, for our interpretation of this effect. The effect of preincubation with amino acids was also observed by Tyler, Piatigorsky and Ozaki (1966).

(5) If eggs are exposed simultaneously to a single unlabeled amino acid and to the labeled mixture, their uptake is strikingly reduced, and incorporation into acid insoluble material is initially reduced in exact proportion to the reduction in uptake ( Table II).

302

F. ROY MACKINTOSH AND EUGF.XE BHI.I.

If eggs are exposed to varying concentrations of 14C ainino acids uptake is not directly proportional to concentration, but incorporation into acid insoluble material is directly proportional to uptake (Fig. 3 ).

When unfertilized eggs of the two species were given one hour pulse labels with 14C aniino acids over an extended period of time, they displayed an essentially constant rate of protein synthesis ( Fig. 4 ) . The incorporation was not only constant on a per milligram protein basis, but was also a constant fraction of amino acid uptake. This constant rate of incorporation was also observed in eggs maintained continuously in 14C amino acids.

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DISCUSSION

Two types of sea urchin cells which are known to be highly active in protein synthesis [those of embryos (Ellis, 1966) and oocytes (Fig. 1 )] have been found to be irreversibly inhibited when returned to puromycin-free sea water after previous exposure to puromycin. In contrast, preparations of mature unfertilized eggs recover to normal rates of protein synthesis after puromycin treatments shorter than 4 hours and do not exhibit signs of irreversible inhibition for at least 6 hours. These observations imply that there is not a detectable fraction of the incorporation of 14C amino acids in preparations of mature unfertilized eggs which can be attributed to either of these classes of highly active cells (oocytes and ovarian fragments, embryos). This finding supports the validity of previously published comparisons of the rate of incorporation in fertilized and unfertilized eggs (Epel, 1967; MacKintosh and Bell, 1967; Bell and MacKintosh, 1967).

A question raised by the foregoing results concerns the basis for the differential response to puromycin which remains to be elucidated.

The experiments on uptake of amino acids and its relation to incorporation of label into proteins establish that surface absorption or other nonspecific uptake is unlikely to be a major factor in this value. The uptake value provides what

PUROMYCIN EFFECT ON ECHINOID EGGS 303

appears to be a self-consistent measure of radioactivity in endogenous amino acid pools under a wide variety of circumstances. Thus the use of this value in verifying the absence of permeability changes (MacKintosh and Bell, 1967; Mac- Kintosh and Bell, 1969) seems justified.

SUMMARY

Protein synthesis in embryos and oocytes is irreversibly inhibited when the cells are returned to puromycin-free sea water after previous exposure to puromycin. In unfertilized eggs, on the other hand, it is not irreversibly inhibited. This effect and others are used to show that mature unfertilized eggs are actively engaged in protein synthesis, and that direct assessment of the relative rate of protein synthesis in mature eggs is not hindered by contamination with a small population of very active cells.

LITERATURE CITED

BELL, E.. AND F. R. MACKINTOSH, 1967. Control of synthetic activity during development Pages 163-174 in A. V. S. deReuck and J. Knight, Eds., Ciba Foundation Sym- posium on Cell Differentiation. J. and A. Churchill, Ltd., London.

BRAY, G. A., 1960. A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem., 1 : 279-285.

ELLIS, C. H., 1966. The genetic control of sea urchin development : A chromatographic study of protein synthesis in the Arbacia punctulata embryo. /. E.\-p. Zool., 163 : 1-21.

EPEL, D., 1967. Protein synthesis in sea urchin eggs : A "late" response to fertilization. Proc. Nat. Acad. Sci., Washington, 57 : 899-906.

LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, 1951. Protein measure- ment with the folin phenol reagent. /. Biol. Chew, 193 : 265-275.

MACKINTOSH, F. R., AND E. BELL, 1967. Stimulation of protein synthesis in sea urchin eggs by prior metabolic inhibition. Biochem. Biophys. Cowinun., 27 : 425-430.

MACKINTOSH, F. R., AND E. BELL, 1969. Regulation of protein synthesis in sea urchin eggs. /. Mol. Biol, 41 : 365-380.

SPIRIN, A. S., AND M. NEMER, 1965. Messenger RNA in early sea urchin embryos: Cytoplasmic particles. Science, 150: 214-217.

STAVY, L., AND P. R. GROSS, 1967. The protein-synthetic lesion in unfertilized eggs. Proc. Nat. Acad. Sci., Washington, 57 : 735-742.

TYLER, A., J. PIATIGORSKY AND H. OZAKI, 1966. Influence of individual amino acids on uptake and incorporation of valine, glutamic acid, and arginine by unfertilized and fertilized sea urchin eggs. Biol. Bull., 131 : 204-217.

TYLER, A., B. S. TYLER AND J. PIATIGORSKY, 1968. Protein synthesis by unfertilized eggs of sea urchins. Biol. Bull., 134 : 209-219.

Reference : Biol. Bull., 139: 304-312. (October, 1970)

CHANGES IN THE EPIDERMAL HISTOLOGY DURING THE

SLOUGHING CYCLE IN THE RAT SNAKE PTYAS KORROS

SCHLEGEL, WITH CORRELATED OBSERVATIONS

ON THE THYROID GLAND

P. F. A. MADERSON, K. W. CHIU1 AND J. G. PHILLIPS

Department of Biology, Brooklyn College, New York 11210 and Department of Pathology,

Massachusetts General Hospital, Boston 02114; Department of Biology, The Catholic

University of America, Washington, D. C. 20017 ; and Department of Zoology,

The University, Hull, Yorkshire, England, U. K.

In an initial description of the histological changes in the snake epidermis associated with periodic skin-shedding, Maderson (1965a) established six arbitrary stages to facilitate reference. Stage 1 — the resting stage — seen in the post-shedding period, was described as a condition in which a single outer epidermal generation was present above a stratum germinativum. Little if any cell proliferation appeared to occur during this stage, but it was suggested that pronounced changes occurred in the innermost living cells later in the cycle. These layers of cells, which appeared to be present throughout the resting stage, were termed the presumptive lacunar tissue and clear layer. Stages 2 through 5 described the sequential formation and maturation of the component parts of the new inner epidermal generation— Oberhautchen, ft- and a-layers. In Stage 6, the pre- shedding condition, the inner generation possessed two or three immature cell layers, which were interpreted as representing presumptive lacunar and clear layer cells. Subsequent studies of other lepidosaurian reptiles (Lillywhite and Maderson, 1968; Maderson, 1966, 1967, 1968; Maderson and Licht, 1967) revealed further details of the structure of the epidermal generation and suggested that the original interpretation (Maderson, 1965a) of the pattern of histogenic activity involved in the laying-down and matura- tion of the lacunar tissue and clear layer was either incorrect, or applicable only to the species Elaphe taeniura.

Sembrat and Drzewicki (1936), Goslar (1958), Lynn (1960, 1970), Mader- son (1965b) and Maderson, Chiu and Phillips (1970) have reviewed the data sug- gesting a relationship between the thyroid gland and periodic skin-shedding in squamate reptiles. Although it is generally held that thyroid hormone stimulates shedding frequency in lizards, while inhibiting it in snakes, there has been no attempt to correlate snake epidermal changes with thyroid gland histology com- parable to Eggert's (1935) study of Lacerta.

The present study of the epidermis and thyroid gland histology of Ptyas korros provides additional data on the histogenesis of the snake epidermal generation, correlates thyroid activity with the described cellular changes, and permits some explanation of the previous experimental results suggesting differences between snakes and lizards in this context.

1 Present address : Department of Biology, Chung Chi College, Shatin, Hong Kong, B. C. C.

304

EPIDERMAL THYROID RELATIONS IN SNAKES 305

MATERIALS AND METHODS

From September 19th through October 12th, 1966, 44 mature male P. korros, Schlegel were purchased in Hong Kong. Some animals were chosen for purchase on the basis of eye texture to ensure representation of all stages of the sloughing cycle (Maderson, 1965a). Following decapitation, the snakes were weighed, and the weight of the abdominal fat determined. A piece of belly skin was fixed in Bouin's fluid and prepared for histological examination as described by Maderson, (1965a).

The thyroid gland was freed from connective tissue, weighed, and fixed in Bouin's fluid for 48 hrs. It was dehydrated in a series of ethyl alcohol, cleared in chloroform, and embedded in 56° C paraffin. Serial sections through the center of the gland were cut at 7 /*, mounted and stained with hematoxylin and eosin.

The 44 thyroid glands were divided into 7 groups according to the stage of epidermal development seen in the skin biopsy from the same animal (see Table I). The follicular cell-height for each group was estimated as follows. The 14—31 (mean 20.2) follicles which fell along the longest axis of the gland were examined. The tallest and shortest cell in each follicle was measured with an ocular micrometer. The average of these two values was taken as the cell height for the particular follicle, and the mean of the cell-height for 14—31 (mean 20. 2) follicles was taken as the cell height for the gland. The average of the cell heights of all glands examined in any one of the 7 groups was taken as indicating the cell height asso- ciated with each of the 7 observable histological conditions of the epidermis.

RESULTS

Epidermal histology

Immediately after shedding, the epidermis of P. korros showed an incomplete outer epidermal generation lying above the stratum germinativum (Fig. 1). The generation consisted of a mature Oberhaittchcn (OBO), /2-layer (/?O) and mesos layer (MO). There was a poorly developed a-layer (AO) and 3-5 layers of presumptive «-cells (PAO). This stage of development is referred to in Table I as Stage 0.

A few days later, the epidermis appeared somewhat different (Fig. 2), and showed a condition referred to in Table I as Stage 1. The a-layer (AO) was then well-developed, and the number of subjacent immature cells reduced by com- parison with Stage 0. Stages 2-4 wherein a complete outer epidermal generation (Maderson, 1967) and part of a differentiating inner generation were seen, resembled exactly similar stages described for Elastic (Maderson, 1965a), and these are referred to in Table I. During Stage 5 (Fig. 3), as the new /3-cells of the inner generation (/?!) finally matured. 8-12 layers of very flattened relatively chromophobic cells were seen just above the columnar, chromophobic cells of the stratum germinativum. These are presumptive mesos cells (see discussion). During Stage 6 (Fig. 4) the ft- and mesos cells became completely mature and the nuclei disappeared from them. Presumptive a-cells (PAT, Fig. 4) appeared and began to mature to form a new a-layer. At the time of shedding ( Stage 6, Table I ) , the epidermis consisted of a complete, mature outer epidermal generation which would be lost from the body. The inner generation (which soon becomes the

306

P. F. A. MADERSON, K. W. CHIU AND J. G. PHILLIPS

OBO

FIGURES 1-4.

EPIDERMAL THYROID RELATIONS IN SNAKES 307

lunctional body surface) consisted of a mature Oberhautchen, ft- and mesos layers, a partially mature a-layer and several layers of presumptive a-cells and exactly resembled the picture described for Stage 0 above.

The animals' external appearance during the cycle changed in a slightly different way from Elaphe (Maderson, 1965a). Eye cloudiness began in P. korros at Stage 4, reached opacity during Stage 5, and cleared while the epidermis showed a Stage 6 histology.

Thyroid acti-i'ity (Table /)

In Table I, thyroid weight in each of the 7 groups is expressed in mg % fat- free body weight. Although some differences are seen, there is no indication that they reflect a cycle of gland activity which can be correlated with the shedding cycle.

Thyroid weight and follicular cell height are not correlated. The cell height was greatest around the time of shedding when the epidermis showed either a Stage 6 condition (9.82 p) or Stage 0 (8.32 ^) , and lowest during Stage 3 (5.91 /*). The follicular cell heights during Stages 1, 2, 3, and 4 were significantly lower than during Stage 0 (about 15-30%, P < 0.05). During Stages 3 through 6 there was a steady rise in epithelial cell height (about 45%, P < 0.05).

Intracellular colloid was seen in the thyroid follicular epithelium in association with all epidermal conditions except Stages 2 and 3. As shown in Table I, the percentage or the number of animals in each instance showing this condition varied ; it was typically found in those animals having thyroids with tall follicular epithelia. \Yhile the presence of intracellular colloid probably indicated that active

FIGURE 1. The epidermis of Ptyas korrus seen one day after sloughing (Stage 0, Table I). There is an incomplete outer epidermal generation above a stratus germinativum ( SG ) . The outer generation has mature /3- (/3O) and mesos (MO) layers. There is a poorly developed a-layer (AO) and three layers of presumptive a-cells (PAO). Oberhautchen (OBO). Hematoxylin and eosin. < 850.

FIGURE 2. The epidermis of Ptyas korros seen during the "perfect resting stage'' (Stage 1, Table I). The j8-layer (/3O) is out of focus at the top of the picture having separated from the underlying tissues due to splitting of the mesos layer (MO) during histological preparation. The a-layer (AO) is now well developed, and the number of layers of living cells between its base and the stratum germinativum (SG) is reduced by comparison with the condition shown in Figure 1. The cell marked X is probably in the process of final maturation and is the homologue of the "clear cell" shown by Roth and Jones (1967). Hematoxylin and eosin. < 780.

FIGURE 3. The epidermis of Ptyas korros showing a very early Stage 5 development. Only the inner generation is shown. Note the characteristic splitting of the cells of the /3-layer (/3l) in this final stage of maturation (cf. Lillywhite and Maderson, 1968). The mesos cells (PMI) are very flattened units and are quite distinct from the nearly mature /3-cells above and first presumptive a-cells (PAI) beneath. Note the columnar shape, chromophobia and oblique orientation of the germinal cells at this time. Oberhautchen (OBI). Hematoxylin and eosin. X 540.

FIGURE 4. The epidermis of Ptyas korros showing a very early Stage 6 development. Only the inner generation is shown. The /3-layer (/3l) is mature and resembles the image shown in Figure 1. The mesos cells (MI) have thickened membranes and the first pycnotic nuclei are seen immediately beneath the /3-layer. There are now three layers of presumptive a- (PAI) cells. When shedding takes place, the inner generation resembles Figure 1 exactly. Hematoxylin and eosin. X 540.

308

P. F. A. MADERSON, K. W. CHIU AND J. G. PHILLIPS

colloid synthesis was taking place, the significance of the variation at different epidermal stages is unknown.

Many of the thyroid glands of epidermal stages 6 and 0 were highly vascnlarized, which suggests that these glands were probahly actively secreting.

DISCUSSION

Since the initial description of the epidermis of Elaphc (Maderson, 1965a), further data on the structure of the squamate epidermal generation have become available (Maderson, 1966, 1967, 1968; Maderson and Licht, 1967; Roth and Jones, 1967). The snake mesos layer was indicated by Maderson (1965a), but

TAHLK I

The weight and the cell-height of the thyroid gland, and the percentage of animals with intmcellular colloid at different skin stages during the sloughing

cycle in Ptyas

			Thyroid gland #	No. of animals
Skin stages	Bodv weight @ (g)	No. of snakes		with intra- cellular
Weighl	Cell height
			mg (%(" )	to
0	223.82 ± 28.23	11	5.24 ± 0.51	8.32 ± 0.40 +	7 (64'Y)
1	181.20 ± 17.69	5	6.24 ± 1.06	6.78 ± 0.58	1
2	233.67 ± 18.48	6	4.92 ± 0.45	6.78 ± 0.29*	0
3	176.00 ± 16.00	2	5.45 ± 0.92	5.91 ± 0.28	0
4	241.89 ± 29.32	9	6.30 ± 0.64	7.17 ± 0.28	4 (44' , )
5	223.33 ± 33.71	6	5.63 ± 0.99	6.90 ± 0.51**	1
6	279.40 ± 18.07	5	7.10 ± 1.87	9.82 ± 1.27	1

# Mean db S.K.

@ Fat-free body weight.

+ Stages 1, 3, and 4 compared with St. 0, P < 0.05.

* Stage 2 compared with St. 0, P < 0.02. Stage 2 compared with St. 6, P < 0.05.

** Stage 5 compared with St. 4, P = not significant.

\\as assumed to be the outermost part of the a-layer. Although the relationship of mesos cells to /i?-cells in terms of protein synthetic capacities is still in doubt, the tissue can now be defined histologically in the snake epidermis. At the onset of Stage 4, presumptive mesos cells are seen as extremely flattened, relatively chromophobic units between the stratum germinativum and the presumptive (3- population [Fig. 4 and plate Ila (Maderson, 1965a)]. Their maturation during the latter part of Stage 4 and Stage 5 is accompanied by a chromophobic appearance of the germinal cells |see Fig. 3 in the present study and in Maderson (1965a, pp. 103-106)]. The mesos layer, like the overlying /?-layer of the inner epidermal generation is always mature prior to shedding. The mature mesos layer in snake epidermal material is nearly always the site of separation of the /3-layer from the subjacent tissues (Maderson and Licht, 1967). The present data from Ptyas korros [supported by observations on the cobra (Chin, Phillips and Maderson, 1969) and

EPIDERMAL THYROID RELATIONS IN SNAKES 309

several other ophidian species (Chin and Maderson, unpublished) | compared with the original study of Etaf/ic lacn'ntra ( Maderson, 1965a) indicate considerahle individual and specific variation in the degree of development of the a-layer at the time of shedding. As indicated previously ( Maderson, 1966) E. tacniura is unusual in its lack of variation in this respect. These new data on the mesos layer and the pattern of differentiation of the a-layer further substantiate the essential morphological similarity of the epidermal systems in the sub-orders of the squamata.

Stage 1 as described here is the homologue of the "one-cell condition" described for Gekko (Maderson, 1966, pp. 40-48) and for Anolis (Maderson and Licht, 1967, p. 159). It is believed that this "perfect resting condition" exists throughout a period of time in which no, or at least, very little proliferative activity takes place in the stratum germinativum. Evidence from a variety of experimental studies indicates that it is variation in the duration of this period which provides the basis for different inter-slough periods in different species or individuals. It is therefore probable that this is one of, if not the most important, points where the hormonal milieu could control shedding periodicity (Maderson, Chiu and Phillips, 1970).

The present data indicate that in Ptyas korros there is a cycle of changes in thyroid epithelial cell height which can be correlated with the changes in epidermal histology associated with periodic skin-shedding. If one accepts the assumption that when the epithelial cells are high, the gland is synthesizing hormone at an enhanced rate and that this hormone is being released into the blood stream, and conversely decreased height indicates relative inactivity of the gland (pp. 129-130, Pickford and Atz, 1957), we can tentatively draw the following conclusion based on the present results. The gland is very active at the time of shedding, activity decreases throughout the subsequent resting phase, reaching a minimum as the first units of the inner epidermal generation become recognizable and then returns to a high level of activity in the latter part of the renewal phase prior to shedding.

The results of his own studies, as well as those of Schaefer (1933), Krockert (1941) and Halberkann (1953; 1954a, b) led Goslar (1964) to propose the following model for the role of thyroid hormone in the control of the snake sloughing cycle. Goslar suggested that although high levels of circulating thyroxine favored germinal proliferation (he eliminated the "perfect resting condition" and obtained a-hyperplasia by injection with thyroid hormone), generation formation, and subsequent shedding could only occur in the presence of a low level of the hormone in the blood stream. He concluded (Goslar, 1964. p. 4) that the hormone inhibited formation of the "Grenzzone" [the clear \ay&r/Oberhautchen complex, the boundary between the outer and inner generations (Maderson, 1967; Maderson and Licht, 1967)]. While this conclusion finds some confirmation in the present results in that the lowest level of thyroid gland activity might be associated with "late" Stage 2-" early" Stage 3 when the clear layer/Oberhautchen complex is first recognizable, it may be that the significant factor is the drop in thyroid hor- mone level during the "late" resting phase. Flaxman, Maderson. Szabo and Roth (1968) showed that generation formation is an intrinsic epidermal capacity dependent on an intraepiclermal feed-back mechanism. It has been argued else- where (Maderson, Chiu and Phillips. 1970) that this mechanism must be activated

,-510 P. F. A. \ I. \DEKSON, K. W. CHIU AND J. G. PHILLIPS

during the late resting [iliase, Otherwise there would not lie a Stage 2 or 3 morpho- li laical condition to identify. To obtain a more dynamic picture of thyroid hormone involvement with shedding in snakes, information on changes in circulating levels throughout the resting phase is needed.

The results of a directly comparable morphological study of Laccrta (Eggert, N35) are quite contrary to the present data. In Lacerta. thyroid activity as judged by histological criteria is low immediately prior to shedding and rises to a maximum immediately afterwards. The level of activity remains high throughout the resting phase, the clear layer/Oberhautchen complex differentiates while the level is maximal, and gland activity diminishes during Stages 3-5 to rise again at the time of shedding. In Gckko <jccko (Chin and Phillips, 1968; Chiu, unpublished data ) there is yet another pattern of thyroid gland activity, showing two peaks of activity during one epidermal cycle. Here, the clear layer/Oberhautchen complex differentiates in a rising titer of thyroid hormone, there is a subsequent drop around the time of shedding, followed by another peak 7 days after shedding, so that most of the resting phase is associated with low thyroxine levels.

\Yhile it is certain that thyroid gland activity does play an important role in controlling shedding frequency in squamate reptiles, it is probable that other endo- crine secretions are equally involved. The presently available data do not permit of the establishment of a model pathway system for either snakes or lizards (see discussion, Maderson, Chiu, and Phillips, 1970). There is no direct information regarding the physiological significance of skin-shedding in squamate reptiles, and indeed there is some doubt as to whether it should be regarded as a consum- mating event in itself or whether it is merely a side effect of other metabolic activi- ties (Maderson, 1965b; Maderson, Chiu and Phillips, 1970j. It is of interest to note therefore that there is good evidence for assuming that it is a more tightly controlled process in some taxa than in others (Salthe and Maderson, 1969). This might indicate that the phenomenon of skin-shedding (in company with factors indirectly associated with it and/or directly controlling it) have been evolutionarily selected for more than once, which could account for known differ- ences in hormonal associations in different taxa.

We wish to thank Dr. W. G. Lynn for critical reading of the manuscript. Thanks are also due to Miss Winnie Tsang for technical assistance and to Mrs. Elizabeth S. Taylor for typing. This work was supported by financial grants from the Nat. Can. Inst. #CA-10844 (P. F. A. Maderson) and from the Nuffield Foundation and Sir Shui-kin Tang (J. G. Phillips).

SUMMARY

1 . Study of the epidermal histology throughout the sloughing cycle in the snake Ptyas korros revealed the existence of a well-defined mesos layer and showed that the a-layer is completed in the immediate post-shedding resting-phase. These observations supplement previous histological studies of the snake epidermis and further substantiate the essential homology of the epidermal generation in these forms with similar units in lizards.

EPIDERMAL THYROID RELATIONS IN SNAKES 311

2. Thyroid weight is not correlated with follicular cell height nor with epidermal cell changes throughout the sloughing cycle.

3. Changes in follicular cell height, indicative of varying levels of gland activity can be correlated with epidermal changes. The shedding complex and the outermost portions of the new inner epidermal generation differentiate during a period of lowest thyroid gland activity ; gland activity is highest around sloughing.

4. These results provide a possible explanation for previous experimental studies of thyroid-sloughing relations in snakes. The problem of the difference between snakes and lizards in this context is discussed.

LITERATURE CITED

CHIU, K. W., AND J. G. PHILLIPS, 1968. Some aspects of the hormonal control of sloughing

in the gecko Gckko gecko L. Proceedings of the 3rd Asia and Oceania Congress

Endocrinology, (A. a Litonjua, Ed.) Part 2 : 298-302. CHIU, K. W., J. G. PHILLIPS AND P. F. A. MADERSON, 1969. Seasonal changes in the thyroid

gland in the male cobra, Naja naja L. Biol. Bull., 136 : 347-354. EGC.EKT, B., 1935. Zur Morphologic und Physiologic der Eidechsen-Schilddnise. 1. Das

jahreszeitliche Verhalten der Schilddriise von Lacerta agilis L., L. riripara Jacq. und

L. muralis Laur. Z. Wiss. Zoo!., 147 : 205-263.

FLAXMAX, B. A., P. F. A. MADERSON, G. SXABO AND S. I. ROTH, 1968. Control of cell differen- tiation in lizard epidermis in vitro. Dcv. Biol.. 18 : 354-374. GOSLAR, H. G., 1958. Die Reptilienhant als endokrines Testohjekt. Endokrlnologie, 36:

279-286. GOSLAR, H. G., 1964. Beitrage zum Hautungsvorgang der Schlangen. Acta Histochcm., 17:

1-60. HALBERKANN, J., 1953. Untersuchungen ztir Beeinflussung des Hautungszyklus der Ringelnatter

durch Thyroxin. Arch. Derma I ol Sypliilo!., 197 : 37-41. HALBERKANN, J., 1954a. Zur hormonalen Beeinflussung des Hautungszyklus der Ringelnatter.

Z. Nahtrforsch., 96: 77-80. HALBERKANN, I., 1954h. Die Hautungsahlauf der Ringelnatter unter Methyl-Thiouracil.

Naturwiss., 41 : 237-238. KROCKERT, G., 1941. Kontinuierliche Hyperthyroidisierung und Epiphysierung an Python

bwittatns. Vita-wine Hormone, 1 : 24-31. LILLYWHITE, H. B., AND P. F. A. MADERSON, 1968. Histological changes in the epidermis of the

sub-digital lamellae of Anolis carolincnsis dguanidae) during the shedding cycle.

J.Morphol. 125: 379-401. LYNN, W. G., 1960. Structure and functions of the thyroid gland in reptiles. Amcr. Midi.

Natur., 64: 309-326.

LYNN, W. G., 1970. The thyroid. In : Biology of the Reptilia. Academic Press, in press. MADERSON, P. F. A., 1965a. Histological changes in the epidermis of snakes during the

sloughing cycle. /. Zoo!., 146 : 98-113. MADERSON, P. F. A., 1965b. The structure and development of the squamate epidermis.

Pages 129-153 in A. G. Lyne and B. F. Short, Eds., The Biology of the Skin and

Hair Growth. Angus and Robertson, Sydney. MADERSOX, P. F. A., 1966. Histological changes in the epidermis of the Tokay (Gckko gecko)

during the sloughing cycle. /. Morphol, 119 : 39-50. MADERSON, P. F. A., 1967. The histology of the escutcheon scales of Gonatodcs (Gekkonidae)

with a comment on the squamate sloughing cycle. Copeia, 1967 : 743-752. MADERSON, P. F. A., 1968. Observations on the epidermis of the Tuatara (Sphenodon

punctatus). J. Anat., 103 : 311-320.

MADERSON, P. F. A., AND P. T.ICHT, 1967. The epidermal morphology and sloughing fre- quency in normal and prolactin injected Anolis carolincnsis (Iguanidae, Lacertilia).

J. Morphol, 123: 157-172.

MADERSON", P. F. A., K. W. CHIT AXM J. G. PHILLIPS, 1970. Endocrine-epidermal relation- ships in squamate reptiles. Mem. Soe. Endocrinol. 18 : 259-284.

312 P. F. A. MADERSON, K. W. CHIU AND J. G. PHILLIPS

l'n KHiKii, G. E., AM) J. W. ATZ, 1957. The Physiology of the I'itnilnry Gland of 1'islicx.

New York Zoological Society, New York, 613 pp. RUTH, S. I., AND W. A. JONES, 1967. The ultrastructure and enzymatic activity of the boa

constrictor (Constrictor constrictor) skin during the resting stage. /. Ultr. Res., 18:

304-323. SALTHE, S. N., AND P. F. A. MADERSON, 1969. Physiological indeterminacy in Anolis. Aincr.

Zool, 9 : 1096. SniAEFER, W. H., 1933. Hypophysectomy and thyroidectomy of snakes. Proc. E.i-p. /?/<//.

Med.,30: 1363-1365. SEMBRAT, K., AND S. DRZEWICKI, 1936. The influence of selachian thyroid upon molting process

of lizards, with some remarks on the skin, the eyes, and the ultimobranchial body of

the thyroidectomized lizards. Zool. Pol., 1 : 119-169.

Reference : Blol Bull., 139: 313-320. (October, 1970)

CYTOLOGICAL AND IMMUNOLOGICAL INVESTIGATIONS OF

SPERM-EGG INTERACTIONS IN SELECTED DECAPODS

(CRUSTACEA) AND LIMULUS POLYPHEMUS

L. (MEROSTOMATAi1

RODNEY C. MOWBRAY,2 GEORGE GORDON BROWN 2 AND CHARLES B. METZ 3

Department of Zoology and Entomology, Iowa State University, Ames, larva;

Institute of Molecular Evolution, University of Miami, Coral Gables, Florida

and The Marine Biological Laboratory, Woods Hole, Massachusetts

The physiological aspects of sperm-egg interactions have been studied exten- sively by many investigators (see Metz and Monroy, 1967 for review). These studies include such topics as the effects of egg substances on specificity of attach- ment, activation of the sperm acrosomal reaction, and release of lytic substances from the acrosome. Many, if not all, of these processes evidently involve gamete surface components; e.g., macromolecules on the surface of the gametes which are believed to interact much like enzyme and substrate or antigen and antibody. Most of this work has been done on echinoderm and amphibian gametes (Metz, 1967 for review) .

The present study deals with gamete specificity and the role of gamete surface components, especially antigens, in the fertilization of several representative species of decapod crustaceans and a xiphosuran, Lhnuhis polyphemus. The morphology of decapod spermatozoa is quite unusual and bizarre (Brown, 1966) as compared to the so-called typical spermatozoa (e.g., sea urchin sperm; Franklin, 1965). The reptantian spermatozoa used in this study are large and non-motile, possess a number of radiating arms, and have a very large and complex acrosome. Initial sperm-egg attachment is polyspermic. These spermatozoa need to be studied physiologically because their interactions with the egg during fertilization should be compared with those of the typical sperm, upon which most previous physiological information is based. In addition, the large size of the decapod sperm facilitates the analysis of its surface components and their behavior during sperm-egg interactions. Finally, since these spermatozoa are non-motile, one of the usual parameters in fertilization, namely sperm motility, is eliminated and the system is correspondingly simplified.

MATERIALS AND METHODS

Live specimens were obtained from three sources: (1) the Marine Biological Laboratory Supply Department, Woods Hole, Massachusetts, (2) the Gulf Speci-

1 Portions of this work were supported by the Fertilixation and Gamete Physiology Training Program at the Marine Biological Laboratory, Woods Hole, Massachusetts CNIH Grant TI-HD-26-07) and by a Small Grant Fund, Iowa State University.

2 Present address : Iowa State University, Ames, Iowa 50010.

3 Present address : University of Miami, Coral Gables, "Florida 33134.

313

314

R. C. MOWBRAY, G. G. BROWN AND C. B. METZ

men Company, Panacea, Florida, and (3) the Florida Marine Biological Specimen Company, Panama City, Florida.

Gametes were obtained from decapods by dissection. Free spermatozoa were obtained from the testes and the seminal receptacle of the female. Spermatozoa contained in spermatophores were obtained from the vas deferens. Mature eggs and oocytes were obtained by maceration of ovarian tissue. Oocytes were con- sidered adequate for experimental use if spermatozoa attached to them in normal proportions (i.e., comparable to the number of spermatozoa attached to normal eggs). Spermatozoa and eggs were obtained from Liiniilns polyphemus males and females by electrical stimulation (Shrank, Shoger, Schechtman and Bishop, 1967).

Antisera were prepared by injecting rabbits subscapularly with an emulsion of

TABLE I

-S ol (i utiscrn f>rt'f>a red, the species of animals used and their source*

Antiserum	Antigen prepared from	Species	Source of animals
Anti-Callinecles	sperm filled	Callinectes sapidus	Gulf Specimen Co.
spermatophores	spermatophores	Rathbun
Anti-Call inectes	seminal receptacle	Callinectes sapidus	Flor. Mar. Biol.
sperm	sperm		Spec. Co.
Anti- Callinectes	eggs	Ca Hi n eel ex sa pid u s	Gulf Specimen Co.
eggs
Anti-Libinia	sperm filled	Libinia emarginata	Marine Bin]. Lab.
spermatophores	spermatophores	Leach
Anti-Pagurus	sperm filled	Pagurus poll icn ris	Marine Biol. Lab.
spermatophores	spermatophores	(Say)
knti-Homarus	vas deferens	Homarus americanus	Marine Biol. Lab.
vas deferens		Milne-Edwards
Anti-Limnlns	sperm	Li in ul us polyphemus	Marine Biol. Lab.
sperm		Linnaeus

* Other species used in this study (Cancer irroratus Say and Ovalipes ocellatus (Herbst) were obtained from the Marine Biological Laboratory.

whole intact sperm and Freund's complete adjuvant (1:3). Table I presents the kinds of antisera prepared, the species used, and the source of the animals. To assure that equivalent amounts of antibody were used in all experiments, the globulin fraction was separated from the whole serum by precipitating with 18% sodium sulfate (Kekwick, 1940). Protein determinations were made on the globulin fractions using the biuret method (Gornall, Bardawill and David, 1949) and the antibody solutions were then adjusted to equivalent concentrations (25 mg protein per milliliter) .

Extracts of sperm and eggs for immunodirrusion experiments were made by homogenizing gametes in a glass homogenizer at 0° C in sea water, 0.85% saline or 0.8 M Tris buffer at pH 7.0. In some cases the gametes were treated with 0.5% sodium deoxycholate to enhance membrane disruption. After homogenization the suspension was freeze thawed in an acetone-dry ice bath to aid in the release of antigens from the membranes. In some cases the suspensions were also treated ultrasonically to break up the cell membranes. This consisted of four, 15 second bursts on a Branson sonifier at 0° C. Cellular debris was removed by centrifuga-

SPERM-EGG INTERACTIONS IN DECAPODS

315

(ion. In most cases the extracts were treated with 0.05% cleoxyribonuclease to digest any DNA present which may bind with proteins and precipitate them. Extracts were made up to a ratio of 1:6 tissue to buffer. Antibody concentration was 25 mg protein per milliliter. The gel was prepared from \% agarose in 0.85% saline with 0.2% sodium azide added as a preservative and 0.05% cadmium chloride added to enhance antigen-antibody precipitate formation.

Some experiments employed papain-digested antibody prepared by the method of Porter (1959) under the following conditions: 100 mg globulin/mg papain (2X crystallized, Sigma Chemical Co.) in 0.1 M potassium phosphate, 0.01 M cystein, 0.002 M disodium EDTA ; pH 7.0, 37° C. Digestion was considered complete (usually after 18-22 hours) when the antibody no longer agglutinated

TABU-: II

Results of cross-insemination experiment reported as the average number of spermatozoa attached per millimeter egg circumference

Spermatozoa	Eggs
Libinia	Callinectes	Cancer	Ovalipes	Homarus	Limulus
Libinia	12.5±3.4*	0.3 ±0.1 5	0	0	0	0
Callinectes	0.4 ±0.22	14.1±3.5	0	0	0	0
Ca ncer	0	0	43. 2 ±6. 5	0	0	0
Ovalipes Uomarus	0 0	0 12.8±2.7	0 9.6±3.3	8.0±2.2 6.0±1.7	0 90.6±10.1	0 0
Limulus	0	0	0	0	0	75.8±9.2

* Standard deviation.

Washed spermatozoa were made up to a 2% suspension. From 10-30 eggs were placed in the well of a spot plate and the excess water removed. Two drops of 2% sperm suspension were added to the well and this mixture was stirred for one minute and then the eggs were washed three times with an excess of sea water. The eggs were then transferred to a microscope slide and observed with phase contrast optics. Ten eggs were chosen randomly and the number of spermatozoa attached to each was determined and recorded. The eggs were not rotated; only the sperm around the egg periphery were counted. The experiment was set up all at one time so that all eggs were treated with the same sperm suspension of each species, etc.

homologous sperm in a 2% suspension. At this time iodoacetamide was added to a final concentration of 0.02 M to inactivate the enzyme. The digests were then dialyzed exhaustively against sea water.

Fluorescein-conjugated antibody was prepared by the method of Riggs, Seiwald. Hurchhalter, Downs and Metcalf (1958). Conditions for this pro- cedure include 0.15 M NaCl, 8% acetone, 0.06 M carbonate-bicarbonate buffer (pH 9.0), 10 mg globulin per milliliter, and 0.05 mg fluorescein isothiocyanate (Nutritional Biochemicals Corp., Cleveland, Ohio) per milligram globulin. This mixture was stirred at 4° C for 18 hours. The conjugated protein fraction was isolated by column chromatography (16 X 2.5 cm column) with 0.1 M phosphate buffer (pTT 7.2) in Sephadex G-25 med. and then dialyzed against sea water for 72 hours. Sperm suspensions (2%) were treated with fluorescein-conjugated antibody for 5 minutes and then washed with sea water. These suspensions were then observed with a Zeiss fluorescence microscope.

316

R. C. MOWBRAY, G. G. BROWN AND C. B. METZ

RESULTS

attachment specificity

'Five different types of experiments were performed on decapod and Limulus gametes. These included cross-insemination, cross-absorption-agglutination, im- munodiffusion, antibody inhibition, and antigen localization experiments. These experiments were designed to test for the following : ( 1 ) the presence of molecular components essential for fertilization on the surface of sperm and egg, (2) the species specificity of such components, (3) the involvement of such components

TABLE 1 1 1 Results of cross-absorption-agghitination experiments

Sera	Spermatozoa
Prepared against sperm	Absorbed with sperm	Libinia	Pagurus	Homarus	Limulus	Ovalipes	Cancer	Callinectes
Control serum		0	0	0	0	0	0	0
Libinia	—	+ + +	0	+	0	0	0	—
	HoiiKirns	+ + +	—	0	— .	• — -	—
Pa gurus	Homarus	0	0	0	0	0	0
Homarus	—	0	-f	-f-f	0	0	(1	- — -
Limulus	Pagiinis	0	0	0 0	+++	0	7	_
	Pa gurus Ca ncer	• —	0 0	•	++	• —	0
Cdllinectes		+	0	+	0	0	+	+++

+ degree of agglutination; 0 = no agglutination ;— = no test performed.

Agglutinations were performed with anti-sperm globulin previously prepared against the sperm of each species. The globulin solutions were all diluted to a concentration of 1.6 mg protein per ml, as this was the concentration at which control globulin no longer agglutinated any of the sperm types. One drop of a 2% sperm suspension was mixed together with one drop of anti-sperm globulin on a microscope slide. All tests included two controls: one treated with sea water and one treated with control globulin. Absorptions were performed by adding whole sperm to the globulin and allowing it to stand for three hours at 5° C.

in sperm-egg attachment, (4) the antigenicity of the components, and (5) the localization of antigenic components with labeled antibody.

Reciprocal sperm-egg crosses were performed in vitro between the following species: Libinia emargmata, Callinectes sapidus, Ovalipes ocellatus, Cancer irroratus, Homarus americamts, and Limulus polyphemus (Table II). Among the crustaceans employed in these experiments, sperm-egg attachment is polyspermic and most if not all of the attached spermatozoa remain visible at the egg surface for several minutes. Accordingly, comparison of the numbers of spermatozoa attached to the egg surface should provide a measure of sperm-egg affinity. The results of the experiments support this view. Two preliminary qualitative experi- ments were performed (results not reported). The results of a third experiment are given in Table II as the average number of spermatozoa attached per egg.

SPERM-EGG INTERACTIONS IN DECAPODS 317

All three experiments agree. The results show that with one exception the attachment of gametes of all the species represented are highly self-specific. The spermatozoa of each species attach only to their own eggs. In the one exceptional case Horn-am s spermatozoa attach to the eggs of three other species, Callinectes, Cancer and Ovalipes. These attachments were less firm than normal and the acrosome often was oriented away from the egg surface. Hoinants eggs failed to cross-attach to the spermatozoa of these three species.

. lufigcuic specificity

Antibodies were used in cross-absorption-agglutination tests on spermatozoa from the following species : Libinia emarginata, Callinectes sapidus, Cancer irroratus.

a

B

FIGURE 1. Immunodiffusion tols; (1) anti-Homarus sperm, (2) anti-Pagurus sperm, (3) anti-Callinectes sperm (4) anti-Libinia sperm, (5) anti-Limulus sperm, (a) Callinectes sperm extract, (b) Homanis sperm extract, (c) Libinia sperm extract, (d) Pagunis sperm extract, (e) Cancer sperm extract, (f) Liinulits sperm extract.

Homarus americanus, Pagunis pollicaris, and Limulus polyphemus (Table III). Three sets of experiments were performed. All antibodies readily agglutinated their own spermatozoa but none of the antibodies agglutinated the spermatozoa of every species. The agglutinations indicate that antigens are on the surfaces of the spermatozoa. Antigens are present over the entire surface since arm to arm, arm to "head" and ''head" to "head" agglutinations occurred. The arm to "head" agglutinations mean that the same antigen appears on the arm and "head." The possibility of arm and "head" specific antigens has not been excluded. The experiments indicate that Llhlnia sperm lias at least two surface antigens, one of which is shared with Homanis sperm. Homarus sperm has at least one antigen which it shares with Pagunis sperm. Lintiiliis sperm has at least three surface antigens, sharing one of them with Pagunis sperm and another with Cancer sperm.

318

R. C. MOWBRAY, G. G. BROWN AND C. B. METZ

This test also indicates that Pagurus sperm shares an antigen with Cancer sperm. .\nl\-CalIincctcs sperm serum agglutinates Libinia, Homarus and Cancer sperma- tozoa. In synopsis, the spermatozoa of Homarus has at least one and Libinia and Pagurus has at least two surface antigens. The sperm of Limulus has at least three surface antigens.

Soluble antigens

Immunodiffusion tests (Fig. 1) were performed on extracts of the same species of spermatozoa as used in cross-absorption-agglutination experiments. The extracted antigens could originate from the surface, the interior or both parts of the spermatozoan. A specific antibody was placed in the center well and sperm

TAHLE IV

Results of inhibition experiments reported us the average number of sperm attached per millimeter of egg circumference

Experiment	Untreated	Libinia emarginata spermatozoa treated with:
Undigested anti-Libinia ~pt-rm serum	Digested anti-Libinia sperm serum	Undigested control serum	Digested control serum	Digested anti-Libinia sperm serum and sheep anti-rabbit globulin serum
1	13.6 ± 3.1	0	0.1 ± 0.10	4.3 ± 1.8	10.3 ± 4.5
2	14.9 ± 3.7	0	0.4 ± 0.15	7.9 ± 2.9	5.1 ± 2.1	0.2 ±0.12

Two drops of a 2% sperm suspension was mixed together with 1 drop of globulin (25 mg protein per ml) in the well of a spot plate. This mixture was agitated for five minutes and then eggs (10-30) were added. After two minutes the excess sperm were washed away and the eggs were examined to determine the numbers of sperm attached. Ten eggs were counted.

extracts in the peripheral wells. Precipitin band formation revealed that each species has at least two soluble antigens. Pagurus (Fig. IB), and Libinia (Fig. ID), spermatozoa each have only two antigens. Homarus (Fig. 1A), Limulus (Fig. IE) and CaUinectes (Fig. 1C) spermatozoa have three antigens. The reac- tion with anti-Pagurus sperm globulin indicates that Pagurus sperm shares one common antigen with Homarus and Libinia spermatozoa (Fig. IB). Likewise, CaUinectes sperm shares one common antigen with Homarus , Libinia, Pagurus, and Cancer spermatozoa (Fig. 1C), and Libinia sperm shares one common antigen with CaUinectes, Homarus, Pagurus, and Cancer spermatozoa (Fig. ID).

Evidence for functional sperm antigens in attachment

Sperm surface antigens can be blocked with specific antibodies to test for their possible role in fertilization. In these experiments Libinia spermatozoa were treated with bivalent or univalent anti-sperm globulin (Table IV). The treated spermatozoa were then mixed with eggs in order to determine their capacity

SPERM-EGG INTERACTIONS IN DECAPODS 319

for attachment. Bivalent anti-sperm globulin treatment reduced the attachment capacity of the sperm. This result \vas probably influenced by the fact that whole antibody agglutinates the sperm into large clumps which effectively prevents them from interacting with the egg. However, spermatozoa treated with univalent, turn agglutinating anti-sperm globulin also tailed to attach to the eggs. Spermatozoa pretreated with digested anti-sperm globulin agglutinate upon subsequent treatment with sheep anti-rabbit globulin (Coombs' or anti-globulin test, Coombs', Mourant and Race. 1945 J. Treatment of spermatozoa with both whole and digested control globulin did not markedly reduce their ability to attach to the egg.

Localization of spcnn antit/cns hy immuno fluorescence

Libinia spermatozoa were treated with fluorescein-labeled anti-L/7>/»/a sperm globulin. Fluorescence was observed over the entire surface of each sperm. No specific area fluoresced more than another. Non-specific staining was checked by treating spermatozoa with fluorescein-labeled control globulin. Such control treated spermatozoa did not fluoresce.

DISCUSSION

The large, non-motile spermatozoa of decapod Crustacea should be unusually favorable for studying the initial stages of sperm-egg attachment and interaction. The present study supports this view. It demonstrates species specificity of sperm- egg attachment, specific sperm surface antigens and, at least in Libinia, apparent involvement of sperm surface antigens in sperm-egg attachment. Thus the decapods resemble the sea urchin and the few other forms (Tyler, 1946; Metz, Schuel and Bischoff, 1964) that have been studied. The present study extends the analysis to the extent that one well defined process, namely sperm-egg attachment, is inhibited by antibody. No clear cut evidence is yet available to specify the fertilization step or steps inhibited by antisperm sera in other metazoa (e.g., Metz, 1967). It may be argued that the conditions of in ritro insemination employed here are suffi- ciently abnormal to produce excessive and pathological polyspermic sperm-egg interaction. However, Hinsch (in preparation) has examined naturally fertilized eggs of Libinia shortly after spawning and finds that these also are highly poly- spermic. It will now be of particular interest to determine if the antibody treated spermatozoa can undergo the acrosomal reaction, in other words if the attachment block is at the pre or post acrosomal reaction stage. In the sea urchin the acrosomal reaction is not affected by antibody pretreatment (Fourtner and Metz, 1967).

The cross-absorption agglutination and precipitin tests show that the crustacean spermatozoa have constellations of surface antigens. The distribution and number of these antigens and their solubility can now be extended using additional inter- specific combinations and appropriate absorbed sera. Additionally, it may be possible to identify and characterize the "attachment" antigen, using specific antibody as a label. Finally, the interesting question of the antigenic relationship between the surface of the reacted acrosome and the rest of the spermatozoa can probably be investigated readily in this material.

320 R. C. MOUT.KAY, G. G. BROWN AND C. H. METZ

Si MATARY

The initial events of sperm-egg interaction have been examined in several crustaceans. These have large non-motile spermatozoa and sperm-egg attachment is polyspermic.

Sperm-egg attachment /;/ ritro is largely species specific among the organisms examined.

Antisperm sera produced in rabbits agglutinates the crustacean spermatozoa. Interspecific agglutinations do occur. Appropriate absorption experiments demon- strate more than one sperm surface antigen.

Spermatozoa pretreated with univalent, non-agglutinating antibody fail to attach to eggs of the species. This indicates that one or more sperm surface antigens are involved in sperm-egg attachment.

Antigens are distributed over the entire sperm surface as shown by the morphology of agglutination and by immunofluorescence.

Two or more soluble antigens can be extracted from crustacean spermatozoa, depending upon species. Some of these antigens give interspecific reactions, others are species specific.

LITERATURE CITED

BROWN, G. G., 1966. Ultrastructural studies of sperm morphology and sperm-egg interaction in

the decapod Calliuectes sctpidus. J. Ultrastruc. Res., 14: 425-440. COOMBS, R. R. A., A. E. MOURANT AND R. R. RACK, 1945. A new test for the detection of weak

and "incomplete" Rh agglutinins. Brit. J . E.rp. PathoL, 26 : 255-266. FOURTNER, C. R., AND C. B. METZ, 1967. Tests for the effects of antihodies on the acrosome

reaction in Arbacia pitnctitlata. Biol. Bull., 133 : 465. FRANKLIN, L. E., 1965. Morphology of gamete membrane fusion and of sperm entry into

oocytes of the sea urchin. /. Cell BioL, 25 : 81-100. GORNALL, A. G., C. S. BARDAWILL AND M. M. DAVID, 1949. Determination of serum proteins

by means of the biuret reaction. /. Biol. Chcm.. Ill : 751 KEKWICK, R. A., 1940. The serum proteins in multiple myelomatosis. Biochcm. J.. 34 :

1248-1257. METZ, C. B., 1967. Gamete surface components and their role in fertilization. Pages 163-224

in C. B. Metz and A. Monroy, Eds., Fertilization, Volume 1. Academic Press, Inc.,

New York, New York. METZ, C. B., AND A. MONROY, 1967. Fertilization, Volume 1. Academic Press, Inc., New

York, New York, 489 pp. METZ, C. B., H. SCHUEL AND E. R. BISCHOFF, 1964. Inhibition of fertilizing capacity of sea

urchin sperm by papain digested non-agglutinating antibody. /. E.rp. ZooL, 155: 261-

272. PORTER, R. R., 1959. The hydrolysis of rabbit gamma-globulin and antibodies with crystalline

papain. Biochcm. J., 73 : 119-126. RIGGS, J. L., R. J. SKIWALD, J. H. BURCHHALTER, C. M. DOWNS AND T. G. METCAI.F, 1958. Iso-

thiocyanate compounds as fluorescent labeling agents for immune serum, .liner. J.

PathoL, 34: 1081-1097. SHRANK, W. W., R. L. SHOGER, L. M. SCHECHTMAN AND D. W. BISHOP, 1967. Electrically

induced spawning in the male and female horseshoe crab, Liinnlits polyphemus: Biol.

Bull., 133 : 453. TYLER, A., 1946. Loss of fertilizing power of sea urchin and Urecltis sperm treated with

"univalent" antibodies vs. antifertilizin. Proc. Soc. E.rp. Biol. Med., 62 : 197-199.

Reference : /?/W. Bull., 139: 321-332. ( ( )rlol>iT. 1070 )

A GENERAL METHOD EOR THE MONOXENIC CULTIVATION

OF THE DAPHXIDAE

JAMES S. MURPHY The Rockefeller University, Neiv York. New York 10021

This laboratory has attempted to obtain culture conditions which should satisfy the members of the family Daphnidae that are commonly found in the lakes of the Northeastern United States. \\& have succeeded with all the commonly available species and have fourteen under monoxenic continuous cultivation, using Chlaniy- domonas reinhardii as the sole food organism.

The classical method for cultivating Cladocera is Banta's stable tea, a pond water extract of horse manure and garden soil (Needham, Gatz and Lutz, 1937). Other successful methods have been developed using mixtures in which either bacteria, protozoa, yeast, or algae are the principal food supply (Mortimer, 1936; Beerstecher, 1952; Murachi and Imai. 1954: Watanabe, Ito and Sasa, 1955; 'Frank. Bolland and Kelly, 1957; Sasa, Kunieda and Tamiya, 1960; Dewey and Parker. 1964). A monoxenic system was developed for D. inagna by Treillard (1924) using rabbit erythrocytes and for Moina macrocopa by Stuart, McPherson and Cooper (1931) using sterile pond water with suspensions of living bacteria. The individual specimens were freed of microorganisms by repeated washings prior to inoculating the food organism. Fritch (1953) showed that Chlamydomonas sp. would support the growth of D. pulc.r if pantothenic acid was added to a system in which bacteria were present. Recently Taub and Dollar (1968) studied the inadequacies of Chlamydomonas reinhardii and Chlorella pyrenoidosa as food for D. pulex.

None of these systems has proved completely satisfactory because of uncon- trollable variation in results. Banta ( 1939) reviewed the problem of "depression" or periods when cultures die out or show a reduced reproduction rate and pointed out that no method was known to prevent the phenomenon. Dewey and Parker (1964) describe the difficulties in obtaining natural water of constant composition free from insecticides and other toxic substances. Anthony D'Agostino and Luigi Provasoli, St. Johns University and Haskins Laboratories (personal communica- tion) have succeeded in devising a dixenic system with Chlamydomonas reinhardii and Sccncdcsmus obliqnus which will support D. inagna in continuous culture. The medium contains several salts and vitamins B12, pantothenic acid and thiamin. The method should prevent the aforementioned problems as the cultures can be isolated from variations in food organisms and changes in the composition of the medium.

MATERIALS AND METHODS

Algae. CJilainvdoinonas reinhardii Indiana U. strain #90 and Scenedesmus obliqnus Indiana U. strain #393 obtained from Dr. Luigi Provasoli were used throughout these experiments as the principal food supply of the Daphnia. They

321

322

JAMES S. MURPHY

I \HI,E I

Composition of the medium (mg/ liter )*

	I'.u^ir iiirilium	Enriched medium
Karly formula	Later (improved! formula
Calcium acetate • X H2O	177.0	59.0	59.0
Potassium penicillin (U.S. P.)	645.0 (l()(i units)	215.0	215.0
Streptomycin sult'ute (U.S. P.)	20.0	7.0	7.0
Bovine albumin, fraction V (Armour)	200.0	67.0	67.0
Calcium pantothcnate	20.0	7.0	7.0
Hi,	0.001	0.0003	0.0003
Thiamin	l.Of	0.6	0.6
Riboflavin		0.03	0.4
Xicotinamidc		0.3	1.3
1- olic acid		3.3	3.3
Biotin		0.3	0.3
Putrescine		0.3	0.3
Choline		0.3	5.0
Inositol		0.6	1 1 .0
Hutner's Trace Elements}		0.3 ml	0.3 ml

* The medium is made up at three times concentration and frozen in 250 ml amounts. When needed it may be thawed, diluted and filtered through a 2 micron porcelain filter cylinder. Con- centrated stock solutions of all the vitamins except biotin, which precipitates, may be stored frozen.

f Only included for a brief time.

J Levine and Ebersolcl's (1958) modification of a trace element mixture designed by Hutner, Provasoli, Schatz and Haskins (1950). We heated the mixture to 100° C and brought it to pH 6 with KOH while hot. The solution is deep green but turns to deep purple on standing. The mix- ture is stable.

were grown on a yeast extract-acetate medium described by Levine and Ebersold ( 1958 ) enriched with one gram per liter of X-Z-Case peptone (Sheffield Chemical Co.) and with agar omitted. The cells were harvested sterilely by low speed centrifugation after 3 or 4 days culture, washed twice in deionized water and added to the Daphnia medium (Table I). A delay of 1-2 hours is allowed before the animals are added to allow time for a rise in pH to occur.

DapJmia. The species that are currently under monoxenic cultivation have been obtained from the following sources. Daphnia magna from the laboratory of Dr. Luigi Provasoli in bacteria-free continuous dixenic cultivation. D. pule.v (four strains), 1. Connecticut Valley Biological Supply Co., Southampton, Massachusetts, 2. General Biological Supply House, Chicago, Illinois, 3. Pine Swamp Pond. Connecticut (Lat. 41°53:63N., Long. 73°24:04W.), 4. Candlewood Lake, Connecticut (Lat. 41°2S:93N., Long. 73°27:63W.), D. catawba, Croton Reservoir, New York (Lat. 41°15:55N., Long. 73°50:54W.), D. pan'iila, Pocantico Lake, New York (Lat. 41°07:06N., Long. 73°50:02W.), D. rctrocurva, Bantam Lake, Connecticut (Lat. 41°42:66N., Long. 73°13:63W.), D. ambigua (two strains), 1. Croton Reservoir, New York, 2. Lake Giles, Pennsylvania (Lat. 41°22:54N., Long. 75°05?90W.), D. laevis, Pine Swamp Pond, Connecticut, D.

MONOXKXK CULTIVATION OF DAI'H XIDAK

323

Candlewood Lake, Connecticut, I>. i/alcatu mendotae (four strains), 1. Swan Lake, New York (Lat. 41 °0(>:62X., Long. 73°49:94W.), (two strains). 2. Candlewood Lake, Connecticut, 3. Croton Reservoir, Xe\v York. Simocephalus serrulatus, Upper Hadlock Pond, Maine (Lat. 44°19:i9N., Long. 68C1728\Y. ) . Scapholeberis umcronata. Swan Lake, New York. Ceriodaphnia rcticulata. Dark Lntry Forest Pond, Connecticut (Lat. 41°47:70X., Long. 7302K82\V. ), C. (jiuninuHjiila. Swan Lake, New York, Moina macrocofm auicricana. ephippeal egg> obtained commercially from John and Ruth Fenneberg, P.O. Kox 1043. Yictor- ville, California, M . inacrocopa auicricana variant, same as above.

Individuals to be isolated are rinsed once or twice and transferred to Falcon 60 >: 15 mm "Tissue Culture" petri dishes containing 10 ml of medium with 2 >: 10" cells/ml Chlamydomonas. Tentative identification is made at this time by stranding the animal on a clean microscope slide. Identifications have been made using Brooks' monograph (1957) for the genus /hif^lniici and Gouklen's monograph ( 1(K>S ) for the Moinidae.

TABLE II

The effect of antibiotics and />n>lri>i on egg dcvclopnicH/ of I), pule.x mnl surnrnl oj

	Deioni/ed di-tilled water	Water with* antibiotic-	Water with* protein	Water with* antibiotics and protein
Egg tested	19	9	24	32
X umber developing into young	18	4	24	26
Number stuck at interface	9	1	0	0
Survival of young for one day	(I	0	16	24
IVr cent survival for one day	()',	12',	67',	7V,

* Concentration same as in Basic Medium (Table I).

Bacteria-free animals were obtained by the following modifications of the methods of Stuart, McPherson and Cooper (1931 ) and of Obreshkove and Fraser (1940). As soon as the progenitrix of the strain to be isolated shows well- developed, eyed embryos, it is transferred six or more times through large droplets of sterile medium (Table I) in the hydrophobia top of the plastic petri dish and left for one hour in a dish with 10 ml of medium. It is then rinsed six more times and the brood pouch of the carapace is opened with sterile needles. Care is taken not to express the gut contents during this procedure but doing so does not necessarily mean failure in obtaining bacteria-free young. The embryos in groups of 1-3 are rinsed twelve or more times in droplets of sterile medium on petri dish tops and placed in sterile medium. The important part of this final washing is to use a new sterile capillary pipette for each transfer step and to carry over with the animals as little medium as possible. This procedure usually yields bacteria- free young. If an original culture is especially heavily contaminated the number of rinses may be doubled. Eggs a few hours old may be used as they will develop and hatch but they can be easily damaged by the washing step.

Moina inacrocopa must be handled differently. The brood sac is closed in this species and the eggs rupture on release. However, satisfactory results are obtained by washing the young immediately after they are released by the mother.

324

JAMES S. MURPHY

Bacteriologically sterile animals are transferred twice weekly into new petri dishes with sterile medium and algae and maintained in constant temperature boxes at either 15° C or 20° C constantly illuminated with two 15 watt cool white fluorescent Imlbs. They may also he kept in screwtop Falcon 30 nil "Tissue Culture Khisks." Sterility was routinely checked using thioglycollate medium. Anaerobes were ruled out both by darklield and phase microscopic examination and by (he culture method of Schaedler, Dubos and Costcllo. ( 1(>(>5 ).

RESULTS Requirement }or cinln'vonic development

Bacteriologically sterile eggs from D. pitle-v, obtained from Connecticut Valley Biological Supply Co., completed embryonic development as well in deionized distilled water as in salt solution. When the young became motile they developed

TABLE III

I'ln- clj'ci I nj I'liriaus sails tested singly and in pairs on siirriral of I ). pulex for more than three days (four animals per test)

	NaCl	KC1	CaCh	MgSO4	PO4 buffer
XaCl	(1	0	4	(1	0
KC1	(1	0	4	0	0
CaCl2	4	4	4	4	4
MgSO4	0	0	4	3 (weak)	(1
PO4 buffer	0	0	4	0	0

The salts were dissolved in (.It-ionized distilled water with bovine albumin, penicillin and streptomycin as in Basic Medium (Table I) with Chlamydomonas and Xccncdcsmus and XaCl 0.004 M, CaCh 0.001 .17. MgSO4 0.0005 M", KC1 0.0001 ,V, Ma phosphate buffer (PH 6.9) 0.002 .V.

a tendency to stick to the air-water interface. This difficult}- was solved by the addition of bovine albumin, fraction V. Table II shows that 0.2 grams per liter accomplished the purpose and that penicillin and streptomycin may be added to the medium to reduce the probability of bacterial contamination.

It had previously been observed that penicillin at 1000 units/ml and that streptomycin at 20 mg/liter were not toxic.

Requirement for juvenile development

The above medium did not support the young even though they were led a mixture of washed Chlamydomonas rcinhardii and Scenedesmus obliqnits whereas a salt mixture allowed continued development. Table III shows that calcium was the necessary factor, for without it, the D. pnle.v died in the next ecdysis. While several calcium salts were tried, calcium acetate was chosen because it is neutral and improves the appearance of the Chlamydomonas. Dilutions lower than 10~4 molar did not support young of D. pnle.r as can be seen from Table IV. Other salts are brought into the medium with the antibiotics and the algae protoplasm.

MONOXENIC CULTIVATION OF DAPHNIDAE

325

TABLE IV

Development from eggs and s it IT i nil for one week of voting of I), pule.x /;; various concentrations of calcium acetate

		Concentration of calcium acetate
		10-6 _u	10-s .u	10-4 M	10-3 u
X umber tested	11	13	14	13	14
Xumber developing	3	10	7	10	10
X umber surviving one week	0	0	0	i	8

Medium consists of bovine albumin, penicillin, and streptomycin as in Basic Medium (Table 1 ) with Chlamydomonas and .Scenedesunts added.

Requirement for fertility

Animals raised to maturity from eggs in the medium as developed to this point, if maintained bacteriologically sterile, survived but produced infertile eggs. These "unproductive" animals constituted the test subjects for the following series of experiments. Table V shows that vitamin B12 increased their fertility. However,

TABLE Y Number of young produced with and without vitamin RI->

	Number females tested	Viable young befoi. Biz 1st clutch	Viable young after 812 2nd clutch
Control without Bjo With Bis	20 19	0 0	10 53

Medium consists of calcium acetate, bovine albumin, penicillin, streptomycin as in Basic Medium (Table I) with Chlamydomonas and Scencdesiuus. Bi2 was added immediately after the first clutch.

the effect was temporary and the animals became '"unproductive" again. In another experiment shown in Table VI, calcium pantothenate (Fritsch, 1953) was used and

TABLE VI

Number of viable young and undeveloped eggs produced with and without calcium pantothenate (20 ing/liter)

		Clutch before pantothenate	Clutch after pantothenate
	Xumber
Medium*	females
	tested	Viable	Undeveloped	Viable	Undeveloped
		young	eggs	young	eggs
Control without Ca
pantothenate	1 1	2	69	5	40 +
With Ca pantothenate	f>	1	42	36	0

* Medium consists of calcium acetate, bovine albumin, penicillin, streptomycin, Bi2, as in Basic Medium (Table I), with Chlamydomonas and Scenedesiints. Calcium pantothenate was added immediately alter the third clutch.

326

I. \.\IKS S. ML'RPHY

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Concentration of calcium pantothenate,mg/liter

FidruK 1. Average number of young per clutch in various concentrations of calcium pantothenate (summary of over 400 clutches).

caused a very striking increase in the number of viable young. Raising the amount of vitamin B1L. 10 or 100 times did not alter the results but leaving it out of the medium produced a sharp reduction in litespan and in the number of young pro- duced. Calcium pantothenate on the other hand had a definite optimal concentration for production of young (see Fig. 1 ), but its presence or absence had no effect on the lifespan which averaged 41 davs from the da}' embryological development began. The animals averaged twelve clutches of eggs apiece regardless of the presence or absence of added pantothenate.

. -llf/ac requirement

A final experiment in this series was done to find out if it was necessary to add both algae to these cultures. Washed eggs of I), pulc.v were started in basic

TABLE VII Effect o/ Chlamydornonas mid Scenedesmus on 1). pulex in basic nicdiuin

	Xo. females	Average number of young
Clutch 1	Clutch 2	Clutch .?
Clilaniydomonas alone Both al^.u-	15 15 15	6.4 (1 8.4	9.9 11.1	14 1.9 18.5

MONOXEXIC CULTIVATION OF UAPHNIDAE

medium (Table I) and fed Chlamydomonas alone, Scencdcsnius alone, or a com- bination of the two. Table YTI shows that cultures with both algae were superior to either alone.

Results Ti'/Y// other species

The above basic medium (Table 1) permitted us to establish four species of Ihiplniiu in bacteria-free continuous cultivation with two algae and another species with one algae. Hy the time this medium was improved (Table I), we had carried D. pu/e.r (Connecticut Valley) 84 generations, D. pulc.v (General Bio- logical) 10 generations, Scapholeberis unicronata 63 generations, Simocephalus serrulatus 40 generations. I>. mat/iia 17 generations, and with Chlamydomonas alone, Moinu niaerocopa 135 generations. On several occasions these cultures

TABLE VI 1 1

.1 ;'<•;•«£<• number of young on the first brood in three media

	D. piiU:\ Connecti- cut Yallt-y	D. pnlr\ General Biological	Moina iiiacrocopa*	Scaphole- beris mucronala	D. iini^iiii	Simo- cephalits serrulatus	D. gali-iila mi-ndotae
( 1 . ) Basic medium with
t\vo ali;, ic (2.i Enriched medium	4.0 (21 )	4.4 (38)	13.3 (80)	2.0 (40)	5.8 (17)	3.2 (20)	0
\vitli i wo alyae earh lornuila	4.6 (33)	5.6 (52)	15.0 (116)	2.7 (84)	8.8 (18)	4.0 (52)	2.7 (25)
' i. ) Enriched medium
with Clila in ydomonas
alone, later formula	5.2 (69)	6.0 (76)	17.7 (175)	2.6 (46) 10.1 (51)	3.7 (24)	2.2 (16)
(4. ) Repeat of number S above	5.8 (28)	6.4 (44)	10.6 (112)	2.8 (41 )	10.0 (20)	4.4 (30)	3.1 (38)

* .\foinn macrocopa was always cnltun-d with Chlamydomonas alone.

Rows 1, 2 and 3 were run simultaneously. The numbers in parenthesis are the total brood- considered in tlie adjacent average.

became contaminated and were immediately reestablished in bacteria-free culture bv isolating and washing embryos or newborn young. When yeast or fungus occurred as contaminants, daily transfer for a few days was usually all that was necessary to leave it behind.

Man}' other species of Ihiphnia were tried in this medium both with and without bacteria with little success. A clone of D. i/aleata uietidotac (Swan Lake) became established in a .'. dilution but only when bacteria and a protozoan (Anisonema] were present. Passages had to include sufficient old medium to quickly re-establish the contaminants. This indicated that it was likely that the medium was deficient rather than toxic. However, no single factor could be tound which would improve the medium enough to support the species D. (jalcata mendotae so we resorted to "shot gun" methods.

Enriched medium, early formula

Immediate success in cultivating D. t/aleafa ineinJatae on algae alone followed adding to the medium a mixture of eight vitamins and Hutner's Trace Elements

JAMES S. MURPHY

(Table I). Table \ III, under the heading "Enriched medium, early formula," demonstrates the effect this medium had on six species of Daphnia. It can be seen that Scapholcberis may not have clone as well but all the others improved as mea- sured by the number of young in their first clutch. Other parameters also showed improvement. They matured slightly sooner, and D. magna particularly in- creased markedly in size and vigor. When tried on other species of Daphnia the medium was unsatisfactory. It would support D. ambigua and D. rctrocnrva but only when the medium was contaminated with bacteria. It did not support Ccriodaphnia reticulata, C. quadramjula, D. parrula, D. cataivba, and D. dubia even when bacteria were present.

Enriched -medium, later formula

At this point a large number of media modifications were tested on the produc- tion of young by D. rctrocurra, D. galcata mcndotac and Simocephahts scrrulatus under unsterile conditions using Chlamydomonas without Scenedeswius. These preliminary experiments indicated that the medium might be improved by increasing the amounts of choline, pyridoxal, inositol, nicotinamide and riboflavin and leaving out Scenedcsmns. The formulation (Table I ) was tested on our seven strains already in bacteria-free serial passage with the type of results shown under "Enriched medium, with Chlamydomonas alone, later formula" (Table VIII). The final row shows the results six months later when we had less difficulty with insecticide. It can be seen that each medium modification causes a definite improve- ment in most strains.

When this final formulation was tried on a wide variety of species of Daphnia it was found to support continuous monoxenic cultivation of eight more. No species of the Daphnidae yet tested has failed to reproduce and become established in the presence or absence of bacteria. All the species develop from isolated eggs and are readily obtained bacteria-free. Fourteen species are now under continuous monoxenic cultivation and doing well. They are listed below in order of increasing difficulty found in establishing and maintaining them in the laboratory. The last two have not been tested on anything but the later formula medium with Chlamydomonas alone.

Current status of strains under cultivation

Mount macrocof>o (two strains) is by far the easiest species to cultivate. It will withstand 0.05 molar salt concentrations, heavy bacterial contamination, and is more resistant to insecticides. It has been maintained bacteria-free for over 200 generations.

Scapholeberis mucronata is also very resistant to insecticides and extremely hardy. The species is small, and while the adults are easy to see because they are deeply pigmented, the newborn young are nearly invisible to the naked eye. It has been maintained for over 200 generations.

D. pulex (four strains) is easy to culture in the laboratory and one strain has been maintained bacteria-free for over 100 generations.

D. magna, although widely used in research, is not by any means as easy to culture as the above. It is also hard to obtain. We have never found it in nature,

MONOXENIC CULTIVATION OF DAPHNIDAE

329

and although the biological supply houses advertise it, they actually supply D. pnlc.v. It is sensitive to insecticide. We have maintained it through 35 and 30 generations. The species was lost once due to insecticide and replenished from a sealed manure- water and algae culture that had supported a small population fur <me year.

Siiiioccplialus scrnilafns is very common in local ponds hut compares with D. inagna in difficulty of maintenance. As soon as the species is obtained bacteria- free it develops the remarkable open-carapace deformity described by Agar ( 1(>13 ). \\'e have not succeeded in preventing this abnormality, but it does not seem to affect their survival and reproduction. It has been maintained for over SO generations.

D. galcata mendoiae is common and easy to maintain in complex medium. Under our conditions either helmeted or unhelmeted animals isolated from nature develop moderately helmeted progeny. It has been maintained for over 30 generations and several strains are under cultivation.

TABLE IX

Comparison of number of young and lifexf>an of Moina macrocopa in iii'o media

		X<>. youii'4	Life-span
	X<>. aiiiiii.il	Average	Range	Average (days)	Range
Basic medium	37	48.7	7-97	10.6	7-15
	63	34.3	4-88	10.4	3-14
Enriched medium
later formula	111	88.4	17-181	11.8	3-20

D. rctrocurva is fairly easy to obtain but more difficult to maintain. All the newborn young of some clones immediately become stuck on the air-water interface and must be sunk daily. They remain moderately helmeted and are most sensi- tive to insecticide. "We have maintained it for 18 generations bacteria free.

D. ambigna is a small species, common and easy to cultivate in the more complex medium. It is sensitive to insecticides. It has been maintained for over 40 generations and repeatedly isolated.

D. parvnla is similar to D. anihi/jua but requires the later formula medium. It also has been maintained for over 40 generations.

D. diibia is not very common in our experience and has the same characteristics as D. retrocurva. We currently have a strain that has gone well for 15 generations and does not get stuck at the interface.

D. catan'ba is fairly common. We have maintained it for 13 generations but it did not adapt easily to our culture conditions. It is very sensitive to insecticides.

Ceriodaphnia quadrangula compares with D. auibigna and pamila. It is harder to remove eggs from Ceriodaphnia females because of their almost spherical body shape. Ceriodaphnia from Swan Lake have large single-spiked fornices in nature and resemble C. lacnstris. but these spikes are lost on cultivation. The spikes are similar to those described by Rzoska (1956) and Zaret (1969). After 8 generations this species was lost due to insecticide and a new isolate has gone 19 more.

330 JAMKS S. MURPHY

D. lae-ris was only obtained bv us recently, having hatched from ephippeal eggs and has gone through 13 generations. It is a large vigorous animal and probably is among the easier to maintain. Krooks ( 1957) has identified this as "Banta's l>. longispina" (page IIS) and it is therefore a well known experimental species.

C. reticulata was obtained onlv recently but has been easy to maintain for 14 generations. As with ('. quadrangula, the eggs are hard to remove from the female. Older females are preferable because thev are much larger than primigravida and have many more eggs.

Effect of medium enrichment on Moina

The effect of the change in medium on the lifespan and total number of young produced by Moina macrocopa is shown in Table IX. The later formula medium produces a marked improvement in both parameters particularly if the extremes are considered. Of the 1 11 animals in the experiment in enriched medium, twenty five or 22% had lifespans of over 15 days. The three experiments reported were done at different times but smaller numbers tested simultaneously show the same general result. We have seen no tendency for the clone of Moina to improve with time alone when the medium is unchanged.

DISCUSSION

The general approach used in this study has been to alternate between modifying the medium to improve the growth of one species and testing the modification on as large a number of species as were currently available. This two-pronged method has the advantage of improving the medium for the species under study and finding new species of Daphnia that will grow in it. Working on numerous types has also allowed us to find better species for a given experiment. The best animal to use for testing a modification seems to be one that will survive in unsterile culture, but which, when monoxenic, will not produce more than an occasional fertile egg. If the correct modification is made, this animal will produce viable young almost immediately. In contrast, a species that dies out yields little information and presents problems of obtaining enough animals, while one that produces a few young mav, in a better medium, merely produce slightly more young, a less critical endpoint. The assumption underlying this approach is that what is good for one animal is good for another. So far, this has been true in all cases except a clone of Scapholeberis which became slightly less productive in the more complex medium. However, it was not necessary to make up a special medium to support this species.

The problem of laboratory contamination with insecticide is most troublesome. If a species is brought into the laboratory and will not survive, the question always arises whether the culture medium is deficient or whether the medium is toxic. Any species that is more sensitive to insecticide than usual will simply seem harder to maintain if the environment is slightly contaminated. The presence of chronic or low level insecticide poisoning is almost impossible to evaluate until sensitivity studies have been made with all the species under cultivation. It is safe to say that all the species reported here have been repeatedly subjected to

MONOXENIC CULTIVATION OF DAPHNIDAE 331

insecticide and Moina and Scapliolchcris in particular have repeatedly survived levels that have killed off most of the others.

The increase in lifespan and production of young of Moina in the enriched medium is remarkable since the strain already was highly productive relative to other species. Even further improvement seems possible since the spread of values for individual animals is very large and the medium has never been modified by direct experimentation with Moina. That a relative deficiency in vitamins should have an effect on the production of eggs and young is not surprising but it is not easy to understand why lifespan should be so markedly affected. Vitamin deficiency in higher animals is known to produce specific disease syndromes and may produce death, but an effect on lifespan has not been recognized. There is suggestive evidence that aged humans are "less resistant than the young to the ill effects of restriction of B complex vitamins" (Horwitt, Liebert, Kreisler and Wittman, 1948, page 106) but that is all.

On the other hand, variation in lifespan with diet is a common occurrence in experiments with various species of the Daphnidae. It may be that the biosynthetic mechanism leading toward egg production is capable of drawing so heavily on the reserves of the animal that upon completion of a clutch of eggs, the animal is thrown into pathological deficiency. It is possible that the nutritional state of the parent will also influence the longevity of the young.

The powerful effect vitamins have on the rate of reproduction of Cladocera and other Crustacea ( Provasoli and Shiraishi, 1959; Shiraishi and Provasoli, 1959; Provasoli and D'Agostino, 1969) may be important in the understanding and con- trol of lake ecology. The evidence of the varying requirements of different species of Daphnia may help explain their distribution in nature.

I wish to thank Dr. Luigi Provasoli for his most valuable advice and for providing some of the organisms used in this study. I am much indebted to Mrs. Nancy Michael, Mrs. Margot Butler, and Miss Marjorie Oftinger for technical assistance and to Mrs. Victoria Murphy. Miss \Yendy Murphy and Miss Carol Murphy for help in the field.

SUMMARY

'Fourteen species of the family Daphnidae have been established under continuous monoxenic cultivation utilizing Chlamydomonas rcinliardii as sole food organism in a medium consisting of calcium acetate, antibiotics, albumin, trace elements and the water soluble vitamins, folic acid, B^, calcium pantothenate, choline, pyridoxal, inositol. thiamin, nicotinamide, riboflavin, biotin and putrescine. The Daphnidae under cultivation include Daplinia iiiat/na, D. f>itlc.\\ D. galcata mendotae, D. laez'is, D. dubia, D. rctrocnrra, D. parrula, D. ainbigita. D. catawba, Moina tnacro- copa, Scaplwleberis inucronata, Simocephalus scrrulatus. Ceriodaphnia reticulata. and C. quadrangula. The requirements for vitamins for some species are more complex than for others. The complete medium is superior for all but Scapholeberis inucronata and markedly increases the lifespan and fertility of Moina macrocopa.

332 JAMES S. MURPHY

LITERATURE CITED

AGAR, W. E., 1913. Transmission of environmental effects from parent to offspring in

Simoccplialus rctnlus. Phil. Trans. K'oy. Soc. London, Series B., 203 : 319-350. BANTA, A. M., 1921. A convenient culture medium for daphnids. Science, 53: 557-558. BANTA, A. M., 1939. Studies on the physiology, genetics, and evolution of some Cladocera.

Carnegie Inst. Wash., Paper No. 39 : 1-285.

BEERSTECHER, E., JK., 1952. The nutrition of Crustacea. l^itamins Hormones, 10: 69-77. BROOKS, T. L., 1957. The systematics of North American Daphnia. Mem. Conn. Acad. Arts

Sci., 13: 1-180. DEWEY, J. E., AND B. L. PARKER, 1964. Mass rearing of Daphnia magna for insecticide

bioassay. /. Econ. Entomol.. 57 : 821-825. FRANK, P. W., C. O. BOLL AND R. W. KELLY, 1957. Vital statistics of laboratory cultures of

Daphnia pulc.r DeGeer as related to density. Physiol. Zool., 30 : 287-305. FRITSCH, R. H., 1953. Die lebensdauer von Daphnia spec, bei verschiedener ernahrung,

besonders bei zugabe von pantothensaure. Z. Wiss. Zool., 157 : 35-56. GOULDEN, C. E., 1968. The systematics and evolution of the Moinidae. Trans. Amcr. Phil.

Soc., New Series 58 : 1-101. HORWITT, M. K., E. LIEBERT, O. KREisLER AND P. WiTTMAN, 1948. Investigations of human

requirements for B-complex vitamins. Bull. Nat. Res. Council, 116 : 1-106. HUTNER, S. H., L. PROVASOLI, A. SCHATZ AND C. P. HASKINS, 1950. Some approaches to

the study of the role of metals in the metabolism of microorganisms. Proc. Amer.

Phil. Soc. ,94: 152-170. LEVINE, R. P., AND W. T. EBERSOLD, 1958. The relation of calcium and magnesium to crossing

over in Chlamydomonas reinliardi. Z. Vcrcrlimigsl., 89 : 631-635. MORTIMER, C. H., 1936. Experimentelle und cytologische untersuchungen iiber den genera -

tionswechsel der Cladoceren. Zool. Jalirb. Abt. Allg. Zool. Pliysiol., 56: 323-388. MURACHI, S., AND T. IMAI, 1954. Studies on the culture of water fleas, Moina macrocopa

Straus, in artificial culture medium. Tohoku J. Agric. Res., 1 : 27-63. NEEDHAM, J. G., P. S. GALTSOFF, F. E. LUTZ AND P. S. WELCH, 1937. Culture Methods for

Invertebrate Animals. Comstock Pub. Co., Ithaca. [Reprinted 1959, Dover Public,

New York.] OBRESHKOVE, V., AND A. W. FRASER, 1940. Growth and differentiation of Daphnia magna

eggs in vitro. Biol. Bull., 78 : 428-436. PROVASOLI, L., AND A. D'AGOSTINO, 1969. Development of artificial media for Artcmia

salina. Biol. Bull., 136 : 434-453. PROVASOLI, L., AND K. SHIRAISHI, 1959. Axenic cultivation of the brine shrimp Artcmia

salina. Biol. Bull., 117 : 347-355. RZOSKA, J., 1956. On the variability and status of the Cladocera Ceriodaphnia cornuta and

Ceriodaphnia rigaudi. Ann. Mag. Nat. Hist., Series 12, 9 : 505-510. SASA, T., R. KUNIEDA AND H. TAMIYA, 1960. Growing Daphnia (water-fleas) with

Chlorclla. J. Gen. Appl. Microbiol., 6 : 252-255. SCHAEDLER, R. W., R. DuBOs AND R. CosTELLO, 1965. The development of the bacterial

flora in the gastrointestinal tract of mice. /. Ex p. Med., 122 : 59-66. SHIRAISHI, K., AND L. PROVASOLI, 1959. Growth factors as supplements to inadequate algal

foods for Tigriopus japonicus. Tohoku, J. Agric. Res., 10 : 89-96. STUART, C. A., M. MCPHERSON AND H. J. COOPER, 1931. Studies on bacteriologically sterile

Moina macrocopa and their food requirements. Pliysiol. Zool., 4 : 87-100. TAUB, F. B., AND A. M. DOLLAR, 1968. The nutritional inadequacy of Chlorella and

Chlamydomonas as food for Daphnia pule.v. Limnol. Occanog., 13 : 607-617. TREILLARD, M., 1924. Sur 1'elevage en culture pure d'un crustace cladocere: Daphnia magna.

C. R. Seanc. Acad. Sci., Paris, 179 : 1090-1092. WATANABE, A., R. ITO AND T. SASA, 1955. Micro-algae as a source of nutrients for daphnids.

/. Gen. Appl. Microbiol., 1 : 137-141. ZARET, T. M., 1969. Predation-balanced polymorphism of Ceriodaphnia cornuta Sars.

Limnol. Oceanog.. 14: 301-303.

Reference: Biol. Bull.. 139: 333-350. (October, 1970)

REPAIR AND REATTACHMENT IN THE BALANIDAE AS RELATED TO THEIR CEMENTING MECHANISM

J. R. SAROYAN, E. LINDNER AND C. A. DOOLEY Mare Island Naval Shipyard, Vallejo, California 94592

An investigation of the attachment of sessile Cirripedia was initiated to aid future studies on fouling problems. The primary objective was the collection of the barnacle adhesive secretion, generally called the cement, for analysis and tests. The observation that detached or injured barnacles produce a cement-like secretion directed the course of the investigation into a study of the anatomy and functions of the cementing organs to determine the nature of this healing secretion.

The young barnacle begins to grow soon after permanent attachment and meta- morphosis have been completed. As is characteristic of the phylum Arthropoda, the body of the barnacle grows in well-defined periods distinguished by the moulting of the exoskeleton. The shell of the barnacle also undergoes cyclic growth (Dar- win, 1854) as evidenced by striations on various parts of the shell : the basis, the opercular valves and the conical portion of the shell composed of plates, for which the term "shell wall" is used here, for simplicity. Of particular interest are the striations on the basis, which form concentric circles and permanently record the basis perimeters of previous growth cycles. Darwin (1854) believed that the periods of shell growth are closely related to the moulting cycles. Our histological studies on the development of the cement ducts (Saroyan, Lindner and Dooley, 1968) seem to support the correlation between growth of the basis and moulting. This study shows that the cement duct network is part of a cuticular membrane and is secreted by epithelium cells. At each moulting, this membrane and the duct network are also moulted but become trapped inside the shell. Since each duct network leads to the basis perimeter at that particular time, the relationship between moulting and growth of the basis is strongly indicated. Costlow and Bookhout (1953, 1956, 1957) and Costlow (1956, 1959), conducted extensive studies on the shell growth of Balanus anipliitrite and B. improvisus] and the elaborate experiments designed particularly to explore the connection between shell growth and moulting indicated no direct correlation. They showed that the shell grows continually, though erratically and independently from the moult- ing (Costlow and Bookhout, 1953, 1956; Costlow 1956, 1959). These findings were based on the outside measurements of the basal perimeter, which also partially include the thickness of the wall plates at the basal edge. Since the perimeter of the basis is overlapped by the edges of the wall, the growth rate of the basis and its relation to the moulting period is not entirely understood.

During growth the leading edges of the calcareous shell grow outward ; thus, the basis and the basal margin of the shell walls become larger in diameter and the shell walls are pushed upward. The shell walls and the basis are tightly connected by numerous muscle tissues around the basal perimeter at the inside joints of the

333

334 J. R. SARD VAN, K. I.INDXKK AND C. A. DOOLKY

leading edges. The contraction of these muscles |>ull the hasis upwards and at the same time the shell wall downwards. Since the hasis is usually firmly cemented to the substratum, the wall is pulled down and presses the basal edge of the shell wall tightly down on the substratum. The shell wall rests upon the growing edge of the hasis, which is a thin, flexible membrane of chitin. regardless of whether or not the species under examination has a calcareous hasis ( Costlow, 1956; Newman, Zullo. and \Yainwright. Il>n7). Consequently, this thin flexible rim of the basis is also pressed very tightly to the substratum leaving only a very slight gap. Due to this peripheral pressure, the basis may grow into the recessions and over the irregularities of a solid substratum, faithfully duplicating its surface structure (Pilsbry, 19H>; Gregg, 194S) and obtaining maximum contact area for adhesion. The forces produced by the growing barnacle enable it to plow away loose deposits, fouling organisms, and detritus or to dig beneath soft materials, such as clay or certain coatings (Barenfanger, 1939; Harris, 1946) in order to reach an underlying solid surface. The barnacle cements itself to the substratum by an adhesive which is secreted at the perimeter of the basis and spreads under it to fill any gap between basis and substratum (Darwin, 1854). Due to the peripheral pressure exerted by the barnacle, the gap to be filled and, therefore, the thickness of the cement layer, is normally less than five microns.

In crowded communities, however, the barnacles may develop abnormally. For example, in such communities Balanus halanoidcs grows into elongated shapes, and Darwin (1854, page 147) observed that sometimes only the shell walls of such specimens reach the substratum, while the noncalcareous basal membrane remains suspended and deeply concave. Darwin found that "thickish roots" were hanging from the basal membrane in the resulting gap. He believed these roots to be cement.

This development form apparently escaped the interest of later investigators since only a few references can be found in the literature on this subject. Crisp (1960) describes specimens which survived complete upward displacement by neighboring barnacles but does not mention any adhesive secretion. Crisp also found that specimens of Balanus balanoides have some limited mobility under lateral pressure by neighboring barnacles and can be moved along the surface of a smooth substratum several centimeters away from the original point of attach- ment. He speculated that the advancing edges form new adhesions as the barnacle gradually undergoes lateral displacement. In a recent article (Newman, Zullo, and Wainwright, 1967, page 170) there is a reference to some unpublished observations, "that Balanus(B) a. ainphitritc Darwin if carefully removed from the substratum without noticeably damaging the wall or calcareous basis, could reattach itself to glass slides, by first cementing and then calcifying itself in place. This is accomplished by protrusion of portions of the mantle from the small spaces that normally occur along the seam between the wall plates and the basis." This short reference, however, neither takes the cementing apparatus into consideration nor goes into detailed explanation of the mechanism of the reattachment process.

The basic anatomy of the cementing apparatus of the Balanidae was described by Darwin (1854). Darwin describes a series of complicated duct networks in a number of species, including Balanus tinfinnabiilnm. Each network originates from a pair of pear-shaped vesicles and after much branching and occasionally

CEMENTING MECHANISM OF BARNACLES 335

rejoining the ducts lead to and terminate in orifices around the basal perimeter or, as in the case of older networks, around the concentric circle \vhich marked the earlier growth cycle. The vesicles are also connected by a channel and form a pair of chains resembling strung beads. Darwin believed that the cementing apparatus is a modified part of the reproductive system and that these vesicles are the remains of degenerated cement glands. Not much later, Krohn (1859) recognized the cementing apparatus as a separate organ, and expressed his doubt about the vesicles being secretory glands. Although some additional studies (Pagenstecher, 1863; Gruvel, 1905; and Thomas, 1944) contributed somewhat to the concept of the cementing apparatus of the Cirripedia. no cement glands of any species of the Balanidae were described until quite recently. Lacombe (1966, 1967, 1968) described the cement gland cells and the cementing apparatus of Balainis tintinnabnlinn, although the description of the duct network deviates somewhat from the accurate description of that of the same species by Darwin (1854) and does not mention the vesicles or their function. \Yhile Lacombe indicates that each separate duct network of B. tintinnabulum develops its own cement gland cells, suggesting a temporary glandular function, in a previous paper (Saroyan, Lindner, and Dooley, 1968), we demonstrated that the gland cells in several other Balanus species develop from the cyprid cement glands independently of the duct network; and the same glands function throughout the whole life of the adult barnacle. YVe also showed that the cement gland cells, although largely inter- twined with the ovarian tissues, are more abundant at the lateral area of the mantle, around the end of the two main channels, which rise from the basis into the mantle tissue in close proximity to the lateral scutal depressor muscle. We demon- strated that the cementing apparatus of these Balanus species is basically identical to that of the Lepadidae, the other family of Cirripedia, namely in having periodically functioning permanent glands on both sides of the mantle ; cement glands consisting of smaller units, which are connected by ducts at a node, where the chemical properties of the secretion are altered ; and only one pair of main channels, which conduct the secretion from the node toward the initial attachment point, where the remains of the cyprid antennules can be found. In the Balanidae, a pair of vesicles form around the basal portion of the main channel in each growing period, thus the main channel runs through the vesicles as a continuous tube (Fig. 20). The cement enters the vesicle through the permeable walls of the main channel portion contained within the vesicle. Under normal conditions, the cement does not follow the main channel beyond the newest vesicle toward the initial attachment point and does not reach the older vesicles. At the newest vesicle, which is usually the closest to the basis perimeter, the cement changes its course to find its way through the duct network to the perimeter. As we showed through the histology of their development, the vesicles do not have any secretory function ; their purpose is mainly the distribution of the cement into the ducts.

We also found (Saroyan, Lindner, and Dooley, unpublished) that the vesicles may be capable of a pumping action ind therefore may be responsible for the transportation of the cement. This pumping action is based upon the barnacle's capability of increasing the pressure inside the mantle cavity by contracting the depressor muscles. As the closed valves depress, they also compress the seawater in the mantle cavity. This pressure increase of the water is transmitted through

336 J. R. SAROYAN, E. LINDNER AND C. A. DOOLEY

the resilient walls of the vesicle to its contents as the vesicle is compressed. The soft, flexible walls of the main channel inside the vesicle collapse from the pressure and shut off the passage back in the main channel. The content of the vesicles therefore is forced into the ducts. \Yhen the pressure in the mantle cavity is released, the resilient vesicle regains its original shape and volume, drawing more fluid in. By size considerations, there is less resistancy in the main channel than in the ducts, therefore the additional fluid will be drawn into the vesicle from the main channel rather than back from the ducts. With such repeated cycles, the secretion can be forced through the duct network quite rapidly with the vesicle serving as a combination of distributing chamber, pump, and checkvalve.

METHODS

Attached adult Balau/ts crcnatus, B. glandula. and B. cariosus from Point Reyes, California, and B. iin pro-visits from Mare Island Strait. California, were collected on Plexiglas and rubber panels in natural environments. Specimens were removed intact from these panels and successfully reattached on glass microscope slides in the laboratory.

Adult barnacles of Balanus crcnatiis, B. glandula, and B. improvisus were also collected on glass microslides in natural environments for microscopic study of the basis and attachment. The specimens were killed, fixed, decalcified, and stained on the original slides. Ten per cent neutral buffered formalin by Lillie (1954) (Hearse, 1961). Baker's (1944) formol-calcium ('Hearse, 1961), and Zenker's fixative (Gray, 1954) were used for fixation. Specimens fixed in Baker's formol-calcium for 24 hours, decalcified in Jenkin's fluid (Hearse, 1961) for 24 hours, and refixed in Baker's formol-calcium for 48 hours provided the most satisfactory results.

For studying barnacles in microtome sections, solid paraffin blocks were exposed at the fouling sites. Barnacles attached to the paraffin blocks were allowed to reach sizes up to 6 mm in diameter before they were fixed, then decalcified while still attached to the paraffin substratum. During the later steps of dehydration, the substratum was dissolved to leave the specimen with all substances between its basis and the substratum intact for embedding and sectioning. These specimens were embedded in Paraplast and cut into 10-micron sections.

For general histology, Mallory's Trichrome was adapted but some additional staining techniques, such as Eosin-Aniline blue and HAS. were also used. For the Mallory Trichrome technique, a shortening of the usual staining and rinsing times (Gray, 1954) was necessary to obtain good results with arthropod tissues. This technique consisted of immersing the preparation for one minute in a one per cent Acid Fuchsin solution, one minute in one per cent phosphotungstic acid, and two minutes in a solution containing two per cent Orange G, two per cent oxalic acid, and ().? per cent Aniline blue WS. The Acid Fuchsin and the phosphotungstic acid solutions each were followed by a one minute rinse, and the final solution by a five minute rinse in distilled water. The preparation was then dipped briefly in 95 per cent alcohol (15 sec) and then put through two absolute alcohol rinses of three minutes each. Two five minute rinses in two changes of xylene preceded the final mounting in Fermount.

CEMENTING MECHANISM OF BARNACLES 337

RESULTS

During the collection of large specimens of Balanus nitlnlns, one of the barnacles suffered a sizable crack in its basis. After 24 hours, an abundant white, opaque, rubbery exudate, or secondary secretion, \vas found which rilled and sealed this injury (Fig. 1). On numerous occasions it has been observed that barnacles sustain injury such as cracks or breaks in their calcareous bases as a result of the forces produced either by themselves or their neighbors. If vital organs are not seriously damaged or if the injury is not too extensive, the barnacle may survive such an accident by repairing the injuries with these secretions. Microscope preparations show that secretions around or in the cracks of the basis have staining characteristics similar to those of the adhesive cement of the barnacles (Figs. 2 and 3).

Secondary secretions were found not only in injured barnacles, but also in specimens of Balanus craiatits and B. glandnla that were partially or completely separated from the substratum. Some of these separations appear to have been caused by excessive force exerted by the barnacle itself in an effort to press the growing edges of the basis close to the substratum. The pressures created at the perimeter may result in lifting and detaching the central portion of the basis. Such bases become concave and the ensuing gap is usually filled with the secondary secretion (Fig. 4).

Microtome sections of these thick layers of secretions often have a cavernous and vertically striated appearance (Fig. 5). This effect is probably caused by the continuous recession of the basis from the substratum. The gap created between basis and substratum is filled with the fluid secretion, but before hardening can take place, the basis continues to recede. The already viscous secretion may then pull threads of material between the two surfaces, thus creating a loose structure. These new gaps are then filled with fresh secretions during the next period; and so the process is continued until the recession either ceases or continues at so rapid a rate that the secretory system is no longer able to supply enough material to fill the gap.

Another type of separation can occur in gregarious communities wrhere the shell walls of neighboring barnacles may be fused together. Since the shell walls grow up from their bases, a faster growing specimen may lift up and detach a slower growing one from the substratum (Fig. 6). The space between the elevated barnacle and the substratum is usually then filled by the opaque secretion. If the gap is too large to be filled, any secretion present may be seen hanging from the basis, indicating that an effort was made by the barnacle to reach the lost sub- stratum to reattach. The rubbery secretion often covers the whole basis in several millimeter thick layers (Fig. 7) concealing the fine structure of the calcareous basal surface. This fine structure exhibiting the well distinguished cement duct network can be seen on the basis of carefully detached specimens such as the ones used in our reattachment experiments (Fig. 8). These barnacles were removed intact from smooth test panels and subsequently reattached to other smooth surfaces, such as glass microscope slides. Successful reattachments wrere observed in specimens regardless of whether they were totally submersed in seawater or suspended in such a way that only the opercular region was suhmersed with the

338

J. R. SAROYAN, E. LINDNER AND C. A. DOOLEY

FIG. 3

FIG. 6

FIGURE 1. Large specimen of Balanus inthilus species suffered a crack in its basis. Within 24 hours, the secretion seen here hardened and repaired the crack.

FIGURE 2. Dark, stained cement in vicinity of (A) circular crack in basis of attached adult barnacle, near (B) initial attachment patch ( Wholemount, Masson-Patay, 40 X).

FIGURE 3. Section through basis of attached adult Balanus crcnatns showing crack in basis filled with cement ( K)-M section, Mallory's Trichrome, 180 X ).

FIGURE 4. Abnormal concave basis on Balanus creuatns specimen detached from solid substratum in the laboratory shows abundant secretion, which represents an attempt by the barnacle to fill the central gap between basis and substratum.

CEMENTING MECHANISM OF BARNACLES 339

basis and the new substratum out of contact with the seawater. The reattach- ments were accomplished by the white opaque substance secreted on the basal surface and spread between basis and substratum (Fig. 9). The closer the contact of these two surfaces, and hence the thinner the secretion layer, the firmer the reattachment appears to be. The reattachment can be so strong that the shell walls and body will break away before the basis can be detached. If the intervening space between basis and substratum is too large to be filled, thick droplets of secretion appear and hang from the basis (Fig. 10). These reattached specimens •can be kept alive indefinitely with proper care and seem to develop normally.

Assuming that the secretion is cement, it could be expected that the secretion would originate from the perimeter of the basis where the newly developed cement duct orifices are located. In general, the secretion does appear at the perimeter, from which it then spreads under the separated or injured area, but centers of secretions can be detected inside the perimeter also (Figs. 9 and 10). In addition, occasionally, an extensive separation or injury occurs isolated within the perimeter and the secretion from the perimeter cannot reach the affected areas in sufficient quantities. Indications are that the barnacle is able to grow new, irregular cement ducts into such a damaged area (Fig. 11). These emergency or secondary cement ducts are larger in diameter than the normal ducts and have few or no bifurcations. Such a duct extends directly from the newest formed vesicle to the damaged area where it ends in an orifice. Normally, only the first vesicle formed after metamorphosis would have such a duct leading from it, as with each succeeding growth period, the duct system becomes increasingly complex in the number of branchings before the final orifices are reached.

The secondary cement ducts usually can he found only in those detached or injured areas where no old primary ducts and orifices can be found as, for example, near the cyprid attachment where the first available duct ends are outside the perimeter of the innermost circle (Fig. 12). There is probably a unique mechanism that enables the barnacle to recognize the need for new ducts and to initiate the growth of these unusual ducts. Likewise, the dissolution of the calcareous matter to permit the growth of new ducts into old sites must somehow occur ; similar processes are known in nature.

In the majority of cases, however, where detachment involves basis areas inside the basis perimeter, the reattaching secretion seems to originate from old primary cement duct ends (Fig. 13). This reuse of old ducts is demonstrated in whole- mounts of the basis where several layers of additional secretion lie around the old duct ends (Fig. 14). The layered appearance of the secretion indicates that there was enough time for one layer to harden before the next one was laid down. Since in the course of normal development, the edge of the growing basis is pressed tightly on the substratum and the cement is spread in a very thin layer, these thicker layers of secretion must have appeared subsequent to the growth period during which the primary secretion took place. Therefore, we consider these thicker layers to be secondary secretions or secondary cement.

FIGURE 5. Microtome section through Balanits crenatus basis area showing three layers of striated secretions (W-fj, section, Mallory's Trichrome, 220 X).

FIGURE 6. Cluster of Balanits crenatus barnacles. Several faster growing specimens have lifted up a slower growing one, which continued to thrive in its elevated position.

J. R. SAROYAN, E. LINDNER AND C. A. DOOLEY

'

FIG. 7

FIG. 10

B

FIG. 12

FIGURE 7. Abundant mass of secretions on basis of Balanns crcnatiis specimen that was naturally detached by other barnacles.

FIGURE 8. Basis of Balaints crenatus after removal from smooth test panel in the laboratory.

FIGURE 9. Basis of specimen in Figure 8 after it reattached to a glass microscope slide in the laboratory.

FIGURE 10. Reattached Bulamts crcinitits specimen. Side A is firmly reattached to a glass microscope slide, while side B is left suspended. Drops of secretion hang from the basis on the side where the barnacle was unable to reach and adhere to the substratum.

CEMENTING MECHANISM OF BARNACLES 341

The quantity of this secondary cement varies according to the size of the animal and the extent of the detachment. It is difficult to estimate the volume of the secondary cement because of its loose, cavernous structure. The volume of the denser primary cement also varies widely according to such factors as the size, age, and growth rate of the barnacle, and the surface characteristics of the substratum. The size and behavior of the very young adult, however, is more uniform. The first adult cement is secreted around the basis perimeter of the first order adult, which metamorphoses from the cyprid. The first order cement ducts are 170 microns long on the average and often not more than two microns in diameter in Balanus crenatus. The cement is spread around the perimeter of the basis of the young adult forming an elliptical ring with the following average measurements :

outside semiaxes/4 == 3(10~2) cm B- 4(10-2) cm

inside semiaxes a == 2.5(10'-) cm &==3.5(10-2) cm thickness d -- 5(1Q-4) cm

The average volume of the cement secreted by the first order adult Balanus crenatus is:

V -- TT (AB-ab)d •- -- 5(1Q-7) cm3

DISCUSSION

It was shown that in most cases an injured or detached barnacle uses the old cement duct system for repair or reattachment. This reuse of old ducts is possible only if the duct system connected to these areas is still functional and the passages are still open. Since the ducts were once filled with primary cement, it would seem that when the cement hardens, the ducts would be plugged with solidified material and thereby rendered useless for subsequent secretion. Darwin (1854), however, found that in Balanus tintinnalnduui the ducts were free of cement. In Balanus crenatus, B. glandula and B. mibilus we found that this was only partially true. We often observed in these species a few cement ducts and vesicles full of hardened cement among the majority of empty ducts. (Fig. 15). It is impossible to offer any positive explanation for the cement-free status of the ducts without a thorough understanding of the hardening mechanism of the barnacle cement. At this time, however, the available information about the chemistry of the barnacle cement is very limited, therefore, we can resort only to theorizations.

Based on fluid mechanical considerations, it can be demonstrated that perhaps the most important physical requirement for the cement at the time of the secretion is low viscosity. According to the Poiseuille law, the pressure drop of a fluid flowing through a duct is directly proportional to the viscosity and the velocity of

FIGURE 11. New, irregular duct growing directly from a (A) vesicle into a (B) damaged area (Wholemount, TriPARS, 180 X).

FIGURE 12. Growth of new, irregularly-shaped ducts originating from (A) vesicles into the (B) damaged initial attachment area (Wholemount, TriPARS, 70 X).

342

J. R. SAROYAN, E. LINDXER AND C. A. DOOLEY

FIG. 13

FIG. 16

FIG. 14

FIG. 17

FIG. 15

FIG. 18

FIGURE 13. Abundant secondary secretions lie around (A) old duct ends, which now contain hardened cement, at the site of a partial detachment of the basis. Newer (B) ducts, which were unaffected by the accident, are empty (\Yholemount, Mallory's Trichrome, 70 X).

FIGURE 14. Old duct ends with new, thick circular layers of cement around them (Whole- mount, TriPARS, 180 x).

FIGURE 15. Microtome section through basis area of Balaims crciiatns that was attached to paraffin shows: (A) Thick irregular layers of cement secretions, (B) empty ducts, (C) duct containing cement, and (I)) substratum ( 10-,u section, Mallory's Trichrome, 180 X).

CEMENTLM; MECHANISM OF BARNACLES 343

the fluid, and the length of the duct and inversely proportional to the fourth power of the radius of the duct.

A/; =

Trr

where A/> == difference in pressure at the duct ends, r? == absolute viscosity of fluid, v ---- volume of fluid delivered per unit time, / = = length of duct, and r -- radius of duct.

It is not yet known how long it takes for the barnacle to secrete the cement, but in a series of yet unpublished experiments, we found already hardened cement within 15 minutes after reattachment. For the sake of the present consideration, permit us to assume that the time interval for the secretion is about one tenth of the hardening time, or t -- 100 sec. This would mean that the velocity of the secretion or the volume of cement delivered in a second by one of the first order ducts is :

V

7

v -- -- 2.5(10-9) cm3 sec-1

I

therefore, A/> *-- lO6^ (dyne cnr-, if -,/ is in poises).

In our example, the numerical value of the additional pressure in dyne cm"2 required to deliver the cement at the arbitrarily selected rate through the narrow ducts is about one million times the viscosity of the cement expressed in poises.

If the cement would have a low viscosity near that of water (/-^ 10~2 poise), the additional pressure would be only: A/> -- 104 dyne cnr2 (or 0.145 psi). But, if the cement had a viscosity more like glycerol or castor oil (-' 10 poise), A/> =: 10r dyne crrr2 (or 145 psi).

These considerations strongly suggest that initially the cement is a fluid of low viscosity which solidifies rapidly after secretion. Preliminary analysis of the hardened cement (Lindner and Dooley, 1969) indicates that the cement consists of mainly organic material and is probably a highly cross-linked polymer. The hardening mechanism of the cement therefore is very likely to be due to a polymerization of monomers (Saroyan, Lindner. Dooley and Bleile, 1970). To

FIGURE 16. The cement precursors are secreted by large, unicellular glands. As more of these glands develop with growth of the barnacle, they are joined to the others by collecting channels, indicated by the arrow, which transport the secretion from each individual gland to the node. The content of the collecting channels does not stain with Mallory's Trichrome (10 fj. section, Mallory's Trichrome, 180 X).

FIGURE 17. The arrow indicates the node where the polymerization is believed to be initiated, because from this point on the channels stain red with Mallory. The red coloration seems to diffuse into the channel from the cytoplasm of the cells composing the node (10 fj. section, Mallory's Trichrome, 180 X ) .

FIGURE 18. The activated cement, which stains intensely red with Mallory, then passes on through the main channel, shown by the arrow, which carries the cement from its poly- merization initiation point to sites of its further distribution (10 n section, Mallory's Trichrome, 180 X).

344

J. R. SAROYAN, E. LINDNER AND C. A. DOOLEY

FIG. 19

FIG. 20

T*v

FIG. 21

FIGURE 19. Perimeter of an inner basis circle with (A) empty duct and (B) funnel- shaped orifice leading to the perimeter. The flushing process, following the cement secretion, has washed both the duct area and (C) a circular ring around the edge of the basis free of (D) cement before hardening took place (Wholemount, Mallory's Trichrome, 220 X).

CEMENTING MECHANISM OF BARNACLES 345

understand the cementing mechanism it would lit- nf importance to know exactly how and where this polymerization is initiated.

There are indications that the cement polymerization is initiated after the secretion has left the cement glands and before it enters the duct network. Those observations (Saroyan, Lindner, and Dooley, 1968) that substances with staining characteristics similar to that of the hardened cement can be found in the main channel, but never in the secretion collecting side channels, and that the chemical characteristics of the secretion appear to be changed abruptly at the joining node of the collecting channels and the main channel suggest polymerization initiation at the node ( Figs. 16, 17, and 18).

Initiation from the environment, namely the seawater, can be ruled out, since it was demonstrated that detached barnacles are also capable of reattachment with their bases and the new substratum out of the seawater. Occasional ducts or vesicles containing hardened cement also make the role of environmental factors questionable, since it is difficult to see how substances from outside could flow back against the cement current through the narrow ducts all the way to the vesicles and mix with the cement to harden it. 'For the same reason, secretions from the shell walls or elsewhere in the organism, but outside the ducts, ion exchange or pH change outside the orifices are unlikely to play an important role in cement hardening.

Initiation inside the ducts, for instance through the duct walls would not offer sufficient basis to explain the presence of cement-like substances further up in the main channel. Also, portions of secretion passing through shorter or longer ducts would be in contact with the initiating surfaces for a shorter or longer time, thus resulting in unevenly activated cement. It is therefore more likely that the polym- erization initiation is restricted to a small portion of the conduits. The node, where the secretion from the scattered cement glands merges into one main channel on each side, is a seemingly ideal site for this.

Using this initiation mechanism, the cement is capable of hardening inside the ducts or vesicles. In order to explain why the majority of the ducts are free of hardened cement, WTC have to consider some mechanism capable of removing the activated secretion from the ducts either after or before the hardening of the cement is completed.

After the hardening of the cement is completed, the most probable method of its removal would be an enzymatic dissolution or reflnidization of the cement, followed by the retention of the altered cement or its absorption through the duct walls. In either case, it is expected that new ducts would be found still containing hardened cement and no cement would be found in the older ducts, since the cement should have been removed from the latter long before. However, we found

FIGURE 20. (A) Vesicles with (B) main channel passing through them. (C) Collapsed main channel portion acting as a checkvalve to prevent vesicle content from backing up. (Wholemounr, Gallego-Garcia, 220 X).

FIGURE 21. Section through basis of an adult Balamts crenatns showing (A) the vesicles and (B) the main channel which passes through them (\2-p. section, Mallory's Trichrome, 200 X ).

346 J. R. SAROYAN, E. LINDNER AND C. A. DOOLEY

that all the cement-containing ducts have been older ones, originating in an earlier growing period. Therefore, there is little evidence to support this theory of removal.

Before the hardening is completed, the already activated cement, while it is still in the early stages of polymerization, can he flushed out and replaced by some nonhardening fluid. The most likely flushing fluid may he the non-activated cement containing its monomers but lacking its catalysts, or deactivated cement containing polymerization inhibitors, or modified cement containing non-reacting derivatives of its monomers. The polymerization can be initiated in two general ways, either "spontaneously" or "externally." Spontaneous polymerization is due to an inherent internal property of the system, in which all the necessary optimum conditions are present. External initiation requires the alteration of an existing condition, such as influencing the energetics, reactivity, or charge distribution of the system, or by adding a polymerization agent or subtracting an inhibitor.

Indications are that the polymerization is initiated externally rather than spontaneously. As was pointed out, the hardening seems to be initiated at the node because no trace of substances with cement-like staining characteristics were found in the collecting side channels, which conduct the secretion from the individual gland cells to the node, but from there on, the main channel invariably contains cement-like traces (Saroyan, Lindner, and Dooley, 1968). The polymerization therefore does not seem to be an inherent property of the secretion of the cement gland cells, but rather externally induced at the node by the mixing of different types of secretions from different types of gland cells or from the node itself by any of the above mentioned mechanism.

In view of these considerations, the cementing process starts in the mantle at the cement gland cells with the secretion of the inactive cement precursor, probably a monomer mixture or solution of low viscosity. The secreted precursor is collected by side channels and directed into the node. Here the secretion becomes activated, possibly by mixing with other secreted substances. The activated mixture merges into the main channel which conducts it from the mantle along the lateral scutal depressor muscle to the basis, where it enters the newest vesicle. From this vesicle the cement is distributed throughout the newly developed duct network and orifices around the enlarged perimeter. The cementing period ends with the secretion of the flushing fluid which displaces the still liquid cement from the ducts. In the course of normal development, this non-hardening flushing sub- stance fills all the ducts, forces the cement out beyond the duct orifices and away from the edges of the basis. The cement hardens in this position under the cuticle of the joint connecting basis and shell walls. In this manner, a ten to forty micron wide circular channel is formed between the edge of the basis and the hardened cement, leaving the flushing fluid contained within this seal (Fig. 19).

After the normal cementing period the vesicle ceases to serve as a fluid trans- porting pump, in conjunction with the main channel, but still acts as an efficient checkvalve that prevents the flushing fluid from backing up and mixing with the new cement. Also, the structure of the vesicle enables it to control the flow of fluids and direct the new cement to where it is needed in the case of injury or detachment.

CEMENTING MECHANISM OF BARNACLES

347

A CEMENTING

, \ INITIAL \

ATTACHMENT \ \ \

SUBSTRATUM \\\ \\

B FLUSHING

\ \ \ \ \ \ FLUID

MAIN CHANNEL

SEPARATION

\\\\\\

V\\

FIGURE 22. A Secretion of cement from cement gland to newest vesicle and duct network during normal development; B. Flushing of duct network following cement secretion; C. Secretion of cement when a separation from substratum has occurred in the region of an old duct.

As was pointed out, the main channel goes through the vesicles as what appears to be a continuous tube rather than as a simple connection (Figs. 20 and 21). That portion of the main channel which is inside the vesicle is probably permeable, permitting transflux into the vesicle, but largely reducing convection and diffusion between the contents of the main channel and the vesicle. As long as the flushing fluid fills the ducts and vesicles, the system remains in balance and no liquid passes through the permeable walls. In the course of normal development, therefore, the new cement does not go beyond the newest vesicle because the rest of the main channel and duct network is filled with the flushing fluid and no room is available for the cement (Fig. 22A). The new cement can enter only the new vesicle and

-US j. R. SAROYAN, E. LINDNER AND C. A. DOOLEY

duct network to be secreted ;it the perimeter. The still liquid cement is displaced by the non-hardening flushing fluid at the end of the cementing period. The cement hardens outside the orifice and seals the flushing fluid inside the ducts (Fig.22B).

However, if the basis separates from the substratum, the cement seal of some duct ends breaks and the flushing fluid drains out of the corresponding ducts and vesicles. In the same fashion, a fracture in the basis would sever some ducts and the flushing fluid would also leak out at the injured site. Due to this leakage, the balance of the system is upset. The pressure then drops on the leaking side of the permeable walls, and the loss of flushing fluid makes room for newly secreted cement. The cement is now allowed to pass further down the main channel until it reaches the vesicle affected by the injury and to pass through the permeable membrane of the main channel. The cement enters the vesicle and into the ducts of the injured network and follows the draining flushing fluid to the site of the injury or detachment (Fig. 22C ) . Since the duct networks of different growing periods are completely isolated from each other except at the vesicles, which are connected only by the main channel, the flushing fluid drains only from duct ends which are affected by the injury and only from those ducts which represent the shortest route between the injury and the corresponding vesicle. Other ducts, unconnected with the injured area, remain filled with the flushing fluid and, hence, the cement bypasses those vesicles which serve the unaffected duct network.

Thus, the vesicle, in conjunction with the main channel not only distributes and transports the cement by means of a pumping action, but also controls and regulates the flow of cement and flushing fluid, as would a valve. The vesicles of different age are situated near each other and are connected by the main channel. This arrangement puts all the vesicles, the corresponding duct networks, and especially the duct ends — regardless of the network to which they belong — almost equidistant from the cement glands. Thus, it is practically as easy to secrete cement through older ducts as through the newest, peripheral duct system. After emergency use, the network is again flushed out and ready for further reuse. In repeated use, however, the flushing process is not always complete because usually larger and irregular amounts of cement are secreted at the emergency locations, thus plugging the orifices. Hence, those reused ducts and vesicles could contain hardened cement.

The authors wish to express their thanks to Dr. Edward Alpen of the Xaval Radiological Defense Laboratory for his advice and interest ; to Mrs. C. D. Kinney and Mr. Ray Krenik of NRDL, and to the Mare Island Photographic Section of San Francisco Bay Naval Shipyard for outstanding photographic work ; and to Mr. Cesar Clavell, Jr., for technical assistance.

SUMMARY

Barnacles, which become partially or totally detached from their substratum in a natural environment, produce a secondary cement secretion. Laboratory experiments demonstrate that the secondary cement can successfully reattach the barnacle to a new substratum. Similar secondary secretion was found at the site

CEMENTING MECHANISM OF BARNACLES 349

of minor injuries to the barnacle basis. The secondary cement usually has a looser, more cavernous structure than the primary cement, but both secretions have simi- lar staining- characteristics.

Microscope preparations indicate that occasionally barnacles are capable of developing new secondary cement ducts leading into the injured or detached areas to secrete secondary cement.

In most cases, however, the existing primary cement duct network is used for the secondary secretion. This is possible only because most of the once used ducts are not plugged by hardened cement, in spite of the fact that the cement can harden inside the ducts. Chemical analysis suggests that the cement is an organic biopolymer and indications are that the cement hardening is initiated inside the organism.

A unique flushing mechanism seems to be responsible for keeping the cement ducts open and ready for reuse. A nonhardening flushing fluid forces the still liquid cement out of the ducts. The cement hardens outside the duct openings sealing the flushing fluid inside the duct network. In case of detachment or injury, the cement seal breaks ; the flushing fluid drains out leaving the duct open for the secondary cement secretion.

The vesicles in conjunction with the main channel control the flow of the flushing fluid and the cement. The permeable wall of the main channel portion inside the vesicle reduces the convection and diffusion between the vesicle and the main channel, thus bypassing of vesicles and duct networks not affected by de- tachment is possible. The wall of the main channel inside the vesicle is also collapsible, thus acting as checkvalve when the vesicle is under pressure and allowing the cement to be pumped only into the ducts toward the secretory orifices.

LITERATURE CITED

BARENFAXGER, C., 1939. Biologische Factoren bei Unterwasser Anstrichen im Meer.

Angewandte Chcinic. 52: 72-75. COSTLOW, J. D., 1956. Shell development in Balaiuts improrisiis Darwin. /. Morphology,

99(2) : 359-415. COSTLOW, J. D., 1959. Effect of carbonic anhydrase inhibitors on shell development and

growth of Balanus improvisus Darwin. Physio!. Zoo/., 32 : 177-184. COSTLOW, J. D., AXTD C. G. BOOKHOUT, 1953. Moulting and Growth in Balanns improvisus.

Biol. Bull., 105: 420-433. COSTLOW, J. D., AND C. G. BOOKHOUT, 1956. Moulting and shell growth in Balaints amphitrite

nweus. Biol. Bull., 110: 107-116. COSTLOW, J. D., AND C. G. BOOKHOVT, 1957. Body growth versus shell growth in Balanus

improi'isus. Biol. Bui!., 113 : 224-232.

CRISP, D. J., 1960. Mobility of Barnacles. Xature, 188: 1208-1209. DARWIN, C., 1851. A Monograph nu the Subclass Cirripedia — The Lepadidae. Ray Society,

London, 400 pp. DARWIN, C., 1854. ./ Monograph on the Subclass Cirripedia — The Ralanidae. Ray Society,

London, 684 pp. GRAY, P., 1954. The Microtomist's l-onnitliiry and Guide. The Blakiston Co., Inc., New

York, 794 pp. GREGG, J. II., 1948. Replication of substrate detail by barnacles and some other marine

organisms. Biol. Bull.. 94 : 161-168.

GRUVEL, A., 1905. Monographic des Cirrhipedes on '/'heenstruees. Masson et. Cie. Paris, 472 pp. HARRIS, J. E., 1946. Report on antifouling research 1942-44. /. Iron Steel Institute, 154

297-333.

350 J. R. SAKOYAN, E. LINDNER AND C. A. DOOLEY

KKOII.N, A., 1859. Beobachtungen iibcr don Zementapparat und dio weiblichen Zeugungsorgane ciniger Cirripedien. Arch, .\\tturficsclt., XXV: 355-364.

I..UOMBE, D., 1966. Glandulas dc ciniento e seus canais em Balanus tintinnabulum (Cirripedia — Balanidae). f'uhlicacao lustitnto ilc Lcsqitisas da Marinha, \~ota tccnica, No. 32: 1-39.

I . M UNI UK, D., 1967. Histoquimica o histofotometria das glandulas cle cimento de Balanus tintinnabulum ( Balanidao — Cirripedia). Publicacao do fnstituto dc Pesquisas dn Mariiilia. No. Oil : 1-29.

LACOMBE, D., 1968. Histologia, histoquimica e ultra estructura das glandulas de ciniento e sou* canais em Haliinns tintinnabulum, Publicacao da Institute dc Pesquisas da Marinha, No. 017: 1-22.

LINDNER, E., AND C. A. DOOI.KY, 1969. Chemical Characterization of Barnacle Cement. I'n- pithl. I\cpt. San l:nui cisco Hay Naval Shipyard, Vallejo, California, Paint Lab. Report No. 69-3, AD856070. (Available from Administrator, Defense Documentation Center, Cameron Station, Alexandria, Virginia 22314.)

NEWMAN, W. A., V. A. ZULLO AND S. A. WAINWRIGHT, 1967. A critique on recent concepts on growth in Balanomorphia (Cirripedia, Thoracica). Crnstoccana 12(2) : 167-178.

PAGENSTECHER, A., 1863. Beitrag zur Anatomic und Entwicklungsgeschichten von Lcpas pectinata. Z. Wiss. Zoo!., 13 : 102.

PEARSE, A. G. E., 1961. Histochcmistry. Theoretical and Applied. Little, Brown, and Com- pany, Boston, 998 pp.

PILSBRY, H. A., 1916. The Sessile Barnacles (Cirripedia) Contained in the Collection of the U. S. National Museum. Includiin/ a Monof/rapli of the American Species. Govern- ment Printing Office, Washington, D. C., 365 pp.

SAROYAN, J. R., E. LINDNER AND C. A. DOOLEY, 1968. Attachment mechanism of barnacles : Fouling prevention, the study of the adhesion of calcareous types of attaching marine organisms. Lnpuhl. /\'ept. San Francisco Bay Naval Shipyard, Vallejo, California, Paint Lab. Report No. 68-1, AD829344. (Available from Administrator, Defense Documentation Center, Cameron Station, Alexandria, Virginia 22314.)

SAROYAN, J. R., E. LINDNER, C. A. DOOLEY AND H. R. BLEILE, 1970. Barnacle Cement: The key to second generation antifouling coatings. Industrial and Engineering Chemistry Product Research and Development, 9(2) : 122.

THOMAS, A. L, 1944. Tegumental glands in the cirripedia Thoracica. Quart. J. Microscop. Sci., 84 : 257-282.

Reference : Biol. Hull.. 139: 351-3o2. < ( )ctober, l"/() )

Till-: APPARENT WATER-PERMEABILITY OF CARCINUS MAENAS

(CRUSTACEA, BRACHYURA, PORTUNIDAE) AS A

FUNCTION OF SALINITY

RALPH I. SMITH 1

! >cpartnicnt of Zoo/ot/y and Ih>i'C Marine Lahonitory (Cullercoats} , Ciiircrsity of Newcastle upon Tyne, lini/lund, ('. K.

There is general evidence (Potts and Parry, 1964, ]>]>. 138-143) that the integumental water-permeability of crustaceans inhabiting fresh and brackish water is less than that of marine species, and the adaptive significance of this in reducing the osmotic work-load is obvious. But whether it is possible for an individual crustacean to alter its own water-permeability as an adaptive physiological response to an environmental change of salinity is not self-evident, and conflicting views have been published. Rudy (1967), using tritiated water (T^O) found adaptive differences in water-permeability among several different species of marine, brackish- water, and freshwater crustaceans, but stated on the basis of his data that the brackish-water, euryhaline, species Palaemonetes variant and Carcinus inocnas could not significantly alter their integumental water permeability. However, I was able to demonstrate on the basis of the uptake of D2O a significant reduction of water-permeability in response to lowered salinity in the very euryhaline crab Rhithropanopeus harrisi ( Smith, 1967 ), and expressed the opinion that Rudy's data on Care hi us did not rule out such a response in the latter crab, although Rudy was correct in that his data did not show a statistically significant reduction of permeabil- ity. Because Carcinus is such an extensively studied animal, it seemed worthwhile to re-examine it by the same D^O method used to demonstrate a water-permeability change in Rhithropanopeus and also in the polychaete worm \crcis dh'crsicolor (Smith. 1970), to provide a basis for comparing Carcinus with less well known species in other waters.

MATERIALS AND METHODS

Specimens of Carcinus of a wide range of sizes and of both sexes were collected in early June on the rocky marine shore near St. Mary's Island, Whitley Bay, Northumberland, in northeastern England. Soft or "paper-shelled" individuals were discarded, as were ovigerous females or crabs lacking one or more claws, or more than a couple of legs. Crabs were maintained at about 15° C at the Dove Marine Laboratory and were adapted for a week or longer in sea water ( S\Y » , and in 75%. 50%. 35% and 25% S\Y. in large plastic boxes provided with stones for shelter. Sea water in these experiments had a chloride concentration of 549 mM/\. Experiments were carried out at the Department of Zoology, University of New- castle upon Tyne ; the adapted crabs needed on a given day were brought to Newcastle in the morning and tested the same day at room temperature (18-19° C I.

1 Author's address: Department of /oniony, L'niversitv of California, Herkdry, California, 94720.

352

RALPH I. SMITH

Kxposure In 5e/< I )L.( ) in various dilutinns of S\V follou cd closely the nu'llmd used l>v Smith (1(H»7) lor Rhithropanopeus except that exposure' time was short- ened tn 15 iitinutes instead ol" 30, since C'arciiuis is considerably more permeable to I)L.O than the inrmer crab. Seawater was diluted bv the addition of local Newcastle pnndwater plus sufficient I )L,( ) ( (>() moles '/< } to yield 75, 50, 35 and 25% SW. Since the addition of the IXO to S\Y caused a dilution to 94% SW, crabs to be tested in this medium were placed in S\Y diluted to 94% SW with deionized water 2-3 hours before the tests. Crabs above 15 g in weight were tested iu a liter of the 5r/r D2O solutions, smaller crabs in 500 ml. At the end of each exposure the crab was removed, quickly dried in a cloth towel, and a few drops of blood drawn by puncture of the arthrodial membrane at a leg base. This

100

</5 90- £ 80

— 50

c

'c 40 g

| 30

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CO

02° Q

i i i i i

345 10

Wet weight in grams

20

75% SW

25-35%SW

J L

30 40 50

FIGURE 1. Curves relating DuO-uptake (as % saturation attained in 15 min ) to wet weight of Cardans plotted by method of least squares. The equations — Uptake = a (weight) (6 — are: 94% SW, Uptake = 57.85 /P"1 "r'L", n = 23; 75% SW, Uptake = 58.45 W<"^0757), »=11; 50% SW, Uptake = 52.07 //''-" I1L"", w = 12; 25-35% SW, Uptake = 48.37 W^'ww\ n = 22.

sample was immediately placed in the large end of a Pasteur pipette, the pipette closed with a small cork, and its tip sealed in a flame. These pipettes, containing samples of blood and medium, were laid on a slide warmer overnight at 50° C to distill the water from the samples into the tips, which projected several inches from the warmer and were cooled by the evaporation of water from a strip of facial tissue paper laid across them with its ends dipping into beakers of water. D^O contents of blood samples, controls, and the respective media were estimated by comparison with drops of standard solutions (0 to 5%, D2O ) in a pair of kerosene/bromobenzene density gradient columns as in Smith's (1967) study of Rhithropanopeus. The method is described in detail in Welsh, Smith, and Kammer (1968, pp. 184— 1S8 ). Values reported are the average of results from the two columns except in a lew instances when one subsample was lost or when too little distillate was obtained for two determinations. In a given day's work, half the crabs used had been adapted to and were tested in a higher salinity, half in a lower. This permitted more effective use of the density gradients and helped to randomize differences in time of adaptation and room temperature.

WATER-PERMEABILITY OF CARCINUS

353

Chloride determinations were made on separate samples from 50 of the nS crabs tested, taken after tbe !)•_.< ) samples by means of disposable capillar\- pipettes ( Drummond "microcaps"), discharged into 1 ml of deioni/ed \\-ater before addition of tbe acid reagent used with the Aminco-Cotlove electrometric chloride titrator.

DL.O-uptake values were recorded as the per cent of the concentration of DoO in the medium attained in the blood in 1 5 minutes, taking the D2O content of the medium as 100%. Since D^O uptake is weight-specific (Smith. 1967), these "% saturation" values (corrected for controls] were treated following the equation, S -- uU'(b~l> in which S -- % saturation at 15 minutes, //" = wet weight in grams, a -- intercept on the ordinate at unit weight, and (/>— 1 i = = slope in double log plot. The calculation of (a) and (/'—I ) \\-as by the method of least squares. For statistical treatment and the calculation of hourly water exchange fractions (K), the values of % saturation were corrected to that of 10 g animals, using the mean (/' — I ) value of all groups. A weight of 10 g was used rather than unit weight

TABLE I

Apparent water-permeability of Cnrcinus as indicated by per cent D^O-saturation in

15 win, with the hourly water exchange fractions (K) and probability (t-test)

that differences are significant. Values adjusted to body weight of 10 g

% sw	n	Mean % sat.	Standard deviation	Standard error	K	P
94	23	44.64	±5.48	1.14	2.36	> 0.05 0.01 < 0.025 > 0.01
75	11	49.54	±5.62	1.69	2.73
50	12	40.44	±4.29	1.24	2.07
25-35	22	35.62	±5.32	1.13	1.76

because 10 g was near the mean weight of the animals used, and so reduced possible errors of extrapolation. The hourly water exchange fraction (K) is given by the equation, K - - (2.3/M Log10 ( 100/1 00-% Sat. ), in which K - - per cent ol body water exchanged per hour (assuming all water is exchangeable), t -- time of exposure to DL»O in hours, % Sat. == c/c concentration of DoO in water of blood at 15 min, referred to external D2O concentration as 100%. The data from 25 and 35% SW, being indistinguishable, were pooled and are shown in Figures 1 and 2 as from 30% SW.

RESULTS

The influx of DL.O into Carciuns is relatively greater in smaller individuals. Curves showing the % saturation of D2O in the blood as a function of weight after a 15-minute exposure to media containing D2O are drawn in Figure 1. The slopes of the curves vary from —0.0757 to —0.1316 averaging —0.1088.

The adjustment of individual % saturation values to a body weight of 10 g provides the data shown in Table I and plotted in Figure 2. The specimens of Carcinns used in these experiments showed the greatest uptake of D^O in 75% SW, with a slight reduction ( P > 0.05 i in (>4' '< SW. and a significant reduction of

354

RALPH I. SMITH

uptake in 50% and 30% SYV (/' ca. 0.01). Calculation of the hourly water exchange fractions ( l\ ) at each salinity yields values of 2.73 in 75% SW and a low value of 1 .7(> in 30/4 S\Y. Like Rhithropanopeus, Carcinns reduces its water exchange or its apparent permeability to water at low salinities.

The chloride concentrations of the blood of Carcinns in the different salinities of these experiments are shown in Figure 3 and in Table Til, line 2. Chloride is strongly hyper-regulated at lower salinities, is isotonic with that of the medium at about 80% SW, and is hypo-regulated in S\Y. By computations which are explained in the Discussion, the net diffusional (osmotic) influx of water into Carcinns at each salinity was calculated (this involved certain assumptions as to

70

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1 std. dev.

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I

i 1

I

I

10 25 50 75

Medium, Percent of Seawater

100

FIGURE 2. Per cent D^O-saturation attained by Carcinits in 15 min as a function of salinity, corrected to a body weight of 10 g (see text). The values in 94% SW are not significantly lower than those in 75% SW (P>0.05), although higher (P<0.05> than those in 50% SW.

the osmotic pressure of the blood) and the results are shown in Table III, line 11. The calculated net water influxes in 50% and 75% SW correspond almost exactly to the volumes of urine produced by Core in us at these salinities, as measured by Shaw (1961) and Binns (1969), shown on lines 13 and 14 of Table III. The calculated net influxes at any salinity are very much higher than those reported by Rudy (1967), but it will be shown in the Discussion that this discrepancy can be largely although not wholly eliminated if the sizes of animals and the tempera- tures are allowed for. The method of calculating net water influx provides for no net influx of water into Carcinns in SW, since the animal is presumed to be iso- osmotic with its medium ; hence the present calculated net influxes and those of Rudy fail to account for the production of urine by Carcinns in SW, as observed by Shaw and by Binns. Likewise, neither the present results nor those of Rudy after

WATER-PERMEABILITY OF CARCINUS

355

30

Medium, Percent of Seawater

50 75

-0.4

200

100

200 300 400

Cl~ of medium in mM/L

500

FIGURE 3. Chloride concentration of blood of Carcinus (left ordinate) as a function of chloride concentration of medium (bottom, Cl in mJ//l ; top, % S\V). The upper broken curve shows assumed osmotic concentration of blood (right ordinate), as used for calculations in text and Table III. Figures below blocks are numbers of animals sampled.

adjustment for size and temperature (Table III, lines 11 and 12) account fully for the large production of urine in 40% SYY measured by Shaw and by Binns (Table III, lines 13 and 14).

DISCUSSION

In discussing the reduced water exchange exhibited by Carcinus and Rhithro- panopcus (Smith, 1967) at low salinities, it should be made clear that an effect and not a mechanism is being described. It is possible that the observed effect is the result of a reduction in the permeability of the integument to water, but in this discussion it is appropriate to use the term "apparent permeability" because no estimate of permeable surface area can be given, and because the effect upon D2O exchange rate could as well result from a reduction in circulation of blood or irriga- tion of the gills as from a reduction in cuticular or epidermal water-permeability.

The small reduction in D2O exchange rate seen in 94% S\Y is significant only between the 5% and 10% levels of probability, but such an effect is physiologically reasonable and may be expected in certain crustaceans, namely those which show hypo-osmotic regulation at higher salinities. Carcinus has not been shown to be a

356 RALPH I. SMITH

hypo-osmotic regulator, but it may be noted (Fig. 3) that the crabs used in the present study hypo-regulated chloride in S\Y, and were isotonic in res]>ect to chloride in about K0f/ S\\ . Although a measure ol osmotic concentration would be more revealing than chloride concentration, this rinding, together with the possibly lower apparent permeability in 94% SW, suggests that water-permeability lowering might be looked for under both hyper- and hypo-saline conditions in crabs which are normally exposed to such extremes in nature. Hypo-osmotic regulation has been reported in a number of brackish- water crabs, including some with terrestrial tendencies (Jones, 1941; Gross, 1964), although Carcimts is reported to be iso-osmotic or slightly hyperosmotic in SW (Robertson, 1960, and others). Rudy (1967) found that Carchnis in his study showed chloride-isotonicity in SW. A clue to the discrepancy between my chloride values and those of Rudy may exist in the finding by Ballard and Abbott (1969) that Callincctcs. a fully aquatic crab of the same family as Carcinus (Portunidae), is hyper-osmotic in SW at 23-24° C, but hypo-osmotic in SW at 28-30° C. Rudy's specimens of Carcinus were adapted and tested at 10° C, mine were adapted at ca. 15° C and tested at 18° C, hence it is possible that his animals were hyper-osmotic, mine iso-osmotic, in S\V. In future studies of Carcinus the relationship between adaptational tem- perature and osmotic concentration of the blood should be critically examined.

In order to compare the present results on Carcinus with those of Smith (1967) on the very small Rhithropanopcus and with those of Rudy (1967) on larger specimens of Carcinus, the earlier data have been recalculated for a body weight of 10 g. In recalculating Rudy's results, the mean (/>— 1 ) value of —0.1088 obtained in the present study has been used. The K values so determined are shown in Table II. Care inns in the present study shows higher water exchange fractions (K) than does Rhithropanopeus; the A"'s for the latter (based on D2O uptake) averaging 39^ those of Carcinus as indicated by the same isotope. This lower water exchange fraction is consistent with the conclusion that the more euryhaline Rhithropanopciis has a lower permeability to water than does Carcinus. When Rudy's data for Carcinus (based on T2O influx at 10° C) are corrected for a weight of 10 g, the K values (Table II) are so far below those of the present study that they have simply been doubled (assuming a QIC of 2) to give values that can be compared with mine obtained at 18-19° C. The Q10 of 2 is close to the mean value of 1.90 found by Evans (1969) for water influx in fish. But even with this temperature correction, Rudy's A' values for Carcinus average only 80% those of the present study.

Are the present data on Carcinus (based on D2O ) any more or less reliable than the data of Rudy based on T^O ? Correction of the latter values by reason- able assumptions about weight and temperature leaves a 2Q% difference in the hourly water exchange fractions, with the A' values based on D2O higher than those based on T2O. It may be significant that A' values for Palaemonetes rarians, obtained by Rudy (1967) using ToO, average only 50^ of the values obtained on this same species by Parry ( 1955) using D2O (Table II ). Sufficient data are not available to permit corrections for weight and temperature but, as Rudy remarks, such corrections would probably not account for the whole of the large discrepancy. Obviously needed are water exchange values based on simultaneous T2O and D2O uptake measurements on the same species in order to decide whether the A' values

WATER-PERMEABILITY OF CARCINUS

35;

TAHI i- I I

Comparisons of hourly water exchange fractions ( l\ ) /nisei/ mi />•,(> an/1 !'•,() niellioils:

Rhithropanopeus harrisi, for weights of I g and Id g, recalculated front Smith (19f>7);

Carcinns maenas/or weight of Id g; Palaemonetes varians of unspecified

weights. See text for rationale of weight and temperature corrections

Animal	Rhithropanopeus hm-i i\i	( ',1/vniHA null' ltd.': (10 yl	Palaemonetes variant (unspcr. weight)
(lg)	(K) g)
Isotope	T2O	D2O	T.,O	D2O	T2O	D2O	T-.O	D2O
Reference	Smith unpub.	Smith 1967	Smith unpub.	Smith 1967	Recalculated from Rudy, 1967	This paper	Rudy 1967	Parry 1955
Temp. °C	18-20°	18-20°	18-20°	18-20°	10°	20°	18-19°	10°	?
120% S\V								0.64	1.61
94-100' ,' S\Y		1.55		0.99	0.98	1.96	2.36
70-75'; S\Y	1.19	1.46	0.71	0.94	0.97	1.94	2.73	0.64	0.95
40-50', S\Y		1.27		0.85	0.90	1.80	2.07
25-35', S\Y		1.05		0.70			1.76
10', S\Y	0.75	1.00	0.47	0.67				0.55
5'c' S\V		0.98		0.65					1.31
\% SYY		0.89		0.00
K (T20)	0.78	0.73	—	0.80	0.50
K (DSO)

yielded 1>y T^O and DL.O represent an isotope effect or result from individual differences in method. Lacking such a simultaneous experiment with the two isotopes of water, the nearest that can he cited is an unpublished experiment (1968) in which 1 repeated the 1967 experiments with Rhithropanopeus, using T2O. \Yithout going into detail except that counts were on equal volumes of blood and medium added directly to the scintillation fluid, the results confirmed the change of apparent permeability with salinity in Rhithropanopeus, and yielded A' values averaging 73-78^ of the K values obtained with D^O (Table II ). This general agreement in the ratio of water exchange values independently arrived at with ToO and DX) in the separate experiments on Rhithropanopeus, Carcinus, and Palaemonetes suggests possibly the presence of an isotope effect causing a faster uptake of D^O than of TL.( ). I Imvever, the magnitude of this effect cannot be precisely stated because the recalculation of Rudy's data on Carcinus has involved a chain of assumptions.

RALPH I. SMITH

Despite possible- isotope effects, there is no reason to suppose that relative water-permeabilities are not lairlv represented in comparative studies using either [)L,() or TjO. Rudy's values for the relative water-permeability of the series of crustaceans he studied using TL.( ) seem valid, and the relative differences in the apparent water-permeability of Carcinus as a function of salinitv on the basis of D2O in the present study likewise appear valid. It is suggested, however, that the water influx indicated by D^>O may be somewhat closer to the actual water influx of the animal. In order to test the reasonableness of the water exchange (K) values obtained on Carcinus, calculations from these values of net diffusional (osmotic) water influx have been made for comparison with reported values of urine production.

Rudy (1967) calculated the water fluxes of Carcinus on the basis of TL,O- exchange fractions (A"), and on the assumptions that the osmolarity of SW is 1.0, that the water content of Carcinus is 70%, and that the osmolarity of its blood can be represented as % SW based on chloride concentration (in effect, as twice the blood chloride molarity). In the following recalculations I have, in general, followed Rudy's method to facilitate comparison, but have approached the prob- lem of blood osmolarity with somewhat different assumptions. This has been necessary because Carcinus in my experiments showed hypotonicity of chloride in SW (Fig. 3 ) and approximate isotonicity of chloride in 80% SW, whereas Rudy found the chloride concentration of the blood of Carcinus in SW to be equal to that of the medium.

Since SW in my experiments had an average chloride concentration of 549 mM/\, its chlorosity is close to 19.1 (/lt (Barnes, 1954) and its freezing point is -1.88° C ( Pantin, 1946), corresponding to an osmolarity of 1.01 osmoles. Rudy's assumption of a 1.0 osmolar concentration of SW is thus reasonable, but to apply the same method to blood is not advisable. Nagel (1934) reported that the chloride concentration of the blood of Carcinus (as XaCl ) accounted for only 88% of the observed freezing-point depression. Lacking a direct measure of osmotic concentration in the animals used in the present study, I have recalculated the osmotic concentration of the blood of Carcinus in 94% SW as equivalent to 460 X (100/88) := 523 m.17 XaCl, an increase of 63 mM/1 over the chloride concentra- tion of 460 mM/1. The chloride concentration of the blood at lower salinities has then been arbitrarily raised by the same absolute amount, and the osmolarity expressed as % SW (Fig. 3 and Table III, lines 3 and 4). The osmotic concen- tration of the blood of Carcinus in 100% SW has been assumed to be equal to that of SW. By the use of such osmotic concentration values, net water influxes have been calculated by the following steps (data in Tables II and III) : e.g., in 30% SW, the mole fraction of water in medium == 55. 56/(55. 56 + 0.30 ) ;= 0.9946 (line 5); the mole fraction of water in blood == 55. 56/(55. 56 + 0.63 ) "0.9888 (line 6) ; the mole fraction difference :: 0.0058 (line 7). This difference accounts for the net water influx, which is equal to 0.0058/0.9946 = 0.58% of the total daily influx (line 10). Assuming a 70% by weight water content in Carcinus and that all water is exchangeable, the total daily water influx -- K X 70% X 24h = 2957% of body weight in water exchanged per day (line 9 ) . AY/ influx is 0.0058 X 2957 : 17.2% of body weight per day (line 11 ). The results of this and similar calcula- tions are shown in Table III, together with the urine volumes of Carcinus as

WATER-PERMEABILITY OF CARCINUS

359

TABI.K III

Calculation of daily net water influxes in Carcinus at different salinities (line 11) for com- parison with urine volume estimates of Shaw, 1961 (line 13) and Binns, 1969 (line 14). The net water influx values in line 12 are recalculated from data of Rudy (1967), adjusted for a body weight of 10 # and 20° C. See text for method of computation

1. Medium, ' ,' sea water	30	50	75	94	100 •
2. Chloride concentration of blood, mA/"/l	282	359	427	too	—
3. Estimated osmotic concen- tration of blood as ni.l/ XaCl 1	345	422	490	523
4. Adjusted osmotic concen- tration of blood as ' \ S\Y	63	77	89	95.3	assume 100
5. Mole fraction water of medium	0.9946	0.9911	0.9867	0.9834	0.9823
6. Mole fraction water of blood	0.9888	0.9863	0.9842	0.9831	0.9823
7. Mole fraction difference	0.0058	0.0048	0.0025	0.0003	0.0000
8. Hourly water exchange fraction ( K)	1.76	2.07	2.73	2.36	assume 2.36
9. Daily water influx (K X 70 X 24) as ' , body weight per day	2957	3478	4586	3965	3965
10. Daily net water influx as ' , of total influx	0.58	0.48	0.25	0.03	nil
11. Daily net influx as r( body weight (present data), tested at 18° C	17.2	16.7	11.5	1.19	nil
12. Daily net influx as % body weight (Rudy, 1967), re- calculated for 20° C	16.8 (40'; S\Y)	—	9.9	—	nil
13. Daily urine volume as % body weight (Shaw, 1961), 16° C	31.3 (40';; SW)	16.5	1 1.1	— •	3.6
14. Daily urine volume as % body weight (Binns, 1969), 9° C	21.1 (4o(; s\Vj	16.9	10.8		4.4

measured by Shaw (1961 ) at ca. 16° C (line 13) and by Binns (1969) at 9° C (line 14), and net water influxes recalculated from Rudy's data (1967) adjusted to a weight of 10 g and 20° C (line 12) .

It does not appear feasible to make a meaningful estimate of the urine volume of Carcinus as a function of temperature. If the osmotic concentration of the blood

360 RALPH I. SMITH

of Cairinns behaves as does that of Cullincctcs ( Ballard and Abbott, 1969), being inversely related to temperature, then a lowering of water exchange (K) at low temperatures might he to an unknown degree counterbalanced by an increased osmotic gradient favoring a higher net water influx and greater urine volume. Obviously needed are determinations of the osmotic concentrations of the blood at the temperature of the experiment rather than or in addition to chloride determina- tions, and direct determinations of urine output as a function of temperature.

The net water influxes and urine volumes shown in Table III support the hypothesis that diffusional (osmotic) entry of water accounts for the urine produc- tion of Carcinns at intermediate salinities (50-70% SW). But if there is no osmotic gradient. Care in us in SW must utilize some form of isotonic water trans- port. Such transport, in the absence of an osmotic gradient and by the expenditure of metabolic energy, is well known in vertebrates, and several possible mechanisms have been postulated (Diamond, 1965). Any mechanism of isotonic water trans- port operative in SW might also operate at lower salinities, and so would increase the net water influx above that calculated on the basis of diffusion. Some such mechanism appears necessary in 30-40% SW, where the calculated difrusional net water influx is well below the reported urine production (Table III). Alterna- tively, one might consider a system such as that suggested by Ussing (1954), in which inner diffusional areas are in series with outer pore-like spaces such that, once a diffusional net influx is established, a bulk flow of water is set up in the "pores" of sufficient velocity to counteract or reduce diffusion in the opposite, outward, direction. Such a system would have the properties of a rectifier or one-way valve, admitting water but restricting the outward diffusion of water below what would be expected on the basis of the water concentration in the blood. The water thus prevented from diffusing out would be available for disposal in the urine, over and above the net diffusional influx calculated from the mole fraction difference between water concentrations in blood and medium. The diffusional influx would be as expected ; what would be reduced is the diffusional efflux.

It is evident that the water economy of Care inns is still incompletely under- stood. Future work could profitably be directed to several problems. Is the apparent reduction of water-permeability with salinity the result of a reduction of circulation, either in the medium bathing gills or gut or in the circulation of the blood, or is it the result of an actual change of integumental permeability? The possibility that permeability reduction may accompany either hyper- or hypo-osmotic regulation seems physiologically adaptive and reasonable, but more critical examination is needed. This should be studied in some crab normally exposed to seasonal hypersaline conditions (Gross, 1961). The problems of correlating the water-permeability figures reported by different workers using ToO and D2O might be solved by simultaneous double-tracer studies of water influx in order to reduce individual operational variation and to evaluate possible isotope effects. In all such work the weights of the animals used must be taken into account and temperature controlled, since hourly water exchange fraction (K) varies with body weight as well as with salinity and temperature, and is thus no more a constant for a species than is respiratory rate.

WATER-PERMEABILITY OF CARCIXUS 361

This work was made possible by a grant of sabbatical leave from the University of California, Berkeley, and the valued support of the John Simon Guggenheim Memorial Foundation. 1 am grateful to Professor and Department Head R. B. Clark and to Professor John Shaw, Department of Zoology, University of New- castle upon Tyne, for their help and interest during my stay. I thank Professor \Y. T. \Y. Potts for helpful criticisms of my earlier presentation of data on Rhithropanopens, and Dr. Paul P. Rudy for discussions of the quantitative differ- ences in our respective papers. I have also benefitted from critical discussion with Mr. Richard "Williams and Dr. Lawrence C. Thompson of the Department of Zoology, and with Professor of Chemistry George C. Pimentel, University of California, Berkeley.

SUMMARY

1. The apparent water-permeability of Carcinns nutcnas, as measured by D2O influx, is 2-3 times higher than that of the more euryhaline crab Rhithropanopeus.

2. Like Rhithropanopeus, Carcinns shows a reduction of water-exchange rate at lower salinities. The highest hourly water-exchange fraction is in 75% S\Y (K -- 2.73). the lowest in 30% S\Y ( K - - 1.76) ; values refer to a crab with wet weight of lOg, at 18° C.

3. The calculated net diffusional (osmotic) water influx is adequate to account for the urine production of Carcinns in 50-70% SW, but does not account for urine production in SW, and only inadequately for the urine produced in 30-40% S\Y. and it seems necessary to postulate some isotonic transport of water.

LITERATURE CITED

BALLARD, B. S., AND W. ABBOTT, 1969. Osmotic accommodation in Callinectes sapidns Rathbun.

Comp. Bioc/u'in. Physio!.. 29: 671-687. BARNES, H., 1954. Some tables for tbe ionic composition of sea water. /. Ex p. Bio!., 31 :

582-588. BIXNS, R., 1969. The physiology of the aiitennal gland of Carcinns inacnas (L.). II. Urine

production rates. /. E.vp. Biol., 51 : 11-16. DIAMOND, J. M., 1965. The mechanism of isotonic water absorption and secretion. S\nip. Soc.

E.rp.Bwl, 19: 329-347. KVAXS, D. H., 1969. Studies on the permeability to water of selected marine, freshwater, and

euryhaline teleosts. /. E.vp. Bin!., 50: 689-703. GROSS, W. J., 1961. Osmotic tolerance and regulation in crabs from a hypersaline lagoon.

Biol. Bull., 121 : 290-301. GROSS, W. J., 1964. Trends in water and salt regulation among aquatic and amphibious crabs.

Biol. Bull.. 127 : 447-466. JONES, L. L., 1941. Osmotic regulation in several crabs of the Pacific coast of North

America. /. Cell. Comp. Physio!.. 18 : 79-92. NAGEL, H., 1934. Die Aufgaben der Exkretionsorgane und der Kiemen bei den Osmoregula-

tion von Carcinns niaenas. Z. Vcrgl. Physio!.. 21 : 468—491. PANTIN, C. F. A., 1946. Microscopical Technique for Zoologists. Cambridge University

Press, 75 pp. PARRY, G., 1955. Urine production by the antennal glands of Palaemonetes I'arians (Leach)

/. E.vp. Biol, 32 : 408-422. POTTS, W. T. W., AND G. PARRY, 1964. Osmotic and Ionic Kc</n!ation in .-Iniinals. Pergamon

Press, 423 pp. ROBERTSON, J. D., 1960. Osmotic and ionic regulation. Pages 317-339 in T. H. Waterman,

Ed., The Physiology of Crustacea, Academic Press, .\\-\v York.

362 RALPH I. SMITH

Ki'Dv, 1'. P., 1967. Water permeability in selected decapod Crustacea. Coinp. Biocliein.

PhysloL, 22 : 581-589. SHA\V, [., 1961. Studies on ionic regulation in Careinus niuenns (L). I. Sodium balance.

7. E.rp. Biol., 38 : 135-152. SMITH, R. I., 1967. Osmotic regulation and adaptive reduction of water permeability in a

brackish-water crab, Rhithropanopeus luirrisi (Brachyura, Xanthidae). Biol. Bull.,

133 : 643-658. SMITH, R. I., 1970. Chloride regulation at low salinities by Nereis dh'crsicolnr. II. Water

fluxes and apparent permeability to water. /. E.rp. Bio!., 53: 93-100. USSING, H. H., 1954. Membrane structure as revealed by permeability studies. Pages 33-42

in J. A. Kitching, Ed., Recent Developments in (.'ell Physiology. Colston Res. Soc.,

Academic Press, New York. WELSH, J. H., R. I. SMITH AND A. E. KAMMER, 1968. The estimation of D2O in water or

blood. Pages 184-188 in J. H. Welsh, R. I. Smith and A. E. Kammer, Laboratory

Exercises in hrrerfcbratc Physiology, Burgess Publishing Co., Minneapolis, Minnesota.

Reference: A1/,./. A1;///.. 139: 3(0-374. ('October. l''7(>)

THE EFFECTS OF ACUTE GAMMA IRRADIATION OX THE BRINK

SHRIMP, ARTEMIA. I. LIFE SPANS AND MALE

REPRODUCTIVE PERFORMANCE '• - ;

RICHARD D. SQUIRED AND DANIEL S. GROSCH (ioicticx Department, \orth Carolina State Cnii'crsity. Ralciyh. North Carolina 27607

The variety of metazoans which have been successfully exploited for genetic studies is quite limited, and the majority of such animals are either insects or vertebrates. The range of metazoans used in assaying the effects of radiation on total reproductive behavior has not been much greater. A broadening of the foundation upon which our knowledge of such effects is based necessitates using organisms which are less well known than the standard laboratory species.

Initial reports of Bowen (1962, 1963a, 1963b, 1964), Groscli (1962), Grosch and Erdman ( 1955 ) indicated that Artcniia is well suited as a laboratory animal for studies of a genetic or radiobiological nature. The primary goal of the present study, the first of a series, was to estimate the overall effects of acute gamma radiation on the fitness components of amphigonic Art curia. Refined measure- ments of genetic damage have had to be postponed until after such an analysis.

The experimental design, and effects on life span and male productivity are reported in this paper. Effects on female productivity shall be the concern of a subsequent paper. This is the first and only report concerning the effects of electromagnetic radiation on male reproductive performance in Artcmia.

MATERIALS AND METHODS

Freshly matured brine shrimp were obtained from two stock #3 culture jars. This stock was derived from commercially obtained California!! cysts, and has been maintained in five-gallon battery jars since 1957 at the Marine Biological Labora- tory, Woods Hole. The jars are kept at room temperatures and allowed to evaporate each winter. The cultures are re-established each spring by adding distilled water to the resultant brine and precipitated salts. The spring culture is thus derived from overwintering cysts and a few adults which may still be alive in the brine.

1 This research was conducted in partial fulfillment of requirements for the senior author's doctoral dissertation at North Carolina State University.

- Financial support was provided the senior author by the Genetics Training Grant ( Public Health Service) for the summer of 1965, and subsequent work was conducted as part of the N. A. S. A. Project NsG-678, Utilization of Habrohraciui and Artcniia as Experimental Materials in Bioastronautic Studies.

3 Paper number 3094 of the Journal Series of the North Carolina State University Agricul- tural Experiment Station, Raleigh, North Carolina.

4 Current Address: Biology Department, Lima Island University, Brooklyn, New York 11201.

363

364

RUM \KI) I). SOIMRK AND DAMKL S. OROSCH

Tlie culture medium consisted of filtered sea water to which was added 50 g ot .sodium chloride ( Fisher Scientific Co.) per liter (Bowen, 1962). Later, after the surviving animals were transported to Raleigh, Xorth Carolina, "Instant Ocean" ( Aquarium Systems, Inc., Wickliffe, Ohio ) was substituted for the sea water.

Holding jars were set up eight days prior to irradiation, and young adults were segregated according to sex tor the next two days. All adults thus obtained were then held for an additional five days. Eight treatment groups of five males each were assigned by use of a random numbers table to provide irradiated males to be mated to untreated females. This procedure was repeated for females, to provide irradiated females to be mated to untreated males. Separate randomizations then assigned an untreated mate to each experimental animal. Pint-size glass jars, each containing 10 fl. oz. of medium, were used tor pair-matings.

For exposure, each treatment group was placed in a small plastic petri dish during exposure.

TABLE I

Alale and fen i it I <' adult life spans in days, with two-tailed t test comparisons of treated versus control animals (maximum range of error in exposure lime:

±0.01 min.)

Level	Dose (kR)	Exposure time (min.)	X ±S.E.	t	x ± s.i':.	t
1	0	0	52.9 ± 8.5	—	45. ft ± 6.2	—
2	0.1	0.20	4Q.O ± 8.7	0.32	43.6 ± 5.5	—
					30.4 ± 5.9a
3	1	1.00	55.8 ± 4.2	0.31	40.9 ± 8.0	0.47
4	1	2.00	55.7 ± 5.5	0.28	28.7 ± 4.6	2.20
5	5	2.00	48.2 ± 8.0	0.40	40.4 ± 5.8	0.61
6	10	2.05	45.8 ± 5.7	0.70	26.7 ± 5.8	2.24
7	50	10.27	15.9 ± 1.2	4.30**	9.9 ± 0.5	5.79***
8	100	20.54	5.9 ± 0.5	5.53***	4.6 ± 0.6	6.63***

" Replication 2 differs significantly from replication 1 ; t == 2.30. ** Significant at 0.01 ; ***Significant at 0.001 .

and given the appropriate dose of gamma radiation from the M. B. L. cesium-137 unit (Table I). There is no significant heat problem with this source, and room temperatures were thermostatically maintained in the middle seventies (°F)

All irradiation was completed in a period of three hours, and the animals were then placed in their assigned pair-mating jars. Each jar was examined daily throughout the entire life span of the treated animal. In the treated-male series, females were held for several days after their mate's death, so that any brood which was "in utero" at his death would also be scored. The dead mate of a treated animal was replaced by an adult which was comparable in age to the dead animal, but bad not yet reached senility. Replacement females were unmated at the time of use. Scoring criteria consisted of debility or death ot the adult animals, and the presence of either cysts or nauplii in the jar.

Female specimens of Artcinia customarily produce a brood every three and one-half to four days. Oviparous broods (cysts) were filtered out. dried and stored

GAMMA RAY KFFKCTS ON ARTEMIA 365

on filter paper. These cysts were later counted and scored for hatchability after hydration in small stender dishes of plain Instant Ocean. Most cysts hatch within 2-1 — IS hours after hydration, hut all hatchahilities were scored daily tor seven days. Viviparous hroods of nauplii were counted immediately.

Broods I and IV were set up in quart jars (each containing 24 fl. oz. of hrine), and raised to adulthood. These hroods wrre then scored for morphological ab- normalities, sex ratio, and the per cent survival.

Each jar was fed daily with a dilute suspension of Fleishmann's baking yeast and evaporated \\ater was replaced hy distilled water. All culture jars received constant overhead fluorescent lighting.

Manipulation of immature and adult animals was greatly facilitated hy use of a commercially ohtained glass meat haster, used as a giant medicine dropper.

Eleven days later, a second replicate was set up in the same manner as just described, again utilizing five animals of each sex at each of the eight dose levels.

The enormous number of cysts produced hy the controls in the treated male experiments precluded a full hatchability analysis for this level, and only a portion of their encysted hroods were so analyzed. Except for this parameter, lull life hi.-tory and reproductive data were obtained for each test animal.

RESULTS

Life span

Means and variances were first established within each replicate, and compared by two-tailed t and F tests. The mean values of two replicates were significantly different for female life span at the 100 R level, and this level was excluded 1rom further analysis. The differences of the other means between replicates within treatment levels were not significant. Some of the differences between variances were significant. Average mean values and pooled variances ( Steel and Torrie, 1960) were obtained independently for the members of each sex within each treatment level. Comparisons between treatment levels were made for each sex, using a two-tailed t test ( Table I ) .

Exposures of 50 and 100 kR significantly reduced male and female life spans. Reductions in female life span approach significance after doses of 2 or 10 kR, and are significant at the 0.10 level. The male and female close-effect curves are linear on a semi log plot for the closes of 10. 50 and 100 kR. although the slopes of these lines are quite different, and demonstrate the consistently greater longevity of the homogametic male.

Male reproductive performance

Xinnl'cr of broods. Comparisons of the average number of fertile broods per male are shown in Table II. \Yhi1e 1 kR did not significantly reduce the number of broods per male. 2 kR caused an evident decrease. The differences between the results from 2, 5. and 10 kR are not significant. After 50 and 100 kR, no broods were produced. For purposes of the present study, fertility was defined as the production of nauplii from either a viviparous brood, or from cysts. The voiding of cysts does not in itself indicate male fertility, since many females are capable of depositing unfertilized cysts (which always fail to hatch). Therefore, only fertile

366

RICHARD I). SOl'lRK AND DANIEL S. (iROSCH

broods were considered in this stud}'. Complete genetic dominant lethality is not distinguishable from sperm inactivation or tailure of copulation.

Reproductive span. The average number of days during which males were able to sire offspring was calculated per treatment level (Table 11 ). Fxamination of the records of individual males indicated three distinct categories, a short repro- ductive span of 11.9 days, a transitional situation, and a long reproductive span of 50.4 days. The short-span animals were found at the 5 and 10 kR irradiation levels. Five of the eight fertile males given 2 1<R also belonged to this group (X -- 1 1 .8 ± 0.9). The remaining three males had a long reproductive span which raised the group's average to the tabulated 2S.4 days. However, two of these three males receiving 2 kR, were temporarily sterile from day 13 to day 29 or 36, after which fertility was regained. At doses below 2 kR, no sterile periods occurred and all males exhibited long reproductive spans.

TABLE 1 1

number of fertile hmols and reproductive spun per irradiate! Artemia mall-, u'llh I lest comparisons (one-tailed) of treated versus control animals

Level	Dose (kR)	Number of fertile broods	Reproductive span (days)	Over-all repro- ductive span
X ±S.E.	t	X ± S.E.	t	X ± S.E.
1	(1	10.4 ± 1.9	—	53.7 ± 8.1	—
2 3	0.1 1	8.2 ± 1.4 9.8 ± 1.1	0.928 0.276	44.1 ± 6.4 52.0 ± 4.1	0.932 0.187	50.4 ± 3.3
4 5 6	2 5 10	4.0 ± 1.4 1.7 ± 0.5 2.2 ± 0.6	2.700* 4.416** 4.121**	28.4 ± 8.7 11.1 ± 1.4 12.6 ± 1.7	2.126* 5.195** 4.951**
11.9 ± 0.7

* Significant at 0.05.

** Significant at 0.01.

On day 46 of replicate 2, the three surviving males of the 5 kR exposure and four surviving males which received 10 kR were scored as aspermic by gross ob- servation. Males at lower levels had cellular material present in their seminal vesicles. However, two of the three males which received 2 kR failed to produce additional nauplii.

Unlike the female response to radiation (data to be presented in paper II of this series) fertility in male brine shrimp appears to be unrelated to life span. A short reproductive span was not correlated with life span.

Cyst hatchability. Hatchability tests were conducted for all oviparous broods. However, analysis was restricted to those encysted broods which ( 1 ) were not completely infertile, (2) contained at least 25 cysts (i.e., the majority), and (3) were not derived from partially viviparous broods. The percentage of hatched cysts was calculated for each brood. The arcsin ^/ % transformation was then obtained for each value (Steel and Torrie, 1960), since it is not legitimate to use the untransformed values for the standard error. Untransformed treatment means and t test comparisons are given in Table III. The number of encysted broods per level which could be utilized varied considerably, and only part of the cysts produced by

GAMMA RAY EFFECTS ON ARTEMIA

367

TAHI.K III

Artemia hate liability per fertile en< yxted brood, u<itli I text / ompansons nf I rented versus control animals. The males received an acute gam ma ray d«\r

		% hatch
Level	Dose (kR)		No. of broods scored
		X	t
1	0	24.3		12
2	U.I	27.4	0.472	27
3	1	15.2	1.583	49
4	2	21.4	0.445	13
5	5	13.7	1.366	3
6	10	6.5	3.200**	13

Significant at 0.01.

unirradiated males were scored. These control data are in close agreement with the control values obtained in the irradiated-female experiment (Squire, in prepa- ration) .

The available sample size at 5 kR is too small to warrant conclusions. Hatch- abilities were significantly reduced after 10 kR. This decrease may be ascribed to : ( 1 ) failure of some eggs to be fertilized, or, ( 2 ) genetic lethals which act prior to the nauplius stage. Thus we consider cyst hatchability as a measure of "dominant" lethal events. We may then calculate the percentage of dominant lethal events as:

% DLE =

% control hatch - - % treated hatch

% control hatch

X 100

This calculation gives an approximate value of 73c/c for 10 kR.

Sun'h'al to adultliood. sc.r ratio, and A\ productivity. Broods I and IV were scored for survival to adulthood and sex ratio. The observed values were highly

TAHI.K IV

Survival to adulthood, sex ratios and morphological abnormalities of A'i animals fathered by gamma irradiated male brine shrimp

		% survival to adulthood		Abnormalities
Level	Dose (kR)		broods produced		Sex ratio % males
X ± S.E.	t	fraction	%
1	0	62.5 ±4.5		14	1/474	0.2	54.6
2	0.1	58.3 ± 5.3	0.579	12	f> 584	1.0	49.5
3	1	69.9 ± 2.1	(1.370)	14	3/564	0.5	52.3
4	2	65.7 db 6.9	(0.378)	8	0/322	0	52.2
5	5	56.2 ± 2.9	1.094	7	6/244	2.5	53.3
6	10	28.2 ± 2.2	4.040**	4	2/50	4.0	60.0

X2 comparison

5.25NS

Significant 0.01 .

RICHARD I). SOU1RE AND DANIEL S. GROSCH

heterogeneous and showed no obvious correlation with ( 1 ) brood size, (2) deposi- tion as a viviparous r.v. oviparous brood, or (3) the length of the post-irradiation period. All viviparous broods were maintained under the same conditions at the M. 15. L., Woods Hole. All oviparous broods were maintained under comparable conditions at Raleigh.

Since the results did not differ greatly between broods 1 and IV, the data were pooled for each treatment level and analyzed on a per-brood basis using a one-tailed t test. After pooling, these values demonstrate a highly significant reduction at 10 kK, but not at lower doses. Sex ratios were not significantly altered at am dose level ( Table IV ).

In addition, some data were obtained tor the reproduction ol animals from brood I. The surviving offspring of 10 kR males died early and were usually sterile. Some of these animals were full-sib mated while others were mated to normal animals. Some were mated to normal animals after their sib died. A total of three fertile broods was produced. At 5 kR, a total of 39 full-sib matings resulted in no viviparous broods and a low frequency of oviparous broods. Adult mortality was still very high. At 2 kR, oviparous broods were more frequent, viviparous broods rare, and adult mortality high. At lower dose levels, oviparous broods were common, while viviparous broods remained relatively rare. Adult mortality was not excessive. Labor and space requirements made it impossible to conduct full life history studies for these X] animals and most cyst hatchabilities were not analyzed. Consequently, observations had best remain on a qualitative basis.

The number of morphological abnormalities in broods I and IV are summarized in Table IV. Four of the level 5 animals were females. One of the level 2 animals was too immature to sex. The other 13 animals were males. The abnormal females consisted of: ( 1 ) one with a pair of male-like claspers and an ovisac full of yolky-appearing material which did not resemble normal eggs, (2) one with a stubby tail and paired ovisacs, (3) one with an undeveloped tubular ovisac and an arched tail, and (4) one with a bulbous distal section of the ovisac. This section is normally thin and tubular, with its long axis parallel to that of the trunk. The axis of this ovisac was ventrally perpendicular to that of the trunk. Abnormalities in the males included eyes which were mosaic for normal pigmentation, shortened or absent claspers, stubby tails, missing caudal furcae, abnormal seminal vesicles, bent tails and apparently fused phyllopodia. Most of these abnormalities were bi- laterally asymmetrical.

DISCUSSION Life spun

These results suggest a threshold for obvious life span effects in the 10 kR region, but data are not available to check additional points in the 10—100 kR range against the linear semi-log plot. Ballardin and Metalli (1966) concluded that the life span values for several unspecified strains of Arteniia may be fitted by an exponential function of the dose after acute exposure of 10, 20, 30, 40, or 50 kR rad. While the curves for diploid and tetraploid females, both bisexual and partheno- genetic, and for tetraploid males show no appreciable shoulder, that for the bisexual diploid males shows a definite shoulder; and these males are more resistant

GAMMA RAY EFFECTS ON ARTEMIA 369

than the other animals. Their abstract does not specify the actual life span values. Grosch and Erdman (1955) also reported males to be more resistant than females, and the data indicated an obvious effect in the 10 kR region. Culture techniques were still sub-optimal in 1955, however, resulting in decreased life span values for both control and treated animals.

The assumption that adult brine shrimp lack tissues with proliferative cells is not valid. Lochheacl and Lochhead (1941 ) found frequent mitotic figures in the hemopoietic organs, and A I. Lochhead (personal communication) has observed occasional mitotic figures in the gut as well. To what extent these somatic divisions may be necessary for adult survival is not known. According to Lochhead and Lochhead (1941), hemopoietic mitoses increase in frequency after physical injury. The blood cells thus produced are phagocytic, and perhaps also perform some nutritive function in oogenesis. Conceivably these cells function in tissue replacement necessary for survival, and consequently Artemia is more sensitive to radiation than the adult form of the holometabolous insects.

The extent to which the nonlocalized centromere of Artemia chromosomes (Stefani, 1963 ) confers somatic cell resistance to radiation damage is not yet known. The retention of radiation-induced fragments over many cell generations has been repeatedly demonstrated in animals which have bolokinetic chromosomes, although mosaic fragment loss may also occur (Barry, Guthrie and Dollinger, 1967; Hughes-Schrader and Ris, 1941 ; Hughes-Schrader and Schrader, 1961 ; Nelson- Rees. 1962; Ris. 1942; Tazima. 1964; Tazima and Onimaru, 1969). As Stefani (1963) pointed out, such a radiation study should be conducted in Artemia. Grosch and Erdman ( 1955 ) noted that a source of radio-resistance mav lie the somatic polyploidy present in some tissues.

Radiation-induced shortening of life span must be viewed as a summation of innumerable deleterious effects on the animal's physiological processes and cannot be ascribed to genetic damage alone. In this species, the female is the heterogametic sex; and it is the heterogametic sex which is usually less resistant to environmental stress and often short-lived.

Male reproductive performance

Reproductive span. Since there are no reports concerning the effects ol electro- magnetic radiation on male reproductive performance in brine shrimp, we are forced to compare Artemia to such distant relations as the insects and mammals. Such comparisons must consider the anatomical, physiological, and genetic differ- ences which separate such diverse organisms. In Artemia, spermatogenesis is continuous and its stages may occur in waves of partially synchronized activity. All stages are present in the mature male ( Fautrez-Firlefyn. 1951). The sperm are aflagellate and may be amoeboid (Fautrez-Firlefyn and Fautrez, 1955). Stefani ( 1963 ) found the chromosomes to be polycentric in the species of Artemia which he studied. Finally, Artemia is aquatic rather than terrestrial.

A dose of 2 kR is enough to destroy or incapacitate the spermatocytes of most organisms. While this dose may allow some sperm cells to be formed, most of these spermatozoa carry dominant genetic defects which cause zygotic death early in embryonic development. In the present study which requires decisions based upon interrupted cyst deposit and failure in larval emergence as well as the absence

RICHARD D. SQUIRE AND DANIKL S. GROSCH

ol naupliar production, sterility ol the male must be assessed in terms of any cessation of visible offspring from bis mate. Nevertheless 2 kR as the sterilizing dose for spermatocytes is consistent with results for some insects, for example Hontby.r ( Sado, 1961 ; Tazima, 1961, 1964). Diptera are more sensitive, with 1.5 kR the upper limit (Riemann. 1967). Mice may be in another class entirely since less than 700 R suffices ( Davies and Evans, 1966 ; Mandl, 1964 ) .

Spermatogonial response to radiation varies considerably according to the organism and the type of spermatogonial cell involved. In the mouse, 300 R destroys "type B," intermediate, and most "type A" spermatogonia. One resistant component of the "type A" population survives, and eventually multiplies to regenerate the germinal epithelium ( Oakberg, 1965). A similar result occurs in Boniby.v after 2 kR (Sado. 1961 ). Spermatogonial LD-50's include about

1 kR for Boinby.r and 23 R for mice.

\Ye conclude that the short fertility period observed in specimens of Artciiiia after 5 and 10 kR represents radio resistant cells which were sperm and perhaps spermatids at the time of treatment. Since the reproductive pattern for most

2 kR males is the same as that for higher doses, the same conclusion seems justified for this level as well. The presence of cellular material in some seminal vesicles of 2 kR males 46 days after treatment, the temporary sterility of two 2 kR males, and the lack of sterility of one 2 kR male all suggest spermatogonial regenera- tion at this treatment level. The failure of some of these males to regain fertility may be due either to high genetic dominant lethality or to low sperm concentration. The latter could result from incomplete testis replenishment by spermatogonia (Gillette. Hopwood, Carlson and Gassner, 1964). This replenishment and subsequent spermatogenesis is believed to have obscured alteration of the reproductive pattern in the 2 kR male which lacked a sterile period. Replenishment probably had an even greater influence at 1 kR, where no sterile period was observed; also some ot the primary spermatocytes may have survived destruction.

Since all surviving males at 5 kR and 10 kR levels were observed to be aspermic on the 46th day after treatment, either all "type A" spermatogonia were destroyed or testis replenishment was insignificant at these doses. The actual duration of the initial fertility period, and hence the subsequent sterility period, is a function of the rate of sperm utilization by the irradiated male. In turn this may depend upon the availability of females and their reproductive cycle. In our experiment, each male was isolated with a single female. If each male had been placed with a fresh receptive female daily, the period of fertility might have been shortened.

Furthermore, the present results were obtained with freshly matured males. Conceivably, slightly older males would contain a greater number of "type A" spermatogonia. If this is the case, more rapid testicular replenishment might result in those males.

Cyst hatcliability. In general, spermatids are believed to be more sensitive than mature spermatozoa to the effects of irradiation (Mandl, 1964). The doses required to induce 99% dominant lethal events in the mature sperm of many insects with monokinetic chromosomes are in the range of 3 kR to 1 1 kR (LaChance, 1967). With holokinetic chromosomes, doses have ranged from 4 kR to 100 kR (LaChance, Schmidt and Bushland, 1967; North and Holt, 1968a, 1968b). Despite considerable differences between insects and shrimp, the esti-

GAMMA RAY EFFECTS ON AKTEMIA 371

mate of 73 (/( dominant lethality at 10 kR seems reasonable. Indeed the dose required for A^rtcuiia seems to be rather low for a holokinetic species, but well within the range of other reported values. Possibly treated spermatids as well as sperm contributed to this value. Such a heterogeneity would be expected to lower the observed resistance values.

The low hatchability value for the 1 kl\ males is probably fortuitous. Upon examination of individual male records, it was found that the average hatchability is no different for the first 15 days than it is for the remainder of the reproductive span. (The untransformed average values were 16.2</£ and 14.9r/r . respectively.) This observation rules out the possibility that the lowered value was due to irradiation effects on meiocytes and earlier stages which were excluded from analysis of the higher levels. If this had been the case, then the average values for hatchability would be expected to be lower in the second period which reflects pre-spermatid stages of treatment.

In Artcuiia, the date of brood deposition cannot be accurately correlated with the cytological stage present at the time of irradiation for several reasons: (1 ) The post-irradiation interval also reflects the reproductive cycle of the female, since only metaphase I eggs are sampled. (2) The normal duration of each spermatogenic stage, the effects of irradiation on the duration of each stage, and the effects of the lower doses of radiation on the survival of these stages are unknown for Artcuiia. (3) The male reproductive system consists of a pair of genital tracts without a common penis. Thus a single fertilization reflects a contribution by one of these tracts, but two successive broods may represent the same tract twice or each tract once. (4) Each testis is a long tubular organ with spermatogonia present along its periphery. A given ejaculate may therefore contain sperm which are heterogeneous in terms of the developmental stage irradiated. Such heterogeneity might be increased by delayed utilization of sperm from males not subjected to sperm exhaustion by multiple mating.

It has been tacitly assumed that the various secretory cells ot the anterior testis have not been damaged. While such cells have been relatively radio-resistant in other animals, no data exist for Artcuiia. A hypothetical threshold at 2 kR for these cells would not be inconsistent with the data, if the observed effect were delayed.

Siiri'h'al to adulthood, sc.v ratio, and X \ productivity. By combining data on survival to adulthood with the information gained for the mortality and re- productive behavior of X, specimens of Artcuiia, we may draw some general conclusions. After an adult male has received 10 kR, most of his potential off- spring are eliminated prior to the naupliar stage. Those which become nauplii show a pronounced mortality rate prior to maturity. Those which reach maturity continue to exhibit a high mortality rate and are almost uniformly sterile. A similar, but less pronounced pattern is indicated for the 5 kR group. Morpho- logical abnormalities are relatively common in both of these groups. The 2 kR level is notable for its lack of demonstrable effect on hatchability, survival to adult- hood, or morphological abnormalities. Effects were still observed in the adult Xi of 2 kR males in terms of reproductive behavior and mortality.

The mosaic nature of morphological abnormalities has often been ascribed to alteration of one-half of a T)X.\ helix following sperm irradiation in other

372 RICHARD I). SOUIRE AND DANIEL S. GROSCH

organisms. \\ e cannot restrict ourselves to this interpretation in Artcniia. The holokinetic nature of the Artcniia chromosome is expected to reduce fragment loss following irradiation. However, small fragments may still disappear from some cell lines, and thus produce a mosaic individual. Recently, Tazima and Onimaru ( 1969) reached the same conclusion in Hoinhy.v where an exponential increase in mosaic frequency was observed in a dose-effect study of irradiated sperm. An earlier report of mosaic fragment loss is that of Xelson-Rees (1962). Incomplete penetrance of induced mutations might also be reflected in asymmetry.

The data for the 10 kR level suggest that the X, is much less fertile than are the irradiated males themselves, a sterility pattern typical of species with holo- kinetic chromosomes. Investigated cases have involved exposure of post-meiotic stages, often mature sperm, to sterilizing doses of radiation. Since the effect of single-break aberrations is minimized by the holokinetic nature of the chromosome, the sterility pattern is believed to reflect multi-break phenomena, and in particular, translocations (Bauer, 1967; Nordenskiold, 1963; North and Holt, 1968a, 1968b; \Yalker and Quintana, 1968). Irregular fragment behavior and inversion heterozygosity may be additional factors, and Nordenskiold (1963) found in the plant Luciila that if a holokinetic chromosome is fragmented into more than two pieces, subsequent recombination with an unfragmented homologue will yield a secondary fragmentation of that homologue. Meiotic segregation may then result in chromosomal imbalance and sterility.

The failure to detect a significant alteration in sex ratio may be due either to a reduced sample size at 10 kR, or to an insufficiently large dose of radiation. Grosch (1962) observed that the sex ratio of Artcniia tended to favor males when the parents were of irradiated ancestry. This tendency was especially pronounced in populations which were on the verge of extinction. Grosch correlated this effect with female heterogamety.

We would like to thank Mrs. Sally Barnes and Mrs. Ann G. Spencer for the technical assistance provided during this experiment.

Sr.MMARY AND CONCLUSIONS

Life spun of both sc.ves

Freshly matured male and female specimens of California!! Artcniia (stock #3) were irradiated with acute doses of 0, 0.1. 1. 2, 5, 10, 50. or 100 kR gamma radiation and pair-mated to untreated animals. Significant reductions in life span resulted after 50 or 100 kR to either sex. Reductions in female life span ap- proached significance after 10 kR. This moderate radiosensitivity can be explained if cell proliferation in some adult tissues improves the probability of individual survival.

Male reproductive performance

Treated males were completely sterile after doses of 50-100 kR. Males which received doses of 5 or 10 kR had an initial fertility period lasting 12 days, followed by permanent sterility. After a dose of 2 kR, 7 out of a sample of 8 males

CAM MA KAY EFFECTS OX ART EMU

demonstrated the saim1 initial period of fertility, followed by a period of sterility. Two of the 7 subsequently recovered their fertility. Xo sterile period occurred after 0.1 or 1 kR. These results are interpreted as indicating spermatogonial stem-cell replenishment of the testicular epithelium in some males after 2 kR and in all males irradiated with 1 kR. It is further hypothesized that all spermatocytes were in some way incapacitated or destroyed following doses above 2 kR.

Hatchability data failed to detect induced dominant lethality in sperm (and perhaps spermatids ) after 2 kR. Induced damage resulted in 73 r/ dominant lethal events at 10 kR. Xo effect on other stages was observed at less than 2 kR.

In the XT generation, a dose of 10 kR reduced survival to adulthood, fertility, and adult life span. Similar, but less pronounced effects were obtained after 2 and 5 kR. These data indicate induced genetic damage. Xo significant alterations were observed in sex ratios.

X, sterility appeared to be greater than that observed for irradiated sperm. This point is being reinvestigated. and would be compatible with the reported holokinetic nature of the .Irtciuia chromosome.

LITERATURE CITED

BALLARDIX, E., AXD P. MKTALLI, 1966. The life span of different strains of Artemia salina

after single and fractionated X-ray exposures. Int. Coin/r. Ruduit. Res.. 3: 28. BARRY, B. D., \V. D. GI'THKIK AND E. .1. DOLLIXCEK, 1967. Evidence of a diffuse centromere

in the European corn borer, Ostrinni nulrilalis (Lepidoptera : Pyralidae). Ann.

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22: 101-125. BO\VEX, S. T., 1962. The genetics of Artemia salimi. I. The reproductive cvcle. Biol. Hull..

122: 25-32. BO\VEX, S. T., 1963a. Tlie genetics of Arlemia siiliini. II. \\ 'liite eye. a sex-linked

mutation. Biol. Bull.. 124: 17-23. BOWEX, S. T., 1963b. The genetics of Artciit'ui salina. III. Effects of X-irradiation and of

freezing u])on cysts. Biol. Bull.. 125 : 431-440. BOWEX, S. T.. 1964. The genetics of Artemia s<ilnni. IV. Hybridization of wild populations

with mutant stocks/ Biol. Bull.. 126: 333-344. DAVIES, D. R., AXU H. J. EVAXS, 1966. The role of genetic damage in radiation-induced cell

lethality. Ad-i\in. Radial. Biol.. 2 : 243-353. FAUTREZ-FlRLEFYN, X., 1951. Etude cvtochimique des acides nucleiques an cours de la

gametogenese et des premiers stades du developpement embryonnaire cbez Artemia

salina L. Arch. Biol.. 62 : 391-438. FAUTREZ-'FIRLEFYN, Xy., AXD J. FAUTREZ, 1955. Etude cytologique et cvtochimique du sperma-

tozoide d' Artemia salina L. Seconde partie : Le noyau, 1'acrosome, le grain, les micro- granules. Arch. Biol.. 66: 195-222. GILLETTE, E. L., M. L. HOPWOOD, W. D. CARLSOX AXD E. X. GASSXEK, 1964. The effect of

X-irradiation of bovine testicles on semen. Radial. Res.. 22: 2o4-275. GROSCH, D. S., 1962. The survival of Artemia populations in radioactive sea water. Biol.

Bull.. 123: 302-316. GROSCH, D. S., AND H. E. ERDMAX. 1955. X-ray effects on adult Artemia. Biol. Bull., 108:

277-282. HuGHES-ScHRADER, S, AXD H. Ris, 1941. The diffuse spindle attachment of coccids,

verified by the mitotic behavior of induced chromosome fragments. /. E.\~p. Zool.. 87 :

429-456. " HuGHES-ScHRADER, S., AXD F. SciiRADER, 1961. The kinetochore of the II eini^lera.

Chromosoma, 12 : 327-350. LACHANCE, L. E., 1967. The induction of dominant lethal mutations in insects by ionizing

radiation and chemicals — as related to the sterile-male technique of insect control. Pages

374 RICHARD D. SQUIRE AND DANIEL S. GROSCH

617-650 in: J. VV. Wright and R. Pal, Eds., Genetics of Insect lectors of Disease.

Elsevier, Amsterdam, Netherlands. I. .\CHAXCE, 1.. E., C. H. SCHMIDT AND R. C. BUSHLAND, 1967. Radiation-induced sterilization.

Pages 147-196 in: W. W. Kilgore and R. L. Doutt, Eds., Pest Control Academic

Press, New York. LOCH HEAD, J. H., AND M. S. LocHHEAD, 1941. Studies on the blood and related tissues in

Arteinia- (Crustacea Anostraca). /. Marshal, 68: 593-632. MANDL, A. M., 1964. The radiosensitivity of germ cells. Bwl Rev.. 39 : 288-371. NELSON-REES, W. A., 1962. The effects of radiation damaged heterochromatic chromosomes on

male fertility in the mealy hug, Planocoecns citri (Risco). Genetics, 47: 661-683. NORDENSKIOLD, H., 1963. A study of meiosis in the progeny of X-irradiated Liizuhi pnrpnren.

Hcrcditas, 49 : 33-47. NORTH, D. T., AXD G. G. HOLT, 1968a. Genetic and cytogenetic basis of radiation-induced

sterility in the adult male cabbage looper, Trichoplusia ni. Pages 391-403 in Sym- posium on Isotopes and Radiation in Entomology, Vienna, 1967: Isotopes and

Radiation in Entomology: Proceedings. International Atomic Energy Agency, Vienna. NORTH, D. T., AXD G. G. HOLT, 1968b. Production of sterile progeny by male cabbage loopers

partly sterilized by radiation. /. Econ. Enionwl, 61 : 928-931.

OAKBERG, E. E., 1965. The effects of dose, dose rate and quality of radiation on the dy- namics and survival of the spermatogonial population of the mouse. Jap. J. Genet..

40 (Suppl.) : 119-127. RIEMAXN, J. G., 1967. A cytological study of radiation effects in testes of the screw-worm

fly, Coelilioin\ia lioininirora.r ( Diptera : Calliphoridae). Ann. Entoinol. Sne. Ainer.,

60 : 308-320. ' Ris, H., 1942. A cytological and experimental analysis of the meiotic behavior of the uni-

valent X chromosome in the bearberry aphid Tamalia ( = Plivllaphis ) caweni (Ckll.).

J. E.rp. Zool.. 90 : 267-330. SADO, T., 1961. Spermatogenesis of the silkworm and its hearing on the radiation induced

sterility. Jap. J. Genet.. 36 ( Suppl.) : 136-151. Syi'iKE, RICHARD D., 1970. The effects of acute gamma irradiation on the brine shrimp,

Artcin'w. II. Female reproductive performance. B\ol. Hull. 139: 375-385.

STEEL, R. G. D., AXD J. H. TORRIE, 1960. Principles and Procedures of Statistics. McGraw- Hill Book Co., Inc., New York, 481 pp. STEFAXI, R., 1963. II centromere non localizzato in Arte/nia salina Leach. Atti Aecad. Naz.

Lined Rend. Cl. Sci. Fis. Mat. Natur.. 35 : 375-378. TAZIMA, Y., 1961. Consideration on tlie changes in observed mutation rates in the silkworm

after irradiation of various stages of gametogenesis. Jap. J. Genet., 36 (Suppl.) : 50-64. TAZIMA, Y., 1964. Tlie Genetics of the Silkii'orm. Logos Press, London, 253 pp. TAZIMA, Y., AND K. OXIMART, 1969. Frequency pattern of mosaic and whole-body mutants

induced by ionizing radiations in post-meiotic cells of the male silkworm. J\Iutat.

Res., 8: 177-190. WALKER, D. W., AND V. QUINTANA, 1968. Inherited partial sterility among survivors from

irradiation-eradication experiment. /. Econ. Entomol, 61 : 318-319.

Reference : Hwl. null.. 139: 375-3S5. ( ( )ctober, 1<>7<))

THE EFFECTS OF ACUTE GAMMA IRRADIATION ON THE BRINE SHRIMP. ARTEMIA. II. FEMALE REPRODUCTIVE PERFORMANCE '•

RICHARD D. SQUIRE*

(ienetics Department, Xorth Carolina State University. Ralcnih, Xorth Carolina 27607

The effects of acute gamma irradiation on adult life span and male reproductive performance were reported in the first paper of this series (Squire and Grosch, 1970), along with the Materials and Methods, and the general rationale behind the experiments. The present paper records the effects on female reproductive performance.

The earlier reports of Grosch and Erdman ( 1955 ) and Grosch and Sullivan (1955) described some of the criteria of x-ray damage to female reproductive performance. However, these data were dependent upon culture techniques which have since been modified. In addition, these reports did not provide an analysis according to the gametogenic stage irradiated. The report of Grosch ( 1962) was restricted to the analysis of populations of irradiated ancestry, and did not concern the treated generation itself.

Cervini and Giavelli (1965), Giavelli (1966), Giavelli and Cervini (!%(>). and Metalli and Ballardin (1962) restricted their studies to fecundity and fer- tility analyses of viviparous broods after oocyte treatment of various parthenogenetic species of Artcniia.

RESULTS Fecundity

The statistical procedures were the same as those described for the life span studies (Squire and Grosch, 1970). Fecundity was defined as the sum total of viviparous (nauplii) and oviparous (cysts) gametes produced by a given female throughout her life. All treatments significantly reduced fecundity (Table I). A dose of 5 kR resulted in greatly reduced fecundity ( X - 43 ) , while doses of 10 50. and 100 kR resulted in total infecundity. Since the mean life span of the 5 kR

1 This research was conducted in partial fulfillment of requirements for the author's doctoral dissertation at Xorth Carolina State University.

- Financial support was provided hy the Genetics Training Grant ( Public Health Service) for the summer of 1965, and subsequent work was conducted as part of the N. A. S. A. Project NsG-678, Utilization of Hahrohrneon and Artemia as Experimental Materials in Bioastronautic Studies.

3 Contribution from the Genetics Department, North Carolina Agricultural Experiment Station, Raleigh, North Carolina. Published with the approval of the Director of Research as Paper No. 309.3 of the Journal Series.

4 Current Address : Biology Department, Long Island University, Brooklyn, New York 11201.

375

376

RICHARD D. SQUIRE

TABLE I

Average In nudity and nuniher of broods per irradiated female, with one-tailed t test comparisons of treated versus control animals

		Fecundity/female	No. of broods 'female
	Dose
^evel	IkR)
		X =F S.E.	t	X T S.E.	t
i	0	1695 T 329	—	10.1 =F 1.8	—
3	1	919 T 216	1.974*	7.0 T 1.3	1.569
4	2	289 =F 134	3.961**	4.2 =F 1.1	3.123**
5	5	43 =F 17	5.021**	1.1 T 0.3	5.800***

* Significant at 0.05.

** Significant at 0.01.

*** Significant at 0.001.

on

fecundity

females was virtually identical to that of the controls, this effect is independent of life span.

The mean differences between replicates within treatment levels were not significant for any dose at 0.05. The F test for variances between replicates within treatment levels was significant for level 5 (5 kR). This difference was due to a single female which produced 1255 gametes, when the group average excluding her contribution was 66. She was 17 standard deviations from the mean, and none of her cysts hatched after the first brood. Accordingly the data from this female were not used in the fecundity analysis. Adjusted means, vari- ances and degrees of freedom were used for comparisons involving this treatment level.

Fertility

A similar analysis was conducted for fertility (Table II), which was defined as the sum total of the nauplii produced as viviparous broods plus those which hatched from oviparous cysts. The difference in variances (but not means) was once again significant for 5 kR. The separate means were 4.6 and 0.2. Appro-

TABI.F 1 1

Average fertility pier female and per cent hatchability per brood, with one-tailed i test comparisons of treated versus control animals

		Fertility/female	% hatchability brood
Level	Dose (kR)
		X T S.E.	i	X	t
1	0	1318 =F 358	—	18.5	—
3	1	718 T 201	1.46 (0.10)	21.0	(0.503)
4	2	64 T 30	3.49**	12.1	1.824*
5	5	2.4 T 1.7	3.68**	2.2	7.412*

Significant at 0.05. Significant at 0.01.

FEMALE REPRODUCTIVE PERFORMANCE

377

priate pooled values were nevertheless obtained for all treatment levels and com- parisons made (Table II i. The lack of significance at 1 kR may be ascribed to the presence of a few females which produced large numbers of cysts with low hatchability. This comparison is significant at the 0.10 level.

As may be seen in Tables I-TT, the average fecundity and fertility per female decreases with increasing dose. The same trend is found when the per female values are computed for average brood size, largest brood size per treatment level, the average of the largest single brood for each of the fecund females, or the average largest brood of the three best females (Table III ).

Cyst hatchability

Hatchability tests for all oviparous broods were conducted and analyzed in

- i .

the same manner as previously described for males. Untransformed treatment

TABLE III

Evidence for a reduction in the number of competent oogonia as a result of gamma irradiation of adult female brine shrimp

Level	Gamma Ray	Avg. size of brood	Largest single brood value in treatment level	Avg. of the largest single brood pro- duced by each female	Avg. fecundity of the largest single brood produced by each of
	(kR)				the 3 most produc-
		Fee.	Pert.	Fee.	Fert.	Fee.	Fert.	tive females
1	0	168	134	485	485	268	240	423
3	1	130	102	374	374	191	179	310
4	2	69	15	217	99	97	26	177
5*	5	57	1	238	18	71	3	133

" The values for level 5 include the exceptionally fecund female described in the text.

means and t test comparisons are given in Table II. While 1 kR did not decrease hatchability. 2 kR and 5 kR did so. The differences of 1 kR vs. 2 kR. and 2 kR rs. 5 kR are also significant.

X umber oj broods

The average number of broods per female was calculated in the same manner as the fecundity data. The reduction in brood number was highly significant at doses of 2 kR and more (Table I). The average interval between broods was calculated per female (Table IV) and found to increase markedly at 2 kR and 5 kR. The average percentage of gametes produced as cysts per female also rises at these levels ( Table I V ) .

Sim'irnl to adulthood and sc.v ratio

The percentage of the nauplii (viviparous or hatched from cysts) to reach maturity was also calculated for broods I and IV. Survival to adulthood mea- sures late dominant lethal events, since, as measured here, it excluded all pre-

378

RICHARD D.

IAIH.K IV

Average brood intervals, l>i-n cnlage of oviparous gametes, and the fraction of encysted broods which failed to hatch after adult females were irradiated

Level	Gamma Rav Dose (kR)	Avg brood interval (days)	Avg % of gametes deposited as cysts female	Fraction of encysted broods which failed to hatch
1	(1	3.74	41.3	1/32
3	1	3.53	38.5	3 11 1
4	2	6.89	86.4	1/23
5	5	9.08	85.7	14 17*

* Nine-tenths of these broods were produced by one exceptional female; see text.

naupliar deaths from analysis. Analysis of brood I was restricted to those broods which were deposited between the fourth and sixth days after treatment, and thus were derived from irradiated oocytes or oocyte-nurse cell complexes. If the time sequence published for the diploid parthenogenetic species from Sete ( Cervine and Giavelli, 1965 ) is applicable to this species, then most of these oocytes \\ere in prophase I at time of treatment, and some may have reached metaphase I. None of these gametes were post metaphase I at time of treatment, since in order to be fertilized the egg's must be in the oviducts when copulation occurs; and such eggs are in metaphase I at that time.

Brood I was analyzed as 2 X 4 contingency tables ( Steel and Torrie, 1960 ) lor survival to adulthood and sex ratio. Survival to adulthood was significantly reduced with increasing dose (P < 0.005). There was no significant change in sex ratio (Table V ).

Brood IV animals were derived from treated oogonia. The survival and sex ratio data are summarized in Table V. None of these values are significant, although the survival value for level 4 might have reached significance with larger

TABLE V

The s arch-til to adulthood and sex ratio of offspring from ten irradiated females per gamma ra v dose level shown

Brood #	Level	Dose (kR)	No. of nauplii	No. of survivors	% survival	Sex ratio % males
h	1	0	157	129	82.2	50.4
	3	1	53	36	67.9	52.8
	4	2	50	21	42.0	52.4
	5	5	18	6	33.0	33.3
X2					40.25**	0.95 NS
[V	1	0	1147	582	50.7	48.7
	3	1	581	252	43.4	49.6
	4	2	55	20	36.4	60.0
	5	5	—	—	—	—
X2					1.04NS	0.18NS

a Brood I data selected for days 4-6 only ; see text. "* Significant at 0.01.

FEMALE REPRODUCTIVE PERFORMANCE

samples. Survival was also analyzed by using t test comparisons of percentage data following the arcsin \ ' , transformation, as well as with the contingency table shown.

Additional observations

Gross observation of females of replicate 1 on the 22nd day after treatment revealed full ovisacs in all cases after 5 and 10 kR. Nevertheless, two of the five females given 5 kR and all of the three surviving 10 kR failed to produce recover- able nauplii or cysts subsequent to this observation. No excretory gland abnor- malities were noted.

Cysts from irradiated females were often orange and translucent, while normal cysts are opaque and brown. Cysts deposited after 5 kR exposures were particu- larly abnormal in this respect, and were also characterized by frequent brittleness ( which resulted in cyst breakage when touched with the dissecting needle during hatchability studies ) and flattened cysts approaching a disc shape. Since all cysts were treated and stored under identical conditions, the abnormality is attributed to experimental treatment, rather than to attendant conditions of the experiment. Broods in which translucent cysts predominated showed poor hatchability and particles resembling cysts occasionally degenerated completely during a brief storage period on filter paper.

Some pair matings were set up from first-brood individuals. Offspring from the 5 kR series failed to reproduce (two full-sib matings plus three females crossed to normal males). Eight full-sib matings from the 2 kR series produced cysts but no nauplii. The offspring from 1 kR females produced both nauplii and cysts. Mortality was high in the 2 kR and 5 kR offspring following sexual maturity. The cysts produced by these Xx individuals were not tested for hatchability in most cases, and full life history data were not obtained.

A few morphological abnormalities were scored in the Xi of treated females. Unfortunately, these animals usually failed to reproduce. The fusion of adjacent appendages to each other, or to genitalia, was the most common trait. Abnormally small eyes or claspers were asymmetrical traits, and abnormal reproductive organs (only one functional ovary), bent tails, missing eyes or claspers were also noted.

DISCUSSION

Factors affecting female reproductive performance

Female fecundity was defined as the sum total of all recoverable gametes produced by a single female throughout her reproductive history. Such a measure is the final result of many interacting factors. Presumably a cohort of oogonia is produced from a smaller number of stem cells. On an average of every three and one-half days, these oogonia then produce a ribbon of cells which in time differen- tiate into an oocyte and a nurse-cell complex. Nurse cells become polyploid and contribute to vitellogenesis. The blood cells have also been suggested to play a role by transporting materials to the ovarian region. Finally, a differentiated oocyte is produced which enters the median ovisac (uterus) and is quickly fertilized. (For a more comprehensive coverage, see papers by Anteunis, Fautrez-Firlefyn and

380 RICHARD D. SQUIRE

Fautrez, t966a, 1('( >(>!>; Bowen, 1962; Cassidy, 1965; Fautrez-'Firlefyn, 1951 ; Loch- head, 1950; Lochhead and Lochhead, 1941, 1967.)

In the author's opinion, .-Irlciiiia oogonia comprise a nonexhausting stem-cell population in the adult female; and a single oogonium may contribute to each successive hrood. According to this view, the stem-cell population would he comprised of "primary oogonia." Kverv three and one-half days, each primary oogonium would divide, producing one primary oogonium and one "secondary oogonium." This secondary oogonium would then undergo a series of mitotic divisions and produce an oocyte-nurse cell complex.

Normally, the second cleavage division occurs ahout five hours after the descent of the eggs into the uterus, and shell formation is initiated at this time in oviparous broods (Fautrez-Firlefyn and Van Dyck, 1961). Unfertilized eggs may also be encysted (Squire, unpublished data). In the case of viviparous broods, the zygotes must differentiate into a swimming nauplius in order to be scored. In the case of oviparous broods, any gamete which passes into the uterus will probably be recovered so long as shell deposition is approximately normal. Cyst hatch- ability, then, is a measure of dominant lethal events. Factors which may con- ceivably affect cyst hatchability include (1 ) failure of fertilization, (2) nutritional inadequacies of the oocyte resulting from damage to the oocyte, to the nutritive cells of the complex, or to general physiological disturbances in the females, (3) im- proper shell deposition, and (4) genetic lethality. Reabsorption, either of gametes prior to fertilization, or of viviparous zygotes after fertilization, might also he a factor in scoring fertility and fecundity.

Fecundity was defined as the total number of recoverable gametes. Thus, it would include (1) any change in the number of oogonia actually present at the time, (2) induced changes in the inherent capacity of those oogonia which were present to produce functional gametes, (3) any nutritional changes which led to the failure of a differentiating oocyte to be recovered (such as the absence of vitellogenesis or reabsorption of the oocyte), (4) reabsorption of viviparous zygotes and (5) failure to score partially developed viviparous zygotes, which had been expelled, prior to their disintegration.

If the concept of an oogonial stem-cell component is correct, then any change in the general health of a female Artcuila may temporarily or permanently alter the number of gametes recovered per brood. Such a decrease could occur without altering the actual number of oogonia present in the ovaries. Although we have no information concerning the effects of irradiation on the physiology of Artcmia, presumably such effects exist, and quite possibly nutrient utilization is altered.

Some information does exist concerning various aspects of brine shrimp nutri- tion. Lochhead and Lochhead (personal communication) reported that oocytes may fail to differentiate in starved animals. They believe that oogonial number is more strongly influenced by nutrition than by age. Subsequent events in our laboratory would tend to support some of their conclusions. Greatly reduced fecundity and stage-specific patterns of larval mortality appeared to be associated with a lack of algae in the diet.

D'Agostino and Provasoli (1968) demonstrated that the inter-relationship of algal diet, salt concentration, other nutritional requirements, and fertility is not

FEMALE REPRODUCTIVE PERFORMANCE 381

a simple one. Reduced salinity resulted in depressed fertility unless specific nutrients were added to their synthetic media.

Fertility, defined as the sum total of the recovered nauplii from viviparous and oviparous broods, combines the factors included in fecundity and hatchability. It should be pointed out that \ve still do not know what factors control the mechanism of cyst deposition. The frequency of cyst production is generally felt to increase as a response to stress, but normal females also produce oviparous as well as viviparous broods. Various environmental factors such as diet, temperature and salinity have been implicated at one time or another.

Fecundity of irradiated females

Female fecundity was progressively reduced at doses of from 1 kR to 5 kR. No recoverable gametes were found at 10 kR or higher doses. The data sum- marized in Tables I-III indicate that the total number of oogonia which success- fully contribute to each brood has been progressively reduced with increasing doses. However, no cytological study of the number of oogonia actually present in these females has been made.

The largest single brood per female, the single largest brood per treatment, and the average of the three largest brood values (on a per-female basis) per treatment group all progressively decrease with increasing dose. This leads me to hypothesize that gonial cell lethality occurs at all doses of 1 kR and above, although the other factors discussed above should not be ruled out.

Typically, the first brood produced by a female Artcinia is relatively small, but subsequent broods are larger. This suggests that the young female may have fewer competent oogonia capable of contributing to each brood, but the number of such oogonia increases rapidly following sexual maturity. Whether this increase is due to recruitment from already existing "dormant" oogonia or to multiplication of stem-cells cannot be determined at the present time. In either case, a differential radio-sensitivity is likely to occur, with actively dividing cells being the most sensitive to damage. Such damage would be expected to give the kind of results reported here.

Although two reports (Grosch and Erdman, 1955; Grosch and Sullivan, 1955) indicated the sterilizing dose for premeiotic and early postmeiotic stages, there have been no reports directly concerned with gonial cell sensitivity in Artemia. The silkworm, Boinby.v, is perhaps the closest biological system with which we can currently make comparisons. This Lepidopteran has 28 pairs of minute and presumably holokinetic chromosomes, as compared to 21 pairs of holokinetic chromosomes in the common Californian race of Arfeiniu. Tazima and Kondo (1963, page 246) concluded that the LD-50 is about 1000 and 2000 R for spermato- gonia and oogonia, respectively, when exposed to Cs-137 acute gamma irradiation. Ballardin and Metalli (1968) state that an oogonial dose of 1 kR had no effect on the fecundity of the diploid parthenogenetic species from Sete, when measured over a 20-day period. Their abstract gave no data concerning this point.

Cyst production and hatchability

The hatchability data show no detectable dominant lethality at 1 kR, although such events do occur at 2 kR and higher doses. The high frequencies of abnormal

RICH ARh I). SQUIRE

cvsts recovered from 5 kR females suggest that the shell glands are damaged by this dose, and lower levels may also have been affected. Such abnormal shell deposition could easily alter the resistance of the embryo to normal environmental conditions such as drying, and thus by reflected in hatchability and fertility data. Since cysts from unfertilized females appear normal, it is concluded that genetic dominant lethals which were induced in the oogoiiia would not affect cyst morphology.

The analysis of hatchability data excluded all encysted broods with zero hatch- ability. This exclusion does not seriously affect the data as summarized in Table 1 1 for the following reasons. Such broods are extremely rare at all levels except 5 kR (Table IV). At this level the average hatchability already approaches zero (X ~ 2.2%). The inclusion of broods with zero hatchability would reduce this value still further, while values for the other levels would remain substantially the same as presented. Excluding such broods is an attempt to separate other domi- nant lethal events from the failure of fertilization. The latter condition will produce broods with zero hatchability.

Repeated failure of copulation often results in the accumulation of several unexpelled egg clutches within the female. Gamete degeneration becomes evident, with pronounced accumulations of yolky material and darkening of the excretory glands (Lochhead and Lochhead, 1941) which may result in temporary or permanent infecundity (M. Lochhead, personal communication). Some females successfully expell numerous egg clutches in the absence of copulation and thus escape this syndrome. Expelled material is usually in the form of cysts, but undeveloped products lacking cyst walls have been recovered. Expulsion of partially developed embryos has also been observed in females after a fractionated dose of 5 kR (1 kR of gamma radiation per day for five days; Squire, unpublished data). These embryos degenerate rapidly at summer temperatures and are easily overlooked. On the other hand, on several filter papers used to separate encysted broods I failed to find cysts after a storage period and suspect that the objects filtered may have been undeveloped eggs without cyst walls.

The average per cent of gametes deposited per female (Table IV) must be viewed with these factors in mind. Nevertheless the difference in values at 2 kR and 5 kR is outstanding. This difference becomes even more remarkable upon observing that at 5 kR all recoverable broods consisted solely of cysts when premeiotic stages were irradiated, and the same wras usually true for the 2 kR series as well. Viviparious broods were frequent after 1 kR and in the controls.

Brood interval and number of broods

The average interval between broods also increased in the 2 kR and 5 kR levels (Table IV). Examination of individual female records suggest this increase was due to the failure of whole broods to be recovered, rather than to a simple lengthening of the interval between broods. This observation cannot be explained simply by complete dominant lethality in the 2 kR series, since most encysted broods showed some hatch ; and a number of viviparous broods were scored. Complete dominant lethality could account for the absence of viviparous broods in the 5 kR series, however, since cyst hatchability approached zero for this group. Failure of fertilization may also explain the 5 kR results, but once again the

FEMALE REPRODUCTIVE PERFOK. \IAXCE

relative infrequency of encysted broods with zero hatchability makes this explana- tion difficult to accept for the 2 kR scries (Table IV ). The observation of Grosch and Erdman (1955, page 2X0 ) bears repeating: "the neighborhood of 2000 R is critical not only for numbers <>t broods produced but on the basis ol inhibition of viviparity."

In earlier research, an acute dose of 2500 R x-days resulted in a cessation of female gamete production, while 2250 R or less did not prevent continued reproduc- tion by cells which were premeiotic at the time of treatment (Grosch and Sullivan, 1955). Acute x-ray doses of 4080 R or more, usually resulted in females with empty uteri, although exceptional females produced a single brood from cells which were prezygotic at the time of irradiation (Grosch and Krdman, 1955). In the present experiment, full uteri were observed at 5 and 10 kl\, although no p'ametes were recovered from 10 kR females and 5 kR females were highlv

o <-> -

infertile. The earlier and recent experiments differed in several factors. In 1955 sea water rather than brine was used. This resulted in a shortened life span for irradiated and control animals, and probably imposed an additional stress as well. The reproductive behavior of treated specimens of Artcinia would thus reflect the combined effects of stress due to irradiation and suboptimal salinity. Squire ( unpublished data) found that the fertility of stock #3 animals cultured in plain Instant Ocean was only 14% that of animals maintained in the improved medium, while Grosch (1962) found that the adaptive values of experimental cultures varied with salinity.

In the present experiment, several 5 kR females were characterized by ex- tremely late dates of brood I deposition. A detailed analysis shows that of the 10 females treated, one produced 1 1 broods, four produced two broods each, two produced one brood each, and two failed to produce anv recoverable broods. In all but one case, these broods were probably oogonia at the time of treatment, suggesting that 5 kR resulted in complete dominant lethality of oocytes irradiated prior to metaphase I.

When the life span data of Grosch and Erdman ( 1955 I are superimposed on the present reproductive histories of various treatment levels, the number of broods per female is quite similar in the two experiments. \Ye may therefore conclude that most, if not all, of the discrepancies between the two experiments stem from differences in life span. As they point out, the first brood deposited in their experiment usually represented cells which were postmeiotic at the time of treatment.

A'j mortality, sc.v ratio, and reproductive performance

Brood I data for survival to adulthood, adult mortality and reproductive pat- terns demonstrate the presence of genetic damage in those animals descended from 2 kR and 5 kR females. Genetic damage at a lower dose has been reported for diploid parthenogenetic Artcniia from Cagliari. A reduction in the fertility of irradiated and X, females followed a 1 kR dose of x-rays to prophase oocytes ( Metalli and Ballardin. 1962). This reduction was significantly greater in the X, than in the treated generation. They obtained similar results with the tetraploid parthenogenetic species from Comacchio. except that the effect was

384 RICHARD D. SQUIRE

less pronounced, and there1 was no signilicanl difference between the Xt and 1 1 ' -ited generations.

Survival to adulthood of nanplii or hatch derived from irradiated oogonia

( brood IV ) demonstrated a decrease in average values with increasing dose, lint

was not significant. Ballardin and Metalli ( 1968 j state that an oogonial dose of

1 kR had no effect on the survival to adulthood of Xi diploid parthenogenetic

Artemia from Sete.

As would be expected, the results of various radiation experiments differ according to the polyploid level and the type of meiosis (regular or variously modi- fied) characteristic of the particular Arteinia species. Additional data from these species will provide a unique opportunity for comparative studies.

The failure of any treatment to seriously affect the sex ratio suggests that the differential segment of the Y-chromosome may not contain many viability loci in the heterogametic female.

I would like to thank Mrs. Sally Barnes and Mrs. Ann G. Spencer for the technical assistance provided during this experiment. The advice and critical review provided by Dr. Daniel S. Grosch was deeply appreciated.

SUMMARY AND CONCLUSIONS

Treated females were completely infecund after doses of 10-100 kR. Reduced fecundity resulted from closes of 1-5 kR. This is ascribed to oogonial lethality, and it is proposed that the Artcinia ovary contains a non-exhausted oogonial stem-cell component which contributes to each successive brood. Additional causes of reduced brood size may be nutritional inadequacies and other physiological damage induced by the treatment. Sterility was almost complete after 5 kR.

Cyst hatchability data revealed no detectable dominant lethality after 1 kR, although such effects did occur after 2 and 5 kR. Some of the failure in hatching reflects probable physiological damage to the shell glands, as well as other physiological and genetic components.

The average number of broods was significantly decreased after 2 and 5 kR. Much of this reduction resulted from the absence of entire broods from the recorded data. Viviparity was also inhibited at these doses.

Data for survival to adulthood, adult mortality and reproductive patterns demonstrate that genetic damage was present in those animals descended from oocytes which had been treated with 2 or 5 kR. Xo definite decrease in survival to adulthood was found in animals descended from treated oogonia after 1 or 2 kR. Sex ratios were not significantly changed with any dose.

LITERATURE CITED

ANTEUNIS, A., N. EAUTREZ-FIKLKFYX AND J. FAUTREZ, 1966a. La structure de ponts inter-

cellulaircs "obtures" et "ouverts" entre oogonies et oocytes dans 1'ovaire d'Artemia

salina. Arch. Bio!., 77 : 645-664. ANTEUNIS, A., N. FATTKEZ-FIKLEFYN AMI J. FAUTREZ, 1966b. L'incorporation de cellules

nourricieres par 1'oocvU- d'- Irtcinin suliuii. Etude an microscope electroiiique. Arch.

Biol, 77 : 665-676.

FEMALE REPRODUCTIVE PERFORMANCE

BALLARDIN, E., AND P. METALLI, 1968. Kstim;iies of SOUK- conii><mcnts of Illness in dipl"i.l

parlhenogenetic Artemia salimi irradiated over several generations. .I//; . Iss. dcncl.

I tul.. 13: 341-345. Mnui'N, S. T., 1962. The genetics of Ar/emia saliua. I. The reproductive rycle. />'/"/. />'»//.,

122: 25-32. CASSIDV, J. D., 1965. Aspects of ultrastructnre and cytochemistry during oogenesis in

. Irtemia salimi. Biol. Hull.. 129 : 402-403. CERVIX i. A., AXD S. GIAVEI.LI, 19d5. Radiosensilivity of different mciolic stages of oocytes in

parlhenogenetic diploid .-Irfcinia saliua Leach. Mntat. l\cs., 2 : 452-456. D'AoosTixo, A. S., AND L. PKOVA.MH.I, 1968. Effects of salinity and nutrients on mono- and

diaxenic cultures of two strains of Artcmia salimi. Biol. Bull., 134: 1-14. FAUTREZ-FIRLEFYX, N., 1951. Etude cytochimique des acides nucleiques au cours de la

gametogenese et des premiers stades du developpement embryonnaire chez Artemia

saliua L. Arch. Biol., 62 : 391-438. F.\rTRE/c-FiKLEKYx, N., AXD F. VAN DYCK, 1961. La culture "iii vitro" de 1'oeuf d'Artemia

salina. Arch. Zool. E.vp. Gen.. 100: 37-42. GIAVELLI, S., 1966. Radiosensitivity of Artcmia oocytes. Int. Con;//-. Radiat. Res. Book

Abstracts, 3 : 92. GIAVELLI, S., AXD A. M. CERVIX:, 1966. La curva di radiosensibilita di ovociti di Artcmia

salina. Aiti Ass. Genet. Hal.. 11 : 292-300. GROSCH, D. S., 1962. The survival of Artemia populations in radioactive sea water. Biol.

Bull.. 123 : 302-316. GROSCH, D. S., AXD H. E. ERDMAX, 1955. X-ray effects on adult Artcmia. Biol. Bull.. 108:

277-282. GROSCH, D S., AXD R. L. SULLIVAX. 1955. X-ray induced cessation of gamete production by

adult female Artcmia. Biol. Bull., 109 : 359. LOCIIIIKAD, \. H., 1950. Artemia, Pages 394-399 in: F. A. Brown, Jr., Ed., Selected Inrcrtc-

hratc Types. John Wiley and Sons, Inc., New York. LOCH HEAD, J. H., AXD AI. S. LOCHHKAD, 1941. Studies on the blood and related tissues in

. Irtonia (Crustacea Anostraca). /. Morphol., 68 : 593-632. LOCH HEAD, J. H., AXD M. S. LOCHHEAD, 1967. The development of oocytes in the brine

shrimp, Artemia. Biol. Bull.. 133 : 453-454. METALLI, P., AXD E. BALLARDIX, 1962. First results on X-ray induced genetic damage in

Artcmia salimi Leach. Atti Ass. Genet. Ital.. 7: 219-231. SIJIIKE, R. D., AXD D. S. GROSCH, 1970. The effects of acute gamma irradiation on the brine

shrimp, Artemia. I. Life spans and male reproductive performance. Biol. Bull.,

139: 363-374.

STEEL, R. G. D., AXD J. H. TORRIE, 1960. Principles and Procedures of Statistics. McGraw- Hill Book Co., Inc., New York, 481 pp. TAZIMA, Y., AND S. KONDO, 1963. Differential radiation-sensilivity of germ cells as a possible

interprelation of sex difference in dose-rate dependence of induced mutation rates in

the silkworm. Pages 237-252 in : F. H. Sobels, Ed., Repair From Genetic Radiation

Damage. The Macmillan Co., New York.

Reference: Biol. />'////., 139: .W. 401. (October, 1('7(»

STUDIES ON Till'. BIOLUMINESCENCE OP" THE MARINE OSTRACOD CRL'STACEAX CYPRIDINA SERRATA1

FREDERICK I. TSUJI, RICHARD V. LYNCH, III AND YATA HANEDA

Department of Biophysics and Microbiology, University of Pittsburgh, and Veterans Hospital, ritlsl>nr(/h, /'ciiiisyl-niiiia, and Yokosuka City Museum, Yokosuka, Japan

At present, at least three species of marine ostracods belonging to the family Cypridinidae are known to be luminous. Among these, the luminescence of only one species has been well-studied. This is Cyfridina hilgendorfii Miiller, 1890, a species found in the coastal waters of Japan. Extensive studies carried out during the past 50 years have yielded detailed knowledge concerning the mechanism of bioluminescence. The organism lives in the sand and comes out to feed at night. When mechanically disturbed, the organism produces a blue luminescence by ejecting luciferin and luciferase into the surrounding sea water from two separate glands. The light-emitting reaction involves the oxidation of luciferin by molecular oxygen, catalyzed by the enzyme luciferase.

The second species is Cypridina noctiluca Kajiyama, 1912. In contrast to C. lii't'/cndorfii, C. noctiluca is a free-swimming pelagic form. It is widely distributed along coastal waters in the western Pacific from southern Japan and Hawaii to Australia and Southeast Asia, and in the Indian Ocean. Haneda (1940) observed C. noctiluca at Palau Island. When the beam of an electric light was directed into the water where large numbers of the organisms were swimming, a bright luminous response was obtained. When a plus ( + ) mark was written on the surface of the water and the light immediately extinguished, a luminous plus ( + ) mark could be observed in the darkness. Other plankton organisms did not give the same response. Haneda (1953) also studied C. noctiluca at Hachijo Island, a subtropical island located approximately 200 kilometers south of Honshu, Japan. The secretory behavior and color of light of C. noctiluca were the same as C. liilyendorfii. Light resulted (positive luciferin-luciferase reaction) when a hot- water extract (luciferin) and a cold-water extract (luciferase) of the organism were mixed. These extracts also gave reciprocal light-emitting cross-reactions with hot- and cold-water extracts of C. hihjcndorfii.

The third species is Vargnla Jian'cyi, recently reported from Jamaica, West Indies, by Seliger and McElroy ( 1965 ) and described by Kornicker and King ( 1965 ). Measurement of the bioluminescence emission spectrum showed a peak at 478 nin, close to the peak of 465 nm for C. hilgendorfii.

1 This work was carried out at Madang, New Guinea, under Program C, ALPHA HELIX 1969 Biological Expedition to New Guinea, Scripps Institution of Oceanography, University of California. It was supported in part by grants from the National Science Foundation (G-274) and the Japan Society for the Promotion of Science under the United States-Japan Cooperative Science Program. This is publication No. 167 of the Department of Biophysics and Micro- biology, University of Pittsburgh.

386

BIOLUMINESCENCE OF C. SERRAT.l 387

The subject of this paper concerns a fourth species, Cv/iridnni s emit a Miillcr. \(>0b. C. scrrata was collected during the recent 1\/Y Al|)lia Helix Biological Expedition to New Guinea. Little is known about the distribution of this organism. The specimens studied by Miiller (1(>0(>) were obtained from three locations: Paternoster Island (Station 40), approximately 11X"'K, 7 S in the Flores Sea, Indonesia; Xorth Ubian (Station 99), 120°26'E, 6°7'X. Philippines; and Sulu Archipelago (Station 109), Philippines. The present finding of C. serrata at Madang, Xew Guinea, suggests that the organism is a widely distributed species in the western Pacific. \Ye present herein the first observations of luminescence in this organism.

MATERIALS AND METHODS

Cypritlina scrrata were collected in a cove in Kranket Island next to Dallman Passage in Madang Harbor, New Guinea. They were also found off Beliau Island and in Binnen Harbor, hut not in such large numbers. C. scrrata were located by shining a flashlight into the water in 2-3 second bursts. This caused elliptical clouds of bright blue luminescence, around 2—15 cm long, to appear in the water, presumably as a result of C. scrrata organisms ejecting luciferin and lucif erase into the sea water.

Collection was carried out by towing two weighted 38-mesh plankton nets (2-3 miles/hour) at a depth of about 1 meter below the surface through waters showing the best flashlight response and emptying them periodically into buckets containing sea water.

Towing distance from shore ranged ironi 2-15 meters along a shore length of approximately 1 kilometer. At high tide, the depth within this area varied between 2-10 meters. An attempt was made to collect C. scrrata by lowering fish heads attached to strings in the water, the same method used for collecting C. hilgendorfii in Japan, but it was unsuccessful. All collections were made between 8-11 PM. Buckets containing the night's collection were strained first through a coarse wire mesh, then through a 74-mesh plankton net screen. When examined microscopically, the filtered material consisted largely of copepods, non- luminous shrimps, siphonophore fragments and C. scrrata. Among these organisms, only C. scrrata was found to luminesce spontaneously. Microscopic counts showed 2000-4000 C. scrrata in a single night's collection. Each such collection was washed with clean sea water, then resuspended in a small volume of sea water and filtered through \Yhatman #2 folded filter paper. The filtered residue was then either freeze-dried or used directly in preparing luciferase. In the latter case, the entire batch was ground with a mortar and pestle, washed with about 40 ml of distilled water into a dialysis bag and dialyzed for 60 hours in an ice box against 4 changes of distilled water. The contents of the bag were then centrifuged for 30 minutes at 1500 X g in an Aloe conical centrifuge. The supernatant was decanted, the precipitate washed twice with 10 nil of distilled water and centrifuged. The supernatants were combined and dialyzed for 48 hours in the refrigerator against 4 changes of distilled water. The contents of the bag were freeze-dried, redissolved in 12 ml of 0.07 .17 sodium phosphate buffer, pH 6.8, dialyzed 16 hours against the same buffer, and put on a 45 cm Sephadex G-200 column equilibrated against

|.\ I. TSUJI. R. V. LYNCH, III AMI) Y. HANEDA

(he buffer. The column was eluled with the buffer and 0.02 nil of each colUrled traction (Iraction volume, <>.3 nil) was assayed lor luciferase artivilv by mixing with 2.0 nil of 0.2 M sodium ])hos]ihate buffer, pi I 0.8, and injecting into a vial containing 0.05 nil of C. hilgendorfii lucifcrin. Light intensity was measured in arbitrary light units using a photomultiplier photometer. The active fractions were combined, dialy/ed (»() hours against many changes of distilled water, and freeze-dried. Further purification of luciferase was not carried out due to limited quantities of the organism.

C. scrrata luciferin used for kinetic and oxygen-requirement experiments was prepared by grinding 0.4 g of the freeze-dried material in 1 5 ml of boiling distilled water in an all-glass homogenizer for 1 minute. The suspension was then quickly cooled in an ice bath while bubbling with 99.99% argon. After centrifuging at 12,000 X g at 4° C for 8 minutes, the clear straw-colored solution of luciferin supernatant was stored under argon in an ice-bath until used. Luciferin used in the chromatographic experiments was prepared separately, by extracting 0.25 g of powdered organisms in 15 ml of absolute methanol for 5 hours in an argon atmosphere. After centrifuging at 12,000 X g at 4° C for 10 minutes, 3 ml of the clear brown supernatant were evacuated to dryness, redissolved in a few drops of absolute methanol, and spotted directly on the Whatman filter paper.

C. hilgendorfii luciferase was prepared in a highly purified form by the method of Tsuji and Sowinski (1961), and in a partially purified form by the same method as for C. scrrata luciferase for comparative purposes.

C. hilgendorfii luciferin was purified by the method of Tsuji (1955), except that the initial extraction of luciferin from the defatted Cypridina powder was carried out with absolute methanol instead of butanol. Nine nil of the final ice-cold butanol solution of luciferin were evacuated to dryness, redissolved in 30 ml of 0.1 N HC1, and used directly. For chromatographic experiments, some of the dry luciferin was redissolved in a few drops of absolute methanol, and used directly.

Chromatography of C. scrrata and C. hilgendorfii luciferins was carried out ac- cording to the method of Tsuji (1955), using Whatman No. 3 MM paper and a solvent mixture of ethyl acetate, ethyl alcohol, and water (5:2:3 by volume). Chromatography was carried out at 23° C for 1 hour and 15 minutes.

Antibody to C. hilgendorfii luciferase was prepared as purified gamma globulin fraction from pooled antisera of rabbits immunized against luciferase as described by Tsuji et al. (1969).

Light intensity was measured using a photomultiplier photometer and a chart recorder.

RESULTS 1 . General observations

Cypridina scrrata organisms are shown in Figure 1 . Two other forms, Cypridina incnnis Muller, 1906, and Melavargula species, of undetermined luminosity, were also collected in small numbers in the same area but were not studied. The specimens were identified (which included dissection studies) by Dr. Louis S. Kornicker of the Smithsonian Institution. Specimens of all three species are on deposit at the V. S. National Museum. The depository numbers

BIOLUMINESCENCE OF C. SERRATA

389

and si/e of two of the specimens of ( '. scrni/a (dissected) arc as follows: 128152, male, length !.<>(> nun, height 0.8n mm; 128153. lemale, length 1.53 mm, height 0.86mm.

The bright bluish luminous clouds, produced by ('. scrnt/a when stimulated with a flashlight (see Discussion section), were beautiful and impressive, and probably represent a unique display among luminous organisms. Short 1 second bursts of light produced relatively few clouds and 2 second bursts produced the maximum number of clouds, approximately 50-100 per cubic meter of water. Spontaneously produced luminous clouds were not observed in the water. The response to a

FIGURE 1. Cypridina scrnita, actual size about 1.6 mm; Madang Harbor, October, 1969.

single stimulation was strikingly uniform : the luminous clouds burst instantly and decayed within 3-4 seconds after the flashlight was turned off. A given water volume usually responded 3-5 times to the flashlight before becoming refractory to further stimulation ; that is, shining light into the water did not elicit any more luminous clouds. The interval between flashlight bursts was 4—5 seconds. A refractory water volume did not respond to further flashlight stimulation, but after remaining in darkness for 20 minutes a few luminous clouds could be produced. Many of the luminous clouds were located 1 meter or more below the surface

J

of the water. Often when these clouds were scooped up with a plankton dip net, a bright blue luminous spot was detected on the net and from it a copious luminous secretion soon began to flow down the side of the net. When the organism was touched or gently teased off the net with a finger for identification, the finger became intensely streaked. These large bright blue spots, often continu- ously luminous, are to be contrasted with the myriads of tiny flashes of light

F. T. TSUJI, R. V. LYNCH, III AXI) Y. HAXHDA

lhat are seen when the net is first raised. The light in the latter instance is primarily due to copepods and siphonophores and soon dies down leaving only the bright blue luminous spots of C. scrrata. The color of light was similar, if not identical, to that of living C. hilgendorfii. \\hen the plankton tow nets were emptied several times into the bucket, the sea water in the bucket turned bright! \- luminous. Such sea water, when passed through filter paper, produced light when mixed with C. hihjciulorfii luciterin. Slight mechanical disturbance of the water in the bucket readily caused C. scrrata organisms to emit a brilliant blue glow, light- ing up the bucket for many seconds. When the water in the bucket was swirled, long trails of blue luminous secretion could be observed.

2.$ Sec.

Time

FIGURE 2. Apparent spontaneous flash recordings of 3 Cypridina scrrata organisms in sea water, recorded with a photomultiplier photometer and strip-chart recorder (Mini-Writer, Watanabe Inst. Co.).

Light response to stimulation was less on moonlit nights. On one moonlit night about 15 individual responses were counted in a 1 hour period, whereas earlier in the month when the moon was dark, thousands of responses could be counted in the same area. On such moonlit nights, several hundred C. serrata could still be collected in an evening by towing plankton nets. These organisms were apparently refractory to light stimulation since only a few responses were obtained in the collection area with a flashlight. These organisms also gave almost no streaking in the collecting net and few spontaneous flashes.

BIOLUMINESCENCE OF C. SERKATA

391

2. Flash- recordings

Collected C. scrrata did not respond to stimulation by flashlight immediately after collection or later in the laboratory. However, they did emit apparent spon- taneous flashes of light which were recorded. A typical set of four flashes is shown in Figure 2. This was made by placing 2 or 4 organisms in a vial containing 10 ml of sea water and monitoring the vial in a photometer. The organisms were isolated with a small glass capillary from a mixture of organisms in a Petri dish with the aid of a low-power microscope. Visual inspection of a vial of C. scrrata

C/9

1 5 Sec.

Time

2.5 Sec.

FIGURE 3. Apparent spontaneous flash recordings of 3 Cypriciina scrrata organisms in sea water showing (A) double flash and (B) possibly triple flash (Mini-Writer, Watanabe Inst. Co.).

organisms showed the flashes to be either point sources of light, or in some cases, long thread-like streaks or jets of luminous secretion which issued from the organisms. A point source of light was about the size of the body of C. scrrata so that the whole organism appeared luminous. The light seemed to be of internal origin and did not show any sign of luminous secretion diffusing into the water. Some of the apparent spontaneous flashes were extremely bright, easily visible to the naked eye in a lighted laboratory room whereas some point sources of light were glows that lasted for many minutes (some were watched for over 30 minutes). The flashes in Figure 2 each show a duration of approximately 1.5 seconds. The

392

F. I. TSUJI, K. V. LYNCH, III AND Y. HANEDA

0 10 20 30 40 50 60 FRACTION NUMBER

FIGURE 4. Elution patterns of Cypridina scrrata luciferase (Curve a), Cypridina hilgcn- dorfii luciferase (Curve &), and mixture of C. serrata and C. hilgendorfii luciferases (Curve c) from Sephadex G-200 column. (Note: overlap of some points.)

shape of the flash curve usually varied from those shown in Figure 2 to some with a double spike. The frequency of flashing was irregular and varied with each batch of organisms. The number of active batches, however, was relatively small. Organisms that did not start flashing within 10 minutes after being placed in the photometer usually did not flash later.

A plot of the logarithm of light intensity (Fig. 2) against time shows that the decay for each curve is exponential, with rate constants, from left to right, of 1.43 sec'1; 0.863 sec'1; pre-spike, 1.13 sec'1 and post-spike, 0.818 sec'1; and 0.946 sec'1. In the third curve from the left, the exponential decay is interrupted by an after- spike, but the after-spike also decays exponentially. The after-spike appears to be a second flash superimposed on the first. It may be clue to triggering in view of the known response of the organism to artificial light. Fig. 3A shows two additional flash recordings with double spikes. Assuming that one animal triggers a second animal, the estimated latency was 500-800 milliseconds for the flash response.

BIOLUMINESCENCE OF C. SERRATA

393

In Figure 3B, which represents .still another flash recording, the curve appears to be a superimposition of 3 flashes.

3. Chromatography of luciferase

Twelve milliliters of partially purified C. scrrata luciferase solution possessing a concentration of 0.40 mg/ml and 12 ml of partially purified C. hilgendorfii luciferase solution possessing a concentration of 0.25 mg/ml were prepared in

CO

CD CD

TIME (Seconds)

FIGURE 5. Luminescence intensity decay curves of Cypridina scrrata luciferin-luciferase mixtures, except as noted. For each decay measurement, 1.0 ml of a solution of luciferase dissolved in 0.1 M sodium phosphate buffer, pH 6.8, was injected with a hypodermic syringe into a mixture of 1.5 ml of luciferin + 1.5 ml of 0.1 M sodium phosphate buffer, pH 6.8, except for curve c, in which luciferase was injected into 0.75 ml luciferin 4- 2.25 ml buffer. Luciferase concentrations: curve a, 2.0 mg/ml; curve b, 1.0 mg/ml; curve c, 0.5 mg/ml; and curves d and c, 0.133 mg/ml. Curve /: 1.0 ml C. hilgendorfii luciferase (1.54X 10~4 mg/ml) injected into 1.5 ml luciferin + 1.5 ml buffer. Curve e is shifted upward 0.2 logarithm units for better comparison. Two separate preparations of C. serrata luciferin were used: one for curves a, b, and c, and another for curves d, e, and / (Bristol Recorder).

394 F. I. TSUJI, R. V. LYNCH, III AND Y. HANEDA

0.07 M sodium phosphate buffer, pH 6.8. On assay with C. hilgendorfii luciferin, the C. s errata luciferase solution possessed a somewhat higher activity than the C. hilgendorfii luciferase solution. Four milliliters of each of these preparations \\ere mixed together, the S ml remainder of the luciferase solutions being frozen. The 8 ml of mixture were dialyzed 16 hours against the same buffer and put on a 90 cm Sc-phadex G-200 column. Each eluted fraction (6.3 ml each) was assayed with ('. hilgendorfii luciferin. One day later the 8 ml of C. serrata luciferase solution were thawed, dialyzed as above, and put on the same column. Still another day later, the C. lulf/endorfii luciferase solution was thawed, dialyzed as above, and put on the same column. The elution curves are shown in Figure 4. All three solutions showed a single activity peak, which appeared at the same place (fraction 42) in the elution diagram. Hold-up volume, measured with 2% dextran blue solution, was 135 ml. Both solutions containing C. hilgendorfii luciferase showed small peaks at the beginning of elution due to what might be a polymerized form of the luciferase. We conclude that C. serrata and C. hilgendorfii luciferases are indistinguishable by gel elution chroinatography.

4. Kinetic measurements

The decay of light intensity in various mixtures of C. serrata luciferin and luciferase was measured. The results are shown in Figure 5, with logarithm of light intensity, which is a measure of reaction rate, plotted against time. In the first experiment, represented by curves a, b, and c, the concentration of luciferin was held constant and the luciferase concentration was varied so as to give concen- trations of C, C/2, and C/4, respectively. In each case, the decay of luminescence followed first order kinetics. The calculated rate constants were 5.67 : : 10~2 sec'1, 3.07:: 10~2 sec"1, and 1.52): 10~2 sec"1, respectively. The corresponding half- times for the decays were 5, 10. and 20 seconds. The rate constants are, therefore, observed to be directly proportional to luciferase concentration. A second experi- ment, represented by curves d and c. was run at a lower luciferase concentration. Luciferase concentration was the same (0.133 mg/ml) but the luciferin concentra- tion of e was one-half that of d. The luminescent reaction initially showed a very high rate of decay, but it soon became first order. The rate constants for d and e were 1.97 X 10~3 sec"1 and 3.34 ) : 10"3 sec"1, respectively. The rate was thus increased by a factor of 1.70 (rate constant <?/rate constant d) on a one-half de- crease in luciferin concentration. The rate constant therefore appears to be dependent on luciferin concentration. Because of a limited supply of C. serrata organisms, the experiment could not be carried out with purified C. serrata luciferin. However, when C. serrata luciferin was replaced with highly purified C. hilgendorfii luciferin, the first order rate constants were found to be directly proportional to luciferase concentration and completely independent of luciferin concentration. In the final experiment, C. serrata luciferin was run against a single concentration of C. hilgendorfii luciferase (1.54>:10~4 mg/ml). The decay curve, /, was typical of the curves previously obtained with a rate constant of 3.00 >: 10~3 sec"1. Immediately after mixing, a high initial rate of decay occurred which was followed quickly by a normal first order decay.

BI( (LUMINESCENCE OF C. SERRATA

395

5. O.vyt/eii requirement

Requirement for oxygen in the luminescent reaction was demonstrated by placing 3.0 ml of C. serrata luciferin diluted with 8.0 ml of 0.1 ]\I sodium phosphate buffer, pH (>.S. in one arm of a mixing apparatus, and 5.0 ml of C. serrata luciferase solution in a second arm. Argon (99.99%) was bubbled through both arms for 12 minutes, then the apparatus was evacuated for 2 minutes with a vacuum pump. The arm containing the luciferin was placed in the cell holder of a photo- multiplier photometer, and the luciferase was then added under vacuum. \'o

f> 80 -i

oc

^

CO

6CH

40-

20-

0

mixed

I

air

\

0

10

20

30

40

50

60

70

TIME (Seconds)

FIGURE 6. Light intensity measurement (tracing of recording) showing requirement for oxygen. Cypridina serrata luciferase and luciferin were mixed in absence of oxygen, then air introduced 17 seconds later. Note the high initial rate of decay ( Lords and Northrup Speedomax Recorder).

light was observed. After 17 seconds, air was admitted into the mixture through a capillary tube. The results are shown in Figure 6. The admission of air produced a bright initial burst of light. The high initial rate of decay was rapidly followed by a normal first order decay. The experiment was repeated with C. hilgendorfii luciferin and luciferase with the same result. The data indicate that oxygen is required by C. serrata in the luminescent reaction.

6. Inhibition b\ antibody

The immunological inhibition experiments are summarized in Table I. Rabbit antibody to C. hilgendorfii luciferase was incubated separately with C . liihjendorfii and C. serrata luciferases, closely matched in activity. After incubation, the residual luciferase activity remaining was determined in the separate mixtures. The antibody is seen to inhibit C. hilgendorfii luciferase to a far greater extent

396

F. I. TSUJI, R. V. LYNCH, III AND Y. IIAXHDA

than C. scrrata luciferase. \\ V conclude that C. scrrata luciferase, while possessing similar catalytic activity, is immunochemically different from C. hilgendorfii luciferase.

7. Chromatography of luciferin

Paper chromatography of C. scrrata and C. hilgendorfii luciferins gave identi- cal Rf values (average of 0.65). We conclude that the luciterins are very similar, if not identical.

DISCUSSION

According to Harvey (1952), the genus Cypridina includes between 20 and 25 species. Among these, two species, Cypridina hilgendorfii and C. noctiluca, are known to he luminous. The luminescence of C. hilgendorfii has been extensively

TABLK I Inhibition of luciferase nclirity hy rabbit antibody

Incubation mixture	Cypriiliuii hilgendorfii	Cypridina serraln
Initial light intensity in arbitrary units	Per cent of control	Initial light intensity in arbitrary units	Per cent of control
Control	42. Of	100	44.SJ	100
Control + 2.0 X lO^mg antibody*	24.0	57	41.0	92
Control + 6.0 X 10 2mg antibody* Control + 18.0 X lO^mg antibody*	7.5 2.0	18 5	28.5 15.0	64 34

* Prepared from pooled antisera of rabbits immunized against C. hilgendorfii luciferase.

f Control consisted of 1.54 X 10~4 mg of purified C. hilgendorfii luciferase dissolved in 1.0 ml of 0.1 M sodium phosphate buffer, pH 6.8.

| Control consisted of 1.33 X 10"1 mg of partially purified C. serrata luciferase dissolved in 1.0 ml of 0.1 M sodium phosphate buffer, pH 6.8. Note: All mixtures were brought up to 1.5 ml final volume with 0.1 M sodium phosphate buffer, pH 6.8, after adding antibody. After 19 hours incubation at 4° C, luciferase activity in a 1.0 ml aliquot was determined as the initial maximal light intensity observed on adding a constant saturating concentration of C. hilgendorfii luciferin

studied and needs little comment. C . noctiluca has been studied on different occa- sions hy Haneda (1940, 1953, 1955). In addition, Cypridina norwegica is reported hy Harvey (1952) to yield extracts that give light-emitting cross-reactions with extracts of C. hilgendorfii. Two other positive luciferin-luciferase cross-reactions have been reported. A close relation, Pyrocypris, a genus belonging to the family Cypridinidae, has been shown hy Harvey (1922) to give a luciferin-luciferase cross-reaction with C. hilgendorfii. A Cypridina species from Jamaica, which emits a bluish yellow or yellowish light, has been found hy Harvey (1924) to cross-react with C. hilgendorfii, which emits a bluish light. Harvey found that C. hilgendorfii luciferase when cross-reacted with Jamaican Cypridina luciferin gave a bluish light, whereas Jamaican Cypridina luciferase when cross-reacted with C. Jiilgcndorfii luciferin gave a yellowish light. The results indicated that the color of light depended on the source of luciferase. More recently, a new

BIOLUMINESCENCE OF C. SERRATA

luminous species, l'ar</nla Jiarrcyi. has been reported from Long Bay, northeast Jamaica, by Seliger and McElroy (1965) and described by Kornicker and King (1965).

The present study illustrates the difficulty that may be encountered in trying to identify an organism that luminesces at sea unless specimens are taken. The evidence in the present instance is not unequivocal, but several reasons suggest that the organisms involved at Madang are Cypridina scrrata. First, there wa> a direct correlation between the flashlight response and the presence of C. scrrata in the water. C. scrrata was absent from areas where the flashlight response was negative. \\ hen the plankton net was towed in such areas, the net brought up only mixtures of copepods, non-luminous shrimps, siphonophores and some dinoflagellates. Xo bright blue luminous spots or C. scrrata organisms were found in the net. Second, the blue color of the luminous cloud was similar to the color of light produced by C. scrrata in the laboratory. This evidence, however, cannot be considered very strong since the color of luminescence of most marine organisms is bluish. Third, the cloud \vas observed to drift in the water during the 3-4 seconds of decay. The size of the cloud was many times greater than any of the luminous organisms collected in the water. These observations are difficult to explain except under conditions in which luminous substances or components that react to produce light are ejected into sea water and afterwards diffuse or are carried by the current. Fourth, the behavior of these organisms toward moonlight was similar to C. hi'ijcndorfd. On moonlit nights, few organisms were found to respond to light stimulation and only a relatively small number appeared in the water. In Japan, the authors have also observed that C. hilgendorfii organisms appear in smaller numbers on moonlit nights.

Another question which naturally arises concerns the origin of luciferin and luciferase in the extracts studied. The freeze-dried material contained both C. scrrata and a mixture of luminous and non-luminous copepods, two closely related ostracods. The luciferins and luciferases from these organisms could possibly give light-emitting cross-reactions with each other and with the luciferin and luciferase of C. hilt/ciitJorfii. However, careful studies conducted by Harvey (1926) seem to rule out this possibility. Harvey showed that the hot- and cold-water extracts of copepods do not give the luciferin-luciferase reaction with each other and do not cross-react with the extracts of Cypridina. Thus, the luciferin and luciferase in this study appear to be of C. scrrata origin.

The response of C. scrrata to light stimulation is strikingly similar to that of C. noctiluca. observed by Haneda (1940). The mechanism wherein- a luminous cloud is produced by C. scrrata is undoubtedly similar to that in C. hilgendorfii. In the latter, luciferin and luciferase are ejected into the water from storage glands, where on diffusion, the light reaction takes place. The elucidation of the light-sensitive response mechanism awaits future study. The finding of the luminescence response system in C. scrrata may be considered significant since light-stimulated luminescence is unknown except for C. noctiluca and fireflies exposed to species-specific flash patterns. Turner ( 1 966 ) , however, states that numerous reports by mariners exist in which luminescence has been observed when light is shone into the sea.

.•>(>X F. I. TSUJI, k. V. LYNCH, III AXI) Y . IIAXKhA

( . scrrala is stimulated to luminesce either mechanically or by artificial light and. in addition, luminesces spontaneously; but C. hilgendorfii is known to be .stimulated only mecbanically. Although ('. sernita did not respond to flashlight stimulation when once ])laced in the collection bucket, it did respond \\hen the water was agitated with the hands. The non-responsiveness ol captive C. scrrala to flashlight stimulation was not further investigated and, therefore, no explanation can be given for the lack of response.

The flash recordings of isolated C. sernita organisms in Figures 2 and 3 suggest that the flash of one organism (produced either spontaneously or by collision with the wall or another organism ) triggers a neighboring organism to flash. This assumption is reasonable because of the known response of C. scrrata to artificial light and by the fact that a flash pattern containing an after-spike is not normally observed with luminous organisms. No comparisons can be made between the C. sen-aid flash and the C. hilgendorfii flash, as no records exist, but the dinoflagellate Noctilnca flash has been investigated by Nicol (1958), Hastings (1959), and Eckert (19(>5, 1967). These results show a flash duration of about 100 milliseconds, a latency of 2-5 milliseconds, and a mean rate constant for the exponential decay of luminescence of 0.088 milliseconds'1. Compared to the C. scrrata flash, the flash duration is about 15 times shorter, the latency is about 200 times shorter, and the decay rate is approximately 100 times faster. Data on the copepod flash, however, are not available in the literature and no comparison can be made. Since the duration of a flash depends on factors such as mixing and concentration of reactants, it would lie difficult to compare a recorded flash with a light-stimulated flash in the ocean, and with light emission from a mixture of luciferin and luciferase.

The luminous cloud, the thread of luminous secretion, and the point source of light, all produced by C. sernita, indicate that the organism is able to control luminescence. It is possible that the quick ejection of a cloud of luminous material serves to propel the organism through the water and provide a screen to escape from a predator. The production of a point source of light appears to indicate a mixing of luciferin and luciferase within the organism or mixing just at the gland orifices through a finely regulated release of luciferin and luciferase. The ability of C. scrrata to control its luminescence would account for the luminous clouds observed in the sea and the flashes observed in the laboratory. "When collecting Vanjnla liurrcyi at Long Hay, Jamaica, in 19(>5 and 1967, Dr. Howard Seliger (personal communication) of the McCollum-Pratt Institute. Johns Hopkins University, observed point source of luminescence similar to ours. The whole body of I7. Jianryi was luminous without any appearance of luminous secretion and he believes that the mixing of luciferin and luciferase takes place by one of the above two mechanisms.

In the reaction between crude C. scrrata luciferin and partially purified luciferase, the decay rate is initially very high. This anomaly was first observed by Amberson (1922) in the C. hilgendorfii luciferin-luciferase reaction. In his early experiments, mixing C. hilgendorfii luciferin and luciferase resulted in a bright initial flash of light, corresponding to a high initial rate of decay, which was then followed by a normal first order decay. The bright initial flash was

BIOLUMINESCENCE OF C. SERRATA

attributed liy Amberson ( 1(>22 i to the active site of the enzyme being free of lucilerin at the outset.

If C. s errata luminescence is clue to a first order reaction, theoretically the rate constant should he independent of luciferin concentration. However, a near doubling of the rate constant occurs when the luciferin concentration is halved (Fig. 5d and e), indicating that the rate constant is dependent on luciferin concentration. The rate constant, however, is independent of luciferin concentra- tion when highly purified (". hilycndorfii luciferin is used. A similar result has also been noted in the C. liilf'/cndorfii luminescent reaction. Amber son (1922), Stevens (1927). Harvey and Snell (1931), and Chase (1956) observed that the first order rate constant increased as the initial luciferin concentration was de- creased. The rate constant increase was about two-fold in the studies of Amberson (1922) and Chase (1956) and five-fold in the experiments of Harvey and Snell (1('31). Subsequently, Chase and Harvey (1942) found that the rate constant showed no change with luciferin concentration if highly purified luciferin and partially purified luciferase were used. They concluded that some impurity in either luciferin or luciferase was responsible for the increase in the rate constants of the earlier workers. In the present work with C. scrrata. the result obtained by using purified C. liiUjcndorfii luciferin appears to indicate that the increase in the rate constant is due to an impurity in the C. scrrata luciferin.

Three other aspects of the study require brief comments. The data (Fig. 6) show clearly that the luminescent reaction of C. scrrata requires oxygen. The same requirement for oxygen was demonstrated many years ago for the C. lii'</cndorfii reaction by Harvey (1917, 1920). The immunochemical results (Table 1) show that C. scrrata luciferase is a related but distinct enzyme from C. hilfjeudurfii luciferase. Tsuji and Haneda (1966) have previously shown that antibody to C. hilf/cndorfii luciferase may be used to distinguish C. hilgen- durfii luciferase from another closely related luciferase. Finally, the identical R/s show that C. scrrata and C. hilf/cndorfii luciferins are very similar, if not the same.

\Ve thank the following individuals for their generous assistance: Mr. Robert Gibson, Territory of Papua and New Guinea, Department of Agriculture, Stock and Fisheries, Madang, for providing a boat and operator employed in the collec- tion of Cypridiua organisms; Dr. J. Woodland Hastings, Harvard University, for the loan of the photomultiplier photometer; Dr. Louis S. Kornicker, Smith- sonian Institution, for identifying the Cypridina specimens and for discussions on Ostracoda; and Dr. John B. Buck, National Institutes of Health, for com- ments on the flash recordings.

SUMMARY

1. The physical appearance and bioluminescence behavior, and light-emitting reaction of the marine ostracod crustacean, Cypriditia scrrata, are described.

2. In the natural environment of the sea. the free-swimming C. scrrata appears to emit almost instantaneously a bright blue luminous cloud when stimulated with artificial light.

400 F. I. TSUJ1, K. V. LYNCH, III AND Y. HANEDA

3. The method of light production, consisting of the ejection of luciferin and luciferase into sea water, and the color of light are similar to that of C. hilgendorfii.

4. In captivity, C. scrrata emits apparent spontaneous flashes of light, whose duration is approximately 1.5 seconds, with an apparent latency of 500-800 milliseconds.

5. C. scrrata luciferase cannot he distinguished trom C. hilgendorfii luciferase hy gel elution chromatography hut may he distinguished immunochemically.

6. The luminescence of C. scrrata is due to a first order reaction, similar to that of C. hilgendorfii. The luciferins and luciferases of hoth organisms cross- react to give light.

7. The luminescence of C. scrra/a. like C. hilgendorfii, is oxygen dependent.

8. C. scrrata luciferin is similar, if not identical, to C. hilgendorfii luciferin when compared by paper chromatography.

LITERATURE CITED

AMBERSON, WILLIAM R., 1922. Kinetics of the bioluminescent reaction in Cvpridina. II.

/. Gen. PhysioL, 4 : 535-558. CHASE, AURIN M., 1956. Effect of luciferin concentration on the rate constant of the

Cypridina luciferase-luciferin reaction. Anat. Rcc., 125: 616-617. CHASE, AURIN M., AND E. NEWTON HARVEY, 1942. A note on the kinetics of Cypridina

luminescence. /. Cell. Corn?. PhysioL, 19 : 242-243. ECKERT, ROGER, 1965. Bioelectric control of bioluminescence in the dinoflagellate Noctilitcit.

Science, 147: 1140-1142. ECKERT, ROGER, 1967. The wave form of luminescence emitted by Noctiluca. J. Gen. PlivsioL,

50: 2211-2237.

HANEDA, YATA, 1940. Bioluminescence. Seirigaku-Shido-Shu, 5: 18-31 (In Japanese). HANEDA, YATA, 1953. Observation on some marine luminous organisms of Hachijo Island,

Japan. Records Occanogr. Works in Japan, 1 : 103-108. HANEDA, YATA, 1955. Luminous organisms of Japan and the Far East. Pages 335-385 in :

F. H. Johnson, Ed., The Luminescence of Biological Systems. Amer. Assoc. Adv. Sci.,

Washington.

HARVEY, E. NEWTON, 1917. Studies on bioluminescence. IV. The chemistry of light pro- duction in a Japanese ostracod crustacean, Cvpridina hilgendorfii, Miiller. Amcr. J.

PhysioL, 42: 318-341. HARVEY, E. NEWTON, 1920. Is the luminescence of Cypridina an oxidation? Amer. J.

PhysioL. 51 : 580-587. HARVEY, E. NEWTON, 1922. Studies on bioluminescence. XIV. The specificity of luciferin

and luciferase. /. Gen. PhysioL. 4 : 285-295. HARVEY, E. NEWTON, 1924. Studies on bioluminescence. XVI. What determines the color

of the light of luminous animals? Amer. J. PhysioL, 70 : 619-623. HARVEY, E. NEWTON, 1926. Additional data on the specificity of luciferin and luciferase,

together with a general survey of this reaction. Amer. J. PliysioL, 77 : 548-554. HARVEY, E. NEWTON, 1952. Bioluminescence. Academic Press, New York, 649 pp. HARVEY, E. NEWTON, AND PETER A. SXELL, 1931. The analysis of hioluminescences of short

duration, recorded with photoelectric cell and string galvanometer. /. Gen. PlivsioL,

14: 529-545. HASTINGS, J. WOODLAND, 1959. Bioluminescence in marine dinoflagellates. Pages 427-434 in:

H. Cjuastler and H. J. Morowitz, Eds., Proceedings of the First National Biophysics

Conference. Yale University Press, New Haven. KAJIYAMA, E., 1912-1913. Study on Ostracoda of Misaki. Zool. .Urn;. (Dobutsugakh

Zasshi), 24(287): 488-492; 24(289): 609-619; 25(291): 1-16 (In Japanese). KORNICKER, Louis S., AND CHARLES E. KING, 1965. A new species of luminescent Ostracoda

from Jamaica, West Indies. Micropaleontology, 11 : 105-110. MULLER, G. W., 1890. Neue Cypridiniden. Zool. Jahrb. Syst.. 5 : 211-252.

BIOLUMINESCENCE OF C. SEKR.lT.l 401

MULLER, G. W., 1906. Die Ostracgclcn der Siftc^a-Expedition. Sihntia /:.r/v<///;V, 30: 1-40. NICOI.. |. A. C., 1958. Observations on luminescence in Noctilucit. J. Mar. Biol. Ass. [7. /\'.,

' 37 : 535-549. SELIGEK, HOWARD H., AND WILLIAM I). MCELROY, 1965. I.i//!it: f'hysica! and Biological

. letion. Academic Press, New York, 417 pp. STEVENS, K. P., 1927. Studies on the amount of light emitted by mixtures of Cypridina

luciferin and luciferase. /. Gen. Physiol., 10 : 859-873. Tsi'ji, FREDERICK I., 1955. Tlie absorption spectrum of reduced and oxidized Cypridina

luciferin, isolated by a new method. Arch. B we hem. Biophys.. 59 : 452-464. TSUJI, FREDERICK I., AND YATA HANEDA, 1966. Chemistry of the luciferases of Cypridina

hilgendorfii and Apoyon ellioti. Pages 137-149 in: F. H. Johnson and Y. Haneda, Eds.,

Bioluminescence in Progress. Princeton University Press, Princeton. TSUJI, FREDERICK I., AND RAYMOND SOWINSKI, 1961. Purification and molecular weight of

Cypridina luciferase. /. Cell. Coinp. Physiol., 58 : 125-129. TSUJI, FREDERICK I., DAVID L. DAVIS AND DAVID H. DONALD, 1969. Chromatographic studies

on spleen and lymph node extracts with antibody activity. J. linini/nol., 102: 519-529. TTKXER, R. J., 1966. Marine bioluminescence. Mar. Ohs.. London, 36: 20-29.

ABSTRACTS OF PAPERS PRESENTED AT THE MARINE BIOLOGICAL LABORATORY

1970

ABSTRACTS OF SEMINAR PAPERS JULY 14, 1970

Inter population variation in slid! components in the stream limpet, Ferrissia rivularis. W. D. RUSSELL-HUNTER, ALBERT J. BURKY AND R. DOUGLAS HUNTER.

In freshwater snails — as in the majority of molluscs — the secreted shells have two principal components: a meshwork of hardened protein and crystalline calcium carbonate (the latter secreted after active uptake directly from environmental water). Earlier work in Scotland showed that in the- euryoecic species, Lyinnaea pcrcgra, the thickness (and mass) of the calcareous shell varies with the calcium available in the waters. Thus, Lymnaca expends about the same energy on shell-making no matter what the environmental hardness. The case of Ferrissia in natural creek populations of upstate New York is strikingly different. As we have reported elsewhere (Science. 155: 338-340, 1967), although these creeks vary in dissolved calcium from 10.4 mg/1 (Black Creek) to 67.6 mg/1 (Limestone Creek), the highly significant differences in shell calcium from 82 mg/g live weight (Fish Creek) to 145 nig/g (Canandaigua Outlet) are not correlated. Anabolic concentration ratios ranged from 1609:1 to 10,615:1 and there was other circumstantial evidence of physiological races.

Recently we have measured total organic carbon and total nitrogen in shells of limpet growth stages from these populations. The noncalcareous component is obviously mostly protein because the C:N ratios are consistently about 3.25:1. Organic carbon in /j.gC per standard shell (3.5 mm) ranges from 7.0 (Canandaigua Outlet), 8.4 (Limestone Creek), 8.7 (Slocum Creek), 9.4 (Fish Creek). 10.2 (Big Bay Creek), 10.3 (Chittenango Creek), to 20.8 (Black Creek). Ranking the protein content of the shells from these seven creeks (in the above order) from lowest 1 to highest 7; and similarly ranking dissolved calcium of the creek waters from lowest A to highest G, and shell calcium from I-VII; we have the following sets (from Canandaigua to Black Creeks) : 1-F-VII, 2-G-III, 3-B-II, 4-C-I, 5-D-VI, 6-E-V, and 7-A-IV. Thus there is neither an inverse relation between shell protein and calcium (expected by an ''adaptive value" hypothesis) nor any direct relationship (expected if both relate to levels of energy turnover). It should be noted that in content of shell protein and of shell calcium (as even more clearly in other measurable characters), the variation within the majority of single populations is very much /r.v.v than the range of variation for the species as a whole. It seems that genetic controls of shell secretion for the two major compo- nents are independent, and that the chances of genetic dispersal among the isolated creek populations of this limpet have resulted in some rather inappropriate shells in certain habitats.

Supported by Grant GM 11693 from the National Institutes of Health.

A fundamental error in /lie </eneral model of genetic selection. ROGER MILKMAN.

The general selection model states that the relative survivorship of alternate genotypes is independent of frequency. For example, the proportion of aa zygotes surviving to reproduce (number of no adults/number of aa zygotes) is said to be in a constant ratio to the proportion of .In zygotes surviving to reproduce. The proportion surviving may change during selection;

402

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 403

the ratio of these proportions does not. ( All conditions are stipulated to remain constant with the exception of the genotype frequencies, which change during the course of selection.)

In constructing a realistic model based on elementary breeding procedures, we find that this ratio is not independent of genotype frequency. For example, consider two genotypic classes, an and Aa, each normally distributed for bean weight with means of 8 and 9 grams, respectively, standard deviation being 1 gram in each case. These distributions (reflecting the impact of additional gene loci and of environmental factors) overlap, and both classes are likely to be represented in the heaviest 59?- of the beans, which are chosen for seed. But as the Aa class increases in frequency, the competitive standard rises — beans must be heavier to make the grade. Mathematically, each common selection threshold is associated with a unique ratio : the proportion of mi zygotes that survive divided by the proportion of Aa zygotes that survive, e.g., 378/9462 -=- 122/538 = 0.176. It can be shown that as the frequency of a superior genotype increases, the rchithr performance of an inferior genotype (cf. the general selection model's adaptive rahtc. or /f) decreases, and this decrease is substantial for a large range of values.

Changes in selection intensity also cause changes in relative .survivorship ; the use of dissimilar standard deviations in the example above would not alter the results materially.

Supported by a National Science Foundation grant to the program for the biological sciences at The University of Iowa.

Ultrastntctiire of the negatively stained spermatozoon of the earthworm. CATHERINE HENLEY.

Spermatozoa from the seminal vesicles of Lutnbricus tcrrcstris were negatively stained with \% PTA, pH 6.8, for 4-10 min, and examined with the Zeiss EM 9A. The preparations had large numbers of intact spermatozoa, in which many details of the structure of acrosome complex, nucleus, mitochondria! middle piece and axoneme closely paralled similar features shown in sectioned material by Anderson and Ellis, and others. In acrosomes interpreted to have undergone a partial acrosome reaction, the acrosome vesicle and rod looked very much like those seen in sections, but the material inside the vesicle had a coiled appearance, rather than a uniform one, and there was always an accumulation outside the acrosome tip of similar-appearing material. In unreactecl acrosomes no internal substructure could be discerned and there was never material at the tip. In the 9 + 2 tail, the microtubules usually fell in rather straight or gently curved configurations, and the singlets were in such close proximity to one another as to appear as one structure. Beta glycogen granules, recently described by Anderson and Personne in sectioned earthworm spermatozoa, were associated with the doublets. Diastase treatment resulted in the disappearance of these granules, but 3-hr incubations of spermatozoa in distilled water at room temperature had no effect on them. However, such prolonged distilled water treatments caused the doublets to fall on the supporting Formvar film in tight coils, while the singlets (usually still attached to one another along most of their lengths) pursued very different courses, along jagged paths with sharp bends, and for long distances. They often assumed remarkably symmetrical arrow-shaped configurations. The cross-connections between the singlets occurred at regular intervals of ca. 150 A and were apparently very strong, as evidenced by the close adherence of the singlets to one another even through turns of 20° or less.

Aided by a grant from the NIH, GM 15311.

JULY 21, 1970

Ilyalnronate and hyalnronidase in the blastema of the regenerating newt limb. BRYAN TOOLE AND JEROME GROSS.

Release of dedifferentiated cells from the structural tissues of the stump and accumulation of these cells in an outgrowing blastema are essential for regeneration of an amputated newt limb. This process requires extensive proliferation and migration of the blastema cells and retention of their dedifferentiated state for a period of approximately two weeks in the case of the adult newt, Diemictylus viridcsccns, studied here.

404 PAPERS 1'KKSKXTKl) AT MARINE BIOLOGICAL LABORATORY

A very marked enhancement of the incorporation of radioactive precursors into hyaluronate has been found in the early blastema as compared to the normal unamputated limb. At 10 day> of regeneration this uptake is most active in the most distal portion of the regenerate but there is a gradation of degree of uptake from zero to 40-fold along the length of the stump. Between 15 and 20 days of regeneration, when the first sign of differentiation of precartilage has appeared in the blastema, there is a drop in the uptake of isotope into liyaluronate but a rise in uptake into chondroitin sulfate, a component of cartilage. These changes continue during the following 30 days of regeneration, and concomitant with them hyaluronidase activity appears in the blastema. This enzyme cannot be detected prior to 15 days or after 50 days of regenera- tion nor in the normal unamputated limb.

It appears, therefore, that the extracellular phase of the early dedifferentiated blastema is rich in hyaluronate and that this macromolecule is removed at the onset of precartilage differentiation.

This investigation was supported by a grant from the National Science Foundation.

Reversible inhihilion of KNA pliogc replication ami macromolecular synthesis l>\ levorphanol. ERIC J. SIMON, DAVID J. GARWES AND JUDITH RAND.

Levorphanol inhibits the production of infectious RNA phages MS2 and Qp by more than 99 per cent. Even at concentrations at which effects on bacterial growth are minimal, phage production is decreased by 85-90 per cent. When the drug is washed away 30 or 60 minutes after infection, phage multiplication begins almost immediately and a normal yield of infectious particles results. Levorphanol is most effective when added before or at the time of infection, but becomes less so with increasing intervals between infection and drug treatment. When added 30 minutes or more after infection levorphanol has no effect on plaque formation. Studies on phage RNA and protein synthesis yielded results similar to those on phage multiplication. When levorphanol is added simultaneously with phage there is no detectable phage RNA nor protein synthesis, while levorphanol addition 30 minutes later results in normal macromolecular synthesis. An alteration in bacterial membrane permeability resulting in exclusion of the drug late in the life cycle of the phage has been ruled out. An effect of levorphanol on an early event in phage reproduction is postulated.

Supported by Grants MH-10227 and AM-01845 from the National Institutes of Health. Eric J. Simon is a Career Scientist of the Health Research Council of the City of New York.

Hydranth re</i/lation in the colonial marine hydroid Podocoryne curm-a. MAX BRAVERMAN.

Unlike the many highly regular colonial hydroids, Podocoryne ciinicn shows no ob- viously visible regularity in the distribution of polyps on the stolons. Since the stolons are completely adherent to the substrate, in this case a microscope slide, an exhaustive description of the colony morphology is contained in a photograph. Analysis of daily photo- graphs of growing colonies shows that statistical regularity does obtain : the number of hydranths and the total length of stolon in a colony tends to constant proportionality.

To determine if this proportionality results from feedback control of young polyp formation, the initiating polyps were removed from young colonies before any new polyps had formed. Removal of the initiating polyp stimulates the formation of new polyps within three days, compared to fed or starved unoperated controls. The difference is much greater, however, between operated and fed controls. A significant difference in hydranth formation between fed and starved controls implicated starvation as a stimulator of polyp formation.

To evaluate the influence of polyp removal independently of that of starvation, polyps were removed from one half of a colony and the colony fed and photographed for six successive days. Since stolon growth on the side from which polyps were removed was the same as on the control side, it could be assumed that the food captured by the control side was equitably distributed and the requisite separation effected. Under these circumstances, a significant difference (P = 0.007) between the number of polyps forming during the two days following removal could be seen. About two times as many polyps formed on the side from which the polyps were removed as on the unoperated control side, indicating localized feedback control of hydranth formation.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 405

JULY 28, 1970

ILvcitatory and inhibitory regulation of efferent nerre activity in the phallic ncri'c (>l the cockroach Periplaneta americana (L). M. E. SPIRA, I. PARNAS AND F. BERGMANN.

Phallic nerve activity was recorded with suction electrodes during electrical stimulation of the ventral nerve cord ( V. N. C.). In all preparations a train of impulses delivered to the Y. N. C. caused up to 600% increase in phallic nerve firing rate for periods of up to 10-15 sec. The increased firing rate is dependent on the stimulus intensity, train duration, and stimulus fre- quency. In a few cases a single pulse was sufficient to cause the increase in firing rate. In these cases the firing rate increased with stimulus intensity. Stimulation of both ipsi and contralateral connectives caused activation of the phallic nerve. However the response to ipsilateral stimula- tion lasted longer and showed a higher frequency. In some preparation a train of stimuli delivered to the Y. N. C. induced a silent period. Application of Eserine (mg/ml) to ganglia of the V. N. C. other than the last abdominal ganglion (A6), did not affect the phallic nerve firing rate. However, application of Eserine to the A6 ganglion caused an immediate increase in the firing rate which is blocked after 3 minutes. Application of d-tubo curarine to the preparation did not alter the spontaneous activity. However under these conditions a train of stimuli delivered to the V. N. C. caused a long lasting silent period (10-20 sec). Application of picrotoxin (mg/ml) to ganglia other than A6, caused no change in phallic nerve activity. Only when picrotoxin was applied to A6 could an increase in phallic nerve activity be observed. This increase persisted for 15 minutes. Later the activity decreased gradually to the normal level. At this stage a single stimulus at the V. N. C. resulted in a burst of activity in the phallic nerve. We suggest that the minimal neuronal network to explain the present results involves spontaneously active phallic motoneurons on which excitatory and inhibitory inputs converge from both the ipsi and contralateral connectives.

Electrical and photochemical sit/ns oj adaptation in the retina of the skate. Raja erinacea and R. oscellata. J. E. BOWLING AND H. RIPPS.

The skate retina appears histologically to contain only rod photoreceptors and should provide, therefore, an ideal tissue for studying the response properties of the scotopic mechanism. In order to determine whether the b-wave of the electroretinogram (ERG) provides a valid index of retinal sensitivity, b-wave and ganglion-cell thresholds were compared during equivalent stages of light- and dark-adaptation. Under all conditions tested, both responses exhibited remarkably similar alterations in sensitivity. For example, on exposing the retina to a moderately bright background illumination there was a period during which neither response could be elicited with intense test flashes. The duration of this silent period depended upon the intensity of the steady background, and lasted for as long as 20 minutes when a significant fraction of the rhodopsin was bleached. However, once retinal excitability returned, thresholds continued to fall until a stable level was reached ; at these levels, the increment threshold was a linear function of background illuminance.

Threshold changes in the ERG and ganglion cell discharge during dark adaptation also followed a parallel time course. After a flash bleach which denuded the retina of about 80 per cent of its rhodopsin content, neither response could be elicited for 10-15 minutes. But following this period of electrical silence, thresholds fell rapidly to within 3 log units of the dark-adapted level. Further recovery of sensitivity was slow, requiring an additional 90 minutes in darkness to reach completion. Fundus reflectometric measurements of rhodopsin regeneration enabled us to correlate changes in pigment concentration with the recovery of threshold that occurred during the slow phase of dark adaptation ; a linear relation was obtained between rhodopsin concentration and log threshold.

Supported by grants (EY-00470, EY-00285, EY-18766) from the National Institutes of Health, and by a Fight For Sight award from the Carrie L. Groedel Fund.

406 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

The role <>j the sodium pump in adaptation in the )ro</ muscle spindle. DAVID I -. \NDO\VNE.

'I'hc mechanism of sensory adaptation in muscle spindles has not been demonstrated previously although visco-elastic models have been proposed whereby the effective stimulus is reduced. Another possible method of reducing the rate of firing of a nerve cell is to hyper- polarize the cell. This could occur /// 7'i'ro by the action of an electrogenic sodium pump.

When a muscle spindle is rapidly stretched and held at a new constant length a train of impulses occurs. If the instantaneous frequency (reciprocal of the interspike interval) is plotted semilogarithmically against time three phases of adaption may be seen. The first phase is the classical dynamic response and the next two are included in the static response. Removing external potassium, adding 10 /u.l/ strophanthidin, or replacing external sodium with lithium all selectively and reversibly block the second phase. As these three conditions would also be expected to block the sodium pump it is concluded that this phase should be closely associated with the sodium pump, which presumably hyperpolarizes the cell in response to the sodium which enters the cell during the generator and action potentials. The observation that it is the second phase which is inhibited supports the division into three phases.

Ultras true titre oj the secondary septa oj Metridium sf>. WALLIS H. CLARK, JR. AND GERTRUDE \Y. HINSCH.

The secondary septa of male Metridium sp., collected at Woods Hole during late July, were examined with both the light and electron microscopes. The endoderm of the basal portions of the septa consists of tubular aggregates of spermatogenic tissue surrounded by occasional interstitial cells. The endoderm is separated from the ectoderm by the mesoglea. The ecto- derm consists of several cell layers, the outermost layer being composed of epithelial cells which face the gastrocoel. The majority of the cells found between the outer epithelial layer and the mesoglea contain homogeneous, secretory-like inclusions. In some instances cells filled with such inclusions appear to be secreting their cytoplasmic contents into the gastrocoel. The epithelial cells contain large Golgi bodies and large, membrane bound aggregates of flocculant material of varying electron density. Septate desmosomes are commonly found between the limiting membranes of these cells. Each of these epithelial cells possesses a single flagellum, the base of which is associated with two centrioles and adjacent cytoplasmic rootlets. The flagella extend from the cell surface and are surrounded by a very fine, electron dense, fibrillar material. Closely associated with this fibrillar material are long undulating microvilli. There are usually 9 micmvilli, though some variation in number, 8 to 10 have been noted.

The function of these fiagellar-villus aggregates is unknown ; however, it seems likely that they may be sensory in nature due to their similarity to the flagella of nematocytes and the sense hairs described in Hydra.

Supported in part by NIH grant Tl-HD-26-08.

AUGUST 4, 1970

Metabolic controls and biological variation. XEAL W. CORNELL.

Although the occurrence of intraspecific physiological variation has been widely recognized, there appears to be few studies and little understanding of the sources of such variation. Con- sequently, an investigation was undertaken concerning possible enzymatic sources of continuous variation in mammals, i.e., the type of variation seen in parameters like growth rate during maturation, basal metabolic rate, and longevity.

From evolutionary considerations it seems unlikely that genetic differences in the catalytic sites of enzymes could account for widespread variation. It is proposed, however, that genetic differences in regulatory properties of enzymes sensitive to feedback control might be- more common in wild populations and could substantially alter overall patterns of metabolism. The proposal is based in part on studies which show that such differences can be induced

PAPERS I'KKSKXTKI) AT M. \RL\K BIOLOGICAL LABORATORY 407

with chemical mutagens, resulting" 1n viable organisms \\ilh significantly altered physiology. Theoretically, the redundancy of controls in. for example, carbohydrate metabolism should make mutations that affect regulatory properties of enzymes more permissible. To illustrate, citric acid, a component of the Kreb's cycle anil also the initial substrate fur fatty acid biosynthesis, acts as an allosteric effector for three different enxymatic reactions. The summed effects of citric acting as an allosteric agent is l<> diminish oxidation of carbohydrates and enhance the biosynthesis of fatty acids (storage) under conditions of high ATP concen- tration.

The consequences of altering the regulatory properties of acetyl-CoA carboxylase, one site affected by citrate, have been evaluated in a computer simulation study. A very small 15- fold) decrease in the Km of acetyl-CoA carboxylase for citrate would result in marked increase in the biosynthesis of fatty acids relative to oxidative metabolism. Experiments will be conducted with denned strains of mice to evaluate the predictions of the simulation model.

Supported by grant HDO3620 from the National Institutes of Health.

.hnilvsis oi the color clunit/es induced by serotonin (5-hydroxytryptamine') and l\'seri/ic acid diethylainide (LS/>) in Hie fiddler crab, Uca pugilator. MILTON

FlNGERMAN AND K. RANGA ]\A(>.

Serotonin in dosages of 0.1 /ug and above per crab evoked pigment dispersion in the erythrophores of eyestalkless and intact Uca. Similar dosages of LSD evoked pigment concen- tration in the erythrophores of intact but not eyestalkless I'ca (light patch variant from Panacea, Florida). LSD and serotonin were ineffective /;; ritn> when assayed on legs isolated from I'ca showing that they do not directly stimulate the erythrophores. In contrast, red pigment-dispersing and pigment-concentrating polypeptide hormones from the eyestalks were effective both in I'itro and in vivo. Assay of extracts of supraesophageal ganglia with the attached circumesophageal connectives from serotonin-injected crabs revealed a marked de- crease in the red pigment-dispersing potency of such extracts compared with controls, indicating that serotonin may have stimulated release of a red pigment-dispersing substance from the neuroendocrine system. In an effort to determine the mode of action of LSD, blood from LSD-treated crabs was assayed for erythrophorotropic activity on eyestalkless crabs. It was found that blood from LSD-treated crabs on a black background evoked more red pigment concentration than did that of the controls. Control crabs on a black background normally contain more red pigment-dispersing hormone than pigment-concentrating hormone. It appears that LSD shifted the hormone balance in the blood in favor of the red pigment-concentrating hormone by either inhibiting the release of red pigment-dispersing hormone or by stimulating the release of red pigment-concentrating hormone. The fact that LSD antagonized the action of serotonin but not that of the red pigment-dispersing polypeptide lends strong support to the view that LSD most likely acts by inhibiting the release of red pigment-dispersing hormone from the neuroendocrine system of the crab. The present studies and the finding of serotonin in the nervous system of Uca ( Spirtes and 'Fingerman, unpublished data) indicate that this indolealkylamine may be normally involved in the control of color changes in this crab.

Supported by Grant GB-7S95X from the NSF.

/:;/.:.• \niatic hydrolysis of the nerve (/uses DFP and Tabun in relation to neri'c junction. FRANCIS C. G. HOSKIN.

The organophosphorus cholinesterase inhibitor, diisopropylphosphorofluoridate (DFP), is hydrolyzed by an enzyme in squid axons and especially in squid head ganglia called "DFPase" with a K,,, = 6.25 X 1(T; .17. Another organophosphorus cholinesterase inhibitor, ethyl N,N- dimethylphosphoramidocyanidate (Tabun), is hydrolyzed only slowly by squid nerve. At 10~L' .17 the rates are 750 ± 9 /umoles DFP and 83 ± 18 /xmoles Tabun hydrolyzed per gram head ganglion per hour. The situation is reversed in rat serum, the rates being 11 ± 1 ^moles DFP and 77 ±10 /xmoles Tabun per milliliter serum per hour. The relative rates of hydrolysis of DFP, of Tabun, and of mixtures of the two by squid nerve indicate that the two substrates compete for a common site on a single enzyme. When a squid axon is bathed in 10~'! M Tabun, this compound penetrates in its active form, attaining about 50% of the outside concentration

40S PAPKKS PKKSKNTKI) AT M. \KI.\K BIOLOGICAL LABORATORY

in 1 hour. This is because, in contrast to DFP, Tabun is only slowly hydrolyzed by squid nerve while, like Dl-T, Tabiin is lipid-solublc. Furthermore, a solution of cholinesterase is com- pletely inhibited by DT"I' at about 10 '; M, but by Tabun at about 10~7 M. With these properties in mind, conduction was measured in squid axons bathed in Tabun or DFP in buffered sea- water. DFP blocks conduction in an intact axon at 5 X 10"" M, whereas Tabun at 10"- M does not block conduction even after a 1-hour exposure to the compound. These results raise the question whether acetylcholinesterase in the conducting- membrane is essential for axonal (induction, and suggest that tin- block obtained with the high concentrations of DFP may have been due to acid formation on enzymatic hydrolysis of DFP. Supported by U.S.P.H.S. grant NS-09090.

Chemistry and biohnj\ <>j nemertine iieiiroto.vnis. WILLIAM R. KEM.

Bacq reported the existence of two neurotoxins, "amphiporine" and ''nemertine," in the nemertine phylum. Both toxins produced convulsions when injected into crustaceans. In addition "amphiporine" also elicited a nicotine-like contracturc of the frog rectus abdominis. A toxin was recently isolated from the lioplonemertine, runnicincrtcs pcrcf/rbia, and un- equivocally identified as anabaseine, 2-(3-pyridyl)-3,4,S,6-tetrahydropyridine. Anabaseine possesses the convulsant and nicotinoid properties of "amphiporine," but has no effect on either the resting and action potentials of the crayfish median giant axon or the compound action potential of the lobster walking leg nerve. The heteronemertine Linens nthcr contains two neurotoxic polypeptides corresponding to Bacq's "nemertine." The polypeptides were partially purified by methanol-acetic acid (95:5) extraction and G-25F Sephadex chromatography (0.1 ^f ammonium acetate-acetic acid, pH 5.0). Crayfish paralytic activity eluted at 1.4 Ve/Vo and 1.7 Ve/Vo. The polypeptides prolong the repolarization phase of the action potential in the lobster walking leg nerve bundle, but lack nicotinic (frog rectus) and hemolytic (human red cells) properties.

Both hoplonemertine (anabaseine) and heteronemertine (polypeptide) toxins are located in the body wall and proboscis integument. Mucus collected from irritated individuals of these species is highly toxic. Presumably these toxins are chemical defences against potential predators.

AUGUST 11, 1970

Mechanisms of lysosomul hvdrolase from phagocytic cells. GERALD WEISSMANN, PETER DUKOR AND ROBERT ZURIER.

Since Metchnikoff, it has been appreciated that phagocytic cells release biologically active substances into surrounding tissues when they engage foreign material. To study mechanisms of enzyme release during phagocytosis in ritro, purified mouse peritoneal cells and polymorpho- nuclear leucocytes from human blood were exposed to particles ( zymosan, latex, opsonised sheep erythrocytes, aggregated albumin). Uptake of undigestible, but not of digestible, materials was associated with release (up to 15% in 2 hrs from macrophages ; up to 25% in 30 min from polymorphs) of lysosomal enzymes: /3-glucuronidase, aryl sulfatase, acid phosphatase, and cathepsin 1); no release of cytoplasmic lactase dehydrogenase was observed during phagocytosis, although freezing or mild sonication readily released this marker. Hydrolase release did not depend upon loss of cellular integrity as judged by dye exclusion or by viability of macrophages in long term cultures. Agents which elevate the level within cells of cyclic AMP (cyclic 3'5' adenosine monophosphate ) retarded (1) extrusion of acid hydrolases (2) uptake of heat aggregated bovine serum albumin (3) degradation of previously ingested heat aggregated bovine serum albumin. Each of these activities could be duplicated by exposing phagocytes to colchicine or vinblastine. The agents which were effective were dibutyryl-cyclic AMP, cyclic AMP with theophylline, prostaglandin Ei with theophylline etc but these needed to be present at concentrations above 10"' M. At lower concentrations cyclic nucleotides markedly enhanced particle uptake and degradation. These studies indicate that cyclic AMP (at pharmacologic concentrations) and microtubule reagents

PAPERS PRESENTED AT MARINE BIOLOGICAL LAIJORATORY 409

can act to block the merger of lysosomes with early phagocytic vacuoles, and furthermore, that their site of action is similar or identical.

Isoclectric focusing of lots t/anin/a (y) crystallins. S. XH;M.\N.

By isoelectric focusing in a pH gradient, the seven components or isomers of rat leu* 7-crystallin have been isolated. In 6M urea, the isoelectric points obtained ranged from pH 8.9 to 7.3. All constituents were identical immunologically, and similar in molecular weights (16-18,000), ultraviolet light extinction ( E! ,,,,,,„, J tm = 18-23 ), relative fluorescences, N- terminal group (alanine), and content of hydrophohic amino acids and ' cystines. The content of charged amino acids (lysine, arginine, aspartic and glutamic acids) differed appreciably. Differences in the exposure of charged side chains could account for the different isoelectric points found. The components with higher isoelectric points were rich in — SH groups (2-3 moles/mole protein), while those with lower isoelectric points contained 2 bonds.

When total 7-crystallin was oxidized in H-Oi., all — SH containing material was precipitated, but the lower isoelectric point constituents remained soluble.

Between 1 and 52 weeks of age, a marked fall in the level* of the three 7*3 with higher i-oelectric points and an increase in the four with lower isoelectric points was found. The incorporation of C"-amino acids in ritro into those 7*5 decreasing in level was diminished, while for those increasing, it was increased. Wearing out or stimulation of long-lived M-RNA molecules known to be present in the lens may regulate this change. 7*5 with higher isoelectric points and — SH constant were predominant in the rat lens nucleus (the oldest portion) where precursors of the urea insoluble (or — SS — linked) protein increase with aging.

Isoelectric focusing profiles of rat, dogfish, cow and human lens 7-crystallins were compared. For dogfish, cow and human 7's, four components each were found. Isoelectric points for the 7*5 of rats ranged from pH 8.9 to 7.3; of dogfish from pH 8.8 to 7.2; of cow from pH 8.1 to 7.4, and of human lenses from pH 7.6 to 6.8. Although most 7 components differed in isoelectric point among these species, a common isoelectric point of one component ( pH 7.4-7.5) was found in all. A common gene, M-RNA, and finally protein chain may exist in the lenses of all of these species. The common protein constituents could be an organ specific 7-crystallin.

Supported in part by the Rochester Eye and Human Parts Bank and U. S. P. H. S. grant #EY0459, of the National Eye Institute.

Intranuclear microtubules dur'nuj f/ro^'tli and division in Tetrahymena pyriformis Gl. WILLIAM D. SULLIVAN, S.J. AND ELINOR M. O'BRIEN.

Intranuclear tubules were observed during growth and division in the macronucleus of the amicronucleate Tetrahymena. In exponential cultures, as the lag phase progressed, from one small packet of 6-10 microtubules (2 ^ in length; 270 A in diameter) there was an increase in number and length of tubules and an unfolding at one end demonstrating the beginning of a lateral growth and a unidirectional growth of the tubules. In synchronized cultures during the first burst of division the tubules were observed to be considerably lengthened (3fi-4fi), parallel to and concentrated at the macronuclear envelope. At no time were they associated with outpocketings and imaginations of the macronuclear envelope except at division when they penetrated the constriction point. In the divided macronucleus tubules extended into the attached SNA and were observed even after complete separation of the SNA from the daughter nucleus.

The changes noted in the morphology and distribution of the microtubules during growth and division confirm previous theories and elicit new theories regarding the role of microtubules in cells. The unfolding of the microtubules at one end during lag phase is consistent with the current theory of self -assembly of the protein subunits and unidirectional growth. The present investigation proposes that the microtubules act as a transport mechanism operable only during division of the cell and even then not in the conventional sense.

The concomitant growth of the microtubules and disintegration of nucleoli as well as the presence of microtubules in the disintegrating nucleoli indicate strongly that the microtubules have their origin in the nucleoli. This would seem to indicate that the protein subunits of

rAi'Kks pRKSKNTFi) AT MARINK BIOLOGICAL LABORATORY

the microtubules arc the same protein subunits of which the nticlcolus is comprised. Preceding this disintegration of the nucleoli the RNA synthesis has been completed and as the nucleoli disintegrate and the microtubules are formed the RNA is included within the protein subunits.

As the macronucleus prepares tor division, the microtubules with RNA extend throughout the length of the nucleus. As the macronucleus divides by constriction the microtubular RNA is deposited in each daughter nucleus and, at the constriction point, into the cytoplasm.

The specific parameters ,ii growth and function, in this particular investigation, therefore, appear as one and the same parameter.

Souic factors controlling reproduction in t/ic spider crab, Libiniu emarginata. GERTRUDE W. HINSCH.

Observations were made on the hormonal control of molting, sexual maturation and breeding in Lihiniii. Mature ovigerous females can be collected during the summer months of June-September. Immature crabs molt to the mature state during August. These have small, white ovaries containing numerous oocytes which show no signs of vitellogenesis. Vitellogenesis is seen in ovaries of crabs newly molted to maturity or those brooding embryos. Mature females collected during the non-breeding season (winter months) have fully developed ovaries.

The normal breeding season can be extended by ablation of the eyestalks of mature females. Oviposition outside of the breeding season occurs following eyestalk removal. There is no indication of further molting in these females. Eyestalk removal in mature females seems not to alter the breeding cycle or production of pheromone which is attractive to males.

Ablation of eyestalks in immature females leads to precocious molting. Rarely, however, do these females molt to maturity. Following eyestalk removal 50 to 51 crabs retained the immature apron and small ovarian size although carapace length was that of mature females.

The results suggest that in Lihiuia. the eyestalk hormone controls molting. In mature females the eyestalk hormone appears not to inhibit ovarian development but to control the release of oocytes from the ovary.

Supported by NIH grants 5-T01-HD00026-07, 5-T01-HD00026-08, 5-T01-HD00026-09.

AUGUST IS, 1<)/0

(hi iinnnniohxjic reactivity to hist o compatibility antii/cns. DARCY B. WILSON.

An unusually large proportion (2-3%) of the lymphocyte population on nonimmunized individuals is capable of being stimulated to proliferate by alien leucocytes of other members of the same species. This mixed lymphocyte interaction ( AILI ) has an immunologic basis since it reflects the immunogenetic status of the cell donors; it is thought to be the outcome of the recognition of homologous histocompatibility ( H ) isoantigens by immunologically com- petent antigen reactive cells (ARCs). However, the large proportion reactive to a single antigen system is an awkward conclusion to accept in view of two constraints of the Clonal Selection Hypothesis of Burnet : — namely, that the range of reactivity of a given ARC is restricted to a single antigenic determinant, and that they occur in low frequency ( 10~r'-10 '; ) in an unimmunized animal.

Assuming that they are monospecific, there are two general possibilities to account for such a high frequency of ARCs: — (1) that they represent an immunologically amplified clone stemming from prior experience of the cell donors with cross-reactive environmental antigens ; and (2) that this particular pool of ARCs is important in some crucial ontogenetic step, that they react to a special class of antigens of limited number, and that their high frequency is brought about by some expansion mechanism under innate genetic control.

Comparison of germ-free-pathogen-free (GFPF) and conventional isogenic Fischer strain rats as cell donors in the MLI lias direct bearing on these two possibilities. Whereas lympho- cytes from both conventional and BFPF are fully reactive to homologous H isoantigens, lymphocytes of conventional rats display some, but significant amounts of reactivity to heterologous H antigens, and lymphocytes from GFPF rats are totally nonreactive to

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 411

heterologous antigens. These findings favor the second alternative. Apparently an individual has a higher proportion of ARCs specific for the antigens of other members of his species than those of other species. De ectahle reactivity by cells of conventional animals to heterol- ogous species antigens may reflect contact with cross-reactive environmental antigens, hut this does not account for reactivity to homologous isoantigens.

An inducible lysin hi Linmhis polyphemus witli similarities to the complement system of rcrteJ>rates: detection, characteristics and dissection from phospJio- lipasc A. HENRY GKWURZ, VANESSA BIRDSEY, DONALD JOHNSON, JEAN

LlNDORKER, KAY TONYNSEND AND ANITA GEWURZ.

Cobra (Ntijtt Jiajc} venom factor (CYF) induces consumption of the six terminal com- plement (C) components in vertebrate serum and induces "passive lysis" of hystanding erythrocytes via C without requirement for antibody or earlier-acting C components. This passive lysis reaction was used to detect C-like activities in several lower vertebrate and invertebrate sera. We now report investigations of a CVF-inducible lysin in Liinulus f>nly- phciints with similarities to the C system of vertebrates.

When optimal CYF is added to mixtures of Liinulus plasma and rabbit erythrocytes (E) in Allen's artificial sea water, marked damage to the E membrane occurs (Step I) which can be visualized as hemolysis and quantified when the cells are resuspended in isotonic saline (Step II). Step I requires high ionic strength (0.63 M NaCl), calcium (9.5 m.l/), time (60 min) and temperature (24° > 37° > 4° C) ; Step II occurs rapidly in absence of divalent cations and higher temperatures.

Cell-phase plasma had somewhat greater activity than serum taken post-coagulation, and cell extracts were inactive. Liinulus plasma retained ability to support lysin formation during 1-5 day storage at 4° C, cycles of freeze-thaw, absorbtions with E and preincubations with 0.04 .17 hydrazine or CVF itself, while activity was blocked by EDTA or 70° C for 1 hr. The activity (85%) partitioned chiefly with the pseudoglobulins during water dialysis. Pre- incubation with endotoxic lipopolysaccharides (LPS) led to substantial titer reductions in selected plasmas, while both CYF and LPS regularly induced substantial titer reductions in whole blood.

CVF also induced phospholipase A activity (PA A) in Liinulus plasma, but PAA seemed dissociable from the CVF-induced hemolysin since it could be generated after preheating plasma to 70° C and partitioned (>9Q%) into the euglobulin fraction. PAA generated during the plasma-CVF interaction therefore seems not sufficient (and perhaps not necessary) for inducible-lysin activity.

We conclude that CVF activates a lysin system in Liinulus plasma with remarkable simi- larities, and certain contrasts, to the C system of man and anticipate that this lysin will provide a marker to help detect progenitor C and related proteins.

Supported by grants from the USPHS (AI-09806-01), American Heart Association, and the Minnesota Medical Foundation. H. G. is an Established Investigator of the American Heart Association.

Enzyme changes associated -lAtli development of bacterial competence. H. T. EPSTEIN.

A working model for the physiological nature of competent cells of Bacillus sitbtilis re- quires growth in a rich medium to make the cells binucleate, amiim acid step-down to stop DNA synthesis and weaken the cell wall, followed by a single cell division step. Incoming DNA is attacked by cell nucleases if suitable restriction signals are not present. Indirect experiments on transfection confirm the drop in DNA polymerase and changes in two nucleases. Direct experiments with sonicates show a 20-fold drop in DNA polymerase activity and an increased activity of a Ca-activated nuclease attacking only single-stranded DNA. Adding Ca to transfection experiments changes phage SP82 transfection in both amount and dependence on DNA concentration; the enzyme is therefore responsible for the nonlinear dependence of SP82 transfection on DNA concentration.

412 PAI'KKS PRESENTED AT MARINE BIOLOGICAL LABORATORY

Macrophage adh'ating protein jroni the sea slar Asterias forbesi coeloinocyle simu- lating effector snl>stances a] the delayed hyper sensitivity response. ROBERT A. PRENDERGAST.

Accumulation of blood borne mononuclear cells in cellular immune reactions is presumed to result from elaboration of chemotactic and macrophage activating substances following the interaction of sensitized lymphocytes with homologous antigen. A protein isolated from the cell sap of the coelomocyte of Asterias forbcsi fulfills many of the criteria for the biological effects of these mediators whose putative function it is to amplify the cellular immune response by vastly increasing the nonspecific inflammatory cell infiltrate at sites of antigen deposition. Of 32,000 molecular weight, this protein migrates as a gamma 1 globulin and has been purified to the extent that a single arc results on immunoelectrophoresis of one protein peak following G75 Sephadex column chromatography of whole coelomocyte cell sap. Intracutaneous injection of as little as 1 microgram of this protein in normal guinea pigs, sheep, mice, rats and rhesus monkeys results in a lesion which is physically, temporally and histologically indistinguishable from the classic delayed hypersensitivity reaction of the tuberculin test in animals previously sensitized to that antigen. Purified peritoneal macrophages free of lymphocyte contamination are inhibited from migrating out of capillary tubes in the presence of the star protein indicating that cross reactivity with some ubiquitous antigen is unlikely as a cause of the skin reactivity seen in all animals thus far tested. Macrophages exposed to this factor either in I'ivo or in t'itro demonstrate an increased content of lysosomes and are capable of inflicting severe damage on underlying L cell or fetal mouse fibroblast monolayer cultures. The star protein itself is not toxic to these indicator cells in the concentration employed (5 micrograms/ml medium). These data suggest that this material acts both as a chemotactic and cell activating factor specifically on the macrophage population of a number of mammalian species, and further that there exists a distinct analogy between the star coelomocyte protein and the mediators of cellular immune reactions in the vertebrates.

GENERAL SCIENTIFIC MEETINGS AUGUST 24-27, 1970

Abstracts in tbis section are arranged alphabetically by antbor. Author and subject references will be found also in the regular volume index, appearing in the December issue.

T. H. Morgan, Richard GoldscJiinidt and the opposition to Mendclian theory 1900- 1940: a chapter in the history of biological ideas. GARLAND E. ALLEN.

It is generally thought that the Mendelian theory was well received after the rediscovery of Mendel's paper in 1900. In reality, the theory faced serious criticism, especially between 1900 and 1915, but even through the 1930's and 1940's. One of the strongest critics before 1910 was T. H. Morgan (1866-1945) whose own work with Drosophila, starting about 1909, convinced him of the value of Mendel's scheme (studies of Morgan's criticisms have been published previously). Another equally strong and more persistent critic was Richard Goldschmidt (1878-1958), long director of the Kaiser Wilhelm Institute and after 1936 Pro- fessor of Zoology at Berkeley. Trained as a descriptive morphologist, Goldschmidt became interested in genetics through his studies of sex determination and the evolution of melanism in the nun and gypsy moths. His physiological theory of sex determination (he saw sex as a balanced state) led Goldschmidt to view heredity as a process. He opposed the Menclelian- chromosome theory of the Morgan school because it emphasized genes as discrete, inviolable units or structures and did not account for their function in chemical or physical terms. To Goldschmidt, Morgan had developed primarily a morphological theory of heredity. While Morgan was aware of the necessity of some day trying to study the physiology of genes, he

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 413

felt that in the years 1915-1930 experimental methods were not available for investigating this problem. Goldschmidt was particularly incensed at "explanations" such as position effect, advanced by Sturtevant in 1925, which seemed to be only pure speculation designed to save the Mendelian hypothesis. Goldschmidt's own concept of the chromosome as a "continuum"- a single long molecule with no discrete genes — was hardly an improvement. But his persistent attacks on the structural theory of Morgan kept alive the very real problem of how genes function in a physiological sense.

The effects of cytochalasin B on cvfuplasmic inurement, clcarat/e. and subsequent development of the squid cnihryo, Loligo pertlei. JOHN M. ARNOLD AND Lois D. WILLIAMS-ARNOLD.

Earlier investigations have demonstrated the existence of a morphogenetic pattern of developmental information in the egg cortex which is transferred to the yolk epithelium (= periblast) prior to organogenesis (Arnold, 1965, 1968). To check the role of cytoplasmic movements in the establishment of this morphogenetic pattern, naturally fertilized embryos were pulsed at various times for ten minutes with 0.2 /ctg/ml of cytochalasin B in dimethylsul- foxide dissolved in sea water. After the pulse, the embryos were returned to normal sea water and development followed. Higher concentrations (2.0 /ug/ml) or longer treatments stopped cytoplasmic streaming in precleavage eggs and blobs of cytoplasm appeared on the surface of the embryos. When embryos were pulsed between first and second meiosis the blastoderm appeared normally but the cleavage pattern became radial rather than bilateral. Cellulation and subsequent development was normal until organogenesis when 40% of the embryos developed abnormalities ranging from a complete lack of recognizable organs to reduced and distorted organs in their usual positions. When pulsed during first mitosis, first furrow formation was prevented or delayed. 78% of the resultant embryos showed abnormalities and of these many more lacked recognizable organs or had organs in abnormal positions. Pulsing at four cells killed many embryos but those that survived had reduced recognizable organs in their proper placement. Pulsing at 32 cells produced 78% abnormal embryos all of which had reduced recognizable organs in their proper placement. Pulsing when the embryo was one-third or three-fifths cellulated produced no significant defects.

It was concluded that cytochalasin B sensitive cytoplasmic movements are important in establishing the blastodisc and positioning the mitotic apparatuses which in turn determine the cleavage pattern. Abnormal cytoplasmic movements lead to an abnormal yolk epithelium which then causes an anomalous pattern of induction resulting in a failure of organogenesis or incompletely differentiated embryos. Thus, the precleavage pattern of cytoplasmic movement seems to determine the organization and position of the organ determining areas in the egg cortex and greatly influence subsequent differentiation of the squid embryo.

This work was supported by NIH grant EY HD 00179.

Effect of fertilization on the calcium and magnesium content of the eggs of Arbacia punctulata. ROOBIK AZARNIA AND EDWARD L. CHAMBERS.

Jelly-free unfertilized eggs were prepared in 0.2% suspension in sea water, divided between two beakers, stirred at 60 rpm, maintained at 20° C, and one of the suspensions inseminated. At intervals duplicate 100 ml samples were removed to Hopkin's tubes, hand centrifuged at 250 X g for 30 sec, the supernatant aspirated, and the volume of eggs plus supernatant fluid in the calibrated tails of the Hopkin's tubes measured using a cathetometer.

Unfertilized eggs in sea water contain on the average 2.98 ± 0.04 fj.m Ca/ml eggs and 21.9 ± 0.3 ^m Mg/ml eggs. Stirring for periods of 11 to 239 minutes, a three minute wash with artificial sea water containing l/10th the normal concentrations of Ca and Mg, or with Ca-free sea water, and treatment with 0.1%- pnmase In remove (he external coals had no effect on the ( 'a and Mg content of unfertilized eggs.

Following fertilization a sharp increase in the Ca content (15% at 3 min) occurs followed by a sharp decrease with the Ca content attaining the unfertilized level by 40 min after insemi- nation. In parallel determinations carried out on eggs washed 3 min in Ca-free, or l/10th

414 I'. \PERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

calcium sea water a 20 to 40% decrease in the Ca content of each of the egg samples was observed compared to the corresponding samples in sea water. The result of this decrease is that the washed eggs exhibited a sharp (15%) lowering of the Ca content at 6 min after fertilization attaining a value 25% less than that of the unfertilized eggs by 40 to 50 minutes after insemination.

With regard to the Mg content, a 3% (but statistically insignificant) increase is observed by 4 minutes after insemination. Thereafter a sharp decrease occurs until by 50 minutes the Mg (ontent is 25% less than that of the unfertilized eggs. Washing the fertilized eggs in lo\\ Ca and Mg sea water had no effect on the Mg content.

The authors acknowledge the able technical assistance of Charlotte B. Vinton. Supported by NSF grant #GB 8054.

. / I'/nis disease of /lie shore crab Cnrcinus mamas. FREDERIK B. BANG.

When blood is taken from a shore crab by a syringe and needle in the presence of an extract of amoebocytes, a clot starts to form in a few seconds, and within a few minutes this contracts on the slide into a tight mass. In the summer of 1969, while at the Station Bio- logique, Roscoff, a crab was found in which typical clotting did not occur. When this non- clotting blood was injected into other crabs, the same disease process was induced 2 to 13 days later. The infection was repeatedly transferred and was manifested by ( 1 ) failure of clotting, (2) reduction in total amoebocyte count, (3) formation of small tight clumps of amoebocytes in the peripheral tissue. The agent was filter passing, and was preserved by freezing of the blood.

Four aspects of this infection have been studied at Woods Hole. (1) Mortality, at least during the first month, is relatively mild and recovery as manifest by a reappearance of the ability to form a firm circular clot is high. Recovered animals are susceptible to reinocula- tion. (2) The agent tested by inoculation of other crabs persists in the blood as long as seven weeks after inoculation and several weeks after the blood has regained the ability to clot. (3) The agent in serum titered to 10". (4) Electron microscopy of infected amoebocytes fixed two days after initiation of signs of infection showed typical virus particles, roughly 100- 150 m/j. which occurred in packages within the cytoplasm, in scattered arrays within the endoplasmic reticulum, and on the surface of the cells. No particles were found in normal amoebocytes with or without clot formation.

Pine structure and infraeellnlar responses oj photoreceptors oj a pelagic tunicate, Salpa. STEPHEN N. BARNES, ANTHONY L. F. GORMAN AND JOHN S.

McREYNOLDS.

Visual cells in chordates, including vertebrates, have been shown to be of the ciliary type. Recent evidence indicates that ciliary type photoreceptors are associated with hyper- polarizing receptor potentials. The urochordate Sulpa, has a lensless eye, arising from the neural ganglion, which is directly accessible to light, being situated on the dorsal surface of the animal beneath the transparent tunic. Sections of the eye examined with an electron microscope show that the visual cells are approximately 10 micra in cross-section and 60 nn'cra in length, and give rise to a randomly organized array of microvilli at one end as their photosensitive structure. No membrane specializations were seen which were derived from cilia. Intracellular recordings were made from these cells with high resistance, KCl-filled microelectrodes. Resting potentials in the dark were low, approximately 10 mv inside nega- tive. The response to illumination was a hyperpolarizing potential change, graded with light intensity, which could be up to 70 mv in amplitude at maximum intensities. The responses were sustained as long as the light was on. No spike activity was seen in these cells. Changes in the voltage drop produced by constant current pulses passed across the cell membrane in dark- ness and during illumination showed that the hyperpolarizing receptor potential is associated with an increase in membrane conductance.

This investigation was supported in part by Training Grant number GM019S1 and Re- search Grant number EY00443 from the National Institutes of Health.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 415

Comparison of melanophorotropic lipoprotein fractious jrom the pituitary of the dogfish. Alustelus canus. and jrom the eycstalk of the fiddler crab, Uca pugilator. CLELMER K. BARTELL, MILTON FIXGERMAX AND JOHN E. STEPHENSON.

Ethanol and 95% methanol : chloroform (2:1) extracts of Uca eyrstalks contain lipoprotein components which, in aqueous media, form low-density micellar lipoproteins that cause dis- persion of pigment in Uca melanophores. At concentrations ahove one eyestalk per animal the activity of both extracts is higher than that of aqueous extracts. The melanophorotropic factor is more stable in methanol: chloroform than in ethanol and will survive filtration on Sephadex LH-20 (methanol: chloroform) with little loss of activity. Extracting in methanol: chloroform both optic ganglia and sinus grands yield melanophorotropic lipoprotein. The density for the active micellar lipoprotein from ethanol extracts is between 1.016 and 1.034 g per ml, for methanol: chloroform, between 1.034 and 1.047 g per ml. When the active material is extracted in methanol: chloroform from lyophilized, chloroform-washed tissue and separated on LH-20 (methanol: chloroform), the micellar lipoprotein density is not changed. A higher density active material which appears to be a pseudomolecular lipoprotein, or apolipoprotein, with a density between 1.06 and 1.20 g per ml has been separated using two diphasic solvent systems — methanol : chloroform: water (2:1:1) for methanol: chloroform extracts and ethanol: chloroform: water (2:1:1) for ethanol extracts. In each system the melanophorotropic mate- rial was recovered in the upper phase, i.e., the methanol: water, or ethanol: water phases. The apolipoprotein from both extracts, when suspended in aqueous media with lipid components from either extract, will associate with micelles having the same density characteristics of the lipoprotein from the whole extract of its origin. The apolipoprotein, therefore, appears to show some specificity. A lipoprotein (density between 1.016 and 1.064 g per ml) that will cause dispersion of pigment in melanophores of dogfish was extracted from Mustelits pituitary using 95% methanol: chloroform (2:1). The presence of melanophorotropic lipoproteins in two animals of diverse phylogenetic origins is evidence that the association between lipids and peptide hormones in cells of endocrine tissue may be a general phenomenon.

Supported by grant GB-7595X from the N.S.F.

Moi'cincnts of ecJiiuocJiroine i/ra>n/les during the early development of Arbacia eggs. Axx M. BELANGER AND RONALD C. RUSTAD.

The echinochrome pigment granules in Arbacia eggs are known to exhibit random saltatory movements before fertilization and migration to the cortex shortly after fertilization. Before the sixteen-cell stage nearly all of the granules leave the region which will become the micromeres. In our experiments this migration occurred during the four-cell stage, leaving a "clear area" on each blastomere.

Incubation at 5° C reversibly inhibited migration to the cortex and formation of "clear areas." The pigment granule movements occurred at the normal times following -/-irradiation of unfertilized or fertilized eggs with doses as large as 250 kR. Colcemid (10~3J/) did not prevent prefertilization saltation, migration to the cortex, or formation of "clear areas." How- ever, the latter was often delayed. Cytochalasin B (1-5 /ug/ml ) reversibly blocked saltation and cortical migration and prevented the formation of "clear areas." Cytochalasin activated the elevation of the fertilization membrane in unfertilized eggs. When activated eggs were washed, pigment granules migrated to the cortex, and "clear areas" were formed at the normal time. When Cytochalasin was added shortly before cytokinesis, division furrows began to form and then regressed, leaving concentrated bands of pigment in the furrow regions.

Since blocking mitosis in prophase with irradiation or in metaphase with Colcemid did not prevent the formation of "clear areas," this phenomenon must be independent of nuclear activity. Since the normal movements of pigment granules occur in the presence of Colcemid, but not Cytochalasin, these movements would appear to be independent of microtubules and dependent on functional microfilaments.

This research was supported by Contract W-31-109-ENG-78 with the U. S. Atomic Energy Commission, Report No. COO-78-233, and by an X. I. H. Training Grant.

416 PAPERS 1'KESENTED AT MARINE BIOLOCK 'AL LABORATORY

The statistical phenot\pe concept, witli applications to skeletal "variation in the mouse. ALLAN BIRNBAUM.

Genetic theory gains conceptual unity, simplicity, and generality with adoption of the concept of a statistical />/(<•;/<>/ v/v, which includes as special cases the two kinds of variation usually considered (qualitative and continuous), and also threshold characters, general multi- nomial classifications, and any others. A statistical phenotype is defined formally simply as a probability distribution over any given set of phenotypes (descriptions). This leads to some new systematic approaches to genetic data analysis, including Mendelian and related interpreta- tions, freed of the usual conceptual restrictions and practical difficulties associated with questions of scaling. It sometimes also provides a useful standpoint for appraising the reality and nature of a possible underlying scale of variation.

The general approach is introduced briefly here; it has been developed independently and more systematically by Collins (Genetics, 1967, 1968, 1969, 1970). It is applied here in detail to the data on skeletal variation in the mouse of Green (Genetics, 1954, 1962), and the results are compared and contrasted with those found by Green by use of the more customary methods. Many of Green's interpretations concerning the adequacy of one-gene and several-gene models are given new support ; for other interpretations, some qualifications or modifications are indicated.

Studies on blood folic acid in the smooth doc/fish, Mustelus canis. T. A. BORGESE

AND L. ClANCI.

Heparinized caudal vein blood, obtained from a single dogfish or pooled from several, was used for determining blood folic acid employing the microbiological assay with LactubaciUus casci as the test organism. Most of the folic acid present in the blood is found in the cells. The mean values and standard errors for whole blood, plasma, and cells from ten experiments were 19.4 ± 4.6, 2.32 ± 0.48, and 72.1 ± 22.0 ng per ml, respectively. Several factors, including suspension medium, plasma concentration, and in z'ii'o maturation of red cells, were shown to influence folic acid levels. Cells washed and resuspended in buffered elasmobranch Ringer's solution, pH 7.2, showed considerably less measurable folate activity than cells washed and resuspended in their own plasma. In each case the cells were reconstituted to the same packed cell volume. The ability of plasma to maintain maximum folate activity in unwashed cells decreased as increasing volumes of plasma were replaced with Ringer's.

After removal of the huffy coat, differential centrifugation separated the red cells into relatively young (less dense) and relatively old (more dense) population. Bioassays on each of these demonstrated that the less dense cells had a higher folate content than the more dense ones.

We conclude that a factor, present in the plasma, is essential for maximum folate activity in the dogfish red cells.

Supported by grants from the Nutrition Foundation, The City University of New York Research Foundation, and Lehman College.

Microspectrophotometry of fist/a! pigment and spectral sensitivity of retinular cells in the crab. Cardmis. M. S. BRUNO, M. I. MOTE AND T. H. GOLDSMITH.

Single rhabdoms from dark-adapted green crabs (Cardans madias} were detached from their surrounding retinular cells and suspended in $%• glutaraldehyde. Transmission spectra were recorded using a 4 X 10 ^m measuring beam incident at right angles to the long axis of the rhabdom. Difference spectra obtained by partial bleaches with red followed by yellow light were almost identical and gave no indication that more than one visual pigment was present. The mean difference spectrum for the total bleach of 23 rhabdoms had Xm:,x at 502-505 nm and had 0.6% ^m~l absorption at the peak.

Spectral sensitivities (reciprocal photons for a 5 mv response) of single retinular cells were measured at 9 wavelengths between 412 and 672 nm in preparations of isolated eyestalks, using intracelluar microelectrodes. Average sensitivity of six cells was found at 495 nm, in agreement with the results on 17 other cells which were held only long enough to measure spectral efficiency (response height for an equal photon spectrum). The spectral sensitivity of

PAPERS PRESENTED AT MARINE BIOUHiU AL LABORATORY 417

the ERG was similar, except that the curve was slightly broader at longer wavelengths, pre- sumably due to the relative transparency of the interommatidial screening pigment in the red. Selective adaptation of single cells with either red or hlue light decreased the sensitivity but rarely changed the shape of the curve from that of the dark-adapted cell. In a few cells, red adaptation caused a decrease in relative sensitivity at the red end of the spectrum. This decrease was associated \\ith slowly occurring changes in sensitivity which made accurate measurements difficult. The time course of these changes suggest that they may have been due to retinomotor phenomena.

This work was supported by USPHS grant EY00222 to Yale University and by a USPHS postdoctoral fellowship to M. I. M.

Pacemaking and phrase-shifting in synchronising fireflies. JOHN BUCK, JAMES F. CASK AND FRANK E. HANSON.

In certain oriental firefly species in which the males habitually flash rhythmically in unison it was found possible to entrain individuals to artificial light flashed in both slower and faster rhythms. In this entrainment the firefly systematically led when paced at a rhythm slower than his spontaneous rhythm and lagged when paced at a faster rhythm. The amount of lead or lag approximated the difference between the pacer period and the firefly's free-running flash period. The firefly's interflash period immediately after the first pacer flash was typically longer or shorter than the pacer period depending on whether or not the flash that would next have occurred, had the firefly persisted in his original rhythm, was inhibited. The phase- shifting effect of the pacer flash was immediate but lasted only one flash cycle — that is, the firefly reverted to his original free-run flashing frequency as soon as the pacer ceased flashing. Such entrainment by artificial light could be explained if each pacer flash in effect resets the firefly's endogenous central nervous pacemaker.

The authors are indebted for the facilities of the 1966 ALPHA HELIX Expedition (Univer- sity of California, San Diego) at Maiwara via Madang, T. P. N. G. Buck acknowledges the assistance of Penrose Fund Grant No. 5017 (American Philosophical Society) and a grant from the National Geographical Society. Case was supported by Contract N00014-69-A- 0200-8006 of the Office of Naval Research.

Comparative physiology of neurally controlled luminescences. J. F. CASE, G. T. REYNOLDS, J. BUCK, J. BURNS AND R. HALVERSON.

The mechanisms of neural control of luminescence have almost totally evaded analysis except in firefly larvae and adults in which adrenergic control seems quite likely. The study of three other systems now suggests that they also are adrenergic.

Railroad worm, Phryxothrix, thoracic and abdominal photophores are neurally excitable and glow in an erratically phasic manner when attached to an eserinized ganglion. Pseudoflashes are induced by re-oxygenation of hypoxic preparations. Isolated photophores glow in nor- adrenaline and amphetamine, but not in acetylcholine. Nor-adrenaline restores glowing in amphetamine exhausted photophores. Midshipman fish, Porichthys, photophores are excitable •:•/<; their peripheral innervation in vivo and in vitro and glow when treated with nor-adrenaline or amphetamine. Acetylcholine and eserine in vivo together induce w?eak glowing but only- after elapse of sufficient time to render a direct effect improbable. The sea pansy, Rcnilla, glows in a sustained fashion in amphetamine or ephedrine but undergoes no luminescence change in nor-adrenaline. Luminescence is inhibited by glutamate without evidence of initial enhance- ment. Thus all probably directly neurally excitable luminescent systems studied to date in some reasonable detail appear to have similar transmitter mechanisms.

Supported by A. E. C Division of Biology and Medicine, Office of Naval Research and the National Science Foundation.

1 he effect of temperature on the effln.v of {-'Ca from tlic ec/t/s of Arbacia punctulata. EDWARD L. CHAMBERS, ROOBIK AZARNIA AND WILLIAM E. McGowAN.

Jelly-free unfertilized eggs were prelabelled in a 1.2% suspension containing 2 /j.c 43Ca/ml, 1 mg streptomycin/ml, pH 8.0, 20° C, stirred at 30 rpm for 12 hours. The eggs were washed

41 (S PAPERS PRESENTED AT MAR IX 1C BIOLOGICAL LABORATORY

free of external '"Ca by repeated addition and decantation of inactive sea water over a two hour period, and then inseminated.

Efflux from the washed unfertilized eggs occurs according to a single term exponential process with a rate constant of 0.0003 min "' (initial efflux for the first several minutes after removal of the unfertilized eggs from labelled sea water was omitted, since this is exceedingly variable and represents desorption from the external coats of the eggs).

Following insemination, efflux occurs according to a double exponential process, with an average rate constant at 20° C for the fast component of 0.074 min"1 (2.5 X 10"12 moles Ca cm"2 sec"1) and 0.0026 min"1 (9 X 10"" moles Ca cm"2 sec"1) for the slow component. At 10° C the rate constants for the fast and slow components are decreased to 0.030 miir1 and 0.00077 min"1, respectively. This gives Qm values for the rate of 4''Ca efflux from the fast component of 2.5, and 3.4 for the slow component, indicating that both phases are linked to the metabolic activity of the egg.

Further evidence that the measured efflux of 4"Ca is intracellular in origin is (T) exposure of washed prelabelled unfertilized eggs to 0.1% pronase and to 0.1 to 0.5 mM lanthanum in bicarbonate and sulfate free sea water do not significantly diminish the ^Ca content, (2) the same double exponential efflux curves were obtained following fertilization whether the eggs had been prelabelled for 50 minutes or for 24 hours, and (3) the decrease in Ca and 45Ca content of fertilized eggs washed in Ca-free sea water, when expressed as per cent of the unfertilized values, are the same.

Supported by NSF grant GB 8054.

Failure oj coelomocytes <>} sonic Asterias torbesi to clump on (/lass. JOHN NORRIS CHILDS TIT.

Clumping of coelomocytes of the starfish Asterias forlicsi involves the formation of loose aggregates of cells and subsequent contraction over three to five minutes to form tight clumps. Clumping was studied in the star since it has a single cell type in a coelomic fluid containing only small amounts of protein. Previous work has shown that a crude extract of coelomocytes clumps these cells in vivo and in an in vitro buffer system of pH less than 5.8 which prevents cells from spontaneously aggregating. Of 300 starfish examined, 31 had cells which failed to clump on glass. These coelomocytes appeared morphologically similar to normal cells and responded normally to the addition of coelomocyte extract in I'ii'o and in vitro. In vitro tests of clump initiating ability of various substances showed this ability to be present in coelomocyte extract and protein isolated from coelomic fluid, but lacking in ATP, ADP and sperm and ova extracts.

There was a correlation between presence of sperm in coelomic fluid and failure to clump. Two early findings showed a ciliated parasitic organism, Orchitophyra stcllarnm, to be present with free sperm in the coelomic fluid of non-clumpers. Upon injecting non-clumpers with coelomocyte extract, sperm seething with ciliates was discharged, establishing a correlation between maleness, infection of testes with Orchitophyra stcllarnm and failure to clump.

In spite of failure to clump spontaneously on glass, the coelomocytes of these non-clumpers did clump in response to the two substances, coelomocyte extract and coelomic fluid protein. A difference observed in the time course of response and final result may indicate a clumping process of several steps, involving a plasma factor initiating loose aggregation and subsequent secretion of a cellular substance promoting tight clumping.

Chanyes in fluorescence oj squid a.vons dur'nuj activity. L. B. COHEN, D. LAN- DO WNE, B. B. SHRIVASTAV AND J. M. RITCHIE.

In an attempt to study changes in membrane structure associated with nerve activity three dyes were applied to the squid giant axon and their fluorescence was monitored during voltage clamp experiments. Signal averaged records of Acridine Orange fluorescence showed a de- crease in fluorescence by about 3 X 10~s when the nerve was hyperpolarized by 50 mv and an increase of the same amount when the nerve was depolarized by 50 mv. Rhodamine B exhibits a similar behavior except that the sign of the change is reversed. As the experiment progresses the amplitude of the change in Rhodamine B fluorescence decreases and then the change

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 419

turns over and is in the opposite direction. The early change is an increase of about 10"' for a 50 inv hyperpolarization and the late change is a decrease of approximately 2 X 10~" for the .same step. With hotli Acridine Orange and Rhodaminc 15 the same results arc ohtained if the dye is micro-injected inside the axon or applied in the outside sea water.

l-analino-8-napthalene stilfnnatc (ANS) shows opposite changes when a]iplied to opposite sides of the membrane. Thus when ANS is mien (injected a 50 niv hyperpolarization causes an increase in fluorescence of about 3 X 10~". When the dye is applied externally this voltage step produces a decrease of the same size. In addition, in records from microinjected axons, one can see a difference between hyperpolarization and depolarization, hyperpolarization being about 30% less effective in causing the change in ANS fluorescence.

The time course of the fluorescence changes follows the voltage with a lag of less than one msec (different lags for different dyes). The changes in fluorescence in these dyes thus seems to be related to the changes in voltage across the axon membrane.

Supported by Public Health Service grants NB 08437 and XI! 08304.

The possible role of hydrogen evanide in the origins of life: the hydrogen cvanide polymers. 1). B. DONNF.R, A. P. LOHO, J. D. \Yos AND J. P. FERRIS.

The chemistry of hydrogen cyanide has been studied with respect to its role in the origins of life. Hydrogen cyanide couples in aqueous media ( pH 8-9) to yield a tetratneric species which serves as an intermediate in the formation of most ot the purines of importance in contemporary biological systems.

Hydrogen cyanide also self condenses to yield polymers ; these reactions occurring under the same conditions that lead to purine synthesis. Polymers containing a repeating subunit (with attendent functionality that imparts to these compounds acidic, neutral, or basic character) have been isolated and separated from one another using Sephadex and ion exchange chromatography. A molecular weight of five hundred is estimated from Sephadex data and sedimentation studies. The structures of the polymers formed are independent of the base used to start the polvmcriza'.ion suggesting that the reaction is initiated by attack of cyanide ion on hydrogen cyanide tetramer.

Hydrolysis in six normal hydrochloric acid degrades twenty to twenty five per cent of the polymers to at least eleven amiiio acids. Neither the amount of amino acids liberated nor the character of the non-degraded portion of the polymers varies significantly when the base used to neutralize the hydrogen cyanide is ammonia, sodium hydroxide, or triethylamine.

The possibility of peptide bonds being present in the polymers has been disproved. Such functionality is inconsistant with some of the physical properties displayed by the polymers. Degradation experiments using pronase and carboxypeptidase A (both of which attack peptide bonds) failed to liberate amino acids. The Biuret test was also negative indicating the absence of peptide bonds.

A new structure is proposed for the polymers consisting of a repeating ring skeleton (which is non-degradable in acid) with labile side chains that are hydrolized to amino acids. The polymers catalize the oxidation of NADH to NAD and resorcinol to o-benzoquinone. Further studies on the structural and catalytic properties of the polymers are in progress.

Supported by grant GP8254 from the National Science 'Foundation and a Research Career Development Award (to J. P. F.) from the National Institute of General Medical Sciences.

S-potentials in the skate retina: intraeellnlar responses during light- and dark- adaptation. ). E. DOYVLIXG AND H. RlI'I'S.

The S-potentials recorded from the all-rod retina of the skate probably originate in the large horizontal cells located immediately below the layer of receptors. These cells were always hyperpolarized by light of any effective wavelength. The adaptive properties of S-units were investigated by measuring changes in sensitivity and D. C. level during light- and dark-adaption. After flash bleaching the retina, the variation in log threshold followed the same time course as observed previously for the ERG h-wave and ganglion cell discharge. The effect of the fla^h on the D.C. level was to maximally hyperpolarize the S-unit for about 10 min during which time no response could be elicited by lest Hashes 8 log units above

420 PAPERS PRESENTED AT MARIXK BIOLOGICAL LABORATORY

absolute threshold; with further time in darkness the !).(.'. level moved hack toward the cell's resting' potential and excitability returned. Subsequently changes in sensitivity exhibited an initial fast (neural) phase and a later slow (photochemical) component, hut neither appeared to he correlated \\ith the temporal course of the changes in I ).C. level.

The increment threshold function measured against steady background fields was also similar to that obtained from more proximal regions of the retina. When the background was sufficiently bright to maximally polarize the S-unit, the cell became unresponsive for periods up to 15 min (depending upon background intensity). However, the cell did not maintain its D.C. level with continuous illumination; as the cell's potential returned toward the resting" level, response amplitude increased, and thresholds decreased to a steady state value.

The waveform and rise and fall times of the S-potentials were markedly different in the light- and dark-adapted states. However, amplitude I'crsns intensity functions determined under light- and dark-adapted conditions for wavelengths from 420 to 620 nm were similar, and the spectral sensitivity curves derived from these data were virtually identical.

Supported by grants ( KY-00470, EY-00285, EY-18766) from the National Institutes of Health, and by a Fight for Sight award from the Carrie L. Groedel Fund.

Differential utilisation of phy to plank ton food resources />v marine ectoprocts. J riMTii \V. DUDLEY.

The general view of filter-feeding by ectoprocts is that they are remarkably alike in the food gathering apparatus, with probable identity in their food resources. Several lines of evidence suggest instead that individual species of ectoprocts found near \Yoods Hole are specialized for making use of the available food resources.

In these species tentacle number varies from 8 to 30, tentacle length from 0.2 to 1.0 mm, mouth diameter from 0.02 to 0.09 mm, and the diameter of the food gathering cone (maximum t'istance across the expanded lophophore) from 0.2 to 1.4 mm. All of these variables are positively correlated, with the greatest number and longest tentacles occurring in species with the largest mouths and food gathering cones. These species would therefore be able to utilize different size fractions of the phytoplankton.

Experimental feeding of micronic beads to two species of Bn</itlu with similar sized zooids showed that though they were consuming beads from within the same size range, the species with the larger mouth was able to consume more large heads, and the species with the faster digestion time was able to consume significantly more beads per zooid.

In view of these observations, it appears that a better initial premise for considering feed- ing by ectoprocts from a given locality is that every species is doing something different from every other. This may he important in considering the diversity of marine ectoprocts, with more than 3000 living species.

Supported by grants from the Hinds Fund, University of Chicago, and the Geological Society of America.

ll'atcr permeability of Fundulus et/t/s. P. B. DUNHAM, A. CASS, J. P. TRIXKAUS AND M. V. L. BENNETT.

FiDnliiliis eggs develop normally in both distilled water and sea water. There is no morphological evidence for a water regulating % mechanism which might he responsible for maintenance of constant volume. Accordingly, we determined tracer water permeability, Pu, of fertilized Fnndiilns eggs from H '-water fluxes at 23° C. For influxes, mid-gastrula eggs from one female were placed in 2:< Holtfreter's medium with tracer. Eggs were removed at intervals, washed in unlabelled medium, and extracted in Bray's solution, which was then counted. Volumes and surface areas were calculated from linear dimensions of the egg hounded by the plasma membrane, assuming the shape to be an oblate spheroid. The saturation level of IP-water, necessary for calculation of k, the rate coefficient of influx, was calculated from the volume ot the egg, assuming 80%, water space. With this assumption, a semilogarithmic plot of the influx data fit well with a straight line. In three experiments, k ranged from 0.15 to 0.17/

I' \PERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 421

hour. I'M. given by the product of k ami tlic volume/surface ratio, ranged from 0.96 to 1.14 • 1(1 '' cm/sec.

I'.tthi.x of 1 1 :-water was measured liy transferring labelled eggs through aliquots of uu labelled medium. The k for efflux was similar to that for influx (0.12/hour after 18 hours ol loading, cS5r/r exchange of egg water content), although there was an indication of series com- partments, since efflux was faster after 1.5 hours of loading (0.17/hour, 20% exchange). The rate of efflux was identical from intact and dechorionated eggs, showing that the plasma memhrane is the limiting harrier for water permeation.

The low Pi. indicates no special mechanism is necessary for regulation of volume. The observed rate of volume change during development of teleost eggs is consistent with the Pn. The PI, of Fitnditlus eggs, 10"" cm/sec, is one of the lowest reported for thin membranes (less than 100 A). Salmon eggs have recently been shown to have a similar Pi, at 5.5° C. The lowest permeability of artificial lipid hilayers is about 10"' cm/sec.

Tlic effect oj cold c.vpositrc on protein synthesis in the he p at q pancreas of the spider crab, Lilnnia emaryinata. I\ KKF.CCA ELLISON.

The spider crab, Lihiniti t'liiiii't/iiitifd, is found from Nova Scotia to the Bay of Mexico. As a species it encounters a wide range of temperatures. The question asked in these experi- ments was whether individuals of this species could be stimulated by cold exposure to undergo compensatory adjustment of metabolism like that reported for certain other poikilotherms.

Ma'e intermolt Lihiniti of medium size ( approx. 150 g) were used. Experimental animals were kept at 10-14° C in running sea water aquaria for 1, 5, 12, and 18 days. Controls were kept at 22° C. Crabs were starved for an appropriate period to match nutritional status of control and experimental animals. Experiments were performed at the same time of day to minimize the effect of circadian rhythms.

Crabs were injected intrapericardially and protein synthesis in the hepatopancreas (a major site of metabolic activity in the non-molting crab) was measured by the incorporation into protein of a mixture of fifteen C" amino acids (5 /xc/crab) after a 30 minute incubation period at 23° C. The ratio of incorporation into protein to free radioactivity in the tissue was taken as the index of protein synthetic capacity. The group of 14 control animals showed a protein synthetic index of 0.28 ± 0.08. Animals kept at 10° C for 1, 5, and 12 days (N = 25 ) did not show significant differences in mean value from the controls, however individual variations were unusually great (standard deviations about 0.17). One group (N = 10, 18 days at 10° C) had a synthetic index of 0.15 ± 0.07 at 20° C indicating loss of metabolic capacity under these experimental conditions. In general, the results indicate no compensatory temperature acclimation in this species, however further studies under other conditions of feeding and handling are needed.

This study was supported by National Science Foundation Grant B8 2326R to Dr. A. Haschemeyer.

In vivo intestinal absorption oj D-glucose in tlic toad fish, Opsanus tan, and flic rainbow trout, Salmo ^airdneri. A. FARMANFARMAIAN AND F. E. DiBsxE-

DETTO.

The uphill transport of sugars and amino acids in the intestine of mammals is Na- dependent. It is useful to investigate the intestinal absorption of nutrients in marine and fresh water fish since the intestinal lumen of these fish may be exposed to high or low sodium concentrations.

In t'i'ro preparations of toadfish everted sacs undergo contraction after excision and are not suitable for absorption studies. Therefore, in vivo procedures were adopted. Fish were weighed and anesthetized in appropriate solutions of MS-222. The midgut was exposed without damage to the circulation. A 3-5 cm segment of the intestine beyond the entry of the bile duct was washed with saline and isolated by means of an anterior and posterior ligature. Solutions of glucose and inulin in the appropriate saline were placed in the segment, mixed, and an initial sample was removed. The abdominal incision was closed properly. The tish were then allowed to revive and swim in a suitable tank or \\ere provided with a How oi

422 PAPERS PRESENTED AT MARINE BIOLOGICAL I. A l!< >U ATORY

oxygenated anesthetic solution over their gills. Tin- absorption was allowed In proceed tor 30 minutes at 20 ± 1° ('. The body cavity was reopened and a terminal sample of the luminal medium and the entire isolated gut segment were removed for analyses. \\'hen an initial solu- tion of 1(1 m. I/ glucose was used, the ancsthcti/ed toadlish gave an absorption rate of 6.83 ± 0.81 jumoles/g fresh gut/hr. The rate for unanesthetized animals ( 6.86 ± 1.17) was not significantly different (/'<0.05). The tissue/medium (T/M) terminal concentration ratio for the former was 0.80 and for the latter was 0.33. The difference was significant and probably due to better circulation in the swimming fish. These ratios did not indicate uphill absorption of glucose. However, at 2 m.l/ initial glucose, the absorption rate dropped to 3.47 ± 0.61 and the T/M was 1.58 indicating that uphill transport of glucose could be observed under those conditions. In trout, at 10 m.l/ initial glucose, a higher absorption rate, 8.89 ± 3.2, was found, and T/M was 0.37.

In part supported by NSF grant GB-8089 and the Research Council of Rutgers University.

.Inalysis oj the chromatophorotropic activity of the central nervous system of the horseshoe crab Limulus polyphemus. MILTON FJNGERMAN, CLELMER K. BAR- TELL AND ROBERT A. KRASNOW.

Extracts of the central nervous system ( CNS ) of the horseshoe crab were known to evoke pigment dispersion in the melanophores of the fiddler crab, Uca. and concentration of the pigment in its leucophores. The present experiments were designed to compare the chromatophorotropic material from Liunilits with that in the eyestalks of the fiddler crab. Ex- racts of Liiinilus CNS cause! melanin dispersion and white pigment concentration in the fiddler crab, ('(•(/ pityildtor, but were not effective in either dispersing or concentrating the pigment in the crab's erythrophores. In contrast, the eyestalks of the fiddler crab are known to contain, in addition to melanin-dispersing and white pigment-concentrating substances, hor- mones that evoke both red pigment-dispersing and pigment-concentrating responses. The pigment-dispersing activities from I 'en eyestalks were eluted from a Bio-Gel P-6 column with an Rr of 0.6 and the pigment-concentrating activities with an Rf of 0.28. In contrast, the melanin-dispersing and white pigment-concentrating activities from Lininlits CNS had relative flow rates of 0.57, which is essentially the same value as that of the pigment-dispersing activities from ("a; eyestalks. Furthermore, the activities from Liiimliis CNS did not separate from each other on a column of Sephadex LH-20 in methanol: chloroform (2:1). When Limulus CNS was first extracted in ethaiml or methanol: chloroform (2:1) rather than directly in saline, both activities were more than three times as large at a concentration of 0.1 equivalent CNS per dose as the corresponding activities obtained with extracts prepared directly in saline. Both activities from Liniitlits CNS were insoluble in acetone and may be due to a single substance.

Supported by Grant GB-7595X from the N. S. F.

The origin of the H</hf producing cells during development of flic Ctcnophorc Mnemiopsis leidyi. GARY FREEMAN AND GEO. T. REYNOLDS.

The origin of the cells which produce luminous products in the radial canals of Mnemiopsis has been studied by removing blastomeres from embryos at early cleavage stages of development and monitoring the ability of the larval fragments that form to produce light with a photo- multiplier and an image intensifier.

The division which forms the eight cell stage produces the first two classes of blastomeres which develop in different ways — the E and M macromeres. The M macromeres are the only class of blastomeres isolated at this stage which develop the ability to produce light. During the next two divisions (16 and 32 cell stages) the M macromeres give off two micromeres which form the contribution of these macromeres to the ectoblast of the embryo. Light production does not occur if the M macromeres are removed at these two stages indicating that the capacity to produce light is not associated with the micromeres formed at these stages.

In a 24 hour old larvae there are four luminous areas which correspond to the radial canals of the four sets of comb plates. The relationship between the M macromeres and

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 423

bioluminescence was studied by removing one or more macromercs at tlie 32 cell sta.^c oi develoinneiit and counting the number of luminous areas which form. The maximum numhei nt luminous areas present is correlated \\ith tin1 immher ot M macromeres which are present at the 32 cell stage.

Single M macromeres isolated from the X, 16 and 32 cell static animals \\ill produce light. However the percentage of cases which produce light declines when the macromeres are isolated from the 16 and 32 cell stages. The relative amount of light produced also declines roughly 500-1000 fold. By increasing the amount of tissue isolated, by culturing two or four M macromeres as a unit from the 16 and 32 cell stages one can increase the percentage of cases that produce light but not the relative amount of light produced. This suggests that an inter- action may take place between the ectoblastic micromeres and the M macromeres which facilitates light production.

.In indue ihlc lysiu in Limulus polyphemus with similarities to the complement system of vertebrates: variations with disease and alterations induced by cobra venom factor in vivo. HENRY GEWURZ, DONALD JOHNSON, VANESSA BIRDSKY. JEAN LINDORFER AND ANITA GEWURZ.

Addition of cobra (Xaja luijc} venom factor (CVF) to Limit/its plasma in vitro activates a lysin system, dissociable from phospholipase A activity (PAA), with remarkable similarities to the complement system of vertebrates. We now report observations of this lysin ;';/ vivo.

Plasma samples from 42 consecutive animals showed a mean titer of 250 units/ml, with notable individual variation (range: < 10-1000). The three most deficient animals (<10, <10, 55, respectively) selectively had < 15,000/mm3 circulating arnoebocytes ( normal = 45, 000/mm3), with decreased and distorted cells recognizable in their gills, suggesting an association between decreased lysin activity and ongoing disease.

In higher vertebrates CVF is a relatively nontoxic factor ( causing only transitory hypo- tension and intravascular hemolysis) which induces sustained suppression of hemolytic comple- ment. Immediately following the intracardiac infusion of 500-1000 units CYF into each of 5 crabs, ( 1 ) CYF-induced lysin activity appeared in the plasma and remained detectable for 1-3 hrs, (2) the peripheral cell count dramatically decreased to 10-40% pre-infusion control values with margination, clumping and finally marked diminution in number of gill amoebocytes, and (3) inducible plasma lysin activity fell to < 10% control values and remained depressed 12-24 hrs. Thus, certain features of CVF-induced interactions of vertebrates were induced in Limit/us along with an exaggerated cytotoxic response.

Bacterial endotoxins induced similar morphologic changes, but failed substantially to alter inducible lysin levels or to induce detectable lysin activity in vivo. Plasma PAA and CVF-inducible PAA reactivities remained normal in each animal cited above, again displaying lack of total identity between inducible lysin and PAA.

We conclude that the CVF-inducible lysin of Linntlns has several features in common with CVF-induced reactivities of vertebrate complement system in vivo, as well as •/;/ vitro.

Supported by grants from the USPHS ( AI-09806-01 ), American Heart Association and the Minnesota Medical Foundation. H. G. is an Established Investigator of the American Heart Association.

Territorial 'it y observed in a population of Tellina agilis (L)ivalvia: Mollusca). WILLIAM H. GILBERT.

Northern dwarf tellin clams, ( Tellina ,n/ilis ) were collected from a tidal flat at Barnstable Harbor, Massachusetts. 36 of these deposit-feeding bivalves (average length of 7 mm) were put in a shallow sediment tray (43X36X4 cm) filled with fine sand containing benthic diatoms upon which the clams feed. The tray was placed in an unshaded, outdoor water table with flowing seawater (15° ±3° C). The tray was removed from the water table twice daily to simulate low tides.

X-ray photographs of the sediment tray were taken at regular intervals, showing the position of each clam in the population without disturbing the sediment. Analysis of the

424 PAPERS 1'KKSKNTKI) AT MAKIXK BIOLOGICAL I. A 1 5OK. \TORY

resulting dispersion patterns indicated significantly imilnnn spacing, uitli each clam main laining a "personal space" of ahont I'l cm"'.

These clams lie mi their left side at a depth n| about .1 cm, extending their inhaleiil si|)lions unto the sediment surface to pick up and ingest sand grains. Kach may hecome aware of its neighbors' location through contact between siphons during feeding. During an ex- perimental period of 25 days, the clams moved around within the sediment. However, most individuals restricted their foraging activity to a given area, seldom intruding into areas occupied by their neighbors.

The major ecological significance of territoriality is that a given area is occupied, more or less exclusively, by a proprietor. Active defense is not the only means of maintaining territories ; mutual avoidance between individuals can produce the same social organization (Marler and Hamilton, Mechanisms of Animal Behavior, Wiley, 1966).

Contribution number 225 from the Systematics-Ecology Program, Marine Biological Laboratory. The Woods Hole Oceanographic Institution provided the use of X-ray facilities. This work was supported by an NSF Graduate Fellowship (tenure at the Univer- sity of Massachusetts, Amherst) and by a Grant-in-aid of Research from Sigma Xi.

Radiothyroidectomy oj the load fish, Opsanus tan. A. R. V. HASCHEMEYER AND V. LAURIE.

Although the evolution of the thyroid gland appears to have been complete with the appearance of the lower vertebrates, no conclusive function for the hormone has been found for most of these forms existing today. For example, several studies have shown that thyroidectomy by surgery or radioiodine produces little or no change in oxygen consumption or growth of fish.

The present work was carried out to ascertain whether the thyroid hormone may play a role in the striking compensation of metabolism which occurs during cold acclimation of fish. Previous studies on the toadfish have shown that liver protein synthesis cold-adapted animals at 10° is 70% greater than that of control animals adapted to 20°, when both are measured at the same temperature. The effect was found to be localized at the stage of addition of amino acid residues to the growing chains, and a sensitive assay for the rate of polypeptide chain assembly /;/ r/Vo was developed. This procedure has now been used to determine liver protein synthetic rate in animals treated with sodium iodide-I':il (25 microcuries/g body weight) for one week prior to a two-week acclimation period at 10°. When assayed for liver polypeptide chain assembly, these animals failed to show the increased rate characteristic of cold-adapted fish.

The fish bodies were scanned for radioactivity using a Geiger counter fitted with a lead collimator. The results showed a high concentration of radioiodine (about 50% of the injected dose) in the pharyngeal region. No significant radioactivity occurred elsewhere. Histological examination showed pycnotic nuclei in the epithelial cells of the thyroid follicles, indicative of radiation damage. Further studies are necessary to determine whether the loss of adaptive capacity in such animals can be reversed by hormone replacement.

This work was supported by Grant B8 2326R from the National Science Foundation.

The acrosome reaction in spermatozoa oj Lumbricus, as revealed bv negative staining. CATHERINE HENLEY.

After negative staining with 1 c/< phosphotungstic acid, pH 6.8, some earthworm sperma- tozoa isolated from the seminal vesicles undergo spontaneous acrosome reactions (see abstract for July 14 seminar, this issue). In the initial stages of these reactions, an amorphous mate- rial is extruded at the anterior tip of the spermatozoon and the structure of the acrosome complex at the proximal region nearest the nucleus is clearly apparent, including a straight, rod-like structure indenting the acrosome vesicle. In spermatozoa in which the acrosome reaction has proceeded to completion, or near-completion, the substructure of this region is no longer visible. The entire acrosome region, except for the tip, is now filled with PTA and appears as an electron-dense area. In the unreacted acrosome, electron-dense PTA does not penetrate and no internal substructure of the acrosome complex is visible, but the details of siirtace structure are clearly defined. An acrosome filament appears at the tip of the com-

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 425

pletelv reacted acrosomc ; this filament is quite rigid and straight, and its length varies from en. 0.71 to 1.2'' microns. There is apparently no lumen within it. At its proximal end, the filament is lodged in a squared-off depression at the end ol the former acrosomc complex region, for approximately 1/7 of its total length. This depression is electron-densCj so that the filament stands out clearly against the dark hackground of I'TA filling the area.

This appears to he the first report of an acrosome reaction in an oligochaete annelid, and of negatively stained reacted acrosoines of any form, although Munn and Barnes recently de- scrihed and illustrated unreacted acrosomes in negatively stained spermatozoa of barnacles.

Aided by a grant from the XIH, GM 15.ill.

Phosphorylation of nuclear protein following fertilisation in Arbacki and Spistila. JOSEPH ILAX AND JUDITH TLAX.

\\'hile studying RNA synthesis in unfertilized and fertilized sea-urchin eggs we ob- served that when unfertilized eggs were incubated for three hours in the presence of 20 /ug/ml of actimomycin D prior to fertilization, the incorporation of P::~ into the tRNA fraction was 10/v that of the control. Analysis of the phenol extracted radioactive material on sucrose gradient revealed that the peak of radioactivity did not coincide with the optical density peak of the tRNA. 'Further purification of the tRNA on a MAK column showed that the tRNA was not labeled. Tine labeled product came oft" the column with the wash. It is soluble in 5% trichloroacetic acid and is precipitated by the addition of 4 volumes of ethanol. The phosphate can be hydrolyzed by treatment in 1 .V NaOH for 1 hour at 90° C. The fraction was solu- bilized by pronase. Upon chromatography phosphoserine was identified as a major P:12 label. A minor component was tentatively identified as phosphothreonine. The label was shown to be in a nucleoprotein which was contaminating the RNA fraction upon phenol extraction. Phosphorylation of nucleoprotein is triggered by fertilization in Arlmciti and is independent of fertilization in Sfisuhi.

Supported by N.S.F. Grant GB-8464.

The peripheral membrane system n\ sea urchin eggs: an electron microscope stiuiv. SADAYTKI IXOUK, GILES H. COUSINEAU, PAUL L. KRUI-A AND ATTILA

BUDAY.

The fine structure of the peripheral membrane system of Arlxiciti puuctulata eggs was studied with an electron microscope using the pre-shadowed surface replica method. The eggs, treated or not with trypsin powder in sea water (0.5 mg/ml) for 10 min at 22° C, were half embedded in a layer of cellulose nitrate. The surface replicas of the exposed upper hemispheres of the eggs were prepared by first shadowing with platinum and carbon-coated. The specimens were then decomposed in 18 .V HUSOi. The peripheral membranes of the eggs, from which the vitelline portion had been digested, were isolated by gentle homogeniza- tion of the eggs and by low speed centrifugation of the brei. The supernatant \vas then passed through a column containing small glass beads. The membranes, adsorbed on the beads, were recovered and washed with distilled water.

Egg surfaces untreated with trypsin, and thus possessing a vitelline membrane, showed an assembly of fine filaments with a thickness of about 20 A. On the other hand, the surface of the trypsin-treated eggs was observed to be made up of filaments with a thickness of approxi- mately 40 A. The peripheral membranes (170-210 A thick), isolated after treating the eggs with trypsin also seemed to have 40. \-thick filaments on both sides. In addition to the filamentous structures disc-like subunits of 190-240 A in diameter and 50-90 A thick were also seen. When these observations are compared with previous findings on peripheral membranes of eggs and sperms of the purple sea urchin and of guinea pig red blood cells, the possibility arises that plasma membranes in general are made up of layers composed of 40 A- thick filaments on both the inner and outer surfaces.

This work was supported by grants no. A-5057, Aoh24 and D-17 from the National Research Council of Canada and hv I'HS grant 1 SO5 RRO7131-01.

420 PAI'KRS I'UKSKXTKI) A.T MAKIXK BIOLOGICAL I, A !',< >RAT< )KY

. / model for predicting I lie influence o\ colony morphology on reproductive potential in the I'l/y/iiin llctoprocta. \\ \RL W. KAUFMANN.

The three most common types ot colony morphology are those which grow linearly, like a vine, those thai are restricted to l\\o dimensions and encrust tlicir substrate, and those that grow in three dimensions as upright hushy forms. Although there are gradations from one form to another, an idealized model of each can he described with tractable mathematical equations. If the asexual hudding rate is constant, the increase in number of zoids as a func- tion of time is a linear equation for one dimensional forms, a quadratic equation for two dimensional forms, and an exponential equation for three dimensional forms. By assuming that each zo'd produces larvae at a constant rate, then equations for the number of larvae as a function of time will he integrals of the colony growth rate equations. Given characteristic hudding rates for each growth form, the equations show that over a short period of time, the one dimensional forms will produce the most larvae, that over an intermediate period of time, the two dimensional forms will produce the most larvae, but that given a long time span, the three dimensional form will outproduce the other two.

Among the many factors which may influence the distribution and abundance of ectoprocts, this model isolates one factor, the effect of colony morphology on larvae production. If the other factors are of lesser importance, then the model would predict that linear growth forms would be favored on substrates which have a short half-life, while substrates progressively more stable would favor the two and three dimensional forms.

T. ). M. Schopf offered advice and laboratory space. The research was supported by grants from the Hinds fund of the University of Chicago and Sigma Xi.

\uclcar localisation of the sialic acid ''activating" enzyme in the unfertilized sea urchin egg. EDWARD I.. KEAX AND WILLIAM E. BRUNER.

The sialic acid "activating" enzyme catalyses the reaction: sialic acid + CTP -*? CMP- sialic acid + PPi . Evidence has recently been presented from our laboratory demonstrating that the subcellular site for this enzyme is the nucleus. In order to investigate this assign- ment of cellular locale further, the distribution of this enzyme between nucleate and non-nucleate portions of the sea urchin egg was studied. Nucleate and non-nucleate segments were obtained by layering washed, unfertilized eggs from Arhacin puuctuhtta in sea water over 0.85 M sucrose and centrifuging for 15 min at 24,000 X g. This relatively mild procedure should minimize artificial redistribution of the enzyme that possibly could result from homogenization. The following specific enzymatic activities (/umoles CMP-sialic acid formed per milligram protein per hour) were [mean — S.P3.M. (number of experiments)]: whole eggs, 0.174 ±0.011 (7); nucleate half. 0.625 ± 0.13 (6) ; non-nucleate half, 0.167 ± 0.023 (6) ; pigment and yolk, 0.0356 ±0.0093 (5); soluble, 0.59. The nucleate area was highly uniform, containing over 95% nucleate segments. The non-nucleate area was not homogeneous and contained from 3 to 10% of the initial amount of whole eggs. The yields of enzyme from these areas were (%) : nucleate half, 54.4 ± 6.3 (6); non-nucleate half, 10.5 ± 4.3 (6); pigment and yolk, 1.84 ±1.1 ( 5 ) ; soluble, 9.3. These observations are consistent with previous findings that the enzyme is located in the nucleus of the cell, and can be regarded as additional evidence supporting this contention. In contrast to the mammalian enzyme, the enzyme in the sea urchin egg is very labile, losing about 80% of its activity after storage at 4° C for 24 hrs.

Supported in part by USPHS EY 00393.

Aggregation and inliihition of aggregation of ainehocytes of the horseshoe crab Limulus polypliemus. DIANNE M. KENNEY, FRANK A. BELAMARICH AND DAVID SHEPRO.

When hemolymph is withdrawn from Limulus polyphemus, the amebocytes rapidly aggre- gate. Rate and extent of aggregation can be measured photometrically by recording changes in turbidity as per cent transmittance.

Amebocyte aggregation is markedly inhibited by high concentrations (60-80 m.l/) of buffered KDTA, and the addition of equivalent concentrations of calcium and/or magnesium do

PAPERS PRESHNTKD AT MARIXK BIOLOGICAL LABORATORY 427

not reverse EDTA inhibition. A "serum" can be prepared by centrifugation of hemolymph in which the cells are allowed to aggregate for a minimum of 15 minutes. This serum, even when diluted 200 times, is capable of restoring full aggregation to EDTA inhibited amebocytes. A supernatant made from an homogenate of amebocytes in 0.51 M Tris-buffered NaCl separated under conditions which minimize aggregation acts in a similar manner. The data indicate that the active substance in serum responsible for reversing EDTA inhibition is released from amebocytes during aggregation. This active substance is non-dialyzable and heat labile, although heat treated cell homogenates retain some activity. Separation of active material from serum and cell homogenate supernatants by gel-filration on G-75 Sephadex produces a single active peak common to both cell homogenate supernatant and serum. The material in this peak absorbs maximally at 270 nm and has an apparent molecular weight of less than 17,000.

Trypsin < 10,200 BAEE units/mg) is active in concentrations of 0.0125% in reversing EDTA inhibition, but bovine thrombin at concentrations up to 20 NIH U/ml is ineffective. Tosyl arginyl methyl ester (TAMe), a competitive inhibitor for trypsin and thrombin, causes inhibition of aggregation similar to that produced by EDTA. Serum in relatively high con- centrations can only partially override TAMe inhibition.

Both N-ethyl maleimide ( NEM) and parachloromercuribenzoate (PMB) inhibit spontane- ous aggregation of amebocytes. Serum is not capable of reversing PMB inhibition even when PMB cells are resuspended in fresh plasma. The addition of cysteine to washed PMB treated amebocytes resuspended in saline results in the formation of aggregates ; the amebocytes appear to cohere without release of granules or formation of pseudopods.

Supported by grants from National Heart Institute HE 10002 and HE 0541 1-11.

\' citral control of myocardial rhythmicity in Pmsycon canaliculatum. K. KUWA- SAWA AND R. B. HILL.

The cardiac nerve was severed near its origin from the right visceral ganglion. Silver stained sections and methylene blue whole preparations revealed no nerve cell bodies along the course of the cardiac nerve nor in the ventricle. Stimulation of the cardiac nerve at fre- quencies increasing from 0.5/sec to 5/sec induced the appearance of ventricular rhythmicity correspondingly increasing in rate and amplitude. Tension was recorded from portions of the ventricular muscle and reached a maximum of 5 g. The resting potential of impaled ventricular fibers amounted to some 50 mv. IJP's of an all-or-none character (estimated synaptic delay 10 to 20 msec) appeared when the cardiac nerve was subjected to stimulation at threshold level for some (inhibitory) axons. The amplitude of individual IJP's amounted to 0.5 to 3 mv and at frequencies of stimulation greater than 3/sec they clearly showed summation resulting in a hyperpolarization. When nerve stimulation was stopped, the summated hyper- polarization was followed by a rebound depolarization. Stronger stimulation of the nerve produced a mixed effect, with hyperpolarization giving way to depolarization. The depolarization could lead to a local depolarizing response or to an action potential with an accompanying contraction. Single contractions could be induced by single strong shocks to the cardiac nerve, but also appeared by rebound at the end of trains of lesser shocks (hyperpolarizing during the train). Afferent activity appeared in the cardiac nerve in response to the application of 5 to 10 g tension to the ventricle, or during spontaneous beating. One might then see a temporal sequence in which the ECG first appeared, to be followed by a contraction in the course of which increased afferent activity built up and then diminished in the cardiac nerve.

\Ye would like to acknowledge grant support from NINDS grant number NS08352.

Tail resolution in .iscidians: Effects of CytocJmlnsin />. JAY LASH, RICHARD A. CLONEY AND RONALD R. MINOR.

During tail resorption in .li/nirmiciiiin constellatwm the caudal epidermis becomes contractile ; this is associated with the alignment of 50 to 70 A cytoplasmic filaments in each contracting cell. In IJoltcnia i-illosa filaments become aligned in the notochordal cells during tail resorption, but not in the epidermis. Cytochalasin B has been shown by T.

42S PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

Schrocdcr and others to selectively disperse the filaments and to disrupt the morphogenetic processes associated with the filaments.

Nine species of ascidians \vere used in experiments with cytochalasin B. The following concentrations (/xg/ml sea water) prevent the initiation of tail resorption in the species listed: Distapliti occiiicntulis ( (1.25-0.50), Diplosoniu iimcdonuhU (0.25-0.50), A. coustcllatinn (0.5-1.0), Ciona intcstinnlis (1.0-2.0), I'crnphnru riridis (1.0-2.0), Botr\Uits schhsscrl (5.0- 7.0), Stychi partita (5.0-10.0). />'. rillosa (5.0-10.0), Mo/uula cilrina (10.0-15.0). The same concentrations block tail rcMupiion once the ])rocess has begun. The effect on tail resorption is reversible if specimens are washed immediately after the tails stop moving into the trunk and if the concentrations are minimal. After prolonged treatment and washing, animals continue metamorphosing, but retain the larval tail. The smooth and cardiac muscle of the trunk and the striated muscle of the tail are not affected by the drug.

A. coiistcllahiin. /). schlosseri and .17. citi'inn are viable for periods up to 7 days at concentrations of 10 /ug/ml, although they are abnormal in appearance and do not increase in size. Ultrastructural analysis shows that in cytochalasin B treated animals the distribution and organization of the filaments is altered ; finely granular material is found where filaments are located in controls. Specimens that have recovered from the effects of the drug have well organized arrays of filaments.

Supported "in part by NS'F grant GB 5394 ( R. A. C) and USPHS HD 00380 (J. L.).

Reversible de-aggregation of alpha-crystallin from the ocular lens of the smooth dogfish Mustelus canis. SIDNEY LERMAN AND ELSA PFKNINGER.

Alpha crystallin, one of the three soluble protein fractions of the ocular lens is a large polymer (molecular weight 0.8-1 X 10") composed of forty or fifty subunits. The de-aggrega- tion of alpha crystallin in 8 M urea and the subsequent recombination of the subunits when the urea was removed was studied by means of ultracentrifugation, ORD and CD spectroscopy and by two immunochemical methods; the Ouchterlony technique and the quantitative complement fixation reaction.

Purified alpha crysallin (obtained by DEAE cellulose column chromatography) was dissolved in 0.11 M Tris buffer containing 0.11 M KG ( pH 7.7.). Ultracentrifugal analysis at 20° C, 48,000 and 60,000 rpm revealed an S3, value of 19.6 for the native protein. ORD studies showed a trough at 231 m^ and a peak at 205 m/u while C.D. analysis revealed a mini- mum at 217 m/j. and a maximum at 196 m^. Identical samples of Alpha crystallin were exposed to 8 M urea (by means of 24 hour dialysis) and the resulting Sa> value was 1.0 while the ORD trough at 231 m/j. disappeared. When these de-aggregated protein samples were dialyzed for 24 hours against a large volume of 0.11 M Tris KC1 buffer (pH 7.7) the subunits apparently recombined. The 82.. value returned to 26 and there was an apparent complete recovery of the Cotton effects both with respect to their location and magnitude.

Ouchterlony studies on the native alpha crystallin and the re-aggregated material showed complete identity while the quantitative complement fixation experiments indicated at least 80-85% recovery of the original antigenic structure following re-aggregation.

Supported by MRC (Canada) Grant MA 3208.

Spontaneous miniature synaptic potentials and (/nautili release of aeetvlclioline in skafe electroplaques. HENRY A. LESTER.

Miniature synaptic potentials (min. PSPs) were recorded with intracellular microelectrodes in cup-shaped skate electroplaques. Cobra toxin and d-tubocurarine blocked both PSPs (evoked by stimulating presynaptic nerves) and min. PSPs. Tetrodotoxin blocked PSPs but not min. PSPs. Min. PSP frequency increased with increases in the KC1 concentration of the bathing solution. These observations suggest that the min. PSPs arise from acetylcholine packets spontaneously released from presynaptic nerve terminals. Normal PSPs had a significantly shorter time course than min. PSPs, because depolarizations greater than 10 mv activate a membrane conductance increase. When reduced by curarizing agents or by reduced calcium concentration ([CV+]), PSP time courses approached those of min. PSPs.

PAPERS PRESEXTKU AT MARIXH BIOLOGICAL LABORATORY

The input resistance (R) of electroplaques for small depolarizations \vas measured with one intracellular electrode for current injection and a second for potential recording. Min. PSP amplitude varied linearly with R, reaching 300 fiv at 50 kfi (the highest R mea- sured). Min. PSPs did not usually appear above the electrode noise for R less than about 20 k!2. A single min. PSP thus represents a peak inward current of about 6 nA ; similar values occur at myoneural synapses.

In the range 0.4-1.0 times normal [Ca++] (5 m.l/ ) , PSP amplitude varied as the fourth power of [Ca"|. This result agrees with vertebrate myoneural and squid synapses. At the lower [Ca++] values, PSP amplitude fluctuated in accordance with the quanta! release hypothesis. The coefficient of variation of the PSP amplitude distribution was determined for two different synaptic inputs (specified by stimulating different presynaptic nerves) to an electroplaque at 0.4 times normal [Ca++|. This procedure gave quantal sizes of 26 /j.\ and 27 fj.\ ; measured min. PSP amplitudes were 76 pv in normal Ca++ and 44 jtv in the test solution. These discrepancies may arise from electrical cable properties of the electroplaque, from non-Poisson PSP statistics, or from giant min. PSPs consisting of several fused quantal packets. One cannot yet conclude that the observed spontaneous min. PSPs constitute the same population which superimpose to form the evoked PSP.

Supported by NIH and NSF grants and by an NIH predoctoral fellowship.

A biochemical comparison of ciliary and fla(/ellar a.roncines from the bay scallop, Aequipecten irradians. R. W. LTXCK.

Dynein, the axonemal ATPase of cilia and flagella, has been characterized by Gibbons. Low ionic strength dialysis or salt extraction of Tctrahyincna cilia produce 14S and 30S dyneins with certain enzymatic differences. 14S dynein has a molecular weight of 600,000; 30S dynein can only be broken down by alkali or brief trypsinization. Reconstitution of 30S (but not 14S) dynein indicates that it comprises the "arms" of the microtubules. Only 14S dynein can K- obtained from echinoid flagella. Possible biochemical and structural differences between cilia and flagella of the same species have now been investigated in Acqnipcctcn. Cilia were obtained from excised gills using three different deciliating media : 70% glycerol, twice con- centrated sea water, and 10% ethanol-10 mM CaCU. After removal of membranes with Triton X-100, all procedures yield axonemes of identical ATPase specific activities. Flagella were prepared by detailing whole sperm. Flagellar and ciliary axonemes were then dialyzed extensively under identical conditions: 1 m.U Tris, 0.1 mM EDTA and 0.1 m.l/ ATP, pH 7.8. In cilia one half of the original ATPase remains bound to the axonemes after dialysis and can only be solubilized by brief trypsinization. Most of the other half is solubilized during the first day and sediments as a 14S component. The B-tubules and one of the central pair also dis- solve, leaving the A-tubule shu/lcts held together in the circular 9-fold configuration by 1000 A periodic linkage fibers. In cross section the outer arms are gone. On SDS-acrylamide gels dynein migrates as two closely spaced bands with molecular weights of approximately 260,000 and 290,000 ± 10%. Flagellar axonemes behave differently, breaking apart on dialysis into unlinked doublet outer fibers from which the central pair and most of the ATPase is removed. Flagellar dynein sediments as a 14S particle and on SDS-a"crylamide gels appears as two components with molecular weights comparable to ciliary dynein.

Supported by XIH Grants GM 15500 and GM 265.

A linear relationship between liglit-indneed current and flash intensity in Limulus central photoreceptors. JOHN E. LISMAX AXD JOEL E. BROWN.

Single photoreceptor cells in Liiiiiilus ventral eye were impaled with two micropipettes and voltage clamped at resting potential. We measured clamping current evoked by 12 msec. Hashes of light. The cell was allowed to dark adapt between flashes. At threshold (i.e., the flash intensity which evokes one "quantum bump" on the average), the average peak current is on the order of 5 na. If the intensity of the flash is increased four times, the peak clamp current increases approximately four times. With successively brighter flashes, the linear relation between flash intensity and peak clamp current holds over a 100-fold range of intensity. That is, the clamp current at 2 log units above threshold appears to be the linear summation

430 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

iif the current from 100 ''quantum bumps." At higher light intensities, the peak current begins to saturate. However, between 2 and 3 log units above threshold, we can find a time on the rising edge of the response such that at all times preceding it, the response at one half the intensity evokes one half the current. At later times, the dimmer light evokes more than half the current.

Thus, for stimuli up to 100 times threshold, the peak light-activated current is the linear summation of quanta! events and moreover, for brighter lights, this linear summation holds for early times during the response, up to at least 1000 times threshold. In addition, we postulate that some mechanism of gain reduction is responsible for the nonlinearity at later times. The latency of this gain reduction decreases with increasing light intensity.

Supported by NIH EY00312 and EY00377.

Large-scale electro phoretic studies oj ullelie variation in Mytilus edulis. ROGER MILKMAN AND LARRY D. BEATY.

In a survey of about 4000 Mytilus cdulis in 24 samples of over 100 individuals each, mostly on or near Cape Cod, allele frequencies were determined relating to three distinct allozymes of leucine aminopeptidase by the Adamkewicz electrophoretic method.

The frequency, />, of the S allele varied considerably over space; the remainder contained M and F in a fairly constant ratio of 2:3. Six southwestern populations at varying depths ( intertidal to 60 feet) and salinities, in a 100 sq mi area including Woods Hole and several islands, were quite uniform, with /> ranging between 0.53 and 0.59. Just northeast of the Cape Cod Canal, /> varied from 0.16 to 0.30 over relatively short distances. Intermediate locations, both on the outer Cape, as well as in the Canal and south of it, ranged from 0.34 to 0.54. The possibility of an East Coast cline for 5" is eliminated by the finding of 0.25 for its frequency in Salisbury, Massachusetts (near New Hampshire), and 0.15 at Sunken Meadow, on the North Shore of Long Island. A substantial deficiency of heterozygotes was found in almost all populations ; this observation and direct evidence suggest the presence of a "silent" allele in addition to the other three.

Just south of the Canal juveniles (0.30) differed strikingly and highly significantly from large, mature mussels of the same sample (0.44). Fluctuations in /> in intermediate locations, even closely spaced and similar ones, seem comparatively high, though few pairs of samples are significantly different. Aside from sampling error, local differences in selection intensity might cause a jagged distribution in any transitional zone where individuals are of diverse geographic and genetic origin, as undoubtedly here.

Modiulns deinissiis, the ribbed mussel, has a remarkably similar LAP polymorphism, though all three allozymes move much faster than those from Mytilus cdulis.

Supported by a National Science Foundation grant to the program for the biological sciences at The University of Iowa.

Luminescence and related fluorescence in coelenterates. JAMES G. MORIN AND GEORGE T. REYNOLDS.

The bioluminescent emission of many coelenterates involves a transfer of energy from the excited state of the emitting molecule (peak emission 460 to 485 nm ) to a second, fluorescent molecule which has an emission from its excited state at 508 nm. These have been termed the photoprotein and the green fluorescent protein, respectively. The fluorescent material can easily be observed with a compound microscope using a high intensity tungsten (or mercury) light source, a blue BG 12 (Leitz) excitation filter between the light and specimen, and a Wratten No. 12 gelatin filter cut to fit into the microscope eyepiece(s). The observed in z>ivo fluorescent spectrum matches closely the in T/Vo luminescent spectrum in those species examined: Obcl'ui t/cnicnlata. O. cuiiiiiiisnralis, O. bicuspidata, Clytia cdicardsi, Rcnilla kollikeri and I'tilosurcits yitcnieyi. The hydroids examined show either discrete green fluorescent cells (photocytes) scattered within the pedicels, stems and stolons of the colonies (<>/x'lia </i-nicitl<it(i, (>. commisuralis, Clytia cdwurdsi and Cinnpaiutlaria calccolifcra) or photocytes concentrated at the tip of the pedicel just below the hydranth (Obelia bicuspidata, O. longissima and l.oi'ciiclhi i/nicilis). The hydromedusae show concentrations of fluorescent

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 431

cells in the tentacular bulbs (Acqitorea, Phialidium and Ohcliu). The pennatulicls show dense aggregations of photocytes within the autozooids either in the lateral portions of the tentacles (throughout in Ptilosarcits and proximally in Kcnillu) or in the column near the oral disc (Stylatula elongata) .

By simultaneous application of fluorescence microscopy and image intensification these green fluorescing cells have been sho\vn to be the exclusive site of luminescence in those animals tested: Obcl'm i/eiiicnlula, O. coiiiinisiiralix, O. bicuspidata, O. longissima, Clytlu cdi^anlxi, .\Ci]itt>rca jorskulcu an 1 Rcnilln kiillikcri. All green fluorescent cells were luminescent and all luminescent cells were fluorescent. There are usually one or more long fluorescent processes in the hydroids with scattered photocytes. Image intensification and concomitant fluorescence of such cells shows that all of the fluorescent areas, including the fine proce^rs, are luminescent. These observations tend to support the generalization that the green fluorescent cells in luminescent coelenterates are the site of the luminescence.

Supported in part by AEC contract AT (30-1 )-3406.

Evidence for gluccu/on biosynthesis and participation of a precursor protein in islets of the anf/lcrfish (Lophius americnnus ) . BRYAN D. XOE AND G. ERIC BAUER.

Glucagon biosynthesis was investigated in the islet tissue of the anglerfish, Lopliius aincricauns. Glucagon was identified in TCA-precipitable, acid alcohol extracts of islets by bioassay, immunoassay, and elution rate after gel filtration. By incubating islets with 14C- and 3H-labeled amino acid pairs, it was shown that the proteins incorporating tryptophan (which is present in anglerfish glucagon) eluted at different rates on gel filtration from those labeled by proline, cysteine and isoleucine (which are incorporated into anglerfish proinsulin and insulin).

After 0 to 60 minutes of in rilro incubation of islets with tryptophan, the acid alcohol extracts contained only one peak of radioactivity on gel filtration. This peak eluted behind the front protein (BSA marker), but preceded proinsulin, indicating a peptide of 9000 or more in molecular weight. Only a small amount of the total protein from the islet extracts eluted in this (post-BSA) region. After two to six hours of incubation with tryptophan, a second major peak of radioactivity appeared. This peak eluted in approximately the same fraction as mammalian glucagon markers. Glucagon radioimmunoassay of column eluates showed that most of the anglerfish glucagon antigenicity was in the region of the second radioactive peak. Incorporation of tryptophan into these proteins was markedly inhibited by cycloheximide, a specific inhibitor of protein synthesis at the ribosome.

The post-BSA (more rapidly-labeled) protein was treated with trypsin. Rechromatography showed a decrease in radioactivity in the initial peak, with the appearance of two new peaks, one of which eluted near the mammalian glucagon marker.

Rigopoulou and others have described a large molecule with glucagon immunoreactivity in extracts of mammalian pancreata. Our data suggest that a similar molecule exists in angler- fish islets and that it may serve as a precursor protein (proglucagon ).

Supported by USPHS Grant 5T01 G MOO 11 4- 14.

Morphological and electrical features of sonic lanje neurons in abdominal (janijlia of Limulus polyphemus Linnaeus. RICHARD A. NYSTROM, RICHARD A. LEVY AND IRVING NADELHAFT.

A cluster of large somata appear at the anterior corner of each abdominal ganglion in Llmnlns. Contrary to the constant morphology and physiology found among neurons in central nervous systems of some molluscs, annelids, and other arthropods, these somata show variance in number (7-11), size (100-250 /JL diameters), and relative positions within the clusters. Intracellular injections of Procion dye reveal neurons with two different branching patterns : the axons of one type exit from the ganglion through the contralateral anterior root nerve while axons of the other type course posteriorly through the ipsilateral connective. Individual neurons have different patterns of input connections from the periphery. Soma membranes possess varying electrical properties. Resting potentials range between 10 and 75 mv

-U2 PAl'KRS PRESENTED AT MARINE BIOLOGICAL LABORATORY

(Mean: 33 + 12 mv). Most cells impaled 1>y an intracellular microelectrode are electrically ^ilent ; these srunc cells have their processes confined to the ventral nerve cord and are probably interiienrons. Other cells display long (12 msec) overshooting spikes (> 30 mv ) while still others display Miiall (<mv) spikes which, apparently, have not invaded the impaled sonia. Some spikes, either small or large, are followed by large hyperpolarizing undershoots. Synaptic potentials are not recorded from these large neurons but are recorded from smaller somata located elsewhere within the ganglion. Concentrations as high as 10 " .17 of acetylcholine, .ylutamate, epinephrine, 5-hydroxytryptamine, 3-hydroxytryptamine, and gamma-aminobutyric acid have no effect on electrical parameters when applied directly to soma membranes. Corre- lations between electrical and morphological properties are limited by the cellular variance within this arachnid species.

This work was supported in part by grants and fellowships to K. A. N. by the National Science Foundation (GB6832) and the National Institute of Neurological Diseases and Stroke ( 1 'F10 NB 1948-01 NSRB and 2 Fll NBO 1948-02 NSRB).

f Differences in blocking frequency oj presynaptic terminals of an a. von innervating different crayfish muscles. I. PARNAS.

A common exciter axon (number 2) innervates the medial and lateral bundles of the deep abdominal extensor muscles of crayfish. This axon evokes large epsps in the medial bundle (DEAM) and relatively small epsps in the lateral bundle (DEAL). The DEAAI generates spikes and twitches to single stimuli to this axon and contraction is fully activated by stimulating at 20/sec. The DEAL requires higher frequencies (40-50/sec). Intracellular recording simultaneously from both muscles shows that activity in DEAM is blocked rapidly on stimulating the axon at 40/sec while activity in DEAL persists at 80/sec. Recovery in DEAM to single pulses is rapid. Extracellular recordings from nerve terminals show that block in DEAM is due to failure of invasion of the spike into the fine branches. Only some branches are blocked at 20-30/sec, but all are blocked at 40/sec. The block is not due to exhaustion of transmitter since reduction of epsp's by increasing Mg to 50 mJl/ does not change the blocking frequency. This finding also excludes secondary effects of the transmitter on lire- and postsynaptic membrane. It appears that the nerve terminals to DEAM have zones of lower safety factor than do those in DEAL.

Work supported by grants to Dr. H. Grundfest from NIH-NINDS (NS-03728 and NS-5328) and grant GB-6988X from NSF. The author wishes to express his gratitude to Prof. Grundfest for his hospitality and help.

Iniai/c intensification and nuii/nctic tape recording system jor microscopic observa- tions of bioluminescence and fluorescence. GEO. T. REYNOLDS AND PAUL BOTOS, JR.

We have previously reported numerous applications of an image-intensifier-microscope system to studies of bioluminescence. The conventional camera used to record previous obser- vations has been replaced by a plumbicon TV camera, which provides a real time display on a monitor while recording on one inch magnetic tape. Provision is made for real time audio recording of data parameters and procedures. Instant replay of an event is possible and successive single frame or slow motion analysis can be made. With a 25 X objective in the microscope, spatial resolution of better than 1 micron is achieved. The plumbicon camera provides 16 millisecond time resolution for dynamic processes. The decision to use a plumbicon rather than a vidicon was based on several factors. The spectral response of the plumbicon provides a better match to the I'll output phosphor of the image intensifier than does that of the vidicon; there is negligible dark current; target readout exhibits a fast response; a larger target field than that of the vidicon permits a more efficient optical coupling; the gamma is close to unity, an important factor in evaluating intensity of light output ; single electrons from the cathode of the image intensifier can be recorded when desired.

The method has been applied to systematic studies of bioluminescence and related fluores- cence in a variety of systems and has provided information on facilitation, fatigue, duration and

PAPERS I'RKSEXTED AT MARINE BIOLOGICAL LABORATORY 433

propagation of the light, in most instances not visible to the unaided eye and not sufficiently intense for direct photographic recording.

Supported by AEC contract AT (.^i-l 1-3406.

Adaptation properties oj st/uid pliotoreeeptors. I I. \KRIS RUTS AND IKWIX M .

SlEGEL.

The cephalopod retina lacks many of the neuronal elements found in vertebrates, i.e., horizontal, bipolar, amacrine and ganglion cells ; thus the graded negative potentials recorded across a small piece of squid eyecup in response to light result primarily from excitation of the retinal receptors. These potentials were used to measure changes in retinal sensitivity during exposure to steady backgrounds of various intensities, and during the course of dark adaptation when the background illumination was extinguished.

In light adaptation, thresholds rose monotonically until a stable level was reached ; with dim backgrounds this process was rapid, and the dark-adapted threshold was quickly re-established when the background light was removed. Intense light adaptation prolonged both phases ; more than 15 min was required for the increment threshold to reach a stable level, and sub- sequent dark adaptation required about 2 hr for the complete return of sensitivity.

Since the rise in threshold produced by relatively dim backgrounds cannot be attributed to a reduced concentration of visual pigment, there is the possibility that depolarization of the receptors is maintained during light adaptation, and is solely responsible for the observed changes in x-nsitivity. That this is not the case was shown in an experiment in which responses were elicited by pairs of brief flashes differing in brightness by 0.6 log unit, delivered in alternating sequence, and covering an intensity range of about 5.5 log units. In all cases the inter-flash interval \vas sufficient to allow the D.C. level to return to its resting potential ; nevertheless, the effect of the brighter flash on the dimmer was to markedly reduce the ampli- tude of the latter.

In the course of some of these experiments it was found that the dark adapted retina produced a biphasic electrical response to an intense flash of light; a positive deflection with a latency of about 5 msec preceded the usual vitreous negative potential. After intense light adaptation which temporarily suppresses the negative potential, the positive transient was revealed as the leading edge of a sustained positive wave the origin of which is unknown.

This study was supported in part by USPHS grants (EY-35,004. EY-00285, and EY-18766 i from the National Institutes of Health.

Xa currents and (7X;1 clnnit/cs during spike electrogenesis o\ eel eiectroplaques. FRANCISCO RUIZ-MAXRKSA.

Normal spike electrogenesis in eel eiectroplaques occurs primarily as a consequence of a transient change in G\a (activation-inactivation cycle). The change in membrane voltage so produced, induces changes in GK (depolarizing K inactivation). Depolarizing K inactivation can be blocked by inducing complete pharmacological K inactivation applying small amounts of BaCU. The changes in membrane conductance during spike electrogenesis in this condition, are only those of the G.\a system, that operates in parallel with the linear Gr.. The values of Gi., EK and E.\a can be easily obtained from I-Y plots ; Cm can be calculated from the time constant of the membrane. With these values, records of membrane action potentials and dv/dt permit the calculation of the time course of Ic, L. and IXa, the sum of the three ( I,,,') being zero. From the calculated values of IN,,, the time course of the change in Gx,, can be obtained. Spikes elicited at different temperatures show that the rise of G.\a is almost unaffected by the changes in temperature while its decay (Na inactivation) has a large tem- perature dependence, confirming previous findings obtained with different techniques.

Supported in part by grants from the NSF (GB6988N) and NINDS-NIH (NB-0372S). to Dr. Harrv Grundfest.

434 PAPERS PRESENTED \T MAKINK BIOLOGICAL LABORATORY

Temporal characteristics oj pacemaker inhibition in Isolated hydranths of Tulm- laria. NORMAN B. RUSHFORTH.

Electrical stimulation of (lie DOS, a conducting system which courses through the stalk and hyclranth of Tnhnldrui. inhihits spontaneous firing in two pacemaker systems of the polyp, the NP system in the distal stalk region and the HP system in the hydranth. Inhibition of the HP system and pacemaker systems in the proximal and distal tentacles and in mature gonophores connected to the hydranth, results from activating the DOS in isolated hydranths. In such preparations the hydranth has hern surgically excised from the stalk to remove NP input to the HP system.

Inhibition of the III' system in isolated hydranths stimulated every 20, 10, 5 or 2.5 sees for 5 minute periods has the following properties. (1) Maximal inhibition occurs between 1 and 2 seconds following a stimulated pulse in the DOS. (2) The degree on inhibition increases with the frequency of stimulation. (3) During the 5 minute period following inhibitory stimu- lation the HP System fires at frequencies significantly greater than prestimulation levels. The level of post-inhibitory excitability is positively correlated with the degree of inhibition during the stimulation period. (4) Complete recovery from inhibition occurs between 12-16 seconds after DOS stimulation.

Inhibition of HP's in isolated hydranths is less complete but longer lasting than inhibition of the NP System in intact polyps.

Supported in part by grants MH-10734 and GM-12302 from the National Institutes of Health.

Semilunar ami other factors influencing hatching jroin cyy-inasscs of Melampus bidentatus •/;/ the field and in tlie laboratory. W. D. RUSSELL-HUNTER, MARTYN L. APLEY AND R. DOUGLAS HUNTER.

Populations of the snail Mehnnpits inhabit higher levels of salt marshes and for adaptive reasons egg-laying, hatching and larval settlement are each confined to cycles of about four days in phase with spring tides. In egg-laying, the semilunar periodicity is an obligate process : stocks of :\fi'liunf>ns brought into the laboratory maintain the summer semilunar pattern without tidal stimuli. In the field another event synchronized with spring high tides is the hatching of free-swimming veligers, which could be obligate (with a rigorous develop- mental timetable) or facultative (dependent on the incidence of tidal submergence).

Laboratory development at 18° C involves first cleavage (5-7 hours after laying), early blastula (12 hours), retained trochophore (4 days), and well-differentiated, active veliger (11 days). In the field, most egg-masses hatch at about 13 days, during spring high tides, yielding enormous numbers of planktonic veligers. In the laboratory, time of hatching proves potentially more flexible, and hatching of veligers can only take place when egg-masses of appropriate age are flooded with seawater. When, after 48 hours of flooding, the terminal age lay between 11.5 and 15 days (both 18° C and 25° C experiments), universally > 90% hatching was achieved. At 18° C, the earliest recorded masses yielding > 90% hatching were at 10.3 days, and the oldest yet recorded yielding 80% at 42 days. In another series of "tidal bathing" experiments (1 hour flooding at intervals of 12.5 hours with 18° C stocks), first and second fl codings had no overt effect and the third produced < 5% or no hatching regardless of age. The fourth flooding was usually completely effective at all appropriate ages with appreciable hatching in 30 minutes and > 98% hatching after 1.5 hours. The total time immersed in these "tidal" experiments was only 4.5 hours (corresponding to field conditions), but it should also be noted that the elapsed time in nil the successful experiments was about 48 hours.

Semilunar field hatching is not obligate. Hatching has flexibility (age 10-24, even 42, days), but must usually result from a ,sr</i/rj;<v of about four floodiin/s in under 50 hours. Being facultative, the process allows better survival and overlap of cohorts but it also re- establishes the synchronization. This is highly significant for later synchronous settlement into appropriate salt-marsh levels.

Supported by Grant GM 11693 from the National Institutes of Health to W. D. R-H., and Research Grant #1116 from the City University of New York to M. L. A.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 435

. / natural experiment in the population genetics of a marine ectoproct suggesting changes in gene frequencies a'//// changes it: lempemtiirc. TIK>M\S J. M. SrnorK AND JAMES!,, (ioocn.

frequencies in colonies of encrusting Schisoporella inincnns \\ere delerniined by electrophoresis of proteins on polyacrylamide gels, with subsequent staining of gels for particular enzymes. From 29 to 47 colonies were collected from 0.5-3 m depth at 5 localities spread over a linear distance of approximately 33 km. From east to west, the localities are (1) Green Pond, about 10 km east of (2) the Marine Biological Laboratory (MBL) Vineyard Sound dock, (3) Sheep Pen Harbor about 1 km from the MBL dock across Woods Hole passage, (4) Robinsons Hole about 13 kms southeast of Woods Hole, and (5) off Cuttyhunk Island, about 10 km southwest of Robinsons Hole.

Eight loci were clearly identified in four enzyme systems. One locus, leucine amino- peptidase 3, is biallelic at each locality. The frequency of the faster moving allele is higher in the west: 0.25, 0.39, 0.40, 0.69 and 0.65. The frequency of the faster allele also is higher in samples dredged from deeper, cooler water in Vineyard Sound west of Martha's Vineyard: 0.50. Summer water temperatures are highest in the east and become lower in the west. Relative to Green Pond, the temperature difference is approximately 1° C lower at the MBL dock and Sheep Pen Harbor, 4° C lower at Robinsons Hole, and 5° C lower at Cuttyhunk. \. niiiconiis grows and breeds in the summer, and is dormant through the winter.

Supported by a grant from the Block Fund, University of Chicago, to Thomas J. M. Schopf.

Effects of 1-methyl adenosine on isolated gonai/s and oocy/es of the starfish; evidence for an ovarian wall niieleosidase. ALLEX W. SCIU'KTX.

The effects a 1-methyl adenosine and 1-methyl adenine on the processes of follicle cell disintegration and oocyte shedding and maturation (germinal vesicle breakdown) were com- pared following in ritro incubation with starfish gonadal fragments or cellular components. Both 1-methyl adenine and 1-methyl adenosine stimulated shedding, maturation and follicle cell disintegration when added to intact gonadal fragments. The 1-methyl adenine was 10-100 times more effective than 1-methyl adenosine in initiating these changes. Isolated oocytes incubated in calcium free sea water however matured only in response to 1-methyl adenine. Addition of ovarian wall tissue to dishes containing 1 -methyl adenosine and immature oocytes produced maturational changes in the oocytes. The incidence of maturation in the isolated oocytes was dependent upon the amount of 1-methyl adenosine present in the dish and these changes occurred first in the area of the gonadal tissue. Ovarian tissue in the absence of 1-methyl adenosine did not induce oocyte maturation. Following the incubation of ovarian wall homogenates with 1 -methyl adenosine, aliquots of this mixture produced matura- tion in isolated oocytes. Ovarian homogenates boiled or treated with proteolytic enzyme (pronase) prior to incubation with 1-methyl adenosine were ineffective in stimulating oocyte maturation. The capacity of 1-methyl adenosine to initiate maturation in isolated oocytes appears to depend upon the ovarian wall for converting 1-methyl adenosine from an inactive to an active product. It is suggested that the ovarian wall tissue(s) contains an enzyme (nucleo- sidase) which converts 1-methyl adenosine to a biologically active substance.

These studies were supported in part by funds from The Population Council ( M70-054c) and the Ford Foundation.

Physiological adaptations of 'Funclulus embryos to varied oxygen tensions. D. R. SHANKLIN AND J. C. SHANKLIX.

Armstrong-Child stages 32-33 of Fitnditlns are distinguished functionally by the appear- ance of vagal inhibition of cardiac activity. Just prior to this the heart is sensitive to light and the oxygen tension of the gas layered over the dish at 20° C. In subdued light, barely sufficient to view heart action, the rate is inversely proportional to oxygen tension. Under air the rate is 27.8 ± 0.3 beats per 15 seconds. A 100 watt incandescent bulb at 25 cm causes a 339?- increase within 5 minutes and a 100% increase by 2.5 hours. The effect is reversible and repeatable, requiring about 45 minutes for recovery. The increase is the same irrespective

436 PAPERS PRESENTED AT MA KINK IHOI.OGICAL LABORATORY

(if oxygen tension, suggesting thai light intensity establishes a specific rate tlircslu ild. Fry have rates about double that of eggs: oxygen layered about 44 per 15 seconds and air layered about nS. This sugrsts that heart rates increase with development, but since morphogenesis is advanced by I00r; oxygen layering, and retarded by 7% oxygen layering, light and oxygen are prohal>l\ independent stimuli to cardiac rate. )'<>llc consumption was approximated by applying volume formulas with the sac as a twice truncated sphere (pericardial sac anteriorly and axial embryo superiorly). 2 mm unfertilized eggs have a volume of 4.2 mm". Residual volumes at 288 hours were 1.63, 2.34, and 2.04 mm" for 7%, 21%, and 100% oxygen, respectively. Yolk consumption is thus minimal for air layered embryos at 1 cm depth. Embryos layered by 7% oxygen went to M() hours without completion of hatching. These had the same yolk volume as the 288 hour eggs. The results indicate Finidiilus embryos are well adapted to current aerohydrospheric conditions. Changes in oxygen tension have significant physiological effects. This work does not indicate the optimum conditions nor when the cardiac effects of oxygen or light begin.

This work was supported by the John A. Hartford Foundation, New York, New York.

Influence of varied o.v\</en tension on fertilisation and on developmental tune in Fundtilus hcteroclitus. J. C. SIIAXKLIX AND 1). R. SIIAXKLIX.

\\ e have suggested oxygen as a driving force in evolution and embryogenesis. Fundulus eggs in 1 cm sea water layered by 7% oxygen in nitrogen, air, and 100% oxygen were main- tained continuously at 20° C. A fourth group was kept at 2 cm under air. Fertilization in 0.25-0.30 cm sea water was 91%; layering at 30 minutes with oxygen at 1 cm had no secondary inhibitory effect. Fertilization rates at 1 cm under 7%, 21%, and 100% oxygen were 33%, 68%, and 27%, respectively. Subsequent survival of developing embryos at 200 hours was 61.5%, 95.5%, and 97.0%, respectively. 7% oxygen retarded all major morphogenetic sequences; up to Armstrong-Child stage 24 by 158% and after onset of circulation (stage 25) by 229%, or 208% totally. 100% oxygen accelerated early cell cleavage but retarded blastula formation and pre-circulatory morphogenesis (stages 21-24). Gastrulation began late but moved faster than in air controls. After stage 25 development to hatching competence took only 75% of normal time, and the total span was thereby shortened to 88%. Switching from air to oxygen layering after onset of circulation resulted in an acceleration of post-circulatory development by 26% calculated against total post-circulatory time and by 42% calculated against the period following the shift. The reverse shift at a similar point delayed hatching competence by 8-11%. Increased depth had a constant effect: pre- and post-circulatory intervals were both increased 60%. The results are compatible with fertilization and embryonic development as sensitive to oxygen tension. At constant temperature more oxygen means more rapid development overall. The effect is mainly mediated through a competent circulation sug- gesting a relation between environmental oxygen and the development of circulation.

This work was supported by the John A. Hartford 'Foundation, New York, New York.

Studies oj nitcleolar A'AVl synthesis in starfish ooeytcs. ELIZABETH L. SHIPPKK, GIOVANNI GIUI-ICE AND ALLKN \V. SCIIUETZ.

The RNA synthetic activity of ovaries and of the nucleoli of oocytes in the germinal vesicle stage were examined in order to explore changes associated with oocyte maturation. Nucleoli from Asterias jorbesi ovaries were isolated using the procedure of Vincent (PNAS, 38: 139. 1952), except for the substitution of 0.01 M Tris buffer pH 7.4 with 0.005 .17 MgCl2 and 0.01 M NaCl for distilled water as the suspension medium. The pellet of nucleoli from 10-15 grams of ovaries which had been incubated for four hours in 20 /iC/ml tritiated uridine ( sp. act. 25 C/mmole) was extracted with SDS, hot phenol and chloroform. The purified RNA was layered onto a 5-20% sucrose gradient and ccntrifugcd for four hours at 39,000 rpm on a Beckman SW-40 rotor. P:!- labelled 26S and 18S RNA from sea urchins were used as markers. TCA insoluble counts were found associated exclusively with RNA of molecular weight less than 1SS.

Whole ovaries were incubated tor four hours in tritiated uridine; half of these were shed with l-methyl adenine. The ribosomal RNA of whole ovaries, mature oocytes and shed

PAPERS PRESENTKD AT MARIXK BIOLOGICAL I. A MORATORY 437

ovaries were extracted using cold phenol. Analysis of these tjradicnts reveals thai (a) the \\hole ovary synthesi/es 2<>S and 1SS RXA as well as large <|nanlities of light RNA species; ih) mature enos are \\ n1hcsi/in» high levels c)|' ](1\\ molecular weight RX'A. hut no detectaliK amount <if lihosomal RNA; and (c) the .shed ovary synthcsi/cs significant amounts of rRNA as well as many other types of intermediate and light RNA.

These data suggest that the nucleolus of oorytes in the germinal vesicle stage can stop the synthesis of rihosomal RXA, and still have associated with it active synthesis of several classes of low molecular weight RXA. Rihosomal RXA appears to he produced in the cells of the ovary with the exception of the fully grown oocytes.

This study was performed in the Fertilization and Gamete Physiology Training Program at the Marine Biological Laboratory ( NIH gram 5-T01-HDO()()2h-0(h and also supported in part by XIH HD03797-02.

HiocJicniical clian</es in the dit/cslii'c (/land of flic fiddler crah, LY;i pugnax, follow- in</ bilateral c\cstalk extirpation. DAVID A. SNYDKR AND JONATHAN 1'. ()KKKN.

Hyestalk extirpation led to 100% molting by surviving animals, thus providing an absolute time scale to which biochemical events were correlated. The mean and median day of molting was thirteen days after clestalking. Digestive glands were homogenized in 0.2 M Tris buffer ( pH 7.X ) and, after incubation in appropriate media, spectrophotometrically assayed for acid and alkaline phosphatases, a-amylase, trypsin, chymotrypsin, glucose, total lipid, and total protein.

Both alkaline and acid phosphatases reached a peak of activity six days after ecdysis. This may he correlated to the deposition of calcium phosphate calcospherites in the cuticle following ecdysis. Total lipid was undetectable from late stage D until ten days after ecdysis when feeding presumably resumed. A decrease in digestive gland lipid observed in late stage D may be due to the secretion of the lipoid. waxy layer of the new cuticle. Glucose reached a maximum concentration at ecdysis. It is suggested that lipid, through its glycerol moiety, may have been converted to carbohydrate (such as glycogen or glucose), which in turn was synthesized into chitin. Chymotrypsin was undetectable at all stages tested. Trypsin and a-amylase reached a peak of activity at ecdysis. The a-amylase activity was proportional to the glucose concentration, a-amylase converts a-1 :4-glucosans (such as glycogen) to dextrins which then may be converted to glucose. The rise in a-amylase activity may be involved with the conversion of glycogen to chitin. If crustaceans can convert lipid to glucosans, an additional source of substrate for a-amylase would be present. The fluctuations in trypsin activity reflect general synthetic activity.

The biochemical changes of the digestive gland represent transfer of stored reserves to blood cells, thence to intra-epidermal connective tissue, and finally to exoskeleton-secreting cells rather than degeneration or loss of functional activity.

Supported by NSF Grant GB-17994 to J. P. G., by NSF Grant GZ-1788 to the Department of Experimental Invertebrate Zoology, Marine Biological Laboratory, and by the Division of Biological and Medical Sciences, Brown University.

Receptive field organization of (/ain/lion cells in the retina of the smooth doi/fish. Mustelus canis. WILLIAM K. STKLL, HKNRY (j. \YA<;.\KR AND MYRON I..

WOLBARSHT.

\\ e have investigated the receptive field properties at threshold of ganglion cells in the moderately dark-adapted, apparently all-rod retina of young (16-24 inch) dogfish. Pieces of isolated eye cup (retina attached) at about 20-22° C were suffused with moist 10(1', oxygen and presented patterned monochromatic light stimuli through a dual-beam optical system including a grating monochromator. Action potentials were recorded extracellular!}- with glass-coated Pt-Ir microelectrodes inserted through the vitreous body, amplified and displayed conventionally.

Most units isolated in the ventral retina responded to a small centered spot of any wavelength \\ith an ON-response, after a latency of about 0.5 second at threshold. Simultaneously presented annuli of inner radius :1 mm elevate the threshold for the ( )X-response or

4SS PAPERS I'RKSKXTKl) AT M \RI.\K BIOLOGICAL LABORATORY

lihil spontaneous <l:irk activity, which is present iiifrc(|ni-iilly. Surli annuli alunt.1. or diffuse light, must often evoke no response in these units, while a luminous bar of appropriate dimcn sjons, intensity and location may evoke also OFF- and ON-OFF-responses. The excitatory and inhibitory regions arc concentric and radially symmetric. The radius ot the excitatory center is about 0.5-1.0 nun according to the change in threshold with the location of a small spot of fixed diameter or with diameter of a spot located in the center of the receptive field. The inhibitory region extends at least from a radius of 1.0 mm to 2.5-5.0 mm, but its full central and peripheral extent has not been determined. The spectral sensitivity of both the excitatory and inhibitory processes is identical ; it is maximal at 500-510 mn but the function appears narrower than expected from corresponding pigment spectra, perhaps because of preferential reflection of light of certain wavelengths by the choroidal tapetum lucidum.

Ganglion cells with different receptive field properties have been observed occasionally but their organization is not yet understood.

Isolation of ne.vin — ///<• linkage protein responsil>/e for maintenance of Hie nine-lohl configuration oj flagellar axonemes. R. F>. STEPHENS.

After detergent removal of membranes, dialysis of cilia or flagella against low ionic strength media generally results in removal of the ATPase dynein and the solubilization of the central pair and matrix, leaving outer fiber doublet microtubules connected either as a ring of nine in cilia or as a sheet of nine in flagella. The nature of the linkage between such outer fibers has proven quite elusive. Some reports describe circumferential material connecting adjacent A-subfibers while others indicate connections running from the A-subfiber of one doublet to the B-subfiber of the next. Axonemes from Strongylocentrotus drocbacliicnsis, .Istcrias sp., and Arbacia pitnchtlata sperm flagella were incubated at 40° C for 2 minutes in 1 m.l/ Tris-HCl, pH 8.0, and 0.1 m!\I MgCl». Such treatment results in depolymerization of the B-subfiber, and removal of dynein, central pair, and matrix proteins. Remaining are nine singlet microtubules connected in parallel by periodic bridges of an apparently extensible mate- rial roughly 200 A wide and spaced at 1000 A ± 10% intervals along the tubules. In these species at least, linkage material thus occurs between adjacent A-subfibers. Depolymerization of A-subfibers by treatment at pH 3 leaves insoluble linkage material. SDS polyacrylamide gel electrophoresis of whole axonemes shows dynein and tubulin bands, a moderately strong- third component, and numerous secondary bands ; electrophoresis of thermally-fractionated A-microtubule-linkage material complex indicates that this third component is greatly enhanced. Electrophoresis of the material remaining after tubule removal shows primarily this third component. The protein represents about 2% of the total axonemal protein and hence less than 1% of the total flagellar protein. The molecular weight by SDS gel electrophoresis is 165,000 ± 10%. The term "nexin" is proposed for this protein, derived from the Latin nc.rns, a tie binding together members of a group.

Supported by NIH Grants GM 15,000 and GM 265.

Species of Renicola (Trematoda) in the kidneys of the yidl, Larus argentatus. HORACE W. STUNKARD.

Stunkard and Shaw (1931) described Ccrcaria parvicaudata n. sp., from Littorina littorca at Woods Hole, Massachusetts. In a later report, Stunkard (1950) gave a more complete description of the species and noted the striking resemblance between C. parvicaudata and a species he had described (1932) as Ccrcaria roscointa n. sp., from Littoriua saxatilis niyro- lincata taken at Roscoff on the Brittany coast of France. Other similar cercariae were discovered in Thais laf>illits, taken near Boothbay Harbor, Maine, and on the north shore of Cape Cod. These cercariae encysted in bivalve mollusks, especially Alytilus edulis, Pectcn irmdians and Gemma i/ci/iiiiti and developed to sexual maturity in the renal tubules of Larus iiri/cntatiis. They were described as a new species, Renicola thaidus Stunkard, 1964. During the summer of 1965, cysts containing metacercariae of C. pa/rzricaiudata were fed to gulls, /-. ari/cntntiis, but no worms were found in the kidneys and attention was directed to other parasites, especially members of the Notocotylidae.

PAPERS PRESENTED AT MARINE BIOU )( 111 \\I. LABORATORY 439

(Marine A'/o/n./v, 3: 306-333, 1969) reported that Ccrcaria roscovita from /,. littorca, taken on the coast of the North Sea near Helgoland, developed in the kidneys of /.. (u-f/cntatns to mature worms that he described as Rcnicnla roscnvitn ( Stunkard, 1932). Warding raised the question of identity between species of Renicola. It is apparent thai C. parricdiiddtn is a species of kcnicola and the reasons for the earlier failure to complete the life-cycle are not clear. The experiments are repeated, but since some two months are required for the worms to mature in the renal tubules, final results are not available. Since C. parvicaudata and C. roscovita infect the same intermediate and definitive hosts on opposite sides of the Atlantic, their identity is probable, but the species is distinct from R. tlundux. Investigation supported by NS'F GB-8423.

Parallel cJiain/cs in tlic thresholds of current induced tensions and Cu-spikes in isolated crayfish muscle fibers. G. SuAREz-KuREz, P. W. BRANDT AND J. P. REUBEN.

Recent evidence indicates that electrical stimuli provide Ca to the contractile system from superficial membrane sites. A parallel relation has been observed between experimentally produced changes in threshold (mv depolarization) for a Ca-activation process ( procaine- induced spikes) and for just detectable tensions evoked by intracellularly applied depolarizing currents, suggesting that membrane Ca-activation is associated with excitation-contraction coupling. Tension thresholds increase linearly with increasing concentrations of Mn ( 1-5 m.V ) . The slope is about 6 mv/m.l/ Mn. The threshold for the procaine induced spikes increases by approximately 4 mv/mJ/ Mn. When Ca,, is increased in the range 13.5 to 150 mM the thresholds for both spikes and tension increase initially with a slope of about 4 mv/10 mM Ca, but the slope decreases at higher concentrations of Ca. SCN (2-200 m.l/), on the other hand, decreases the threshold values for the spikes and the tension. In both cases the function is logarithmic, the thresholds decreasing as SCN is increased (—4 mv/log SCN). Thus, the changes induced by the three agents obey different functions but in each case the effects on the thresholds for activation of Ca-spikes and tension development are parallel.

This work was supported by grants to Dr. H. Grundfest from NIH-NINDS ( NS 03728) and NSF (GB 6988X ). Dr. Suarez-Kurtz was a Special Fellow-USPHS.

Distribution and metabolism oj J-mcthyl adeninc in the starfish dnr'nuj shedding and oocyte maturation in vitro. BRYAN TOOLE, SHIRLEY HULL, ELIZABETH L. SHJPPEE AND ALLEN W. SCHUETZ.

The incorporation of 3H-l-methyl adenine into ovarian fragments and into isolated oucyte> of Astcrias forhcsi has been examined as a means of providing information about the action of 1 -methyl adenine in inducing shedding and maturation of starfish oocytes.

Continuous uptake of isotope occurred for a period of 2-4 hours into pieces of ovary ex- posed to 3H-1 -methyl adenine in filtered sea water. As much as 60% of this isotope was incorporated after the processes of shedding and maturation were complete, and at high doses of 1 -methyl adenine the amount incorporated was 20-30 times that needed for these processes to occur. The proportion of uptake into eggs and ovary wall was approx. 1:40 even though the proportion of the two components by weight is 5:1. Autoradiography performed after uptake of isotope for periods of 5-70 minutes revealed a progressive accumulation of grains over the outer epithelial layer of the ovarian wall, with virtually no grains elsewhere.

At least 90% of the isotope taken up by the ovary wall was dialysable and no isotope was recovered in hot or cold trichloroacetic acid precipitates. The dialysable material was found to have the same capacity to induce maturation of isolated oocytes as 1-methyl adenine.

The major proportion of isotope incorporated into oocytes, either isolated or within the ovary, was also found to be dialysable and no incorporation into nucleic acid or protein was obtained.

The presence of dinitrophenol prevented the shedding and maturation of oocytes by 1-methyl adenine but the uptake of aH-l-methyl adenine into the ovary wall or into the oocytes was not affected. Preincubation with dinitrophenol followed by washing did not inhibit the action

440 I'AI'KRS I'UKSKN'TKI) AT MAUINK BIOLOGICAL LABORATORY

dl the hormone. It is concluded thai either (i) the uptake oi 111 methyl adenine in n<> \\a\ irtlects ilic physiological action ot thr substance; or < ii ) dinitrophenol is acting at a step in these processes subsequent to the incorporation of 1 -methyl adenine.

Support was provided by MM (grant 5-T01-HD00026-09 and M I >037n7 (>_' ) and The I 'opulation C< mncil.

Permeability and structure of cellular junctions in the starfish einhrvo. JOSEPH T. TTPPER, KARL S. WITTMAN AND Jonx \Y. SAKNDERS, JR.

Electrical coupling between cells of the starfish embryo, . -Ixtcriux jurbcsi, arises at the 32-cell stage. This is in contrast to several other embryos which exhibit cell to cell communi- cation much sooner, c.y., between the first two daughter cells and all cells thereafter in amphibians. Coincident with the onset of cellular coupling at the 32-cell stage is the formation of surface membrane junctions between adjacent cells of the embryo. At the 16-cell stage there are no visible junctions at the electronmicroscope level and the cells are separated by rather wide gaps ( 1000 A to 1 /j, ) . Electronmicroscope observations on the cellular junctions arising at the 32-cell stage show them to possess characteristics of both tight and gap junctions, e.g., 130- 160 A width, pentilaminar structure when stained after dehydration. However, due to prob- lems of fixation of the embryos, enough detail in structure is not present to distinguish between these two types of junction, one of which is strongly implicated in electrical transmission between cells, i.e., the gap junction. The permeability of these junctions to larger molecules has been studied by iontophoretic injection of the fluorescent dye Procion Yellow ( MW ~~500). Cells injected at the 16-cell stage have been observed to continue division to the 32-cell stage, which serves as an indication of cell integrity. However, the dye is confined to the two daughter cells, even though the embryos show electrical communication at this stage. The dye is also confined to daughter cells at the 64-cell stage. No observations have been possible beyond this stage due to difficulty in exact dye localization. It should he noted that Procion has been shown to bind membranes of the crayfish axon. Such binding could reduce its mobility across junctions. However, we have not observed any preferential localization of the dye at membranes of the starfish cells. It is interesting to speculate that such junctions may, at a later time, exhibit permeability to larger molecules and that this selectivity may play a role in development.

Supported by grants NBC) 07681 and H DO 3734-02 from NIH and GB 6638X from NSF.

DNA synthesis dnrhui regeneration in Tubularia. KENYON S. TWEEDELL.

The relation of cellular activity to primordium formation during hydranth regeneration in the hydroid Tubularia is obscure. In order to determine the site and time of DNA synthesis during regeneration, a series of regenerates were incubated in H3 thymidine for various intervals after hydranth amputation. In another series of experiments regenerates of Tubularia spectabilis and Tubularia crocca were sampled after isotope injection at succeeding stages of regeneration. A 1 to 2 /xl quantity of H3 thymidine (0.3 to 1 /uc/stem) was injected at different stages (from 3 hours post amputation until after new hydranth formation). After ' to 1 hour pulses, the regenerates were fixed and examined by autoradiography.

Uptake was slight in all stages of regenerating stems when they were exposed to 1-2.5 Ate/ml of Ha thymidine in the culture fluid. Apparently the isotope cannot penetrate the perisarc unless an opening is maintained.

In the injected series of 1 hour pulse during the first 3 hours after amputation showed uniformly scattered nuclear uptake in both the epidermis and the gastrodermis. Further injections at 2 hour intervals from 4.5 to 15 hours after amputation gave relatively little labeling to any parts of the regenerates. Mass cell migration has taken place by this time.

From 17 hours after amputation, when the two banded hydranth primordium first appears in T. spechihilis, nuclear uptake was again evident in both the hydrocaulus and in the primordium. Thymidine uptake was found in both epidermis and gastrodermis in all regenerative stages but qualitative and quantitative differences were noted along the stem. At the proximal end, the label was more heavily concentrated in the epidermis and uptake diminished toward the

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 441

distal cud. Tlic gastrodermis uas only moderately labeled at the proximal cud and along the hydrocauhis hut uptake increased distally.

In the primordium nuclear uptake uas most c\ idcnt in the gastrodermis and relatively little \v:is seen in the epidermis except during the late tentacle formation. .As regeneration neared c<ini])letion, the lower hydrocaulns was still labeled; in the hydranth uptake was concentrated in the basal hypostome and peduncle. Within the first day post emergence incorporation was limited to the hydrocaulus.

.-Ihsorhance inelfiiu/ profile analysis of DNA and chroinatin in somatic cells and sf>cnn of the spider crah, Libinia emarginata. JACK C. Y.\r<;Tix AND GERTRUDE W. H i x si 1 1 .

Recent studies have shown that crustacean nuclear DNA from a variety of tissues of various species bands in the analytical ultracentrifuge in at least three different zones, corre- sponding to the main band, an (A+T)-rich satellite and a (G+C)-rich satellite. The present study examines total testis DNA, isolated sperm DNA and sperm chromatin, with special reference to DNA heterogeneity and the presence of sperm nuclear basic proteins.

Absorbance melting profiles of isolated total testis DNA in SSC/10 show two distinct T,,, values: 48.5° C (7.7% of the total hyperchromicity, presumably representing the (A+T)- rich satellite reported in closely-related species) and 70.0° C (88.9% of the total hyperchromicity. representing the main band DNA). An additional transition occurs at about 90° C (3.4% of the total hyperchromicity) and presumably represents the (G+C)-rich satellite. This latter transition often appears biphasic, suggesting heterogeneity, although this is not yet certain.

A technique is described for isolation and purification of sperm (99%+ pure), and for fractionation of these cells into highly purified acrosomes and chromatin. Absorbance melting profiles for sperm chromatin and for sperm chromatin treated extensively with pronase are identical, suggesting that basic proteins are not associated with sperm chromatin. Of special interest is the finding that these profiles reveal a lack of the (A-rT)-rich satellite in deproteinized sperm chromatin. In order to determine if this apparent loss is due merely to the chromatin isolation technique, sperm DNA was isolated by a modification of the Marmur procedure, with identical results. It is suggested that the loss of the (A+T)-rich satellite occurs during sperrniogenesis, as Feulgen photometric analysis of spermatid nuclear DNA content in another crab, Einerita aihil/n/a. has revealed a 6-9% loss in nuclear DNA during sperrniogenesis.

We hypothesize that the (A + T)-rich satellite in crustaceans represents a highly amplified gene (or genes) which appears during development and differentiation of somatic cells. The significance of these results remains to be established.

This study was performed in the Fertilization and Gamete Physiology Training Program at the .Marine Biological Laboratory (NIH grant 5-T01-HD00026-(!'M.

Fluorescence analysis oj the e.rcitatiou process in nerre nsin</ hydrophobic probes. . \KIRA \YATANABE, ICHIJI TASAKI AND MARK HALLKTT.

A hydrophobic probe, 2-p-toluidinylnaphthalene-6-sulfonate (TNS), was injected into a squid giant axon and fluorescence properties of the axon were examined under illumination with near visible ultraviolet light (365 nm wave length). The axon was found to fluoresce with an emission spectrum spreading from about 410 to 560 nm, the emission maximum being around 450 nm. When such a TNS-stained axon was stimulated, the intensity of the fluorescent light between 420 and 480 nm was found to decrease slightly. This finding suggests that the process of nerve excitation involves a transition of the membrane from a hydrophobic to a hydrophilic state. When the incident (i.e.. exciting) UY light was polarized in the direction either parallel or perpendicular to the longitudinal axis of the axon, the fluorescent light contribution to the transient decrease during nerve excitation was nearly completely polarized, indicating that the axonal membrane has a rigid, crystalline structure. When the stimulation was preceded by weak anodal polarization the magnitude of the optical response was greatly enhanced. Probably anodal polarization increased the number of the probe molecules at hydrophobic sites in the axon membrane. The fluorescence intensity of TNS-stained axons

442 PAI'KKS Pk'KSKNTKI) AT Al. \KI\K BIOLOGICAL LABORATORY

\\.is examined under voltage-clamp. With depolari/ing clamping pulses, the magnitude of the transient decrease in fluorescence was found to vary with the loss of membrane impedance. Tetrodotoxin delayed the time-course and often reduced the magnitude of fluorescence changes. The significance of Ihesr experimental findings are discussed.

Synaf>tic ori/ani:::ation oj the oculomotor nucleus: a comparative electron micro- scopic study. STEPHEN G. \\'.\\ MAN AND GEORGE I). PAPPAS.

Electron microscope studies of the oculomotor nuclei of five vertebrates (Chiloinyctcnts scJiocfifi, Rana f>if>icns. .Inolis carolincnsis, Fclis doiuesticu, and Macufti iin/llata) reveal that electrotonic synapses may be a general feature of the inframammalian oculomotor nucleus. Oculomotor neurons in the spiny boxfish (Chilomycterus) have one or a few large dendrites which ramify in the tegmental neuropil. A single presynaptic fiber may establish many en passant synapses with a postsynaptic cell, and may penetrate into indentations in the cell sur- face. A'aiitt oculomotor nucleus contains similar motoneurons and smaller multipolar inter- neurons. There is a specific relationship of some axonal processes with subsynaptic cisterns. Axodendritic synapses predominate on the multipolar neurons in chameleon (AnoHs*) oculo- motor nucleus. Subjunctional bodies are present. The presence of close membrane appositions in the teleost oculomotor nucleus has previously been correlated with electrotonic coupling. Close appositions are present in Rana and Anolis, so that electrotonic coupling prob- ably occurs in these species. Presynaptic terminals at the electrotonic synapses, which often arise at nodes of Ranvier, contain both clear and dense core vesicles. In contrast, gap junctions were not found at synapses on the multipolar neurons in lateral divisions of cat and monkey oculomotor nuclei. Presynaptic terminals in these nuclei are large, and establish axosomatic, axodendritic, and spine synapses. The presence of axo-axonic synapses suggests the operation of presynaptic inhibition. Vesicle distributions at synapses in each of the species examined can be distinctly non-random. Vesicle populations often exhibit a sharp boundary located along an extension of the axon cylinder, suggesting that intra-axonal filamentous networks and/or axoplasmic flow may act as a harrier to vesicle diffusion.

Supported in part by grants (NB-07512, 5T5-GM-1674) from the National Institutes of Health.

Only decreases in illumination elicit spike responses in the siphonal photoreceptov system of the hard-shell clam, Mercenaria nitrcenaria. MICHAEL L. WIKDER-

HELD AND EDWARD F. MAcNlCHOL, JR.

This clam responds behaviorally to dimming of light by retracting the siphon and closing the shell. Presumably this is a protective reflex used to withdraw the siphon when a predator's shadow passes over. We have recorded spike responses from axons in the siphonal nerve which could mediate this reflex.

Experiments were performed on the isolated siphon, posterior adductor muscle and visceral ganglia maintained in sea water. The siphonal nerve was cut near the ganglion and small strands were sucked into a 50 /JL suction electrode. Spikes from single axons were isolated with a window circuit. Responses were obtained by illuminating the yellow-pigmented region of the inner siphon wall. Light with a spectral peak near 510 nm was used throughout, since earlier experiments showed this system to be maximally sensitive at about this wavelength.

We have observed only off-responses, never responses to the onset of illumination. Using a small shadow, the receptive area of a single axon can be localized to a region of approxi- mately 85 fj., indicating that the receptor cells which these axons innervate probably lie near the surface of the inner siphon wall.

The off-response appears to grow monotonically with the duration of illumination, at least up to durations of 500 sec. The off-response to a flash of light is dependent on the amount of previous illumination. In a dark-adapted preparation, the first spike after a flash may not occur for 5-10 sec whereas, with an identical flash, the light-adapted latency can be less than 100 msec. The response to a standard flash can he reduced or eliminated by a steady background light. The flash response is enhanced immediately after the background is terminated and

PAPERS PRESENTED AT MARINE BIOLOGIC AI. LABORATORY 443

then decays back to the pre-background level. This decay time varif.s from a few seconds In several minutes, depending on the intensity and duration o{ the background.

Both authors are from the National Institute of Neurological Diseases and Stroke, National Institutes of Health, P.ethrsda, Maryland 20014.

Structure and function <>\ inicros/^iridian spores. KARL \YKIDXKR AND WILLIAM TRACER.

Microsporidian sporozoa are small eucaryotes suited for intraccllular environments such as muscle, gut and nerve cells. They induce chronic to acute infections in most major animal categories, including man, but are particularly well known as parasites of arthropods and fish. In the genus Xosanu the resistant spores characteristically extrude a fine filament when ingested bv an appropriate host. It is thought that this filament serves to inject infective material into a host cell, but little is known as to the mechanism of extrusion or the nature of the early infective stages.

This ultrastructural study featured the spores of two species, Xoseina lophii from the cranial ganglia of the anglerfish (Lophnis amcricaiuis) and Noscnta sp. from the muscle tissue of the blue crab (Calliuccfcs sup id us). N. lophii has a spore about 3 n long X 1 /* wide containing a conspicuous nucleus about 0.7 /JL in diameter, cytoplasm with numerous free ribosomes and an elaborate polar arrangement of pleated membranes (polaroplast) that are continuous with the outer of the two envelopes of the tightly coiled polar filament. The spore wall consists of a thick (0.1 /j.) electron-lucent inner layer surrounded by a denser, thinner layer (0.02 M).

Spores of \'osc/iui sp. were induced to extrude their filaments by pretreatment for 1 hr with either 0.1 N KOH or Michaelis veronal acetate buffer ( pH 9) followed by immersion in a tissue culture medium (199 or Eagle's MEM). Extrusion reached a peak within 15 minutes. Spores with an attached extruded filament were empty except for a few membrane profiles. The extruded filaments had two envelopes with the same diameter as in the pre-extruded state.

Thus the structure of microsporidian spores fulfills two functions: (1) protection by means of the thick wall and outer coat; (2) extrusion by an explosive mechanism projecting the spore's vital contents through the polar filament into a host cell.

Supported by a grant (5T01-A1-00192) from the National Institute of Allergy and Infectious Diseases.

Effect of puromycin on the cirowth pattern of Campanularia flexuosa. KARL S.

WlTTMAN.

Growth of Canipannhiria flc.ruosa occurs through elongation of the stolon and develop- ment of hydranths from hydrocauli. Newly differentiated hydranths have a life span of about one week and are resorbed with the products of regression available for further colonial growth. The objective of this study was to determine the effects of puromycin on stolon elongation and the hydranth differentiation-regression cycle.

Colonies were mainained in culture on glass slides suspended in filtered sea water at 17° C and fed daily with newly hatched Artcinia nauplii. Healthy cultures were placed in petri dishes containing either sea water alone or 20 /ug/ml puromycin in sea water for periods from 10 hours to seven days.

The rate of stolon elongation of puromycin-treated cultures was significantly lower than that of control cultures. Growth rate reduction was noticeable between 12 and 24 hours after puromycin treatment was begun. Within 24 hours afer removal of puromycin normal stolon growth was resumed. The length of treatment did not influence the recovery period.

All mature hydranths of puromycin-treated cultures entered the regression phase simul- taneously within 24 hours after drug administration, while controls maintained their usually low rate of hydranth regression. Treated hydranths in stages of differentiation regressed instead of completing development. No new hydranths were formed during puromycin treatment. Within 24 hours after removal of puromycin hydranth differentiation resumed at rates similar to controls.

444 PAl'KKS I'kFSFXTFU AT MARINE BIOLOGICAL LABORATORY

This work suggests that pnromycin acts by (1) inhibiting the process of differentiation p<»sibly by depressing synthesis of protein and (2) reducing the immediate reuse of the products of regression.

This study was supported by a Faculty Fellowship and (irant-in-Aid from the State University of New York Research Foundation.

The elioroid retes oj seu[>, Stenotonnts versicolor, anil striped bass, Roccus saxa-

tilis. ROBERT C. WOLLKY AND D. EUGENE COPELAND.

Two retes commonly found in teleost fishes — the swimbladder rete and the choroid rete of the eye — are involved in oxygen concentration by countercurrent multiplication. The swimbladder rete of the striped bass (Roccus su.rcttilis ), scup (Stenotonuis rcrsicolor}, and eel (Aiiiinilla rustrnta), as well as the choroid rete of the striped bass and scup (the eel has no choroid rete) were studied by light and electron microscopy. All three swimbladder retes exhibited cross section features characteristic of a rete mirabile : densely packed array of uniformly sized capillaries with regular interdigitation of afferent and efferent elements. The choroid rete of the bass eye was composed throughout its length of irregularly shaped, thin walled capillaries which were loosely packed and irregularly arranged. Scup choroid rete contained small, thick walled, irregularly shaped capillaries in its proximal regions. In the distal portions the capillaries assumed two different sizes, part remaining small and thick walled, the rest becoming large, irregular and thin walled. The retes of both the eye and the swimbladder are structured to provide countercurrent flow of blood. The rete of the eye, however, possesses vessels that on the average are larger, more thin walled, and more irregularly arranged than in the rete of the swimbladder. So much so that in some instances (striped bass particularly) the venous return seems to be in the form of open sinusoidal spaces. This could permit a more slow, turbulent flow of blood. The countercurrent principle would still apply but the efficiency of the system would be less. On the other hand, there would be more time for active secretory exchange (s) to occur.

This research was supported by grant in aid from NIH (GM-06836), NSF (GB-676) and training grant from NIH (GM-669).

How protein coloration modifies interactions J^efu'een lenses and IT lit/lit. S. ZIGMAN, T. YULE AND G. GRIKSS.

The UY light (365 m/j.} absorbed and the fluorescence (360 m,u/45() m/j.) emitted by normal and artificially colored dogfish lenses and extracted proteins were compared. The un- treated dogfish lens (1 cm diameter) absorbed 50% of this light, and the relative (to water) fluorescence was 64. When the absorptions and fluorescences of the purified a, /3, and 7-crystal- lins were added up and extrapolated to their concentrations within the lens (30% protein), the values obtained were far in excess of these in the intact lens. Protein-protein interactions within the highly structured lens appear to minimize absorption and fluorescence by it. /3- crystallin exhibited the greatest absorption and fluorescence of the three crystallins.

When whole dogfish lenses, homogenates or purified crystallins were exposed to 400 jiiW/cnr of 365 m/j. light for 18 lirs in the presence of 0.1% tyrosine, L-dopa, tryptophane, or p-aminobenzoic acid (PABA) at neutral pH, the reagents themselves, the lenses and the crystallins were stained various shades of yellow (tryptophane), brown (PABA), and black (tyrosine, L-dopa). Lenses and crystallins were also stained during soaking in colored solutions of these irradiation products. Reducing agents (ascorbic acid, dithicorythritol) prevented such color formation in UV light, but could not reverse it. The color could not be removed from the lens proteins, which even leached it out of concentrated solutions. The same color was caused by exposure of these reagents to sunlight and fluorescent light. A

PAPERS PRESENTED AT MARIXK BIOLOGICAL LABORATORY 445

100% + increase in the 365 HIM absorption and 360/450 ni/x fluorescence of whole lenses or purified crystallins resulted from reaction with tryptophane and PAR A under UY light. The greatest increase was found in the •y-crystallin fraction.

The development of colored lenses in animals normally exposed to near UV light may protect the retina from the damage of irradiation by this incri-a.se in absorption and fluorescence. In the dogfish, rat and rabbit, the lens is normally colorless; in humans and certain diurnal animals, it becomes yellow to brown with aging. The formation of this color is probably a chemical adaptation in the lenses of animals exposed to near UY light ( i.e., sun- light) which converts some of this light into fluorescence. A lower intensity of UV light thus reaches the retina thereby reducing possible damage to cells (rods, pigment epithelium) sensitive to it.

Supported by the Rochester Eye and Human Parts Bank and USPHS Grant EY 045'' of the Na'ional Eye Institute.

Vol. 139, No. 3 December, 1970

THE

BIOLOGICAL BULLETIN

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY

PHYSIOLOGY OF THE REPRODUCTIVE CYCLE IN THE COCKROACH BYRSOTRIA FU MI GAT A (GUERIN)

R. H. EARTH, JR. AND WILLIAM J. BELL* Department of Zoology, University of Texas, Austin, Texas 78712

Previous studies on reproduction in cockroaches and in certain other insects have implicated juvenile hormone in the control of various processes occurring dur- ing the female reproductive cycle: yolk formation (reviews: Wigglesworth, 1964; Engelmann, 1968), vitellogenic blood protein secretion (Coles, 1964; Minks, 1967; Bell, 1969a; Engelmann, 1969; Scheurer, 1969a), colleterial gland activity (Willis and Brunet, 1966; Bodenstein and Shaaya, 1968; Shaaya and Bodenstein, 1969) and sex pheromone secretion (Earth, 1962, 1968; Emmerich and Earth, 1968). These processes have not been investigated, however, with regard to their integra- tion in the reproductive cycle. It is the purpose of this communication to describe the reproductive cycle of Byrsotria fumigata, an ovoviviparous cockroach, and to report observations on the factors which control the initiation and termination of these four processes during the cycle.

The cycle of B. fumigata differs from that of the more primitive oviparous forms in that the oothecae are oviposited into a brood sac and incubated within the female until hatching rather than being produced at frequent intervals and ovi- posited externally (Fig. 1). Oocyte development and the other reproductive proc- esses characteristic of the preoviposition period are inhibited during the lengthy term of pregnancy ; after parturition the various reproductive processes recommence. Owing to the greater complexity of their cycle, the ovoviviparous cockroaches are more interesting than oviparous species from the viewpoint of the physiological control mechanisms operating in the regulation of the cycle. Female reproductive cycles in cockroaches have been discussed in relation to sexual behavior (Barth, 1968) and also with regard to the evolution of the cycle (Roth, 1970). The ovi- parous type of cycle, as exemplified by Periplaneta americana, has been treated by Bell (1969b) with special reference to oocyte development.

1 Present address : Departments of Entomology and of Physiology and Cell Biology, Univer- sity of Kansas, Lawrence, Kansas 66044.

447

Copyright © 1971, by the Marine Biological Laboratory Library of Congress Card No. A38-518

448

R. H. EARTH, JR. AND WILLIAM J. BELL

OVIPAROUS (e.g. Periplanela, Superfamily Blattoidea)

OVOVIVIPAROUS(e.g. Byrsofria, Blaberidae, Blaberoidea)

Adult molt

Sexual

maturation

1st preoviposition period

Oocyte development-, sexually receptive-, C. A. active

Ovulation

Unreceptive Oviposition

2nd preoviposition period

Oocyte development; sexually receptive; C.A. active

Ovulation

etc.

Adult molt

Sexual

maturation

1st preoviposition period

Oocyte development-, sexually receptive-, C.A. active

Ovulation and Oviposition

Pregnancy

No oocyte development; unreceptive ; C.A. inactive

Parturition or abortion

2nd preoviposition period

Oocyte development; often receptive; C.A. active

Ovulation and Oviposition

etc.

FIGURE 1. Reproductive cycles of cockroaches.

METHODS AND MATERIALS 1. Insect material

B. jumigata nymphs were maintained in plastic cages with access to food and water as described previously by Earth (1964). All females used in the experi- ments were removed from the cages within 24 hours after they emerged as adults

REPRODUCTIVE CYCLE IN BYRSOTRIA

449

(designated day 0) and were thereafter housed individually in 250 ml beakers. The females were mated between days 12 and 16 after emergence and spermato- phore insertion was used as a criterion for successful mating. Experimental females were bled and dissected within 6 hours following the assay for pheromone, and the blood was stored at —5° C. The clotted cells were separated from the serum by allowing the serum to seep out of the clot in a horizontal test tube. Oocytes and colleterial glands were excised from dissected females and extracted as described below.

2. Pheromone assay

The sex pheromone assay, previously described by Barth (1961, 1962), con- sisted of removing a filter paper from a beaker containing a female and placing it in or suspending it a few inches above a container of males for two minutes. If the female was secreting pheromone (absorbed by the filter paper) the paper elicited courting responses in groups of adult males. In the present study an additional assay for pheromone secretion was used ; the virgin female herself was placed in or held a few inches above a container of males. In this way the pres- ence of the pheromone on the cuticle of the female could be assayed by the male response.

3. Yolk deposition

The criteria used to measure the initiation and progress of yolk deposition were the appearance of refractile yolk spheres in the oocyte cortex, appearance of sig- nificant amounts of extractable protein in the oocytes and changes in oocyte volume (Bell, 1969b).

4. Vitellogenin secretion

The Oudin (1948, 1952) technique was used to resolve the resulting antigen- antibody reactions when blood or yolk fluid was reacted with an antiserum con- taining antibodies homologous to the yolk antigens. The antiserum was derived from the blood of rabbits which had been injected with the proteinaceous portion of B. fumigata yolk fluid. The yolk extracts and the antiserum were prepared

TABLE I

Concentration of yolk antigens in cockroach blood relative to the concentration in a yolk fluid standard

	Antigen (% concentration)
	A	B	C	D
Yolk fluid	100.00	100.00	100.00	100.00
Ovariectomized female (day 16)	4.00	10.90	86.40	100.00
Adult female (day 15)	1.20	4.30	101.05	118.00
Pregnant female	0.04	0.03	93.70	87.00
Adult male (day 15)	0.00	0.00	101.05	117.00
Female nymph	0.00	0.00	94.00	94.00

450

R. H. EARTH, JR. AND WILLIAM J. BELL

using techniques previously described for the preparation of an antiserum against yolk proteins of Periplaneta anicricana (Bell, 1970).

Glass tubes (6 X 0.5 cm) were half-filled with diluted antiserum mixed with agar (1 : 10). When the antiserum-agar mixture was solidified, yolk fluid or cock- roach blood was layered on the tubes and a topping of mineral oil was added to prevent evaporation. As the antigens diffused into the antiserum-agar, four pre- cipitin bands with sharp leadings edges were observed. Of the four bands, 2 were of light density (antigens C and D) and resulted from precipitin tests with yolk fluid or blood from male, female or immature cockroaches. Table I shows that the

E E

3

o

r oo

c o

rt

i_

O)

OJ

o

c to

- Q

Yolk Fluid Dilutions: IOO% 10

O-l

o-oi

Day 15 1 Blood

Pregnant ? Blood

Blood =

^^^^^^W

Nymph 1 Blood

n

Day 15

o*Blood

|:;i; antigen A

antigen B

FIGURE 2. Distance of migration by precipitin bands in Oudin tubes containing antiserum absorbed with male blood and mixed with agar.

concentration of antigens C and D is similar in yolk fluid and blood from males, females and nymphs. Added to the fact that antigens C and D do not accumulate in ovariectomized female blood and are not selectively sequestered by the oocytes (Table I), these data suggest that antigens C and D are not female-specific vitello- genic blood proteins, but occur as minor components of the yolk.

Antigens A and B which produce the two dense precipitin bands, on the other hand, are taken up by the oocytes over a considerable concentration barrier and accumulate in the blood of ovariectomized females. Moreover, these antigens are female-specific, occur only in adult, vitellogenic females and, as indicated by their dense bands of precipitation in Oudin tubes, comprise a major portion of the yolk protein complement. In view of these findings, the antigens responsible for the

REPRODUCTIVE CYCLE IN BYRSOTRIA

451

formation of precipitin bands A and B are referred to here as vitellogenins. This term was applied by Pan, Bell and Telfer (1969) to designate a functionally dis- crete group of insect blood proteins which are female-specific and which are selec- tively sequestered by the oocytes.

Figure 2 illustrates the results of using the Oudin test to measure the concen- tration of vitellogenins A and B in B. fumigata blood or serially diluted yolk fluid (in this case the antiserum was previously absorbed with male blood in order to remove antibodies homologous to antigens C and D). It has been shown by Oudin that a linear relationship exists between the logarithm of the antigen concentration and the value K (distance of migration [mm] by the leading edge of a precipitin band divided by the square root of time [min]). With regard to the above rela- tionship, the distance of migration of the precipitin bands in tubes containing serial dilutions of yolk fluid were measured and these distances were divided by the square root of time ; the resulting K values were plotted against the concentration

u

c o

O

100

10

0.1

0.01

O)

o

.0 2

.0 4

.0 6

.0 8

K

.1 o

.1 2

.1 4

FIGURE 3. Logarithm of yolk fluid concentration plotted against K values [distance of migration of precipitin bands (in mm) divided by the square root of the time interval (in minutes) ].

of yolk fluid (Fig. 3). The rates of migration of the bands in tubes layered with blood from 15-day females, pregnant females and 16-day ovariectomized females were also measured and the concentration of the vitellogenins in these solutions, relative to their concentrations in the yolk fluid, was ascertained by referring to the standard curve shown in Figure 3. Table I shows the concentration of the vitello- genins in the solutions tested.

In the studies presented in this paper, the relative concentrations of the vitello- genins were measured in the blood of females during the reproductive cycle in order to ascertain the relationship between the yolk precursor secretion cycle and other reproductive processes.

5. Colleterial gland glucoside activity

The spectrophotometric glucoside assay of Willis and Brunei (1966) was em- ployed to measure the secretory activity of colleterial glands. The left colleterial

452

R. H. EARTH, JR. AND WILLIAM J. BELL

glands were excised and homogenized in 2 ml of saturated ammonium sulfate. 0.2 ml of the supernatant was mixed with 2.8 ml of distilled water and the solutions were read against appropriate blanks in a Carey recording spectrophotometer.

Colleterial gland extracts contained the glucoside (Gl-A) which was previously characterized by paper chromatography (Stay and Roth, 1962) and was found to have an absorption spectrum at 2770 A. A second component, with a peak of 2850 A, was observed in glands excised from newly emerged females (Days 0 to 8). Glucoside units (a change in O.D. of 0.001) were measured in colleterial glands from females during the reproductive cycle.

12

*> 10 £

UJ ,

O

>

LU

I— >

O

10 12 14 16 18

DAYS AFTER EMERGENCE

20

22 24

26

FIGURE 4. Changes in oocyte volume during the first preoviposition period. Vertical lines are standard deviations. Each point represents the average of 10 to 20 females.

The width of the left colleterial gland tubules was also used as an indication of gland activity. In newly emerged females the gland tubules are white and have an average width of 0.1 mm, while in vitellogenic females the tubules are bluish green and have an average width of 0.6 mm.

RESULTS 1. Yolk deposition

The onset of yolk deposition occurs on about day 5 after ecdysis; the basal oocytes are characterized by detectable quantities of extractable vitellogenin, an increase in volume and the appearance of refractile yolk spheres in the oocyte cortex. Rapid deposition of vitellogenic blood proteins occurs during days 14 to 22, followed by a period of less intense deposition and terminating completely by day 26. Oocytes with an average volume of 10 mm3 were ovulated between days 23 and 30 (average of 26) after emergence (Fig. 4). Employing essentially similar culture conditions and techniques as in the present study, Roth and Stay (1962)

REPRODUCTIVE CYCLE IN BYRSOTRIA

453

reported that in their colonies the mated females oviposited between days 26 and 41 (average of 32.4). This apparent reduction in the period required for oocyte maturation in insects cultured over a long period of time has also been observed in Schistocerca yrcgaria (L. Hill, University of Sheffield, personal communication). Female B. fitinigata are normally receptive to courting males between days 10 and 30 after emergence. In females which fail to mate, the oocytes often fail to develop or may be resorbed after partial development ; moreover, nearly all virgins (including those which do oviposit) lack the synchronous oocyte growth observed in mated females. Virgin females which oviposit do so slightly later than mated females (Roth and Stay, 1962).

2. Vitellogenin secretion

The blood of females on day 4 or 5 after emergence contains slight, but un- measurable quantities of vitellogenin (less than 0.001% relative to the yolk fluid standard), suggesting that these yolk precursors are present, but only in trace amounts. Significant levels of both vitellogenins appear in the blood of most females on day 7 or 8. Antigen B is detected about 24 hours prior to the appear- ance of antigen A, and throughout the cycle antigen B occurs in higher concentra-

12 DAYS

16 AFTER

20 24

EMERGENCE

28

30

FIGURE 5. Changes in blood vitellogenin concentration during the first preoviposition period. Vertical lines are standard deviations. Each point represents an average of 10 to 15 females.

454 R. H. EARTH, JR. AND WILLIAM J. BELL

tion than antigen A (relative to their concentration in the yolk fluid standard). From the time of their first appearance in female blood the vitellogenins increase synchronously to a peak on day 16 (Fig. 5), drop to a low level by day 18, increase to a second peak at day 22 and then decrease in concentration until ovulation. Finally, in most females the vitellogenins disappear entirely from the blood during pregnancy ; indeed, by the tenth day of pregnancy the vitellogenins could not be detected in 80% of the females tested.

The trough in the vitellogenin curve between days 17 and 20 corresponds to the period of most intense yolk investment. During this period the oocytes increase in volume by 4.00 mm3, an increment which is greater than that of the previous or the succeeding 4 day period. Secondly from days 20 to 24 the rate of increase in oocyte volume is substantially less than during any other period in the vitello- genic cycle ; this hiatus in yolk deposition may account for the observed replenish- ment of blood vitellogenin which reaches a peak on about day 22. Resorption of some basal oocytes which commonly occurs even in mated females towards the end of the vitellogenic period, may also contribute to the second peak in vitellogenin concentration.

Measurements were made of the vitellogenin concentration in the blood of ovariectomized females. The accumulation of the protein yolk precursors in cas- trated females (which reaches a concentration of 4.0% [A], and 10.9% [B] at day 16) gives credence to the role of these proteins in yolk deposition ; moreover, the observation that the vitellogenin concentration of ovariectomized females de- clines to 1.4% [A], and 4.6% [B] at the time when normal females are initiating egg incubation suggests that the observed decreasing concentrations of blood vitellogenin towards the end of vitellogenesis is due to processes in addition to the removal of these proteins from the blood by yolk-forming oocytes.

3. Pheromone production

On the basis of the filter paper assay it was reported that 90% of normal virgin females initiated pheromone production between 10 and 30 days after the imaginal molt (Earth, 1961, 1962). However, it was also noted that females maintained with males from emergence occasionally mated as early as day 4 (Barth, 1962). This observation together with other studies demonstrating the importance of the sex pheromone for the release of male courtship behavior (Barth, 1964) led to the con- clusion that the pheromone is very likely present on the body surface of the female at an earlier age than that at which it is detectable on filter paper. For this reason the pheromone assay employing the female as described above was extensively employed in these studies. When introduced into a chamber of males, the test females usually showed the immobilization reaction (flattening against the substratum and with- drawing the antennae laterally beneath the pronotal shield — Barth, 1964), or less commonly attempted to escape by hiding under a nearby object. Such females were invariably sexually unreceptive and behaviorally were essentially as inert as filter papers, hence any stimuli presented to the males are highly likely to have been chemical alone.

Table II indicates that by day 4 a sufficient quantity of pheromone was present on the cuticle of 5% of the females tested to elicit a courting response by adult males. On day 10, 94% of the females placed in male cages elicited the male court-

REPRODUCTIVE CYCLE IN BYRSOTRIA

455

TABLE II Initiation of pheromone production*

	Days after emergence
	1	2	3	4	5	6	7	8	9	10	11	12
% of females with detectable
cuticular pheromone	0	0	0	5	17	32	52	74	84	94	100	100

* Represents data from 65 females.

ing response. It is clear that by means of this assay procedure, data can be ob- tained which bring the initiation of pheromone secretion into line with the initiation of other reproductive processes at the onset of the reproductive cycle. Earth (1970) reported that the pheromone is a product of some portion of the female genital tract other than the colleterial glands ; thus according to our current hypothe- sis, the pheromone is secreted through the genital opening and is selectively ad- sorbed onto the surface of the cuticle. It is also adsorbed onto the surface of other objects in the vicinity (i.e., filter papers). Whether such objects possess sufficient pheromone activity to stimulate the males depends upon the rate of pheromone output by the female relative to the evaporation and/or breakdown rate of the pheromone. Output rates seem to vary considerably and may be characteristic of individual females (Earth, 1962).

As reported by Earth (1968) and as shown in Table III, pheromone produc- tion ceases within a few days after mating. In some individuals it may cease as early as the first day and in no case has it been observed to continue beyond the third day following mating. By contrast virgin females continue to produce the pheromone until they ovulate; moreover, if they fail to ovulate on schedule (Roth and Stay, 1962), they may continue to produce sex pheromone for several weeks.

A related event of importance which occurs early in the reproductive cycle of the female is the onset of sexual receptivity. Previous work on sexual receptivity suggests that in contrast to the other four processes under discussion, it is not regu-

TABLE III

Cessation of pheromone production by females after mating*

	Age
Number of females tested	(in days after mating)	% females with positive pheromone
	when first tested
66	Of	100
4	1	0
31	2	0
16	3	331
15	4	0

* Filter paper assay used in all tests. Females mated at 10-16 days of age one day after demonstrating pheremone production by means of the filter paper assay. f Indicates the day on which females were mated. j These females all ceased pheromone production on day 4 after mating.

456

R. H. EARTH, JR. AND WILLIAM J. BELL

lated directly by the corpora allata (Roth and Earth, 1964; Barth, 1968; Engel- mann and Barth, 1968). The results of the female pheromone assay above indicate that the female herself is not receptive at the time when sex pheromone on the stir- face of her cuticle makes her attractive to males. In view of our interest in the sequence of reproductive events we continued to test a small number of active females daily for signs of receptivity. All six animals so tested showed antenna- tion and mounting of displaying males within the two minute test period on the second day following appearance of pheromone on the cuticle. Thus females appear to reach a high level of sexual receptivity about 2 days after the onset of pheromone secretion. It may be noted that this is likely to be several days before the average onset date for pheromone secretion as determined by the filter paper assay. Herein lies the probable explanation for the accounts in the literature of mating in B. fiunigata females prior to the onset of pheromone production (Barth, 1962 ; Roth and Stay, 1962).

z

D

HJ Q

cn o u

D

8OO -

eoo -

4oo -

200 -

12 16 2O

DAYS AFTCR EMERGENCE

24

28

FIGURE 6. Changes in left colleterial gland glucoside concentration during the first pre- oviposition period. Each point represents the average glucoside units measured for the pooled glands of 5 females.

4. Colleterial gland activity

The left colleterial gland of B. fumigata secretes the structural protein of which the ootheca is composed and a glucoside of the phenolic tanning agent (Stay and Roth, 1962). Synthesis and storage of the protein is indicated by a bluish green color and increase in width of the gland tubules. The appearance of color and increase in colleterial gland size was observed to occur between days 4 and 6. In addition to the glucoside with an absorption peak of 2770 A, the glands contained an unknown substance with an absorption peak of 2850 A ; this unknown was present in the left colleterial gland extracts (in saturated ammonium sulfate) until between days 6 and 8 at which time the unknown was replaced by the glucoside.

REPRODUCTIVE CYCLE IN BYRSOTRIA 457

The presence of the left colleterial gland glucoside of B. fumigata (Gl-A), identified chromatographically by Stay and Roth (1962), was first detected between days 6 and 8 after adult ecdysis. The units of activity increased linearly (Fig. 6) in mated females until a leveling off point occurred on about day 24. In virgin females the individual variation was very large owing to the low concentration of glucoside observed in many of these animals after day 15.

Following ovulation and oviposition there was a marked decline in glucoside activity, but some glucoside is apparently retained by the colleterial glands through- out the pregnancy period. It is interesting to note that the average period of pregnancy (53.1 days) observed in these studies is substantially shorter than the average pregnancy period of 76.2 days recorded by Roth and Stay (1962) ; these findings provide further evidence for the acceleration of reproductive processes in insects cultured for a number of generations.

DISCUSSION

Four reproductive processes in the cockroach B. fumigata are known to be influenced by juvenile hormone (Bell and Earth, 1970). These processes appear to be initiated simultaneously in females on about the fifth day after adult emergence. An attempt was made in the present study to determine \vhether the initiation of any one process consistently preceded any other, but this was shown not to be the case.

A number of analogies between the reproductive cycle of B. fumigata and those of other cockroach species are revealed by the study reported here. The crucial difference between the two major cockroach groups is that the cycle of the family Blaberidae includes a period of pregnancy which is absent from the cycle of the superfamily Blattoidea and the family Blattellidae of the superfamily Blaberoidea (classification according to McKittrick, 1964) (see Fig. 1). This difference brings about obvious changes in the schedule of yolk formation, vitellogenin secretion, colleterial gland function and pheromone secretion ; in P. americana, for example, the cycle of vitellogenin secretion and sequestration by the oocytes is continuous owing to the absence of a pregnancy period (Bell, 1969b). Among several con- sequences of a continuous cycle as exemplified by P. americana is the elimination of a peak in blood vitellogenin found in many B. fumigata females at the termination of yolk formation. In P. americana the second (penultimate) set of oocytes, which begins development prior to the termination of development in the first (basal) set, accumulates any vitellogenins which are not sequestered by the basal oocytes. On the other hand in B. fumigata there is a small quantity of blood vitellogenin which is not utilized and which remains in the blood during the first 10 to 20 days of pregnancy.

Another consequence of having continuous reproductive cycles and probably continuous juvenile hormone secretion, is that vitellogenin secretion and yolk depo- sition do not occur periodically, but seem to be maintained constantly throughout the reproductive life of the female (Bell, 1969b). Colleterial gland activity, on the other hand, proceeds somewhat differently for reasons of cyclic demand ; during ootheca formation in P. americana the colleterial gland glucoside activity is reduced by 75%, but the supply is replenished within 2 days after ootheca formation (Willis and Brunet, 1966).

458 R. H. EARTH, JR. AND WILLIAM J. BELL

In B. jumigata the onset of pregnancy brings abont a concomitant termination of vitellogenin secretion and yolk deposition; the same is true in Leucophaea maderae, another oviviparous species (Engelmann and Penney, 1966). The colle- terial gland glucoside activity is reduced by 78% during ootheca formation in B. jumigata, but it is not until parturition that the gland begins to fill again ; parturi- tion is also the point in the reproductive cycle at which yolk deposition and vitello- genin secretion are again initiated.

Pheromone activity is first associated with the cuticle of day 4 females and may ultimately be detected on filter papers in beakers housing day 10 or older females. It is not known whether pheromone transfer to the filter papers is due to an in- creased pheromone output by the females or to a change in the structure of the pheromone molecule. The absence of a precise correlation between the pheromone secretion schedule and the schedule of female receptivity, as observed in our study, substantiates earlier work on B. jumigata, Nauphoeta cinerea and L. maderae (Roth and Barth, 1964; Barth, 1968), suggestive of different control mechanisms for the two processes.

The properties and functions of the two B. jumigata vitellogenic blood proteins suggest that they are analogous to similar proteins observed in L. maderae (Dejmal and Brookes, 1968; Engelmann and Penney, 1966; Engelmann, 1969), and P. americana (Bell, 1969b, 1970). In all three cases the vitellogenins require a high ionic strength for solubility and are selectively incorporated from the blood by yolk- forming oocytes. In the three species mentioned above, the two primary yolk com- ponents can be extracted only if the medium contains a salt concentration of at least 0.4 M NaCl. Whereas the vitellogenins are the primary yolk constituents, other protein components do exist in cockroach yolk as demonstrated electrophoretically or immunochemically by Adiyodi and Nayar (1967), Bell (1970), Nielsen and Mills (1968) and Scheurer (1969b). These proteins, however, are present in relatively small amounts and do not exceed their relative concentration in blood ; hence they are not selectively sequestered by the oocytes.

With regard to the hormonal effect, it should be noted that even though the onset of juvenile hormone secretion is thought to occur at about the same time as the initiation of the various reproductive processes, it is impossible to determine at this time whether the hormone acts directly upon the ovaries (yolk deposition), fat body (yolk precursor synthesis), colleterial glands and pheromone gland; indeed, until the molecular effects of the hormone are further elucidated we will not know the precise relationship between the effector sites of juvenile hormone and the processes which we have studied. Specific effects of the hormone will be the topic of subsequent papers in this series.

The authors are grateful for the technical assistance of Mrs. Carol Traver. This work was supported by research grants from NIH (2T01-GM00837-07) and NSF (GB-7466).

SUMMARY

1. Four reproductive processes (vitellogenin secretion, yolk deposition, colle- terial gland activity and pheromone production) are initiated on about the fifth day

REPRODUCTIVE CYCLE IN BYRSOTRIA 459

after adult ecdysis. There is no apparent sequence of initiation of these processes except that vitellogenin secretion either precedes or occurs concomitantly with the initiation of yolk deposition.

2. Two blood proteins, which are female-specific yolk precursors (vitellogenins A and B), reach a concentration peak on day 16 and then decrease in response to rapid yolk deposition. A second peak on day 22 is thought to represent yolk precursors which were released to the blood as a result of the resorption of some basal oocytes. The vitellogenins are either absent or occur in trace quantities in the blood of pregnant females.

3. Sex pheromone is detectable on the surface of the cuticle by day 10 in 94% of the females tested. Following mating pheromone production is drastically curtailed.

4. Glucoside activity in colleterial glands increases almost linearly from day 8 to 24. After ootheca formation, which involves the utilization of colleterial gland secretion, there is a 78% reduction in glucoside activity.

5. The sequence of events in the reproductive cycle of females of B. fumigata is compared with that of other cockroaches.

LITERATURE CITED

ADIYODI, K. G., AND K. K. NAYAR, 1967. Haemolymph proteins and reproduction in Periplaneta amerlcana: the nature of conjugated proteins and the effects of cardiac-allatectomy on protein metabolism. Biol. Bull, 133: 271-286.

EARTH, R. H., JR., 1961. Hormonal control of sex attractant production in the Cuban cock- roach. Science, 133: 1598-1599.

EARTH, R. H., JR., 1962. The endocrine control of mating behavior in the cockroach, Byrsotria fumigata (Guerin). Gen. Comp. Endocrinol., 2: 53-69.

EARTH, R. H., JR., 1964. The mating behavior of Byrsotria fumigata (Guerin) (Blattidae, Blaberinae). Behaviour, 23: 1-30.

EARTH, R. H., JR., 1968. The comparative physiology of reproductive processes in cockroaches. Part I. Mating behaviour and its endocrine control. Advan. Reprod. PhysioL, 3: 167- 207.

EARTH, R. H., JR., 1970. Pheromone-endocrine interactions in insects. Mem. Soc. Endocrinol., 18: 373-404.

BELL, W. J., 1969a. Dual role of juvenile hormone in the control of yolk formation in Peri- plancta amcricana. J. Insect PhysioL, 15: 1279-1290.

BELL, W. J., 1969b. Continuous and rhythmic reproductive cycle observed in Periplaneta amerl- cana (L.). Biol. Bull, 137: 239-249.

BELL, W. J., 1970. Demonstration and characterization of two vitellogenic blood proteins in Periplaneta americana: an immunochemical analysis. /. Insect PhysioL, 16: 291-299.

BELL, W. J., AND R. H. BARTH, JR., 1970. Quantitative effects of juvenile hormone in the cock- roach Byrsotria fumigata (Guerin). /. Insect PhysioL, in press.

BODEN.=TEIN, D., AND E. SHAAVA, 1968. The function of the accessory sex glands in Periplaneta amcricana (L.) I. A quantitative bioassay for juvenile hormone. Proc. Nat. Acad. Sci.,59: 1223-1230.

COLES, G. C, 1964. Some effects of decapitation on metabolism in Rhodnius prolixiis Stal. Nature, 203: 323-324.

DEJMAL, R. K., AND V. J. BROOKES, 1968. Solubility and electrophoretic properties of ovarial protein of the cockroach, Leucophaca maderae. J. Insect PhysioL, 14: 371-381.

EMMERICH, H., AND R. H. BARTH, JR., 1968. Effect of farnesyl methyl ether on reproductive physiology in the cockroach, Byrsotria fumigata (Guerin). Z. Naturforsch., 23b: 1019-1020.

ENGELMANN, F., 1968. Endocrine control of reproduction in insects. Ann. Rev. Entontol., 13: 1-26.

460 R. H. EARTH, JR. AND WILLIAM J. BELL

ENGELMANN, F., 1969. Female-specific protein: biosynthesis controlled by corpus allatum in Lettcophaca maderae. Science, 165: 407-409.

ENGELMANN, F., AND R. H. EARTH, JR., 1968. Endocrine control of female receptivity in Leuco- phaea maderae (Blattaria). Ann. Entomol. Soc. Amer., 61: 503-S05.

ENGELMANN, F., AND D. PENNEY, 1966. Studies on the endocrine control of metabolism in Leucophaea maderae. 1. The haemolymph proteins during egg maturation. Gen. Comp. Endocrinol, 7: 314-325.

McKiTTRicK, F. A., 1964. Evolutionary studies on cockroaches. Cornell Univ. Agr. Exp. Station Mem., No. 389: 1-197.

MINKS, A. K., 1967. Biochemical aspects of juvenile hormone action in the adult Locusfa migratoria. Arch. Neerl. ZooL, 17: 175-258.

NIELSEN, D. J., AND R. R. MILLS, 1968. Changes in electrophoretic properties of haemolymph and terminal oocyte proteins during vitellogenesis in the American cockroach. /. Insect Physiol, 14: 163-170.

OUDIN, J., 1948. L'analyse immunochemique qualitative; methodes par diffusion des anti- genes au sein de rimmunoserum precipitant gelose. Ann. Inst. Pasteur (Paris}, 75: 30-52.

OUDIN, J., 1952. Specific precipitation in gels and its application to immunochemical analysis. Methods Mcd. Res. 5: 335-378.

PAN, M. L., W. J. BELL AND W. H. TELFER, 1969. Vitellogenic blood protein synthesis by insect fat body. Science, 165: 393-394.

ROTH, L. M., 1970. Evolution and taxonomic significance of reproduction in Blattaria. Ann. Rev. Entomol., 15: 75-96.

ROTH, L. M., AND R. H. EARTH, JR., 1964. The control of sexual receptivity in female cock- roaches. /. Insect Physiol., 10 : 965-975.

ROTH, L. M., AND B. STAY, 1962. A comparative study of oocyte development in false ovovivi- parous cockroaches. Psyche J. Entomol. (Cambridge, Massachusetts'), 69: 165-208.

SCHEURER, R., 1969a. Endocrine control of protein synthesis during oocyte maturation in the cockroach Leucophaea maderae. J. Insect Physiol., 15: 1411-1419.

SCHEURER, R., 1969b. Haemolymph proteins and yolk formation in the cockroach, Leucophaea maderae. J. Insect Physiol., 15: 1673-1682.

SHAAYA, E., AND D. BODENSTEIN, 1969. The function of the accessory sex glands in Pcri- planeta americana (L.) II. The role of the juvenile hormone in the synthesis of pro- teins and protocatechuic acid glucoside. /. Exp. ZooL, 170: 281-292.

STAY, B., AND L. M. ROTH, 1962. The colleterial glands of cockroaches. Ann. Entomol. Soc. Amer. ,55: 124-130.

WIGGLESWORTH, V. B., 1964. Hormones in growth and reproduction. Advances in Insect Physiology, 2: 247-336.

WILLIS, T. H.,AND P. C. J. BRUNET, 1966. Hormonal control of colleterial gland secretion. /. Exf. £iW.. 44: 363-378.

Reference : Biol. Bull., 139: 461-475. (December, 1970)

EXPERIMENTAL STUDIES ON THE ENDOCRINOLOGY AND

REPRODUCTIVE BIOLOGY OF THE VIVIPAROUS POLY-

CHAETE ANNELID, NEREIS LIMNICOLA JOHNSON J

DENIS G. BASKIN2 AND DAVID W. GOLDING 3

Department of Zoology and its Cancer Research Genetics Laboratory, University of California, Berkeley, California

The reproductive biology of relatively few Northeastern Pacific nereids has been described: Nereis linmicola (as Neanthes lighti} (Smith, 1950), A7. vexillosa (Johnson, 1943), N. gnibei (Reish, 1954; Schroeder, 1968), N. caudata (Reish, 1957), and Micronereis nanahnoensis (Berkeley and Berkeley, 1953). Gould and Schroeder (1969), Schroeder (1967, 1968), and Baskin (1970) have reported on experimental studies related to endocrine control of reproduction and development.

The reproductive endocrinology of N. limnicola is of interest for a number of reasons. This viviparous species has a close relationship with, and is virtually indistinguishable from, the oviparous Nereis diversicolor (Smith, 1958), the sub- ject of previous studies on the hormonal control of reproduction (Durchon, 1952; Clark and Ruston, 1963 ; Durchon and Boilly, 1964 ; Durchon and Dhainaut, 1964 ; Durchon and Dhainaut-Courtois, 1964; Durchon and Porchet, 1970). The em- bryology and reproductive biology of A7. limnicola has been studied by Smith (1950). These viviparous worms are self-fertilizing hermaphrodites; fertilization occurs in the coelom, where development proceeds until the larvae are 4—5 mm in length and have approximately 20-30 pairs of parapodia. Parturition occurs by ruptures of the body wall of the adult. This reproductive pattern is unique among nereids (Smith, 1958) and raises the possibility of endocrine regulation of vivi- parity.

Both somatic and gametic maturation are known to be hormonally controlled in nereids (see reviews by Clark, 1965, 1969; Hauenschild, 1965 ; Durchon, 1967). The primary oocytes are shed from a proliferative epithelium and grow within the coelomic cavity (Dales, 1950; Durchon, 1952). Once the oocytes have reached a critical diameter, removal of the brain (supraesophageal ganglion) may result in a phase of rapid oocyte growth, although this response is variable in different species (Durchon, 1952, 1956; Clark and Ruston, 1963; Hauenschild, 1965, 1966; Dhainaut and Porchet, 1967; Malecha, 1967; Schroeder, 1968). Typically, somatic and gametic maturation coincide, whether somatic maturation consists of metamor- phosis into the epitokous heteronereid form, or in the less spectacular modifications characteristic of atokous species, and it is thought that both processes are con-

study was supported by a Predoctoral Fellowship from the U. S. P. H. S. to Denis G. Baskin, and NSF Grant GB-6424 to Dr. Howard A. Bern.

2 Present address : Department of Anatomy, Albert Einstein College of Medicine, Bronx, New York 10461.

3 Present address : Department of Zoology, University of Newcastle-upon-Tyne, England, U. K.

461

462 DENIS G. BASKIN AND DAVID W. GOLDING

trolled by the declining titer of a single hormone of presumed cerebral origin (Golding, 1967c). However, several observations by Smith (1950), as well as by the present authors indicate that the temporal relationships of the gametic and somatic aspects of maturation in N. limnicola differ from the typical nereid pattern.

The source of the maturation-inhibiting hormone in nereids has been presumed to be neurosecretory cells of the supraesophageal ganglion (see Gabe, 1966; Gold- ing, 1967b; Dhainaut-Courtois, 1968b, for reviews). More recently, however, it has been suggested that the infracerebral gland, a possible neuroendocrine complex on the ventral surface of the cerebral ganglion, might be the source of this factor (Dhainaut-Courtois, 1968a; Golding, Baskin and Bern, 1968). This complex is composed of an epithelium that contains two principal cell types : ( 1 ) the promi- nent a cells, which in transverse section give the gland the configuration of a columnar epithelium and whose cytoplasm is devoid of electron-dense granules ; (2) the relatively scarce b cells, which are irregular in shape and which contain electron-dense cytoplasmic granules. Neurosecretory axons originating from within the brain pass through the fibrous brain sheath and are found amongst the cells of this epithelium. The endings of other neurosecretory axons are found adjacent to the inner surface of the fibrous sheath that encapsulates the brain and separates it from the epithelium.

Since previous work on nereid reproductive endocrinology had been carried out on oviparous species, it was of special interest to determine if endocrine control of gamete maturation in N. limnicola, a viviparous species, conforms to the general nereid pattern. Golding, Baskin and Bern (1968) observed that the infracerebral gland epithelium of N. limnicola was thicker than that of other nereids, and specu- lated that the unusually well-developed infracerebral gland of N. limnicola might be related to the viviparous reproductive pattern of this species.

The present paper reports on several experiments related to the endocrine con- trol of reproductive maturation in N. limnicola. Furthermore, possible endocrine influence on development of larvae within the coelom was studied. Finally, parts of a severed brain were implanted into decerebrate worms in order to examine the possible relationship of the infracerebral gland to production of the maturation- inhibiting hormone.

MATERIALS AND METHODS

Specimens of N. limnicola were collected at Lake Merced in San Francisco, California. They were gradually adapted to 25% sea water at 14° C in the labora- tory for several days before being examined for gametes. This procedure was followed since worms adapted to lower salinities usually did not survive anesthesia. The choice of 25% sea water was made because this salinity falls within the range in which N. limnicola regulates the osmotic concentration of its coelomic fluid at levels comparable to that when the worms are adapted to Lake Merced water, which has a salinity of 0.5% sea water and is considered to be fresh (Oglesby, 1965). Each worm was removed from the water and blotted on filter paper. All coelomic fluid samples were of approximately identical volume, and were taken using a fine-tipped capillary tube and examined under a coverslip in a drop of mineral oil. All measurements were taken at 100 X magnification, and oocyte diameters were measured with an ocular micrometer. As several size classes of

ENDOCRINOLOGY OF VIVIPAROUS NEREID 463

oocytes were usually present in an individual, only the diameters of the largest oocytes found in the sample were utilized, following the procedure used by Schroeder (1968). Terminology for the developmental stages and larvae follows Smith (1950).

The quantity of motile sperm present in each worm was evaluated on an ordinal scale of 0 to 5 as follows:

0 no motile sperm present in entire sample.

1 less than 10 motile sperm present in entire sample.

2 less than 10 motile sperm present in an average field.

3 11-20 motile sperm present in an average field.

4 21-50 motile sperm present in an average field.

5 more than 50 motile sperm present in an average field.

Because the sampling procedure usually required several days, the worms were held in a refrigerator (6° C) until all specimens used in an experiment had been examined.

All of the worms used in each experiment, including the intact controls, were anesthetized in 5% ethanol in 25% sea water. Decereb ration was carried out as described by Golding (1967a). In the case of decerebrate worms which received an implanted brain, the excised brain was inserted into the coelom by pushing it posteriorly through the wound resulting from the brain removal operation. In experiments in which portions of the brain were implanted, the excised brain was placed on filter paper under a dissecting microscope and the cut made with fine- tipped scissors. The desired portion of the brain was then implanted into the coelom as described above. Single parapodia were removed from each worm to identify individuals. The jaws of worms with intact brains were snipped off at the base to prevent cannibalism. All wounds sealed themselves by contractions of surrounding tissues. After the operations, the worms were placed in 25% sea water which had been filtered, boiled, and cooled, and to which 140 mg/liter strepto- mycin sulfate (Upjohn) had been added. The worms were maintained in plastic refrigerator dishes containing several layers of glass tubes (as described by Golding, 1967a) at a constant 18° C, and were not fed during the experiments. In all cases, the worms appeared healthy and vigorous up to the completion of the experiments.

The results were analyzed statistically using the non-parametric Fisher exact probability, Chi-square, and Mann-Whitney U tests from Siegel (1956).

The brains used for histology were fixed in Kelly's fluid, sectioned at 5 ju, in Paraplast, and stained with paraldehyde fuchsin (Clark, 1955).

RESULTS The relationship of somatic and gametic maturation

Several somatic changes that are associated with reproductive maturation of other nereids appear to be delayed in N. limnicola. The extensive histolysis of body musculature, which characteristically accompanies nereid gametic maturation (Defretin, 1949), does not coincide with sperm and oocyte maturation in this spe- cies. Our observations indicate that muscular histolysis in N. limnicola occurs after the gametes have matured and is correlated with the presence of advanced

464

DENIS G. BASKIN AND DAVID W. GOLDING

larval stages in the coeloni. Furthermore, as reported by Smith (1950), the coelomocytes are abundant in the coelomic fluid of worms with mature oocytes, and the coelomocyte concentration does not decline until after cleavage stages are present in the coelom.

Another aspect of somatic maturation that is associated with the reproductive maturity of nereids is the loss of regenerative ability. Adult worms regenerate a pygidium but only rarely regenerate lost segments (Golding, 1967c). In order to determine if this aspect of maturation is delayed in N. limnicola, six specimens containing mature oocytes ( 130-160 /x in diameter), but no larvae, were kept for three weeks after removal of about one-half of their posterior segments. Each worm regenerated a pygidium but did not regenerate any segments. These results indicate that regenerative growth does not occur during the later stages of gamete maturation of N. limnicola.

TABLE I

The effect of decerebration on gamete maturation

	Experiment 1*	Experiment 2**
Group	Number of worms	Worms with larvae	Number of worms	Worms with larvae	Worms with motile sperm	Motile
						sperm
	Initial	Final	Initial	Final	Initial	Final	Initial	Final	Initial	Final	score
Decerebrate	15	14	0	12	18	18	0	16	3	17	3.2
Intact	15	12	0	6	18	17	0	2	2	11	2.1
Implant	15	14	0	1	18	18	0	4	1	11	1.6

* 19 days. ** 3 weeks.

Effect of decerebration on gamete maturation

Experiment 1. Forty-five animals were separated into three groups of equal size. A sample of coelomic fluid was obtained from each animal and both the maximum and mean oocyte diameter was determined for each worm. The absence of sperm was confirmed for each specimen. The three groups were subjected to the following treatments, respectively: Intact, brain left in situ; Decerebrate, brain removed ; Implant, brain removed and implanted into the coelom.

The groups were maintained under identical conditions. After 19 days, further samples of coelomic contents were obtained, and the presence or absence of larvae was noted. The results, summarized in Table I, showed that a significantly greater number of Decerebrate worms contained larvae than did the Implant group (Fisher, P < 0.005). The Intact and Implant groups also differed in this respect (Fisher, P < 0.05), but the difference between the Decerebrate and Intact groups was not significant.

Experiment 2. In a second experiment, the coelomic contents of 54 worms were examined and three groups of equal size were established as in experiment 1 : Intact, Decerebrate, and Implant. Worms at various stages of maturity were apportioned equally among the three groups. Each group was maintained in a

ENDOCRINOLOGY OF VIVIPAROUS NEREID 465

separate container under identical conditions. After eight days, a small sample of coelomic fluid was taken from each worm and examined for sperm. Three weeks after the initial sample, each worm was re-examined for oocytes, sperm and larvae.

The results of experiment 2 are shown in Table I. Clearly, significantly more specimens of the Dcccrebratc group contained larvae than either the Intact or Implant groups (Fisher, P < 0.001). There was no difference between the Implant: and Intact groups in this respect. Hence, the precocious appearance of coelomic larvae, which followed brain removal, did not occur if a brain had been implanted into the coelom of the decerebrate worm.

There was no difference among the three groups with respect to the presence of motile sperm after eight days. After three weeks, however, a greater propor- tion of individuals in the Decerebrate group had motile sperm as compared to the Intact and Implant groups (Fisher, P < 0.05). The average scores for quantity of sperm, as shown in Table I, indicate that the Decerebrate worms produced more sperm compared to the Implant worms ( Mann-Whitney, P < 0.01). Although the Decerebrate group also produced more sperm than did the Intact specimens, the scores were not significantly different. Nevertheless, the only strictly comparable groups are the Decerebrate and the Implant, and the results indicate that brain removal results in the production of abnormally more sperm as well as precocious sperm maturation.

Histology of the implanted brains

At the conclusion of experiment 2, the brain was recovered from the coelom of each worm in the Implant group. In each case, the ganglion, which was implanted at the level of the prostomium, was located in the posterior region of the body, and was floating unattached in the coelomic fluid. The brains were intact and showed no pronounced allometric changes, and the eyes were in a relatively normal position. Histological examination revealed that the infracerebral gland epithelium was normal in appearance. A comparison of the implanted and in situ brains revealed only subtle differences in the appearance of this epithelium (Fig. 1). There was the impression that, in the case of implanted brains, the b cells were more numerous, since they seemed to stain slightly more intensely. However, the differences were not evaluated quantitatively. The a cells did not exhibit unusual hypertrophy or atrophy, nor did they show significant storage or depletion of cytoplasmic inclu- sions, as compared with the in situ brains, although the a cells of implanted brains sometimes showed a slight peripheral deposition of granular material (Fig. 1A). Further, the implanted brains showed no unusual difference in the stainability of the cells and fiber tracts within the cerebral ganglion.

Effect of decerebration on viviparous development

Three groups of 13 worms each were established as described in the previous experiments : Decerebrate, Intact and Implant. A parapodium was removed from each worm for purposes of identifying individuals. Initially, all worms had devel- opmental stages present in the coelom ; the oldest stages present varied from gas- trulae to second-cirrus larvae. However, each group had about the same distribu-

DENIS G. BASKIN AND DAVID W. GOLDING

br

br

B

ENDOCRINOLOGY OF VIVIPAROUS NEREID

467

tion of developmental stages. Following the operations, all of the worms were maintained in the same dish containing aerated 25 % sea water at 18° C.

After 21 days, the worms were re-examined for larvae; the results are shown in Table II. Coelomic larvae were present in only 45% of the Intact group and 66% of the Implant group, whereas all survivors of the Decerebrate group con- tained larvae. The Decerebrate group thus had a greater proportion of individuals which still had coelomic larvae as compared to the Intact (Fisher, P C 0.01) and Implant (Fisher, P < 0.05) groups; there \vas no significant difference between the latter two groups. However, of those worms which contained larvae in each group, all had larvae and juveniles up to 30 setigers. No worms underwent parturition.

TABLE II The effect of decerebration on viviparous development

	Number of worms		Number of worms*
Group		Stages present at conclusion*
Initial	Final*	With larvae	Without larvae
Decerebrate	13	11	19-30 setigers	11	0
Intact	13	11	26-29 setigers	5	6
Implant	13	12	24-30 setigers	8	4

* After three weeks.

Effect of implantation of parts of brains on gamete maturation in decerebrate worms

After initially sampling the coelomic fluid contents of 76 worms, the following groups were established :

Decerebrate Brain removed (15 worms),

Intact Implant Brain excised and implanted into the coelom (16 worms), Dorsal Implant Dorsal part of the excised brain implanted (15 worms), Ventral Implant Ventral part of excised brain implanted (15 worms), Dorsal and Ventral Implant Brain cut and both the dorsal and ventral parts implanted (15 worms).

Initially, all worms contained oocytes, and the maximum oocyte diameters varied from 50 ^ to 139 p., but in each group the distribution of worms at various stages of maturity was similar. Sperm were not present in any of these worms. The brain was removed from each worm and, wrhere appropriate, cut in a horizontal plane in order to separate the ventral region, on which is located the infracerebral gland epithelium, from the dorsal part, in which are located many neurosecretory cells. The desired portions were then implanted into the coelom.

FIGURE 1. A. This infracerebral gland epithelium is from a brain recovered after three weeks of implantation into a decerebrate worm. B. This infracerebral gland epithelium is from a normal in situ brain ; br, brain ; leg, infracerebral gland epithelium a cells. The arrows point to b cells. (Both A and B : Helly, paraldehyde fuchsin, 500 X.)

468

DENIS G. BASKIN AND DAVID W. GOLDING

TABLE III

The effect of implanting parts of a brain on gamete maturation in decerebrate worms

	Number of worms	Coelomic fluid contents*
Group
	Initial	Final*	Oocytes	Larvae	Neither
Decerebrate	15	15	0	11	4
Intact implant	16	16	14	1	1
Dorsal implant	15	12	4	6	2
Ventral implant	15	14	3	9	2
Dorsal and ventral implant	15	15	6	9	0

* After 19 days.

After 19 days, each worm was examined for oocytes and larvae. The results are summarized in Table III. The Decerebrate group had significantly more worms with larvae than the Intact Implant group (Chi-square, P < 0.001). The Dorsal, Ventral, and Dorsal and Ventral Implant groups were similar in having several worms containing larvae, and thus resembled the Decerebrate group in this respect ; however, they differed from the Decerebrate group in that each of the former had several worms containing oocytes, but no larvae. The Intact Implant group had significantly fewer worms with larvae than did those of the Dorsal Im- plant and Ventral Implant groups (Chi-square, P < 0.01).

It is conceivable that differences could exist between the groups as to the developmental progress of individual worms, since all stages from cleavage to second-cirrus larvae are included in the category, "larvae." This possibility was examined by considering only the worms containing either oocytes or larvae (or both). Although larvae of several developmental stages (as well as oocytes) were often present in an individual, the most advanced stage present was considered an index of developmental progress. The most advanced stages present in individual worms of each group were tabulated and expressed as a percentage indicating the relative proportion of those individuals within each group (Table IV). The data suggest that the decerebrate worms were more advanced with respect to the devel- opmental progress of their coelomic larvae as compared to the other groups, and this impression is strengthened if the groups are compared as to the proportion of each group that reached selected developmental stages, as shown in Figure 2.

TABLE IV

The percentage of worms containing oocytes or larvae at selected developmental stages for each group in Table III

Group	Oocytes	Cleav- ages	Gast- rulae	Troch- ophore	3-segment larvae	Tentacle- bud larvae	2nd-cirrus larvae
Decerebrate	0	0	10	27	18	27	18
Intact implant	93	7	0	0	0	0	0
Dorsal implant	40	0	0	20	10	30	0
Ventral implant	26	8	33	8	17	0	8
Dorsal and ventral implant	40	0	0	13	27	20	0

ENDOCRINOLOGY OF VIVIPAROUS NEREID

469

u

GROUP

100

_

t£g Intact Implant

90

-

LiJ Decerebrate

KVI Dorsal Implant

80

-

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till Ventral Implant

:::::

^»«

L/4 Dorsal & Ventral

70

"

1 1 1

Implant

60

-

~

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~

1

H

^

50 40

-

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1

30

-

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"

1 n —

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-

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(

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Cleavage Gastrula Trochophore 3-Segment Tentacle-Bud 2nd-Cirrus

DEVELOPMENTAL STAGES

FIGURE 2. This figure compares the developmental progress of the groups in the brain- cutting experiment. The horizontal axis represents larval stages in sequence of development. The vertical axis represents the percentage of worms, in each group, containing larvae which had advanced to the stages indicated. The percentages are based on the worms that contained oocytes, larvae, or both, for each group, and are adapted from Table IV.

A greater proportion of the Decerebrate worms contained larvae which had pro- gressed to each of the selected developmental stages, as compared to the other four groups. Thus, of the worms considered, 90% of the Decerebrate group contained larvae which had progressed to the trochophore stage, as compared with 60% for the Dorsal Implant and Dorsal and Ventral Implant groups and 33% for the Ven- tral Implant group. The one worm which had larvae in the Intact Implant group contained cleavage stages ; the remaining members of this group contained oocytes. Although the actual numbers are somewhat small for comparison, the data indicate that the larvae of worms in the Decerebrate group showed the most developmental progress, the Intact Implant worms showed the least progress, and the three groups which received implants of the brain fragments were intermediate, in this respect. Of the latter three groups, the Ventral Implant showed the least developmental progress, since a smaller proportion of its members had larvae which had progressed to trochophore or later stages.

DISCUSSION

In many nereids, gametic maturity is accompanied by a profound somatic meta- morphosis into an epitokous swimming form known as a Heteronereis (Clark,

470 DENIS G. BASKIN AND DAVID W. GOLDING

1961). However, even in species that reproduce in the atokous form, several somatic changes are correlated with reproductive maturation. Dales (1950) has described these changes for N. divcrsicolor, an atokous, oviparous species. The coelomocytes fill the body cavity during the early phase of sexual development and almost disappear during the final stages of gametic maturation as the coelom becomes occluded with mature oocytes. Histolysis and dedifferentiation of the musculature have rendered the body wall thin and fragile at the time of spawning.

N. Hvnnicola, in common with N . divcrsicolor, reproduces in the atokous form. However, the temporal relationship between events characteristic of somatic and gametic maturation differ significantly in the viviparous N. limnicola from the general nereid pattern. Histolysis of body musculature does not coincide with the maturation of the gametes. Furthermore, the coelomocytes fill the coelomic cavity of worms containing mature gametes and the coelomocyte concentration does not decrease until after cleavage stages are present in the coelom (Smith, 1950). The body musculature undergoes histolysis during the period when advanced larval stages are present in the coelom. The coelomocyte concentration decreases sig- nificantly by the time of parturition. Thus, the muscle histolysis and the reduction of coelomocytes are delayed to a significant extent with respect to the maturation of the gametes, and occur in correlation with the intracoelomic development of the offspring rather than in association with the final stages of gamete maturation, as is the case with oviparous nereid species.

In N. limnicola the gametic and somatic aspects of maturation are out of phase, and this shift can be interpreted in the context of present views of the endocrine integration of nereid reproductive development. Both somatic and gametic maturational changes occur simultaneously in other nereid species, and the declining titer of a single inhibitory hormone is thought to control both processes (reviews by Clark, 1965; Durchon, 1967). The lack of synchrony between these processes in N. limnicola raises the possibility that the gametic and somatic aspects of matura- tion may be controlled by separate inhibitory hormones which are withdrawn simul- taneously in an oviparous nereid but withdrawn sequentially in N. limnicola, a viviparous species. An alternative and more plausible explanation would attribute this lack of synchrony to different sensitivities of the respective processes to the declining titer of a single inhibitory factor. Thus, somatic maturation may have become more sensitive to the lowr hormone levels at sexual maturity and thus be delayed with respect to gamete maturation, or the gametes may have developed less sensitivity to the inhibitory hormone and mature precociously with respect to the onset of the somatic changes. It is not possible to decide on one of these alternatives on the basis of present knowledge.

The condition of having the somatic and gametic maturational changes occur out of phase is interpreted as a variation of a basic endocrine control mechanism found in oviparous nereids. In the case of N. limnicola, this control is adapted as a specialization for a viviparous mode of reproduction. Thus, the endocrine inte- gration of the process of muscle histolysis, which prepares the sexually mature oviparous nereid for spawning, is modified in N. limnicola to prepare the worm for parturition.

With respect to hormonal control of gamete maturation, N. limnicola does not appear to depart from the general nereid pattern. The two decerebration experi- ments demonstrate that brain removal results in the premature appearance of larvae

ENDOCRINOLOGY OF VIVIPAROUS NEREID 471

in the coelom. In experiment 1, the only two strictly comparable groups are the Decerebrate and Implant, and these give clear indication of an inhibitory influence on gamete maturation and, therefore, indirectly on larval production. The differ- ence between the Intact and Implant groups is difficult to interpret, although com- parable differences have been reported with respect to the progress of regeneration in N. diversicolor with intact and implanted brains (Golding, 1967a). This differ- ence is not confirmed by the larger scale experiment 2, but this latter experiment does confirm the results of experiment 1 which demonstrate that the supraesophageal ganglion exerts an inhibitory endocrine control over maturation of the gametes.

Numerous studies have shown that gamete maturation is inhibited by a hor- mone presumably secreted by the brain, and this inhibition has been described in males (Durchon and Schaller, 1964; Malecha, 1967) and females (Clark and Ruston, 1963; Durchon and Dhainaut, 1964; Durchon and Boilly, 1964; Hauen- schild, 1966; Dhainaut and Porchet, 1967) among several species of oviparous nereids. It is not surprising, therefore, that hormonal inhibition of both sperm and oocyte maturation occurs simultaneously in N. limnicola, a self-fertilizing hermaphrodite in which the eggs and sperm develop simultaneously within the coelom of an individual. The maturation-inhibiting hormone is considered identical to the endocrine factor necessary for regeneration of immature worms (Golding, 1967c). Therefore, the observation that sexually-mature N. limnicola does not regenerate is further evidence that, relative to immature worms, the titer of the maturation-inhibiting hormone is low in worms with mature gametes.

There has been some doubt regarding the viability of oocytes whose growth had been accelerated in response to decerebration, since previous workers have failed to obtain normal development following fertilization of these oocytes (Choquet, 1962; Clark and Ruston, 1963). However, the present results demonstrate that the precociously-mature oocytes of N. liinnicola, produced in response to decere- bration, are capable of normal development. It is interesting that normal, mature oocytes of N. limnicola resemble the abnormal oocytes obtained by Clark and Ruston (1963) following decerebration of N. diversicolor. In both cases, the oocytes lack the dense accumulation of yolk droplets characteristic of most nereid oocytes, which are normally fertilized externally. However, in N. limnicola these oocytes are fertilized in the coelom, and the coelomic milieu undoubtedly provides the nutrients and other factors necessary for the successful growth and development of the embryos. It has been suggested that the infracerebral gland a cells may be involved in this respect (Baskin, 1970).

Smith (1950) reported that the oocytes of N. limnicola (from a different popu- lation than those used in this study) normally matured at a diameter of 120-170 ju,, and our observations indicated that in the worms from Lake Merced, they were fertilized at about 150-160 /A. In the Decerebrate group of experiment 2, the oocytes of worms whose largest oocytes were between 73 and 128 /A underwent accelerated growth as evidenced by the precocious appearance of larvae in the coelom. It could not be determined, however, whether the oocytes had attained a normal size at fertilization, or whether they were fertilized at a smaller diameter. One decerebrate worm, whose largest oocyte were initially about 86^, had 148 ^ oocytes after three weeks. Many of the worms which contained early embryonic stages also had oocytes of 140-160 /A. These observations suggest that the oocytes accelerated growth to a relatively normal size before being fertilized, rather than

472 DENIS G. BASK IN AND DAVID W. GOLDING

being fertilized at a smaller diameter. It would be of inteerst to know how early the oocytes of this species can be accelerated by decerebration and still be capable of normal development.

Decerebration of worms with coelomic larvae did not adversely affect the vivi- parous embryonic development and growth. In fact, the Decerebrate group had a greater proportion of worms containing larvae as compared to the Intact and Im- plant groups. However, the number of larvae produced by each worm was not recorded, and it is thus conceivable that the groups differed as to the number of larvae produced per worm. The significance of not finding larvae in some of these worms at the conclusion of the experiment is not understood. It is unlikely that the adults underwent parturition, since this event is catastrophic and the adult usually dies. The worms were not fed during the experiment, and it is possible that the larvae were resorbed, since larval resorption has been previously reported in this species (Smith, 1950). Although this question is not resolved, the phe- nomenon does not alter the general conclusion that larval development per se does not require the presence of the cerebral ganglion of the adult.

The results of the experiments in which parts of a brain were implanted into decerebrate worms are significant in view of the problems regarding the presumed source of the cerebral endocrine activity. Previous experimental work has localized this endocrine activity to the posterior region of the cerebral ganglion (Durchon and Dhainaut-Courtois, 1964; Hauenschild, 1966; Golding, 1967b), and attention has been focused upon presumed hormonogenic neurosecretory cells. In contrast, Golding, Baskin and Bern (1968) and Dhainaut-Courtois (1968a) have suggested that the infracerebral gland, which is located on the postero-ventral surface of the ganglion, may fulfill this endocrine role. The present histological observation that the infracerebral gland epithelium was present and normal in appearance on im- planted cerebral ganglia that had been secreting the maturation-inhibiting hormone, is compatible with the latter view.

Parts of a brain were implanted into decerebrate hosts to determine whether the source of the maturation-inhibiting hormone is associated with the dorsal region, in which are located many neurosecretory cells, or the ventral region, on which is located the infracerebral gland epithelium. Of the three groups which contained implanted brain fragments, developmental progress was essentially similar in each. As compared to the Dorsal Implant and Dorsal and Ventral Implant groups, the Ventral Implant group showed slightly less developmental progress, which might indicate that the ventral region of the brain exerts a stronger inhibi- tory effect on maturation. But an equivalent effect was not obtained when a ventral and dorsal part were implanted together ; thus, any conclusions about a differential influence of the dorsal and ventral regions, respectively, are considered tentative.

The inhibitory effect of the brain fragments was not equivalent to that of an intact ganglion, even where dorsal and ventral fragments of the same brain were implanted together. But neither wrere the fragments totally without effect, since all three groups which received an implanted brain fragment showed evidence of developmental retardation when compared to the decerebrate group. This retarda- tion is considered an indirect effect stemming from the inhibition of oocyte matura- tion, and does not suggest a direct inhibitory influence on larval development.

ENDOCRINOLOGY OF VIVIPAROUS NEREID 473

Although this experiment did not localize the source of the hormone, the results are consistent with the hypothesis that neurosecretory cells of the brain and the cells of the infracerebral gland epithelium may form a functional unit or system for the production of the maturation-inhibiting principle. The physical integrity of this system was apparently disrupted by transecting the brain so as to separate the infracerebral gland epithelium from its neurosecretory component, resulting in the reduction, but perhaps not complete absence, of hormone secretion.

This interpretation does not conflict with the view that the infracerebral gland cells may be endocrine, although their significance in this respect is incompletely understood. While the a cells of the infracerebral gland epithelium have been compared in their ultrastructure to that of endocrine cells of the insect corpus allatum (Dhainaut-Courtois, 1968a), it has been concluded elsewhere that the a cells are probably not the source of known endocrine principles (Baskin, 1970). However, the granule-filled b cells, which resemble protein-secreting gland cells in their ultrastructure, are most differentiated in immature worms and degenerate in older worms (Dhainaut-Courtois, 1968a). Since this degeneration parallels the disappearance of the maturation-inhibiting hormone, it is tempting to speculate that the b cells may be associated with the secretion of this hormone.

The functional significance of the infracerebral gland epithelium and its relation- ship to neurosecretory activity of the brain remain to be elucidated. Nevertheless, future investigations on nereid endocrinology must consider the possible importance of this system to neuroendocrine regulation in these worms.

The authors are grateful to Dr. Ralph I. Smith, Dr. Howard A. Bern, and Dr. Rudolph L. Pipa for their support, help, and critical discussions with various aspects of the study and manuscript.

SUMMARY

1. The relationship of gametic and somatic maturation in the atokous polychaete, Nereis liinnicola, differs significantly from the general nereid pattern. In this viviparous species muscle histolysis and the decrease in the coelomocyte concentra- tion are delayed with respect to the onset of gamete maturation, and occur, instead, in association with the intracoelomic development of the offspring. However, worms with mature gametes are unable to regenerate lost caudal segments.

2. The cerebral ganglion of N. liinnicola exerts an inhibitory influence on the maturation of sperm and oocytes. Brain removal results in the precocious appear- ance of normal larvae in the coelom. This effect is prevented by implanting a brain into the coelom of the decerebrate worms.

3. Histological examination of brains that had been implanted for three weeks revealed that the infracerebral gland epithelium was present and normal in appearance.

4. Decerebration of worms containing coelomic larvae did not delay or accel- erate larval development.

5. The effect of implanting parts of brains on gamete maturation of decerebrate worms was studied, and the results indicated that the dorsal part of the brain, which contains many neurosecretory cells, and the ventral part of the brain, on

474 DENIS G. BASKIN AND DAVID W. GOLDING

which is located the infracerebral gland epithelium, had approximately equivalent inhibitory activity when implanted alone or together, but this inhibitory effect on oogenesis was not as great as that produced by an implanted intact brain. These results are interpreted to mean that the infracerebral gland epithelium and neuro- secretory cells of the brain may form a neuroendocrine system, the integrity of which is essential for secretion of the maturation-inhibiting hormone at normal levels.

LITERATURE CITED

BASKIN, D. G., 1970. Studies on the infracerebral gland of the polychaete annelid, Nereis liin-

nicola, in relation to reproduction, salinity and regeneration. Gen. Comp. Endocrinol.,

in press. BERKELEY, E., AND C. BERKELEY, 1953. Micronereis nanaimoensis n. sp. : with some notes on

its life history. /. Fish. Res. Board Can., 10: 85-95. CHOQUET, M., 1962. Effet inhibiteur de 1'hormone cerebrale sur 1'evolution des cellules sexuelles

chez Nereis pelagica L. (Annelide Polychete). C. R. Soc. Biol., 156: 1112-1114. CLARK, R. B., 1955. The posterior lobes of the brain of Nephtys and the mucus glands of the

prostomium. Quart. J. Microscop. Sci., 96: 545-565.

CLARK, R. B., 1961. The origin and formation of the heteronereis. Biol. Rev., 36: 199-236. CLARK, R. B., 1965. Endocrinology and the reproductive biology of polychaetes. Oceanogr.

Mar. Biol. Ann. Rev., 3: 211-255. CLARK, R. B., 1969. Endocrine influence in annelids. Gen. Comp. Endocrinol., Suppl. 2: 572-

581. CLARK, R. B., AND R. J. G. RUSTON, 1963. The influence of brain extirpation on oogenesis in

the polychaete Nereis diver sicolor. Gen. Comp. Endocrinol., 3: 529-541. DALES, R. P., 1950. The reproduction and larval development of Nereis diversicolor O. F.

Miiller. /. Mar. Biol. Ass. U. K., 29: 321-360. DEFRETIN, R., 1949. Recherches sur la musculature des Nereidiens au cours de 1'epitoquie, sur

les glandes parapodiales et sur la spermatogenese. Ann. Inst. Oceanogr., 24: 117-257. DHAINAUT, A., AND M. PORCHET, 1967. Evolution ovocytoire en 1'absence d'hormone cerebrale

chez Perinereis cultrifcra Grube (Annelide Polychete). C. R. Acad. Sci. Paris, 264D :

2807-2810. DHAINAUT-COURTOIS, N., 1968a. Contribution a 1'etude du complexe cerebrovasculaire des

nereidiens. Cycle evolutif des cellules infracerebrales de Nereis pelagica L. (Anne- lide Polychete) ; etude ultrastructurale. Z. Zellforsch., 85: 466-482. DHAINAUT-COURTOIS, N., 1968b. fitude histologique et ultrastructurale des cellules nerveuses

du ganglion cerebral de Nereis pelagica L. (Annelide Polychete). Comparaison entre

les type cellulaires I-IV et ceux decrits anterieurement chez les Nereidae. Gen. Comp.

Endocrinol., 11: 414-443. DURCHON, M., 1952. Recherches experimentales sur deux aspects de la reproduction chez les

Annelides Polychetes : L'epitoquie et la stolonisation. Ann. Sci. Natur. Zool., 14:

118-206. DURCHON, M., 1956. Role du cerveau dans la maturation genitale et le declenchement de

1'epitoquie chez les nereidiens. Ann. Sci. Natur. Zool., 18: 269-273.

DURCHON, M., 1967. L'Endocrinologie des Vers ct des Mollusqucs. Masson, Paris, 241 pp. DURCHON, M., AND B. BOILLY, 1964. fitude ultrastructurale de Pinfluence de 1'hormone cere- brale des Nereidiens sur le developpement des ovocytes de Nereis diversicolor O. F.

Miiller (Annelide Polychete) en culture organotypique. C. R. Acad. Sci. Paris, 259:

1245-1247. DURCHON, M., AND A. DHAINAUT, 1964. Influence de 1'hormone cerebrale des nereidiens sur

la croissance des ovocytes. fitude en culture organotypique. C. R. Acad. Sci. Paris,

259: 917-919. DURCHON, M., AND N. DHAINAUT-COURTOIS, 1964. Sur la localisation du centre hormonale

inhibiteur de la maturation genitale male dans le cerveau de Nereis diversicolor O. F.

Miiller. (Annelide Polychete). C. R. Soc. Biol., 158: 550-554.

ENDOCRINOLOGY OF VIVIPAROUS NEREID 475

DURCHON, M., AND M. PORCHET, 1970. Dosage de 1'activitie endocrine cerebrale au cours du

cycle genital femelle chez Nereis dii'crsicolor O. F. Mailer (Annelide Polychete).

C. R. Acad. Sci. Paris, 270D: 1689-1691. DURCHON, M., AND F. SCHALLER, 1964. Rechcrches endocrinologiques en culture organotypique

chez les annelides polychetes. Gen. Comp. EndocrinoL, 4: 427-432. GABE, M., 1966. Neurosecretion. Pergamon, New York, New York, 872 pp. GOLDING, D. W., 1967a. Neurosecretion and regeneration in Nereis. I. Regeneration and the

role of the supraesophageal ganglion. Gen. Comp. EndocrinoL, 8 : 348-355. GOLDING, D. W., 1967b. The diversity of secretory neurones in the brain of Nereis. Z. Zell-

forsch.,82: 321-344. GOLDING, D. W., 1967c. Endocrinology, regeneration and maturation in Nereis. Biol. Bull.,

133: 567-577. GOLDING, D. W., D. G. BASKIN AND H. A. BERN, 1968. The infracerebral gland — A possible

neuroendocrine complex in Nereis. J. Morphol., 124: 187-216. GOULD, M. C., AND P. C. SCHROEDER, 1969. Studies on oogenesis in the polychaete annelid

Nereis grubei Kinberg. I. Some aspects of RNA synthesis. Biol. Bull., 136: 216-255. JOHNSON, M. W., 1943. Studies on the life history of the marine annelid Nereis vcxillosa.

Biol. Bull, 84: 106-114. HAUENSCHILD, C., 1965. Die endokrine Steuerung de Fortpflanzung bei Anneliden. Arch.

Anat. Microscop. Morphol. Exp., 54: 429-452. HAUENSCHILD, C., 1966. Der hormonale Einfluss des Gehirns auf die sexuelle Entwicklung bei

detn Polychaeten Platyncrcis dumerilii. Gen. Comp. EndocrinoL, 6: 26-73. MALECHA, J., 1967. Transformation heteronereidienne et gametogenese chez Nereis sitccinea

(Leukart) (Annelide Polychete). C. R. Acad. Sci. Paris, 265D: 613-615. OGLESBY, L. C., 1965. Steady-state parameters of water and chloride regulation in estuarine

nereid polychaetes. Comp. Biochcm. Physio!., 14: 621-640. REISH, D. J., 1954. The life history and ecology of the polychaetous annelid Nereis grubei

(Kinberg). Allan Hancock Found. Occas. Papers, 14: 1-75. REISH, D. J., 1957. The life history of the polychaetous annelid Neanthcs caudata (delle

Chiaje), including a summary of development in the family Nereidae. Pacific Sci.,

11: 216-228.

SCHROEDER, P. C., 1967. . Morphogenesis of epitokous setae during normal and induced meta- morphosis in the polychaete annelid Nereis grubei (Kinberg). Biol. Bull., 133: 426-

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from California. Pacific Sci., 22: 476-481. SIEGEL, S., 1956. Nonparainetric Statistics for the Behavioral Sciences. McGraw Hill, New

York, 312 pp. SMITH, R. I., 1950. Embryonic development in the viviparous nereid polychaete, Ncanthes

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SMITH, R. L, 1958. On reproductive pattern as a specific characteristic among nereid poly- chaetes. Syst. ZooL, 7 : 60-73.

Reference : Biol. Bull., 139: 476-484. (December, 1970)

INTERACTION OF FOOD AND PHOTOPERIOD IN THE TERMI- NATION OF LARVAL DIAPAUSE IN CHAOBORUS AMERI- CANUS (DIPTERA: CULICIDAE) 1

WILLIAM E. BRADSHAW2

The University of Michigan, Ann Arbor, Michigan 48104

The role of food in the termination of diapause is probably without significance in the many diapausing insects which are inactive and which seldom, if ever, feed. There are, however, certain nematocerous Diptera which overwinter as larvae and are capable of both movement and feeding. Indeed, studies concerning the photo- periodic induction of development in diapausing larvae of the chironomids Metrioc- ncinus (Paris and Jenner, 1959) and Chironomus (Engelmann and Shappirio, 1965) or of the culicids Anopheles barberi (Baker, 1935), Anopheles bifurcatus (Vinogradova, 1964), Anopheles plumb eus (Vinogradova, 1962), Aedes triseriatus (Love and Whelchel, 1955), and Toxorhinchites rutilits (McCrary and Jenner, 1965) involved the effects of photoperiod on fed animals only. Studies concerning the overwintering larvae of the culicid Chaobonts americanus (Bradshaw, 1969) considered the effects of photoperiod on both fed and starved animals. The assess- ment of the contribution of food is difficult, if not impossible, in filter feeders or detritus eaters like the chironomids or the culicine and anopheline mosquitoes. Chaoborine and toxorhinchitine mosquitoes, on the other hand, are carnivores. In Chaoborus, food and photoperiod have been shown to interact synergistically to effect the termination of larval diapause (Bradshaw, 1969). Furthermore, develop- ment in Chaobonts is proportional to the number of long days with food. Chao- borus, therefore, is conveniently adapted for the study of the food component in the termination of diapause.

MATERIALS AND METHODS Animals and general conditions

All experiments in this research involve large yellow larvae of Chaoborus ameri- canus Johannson caught in George Pond, a small kettle hole in the center of the University of Michigan's George Reserve, near Pinckney, Michigan, during Janu- ary and February, 1967 and 1968. The larvae were transferred on the day of capture to maintenance conditions of short day (8L, 16D) and 5° C without food. Large numbers of larvae were kept in open gallon jars under these conditions until the starting date of the experiment at which time they were removed, the large yellow larvae, known to be the fastest responding morphs in the population (Brad- shaw, unpublished observations), were sorted from this stock, warmed to 25° C,

1 Aided by N I H Grant 5T01-GM00989.

2 Present address : Harvard Biological Laboratories, 16 Divinity Avenue, Cambridge, Massachusetts 02138.

476

INTERACTION OF FOOD AND PHOTOPERIOD

477

and placed on the experimental conditions outlined below. Food in all cases con- sisted of laboratory larvae of the mosquito, Culex pipiens, provided in saturating amounts unless otherwise stated. All the experiments were run at 25° C under either long day (16L, 8D), or short day (12L, 12D) conditions.

Statistical methods

Standard methods (Snedecor, 1956, Ch. 6, 7) were used to derive regression lines, regression coefficients (r), and the probabilities of correlation (P). Per- centage data were also analyzed by computation of the normal variate as outlined by Ractliffe (1967, pages 136-137).

Comparisons of sample means were made either by the standard t test or by a modification of Duncan's Multiple Range Test (Duncan, 1955). Analysis of variance was carried out with raw data only when appropriate as demonstrated by Bartlett's test for homogeneity of variance (Snedecor, 1956, pages 285-289) ; other- wise, arcsin-per cent-square-root or log transformations were tried until homo- geneity of variance was achieved. If the value obtained for F was significant at the 5% level of confidence, then a variation of Duncan's Multiple Range Test was employed to find significant differences between means (D}. In the present study, instead of ranking n means according to their magnitudes, Duncan's Studentized value for comparison at the nth rank is used to derive the significant difference (D). This method is more conservative than either the commonly used Least Significant Difference or Duncan's original method but embodies the convenience of the former.

EXPERIMENTAL RESULTS Effect of various combinations of food and daylength on the large yellow larvae

Large yellow larvae were exposed to short day without food starting 2-2-68 and 3-31-68, to long day without food starting 2-2-68 to 5-11-68 (5 replicates), to short day with food starting 2-2-68 to 5-11-68 (6 replicates), and to long day with food starting 2-2-68 to 5-11-68 (4 replicates). They were exposed to experimental conditions for at least 30 days unless all larvae had either developed or died prior to that time.

As shown in Table I, short day without food failed to elicit any substantial pupation in the large yellow larvae. Either long day without food or short day

TABLE I

Developmental response of large yellow larvae to various combinations of food and daylength in n replicates of 50 larvae each (mean per cent ± standard deviation)

	Per cent Pupation	n
After 15 days	After 30 days
Long day fed Long day starved Short day fed Short day starved	81.0 ± 11.2 6.0 ± 4.4 14.7 ± 13.8 0.0 ± 0.0	89.8 ± 2.7 6.4 ± 4.1 16.3 ± 13.4 0.0 ± 0.0	4 5 6 2

478 WILLIAM E. BRADSHAW

with food elicited some pupation but never more than 20% for the former or 40% for the latter. Long day with food, on the other hand, elicited substantial pupation, even after only 15 days. Thus, while either food or long day photoperiod is neces- sary for some development, a substantial response is not evoked unless the food and long day stimuli occur simultaneously. It would further appear that 90% of all potential development is realized after only 15 days experimental time. For rea- sons of convenience, 15 days was therefore chosen as the standard assay period to be used in subsequent experiments.

Effect of daylength on total amount of food consumed

On 4—18-68, two groups of 50 diapausing larvae were placed in ^ oz jars, one larva per jar. One group was placed on long day, the other on short day. Each Chaoborus larva was offered twelve Culex per day for five days. Thereafter, the food was removed and the Chaoborus observed for ten additional days. Develop- ment was then scored and the experiment terminated. To increase homogeneity of food particle size, only those mosquito larvae which passed through a 2 X 2 mm mesh but not a 1 X 1 mm mesh were used. Each day, the number of mosquito larvae eaten was tabulated, the remainder removed, and twelve fresh larvae added.

Of the 60 mosquitoes offered, each Chaoborus larva ate an average of 30 on long day and 32 on short day. On long day, of 45 surviving individuals, the 44 pupating larvae ate an average of 30 mosquitoes while the only non-developing larva ate 36. On short day, of 49 surviving individuals, both the 19 pupating and the 30-non-developing larvae ate an average of 32 mosquitoes. Daylength, there- fore, has little or no effect on the amount of food consumed.

Effect of food or long day on sustaining development

The following experiments are designed to determine whether long day alone or food alone following long day with food is stimulatory, neutral, or inhibitory with respect to development induced by long day with food. A series of 10 oz jars was each provided with 50 diapausing larvae. These groups of larvae were then exposed to 0, 1, 2, 3, or 4 long days with food after which time they were placed on short day without food (experiment started 4—18-68 and replicate started 5-11- 68), on long day without food (experiments started 4—18 — 68 and replicate started 5-11-68), or on short day with food (experiment started 5-11-68) for a total of 15 days experimental time after which development was scored and the experiment terminated.

In all experiments, there was a significant correlation between the number of days exposure to long day with food and the amount of subsequent development (Fig. 1). All exposures to one long day with food elicited more development than the control, i.e., 15 short days without food (Fig. 1A), 15 long days without food (Fig. IB), or 15 short days with food (Fig. 1C). Furthermore, a significant difference was noted between the responses to any two day increment in exposure to long day with food, i.e., 3 or more days exposure to long day with food elicited significantly more development than 1 ; 4 days elicited more than 2 or 1, etc. (Duncan's Multiple Range Test after arcsin transformation : F = 47.67 ; P < 0.01 ; D= 11.0).

INTERACTION OF FOOD AND PHOTOPERIOD

479

After subtracting out the control values, there was no significant difference be- tween the amount of development elicited by short day without food, long day with- out food, or short day with food following long day with food (analysis of variance: F = 1.2; P > 0.25). It would thus appear that neither long day nor food has any ability to retard or sustain development once it has been initiated.

r a b

80^

z

O 60-

40-

20 —

.91 * 1.6 1 2.8

.93 *

9.6

1 2.6

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16.8 21.6

1 3.0 9.6

(A) SD-S

(B) LD-S

.9 8 *

7.6

1 7.2

(C) SD-F

_J

01234 01234 01234

DAYS LONG DAY FED

FIGURE 1. Ability of long day and food to sustain development. Diapausing larvae were exposed to long day with food for 0-4 days after which they were placed on short day and starved (A), long day and starved (B), or short day and fed (C), in experiments starting 4-18-68 (solid line) or 5-11-68 (shaded outline). Abbreviations are: r, coefficient of correla- tion between days long day fed and and per cent pupation ; *, significant correlation ; ** highly significant correlation ; a and b, constants in the regression equation : % pupation = a + b (days long day fed) ; SD, short day; LD, long day; F, fed; S, starved.

Persistent effects of long day

This experiment was designed to test whether previous exposure to long day without food would enhance the developmental response of larvae subsequently exposed to short day with food. Diapausing larvae were placed in a series of 10 oz jars, 50 animals to a jar. They were then exposed to long day without food for 0 (control), 1, 2, 3, 4, 5, or 6 days after which they were transferred to short day and provided an excess of food for a total experimental time of 15 days after which development was scored and the experiment was terminated. The initial experi- ment starting 5-11-68 was repeated with fresh animals starting 6-1-68.

Unlike the response to long day with food (Fig. 1), there was no correlation between the duration of exposure to long day without food and the amount of development elicited (Fig. 2, solid line) (after subtracting out the control values: r — 0. 14; P > 0.85 for 10 df). While there were no significant differences among responses elicited by 1, 2, 3, 4, 5, and 6 days exposure to long day without food,

480

WILLIAM E. BRADSHAW

2 and 6 days exposure elicited a significantly greater amount of pupation than did the control (Duncan's Multiple Range Test after arcsin transformation: F — 5.00; P < 0.05; D --- 12.8%). These results suggest the photoperiodic information may be accumulated to some extent by C. americanus but that the synergistic interaction of food and photoperiod requires both parameters simultaneously rather than sequentially.

Persistent effects of food

The following experiments reciprocate the ones above and are designed (1) to test whether trophic information is stored by C. americanus and (2) to substantiate the inference that the synergistic interaction of food and long day requires simul- aneous input. For this purpose, diapausing larvae were placed in a series of 10 oz jars, 50 animals to a jar. They were then exposed to short day with an excess of food for 0 (control), 1, 2, 3, 4, 5, or 6 days after which time the food was removed and the Chaobonts larvae transferred to long day conditions without food for a total experimental time of 15 days. The initial experiment starting 4-18-68 was repeated in duplicate, both of the latter experiments starting 5-11-68.

50-

DAYS

123456 SD-F BEFORE LD-S '//////////,

LD-S BEFORE SD-F

J

FIGURE 2. Developmental response of larvae on short day with food after 0-6 days exposure to long day without food (average of two replicates) and of larvae on long day without food after 0-6 days exposure to short day with food (average of three replicates) ; ordinate, per cent pupation ; other abbreviations as in Figure 1.

As in the case of long day followed by food, there was no correlation between the duration of exposure to food and the amount of development elicited (Fig 2, diagonal shading) (after subtracting out the control values: r = 0.07; P > 0.99). On the other hand, all of the experimental values were significantly greater than the controls, even though they were not significantly different from each other (Duncan's Multiple Range Test after arcsin transformation: F = 3.03; P < 0.05; D = 13.2%). These results confirm the concept that the synergistic response to food and long day requires simultaneous rather than sequential input of these factors.

Inspection of the larval mortality (Fig. 3A, solid dots) and pupal survivorship (Fig. 3B) after 15 days experimental time reveals that both are highly significantly

INTERACTION OF FOOD AND PHOTOPERIOD

481

correlated with the number of days feeding on short day. This observation indi- cates that food is stored — as long as 15 days — and may be called upon to support larval life or adult development.

The following experiments, designed to further elucidate the long-term effects of food, employed the duplicate experiments started on 5-11-68 as described above. At the end of 16 days total experimental time, the duplicates were split into two groups of 7 experimental populations each, having received from 0 to 6 days prior feeding. The sample size in each experimental population then consisted of that number of larvae alive and undeveloped at the end of 16 days. One group was given a one day food pulse, the other a four day food pulse, both under long day conditions. After pulse, the larvae remained on long day without food until a total of 31 days experimental time had elapsed. Larval mortality, pupal survivorship, and amount of development were then scored and the experiment terminated.

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(A)

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r = .89 P<.01 --D 1 r = .84 P<.05 —04

01 23456

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DAYS PRIOR FEEDING

FIGURE 3. Larval mortality (A) and pupal survivorship (B-C) after 0-6 days exposure to food on short day followed by continuous long day without food. Percentages after 15 days experimental time are shown by solid dots •. On day 16, duplicate experimental groups were provided a one day (Q) or a four day (O) food pulse on long day. The food was removed after this time and the larvae again placed on long day without food. Per cent larval mortality and per cent eclosion were scored on the 31st day, using the number of living, undeveloped larvae on day 16 as the sample size.

Among both the one day and four day food pulsed larvae, there was a significant correlation between prior feeding ten to thirty days beforehand and larval mortality (Fig. 3A) or pupal survivorship (Fig. 3C). Development in response to a one day or a four day food pulse also appeared to be proportional to the number of days prior feeding when per cent response was calculated on the basis of the number of larvae alive at the initiation of the food pulse experiments (i.e., the 16th day). But. if percentage development is calculated on the total number of this sample size surviving until the 31st day (n = number of living larvae + number of pupae), then development was no longer proportional to the number of days prior feeding (r — 0.47 ; P > 0.05). It would thus appear that food stored from remote feeding is capable of maintaining larval life or sustaining adult development but does not contribute to the termination of diapause in C. americanus.

482 WILLIAM E. BRADSHAW

DISCUSSION

Food very clearly is accumulated and stored by the larvae ; the effects of this stored food are readily seen contributing to larval survivorship (Fig. 3A) and to adult development (Fig. 3B, C). Yet, these same nutritional reserves do not enable the larvae to respond developmentally to long day in the absence of con- tinued feeding. The apparent inconsistency is easily explained by ascribing a dual role to food in the developmental process. First, food is functioning as an energy source ; second, food, or feeding, is acting as a physiological trigger to initiate development. Without long day photoperiod, food still increases the nutritional plane of the larvae but is unable to trigger substantial development. Thus, larvae exposed to food on short day and then exposed to long day without food do not show an increasing response related directly to the number of days prior feeding, nor do they show a food independent synergistic response to long day without food after a certain "critical number" of days prior feeding. Moreover, when careful comparison of food intake is made, it becomes clear that the above failure to respond to feeding on short day is not due to a short day repression or long day stimulation of feeding.

The simplest nutrition-independent explanation of food as a physiological trigger would be stimulation of a neuroendocrine reflex by food as an environmental cue. This model is not novel in endocrine control of development but is paralleled by the control of moulting in the bugs Rhodnius (Wigglesworth, 1933) and Cime.r (Kemper, 1931). In Rhodnius, the moulting stimulus is initiated by stretch receptors on the intersegmental muscles of the abdomen which is sizeably distended after a blood meal (Wigglesworth, 1934). Undoubtedly, Rhodnius derives nutri- tion from these blood meals since (1) the moult occurs within a constant time interval after a normal blood meal, regardless of the duration of the fasting period prior to the meal and (2) the animal may continually consume small amounts of blood and yet refrain from moulting indefinitely. The important point is that the induction of moulting by food is effected by independently of these nutritional benefits.

If food in Chaobonis is also acting via a neuroendocrine reflex independently of nutritional benefits, then not only should the persistent effects of food stimulation be of short duration as seen above (Fig. 2), but should also be of the same order of magnitude as the persistent effects of long day stimulation. The response of animals exposed to 1 to 6 long days without food before exposure to short day with food bears out this argument (Fig. 2, solid line).

A paradox remains both in the experiment exposing animals to short day with food before long day without food, and in the experiment exposing animals to long day without food before short day with food : some or all experimental animals respond significantly more than do the controls, even though both food and long day only interact synergistically when they occur simultaneously This problem may be clarified if one ascribes a physiological latency to both food and long day. Such a latency is not especially new since diapause in many insects may be cued by stimuli perceived in previous instars or even the previous generation. Thus, in the present case, while the experimental design implies that diapausing larvae are receiving 1-6 clays feeding prior to long day, a lag in the larvae themselves gives the physiological impression that food is being perceived from 2-7 days. In all the experimentals, but not in the controls, there will then be one day's overlap during

I\TKK\( TIOX ()K FOOD AND PH( >T< H'KKK )l >

which the larvae receive hnth the latent food stimulus and the newly impressed long day stimulus; hence, one day's worth of synergistic response.

Essentially, this model defines the period of physiological latency as that time during which synergistic interaction of food with long day is taking place. Thus, the actual duration of food latency is the number of days on long day with food necessary to induce development equal to the average development resulting from 1-6 days feeding on short day before exposure to long day without food. The latter value is 29(/< development for the experiment starting 4-18-68 and 32% development for the experiment starting 5-11-68. The regression equations for development as a function of day.s long day fed before long day starved are (for experiments started 4-18-68 and 5-11-68, Fig. IB) :

4-18: % development:: 17+ 13.0 Mays long day fed) 5-11: % development -- 22 + 9.6 (days long day fed)

Solving the equations for the parameter (days long day fed), given 29% and 32% development, the latent period of food is 0.9 days for both 4-18 and 5-11. Simi- larly, the latent period of long day is found from the average pupation induced by 1-6 days long day without food before exposure to short day with food in experi- ments started 5-11-68 and 6-1-68, 427r and 34% development, respectively. The regression equation for the only estimate of inductive capacity of long day with food before exposure to short day with food on 4-18 (Fig. 1C) is:

% development -- 8 + 17.2 (days long day fed)

yielding latent periods of 1.5 and 2.0 days. These latter values should be regarded only as estimates since the pertinent experiments were not started concurrently and since this source of error was compounded because the experiments were in progress during that time of year when the effects of long day were declining and those of food increasing (Bradshaw, 1969). Nonetheless, it can be concluded that the latent period of food probably does not exceed that of long day. The latent periods of these two inductive cues thus appear to be similar and completely consistent with the concept of nutritional independence of food in the inductive process. It should be noted that nothing here is meant to imply that food does not make a substantial, long-term contribution to the developing pupa or adult (Fig. 3) ; the important point to be made again is that this nutritional use of food is independent of the use of food as an environmental signal.

Appreciation is extended to Billy E. Frye, Christina M. Holzapfel, and David G. Shappirio for their consideration and advice concerning the research here presented.

SUMMARY

1. Food and long day photoperiod interact synergistically to effect the termina- tion of larval diapause but the in] nit of food and long day must be simultaneous rather than sequential for the synergistic effect to take place,

2. Day length doss not affect food consumption.

484 WI1.1.I AM K. I'.KADSHAW

3. Neither food alone nor long day alone is capable of augmenting development initiated by long day with food ; likewise, short day without food does not appear to retard development once it has been initiated.

4. Food is nutritionally capable of affecting larval and pupal survivorship for up to several weeks bur its capacity to interact synergistically with long day to induce development persists for only a day.

5. The capacity of long day to interact synergistically with food to induce devel- opment persists for H to 2 days.

6. Food, therefore, is probably acting via some neuroendocrine reflex as an environmental cue independently of its nutritional contribution to the overwinter- ing larva or to the resultant pupa and adult.

LITERATURE CITED

BAKER, F. C, 1935. The effect of photoperiodism on resting tree-hole mosquito larvae. Can. Eutomol., 67: 149-153.

BRADSHAW, W. E., 1969. Major environmental factors inducing the termination of larval dia- pause in Chaoborus amcrlcanus Johannsen (Diptera: Culicidae). Biol. Hull., 136: 2-8.

DUNCAN, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42.

ENGELMANN, W., AND D. G. SHAPPIRIO, 1965. Photoperiodic control of the maintenance and termination of larval diapause in Chirononms teutons. Nature, 207: 548-549.

KEMPER, M., 1931. Beitrage zur Biologic der Bettwanze (Cimcx Lccturlarms L.). II. Uber die Hautung. Z. Wiss. Biol., Abt. A., 22: 53-109.

LOVE, G. J., AND J. G. WHELCIIEL, 1955. Photoperiodism and the development of Acdcs tri- seriatus (Diptera: Culicidae). Ecology, 36: 340-342.

McCRARY, A. B., AND C. E. JENNER, 1965. Influence of daylength on sex ratio in the giant mosquito, Toxorhinchites rutilns, in nature. Amer. Zool., 5: 206.

PARIS, O. H., AND C. E. JENNER, 1959. Photoperiodic control of diapause in the pitcher plant midge, Metriocncmus knabi. Pages 601-624 in R. B. Withrow, Ed., Photoperiodism and Related Plicnomena in Plants and Animals. American Association for the Ad- vancement of Science, Washington, D. C.

RACTLIFFE, J. F., 1967. Elements of Mathematical Statistics. Oxford University Press, Lon- don, 224 pp.

SNEDECOR, G. W., 1956. Statistical Methods. Iowa State University Press, Ames, 534 pp.

VINOGRADOVA, E. B., 1962. Role of photoperiodism in seasonal development of tree-hole malarial mosquito Anopheles plumbcus Steph. (Diptera, Culicidae). Doklady Aka- demii Nauk Soyuz Sovetskikh Sotsialisticheskikh Respublik, 142: 481-482. (AIBS Transl.)

VINOGRADOVA, E. B., 1964. The ecological regulation of the seasonal cycle in the malarial mos- quito Anopheles bifurcatus L. (Diptera, Culicidae). Doklady Akademii Nauk Soynz Sovetskikh Sotsialisticheskikh Rcspublik, 151: 1204-1206. (AIBS Transl.)

WIGGLESWORTH, V. B., 1933. The physiology of the cuticle and of ecdysis in Rhodnius prolixus (Triatomidae, Hemiptera) ; with special reference to the function of the oenocytes and the dermal glands. Quart. J. Microscop. Sci., 76: 269-329.

WIGGLESWORTH, V. B., 1934. The physiology of ecdysis in Rhodnius prolixus (Hemiptera). II. Factors controlling moulting and "metamorphosis." Quart. J. Micrnscop. Sci., 77: 191-222,

Reference : /*/<•/. Hull.. 139: 485-4'M. ( 1 K-cemkT. 1970)

DIXENIC CULTURE OK DAPHNIA MAGNA, STRAUS

ANTHONY S. D'AGOSTINO AND LUIGI PRO VA SOLI 1

St. John's University, Biology Dcpt., New York 11432 and Haskins Laboratories, 165 Prospect Street, Nciv Haven, Conn. 06520

Banta (1921) demonstrated that Daphnia could be grown agnotobiotically \vitli relative ease. Since then, rearing was improved by replacing horse-manure infu- sion with unialgal cultures (Lefevre, 1942). Large populations of D. magma were maintained for 18 months in artesian well water, and fed with yeast and Scenc- dcsmus ; these cultures were stable and supplied abundant progeny of known age, genetic homogeneity and vigor, for insecticide bio-assays (Dewey and Parker, 1964). Attempts to replace pond water with synthetic salt solutions have met with limited success (Anderson, 1945; Freeman, 1953; Boyd, 1957; Taub and Dollar. 1964). Dewey and Parker (1964) reported that under the most favorable condi- tions (Wesson's (1932) salt mixture in distilled water) fecundity gradually de- clined and reproduction ceased at the 32nd generation.

Taub and Dollar (1968) re-explored the dependence of alga-fed Daphnia piilc.v on water biologically conditioned in aquaria (BCW). They concluded that algae grown in defined media were deficient in nutritional factors needed by Daphnia. Presumably "BCW" contained unidentified substances, which fostered normal development and ovulation of Daphnia.

Since this problem seemed nutritional, we axenized D. magna and fed it on pure cultures of algae to define conditions for sustained culture.

MATERIAL AND METHODS Crude cultures

Agnotobiotic D. magna strain #10 was kindly supplied by Dr. F. Taub, Col- lege of Fisheries, Seattle, Washington. Stock cultures were maintained in covered "storage dishes" (Corning #3250), 30 individuals in 200 ml of DM2, a synthetic medium containing (w/100 ml dist. FLO) : KC1, 5 mg; MgSO4-7FLO, 4 mg; Ca (as C1-), 2 mg; K2HPO4, 0.6 mg; KH2PO4, 0.6 mg; NaNO,, 5 mg; NaSiO,- 9H,O, 2 mg; Fe (as Cl'), 0.05 ,xg; Metals PIT, [1 ml of P II metals contains: ethylenediaminetetraacetic acid (as Na.,), 1 mg; Fe (as Cl), 0.01 mg; B (as H3BO3), 0.2 mg; Mn (as Cl), 0.04 mg; Zn (as Cl). 5 ^g; Co (as Cl), 1 /*g; (Provasoli, McLaughlin and Droop, 1957)] 1 ml; Metals S II, [1 ml of S II metals contains: Br (as Na), 1 mg; Sr (as Cl), 0.2 mg; Rb (as Cl), 0.02 mg ; Li (as Cl), 0.02 mg; Mo (as Na salt), 0.05 mg ; I (as K), 1 ^g; V (as NH4VCU. 1 ju.g; (Provasoli, McLaughlin and Droop, 1957)1 1 ml; vitamin B12, 1 ju.g; and thiamine HC1, 10 /xg; adjusted to pH 7.0.

1 This work was aided by Contract Nonr 4062 with the Office of Naval Research and research grant GB-4860 of the National Scii-nce Foundation.

485

486 ANTHONY S. D'AGOSTTNO AND LUIGI PROVASOT.I

They were fed axenically grown Scenedesmus obttquus (Indiana Collection No. 393) and Chlamydomonas reirihardi, minus strain (Indiana Collection No. 90V Continuous cultivation was kept by subculturing the first 30 newborn nauplii of each generation.

Transfer dishes were prepared 8 days previously by inoculating DM., with algae grown on DA medium. [DA medium: mg/100 ml: Na:,- citrate -2H,O, 2.0; MgSO,-7H,A 2.0; K2HPO4, 2.0; Fe (as SO4), 0.2; Thiotone (Baltimore P,iol. Lab), 60.0; Trypticase (Baltimore Biol. Lab), 16.0; yeast extract (Difco), 50; pH 6.5 (DA -Medium A, Table 2, p. 840, Provasoli and Pintner. 1953).] Addi- tion of DA to DML, promoted initially rapid growth of bacteria which soon sub- sided and was succeeded by algal growth. Mortality was high if newborn Daphnia were inoculated during the period of bacterial growth.

Antibiotics

Serial washings of nauplii from crude cultures failed to eliminate bacteria. Antibiotics were tried after determining their effectiveness on the bacterial flora association with Daphnia. Samples of water from crude cultures were diluted with an equal volume of Dextrose Broth (Difco) ; 4 hours later 1-ml aliquots were spread on solidified nutrient agar (Difco). One hour later, surplus fluid was removed and antibiotic sensitivity discs (Difco) were pressed on the agar surface. Zones of inhibition, at room temperature, were recorded at 24, 48 and 72 hours. No one antibiotic completely inhibited bacterial growth. Streptomycin sulfate and chloromphenicol were the most effective ; however, resistant strains developed within the zones of inhibition in all cases.

Antibiotics effective against the micro-organisms were tested for toxiciity to D. niagna. Nauplii or adults of Daphnia were exposed to varying concentrations of antibiotics in 10 ml of DM (-- DM., without thiamine and B]2). Organic sub- stances were excluded to avoid excessive growth of resistant bacteria. The anti- biotics were dispensed from fresh Seitz-filtered stock solutions. Chlamydomonas rcinhardi and Scenedesmus obliquits were added as food. The cultures were ob- served twice daily. Inhibitory concentrations of antibiotics paralyzed the second antennae of the Daphnia, the animals settled to the bottom of the tube and soon died.

Single antibiotics permitting survival of adults and larvae for > 2 days were (/xg/nil) : chloramphenicol 25; kanamycin 100; nalidixin 50; neomycin 20; poly- mixin 7.5 (1000 fig ~ 7760 units) penicillin 500 (1000 /ig =; 1650 units) ; strepto- mycin-SO4 100; tetracycline 10; trichomycin 50.

Several mixtures designed to suppress a wide bacterial spectrum were pre- pared and tested. Chloramphenicol, kanamycin, nalidixin and polymixin were omitted because few Daphnia exposed to them survived. The concentration of penicillin was kept below 450 /j.g/ml because larger doses changed the pH of the media to < 5.6 — a pH not tolerated by Daphnia in these media.

A.venisation of Daphnia magna

In crude cultures D. ina</na nauplii became females after 8 days and deposited their first parthenogenetic brood (4-6 larvae per female). Seven-day-old gravid females with eggs ready to hatch were transferred to tubes containing 10 ml of

DIXENIC CULTURE OF DAPHAI.l 487

sterile DM and 0.5 ml of antibiotic mix I) [antibiotic mix 1) contained (/^g/ml) : neomycin 10; streptomycin 1500; penicillin 6000 (1000 p.g = - 1650 units); tetra- cline 400]. This concentration, although lethal to adults, allowed 2-3-day survival —an ample time for release of neonates.

To prevent fecal accumulation and overgrowth by bacteria, the females were transferred everv 15 minutes to fresh tubes similarly prepared. Usually the nauplii hatched during the same working day ; if not, gravid females were left over- night in DM and 0.25 ml/10 ml of antibiotic mix D. Alternatively, sublethal doses of antibiotic mixtures (0.1 ml/10 nil), were used; obviously more rinses were needed to remove by dilution the bacterial flora.

Neonates collected within minutes after deposition were washed through 10 serial baths ( 10 ml DM +0.25 ml antibiotic mix D). The animals remained in each bath 10 minutes. After the 10th wash, sterility was tested by transferring them into DA liquid medium for \ hour [This tube was incubated in the dark at 28° C. If infected, visible turbidity appeared before algal growth could mask it. Darkfield microscopy was also used to detect infectants.J ; then they were distributed singly into tubes with 10 ml of DM2. Several food algae were added aseptically alone and in combinations to find an adequate food for axenic cultures.

Maintenance of axenic stock cultures

Bacteria-free nauplii developed into fertile females readily when fed ^'. obliquus and C. rci-nhardi grown in DA medium. To maintain dixenic cultures, filial gen- eration nauplii were collected and washed three times in 10 ml of DA liquid me- dium ; the last wash tube served as sterility test. The washed larvae were distrib- uted singly in screwcap tubes (20 X 125 mm Pyrex) containing 10 ml of DM2 and inoculated with 0.5 ml each of dense cultures of the two algae grown in liquid DA. The tubes were incubated at room temperature in racks illuminated (200 ft-c) continuously by white fluorescent lamps. This initial inoculum of algae sufficed to feed the larvae and to produce ample algae for feeding ad libitum the adult female for 20 days if each newborn brood was withdrawn within a day of deposition. Every 30 or so generations, records of survival, developmental time, and fecundity were made on a set of seven tubes. Survival of nauplii was 90-100% ; of these, 90-95% became fecund females in 8 days. The average production was 29.7 nauplii per female life-span. After the 20th day, the algal population became too small to support Daphnia fertility. Over 200 generations of Daphnia have been obtained to date without lessening of vitality.

Several variables that may have influenced cultures were studied. In each case nauplii were transferred consecutively into six 10-ml tubes of DM to minimize carry-over of nutrients and to eliminate the algae, before being inoculated into the experimental tubes.

RESULTS

Experience with mineral media for lake algae indicates that they require dilute media (Chu, 1942; Rodhe, 1948; Provasoli and Pintner, 1953). Although optimal growth depends, roughly, on Ca/Mg, Na/K and divalent/monovalent ratios being optimal, planktonic algae adjust well to wide variations provided that tolerance limits in respect to total solid concentrations are not approached (Provasoli, McEaughlin

488 \\TIIONY S. D'Ar.OSTINO AND LU1G1 PROVASOLI

and Pintner, 1954). Clm (1942) simply diluted the old medium of Benecke, then determined the best ratios for planktonic algae. Murachi and Imai (1954) found that a slightly modified Bristol solution diluted 10 X was satisfactory for Moina macrocopa.

Several dilute media for freshwater algae were tried. Various modifications and combinations of the more promising media led to medium DM (=- DM2 minus vitamins), which supported good algal growth without injuring Daphnia. These trials were done with bacterized cultures of Daphnia. Under such conditions D. magna fed Scenedesmus survived only a few generations. Other food organisms were tried. The combination 5'. obliquus and Chlamydomonas reinhardi seemed best: it allowed 30 non-axenic generations before fertility decreased. [These two algae were grown in DM medium and inoculated in new DM a few days before transferring the newborn Daphnia of the next generation.]

In several cases, for lack of well-grown algal cultures, we used, as inoculum algal cultures from the culture collection which were grown on agar slants of DA medium ; the organic components do not harm algal growth and permit detection of contamination. Because of the organic content of the medium, care was taken at first to remove the algae without digging into the agar since carry-over of nu- trients might lead to excessive bacterial growth. On the contrary, introduction of DA agar flakes resulted in better growth of Daphnia. This observation became useful when fertility of Daphnia declined ; large flakes of DA agar were added with the algal inoculum and restored fertility. Later on 0.5 ml of each algal culture grown in DA broth was used as inoculum. Thirty additional non-axenic genera- tions were obtained before maintenance of the bacterized strain was discontinued.

Meanwhile axenic newborn specimens of D. niagna were obtained. Several potential food organisms were tried which had been grown on DA agar. Inoculated axenically into DM2 medium, Chlorella vulgaris and C. clipsoidea supported devel- opment to young females but not reproduction ; Navicula pelliculosa and Saccharo- myces cerivisiae allowed maturation of adults and egg production, but the eggs never hatched. Scenedesmus quadricaudatum and Chlamydomonas moewusii per- mitted only survival of larvae (1-3) days). 3\ obliquus and C. reinhardi, used singly, supported growth up to young females; increasing the light from 100 to 200 ft-c resulted in adult females but no reproduction. 6\ obliquus at 200 ft-c pro- duced substances which thickened the medium ; as a result, the specimens of Daphnia were immobilized and soon died. D. magna fed on the combination 6". obliquus and C. reinhardi in DM, gave adult females at 100 ft-c and finally nauplii at 150 and 200 ft-c. Surprisingly the presence of C. reinhardi resulted in no gelling of the medium by 6\ obliquus.

Ten generations of D. magna were obtained in DM2 with the 2 algae as food, then fertility fell off sharply. By that time we had found with bacterized cultures that the addition of DA (1 ml/10 ml medium) restored fertility. This held even under germ-free conditions. We are now a the 200th germ-free generation ; mean generation time 8.5 days ; mean survival of fecund females 20-22 days ; mean new- born production per female life-span 30 (Table I).

Agitation and higher concentrations of nitrates and phosphates were tried to eliminate the possibility that the organic enrichment simply made good a deficiency of the medium in these nutrients. In media without vitamins (DM), increase in

DIXENIC CULTURE OF DAPHN1A

489

total phosphates up to 4 mg'/o and nitrates to 40 mg/'o resulted in ovigerous females (as opposed to young females with lower concentrations of N and P ) but the eggs generally did not hatch and the few nauplii obtained were sickly. Addition of vitamin B12 and thiamine (DM2), DA or liver extract resulted in viable nauplii. Several modifications of DM2 were then tried (Table I). Higher N and P and/or the addition of an N -containing pll buffer failed to support more generations even in the presence of B12 and thiamine.

TABI.L I

Modification of Basal media (food algae S. obliquus and C. reinhardi)*

	Media designation (mg% ; w/v)
	DM2	DMs	DM2 +DA	DM6	DM6 -hG	DM6	DM8
P (as K2HPO4 and KH2PO4)	0.24	0.24	0.24	0.24	0.24	0.81	0.81
N (as NaNO3)	0.82	0.82	0.82	0.82	0.82	3.3	3.3
N (in 30 mg' 0 glycylglycine buffer)		6.3
Thiamine (jug'.'o, w/v)	10	10	10		6.3		10
Bl2 (jug' o, W/V)	0.1	0.1	0.1				0.1
Yeast extract			0.5
Thiotone (Baltimore Biol. Lab.)			6.0
Trypticase (B.B.L.)			1.6
Daphnia magna
Generations**	12	12	>200	1	1	1	10
Generation time***	9.8	8.6	8.5	9	9	9	12.9
			[6-11]
Mean new born/9*:	15.6	23	29.7	9	5	2	21.3
			[10-66]

* Algae grown separate!}- in various liquid media and fed to Daphnia being cultured serially in corresponding media.

** Consecutive generations obtained.

*** Days needed by a newborn nauplius to become a fertile female.

**** Average of several trials.

Since the only factors inducing sustained, if limited fertility were vitamin B12 and thiamine, other vitamins were tried as replacements of the organic enrichment of the DA medium. Since the growth and fertility of Daphnia apparently depended upon the composition of the medium in which the algae were grown, depleted algae grown serially in a medium without vitamins (medium DM) when inoculated with Daphnia nauplii in media with vitamins might show a substantial difference in number of I<\ nauplii. This was indeed so: the effect of various vitamins was evi- dent in 3 weeks. The only vitamin which significantly increased the Ft nauplii was B12. Combined with B]2 and thiamine, pantothenate increased nauplii produc- tion (Experiment A, Table II) ; pyridoxine was occasionally effective.

To confirm these short-term results, in Experiment B (Table II), the algae were again grown without vitamins (1)M7) and subjected to the experimental variables, reduced after the \'\ to one optimal concentration for each vitamin. The

490

ANTHONY S. D'AGOSTINO AND LUIGI I'K< )Y \SOLI

TABLE II

Effect of vitamins (No. nauplii/

Vitamin addition G*g%)	Exp. A F! only	Exp. B	Exp. ct
Fi	F2	F3	F4	Fs	F6	F?	F8	F9	Fio	Fl6	Fv>
Mineral base (DMv)	2	0.7	0											0
DM: + Bi2 0.05		8.3	9	discontinued
DM? + 8:2 0.1	18	21	26	24	26	18	8	1.7	0.3	0				[26
DM7 + Bi2 0.2		19	22	discontinued
DM? + thiamine 25		0
DM? + thiamine 50	2.5	4.5	0											0
DM? + thiamine 100		2.3	0
DM? + pantothenate
12**		0
DM? + pantothenate
25**	0	0												0
DM? + pantothenate
50**		1.3	0
DM7 + Bu 0.1 + thia-
mine 50	20	23	24	22	15	11.3	7	0						23
DM? + 612 0.1 + pan-
tothenate 25	32	25	22	17.3	3	0								31
DM? -j- 812 + thiamine
+ pantothenate
25***	44	33	29	35	32	28	36	31	31	28	31	26	28	45
DM7 + 812 + thiamine
+ pantothe-
nate + pyri-
doxine****								28	32	30	33	26	31
DM7 + BnO.l + thia-
mine 50 +
DA (1ml/ 10)		29	38	41	28	37	38	36	37	31	31	31	33

* Each value in table is the average of triplicate tubes.

** Ca- pantothenate was filter-sterilized and added aseptically.

*** The No. nauplii given for Fi-F& are for the aseptic addition of 25 ng% °f Ca- pantothenate. From Fe on, the values are for 250 j*g% Ca- pantothenate autoclaved with the medium. The generations Fe-Fio were run in duplicate series (i.e., these 2 concentrations of pantothenate) of triplicate tubes ; the average number of nauplii per female was almost identical indicating that autoclaving resulted in a high inactivation of pantothenate. The series with aseptic ad- dition of pantothenate was discontinued at the Fn which became infected.

**** This series was inoculated with nauplii, of the Ft in Bu + thiamine + Ca- pantothenate.

)• See text.

previous results were confirmed : only B12 + thiamine + pantothenate supported fertility beyond the F8, and the average number of nauplii per female life-span was similar but consistently lower than with DM7 + B12 + thiamine + DA (1 ml/10 ml) — a combination similar to DM2 + DA which supported over 200 aseptic gen- erations. Most of the effect of the organic enrichment could thus be replaced.

The fact that in these experiments the algae and Daphnia were grown in the same medium made it difficult to discern whether the organic enrichment (or the vitamins) acted directly on the Crustacea or via the algae. To rule out the possi- bility that Daphnia fertility depended upon direct uptake of organics from the medium, the algae were grown separately on agar media to avoid carry-over of enrichment, then fed to Daphnia, in a completely mineral medium (DM). The original experiment (Table I) of comparing DM, and DM2 -f DA was repeated: C. reinhardi and S. obliqniis were grown seperately on DM2 and in DA solidified with 2% agar. When grown, a loopful of each alga was scraped from the agar surface, avoiding removal of agar pieces, and inoculated with one Daphnia nauplius in a tube of DM (Experiment C, Table II). Additional loopfuls of algae were added if needed. The line fed on algae grown in I)ML, failed to reproduce beyond the llth generation. The line fed on algae grown in DA showed undiminished fertility (the experiment was discontinued at the 20th generation), duplicating the results obtained when liquid DA was added directly to the algae-crustacean culture.

niXKXU CULTTKK OF D.II'HNIA

TABLE III

Effects of algal preconditioning

Experiment	Medium used for culturing	Average results*
algal inoculum	algae + Daphnia	Nauplii/9	Generation time (days)	No. generations averaged
Mineral part	Enrichment	Mineral	Enrichment
B	DM? liquid (no vitamin-)	0	DM7 + DM7 + DMv	Bit + thia. + pantoth. Bu + thia. r + DA medium 0	31 34 0.7	12.6 13.7 17	15 15 1
C	DM agar2 0	Bi2 + thia. DA agarf	DM DM	0 0	18.4 19.5	S..S 8.6	11 20
Culture maintenance	0	DA liquid	DM	Biz + thia. + I ml/ 10 DA	29.7	8.5	50

* Average of data from one or more generations; for each generation the results of triplicate tubes were also a\ n- aged.

I" Algal cells only were removed from agar and served as inoculum.

DISCUSSION

Numerous attempts to replace pond water \vith defined salt solutions for con- tinuous cultures of daphnids have failed ( at best only 30 consecutive generations of D. inayna. were obtained by Dewey and Parker). We experienced a similar failure in DM medium. Later results show that for continuous cultures of Z). magnet, choice of food organisms and, especially, provision of an organic enrichment are far more important than the composition of the mineral medium; our medium DM does not differ very much from medium 37 of Taub and Dollars (1968).

Fourteen species of Cladocera are now in monoxenic continuous culture with C. rcinhardi as food organism in a unique medium consisting of Ca acetate, albumin, water-soluble vitamins, trace metals, and distilled water (Murphy, 1970) ; no ad- vantage was found in adding other mineral salts ! An explanation of this astonish- ing feat is simply that C. reinliardi is a collector of essential minerals (see composi- tion in table 5, page 615, Taub and Dollar, 1968).

It is not surprising, therefore, that other daphnids thrive on DM2 enriched with pantothenate ; many bacterized generations of D. galcata uicndotae (J. S. Suffern, Biology Dept., Yale University, personal communication) and >10 bacteria-free generations of D. pulex and S. nmcronata (D. E. Conklin. Raskins Labs., per- sonal communication) were obtained without loss of fertility when fed in this medium on C. rcinhardi and 6\ obliquus.

Addition of vitamins to inadequate algal food had also been beneficial for Tigriopus japonicus (Shiraishi and Provasoli, 1959) but these findings were based on only a few generations. The results of Murphy attest that addition of vitamins is essential in supplementing the nutritive value of one alga for several daphnids. As in our experiments, vitamin B12 and Ca pantothenate had a decisive effect on female fertility and viability of nauplii, and permitted, with the addition of thiamine (-- his basal medium), over 40 generations of Da[>lniia f>nlc.r, Scapholeberis niucro-

492 ANTHONY S. D'AGOSTINO AND I. IK, I 1'ROVASOI.I

nata and Simocephalus serrulatus fed on C. rcinhardi and .V. ubliqiius. However this medium failed to support continuous culture of other daphnids until a mixture of 8 additional vitamins was added. This addition improved also the number of nauplii produced in the 1st brood of the above 3 species. In other experiments C. rcinhardi could be employed as the sole food organism for 14 species by increas- ing the concentration of choline, pyridoxal, inositol, riboflavin, and nicotinamide. In this medium, D. magna, S. serrulatus and D. retrocurva were the most difficult to maintain, indicating that some daphnids may be nutritionally more exacting than others.

Previous experiments on Artetnia (D'Agostino and Provasoli, 1968; Provasoli and D'Agostino, 1969) gave circumstantial evidence that organic enrichment influ- enced the fertility of Artemia (i.e., number of generations) not directly but via the algae. The results with D. magna grown in mineral media and fed with algae which had been grown separately on organic enrichments or vitamins (Experiment C) seem to finally prove this hypothesis.

Experiments B and C support another hypothesis, i.e., that the enrichments modify the nutritive value of the algae for Daphnia. Generation time (i.e., days needed for a nauplius to become a fertile female) depended upon the kind of medium in which the algae used as inoculum were grown (Table III).

If the 2 algae were pregrown for several transfers in mineral media and inocu- lated in a rich medium with one Daphnia nauplius (Experiment B), the Daphnia generation time was long (12-13 days) but the number of nauplii per female was high (30-34). Conversely, if the algae were pregrown in organic media (DA or DM2) and inoculated in mineral media (Experiment C) the generation time was short (8.5 d) and nauplii production per female was low (18-20).

The preconditioned algal inoculum, once transferred in a new medium with one Daphnia nauplius, starts to reproduce logarithmically ; the medium greens. Simul- taneously, the nauplius as it grows to a fertile female, grazes more algae. The algae, as they divide in the new medium, change in physiology and storage products, either losing gradually their nutritional value (if preconditioned in organics and inoculated in mineral media) or gaining nutritional value in the converse experi- ment. As it happened, the generation time should therefore be influenced by the type of medium in which the algae were pregrown, and production of nauplii by the type of medium in which the algae have been inoculated with the Daphnia nauplius. Since the difference in generation time between experiments B and C was of 4.5 days, the size of algal inoculum and/or rate of division of the algae were probably high. A closer equilibrium between grazing rate and algal division rate- grazing rate tending to nullity the positive or negative nutrient effect of algal divi- sion— should result in a much smaller differential in generation time. Naturally, the differential may also indicate that the medium-mediated physiological changes in the algae leading to a different cell composition are slow. The short generation time and high nauplii production obtained for 200 generations when the algae were pregrown (DA) and inoculated (DM2 + DA) in organic enriched media con- firms experiments B and C.

That the enrichment acts via the algae and not directly on the Crustacea is further substantiated by the recent demonstration that Crustacea are extremely inefficient in the uptake of solutes (Anderson and Stephens, 1969) and that because

DIXENIC CULTURE OF DAPHNl. 1 493

of this, Artemia salina, when grown in vitro on artificial food, is unable to grow unless the major nutrients are presented as particles (Provasoli and D'Agostino, 1969).

Since the results of Murphy (1970), Lewis (1967) and of our group indicate that widely differing genera of freshwater, euryhaline and hyperhaline filter-feeding Crustacea behave similarly, it seems probable that addition of vitamins to 2- or 3- membered algae-crustacea cultures might permit continuous cultivation of Crustacea which so far have proved difficult to grow.

SUMMARY

1. Daphnia magna can be grown for at least 200 generations, axenically or in crude cultures, in a defined mineral medium, enriched with vitamin B1:, and thia- mine and 1 ml/100 of a dilute organic medium, when fed with Chlamydomonas rclnhardi and Scenedestnus obliquus.

2. The organic enrichment is essential for maintaining continued fertility of D. magna.

3. The organic enrichment can be replaced by the addition of pantothenic acid to vitamin B12 and thiamine without lowering the fertility of D. magna.

4. The organic enrichment (or the vitamin mixture) does not act directly on D. magna but via the algae by changing their nutritional value for Daphina.

5. Addition of vitamins to the medium in which the algal food is grown with Crustacea may allow continuous cultures of herbivorous Crustacea which are con- sidered difficult to grow.

LITERATURE CITED

ANDERSON, B. G., 1945. The toxicity of DDT to Daphnia. Science. 102: 539. ANDERSON, J. W., AND G. C. STEPHENS, 1969. Uptake of organic material by aquatic inverte- brates. VI. Role of epiflora in apparent uptake of glycine by marine crustaceans.

Mar. Biol. ,4: 243-249.

BANTA, A. M., 1921. A convenient culture medium for Daphnids. Science. 53: 557. BOYD, J. E., 1957. The use of Daphn'ui nniinia in the microbioassay of insecticides. Ph.D. thesis.

Pennsylvania State University, 205 pp. CHU, S. P., 1942. The influence of the mineral composition of the medium on the growth of

planktonic algae. I. Method and culture media. /. Ecol., 30: 284-325. D'AGOSTINO, A., AND L. PROVASOLI, 1968. Effects of salinity and nutrients on mono- and

dixenic cultures of two strains of Artemia salina. Biol. Bull.. 134: 1-14.

DEWEY, J. E., AND B. L. PARKER, 1964. Mass rearing of Daphnia magna for insecticide bio- assay. /. Econ. Entomol., 57: 821-825. FREEMAN, L., 1953. A standardized method for determining the toxicity of pure compounds to

fish. Sewage hid. Wastes, 25: 845-848. LEFEVRE, M., 1942. L'utilization des algues d'eau douce par les cladoceres. Bull. Biol. Fr.

Belg., 76: 250-270. LEWIS, A. G., 1967. An enrichment solution for culturing the early developmental stages of the

planktonic marine copepod Enchacta japonica Marukawa. Limnol. Occanog. 12: 147-

148. MURACHI, S., AND T. IiiAi, 1954. Studies on the culture of water fleas, Moina macrocopa

Straus, in artificial culture medium. Tohoku J. Agric. Res., 5: 27-36. Mt'RFHY, J. S., 1970. A general method for the monoxenic cultivation of the Daphnidae. Biol.

Bull., 139: 321-332. PROVASOLI, L., AND A. S. D'AGOSTINO, 1969. Development of artificial media for Artcmia

salina. Biol. Bull., 136: 434-453.

494 ANTHONY S. J)'AC,( >STI \( ) A.ND l.l'Mil I'KOVASOLI

PROVASOLI, L., AND I. J. I'INTNKR, 1953. Ideological implications of in vitro nutritional require- ments of algal flagellates. Ann. Nczv York Acad. Sci., 56: 839-851.

PROVASOLI, L., J. J. A. MCLAUGHLIN AND M. R. DROOP, 1957. The development of artificial media for marine algae. Arch. Mikrobiol., 25: 392-428.

PROVASOLI, L., J. J. A. MCLAUGHLIN AND 1. J. PINTNER, 1954. Relative and limiting concen- trations and major mineral constituents of the growth of algal flagellates. Trans. New York Acad. Sci. Series II, 16: 412-417.

RODHE, W., 1948. Environmental requirements of fresh-water plankton algae. Svmb. Bui. Upsal. 10: 1-149.

SHIRAISHI, K., AND L. PROVASOLI, 1959. Growth factors as supplement to inadequate algal foods for Tigriopns japonicus. Tolmkn J. A</r. Res., 10: 89-96.

TAUB, F. B., AND A. M. DOLLAR, 1964. Chlorella-Daphnia food-chain study: The design of a compatible chemically defined culture medium. Liinnol. Occanog., 9: 61-74.

TAUB, F. B., AND A. M. DOLLAR, 1968. The nutritional inadequacy of Chlorella and Chlainydo- nwnas as food for Daplniia pnlcx. Liiiiuol. Occanog., 13: 607-617.

WESSON, L. G., 1932. A modification of the Osborne Mendel salt mixture containing only inor- ganic constituents. Science, 75: 339-340.

Reference: liwl. Hull.. 139: 495-501. (December, 1970)

SENSITIVITY OF ECHOLOCATION IN CAVE SWIFTLETS

DONALD R. GRIFFIN AND RODERICK A. SUTHERS

The Rockefeller University it ml AY.c1 York Zoological Society, New York, New York and Indiana University, Bloomington, Indiana 47401

The specializations of bat> nt" tin- .suborder Microchiroptera for echolocation have naturally suggested that nocturnal or cave dwelling birds might orient themsel\<-- in the same way. Numerous preliminary tests of several species (mostly unpub- lished) have yielded negative results, and only two avian genera have been clearly demonstrated to use echolocation. These are the oilbirds of South America. Stcatoniis caripcnsis (Griffin, 1954), and certain species of the genus Collocalia, the cave swiftlets of Southeast Asia (Griffin. 1958; Novick, 1959; Medway, 1950. 1967). The orientation sounds of both species are brief audible clicks lasting a few milliseconds. They tend to have a peak of energy between two and eight kHz. but also a very broad acoustic spectrum extending to ultrasonic frequencies. The clicks are emitted primarily in dim light or darkness and increase in repetition rah- like those of bats and cetaceans when difficult orientation problems arise. A very similar sort of echolocation is practiced by bats of the genus Rousettus, the only known case in the suborder Megachiroptera.

Although oilbirds and swiftlets can certainly avoid large obstacles by echoloca- tion, there is very little evidence concerning their proficiency, and we know almost nothing about the minimum size of object that can be detected. In contrast consid- erable data on this point are available from bats of the suborder Microchiroptera (Griffin, 1958; Suthers, 1965, 1967; Schnitzler, 1966). Griffin. Novick. and Korn- field (1958) measured the size of cylindrical obstacles that could be detected by a single individual Rouscttns aegypticus. With sufficient practice this animal could detect wires as small as 0.46 millimeter diameter at distinctly above the chance level. Rousettus uses as orientation sounds audible clicks that are similar to those of Collocalia and Steatoniis, but in view of evidence that most small mammals can hear well at ultrasonic frequencies (Rails, 1965) it is difficult to ascertain which part of the broad frequency spectrum emitted by Rousettus generates the echoes by which it detects small obstacles. Medway (1967) studied the ability of Collocalia fuciphaga to avoid vertical wooden rods one centimeter square spaced 15 cm apart. These birds with a wingspan of about 27 cm showed no ability to avoid these obstacles when first encountered in darkness. But they maneuvered between them almost perfectly in the light. Their performance in the dark improved on successive flights, but the experiment did not demonstrate how much of this improvement resulted from learning the positions of the obstacles and how much from echoloca- tion. The many species of Collocalia appear to differ widely in their use of dark- caves and their reliance on echolocation.

During the 1969 ALPHA HELIX expedition to New Guinea we studied the orientation sounds of Collocalia vanikorcnsis granti, and carried out preliminary experiments that indicate the approximate threshold size of cylindrical obstacles

495

DONALD R. GKIFK1N AND KODKK'K K A. Sl/TIIKUS

detectable hv echolocation. This species nests in a totally dark chamber of a cave near Amele, south of Madang, which we visited twice in June, 1969, for pre- liminary observations. Local opposition of a quasi-religious nature prevented ex- tensive studies of this colony, but through the kindness of Dr. Kiro Kikkawa three birds of the .same species were obtained in mist-netting operations near Omuru within a few km of this cave.

METHODS

The obstacle avoidance tests were conducted in a dark chamber constructed from a double layer of heavy, opaque, black plastic. This was shaded and sheltered from rain by a fly of the same material below a thatched roof. A small air conditioner was used to maintain a temperature of approximately 26-28° C. The tests were conducted both in the daytime and after dark, but the chamber was sufficiently light- tight that even in bright sunlight, and when we were fully dark adapted, we could not see large white objects. All test obstacles were small wires or rods extending vertically from floor to ceiling and spaced 40 cm apart horizontally. With the lights off they were quite invisible. The chamber was 4.9 meters long, 2.9 meters wide and 2.1 meters high with its long axis approximately east-west. Near the center, 2.3 meters from the west end, a wooden frame around the walls, floor, and ceiling reduced the height to 1.86 meters. On this frame we mounted a series of cords and pulleys from which obstacles were mounted and shifted horizontally in position while maintaining their horizontal spacing. The obstacles mounted on this frame will be referred to below as the middle obstacles. Three additional rows of vertical obstacles were hung from hooks in the ceiling at 1.5. 2.4. and 3.9 meters from the west end of the chamber. These could also be shifted between the fixed positions of hooks in the ceiling, but this type of shifting was less convenient and was carried out less often. The obstacles in the middle plane were fastened by rubber bands at their tops and bottoms while those in the other three rows were held straight by light weights. The swiftlets almost always flew at least 30 cm below the ceiling and at an even greater distance above the floor, both in the light —where they could easily be seen — and in darkness where we could locate them by their audible orientation sounds. All trials considered below consisted of flights through the obstacle planes at 30 cm or more from Avails, or ceiling. The smallest obstacles tested were supported by attachments of the same size as the larger obstacles. In view of the poor performance with the smallest obstacles tested we did not take stringent precautions against the possibility that the echolocation achieved by these birds was based on echoes from the weights stretching the obsta- cles at the bottoms of three rows, or the attachments of the ceiling.

Cave swiftlets are delicate birds which are difficult to maintain in captivity in good physical condition for more than a day or two, even though they were force- fed food mixtures suitable for insectivorous birds. All experiments considered below involved swiftlets that had been in captivity only one or twro days and appeared approximately as vigorous and adept at avoiding obstacles as when first brought into the flight chamber. All data from these four birds during this period are included in Table I except for times when they would, after several minutes of flight, temporarily appear weak or refuse to fly the length of the flight chamber. One of us held each bird in the hand roughly 30-40 cm below the ceiling and

!•( IK (LOCATION IN CAVK SWIFTLETS

497

released it only after the lights wen- extinguished. Tn most cases one of us watched with the infrared viewer from the opposite end of the flight chamber in the hope of observing the bird's flight path. This instrument afforded a clear view of the moving bird in only a minority of the flights, but careful listening to the orienta- tion sounds sufficed to show the bird's approximate flight path, and as soon as it had passed through one to four barrier planes we switched on our flashlights and inspected the obstacles immediately to detect vibration or other motion. Unfor- tunately the sound of contact with ob.stacles could not be used, as we have com- monly done with bats, because it was masked by the orientation sounds of the birds. Since no test was begun until all obstacles had again come to rest, and since even a light brush of a swiftlet's wing set the obstacles in evident motion, we feel con- fident that we detected most contacts with obstacles.

TABLE I

Obstacle avoidance scores of Collocalia vanikorensis grant! flying through an array of vertical wires and rods. N indicates nuinhcr of trials, ' ',, M per cent misses. All flights in darkness or with the bird blindfolded. The flights marked "Shifted" ivere those immediately after the obstacles had been moved horizontally b\ several centimeters.

		2 mm plastic	6..? mm	8 mm
		covered win--	iron rods	plastic tuhiiiK
Uiid No.	I >ate
		2V	', i/	N	', u	N	', i/
1	23 July	20	50',
	23 July (blindfolded)	56	46^;			16	63<?<
2	23 July	82	48',			20	so' ;
	24 Julv	26	42' ,	17	76',	20	70',
3	25 July	55	31',	31	74',	31	58' ,
4	29 Julv	68	40',	29	83 f-f	30	77',
	29 Julv (shifted)	2	50r;	12	92',	10	70',
Total July 24-29	151	37<%	89	80',	91	68 ';
Total of all birds		309	43',	89	80' ;	127	689?

The positions of the obstacles, particularly those in the middle row, were shifted horizontally from time to time without any noticeable effect on the birds' success at avoiding them. An especial effort was made to do this with bird Xo. 4. Tt was enclosed in the cupped hands while obstacles were shifted and released only after the lights had been turned off. The performance of this bird was somewhat better during flights immediately after such a shift in obstacle position. It thus seems unlikely that learning of the position of obstacles (Griffin, 1058. pp. 162-167; Neuweiler and Moehres, 1967) had any appreciable effect in these experiments. The two millimeter obstacles were shifted less often than the larger ones, so that the birds had a better opportunity to learn their positions, but this did not elimi- nate the substantial difference in avoidance of the larger and smaller cylinders.

The orientation sounds of two swiftlets were recorded as they flew in the dark- ened chamber with a Roberts model 6000 stereo tape recorder and Electro Voice model 644 microphones. The frequency response of this system was limited by the microphone which was flat within about ±3 dB from 700 to 12,000 Hz and down about 10 dB at 17,000 Hz. Portions of this recording were displayed on an

DONALD K. (jKII-'HX AND l\'< >I >KK ICK A. SUTHERS

oscilloscope and photographed for further analysis. Spectrographs of a numher of clicks were also made with a Kay Electric Co. model 6061B sound spectrograph.

RESULTS

Obstacle avoidance scores for all four hirds under all conditions tested are included in Table I for the sake of completeness, but there do not seem to be any significant differences between birds or conditions. Consequently the overall aver- ages provide the most reliable picture. All data are pooled as in the final line of the table, but the last three days are also listed separately because on those days all three birds were tested with all sizes of obstacle. The results are consistent in showing a large difference between 2 millimeter and 6.3 millimeter obstacles and a somewhat poorer performance with the 8 millimeter plastic tubing. The flight paths of these birds were too variable and insufficiently well observed to allow anv accurate calculation of chance scores, but the poorest performers registered about 30 to 40% misses. When we watched the birds with the infrared viewer our impression was that the two larger sizes of obstacles were avoided by turns and dodging maneuvers. But the resolution of this instrument under the conditions of our experiments was too poor to justify much confidence in this observation.

»»M»I» . r^}+tiin>i 1 1 Hi

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FIGURE 1. Oscillographic display of clicks emitted by Collocalia vanikorensis; (a) most commonly emitted type consisting of double click; (b) less common type composed of three high amplitude portions. Arrows indicate two initial high amplitude portions of envelope which are responsible for double vertical bars on sonagrams in Figure 2a. Bar equals 10 msec.

The poorer performance with 8 millimeter plastic tubing than with 6.3 milli- meter iron rods is somewhat puzzling, since the echoes from the larger cylinders must have been more intense. Our impression was that motion of the iron rods after a light touch was more difficult to observe and more quickly damped out. Thus we feel the most likely explanation for the drop from 80 to 68% misses was that we failed to detect motion of the 6.3 mm rods after some light touches.

Each orientation sound typically consisted of a moderate amplitude peak a few milliseconds long, followed after several milliseconds by an appreciably higher in- tensity peak about -I to 8 msec in duration ( Fig. la). This second high amplitude peak continued as a gradually attenuated complex waveform which disappeared into the noise level of the recording after about 30 to 50 msec. Much of this

ECHOLOCATION IN CAVE SWIFTLETS

199

terminal portion was probably due to echoes from the chamber walls, floor or ceil- ing. The double nature of each click was clearly audible when the tape recording was reproduced at one-eighth its original speed. Occasional pulses also showed a third high amplitude peak (Fig. Ib). Click intervals (measured as the time between the maximum amplitude portions of successive clicks, rather than the silent intervals between them) for 477 clicks comprising several flights averaged

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FIGURE 2. Sound spectrographs of eight successive orientation clicks of a single C. vani- korensis flying in the dark. Approximate sensitivity of recording system uniform within ±3 dB from 0.7 to 12 kHz and down 10 dB at 17 kHz; (a) wide band width display (300 cps at 3 dB down) showing temporal relationships; (b) intensity contour plot indicating relative in- tensity by 6 dB steps between contours. Darkest contours represent most intense sounds; (c) narrow band width display (45 cps at 3 dB down) giving better frequency resolution but blurring temporal relationships and losing some lower intensity portions.

500 DON A 1. 1) R. ( IK 1 I<M- IN AND RODERICK A. SUTHERS

1 16 msec with a minimum of 48 msec and a maximum of 358 msec, corresponding to repetition rates of about 3 and 20 clicks per second, respectively.

The double nature of the clicks is readily visible in the spectrograms (Fig. 2) which also indicate the distribution of sound energy in each click. Tt is evident that clicks of C. vanikorensis have a wide frequency spectrum most of which appears to lie within the human audible range. The highest intensity sound occurs between 4.5 and 7.5 kHz, but as in all impulsive clicks there is appreciable sound energy at higher frequencies. A few clicks showed small amounts of acoustic energy as high as 16 kHz, the upper frequency limit of the spectrogram, but this is uncommon. Very faint low frequency portions of the clicks extend downward to about 2 kHz.

DISCUSSION

Medway (1959) reported most of the acoustic energy of clicks emitted by CoUocalia maxima loivi occurred between 2 and 4.5 kHz with very little energy below 1.5 or above 5.5 kHz. Novick (1959) found the principal frequency of CoUocalia brcvirostris imicolor to lie between 4 and 5 kHz but noted many over- tones. These apparent interspecific differences may in part reflect different sensi- tivities of the various tape recorders and microphones to high frequencies. Tt does however appear that clicks produced by C. vanikorensis contain relatively more high frequency components than do those of other CoUocalia thus far studied. The presence of these high frequencies could theoretically improve the bird's ability to detect echoes from small objects. It is interesting to note that Medway (1967) found C. jiiciphacia. which emits clicks with frequencies principally in the range between 1.5 and 4.5 kHz, unable to echolocate a barrier of wooden rods 1 cm2 in cross sec- tion, whereas our data indicate C. vanikorensis regularly echolocates iron rods 6.3 mm in diameter.

While it would clearlv be desirable to obtain considerably more extensive data involving more birds, a wider range of obstacle sizes and spacings, and more trials, these results are of interest because no previous investigations have included even such approximate determinations of the threshold sizes of obstacles which CoUocalia can detect by echolocation. Under more or less favorable conditions the threshold size of obstacles may vary to some extent, but it seems unlikely that it would be far outside the interval between 2 and 6 millimeter diameter cylindrical obstacles.

Although the auditory sensitivity of Colloralia has not been studied directlv, the extensive data on other birds ( Schwartzkopff. 1968; Konishi. 1969) suggest that they are not likely to have high auditory sensitivity extending above 20 kHz. Tn the absence of direct evidence, it seems most likelv that CoUocalia, like all other

j

birds that have been adequately studied, have a frequency range of hearing roughly comparable to our own. Tt also seems likely, though not absolutely certain, that C. vanikorensis employs orientation sounds without useful components above the range of human hearing. If so, the wavelengths of sound used by these birds for echolocation are also available to blind men attempting echolocation based on audible sounds. This implication of the still verv poorly understood acuity of echolocation in CoUocalia justifies further and more intensive study of the questions discussed in this paper.

ECHOLOCATION IN CAVE SWIFTLETS 51 1 1

The support of the National Science Foundation is gratefully acknowledged, including both the 1969 ALPHA HELIX Expedition, and grant GB 7155 to the New York Zoological Society.

SUMMARY

The acuity of echolocation in Collocalia vanikorensis granti was studied by measuring the success of freshly captured birds in avoiding cylindrical obstacles in total darkness. The percentage of misses varied from approximately 40% with 2 millimeter insulated wires to 80% with 6.3 millimeter iron rods and 68>% with 8 millimeter plastic tubing. The orientation sounds used for echolocation are clicks with the major energy between 4.5 and 7.5 kHz. Although the frequency range of hearing in these birds has not yet been measured, it seems probable that they can echolocate obstacles as small as 6 millimeter diameter rods by means of fre- quencies within the range of human hearing.

LITERATURE CITED

GRIFFIN, D. R., 1954. Acoustic orientation in the oil bird, Stcatontis. Proc. Nat. Acad. Sci.,

39: 884-893. GRIFFIN", D. R., 1958. Listening in the Dark. Yale University Press, New Haven, Connecticut,

413 pp. GRIFFIN, D. R., A. NOVICK AND M. KORNFIELD, 1958. The sensitivity of echolocation in the

fruit bat Rousettus. Biol. Bull., 115: 107-113. KONISHI, M., 1969. Hearing, single-unit analysis, and vocalizations in songbirds. Science, 166:

1178-1181.

MEDWAY, LORD, 1959. Echolocation among Collocalia. Nature, 184: 1352-1353. MEDWAY, LORD, 1967. The function of echonavigation among swiftlets. Anim. Behav., 15:

416-420. NEUWEILER, G., AND F. P. MOEHRES, 1967. The role of spatial memory in the orientation.

Pages 129-140 in R-G. Busnel, Ed., Animal Sonar Systems. Laboratoire de Physio- logic Acoustique, Jouy-en-Josas, France.

NOVICK, A., 1959. Acoustic orientation in the cave swiftlet. Biol. Bull, 117: 497-503. RALLS, K., 1965. Auditory sensitivity in mice: Pcromyscus and Mtts musculus. Anim. Behav.,

15: 123-128. SCHNITZLER, H-U., 1967. Discrimination of thin wires by flying horseshoe bats (Rhinolophi-

dae). Pages 69-87 in R-G. Busnel, Ed., Animal Sonar Systems. Laboratoire de

Physiologic Acoustique, Jouy-en-Josas, France. SCHWARTZKOPFF, J., 1968. Structure and function of the ear and of the auditory brain areas

in birds. Pages 41-59 in A. V. S. Reuck and J. Knight, Eds., Ciba Symposium on

Hearing Mechanisms in Vertebrates. Churchill, London.

Si IHKKS, R. A., 1965. Acoustic orientation by fish-catching bats. /. Exp. Zool, 158: 319-348. SUTHERS, R. A., 1967. Comparative echolocation by fishing bats. /. Mammal, 48: 79-87.

Reference: Biol. Bull., 139: 502-500. (December, 1970)

THE ROLE OF AMOEBOCYTES IN THE REGENERATION OF

SHELL IN THE LAND PULMONATE, EUPLECTA

IN DIC A (PFIEFFER)

S. P. KAPUR AND A. SEN GUPTA Department of Zonlnrjy, Punjab University, Chandigarh, India

The presence of amoebocytes at the site of shell regeneration in gastropods has been observed by several workers. But opinions and observations differ as to the manner in which the amoebocytes participate in the repair process. Burning (1957) states that in Hcli.v aspcrsa amoebocytes appear along cracks in the regenerating membrane due to irritation and he dismisses them as unconnected to shell repair. McGee-Russell (1954) also noted in regenerating snails the presence of some cells in the extrapallial fluid that originated from mantle epithelium but he did not assign them any role in the repair process. Abolins-Krogis (1963, 1968) opines that during shell repair, amoebocytes transfer large amounts of repairing materials like proteins and calcium from the yellow body cells and digestive gland cells to the actual site of shell repair, liberate it there and withdraw from the site. But some of the cells become accidentally trapped within the calcifying plates of the shell. To complicate matters further, studies of Kapur and Gibson (1967) on the normal development of shell in Helisoina dnryi ciidiscus point to the appearance of amoebo- cytes on the ostracum of the shell in the juvenile snails and that later these form multinucleate organic plates. These have been suggested to be directly involved in the calcification of the adult shell.

With such divergent views on the role of amoebocytes in the repairing as well as normal shells, the controversy is wide open for more studies. Also since studies on shell repair in gastropods have almost exclusively been on a single land snail, Helix, it was decided that new investigations should be carried out in other genera of land pulmonates. The present communication deals with the regeneration of shell in Euplecta indica (Pfeiffer), a terrestrial pulmonate from Chandigarh, India.

MATERIALS AND METHODS

Specimens of Euplecta indica were collected from a garden in Chandigarh. They were maintained in a terrarium at 21° C and were fed on cabbage leaves. Seven groups of thirty snails each were used in this study. A hole, about 3 to 4 mm in diameter, was made in the shell of each snail, some distance behind the shell aperture. It was then covered with a piece of glass coverslip, using warm paraffin wax for pasting. The snails deposited the regenerating shell material on the undersurface of the coverslips. At ten different intervals, ranging from 1 hour to 108 hours, the coverslip bearing the regenerate material was removed from the shell, placed in the required fixative and processed accordingly. Regenerate materials obtained thus at different intervals from each group of 30 snails were stained with the following techniques: group 1. haematoxylin and eosin ; group 2, toluidine blue;

502

ROLE OF AMOKIUH YTHS IN SHELL KKI'AIK 503

group 3, alcian blue-Delafield haematoxylin ; group 4, mercuric bromophenol blue (Hg-BPB) ; group 5, periodic acid-Schiff (PAS) with and without diastase diges- tion; group 6, Sudan black B and group 7, Feulgen test. Regenerate samples from 1 to 4 and 7 were fixed in 10% neutral formaline, from group 5 in Bouin's fluid and from group 6 in formol-calcium. Some neutral formalin-fixed samples of shell regenerate were tested for the presence of calcium, using the von Kossa tech- nique. In group 3, Delafield haematoxylin was used as a nuclear counterstain instead of the recommended neutral red because batches of this dye then available were not compatible with the alcian blue technique. All the histochemical tech- niques were carried out as described by Pearse (1961). The regenerate-bearing pieces of coverslip after staining, dehydration and clearing were mounted as whole mounts in Canada balsam, with the regenerate-bearing surface facing upwards. Samples from group 6 were similarly mounted in glycerine jelly.

It was observed during this study that given everything equal, including size of animals, their time of collection and feeding, the rate of shell repair varied between individuals. The timings given are not rigid invariables, but represent the average conditions seen in the greatest number of individuals in all the groups used in this study.

OBSERVATIONS

The process of shell repair is initiated by the deposition of a glistening, trans- lucent regeneration membrane, one hour after shell damage. This membrane stains uniformly blue with haematoxylin, is uniformly PAS positive with and without diastase digestion, is slightly metachromatic in toluidine blue, stains bluish-green in alcian blue, gives a positive test with Hg-BPB and is also weakly sudanophil. No localization of staining was observed by any of the above techniques, as the regeneration membrane is homogeneous and structureless. The above mentioned tests show the presence of neutral mucopolysaccharides, glycoproteins, mucopro- teins, acid mucopolysaccharides, proteins and small amounts of lipids.

One hour after the start of regeneration, amoebocytes suddenly make their appearance on the regenerating membrane, continue to increase in number until at six hours their number reaches the highest peak (Fig. 1). Two types of amoebo- cytes were observed and we designate them as the a-type and the b-type. Nuclei of a-type cells are deeply basophilic with a compact coarsely granular meshwork of chromatin. Nuclei of b-type cells are vesicular, with scattered chromatin granules interlinked by fine fibrils. The nucleo-cytoplasmic ratio of the two kinds of cells also differs : nuclei of a-type cells occupy a third or a quarter of the total cell volume whereas nuclei of the b-type cells occupy more than half the extent of the cell (Fig. 2). Further, the a-type amoebocytes are about half the size of the b-type amoebocytes, but occasionally acquire the full size of the b-type amoebocytes. Occasionally the nuclei of some a-type amoebocytes possess U, V, J or dumbbell shapes. Possibly the nuclei of these cells are undergoing necrosis (Fig. 6).

The nuclei of both types of cells showed the usual basophilia with haematoxylin and toluidine blue. Besides, the chromatin granules in both types of nuclei show metachromasia with toluidine blue and also stain positively with alcian blue. The general cytoplasm of both cells is eosinophilic toluidine blue positive and alcian blue positive. The cytoplasmic granules of the a-type amoebocytes show metachromasia

504

S. P. KA1TR AND A. SKN GUPTA

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ROLE OF AMOEBOCYTES IN SHELL REPAIR 5<J5

with toluidine blue. Calcium is present in the cytoplasm of both types of amoebo- cytes in the form of granules (Fig. 3). Nuclei of both cell-types give a positive test with Hg-BPB but the nucleus of the a-type does so more intensely, while the nucleoplasm of the b-type remains unstained. The cytoplasm of both cell-types is also positive to Hg-BPB but in the b-type cells, a narrow perinuclear zone remains unstained. Nuclei of both cell-types are negative to Sudan black B staining but their cytoplasm is diffusely positive. Some sudanophilic granules are present in the perinuclear zone which as stated above, gives no response to Hg-BPB staining. Nuclei of both cell-types are Feulgen positive.

The preceding observations relate to the amoebocytes that have just arrived at the regeneration site. But soon, about an hour after their arrival, the amoebocytes cluster and start losing their identity (Fig. 4). It appears that their outer cell membranes rupture and their cytoplasm precipitates, leaving their nuclei in a free, denuded state (Fig. 5). Such patches of precipitated cell material exhibit the same staining reactions as in the intact amoebocytes. The}- give positive results for neutral mucopolysaccharides, glycoproteins, mucoproteins, acid mucopolysaccharides, proteins and lipids. The free nuclei are seen as long as 83 hours after the precipi- tation of the amoebocytes. However, gradually, these nuclei become pycnotic, diminish in size and finally disintegrate. Such disintegrated nuclear material along with the precipitated cytoplasm is still Feulgen positive. The a-type cells with U, V, J and dumbbell-shaped nuclei also behave in the same manner (Fig. 6).

The site of repair now has a regeneration membrane bearing large numbers of intact amoebocytes and also patches of precipitated cells which had arrived earlier. In addition, numerous fine tracts of acid mucopolysaccharides are observed. These are possibly derived from the secretion products present in the extrapallial fluid which is constantly bathing the regenerating material.

The first indication of the initiation of the calcifying process of the regenerate is given by the appearance of small crystals of calcium carbonate on top of or along the precipitated cytoplasm of the amoebocytes and the fine tracts of acid mucoploy- saccharides. These crystals are in spheritic double-fan shaped arrays (Fig. 7). Thus the materials contributed by the amoebocytes serve as the calcifying sub- stratum. As crystallization proceeds, a steady decline in the metachromasia of the organic substratum is noted. The regeneration plate thus comes to have sev- eral calcifying tracts showing crystallization of calcium carbonate, coincidental with the tracts of cellular precipitation. The process of arrival of amoebocytes and their precipitation is a continued one, occurring simultaneously with crystal formation from this point onwards. Thus the precipitation of the amoebocytes contributes to

FIGURE 1. Two-hour regenerate: amoebocytes on the regeneration membrane ; toluidine blue.

FIGURE 2. Six-hour regenerate : a- and b-type amoebocytes. Note the difference in the nuclei of the two cell types ; toluidine blue.

FIGURE 3. Four-hour regenerate : note the calcium granules in the cytoplasm of the amoe- bocytes ; von Kossa-neutral red.

FIGURE 4. Six-hour regenerate : intact cells at right, precipitated cells on the left of the picture ; toluidine blue.

FIGURE S. Six-hour regenerate: free nuclei lying in the precipitated cytoplasm; alcian blue-Delafield haematoxylin.

FIGURE 6. Six-hour regenerate : free pycnotic nuclei of various shapes ; one intact b-type amoebocyte can be seen ; toluidine blue.

506

S. r. KAl'UR AND A. SEN GUT'I \

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ROLE OF \M<)KM<KTTKS IN SI1KLL KKI'AIU

the formation of the organic ostracum upon which the growth of the mineral fraction of the shell proceeds. The growing calcium carbonate crystals soon acquire a tabular form (Fig. 8).

As these crystals grow larger, they meet adjacent growing tabulae at their peripheries and coalesce with them (Fig. 9). Small acicular crystals appear in parallel and in spheritic aggregates in the spaces between such intergrowing tabulae (Fig. 10). A second layer of crystals is initiated on top of the first layer even before the latter is completed. Thus the regenerate grows in extent as well as in thickness at the same time.

At 24 hours the regenerate consists of an almost complete sheet of tabulae, -with most of the intervening spaces filled up by parallel and spheritic aggregates of crystals (Fig. 11). New batches of amoebocytes continue to arrive and precipitate, the only difference now being that whereas the first batches arrived and precipitated on the regeneration membrane, the later arrivals precipitate upon the crystalline layers. Successive layers of crystals are laid down until in about a week's time the regenerate approaches the original shell in thickness.

In surface view the regenerate appears as a thick conglomerate of crystals. It actually consists of several indistinctly separated crystalline sheets, each of which may be seen in a different plane of focus at the edges.

The fully regenerated shell is intimately fused with the original shell and cannot be easily separated from it. The regenerate lacks a periostracum, which is present in the normal shell. It also differs from the latter in its random arrangement of crystals and in its partly cellular origin, of which the normal shell presents no evidence.

DISCUSSION

It has been observed in this study that within an hour of shell damage, a homo- geneous, thin, translucent membrane appears over the damage area of the shell. This has probably come about by the precipitation of secreted organic substances present in the extra-pallial fluid. To this extent shell regeneration is a physico- chemical process. But we cannot agree with workers like Manigault (1933) and McGee-Russell (1954) on Helix that the entire regeneration of shell is brought about in this manner. On the other hand observations of Wagge (1951) also on Helix that the regeneration membrane is formed exclusively by the amoebocytes which arrive at the site to deliver proteins and calcium are not tenable here. What we find is that once the precipitated membrane is formed at the beginning of regen- eration, it acts as a scaffolding for the amoebocytes to perform their role in regeneration.

FIGURE 7. Seven-hour regenerate : initial double-fan shaped and spheritic crystal aggregates in association with the cellular precipitate and two free pycnotic nuclei ; toluidine blue.

FIGURE 8. Nine-hour regenerate: growing tabloid crystals on the organic ostracum.

FIGURE 9. Ten-hour regenerate : intergrowth of tabulae. Note coalescence at the margins of the crystals.

FIGURE 10. Twelve-hour regenerate : parallel and spheritic crystal aggregates appearing in the spaces between the tubulae.

FIGURE 11. Twenty-four-hour regenerate: note that the spaces between the tabulae are nearly filled by the parallel and spheritic aggregates of crystals.

508 S. I'. KAJ'UK AM) A. SEN GUPTA

Further it is observed that an hour after the start of shell regeneration, large numbers of amoebocytes arrive at the newly formed regeneration membrane. But unlike observations nf \Yagge (1(»51) it is found that these cells do not merely deliver their pay loads and then withdraw from the membrane. Instead, it appears that the amoebocytes lose their identity, their outer cell membranes become dis- organized and their cytoplasm precipitates, leaving free nuclei. In fact the amoebo- cytes arrive in such large numbers that the original regeneration membrane is no longer seen. Histochemical tests on the intact and disorganized cells reveal the presence of large amounts of protein, PAS positive mucopolysaccharides and acid mucopolysaccharides. These substances are necessary components of most calci- fying matrices in animals. Besides, calcium granules were also detected in the cytoplasm of the amoebocytes. These findings are in agreement with those of Abolins-Krogis on Helix (1960, 1963 and N(>8) that amoebocytes carry histo- chemical substances necessary for calcification from different organs of the body to the site of regeneration. We further find that not only are these substances transferred to the regeneration site, but also that the cytoplasm of the amoebocytes contributes predominantly to the formation of the organic ostracum on which the calcification processes are initiated.

The calcification of the regenerate is initiated by the appearance of small double- fan shaped spheritic aggregates of crystals. These initial crystals have also been observed by Abolins-Krogis (1968) in her studies on Helix, although she invests their structure with a large amount of organic content.

The metachromasia noticed prior to the beginning of crystallization is seen to recede in the later stages of calcification. This may be indicative of the binding of calcium by acid radicles on the acid mucopolysaccharide molecules (Tanaka and Hatano, 1955; Horiguchi, 1956; Kado, 1960) which are in association with the protein matrix. As the tabloid crystals grow large, they fuse with adjacent crystals and tend to form a continuous sheet. New batches of amoebocytes continue to arrive and settle over the crystalline sheets and thin films of organic material which is probably continually precipitated from the extrapallial fluid. Soon the amoebo- cytes become disorganized, spill over their contents and start calcification of another crystalline sheet once again.

Although no cellular involvement is noticeable in the structure of the adult shell of Euplecta, the involvement of amoebocytes in the calcification of developing and adult shell is known from the studies of Kapur and Gibson (1967) and Kapur and Bansal (in preparation). In fact, Kapur and Gibson found precipitation of the newly arrived amoebocytes on the protoconch and subsequent initiation of calci- fication in Hclisoina duryi cudiscus. The disruption of amoebocytes at the site of shell regeneration reminds us of a rather similar behavior of leucocytes in wound healing in vertebrates.

The authors are grateful to Professor G. P. Sharma, Head of the Department of Zoology, Pan jab University, for encouragement and for providing the necessary laboratory facilities during the course of this work.

ROLE OF AMOEBOCYTES IN SHELL REPAIR 509

SUMMARY

An hour after the damage of the shell in Enplccta indie a (Pfieffer) a thin, structureless, basophilic regeneration ineinhrane appears. Thereafter, a large num- ber of amoebocytes precipitate on this membrane. Soon these break up and con- tribute their cytoplasmic contents to the regenerate. This is positive for glyco- proteins, mucoproteins, neutral tnucopolysaccharides, acid mucopolysaccharides, proteins, calcium and some lipids. Also, tracts of acid mucopolysaccharides pre- cipitate from the extrapallial fluid and are deposited on the regenerate.

Calcification is initiated by the appearance of seed crystals of calcium carbonate on top of or along areas of cellular precipitation. This leads to a progressive decrease in metachromasia. The crystals grow into large tabular forms, coalesce with the adjacent tabulae and form a calcified sheet of regenerated material. The spaces between the tabulae are later packed by small crystal aggregates of various shapes. More and more amoebocytes continue to arrive, settle on the calcified sheets along with precipitated organic matter from the extrapallial fluid, break up and repeat the calcifying process until a regenerate of the same thickness as the normal shell is formed.

LITERATURE CITED

ABOLINS-KROGIS, A., 1960. The histochemistry of the hepatopancreas of Helix poinatia (L.) in

relation to the regeneration of the shell. Arkiv. ZooL, 13: 159-201. ABOLINS-KROGIS, A., 1963. The histochemistry of the mantle of Helix poinatia (L.) in relation

to the repair of the damaged shell. Arkiv. ZooL, 15: 461-474. ABOLINS-KROGIS, A., 1968. Shell regeneration in Helix pomatia with special reference to the

elementary calcifying particles. Symp. ZooL Soc. London, 22: 75-92. BURNING, W. C, 1957. Repair of a defect in the shell of the snail, Helix aspcrsa. J. Bone and

Joint Sitrg., 39 A (2) : 377-393. HORIGUCHI, Y., 1956. Biochemical studies on Pteria (Pinctada} martcnsii (Dunker) and Hyri-

opsis schh-gclii (v. Martens). II. Separation of crude sulfomucoplysaccharides from

various tissues of Pteria (Pinctada) martcnsii (Dunker) and Hyriopsis schlegclii (v.

Martens) . Bull. Jap. Soc. Sci. Fish., 22 : 463-466. KADO, Y., 1960. Studies on shell formation in molluscs. /. Sci. Hiroshima Univ. Ser. B, Div.

J., 19: 163-210. KAPUR, S. P., AND M. A. GIBSON, 1967. A histological study of the development of the mantle

edge and shell in Hclisoma duryi cudiscus (Pilsbry). Can. J. ZooL, 45: 1169-1181. MANIGAULT, P., 1933. La tension du gaz carbonique, condition de la regeneration de la coquille

chez les Helix. Trav. Sta. Biol. Roscoff., 11 : 51-59. McGEE-RussELL, E. S., 1954. A cytological study of tissues concerned in secretion of shell in

the snail, Helix aspcrsa. Ph.D. thesis, Oxford University. PEARSE, A. G. E., 1961. Histochemistry: Theoretical and Applied. ]. and A. Churchill Ltd.,

London, 998 pp. TANAKA, S., AND H. HATANO, 1955. Biochemical studies on the pearl oyster, Pinctada martcnsii.

IV. On the uptake of radioactive calcium by pearl oyster and its deposition on the pearl

and the shell. /. Chcm. Soc. Japan, 76: 602-605.

WAGGE, L. E., 1951. The activity of amoebocytes and of alkaline phosphatases during the re- generation of the shell in the snail, Helix aspcrsa. Quart. J. Microscop. Sci., 92:

307-321.

Ki.-kmK.-e: liiol. Hull., 139: 510-519. (December, 1970)

"DISTANCE EFFECT" IN PIGEON ORIENTATION :

AN EVALUATION

WILLIAM T. KEKTON Section of Neurobiology and Bchurior, Cornell University, Itlmca, New York 14850

In several papers, Matthews (1955, 1963) has reported that his pigeons exhibit poorer orientation when released at intermediate distances than when released at very short distances or at long distances. He interprets this "distance effect" as indicating that the pigeons' system of navigation is not sufficiently accurate to be used effectively at distances less than about 80 km. Matthews explains the accurate vanishing hearings of his pigeons at very short distances as probably being due to pilotage by familiar landmarks rather than to true navigation. He suggests that at intermediate distances the birds are too far from home to use landmarks but still too close to use accurately their bicoordinate navigation system, hence their poor homeward orientation.

Mittelstaedt (in Schmidt-Koenig, 1963a, 1963b) examined orientation as a function of distance in terms of the homeward component (i.e., the component of the mean vanishing vector that is in the homeward direction), but the results of his analysis of the initial orientation of naive pigeons in Germany were inconclusive. An analysis by Schmidt-Koenig (1965, page 244) of the orientation of Matthews' experienced pigeons in England provided no convincing evidence of a distance effect, nor was convincing support for such an effect obtained in an elaborate series of releases conducted by Schmidt-Koenig (1963) in North Carolina. Later, however, Schmidt-Koenig conducted extensive tests of the "distance effect in experienced pigeons flown in both North Carolina (1964, 1966) and Germany (1968), and published results that seemed to support Matthews' ideas. He found high values of the homeward component at distances less than about 19 km and greater than about 96 km ; between these distances there was a zone of poor orientation. Wall- raff (1967) reported similar results with untrained pigeons released on their first homing flights in Germany.

If the distance effect is a general characteristic of pigeon homing, it has impor- tant implications for the nature of the navigation system used by these birds. It would be consistent, for example, with a system such as the sun-arc hypothesis of Matthews (1953), which would be effective at short distances only if the birds could determine the sun's position with far greater precision than most workers have believed possible. Matthews (1968) has recently relied heavily on the distance effect in interpreting much of the published data on pigeon homing.

Because of the theoretical importance of the distance effect if it really exists, and because I have doubted its general occurrence in view of the very good home- ward orientation regularly exhibited by both our untrained (Keeton and Gobert, 1970) and our experienced (Keeon, 1969) pigeons at the intermediate distances where orientation should be poorest, my colleagues and I conducted, during 1'HiS

510

"blSTANCK KI-FECT" IN ORIENTATION 511

and 1969, an extensive series of test releases to determine whether any distance effect is evident in the performance of our pigeons.

.\ I KT 1 1 ODS

Test releases were performed at various distances north, east, south, and west of the Cornell pigeon lofts at Ithaca, New York. More particularly, the release sites were: North— 5.9, 10.0, 26.8, 49.0, 84.6, 143.3, 200.3 km; East— 5.7, 14.9, 33.5, 60.9, 91.1, 129.8, 164.2, 204.2 km; South— 5.4, 12.1, 20.5, 32.6, 47.3, 49.5, 84.5, 124.6, 188.3 km; West— 9.0, 17.8, 33.9, 47.2, 70.3, 119.9, 155.9, 177.7 km. At all but the most distant sites, a minimum of three test releases (usually more) were conducted, using different birds, and on different days. Except in 12 in- stances, at least 10 single-tossed birds (mean = : 14.7) were used in each test release. Included in the analysis reported here are the results of a total of 172 test releases involving a total of 2525 single tosses of birds.

All birds used in the test releases had had previous homing experience from all four cardinal directions. For releases at 20 km or less, the birds' previous experi- ence had included single-toss flights from distances this great or greater. For re- leases at more distant sites, the birds had previously made single-toss flights from distances at least as great as that of the next nearer site in the series.

In each test release, the birds were tossed individually from the hand, the directions in which they were pointed at the toss being randomized. Each bird was observed with 10 X 50 binoculars until it vanished from sight, and a compass bear- ing for the vanishing point was recorded to the nearest 5 degrees. The interval between toss and vanish was timed with a stop watch. No bird was released until the previous bird had been out of sight at least 5 minutes.

The circular mean (both direction and vector length) of the vanishing bearings of each release was calculated by vector analysis, following the procedure outlined by Batschelet (1965). Bearings were tested for randomness by means of the Rayleigh test (Batschelet, 1965). The homeward component was calculated as proposed by Mittelstaedt (in Schmidt-Koenig, 1963a. 1963b) : h -- a -cos (a -- /?). where a is the length of the mean vector, a is the mean direction, and /? is the home direction. The homeward directional component was calculated bv the equation : d = cos (a — /?). Values for both the homeward component and the homeward directional component may range from -1 to +1; values for the length of the mean vector may range from 0 to -f 1.

For each of the four directions, the homeward component (HC), homeward directional component (HDC), and length of mean vector (LMY) were plotted as a function of the distance of the release sites from the loft. Tn each case, the curves are based on the arithmetic means of the corresponding values obtained from all tests at each site.

Since it seemed possible that the length of the vanishing intervals (elapsed time between toss and vanish) might indicate something about the relative difficulty of orienting at different release sites, the mean vanishing interval for each release was calculated, and then the means of the means for all tests at each site were plotted as a function of distance.

512

\\ ILLIAM T. KKKTON

Ml"

0 " 10 10

0 10 20 30 40 50

n"~ 70 80 90 100 fiO 120 130

"?0 80 90 100 110 120 130 140 150 .60 170 180 '90 200 310 220

1.00

'

.'

0 10 20 30 40 50 60 70 80 90 100 "0 120

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 «*

100

.80

-.40

01" 30 40 50 60 70 80 90 100 HO 120

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200

FIGURE 1, A-C.

"I>ISTAXCK KFKKC T" IX < )KI KNTATION

513

30 40 50 60 70 80 90 100 110 120

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 ISO 190 200 210

•

•

SUMMARr AIL DIRECTIONS

10 10 20

20 30 40

40 60 70

50 60 70

80 90 100 110 120

90

140 150

100 160 170

110

FIGURE 1. Graphs of the homeward component (solid curves), homeward directional com- ponent (dotted curves), and length of mean vector (dashed curves) plotted against distance of release from the home loft. The black dots indicate the values of the homeward component for the individual test releases ; the curve connects the arithmtic means of the values for each site ; A, releases from north of the loft ; B, releases from the east ; C, releases from the south ; D, releases from the west ; E, summary of all releases. The value indicated by X at 15.1 km in graph B is the mean homeward component for five releases at the alternate site discussed in the text.

RESULTS

Figures 1A, IB, 1C, and ID show the results of our releases from north, east, south, and west of the Cornell lofts, respectively. Figure IE is a graph of the results of averaging the values from the four directions.

Not only do our graphs of the homeward component show little resemblance to those based on Schmidt-Koenig's data, but also they show few resemblances among themselves. There is no indication that the homeward component varies consist- ently as a function of distance in the manner suggested by Matthews, Schmidt-

514

YVII.U AM T. KKKTON

Koenig, and \\'allralT. More specifically, the poor orientation reported by those authors for intermediate distances is not seen — indeed the average of the homeward components for 27-34 km (17-21 miles) is 0.67, a remarkably high value; it is difficult to imagine that significantly better orientation could be obtained consistently at any distance.

That the good orientation at intermediate distances shown by the experienced birds used in the graphed releases is not simply a result of training is demonstrated by the equally good orientation at these distances regularly exhibited by our first- flight pigeons. Figure 2 shows examples of typical releases of such birds at roughly

0

FIGURE 2. Examples of vanishing bearings of first-flight pigeons (ones never before taken away from the loft) released under sunny conditions: A, Harford, New York, 9.4 miles from the loft, April 25 and May 5, 1969, home bearing 276°, mean bearing 283° (nonrandom, P • 0.0001) ; B, Burdett, New York, 21.1 miles, October 23, 1968, home bearing 91°, mean bearing 101° (nonrandom, P- 0.002); C, near Locke, New York, 16.6 miles, October 23, 1968, home- bearing 171°, mean bearing 205° (nonrandom, P- 0.005) ; D, Fleming, New York, 30.4 miles, December 12, 1968, home bearing 164°, mean bearing 139° (nonrandom, P = 0.0007). [In this figure and in Figures 4 and 5, the home direction is indicated by a dashed arrow, true north by a thin line at the top of the circle, and the mean vector by a solid arrow whose length is in- versely proportional to the extent of scatter (the arrow would reach the periphery of the circle if there were no scatter, i.e., if all the birds vanished in exactly the same direction, and thi_- arrow would have 0 length if the vanishing bearings were uniformly scattered around the com- pass). Each symbol on the periphery of the large circle represents the vanishing bearing of one bird.]

"DISTANCE EFFECT" IN ORIENTATION

515

16, 32, and 48 km (10, 20, and 30 miles) ; other examples have been published elsewhere (Keeton and Gobert, 1970).

Figure 3 shows mean vanishing intervals plotted against distance. The curves for the four cardinal directions show few resemblances among themselves. Except that for all directions vanishing intervals tend to be short at near (4—20 km) release points, there is no consistent relationship between vanishing interval and distance. In this regard, our results agree with those of Schmidt-Koenig (1966).

DISCUSSION

In the published studies of distance effect, only the homeward component has been used as a measure of orientational performance. But the homeward compo- nent is not a particularly good measure since it is sensitive both to the deviation of the mean vector from the true home direction and to the extent of scatter of the bearings. Thus a homeward component of 0 could result from many fundamentally different distributions of the vanishing bearings, including two extreme distribu- tions that are the opposites of each other — a circularly uniform distribution (i.e., one with no mean vector) or a distribution in which all bearings deviate 90° in the same direction from home (i.e., one as different from circular uniformity as a dis- tribution can possibly be, but with the mean vector oriented at right angles to home). Clearly these distributions would have very different biological meanings, yet the homeward component permits no distinction between them.

Because of the desirability of distinguishing between the effects of mean direc- tional error and the effects of scatter, the graphs in Figure 1 show, in addition to

10

V.

110

100

120

130

0 10 20 V 40 50 60 80 90

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 KM.

FIGURE 3. Graph of mean vanishing intervals (in minutes) plotted against distance of

release from the home loft; Xorth, solid curve; East, dashed curve; South, dotted curve; West, dot-dashed curve.

516

WILLIAM T. KEETON

the homeward component, the homeward directional component and the length of the mean vector. The homeward directional component is a function only of the deviation of the mean vector from the Imme direction, and the length of the mean vector is a function only of the extent of scatter of the bearings. Hence these two indices give independent measures of the two variables that together determine the homeward component.

The figures indicate that the homeward directional component and the length of the mean vector vary independently. Thus, in Figure 1A it can be seen that the rises and falls of the homeward component curve at the shorter distances are clue almost entirely to variations from site to site in the degree of scatter of the bearings, the accuracy of the mean bearings remaining nearly constant, whereas at the longer distances the shape of the curve is determined by variations in the accu- racy of the mean bearings, the degree of scatter remaining nearly constant. In

Q

o9

Q

B

FIGURE 4. Release (A) showing very little scatter of the vanishing bearings but a mean deviating appreciably from the home direction, contrasted with a release (B) with much more scattered bearings but a more homeward directed mean ; A, Berry Hill Fire Tower, New York, 37.9 miles from the loft, October 14, 1968, home bearing 258°, mean bearing 299° (nonrandom, P < 0.0001) ; B, near New Berlin, New York, 56.6 miles, July 30, 1967, home bearing 257°, mean bearing 256° (nonrandom, P < 0.0001).

Figure IB, the similar values for the homeward component at the release sites 61 km and 91 km from the loft obscure the fact that at the 61 km site the mean vector deviates considerably from the home direction (mean HDC — 0.56) but the scatter of the bearings is minimal (mean LMV = 0.87), whereas at the 91 km site the mean vector is oriented very accurately toward home (mean HDC = 0.86) but the scatter of the bearings is greater (mean LMV = 0.67) ; i.e., the similar values for the homeward component at the two sites result from quite different behavior by the pigeons (Fig. 4). Similarly, a sharp drop in the value of the homeward component is seen at both the 143 km north site (Fig. 1A) and the 120 km west site (Fig. ID), but the behavior of the pigeons at the two sites is fundamentally different. At 120 km west (Fig. 5A), the birds vanish randomly or nearly ran- domly (mean LMV = 0.44) but sometimes with a slight tendency to go in the opposite direction from home (mean HDC: -0.44), whereas at 143 km north (Fig. 5B), the birds always vanish nonrandomly and consistently show very little

"DISTANCE EFFECT" IN ORIENTATION

517

scatter in their bearings (mean LMV =0.81) but the mean vector is always oriented considerably west of the true home direction (mean HDC = 0.39).

Some re-analysis of Schmidt-Koenig's data in terms of homeward directedness of the mean vector and scatter of the bearings is possible. Thus, for example, if we look at the original data of Schmidt-Koenig (1966, page 41) from which Wall- raff (1967, page 340) graphed the homeward component of Schmidt-Koenig's releases from the west, we discover that the nearly identical values for the home- ward component shown for the releases at 30 km and 40 km in Wallraff's curve result from very different behavior on the part of the pigeons. The homeward component of 0.06 for 30 km is the average of the values (+0.64 and —0.51) for two releases both of which had bearings that were distributed nonrandomly (P < 0.01) but in which the mean vectors were oriented in nearly opposite directions, one homeward and one away from home. By contrast, the homeward component

000

cP

FIGURE 5. Release (A) showing very great scatter of the vanishing bearings, contrasted with a release (B) with little scatter but a mean deviating by a large angle from the home direction ; A, Jersey Hill Fire Tower, New York, 74.5 miles from the loft, August 2, 1968, home bearing 85°, mean bearing 269° (random, P — 0.287) ; B, Castor Hill Fire Tower, New York, 89.1 miles, July 31, 1969, home bearing 200°, mean bearing 272° (nonrandom, P = 0.0005).

of 0.04 for 40 km is the average of the values (+0.23 and —0.14) for two releases both of which yielded random bearings. Thus emphasis on the homeward com- ponent has resulted in lumping together as similar examples of poor homeward orientation at intermediate distances values resulting from fundamentally different behavior. (Values for distances and for homeward components mentioned here are estimated from Schmidt-Koenig's (1966) figure and are thus only approxi- mate.)

Wallraff's (1967, page 341) graph of distance effect in his own first-flight pig- eons permits re-analysis in terms of homeward directedness of the mean vector and scatter of bearings. The results indicate that his data do not support the idea that orientation improves with distance. Thus his graph of releases from the east shows a rising homeward component at distances beyond 20 km, but this "improvement" turns out to be due largely to increased scatter in the bearings rather than to a more homeward-directed mean vector. In all but the first of his east releases, the

518 WILLIAM T. KEETON

mean vector is oriented away from home, hence the increasing scatter of his birds' bearings with distance results in a higher real value (but lower absolute value) for the homeward component. In short, there seems to be a trend toward randomness rather than toward improved homeward orientation.

The values for the homeward component obtained at one release site may differ significantly from those obtained at another site located approximately the same distance and direction from the home loft. For example, the mean homeward component at our 14.9 km east site (Fig. IB) is 0.79, indicating very good home- ward orientation. After conducting a series of five releases at this site (where the home direction is 247°), we performed a series of five releases at an alternate site 15.1 km east of the loft (home direction, 276°), and obtained a mean homeward component of 0.37, indicating much poorer orientation. Clearly the shape of the homeward component curve shown in Figure 2B would be quite different if the results at this alternate site were substituted for those at our usual site. The home- ward component appears to depend upon peculiarities of the individual release sites that are not correlated with distance from the loft. Depending upon ones selection of sites, curves of many different shapes could be obtained when the homeward component is graphed as a function of distance.

It follows from this type of analysis that the homeward component is not a very useful basis for comparing pigeons' orientational behavior at different release sites. Much more meaningful information is provided by the combination of the home- ward directional component and the length of the mean vector. However, our data indicate that neither of these varies consistently for our birds with the distance of the release site from the loft. Graue (1970) has recently also failed to find a dis- tance effect in the orientation of his birds in Ohio. The distance effect does not appear to be a general phenomenon, and hence its theoretical implications are doubtful.

I wish to express my thanks to all of the following people who helped conduct the releases reported in this paper: Andre Gobert, A. Irene Brown, Lorraine Pak- kala, Donald Windsor, Judith R. Alexander and Howard French. This research was financed by grant No. GB-13046X from the National Science Foundation.

SUMMARY

1. Matthews (1955, 1963), Schmidt-Koenig (1964, 1966, 1968), and Wallraff (1967) have reported that pigeons orient toward home best when released close to the loft or at a long distance from it ; they report poor orientation at intermediate distances, and suggest that this has important implications for the nature of the navigational system used by birds.

2. We have failed to detect any such distance effect in 172 test releases utilizing 2525 single-tossed pigeons from the Cornell lofts. When the homeward component is plotted against distance, we obtain dissimilar curves for the four cardinal direc- tions. Furthermore, the values of the homeward component may be quite different at two release sites approximately the same distance and direction from the loft.

"DISTANCE EFFECT" IN ORIENTATION 519

3. We obtained particularly good orientation at the intermediate distances where, according to the distance effect, it should be poorest. This was true not only of experienced birds but also of first-flight youngsters.

4. Analysis of our data, as well as re-analysis of some of the published data of others, indicates that a much clearer picture of the behavior of the birds is given by the combination of the homeward directional component and the length of the mean vector. This permits segregation of the two factors that together determine the homeward component. These two measures vary independently, and neither shows the distance effect.

5. I conclude that the distance effect is not a general phenomenon.

LITERATURE CITED

BATSCHLET, E., 1965. Statistical Methods for the Analysis of Problems in Animal Orientation and Certain- Biological Rhythms. American Institute of Biological Sciences, Wash- ington, D. C., 57 pp.

GRAUE, L. C., 1970. Orientation and distance in pigeon homing (Cohtmba lii'ia). Anim.Behav., 18: 36-40.

KEETON, W. T., 1969. Orientation by pigeons: Is the sun necessary? Science, 165: 922-928.

KEETON, W. T., AND A. GOBERT, 1970. Orientation by untrained pigeons requires the sun. Proc. Nat. Acad. Sci., 65: 853-856.

MATTHEWS, G. V. T., 1953. Sun navigation in homing pigeons. /. Exp. Biol., 30: 243-267.

MATTHEWS, G. V. T., 1955. Bird Navigation. Cambridge University Press, Cambridge, 141 pp.

MATTHEWS, G. V. T., 1963. The orientation of pigeons as affected by the learning of landmarks and by the distance of displacement. Anim. Behav., 11: 310-317.

MATTHEWS, G. V. T., 1968. Bird Navigation. [2nd Ed.] Cambridge University Press, Cam- bridge, 197 pp.

ScHMiDT-KoENic, K., 1963a. Neure Aspekte iiber die Orientierungsleistungen von Brieftauben. Ergeb. Biol. ,26: 286-297.

ScHMiDT-KoENiG, K., 1963b. On the role of the loft, the distance and site of release in pigeon homing (the "cross-loft experiment"). Biol. Bull., 125: 154-164.

SCHMIDT-KOENIG, K., 1964. Initial orientation and distance of displacement in pigeon homing. Nature, 201: 638.

SCHMIDT-KOENIG, 1965. Current problems in bird orientation. Pages 217-218 in E. Hinde, K. Lehrman and E. Shaw, Eds., Advances in the Study of Behavior, Volume 1. Aca- demic Press, New York.

SCHMIDT-KOENIG, K., 1966. Uber die Entfernung als Parameter bei der Anfangsorientierung der Brieftaube. Z. J'crgl. Physiol., 52: 33-55.

SCHMIDT-KOENIG, K., 1968. Entfernung und Genauigkeit der Anfangsorientierung von Brief- tauben. Z. Vergl. Physiol., 58: 344-346.

WALLRAFF, H. G., 1967. The present status of our knowledge about pigeon homing. Proc. 14th Int. Ornithol. Con,/r.. 14: 331-358.

NOTE ADDED IN PROOF

While this paper was in press, Wallraff (1970, Z. Tierpsychol., 27: 303-351) published an analysis of his data in terms of the accuracy of the mean bearing and the length of the mean vector (see his page 340). Also, Schmidt-Koenig (1970, Z. Vergl. Physiol., 68: 39-48) has published further data from his studies of distance effect in Germany. Although he con- tinues to report a distance effect in the homeward component, he does not find the same effect in either vanishing interval or, more importantly, homing speed. This suggests that his birds released at intermediate distances are correcting their bearings soon after being lost from sight, while they are still within the "zone of disorientation." If this is so, it contradicts the hypothesis that the birds must be either close to home or very distant from home to orient accurately.

Reference : Biol. Bull, 139: 520-538. (December, 1970)

CONTROL OF MOLTING IN MANDIBULATE AND CHELICERATE

ARTHROPODS BY ECDYSONES

A. KRISHNAKUMARAN AND H. A. SCHNEIDERMAN

Department of Biology, Marqucttc University, Miki'aiikcc, Wisconsin 53233 and Developmental

Biology Laboratory, School of Biological Sciences, University of California, Irvine,

California 92664 and Marine Biological Laboratory, Woods Hole,

Massachusetts 02543

Ecdysone and related steroids are the molting hormones of insects (cf. Novak, 1966; Kaplanis, Thompson, Robbins and Bryce, 1966; Kaplanis, Thompson, Yama- moto, Robbins and Louloudes, 1967 ; Thompson, Kaplanis, Robbins and Yamamoto, 1967; Williams, 1968). When 0.5 to 5 /zg of these compounds are injected into isolated pupal abdomens of Cynthia moths, for example, they cause prompt devel- opment and the pupal abdomen molts into the corresponding fragment of an adult abdomen (Williams, 1968). The sequence of events triggered off by ecdysones in insects, which are collectively termed "molting," includes separation of the epi- dermis from the old cuticle (apolysis), the secretion of molting fluid, the secretion of a new cuticle, the digestion and resorption of part of the old cuticle, and the shedding of the old cuticle (ecdysis). Similar events occur in crustaceans during a normal molt cycle (Passano, 1960), but the control agents have not yet been identified. Evidence that ecdysones might be involved came from studies of Carlisle (1965) which showed that ecdysone extracts from different crustaceans and locusts caused molting in the crab Carcinus inacnas from which Y-glands had been extirpated. Furthermore, ecdysones identical in their chemistry to those of insects were isolated from crustaceans (Hocks, Schulz and Karlson, 1967; Hamp- shire and Horn, 1966 ; Galbraith, Horn, Middleton and Hackney, 1968) and Lowe, Horn and Galbraith (1968) reported that injection of ecdysterone (== 20 hydroxy ecdysone or /?-ecdysone) isolated from the crustacean, Jasus lalandi, caused shorten- ing of the interecdysial period in eye-stalkless crayfish, Procambarus sininlans. However, when we began these experiments, no reports existed of the successful induction of molting in arthropods other than insects by purified ecdysones.

The first example of ecdysone-induced molting in arthropods other than insects was the induction of molting in an isopod, ArmadilKdmm vulgare, by ecdysterone (Krishnakumaran and Schneiderman, 1968, 1969). About nine days after receiving 150 /zg/g of ecdysterone, the epidermis of the posterior parts of those isopods under- went apolysis and secreted a new cuticle and, two to three days later, underwent normal ecdysis. This was followed by secretion of a new cuticle and ecdysis of the anterior part, after which the animals resumed normal activity. The fact that both insects and crustaceans used ecdysones suggested that all mandibulate arthropods might use the same molecule for the control of molting. It was unclear, however, whether the other major subdivision of arthropods, the chelicerates, also use the ecdysones to control molting. The data in this report demonstrates that ecdy- sterone and several other ecdysones induce molting in crustaceans. These include

520

ECDYSONE EFFECTS ON ARTHROPODS 521

cyasterone, ponasterone A, a ecdysone, inokosterone and an ecdysone analogue, ft SEA 1. The data also show that diverse chelicerate arthropods including spiders, tarantulas and horseshoe crabs can be caused to molt by the injection of ecdysterone. The results also demonstrate that injected ecdysones cause a similar pattern of cuticle formation and molting in crustaceans, insects and arachnids. The possible evolutionary significance of a common path for the chemical control of molting in arthropods is discussed.

A preliminary account of these results was published earlier (Krishnakumaran and Schneiderman, 1968). In addition, Wright (1969) has recently demonstrated that ecdysone causes molting in. ticks.

MATERIALS AND METHODS Experimental animals

Two species of crayfish of the genus Procambarns were used. One was collected locally in northeastern Ohio and the other was purchased from Schettle Biologicals, Stillwater, Minnesota. The Ohio forms weighed between 3 to 12 grams while Minnesota forms weighed between 10 to 22 grams. The crayfish were maintained in the laboratory in shallow aquaria containing tapwater. The temperature and photoperiod were those of the laboratory (24-26° C and 12-15 hrs of light). Elodea served as food and also helped in oxygenation. In addition, the animals were fed meat once every two weeks. They survived for long periods under these conditions and several of them molted spontaneously in early summer and late fall. These ecdysed normally and survived the molt, provided that they were isolated from their cannibalistic neighbors when they were newly ecdysed. The crayfish were maintained in the laboratory for several days before they were used for the experiments. This helped in determining the normal rate of molting for that season of the year. In certain seasons (e.g., spring), it was necessary to wait until the crayfish molted once in the laboratory before they could be used.

Another crustacean used in this series of experiments was the fiddler crab, Uca pug Hat or. Specimens of U. pugilator were collected off Woods Hole and shipped to Cleveland in August where they were maintained in sand troughs containing "instant ocean," an artificial seawater (Aquarium Systems, Inc., Cleveland, Ohio). They were fed periodically on crab meat and survived for several months. Crabs in the process of molting could be recognized by their pale carapace and lethargic movements, and were isolated individually in small transparent plastic containers containing sand and instant ocean. If such crabs failed to ecclyse or died within a week after isolation, they were peeled and examined to determine whether or not they had secreted a new cuticle.

Three different chelicerate arthropods were also used. The horseshoe crab, Limulits polyphemus, the spider, Arancns cornntiis. and the tarantula, Dugesiella hentzi. Specimens of L. polyphemus were collected off Woods Hole, Massachu- setts in July and were maintained either in running sea water tanks at the Marine Biological Laboratory there, or in an aquarium containing "instant ocean." Ani- mals weighing 30 to 50 g each or 3 to 12 g each were used. No food was given during the experiment except for debris that might have been brought in by the running sea water and from the seaweed and colonial invertebrates present in the

522 A. KRISHNAKUMARAN AND H. A. SCHNEIDERMAN

aquarium. Animals were maintained at 19° C and received approximately 12 to 15 hrs of daylight. No effort was made to regulate photoperiod. Such animals survived several months under these conditions and several were still alive after a year in the artificial sea water.

The spider Araneus and the tarantula Dugesiella were obtained from Carolina Biological Supply, Inc. Araneus were collected in Oregon and were maintained individually in 30 ml plastic vials with screw caps. They weighed between 100 to 400 mg each and were sexually mature as evidenced by the production of egg cocoons. Tenebrio pupae were given as food weekly. The animals were kept in the dark at 18° C. These animals displayed no external morphological signs of molting prior to ecdysis. However, most of the spiders that ultimately secreted a new cuticle, stopped feeding and spun a molting pad which consisted of a few threads attached to the wall of the vial. Normally, these animals spin an orb web.

Tarantulas were maintained individually in glass tanks, the floor of which was covered with gravel. A cardboard tube served as a hiding place. They weighed between 8 to 12 g and both sexes were used. Live crickets and Tenebrio pupae were given as food weekly.

Hormones and other reagents

Ecdysterone and inokosterone were obtained from Rohto Pharmaceuticals, Osaka, Japan or Mann Research Laboratories, New York, a ecdysone wras pro- vided by Dr. P. Hocks, Schering, A. G. Berlin, ponasterone A was provided by Dr. John Pollard of Calbiochem. Cyasterone was provided by Professor C. M. Williams and came from the laboratory of Professor T. Takemoto, Tohoku Uni- versity, Sendai, Japan. Drs. W. Robbins and Malcolm Thompson of the USDA at Beltsville, Maryland, provided the four ecdysone analogues: (3 SEA-1 (A7-5/3- cholestene-2/3, 3/3, 14a-triol-6-one), (3 SEA-4 (A7-5£-cholestene-2/3, 3/3-diol-6-one), /3 SEA-12 (A7-5£-sitostene-2£, 3/3, 14«-triol-6-one) and a SEA-1 (A7-5a-cholestene- 2ft, 3/3, 14a-triol-6-one). Cholesterol and beta sitosterola were purchased from Nutritional Biochemicals, Cleveland, Ohio. Tritiated thymidine (Schwartz Bio- research) was employed at a concentration of 1 me/ml with a specific activity of 1.9 c/m Mole.

Ecdysterone and inokosterone were dissolved in insect Ringer (Ephrussi and Beadle, 1936), 10% ethanol or crustacean Ringer (Pantin's Ringer according to Marine Biological Laboratory Formulary, M.B.L., Woods Hole, Massachusetts) as the case may be. Cyasterone and ponasterone were dissolved in W% ethanol, and a ecdysone in 20 % ethanol. The ecdysone analogues were suspended in 20% ethanol, whereas the cholesterol and beta sitosterol were either dissolved in absolute ethanol or suspended in 50% ethanol.

Experimental procedures

Animals were anaesthetized in crushed ice and/or carbon dioxide. The mate- rials were injected via a glass needle or 31 gauge steel needle using a Hamilton microliter syringe or a microburette. The volume of the material injected was usually less than 2 to 4% of the weight of the animal. Animals were kept for 2 to 4 hours at 5° C after injection before they were returned to the temperature

ECDYSONE EFFECTS ON ARTHROPODS 523

at which they were normally maintained. Tritiated thymidine was injected at a dose of 10 ju.c/g live weight of the animal. The isotope was administered at specific times after the injection of ecdysterone and was allowed to circulate for specific periods of time before the animals were killed. Animals to be killed were sliced in half and plunged immediately into Benin's fluid and processed to make 5 to 6 ^ thick paraffin sections. Autoradiographic methods were similar to those described earlier (see Krishnakumaran, Berry, Oberlander and Schneiderman, 1967).

Each experiment was repeated at least once, and in several cases two or three times. In all cases the results obtained were comparable and the results of typical experiments are shown in the tables.

RESULTS 1. Effects of ecdysterone on Procambarus

In the first series of experiments, ecdysterone dissolved in Ringer solution was injected to a final concentration of 20 //.g/g live weight. The data in Table I reveal that all the experimental animals molted within 10 days of the injection. The molt was abnormal, and in no case was there successful ecdysis and survival of the experimental animals. However, the animals did undergo apolysis and secreted a new cuticle which became obvious when the old cuticle was peeled away. All of the animals showed a swelling between the cephalothoracic shield and the abdomen. In a few cases the cephalothoracic shield separated from the new cuticle near its junction with the abdominal tergites.

TABLE I

Effect of ecdysterone on molting in the crayfish, Procambarus sp.

Number of Per cent that molted

Chemical and dose animals within a month

Uninjected control	10	10
Ethanol 10% 4 Ml/g	10	0
Cholesterol 20 Aig/g	10	0
Ecdysterone 20 Mg/g	10	100*
10 Mg/g	10	100*
6 Mg/g	10	100*
3 iug.'g	10	lOOf

Animals weighed between 12 and 20 grams each. * Molted within 10 days after injection. f Molted within 14 days after injection.

In insects it is known that high doses of ecdysones are pathological and fre- quently cause the death of the injected animals (Kobayashi, Takemoto, Ogawa and Nishimoto, 1967; Williams, 1968). Presuming that the mortality observed in the preceding experiment may have been the result of a pathologically high dose of ecdysterone, lower concentrations of ecdysterone were injected. In one series of experiments we injected 3, 6. or 10 ftg/g ecdysterone. Table I shows that even 3 jug/g of ecdysterone induced molting in Procambarus. However, even after such a low dose, almost all of the experimental animals died during ecdysis. Only two

524 A. KKISIIX AKUMARAN AND H. A. SCHNEIDERMAN

of the crayfish which received 3 pg/g ecdysterone completed ecdysis and survived. Even these were able to shed spontaneously only the caphalothoracic shield, and the remaining cuticle had to be peeled away.

Although between 3 and 20 //.g/g of ecdysterone were effective in inducing molt- ing processes, there were distinct differences in the response to low and high doses. Thus an injection of 6, 10, or 20 ju,g/g of ecdysterone resulted in apolysis and secretion of a new cuticle within ten days, whereas animals injected with 3 pg/g took about fourteen days to secrete a new cuticle. Crayfish receiving lower doses of ecdysterone also differed in the size of the gastroliths formed and the extent of digestion of the old cuticle. Gastroliths were best developed and the digestion of old cuticle most pronounced in crayfish that received 3 jug/g ecdysterone. The gross structure and histochemistry of the cuticles deposited in response to different closes of ecdysterone were identical, but certain morphological features of the cuticle were affected by the dose. Thus, the newly-deposited cuticle was similar to normal cuticle and contained a non-chitinous epicuticle, a chitinous, lamellated endocuticle and polyphenolase. However, animals that received 20 fig/g ecdysterone secreted a much thinner new cuticle than those that received lower doses of the hormone. Also, the bristles and hairs on the uropods and the branchial gills were shorter and ill-formed in all experimental animals except those that received only 3 //g/g ecdysterone.

An additional effect of high doses of ecdysterone was its inhibition of regenera- tion of appendages. Crayfish that had lost some of their appendages (usually walk- ing legs) several weeks before hormone treatment, were injected with 20 p-g/g ecdysterone. These animals molted promptly, between 7 and 10 days after they received the hormone, without regenerating the lost appendages. However, similar animals that received 2 to 3 ^g/g ecdysterone formed a small regenerate. In con- trast, crayfish whose appendages were amputed only 7 to 10 days prior to the injection of ecdysterone failed to regenerate even after treatment with 2 to 3 /^g/g ecdysterone. These results are reminiscent of the situation in the wax moth GaUeria (Madhavan and Schneidennan, 1969) where regeneration of imaginal wing discs in the last larval instar is promoted by low doses of ecdysone, but fails to occur after injecting high doses. In the crayfish, as in GaUeria, ecdysone may be necessary for regeneration, but when applied in high doses it provokes molting so promptly that there is insufficient time for the cell divisions necessary for regenera- tion.

2. Histological and autoradiographic studies of the crayfish epidermis under the influence of ecdysterone

To determine when the various events of the molt cycle occurred in the epidermis of the crayfish under the influence of ecdysterone, Procambarus was injected with 2 ju.g/g ecdysterone, and three animals were killed and examined at 4, 24, 48, 96, and 144 hours after the injection of the hormone. Animals were processed for his- tological study and 4 to 5 micron thick paraffin sections were prepared and stained with Mallory's triple stain or Meyer's haemalum and eosin. A study of these sec- tions revealed that the old cuticle had apolysed from the epidermis about 48 hrs after the injection of hormone. By 96 hrs after injecting the hormone, a new cuticle, approximately 8 to 10 /A thick in the tergite region, had already been secreted.

ECDYSONE Kl-TKlTS ON ARTHROPODS 525

At this time the nuclei, which began to enlarge at the time of apolysis, were greatly enlarged. Associated with this nuclear enlargement was an increase in the size and basophilia of the epidermal cells. Events which occur after 96 hrs are difficult to analyze in the present experiments because they may have been associated with the pathological changes connected with the imminent death of the animals. Six days after the injection of ecdysterone, the epidermis in some of the crayfish showed further changes, such as a decrease in cytoplasmic volume. They resem- bled to some extent intermolt epidermis, except that in the normal intermolt animal the nuclei of epidermal cells are compact and lack chromatin granules.

Analysis of DNA synthesis in epidermis was undertaken next. For this pur- pose crayfish were injected with ecdysterone at a dose of 2 /*g/g and immediately thereafter, or 4, 24, 48, or 96 hrs later, tritiated thymidine (10 /*.c/g) was injected. Individuals were killed both 2 and 24 hrs after the injection of isotope. None of

TABLE II

Effect of ecdysone analogues and steroids on molting in the crayfish, Procambarus sf>.

Number of Per cent that

Chemical and dose animals molted

10',, ethanol 3 /xl/g	10	0
Ecdysterone 6 jug/g	10	100
laokosterone 6 yug/g	6	100
Cholesterol 6 Mg/g	16	0
Cholesterol 20 ^g/g	15	0
13 SEA- 1 50 Mg/g	6	50
a SEA-1 50 yug/g	6	0
13 SEA-4 50 Mg/g	6	0
13 SEA- 12 50Mg/g	6	0
(3 Sitosterol 20 Mg/g	6	0

Animals weighed between 12 and 20 grams each.

the epidermal cells incorporated tritiated thymidine into their nuclei, although blood cells engaged in extensive DNA synthesis during this period, and many well-labelled blood cells were seen. Apparently, the ecdysterone at the levels used in these experiments does not induce DNA synthesis in the epidermis of these crayfish, although it induces molting. It is noteworthy also that muscles, nerve cells, and connective tissue failed to synthesize DNA.

3. The specificity of ecdysterone in inducing molting

Does ecdysterone induce molting in these crayfish because it is either the normal molting hormone or an analogue of the normal hormone, or, is it possible that its effects represent a nonspecific pharmacological action of steroids? To test this possibility, we injected crayfish with various sterols, such as cholesterol and beta sitosterol or ecdysone analogues such as ft SEA-1, a SEA-1, ft SEA-4, and ft SEA- 12. These agents have very little or no ecdysone effects in insects. The result> are recorded in Table II. Crayfish injected with 6 or 20 /tg/g cholesterol or 20 /xg/g sitosterol or 50 //g/g « SEA-1, ft SEA-4, or ft SEA-12 did not molt. These data suggest that the effects of ecdysterone are not due to a nonspecific pharma-

526

A. KRISHNAKUMARAN AND H. A. SCHNE1DKKMAN

cological effect of steroids. Even chemicals closely related to the ecdysones, such as a SEA-1, ft SEA-4 and ft SEA-12, do not induce molting (see Table II).

The only chemical that showed any molt-inducing effect in Procambarus was ft SEA-1, which is structurally similar to the ecdysones except for the absence of hydroxyl groups on the side chain. This substance has ecdysone-like effects on the house fly and Calliphora (Robbins, personal communication). In Procambarus it caused molting in 50% of the crayfish into which it was injected. These animals developed fully-formed gastroliths, more-or-less completely digested their old cuticle and underwent spontaneous partial ecdysis, after which they died. Unlike ecdy- sterone, which induced prompt molting, ft SEA-1 caused animals to molt 3 to 4 weeks after they received an injection.

4. Effects of phytoecdysones on Procambarus

From the preceding experiments it appears that ecdysterone induces molting in crayfish by specific hormonal action rather than by some nonspecific pharmacologi- cal effect. Is this activity limited to ecdysterone which normally occurs in crusta- ceans, or do the other zooecdysones and phytoecdysones that cause molting in insects also induce molting in crayfish? To test this, a ecdysone, inokosterone, ponasterone A and cyasterone were injected into Procambarus. An injection of 3 fj-g/g of any of these ecdysones induced molting in these crayfish. But as Table

TABLE III Effects of different ecdysones on molting in the crayfish, Procambarus sp.

		Per cent that molted during the	Cumulative
		period indicated (approximate)	% of cray-
Treatment	Number of animals		fish that molted dur-
		7-10 days	10-14 days	14-21 days	ing the 2 1 day period
Uninjected controls	25	0	0	0	0
Controls injected with 10% ethanol 3 n\/g	10	0	0	0	0
Ecdysterone 3 Mg/g	7	14	86	0	100
a Ecdysone 3 pg/g	15	56	7	7	66
Inokosterone 3 Mg/g	14	21	56	7	86
Ponasterone A 3 Mg/g	15	14	35	0	46
Cyasterone* 3 Mg/g	10	10	10	10	30

Animals weighed 3 to 20 grams.

Most of the animals died in the process of molting.

* Higher doses, such as 6 Mg/g and 10 Mg/g, induced molting in 100% of experimental

als.

Ill shows, there were differences in the percentage of animals that responded and the time required for a response. Cyasterone was the least effective and induced molting in only 30% of the animals. In contrast, ecdysterone was the most effec- tive and induced molting in all of the experimental animals. Inokosterone, a ecdy- sone and ponasterone were intermediate in effectiveness with 86%, 66% and 46% of the animals molting, respectively. \Yhen higher doses of cyasterone (6 and 10 /xg/g) were injected, all of the treated crayfish molted, indicating that the response was dose-dependent.

ECDYSONE EFFECTS ON ARTHROPODS

527

Another distinguishing feature of the different ecdysones was the interval be- tween the time of injection of hormone and the induction of the molt. An injection of 3 /tg/g of a. ecdysone caused 56% of the animals to molt between 7 and 10 days, whereas in the case of ponasterone, inokosterone and ecdysterone, most of the animals molted between 10 and 14 days after injection. The reasons for this difference are not obvious ; it is possible that a ecdysone is the true hormone with a short half-life, while other ecdysones may be less effective, but more stable, degrada- tion products or analogues of a ecdysone (cf. King and Siddall, 1969).

Almost all of the crayfish that had been induced to molt experimentally, died after secreting a new cuticle. In an effort to increase their survival, we removed their eye stalks. The rationale behind this maneuver is the fact that eye stalks are known to produce and store a molt-inhibiting agent (cf. review by Passano, 1960). The site of action of this agent is not known, but if it acted upon epidermal cells, then its removal might permit better survival after ecdysis. Eye stalks were removed the day prior to, or immediately after, the injection of 3 /xg/g of ecdy- sterone. However, survival after the molt was not increased.

5. Effects of ecdysterone on Uca pugilator

Another experiment tested the effects of ecdysterone on a marine crustacean, Uca pugilator, a semi-terrestrial fiddler crab. Fiddler crabs received 20 ttg/g of ecdysterone dissolved in 4 /xl of Ringer solution. Controls received 4 p\ of Ringer solution or no injection. The results, recorded in Table IV, show that 90% of

TABLE IV

Effect of ecdysterone on the induction of molting in the fiddler crab, Uca pugilator

		Per cent that spontaneously ecdysed during the specific period (*) or re-	Cumulative
Treatment	Number of animals	vealed a new cuticle after peeling	that molted during the 30
					day period
		0-10 days	10-20 days	20-30 days
Uninjected controls	15	0	7*	7*	14
Controls injected with 4 jul/g Ringer	30	10*	3*	3*	16
Experimental injected with 20 /ug/g
ecdysterone in 4 //' Ringer	30	0	83	7	90

the experimental animals molted within 30 days, whereas only 16% of the injected controls and 14% of the uninjected controls molted during this period. Unlike the controls which underwent normal ecdysis, the experimental animals never underwent spontaneous ecdysis, although they showed apolysis, deposition of a ne\v cuticle, secre- tion of molting fluid and partial resorption of the old cuticle. The experimental ani- mals that had deposited a new cuticle could easily be recognized by their pale color and lethargic movements. When such crabs were peeled, their newr cuticle was re- vealed, but they immediately died. Even if left for 10 days after the first appear- ance of pale color, they failed to undergo spontaneous ecdysis. Another feature of the experimental molt was that a large percentage of the animals responded within the same short span of time ; namely, 14 to 18 days after injection of ecdysterone.

528

A. KRISHNAKUMARAN \M> II. A. SCHNEIDERMAN

The cuticle formed in response to the injection of ecdysterone was similar in general appearance to normal cuticle and possessed epicuticle, a lamellated endo- cuticle and polyphenolase. However, experimentally-induced cuticle differed from the normal cuticle in several respects. In general, the induced cuticle was thinner than the normal cuticle. This may be in part the result of premature death, but appeared to be associated more with the artificial induction of molting. Another conspicuous difference was in the nature of the tubercles on the lateral borders of the dactylus and claw and the anterior ventral margins of the carapace. The tubercles in the experimental animals were much smaller and ill-formed. However, their distribution and orientation was identical to the pattern found in the old cuticle. Similarly, the bristles and sensory hairs at the borders of the appendages and on the general cuticular surface were short and ill-formed. In normal animals, the cuticle on the two sides of the maxilliped differ in thickness: the external surface has a thick cuticle (20 to 25 p.}, whereas the internal surface has a thin cuticle (4 io (jfj.). In experimentally-treated animals the cuticle on both sides of the maxilli- ped is of the same thickness (4 to 6jn). In addition, in normal animals, the external surface of the maxilliped bears tubercles, while in the experimentally- treated animals, there were either no tubercles or small tubercles.

TABLE V

Effect of ecdysterone on molting in Araneus cornutus

		PCI cent that molted during tin;	Cumulative %
	Number	period indicated	of spiders that
Treatment	of		molted during
	animals					the 29-day
		0-8 days	8-15 days	15-22 days	22-29 days	period
Controls injected with 4 ^1
of Ringer	24	8*	0	4*	0	13
Experimental injected with 2U //g
of Ecdysterone in 4 /xl Ringer	29	7*	28	14	14	62

* Survived after spontaneous ecdysis. Others were peeled to determine whether they had molted.

Control and experimental groups contained 29 and 30 spiders each, respectively. One of the experimental and 5 of the controls died within the first three days after injection and are not included in the data. Sixteen controls and six experimental survived for the duration of the ex- periment but did not show any signs of molting.

6. Effects of ecdysterone on Araneus cornutus

In the first series of experiments with the spider Araneus, animals were chilled on crushed ice and injected with 20 /xg/animal ecdysterone in 4 jul of Ringers. The animals weighed approximately 150 mg, and thus the average dose of hormone was about 130 |U.g/g. Controls received 4 /J of Ringer solution. Results reported in Table V show that 62% of the experimental molted in the 4-week period after injection, whereas only 13% of the controls molted. Unlike the controls which survived spontaneous ecdysis and continued normal life, the experimentals under- went apolysis and deposited a new cuticle, but then died. This was true of all the experimentally-treated animals that molted except for two that molted within

ECDYSONE EFFECTS ON AK I MKoPODS

529

FIGURE 1. Surface view of cuticle from opisthosomal tergite in the spider, Arancux cor- initits induced to molt by injection of ecdysterone. Xote the short bristles and the inconspicuous ridges.

FIGURE 2. Same view as above from a control spider. The bristles are much longer and the ridges are conspicuous.

FIGURE 3. Cross section of telson from ecdysterone-injected Limulus showing the newly formed cuticle (arrow) inside the old cuticle.

FIGURE 4. Cross section of telson from control Lininlus injected with Ringer.

A. KRISHNAKUMARAN AND H. A. SCHNEIDERMAN

the first eight days after receiving the injection of ecdysterone. Probably these had already begun spontaneous ecdysis before the hormone injection.

Frequently it was possible to recognize spiders that were about to secrete a new cuticle and molt by the fact that they ceased to feed and spun a molting pad rather than the normal orb web. Control spiders underwent spontaneous ecdysis, usually within a week after they ceased feeding and spun a molting pad, whereas the experi- mental spiders died. When the dead, hormone-treated spiders were peeled, it became evident that they had secreted a new cuticle. However, although the cuticle secreted after the injection of ecdysterone resembled normal cuticle in the arrangement of the basic cuticular layers and its staining properties, it was abnormal, particularly in its surface pattern and in the morphology of spines and bristles. While normal cuticle bears conspicuous ridges on its outer surface, in the experi- mental cuticles, the ridges were either shallow or absent. Spines which are long in normal cuticles were much reduced in length in the experimentals and frequently did not rise above the surface of the cuticle. However, the sockets bearing these spines appeared to be normal in number and distribution in experimental animals (Figs. 1 and 2).

In a second series of experiments, ecdysterone was dissolved in absolute methanol to a final concentration of 5 /^g/ju.1 and applied topically to the surface of the abdomen. Each spider received either 4 /j.\ of this methanolic solution or was dipped in a methanolic solution. The controls received either the same amount of absolute methanol or were dipped in methanol. Whereas 50% of the ecdysterone- treated spiders molted in a three week period, only 15% of the controls molted during this period. However, the mortality was much higher than after injection of ecdysterone in Ringer solution.

7 . Effects of ecdysterone on Dugesiella hentzi

The primitive tarantula spider, Dugesiella, was used in the following experi- ments. A group of four tarantulas (2 males and 2 females), weighing between 8 and 12 g each, were injected with 200 ^g of ecdysterone in 40 /A of Ringer solution, an effective dose of 16 to 25 /xg/g. Controls (2 males and 2 females) received 40 fj.\ of Ringer solution. Both groups remained active and fed normally, but neither the experimentals nor the controls molted during the 60-day period they were observed. Two months after the initial injections, three of the experimental ani- mals were injected with a larger dose of ecdysterone — 50 jug/g — and the fourth was injected with 100 /*g/g. Controls received corresponding amounts of Ringer solution. Seven days after receiving the second injection of ecdysterone, one of the experimental animals, which received 50 /xg/g, died. Dissection revealed that the animal had undergone apolysis but there were no signs of a new cuticle. Twenty days after the second hormone injection, another of the tarantulas under- went spontaneous ecdysis and survived. It secreted a normal cuticle replete with spines and bristles. (This same animal imderwent a normal, uninduced. spon- taneous molt ten months later and is alive at the time of writing). Between 22 and 28 days after the second hormone injection, the other two hormone-treated animals also molted. These showed both apolysis and the secretion of a new cuticle, but did not undergo spontaneous ecdysis. Like the old cuticle, the new cuticle possessed a distinctive fuchsinophilic epicuticle and an aniline blue-stained

ECDYSONE EFFECTS ON ARTHROPODS

531

lamellated endocuticle. However, it differed from the normal cuticle in the absence of bristles and spines. The controls survived for several months without molting. One of these was killed two months after the second injection of Ringers and there were no signs of initiation of a molt. Since the molts in all four experimental ani- mals were induced in September, three months after the normal natural period of molting in these animals, and since none of the four controls molted spontaneously during this period, it seems clear that the injection of ecdysterone induced the molt.

8. Attempts to demonstrate effects of ecdysterone on isolated abdomens of spiders

One sure way to determine whether ecdysterone is responsible for the induction of molting in insects has been the testing of the hormones on isolated abdomens which are devoid of the major endocrine glands. The morphology of spiders ap- peared to make them particularly amenable to such a surgical maneuver. Although we do not know the source of the molting hormone in the spiders, we assumed that the glands responsible for molting are not located in the abdomen. With this gra- tuitous assumption in mind, we prepared a number of isolated abdomens of Araneus and Dugeslella and some other common spiders by ligating the narrow waist-like opisthosoma. The anterior halves of these animals were excised and, after apply- ing penicillin, streptomycin and phenylthiourea, the wound was sealed with paraffin. These isolated abdomens survived for a week to ten days. However, injection of 20 /jig of ecdysterone failed to cause molting. These unsuccessful results are

TABLE VI Effects of ecdysterone on molting in Limulus polyphemus

	Number	Per cent showing
Treatment	of	signs of molt	Remarks
	animals	within one month
Uninjected controls	50	2	Only apolysis
Controls injected with 8 n\ of Crustacean
Ringer	6	0	—
Experimental injected with 40 ng/g	5	100	Secreted new cuticle but
ecdysterone in 8 y\ Ringer			had to be peeled

Animals weighed between 35 and 50 grams each and were kept in running sea water.

reported only because this may be the first report of such surgical operations on spiders and may prove useful for some other study. The negative results we ob- tained may reflect the fact that we performed only a limited number of operations.

9. Effects of ecdysterone on Limulus polyphemus

The horseshoe crab, Limulus is a relict merostomate arachnoid, only remotely related to modern arachnids, such as spiders and scorpions. Living genera of horseshoe crabs resemble closely the ancient genus Paleolimulus which lived some 200,000,000 years ago in the Permian. Their closest relatives are thought to be the extinct eurypterids and trilobites. Large specimens of Limulus which weighed 35 to 50 grams each. wrere injected with 40 //.g/g ecdysterone dissolved in crustacean Ringer. Controls were either injected with a corresponding amount of Ringer or

532 A. KRISHNAKUMARAN AND H. A. SCHNEIDERMAN

were uninjected. The results recorded in Table VI show that all experimentals molted. Only one of the fifty uninjected controls and none of six injected controls showed any signs of molting. Even the one control animal that showed signs of molting, underwent only partial apolysis. In Limulus, as in the other animals studied in this report, the experimentally-induced molt was different from a normal molt. The animals underwent apolysis and became paler in color but they never spontaneously shed their cuticles. Microscopic examination of such animals after peeling the old cuticle confirmed that the new cuticle had the same cuticular layers as the old cuticle (Figs. 3 and 4). Treatment of a second batch of smaller speci- mens of Limulus (3 to 10 grams) gave similar results.

These two series of experiments were performed at the Marine Biological Labora- tory, Woods Hole, Massachusetts, where the specimens of Limulus were kept in tanks of running sea water. When the experiments were repeated in the laboratory at Cleveland, Ohio, using artificial sea water in a circulating system, they were unsuc- cessful. However, all 25 of the ecdysterone-injected specimens of L. polyphemus died within four weeks, whereas 60% of the 25 controls injected with Ringer sur- vived for more than four months. None of the controls or the experimental animals deposited a new cuticle.

DISCUSSION 1. Ecdysones as the true molting hormones of all arthropods

The data show that ecdysterone causes molting in diverse chelicerate and man- dibulate arthropods. The effects appear to be true hormonal effects and not non- specific effects of steroids. For example, less than 3 /tg/g of ecdysterone caused molting in crayfish, whereas doses up to 50 //-g/g of steroids such as cholesterol, beta sitosterola and the ecdysone analogues (/3 SEA-4, ft SEA-12), which are hormonally inactive in house fly and CalUphora assays, have no effect in crayfish. A second reason for believing that the ecdysones are the true molting hormones of these arthropods is the occurrence of « ecdysone, deoxyecdysone, ecdysterone and at least two other ecdysones in crustacean (Galbraith et al., 1968; King and Siddall, 1969; Faux, Horn, Middleton, Fales and Lowe, 1969). Although their presence in chelicerate arthropods has not been uncovered thus far, it seems likely that ecdy- sones occur in these arthropods as well. Thirdly, the dose required to induce molt- ing in insects (Williams, 1968 ; Krishnakumaran, Granger and Schneiderman, 1970) and crustaceans (present results) is in the same physiological range.

A fourth line of evidence that supports the view that ecdysones are the natural molting hormones of arthropods other than insects is the abnormality of the cuticle deposited in response to ecdysone treatment. The experimentally-induced cuticles of spiders and crabs show several features which are similar to the pathological effects of high doses of ecdysones in insects. Such pathological effects in insects have been termed "hyperecdysonism" by Williams (1968) and appear to represent the first example of hyperhormonism in invertebrates. In Lepidoptera, these fea- tures include short, ill-formed bristles and scales, a decreased number of bristles and scales, an abnormal cuticular texture, and have been attributed to a telescoping of the normal sequence of synthetic events in which epidermal cells engage during the process of cuticle deposition. In the present experiments similar abnormalities

ECDYSONE EFFECTS ON ARTHROPODS 533

were found in spiders (fewer and shorter hairs and bristles) and in fiddler crabs (reduced size of knobs on dactylus and on the anterior ventral surface of the cara- pace). These abnormalities appear to be caused by the abnormal dosage and timing of the application of the hormones : Normally ecdysone is released gradually, but in these experiments it was applied all at once. It seems that such "hyper- ecdysonic" effects would be expected only if ecdysterone were either a true natural molting hormone or of similar structure to the true molting hormone. The follow- ing facts also support this opinion.

If ecdysone caused some general pathological effect which resulted in abnormal cuticles, it might be effective even after the process of molting had begun. This is not the case. Injection of a high dose of ecydysterone after the initiation of molting has no "hyperecdysonic" effects in insects. The same appears to be true of the spiders which molted during the first eight days after injection of the ecdysterone. These spiders ecdysed spontaneously and produced a normal-looking cuticle, appar- ently because molting had been initiated prior to the injection. Thus, it appears reasonable to presume that the action of ecdysterone in the crayfish, and possibly in the other arthropods, is a true hormonal action.

\Yhat are the targets of ecdysone? In insects the targets of ecdysone include the epidermis (Wigglesworth, 1957) midgut (Piepho, Holz and Jung, 1964) nervous system (Pipa, 1969), imaginal discs (Madhavan and Schneiderman, 1969), several internal organs (Sehnal and Schneiderman, 1970) and sometimes the ecdy- sial glands (Schneiderman and Gilbert, 1964). The target tissues of ecdysones in other arthropods are probably the same, but certainly include the epidermis, for the following reasons. It is unlikely that the ecdysones cause molting in these diverse arthropods by activating the animal's own ecdysial glands, for if this were the case, one would not expect abnormal cuticles. When the ecdysial glands of insects are activated, by whatever means, normal cuticles are produced, provided one does not inject excess amounts of ecdysones. The abnormalities produced in the cuticle of these diverse arthropods by excessive amounts of ecdysone, suggest a direct action of the ecdysones on the epidermal cells. Apparently, at least the chitogenous epi- thelium of most arthropods is capable of responding to insect ecdysones. This implies that the final common path of the control of molting in most arthropods is the same, involving an ecdysone or ecdysone-like molecules and the associated receptor sites in the epidermal cells. Although definitive evidence for true homol- ogy must await the identification of the ecdysial glands in, and the isolation of ecdysones from chelicerate arthropods, the evidence presented in this report points to such homology.

The nature of the primary action of ecdysones on insect epidermal cells remains to be identified (cf. discussion in Krishnakumaran et al., 1967). However, the results of the autoradiographic experiments on crustaceans reported here, which demonstrate that the epidermal cells of crayfish can secrete a new cuticle without first engaging in DNA replication, emphasize that the fundamental role of ecdysones as molting hormones may be uncoupled from any role they have have as growth hormones. Similar molts without DNA replication have been observed in adult insects (Krishnakumaran and Schneiderman, 1964; Krishnakumaran et al., 1967) and in insects treated with high doses of ecdysones (Krishnakumaran, Granger and Schneiderman, 1970). It remains to be demonstrated when DNA replication nor- mally occurs in the interecdysial period of crustaceans.

534 A. KRISHNAKUMARAN AND H. A. SCHNEIDERMAN

It is of interest that ecdysterone caused behavioral effects in addition to its effects on the cuticle. Spiders repsoncled to ecdysterone by spinning a molting pad before any obvious changes in the cuticle were evident. Whether these are direct effects on the nervous system, analogous to those caused by sex hormones in verte- brates, or some indirect effects remains to be proven.

2. Relative activities of different ecdysones

In insects and in crayfish the active ecdysones were effective at doses varying between 0.5 to 5 /xg/g live weight. In chelicerate arthropods, higher doses appeared to be necessary. Injections of about 40 to 50 /^g/g were required to initiate molt- ing in tarantulas and in Li nut Ins. Perhaps the specific ecdysones employed by chelicerates are different from their counterparts in insects and crustaceans.

From a study of the relative activities of the five different ecdysones, it appears that ecdysterone is the most active in crustaceans, followed by inokosterone, a ecdy- sone and ponasterone A, with cyasterone being the least active. This contrasts with observations on some insects where cyasterone is among the most active of the ecdysones. For example, in the lepidopteran, Samia cynthia, the relative activi- ties of the ecdysones are : cyasterone > ponasterone A > a ecdysone > ecdysterone > inokosterone (Williams, 1968). It is of interest also that ft SEA-1, which is the least active of the active ecdysone analogues in insects, is also the least active ecdy- sone for crustaceans.

From the analysis of all the dose-effect data presently available to us, it appears that almost all of the ecdysones which are active in one group of arthropods will have some activity in other groups. Whether injected ecdysones actually affect target cells themselves, or are metabolically converted into other ecdysones which affect target cells, is unknown. Indeed, even in insects, the role of the interconver- sion of the several ecdysones is still unclear (r/., for example, King and Siddall, 1969).

3. Survival after experimental molt

Insects which are caused to molt by injections of ecdysones commonly fail to survive. This is true also of most of the other arthropods examined here. In fact, only two each of the crayfish and spiders and one of the tarantulas which were caused to molt by ecdysones, survived for long periods after the molt. It is possible that the experimentally-induced molts caused death because of hyper- ecdysonism or because of the absence of certain necessary preparations for molting controlled by other hormones (such as the brain).

What are the probable causes of death in ecdysone-induced molts? In insects abnormal molting results in a defective cuticle in which wax layers may be incomplete and animals die because of desiccation (cf. review by Schneiderman, Krishnakumaran, Bryant and Sehnal, 1969). In other arthropods, defects in cuticle may have manifold effects. In crayfish, such defects may decrease their waterproofing and make them subject to dilution by fresh water. Both in spiders and in crustaceans, the presence of the old cuticle plus molting fluid over the book- lungs or gills will decrease the effectiveness of these respiratory organs by increasing the distance across which gases must diffuse. Undoubtedly, there are other causes

ECDYSONE EFFECTS ON ARTHROPODS 535

of death, but it appears likely that many are associated with cuticle. Ecdysones themselves do not appear to be toxic unless they cause molting, and animals which received injections of ecdysone rarely died except in the process of molting.

The only crayfish that survived were those that received a low dose of ecdy- sterone. Perhaps this activated their own ecdysial glands, or the dose was in the physiological range. The crayfish that died showed abnormal calcium resorption as indicated by poor formation of gastroliths and incomplete resorption of the old cuticle. These crayfish never shed their cuticles, possibly due to a failure in the absorption of water which normally precedes ecdysis.

These results are in marked contrast to those we obtained with terrestrial isopods in which normal molting was induced in more than half the animals by injecting ecdysterone (Krishnakumaran and Schneiderman, 1969). What are the reasons for the survival of these isopods and the death of other hormone- treated arthropods? One reason may be the peculiar way in which isopods molt. Posterior and anterior halves of these pill bugs undergo ecdysis consecutively, 3 to 4 days apart. This may involve either (a) a mechanism to remain insensitive to high levels of ecdysones circulating in the hemocoel, or (b) a con- trolling device that regulates the time of response of the chitogenous epithelium or (c) a mechanism to inactivate the excess ecdysone or (d) a combination of the above. Such a mechanism would prevent any hyperecdysonic effects and thus permit better survival. Another possibility is that the ecdysone activates the animals' own ecdysial glands.

4. Phylogenetic considerations

The fact that the ecdysones are capable of inducing molting, not only in insects but also in diverse crustaceans and chelicerates, suggests close similarity or even identity of the mechanisms that control molting in arthropods. If this proves to be true, it may throw some light on the phylogeny of the arthropods. The similarity in the chemistry of the molting hormone, and by implication the receptor sites in the chitogenous epithelium, strongly suggests the common ancestry of all arthro- pods. Manton (1964) and earlier Tiegs and Manton (1958) contend that the arthropods are polyphyletic in origin. They argue that the similarities in the structure and chemistry of the cuticle (see Richards, 1951; Krishnakumaran, 1961) independently evolved by convergence. The fact that the mechanisms controlling secretion of the cuticle are also homologous, makes such an argument unlikely. The convergence hypothesis becomes even less tenable when one adds the fact that the ecdysial glands of both insects and crustaceans have similar origins from the ectoderm of the embryonic cephalic region (see Jenkin, 1962; Herman, 1967). We have no knowledge of the location, structure, and origin of the ecdysial glands in the chelicerates. Should the ecdysial glands of chelicerates prove to be of epidermal origin and arise from the prosomic region, it would establish beyond all reasonable doubt that the arthropods are truly a homogeneous group with a monophyletic origin.

However, if it turns out that ecdysones are present and function in a wide variety of invertebrates such as annelids, nematodes, priapulids and related aberrant schizocoelic groups, then it is still possible that different arthropod groups may have evolved independently from several diverse post-annelid, prearthropod

536 A. KRISHNAKUMARAN AND H. A. SCHNEIDERMAN

ancestors. It will be of interest to investigate the distribution and effects of ecdysones in annelids and other groups.

This research was supported in part by research grants HE 06320 and HE 10082 from the United States Public Health Service.

SUMMARY

1. The ability of ecdysones to induce molting in arthropods other than insects was examined in representatives of both the mandibulate and chelicerate subphyla.

2. All five ecdysones tested caused molting in the fresh-water crayfish, Procambarus. Their relative activities were : ecdysterone > inokosterone > a ecdy- sone > ponasterone A > cyasterone. Doses as low as 3 /xg/g of ecdysterone caused 100% of all test crayfish to undergo apolysis and secrete a new cuticle within 14 days after injection, but only in a few cases did the animals shed their old cuticles spontaneously after experimental treatment. At higher doses the new cuticle was thinner than normal and had abnormal bristles. The stimulation of molting was specific for ecdysones and was not copied by a variety of ecdysone analogues or other steroids.

3. Histological and autoradiographic studies revealed that ecdysterone at the levels used in these experiments caused molting in crayfish without DNA replica- tion.

4. Ecdysterone also caused molting in the marine fiddler crab, Uca pugilator.

5. Ecdysterone caused molting in several chelicerate arthropods including the spider, Araneus cornutus, the tarantula, Dugesiella hentsii and in the horseshoe crab, Limulus polyphetnus, which is among the most primitive of all living arthropods.

6. In spiders the ecdysone caused behavioral effects before any obvious changes in the cuticle were evident.

7. In almost all cases, molts induced by ecdysone were characterized by abnormal cuticles similar to those produced by injections of ecdysones in insects, a result which suggests a direct action of ecdysone on the epidermal cells. Most of the experimental animals failed to survive the molt and few underwent spon- taneous ecdysis. These effects probably result from the abnormal delivery of a large amount of hormones in one dose, in contrast to the gradual release of hormone in situ.

8. From an analysis of all of the dose-effect data, it is concluded that almost all of the ecdysones which are active in one group of arthropods will have some activity in other groups. Since spiders and horseshoe crabs require doses about ten times as high as those needed for mandibulate arthropods, the specific ecdy- sones employed by the chelicerates may differ from their counterparts in insects and crustaceans.

9. The evidence suggests that ecdysones are the normal molting hormones of all arthropods and supports the view that arthropods have a common ancestry and are not a polyphyletic group.

ECDYSONE EFFECTS ON ARTHROPODS 537

LITERATURE CITED

CARLISLE, D. B., 1965. The effects of crustacean and locust ecdysone on moulting and pro-

ecdysis in juvenile shore crabs, Carcinus maenas. Gen. Comp. Endocrinol., 5: 366-372. EPHRUSSI, B., AND G. W. BEADLE, 1936. A technique of transplantation for Drosophila.

Aincr. Natur., 70: 218-225. FAUX, A., D. H. S. HORN, E. J. MIDDLETON, H. M. PALES AND M. E. LOWE, 1969. Molting

hormones of a crab during ecdysis. Chcm. Comm., no. 4: 175-176. GALBRAITH, M. N., D. H. S. HORN, E. J. MIDDLETON AND R. J. HACKNEY, 1968. Structure

of deoxy crustecdysone, a second crustacean molting hormone. Chein. Comm., no. 2 :

83-84.

HAMPSHIRE, F., AND D. H. S. HORN, 1966. Structure of crustecdysone, a crustacean molt- ing hormone. Chcm. Comm., no. 2 : 37-38.

HERMAN, W. S., 1967. The ecdysial glands of arthropods. Int. Rev. Cytol., 22: 269-347. HOCKS, P., G. SCHULZ AND P. KARLSON, 1967. Die Struktur des Ecdysons. Naturwissen-

schaften, 54: 44-55. JENKIN, P. M., 1962. Animal Hormones; A Comparative Survey, Part 1. Pergamon Press,

Oxford and New York, 310 p. KAPLANIS, J. N., M. J. THOMPSON, W. E. ROBBINS AND B. M. BRYCE, 1967. Insect hormones:

alpha ecdysone and 20 hydroxy-ecdysone in Bracken fern. Science, 157 : 1436-1438. KAPLANIS, J. N., M. J. THOMPSON, R. T. YAMAMOTO, W. E. ROBBINS AND S. J. LOULOUDES,

1966. Ecdysones from the pupa of the tobacco hornworm, Manduca sexto, (Johannson).

Steroids, 8(5) : 605-623. KING, D. S., AND J. B. SIDDALL, 1969. Conversion of a-ecdysone to /3-ecdysone by crustaceans

and insects. Nature, 221 : 955-966. KOBAYASHI, M., T. TAKEMOTO, S. OGAWA AND N. NISHIMOTO, 1967. The molting hormone

activity of ecdysterone and inokosterone isolated from Achyranthis radix. J. Insect.

PhysioL, 13: 1395-1399. KRISHNAKUMARAN, A., 1961. The arthropod cuticle. The Presidency College Zoology Mag.

(Madras, India), 8: 23-38. KRISHNAKUMARAN, A., AND H. A. SCHNEIDERMAN, 1964. Developmental capacities of the

cells of an adult moth. /. Exp. Zool., 157: 293-307. KRISHNAKUMARAN, A., AND H. A. SCHNEIDERMAN, 1968. The chemical control of molting

in arthropods. Nature. 220 : 601-602. KRISHNAKUMARAN, A., AND H. A. SCHNEIDERMAN, 1969. Induction of molting in Crustacea

by an insect molting hormone. Gen. Comp. Endocrinol., 12: 515-518. KRISHNAKUMARAN, A., R. E. GRANGER AND H. A. SCHNEIDERMAN, 1970. Reprogramming

and cell division in insect epidermal cells. IVUhclm Roit.v' Arch. Entzvickhtngsmech.

Organisincn., in press. KRISHNAKUMARAN, A., S. J. BERRY, H. OBERLANDER AND H. A. SCHNEIDERMAN, 1967.

Nucleic acid synthesis during insect development. II. Control of DNA synthesis in

the Cecropia silkworm and other saturniid moths. /. Insect. Pliysiol., 13 : 1-57. LOWE, M. E., D. H. S. HORN AND M. N. GALBRAITH, 1968. The role of crustecdysone in

molting crayfish. E.rpcrientio, 24: 518-519.

MADHAVAN, K., AND H. A. SCHNEIDERMAN, 1969. Hormonal control of imaginal disc regenera- tion in Gallcria mcllonella (Lepidoptera). Biol. Bui!., 137: 321-331. MANTON, S. M., 1964. Mandibular mechanisms and the evolution of arthropods. Phil. Trans.

Roy. Soc. London, Scries B., 247 : 1-183.

NOVAK, V. J. A., 1966. Insect Hormones. [3rd (1st English) ed.] Methuen, London, 478 pp. PASSANO, L. M., 1960. Molting and its control. Pages 473-536 in T. H. Waterman, Ed.,

The Physiology of Crustacea, Volume 1. Academic Press, New York. PIEPHO, H., I. HOLZ AND U. JUNG, 1964. Hormonal Verjungung des Mitteldarmes. Natnr-

n'issencJiaftcn, 51 : 250. PIPA, R. L., 1969. Insect neurometamorphosis. IV. Effects of the brain and synthetic ecdysone

upon interganglionic connective shortening in Galleria mellonclla (L.) (Lepidoptera).

/. Exp. Zool., 170: 181-192.

538 A. KRISHNAKUMARAN AND H. A. SCHNEIDERMAN

RICHARDS, A. G., 1951. The Integument of Arthropods; the Chemical Components and their Properties, the Anatomy and Development, and the Permeability. University of Minnesota Press, Minneapolis, Minnesota, 411 p.

SCHNEIDERMAN, H. A., AND L. I. GILBERT, 1964. Control of growth and development in insects. Science, 143: 325-333.

SCHNEIDERMAN, H. A., A. KRISHNAKUMARAN, P. J. BRYANT AND F. SEHNAL, 1969. Endo- crinological and genetic strategies in insect control. Pages 14-25 in R. E. Krauss, Ed., Proceedings of a Symposium on potentials in Crop Protection. New York Agri- culture Experimental Station. Geneva, New York.

SEHNAL, F., AND H. A. SCHNEIDERMAN, 1970. Action of ecdysone on Galleria larvae. I. In- duction of molting, ll'ilhclm Rou.v' Arch. Entivicklungsmcch. Organismen., in press.

TIEGS, O. W., AND S. M. MANTON, 1958. The evolution of the Arthropoda. Biol. Rev., 33; 255-337.

THOMPSON, M. J., J. N. KAPLANIS, W. E. ROBBINS AND R. T. YAMAMOTO, 1967. 20, 26-di- hydroxyecdysone, a new steroid with molting hormone activity from the tobacco hornworm, Manduca se.vta (Johannson). Chem. Comin., 13: 650-653.

WIGGLESWORTH, V. B., 1957. The action of growth hormones in insects. Symp. Soc. Exp. Biol., 11 : 204-227.

WILLIAMS, C. M., 1968. Ecdysone and ecdysone-analogues : their assay and action on dia- pausing pupae of the Cynthia silkworm. Biol. Bull., 134: 344-355.

WRIGHT, J. E., 1969. Hormonal termination of larval diapause in Dermacentor albipictus. Science, 163: 390-391.

Reference : Biol. Bull, 139 : 539-548. (December, 1970)

GASTRULATION IN THE TURBELLARIAN HYDROLIMAX GRISEA

(PLATYHELMINTHES; PLAGIOSTOMIDAE) : FORMATION

OF THE EPIDERMAL CAVITY, INVERSION

AND EPIBOLY1

W. DONALD NEWTON Department of Zoology, University of North Carolina, Chapel Hill, North Carolina 27514

Gastrulation of the commensal turbellarian, Paravortex gemellipara, was de- fined by Ball (1916, p. 507) in the statement: "If by the process of gastrulation is meant the infolding or overgrowth of entoderm by ectoderm, then true gastrula- tion occurs. . . ." Ball's definition covers all processes, direct or indirect, by which ectoderm achieves its definitive position in the developing turbellarian. Entolecithal embryos of acoels and of polyclads gastrulate by epiboly or overgrowth by micro- meres of yolk-bearing macromeres and their derivatives (Bresslau, 1933; Kato, 1940, 1968). Ectolecithal embryos of rhabdocoels, alloeocoels and triclads gas- trulate in various ways, all of which may well represent adaptations to or modifica- tions by the yolk cells which surround the developing embryo (Kato, 1968).

According to Ball (1916) the first step in the gastrulation of the ectolecithal embryo of P. gemellipara is the differentiation of the ectoderm. The outer cells of the embryonic mass become flattened and stretch posteriorly to entrap the endoder- mal cells which have enveloped the yolk cells. Ball concluded that gastrulation of P. gemellipara occurs by epiboly. Bresslau (1904) had earlier observed a similar process in Mesostoma. As cited by Hyman (1951, p. 175), Bresslau ob- served that the peripheral cells of the venter of the embryo of Mesostoma ". . . arrange into a surface epithelium, which is the ventral epidermis and which gradually spreads dorsally to enclose the dorsal yolk mass." The gastrulation of Monocelis fusca is accomplished by the differentiation and spreading of eight blastomeres (six Hiillsellen, two Vitellocytophagen) , which form a peripheral embryonic epithelium (Giesa, 1966). The Vitellocytophagen, at the vegetal pole, engulf yolk cells, and are subsequently overgrown by four spreading dqiiatorialen Hiillsellen. The vegetal Hiilhcllen and additional Hiljzellen transfer the remaining yolk cells into the body of the embryo. The definitive epidermis of M. jiisca is formed when blastomeres move into the embryonic epithelium from the embryonic mass. The blastomeres differentiate into the epidermal cells, the nuclei of which sink back into the parenchyma (embryonic mass) of the turbellarian.

Gastrulation of triclad embryos involves the formation of transitory structures which do not contribute to the definitive organism (Bresslau, 1933; Kato, 1968). Blastomeres. form a thin outer provisional ectoderm around the central yolk syncytium after the yolk cells aggregate. At one point on this provisional

1 Research performed in partial fulfillment of the requirements for the degree Doctor of Philosophy, in the Department of Zoology, University of North Carolina at Chapel Hill, under the direction of Dr. Donald Paul Costello and Dr. Catherine Henley.

539

540

W. DONALD NEWTON

ectodermal sac a transitory pharynx forms by accumulation of blastomeres. A thin-walled cavity forms at the inner end of the temporary pharynx. The cavity is lined by a thin layer of cells, the internal membrane. The external cellular yolk cells are sucked through the transitory pharynx into the thin-walled cavity, now termed the temporary intestine. Between the wall of the temporary intestine and the provisional ectoderm, the definitive epidermis differentiates from proliferating blastomeres and spreads to replace the degenerating cells of the provisional outer and inner membranes.

A remarkable process of gastrulation has been observed in a series of studies on the developmental cycle of Hydrolimax grisea. Hydrolimax is the only fresh- water member of the alloeocoel family Plagiostomidae reported from North America (Hyman, 1938). First described by Haldeman (1843), Hydrolimax was rediscovered by Hyman (1938) in collaboration with Ulric Dahlgren of

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FIGURE 1. Cleaving embryo of Hydrolimax grisca enveloped by radially arranged (and closely adhering) yolk cells. Note the arrangement of the blastomeres : the smaller blasto- meres are situated in the center of the embryo (embryonic mass) ; Costello's fixative, 4° C; Ib, large blastomere ; sb, small blastomere ; ycm, yolk-cell mass ; yen, yolk-cell nucleus.

GASTRULATION OF HYDROLIMAX GRISEA

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FIGURE 2. Older embryo : a solid mass of blastomeres situated in the center of the yolk-cell mass. Slightly older embryos are bilobed ; Heath's polyclad fixative, 4° C ; em, embryonic mass.

Princeton University. Hyman (1938) gave an account of the morphology and taxonomy and reviewed the scant literature of this turbellarian. A review of the more recent literature and a study of the oogenesis of Hydrolwnax is presented in an earlier paper (Newton, 1970).

MATERIAL AND METHODS

The cocoons of Hydrolimax are characteristically red-brown in color and surprisingly large (up to 2 mm in diameter) for the size of the adult turbellarian (4 to 10 mm in length; 1 to 2 mm in width), as noted by Hyman (1938). They are deposited on hard surfaces, the underside of rocks, leaves, on bark of submerged tree trunks and rarely on emergent plants. The cocoons are deposited in the early spring, late March or mid-April, to June.

The material for this study was collected from two sources, Little Creek, east of Chapel Hill, and Morgan Creek, below University Lake impoundment west of Chapel Hill, North Carolina. Prior to fixation the cocoon capsules were

542

W. DONALD NEWTON

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FIGURE 3. Embryo with expanding epidermal cavity lined by lightly-staining epidermal cells. The embryo is approaching the surface of the yolk-cell mass ; Worcester's fixative, 25° C ; ec, epidermal cavity ; ep, epidermis.

punctured with insect pins to permit penetration of fixatives, as the capsule proved impermeable to the fixatives. During washing, the cocoons were opened up more to aid in washing, dehydration and infiltration. The specimens were placed in stender dishes and flooded with one of the following fixatives : Bouin's, Allen's, Worcester's, Heath's polyclad fixative and D. P. Costello's modification of Heath's fluid (sat. mercuric chloride, 81 parts; formalin, C. P., 9 parts; glacial acetic acid, 5 parts). The fixatives were used at initial temperatures of 25° C and 4° C. In addition some material was fixed in Bouin's at 37° C. The cocoons remained in the fixatives from 4 to 12 hours. Material fixed in Bouin's and Allen's fluids was washed in 70% ethanol until picric acid no longer leached out. Material fixed in Worcester's, Heath's and Costello's fluids was washed in several changes of distilled water for a period slightly longer than the time of fixation.

GASTRULATION OF HYDROLIMAX GRISEA

543

The cocoons were dehydrated, cleared, and infiltrated and embedded in filtered Paraplast (M. P. 56-57° C). Serial sections, at 8 /j,, were cut. The sectioned material was stained with Heidenhain's iron-haematoxylin according to the procedures and recommendations of McClung and Conn (1937). Material fixed in Bouin's and Allen's fluids was more difficult to extract, taking longer, and, in the final stages of extraction, more difficult to control. Drawings were made with the aid of a camera lucida attached to a Spencer student microscope with a 4 mm objective and 15 X ocular.

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FIGURE 4. Epidermal cavity has expanded, forcing the overlying yolk cells aside. The epidermis nearest the surface of the yolk-cell mass will thin and become perforated. Note increased number of epidermal cells; Worcester's fixative, 25° C.

544

W. DONALD NEWTON

Some shattering of sections by cocoon capsule fragments did occur, but, in general, excellent serial sections were obtained.

OBSERVATIONS

Hydrolimax grisea, an hermaphroditic animal, practices mutual insemination by injection of spermatozoa through the wall of the genital atrium into the adjacent parenchyma (Hyman, 1938; Kepner, Stirewalt and Ferguson, 1941; Stirewalt, Ferguson and Kepner, 1942). The spermatozoa make their way through

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FIGURE 5. Epidermal cavity has collapsed and the embryo is in the process of inversion. The embryo rises out of and spreads over the yolk-cell mass (arrows) ; Worcester's fixative, 25° C; br, brain.

GASTRULATION OF HYDROLIMAX GRISEA

545

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FIGURE 6. Inverted embryo of Hydrollmax. The lightly-staining epidermis is spreading posteriorly to entrap the yolk-cell mass; Bouin's fixative, 25° C.

the parenchyma of the adult turbellarian to the oocytes (Hyman, 1938). At no time do the spermatozoa enter into the oviduct, as I have observed in studies of many sectioned specimens of mature Hydrolima.r. As described earlier (Newton, 1970), the oocytes are fertilized prior to breakdown of the germinal vesicle. The fertilized oocytes migrate to the common genital atrium where 9 to 36 of them are incorporated, along with many yolk cells, into the cocoon. At the time of cocoon formation, the oocytes are in meiotic prophase I (Newton, 1970).

Prior to the maturation divisions of the eggs, the yolk cells are spherical and loosely arranged around the oocytes. By meiotic metaphase I, the yolk cells elongate and become radially arranged around the egg. They adhere closely to the egg and maintain a closed envelope around the developing embryo (Figs.

546 W. DONALD NEWTON

1-5). Cleavage of the egg occurs within this enveloping yolk-cell mass. The yolk cells follow or push into the cleavage furrows, apparently causing the wide separa- tion of the blastomeres. In the late cleavage stages of the embryo of Hydrolimax a pattern of cell distribution emerges. The blastomeres, at first scattered among the yolk cells, aggregate toward the center of the yolk-cell mass, with the smaller blastomeres oriented toward the center of the embryo (Fig. 1). Cleavage con- tinues until the embryo is a solid mass of cells (Fig. 2). In embryos slightly older than that of Figure 2 the mass of blastomeres, the main embryonic mass, is bilobed.

The cytoplasm of the blastomeres in the center of the embryo becomes less basophilic than the cytoplasm of surrounding blastomeres. A cavity appears among these lightly-staining cells and expands (Fig. 3). The cells which line the expand- ing cavity are derived from the lightly-staining blastomeres. These cells lining the cavity are the epidermal ( cctodermal] cells of Hydrolima.v. At stages of develop- ment slightly later than that of Figure 4, the cells lining the cavity are, at the light microscope level, similar in many respects to the epidermal cells of the adult Hydrolimax. It is this similarity which first drew my attention to the origin of the epidermis of this turbellarian. Because the cavity is lined by epidermis, it will be called the epidermal caint\. The epidermal cells flatten and spread as the cavity continues to expand. They increase in number, apparently through recruitment from the main embryonic mass, as no mitoses are seen among the epidermal cells.

The embryo approaches the surface of the enveloping yolk cell mass (Fig. 4). At this stage the embryo may be described as a mass of cells to one side of which is the expanding epidermal cavity. In Figure 4 the epidermal cavity is close to the surface of the yolk-cell mass and the main embryonic mass is centrally disposed. That portion of the embryo near the surface of the yolk-cell mass consists of a single layer of epidermis with a few associated embryonic cells. The covering yolk cells withdraw or are pushed aside by the expanding epidermal cavity. The epidermis thins and becomes perforated ; the opening which appears unites the epidermal cavity with the environment within the cocoon. The epidermal cavity becomes obliterated by compression of the embryo by other embryos and yolk-cell masses within the cocoon (Fig. 5).

The epidermal cells spread out from the opening of the collapsed epidermal cavity as the embryo rises out of the yolk-cell mass. The embryo is thus com- pletely inverted and comes to lie to one side of the yolk-cell mass (Fig. 6). During inversion and afterward, the epidermis, accompanied by a few internal embryonic cells, stretches posteriorly to cover and entrap the yolk-cell mass. Gastrulation in Hydrolima.v is thus completed. At the close of gastrulation the embryo is bounded by a single layer of epidermal cells, beneath which are scattered the embryonic cells which accompanied the epidermis during epiboly. The anterior end of the embryo contains the main mass of embryonic (parenchymal) cells in which the brain (rf. Figs. 5 and 6) and pharynx are differentiating and from which will develop other definitive organs of the adult.

DISCUSSION

The gastrulation of Hydrotimax, as described, is apparently unique. Com- pared with the gastrulation of other turbellarians, however, it parallels a two- part pattern of (1) differentiation of the epidermis and (2) movement of the

GASTRU.ATION OF IIYDROLTM.IX (',RISEA -S47

epidermis to its definitive location (cf. Ball, 1916). The differentiation of the epidermis of H. grisea is manifested by a loss of basophilia by the ectodermal cells in the center of the embryonic mass and by the formation of the epidermal cavity among the epidermal cells.

Unique to H. grisea is the differentiation of the epidermal cells in the center of the embryo. In Paravortex (Ball. 1916) and in Mesostoma (Bresslau, 1904) the epidermal cells appear on one side of the embryonic mass, after the embryo has moved to one side of the yolk-cell mass. They flatten and spread, entrapping yolk cells and parenchymal cells. Bresslau's (1904) study of the embryonic de- velopment of Plagiostomum girardi revealed an interesting pattern of grastrulation as related to the early cleavage and distribution of blastomeres : (1) The micro- meres are always directed toward the capsule wall of the cocoon. (2) The epidermis arises on the side of the embryo facing the capsule wall. The prospec- tive epidermal cells migrate to the surface of the yolk-cell mass and differentiate, flattening and spreading. The central disposition of the small blastomeres of the embryo of H \dro1imax suggests a similar pattern : The small blastomeres are di- rected toward the center of the embryo. The epidermis arises in the center of the embryonic mass. However, studies of the lineage of the epidermal cells of Hydrolimax are incomplete.

In Hydrolimax, the formation and expansion of the epidermal cavity are the initial steps of gastrulation. The epidermal cavity is not a blastocoel (cf. Giesa. 1966). The expansion of the epidermal cavity apparently pushes the yolk cells aside and forces the epidermal cells to the outer surface of the yolk-cell mass where they can participate in the subsequent steps of gastrulation, inversion and epiboly. Inversion in the case of Hydrolimax is accomplished when the embryo, displaced from the center of the yolk-cell mass, opens out onto the surface of the yolk-cell mass. The inversion as described for Hydrolimax is not comparable to "inversion" as it occurs in certain sponge larvae : the flagellated choanocvtes invert through the osculum of the larval sponge, forming an amphiblastula. The choanocvtes assume their definitive position within the body of the sponge during gastrulation by a process of imagination (Okada, 196S).

The process of gastrulation of the embryo of Hydrolimax can perhaps be more appreciated and better understood by considering the environment in which the embryo develops : as a pre-meiotic oocyte, and during meiosis, cleavage, and early gastrulation, the developing turbellarian is surrounded by volk cells which contribute nothing directly to the definitive organs. The yolk cells provide nutriment to the embryo and to the juvenile turbellarian after it emerges from the cocoon. Each embryo in the cocoon is surrounded by its own yolk-cell mass. The yolk cells adhere closely to the egg. They push into the cleavage furrows and apparently cause or contribute to the initial scattering of the blastomeres through the yolk-cell mass. The blastomeres reaggregate within the center of the yolk-cell mass- — by what process or force remains to be discovered. There the blastomeres organize and/or differentiate for the task of inversion, which is accomplished to a large extent by the expanding epidermal cavity.

I wish to thank Dr. Donald Paul Costello, Dr. Catherine Henley and "Mr. Donald E. Kent for advice during the course of this study. I am grateful

548 W. DONALD NEWTON

to Dr. Louise Bush of Drew University for reading an earlier version of the manuscript and for providing information on the taxonomy and literature of the plagiostomids.

SUMMARY

1. Three events characterize the gastrulation of Hydrolimax grisea: formation of the epidermal cavity, inversion, and epiboly.

2. The epidermal cavity, the outstanding feature of gastrulation in this animal, forms in the center of the embryo among the lightly-staining cells of the future epidermis. The cavity expands, pushing aside the yolk cells which surround the embryo. Eventually the cavity becomes open at the surface of the yolk-cell mass. It is subsequently obliterated by compression from the other embryos within the cocoon.

3. Inversion begins when epidermal cells, which lined the epidermal cavity, spread around the yolk-cell mass from the opening of the collapsed cavity. The embryonic mass rises from the center to the surface of the yolk-cell mass, con- tributing to the progress of the inversion.

4. When the embryo is located to one side of the yolk-cell mass, epiboly begins. The epidermal cells stretch posteriorly to entrap the yolk-cell mass. A few parenchymal cells accompany the spreading epidermis.

LITERATURE CITED

BALL, STANLEY C., 1916. The development of Paravorte.r f/cmellipara. J. Mnrphol., 27 : 453-558.

BRESSLAU, ERNST, 1904. Beitrage zur Entwicklungesgeschichte der Turbellarien I. Die Entwicklung der Rhabdocolen uncl Alloeocolen. Z. Wiss. Zoo!., 76: 213-332.

BRESSLAU, ERNST, 1933. Turbellaria. Pages 52-293 in W. Kukenthal and T. Krumbach, Eds., Handbuch der Zoologic, 2 : 52-293.

GIESA, SIEGFRIED, 1966. Die Embryonalentwicklung von Mnnocclis fusca Oerstad (Turbellaria, Proseriata). Z. MorphoL 6kol. Tiere, 57: 137-230.

HALDEMAN, S. S., 1843. Description of two new species of Cypris, and a genus of Sterel- mintha presumed to be new. Proc. Acad. Nat. Scl. Philadelphia, 1 : 166.

HYMAN, LIBBIE H., 1938. North American Rhabdocoela and Alloeocoela. II. Rediscovery of Hydrolimax grisea Haldeman. Amcr. Mus. Nov., 1004 : 1-19.

HYMAN, LIBBIE H., 1951. The Invertebrates II. Platyheluuntlics and Rliynchocoela. McGraw- Hill Book Co., Inc., New York, 550 pp.

KATO, KOJIRO, 1940. On the development of some Japanese polyclads. Jap. J. Zool., 8 : 537-573.

KATO, KOJIRO, 1968. Platyhelminthes. Pages 125-143 in M. Kume and K. Dan, Eds., [trans, by J. C. Dan], Invertebrate Embryology. NOLIT, Belgrade.

KEPNER, WILLIAM A., M. A. STIREWALT AND F. F. FERGUSON, 1941. A new turbellarian (Alloeocoela) from Beaufort, North Carolina, Plagiostomnm dahlgreni, n. sp. /. Elisha Mitchell Sci. Soc., 57 : 253-260.

McQ-UNG, C. E., AND H. J. CONN, 1937. Stains and Staining. Pages 537-614 in C. E. McClung, Ed., Handbook of Microscopical Technique. Paul B. Hoeber, New York.

NEWTON, W. DONALD, 1970. Oogenesis of the freshwater turbellarian Hydrolimax grisea (Platyhelminthes; Plagiostomidae) with special reference to the history of the super- numerary asters and central bodies. /. Morphol., 132 : 27-46.

OKADA, KIICHIO, 1968. Porifera. Pages 71-85 in M. Kume and K. Dan, Eds. [trans. J. C. Dan], Invertebrate Embryology. NOLIT, Belgrade.

STIREWALT, M. A., F. F. FERGUSON AND W. A. KEENER, 1942. Two new Turbellaria (Alloeo- coela) from Beaufort, North Carolina, belonging to the new genus, Pregermarium. J. Elisha Mitchell Sci. Soc., 58: 69-78.

Reference : Biol. Bull., 139: 549-556. (December, 1970)

COELOMOCYTE AGGREGATION IN CU CUM ARIA FRONDOSA:

EFFECT OF ETHYLENEDIAMINETETRAACETATE,

ADENOSINE, AND ADENOSINE NUCLEOTIDES

I'. B. NOBLE i

Institute for Cell Studies, Faculty of Medicine, and the Marine Sciences Research Laboratory, Memorial University of Newfoundland, St. John's, Nezvfoundland

Coelomocytes, a collective name for cells existing within the coelomic fluid of echinoderms, rapidly aggregate upon removal from the coelomic cavity. Descrip- tive accounts of the aggregation phenomena have been given by various workers (Bookout and Greenburgh, 1940; Boolootian and Giese, 1959; Endean, 1966), and a striking feature is the morphological change which occurs in one of the coelomocyte types, the bladder amoebocyte, and which appears to involve extensive membrane rearrangement.

Ethylenediaminetetraacetate (EOT A) was first used as a disaggregating agent by Anderson (1953) and is thought to act by chelating with divalent cations, in particular, calcium and magnesium, which, at the turn of the century, were shown to be of importance in cell aggregation (Roux, 1894).

Recently, much interest has been centered around the effects of adenosine and adenosine nucleotides on the aggregating behavior of cells ; for example, adenosine diphosphate (ADP) has been shown to enhance platelet aggregation which can be reversed by adenosine or 2-chloroadenosine (Born and Cross, 1963), and adenosine triphosphate (ATP) has been found to inhibit the aggregation of embryo chick fibroblast cells (Knight, Jones, and Jones, 1966). Adenosine nucleotides have been implicated as playing an important physiological role in cellular adhesive and aggregating mechanisms which appear to reside at the cell surface (Jones, 1966).

In this study an attempt has been made to measure quantitatively the effects of EDTA, adenosine, and adenosine nucleotides upon coelomocyte aggregation in the holothurian, Cucumaria frondosa.

MATERIALS AND METHODS

Sea cucumbers weighing between 238 g and 425 g were obtained from Logy Bay, Newfoundland. They were kept in running sea water of temperature range 1° C to 3° C during the course of these experiments, which lasted from January to April. All needles and glassware coming into contact with coelomic fluid were siliconized with Siliclad (Clay-Adams). The substances to be tested for their effect upon coelomyocyte aggregation were used at the following concentrations: EDTA, 6 X 10-3M; ATP, ADP, and adenosine monophosphate (AMP), all at 5 X W'*M. They were dissolved in an artificial sea water (ASW), calcium and magnesium free, of the following composition : sodium chloride, 28.326 g ;

1 Postdoctoral Fellow of the Medical Research Council of Canada.

549

550 P. B. NOBLE

sodium sulfate, 3.917 g; potassium chloride, 0.664 g; sodium bicaronate, 0.192 g; and potassium bromide, 0.096 g per liter of double-distilled water. Calcium- and magnesium-free sea water was used because EDTA and, to a lesser degree, ATP and ADP chelate with these divalent cations. If these compounds were dissolved in ordinary sea water, it would be difficult to assess what effective concentration of these compounds was coming into contact with coelomic fluid and coelomocytes.

Particluate matter was removed from the solutions by filtering through a milli- pore filter of 0.45 fi pore size. The pH of the solutions was adjusted to either pH 6.0 or pH 7.8 using 0.1 N HC1 and 0.1 AT NaOH, respectively, and was checked prior to each experiment. The pH of the resulting mixture of coelomic fluid and test solution was not measured.

Coelomcyte samples were obtained by piercing the dorsal body wall with a 22-gauge needle and withdrawing 0.1 ml of coelomic fluid into a disposable tuber- culin syringe containing 0.1 ml of one of the test solutions. The two solutions were mixed by moving the plunger in the syringe gently to-and-fro several times. A drop of fluid was then placed in a chamber which was made as follows : two strips of Parafilm (Fisher), 1 cm apart, were placed on a microscope slide, and a glass coverslip was carefully lowered on top of the drop of fluid, with its edges resting upon the strips of Parafilm. The resulting chamber was sealed with Vaseline. At no time was the drop of fluid allowed to come into contact with either the Parafilm strips or Vaseline seal. The strips of Parafilm prevented the weight of the coverslip from causing damage to the cells.

Twenty minutes after the initial withdrawal of the coelomic fluid, the percent- age of all cells that were not associated with a cell aggregate, that is, the free cells, was determined in the chamber. Of these free cells, the ratio, expressed as a percentage, of the number of bladder amoebocytes to the sum of bladder and filiform amoebocytes was enumerated in order to assess the tendency of the cells to undergo morphological change. The number of cell aggregates and the number of cells per aggregate were recorded as an indication of the intensity of the cellular aggregation process.

The chambers, solutions, needles, and syringes were kept at 4° C, except when the sample was withdrawn and the counting of the cells was done in the chambers. A minimum of 200 cells, both free and in aggregates, were counted. The same six animals were used for each solution at each pH value.

RESULTS

The effects of EDTA, ADP, and ATP upon coelomocyte aggregation are sum- marized in Table I. Of all the substances tested, only EDTA at pH 6.0 prevented both the morphological change from the bladder to filiform amoebocyte and the aggregation of coelomocytes. EDTA at pH 7.8 largely prevented cell aggregation but did not prevent the morphological change. Adenosine triphosphate at pH 7.8 appeared to enhance the morphological change of bladder to filiform amoebocyte. The results obtained with the remaining substances were not statistically significant from those obtained with ASW (Ca++ and Mg++ free) controls. The effect of pH alone upon coelomocyte aggregation was not found to be statistically significant when the data obtained for ASW (Ca++ and Mg+f free) at pH 6.0 and pH 7.8 were compared (P > 0.05).

COELOMOCYTE AGGREGATION

551

TABLE I

Effects of EDTA, adenosine and adenosine nucleolides upon coelomocyte aggregation

	PH	Of all cells % free	Of free cells B/B + F X lOOf
\s\V*	6	65.82 ± 5.81	7.67 ± 2.27
	7.8	39.54 ± 9.72	4.98 ± L.53
ASW*	6	89.7U ± 5.45**	93.87 ± 2.26***
+ EDTA 6 X 10- • .17	7.8	79.76 ± 7.39**	10.37 ± 2.04
ASW*	6	53.64 ± 9.50	7.98 ± 2.91
+ Adenosine 5 X 10~4 M	7.8	58.67 ± 7.82	1.83 ± 1.15
ASW*	6	54.25 ± 6.K)	4.36 ± 3.09
+ AM!' 5 ( 10-' .17	7.8	52.88 ± 8.03	1.22 ± 0.88
ASW*	6	46.84 ± 11.24	6.72 ± 3.87
+ A DP 5 X 10~4 M	7.8	51.63 ± 9.04	0.68 ± 0.45
ASW*	6	56.18 ± 8.06	3.64 ± 2.71
+ ATP 5 X 10"4 .17	7.8	47.31 ± 3.50	0.21 ± 0.20**

t Bladder anioebocyte/bladder + filiform amoebocyte X 100.

* ASW— Artificial sea water Ca++ Mg++ free.

** 0.01 > 1> > 0.001 significant 1.,

*** P < 0.001 highly signihcant/Ulth res^ect to ASU at appropriate pH

\lc.in ± S.E.M. 6 animals.

The percentage of all the cells counted that were found in a given aggregate is shown in Figure 1. The intensity of the cell aggregation process was found to be minimal with EDTA at pH 6.0, and this rinding is in keeping with the results described for EDTA at pH 6.0 upon the morphological change and cell aggrega- tion. The very small number of aggregated cells obtained with EDTA at pH 6.0 were bladder amoebocytes, which were probably adjacent to one another purely by chance in the counting chamber and therefore were counted as a cell aggregate.

Coelomocyte morphology

It is relevant to this study to describe the more predominant coelomocyte types and to present the morphological changes which occur prior to and during cell aggregation. Figures 2-11 show these morphological changes as well as the major coelomocyte types.

The bladder amoebocyte (Figs. 2 and 3) is 15 ^-20 p. in diameter and possesses petaloid or bladder-like lobes which appear to undergo extensive morphological rearrangement to what is known as the filiform amoebocyte (Figs. 4 and 5) as a prerequisite to cell aggregation. Morula cells, appoximately 10 fj. in diameter, are capable of movement and are characterized by several refractile granules within their cytoplasm when viewed with phase-contrast (Fig. 6). Figures 7 to 9 show motile cells which can exhibit a variety of different forms, and these have been called "lymphocytes" (Hetzel, 1963) because of their morphological and staining

552

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COELOMOCYTE AGGREGATION

553

FIGURE 2. All photographs phase-contrast ; Bladder amoebocyte, X 400. FIGURE 3. Bladder amoebocyte, X 1000. FIGURE 4. Filiform amoebocyte, X 400. FIGURE 5 Filiform amoebocyte, X 1000. FIGURE 6. Morula cell, X 1000. FIGURES 7, 8 and 9. Lymphocytes, X 1000. FIGURES 10 and 11. Typical coelomocyte aggregation, X 400.

354 P. B. NOBLE

similarities to mammalian lymphocytes. This is an unfortunate choice of termi- nology since it implies that these cells are analogous to mammalian lymphocytes which are known to be responsible for cell mediated and humoral immune mecha- nisms, properties \vhich have not yet been shown to be possessed by echinoderm "lymphocytes". It is important to note that lymphocytes are notoriously deficient iu morphological details, a fact contributing to the difficulties encountered in higher animals or associating the many physiological properties observed with this morphologically classified type of cell. Until such time that the known functions of echinoderm "lymphocytes" will suggest a functional terminology, a term should be used which denotes our ignorance regarding the functions of these cells and not one which implies properties as yet unproved.

Typical coelomocyte aggregates are shown in Figures 10 and 1 1 .

DISCUSSION

It has been shown that EDTA at pH 6.0 prevents both the morphological change and cell aggregation. At this pH, EDTA is reported to have a low stability coefficient for binding calcium ions, the greatest binding capacity for calcium being at pH 8.0 (Curtis, 1967). In this study, EDTA at pH 7.8 pre- vented aggregation but did not prevent the morphological change from bladder to filiform amoebocyte. These differences suggest that the cell aggregation phenomena can be divided into two different stages : one possibly requires calcium ions for the actual aggregation process, while the morphological change, and con- sequently cell aggregation, is prevented either by the removal of some other material which is chelated by EDTA at pH 6.0 or by EDTA reacting directly with the cell membrane or some other cellular component. In this regard it is pertinent to recall that L. Weiss (I960) pointed out that effective disaggregation of cells by EDTA is not proof that it does so by chelating with calcium ions.

Jones (1966) has recently put forward a unifying hypothesis of cell adhesion linking the adenosine nucleotides with contractile and relaxing properties of an actomyosin-like protein with ATPase activity located at the cell surface. For example, levels of ATP and ADP. by governing the physiological state of the contractile protein could, by initiating conformational changes in the membrane, bring about changes in the distribution of charges at the cell surface and hence alter adhesiveness and aggregating ability of cells. Evidence has been obtained using a variety of different cellular systems to support this concept. For example, glycerol-extracted cells of non-muscular origin, contracted in the presence of exo- genous ATP (Hoffman-Berling, 1954), and contractile proteins have been isolated from many different cell tvpes (Hoffman-Berling, 1956; Bettex-Galland, and Luscher, 1959; Loewy, 1952) ; ATPase activity has also been demonstrated in membranes of cells (Essner, Novikoff, and Masek, 1958; Novikoff, 1960).

No effect of adenosine or adenosine nucleotides upon coelomocyte aggregation was found in this study, with the possible exception of ATP at pH 7.8, which appeared to enhance the morphological change. One explanation of this latter result is that ATP provided an additional source of energy necessary for the membrane rearrangement which occurs in the bladder amoebocvte. An attempt was made to use a potentially more sensitive method of observing the effects of adenosine and adenosine nucleotides upon coelomocyte aggregation, that is, the

COELOMOCYTK \< ,< ,RK< , \TION

turbiclimetric method of Born and Cross (1963) which has been used extensively to follow aggregation of mammalian cells. However, in order to standardize the number of coelomocytes per cubic millimeter of fluid it was necessary to prevent aggregation by using EDTA at pH 6.0. Subsequent addition of adenosine and adenosine nucleotides did not produce aggregation as measured by a decrease in optical density and by direct microscopical observation. This finding is. of course, in keeping with the data presented in this study. The interpretation of this result is complicated, however, by the observation of P.orn and Cross (1963), who showed that platelets would not aggregate in response to ADP if the cells were in plasma which contained EDTA as an anticoagulant. One could argue that the presence of EDTA prevented any subtle effects of adenosine and adenosine nucleotides upon aggregation from being detected.

That adenosine and adenosine nucleotides, when used at concentrations which are effective in modifying avian and mammalian cell aggregation, fail to modify coelomocyte aggregation raises the question as to whether or not the mechanism of coelomocyte aggregation is similar to that postulated for other cell aggregating systems (Jones, 1966). To answer fully this question, studies are needed to see if contractile and relaxing proteins having ATPase activity can be isolated from coelomocyte membranes.

The result obtained with EDTA at pH 6.0 is also contrary to the known effects of EDTA at this pH upon mammalian and other cell aggregating systems. Many reports on the failure of EDTA to prevent cell aggregation and the en- chancement of cell dispersion have been attributed to its poor binding capacity for calcium ions at this pH ( Curtis, 1967). The mechanism by which EDTA at pH 6.0 stabilizes bladder amoebocvte membranes warrants further investigation.

The author is indebted to Dr. K. "B. Roberts. Professor of Physiology, for his advice and encouragement and critical reading of the manuscript. My thanks also go to Dr. F. A. Aldrich, Director of the Marine Sciences Research Labora- tory, who so generously granted facilities for this work.

SUMMARY

Cells found within the coelomic cavity of the sea cucumber. Cucuinaria frondosa, aggregated rapidly upon removal from the body. This cell aggregation is normally characterized by a morphological change in the bladder amoebocvte which becomes a filiform cell. A method has been devised whereby the effects of EDTA, adenosine, and adenosine nucleotides could be observed upon coelomocyte aggregation. It was found that EDTA at pH 6.0 in ASW (Ca++ and Mg" free) prevented the morphological change from bladder to filiform amoebocvte as well as cell aggregation. EDTA at pH 7.8 in ASW (Ca++ and Mg++ free) did not prevent the morphological change although it did prevent cell aggregation. P>ased on the different chelating affinities of EDTA for calcium ions at these two dif- ferent pH values, it is suggested that coelomocyte aggregation has two components : (1) the morphological change, which is Ca++ independent and possibly requires the removal of some other cation or is a direct effect of EDTA upon some

556 P. R NOBLE

cellular component, and (2) the cell aggregation itself which appears to be Ca+l dependent.

Adenosine and adenosine nucleotides in general did not appear to influence coelomcyte aggregation. These results raise the question as to whether or not the mechanism of coelomocyte aggregation is similar to and can he described in the same terms as the mechanism currently used to explain avian and mammalian cell aggregation.

LITERATURE CITED

ANDERSON, N. C., 1953. The mass isolation of whole cells from rat liver. Science, 117:

627-628. BETTEX-GALLAND, M., AND E. F. LUSCHER, 1959. Extraction of an actomyosin-like protein

from human thrombocytes. Nature, 184: 276-277. BOOKHOUT, C. G., AND N. D. GREENBURG, 1940. Cell types and clotting reactions in the

echinoid, Mellita quinquiesperforata. Biol. Bull., 79 : 309-320. BOOLOOTIAN, R. A., AND A. C. GIESE, 1959. Clotting of echinoderm coelomic fluid. /. Exp.

Zool, 140: 207-229. BORN, G. V. R., AND M. J. CROSS, 1963. The aggregation of blood platelets. /. Physiol.,

108: 178-195. CURTIS, A. S. G., 1967. The Cell Surface: It's Molecular Role in Morphogenesis. Logos

Press, London, 129 pp. ENDEAN, R., 1966. The coelomocytes and coelomic fluid. Chapter 13 in R. A. Boolootian,

Ed., The Physiology of Echinodcrmata. Interscience Publishers, New York. ESSNER, E., A. B. NOVIKOFF AND B. MASEK, 1958. Adenosine triphosphatase and 5' nucleo-

tidase activities in the plasma membrane of liver cells as revealed by electron micro- scopy. /. Biophys. Biochem. Cytol., 4: 711-716. HETZEL, H. R., 1963. Studies on holothurian coelomocytes — 1 A survey of coelomocyte

types. Biol. Bull, 125 : 289-301. HOFFMAN-BERLING, H., 1954. Adenosintriphosphate abs betriebsstoff von zellbewegungen.

Biochem. Biophys. Acta, 14: 182-194. HOFFMAN-BERLING, H., 1956. Das kontraktile eiweiss undifferenzierter zellen. Biochem.

Biophys. Acta, 19: 453-463.

JONES, B. M., 1966. A unifying hypothesis of cell adhesion. Nature, 212 : 362-365. KNIGHT, V. A., B. M. JONES AND P. C. T. JONES, 1966. Inhibition of the aggregation of

dissociated embryo-chick fibroblast cells by adenosine triphosphate. Nature, 210 :

1008-1010. LOEWY, A. G., 1952. An actomyosin-like substance from the plasmodium of a myxomycete.

/. Cell Comp. Physiol., 40: 127-156. NOVIKOFF, A. B., 1960. Enzyme localization in tumor cells. Pages 219-267 in Cell Physiology

of Neoplasia. University of Texas Press, Austin. Roux, W., 1894. Uber den "Cytotropismus" der furchungszellen des grasfrosches (Rana

fttsca). Arch. Entwick lung sine ch. Organismen, Wilhclm Roux, 1: 43-68. WEISS, L., 1960. The adhesion of cells. Int. Rev. Cytol., 9: 187-225.

Reference: Biol. Bull, 139: 557-573. (December, 1970)

LARVAL DEVELOPMENT OF PAGURUS ANNULIPES (STIMPSON,

1862) AND PAGURUS POLLICARIS SAY, 1817

REARED IN THE LABORATORY

CARL F. NYBLADE

Department of Zoology, University of Washington, Seattle, Washington 98105

The hermit crabs Pagurus annulipes (Stimpson, 1862) and P. pollicaris Say, 1817 are common hermits found in shallow subtidal water from Massachusetts south to Florida. The southern distribution of P. annulipes reaches some distance north of Miami on the east coast of Florida and on the west coast of Florida extends from perhaps central or northwestern Florida westward along the perim- eter of the Gulf of Mexico at least to Texas (Provenzano, 1959 and personal communication; Rouse, 1969). P. pollicaris, perhaps a subspecies, is also found along the northern perimeter of the Gulf of Mexico from western Florida to Texas (Provenzano, 1959). In the Woods Hole, Massachusetts region these two species and P. longicarpus Say comprise the shallow-water hermit crab fauna.

The ovigerous season of P. annulipes extends from about May to the end of September and completely overlaps that of P. longicarpus. The season of P. pollicaris is from early spring to the end of June. These seasons have been deter- mined by examination of adults and of limited plankton samples during the summers of 1967 and 1968.

A review of the literature on larvae of members of the genus Pagurus has been given by Coffin (1960). All recent literature is cited by Roberts (1970) with the exception of Forss and Coffin, 1960; Kurata, 1964 and 1968; and Greenwood, 1966.

This paper deals with the external anatomy of P. annulipes and P. pollicaris larvae. Utilizing the recent description of P. longicarpus larvae (Roberts, 1970), two keys to the larvae of the shallow-water hermit crabs found in the Woods Hole, Massachusetts region are given, one based on larval pigmentation and the other on larval external anatomy. Comparisons are made with other species of the genus Pagurus in the context of examining current views on groups within the

genus.

MATERIALS AND METHODS

Ovigerous females were obtained from the Marine Biological Laboratory Supply Department of Woods Hole, Massachusetts during the summers of 1967 (P. annulipes) and 1968 (P. pollicaris). They were maintained in beakers until the eggs hatched. Larvae were transferred writh a large bore pipette to small beakers and reared individually (P. annulipes) or in groups of 5 (P. polli- caris). Larvae were maintained at room temperature (24 ± 2° C) and at 16 ±0.5° C (some P. pollicaris) in a controlled environment chamber with con- stant light. Water was changed and Artemia nauplii added as the culturing

557

558 CARL F. \Ym.\[>K

conditions dictated, usually daily or every other day. Larvae were examined daily to determine duration of each stage. Because no P. pollicaris successfully molted to the megalopa, duration for the fourth zoeal stage, was determined by assuming a successful molt. In addition to laboratory-reared specimens the verbal description of P. annulipes larvae utilized specimens obtained from the plankton collected just off the Bureau of Commercial Fisheries Laboratory, Woods Hole, Massachusetts.

Both fresh and preserved (70% ethanol) larvae were dissected with micro- needles under the dissecting microscope. Details were checked using dark field illumination. Verbal descriptions were based on 3 to 10 specimens except for carapace and total length. Depending on the stage, 4—108 specimens of P. annulipes (all planktonic) and 1 specimen of P. pollicaris were measured to determine lengths. Megalopal appendages of P. pollicaris were dissected out from incomplete molts, somewhat distorted and not fully expanded but adequate for description. Drawings were done from single specimens, using a camera lucida to give overall proportions with detail filled in by eye. Chromatophore patterns were determined from living larvae and larvae freshly killed in glycerine.

Throughout the descriptions the terminology of Roberts (1970) has been followed and the following abbreviations have been used: Al — antennule, A2— antenna, Mn — mandible, MX 1 — maxillule, MX 2 — maxilla, Mxp 1 to 3 — first to third maxilliped, P 1 to 5 — first to fifth pereiopod, PI 2 to 5 — second to fifth pleopod, U-— uropod. The verbal descriptions have been abridged to report only- variations of a given character among specimens, i.e., information not illustrated in the figures.

RESULTS

As in other species of the genus, Pagurus annulipes and P. pollicaris have 4 zoeal stages and a megalopa. No variation in the number of stages was observed and no living prezoeae were observed.

Pagurus annulipes (see Figs. 1-4} : Zoea I

Duration 5.4 days (N • =21).

Carapace length 0.9-1.3 mm, av. 1.1 mm (N = 108).

Total length 1.9-2.4 mm, av. 2.2 (N -. - 108).

Carapace without processes except for posterolateral spines. Rostrum unorna- mented, long, drawn out to a thin point, curved slightly ventrally, slightly longer than the antennae, which are longer than the antennules. Eyes sessile. Six abdominal segments, the sixth fused to the telson. Segments 2 through 5 with 2 pairs of posterodorsal and 1 pair of ventrolateral small spines, the ventrolateral spines of segment 5 only slightly longer than the others. Pleopod buds and uropods absent.

Red chromatophores predominate. One complex present around mouthparts extending from the base of the antennules posteriorly on each side of the labrum to the base of the mandibles. A single large red chromatophore on midlateral portion of each side of carapace ; a large red chromatophore in abdominal segments 2 and 3 ; and a complex in segments 5 and 6-telson. One small medial yellow

P. ANNULIPES \\'D POLLICARIS LARVAE

FIC.URE 1. Payurits annulipcs; a. antennules, b. antennae, c. mandibles of zoeal stages I-IV and megalopa.

chromatophore in abdominal segment 6-telson. One large bar-shaped yellow chromatophore in cardiac region of thorax. Diffuse yellow pigment over dark pigment of eye.

A 1 — \Yith 1 long, 2 medium and 1 to 3 short terminal aesthetascs (A 1 similar in zoea II).

A 2 — Scale with 8 plumose setae, 1 or 2 small setae medially.

MX 1 — Basal endite produced into 2 strong spines with 1 to 3 cuneate ^pinnies each.

560

CAKI. F. X'YP.LADE

2. I'di/itnts annulipcs; a. maxillules, b. maxillae, c. first maxillipeds of zoeal stages I-IV and megalopa.

MX 2 — Distal lobe of coxal endite with 3 or 4 terminal setae (similar in zoea II, III, IV). Endopocl unsegmented with 2 or 3 subterminal setae.

Mxp 1 — Basis with 5 to 6 long and 2 or 3 short medial setae (similar in zoea II, III, IV).

P. slNNUI //'/-.V AND POLLICAR1S LARVAE

561

b

FIGURE 3. Pagurus anmilipcs; a. second maxillipeds of zoeal stages I-IV and megalopa, b. third maxillipeds of zoeal stages II-IV and megalopa, c. second through fifth pleopods of megalopa.

Zoea II

Duration 3.4 days (N • - 14).

Carapace length 1.3-1.7 mm, av. 1.5 mm (N

Total length 2.3-3.1 mm, av. 2.9 (N = 40).

= 40),

562

CAR] !•. \YIM..\DE

FIGURE 4. Pagurus aumilipcs; telsons of zoeal stages I-IV and megalopa, with uropods of zoeal stages III and IV and megalopa.

MX 1 — Basal endile with 4 strong spines, the 3 largest each hearing 2 or 3 cuneate spinules (similar in xoea III).

Mxp 3 — Endopod bud with 1 or 2 terminal setae.

Zoca III

Duration 4.0 days (N--7}.

Carapace length 1.5-1.8 mm. av. 1.7 mm (N • ~- 10). Total length 2.8-3.2 mm, av. 3.0 mm (N • - 10). Sixth abdominal segment distinct, with uropods.

P. ANNULIPES AND POLLICARIS LARVAE 563

Pigmentation unchanged except for the appearance of small yellow chromato- phores, 1 at the base of each eye and 1 medially in abdominal segment 2.

A 1 — Outer ramus with 1 long and 2 or 3 shorter terminal aesthetascs plus 2 or 3 short setae. Distal end of peduncle with 1 to 3 small setae.

Zoea IV

Duration 2 days (N = 1).

Carapace length 1.9-2.1 mm (N = 3).

Total length 3.7-3.9 mm (# = 3).

Carapace, rostrum and abdomen segment number unchanged. Pleopod buds present.

Pigmentation unchanged except for the appearance of a yellow chromatophore at the base of each antennule.

A 1 — Outer ramus with 1 long and 3 or 4 shorter terminal aesthetascs and 1 or 2 smaller setae also with 4 or 5 median aesthetascs. Distal end of peduncle with 2 or 3 small setae.

MX 1 — Coxal endite with 6 or 7 stout setae. Endopod with 2 or 3 terminal non-plumose setae.

Megalopa

Duration not determined.

Carapace length 1.3-1.7 mm (^ = 4).

Total length 2.7-3.2 mm (N = 4).

Carapace nearly like adult. Posterolateral spines gone. Rostrum reduced and rounded. Eyes stalked with eye scales present. Abdomen with 6 segments, slightly asymmetric.

Pigmentation greatly elaborated. Of particular interest is incipient banding of the second and third pereiopods, suggesting the adult pattern.

A 1 — Outer ramus terminated with 1 long and 2 or 3 short setae. Inner ramus with 5 or 6 short terminal setae.

A 2 — Flagellum of 10 to 14 segments.

MX 2 — Proximal lobe of coxal endite with 5 or 6 setae. Proximal lobe of basal endite with 7 to 10 setae, distal lobe with 7 to 11 terminal and 2 sub- terminal setae. Scaphognathite with 35 or 36 plumose setae.

Mxp 1 — Coxal endite with 4 or 5 setae.

P 4— Subchelate, propodus with a single row of 5 to 7 tubercles representing adult rasp, dactylus with 1 to 3 tubercles.

PI 2 to 4 — Exopod with 8 long and 1 (2) short plumose seta.

U — Exopod with 12 or 13 plumose and 3 to 5 shorter non-plumose setae and with 11 to 13 tubercles. Endopod with 2 or 3 non-plumose setae and with 4 or 5 tubercles.

Pagurus pollicaris (see Figs. 5-10} : Zoea I

Duration at room temperature 3.8 days (N = 26), at 16° C 11.6 (N - - 17). Carapace length 1.6 mm, total length 2.8 mm.

562

CAR I. P. XYMI.ADE

FIGURE 4. Pac/urus aunulipcs; telsons of zoeal stages I-IV and megalopa, with uropods of zoeal stages III and IV and megalopa.

MX 1 — Basal endite with 4 strong spines, the 3 largest each hearing 2 or 3 cuneate spinules (similar in zoea III).

Mxp 3 — -Endopod bud with 1 or 2 terminal setae.

Zoca III

Duration 4.0 days (N--7).

Carapace length 1.5-1.8 nun, av. 1.7 mm (N • - 10). Total length 2.8-3.2 mm, av. 3.0 mm (N •• - 10). Sixth abdominal segment distinct, with uropods.

P. ANNULIPES AND POLLICARIS LARVAE

563

Pigmentation unchanged except for the appearance of small yellow chromato- phores, 1 at the base of each eye and 1 medially in abdominal segment 2.

A 1 — Outer ramus with 1 long and 2 or 3 shorter terminal aesthetascs plus 2 or 3 short setae. Distal end of peduncle with 1 to 3 small setae.

Zoea IV

Duration 2 days (N = 1).

Carapace length 1.9-2.1 mm (N = 3).

Total length 3.7-3.9 mm (N = 3).

Carapace, rostrum and abdomen segment number unchanged. Pleopod buds present.

Pigmentation unchanged except for the appearance of a yellow chromatophore at the base of each antennule.

A 1 — Outer ramus with 1 long and 3 or 4 shorter terminal aesthetascs and 1 or 2 smaller setae also with 4 or 5 median aesthetascs. Distal end of peduncle with 2 or 3 small setae.

MX 1 — Coxal endite with 6 or 7 stout setae. Endopod with 2 or 3 terminal non-plumose setae.

Megalopa

Duration not determined.

Carapace length 1.3-1.7 mm (N = 4).

Total length 2.7-3.2 mm (N = 4).

Carapace nearly like adult. Posterolateral spines gone. Rostrum reduced and rounded. Eyes stalked with eye scales present. Abdomen with 6 segments, slightly asymmetric.

Pigmentation greatly elaborated. Of particular interest is incipient banding of the second and third pereiopods, suggesting the adult pattern.

A 1 — Outer ramus terminated with 1 long and 2 or 3 short setae. Inner ramus with 5 or 6 short terminal setae.

A 2 — Flagellum of 10 to 14 segments.

MX 2 — Proximal lobe of coxal endite with 5 or 6 setae. Proximal lobe of basal endite with 7 to 10 setae, distal lobe with 7 to 11 terminal and 2 sub- terminal setae. Scaphognathite with 35 or 36 plumose setae.

Mxp 1 — Coxal endite with 4 or 5 setae.

P 4— Subchelate, propodus with a single row of 5 to 7 tubercles representing adult rasp, dactylus with 1 to 3 tubercles.

PI 2 to 4 — Exopod with 8 long and 1 (2) short plumose seta.

U-— Exopod with 12 or 13 plumose and 3 to 5 shorter non-plumose setae and with 11 to 13 tubercles. Endopod with 2 or 3 non-plumose setae and with 4 or 5 tubercles.

Pagurus pollicaris (see Figs. 5-10) : Zoea I

Duration at room temperature 3.8 days (N = 26), at 16° C 11.6 (N = 17). Carapace length 1.6 mm, total length 2.8 mm.

564

CARL F. NVBLADE

FIGURE 5. Pagnrus pollicaris; zoeae I-IV, lateral view. Open circles represent yellow chromatophores ; stippled, red.

Yellow chromatophores predominate. Three small yellow chromatophores on posterior half of rostrum. Red chromatophore complex from the hase of the antennule posteriorly on each side of the lahrum to the base of the mandibles. Yellow chromatophore towards base of each antennae. Two yellow chromatophores towards the outer margin of the bases and 1 towards the inner margin of the exopods of the first maxillipeds. One yellow chromatophore towards outer margin of the bases and 1 towards the inner margin of the exopods of the second maxillipeds. Carapace with 1 or 2 pairs of yellow chromatophores between the eyes, with single yellow chromatophore on midlateral edge of each side, with group of 4 to 6 yellow chromatophores towards posterolateral edge on each side, with 1 yellow chromatophore towards each posterolateral spine, and with a single yellow chromatophore at posteromedial edge. Abdominal segment 1 with 1 or 2 dorsal posteromedial yellow chromatophores, segment 2 with single or pair of posteromedial and a pair of posterolateral yellow chromatophores, seg- ments 3 and 4 with pair of posterolateral yellow chromatophores, segment 5 with 3 pairs of posterolateral yellow chromatophores, segment 6-telson with pair of anteromedial red chromatophores followed posteriorly by 4 pairs of yellow

P. ANNULIPES AND POLLICARIS LARVAE

565

0.2mm

FIGURE 6. f'ni/nnis pollicaris; a. antennules, h. antennae of zoeal stages I-IY and megalopa.

chromatophores plus a pair of posterolateral yellow chromatophores. Diffuse yellow pigment over dark pigment of eyes.

A 1 — 3 or 4 terminal setae.

MX 1 — Basal enclite produced into 2 strong spines with 2 or 3 cuneate spinnules each.

566

CARL F. NYBLADE

0.2mm

0.2mm

FIGURE 7. Pagums pollicaris; a. mandibles, b. maxillules, c. maxillae of zoeal stages I-IV and megalopa.

Mxp 1 — Basis with 6 long and 3 or 4 short medial setae (Mxp 1 similar in zoea II, III, IV). Endopod segment 1 with 3-4 setae.

Mxp 2 — Basis with 2 or 3 short medial setae (similar in zoea II, III, IV). Endopod segment 1 with 1 stout and 1 or 2 finer setae.

Zoea II

Duration at room temperature 3.2 days (N - 46), at 16° C 10.8 (N Carapace length 1.8 mm, total length 3.2 mm.

= 11)

P. ANNULIPES AND POLLICARIS LARVAE

567

FIGURE 8. Pagurns pollicaris; a. first maxillipeds, b. second maxilipeds of zoeal stages I-IV and megalopa.

Pigmentation unchanged except for the addition of 1 or 2 posterior yellow chromatophores in each eye and of a median dorsal red bar-shaped chromatophore in abdominal segment 2.

A 1 — Outer ramus with 2 or 3 short setae. Distal end of peduncle with 1 or 2 small setae on outer edge (similar in Zoea III).

MX 1 — Basal endite with 4 strong spines bearing 0 to 3 cuneate spinules (similar in Zoea III).

Zoea III

Duration at room temperature 3.6 days (N = 33), at 16° C 13.8 (N Carapace length 2.1 mm; total length 3.5 mm.

-9).

568

CARL F. NYBLADE

0.2mm

FIGURE 9. Paynnis pollicaris; a. third maxillipeds of zoeal stage I-IV and megalopa, b. second through fifth pleopods of megalopa.

Pigmentation unchanged except for the addition of 1 anterior yellow chroma- tophore in each eye.

A 1 — Outer ramus with 3 or 4 setae.

Zoea IV

Duration at room temperature 4.6 days (N - • 15), at 16° C 20.5 (N = 2). Carapace length 2.2 mm, total length 3.3 mm.

Pigmentation unchanged.

A 1— Outer ramus with 3 incipient segments ; segment 3 with 5-6 terminal aesthetascs. Distal end of peduncle with 2 or 3 small setae.

P. ANNULIPES AND POLLICARIS LARVAE

569

FIGURE 10. Pay urns pollicaris; telsons of zoeal stages I-IV and megalopa, with uropods of zoeal stages III and IV and megalopa.

MX 1 — Coxal endite with 7 or 8 stout setae.

MX 2 — Proximal lobe of basal endite with 5 or 6 terminal setae and 1 medial.

Megalopa

Duration, carapace and total lengths not determined.

570 CARL F. NYBLADE

Pigmentation not able to be determined, presumed to be predominantly yellow chromatophores. Aesthetasc number on A 1 not able to be determined.

A 2 — Flagellum of 12 to 14 segments.

MX 2 — Proximal lobe of coxal endite with 8 to 10 terminal setae, distal lobe with 10 to 12. Proximal lobe of basal endite with 8 to 11 terminal setae, distal lobe with 8 to 12. Endopod unsegmented with 7 to 12 terminal setae.

Mxp 1 — Exopod with 4 or 5 short plumose setae.

Mxp 2 — Endopod with scattered short setae, 7 to 9 terminally. Exopod with 6 or 7 short terminal plumose setae.

Mxp 3 — Exopod with 6 or 7 terminal plumose setae.

P 4- — Subchelate, propodus with single row of 6 or 7 tubercles representing adult rasp, dactylus with 2 or 3 tubercles.

P 5 — Propodus with tubercles in 4 rows totalling 14 to 16, dactylus with 3 or 4 tubercles and several short setae.

PI 2 to 4 — Exopod with 9(10) plumose setae.

U — Exopod with 10 or 11 long plumose setae and 8 to 12 shorter non-plumose setae, with 19 to 26 lateral and terminal tubercles. Endopod with 4 or 5 non- plumose setae, with 5 to 7 tubercles.

Larval key to three species of the genus Pagurus found in the Woods Hole, Massachusetts region

All species pass through 4 zoeal stages and 1 megalopa. Key to Larval stages

1. Carapace without long rostrum, maxillipeds for feeding, 5 pairs of segmented

functional pereiopods megalopa

Carapace with long rostrum, maxillipeds with natatorial setae, pereiopods un- segmented or weakly segmented rudiments 2

(zoea)

2. Uropods present 3

Uropods absent 4

3. Uropod with exopod free from basis fourth zoea

Uropod unsegmented third zoea

4. Telson with 12 articulated plumose setae, third maxilliped functional with na- tatorial setae second zoea

Telson with 10 articulated plumose setae, third maxilliped rudimentary without setae first zoea

Key to Species

For living or freshly killed larvae. Zoeae and Megalopae

1. Few scattered mostly red chromatophores P. longicarpus

Numerous chromatophores 2

2. Red chromatophores predominate : very large red chromatophore on each mid- lateral portion of carapace and large red chromatophore in abdominal segments 2-3, megalopal pereiopods 2 and 3 with incipient red banding P. annulipes

P. ANNULIPES AND POLLICARIS LARVAE 571

Yellow chromatophores predominate : many on carapace at least 1 pair per abdominal segment, and one on posterolateral sides of telson P. pollicaris

For preserved larvae. Zoeae

1. Ventrolateral spines of abdominal somite 5 long, almost reaching telson

P. longicarpus

Ventrolateral spines of abdominal somite 5 medium, not much larger than those of other somites 2

2. Maxillulary endopod with 3 terminal setae, telson process 4 of zoea III and

IV longest P. pollicaris

Maxillulary endopod with 2 terminal setae, telson process 4 of zoea III and IV very small P. annulipes

Megalopae

Telson formula 4 + 4 : all processes long articulated plumose setae

P. annulipes

Telson formula 5 + 5 : process 1 minute spine, processes 2 to 5 long articulated

plumose setae P. longicarpus

Telson formula 8 + 8: processes 1, 3, and 4 small spines, process 2 a fine hair, and processes 5 to 8 long articulated plumose setae P. pollicaris

DISCUSSION

In 1903 Thompson illustrated from plankton material the larval stages of what he considered to be Pagurus longicarpus, although he was aware of a possible con- fusion with P. annulipes. In comparing the preceding description of P. annulipes with Thompson's figures, the following differences are noted : third zoea slightly smaller than Thompson's ; antennal scale setation of zoea I to IV 8 long and 1 to 2 short, 9 long and 1 short, 10 long, 10 long in this study compared with 9 long and 1 short for all four zoeal stages in Thompson's ; maxillulary basal endite of zoea II with 4 spines compared with 3 spines in Thompson's ; maxilliary scap- hognathite setation of zoea II and III 7 and 9 compared with 6 and 10 in Thompson's; first and second maxilliped exopod setation of zoea II 7 compared with 6 in Thompson's; megalopal pleopod 2 to 5 setation 9 (10), 9 (10), 9 (10), 8 compared with 7, 7, 9, 7 in Thompson's. These differences are minor when compared with the overwhelming smilarity between his figures and this description. It is clear that Thompson in reality figured the larval stages of P. annulipes, not P. longicarpus (see Roberts, 1970). Although Thompson states that larvae of both species are found in plankton samples taken in Woods Hole during the summer, confusion ' arose from his assumption that, since the adults of P. longicarpus are considerably larger than those of P. annulipes, the larvae of P. longicarpus likewise would be larger. Just the opposite is the case ; P. annulipes larvae are the larger.

Roberts (1970) has made a detailed comparison of P. annulipes and P. longi- carpus in his Table II. In addition to the differences which he noted are the following : P. annulipes zoeae have a large bar-shaped yellow chromatophore in

572 CARL F. NYBLADE

the cardiac region of the thorax, P. annulipes maxilliped exopod setation of zoea III is 7 compared with 8 in P. longicarpus, the nropod exopod of P. annulipes zoea IV has 1 terminal spur compared with 1 or 2 in P. longicarpus, and the megalopa of P. annulipes does not have a minute lateral spine on each side of the telson.

MacDonald, Pike and Williamson (1957) divide Pagurus larvae into two groups, A and B, separated by 9 zoeal and megalopal characteristics (see Roberts, 1970, Table III). Pike and Williamson (1960) propose a third group C, which shares 2 of the 9 characteristics with group A larvae, 5 with group B, and which has 3 similar to neither group. Roberts (1970) proposes a fourth group D for P. longicarpus, which shares 3 of the 9 characteristics with group A larvae, 4 with B, and in 2 is not similar to group A, B or C. P. annulipes larvae differ from those of group B in 3 respects : possessing a yellow chromatophore over the stomach region as in group A, having medium length lateral spines on abdominal segment 5 as in group C and having a megalopa with antennae about equal in length to the major cheliped as in group D. From the preceding description of P. poUicaris larvae it is seen that they differ from group B also in 3 respects : having telson process 4 of zoea III and IV long and fused with the telson as in group A, possessing medium length lateral spines on abdominal somite 5 as in group C and having a unique antennal scale setation.

Several points need to be made pertaining to the validity and value of present grouping within the genus Pagurus. Characteristics used to distinguish the groups A, B, C and D are those observable without complete dissection of the larval append- ages. Although not useful for quick identification, less easily observable character- istics of the appendages may be of equal or greater taxonomic significance. Another point is that complete larval descriptions for only 8 and partial descriptions for 9 Pagurus species have been reported (John C. Markham, School of Marine and Atmospheric Sciences, University of Florida, personal communication), out of more than 180 described species (Gordon, 1956). The 3 species most recently described would indicate the necessity of erecting a new group for each or of concluding that the presently proposed groups are neither taxonomically meaningful nor useful in facilitating plankton identification. The latter view is held here.

I am indebted to my wife, Beth, and Hank Roberts for technical help with the manuscript and figures and to Drs. David Egloff, Anthony Provenzano, Jr., Paul Illg, and Morris Roberts, Jr., for their critical reading of the manuscript. During the course of this study I was the recipient of a Public Health Service summer training grant, a Woods Hole Oceanographic Institution Summer Fellow- ship, a National Science Foundation Graduate Fellowship, and a Danforth Foundation Graduate Fellowship.

SUMMARY

1. The external anatomy of 4 zoeal stages and a megalopa of Pagurus annulipes and P. poUicaris is described from laboratory-reared specimens.

2. Keys to the shallow-water hermit crab larvae of the Woods Hole, Massa- chusetts region are given, based on larval pigmentation and on external anatomy.

P. ANNULIPES AND POLLICARIS LARVAE 573

3. Comparison with Thompson's (1903) figures shows that he illustrated P. annulipes, not P. longicarpus.

4. Comparisons with other larvae of the genus Pagurus are made in context of an examination of currently proposed groups within the genus. The validity and usefulness of these groups is seriously questioned.

LITERATURE CITED

COFFIN, H. G., 1960. The ovulation, embryology, and developmental stages of the hermit

crab, Pagurus samuclis Stimpson. U'alla Walla Coll. Publ., 25: 1-30. FORSS, C. A., AND H. G. COFFIN, 1960. The use of the brine shrimp nauplii Artcmia salina

as food for the laboratory culture of decapods. Walla Walla Coll. Publ., 26: 1-15. GORDON, J., 1956. A bibliography of pagurid crabs, exclusive of Alcock, 1905. Bull. Amcr.

Mus. Natur. Hist., 108: 257-352. GREENWOOD, J. G., 1966. Some larval stages of Pagurus no-cac-zealandiae (Dana), 1852.

(Decapoda, Anomura). New Zealand J. Sci., 9: 545-558. KURATA, H., 1964. Larvae of decapod Crustacea of Hokkaido. 5. Paguridae (Anomura).

Bull. Hokkaido Regional Fish. Res. Lab., 29: 24-48. KURATA, H., 1968. Larvae of Decapoda Anomura of Arasaki, Sagami Bay — I. Pagurus

samuclis (Stimpson) (Paguridae). Bull. Tokai. Regional Fish. Lab., Tok\o., 55:

265-270. MACDONALD, J. D., R. B. PIKE AND D. I. WILLIAMSON, 1957. Larvae of the British species

of Diogenes, Pagurus, Anapagurus and Lithodes (Crustacea, Decapoda). Proc. Zoo/.

Soc., London., 128: 209-257. PIKE, R. B., AND D. I. WILLIAMSON, 1960. Larvae of decapod Crustacea of the families

Diogenidae and Parguridae from the Bay of Naples. Pubbl. Sta. Zoo/. Napoli., 31 :

493-552. PROVENZANO, A. J., JR., 1959. The shallow water hermit crabs of Florida. Bull. Mar. Sci.,

9: 349-420. ROBERTS, M. H., JR., 1970. Larval development of Pagurus longicarpus Say reared in the

laboratory, I. Description of larval instars. Biol. Bull., 139: 188-202. ROUSE, W. L., 1969. Littoral Crustacea from Southwest Florida. Quart. J. Florida Acad.

Sci., 32: 127-152. THOMPSON, M. T., 1903. The metamorphosis of the hermit crab. Proc. Boston Soc. Natur.

Hist., 31 : 147-209.

Reference : Biol Bull, 139: 574-579. (December, 1970)

EFFECTS OF SUBZERO TEMPERATURES AND TRAWLING STRESS

ON SERUM OSMOLALITY IN THE WINTER FLOUNDER

PSEUDOPLEURONECTES AMERICANOS

BRUCE L. UMMINGERi

Department of Biology, Yale University, New Haven, Connecticut

Since the freezing point of sea water (-1.8° C) is approximately one degree Centigrade lower than the freezing point of the serum of most marine teleosts (-0.7° C to -0.8° C), the possibility exists that these fish may encounter temperatures that would freeze their blood. Most arctic (Scholander, van Dam, Kanwisher, Hammel and Gordon, 1957; Eliassen, Leivestad and Miller, 1960; Gordon, Amdur and Scholander, 1962; Leivestad, 1965), Antarctic (DeVries and Wohlschlag, 1969; R. N. Smith, personal communication) and temperate zone (Scholander, van Dam, Kanwisher, Hammel and Gordon, 1957; Umminger, 1969a, b) fish avoid freezing at subzero temperatures by existing in a supercooled state. However, a few species of Antarctic fish of the genus Trematomus have been reported to lower the freezing point of their plasma to make it isosmotic with sea water (DeVries and Wohlschlag, 1969; Potts and Morris, 1968). Pearcy (1961) has also reported that the winter flounder, Pseudopleitronectes americanus, forms an "antifreeze" at subzero temperatures by lowering the freezing point of its serum. These data on supercooling and "antifreeze" formation in fish are summarized in Table III. Since P. americanus is the only marine teleost from the temperate zone that has been reported to form an "antifreeze' in winter, the present investigation was undertaken in an attempt to confirm these observations.

MATERIALS AND METHODS

The studies on temperature acclimation presented in this paper utilized labora- tory-acclimated fish. Ten adult specimens of P. americanus were caught by trawl in Long Island Sound near Stonington, Connecticut, on January 3, 1967. Four of these fish were subsequently maintained in the laboratory on a photo- period of eight hours of light per day at 15° C for five weeks; one fish was kept at 4° C for five weeks; three fish were kept at 4° C for four weeks and at -1.0° C for seven days; and one fish was maintained at 4° C for four weeks and at -1.5° C for seven days. The tenth fish was acclimated to 4° C for four weeks and transferred to water at -1° C; the temperature was gradually lowered to -1.8° C and the fish died. At autopsy, the tails of flounder lightly anesthetized with tricaine methanesulfonate (MS 222) were severed and the free-flowing blood was collected from the caudal artery. After centrifugation of the clotted blood, the serum was frozen at -20° C for later determinations of serum osmolality.

1 Present address : Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221.

574

FLOUNDER AT SUBZERO TEMPERATURES

575

TABLE I

Effect of temperature on serum osmolality of P. americanus

Temperature of acclimation	Fish no.	Sex	Serum osmolality (mOsm/liter)	Serum freezing point	Amount serum supercooled
15° C	1	M	373	-0.69° C	o°c
	2	M	423	-0.79° C	o°c
	3	M	403	-0.75° C	0° C
	4	M	355	-0.66° C	u°c
	Mean ± S.E.		389 ± 15	-0.72° C ±
				0.02° C
4° C -1.0° C	5 6	M F	,554, 405	-0.66° C -0.75° C	0° C 0.25° C
	7	F	356	-0.66° C	0.34° C
	8	F	397	-0.73° C	0.27° C
	Mean ± S.E		386 ± 15	-0.71° C ±	0.29° C ±
				0.02° C	0.02° C
-1.5° C	9	M	420	-0.78° C	0.72° C

Total osomolality, in reference to standard sodium chloride solutions, was esti- mated with a Mechrolab (new Hewlett-Packard) vapor-pressure osmometer equipped with Hamilton microsyringes. Statistical significance was determined using Student's t test.

In addition to the experiments on thermal acclimation, an investigation into the effects of trawling on serum osmolality was undertaken using ten freshly caught specimens of P. americanus. These fish were captured by trawl in water at 15° C from Long Island Sound near Old Saybrook, Connecticut, on October 14, 1966. The freshly caught fish were transported to the Essex Marine Laboratory in large steel wash buckets and autopsied at 15-minute intervals. Serum osmolality was determined (using the methods described above) for five fish killed 1-2 hours after capture and for five fish killed 5-6 hours after capture. There was no period of laboratory acclimation for these fish.

TABLE II

Effect of time after capture with trawl on serum osmolality of P. americanus

Group	Minutes after capture	Serum osmolality	Sex
Killed about 1-2 hours after	60	400	M
capture	75	392	M
	90	417	F
	105	412	F
	120	440	F
	Mean ± S.E.	412 ± 8
Killed about 5-6 hours after	285	447	M
capture	300	448	M
	315	421	F
	330	531	F
	345 r	460	M
	Mean ±^S.E.	461 ± 18*

* Significantly different from group killed 1-2 hours after capture (P < 0.05).

576

BRUCE L. UMMINGER

TABLE III

Supercooling and "antifreeze" formation in marine teleosts

Family and species	Water temperature	Freezing point of serum	Amount serum supercooled	Reference
FAMILY ANARHICHADIDAE
Anarhichas minor	-1.5° C	-0.80° C	0.70° C	Eliassen, Leivestad
				and Mjzlller, I960
FAMILY COTTIDAE
Callus scorpius	-1.5° C	-0.86° C	0.64° C	Eliassen, Leivestad
				and Miller, 1960
Gymnacanthus tricuspis	-1.73° C	-0.93° C	0.80° C	Scholander, van Dam,
				Kanwisher, Ham-
				mel and Gordon,
				1957
Icelus spatula	-1.73° C	-0.96° C	0.77° C	Scholander, van Dam,
				Kanwisher, Ham-
				mel and Gordon,
				1957
Myoxocephalus scorpius	-1.73° C	-1.44° C	0.29° C	Scholander, van Dam,
				Kanwisher, Ham-
				mel and Gordon,
				1957
	-1.7° C	-1.25° C	0.45° C	Gordon, Aindur and
				Scholander, 1962
FAMILY CYCLOPTERIDAE
Cyclopterus lumpus	-1.5° C	-0.88° C	0.62° C	Eliassen, Leivestad
				and M011er, 1960
Liparis koefoedi	-1.73° C	-0.91° C	0.82° C	Scholander, van Dam,
				Kanwisher, Ham-
				mel and Gordon,
				1957
Liparis sp.	-1.9° C	-0.92° C	0.98° C	DeVries, 1970
FAMILY CYPRINODONTIDAE
Fundulus heteroditus	-1.5° C	-0.80° C	0.70° C	Umminger, 1969a
FAMILY GADIDAE
Boreogadus saida	-1.73° C	-1.02° C	0.71° C	Scholander, van Dam,
				Kanwisher, Ham-
				mel and Gordon,
				1957
Cadus morhua	-1.5° C	-0.80° C	0.70° C	Eliassen, Leivestad
				and M011er, 1960
	-1.4° C	-0.76° C	0.64° C	Leivestad, 1965
Gadus ogac	-1.73° C	-1.47° C	0.26° C	Scholander, van Dam,
				Kanwisher, Hani-
				mel and Gordon,
				1957
	-1.7° C	-0.94° C	0.76° C	Gordon, Amdur and
				Scholander, 1962
Microgadus tomcod	-1.5° C	-0.98° C	0.52° C	Gordon, Amdnr and
				Scholander, 1962
FAMILY NOTOTHENIIDAE
Notothenia larseni	-1.87° C	-1.51° C	0.36° C	DeVries and Wohl-
				schlag, 1969
Notothenia neglecta	-1.9° C	-1.08° C	0.82° C	Smith, 1968

FLOUNDER AT SUBZERO TEMPERATURES

TABLE III. — (Continued)

577

Family and species	Water temperature	Freezing point of serum	Amount serum supercooled	Reference
Notothenia rossii	-1.9° C	-1.07°C	0.83° C	Smith, 1968
Trematomus bernacchii	-1.73° C	-1.95° C	0° C*	Potts and Morris,
				1968
	-1.9° C	-1.98° C	0°C*	DeVries, 1970
Trematomus borchgrevinki	-1.9° C	-2.07° C	0° C*	DeVries, 1970
Trematomus hansoni	-1.9° C	-2.01° C	0° C*	DeVries, 1970
Trematomus loennbergi	-1.9° C	-1.83° C	0.07° C	De Vries, 1970
Trematomus newnesi	-1.8° C	-1.01° C	0.79° C	Smith, 1968
FAMILY PLEURONECTIDAE
Drepanopsetta platessoides	-1.5° C	-0.93° C	0.57° C	Eliassen, Leivestad
				and Mpller, 1960
Pseudopleuronectes americanus	-0.80° C	-1.15° C	0° C*	Pearcy, 1961
	-1.0° C	-0.71° C	0.29° C	present paper
	-1.5° C	-0.78° C	0.72° C	present paper
FAMILY ZOARCIDAE
Lycodes turneri	-1.73° C	-0.97° C	0.76° C	Scholander, van Dam,
				Kan wisher, Harn-
				mel and Gordon,
				1957
Rhigophilia dearborni	-1.9° C	-1.52° C	0.38° C	DeVries, 1970

Antifreeze present; freezing point of serum lower than temperature of water.

RESULTS

As indicated in Table I, the average serum osmolality of specimens of P. americanus acclimated to -1° C was not significantly different from the serum osmolality of fish acclimated to 15° C. The serum osmolality of flounder at -1° C was 388 mOsm/liter which corresponds to a serum freezing point of -0.71° C. These fish formed no "antifreeze," being supercooled by 0.29° C. Similarly, the serum osmolality of a single fish held at -1.5° C was 420 mOsm/liter ; this fish was supercooled by 0.72° C. A single fish was placed into water at -1° C and the temperature \vas gradually lowered to -1.8° C over a seven-day period. At this temperature, ice began to form at the surface of the sea water and tiny crystals of ice began to circulate throughout the water in the aquarium. The presence of these crystals of ice caused the fish to freeze solid and die ; presumably, the ice crystals seeded the supercooled blood, causing it to freeze.

Studies on freshly caught flounder (Table II) at 15° C showed that serum osmolality increased from 412 mOsm/liter in fish killed 1-2 hours after capture to 461 mOsm/liter in fish killed 5-6 hours after capture. Not only did serum osmolality increase significantly with time after capture, but also the serum osmolality of the freshly caught fish at 15° C (437 mOsm/liter) was significantly higher than the serum osmolality of fish acclimated to the laboratory at 15° C (389 mOsm/liter).

DISCUSSION

Pearcy (1961) found that specimens of P. americanus living in the Mystic River estuary in Connecticut during the winter of 1958-59 often encountered

578 BRUCE L. UMMINGER

temperatures as low as -0.8° C. These winter fish had an average serum freezing point of -1.15° C. In contrast, the serum freezing point for fish collected in the summer was -0.63° C. In the present investigation, the serum freezing point (-0.71° C) of cold-acclimated winter fish is more similar to the summer than to the winter values reported by Pearcy (1961). What, then, accounts for the discrepancy in the data from these two studies ?

The only obvious difference in the procedures used in the two investigations concerns the handling and capture of the fish. Pearcy (1962) used freshly caught flounder that may well have been stressed by the trawling, whereas the present investigation deals with fish that had been acclimated to the laboratory for several weeks. The present study shows that capture by trawl can elevate the serum osmolality of P. americanus. Therefore, the trawling procedures employed by Pearcy (1961) may account for the exceedingly high values he obtained for serum osmolality in winter fish. Similar studies by Slicher, Pickford and Pang (1966) on Fundnlus heteroclitus showed that the serum osmolality was elevated in fish unaccustomed to handling when compared with fish "trained" to be fa- miliar with the handling procedures. Therefore, Pearcy 's account of "antifreeze" formation in the winter flounder at low temperatures was probably due to an increased serum osmolality produced by trawling stress during capture.

Another possible reason for the discrepancy in these two studies is that there may be yearly variations in the ability of the flounder to produce its "anti- freeze." For example, Gordon, Amdur and Scholander (1962) were unable to find the high serum osmolalities reported by Scholander, van Dam, Kanwisher, Hammel and Gordon (1957) in two species of arctic fish. Gordon, Amdur and Scholander (1962) concluded that the difference in the serum osmolality measured during different years was real and that the amount of "antifreeze" added to the blood during the winter was extremely variable from year to year. This may well be the case with the winter flounder also.

Pearcy (1961) also found that winter flounder kept in the laboratory would freeze and die at temperatures between -1.0° C and -1.5° C. These lethally low temperatures were similar to the serum freezing points of freshly captured winter fish. Pearcy concluded that the flounder could not survive temperatures much below -1.15° C since this was the freezing point of the serum. Apparently, these data show that winter flounder cannot exist in a supercooled state. However, the present study shows that the winter flounder definitely can survive in a super- cooled state. The fish freeze and die only when ice crystals are present in the water to nucleate the supercooled blood. In nature, surface ice is probably seldom encountered because winter flounder are bottom fish that often cover themselves with mud or sand. The ability of the winter flounder to avoid freezing by existing in a supercooled state (as reported in this paper) is the same mechanism utilized by the majority of cold-hardy marine teleosts (Table III) in surviving the sub- zero cold.

Much of this paper is part of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Yale University. The work was supported by traineeships from the National Aeronautics and Space Administration and the National Science Foundation awarded through the De- partment of Biology at Yale University. Supplies and equipment were provided

FLOUNDER AT SUBZERO TEMPERATURES 579

by Professor Grace E. Pickford through grants from the National Science Founda- tion. I wish to express my appreciation to Professor Pickford for the stimulation and guidance that made this study possible.

SUMMARY

1. The average serum osmolality of winter flounder, Pseudopleuronectes americamts, acclimated in the laboratory to -1° C (386 mOsm/liter) was not significantly different from the serum osmolality of fish at 15° C (389 mOsm/liter).

2. Winter flounder survived temperatures as low as —1.5° C in a super- cooled state. When ice crystals were present in the sea water at -1.8° C, the fish froze and died due to nucleation of the supercooled blood.

3. The serum osmolality of freshly caught flounder increased significantly with time after capture (up to six hours) and was significantly higher than the serum osmolality of laboratory acclimated fish at the same temperature.

4. A previous account of "antifreeze" formation in the winter flounder at low temperatures was probably due to an increased serum osmolality produced by trawling stress during capture.

LITERATURE CITED

DEVRIES, A. L., 1970. Freezing resistance in Antarctic fishes. Pages 320-328 in M. Holdgate, Ed., Antarctic Ecology, Volume 1. Academic Press, New York.

DEVRIES, A. L., AND D. E. WOHLSCHLAG, 1969. Freezing resistance in some Antarctic fishes. Science, 163 : 1073-1075.

ELIASSEN, E., H. LEIVESTAD AND D. MILLER, 1960. Effects of low temperatures on the freezing point of plasma and on the potassium/sodium ratio in muscles of some boreal and subarctic fishes. Arbok. Univ. Bergen, Mat.-Naturv., No. 14 : 1-24.

GORDON, M. S., B. H. AMDUR AND P. F. SCHOLANDER, 1962. Freezing resistance in some northern fishes. Biol. Bull., 122 : 52-62.

LEIVESTAD, H., 1965. Supercooling and osmoregulation in teleosts of the boreal and sub- arctic. Intl. Comm. Northwest Atlantic Fish. Spec. Publ., 6: 747-752.

PEARCY, W. G., 1961. Seasonal changes in osmotic pressure of flounder sera. Science, 134: 193-194.

PEARCY, W. G., 1962. Ecology of an estuarine population of winter flounder, Pseudopleuro- nectes americanus (Walbaum) III. Distribution, abundance, growth, and production of juveniles; survival of larvae and juveniles. Bull. Bingham Oceanogr. Coll., 18(1): 39-64.

POTTS, D. C., AND R. W. MORRIS, 1968. Some body fluid characteristics of the Antarctic fish, Trematomus bernacchii. Mar. Biol., 1 : 269-276.

SCHOLANDER, P. F., L. VAN DAM, J. W. KANWISHER, H. T. HAMMEL AND M. S. GORDON, 1957. Supercooling and osmoregulation in arctic fish. J. Cell. Comp. Physiol., 49 • 5-24.

SLICHER, A. M., G. E. PICKFORD AND P. K. T. PANG, 1966. Effects of "training" and of volume and composition of the injection fluid on stress-induced leukopenia in the mummichog. Prog. Fish-Cult., 28 : 216-219.

SMITH, R. N., 1968. The biochemistry of freezing resistance of some Antarctic fish. (Per- sonal communication of a paper presented to the Scientific Committee on Antarctic Re- search Symposium on Antarctic Ecology, Cambridge, 1968.)

UMMINGER, B. L., 1969a. Physiological studies on supercooled killifish (Fundulus hetero- clitus} I. Serum inorganic constituents in relation to osomtic and ionic regulation at subzero temperatures. /. E.rp. Zool., 172 : 283-302.

UMMINGER, B. L., 1969b. Physiological studies on supercooled killifish (Fundulus hcteroclitus) II. Serum organic constituents and the problem of supercooling. /. Exp. Zool., 172 : 409-424.

INDEX

Absorption of D-glucose in vivo in the toad- fish and the rainbow trout, 421 Abstracts of papers presented at the Marine

Biological Laboratory, 402 Acetylcholine and synaptic potentials in skate

electroplaques, 428 Acetylcholinesterase activity in eserine-treated

ascidian embryos, 239 Acrosome reaction in spermatozoa of Lnin-

bricns, 424

Activity, chromatophorotropic, of the central nervous system of the horse-shoe crab, 422 Adaptation, light- and dark-, in the skate retina, intracellular responses during, 419 retinal, in the skate, electrical and photo- chemical signs of, 405 of Fundulus embryos to oxygen tensions,

435

properties of squid photoreceptors, 433 Adaptations, behavioral, of amphibious fishes,

115 Adenine, 1 -methyl, in the starfish, distribution

and metabolism of, 439 Adenosine, effect on coelomocyte aggregation

in Cncuruaria frondosa, 549 nucleotides, effect on coelomocyte aggrega- tion, in Ciicumaria frondosa, 549 1 -methyl, effect on gonads and oocytes of

the starfish of, 435 Adhesive used in repair and reattachment in

barnacles, 333

Aequipecten irradians, a biochemical com- parison of ciliary and flagellar axonemes of, 429 Aggregation of amebocytes of the horseshoe

crab, 426

of coelomocytes in Cucninaria frondosa, effect of ethylenediamine-tetraacetate, adenosine, and adenosine nucleotides on, 549

of some starfish coelomocytes, 418 Airbreathing, control of in fishes, 115 Aliphatic sesquiterpenes, specific juvenile hor- mone effects in 222 Allelic variation in MytUns, electrophoretic

studies of, 430

ALLEN-, G. E. T. H. Morgan, Richard Gold- schmidt and the opposition to Mendelian

theory 1900-1940 : a chapter in the history of biological ideas, 412

Alpha-crystallin from the ocular lens of the

dogfish, reversible de-aggregation of, 428

Amoebocytes, role in the regeneration of shell

in the land pulmonate, 502 and shell repair in the land pulmonate, 502 of the horseshoe crab, aggregation of, 426 Anabaseine, chemistry and biology of, 408 Annelid, endocrinology and reproductive biol- ogy of, 461

Annual Report of the Marine Biological Lab- oratory, 1

Antigens, histocompatibility, immunologic re- activity to, 410 APLEY, M. L. See W. D. RUSSELL-HUNTER

434 Arbacia, phosphorylation of nuclear protein

following fertilization in, 425 eggs, movements of echinochrome granules

during the early development of, 415 Arbacia punctulata, effect of temperature on the efflux of calcium from the eggs of, 417 eggs, effect of fertilization on the calcium

and magnesium content of, 413 eggs, peripheral membrane system of, 425 nuclear localization of sialic acid in the

unfertilized eggs of, 426 uptake and use of amino acids by, 296 ARNOLD, J. M. AND L. D. WILLIAMS-ARNOLD. The effects of cytochalasin B on cyto- plasmic movement, cleavage, and subse- quent development of the squid embryo, Loligo pcalei, 413 Artemia, effects of gamma irradiation on, 363

375

female reproductive performance of, 375 life spans and male reproductive perform- ance of, 363 Arthropod molting, control of by ecdysones

520 Ascidian embryos, eserine-treated, acetylcho-

linesterase activity in, 239 Ascidians, effects of Cytochalasin B on tail

resorption in, 427 Assay for ecdysone, evaluation of "CaUiphora

Test" as, 138

Asterias forbcsi, permeability and structure of cellular junctions in embryos of, 440

580

INDEX

581

coelomocyte simulating effector substances of the delayed hypersensitivity response, macrophage activating protein from, 412

coelomocytes, failure to clump on glass by, 418

oocytes, studies of RNA synthesis in, 436 Axon in crayfish muscles, differences in block- ing frequency of presynaptic terminals of, 432

Axonemes, ciliary and flagellar, from the bay scallop, a biochemical comparison of, 429

flagellar, isolation of linkage protein respon- sible for maintenance of nine-fold con- figuration of, 438 Axons, squid, changes in fluorescence of, 418

AZARNIA, R. AND E. L. CHAMBERS. Effect of

fertilization on the calcium and mag- nesium content of the eggs of Arbacia punctulata, 413 See E. L. CHAMBERS, 417

B

Bacterial competence, enzyme changes asso- ciated with development of, 411

Balanidae, comparative study of the cement glands in, 164

Balanus, comparative study of the cement

glands of, 164 repair and reattachment in, 333

BANG, F. B. A virus disease of the shore crab Carcinus macnas, 414

Barnacles, comparative study of the cement

glands in, 164 repair and reattachment in, 333

BARNES, S. N., A. L. F. GORMAN AND J. S. McREYNOLDS. Fine structure and intra- cellular responses of photoreceptors of a pelagic tunicate Salpa, 414

BARTELL, C. K., M. FINGERMAN AND J. E. STEPHENSON. Comparison of melano- phorotropic lipoprotein fractions from the pituitary of the dogfish, Mustelus camts, and from the eyestalk of the fiddler crab, Uca pugilator, 415 See M. FINGERMAN, 422

EARTH, R. H., JR. AND W. J. BELL. Phys- iology of the reproductive cycle in the cockroach Byrsotria jumiqata (Guerin), 447

BASKIN, D. G. AND D. W. GOLDING. Experi- mental studies on the endocrinology and reproductive biology of the viviparous polychaete annelid. Nereis limnicola John- son, 461

BAUER, G. E. See B. D. NOE, 431

BEATY, L. D. See R. MILKMAN, 430

BELAMARICH, F. A. See D. M. KENNEY, 426

BELANGER, A. M. AND R. C. RUSTAD. Move- ments of echinochrome granules during the early development of Arbacia eggs, 415

BELL, E. See F. R. MACKINTOSH, 296 BELL, W. J. See R. H. EARTH, JR., 447 BENNETT, M. V. L. See P. B. DUNHAM, 420 BERGMANN, F. See M. E. SPIRA, 405 BERNAL, P. See A. W. EBELING, 115 Bioluminescence and fluorescence, microscopic

observation of, 432

Bioluminescence of Cypridina serrata, 386 BIRDSEY, V. See H. GEWURZ, 411, 423 BIRNBAUM, A. The statistical phenotype con- cept, with applications to skeletal variation in the mouse, 416

Blood folic acid in the dogfish, studies on, 416 BORGESE, T. A. AND L. CiANCi. Studies on blood folic acid in the smooth dogfish, Mustelus canis, 416

BOTOS, P., JR. See G. T. REYNOLDS, 432 Brachyura, water permeability of, 351 BRADSHAW, W. E. Interaction of food and photoperiod in the termination of larval diapause in Chaoborus americanus (Dip- tera :Culicidae), 476

BRANDT, P. W. See G. SUAREZ-KUREZ, 439 BRAVERMAN, M. Hydranth regulation in the colonial marine hydroid Podocoryne car- nca, 404

BROWN, G. G. See R. C. MOWBRAY, 313 BROWN, J. E. See J. E. LISMAN, 429 BRUNER, W. E. See E. L. KEAN, 426 BRUNO, M. S., M. I. MOTE AND T. H. GOLD- SMITH. Microspectrophotometry of visual pigment and spectral sensitivity of retin- ular cells in the crab, Carcinus, 416 BUCK, J., J. F. CASE AND F. E. HANSON. Pacemaking and phase-shifting in syn- chronizing fireflies, 417 See J. F. CASE, 417 BUDAY, A. See S. INOUE, 425 BURKY, A. J. See W. D. RUSSELL-HUNTER,

402

BURNS, J. See J. F. CASE, 417 Busycon canaliculatum, neural control of myo-

cardial rhythmicity in, 427 Byrsotria fumigata, physiology of the re- productive cycle in, 447

Calcification in echinoderms, 151 Calcification in the land pulmonate, 502 Calcium and magnesium content of Arbacia

eggs, effect of fertilization on, 413 from Arbacia eggs, effect of temperature on

the efflux of, 417

582

INDEX

spikes in crayfish muscle fibers, parallel changes in the thresholds of current in- duced tensions and, 430

Calcium-45 incorporation in vitro by regener- ating spines of S. pupiiratus, effects of temperature and diamox on, 151

"Calliphora Test," evalution of as an assay for ecdysone, 138

Campawularia flcxuosa, effect of puromycin on the growth pattern of, 443

Canals, study of in some balanid barnacles, 164

Curassius aurahts, effects of superabundant oxygen on thermal tolerance of, 229

Carbonic anhydrase, effects of an inhibitor (Diamox) of, on calcification in regener- ating sea urchin spines, 151

Carcimis maenas, a virus disease of, 414 microspectrophotometry of visual pigment and spectral sensitivity of retinular cells in, 416 water permeability of, 351

CASE, J. F., G. T. REYNOLDS, J. BUCK, J. BURNS AND R. HALVERSON. Compara- tive physiology of neurally controlled luminescences, 417 See J. BUCK, 417

CASS, A. See P. B. DUNHAM, 420

Cecropia silkworm, origin, distribution and fate of the molting fluid proteins of, 277

Cellular junctions in the starfish embryo, permeability and structure of, 440

Cement duct network in barnacles, 333

Cement glands in some balanid barnacles, a comparative study of, 164

Cement glands of barnacles, 164, 333

Cement hardening mechanism in barnacles, 333

Cementing apparatus of barnacles, 164, 333

Cementing mechanism in balanidae, 333

CHAMBERS, E. L., R. AZARNIA AND W. E. McGowAN. The effect of temperature on the efflux of 45ca from the eggs of Arbacia punctulata, 417 See R. AZARNIA, 413

Chaoborus amcricanus, interaction of food and photoperiod in the termination of larval diapause in, 476

Chelicerate arthropods, control of molting in by ecdysones, 520

Chesapeake Bay jellyfishes, observations on and toxins of, 180

CHILDS, J. N. III. Failure of coelomocytes of some Asterias forbesi to clump on glass, 418

CHIU, K. W., W. G. LYNN AND J. P. LEICHNER. Environmental temperature

and thyroid activity in the lizard, Scelo-

ponis occidentalis, 107 See P. F. A. MADERSON, 304 Clilainydoiuonas reinliardii, sole food organism

in the monoxenic cultivation of daphnidae,

321

Choroid retes of scup and striped bass, 444 Chromatophorotropic activity of the central

nervous system of the horseshoe crab,

422

Chrysaora (Dactylometra) quinqitecirrha, ob- servations on and toxins of, 180 CIANCI, L. See T. A. BORGESE, 416 Cilia, changes following negative staining of

microtubules of, 265 Ciona intestinalis embryos, acetylcholin-

esterase activity in, 239 Cirripedia, comparative study of the cement

glands in, 164

repair and reattachment in, 333 CLARK, W. H., JR. AND G. W. HINSCH.

Ultrastructure of the secondary septa of

Mctrtdintn sp., 406 Clingfish, amphibious Chilean, emersion of,

115

CLONEY, R. A. See J. LASH, 427 Clytia, hydroids, life cycle of, 203 Cnidaria : toxin of jellyfishes from Chesapeake

Bay, 180 Cobra venom factor, variations in Liimihts

inducible lysin induced by, 423 Cockroach, physiology of the reproductive

cycle in, 447 regulation of nerve activity in the phallic

nerve of, 405

Coelenterata : toxin of jellyfishes from Chesa- peake Bay, 180 Coelenterates, luminescence and fluorescence

in, 430 Coelomocyte aggregation in Cucumaria fron-

dosa, effect of ethylenediamine-tetraace-

tate, adenosine, and adenosine nucleotides

on, 549 morphology, changes during aggregation,

549 Coelomocytes of some starfish, failure to

clump on glass, 418 COHEN, C. See Y. LENSKY, 277 COHEN, L. B., D. LANDOWNE, B. B. SHRI-

VASTAV AND J. M. RITCHIE. Changes in

fluorescence of squid axons during activ- ity, 418 Cold exposure, effect on protein synthesis in

the hepatopancreas of the spider crab of,

421 Collocalia vanikorcnsis granti, sensitivity of

echolocation in, 495

INDEX

583

Colony morphology, influence on reproductive potential in Ectoprocta of, 426

Coloration, protein, modification of interac- tions between lenses and UV light by, 444

Competence, bacterial, enzyme changes asso- ciated with development of, 411

Complement system of vertebrates, similarities of inducible lysin in Limuhis to, 423

Continuous culture of Daphnia, 485

Control, neural, of luminescence, comparative physiology of, 417

COPELAND, D. E. See R. C. WOLLEY, 444

CORNELL, N. W. Metabolic controls and bio- logical variation, 406

COUSINEAU, G. H. See S. INDUE, 425

Crassostrca virginica, filtration of particles from suspension by, 248

Crayfish muscle fibers, parallel changes in thresholds of current induced tensions and calcium spikes in, 439

differences in blocking frequency of pre- synaptic terminals of an axon of, 432

Crustacea, description of larval instars of a

species of decapod 188 repair and reattachment in, 333 water permeability of, 351

Crustacean, bioluminescence of, 386

Ctenophore, origin of light producing cells during development of, 422

Ciicninaria frondosa, effect of ethylenediamine- tetraacetate, adenosine, and adenosine nucleotides on coelomocyte aggregation in, 549

Cyanide, hydrogen, polymers and the origin of life, 419

Cyasterone, see ecdysones, 520

Cypridina serrata, bioluminescence of, 386

Cytochalasin B, effect on cytoplasmic move- ment, cleavage and subsequent develop- ment of the squid embryo by, 413

Cytological investigations of sperm-egg inter- actions in decapods and Linnilus, 313

Cytology of some balanid branacles, 164

D

D'AGOSTINO, A. S. AND L. PROVASOLI. Dixenic culture of Daphnia magna, Straus, 485

Daphnia magna, dixenic culture of, 485 Daphnidae, monoxenic cultivation of, 321 Decapoda, description of larval instars of a

species of, 188

Decapods, cytological and immunological in- vestigations of sperm-egg interactions in, 313

Development, early, of Arbacia eggs, move- ment of echinochrome granules during, 415

of the ctenophore, origin of light producing

cells during, 422 time and fertilization in Fitiidiiliis, influence

of oxygen tension on, 436 DFP, in relation to nerve function, enzymatic

hydrolysis of, 407 D-glucose absorption in vivo in the toadfish

and the rainbow trout, 421 Diamox, effect on incorporation of Calcium-45

in vitro by regenerating spines of S.

pnrpuratus, 151

Diapause, larval, in Chaoborus, 476 DiBENEDETTo, F. E. See A. FARMANFAR-

MAIAN, 421 Diemictylus viridescens, hyaluronidase in the

blastema of the regenerating, 403 Differentiation of epidermal cells in turbel-

larians, 539

Digestive enzymes in Parancmcrtcs peregrina,

92

Digestion, site and sequence of, of Par- ancmcrtes peregrina, 92

Director's Report, 1969-1970, 7

"Distance effect" in pigeon orientation, 510

Distribution of molting fluid proteins of the cecropia silkworm, 277

Dixenic culture of Daphnia magna, 485

DNA and chromatin in somatic cells and

sperm of the spider crab, 441 synthesis during regeneration in Tubularia, 440

Dogfish, studies on blood folic acid in, 416 lens, protein coloration modification of interactions between lenses and UV light in, 444

retina, receptive field organization of ganglion cells in, 437

DONNER, D. B., A. P. LOBO, J. D. Wos AND J. P. FERRIS. The possible role of hy- drogen cyanide in the origins of life : the hydrogen cyanide polymers, 419

DOOLEY, C. A. See J. R. SAROYAN, 333

DOWLING, J. E. AND H. Ripps. Electrical and photochemical signs of adaptation in the retina of the skate, Raja crinacea and R. oscellata, 405

S-potentials in the skate retina : intracellular responses during light- and dark-adapta- tion, 419

DUDLEY, J. W. Differential utilization of phy- toplankton food resources by marine ecto- procts, 420

DUKOR, P. See G. WEISSMAN, 408

DUNHAM, P. B., A. CASS, J. P. TRINKAUS AND M. V. L. BENNETT. Water perme- ability of Fundiilus eggs, 420

584

INDEX

E

Earthworm, ultrastructure of spermatozoon

of, 403 EBELING, A. W., P. BERNAL AND A. ZULETA.

Emersion of the amphibious Chilean cling-

fish, Sicyases sangitineus, 115 Ecdysone, ".Calliphora Test" as an assay for,

138 Ecdysones, control of molting in arthropods

by, 520 Ecdysterone = /3-Ecdysone 20-hydroxy-

ecdysone, see ecdysones, 138, 520 Echinochrome granules, movement during

early development of Arbacia eggs of, 415 Echinoderms, calcification in, 151 Echolocation in cave swiftlets, sensitivity of,

495 Ectoprocta, marine, a natural experiment in

the population genetics of, 435 influence of colony morphology on reproduc- tive potential in, 426 marine, differential utilization of phyto-

plankton food resources, 420 Effect of vitamins on fertility of Daphnia, 485 Eggs, Fundulns, water permeability of, 420 Electric counter, use in particle filtration ex- periments, 248 Electrogenesis of eel electroplaques, sodium

currents during, 433 Electrophoresis of allelic variation in Mytilus

ednlis, 430 of proteins to determine gene frequencies in

Schisoporella, 435 Electroplaques, eel, sodium currents during

spike electrogenesis of, 433 skate, synaptic potentials and acetylcholine

in, 428 ELLISON, R. The effect of cold exposure on

protein synthesis in the hepatopancreas of

the spider crab, Libinia emarginata, 421 Embryos, ascidian, acetylcholinesterase activ- ity in, 239

Emersion of the clingfish, 115 Endocrinology of Nereis, experimental studies

on, 461

Endopeptidases in Paranemertes peregrina, 92 Enzymatic hydrolysis of DFP and Tabun in

relation to nerve function, 407 Enzyme changes associated with development

of bacterial competence, 411 Epiboly, formation of in Hydrolimax grisea,

539 Epidermal cavity, formation of in Hydrolimax

grisea, 539 EPSTEIN, H. T. Enzyme changes associated

with development of bacterial competence,

411

Ethylenediaminetetraacetate, effect on coelo- mocyte aggregation in Cucumaria jron- dosa, 549

Euplecta indica, role of amoebocytes in re- generation of shell in, 502

Excitation process in nerve, fluorescence ana- lysis of, 441

Exopeptidases in Paranemertes peregrina, 92

Expenditure and income, Marine Biological Laboratory, 73

Extracellular and intracellular proteolysis in Parancmcrles peregrina, 92

Eyestalk extirpation, biochemical changes in the digestive gland of the fiddler crab following, 437

of the fiddler crab, comparison of melano- phorotropic hormones from the pituitary of the dogfish with those from, 415

FARMANFARMAIAN, A. AND F. E. DiBENE- DETTO. In vivo intestinal absorption of D-glucose in the toadfish, Opsanus tan, and the rainbow trout, Salmo gairdncri, 421

Fat in Paranemertes peregrina, 92

Fate of the molting fluid proteins of the ce- cropia silkworm, 277

Feeding behavior of Paranemertes peregrina, 80

Female reproductive performance in Artemia, 375

FERRIS, J. P. See D. B. DONNER, 419

Fcrrissia riznilaris, interpopulation variation in shell components in, 402

Fertilization, effect on calcium and mag- nesium content of Arbacia eggs of, 413 and developmental time in Fundulus, influ- ence of oxygen tension on, 436 in Arbacia and Spisula, 425

Filtration of particles from suspension by the American oyster, 248

FINGERMAN, M. AND K. R. RAO. Analysis of the color changes induced by serotonin (5-hydroxytryptamine) and lysergic acid diethylamide (LSD) in the fiddler crab, Uca pugilator, 407

C. K. BARTELL AND R. A. KRASNO. Anal- ysis of the chromatophorotropic activity of the central nervous system of the horseshoe crab Limulus polyphemus, 422 See C. K. BARTELL, 415

Fireflies, synchronizing, pacemaking and phase-shifting in, 417

Fishes, amphibious, 115

Flagella, changes following negative staining of microtubules of, 265

INDEX

585

Flagellar axonemes, isolation of linkage pro- tein responsible for maintenance of nine- fold configuration of, 438

Fluorescence analysis of the excitation proc- ess using hydrophobic probes, 441 and bioluminescence, microscopic observa- tions of, 432

and luminescence in coelenterates, 430 of squid axons during activity, changes in. 418

Folic acid, blood, in the dogfish, studies on, 416

Food, interaction in termination of larval dia- pause in Cliaoborus amcricanns, 476 preferences of Paranemertes peregrina, 80 reserves of Paranemertes peregrina, 92

FRAENKEL, G., AND J. ZDAREK. The evalua- tion of the "Calliphora Test" as an assay for ecdysone, 138

FREEMAN, G. AND G. T. REYNOLDS. The origin of the light producing cells during development of the Ctenophore Mnemiop- sis Icidyi, 422

FROMSON, D. AND J. R. WHITTAKER. Acetyl- cholinesterase activity in eserine-treated ascidian embryos, 239

Fundnhis, adaptations to varied oxygen ten- sions of embryos of, 435 eggs, water permeability of, 420

Funduhis Iicteroclitus, influence of varied oxy- gen tension of fertilization and develop- mental time in, 436

Gamma crystallins, lens, isoelectric focusing

of, 409 Gamma irradiation, effects on the brine shrimp

of, 363, 375

GARWES, D. J. See E. J. SIMON, 404 Gastrulation in Hydrol'nnax grisca, 539 Genetic selection, fundamental error in the

model of, 402 Genetics, population, of a marine ectoproct,

a natural experiment in, 435 of Mytilus, 430

GEWURZ, A. See H. GEWURZ, 411, 423 GEWURZ, H., V. BIRDSEY, D. JOHNSON, J.

LlNDORFER, K. TOWNSEND AND A.

GEWURZ. An inducible lysin in Limulus polyphenms with similarities to the com- plement system of vertebrates : detection, characteristics and dissection from phos- pholipase A, 411

D. JOHNSON, V. BIRDSEY, J. LINDORFER AND A. GEWURZ. An inducible lysin in Limulus polyphcmus with similarities to the complement system of vertebrates :

variations with disease and alterations induced by cobra venom factor in vivo, 423

GIBSON, R. The nutrition of Paranemertes peregrina, (Rhynchocoela : Hoplonemer- tea). II. Observations on the structure of the gut and proboscis, site and se- quence of digestion, and food reserves, 92 GILBERT, W. H. Territoriality observed in a population of Tcllina afjilis (Bivalvia: Mollusca), 423

Gills and airbreathing in the clingfish, 115 Glucagon biosynthesis in the islets of the

anglerfish, 431

Glycogen in Paranemertes peregrina, 92 Goldfish thermal tolerance, effects of super- abundant oxygen on, 229 GOLDING, D. W. See D. G. BASKIN, 461 Goldschmidt, Richard and the Mendel ian

theory, 412

GOLDSMITH, T. H. See M. S. BRUNO, 416 Gonads and oocytes of the starfish, effects of

1 -methyl adenosine on, 435 GOOCH, J. L. See T. J. M. SCHOPF, 435 GORMAN, A. L. F. See S. N. BARNES, 414 GREEN, J. P. See D. A. SNYDER, 437 GRIESS, G. See S. ZIGMAN, 444 GRIFFIN, D. R. AND R. A. SUTHERS. Sensi- tivity of echolocation in cave swiftlets, 495 GROSCH, D. S. See R. D. SQUIRE, 363 GROSS, J. See B. TOOLE, 403 GUIDICE, G. See E. L. SHIPPEE, 436 Gut and proboscis, structure of, of Parane- mertes peregrina, 92

H

HALLETT, M. See A. WATANABE, 441 HALVERSON, R. See J. F. CASE, 417 HANEDA, Y. See F. I. TSUJI, 386 HANSON, F. E. See J. BUCK, 417 HASCHEMEYER, A. E. V. AND V. LAURIE. Radiothyroidectomy of the toadfish, Opsanus tan, 424 Hatching of Mclainpus bidcntatus, influence

of semilunar factors on, 434 HAVEN, D. S. AND R. MORALES-ALAMO. Filtration of particles from suspension by the American oyster Crassostrea i'ir- iiiniea, 248

HEATFIELD, B. M. Calcification in echino- derms : effects of temperature and diamox on incorporation of calcium-45 in vitro by regenerating spines of Strongylocen- trotus pitrpuratits, 151 HEJNO, K. See K. SLAMA, 222 HENLEY, C. Changes in microtubules of cilia and flagella following negative staining with phosphotungstic acid, 265

586

INDEX

Ultrastructure of the negatively stained

spermatozoon of the earthworm, 403 The acrosome reaction in spermatozoa of Lumbricus, as revealed by negative stain- ing, 424

Hepatopancreas of the spider crab, effect of cold exposure on protein synthesis in, 421

Hermit crabs, larval development of, 557

HILL, R. B. See K. KUWASAWA, 427

HINSCH, G. W. Some factors controlling reproduction in the spider crab, Libinia einarginata, 410 See W. H. CLARK, JR., 406 See J. C. VAUGHN, 441

Histochemistry of Euplecta indica, 502

Histocompatibility antigens, immunologic re- action to, 410

Histology, epidermal, in the rat snake, changes

in, 304 of some balanid barnacles, 164

Homing in pigeons, 510

Hormones, insect, effects in aliphatic sesqui- terpenes, 222

HOSKIN, F. C. G. Enzymatic hydrolysis of the nerve gases DFP and Tabun in rela- tion to nerve function, 407

HULL, S. See B. TOOLE, 439

HUNTER, R. D. See W. D. RUSSELL-HUNTER, 402, 434

Hyalophora cecropia, origin, distribution and fate of molting fluid proteins of, 277

Hyaluronidase in the blastema of the regener- ating newt limb, 403

Hydrogen cyanide polymers and the origins of life, 419

Hydroids, hydranth regulation in, 404 in culture, 203

Hydrolase, lysosomal, from phagocytic cells, mechanism of, 408

Hydrolimax grisea, formation of epidermal cavity, inversion and epiboly in, 539

Hydromedusa, Phialidium grcgarium, life cycle in the laboratory of, 203

Hydrophobic probes, fluorescence analysis of the excitation process in nerve using, 441

Hypersensitivity response, delayed, macro- phage activating protein from the sea star coelomocyte simulating effector sub- stances of, 412

ILAN, JOSEPH AND JUDITH ILAN. Phosphory- lation of nuclear protein following fertili- zation in Arbacia and Spisula, 425 ILAN, JUDITH. See JOSEPH ILAN, 425 Image intensification for microscopic observa- tions of bioluminescence and fluorescence, 432

Immunological investigations of sperm-egg

interactions in decapods and Limulus, 313

reactivity to histocompatibility antigens, 410

Income and expenditure, Marine Biological Laboratory, 73

Inhibition, pacemaker, in hydranths of Tubu- laria, temporal characteristics of, 434

Inokosterone, see ecdysones, 520

INDUE, S., G. H. COUSINEAU, P. L. KRUPA AND A. BUDAY. The peripheral mem- brane system of sea urchin eggs : an elec- tron microscope study, 425

Intensification, image, for microscopic obser- vations of bioluminescence and fluores- cence, 432

Intracellular responses of photoreceptors of a tunicate, 414

Inversion, formation of in Hydrolimax grisea, 539

Islet tissue of the anglerfish, glucagon bio- synthesis in, 431

JAROLIM, V. See K. SLAMA, 222

Jellyfishes from Chesapeake Bay, observations

on and toxins of, 180 JOHNSON, D. See H. GEWURZ, 411, 423 Junctions, cellular, in the starfish embryo,

permeability and structure of, 440 Juvenile hormone effects, specific, in aliphatic

sesquiterpenes, 222

K

KAPUR, S. P. AND A. SEN GUPTA. The role of amoebocytes in the regeneration of shell in the land pulmonate, Euplecta indica (Pfieffer), 502

KAUFMANN, K. W. A model for predicting the influence of colony morphology on reproductive potential in the Phylum Ectoprocta, 426

KEAN, E. L. AND W. E. BRUNER. Nuclear localization of the sialic acid "activating" enzyme in the unfertilized sea urchin egg, 426

KEETON, W. T. "Distance effect" in pigeon orientation: An evaluation, 510

KEM, W. R. Chemistry and biology of nemer- tine neurotoxins, 408

KENNEY, D. M., F. A. BELAMARICH AND D. SHEPRO. Aggregation and inhibition of aggregation of amebocytes of the horseshoe crab Limulus polyphemus, 426

KRASNOW, R. A. See M. FINGERMAN, 422

KRISHNAKUMARAN, A. AND H. A. SCHNEIDER- MAN. Control of molting in mandibulate and chelicerate arthropods by ecdysones, 520

INDEX

587

KRUPA, P. L. See S. INOUE, 425

KUWASAWA, K. AND R. B. HILL. Neural con- trol of myocardial rhythmicity in Busy- con canaliculatum, 427

LACOMBE, D. A comparative study of the cement glands in some balanid barnacles (Cirripedia, Balanidae), 164

LANDOWNE, D. The role of the sodium pump in adaptation in the frog muscle spindle, 406 See L. B. COHEN, 418

Lams argentatus, species of Rcnicola in the kidneys of, 438

Larval development of hermit crabs, 188, 557 of Pagunts annulipes and Pagtirus pollicaris

reared in the laboratory, 557 of Pagums longicarpus, 188

Larval diapause in Chaoborus, termination of, 476

Larval instars of Pagurus longicarpus, de- scription of, 188

LASH, J., R. A. CLONEY AND R. R. MINOR. Tail resorption in ascidians : effects of Cytochalasin B, 427

LAURIE, V. See A. E. V. HASCHEMEYER, 424

LEICHNER, J. P. See K. W. CHIU, 107

Lens, ocular, of the dogfish, reversible de- aggregation of alpha-crystallin from, 428

Lens gamma crystallins, isoelectric focusing of, 409

Lenses and UV light, protein coloration modi- fication in interactions between, 444

LENSKY, Y., C. COHEN AND H. A. SCHNEIDER- MAN. The origin, distribution and fate of the molting fluid proteins of the cecropia silkworm 277

LERMAN, S. AND E. PFENINGER. Reversible de-aggregation of alpha-crystallin from the ocular lens of the smooth dogfish Mustelus cants, 428

LESTER, H. A. Spontaneous miniature synap- tic potentials and quantal release of acetylcholine in skate electroplaques, 428

Levorphanol, reversible inhibition of RNA phage replication and macromolecular syn- thesis by, 404

LEVY, R. A. See R. A. NYSTROM, 431

Libinia cmarginata, control of reproduction in

410 DNA and chromatin in somatic cells and

sperm of, 441

effect of cold exposure on protein synthesis in the hepato-pancreas of, 421

Life cycle of Phialidium gregarium in the laboratory, 203

Life spans and male reproductive performance

of Artcmia, 363 Liinulus polyphcmus, aggregation of amebo-

cytes of, 426 chromatophorotropic activity of the central

nervous system of, 422 cytological and immunological investigations

of sperm-egg interactions in, 313 inducible lysin in, 411

inducible lysin, similarities to the comple- ment system of vertebrates of, 423 morphological and electrical features of large neurons in abdominal ganglia of, 431 ventral photoreceptors, a linear relationship between light-induced current and flash intensity in, 429

LINCK, R. W. A biochemical comparison of ciliary and flagellar axonemes from the bay scallop, Aequipecten irradians, 429 LINDNER, E. See J. R. SAROYAN, 333 LINDORFER, J. See H. GEWURZ, 411, 423 LISMAN, J. E. AND J. E. BROWN. A linear relationship between light-induced cur- rent and flash intensity in Liinulus ven- tral photoreceptors, 429

Lizard, environmental temperature and thy- roid activity in, 107 LOBO, A. P. See D. B. DONNER, 419 Loligo pealei, effects of cytochalasin B on cytoplasmic movement, cleavage, and sub- sequent development of the embryo of, 413 Lophius americamis, glucagon biosynthesis in islets of, 431

Low temperature acclimation in the winter

flounder, 574 LSD, analysis of the color changes induced

in the fiddler crab by, 407 Luiubricus, acrosome reaction in spermatozoa

of, 424

ultrastructure of spermatozoon of, 403 Luminescence, neurally controlled, compara- tive physiology of, 417 and fluorescence in coelenterates, 430 during development of the ctenophore, 422 LYNCH, R. V. III. See F. I. TSUJI, 386 LYNN, W. G. See K. W. CHIU, 107 Lysin, inducible, in Liinulus polyphcnms, 411 Lysosomal hydrolase from phagocytic cells, mechanisms of, 408

M

MACKINTOSH, F. R. AND E. BELL. Reversible response to puromycin and some char- acteristics of the uptake and use of amino acids by unfertilized sea urchin eggs, 296

MACNICHOL, E. F., JR. See M. L. WIEDER- HELD, 442

588

INDEX

, P. F. A., K. W. CHIU AND J. G. PHILLIPS. Changes in the epidermal his- tology during the sloughing cycle in the rat snake Ptyas korros Schlegel, \\itli correlated observations on the thyroid gland, 304

Magnesium and calcium content of Arbacia eggs, effect of fertilization on, 413

Male reproductive performance of Artonia, 363

Mandibulate arthropods, control of molting by ecdysones in, 520

Marine Biological Laboratory, annual report of, 1

McGowAN, W. E. See E. L. CHAMBERS, 417

McREYNOLDS, J. S. See S. N. BARNES, 414

Mclampus bidcntatus, influence of semilunar factors on hatching of, 434

Melanophorotropic hormones from the pitui- tary of the dogfish and the eye-stalk of the fiddler crab, comparison of, 415

Membrane system, peripheral, of sea urchin eggs, 425

Mcrccnaria mcrccnaria, spike responses in the photoreceptor system of, 442

Metabolic controls and biological variation, 406

Metridium sp., ultrastructure of the secondary septa of, 406

METZ, CHARLES B., memorial for Albert

Tyler, 9 see R. C. MOWBRAY, 313

Microsporidian spores, structure and function of, 443

Microtubules, intranuclear, during growth and division in Tetrahymena pvrifonnis, 409

Microtubules of cilia and flagella, changes in following negative staining of, 265

MILKMAN, R. A fundamental error in the

general model of genetic selection, 402 AND L. D. BEATY. Large-scale electro- phoretic studies of allelic variation in Mytilus cdulis, 430

MINOR, R. R. See J. LASH, 427

Mnemiopsis Icidyi, origin of the light pro- ducing cells during development of, 422

Model of genetic selection, fundamental error in, 402

Molting fluid proteins, origin, distribution and fate of, 277

Molting in arthropods, control of by ecdy- sones, 520

Monoxenic cultivation of Daphnidae, 321

MORALES-ALAMO, R. See D. S. HAVEN, 248

Morgan, T. H. and the Mendelian theory, 412

MORIN, J. G. AND G. T. REYNOLDS. Lumi- nescence and related fluorescence in co- elenterates, 430

MOTE, M. I. See M. S. BRUNO, 416

MOWBRAY, R. C., G. G. BROWN AND C. B. METZ. Cytological and immunological in- vestigations of sperm-egg interactions in selected decapods (Crustacea) and Limulus polyphemus L. (Merostomata), 313

Mucopolysacharides (acid) in some balanid barnacles, 164

MURPHY, J. S. A general method for the monoxenic cultivation of the Daphnidae, 321

Muscle fibers, crayfish, parallel changes in thresholds of current induced tensions and calcium spikes in, 439

Muscle spindle, frog, role of the sodium pump in adaptation in, 406

Muscles in some balanid barnacles, 164

Afustclus canis, receptive field organization of

ganglion cells in the retina of, 437 reversible de-aggregation of alpha-crystallin

from the ocular lens of, 428 studies on blood folic acid in, 416 comparison of melanophorotrophic hor- mones from the eyestalk of the fiddler crab with those from the pituitary of, 415

Mutual insemination in turbellarians, 539

Myocardial rhythmicity in Busycon canalic- lilatum, neural control of, 427

Mytilus ediilis, electrophoretic studies of allelic variation in, 430

N

NADELHAFT, I. See R. A. NYSTROM, 431

Natural and synthetic materials with insect hormone activity, 222

Nemertine neurotoxins, chemistry and bio- logy of, 408

Nereid escape response, 80

Nereis limnicola, endocrinology and reproduc- tive biology of, 461

Nerve activity in the phallic nerve of the cockroach, 405

Neural control of myocardial rhythmicity in Busycon canallcitlatinu, 427

Neurally controlled luminescence, comparative physiology of, 417

Neurons, large, in abdominal ganglia of Limulus, morphological and electrical features of, 431

Neurotoxins, nemertine, chemistry and bio- logy of, 408

NEWTON, W. D. Gastrulation in the turbel- larian Hydrolimax grisca (Platyhel- minthes; Plagiostomidae) : Formation of

INDEX

589

the epidermal cavity, inversion and epi- boly, 539

Nexin, isolation of from flagellar axonemes, 438

NOBLE, P. B. Coelomocyte aggregation in Cucumaria frondosa: Effect of ethylene- diaminetetraacetate, adenosine, and ade- nosine nucleotides, 549

NOE, B. D. AND G. E. BAUER. Evidence for glucagon biosynthesis and participation of a precursor protein in islets of the anglerfish (Lophius amcricaints), 431

Noseina, structure and function of micro- sporidian spores in, 443

Nuclear protein following fertilization in Arbacia and Spisitla, phosphorylation of, 425

Nucleosidase, ovarian wall, in the starfish, evidence for, 435

Nutrition of Paranemertes pcrcgrina, 80, 92

NYBLADE, C. F. Larval development of Pag- itms anmilipes (Stimpson, 1862) and Pagurus pollicaris Say, 1817 reared in the laboratory, 557

NYSTROM, R. A., R. A. LEVY AND I. NADEL- HAFT. Morphological and electrical fea- tures of some large neurons in abdominal ganglia of Limulus polvplicmus Linnaeus, 431

O'BRIEN, E. M. See W. D. SULLIVAN, S. J.,

409 Oculomotor nucleus, synaptic organization of,

442 Oocytes, starfish, studies of RNA synthesis

in, 436 Opsanus tan, absorption of D-glucose in, 421

radiothyroidectomy of, 424 Orientation, pigeon, "distance effect" in, 510 Origin of molting fluid proteins of the

cecropia silkworm, 277

Origins of life and hydrogen cyanide poly- mers, 419

Osmoregulation in the winter fluonder, 574 Ostracod, bioluminescence of, 386 Ovaries in some balanid barnacles, 164 Oxygen, superabundant, on thermal tolerance

of goldfish, effect of, 229 Oyster, American, filtration of particles from

suspension by, 248

Pacemaker inhibition in hydranths of Tubu- laria, temporal characteristics of, 434

Pacemaking and phase-shifting in synchroniz- ing fireflies, 417

Pagurus annulipcs, larval development of 557

Pagurus lonc/icarpits reared in the laboratory, larval development of, 188

Pagurus pollicaris, larval development of, 557

PAPPAS, G. D. See S. G. WAXMAN, 442

Paranemertes pcrcgrina, nutrition of, 80, 92

PAKNAS, I. Differences in blocking frequency of presynaptic terminals of an axon in- nervating different crayfish muscles, 432 See M. E. SPIRA, 405

Peripheral membrane system of sea urchin eggs, 425

Periplancta amcricana, regulation of nerve activity in the phallic nerve of, 405

Permeability, water, of Carcimis macnas as a

function of salinity, 351 water, of Fundulus eggs, 420 and structure of cellular junctions in the starfish embryo, 440

PFENINGER, E., See S. LERMAN, 428

Phase-shifting and pacemaking in synchro- nizing fireflies, 417

Phenotype, statistical, concept applied to skeletal variation in the mouse, 416

Phialidhim grcgarium, life cycle in the labora- tory of, 203

PHILLIPS, J. G. See P. F. A. MADERSON, 304

Phospholipase A in Limulus, detection, char- acteristics and dissection of inducible lysin from, 411

Phosphorylation of nuclear protein following fertilization in Arbacia and Spisitla, 425

Phosphotungstic acid, changes in microtubules of cilia and flagella following negative staining with, 265

Photoperiod, interaction in termination of larval diapause in Chaoborus americanus, 476

Photoreceptor system of the hard-shell clam, 442

Photoreceptors, ventral, of Limulus, a linear relationship between light-induced cur- rent and flash intensity in, 429 of a tunicate, ultrastructure and intracellular

responses of, 414 of squid, adaptation properties of, 433

Physiology of the reproductive cycle in the cockroach, 447

Phytoplankton food resources, differential utilization by marine ectoprocts of, 420

Pigeon orientation, "distance effect" in, 510

Pituitary of the dogfish, comparison of mel- anophorotropic hormones from the eye- stalk of the fiddler crab with those from, 415

Platynereis bicanaliculata, food choice of Paranemertes percgrina, 80

Podocoryne carnca, hydranth regulation in, 404

590

INDEX

Polychaete, viviparous, endocrinology and re- productive biology of, 461

Polymers of hydrogen cyanide and the origins of life, 419

Ponasterone, see ecdysones, 520

Population genetics of a marine ectoproct, a natural experiment in, 435

Population genetics of Mytilus, 430

Portunidae, water permeability of, 351

POWELL, W. A. See N. E. RICE, 180

PRENDERGAST, R. A. Macrophage activating protein from the sea star Astcrias jorbcsi coelomocyte simulating effector substances of the delayed hypersensitivity response, 412

Presynaptic terminals of an axon innervating crayfish muscles, differences in blocknig frequency of, 432

Proboscis of Paranemertes pcrcgrina, 80, 92

Protein synthesis in the hepatopancreas of the spider crab, effect of cold exposure on, 421

Proteins, molting fluid, of the silkworm, origin, distribution and fate of, 277

PROVASOLI, L. See A. S. D'AGOSTINO, 485

Pscudoplcuronectes americawus, effects of sub- zero temperatures and trawling stress on serum osmolality in, 574

Ptyas korros, changes in epidermal histology during sloughing cycle in, 304

Pulmonate, land, role of amoebocytes in shell regeneration in, 502

Puromycin, effect on growth pattern of Campanularia flexuosa of, 443

Puromycin, reversible response by unfertilized sea urchin eggs to, 296

R

Radiothyroidectomy of the toadfish, 424 Raja crinacea and R. osccllata, adaptation in

the retina of, 405 RAND, J. See E. J. SIMON, 404 RAO, K. R. See M. FINGERMAN, 407 Rat snake, changes in epidermal histology dur- ing sloughing cycle in, 304 Reattachment and repair in the balanidae, 333 Regeneration, newt limb, hyaluronidase in the

blastema of, 403

in Tubularia, DNA synthesis during, 440 of calcined tissues in the land pulmonate,

502

of shell in Euplecta indica, role of amoebo- cytes in, 502

Rcnicola in the kidneys of the gull, 438 Repair and reattachment in the balanidae, 333

of molluscan shell, 502

Replication, RNA phage, reversible inhibition of by levorphanol, 404

Report of the Director, 1969-1970, 7 Reproductive biology of Nereis, experimental

studies on, 461

Reproductive cycle in the cockroach, physiol- ogy of, 447

Reproductive performance in Artcmia, 363, 375 Reproductive potential in Ectoprocta, influence

of colony morphology on, 426 Reproduction in the spider crab, control of,

410 Resorption, tail, in ascidians, effects of Cyto-

chalasin B on, 427

Respiration, cutaneous, of the clingfish, 115 Respiratory gas exchange in clingfish, 115 Retes, choroid, of scup and striped bass, 444 Retina, skate, intracellular responses during light- and dark-adaptation in S-potentials in, 419 of the dogfish, receptive field organization

of ganglion cells in, 437 Retinal adaptation in the skate, electrical and

photochemical signs of, 405 REUBEN, J. P. See G. SUAREZ-KUREZ, 439 Reversible response to puromycin by unfertil- ized sea urchin eggs, 296

REYNOLDS, G. T. AND P. BOTOS, JR. Image intensification and magnetic tape record- ing system for microscopic observations of bioluminescence and fluorescence, 432 See J. F. CASE, 417 See G. FREEMAN, 422 See J. G. MORIN, 430 Rhythmicity, myocardial, in Busycon canali-

culatum, neural control of, 427 RICE, N. E. AND W. A. POWELL. Observa- tions on three species of jellyfishes from Chesapeake Bay with special reference to their toxins. I. Chrysaora (Dactylo- inctra) quinquccirrha, 180 RIPPS, H. AND I. M. SIEGEL. Adaptation

properties of squid photoreceptors, 433 See J. E. DOWLING, 405, 419 RITCHIE, J. M. See L. B. COHEN, 418 RNA synthesis in starfish oocytes, studies of,

436

ROBERTS, M. H., JR. Larval development of Pagjirus longicarpits Say reared in the laboratory. I. Description of larval in- stars, 188

Roccus saxatilis, choroid retes of, 444 ROE, P. The nutrition of Paranemertes pcre- grina (Rhynchocoela : Hoplonemertea). I. Studies on food and feeding behavior, 80

RoosEN-RuNGE, E. C. Life cycle of the hy- dromedusa Phialidium gregarium (A. Agassiz, 1862) in the laboratory, 203

INDEX

591

RUIZ-MANRESA, F. Na currents and GN, changes during spike electrogenesis of eel electroplaques, 433

RUSHFORTH, N. B. Temporal characteristics of pacemaker inhibition in isolated hy- dranths of Tnbiilaria, 434

RUSSELL-HUNTER, W. D., M. L. APLEY AND R. D. HUNTER. Semilunar and other fac- tors influencing hatching from egg- masses of Mclampus bidentatus in the field and in the laboratory, 434 A. J. BURKY AXD R. D. HUNTER. Inter- population variation in shell components m the stream limpet, Ferrissia rivularis 402

RUSTAD, R. C. See A. M. BELANGER, 415

Salinity, water-permeability of Carcinus

maenas as a function of, 351 Salmo gairdneri, absorption of D-glucose in,

Salpa, ultrastructure and intracellular re- sponses of photoreceptors of, 414

SAROYAN-, J. R., E. LINDNER AND C. A. DOOLEY. Repair and reattachment in the Balanidae as related to their cementing mechanism, 333

SAUNDERS, J. W., JR. See J. T. TUPPER, 440

Sceloporus occidcntalis, environmental tem- perature and thyroid activity in, 107

SCHNEIDERMAN, H. A. See A. KRISHNAKU-

MARAN, 520

See Y. LENSKY, 277

SCHOPF, T. J. M. AND J. L. GOOCH. A nat- ural experiment in the population genetics of a marine ectoproct suggesting changes in gene frequencies with changes in tem- perature, 435

SCHUETZ, A. W. Effects of 1 -methyl adeno- sme on isolated gonads and oocytes of the starfish; evidence for an ovarian wall nucleosidase, 435 See E. L. SHIPPEE, 436 See B. TOOLE, 439

Sea urchin eggs, unfertilized, reversible re- sponse to puromycin by, 296 uptake and use of amino acids by, 296 Secretion used in repair and reattachment in

barnacles, 333

Secretory cells in some balanid barnacles, 164 Selection, genetic, fundamental error in the model of, 402

Semilunar factors, influence on hatching of Melampus bidentatus in the field and the laboratory, 434

SEN GUPTA, A. See S. P. KAPUR, 502

Serotonin, analysis of the color changes in- duced in the fiddler crab by, 407 Serum osmolality in the winter flounder, ef- fects of subzero temperatures and trawl- ing stress on, 574

SHANKLIN, D. R. AND J. C. SHANKLIN. Physiological adaptations of Funduhts em- ^ bryos to varied oxygen tensions, 435 See J. C. SHANKLIN, 436 SHANKLIN, J. Q AND D. R. SHANKLIN, In- fluence of varied oxygen tension on fer- tilization and on developmental time in Fundulus heteroclitus, 436 ^ See D. R. SHANKLIN, 435 Shell components in Ferrissia, interpopulation

variation in, 402 regeneration in the land pulmonate, role of

amoebocytes in, 502 SHEPRO, D. See D. M. KENNEY, 426 SHIPPEE, E. L., G. GIUDICE AND A. W. SCHUETZ. Studies of nucleolar RNA syn- thesis in starfish oocytes, 436 See B. TOOLE, 439 Shrimp, brine, effects of gamma irradiation

on, 363, 375

SHRIVASTAV, B. B. See L. B. COHEN, 418 Sialic acid in the unfertilized sea urchin egg,

nuclear localization of, 426 Sicyases sanguinens, emersion of, 115 SIEGEL, I. M. See H. RIPPS, 433 Silkworm, cecropia, origin, distribution, and fate of the molting fluid proteins of 277 SIMON, E. J., D. J. GARWES AND J. RAND. Reversible inhibition of RNA phage repli- cation and macromolecular synthesis by levorphanol, 404 Skate, electrical and photochemical signs of

adaptation in the retina of, 405 electroplaques, synaptic potentials and acetyl-

choline in, 428

retina, S-potentials in intracellular re- sponses during light- and dark-adaptation in, 419

SLAMA, K., K. HEJNO, V. JAROLIM AND F. SORM. Natural and synthetic materials with insect hormone activity. 5. Specific juvenile hormone effects in aliphatic ses- quiterpenes, 222 Sloughing cycle in the rat snake, changes in

epidermal histology during, 304 SMITH, R. I. The apparent water-permeabil- ity of Carcinus maenas (Crustacea, Bra- chyura, Portunidae) as a function of salinity, 351

SNYDER, D. A. AND J. P. GREEN. Biochemical changes in the digestive gland of the fid- dler crab, Uca pugnax, following bilateral eyestalk extirpation, 437

592

INDEX

Sodium currents during spike electrogenesis

of eel electroplaques, 433 pump, role of in adaptation in the frog muscle spindle, 406

SORM, F. See K. SLAMA, 222

Spectral sensitivity of retinular cells in the crab, microspectrophotometry of, 416

Sperm and somatic cells of the spider crab, DNA and chromatin in, 441

Sperm-egg interactions in decapods and Linm- lus, cytological and immunological investi- gations of, 313

Spermatozoa of Lumbricus, acrosome reaction

in, 424 ultrastructure of, 403

Spider crab, control of reproduction in, 410

Spines, regenerating, of S. piirpuratns, effects of temperature and diamox on incorpora- tion of calcium-45 in vitro by, 151

SPIRA, M. E., I. PARNAS AND F. BERGMANN.

Excitatory and inhibitory regulation of

efferent nerve activity in the phallic nerve

of the cockroach Pcriplancta amcricana

_ (L), 405

Spisnla, phosphorylation of nuclear protein following fertilization in, 425

Spores, microsporidian, structure and func- tion of, 443

Squid, effects of cytochalasin B on cytoplasmic movement, cleavage and subsequent devel- opment of the embryo of, 413 axons, changes in fluorescence of, 418 photoreceptors, adaptation properties of, 433

SQUIRE, R. D. The effects of acute gamma irradiation on the brine shrimp, Artcmia. II. Female reproductive performance, 375 AND D. S. GROSCH. The effects of acute gamma irradiation on the brine shrimp, Artemia. I. Life spans and male repro- ductive performance, 363

Starfish, distribution and metabolism of 1- methyl adenine in, 439

Statistical phenotype concept applied to skele- tal variation in the mouse, 416

STEINBACH, H. BURR, Director's Report, 7

STELL, W. K., H. G. WAGNER AND M. L. WOLBARSHT. Receptive field organization of ganglion cells in the retina of the smooth dogfish, Mustclus canis, 437

Stenotomus versicolor, choroid retes of, 444

STEPHENS, R. E. Isolation of nexin — the link- age protein responsible for maintenance of the nine-fold configuration of flagellar axonemes, 438

STEPHENSON, J. E. See C. K. BARTELL, 415

Stimulation and control of airbreathing in fishes, 115

Stinging cells of jellyfishes from Chesapeake

Bay, 180 Stnniciylocentrotus purpuratiis, uptake and use

of amino acids by, 296 Strongylocentrotus purpwatus regenerating

spines, effects of temperature and diamox

on incorporation of calcium-45 in vitro by,

151 STUNKARD, H. W. Species of Rcnicola (Tre-

matoda) in the kidneys of the gull, Larus

argaitatits, 438

SUAREZ-KUREZ, G., P. W. BRANDT AND J. P.

REUBEN. Parallel changes in the thresh- olds of current induced tensions and Ca- spikes in isolated crayfish muscle fibers, 439

SULLIVAN, W. D., S.J. AND E. M. O'BRIEN. Intranuclear microtubules during growth and division in Tetrahymena pyriformis Gl., 409

Supercooling in fish, 574

SUTHERS, R. A. See D. R. GRIFFIN, 495

Swiftlets, cave, sensitivity of echolocation in 495

Synaptic organization of the oculomotor nu- cleus, 442

potentials and acetylcholine in skate electro- plaques, 428

Synthesis, DNA, during regeneration in Tiibu- laria, 440

Synthesis, macromolecular, reversible inhibi- tion of by levorphanol, 404

Synthesis, RNA, in starfish oocytes, studies of, 436

Synthetic and natural materials with insect hormone activity, 222

Tabun, in relation to nerve function, enzymatic hydrolysis of, 407

Tail resorption in ascidians, effects of Cyto- chalasin B on, 427

TASAKI, I. See A. WATANABE, 441

Taxonomy of Phialidiwm gregarium, 203

Tellina agilis, territoriality observed in a population of, 423

Temperature, effect on incorporation of cal- cium-45 in vitro by regenerating spines of Strongylocentrotus pjirpurahis, 151 effect on the efflux of calcium from Arbacia

eggs of, 417 environmental, and thyroid activity in a

lizard, 107

subzero, effect on serum osmolality in the winter flounder, 574

Terminals, presynaptic, of an axon innervating crayfish muscle, differences in blocking frequency of, 432

INDEX

593

Territorial ity in a population of Tellina, 423 Tetrahyincna pyriforviis, intranuclear micro- tubules during growth and division in, 409 Thermal tolerance of goldfish, effects of super- abundant oxygen on, 229

Thyroid activity and environmental tempera- ture in a lizard, 107 Toadfish, radiothyroidectomy of, 424 TOOLE, B. AND J. GROSS. Hyaluronate and hyaluronidase in the blastema of the re- generating newt limb, 403 TOOLE, B., S. HULL, E. L. SHIPPEE AND A. W. SCHUETZ. Distribution and metabolism of 1-methyl adenine in the starfish during shedding and oocyte maturation in vitro, 439

TOWNSEND, K. See H. GEWURZ, 411 TRACER, W. See E. WEIDNER, 443 Trawling stress, effect on serum osmolality in

the winter flounder, 574 Trematoda in the kidneys of the gull, 438 TRINKAUS, J. P. See P. B. DUNHAM, 420 TSUJI, F. L, R. V. LYNCH, III AND Y. HA- NEDA. Studies on the bioluminescence of the marine ostracod crustacean Cyprldina scrrata, 386

Titbitlaria, DNA synthesis during regenera- tion in, 440

temporal characteristics of pacemaker inhi- bition in hydranths of, 434 Tunicate, ultrastructure and intracellular re- sponses of photoreceptors of, 414

TUPPER, J. T., K. S. WlTTMAN AND J. W.

SAUNDERS, JR. Permeability and struc- ture of cellular junctions in the starfish embryo, 440

Turbellarian, gastrulation in, 539

TWEEDELL, K. S. DNA synthesis during re- generation in Tiibularia, 440

TYLER, ALBERT, memorial of, 9

U

Uca pngilator, analysis of the color changes

induced by serotonin and LSD in, 407 comparison of melanophorotropic hormones from the pituitary of the dogfish with those from the eyestalk of, 415 Uca pugna.v, biochemical changes in the diges- tive gland following eyestalk extirpation of, 437

Ultrastructure of intranuclear microtubules during growth and division in Tetrahy- mena pyriformis, 409 of photoreceptors of Salpa, 414 of spermatozoa of Lumbricus, 424 of spermatozoon of the earthworm, 403 of the peripheral membrane system of sea urchin eggs, 425

of the secondary septa of Mctridium sp., 406 of the synaptic organization of the oculo- motor nucleus, 442

UMMIXGER, B. L. Effects of subzero tempera- tures and trawling stress on serum osmo- lality in the winter flounder, Pseudopleu- ronectes americanus, 574

Variation, allelic, in Mytilus, electrophoretic

studies of, 430

biological, and metabolic controls, 406 interpopulation, in shell components in Fer-

rissia, 402

skeletal, in the mouse, statistical phenotype concept applied to, 416

VAUGHN, J. C. AND G. W. HINSCH. Absorb- ance melting profile analysis of DNA and chromatin in somatic cells and sperm of the spider crab, Libinia emarginata, 441

Venom of Parancmertes percgrina, 80

Virus disease of the shore crab Carcimis tnaenas, 414

Visual pigment of retinular cells in the crab, microspectrophotometry of, 416

Vitamins and sustained fertility of Daphniu,

485

(water soluble) used for the monoxenic cul- tivation of Daphnidae, 321

W

WAGNER, H. G. See W. K. STELL, 437

WATANABE, A., I. TASAKI AND M. HALLETT. Fluorescence analysis of the excitation process in nerve using hydrophobia probes, 441

Water permeability of Carcimis maenas as a

function of salinity, 351 of Fundulus eggs, 420

WAXMAN, S. G. AND G. D. PAPPAS. Synaptic organization of the oculomotor nucleus : a comparative electron microscopic study, 442

WEATHERLEY, A. H. Effects of superabun- dant oxygen on thermal tolerance of gold- fish, 229

WEIDNER, E. AND W. TRACER. Structure and function of microsporidian spores, 443

WEISSMANN, G., P. DUKOR AND R. ZURIER. Mechanisms of lysosomal hydrolase from phagocytic cells, 408

WHITTAKER, J. R. See D. FROMSON, 239

\VlKDERHELD, M. L. AND E. F. MAcNlCHOL,

JR. Only decreases in illumination elicit spike responses in the siphonal photore- ceptor system of the hard-shell clam, Mcrcenaria mercenaries, 442

594

INDEX

WILLIAMS-ARNOLD, L. D. See J. M. ARNOLD, 413

WILSON, D. B. On imrnunologic reactivity to histocompatibility antigens, 410

Winter flounder, effects of subzero tempera- tures and trawling stress on serum osmo- lality in, 574

WITTMAN, K. S. Effect of puromycin on the growth pattern of Campanularia flcxnosa, 443

WITTMAN, K. S. See J. T. TUPPER, 440

WOLBARSHT, M. L. See W. K. STELL, 437

WOLLEY, R. C. AND D. E. COPELAND. The

choroid retes of scup, Stenotomus ver si- color and striped bass, Roccus saxatilis, 444 Wos, J. D. See D. B. DONNER, 419

Yolk-cell mass in Hydrolimax grisca, 539 Yolk cells and cleavage furrows in Hydro- limax grisea, 539 YULE, T. See S. ZIGMAN, 444

ZDAREK, J. See G. FRAENKEL, 138

ZIGMAN, S. Isoelectric focusing of lens gamma

(7) crystallins, 409 T. YULE AND G. GRIESS. How protein

coloration modifies interactions between

lenses and UV light, 444 ZULETA, A. See A. W. EBELING, 115 ZURIER, R. See G. WEISSMANN, 408

Volume 139

Marine Biological Laboratory

L.I BRARY

SEP 4 1970 WOODS HOLE, MASS.

Number 1

THE

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MBL WHOI LIBRARY

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