THE
BIOLOGICAL BULLETIN
PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
JOHN M. ANDERSON, Cornell University J. LOGAN IRVTN, University of North Carolina
DAVID W. BISHOP, Carnegie Institution of JOHN H. LOCHHEAD, University of Vermont
Washington y L LoosANOFF n> s> Fish and Wiidiife
JAMES CASE, University of California, Service
Santa Barbara L. H. KLEINHOLZ, Reed College
JOHN W. GOWEN, Iowa State College BERTA SCHARRER, Albert Einstein College of
SALLY HUGHES-SCHRADER, Duke University Medicine
LIBBIE H. HYMAN, American Museum of WM. RANDOLPH TAYLOR, University of
Natural History Michigan
DONALD P. COSTELLO, University of North Carolina
Managing Editor
VOLUME 123
JULY TO DECEMBER, 1962
/ * ^O \
Yt/e2N^«
1 «S Y S,
Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE 8C LEMON STS.
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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,
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Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London,
W. C. 2. Single numbers $2.50. Subscription per volume (three
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Communications relative to manuscripts should be sent to the
Managing Editor, Marine Biological Laboratory, Woods Hole,
Massachusetts, between June 1 and September 1, and to Dr.
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during the remainder of the year.
Second-class postage paid at Lancaster, Pa.
LANCASTER PRESS, INC., LANCASTER, PA.
CONTENTS
No. 1. AUGUST, 1962
PAGE
Annual Report of the Marine Biological Laboratory 1
ARNOLD, JOHN M.
Mating behavior and social structure in Loligo pealii 53
BLACK, ROBERT E.
Respiration, electron-transport enzymes, and Krebs-cycle enzymes in
early developmental stages of the oyster Crassostrea virginica 58
BLACK, ROBERT E.
The concentrations of some enzymes of the citric acid cycle and electron
transport system in the large granule fraction of eggs and trochophores
of the oyster, Crassostrea virginica 71
CHUANG, S. H.
Feeding mechanism of the echiuroid, Ochetostoma erythrogrammon
Leuckart & Ruepell 1828 ^ 80
CHUANG, S. H.
Sites of oxygen uptake in Ochetostoma erythrogrammon Leuckart &
Ruepell (Echiuroidea) 86
ENGELS, WILLIAM L.
Day-length and termination of photorefractoriness in the annual testicu-
lar cycle of the transequatorial migrant Dolichonyx (the bobolink) .... 94
FARMANFARMAIAN, A., AND JOHN H. PHILLIPS
Digestion, storage, and translocation of nutrients in the purple sea urchin
(Strongylocentrotus purpuratus) 105
FlNGERMAN, MlLTON, R. NAGABHUSHANAM AND LORALEE PHILPOTT
Photomechanical responses of the proximal pigment in Palaemonetes and
Orconectes 121
HARVEY, ETHEL BROWNE
Prorlavin and its influence on cleavage and development 132
KUENZLER, EDWARD J., AND BOSTWICK H. KETCHUM
Rate of phosphorus uptake by Phaeodactylum tricornutum 134
LANDERS, WARREN S., AND RICHARD C. TONER
Survival and movements of the flatworm, Stylochus ellipticus, in dif-
ferent salinities and temperatures 146
MORRISON, PETER
Body temperatures in some Australian mammals. III. Cetacea (Me-
gaptera) 154
MOULTON, JAMES M.
Intertidal clustering of an Australian gastropod 170
PROVENZANO, ANTHONY J., JR.
The larval development of Calcinus tibicen (Herbst) (Crustacea,
Anomura) in the laboratory 179
81791
iv CONTENTS
SUGIURA, YASUO
Electrical induction of spawning in two marine invertebrates (Urechis
unicinctus, hermaphroditic Mytilus edulis) 203
GIFFORD, CHARLES A.
Some observations on the general biology of the land crab, Cardisoma
gnanhumi (Latreille), in south Florida 207
No. 2. OCTOBER, 1962
ARMITAGE, KENNETH B.
Temperature and oxygen consumption of Orchomonella chilensis (Heller)
(Amphipoda : Gammeroidea) 225
BAYLOR, EDWARD R., AND WILLIAM E. HAZEN
The analysis of polarized light in the eye of Daphnia 233
HAZEN, WILLIAM E., AND EDWARD R. BAYLOR
Behavior of Daphnia in polarized light 243
BRANDOM, WILLIAM FRANKLIN
Karyoplasmic studies in haploid, androgenetic hybrids of California
newts 253
BROWN, FRANK A., JR.
Responses of the planarian, Dugesia, and the protozoan, Paramecium,
to very weak horizontal magnetic fields 264
BROWN, FRANK A., JR.
Response of the planarian, Dugesia, to very weak horizontal electro-
static fields 282
CLEGG, JAMES S.
Free glycerol in dormant cysts of the brine shrimp, Artemia salina, and
its disappearance during development 295
GROSCH, DANIEL S.
The survival of Artemia populations in radioactive sea water 302
KLEINHOLZ, L. H., H. ESPER, C. JOHNSON AND F. KIMBALL
Neurosecretion and crustacean retinal pigment hormone : assay and
properties of the light-adapting hormone 317
MAIRS, DONALD ¥., AND CARL J. SINDERMANN
A serological comparison of five species of Atlantic clupeoid fishes 330
v MATHEW, A. P.
Reproductive biology of Lychas tricarinatus (Simon) 344
MENDOZA, GUILLERMO
The reproductive cycles of three viviparous teleosts, Alloophorus
robustus, Goodea luitpoldii and Neoophorus diazi 351
MUN, A. M., P. TARDENT, J. ERRICO, J. D. EBERT, L. E. DELANNEY AND
T. S. ARGYRIS
An analysis of the initial reaction in the sequence resulting in homologous
splenomegaly in the chick embryo 366
SASTRY, A. N., AND R. WINSTON MENZEL
Influence of hosts on the behavior of the commensal crab Pinnotheres
maculatus Say 388
SIMPSON, MARGARET
Reproduction of the polychaete Glycera dibranchiata at Solomons,
Maryland 396
CONTENTS v
SIMPSON, MARGARET
Gametogenesis and early development of the polychaete Glycera di-
branchiata 412
TWEEDELL, KENYON S.
Cytological studies during germinal vesicle breakdown of Pectinaria
gouldii with vital dyes, centrifugation and fluorescence microscopy. . . . 424
MILLER, JAMES A., JR., LORALEE L. PHILPOTT AND C. LLOYD CLAFI
Oxygen uptake in short pieces of Tubularia stems 450
Abstracts of papers presented at the Marine Biological Laboratory 461
No. 3. DECEMBER, 1962
ANSELL, ALAN D.
Observations on burrowing in the Veneridae (Eulamellibranchia) 521
DAN, JEAN C.
The vitelline coat of the Mytilus egg. I. Normal structure and effect
of acrosomal lysin 531
ENGELS, WILLIAM L.
Migratory restlessness in caged bobolinks (Dolichonyx oryzivorus, a
transequatorial migrant) 542
GREGG, JOHN R.
Anaerobic glycolysis in amphibian development 555
HERMAN, SIDNEY S.
Spectral sensitivity and phototaxis in the opossum shrimp, Neomysis
americana Smith 562
JENNINGS, J. B.
Further studies on feeding and digestion in triclad Turbellaria 571
LASHER, R., AND R. RUGH
The "Hertwig Effect" in teleost development 582
OSBORNE, PAUL J., AND A. T. MILLER, JR.
Uptake and intracellular digestion of protein (peroxidase) in planarians 589
PAINE, ROBERT T.
Filter-feeding pattern and local distribution of the brachiopod Discinisca
strigata 597
READ, KENNETH R. H.
The hemoglobin of the bivalved mollusc, Phacoides pectinatus Gmelin. 605
RUCK, PHILIP
On photoreceptor mechanisms of retinula cells 618
SKINNER, DOROTHY M.
The structure and metabolism of a crustacean integumentary tissue
during a molt cycle 635
STEPHENS, G. C.
Uptake of organic material by aquatic invertebrates. I. Uptake of glu-
cose by the solitary coral, Fungia scutaria 648
YULES, RICHARD B.
Responses from a proprioceptive organ of the crab, Sesarma reticulatum,
during the molt cycle 660
Vol. 123, No. 1 August, 1962
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
THE MARINE BIOLOGICAL LABORATORY
SIXTY-FOURTH REPORT, FOR THE YEAR 1961 — SEVENTY-FOURTH YEAR
I. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST 15, 1961) .... 1
STANDING COMMITTEES
II. ACT OF INCORPORATION 4
III. BY-LAWS OF THE CORPORATION 4
IV. REPORT OF THE DIRECTOR 6
Addenda :
1. The Staff 7
2. Investigators, Lalor and Lillie Fellows, and Students 10
3. Fellowships and Scholarships 21
4. Tabular View of Attendance, 1957-1961 21
5. Institutions Represented 22
6. Evening Lectures 24
7. Shorter Scientific Papers (Seminars) 24
8. Members of the Corporation 25
V. REPORT OF THE LIBRARIAN 45
VI. REPORT OF THE TREASURER 46
I. TRUSTEES
GERARD SWOPE, JR., President of the Corporation, 570 Lexington Ave., New York City
A. K. PARPART, Vice President of the Corporation, Princeton University
PHILIP B. ARMSTRONG, Director, State University of New York, Medical Center at
Syracuse
C. LLOYD CLAFF, Clerk of the Corporation, Randolph, Mass.
JAMES H. WICKERSHAM, Treasurer, 530 Fifth Ave., New York City
EMERITI
W. C. CURTIS, University of Missouri
PAUL S. GALTSOFF, Woods Hole, Massachusetts
E. B. HARVEY, Woods Hole, Massachusetts
M. H. JACOBS, University of Pennsylvania School of Medicine
MARINE BIOLOGICAL LABORATORY
F. P. KNOWLTON, Syracuse University
CHARLES W. METZ, Woods Hole, Massachusetts
W. J. V. OSTERHOUT, Rockefeller Institute
CHARLES PACKARD, Woods Hole, Massachusetts
A. C. REDFIELD, Woods Hole Oceanographic Institution
LAWRASON RIGGS, 74 Trinity Place, New York 6, N. Y.
TO SERVE UNTIL 1965
ERIC G. BALL, Harvard Medical School
D. W. BRONK, Rockefeller Institute
MAC V. EDDS, JR., Brown University
RUDOLF KEMPTON, Vassar College
I. M. KLOTZ, Northwestern University
ARNOLD LAZAROW, University of Minnesota Medical School
ALFRED H. STURTEVANT, California Institute of Technology
GEORGE WALD, Harvard University
TO SERVE UNTIL 1964
C. LALOR BURDICK, The Lalor Foundation
E. G. BUTLER, Princeton University
K. S. COLE, National Institutes of Health
S. KUFFLER, Harvard Medical School
C. B. METZ, Oceanographic Institute, Florida State University
ROBERTS RUGH, College of Physicians and Surgeons, Columbia University
G. T. SCOTT, Oberlin College
E. ZWILLING, Brandeis University
TO SERVE UNTIL 1963
L. G. EARTH, Columbia University
JOHN B. BUCK, National Institutes of Health
AURIN M. CHASE, Princeton University
SEYMOUR S. COHEN, University of Pennsylvania School of Medicine
DONALD P. COSTELLO, University of North Carolina
TERU HAY ASH i, Columbia University
DOUGLAS A. MARSLAND, New York University, Washington Square College
H. BURR STEINBACH, University of Chicago
TO SERVE UNTIL 1962
FRANK A. BROWN, JR., Northwestern University
SEARS CROWELL, Indiana University
ALBERT I. LANSING, University of Pittsburgh Medical School
WILLIAM D. MCELROY, Johns Hopkins University
C. LADD PROSSER, University of Illinois
S. MERYL ROSE, Wesleyan University
MARY SEARS, Woods Hole Oceanographic Institution
ALBERT TYLER, California Institute of Technology
TRUSTEES
EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES
GERARD SWOPE, JR., ex officio, Chairman I. M. KLOTZ, 1964
JAMES H. WICKERSHAM, ex officio KENNETH S. COLE, 1963
ARTHUR K. PARPART, ex officio STEPHEN KUFFLER, 1963
P. B. ARMSTRONG, ex officio JOHN BUCK, 1962
MAC V. EDDS, JR., 1964 ALBERT I. LANSING, 1962
THE LIBRARY COMMITTEE
MARY SEARS, Chairman C. LADD PROSSER
SEYMOUR S. COHEN IRVING M. KLOTZ
MARTIN LUBIN KENYON TWEEDELL
THE APPARATUS COMMITTEE
ALBERT I. LANSING, Chairman DAVID POTTER
CLIFFORD HARDING HOWARD SCHACHMAN
ARNOLD LAZAROW RALPH H. CHENEY
THE SUPPLY DEPARTMENT COMMITTEE
RUDOLF T. KEMPTON, Chairman RICHARD SANBORN
SEARS CROWELL MAC V. EDDS, JR.
GEORGE SCOTT W. J. ADELMAN
THE INSTRUCTION COMMITTEE
JOHN B. BUCK, Chairman RICHARD C. STARR
TERU HAYASHI ROBERT K. CRANE
BOSTWICK KETCHUM EUGENE COPELAND
THE BUILDINGS AND GROUNDS COMMITTEE
EDGAR ZWILLING, Chairman DANIEL GROSCH
MORRIS ROCKSTEIN STEPHEN KUFFLER
JAMES CASE DE\VITT STETTEN
THE RADIATION COMMITTEE
G. FAILLA, Chairman (Deceased) WALTER L. WILSON
ROGER L. GREIF WALTER S. VINCENT
CARL C. SPEIDEL
THE RESEARCH SPACE COMMITTEE
PHILIP B. ARMSTRONG, Chairman MAC V. EDDS, JR.
ARTHUR K. PARPART WILLIAM D. MC£LROY
4 MARINE BIOLOGICAL LABORATORY
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 tlie Commonwealth.
III. BY-LAWS OF THE CORPORATION OF THE MARINE
BIOLOGICAL LABORATORY
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 President, Vice 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 eight Trustees to serve four years, and shall transact such
other business as may properly come before the meeting. Special meetings of the mem-
bers 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 By-laws, no notice of the Annual Meeting need be given. Notice of any special
BY-LAWS OF THE CORPORATION 5
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 fifteen (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 the Annual
Meeting of the Corporation at the Laboratory in Woods Hole, Mass. Special meetings
of the Trustees shall be called by 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-two 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. In addition, there shall
be two groups of Trustees as follows :
(B) Trustees ex officio, who shall be the President and Vice President of the Cor-
poration, the Director of the Laboratory, the Associate 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
Emeritus 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-
poration; they shall elect a President of the Corporation who shall also be Chairman of
the Board of Trustees and who shall be elected for a term of five years and shall serve
until his successor is selected and qualified ; and shall also elect a Vice President of the
Corporation who shall also be the Vice Chairman of the Board of Trustees and who shall
be elected for a term of five years and shall serve until his successor is selected and
qualified; they shall appoint a Director of the Laboratory; and they may choose such
other officers and agents as they may think best ; they may fix the compensation and define
the duties of all the officers and agents ; 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 offices. 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.
6 MARINE BIOLOGICAL LABORATORY
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 By-laws may be altered at any meeting of the Trustees, provided that the
notice of such meeting shall state that an alteration of the By-laws will be acted upon.
IV. REPORT OF THE DIRECTOR
To: THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY
Gentlemen :
I submit herewith the report of the seventy-fourth session of the Marine Bio-
logical Laboratory.
1. Plant Additions
During the past winter six cottages were built on the Devil's Lane Tract with
funds which were generously granted the Laboratory by the James Foundation of
New York. These are winterized cottages which can be available throughout the
year to visiting scientists at the Laboratory. This significant addition to the
Laboratory's housing will help to ease a difficult situation.
2. Systematics-Ecology Program
Dr. Melbourne R. Carriker has been appointed Director of the Systematics-
Ecology Program, the appointment to become effective on September 1, 1962.
Dr. Carriker took his Bachelor of Science degree at Rutgers in 1939 and his
Doctor of Philosophy degree at the University of Wisconsin in 1943. He has
been a member of the zoology staffs at Rutgers University and at the University
of North Carolina and has served as Supervisory Fishery Research Biologist at
the Oxford, Maryland, Biological Laboratory of the Bureau of Commercial Fish-
eries. The program under Dr. Carriker's direction will be financed in part by a
grant from the Ford Foundation.
3. Personnel Changes
This past summer Dr. Eugene P. Odum completed his five-year term as head
of the training program in Marine Ecology and is being succeeded by Dr. John H.
Ryther. Dr. J. Woodland Hastings takes charge of the training program in Physi-
ology, succeeding Dr. W. D. McElroy. Dr. C. B. Metz will head up the newly
established training program in Fertility Problems and Dr. James D. Ebert will
take over the direction of the Embryology training program. The Laboratory has
been most fortunate in the men it has enlisted as heads of its various training
programs.
REPORT OF THE DIRECTOR
4. Naming of Buildings
At its midwinter meeting the Board of Trustees named its two main laboratories
in honor of two former Directors of the Laboratory. The main laboratory will
be known as the Lillie Building in honor of Frank R. Lillie, Director from 1908
to 1926 and President of the Corporation from 1925 to 1942. Dr. Lillie is in a
large measure responsible for the modern development of the Laboratory. The
new laboratory building will be the Whitman Building in honor of Charles O.
Whitman, Director from 1888 to 1908, who guided the destinies of the Laboratory
through a very critical period of its development. Suitable plaques will be placed
on these buildings recording this action of the Board of Trustees.
5. Grants, Contracts and Contributions in Support of Laboratory Activities, in-
cluding Training Grants
The total income from these services of support amounted to $302,716 in
1961. This represents 31% of the $988,172 total income and is made up of support
from the following :
Training grants from NIH and NSF, support for regular research activities
from NIH, NSF, AEC and ONR and gifts from the MBL Associates, Josephine C.
Crane Foundation, The Rockefeller Foundation, and the following pharmaceutical
companies: The Merck Co. Foundation, C.I.B.A. Pharmaceutical Products, Inc.,
Abbott Laboratories, Schering Foundation Inc., Eli Lilly and Company, The
Upjohn Company, Wallace Laboratories and the Olin Mathieson Chemical Cor-
poration Charitable Trust.
6. Deaths
During the course of the year the Laboratory lost two of its very eminent
members through death, Dr. Otto Loewi and Dr. G. Failla. Both of these scientists
conferred distinction on the Laboratory through their membership in the Corpora-
tion and their scientific activities through many summers of research activity at
the Laboratory.
Also we must note the passing of Mr. Alton J. Pierce of the technical staff
who was highly regarded for his kind good nature, cooperativeness and technical
skill.
Respectfully submitted,
PHILIP B. ARMSTRONG
Director
ZOOLOGY
I. CONSULTANTS
F. A. BROWN, JR., Professor of Zoology, Northwestern University
LIBBIE H. HYMAN, American Museum of Natural History
ALFRED C. REDFIELD, Woods Hole Oceanographic Institution
MARINE BIOLOGICAL LABORATORY
II. INSTRUCTORS
CLARK P. READ, Professor of Biology, Rice University, in charge of the course
BERNARD L. STREHLER, Chief, Cellular and Comparative Physiology, Division of Geron-
tology, National Institutes of Health
RICHARD C. SANBORN, Professor of Zoology, Purdue University
JAMES CASE, Associate Professor of Zoology, State University of Iowa
EARL SEGAL, Assistant Professor of Biology, Rice University
CHARLES E. JENNER, Professor of Zoology, University of North Carolina
W. D. RUSSELL HUNTER, Department of Zoology, University of Glasgow, Scotland, U. K.
III. ASSISTANTS
DAVID C. GRANT, Yale University
STEPHEN SMITH, Wesleyan University
EMBRYOLOGY
I. INSTRUCTORS
NELSON T. SPRATT, JR., Professor of Zoology, University of Minnesota, in charge of the
course
PHILIP GRANT, Assistant Professor of Pathobiology, The Johns Hopkins University
JOHN W. SAUNDERS, JR., Professor of Zoology, Marquette University
TORE HULTIN, Wenner-Grens Institute, Stockholm, Sweden
AARON MOSCONA, Professor of Zoology, University of Chicago
LAURENS RUBEN, Assistant Professor of Biology, Reed College
II. LABORATORY ASSISTANTS
JOHN ARNOLD, Oberlin College
RICHARD WHITTAKER, Yale University
PHYSIOLOGY
I. CONSULTANTS
MERKEL H. JACOBS, Professor of Physiology, University of Pennsylvania
OTTO LOEWI, Professor of Pharmacology, New York University School of Medicine
ARTHUR K. PARPART, Professor of Biology, Princeton University
ALBERT SZENT-GYORGYI, Director, Institute for Muscle Research, Marine Biological
Laboratory
II. INSTRUCTORS
W. D. McELROY, Director, McCollum-Pratt Institute, Johns Hopkins University; in
charge of the course
PHILIP E. HARTMAN, Associate Professor of Biology, Johns Hopkins University
TIMOTHY H. GOLDSMITH, Yale University
HOWARD K. SCHACHMAN, Virus Laboratory, University of California, Berkeley
ROBERT B. LOFTFIELD, Massachusetts General Hospital
ANDRE JAOENDORF. McCollum-Pratt Institute, Johns Hopkins University
J. WOODLAND HASTINGS, Assistant Professor of Biochemistry, University of Illinois
REPORT OF THE DIRECTOR 9
III. LABORATORY ASSISTANT
Luis OTERO, University of Puerto Rico, Rio Piedras
BOTANY
I. CONSULTANT
WILLIAM RANDOLPH TAYLOR, Professor of Botany, University of Michigan
II. INSTRUCTORS
RICHARD C. STARR, Professor of Botany, Indiana University, in charge of the course
WALTER R. HERNDON, Associate Professor of Botany, University of Alabama
JOHN M. KINGSBURY, Associate Professor of Botany, Cornell University
TYGE CHRISTIENSEN, Institut for Sporeplanter, University of Copenhagen
III. LABORATORY ASSISTANTS
AUSTIN BROOKS, Department of Biology, Wabash College
WAYNE NICHOLS, Department of Biology, University of Alabama
ECOLOGY
I. CONSULTANTS
PAUL GALTSOFF, U. S. Fish and Wildlife Service, Woods Hole
ALFRED C. REDFIELD, Woods Hole Oceanographic Institution
BOSTWICK H. KETCHUM, Woods Hole Oceanographic Institution
EDWIN T. MOUL, Rutgers University
CHARLES E. JENNER, University of North Carolina
HOWARD T. ODUM, University of Texas
II. INSTRUCTORS
EUGENE P. ODUM, Alumni Foundation Professor of Zoology, University of Georgia, in
charge of the course
JOHN H. RYTHER, Marine Biologist, Woods Hole Oceanographic Institution
HOWARD L. SANDERS, Woods Hole Oceanographic Institution
WALTER R. TAYLOR, Chesapeake Bay Institute, Johns Hopkins University
III. LABORATORY ASSISTANTS
ELIJAH V. SWIFT, Johns Hopkins University
WILLIAM S. BROUGHTON, University of Georgia
1. THE LABORATORY STAFF
MRS. DEBORAH LAWRENCE HARLOW, Librarian ROBERT KAHLER, Superintendent,
CARL O. SCHWEIDENBACK, Manager, Supply Buildings and Grounds
Department ROBERT B. MILLS, Manager, DC-
IRVINE L. BROADBENT, Office Manager partment of Research Service
10 MARINE BIOLOGICAL LABORATORY
GENERAL OFFICE
MRS. LILA S. MYERS MRS. MARION C. CHASE
MRS. VIVIEN R. BROWN MRS. VIVIAN I. MANSON
MRS. VIRGINIA M. MOREHOUSE MRS. RUTH MAYO
LIBRARY
NOREEN RICHMOND TEENA B. CHASE
DORIS RICKER ALBERT K. NEAL
MAINTENANCE OF BUILDINGS AND GROUNDS
ROBERT ADAMS ELIZABETH KUIL
ELDON P. ALLEN DONALD B. LEHY
FRANCIS CAVANAUGH RALPH H. LEWIS
MANUEL P. DUTRA RUSSELL F. LEWIS
STANLEY ELDREDGE ALAN G. LUNN
GARDNER F. GAYTON ELLEN T. NICKELSON
ROBERT GUNNING JAMES S. THAYER
WALTER J. JASKUN ROBERT H. WALKER, JR.
DEPARTMENT OF RESEARCH SERVICE
GAIL M. CAVANAUGH LOWELL V. MARTIN
CAROLINE McDANiEL FRANK E. SYLVIA
SEAVER R. HARLOW
SUPPLY DEPARTMENT
DONALD P. BURNHAM BRUNO F. TRAPASSO
ARNOLD BOWDEN JOHN J. VALOIS
ROBERT O. LEHY JARED L. VINCENT
ROBERT M. PERRY HALLETT S. WAGSTAFF
MRS. PATRICIA TAVARES
2. INVESTIGATORS; LALOR AND LILLIE FELLOWS; AND STUDENTS
Independent Investigators, 1961
ADAMS, RALPH G., Physicist, National Institutes of Health
ADELBERG, EDWARD A., Professor of Bacteriology, University of California
ADELMAN, WILLIAM J., JR., Physiologist, National Institutes of Health
ALJURE, EMILIO, Universidad del Valle, Cali, Colombia
ALLEN, M. JEAN, Professor of Biology, Wilson College
ARMSTRONG, PHILIP B., Professor of Anatomy, State University of New York College of
Medicine, at Syracuse
ATWOOD, KIMBALL C., Professor of Microbiology, University of Illinois
AUSTIN, C. R., Member of Research Staff, National Institute for Medical Research, London
BAIRD, SPENCER L., Associate, Institute for Muscle Research
BARTH, L. G., Professor of Zoology, Columbia University
BAYLOR, MARTHA B., Marine Biological Laboratory
BENNETT, MICHAEL V. L., Assistant Professor of Neurology, College of Physicians and
Surgeons
BERNSTEIN, MAURICE H., Assistant Professor of Anatomy, Wayne State University
BEUKERS, ROBERT, Staff Member, Technological University, Delft, Netherlands
REPORT OF THE DIRECTOR 11
BINSTOCK, LEONARD, Electronic Engineer, National Institutes of Health
BLAKE, JOHN M., Chief Biologist, Aquacultural Research Corporation
BLUM, JOHN L., Professor of Biology, Canisius College
BOSLER, ROBERT, Dept. of Pharmacology, Harvard Medical School
BROWN, FRANK A., JR., Morrison Professor of Biology, Northwestern University
BRYANT, S. H., Assistant Professor of Pharmacology, University of Cincinnati, College oi
Medicine
BURKE, JOSEPH A., Assistant Professor of Biology, Loyola College
CAMPBELL, JAMES W., Instructor in Biology, Rice University
CARLSON, FRANCIS D., Professor of Biophysics, Johns Hopkins University
CASE, JAMES, Associate Professor of Zoology, University of Iowa
CHAET, ALFRED B., Associate Professor of Biology, American University
CHANDLER, WILLIAM K., Medical Officer, National Institutes of Health
CHENEY, RALPH HOLT, Professor of Biology, Brooklyn College
CHILD, FRANK M., Assistant Professor of Zoology, University of Chicago
CHRISTENSEN, A. KENT, Instructor in Anatomy, Harvard Medical School
CHRISTENSEN, TYGE, University of Copenhagen, Institute of Thallophyta
CLAFF, C. LLOYD, Research Associate in Surgery, Harvard Medical School
CLARK, ARNOLD M., Professor of Biological Sciences, University of Delaware
COLE, KENNETH S., Chief, Laboratory of Biophysics, National Institutes of Health
COLLIER, JACK R., Marine Biological Laboratory
COLWIN, ARTHUR L., Professor of Biology, Queens College
COLWIN, LAURA HUNTER, Queens College
COOPERSTEIN, SHERWIN J., Associate Professor of Anatomy, Western Reserve University
COPELAND, EUGENE, Professor of Zoology, Tulane University
COSTELLO, DONALD PAUL, Kenan Professor of Zoology, University of North Carolina
CRANE, ROBERT K., Associate Professor of Biological Chemistry, Washington University
Medical School
CROWELL, SEARS, Associate Professor of Zoology, Indiana University
CSAPO, ARPAD I., Associate Professor, Rockefeller Institute
DALTON, JOHN C., Assistant Professor of Biology, University of Buffalo
DETTBARN, WOLF-DIETRICH, Assistant Professor of Neurology, College of Physicians and
Surgeons
DIAMOND, JACK, Harvard Medical School
EDDS, MAC V., JR., Professor of Biology, Brown University
FAILLA, G., Senior Physicist Emeritus, Argonne National Laboratory
FERGUSON, JAMES J., JR., Assistant Professor, University of Pennsylvania
FISCHER, SIEGMUND, Associate in Research, Albert Einstein College of Medicine
FITZPATRICK, THOMAS B., Professor of Dermatology, Massachusetts General Hospital
FRAZIER, HOWARD S., Massachusetts General Hospital
FURSHPAN, EDWIN J., Associate in Neurophysiology, Harvard Medical School
GAINER, HAROLD, Research Associate, College of Physicians and Surgeons
GILMAN, LAUREN C., Associate Professor of Zoology, University of Miami
GIRARDIER, LUCIEN, Research Associate, College of Physicians and Surgeons
GLADE, RICHARD W., Assistant Professor of Zoology, University of Vermont
GOLDRING, IRENE P., Assistant Professor of Research Surgery, Albert Einstein College of
Medicine
GOLDSMITH, TIMOTHY H., Yale University
GRANT, PHILIP, Assistant Professor of Pathobiology, Johns Hopkins University School of
Hygiene
GREIF, ROGER L., Associate Professor of Physiology, Cornell University Medical College
GROSCH, DANIEL S., Professor of Genetics, North Carolina State College
GROSS, PAUL R., Associate Professor of Biology, New York University
GRUNDFEST, HARRY, Professor of Neurology, College of Physicians and Surgeons
GUTTMAN, RITA, Associate Professor of Biology, Brooklyn College
GWILLIAM, GILBERT F., Assistant Professor of Biology, Reed College
HAGINS, WILLIAM A., Physiologist, National Institutes of Health
HAGIWARA, SUSUMU, Professor of Zoology, University of California
12 MARINE BIOLOGICAL LABORATORY
HARDING, CLIFFORD V., Assistant Professor of Physiology, College of Physicians and Surgeons
HARTMAN, PHILIP E., Associate Professor of Biology, Johns Hopkins University
HARVEY, ETHEL BROWNE, Marine Biological Laboratory
HASTINGS, J. WOODLAND, Assistant Professor of Biochemistry, University of Illinois
HATHAWAY, RALPH R., Oceanographic Institute, Florida State University
HAYASHI, TERU, Professor of Zoology, Columbia University
HEGYELI, ANDREW, Institute for Muscle Research, Marine Biological Laboratory
HENLEY, CATHERINE, Research Associate, University of North Carolina
HERNDON, WALTER R., Associate Professor of Biology, University of Alabama
HERVEY, JOHN P., Senior Electronic Engineer, Rockefeller Institute
HERZOG, WALTER, Post Doctoral Fellow in Pharmacology, University of Cincinnati College of
Medicine
HIGMAN, HENRY B., College of Physicians and Surgeons
HIRSHFIELD, HENRY I., Associate Professor of Biology, Washington Square College
HOSKIN, FRANCIS C. G., Assistant Professor of Neurology, College of Physicians and Surgeons
HULTIN, TORE, Wenner-Grens Institute, University of Stockholm
HUNTER, W. D. RUSSELL, Lecturer in Zoology, University of Glasgow, Scotland
HURWITZ, JERARD, Associate Professor of Microbiology, New York University School of
Medicine.
ISENBERG, IRVIN, Institute for Muscle Research, Marine Biological Laboratory
JAGENDORF, ANDRE, Associate Professor, Johns Hopkins University
JANISZEWSKI, LESZEK, Master of Biology, N. Copernicus University, Poland
JENNER, CHARLES E., Professor of Zoology, University of North Carolina
KAMINER, BENJAMIN, Institute for Muscle Research, Marine Biological Laboratory
KANE, ROBERT E., Assistant Professor of Cytology, Dartmouth Medical School
KATZ, GEORGE M., Research Associate, Columbia University
KEMPTON, RUDOLF T., Professor of Zoology, Vassar College
KEOSIAN, JOHN, Professor of Biology, Rutgers, The State University
KINGSBURY, JOHN M., Associate Professor of Botany, Cornell University
KISHIMOTO, UICHIRO, National Institutes of Health
KLEINHOLZ, LEWIS H., Professor of Biology, Reed College
KRANE, STEPHEN M., Associate in Medicine, Harvard Medical School
KUFFLER, STEPHEN W., Professor of Neurophysiology, Harvard Medical School
LANDAU, JOSEPH V., Chief, Oncology Section, VA Hospital, Albany, New York
LANSING, ALBERT I., Professor of Anatomy, University of Pittsburgh School of Medicine
LAZAROW, ARNOLD, Professor of Anatomy, University of Minnesota
LEAF, ALEXANDER, Associate Professor of Medicine, Harvard Medical School
LERMAN, SIDNEY, Assistant Professor of Ophthalmology, University of Rochester
LEVY, MILTON, Professor of Biochemistry, New York University College of Dentistry
LOCH HEAD, JOHN H., Professor of Zoology, University of Vermont
LOEWENSTEIN, WERNER R., Associate Professor of Physiology, College of Physicians and
Surgeons
LOFTFIELD, ROBERT B., Associate Biochemist, Massachusetts General Hospital
LONDON, IRVING M., Professor of Medicine, Albert Einstein College of Medicine
LORAND, L., Associate Professor of Chemistry, Northwestern University
DE LORENZO, A. J., Director Anatomical and Pathological Laboratories, Johns Hopkins Uni-
versity
LOVE, -WARNER E., Assistant Professor of Biophysics, Johns Hopkins University
MAHLER, H. R., Professor of Chemistry, Indiana University
MARSLAND, DOUGLAS, Professor of Biology, Washington Square College
MATEYKO, G. M., Assistant Professor of Biology, Washington Square College
MCELROY, WILLIAM D., Director, McCollum-Pratt Institute, Johns Hopkins University
METZ, CHARLES B., Professor of Zoology, Florida State University
METZ, CHARLES W., Emeritus Professor of Zoology, University of Pennsylvania
MIDDLEBROOK, WILLIAM R., Institute for Muscle Research, Marine Biological Laboratory
MILKMAN, ROGER D., Associate Professor of Zoology, Syracuse University
MILLER, FAITH S., Assistant Professor of Anatomy, Tulane University
MILLER, JAMES A., Professor of Anatomy, Tulane University
REPORT OF THE DIRECTOR 13
MOORE, RICHARD O., Professor, Ohio State University
MORRILL, JOHN B., Assistant Professor, Wesleyan University
MOSCONA, A. A., Professor of Zoology, University of Chicago
MULLINS, LORIN J., Professor of Biophysics, University of Maryland
MUSACCHIA, X. J., Associate Professor of Biology, Saint Louis University
NACE, PAUL FOLEY, Professor of Biology, McMaster University
NAGI, TOSHIO, Research Associate, University of Illinois
NELSON, LEONARD, Associate Professor of Physiology, Emory University
ODUM, EUGENE P., Professor of Zoology, University of Georgia
PALINCSAR, EDWARD E., Assistant Professor of Biology, Loyola University
PARKER, JOHNSON, Assistant Professor of Plant Physiology, Yale University
PARPART, ARTHUR K., Professor of Biology, Princeton University
PENMAN, SHELDON, Assistant Professor of Physics, College of Physicians and Surgeons
PERSON, PHILIP, Chief, VA Hospital, Brooklyn
PETRIE, ASENATH, Research Associate, Harvard University and London University
POTTER, DAVID, Associate in Neurophysiology, Harvard Medical School
PROSSER, C. LADD, Professor of Physiology, University of Illinois
RAPPORT, MAURICE M., Professor of Biochemistry, Albert Einstein College of Medicine
READ, CLARK P., Professor of Biology, Rice University
REBHUN, LIONEL I., Assistant Professor of Biology, Princeton University
REUBEN, JOHN P., Research Associate, College of Physicians and Surgeons
RIESER, PETER, Head, Department of Biology, St. John Fisher College
ROCKSTEIN, MORRIS, Associate Professor of Physiology, New York University College of
Medicine
ROSE, S. MERYL, Professor of Biology, Wesleyan University
ROSENBERG, EVELYN K., Associate Professor, New York University
ROSENBERG, PHILIP, Columbia University, College of Physicians and Surgeons
ROTH STEIN, HOWARD, College of Physicians and Surgeons
ROWLAND, LEWIS P., Assistant Professor of Neurology, College of Physicians and Surgeons
RUBEN, LAUREN s, Assistant Professor of Biology, Reed College
RUCK, PHILIP R., Assistant Professor of Biology, Tufts University
RUGH, ROBERTS, Associate Professor of Radiology, College of Physicians and Surgeons
RUSTAD, RONALD C., Assistant Professor of Physiology, Florida State University
SANBORN, RICHARD C., Professor of Zoology, Purdue University
SANDERS, HOWARD L., Woods Hole Oceanographic Institution
SAUNDERS, JOHN W., Professor of Biology, Marquette University
SCHACHMAN, HOWARD K., Professor of Biochemistry, University of California
SCHARRER, ERNST, Professor of Anatomy, Albert Einstein College of Medicine
SCHOFFENIELS, ERNEST, Assistant Professor of Neurology, College of Physicians and Surgeons
SCOTT, ALLAN, Professor of Biology, Colby College
SCOTT, SISTER FLORENCE MARIE, Professor of Biology, Seton Hill College
SCOTT, GEORGE T., Chairman, Department of Biology, Oberlin College
SEGAL, EARL, Assistant Professor of Biology, Rice University
SELIGER, HOWARD H., Research Associate, McCollum-Pratt Institute, Johns Hopkins University
SENFT, ALFRED W., Marine Biological Laboratory
SENGEL, PHILIPPE, College de France
SIMMONS, JOHN E., Rice University
SJODIN, RAYMOND A., Associate Professor of Biophysics, University of Maryland
SLIFER, ELEANOR H., Professor of Zoology, State University of Iowa
SMELSER, GEORGE K., Professor of Anatomy, Columbia University
SONNENBLICK, BENJAMIN, Professor of Biology, Rutgers, The State University
SPECTOR, ABRAHAM, Instructor, Howe Laboratory, Harvard Medical School
SPEIDEL, CARL C., Professor of Anatomy, University of Virginia
SPINDEL, WILLIAM, Associate Professor of Chemistry, Rutgers University
SPRATT, NELSON T., Chairman, Department of Zoology, University of Minnesota
SPYROPOULOS, CONSTANTINE S., Neurophysiologist, National Institutes of Health
STARR, RICHARD C., Professor of Botany, Indiana University
STEFFENSEN, DALE M., Associate Professor of Botany, University of Illinois
14 MARINE BIOLOGICAL LABORATORY
STEINBACH, H. BURR, Professor of Zoology, University of Chicago
STEINHARDT, JACINTO, Director, Operations Evaluation Group, Massachusetts Institute of
Technology
STETTEN, DEWirr, JR., Associate Director, NIAMD, National Institutes of Health
STONE, WILLIAM, JR., Director, Ophthalmic Plastics Laboratory
STREHLER, BERNARD L., Chief, Cellular and Comparative Physiology Section, National Institutes
of Health
STRITTMATTER, PHILIPP, Assistant Professor of Biochemistry, Washington University
SUSSMAN, MAURICE, Professor of Biology, Brandeis University
SZABO, GEORGE, Associate in Anatomy, Harvard Medical School
SZENT-GYORGYI, ALBERT, Director, Institute for Muscle Research, Marine Biological Laboratory
SZENT-GYORGYI, ANDREW, Head of Muscle Section, Institute for Muscle Research, Marine
Biological Laboratory
TASAKI, ICHIJI, Chief, Special Senses Section, National Institutes of Health
TAYLOR, ROBERT E., Physiologist, National Institutes of Health
TAYLOR, WILLIAM RANDOLPH, Professor of Botany, University of Michigan
TAYLOR, W. ROWLAND, Assistant Professor of Oceanography, Johns Hopkins University
TEORELL, TORSTEN, Professor, University of Uppsala
DE TERRA, NOEL, Research Associate, Rockefeller Institute
TORCH, REUBEN, Associate Professor of Zoology, University of Vermont
TRAMS, EBERHARD G., National Institutes of Health
TRAVIS, DAVID MONROE, Assistant Professor of Pharmacology, University of Florida
TROLL, WALTER, Associate Professor, New York University Aledical Center
TUNIK, BERNARD D., Associate Professor, State University of New York, Long Island
TWEEDELL, KENYON S., Assistant Professor of Biology, University of Notre Dame
DEVILLAFRANCA, GEORGE W., Associate Professor of Zoology, Smith College
VILLEE, CLAUDE A., Associate Professor of Biological Chemistry, Harvard University
VINCENT, WALTER S., Assistant Professor of Anatomy, Upstate Medical Center, State Uni-
versity of New York
WALD, GEORGE, Professor of Biology, Harvard University
WARREN, LEONARD, National Institutes of Health
WEBB, H. MARGUERITE, Associate Professor of Biological Sciences, Goucher College
WHITING, ANNA R., University of Pennsylvania
WICHTERMAN, RALPH, Professor of Biology, Temple University
WIERCINSKI, FLOYD J., Associate Professor of Biological Sciences, Drexel Institute of Tech-
nology
WILBER, CHARLES G., Dean of the Graduate School, Kent State University
WILSON, WALTER L., Associate Professor of Physiology, University of Vermont
WITTENBERG, JONATHAN B., Associate Professor of Physiology, Albert Einstein College of
Medicine
WOLFF, ETIENNE, Professor au College de France
WOLSKY, ALEXANDER, Professor of Experimental Embryology, Fordham University
WRIGHT, PAUL A., Associate Professor of Zoology, University of New Hampshire
WURTZ, ROBERT, University of Michigan
WYTTENBACH, CHARLES R., Instructor in Anatomy, University of Chicago
ZIGMAN, SEYMOUR, Research Associate, Ophthalmic Plastics Laboratory
ZIMMERMAN, ARTHUR M., Assistant Professor, State University of New York, Downstate
Medical Center
ZWILLING, EDGAR, Professor of Biology, Brandeis University
Lalor Fellows, 1961
WOLFF, ETIENNE, College de France
CHRISTENSEN, A. KENT, Harvard Medical School
HATHAWAY, RALPH, Florida State University
SENGEL, PHILIPPE, College de France
SENGEL, M. LOUISE, College de France
WARREN, LEONARD, National Institutes of Health
REPORT OF THE DIRECTOR 15
Lillie Fellow, 1961
AUSTIN, C. R., National Institute for Medical Research, London
Grass Fellows, 1961
GRAFSTEIN, BERNICE, McGill University
HALL, ZACH, Emory University
HERZOG, WALTER, University of Cincinnati College of Medicine
WURTZ, ROBERT, University of Michigan
Beginning Investigators, 1961
ADYE, JAMES C., Johns Hopkins University
BAUER, G. ERIC, University of Minnesota
BEAMES, CALVIN G., JR., Rice University
BITO, LASZLO, Columbia University
BRINLEY, F. J., JR., Johns Hopkins Medical School
BROWN, JOEL E., Massachusetts Institute of Technology
CHERVIN, PAUL N., University of Vermont
CHRISTIANSEN, JANICE H., Temple University School of Medicine
DOOLITTLE, RUSSELL F., Harvard University Medical School
DUNHAM, PHILIP B., University of Chicago
ECKERT, ROGER O., Harvard University
EISENBERG, ROBERT S., Harvard College
FIELD, JAMES B., National Institutes of Health
Fox, STEPHEN S., University of Michigan
GASSELING, MARY T., Marquette University
HUMPHREYS, TOM DANIEL, University of Chicago
ISHIKO, NOBUSADA, Kumamoto University Medical School
JACKSON, JAMES A., Western Reserve University
KITAZUME, YOSHIJI, University of Kobe, Japan
LEIBERMAN, PAUL M., University of Vermont
MOORE, RICHARD O., Ohio State University
MORAN, JOSEPH F., Russell Sage College
SCHLESINGER, ROBERT M., National Institutes of Health
SCHUEL, HERBERT, Florida State University
SHEPHARD, DAVID C., University of Chicago
SLAYMAN, CLIFFORD L., Rockefeller Institute
SRINIVASAN, DOBLI, Columbia University
STONE, AUDREY L., National Institutes of Health
WHEELER, MAYNARD B., Columbia University
Research Assistants, 1961
ALEXANDER, DOUGLAS GORDON, University of North Carolina
ALLAWAY, ELIZABETH, Mount Holyoke College
ARNOLD, JOHN M., Oberlin College
ATLAS, MERYL, State University of New York, Upstate Medical Center
BARNWELL, FRANKLIN H., Northwestern University
BART, KENNETH, State University of New York, Upstate Medical Center
BENNETT, JOHN E., Purdue University
BENZINGER, ROLF H., Johns Hopkins University
BOLEYN, BRENDA J., Harvard Medical School
BOOKER, VIRGINIA TOD, Smith College
BOWERS, WILLIAM E., College of Physicians and Surgeons
BRADBURY, JACK W., Reed College
BREWER, JOHN M., Johns Hopkins University
16 MARINE BIOLOGICAL LABORATORY
BROOKS, AUSTIN, Wabash College
BROUGHTON, WILLIAM S., University of Georgia
BYRNE, SYLVIA A., Syracuse University
CARLIN, IRA S., Plymouth, Mass.
CECCARIM, COSTANTE, St. Peter's College
CICAK, ANNA, Albert Einstein College of Medicine
CLARK, ELOISE E., Columbia University
COHN, DUNELL E., Oak Ridge, Tennessee
CORABI, MARY, Brooklyn, New York
CORNELL, KENNETH, Institute for Muscle Research, Marine Biological Laboratory
Cox, ELEANOR, University of Texas
CROSBY, GAYLE M., Brandeis University
CROWE, PRISCILLA ANN, Seton Hill College
DE LA CRUZ, ARMANDO, University of the Philippines
DEAIuTH, SIMON H., College of Physicians and Surgeons
DEWEL, WILLIAM C., Wesleyan University
DOUGHERTY, WILLIAM J., Princeton University
EDWARDS, JACKIE, University of Alabama
EIGNER, ELIZABETH ANN, Massachusetts General Hospital
EMMONS, LOUISE H., North Carolina State College
ERSKINE, MRS. LOUISE, Institute for Muscle Research, Marine Biological Laboratory
EVANS, THOMAS EDWARD, Florida State University
FEIBEL, ROBERT MARKS, University of Cincinnati Medical College
FISHER, SYLVIA SUE, Saint Louis University
FLATHERS, ANN R., University of New Hampshire
FORAN, ELIZABETH, Smith College
FRANCISCO, ANNE S., Agnes Scott College
FRIEDMAN, SUE, Smith College
VAN GELDER, Nico M., Harvard Medical School
GINSBERG, ALLEN, Brooklyn Veterans Administration Hospital
GINSBERG, MYRON D., Wesleyan University
GRABSKE, ROBERT JEROLD, University of Kansas
GRANT, DAVID C., Yale University
GRANT, ROBERT J., Columbia University
GRIGGS, ROBERT C., University of Delaware
GRIMES, MARIAN, Western Reserve University
GURRY, SUSAN M., Smith College
GUTKNECHT, JOHN, University of North Carolina
HABAS, LINDA B., University of Illinois
HALL, PRUDENCE J., Princeton University
HARDY, DONNA JOY, Johns Hopkins University
HAYWARD, GEORGE EDWARD, Washington University
HECK, HENRY D'ARCY, Princeton University
HINSHAW, CAROLYN Jo, Indiana University
HIRSCH, REGINA, New York State University, Downstate Medical Center
HUFNAGEL, LINDA A., University of Vermont
JACKSON, THOMAS JOHN, Jefferson Medical College
KARSTADT, MYRON, Queens College
KAYSER, ELEONORE, McCollum-Pratt Institute, Johns Hopkins LTniversity
KENT, BARBARA, New York City
KIELICH, J. C., Massachusetts Eye and Ear Infirmary
KIMBALL, FRANCES, Reed College
KITZMAN, WILLIAM B., Rice University
KUSANO, KIYOSHI, Tokyo Medical-Dental University
LAMBORG, MARVIN R., Hunting Laboratories, Massachusetts General Hospital
LANG, NORMA J., Indiana University
LAUFENBERG, HENRY J., JR., Hahnemann Medical College
LEE, THOMAS W., Rice University
REPORT OF THE DIRECTOR 17
LEITNOR, LEO G., National Institutes of Health
LENOX, MARILYN, Muskingum College
LEVIN, STEPHEN, Wesleyan University
LIEBERMAN, IRVING, University of Pittsburgh School of Medicine
LILLIBRIDGE, JACQUELINE S., State University of New York, Downstate Medical Center
LINDALL, ARNOLD, JR., University of Minnesota
MALINOU, SHELDON H., New York University College of Dentistry
MARBURG, KENNETH, Johns Hopkins University
MAREN, PETER H., University of Florida College of Medicine
MCCARTHY, JOAN, Colby College
MCDONALD, KAY, Rice University
MCLAUGHLIN, JANE A., Institute for Muscle Research, Marine Biological Laboratory
MILLER, RICHARD L., University of Chicago
MORRISSEY, JAMES D., Upstate Medical Center, State University of New York
NAUMANN, CHRISTINE, Smith College
NEWTON, SANDRA, Rice University
NICHOLS, HERBERT W., University of Alabama
NOVAK, ROBERT L., University of Delaware
NYSTROM, RICHARD A., University of Illinois
OLIVO, RICHARD F., Columbia University
ORLANDO, JOSEPH C., Loyola College
OTERO-VILARDEBO, Luis R., University of Puerto Rico
PAX, RALPH A., Purdue University
PERRY, BARBARA, Institute for Muscle Research, Marine Biological Laboratory
PHILPOTT, CHARLES W., Tulane University
PHILPOTT, LORALEE L., Tulane University
PHILPOTT, DELBERT, Institute for Muscle Research, Marine Biological Laboratory
PODLESKI, THOMAS, College of Physicians and Surgeons
POWERS, JOSEPH A., Wesleyan University
PURPLE, RICHARD L., Rockefeller Institute
RANDALL, VIRGINIA D., Jackson College
RAY, FRANCES, New York University
RODGERS, PATRICIA, State University of New York, Downstate Medical Center
ROSE, JEANNETTE, Vassar College
ROSE, ROBERT, American University
ROSENBLUTH, RAJA, Columbia University
RUNK, RUTH C., Institute for Muscle Research, Marine Biological Laboratory
SANDLIN, RONALD A., National Institutes of Health
SCHROEDER, THOMAS E., Northwestern University
SCHUR, MATTHEW A., Oberlin College
SCOTT, NANCY J., University of Vermont
SHEPPARD, DAVID ELSON, Johns Hopkins University
SHERIDAN, WILLIAM FRANCIS, University of Florida
SMITH, MARIE F., Johns Hopkins University
SMITH, STEPHEN D., Wesleyan University
SPENCER, JOYCE, Harvard Medical School
SPIEGEL, JEANETTE, National Institutes of Health
STAHL, MRS. RUTH C., Johns Hopkins University
STEVENS, JEAN M., Reed College
SWENEY, LAURA A., University of Minnesota
SWIFT, ELIJAH, Johns Hopkins University — Chesapeake Bay Institute
SZENT-GYORGYI, EVE, Institute for Muscle Research, Marine Biological Laboratory
SZENT-GYORGYI, GYULA, Institute for Muscle Research, Marine Biological Laboratory
SZENT-GYORGYI, MARTA, Institute for Muscle Research, Marine Biological Laboratory
WATKINS, DUDLEY T., Western Reserve Medical School
WATTERS, CHRISTOPHER, University of Notre Dame
WEINSTOCK, MICHAEL S., American University
WEIS, PEDDRICK, New York University College of Dentistry
18 MARINE BIOLOGICAL LABORATORY
WESTHOFF, D. DOUGLAS, Saint Louis University
WHITTAKER, J. RICHARD, Yale University
WILLIAMS, GEORGIA J., Wilson College
WOOD, BARRY F., Syracuse University
YANOSIK, HAROLD JON, National Institutes of Health
Library Readers, 1961
ADAMS, ELIJAH, Professor of Pharmacology, Saint Louis University School of Medicine
BALL, ERIC G., Professor of Biological Chemistry, Harvard Medical School
BODANSKY, OSCAR, Chief, Sloan-Kettering Institute for Cancer Research
BUCK, JOHN, Physiologist, National Institutes of Health
BURBANCK, W. D., Professor of Biology, Emory University
BUTLER, ELMER G., Osborn Professor of Biology, Princeton University
CHASE, AURIN M., Associate Professor of Biology, Princeton University
CLEMENT, A. C., Professor of Biology, Emory University
CLIFFORD, SISTER ADELE, Professor of Biology, College of Mount St. Joseph
COHEN, SEYMOUR S., Professor of Biochemistry, University of Pennsylvania
DAVIS, BERNARD D., Professor of Bacteriology, Harvard Medical School
EISEN, HERMAN N., Professor of Microbiology, Washington University
FLESCH, PETER, Associate Professor of Research Dermatology, University of Pennsylvania
FRIES, E. F. B., Professor, City College of New York
GABRIEL, MORDECAI L., Associate Professor of Biology, Brooklyn College
GINSBERG, HAROLD S., Chairman, Department of Microbiology, University of Pennsylvania
GUREWICH, VLADIMIR, Associate Physician, Cornell Division, Bellevue Hospital
HOBERMAN, HENRY D., Professor of Biochemistry, Albert Einstein College of Medicine
HOTCHIN, JOHN E., Assistant Director, New York State Department of Health
HURWITZ, CHARLES, VA Hospital, Albany
ISSELBACHER, KURT J., Assistant Professor of Medicine, Harvard Medical School
JACOBS, M. H., Emeritus Professor of General Physiology, University of Pennsylvania
KABOT, ELVIN A., Professor of Microbiology, College of Physicians and Surgeons
KARUSH, FRED, Professor of Microbiology, University of Pennsylvania School of Medicine
KLEIN, MORTON, Professor of Microbiology, Temple University
KLOTZ, IRVING M., Professor of Chemistry, Northwestern University
KNOBIL, ERNST, Assistant Professor of Physiology, Harvard University
LEVINE, RACHMIEL, Professor of Medicine, New York Medical College
LINEAWEAVER, THOMAS H., Woods Hole, Massachusetts
LOWENFELD, IRENE E., Research Assistant in Ophthalmology, College of Physicians and
Surgeons
LOWENSTEIN, OTTO, Research Associate in Ophthalmology, College of Physicians and Surgeons
MALKIEL, SAUL, Research Associate, Harvard Medical School
MCDONALD, SISTER ELIZABETH SETON, Professor of Biology, College of Mt. St. Joseph
MclNTiRE, F. C., Head of Biochemical Research, Abbott Laboratories
NOVIKOFF, ALEX B., Research Professor, Albert Einstein College of Medicine
PULLMAN, BERNARD, Institut de Biologic Physico-Chimique, Paris
REEVES, ROBERT BLAKE, Assistant Professor of Zoology, Cornell University
ROTH, JAY S., Professor of Biochemistry, University of Connecticut
SANDEEN, MURIEL I., Assistant Professor of Zoology, Duke University
SCHLAMOWITZ, MAX, Associate Cancer Research Scientist, Roswell Park Memorial Institute
SPIEGEL, MELVIN, Assistant Professor of Zoology, Dartmouth College
URETZ, ROBERT B., Assistant Professor of Biophysics, University of Chicago
WAINIO, WALTER W., Professor of Biochemistry, Rutgers, The State University
WHEELER, GEORGE E., Assistant Professor of Biology, Brooklyn College
WILSON, IRWIN B., Associate Professor of Biochemistry, College of Physicians and Surgeons
YNTEMA, CHESTER L., Professor of Anatomy, State University of New York, Upstate Medical
Center
ZORZOLI, ANITA, Associate Professor of Physiology, Vassar College
REPORT OF THE DIRECTOR 19
Students, 1961
All students listed completed formal course program, June 19 to July 29th. Asterisk
indicates students completing Post Course Research Program, July 30 to September 2nd.
ECOLOGY
*MARY A. ASHCRAFT, Wilson College
*ROBERT J. BARSDATE, University of Pittsburgh
CAROL A. BAUMANN, Chatham College
*L. LEHR BRISBIN, Wesleyan University
CHARLES H. BUTTERFIELD, Chevy Chase, Maryland
DAVID A. DOBBINS, University of Minnesota
*ROGER W. DOYLE, Dalhousie University
*DIRK FRANKENBERG, Emory University
*THOMAS A. GAUCHER, Narragansett Marine Laboratory
JOHN GUTKNECHT, University of North Carolina
*WILLIAM T. HALL, Fordham University
GEORGE HAYWARD, Washington University
CONRAD A. ISTOCK, University of Michigan
MARVIN P. KAHL, University of Georgia
*WALTER E. KNOX, Drew University
BARBARA MARCH, Marquette University
*ELBA L. MAS, Yale University
*FERMIN SAGARDIA, Rutgers University
*JUDITH A. SHULMAN, Cornell University
*SANDRA ELIZABETH WAGNER, Vassar College
*MARGARET J. WALDREP, University of Alabama
ROBERT G. WETZEL, University of California
BOTANY
MARILYN L. ALBERT, University of Texas
GEORGE C. CARROLL, Swarthmore College
*CHARLES F. CLELAND, Wabash College
ELEANOR Cox, University of Texas
* WILLIAM H. DARDEN, JR., University of Alabama
VICTOR EMANUEL, University of Texas
*ROBERT B. GORDON, University of California
*DANA G. GRIFFIN, III, Texas Technological College
*R. DON GROOVER, University of Alabama
*JEAN HATCH, Vassar College
RAYMOND W. HOLTON, University of Michigan
ROBERT HOSHAW, University of Arizona
*PATRICIA MANCO, Clark University
ROBERT McCLARY, Indiana University
JOHN C. MESSENGER, Yale University
*WILLIAM R. RAYBURN, Washington University
ALVIN REEVES, II, Indiana University
*MICHAEL J. WYNNE, Washington University
*JOANNE R. ZIEGLER, Cornell University
PHYSIOLOGY
*ERIC R. BISCHOFF, Washington University
RICHARD C. BLINKOFF, Rockefeller Institute
IAIN BOWMAN, National Institutes of Health
*JOHN W. BREWER, Johns Hopkins University
20 MARINE BIOLOGICAL LABORATORY
EDWARD A. BRUNNER, Hahnemann Medical College
*GRACIELA C. CANDELAS, University of Puerto Rico
EUGENIE J. DUBNAU, Columbia University
PAUL T. ENGLUND, Rockefeller Institute
HOWARD L. GILLARY, Oberlin College
*ALAN HOOPER, Oberlin College
*EowiN F. HUMPAL, JR., University of Minnesota
*ANNA E. KAMMER, University of California
ALEXANDER KEYNAN, Israel Institute for Biological Research
*LAWRENCE M. LICHTENSTEIN, Johns Hopkins University
Lu-Ku Li, Princeton University
*HARRY J. MERSMANN, St. Louis University
*WILLIAM M. MITCHELL, Johns Hopkins University
*THOMAS A. MURPHY, Yale University
*KENNETH W. PERRY, JR., Syracuse University
*JACOB LEE RAAB, University of Chicago
*ELIZABETH RITTENHOUSE, University of Michigan
HARRIETTE C. SCHAPIRO, University of Miami
JOSEPH P. SENFT, University of Buffalo
MARTHA R. SHEER, St. Louis University School of Medicine
HARRY W. TABER, University of Rochester
KENNETH S. WARREN, National Institutes of Health
CHARLES D. YEGIAN, University of California
C. RICHARD ZOBEL, Johns Hopkins University
EMBRYOLOGY
ALLAN L. ALLENSPACH, Iowa State University
ALVIN J. CLARK, Columbia University
REV. RICHARD T. CLEARY, Johns Hopkins University
* STILES D. EZELL, JR., Bryn Mawr College
*PAUL E. FELL, Stanford University
ELLEN FISHER, Mt. Holyoke College
*LINDA GARRICK, Goucher College
HENRY B. GARRISON, Yale University
LUIGI GIACOMETTI, Brown University
DONALD S. GORMAN, Harvard University
*BERNICE GRAFSTEIN, McGill University
*JEROME GROSS, Massachusetts General Hospital
*JOHN L. KELLAND, Princeton University
JANOS LANYI, Harvard University
HARRIS I. LEHRER, Brandeis University
MICAL E. MIDDAUGH, University of Minnesota
*FERNANDO L. RENAUD-RENAUD, University of Chicago
*GRETCHEN SCHABTACH, Carnegie Institution of Washington
*ROBERT L. SEARLS, Brandeis University
*SIDNEY B. SIMPSON, JR., Tulane University
*RICHARD G. SKALKO, University of Florida
INVERTEBRATE ZOOLOGY
EVELYN ALIFERIS, University of Massachusetts
*PETER B. ARMSTRONG, University of Rochester
*ROLAND H. BAGBY, University of Illinois
RUTH R. BENNETT, Tufts College
SHIRLEY BRODY, University of Rochester
*ALBER H. CASS, JR., Dartmouth College
REPORT OF THE DIRECTOR 21
RICHARD H. COLBY, Massachusetts Institute of Technology
ALBERT J. CORKILL, De Paul University
MICHAEL W. Dix, Harvard University
JACK D. DONAHUE, Columbia University
DONNA C. EMRICH, Wilson College
*HECTOR R. FERNANDEZ, University of Miami
*JAMES H. FUNSTON, Earlham College
RAY H. GAVIN, Howard University
*MARILYN GOLDSMITH, Brown University
*V. ANN HALE, McGill University
SISTER MARY A. HANDY, University of Notre Dame
HERMAN B. HARTMAN, American University
SISTER MARY C. HEROLD, Fordham University
Avis G. HULL, Drew University
ASTRID KODRIC, D'Youville College
OMER R. LARSON, University of Minnesota
ARTHUR C. LERNER, Lafayette College
*ELLEN M. LEVINE, Washington Square College
WINTER P. LUCKETT, University of Missouri
*JAMES S. McDANiEL, University of Oklahoma
JOYCE T. McKEE, New York University
ROBERT ALLEN MENZIES, University of Florida
ALICE T. O'MALLEY, Clark University
KAY EILEEN SAEGER, University of Illinois
HOWARD A. SCALZI, Washington and Jefferson College
*JACK L. SCHWADE, Rice University
DOROTHY S. SEARLE, Oberlin College
PHILIP J. SKEHAN, JR., Syracuse University
*HELEN STOUT, Radcliffe College
CLARENCE E. STYRON, JR., Davidson College
WILLIAM H. TALBOT, Rockefeller Institute
MARGARET W. TRYON, Wheaton College
MARTHA E. WELSH, Oberlin College
ROBERT S. WILCOX, University of Oklahoma
3. FELLOWSHIPS AND SCHOLARSHIPS, 1961
Lucretia Crocker Scholarship :
ELEANOR Cox, Botany Course
ROBERT G. WETZEL, Ecology Course
The Merkel H. Jacobs Scholarship:
EDWIN F. HUMPAL, JR., Physiology Course
WILLIAM MITCHELL, Physiology Course
The Edwin Grant Conklin Memorial Scholarship:
SIDNEY B. SIMPSON, JR., Embryology Course
The Emma Coote Drew Memorial Scholarship:
TOM MURPHY, Physiology Course
4. TABULAR VIEW OF ATTENDANCE 1957-1961
1957 1958 1959 1960 1961
INVESTIGATORS— TOTAL 326 410 427 458 458
Independent 186 203 215 231 224
Under Instruction 23 39 45 42 32
Library Readers 42 54 51 50 49
Research Assistants 75 114 116 135 151
22
A1ARINE BIOLOGICAL LABORATORY
STUDENTS — TOTAL 139
Invertebrate Zoology 55
Embryology 27
Physiology 30
Botany 18
Ecology 9
TOTAL ATTENDANCE 465
Less persons represented as both
investigator and student 3
INSTITUTIONS REPRESENTED — TOTAL 129
By investigators 94
By students 35
SCHOOLS AND ACADEMIES REPRESENTED
By investigators 1
By students 5
FOREIGN INSTITUTIONS REPRESENTED 16
By investigators 11
By students 5
138
55
22
27
18
16
548
142
110
74
26
20
6
134
49
23
27
20
15
561
143
98
73
2
12
38
29
9
122
43
20
28
18
13
580
144
83
61
5
2
14
11
3
130
40
21
28
19
22
586
1
132
107
70
28
21
7
5. INSTITUTIONS REPRESENTED, 1961
University of Alabama
Albert Einstein College of Medicine
American University
Argonne National Laboratory
Arizona University
Brandeis University
Brooklyn College
Brown University
University of Buffalo
University of California
Canisius College
Carnegie Institution of Washington
Chatham College
University of Chicago
University of Cincinnati
City College of New York
Clark University
Colby College
College of Physicians and Surgeons
Columbia University
University of Connecticut
Cornell University
Cornell University Medical School
Dartmouth College
Dartmouth Medical School
University of Delaware
DePaul University
Drew University
Drexel Institute of Technology
Duke University
D'Youville College
Earlham College
Emory University
University of Florida
Florida State University
Fordham University
University of Georgia
Goucher College
Hahnemann Medical School
Harvard University
Harvard University Medical School
Howard University
University of Illinois
Indiana University
Institute for Muscle Research
Iowa University
Iowa State University
Jackson College
Jefferson Medical College
Johns Hopkins University
University of Kansas
Kent State University
Lafayette College
Loyola College
Marquette University
University of Maryland
University of Massachusetts
Massachusetts Eye and Ear Infirmary
Massachusetts General Hospital
Massachusetts Institute of Technology
Medical College of Virginia
University of Miami
University of Michigan
University of Minnesota
University of Missouri
Mount Holyoke College
Mt. St. Joseph on the Ohio
Muskingum College
REPORT OF THE DIRECTOR
23
University of New Hampshire
New York State University, College of
Medicine at Syracuse
New York State University, College of
Medicine at Brooklyn
New York University
New York University, Bellevue Medical
Center
New York University School of Dentistry
New York University, Washington Square
College
New York State Department of Health
North Carolina State College
University of North Carolina
Northwestern University
Notre Dame University
Oberlin College
Ohio State University
University of Oklahoma
University of Pennsylvania
Pennsylvania University Medical School
University of Pittsburgh Medical School
Princeton University
Purdue University
Queens College
Radcliffe College
Reed College
University of Rhode Island
Rice University
University of Rochester
Rockefeller Institute
Roswell Park Memorial Institute
Russell Sage College
Rutgers University
St. John Fisher College
St. Louis University
St. Peter's College
Seton Hill College
Single Cell Research Foundation
Sloan-Kettering Institute
Smith College
Stanford University
Swarthmore College
Syracuse University
Temple University
Texas Technology College
Tufts University
Tulane University
U. S. Fish and Wildlife Service
U. S. Public Health Service
Vassar College
Veterans Administration Hospital at Albany
Veterans Administration Hospital at
Brooklyn
University of Vermont
University of Virgina
Wabash College
Washington University
Washington University Medical School
Washington and Jefferson College
Wayne State University
Wesleyan University
Western Reserve University
Wilson College
Woods Hole Oceanographic Institution
Yale University
FOREIGN INSTITUTIONS REPRESENTED
University of Puerto Rico
Dalhousie University, Canada
McGill University, Canada
Israel Institute for Biological Research
National Institute for Medical Research,
London
Technilogical University, Delft, Netherlands
Institut for Sporeplanter, Copenhagen
University of the Philippines
University of Geneva, Switzerland
Czechoslovak Academy
University of Glasgow
Kumamoto University Medical School, Japan
N. Copernicus University, Poland
University of Witwatersrand, Johannesburg,
South Africa
University of Kobe, Japan
Tokyo Medical and Dental University
AlcMaster University, Canada
London University
College de France
University of Uppsala, Sweden
SUPPORTING INSTITUTIONS AND AGENCIES
Associates of the Marine Biological
Laboratory
Atomic Energy Commission
Josephine B. Crane Foundation
The Grass Foundation
The Lalor Foundation
George Frederick Jewett Foundation
The Merck Company Foundation
National Institutes of Health
National Science Foundation
Office of Naval Research
The Rockefeller Foundation
Swope Gift Corporation
24
MARINE BIOLOGICAL LABORATORY
CORPORATE ASSOCIATES
Abbott Laboratories
CIBA Pharmaceutical Products Inc.
Carter Products, Inc.
Eli Lilly and Company
Olin Mathieson Chemical Corporation
Charitable Trust
Schering Foundation, Inc
E. R. Squibb and Sons
The Upjohn Company
Wallace Laboratories
6. FRIDAY EVENING LECTURES, 1961
June 30
C. R. AUSTIN "Variety in mammalian egg nuclei"
July 7
DANIEL E. KOSHLAND "Protein structure and enzyme specificity"
July 14
J. Z. YOUNG "The visual and statocyst systems of cephalopods"
July 21
J. Z. YOUNG "The learning systems of Octopus"
July 28
BENJAMIN ZWEIFACII "Reticulo-endothelial system in relation to adap-
tive reactions"
August 4
DAVID H. HUBEL "The eyes, the brain and perception"
August 11
ETIENNE WOLFF "Some aspects of the principle of competition in
the developing limb of the chick embryo"
August 18
EDWIN CHARGAFF "Sequence problems in the deoxyribonucleic acids"
August 25
WILLIAM P. JACOBS "Compensatory growth, cell differentiation and
flowering as analyzed with the aid of formal
rules of proof"
7. TUESDAY EVENING SEMINARS, 1961
July 11 ROGER MILKMAN
LIONEL I. REBHUN
DONALD P. COSTELLO
July 18 RUSSELL DOOLITTLE
R. H. CHENEY
C. C. SPEIDEL
"Temperature adaptation in Drosophila pupae"
"Endoplasmic reticulum in aster formation"
"The orientation of centrioles in dividing cells
and its significance"
"The nature of lamprey eel fibrinopeptide mate-
rial"
"Equivalent dosage effects of ultraviolet and
x-ray irradiation of Arbacia gametes, as
recorded by cinephotomicrography"
"Time-lapse cinephotomicrographs illustrating
abnormalities of viscosity, density, and cleav-
age in developing sea urchins derived from
various fertilization combinations of irradi-
ated gametes"
REPORT OF THE DIRECTOR 25
July 25 A. KENT CHRISTENSEN "Fine structure of an unusual sperm in the
flatworm Plagiostomum"
ALEX B. NOVIKOFF "Observations on the golgi apparatus and re-
lated lysosomes"
BERTA SCHARRER "Functional analysis of the corpus allatum of
the insect, Leucophaea maderae, with the
electron microscope"
August 8 EUGENE COPELAND "Ultrastructure of teleost swim bladder"
PHILIP B. DUNHAM "The physiological basis of acclimation of Tet-
rahymena to high NaCl medium"
EUGENE P. ODUM "Excretion rate of radio-isotopes as indices of
metabolic rates in nature : biological half-life
of zinc-65 in relation to food consumption,
growth and reproduction in arthropods"
August 15 PAUL WEISS "Motion picture records of cell interactions:
responses of different cell types to medium,
to substratum, and to each other"
August 22 EMILIENNE WOLFF "In vitro culture of human tumors on explants
ETIENNE WOLFF of chick embryonic organs"
8. MEMBERS OF THE CORPORATION, 1961
LIFE MEMBERS
BRODIE, MR. DONALD M., 522 Fifth Avenue, New York 18, New York
CARVER, DR. GAIL L., Mercer University, Macon, Georgia
COLE, DR. ELBERT C., 2 Chipman Park, Middlebury, Vermont
COWDRY, Dr. E. V., Washington University, St. Louis, Missouri
CRANE, MRS. W. MURRAY, Woods Hole, Massachusetts
HESS, DR. WALTER, 309 Aiken Street, Rock Hill, South Carolina
KNOWLTON, DR. F. P., c/o Mr. G. L. Gravett, Jamesville R D #2, New York
LEWIS, DR. W. H., Johns Hokpins University, Baltimore, Maryland
LOWTHER, DR. FLORENCE DEL., Barnard College, New York City, New York
MALONE, DR. E. F., 6610 North llth Street, Philadelphia 26, Pennsylvania
MEANS, DR. J. H., 15 Chestnut Street, Boston, Massachusetts
MEDES, DR. GRACE, 303 Abington Avenue, Philadelphia 11, Pennsylvania
MOORE, DR. J. PERCY, University of Pennsylvania, Philadelphia, Pennsylvania
PAYNE, DR. FERNANDUS, Indiana University, Bloomington, Indiana
PLOUGH, Dr. H. H., Amherst College, Amherst, Massachusetts
PORTER, DR. H. C., University of Pennsylvania, Philadelphia, Pennsylvania
RIGGS, MR. LAWRASON, 74 Trinity Place, New York 6, New York
SCOTT, DR. ERNEST L., Columbia University, New York City, New York
SCHRADER, DR. SALLY, Duke University, Durham, North Carolina
TURNER, DR. C. L., Northwestern University, Evanston, Illinois
WAITE, DR. F. G., 144 Locust Street, Dover, New Hampshire
WALLACE, DR. LOUISE B., 359 Lytton Avenue, Palo Alto, California
WARREN, DR. HERBERT S., 610 Montgomery Avenue, Bryn Mawr, Pennsylvania
REGULAR MEMBERS
ABELL, DR. RICHARD G., 7 Cooper Road, New York City, New York
ADAMS, DR. A. ELIZABETH, Mount Holyoke College, South Hadley, Massachusetts
26 MARINE BIOLOGICAL LABORATORY
ADELMAN, DR. WILLIAM J., Department of Neurophysiology, National Institutes of
Health, Bethesda 14, Maryland
ADDISON, DR. W. H. F., 286 East Sidney Avenue, Mount Vernon, New York
ADOLPH, DR. EDWARD F., University of Rochester, School of Medicine and Den-
tistry, Rochester, New York
ALBERT, DR. ALEXANDER, Mayo Clinic, Rochester, Minnesota
ALLEN, DR. M. JEAN, Department of Biology, Wilson College, Chambersburg,
Pennsylvania
ALLEN, DR. ROBERT D., Department of Biology, Princeton University, Princeton,
New Jersey
ALSCHER, DR. RUTH, Department of Physiology, Manhattanville College, Purchase,
New York
AMATNIEK, DR. ERNEST, Department of Neurology, College of Physicians and
Surgeons, New York 32, New York
AMBERSON, DR. WILLIAM R., Woods Hole, Massachusetts
ANDERSON, DR. J. M., Department of Zoology, Cornell University, Ithaca, New
York
ANDERSON, DR. RUBERT S., Medical Laboratories, Army Chemical Center, Mary-
land (Send mail to Box 632, Edge wood, Maryland)
ARMSTRONG, DR. PHILIP B., Department of Anatomy, State University of New
York College of Medicine, Syracuse 10, New York
ARNOLD, DR. WILLIAM A., Division of Biology, Oak Ridge National Laboratory,
Oak Ridge, Tennessee
ATWOOD, DR. KIMBALL C, 702 West Pennsylvania Avenue, Urbana, Illinois
AUSTIN, DR. MARY L., Wellesley College, Wellesley, Massachusetts
AYERS, DR. JOHN C., Department of Zoology, University of Michigan, Ann Arbor,
Michigan
BAITSELL, DR. GEORGE A., Osborn Zoological Laboratories, Yale University, New
Haven, Connecticut
BALL, DR. ERIC G., Department of Biological Chemistry, Harvard University
Medical School, Boston 15, Massachusetts
BALLARD, DR. WILLIAM W., Department of Zoology, Dartmouth College, Hanover,
New Hampshire
BALTUS, DR. ELYANE, Laboratory of Animal Morphology, Brussels, Belgium
BANG, DR. F. B., Department of Pathobiology, Johns Hopkins University School
of Hygiene, Baltimore 5, Maryland
BARD, DR. PHILLIP, Johns Hopkins Medical School, Baltimore, Maryland
EARTH, DR. L. G., Department of Zoology, Columbia University, New York 27,
New York
EARTH, DR. LUCENA, Department of Zoology, Barnard College, New York 27,
New York
BARTLETT, DR. JAMES H., Department of Physics, University of Illinois, Urbana,
Illinois
BAYLOR, DR. E. A., Woods Hole Oceanographic Institution, Woods Hole, Mas-
sachusetts
BAYLOR, DR. M. B., Marine Biological Laboratory, Woods Hole, Massachusetts
BEAMS, DR. HAROLD W., Department of Zoology, State University of Iowa, Iowa
City, Iowa
REPORT OF THE DIRECTOR 27
BECK, DR. L. V., Department of Pharmacology, Indiana University School of Ex-
perimental Medicine, Bloomington, Indiana
BEHRE, DR. ELINOR M., Black Mountain, North Carolina
BENNETT, DR. MICHAEL V., Department of Neurology, College of Physicians and
Surgeons, New York 32, New York
BENNETT, DR. MIRIAM F., Department of Biology, Sweet Briar College, Sweet
Briar, Virginia
BERG, DR. WILLIAM E., Department of Zoology, University of California, Berkeley
4, California
BERMAN, DR. MONES, Institute for Arthritis and Metabolic Diseases, National Insti-
tutes of Health, Bethesda 14, Maryland
BERNHEIMER, DR. ALAN W., New York University College of Medicine, New
York 16, New York
BERNSTEIN, DR. MAURICE, Department of Anatomy, Wayne State University Col-
lege of Medicine, Detroit 7, Michigan
BERTHOLF, DR. LLOYD M., Illinois Wesleyan University, Bloomington, Illinois
BEVELANDER, DR. GERRIT, New York University School of Dentistry, 477 First
Avenue, New York 16, New York
BIGELOW, DR. HENRY B., Museum of Comparative Zoology, Harvard University,
Cambridge 38, Massachusetts
BISHOP, DR. DAVID W., Department of Embryology, Carnegie Institution of Wash-
ington, 115 West University Parkway, Baltimore 10, Maryland
BLANCHARD, DR. K. C., Johns Hopkins Medical School, Baltimore, Maryland
BLOCK, DR. ROBERT, 518 South 42nd Street, Apt. C7, Philadelphia 4, Pennsylvania
BLUM, DR. HAROLD F., Department of Biology, Princeton University, Princeton,
New Jersey
BODANSKY, DR. OSCAR, Department of Biochemistry, Memorial Cancer Center,
444 East 68th Street, New York 21, New York
BODIAN, DR. DAVID, Department of Anatomy, Johns Hopkins University, 709
North Wolfe Street, Baltimore 5, Maryland
BOELL, DR. EDGAR J., Osborn Zoological Laboratories, Yale University, New
Haven, Connecticut
BOETTIGER, DR. EDWARD G., Department of Zoology, University of Connecticut,
Storrs, Connecticut
BOLD, DR. HAROLD C., Department of Botany, University of Texas, Austin, Texas
BOREI, DR. HANS G., Department of Zoology, University of Pennsylvania, Phila-
delphia 4, Pennsylvania
BOWEN, DR. VAUGHAN T., Woods Hole Oceanographic Institution, Woods Hole,
Massachusetts
BRADLEY, DR. HAROLD C., 2639 Durant Avenue, Berkeley 4, California
BRIDGMAN, DR. ANNA J., Department of Biology, Agnes Scott College, Decatur,
Georgia
BRONK, DR. DETLEV W., Rockefeller Institute, 66th Street and York Avenue, New
York 21, New York
BROOKS, DR. MATILDA M., Department of Physiology, University of California,
Berkeley 4, California
BROWN, DR. DUGALD E. S., Department of Zoology, University of Michigan, Ann
Arbor. Michigan
MARINE BIOLOGICAL LABORATORY
BROWN, DR. FRANK A., JR., Department of Biological Sciences, Northwestern Uni-
versity, Evanston, Illinois
BROWNELL, DR. KATHERTNE A., Department of Physiology, Ohio State University,
Columbus, Ohio
BUCK, DR. JOHN B., Laboratory of Physical Biology, National Institutes of Health,
Bethesda 14, Maryland
BULLINGTON, DR. W. E., Randolph-Macon College, Ashland, Virginia
BULLOCK, DR. T. H., Department of Zoology, University of California, Los Angeles
24, California
BURBANCK, DR. WILLIAM D., Emory University, Box 834, Atlanta 22, Georgia
BURDICK, DR. C. LALOR, The Lalor Foundation, 4400 Lancaster Pike, Wilmington,
Delaware
BURKENROAD, DR. M. D., c/o Lab. Nal. de Pesca, Apartado 3318, Estofeta #1,
Olindania, Republic of Panama
BUTLER, DR. E. G., Department of Biology, P. O. Box 704, Princeton University,
Princeton, New Jersey
CAMERON, DR. J. A., Baylor College of Dentistry, Dallas, Texas
CANTONI, DR. GIULLIO, National Institutes of Health, Mental Health, Bethesda 14,
Maryland
CARLSON, DR. FRANCIS D., Department of Biophysics, Johns Hopkins University,
Baltimore 18, Maryland
CARPENTER, DR. RUSSELL L., Tufts University, Medford 55, Massachusetts
CARSON, Miss RACHEL, 11701 Berwick Road, Silver Spring, Maryland
CASE, DR. JAMES, Department of Zoology, State University of Iowa, Iowa City,
Iowa
CATTELL, DR. McKEEN, Cornell University Medical College, 1300 York Avenue,
New York 21, New York
CATTELL, MR. WARE, Cosmos Club, Washington 5, D. C.
CHAET, DR. ALFRED B., Department of Biology, American University, Washington
16, D. C.
CHAMBERS, DR. EDWARD, Department of Physiology, University of Miami Medical
School, Coral Gables, Florida
CHANG, DR. JOSEPH J., Edward Zintl Institute, Hochschostr. 4, Darmstadt,
Germany
CHASE, DR. AURIN M., Department of Biology, Princeton University, Princeton,
New Jersey
CHENEY, DR. RALPH H., Biological Laboratory, Brooklyn College, Brooklyn 10,
New York
CLAFF, DR. C. LLOYD, 5 Van Beal Road, Randolph, Massachusetts
CHILD, DR. FRANK M., Department of Zoology, University of Chicago, Chicago 37,
Illinois
CLARK, DR. A. M., Department of Biological Sciences, University of Delaware,
Newark, Delaware
CLARK, DR. ELOISE E., Department of Zoology, Columbia University, New York
27, New York
CLARK, DR. E. R., 315 S. 41st Street, Philadelphia 4, Pennsylvania
CLARK, DR. LEONARD B., Department of Biology, Union College, Schenectady,
New York
REPORT OF THE DIRECTOR 29
CLARKE, DR. GEORGE L., Biological Laboratories, Harvard University, Cambridge
38, Massachusetts
CLELAND, DR. RALPH E., Department of Botany, Indiana University, Bloomington,
Indiana
CLEMENT, DR. A. C., Department of Biology, Emory University, Atlanta 22,
Georgia
COHEN, DR. SEYMOUR S., Department of Biochemistry, University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania
COLE, DR. KENNETH S., (NINDB), National Institutes of Health, Bethesda 14,
Maryland
COLLETTE, DR. MARY E., 34 Weston Road, Wellesley 81, Massachusetts
COLLIER, DR. JACK R., Marine Biological Laboratory, Woods Hole, Massachusetts
COLTON, DR. H. S., Box 601, Flagstaff, Arizona
COLWIN, DR. ARTHUR L., Department of Biology, Queens College, Flushing, New
York
COLWIN, DR. LAURA H., Department of Biology, Queens College, Flushing, New
York
COOPER, DR. KENNETH W., Department of Cytology, Dartmouth Medical School,
Hanover, New Hampshire
COOPERSTEIN, Dr. Sherwin J., Department of Anatomy, Western Reserve Uni-
versity Medical School, Cleveland, Ohio
COPELAND, DR. D. E., 5820 Hurst Street, Apartment B, New Orleans 18, Louisiana
COPELAND, DR. MANTON, Bowdoin College, Brunswick, Maine
CORNMAN, DR. IVOR, Hazelton Laboratories, Box 333, Falls Church, Virginia
COSTELLO, DR. DONALD P., Department of Zoology, University of North Carolina,
Chapel Hill, North Carolina
COSTELLO, DR. HELEN MILLER, Department of Zoology, University of North Caro-
lina, Chapel Hill, North Carolina
CRANE, MR. JOHN O., Woods Hole, Massachusetts
CRANE, DR. ROBERT K., Department of Biological Chemistry, Washington Uni-
versity Medical School, St. Louis, Missouri
CROASDALE, DR. HANNAH T., Dartmouth College, Hanover, New Hampshire
CROUSE, DR. HELEN V., Department of Botany, Columbia University, New York
27, New York
CROWELL, DR. P. S., JR., Department of Zoology, Indiana University, Bloomington,
Indiana
CSAPO, DR. ARPAD I., Rockefeller Institute, 66th Street and York Avenue, New
York 21, New York
CURTIS, DR. MAYNIE R., Box 1015, University of Miami, South Miami, Florida
CURTIS, DR. W. C., University of Missouri, Columbia, Missouri
DAN, DR. JEAN CLARK, Misaki Biological Station, Misaki, Japan
DAN, DR. KATSUMA, Misaki Biological Station, Misaki, Japan
DANIELLI, DR. JAMES F., Department of Zoology, King's College, London, England
DAVIS, DR. BERNARD D., Harvard Medical School, 25 Shattuck Street, Boston 15,
Massachusetts
DAWSON, DR. A. B., Biological Laboratories, Harvard University, Cambridge 38,
Massachusetts
DAWSON, DR. J. A., 129 Violet Avenue, Floral Park, Long Island, New York
30 MARINE BIOLOGICAL LABORATORY
DEANE, DR. HELEN W., Albert Einstein College of Medicine, New York 61, New
York
DILLER, DR. IRENE C, Institute for Cancer Research, Fox Chase, Philadelphia 11,
Pennsylvania
DILLER, DR. WILLIAM F., 2417 Fairhill Avenue, Glenside, Pennsylvania
DIXON, DR. FRANK J., Head of Division of Experimental Pathology, Scripps Clinic
and Research Foundation, 476 Prospect Street, La Jolla, California
DODDS, DR. G. S., West Virginia University School of Medicine, Morgantown,
West Virginia
DOLLEY, DR. WILLIAM L., Trevillians, Virginia
DOTY, DR. MAXWELL S., Department of Biology, University of Hawaii, Honolulu,
Hawaii
DURYEE, DR. WILLIAM R., Department of Pathology, George Washington Uni-
versity School of Medicine, 2300 K Street, N. W., Washington 7, D. C.
EDDS, DR. MAC V., JR., Department of Biology, Brown University, Providence 12,
Rhode Island
EDWARDS, DR. CHARLES, Department of Physiology, University of Minnesota,
Minneapolis 14, Minnesota
EICHEL, DR. HERBERT J., Hahnemann Medical College, Philadelphia, Pennsylvania
EISEN, DR. HERMAN, Department of Medicine, Washington University, St. Louis,
Missouri
ELLIOTT, DR. ALFRED M., Department of Zoology, University of Michigan, Ann
Arbor, Michigan
ESSNER, DR. EDWARD S., Department of Pathology, Albert Einstein College of
Medicine, New York 61, New York
EVANS, DR. TITUS C, State University of Iowa College of Medicine, Iowa City,
Iowa
FAILLA, DR. G., Building 203, Argonne National Laboratory, Argonne, Illinois
FAURE-FREMIET, DR. EMMANUEL, College de France, Paris, France
FERGUSON, DR. F. P., Division of General Medical Sciences, National Institutes of
Health, Bethesda 14, Maryland
FERGUSON, DR. JAMES K. W., Connought Laboratories, University of Toronto,
Ontario, Canada
FIGGE, DR. F. H. J., University of Maryland Medical School, Lombard and Green
Streets, Baltimore 1, Maryland
FINGERMAN, DR. MILTON, Department of Zoology, Newcomb College, Tulane
University, New Orleans 18, Louisiana
FISCHER, DR. ERNST, Department of Physiology, Medical College of Virginia,
Richmond, Virginia
FISHER, DR. JEANNE M., Department of Biochemistry, University of Toronto,
Toronto, Canada
FISHER, DR. KENNETH C., Department of Biology, University of Toronto, Toronto,
Canada
FORBES, DR. ALEXANDER, Biological Laboratories, Harvard University, Cambridge
38, Massachusetts
FRAENKEL, DR. GOTTFRIED S., Department of Entomology, University of Illinois,
Urbana, Illinois
FREYGANG, DR. WALTER H., JR., Box 516, Essex Fells, New Jersey
REPORT OF THE DIRECTOR 31
FRIES, DR. ERIK F. B., Box 605, Woods Hole, Massachusetts
FRISCH, DR. JOHN A., Canisius College, Buffalo, New York
FURSHPAN, DR. EDWIN ]., Department of Neurophysiology, Harvard Medical
School, Boston 15, Massachusetts
FURTH, DR. JACOB, 183 Cleveland Avenue, Buffalo, New York
FYE, DR. PAUL M., Woods Hole Oceanographic Institution, Woods Hole, Mas-
sachusetts
GABRIEL, DR. MORDECAI, Department of Biology, Brooklyn College, Brooklyn 10,
New York
GAFFRON, DR. HANS, Department of Biology, Florida State University, Conradi
Building, Tallahassee, Florida
GALL, DR. JOSEPH G., Department of Zoology, University of Minnesota, Minne-
apolis 14, Minnesota
GALTSOFF, DR. PAUL S., Woods Hole, Massachusetts
GILMAN, DR. LAUREN C, Department of Zoology, University of Miami, Coral
Gables, Florida
GINSBERG, DR. HAROLD S., Department of Microbiology, University of Pennsyl-
vania School of Medicine, Philadelphia 4, Pennsylvania
GOLDSMITH, DR. TIMOTHY H., Department of Zoology, Yale University, New
Haven, Connecticut
GOLDSTEIN, DR. LESTER, Department of Zoology, University of Pennsylvania,
Philadelphia 4, Pennsylvania
GOODCHILD, DR. CHAUNCEY G., Department of Biology, Emory University, At-
lanta 22, Georgia
GOODRICH, DR. H. B., Department of Biology, Wesleyan University, Middletown,
Connecticut
GOTSCHALL, DR. GERTRUDE Y., Rockefeller Institute, 66th Street and York Avenue,
New York 21, New York
GRAHAM, DR. HERBERT, U. S. Fish and Wildlife Service, Woods Hole, Mas-
sachusetts
GRAND, MR. C. G., Cancer Institute of Miami, 1155 N. W. 15th Street, Miami,
Florida
GRANT, DR. PHILIP, Department of Pathobiology, Johns Hopkins University
School of Hygiene, Baltimore 5, Maryland
GRAY, DR. IRVING E., Department of Zoology, Duke University, Durham, North
Carolina
GREEN, DR. JAMES W., Department of Physiology, Rutgers University, New
Brunswick, New Jersey
GREEN, DR. MAURICE, Department of Microbiology, St. Louis University Medical
School, St. Louis, Missouri
GREGG, DR. JAMES H., Department of Biological Sciences, University of Florida,
Gainesville, Florida
GREGG, DR. JOHN R., Department of Zoology, Duke University, Durham, North
Carolina
GREIF, DR. ROGER L., Department of Physiology, Cornell University Medical Col-
lege, New York 21, New York
GRIFFIN, DR. DONALD R., Biological Laboratories, Harvard University, Cam-
bridge 38, Massachusetts
32 MARINE BIOLOGICAL LABORATORY
GROSCH, DR. DANIEL S., Department of Genetics, Gardner Hall, North Carolina
State College, Raleigh, North Carolina
GROSS, DR. PAUL, Department of Biology, New York University, University
Heights, New York 53, New York
GRUNDFEST, DR. HARRY, Columbia University, College of Physicians and Surgeons,
New York 32, New York
GUDERNATSCH, DR. FREDERICK, 41 Fifth Avenue, New York 3, New York
GUTTMAN, DR. RITA, Department of Physiology, Brooklyn College, Brooklyn 10,
New York
HAJDU, DR. STEPHEN, National Institutes of Health, Bethesda 14, Maryland
HALL, DR. FRANK G., Department of Physiology, Duke University Medical School,
Durham, North Carolina
HAMBURGER, DR. VIKTOR, Department of Zoology, Washington University, St.
Louis, Missouri
HAMILTON, DR. HOWARD L., Department of Zoology, Iowa State College, Ames,
Iowa
HANCE, DR. ROBERT T., RR #3, 6609 Smith Road, Loveland, Ohio
HARDING, DR. CLIFFORD V., JR., 300 Knickerbocker Road, Tenafly, New Jersey
HARNLY, DR. MORRIS H., Washington Square College, New York University, New
York 3, New York
HARTLINE, DR. H. KEFFER, Rockefeller Institute, 66th Street and York Avenue,
New York 21, New York
HARTMAN, DR. FRANK A., Ohio State University, Hamilton Hall, Columbus, Ohio
HARVEY, DR. ETHEL BROWNE, Marine Biological Laboratory, Woods Hole, Mas-
sachusetts
HASTINGS, DR. J. WOODLAND, Division of Biochemistry, University of Illinois,
Urbana, Illinois
HAUSCHKA, DR. T. S., Roswell Park Memorial Institute, 666 Elm Street, Buffalo
3, New York
HAXO, DR. FRANCIS T., Division of Marine Botany, Scripps Institution of Ocean-
ography, University of California, La Jolla, California
HAYASHI, DR. TERU, Department of Zoology, Columbus University, New York
27, New York
HAYDEN, DR. MARGARET A., 34 Weston Road, Wellesley 81, Massachusetts
HAYWOOD, DR. CHARLOTTE, Mount Holyoke College, South Hadley, Massachusetts
HENDLEY, DR. CHARLES D., 615 South Avenue, Highland Park, New Jersey
HENLEY, DR. CATHERINE, Department of Zoology, University of North Carolina,
Chapel Hill, North Carolina
HERNDON, DR. WALTER R., Biology Department, University of Alabama, Univer-
sity, Alabama
HERVEY, DR. JOHN P., Box 735, Woods Hole, Massachusetts
HIATT, DR. HOWARD H., Department of Medicine, Harvard Medical School, Boston
15, Massachusetts
HIBBARD, DR. HOPE, Department of Zoology, Oberlin College, Oberlin, Ohio
HILL, DR. SAMUEL E., 135 Brunswick Road, Troy, New York
HIRSHFIELD, DR. HENRY I., Department of Biology, Washington Square College,
New York 3, New York
REPORT OF THE DIRECTOR
HISAW, DR. F. L., Biological Laboratories, Harvard University, Cambridge 38,
Massachusetts
HOADLEY, DR. LEIGH, Biological Laboratories, Harvard University, Cambridge 38,
Massachusetts
HODES, DR. ROBERT, Department of Pediatrics, Mount Sinai Hospital, New York
29, New York
HODGE, DR. CHARLES, IV, Department of Biology, Temple University, Philadelphia,
Pennsylvania
HOFFMAN, DR. JOSEPH, National Heart Institute, National Institutes of Health,
Bethesda 14, Maryland
HOGUE, DR. MARY J., University of Pennsylvania Medical School, Philadelphia 4,
Pennsylvania
HOLLAENDER, DR. ALEXANDER, Biology Division, Oak Ridge National Laboratory,
Oak Ridge, Tennessee
HOLZ, DR. GEORGE G., JR., Department of Zoology, Syracuse University, Syracuse,
New York
HOPKINS, DR. HOYT S., 59 Heatherdell Road, Ardsley, New York
HUNTER, DR. FRANCIS R., University of the Andes, Calle 18-a Carrera 1-E,
Bogota, Colombia, South America
HUTCHENS, DR. JOHN E., Department of Physiology, University of Chicago, Chi-
cago 37, Illinois
HYDE, DR. BEAL B., Department of Plant Sciences, University of Oklahoma, Nor-
man, Oklahoma
HYMAN, DR. LIBBIE H., American Museum of Natural History, Central Park
West at 79th Street, New York 24, New York
IRVING, DR. LAURENCE, U. S. Public Health Service, Anchorage, Alaska
ISENBERG, DR. IRVIN, Institute for Muscle Research, Marine Biological Laboratory,
Woods Hole, Massachusetts
ISELIN, MR. COLUMBUS O'D., Woods Hole, Massachusetts
JACOBS, DR. M. H., University of Pennsylvania School of Medicine, Philadelphia 4,
Pennsylvania
JACOBS, DR. WILLIAM P., Department of Biology, Princeton University, Princeton,
New Jersey
JENNER, DR. CHARLES E., Department of Zoology, University of North Carolina,
Chapel Hill, North Carolina
JOHNSON, DR. FRANK H., Department of Biology, Princeton University, Princeton,
New Jersey
JONES, DR. E. RUFFIN, JR., Department of Biological Sciences, University of
Florida, Gainesville. Florida
JONES, DR. RAYMOND F., Department of Biology, Princeton University, Princeton,
New Jersey
KAAN, DR. HELEN W., Marine Biological Laboratory, Woods Hole, Massachusetts
KABAT, DR. E. A., Neurological Institute, College of Physicians and Surgeons,
New York 32, New York
KANE, DR. ROBERT E., Department of Cytology, Dartmouth Medical School, Han-
over, New Hampshire
34 MARINE BIOLOGICAL LABORATORY
KARUSH, DR. FRED, Department of Pediatrics, University of Pennsylvania, Phila-
delphia 4, Pennsylvania
KAUFMANN, DR. B. P., Carnegie Institution, Cold Spring Harbor, Long Island,
New York
KEMP, DR. NORMAN E., Department of Zoology, University of Michigan, Ann
Arbor, Michigan
KEMPTON, DR. RUDOLF T., Department of Zoology, Vassar College, Poughkeepsie,
New York
KEOSIAN, DR. JOHN, Department of Biology, Rutgers University, Newark 2, New
Jersey
KETCHUM, DR. BOSTWICK H., Woods Hole Oceanographic Institution, Woods
Hole, Massachusetts
KILLE, DR. FRANK R., State Department of Education, Albany 1, New York
KIND, DR. C. ALBERT, Department of Zoology, University of Connecticut, Storrs,
Connecticut
KINDRED, DR. J. E., University of Virginia, Charlottesville, Virginia
KING, DR. ROBERT L., State University of Iowa, Iowa City, Iowa
KINGSBURY, DR. JOHN M., Department of Botany, Cornell University, Ithaca,
New York
KISCH, DR. BRUNO, 845 West End Avenue, New York City, New York
KLEIN, DR. MORTON, Department of Microbiology, Temple University, Philadel-
phia, Pennsylvania
KLEINHOLZ, DR. LEWIS H., Department of Biology, Reed College, Portland 2,
Oregon
KLOTZ, DR. I. M., Department of Chemistry, Northwestern University, Evanston,
Illinois
KOLIN, DR. ALEXANDER, Department of Biophysics, California Medical School,
Los Angeles 24, California
KORR, DR. L M., Department of Physiology, Kirksville College of Osteopathy,
Kirksville, Missouri
KRAHL, DR. M. E., Department of Physiology, University of Chicago, Chicago 37,
Illinois
KRANE, DR. STEPHEN M., Massachusetts General Hospital, Boston 14, Mas-
sachusetts
KRAUSS, DR. ROBERT, Department of Botany, University of Maryland, Baltimore,
Maryland
KREIG, DR. WENDELL J. S., 303 East Chicago Avenue, Chicago, Illinois
KUFFLER, DR. STEPHEN W., Department of Pharmacology, Harvard Medical
School, Neurophysical Laboratory, Boston 15, Massachusetts
KUNITZ, DR. MOSES, Rockefeller Institute, 66th Street and York Avenue. New
York 21, New York
LACKEY, DR. JAMES B., Box 497, Melrose, Florida
LAMY, DR. FRANCOIS, Department of Anatomy, University of Pittsburgh School of
Medicine, Pittsburgh 13, Pennsylvania
LANCEFIELD, DR. D. E., Queens College, Flushing, New York
LANCEFIELD, DR. REBECCA C., Rockefeller Institute, 66th Street and York Avenue,
New York 21, New York
REPORT OF THE DIRECTOR 35
LANDIS, DR. E. M., Harvard Medical School, Boston 15, Massachusetts
LANSING, DR. ALBERT I., Department of Anatomy, University of Pittsburgh Medi-
cal School, Pittsburgh 13, Pennsylvania
LAUFFER, DR. MAX A., Department of Biophysics, University of Pittsburgh, Pitts-
burgh 13, Pennsylvania
LAVIN, DR. GEORGE I., 6200 Norvo Road, Baltimore 7, Maryland
LAZAROW, DR. ARNOLD, Department of Anatomy, University of Minnesota Medical
School, Minneapolis 14, Minnesota
LEDERBERG, DR. JOSHUA, Department of Genetics, Stanford Medical School, Palo
Alto, California
LEE, DR. RICHARD E., Cornell University College of Medicine. New York 21,
New York
LEFEVRE, DR. PAUL G., University of Louisville School of Medicine, Louisville,
Kentucky
LEHMANN, DR. FRITZ, Zoologische Inst., University of Berne, Berne, Switzerland
LEVINE, DR. RACHMIEL, Michael Reese Hospital, Chicago 16, Illinois
LEVY, DR. MILTON, Department of Biochemistry, New York University School of
Dentistry, New York 10, New York
LEWIN, DR. RALPH A., Scripps Institution of Oceanography, La Jolla, California
LEWIS, DR. IVEY F., 1110 Rugby Road, Charlottesville, Virginia
LING, DR. GILBERT, 307 Berkeley Road, Merion, Pennsylvania
LITTLE, DR. E. P., 216 High Street, West Newton, Massachusetts
LLOYD, DR. DAVID P. C, Rockefeller Institute, 66th Street and York Avenue, New
York 21, New York
LOCHHEAD, DR. JOHN H., Department of Zoology, University of Vermont, Burling-
ton, Vermont
LOEB, DR. R. F., 950 Park Avenue, New York 28, New York
LOEWENSTEIN, DR. WERNER R., Department of Physiology, College of Physicians
and Surgeons, New York 32, New York
LOEWI, DR. OTTO, 155 East 93rd Street, New York City, New York
LOFTFIELD, DR. ROBERT B., Massachusetts General Hospital, Boston, Massachusetts
LORAND, DR. LASZLO, Department of Chemistry, Northwestern University, Evans-
ton, Illinois
DELORENZO, DR. ANTHONY, Anatomical and Pathological Research Laboratories,
Johns Hopkins Hospital, Baltimore 5, Maryland
LOVE, DR. Lois H., 1043 Marlau Drive, Baltimore 12, Maryland
LOVE, DR. WARNER E., 1043 Marlau Drive, Baltimore 12, Maryland
LUBIN, DR. MARTIN, Department of Pharmacology, Harvard Medical School,
Boston 15, Massachusetts
LYNCH, DR. CLARA J., Rockefeller Institute, 66th Street and York Avenue, New
York 21, New York
LYNN, DR. W. GARDNER, Department of Biology, Catholic University of America,
Washington 17, D. C.
MACDOUGALL, DR. MARY STUART, Mt. Vernon Apts., 423 Clairmont Avenue,
Decatur, Georgia
McCANN, DR. FRANCES, Department of Physiology, Dartmouth Medical School,
Hanover, New Hampshire
36 MARINE BIOLOGICAL LABORATORY
McCoucn, DR. MARGARET SUMXYALT, University of Pennsylvania Medical School,
Philadelphia 4, Pennsylvania
MCDONALD, SISTER ELIZABETH SKTON, Department of Biolnoy. College of Mt. St.
Joseph, Mt. St. Joseph, Ohio
MCDONALD, DR. MARGARET H., Carnegie Institution of Washington, Cold Spring
Harbor, Long Island, New York
MCELROY, DR. WILLIAM D., Department of Biology, Johns Hopkins University,
Baltimore 18, Maryland
MAAS, DR. WERNER K., New York University College of Medicine, New York
City, New York
MAGRUDER, DR. SAMUEL R., Department of Anatomy, Tufts Medical School, 135
Harrison Avenue, Boston, Massachusetts
MANWELL, DR. REGINALD D., Department of Zoology, Syracuse University, Syra-
cuse 10, New York
MARSHAK, DR. ALFRED, Department of Radiology, Jefferson Medical College,
Philadelphia 7, Pennsylvania
MARSLAND, DR. DOUGLAS A., New York University, Washington Square College,
New York 3, New York
MARTIN, DR. EARL A., Department of Biology, Brooklyn College, Brooklyn 10,
New York
MATHEWS, DR. SAMUEL A., Thompson Biological Laboratory, Williams College,
Williamstown, Massachusetts
MAYOR, DR. JAMES W., 8 Gracewood Park, Cambridge 38, Massachusetts
MAZIA, DR. DANIEL, Department of Zoology, University of California, Berkeley 4,
California
MEINKOTH, DR. NORMAN A., Department of Biology, Swarthmore College,
Swarthmore, Pennsylvania
METZ, DR. C. B., Oceanographic Institute, Florida State University, Tallahassee,
Florida
METZ, DR. CHARLES W., Box 714, Woods Hole, Massachusetts
MIDDLEBROOK, DR. ROBERT, Institute for Muscle Research, Marine Biological Lab-
oratory, Woods Hole, Massachusetts
MILKMAN, DR. ROGER D., Department of Zoology, Syracuse University, Syracuse
10, New York
MILLER, DR. J. A., JR., Department of Anatomy, Tulane University Medical School,
New Orleans 18, Louisiana
MILNE, DR. LORUS J., Department of Zoology, University of New Hampshire,
Durham, New Hampshire
MOE, MR. HENRY A., Guggenheim Memorial Foundation, 551 Fifth Avenue, New
York 17, New York
MONROY, DR. ALBERTO, Institute of Comparative Anatomy, University of Palermo,
Italy
MOORE, DR. GEORGE M., Department of Zoology, University of New Hampshire,
Durham, New Hampshire
MOORE, DR. JOHN A., Department of Zoology, Columbia University, New York 27,
New York
REPORT OF THE DIRECTOR 37
MOORE, DR. JOHN W., Laboratory of Biophysics, NINDB, National Institutes of
Health, Bethesda 14, Maryland
MORRILL, DR. JOHN B., JR., Department of Biology, Wesleyan University, Middle-
town, Connecticut
MOUL, DR. E. T., Department of Botany, Rutgers University, New Brunswick,
New Jersey
MOUNTAIN, MRS. J. D., Charles Road, Mt. Kisco, New York
MULLINS, DR. LORIN J., Department of Biophysics, University of Maryland School
of Medicine, Baltimore 1, Maryland
MUSACCHIA, DR. XAVIER, JR., Department of Biology, St. Louis University, St.
Louis 4, Missouri
NABRIT, DR. S. M., President, Texas Southern University, 3201 Wheeler Avenue,
Houston 4, Texas
NACE, DR. PAUL FOLEY, Department of Biology, Hamilton College, McMaster
University, Hamilton, Ontario
NACHMANSOHN, DR. DAVID, Columbia University, College of Physicians and Sur-
geons, New York 32, New York
NAVEZ, DR. ALBERT E., 206 Churchill's Lane, Milton 86, Massachusetts
NELSON, DR. LEONARD, Department of Physiology, Emory University, Atlanta 22,
Georgia
NEURATH, DR. H., Department of Biochemistry, University of Washington, Seattle
5, Washington
NICOLL, DR. PAUL A., Black Oak Lodge, RR #2, Bloomington, Indiana
Niu, DR. MAN-CHIANG, Department of Biology, Temple University, Philadelphia,
Pennsylvania
NOVIKOFF, DR. ALEX B., Department of Pathology, Albert Einstein College of
Medicine, New York 61, New York
OCHOA, DR. SEVERO, New York University College of Medicine, New York 16,
New York
ODUM, DR. EUGENE, Department of Zoology, University of Georgia, Athens,
Georgia
OPPENHEIMER, DR. JANE M., Department of Biology, Bryn Mawr College, Bryn
Mawr, Pennsylvania
OSTERHOUT, DR. W. J. V., Rockefeller Institute, 66th Street and York Avenue,
New York 21, New York
OSTERHOUT, DR. MARION IRWIN, Rockefeller Institute, 66th Street and York
Avenue, New York 21, New York
PACKARD, DR. CHARLES, Woods Hole, Massachusetts
PAGE, DR. IRVINE H., Cleveland Clinic, Cleveland, Ohio
PARPART, DR. ARTHUR K., Department of Biology, Princeton University, Princeton,
New Jersey
PASSANO, DR. LEONARD M., Osborn Zoological Laboratories, Yale University, New
Haven, Connecticut
PATTEN, DR. BRADLEY M., University of Michigan School of Medicine, Ann Arbor,
Michigan
PERKINS, DR. JOHN F., JR., Department of Physiology, University of Chicago,
Chicago 37, Illinois
38 MARINE BIOLOGICAL LABORATORY
PERSON, DR. PHILIP, Chief, Special Dental Research Program, VA Hospital,
Brooklyn 9, New York
PETTIBONE, DR. MARIAN H., Department of Zoology, University of New Hamp-
shire, Durham, New Hampshire
PHILPOTT, MR. DELBERT E., 496 Palmer Avenue, Falmouth, Massachusetts
PICK, DR. JOSEPH, Department of Anatomy, New York University, Bellevue Medi-
cal Center, New York 16, New York
PIERCE, DR. MADELENE E., Department of Zoology, Vassar College, Poughkeepsie,
New York
POLLISTER, DR. A. W., Department of Zoology, Columbia University, New York
27, New York
POND, DR. SAMUEL E., 53 Alexander Street, Manchester, Connecticut
POTTER, DR. DAVID, Department of Neurophysiology, Harvard Medical School,
Boston 15, Massachusetts
PROCTOR, DR. NATHANIEL, Department of Biology, Morgan State College, Balti-
more 12, Maryland
PROSSER, DR. C. LADD, Department of Physiology, Burrill Hall, University of Illi-
nois, Urbana, Illinois
PROVASOLI, DR. LUIGI, Haskins Laboratories, 305 E. 43rd Street, New York 17,
New York
RAMSEY, DR. ROBERT W., Medical College of Virginia, Richmond, Virginia
RANKIN, DR. JOHN S., Department of Zoology, University of Connecticut, Storrs,
Connecticut
RANZI, DR. SILVIO, Department of Zoology, University of Milan, Milan, Italy
RATNER, DR. SARAH, Public Health Research Institute of the City of New York,
Foot of East 15th Street, New York 9, New York
RAY, DR. CHARLES, JR., Department of Biology, Emory University, Atlanta 22,
Georgia
READ, DR. CLARK P., Department of Biology, Rice University, Houston, Texas
REBHUN, DR. LIONEL I., Department of Biology, Box 704, Princeton University,
Princeton, New Jersey
RECHNAGEL, DR. R. O., Department of Physiology, Western Reserve University,
Cleveland, Ohio
REDFIELD, DR. ALFRED C., Woods Hole, Massachusetts
RENN, DR. CHARLES E., 509 Ames Hall, Johns Hopkins University, Baltimore 18,
Maryland
REUBEN, DR. JOHN P., Department of Neurology, College of Physicians and Sur-
geons, New York 32, New York
REZNIKOFF, DR. PAUL, Cornell University Medical College, 1300 York Avenue,
New York 16, New York
RICHARDS, DR. A., 2950 E. Mabel Street, Tucson, Arizona
RICHARDS, DR. A. GLENN, Department of Entomology, University of Minnesota,
St. Paul 1, Minnesota
RICHARDS, DR. OSCAR W., American Optical Company, Research Center, South-
bridge, Massachusetts
ROCKSTEIN, DR. MORRIS, Department of Physiology, New York University College
of Medicine, New York 16. New York
REPORT OF THE DIRECTOR 39
ROGICK, DR. MARY D., College of New Rochelle, New Rochelle, New York
ROMER, DR. ALFRED S., Harvard University, Museum of Comparative Zoology,
Cambridge 38, Massachusetts
RONKIN, DR. RAPHAEL R., Department of Physiology, University of Delaware,
Newark, Delaware
ROOT, DR. R. W., Department of Biology, College of the City of New York, New
York City, New York
ROOT, DR. W. S., Department of Physiology, Columbia University, College of
Physicians and Surgeons, New York 32, New York
ROSE, DR. S. MERYL, Department of Biology, Wesleyan University, Middletown,
Connecticut
ROSENBERG, DR. EVELYN K., Department of Pathology, New York University,
Bellevue Medical Center, New York 16, New York
ROSENBLUTH, Miss RAJA, Department of Zoology, Columbia University, New
York 27, New York
ROSENTHAL, DR. THEODORE B., Department of Anatomy, University of Pittsburgh
Medical School, Pittsburgh 13, Pennsylvania
ROSLANSKY, DR. JOHN, Department of Biology, Princeton University, Princeton,
New Jersey
ROTH, DR. JAY S., Department of Zoology and Entomology, University of Con-
necticut, Storrs, Connecticut
ROTHENBERG, DR. M. A., Scientific Director, Dugway Proving Ground, Dugway,
Utah
RUGH, DR. ROBERTS, Radiological Research Laboratory, College of Physicians and
Surgeons, New York 32, New York
RUNNSTROM, DR. JOHN, Wenner-Grens Institute, Stockholm, Sweden
RUTMAN, DR. ROBERT J., General Laboratory Building, 215 S. 34th Street, Phila-
delphia, Pennsylvania
RYTHER, DR. JOHN H., Woods Hole Oceanographic Institution, Woods Hole,
Massachusetts
SANBORN, DR. RICHARD C., Department of Biological Sciences, Purdue University,
Lafayette, Indiana
SANDEEN, DR. MURIEL I., Department of Zoology, Duke University, Durham,
North Carolina
SAUNDERS, DR. JOHN, Department of Biology, Marquette University, Milwaukee
3, Wisconsin
SAUNDERS, MR. LAWRENCE, West Washington Square, Philadelphia 5, Pennsyl-
vania
SCHACHMAN, DR. HOWARD K., Department of Biochemistry, University of Cali-
fornia, Berkeley 4, California
SCHARRER, DR. ERNST A., Department of Anatomy, Albert Einstein College of
Medicine, New York 61, New York
SCHLESINGER, DR. R. WALTER, Department of Microbiology, St. Louis University
School of Medicine, 1402 South Grand Boulevard, St. Louis 4, Missouri
SCHMIDT, DR. L. H., Christ Hospital, Cincinnati, Ohio
SCHMITT, DR. FRANCIS O., Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts
40 MARINE BIOLOGICAL LABORATORY
SCHMITT, DR. O. H., Department of Physics, University of Minnesota, Minne-
apolis 14, Minnesota
SCHNEIDERMAN, DR. HOWARD A., Department of Biology, Western Reserve Uni-
versity, Cleveland, Ohio
SCHOLANDER, DR. P. F., Scripps Institution of Oceanography, La Jolla, California
SCHOTTE, DR. OSCAR E., Department of Biology, Amherst College, Amherst,
Massachusetts
SCHRAMM, DR. J. R., Department of Botany, Indiana University, Bloomington,
Indiana
SCOTT, DR. ALLAN C, Colby College, Waterville, Maine
SCOTT, DR. D. B. McNAiR, Botany Annex, Cancer Chemotherapy Laboratory,
University of Pennsylvania, Philadelphia 4, Pennsylvania
SCOTT, SISTER FLORENCE MARIE, Seton Hill College, Greensburg, Pennsylvania
SCOTT, DR. GEORGE I., Department of Zoology, Oberlin College, Oberlin, Ohio
SEARS, DR. MARY, Woods Hole Oceanographic Institution, Woods Hole, Massa-
chusetts
SELIGER, DR. HOWARD H., McCollum-Pratt Institute, Johns Hopkins University,
Baltimore, Maryland
SENFT, DR. ALFRED W., Woods Hole, Massachusetts
SEVERINGHAUS, DR. AURA E., Department of Anatomy, College of Physicians and
Surgeons, New York 32, New York
SHANES, DR. ABRAHAM, Department of Pharmacology, University of Pennsyl-
vania School of Medicine, Philadelphia 4, Pennsylvania
SHAPIRO, DR. HERBERT, 5800 North Camac Street, Philadelphia 41, Pennsylvania
SHAVER, DR. JOHN R., Department of Zoology, Michigan State University, East
Lansing, Michigan
SHEDLOVSKY, DR. THEODORE, Rockefeller Institute, 66th Street and York Avenue,
New York 21, New York
SICHEL, DR. FERDINAND J. M., University of Vermont, Burlington, Vermont
SICHEL, MRS. F. J. M., 38 Henderson Terrace, Burlington, Vermont
SILVA, DR. PAUL, Department of Botany, University of California, Berkeley 4,
California
SLIFER, DR. ELEANOR H., Department of Zoology, State University of Iowa, Iowa
City, Iowa
SMELSER, DR. GEORGE K., Department of Anatomy, Columbia University, New
York 32, New York
SMITH, DR. DIETRICH C., Department of Physiology, University of Maryland
School of Medicine, Baltimore, Maryland
SMITH, MR. HOMER P., General Manager, Marine Biological Laboratory, Woods
Hole, Massachusetts
SMITH, MR. PAUL FERRIS, Marine Biological Laboratory, Woods Hole, Massa-
chusetts
SMITH, DR. RALPH I., Department of Zoology, University of California, Berkeley
4, California
SONNEBORN, DR. T. M., Department of Zoology, Indiana University, Bloomington.
Indiana
SONNENBLICK, DR. B. P., Rutgers University, 40 Rector Street, Newark 2, New
Jersey
REPORT OF THE DIRECTOR 41
SPEIDEL, DR. CARL C, Department of Anatomy, University of Virginia, University,
Virginia
SPIEGEL, DR. MELVIN, Department of Zoology, Dartmouth College, Hanover,
New Hampshire
SPRATT, DR. NELSON T., Department of Zoology, University of Minnesota, Minne-
apolis 14, Minnesota
SPYROPOULOS, DR. C. S., Bldg. 9 — Rm. 140, National Institutes of Health, Beth-
esda 14, Maryland
STARR, DR. RICHARD C., Department of Botany, Indiana University, Bloomington,
Indiana
STEINBACH, DR. H. BURR, Department of Zoology, University of Chicago, Chicago
15, Illinois
STEINBERG, DR. MALCOLM S., Department of Biology, Johns Hopkins University,
Baltimore 18, Maryland
STEINHARDT, DR. JACINTO, Director of Operations Evaluation Group, Massa-
chusetts Institute of Technology, Cambridge, Massachusetts
STEPHENS, DR. GROVER C., Department of Zoology, University of Minnesota,
Minneapolis 14, Minnesota
STETTEN, DR. DE\VITT, Director in Charge of Research, NIAMD, National In-
stitutes of Health, Bethesda 14, Maryland
STETTEN, DR. MARJORIE R., NIAMD, National Institutes of Health, Bethesda 14,
Maryland
STEWART, DR. DOROTHY, Rockford College, Rockford, Illinois
STOREY, DR. ALMA G., Department of Botany, Mount Holyoke College, South
Hadley, Massachusetts
STONE, DR. WILLIAM, JR., Ophthalmic Plastics Laboratory, Massachusetts Eye
and Ear Infirmary, Boston, Massachusetts
STRAUS, DR. W. L., JR., Department of Anatomy, Johns Hopkins University
Medical School, Baltimore 5, Maryland
STREHLER, DR. BERNARD L., Cellular and Comparative Physiology Section, Na-
tional Institutes of Health, Bethesda 14, Maryland
STRITTMATTER, DR. PHILIPP, Department of Biological Chemistry, Washington
University Medical School, St. Louis, Missouri
STUNKARD, DR. HORACE W., American Museum of Natural History, Central
Park West at 79th Street, New York 24, New York
STURTEVANT, DR. ALFRED H., California Institute of Technology, Pasadena 4,
California
SUDAK, DR. FREDERICK N., Department of Physiology, Albert Einstein College
of Medicine, New York 61, New York
SULKIN, DR. S. EDWARD, Department of Bacteriology, University of Texas, South-
western Medical School, Dallas, Texas
SWOPE, MR. GERARD, JR., 570 Lexington Avenue, New York 22, New York
SZENT GYORGYI, DR. ALBERT, Institute for Muscle Research, Marine Biological
Laboratory, Woods Hole, Massachusetts
SZENT GYORGYI, DR. ANDREW G., Institute for Muscle Research, Marine Biological
Laboratory, Woods Hole, Massachusetts
TASAKI, DR. ICHIJI, Laboratory of Neurophysiology, NINDB, Bethesda 14.
Maryland
42 MARINE BIOLOGICAL LABORATORY
TASHIRO, DR. SHIRO, University of Cincinnati Medical College, Cincinnati, Ohio
TAYLOR, DR. ROBERT E., Laboratory of Neurophysiology, NINDB, Bethesda 14,
Maryland
TAYLOR, DR. WM. RANDOLPH, Department of Botany, University of Michigan,
Ann Arbor, Michigan
TAYLOR, DR. W. ROWLAND, Department of Oceanography, Johns Hopkins Uni-
versity, Baltimore, Maryland
TE\¥INKEL, DR. Lois E., Department of Zoology, Smith College, Northampton,
Massachusetts
TOBIAS, DR. JULIAN, Department of Physiology, University of Chicago, Chicago,
Illinois
TRACY, DR. HENRY C, General Delivery, Oxford, Mississippi
TRACER, DR. WILLIAM, Rockefeller Institute, 66th Street and York Avenue, New
York 21, New York
TRINKAUS, DR. J. PHILIP, Department of Zoology, Osborn Zoological Laboratories,
Yale University, New Haven, Connecticut
TROLL, DR. WALTER, Department of Industrial Medicine, New York University
College of Medicine, New York 16, New York
TWEEDELL, DR. KENYON S., Department of Biology, University of Notre Dame,
Notre Dame, Indiana
TYLER, DR. ALBERT, Division of Biology, California Institute of Technology,
Pasadena 4, California
URETZ, DR. ROBERT B., Department of Biophysics, University of Chicago, Chicago,
Illinois
DE VILLAFRANCA, DR. GEORGE W., Department of Zoology, Smith College. North-
ampton, Massachusetts
VILLEE, DR. CLAUDE A., Department of Biological Chemistry, Harvard Medical
School, Boston, Massachusetts
VINCENT. DR. WALTER S., Department of Anatomy, State University of New
York School of Medicine, Syracuse 10, New York
WAINIO, DR. W. W., Bureau of Biological Research, Rutgers University. New
Brunswick, New Jersey
WALD, DR. GEORGE, Biological Laboratories, Harvard University, Cambridge 38,
Massachusetts
WARNER, DR. ROBERT C., Department of Chemistry, New York University College
of Medicine, New York 16, New York
WATERMAN, DR. T. H.. Department of Zoology, 272 Gibbs Research Laboratory,
Yale University, New Haven, Connecticut
WEBB, DR. MARGUERITE, Department of Physiology and Bacteriology, Goucher
College, Towson, Baltimore 4, Maryland
WEISS, DR. PAUL A., Laboratory of Developmental Biology, Rockefeller Institute,
66th treet and York Avenue, New York 21, New York
WENRICH, DR. D. H., Department of Zoology, University of Pennsylvania, Phila-
delphia 4, Pennsylvania
WERMAN, DR. ROBERT, Institute of Psychiatric Research, University of Indiana
Medical Center, 1100 W. Michigan Street, Indianapolis 7, Indiana
REPORT OF THE DIRECTOR 43
WHEDON, DR. A. D., 21 Lawncrest, Danbury, Connecticut
WHITAKER, DR. DOUGLAS M., Rockefeller Institute, 66th Street and York Avenue,
New York 21, New York
WHITE, DR. E. GRACE, 1312 Edgar Avenue, Chambersburg, Pennsylvania
WHITING, DR. ANNA R., Department of Zoology, University of Pennsylvania,
Philadelphia 4, Pennsylvania
WHITING, DR. PHINEAS, Department of Zoology, University of Pennsylvania,
Philadelphia 4, Pennsylvania
WICKERSHAM, MR. JAMES H., 530 Fifth Avenue, New York 36, New York
WICHTERMAN, DR. RALPH, Department of Biology, Temple University, Phila-
delphia, Pennsylvania
WIEMAN, DR. H. L., Box 485, Falmouth, Massachusetts
WIERCINSKI, DR. FLOYD J., Department of Biological Sciences, Drexel Institute
of Technology, 32nd and Chestnut Streets, Philadelphia 4, Pennsylvania
WIGLEY, DR. ROLAND L., U. S. Fish and Wildlife Service, Woods Hole, Massa-
chusetts
WILBER, DR. C. G., Dean, Graduate School, Kent State University, Kent, Ohio
WILLIER, DR. B. H., Department of Biology, Johns Hopkins University, Baltimore
18, Maryland
WILSON, DR. J. WALTER, Department of Biology, Brown University, Providence
12, Rhode Island
WILSON, DR. WALTER L., Department of Physiology, University of Vermont
College of Medicine, Burlington, Vermont
WITSCHI, DR. EMIL, Department of Zoology, State University of Iowa, Iowa
City, Iowa
WITTENBERG, DR. JONATHAN B., Department of Physiology and Biochemistry,
Albert Einstein College of Medicine, New York 61, New York
WRIGHT, DR. PAUL A., Department of Zoology, University of New Hampshire.
Durham, New Hampshire
WRINCH, DR. DOROTHY, Department of Physics, Smith College, Northampton,
Massachusetts
YNTEMA, DR. C. L., Department of Anatomy, State University of New Yrork
College of Medicine, Syracuse 10, New York
YOUNG, DR. D. B., Main Street, North Hanover, Massachusetts
ZIMMERMAN, DR. A. M., Department of Pharmacology, State University of New
York, Downstate Medical Center, Brooklyn 3, New York
ZINN, DR. DONALD J., Department of Zoology, University of Rhode Island, King-
ston, Rhode Island
ZIRKLE, DR. RAYMOND E., Department of Radiobiology, University of Chicago.
Chicago 37, Illinois
ZORZOLI, DR. ANITA, Department of Physiology, Vassar College, Poughkeepsie,
New York
ZWEIFACH, DR. BENJAMIN, New York University Bellevue Center, New York 16,
New York
ZWILLING, DR. EDGAR, Department of Biology, Brandeis University, Waltham
54, Massachusetts
44
MARINE BIOLOGICAL LABORATORY
ASSOCIATE MEMBERS
ALTON, DR. AND MRS. BENJAMIN H.
ARMSTRONG, DR. AND MRS. P. B.
BACON, MRS. ROBERT
BAITSELL, MRS. GEORGE
BALL, MRS. ERIC
BARBOUR, MR. Lucius H.
BARTOW, MR. AND MRS. CLARENCE
BARTOW, MRS. FRANCIS D.
BARTOW, MR. AND MRS. PHILIP K.
BELL, MRS. ARTHUR W.
BRADLEY, MR. AND MRS. ALBERT L.
BRADLEY, MR. AND MRS. CHARLES
BROWN, MRS. THORNTON
BURDICK, DR. C. LALOR
BURLINGAME, MRS. F. A.
CAHOON, MRS. SAMUEL, SR.
CALKINS, MRS. GARY N.
CALKINS, MRS. G. NATHAN, JR.
CALKINS, MR. AND MRS. SAMUEL W.
CARLTON, MR. AND MRS. WINSLOW
CLAFF, DR. AND MRS. C. LLOYD
CLARK, DR. AND MRS. ALFRED HULL
CLARK, MRS. LEROY
CLARK, MR. AND MRS. W. VAN ALAN
CLOWES, MR. ALLEN W.
CLOWES, MRS. G. H. A.
CLOWES, DR. AND MRS. G. H. A., JR.
COLTON, MR. AND MRS. H. SEYMOUR
COWDRY, DR. AND MRS. E. V.
CRANE, MR. AND MRS. BRUCE
CRANE, MR. JOHN
CRANE, Miss LOUISE
CRANE, MRS. MURRAY
CRANE, MR. STEPHEN
CRANE, MRS. W. CAREY
CROSSLEY, MR. AND MRS. ARCHIBALD M.
CROWELL, MR. AND MRS. PRINCE S.
CURTIS, DR. AND MRS. W. D.
DANIELS, MR. AND MRS. F. HAROLD
DAY, MR. AND MRS. POMEROY
DRAPER, MRS. MARY C.
DREYER, MR. AND MRS. FRANK A.
ELSMITH, MRS. DOROTHY
ENDERS, MRS. FREDERICK
EWING, MR. AND MRS. FREDERIC
EWING, MR. WILLIAM
FAY, MR. AND MRS. HENRY H.
FISHER, MR. AND MRS. B. C.
FRANCIS, MRS. LEWIS H., JR.
FROST, MRS. FRANK J.
GALTSOFF, MRS. PAUL S.
GlFFORD, MR. AND MRS. JOHN A.
GlFFORD, MR. AND MRS. PROSSER
GlLCHRIST, MR. AND MRS. JOHN M.
GlLDEA, DR. AND MRS. E. F.
GREEN, Miss GLADYS M.
GULESIAN, MRS. PAUL J.
HAIG, MRS. R. H.
HAMLEN, MR. AND MRS. J. MONROE
HARRELL, MR. AND MRS. JOEL E.
HARRINGTON, MR. AND MRS. ROBERT
HARVEY, DR. ETHEL B.
HERRINGTON, MRS. A. W. S.
HERVEY, DR. AND MRS. JOHN P.
HlRSCHFELD, MRS. NATHAN B.
HOUSTON, MR. AND MRS. HOWARD
JEWETT, MRS. G. F.
JOHLIN, MRS. JACOB M.
KEITH, MR. AND MRS. HAROLD C.
KING, MR. AND MRS. FRANKLIN
KOLLER, MR. AND MRS. LEWIS
LAURENCE, MR. AND MRS. THOMAS E.
LEMANN, MRS. BENJAMIN
LlNEAWEAVER, MR. THOMAS, III
LOBB, MRS. JOHN
LOEB, DR. AND MRS. ROBERT F.
MCCUSKER, MR. AND MRS. PAUL J.
MCKELVY, MR. JOHN E.
MARSLAND, MRS. DOUGLAS A.
MARVIN, MRS. WALTER T.
MAST, MRS. S. O.
MEIGS, DR. AND MRS. J. WISTER
MITCHELL, MRS. JAMES McC.
MIXTER, MRS. W. JASON
MOSSER, MRS. BENJAMIN D.
MOTLEY, MRS. THOMAS
NEWTON, Miss HELEN
NICHOLS, MRS. GEORGE
NIMS, MRS. E. D.
PACKARD, MRS. CHARLES
PARK, MR. AND MRS. M. S.
PENNINGTON, Miss ANNE H.
REDFIELD, DR. AND MRS. ALFRED C.
REZNIKOFF, DR. AND MRS. PAUL
REPORT OF THE LIBRARIAN
45
Ri<;<;s, MR. AND MRS. LAWRASON
R i YIN us, MRS. F. M., JR.
ROBINSON, DR. MILES
RUDD, MR. AND MRS. H. \Y. Ihvicirr
SANDS, Miss ADELAIDE G.
SAUNDERS, MR. AND MRS. LAWRENCE
SHIVERICK, MRS. ARTHUR
SINCLAIR, MR. AND MRS. W. RICHARD-
SON
SPEIDEL, DR. AND MRS. CARL
STONE, MR. AND MRS. LEO
STONE, MR. AND MRS. S. M.
STRAUSS, DR. AND MRS. DONALD B.
STUNKARP, MRS. HORACE W.
SWIFT. MR. E. KENT
Sworn, MR. AND MRS. GERARD, JR.
SWOPK, Miss HENRIETTA
TOMPKINS, MR. AND MRS. B. A.
WEBSTER, MRS. EDWIN S.
WHITELEY, Miss MABEL W.
WlCKERSHAM, MR. AND MRS. JAMES H.
WlLHELM, DR. AND MRS. HlLMER J.
WILLISTON, MR. SAMUEL
WILLISTON, Miss EMILY
WILSON, MRS. EDMUND B.
WOLFINSOHN, MRS. WOLFE
V. REPORT OF THE LIBRARIAN
In 1961, the Library received 1730 current journals, 59 new titles having been
added during the year. Of the total, the Marine Biological Laboratory subscribed
to 509, received 648 in exchange and 192 as gifts. The Woods Hole Oceano-
graphic Institution subscribed to 115, received 204 in exchange and 62 as gifts.
The Laboratory purchased 63 books, received 109 complimentary copies (8
from authors and 101 from publishers) and accepted 62 miscellaneous gifts.
The Institution purchased 67 books and received 22 as gifts. The total number
of books accessioned totalled 323.
Through purchase, exchange, and gift, the Laboratory completed 16 journal
sets and partially completed 25. The Institution completed 7 sets and partially
completed 8. There were 2647 reprints added to the collection, of which 1809
were of current issue.
The Library now contains 78,800 bound volumes and 219,099 reprints. There
were 535 journal volumes sent out on interlibrary loan and 49 borrowed. About
1200 volumes were bound.
Books and reprints were presented by Drs. Irvine H. Page, John P. Hervey,
Kirk Bryan arid Roberts Rugh. A set of the Catholic Encyclopedia was given by
St. Joseph's College. The Library extends grateful acknowledgment to these
generous donors.
In September the Library purchased a photocopying machine. This is for
use in supplying (at cost) short articles requested on interlibrary loan. This
service eliminates sending out valuable publications and decreases the wear and
tear brought about by interlibrary loans. From September through December,
52 requests were filled by photoprints.
The circulation of books and reprints increased greatly, thus emphasizing the
trend of growth established during the last few years. The Library facilities are
now available, throughout the winter months, to local doctors and to college and
high school students. Expansion and improvements are now being planned and
the staff looks forward to a year of progress.
Respectfully submitted,
DEBORAH L. HARLOW,
Librarian
46 MARINE BIOLOGICAL LABORATORY
VI. REPORT OF THE TREASURER
The market value of the General Endowment Fund and the Library Fund at
December 31, 1961, amounted to $2,025,139 as against book value of $1,193,853.
This compares with values of $1,796,571 and $1,146,393 at the end of the pre-
ceding year. The average yield on the securities was 3.37% of the market value
and 5.72% of book value. The total uninvested principal cash in the above
accounts as of December 31, 1961, was $1,932. Classification of the Securities held
in the Endowment Funds appears in the Auditor's report.
The market value of the pooled securities as of December 31, 1961, was
$373,641 with uninvested principal cash of $283.27; the market value at December
31, 1960 being $333,218. The book value of the securities in this account was
$293,068 on December 31, 1961, compared with $274,294 a year earlier. The
average yield on market value was 3.39% and 4.32% of book value.
The proportionate interest in the Pool Fund Account of the various Funds
as of December 31, 1961, is as follows:
Pension Funds 26.109%
General Laboratory Investment 52.177
Other :
Bio Club Scholarship Fund 1 .494
Rev. Arsenius Boyer Scholarship Fund 1.829
Gary N. Calkins Fund 1.711
Allen R. Memhard Fund 332
F. R. Lillie Memorial Fund 5.766
Lucretia Crocker Fund 6.243
E. G. Conklin Fund 1 .056
M. H. Jacobs Scholarship Fund 753
Jewett Memorial Fund 557
Anonymous Gift 1.973
Donations from the M.B.L. Associates for 1961 were $4,330 as compared
with $4,320 for 1960. Unrestricted gifts from foundations, societies and com-
panies amounted to $36,348.
The James Foundation made a gift of $50,000 towards the building of addi-
tional housing units in Devil's Lane which will be ready for occupancy in June,
1962.
We are administering 15 grants for investigators in addition to those grants
made directly to the Marine Biological Laboratory for general support and train-
ing courses. The amounts of grants vary in accordance with the investigator's
project of research. An amount of 15% based on amount expended is allowed
the Laboratory as overhead.
The Lillie Fellowship Fund with a market value of $96,705 and a book value
of $92,464, as well as the investment in the General Biological Supply House with
a book value of $12,700, is carried in the Balance Sheet, item "Other Invest-
ments." The General Biological Supply House for the fiscal year ended June
30, 1961, had a profit after taxes of $312,000 as compared to $314,034 in 1960
and $303.300 in 1959 and $218,210 in 1958, and $123.430 in 1957. In the fiscal
REPORT OF THE TREASURER 47
year 1961, the Marine Biological Laboratory received dividends from the General
Biological Supply House of $33,020 as against $30,480 in 1%0 and $30,480 in
1959 and $25,400 in 1958.
Following is a statement of the auditors :
To the Trustees oj the Marine Biological Laboratory, Woods Hole, Massachusetts:
We have examined the balance sheets of the Marine Biological Laboratory as
of December 31, 1961 and 1960, the related statements of operation expenditures,
income and current fund for the years then ended, and statement of funds for
the year ended December 31, 1961. Our examination was made in accordance
with generally accepted auditing standards, and accordingly included such tests
of the account records and such other auditing procedures as we considered
necessary in the circumstances.
In our opinion, the accompanying financial statements present fairly the
assets, liabilities and funds of the Marine Biological Laboratory at December
31, 1961, and the results of its operations for the year then ended.
Boston, Massachusetts
March 16, 1962 LYBRAND, Ross BROS. & MONTGOMERY
JAMES H. WICKERSHAM,
Treasurer
48 MARINE BIOLOGICAL LABORATORY
MARINE BIOLOGICAL LABORATORY
l'> A LANCE SHEETS
December 31, 1961 and 1960
Ini'cstiiicnts
1961 1960
Investments held by Trustee :
Securities, at cost (approximate market quotation 1961— $2,025,000) $1,193,853 $1,146,393
Cash 1,932 255
1,195,785 1,146,648
Investments of other endowment and unrestricted funds :
Pooled investments, at cost (approximate market quotation 1961 —
$373,641) less $5,728 temporary investment of current fund cash 287,340 268,566
Other investments 138,546 137,742
Cash 12,764 10,839
Accounts receivable 41 21
$1,634,476 $1,563,816
Plant Assets
Land, buildings, library and equipment (note) 4,795,960 3,280,059
Less allowance for depreciation (note) 1,189,121 1,142,879
3,606,839 2,137,180
Construction in progress 105 1,455,811
Cash 82,042
U. S. Government obligations, at cost :
$50,000 Treasury certificates, due 5/15/62 50,000
$3,656,944 $3.675,033
Current Assets
Cash 65.623 77,546
Temporary investment in pooled securities 5.728 5,728
Accounts receivable (U. S. Government, 1961—
$20,129; 1960— $43,443) 49,290 59,889
Inventories of specimens and Bulletins 43,712 47,641
Prepaid insurance and other 6,870 16,778
$ 171,223 $ 207,582
REPORT OF THE TREASURER 49
MARINE BIOLOGICAL LABORATORY
BALANCE SHEETS
December 31, 1961 and 1960
Endowment Funds
1961 1960
Endowment funds given in trust for benefit of the Marine Biological Lab-
oratory $1,195,785 $1,146,648
Endowment funds for awards and scholarships :
Principal 126,302 126,302
Unexpended income 9,600 7,285
135,902 133,587
Unrestricted funds functioning as endowment 206,378 206,378
Retirement fund 81,790 71,449
Pooled investments — accumulated gain 14,621 5,754
$1,634,476 $1,563,816
Plant Liability and Funds
Funds expended for plant, less retirements 4,796,065 4,668,475
Less allowance for depreciation charged thereto 1,189,121 1,142,879
3,606,944 3,525,596
Unexpended plant funds 50,000 82,042
3,656,944 3,607,638
Accounts payable 67,395
$3,656,944 $3,675,033
Current Liabilities and Funds
Accounts payable 30,337 41,106
Unexpended research grants 52,837 51,726
Unexpended balances of gifts for designated purposes 8,878 9,663
Current fund 79,171 105,087
$ 171,223 $ 207,582
Note — The Laboratory has since January 1, 1916, provided for reduction of book
amounts of plant assets and funds invested in plant at annual rates ranging from 1% to 5%
of the original cost of the assets.
50 MARINE BIOLOGICAL LABORATORY
MARINE BIOLOGICAL LABORATORY
STATEMENTS OF OPERATING EXPENDITURES, INCOME AND CURRENT FUND
Years Ended December 31, 1961 and 1960
Operating Expenditures
1961
1960
Research and accessory services $ 268,193 $ 250,578
Instruction 158,780 219,234
Library and publications 67,189 61,462
Direct costs on research grants 177,938 182,899
672,100 714,173
Administration and general 83,366 70,037
Plant operation and maintenance 118,993 117,980
Dormitories and dining 160,838 162,713
Additions to plant from current income 27,210 78,654
1,062,507 1,143,557
Less depreciation included in plant operation and dormitories and
dining above but charged to plant funds 48,419 48,086
1,014,088 1,095,471
Income
Research fees 102,081 56,408
Accessory services (including sales of biological specimens, 1961—
$43,045, 1960— $48,817) 86,790 151,109
Instruction fees 27,730 23,905
Grants for instruction and research training 148,078 185,571
Library fees, Bulletin subscriptions and other 39,575 35,174
Reimbursements and allowances for direct and indirect costs on research
grants 195,716 221,197
Dormitories and dining income 123,231 105,086
723,201 778,450
Gifts used for current expenses 40,468 48,300
Grants used for current expenses 1 14,170 143,018
Investment income used for current expenses 110,333 105,066
Total current income 988,172 1,074,834
Excess of operating expenditures over current income 25,916 20,637
Current fund balance January 1 105,087 125,724
Current fund balance December 31 $ 79,171 $ 105,087
REPORT OF THE TREASURER
51
MARINE BIOLOGICAL LABORATORY
STATEMENT OF FUNDS
Year Ended December 31, 1961
Balance Gifts and Invest- Used for Other Balance
Jan. 1, Other ment Current Expendl- Dec. 31,
1961 Receipts Income Expenses turcs 1961
Invested funds $1,563,816 $76,274 $116,909 $108,863 $13,660 $1,634,476
Unexpended plant funds . $ 82,042 63,290 95,332 $ 50,000
Unexpended research
grants $ 51,726 468,319 467,208 $ 52,837
Unexpended gifts for
designated purposes . $ 9,663 40,678 40,468 995 $ 8,878
Current fund $ 105,087 25,916 $ 79,171
$648,561 $116,909 $642,455 $109,987
Gifts 103,968
Grants for research, train-
ing and support .... 468,319
Net gain on sales of
securities 58,004
Appropriated from current
income and other . . . 18,270
$648,561
Expended for construction
of new building .... 95,332
Scholarship award 2,700
Payments to pensioners . . 10,960
Other 995
$109,987
52
MARINE BIOLOGICAL LABORATORY
MARINE BIOLOGICAL LABORATORY
SUMMARY OF INVESTMENTS OF ENDOWMENT FUNDS
December 31, 1961
Securities held by Trustee :
General endowment fund:
Investment
U. S. Government securities $
Corporate bonds
Cost
35,110
514,520
% of
Total
3.6
52.0
Market
Quotations
$ 35,963
501,318
% of I
Total
2.1
29.8
ncome
1961
$ 1,651
$ 20,625
Preferred stocks
84,778
8.6
73,012
4.4
3,283
Common stocks
354,366
35.8
1,069,462
63.7
31,399
988,774
100.0
1,679,755
100.0
56,958
General Educational Board endowment
fund:
U S Government securities
31,040
15.1
31,853
9.2
1,491
Other bonds
86,679
42.3
87,550
25.3
3,125
Preferred stocks
26,745
13.0
25,312
7.3
1,160
Common stocks
60,615
29.6
200,669
58.2
5,649
205,079
100.0
345,384
100.0
11,425
Total securities held by
Trustee $1,193,853
$2,025,139
Investments of other endowment and un-
restricted funds :
Pooled investments :
U. S. Governmen
Corporate bonds
Preferred stocks
Common stocks
Other investments :
U. S. Government securities $ 7,000
Other bonds 47,906
Preferred stocks 3,728
Common stocks 46,530
Real estate 33,382
138,546
Total investments of other en-
dowment and unrestricted
funds $ 431,614
Total investment income
Custodian's fees charged thereto
Income of current funds temporarily invested
in pooled securities
$ 68,383
securities ....
15,111
5.2
15,027
4.0
146
134,718
46.0
134,474
36.0
6,928
192
143,239
48.8
224,140
60.0
5,408
$ 293,068 100.0 $ 373,641 100.0 $ 12,674
$ 350
1,998
131
34,195
36,674
$ 49,348
$117,731
(577)
(245)
Investment income distributed to funds
$116,909
MATING BEHAVIOR AND SOCIAL STRUCTURE
IN LOLIGO PEALII
JOHN M. ARNOLD
Department of Zoology, University of Minnesota, Minneapolis 14, Minnesota
In the course of studies on embryological development of the squid, Loligo
pealii, observations of the social behavior and mating reactions of this species were
made. Observations made in the laboratory were confirmed by watching squid in
their natural habitat. These studies have revealed a method for artificially stimu-
lating mating and subsequent egg-laying. It is thus possible to have a reliable
source of squid eggs, at convenient times, in the laboratory. Drew (1911) made
extensive observations on copulation and egg-laying in Loligo pealii but, to the
author's knowledge, nothing has been recorded on mate selection and social
structure in this species.
Squid are particularly suitable for a study of social behavior for a number of
reasons. They are pelagic and gregarious. Since there are no territorial separa-
tions, there is ample opportunity for a maximum of intraspecific interaction and,
therefore, for the development of a social structure. They possess a moderate
amount of intelligence which would enhance the establishment of social structure.
Other than a few scattered observations on the Octopoda, very little has been
published on the social behavior of the Cephalopoda. This paper is an attempt to
show that Loligo pealii has a social structure concerned with its mating behavior.
MATERIALS AND METHODS
The animals used in this study were captured in fish traps in the vicinity of
Woods Hole or Barnstable, Massachusetts, in the summers of 1960 and 1961.
Late in the summer of 1961 some animals were used that had been captured in fish
traps off the coast of Rhode Island. There seemed to be some minor differences in
these two populations, indicated by their interaction, but their intra-population
interaction was the same. The animals were all sexually mature but varied in
mantle length from about six to ten inches. The males appeared to be slightly
larger than the females. The animals were obtained from the Supply Department of
the Marine Biological Laboratory, usually shortly after their capture and at most
one day thereafter. They were kept in a 2\ by 6 foot fiberglass tank filled to a
depth of 12 inches with rapidly running sea water. Usually from four to ten
animals were kept in such a tank at one time without apparent overcrowding.
Usually more males were present than females, although in some cases the situation
was reversed. The animals were fed about two Fnndulus heteroclitus per squid,
usually daily. Under these conditions the squid survived about five days. In one
case a male survived for seventeen days in a tank that was undisturbed except for
periodic feeding.
Males and females are easily distinguished by the presence of the white testis in
53
54 JOHN M. ARNOLD
the male, the slimmer outline of the males, and the presence of the bright orange
accessory nidamental gland visible through the mantle of the female. Individuals
• >f either sex could be distinguished by their size or by characteristic wounds and
srars on their mantles.
The animals were stimulated with a naturally laid egg mass tied to a cotton
cord and lowered into one corner of the tank. An artificial egg mass was con-
structed of water-filled tubing made of polyethylene sheet fused together at the
edges. A small amount of phenol red was added to the water used in the tubing to
give it an orange color like that of the egg mass. Other animals were introduced to
an existing group by slowly submerging a bucket containing the new animals into
the tank and allowing them to swim out. The observations of squid in nature were
made from a fixed wharf or an anchored boat in shallow water after dark. These
observations were made with the assistance of a fixed incandescent lamp.
OBSERVATIONS
Initiation of sexual behavior
Normally, the captive squid swim parallel to each other in a small school moving
back and forth in synchrony. There is no apparent social order to their position
in these small schools. The males and females move about in the tanks paying no
apparent attention to each other. Since these animals were chosen randomly from
a much larger group (about 30-50), any prior social pattern is assumed to have
been broken. There seems to be no particular dominance in feeding behavior since
any Fundulus offered is taken by the nearest squid. No other evidence of any
social pattern has been observed and if any such structure existed at this time it was
latent. Therefore, it is assumed that mate selection had not taken place in this
condition. In these circumstances a stimulus could be presented and characteristic
responses observed.
If a naturally laid egg mass was tied to a string and placed in one corner of the
tank, the squid almost immediately "broke formation" and investigated the egg
mass. This response began when the animals swam rapidly toward the egg mass,
formed their arms into a cone, and pointed at the egg mass. Occasionally the egg
mass would be flushed with spurts of water from the funnel of an approaching squid.
One animal after another would dart up to the egg mass and "feel" it with its arms.
Then each would rapidly dart away and rejoin the group. Two individuals would
occasionally approach the egg mass at the same time. Both males and females
would show this response which would occur within about twenty seconds after the
introduction of the egg mass.
This stimulus seemed to be completely visual because of the speed with which
the squid responded. This hypothesis was checked by using an artificial egg mass
of polyethylene tubing. This artificial egg mass elicited the same response as a real
egg mass. The animals investigated and felt it but responded abnormally by flush-
ing it repeatedly with spurts of water from their funnels and by swimming excitedly
to and fro. However, this was still followed by normal mate selection and the
establishment of a hierarchy. Usually the egg string would be held in the arms for
several minutes and then dropped on the bottom of the tank. In two cases egg
capsules were actually laid on the artificial egg mass although the female made
MATING BEHAVIOR IN LOIJGO PEALII
several approaches before finally attaching the egg string. Further evidence of the
visual nature of this stimulus can he drawn from the fact that in the absence of an
egg mass, egg strings would be attached to anything that resembled an egg mass.
Several times egg strings have been deposited on the arms of a dead squid left in
the tank. In cases of deprivation of stimulus the squid will even investigate the
extended fingers of a human hand placed in the tank (not recommended).
The stimulus worked best on squid that had not been known to have bred
recently, hence the necessity of getting the animals as soon as possible after their
capture. If deprived of stimulus for a long period of time, females would eventually
drop egg strings but made no apparent attempt to form an egg mass unless a nucleus
of egg strings accumulated by chance at the tank drain.
This response to an egg mass has been observed in nature by dangling an egg
mass on a string in front of a school of squid. Egg-laying has occurred in these
egg masses.
Establishment of a hierarchy
The investigative behavior was followed by dominance determination behavior.
This began by the males raising one median arm a few centimeters above the rest
of the arms and waving it. Sham battles usually followed in which the males
rushed at each other but did not actually touch each other. At the same time they
developed dark brown lateral areas at the base of the arms. This color pattern
seemed to be characteristic of sexually aroused male squid. During this time a
given male would place himself between the rest of the group and a female of his
choice. Any approaching male would be threatened by a waving of the median
arm. If the approaching male was persistent, he would be driven off by rushes from
the selecting male. In cases where the intruder was extremely insistent, the two
males would sometimes bump tails and display characteristic dark colored spots
along the lateral margins of the fins. These spots were not observed at any other
time on any male. This has been interpreted as a further warning sign. In a
relatively few cases there was actual contact between two males. In these cases the
males rushed at each other and one grasped the other around the mantle. In three
observed cases the arms interlocked and in one case the tip of an arm was bitten off.
This behavior continued between all the males in the tank until one male was estab-
lished as the dominant male. Other males appeared subordinate to this male. The
subordinate males each selected a female and would undergo similar combat among
themselves. An individual's position in the social structure seemed roughly
correlated with its size. Since there were fewer females than males in the tank,
a few males did not have mates. Only rarely did one of the mateless males succeed
in displacing a mated male. The squid taken from the traps at Rhode Island
seemed to be more aggressive and would challenge and displace larger squid taken
from the Cape Cod population. As the physical conditions of a male deteriorated
the same social structure remained until he reached a completely defenseless state
and another male could take over his female. If a new male was introduced to a
group that had established a social structure he would be immediately challenged
by the other males until he was integrated into the social structure. This would
often result in a displacement of an established male and the displaced male would
then displace his subordinate or, if in poor physical condition, be relegated to the
56 JOHN M. ARNOLD
mateless group. Only rarely did any male attempt to change its mate
spontaneously.
The behavior of the females was less active. Normally a female would show
slight avoidance of a male but seemed to have no mate preference. While mate
selection and challenging went on between males, females paid no apparent attention
to the males. Once the social structure among males had been established the
males attempted copulation with the females. Normally a female would resist by
darting away quickly when the male attempted copulation. After a short interval
the female usually accepted the male's advances and copulation followed.
Copulation and egg-laying
The behavior and events during copulation have been well described by Drew
(1911) and only details not recorded there will be mentioned here. The male
swam parallel to the female and moved back and forth exactly at the same time (as-
suming no other male intruded). The spots at the base of the arms of the male
became intense. Occasionally copulation was preceded by the male reaching out
with one arm and lightly touching the female on the mantle behind the head. There
were two methods of copulation observed. Most common was the lateral method
in which the male paralleled the female and grasped her around the mantle behind
the head, the hectocotylized arm was placed into his own mantle, spermatophores
picked up, and then transferred to the female by way of her funnel. This took
about ten seconds. The second method of copulation involved a meeting of the
male and female head to head and probably resulted in a transfer of the spermato-
phores to the buccal pouch. This method was infrequently observed.
Occasionally a female would strongly resist a male and would not permit copula-
tion at all. This was usually done by swimming rapidly away or by struggling
when the male grasped her. Rarely, if a male was very aggressive, the female
would actively resist by grasping the male with her arms. On three occasions
females have propelled themselves out of the tank in an attempt to escape an aggres-
sive male. In cases where the females outnumbered the males a polygamous rela-
tionship would develop. In two cases, when males were in reduced physical
condition, the females took the aggressive role and attempted to force copulation by
grasping the male about the mantle. In one case an aggressive female could get
no response and finally ate part of the male. Several times males displayed
necrophilia (Daveian behavior) when no living females were available to them.
Copulation was usually followed by egg-laying. Drew (1911) has given an
elaborate account of egg-laying and all that will be mentioned here is a brief account
for the sake of completeness. After copulation the female approached the egg mass
and again investigated it. An egg string was then passed from the funnel to the
arms which encircled it. The female approached the egg mass and reached into
the center and manipulated the egg string into place. One end of the egg string is
free of eggs, narrower, and denser in composition. This end was intertwined in
the egg mass with the tips of the arms so that the string was firmly enmeshed. If
during this time the string was dropped the complete operation was continued as if
nothing abnormal had happened. The female quickly retreated from the egg mass
following the attachment of the egg string. A new egg string was then passed up
MATING BEHAVIOR IX LOLIGO PEALII 57
to the arms and the process repeated. In one case, seven females added 26 egg
strings to one small egg mass of twenty strings in thirteen minutes.
DISCUSSION
The major points of this paper are the nature of the stimulus of reproductive
behavior and the resultant social structure. The stimulus was no doubt visual
because of the speed at which the response occurred and the fact that objects having
a resemblance to egg masses would cause such a response. It is the author's
opinion that this stimulus elicits sexual behavior. This would explain the fact that
egg masses are often found attached to Fucus or similar sea weeds. Several fe-
males deposit their egg strings on a common egg mass which agrees with this
hypothesis. The report of vast beds of squid eggs off the California coast could
also be explained by such a hypothesis (McGowan, 1954). Undoubtedly, there
must be another stimulus that elicits the original mating that results in the forma-
tion of the original egg mass. This egg mass then elicits mating behavior in other
squid. The nature of the original stimulus remains unknown and it is possible that
this original mating occurs spontaneously.
The social structure in the males appears to be a classical peck order with an
establishment of a dominant male able to resist all other males, and of a series of
subordinate males. These subordinate males are able to resist males of a lower
position but may be displaced by a male of higher position.
The author wishes to thank Mr. Robert O. Lehy of the Marine Biological
Laboratory Supply Department for his cooperation with this project, and Dr. N. T.
Spratt, Dr. R. K. Josephson, and Mr. R. B. Forbes for reading and criticizing this
manuscript.
SUMMARY
1. Observations of Loligo pealii have shown the egg mass can stimulate sexual
behavior. This stimulus apparently has a visual basis.
2. This stimulus is followed by establishment of a social hierarchy and by mate
selection by the males. The males exhibit warning displays, sham battles, and mate
protection during this time.
3. Normally the females respond passively but occasionally they will take an
aggressive role.
4. This mating behavior results in copulation and egg-laying; thus a method
for obtaining naturally laid eggs has been revealed.
LITERATURE CITED
DREW, G. A., 1911. Sexual activities of the squid Loligo pealii. I. /. Morph., 22 : 327-360.
McGowAN, J. A., 1954. Observations on the sexual behavior and spawning of the squid Loligo
opalescens at La Jolla, California. Calif. Fish. Game, 40 : 47-54.
RESPIRATION, ELECTRON-TRANSPORT ENZYMES, AND
KREBS-CYCLE ENZYMES IN EARLY DEVELOPMENTAL
STAGES OF THE OYSTER CRASSOSTREA VIRGINICA1
ROBERT E. BLACK
Department of Biology and Virginia Institute of Marine Science,2
College of William and Mary, Williamsburg, Virginia
A marked increase in the rate of respiration during early development has been
noted in many different organisms (see Brachet, 1950; Boell, 1955). In most
cases the changes in respiratory enzymes which might contribute to the respiratory
increases have not been fully studied. Of particular interest in this connection are
studies which have dealt with enzymes of the tricarboxylic acid (citric acid) cycle,
as well as those investigations of the enzymes involved in the transfer of electrons
between substrates and oxygen, via the systems which oxidize reduced pyridine
nucleotides and succinic acid.
Parallels between the increase in respiration and that of cytochrome oxidase
have been noted in the grasshopper (Bodine and Boell, 1936; Allen, 1940), the
salamander (Boell, 1945), and the chick (Albaum and Worley, 1942; Albaum et a!.,
1946; Levy and Young, 1948). In Xenopus laevis Boell and Weber (1955) have
reported an increase in cytochrome oxidase beginning during cleavage. This is in
contrast to the data of Spiegelman and Steinbach (1945) on the developing eggs of
Rana pipiens and to those of Petrucci (1957) on cytochrome oxidase during the
early development of Bufo bujo. In the developing salamander increases in the
activity of succinoxidase have been shown to be similar to those of cytochrome
oxidase (Boell, 1948; Krugelis et al, 1952).
Several enzymes of the tricarboxylic acid cycle have been studied in embryos of
the chick between the ages of 2 and 6^ days of incubation by Mahler, Wittenberger
and Brand (1958). Aconitase, isocitric dehydrogenase, alpha-keto glutaric de-
hydrogenase, succinic dehydrogenase, malic dehydrogenase, and fumarase all in-
creased at rates equal to or above that of total embryonic protein up to three days
of incubation ; following this time only malic dehydrogenase was found to accumu-
late as rapidly as total protein. On the basis of assays in homogenates these authors
concluded that the oxidation of pyruvate by enzymes of the citric acid cycle is
probably not the rate-limiting factor in the embryonic respiration. Brand and
Mahler (1959) have obtained similar results in the chick in an investigation of
enzymes oxidizing reduced diphosphopyridine nucleotide. Diaphorase, DPNH-
oxidase, and cytochrome oxidase all increased in specific activity until the fourth
day of development, after which their rate of accumulation was exceeded by that of
other embryonic protein.
In eggs of marine invertebrate animals few studies have been made of changes in
1 This investigation was supported by a grant (G-9847) from the National Science
Foundation.
2 Contribution No. 116.
58
ENZYMES IX OYSTER EMBRYOS 59
respiratory enzymes during development. In the sea urchin Gustafson and Hassel-
berg (1951) found that the activities of succinic dehydrogenase and malic dehy-
drogennse increased after the mesenchyme-blastula stage. Deutsch and Gustafson
(1952) found lower activities of cytochrome oxidase in homogenates of blastulae
than in those of cleaving eggs. In an attempt to relate respiratory changes to
changes in enzymes, Runnstrom (1930, 1956) has postulated that the terminal
oxidase system is present but relatively inactive until the swimming, larval stage is
reached in the sea urchin. Black and Tyler (1959) have reached a similar con-
clusion for eggs of Urechis caupo and Strougylocentrotus purpuratus on the basis
of relative rates of oxidation of carbon monoxide in the light and dark by these
developing embryos.
In view of the importance of enzymes of the citric acid cycle and the terminal
electron-transport system in the respiration of most animals, it is of interest to
extend the present knowledge concerning developmental changes in these enzymes
to embryos of marine invertebrate animals other than the sea urchin. In the present
report, therefore, data on changes in the respiration and in the activities of several
respiratory enzymes of early embryos of the oyster, Crassostrca virginica, are
presented. Cleland (1951) has demonstrated the presence of most enzymes of the
Krebs citric acid cycle in homogenates of oyster eggs, since he obtained respiratory
stimulation in such homogenates after the addition of intermediate substrates of
this cycle. In addition, succinoxidase and cytochrome oxidase have been shown to
be present in the same material by Cleland. The present study is the first in which
the existence of a DPNH-oxidase system has been demonstrated and in which
direct methods have been used to indicate the presence of enzymes of the Krebs
cycle in oyster embryos.
MATERIALS AND METHODS
Oysters were collected during the spawning season from pilings at Gloucester
Point, Virginia, and they were stored in a refrigerated river-water system, in which
the salinity was 18 to 20 ppm. To obtain gametes, the animals were opened, and
portions of the gonads were removed and shaken gently in the water. The eggs
were washed by settling and inseminated. The fertilized eggs were washed several
times by settling until excess sperm had been removed. The eggs were cultured in
a rotating flask at 20° C. in water of the above salinity containing 0.005 M glycyl-
glycine buffered to pH 7.8. Penicillin (100 units per ml.) and streptomycin (50
micrograms per ml.) were added to all cultures to retard bacterial growth. The
concentration of eggs in the rotating flask never exceeded 107 per liter.
Since it was difficult to ascertain the percentage of fertilization immediately,
eggs were removed for the first measurements of respiration and/or enzyme activity
immediately after the first cleavage (about 1-1-^ hours following fertilization). In
all experiments reported the percentage of cleaving eggs was better than 85%.
Later times at 20° C. and stages used for subsequent measurements were as follows :
8-10 hours (swimming blastula), 23-25 hours (trochophore), and 48-50 hours
(early veliger). Only swimming embryos were used for measurements in blastula,
trochophore, and veliger stages.
The eggs or embryos removed for analysis were washed by centrifugation and
suspended in a known volume of sea water (25-50 ml.), so that aliquots could be
60 ROBERT E. BLACK
taken for counting. From the suspension, five aliquots of 0.5 or 1.0 ml. were taken,
utul these were each diluted to 25 or 50 nil. From each diluted aliquot two samples
of 0.5 or 1.0 ml. were counted, making a total of ten counts (1,000 to 2,000
embryos) in all.
In one series of experiments measurements of respiration were made on living
embryos and they were then homogenized in dilute phosphate buffer for measure-
ments of succinic dehydrogenase, DPNH-oxidase, and cytochrome oxidase. For
measurements of respiration the embryos were suspended in dilute sea water con-
taining 0.005 M glycylglycine, pH 7.8, plus penicillin and streptomycin as indicated
above. Aliquots of 2.0 ml. were measured into duplicate Warburg vessels of 15-ml.
capacity and respiration was measured at 25° C. for one hour. The embryos were
then rinsed carefully into centrifuge tubes, packed by centrifugation, resuspended in
10 volumes of cold 0.033 M phosphate, pH 7.4, and homogenized with a syringe
and No. 20 needle. The homogenate was made to a known volume ( 1 to 5 ml. )
in a calibrated vessel, and aliquots were withdrawn for the determination of
the activities of cytochrome oxidase, DPNH-oxidase, and succinic dehy-
drogenase. Measurements of these activities were completed within 1| hours after
homogenization.
In other series of experiments measurements of the activities of TPN-specific
isocitric dehydrogenase and alpha-ketoglutaric dehydrogenase and of malic dehy-
drogenase and aconitase were performed. For assaying the first two enzymes,
embryos were homogenized in a solution containing 0.3 M sucrose and 0.05 M Tris
(hydroxymethyl) aminomethane, pH 7.4. The latter two enzymes were assayed in
homogenates made in 0.03 M Tris, pH 7.4. All measurements were completed
within 1^ hours after homogenization.
All of the measurements of enzyme activity were performed at 25° C. With the
exception of cytochrome oxidase, which was assayed manometrically, the activities
of all enzymes were determined by spectrophotometric methods. For the latter
assays, 3.0-ml. silica cuvettes of 1-cm. light path were used in a Beckman Model DU
spectrophotometer. Between readings the cuvettes were incubated in a water bath
at 25° C. or at room temperature of 25°.
All assays were performed at substrate concentrations which allowed initial rates
to follow zero-order kinetics. Assays were always performed at two or more levels
of homogenate concentration. This provided continuous assurance that reaction
rates were directly proportional to the concentration of homogenate in the reaction
mixtures. In general, duplicate determinations were made at one concentration of
homogenate and a single determination was made at half this concentration. Analy-
ses in which the reaction rate was not proportional to homogenate-concentration
or in which the rate of reaction was not constant were discarded. Differences
between rates recorded for any pair of duplicates seldom exceeded 10% of the mean
rate for the pair. Details of the assay methods for the enzymes are listed below.
(Homogenate percentages given below are approximate, based on volumes of
packed embryos.)
Cytochrome oxidase. Manometric method of Schneider and Potter (1943).
Warburg vessels contained 0.02 M ascorbic acid, pH 7.4, 2 X 10~* M cytochrome
c (based on M. W. of 16,000), 0.067 M phosphate, pH 7.4, 6 X 10~4 M aluminum
chloride, and 0.5 to 1.5 ml. of 5% homogenate. Assays were made at three levels
of homogenate concentration, and the auto-oxidation rates of ascorbic acid were
ENZYMES IN OYSTER EMBRYOS 61
obtained by extrapolating the rates of oxygen uptake to zero homogenate concen-
tration. After equilibration, readings were taken for 5-10 minutes before tipping
in 2 X 10~3 M cytochrome c from the side arm. The initial rate of activity was
calculated on the basis of three readings taken at 5-minute intervals after the addi-
tion of cytochrome c from the side arm. The endogenous oxygen uptake was ob-
tained by subtracting the low rate of auto-oxidation of ascorbic acid in the absence
of cytochrome c from the rate of uptake in the vessels before the cytochrome c was
added to the main chamber. The endogenous rates of oxygen uptake were usually
less than 5% of the rates in the presence of cytochrome c. The auto-oxidation
rates of ascorbic acid when both cytochrome c and homogenate were present varied
between 60 and 90 microliters per hour in different experiments.
DPNH-o.vidase. Spectrophotometric method of Brand and Mahler (1959).
The cuvettes contained 1.7 X 10~4 M reduced diphosphopyridine nucleotide
(DPNH), 3 X 10-6 M cytochrome c, 0.05 M phosphate, pH 7.4, and 0.05 to 0.2
ml. of 10% homogenate. The blank cuvette contained all components except
homogenate. After the addition of all substances to blank and experimental cells,
they were incubated for 10 minutes at 25° C. before the first reading was taken.
Thereafter readings were taken at 340 millimicrons at 2- to 4-minute intervals for
12 to 20 minutes. No endogenous activity was subtracted from the rates, since
controls in several experiments showed that after the initial incubation the A340 of
homogenate plus cytochrome without added DPNH remained constant. Because
of the high absorption of reduced DPN, the use of blank cuvettes without substrate
was not feasible.
Succiinc dehydrogcnase. Spectrophotometric method of Slater and Bonner
(1952). The experimental cuvettes contained 0.01 M sodium cyanide, 10~3 M
potassium ferricyanide, 0.02 M sodium succinate, 0.1 M phosphate, pH 7.4, and 0.1
to 0.4 ml. of 10% homogenate. The blank cuvette contained only homogenate in
0.1 M phosphate. After the addition of all components the cuvettes were incubated
at 25° C. for 10 minutes before the first reading was taken. The absorbence at 410
millimicrons was measured at 5- or 10-minute intervals for 25 to 40 minutes.
Isocitric deJiydrogenase. Spectrophotometric method of Ochoa (1948). The
experimental cuvette contained 0.02 M Tris (hydroxymethyl) aminomethane, pH
7.5, 7 X 10-* M isocitrate, 5 X 1Q-3 M sodium cyanide, 4.5 X 10"5 M triphospho-
pyridine nucleotide (TPN), 6 X ICh4 M MnCU, and 0.1 or 0.2 ml. of 10% homog-
enate. The blank contained all components except isocitrate. Readings at 340
millimicrons were begun immediately after the addition of homogenate to both blank
and experimental cells, and they were continued at 2- or 3-minute intervals for
8-10 minutes.
Alpha-ketoglutaric deJiydrogenase. Method of Sanadi and Littlefield (1951).
Experimental cuvettes contained 0.1 M phosphate, pH 7.7, 2.5 X 10~3 M co-
carboxylase, 5 X 10'* M sodium cyanide, 8.6 X 10~5 M 2.6-dichlorophenolindo-
phenol, 3.5 X 10~3 M magnesium chloride, 6.7 X 10~3 M alpha-ketoglutarate, pH
7.7, and 0.1 or 0.2 ml. of 10% homogenate. The blank contained all components
except substrate. The readings were begun immediately after the addition of
homogenate, and 4 readings at 600 millimicrons were taken at 2-minute intervals.
In one experiment ferricyanide was used as the hydrogen acceptor in the assay of
this enzyme by the method of Stumpf et at. (1947). The cuvettes contained simi-
lar amounts of all components, except that 10~3 M potassium ferricyanide was
62 ROBERT E. BLACK
substituted for the 2,6-dichlorophenolindophenol, and the decrease in optical density
was read at 410 millimicrons for 25-30 minutes.
Fumarase. Spectrophotometric method of Racker (1950). Experimental
cuvettes contained 0.05 M malate in 0.05 M phosphate, pH 7.4; blank cuvette
contained only phosphate. After addition of 0.05 or 0.1 ml. of 10% homogenate to
both blank and experimental vessels, the absorbence at 240 millimicrons was meas-
ured at 2-minute intervals for 10 minutes.
Malic dehydrogenase. Method of Mehler et at. (1948). The experimental
cuvette contained 0.02 M Tris buffer, pH 7.5, 1Q-3 M sodium cyanide, 5.1 X 10~5 M
oxalacetate, pH 7.5, 1.7 X 10~4 M DPNH, and 0.05 to 0.1 ml. of \% homogenate.
The substrate was added last, and readings were taken at 340 millimicrons at 15-
second intervals for one minute. The oxalacetate solution was prepared just before
use and kept on ice. Not more than 10 minutes elapsed between the preparation of
this substrate and the enzyme assay. The amounts of pyruvic acid formed by spon-
taneous decarboxylation of the oxalacetate were considerd to be negligible. At least
four determinations of enzyme activity were always made.
Aconitase. Method of Racker (1950). The experimental cuvettes contained
0.03 M citrate in 0.05 M phosphate, pH 7.4, and 0.05 or 0.1 ml. of 10% homogenate.
The blank cuvette contained only homogenate and buffer. The increase in ab-
sorbence at 240 millimicrons was determined at 2-minute intervals between 5 and 15
minutes after the start of the reaction.
The following values (in cm.2/mole X 106) were used for the molar extinction
coefficients of the substances used in the assays : DPNH and TPNH, 6.22 at 340
millimicrons (Horecker and Kornberg, 1948), cis-aconitate, 3.30 at 240 millimicrons
(Racker, 1950), ferricyanide, 1.00 at 410 millimicrons (Strittmatter and Velick,
1956), and 2,6-dichlorophenolindophenol, 18.5 at 600 millimicrons (Sanadi and
Littlefield, 1951).
The sources of materials used in the assays were : Tris (hydroxymethyl)
aminomethane, EDTA, citric acid, succinic acid, and ascorbic acid, Will Corpora-
tion; potassium ferricyanide and 2,6-dichlorophenolindophenol, Fisher Chemical
Company; DPN (95-100%), DPNH (Type I, 90-95%), and thiamine pyrophos-
phate (cocarboxylase, 80-90%), Sigma Chemical Company; and cytochrome c
(0.34% iron), trisodium isocitrate, alpha-ketoglutaric acid, and oxalacetic acid,
Nutritional Biochemicals Corporation.
Stock solutions of coenzymes and substrates used in the assays were made and
frozen in several small batches, so that only one or two thawings were necessary in
using each batch. Stock solutions of other chemicals were stored at 2-4° C.
Before beginning each experiment, sufficient quantities of all solutions were made
so that, with the exception of the assay for malic dehydrogenase, all determinations
during any 2-day experiment were made from the same stock solutions. The con-
centrations of the pyridine nucleotide coenzymes were determined at intervals dur-
ing some 2-day experiments to ascertain whether decomposition of these materials
had occurred during storage.
RESULTS
Respiration. Cleland (1950) has reported that in the eggs of Ostrca respiration
rises at least until the blastula stage. To this author's knowledge, no other investi-
gation of respiratory changes during early development of the oyster has been
ENZYMES IN OYSTER EMBRYOS
63
made. The results of the present series of respiration measurements are presented
in Table I. The rate of respiration of the blastula was found to be about three
times that of the egg at the first cleavage. A further three-fold increase in rate oc-
curred between the blastula stage (9 hours) and the trochophore stage (24 hours).
The respiratory rate of the two-day-old larva (early veliger) was not found to be
significantly different from that of the trochophore. The levelling-off of respira-
tory rate at the trochophore stage is most likely not due to starvation, since oyster
embryos cultured at 20° apparently do not begin feeding until they are about 60
hours old (Amemiya, 1926).
TABLE I
Rales of respiration of developing eggs of Crassostrea virginica. The values represent micro-
moles of oxygen taken up per minute by one million embryos. Numbers in paren-
theses in headings refer to hours after fertilization. Embryos were grown
at 20° C., and respiration was measured manometrically at 25° C.
for periods of about one hour
Expt.
Cleaving eggs
(1-li hrs.)
Blastulae
(8-10 hrs.)
Trochophores
(23-25 hrs.)
Early veligers
(48-50 hrs.)
1
0.027
0.076
0.180
2
0.024
0.045
0.153
3
0.021
0.219
4
0.020
0.109
0.162
5
0.159
0.139
6
0.215
0.142
7
0.182
0.179
8
0.284
0.234
9
0.203
0.181
10
0.029
0.067
0.100
Average
0.024
0.074
0.195
0.163
±0.003
±0.023
±0.039
±0.042
Succinic dehydrogenase, DPNH-o.vidase, and cytochrome oxidase. In meas-
uring the activities of these enzyme systems it was desirable to determine the condi-
tions under which maximal rates of electron transfer could occur in homogenates.
In preliminary experiments the effects of pH, buffer concentration, and substrate
concentration on the activities of the succinic dehydrogenase and DPNH-oxidase
were tested. Variation of pH between 7.2 and 7.8, of succinate between 0.01 and
0.04 M, of phosphate between 0.05 and 0.15 M, and of ferricyanide between 1Q-3
and 2 X 10"3 M had but little effect on the activity of succinic dehydrogenase. In
one experiment the rate of reduction of cytochrome c by a homogenate of fertilized
eggs was compared with the rate of ferricyanide reduction, when the homogenate
was oxidizing succinate. The rate of transfer of electrons to cytochrome c was
measured by determining manometrically the activity of the succinoxidase system,
in which succinic dehydrogenase is the rate-limiting factor, when cytochrome c is
added to the system. This determination, made by the method of Schneider and
Potter (1943), indicated that the cytochrome was reduced at a rate about one-third
that of the ferricyanide (measured by the spectrophotometric method). This re-
sult is similar to that of Green et al. (1955), who found that highly purified prepara-
tions of the succinic dehydrogenase complex from beef-heart mitochondria reduced
64
ROBERT E. BLACK
TABLE II
Activities of succinic dehydrogenase, DPNH-oxidase, and cytochrome oxidase in homogenates
of embryos of the oyster. Homogenates were made in 0.03 M phosphate, pH 7.4.
Assays were performed at 25° C. See text for details of assay systems.
Values represent micromoles of substrate (ferricyanide, DPNH,
or oxygen] utilized per minute by one million embryos
Expt.
Cleaving egg
Blastula
Trochophore
Veliger
Succinic dehydrogenase
1
0.190
0.160
0.135
2
0.155
0.090
0.155
3
0.170
0.175
4
0.110
0.185
0.175
6
0.185
0.155
7
0.105
0.130
8
0.255
0.135
9
0.135
0.145
10
0.098
0.126
0.085
Average
0.145
0.140
0.164
0.130
±0.035
±0.036
±0.042
±0.024
DPNH-oxidase
1
0.111
0.098
0.116
2
0.131
0.088
0.077
3
0.118
0.121
4
0.081
0.130
0.097
6
0.129
0.083
7
0.134
0.139
8
0.210
0.125
9
0.167
0.085
10
0.115
0.074
0.078
Average
0.111
0.098
0.144
0.102
±0.017
±0.026
±0.035
±0.025
Cytochrome oxidase
1
0.383
0.445
0.368
2
0.408
0.389
0.578
3
0.362
0.339
4
0.362
0.618
0.492
6
0.337
*
7
0.300
0.177
8
*
0.215
9
0.535
0.284
10
0.393
0.325
0.330
Average
0.382
0.444
0.421
0.252
±0.018
±0.106
±0.122
±0.059
* Activity not proportional to concentration of homogenate.
ENZYMES IN OYSTER EMBRYOS 65
ferricyanide more rapidly than cytochrome c and also more rapidly than several
other artificial electron-acceptors.
In preliminary experiments with DPNH-oxidase, it was found that this enzyme
system was inhibited about 30% by a 2-fold excess of DPNH or by a 2-fold excess
of cytochrome c. These effects have also been reported for the DPNH-oxidase sys-
tem of chick embryos by Brand and Mahler ( 1959) . The optimum pH for this sys-
tem was found to be 7.4. The activity of the system was decreased in phosphate
concentrations below 0.05 M, and occasionally was found to be somewhat higher in
0.1 M than in 0.05 M buffer ; however, the latter effect was not consistently obtained.
In several experiments the activity of the DPNH oxidase was shown to be inhibited
95% or more by 10~3 M cyanide. In the absence of added cytochrome, the DPNH
oxidase activity was very low in most experiments.
Since the activity of cytochrome oxidase was always greater than the combined
activities of the two systems above, no special effort was made to determine the
conditions for maximal activity of this enzyme. In preliminary experiments it was
found that neither the concentration of cytochrome c nor that of ascorbic acid was a
limiting factor in the assays. The effects of variation of pH or buffer concentra-
tion were not tested.
The results of measurements of these components of the electron transport sys-
tem are presented in Table II. Since relatively large amounts of homogenate were
required for the assays, the experiments were divided into two series. In the first,
measurements were made betwen the first cleavage and 24 hours ; in the second the
rates of enzyme activity in 24- and 48-hour stages were compared. No consistent
change was observed in the activity of succinic dehydrogenase or of DPNH oxidase
during development to the veliger. (The average value for DPNH oxidase at 24
hours is 30-40% higher than those in the blastula and veliger; this difference is
considered to be too small to be significant in view of the variation between values
obtained at each stage in different experiments. )
Cytochrome oxidase is also nearly constant in activity up to the trochophore
stage. A considerable decrease in the activity of this enzyme between 24 and 48
hours was found in most experiments ; moreover, in one measurement at each of
these stages the activity was not proportional to the concentration of homogenate.
These findings may indicate the presence of an inhibitor of this enzyme in late
stages, as suggested by Deutsch and Gustafson (1952) for homogenates of sea
urchin blastulae. A slight clumping of homogenates of trochophores and veligers
usually occurred in the presence of 2 X 10~4 M cytochrome c. This did not appear
to affect the relationship between concentration of homogenate and enzyme activity
in most manometric experiments. In lesser concentrations, such as those used in
the assay of DPNH oxidase, no clumping of the homogenates occurred.
A comparison of respiratory rates during development (Table I ) with the
enzyme activities reported in Table II shows that the terminal enzyme systems must
transfer electrons at an increasing rate as development progresses in order to ac-
count for the increase in respiration. Such an increased rate of transfer could be
due to the greater rate of production of succinate and reduced pyridine nucleotides,
or it could be a result of the synthesis of some rate-limiting component of the
terminal system, such as cytochrome c. Cytochrome c was not a limiting factor in
any of the assay-systems ; it is therefore not possible from present data to determine
66
ROBERT E. BLACK
whether it is rate-limiting for the respiration at any stage of development. Data
presented in the next section indicate that at least two of the enzymes of the citric
acid cycle do increase in activity during one phase of development.
Isocline dehydrogenase and alpha-ketoglutaric dehydroyenase. The levels of
activity of these enzymes at each embryonic stage investigated are listed in Table III.
In contrast to the terminal enzymes, a marked increase in the activity of isocitric
dehydrogenase was observed between 9 and 24 hours in all experiments. Thus in
the trochophore the level of this enzyme is about three times the level in the blastula.
This increase is about equivalent to the increase in respiration during the same
TABLE III
Activities of isocitric and alpha-ketoglutaric dehydrogenase s in homogenates of oyster embryos.
Homogenates were made in 0.3 M sucrose and 0.05 M Tris (hydroxymethyl) amino-
methane, pH 7.4. Assays were performed at 35° C. (for details see text}.
Values represent micromoles of substrate utilized per million
embryos per minute
Expt.
Cleaving egg
Blastula
Trochophore
Veliger
Isocitric dehydrogenase
11
0.080
0.070
0.330
0.100
12
0.069
0.210
0.150
13
0.050
0.043
0.190
0.140
14
0.104
0.131
0.146
0.289
15
0.083
0.088
0.284
0.277
16
0.098
0.109
0.272
0.256
Average
0.097 ± 0.024
0.088 ± 0.030
0.239 ± 0.060
0.202 ± 0.074
Alpha-ketoglutaric dehydrogenase
11*
0.084*
0.220*
0.290*
13
0.024
0.018
0.055
0.033
14
0.021
0.021
0.038
0.038
15
0.028
0.020
0.050
0.058
Average**
0.024 ± 0.003
0.020 ± 0.001
0.048 ± 0.007
0.043 ± 0.011
* Assayed with ferricyanide.
** Data for 2,6 dichlorophenolindophenol only.
period. In no case was any increase in this enzyme observed prior to the blastula
stage. In some experiments, decreases in the activity of isocitric dehydrogenase
were found between 24 and 48 hours. The activity of this enzyme was nearly lOCK/c
higher in sucrose homogenates than in homogenates made in 0.03 M Tris. The
addition of sucrose to homogenates made in dilute buffer also enhanced the activity
by as much as 50%. The reason for this effect is not known.
The levels of activity of alpha-ketoglutaric dehydrogenase. shown in Table IV.
indicate that approximately a 2.5-fold increase in the activity of this enzyme occurs
between 9 and 24 hours of development. As in the case of isocitric dehydrogenase,
little change in the activity of this enzyme occurs during cleavage or during develop-
ment from trochophore to veliger. In one experiment the rate of reduction of ferri-
ENZYMES IN OYSTER EMBRYOS
67
cyanide was used as a measure of the activity of this enzyme. This hydrogen
acceptor was reduced at a rate which was 5-10 times higher than that of the
2,6-dichlorophenolindophenol ; however, the high molar extinction of the latter made
it more desirable for assays of homogenates with low activities. The assays were
complicated by the high rates of endogenous reduction of this dye, and by the rapid
loss of enzyme activity with either acceptor. In most homogenates prepared in
dilute Tris or phosphate buffer, the activity of alpha-ketoglutaric dehydrogenase was
too low to measure. Data are therefore presented only for homogenates made in
buffered sucrose.
TABLE IV
Activities of malic dehydrogenase and aconitase in homogenates of oyster embryos. Homoge-
nates were made in 0.03 M Tris buffer, pH 7.4. Assays were performed at 25° C.
Values represent micromoles of substrate utilized per minute by
one million embryos
Expt.
Cleaving egg
Blastula
Trochophore
Veliger
Malic dehydrogenase
17
9.2
9.3
15.6
10.0
18
10.0
14.5
12.0
19
8.5
10.5
10.3
12.6
20
7.0
8.2
7.7
8.0
21
8.0
9.1
7.2
9.3
22
8.5
8.1
7.0
6.0
Average
8.5 ± 0.9
9.2 ± 0.8
10.4 ± 3.5
9.7 ± 2.2
Aconitase
17
0.068
0.082
0.123
0.106
18
0.082
0.080
0.110
0.090
19
0.080
0.088
0.120
0.090
20
0.095
0.110
0.063
0.104
21
0.100
0.110
0.065
0.070
Average
0.085 ± 0.011
0.094 ± 0.013
0.096 ± 0.026
0.093 ± 0.013
Fumarase, malic dehydrogenase and aconitase. Efforts to measure fumarase
in whole homogenates, prepared in either sucrose or in dilute buffer, were unsuc-
cessful. Although the enzyme was found to be present in all stages by the spectro-
photometric method, its activity was extremely low and was not proportional to the
homogenate concentration in most cases. In contrast to fumarase, the activity of
malic dehydrogenase is extremely high in all stages of development, being at least 20
times that of any other enzyme measured (Table IV). High activities have been
noted for this enzyme in other animal tissues (cf. Krebs and Lowenstein, 1960).
Little change in activity was found during development to the veliger stage.
The presence of aconitase in all stages of development is of interest, since this
enzyme has been reported to be absent from adult oyster-mantle (Jodrey and
Wilbur, 1955). As in the case of malic dehydrogenase, no change in activity occurs
during any phase of early development (Table IV). Neither the activity of
68 ROBERT E. BLACK
aconitase nor that of malic dehydrogenase was affected hy the composition of the
medium used for homogenization ; the data are therefore given for homogenates
made in 0.03 HI Tris buffer.
DISCUSSION
The data reported above indicate that : ( 1 ) although respiration increases 3-fold
during cleavage, little change in activity of any of the enzymes of the citric acid
cycle occurs before the blastula stage is reached; (2) a further 3-fold increase in
respiration between blastula and trochophore stages is paralleled by increases in two
enzymes of the Krebs cycle; and (3) between trochophore and veliger stages there
is little change in respiration or in enzyme activity, with the possible exception of a
decrease in cytochrome oxidase. With regard to the period of cleavage, the results
are similar to those of Gustafson and Hasselberg (1951) for the sea urchin and to
those of Spiegelman and Steinbach (1945) and Petrucci (1957) for two amphibians.
Following the blastula stage of the oyster marked increases were found in the
activities of isocitric dehydrogenase and alpha-ketoglutaric dehydrogenase. Such
increases in activity of two enzymes which are "biochemically adjacent" in the citric
acid cycle are of considerable interest, and it would be desirable to determine
whether the changes occur simultaneously or sequentially.
No change in activity of most enzymes of the Krebs cycle or of the electron-
transport system occurs during development to the veliger stage in the oyster. This
finding is in contrast to that of Gustafson and Hasselberg (1951) for the sea urchin,
in which both succinic dehydrogenase and malic dehydrogenase increase 4- to 5-fold
between blastula and pluteus stages. These authors have postulated that an in-
crease in the number of mitochondria is responsible for changes in activity of these
and other enzymes during this period of development. Counts of granules exhib-
iting the staining properties of mitochondria in developing sea urchins have given
supporting evidence for this hypothesis, since the number of these granules ap-
parently increases during about the same period of development (Gustafson and
Lenicque, 1952; Shaver, 1956).
In the unfertilized egg of the oyster Cleland (1951) has obtained evidence for
the localization of succinic oxidase and cytochrome oxidase in cytoplasmic granules,
and he has shown that removal of the granules from homogenates by centrifugation
drastically reduces the ability of the homogenates to take up oxygen in the presence
of substrates of the Krebs cycle. This latter finding may be simply due to the
removal of the terminal electron-transport systems, which would reduce the respira-
tion of the homogenates even in the presence of substrate. Since his results clearly
indicate the localization of succinic oxidase and cytochrome oxidase in the large
granules, it is possible to conclude from the present study that any changes in
number of granules during development of the oyster are not accompanied by
changes in these terminal enzymes. Data on the concentrations of other enzymes
of the citric acid cycle in the large granule fraction from homogenates of eggs and
larvae are presented in an accompanying report (Black, 1962).
The author is indebted to Mr. James Egan and to Miss Lynn Search for their
technical assistance during this investigation. A preliminary report of these results
has been previously published (Black, 1960).
ENZYMES IN OYSTER EMBRYOS 69
SUMMARY
1. Measurements of respiration, cytochrome oxidase, and the DPNH oxidase
system, as well as five enzymes of the citric acid cycle, aconitase, isocitric dehy-
drogenase, alpha-ketoglutaric dehydrogenase, succinic dehydrogenase, and malic
dehydrogenase, have been made in oyster embryos between the first cleavage and
the early veliger stage. The rate of respiration increases 9-fold to the trochophore
stage and levels off until the veliger stage is reached. Succinic dehydrogenase,
DPNH oxidase, malic dehydrogenase and aconitase were not found to change ap-
preciably during development to the veliger. Cytochrome oxidase showed no sig-
nificant change prior to the trochophore stage, but decreases were found in this
enzyme after this stage. Isocitric dehydrogenase and alpha-ketoglutaric dehy-
drogenase were found to increase, paralleling the increase in respiration, between the
blastula and trochophore stages. Following this stage, these enzymes remain con-
stant in activity up to the veliger stage.
2. The results are shown to be in contrast to those obtained for the sea urchin
by other workers. The possible relationships between changes in enzyme activities
and increases in respiratory rate are considered.
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THE CONCENTRATIONS OF SOME ENZYMES OF THE CITRIC ACID
CYCLE AND ELECTRON TRANSPORT SYSTEM IN THE LARGE
GRANULE FRACTION OF EGGS AND TROCHOPHORES
OF THE OYSTER, CRASSOSTREA VIRGINICA l
ROBERT E. BLACK
Department of Biology and Virginia Institute of Marine Science,2
College of William and Mary, Williamsburg, Virginia
In an accompanying report Black (1962) has shown that considerable changes
in the relative activities of several enzymes of the tricarboxylic acid cycle occur
during early development of the oyster. Thus, between the blastula and trocho-
phore stages two enzymes, TPN-specific isocitric dehydrogenase and alpha-keto-
glutaric dehydrogenase, increase 2- to 3-fold, roughly paralleling the increase in
respiration during this same period. Five other enzymes were found not to change
appreciably during development to the trochophore. These were: aconitase, suc-
cinic dehydrogenase, malic dehydrogenase, DPNH oxidase, and cytochrome oxi-
dase. Following the trochophore stage cytochrome oxidase decreased somewhat in
activity, while the other enzymes remained constant.
With the exception of isocitric dehydrogenase, most of the enzymes studied are
known to be present in considerable concentration (though not usually localized)
in the mitochondrial fraction of vertebrate tissues. The relationship of enzymic
changes to the possible biochemical differentiation of these respiratory granules
must therefore be considered. Previous investigations pertaining to this phenome-
non include the finding that the content of cytochrome oxidase increases in mito-
chondria of differentiating rat muscle (Shen, 1955), the work reported by Weber
and Boell (1955) and Boell and Weber (1955) in which an increase in the content
of cytochrome oxidase and succinoxidase in mitochondria of Xenopus laevis was
found during development, and the investigation of Mahler, Wittenberger and
Brand (1958) in which changes in the relative activities of several respiratory en-
zymes were found to occur in the large granule fraction of homogenates of the chick-
between 2 and 6^ days of development.
In eggs of marine invertebrates few studies have been made of the distribution
of respiratory enzymes in the various cell fractions which can be obtained from
homogenates. In the sea urchin mitochondria isolated in sucrose from unfertilized
eggs have been shown to contain succinoxidase and cytochrome oxidase (Maggio
and Ghiretti-Magaldi. 1958). The latter enzyme undergoes a 30% increase in the
mitochondria at fertilization, but shows no further change until the blastula stage is
reached (Maggio, 1959). Cytochemical studies of eggs of various invertebrates,
in which respiratory enzymes (usually indophenol oxidase and succinic dehy-
1 This investigation was supported by a grant (G-9847) from the National Science
Foundation.
2 Contribution No. 117.
71
72 ROBERT E. BLACK
drogenase) have been reported to be associated with granules, are reviewed by
Brachet (1960). In eggs of the oyster Cleland (1951) has studied oxygen uptake
by homogenates in the presence of substrates of the citric acid cycle, and has found
that removal of the large granules by centrifugation results in a decreased ability of
the homogenates to respire in the presence of such intermediates. In addition, he
has shown that succinoxidase and cytochrome oxidase are localized in the large
granules.
In the present study an attempt has been made to determine the distribution of
several enzymes involved in aerobic respiration between the "large-granule" fraction
(yolk, mitochondria and other granules) and the "supernatant" fraction (sub-
microscopic and soluble elements) derived from sucrose homogenates of fertilized
eggs and trochophores of the oyster, Crassostrea virginica. Several changes in
enzyme distribution and in enzyme content of the large granules will be shown to
occur during this developmental period.
MATERIALS AND METHODS
The oysters used in this study were collected by dredging from the Rappahan-
nock River and stored in trays at Gloucester Point, Virginia. The eggs were
removed from gonads, inseminated, and cultured by methods described previously
(Black, 1962). The stages used for homogenization were cleaving eggs (1^ hours
after fertilization) and late trochophores (20 hours after fertilization when cultured
at 22° C.). The eggs and larvae were collected by centrifugation and homogenized
in 10-15 volumes of 0.55 M sucrose buffered with 0.05 M Tris (hydroxy methyl)
aminomethane, pH 7.35. This medium is approximately isotonic to the dilute
sea water in which eggs were grown. The addition of as much as 5% poly vinyl
pyrrolidone to the medium caused considerable clumping of the granules in homog-
enates; this component was therefore omitted from the homogenization mixtures.
Homogenization of cleaving eggs was performed by forcing the suspension through
a 22-gauge needle ; usually 3-5 minutes were required for complete disruption of the
eggs. Partial homogenization of trochophores required 15-20 minutes of this
treatment, or two minutes of blending in a Lourdes multimixer at 16,000 rpm. No
attempt to achieve complete homogenization of the trochophores was made. The
homogenization and all subsequent operations were carried out at 0° C.
The homogenates were divided into two aliquots of 2 ml. each, and each aliquot
was centrifuged for 10 minutes at 1,000 X gravity in a Lourdes Model LR refrig-
erated centrifuge containing a swinging bucket rotor. The sediment from the first
centrifugation was washed once with 2 ml. of buffered sucrose, and the two super-
natant fractions were combined in each of the two aliquots. The washed, low-
speed sediment contained some nuclei but consisted chiefly of whole cells and
embryonic coats, and it was discarded from both aliquots.
Both aliquots of the homogenate, minus nuclei, whole cells, and embryonic coats,
were centrifuged at 18,000 X gravity for one hour in order to sediment the gran-
ules. This centrifugal force was near the maximum which could safely be obtained
with the rotor available. In preliminary experiments a centrifugation time of one
hour was found to be barely sufficient to sediment nearly all of the visible granules
as well as all of the succinic dehydrogenase and DPNH oxidase from homogenates
of fertilized eggs. In the preliminary experiments, the supernatant fluid recovered
ENZYMES IN GRANULES OF OYSTER EMBRYOS 73
from this high-speed centrifugation of egg homogenates was centrifuged for an
additional hour at the same force. This second treatment failed to cause the sedi-
mentation of a usable quantity of granules.
The high-speed sediment in one of the aliquots of homogenate was resuspended
in 4 ml. of buffered sucrose and washed by centrifugation for an additional hour.
The other aliquot served as a control, in which the sediment was merely resuspended
in the original supernatant fluid. This aliquot, labelled "whole homogenate"
(minus nuclei), was centrifuged again during the washing of the granules. Super-
natant fractions from the first and second centrifugal treatments of the "experi-
mental" aliquot were combined, and the volume was noted. The washed granules
were diluted to 4.0 ml. in buffered sucrose. Enzyme determinations were then
made on the washed granules, the combined supernatant fractions, and the "whole
homogenate" (combined granule and supernatant fractions). In addition, the
fatty fraction found at the top of the first supernatant portion of the fertilized eggs
was removed with a spatula, suspended in 1 ml. of buffered sucrose, and assayed
for all enzymes. Since no enzyme was found to be concentrated in this fraction,
the assays were not repeated on the fat from homogenates of trochophores.
The enzymes measured in the separate fractions were : aconitase, isocitric dehy-
drogenase, alpha-ketoglutaric dehydrogenase, succinic dehydrogenase, fumarase,
malic dehydrogenase, DPNH oxidase (with and without added cytochrome c} , and
DPNH-cytochrome c reductase. Simultaneous assays of each enzyme were usually
conducted on corresponding fractions from three separate batches of eggs or
embryos. Each experiment was repeated once, so that all of the reported values
represent the averages of data which were obtained from at least six batches of eggs
or embryos. Usually not more than one or two enzymes could be measured in any
one experiment since it was desirable to complete the determinations within 1 or \\
hours after preparation of the fractions. Succinic dehydrogenase was measured in
all of the granule preparations for use as a reference enzyme.
Spectrophotometric methods used in assaying all of the above enzymes except
DPNH-cytochrome c reductase have been listed previously (Black, 1962). The
latter enzyme was determined by the method of Strittmatter and Velick (1956).
The cuvettes contained 10~4 M cytochrome c, 10 3 M sodium cyanide, 1.7 X 10~4 M
DPNH, 0.05 M phosphate, pH 7.4, and 0.1 or 0.2 ml. of homogenate or homog-
enate-fraction in a total volume of 3.0 ml. The changes in A550 were followed at
15-second intervals for one or two minutes after the addition of enzyme. Assays
of all of the enzymes were performed at 25° ± 1° C.
RESULTS
Microscopic examination of homogenate fractions. No serious effort was made
to characterize any component of the large-granule fraction. Under oil phase the
granules were seen to consist of both spherical and rod-shaped bodies of a wide
range of sizes. Attempts to stain with Janus green were partially successful, as
determined on masses of granules which were collected by centrifugation after
staining; however, it was difficult to observe staining of individual granules by use
of the microscope. The supernatant fraction was not found to possess visible
granules under ordinary lighting or phase ; however, under dark-field illumination
small light-scattering particles could be seen.
74
ROBERT E. BLACK
Enzyme distributions in granule and supernatant fractions. The percentages of
total enzyme activities recovered in the large granules of eggs are listed in Table I,
column 2. These percentages are based on the sums of the activities recovered in
both fractions. In the egg, fumarase, malic dehydrogenase, and DPNH-cytochrome
c reductase are found almost entirely in the supernatant fluid, whereas appreciable
percentages of all of the other enzymes are found in the granules. The electron-
transport enzymes, succinic dehydrogenase and DPNH oxidase, are recovered al-
most exclusively in the granules, as might be expected if this fraction contains
TABLE I
Distribution and total recovery of respiratory enzymes in homogenate-fractions of fertilized eggs and
trochophores. Homogenates in 0.55 M sucrose containing 0.05 M Tris, pH 7.35, were freed
of nuclei and centrifuged at 18,000 X gravity for one hour. The sediment was washed
once by the same treatment and the two supernatant fractions were combined.
Whole homogenates were also freed of nuclei and whole cells for
comparison with above fractions. Standard deviations
are based on six determinations, in sep-
arate batches of embryos, of each value
Percentage of total recovered
enzyme present in granules
Percentage recovery of enzyme in
based on 100% for the
granules plus supernatant fluid
sum of the activities
based on 100% for
Enzyme
in granules and super-
the whole homogenate
natant fraction
Fertilized
egg
Trochophore
Fertilized
egg
Trochophore
Aconitase
37.5 ± 9.1
25.7 ± 5.0
88.7 ± 8.1
108.6 ± 27.3
Isocitric dehydrogenase
65.1 ± 4.9
63.3 ± 1.6
80.5 ± 12.8
91.3 ± 18.0
Alpha-ketoglutaric dehydrogenase
63.9 ± 6.7
100.0 ± 0.0
Succinic dehydrogenase
100.0 ± 0.0
67.1 ± 2.0
77.6 ± 13.4
98.9 ± 8.9
Fumarase
3.0 ± 1.3
16.1 db 3.5
302.8 ± 128.6
135.9 ± 15.0
Malic dehydrogenase
8.5 ± 2.4
11.2 ± 2.2
89.5 ± 9.1
112.6 ± 11.7
DPNH oxidase (without
cytochrome c)
92.0 ± 1.6
46.9 ± 5.8
84.0 ± 13.2
87.3 ± 15.6
DPNH oxidase (with added
cytochrome c)
92.1 ± 2.9
60.1 ± 5.3
84.7 ± 6.1
81.9 ± 10.6
DPNH-cytochrome c reductase
6.3 ± 1.8
23.9 ± 4.8
123.7 ± 14.5
94.5 ± 15.7
nearly all of the mitochondria. The finding that an active DPNH-cytochrome c
reductase is almost entirely localized in the supernatant fraction from egg homog-
enates is of interest, since this enzyme has been reported to be present in high con-
centration in the microsomal fraction of mammalian liver (see Strittmatter and
Velick, 1956). None of the enzymes listed were found to be present in quantity
in the fatty fraction of the egg.
The average per cent recovery of each enzyme in the granule fraction of trocho-
phores is given in Table I, column 3. Major increases in the percentages of alpha-
ketoglutaric dehydrogenase, fumarase, and DPNH-cytochrome c reductase re-
covered in the granules are found when these data are compared with those which
were obtained for egg homogenates. Alpha-ketoglutaric dehydrogenase appears to
be localized in the granules of trochophores, and the percentage of fumarase and
ENZYMES IN GRANULES OF OYSTER EMBRYOS 75
DPNH-cytochrome c reductase in the trochophore granules are 4 to 5 times higher
than in the granules obtained from eggs. In contrast to these enzymes, succinic
dehydrogenase and DPNH oxidase in the trochophore homogenates were found
to be distributed between the granule and the supernatant fractions, so that about -I
of the total recovered enzyme was found in the supernatant fluid in each case. Cen-
trifugation of the trochophore homogenates for 2 hours at 18,000 X gravity did not
result in increased sedimentation of either enzyme.
In Table 1, columns 4 and 5, the sums of the recovered enzyme activities in the
separate fractions are expressed as percentages of the activities found in the "whole
homogenates" (minus nuclei). With the exceptions of fumarase and DPNH-
cytochrome c reductase, the recoveries of most enzymes are somewhat higher in
the trochophore fractions than in those of fertilized eggs. This difference may be
an indication that there are substances in whole homogenates of trochophores which
inhibit enzyme activities, or that the enzymes in the separated fractions from tro-
chophores are somewhat more stable than in those from eggs. Because of the high
endogenous activity of whole homogenates with the dye, 2, 6-dichlorophenolindo-
phenol, the activities of alpha-ketoglutaric dehydrogenase were not measured in the
whole homogenates. Endogenous reduction of this dye was almost negligible in
the separated fractions.
The extremely high recovery of fumarase in separated fractions of eggs and
trochophore homogenates is of particular interest. As noted previously (Black,
1962), fumarase activity is extremely variable in whole homogenates of all stages.
In the separated fractions of eggs, nearly all of the activity was present in the super-
natant fluid; the inhibition in whole homogenates therefore appears to be caused
by the presence of the granules. Since the inhibition is obtained when either
fumarate or malate is used as the substrate, it does not appear to be a result of
any competing reaction which might be catalyzed by the granules. A marked
reduction in the total recovery from separate fractions is observed in the trocho-
phore ; this may indicate that trochophore granules inhibit the enzyme to a lesser
extent than granules from eggs. A calculation of the total fumarase activity in
eggs has been made from the data obtained on the separate fractions. This calcu-
lation shows that one million eggs have sufficient enzyme to convert 0.442 ± 0.098
micromoles of malate to fumarate per minute. The ratio of total fumarase to total
succinic dehydrogenase in the trochophore is not appreciably different from that in
the egg; fumarase activity therefore probably does not change during this period
of development.
An excessive recovery of DPNH-cytochrome r reductase is also found in
separated fractions of the egg, but not in those of the trochophore (Table I). A
slight inhibition of this enzyme by the granules of the egg again appears to be
responsible for the high recovery. The total activity of this enzyme in all fractions
of the egg is calculated to be sufficient to reduce 0.448 ± 0.056 micromoles of
cytochrome c per minute per million eggs, and the ratio of total reductase to total
DPNH oxidase is the same in eggs and trochophores.
Ratios of enzyme activities in granules. The findings summarized in Table I,
together with the data available from assays of enzymes in whole homogenates
(Black, 1962), indicate that changes in the relative activities of respiratory enzymes
in the large granules must occur during development. In order to determine more
76
ROBERT E. BLACK
precisely the extent of the changes in the granules, measurements of the ratios of
the activities of these enzymes to that of succinic dehydrogenase have been made
on the granule fractions prepared from eggs and from trochophores. The average
ratios obtained are presented in Table II. The averages have been calculated from
data obtained in 6 to 10 separate determinations of each ratio. The only enzyme
which was found to be constant in the two stages in comparison to the reference
enzyme was DPNH oxidase in the presence of added cytochrome c. This enzyme
system had an activity which was almost exactly equal to that of succinic dehydro-
genase in the granules of both eggs and trochophores (ferricyanide was used as
the electron acceptor in all assays of the reference enzyme). In the absence of
TABLE II
Ratios of activities of respiratory enzymes to that of succinic dehydrogenase in granules of fertilized
eggs and trochophores. Ratios are expressed as micromoles of substrate utilized per minute
divided by micromoles of ferricyanide reduced per minute by succinic dehydrogenase.
All ratios were determined at 25° C. Standard deviations are based on 10
determinations of each ratio for isocitric dehydrogenase and 6
determinations of all other ratios
Enzyme
Fertilized egg
Trochophore
Per cent
change
in ratio
't'
Aconitase
0.431 ± 0.160
0.606 ± 0.155
+41
2.33*
Isocitric dehydrogenase
1.350 ± 0.370
2.530 ± 0.460
+ 88
7.00**
Alpha-ketoglutaric dehydrogenase
0.083 ± 0.013
0.170 ± 0.050
+ 105
3.78*
Succinic dehydrogenase
1.0
1.0
Kumarase
0.196 ± 0.138
0.930 ± 0.430
+382
3.61*
Malic dehydrogenase
8.16 ±3.30
23.50 ± 5.90
+ 188
5.01**
DP.\H oxidase (without added
cytochrome c)
0.524 ± 0.088
0.370 ± 0.077
-29
9.06**
DPNH oxidase (with added
cytochrome c}
0.990 ± 0.080
1.020 ± 0.240
+3
0.08
DPNH-cytochrome c reductase
0.343 ± 0.130
1.560 ± 0.350
+355
14.31**
*P < 0.005
** P < 0.001
added cytochrome the activity of DPNH oxidase in the egg granules was about i/o
of the maximum activity. A decrease of 29% in the activity of the unstipplemented
DPNH oxidase system, relative to that of the reference enzyme, was found in the
trochophore granules ; presumably a loss of endogenous cytochrome c from the
granules was responsible for this change.
With the exception of the above enzyme system, all of the enzymes investigated
were found to have considerably higher activities, relative to that of succinic dehy-
drogenase, in the trochophore granules than in those of fertilized eggs. The per-
centage increases in ratio range from 41 for aconitase to 382 for fumarase (Table
II). An analysis of the data given in Table II shows that the probability is less
than 0.005 that the differences found between the egg and trochophore granules for
all enzymes except cytochrome-supplemented DPNH oxidase are due to random
variations in ratios.
ENZYMES IN GRANULES OF OYSTER EMBRYOS 77
DISCUSSION
The differences in enzyme ratios in the granules between the two stages investi-
gated are undoubtedly related to changes in distribution and in total amounts of the
enzymes. These may be summarized as follows: (1) about % of the succinic
dehydrogenase and DPNH oxidase are present in the supernatant fraction of the
trochophores, whereas these enzymes are almost entirely localized in the granules
of eggs (Table I) ; (2) in contrast to these enzymes, higher proportions of alpha-
ketoglutaric dehydrogenase, fumarase, malic dehydrogenase, and DPNH-cytochrome
c reductase are associated with the granules of trochophores than with the granules
of eggs (Table I) ; and (3) a 170% increase in the total activity of isocitric dehydro-
genase and a 140% increase in that of alpha-ketoglutaric dehydrogenase are found in
whole homogenates during development (Black, 1962). The distribution of iso-
citric dehydrogenase between the two fractions is the same in eggs and trocho-
phores, about 63-65% of this enzyme being present in the granules. The increase
in relative activity of this enzyme in the granules should therefore be at least 170%.
In the case of alpha-ketoglutaric dehydrogenase a change in distribution apparently
occurs during development, so that 100% of the activity is recovered in the trocho-
phore granules, whereas only 64% is present in the egg granules. The percentage
increase in the granules should therefore amount to (140 X 100/64) or at least
210%. The changes actually found in these two enzymes are only about half as
great as the predicted changes (Table II). One possible explanation for these dis-
crepancies is that selective destruction of some enzymes might occur in one of the
homogenate-fractions of either developmental stage, giving erroneous values for
distribution of the enzymes or for the ratios of activities in the granules. This pos-
sibility seems especially applicable to alpha-ketoglutaric dehydrogenase, since the
activity of this enzyme was always found to decline very rapidly during the assays.
In evaluating the data given in Tables I and II it is necessary to consider
possible artifacts other than the one given above. The presence of succinic dehydro-
genase and DPNH oxidase in the supernatant fraction of the trochophores could
have been caused by disruption of mitochondria during homogenization ; however,
such disruption of mammalian mitochondria usually results in the solubilization of
many of the enzymes of the citric acid cycle (see Hogeboom, 1954). In the tro-
chophore aconitase is slightly less concentrated in the granules than in the egg,
and some loss of cytochrome c from the trochophore granules also appears to be
probable. Except for these two enzymes and the terminal enzymes mentioned
above, however, no enzyme investigated is less concentrated in the trochophore
granules than in the egg granules. Since the total amounts of succinic dehydro-
genase and DPNH oxidase do not change during development, there appear to be
only two possible ways to account for their presence in the supernatant fraction of
the trochophore. These are: (1) a specific loss (either natural or artificial) of the
electron-transport enzymes from the granules of the trochophore without corre-
sponding losses of other enzymes; and (2) natural or mechanical splitting of the
granules of the trochophore in such a way that the submicroscopic fragments retain
a full complement of enzymes. In the latter case enzymes of the citric acid cycle
might be associated with succinic dehydrogenase and DPNH oxidase in submicro-
scopic particles which were not recovered in the granule-fraction of the trochophore.
The first possibility seems unlikely in view of the results other workers have ob-
78 ROBERT E. BLACK
taiticd with mammalian mitochondria — if % of the terminal enzymes are lost from
the granules then one would expect that even larger proportions of the other
enzymes would be lost. In any case the relative increases in the granule-fraction
of all of the enzymes except aconitase are too large to be accounted for by the
selective loss of the reference enzyme from the granules.
The simplest interpretation of the data given in Table II is that isocitric dehy-
drogenase, alpha-ketoglutaric dehydrogenase, fumarase, malic dehydrogenase, and
DPNH-cytochrome c reductase increase in the large granules during development
to the trochophore. The increases in some of the enzymes are a result of the in-
corporation of existing enzyme molecules into the granules. This appears to be
true for fumarase and DPNH-cytochrome c reductase and possibly also for alpha-
ketoglutaric dehydrogenase and malic dehydrogenase. The increases in other en-
zymes (isocitric and alpha-ketoglutaric dehydrogenases) result from the incorpora-
tion of newly-synthesized enzyme molecules into the granules or from their actual
synthesis by the granules. One possible result of such changes in the granules is
an increase in their capacity for catalyzing oxidations via the citric acid cycle. This
may be related to the large increase in respiration which occurs during development.
It is interesting to speculate on the possibility that the populations of granules in
the egg and trochophore are heterogeneous with respect to the enzyme content of
individual granules. Thus the relative changes noted in Table II might represent
increases in the number of large granules containing high concentrations of some
enzymes (fumarase and DPNH-cytochrome c reductase, for example) but low
concentrations of others. If such heterogeneity exists it may be expected that a
diversity of aerobic metabolic pathways also exists in the embryo. In marine
animals several workers have obtained cytochemical evidence for the existence of
more than one kind of enzyme-containing granule in the egg ; however, few of the
enzymes investigated are directly involved in respiration. These findings are
reviewed by Pasteels (1958).
SUMMARY
1. The enzymes aconitase, TPN-specific isocitric dehydrogenase, alpha-keto-
glutaric dehydrogenase, succinic dehydrogenase, fumarase, malic dehydrogenase,
DPNH oxidase, and DPNH-cytochrome c reductase have been assayed in two
fractions, large granules and supernatant, prepared from sucrose homogenates of
fertilized eggs and trochophores of the oyster, Crassostrea virginica. Succinic dehy-
drogenase and DPNH oxidase are almost completely localized in the granules of
the fertilized egg, but in the trochophore about % of each enzyme is found in the
supernatant fraction. High percentages of aconitase, isocitric dehydrogenase, and
alpha-ketoglutaric dehydrogenase are found in the granules of both stages ; the
latter enzyme appears to be localized in the trochophore granules. Fumarase,
malic dehydrogenase and DPNH-cytochrome c reductase are almost absent from
granules of the egg, but considerable proportions of these enzymes are found in
trochophore-granules.
2. Ratios of enzyme activities in the granules relative to that of succinic dehy-
drogenase have been determined at the two developmental stages. All of the en-
zymes except DPNH oxidase increase in activity, relative to the reference enzyme,
in the granules during development to the trochophore stage. The activity of
ENZYMES IN GRANULES OF OYSTER EMBRYOS 79
DPNH uxidase in the presence of cytochromc c is about the same as that of the
succinic dehydrogenase in both stages. These changes appear to indicate that
differentiation of the population of respiratory grannies occurs during development
of the oyster.
LITERATURE CITED
BLACK, R. E., 1962. Respiration, electron-transport enzymes, and Krebs-cycle enzymes in early
developmental stages of the oyster, Crassostrea virginica. Biol. Bull., 123: 58-70.
BOELL, E. J., AND R. WEBER, 1955. Cytochrome oxidase in mitochondria during amphibian
development. Ex p. Cell Res., 9: 559-567.
BRACKET, J., 1960. The Biochemistry of Development. Pergamon Press, New York.
CLELAND, K. W., 1951. The enzymatic architecture of the unfertilized oyster-egg. Australian
J. Exp. Biol. and Med. Set., 29: 34-45.
HOGEBOOM, G. H., 1954. The isolation and biochemical properties of liver mitochondria. In:
Fine Structure of Cells, VIII Congress of Cell Biology, Leiden. Interscience Pub-
lishers, New York.
MAGGIO, R., 1959. Cytochrome oxidase activity in the mitochondria of unfertilized and
fertilized eggs. Exp. Cell Res., 16: 272-278.
MAGGIO, R., AND A. GHIRETTI-MAGALDI, 1958. The Cytochrome system in mitochondria of
unfertilized sea urchin eggs. Exp. Cell Res., 15: 95-102.
MAHLER, H. R., M. H. WITTENBERGER AND L. BRAND, 1958. Biochemical studies of the
developing avian embryo. II. Enzymes of the citric acid cycle. J. Biol. Clicm., 233:
770-782.
PASTEELS, J. J., 1958. Comparative cytochemistry of the fertilized egg. In: A Symposium
on the Chemical Basis of Development, ed. by W. D. McElroy and B. Glass. Johns
Hopkins Press, Baltimore.
SHEN, S. C., 1955. Enzyme development as ontogeny of specific proteins. In: Biological
Specificity and Growth, ed. by E. G. Butler. Princeton Univ. Press, Princeton, X. J.
STRITTMATTER, P., AND S. F. VELICK, 1956. A microsomal Cytochrome reductase specific for
diphosphopyridine nucleotide. /. Biol. Chem., 221: 277-286.
WEBER, R., AND E. J. BOELL, 1955. Uber die Cytochromoxydase-aktivitat der Mitochondrien von
fruhen Entwicklungsstadien des Krallenfrosches (Xenopus laevis Daud.). Rev. Snisse
Zool, 62: 260-268.
FEEDING MECHANISM OF THE ECHIUROID, OCHETOSTOMA
ERYTHROGRAMMON LEUCKART & RUEPPELL, 1828
S. H. CHUANG
Dcpt. of Zoology, University of Singapore, Singapore
The feeding mechanism of both Urcchis caiipo and Echiurus echiurus has been
described. The former (Fisher and MacGinitie, 1928) secreted a mucus tube
2-8 inches long, the open upper end of which was fastened to the burrow near its
opening, while the lower end remained attached to the body. This tube filtered
the water flowing through the burrow, became loaded with food particles and was
subsequently swallowed. Echiurus echiurus fed intermittently in aquaria (Gislen,
1940). Periods of feeding of 1-2 days alternated with rest periods lasting several
days. During each feeding period food collection occurred at intervals of 20
minutes to a couple of hours. The proboscis emerged from the burrow in feeding,
as Wilson (1900) had previously noted, with its distal margin facing anteriorly
and dorsally towards the substratum to gather food particles and transfer them to
the ventral surface where they were glued together with mucus and carried by
ciliary currents towards the mouth.
The feeding mechanism of Ochetostoina is unknown, except for the observation
of Sluiter (1884) that in the shallow water-covered part of the beach at Billiton
the proboscides of 0. erythrogrammon moved slowly on the sand to shovel up sand
and organic matter on to the proboscis groove and convey them to the mouth.
In the present study observations were made on Ochetostoina specimens both
in their natural habitat on the beach and also in the laboratory.
MATERIALS AND METHODS
Ochetostoma erythrogrammon occurs in large colonies between mean low water
neaps and mean low water springs in the intertidal sandy mud of Singapore and
neighboring islands. An opening, 3 mm. in diameter, through which the proboscis
may emerge in feeding, leads into a U-shaped burrow of 1 cm. diameter. The
burrow consists of two vertical or oblique tunnels each 20 cm. long connected by
a horizontal tunnel 25-45 cm. long. Although the burrows can be located by the
proboscides above the surface at ebb tide, digging up the animals without damage
is not easy because of the difficulty of locating the direction of the horizontal tunnel
due to blocking up of the other opening of the burrow by a plug of sand or mud.
Sluiter (1884) reported the ease with which the intact animal was obtained
by pressing the foraging proboscis on the sand with the fingers and digging up the
trunk with the other hand at Billiton. Due presumably to the less muddy sub-
stratum around Singapore, this method always resulted in autotomy.
Aquaria were set up by using sandy mud from the same bed from where the
specimens were collected. Fine carborundum powder was used to trace the ciliary
currents on the proboscis under the binocular microscope.
80
FEEDIXC MECHANISM OF OCHETOSTOMA *1
RESri.TS AND I )lS(TSSKt\
At Pulau Hantu, a sandy island south of Singapore, which rises one meter
above the highest spring tide, the substratum from mid-tide level downwards consi.-^
of greyish to purplish impervious clay with an overlying layer of coarse muddy sand
2-5 cm. thick. At ebb tide the water retained in the interstices of the sand at the
higher shore levels slowly drains along and thus wets the sandy crust above the clay
subsoil of the lower shore. At every ebb tide, irrespective of the time of day or
night, Ochetostoma erythrogrammon feeds by extending its proboscis out of the
burrow with the dorsal surface touching the substratum to collect sand and detritus
with the dorsally turned distal region of its proboscis. When the surface sand
and detritus near the mouth of the burrow are taken up, the proboscis extends
further in approximately the same direction. The fully extended proboscis, ex-
ceeding 25 cm. in length in some specimens, becomes thin and narrow. Its entire
ventral surface is covered with sand grains, detritus and extruded mucus (Fig. 1).
K 1. Ochetostoma erythrogrammon. The proboscis, stretched horizontally across fig-
ure and loaded with sand and detritus, forages on the wet sand outside the burrow (arrow)
at ebb tide. Tracks on sand above burrow indicate previous excursions of proboscis
The smaller particles move towards the mouth along the length of the proboscis
but the larger sand grains seem to remain stationary until muscular contractions
of adjacent parts of the proboscis move them on. When poked with a stick the
fully extended proboscis withdraws, discarding the collected sand grains and detritus
at the mouth of the burrow. After an interval of time the proboscis re-emerges
to extend in a different direction from the one previously taken. At the end of
the low tide several tracks indicating foraging excursions of the proboscis may
be seen radiating from the opening of the burrow (Fig. 1 ).
On more than ten visits to Pulau Hantu during ebb tide proboscides of Ocheto-
stoma foraging on the wet sand were observed. That the same specimens protruded
their proboscides at every ebb tide is demonstrated by the following observations.
During ebb tide at 3:30 A.M. on 27th August 1961 the burrows through which
S. H. CHUANG
19 proboscides protruded were marked by iron rods driven into the clay near the
opening of the burrow. During the next ebb tide at 5 :45 P.M. on the same day
8 proboscides emerged from the marked burrows to forage in brilliant sunshine.
They returned into the burrows when rising tide flooded the openings. This
method of feeding occurs only when the surface sand is wet enough, since it also
occurs on the wet muddy shore of the west coast of Singapore Island, but not on
the crumbly, porous, well-drained muddy sand of an adjacent island where a bed
of Ochetostoma is also found. Stephen and Robertson (1952) also reported the
presence of tracks radiating from one side of the opening of Ochetostoma burrow
on the sandy shore at Mbweni, Zanzibar.
The proboscis underwent frequent changes of shape. It rolled itself up into an
almost closed cylinder by apposition of the lateral margins. It flattened out into
a long thin ribbon during feeding. It shortened to less than a fifth of the length
of the fully contracted trunk or extended to more than four times the length of
the relaxed trunk. The width varied between 3 and 11 mm. Observations under
the binocular microscope showed that the ventral surface changed frequently from
a plane to a concave shape. Moreover, longitudinal troughs and transverse grooves
on the ventral surface and puckers along the lateral margins appeared and dis-
appeared in various regions according to the degree of contraction of the under-
lying muscles.
The cilia lined only the ventral surface of the proboscis and were of uniform
length of 11-13 //. in the living state, unlike those of Echiurus echinnts, in the
proboscis of which Gislen (1940) reported larger cilia on the tip, lateral margins
and the "eminence."
As regards the ciliary currents the ventral surface of the proboscis can be
subdivided into three regions, namely the distal, middle and proximal regions.
The distal region had in its distal 4 mm. or so only posteriorly directed ciliary
currents. These also occurred in the middle of its proximal 4 mm., where they
were flanked by postero-medially directed ciliary currents (Fig. 2a). During
feeding the extremely mobile distal region turned dorsal ly to explore the substratum
and pick up participate matter. It was also used for digging a burrow.
The middle region, which formed the greater part of the proboscis and varied
in length with the degree of extension, had medially directed ciliary currents along
the lateral fields. There were posteriorly directed ciliary currents in the middle
flanked by postero-medially directed ciliary currents ( Fig. 2b ) .
The proximal region, almost as mobile as the distal region, had a tract of
posteriorly directed ciliary currents along the middle. This tract was flanked by
a narrow tract of postero-medially directed ciliary currents. Lateral to this was a
tract of medially directed ciliary currents. In addition to these there were several
narrow tracts of cilia beating laterally outwards along the thickened rim. The
rim on each side puckered up into folds and valleys. By referring to the pigments
on the rim it was possible to observe that the folds and valleys were not fixed in
position but could vary. The valleys at any particular moment could become folds
at the next. As a result of this arrangement the movement of participate matter
along the rim could be outward or inward according to whether the outwardly
beating or the inwardly beating tracts of cilia were oriented at the top of the fold.
In the laboratory an intact specimen extended its proboscis 4-7 cm. with the
FEEDING MECHANISM OF OCHETOSTOMA
83
a
B
en
;, * ^^sasiatias* *
^L^-Jr^^^
FIGURE 2. Ciliary currents (small arrows) and paths (large arrows) of accepted and
rejected particles on the proboscis of Ochetostoma erythrograiiuiwn. a and b, ventral view of
distal and middle regions, respectively, c, ventral view of proximal region rejecting particles;
B — bulge, d, antero-ventral view of proximal region accepting particles into the mouth
funnel (F).
dorsal surface touching the bottom of the waxed tray. The distal part of the
proboscis swung from side to side and its ventral surface faced dorsally to scour
the substratum and pick up particles of carborundum added. These were coated
with mucus and carried in the median tract of posteriorly directed ciliary currents
through the expanded proximal region into the mouth (Fig. 2d). \Yhen particles
of clean sand 1 mm. or more in diameter approached the proximal region, this soon
partially rolled up into a cylinder and became dorso-ventrally depressed. A bulge
also formed 2-3 mm. anterior to the mouth. The passage into the funnel-shaped
proximal region was thus blocked. These large particles therefore moved poste-
riorly across or alongside the anterior part of the bulge, over the rim of the
proboscis ventrally (Fig. 2c) and were rejected.
The equivalent of the bulge of Ochetostoma in Echhtnis cclunnts is presumably
the ridge or "eminence," since Gislen ( 1940) found that it could bulge or sink into
a furrow. He believed that the peristaltic movements of the eminence helped to
move the mucus thread down the mouth-funnel. Two ventral lips or swellings
of the proximal region of the proboscis fitted into the depressions on either side of
84 S. H. CHUANG
the eminence to prevent larger particles from entering the mouth in Echiurus
i'< hhinis. In this and in Ochetostoum erythrogrammon the rejection mechanism
is therefore muscular and differs only in small details. Fisher and MacGinitie
(1928) observed that large particles were rejected when the mucus tithe was being
swallowed by Urcchis caupo but the details of this rejection mechanism were not
described.
Intact specimens placed in aquaria with muddy-sand bottom built U-shaped
tunnels by forcing the distal region of the proboscis into the sand and working out
a hole with it while the trunk meantime lay prostrate on its side or dorsal surface.
The proboscis disappeared into the hole dragging the trunk after it as in I'rcchis
caupo (Fisher and MacGinitie, 1928). The ventral setae were not used in digging
the hole in Ochetostouia. In Urechls they were used in enlarging the tunnel by
scraping off material from the sides (Fisher and MacGinitie, 1928). Echiiints
echiums, however, performed digging movements alternately with the ventral
setae at the rate of 6-9 times per minute and the stiffened anterior end of the trunk,
while the proboscis remained inactive. Gislen (1940) found that the anterior end
of the trunk entered the excavated hole dragging the proboscis along, and illus-
trated (text-figure 10 at page 15) the posterior end of the trunk and the distal tip
of the proboscis remaining outside at one stage of digging a burrow. In spite of
the closer systematic relationship between Ochetostouia and Echinrns, the former
resembled more closely the more distant relative I'rcchis caupo in its digging
behavior.
When the surface of the aquarium substratum was under 3-4 cm. of water, the
proboscis in Ochetostouia remained inside the burrow and performed feeding move-
ments by exploring and picking up particles along the wall of the burrow with the
distal region of the proboscis. After some time the animal turned around so that
the proboscis could explore and collect particles from the other end of the burrow.
Echiunts also put out part of its proboscis while still submerged under water to
collect food particles of the aquarium bottom (Wilson, 1900; Gislen, 1940) and leave
distinct tracks (Gislen, 1940). Some time after the water in the aquarium was
siphoned out, Ochetostouia extended its proboscis out of the burrow to feed in the
same manner as observed under natural conditions on the beach. The collection
of food particles with the proboscis inside or outside the burrow is presumably the
usual method of feeding in all echiuroids, since I'rcchis caupo also gathered sedi-
ment with its proboscis while lying outside the burrow in an aquarium (Fisher,
1946). Due to the reduced size of the proboscis in Vrcchis caupo, an alternative
method involving filtration of food particles with mucus tube was developed. Xo
mucus tube of the type secreted by I'rcchis caupo was formed in Ochetostouia.
SUMMARY
1. Ochetostouia crythroyramuwn built U-shaped burrows between mean low
water neaps and mean low water springs in the intertidal sandy mud of Singapore
and neighboring islands.
2. At ebb tide the proboscis emerged from the burrow in wet beaches to collect
and swallow sand grains and detritus from the surface of the shore.
3. The ciliary currents on the proboscis and the course of the accepted and
rejected particles were described.
FEEDING MECHANISM OF OCHETOSTOMA
4. The feeding mechanism of Ochetostoma erythrogrammon was compared with
those of Echinrus cchinrus and L'rcchis caupo.
LITERATURE CITED
FISHER, W. K., 1946. Echiuroid worms of the North Pacific Ocean. Proc. U. S. A '<//. Mus..
96: 215-292.
FISHER, W. K., AND G. E. MACGINITIE, 1928. The natural history of an echiuroid worm.
Ann. Mat,. Nat. I list.. So: 10, 1: 204-213.
GISLEN, T., 1940. Investigations on the ecology of Echhints. Lands L'nir. Arsskt:. nc-a' ser.,
36: 1-39.
SLUITER, C. P., 1884. Beitraege zu der Kenntnis der Gephyreen aus dem malayischen Archipel,
3. Mittheil. Natiiurk. Tijdschr. Ncd.-Ind., 43: 26-88.
STEPHEN, A. C., AND J. D. ROBERTSON, 1952. A preliminary report on the Echiuridae and
Sipunculidae of Zanzibar. Proc. Roy. Sue. Edinb.. Sect. H, 64: 426^40.
WILSON, C. B., 1900. Our North American echiurids. Bwl. Bull., 1: 163-178.
SITES OF OXYGEN UPTAKE IN OCHETOSTOMA
ERYTHROGRAMMON LEUCKART & RUEPPELL
(ECHIUROIDEA)
S. H. CHUANG
Hcpt. of Zoology, University of Singapore, Singapore
In the echiuroid Urcchis canpo inhalations and exhalations of sea water by the
muscular cloacal chamber during respiration occurred through the anus (Fisher and
MacGinitie, 1928b ) . These authors pointed out that the peristaltic movements
passing along the trunk of this worm not only renewed the water in its burrow but
also moved that in the respiratory chamber of the gut. Redfield and Florkin
(1931) observed that in Urechis the oxygen in the water enclosed within the burrow
and in the blood was insufficient to maintain the normal metabolic rate for the
duration of the low tide, during which the hemoglobin of the blood might be ex-
pected to transport an adequate supply of oxygen to the organs of the body. Hall
(1931) found that the oxygen consumption of Urcchis caupo was comparable to
that of related forms.
Ochetostonia crythrograiinnon in many poorly drained beaches in the tropics
also feeds during low tide by protruding its proboscis outside the burrow. The
reduced availability of oxygen in the burrow at ebb tide, the small diameter of the
hindgut. and the irregular, infrequent and small outflows from the anus of speci-
mens in burrows built along the glass wall of the aquarium in the laboratory suggest
that the anus may not be the sole organ of respiration.
In the present study the oxygen uptake of entire specimens and of parts of the
body of Ochetostonia crythrograininon was determined.
MATERIALS AND METHODS
Specimens of Ochetostonia erythrogrammon from the intertidal muddy sand of
the west coast of Singapore Island, where feeding also occurs during ebb tide, were
starved for 3-5 days to allow the faecal pellets to be completely voided, and their
oxygen consumption was determined in a closed bottle. As a precaution against
excessive peristaltic movements each specimen was confined in a cylindrical bag 1
cm. in diameter and 6-8 cm. long, to which it was acclimatized for one day. This
bag of nylon netting of 81 meshes per sq. cm. was slipped, together with the enclosed
specimen, into a bottle of about 175-milliliter capacity, the actual volume of which
was previously determined.
The natural sea water used was filtered into a jar with a capacity of 13 liters
and thoroughly aerated before it was covered with a thick layer of oil and siphoned
into individual bottles containing the experimental animals. To ensure that the
water siphoned into the bottles was not in contact with air, the water in the bottle
was retained only after an amount of water equivalent to twice the volume of the
bottle had passed through. The experiments were carried out at 19.3, 19.5, 19.9
86
SITES OF OXYGEN UPTAKE IN OCHETOSTOMA 87
and 20.0° C. ± 0.1° C. Each bottle containing the experimental animal was turned
at half-hourly intervals to ensure thorough mixing.
To prevent cloacal respiration a nylon rod of suitable diameter was inserted into
the cloaca via the anus and secured by ligating the posterior tip of the trunk around
it. This treatment did not seem to adversely affect the specimens even after more
than 9 hours of anal blockade, since they survived when the rod was released by
cutting away the ligature.
The proboscis was easily detached by gently squeezing with a pair of fine forceps
its attenuated junction with the trunk. Autotomy of the proboscis occurs in nature
and contraction of the circular muscle of the trunk at this junction prevents bleed-
ing. The detached proboscis continues to move for several days with its cilia still
beating. Its oxygen uptake was determined immediately after its separation from
the trunk.
The oxygen content was determined by the modification of Fox and Wingfield
(1937) of the Winkler method using phosphoric acid. A blank control was run at
the same temperature with every batch of water to find out the amount of oxygen
consumed by microorganisms present in the sample of filtered water. This amount
was very small for the duration of the experiments and was deducted from the
amount of oxygen consumed by the specimens. The fixed tissues of Ochetostonia
comprised the proboscis, body wall with attached nephridia and the gut wall drained
of its contents. Wet weight refers to their weight after blotting with filter paper,
and dry weight, after drying for 24 hours at 100° C. in an oven. The coelomic
corpuscles were not included.
To record the peristaltic movements of the trunk the apparatus used was based
on the same principle as the one devised by Wells (1951) but with the following
modifications to suit the weak movements of Ochetostonia, namely the use of ( 1 ) a
light rubber bung for float, (2) a weak spring attached to a lever with writing
point to counterbalance the float and (3) a plastic U-tube of about 8-10 mm. diam-
eter to exactly fit the trunk diameter of the worm.
To record the quantity of water pumped by the worm the inlet end of the plastic
U-tube of an apparatus based on the one used by Hall (1931) for Urechis had to
be submerged below the surface of the water before any water could be pumped out
by the weak peristaltic movements of Ochetostonia.
RESULTS AND DISCUSSION
The movements of Ochetostonia likely to influence the oxygen consumption con-
siderably are peristaltic and antiperistaltic movements of the trunk and the move-
ments of the proboscis. Ochetostonia erythrogrammon, the biggest specimen of
which barely weighs 10 gm., is a small echiuroid compared with Urechis. The
peristaltic movements of the trunk, which is less muscular than that of Urechis,
were weak and did not displace a large enough volume of water. Only 0-25 cc.
was pumped irregularly over a period of three hours ; this is less than the quantity
pumped by Urechis in one minute (Hall, 1931 ). In Ochetostouia reared in aquaria
in the laboratory peristaltic waves at the rate of four per minute may pass through
the trunk. Each peristaltic movement causes a stream of water to issue from the
opening of the burrow facing the posterior end of the body. These peristaltic move-
ments serve to renew the water of the burrow for respiratory and feeding purposes.
88
S. H. CHUANG
The tracing in Figure 1A shows a spell of regular peristalsis at the rate of about
three per minute, while Figure IB shows some irregular peristaltic movements oc-
curring in another specimen. A series of peristalses is usually succeeded by a rest
period of variable duration.
Hall (1931) showed that the oxygen consumption of Urechis in U-tubes bore no
consistent relation to oxygen partial pressure at least over the range of 138.2 to 93.3
mm. Hg. The range of oxygen tension encountered by Ochetostoma in nature
must be considerable from flood tide to ebb tide. Under experimental conditions
the range of 5.08 to 3.20 cc. oxygen per liter at the onset of the experiments is within
the usual range encountered by the animals and the experiments were continued
FIGURE 1. Record of peristaltic movements of Ochetostoma erythrogrammon. Read from
left to right. Time : one division per minute. Each convex (upward) part of the curve repre-
sents the passage of a peristaltic wave along the trunk. A. Record of a specimen with regular
peristaltic movements. B. Record of another specimen with peristaltic movements occurring at
irregular intervals.
until the oxygen consumed amounted to about 25c/c of the original amount except in
specimen 12 where the experiment was continued until the oxygen content dropped
to 1.1 cc. /liter. In this case the consumption was not far below the mean, indicat-
ing that a fall in oxygen tension did not materially affect oxygen uptake.
In Table I the blocked anus of specimens 1-8 was released at the start of the
second period. In specimens 9-12 the anus was blocked at the start of the second
period. With the exception of specimens 8 and 9, the rate of oxygen uptake was
greater during the second period irrespective of the state of the anus and the oxygen
tension. Since Hall (1931) showed that the oxygen consumption in Urechis caupo
almost doubled with increased activity, presumably there was a tendency towards
increased activity during the second period, making it difficult to assess the true
effects of blocking the anus. Although the peristaltic movements of 0. crytli-
rogrcnnnwn were subdued by confinement in a nylon bag, it was not possible to
SITES OF OXYGEN UPTAKE IN OCHETOSTOMA
89
TABLE I
Rate of oxygen consumption of Ochetostoma erythrogrammon
Specimen
Duration
per period
Animal with blocked anus
Normal animal
No.
Weight in grains
Initial <>:
content
Oxygen uptake
based on
Initial O2
content
Oxygen uptake
based on
wet
dry
wet
weight
dry
weight
wet
weight
dry
weight
hrs.
cc./liter
i . , Kin./hr.
cc./gm./hr.
cc. /liter
CC. Kin. lir.
cc./gm. hi.
Peri
)d I
Peril
,d II
1
0.5585
0.0845
2
4.32
0.0710
0.4691
4.00
0.0715
0.4725
2
0.9024
0.1440
2
4.32
0.0580
0.3633
4.00
0.0635
0.3802
3
0.6577
0.0947
4
4.84
0.0512
0.3556
4.82
0.0633
0.4395
4
0.5041
0.0939
4
4.84
0.0761
0.4084
4.80
0.0962
0.5163
5
0.8646
0.1437
4
4.84
0.0571
0.3436
4.80
0.0660
0.3969
6
0.6347
0.1158
4
4.84
0.0618
0.3388
4.74
0.0827
0.4531
7
0.5965
0.1025
4
4.84
0.0826
0.4808
4.74
0.1030
0.5993
8
0.5928
0.0772
4
4.84
0.0613
0.4704
4.74
0.0513
0.3943
Period II
Period I
Q
1.5515
0.2497
4
5.08
0.0358
0.2225
4.49
0.0369
0.2295
10
0.6624
0.1085
4
5.08
0.1330
0.8120
4.49
0.1021
0.6231
11
0.7096
0.1145
4
4.85
0.1134
0.7031
4.49
0.0539
0.3343
12
0.9370
0.1621
01 Ql
V2, 02
4.64
0.0697
0.4031
4.00
0.0542
0.3135
Mean uptake :
0.07258
OA-L7ft
0.07038
0.4294
ensure that the activity was of equal intensity between the various experimental
periods.
In cases where the oxygen uptake fell after blocking- of the anus, the fall was
small, however, indicating that the cloaca and hindgut in 0. erythrogrammon are of
no respiratory significance in contrast to Urcchis canpo (Fisher and MacGinitie,
1928b; Redfield and Florkin, 1931).
Hall (1931) pointed out the large amount of blood present in Urccliis and its
inclusion in the weight of tissues in the calculation for the rate of oxygen uptake
would give a low value. Similarly, in 10 specimens of 0. erythrogrammon the
blood and gut fluid averaged 82% of total weight of the animal against 35 c/< for
Urcchis caupo (Hall, 1931 ) and were not included in the calculation of oxygen
consumption.
Table II shows that both trunk and proboscis, when separated from each
other, consumed oxygen. With the exception of specimens 1 and 14, the trunk
consumed more oxygen than the entire animal during the first experimental period,
thus demonstrating the variability of oxygen uptake and establishing the trunk as
the chief respiratory organ in this species. The higher uptake of the proboscis-less
trunk was probably due to increased activity of the trunk after the loss of the
proboscis. Hence in addition to its role in pumping and renewing the water in
the burrow for respiratory and feeding purposes, the trunk of O. erythrogrammon
also serves as a respiratory surface for oxygen uptake, for which it is well suited be-
90
S. H. CHUAXG
TABLE 1 1
Rate of oxygen consumption of the trunk and proboscis of Ochetostoma erythrogrammon
in cc./gm./hr.
Speci-
men
No.
Trunk
wet
weight
Probos-
cis wet
weight
Duration
of period
Period I: Normal animal
Period II: Trunk separated from proboscis
Initial €>•>
content
Oxygen uptake
based on
Initial O2
content
Trunk 62 uptake
based on
Proboscis Ch
uptake based on
wet
weight
dry
weight
wet
weight
dry
weight
wet
weight
dry
weight
gm.
gm.
hrs.
cc. /liter
cc.
cc.
cc. /liter
cc.
cc.
cc.
cc.
1
0.3721
0.1864
2
4.00
0.0715
0.4725
3.40
0.0976
0.5430
0.0458
0.4853
2
0.5678
0.3346
2
4.00
0.0635
0.3802
3.40
0.1212
0.6226
0.0700
0.6996
3
0.3963
0.2614
2
3.32
0.0544
0.3781
3.20
0.1007
0.5443
0.0365
0.4455
4
0.4515
0.0526
2
3.32
0.0786
0.4222
3.20
0.1198
0.6152
0.1131
0.9916
5
0.6651
0.1995
2
3.32
0.0341
0.2054
3.20
0.0576
0.3015
0.0158
0.1910
6
0.5105
0.1242
2
3.32
0.0669
0.3666
3.20
0.0886
0.4416
0.0843
0.7817
13
0.4454
0.2102
2
4.32
0.0719
0.4457
4.00
0.1313
0.6667
0.0545
0.6367
14
0.5174
0.2741
3
3.90
0.1302
0.8280
4.89
0.1513
0.8062
0.0463
0.4627
15
0.8285
0.3020
3
3.49
0.0345
0.2400
5.08
0.0507
0.3073
0.0453
0.5240
Mean uptake :
0.0672
0.4123
0.1021
0.5387
0.0568
0.5798
cause of the following reasons : firstly, the thinness of the hody wall, which is a
common feature of the genera Ochetostoma and Thalassema, facilitates diffusion
of oxygen. Secondly, the large surface area is further increased by elongation of
the trunk usually seen in specimens inside the burrows in laboratory aquaria.
Thirdly, the presence of a large quantity of body fluid and haemoglobin-containing
coelomic corpuscles continually agitated by peristaltic movements.
The oxygen uptake of the detached proboscis in 9 specimens averaged 17.2%
(range: 7.6-25.4%) of the combined uptake of detached trunk and proboscis. The
proboscis is therefore an accessory but not indispensable respiratory organ, since
proboscis-less trunks survive indefinitely. Because of the extensibility of both
trunk and proboscis it is difficult to compare their available respiratory surface.
Due to the different degrees of hydration between body wall and proboscis, the
average oxygen uptake of the trunk was twice that of the proboscis per gm. wet
weight, although on the basis of dry weight the average uptake was approximately
equal (Table II ).
It is obviously an advantage to the proboscis in being able to respire inde-
pendently of the trunk, since at ebb tide when the proboscis is fully extended forag-
ing on the surface of the wet sand its actively moving distal tip is some 25 cm.
away from the trunk that lies in the oxygen-depleted water inside the burrow. The
presence of a certain amount of coelomic fluid and coelomic corpuscles inside the
proboscis during full extension presumably increases its efficiency as a respiratory
organ. This respiratory function explains the survival of the proboscis several
days after its severance from the trunk.
An indirect evidence in support of the respiratory function for the proboscis is
SITES OF OXYGEN UPTAKE IN OCHETOSTOMA 91
the length and extensibility of the proboscis in the genus Ochctostoina and the
presence of gill-like processes along the ventral margins in the proximal part of the
proboscis in 0. arkati (Prashad, 1935; Wesenberg-Lund, 1959 ) and O. atlantidei
(Wesenberg-Lund, 1959). These processes, Wesenberg-Lund (1959) suggested,
may function as a respiratory organ. The large oxygen uptake by the proboscis in
0. erythrograinnwn suggests respiratory function for the entire available external
surface of the proboscis. It would be interesting to know whether the outer row of
processes in 0. atlantidei would disappear with full extension of the proboscis; the
inner row in O. atlantidei and the processes in 0. arkati may well be mere folds and
presumably disappear with full extension, since in 0. erythrogranunon similar folds
or processes occur transiently along the ventral margin when the proboscis contracts
but disappear with full extension.
Although a study of the relative importance of the different sites of oxygen up-
take in Urcchis caupo is lacking, available evidence, such as the presence of a long,
large, inflatable hindgut and cloaca (Fisher and MacGinitie, 1928a; Fisher, 1946)
and the occurrence of inhalations and exhalations through the anus (Fisher
and MacGinitie. 1928b; Redfield and Florkin, 1931; Hall, 1931; Fisher, 1946),
points to the importance of the hindgut as a respiratory organ. Redfield and
Florkin (1931) observed antiperistalsis of UrecJiis hindgut. obtained 25-35 cc.
of water at a single discharge during anal exhalation and found that this water
contained less oxygen but more carbon dioxide than aquarium water outside
the body. They believed that the thick body wall of Urcchis must absorb a rela-
tively small amount of oxygen in comparison with the hindgut. The relative im-
portance of the sites of respiratory exchange thus differs between the echiuroids,
TABLE III
Rates of oxygen consumption of some annelids, echiuroids and a sipunculoid
Oxygen consumption
Animal Author cc./gm./hr.
Tubifex Brazda (1939) 0.2
Schizobranchia insignis Dales (1961) 0.1920
Ochetostoma erythrogratnmon Present author 0.0692
Bispiravoluticornis Zoond(1931) 0.0573
Sabella Wells (1952) 0.0488
Lnmbricus terrestris Johnson ( 1942) 0.045
Myxicola Wells ( 1952) 0.0398
Sipunculus niidus Cohnheim (from Krogh, 0.0313-0.0688
1916)
Arenicola marina Borden (1931) 0.031
Nereis virens Bosworth, O'Brien and 0.026
Amberson (1936)
Hirudo Heilbrunn (1952) 0.023
Urechis caupo Hall (1931) 0.0198
Glycera siphonostoma Montuori (from Krogh, 1916) 0.0146
Chaetopterus pergamentacens Bosworth, O'Brien and 0.0078
Amberson (1936)
l>2 S. H. CHUANG
Urccliis and Ochetostoma, Presumably, in Urcchis the thick body wall prevents
rapid diffusion of oxygen and the small size of the proboscis offers only a small
respiratory surface. Apart from members of the genus Urcchis, only Nellobia
eusoitia has a large hindgut and cloaca (Fisher, 1946), which may have a respira-
tory function. All other known echiuroids have a slender coiled hindgut and small
cloaca, which are obviously not adapted for efficient respiratory function, but they
have a thin and presumably respiratory body wall.
Krogh (1916) pointed out the difficulty of comparing the metabolism of different
invertebrate animals and that a fair comparison could not be made on the basis of
fresh weight because of the enormous differences in the composition of the various
animals. Although dry weight offers a better basis for comparisons, the presence
of varying amounts of reserve material, skeletal and other inactive tissues also ren-
ders this far from ideal (Krogh, 1916). When the oxygen consumption of O.
erythrogrammon based on wet weight of fixed tissues (i.e. minus coelomic fluid and
gut fluid) is compared with annelids, sipunculoids and Urcchis, it occupies a position
near the top in the descending series of rates shown in Table III.
SUMMARY
1. The oxygen consumption of Ochetostoma erythrogrammon averaged 0.0692
cc. per hour per gram of wet weight of fixed tissue.
2. After blockade of the anus the oxygen uptake did not diminish, indicating that
the cloaca and hindgut have no significant respiratory function.
3. Both trunk and proboscis took up oxygen after separation, the latter con-
suming oxygen averaging \7c/c of the combined uptake of trunk and proboscis.
4. The relative importance of cloaca and hindgut. trunk, and proboscis as
respiratory organs was discussed.
5. The oxygen consumption of O. erythrogrammon was compared with related
animals.
LITERATURE CITED
BOKDEN, M. A., 1931. A study of the respiration and of the function of haemoglobin in
Planorbis corncus and Arcnicola marina. J. J\far. Biol. Assoc., (n.s.) 17: 709-738.
BOSWORTII, M. W., H. O'BRIEN AND W. R. AMBERSON, 1936. Determination of the respiratory
quotient in marine animals. /. Cell. Conip. Physiol., 9 : 77-87.
BRAZDA, F. G., 1939. Respiratory exchange of the fresh water annelid, Tttbifc.i: Proc. Soc.
E.i-p. Biol.. 42: 734-736.
DALES, R. P., 1961. Observations on the respiration of the sabellid polychaete Schisobranchia
insii/nis. Biol. Bull.. 121 : 82-91.
FISHER, W. K., 1946. Echiuroid worms of the North Pacific Ocean. Proc. V . S. Natl. Mits..
96 : 215-292.
FISHER, W. K., AND G. E. MAC&NITIE, 1928a. A new echiuroid worm from California. Ann.
Mag. Naf. Hist.. Set: 10, 1 : 199-204.
FISHER, W. K., AND G. E. MACGINITIE, 1928b. The natural history of an echiuroid worm.
Ann. Mail. Nat. Hist., Ser. 10, 1 : 204-213.
IMIX, H. M., AND C. A. Wi. \CKIKI.I), 1937. The activity and metabolism of poikilothermal ani-
mals in different latitudes. II. Proc. Zool. Soc. London, 107: 275-282.
HAI.I., V. E., 1931. The muscular activity and oxygen consumption of L'rccliis caupo. Biol.
Bull.. 61 : 400-416.
L. V., 1952. An Outline of General Physiology. 3rd ed. 818 pp. Saunders,
Philadelphia,
SITES OE OXYGEN UPTAKE IX OCHETOSTOMA 93
|(in\so\, M. I.., 1942. The respiratory function of the haemoglobin of the earthworm. ./.
/:>/>. />'/,>/., 18: 2(>n 277.
KROGH, A., I'Mh. The Respiratory Exchange of Animals and Man. 173 pp. Longman-.
Green, London.
PRASHAD, B., 1935. On a collection of echiuroids of the genus 'ihulaxscnni Lamarck in the
Indian Museum, Calcutta. l\cc. Indian Mus.. 37 : 39-43.
KEDFIELD, A. C., AND M. FLOKKI.X, 1931. The respiratory function of the blood of / ><-<7n'.v
cunpo. Riol. Hull., 61 : 185-210.
\YKI.LS, G. P., 1951. On the behaviour of Sahclla. J'roc. l\'o\<. Sac. London, Scr. H. 138:
278-299.
WELLS, G. P., 1952. The respiratory significance of the crown in the polychaete worms Stil>clla
and My.ricola. Proc. Roy. Soc. London. Scr. B, 140: 70-82.
WESENBERG-LuND, E., 1959. Sipunculoidea and Echiuroidea from Tropical \Yest Africa.
Atlantidc Report, 5: 177-210.
ZOOXD, A., 1931. Studies in the localisation of respiratory exchange in invertebrates. II. The
branchial filaments of the sabellid, Rispiru I'oluticoniis. J. /:.r/>. Riol., 8 : 258-262.
DAY-LENGTH AND TERMINATION OF PHOTOREFRACTORINESS
IN THE ANNUAL TESTICULAR CYCLE OF THE
TRANSEQUATORIAL MIGRANT DOLICHONYX
(THE BOBOLINK) 1
WILLIAM L. ENGELS
Department of Zooloc/y, University of North Carolina, Chapel Hill, N. C.
At the end of a reproductive season the testes of hirds undergo a regression,
which results in minute, inactive gonads composed of small tubules formed almost
entirely of spermatogonia, the tubules separated by masses of undifferentiated inter-
stitial tissue. In those passeriform birds in which testicular recrudescence is under
photoperiodic control, periods of long day-length fail not only to prevent the occur-
rence of this regression but fail to stimulate recrudescence after the regression is
completed. Hence, this period of the annual cycle is known as the photorefractory
phase (the "preparatory phase" of Wolfson, 1958, p. 372). After some weeks of
exposure to short photoperiods this refractoriness disappears ; thereafter, long
photoperiods again stimulate the mechanism which produces testicular
recrudescence.
Among transequatorial migrants the existence of a photoperiodic mechanism,
including a refractory period, has so far been demonstrated only in the bobolink.
Dolichonyx oryzivorus (Engels, 1959, 1961; Wolfson and Westerhoff, 1960). It
has been shown (Engels, 1961) that (1 ) exposed to the natural day-lengths of the
northern hemisphere, as experienced by such temperate zone migrants as Junco
hyeinalis and Zonotrichia albieollis, the testicular cycle of Dolichonyx develops
ultimately (April) approximately in normal phase; (2) Dolichonyx is able to over-
come naturally induced, autumnal refractoriness on longer photoperiods (12 hours)
than can at least some populations of Junco and Zonotrichia; but (3) the rate of
response to long photoperiods (14 hours) following termination of refractoriness is
slower in Dolichonyx than in the other two forms. However, as was pointed out
(Engels, 1961, p. 146). the photoperiods used in these earlier experiments to re-
lease refractoriness were considerably shorter, and the duration of treatment con-
siderably longer, than birds could be expected to experience in nature in a migration
from the northern to the southern hemisphere soon after the September equinox.
The experiments now to be reported upon were designed ( 1 ) to compare
Dolichonyx to north temperate zone migrants with respect to the timing of termina-
tion of refractoriness when exposed to the natural day-lengths of the north tem-
perate zone, and (2) to determine the capacity of Dolichonyx to overcome refrac-
toriness when exposed to photoperiods more nearly comparable to those normally
experienced in post-nuptial transequatorial migration. Since only meager and
scattered information on the timing of the southward transequatorial passage of
Dolichonyx can be found in the literature, special effort was made to establish the
1 Research supported in part by a grant from the National Science Foundation (G-6163).
94
PHOTOREFRACTORINESS IN BOBOLINKS 95
pertinent facts. The details which are presented below on autumnal migration in
South America were obtained mostly from specimens in the collections of major
museums in the United States.
MATKRIALS AND MKTIIODS
Twenty-one adult male bobolinks (Dolieliony.v oryzirorns ) , all of which had
experienced the natural day-lengths of the northern hemisphere during the preced-
ing summer, were used in the experiments. Two were captured near Wilmington.
North Carolina, in September, a few weeks before experimental treatment was
begun ("autumn captures") ; fifteen were captured near Gainesville, Florida,- in
early May of the year of experimental treatment ("spring captures" ) ; four had been
in captivity one to two years ("second-year experimentals" ).
The birds were kept in an outdoor aviary, exposed to the natural day-lengths of
Chapel Hill (Lat. 36° N.), until experimental illumination was begun at various
times from October 2 to November 28. At the beginning of artificial lighting they
were confined individually in small cages (each about 22 cm. X 25 cm. X 40 cm. )
Each cage was furnished with a food hopper and two 100-cc. water-tubes. Food
consisted of a mash formulated as a complete diet for egg-laying "game" birds ; a
small amount of soluble terramycin was added to the water. The lights used to
provide the experimental photoperiods were automatically switched on and off by
electrically operated time-switches. Eight different lighting schedules were used ;
details of the schedules, including light intensity, are given below. Light intensity
was measured at perch-level.
The birds were examined weekly. Testicular recrudescence was determined by
the development of black pigment in the beak, especially evident in the "mandible" ;
this pigmentation is caused directly by the male sex hormone (Engels, 1959). In
seven bobolinks which were killed, during the winter of 1961-62, within a few days
to a maximum of two weeks following the first appearance of this pigmentation, the
testes averaged 179 mm.3 per bird in volume (range, single testis. 32.5 mm.3 to
131.5 mm.3) (previously unpublished data). The volume of an inactive testis. in
males with light-colored beaks, is less than 2 mm.3
RESULTS
1. Termination of refractoriness under natural day-lengths of Lat. 36° N.
(Figure 1 )
Five groups of birds, two to four in each group, were used in this series of
experiments. Group A (Group E of Engels, 1961, p. 143) consisted of two
"autumn captures" removed from the aviary to an indoor, light-tight compartment
on October 2 and exposed thereafter to constant daily 14-hour photoperiods (white
fluorescent lights, intensity about 90 foot-candles. ) Neither of these birds had
developed beak pigmentation by late May, when observations were terminated.
Group B consisted of four birds, all "2nd year experimentals." They were ex-
posed to natural day-lengths from late May until November 28, after which white
2 This study could not have been made at this time except for the kindness of Cameron E.
Gifford, University of Georgia (presently at Earlham College, Richmond, Indiana), who gen-
erously made these birds available to me after I had failed in attempts to capture some in
North Carolina during the spring migration of 1961.
96
WILLIAM L. EX(,KLS
-15
-14
35°S"
»A,C,B -
Dotes by which beak
became black
A - None (2)
B - Feb. 1,28, Mar. 8, 15
C- Feb. 28, War. 8(2)
D- Jan. 8(2)
E -Jan. 12, 18
SEPT
OCT
NOV
DEC
JAN
FEB
MAR
FIGURE 1. Experiments to determine the time of termination of photorefractoriness in
bobolinks exposed to the natural day-lengths of Lat. 36° N. At various times between
October 2 and November 28, day-length was abruptly increased either to 14 hours daily or to
a simulation of the daily change in day-length (sunrise-sunset) occurring at that time in the
southern hemisphere. For this treatment groups A and E were moved, at the times indicated,
to light-tight compartments indoors ; the other groups remained outdoors, where the artificial
lighting was superimposed on the natural day-length. The development of black pigment in
the beak is evidence of testicular recrudescence.
fluorescent lights ( which insured a minimum intensity of 30 to 35 foot-candles )
provided 14-hour daily photoperiods (5:15 AM-7:15 PM ) ; these birds remained
in the outdoor aviary, hence during the dark period were exposed to approximately
normal light of the night sky. Beak pigmentation indicative of testicular recrudes-
cence developed first in one bird during the week ending February 1, in the other
three before March 15.
Group C consisted of three "spring captures" ; they were subjected to exactly
the same light schedules and other conditions as Group B except that the 14-hour
photoperiods were begun almost two weeks earlier, on November 15. Beak pig-
mentation developed during the last week of February and first week of March.
Group D consisted of two "spring captures" ; they were caged in an outdoor
aviary where, beginning November 8, incandescent lamps (intensity about 45 foot-
candles ) provided photoperiods which approximated the changing sunrise-sunset
day-lengths normally occurring during the months of November to March at Lat.
45° S. The abrupt change in photoperiod on the first day was from 11^ hours to
14i hours; the photoperiod then increased gradually to more than 15^ hours in
PHOTOREFRACTORINESS IN BOBOLINKS 97
December. Both birds developed the nuptial pigmentation of the beak during the
first week of January. (These lights were automatically switched on and off by
an "astronomical-dial" time switch, geared to the daily changes in time of sunrise
and of sunset at Lat. 45°, manufactured by the Sangamo Electric Co., Springfield,
Illinois.)
Group E also consisted of two "spring captures." They were removed from an
outdoor aviary on November 1 and thereafter subjected indoors to photoperiods
corresponding to the changing sunrise-sunset day-lengths of Lat. 35° S. Beak pig-
mentation developed during the second and third weeks of January. ( White fluo-
rescent lights, intensity about 90 foot-candles, switched on and off by an "astronomi-
cal-dial'' clock were used ; between photoperiods the birds were in absolute
darkness.)
General conclusion
The mechanism which stimulates testicular recrudescence in bobolinks is refrac-
tory to long photoperiods in early autumn, at least until October 1 ; when birds
are held captive in the northern hemisphere, exposed to the natural day-lengths of
Lat. 36° N., photorefractoriness is terminated sometime during October, definitely
by November 1.
2. The southtvard migration of bobolinks and flic day-lengths experienced by them
during migration (Figures 2, 3 )
Oberholser (1920) brought together data from a number of localities in the
United States, gathered over various periods of years, which give for each locality
an average "first date seen" and an average "last date seen," that is, average dates
of arrival and departure. These data for the eastern United States in autumn are
incorporated in the accompanying chart of latitudinal distribution (Fig. 2). Since
1947 "Audubon Field Notes," in an annual review of autumnal migration of North
American birds, provides some additional data for points within the United States.
Unfortunately, records chiefly only of an unusual nature (exceptionally early or
exceptionally late observations ) are published here, but there have been a feu-
notices on peaks of abundance. All the "Audubon Field Notes" records, through
1960, are also represented in Figure 2. No such data are available for the migra-
tion south of the United States. Through the kind cooperation of a number of
individuals and institutions T have been able to locate, in museums of this country,
89 specimens taken south of the United States and to compile the data on locality
and date of collection.3 Thirty-seven of these specimens were taken in September,
October, and November. Together with the Oberholser and the "Audubon Field
3 I am grateful to the following individuals and institutions for lending specimens for im-
personal examination and/or for supplying the "lahel data" on specimens not seen by me :
Dean Amadon, American Museum of Natural History (New York) ; Kenneth C. Parkes,
Carnegie Museum (Pittsburgh); Emmet R. Blake, Chicago Natural History Museum; R. A.
Paynter, Jr., Museum of Comparative Zoology (Harvard) ; Harrison B. Tordoff, Museum of
Zoology, University of Michigan ; James Bond, Philadelphia Academy of Sciences ; and P. S.
Humphrey and Mary A. Heimerdinger, Peabody Museum of Natural History (Yale).
The following reported that their collections lacked specimens of Dolichonyx taken south
of the United States : Alden H. Miller, Museum of Vertebrate Zoology, University of Cali-
fornia; Donald Hoffmeister, Natural History Museum, University of Illinois; E. Raymond
Hall, Natural History Museum, University of Kansas; George H. Lowery, Museum of Natural
Science, Louisiana State University; and H. G. Deignan, U. S. National Museum (Washington).
98
WILLIAM L. ENGELS
-50°N
* i i i i i
X
1 1 1 I 1 1
II 111 1
X
< X
Ar\° i
v XX
T X
T
• ^ *
-30° •
• X
» • • •/
• «p
X
* ** x r
X
•
-20°
• t
-
-10°
* i«|
t • t t
t
- 0°
~
-10°
-
-20°
. + 4 *
•30°S
AUGUST
SEPTEMBER
1 1 1 1 1 t
OCTOBER
NOVE MBER
1 1 t 1 1 1
FIGURE 2. Latitudinal distribution of bobolinks from August through November. The
horizontal lines approximate the southern border of the breeding grounds (Lat. 40° N.) and
the northern border of the "wintering" grounds (Lat. 8° S.). Plain circles are from published
sight records, mostly from Audnbon Field Notes (vols. 1-14, 1947-1960) ; squares indicate
"peaks of abundance" reported in the same journal; + and X indicate annual average dates of
arrival and departure, respectively (average dates of "first seen" and "last seen" ; data from
Oberholser, 1920) ; circles with vertical lines indicate museum specimens (line above circle
= male, below = female). The major part of the "wintering" grounds lie below about Lat.
14° S. (in Peru, Bolivia, Brazil [Matto Grosso], Paraguay and northern Argentina), but the
species occurs regularly between November and March in the Trujillo and Lima districts of
Peru, on the western slope of the Andes, at Lat. 8°-12° S. (Koepcke, 1961).
Notes" data, these records give a reasonably clear picture of the southward migra-
tion of bobolinks (Fig. 2). The breeding grounds, north of Lat. 40° N., are
usually emptied by the end of the second or third week of September. Meanwhile,
some individuals (which must have started their journey in August) have at that
time already made the trans-Gulf or trans-Caribbean passage and are in Central
or South America at Lat. 8°-ll° N. Although some individuals may still be in
the United States in late October, or exceptionally even in early November, some
have reached the '"wintering" area below Lat. 8° S. at least by November 1. In
Figure 3 the same latitudinal distribution data are plotted against day-length (sun-
rise-sunset plus morning and evening civil twilight). The arrow drawn through
this figure gives a rough approximation of the day-lengths experienced by an
"average" bobolink during the southward transequatorial migration. Whatever
the degree of validity of this approximation, there are certain limits on the day-
length cycle which migrating bobolinks may experience, limits which are imposed
PHOTOREFRACTORINESS IN? BOBOLINKS
99
1 1 1 1 r
~n
oe
20°N
40°N
AUGUST
SEPTEMBER
OCTOBER
NOVEMBE R
FIGURE 3. Day-lengths (including civil twilight) experienced by bobolinks from August
through November. Same data and symbols as in Figure 2. The arrow roughly approximates
the migration of an "average" bobolink. See text for discussion of limiting factors. (Day-
length data from : Tables of Sunrise, Sunset and Twilight : Supplement to the American
Ephemeris, 1946; U. S. Naval Observatory. Government Printing Office, Washington, D. C.)
by the changes in day-length at different latitudes, some of which also are shown in
this figure. Any birds which might reach the equator during the third week of
September would experience at about that time their shortest day-length, about 12
hours 45 ± 4 minutes (sunrise-sunset plus civil twilight) ; thereafter they would be
exposed to gradually increasing day-lengths (until the December solstice). Any
birds still north of the equator on October 1. at whatever latitude, would at that
time also experience day-lengths of about 12 hours 45 ± 4 minutes. Day-lengths
continue to decline in the northern hemisphere until the December solstice but at
a progressively lower rate the lower the latitude. As a consequence of this phe-
nomenon, and as is evident in Figure 3, a southwardly migrating bird begins to
experience a progressive increase in day-length while still north of the equator in
October, an even greater increase in November ; it is again exposed to a little more
than 12^ hours of day-light when it reaches the equator. South of the equator, of
course, day-lengths then everywhere are increasing above that level.
3. Termination of refractoriness under day-lengths comparable to those experienced
in transequatorial migration (Figure 4)
In addition to the previously described Group A, three groups of bobolinks, three
birds in each group, were used in this series of experiments. All of these nine
birds were "spring captures" which had spent the summer in an outdoor aviary,
exposed to the natural day-lengths of Lat. 36° N. As with Group A, they were
brought indoors at the beginning of October into light-tight, ventilated compart-
ments and exposed there to artificial photoperiods under white fluorescent lights.
Between photoperiods they were in absolute darkness.
100
WILLIAM L. ENGELS
-15
Dates by which beak
became black
A- None (2)
F- Feb. 12,19, Mar. 19
G - Dec. 7, Feb. 19
H- Jan. 29, Feb. 2,12
a
o
IT
LLl
a.
o
36° N
-13
-12
AUG
1
SEPTEMBER
1
OCTOBER
NOVEMBER DEC
FIGURE 4. Experiments to determine length of photoperiod, and duration of treatment, which
may release bobolinks from photorefractoriness during October and November. On October 2
ten birds were removed, from the natural day-lengths (including civil twilight) of Lat. 36° N.,
to constant daily photoperiods of different lengths, in four groups. Development of black
pigment in the beak indicates testicular recrudescence. The dotted line is a rough approximation
of day-length experienced by an "average" bobolink during southward, post-nuptial migration
(cf. Figure 3).
Group F
These three birds were exposed to 12f-hour photoperiods (light intensity about
90 foot-candles) for six weeks; on November 13 the photoperiod was increased to
14 hours. Two birds developed beak pigmentation in early and mid-February, the
last in the third week of March.
Group G
As in the previous group the initial photoperiods were 12 J hours (light intensity
90 foot-candles), but these were continued for only five weeks; the 14-hour photo-
periods were begun on November 6. One bird developed beak pigmentation sur-
prisingly early, during the first week of December, another not until the third week
of February. The third bird accidentally hung itself in the cage during the period
December 22-26. At the time the accident was discovered the lower beak was
discolored, but the testes were minute.
Group H
The initial photoperiods were 12^ hours (light intensity about 45 foot-candles)
and they were continued for only four weeks. On October 30 the photoperiods
were increased to 14 hours. One bird developed beak pigmentation during the last
week in January, the other two in early February.
General conclusion
The post-nuptial photorefractoriness exhibited by bobolinks at the beginning of
October can be terminated by only five weeks of relatively long, 12f-hour, photo-
PHOTOREFRACTORINESS IN BOBOLINKS 101
periods or by only four weeks of 12|-hour photoperiods. These lighting schedules
approximate the day-lengths experienced by bobolinks in post-breeding migration.
DISCUSSION
Bartholomew (1949) pointed out that photorefractoriness might play an im-
portant role in regulating the timing of the annual recrudescence of the testes in
Passer doinesticus. It may be a significant factor in many passeriform birds in
which the annual testicular cycle is controlled by photoperiodism (Wolfson, 1952 ;
Wolfson, 1958). It assures that the gonad-stimulating mechanism does not again
become activated, following regression and reconstitution of the inactive testes, un-
til middle or late autumn when days are short, and continue to shorten, and the
photoperiodic stimulus is therefore weak at best. Recrudescence of the testes, and
the appearance of male sexual behavior, with ultimate development and release of
motile spermatozoa, is thus suitably delayed.
In the absence of experimental evidence it was not easy to fit transequatorial
migrants into this picture, because in post-nuptial migration they pass directly from
the shortening days of the northern hemisphere autumn into the lengthening days
of the southern hemisphere spring, never experiencing the retarding effect of the
short days of winter. This consideration leads logically to the question of the
photorefractory phase in such migrants. Bissonnette (1937) had suggested that
"prolonged refractory periods . . . would supply the necessary delay to prevent
even transequatorial migrants from breeding in their southern range" (p. 263).
Farner (1954) postulated for transequatorial migrants "a characteristically longer
refractory period" (p. 29). Wolfson (who since 1958 has preferred the term
"preparatory phase") has spoken of the relation between day-length and the photo-
refractory phase as the "main problem" in equatorial and transequatorial migra-
tion and of the regulation of this phase as the "critical problem" (Wolfson, 1959,
pp. 706-7; Wolfson, 1960, p. 785). Wolfson and Westerhoff (1960), in a report
on some preliminary experiments with bobolinks, suggested that in this species, as
compared with temperate zone species, a longer period of short days may be re-
quired in the regulation of the preparatory phase.
In all temperate zone species so far investigated, the refractory period is ter-
minated in nature in middle to late autumn, that is, variously between mid-October
and mid-November or even early December (published data summarized by Farner,
1954; Farner, 1959; Wolfson, 1958). It seems evident from our first series of
experiments that, under comparable conditions (i.e., the natural day-lengths of
middle latitudes in the northern hemisphere), photorefractoriness in Dolichony.v
may be terminated as early as November 1. Since normal reproductive activity in
the preceding season had been suppressed in these captive birds, testicular regres-
sion may have been accelerated, leading possibly to an earlier termination of refrac-
toriness. However, in some temperate zone species refractoriness persists until mid-
November and in at least one species until early December (Zonotrichia albicollis,
Lat. 42° N., Shank, 1959). Thus, even if we allow two to five weeks for a possible
effect of the celibacy imposed on our captive birds, it would seem that Dolichonyx
does not differ markedly from temperate zone migrants with respect to the timing
of release from refractoriness in the northern hemisphere autumn.
102 WILLIAM L. ENGELS
The present experiments also permit comparison of Dolichonyx and temperate
zone migrants in two other respects, namely, the length of the short days effective
in terminating refractoriness, and also the number of such short days required. In
previously reported experimental studies on the regulation of photorefractoriness
by short days, photoperiods longer than 12 hours have not been employed (except
by Wolfson and Westerhoff, 1960, for Dolichonyx). However, in at least some
populations of Junco hy emails and Zonotrichia albicollis, even eight weeks of ex-
posure to 12-hour photoperiods, beginning October 1, does not release refractori-
ness (Engels, 1961). In another population of Z. albicollis, studied by Shank
(1959), five weeks of 12-hour photoperiods, beginning October 1, failed to ter-
minate refractoriness in any of nine males tested ; thirty-one days of 9-hour photo-
periods failed for five of eight males tested. In the present experiments, refractori-
ness in Dolichonyx was terminated by only four weeks of 12^-hour photoperiods,
and also by five weeks of 12f-hour photoperiods, both beginning October 1. There-
fore, the suggestion of Wolfson and Westerhoff, mentioned above, that Dolichonyx
may require a longer period of short days to complete the "preparatory" phase,
seems to be inapplicable. (Termination of refractoriness by November 1 under the
influence of natural day-lengths at Lat. 36° N. also argues against this idea.) It
may be suggested rather that in Dolichonyx one of the adjustments of the photo-
periodic mechanism to transequatorial migration lies in the capacity to overcome
refractoriness on relatively longer days, up to at least 12f hours.
Another adjustment, to the long days experienced between breeding seasons
during the southern hemisphere summer, was indicated by earlier studies ( Engels,
1961) which showed that, when bobolinks, j uncos and white-throated sparrows were
subjected to identical effective treatment for termination of refractoriness and
stimulation of the gonad, testicular recrudescence in the bobolinks lagged several
weeks behind recrudescence in the other two forms. It was suggested, at the
time (p. 145), that this retardation of the bobolink cycle might be explained simply
as evidence of a very slow rate of response (to 14-hour photoperiods) following
the termination of refractoriness. An interesting alternative explanation might be
that, during the period of exposure to shorter days, the photorefractory phase of
Dolichonyx is not actually terminated (Engels, 1959, p. 764) but reaches a point
where "longer days [no longer] prevent, but perhaps delay [its] completion"
(Wolfson, I960, p. 785 ). It was hoped initially that the present experiments would
throw light on this question but, among other deficiencies, the number of birds used
was too small to give the required information. It will be interesting to test the
idea with experiments of a different design.
Examination of museum specimens indicates that the black pigmentation of the
beak, which we used as a criterion of testicular recrudescence in bobolinks, in nature
does not develop until April, when northward migration already is underway.
Thus, in all of our experiments the development of this pigmentation was greatly
accelerated, occurring in December, January, February or early March, even when
we attempted to approximate, after October 1, the day-lengths expected to be en-
countered during the autumnal migration, followed by an approximation of the
average day-length of the southern hemisphere summer. Obviously, much re-
mains to be learned about the regulation of the natural timing of the testicular cycle
in this transequatorial migrant.
PHOTOREFRACTORINESS IX BOBOLINKS 103
SUM MAKY
1. The testicular cycle of Dollchonyx oryzivorns, a bird which breeds above Lat.
40° N. and winters below Lat. 8° S., exhibits a photorefractoriness in early autumn,
which is maintained by constant daily 14-hour photoperiods (experiment begun
October 2).
2. Some individuals which had been held captive outdoors, exposed to the
natural day-length of Lat. 36° N., were shifted to experimental, long photoperiods
at various times between November 1 and November 28. Within a few months all
of them developed the characteristic black beak pigmentation indicative of testicular
recrudescence. From these results it is concluded that, under the influence of
autumnal day-lengths of middle latitudes of the northern hemisphere, refractoriness
is terminated by November 1 and that Dolichanyx thus does not differ appreciably
from temperate zone species in the timing of this event under these conditions.
3. From published data on the time of autumnal migration within the United
States and from data furnished by museum specimens collected south of the United
States, an approximation of the cycle of day-lengths experienced by migrating bobo-
links is constructed, which indicates that an "average" bobolink may experience in
autumn only a few weeks of day-lengths less than about 12f hours.
4. Beginning October 2, when captive bobolinks were experiencing natural day-
lengths of about 12 hours 41 minutes, some were exposed to constant daily photo-
periods of 12^ hours for four weeks, others to 12^-hour photoperiods for five or six
weeks, after which the photoperiod was increased to 14 hours. Testicular re-
crudescence occurred in all. It is concluded that photorefractoriness can be over-
come in this species by only a few weeks of exposure to photoperiods which in
length are comparable to those it encounters in transequatorial migration but which
are longer than those which maintain refractoriness in such temperate zone forms as
Junco hyemalis and Zonotrichia albicollis. The number of shorter days required
for the termination of refractoriness seems to be of the same general order of
magnitude as for Junco and Zonotrichia.
LITERATURE CITED
BARTHOLOMEW, G. A., JR., 1949. The effect of light intensity and day length on reproduction
in the English sparrow. Bull. Mus. Comp. Zooi, 101: 433-476.
BISSONNETTE, T. H., 1937. Photoperiodicity in birds. Wilson Bull., 49: 241-270.
ENGELS, W. L., 1959. The influence of different daylengths on the testes of a transequatorial
migrant, the Bobolink (Dolichonyx orysivorns). In: Photoperiodism and Related
Phenomena in Plants and Animals (pp. 759-766). R. Withrow, Ed. Publ. No. 55,
Amer. Assoc. Adv. Sci., Washington, D. C.
ENGELS, W. L., 1961. Photoperiodism and the annual testicular cycle of the bobolink (Doli-
chon\x or\zii'orus), a transequatorial migrant, as compared with two temperate zone
migrants. Biol. Bull., 120: 140-147.
EARNER, D. S., 1954. Northward transequatorial migration of birds. Sci. Rev. (Netv Zealand),
12: 29-30.
EARNER, D. S., 1959. Photoperiodic control of annual gonadal cycles in birds. In: Photo-
periodism and Related Phenomena in Plants and Animals (pp. 717-750). R. Withrow,
Ed. Publ. No. 55, Amer. Assoc. Adv. Sci., Washington, D. C.
KOEPKE, M., 1961. Birds of the western slope of the Andes of Peru. Amer. Mus. Xoritates,
no. 2028, p. 24.
OBERHOLSER, H. C., 1920. The migration of North American birds. Bird Lore, 22: 213-217.
104 WILLIAM L. ENGELS
SHANK, M. C., 1959. The natural termination of the refractory period in the slate-colored
junco and in the white-throated sparrow. Auk, 76: 44-54.
WoLFSON, A., 1952. The occurrence and regulation of the refractory period in the gonadal
and fat cycles of the Junco. ./. /:.r/>. Zool.. 121: 311-325.
WOLFSON, A., 195S. Regulation of refractory period in the photoperiodic response's of the
white-throated sparrow. ./. /-;.r/>. Zool., 139: 349-380.
WOLFSON, A., 1959. The role of light and darkness in the regulation of spring migration and
reproductive cycles in birds. In: Photoperiodism and Related Phenomena in Plants
and Animals (pp. 679-716). R. Withrow, Ed. Publ. No. 55, Amer. Assoc. Adv. Sci.,
Washington, D. C.
WOLFSON, A., 1960. Role of light and darkness in the regulation of the annual stimulus for
spring migration and reproductive cycles. Proc. Xlltli International Ornithological
Congress, Helsinki, 1958, pp. 758-789.'
WOLFSON, A., AND T. R. WESTERHOFF, 1960. Photoperiodic regulation of the preparatory phase
of the annual gonadal cycle in a transequatorial migrant, Dolichon\x or\zh>orus.
Anat. Rcc., 137: 402.
DIGESTION, STORAGE, AND TRANSLOCATION OE NUTRIENTS
IN THE PURPLE SEA URCHIN ( STRONGYLOCEXTROTUS
PURPURATUS)1
A. FARMANFARMAIAN 2 AND JOHN H. PHILLIPS
Department of Bacteriology, University of California, Berkeley 4, California
The internal transport of nutrients in echinoderms has been a matter of interest
since 1809 when the French Institute offered a prize for a description of the
"circulatory" system of asteroids, echinoids, and the holothuroids (Tiedemann,
1816). Since that time the anatomy of various echinoderms has been studied by
numerous investigators, prominent among whom were Perrier, Hamann, and
Cuenot. The accumulated knowledge of the Phylum Echinodermata was presented
in a treatise by Hyman in 1955.
A survey of this literature indicates a general recognition of three fluid systems,
i.e., the perivisceral fluid, the water vascular system, and the haemal system. How-
ever, the roles of these systems in the translocation of food remain obscure.
The vessels of the water vascular system and the sinuses of the haemal system
are very narrow and delicate. Sampling of the fluids that they contain is ex-
tremely difficult. Therefore, examination of transport in these systems has been
limited to microscopic observations of the movement of objects within the vessels
or sinuses, particles of injected dyes or the naturally present coelomocytes being
the objects observed (Perrier, 1875; Kawamoto, 1927; see also Hyman, 1955).
The perivisceral fluid, which bathes the internal organs, is relatively large in quan-
tity and more accessible to sampling and subsequent examination. This fluid from
representatives of the more conspicuous classes, namely the holothuroids, asteroids,
and echinoids, has been subjected to physiological and biochemical analysis (Jacob-
sen and Millott, 1953; Lasker and Giese, 1954; Boolootian and Giese, 1958; Far-
manfarmaian, 1959; see also Hyman, 1955). Some observations in these studies
have resulted in the assignment of various possible functions to the variety of coelo-
mocytes which are to be found in this fluid as well as in the tissues of the animals
(Hyman, 1955; Stott, 1955; Boolootian and Giese, 1958).
The suggestion that these cells may be involved in nutrient transport is based
on very little experimental evidence. Phagocytosis of foreign materials such as
carbon, carmine, and fat particles and their deposition in certain tissues may or
may not simulate aspects of natural nutrient transport. Since phagocytic cells are
known to ingest such non-nourishing and inert particles as polystyrene latex
spherules with subsequent migration in vitro (Sbarra and Karnovsky, 1959, 1960),
no a priori significance may be attached to such processes when they are observed
in vivo. Even studies involving the injection of materials such as sugars or iron
saccharate (Lasker and Giese, 1954; Stott, 1955), though useful under special
1 This study was supported by National Science Foundation Grant No. G-10867.
2 Present address : Department of Biology, University of Shiraz, Shiraz, Iran.
105
106 A. FARMANFARMAIAN AND JOHN H. PHILLIPS
circumstances, cannot be regarded as good indicators of the natural process of
transport following the digestion and absorption of food. The considerations men-
tioned above indicate the desirability of a systematic study of the translocation of
nutrients following natural absorption. Knowledge of this phenomenon is pre-
requisite to the understanding of much of the biology of echinoderms. It has been
possible to carry out such a study through the feeding of algae labelled with carbon-
fourteen to sea urchins.
MATERIALS AND METHODS
The purple sea urchin, Strongylocentrutns purpitratus, was chosen for these
investigations. Animals with a test diameter of 4—7 cm. were starved for five to
eight weeks prior to experimental feedings. The animals were maintained in well-
aerated sea water at 15° C. in the laboratory.
The red alga, Iridaca flaccidurn, was used as food in the experiments. (The
name Iridophycus has been used to describe this genus also.) This material was
chosen as food because it is abundant in the habitat of the urchin throughout the
year; it constitutes one of the animal's natural foods as judged by the examination
of gut contents in the field ; and it is capable of maintaining urchins in good condi-
tion for over a year in laboratory aquaria when used as the sole source of food.
Much of the biochemistry of this alga is known (Hassid, 1936; Bean ct al., 1953;
Bean and Hassid, 1955).
Cut discs of the alga were labelled with C14 according to the method of Bean
ct al. (1953), using the gas-sealed apparatus, which they describe, as a photo-
synthesizing chamber. One to three grams of labelled alga could be prepared in
this manner. Amounts of the alga less than one gram were labelled in sealed vials
in a sea water solution of NaHC14O3 at pH 8. In either case the amount of
C^CX available to the alga was empirically adjusted to give 2-4 X 104 counts per
minute/mg. wet weight of alga. The photosynthetic assimilation of C14(X was
allowed to proceed for 10 hours at about 18° C. The algal discs were sampled in
several places to determine their specific activity. The discs were then drained
and weighed carefully before being fed to the animals.
The animals were fed in sealed jars maintained at 15° C. ; see Figure 1. These
jars were provided with capillary air inlets which opened below the sea water and
vacuum outlets from the gas phase. It was thus possible to draw of! metabolic
C14O2 into a Ba(OH)2 trap and provide the animal with continuous aeration
without contaminating the laboratory. The animals were never allowed more than
five hours of feeding time. After the desired period of feeding, the remainder of
the algal disc was removed and weighed as before.
All of the tissues were freshly sampled in such a manner as not to contaminate
one another. This was achieved by careful dissection, the use of several sea water
washes, and frequent changes of dissecting instruments. Duplicate samples were
taken in all cases. When the final specific activity of duplicate samples differed
by more than 10^, the samples were rejected. Rejection occurred most frequently
when the gonads were ripe and spawning took place during sampling. Other
sources of error responsible for large differences in duplicates were the difficulty
of weighing fresh tissue to constant weight and contamination during dissection.
SEA URCHIN NUTRIENT TRANSLOCATION
107
FIGURE 1. Arrangement of feeding jars in which sea urchins were fed radioactive algae.
The perivisceral fluid was sampled by a one-milliliter syringe through the
peristomeal membrane and directly from the main coelom. A volume of \Q°/c
solution of ethylene diamine tetraacetic acid at pH 8.0 equal to the sample volume
was used as anticoagulant. The cells and plasma were separated by centrifugation
as desired.
Since the specific activities of various tissues were to be compared, it was
necessary to use a homogeneous suspension or solution of the tissues. A high
108
A. FARMANFARMAIAN AND JOHN H. PHILLIPS
degree of homogeneity was attained by digesting samples of tissue in NaOH with
the aid of 30/{, H..O., and heat. Soft animal tissue samples of 10-20 mg. and
100-200 mg. samples of test wall were placed in graduated centrifuge tubes and
covered to the 0.5-ml. mark with 1 J\l NaOH in sea water. Algal samples
were covered with 10 J\I NaOH during the digestion and subsequently diluted
with sea water in a manner calculated to keep the concentration of salts uniform.
Time of digestion and the amount of H.O., used were as needed. Unless otherwise
stated, the activity of the peri visceral fluid was determined without digestion.
Samples of digests were placed on stainless steel planchets, dried, and counted
in a Nuclear Chicago gas-flow counter, model D-47 with mica window, equipped
with scaling unit model 161 A, sample changer model c-HOB, and printing timer
model c-lllB. All errors in the counting procedure were corrected by the methods
of Calvin et al. (1949) and Kamen (1957). Particular attention was paid to
errors in geometry and absorption because of the high salt content of the samples.
Paper chromatograms were analyzed by an Actigraph model c-lOOA combined
with sealer model 1620A and recorder R1000.
RESULTS AND DISCUSSION
Digestion. The digestive tube of the purple sea urchin consists of a buccal
pouch, a short pharynx, and an esophagus that enters the first convolution of the
tube. This portion of the tube immediately below the esophagus will be referred
to as the stomach. The stomach opens into the second convolution, referred to
here as the intestine, and the latter terminates in a short rectum which opens to
the outside via an anus. The stomach and intestine each have five festoons which
will be designated one to five in sequence from mouth to anus.
The conversion of algal C14 into animal C14 was determined in the following
manner : the specific activity of the alga pieces fed and the specific activity of the
contents of the digestive tubes of animals sacrificed at various times were deter-
mined. These results are presented in Table I, and show that conversion efficiency,
as is indicated by efficiency of digestion and absorption, is about 90% when the
animal is fed a limited, 20 to 100 mg. wet weight, amount of Iridaea. Since the
TABLE I
Conversion efficiency of CH-labelled substances from Iridaea tissue into sea urchin tissue
C/M/mg. of dry digest of
Days after start
of feeding
% Activity
incorporated
Nature of gut content
Algae fed
Gut content
1
526
38.6
93
Bite form
No bags
No bacterial enrichment
2
490
50.3
90
As above
3
555
36
93
Bag form
Bacterial enrichment
4
641
53
91
Bag form
High bacterial enrichment
9
294
33
89
As above
SEA URCHIN NUTRIENT TRANSLOCATION 109
digestive efficiency remains nearly the same throughout the interval of one to nine
days after a limited amount of food is ingested, the results suggest that digestion
and absorption of at least the labelled portion of the food occurs primarily on the
first day, and thereafter the remnants of food are on their way to defecation. Since
the food has not passed the fourth festoon of the stomach by the second day, the
esophagus, the stomach, or both would appear to be the main site of digestion in
the animal. The algal material in the first four festoons of the stomach is essen-
tially in bite form and free of bacterial enrichment. As is indicated in Table I,
bacteria become conspicuous by the third day when the material has passed the fourth
and fifth festoons of the stomach. At this point the material has been converted
from the bite form to the bag form. These different kinds of gut contents are
pictured in Figure 2. These observations suggest that the digestion of Irldaca in
1 c*v.
*
•
k
FIGURE 2. Pieces of Iridaca removed from various parts of the digestive tube. The transition
from bite form from the stomach to bag form from the intestine is seen from left to right.
the stomach is not dependent on bacterial action and is more likely under the influ-
ence of digestive enzymes secreted by the sea urchin.
This suggestion was confirmed by examining the digestive ability of extracts
of esophagus, stomach, and intestine. The tissues, washed free of gut contents,
were homogenized in sea water and freed of large tissue fragments by centrifugation.
Washed soaked agar and washed bite-sized pieces of Iridaea were used as sub-
strates. Toluene was added to prevent bacterial growth. Reducing sugar was
110 A. FARMANFARMAIAN AND JOHN H. PHILLIPS
TABLE 1 1
Reducing sugar liberated by tissue extracts*
Agar Iridaea
Extract of esophagus 76 ^tgf 60 fig
Extract of stomach 160 80
Extract of intestine 80 40
Pooled extract 240 80
No extract 56 40
"Twelve-hour incubation at 15° C. ; 50 nig. of agar and Iridaea used as substrates. The
amounts of tissue extract of esophagus, stomach, and intestine were 5, 71, and 78 ng protein.
The pooled extract was a preparation containing equivalent amounts of each of the extracts.
All values were corrected for the contribution of reducing material in the extracts.
t Values equivalent to jug glucose.
determined after incubation by the method of Park and Johnson (1949). Table II
shows the results and conditions of incubation. It should be noted that some reduc-
ing material is solubilized from Iridaea and agar under the conditions of incubation.
However, larger amounts appeared in solution after exposure to extracts of stomach
or esophagus. The extract of intestine when tested by itself did not liberate addi-
tional reducing material from Iridaea. Whether or not it contributes to the mate-
rial released by the extract pool remains to be determined. Both the stomach and
the esophagus appear to possess digestive enzymes. Since the amount of mate-
rial in the extract of esophagus is only one-fifteenth the amount in the other two
extracts, the results suggest that the esophagus may contain appreciable amounts of
digestive enzymes. The presence of enzymes capable of hydrolyzing agar is
definitely indicated.
Lasker and Giese (1954) reported the presence of enzymes in extracts pre-
pared from the whole digestive tube of the purple sea urchin. Enzymes capable of
digesting casein, starch, and iridophycin, a galactan prepared by Hassid from
Iridaea, were detected. Eppley and Lasker (1959) have demonstrated alginase and
algin depolymerase activity in the digestive tract of this animal. The failure of
Lasker and Giese (1954) to detect agar-digesting enzymes may have been due to
their use of agar warmed to 37-40° C. Since this temperature is more than 10
degrees above the lethal temperature of the animal, it may result in inactivation of
the enzymes responsible for agar digestion.
While the Aristotle's lantern of the sea urchin is a magnificent masticatory ap-
paratus that is capable of reducing the alga to small pieces before it reaches the
esophagus, the final disintegration of algal structure by the subsequent action of
digestive enzymes has not been observed. There is no question that the bacteria
from the intestine of the sea urchin can attack algae, but their role in the digestion of
that part of the food which is normally assimilated may not be significant. When
Iridaea is made available to the sea urchin in plentiful quantities, the animal often
feeds continuously and defecates rapidly. LTnder these conditions the feces are
usually in bite form and without bacterial enrichment. The disintegration of algal
structure observed in the distal festoons of the intestine when defecation is delayed
may provide additional assimilable material. The uptake of such material by the
animal would not have been observed in the experiments reported here since
labelling of the structural elements of the algal cells would have to have been ac-
SEA URCHIN NUTRIENT TRANSLOCATION
111
complished during their growth. However, the lack of participation of micro-
organisms in the digestion of the labelled portion of the alga is further indicated by
studies in which bacterial action was inhibited by antibiotics. C14-labelled algal
material kept in strong solutions of streptomycin and penicillin prior to feeding gave
the same digestive efficiency and no bacterial enrichment of the feces.
That the microorganisms observed in feces do not constitute forms unique to
the urchin is suggested by the following observations : finely chopped Iridaca in
sterile sea water was incubated for four days in the dark at 15° C. Similar prepara-
tions were inoculated with bacteria-enriched contents of the rectum. Both series
yielded a grossly similar collection of bacteria and protozoa. These preliminary
experiments suggest that the fauna and flora of the sea urchin gut may represent the
symbionts of the ingested alga. Mastication and digestion by the sea urchin render
the material more susceptible to attack by these microorganisms and results in the
bacterial and protozoan enrichment so often observed in the intestine of the animal.
Storage of digested jood. From studies on the reproductive physiology and
biochemistry of the purple sea urchin, the site of food storage has become a point
of controversy (Giese et al., 1958). It was, therefore, desirable to determine the
site of deposition of C14-labelled compounds in the animal. The overall specific
activity of the animal was calculated in the following manner :
Specific Activity of animal
(total activity of ingested algal material) (conversion efficiency)
(total wet weight of the animal in mg.)
The specific activities of various tissues of the sea urchin were determined and
expressed in relation to the overall activity of the animal, the latter being given
a value of 1.00. These results are presented in Table III. The esophagus and the
first festoon of the stomach appear to be the main sites of nutrient storage. The
drop in activity of the walls of the digestive tract by the seventh day after feeding
was not accompanied by a general shift of material to all other tissues. It is well
TABLE III
Specific activity of various tissues relative to the calculated overall specific activity of the
animal in counts/ minute /mg. wet weight
Days after feeding
i
2
3
7
Tissue
Whole animal
1.0
1.0
1.0
1.0
Body wall
interambulacral
0.1
0.3
0.1
0.3
ambulacral
0.3
0.5
0.2
0.5
Gonad
1.6
1.0
0.8
1.3
Esophagus
20.4
60.0
26.3
21.2
Stomach
Festoon 1
37.5
86.0
47.0
27.3
Festoon -1
7.(>
14.5
5.2
11.8
Intestine
Festoon 1
6.2
15.5
3.5
10.5
Festoon 4
2.3
12.0
1.6
4.7
112
A. FARMANFARMAIAN AND JOHN H. PHILLIPS
established that a starving sea urchin will resorb its gonads, but there is no indica-
tion that the gonads constitute the natural storage organ of these animals. A recent
study of Giese (1961 ) indicates that lipid is the main reserve food of Strongvloccn-
trotus purpuratus, S. jranciscanus, and Alloccntrotus fragilis. The lipid is stored in
the wall of the intestinal tract and is observed to decrease in amount during starva-
tion. In the asteroids the hepatic caeca have been shown to be the organs of stor-
age (Farmanfarmaian et al., 1958; Anderson, 1953). Anatomically these are
diverticula of the digestive tract, and the festoons of the sea urchin digestive tract
may be compared to them in function.
TRANSLOCATION OF NUTRIENTS
1. The haemal system
In general this system consists of poorly defined sinuses often filled with red
coelomocytes. No movement of fluid within any part of the system has been ob-
served even though a rhythmic beat may be seen in the outer sinus of the stomach
and its collateral sinuses. In spite of careful attempts, the fluid could not be
sampled without contamination. Therefore, the role of the haemal system in the
transport of nutrients was assessed by indirect means. C14-labelled material ap-
pears in the gonads even on the first day after feeding; see Table III. Each gonad
FKITKE 3. The aboral haemal ring and its short sinuses which penetrate the gonads. The
arrow indicates one of the sinuses. Photograph is of a fresh specimen enlarged two times.
SEA URCHIN NUTRIENT TR. \\SI.OCATIOX
113
is penetrated l>v only <>w sinus from tin' aboral haemal ring. Therefore, it was
possible to sever this sinus and compare the specific activity of a gonad thus iso-
lated from the haemal system with its normal neighbor; see Figure .^. This delicate
operation was accomplished by carefully drilling a half-millimeter hole just oral to
the gonopore in the center line of the interambulacral region. Under a dissecting
microscope equipped with a strong spotlight, the haemal sinus and the gonoduct of
the gonad were seen just inside the test wall. These tubes could then be gently
lifted up by means of a finely bent needle and severed by a microscalpel made from
a piece of razor blade. The hole was closed with a fine wooden plug covered with
Vaseline. The operation did no apparent harm to the animal nor altered its behavior
in any observable manner. The cut tubes constricted and healed within 24 hours,
and the connection between the aboral haemal ring and the gonad began to
regenerate by about the tenth day after the operation.
Twenty-four hours after the operation, animals were fed C14-labelled Iridaea,
and the specific activity of the gonads was determined at intervals following feeding.
The results are presented in Table IV. In all cases the isolated gonad and its
TABLE IV
Comparison of the specific activity of normal gonad with neighboring gonad whose
connection to the aboral haemal ring was experimentally severed
C/M/mg. wet weight
Days after
feeding
Sex
Gonad
gravidity
Animal wet
weight in
grams
Overall
Gonad
Gonad
animal
experimental
control
1
9
Ripe
31
160
275
253
2
9
Ripe
12
24
26
25
3
d1
Ripe
25
230
177
181
7
tf
Ripe
25
42
54
55
neighbor contained essentially the same amount of activity, irrespective of sex or
number of days after the start of feeding. The active materials, therefore, must
have arrived via routes other than the haemal system.
These experiments were carried out on animals with ripe gonads. The possibil-
ity that the haemal system plays a special role in nutrient transport to the gonads
during their period of buildup, i.e., August through November (Giese et al.. 1958),
must be examined by experimentation. However, data presented in a later section
indicate the more general route of nutrient transport.
The function and microanatomy of the haemal system remain an enigma. This
collection of sinuses does not appear to constitute a circuitous system, nor does it
appear to have, functionally speaking, a point of origin or terminus. The enigma
is no less striking when other classes of the phylum are considered (Hyman. 1955 ).
The haemal system may be the vestige of a true transport system in the ontogeny
or phylogeny of echinoderms.
2. The water vascular system
This system consists of a well-defined water vascular ring, the stone canal, and
the five radial canals that penetrate the ambulacra! regions through the auricles and
114
A. FARMANFARMAIAN AND JOHN H. PHILLIPS
thence send side branches to ampullae of the podia. Although coelomocytes may be
observed to move within the lumen of the radial canals, there is no clear direction
of flow. There is good evidence to support the suggestion that the canals of the
water vascular system maintain the hydrostatic pressure required for the operation
of the podia and their ampullae (Cuenot, 1948; Hyman, 1955).
Because of the difficulty encountered in attempts to sample the fluid of this sys-
tem, an indirect method similar to that used for the haemal system was adopted.
FIGUKK 4. A view of the oral side of the animal from within. The lantern has been pushed
to one side. The arrow points to the radial haemal sinus and the radial water canal just prior
to their entrance into the orifice of the auricle. Photograph of fresh specimen twice enlarged.
The operation was considerably simpler because the radial haemal sinus and the
radial water canal adhere firmly to the peristomeal membrane just prior to their
entrance into the auricle. (See Fig. 4.) A 1-mm. incision through the soft
peristomeal membrane across the center line of the ambulacral region exposed these
tubes. The tubes were severed as described in the operation on the haemal system.
Healing and regeneration are about the same as those described for the haemal
system. Twenty-four hours after the operation, animals were fed C ^-labelled
Iridaea, and the specific activity of the ambulacral areas determined in the usual
manner. The results of these experiments are presented in Table V. No sig-
nificant difference was noted between the ambulacral regions isolated from the radial
haemal sinus and water canal and neighboring ambulacra which were left connected.
SEA URCHIN NUTRIENT TRANSLOCATIOX 115
TABU. V
Comparison of the specific activitv of normal ambulacrum with neighboring ambulacrum
whose radial water canal and haemal si mix were experimentally severed
at the auricle
Days after feeding
Animal wet weight
in grams
C/M/mg. wet weight
Overall
animal
Ambulacrum
experimental
Ambulacrum
control
1
31
160
53
55
2
32
24
14
13
3
29
28
13
12
7
25
42
22
22
It seems reasonable to conclude that the water vascular system does not play a
significant role in the transport of digested food under these circumstances. These
experiments provide additional evidence for the lack of importance of the haemal
system in this process.
3. The perivisceral fluid
The perivisceral fluid occupies the main coelomic chamber of the sea urchin and
is kept in circulation by the cilia of the epithelial lining of the coelom. All the
internal organs of the animal are bathed by this fluid. Numerous cells, the coelomo-
cytes, of seven different kinds may be observed in this fluid (Boolootian and Giese,
1958). The plasma phase of this fluid contains low levels of nitrogenous com-
pounds, carbohydrates, and possibly fats (Giese et al., 1958; see also Hyman, 1955)
and has essentially the same salt composition as sea water.
Since there are several milliliters of this fluid in the main coelom which may
be tapped by a syringe via the soft peristomeal membrane, it was possible to sample
this fluid directly and determine the level of activity at various intervals after the
start of feeding. Figure 5 shows the results of a series of such experiments. The
labelled substances reach a peak level within six hours and then decrease in concen-
tration to a fairly constant level within the first 24 hours. Thereafter the level is
generally maintained for at least nine days, the period during which samples were
usually taken and examined. The general shape of the graphs in Figure 5 was
independent of animal size and quantity of C14-containing material fed.
In studies on the respiration of the purple sea urchin, it was demonstrated that
the perivisceral fluid serves as a medium for respiratory gas exchange (Farman-
farmaian, 1959). It has also been demonstrated that the oxygen uptake of a
starved urchin may increase by as much as 50^ following feeding (unpublished
data ) . The possibility that the peak levels depicted in Figure 5 are due to a gush of
respiratory C^CX was tested in the following way: duplicate sets of samples wrere
taken from an animal. One set was acidified to pH 2 in order to convert any C14O2
to a volatile form. After drying, the activity in both sets was determined. Figure
6 shows the result of this experiment. The activity found in the perivisceral fluid
is not due to respiratory C14O.,. which must be present at any given time at a
negligible level.
116
A. FARMANFARMAIAN AND JOHN H. PHILLIP^
Rate of Appearance of Cl4-labeled Substances in Perivisceral
Fluid of Four Sea Urchins. Time after Start of Feedings
I I I 1 _ y / I
3579
Hours Days
FIGURE 5.
/ / i
2 6 10 14
Hours Days
The rise and fall in the activity of the perivisceral fluid suggested two
hypotheses :
a. The transfer of nutrients from the digestive tube to the perivisceral fluid is
controlled by some mechanism, e.g. neurosecretions. When a starved animal is
fed, nutrients are rapidly mobilized, and a peak level of activity in the perivisceral
fluid is observed. Within the first day after feeding, the tissues of the animal
attain a state of relative sufficiency, and a feedback mechanism reduces and main-
tains the level of mobilized nutrients in a steady-state.
b. The peak level of activity in the perivisceral fluid is due to one or more
labelled substances which are rapidly released and transferred to the perivisceral
fluid by passive or active diffusion across the wall of the digestive tube. Since the
quantity of algal material fed is restricted, the quantity of these diffusing sub-
stances is also limited. An initial peak is to be expected, and when the labelled
substances are absorbed by the tissues, a steady-state is attained. In the steady-
state, nutrients are mobilized from the reserves at the same rate as they are con-
sumed by the tissues, or the steady-state is maintained due to some control
mechanism.
These two hypotheses were tested by a series of experiments. Animals were
SEA URCHIN NUTRIENT TRANSLOCATION
117
.14
-
Level of C Activity in Perivisceral Fluid
before and after Acidification
Untreated Perivisceral Fluid
Perivisceral Fluid Acidified
to pH 2
O
O
4567
Hours
FIGURE 6.
8
I 2 3
Days
fed labelled algae in the usual manner and the activity in the perivisceral fluid was
determined. On the second day after establishment of the steady-state, the animals
were fed again and the level of activity in the perivisceral fluid was again followed.
Figure 7 shows the results of one of these experiments. If the first hypothesis
were correct, a second peak would not have been expected. The first meal would
have been expected to correct any nutrient deficiency of the tissues. Since a second
peak was observed, the starvation preceding feeding was not responsible for the
peak. The second hypothesis would appear to be more tenable, with the peak level
representing material which rapidly diffuses from the gut into the perivisceral fluid.
The possible involvement of a control mechanism in maintenance of the steady-state
cannot be determined by these experiments and will require further study.
The role played by the coelomocytes in the transport of nutrients was deter-
mined by measuring the partition of activity between the plasma and cells of the
perivisceral fluid at various intervals after the start of feeding. In order to achieve
proper geometry for counting and to obtain comparable data, both the cells and the
plasma were digested after separation and appropriate dilutions of the digests were
pipetted onto the planchets for counting. Figure 8 presents the results and indi-
cates that during the peak of the activity nearly all of the activity is in the plasma
phase. Within one day from the start of feeding, as the steady-state is approached,
118
A. FARMANFARMAIAN AND JOHN H. PHILLIPS
J4
Rate of Appearance of C -labeled Substances in Perivisceral
Fluid after 2 Consecutive but Separate Feedings
o 1500
CD
O
CO
CD
Q_ 1000
1
O
\
50°
CO
~ci
ZJ
O
o
Start of
Second Feeding
i i i
/ /i i / /
i i i i i i
4
8 10
I 2 50 52 54 56 58
Hours
Days
Hours
FIGURE 7.
the coelomocytes become the more heavily labelled phase. Table VI presents a set
of similar results obtained with additional animals. At the peak level of activity
more than 90% of the label is in the plasma. When the activity in the peri visceral
fluid levels off, less than 50% of the activity remains in the plasma. Since coelomo-
cytes, particularly the red eleocytes (Boolootian and Giese, 1958) may be observed
in the wall of the digestive tube and other tissues, the presence of label in these cells
adds support to the view that normal transport of nutrients from the site of reserves
to other tissues may be partially achieved via the agency of coelomocytes. The
above results do not, however, preclude the possibility that the labelled material in
TABU-: VI
Distribution of Cl4-labelled substances in the plasma and the coelomocytes of the perwisceral
fluid of four sea urchins
Animal
Sampled at
Counts/Min/O.l ml. of
% Activity
in plasma
Perivisceral
fluid
Plasma
A
B
C
Peak of activity
Peak of activity
"Leveled-off" activitv
843
107,?
186
763
1070
36
90.5
99.5
19.3
D
"Leveled-off" activity
250
116
46.5
SEA URCHIN NUTRIENT TRANSLOCATION
119
Rate of Appearance and Distribution of Cl4-labe!ed
Substances in Plasma and Coelomocytes of
Perivisceral Fluid. Time after Start of Feeding
& — A Plasma
° — ° Coelomocytes
3 5 7 10
30 35
7 f
23456
Hours
Days
FIGURE 8.
these cells may be attributed to their own nutritional requirements ; their appear-
ance in the tissues of the animal may serve other functions.
Attempts were made to identify the labelled compounds found in the perivisceral
fluid. Nearly 90% of the activity observed during the peak period was accounted
for by one substance which was identified as galactose by its chromatographic be-
havior in three different solvent systems, conversion to mucic acid, and oxidation by
galactose dehydrogenase (Block ct al., 1958; Doudoroff, personal communication).
Free galactose is not a major constitutent of Iridaca tissue. The form of galactose
most heavily labelled under the conditions of labelling used here is galactosylglycerol.
None of this material could be detected in the perivisceral fluid. This galactoside is
apparently hydrolysed by enzymes of the urchin gut.
After the peak level of activity is replaced by the establishment of a steady-state,
the C14 is distributed among several compounds. Both carbohydrates and amino
acids possess activity. Because the level of activity is very low, final identification
of these compounds will require microtechniques which have not yet been attempted.
SUMMARY
1. In the purple sea urchin the digestion and absorption of the C14-labelled con-
stituents of the alga, Iridaca, occur mainly in the esophagus and adjacent festoons of
the stomach.
2. The fauna and flora of the sea urchin gut do not appear to be involved in
this digestive process.
3. The absorbed materials are stored mainly in the wall of the gut.
4. During absorption there is a diffusion of labelled material into the plasma of
120 A. FARMANFARMAIAN AND JOHN H. PHILLIPS
the perivisceral fluid. A peak level is reached around the sixth hour after the start
of feeding. Galactose accounts for 90% of this material and must have been liber-
ated enzymatically from galactose-containing compounds such as galactosylglycerol.
5. The peak level of activity is replaced by a prolonged interval in which the
level of activity is reduced but quite constant. The radioactivity is distributed over
a variety of compounds including both amino acid and carbohydrates.
6. Translocation of nutrients is accomplished by the perivisceral fluid. No
evidence for the participation of either the haemal or water vascular systems could
be demonstrated.
LITERATURE CITED
ANDERSON, J. M., 1953. Structure and function in the pyloric caeca of Astcrias forhcsi. Biol.
Bull., 105: 47-61.
BEAN, R. C., E. W. PUTNAM, R. D. TRUCCO AND W. Z. HASSID, 1953. Preparation of C"
labelled d-galactose and glycerol. J. Biol. Chcin., 204: 169-173.
BEAN, R. C., AND W. Z. HASSID, 1955. Assimilation of C"O2 by a photosynthesizing red alga,
Iridophycus flacciduin. J. Biol. Chan., 212: 411-425.
BLOCK, R. J., E. L. DURRUM AND G. ZWEIG, 1958. A Manual of Paper Chromatography and
Paper Electropboresis. Academic Press, Inc., New York.
BOOLOOTIAN, R. A., AND A. C. GiESE, 1958. Coelomic corpuscles of echinoderms. Biol. Bull.,
115: 53-63.
CALVIN, M., C. HEIDELBERGER, J. C. REID, B. M. TOLBERT AND P. F. YANKWICK, 1949. Isotopic
Carbon. John Wiley and Sons, New York.
CUENOT, L., 1948. Anatomic, ethologie et systematiques des echinodermes. hi: Traite de
zoologie, P. Grasse, editor, vol. XL
EPPLEV, R. W., AND R. LASKER, 1959. Alginase in the sea urchin, Strongylocentrotus pnrpura-
lus. Science, 129: 214-215.
FARMANFARMAIAN, A., 1959. The respiratory surface of the purple sea urchin {Strongylocen-
trotus pitrpnratus) . Doctoral Dissertation. Stanford University, California.
FARMANFARMAIAN, A., A. C. GIESE, R. A. BOOLOOTIAN AND J. BENNETT, 1958. Annual repro-
ductive cycles in four species of west coast starfishes. /. E.rf>. Zoo/., 138 : 355-367.
GIESE, A. C., 1961. Further studies on Alloccntrotus fnu/ilis. a deep-sea echinoid. Biol. Bull.,
121: 141-150.
GIESE, A. C, L. GREENFIELD, H. HUANG, A. FARMANFARMAIAN, R. A. BOOLOOTIAN AND R.
LASKER, 1958. Organic productivity in the reproductive cycle of the purple sea urchin.
Biol. Bull.. 116: 49-58.
HASSID, W. Z., 1936. Carbohydrates in Iridaca laminaroidcs (Rhodophyceae). Plant Phvsiol.,
11: 461-463.
HYMAN, L. H., 1955. The Invertebrates. Vol. IV. McGraw-Hill Book Co., Inc. New York.
JACOBSON, F., AND N. MILLOTT, 1953. Phenolases and melanogenesis in the coelomic fluid of
Diadcina. Proc. Roy. Soc. London, Scr. B, 141 : 231-247.
KAMEN, M. D., 1957. Radioactive Tracers in Biology. Academic Press Inc., New York.
KAWAMOTO, N., 1927. Anatomy of Caudina chilcnsis. Tohoku Unh'. Sci. Refits. Scr. 4, Biol.,
2 : 239-265.
LASKER, R., AND A. C. GIESE, 1954. Nutrition of the sea urchin, Strongylocentrotus pnrpura-
his. Biol. Bull.. 106: 328-340.
PARK, J. T., AND M. J. JOHNSON, 1949. A submicrodetermination of glucose. J. Biol. Chcin.,
' 181: 149-151.
PERRIER, E., 1875. Appareil circulatoire des oursins. Arch. Zool. Ex p. lrr Scric, 4: 605-643.
SBARRA, A. J., AND M. L. KARNOVSKY, 1959. The biochemical basis of phagocytosis I. /. Biol.
Chcin., 234: 1355-1362.
SBARRA, A. J., AND M. L. KARNOVSKY, 1960. The biochemical basis of phagocytosis II. /. Biol.
Chcm., 235: 2224-2229.
STOTT, F. C., 1955. The food canal of the sea urchin I'.cliinits csculcntus L. and its functions.
Proc . Zool. Soc. Lond.. 125 : 63-86.
TIEDEMANN, F., 1816. Anatoiiiie der Rohren-Holothurie des pomeranzfarbigen Seesterns uncl
Stein- Seeigels. Landshut, Joseph Thomannschen Buchdruckerei.
PHOTOMECHANICAL RESPONSES OF THE PROXIMAL PIGMENT
IN PALAEMONETES AND ORCONECTES '
MILTON FINGERMAN, R. NAGABHUSHANAM AND LORALEE PHILPOTT
Department of Zoology, Ncuromb College, Tulane University, New Orleans 18, Louisiana,
and Marine Biological Laboratory, Woods Hole, Massachusetts
Photomechanical adaptation of the crustacean compound eye in response to
changes in illumination depends upon the behavior of the distal, proximal, and re-
flecting retinal pigments. The most recent review of the literature dealing with
these pigmentary effectors was written by Kleinholz (1961). Much of the avail-
able information relates to the retinal pigments of the prawn Palacmoncles rulgaris.
However, many of the investigators of retinal pigments in Palaemonetes and other
crustaceans concerned themselves with the distal pigment only. Consequently, little
is known about the physiology of the proximal and reflecting pigments. Parker
(1896, 1897) was the first investigator to describe in a detailed manner the move-
ments of the retinal pigments in Palaemonetes. Welsh (1930) later continued the
study. He observed that migration of the distal pigment in light-adapting eyes
was the result of shortening of the distal pigment cells. Sandeen and Brown (1952)
found that the position maintained by this pigment in Palaemonetes is a function
of the brightness of the visual field, not a true albedo response.
As far as endocrine studies are concerned, in 1936 Kleinholz reported that
eyestalk extracts of Palaemonetes, when injected into dark-adapted prawns, caused
light-adaptation of the distal and reflecting pigments but had no effect on the
proximal pigment. More recently, evidence was obtained for a principle that dark-
adapted the distal pigment. Brown, Fingerman and Hines (1952) observed that
the response of the distal pigment to a light stimulus depended upon the light
history of the prawns. The distal pigment of specimens kept in darkness over-
night showed a much greater degree of light-adaptation in response to one minute
of bright illumination than did this pigment in prawns preconditioned in dim light
To explain this difference in response, the investigators postulated that a dark-
adapting hormone was available to the prawns that had been exposed to the pre-
conditioning low illumination but not to those that had been in darkness. Webb
and Brown (1953) and Brown, Webb and Sandeen (1953) interpreted similar
experiments in the same manner. Brown, Hines and Fingerman (1952) were able
to increase the rate of dark-adaptation by injecting eyestalk extract into prawns at
the time they were placed in the darkroom. Fingerman, Lowe and Sundararaj
(1959) later were able to produce, by injection of eyestalk extracts, a dark-adapta-
tional response in prawns maintained in light. Nothing further, however, has been
done with the proximal and reflecting retinal pigments of Palaemonetes.
Information about the retinal pigments of the other crustacean used in this
investigation, Orconectes clypeatits. is meager. The distal pigment of this crayfish
is regulated by dark-adapting and light-adapting principles, just as in Palaemonetes
1 This investigation was supported in part by Grant No. B-838 from the National Institutes
of Health.
121
122 MILTON FINGERMAN, R. NAGABHUSHAXAM AND LORALEE PHILPOTT
(Fingerman, Mobberly and Sundararaj, 1959). The proximal and reflecting pig-
ments of this organism have not been studied.
The primary objective of the present series of experiments was to obtain more
information about migration of the proximal pigment in Palaenionctes vulgar Is,
especially the controlling mechanism. Kleinholz (1961) has suggested the possi-
bility that this pigment is an independent effector. Among the experiments were
some designed to determine ( 1 ) rates of migration of the proximal pigment under
different experimental conditions, and (2) the character of the response of this
pigment to a series of intensities of illumination. An experiment was also per-
formed with Orconcctcs, by way of comparison with Palaenionctes, in order to
determine whether the control mechanism could be the same for the proximal
pigment of both species.
MATERIALS AND METHODS
Specimens of the prawn Palaetnonetes rulgaris were obtained in the Woods
Hole area weekly during the summer of 1960 through efforts of personnel from
the Supply Department, Marine Biological Laboratory. We are extremely in-
debted to these individuals. The prawns were kept in large aquaria with running
sea water. The crayfish. Orconectes clyf>catus, were collected during the spring
of 1961 in roadside ditches at Hickory, Louisiana, and were maintained in aquaria
that contained aerated tap water. All of the experiments were performed at
22-24° C.
In order to determine accurately the position of the proximal retinal pigment,
eyestalk sections, 20 /A thick, were prepared. In two of the experiments the position
of the distal retinal pigment, also apparent in the sections, was of interest. The
specimens were killed by immersion in boiling water for 10-15 seconds, thereby
rapidly stopping additional migration of the retinal pigments. Both eyestalks were
then removed from each specimen and placed in Bouin's solution until paraffin
sections could be prepared. With the aid of a compound microscope, ocular
micrometer, transmitted light, and reflected light, the positions of the pigments
relative to the basement membrane could be precisely determined. Reflected light
was used as an aid in distinguishing between the proximal and reflecting pigments.
The difficulty of distinguishing between these pigments when transmitted light
alone is used was noted by Kleinholz (1936). For the sagittal sections three meas-
urements were made, just as was done by de Bruin and Crisp (1957) with the eyes
of European crustaceans: (A) distance from outer corneal surface to distal edge
of proximal pigment, (B) distance from outer corneal surface to distal edge of
distal pigment, and (C) distance from outer corneal surface to basement mem-
brane. The position of the basement membrane is constant. The ratio A/C was
called the proximal pigment index ; B/'C, the distal pigment index. L^se of ratios
minimized the effects of size differences among the specimens.
EXPERIMENTS AND RESULTS
Palaenionctes vnhjaris
Times required for li(/ht-adaf>tatio)i and dark-adaptation of flic proximal retinal
pigment
The object of this set of experiments was to determine the length of time
required for migration of the proximal retinal pigment of Palacnionetcs from the
PROXIMAL RKTIXAL PIGMENT
123
light-adapted position to the dark-adapted one and back again. One group of
prawns was placed in a darkroom for two hours and another group in white pans
was exposed for two hours to an illumination of 560 ft. c. In a preliminary experi-
ment this combination of background and intensity was sufficient to cause maximal
light-adaptation of the proximal pigment. At the end of the two hours of pre-
conditioning, 10 animals from each group were killed. The animals remaining in
the darkroom were then exposed to 560 ft. c. while on a white background and the
prawns that had been in light were put in the darkroom. At 15-minute intervals
10 animals from each group were preserved. The experiment was performed
twice.
1.0
x
LJ
Q
Z 0.9
z
u
5
o
Q.
<0.8
X
O
C£
PL
0.7
B
15
30 45 0
MINUTES
15
30
FIGURE 1. Relationships between the average proximal pigment index of Palaemonetes and time
in minutes following transfer from light to darkness (A) and from darkness to light (B).
The means of the proximal pigment indexes are presented in Figure 1 . Each
point in the figure represents the average index of 20 eyestalks, each from a different
prawn. Inspection of Figure 1 reveals that dark-adaptation required 45 minutes
and light-adaptation 30 minutes. In a fully dark-adapted eye the proximal pigment
index was 1 .0 ; all of the proximal pigment had migrated proximal to the basement
membrane. The proximal pigment index of a light-adapted eye was about 0.735.
The mean maximal distance the proximal pigment had migrated distally in 38 fully
light-adapted eyes was 92 /*. The mean distance from the outer corneal surface to
the basement membrane in the same 38 eyestalks was 347 /*.
124 MILTON FINGERMAX, R. XAGAUHL'SHAXAM AXD LORALEE PHILPOTT
Relationship hetween lit/lit intensity and pro.vinial piyinent index oj prawns on black
and on white backgrounds
The objective of this experiment was to determine the manner in which the
proximal pigment responds to different intensities of illumination and shades of
background. To accomplish this purpose prawns in black and in white pans were
exposed for two hours to one of a series of incident illuminations between 0.55 and
2230 ft. c. The latter intensity was sunlight. The other intensities were obtained
by placing the containers of prawns at appropriate distances from the sources of
illumination. Each intensity was measured with a General Electric photometer that
had been calibrated at the Department of Physics, Newcomb College. Water in
the pans exposed to the higher intensities was changed frequently to avoid heating
of the prawns.
Inspection of the proximal pigment indexes revealed that at each illumination
the pigment was in a more light-adapted position in prawns in white pans than
in black ones. To learn whether this difference was a true background response
depending upon the albedo, ratio of incident to reflected light, or simply a response
to the intensity of reflected light, the fractions of the incident light reflected from
the black and the white backgrounds had to be determined. The white background
reflected one-half of the incident light but the black background reflected merely
%0 °f tne incident illumination. Then, on the basis of these data, the intensities
of incident light were converted to intensities of reflected light. The mean proximal
pigment indexes were subsequently plotted in Figure 2 against the logarithms of
the reflected light intensities. Each point represents the mean of 12-20 indexes,
the average number being 16.9. If the response depended on the albedo alone,
0.95
u 0.90
Q
o
D.
0.80
O 075
rr
Q.
0.70-
•o-
I
1
I
-3 -2 - I
REFLECTED
0123
LIGHT INTENSITY, FT C.
FIGURE 2. Relationship between the average proximal pigment index of I'uliicimnu'tcs and
the logarithm of the reflected light intensity. Circles, \vliite background ; dots, black background.
See text for complete explanation,
PROXIMAL RETIXAL PIGMENT 125
thru the data would have- fallen along two distinct curves with no overlap of the
indexes obtained from prawns on each of the two backgrounds. Such a situation
is characteristic of chromatophore responses to background. However, inspection
of Figure 2 revealed that the data fell more naturally along one curve. In tin-
range of reflected light intensities between 0.55 and 12.5 ft. c. there was considerable
overlap of the data. Maximal light-adaptation occurred at a reflected illumination
of about 140 ft. c.
One must conclude from these data, therefore, that the position of the proximal
pigment depends upon the intensity of illumination reflected from the surroundings
rather than the albedo. Furthermore, the proximal pigment is functional over a
wide range of light intensities and can be maintained in positions intermediate
between the fully light-adapted and dark-adapted ones.
Relationships between (a) time in light and time required for re-dark-adaptation
and ( b ) time in darkness and time required for re-light-adaptation
The object of this set of experiments was to learn whether the rates of light-
and dark-adaptation of the proximal pigment can be altered by appropriate stimuli
or whether these rates are independent of the light history of the animals. For use
in the first set of experiments 16 white enameled pans, containing 10 animals each,
were placed in the darkroom for two hours to assure maximal dark-adaptation of
the proximal retinal pigment. At the end of the two hours, the prawns in one
pan were killed. Subsequent observation of the eyestalks of the latter prawns
revealed their average proximal retinal pigment index was 1.0, i.e. dark-adaptation
of the proximal pigment had occurred. The remaining 15 pans were placed under
an illumination of 250 ft. c. After five minutes in light, four of the pans were
returned to the darkroom, four more were returned after 15 minutes in light, and
another four after 45 minutes of illumination. With the return of each group of
four pans to the darkroom, the prawns in a fifth pan were killed. The prawns in
one of the pans from each of the three groups were preserved 10. 20, 40. and 60
minutes after their group had been placed in the darkroom the second time. This
experiment was clone twice and the data were qualitatively the same. The observed
pigment indexes are presented in Figure 3 A where each point represents the mean
of 12-20 indexes, the average being 16.5. Zero time is the onset of the illumination
period.
The proximal pigment of the prawns that had been in light for five minutes
continued to light-adapt for at least 10 minutes after they had been put in the
darkroom again. The indexes presented for the prawns at the end of the five
minutes of illumintaion and after 10 minutes in darkness represent the means of
19 and 20 values, respectively. The subsequent rate of re-dark-adaptation was
less than maximal. The proximal pigment of the prawns that had been in light 15
and 45 minutes slowly began to dark-adapt, but the rate increased with time.
The proximal pigment of prawns that had been illuminated 45 minutes was more
nearly dark-adapted after 60 minutes than it was in either the 5- or 1 5-minute
groups after each had been in the darkroom for an hour.
The reciprocal experiment was performed next. Sixteen white enameled pans,
each containing 10 prawns, were exposed to an illumination of 250 ft. c. for two
hours. At the end of this period of conditioning, prawns from one pan were killed
126 MILTON FINGERMAN, R. NAGABHUSHANAM AND LORALEE PHILPOTT
0.9
o.e
Q
Z
h
z
UJ
2
O
Q.
07
0.9
O 0.8
o:
D.
0.7
0.6 -
20
40
60
MINUTES
80
100
120
FIGURE 3. Relationships between mean proximal pigment index of Palaemonetes and time
in minutes. A, dark-adapted prawns illuminated and then returned to the darkroom ; B, light-
adapted prawns put in the darkroom and then returned to light. Circles, 5 minutes ; dots,
15 minutes; half-filled circles, 45 minutes in light (A) or dark (B). The dashed lines connect
the initial indexes of each group of prawns.
and the eyestalks preserved. The remaining 15 pans were placed in the darkroom.
After five minutes in the dark, four of the pans were returned to an illumination
of 250 ft. c., four more were returned after 15 minutes, and another four after 45
minutes. With the return to light of each group of four pans, the animals in a fifth
pan were killed. The position of the pigment 10, 20, 40. and 60 minutes follow-
ing the return of each group to light, was determined by fixing the eyestalks of the
animals from one pan at the appropriate intervals. The experiment was repeated.
The averaged data are presented in Figure 3B where the mean number of indexes
represented by each point ranges from 14 to 20, the average being 17.6. The rate
of re-light-adaptation was a direct function of the time spent in darkness ; the
longer animals were kept in the dark (after having been previously light-adapted),
the more rapid was the rate of re-light-adaptation.
PROXIMAL RETINAL PIGMENT
127
Influence of lit/lit history on the response oj /lie retinal pit/iuenls to a hii/h intensity
of illumination jor one minute
The aim of this set of experiments was to determine the character of the response
of both the distal and proximal pigments to one minute of high intensity illumina-
tion in prawns kept overnight (a) in darkness and (h) in the stock aquarium.
The prawns that had been in the stock aquarium overnight had been exposed to the
gradually increasing illumination of dawn. At 5:00 A.M. on the morning the
experiment was performed, 30 animals from the stock aquarium were divided
equally among three white enamelled pans and exposed for one minute to an illumi-
nation of 250 ft. c. After this bright stimulus the animals in one pan were killed
0.6
0.5
0.4
0.3
0.2
1.0
0.9
o:
0.8
0.7
0
HOURS
FIGURE 4. Relationships between time in hours and (A) average distal pigment index
(D. P. I.) and (B) average proximal pigment index (P. P. I) of Palacmanclcs kept in darkness
overnight (circles) and of prawns kept overnight in the stock aquarium where they were
exposed to dawn ('dots). At 5 A.M. both groups were exposed to an illumination of 250 ft. c.
for one minute.
128 MILTON FINGERMAN, R. NAGABHUSHAXAM AND LORALEE PHILPOTT
and their eves preserved. The remaining pans in the meanwhile were placed in
the darkroom. After 30 minutes in darkness, the animals from one pan were
killed. The remaining 10 prawns were killed after 60 minutes. Also at 5 A.M.,
six white enameled pans, containing 10 prawns each, were taken from the dark-
room after having been there since 5 P.M. the previous evening and were exposed
for one minute to the same 250 ft. c. The animals in one pan were killed imme-
diately. The remaining five pans were returned to the darkroom. Prawns in one
of the pans in the darkroom were killed 30, 60, 90, 180, and 300 minutes after the
return to the darkroom. This experiment was performed twice.
After the eyestalks were sectioned, the indexes of the distal and proximal pig-
ments were determined. The averaged data were used in the preparation of Figure
O:
Ci
0.16
0.14
0.12
0.10
0.08
0.06
1.00
0.95
090
- 0.85
a:
080
0.75
0.70
0.65k
B
HOURS
FIGURE 5. Relationship between time in hours and (A) average distal pigment index
(D.P.I.) and (B) average proximal pigment index (P.P.I.) of Orconectes kept in darkness
overnight (circles) and of prawns illuminated overnight under one ft. c. (dots). At 5 A.M.
both groups were exposed to an illumination of 250 ft. c. for one minute.
PROXIMAL RETINAL PIGMENT 129
4 where each point represents 13-20 indexes, the mean being 17.4. It is apparent
from inspection of this figure that re-dark-adaptation of the distal and proximal pig-
ments occurred at a very slow rate in the prawns kept in darkness overnight ; one
minute of bright light produced a light-adaptational response of both pigments that
required about five hours to subside. On the other hand, dark-adaptation of both
pigments occurred at the maximal rate in the Palaemonetes that had been exposed
to dawn.
Orconectes clypeatus
Influence of light-history on the response of the distal and proximal pigments to a
light stimulus of one minute duration
This experiment, performed twice, was essentially the same as the one described
immediately above with the single change in protocol having been that one group
of the crayfish was exposed to an illumination of one ft. c. from 5 P.M. to 5
A.M. rather than the gradually increasing illumination associated with dawn. The
objective of this experiment was to learn whether under similar experimental cir-
cumstances the proximal and distal pigments of Orconectes would respond in the
same fashion as the pigments of Palaemonetes. If such a situation was observed,
then presumably the controlling mechanism would be similar in both organisms.
The averaged data for the distal and proximal pigments are presented in Figure 5
where each point represents the mean index for 20 eyestalks. The mean distance
from the outer corneal surface to the basement membrane in 40 eyestalks was 318 p..
Inspection of the figure reveals that the proximal and distal pigments of the crayfish
that had been in darkness overnight responded with a light-adaptational response
that lasted for at least three hours, whereas the pigments of the Orconectes illumi-
nated all night dark-adapted more rapidly. The distal pigment of the latter group
showed a light-adaptational response in darkness whereas the proximal pigment
dark-adapted in one hour.
DISCUSSION
The experiments described above provide some basic information concerning
the proximal retinal pigment of Orconectes and especially Palaemonetcs. Parker
(1896, 1897) had reported that dark-adaptation of the proximal pigment of Palae-
monetes required 45-60 minutes ; light-adaptation, 30-45 minutes. Dark- and
light-adaptation of the proximal pigment in the Palaemonetes used in the present
investigation required 45 and 30 minutes, respectively (Fig. 1). The latter times
were the same as the minimal values presented by Parker. Repetition of Parker's
experiment seemed justified in view of the lack of agreement some investigators have
noted with Parker's (1896, 1897) data on migration of the distal pigment of Palae-
monetcs. Parker observed that light- and dark-adaptation of the distal pigment
required 90-105 and 105-120 minutes, respectively. Welsh (1930) later reported
that light-adaptation occurred in 40-50 minutes and dark-adaptation in 80-90 min-
utes when observed in living specimens, but when the pigment was observed in
fixed and sectioned eyestalks the corresponding times were 50-60 and 90-120
minutes. On the other hand, Sandeen and Brown (1952) stated that light-adapta-
tion of the distal pigment in Palaemonetes required about 95 minutes and dark-
adaptation 60 minutes. De Bruin and Crisp (1957) found that light-adaptation of
the proximal pigment in the mysid Praunus flc.ruosiis and the prawns Palacmon
130 MILTON FINGERMAN, R. NAGABHUSHANAM AND LORALEE PHILPOTT
serratus and Pandalus montagui required 4, 4, and 4-6 minutes, respectively, under
an illumination of 1.1 ft. c. Light-adaptation of the distal pigment in the respective
organisms required 20, 90, and 40 minutes. As in Palaemonetes, more time was
required for light-adaptation of the distal pigment in Palaemon, Praumis, and Pan-
dalus than for the proximal pigment.
The conclusion that the position of the proximal pigment in Palaemonetes is a
function of the reflected light intensity and not the albedo is the same as that of
Sandeen and Brown (1952) who studied the distal pigment of the same species.
The black background merely serves to decrease the intensity of illumination
reflected onto the eyes. This observation is not too surprising when one considers
that specimens of Palaemonetes, when swimming, extend their eyestalks at right
angles to the body. Because of this swimming posture ( 1 ) shielding of the
ommatidia by the body is minimal and (2) most of the ommatidia are stimulated
by light reflected directly from the bottom and walls of the container and indirectly
by internal reflections at the water-air interface. Very few of the facets appear
to receive stimulation directly from the light source, especially when the illumination
is a point source.
The results of the experiments concerned with alterations in the rate of dark-
adaptation (Figs. 3 A, 4, 5) indicate that the proximal pigments of Palaemonetes
and Orconectes are not independent effectors, responding directly to illumination,
but rather are under endocrine control. The proximal pigment of prawns that had
been in light for only five minutes continued to light-adapt after they were put in
the darkroom again (Fig. 3A). This response is strong indication of the release
of a light-adapting principle in response to the dark-to-light change.
The striking difference in behavior of the proximal pigment observed when the
effect of one minute of bright light was determined with animals maintained in
darkness overnight and prawns exposed to the increasing illumination of dawn
(Fig. 4, Palaemonetes} or to a low intensity of illumination all night (Fig. 5,
Orconectes} can also be readily interpreted in terms of endocrine regulation of this
pigment. The results presented for the distal pigment in Figures 4 and 5 are
similar to findings of Brown, Fingerman and Hines (1952) and Webb and Brown
(1953) for the distal pigment of Palaemonetes. The data for the distal pigment of
Palaemonetes were included to illustrate the similarity in behavior of this pigment
and the proximal pigment. One possible explanation of the behavior of the proxi-
mal pigment in Palaemonetes and Orconectes kept in darkness overnight is that a
large quantity of light-adapting hormone was released as a result of the one minute
of bright light. This hormone presumably accumulated in darkness. In the
prawns exposed to illumination prior to 5 A.M. a faster rate of dark-adaptation was
observed. Presumably, the light-adapting principle did not accumulate in illumi-
nated prawns. Brown, Fingerman and Hines (1952) and Fingerman and Mob-
berly (1960) found that the distal pigment light-adapting hormone accumulated in
specimens kept in darkness. A second possible explanation for the rapid rate of
dark-adaptation of the second group of prawns is that the low intensity of illumi-
nation was responsible for providing the animals with the ability to dark-adapt
rapidly by stimulating production and storage of dark-adapting hormone. How-
ever, the experiments do show that the light history of the animals greatly influenced
their ability to light- and dark-adapt. This observation makes it extremely difficult
PROXIMAL RETINAL PIGMENT 131
to accept the hypothesis that the proximal pigment of Palacmonetes is an inde-
pendent effector. More likely, the proximal pigment is controlled by at least one
blood-borne principle, and conceivably light-adapting and dark-adapting hormones
may both be involved.
SUMMARY AND CONCLUSIONS
1. Light-adaptation of the proximal retinal pigment of Palaemonetes required
30 minutes; dark-adaptation, 45 minutes.
2. The proximal pigment of Palaemonetes more closely approached the light-
adapted position when the prawns were on a white background than on a black
background under the same incident light intensity. The black background func-
tioned merely to decrease the brightness of the visual field.
3. The proximal pigment in specimens of Palaemonetes and Orconectcs kept
overnight in darkness showed a greater light-adaptational response after exposure
to a high-intensity light stimulus than specimens that had been under a low intensity
of illumination prior to the bright light.
4. Evidence was presented in support of the hypothesis that light-adaptation
of the proximal retinal pigment of Palaemonetes, following a dark-to-light change,
is due to discharge of light-adapting hormone.
5. The data were discussed in relation to the findings of other investigators.
LITERATURE CITED
BROWN, F. A., JR., M. FINGERMAN AND M. N. HINES, 1952. Alterations in the capacity for
light and dark-adaptation of the distal retinal pigment of Palaemonetes. Phvsiol. Zool.,
25: 230-239.
BROWN, F. A., JR., M. N. HINES AND M. FINGERMAN, 1952. Hormonal regulation of the distal
retinal pigment of Palacmonetes. Biol. Bull., 102: 212-225.
BROWN, F. A., JR., H. M. WEBB AND M. I. SANDEEN, 1953. Differential production of two
retinal pigment hormones in Palaemonetes by light flashes. 7. Cell. Comp. Physiol.,
41: 123-144.
DE BRUIN, G. H. P., AND D. J. CRISP, 1957. The influence of pigment migration on vision of
higher Crustacea. /. Ex per. Biol, 34: 447-463.
FINGERMAN, M., AND W. C. MOBBERLY, JR., 1960. Investigation of the hormones controlling
the distal retinal pigment of the prawn Palaemonetes. Biol. Bull., 118: 393-406.
FINGERMAN, M., M. E. LOWE AND B. I. SUNDARARAJ, 1959. Dark-adapting and light-adapting
hormones controlling the distal retinal pigment of the prawn Palacmonetes vulgaris.
Biol. Bull., 116: 30-36.
FINGERMAN, M., W. C. MOBBERLY, JR. AND B. I. SUNDARARAJ, 1959. Hormonal regulation of
the distal retinal pigment of crayfishes, and the effects of long exposure to light and
darkness. Amcr. Midi. Nat., 62: 429-439.
KLEINHOLZ, L. H., 1936. Crustacean eye-stalk hormone and retinal pigment migration. Biol.
Bull., 70: 159-184.
KLEINHOLZ, L. H., 1961. Pigmentary Effectors. /;;: "The Physiology of Crustacea," T. H.
Waterman, ed., Vol. II, pp. 133-169. Academic Press Inc., New York.
PARKER, G. H., 1896. Pigment migration in the eyes of Palaemonetes. A preliminary notice.
Zool. Ann.. 19: 281-284.
PARKER, G. H., 1897. Photomechanical changes in the retinal pigment cells of Palaemonetes,
and their relation to the central nervous system. Bull. Mus. Comp. Zool., 30: 275-300.
SANDEEN, M. I., AND F. A. BROWN, JR., 1952. Responses of the distal retinal pigment of
Palaemonetes to illumination. Physiol. Zool., 25: 223-230.
WEBB, II. M., AND F. A. BROWN, JR., 1953. Diurnal rhythm in the regulation of the distal
retinal pigment in Palaemonetes. J. Cell. Comp. Physiol., 41: 103-122.
WELSH, J. H., 1930. The mechanics of migration of the distal pigment cells in the eyes of
Palacmonetes. /. E.vpcr. Zool., 56: 459-494,
PROFLAVIN AND ITS INFLUENCE ON CLEAVAGE
AND DEVELOPMENT1
ETHEL BROWNE HARVEY
Department of Biology, Princeton University, Princeton, N. J.. and the Marine
Biological Laboratory, Woods Hole, Mass.
Proflavin is one of the acridine dyes and has been shown (Lerman, 1961) to
combine directly with nucleic acids ; the combined product becomes highly photo-
sensitive. It is a very effective mutagen. One would suppose that a study of the
influence of proflavin on the mitotic figure of the Arbacia egg might, from the nature
FIGURES 1, 2. Normal cells, showing regular distribution of pigment granules.
FIGURES 3 to 9. Pigment granules in segregated masses.
1 It has long been the policy of The Biological Bulletin not to accept very short papers or
brief notes. Because of the distinguished contributions of Dr. Ethel Browne Harvey to the
Marine Biological Laboratory, an exception is being made in this case. — Editor.
132
INFLUENCE OF PROFLAVIN ON DEVELOPMENT 133
of proflavin, give interesting results. Light is necessary for the action of proflavin ;
in the light as well as in the dark, cleavage goes on normally, whether proflavin is
present or not. In the light as well as in the dark, proflavin was found to have no
influence in producing cleavage without the mitotic figure, as I had thought pos-
sible ( 1960) . So far I have found no influence of proflavin on the mitotic figure. It
does, however, cause a delay in cleavage of one or two hours, and often inhibits
cleavage completely.
The amount of proflavin to be used to give the best results has been found to
be 10 micrograms per cc. of sea water. This should be made up frequently and kept
in the refrigerator. If not subjected for too long a period (over 50 minutes?) or
in too great a strength the action is reversible. The strength should be not greater
than 10 micrograms per cc. of sea water.
The most striking effect of the proflavin is to cause a concentration of the red
pigment granules into two or more large masses of granules. In the normal egg,
the pigment is scattered throughout the cells as small pigment granules. These
remain, in eggs treated with proflavin, as segregated masses, becoming more nu-
merous with time, without any tendency to combine. Even when the unfertilised
eggs are treated with proflavin, cleavage (after fertilization) is delayed and ab-
normalities occur, and the pigment is concentrated in masses in the cleavage cells.
The accompanying drawings, made by Eve Chambers, Woods Hole, Mass., show the
pigment granules in the normal unfertilized egg scattered throughout the cytoplasm
(Figs. 1, 2) and later (Figs. 3 to 9) aggregated in masses in the somewhat irregular
cleavage cells. Viscosity is greatly increased by proflavin, as found also by
Lerman (1961).
LITERATURE CITED
BEINERT, H., 1961. Some comments on flavin and flavoprotein complexes and semiquinones.
In: A Symposium on Light and Life, edited by W. D. McElroy and B. Glass. Balti-
more, The Johns Hopkins Press.
HARVEY, E. B., 1960. Cleavage with nucleus intact in sea urchin eggs. Biol. Bull., 119: 87-89.
LERMAN, L. S., 1961. Structural considerations in the interaction of DNA and the acridines.
/. Molec. Biol., 3 : 18-30.
VELICK, S. F., 1961. Spectra and structure in enzyme complexes of pyridine and flavin nucleo-
tides. In: A Symposium on Light and Life, edited by W. D. McElroy and B. Glass.
Baltimore, The Johns Hopkins Press.
RATE OF PHOSPHORUS UPTAKE BY PHAEODACTYLUM
TRICORNUTUM 1
EDWARD J. KUENZLER AND BOSTWICK H. KETCHUM
Woods Hole Oceanographic Institution, Woods Hole, Massachusetts
Research concerning phosphorus relations in unicellular algae has been active
and profitable in several fields — oceanography, limnology, ecology, plant physiology,
and biochemistry (Kamen and Spiegelman, 1948; Rice, 1953; Ketchum, 1954;
Arnon, 1956; Bradley, 1957; Krauss, 1958; Steele, 1959; and many others). Two
problems that have not yet been adequately studied are the uptake of phosphorus by
given species of algae under controlled conditions, and the influence of algal popula-
tions on the recycling rates of phosphorus in whole communities. These problems
are closely related, and ecologists recognize that the recycling rate is as fundamental
a parameter of a community as the absolute abundance of algae and of nutrient phos-
phorus (Ketchum et al., 1958). As an approach to the problem of measuring
community recycling rates, we measured the rate at which Phaeodactylum tricornu-
tum Bohlin (previously called Nitzschia clostcrium forma minutissima Allen and
Nelson) (Lewin, 1958) accumulated both the abundant phosphate in fresh medium
and the scant phosphate of P32 carrier.
The authors wish to express their appreciation to Mr. N. Corwin for performing
many of the chemical phosphorus analyses ; to Dr. V. T. Bowen for use of his radio-
isotope laboratory facilities and for criticism of the manuscript ; and to Dr. R. R. L.
Guillard for criticism of the manuscript.
METHODS
Chemical analysis. The participate (cellular) fraction was separated from the
dissolved phosphorus fractions by filtering samples of algal culture through either a
BaSO4 precipitate deposited on a sintered-glass filter funnel, or a millipore mem-
brane filter, pore size 0.8 /*, previously washed by drawing 100 ml. of 10% HC1
and several copious rinses of distilled water through it. Inorganic phosphate in
samples of filtrate was determined by the Deniges-Atkins colorimetric method as
described by Wooster and Rakestraw (1951). The color was measured in a
photoelectric colorimeter (Ford, 1950) using a red filter (Corning 2408) and a 9- or
29-cm. light path. Total dissolved phosphorus in other samples of filtrate and
cellular phosphorus caught upon the BaSO4 precipitate or membrane filter were
determined by the Harvey (1948) method as modified by Ketchum et al. (1955)
using a 29-cm. path length in the photometer. Phosphorus concentration was calcu-
lated by use of appropriate factors obtained by calibration with standard solutions.
1 Contribution No. 1192 from the Woods Hole Oceanographic Institution. This investiga-
tion was supported in part by the U. S. Atomic Energy Commission under Contract AT (30-1)-
1918 and by Office of Naval Research under Contract Nonr 2196(00).
134
PHOSPHORUS UPTAKE BY PHAEODACTYLUM
135
Strickland and Parsons (1960) reported the limit of sensitivity of the phosphate
method to be about 0.08 //.g. at P/l (/*g. at P/l — ;u,M) ; the method for total phos-
phorus has about the same limit.
Radioisotope analysis. The dissolved P32 was separated from the cellular P32
fraction by filtering a 10-ml. portion through a membrane filter. The cells on the
filter were not washed because such treatment leaches phosphorus from cells (Rice,
1953). To estimate the P32 held by sorption on the filter, we made "sorption"
blanks by placing two or three membrane filter discs together on the filter holder so
that the cells were caught on the upper disc and only the filtrate passed through the
lower discs. The filter disc with the cells, the sorption discs, and 1-ml. aliquots of
the filtrate were dried on separate planchets for counting with an end-window G-M
detector connected to a sealer. Corrections were not necessary for self-absorption,
for geometry of the detector, nor for coincidence losses, but corrections were made
for decay when applicable.
The amount of vacuum applied to draw the water through the filter was one
source of error in determining the P32 distribution between water and cells. To
evaluate this, part of a culture of Phaeodactylum was freed of living cells by cen-
trifuging it and then adding a drop of formalin to the centrifugate to kill any residual
cells. To this, and to another portion of the culture containing living cells, equal
amounts of P32 were added. Both were placed in the dark for more than an hour
to permit equilibration with the P32. When the centrifugate without living cells
was drawn through three consecutive filters, only about 3% of the radioactivity was
found on the filters, and most of that on the upper one (#1, Table I). Higher
vacuum resulted in somewhat lower amounts of activity on the filters, presumably
because less interstitial water remained. When the culture of living cells was
filtered, practically all of the P32 was in the cells and was therefore retained by the
top filter. The amount of activity in the filtrate increased with increased vacuum,
and the activity on the sorption filters (#2 and 3) was quite variable (Table I).
Although the amount of P32 on the sorption filters from the culture was much lower
than that from the cell-free centrifugate, it was large compared to the P32 in the
filtrate. The increasing P32 in the filtrate with intensified suction suggested physical
damage to the cells, with labeled particles and soluble fractions being caught on the
TABLE I
Radioactivity detected on membrane filters and in the filtrate at different degrees of vacuum
expressed as the differential between atmospheric and flask pressures. Values are
counts per minute (cpm) in 10 ml. of centrifugate or culture
Vacuum
(mm. Hg) :
50
100
200
250
360
510
660
Centrifugate:
Filter # 1
460
450
330
2
140
110
110
3
140
120
90
Filtrate
20,700
19,600
20,700
Culture:
Filter # 1
18,200
18,200
18,400
18,000
18,200
18,600
2
4.7
9.5
6.2
6.5
3.1
2.4
3
4.4
5.3
2.8
1.4
2.0
1.9
Filtrate
4.0
8.0
5.0
19
11
25
136
EDWARD J. KUENZLER AND BOSTWICK H. KETCHUM
lower millipore filters and passing through with the filtrate. The measured ac-
tivity in the filtrate of experiments I to V (Table II) is probably an overestimate
because the importance of gentle vacuum was not then known ; the suction used was
usually in the range 250-500 mm. Hg. In experiments VI to X the pressure differ-
ential was kept below 50 mm. Hg. Because of the release of some phosphorus
from the cells during filtration, the radioactivity on the sorption blank was added to
the activity of the cells.
TABLE II
Comparison of concentration of phosphate in culture medium as measured chemically with
that calculated from the minimum concentration of P32 during the experiment.
minimum filtrate P32
Calculated P04 = - - X Total P
total P32
Chemical analysis 0*M)
P32 (cpm/10 ml.)
Calculated
Experiment
Filtrate*
P04
Total P,
cellular and
dissolved
Filtrate
minimum
Total,
cellular and
dissolved
nitrate
PO4
(MM)
I
.11
1.77
10
4,180
.0042
II
.15
2.42
7
7,730
.0022
III
.16
4.06
4
7,890
.0021
IV
.16
4.79
8
7,720
.0050
V
.11
8.43
110
7,620
.12
VI
(.41)
1.37
32
47,500
.00093
VII
(.36)
1.62
23
52,000
.00072
VIII
(.50)
2.19
45
51,500
.0019
IX
(.50)
3.25
83
199,000
.0014
X
(-41)
5.63
80
203,000
.0022
* The results in parentheses are not considered dependable because of unusually large blanks.
Measurement of phosphate concentration by isotope partition. The partition of
P32 between cells and medium was used to measure the dissolved phosphate concen-
tration when it was near or below the limit of sensitivity of chemical analysis. As-
suming that algae absorb P32O4 and P31O4 to the same degree, and that they do not
release labeled phosphorus compounds other than orthophosphate, at equilibrium the
ratio dissolved P32/total P32 should equal the ratio dissolved PO4/total P. There-
fore, dissolved PO4 = (total P) (dissolved P32)/( total P32). This calculation un-
doubtedly overestimates the real concentration for the following reasons : ( 1 ) equi-
librium, and hence the minimum dissolved P32, may not have been reached when the
experiment was terminated; (2) even gentle suction may damage some cells, re-
leasing labeled phosphorus; and (3) the assumption of negligible release of labeled
soluble organic phosphorus compounds during even short experiments may be false.
The dissolved PO4 calculated from P32 in nine cultures equilibrated in the dark was
less than 4% of the value obtained by chemical analysis (Table II). Only in ex-
periment V do the two values agree closely.
These results suggest that in media equilibrated with P-deficient algae the major
part of the PO4 measured chemically was liberated from acid-labile phosphorus
compounds in the filtrate by the acid-molybdate reagent during the chemical analy-
sis, and that only a small fraction was present as free phosphate ions in the whole
PHOSPHORUS UPTAKE BY PHAEODACTYLUM 137
culture. The values in Table I show that phosphate from cells damaged during
filtration would have been too low to detect chemically. We took the values
derived from isotope partition to be the real phosphate concentrations and used
them for further calculations.
EXPERIMENTAL RESULTS
Uptake from phosphate-rich media. The net uptake of phosphorus by a pure
culture of Phaeodactylum was studied by analytical chemical techniques. A culture
was grown in constant light in "f-medium" (Guillard and Ryther, 1961) (Table
III) until cell multiplication had reduced the phosphate concentration in the medium
and the phosphorus content of the cells to a low level (4.2 X 10~15 mole/cell).
Ten-mi, portions of this culture were added to each of four flasks containing 480 ml.
of "f-medium" with various phosphate additions, giving 44 X 107 cells/liter in each
flask. The dissolved phosphate concentrations were 8, 16, 32, and 80 ju,M (flasks
A, B, C, and D, respectively). The flasks were illuminated (400 foot-candles) at
20° C., and 10-ml. samples were filtered periodically to measure dissolved and par-
ticulate phosphorus. The sensitivity and standard deviations were about 1 /*M in
samples containing low concentrations of phosphorus because of a 20-fold dilution
during the analyses ; at the highest concentrations the standard deviations were as
much as 5 /xM because dilutions as great as 100-fold were necessary. The cells
were counted periodically using a Spencer Bright-Line counting chamber.
TABLE III
Composition of "f-medium" (Guillard and Ryther, 1961}
NaNO3 150 mg. (1.76 mM)
NaH2PO4-H2O
Fe sequestrene*
Na2SiO3-9H2O
Vitamins:
Thiamin-HCl 0.2 mg.
Biotin 1.0 /zg
Biz 1.0 fig
Trace metals :
CuSO4-5H2O 0.0196 mg. (0.005 mg. Cu or 0.079 MM)
ZnSO4-7H2O 0.044 mg. (0.01 mg. Zn or 0.153 MM)
CoCl2-6H2O 0.020 mg. (0.005 mg. Co or 0.085 /uM)
MnCl2-4H2O 0.360 mg. (0.1 mg. Mn or 1.83 MM)
Na2MoO4-2H2O 0.013 mg. (0.005 mg. Mo or 0.052 MM)
Sea water To one liter
* Sodium iron salt of ethylene dinitrilo tetraacetic acid (EDTA). Ferric chloride and EDTA
or the sodium salt of EDTA can be mixed to give the same amounts of iron and the chelator;
adjust pH to about 4.5. Ferric sequestrene is made by Geigy Industrial Chemicals, Saw Mill
River Road, Ardsley, New York.
The time course curves of phosphate uptake by phosphorus-poor Phaeodactylum
(Fig. 1A) are similar in shape, but the period of accumulation was much extended
at high initial phosphate concentrations. Whereas it took 6 days before the phos-
phate was depleted in flask D, it was depleted in two hours or less in flask A. In
. .
10 mg. (72.5 MM)
10 mg. (1.3 mg. Fe or 23.3 /zM)
30 mg. (3 mg. Si or 106 juM)
138
EDWARD J. KUENZLER AND BOSTWICK H. KETCHUM
80 •
6 8 10 12 14 16 18 20
HOURS
8 10 12 14 16 18 20
DAYS
FIGURE 1. The changes of dissolved phosphate in the media (1A) and of intracellular
phosphorus (IB) when Phacodactylum was grown in media of different initial phosphate con-
tent. The initial cell count was 44 X 10T cells/liter and the initial phosphate concentrations
were 8, 16, 32 and 80 /*M, for A, B, C, D, respectively.
PHOSPHORUS UPTAKE BY PHAEODACTYLUM
139
all cultures the phosphate was not significantly different from zero after 13 clays.
The phosphorus content of the cellular fraction (Fig. IB) was the inverse of the
phosphate content. The apparent increase in total phosphorus concentration at
the end of the experiment is consistent with the usual rate of evaporation of water
from media. The difference between total dissolved phosphorus and dissolved
phosphate, frequently referred to as dissolved organic phosphorus, was never a
significant amount in A or B, but reached a maximum of about 8 ju,M in flasks C and
D at 6 and 12 hours, respectively. In all cultures it was not significantly different
from zero after the sixth day.
The quantity of phosphorus per cell, shown in Figure 2, reached a maximum in
two to 12 hours, the peak occurring later in the flasks with higher initial phosphate
10
20 30
HOURS
40
6 8 10 12 14 16 18 20
DAYS
FIGURE 2.
Phosphorus concentration of Phaeodactylum as a function of time after inoculation
into media with varying initial phosphate concentrations as in Figure 1.
concentrations. The maximum concentration per cell in flasks A, B, and C was
nearly a function of the initial phosphate concentration in the medium because these
cultures were able to remove almost all of the phosphate before appreciable cell
multiplication occurred. The number of cells in flask D, however, nearly doubled
in the first 24 hours, at which time about half of the phosphate still remained in
solution. These cells reached their peak concentration in 12 to 24 hours. The later
decreases in phosphate per cell resulted from continuing cell multiplication.
The increase from 4 to 66 X 10~15 mole/cell (flasks C and D), a factor of 16, in
12 hours is remarkable. Part of the increase per cell may be attributed to cell en-
largement prior to division, but much of the increase must represent a greater con-
centration of phosphorus in the protoplasm. Negligible uptake by adsorption was
shown by an experiment described below. As growth of the culture continued, the
140
EDWARD J. KUENZLER AND BOSTWICK H. KETCHUM
intracellular concentration declined until, at about 2 X 10 15 mole/cell in flask A,
Phaeodactylum became phosphorus-deficient and stopped multiplying. In the other
flasks cell division continued at a slow rate1 up to 20 days. The final cell counts
varied from 400 to 960 X 107 cells/liter.
The rates of phosphate uptake by Phaeodactylum were neither linear, owing to
removal of a constant quantity of phosphate per unit time, nor exponential, owing
to removal of a constant proportion per unit time, but were intermediate. The
linear, or mean, rates are simply the uptake of phosphate per cell divided by the
duration of each sampling interval. The exponential rate at the beginning of each
interval was calculated by
k =
\\\Pt -- In
n-t
and
v = k-P0
(1)
(2)
where P0 and
are the phosphate concentrations in solution at the beginning and
end of a sampling interval, / is time in minutes, n is the cell density (number per
volume), k is the velocity constant, and v is the instantaneous initial uptake rate.
Both k and v are negative. The exponential and linear rates are shown in Table IV.
TABLE IV
Rates of phosphate uptake by Phaeodactylum in the light (400 fc) in media of high phosphate
concentration. The first column for each experiment is the instantaneous initial
rate calcitlated assuming an expontential rate of phosphate uptake;
the second column is the mean rate assuming a linear uptake
during the interval. Values are 10~17 mole /cell -minute
A
B
c
D
Initial POt G*M) :
Approx. time
8
16
32
80
(hours)
Initial
Mean
Initial
Mean
Initial
Mean
Initial
Mean
0-2
(32)*
13
24
17
15
13
13
12
2-6
—
—
(13)*
6
10
8
14
12
6-12
• —
—
—
—
12
6
5
4
12-24
—
—
—
—
(D*
1
3
3
* Undependable because of extremely low final phosphate concentration.
The agreement between flasks A to D during each time interval demonstrates that in
all four experiments the rate of supply of PO4 to the cells did not limit the uptake
rates as long as PO4 was chemically detectable. The rates were thus more de-
pendent upon the physiological condition of the cells than upon the PO4 concentra-
tions. The decreasing rates in C and D as the cells became glutted (Fig. 2) after
about 6 hours are also apparent.
The uptake of P32 by living, phosphorus-poor Phaeodactylum was compared to
that of an equally dense suspension of chloroform-killed cells in darkness at 22° C.
Separation by filtration showed that during the first hour the living cells accumu-
lated 93% of the added P32 whereas the chloroform-killed cells took up only
PHOSPHORUS UPTAKE BY PHAEODACTYLUM
141
The living cells continued their uptake, but the dead cells changed little during the
next two hours. On the basis of this experiment and others in which cells were
killed by heat, toluene, or chloroform, it was concluded that physical sorption by
the cells was not an important factor in the rapid uptake of phosphorus by algal
cultures.
Ketchum (1939a) showed Phaeodactylum tricornutum to be capable of accumu-
lating 25 X 10~15 mole/cell in 48 hours of darkness. This value is an order of
magnitude lower than the values in Table IV. The highest net uptake rates re-
ported for Phaeodactylum by Ketchum (1939b), when the cells were added to
medium containing about 1 ^M phosphorus and illuminated, were about 20% of the
rate of experiment A (Table IV) during the first two hours. His lower rates may
be the result of the lower phosphate concentrations, the lower experimental tempera-
ture, the longer sampling interval, and perhaps a different physiological condition
of the cells.
Uptake from phosphate-poor media. The rates of uptake at low phosphate con-
centrations were determined by adding P32 as phosphate to equilibrated cultures and
measuring the rate of change of radioactivity in the medium. Sea water medium
was prepared with five different concentrations of phosphorus and one-tenth the
concentration of other nutrients of "f-medium." The phosphorus and iron were
added aseptically after autoclaving to minimize formation of precipitates because
such precipitates rapidly remove P32 from the water. Silicon was omitted from
the medium since it does not limit growth of Phacodactylnin. Equal inocula from
a phosphorus-poor culture were added to 500 ml. of each of the five media. The
flasks were placed in darkness at 20° C. for 24 hours. Cell counts were made for
each flask. Triplicate 100-ml. samples were then removed, and the cells were
separated by filtration for phosphate, total dissolved phosphorus, and cellular phos-
phorus determinations. Practically all of the phosphorus at this time was in the
cells (Table V). Radioactive phosphate was added to the remainder of each
culture and they were kept in darkness at 21-25° C.
TABLE V
Phosphorus concentrations, cell counts, and uptake rates of Phaeodactylum in the dark
in media of low phosphate concentrations
Phosphorus concentration
Experiment
as PO4 in
as cells in
within cells
Total P32
(105Cpm/l)
Cell count
(10' cells/1)
Uptake rate
(10~17 mole/cell min.)
nitrate
culture
(1(T16 mole/
(MM)
(MM)
cell)
I
0.054
1.8
6.1
4.4
29
3.0
II
0.052
2.4
10 1
8.2
23
2.7
III
" 0.052
4.1
181
8.5
23
2.2
IV
" 0.055
4.8
19 1
7.8
25
3.0
V
~ 0.17
8.3
34
8.4
24
0.97
VI
^ 0.011
1.2
8.2
48
15
1.3
VII
0.011
1.5
9.4
52
16
1.1
VIII
0.012
2.0
12
52
16
1.1
IX
0.041
3.1
19
200
16
3.2
X
0.042
5.5
34
210
16
3.0
142 EDWARD J. KUENZLER AND BOSTWICK H. KETCHUM
Ten-nil, portions of the culture were filtered periodically during the next three
hours and the P32 content of both the cells and the water was determined. The up-
take of P32 by the cells proceeded smoothly and rapidly, the time course curves being
logarithmic. It was from these data that we concluded that the chemical method of
analysis overestimated the low concentrations of dissolved phosphate in the medium.
The final equilibrium value in these cultures is given in the last column of Table II.
This value plus the amount of carrier phosphate added with the P32 gives the origi-
nal phosphate concentration listed in Table V. This assumes that the cells had
reached equilibrium after the 24-hour dark period and reached the same equilibrium
after assimilating the radio- and carrier phosphate during the three-hour experi-
mental period. The amount of carrier phosphate added (0.01-0.05 /xM) was large
compared to the dissolved phosphate but small compared to the total phosphorus
in the culture.
The rate of phosphate uptake was computed by a formula derived from (1)
and (2) :
in which P0* and Pf* are the P32 concentrations in solution at the beginning and end
of the interval and Pn is the initial total dissolved phosphate content. The other
symbols are as previously defined. The rates computed for the first 7-9 minutes of
the experiment are given in Table V which also gives the total activity and the cell
count for each culture.
The rates of uptake from the low phosphate concentrations by these cells are an
order of magnitude less than those in Table IV where the cells were lower in phos-
phorus content and the dissolved phosphate concentrations were much greater. The
rates at low concentrations showed no definite trend related to either the phosphate
concentration or to the phosphorus content of the cell.
DISCUSSION
The two experiments described were designed to measure phosphate uptake
under contrasting conditions. In the first experiment (Table IV) phosphorus-
deficient cells were transferred to a medium containing large amounts of phosphate
relative to the amount in the cells. The different amounts formed a source of
supply for periods ranging from about two hours to six days. The initial assimila-
tion, when growth and cell division were negligible, increased the phosphorus con-
tent of the cells which were, thus, recovering from their deficiency. At longer times,
which depended on the amount of phosphate made available, the content per cell
decreased because of cell division. In this experiment the amount of phosphorus in
the cell varied from 2 to 66 X lO'15 mole/cell, emphasizing again the wide range of
variation which can be induced by varying the external conditions. The initial
rates of assimilation under these conditions varied from 12 to 24 X 10~17
mole/cell -min. and decreased as the phosphate was removed from solution.
In the second experiment (Table V) phosphorus-deficient cells were allowed to
assimilate varying amounts of added phosphorus in the dark so that at the start of
the experimental period practically all of the phosphorus was in the cells, their
content being from 6.1 to 34 X 10"15 mole/cell. Radio- and carrier phosphate was
PHOSPHORUS UPTAKE BY PHAEODACTYLUM 143
added in amounts that increased the low concentrations of dissolved phosphate hut
that were small compared to the phosphorus in the cells. Under these conditions
the uptake of phosphate varied from about 1 to 3 X 10~17 mole/cell- min. with no
apparent relationship to either the cell phosphorus or the dissolved phosphate. Al-
though the available phosphate concentration in the two experiments varied initially
by nearly four orders of magnitude, the rates of assimilation differed by only one.
Phosphorus is not only assimilated rapidly by deficient cells, but intracellular
phosphorus is also constantly exchanged with that in the water (Kamen and
Spiegelman, 1948; Goldberg et al., 1951; Rice, 1953; and Knauss and Porter,
1954). It was not possible to separate the two mechanisms, uptake and exchange,
in the present experiments. The results given in Table V for the assimilation from
low phosphate medium were calculated assuming all of the change was uptake with
no exchange. A calculation was also made assuming that all can be attributed to
exchange, with no net uptake (Russell, 1958), and this gave almost exactly the
same rates.
Phaeodactyluin stops dividing when the intracellular phosphorus falls to about
2 X 10~15 mole/cell. During its most active growth a population can double in
about 18 hours of continuous illumination. The lowest rate of uptake we have
measured, 10~17 mole/cell- min., would permit a phosphorus-deficient cell to double
its phosphorus content in 200 minutes, or slightly over three hours. Since this
rate of uptake was from very low phosphate concentrations in the medium, it seems
unlikely that the rate of assimilation of phosphorus would ever limit the rate of
growth of a phytoplankton population in nature. The total supply could, of course,
determine the final size of the population.
The rate of uptake under the different conditions must reflect the physiological
state of the cells and must ultimately be dependent upon both the supply of phos-
phorus in the cells and in the medium, although these relationships are not con-
clusively demonstrated in our experiments. On the other hand, if availability of
intracellular energy and enzyme reserves, needed for the work of active transport
through the cell membrane, strongly influences the uptake rate, then darkened cells
can have rates nearly those of illuminated ones if they can draw upon previously
stored reserves. Odum ct al. (1958) similarly showed that illumination had little
effect upon the P32 uptake of benthic algae.
Munk and Riley (1952) showed theoretically that small cells should absorb
nutrients more rapidly than larger ones, and Odum et al. (1958) demonstrated that
filamentous or thin benthic algae absorb P32 much faster than the more massive
algae. There were, undoubtedly, some differences in cell size and surface-to-
volume ratio in Phaeodactvltnn, but these differences probably affected the uptake
rates only slightly.
In Phaeodactylnni the chromatophore shrinks to one-third or less of its original
length as the cell becomes phosphorus-deficient. Chlorophyll measurements in
other experiments (unpublished) prove that the absolute amount of chlorophyll
per cell declines markedly in phosphorus-deficient cells (cj. Ketchum ct al., 1958).
Uptake rates per unit of protoplasm, cell nitrogen, or chlorophyll may prove to be
more meaningful than per cell, especially when comparing the rates of algae of
widely differing sizes or when studying natural mixed populations. In experi-
ments VI-X, chlorophyll concentration was measured; it was initially 7.1 ±0.6
144 EDWARD J. KUENZLER AND BOSTWICK H. KETCHUM
X 10~14 g. Chi A/cell, and the uptake rates on this basis range from 1.6 X 10'4 to
4.5 X lO'4 mole P/g. Chi A-min.
Phaeodactylum is able to remove phosphate to levels below the sensitivity of the
chemical analytical method. The lowest concentration, determined from P32 re-
maining in solution, was 7.2 X 1O10 M. This confirms the often quoted ability of
algal cells to concentrate phosphorus greatly. We have not measured the volume
of Phaeodactylum cells, but Ketchum and Redfield (1949) give a dry weight per
cell of 2.32 X 10'11 grams. Assuming 20% dry matter in the cells and a density
of 1, the volume of cells in experiments VI-X (Table V) would be 1.9 X 10~2
ml. /I. Lewin et al. (1958) found very similar weights and volumes in fusiform
Phaeodactylum. The concentration of phosphorus within the cells varied from
0.08 to 0.3 M. These were in equilibrium with external concentrations of 7.2 to
22 X 10~10 M at the end of the experiment (Table II), producing concentration
factors of about 10s. Under these conditions most of the intracellular phosphorus
probably is firmly bound in the cells with only a very small fraction present as free
phosphate ions.
The physiological condition of natural phytoplankton with regard to nutrients
is still difficult to assess. The uptake rate even at low concentrations is high
enough so that phosphorus should never be limiting in any waters with chemically
detectable phosphate concentrations. Our experiments have shown, however, that
the chemical method in use measures some materials which are not treated as free
phosphate ions by the living cell. Also, Phaeodactylum can accumulate thirty times
as much phosphorus as the minimum required for cell division and may reduce the
concentration of phosphate to undetectable levels in the water while the cells are still
relatively phosphorus-rich. Such cells can continue to divide, in the light, with
no further phosphorus accumulation. Inability to measure phosphate in sea water,
then, can not be taken as evidence that it is limiting population growth or organic
production.
SUMMARY
Portions of a phosphorus-deficient culture of Phaeodactylum tricornutum Bohlin
were dispensed into fresh media containing phosphate concentrations from 8 to 80
ju,M. The instantaneous initial phosphate uptake rates were 12 to 24 X 10~17 mole/
cell-min. The concentrations of phosphorus in the cells extended from a high of
66 X 10~15 mole/cell after 12 hours' exposure to phosphate-rich (32 and 80 ^M)
media to a low of 2 X 10~15 mole/cell when PO4 depletion of the medium limited
further growth. In another experiment Phaeodactylum was prepared with varying
intracellular P concentrations in media with very low PO4 concentrations. Radio-
active phosphate was then added, the time course of P32 distribution was followed,
and the rate of phosphate uptake was calculated. The initial rates ranged from
10"17 to 3 X 10^17 mole/cell- min. These were about one order of magnitude less
than the uptake rates during the first two hours of the first experiment, even though
the PO4 concentrations were two to four orders of magnitude lower. Radio-
isotope analysis showed that Phaeodactylum decreased the phosphate in the medium
to as little as 7.2 X 10 1(1 .17, a concentration much below the limit of sensitivity
of the chemical analytical method.
PHOSPHORUS UPTAKE BY PHAEODACTYLUM 145
LITERATURE CITED
ARNON, D. I., 1956. Phosphorus metabolism and photosynthesis. Ann. Rev. Plant Physiol.,
7: 325-354.
BRADLEY, D. F., 1957. Phosphate transients in photosynthesis. Arch. Biochctn. Biophys., 68:
172-185.
FORD, W. L., 1950. Seagoing photoelectric colorimeter. Analyt. Chem., 22: 1431-1435.
GOLDBERG, E. D., T. J. WALKER AND A. WHISENAND, 1951. Phosphate utilization by diatoms.
Biol Bull, 101: 274-284.
GUILLARD, R. R. L., AND J. H. RYTHER, 1961. Studies of marine planktonic diatoms. I.
Cyclotella nana (Hustedt) and Detonula confervacea (Cleve) Gran. Canad. J. Micro-
biol. (In press.)
HARVEY, H. W., 1948. The estimation of phosphate and of total phosphorus in sea waters.
/. Mar. Biol. Assoc., 27: 337-359.
KAMEN, M. D., AND S. SPIEGELMAN, 1948. Studies on the phosphate metabolism of some uni-
cellular organisms. Cold Spring Harbor Symp. Quant. Biol., 13: 151-163.
KETCH UM, B. H., 1939a. The development and restoration of deficiencies in the phosphorus and
nitrogen composition of unicellular plants. /. Cell. Comp. Physiol., 13: 373-381.
KETCHUM, B. H., 1939b. The absorption of phosphate and nitrate by illuminated cultures of
Nitsschia closterium. Amer. J. Bot., 26: 399-407.
KETCHUM, B. H., 1954. Mineral nutrition of phytoplankton. Ann. Rev. Plant Physiol, 5:
55-74.
KETCHUM, B. H., AND A. C. REDFIELD, 1949. Some physical and chemical characteristics of
algae grown in mass cultures. /. Cell Comp. Physiol, 33: 281-300.
KETCHUM, B. H., N. CORWIN AND D. J. KEEN, 1955. The significance of organic phosphorus
determinations in ocean waters. Deep-Sea Res., 2: 172-181.
KETCHUM, B. H., J. H. RYTHER, C. S. YENTSCH AND N. CORWIN, 1958. Productivity in relation
to nutrients. Rappt. Cons. Explor. Mer, 144: 132-140.
KNAUSS, H. J., AND J. W. PORTER, 1954. The absorption of inorganic ions by Chlorella
pyrenoidosa. Plant Physiol, 29: 229-234.
KRAUSS, R. W., 1958. Physiology of the fresh-water algae. Ann. Rev. Plant Physiol, 9:
207-244.
LEWIN, J. C., 1958. The taxonomic position of Phaeodactylum tricornutum. J. Gen. Micro-
biol, 18: 427-432.
LEWIN, J. C., R. A. LEWIN AND D. E. PHILPOTT, 1958. Observations on Phaeodactylum tri-
cornutum. J. Gen. MicrobioL, 18: 418-426.
MUNK, W. H., AND G. A. RILEY, 1952. Absorption of nutrients by aquatic plants. J. Mar.
Res., 11: 215-240.
ODUM, E. P., E. J. KUENZLER AND M. X. BLUNT, 1958. Uptake of P32 and primary productivity
in marine benthic algae. LitnnoL Oceanog., 3: 340-345.
RICE, T. R., 1953. Phosphorus exchange in marine phytoplankton. Fish. Bull, U. S. Xo. 80,
54: 77-89.
RUSSELL, J. A., 1958. The use of isotopic tracers in estimating rates of metabolic reactions.
Perspect. Biol Med., 1: 138-173.
STEELE, J. H., 1959. The quantitative ecology of marine phytoplankton. Biol. Rev., 34: 129-158.
STRICKLAND, J. D. H., AND T. R. PARSONS, 1960. A manual of sea water analysis. Fish. Res.
Bd. Canada Bull. No. 125. P. 41.
WOOSTER, W. W., AND N. W. RAKESTRAW, 1951. The estimation of dissolved phosphate in sea
water. /. Mar. Res.. 10: 91-100.
SURVIVAL AND MOVEMENTS OF THE FLATWORM, STYLOCHUS
ELLIPTICUS, IN DIFFERENT SALINITIES AND TEMPERATURES
WARREN S. LANDERS AND RICHARD C. TONER
U. S. Bureau of Commercial Fisheries, Biological Laboratory, Milford, Connecticut
The flatworm, Stylochus ellipticus, an experimentally-proven predator of oysters
(Loosanoff, 1956), is one of the most abundant marine polyclads along the Atlantic
and Gulf coasts of the United States (Hyman, 1940) and, consequently, may be one
of the most important oyster enemies. A review of the literature offered by Hopkins
(1949, 1950) showed that there is little published information regarding its physio-
logical behavior. However, Pearse and Wharton (1938) in their report on the
physiology of the related species, Stylochus inimicus, from Apalachicola Bay, Flor-
ida, included some observations on Eustylochus mcridionalis (Stylochus ellipticus)
from the same area. They found that S. ellipticus could survive a slow decrease in
salinity from 32 parts per thousand to as low as 2.9 ppt, but died in salinities below 6
ppt if the decrease was abrupt. They also reported that the worms became dormant
at temperatures below 7° C. These circumstances suggested that some environ-
mental control of 5. ellipticus might exist in certain areas in cold climates, especially
where low salinity and low temperature coincide.
Since information on the physiological behavior of S. ellipticus in the northern
part of its range is lacking, our studies were initiated to observe the behavior of
adult worms (10 to 18 millimeters long) of Milford Harbor, Connecticut, at differ-
ent salinities and temperatures, with emphasis on observations at low temperatures
and salinities. While the salinity of Long Island Sound proper usually fluctuates
within a narrow range of approximately 26 to 28 ppt, oyster beds located in estuaries,
salt water ponds and rivers may at times, especially in the early spring, be exposed to
water that is almost fresh. The annual range of water temperature in this latitude is
from about — 1° C. to approximately 25° C.
Effects of salinity
Observations were made on survival of the worms in different salinities and the
effect of these salinities on movement of these worms. "Righting time," i.e., the
time required for a worm to return to normal position after having been turned
ventral side up, was used as a quantitative measure. Righting times were deter-
mined frequently at each salinity tested ; however, more observations were made at
the lower salinities, where individual variations in righting time were largest, than
at the higher salinities. All observations were made in the laboratory at room tem-
perature (18° C. to 22° C. ) in standing water, which was changed twice a week.
The low salinities were made by diluting sea water from Milford Harbor (about 27
ppt) with tap water demineralized by a Barnstead BD-2 apparatus.
In the first experiment the effects of an abrupt decrease in salinity on survival
and righting time of 6". ellipticus were investigated. Groups of 10 worms each were
transferred directly from Milford Harbor water to enamel pans, arranged in pairs,
146
MOVEMENTS OF STYLOCHUS
147
each containing six liters of water of the following salinities: 25, 22.5, 20, 17.5, 15,
12.5, 10, 7.5, 5 and 2.5 ppt and fresh water. Two groups, each containing 10 worms
placed in undiluted water from Milford Harbor, served as controls.
Worms transferred directly to salinities as low as 7.5 ppt survived this abrupt
change. Those transferred to salinities of 10 ppt showed no distress at any time,
30
25
20
CL
Q.
> 15
t
2
<
tf)
10
10 20
TIME IN SECONDS
30
40
FIGURE 1. Average righting time of Stylochus ellipticus from Milford Harbor, Conn, in
different salinities at room temperature (18°-22° C.).
148
WARREN S. LANDERS AND RICHARD C. TONER
while in a salinity of 7.5 ppt some worms, during the first two clays, lost color, se-
creted slightly more mucus than is normal and were sluggish. The worms trans-
ferred to 5 ppt were similarly affected, but for a longer time. Four of these died
during the first week after transfer, but the survivors eventually ceased to show
symptoms of distress and assumed a normal appearance. All of the worms trans-
ferred directly to 2.5 ppt and fresh water died within a few hours. However, when
some that had become acclimated to a salinity of 5 ppt were transferred to 2.5 ppt,
they showed no signs of distress and remained alive and active.
After all symptoms of distress had disappeared in the low salinities, righting
times were determined for worms of all groups. The average righting time ranged
from 10 to 13 seconds in all salinities, except 5 ppt and 2.5 ppt. In 5 ppt it was 22
seconds and increased to 37 seconds in 2.5 ppt (Fig. 1).
In a second experiment, survival of worms in salinities below 5 ppt was deter-
mined after they had been conditioned at intermediate salinities. Three groups of
100 worms each were conditioned for two weeks ; one group, at a salinity of 15 ppt ;
30
25
20
o
EC
LU
LU
10
20
40 60
TIME IN SECONDS
80
100
120
FIGURE 2. Average righting time of Styloclnis ellipticus from Milford Harbor, Conn., at
different temperatures in a salinity of about 27 ppt.
MOVEMENTS OF STYLOCHUS
149
30
25
20
UJ
tr
<r
UJ
10
10
20
30 40
MM PER MINUTE
50
60
70
FIGURE. 3.
Average locomotion rate of Stylochus ellipticus from Milford Harbor, Conn., at
different temperatures in a salinity of about 27 ppt.
another group, at 10 ppt; and the third, at 5 ppt. Early in the conditioning period
16 of the worms in 5 ppt died, but the remainder of this group became acclimated.
None of the worms died or showed distress in either 15 ppt or 10 ppt. At the end
of the two weeks of conditioning, groups of ten worms from each conditioning salinity
were placed in duplicate enamel pans, each containing six liters of standing water
of the following salinities : 2.5, 1.5 and 0.5 ppt and fresh water. Two groups of ten
worms each, placed in undiluted Milford Harbor water, served as controls.
Conditioning at intermediate dilutions had little effect on the ability of these
worms to survive in low salinities. By the end of the fourth day all the worms con-
ditioned for two weeks at 15 ppt and then transferred to salinities of 2.5 ppt and
lower had died. Worms conditioned at 10 ppt had died by the end of the seventh
day after transfer. Those conditioned at 5 ppt, and transferred to 1.5 ppt and 0.5
ppt and fresh water, also were dead by the end of the seventh day. Twelve of the
20 worms conditioned at 5 ppt and transferred to 2.5 ppt died by the end of the
seventh day, but the remaining eight were alive and healthy when the experiment
ended. Only one of 20 worms in the control pans died during the experiment.
150 WARREN S. LANDERS AND RICHARD C. TONER
60
50
40
tr. 30
UJ
O.
20
10
10 15
TEMPERATURE
20
25
30
FIGURE 4. Average locomotion rates of Stylochns ellipticus from Milford Harbor, Conn., and
from Apalachicola Bay, Florida at similar water temperatures.
Apparently, the lowest salinity in which S. ellipticus of Milford Harbor can survive,
even with prior conditioning at an intermediate salinity, is about 2.5 ppt.
Effects of temperature
The effects of temperature on righting time and "locomotion rate," i.e., the
rate of forward movement, of 6". ellipticus were observed in running sea water at 30°,
25°, 20°, 17.5°, 15°, 12.5°, 10° and 7.5° C. The water temperatures were main-
tained within ± 1 ° C. of the desired levels by mixing, in different proportions, heated
and unheated Milford Harbor water.
Three groups of ten worms each were allowed to adjust to a selected tempera-
ture for three days. Within the next week three separate observations were made
on righting time and locomotion rate of all of the worms. At the end of this time
the water temperature was changed, the worms were allowed to adjust to the new
temperature, and a new series of observations was made. Locomotion rate was
MOVEMENTS OF STYLOCHUS
151
determined by noting the length of time needed for each worm to move 40 mm., with-
out stopping, in a straight line.
The average righting time at 20°, 25° and 30° C. was fairly constant at 7 to 9
seconds, but increased to 17 seconds at 17.5° C. and remained at approximately this
level at temperatures down to and including 12.5° C. At 10° C. righting time in-
creased to 30 seconds and rose sharply to 116 seconds at 7.5° C. (Fig. 2).
The average locomotion rate varied somewhat erratically at different tem-
peratures, but tended to decrease with decreasing temperature. From a rate of
over 50 millimeters per minute at 20° C. and higher it dropped to approximately
10 mm. /mm. at 7.5° C. (Fig. 3). A comparison of average locomotion rates ob-
served in this experiment with similar data reported by Pearse and Wharton (1938)
for 5\ cllipticus from Apalachicola Bay showed that, at the same temperatures loco-
motion rates of Milford Harbor worms were consistently higher than those of Florida
worms (Fig. 4). This difference may indicate the existence of physiological races
within this species of flatworm.
120
100
80
01
o
z
o
o
UJ
C/)
UJ
5
\-
60
40
20
j_
10 15
SALINITY IN
20
25
30
PPT
FIGURE 5. Average righting time of Stylochus ellipticus from Milford Harbor, Conn, at
different salinities and temperatures.
152 WARREN S. LANDERS AND RICHARD C. TONER
Combined effects of temperature and salinity
The combined effects of temperature and salinity on survival and movement of
5\ ellipticus were also studied. Again, righting time was used as a criterion of
movement. Duplicate groups of five worms each were held in polyethylene boxes in
three liters of standing water of different salinities. These boxes were kept in
a water bath where the desired temperatures were maintained.
The procedure used to study the combined effects of temperature and salinity on
the righting times of worms was similar to that used in observing the effect of tem-
perature alone, i.e., worms in all salinities were held at the same temperature until
their righting times had been determined, then the water temperature was changed
and the observations repeated. Using this procedure righting times were obtained
in Milford Harbor water at a salinity of about 27 ppt and also in salinities of 15, 10,
7.5 and 5 ppt at temperatures of 20°, 15°, 10° and 7.5° C. Worms tested at 5 ppt
were conditioned to this salinity prior to starting the experiment.
Although worms showed some movement in all combinations of temperature and
salinity tested, the effects of low temperature and salinity in slowing down their
movements became more apparent as the two factors were progressively lowered, and
the depressing effects of each factor on their movement tended to reinforce one
another (Fig. 5). For example, at 20° C. worms in all salinities from Milford Har-
bor water to that of 7.5 ppt had approximately the same average righting time, rang-
ing from 11 to 15 seconds, but worms in 5 ppt required an average of 21 seconds to
turn over, reflecting primarily the adverse effect of low salinity.
At 15° C. the average righting times of worms in 7.5 ppt and higher salinities
were again about equal, but had increased slightly to approximately 18 seconds, due
to the effect of the lower temperature. In 5 ppt, however, righting time increased
more sharply than expected. For example, at normal salinity a decrease in tem-
perature to 15° C. had no appreciable effect on righting time, and at room tempera-
ture lowering of the salinity to 5 ppt had only increased righting time to 22 seconds,
but when the temperature was reduced to 15° C. and the salinity to 5 ppt, simul-
taneously, righting time increased to 79 seconds. The combined effect was, there-
fore, greater than could have been predicted from the effects of these factors when
studied separately.
At 10° C. worms in all salinities were affected by lowered temperature, while
those in salinities of 5, 7.5 and 10 ppt showed the exaggerated effect of a combina-
tion of low temperature and salinity. For example, in Milford Harbor water and in
15 ppt the average righting time was 32 and 35 seconds, respectively, while at 10 ppt
it was 48 seconds, 54 seconds at 7.5 ppt and 103 seconds at 5 ppt.
At a temperature of 7.5° C. the worms in all salinities had slowed their move-
ments still further, but the same pattern of increase in righting times was noted.
In many areas where oyster cultivation is carried on, pronounced variations occur
in either temperature or salinity or both. If the rate of predation of flatworms
on oysters is closely related to the worms' ability to move about, our studies indicate
that predation by these worms must vary considerably at different seasons of the
year, especially in a cold climate. Even though their predatory activities may be
curtailed by low temperature or low salinity, their ability to survive these adverse
conditions makes them a serious threat wherever oyster culture is practiced.
MOVEMENTS OF STYLOCHUS 153
We wish to express our appreciation to Dr. V. L. Loosanoff for suggesting this
problem and for his critical review of the manuscript. We also wish to thank Mr.
Harry C. Davis for his helpful suggestions in the preparation of this paper, Mr.
Manton Botsford for the illustrations and Miss Rita Riccio for her careful editing.
SUMMARY
1. Stylochus ellipticus from Milford Harbor, Connecticut, survived abrupt trans-
fer from a salinity of about 27 ppt to salinities as low as 7.5 ppt at room temperature.
Those transferred directly to 5 ppt suffered a mortality of 20(/c but all worms died
when placed directly in 2.5 ppt and fresh water. However, worms that acclimated to
5 ppt survived subsequent transfer to 2.5 ppt.
2. Righting time of 6". elliptic us at room temperature remained constant at 12 to
15 seconds in salinities ranging from about 27 ppt to 7.5 ppt but increased to 22
seconds in 5 ppt and to 37 seconds in 2.5 ppt.
3. Righting time of vS". ellipticus in a salinity of about 27 ppt was approximately 8
seconds at temperatures of 20°, 25° and 30° C., 16 seconds at 12.5°, 15° and
17.5° C., 30 seconds at 10° C. and 116 seconds at 7.5° C.
4. Locomotion rate of .9. ellipticus varied erratically with temperature but gen-
erally decreased with temperature decreases below 20° C. It exceeded 50 milli-
meters per minute at temperatures of 20° C. and higher but decreased to 10
mm./min. at 7.5° C.
5. At the same temperatures S. ellipticus from Milford Harbor moved faster
than reported for the same species from Apalachicola Bay, Florida. This observa-
tion suggests that there may be physiological races within this species of flat worm.
6. When temperature and salinity were lowered simultaneously righting time
of 5". ellipticus was frequently longer than the combined righting times obtained when
the two factors were observed separately.
LITERATURE CITED
HOPKINS, SEWELL H., 1949. Preliminary survey of the literature on Stylochus and other flat-
worms associated with oysters. Texas A. and M. Res. Found. Proj. Nine, 1-16.
HOPKINS, SEWELL H., 1950. Addendum to "Preliminary survey of the literature on Stylochus
and other flatworms associated with oysters." Texas A. and M. Res. Found. Proj.
Nine, 1-4.
HYMAN, LIBBIE H., 1940. The polyclad flatworms of the Atlantic coast of the United States and
Canada. Proc. U. S. Nat!. Museum. 89(3101) : 449-495.
LOOSANOFF, V. L., 1956. Two obscure oyster enemies in New England waters. Science, 123 :
1119-1120.
PEARSE, ARTHUR S., AND GEORGE W. WHARTON, 1938. The oyster "leech," Stylochus iniinicits
Palombi, associated with oysters on the coast of Florida. Ecol. Monogr., 8: 605-655.
BODY TEMPERATURES IN SOME AUSTRALIAN MAMMALS.
III. CETACEA1 (MEGAPTERA)
PETER MORRISON
School of Physiology, University of Queensland, Brisbane, and Departments of Zoology and
of Physiology, University of Wisconsin, Madison 6, Wisconsin
Whales have always attracted popular interest both as the largest animals and
as a valuable object in commerce, but their size and wide range make approach
difficult and have discouraged the accumulation of reliable information about them.
Although their unique size lends interest to any aspect of their life, the topic of
body temperature to be considered here bears on a point of particular interest in
view both of the general relations between energy production, mass, and heat
dissipation in mammals, and the special problems of aquatic mammals (Scholander
and Schevill, 1955).
Because of "technical" difficulties, body temperatures have only conveniently
been measured on dead whales, although advances in telemetry may offer another
approach in the future. Ordinarily one would consider a measurement obtained
an hour after death to be quite questionable and one obtained ten hours after
death to be of no value at all. However, the enormous bulk of the whale greatly
reduces heat dissipation. The covering layer of blubber constitutes an effective
insulation and this, together with the high heat capacity of the system and the
low relative surface area, greatly reduces heat loss. It has been estimated that
a 24 M. (122-ton) whale has a surface of 275 M.2 or only 23 cm.2/kg. (Laurie,
1933). This is a tenth of the value in man and less than a hundredth of that in a
mouse. Of course, in the living animal most of this reduction in surface is
compensated for by the (presumed) lowrer metabolic rate in the whale, so that
the metabolic output per unit of body surface may differ by no more than a factor
of three in the largest and smallest mammals.2
In practice, it has been long known that whales do cool slowly after death.
For example, Guldberg (1885) reported a temperature of 34° in a fin whale
three days after death. Cockrill (1951) described a whale fillet of about three
tons which was towed for 21 hours in antarctic waters with a resultant fall of
only 1° F. in deep temperature. Ash (personal communication) has "observed
1 This study was carried out with assistance from the Guggenheim Foundation and the
U. S. Educational Foundation in Australia, and would not have been possible without the
wholehearted assistance of Dr. Victor Macfarlane of the School of Physiology at Brisbane.
I am indebted to Dr. Masaharu Nishiwaki of The Whales Research Institute (Tokyo), to Dr.
J. G. Sharp of The Low Temperature Research Station (Cambridge) and to Dr. C. E. Ash
of United Whalers Ltd. (London) for supplying very interesting unpublished records of whale
temperatures ; and to Dr. Clinton Woolsey for similar data on a porpoise. I also appreciate
the kindness of Dr. William E. Schevill in checking the manuscript and nomenclature.
2 This represents the difference between the two weight functions for surface (A = kWea7)
and metabolism (M = kW'™) so that M/A = kW0'"" '. Taken over the shrew-to-whale weight-
span (3— MO8 g.) this amounts to a factor of 3X. Over the mouse-to-elephant span
(20-^4X 106g.) the factor is 2.2.
154
BODY TEMPERATURE IN MEGAPTERA 155
a fillet of meat (longissimus dorsi) maintain a steady temperature of 35° C. in the
interior for 15 hours post mortem." Robinson et al. (1953) summarized data
to show (p. 6) that "up to 24 hours after death, whale muscle does not lose heat
to a significant extent." Indeed, Irving and Krogh (1954) have reported that
even in the very much smaller (though well insulated) reindeer and caribou, and
under the extreme ambient conditions of —45° C. in strong wind, there was no
demonstrable fall in deep body temperature in the hour following death.
MATERIALS AND METHODS
Observations wrere made on 20 recently killed humpback whales (Megaptera
novaeangliae) at coastal whaling stations on Moreton Island in southern Queens-
land just off Brisbane and in Byron Bay in northern New South Wales, the
easternmost point in Australia. We are indebted to the respective concerns.
Whale Products, Ltd. and the Byron Whaling Co., Ltd., for their cooperation in
making these studies possible. Measurements on four whales (No. 512, X, Y.
and Z) were kindly made by Dr. K. C. Robbins of the CSIRO. At both these
locations whales are killed close offshore from small vessels similar to those used
in the larger antarctic whaling operations. But instead of being picked up by
a large "factory" ship they are towed to fixed shore installations where the
carcasses are cut up and rendered. The period between death and disposal varies
from one to sixteen hours, depending on the towing distance, the weather condi-
tions, and the work load. These whales ranged from 35 to 45 feet in length and
were considered to carry roughly a ton of weight for each foot in length.
Temperatures were measured with copper-constantan thermocouples using
a Cambridge thermocouple-potentiometer unit. This provided connections for
manual switching between six thermocouples. One of these was used as a refer-
ence junction for calibration of the instrument against a mercury thermometer in
a small Dewar flask. The other five were fixed at twelve-inch intervals along a
one-inch stainless steel tube. The plastic-coated thermocouple \vires were further
protected by an outer small plastic tube, which was led down the inside of the probe
and brought out at the appropriate point through a small hole. The junction was
thus exposed on the outer surface of the probe (although still protected by the
plastic cover), and the far end was run through another hole and secured inside
by knotting. A demountable harpoon head with tempered cutting edges was fixed
to the shaft to allow penetration through the tissues. The first thermocouple lay
two inches above the head, and the fifth, four feet higher ; but all thermocouple
wires were of the same length. In preliminary tests it was not possible to check
the thermocouples against one another to better than 1° C. But on two occasions
during measurements on whales the heart was penetrated so that the hollow
thermocouple probe carried a stream of blood out of the body. Under these condi-
tions, values for the several couples all check each other within 0.1° C.
In one series of measurements, made from the deck of the whale chaser im-
mediately after capture, approach was limited to the more posterior regions and
insertions could be made only with great difficulty. Because of unfavorable
weather conditions, the carcass rose three to six feet with each swell, and on
several occasions the one-inch steel probe was bent to a right angle against the side
of the vessel. Most of the measurements, however, were carried out after the
156
PETER MORRISON
whales had been towed ashore and under these circumstances the probe could be
readily positioned in any part of the body. Insertions were normally made from
the ventral surface near the midline, with the last thermocouple set just below
the surface, or when further penetration was stopped by bone. The various
insertion positions are diagramed in Figure 1.
FIGURE 1. Humpback whale profile showing axial positions for thermometer insertions.
F, flipper ; U, umbilicus ; G, genital opening.
RESULTS
In each case, values from the four or five thermocouples provided a tempera-
ture profile through the whale. The anticipated sequence, that most commonly
36-
34-
32-
30-
28-
HUMPBACK WHALE No. 502
DEAD 13 HOURS
38 FT. <$
1 1^
2 4
VENTRAL INSERTION DEPTH IN FEET
FIGUKE 2. Temperature-depth profiles at various axial positions in whale No. 502,
showing normal gradients.
BODY TEMPERATURE IN MEGAPTERA
157
observed, found the innermost couple (#1) or perhaps the next (#2) at the highest
temperature, with the other values decreasing uniformly as the surface was
approached. A representative set of such data is shown in Figure 2.
However, sometimes quite abrupt changes were observed between adjacent
regions; and even reverse gradients were seen (Fig. 3). The reason for these
marked changes in temperature was not immediately evident, although a some-
what similar lability was described by Nishiwaki (personal communication) in
successive measurements on whales taken during Japanese operations in the Ant-
arctic. Substantial changes in temperature either over short distances or short
time periods seem incompatible with either a thermoregulated or an inert body
of this bulk. The explanation appears to lie in changes which occur during
capture. At this time, the whale is first secured by a harpoon with an attached
line; and a subsequent shot carrying an explosive charge kills the animal.
Sometimes several such shots may be required. These wounds can admit sea
water into the animal ; and during their death throes quantities of sea water may
be ingested, taken into the rectum or vagina, or aspirated into the lungs." Accord-
ingly, the temperature profile through positions III and V in Figure 3 must
reflect the presence of a mass of cold water, in this case at a depth of 1.5 to 2.5
feet from the surface.
36-
34-
o
32-
30-
28-
HUMPBACK WHALE No. 499
DEAD II HOURS
43 FT.
I
2
i
4
VENTRAL INSERTION DEPTH IN FEET
FIGURE 3. Temperature-depth profiles at various axial positions in whale No. 499,
showing inverted gradients.
3 Such circumstances were described by men of the flensing crew.
158
PETER MORRISON
Body temperatures also varied markedly with the location of the insertion
along the body axis (Fig. 4). Highest values were found to the posterior, and
a point of insertion midway between the umbilicus and the genital opening con-
sistently gave the highest readings. At this point, the inner thermocouples would
lie in or near the large muscle masses along the vertebrae. Mean maximum
values at the various insertion positions are shown in Figure 4. Values at the
level of the heart (position II) were somewhat higher than the next point to the
rear, but averaged 2° lower than the maximum for the animal (position V).
36-
o
o
z
.0)34-
32-
-500
10 20 30
AXIAL POSITION FROM NOSE IN FEET
40
FIGURE 4. Maximum body temperatures at various axial positions in four individuals.
Heavy curve through crossed circles represents an average for all values calculated from means
for pairs of values from two axial positions (all axial positions were not measured on all
whales).
Complete data for these maximum temperatures along the axis are presented
in Table I. Data for the entire series of whales, including the maximum tempera-
ture observed anywhere in the body, are summarized in Table II. No relation is
seen between body temperature and either sex or length. Nor is there a correla-
tion with the time after death, mean values after 2, 8, and 14 hours differing by
only 0.2° C.
Average values for all points in each transect are presented in Table III. It
is of interest that, even though position V always produced the highest single
temperature, the mean value along this transect was lower than that at either the
genital or the umbilical position on each side.
BODY TEMPERATURE IN MEGAPTERA
159
TABLE I
Maximum temperatures at various axial positions
Whale
I & II
in
IV
V
VI & VII
492
35.2
35.6
35.6
493
34.8
34.4
34.1
35.9
494
35.2
498
32.1
36.3
499
34.3
33.0
35.5
34.9
500
34.2
32.5
36.3
501
33.7
33.5
35.8
33.5
502
32.6
31.5
35.3
34.6
503
34.9
35.1
35.3
512
36.1
X
36.2
Y
36.8
Z
37.1
B
34.5
36.1
35.3
35.4
C
35.0
D
34.0
33.7
34.5
G
37.3
37.7
H
36.8
I
35.7
35.8
36.3
34.3
J
34.9
34.9
34.2
Mean values
34.4
33.9
35.5
35.8
35.2
(ID
(ID
(4)
(14)
(ID
Ave. depth (ft.)
2.7
3.8
3.7
3.3
2.8
DISCUSSION
This study has shown that there may be considerable variation in temperature
in different parts of the whale's body as measured after capture by commercial
procedures. Some of this variation appears to be an artifact resulting from
the intake of cold sea water into the body. However, one may minimize this
error, as was done, by multiple testing to identify and avoid any "cold spots."
But, with this precaution taken, there still remain substantial differences in the
maximum temperatures along the body axis. Such axial temperature gradients
are not uncommon among other mammals and may be quite prominent in some
special situations such as arousal from hibernation ; but the region of highest
temperature is always near the heart and liver, with values declining to the rear.
In the whale we see the reverse situation, with the higher values at the rear
and lower values to the front. Values in the heart itself are not the lowest, but
average 1.5° C. below the maximum. This may reflect a different balance in this
large animal between the heat production in the muscle mass and that in the
visceral organs. In considering this question, it would be very useful to have
values for brain temperatures in whales and to have axial distributions in smaller
cetaceans, but neither are available. However, measurements on the small killer
whale (Orcinus) by Portier (1908) and on the seal (Phoca) by Scholander et al.
(1942) gave higher brain temperatures than visceral temperatures. Another
possibility to account for this axial gradient is extra heat production by the
160
PETER MORRISON
TAHLK II
Maximum hmly temperatures in humpback whales
Whale
Sex
Length ft.
Hours dead
•
TB °C.
B
c?
37
5
36.1
C
0.5
35.0
D
0.5
34.5
G
1
37.7
H
0.8
36.8
I
5.5
36.3
J
2.3
34.9
2.2
35.9(7)**
492
tf
40
8
35.6*
493
<?
35
7
35.9*
494
9
33
7
35.2*
503
<?
35
7
35.3*
X
9
34
8
36.2
Y
9
33
8
36.8
Z
9
36
8
37.1
7.6
36.1(7)***
512
9
38
17
36.1
498
rf1
41
11
36.3
499
9
43
11
35.5
500
9
42
18
36.3
501
c?
39
14
35.8
502
rf1
38
13
35.3
14
35.9(6)***
36.0(20)
* "U-G" position not measured; may average 0.4'
** Whales from Byron Bay.
*** Whales from Moreton Island.
low.
TABLE III
Average body temperatures at various axial positions*
Whale
I or II
Flippers**
ill
F.-U.
IV
Umbilicus
V
U.-G.
VI
Genital
492
493
(35.2)
34.4
35.1
33.6
35.4
33.5
35.3
494
34.8
498
30.8
35.7
499
32.7
31.5
32.5
34.9
500
32.9
501
502
503
(33.7)
(32.2)
34.1
32.8
31.1
34.7
32.4
33.8
32.1
33.4
34.3
33.3
33.0
34.5
33.8
34.2
* 250 temperatures on 21 whales.
** Parenthesized values at flippers ; others 2-3 feet behind at heart.
BODY TEMPERATURE IN MEGAPTERA
161
intestinal bacteria. In the largest land mammal, the elephant, the rectal tempera-
ture is suggested to be higher than the rest of the body because of this effect
(Benedict, 1936). However, as carnivores, the whales should be less subject to
this influence; and Robinson ct al. (1953) report a very low bacterial content
in the feces, perhaps 10~3-10~6 times that in most fecal material. But, whatever
the bacterial calorification, its effect would be enhanced by the large volume
available and the whale's lower metabolic rate.
The data of Nishiwaki referred to above show some striking declines in
temperature over relatively short periods. Indeed, the average decline for a series
of whales is almost 3°/hr. (Fig. 5). It seems impossible to account for this
except as a local cooling effect of water taken into the body at the point of
measurement.
Equally striking are three curves where the temperature increased sharply
instead of decreasing. One wonders if these may represent the phenomenon of
20
HOURS POST MORTEM
FIGURE 5. Temperature changes in dead whales from data of M. Nishiwaki (personal
communication). Heavy curve and crossed symbols represent average of all data. Other
curves illustrate rapid transitions of temperature.
162
PETER MORRISON
"burning," a sharp increase in the dead whale's temperature described by whalers,
which may be attributable to an explosive bacterial proliferation through the tissues.
Under certain conditions, anaerobic bacteria may be widely distributed throughout
the animal, apparently carried from the gut through the blood stream (Robinson
ct a/., 1953). I know of no actual temperature measurements which describe this
phenomenon, although Kanwisher and Leivestad (1957) have reported an increase
of 0.3°/hr. over an 8-hour period in one whale. The observed increases of 4 to
5°/hr. (Fig. 5) represent about 1 cc. O2/g. hr. or more than ten times the
(predicted) basal heat production of the whale. The observed maxima near 40°
(Fig. 5) may seem rather low to justify the impression of "burning," but it should
be kept in mind that such comments are made by men acclimated to polar conditions.
A temperature field of this size and intensity set in an icy sea could well feel
uncomfortably hot to these observers.
TABLE IV
Whale temperatures by F. H. Addison*
Hours dead**
TB***
Rangef
Valuestt
1-4
34.5
32-38
54
5-6
34.5
33-38
51
7-8
34.6
32-37
87
9-10
34.7
33-37
90
11-12
34.8
33-37
81
13-14
34.8
33-37
46
15-17
35.1
33-37
81
1-17
34.7
32-38
490
* Unpublished Ministry of Food Report, "Antarctic Whaling Expedition, 1949-50";
personal communication from Dr. J. G. Sharp.
:* Observations made on deck of factory ship.
"The highest meat temperature is invariably obtained of the longissimus dorsi, the best
site being the middle part of the muscles adjacent to the lumbar vertebrae."
f The wide range reflects the large samples. These data showed a normal distribution with
a standard deviation of 0.9° C. (See Fig. 6.)
ft The bulk of these measurements (92%) were on Fin Whales, but included 27 values for Bine
Whales and 14 for Humpback Whales.
The observations of Addison represent a beautiful series of data to show how
constant the body temperature can be in whales (Table IV). The number of
these measurements (490) is more than three times that of all the other values
identified in the literature. In these measurements on muscles from animals
which reached the factory ship at times ranging from one to seventeen hours after
death, no fall in temperature at all was seen and actually a small increase
(0.033° C./hr.) was observed. This sequence also involved the transition through
rit/or from "dry" meat to "wet" meat, and the increase of 0.6° C. in temperature
is attributable to the breakdown of the phosphate energy reserve in the muscle.
When values for "wet" and "dry" meat were plotted separately, neither showed
any change in temperature with time.
The range of observed values in this series (89-101° F. or 31.7-38.3° C.)
seems large but only reflects the large sample size. A frequency polygon of these
BODY TEMPERATURE IN MEGAPTERA
163
120-
ADDISON (SHARP) WHALE
TB DATA
100
FIGURE 6. Frequency distribution polygon for longissimus dorsi temperatures from data
of F. A. Addison (personal communication from J. G. Sharp). Heavy curve, all values;
dotted curve, "dry" meat (before rigor) ; dashed curve, "wet" meat (after rigor) : ±1 S. D.
: 68% ; ±2 S. D. ---- 94% ; ±3 S. D. : : 99.6%. The terms "dry" and "wet" are practical
expressions describing meat which is respectively firm and stick}' (before rigor) or wet and
sloppy (after rigor) to the touch. A third term, "rubbery," relates to muscle actually in rigor
mortis (Sharp and Marsh, 1953).
data is given in Figure 6 for the total data as well as for the "wet" and "dry"
samples. This transformation has an evident, but lesser, influence on the spread
of the data, the principal variability being, apparently, differences in initial tem-
peratures in the whales. The standard deviation of the values, 0.9°, represents
considerable variation but not more than is seen in many other mammals.
The data of Zenkovic (1938) are of particular interest because he measured five
different species and further was able to make observations on five individuals
which were still alive, although seriously wounded. Because this paper is difficult
of access, the results are summarized in Table Y. Ordinarily, values from living
animals would be given precedence over values from dead animals. But we have
just considered this problem at length to conclude that in the whale this is not so.
However, Zenkovic's five values on still living animals are distinctly higher than
his other values, including one taken less than an hour after death. Our best
judgment is that these "live" values are not representative, but are the result of
the continuing exertions of the mortally wounded whale, which is, of course, only
approached when it has been completely exhausted and is almost dead. This is
riot to say that these temperature levels are necessarily outside the normal distribu-
164
PETER MORRISON
TABLE V
Whale temperatures from Zenkovic ( 1938) *
Hours after
death
Sperm
Grey
Fin
Humpback
Blue
Mean
Hours after
death
0
38.2 (2)
38.4 (2)
38.1(1)
38.3 (5)
0
1-1
36.5 (3)
36.7(12)
1-11
i-U
36.4 (6)
37.1 (3)
ii-2
36.7 (3)
36.3(4)
36.5(12)
11-4
2-3
36.6 (3)
3-4
36.2 (2)
5-71
35.5 (6)
35.9 (9)
5-9
7f-9
36.3 (3)
9-13
35.5 (3)
12-16
35.7(3)
35.6 (6)
9-16
0<TB^4
36.7 (3)
36.6(12)
36.8 (5)
36.3(4)
36.6(24)
0<TB
35.9 (9)
36.5(15)
36.3 (8)
36.3(4)
35.7(3)
36.2(39)
TB^4
37.3 (5)
36.6(12)
37.3 (7)
36.7(5)
36.9(29)
mean TB
36.3(11)
36.5(15)
36.7(10)
36.7(5)
35.7(3)
36.1(44)
"For live whales, insertion was either in the animals side nearer the belly, almost at the
beginning of the whales side folds; or sometimes in the belly depending on the position in which
the whale was made fast to the boat."
"Thermometer No. 2 was completely immersed to a depth of 30-40 cm in the body and ex-
tracted with a line of veins. The thermometers were kept in the animals body for 10 minutes".
TABLE VI
Whale temperatures by M. Nishiwaki*
Species
Sex
Length ft.
Chase hr.
Hours post-
mortem
TB**
(Range)
#
Blue
<?
75-79
<1
33(30-36)
6
9
70-87
<1
31(27-35)
7
Fin
rf1
62-71
<1
29(25-31)
6
9
67-74
<1
32(29-36)
5
<0.1
31.0(25-36)
21
<0.2
<0.3
29(25-34)
7
0.25
<0.3
33(30-35)
3
0.5
<0.3
32(29-35)
4
1-2
<0.3
32(30-35)
3
5
<0.3
36
1
* Taken during the Japanese Antarctic Whaling Expedition, 1948-49 (personal communi-
cation).
** Values represent measurements in which a thermometer probe was inserted 60-70 cm.
(2£ ft.) into the "trunk, the portion immediately below the base of the flipper." "It is, therefore,
probable that the bulb rested in the abdominal cavity in some of the measurements." In five
individuals "trunk" values averaged 4° higher than "tail" values in which "the bulb of the ther-
mometer was forced 40-50 cm. (1£ ft.) into the portion of the lateral side of the whole body below
the dorsal fin."
BODY TEMPERATURE IN MEGAPTERA
165
tion for whales, since Addison's range of values went as high as 38°. We could,
then, take this as an example of the effect of vigorous activity on body temperature
in the whale, but would not accept these values as representative of the average
temperature in this animal.
Nishiwaki's data (Table VI) may also be looked at in this regard. Comparison
of animals chased for times ranging from ten minutes up to five hours suggests
some increase, but the variability is too great for conviction. And, of course, an
animal could well reflect considerable prior activity, even though it were chased
for only a short time. In both these studies average values for the several species
showed no significant differences, nor were differences due to sex or size apparent.
Whale temperatures from all available sources are summarized in Table VII
and average 35.4° C. for twelve authors. The greater number of these values
were specifically taken in the dorsal musculature and average 34.6° C. for 547
individuals (34.5° C. for eight authors). This is a distinctly lower mean than
that of the residual group which includes the present study, 36.2° C. for 57
individuals (36.9 for four authors). This suggests that, although consistent high
values are found in the back muscle, it is not the warmest spot in the whale body.
In the porpoise (Delphinus), Richard and Neuville (1897) found the viscera to
be 0.3° C. warmer than the dorsal muscle mass.
This overall mean value of 35.4° C., or even the latter mean of 36.2° C. for
TABLE VII
Average body temperatures in whales, after various observers
Observer
Species
Hours
dead
TB °C.
Values
Location
Scoresby, 1820
Bowhead
38.8
(1)
Blood
Guldberg, 1900
(Sperm)*
(40.0)*
Guldberg, 1885
Blue
2
35.4
(1)
"Back flesh"
Laurie, 1933
Blue, fin
35.1
(30)
Long, dorsi
Zenkovic, 1938
Humpback, fin,
1-16
36.2
(35)
sperm, blue
gray
Humpback, fin,
alive
(38.3)
(5)
sperm
Aaser, 1944
1-24
33.1
(18)
Inside muscle
Parry, 1949
Blue
1
35.5
(3)
Epaxial muscle
Cockrill, 1951
(31-34.5)
9" in fillet
Robinson et al., 1953
Blue, fin
0-24
33.4
(26)
Inside muscle
Kanwisher and
Fin
36.6
(1)
Muse, and body-
Leivestad, 1957
cavity
Addison**
Fin
1-16
34.7
(490)
Long, dorsi
Ash**
-15
35
(1)
Long, dorsi
Nishiwaki**
Blue, fin
1
(25-36)
(24)
Sharp**
0.4
34.4
(1)
Long, dorsi
This study
Humpback
0.5-18
36.0
(20)
See text
* Guldberg (1900) cited Beal (1839) for this value and species, but Beal only refers to this
value as an upper limit for cetaceans. Desmoulins (1822), whom Beal cited for his statement,
apparently arrived at the value of 40° C. by arbitrarily adding on 1-4° C. for presumed cooling to
the values of Scoresby (1820) for a narwhale and a baleen whale (Tables VII and VIII).
** Personal communication.
166
PETER MORRISON
whales, is well below the averages of 37.8° C. for 56 temperate mammal species
(Morrison and Ryser, 1952) and of 38.3 for 21 Alaskan mammal species (Irving
and Kmgh, 1954). It is of ink-rest to see if this is also true of smaller cetaceans,
i.e., whether the low body temperature relates to the large size or to the order of
Cetacea. Table VIII summari/es the temperatures available for smaller cetaceans.
Values from nine authors range from 35.6 to 37.8° C. and average 36.5° C.
(36.7 for 13 individuals). It might be suggested that abnormally high values
TABLE VIII
The body temperature of some smaller cetaceans^
Reference
Species
Turn °C.*
Site
Notes
Richer (1672)
marsoiiin
"Scarcely less
warm than
land ani-
mals"'1
Abdomen
Boerhaave
(1741)***
fishes with lungs
"As other
mammals"
Broussonet (1785)
marsoiiin
35. 6f
Neck wound
Bleeding heavily
Scoresby (1820)
Monodon monoceros
36.1
"Blood"
15'; dead 90 min.
Davy (1826)
porpoise
37.8
Liver
Live on deck, at
lat. 8°
Richard and
Neuville (1897)
Delphinus delphis
35.6
(35.3)
Rectum, ab-
domen
Dorsal muscle
Harpooned
mass
Grieg (1907)
Orcinus gladiator
37.1*
Muscle
Harpooned,
dragged on shore
Portier (1908)
Orcinus gladiator
36.6
(36.9)
Rectum, liver,
vagina
Brain
Jolyet (1893)ft
Tursiops truncatus
37.0
Wislocki (1933)
Tursiops truncatus
36.0
Stranded (?)
YVoolseyftt
Tursiops truncatus
37.0
Rectum
Restrained out of
water for 2 hrs.
| Early interest in the temperature of aquatic mammals is notable. Martine (1740), who
has been cited as the first reliable authority in medical thermometry, devoted more space to this
group than to all the other homeotherms except man.
* All values represent single individuals except Grieg ( =5).
** Apparently no thermometer used.
*** Probably not an original observation.
t Calculated from original value of 28.5 taken as °R.
tt Not clear if this is an original measurement.
ttt Personal communication.
BODY TEMPERATURE IN MEGAPTERA
167
will be obtained from cetaceans restrained <>nt <>f water. If the three such refer-
ences are eliminated, the average for the remainder is 3(>.2° ('. (six authors and
individuals ) .
We might look for a more general correlation of low body temperature with
an aqnatic mode of life. Values in Table IX for two carnivores average 37.2
(12 individuals) and for seven pinnipeds average 37.3 (70 individuals). These
means are closer to the general averages for mammals but are, however, distinctly
below a mean value of about 38.5° C. for their terrestrial relatives in the Carnivora
which have higher-than-average body temperatures (Morrison and Ryser, 1952;
Irving and Krogh, 1954). One further specific comparison of interest relates
the marine polar bear to its terrestrial relatives. Twelve values for black and
TABLE IX
Body temperatures in marine Carnivora and Pinnipedia
Species
TBin °C.
Site
Author
"Sea Calf"
(38.9)
abdomen
Marline (1740)
Phoca vitulina
37.8 (24:3)*
liver, abd.
Scholander et al, (1Q42)
(38.4) (7:2)
brain
Erignathus barbatus
37.2 (5:5)ft
rectal
Irving and Krogh (1954)
Halichoerus grypus
36.5 (6:1)
Scholander (1940)
Mirounga angustirostris
36.0 (13:13)***
rectal
Bartholomew (1954)
Mirounga leonina
37.8
Aretas (1951)
Callorhinus ursinus
37.4 (32:32)
heart
Hanna (1924)
Callorhinus ursinus
37.7 (13:13)**
Bartholomew and VVilke (1956)
E u m etopias jubata
38.5 (2:2)f
rectal
Irving and Krogh (1954)
Enhydra lutn's
38.5 (1:1)
rectal
Irving and Krogh (1954)
Enhydra lutris
36.8 (6:6)f
Stullken and Kirkpatrick (1955)
Thalarctos maritimus
37.5 (3:3)f
Anon. (1827)
Thalarctos maritimus
37.3 (2:2)f
viscera
Irving and Krogh (1954)
* Lost 2.5° during dive; parenthesized numbers show measurements and individuals.
** Gained up to 4° during activity on land.
***Lost 2. 2° at night (5:5).
f Shot.
brown bears average 37.9° C. or 0.5° C. above the level of the polar bear (Irving
and Krogh, 1954; Hock, 1957). Although the difference is small, it appears
statistically significant (t -- 3.0). In sum, therefore, all of these group means do
support the association of aquatic life with a reduction in body temperature.
SUMMARY
1. A series of some 250 body temperature measurements were made on 20
humpback whales (Mcgaptera novaeangliae) from the east coast of Australia. The
distribution in the animal was plotted by means of a series of ventro-dorsal tempera-
ture profiles. Inverted temperature profiles were sometimes found, indicating the
presence of internal masses of cold water and offering an explanation for the
aberrant temperature values sometimes reported for whales.
2. Maximum values were found posteriorly near the umbilicus and the genital
168 PETER MORRISON
opening, and at a depth of 3.3 feet. The average was 36.0° ; and there was no
correlation with sex, size (33-40 ft.), or time after death (0.5-18 hrs.). This
body temperature is close to the mean of literature values for whales (35.8°)
and, as well, for smaller cetaceans (36.4°), but is appreciably below that for the
Pinnipedia (37.3°).
LITERATURE CITED
AASER, C. S. S., 1944. Rapport over hvalkj0ttunders0kelser sommeren 1943. Norsk. I'ct.
Tidsskr., 56: 33-62.
ANON., 1827. Temperature de quelques Animaux du Nord, prises au Port Bowen ( Extrait <lu
dernier Voyage du Capitaine Parry). .-Inn. de Cliiin. ct de Phys. (2nd scr.), 34: 111.
ARETAS, R., 1951. L'elephant de mer (Mirounga Iconina (L)). Mammalia, 15:105-117.
BARTHOLOMEW, G. A., JR., 1954. Body temperature and respiratory and heart rates in the
northern elephant seal. /. Mamm., 35: 211-218.
BARTHOLOMEW, G. A., AND F. WILKE, 1956. Body temperature in the northern fur seal,
Callorhinus ursinus. J . Mamm., 37 : 327-337.
BEALE, T., 1839. The Natural History of the Sperm Whale. Van Voorst, London. 393 pp.
BENEDICT, F. G., 1936. The physiology of the elephant. Carnegie Institution of Washington.
Publication No. 474. 302 pp.
BOERHAAVE, H., 1741. Elementa Chemiae, 2d ed. (translated by Peter Shaw from original
Latin). Longman, London.
BROUSSONET, 1785. Memoire pour servir a 1'histoire de la respiration des poissons. Mem. (de
Mathcmatiquc ct de Physique) de I'Acad. Roy. des Sci., Paris, 174-196.
COCKRILL, W. R., 1951. Antarctic pelagic whaling. Vet. Rec., 63: 111-124.
DAVY, J., 1826. Observations on the temperature of man and other animals. III. Of the
temperature of different kinds of animals. Edin. Phil. J., 14 : 38-46.
DESMOULINS, A., 1822. Diet, class. d'Hist. Nat., 2: 159.
GRIEG, J., 1907. Hvalernes legemstemperature. Naturen, 31 : 125-126.
GULDBERG, G. A., 1885. tlber das Centralnervensystem der Bartenwale. Forlmndluujcr i
Vidcnskabs-sclskabet i, Christiania, Aar 1885, No. 4. 154 pp.
GULDBERG, G. A., 1900. Nyt mag a sin for naturvidenskabcrnc, 38 : 65-70.
HANNA, G. D., 1924. Temperature records of Alaska fur seals. Amcr. J. Physiol., 68: 52-53.
HOCK, R. J., 1957. Hibernation in Cold Injury. Trans. 5th Conf. Josiah Macy Found., pp.
61-133.
IRVING, L., AND J. KROGH, 1954. Body temperatures of arctic and subarctic birds and mam-
mals. /. Appl. Physiol., 6: 667-680.
JOLYET, F., 1893. Recherches sur la respiration des cetaces. C. R. Soc. Biol., 45 : 655-656.
KANWISHER, J., AND H. LEIVESTAD, 1957. Thermal regulation in whales. Nonvegian Whalimj
Gazette, 46 : 1-5.
LAURIE, A. H., 1933. Some aspects of respiration in blue and fin whales. Discovery Reports,
7 : 363-406.
MARTINE, 1740. Essays Medical and Philosophical. A. Millar, London. 376 pp.
MORRISON, P. R., AND F. A. RYSER, 1952. Weight and body temperatures in mammals.
Science, 116: 231-232.
PARRY, D. A., 1949. The structure of whale blubber, and a discussion of its thermal properties.
Quart. J. Micr. Sci., 90: 13-25.
PORTIER, P., 1908. Temperature de vertebres mar ins, en particular des poissons du groupe des
thons. C. R. Soc. Biol., 64: 400-402.
RICHARD, J., AND H. NEUVILLE, 1897. Sur quelques cetaces observes pendant les campagnes
du yacht Princess-Alice. Mem. Soc. Zoo/, de France, 10: 100-109.
RICHER, - — , 1672. Observations astronomiques et physiques faites en 1'isle de Ca'ienne. Mem.
A cad. Sci., 7 : 230-326.
ROBINSON, R. H. M., M. INGRAM, R. A. M. CASE AND J. G. BENSTEAD, 1953. Whalemeat :
Bacteriology and Hygiene. Dept. Sci. and Indust. Res., Food Invest. Special Report
No. 59. London. 56 pp.
BODY TEMPERATURE IN MEGAPTERA 169
Sc IIOLANDER, P. F., 1940. Experimental investigation on the respiratory function in diving
mammals and birds. Hralradets Skifter, No. 22. 1-131.
SCHOLANDER, P. F., L. IRVING AND S. W. GRiNNELL, 1942. On the temperature and metabolism
of the seal during diving. /. Cell. Couip. Physiol., 19: 67-78.
SCHOLANDER, P. F., AND W. E. SCHEVILL, 1955. Countercurrent vascular heat exchange in
the fins of whales. /. Afipl. I'liysiol., 8: 279-282.
SCORESBY, W., 1820. An account of the arctic regions. Constable, Edinburgh, 1 : 477.
SHARP, J. G., AND B. B. MARSH, 1953. Whalemeat: Production and Preservation. Dept. Sci.
and Indust. Res., Food Invest. Special Report No. 58. London. 47 pp.
STULLKEN, D. E., AND C. M. KIRKPATRICK, 1955. Physiological investigation of captivity
mortality in the sea otter (Enhydris littris). Transactions of the Twentieth N. Amer.
Wildlife Conference, 476-494.
WISLOCKI, G. B., 1933. Location of the testes and body temperature in mammals. Quart. AVr1.
Biol., 8 : 385-396.
ZENKOVIC, B. A., 1938. The temperature of whales. C. R. de I'Acad. dcs Sci. de I'URSS,
18: 685-687.
INTKRTIDAL CLUSTKR1 N( i OF AN AUSTRALIAN (iASTROI'OU '• -
JAMES M. MOULTON
Department of Biul/n/y, Bowdoin College, Brunswick, Maine
The migrations and adaptive behavior of intertidal gastropods have received in-
creasing attention in recent years (Abe, 1955; Jenner, 1958; Anderson, 1961;
Fraenkel, 1961; Kornicker, 1961; Sindermann, 1961), but the life histories of
cerithiid mollusks are little known (Anderson, 1960), and the small gastropods of
northern Australia have received little attention generally (Laseron, 1956; Mc-
Michael, 1960). The behavior of a Cerithium here described may be one aspect of
acclimation to drying conditions and perhaps of high temperature acclimation in a
tropical intertidal gastropod (see recent review of Segal, 1961).
Clustering and dispersal in rhythm with the tides, of a population of the cerithiid,
Cerithium Clypeomorus uwnilifcnini Kiener, occurs on tropical Heron Island in the
Capricorns off the Queensland coast of Australia. The observations recorded here
were made between October, 1960, and January, 1962, principally in October through
December, 1960, while the author was a Fulbright scholar in the Department of
Zoology at the University of Queensland.
The ecology and distribution of intertidal organisms in relation to the geography
and tides of Heron Island have been discussed by Endean et al. (1956). The Island
is at present bordered on its southwestern and northeastern shores by formations of
beachrock, a consolidated calcareous deposit of uncertain chemical origin (Revelle
and Emery, 1957; Kaye, 1959); beachrock distribution at Heron Island has pre-
sented a changing picture over the last 125 years (Saville-Kent, 1893, pp. 94-95,
106-108; Steers, 1938).
BEHAVIOR OF CERITHIUM AT HERON ISLAND
The Heron Island population of Cerithium (Fosberg ct al., 1961) inhabited
chiefly during October and November of 1960 a relatively smooth beachrock plateau
on the western tip of the Island ( Fig. 1 ) . During high tide the animals were
1 Contribution No. 1265 from the Woods Hole Oceanographic Institution.
- This study was made while the author was a Fulbright scholar in the Department of Zool-
ogy at the University of Queensland during 1960-1961. His work was also supported by the
Woods Hole Oceanographic Institution, by NSF Grant G-4403, and by a John Simon Guggen-
heim Memorial Fellowship, for all of which he would express deep appreciation. He is much
indebted to the Great Barrier Reef Committee for use of the facilities of the Heron Island Marine
Research Station and to the Department at the University of Queensland for its generous
hospitality.
Dr. D. F. McMichael of the Australian Museum, Sydney, kindly identified the species of
Cerithium.
Professor W. Stephenson of the University of Queensland and Miss Isobel Bennett of the
University of Sydney indicated that they had earlier observed the behavior of Cerithium here
described.
Mr. H. F. Manning, caretaker of the Heron Island Station, very kindly carried on observa-
tions on CeritliittDt distribution following the author's visit.
170
INTERTIDAL GASTROPOD CLUSTERING
171
1
FIGURE 1. Appearance at low tide of the Heron Island Ccrithinin plateau (right center) on
November 24, 1960. Algal scum had accumulated, graying most of the plateau.
FIGURE 2. A portion of the Cerithinin population on the bottom at high tide on October 15,
showing scattered distribution characteristic of tidal feeding. A clustering site of the preceding
low tide is encircled in the left foreground.
172 JAMES M. MOULTON
homogeneously distributed over the bottom in the intertidal zone (Fig. 2) ; during
low tide they were tightly clustered in roughly circular groups, generally of a few
hundred to a few thousand individuals, on the open rock face (Fig. 3) ; an occasional
isolated individual and clusters of a few to several snails occurred. Cerithium also
clustered in sandy crevices and pits of rougher beachrock. Clustering occurred
on the ebbing tide both night and day.
When on an incoming tide the water column attained 10 cm. over a Cerithinin
cluster, individuals began to move out from the edges of the cluster (Fig. 4). The
clusters continued to disperse until the relatively homogeneous distribution of high
tide over the feeding zone had been attained. On the ebbing tide, Cerithium began
abruptly streaming toward various foci on the rock plateau under a water column of
about 50 cm. ; at 40 cm. the foci were clearly apparent with streams of converging
Cerithium radiating around them ; at 30 cm., the clusters were tightly formed
(Fig. 5).
Observation of the behavior of marked individuals (shells marked with yellow
enamel) of two clusters and of marked clustering sites in October, 1960, demon-
strated that individual clusters were formed on successive tides of different compo-
nents of the population, the painted individuals becoming increasingly dispersed
during the period of observation; clustering sites varied on successive tides, there
being no apparent predilection for specific sites.
Through November and December of 1960, the beachrock plateau occupied by
the Cerithium population changed in character; a grey, silty scum of a blue-green
alga (probably an Oscillatoria — R. Wood, personal communication) accumulated
over the plateau. The Cerithium population became gradually restricted to sandy
crevices and finally by mid-January, 1961, Cerithium had abandoned the plateau, and
were beginning to concentrate at the southeastern end of Heron Island (Miss J.
Badham, personal communication). Figures 3 and 6 contrast the appearance of
similar areas of the plateau on October 17 when clustering was at its height and
December 16, when the Cerithium population had become entirely restricted during
low tide to sandy crevices in the plateau.
By October of 1961 the Cerithium population was concentrated in Shark Bay on
the southeastern corner of Heron Island clustering and feeding over a sandy area
above the nearby beachrock which at this time was apparently clear of algae (H. F.
Manning, personal communication). Similar migrations have apparently occurred
in the past; thus, Stephenson and Searles (1960) found a marked drop in the
Cerithium population inhabiting experimental plots of beachrock near the Heron
Island Marine Research Station between October 1, 1959, and January 12, 1960 — a
period corresponding to that of departure of the population from the western
plateau in late 1960.
Clustering and dispersal occur experimentally in the absence of tidal rhythms.
The single cluster removed to a shallow pan submerged in a pool of sea water behaves
like a larger population ; in shallow depths imitative of a falling tide, aggregation
into a number of clusters occurs over a period of 24 minutes (Fig. 7). Subsequent
lowering of the pan to a depth of 13 inches results in a gradual breaking up of the
formed clusters as a search for food is initiated (Fig. 7). That is, changes in depth
bring about distributions similar to those obtained over a normal tide cycle, inde-
pendently of tidal rhythms. Tidal rhythms of physiological processes observed in
IXTERTIDAL GASTROPOD CLUSTERING
173
FIGURE 3. Clustered Cerithium exposed intertidally on October 15. Each black cluster con-
tains from several to over a thousand individuals.
FIGURE 4. Individual Cerithium leaving clusters on the incoming tide on October 15.
174
JAMES M. MOULTON
PBT
|'"K;UKK 5. ReaggregaU-d and clustering Ccrithiiuit (dark patches) on a falling tide, still
submerged, on October 15.
FIGURE 6. The Ccrithiitin plateau in late November when Ceritliiinn had retreated to sand-
filled crevices, accompanying progressive accumulation of scum on the plateau. (Compare
with Figure 3.)
INTERTIDAL GASTROPOD CLUSTERING
175
>' >-
*•
4
2min.
t .
24min.
1.8in. 56min. 13in.
OOmjn.after lowering)
Cerjthium Distribution and Water Level
FIGURE 7. The distribution of a single cluster of Ccrithium in a 12 inch X 14 inch enamel pan of
sea water at various times and depths after immersion.
other mollusks (Rao, 1954) are, however, suggestive that Cerithium may respond
in other ways to the tide cycle than by clustering and aggregation.
COMPOSITION OF THE POPULATION
The Cerithium population in late 1960 was comprised chiefly of two varieties of
the same species : a dark gray translucently shelled form with a spire of lighter
color than the major whorl, and a lighter colored, heavier shelled, less common form
with a thickened lip similar in these details to the knobbed cerithium (C. cacrulcnm)
of eastern Africa. Intermediates between the two types occur.
Size distribution of a cluster of 1377 individuals between 6 and 15.5 mm. in
length is shown in Figure 8. Periodic recruitments to the population, presumably
SIZE FREQUENCY IN CERITHIUM CLUSTER
Heron Islond XI/9/60
10
10 11 12
Length in millimeters
FIGURE 8. Size distribution within a single cluster of 1377 individuals collected
on November 9, 1960.
176 JAMKS M. MOULTON
of seasonal occurrence in view of their regular periodicity, are indicated by breaks
in the spectrum of distribution at just above each millimeter mark.
In late 1960 there were no cerithiid egg filaments on the plateau (described by
Ostergaard, 1950, for Clava obeliscus), nor did cerithiid veligers, described by
Lebour (1944) for C. ferrugineum, occur in plankton towed in October and Novem-
ber in the channel between Heron Island and Wistari Reef ; in fact veligers of any
type were scarce. The smallest individual collected on the plateau during late 1960
measured 4 mm. in length.
Males bore active gametes, but ovaries were poorly developed and no ripe eggs
were found in a few hundred females examined. Sex ratios were inconstant in
clusters. In short, clustering seemed not related to reproductive activity. The
reproductive habits of cerithiids vary considerably, as between species of Cerithiopsis
(Lebour, 1933) and Cerithium ferrugineuw (Lebour, 1944).
A variable proportion of the Ccrithiuni population on the western plateau of
Heron Island was parasitized by larval bird flukes, notably by a heterophysid
opisthorchioid (Dr. John Pearson, personal communication) characterized by a
cercaria with a large chocolate-brown tail ; several other species occurred. Between
November 18 and 23, 1960, 7% and 45% of small samples (60 each) of the thin- and
thick-shelled forms, respectively, were parasitized, and in early January Miss J.
Badham, an undergraduate of the University of Queensland, reported finding 2Z% of
a random sample of 50 animals from the western part of Heron Island (where shore
birds, especially the silver gulls, were most numerous) were infested with trematode
larvae. By this time, a population of Cerithhnn had begun to accumulate at Shark-
Bay, and here only 1 to 2% of a sample of 30 were infested. Shore birds were of
far less common occurrence in this area. Sindermann (1961) has reported that
larval trematode infestation inhibits migrations of two north temperate gastropods.
DISCUSSION
Whatever the factors that influence the choice of clustering site and clustering
itself, it seems likely that the habit is an adaptation of Ccrithhun to drying conditions
and high temperatures on tropical beaches, and that it falls into the habitat category
of ecologically important homeostatic mechanisms suggested by Bullock (Segal,
1961). The clustering habit effectively decreases air temperature to which
Cerithhnn is exposed at Heron Island at low tide during the daytime (30.6° C. on
the sand surface; 27.8° in the center of a cluster on January 4, 1962 — H. F. Man-
ning, personal communication). However, water temperature may exceed air
temperature by a degree or two over the clusters ; and L. neritoidcs withstands tem-
peratures up to 47° C. in air (Fraenkel, 1961). It seems likely that prevention of
drying is a more significant result of clustering than is a lowering of temperature.
It is suggested that clustering is a behavioral adaptation to tropical conditions, super-
imposed perhaps over physiological adaptations which have been demonstrated in
other mollusks (Fraenkel, 1961; Segal, 1961).
Clustered individuals are always damp, and clusters taken to the laboratory,
spread and allowed to dry during the first marking experiments, died rapidly.
Within a cluster there is always some degree of movement of individuals, resulting
in a slow turnover of the group. Further, a cluster contains a varying amount of
damp sand or silt, and the shells of many individuals are decorated with small bits
INTERTIDAL GASTROPOD CLUSTERING 177
of green alga (probably an Ulva — R. Wood, personal communication). These fac-
tors combine to retain a moist environment for clustered Cerithium during low tide.
Consistent responses to height of the water column are suggestive that Cerithium
possesses a hydrostatic mechanism which helps to determine whether the animals
shall be clustered or dispersed. In behavioral terms one can envisage that the
falling tide induces a positive barokinesis (increased rate of movement due to falling
pressure) and clustering due to thigmotaxis (a search, induced by the falling tide,
for contact surfaces which may be conducted along slime trails laid down by other
animals, the search continuing until clustering results). According to this hy-
pothesis, following the exposure period of low tide, barokinesis induced by an in-
creasing water column or increased activity induced by the moisture of the incoming
tide may induce the animals to terminate clustering in the search for food (see
Carthy, 1958). A remarkable degree of sensitivity to the water column has been
demonstrated in a crustacean (Enright, 1960), and there is some evidence that
copepod sex ratios may be influenced by hydrostatic pressure (Vacquier, 1962).
Analogous aggregations of intertidal gastropods occur in northern waters with
the onset of cold weather. Thus, in the Gulf of Maine and on the shores of Massa-
chusetts' Cape Cod, populations of Nassarius obsoletus aggregate in dense concen-
trations (Jenner, 1958), probably preceding a general movement to deeper water
from the intertidal mudflats (Sindermann, 1961). Rock face types such as the
littorinids and Thais of the Maine coast have been observed to aggregate in clusters
on the rock faces before moving into submerged positions in deep crevices ; with
the approach of warmer weather the movement is reversed as the animals reoccupy
exposed situations. In all of these cases, however, the tidally rhythmic aggregations
and dispersals demonstrated by Cerithium are lacking.
SUMMARY
1. A striking rhythmical clustering and dispersal of a population of Cerithium
Clypeomorus monilifcrum Kiener on Heron Island in the Capricorns of Australia is
described.
2. A sequence of events preceding a seasonal migration of the concentrated
Cerithium population is reconstructed.
3. The role of the described habit as an ecologically important homeostatic
mechanism is discussed.
LITERATURE CITED
ABE, N., 1955. Colony formation of a limpet, Acmaea dorsursa Gould, and variation of level
of the colony. Bull. Mar. B'wl Sta. Asanntshi, 7: 127-132.
ANDERSON, D. T., 1960. The life histories of marine prosobranch gastropods. J. Malacological
Soc. Aust., No. 4, Nov. 13, 1960.
ANDERSON, D. T., 1961. The reproduction and life history of the gastropod Bembichtm nanttin
(Lamarck, 1822) (Fam. Littorinidae). Ansf. J. Sci., 24: 242.
CARTHY, J. D., 1958. An Introduction to the Behaviour of Invertebrates. George Allen &
Unwin Ltd. Bristol.
ENDEAN, R., W. STEPHENSON AND R. KENNY, 1956. The ecology and distribution of intertidal
organisms on certain islands off the Queensland coast. Aust. J. Mar. Frcshw. Res., 7:
317-342.
ENRIGHT, J. T., 1960. Pressure sensitivity of an amphipod. Science, 133: 758-760.
FOSBERG, F. R., R. F. THORNE AND J. M. MOULTON, 1961. Heron Island, Capricorn Group,
Australia. Atoll Research Bull., No. 82, Dec. 31, 1961.
178 JAMES M. MOULTON
FRAENKEL, G., 1961. Resistance to high temperatures in a Mediterranean snail, Littorina
neritoides. Ecology, 42: 604-606.
JENNER, C. E., 1958. An attempted analysis of schooling behavior in the marine snail
Nassarius obsolctits. Biol. Bull., 115: 337-338.
KAYE, C. A., 1959. Quatenary shoreline changes, Puerto Rico. Coastal geology of Puerto Rico.
U. S. Geol. Survey, Professional Paper 317-B.
KORNICKER, L. S., 1961. Observations on the behavior of the littoral gastropod Terebra sallcana.
Ecology, 42: 207.
LASERON, C. F., 1956. The family Cerithiopsidae (Mollusca) from the Solanderian and
Dampierian zoogeographical provinces. Aust. J. Alar. Freshiv. Res., 7: 151-182.
LEBOUR, M. V., 1933. The life histories of Cerithiopsis tubercularis (Montagu), C. barleci
Jeffreys, and Triphora pcrversa (L.). /. Mar. Biol. Assoc., 18: 491-498.
LEBOUR, M. V., 1944. The eggs and larvae of some prosobranchs from Bermuda. Proc. Zool.
Soc. London, 114: 462-489.
McMiCHAEL, D. F., 1960. Shells of the Australian Sea-Shore. Jacaranda Press. Brisbane.
OSTERGAARD, J. M., 1950. Spawning and development of some Hawaiian marine gastropods.
Pacific Sci., 4, No. 2: 75-115.
RAO, K. P., 1954. Tidal rhythmicity of rate of water propulsion in Mytilus and its modifiability
by transplantation. Biol. Bull, 106: 353-359.
REVELLE, R., AND K. O. EMERY, 1957. Chemical erosion of beach rock and exposed reef rock.
U. S. Geol. Survey, Professional Paper 260-T.
SAVILLE-KENT, W., 1893. The Great Barrier Reef of Australia : Its Products and Potentialities.
W. H. Allen and Co., Ltd. London. (Pp. 106-108; 94-95.)
SEGAL, E., 1961. Acclimation in molluscs. Amer. Zoologist, 1: 235-244.
SINDERMANN, C. J., 1961. The effect of larval trematode parasites on snail migrations. Amer.
Zoologist, 1: 389.
STEERS, J. A., 1938. Detailed notes on the islands surveyed and examined by the geographical
expedition to the Great Barrier Reef in 1936. Reports of the Great Barrier Reef
Comm., 4, part 3: 51-104.
STEPHENSON, W., AND R. B. SEARLES, 1960. Experimental studies on the ecology of intertidal
environments at Heron Island. I. Exclusion of fish from beach rock. Aust. J. Mar.
Frcshw. Res., 2 : 241-267.
VACQUIER, V., JR., 1962. Hydrostatic pressure has a selective effect on the copepod Tigriopns.
Science, 135: 724-725.
THE LARVAL DEVELOPMENT OF CALCINUS TIBICEN (HERBST)
(CRUSTACEA, ANOMURA) IN THE LABORATORY1
ANTHONY J. PROVENZANO, JR.
Institute of Marine Science, University of Miami, Miami 49, Florida
Since the first studies on hermit crab development in the early 19th century,
workers have had to rely on reconstructions of developmental sequences from plank-
tonic material. In some instances it has been possible to maintain larvae taken from
the plankton through one moult in the laboratory and thus to tie together series of
larvae believed to represent single species. This was the method used by Thompson
(1904) in his classic work on Pagurus development, and by more recent workers as
well (MacDonald, Pike and Williamson, 1957; Pike and Williamson, 1960;
Dechance, 1961). The reconstruction method may be used profitably only in waters
with restricted faunas. Planktonic larvae from any but the most intensively studied
areas can seldom be identified with certainty. In tropical waters which are generally
less well studied and in which there are usually several times the number of species
and genera found in temperate seas, the reconstruction technique is much less useful.
Although the tropical Western Atlantic contains the second richest pagurid fauna
in the world (Wass, unpubl. ; Provenzano, 1959, 1961a, 1961b) there is not a single
species of the family Diogenidae in the West Indies for which the larval development
is known. Excepting two species of Paguristes which have abbreviated development
(Hart, 1937; Pike and Williamson, 1960) no hermit crabs of that family have
previously been reared in the laboratory from the first zoea to the post-larval stage.
Fortunately, in recent years advances have been made in the development of
techniques for rearing several decapod groups in the laboratory. Broad (1957a,
1957b) and Dobkin (in press) have been able to rear caridean shrimps. Costlow
and Bookhout (1959, 1960a, 1960b, 1961a, 1961b), Knudsen (1958, 1959a, 1959b),
Hart (1960) and Chamberlain (1961) have been successful with a variety of
brachyuran crabs. Forss and Coffin (1960) pointed out the applicability of a
method for rearing several decapod groups and Coffin (1960) was able to rear a
species of the hermit crab family Paguridae. At this laboratory a terrestrial hermit
crab of the family Coenobitidae has been successfully reared to metamorphosis
(Provenzano, in press).
Until very recently there have been no descriptions of larvae of the tropical hermit
crab genus Calcinus. According to Pike and Williamson (1960), the glaucothoe
stage described and figured by Bouvier (1922) and attributed by him to Clibanarius
may be the postlarva of Calcinus ornafus (Roux) of the Mediterranean. Bourdil-
lon-Casanova (1960) described and figured a zoea taken from the plankton, which
she believed to be the first stage of Calcinus ornatus. Pike and Williamson (1960)
1 Contribution no. 395 from the Marine Laboratory, University of Miami, Florida. This
work was supported by National Institutes of Health grant no. RG-7166(A) and National Science
Foundation grant no. G-16298, and the present paper constitutes a scientific report to those
agencies.
179
180 ANTHONY J. PROVENZANO, JR.
were able to obtain the first zoea of C. ornatus from a laboratory hatching and by
holding identical larvae from the plankton through one moult in the laboratory they
were able to obtain the second stage. Similarly they obtained and described five
zoeal stages and one glaucothoe stage for that species.
In the Western Atlantic only two species of Calcinus are known to occur (Pro-
venzano, 1960). Lewis (1960) gave a very brief description and a figure for the
first zoea of Calcinus tibicen (Herbst) but neither is sufficiently detailed for use in
identification. No other reports on larvae of this genus are known. The present
paper describes the larval development of Calcinus tibicen in the laboratory.
MATERIALS AND METHODS
Ovigerous females of Calcinus tibicen were collected at Bear Cut, Biscayne Bay,
Florida. In the laboratory they were kept in large fingerbowls containing filtered
sea water until hatching occurred. In the first experiment, conducted from 7 June
1960 to 3 August 1960, approximately 40 larvae were removed by means of a wide-
bore pipette to plastic compartmented trays, five larvae to each compartment of
about 50 cc. capacity. Every second day larvae were transferred to corresponding
compartments in fresh trays containing filtered sea water and freshly hatched
Artemia nauplii as food. Temperature of the standing sea water during this period
gradually increased from 26.5° C. to 30.3° C. with diurnal fluctuations of about 1° C.
Salinity samples taken at each change of water were titrated and values ranged from
31.1 ppt to 35.0 ppt. The second experiment ran from 17 April 1961 to 7 June 1961.
Approximately 150 larvae were placed singly in individual compartments contain-
ing filtered sea water and freshly hatched Artemia. A number was assigned to each
compartment and every second day larvae were transferred to correspondingly num-
bered compartments in freshly prepared trays. By daily examination of the trays
for exuviae and for dead animals, and by preserving exuviae of each larva in an ap-
propriately numbered vial it was possible to follow the complete history of each
specimen. Temperatures ranged from 26.0° C. to 29.0° C. during the experiment.
Salinity was semi-controlled by storage of filtered sea water in five-gallon carboys.
Three lots of water were used during the development of these larvae, according to
the following schedules : from 17 April to 12 May, salinity of 35.52 ppt during stages
I-V; from 12 May to 22 May, salinity of 37.41 ppt during stages V, VI, some VII,
and glaucothoe ; and from 22 May to termination of the experiment, salinity was
37.07 ppt and affected stages VII, VIII, and some glaucothoes. No attempt was
made to control illumination other than to prevent direct sunlight from falling onto
the transparent boxes.
Larvae and exuviae were preserved in 5-7% sea water-formalin buffered with
hexamethylene tetramine or were rinsed in fresh water and stored in 70% ethyl alco-
hol. After staining in Mallory's acid fuchsin red, specimens and exuviae were dis-
sected in 85% lactic acid and appendages were mounted in Hoyer's medium. Draw-
ings of whole larvae were made from live, immobilized animals or from freshly killed
specimens. A stereoscopic dissecting microscope with magnifications up to 36 X was
used for dissections and for making camera lucida drawings of whole animals. En-
largements of appendages and other parts were drawn using a camera lucida with a
monocular microscope at 90 X and details were checked at 450 X .
LARVAL DEVELOPMENT OF CALCINUS 181
The descriptive portion of the text was based chiefly on specimens under the
microscope at the time of description. Usually appendages of at least two or three,
sometimes five or six, different animals were examined during preparation of the
descriptions. Notes made on additional material were incorporated later.
Setae were drawn as they appeared with respect to number, position, and length
but setules when present were illustrated semi-diagrammatically. In most illus-
trations of appendages the setules are shown as somewhat shorter and much less
numerous than in the specimens.
Duration refers to the time range between moults for animals which successfully
passed through a given stage. This "normal" duration was often exceeded by larvae
which died without moulting to the subsequent stage and such data are not included
in the values for duration.
Measurements were made with the aid of an ocular micrometer. Total length
(TL) was measured from the tip of the rostrum to the posterior border of the telson,
exclusive of all telson processes. Length of carapace (CL) was measured from the
tip of the rostrum to the most posterior lateral margin of the carapace including the
lateral spines. The numbering of telson processes follows the system of Pike and
Williamson (1960) not MacDonald, Pike and Williamson (1957).
The females from which larvae were obtained are on deposit in the Marine
Museum of the Institute of Marine Science, University of Miami.
This work was initiated during a pilot study supported by the National Institutes
of Health, U. S. Public Health Service, and was completed with support of the
National Science Foundation. I am indebted to both those agencies. I would like
to thank Sheldon Dobkin and Dr. Gilbert L. Voss for criticisms of the manuscript.
RESULTS
The number of zoeal stages through which Calcinus tibicen may pass in the lab-
oratory before moulting to the glaucothoe stage is variable. The glaucothoe was
obtained after six, seven or eight zoeal instars. The principal features of each
stage are as follows :
First Zoea. Figs. 1,1; 2, I.
Size. TL 1.9 mm. ; CL 1.3 mm.
Duration. From five to eight days.
The carapace has a prominent carinate rostrum and there is a large corneous
submarginal spine postero-laterally on each side of the carapace. The eyes are
immobile. The fifth abdominal somite has a prominent very slightly curved medio-
dorsal spine and a smaller lateral spine on each side of the same somite. There is
a dorso-lateral pair of fine hairs on somites two, three and four which are not noticed
on later stages. The sixth somite is fused to the telson (Fig. 3, I) which is broader
than long, notched medially, and armed with seven processes on each side. The
first or outermost process is apparently fused to the telson, but a line of demarcation
may be seen. The second process is a fine hair bearing setules. Processes three to
seven are strong plumose spines articulated with the telson. There is a red pigment
182
ANTHONY J. PROVEXZANO, JR.
FIGURE 1. Calcinus tibicen. Zoeal stages I-VI, dorsal view.
spot at about the mid-point of the antennule and diffuse orange-red pigment over
the thorax, but under the carapace. In some specimens there is a diffuse bluish
pigment on the telson.
The antennule (Fig. 4, I) terminates in at least one aesthete and four other
processes. There is a very prominent subterminal plumose seta.
The antennal endopodite is fused to the basipodite and bears two long terminal
plumose setae and one subterminal seta less than one-half as long as the others (Fig.
5, I). A short, toothed spine is present on the ventral surface of the basipodite.
The antennal scale is about three times longer than wide, slightly longer than the
endopodite, is concave on the lateral margin and bears a terminal tooth. Sub-
terminally there is a short hair followed by nine, rarely eight, longer plumose setae.
The mandibles are dissimilar, simple toothed processes.
The three-segmented endopodite of the maxillule (Fig. 6, I) has three terminal
setae with another distally on the second segment. The proximal endite of the basi-
podite has six setae of which four are branched, two simple. The distal endite
terminates in two large spines and two non-plumose setae.
LARVAL DEVELOPMENT OF CALCINUS
183
FIGURE 2. Calciniis tibicen. Zoeal stages I-VI, lateral view.
The maxilla (Fig. 7, I) bears six or seven setae on the proximal lobe of the coxal
endite, four on the distal lobe. There are four setae on both proximal and distal
lobes on the basal endite. The bilobed unsegmented endopodite bears four setae,
the scaphognathite five plumose setae.
The first maxilliped (Fig. 8, I) has a prominent curved seta upon a papilla
184
ANTHONY J. PROVENZANO, JR.
FIGURE 3. Calcinus tibicen. The telson, zoea I- VI.
proximally on the medial margin of the basipodite, with about nine additional setae
along the medial margin. The exopodite is composed of two indistinct segments
and terminates in four long plumose setae. The endopodite is five-segmented, the
terminal segment bearing four plumose setae apically with a fifth proximally. The
LARVAL DEVELOPMENT OF CALCINUS
185
FIGURE 4. Calcinns tibicen. The antennule, zoea I through glaucothoe.
186
ANTHONY J. PROVENZANO, JR.
E
E
in
•
O
FIGURE 5. Cakinus tibicen. The antenna, zoea I through glaucothoe.
antepenultimate segment bears a single distal hair medially. The other segments
bear a pair of setae medially. There is a row of very fine setules along the lateral
margin of the endopodite.
The second maxilliped (Fig. 9, I) has only three setae on the medial margin of
LARVAL DEVELOPMENT OF CALCINUS
187
II
0.5 mm
V VI
FIGURE 6. Calcinns tiblcen. The maxillule, zoea I through glaucothoe.
the basipodite. The exopodite has four long plumose terminal setae. The endo-
podite is only four-segmented, the terminal segment having four apical setae and one
seta subdistally. The other segments each have a pair of distal setae medially.
The third maxilliped (Fig. 10, I) is a uniramous rudiment.
Second Zoea. Figs. 1, II ; 2, II.
Size. TL 2.5-2.6 mm. ; CL 1.5 mm.
Duration. From three to five days.
Mobile eyes serve to distinguish the second zoea from the first. The dorsal
spine of the fifth abdominal somite is still very prominent, much larger than the
laterals. The sixth somite is still fused to the telson which bears an extra pair of
processes medially (Fig. 3, II). The outermost process on each side of the telson is
clearly not fused to the telson. In addition to the same red-orange coloration as
noted for the previous stage, there is diffuse blue on the anterior carapace ventrally
and a blue cast to the carapace dorsally, especially in the cardiac region.
The antennule (Fig. 4, II) has five or six terminal processes, at least one of
which is an aesthete. There is a large plumose seta subterminally and a pair of small
fine setae at about the same distance but on the opposite side.
The antenna (Fig. 5, II) is essentially unchanged in form and setation. There
188
ANTHONY J. PROVENZANO, JR.
FIGURE 7. Calcinus tibiccn. The maxilla, zoea I through glaucothoe.
is a small tooth laterally on the basal portion in addition to the spine at the base of
the endopodite.
The mandible is unchanged to any notable degree.
The segmented endopodite of the maxillule (Fig. 6, II) is unchanged and the
coxal endite still has four feathered setae and three simple setae but the basal endite
now has four large teeth and two setae.
The maxilla (Fig. 7, II) has seven setae upon the proximal lobe of the coxal
endite and four setae on the distal lobe. Apparently there are five on the proximal
lobe of the basal endite and four on the distal lobe. The endopodite has two proxi-
mal and three distal setae. The scaphognathite has six or seven short plumose setae.
The first maxilliped (Fig. 8, II) has six plumose natatory setae on the exopodite.
The endopodite still has five setae on the terminal segment and there are two distally
LARVAL DEVELOPMENT OF CALCINUS
189
FIGURE 8. Calcinus tibicen. The first maxilliped, zoea I through glaucothoe.
190
ANTHONY J. PROVENZANO, JR.
G
FIGURE 9. Calcinus tiblcen. The second maxilliped, zoea I through glaucothoe.
LARVAL DEVELOPMENT OF CALCINUS
191
0.5 mm
IV
VI ^ G
FIGURE 10. Calcinus tibicen. The third maxilliped, zoea I through glaucothoe.
on the medial side of the penultimate segment. The antepenultimate segment bears
one seta medially and one laterally. The two proximal segments each bear one seta
laterally and two medially. There are eight or nine setae along the medial margin
of the basipodite in addition to the proximal hooked process.
The second maxilliped (Fig. 9, II) has six natatory setae on the exopodite. The
four-segmented endopodite bears five setae on the terminal segment and on each of
the two next proximal segments there are three setae, one of them laterally, two
medially. The most proximal segment has only two setae medially. There are
three setae on the medial margin of the basipodite.
192 ANTHONY J. PROVENZANO, JR.
The third maxilliped (Fig. 10, II) is a jointed uniramous appendage with five
plumose terminal setae.
Third Zoea. Figs. 1, III; 2, III.
Size. TL 3.0 mm.; CL 1.7-1.8 mm.
Duration. About five days, from approximately the 12th day after hatching to the
17th day.
Aside from increase in size there is no major change in form of the cephalothorax.
The fifth abdominal somite still bears a prominent medio-dorsal spine and a pair of
lateral spines. The sixth abdominal somite is no longer fused with the telson and
bears a postero-medio-dorsal spine as well as unjointed uropods (Fig. 3, III). The
exopodite of the uropod bears usually eight, sometimes seven, plumose setae and
has two inconspicuous hairs situated medio-ventrally while the endopodite of the
uropod is a simple unarmed bud. The telson is armed laterally with an unfused
hairless tooth. The second process as in earlier stages is an inconspicuously plumose
hair and medially to this third process is a feathered articulated spine. The fourth
process has become a large unarmed fused spine. Processes five to nine are articu-
lated plumose setae. The total count of telson processes is 9 + 9.
The antennule (Fig. 4, III) is segmented. The terminal segment bears two
large and three fine processes while the basal peduncle bears three large plumose
setae and two or three very small setules.
The antennal scale (Fig. 5, III) bears about 12 medial plumose setae. The
articulated endopodite has a single terminal seta. The armed spine and the hooked
tooth at the base of the endopodite and scale are still present.
The mandible has a few more very small teeth on the cutting edge.
The maxillule (Fig. 6, III) is essentially unchanged.
The maxilla (Fig. 7, III) appears unchanged except for the scaphognathite which
may have up to 11 plumose setae.
The first maxilliped (Fig. 8, III) has six natatory setae on the exopodite. The
five-segmented endopodite has four terminal setae and one proximal seta on the
ultimate segment. The penultimate segment bears a pair of setae medially. The
antepenultimate segment bears one medial and one lateral, while the next two
proximal segments each bear one seta laterally and one large and one small medially.
There are three groups of about three setae on the medial margin of the basipoclite
and two setae near the proximal hooked process.
The second maxilliped (Fig. 9, III) also has six natatory setae on the exopodite.
The four-segmented endopodite has on the distal segment four terminal setae and
one proximal seta. The penultimate segment bears two setae medially, one laterally
as does the antepenultimate segment, but the first segment bears only two medial
setae. There are three or four setae on the medial margin of the basipodite.
The third maxilliped (Fig. 10, III) bears five or six natatory setae.
Fourth Zoea. Figs. 1, IV; 2, IV.
Size. TL 3.8-4.2 mm. ; CL 2.0-2.4 mm.
Duration. Usually four or five days, rarely six.
The spines of the fifth abdominal somite have become relatively smaller, especially
the lateral. The telson armature (Fig. 3, IV) may be similar to that of the preced-
LARVAL DEVELOPMENT OF CALCINUS 193
ing stage but in six of seven specimens taken at random and examined, a very small
medial articulated spine was also present. The most readily noted characteristic <>!"
this stage is the changed armature of the now articulated uropods. The exopodite
of the uropod has a strong curved outer spine with nine or ten plumose setae along
the inner margin of the uropod. The endopodite hears four or five plumose setae.
The exopodite has two medio-ventrally placed very inconspicuous hairs ; the
endopodite has one.
The antennule (Fig. 4, IV) has its terminal segment ending in two large and
four small sensory processes. At the distal end of the proximal segment there are
four small setules and four large plumose setae.
The antenna (Fig. 5, IV) is basically unchanged but now there are 13-15 setae
in addition to the terminal tooth on the antennal scale. The endopodite still
terminates in a single seta.
The mandible shows no essential change.
The maxillule (Fig. 6, IV) has on the coxal endite three or four curved plumose
setae and a similar number of simple setae. The basal endite has usually five but
occasionally six strong teeth plus two setae. The endopodite is unchanged.
The maxilla (Fig. 7, IV) has from 9-12 plumose setae on the scaphognathite.
The endopodite bears four large setae and one smaller. The proximal lobe of the
coxal endite bears seven to eight setae ; the distal lobe, four. The proximal lobe
of the basal endite bears four ; the distal lobe also four.
The first maxilliped (Fig. 8, IV) still bears a prominent curved seta at the
proximal medial corner of the basipodite and distally there are two, three and two
setae along that margin. The exopodite bears six natatory setae. The five-seg-
mented endopodite has five setae on the terminal segment, a pair distally on the
penultimate segment, and one laterally, one medially on the antepenultimate segment.
There are two medial and one lateral setae distally on the next segment and again
on the most proximal segment.
The second maxilliped (Fig. 9, IV) also bears six natatory setae on the
exopodite. On the four-segmented endopodite there are five setae terminally, two
medially and one laterally on both the penultimate and antepenultimate segments and
only two medial setae on the proximal segment.
The third maxilliped (Fig. 10, IV) is a jointed uniramous appendage with
sometimes four, more usually five or six natatory setae.
Fifth Zoea. Figs. 1, V; 2, V.
Size. TL 4.3-4.9 mm. ; CL 2.3-2.7 mm.
Duration. Usually five days, occasionally four or six.
This stage differs little in gross appearance from the preceding stage, but the
length of the telson is now about equal to the width and there is an articulated medial
telson spine in most specimens, making the armature formula 9+1+9 (Fig. 3, V ).
The exopodite of the uropod retains the curved outer spine and now bears 10-11
plumose setae while the endopodite bears from six to seven. The exopodite bears
four inconspicuous setae ventrally.
The antennule (Fig. 4. V) has about four terminal sensory aesthetes. There are
usually three, rarely four large plumose setae distally on the basal segment and
194 ANTHONY J. PROYEXZAXO, JR.
another large seta with three or four very fine processes stilxlistally. A simple lobe
arises from the same distal area of the basal segment or peduncle.
The antenna ( Fig. 5, Y) is basically unchanged, the scale bearing 14-10 plumose
setae. The endopodite reaches two-thirds the length of the scale and terminates
with one or two processes.
The mandible is essentially unchanged, having added a few fine teeth.
The maxillule ( Fig. 6, Y ) has four curved plumose setae on the proximal endite
and four simple setae. On the distal endite there are five to seven strong teeth and
two setae. The endopodite is unchanged.
The maxilla (Fig. 7, Y ) bears 13-19 plumose setae on the scaphognathite. The
endopodite bears three plus three setae. The distal lobe of the basal endite bears
four setae, the proximal lobe five ; the distal lobe of the coxal endite bears four, the
proximal lobe 9—1 1 setae.
TABLE I
Characters of a "terminal" zoea VI and a "non-terminal" zoea VI
Larva no. 49 Larva no. 1 1 7
Leg buds very well developed very small
Pleopod buds present not present
Telson armature 9 + 1+9 10 + 10
Telson L/YY ratio 2.4/1.8 2.2/1.7
I'ropod setae
exopodite 13-14 12
endopodite 9 7
The first maxilliped (Fig. 8, V) has groups of about two, three, three and two
setae on the medial margin in addition to the curved proximal process and a single
seta near it. The exopodite bears six natatory setae, of which four are situated on a
partially distinct distal segment. The terminal segment of the endopodite bears five
setae. The penultimate segment bears two medial setae distally. The antepenulti-
mate segment bears one seta medially and another laterally. The two proximal
segments each bear two medial setae and one lateral seta.
The second maxilliped (Fig. 9, Y) has three setae on the medial margin of the
basipodite. The exopodite bears six or seven natatory setae. The endopodite is
similar to that of the preceding stage.
The third maxilliped (Fig. 10, V ) is uniramous and bears seven or eight natatory
setae. There may be the beginning of an endopodal lobe.
Sixth Zoea. Figs. 1, VI; 2, VI.
Size. TL 4.8-5.6 mm. ; CL 2.6-3.3 mm.
Duration. Usually four or five, rarely six days.
Larvae in the sixth zoeal stage may produce a glaucothoe directly or may moult
into a seventh zoeal stage. The degree of apparent difference between stage YI and
stage Y or between two stage YI larvae is related to the future fate of the larva.
For example, some characters of two stage YI larvae which died without moulting
are shown in Table I.
Larva no. 49, had it survived, probably would have produced a glaucothoe, judg-
ing from results with other larvae. Larva no. 117 would have produced another
zoeal stage, the form of which was distinguishable beneath the cuticle. In the
LARVAL DEVELOPMENT OF CALCINUS
195
present study three classes of stage VI larvae were observed but were not always
distinguished before termination of the experiment. Some moulted directly from
stage VI to the glaucothoe; some moulted from VI to VII and then to glaucothoe ;
some moulted from VI to VII to VIII and then to glaucothoe. The following
description of stage VI is based primarily on a series (no. 65) in which the
glaucothoe was produced directly from this stage.
The telson (Fig. 3, VI ) is distinctly longer than wide.
The antennule (Fig. 4, VI) bears three large and several smaller terminal
aesthetes. There are three subterminal pairs of processes. The lobe at the joint
of the terminal and basal segments extends nearly to the end of the terminal segment.
There are three small setae and four large plumose setae at the distal end of the basal
segment, with another large one (or two in some specimens) more proximally.
TABLE II
Characters of several stages in a series producing the glaucothoe after six zoeal stages.
(Larva no. 65)
Stage
III
IV
V
VI
Telson L/YV
1.4/2.0
1.8/2.0
2.1/2.0
2.4/2.0
Telson armature
9+9
9 + 9
9 + 1+9
9 + 1+9
lYopod setae
exopodite
8
10
12
14
endopodite
0
5
7
9
The antennal scale (Fig. 5, VI) bears 16 or 17 plumose setae while the endo-
podite which is now two-segmented exceeds the scale in length and terminates in a
single hair (but occasionally in three, of which two are small and inconspicuous).
The mandible is not changed notably. The bud of a mandibular palp could not
be distinguished in available material.
The maxillule (Fig. 6, VI) is unchanged with respect to the endopodite but the
basal endite has five apparently articulated and two non-articulated teeth and two
simple setae. The coxal endite bears four larger curved plumose setae and five
smaller simple setae.
The maxilla (Fig. 7, VI) upon the proximal lobe of the coxal endite bears 13
setae, on the distal lobe four. The proximal lobe of the basal endite bears eight,
the distal lobe four or five setae. The endopodite bears five setae and the
scaphognathite has about 19-20 plumose setae.
The first maxilliped (Fig. 8, VI) still bears the curved proximal process on the
basipodite with additional setae along the medial margin. The exopodite bears six
natatory setae. The five-segmented endopodite has the same arrangement of setae
as in the preceding stage.
The second maxilliped (Fig. 9, VI ) bears three setae on the medial margin of
the basipodite and the exopodite bears seven natatory setae. The armature of the
four-segmented endopodite is similar to that of the preceding stage.
The third maxilliped (Fig. 10, VI ) may lie uniramous in a long developmental
sequence. When the sixth zoea is the penultimate zoea in a series the basipodite
may be slightly swollen. When it is the last zoea. the third maxilliped may be
biramous, the endopodite being represented by a long lobe arising from the proximal
196
ANTHONY J. PROVKX/AXO, JR.
FIGURE 11. Calcimis tibiccn. The glaucothoe, lateral view (above) and dorsal view (below).
portion of the basipodite and bearing two or three setae. The exopodite in a termi-
nal stage VI usually bears eight natatory setae.
Zoeal stages VII and VIII.
These stages, both or the first of which were sometimes passed through prior to
attainment of the glaucothoe, are the result of non-uniform rates of internal growth
coupled with a more or less regular moulting cycle. There were no essential addi-
tions or changes from preceding stages except that where the preceding stages were
somewhat retarded as regards degree of setation, these later stages appeared to make
FIGURE 12. Calcimis tihiccn. The telson and uropods of the glaucothoe (G)
and the first crab (C).
LARVAL DEVELOPMENT OF CALCINUS
197
FIGURE 13. Calciinis tibiccn. Glaucothoe. Pi, P3, Pt, P3, first, third, fourth and fifth
pereipods ; tndl, mandible; p!2, p!3, p!4, pi,-,, the pleopods of the second, third, fourth and fifth
abdominal somites.
198 ANTHONY J. PROVENZANO, JR.
up for it. Thus in one stage VIII specimen while the telson ratio of 2.6/1.4 indi-
cated a slightly more elongate telson than in the final zoea of a series in which only
six zoeal stages were found, the same animal had a telson armature count of 1 1 + 1 1
processes and the uropods bore 14-15 setae on the exopodites, 9-10 setae on the
endopodites as in terminal stage VI. The antennal scale of a stage YIII may
also resemble that of a terminal stage VI, having 16 plumose setae. In a series with
eight zoeal stages the earlier stages at least as far back as stage III were somewhat
less advanced than the corresponding stage of a shorter series. Table II summarizes
changes of a few characters within one series.
Glaucothoe. Figs. 11, 12G, 13.
Size. TL 3.5-3.9 mm. ; CL 1.4-1.6 mm.
Duration. This stage was attained 32^1-0 days after hatching for larvae which
passed through only six zoeal stages, and after 39-42 days for larvae which
passed through eight zoeal stages. Most specimens of the 1961 series died
within 48 hours after attaining this stage. One glaucothoe lived for six days
until killed accidentally. In the 1960 series one specimen attained the glaucothoe
on the 37th day after hatching but lost one leg during transfer operations on the
47th day. Nevertheless the animal survived, regenerated the leg, and on the
57th day after hatching moulted to the first crab stage.
In C. tibicen the carapace has lost the postero-lateral spines and is now divided
into sections. The rostrum is blunt but well developed. The pereiopods are free
and functional, the chelipeds being subequal and the other legs symmetrical. The
abdomen is not quite twice the length of the cephalothorax. The abdomen bears
biramous pleopods on somites two to five, but only the exopodite of each carries setae.
The telson (Fig 12, G) is subrectangular, not indented as in the crab (Fig.
12, C), and may bear from 9-15 plumose setae terminally with two pairs laterally.
Glaucothoes attained after zoea VI had 11 setae, one attained after VIII had 15
setae along the posterior margin. There may be a few pairs of smaller setae medio-
dorsally on the telson as well. The uropods are changed somewhat from the preced-
ing zoeal stage and now bear corneous nodules along the posterior margin in addition
to plumose setae. The exopodite may bear from 20-23 plumose setae plus a few
very fine simple hairs. The endopodite may bear from 13 to 15 plumose setae as
well as a few simple hairs.
The eyes, together with the eyestalks, are about two times longer than wide and
are widest at the eyes. There are no ocular scales apparent. (Note : In the first crab
stage the eyestalk is wider than the eye and there is a simple, acute scale at the base
of each. )
The antennule (Fig. 4, G ) is now distinctly biramous with three segments com-
posing the internal or ventral ramus. The terminal segment may have up to nine
small setae, the others two or three. The external or dorsal flagellum which arises
from the same peduncular segment is composed of five articles. The most proximal
is unarmed, the terminal segment bears a few fine setae. The intervening segments
each bear a number of aesthetes.
The antenna (Fig. 5, G) reaches to the tips of the cheliped and has a scale at the
base very different from the zoeal scale. In the endopodite there are two peduncular
segments followed by about 10 flagellary segments, each of which bears a few short
setae.
LARVAL DEVELOPMENT OF CALCINUS 199
The mandible (Fig. 13, mdl) has a cuplike grinding surface and a three-seg-
mented palp, the distal end of which hears 12-13 short setae.
The maxillule (Fig. 6, G) has lost the segmentation of the endopodite which
now appears as a simple palp hearing a few setae. The basal endite bears a greatly
increased number of stout spines and setae, about 17-21, distally and a pair proxi-
mally. The coxal endite also bears a greatly increased number of setae.
The maxilla (Fig. 7, G) bears a double row of setae on the proximal lobe of the
coxal endite, about seven on the distal lobe. There is a large number of setae on
the lobes of the basal endite but the endopodite no longer bears any setae. The
scaphognathite is very well developed and has approximately 65 short plumose setae.
The first maxilliped (Fig. 8, G) is radically changed. The exopodite is short
and broad, bearing seven plumose setae along the exterior margin. The endopodite
is unsegmented and is devoid of setae. The basipodite appears bilobed and bears
about 18 setae on the distal lobe and about six on the proximal lobe.
The second maxilliped (Fig. 9, G ) is least changed of all the mouthparts. The
exopodite is clearly unsegmented distally.
The third maxilliped (Fig. 10, G) is biramous and bears about six to eight
plumose setae terminally on the exopodite. The ultimate and penul Jmate segments
of the endopodite are heavily armed with setae, some of which bear setules. The
antepenultimate segment has a row of distal setae while the proximal segment bears
only three or four simple setae. The ischium has a row of fine tubercles or teeth
along the medial margin.
The chelipeds are about equal in size, each with a few setae. The second and
third pereiopods (Fig. 13) have the dactyli about half the length of the propodi.
There are a few setae on the dactyl and four horny spines ventrally in addition to the
corneous terminus. There are setae and a couple of short spines distally on the
propodus. The fourth pereiopod is non-chelate and the propodus has a double row
of about 15 corneous granules on the latero-ventral surface. The fifth pereiopod
bears granules on both dactyl and propodus and very long setae are present on these
segments.
The pleopods are biramous, the inner ramus being unarmed. The pleopod of the
fifth somite is shorter than the preceding ones. The number of setae is eight on the
pleopod of the second somite, nine on the others.
DISCUSSION
The first stage larva attributed by Bourdillon-Casanova (1960) to Calchms
ornatns (Roux ) bears very close resemblance to that described by Pike and William-
son (1960) from a laboratory hatching. There are minor differences in that the
antennal scale has 1 1 plumose setae and the short seta of the antennal endopod is
half as long as the others according to Bourdillon-Casanova, whereas Pike and
Williamson reported nine plumose setae on the scale and the short seta as one fourth
as long as the others. If these are in fact objective differences they may well be
within range of variability for a single species as indicated in the present paper, but
evaluation of such characters is hampered by the general lack of detailed descrip-
tions of hermit crab larvae. The antennule of C. ornatns was not figured by Pike
and Williamson, but Bourdillon-Casanova showed it terminating with three aesthetes
and three setae in addition to a large plumose subterminal seta. In C '. tibiccn I was
200 ANTHONY J. PROVENZANO, JR.
able to distinguish with certainty only one aesthete and four other processes which
may have been aesthetes.
The combination of prominent postero-lateral carapace spines and the presence
on the fifth abdominal somite of a single medio-dorsal spine and a pair of lateral
spines is common to both species and may be unique to the genus, but Pike and
Williamson also attributed an unidentified larva lacking the fifth abdominal medio-
dorsal spine to Calchuts. In the first zoea of C. ornatus the medio-dorsal spine of
the fifth abdominal somite is shorter than the pair of lateral spines, whereas in C.
tibicen it is distinctly longer. The very small lateral spines or teeth on the second,
third and fourth abdominal somites of C. ornatus were not distinguished in C. tibicen.
A character of questionable status is the endopodite of the maxillule which ac-
cording to Pike and Williamson is two-segmented in C. ornatus and which in other
Diogenidae never has more than two segments. In C. tibicen this endopodite ap-
peared to have three segments in the specimens for which dissection of the appendage
was successful. Hart (1937) reported three segments for the maxillulary endo-
podite of Paguristes turgidus and Orlamunder (1942) showed three segments for
Birgus latro. Coenobita clypcatus also has been shown to have a three-segmented
endopodite (Provenzano, in press). Both these latter species are members of the
family Coenobitidae, presumably derived from the same line as the Diogenidae to
which Calcinns belongs.
Comparison of later zoeal development of C. tibicen with that of other species of
Calcinns is restricted to the work of Pike and Williamson. Those authors obtained
their first stage larvae from a laboratory hatching and the subsequent stages from the
plankton. They did not illustrate completely each stage including appendages but
did describe certain essential features of each stage. Size of first stage larvae is
similar for the two species, but the fifth and sixth zoeae of C. tibicen are slightly
larger than the fifth zoea of C. ornatus. Other characters mentioned by those
authors for later zoeal stages do not differ significantly from those of C. tibicen, with
the exception that the postero-medio-dorsal spine on the sixth abdominal somite of
C. tibicen is not certainly indicated in the illustrations of the later stages of
C. ornatus. In both species the telson becomes more elongate during zoeal
development.
The glaucothoes of the two species are very similar in gross appearance and this
similarity may extend to details. Appendages are not illustrated or described in
detail by Pike and Williamson but Bouvier (1922) for Glaucothoe griinaldi gave
enlarged figures of the first, second, and fourth pereiopods, the antennule and an-
tenna and the tail fan in addition to a figure of the whole animal. Between that
form and the glaucothoe of C. tibicen there are a few minor differences which if later
confirmed may prove to be significant at the specific level. Such differences include
number of spines on the ventral margin of the ambulatory dactyl and relative lengths
of some antennal segments, as well as certain setation. In all important respects,
however, Glaucothoe griinaldi resembles the two glaucothoes certainly known for
Calcinus and in fact, as Pike and Williamson have already indicated, Bouvier's form
may belong to C. ornatus. Pike and Williamson stated that the glaucothoe of C.
ornatus has the pleopods (each?) armed on the outer ramus with nine plumose
setae, where in C. tibicen there are only eight on the pleopod of the second abdominal
somite, nine on the others.
Insofar as the description and illustration of C. ornatus and Glaucothoe griinaldi
LARVAL DEVELOPMENT OF CALCINUS 201
indicate, the similarities between glaucothoes of several species may be much more
significant than any differences. Until now glaucothoes of two species within one
genus of Diogenidae have not been compared in detail. Rather the few descriptions
available were for species of hermit crabs in different genera, and differences between
most of these seemed to be quite distinctive. Whether those differences were generic
in significance or merely specific could not be ascertained, especially since in
Paguristes, the only diogenid genus for which larvae of more than one species were
known, there seem to be notable differences between the larvae. Recently it was
shown that zoeae and glaucothoes of two species of Coenobita are more similar to
each other than to those of other genera of hermit crabs (Provenzano, in press).
The present study also confirms that two species of a genus of diogenid hermits even
as larvae bear more resemblance to each other than to members of other genera, a
long accepted hypothesis for which there has been little direct evidence.
SUMMARY AND CONCLUSIONS
1. Larvae of Calciinis tibiccn (Herbst) were reared in the laboratory from
hatching to the post-larva on a diet of Artcmia nauplii. Temperatures ranged from
26° to 30° C. Salinity in one experiment fluctuated between 31 ppt and 35 ppt and
in the second experiment salinity was maintained between 35.5 ppt and 37.1 ppt.
Under these conditions the zoeal phase of development was completed in 32 to 42
days. The number of zoeal stages in the laboratory development of this species is
variable, the glaucothoe being attained after six, seven or eight zoeal stages.
2. Six zoeal stages and the glaucothoe have been described and illustrated. The
only two species of Cold nits for which zoeae are certainly known share the appar-
ently unique features of a pair of prominent submarginal postero-lateral spines on
the carapace and a medio-dorsal and pair of lateral spines on the fifth abdominal
somite. The species apparently differ in that C. tibiccn has the postero-medio-dorsal
spine of the fifth abdominal somite longer than the laterals, a postero-medio-dorsal
spine is present on the sixth abdominal somite of zoea III and older larvae, the
maxillulary endopodite is three-segmented, and there are no minute lateral spines
on abdominal somites two to four.
3. Adequate comparisons of the zoeal appendages and glaucothoe characters of
C. tibicen with those of other species are not possible until more detailed descriptions
are available, but the information which has been published indicates close similarity
of larval characters of species within a genus.
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ELECTRICAL INDUCTION OF SPAWNING IN TWO MARIXK
INVERTEBRATES (URECHIS UNICINCTUS,
HERMAPHRODITIC MYTILUS EDULIS)
YASUO SUGIUKA
Misaki Senior Hii/h School, Miitra-Shi, Kanagawa Prefecture, Japan
The investigations of Iwata (1950) and Harvey (1952) have proved electrical
stimulation to he a very effective means for inducing spawning of sexual cells in sea
urchins. Recently, Kuhota (1962) has reported the same effect in an insect
(dragon-fly ). Inspired hy their success, the author tested the applicability of this
method to various shore invertebrates, two successful cases of which will be briefly
reported.
I. Urccliis nnicinctits
MATERIALS AND METHODS
By the ordinary practice, for obtaining reproductive cells of Urcchis, either the
body wall must be cut open or the eggs must be collected directly from the opening
of the gonoduct by a fine pipette (Newby, 1932). When the electrical method is
used, it is possible to obtain with ease any desired quantity of reproductive cells, and
this can be applied repeatedly to the same individual, if necessary, since spawning
lasts only during the stimulation. Moderate stimulation of the adults causes no
harmful effects in the later development of the gametes.
The spawning season of Urcchis itnicinctus is said to be from October to March
(Hiraiwa and Kawamura, 1936; Ohkawa, 1958; Sakiyama, 1958). At Kisarazu,
Chiba Prefecture, where this study was performed, the rate of occurrence of sex-
ually mature individuals remains fairly high even in April. Twenty animals
(about 10 cm. in length in contracted state) were used.
One individual at a time is placed in a vessel (155 mm. in diameter, 33 mm. in
depth) filled with sea water, and a pair of Ag-electrodes (1 mm. in diameter) are
dipped vertically into the sea water 20 mm. apart across the body axis of the worm.
The temperature and the specific gravity of the sea water are adjusted to 16.0-20.0°
C. and 1.0209-1.0232, respectively, and alternating current (30 volts) is applied for
various periods.
RESULTS
As soon as the stimulation is applied, the animal contracts quickly and in many
cases a large swelling appears in the front of the body. About 4-20 seconds after
stimulation, the animal begins to discharge reproductive cells forcibly through the
openings of the gonoducts (Fig. 1). There is no conspicuous difference between
females and males in their reaction to electrical stimulation.
On cessation of stimulation, the animal relaxes and after a while spawning also
stops, a mass of sexual cells being deposited on the bottom of the vessel. A con-
203
204
YASUO SUGIURA
siderable amount of eggs or sperm can be obtained by a single discharge ranging
from 5 to 35 seconds.
Over-stimulation lasting more than 30 seconds should be avoided, since this
causes prolonged contraction or occasionally even death. The time required for
recovery from the stimulation-induced contraction varied widely, from several
minutes to several hours. Intermittent stimulations of very short duration seem
to act excessively : after three stimulations of 6 seconds each, given 3-5 minutes
apart, although all the animals discharged, 20% of them failed to recover from
contraction.
FIGURE 1. Urcchis iinicinctits spawning under electrical stimulation.
If due caution is paid to the duration of, and the intervals between, stimulations,
one to two stimulations a day can be given for 6 consecutive days to animals fully
charged with reproductive cells. In one case, animals which had been kept for 11
days in captivity and made to spawn nine times, still discharged eggs which
developed on insemination, to normal trochophores.
II. Hermaphroditic i\f \Hlus cdulis
MATERIALS AND METHODS
Mytiliis cdulis is normally dioecious, and hermaphroditic individuals can be
found only very rarely. Among some 400 individuals examined by the author at
ELECTRICAL INDUCTION OF SPAWNING
205
Kisarazu, Chiba Prefecture, four hermaphroditic mussels were met with (Fig. 2).
They were detected by the colour of the gonads and the condition of the follicles, and
further checked by smear preparations of the gametes.
With respect to the induction of spawning hy electrical stimulation of Mylilits,
Iwata's report is availahle (1949). In 1951, he further showed that discharge could
he induced in excised parts of the mantle. Since i\Iytilits eggs are unfertilizahle as
taken from excised ovaries, requiring to he at least quasi-normally spawned, this
method of Iwata's was used to obtain gametes from such hermaphroditic individuals.
Pieces of the mantle about 5 mm.2, containing either ovary or testis, were cut out
of a hermaphroditic individual. Each piece was stimulated separately by 40 volts
a. c. for 15 seconds with a pair of Ag-electrodes 1 mm. in diameter, placed 20 mm.
FIGURE 2. Hermaphroditic gonad.
apart in a vessel of 72 mm. diameter filled with sea water to a depth of 5 mm. After
a latent period of about 30 minutes (19° C.), eggs and sperm were spawned; these
were used for cross-fertilization in various combinations. In no case did eggs to
which sperm was not added show any development, indicating that they were not
contaminated with sperm during spawning.
RESULTS
Self- and cross-fertility
Separately obtained gametes were mixed in various combinations and the per-
centages of resulting fertilization were counted. The results are summarized in
Table I. Later development of such zygotes was followed; in all four hermaphro-
ditic individuals, self-fertilization zygotes were found to develop at the normal pace,
and the embryos showed no difference whatsoever from out-bred controls.
Gametes were sometimes obtained from an ovary and a testis situated in the
mantels of opposite valves and in other cases they occurred together on the same side;
206 YASUO SUGIURA
I AHI.K I
l'\'i'tilizul>ililv of ^uncles from hermaphroditic mussels
Combination Fertilization percentage
Herm. egg X herni. sperm 92.8%
Normal egg X henn. sperm 94.0%
Herm. egg X normal sperm 93.2%
Normal egg X normal sperm 96.0%
in both cases the results were identical. In one mussel, a piece of the mantle con-
taining gonads of both sexes was stimulated and normal larvae were obtained
without further insemination.
These facts lead to the conclusion that the gametes of hermaphroditic Mytilus
cdulis are capable of self-fertilization which produces perfectly normally developing
larvae.
The author wishes to acknowledge his indebtedness to Prof. K. Dan of Tokyo
Metropolitan University for his encouragement and advice during this work. This
research was carried out when the author was at the Kisarazu Fisheries Laboratory
of Tokyo University of Fisheries. Thanks are due to Prof. M. Katada, director of
the Laboratory, and Mr. I. Shimizu of Funabashi Senior High School, who
cooperated with the author in this work.
SUMMARY
1. Electrical stimulation of spawning was tried on Urcchis iinichictus and
hermaphroditic M \tilns cdulis.
2. Urcchis reacts immediately, and if over-stimulation is guarded against,
samples can be repeatedly obtained from the same individual, which would be very
advantageous for some kinds of experiments.
3. Excised pieces of hermaphroditic Afytihts were stimulated electrically to
spawn ; the gametes so obtained showed perfect self-fertility.
LITERATURE CITED
HARVEY, E. B., 1952. Electrical method of "sexing" Arbacia and obtaining small quantities of
eggs. Biol. Bull,, 103: 284.
HIKAIWA, Y., AND T. KAWAMURA, 1936. Relation between maturation division and cleavage in
artificially activated eggs of Urcchis unicinctiis. Biol. Bull.. 70: 344-351.
INVATA, K. S., 1949. Spawning of Mytilus cdulis. (2) Discharge by electrical stimulation.
Bull. Jap. Sue. Sci. Fish., 15: 443-446. (in Japanese)
IWATA, K. S., 1950. A method of determining the sex of sea urchins and of obtaining eggs by
electric stimulation. Annot. Zoo/. Jap.. 23: 39-42.
IWATA, K. S., 1951. Spawning of Mytilus cdulis. (5) A method to obtain mature eggs from
mantle piece. Bull. Jap. Soc. Sci. Fish., 17: 15-18. (in Japanese)
KI-BOTA, T., 1962. An electrical method of forcing the damselfly (Odonata) to oviposit. Zool.
May., 71. (in Japanese)
NEWBY, W.W., 1932. The early embryology of the echiuroid, Urcchis. Biol. Bull., 63: 387-399.
OHKAWA, M., 1958. Studies on the fertilization in eggs of the echiuroid worm, Urcchis
i/iiicinctiis. (3) On the growth of egg-cells suspended in the body fluid. /. Yokohama
Municipal Univ. Scr. C-25, 95: 1-13. (in Japanese)
SAKIYAMA, F., 1958. Rearing experiments of Urcchis larvae. Nat. Sci. Rep. Ochanomisu
Univ., 9: 47-56.
SOME OBSERVATIONS OX THE GENERAL BIOLOGY OF THE
LAND CRAB, CARDISOMA GUANHUMI (LATREILLE),
IN SOUTH FLORIDA '
CHARLES A. GIFFORD
The Institute of Marine Science, University of Miami, Miami 49, Florida
Land crabs of the genus Cardisoma, Family Gecarcinidae, are an important ele-
ment of the fauna of many tropical coastal and estuarine areas. The genus is
circum-equatorial, with different species on the east and west coasts of each continent.
Cardisoma gnanhnmi was described by Rathbun (1918). Its range includes the
east coast of America, from Florida to Brazil, and the Caribbean Islands.
Despite the large numbers in which Cardisoma occurs in or near many densely
populated areas, and its spectacular colors, migrations, and swarming, it has been
largely neglected by zoologists. Brief descriptions of its ecology and behavior were
given by Pearse (1915 ) and by de Oliviera (1946). Pearse (1934) found its blood
to be hypo-osmotic to normal sea water. The eggs and first zoeal larva of the \Yest
African species, C. aniiatiini, were described by Cannon (1923). Taxonomic
descriptions and reports of occurrence are more frequent and sometimes include brief
notes on habits and ecology.
Some aspects of the physiology of blood regulation of C. guanhumi have been
studied in this laboratory and will be published elsewhere. The following observa-
tions were made during the collection and maintenance of a stock of animals in the
laboratory. Since Florida is at the northern limit of the animal's range, these
observations may not apply to the species as a whole.
GENERAL DESCRIPTION
C. guanhumi was described in detail by Rathbun (1918). As an adult it some-
what resembles Lea in general shape; its eyes are widespread, large and on fairlv
long stalks, and one cheliped of adult males is enlarged. Cardisoma. however, is
much larger than Uca. Adult males weighing 500 grams, with carapace widths of
10-11 cm., are not uncommon. Crabs weighing 4-5 grams are also common,
although more difficult to capture.
HABITATS
Geographic range
C. guanhumi has been seen by the author, or reported to him by competent ob-
servers, from Yero Beach on the central east coast of Florida around the tip of the
peninsula, in the Florida Keys, and along the Gulf coast as far north as Tampa. It
1 Contribution No. 396 from The Marine Laboratory, University of Miami.
This study uas supported in part by Grant No. NSF G-7044 from the National Science
Foundation.
207
208 CHARLES A. GIFFORD
was reported from Louisiana (Behre, 1949), and has been seen l>y the author at
Ivockport, Texas. It has IK it been seen more than eight kilometers inland.
Salinity raiujc and tolerance <>j desiccation
In south Florida the salinity range of the surface water nearest to Cardisoma
burrows varies from fresh water (Cl -- 0.4 mM/L.) to slightly concentrated sea
water (Cl about 600 m.U/L.). This range, and its effect on blood composition, will
be discussed in greater detail in later papers. Crabs have been reported on the
ocean bottom several hundred meters offshore in the Florida Keys (L. Greenfield,
personal communication). During extended droughts crabs living inland feed on
plants on the bottoms of fresh-water drainage ditches and canals. Individual Cardi-
soma have been kept completely immersed in a running sea water aquarium for six
months, but tolerance of extended immersion in large volumes of fresh water has
not been tested.
When permitted, Cardisoma spends most of its time out of water. In a box
containing a shallow pan of water, C. gitanlnimi will approach the pan, dip its small
chela in the water, then touch the moistened chela to the borders of the buccal cavity
and the maxillipeds. Over the salinity range from tap water to 150% sea water, the
crab then enters the pan, lowers its body until the ventral margin of the carapace is
immersed, and fills its gill chambers. It then leaves the pan, and within a few
minutes drains the gill chambers by elevating the front of the carapace. The latter
is firmly attached posteriorly, but it is free to lift slightly at the anterior end, an
action which allows most of the water to drain from the gill chambers. A recording
device measuring the frequency and duration of such immersions by a crab given
access to 0.5% sea water showed that most occurred at about the time of sunrise
and sunset. The average time of immersion was about two hours per day under
the conditions of the test.
Cardisoma can live for many days in moist air, but only for about two days under
severe desiccation. A group of eight crabs contained 60-70% water (on original
weight basis) and lost 10-15% of their original weight at the time of death by
desiccation.
Colonies
In south Florida Cardisoma lives in two major types of colonies. In one of
these, the burrows are located in flat ground not immediately adjacent to free surface
water. These may be either among mangroves, in open fields of tall grass, or in
open hardwood groves. Among mangroves the burrows are usually on ridges of
comparatively high ground. The burrows extend to ground water, which may be
from one-third to two meters below the surface. The upper part of the burrow is
generally vertical or nearly so. In local colonies of this type, burrow density may
average one per square meter over areas of several hectares. These colonies are
generally within half a kilometer of some kind of free surface water. Cardisoma
burrows have been found in many types of soil, but the largest colonies of this type
are found in Perrine marl (described by Gallatin, 1958).
In the other type of colony, the burrows are located in the banks of drainage
ditches or canals, near the edges of fresh-water streams or ponds, or in hard soil near
GENERAL BIOLOGY OF THE LAND CRAB 209
salt water. The burrow can be either in the vertical face of the bank extending
horizontally into it, or on level ground immediately adjacent to the bank, when the
burrow tends toward the vertical. Burrow density frequently exceeds one per
square meter in this type of colony. Quite often the burrows are as close together
as they can be without inter-connecting or collapsing.
Both habitats are shared with other animals. Raccoons (Procyon lot or) are
common in the first type, and may be an important predator of Cardisoma. Gray
squirrels (Citellus carolinensis'), rabbits (Sylvilagus sp.), and rats (Rattits sp. )
also occur there. The blue crab, Callincctcs sapidus, is occasionally seen in the
ditches, as are prawns of the genera Palaetnontes and Macrobrachium, and the cray-
fish, Procambarus allcnii. On the seaward border of its range Cardisoma is
sympatric with several species of Uca, and its habitat may border that of Ocypode
alb leans.
LIFE CYCLE
Mating, oi'iilation, and fertilization
The time, place, and manner of copulation are unknown. The sperm are carried
by the female in a spermatheca, and fertilization is internal. In seven animals the
egg mass weighed 11.9% (±2.2) of the body weight. In five females the egg
cluster, just prior to spawning, contained 19,000 to 20,000 fertilized eggs/gm. of egg
cluster. A female weighing 160 grams would thus release about 370,000 eggs at
each spawning. The diameters of ten eggs ranged from 430 to 440 mu,
The spawning period extends from June or early July to December, with a peak
in October and November. Spawning occurs in waves which appear to have a lunar
or twice monthly rhythm. These are described below, under migrations. A more
detailed report of the female reproductive cycle is in preparation.
Adult females generally change color from blue to yellowish-white, at about the
time of the first ovulation of the season, and the lighter color generally persists
through the season. The females which have spawned are thus effectively tagged.
Ovaries of such crabs, captured between the July and August, 1961, spawning
periods, were again beginning to mature, suggesting that individual females may
spawn more than once during a single season.
Cyclic changes in the male reproductive system are either absent or less pro-
nounced than the female.
Fertilized eggs were carried on abdominal appendages for ten days by one female
crab which ovulated in captivity. When first deposited, the egg mass is black, com-
pact, and shiny. As development proceeds, it becomes loose and ragged, and light
brown in color. The embryos, by then, have reached the pre-zoea or zoea stage.
At this time the females generally start migrating to salt wrater to shed their young,
seldom traveling more than five to six kilometers. This distance must be accom-
plished in one night, or at most two, because if ovigerous females at this stage are
captured and held overnight, the egg mass usually disintegrates. The females
seek cover near the water's edge and periodically enter the water and release the eggs
or larvae by rapid fanning movements of the abdomen.
Attempts to rear the larvae have been unsuccessful, and the foods suggested by
Costlow and Bookhout (1960) do not sustain development. The larvae may ingest
yeast cells or other microorganisms and produce fecal pellets or strings, but only a
210
CHAKLKS A. GIFFORU
few have survived the first molt. The survivors spent 5-7 days in the first zoeal
stage. Cannon's drawings (1923) of the eggs and first zoeal stage of the West
African species, C. armatnm, closely resemble the corresponding stages of C.
giianliuini.
Attempts to hatch C. guanlutini eggs in drainage ditch water brought to the lab-
oratory were unsuccessful. Only a few eggs hatched, and these were pre-zoeal
which soon died. When tested in increments of 10% sea water, both hatching and
survival improved with increasing salinity. All eggs hatched in 40% sea water
(diluted with drainage ditch water), but the larvae lived only a few days. The
percentage of eggs which hatched declined progressively in salinities over 50% sea
water, but the larvae survived longest (7-10 days) in 80-90% sea water. This was
also true of larvae hatched in 40% sea water and transferred to 90% sea water. The
C. guanhumi — Chelae
Minor
FIGURE 1. Shapes of major and minor chelae of C. guanhumi. Distribution of types of
major chelae with growth stage is explained in the text.
main spawning period occurs at the height of the rainy season, when littoral salinity
may be decreased by run-off. The ability of the larvae to hatch and survive in
brackish water may thus have adaptive value.
The duration of the larval period is unknown, but it may be several months. In
May, 1961, a group of very small crabs (<5 grams) appeared on an exposed drain-
age ditch bank a kilometer or so inland. Ovigerous females had not been seen
since December, 1960, five months previously. It would appear that it had taken
these specimens at least five months to develop from zoea larvae to five-gram crabs,
or to have colonized the area from some other center.
Post-larval life
In its growth from small crab to adult, Cardisouia undergoes a series of morpho-
logical changes. Post-larval life can be divided into three stages, juvenile, transi-
tional, and adult on the basis of the following characteristics : ( 1) shape and size of
the major chela; (2) shape of the carapace.
Juvenile: In the smallest crabs captured (<5 grams), the- sexes can be differen-
tiated only by retracting the abdomen, at this stage resembling that of the adult male,
and identifying the female genital pores on the under surface of the third segment of
GENERAL BIOLOGY OF THE LAND CRAB
211
the cephalo-thorax, or the male copulatory organs on the first abdominal segment.
The chelipeds are unequal in both males and females, with the major one resembling
that of the adult male (see Fig. 1). The minor chelipeds are similar in the two
sexes, but as growth proceeds the major ones come to differ in both size and shape.
As is shown in Figure 2, the shape of the carapace changes with growth. The
ratio, carapace width/carapace length is fairly constant throughout life (C.W./C.L.
: 1.222 ± 0.022 for 73 male and female crabs weighing from 3 to 500 grams), but
the junction between dorsal and lateral surfaces becomes rounded and the gill
chamber covering bulges out farther to the side. This change in shape is further
5 gms.
150 gms.
5 cm.
-
\
.450 gms.
C.guanhumi- Carapace Shape
FIGURE 2. Differences in carapace shape of male C. giianhwni of different live weights. The
carapace is often asymmetrical, bulging out farther on the side toward the major cheliped.
illustrated in Figure 3, in which the ratio of carapace width to orbital width (the
distance between the tips of the innermost of the two epibranchial teeth projecting
from the lateral borders of the orbits) is plotted against the logarithm of weight.
Mean values of this ratio for both male and female crabs increase linearly from 1.12
at five grams to about 1.18 at forty grams.
At succeeding molts the female abdomen widens, becoming first triangular and
then semi-elliptical, and the ratio C.W./O.W. increases. By the time the body
weight reaches 40 grams the sexes can be distinguished by abdominal shape. The
chelipeds of both sexes still resemble those of the adult female. The junction of the
dorsal and lateral carapace surfaces remains angular, and is defined by a ridged
suture. Attainment of 40 grams live weight marks the end of the juvenile period.
Color also changes with growth ; these changes are described below.
Transitional: The beginning of the transitional period, at about 40 grams live
weight, is marked by the beginning of major sexual dimorphism, and by an abrupt
212
CHARLES A. GIFFORD
increase in the width of the carapace compared to orbital width (Figs. 2 and 3).
Below 50 grams the mean values of the ratio C.W./O.W. are similar for males and
females. Between 50 and 200 grams the mean values of C.W./O.W. are con-
sistently higher for males, although standard deviations overlap. Above 200 grams,
values for both sexes are erratic and displaced above the lines established between 50
and 200 grams.
1.40
i
Q
^1.30
H
00
cr
o
UJ
§1.20
1.12
o o o
°o &>
o A
o A
o A
I
I I I
10 20 50
LIVE WEIGHT(gms)
100
200
500
FIGURE 3. The ratio, carapace width/orbital width, plotted against log live weight. Circles
represent mean values for from 7 to 22 males in 10-gm. weight increments. Triangles represent
similar groups of females. Standard deviations averaged ± 0.022 for all groups. Below 50 gm.
both sexes are represented by circles.
Other morphological changes which occur in the transitional period are a gradual
rounding of the junction of the dorsal and lateral carapace surfaces, the loss or
diminution of the second epibranchial tooth (this occurs earlier and more consistently
in males), and, in the male, the enlargement and change of shape of the major cheli-
ped. Transitional males sometimes have identical chelipeds, or, if they are unequal
in size, the major one may have the same shape as the minor. The range of shapes
is shown in Figure 1. The major cheliped of the male is generally recognizably
different from that of the female when body weight reaches 120 grams, but great
enlargement of it is rare in crabs weighing less than 200 grams.
Adult: There is no sharp morphological change from transitional to adult. The
major change in outward appearance is in color, which is described in a later sec-
GENERAL BIOLOGY OF THE LAND CRAB
213
tion. The morphological changes described above are gradual, and, from the
limited observations made, do not occur in any particular order. Females can
attain sexual maturity and spawn at any weight over 40 grams. The size at which
males achieve sexual maturity is unknown.
Handedness
The major chela can occur on either the right or left side of the animal. Of 562
crabs examined, 302, or 53.7%, were right-handed; 244, or 43.4% were left-handed,
and 16, or 2.1% had equal-sized chela.
Molting
The difficulty of ascertaining the molting habits of Cardisoma has been mentioned
by Schmidt (1934). Although empty carapaces are often found in the crab
TABLE I
Distribution of color phases in different weight and sex groups of Cardisoma guanhumi
(Total number of crabs examined: male, 454, female, 309)
Color Phase
(Number of cases)
Juvenile
Transitional
Adult
Weight
(gm.)
Male
Female
Male
Female
Male
Female
0-39.9
27
20
0
0
0
0
40.0-79.9
0
0
0
12
4
5
80.0-129.9
0
0
7
8
28
38
130.0-189.9
0
0
2
23
52
125
Over 190.0
0
0
0
0
325
78
colonies, no intact cast shell has been seen. One juvenile crab has been forced
to molt in captivity by removal of four pereiopods followed by continuous feed-
ing. Destalked crabs have not lived more than three weeks after operation, and
none of these has shown any outward sign of approaching molt before death.
One crab with an uncalcified carapace, but with hardened mandibles and legs, was
captured as it left its burrow and entered a salt water pond. Many "new-looking"
crabs, with bright colors, unscratched carapaces, and no missing appendages, were
seen when the crabs first became active in the spring of 1961. Molting probably
occurs in the depths of the burrow.
Coloration
In juvenile C. guanhumi the dorsal surface of the carapace is tan or brown with
scattered small purple spots over the visceral mass. The orbits and eyestalks are
purple, and the sides of the carapace and the tips of the chelae are white. The walk-
ing legs and the proximal segments of the chelipeds can be any combination of light
blue, pale orange, and tan. Transitionals are similar to juveniles, but the colors are
214
CHARLES A. GIFFORD
more intense. The orbital purple hand extends laterally and posteriorly over the
gill chambers, and down over the sides of the carapace. The middle segments of
the pereiopods are purple. In both juveniles and transitionals, the ischium of the
third maxilliped is white and all the more distal segments are bright orange red. An
orange band frequently extends around the lower edge of the carapace, especially
at its posterior margin, and over the proximal ends of the meri of the walking legs.
Adults are generally blue, but may be mottled blue and white or blue and dull
yellow, or any shade between completely white and completely dull yellow. The
chelae are white or yellowish-white. The maxillipeds are blue, white, or yellow,
matching the rest of the crab.
TABLE 1 1
Sex and color distribution of Cardisonia guanhumi collected near the bay shore during a
migration, August 30, 1961
Color
Male
Female
No. of crabs
% of males
% of total
No. of crabs
% of females
% of total
Blue
121
94.5
39.4
121
67.7
39.4
White
1
0.8
0.4
32
17.9
10.4
Purple
Brown
5
1
3.9
0.8
1.6
0.4
22
4
12.3
2.2
7.2
1.3
128
41.8
179
58.3
The distribution of color stages of C. guanhumi with weight and sex is given in
Table I, and the distribution of color in a group of migrating crabs is given in
Table II. Most crabs develop the adult color pattern by the time they weigh 80-90
grams, but some do not until they reach 180 grams. A higher proportion of these
are female. Females generally have the adult color scheme before reaching sexual
maturity.
These color patterns are achieved by different combinations of the effects of
pigments embedded in the shell and of epidermal chromatophores. Briefly, juveniles
use both means of coloration, shell pigments are more important in transitionals, and
adult coloration is exclusively due to pigments in epidermal chromatophores.
The pigment of the shell occurs just below the epicuticle. In histological sections
this layer stains differently than the rest of the shell. Two types of shell pigment
have been observed in C. guanhumi: a continuous brown, orange, or yellow layer
most prominent in juveniles, and a stratum of discrete round or irregularly shaped
spots of purple or violet pigment, which may or may not be superimposed on the
brown continuous layer. The latter is most evident on the carapace of transitionals.
Where the two types occur together, as on the dorsal surface of the carapace of
juveniles and transitionals, the purple spots extend farther into the shell than the
brown layer does. In the orbits and on the eyestalks of juvenile and transitional
crabs the purple spots occur alone, and merge into a continuous layer. These pig-
ments are absent in adults. Since a series of shells has been found containing
gradually decreasing amounts of pigment, more than one molt may be required
for their removal.
GENERAL BIOLOGY OF THE LAND CRAB
215
Monochromatic red and black chromatophores can be seen in the pereiopods and
gill chamber linings of juvenile C. guanhumi. These are embedded in a diffuse
layer of white pigment that may or may not be in chromatophores ; it occurs in fine
filaments, but has not been seen to contract. In intact crabs in daylight both colors
of chromatophores are contracted. The red chromatophores are much smaller than
the blacks, and about six times as numerous. In darkness, or following eyestalk
removal, both types expand, but even when fully expanded the blacks do not
overlap.
IO3
I960 , June
0
July
0
Aug.
0 O
Sept. Oct.
'0
Nov. Dec.
b b b
IO2
—
Spawning
10
10
-
y
1
K
No ._,)
Data
/ 1 /
_
A
Non- spawning
c»
o •"
o
O
O
O
o o
0 0
*,o»
0)
EIO2
•2
—
1
Spawning
•o
O)
JlO
!
J
I//, Jv
/ /
II / Illl
I/// ill) III
\n
// Illi/n
Ik/ v 1 / /
—
/ /
w
v/
7
/// //// / A A / /
114 I02
IO3
—
Non-spawning
IO4
—
1961 i June
July
1
Aua. Seot
i Oct.
i Nov. i Dec. i
FIGURE 4. The frequency and intensity of migrations of C. guanhumi in the periods from
May to December, 1960 and 1961. Diagonal marks indicate dates when no migrating
crabs were seen. X indicates dates when no crabs were seen. Circles indicate dates of full
moon. In 1960 numbers greater than IO3 were not estimated.
In transitionals the chromatophores are obscured by the shell pigments described
above.
Adult C. guanhumi are all deep blue when they first become active at the end
of the dry season in late May. This color is due to a dense layer of expanded black
chromatophores closely applied to the inner surface of the membranous layer of the
shell. This layer can be observed, either in histological section as in Figure 5, or
by removing and opening a walking leg and viewing the exposed epidermis perpen-
dicularly from its inner surface by transmitted light. In deep blue crabs only
expanded black chromatophores can be seen, embedded in a filamentous white
pigment similar to that seen in juveniles.
Variations in adult color appear during the summer and fall. At some time
216
CHARLES A. GIFFORD
FIGURE 5. Section of the epidermal lining of the gill chamber (X 225) showing a dense
layer of expanded black chromatophores immediately under the membranous layer of the shell.
The calcified layers of the shell were removed to facilitate sectioning. The separation. between the
membranous layer and the epidermis is an artifact of fixation. The large cells embedded in the
epidermis are not chromatophores. The outward color of the crab was blue.
FIGURE 6. The epidermis of a walking leg viewed from its inner surface by transmitted
light. (X140). The black chromatophores are withdrawn from the membranous layer and
contracted. A layer of expanded yellow chromatophores is located closest to the shell. The
outward color was yellow.
FIGURE 7. Same as Figure 6 ( X 140), but with both black and yellow chromatophores
nearly contracted. The outward color was white.
GENERAL BIOLOGY OF THE LAND CRAB 217
l>d ween ovulation and spawning, most females turn from the normal deep blue to
either white or yellow. Intergrades between all three colors have been seen in
ovigerous females. When examined as described above, the epidermis of these crabs
displays both yellow and black chromatophores in different stages of expansion. In
some preparations faint white contracted chromatophores can be seen on the inward
side of the blacks when the preparation is viewed with reflected light. The vellow
chromatophores resemble the blacks in number and size, but are separate from them
and located closer to the inner surface of the shell. Unless both types are fully ex-
panded the yellow chromatophores can be seen through or between the blacks.
Various combinations of expansion of the two types are shown in Figures 6 and 7.
They account for the color stages between blue and yellow. In females the whole
surface of the crab is usually more or less the same color. While deep blue or bright
yellow crabs do not undergo rapid color changes, even when destalked, intermediate
color stages can change from blue-gray to yellowish white in an hour or so. The
percentage of white females in the population increases after each spawning period.
Males also change from blue to white or yellow, but only in the fall, and then not
as frequently as females do. While completely white or yellow males have been
seen, blue males mottled with patches of white or dull yellow, or with one or more
white legs, are more common.
In a small fraction (less than 6%) of the adult population, the dorsal surface of
the carapace and the distal segments of the legs are dark brown or orange-brown.
The sides of the carapace and the upper segments of the walking legs are lighter
brown, and frequently have a faint lavender tone. The brown color is in the shell
and the epidermal pigments do not show through, hence females of this group do
not change color when they spawn. The eyes are always brown or yellow in contrast
to the normal black. The migratory pigments seem to be withdrawn or absent.
The chelipeds of adult males of this color pattern are often equal in size and shape,
in contrast to the normal asymmetry. The outer margins of all but the dactyls of
the walking legs have many conspicuous clusters of long, fine, black bristles. Juve-
niles and transitionals of this group are bright orange-red all over, in contrast to the
normal purple-brown-orange color scheme. At maturation the third maxillipeds
of this group change from orange to brown. Ovigerous females of this aberrant
group have been found during the normal spawning periods, but the following
slight differences from normal spawning have been observed. In the first spawning
period of 1961 (in late June) over half of the females captured during the first two
days were of this type, although they constitute only about 2% of the female popula-
tion. In the third spawning period ( in late August ) several ovigerous crabs of this
type were captured several days after the normal spawning migration ended. None
were seen during the main spawning periods in September and October of 1961.
HABITS
Migration and swarming
The observation area extends roughly thirteen kilometers along Biscayne Bay
and from three to five kilometers inland. It has a dense crab population, and
contains a variety of habitats.
Exploratory daylight trips were made to locate areas of the most dense popula-
tion and to capture crabs for use in the laboratory. It was soon found that crabs in
218 CHARLES A. GIFFORD
colonies dispersed through fields and woods could be captured only by trapping,
which was non-selective and slow. Subsequent effort was concentrated on the canal
bank colonies, which are usually bordered by some sort of road. Crabs can be cap-
tured easily at night in these roads because automobile headlights or a powerful flash-
light seem to dazzle or confuse them. On crab-collecting trips, the number of crabs
moving, the types of activity, and the sex, size, and* color of the crabs captured were
recorded. Migrations were detected by observing the numbers of crabs crossing
the roads near the colonies or accumulating in large numbers in different parts of
the area. A fixed route was followed, and observation and collecting trips were
generally made within three hours after sunset. Individuals were not tagged, so
this method can only detect mass migrations. As a pattern of activity began to
emerge, specific observation trips were made to determine types of activity,
periodicity, and the number of crabs participating.
In south Florida C. guanhumi is more active in the wet season (May to Decem-
ber) than in the dry season. This pattern can be modified by unseasonal rains or
droughts, but during February, March, and April the crab tends to stay in its burrow
and feeding is greatly reduced or stopped. At both ends of the dry season more
crabs can be seen feeding under water than on land. During this period of decreased
activity, crabs often can be seen just inside the burrow entrance. The burrow en-
trances are generally kept open and free of debris, or closed a few centimeters from
the surface with mud brought up from underground. The number of closed burrows
increases in the dry season, but they are never all closed or all open.
During the rainy season Cardisoma displays several types of activity. Crabs in
the canal bank colonies are generally nocturnal, but in one wooded field colony this
is reversed : crabs are active during the clay and inactive at night. At the peak of
the largest mass migrations crabs also keep moving during the day, but in both cases
of daylight activity they seek shade at mid-day.
Crabs may remain in the burrow, feed within a few feet of the burrow, join
other crabs in swarming within 50 feet or so of the canal bank, or join other crabs
in mass migrations. The intensity and duration in time of the major episodes of
swarming and migration in 1960 and 1961 are shown in Figure 4. These periods
can be divided into two categories : spawning migrations involving only ovigerous
females, and mass migrations or swarming involving a cross-section of the whole
adult population.
The spawning season extends from late June to early December, but is not
continuous. Spawning occurs in sharp peaks near the time of the full moon. The
number of ovigerous females recorded in each period increases to a maximum in Sep-
tember and October, then declines. During each period ovigerous females appear
simultaneously over the whole inland portion of the study area four to six days before
the full moon. Concentration may vary considerably from one place to another.
During the following nights the number of ovigerous females moving increases,
generally reaching a peak between one night before the full moon and one night
after. By the second night of the period large numbers begin to accumulate at the
bay shore. By the night of the full moon many of these have spawned and a few are
beginning to move back inland.
The spawning period ends abruptly. Usually only a few ovigerous females
could be found in the study area 48 hours after the peak of a spawning period. The
gradual termination of the October, 1961, period may have been due to a sudden
GENERAL BIOLOGY OF THE LAND CRAB 219
20° F. drop in temperature which occurred two days lieforc the full moon. Re-
cently spawned females can he seen crossing roads parallel to the hay shore for sev-
eral nights after the end of the spawning period.
Semi-lunar spawning periods also occur, hut these are not as consistent, as
intense, or of as long duration as the lunar peaks. In 1(^>0 they were recorded
midway in the lunar cycle in Octoher and November. In 1961 they occurred from
J J •f
August through December, the end of the observation period. In Octoher, 1961, a
few (<5) ovigerous females were found on each of four census trips between the
semi-lunar and lunar spawning periods. On the first three nights of the October
lunar spawning period the numbers of ovigerous females seen were 83, 296, and an
estimated 8000, respectively.
In the canal bank colonies activities of crabs range from complete inactivity to
mass migration. Nights on which a standard trip through the study area revealed
100 or more non-ovigerous crabs crossing roads 50 yards or more from the nearest
colony, or congregating in areas of low burrow density, have been arbitrarily called
non-spawning migrations, and are recorded as such in Figure 4.
Four non-spawning migrations occurred in 1960 and two in 1961. All of these
started at the time of, or shortly after, a spawning migration. The two large non-
spawning migrations in 1961 were most closely observed and followed a common
pattern.
While the spawning migration is in progress, transitional and adult males and
non-ovigerous females congregate at the inland ends of some of the drainage ditches
and canals leading back from the bay. At about the time that spawning ends, they
leave the canal heads and radiate out, many of them going inland. Two or three
days later large numbers appear along the bay edge. They are active during the
day, and at low tide they can be seen crossing the exposed mud flats and entering
the water. They have also been seen in the upper branchs of mangrove trees at
this time, although they do not normally climb trees. Within a day or two the
swarm at the bay edge disappears, and for several nights following large numbers of
crabs can be seen crossing inland roads running parallel to the bay. A few crabs
. could always be seen along the canals, and one wooded field colony has always had
a crab in every burrow inspected while the migrations were in progress.
The first part of this sequence, congregation at certain canal and ditch heads
and swarming in adjacent roads and fields, occurred during all of the spawning
migrations in 1961, but only developed into mass non-spawning migrations after
the July and August spawning migrations.
The sex and color distribution in a sample (307 crabs) taken near the bay just
after the peak of the August, 1961, migration is given in Table II. Some recently
spawned (white) females were found, along with transitionals (purple) of both
sexes but the largest fraction consisted of equal numbers of adult (blue) males and
females.
Spawning migrations differ from non-spawning in occurring more regularly and
uniformly over the whole study area, in being of shorter duration, and in beginning
and ending more abruptly.
DISCUSSION
Physiological, anatomical, and behavioral adaptations of crustaceans to terrestrial
life have been reviewed by Edney (1960). ''Terrestrialness" among decapods
!20 CHARLES A. GIFFORD
from generally intertidal genera like Sesanna, Pachygrapsus, and Uca to
forms such as Biri/ns and Gecarcinits which enter water only to spawn, and perhaps
to molt. C. guanhumi varies in terrestrialness with the location of its colony and
with the season. Nearly all C. (jitanhnini Imrrows extend down to ground water,
on which crahs in inland colonies depend to replenish water losses. They must
spawn in the sea, but otherwise they exist as land animals. Crahs living near water
frequently enter it to feed, to avoid capture, and perhaps to migrate. In the low-
lying coastal and estuarine areas which it inhabits, C. guanhumi's retention of am-
phibious ability and its independence of salinity greatly increase its food supply and
provide protection from predators and climatic extremes.
A definitive estimate of C. guanhumi' s ecological importance is beyond the scope
of this paper. Its ability to starve for long periods complicates estimations of its
food requirements. Even if these are low, the observation that its biomass ap-
proaches two metric tons/hectare in inland colonies, and may be higher along
waterways, would seem to make it an important animal in both of these habitats.
Cardisoma is exceptionally accessible for observation. Its habits of daylight
activity in inland colonies and its periodic daylight migrations, when hundreds or
thousands of individuals may be visible at one time, make the occurrence of color
and growth stages, sexual dimorphism, and an aberrant morphological minority
readily apparent.
C, guanhumi also seems unusual among crustaceans in its lunar spawning be-
havior. In this context spawning is defined as the release of larvae from their
attachment to the female ; ovulation as the release of mature ova from the ovary,
followed immediately by their fertilization, acquisition of a chitinous covering and
attachment to pleopod hairs ; maturation as the growth and yolk accumulation of
ova in the ovary.
Lunar swarming in the prawn Anchistoides antiguensis, and lunar periodicity of
color changes and motor activity in crabs have been reviewed or reported by Brown
(1961a), Hauenschild (1960), and Bennett ct al. (1957). None of these mention
lunar spawning. Korringa (1947, 1957) cites many instances of lunar or semi-
lunar spawning periodicity in polychaetes, molluscs, the grunion, Lcurcsthes tennis,
and the chironomid, Clunio marina, but does not mention lunar spawning in crus-
taceans. Caiman (1911) quotes Andrews' and Stebbing's (reference not given)
descriptions of spawning migrations by the gecarcinid crabs Gecarcoidae lalandii
from Christmas Island, and Gecarciniis ruricola from the West Indies. In both
species spawning is said to be simultaneous, i.e., a mass spawning migration occurs,
but this happens only once a year, during the rainy season. Andrews further stated
that another Christmas Island gecarcinid, Cardisoina hirtipes, was not observed to
enter mass spawning migrations.
Whether lunar spawning is rare in crustaceans or whether other forms have not
been observed as closely as C. guanhumi is unknown, but its occurrence in this
species seems reasonably certain. In the five-month 1961 spawning season 90%
of all ovigerous females observed were seen in the six three-day periods preceding
the full moons. A peak occurred one day before the full moon in the first four of
these periods, and three days before in the last two. Smaller mid-period peaks also
occurred.
Embryos taken from females migrating to the sea are generally near the same
GENERAL BIOLOGY OF THE LAND CRAB 221
stage of development, implying a common ovulation date for most of the females
spawning in a given period. It seems less likely that crabs can influence the rate
of embryological development after the eggs are fertilized and attached to abdominal
appendages, than that they can influence the rate of ovarian maturation or hold the
mature ova to be released under the influence of a common external stimulus.
Numerous instances of the latter in marine invertebrates are given by Giese (1959a,
1959b). Separate control (at least partly hormonal) of maturation and ovulation in
Uca pngilator is suggested by the observation of Brown and Jones (1949) that eye-
stalk removal outside the normal spawning period is followed by ovarian maturation
but generally not by normal ovulation and oviposition. Only one captive C.
giianludiii has ovulated, and that was within a few days of capture. The simplest
explanation of C. guanhumi's synchronized cyclic spawning consistent with these
observations is that maturation is induced and roughly synchronized by a combina-
tion of internal conditions with one or more external factors, and that ovulation
(hence spawning) is triggered by some external change occurring twice in each
lunar cycle. Speculation on the nature of the external stimuli is not profitable in
the absence of additional information.
Changes in overall coloration and shape with increasing size, similar to those
occurring in C. guonJutnii, have been reported for Uca (Crane, 1941a) and for
Ocypode (Crane, 1941b; Cowles, 1906). Adults of both genera were said to have
little shell pigment and to undergo physiological color change, either as a part of
courting behavior (Uca} or in response to changes in background, illumination, or
temperature (Ocypode).
The color change from blue to white or yellow, which occurs in ovulating C.
giianhumi, appears similar to the morphological color change described by Brown
(1934), in that the amount of yellow pigment in the epidermis increases. The gill
chamber covering of C. guanJuuni can be cut away with its epidermal lining intact.
If the latter is stripped from the shell, much of the epidermal pigment remains behind.
In deep blue crabs only black pigment can be obtained in this way, while in crabs
ranging from blue-white to yellow, increasing amounts of yellow pigment appear.
In completely yellow crabs the pigment adhering to the shell is almost all yellow,
although much pigment can be rinsed from the epidermis. Completely yellow crabs
may stay that way for months, without further change, but white crabs can undergo
physiological color change. Ovigerous females are frequently blue or blue-gray
when captured at night, but lighten in an hour or so if exposed to artificial light.
They are almost invariably white or yellow the following morning.
The literature concerning hormonal control of physiological color changes is
vast (Brown, 1961b; Kleinholz, 1961 ; Carlisle and Knowles, 1959), but very little
has been done on morphological changes. It would seem that C. guanhumi, in
which large numbers of crabs undergo such changes simultaneously at predictable
times, would provide exceptional material for further study of them. The control of
the various stages in the reproductive cycle might also be profitably investigated.
SUMMARY AND CONCLUSIONS
1. Observations on the general biology of the land crab, Cardisoma gnanhnnii, in
southern Florida are presented, touching upon its geographical range, habitats, life
cycle, growth, color changes and spawning and non-spawning migrations.
CHARLES A. GIFFORD
2. C. (juanhitnii may be nearly terrestrial, entering water only to replace water
losses and to spawn, or it may be amphibious, entering either fresh or salt water to
feed, to escape predators and climatic extremes, and perhaps to migrate.
3. Post-larval C. gnanliumi pass through three growth stages, juvenile, transi-
tional, and adult, which are defined on the basis of morphological and color changes.
4. A small minority (about 6%) of the population differs markedly in pigmen-
tation, and to a lesser extent morphologically and in spawning behavior, from the
normal.
5. C. guaiihiimi migrates to salt water to spawn in sharp lunar and semi-lunar
peaks preceding the new and full moons between June and November. Non-spawn-
ing migrations, involving both males and nonovigerous females, sometimes follow
the spawning migrations, but are less well-defined and may be localized.
6. Adult color is due to epidermal chromatophores. Females change from the
normal adult blue to white or dull yellow at the time of ovulation. The overall
change is morphological, involving production of yellow chromatophore pigment.
Physiological color changes, absent at the extremes, can occur while this change is
in progress.
7. Males may undergo similar color changes in autumn, but such changes are
less frequent and usually incomplete.
8. Once attained, the yellow color is apparently retained through the fall and
winter.
9. These observations are discussed, and some tentative explanations are given.
LITERATURE CITED
BEHRE, E. H., 1949. Notes on the occurrence of Cardisorna gitanhitmi Latreille at Grand Isle,
Louisiana. Proc. La. Acad. Sci., 12: 19-22.
BENNETT, M. F., J. SHRINER AND R. A. BROWN, 1957. Persistent tidal cycle of spontaneous
motor activity in the fiddler crab, Uca puga.r. Blol. Bull., 112: 267-275.
BROWN, F. A., JR., 1934. The chemical nature of the pigments and the transformations re-
sponsible for the color changes in Palacuwnctes. Biol. Bull., 67: 365-380.
BROWN, F. A., JR., 1961a. Physiological rhythms. In: Tbe Physiology of Crustaceans, Vol.
2, T. H. Waterman, Ed., Academic Press, New York and London : 401-430.
BROWN, F. A., JR., 1961b. Chromatophores and color changes. In: Prosser, C. L., and F. A.
Brown, Jr., Comparative Animal Physiology. 2nd Ed., W. B. Saunders & Co.,
Philadelphia.
BROWN, F. A., JR., AND G. JONES, 1949. Ovarian inhibition by a sinus gland principle in the
fiddler crab. Biol. Bull., 96: 228-232.
CALMAN, W. J., 1911. The Life of Crustaceans. Methuen and Co., London.
CANNON, H. G., 1923. A note on the zoea of the land crab, Cardisotnu annatuiu. Proc. Zool.
Soc., London, 1923: 11-14.
CARLISLE, D. B., AND F. KNOWLES, 1959. Endocrine control in Crustaceans. Cambridge
Monogr. in Exp. Biol., 10: 1-117, Cambridge University Press, Oxford.
COSTLOW, J. D., AND C. G. BooKHOUT, 1960. A method for developing brachyuran eggs in vitro.
Liiinwl. and Ocainoyr., 5: 212-215.
COWLES, R. P., 1906. Habits, reactions, and associations in Ocypodc urciuiria. Carnegie Inst.
Wash. Publ. No. 103. Papers fnnn the Turtiu/as Lahomtory, 2: 1-41.
CKANK, T., 1941a. Crabs of the genus Ui'ii from the west coast of Central America. Zoologica,
'26: 145-208.
CRANE, J., 1941b. On the growth and ecology of crabs of the genus ( Vv/'<></i'. Zo.ologica, 26:
297-310.
EDNEY, E. B., 1960. Terrestrial adaptations. In: The Physiology of Crustaceans, Vol. I, T. H.
Waterman, Ed., Academic Press, New York and London : 367-388,
GKNKRAL BIOLOGY OF THE LAX I) CRAP,
GALLATIN, M. H., 1958. Soil Survey — Dade County, Florida. U.S.D.A. Soil Conservation
Service, Series 1947, No. 4.
GIESE, A. C, 1959a. Reproductive cycles of some west coast invertebrates. A.A.A.S. Puhl. Xo.
55 : 625-638.
GIESE, A. C., 1959b. Annual reproductive cycles in marine invertebrates. Ann. Rc-i'. Phvsiol.,
21: 547-576.
HAUENSCHILD, C., 1960. Lunar periodicity. Cold Sf>riii</ Harbor Svmp. Omint. ttiol., 25:
491-497.
KLEINHOLZ, L. H., 1961. Pigmentary effectors. In: The Physiology of Crustacea, Vol. 2,
T. H. Waterman, Ed., Academic Press, New York and London: 133-170.
KORRINGA, P., 1947. Relations between the moon and periodicity in the breeding of marine
animals. Ecol. Monoyr., 17: 347-381.
KORRINGA, P., 1957. Lunar periodicity. Mem. Gcol. Soc. Aincr., 67: 917-934.
DE OLIVIERA, L., 1946. Ecological studies on the edible crabs Uca and Guaiamu, Cardisaina
(/Hanhuini, and Ucidcs cordatus. Mem. hist. Oswaldo Cnts (Brazil), 44: 295-322.
PEARSE, A. S., 1915. An account of the Crustacea collected by the Walker expedition to Santa
Marta, Colombia. Proc. U. S. Nat. Mus., 49(2133) : 531-556.
PEARSE, A. S., 1934. Freezing points of bloods of certain littoral and estuarine animals. Papers
from the Tortuyas Laboratory, 28: No. 435: 93-102.
RATHBUN, M. J., 1918. The Grapsoid crabs of America. Bull. U. S. Nat. Mus., 97: 1-461.
SCHMIDT, W. L., 1934. Crustaceans. In: Smithsonian Scientific Series, Vol. 10, Shelled In-
vertebrates, C. G. Abbot, Ed., Smithsonian Institution Series, Inc., New York: 89-248.
Vol. 123, No. 2 October, 1962
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
TEMPERATURE AND OXYGEN CONSUMPTION OF
ORCHOMONELLA CHILENSIS (HELLER)
(AMPHIPODA: GAMMEROIDEA)
KENNETH B. ARMITAGE
Department of Zoology, The University of Kansas. Lawrence, Kansas
0. chilensis was collected by several antarctic expeditions and probably is ant-
arctic circumpolar (Shoemaker, 1945). The species has previously been divided
into several forms or into several separate species ; the antarctic populations were
named Orchomenopsis rossi A. O. W. (Walker, 1903). Chilton (1912) con-
cluded that Orchomonella chilensis is a widely distributed and variable species. It
was originally described from Chile and has been collected from both the North
and South Atlantic Oceans.
Walker (1907) reported 0. chilensis was present in McMurdo Sound in
enormous numbers from May through October and disappeared from the traps
between October 25 and January 27, the time of the antarctic summer. However,
the animals were readily collected in traps baited with seal meat and placed at a
depth of 240 m. off Cape Armitage during the antarctic summer of December, 1960,
and January, 1961.
Walker (1907) reported that this amphipod could exist only in water a little
above freezing. Because of the recent studies of metabolic compensation in
poikilothermic animals, it seemed of interest to measure the relationship between
temperature and oxygen consumption of an animal that lives the entire year at a
temperature near —1.8° C.
This research was supported by the National Science Foundation under Research
Grant 13231. Mr. Hugh B. House assisted in the laboratory work. Dr. Gunther
Schlager assisted with the statistical analysis of the data. The experiments were
conducted in the Biology Laboratory, NAF, McMurdo, Antarctica.
MATERIALS AND METHODS
Animals collected from traps were returned to the laboratory in sea water in
insulated cans. The animals were kept in aerated sea water in a constant tempera-
ture cabinet at —1.8° C. About 24—48 hours prior to a run, the animals were
placed in a refrigerator or water bath at the same temperature as that of the run.
225
Copyright © 1962, by the Marine Biological Laboratory
226
KENNETH B. ARMITAGE
However, because of the lack of facilities for temperature control, the animals were
subjected to considerable variation in temperature at temperatures exceeding 2° C.
Wide-mouth bottles of about 60 cc. capacity were used as respirometers. Fresh,
filtered sea water was aerated for 12 hours prior to a run. Sufficient water was
stirred in a large beaker to fill 24 bottles. Three bottles were used as controls and
one animal was placed in each of the remaining 21 bottles. All bottles were closed
with a glass stopper and checked for air bubbles. The bottles were placed in a
rack and the rack placed in a covered water bath of the proper temperature. Tem-
perature of the water bath varied about 0.1° C. Most runs were 4 hours, but some
were 6 or 8 hours.
At the end of each run, the amount of dissolved oxygen in each bottle was
determined by means of the unmodified Winkler method. The difference between
the amount of oxygen in a bottle with an animal and the mean of the three controls
TABLE I
Oxygen consumption and body size of Orchomonella chilensis (Heller)
Tempera-
ture, ° C.
Mean wet
weight,
nearest nig.
Oxvgen consumption
(Ml./gm./hr.)
Coefficients
O:TK
O«:W
W
X
S.K.x
b
S.E.j,
,.**
fc-i
S.E.6-1
r**
-1.8
67
128.3
0.17
0.455
0.103
0.408
-0.470
0.093
-0.455
0
87
117.7
0.15
0.471
0.058
0.631
-0.530
0.059
-0.675
2
86
141.2
0.16
0.462
0.106
0.403
-0.467
0.091
-0.459
4
75
124.3
0.16
0.665
0.083
0.630
-0.353
0.099
-0.339
6
71
146.8
0.17
0.642
0.107
0.519
-0.309
0.108
-0.278
8
71
158.8
0.14
0.604
0.061
0.707
-0.393
0.061
-0.543
10
70
231.0
0.10
0.571
0.078
0.596
-0.448
0.062
-0.590
12
75
176.8
0.15
0.497
0.069
0.587
-0.589
0.090
-0.553
All values of r highly significant. Test for heterogeneity of ;•: null hypothesis, all r's are from
the same population. For O->:T<F, \- = 16.92, d.f. = 7, .02 > P > .01. Reject null hypothesis.
O-..-W
— — , x2 = 20.57, d.f. = 7, .01 > P > .001. Reject null hypothesis.
was the amount of oxygen used. Each animal was blotted dry and its wet weight
determined on a Mettler Type B5 analytical balance. Because no brooding female
amphipods were found, the sex of the animals was not determined.
A preliminary survival experiment indicated that the animals were intolerant to
temperatures above 15° C. Therefore, runs were made at —1.8° C., 0° C. and at
2° intervals through 12° C. At 12° C., there was considerable mortality in the
respirometers and it was not feasible to make determinations of oxygen consumption
at higher temperatures. The oxygen consumption of 100 animals was determined
at each temperature ; 800 animals were used overall.
Two sources of error should be noted. No attempt was made to control activity.
There was no control over the amount of feeding by the animals except they had not
fed for at least 24 hours prior to being used. In addition, the animals were not
acclimated to the experimental temperatures. Thus the R-T curve is acutely
OXYGEN CONSUMPTION OF ORCHOMONELLA 227
measured. Probably the animals lived for several months at -1.8° C. or slightly
warmer before they were brought into the laboratory.
OXYGEN CONSUMPTION AND SIZE
The consumption of oxygen per unit time is a function of size as expressed in
the following equation :
(1) 02 = aW»,
where W — weight and a and b are constants. Equation ( 1 ) may be divided by W
to produce a weight-specific respiration :
• (2) ! = «W>-'.
In these equations, 6 and b~l are regression coefficients. For crustaceans, b is gen-
erally between 0.67 and 1.0 and 6~1 is usually between —0.05 and —0.40 (Wolve-
kamp and Waterman, 1960).
The regression coefficients of double-logarithmic plots were determined by the
method of least squares. Additional statistics calculated were the standard errors
of 6 and 6-1 and the coefficients of correlation (r) (Table I). A different b (Fig. 1 )
or 6~1 was obtained at each temperature. Because the standard errors were so
variable, the regression lines of b were tested for homogeneity by an analysis of
covariance (Steel and Torrie, 1960; p. 319). The null hypothesis, there is no
difference in regression coefficients for log oxygen consumption against log weight
at the 8 temperatures, was tested by means of the F test. Because F — 5.407
(/><.01), the null hypothesis was rejected and it was concluded that the regression
lines were different from one another. However, there was considerable hetero-
geneity in the unexplained SS at 8°, 10° and 12° C. Therefore, a second test for
homogeneity was made, omitting the three higher temperatures. Because F — 5.66
(/><.01), it was concluded that the regression slopes were heterogeneous.
Heterogeneity in regression slopes was found in Artemia salina (Conover, 1960).
in the snails Lymnaca palnstris and L. pcregcr (Berg and Ockelmann, 1959) and
in the crabs Uca pugnax and U. rapa.v (Vernberg, 1959). Vernberg and Conover
demonstrated a direct effect of temperature on b or b~l. Berg and Ockelmann
demonstrated a seasonal shift in b ; when the animals were tested at 18° C., the slope
of the regression line was greater in June than in August. The authors suggested
that the seasonal variation might be caused by a comparatively greater increase
of oxygen consumption by the larger animals during the season of reproduction.
However, in this regard, Rao and Bullock (1954) showed that habitat temperature
of the animal prior to study could affect O10 and that Qu, commonly increases
with increasing size over the range of normal physiological activity. Presumably
the differences in habitat temperatures might account for the differences reported
by Berg and Ockelmann, although it is not unlikely that several interacting factors
were present.
Temperature seems more likely to be the primary factor in explaining the
variation of b in O. chilcnsis. The animals were collected from an environmental
temperature that probably varied less than 1° C. for several months, nor were the
animals reproducing. Although Qin was not calculated, it is evident from Figure 2
228
KENNETH B. ARMITAGE
40. Or
300
20 0
•^-10.0
o
t
8.0
60
(O
O 5.0
O
3.0-
2.0-
10.0
20.0 30,0 40.0 50.0 1000
WEIGHT (mg)
200.0 250.0
FIGURE 1. The relationship between oxygen consumption and body size in Orchomonella
chilcnsis (Heller). Temperature is degrees Celsius (C).
that Q:o varied with size. Small animals greatly reduced oxygen consumption at
4° C. as compared with 2° C. With increasing size, the amount of reduction
decreased and the largest animals increased consumption. Another trend appeared
between 6° and 8°, the smallest animals increasing and the largest animals
decreasing consumption.
These trends are supported by the values of r. Because the distribution of r's
is asymmetrical, all r's were converted to the normally distributed r and the z's
tested for homogeneity. The null hypothesis, all r's are from the same population,
was rejected (Table I). Therefore, it is evident that the relationship, On'.W, is
influenced by temperature. The rr's were tested for significant differences (Table
II). Significant differences, among others, occurred between 2° and 4° C. and
OXYGEN CONSUMPTION OF ORCHOMONELLA
229
30
20
O 10
Q_
ID
CO
O
O
CM
O
3J
JL
_L
-1.8
468
TEMPERATURE C°
10 12
FIGURE 2. The relationship between oxygen consumption and temperature for different
sized Orchomonella chilcnsis (Heller).
between 6° and 8° C. Similar results were obtained comparing the r's of the
relationship Q,,/W'.W. However, the distribution of significant differences among
s's of the weight-specific rate: weight values differed from the distribution of sig-
nificant differences among s's of the rate: weight values. Although the general
conclusion that temperature affects the rate: weight relationship does not depend
on the method of evaluating rate, the specific effects of temperature do depend
on the method of analysis.
According to von Bertalanffy and Krywienczyk (1953), if b — 0.66, metabolism
is proportional to surface; if 6 = 1.0, metabolism is proportional to weight; if
230
KENNETH B. ARMITAGE
6 > 0.66, but < 1.0, metabolism is intermediate. The values of 6 for O. chilcnsis
indicate several metabolic types in the population including types not included in
the above scheme. Locker (1961), however, believes that metabolism is basically
related to surface, but this relationship can be modified by factors such as
temperature.
RATE-TEMPERATURE CURVE
The shape of the R-T curve (Fig. 3) will depend, in part, on the size of the
animals used and will reflect the composition of the population at the time the
TABLE 1 1
Differences between r's converted to z. All differences exceeding the LSD marked
with an asterisk
Rate: Weight (Oi:W)
0 C.
-1.8
0
2
4
6
8
10
12
-1.8
0
.310*
.006
.308*
.142
.448*
.254
.240
0
0
.316*
.002
.168
.138
.056
.070
7
0
.314*
.148
.454*
.260
.246
4
0
.167
.140
.055
.068
6
0
.306*
.112
.098
8
0
.194
.208
10
0
.014
12
0
Weight-specific Rate: Weight
W
-1.8
0
.329*
.005
.137
.206
.117
.187
.131
0
0
.324*
.466*
.534*
.212
.142
.198
2
0
.142
.211
.112
.182
.126
4
0
.069
.254
.324*
.268
6
0
.323*
.392*
.336*
8
0
.069
.013
10
0
.056
12
0
Significant differences among s's: / = c,
/ /
z-> / \
/ \
- —,
n — 3
— 0.282.
animals were collected. For example, oxygen consumption was higher at 0° C.
than at —1.8° C. for each size class (Fig. 2), but the rate at 0° C. was lower than
at —1.8° C. (Fig. 3) because the population at —1.8° C. averaged 20 ing. less in size
(Table I). However, the decrease of oxygen consumption at 4° C. occurred in
all except the largest members of the population (which were rare) and therefore
seems characteristic of the population in general. This decrease appears to be an
adaptation to high temperatures. The adaptation lies in the vertical displacement of
the R-T curve and suggests a change in Q10. This change might be brought about
by a shift in the control of metabolic reactions to an alternate enzymatic pathway.
OXYGEX CONSUMPTION OF ORCHOMONELLA
231
Following the shift between 2° and 4° C., the rate of oxygen consumption increased
and beyond 8° metabolic control was inadequate. Under acclimated conditions,
the animals might be fairly good regulators to 6° or 8° C., but would likely be
conformers in any event at temperatures above 8° C. One might not expect such a
high degree of regulation in a species living at such a narrow range of environmental
temperature. It would be interesting to (determine the response pattern of the
species in other parts of its geographical range, as the R-T curve of the antarctic
population may result from the genetic history of populations that previously lived in
waters with wider fluctuations of temperature than now seem to occur in McMurdo
Sound.
The levels of oxygen consumption were about the same as arctic amphipods
(Scholander et al., 1953) and were higher than in amphipods of temperate regions
(Wolvekamp and Waterman, 1960), when the differences in experimental tem-
250 -
|«200
C
o
a.
E
150
o
o
c
a>
e»
>»
•x.
O
lOOll—
-1.8
I
4 6
Temperature - C°
8
10
12
FIGURE 3.
The acutely determined R-T curve for populations of
Orchomonella chilensis (Heller).
peratures are eliminated by extrapolating the curves of temperate species downward
or projecting the slope between 8° and 10° of O. cliilcnsis upward. Thus, O.
chilensis clearly demonstrates metabolic compensation (Bullock, 1955).
SUMMARY
1. The rate of oxygen consumption of the antarctic amphipod Orchomonella
chilensis (Heller) was determined over a graded temperature series from -1.8°
to 12° C.
2. The regression coefficients of double-log plots of rate: weight and weight-
specific rate: weight were temperature-dependent. The correlation coefficients
KENNETH B. ARMITAGE
between size and rates of consumption were highly significant and varied significantly
with temperature. Q10 varied with size.
3. The acutely determined R-T curve shows some regulation between —1.8°
and 6° C. Metabolic compensation was evident.
LITERATURE CITED
BERG, K., AND K. W. OCKELMANN, 1959. The respiration of freshwater snails. /. Exp. Biol.,
36: 690-708.
VON BERTALANFFY, L., AND J. KRYWIENCZYK, 1953. The surface rule in crustaceans. Amer.
Nat., 87: 107-110.
BULLOCK, T. H., 1955. Compensation for temperature in the metabolism and activity of
poikilotherms. Biol. Rev., 30: 311-342.
CHILTON, C., 1912. The Amphipoda of the Scottish National Antarctic Expedition. Trans.
Roy. Soc. Edinburgh, 48: 455-520.
CONOVER, R. J., 1960. The feeding behavior and respiration of some marine planktonic Crustacea.
Biol. Bull, 119: 399-415.
LOCKER, A., 1961. Das Problem der Abhangigkeit des Stoffwechsels von der Korpergrosse.
Natunviss., 12: 445-449.
RAO, K. P., AND T. H. BULLOCK, 1954. Qw as a function of size and habitat temperature in
poikilotherms. Amcr. Nat., 88: 33-44.
SHOEMAKER, C. R., 1945. Amphipoda of the United States antarctic service expedition 1939-41.
Rep. Sci. Res. U. S. Ant. Ser. Exp. 1939-1941, 289-293.
SCHOLANDER, P. F., W. FLAGG, V. WALTERS AND L. IRVING, 1953. Climatic adaptation in arctic
and tropical poikilotherms. Physiol. Zoo/., 26: 67-92.
STEEL, R. G. D., AND J. H. TORRIE, 1960. Principles and Procedures of Statistics. McGraw-
Hill Book Co., Inc., N. Y.
VERNBERG, F. J., 1959. Studies on the physiological variation between tropical and temperate
zone fiddler crabs of the genus Uca. II. Oxygen consumption of whole organisms.
Biol. Bull., 117: 163-184.
WALKER, A. O., 1903. Amphipoda of the "Southern Cross" antarctic expedition. /. Linn. Soc.,
29: 38-64.
WALKER, A. O., 1907. Crustacea III — Amphipoda. National Antarctic Expedition 1901-1904.
Natural History. London.
WOLVEKAMP, H. P., AND T. H. WATERMAN, 1960. Respiration, pp. 35-100. In: T. H. Waterman
(ed.), The Physiology of Crustacea. Academic Press, New York.
THE ANALYSIS OF POLARIZED LIGHT IN THE EYE OF DAPHNIA1
EDWARD R. BAYLOR AND WILLIAM E. HAZEN
ll'oods Hole Oceanographic Institution, Woods Hole, Mass., and
The College, University of Chicago, Chicago 37, Illinois
The operation of any polarization detector involves : ( 1 ) separation of incident
light into two vectors perpendicular to each other and to the direction of propagation
of the light beam, (2) suppression of one vector, (3) intensity estimation of the
remaining vector. In physical instruments polarization analysis depends on rotation
of the analyzer around the direction of propagation of the light beam. In the
biological systems found in arthropods, polarization analysis depends on the
possession of a radial array of analyzers, whether this is the rhabdomere-retinula
cell complex or the many different corneal and lens surfaces of the compound eye of
arthropods. Such an array of analyzers permits simultaneous comparison of
polarized intensities present at all azimuths about the direction of propagation of
the light beam. These comparisons may be between different ommatidia, with the
receptor system of each ommatidium acting as a unit, or between parts of the receptor
system of a single ommatidium.
Three models have been proposed to account for the orientation of animals to
polarized light, of which two are intra-ocular, the third extra-ocular. The three
models are: (1) the Brewster-Fresnel model in which one or more refractions and
reflections at corneal or lens surfaces serve to alter preferentially the intensity of
light polarized parallel to the plane of incidence; (2) dichroic filters (the rhab-
domeres) with the fast axes tangential to the radii of the array of niters; (3) the
reflected brightness pattern in which the intensity of light reflected or scattered from
the environment is greater perpendicular to the light polarization plane.
The Brewster-Fresnel refraction model relying upon a single refraction was
proposed by Stephens, Fingerman and Brown (1953) for the Drosophila eye. The
Brewster-Fresnel reflection model relying upon internal reflection from a lenticular
surface was proposed by Baylor and Smith (1953) for Daphnia. The presence
of dichroic filters in the eye of the bee was suggested by Autrum (see von Frisch,
1950) and has been supported by Stockhammer (1956, 1959). The environmental
reflection pattern as an orientation stimulus was suggested by Baylor and Smith
(1958).
Values of the theoretical light intensities calculated from the Fresnel equations
are compared with actual measurements through two surfaces of the daphnid cone
lens. The data presented here support the first of these intra-ocular models for
the eye of Daphnia pulex (de Geer).
MATERIALS AND METHODS
The measurements were made on the lenses of freshly killed daphnids mounted
in water under a coverslip on a microscope slide and examined at 500 X under a
1 Contribution No. 1236 from the Woods Hole Oceanographic Institution, Woods Hole,
Mass. Supported in part by a grant from the National Institutes of Health.
233
234 EDWARD R. BAYLOR AND WILLIAM E. HAZEX
Leitz Ortholux microscope with a trinocular head. A rotatable type N Polaroid
filter was interposed in the light beam beneath the microscope condenser. A 10 X
ocular fitted with a field stop diaphragm replaced the camera on the trinocular head
and was coupled to a photomultiplier photometer (Photovolt model No. 501 M) for
measuring light intensities. The field stop diaphragm placed at the image plane
of the ocular restricted the field of view of the photomultiplier to a circle 10 ju. in
diameter. The center of the circle was coincident with the intersection of crosshairs
in one of the viewing oculars to permit location of the desired area.
The change in light transmission through the carapace adjacent to the eye was
measured as the Polaroid was rotated through 90° from a position parallel to the
preferred transmission plane of the microscope to one perpendicular to that plane.
Individual lenses were aligned with the preferred transmission plane of the
microscope and the change in transmission of refracted light was again measured
with the Polaroid in two positions. These measurements were repeated with the
long axis of the lens rotated 90°.
RESULTS AND DISCUSSION
The measurements from 24 cone lenses from 6 different eyes are summarized in
Table I. Columns 2 and 5 contain data based on the measured intensities of light
transmitted through the carapace alone. These percentages are calculated by
dividing the reading of the photometer with the Polaroid in the easl^west-ppsition
by the reading with the Polaroid in the north-south position (NS/EW X 100),
where EW is the preferred transmission plane of the optical system of the
microscope. This always gives a calculated value of less than 100. A comparison
of columns 2 and 5 shows the difference due to the carapace alone, because the
carapace has been turned through ninety degrees. The differences measured in
this manner are small, and are probably random, indicating, the carapace is not an
effective polarization analyzer. A comparison of columns /and "2, and columns 5
and 6 shows not only the difference between the effectiveness of the lens and the
effectiveness of the carapace but also that the preferred transmission plane of
the hemispherical lens is parallel to the long axis of the cone. In columns 2 through
4, with the lens oriented NS, the average transmission of the background, 77.9%, is
exceeded by that of the lens, 81.3%, a difference of 3.4% . This is because the lens
transmits a greater proportion of the light when the Polaroid is in the NS position,
that is, parallel to the lens axis. In columns 5 through 7 the reverse is true : the
lens faces EW, its preferred plane is EW and therefore the fraction NS/EW
becomes smaller when the Polaroid is turned from EW to NS, that is, perpendicular
to the lens axis. The mean observed change is from 77.3% to 74.2%, a difference
•of 3.1%. Columns 4 and 7, taken by themselves, measure the change in intensity
•corrected for the lens system of the microscope, with the difference in column 4
being positive, that in column 7 negative. Of the 48 readings, four differ in a
•direction opposite to the expected direction. The mean difference of all the
measurements, correcting for the difference in sign, is 325% and lies between 2.Q%
and 4.5% with a probability greater than .99. This difference is consistent with
the calculated values for the Brewster-Fresnel reflection model. Reference to
Figure 1 shows that measurements have been made on light diffracted both at point
B and at point D.
POLARIZED LIGHT ANALYSIS IN DAPHXIA
235
TABLE I
Measured values of light intensity
Lens NS
Lens EW
% transmission
of background
% transmission
of lens
Difference
% transmission
of background
% transmission
of lens
Difference
1
81.00
83.75
2.75
76.50
71.59
-4.91
2
76.50
85.00
8.50
77.00
75.00
-2.00
3
77.00
82.24
5.24
77.00
73.74
-3.26
4
77.00
80.62
3.62
76.50
72.83
-3.67
5
78.00
96.67
18.67
77.00
75.38
-1.62
6
77.70
78.57
0.87
77.50
73.38
-4.12
7
80.00
82.02
2.02
77.00
73.38
-3.62
8
76.00
82.43
6.43
77.50
74.52
-2.98
9
76.00
83.78
7.78
78.50
72.29
-6.21
10
77.00
80.72
3.72
78.00 75.66
-2.34
11
78.00
80.49
2.49
77.50 69.87
-7.63
12
77.00
81.32
4.32
77.00 73.97
-3.03
13
79.00
79.89
0.89
78.50 75.29
-3.21
14 78.00
81.71
3.71
78.00 74.29
-3.71
15
78.00
80.00
2.00
78.00 73.54
-4.46
16 76.00
75.00
-1.00
78.00 75.34
-2.66
1 7 79.00
82.56
3.56
78.50 74.26
-4.24
18
79.00
78.87
-0.13
77.50
78.31
+0.81
19 77.00
79.76
2.76
75.50
74.71
-0.79
20 76.00
76.68
0.68
78.50 75.58
-2.92
21
78.00
83.33
5.33
78.50
76.37
-2.13
22
77.00
77.95
0.95
77.00 76.16
-0.84
23
82.00
77.78
4.22
77.00 72.63
-4.37
24
80.00
81.08
.108
74.00
73.85
-0.15
Mean
77.92
81.34
3.42
77.33
74.25
-3.08
Table II summarizes the calculated intensities for rays incident between 10° and"
80° on the external surface of the daphnid cone lens (angle 0 in Figure 1) and
whose plane of polarization is either parallel or perpendicular to the long axis of
the lens. For these calculations the index of refraction is assumed to be 1.53 and
that of the blood 1.33, yielding a relative index of refraction of 1.15. Since the
tangent of the angle of maximum polarization equals the relative refractive index,,
this angle is 49° for the external surface and 41° for the internal surface. To*
simplify the optical calculations and discussion, only those rays confined to a single
plane are considered throughout the paper. This plane bisects the conical figure
of the lens and is identical with the plane of incidence, the latter of which is defined
by the incident ray and the perpendicular at the point of incidence. As is customary,
the polarization plane is described as either parallel or perpendicular to the plane
of incidence. For example, AB parallel means the light beam AB, which is
parallel to the plane of incidence at B in Figure 1. In Table II, columns 2 through
7 contain values of reflected and refracted intensities corresponding to the labeled
portions of the light paths in Figure 1. These values are referred to an intensity
of 100 in the incident ray, AB.
236
EDWARD R. BAYLOR AND WILLIAM E. HAZEN
FIGURE 1. Diagram of a single cone lens showing the light path ABDF for the Brewster-
Fresnel internal reflection model. Light from A is incident at B with the angle of incidence
lahelled 0.
Columns 2 and 3 show the small differences which exist after the initial reflection
and refraction at B ; columns 4 and 5 give the somewhat greater differences occur-
ring after two refractions at B and at D. The differences between DE parallel and
DE perpendicular are given in column 9 and correspond to the measured differences
of Table I. The maximum calculated difference at 70° is 3.28% while the mean
obsemcd difference is 3.0%. That these values are in such good agreement is
TABLE II
Calculated values of light intensities
1
Angle of
incidence
2
BDi
3
BD,,
4
DEi
5
DEM
6
DFi
7
DF,,
8
DFi/DFn
9
DEi-/DE,i
10
86.16
86.21
74.24
74.32
0.45
0.40
1.125
00.08
20
85.02
85.20
72.28
72.59
0.52
0.32
1.625
00.31
30
82.90
83.34
68.72
69.46
0.66
0.21
3.143
0.71
40
79.34
80.24
62.96
64.38
0.95
0.07
13.57
1.44
50
73.36
74.89
53.82
56.09
1.51
0.15
10.03
2.27
60
63.26
65.72
40.02
43.20
2.65
0.30
8.32
3.18
70
46.26
49.68
21.40
24.68
4.73
1.77
2.67
3.28
80
20.43
24.53
4.17
06.02
6.13
4.80
1.27
1.8
The value of the incident ray, AB, is 100.
POLARIZED LIGHT ANALYSIS IN DAPHNIA
237
largely chance because any single measurement in Table I gives an average value for
many degrees of incidence. The fact that any difference whatsoever can be shown
in Table I probably means that (1) the index of refraction is higher than the
10
20 30 40 50 60 70 80
ANGLE OF INCIDENCE
90
FIGURE 2. Ratios of vector in tensities resolved at corneal interfaces. Curve A is DEn/DEi.
Curve B is calculated from data of Stephens, Fingerman and Brown (1953) and corresponds to
BDu/BDi for a cornea-air interface. Curve C is DFi/DFn. The subscript J_ means the ray
is polarized perpendicular to the plane of incidence and the subscript || means the ray is polarized
parallel to the plane of incidence.
238 EDWARD R. BAYLOR AND WILLIAM E. HAZEN
assumed value of 1.53 and (2) the optical surfaces of the cone lens are not
hemispherical and therefore the overall efficiency of analysis is higher. A more
effective polarization analyzer could be realized by altering the shape of the optical
surfaces so that more of the incident rays would meet the surface close to the
polarizing angle. The shapes of cone lenses will be discussed later.
The effectiveness of the resolution of the refracted ray into two vectors is shown
in Figure 2, where curve A is the plot of log (DE parallel/DE perpendicular).
Contrasted with this is the vector resolution of the reflected ray, curve C, log (DF
perpendicular/DF parallel), where the ratio of vector intensities is very high,
especially near the polarizing angle. At the polarizing angle internal reflection
of the parallel ray diminishes to zero and therefore the ratio approaches infinity.
In this way the small differences which were measured for Daphnia can be asso-
ciated with an effective analysis within the eye. It should be emphasized, however,
that the absolute intensity of rays at the receptor in this model is low.
The effectiveness of any light polarization analyzer depends upon its ability to
separate the incident light into two vectors and to present these two vectors for
intensity measurement. The ratio of the intensities of the two vectors is thus a
measure of the effectiveness of the polarization analyzer. The calculations in
Table II permit a comparison of the effectiveness of the two different kinds of
Brewster-Fresnel models which have been proposed. The first model depends
upon a single refraction at the corneal surface and was proposed for the eye of
Drosophila by Stephens, Fingerman and Brown (1953). The effectiveness of
this model in resolving the incident light into two vectors depends on a high
relative index of refraction characteristic of an air-cornea interface but not of a
water-cornea interface. The ratios of the different vector intensities resolved by a
single refraction at the cornea-air interface and by two refractions at the cornea-
water interface are shown in curves A and B of Figure 2. Comparison of these
two curves with curve C of the same figure shows unequivocally that refraction is
not as effective as reflection for polarization analysis. It remains to be seen
whether beams of light incident on the terrestrial arthropod cornea at high angles
proceed by multiple reflection to the light-sensing apparatus. A sequence of such
internal reflections (at less than the critical angle) would be a very effective light
polarization analyzer. The observations of de Vries and Kuiper (1958) for
Diptera and Waterman (1954) for Limulus, that ommatidla are sensitive to light
incident at high angles, might be thought of as lending credence to this view.
However, Waterman's (1954) work relating intensity threshold to angles of
incidence raises serious doubts concerning the Brewster-Fresnel refraction model in
the natural habitat because the intensity threshold for light incident near the polariz-
ing angle is approximately 100 times greater than that of light normal to the
surface. The confusion of polarized intensities with non-polarized intensities and
the obscuring of any particular polarized light stimulus seem inevitable with this
model unless this eye possesses an ability to distinguish \% brightness differences.
The second Brewster-Fresnel analyzer model proposed by Baylor and Smith
(1953) involves the somewhat unorthodox light path ABDF of Figure 1, which
requires the light to be incident at the cornea-blood interface twice, once on enter-
ing at B and again on being reflected at D. The ratios of orthogonally polarized
intensities reflected from D are plotted in curve C of Figure 2 where they give a
POLARIZED LIGHT ANALYSIS IN DAPHXIA
239
somewhat exaggerated impression of the effectiveness of this light polarization
analyzer when the ratio of intensities goes to infinity at the polarization angle. The
operation of this model may be seen in three dimensions in Figure 3. In Figure 3
the cone lenses are depicted on xyz coordinates to represent a solid figure. A ray of
light parallel to the y axis and polarized parallel to the z axis is incident on the
surmounting hemisphere of each of the cone lenses. The intensities resulting from
subsequent refractions and reflections are summarized on the figure and were taken
from the 60° line of Table II where the ratio of the intensities at the light-sensing
apparatus is approximately 8 to 1.
Microscopic observations of the compound eye of Daphnia pulex reveal that the
cone lens is not a circular solid cone of 45° surmounted by a hemisphere. Con-
FIGURE 3. Three-dimensional diagram of the Brewster-Fresnel internal reflection model
showing two cone lenses at right angles. Light rays are incident parallel to the y axis with an
intensity of 100, and polarized parallel to the yz plane. The numbers represent calculated
intensities at the various parts of the light path.
siderable variation in shape and contour is observed in the lenses of the eyes
studied. In particular, one type of cone lens has a rather special shape in which the
contours exhibited are of considerable theoretical interest because they are com-
parable to those predicted and drawn on paper from simple geometrical optical
considerations. Starting with the knowledge that the light-sensing apparatus lies
at the tip of the cone lens and with the constraint that the angle of the cone should be
approximately 45° we may reconstruct the light path FDBA of Figure 1 through
the cone lens step by step, starting at the apex and working backward to the outside
240
EDWARD R. BAYLOR AND WILLIAM E. HAZEN
100 200
MICRONS
B
C
FIGURE 4. A, Outlines of selected cone lenses of Daphnia pulcx. B, Composite tracing of
photographs of three serial frontal sections through the compound eye of Daphnia pulcx. Optic
nerve protrudes from the center toward the 100-micron mark of the scale. C, Constructed lens
with three light rays.
of the lens. A series of rays 5 to 10° apart are drawn from the apex toward the
open end of the cone. For maximum efficiency of polarization detection each of
these rays should be reflected from the periphery of the lens at the polarizing angle.
Therefore, at the open end of the cone we construct across each ray a line which
intersects the ray at this polarizing angle. The distances along the rays from the
apex to the intersections are adjusted so that the constructed lines intersecting
the rays produce a smooth curve. At each intersection of a ray with the curve
POLARIZED LIGHT ANALYSIS IN DAPHNIA 241
so produced a line is drawn perpendicular to it to permit construction of the
reflected ray DB of Figure 1. The reflected rays are extended across the long axis
of the cone toward B. A second intersecting surface is constructed across the
rays to form a smooth curve which refracts all rays outward into a parallel bundle.
The two smoothed theoretical surfaces are then joined across the base of the cone
to complete the constructed figure. The completed figure (Fig. 4 C) cannot
be superimposed on any photographs of cone lenses (Fig. 4 A and B) but provides
a better approximation to the actual figure than does a hemisphere. These observa-
tions are consistent with the hypothesis that the dioptric contours of some cone
lenses are specialized for polarizing angle reflection of light beams traveling at
right angles to the long axis of the cone. The observations are also consistent
with the intensity ratios measured in the light beam DE of Figure 1 and sum-
marized in Table I, which are higher than anticipated from the calculations in
Table II. The observed dioptric contours may also serve to decrease the intensity
of ambient light incident parallel to the long axis of the cone. Figure 4 shows a
constructed lens with outline drawings of selected lenses. Changes of lens shape as
a result of fixation appear to be small when photographs of fixed material are
compared with those of living material.
It is difficult to see how any of the models for light polarization plane detection
operate effectively in a natural situation where the intensity of polarized light with
a particular direction of propagation incident upon a receptor is masked by and
confused with the intensity of light, whether polarized or non-polarized, from all
other sources. When this happens the receptors must be able to distinguish
intensity differences of a few per cent if orientation is to be precise. If we assume
the model to be a perfect detector in the sense that the NS detector receives all
light polarized in the NS plane and rejects all light polarized in the EW plane, it
is still subject to confusion by ambient non-polarized light. Even with 100%
polarized light the intensity ratios present for comparison in the Brewster-Fresnel
external reflection model of Stephens et al. are not as great as 2:1, whereas the
Brewster-Fresnel internal reflection model and the Autrum model have a maximum
theoretical intensity ratio of infinity. The Brewster-Fresnel internal reflection
model proposed for the daphnid cone lens appears especially vulnerable to the
criticisms outlined above because such a small percentage of the incident polarized
light is transmitted to the light-sensing apparatus. Therefore, it might be assumed
that the reflected brightness pattern is the sole orienting stimulus. That it is not has
been shown by Baylor and Smith (1953) and by Waterman (1960) who showed
that orientation remained in spite of careful filtration of the water. In a separate
experiment Smith and Baylor (1960) used a small half- wave plate umbrella to
rotate the polarization plane of only the light directly incident on the daphnid
without altering the reflection pattern. Here the daphnid oriented only to the
polarized light plane incident from overhead unless the water was deliberately
made turbid by addition of yeast. The function of polarized light responses in
nature remains to be demonstrated and the possibility should not be ignored that
many cases of polarized light responses may be only laboratory curiosities.
We wish to acknowledge the contributions of Prof. Frederick E. Smith of the
University of Michigan with whom studies on the geometric optics of Daphnia
242 EDWARD R. BAYLOR AND WILLIAM E. HAZEN
inagna lenses were begun. Also, we wish to acknowledge a similar set of
calculations by G. Schreuder-van Zanten and J. W. Kuiper in a manuscript sent
to us by Prof. Kuiper.
SUMMARY
1. Three models suggested to account for the ability of arthropods to detect the
plane of linear polarized light are characterized.
2. Measurements of polarized light refracted through the cone lens of Daphnla
pul ex are summarized.
3. These measurements are compared with calculated intensities derived from
one of the three models.
4. The shape of the cone lens of Daphnla and the specialization of their contours
for polarization analysis are suggested.
5. The operation of the various models in nature is criticized.
LITERATURE CITED
BAYLOR, E. R., AND F. E. SMITH, 19S3. The orientation of Cladoccra to polarized light.
Amer. Nat., 87: 97-101.
BAYLOR, EDWARD R., AND FREDERICK E. SMITH, 1958. Extra-ocular polarization analysis in the
honey bee. Atiat. Rec., 132: 411.
VON FRISCH, K., 1950. Die Sonne als Kompass im Leben der Bienen. Experientia, 6: 210-221.
SMITH, FREDERICK E., AND EDWARD R. BAYLOR, 1960. Bees, Daphnia and polarized light.
Ecology, 41: 360-363.
STEPHENS, GROVER C., MILTON FINGERMAN AND F. A. BROWN, JR., 1953. The orientation of
Drosophila to plane polarized light. Ann. Ent. Soc. of Amer.y 46: 75-83.
STOCKHAMMER, K., 1956. Zur Wahrenemung der Schwingsrichtung linear polarisierten Lichtes
bei Insekten. Zcitschr. vergl. Physiol., 38: 30-83.
STOCKHAMMER, K., 1959. Die Orientierung nach der Schwingungsrichtung linear polarisierten
Lichts und ihre sinnesphysiologischen Gnmdlagen. Erg. der BioL, 21 : 23-56.
DE VRIES, HESSEL, AND JAN W. KUIPER, 1958. Optics of the insect eye. Annals New York
Acad. Scl, 74: 196-203.
WATERMAN, T. H., 1954. Directional sensitivity of single ommatidia in the compound eye of
Limutus. Proc. Nat. Acad. Sci., 40: 252-257.
WATERMAN, T. H., 1960. Interaction of polarized light and turbidity in the orientation of
Daphnia and Mysidinm. Zeitschr. vergl. Physiol., 43: 149-172.
BEHAVIOR OF DAPHNIA IN POLARIZED LIGHT1
WILLIAM E. HAZEN AND EDWARD R. BAYLOR
The College, University of Chicago, Chicago 37f Illinois, and Woods Hole
Oceanographic Institution, Woods Hole, Mass.
Three models have been proposed to account for the apparent ability of animals
to perceive the plane of vibration of polarized light. Two of the proposed models
are intra-ocular, the third is extra-ocular. The three models are : ( 1 ) a radial
array of dichroic filters (rhabdomeres) with their fast axes tangential to the radii
of the array; (2) the Brewster-Fresnel models in which one or more refractions
and reflections at corneal or lens surfaces serve to diminish preferentially the
intensity of light polarized parallel to the plane of incidence; (3) the reflected
brightness pattern in which the intensity of light reflected and scattered from the
environment is least parallel to the polarization plane and greatest perpendicular
to the polarization plane.
Two Brewster-Fresnel models have been proposed. The Brewster-Fresnel
reflection model relying upon a single refraction was proposed by Stephens, Finger-
man and Brown (1953) for the Drosophila eye. The Brewster-Fresnel reflection
model relying upon internal reflection from a lenticular surface was proposed by
Baylor and Smith (1953) for Daphnia. That daphnids utilize an intra-ocular
analyzer in clear water was established by Baylor and Smith (1960) using half-
wave plates to distinguish between intra-ocular and extra-ocular polarization
analyzers. These experiments corroborated their earlier findings (Baylor and
Smith, 1953) as well as those of Waterman (1960). To test the Brewster-Fresnel
internal reflection model, Baylor and Hazen (1962) conducted optical analyses of
the lenses of Daphnia pulex (de Geer), including a microphotometric study of
polarized light transmitted by the lenses. Their results are in agreement with
the Brewster-Fresnel internal reflection model. The present paper examines the
consequences of this model on the behavior of daphnids under polarized light.
We assume that in its response to polarized light, the daphnid moves so that
the rhabdomeres of the forward ommatidia receive maximum light intensity. If
this assumption is true, then the addition of light to the lateral ommatidia should
disrupt the precision of the daphnid response to the polarization plane. The degree
of disruption should be proportional to the amount of light added to the lateral
ommatidia.
For polarization detection, the intensity of light at the rhabdomeres is maximum
when the polarization plane of the incident light is perpendicular to the long axis
of the cone. The forward and lateral ommatidia are perpendicular to each other
in a horizontal plane, and therefore present mutually perpendicular planes of
incidence to a vertical beam of light, as in Figure 1. The Fresnel equations require
that whenever the forward-directed ommatidium has a maximum intensity at the
1 Contribution No. 1264 from the Woods Hole Oceanographic Institution. This research
was supported by a grant from the National Institutes of Health.
243
244
WILLIAM E. HAZEN AND EDWARD R. BAYLOR
rhabdomeres, then the laterally directed ommatidium has a minimum intensity at
its rhabdomeres; the ratio of the intensity is approximately 8:1. Experimentally
changing this ratio by directing horizontal light beams at the lateral ommatidia of
a population of daphnids already orienting to a vertical beam of polarized light
should produce an orientation in which the number of animals directed to the
stimulus of the lateral beam is the same as the number directed by the vertical beam.
We report here three sets of experiments. The first, with nonpolarized light,
shows how a population of Daphnia oriented to two horizontally opposed light
beams (AB in Fig. 2} changes orientation upon the addition of a second pair of
horizontally opposed light beams (CD in Fig. 2) perpendicular to the first. This
experiment tests the validity of the primary assumption on which the Brewster-
Fresnel internal reflection model rests, i.e., that positive phototaxis is guided by
FIGURE 1. Three-dimensional diagram of the Brevvster-Fresnel internal reflection model,
showing two cone lenses at right angles. Polarized light is incident from above with an
incident value of 100. The plane of polarization is parallel to the YZ plane. Numbers represent
intensities at various parts of the light paths.
maximum intensity reception in the forward ommatidia. The second set of
experiments shows how the orientation to a vertical polarized beam is altered by
a pair of horizontally opposed beams parallel to the plane of polarization ; in this
experiment the lateral beam illuminates the lateral ommatidia of those animals
responding to the plane of polarization. The third set of experiments shows how
daphnids which appear to be primarily photonegative nevertheless have a secondary,
weaker positive phototaxis which operates at right angles to the primary and
vigorously negative phototaxis.
DAPHNIA IN POLARIZED LIGHT
PROCEDURE
245
Experimental animals were from a laboratory culture of Daphnia pule.v
(de Geer) grown under constant light and fed a mixture of algae and yeast daily.
Approximately a hundred of these animals were placed in filtered water in a lucite
tank one foot on each side and shielded from stray light in a darkened room. A
projection lamp with lenses and a polarizer hung four feet above the tank and
provided a linearly polarized light beam. A black shield prevented light from
shining on the sides of the tank and being reflected from them. The irradiance of
this beam on the tank was approximately 100 foot lamberts. Two opposed, matched
projection lamps were placed so that their beams were parallel to the plane of
polarization; a second pair was placed perpendicular to the first (Fig. 2). The
brightness of these lamps could be varied with neutral density filters or by a variable
FIGURE 2. Diagram of test tank and its illumination. AB and CD are pairs of hori-
zontally opposed light beams ; E is a vertical beam polarized in ABE plane. CD is perpendicular
to AB.
transformer. Light intensities were measured with a model 501 M Photovolt
photometer.
To record the orientation of the swimming animals during a test, a time exposure
photograph of three seconds was made. The path of each moving daphnid in the
field was represented by a line on the photograph. The directions of these lines
were measured with a protractor, and the measurements were grouped into twelve
intervals of 15° each. The midpoints of these intervals were 0° = (180°, line AB
in Fig. 2), 15°, 30°, 45°, . . . 165°. The 0° azimuth was parallel to the plane of
polarization of the overhead light beam (E in Fig. 2) and also to one pair of hori-
246
WILLIAM E. HAZEN AND EDWARD R. BAYLOR
zontally opposed light beams (AB in Fig. 2). In the experiment without the
overhead polarized light two pairs of horizontally opposed light beams were em-
ployed, one at the 0° azimuth and the other at the 90° azimuth. In another set of
experiments in which only side lights were used, the single pair of opposed horizontal
beams was parallel to the 90° azimuth.
For convenience in discussing and manipulating the data we may calculate an
45° 90° 135°
AZIMUTH OF PATHS
180'
FIGURE 3. The per cent of a population of Daphnia oriented at various azimuths relative
to horizontal, nonpolarized light beams. Solid line represents the data from a single pair of
opposed light beams parallel to the 90° azimuth. Dotted line represents data from two pairs of
opposed beams perpendicular to each other, one pair parallel to the 0° azimuth, the other pair
parallel to the 90° azimuth.
DAPHNIA IN POLARIZED LIGHT 247
index of the angular orientation relative to any given azimuth from the following
relation :
j) p
•L8 ~ ' 1 0-f90 i • i r
•75 — ; — -jc- - = 1. ()., the index ot angular orientation:
where Py is the ratio between paths parallel to the azimuth heading Q and all paths,
and P0+9o° is the ratio of paths parallel to the azimuth heading 6 + 90° and all
paths. The measurements of orientation were grouped by 15° intervals as stated
above. In presenting the data, running averages of three groups are used and thus
orientation for a given angle includes all organisms oriented within 22.5° of that
angle. Since each ommatidium subtends an angle of 50°. greater precision of
orientation implies some integration of receptor information. The index of
orientation can vary from plus one to minus one with zero being an indication of
equal amounts of behavior in both directions, which would include random behavior.
RESULTS
The solid line of Figure 3 shows the orientation of 141 Daplmia to a single pair
of horizontally opposed light beams. The response, with maxima at 75° and 105°,
is approximately parallel to the light beams which are directed along the 90° azimuth.
The average index of orientation at these two peaks is 0.87. The lower index of
orientation at 90° is unexplained but has been reproduced in several experiments.
The dotted line shows the orientation of daphnids to four matched lights 90° apart
in the horizontal plane. The responses to the two perpendicular pairs of beams
are parallel to the beams and are nearly equal with an index of orientation of —0.09.
Results of the experiment with one pair of opposed beams, described by the solid
line of Figure 3, appear to support the assumption that daphnids possess a positive
phototaxis and orient by maintaining maximum light intensity in the forward
ommatidia.
The experiment with two pairs of opposed beams shows that when the front
and lateral ommatidia are equally illuminated, the population of daphnids has equal
numbers of animals orienting to each pair of opposing beams and thereby further
supports the assumption that positive phototaxis is guided by maintenance of
•maximum intensity in the forward ommatidia. The data show that the daphnid
compound eye does not act simply as a receptor consisting of a large number of
parts, each obeying a cosine2 law, where the intensity at the receptor will be equal
to some constant times the cosine2 of the angle of incidence. If the daphnid eye
did obey the cosine2 law, the 4-beam experiment would produce random results.
That the daphnid eye could not obey the cosine2 law is also clear because each
ommatidium subtends an angle of approximately 50°, thereby limiting the angle
through which each ommatidium can receive light directly.
We know from the 4-beam experiment what the response of a population of
•daphnids is when the front and lateral ommatidia are equally illuminated. What,
then, will be the effect of an overhead polarized beam in combination with one pair
of horizontally opposed lateral beams which can be varied in intensity to produce
various ratios of overhead polarized light intensity to lateral nonpolarized light
'intensity?
248
WILLIAM E. HAZEN AND EDWARD R. BAYLOR
30
20
i
10
0
30
\ \
-N-105
\R-200-\
x* 78
'P«O.OOOI
I.O.+0.84
CONTROL
N-II2
" IOI —|
P«O.OOOI
I .0.+0.9I
B
CJ
20
Uj
K
Uj
10
0
30
T \
N- 71
IR-40'1
I
A
/\
\
20
10
0
P< O.OOOI
1. 0. +0.6 1
I I
N-92
IR-100 = 1
xz 77
' P« 0.000 1
I-0.-fO.78
A
N-50
IR-20: 1
X* 12
P<0.25
1 .0.+0.03
— N-76
X2 18 /
P<0.05 /
I.O.-0.047 i
30 60 90 120 150 180 0
DEGREES
30 60 90 120 150
DEGREES
180
FIGURE 4. Orientation of Daphnia at various azimuths relative to the plane of polarization
(0-180°) at six different ratios of intensities of vertical to horizontal light beams. The vertical
beam is polarized and at a constant intensity. The pair of horizontal, opposed beams is parallel
DAPHNIA IN POLARIZED LIGHT
249
TABLE I
A summary of data
Intensity ratio
Number of paths
measured
Chi"
P
Index of orientation
Control
112
101
.0001
+0.91
200:
105
78
.0001
+0.84
100:
92
77
.0001
+0.78
40:
71
56
.0001
+0.61
20:
50
12
0.25
+0.03
10:
99
15
0.10
-0.02
5:1
76
18
0.05
-0.047
2 Beams
136
-0.87
4 Beams
176
0.09
Figure 4 shows six graphs of the response of Daphnia to different intensities of
lateral light, the overhead polarized beam remaining constant. The ratios of the
overhead polarized intensities to side nonpolarized intensities (IR in Fig. 4) were
chosen so that some were higher than the balance point ratio predicted on a
theoretical optical basis, and some were lower. Each graph is plotted with the
data from a control experiment, shown as a dotted line, in which only the vertical
polarized beam is present. The data on the graphs are summarized in Table I.
In the control, Chi square for the null hypothesis that the direction of swimming
is random is 101, giving a probability much less than 1 in 10,000 that the behavior
is random. The Chi square tests for the different intensities of lateral light are
included in Figure 4. An examination of the graphs in sequence from that
showing an intensity ratio (IR) of 200:1 to that of 5:1 shows a gradual change
in orientation. At 200:1 and 100:1 the effect of lateral light intensity is minimal.
At 20:1 the Chi square test gives a probability of the orientation being different
from random orientation of only 0.25, showing that at this ratio the intensity
apparent to the animal is nearly the same parallel and perpendicular to the plane
of polarization. At the ratio of 5 : 1 the taxic response is oriented more toward the
lateral light than it is to the stimulation offered by the polarized beam.
The calculated ratio of overhead polarized intensity to side nonpolarized in-
tensities produced at the rhabdomeres of two perpendicular ommatidia by a vertical
beam of polarized light is 8: 1, as shown in Figure 1. The index of orientation with
this ratio is +0.91. Throughout the range of intensity ratios of vertical to
horizontal illumination there is a graded response. This graded response is best
seen in Figure 5 where the index of orientation is plotted against the intensity ratios.
Because the magnitude of the electroretinogram is proportional to the logarithm
of the intensity of the stimulating light (Hartline, 1930), the intensity ratios of
Figure 5 were plotted as logarithms. The data points seem to fall in a straight line
to the plane of polarized light and varies in intensity. Abbreviations : N is number of animals
used; IR is the intensity ratio of overhead polarized to lateral nonpolarized light beams; x2 is
Chi square value ; P is the probability that the orientation is random ; I.O. is index of
orientation.
250
WILLIAM E. HAZEN AND EDWARD R. JJAYLOR
100.0
IS
10.0-
1.0
-1.0 -0.5 0 -HO. 5 +1.0
INDEX OF ORIENTATION
FIGURE 5. The degree of orientation at various intensity ratios of vertical to horizontal
light beams. The scale of the intensity ratios is logarithmic.
which was drawn by eye. We interpret the intercept of this line with the zero
value of the abscissa to be the ratio at which the intensities in the lateral and forward
ommatidia are equal. From the graph this value is approximately 15:1. By theory
this ratio should be approximately 8:1 as calculated for Figure 1. In view of the
uncertainty of the assumptions made concerning the index of refraction and the
shape of the lenses we think this discrepancy is small.
The data do not, of course, distinguish between the Brewster-Fresnel internal
reflection model and all other models. A single refraction with the light path
direct to visual pigments (Stephens ct al., 1953) would produce maximum intensity
DAPHNIA IN POLARIZED LIGHT 251
in lateral ommatidia when daphnids orient perpendicular to the polarization plane.
On the other hand, a refraction followed by a reflection would produce maximum
intensity in the forward ommatidia when daphnids are similarly oriented. If
responses to polarized light are based on the same physiological mechanisms as
positive phototaxes (and the 4-beam experiment strongly supports this hypothesis),
then the Brewster-Fresnel refraction model of Stephens et al. is ruled out for
daphnids, but the Brewster-Fresnel internal reflection model is not ruled out.
In a further attempt to test the assumption that the orientation to polarized
light is essentially a phototaxic response in which the forward ommatidia are kept
bright, we studied the behavior of daphnids made photonegative by drugs or by
ultraviolet light. In these animals we expected to find orientation parallel to
the plane of polarization rather than perpendicular, but this expected orientation
did not occur. The failure of photonegative daphnids to orient parallel to the
polarization plane has constituted a major criticism of the Brewster-Fresnel
reflection model (personal communication from Colin Pittendrigh and from Rudolph
Jander). Clearly, we must resolve this apparent paradox or abandon the model
altogether.
The paradox may be resolved if two separate and distinct phototaxes are
involved. The primary and obvious phototaxis response of pilocarpine-treated
daphnids to an intense parallel light beam is a vigorous negative phototaxis. The
secondary phototaxic response of these animals is a weakly positive phototaxis
to any dim light beam perpendicular to the intense beam. This paradoxical behavior
of daphnids is consistent with their possession of two separate photoreceptors having
quite different functions (Baylor and Smith, 1957) : the compound eye is sensitive
to polarized light, whereas the naupliar eye appears to control the sign of phototaxis
and geotaxis in response to a number of chemical and physical factors of the
environment. The behavior of daphnids in the natural habitat shows an obvious
adaptive value for these two distinct and separate phototaxes executed approximately
at right angles to each other. A negative and a positive phototaxis to the sun
are presumably useful for guiding vertical migration, and at the same time a positive
phototaxis for light scattered from phytoplankton or other food particles permits
food-finding during the day when daphnids are photonegative. In daphnids made
vigorously photonegative by treatment with 10~6 M pilocarpine, the change in
behavior produced by adding a horizontal beam to the vertical polarized beam was
compared with the same experiments in which the animals were untreated and
photopositive. Results of preliminary experiments show no significant difference
between vigorously photonegative drug-treated daphnids and untreated photo-
positive daphnids. The data points from these experiments fall on the curve of
Figure 5.
We are hopeful of finding another drug which will reverse the secondarily
positive phototaxis normally associated with finding food. When this is done we
may then anticipate that such animals treated in this way will orient parallel to
the polarization plane of an overhead light.
SUMMARY
1. Daphnids illuminated by a single vertical beam of polarized light swam
approximately perpendicular to the polarization plane.
252 WILLIAM E. HAZEN AND EDWARD R. BAYLOR
2. Daphnids illuminated by a single pair of opposed horizontal beams of light
oriented toward the brighter light of the pair.
3. Daphnids illuminated by two pairs of opposed horizontal beams set at right
angles to each other swam in the beam of the brighter pair of light beams.
4. Daphnids illuminated simultaneously by three beams (one polarized and
coming from overhead, the other two nonpolarized and horizontally opposed,
parallel to the polarization plane of the overhead light) responded to the overhead
polarized light when its intensity was greater than 20 times that of the horizontal
beams. When the intensity of the overhead beam of polarized light was less than
20 times that of the horizontal beams, the daphnids responded to the horizontal
opposed beams instead of the polarized beam from overhead.
5. The changes in behavior induced by various intensity combinations of over-
head and horizontal light beams were in good agreement with the changes predicted
from daphnid eye structure.
6. Daphnids exhibiting drug-induced negative phototaxis were shown to
possess simultaneously a secondary weak positive phototaxis always executed at
right angles to the negative phototaxis. This weak positive phototaxis at right
angles to the negative phototaxis is proposed to account for photonegative daphnids
which orient perpendicular to the polarization plane of a vertical beam of light.
LITERATURE CITED
BAYLOR, E. R., AND W. E. HAZEN, 1962. The analysis of polarized light in the eye of Daphnia.
Biol. Bull., 123: 233-242.
BAYLOR, E. R., AND F. E. SMITH, 1953. The orientation of Cladocera to polarized light.
Amcr. Nat., 87: 97-101.
BAYLOR, E. R., AND F. E. SMITH, 1957. Diurnal migration of plankton crustaceans. In:
Recent Advances in Invertebrate Physiology, ed. by B. T. Scheer. Univ. of Oregon
Press.
BAYLOR, E. R., AND F. E. SMITH, 1960. Bees, Daphnia, and polarized light. Ecologv, 41 :
360-363.
HARTLINE, H. K., 1930. Dark adaptation of the eye of Limultis, as measured by its electric
response to illumination. /. Gen. Physiol., 13: 379-389.
STEPHENS, G. C, M. FINGERMAN AND F. A. BROWN, JR., 1953. The orientation of Drosophila
to plane polarized light. Ann. Entomol. Soc. Amer., 46: 75-83.
WATERMAN, T. H., 1960. Interaction of polarized light and turbidity in the orientation of
Daphnia and Mysidiu-m. Zeitschr. vergl. Physiol., 43: 149-172.
KARYOPLASMIC STUDIES IN HAPLOID, ANDROGENETIC HYBRIDS
OF CALIFORNIA NEWTS l
WILLIAM FRANKLIN BRANDOM 2, s
Department of Biological Sciences, Stanford University, Stanford, Calif.
The combination of a nucleus of one species acting in the cytoplasm of another
is theoretically ideal for the study of the roles of the nucleus and cytoplasm in the
differentiation of characters which distinguish the species. The means for achieving
interspecific karyoplasmic combinations has been by heterospermic fertilization of
eggs devoid of active maternal chromosomes. The preponderance of interspecific
karyoplasmic hybrids in amphibians has been androgenetic haploids (Fankhauser,
1955; Moore, 1955). Although repeatedly attempted in the past, these haploids
typically die prior to the appearance of recognizable species characters. Of 21
interspecific androgenetic, haploid hybrid combinations enumerated by Fankhauser
(1955), none developed to a stage permitting an analysis of the relative influence of
the foreign haploid nucleus or the cytoplasm on a specific character.
In a classic experiment, Hadorn (1936) overcame the difficulty of rearing haploid
hybrids by grafting haploid tissue of Triton palniatus cytoplasm and T. cristatus
nucleus to diploid homospermic T. alpestris hosts. The postmetamorphic skin of
palniatus is characterized by projections formed by flattened epidermal cells; the
skin of cristatus is smooth. The grafted haploid hybrid skin on metamorphosed
alpestris hosts possessed projections typical of palmatus, the cytoplasmic donor in
the hybrid merogon. This species character, although it appears late in develop-
ment, has been considered to be "determined" in the egg cytoplasm prior to insemi-
nation, i.e., the character is an expression of the genotype of the diploid oocyte
from which the egg was derived. As was recognized by Hadorn, a complicating
factor in this experiment is that the epidermis of alpestris, the diploid host, also
forms skin protuberances.
Dalton (1946) produced hybrid merogons of Taricha (Trititrus) rivularis
cytoplasm and T. torosa nucleus. The two species differ strikingly in larval pigment
patterns. Dalton transplanted haploid hybrid merogonic neural crest to diploid
torosa hosts. The transplanted haploid hybrid tissue produced a pigment pattern
essentially like that of torosa, the nuclear contributor. However, an early influence
of the cytoplasmic donor, rivularis, was manifested in the rate of melanization and
distribution of the pigment cells.
The circumvention of the early demise of haploid hybrid tissue by transplantation
to diploid embryos has been of value, but in order to rule out the possibility of any
1 Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy
in the Department of Biological Sciences, Stanford University, Stanford, California.
2 Supported in part by Fellowship CF-7379 from the National Cancer Institute, U. S.
Public Health Service.
3 Present address : Department of Zoology, Newcomb College of Tulane University, New
Orleans, Louisiana.
253
254
WILLIAM FRANKLIN BRANDOM
TOROSA
RIVULARIS
TOROSA
1 1
FIGURE 1. Drawings of larval pigment patterns and balancers : a, homospermic diploid
T. torosa (paternal nuclear contributor) ; b, homospermic diploid T. rivularis (maternal
cytoplasmic contributor) ; c, androgenetic, haploid hybrid. Schematic karyoplasmic constitutions
depicted on the left; camera lucida drawings of diploid (22) and haploid (11) chromosome
complements on the right.
influence of the host tissues on the differentiation of species characters, it is still
desirable to obtain whole haploid hybrid larvae. During the course of an experiment
designed to study gene-dosage effects in polyploid hybrids (Brandom, 1960), two
of 54 larvae from Taricha rivularis eggs fertilized with T. torosa sperm were
haploids. Both developed to stages where distinctive species characters were
readily visible. The parent species are advantageous for karyoplasmic studies by
virtue of their distinctive larval pigment patterns and the formation of viable diploid
hybrids. The larval melanophores of rivularis are distributed over the lateral and
dorsal body surfaces (Fig. Ib), whereas those of torosa are confined primarily to
compact dorsal bands (Fig. la). The diploid hybrid is intermediate to the parent
species. Another character, the balancer, is always fully developed in torosa, but
rudimentary or absent in rivularis.
The results of a few selected experiments bearing on the analysis of the localiza-
tion of factors which direct the differentiation of species characters are summarized
in Table I. The conflicting results of these experiments stress the need for further
experimentation that might aid in the clarification of the karyoplasmic problem.
The present report deals with this problem.
MATERIALS AND METHODS
The methods employed have been described in detail elsewhere (Brandom,
1960). Eggs of Taricha rivularis were fertilized by sperm of T. torosa, heat-
shocked at 35° to 37° C. for ten minutes, and returned to room temperature. Those
KARYOPLASMIC STUDIES IN NEWTS
255
FABLE I
The role of the nucleus and the cytoplasm in the
determination of specific characters
Organisms
Author?
Localization of the factors for the deter-
mination of specific characters
Nucleus
Cytoplasm
Haploid, androgenetic hybrids
Sea urchins
Amphibians
Boveri, 1889
Horstadius, 1936
von Ubisch, 1953
Hadorn, 1936
Dalton, 1946
Sambuichi, 1952
Humphrey and Fankhauser, 1957
+
+
Diploid, androgenetic and nuclear-transplant hybrids
Insects
Amphibians
Astaurov and
Ostriakova-Varshaver, 1957
Sambuichi, 1957
McKinnell, 1960
Gurdon, 1961
* Of historical interest; results later re-interpreted (Boveri, 1918).
** Partial, early cytoplasmic effect.
embryos which survived to larval stages were tail-clipped, fixed in Bourn's fluid,
the tail-tips stained in Mayer's acid haemalum and mounted whole. In addition to
direct chromosome counts in the tail-tips, heteroploidy was confirmed by the
comparison of nuclear and cell size in whole-mount tail-tips and microsections.
RESULTS
Heat-treated rivularis eggs (4,369) fertilized with torosa sperm yielded two
haploid, androgenetic hybrids which developed to mid-larval stages (T witty stage
39; see Rugh, 1962). Although exceptional homospermic haploids have been
reared to more advanced stages (Baltzer, 1922; Fankhauser, 1937, 1938), these
are the first haploid, androgenetic hybrid amphibians to develop to a stage where
species characters are discernible.
The controls for this experiment were larvae of the two homospermic diploid
species and diploid rivularis /torosa hybrids. In addition, homospermic haploid
torosa were obtained in heat-shock experiments conducted prior to the hybrid
experiments. The haploid homospermic larvae have a normal torosa pigment
pattern (see Dalton, 1946, p. 195). No homospermic heat-treated rivularis eggs
have developed into haploids (Brandom, 1960). However, it may be assumed,
256
WILLIAM FRANKLIN BRANDOM
O
G
0
O
O
KARYOPLASMIC STUDIES IN NEWTS 257
based on other homospermic haploid experiments, that the pigment pattern of
haploid rivularis larvae would not be qualitatively altered.
The two haploids did not differ noticeably from control larvae in cleavage rates.
Marked developmental difficulties were first noted in yolk-plug and neurula stages.
Large yolk-plugs persisted up to early tail-bud stages, and the neural folds closed
irregularly. Yolk extrusion was observed through wounds in the ventral body
wall of both haploid larvae. In early tail-bud larvae pronounced edema in the
heart, gill, and forelimb-bud regions remained until the time when the embryos
either died or were fixed. Alleviation of fluid pressure by surgical means did not
materially reduce the edemic condition. The fluid imbalance and dwarf appearance
of our haploids are two of the characteristics normally associated with the haploid
amphibian syndrome. Microcephaly occurred in one haploid, but in the other the
head was near-normal when the animal was fixed. No early localized breakdown
in head mesenchyme, a difficulty previously noted in some haploid hybrids of
European Triton (Baltzer, 1930), was found in our material.
1. Tissue and organ architecture
One haploid hybrid ceased development after the appearance of larval species
characters but deteriorated before it could be fixed for sectioning; the other was
fixed in good condition. The nuclei and cells of the haploid hybrid were smaller
and more numerous than those in comparable areas in the diploid controls. This
is illustrated by the outline drawings of tissues in the tailtips (Fig. 2) and micro-
sections (Fig. 3). Limited nuclear pyknosis was observed in the brain but the
haploid central nervous system contained mostly normal cells. The notochord was
bi- and tripartite in some regions ; anteriorly it was single, posteriorly it became
progressively divided by thickening partitions into two and then three divisions.
Duplication of the notochord was previously reported in homospermic haploid
torosa larvae (Dalton, 1940).
The kidney tubules in the haploid were more numerous and contained larger
lumina than those of the diploid control. It is not known whether there is a
functional relationship between the abnormalities of the kidneys and the fluid
imbalance. Rafferty (1961) concludes from homoplastic transplantation experi-
ments (haploid to diploid and diploid to haploid kidney transplants) that factors
other than the haploid kidney are likely to be involved in the fluid imbalance
syndrome.
The shape of cells in the lens of the eye and the nephric duct is more cuboidal
than comparable cells in the diploid control (Fig. 3). The tendency of haploids to
approximate normal organ and body size in the face of decreased nuclear and cell
size is partially achieved by a compensatory adjustment in cell shape and cell number
(Fankhauser, 1955). As might be expected on the basis of observations of
homospermic haploids, the architecture of the heterospermic haploid cells is
subordinated to the achievement of near-normal organ size.
FIGURE 2. Drawings of nuclei from larval tailtips of diploid and haploid hybrids of T.
riimlaris ¥ /< T. torosa <$ : a-e, diploid ; a'-e', haploid. Reading from top to bottom : epidermal
interphase nuclei; mesenchyme cell nuclei; nuclei of lateral-line organs; epidermal glands (dotted
outline) and nuclei; red blood cell nuclei (absent in haploids) X 540.
258
WILLIAM FRANKLIN BRANDOM
a
c
O.lmm
FIGURE 3. Projection-drawings of microsections of diploid (a-e) and haploid (a'-e')
hybrids of T. rivularis ? X T. torosa $ : a, a', low-power drawings of myelencephalon ; b, b',
nuclei from shaded areas of a and a' ; c, c', cells and nuclei of glands of the epidermis ; d, d',
nuclei of peripheral layer of the lens ; e, e', nephric ducts.
KARYOPLASMIC STUDIES IX NEWTS 259
2. Balancer
As is characteristic for mountain stream-dwelling salamander larvae, the
balancer is either rudimentary or absent in rivularis, whereas in torosa this organ
is fully developed (Twitty, 1936). The balancer is always present in the diploid
hybrid of rivularis $ X torosa <$, although it may be reduced in comparison with
homospermic diploid larval torosa. In the heterospermic haploids, the balancer
was fully developed (Fig. 1). Thus, the torosa nucleus acting in rivularis cyto-
plasm directed the development of this organ into a strictly nuclear-donor structure.
3. Larval pigment pattern
The banded arrangement of the larval melanophores of the haploids was
dominantly like that of the paternal nuclear contributor, torosa (Fig. 1). A few
melanophores were visible on the flanks, but they were not in excess of those found
in homospermic torosa larvae. Although a slight effect of the rivularis cytoplasm
cannot be ruled out as a possibility, the random arrangement of the few melanophores
on the yolk area can be ascribed to physical disturbances of the larval pigment
pattern as a consequence of the extreme ventral and lateral body swelling. In
support of the latter alternative, no strong evidence of rivu!aris-\i\<e early pigmenta-
tion was observed.
DISCUSSION
Although the present report is concerned with nuclear-cytoplasmic haploid
hybrids, several experiments involving diploid nuclei of one species acting in the
cytoplasm of another bear on the problem of the differentiation of species characters.
Astaurov and Ostriakova-Varshaver (1957) reported the first adult karyoplasmic
hybrids. Diploid, androgenetic hybrids of Bomby.r mandarina and B. inori were
obtained by temperature shock and x-ray treatment to fertilized eggs. The parent
species differ in distinctive morphological characters. Mandarina caterpillars are
of dark markings while mori caterpillars have different markings depending on the
race. Mandarina moths are dark greyish-brown, while those of mori are white
or cream-colored. In the mandarina cytoplasm plus mori nucleus combination, the
species characters were all like those of mori. The cytoplasm did not visibly affect
the species characters of the hybrid. None of the hybrids of mori cytoplasm plus
mandarina nucleus developed to images, but four individuals were typically nuclear-
like in body size, larval markings, and other characters.
Employing the nuclear transplantation technique of Briggs and King (1953),
Sambuichi (1957) transplanted diploid nuclei of Rana nigromaculata brevipoda into
enucleated eggs of R. n. nigromaculata. Larval character differences in these two
subspecies include tadpole color, labial tooth formula, and shape of the tail. The
young metamorphosed frogs differ in dorsal and ventral color pattern. The diploid
hybrids are intermediate to the parents in all the characters. With one exception,
the embryos, tadpoles, and young frogs of nigromaculata cytoplasm plus brevipoda
diploid nucleus contained only characters of the nuclear-donor subspecies. The
exceptional individual later became brevipoda-\ike.
McKinnell (1960) transplanted nuclei of kandiyohi dominant-mutant Rana
pipiens into wild-type Rana pipiens egg-cytoplasm. Three of the intraspecific
260 WILLIAM FRANKLIN BRANDOM
karyoplasniic chimeric tadpoles underwent metamorphosis and each had pigment
patterns similar to the nuclear donor, kandiyohi.
Gurdon (1961) transplanted nuclei between two subspecies of Xenopus laevis
(X. I. lamis and X. 1. inctorianits). The two subspecies differ in the time of
appearance of the larval body and anal melanophores and in postmetamorphic color
and color patterns. The nuclear transplant larvae and frogs all showed the
distinguishing characteristics of the subspecies which provided the nucleus.
Returning to the haploid experiments, Boveri (1889) first attempted combining
the nucleus of one species with the cytoplasm of another by fertilizing egg frag-
ments of Sphaerechinus granularis with sperm of Parechinus microtuberaculatus.
Boveri's pioneer work on sea urchins was criticized on several counts by Morgan
(1895) and Seeliger (1896) and, upon repeating his earlier experiments, he showed
that it was not possible to produce viable haploid hybrid merogons that would
develop beyond gastrulation (Boveri, 1918). The limited development of whole
haploid, androgenetic hybrid sea urchin embryos was partially overcome by
Horstadius (1936). He surgically combined the presumptive skeletal micromeres
of haploid Paracentrotus 5 X Psammechinus <§ hybrid with homospermic ectodermal
and endodermal cells of Paracentrotus. In these germ-layer chimeras, the larval
skeleton resembled the species which furnished the nucleus of the skeletal cells.
More recently, von Ubisch (1953) obtained good merogonic hybrid plutei of
Sphaerechinus cytoplasm plus Psammechinus (or Paracentrotus} nucleus. Skeletal
characteristics and ciliated bands of the merogons all showed characters of the
species which contributed the nucleus.
Finally, Humphrey and Fankhauser (1957) produced intraspecific haploid
hybrids between wild, dark (DD) and recessive, white (dd) axolotls by cold-shock
treatment of fertilized eggs. The embryos were predominantly white haploids, the
recessive color of the males and therefore of androgenetic origin. Only one dark
haploid was obtained, presumably of gynogenetic origin.
Concerning temperature shock as a means of inducing androgenesis, Book
(1945) has proposed that cold shock, if it affects the egg when it is in the second
anaphase, may cause a paralysis of the spindle. According to this hypothesis, the
egg chromosomes remain in the anaphase without being able to reorganize a resting
nucleus. A return to normal temperature activates the sperm nucleus ; the egg
nucleus anaphase configuration does not have the same attraction for the sperm
nucleus as does the metaphase, and the result is that the centrosome of the sperm
nucleus divides, resulting in a haploid embryo with paternal chromosomes. The
mode of elimination of the maternal chromosomes in the androgenetic hybrids
reported herein is not known. However, since both of our haploids were andro-
genetic and all of Humphrey and Fankhauser's (1957) axolotls were androgenetic
except one, some such mechanism may be operating in the great majority of
haploids derived from temperature-shocked amphibian eggs.
The architecture of the heterospermic haploid tissues was the same as was
observed in homospermic haploids (Fankhauser, 1955). Compared to the diploid
hybrid controls, the cells of the heterospermic haploids are greater in number but
smaller in volume (Figs. 2 and 3). Adjustment in cell shape in single-layered
tissues and organs in order to maintain near-normal organ size also agrees with
prior observations on homospermic haploids (Fankhauser, 1945).
KARYOPLASMIC STUDIES IN NEWTS 261
The embryological basis for specific larval pigment patterns has been extensively
investigated by extirpation, transplantation, and tissue culture experiments (Twitty,
1945, 1949; Twitty and Niu, 1948, 1954). Both in situ and when explanted in
coelomic fluid, torosa melanophores migrate out, become highly melanized, and
then secondarily reaggregate. Under the same conditions rivularis melanophores
neither differentiate as fully nor reaggregate as strongly as do those of torosa. The
two species also differ in the number of larval chromatophores ; rivularis melano-
phores are more numerous than torosa. These and other findings of Twitty and
his co-workers permit us to consider the qualitative changes in the larval pigment
patterns in the haploid hybrids as due to quantitative, gene-mediated changes in
the pigment cells themselves.
A genetic effect of the single torosa genome acting in rivularis cytoplasm was
discernible in the number of larval melanophores. Although difficult to quantitate
because of the secondary banding, there were fewer melanophores in the haploid
hybrids than in the diploid rivularis /torosa larvae. This suggests that the nuclear-
donor species (torosa) is exercising a strong action that tends to override a typical
consequence of haploidy. Ordinarily, the number of larval pigment cells is greater
in homospermic haploids than in homospermic diploids (Fankhauser and Schott,
1952).
The melanophores in the two haploid hybrids were densely pigmented like
those of homospermic, diploid torosa larvae. Hence, a diminishing effect on the
melanization of the larval melanophores by the rivularis cytoplasm was not seen.
Interpreted in the light of Twitty's findings, the higher grade of differentiation of
the haploid pigment cells (visibly manifested by their highly melanized state)
qualitativly affected the pigment pattern. The aggregation into dense dorsal bands
in homospermic torosa is due to the retraction of intercellular processes and occurs
only with the attainment of advanced melanophore differentiation characteristic for
this species (Fig. la). The larval pigment pattern of the haploid, androgenetic
hybrids indicates that the torosa nucleus was the locus of the factors which
determined this larval species character (Fig. Ic).
The fully developed balancer in the heterospermic haploid (which is absent or
rudimentary in rivularis} emphasizes the strong directive influence of the torosa
nucleus in the progressive acquisition of this species character.
The lack of species characters of the cytoplasmic donor, rivularis, does not
exclude the possibility that the cytoplasm produced profound but unseen effects on
the propigment and balancer cells before stages when these cells were well dif-
ferentiated, and subsequently assumed the larval pigment pattern and balancer
characteristic for the nuclear-donor species. These results do show that the
cytoplasm does not materially affect the specific characters of whole haploid
rivularis /torosa hybrids during those stages when the visual recognition of species
characters can be made.
The author is grateful to Dr. Victor C. Twitty for direction, support, and
encouragement during this investigation. Thanks are also due Dr. Gerhard
Fankhauser for appraising the manuscript in its incipient form.
262 WILLIAM FRAXKLIX BRAXDOM
SUMMARY
Two species of West Coast newts differ strikingly in larval pigment patterns.
Taricha torosa has a banded arrangement of the larval melanophores ; in T. rivularis
the larval melanophores are dispersed. Torosa is also characterized by a well
developed balancer, whereas in rivularis the balancer is either absent or rudimentary.
1. Two of 54 heat-shocked, interspecific hybrids of T. rivularis $ X T. torosa £
were haploids. The two haploids are the first amphibian androgenetic, haploid
hybrids to develop to stages where species characters could be observed.
2. The tissue and organ architecture of the heterospermic haploids conform to
prior findings in homospermic haploids. The nuclei and cells are smaller and more
numerous than in the diploid controls. A compensatory adjustment in cell shape
as well as cell number was observed in single-cell layered organs.
3. The balancer was fully developed in the heterospermic haploids, thus indi-
cating a strong directive influence of the nucleus (torosa} in the formation of
this organ.
4. The larval pigmentation was dominantly like the nuclear-donor species in
the number, degree of melanization, and pattern formation of the melanophores. No
evidence was found of an influence on pigmentation by the cytoplasmic-donor species.
5. The above findings are discussed in relation to other studies on the roles of the
nucleus and the cytoplasm in the differentiation of species characters.
LITERATURE CITED
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genesis in silkworms as a means for experimental analysis of the nucleus-cytoplasm
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BALTZER, F., 1922. Ueber die Herstellung und Aufzucht eines haploiden Triton taeniatus.
Vcrhandl. Schwciz. Natitrforsch. Ges., Bern, II Teil, 248-249.
BALTZER, F., 1930. Ueber die Entwicklung des Triton-merogons Triton taeniatns (?)
X cristatus (J1). Rev. Snissc Zoo/., 37: 325-332.
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Sitzbcr. Gcs. Morph. Physiol., Milnchcn, V.
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merogonischer und partiell-merogonischer Seeigelbastarde. Arch. f. Entw., 44: 417-471.
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Zool., 143: 323-345.
BRIGGS, R., AND T. J. KING, 1953. Factors affecting the transplantability of nuclei of frog
embryonic cells. /. Exp. Zool, 122: 485-506.
DALTON, H. C., 1940. Experiments on the development of haploid salamander embryos.
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salamander, Triturus pyrrhogastcr. Genetics, 22: 192.
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KARYOPLASMIC STUDIES IN NEWTS 263
FANKHAUSER, G., AND B. W. SCIIOTT, 1952. Inverse relation of number of melanophores to
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105-120.
GURDON, J. B., 1961. The transplantation of nuclei between two subspecies of Xenopus laevis.
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RESPONSES OF THE PLANARIAN, DUGESIA,
AND THE PROTOZOAN, PARAMECIUM, TO VERY WEAK
HORIZONTAL MAGNETIC FIELDS1
FRANK A. BROWN, JR.
Department of Biological Sciences, Northwestern University, Evanston, Illinois
The question of whether living things are sensitive to terrestrial magnetism has
undoubtedly fleeted through the minds of innumerable persons since this geophysical
factor first became known. But neither the naturalist, observing the behavior
of organisms in the field during their continuing responses to the myriads of more
obvious physical factors, nor the experimental biologist, casually testing the response
of living things to artificial magnetic fields, even very strong ones, found any
consistent evidence that living creatures perceived this weak terrestrial force. It
is a common observation that animals in nature may come to bear at any given
moment apparently all possible compass relations in their bodily orientation ;
orientation of the normal resting or foraging animal to the horizontal component
of magnetic field would be expected generally to be of no adaptive consequence.
In recent years, however, two kinds of phenomena have come to the forefront in
biological research which are exceedingly difficult, in some instances impossible,
to account for in any orthodox physiological terms. These two phenomena are
the apparent persistence, in rigorously controlled constancy of all the more obvious
factors of the physical environment, of biological senses of time and space in their
terrestrial relationships. The first includes the mechanism for timing the well-
known persistent daily, tidal, monthly, and annual periodisms. The second involves
the still completely mysterious "map sense" or capacity to localize position in space
which is so evident in a wide spectrum of kinds of animals capable of "homing."
Also demanding a rational explanation is the means by which organisms, even
when screened from fluctuations of every obvious weather-related factor, still display
variations in metabolic rate, even of quite substantial magnitudes, correlated with
the essentially aperiodic weather disturbances (Brown, 1959, 1960). There
remains no reasonable doubt that organisms are sensitive to some subtle geophysical
factors which pervade the ordinary "controlled" conditions of the physiology labora-
tory. Fluctuations in these unidentified factors must bear information related to
weather changes, as well as to terrestrial time and space.
In a series of recent experiments, it has been demonstrated that the marine mud-
snail, Nassarius obsoletus, is able to perceive small changes in strength of the
horizontal component, H, of a magnetic field close in strength to that of the earth's
natural field (Brown, Brett, Bennett and Barnwell, 1960; Brown, Webb and Brett,
1960; Barnwell and Webb, 1961). The snail can, furthermore, distinguish between
directions of the fields, both the earth's and weak ones produced by bar magnets
1 This study was aided by a grant from the National Science Foundation, G- 15008, a grant
from the National Institutes of Health, RG-7405, and by a contract between the Office of Naval
Research, Department of Navy and Northwestern University, 1228-03.
264
RESPONSE TO WEAK MAGNETIC FIELD
265
FIGURE 1. The apparatus employed in the study of response of Dugesia to magnetism,
showing arrangement of (A) 7s -watt opalescent lamp; (B) sleeved light-conducting glass
tubes; (C) Petri dish centered over a polar coordinate grid.
(Brown, Bennett and Webb, 1960; Brown and Webb, 1960; Brown, 1960; and
Brown and Barnwell, 1961). The character of the response to magnetic field
exhibits rhythmic changes that are regulated by the solar-day and lunar-day "clocks"
of the snails, and by a synodic monthly one. The following study was conducted ta
learn whether such responsiveness was of wider biological distribution.
METHODS AND MATERIALS
The common, small flatworm, Dugesia dorotocephala, was selected as an animal
possessing convenient behavioral characteristics for study, and simultaneously being
non-marine and phylogenetically very distantly related to the snails. The worms
were collected twice a year, in early October and in June, from a small spring-fed
stream in Fox River Grove, Illinois. They were maintained in darkened containers
in the laboratory, and fed liver twice weekly.
The apparatus consisted of a 3f-inch glass Petri dish centered over a polar
coordinate, paper grid (Fig. 1). This apparatus was set inside a black-lined
wooden cabinet ; use of ferromagnetic materials was carefully avoided in the whole
assembly. The apparatus was continuously illuminated from a 0.5-inch circular
source about 16 inches directly above the center of the grid, provided with a 7^-watt
opalescent lamp which yielded an illumination of about five lux at the Petri-dish
level. A second small light provided a weak horizontal source of illumination
266
FRANK A. BROWN, JR.
parallel to the zero axis of the grid, and a third, horizontal weak light source
provided illumination parallel with the 90° axis, from the right side. The hori-
zontal light sources were onion-skin paper-covered ends of black-sleeved, 10-mm.
solid glass rods leading light into the cabinet from a 71 -watt opalescent lamp attached
rigidly to the outside of the cabinet. To minimize other, uncontrolled illumination
sources, the experiments were conducted in a darkened room.
In operation, a planarian was quickly oriented with a fine brush toward the zero
axis of the polar grid, just short of its origin, as illustrated in Figure 1. The
deviation in worm path from the initial direction was then recorded in terms of
the point, to the nearest 5°, at which the worm crossed the circular arc one inch
from the origin. Measurement of the times to reach the one-inch line was found
for 22 paths to average 15.45 seconds; standard deviation was ±2.02 seconds.
There were no obvious orienting cues in the system other than the two horizontal
light sources. The single vertical light source provided no cue. Any uninten-
NORTH -DIRECTED 4 GAUSSES NORTH -DIRECTED EARTH'S FIELD
EAST-DIRECTED
4 GAUSSES
Z +
<
2$
A.
0
B.
c.
25°
20°
20°
0
,5°
15°
r
10°
o °
' ,
'
10°
0
O <
o
5°
o
0 o o
o
0
o •
o
• 0
o
• o • o
0 ° 0 0 o
0 "
0 0
o
0 0
0
• 8 c ' 8 ?
5°
0
O 0
0 .
Q_
• • o
~ • 0
0
o o
° o
o
• 0
« ° °
0 0
5°
1 ° *
O (
8 o *
o
s
O
5°
• 0
o °
•
o
• 0
o •
° 0 0 C
10°
.
O
10°
15°
»
•
15°
-\o
±90
±I80°0 ±90°
ELONGATION OF MOON
±180 0
FIGURE 2. The mean angular path of initially N-directed planarians as a function of
elongation of the moon (0° = New Moon; 180° = Full Moon); with (A) a North-oriented
magnetic field of 4 gauss, (B) the earth's field alone and (C) an experimental field of 4 gauss,
-with South pole directed Eastward. Solid circles, first experimental series (November and
December) ; open circles, repetition of experiment (January).
tional bias due to asymmetry of any other factors, such as extraneous reflected
illumination, remained essentially unchanged throughout the period of
•experimentation.
Beneath the apparatus were calibrated slots into which an 18-centimeter Alnico
bar magnet could be placed horizontally at distances to produce any one of a
•series of horizontal field strengths, with the south pole rotatable to any desired
compass direction to supplement, subtract from, or otherwise modify, the natural
horizontal field.
It should be recalled at this time that by convention of physicists, the earth's
northern pole is a magnetic S pole and the southern pole, a magnetic N one.
RESPONSE TO WEAK MAGNETIC FIELD
267
ORIENTATION OF NORTH-BOUND WORMS IN A SINGLE, VERTICAL-LIGHT FIELD-
During about a two-month period from November 8, 1960, through January 2r
1961, 4677 planarian paths were recorded,2 2172 in the earth's field alone and 1766
in a four-gauss field, with the S pole directed East. The apparatus was at all times
directed magnetic north. A single horizontal light, that one parallel to the zero*
axis of the grid, was used. It was left on only until the moment the worm
reached the origin. It was then extinguished. The observations were made on
about 24 occasions, never more than one series on any given day, distributed over
the whole period. In very few cases, with too small sample sizes on a given day,
the data were combined with those of the succeeding day. The observations were
always made sometime between 9 A.M. and 3 P.M., to minimize any daily rhythmic
change that might occur, comparable to that previously demonstrated for the snails.
For each sample series 45 to 140 paths were recorded for each in the earth's field
alone -and in the E-W four-gauss experimental field.3 On six occasions over the
TABLE I
Planarian paths
No magnet
E. W. magnet
N. S. magnet
N
Mean
a
N
Mean
a
N
Mean
<r
Nov. 25-6
82
+ 10.00
38.2
85
+2.60
25.0
85
- 3.24
35.1
Nov. 28
83
+ 9.50
32.2
59
+2.55
23.1
53
+ 4.06
39.9
Nov. 29
51
+ 10.20
29.6
46
+4.80
26.5
55
-10.45
43.7
Nov. 30
98
+ 6.42
36.2
77
+0.58
25.6
89
+ 5.17
39.4
Dec. 1-2
140
+ 8.03
33.4
72
+2.43
19.9
77
+ 5.20
47.0
Dec. 3
57
+ 5.80
36.6
47
+3.51
22.4
54
+ 2.32
35.7
a
ff
<T
Range 29.6 - 38.2°
Range 19.9 - 26.5°
Range 35.1 --47.0°
M = +8.25 ± 1.53°
M = +2.68 ± 1.22°
M = -0.85 ± 2.00°
<r = 34.6 ± 1.08°
o- = 24.0 ± 0.87°
<r = 40.6 ± 1.42°
period November 25 through December 3, and on seven occasions from December
19 through January 2, each observational series included the recording of paths
under three conditions. These were in the aforementioned two fields, and now, in
addition, in a four-gauss field with the S pole directed north. This last field
augmented the earth's natural 0.17-gauss north-directed horizontal one. The total
number of paths observed in the N-S field was 739.
2 The author wishes to acknowledge his indebtedness to the several persons, especially to
Young H. Park, Bertil S. Thunstrom, and Andrew Bertagnolli, who devoted many hours to
acquiring the data of this study.
3 The stated horizontal field strengths in this report are the fields present at the level of
the worms when the bar magnet is oriented to oppose the earth's own horizontal vector and
differs from these in the expected manner as it is rotated to other directions in the earth's 0.17-
gauss field. The values are accurate only to about ±10% as a consequence of pole-strength
variations among the individual magnets employed. The field strengths were initially computed,
but later were confirmed with a Rawson gaussmeter.
268
FRANK A. BROWN, JR.
A second and more systematic study, involving 3493 worm-paths, 1186 in the
earth's field, 1181 in the N-S, and 1126 in the E-W magnetic fields, was conducted
between December 28, 1960, and January 31, 1961. There were 29 observational
series, involving 29 different days ; each series included observations under each
of the three conditions of experimental fields.
The mean path of the worms in solely the earth's field exhibited throughout the
three-month period a synodic monthly fluctuation, the worms turning maximally
counterclockwise at the time of new moon and maximally clockwise over the fort-
night centered on full moon. In Figure 2B, the solid circles indicate the mean
path in degrees as a function of elongation of the moon (angle between sun-earth
and moon-earth axes) for the worms in the earth's field for the first experimental
study, and the open circles for the repetition of the experiment, or second study.
In Figure 2C are the mean paths at the same times as measured for the four-gauss
E-W experimental field, and in Figure 2A for the N-S experimental field. In every
20-
u
Ld
D 40
a
LJ
cr
^20
'RT
FIELD
FIELD
-75° -50*
DUGESIA
-25° 0 +25"
PATH A NGLE
+ 50* +75*
4-GAUSS FIELD
FIGURE 3. Frequency distributions of planarian paths in the 4-gauss N-S field (upper) and
E-W field (lower), during the period November 25-December 3, 1960 (see Table I), a period
during a late autumn full-moon semi-month.
single daily series in the first set of experiments, when the worms were turning
clockwise in the earth's field, the experimental E-W field effected less clockwise
turning, and when the worms were turning counterclockwise, the E-W field effected
less counterclockwise turning. This is illustrated, in part, in Table I for the
relatively stable values obtained during the full-moon semi-month, November 25-
December 3.
The worms were apparently able to distinguish between the E-W and N-S four-
gauss fields on the one hand, and between either of these and earth's field on the
other, as is evident both from inspection of Figure 2 and from the statistical analyses
shown in Table I. In Table I, it is seen that the mean paths of the worms in the
E-W and N-S fields were not significantly different from one another, but the
standard deviations were clearly so. The increased standard deviation in the
N-S field over the E-W one reflected a conspicuous increase in turning, but now
RESPONSE TO WEAK MAGNETIC FIELD
269
both clockwise and counterclockwise. The effect is evident from comparison of
the two frequency distributions in Figure 3.
The planarian orientational response in the earth's field alone appeared, during
this study, to be a function of elongation of the moon, over nearly three 360° cycles.
The relationship seemed to be roughly symmetrical about 0°. The limits of clock-
wise and counterclockwise response appeared, within the error of measurement, to
be phase-synchronized with the upper and lower lunar transits.
From Figure 2, it is evident without need of recourse to statistical analysis that
the monthly cycle, so obvious in the earth's magnetic field alone, was partially
suppressed by the 4-gauss E-W field, and abolished by the 4-gauss N-S one.
JAN 1961
FEB
ICf
ICf
15
25°
35°
I AUG
I OCT
if
25°
35°
DEC
I JAN 1963 I FEB
25°
35°
FIGURE 4. Variations in the character of the monthly orientation rhythm of Dugesia over
about a 17-month period. From November through January, 1960, the worms were in a
single vertical light field ; each point was the average of 45 to 140 paths. For the remainder
of the time, the worms were in a three-light field with each point the average of 15 paths.
MONTHLY RHYTHM IN NORTH-BOUND WORMS
The monthly variation in the path of the North-directed planarians during late
morning and early afternoon hours was followed for more than an additional year.
Data were obtained from late March through August by sorting out the numerous
control samples of North-bound worms in the earth's field alone from experiments
involving responses of Dugesia to modified fields. The latter included (1)
changes in compass direction in the earth's field, (2) rotation of a horizontal, experi-
mental 10-gauss field while animals remained continuously magnetic-North directed,
and (3) differentiation of magnetic axes of horizontal fields as related to hori-
270 FRANK A. BROWN, JR.
zontal field-strength. These latter results have been reported earlier (Brown,
1962a) in a preliminary account, but will be described in more detail below.
For all these later experiments from April, 1960, onward, the illumination was
different from that of the initial experiments. The worm's orientation was observed
in a steady three-light field ; the three lights were at 90° to one another in the
arrangement described earlier.
The mean path of the worms always involved counterclockwise turning in
response to the weak light on the right. This illumination pattern was adopted
because the variance of paths was found to be less. Each sample now comprised
always the mean of only 15 worm paths.
In Figure 4 are shown the mean paths in degrees plotted against day of the
year and phase of moon from November 10, 1960, through to April 14, 1962. The
clear monthly cycle, with maximum counterclockwise turning on the day of new
moon and maximum clockwise turning about the time of full moon, is again
evident from November 10 through January 31. Here each point is the mean of 45
to 140 worm paths. No data including mean paths of worms North-bound in the
earth's field alone were obtained between January 31 and March 31.
North-bound worms in the earth's field alone exhibited a monthly variation
between March 31 and about the middle of May, but now the mean path of the
worms, in the three-light field, was steadily to the left and the turning relationship
to lunar phase was the mirror image of the earlier observations during the
preceding late fall and winter. Scatter of the mean paths was substantially greater
than during the preceding period of study.
By early June, there was a suggestion of a tendency for a maximum in clockwise
turning to occur near the times of both new and full moon. By the latter part of
June, the scatter of the mean paths had become even greater and continued so for
three or four months. There was suggestion from inspection of the data, however,
of a tendency for counterclockwise turning to be greater over the quarters of the
moon than over the times of new and full moon. A quantitative analysis of the
paths from June 15 to August 31 proved those for the seven-day periods centered
on the moon's quarters to be —26.48 ± 0.384 (N = 157) and those for seven-day
periods centered on new and full moon to be —24.21 ± 0.393 (N — 136). The
difference between these two was highly significant (t = 4.1). In other words,
there appeared to be a low amplitude semi-monthly fluctuation during the summer
with maximum clockwise turning at both new and full moon.
By late August, there was an abrupt inversion in lunar relationship to yield a
maximum in counterclockwise turning at full moon, and another near new moon
early in September. There was thereafter a gradual return to a clear monthly
fluctuation with maximum left turning at new moon and right turning at full
moon. The monthly fluctuation became progressively more sharply defined, with
scatter of mean paths reduced, between September and November. By the latter
time, the overall form and phase relations of the monthly variation had become, and
remained through the winter, qualitatively like those which obtained for the cor-
responding months of the preceding year.
By early March, there was an abrupt alteration in phase to give a maximum in
clockwise turning at new moon. This inversion, which seems to have been
anticipated during the preceding two months, judging from the gradual decreasingly
RESPONSE TO WEAK MAGNETIC FIELD
271
DUGESIA
0
15°
0
0
^8
0
o
10°
0
00
OQgO
o
0
o
0
o
0
CO
6*
5°
8
o
I
0
0
•wJ
O
o:
CD
0
h-
0
o
0
o
z
o
o
o
u
0
0
0
0 5°
CC
cf
oo
u.
o o
OQO
o
i ,
0
cP
0
LL-
5 "°
r? O
0
15°
§
o
0
20°
-
0
25°
-
1 1
1
1
N
DIRECTION OF 10-GAUSS
W
FIELD
FIGURE 5. Difference between the mean paths of Dugcsia, initially directed Northward in
the earth's field, and the comparable paths when experimental, horizontal 10-gauss fields, with
South-pole directed in each of four compass directions, are superimposed. Path angle is
expressed as difference from interpolated controls in the earth's field alone.
272 FRANK A. BROWN, JR.
strong left turning at new moon, suggested the initiation of a mirror-imaging of
a monthly cycle during April to June, comparable to that observed the corresponding
months of the preceding year. The variations in form of the monthly rhythm clearly
suggest an annual component.
ORIENTATION TO A TEN-GAUSS HORIZONTAL FIELD
Between March 31 and May 5 the response of Dugesia to a 10-gauss horizontal
field was determined as the field was rotated by 90° intervals while the orientation
apparatus itself remained steadily directed magnetic North. On 20 mornings series
were run consisting of each of four directions of the experimental field, with a
"control" in the earth's field alone following each 10-gauss exposure. The effects
of the 10-gauss field, with S-pole directed N, E, S, and W, expressed as differences
from the average path of the four controls in the same-day series, is illustrated in
Figure 5. The worms clearly differentiated between parallel and right-angle fields,
and between N- and S-directed fields.
A COMPASS-DIRECTION EFFECT IN EARTH'S FlELD ALONE
Another experiment with Dugesia was very informative. This was performed
during the period from June 15 through September 12. It involved simply ro-
tating the orientation apparatus by 90° intervals in a darkened room, in the earth's
field alone. In this experiment, performed on 30 different afternoons distributed
over the three-month period, each series included all four compass directions, in
shuffled order, followed at once (in every instance but one) by a repeat of the
four, again shuffled. When average path for each compass-direction was computed
as the difference from the mean for the four directions of that particular group of
four, the results shown in Figure 6 were obtained. The worms in the earth's field
alone clearly distinguished between N-S and E-W orientations. However, the
results obtained in the earth's horizontal field of 0.17 gauss and illustrated in
Figure 6 are essentially the mirror-image of those depicted in Figure 5, which are
the results from the rotation of the 10-gauss field. The significance of this differ-
ence was clarified from the results of the following experiment that was run
concurrently.
RELATION OF HORIZONTAL FIELD-STRENGTH TO ORIENTATION
This experimental series was run on 24 mornings distributed uniformly over
the three summer months. In this one, in shuffled order but always starting and
ending with a control, and with a third control midway in the series, were experi-
mental horizontal magnetic fields of four strengths — 0.25, 2.0, 5.0, and 10.0 gauss,
with South pole directed North and West. Figure 7 illustrates the results obtained
by taking the difference between N and W. It is clear that between the three
weaker fields on the one hand and the 10-gauss one on the other, there is a re-
versal in relative left-turning influence. The difference between response to N-
directed and W-directed fields increases with a positive sign up to some limit, as
the experimental field increasingly overrides the earth's, but then abruptly adopts
a negative sign somewhere between 5 and 10 gauss.
RESPONSE TO WEAK MAGNETIC FIELD
273
8°
I
H
z
<
LJ
u.
u.
8
DUGESIA
0
0
o
8
N
0
0
8
&
rO
O
00
o
o
O
O
O
O
O
3°°
8
E S
COMPASS DIRECTION
00
o
o
o
CO
0©0
8
o
o
CD
o
W
FIGURE 6. Difference between mean path of planarians for each four compass directions
and the mean path in the same series for all four directions taken together, as the orientation
apparatus is rotated to each of the four compass directions, in shuffled order, in the earth's
field alone.
274
FRANK A. BROWN, JR.
o
-J
LJ
O
LJ
h-
o
u
a
Q
I
I
QC
O
1
L.
U.
Q
" DUGESIA
15°
o
00
o
10
-
oo
0
0
0
0
0
5°
Go 0
80
<0
9^ °
oo
o
0
o
00
0
00
c®> °
8
8U
0
0
0
0 0 000
0
5°
o
§° 8 &
o
o
0
O R r>
10°
* ^
0
0
o o
8
15°
0
0
20°
o
0
1 1 1
1
0.25
2. 5.
WEST-DIRECTED
FIELD
10.
(GAUSS)
FIGURE 7. Difference between the mean paths of Dugesia initially directed Northward in
the earth's field and simultaneously subjected to experimental N-directed fields to supplement
the earth's to yield the values indicated, and the mean path resulting from rotation of the
supplementing magnet 90° in a counterclockwise direction.
RESPONSE TO WEAK MAGNETIC FIELD
275
RESOLUTION OF MAGNETIC FIELD DIRECTION
One final experimental study was conducted with Dugesia between June 20 and
August 16, 1961. Two observers were involved, working concurrently. The ex-
periment was performed on 21 different mornings distributed over the two-month
period. For each daily series 15 worms were observed moving compass-North
under each of 11 conditions presented in shuffled order. The observers were
wholly uninformed of the conditions which obtained at the time of their observations.
The eleven conditions included seven in which a 5-gauss horizontal field was
presented at each of seven orientations at 15° intervals from S-pole directed North
to S-pole directed West, and four in which the magnet was removed and the worms
moved North in the earth's field alone.
2!?
\-
<
Q.
LJ
27°
29"
30^
CONTROLS
0"
N
30^
60
75C
90°
W
DIRECTION OF S - POLE (5 GAUSS)
FIGURE 8. Open circles illustrate the relationship of mean path to magnet orientation
for magnetic-N-directed Dugesia in a three-light field and subjected to an experimental 5-gauss
field with S-pole changed by 15° intervals from North to West. The means for each of four
successive controls in the series, for the earth's field alone, which were interpolated in random
order in each experimental series, are indicated by the solid circles. Standard errors of the
means are shown.
276
FRANK A. BROWN, JR.
TABLE II
Path deviations from controls on same day
Angle
M
S.E.
X
Variance
0°
+ 1.50°
±0.97
42
34.4°
15°
+ 1.14°
±0.76
42
23.0°
30°
-0.36°
±0.65
42
17.1°
45°
-1.48°
±0.77
42
23.6°
60°
-2.63°
±0.83
42
26.4°
75°
-3.84°
±0.78
42
24.6°
90°
-4.46°
±0.85
42
27.4°
The mean paths of experimentals and controls for the two-month period are
plotted against the conditions, in Figure 8, together with standard errors of the
means. It is evident that the mean path of the worms was a function of the angle
of the experimental horizontal field.
The standard errors are relatively large, in some measure a consequence of
systematic fluctuations in paths of all worms, both controls and experimentals, from
day to day. These latter fluctuations included a highly significant semi-monthly
component. Consequently, it was not surprising to find, as shown in Table II, that
when the mean paths of the experimentals were treated as deviations from the mean
path for the four controls in the same series, significantly smaller errors were
observed.
Two other facts were notable. As shown by Table III, the variance of the 42
mean paths in an experimental magnetic field was in every instance substantially
greater than for any one of the four controls. The presence of a 5-gauss field sig-
nificantly (P < .005) increased variance over that of controls. And whether one
deals with variances of the actual mean paths (Table III) or variances of devi-
ations from control paths (Table II), minimum variance is observed in this ex-
periment when the worms tend to move in a path most nearly parallel with the
5-gauss horizontal field. The differences in Table II between the variances at 30°
and 0° are statistically significant as determined by the test (P < 0.01), as is also
that between 30° and 90 °^ (P < .05).
TABLK 1 1 1
Variances and mean paths
With magnet
Orientation
Variance
Mean path
Variance
Mean path
0°
36.20°
23.5°
I 19.08°
24.7°
15°
34.46°
23.9°
30°
31.05°
25.4°
11 26.80°
25.1°
45°
44.40°
26.5°
60°
32.82°
27.9°
III 22.57°
25.2°
75
40.06°
28.9°
90°
44.20°
29.5°
IV 25.95°
25.1°
Controls
RESPONSE TO WEAK MAGNETIC FIELD
277
RESPONSE OF PARAMECIUM TO A 1.3-GAuss FIELD
It was of interest to learn whether a single-celled form exhibited such orienta-
tional responses. Paramecium caitdatnm was permitted to escape from the exit
of a magnetic-South-directed, minute, funnel-shaped, aluminum corral set in the
center of a 3!f-inch round Petri dish containing water 2 mm. deep. The corral
exit was carefully entered over the origin of a polar coordinate paper grid (Fig.
9A). The grid was, in turn, set on the platform of a stereoscopic microscope and
illuminated weakly from below by a 7 \ -watt incandescent lamp with opalescent
glass. Between the lamp and the microscope platform was a water filter for heat
absorption, and an opaque screen with a circular opening carefully centered under
the corral exit. The whole apparatus was placed in a darkened enclosure. With
this arrangement, the emerging paramecia were clearly silhouetted for observation.
A.
-WITH MAGNET
-WITHOUT MAGNET
400-
300-
O
UJ
O
LJ
Q.
0 0
*
0 °
•n
0
+
t
, O
8
0
0
D
o •
• o
• -
0
0 •
V
D *0 "0 '0 I
200
100
EUONCATION Of MOON (RE: ±46°)
40° 80 120°
PATH ANGLE
160°
200
FIGURE 9. (A) Orientation of the apparatus and of the experimental bar magnet for the
Paramecium study, illustrating mean paths in the natural and experimental field, the range of
mean paths in the apparent monthly cycle, and the standard deviations of the paths. (B) The
distribution of paths of Paramecium in the earth's magnetic field alone (solid line) is compared
with the distribution when the experimental 1.3-gauss, E-directed field is superimposed (broken
line). For purposes of this illustrated comparison, the values for the controls were increased
proportionately to make the total number equal to that of the experimental series. (C) The
relationship between mean path and elongation of moon treated as deviations from the fourtli
day after new moon.
Each experiment consisted of alternating observations of ( 1 ) a few paramecia
making their exit in the earth's magnetic field alone with (2) a few fully comparable
exits when the magnetic field was altered by an 18-centimeter Alnico bar magnet,
placed horizontally and centered directly below the exit with S-pole pointed East.
278 FRANK A. BROWN, JR.
The distance of the magnet was such that the strength of the horizontal East-di-
rected component was 1.3 gauss. This horizontal strength is about eight times
the earth's H component ; the total field is only two to three times the earth's total
field, F. Although the microscope base was predominantly constructed of non-
ferromagnetic materials, two pairs of steel screws symmetrically placed two to
three inches east and west were present and the microscope arm was of ferrous
metal. However, that the earth's field and experimental 1.3-gauss field were not
significantly distorted was assured by placing a small compass in the place of the
corral. The angle of deviation of the paths from the initial southward course was
determined by the point at which the animal crossed a circle perimeter with 0.5-
inch radius, centered at the origin of the grid.
Between the dates February 14 and March 9, 1961, a total of 3774 individual
paths were observed, 1762 in the earth's field and 2012 in the experimental, mag-
netic field. These data were obtained exclusively between the hours 8:30 A.M.
and 4:30 P.M., but chiefly between 2:30 and 4:30 P.M. The observations were
made on 12 different days during the 24-day period of study.
There was a strong positive correlation between the mean paths of the samples
run in the experimental magnetic field and the control samples on the same day
(r = 0.86 ± 0.09). There was also a highly significant difference between the
variances of paths under the two magnetic conditions. The stronger, imposed
East-West field produced a highly significantly greater amount of deviation of
paths from South, both clockwise and counterclockwise. Expressed as standard
deviation of paths, a value of 37.7 ± 0.638° was found for the earth's field and
41.3 ± 0.656° for the stronger, E-W oriented one. There was, therefore, clearly
an influence, highly statistically significant, of the field-strength change, whether
one ascertained probabilities by the F or t test (F = 1.20; t = 3.93).
Analysis of the data indicated that there was no significant difference between
the mean path of those paramecia in the 0.17-gauss South-directed earth's field and
of those in the East-directed 1.3-gauss field.
However, the comparative distributions of frequencies of paths for paramecia in
the 1.3-gauss E-W field and in the earth's South-directed field alone are shown in
Figure 9B. Using a Chi-square test to measure the probability that the two
samples were drawn from the same population, a value of x2 — 29.95 with 9 de-
grees of freedom was obtained (P < .001). Inspection of the figure suggests this
highly significant difference to be a consequence in large measure of an overall
shift of the crown of the distribution curve for the animals in the experimental
field to the left of that of the controls in the earth's alone.
In view of the previously demonstrated synodic monthly fluctuation in mean
path of both mud-snails and planarians, the Paramecium data were examined for
the possible existence of a comparable periodism. Inspection of the mean paths as
a function of time revealed a distinct suggestion that the paramecia, too, displayed
a monthly fluctuation. The inspection suggested that maximum clockwise turn-
ing was occurring for paramecia about four days after new moon (Feb. 19) and
maximum counterclockwise turning about four days after full moon (March 6).
In fact, computed correlations, with elongation of the moon considered as an
intrinsic time series, corroborated this suggestion. With elongation of the moon
expressed as ± ISO-degree deviation from four days after new moon (+48.8°) the
RESPONSE TO WEAK MAGNETIC FIELD 279
value of the coefficient of correlation, r, was 0.76 ± 0.09, N -- 24. This was
higher than that found in any other phase relationship with respect to the natural
monthly cycle (Fig. 9C).
Since only a 24-day period (ca. 290°) was involved in the study, it is obviously
not possible to conclude with great confidence that the period of this long-cycle
fluctuation in mean path \vas, indeed, a monthly one. However, that it probably
was a monthly one is suggested since extremes of both clockwise and counterclock-
wise response appeared to occur within the 24-day period, and the interval between
the estimated maximum clockwise and the estimated maximum counterclockwise
turning seemed clearly consistent with it being 180°. Indirect support for such a
cycle is considered to come from the now far better established occurrence of this
period in comparable orientations of the two other previously investigated species.
Just as for the snail and planarian, one very conspicuous influence of magnetic
field is upon the turning tendency in the field, without respect to whether it is
clockwise or counterclockwise. It seems probable that the character of response
of paramecia to an increase in magnetic field will be found, as in the other two kinds
of animals, to be functions of (1) times of lunar and solar days, and their inter-
ference derivative, the synodic month, and (2) the direction of the H-component
of magnetism with relation to the long axis of the body.
DISCUSSION
Several considerations were involved in planning the present investigation.
First, to be of significance under natural conditions, the organism must exhibit
responsiveness to field strengths of the order of magnitude of the earth's. Any
perceptive system of this sensitivity could well be expected to display little or no
resolving power for fields differing greatly in strength from the earth's. There-
fore, only weak fields were investigated. Secondly, to be maximally adaptive the
organism would be expected to be able to differentiate the compass direction of
these very weak fields. Thirdly, any response obtained might be expected to vary
in a "clock-regulated" manner. And lastly, to account for a number of still un-
explained biological phenomena, the responses must be postulated capable of sign
reversal. For this study, the orientation of whole organisms was considered to
constitute the most sensitive method for assaying any possible biological resolution
of magnetic field strength and direction.
To reduce the problem to its simplest form we attempted to learn the nature of
orientational tendencies or pressures in samples of a population subjected initially
to enforced orientation in a highly restricted unit of space, to horizontal magnetic
fields, both natural and experimental. As we have seen, the general method con-
sisted in inducing, or permitting, organisms initially to travel in an arbitrarily de-
cided magnetic compass direction in the earth's natural field and in experimentally
altered magnetic fields, and assaying the amount the animal's paths have deviated,
clockwise or counterclockwise, from the initial path after an arbitrary constant
short distance was traversed.
In this present study the experimental fields that were used were only those
obtained in the number 2 position of a straight bar magnet, in order that maximum
simplicity could be achieved. By this means it was possible to alter at will the
horizontal components of magnetism without significant change in the ambient
280 FRAXK A. BROWN, JR.
vertical component, which throughout the experimental studies remained the earth's
natural one. Furthermore, with the path of the worms being assayed for only a
relatively short distance over the number 2 position, and in a plane parallel to that
occupied by the magnet, insignificant field-strength differences were present within
any given experimental field. The earth's magnetic field is essentially a homo-
geneous one. The fields that were employed in this study were similarly relatively
homogeneous. The field gradient was less than % gauss per centimeter.
In those experiments in which the magnet was rotated in a horizontal plane in
the earth's field there was a change not only in direction of the imposed horizontal
component, but there was also a difference in its strength as the magnet's contribu-
tion supplemented or antagonized the geomagnetic one. No attempt was made to
compensate for this. For the 10-gauss field, for example, this involved about a
34% range and for the 5-gauss one, nearly a 7% one. However, these field-strength
differences can not alone account for the resolution of the direction of horizontal
vector by the organisms since field-strength differences many times larger than
these small percentages did not duplicate the influences of small changes in field
orientation. Experiments are now in progress which are expected to provide
information as to the relative roles of changes in the strength and direction of the
horizontal vector.
The problem of resolving organismic responses to weak magnetic fields is
compounded by the recent discovery that mud-snails are extraordinarily sensitive
to differences in the horizontal vector of electrostatic field (Webb, Brown and
Schroeder, 1961). Furthermore, Dugesia too has such responsiveness and dis-
plays a "compass direction effect" in response to very weak electrostatic fields which
is, at least in good measure, independent of the magnetic-compass response ( Brown,
1962b). In the present studies, no attempt was made to control the ambient geo-
electrostatic field and its changes.
The implications of findings such as the ones reported here, and ones described
earlier for the mud-snail, Nassarius (Brown, Brett, Bennett and Barnwell, 1960;
Brown, Webb and Brett, 1960; Brown, Bennett and Webb, 1960), are great not
only in providing an additional parameter to contribute toward the solution of such
stubborn problems as those of living clocks and navigational systems of organisms,
but also for the problem of regulation within living systems in general. With bio-
logical systems possessing astounding sensitivity to such weak magnetic fields, the
possibility exists that magnetism may normally play a role in general, organismic
integration, either directly or through the biological clock system.
The kinds of magnetic responses described here for Dugesia appear not to be
specific for this flatworm but simply to represent a general property of living
things. The potential of such a sensitivity, with capacity to resolve strength and
directional changes, when incorporated into adaptive behavioral systems as an
informational input seems tremendous. Search for possible important adaptive
roles of these extraordinary biomagnetic sensitivities will probably be very
rewarding.
SUMMARY
1. The orientational response of the planarian, Dugesia, at a given time of solar
day undergoes what appears to be a semi-monthly or monthly fluctuation, probably
RESPONSE TO WEAK MAGNETIC FIELD 281
a consequence of the possession of a lunar-day rhythm in response to some compass-
directional factor.
2. The monthly rhythm in Dugesia is modifiable by a weak magnetic field.
3. The monthly rhythm appears to undergo an annual modulation.
4. Dugesia exhibits a response to weak magnetic fields in the range of 0.17 to
10 gauss.
5. Dugesia differentiates between a horizontal field parallel to the long axis of
the body and a field at right angles, and between N and S poles, and, furthermore,
is able to resolve intermediate angular orientations of field with remarkable
precision.
6. The response of Dugesia alters its character in passing from a field close to
-the earth's strength to one as little as 10 gauss, suggesting the perceptive mechanism
to be specifically adapted to such a weak field as the geomagnetic one.
7. There is suggestive evidence that the protozoan Paraincciinu also responds
to very weak magnetic fields.
8. Some possible roles for organisms of such astounding responsiveness to very
weak magnetic fields are discussed briefly.
LITERATURE CITED
BARNWELL, F. H., AND H. M. WEBB, 1961. Responses of the mud-snail, Nassarius, to experi-
mental reversals in direction of very weak magnetic fields. Biol. Bull., 121: 381.
BROWN, F. A., JR., 1959. Living clocks. Science, 130: 1535-1544.
BROWN, F. A., JR., 1960. Response to pervasive geophysical factors and the biological clock
problem. Cold Spring Harbor Symp. Quant. Biol., 25: 57-71.
» BROWN, F. A., JR., 1962a. Extrinsic rhythmicality : A reference frame for biological rhythms
under so-called constant conditions. Ann. Nezv York Acad. Sciences (in press).
BROWN, F. A., JR., 1962b. Response of the planarian, Dugesia, to very weak horizontal elec-
trostatic fields. Biol. Bull., 123: 282-294.
BROWN, F. A., JR., AND F. H. BARNWELL, 1961. Organismic orientation relative to magnetic
axes, in responses to weak magnetic fields. Biol. Bull., 121 : 384.
BROWN, F. A., JR., M. F. BENNETT AND H. M. WEBB, 1960. A magnetic compass response of
an organism. Biol. Bull.. 119: 65-74.
BROWN, F. A., JR., W. J. BRETT, M. F. BENNETT AND F. H. BARNWELL, 1960. Magnetic response
of an organism and its solar relationships. Biol. Bull., 118: 367-381.
BROWN, F. A., JR., AND H. M. WEBB, 1960. A "compass-direction effect" for snails in constant
conditions, and its lunar modulation. Biol. Bull., 119: 307.
BROWN, F. A., JR., H. M. WEBB AND W. J. BRETT, 1960. Magnetic response of an organism and
its lunar relationships. Biol. Bull., 118: 382-392.
WEBB, H. M., F. A. BROWN, JR. AND T. E. SCHROEDER, 1961. Organismic responses to differ-
ences in weak horizontal electrostatic fields. Biol. Bull., 121: 413.
RESPONSE OF THE PLANARIAN, DUGESIA, TO VERY WEAK
HORIZONTAL ELECTROSTATIC FIELDS1
FRANK A. BROWN, JR.
Department of Biological Sciences, Northzvcstern University, Evanston, Illinois
A deep-seated, persistent, rhythmic nature, with periods identical with or close
to the major natural geophysical ones, appears increasingly to be a universal bio-
logical property. Striking published correlations of activity of hermetically sealed
organisms with unpredictable weather-associated atmospheric temperature and
pressure changes, and with day to day irregularities in the variations in primary
cosmic and general background radiations, compel the conclusion that some, normally
uncontrolled, subtle pervasive forces must be effective for living systems. The
earth's natural electrostatic field may be one contributing factor.
A number of reports have been published over the years advancing evidence
that organisms are sensitive to electrostatic fields and their fluctuations. More
recently Edwards (1960) has found that activity of flies was reduced by sudden
exposures to experimental atmospheric gradients of 10 to 62 volts/cm., and that
prolonged activity reduction resulted from gradient alternation with a five-minute
period. In 1961, Edwards reported a small delay in moth development in a
constant vertical field of 180 volts/cm., but less delay when the field was alternated.
The moths tended to deposit eggs outside the experimental field, whether constant
or alternating, in contrast to egg distribution of controls. Maw (1961), studying
rate of oviposition in hymenopterans, found significantly higher rates in the insects
shielded from the natural field fluctuations, whether or not provided instead with a
constant 1.2 volts/cm, gradient, than were found in either the natural fluctuating
field, or in a field shielded from the natural one and subjected to simulated weather-
system passages in the form of a fluctuating field of 0.8 volts/cm.
A study in our laboratory early in 1959 (unpublished) by the late Kenneth R.
Penhale on the rate of locomotion in Dugesia suggested strongly that the rate was
influenced by the difference in charge of expansive copper plates placed horizontally
in the air about six inches above and closely below a long horizontal glass tube of
water containing the worms. Locomotory rates in fields of 15 volts cm. (+ beneath
the worms) were compared with those in fields between equipotential plates. The
fields were obtained with a Kepco Laboratories, voltage-regulated power supply. A
comparable study with the marine snail, Nassarius, by Webb, Brown and Brett
(1959), employing a Packard Instrument Co., high-voltage power supply, con-
firmed the occurrence of such responsiveness to vertical fields of 15 to 45 volts/cm.,
and advanced evidence that the response of the snails displayed a daily rhythm.
More recently, it was demonstrated that mud-snails, even while submerged
in sea water, were able to resolve a horizontal field difference of 2 volts/cm, in the
1 This study was aided by grants from the National Science Foundation, G- 15008, and the
National Institutes of Health, RG-7405, and by a contract between the Office of Naval Research,
Department of Navy, and Northwestern University, 1228-03.
282
RESPONSE TO ELECTROSTATIC FIELD
air at right angles to their bodies, and to exhibit a characteristic orientational
response (Webb, Brown and Schroeder, 1961). The fields were obtained with B
batteries. The snails appeared able also to distinguish the direction of the very
weak gradient across their bodies. The character of the electrostatic response^was
altered simply by changing from South to East the compass direction in the earth's
field in which the response was assayed. There seemed to be an influence upon the
electrostatic response, by some natural force the effectiveness of which altered with
geographical orientation of the organisms.
The following study was made in order to determine whether a comparable
sensitivity to very weak electrostatic fields obtains for a common fresh-water
planarian, and if so, to learn more concerning its properties.
METHODS AND MATERIALS
The turning of planarian worms, Dugesia dorotocephala, was assayed as they
moved forward from an initially enforced orientation in a weak three-light field
(Brown, 1962). The three light sources were (1) directly vertical to the initial
point in the path, (2) in the horizontal plane directly behind the initially oriented
worm, and (3) horizontally 90° to the right of the starting point. In response to
this configuration of illumination, the mean path of samples of the worm population,
photonegative, always included turning of the worms to the left. The strength
of left-turning response was rendered quantitative by recording, to the nearest 5°,
the points at which the worms crossed the arc of a circle of one-inch diameter
centered at the starting point of the worm. Clockwise turning of the individuals
was recorded in positive degrees of arc and counterclockwise turning by negative
degrees of arc (Fig. 1) from a mean path directly forward (0°).
The effects of horizontal electrostatic-field gradients were determined by compar-
ing the mean values for 15-path samples in field gradients modified by rendering two
aluminum plates in air (Fig. 1), to right and left, equipotential or with potential
difference of 45 volts with + to right or + to left. The effects were studied under
experimental conditions in which other variables included : ( 1 ) magnetic compass
direction of the initial, enforced orientation of the worm, which was modified by
rotating the whole apparatus to the desired compass direction ; (2) time of day the
experiment was conducted; and (3) experimental alteration of the natural magnetic
field by a horizontal bar magnet centered an appropriate distance beneath the
apparatus.
In practice, the worms were placed in a 35 -inch glass Petri dish in 0.5 cm.
of water and the dish centered on polar-coordinate paper. This was set upon the
floor of a blackened wooden box. The upper portion of one side of the box was
open to the observer. In the roof of the box, 16 inches high, was a small light
source. The horizontal light sources were onion-skin-covered ends of 10 mm. solid
glass tubing, enclosed in black shielding. Through these light was transmitted
into the box from a 7i-watt incandescent lamp firmly attached to the outside of the
box. Symmetrically to right and left of the Petri dish were large 7x9 inch
aluminum plates, sandwiched between glass plates darkened with flat black paint.
The level of the worm starting-point was close to an axis between the centers
(horizontal and vertical) of the two plates. The aluminum plates were about 8
inches apart, thus giving about a 2-inch air space between each plate and the
284
FRANK A. BROWN, JR.
N
\
\
\
\
\
FIGURE 1. The apparatus organization employed in this study, including Petri dish, polar
coordinate grid, and aluminum plates. Three-light arrangement is not illustrated. Broken
circular line indicates apparatus rotatable relative to earth's geographic field.
RESPONSE TO ELECTROSTATIC FIELD 285
Petri dish. The whole visual environment of the orienting worms comprised a
rigid system which was constant as the apparatus was rotated in the earth's field.
This apparatus was always used in a darkened cubicle to minimize extraneous
illumination. As many as four identically constructed pieces of equipment were
in operation concurrently, and sequentially, during the course of the study.
The overall average field gradient contributed by the plates was about 2 volts
per cm. when these were connected with a 45-volt B battery, with the polarity
alterable by a pole-reversing switch. When the battery was disconnected by a toggle
switch (SPOT) the plates were simultaneously directly interconnected to assure
their equipotential state.
The experiments in any given series were conducted at the same time each day to
avoid any complicating factor introduced by a daily rhythm. In addition, each
experiment extended over two or more months to randomize any lunar daily
influence which might obtain comparable to those well-established to occur for
response to very weak magnetic fields.2
RESULTS
Response of Soittli-dircctcd worms in the morning: The first series of experi-
ments involved the responses of worms initially always directed magnetic South
in the earth's field. The observations were made sometime between 8 :30 and 11 A.M.
and consisted of two groups of four assays each. Each of the two groups included
the determination of the mean paths of 15-worm samples under each of four condi-
tions, two controls (equipotential plates) and two experimentals, + to left (+L)
and + to right (+R). The order of the four was selected arbitrarily and differed
steadily from one group to the next throughout the two-month experimental period,
September 20 through November 17, 1961. The charge across the plates was
altered by a person other than the observer. The observer was never informed as
to conditions in effect until each day's double series was completed.
The results of this experiment, in which the difference between each, +R and
+ L, from the mean of the two controls in the group was computed, are plotted in
Figure 2. Were the worms incapable of differentiating between the equipotential
plates and those possessing a potential difference, the average difference between
these would be zero, and the points would be expected to vary randomly about
zero. As is evident from inspection of Figure 2, the mean was highly significantly to
the right of zero. The mean was +2.342 ± 0.342° (t = 6.87, N = : 152, P < 1Q-10).
These results leave no reasonable doubt that the change from equipotential plates to
the 45-volt difference was effecting a mean clockwise turning of Dugesia.
The relation of response to compass direction: The foregoing experiment was
repeated during the period October 24, 1961, through February 27, 1962, initially
by observers different from the one concerned in the first experiment. Five different
observers, employing four sets of equipment, eventually contributed to the data.
Again, the observations were always made between 8:30 and 11 A.M. But now
the effects of the 45-volt difference between the plates, expressed as difference from
the equipotential plates, were determined not simply with South-directed apparatus,
2 The author wishes to acknowledge here his appreciation to a number of persons,
particularly Young H. Park, Sam D. Park, Polly Merrill, Stephanie Struggles, and Gertrude
L. Siegel, who devoted many hours to acquiring the data for this study.
286
FRANK A. BROWN, JR.
but with apparatus directed in each of eight compass directions. A single observer
on any given day would arbitrarily select one of the compass directions. Otherwise
the observations were made just as in the first experimental series. All observers
contributed to data from each compass direction. The observers were now in-
formed, however, as to the experimental conditions obtaining, but four of the five
observers were uninformed of previous work, or even of the nature of the problem.
The results of the four-month study are summarized in Table I, and the f requency
distribution for each compass direction illustrated in Figure 3.
There was reasonably good confirmation of the earlier South-directed "un-
informed" experimental series. However, a clear compass-direction effect was
now evident. When the apparatus was southerly directed, the increase in potential
gradient turned the worms clockwise, when northerly directed, counterclockwise,
with graded differences between these directions. More particularly (see Figure 4),
the results suggested that the axis of the compass-direction effect was a S-SE to
X-XW one instead of a magnetic X-S one.
Relationship of response to time of day: While this last experimental study was
under way data were being gathered from occasional comparable series run between
2 and 6 P.M., commencing on September 15, 1961. These provided a strong
•HO0
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oo
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o
. • °% ' % • °. S°o°S °» .
oo- o °^on
°o °°° o o ° coo ° 0 o § ^
LJ
cr
o°0 ° °°°° !°° 0°oo°o°
0 00 0 ° 0
°00°0 00 o.
o 0°° 0 o
I
0 ° o o °
00 00
H
o
<
a -5°
o o
"o o
z
0
<
o
u
2-,o°
o
-,5-
0
1 1 1 1 1 1
30 10 20 30 10
SEPT. OCT. NOV.
10 20
FREQ.
FIGURE 2. The differences between path of worm samples (15 paths) in a 2-volt/cm. right-angle
gradient and path in the same experimental series in an equipotential field.
RESPONSE TO ELECTROSTATIC FIELD
287
TABLE I
Morning paths
Direction
Mean
S.E.
t
N
Probability
N
-1.325
±0.605
2.13
64
<.05
NE
-1.125
±0.354
3.09
76
<.005
E
+ 1.330
±0.433
2.92
56
<.005
SE
+ 1.950
±0.498
3.92
84
<.001
S
+ 1.805
±0.549
3.29
86
<.005
sw
+2.275
±0.476
4.74
80
<.001
w
-0.920
±0.502
1.41
78
<.20
NW
-1.350
±0.424
3.18
80
<.005
suggestion that afternoon values were not showing the same form of compass-
direction relationship as the morning ones. Instead, the results suggested that
there was an inversion of the compass-direction effect. This question was eventually
pursued more systematically and studied until May 1, 1962. It is evident from
Table II and Figure 4 that for two directions for which moderately extensive data
were obtained, NW and S, the electrostatic field effect shows a clear inversion of
the afternoon values relative to the morning ones. That the afternoon responses for
each direction were different from the morning ones was far more clearly apparent
15
00 10
LJ
u
O
LJ
a:
§
0
N
NE
SE
S
SW
w
NW
LJ
CO
Z o
O 5
a.
CO
LJ
* 1C?
15°
J
]
]
COMPASS
DIRECTION
FIGURE 3. Frequency distributions of the differences between paths of worm samples in a
2-volt/cm. right-angle gradient and path in the same experimental series in an equipotential
field for each of eight compass-directional orientations.
288
FRANK A. BROWN, JR.
N
FIGURE 4. Comparison of the compass-direction effect upon response to electrostatic
gradient for morning (dashed line) and afternoon (dotted line) hours. Degree of left turning
is indicated by concentric circles inside heavily inked one, and right turning by concentric
circles outside of it.
upon finding 6 of the 8 directional differences (NE, E, SE, S, SW, and NW)
statistically significant.
Influence of experimental magnetic-field changes on response: Since there was
conspicuously a compass-directional relationship of the character of the response to
the 2 volt/cm, change in electrostatic field it became of interest to learn whether this
was directly dependent upon responsiveness of the worms to the magnetic field.
Consequently, an additional experiment was conducted between February 17 and
April 11, 1962. This comprised, until March 13, observing in the morning the
effects upon mean paths of the electrostatic-field difference for North- and South-
directed planarians and for North-directed ones under experimental conditions of
an artificial magnetic field differing from the earth's only in that the natural 0.17-
RESPONSE TO ELECTROSTATIC FIELD
289
TABLE II
Afternoon responses
Direc-
tion
Mean
S.E.
t
N
Prob.
Difference
from A.M.
S.E. cliff.
t
N
Prob.
N
- .12
±0.48
0.25
42
<.9
+ 1.20
±0.772
1.55
106
<.20
NE
+ .37
±0.53
0.70
51
<.5
+ 1.52
±0.634
2.40
127
<.02
E
+ .33
±0.25
1.32
178
<.2
-1.00
±0.499
2.00
234
<.05
SE
- .20
±0.46
0.17
42
<.9
-2.15
±0.678
3.17
126
<.005
S
- .92
±0.46
2.00
62
= .01
-2.73
±0.716
3.81
148
<.001
SW
- .57
±0.54
1.05
40
= .3
-2.85
±0.720
3.96
120
<.001
W
+ .12
±0.56
0.21
41
<.30
+ 1.04
±0.752
1.38
108
<.20
NW
+ 1.44
±0.38
3.79
60
<.001
+2.79
±0.569
4.90
140
<.001
gauss North-directed horizontal component of the field was experimentally reversed
to become a 0.2-gauss, South-directed one. Upon the basis of response to magne-
tism, the worms should now receive stimulation closely similar to that normally
experienced by South-directed worms. On March 13, one additional condition was
added to the series, namely South-directed worms in the earth's field were given
experimentally (as a 0.2-gauss field) essentially the magnetic equivalent of a
North-directed route.
In practice, each series comprised, in random order, pairs of observations under
each of the three or four conditions, with the order, equipotential to non-equipo-
tential plates, in each pair determined by the flip of a coin. The three observers
of the worms were uninformed of the order for each pair.
The results of this experiment are shown in Table III. The experiments
showed the same qualitative difference between North and South in the earth's
field that had been observed in the earlier series similarly conducted in the morning.
Evident from the table is the fact that compass-North-directed worms given a South-
directed experimental magnetic field, N(S), did not come to behave like South-
directed ones, S. Indeed, the experimental reversal of the magnetic field even
augmented the characteristic counterclockwise, North-directed response, N. The
difference between N and S responses was 1.496 ± 0.546° (t = 2.74). The differ-
ence between N(S) and S responses was 2.68 ± 0.522° (t = 5.13). In fact, the
difference between N and N(S), 1.16 ±0.501°, was itself statistically significant
The difference between S and S,N, is, unlike the difference between N and
N(S), in the direction to be expected were the compass-direction effect to result
TABLE III
Influence of experimental magnetic-field reversal
Direction
Mean
Standard error
t
N
Probability
N
-0.493
±0.372
1.325
93
<.2
N(8)
-1.65
±0.336
4.91
93
<.001
S
+ 1.03
±0.400
2.57
93
<.02
S(Nl
-0.22
±0.524
0.42
50
<.7
290
FRANK A. BROWN, JR.
from au influence of magnetism. Although the sample of S(N) is only about half
the size of the other three experimental conditions, due to the late addition to the
series, the difference of 1.25 ± 0.658° suggests that a comparable statistically sig-
nificant difference would have been demonstrated had the sample been larger.
It is clear that experimental reversal of the magnetic field did not reverse the
relative differences in path as did a change in compass direction. The introduction
of the weak magnetic fields for each of the two directions simply displaced the
response to the weak electrostatic field, either to a less positive orientational one
(S) or a more negative one (N). In other words, the effect of the reversed
20
0
z
Ld
D
O
u
a:
LL.
o
10
A
u
n
B
nn
n n
-10
DIFFERENCE
FIGURE 5. Comparison of the frecjuency distributions of the +R effect minus +L effect
for (A) the period November 20, 1961 through March 31, 1962, and (B) the periods September
15 through November 17, 1961 and April 1 through May 1, 1962.
magnetic fields appeared to be simply to displace about 1 ° to the left the electrostatic
response-pattern related to compass directed without altering its form. Therefore,
this particular compass-direction effect of electrostatic response is in large measure
independent of the previously described geomagnetic compass-response of snails
and planarians (Brown, Bennett, and Webb, 1960; Brown, 1962).
Resolution of field direction across body: The data were searched for evidence
concerning whether or not Dugesia was able to distinguish between a potential
RESPONSE TO ELECTROSTATIC FIELD 291
difference with positive charge to right and one with positive charge to left. Assum-
ing no capacity of the worms to distinguish between these two conditions, the
difference between the two should average zero, and there should be a random
distribution of values about zero.
When, however, the morning differences between the effects of the two field
orientations, +R minus +L, were examined for all eight compass directions for
the whole period of study, October 24 through February 27, a difference of
+0.821 ± 0.277° was obtained (t = 2.96; N = 297; P<.005). Furthermore,
within this four-month period the data departed significantly in both directions from
a random distribution in time. For example, from December 4 to 23, inclusive,
the mean was —1.31 ± 0.51° ; N = 44, P < .02 ; whereas from December 26 through
February 10, it was much more strongly significant and positive +1.535 ± 0.408° ;
N = 170, P < .001. The data suggested that for the former period the worms were
distinguishing between the two directions of the field and turning away from +R,
and during the latter, were distinguishing and turning toward the +R.
A second kind of suggestion that the worms were able to distinguish the two
field-directions came from a comparison of the values of the differences between
effects of the two fields, +R minus +L, for the morning and afternoon experi-
mental series. The mean difference computed from all data for the afternoon
indicated they were turning away from +R, though not statistically significantly so.
When the frequency distributions of all the afternoon values for the colder months
(November 17 through March 31) were plotted, a bimodality was suggested
(Fig. 5A). It thus appeared again that two kinds of response were evident to +R.
There was either (1) a tendency to turn weakly toward it, or (2) a tendency to
turn more strongly away from it.
This bimodality was significantly less apparent for all the afternoon values
obtained for warmer months (September 15 through November 15, 1961 ; April 1
through May 1, 1962) ; the frequency distribution for these two periods is shown
in Figure 5B. A Chi-square test for significance of a difference between the two
populations of values depicted in Figure 5 gave x2 — 32.05, when scattered
peripheral values were combined to render N = 12, or P < .003. Such a difference
would not be expected unless +R and +L could elicit different responses by the
worms. A suggestion of the occurrence of two signs of responses was present also
in the frequency distribution of the morning data.
A third kind of evidence pointing to ability of the worms to distinguish between
the two field orientations came from a study of differences in variances of the values
TABLE IV
Variances of +R effect minus +L effect
Direction Var. through May 1 N through May 1
N 21.85 49
NE 23.20 59
E 13.54 111
SE 17.71 63
S 22.86 150
SW 27.20 58
W 21.45 52
N\V 12.95 72
292 FRANK A. BROWN, JR.
of +R effect minus +L effect, with compass direction for all data. These are
presented in Table IV. Variance differs in a statistically significant manner with
compass direction. ]t reaches its highest values in the SW- and NE-directed
series with minima for E and NW ones. Significance is readily demonstrable by the
F test for differences between variances, between minima and maxima in this
compass-direction effect (e.g., K to NE, P < 0.05 ; E to SW, P < .01 ; NW to SW,
P < .01 ; NW to NE, P < .05). It is self-evident that such differences with
compass direction could not be expected were +R to be physiologically indis-
tinguishable from +L.
The evidence, taken as a whole, suggests therefore that the relative responses of
the worms to +R and +L vary with time, with geographic orientation of the worms,
and with hour of the day.
DISCUSSION
In the study which is reported here, the exact values of the fields to which the
animals were subjected were never known. The natural field was unquestionably
reduced substantially and maximally in the horizontal axis connecting the two equi-
potential plates, and minimally in all axes at right angles to this, including both
horizontal and vertical ones. The important thing for this study was that whatever
horizontal potential gradient remained at right angles to the initial path of the worms,
the experimental gradient in one direction added 2 volts per cm. to that field, and in
the other direction subtracted this amount. By such means it was possible, there-
fore, to determine whether the animal could resolve such small changes.
The orientation of the worms in the experiment was observed while they were
submerged in tap-water whose source was Lake Michigan. Such water is, relative
to the surrounding air, a good conductor. Therefore, the overall electrostatic
gradient to which the worms were directly subjected was far smaller than the 2
volts/cm, gradient in the air. The value can be estimated to be 6 to 8 orders of
magnitude below that in the air as a consequence of the "Faraday-cage effect" of the
worm's ambient aqueous medium. To exhibit such responses as the worms did in
these experiments would require a sensitivity to essentially static electric gradients
of the order of fractions of a microvolt per centimeter.
The significance of this demonstrated sensitivity for animals is apparent. In
speculations on the mechanism involved in the reported responses of insects to at-
mospheric gradients, surface charge has been importantly considered (Edwards,
1960). In the light of the "Faraday-cage action" of every organism's body as it
behaves as a volume conductor, it has been difficult to believe that the minute resid-
ual gradients within the organism, correlated with the larger atmospheric gradients,
could result in any response of individual cells or organs located protectively inside
the external boundaries of the organism. The studies with the worms, and earlier
studies with the marine snails submerged in sea water (even a slightly better con-
ductor), have proven there exists cellular sensitivity adequate to require a recon-
sideration of the mechanism of response in such terrestrial organisms as the insects,
hamsters (Schua, 1954) and even man (Frey, 1952).
Sensitivities of the order of those established by this study provide one means
for an influence of weather-system changes on organisms. Such meteorological
changes are not uncommonly accompanied by electrostatic fluctuations more than
RESPONSE TO ELECTROSTATIC FIELD IV 3
one hundred times as great as the experimental ones employed in this study. An
innate ability of living things to interpret specific parameters of electrical change in
their environment may prove to be a partial explanation of apparent forewarnings
some organisms have appeared to receive relative to meteorological disturbances.
Ability to resolve small differences in strength ©f horizontal vectors of atmos-
pheric electrostatics, and their direction as well, can contribute as a navigational
aid. This would comprise an electrostatic "compass." Such a compass may be
used along with other aids, such as response to magnetic field and visual responses,
including use of celestial references.
The earth's atmosphere displays periodic variations in diverse electrical param-
eters. These relate importantly to movements to the earth with respect to sun
and moon. Ability to resolve strength, direction, and frequency and amplitude of
oscillations in electrostatic field, can theoretically provide an organism with a means
of deriving valuable information as to the period lengths of the natural geophysical
rhythms. Both local-time and universal-time components are present in these
fluctuations. Responsiveness to electrostatic fields may possibly be one of the nor-
mally contributing factors to the timing system of the extraordinary clocks of animals
and plants.
Such sensitivity of a protoplasmic system to an electric field as appears to be
present renders it probable that protoplasm is far more sensitive to electromagnetic
fields of radio-frequency than has generally been conceded, or even reported, up to
the present. This possibility is further supported by the correspondingly great
sensitivity to extremely weak magnetostatic fields reported elsewhere. It is con-
ceivable that failure to disclose such perceptivity may commonly be a consequence of
an inability, to date, to discover an invariable kind of response by the organism to
such a stimulus.
The complexity of the response mechanism of the planarians to electrostatic fields
as revealed by these studies, and the relationships of the response to both temporal
and spatial orientation, certainly suggest the hypothesis that responsiveness to this
factor plays still undisclosed and important roles in the lives of terrestrial creatures.
SUMMARY
1. The planarian Dugcsiu is able, even while in water, to perceive a change of
2 volts/cm, in electrostatic gradient in the surrounding air.
2. There is reason to presume that in order to show this response the organism
is responding to differences in ambient static gradient of the order of fractions of a
microvolt per cm.
3. The strength and character of worm response to a right-angle potential change
are related to the direction the worm is oriented in the earth's geographic field, and
to time of day.
4. A field-change in South-bound worms in the morning effects clockwise turn-
ing. A similar field-change for North-bound worms effects counterclockwise turn-
ing. In the afternoon the relationship of electrostatic response to geographic
direction is essentially the mirror-image of that of the morning.
5. Dugesia is able to distinguish the direction of a gradient across its body.
6. A few of the possible significances of these findings are discussed briefly.
294
FRANK A. BROWN, JR.
LITERATURE CITED
BROWN, F. A., JR., 1962. Responses of the planarian, Dngcsia, and the protozoan, Paramecium,
to very weak horizontal magnetic fields. Biol. Bull., 123: 264-281.
BROWN, F. A., JR., M. F. BENNETT AND H. M. WEBB, 1960. A magnetic compass response of an
organism. Biol. Bull., 119: 65-74.
EDWARDS, D. K., 1960. Effects of artificially produced atmospheric electrical fields upon the
activity of some adult Diptera. Canad. J. Zool, 38: 899-912.
EDWARDS, D. K., 1961. Influence of electrical field on pupation and oviposition in Nepytia
Phantasmaria Stkr. (Lepidoptera: Geometridae). Nature, 191: 976, 993.
FREY, W., 1952. Der atmospharische Vertikalstrom. Arch. Kreislaufforsch, 18: 129-132.
MAW, M. G., 1961. Suppression of oviposition rate of Scambus blolianac (Htg) (Hymenop-
tera : Ichneumonidae) in fluctuating electric fields. Canad. J. Entom., 93 : 602-604.
SCHUA, L. F., 1954. Wirken luftelektrische Felder auf Lebenwesen? Umschau Wiss. Tech.,
54: 468-469.
WEBB, H. M., F. A. BROWN, JR. AND W. J. BRETT, 1959. Effects of imposed electrostatic field
on rate of locomotion in Ilyanassa. Biol. Bull., 117: 430.
WEBB, H. M., F. A. BROWN, JR. AND T. E. SCHROEDER, 1961. Organismic responses to differ-
ences in weak horizontal electrostatic fields. Biol. Bull., 121: 413.
FREE GLYCEROL IX DORMANT CYSTS OF THE BRINE SHRIMP
ARTEMIA SALINA, AND ITS DISAPPEARANCE
DURING DEVELOPMENT1
JAMES S. CLEGG -
Dcpurtmcnt of Biology, The Johns Hopkins University, Baltimore 18, Maryland
During part of a previous study on the stored carbohydrates of various dormant
organisms (Clegg and Filosa, 1961), large amounts of a carbohydrate-like sub-
stance were observed in extracts of Artemia salina cysts. On the basis of mobility
and reactivity on paper chromatograms, this substance appeared to be glycerol.
Since large amounts of free glycerol have been shown to accumulate during dia-
pause, and in other hypometabolic stages of certain insects (cf. Salt, 1961), a more
thorough analysis was undertaken. In addition, although several studies have
been made of the chemical components of these cysts, no mention has been made of
glycerol (the extensive literature on Artemia has recently been cited by Dutrieu,
1960). The present report deals chiefly with the identification of free glycerol in
Artemia cysts and the changes in its concentration during development. A pre-
liminary report on the presence of glycerol in Artemia cysts has been published
(Clegg and Evans. 1962).
MATERIALS AND METHODS
Dried cysts of Artemia, which are embryos in the early stages of development
covered by a chitinous shell (Dutrieu, 1960), were obtained as a gift from the Brine
Shrimp Sales Co., Inc., Hayward, California. Unless designated otherwise, the
cysts used were collected in the fall of 1960 from the evaporating ponds near
Hayward, and analyses of these cysts were begun in the summer of 1961. They
were washed briefly with distilled water to remove any empty shells, and were then
dried at room temperature for at least twenty days before use. Over 70% of these
cysts produced active nauplii when incubated in sea water at 24-26° C.
For the isolation of glycerol, about 5 g. of cysts were homogenized in a Ten
Broeck homogenizer with 30 ml. of 80% ethanol. The homogenate was filtered
and the filtrate decolorized with Norit (1% w/v). After removal of the Norit, the
clear filtrate was concentrated under reduced pressure and then extracted with
benzene. The organic phase was discarded and the remaining solution was puri-
fied by paper chromatography (Evans and Dethier, 1957). The combined eluates
from the chromatographic separation were concentrated by evaporation at 50° C.,
and then dried over CaCL to a viscous syrup (about 300 mg.) which then was used
for the identification studies.
For quantification of glycerol, 40-80 mg. of cysts were homogenized in 1.0 ml.
of 80% ethanol ; the homogenate was transferred to a graduated centrifuge tube
1 Supported by a grant (E-2358) from the Public Health Service.
- Present address : Department of Zoology, University of Miami, Coral Gables 46, Florida.
295
296 JAMES S. CLEGG
with four 1-ml. washings, and the volume was made up to 5.0 ml. with distilled
water. After centrifuging for 15 minutes at 3000 rpm., aliquots were taken for
determination of glycerol in the supernatant by the colorimetric method of Lambert
and Neish (1950) as modified by Burton (1957). In preliminary experiments,
the glycerol was first isolated and identified by paper chromatography and then
eluted for quantification. It was found later that results obtained by this method
differed from direct determinations on the supernatant by not more than 3%. As
a result, direct colorimetric determinations were carried out on the supernatant.
Trehalose was determined by the anthrone method of Dimler et al. (1952) after
its isolation from the supernatant by paper chromatography (Clegg and Evans,
1961). The pellet from the ethanol extraction was analyzed for polysaccharide by
re-homogenizing in 5.0 ml. of 5% trichloroacetic acid, centrifuging, and using an
aliquot of the supernatant for the anthrone method of Dimler et al. (1952). Results
obtained by this procedure were similar to those obtained by conventional alkali ex-
traction and alcohol precipitation methods. This material, when hydrolyzed by
acid, yielded only glucose, as judged by paper chromatography, and will be referred
to as glycogen in the present study.
To obtain the nauplii, a known amount of cysts (40-80 mg.) was incubated in
a Petri dish containing filtered sea water at 24-26° C. In all cases, the nauplii were
collected within three hours of emergence from the cysts and were separated from
the mixture of developing cysts and shells by virtue of the fact that the nauplii were
positively phototactic while the cysts and shells floated on the surface. The nauplii
were pipetted from the medium, filtered, and washed with distilled water. They
were then dried to constant weight and analyzed by the same methods given for the
cysts. The empty shells were collected and analyzed after 96 hours of incubation.
Average weights of the cysts, shells, and nauplii were obtained by placing 50 to
100 individuals on a pre-weighed coverslip, drying to constant weight, and re- weigh-
ing on a Mettler Micro Balance (sensitive to about 1
RKSULTS AND DISCUSSION
Identification of glycerul
The substance in question migrated on paper chromatograms with authentic
glycerol in the following solvent systems (v/v) : (1) water-saturated ethyl acetate;
(2) n-butanol, ethanol, acetone, water (5:4:3:2); (3) chloroform, ethanol (8:2) ;
(4) ethyl acetate, ethanol, water (12:2:1) ; and (5) n-propanol, ethyl acetate, water
(7:1:2). When mixtures of the substance and authentic glycerol were chromato-
graphed, no separation was observed in these solvent systems. Positive identifica-
tion of the substance as glycerol was established by preparation of the tribenzoate
derivative (Segur, 1953). The product, recrystallized from 90% ethanol, had a
melting point of 71-72.5° C. The tribenzoate prepared from authentic glycerol had
a m.p. of 71.5-72.5° C., and the mixed m.p. was 72-73° C.
Levels of glycerol and glycogen in the cysts and nauplii
Dutrieu (1960) has shown that net glycogen synthesis occurs in Artemia during
the transition from the dormant cyst to the active nauplius. Net glycogen synthesis
also occurs after diapause is broken in the eggs of the silkworm, Bombyx tnori
(Chino, 1957), and glycerol and sorbitol were shown to be its precursors (Chino,
GLYCEROL IN ARTEMIA CYSTS
297
TABLE I
Glycerol and glycogen content of the cysts, and nauplii of newly emerged Artemia
Stage
Per cent of the dry weight
Glycerol
Glycogen
MeaniS.E.
No.
MeaniS.E.
N'o.
Cyst
Shell
Embryo
Newly emerged nauplius
4.91 ± 0.42
0.19
6.30* ± 0.48
4.85 ±0.21
(9)
(3)
(9)
(13)
1.13 ±0.09
0.04
1.86* ± 0.14
15.1 ±0.2
(8)
(3)
(8)
(6)
* 1 mg. cysts = 0.78 mg. embryo (Table II); 4.91 -f- 0.78 = 6.30% glycerol of the embryo
dry weight.
1958). Therefore, studies were undertaken to determine whether or not glycerol
was converted to glycogen following the termination of dormancy in Artemia.
Sorbitol, incidentally, was not found in these cysts (limit of detection = 0.2% of the
dry weight).
Table I summarizes the results obtained by incubating cysts in 2% NaCl. Glyc-
erol was present in the dried cysts before incubation to the extent of about 5% of
the dry weight and, on the basis of cyst dry weight, no decrease was measured during
the transition from cyst to nauplius. Similar values were obtained by homogen-
izing the cysts in distilled water at 0-4° C. This indicated rather strongly that the
amount of glycerol found was present as free glycerol in the cyst. The small values
given for the shells are maximal since a few undeveloped cysts might also be present
in the shell fraction. In addition, it should be pointed out that the assay system used
is not wholly specific for glycerol, so these low values may not be glycerol at all. In
any event, it was clear that most, if not all, of the glycerol was confined to the embryo..
In order to compare the glycerol levels in the embryo with those in the nauplius it
was first necessary to estimate the weight of the embryo. This was so because the
shell, constituting a large percentage of the cyst weight, was shed when the nauplius
emerged, and would not be used as a basis for estimating glycerol levels in the
nauplius. This information, given in Table II, indicated that the embryo consti-
tuted about 78% of the cyst dry weight. This figure was then used to calculate the
concentration of glycerol in the embryo, shown in Table I as over 6% of the dry-
TABLE II
Dry weights of the cyst, shell, and nanplius
Mean weight
Stage
Per cent of the
cyst weight
Mg- dbS.E.
No.
Cyst
2.55 ± 0.03
(7)
100
Shell
0.57 ± 0.01
(7)
22
Embryo
1.98 ± 0.03
(7)
78
Newly emerged nauplius
1.93 ±0.16
(8)
—
298
JAMES S. CLEGG
weight. Even on this basis the glycerol content decreased by only about 1.5% of the
dry weight during the formation of the nauplius. At the same time, glycogen levels
increased by about 14% of the weight, on the basis of cyst and embryo dry weight,
as shown in Table I. Clearly, the small decrease observed in the glycerol content
could not account for the amount of glycogen synthesized. Therefore, the source of
most of this glycogen, unlike Bombyx eggs, was not glycerol. It would appear from
the study of Dutrieu (1960) that trehalose, a non-reducing disaccharide of glucose,
o
W 4
o:
Q
u_
O
UJ
o
(r
LU
a.
UJ
o
o
UJ
o
>-
_i
o
o
— o Incubated in sea water
— • " " " " + yeast
I
I
I
036 12 20 24 48 72
TIME AFTER EMERGENCE FROM THE CYST
(HOURS )
FIGURE 1. Glycerol content of fed (•) and unfed (o) nauplii as a function of time
after emergence from the cyst.
GLYCEROL IN ARTEMIA CYSTS 299
might be the chief substrate for the glycogen synthesis observed during the develop-
ment of Artemia. This aspect will be considered in a future publication. The
values given in Table I for glycogen concentrations in the nauplius are more than
twice those reported by Dutrieu (1960). There are several obvious possible
explanations for this difference.
The results given above indicated that glycerol was not being used either as an
important source of energy during development or as a major substrate for glycogen
synthesis. Accordingly, the fate of glycerol in the nauplius was examined.
Glycerol levels as a Junction of nauplius age
A large number of newly emerged nauplii was collected as described and divided
into two groups. One group was incubated in filtered sea water and the other in
sea water containing 1 mg. of dried yeast per ml. as a source of food. After various
periods of incubation the nauplii were analyzed for glycerol content. The averaged
results of three separate experiments are given in Figure 1 . The amount of glycerol
in the nauplii decreased sharply during the first 24 hours of incubation and then re-
mained at a very low, constant level. These latter values are probably due to the
presence of small amounts of non-glycerol substances that produce color with the
reagents, since glycerol could not be detected in 72-hour-old nauplii when these ex-
tracts were analyzed by paper chromatography (limit of detection = 0.2% of the dry
weight). Since the rapid decrease in glycerol content was observed in fed and un-
fed nauplii it seems that glycerol disappearance is not influenced by nutrition. Com-
parisons of the glycogen content of these two groups of nauplii were not made since,
in the case of those incubated with yeast, the amount of glycogen present in the gut
lumen, due to the presence of ingested yeast, was uncontrollable. Consequently, it
is not known whether the decrease in glycerol is accompanied by an increase in gly-
cogen. Because the nauplii are so small, attempts have not yet been made to fol-
low the metabolic fate of injected radioactive glycerol. The present results do show,
however, that glycerol essentially disappears during the first day following emergence
from the cyst.
Glycerol, trehalose, and glycogen contents of aged cysts
Next, the effect of source, age, and storage condition on the carbohydrate com-
position of the cyst was examined. These aged cysts, and a brief resume of their
history, were generously supplied by Mr. Maurice Rakowicz of Brine Shrimp Sales
Co., Inc., Hay ward, California. At least 200 mg. of cysts from each group were
analyzed for trehalose, glycerol, and glycogen content by the methods described
above. Dutrieu (1960) has shown that trehalose and small amounts of glucose are
the main alcohol-soluble sugars present in Artemia cysts and this has been confirmed
in the present study. The per cent hatch was obtained by incubating at least 500
cysts from each of the groups for 72 hours in sea water at 24-26° C., and then
counting the number of viable nauplii produced. The results, summarized in Table
III, showed that the trehalose content of these several groups was quite constant,
whereas the glycogen and glycerol contents showed considerable variation. The
most striking difference between these groups was the per cent hatch, none of the
cysts producing viable nauplii in the 1938 group. Noteworthy was the increased
viability of those cysts stored in racuo compared with those stored in air since 1951.
JAMES S. CLEGG
TABLK III
Glycerol, glycogen, and trehalose contents of aged cysts
Per cent of the dry weight
Origin of cysts and date collected
Storage
Average %
hatch
Glycerol
Trehalose
Glycogen
San Francisco, 1961
air
4.91
14.27
1.13
73
San Francisco, 1951
air
2.48
16.49
1.18
5
San Francisco, 1951
vacuum
2.49
17.29
1.65
62
San Francisco, 1938
air
2.45
14.68
1.05
0
Great Salt Lake (Utah), 1951
air
4.73
15.09
2.67
4
The fact that cyst viability greatly decreased with aging in air, while the trehalose
and glycogen content did not appear to diminish appreciably, suggests that a source
of energy is not the limiting factor determining viability during aging for long
periods. These findings also emphasize the "metabolic dormancy" of these cysts, at
least with respect to carbohydrate metabolism. For the present, however, the main
conclusion derived from these results was that trehalose, glycogen, and glycerol are
the normal and principal carbohydrates of dormant Artemia embryos. A detailed
study is presently being made to determine the origin of glycerol in the embryo, the
metabolic fate of glycerol in the nauplius, and the role of glycerol and trehalose in
the dormancy of Artemia cysts.
I express my thanks to Dr. David R. Evans for a critical reading of the
manuscript.
SUMMARY
1. Free glycerol was identified as a major carbohydrate component of the dor-
mant cysts of Artemia salina.
2. The amount of glycerol present in cysts aged for a year in the dry state was
found to be about 5% of the cyst weight, and was shown to be restricted to the
embryonic part of the cyst.
3. Glycerol content decreased slightly during the formation of the nauplius and
then rapidly decreased to a very low level after the nauplius emerged from the cyst.
The decrease in glycerol content could not account for the synthesis of glycogen
during formation of the nauplius.
4. The glycerol, trehalose, and glycogen contents, and the viability of cysts aged
up to 28 years were determined.
LITERATURE CITED
BURTON, R. M., 1957. The determination of glycerol and dihydroxyacetone. Methods in
Ensynioloiiy, 3: 246-248. Academic Press, N. Y.
OIINO, H., 1957. Carbohydrate metabolism in the diapause egg of the silkworm, Bombyx mori.
I. Diapause and the change of glycogen content. Embryologia, 3: 295-316.
CHINO, H., 1958. Carbohydrate metabolism in the diapause egg of the silkworm, Bombyx mori.
II. Conversion of glycogen to sorbitol and glycerol during diapause. J. Ins. Physiol.,
2: 1-12.
CLKGO, T. S., AND D. R. EVANS, 1961. The physiology of blood trehalose and its function during
' flight in the blowfly. /. £.r/>. B'wl, 38: 771-792.
GLYCEROL IN ARTEMIA CYSTS 301
CLEGG, J. S., AND D. R. EVANS, 1962. Free glycerol in dormant cysts of the brine shrimp,
Artcmia salina. Amcr. Zool. (abstract) in press.
CLEGG, J. S., AND M. F. FILOSA, 1961. Trehalose in the cellular slime mould Dictyostelium
mucoroides. Nature, 192: 1077-1078.
DIMLER, R. J., W. C. SCHAEFER, C. S. WISE AND C. E. RIST, 1952. Quantitative paper
chromatography of D-glucose and its oligosaccharides. Analyt. Chcm., 24: 1411-1414.
DUTRIEU, J., 1960. Observations biochemiques et physiologiques sur le developpement d'Artcmia
salina Leach. Arch. Zool. Exp. ct Gen., 99: 1-128.
EVANS, D. R., AND V. G. DETIIIER, 1957. The regulation of taste thresholds for sugars in the
blowfly. /. Ins. Physio!., 1: 3-17.
LAMBERT, M., AND A. C. NEISH, 1950. Rapid method for estimation of glycerol in fermentation
solutions. Canad. J. Res. B, 28: 83-89.
SALT, R. W., 1961. Principles of insect cold hardiness. Ann. Rev. Ent., 6: 55-75.
SEGUR, J. B., 1953. In: Glycerol (C. S. Miner and N. N. Dalton. editors). New York,
Rheinhold Publishing Corp., p. 174.
THE SURVIVAL OF ARTEMIA POPULATIONS IX RADIOACTIVE
SEA WATER
DANIEL S. GROSCHL 2
Genetics Department, North Carolina State College, Raleigh, North Carolina
With salt-water organisms there have been few attempts to check conclusions
based on data from the more traditional species used in radiation genetics. Possi-
bly this traces to difficulties in maintaining stocks of known ancestry, although we
have met no serious maintenance problems with strains of Artemia. The brine
shrimp thrives without running water and thus we avoid escape of zygotes or the
loss of floating gametes. Furthermore, their ability to cope with environmental
stresses, including ionic and osmotic changes (Lochhead, 1941), suggested that
Artemia would be ideal for experiments in which radioisotopes would be added to
sea water.
The persistence of mass cultures and the fitness components obtained from pair
mating tests are reported below for Artemia whose ancestors survived sea water to
which either P32 or Zn65 had been added. These isotopes were used because radi-
ation ecologists have shown particular concern about their presence in the vicinity
of nuclear reactors and atomic test sites (Gong, et al., 1957; Davis, 1958; Davis
et al., 1958). For comparison and contrast, data from experimental populations
whose ancestors had received acute exposures to x-rays are also included.
Considering fitness to be measurable as the number of mature offspring left by
tested parents, we obtained a basis for comparison between descendants of control
and irradiated Artemia. There is no evidence of increased fitness over controls for
any experimental population in sea water, diluted sea water or brine.
MATERIALS AND METHODS
Stock origin and maintenance
Our Artemia salina stocks originated from commercial dry cysts of the diploid
amphigonic California strain. Although mass culture techniques were explored
earlier (Grosch and Erdman, 1955), the oldest cultures extant date from 1957.
One of these, number 3 used in the present study, has been maintained from its be-
ginning in the same 5-gallon rectangular battery jar. Additional available control
cultures were begun in 1959 in the same cylindrical gallon jars now containing them.
Of the several available, number 8 has been used in the present study. Control
maintenance has not been a problem. In fact, five control cultures have been dis-
carded due to limitations in space. All control cultures were started from several
1 The U. S. Atomic Energy Commission has provided funds to support summer assistants
at the Marine Biological Laboratory, Woods Hole. For successive summers, the assistants
were Molly Plumb, Sally Corlette, Barbara Thomas Stone, and Louise Emmons.
2 Published with the approval of the North Carolina Agricultural Experiment Station as
Paper No. 1465 of the Journal Series.
302
SURVIVAL OF ARTEMIA POPULATIONS
303
hundred dry cysts, and as many as 300 well developed Artemia have been counted in
a gallon control at the height of the summer. Earlier, in June, the first group to
mature tends to be somewhat smaller, numbering 50 to 100.
Using ten pairs of adults per three liters of sea water seemed the most feasible
approach to setting up radioisotope experiments. A series of doses can be instituted
simultaneously without endangering persistence of the control culture from which
the pairs of adults are removed. Culture #3 provided the parents for all experi-
mental cultures to date. Table I summarizes these cultures and the nature of their
treatments.
TABLE I
Inception and subsequent history of three-liter experimental cultures of Artemia. T = tested
by pair matings. DNS = did not survive. ? = survival questionable
1958
1959
I960
1961
1962
juc. P32 added
30 A
T
T
T
testing
B
T
T
too few to test
DNS
30 MC. P32 added
DNS
30 MC. P32 added
DNS
30 fie. P32 added
40
60
90
T
T
T
DNS
120
DNS
Ate. P32 added
30
90
too few to test
T
testing
120
became extinct
150
DNS
200
DNS
450
DNS
r, x-rav
1000
T
T
testing
2500
DNS
3000
DNS
r, x-ray
1000
2500
DNS
3000
DNS
yuc. Zn65 added
too few to test
30
T
60
DNS
90
DNS
120
DNS
MC. Zn63 added
30
?
60
DNS
90
DNS
120
DNS
304 DANIEL S. GROSCH
In 1958, P32 in phosphate form was added to a series of three-liter (3-L. ) cul-
tures at the following levels: 30, 40, 60, 90 and 120 /*c. The 30 /*c./3-L. culture
gave rise to two subcultures known as "A" and "B" which differ by one generation.
In August, when Fr> larvae became evident, the F2 parents were removed to another
3-L. jar where they produced cysts which overwintered. This culture was desig-
nated "A." The culture derived from the cysts produced by the F3 remaining in
the original jar has been known as "B."
In 1959 duplicate experiments were set up at 30, 90, and 120 /AC./3-L. In addi-
tion higher doses were given to check on the suspected limits of tolerance: 150, 200,
and 450 p£. In 1959 and each successive year descendants from the 1958 30-//,c.
dose were subcultured and given a repeated dose of 30 p.c. of P32
Zn65 in chloride form was added to four 3-L. cultures in 1960 at the following
levels: 30, 60, 90, and 120 /xc. This was repeated in 1961.
The x-ray exposures were made in 1959 and 1960. Each year, ten pairs of
adults were given three doses each from the Woods Hole generator. It operated at
30 ma. and a 200 Kv. peak with an inherent filter equivalent to 0.2 mm. of Cu. De-
livered in a few minutes, the acute doses were 1000 r, 2500 r, and 3000 r, respec-
tively below, near and above the dose found sterilizing for adult females (Grosch
and Erdinan, 1955; Grosch and Sullivan, 1955). All cultures have remained in-
doors, shelved near, but not in, windows which receive sunlight for half of the day.
The cultures were untouched from September until June. During this winter
period, the water gradually evaporated until only an inch of saturated brine re-
mained, along with crystalline salt deposits and colorless algal debris. Persistent
adults were seen only occasionally. The cultures were reconstituted from the cysts
deposited on the sides of the container upon filling with distilled water to the
original high water line. The salts dissolved with stirring.
In general the procedure followed a natural sequence of events described by
Boone and Baas-Becking (1931) for California salterns, where "winter eggs" are
left along the high water marks, to "swell and burst" in the spring when freshets
dissolve the salt crust and the environment reaches a favorable salinity. In labora-
tory cultures it has seemed necessary to remove any large masses of putrefying algae
soon after emergence of the Artemia larvae. Earlier removal may not be advantage-
ous because some cysts can be trapped in the mass.
Pair mating tests
In order to study reproductive capacity and adult life span, pairs of young adults
were moved to quart jars from the large mass cultures upon reaching sexual
maturity. Transfer was by dipping or pouring because adults are easily injured
by pipetting. The pair matings were inspected daily until the death of both animals.
Upon their appearance, broods of live young were counted and removed to separate
containers, to determine their ability to complete development. If cysts appeared,
they were filtered from the culture, dried, counted and resuspended in dilute sea
water for hatchability determinations. When broods reached sexual maturity, the
offspring were counted again and sexed.
Control data gave us reason to believe that quart jars are entirely adequate for
survival records, but to make sure, crowding experiments were performed with
much smaller 4-ounce jars. The experiments, repeated three times, involved a
SURVIVAL OF ARTEMIA POPULATION'S 305
series of 2, 4, 8, 16, 32, 64, 128, 256 nauplii per 4 ounces of sea water. Results of
crowding were evident when groups involved more than 32, but this took the form
of repressed growth and delayed maturity rather than death. A feedback phenome-
non, such as reported by Rose (1960) for fish and amphibia, is suggested.
During the summer all cultures were fed daily with yeast suspension, roughly
at the rate of one drop per adult, added to the culture water. In addition they ate
the volunteer algae present in the cultures. In fact pair matings and their offspring
were maintained under constant illumination from banks of fluorescent tubes as
customary in algae culture. The temperature on warm days reached 28° C. under
such circumstances, at night and on cool days it fell off a degree or two. The tem-
perature for mass cultures elsewhere in the room varied more than this during the
growing period, averaging 25° C. but rising to 30° for afternoons when sun reached
the cultures and falling to 20° on cool nights. This is much like the range in
temperatures experienced by Bowen's (1962) cultures.
Pair mating tests were performed in sea water at the convenient specific gravity
of 1.02 until 1961 when the comparisons were also made at higher and lower specific
gravities within the range of adaptation found by earlier investigators (Jensen,
1918 ; Bond, 1932). Sea water was diluted to a specific gravity of 1.01 with distilled
water. For high salinities NaCl was stirred into sea water to raise the specific
gravity to 1.07 and 1.12. Adults typically survived transfer directly to 1.01 (lower)
or 1.07 (higher) specific gravities, but rarely survived transfer to the 1.12 brine.
Therefore gradual conditioning was attempted by daily additions of twelve succes-
sive equal doses of salt until a specific gravity of 1.12 was reached. However, only
about 10% of the young adults used survived such conditioning.
RESULTS
Survival of cultures
Control cultures have been prolific and maintain themselves without difficulty.
On the other hand, experimental cultures may be sparsely populated and those
experiencing higher levels of radiation quickly trend to extinction. This results
from reproductive failure rather than any other obvious influence on the treated
adults. A summarv of cultures begun and those which failed to survive is given by
Table I.
In the P32 series, 3-L. cultures above 90 ^c. have failed to survive. This limit
was demonstrated for both the 1958 and 1959 series of experiments. The most per-
sistent case was a sparse population in a 120-^u.c. jar which survived the 1959-60
overwintering but during the summer of 1960 did not expand successfully. Sub-
cultures of 30-fj.c. experiments have not survived a repeat dose of 30 /j.c. of P32.
Furthermore, after 14—15 generations, the 1958 series of P32 cultures have entered
a period of decline and seem to be on the verge of extinction. Cultures of the 1959
series which have gone through only nine generations appear to be in better
condition.
In the Zn65 series Artemia cultures have survived only the lowest dose, 30 ju.c./
3L. In the x-ray series persistent cultures have not been obtained from Artemia
receiving more than 1000 r. In one culture from Artemia which had received
1000 r, nine generations have now elapsed. In 1960 the culture whose ancestors
received 1000 r of x-ray appeared superior to the culture begun at the same time
306
1959
DANIEL S. GROSCH
I960 1961
30 r-
20
CO
10
\\
\\
\\
\\
\\
\\
\\
\\
\\
\\
C-3
FROM
90 C3
1958
—
I
—i
\
\
s
^
S
\
\
\
\
\
\
\
\
\
\
\
\
\
\
S
S
\
,.
A B
30 9O X
01
in
Oi
\
\
\\
\\
\\
\\
\\
\\
ss
\\
\\
\\
\\
\\
\\
\\
\\
\\
\\
\\
C 3 30 90 X C 8 90
O)
m
in
en
FIGURE \. Survival of adult Artcmia after transfer to quart jars for pair mating tests.
The summer in which the data were obtained is identified by the year shown at the top ; the
cultures from which the animals were taken are designated along the bottom of the figure.
C indicates control ; X stands for x-rayed ; 30 and 90 refer to the pc. of P32 added to respective
3-liter cultures at the start of the experiments. Female survival is given by the right bar of
each pair.
incorporating 90 p.c. of P32. The x-ray culture provided adequate numbers of pairs
for testing during a period when the P:i2 jar was too sparsely populated.
Duration of life
The average number of days between their transfer to quart jars and the death
of members of mated pairs of Artemia is taken as a measure of adult life span (Fig.
1). Typical standard errors associated with these values range from 2.40 to 2.49
days for males and from 3.12 to 3.65 days for females. In 1959, experimental ani-
mals lived as long as or longer than the controls from culture #3. In subsequent
years, individuals whose ancestors were subjected to radiation tended to die sooner
than #3 controls. In 1961 an additional control, #8, was sampled. This lias been
maintained in exactly the same size and shape of jar as all experimental cultures.
As shown, the adults withdrawn from #S lived even longer than those from #3.
Therefore, size and shape of container are ruled out as influences in poor life-span
of experimental adults.
SURVIVAL OF ARTEMIA POPULATIONS
307
30 i-
1.07
1.01
20
CO
SP. GR.
A
C-3 30
C-3 30 X
FIGURE 2. Survival of adult Artcwia used for pair mating tests in sea water to which NaCl
had been added (1.07 sp. gr.) and in diluted sea-water (1.01 sp. gr.). The respective culture
supplying the animals is designated along the bottom : C-3 indicates control #3, A 30 refers to
30 /ic. added to a 3-L. culture in 1958, X stands for the culture whose original parents were
x-rayed in 1959. Female survival is given by the right bar of each pair.
Pair matings from the 1958 cultures receiving 90 ^c. of P32 have given relatively
consistent life span records in successive years. This is true also for the duplicate
experiment begun in 1959. Furthermore, similar life spans have been obtained
from the acute dose of 1000 r of x-rays. On the other hand, the cultures derived
from a 30-/*c. P32 treatment have varied over the years of study. Recently adult
life span has proved brief in ordinary sea water. Additional results which are not
included on the figure are for the 30-^c. Zn65 culture begun in 1960. Average
survival of males was 18.6 ± 3.95, and 19.3 ± 2.28 days for females.
When brine of 1.07 specific gravity was used for tested pairs, adult life span
was prolonged. The proper comparison is between the 1.07 results of Figure 2
and the 1961 results of Figure 1 for the same three cultures. The standard errors
for Figure 2 values are within the range stated above for Figure 1 values.
In brine of higher specific gravity, 1.12, the two pairs of control adults which
survived conditioning had lengthy spans of life: 31 and 36 days for males, and 28
and 24 days for females. P32 animals did not do as well. Four conditioned pairs
308
DANIEL S. GROSCH
averaged 22.5 and 13.2 days for males and females, respectively. From the x-ray
population, the one pair conditioned died after only 11 (female) and 15 (male) days.
Figure 2 also presents survival of adults in dilute sea water of 1.01 specific
gravity. These values are better but do not impressively exceed the 1961 sea- water
results for the same culture (Fig. 1). It is impossible to run all tests simultane-
ously so some improvement might be due to increased experience of the assistant.
However, in this case less variability might be expected. Instead, standard errors
exceed 4 days, and for males of control #3 is a high 5.22 days.
Components of fitness
Life span is part of the story, but it is possible to examine the various aspects of
reproductive failure more directly. The summaries in Tables II and III indicate
whether mated pairs are likely to give rise to sexually mature offspring. Adaptive
values epitomize the reproductive efficiency of a genotype in a certain environment.
TABLE II
Reproductive behavior of Artemia cultures as revealed by pair mating studies in sea water
Cultures treated
Number of
Zygotes
voided *
% Survived
Mature adults
Adaptive
indicated
pair
Per pair
Per brood
to adult
per pair
value
Results of
pair matings
in 1959
Control #3
1.2
176.9
81.7
24.3
43.0
1.00
30V-/3L. 1958
"A"
0.9
31.6
27.4
24.4
7.7
0.18
"B"
0.7
85.1
52.7
26.0
22.1
0.51
90>c./3L. 1958
0.6
31.8
39.8
20.0
6.4
0.15
Results of
pair matings
in 1960
Control #3
2.6
387.4
149.0
43.1
156.97
1.00
"A"
1.6
165.9
103.7
27.6
45.79
0.29
"B"
2.6
381.9
146.9
31.5
120.30
0.77
90 Mc. 1958
1.3
110.1
84.7
30.3
33.36
0.21
X- ray
1000 r. 1959
1.0
106.5
106.5
31.2
33.32
0.21
Results of
pair matings
in 1961
Control #3
2.2
179.5
81.6
50.3
90.3
1.00
Control #8
2.4
164.6
68.6
76.5
125.9
1.39
"A"
0.3
12.8
42.7
57.6
8.7
0.10
90 MC. 1958
0.2
6.1
30.5
0
0
0
X-ray 1959
0.5
19.93
39.9
14.5
2.9
0.03
90Mc./3L. 1959
0.3
7.08
28.3
32.7
2.3
0.03
Xn66
30juc./3L. 1960
1.17
59.2
50.6
72.6
42.9
0.58
Zygotes voided is used to refer to the number of nauplii and cysts deposited.
SURVIVAL OF ARTEMIA POPULATIONS
TABLE III
Reproductive behavior of Artemia cultures revealed in 1961 by pair mating studies in salt
waters of higher and lower specific gravities than sea water
309
Cultures tested
Number of
broods per
pair
Zygotes voided *
' , Survived
to adult
Mature adults
per pair
Adaptive
value
Per pair
Per brood
Specific gravity 1.01
Control #3
2.2
228.6
103.9
47.4
108.3
1.00
"A" 1958
0.8 20.5
24.6
26.4
5.4
0.05
X-ray 1959
1.1
51.2
46.6
62.8
32.2
0.30
Specific gravity 1.07
Control #3
2.8
182.5
65.3
51.7
94.3
1.00
"A" 1958
1.9
107.3
60.4
61.9
66.4
0.70
X-ray 1959
2.1
85.4
41.0
69.0
58.9
0.62
* X umber of nauplii and cysts deposited.
As defined (Dobzhansky, 1951) adaptive value is the relative capacity of carriers
of a given genotype to transmit their genes to the gene pool of the following genera-
tions. On this basis we have taken our evidence of the average number of mature
progeny produced per pair, assigned the unit value to control #3 and made the
pertinent comparisons within each year.
Aspects in which a cultured population is deficient are revealed in these experi-
ments where all voided zygotes and all adults developing therefrom are counted.
Table II presents results for pair mating tests in three successive years, using sea
water of 1.02 specific gravity. Table III presents data from 1961 experiments at
lower and higher specific gravities.
Experimental populations have never approached the controls in their produc-
tion of live offspring. "B" came closest in 1960 when as many broods were de-
posited and the number of zygotes per brood was only slightly lower. However,
the curbing influence was revealed in fewer offspring surviving to adulthood. Sub-
sequently decline of this culture has been so drastic that in 1961 it did not produce
enough adults to allow pair mating tests. Adequate numbers of adults for the
1961 pair mating tests were provided by the survivor of the highest P;j2 level of
1958, 90 juc./3L., but Table II demonstrates poor performance in all the aspects
considered. The culture which gave an adaptive value of zero in 1961 tests did not
survive overwintering to 1962.
A small number of broods, a form of infecundity, has appeared for various ex-
perimental cultures tested by pair matings. At the same time, the decrease in the
number of zygotes per brood may not be severe. Indeed, in 1961 "A" was produc-
ing considerably larger broods than it was in 1958 and survival to adulthood was
better than for #3 control, yet relatively few adult offspring were obtained, chiefly
because parents which were fertile produced only single broods. Earlier indica-
tions of the importance of fecundity came in l*7^ when experimentals and controls
310
DANIEL S. GROSCH
differed only slightly in larval lethality, and in 1960 when there was little difference
in larval lethality among various experimental groups. Finally in 1961, larval
survival for "A" and for Zn65 pair mating tests surpassed that for the #3 control
although neither adaptive value at all approaches the control value.
Table III demonstrates that Artemia react differently in their reproductive
abilities when the specific gravity of the medium was made higher or lower than the
convenient 1.02 of sea water. Particularly notable were the improvements in all
reproductive aspects for "A" and the x-ray parents when brine of 1.07 specific
gravity was used. Improvements, except in number of zygotes per brood, were
also seen for control #3.
On the other hand, brine of 1.12 specific gravity did not improve the reproductive
ability of Artemia. One or more broods were produced for the few pairs condi-
tioned to this salty brine, but survival to adulthood was poor: 26.1% for controls,
9.3% for "A" and zero for larvae from x-ray parents. The number of offspring per
brood was not good : 43, 26, and 14.5, respectively.
Hatchability of cysts
Although cysts occur regularly in the mass cultures which evaporate slowly
during the winter, under the conditions of the pair mating tests, "winter eggs"
appeared only occasionally. When obtained they formed the last brood or a part
of it and contributed only a small fraction of the total zygotes voided. Table IV
presents emergence or hatchability of the cysts, along with the number of mated
pairs producing cysts. On the basis of the 1960 records it might seem that when
females have a longer life span (Fig. 1) they are more likely to deposit an encysted
brood, although this is not borne out by our subsequent experience. For example
Control #8 in 1961 averaged 24+ days for female survival but deposited no cysts.
Furthermore in brine of 1.07 specific gravity only 2 of 10 #3 control females
deposited cysts, although the average survival for both sexes was 26-27 days.
If there is no sperm storage (Bo wen, 1962) male life span could be a limiting
factor on cyst deposition. However, from this standpoint the 1960 #3 control
would drop back to 25 days for effective pair survival, which is not significantly
different from the #8 1961 control and shorter than the 1961 #3 control values in
brine (1.07 specific gravity).
TABLE IV
Cyst deposit and emergence from cysts
Source
1959
I960
1961
% emerged
No. laying
cysts per
15 mated
% emerged
No. laying
cvsts per
20 mated
% emerged
No. laying
cysts per
10 mated
Control #3
41.9
6
46.4
17
46.0
6
30 MC. (A
15.4
1
29.2
12
0
pw |B
24.9
3
47.5
16
90 Mc. P32 1958
18.1
1
58.5
6
0
X-ray
49.5
8
28.4
2
Zn66
45.5
1
SURVIVAL OF ARTEMIA POPULATIONS
311
In spite of the inconsistent deposit of cysts by females maintained in isolated
jars under continuous illumination, some insight is provided concerning survival of
mass cultures. In 1960, the year when a large proportion of pairs produced cysts,
tests of three out of four experimental cultures showed hatchability above the
control values. On this basis hatchability does not seem to be a major influence
upon survival of a culture.
Control values between 40 and 50% hatchable cysts are not unexpected in un-
selected samples. Un selected commercial samples of cysts from natural populations
may give even lower hatchability. Flotation or some other method of eliminating
deficient or empty cysts seems necessary to improve hatchability.
Sc.r ratio
A subtle difference between populous cultures and those which appear headed
for extinction is revealed by summarizing the sex ratios of offspring reaching
maturity. A vulnerability of females in treated populations suggests the segrega-
tion of deleterious induced recessives in the heterogametic sex (although the author
is aware that the question of female heterogamety in Artemia is still controversial ) .
Table V demonstrates that the sex ratio tends to favor males when the parental
pairs tested are drawn from cultures whose ancestors were irradiated. In eight
out of ten sets of pair mating tests the control value for the particular year is
exceeded. Chi square determinations provide significant values for 1960 "A" and
TABLE V
Sex ratios given as the ratio of males to females, and chi square values
calculated from the original data
Origin of parents
1959
x-
I960
x"
1961
x-
Control #3
.82
.74
.91
P32 30 MC. A
1.25
2.069
.92
4.650*
1.31
3.450
P32 30 MC. B
1.00
1.031
1.15
16.987**
P32 30 MC. 1958
.72
.124
.83
.907
X-rav 1959
.68
.176
1.22
.860
Zn6S 30 MC.
1.02
1.380
Control #8
.95
.437
1.07 sp. gr. brine
Control #3
.91
A 1958
1.34
9.903**
X-ray 1959
.93
.006
1.01 sp. gr. dilute sea water
Control #3
.88
A 1958
1.40
3.262
X-ray
.87
.0004
* = significant.
* = highly significant.
312 DANIEL S. GROSCH
"B" results as an indication that deviations are more than subtle in those cases.
Note that in controls, more adult females were produced than males, while
experimental cultures favor males.
At both higher and lower specific gravities the sex ratio favors males in tests of
Culture A originally derived from ancestors exposed to 30 /AC. of P32 in 1958. The
chi square value for the 1.07 specific gravity test is highly significant. The culture
from x-rayed ancestors, which at present is more prolific than "A", shows sex
ratios not significantly different from those of #3 controls run at the same specific
gravities.
DISCUSSION
The fact that Artcmia cultures derived from radioisotope- and x-ray-exposed
ancestors are doing poorly may be viewed from several aspects although the problems
of waste disposal, ecological disturbance and population genetics are interrelated.
In practice, where isotopic concentrations have been determined in the environs
of the Hanford, Washington, nuclear plant, concentrations in the effluent water are
much lower than the levels used for our experiments. Bustad (1960) reports
2 X 10~8 /U.C./CG. for P32 and 1 X 10~7 /xc./cc. for Zn65. These are activation products
rather than discharged wastes. Another example is White Oak Lake which
received effluents from Oak Ridge, Tennessee. Wastes here include fission products
and transuranic elements, yet the average concentration in the water was estimated
at 10~3 /xc./cc., lower by at least a factor of ten than any of our experiments.
On the other hand, a document considered when the experiments were planned
(NAS-NRC, 1959) gave a maximum permissible concentration of Zn65 in drinking
water, 6 X 10~2 //,c./cc. or 180 /AC./3L., a level twice that at which Artcmia can
persist, and six times that which makes population survival difficult. The gen-
eralized concentration factor employed for invertebrates provided a more acceptable
value of 1.2 X 10"* /AC./CC. as the permissible sea water concentration for Zn65.
In contrast, even without the invertebrate concentration factor, the MPC of P32 in
drinking water (2 X 10~* /tc./cc.) was placed well below any level yet studied with
Artcmia populations. The recommendations were based on Handbook 52 of the
National Bureau of Standards, now superseded by Handbook 69 in which permis-
sible levels have been reduced for many isotopes.
In waters studied by ecologists it was the highest trophic levels which were
damaged. Although species of fish were disappearing from White Oak Lake, and
shortened life span and poor growth were reported for others, populations of
aquatic insects were able to survive in spite of impressive concentration factors
(Buchsbaum, 1958). Enormous doses of radiation may be necessary to destroy
completely a primary trophic level such as an algal-protozoan community. No
significant physiological or morphological damage to marine algae was demonstrated
after the Bikini atomic tests (Blinks, 1952), although damage to the hereditary
mechanism was not assessed. Doses such as those employed in the present experi-
ment apparently seem in the range necessary to interfere with the primary consumers
of the second trophic level, Arteniia for example. Furthermore, the approach of
the population geneticist is needed to reveal the nature and extent of the damage.
Experimental Artcmia showing no visible evidence in numbers or appearance of
individuals for one or several generations, may carry hidden genetic damage
responsible for subsequent decline to a dangerously small population.
SURVIVAL OF ARTEMIA POPULATIONS 313
Diptera have been the preferred material for such research, even for estimating
genetic damage from the Caroline Islands atomic tests (Stone et al., 1957 ; Stone and
Wilson, 1958). Experimental procedures included population sampling by brother-
sister matings. Reproductive performance, studied under laboratory conditions,
revealed that direct irradiation and fallout damaged Drosophila ananassae popula-
tions severely. Many mutants and gene combinations interfered with development
to adulthood, a difficulty demonstrated again in the present Artemia experiments.
In spite of viability problems, the Drosophila populations have managed to return
to normal reproductive performance, presumably through the operation of natural
selection. The flies required from 26 to 161 generations to achieve reproductive
recovery. Little more than half the lower number of generations has elapsed for
the oldest Artemia culture. It will be interesting to see whether any of the
irradiated Artemia populations can accomplish a recovery to normal levels of
reproductive performance.
For D. ananassae no consistent relation of egg counts to genotype was detected
(Stone et al., 1957) although survival-extinction predictions for D. melanogaster
are based in part upon fecundity (Wallace and Dobzhansky, 1959). Since the
maximum number of possible offspring depends upon the number of functional eggs
produced, there is a certain number of eggs required per female if the population is
not to become extinct when exposed to a given amount of radiation. Fecundity as
well as zygote viability is under genetic control and subject to irradiation damage,
so that two dose-dependent aspects of survival are interrelated. With the excep-
tion of "B" in 1960, our experimental Artemia cultures have shown poor fecundity
from the beginning. Possibly in a viviparous animal this matter is more serious
than in an oviparous form. Insurmountable crises in development may occur
which result in elimination of the zygote before deposit. Indeed, our category of
"zygotes voided" may really reflect early embryo death and resorption as well as egg
productivity. The cysts, which are often incorrectly called "eggs," are really
embryos as far along as the blastula stage.
The price paid for the elimination of detrimental and lethal factors from a
population is death of individuals, actual or potential. Our Artemia populations
may now be paying this price. Controversy exists concerning (a) the retention
of seemingly deleterious chromosomes for virtue of their characteristics in hetero-
zygous individuals (Wallace, 1956), and (b) whether ambivalent mutants exist
which impair fitness when homozygous but improve that of their heterozygous
carriers (Wallace and Dobzhansky, 1959). High adaptive values for irradiated
Drosophila populations have been reported (Wallace and King, 1951 ; Wallace,
1951), and the adaptive value for one acutely irradiated population even exceeded
control values. In this case an x-ray dose of 1000 r was delivered to females and
seven times that dose to males. In contrast to the Drosophila results, experimental
cultures of Artemia whose ancestors received 1000 r. to both sexes are clearly
inferior to control populations. Indeed, for experimental Artemia, none of the
adaptive values approach the high values reported for Drosophila. However, here
again a comparable number of generations has not elapsed. By 1956, Wallace's
populations had been followed for 150 generations; by 1959. 200 generations had
elapsed. In addition there are a number of other features, such as size of organism
and irradiation in water vs. air, which complicate a comparison. Furthermore
314 DANIEL S. GROSCH
Wallace's Drosophila populations involved a contrived genetic background, an inten-
tional isogenicity not readily obtainable with other organisms. Also, from a
cytological standpoint it may be significant that Drosophila has a small number of
chromosomes, some of which are long, possibly an ideal situation for fixation of
chromosomal polymorphism. In contrast, Artemia has a large number of short
chromosomes.
Selection experiments clearly indicate the accumulation of genetic lethals in
irradiated laboratory stocks of Drosophila (Muller, 1950), but Wallace argued that
fitness of a population consisting mainly of heterozygous individuals may be excel-
lent, provided the population is large enough so that segregation of detrimental
homozygotes will not threaten its existence. Perhaps our populations of several
hundred Artemia are dangerously small, but this reflects our decision to devote
facilities and efforts to a number of cultures encompassing a range of treatments,
rather than to a few enormous populations which might have been given treatments
too low for sharply defined comparisons. Actually, results from populations of
limited size may be especially pertinent for practical considerations in other
organisms. Although seasonally dense populations of Artemia occur in some
salterns, such cases may be exceptional in present day ecology. Field studies have
shown that most of the species present in a locality are represented by only a few
individuals (Williams, 1953).
Doubt has been cast on improvement resulting from irradiation through a
neoclassical version of heterosis. If mutations increasing the viability of the
heterozygote are not demonstrable under favorable conditions for their detection,
they are not too helpful an explanation of conditions in natural and experimental
populations (Muller and Falk, 1961). Only decreases in the average viability of
an otherwise homozygous Drosophila melanogaster genotype were obtained for
radiation-induced mutations in heterozygous and unselected conditions (Falk, 1961).
Furthermore, in laboratory conditions no significant influences on heterozygote
viability were demonstrable for D. willistoni lethals, whether natural or induced (da
Cunha et al., 1959). In plants, Stadler's (1932) pessimism about the damaging
aspects of radiation-induced mutation is traditional, although for cultivated crops
desirable traits may emerge from irradiated populations under the practice of arti-
ficial selection (Gustafsson, 1947; Sparrow and Singleton, 1953; Konzak, 1954;
Gregory, 1956). Finally, to date, only detriment has been demonstrated for
Artemia cultures descended from irradiated ancestors.
A notable point concerning Artemia biology has emerged from these studies. In-
creased life span and improved reproductive performance in brine (1.07 specific
gravity) indicate favorable aspects in addition to a lack of predators (Lochhead,
1941) in the niche with which Artemia is associated. Other recent investigators
feel it desirable to culture Artemia in water saltier than sea water. Bowen's (1962)
standard procedure is to add NaCl as we have done. After trials with different
concentrations, Goldschmidt (1952) adopted a standard specific gravity of 1.04
obtained by evaporation.
SUMMARY
1. Results are presented for four years of study on the survival of Artemia
cultures when ancestors have been exposed to a series of doses of either radioisotopes
SURVIVAL OF ARTEMIA POPULATIONS 315
-or x-rays. Cultures were begun by transferring 10 pairs of adults from a control
•culture to a 3-liter jar of sea water. Ordinarily, within a generation this gives rise
to a culture of several hundred animals.
2. Three-liter cultures did not persist if more than 90 [*.c. of P32 or more than
30 /j.c. of Zn65 have been added. Subcultures of 30 ;u,c. of P32 per three liters did
not survive a second dose of 30 //.C./3L. Also, cultures failed if 2000 r or more
of x-rays were delivered to the 10 pairs of adults used to institute the culture.
3. The treatments investigated had no obvious effect upon the original adults.
Decline and extinction of the cultures occurred at the first or subsequent generations
•of offspring.
4. In order to assess reproductive failure, pairs when sexually mature were
transferred from the 3-L. cultures to quart jars. All zygotes voided were counted
and hatchability was determined for any cysts deposited. Each brood was trans-
ferred to a separate container. Progeny surviving to adulthood were counted again
and sexed.
5. (a) Decrease both in number of zygotes voided and in survival to adulthood
contributed to low adaptive values for experimental organisms.
(b) The sex ratio among offspring tends to favor females in control and
males in experimental material.
6. Routinely the convenient specific gravity of 1.02 has been used for pair
matings and spring reactivation of mass cultures. In 1961 pair mating tests were
run in dilute sea water of 1.01 specific gravity and in sea water to which NaCl had
been added to reach a specific gravity of 1.07. Both life span and reproductive be-
havior were improved in brine of 1.07 specific gravity. However, attempts to con-
dition adults to saltier brine of 1.12 specific gravity were rarely successful and re-
productive performance of the few shrimp conditioned was poor. Evidently there
is an optimum brine range for Artemia, involving more fundamental biological
aspects than previously reported.
LITERATURE CITED
BLIXKS, L. R., 1952. Effects of radiation in marine algae. /. Cell. Comp. Phvsiol., 39 Suppl.
2: 11-18.
BOXD, R. M., 1932. Observations on Artcmia "franciscana" K. especially on the relation of
environment to morphology. Int. Rev. der qes. Hvdrobiol. u. Hydrographie, 28:
117-125.
BOOXE, ELEANOR AND B. G. M. BAAS-BECKING, 1931. Salt effects on eggs and nauplii of
Artemia salina L. /. Gen. Physiol., 14: 753-763.
BOWEN, SARANE T., 1962. The genetics of Artemia salina. I. The reproductive cycle. Biol.
Bull, 122: 25-32.
BUCHSBAUM, R., 1958. Species response to radiation: radioecology. 124-141, Radiation
Biology and Medicine. Ed. by W. D. Claus. Addison-Wesley Publ. Co., Reading,
Mass.
BUSTAD, L., 1960. Significance of nuclear industry effluents in animal populations. Chapt. 17,
Symposium on Radioisotopes in the Biosphere. Univ. of Minnesota Press, Minneapolis.
DA CUNHA, A. B., J. S. DE TOLEDO, C. PAVAN, H. L. DE SOUZA, H. E. MELARA, N. GABRUSEWYCZ,
M. R. GAMA, M. L. PIRES DE CAMARGO AND L. C. DE MELLO, 1959. A comparative
analysis of the effects of natural and of radiation-induced lethals in heterozygous indi-
viduals and of their frequencies in natural populations of Drosophila willistoni.
Progress in Nuclear Energy, Series VI, 2: 359-363.
DAVIS, J. J., 1958. Radioisotopes in Columbia River organisms. Radiation Res., 9: 105-106.
316 DANIEL S. GROSCH
DAVIS, J. J., R. W. PEKKINS, R. F. PALMER, W. C. HANSON AND J. F. CLINE, 1958. Radio-
active materials in aquatic and terrestrial organisms exposed to reactor effluent water.
Second U. N. Int. Conf. Peaceful Uses of Atomic Energy, 18: 423-428.
DOBZHANSKY, T., 1951. Genetics and the Origin of Species. 3rd Edition Revised. Columbia
University Press, New York.
FAI.K, R., 1961. Are induced mutations in Drosophila overdominant ? II. Experimental results.
Genetics, 46: 737-757.
GOLDSCHMIDT, ELIZABETH, 1952. Fluctuation in chromosome number in Artemia salina. J.
Morph., 91: 111-131.
GONG, J. K., W. H. SHIPMAN, H. V. WEISS AND S. H. COHN, 1957. Uptake of fission products
and neutron induced radionuclides by the clam. Proc. Soc. Exp. Biol. Med., 95: 451-454.
GREGORY, W. C., 1956. Induction of useful mutations in the peanut. Brookhaven Symposium
in Biology, 9: 177-190.
GROSCH, D. S., AND H. E. ERDMAN, 1955. X-ray effects on adult Artcmia. Biol. Bull., 108:
277-282.
GROSCH, D. S., AND R. L. SULLIVAN, 1955. X-ray induced cessation of gamete production by
adult female Artcmia. Biol. Bull., 109: 359.
GUSTAFFSON, A., 1947. Mutation in agricultural plants. H credit-as. 33: 1-100.
JENSEN, A. C., 1918. Some observations on Artcmia qracilis the brine shrimp of Great Salt
Lake. Biol. Bull., 34: 18-32.
KONZAK, C. F., 1954. Stem rust resistance in oats induced by nuclear radiation. Agron. J.,
46: 538-540.
LOCHHEAD, J. H., 1941. Artcmia, the brine "shrimp." Turto.r Nnt's, 19: 41-45.
MULLER, H. J., 1950. Radiation damage to genetic material. Atncr. Sci., 38: 33-59; 399-425.
MULLER, H. J., AND R. FALK, 1961. Are induced mutations in Drosophila overdominant?
Genetics, 46: 727-735.
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posal into Atlantic and Gulf coastal waters. Publ. No. 655. Washington, D. C.
NATIONAL BUREAU OF STANDARDS, U. S. DEFT. COMMERCE. Handbook 52, 1953. Handbook 69,
1959. Washington, D. C.
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SPARROW, A. H., AND W. R. SINGLETON, 1953. The use of radiocobalt as a source of gamma
rays and some effects of chronic irradiation on growing plants. Amer. Nat., 87: 29-48.
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Congress Genetics, 1 : 274-294.
STONE, W. S., M. R. WHEELER, W. P. SPENCER, FLORENCE D. WILSON, JUNE T. NEUEN-
SCHWANGER, T. G. GREGG, R. L. SEECOF AND C. L. WARD, 1957. Genetic studies of
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WALLACE, B., 1951. Genetic changes within populations after x-irradiation. Genetics, 36:
612-628.
WALLACE, B., 1956. Studies on irradiated populations of Drosophila mclanogastcr. J. Genetics,
54: 280-293.
WALLACE, B., AND T. DOBZHANSKY, 1959. Radiation, Genes and Man. Henry Holt and Co.,
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WALLACE, B., AND J. C. KING, 1951. Genetic changes in populations under irradiation. Amer.
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WILLIAMS, C. B., 1953. The relative abundance of different species in a wild animal population.
/. Animal Ecology, 22: 14-31.
XEUROSECRETION AND CRUSTACEAN RETINAL PIGMENT
HORMONE: ASSAY AND PROPERTIES OF THE LIGHT-
ADAPTING HORMONE 1
L. II. KLEIXHOLZ, H. ESPER, C. JOHNSON AND F. KIMBALL
The Biological Laboratories, Reed College, Portland 2, Oregon; Marine Biological Laboratory,
Woods Hole, Mass.; Kristineberg Zoological Station, Fiskebackskil, Szvedcn;
Zoological Station, Naples, Italy
Within the past three decades, hormones from the X-organ-sinus-gland complex
of the crustacean eyestalk have been shown to participate in a variety of physio-
logical systems : color change, photomechanical movements of retinal pigments, hy-
perglycemia under stressing conditions, molt, regeneration, and ovarian growth.
Such physiological effects have heen reviewed in detail by a number of contributors
to a study of the physiology of Crustacea (Bliss, 1960; Charniaux-Cotton, 1960;
Florkin, 1960; Kleinholz, 1961 ; Passano, 1960; and Welsh, 1961). It is apparent
from these reviews that the physical and chemical properties of the reported active
principles are not well known, although such information would be valuable in re-
solving the number of different hormones responsible for the variety of physiologi-
cal effects obtained with crude extracts of eyestalks. The erythrophore-concentrat-
ing hormone has been the only one reported as a purified preparation (Edman,
Fange and Ostlund, 1958) but no thorough tests have been made either of its
chemical or physiological homogeneity ; this preparation shows no activity in light-
adapting distal retinal pigment (Kleinholz, 1958; Kleinholz et al., 1962). Knowl-
edge of the properties of these eyestalk hormones would be helpful not only in indi-
cating their chemical nature but also in their separation, purification and subsequent
chemical identification.
Such anticipated separation and purification attempts will require assay methods
for each of the active principles being investigated. Abramowitz (1937) has de-
scribed a biological assay of chromatophore hormone based on the melanophore of
Uca, while Sandeen (1950) and Fingerman (1956) have measured erythrophore
responses to hormone by methods that might be developed into an assay procedure.
It has been shown that the distal retinal pigment of Palacnwn adspcrsus (Kleinholz
and Knowles, 1938) and of Palacnwnctcs vulgaris (Sandeen and Brown, 1952) as-
sumes positions intermediate between the extremes of light- and of dark-adaptation
related to intensity of illumination. Since a range of concentrations of injected
eyestalk extract produces a similar graded response of the distal retinal pigment in
Palaemon (Kleinholz, 1938), it is postulated that normal photomechanical move-
ment of these effectors may be regulated by the amount of hormone liberated into the
circulatory system, and that an assay for this hormone could be devised on the basis
of these observations.
1 Aided by grants to L. H. K. from the National Science Foundation (G-3986) and from the
National Institutes of Health (B-2606). Some of the results reported here have been
described in preliminary abstracts, Kleinholz and Kimball (1961) and Kleinholz et al. (1961).
317
318
L. H. KLEIXHOLZ, H. ESPER, C. JOHXSON AXD F. KIMBALL
x.30r
LU
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LU
I
Q_
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o:
en
5.00
-1.0
0 1,0
LOG. CONC.ES/ML
2,0
o
z
LU
Z,
o
QL
.20
P- 10
LU
in
5.00
-10
-0.5 0
LOG. CONC.ES/ML
0.5
CRUSTACEAN RETINAL PIGMENT HORMONE 319
The present report describes dosage-response relations for the light-adapting
retinal pigment hormone, and several properties of this principle. Both kinds of
examination were undertaken as necessary preliminaries to a systematic attempt at
isolating the hormone in pure form.
MATERIALS AND METHODS
The decapod crustaceans, Libinia cuiarginata Leach (males, weighing 500-550
grams), Palaemonetes vulgaris Say (not selected by sex but including a large pro-
portion of ovigerous females, 35-40 mm. rostrum-telson length) and Carcinns
maenas Linnaeus (males, approximately 5 cm. in maximum carapace width) were
donor species whose eyestalks were used to construct dosage-response curves for
the distal retinal pigment. Palaemonetes mlgaris - was the test animal for the first
two donor species and Palaemon adspersus - for the third. Eyestalks from the
light-adapted donor species were triturated with a small amount of reagent-grade
sand and were extracted with measured amounts of solvent (distilled water for
Palaemonetes eyestalks, sea water for the others). The tissue suspensions were
centrifuged and the supernatants injected into test animals within an hour after the
extractions were begun.
Test and control animals, isolated in individual containers, were dark-adapted
for 3-10 hours before injection. At timed intervals, 0.05 ml. of the prepared ex-
tract was injected into a test animal by the dim light from a red lamp; uninjected
control animals were exposed to the same light for comparable periods. Forty-five
minutes after injection (Welsh, 1930; Kleinholz. 1936, 1938), response of the
distal retinal pigment cells was measured. The slight modification of the Sandeen
and Brown (1952) method of recording the response as a "distal retinal pigment
index" (DRPI) has been described (Kleinholz ct al, 1962). Briefly, the ratio
of two measurements (distance from the cornea to the distal margin of the distal
retinal pigment, and distance from the cornea to the proximal margin of the dorsal
pigment spot shown in Figure 1) furnishes the DRPI. The dosage-response
curves are based on a minimum of 10 injected test animals (i.e., 20 retinas) for each
concentration of extract.
Stability of distal retinal pigment light-adapting hormone (DRPLH) to dry-
ing, heating and freezing was examined. An extract of 10 eyestalks of Libinia in
1 ml. distilled water was heated for 2 minutes at 100° C. and centrifuged. Three
100-/xl. aliquots of the supernatant were applied to strips of filter paper and dried in
a stream of warm air; the paper strips were then stored under vacuum at 20° C.
After 1, 6, and 20 days of storage one of the paper strips was eluted for 2 hours
with 0.5 ml. distilled water. The eluates, equivalent to concentrations of 2 eye-
stalks per 1 ml., were tested for activity by injection into dark-adapted Palaemonetes.
2 The systematic nomenclature of these crustaceans has recently undergone revision. The
new names were also used in the first report in this series, Kleinholz ct al. (1962).
FIGURE 1. Regression of distal retinal pigment index (response) on logarithm of eyestalk
concentration of injected extracts. The upper figure is for Palaemonetes eyestalk extract, with
the standard error of the estimate shown in broken lines. The inset drawing of an eyestalk shows
the measurements made for calculating the DRPI. The lower figure is for Libinia eyestalk
extract. The test species for both figures is P. r
320 L. H. KLEINHOLZ, H. ESPER, C. JOHNSON AND F. KIMBALL
The effect of heat on activity of retinal pigment hormone was examined by com-
paring DRPI responses produced by extract of eyestalks dried 2 hours at 110° C.
with responses given by extracts prepared from fresh, unheated eyestalks of the
same donors. Ablated eyestalks, one from each of 15 light-adapted Palaemonetes,
were placed in the drying oven. The remaining eyestalks, removed immediately
thereafter, were ground and extracted in 1.5 ml. distilled water, centrifuged, and the
supernatant injected into dark-adapted Palaemonetes. The oven-dried eyestalks
were then similarly extracted and tested.
Hormone activity in frozen-dried eyestalks was compared with that in oven-
dried eyestalks. One eyestalk from each of 20 Palaemonetes was collected and
frozen in a small boat of aluminum foil kept on solid CO,, while the second eye-
stalks from these donors were dried at 115° C. The frozen eyestalks were lyophi-
lized, after which both sets of eyestalks were stored at room temperature in a
vacuum desiccator over anhydrous CaSO^. On the following day extracts were
prepared in concentrations of 20 eyestalks per 1 ml. and were tested.
Solubility of retinal pigment hormone in ethanol and in acetone was determined.
Sets of eyestalks from light-adapted Palaemonetes were dried for 3-7 hours at
115° C. and stored in a vacuum desiccator over anhydrous CaSO4 until used. One
set of dried eyestalks was homogenized and extracted with distilled water ; the
second set of contralateral eyestalks from the same donors was extracted with 100%
ethanol that had been dried for the preceding 24 hours over CaO. After centrifu-
gation, each residue was washed with its appropriate solvent. The combined ethanol
supernatants were evaporated to dry ness at 115° C., and the residue dissolved in
distilled water. The homogenized tissue remaining after ethanol extraction was
then extracted with distilled water. The final extracts thus represented the original
control aqueous extract, the ethanol-soluble extract, and the ethanol-insoluble ex-
tract, all now in aqueous solutions whose concentrations were adjusted to 10 eye-
stalks per 1 ml. Activity of the 100% ethanol fraction was measured on two
successive days, the extracts being stored at —20° C. in the interim. The same
procedure was used to extract dried eyestalks with 95% ethanol, and with acetone
containing 1% glacial acetic acid.
Dialyzability of retinal pigment hormone was tested with Visking cellophane
tubing. Distilled water extracts of Libinia eyestalks were heated for 2 minutes at
100° C., centrifuged, and 1 ml. of the supernatant, equivalent to 10 eyestalks, was
dialyzed at 10° C. against 1 ml. of distilled water. Samples of the dialysate were
injected into test Palaemonetes after 3 and 24 hours of dialysis ; the contents of the
cellophane bag were also tested for activity after 24 hours of dialysis.
Gradual inactivation of retinal pigment hormone, found to occur in freshly pre-
pared extracts allowed to remain at room temperature, was suspected of being
mediated by tissue enzymes, and the rate of inactivation was examined from this
aspect. An extract of 10 eyestalks of Palaemonetes in 1 ml. of filtered sea water
was prepared; immediately after centrifugation a portion of the supernatant was
tested at "zero" hours by injection into 5 dark-adapted Palaemonetes. The rest of
the supernatant solution, kept at about 25° C., was tested for activity at intervals
thereafter of 3, 6, 10.5 and 12 hours. Two modifications in procedure were made
to minimize the possible role of micro-organisms in the sea water. In one modifi-
cation an extract of 40 Palaemonetes eyestalks in 4 ml. of distilled water was divided
CRUSTACEAN RETINAL PIGMENT HORMONE 321
into two parts, one of which was heated at 100° C. for 1 minute. The extracts
were then centrifuged and both supernatants tested for activity at "zero" hours.
The two solutions, kept at 25-27° C., were tested again 12 and 24 hours afterwards.
In the second modification, the supernatant of a centrifuged extract containing 40
Palaemonetes eyestalks in 2 ml. of distilled water was divided into two equal por-
tions. One sample was diluted with an equal volume of distilled water, while to
the second was added an equal volume of antibiotic solution (10 mg. Parke-Davis
crystalline Penicillin G-potassium and 10 mg. Squibb Mycostatin in 50 ml. distilled
water). The two extracts and a control consisting of the antibiotic solution were
injected into groups of 5 test Palaemonetes at "zero" hours ; a second test of the
two eyestalk extracts was made 1 1 hours later. The amount of inactivation of the
DRPLH was calculated from :
100% - (DRP!.'.-DRP!; x 10°) = % inactivatio"
where DRPIt = the average DRPI produced by unheated extract at the various
intervals after its preparation ; DRPI0 = the average DRPI produced either by ex-
tract tested at "zero" hours or by heated extract; DRPIC == the average DRPI.
0.050, found for a large series of dark-adapted, uninjected control Palaemonetes.
The optimum pH for this inactivation was determined and distribution of the
enzyme in a variety of tissues was examined. A stock enzyme solution was pre-
pared by homogenizing 200 Palaemonetes eyestalks in an ice bath and by extracting
the homogenate with small amounts of iced \% Nad solution. The supernatant,
after centrifugation, was dialyzed for 20-24 hours at 3° C. against three changes of
1.5 liters of 1% NaCl, to remove retinal pigment hormone, and was made to a
volume of 2 ml. The retinal pigment hormone substrate was a partially-purified
preparation containing the equivalent of 200 eyestalks of Palaemonetes per 1 ml.
Both preparations were stored at —20° C., samples being removed as needed from
the thawed solutions. For subsequent tests, 0.3 ml. of distilled water and 0.1 ml.
of the enzyme preparation were added to each of two centrifuge tubes, one of the
tubes being heated for 2 minutes at 100° C. to denature the enzyme and serve as a
control. Appropriate buffer, 1.5 ml., and 0.1 ml. of the retinal pigment hormone
preparation were then added to each tube and the mixtures incubated for 6 hours
at 38° C. After centrifugation and checking the pH of the supernatants, hormonal
activity was tested, the supernatant containing the undenatured enzyme being in-
jected first, generally within 15 minutes after removal from the incubator. The
amount of inactivation of the DRPLH was calculated as described above. The
buffers used were: 0.1 M succinate, 0.2 M borate, and 0.2 M Tris maleate, to
provide a series of p'H concentrations ranging from 5.1 to 9.1.
A number of tissues other than Palaemonetes eyestalks were examined for the
presence of this hormone-inactivating enzyme. Preparation of the enzyme extract
from these tissues was made as described above for eyestalks ; quantitative details
are summarized in Table II. A volume of tissue brei was placed in each of two
tubes, one of which was heated to denature the enzyme. To both tubes were added
1.5 ml. of 0.2 M Tris maleate buffer at pH 7.4 and 0.1 ml. of retinal pigment hor-
mone solution. After incubation for 6 hours at 38° C. the mixtures were centri-
fuged, and the supernatant tested for activity.
322
L. H. KLEINHOLZ, H. ESPER, C. JOHNSON AND F. KIMBALL
The effect of proteolytic enzymes on retinal pigment horm< me activity was tested
with several preparations. Aqueous extracts of eyestalks in known concentration
were heated briefly in a boiling water bath to coagulate eyestalk debris. The super-
natant, after centrifugation, was divided into two equal portions, enzyme being added
to one while the other served as a control. Incubation at 35-38° C. was for varying
periods (Table III), after which both mixtures were immersed in a 100° C. water
bath for 1-2 minutes, centrifuged, and the activity of the supernatants tested. At
Naples, extract was prepared from eyestalks of Palaemon scrratus and tested on
Palaemon .viphias; at Woods Hole, donor and test species were Palaemonetes vul-
garis. Salt-free crystalline trypsin and chymotrypsin (Worthington Biochemical
Co.) and a crystalline chymotrypsin (Armour and Co.) containing 50^0 ammonium
sulfate were the enzvmes used.
OBSERVATIONS
1. Dosage-response relations
Eyestalk extracts of Palaemonetes give the following average DRPI values and
the calculated standard deviations when injected into dark-adapted Palaemonetes in
TABLK I
Properties of the light-adapting distal retinal pigment hormone. Activity tests were made on
dark-adapted Palaemonetes vulgaris, as described in the text. DRPI, average distal
retinal pigment index for a test group and the standard deviation; ES, eyestalks.
Eyestalk extract
Results
Donor species and
concentration
Treatment
Experimental
DRPI ±S.D.
Control
DRPI ±S.D.
P. vulgaris:
10 ES/ml.
Oven-dried ES vs. control fresh ES
0.215 ± 0.01
0.200 ±
0.02
P. vulgaris:
5 ES/ml.
Oven-dried ES vs. control fresh ES
0.143 ± 0.04
0.181 ±
0.03
P. vulgar is:
20 ES/ml.
l.yophil. ES vs. oven-dried control ES
0.222 d= 0.01
0.229 ±
0.01
P. vulgaris:
10 ES/ml.
100%-EtOH-sol. extract vs. H,O-ex-
tract of oven-dried ES
100%-EtOH-insol. extract vs. H=O-
extract of oven-dried ES
0.090 ± 0.01
0.214 ± 0.01
0.227 ±
0.227 ±
0.01
0.01
P. vulgaris:
10 ES/ml.
95%-EtOH-sol. extract vs. H2O-extract
of oven-dried ES
0.161 ± 0.04
0.182 ±
0.05
Acetone-HAc-sol. extract vs. H2O-ex-
0.049 ± 0.01
0.225 ±
0.01
P. vulgaris:
10 ES/ml.
tract of oven-dried ES
Acetone-HAc-insol. extract vs. H^O-
extract of oven-dried ES
0.215 ± 0.01
0.225 ±
0.01
L. emarginatu:
pre-dialysis
10 ES/ml.
Dialysate after 3 hrs.
Dialysate after 24 hrs.
Dialysate after 22 hrs.
0.113 ± 0.02
0.196 ± 0.06
0.215 ± 0.02
CRUSTACEAN RETINAL PIGMENT HORMONE
323
TABLE I — (Continued)
Eyestalk extract
Results
Donor species and
concentration
Treatment
Experimental
DRPI ±S.D.
Control
DRPI ±S.D.
Inactivation at 25° C. :
0 hrs.
0.189 ± 0.04
P. mil gar is:
3 hrs.
0.144 ± 0.04
10 ES/ml.
6 hrs.
0.114 ± 0.03
10.5 hrs.
0.084 ± 0.02
12 hrs.
0.049 db 0.01
Inactivation of unheated extract vs.
P. vulgar is:
10 ES/ml.
control heated extract:
0 hrs.
12 hrs.
0.208 ± 0.02
0.118 ± 0.04
0.216 ± 0.01
0.215 ± 0.01
24 hrs.
0.100 ± 0.02
0.200 ± 0.02
Inactivation of extract + antibiotic vs.
P. vulgar is:
control without antibiotic
10 ES/ml.
0 hrs.
0.208 ± 0.02
0.199 ± 0.01
11 hrs.
0.077 ± 0.01
0.062 ± 0.02
pH optimum of inactivation ; unheated
extract vs. extract with enzyme de-
natured :
P. vulgar is:
ca. 20 ES/ml.
pH 5.1
pH 6.0
pH 6.7
0.171 ± 0.03
0.179 ± 0.02
0.133 ± 0.04
0.178 ± 0.03
0.226 ±0.01
0.195 ± 0.02
pH 7.3
0.104 ± 0.03
0.205 ± 0.02
PH 8.0
0.131 ± 0.03 0.222 ± 0.02
pH 9.1
0.155 ± 0.04
0.161 ± 0.04
the concentrations shown: 30 eyestalks per ml. — 0.223 ± 0.018; 20 eyestalks per
ml. == 0.205 ±0.019; 10 eyestalks per ml. == 0.190 ± 0.031 ; 5 eyestalks per ml.
= 0.161 ± 0.029; 2 eyestalks per ml. == 0.127 ± 0.036; 1 eyestalk per ml. == 0.120
±0.022; 0.5 eyestalk per ml. = 0.070 ± 0.015 ; 0.2 eyestalk per ml. == 0.061
± 0.019. The relation between these data is linear when concentration of injected
eyestalk extract is plotted on a logarithmic scale (Fig. 1). The equation for this
relation is : Y - 0.108 + 0.077 log A', where Y is the average DRPI for 10 test
animals, and X is the concentration of the injected extract, within the limits of the
tipper and lower thresholds. The standard error of the estimate is ±0.008 DRPI.
Extracts of Libinia eyestalks tested on Palaemonetes result in the following
average DRPI values and their standard deviations: 2 eyestalks per ml. = 0.235
± 0.015 ; 1 eyestalk per ml. == 0.197 ± 0.039; 0.5 eyestalk per ml. == 0.168 ± 0.053 ;
0.25 eyestalk per ml. = 0.137 ± 0.022 ; 0.125 eyestalk per ml. = 0.101 ± 0.011.
The upper threshold concentration is about 2 eyestalks per ml., because the next
higher concentration tested, 4 eyestalks per ml., gives a DRPI of 0.237 ± 0.019.
The linear relation resulting from a plot of the average response against the loga-
rithm of concentration has for its equation: Y — 0.200 + 0.107 log A', with the
standard error of the estimate being ±0.007 DRPI.
324
L. H. KLEINHOLZ, H. ESPER, C. JOHNSON AND F. KIMBALL
TABLE II
Inactlvation of retinal pigment hormone by tissue brei. DRPIU, average distal retinal pigment
index of unheated extract and its standard deviation; DRPI;,, average distal retinal
pigment index of Jieated control extract and the standard deviation.
Species
Tissue usc'l
kb* TO
Saline vol.
used for
making tissue
brei
Brei vol. in
incubated
mixtures
DRPIu
DRPIh
Inactiva-
tion
Heart; wet wt.
2 ml.
0.2 ml.
0.140 ± 0.04
0.205 ± 0.03
41%
= 0.6 gm.
Vas deferens from
2 ml.
0.4 ml.
0.050 ± 0.01
0.212 ± 0.03
100%
2 males
Libinia
Thoracic muscle
4 ml.
0.4 ml.
0.061 ± 0.01
0.195 ± 0.03
92%
emarginata
= ca. 1 gm. wet
wt.
Hypodermis from
2.5 ml.
0.4 ml.
0.055 ± 0.02
0.232 ± 0.02
97%
2 carapaces
Blood ; 3 ml.
0 ml.
0.4 ml.
0.213 ± 0.03
0.215 ± 0.01
1%
Palaemonetes
Ventral nerve
1 ml.
0.4 ml.
0.093 ± 0.02
0.197 ± 0.03
72%
vulgaris
cord; 35 animals
Pandalus
50 eyestalks,
C f\f\
2.5 ml.
0.2 ml.
0.151 ± 0.03
0.223 ± 0.02
42%
borealis
= ca. 500 mg.
t
dry wt.
Mercenaries
Adductor muscle,
4 ml.
0.4 ml.
0.059 ± 0.01
0.218 ± 0.03
95%
inercenaria
wet wt. = 2 gm.
Similar tests with extracts of Cardnus eyestalks on P. adspcrsus as test animal
yield the following DRPI responses : IS eyestalks per ml. = 0.188 ± 0.025 ; 10 eye-
stalks per ml. = 0.196 ± 0.029; 5 eyestalks per ml. = 0.190 ± 0.031 ; 2.5 eyestalks
per ml. = 0.178 ± 0.027 ; 1 eyestalk per ml. == 0.134 ± 0.015 ; 0.5 eyestalk per ml.
= 0.129 ± 0.020 ; 0.2 eyestalk per ml. == 0.048 ± 0.012 ; 0.1 eyestalk per ml. = 0.035
± 0.004. The upper threshold concentration seems to be about 5 eyestalks per ml.
If these responses are plotted as a function of the logarithm of concentration of the
injected extract the equation for the resulting linear relation is : Y — 0.134 -f 0.096
log X, with the standard error of the estimate being ±0.015 DRPI.
2. Stability, solubility and dialysability of the hormone
Samples of aqueous extract of Libinia eyestalks, dried on filter paper strips and
stored in vacuum, retain most of their activity. This is shown by average responses
of 0.210 ± 0.02; 0.150 ± 0.04; and 0.170 ±0.02 obtained when eluates from such
paper strips made 1, 6, and 20 days, respectively, after storage are tested by injec-
tion. The concentration of the eluates (on the assumption that complete elution of
hormone had occurred) was 2 eyestalks per ml.; the responses can be compared
with the dosage-response curve for Libinia in Figure 1. A better controlled ex-
amination of the effects of drying on stability of retinal pigment hormone is shown
in the next group of experiments. Extracts prepared from oven-dried Palaemo-
netes eyestalks give responses only slightly different from those produced by con-
CRUSTACEAN' RETINAL PIGMENT HORMONE
325
TABLE III
Effect of proteolytic enzymes on activity of retinal pigment hormone. Prepared extracts, after
heat treatment, were divided into two portions, enzyme being added to one and the other
serving as control. The animals used were: P.s., Palaemon serratus; P.v.,
Palaemonetes vulgaris; P.x., Palaemon xiphias. The crystalline enzymes
used were: T, trypsin, and C, chymotrypsin; the designation in parentheses
indicates the commercial source given in "Afethods." Results are
shown as DRPI, average distal retinal pigment index with
the standard deviation, and the percentage
inactivation, calculated as described.
Eyestalk extract
Enzyme treatment
Results
Donor
Cone.
Enzyme
Cone.
Incubation
Test
species
DRPI ±S.D.
Inacti
vation
Enzyme
Control
P.v.
P.v.
P.s.
P.v.
P.v.
10 ES/ml.
10 ES/ml.
3 ES/ml.
10 ES/ml.
10 ES/ml.
T (WBC)
T (WBC)
C (ARM)
C (WBC)
C (WBC)
10 mg./ml.
5 mg./ml.
5 mg./ml.
8 mg./ml.
8 mg./ml.
12 hrs. at 35° C.
11 hrs. at 35° C.
19 hrs. at 37.5" C.
4 hrs. at 37.5° C.
13 hrs. at 37° C.
P.v.
P.v.
P.x.
P.v.
P.v.
0.136 ±0.03
0.122 ±0.03
0.097 ± 0.02
0.098 ± 0.02
0.075 ±0.01
0.207 ±0.02
0.193 ±0.01
0.200 ±0.01
0.204 ± 0.03
0.208 ±0.01
45%
50%
70%
69%
84%
trol extracts of fresh eyestalks ; similarly, extracts of lyophilized evestalks result in
test indices much like those obtained with oven-dried eyestalks (Table I).
Solubility studies, with precautions taken to avoid moisture in the solvents and
in the eyestalk tissue, show little or no activity extracted by 100% ethanol or by
acetone containing \% glacial acetic acid. On the other hand, 95% ethanol does
extract active material from oven-dried eyestalks. Some loss in activity occurred
when the 100%-ethanol series (the ethanol-soluble, the ethanol-insoluble and the
control aqueous extract) was thawed and tested after storage at —20° C. ; the respec-
tive DRPI were 0.071 ± 0.01, 0.123 ± 0.04, and 0.188 ± 0.02, and an insoluble
residue was present in each thawed preparation.
The hormone readily passes through a cellophane membrane, three hours of
dialysis being sufficient to indicate the presence of activity in the dialysate (Table
I ) . After 24 hours of dialysis, the tested dialysate produced a maximum response
(compare with the dosage-response curve for Libinia in Figure 1).
3. Enzymatic inactivation
Spontaneous inactivation of retinal pigment hormone occurs regularly in ex-
tracts of fresh eyestalks, although the rate and degree of inactivation may be vari-
able. Inactivation in one such experiment is shown in Table I, where the average
DRPI, 0.189, of the freshly prepared eyestalk extract at 0 hours declines progres-
sively until practically no activity remains 12 hours afterward. The rate of such
inactivation is shown in Figure 2, with the percentage of inactivation being calcu-
lated as described in the section on methods.
A possible enzymatic basis for this inactivation is showrn by tests with identical
extracts, one of which is heated at 100° C. for 1 minute and serves as control for the
unheated extract (Table I). The unheated extract results in an average DRPI of
0.208 at 0 hours and an average DRPI of 0.100 after 24 hours at 25-27° C., while
the average activity values obtained with the heated control extract are not ap-
326 L. H. KLEINHOLZ, H. ESPER, C. JOHNSON AND F. KIMBALL
preciably changed under these conditions. Eyestalk extracts of Palacmon serratns
(5 per ml.) were tested in like manner on dark-adapted Palacmon .viphias. The
extracts were divided into two portions, one being heated for 2 minutes at 100° C.,
and were then incubated at 37° C. for 15 hours. The average DRPI obtained with the
unheated extract is 0.104 ± 0.03 (21 test animals), while that from heated extract
is 0.193 ± 0.02 (17 test animals). The average DRPI for 26 dark-adapted, unin-
jected control P. xiphias is 0.052. The calculated percentages of inactivation are
68% for the Palaemonctes test and 63% for the Palacmon test, although incubation
temperatures and experimental periods were not identical in the two cases.
Addition of antibiotic compounds to eyestalk extracts before incubation does not
prevent loss of hormone activity. A curve constructed from the data in Table I
over the range pH 5.1-9.1 shows the optimum for this inactivating enzyme to be
pH 7.5. A summary of results from examination of a variety of tissues (Table II)
shows that this enzyme is present in all tissues tested except blood.
Incubation of eyestalk extracts for different periods and with varying concentra-
tions of trypsin or of chymotrypsin was made in a number of experiments, five of
which are summarized in Table III. Nearly 50% of the activity originally present
is inactivated by trypsin, while chymotrypsin brings about between 70-85%
inactivation.
DISCUSSION
The construction of dosage-response curves for the light-adapting distal retinal
pigment hormone makes available a quantitative biological assay method for this
hormone. The accuracy with which such assay can be made, however, will prob-
ably depend upon standardization of the procedure in the individual laboratory.
The average DRPI values we obtain with control extracts of eyestalks from
Palaemonetes and from Libinia show good agreement with those read from the
dosage-response curves. Our suggested assay procedure is to obtain by serial
dilution of the "unknown" the concentration producing an average DRPI slightly
below the upper threshold response of the test animals. The average DRPI for
this dilution and those obtained with two additional dilutions below this upper
threshold concentration can then be substituted in the equation for the standard
dosage-response curve to find their equivalent concentrations. Calculation of the
average concentration of eyestalks in the original extract readily follows. It is
evident from the examples reported here that the equations for such standard
curves may vary with the species of the eyestalk donor and of the test animal,
and it will therefore be necessary to construct such a standard curve for the
particular species used.
The interconvertibility of such information from one laboratory to that from
another would be aided by defining a physiological unit of hormone activity. For
the present this can be done with data resulting from tests with Palaemonctes
reported here. We therefore define the Palamontcs unit for distal retinal pigment
hormone as that concentration of eyestalks which, when injected into a minimum of
10 dark-adapted Palaemonetes vulgar is, measuring 35-40 mm. from rostrum to
telson, yield an average DRPI of 0.150, this point being selected because it is about
mid-way between the upper and lower threshold concentrations on the standard
dosage-response curve. By this definition, 1 Palaemonetes unit is contained in
Palaemonetes extracts having a concentration of 3.5 eyestalks per 1.0 ml. or in
CRUSTACEAN RETINAL PIGMENT HORMONE
327
Libinia extracts having a concentration of 0.34 eyestalks per 1.0 nil. After retinal
pigment hormone has been isolated in pure form, dosage-response relations of what-
ever species were being used could be compared with the homogeneous preparation
as a reference.
The stability and solubility properties described above show that eyestalks retain
retinal pigment hormone activity after being oven-dried or lyophilized. This, and
Carlson's (1936) report of chromatophorotropic activity in eyestalks dried and
stored over a long period, have been useful in collecting and preparing quantities of
eyestalk material for purification. We initially observed some loss in activity,
accompanied by the formation of a precipitate, in fractionated eyestalk extracts
thawed after storage at —20° C., and have therefore avoided repeated freezing and
thawing of such preparations.
The in vitro inactivation by tissue extracts and by proteolytic enzymes point out
additional interesting features of retinal pigment hormone. The variety of tissue
extracts which inactivate the hormone, a pH optimum of about 7.5 for such
inactivation, and the fact that the ability to destroy hormonal activity is thermolabile
indicate a widely-occurring enzyme or group of enzymes. Whether such an
enzyme system has an in vivo role in degrading hormone in the normal physiology
of the retinal effectors is not known. Similar inactivation of chromatophorotropic
hormone, first reported by Carstam (1951) for epidermis and later by Perez-
Gonzalez (1957) and by Stephens and Green (1958) for a number of other
HOURS
FIGURE 2. Rate of inactivation of distal retinal pigment hormone by enzyme in an eyestalk
extract which was allowed to remain at room temperature for 12 hours. Extract was prepared
from eyestalks of Palacmonctes.
328 L. H. KLEINHOLZ, H. ESPER, C. JOHNSON AND F. KIMBALL
crustacean tissues, may also explain apparent differences in hormone activity
reported between boiled and unboiled eyestalk extracts.
The reduction by trypsin and by chymotrypsin of retinal pigment hormone
activity described here was also confirmed by Fingerman and Mobberly (1960),
after personal communication to them of our results. They too obtain partial loss
of hormone activity in their trypsin-treated preparations. We observe a greater
amount of inactivation of the retinal pigment hormone by chymotrypsin than by
trypsin, but, because we do not yet know with any certainty the chemical nature of
retinal pigment hormone, discussion of differences between trypsin and chymotrypsin
in their proteolytic action on specific substrate linkages would be little more than
speculation at this time. Such differences between trypsin and chymotrypsin may
be due to the presence in crude eyestalk extracts of substances differentially inhibit-
ing the two enzymes. Knowles et al. (1956) explain the failure of trypsin to
inactivate chromatophorotropic hormone in eyestalk extract of Palaemon as prob-
ably due to inhibitory substances in extract of whole eyestalks, since electro-
phoretically separated chromatophorotropin is readily inactivated by trypsin. In
vitro inactivation of chromatophorotropic hormone of Uca by trypsin, chymotrypsin,
and papain has been reported (Perez-Gonzales, 1957; Stephens and Green, 1958).
On the basis of these and other properties, it has been suggested that the
activity of chromatophorotropins is dependent on the presence of peptide bonds in
the hormone, but the known esterase activity of trypsin and chymotrypsin do not
permit this identification with assurance. Similar properties of the retinal pigment
hormone, such as small molecular size, thermostability, inactivation by tissue extracts
(peptidases?) and by proteolytic enzymes may indicate linkages common to the
molecular structure of the two groups of hormones. The partial degradation
by trypsin and the gradual "spontaneous" inactivation occurring in eyestalk extracts
imply that portions of the hormone molecule may not be essential to physiological
activity of retinal pigment hormone. This too must remain as speculation until such
properties can be examined in highly purified preparations of the hormone.
SUMMARY
1. A standard assay for the content of light-adapting distal retinal pigment
hormone in crustacean eyestalk extracts is described. Linear regression equations for
the relation between response of the retinal effectors of test Palaemonetes and con-
centration of eyestalk extract from Palaemonetes and from Libinia have been
calculated.
2. A Palaemonetes unit of this hormone is defined as that concentration of
eyestalk extract, injected into a minimum of 10 dark-adapted P. vulgaris measuring
35-40 mm. in rostrum-telson length, which will result in an average distal retinal
pigment index of 0.150. For the Palaemonetes and Libinia used in this study 1
Palaemonetes unit is equivalent respectively to concentrations of 3.5 and 0.34
eyestalks per 1.0 ml.
3. Thermostability, small molecular size, complete or partial inactivation by
tissue extracts (peptidases?) and by crystalline trypsin and chymotrypsin are
properties of the hormone consistent with a possible peptide structure.
CRUSTACEAN RETINAL PIGMENT HORMONE 329
LITERATURE CITED
ABRAMOWITZ, A. A., 1937. The chromatophorotropic hormone of the Crustacea : standardization,
properties and physiology of the eye-stalk gland. Biol. Bull., 72: 344-365.
BLISS, D. E., 1960. Autotomy and Regeneration. In: The Physiology of Crustacea (T. H.
Waterman, ed.) vol. 1 : 561-589. Academic Press, New York.
CARLSON, S. P., 1936. Color changes in brachyuran crustaceans, especially in Uca pugilafor.
Kgl. Fysiograf. Sallskap. Lund Fork., 6 : 1-18.
CARSTAM, S. P., 1951. Enzymatic inactivation of the pigment hormone of the crustacean sinus
gland. Nature, 167: 321-322.
CHARNIAUX-COTTON, H., 1960. Sex Determination. In: The Physiology of Crustacea (T. H.
Waterman, ed.) vol. 1: 411-447. Academic Press, New York.
EDMAN, P., R. FANGE AND E. OSTLUND, 1958. Isolation of the red pigment concentrating
hormone of the crustacean eyestalk. In: Zweites Internationales Symposium uber
Neurosekretion (W. Bargmann, B. Hanstrom and E. Scharrer, eds.) pp. 119-123.
Springer, Berlin.
FINGERMAN, M., 1956. Physiology of the black and red chromatophores of Callinectes sapidus.
J. Exp. Zoo!., 133: 87-105.
FINGERMAN, M., AND W. C. MOBBERLY, JR., 1960. Investigation of the hormones controlling the
distal retinal pigment of the prawn Palaemonctcs. Biol. Bull., 118: 393-406.
FLORKIN, M., 1960. Blood Chemistry. In: The Physiology of Crustacea (T. H. Waterman,
ed.) vol. 1 : 141-159. Academic Press, New York.
KLEINIIOLZ, L. H., 1936. Crustacean eye-stalk hormone and retinal pigment migration. Biol.
Bull, 70: 159-184.
KLEINHOLZ, L. H., 1938. Studies in the pigmentary system of Crustacea. IV. The unitary
versus the multiple hormone hypothesis of control. Biol. Bull., 75: 510-532.
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and E. Scharrer, eds.) pp. 110-112. Springer, Berlin.
KLEINHOLZ, L. H., 1961. Pigmentary Effectors. In: The Physiology of Crustacea (T. H.
Waterman, ed.) vol. 2: 133-169. Academic Press, New York.
KLEINHOLZ, L. H., P. R. BURGESS, D. B. CARLISLE AND O. PFLUEGER, 1962. Neurosecretion
and crustacean retinal pigment hormone : distribution of the light-adapting hormone.
Biol. Bull, 122: 73-85.
KLEINHOLZ, L. H., H. ESPER, C. JOHNSON AND F. KIMBALL, 1961. Characterization and partial
purification of crustacean eyestalk hormones. Amer. Zoologist, 1: 366.
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pigment hormone. Biol. Bull., 121: 395.
KLEINHOLZ, L. H., AND F. G. W. KNOWLES, 1938. Studies in the pigmentary system of
crustaceans. III. Light-intensity and the position of the distal retinal pigment in
Leandcr adspcrsits. Biol. Bull., 75 : 266-273.
KNOWLES, F. G. W., D. B. CARLISLE AND M. DUPONT-RAABE, 1956. Inactivation enzymatique
d'une substance chromactive des Insectes et des Crustaces. C. R. Acad. Sci. Paris,
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Waterman, ed.) vol. 1: 473-536. Academic Press, New York.
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A SEROLOGICAL COMPARISON OF FIVE SPECIES
OF ATLANTIC CLUPEOID FISHES
DONALD F. MAIRS * AND CARL J. SINDERMANN
U. S. Bureau of Commercial Fisheries Biological Laboratory, Boothbay Harbor, Maine
The potential role of serology in fishery biology has received increased attention
during the past decade (Gushing, 1952, 1956; Ridgway, 1957; Ridgway, Gushing
and Durall, 1958; Sindermann, 1958; Suzuki, Shimizu and Morio, 1958; O'Rourke,
1959; Sindermann and Mairs, 1959; O'Rourke, 1960; Ridgway and Klontz, 1960V
While most of this recent interest has been stimulated by application of red cell
techniques to the identification of subpopulations, use of the varied methods
available to serology can provide information on any taxonomic level.
Comparative serological studies of serum proteins began over half a century ago
with the work of Nutall (1901) and have been pursued vigorously in recent decades
ty Boyden (1926, 1942, 1943, 1954), Gemeroy (1943), Leone (1949, 1950, 1954),
Leone and Pryor (1954), Leone and Wiens (1956) and others. This work has
demonstrated the utility of serum proteins in systematic studies, especially of
higher taxonomic groups. Quantitative examination of the serological relation-
ships of several species within a genus or family have been made (Stallcup, 1954;
Leone and Wiens, 1955). Clear distinctions of species are possible with highly
specific antisera ; even species hybrids, such as the hinny and mule, may be
distinguished from parental species (Boyden, 1942, 1953). Comparative sero-
logical studies using erythrocyte antigens have been most useful in distinguishing
geographic groups, strains, subpopulations or races within a species (Owen, Stor-
mont and Irwin, 1947; Moody, 1948; Ridgway and Klontz, 1960; and others).
Irwin, Cole and Gordon (1936) and Irwin (1938, 1955) determined serological
relationships of avian species and species hybrids using cellular antigens.
The present investigation was undertaken in 1958 to clarify the natural relation-
ships of several clupeoid species from the western North Atlantic, as well as to
establish a serological baseline for concurrent studies of intraspecies groups of
herring. The following clupeoids were compared : alewife, Alosa pscudoharcngns
(Wilson) ; blueback herring, Alosa acsth'alis (Mitchill) ; American shad, Alosa
sapidissima (Wilson); Atlantic herring, Clupca horoigus harengus Linnaeus;
Atlantic menhaden, Brevoortia tyrannus (Latrobe). Four methods were used:
(1) precipitin tests read photoelectrically with a Libby photronreflectometer ; (2)
precipitin tests visualized by agar diffusion; (3) erythrocyte agglutination with
absorbed antisera; and (4) paper electrophoresis. This composite approach
permitted scrutiny of species relationships from several serological viewpoints.
MATERIALS AND METHODS
Blood samples were collected from fish caught in commercial trap nets on the
New Jersey coast. The sampling was done in the spring, summer and fall of 1958,
1 Present address : Fish Control Laboratory, La Crosse, Wisconsin.
330
SEROLOGY OF CLUPEOID FISHES 331
spring and winter of 1959, and spring of 1960, using the cardiac puncture method
described by Perkins (1957). Samples were held at approximately 4° C. until
clotting had occurred ; the serum was then decanted and either used immediately or
stored at —20° C. Cells for antiserum absorptions and agglutination tests were
washed from the clots remaining.
1. Photronreflectometer
Since photronreflectometer determinations and agar diffusion are but different
methods of visualizing precipitin reactions, the same rabbit antisera could be used
for both. Pooled serum samples constituted the antigens for each species of fish
tested. Antisera were prepared in rabbits by six or twelve subcutaneous injections
of .5 ml. of antigen given on alternate days. Only a single series of antigen injec-
tions was given, to obtain as specific antisera as possible, in accord with findings
of Wolfe and Dilks (1946) and Leone (1952). This resulted in antisera of some-
what lower titer, which are subject to relatively larger experimental error when
tested than are strongly reacting antisera. However, an attempt was made to
reduce any such error by repeating tests of each serum-antiserum combination.
Photronreflectometer determinations were made with a 20-minute reaction time,
which provides greatest separation of antigens from closely-related species (Leone
1950). Turbidimetric precipitin tests were carried out following the methods
outlined by Boyden and DeFalco (1943) and Leone (1949). Constant amounts
of antiserum were added to doubling dilutions of antigen, 1 :250 to 1 : 625,000. This
provided a titration curve with a peak at optimal proportions of the reactants, and
with slopes to extreme antigen excess on one side and extreme antibody excess on
the other. Whole curve comparisons of reactions were made, using summated
turbidities of all the antigen concentrations tested, this summation being proportional
to the area subtended by the curve. The homologous reaction was designated as
100%, so the summated turbidity (relative area) of the curve obtained with each
heterologous antigen could thus be related to the homologous reaction as a per cent.
Since five species were compared, five curves were obtained for each antiserum..
2. Agar diffusion
The agar diffusion technique was essentially the method originally described
by Ouchterlony in 1948, and since used by Bjorklund (1952a, 1952b), Leone,.
Leonard and Pryor (1955), Gasman (1958), Wilson (1958), Morrill (1959),
Ridgway (personal correspondence) and others. A medium of 1.5% Difco agar
and .72% sodium chloride was prepared and a basal layer of 8 ml. poured into a flat-
bottomed Petri dish. After the basal layer hardened, cylinders of glass tubing (1
cm. in length, 4 mm. inside diameter) were placed in position, one in the
center for antiserum and others set equidistantly around it, for the sera of the five
species. The center of each serum receptacle was 1.5 cm. from the center of the
antiserum receptacle. After a second S-ml. layer of medium had been added and
hardened, undiluted antisera and pooled sera were placed in the cylinders in
amounts of .25 ml. per receptacle. The plates were then sealed with masking taper
labelled, and incubated at a temperature of 35.5° C. for seven days. Reading
of the precipitate patterns was visual, although photographs were made of some
plates.
332 DONALD I-". MAIRS AND CARL J. SINDERMANN
3. Erythrocyte agglutination
Three rabbits were immunized with pooled washed cells of each species tested.
Six or nine .5-ml. doses were injected subcutaneously on alternate days. Trial
bleedings were made 10 days after the last injection and if the antiserum titer was
adequate, food was withheld and the animals bled terminally on the following day.
Antisera were frozen in 3-ml. aliquots at —20° C. until absorptions and cell
agglutination tests were carried out.
Cells of each clupeoid species used for antiserum absorptions were washed three
times in 1.5% saline, and approximately equal amounts of erythrocytes from all
individuals in the sample were pooled. Antiserum was diluted 1:4 and added
to pooled cells in proportions of four parts antiserum to one part cells. After
10 minutes of absorption the cell-antiserum suspension was centrifuged and the
absorbed antiserum tested against a previously removed aliquot of cells used for
absorption. Absorptions of antisera to all five species with each of the five
erythrocyte pools were done simultaneously, and one absorption was usually suffi-
cient to remove all antibodies reactive with the absorbing cells. Agglutination tests
were then carried out, using all possible combinations of absorbed antisera and cells
from each of the five species. Each agglutination test used .2 ml. absorbed anti-
serum and .05 ml. of 4% cell suspension. Readings were taken after 15 minutes
of incubation at room temperature and 30 seconds' centrifugation.
-/. Electrophoresis
Paper electrophoresis was carried out with a Spinco Model R system for six
hours at 15 milliamperes, using a veronal buffer of pH 8.6 and ionic strength of
0.05. With each run, a sample of human serum was used as a control. Each
sample consisted of .01 ml. of serum. Representative curves were obtained by
use of a photoelectric densitometer.
RESULTS
1. Photronre fleet ometer
The results of serological comparisons of five species of clupeoids, using the
photronreflectometer with one series of highly specific antisera, are summarized in
Figure 1. Comparative values for reactions of each antiserum are in vertical
columns beside each curve. Heterologous antigen reactions are presented in per
cent of the homologous reaction. Reciprocal relationships show good agreement
in most cases, especially in view of the fact that each antiserum must be considered
.as a separate entity as far as its specificity (discriminating capacity) is concerned.
The serological relationships of the five species presented in Figure 1 are
obviously not linear, but may be expressed satisfactorily in a three-dimensional graph
(Fig. 2) based on "serological distances" calculated by subtracting the per cent
heterologous reaction from 100 (Boyden, 1926). This converts the relative cor-
respondence of that particular antigen to a "relative distance" value. With this
method antigens closely related to the homologous antigen will be relatively close
to it, while those antigens of increasing dissimilarity will be increasingly far from
the homologous. For example, in Figure 1 the serum proteins of the alewife gave
a reaction that was 90% of the homologous reaction between blueback serum and
SEROLOGY OF CLUPEOID FISHES
333
ANTI-ALEWIFE
ALEWrfE 100%
— « — BL'JEBACK 69%
.. SHAD 70%
MENHADEN 46%
ANTI-BLUEBACK
ANTIGEN DILUTION
ANTIGEN DILUTION
ANTI -SHAD
ANTIGEN DILUTION
A'JTI -MENHADEN
ENMADEN IOO%
40 53%
LUE8ACIC 51%
23456783
ANTIGEN DILUTION
ANTI-HERRING
— — — HERAIN6 IOO%
-—• — -BLUtBACK 32%
ALEwiFC 31%
ANTIGEN DILUTION
FIGURE 1. Precipitin curves derived from reactions of serum antigens of
five clupeoid species with antisera prepared to each species.
334
DONALD F. MAIRS AXD CARL J. SINDERMANN
FIGURE 2. Three-dimensional representation of the present relationships of five clupeoid species,
as determined from precipitin tests read with the photronreflectometer.
anti-blueback serum. The relative serological distance between blueback and alewife
with this antiserum is thus 10. However, the reaction of herring serum proteins
with the anti-blueback serum was only 27% of the homologous, so the relative
serological distance between blueback and herring with this antiserum is 73. All
relative distance values between any two species were averaged in preparation of
Figure 2, and each species locus was determined by average distance values from
each of the other species.
The data indicate that blueback and alewife have a high degree of serological
correspondence ; that shad are closer to the blueback-alewife complex than to either
menhaden or herring ; and that menhaden and herring are remote from all others,
with herring consistently at the greatest serological distance from the other four
species.
The numerical relationship values obtained are not fixed, but reflect results ob-
tained with the particular antisera used. The relative positions of species with re-
SEROLOGY OF CLUPEOID FISHES 335
spect to one another should, however, remain relatively constant. Boyden, DeFalco
and Gemeroy (1951) and Boyden (1953) have demonstrated that despite varia-
tions in specificity and systematic range of several antisera of the same kind, a con-
sistent placement series will emerge for the species tested. This was demonstrated
in the present work by a second series of antisera with lower specificity obtained
by deliberately prolonged injections extending over a one-month period. Such anti-
sera were in most instances less specific, but no variations in relative placement of
the five species occurred. Serological distances separating heterologous and homol-
ogous species were reduced in most cases, but relative positions and placements
were similar.
2. A gar diffusion
Clear patterns of precipitate were obtained with each of the antisera used. Al-
though minor differences were noticed in the discriminating capacity of different
rabbits immunized against the same species, the reproducibility was high with re-
spect to number and position of precipitate bands in the gel. Diagrammatic sketches
of typical patterns appear in Figure 3.
Since results of agar plate tests are very difficult to quantitate, they are not as
useful for the determination of exact serological distances between species as the
photronreflectometer. However, since closely related species display similar re-
actions and share some precipitate bands in reactions of identity, it is possible to get
a good general idea of the relationships involved.
Alewife and blueback sera showed very similar reciprocal reactions. Shad
serum reacted strongly to antisera prepared against alewives and bluebacks and
shared some precipitate bands with these species, but the reciprocal reactions were
somewhat weaker ; thus, in the overall picture shad must be placed slightly farther
from alewives and bluebacks than these two species are from one another. Men-
haden and herring appear to stand somewhat apart from the alewife-blueback-shad
complex, but the reactions of menhaden showed a closer relationship to that group
than did those of herring. Apparently, menhaden and herring have very little
taxonomic affinity, for antisera prepared against either of these species evoked
only the faintest traces of precipitate from sera of the other, and these trace reac-
tions were ahvays of the type representing complete non-identity of antigens.
The results of the agar diffusion tests substantiate the more precise picture of
relationships shown by the photronreflectometer. The positions of the species are
in the same orientation as those deduced from the turbidimetric measurements.
3. Erythrocytc agglutinations
Tests were made with reagents obtained by absorbing each antiserum with cells
of each of the five species. The reactions (Table I) represent composites of three
separate tests, each using different samples of fish blood and a different antiserum.
Results are recorded conventionally in descending order from ( + + + + ), repre-
senting complete agglutination, to ( — ) representing no agglutination.
The reduction in strength of agglutinations after absorptions indicates that all
antisera contained substantial amounts of cross-reactive antibodies. Alewife and
blueback cells shared many antigens, so that absorptions with either gave very
similar but not identical reactions. Shad and menhaden shared some antigens, so
336
DONALD F. MAIRS AND CARL J. SINDERMANN
ANTI-MENHADEN
ANTI-HERRING
FIGURE 3. Diagrams of reactions in representative agar diffusion plates for each of the five
clupeoid species. A = alewife, B = blueback, H = herring, M = menhaden, S = shad.
SEROLOGY OF CLUPEOID FISHES
337
TABLH I
Reactions of erythrocytes of five clupeoid species with absorbed antisem
Agglutination reactions
Antiserum
Cells used in
absorptions
Alewife
Blueback
Herring
Menhaden
Shad
Alewife
Alewife
—
—
—
—
—
Blueback
+
—
+
—
—
Herring
+ + +
+ +
—
—
+
Menhaden
+ + +
+ +
+ +
—
—
Shad
+ +
+
+ +
—
—
Unabsorbed
+ + +
+ +
+ + +
+
+ +
Blueback
Alewife
—
—
+
—
_
Blueback
—
—
—
—
—
Herring
+ +
+
—
—
—
Menhaden
+ +
+
+ + +
—
—
Shad
+ +
+
+ +
—
—
Unabsorbed
+ + + +
+ + +
+ + + +
+
+ +
Herring
Alewife
—
—
+
—
_
Blueback
+
—
+ +
—
—
Herring
—
—
—
—
—
Menhaden
+
+
+ + +
—
—
Shad
—
—
+ +
—
—
Unabsorbed
+ + + +
+ +
+ + +
+
+ +
Menhaden
Alewife
—
—
+ +
+
+
Blueback
+
—
+ +
+
+
Herring
+
+
—
+
+
Menhaden
—
—
—
—
—
Shad
+
+
+ +
+
—
Unabsorbed
+ + +
+ +
+ + +
+ +
+ +
Shad
Alewife
—
—
+ + +
+
+ +
Blueback
+
—
+ + +
+ +
+ +
Herring
+
—
—
+ +
+ +
Menhaden
+ + +
+ +
+ + +
—
+
Shad
—
—
—
—
—
Unabsorbed
+ + +
+ +
+ + +
+ + +
+ + +
that absorptions gave a somewhat similar pattern of reactions, although the relation-
ship was by no means as close as that of alewife and blueback.
4. Electrophoresis
Electrophoretic analyses were made with both pooled and individual sera. Indi-
vidual sera appeared in all cases to give better definition of components, although
they conformed to the general pattern of the pools. Considerable intraspecific
variation in patterns was evident, but a generalization of each species pattern was
possible. The curves derived from these generalized patterns are depicted in
Figure 4, as determined by densitometer readings ; they are, however, not intended
to illustrate absolute species specificity. The numbers designating each peak are
338
DONALD F. MAIRS AND CARL J. SINDERMANN
ALEWIFE
t HI
BLUEBACK /
HI I
SHAD
tozm
MENHADEN /
HERRING
m
FIGURE 4. Representative paper electrophoresis diagrams for five clupeoid species as determined
hy densitometer readings. Dotted line represents human serum used as control.
SEROLOGY OF CLUPEOID FISHES 339
used for ease in description and do not denote identity of components between
species.
The alewife and blueback sera displayed very similar electrophoretic patterns,
each with three major fractions. The main difference between the two species was
found in the least mobile fraction ; in alewives this fraction was less mobile than
human beta globulin, whereas in bluebacks it migrated further than human beta
globulin. Both species had a weak fraction of the same mobility as human alpha-2
globulin, and a fraction of slightly less mobility than human albumin. In some
specimens of alewife, a weak fourth fraction appeared, intermediate in mobility
between the human alpha-2 and beta globulin fractions.
The shad samples tested displayed a pattern of four fractions. The least mobile
of these did not migrate as far from the point of application as human beta globulin.
A strong fraction with slightly more mobility was present in all specimens, as were
moderate fractions .with slightly more mobility, respectively, than human alpha-2
and alpha- 1 globulins.
The pattern shown by the menhaden sampled had two fairly strong fractions.
One of these was identical in mobility to human beta globulin, and one was inter-
mediate between human alpha- 1 globulin and albumin.
Three moderately strong fractions were obvious in the patterns of the sea
herring tested. The least mobile migrated a shorter distance than human beta
globulin, while the others travelled the same distances, respectively, as human
alpha-2 globulin and albumin. In some fish of this species a fourth fraction
appeared which was intermediate between human alpha- 1 globulin and albumin.
DISCUSSION
1. Photronrcflectometer
The clear differentiation of five clupeoid species with this precipitin technique
read photoelectrically offers tempting possibilities for future studies. It would be
interesting, with the background of the present data, to compare other morphologi-
cally similar but geographically isolated clupeoid species. Suspected hybrids, such
as those between Alosa psendoharcngns and A. acstivalis might also be examined
serologically. Comparison of populations or "races" of cosmopolitan species, such
as Clupea harengus, with serum techniques might also prove instructive.
The limitations of such studies must be kept in mind. Results of serological
examinations would not be considered as the sole criterion for taxonomic conclu-
sions, but should be evaluated together with morphological and other data to pro-
vide as broad a base as possible for such conclusions. Information about the
possible influences of environmental and physiological changes on serological re-
actions should be obtained. It should also be emphasized that results will indicate
present serological relationships, but will give only indirect information about the
evolutionary history of the species concerned. Despite such limitations, serum
techniques offer possibilities in fisheries research that should be explored as vigor-
ously as other techniques for the understanding of natural relationships.
2. A gar diffusion
Further experimentation should be conducted with agar diffusion to determine
its usefulness in studies on fish populations. Absorption of antisera to remove spe-
340 DONALD K MAIRS AND CARL J. SINDERMAXX
cies antibodies would probably be a virtual necessity for work at the intraspecific
level.
The advantage of this type of test obviously lies in the fact that the total precipi-
tate is resolved into its component antigen-antibody reactions, which are subject
to direct visual observation. While this permits a high degree of qualitative differ-
entiation and gives a good idea of general species relationship, it is very difficult
to measure exactly the total amount of precipitate formed. For this reason, it is
best to leave the final calculation of relative serological distances between species to
turbidimetric methods, keeping in mind the fact that rabbits or other experimental
animals are not the exclusive and final arbiters of natural relationships.
3. Erythrocytc agglutination
The use of pooled cells to absorb antisera to all five clupeoids, and the subse-
quent testing of such pools with reagents obtained by absorptions, have provided a
criterion for determining the relationships of five clupeoid species. It would be
instructive to apply this test to additional clupeoid species in other regions.
Another logical extension of this work would be determination of the distribution
of similar or identical antigens in individual blood samples of the five species. In-
dividual differences have been noted in the present work for all five species, but,
except for herring, blood group systems have not been proposed. The use of pooled
cells masks individual differences, and it is obviously less precise than a study of
discrete antigens with specific reagents. To test the utility of the single antigen
approach, individual samples of all species were tested with the reagents used in
routine examination for the C blood group antigen of herring (Sindermann and
Mairs, 1959). Positive reactions w-ere obtained with most alewives, half of the
bluebacks, a few shad, and no menhaden, indicating the existence of the same or at
least a closely related antigen in species other than herring. Similar tests with other
specific reagents could create a mosaic of reactions that would provide a clearer
picture of the affinities disclosed by the present study. The utility of such an ap-
proach has already been demonstrated with certain mammalian species by Stormont
and Suzuki (1958).
4. Electrophorcsis
Although it was possible to construct electrophoretic patterns characteristic of
the five species of clupeoids, the intraspecific variations encountered in this study
and in an electrophoretic examination of herring populations (Mairs and Sinder-
mann, 1960) indicate that great caution should be employed in the establishment of
specific patterns for teleostean species. It has been shown that the component
fractions of serum display variability in both quantity and electrophoretic mobility
depending on such factors as disease, age. sex and starvation (Moore, 1945; Des-
sauer and Fox, 1956; Drilhon ct al., 1956; Sindermann and Mairs, 1958). This
variability could lead to significant overlap and confusion of patterns, especially
among closely related species. In many cases, it is doubtless possible to character-
ize a high proportion of the electrophoretic patterns in a sample, but proposal of a
species-specific pattern would have to follow testing of a large number of individu-
als, with close reference to maturity, disease, and any other physiological or environ-
mental factor known or likely to affect electrophoretic characteristics. Only in
SEROLOGY OF CLUPEOID FISHES 341
this way could a species "norm," if such exists, he properly defined and an insight
gained on the true significance of the variations encountered.
The present study indicates that, while paper electrophoresis may have some
general usefulness in fish taxonomy, immunological techniques are preferable for
precise differentiation and attempts at determining natural relationships.
SUMMARY AND CONCLUSIONS
1. An investigation of the serological relationships of five species of clupeoid
fishes was made by four methods: (a) photronreflectometer, (b) agar diffu-
sion, (c) erythrocyte agglutination with absorbed antisera, and (d) paper
electrophoresis.
2. Agar diffusion enabled qualitative differentiation of the species tested, while
the photronreflectometer provided a quantitative measure of relative serological
distances between species ; results from the two methods were in good agreement.
3. On the basis of results obtained by the photronreflectometer and agar diffu-
sion methods, the following species relationships are indicated : alewives and blue-
backs are very closely related ; shad lie quite close to alewives and bluebacks, but
farther from them than alewives and bluebacks are from one another ; menhaden and
herring are further removed from the alewife-blueback-shad complex, with men-
haden closer to it than herring; herring are comparatively remote from the other
four species.
4. Generalized electrophoretic patterns were found for each clupeoid species.
However, because of intraspecies variability, paper electrophoresis does not seem
to be as useful a procedure for determining relationships of fishes as immunological
methods. Species-specific patterns should be proposed only after large numbers
of individuals representing both sexes have been sampled under different physio-
logical conditions.
5. Absorptions of rabbit antisera with pooled erythrocytes of each of the five
clupeoid species indicated that alewife and blueback were antigenically very similar ;
menhaden and shad were antigenically somewhat similar, although not as close as
alewife and blueback.
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REPRODUCTIVE BIOLOGY OF LYCHAS
TRICARINATUS (SIMON)1
A. P. MATHEW
Department of Zoology, Mar Ivanios College, Trivandrum, India
In a previous paper on the embryonic nutrition in Lyclias tricarinatus (Mathew,
1960), I had drawn attention to a curious phenomenon of arrest of development in
internal embryos in this scorpion. As this phenomenon was an unusual one, fur-
ther studies were made on it and this paper embodies the results of these investi-
gations.
MATERIALS AND METHODS
Gravid females of L. tricarinatus were collected almost every month from Muk-
kunni hills near Trivandrum (only during the wet season) and from Nagercoil
and adjoining hills like Asambu and Thadikarankonam in Kanyakumari District of
Madras State. Specimens were also collected from Vaikom and Moovatupuzha in
North Travancore. Suitable cages were improvised for them and they were reared
in these and observations were made in each case as to the delivery of young. As
occasions demanded, the specimens were killed and the conditions of the ovary ex-
amined, and all these ovaries were preserved and labelled for future study.
OBSERVATIONS
The ovary consists of a pair of longitudinal tubes on each side, each pair con-
nected together by five transverse tubes forming four quadrilateral meshes on each
side ; so, unlike other scorpions, here the female system has almost the same arrange-
ment of tubules as in the male system. However, at the hindermost part, the
inner longitudinal tubes of either side are fused so that this hind extremity of the
ovary alone resembles the ovarian network of other scorpions. The developing
embryos are inside the ovarian tubes as typical of the Buthidae. All along the
outside of the tubes there are eggs in different stages of growth, in the tiny bubble-
like follicles. Among these are a few which are larger than the others. The eggs
in them are surrounded by thick envelopes. They have already begun to develop
and in some, well developed blastoderms may be seen. Does this mean that while
one set of embryos has already begun to develop inside the ovarian tubes, other sets,
too, are developing, so that at one time there can be young of different stages of de-
velopment in the ovarian tube? On morphological and physiological grounds, this
is an impossibility and does not happen in any scorpion studied so far. Since
1 This work was supported by a grant from the Council of Scientific and Industrial
Research, Government of India. Financial aid from the Ministry of Scientific and Cultural
Affairs, Government of India, for the purchase of some items of equipment, is also acknowledged.
I also express my gratitude to Dr. A. D. Lees of the Agricultural Research Council,
Cambridge, for having read through the manuscript and given helpful suggestions.
,44
REPRODUCTIVE BIOLOGY OF LYCHAS 345
ovarian tubes themselves are the embryo-ducts, if there are embryos of different
stages of development in these tubes, when some of the embryos mature and are
extruded, they will push out the younger immature embryos too.
Ovarian tubes in which the embryos are fully mature also have eggs in different
stages of growth, in tiny bubble-like follicles, with a few larger follicles enclosing
segmenting zygotes or fully formed blastoderms, on the outside; just the same as
seen in the earlier stages. Evidently they have remained all through the develop-
ment of the internal embryos without any change. This would indicate that these
zygotes and early embryos outside the ovarian tube are held in arrested state of
growth, or in a condition resembling diapause, until the extrusion of the developed
embryos. After the extrusion of these developed embryos, the suppressed embryos
might get pushed into the ovarian tube and resume their course of development. If
this should happen, the scorpion, after giving birth to one brood, must be able to
develop another brood of embryos without fresh copulation. To test if this actually
happens a few breeding experiments were conducted and observations were made.
BREEDING EXPERIMENT
Six gravid females were selected from a collection from the field and reared in
artificial cages providing as nearly natural conditions as possible — one female in a
cage. They were properly fed, given insects and water, and observations were
recorded.
Female A
Collected and caged 3-5-1958
Delivered the young 9-5-1958
Young moulted 14-5-1958
left the mother 19-5-1958
After delivery the mother was kept in the cage and properly looked after. It
grew well and after about a month was seen with the abdomen swollen as if it was
becoming gravid again. There was no male scorpion anywhere in the vicinity. A
little later the abdomen was still more distended and through the thin translucent
sterna, the internal embryos could be seen quite clearly. There was no more doubt
as to its second pregnancy. On June 20th, i.e., 41 days after the previous extru-
sion of young, a second brood came out and the young took their position on the
mother's back as usual.
Delivery of the second brood 20-6—1958
Young moulted 26-6-1958
Young left the mother 1-7-1958
The female was then transferred to another cage and carefully looked after for
another month. By this time it was showing considerable weakness and it was not
possible to keep it alive any further. So it was killed on the 28th of July, 1958, and
the ovary was examined. There were 27 developing embryos inside, which were
fairly advanced and the black pigmented eyes were conspicuous.
With three of the other specimens this experiment was repeated under
similar conditions and the above observations were confirmed. Since then
346 A. P. MATHEW
these observations were further repeated and confirmed in 1960 and again in
1961 with fresh specimens obtained mostly from Nagelcoil. From these
rearing experiments, the following conclusions can be drawn : ( 1 ) Lychas
tricarinatus can, after one delivery, produce at least three successive broods
of young when isolated from all possibility of contact with a male. (2) The
time elapsing between one delivery and the next is about 40-42 days. This
may be taken as the approximate period of gestation. Since the period of gestation
can be approximately determined, counting from the time of one delivery, it is
possible to keep females in captivity and study the stages of development of embryos
within specified periods of time. So a few gravid females were kept in captivity and
after one delivery, the embryos were examined and studied at different intervals. In
all these there were growing embryos developing at the normal rates, as could be
tested by the condition of the embryos at definite periods in the different specimens
examined. The development and the stages attained as seen in these studies will
be described elsewhere, dealing with the embryology of Lychas.
The series of breeding experiments described above show conclusively that this
scorpion does produce successive broods without pairing after each brood has been
extruded. How are the new embryos formed? The following possibilities may be
suggested, but each has to be tested and proved.
(1) It may be that there are slight variations in the developmental stages at-
tained by embryos of a brood in the uterine tube and the most mature are extruded
first and the others are retained till they too are fully developed, to be extruded as
the second brood, and so on.
(2) Parthenogenesis.
(3) Sperm received at one copulation are preserved in the spermathecae, viable
and capable of fertilising eggs for future broods.
(4) After once pairing, numerous eggs are fertilised but only a few of these
can develop in the limited space of the uterine tubes (ovarian tubes). These few
alone pass into the uterine tubes and continue development while the others are
kept in an arrested state of development till the developing brood is extruded.
Regarding the first possibility, suggested above, we may note the following facts
and observations : ( 1 ) Opening gravid females, we do not notice such disparity in
stages of the development of individuals of a brood. In early stages a slight
disparity may appear but by the time the embryos have attained about half maturity,
all are of the same stage except rarely a diseased or injured individual which fails
to be extruded. (2) When a female is opened soon after extrusion of a brood, no
younger stages are seen to be retained in the ovarian tubes but they are completely
empty and lie flabby in the body cavity ; all eggs and zygotes on the ovarian tubes
are on the outside only.
Occasionally, when several specimens are opened, one comes across a dead
embryo still retained in some part of the ovarian tube. But this is a diseased or
dead one which could not be extruded and so has been retained there to undergo
gradual histolysis and absorption. These have been observed in other scorpions
too, such as Hetcrometrus (Mathew, 1956). This may cause obstruction for the
other embryos, but because of the retiform arrangement of the ovarian tubes provid-
ing several alternating pathways, no such blockade actually takes place ; all viable
embryos escape.
REPRODUCTIVE BIOLOGY OF LYCHAS 347
These considerations help us to rule out the first possibility.
The second possibility suggested is parthenogenesis. It is known to take
place under special conditions in many arthropods, but one indication for partheno-
genesis is the absence or rarity of males. With regard to Lychas, however, this
condition does not hold. Estimating the proportion of the sexes from collections
that have been made during these four years, one can say that in number the sexes
are almost equal — sometimes there may be a slight predominance of females. At
any rate it can be most confidently stated that there is no scarcity of males.
Again, if development is parthenogenetic there is no need for the early formation
of zygotes and blastoderms ; the development processes may well start after one
brood has been extruded.
The third possibility suggested is that viable sperm may be retained in the
spermatheca to be used over and over again. This is not an impossibility but
microscopic examination of the contents of the seminal vesicles has failed to show
sperms held in reserve. Also, as already observed with reference to partheno-
genesis, if there is sperm held in reserve for future fertilisation, why the early
formation of zygotes and blastoderms?
The last suggestion, which is in agreement with all the observed phenomena,
appears to be most satisfactory.
(1) The ovarian tubules of a female which has recently extruded a brood of
young possess numerous tiny bubble-like follicles in which there are young ova or
zygotes or blastoderms, the latter two being distinguishable by their larger size
and being covered over by a thick pigmented shell, the "chorion."
(2) In the ovarian tubes, examined a week later, we find that some of these
blastoderms enclosed in the "chorion" have sunk into the ovarian tube while the
zygotes that are still outside begin segmentation and develop into blastoderms.
Only a limited number of embryos can sink into the ovarian tube and the number
appears to be roughly controlled by the extent of space available on the ovarian tube.
In an ovarian tube with well developed internal embryos, the swellings are seen
more or less evenly spaced, thus giving it the characteristic moniliform appearance.
(3) All through the subsequent development, when the internal embryos are
growing to maturity, the external zygotes and blastoderms remain without any
further development. They remain dormant till this brood is extruded.
EMBRYONIC DIAPAUSE IN LYCHAS TRICARINATUS
This arrested development described above, as seen in Lychas tricarinatits, may
be taken to be a special type of diapause of an internally developing embryo. It
would be interesting to enquire into the probable mechanism of the initiation and
final release of this "diapause." The special development of certain glandular
structures synchronising with the initiation and release of diapause makes it highly
probable that these are under hormonal control. The following observations of the
developmental cycle, accompanied by the enlargment or breakdown of the glandular
structures concerned, are illuminating.
Stage 1. In Lychas, the development of the embryo up to the establishment of
the germinal layers take place in the follicles outside the ovarian tube. The zygotes
and blastoderms are surrounded by a thick dark pigmented shell or "chorion"
(Fig. 1,A).
348
A. P. MATHEW
1
KEY TO LETTERING
A, large follicle with blastoderm surrounded by chorion.
Al, egg with blastoderm sinking into the ovarian tube.
A2, "empty chorion" from which the embryonic rudiment has passed out. Develops into corpus
luteum 2, forming the embryonic feeding mechanism.
B, small follicles with eggs (earlier).
C, cells at the base of the follicle become glandular corpus luteum 1.
D, the cast off chorion and stalk — corpus luteum 3.
E, embryo.
Mg, midgut of the embryo.
St, stalk of the feeding mechanism connected with the embryonic gut.
FIGURE 1. Part of the ovarian tube (early) bearing follicles enclosing blastoderms or eggs.
FIGURE 2. A follicle with blastoderm sinking into the ovarian tube (Al), causing the part
of the ovarian tube to bulge a little. At the site of the insinking (C, C) the outer cells begin
to enlarge and become glandular.
FIGURE 3. The embryonic rudiment (E) is pushed into the ovarian tube from the ruptured
"chorion" (A2), which is at the same time pushed upwards, everting the invaginated outer layer
at the site of the insinking of the egg. The cells of this portion (C) have now become enlarged
and glandular. At this stage they form button-like thickenings on the swollen parts of the
ovarian tube in which are the developing embryos.
FIGURE 4. The upward movement of the chorion (A2) continues. It pushes itself out of
the inner layer and projects out, covered over by the everted glandular cells of the follicle (C).
REPRODUCTIVE BIOLOGY OF LYCHAS 349
Stage 2. After extrusion of a brood, these follicular embryos surrounded by the
"chorion," which appears to be developed out of follicular cells, sink into the ovarian
tube (Fig. 2, Al). The site of the insinking marking the base of the follicle soon
develops a spongy texture and becomes glandular (C in the figures). Soon this
assumes the form of a button-like papilla with enlarged glandular cells. This
structure may be regarded as corpus luteum 1. The term "corpus luteum" has been
used by Laurie (1890) for the empty follicle in Euscorpius after the embryo has
passed into the ovarian tube for further development. He surmised that the
hormone from this corpus luteum prevents the development of new embryos. Here,
however, it is not one structure that develops secretory properties in connection
with the developmental cycle, but at least three different parts of follicular origin
serve this function ; these structures are here tentatively called corpus luteum 1 , 2
and 3.
Stage 3. In subsequent development when the "embryonic rudiment" is passed
out of the "chorion" into the ovarian tube, the partially empty "chorion" is pushed
upwards through the spongy structure, that has been termed above corpus luteum 1 ,
and breaking through it, gets exposed in the body cavity as a tiny rounded dark
brown body, A2, retaining connection with the gut of the developing embryo (Fig.
5). This body with its stalk (St.) develops into the queer "feeding mechanism"
which has been described elsewhere (Mathew, 1960). This structure, which also
is of follicular origin, has been regarded as a corpus luteum and may be distinguished
as corpus luteum 2 (A2). When this structure is fully formed, corpus luteum 1 is
seen to form a spongy globular mass surrounding its stalk (Fig. 5. C.). As the
embryo grows, it is seen to be fed with the rich secretion of the feeding mechanism,
directly passed into the midgut of the embryo.
Stage 4. When the growth of the embryo is completed and it is about to be
extruded, the connection with the midgut is constricted off where it joins the
midgut and the whole stalk, tipped with the dark globular "chorion," slips off and
drops into the mother's body cavity during parturition (Fig. 6).
Stage 5. Post-partum changes. The discarded feeding mechanism with its
stalk (D), instead of immediately degenerating, develops into a club-shaped glandu-
lar organ, apparently actively functioning for some time. This may be called
corpus luteum 3. Soon after, however, this structure begins to atrophy, undergoing
histolysis.
DISCUSSION
From the above observations, it will be seen that corpus luteum 1 is formed
immediately after the developing embryos have sunk into the ovarian tube. No
more of the embryos sink in after this and so it is suggested that its secretion has the
property of inhibiting further development of the follicular embryos : they remain
outside the ovarian tube without sinking in and their growth appears to be ar-
rested. In other words, they are in a state of "diapause" in which they remain till
the developing embryos are extruded.
FIGURE 5. Finally the chorion (A2) breaks through the ovarian wall completely and
projects out freely; the glandular cells (C) form a collar around the stalk (St) connecting the
chorion with the gut of the embryo.
FIGURE 6. When parturition takes place, the chorion (A2) with its stalk (St) is
separated off and left in the body cavity. There they develop into club-shaped bodies (D).
350 A. P. MATHEW
After the extrusion of the young, the feeding mechanism, consisting of the
globular "chorion" and its club-shaped stalk, are released from the ovarian wall
and drop into the body cavity. These curious bodies rapidly develop into glandular
structures of a third type, called above corpus luteum 3. These are really spent
structures which have served their purpose and are to disintegrate normally. But
instead of that, they develop into glandular structures functioning actively for a
time. To the secretion of this we can attribute only one function — that of "re-
viving" the embryos from their "diapause" condition, for, soon after they are re-
leased into the body cavity, fresh external embryos begin to sink in and continue
development. At the same time it is significant that the glandular cells of corpus
luteum 1, whose secretion was suggested to prevent further insinking of embryos,
have shrunk up and become almost indistinct.
Soon after the "diapausing" or "resting" embryos have started their further
course of development, we find that these bodies, corpus luteum 3, break up and
disappear.
Here we have a singular instance of embryonic diapause in an internally de-
veloping egg. In insects, embryonic diapause is fairly well known ; it occurs more
commonly at stages more advanced than the blastoderm stage. But all these eggs
are outside and their "diapause" and later its termination appear to be controlled
by the influences of the external environment (Lees, 1955). In this scorpion, how-
ever, diapause occurs at the blastoderm stage or earlier ( ?), but never later. Fur-
ther development of the blastoderm can take place only after it sinks into the ovarian
tube, and later development appears to be an uninterrupted process. Naturally, the
blastoderm stage is the most suitable period for an arrest of growth.
It is possible to look upon diapause in Lychas, also, as controlled by the environ-
ment— the maternal body cavity in which development takes place. Naturally it
has not to depend on a precarious external environment but is under the influence
of a well regulated internal environment changing rhythmically in correspondence
with a reproductive cycle.
SUMMARY
1 . An unusual instance of embryonic diapause in a scorpion is described. Among
the eggs that are fertilised at a time, only a few sink into the ovarian tube and con-
tinue to develop to maturity while the others remain in a state of arrested growth or
diapause, until the extrusion of the first batch of embryos. Further insinking of the
eggs appears to be prevented by the secretion of corpus luteum 1.
2. After the extrusion of the first batch of embryos the diapausing embryos get
revived and sink into the ovarian tube to give rise to the next brood. The termina-
tion of the "diapause." also, appears to be in response to a secretion from another
glandular structure, termed here corpus luteum 3. This incidentally explains how
Lychas can produce two or three broods in succession without undergoing repeated
copulation.
LITERATURE CITED
LAURIE, M., 1890. Embryology of the scorpion, Euscorpins italicus. Quart. J. Mlcr. Sci.. 31 :
105-141.
LEES, A. D., 1955. The Physiology of Diapause in Arthropods. Cambridge University Press.
MATHEW, A. P., 1956. Embryology of Hctcromctrns scaber. University of Travancorc,
Zoology Memoirs, I: 1-111.
MATHEW, A. P., 1960. Embryonic nutrition in Lychas tricarinatus, J. Zool. Soc. India, 12:
220-228.
THE REPRODUCTIVE CYCLES OF THREE VIVIPAROUS
TELEOSTS, ALLOOPHORUS ROBUSTUS, GOODEA
LUITPOLDII AND NEOOPHORUS DIAZI 1
GUILLERMO MENDOZA
Biology Department, Grinncll College, Grinnell, lozva
Extensive taxonomic and descriptive work has been done on the Goodeidae, a
family of fresh-water viviparous cyprinodonts from Mexico, but very little is known
about their reproductive cycles. Except for a detailed three-year laboratory study
on Neotoca bilincata (Mendoza, 1939), no other major study on the reproductive
cycles of the goodeids has been made either in the laboratory or in the field. The
only other principal source of information on the breeding cycles in the family is in
Meek (1904), in which a brief and inadequate statement is included in the taxonomic
description of each species. Miscellaneous information on the reproductive cycles
of the goodeids also is scattered in many of Turner's articles but he made no detailed
study of any one species.
Because of the scarcity of this information, it was proposed to make an analysis,
from field specimens, of the female reproductive cycles of three species in the family.
This study concerns the duration of the female cycle, the number and size of broods,
the length of the gestation period, etc. This is the first intensive study that has been
made of the reproductive cycles from specimens collected in their natural habitat.
The writer is greatly indebted to Sr. Aurelio Solorzano Preciado, Director of the
Estacion Limnologica in Patzcuaro, Michoacan, and to Juan Piza M., one of the
attendants at the station, for their unending cooperation in helping the writer to
make many of these collections.
MATERIALS AND METHODS
This study concerns three members of the family Goodeidae: Alloophorus robus-
tus (Bean), Goodca luitpoldii (Therese von Bayern and Steindachner), and Neo-
ophonis diazi (Meek). The three species were collected from Lake Patzcuaro.
They were chosen because they inhabit the same lake and because it was possible to
get a continuous supply of adults throughout the year.
Mature Alloophorus and Goodea females normally range from 90-110 mm. in
length, although exceptionally large specimens of each species may exceed 130 mm.
Mature females of Neoophorus range from 70-90 mm. in length.
The writer relied entirely on local fishermen for aid in collecting specimens.
Because of the exuberant growth of reeds, lilies and other vegetation near the shore,
the local fishermen use the much-publicized "butterfly" nets as scoops and work from
their small canoes in groups of three to five. In the open lake the fishermen use
large seines that measure 300 feet or more in length.
1 This study was supported by Grants G5114 and G16726 of the National Science Foundation.
351
352 GUILLERMO MENDOZA
This study is based on a total of 3261 females collected during 1957; approxi-
mately 50 females of each species were collected twice each month throughout the
year. Specimens were divided as follows: 1010 Alloophorus, 1117 Goodea and
1134 Ncoophorus. Similar collections, numbering more than 6000 females, were
made during 1956 and 1958 but these were used for reference and comparison pur-
poses only. The average size of all collections was 47 ovaries. Some females were
collected alive in the lake and others were purchased in the local market since fresh
fish were available at least once per week. The fishermen normally bring in only
mature adults; they show no preference for either sex or stage of gestation.
Alloophorus and Neoophorus normally are collected along the shore of the lake ;
Goodea usually is collected in open water but may prefer the shore during the
breeding season. Lastly, in obtaining specimens in the market, a definite effort was
made not to select only large gravid specimens but to take all females, regardless of
size or stage of gestation.
In these viviparous species, the ovary is a single structure, compact, spindle-
shaped, hollow, and continuous caudad with the oviduct which in turn opens to the
outside at the genital pore. The ovary has ovigerous tissue but it also acts as a
uterus, for all development from fertilization to birth occurs in the ovarian lumen.
When the young are ready for birth, they escape from the sacculated ovary and
emerge as free-swimming forms.
Upon collection, each ovary was removed and preserved either in formalin or
special fixatives such as Bouin's or Zenker's fluids. All ovaries collected were
preserved, regardless of the stage of gestation, and placed in one of the following-
categories : immature ovaries, resting ovaries, ovaries with growing eggs, ovaries
with free eggs, ovaries with embryos in different stages of development, and post-
partum ovaries. On classifying the gonads into different stages, relatively little
difficulty was encountered. Ovaries without free eggs or young were dehydrated
and cleared in cedarwood oil. The gonads then were examined with a stereoscopic
microscope and classified into the proper stage. Ovaries with embryos in actual
stages of development were placed into one of twelve classes according to size. The
smallest group ranged from 3.5-5.0 mm. in length and successive groups or classes
were formed at increments of 2.5 mm. (e.g. 5.1-7.5 mm., 7.6-10.0 mm., etc.) ; the
largest group was 30.1-32.5 mm. (in Goodea).
STAGES OF GESTATION
The data on the three species are given concurrently, that is, comparable stages
of development are considered at one time for all three forms. The principal
description is based on Alloophorus and is followed in turn by the ones on Goodea
and Neoophorus.
Immature ovaries
Immature ovaries in all three species measured from 1-2 mm. in maximum width
by 15-20 mm. in length. The typically immature ovary has delicate external walls
and internal folds ; eggs vary up to 350 p. and are densely packed in the anterior
half or two-thirds of the gonad. Measurement of the diameter of the small ovaries
was made with a micrometer eyepiece in a stereoscopic microscope ; the length and
diameter of the larger ovaries were measured with a millimeter ruler.
REPRODUCTION IN VIVIPAROUS TELEOSTS
353
5
°
80
60
40-
2O
5«0
^ 60
k!
ft 40-
£
k 20
f°
^ 80
§ 60
^40
k.
^. 20
I
,.-•-, GOODEA LUITPOLDII
-IMMATURE
RESTING
POST-PA RTUM
ALLOOPHORUS ROBUSTUS
NEOOPHORUS DIAZ/
COLLECTION DATES
FIGURE 1. The occurrence during the year of females of the three species in immature,
resting and post-partum stages. The number of ovaries in different stages appears as percentage
figures of the day's collection.
354 GUILLERMO MENDOZA
Xormally, few immature females were collected. Figure 1 shows that from
January to April the number of these ovaries in Alloophorus is high, between 54.8
and 67.3% of the individual collections; thereafter the number drops markedly and
remains low during the rest of the year. After spring very few immature females
were captured. Figure 1 indicates that in Goodca immature ovaries did not fol-
low quite the same pattern of appearance as those of Alloophorus. In Ncoophorus,
on the other hand, immature females appeared uniformly throughout the year,
usually forming 10% or less of the collections. At no time were immature ovaries
in this species as abundant nor did they have the pre-season high so clearly evident
in Alloophorus.
Resting ovaries
Resting ovaries are mature but do not contain young. These ovaries vary from
2-3 mm. in diameter and up to 20 mm. in length in Neoophorus and 20-30 mm. in
length in Goodea and Alloophorus. The external wall and internal folds are very
thick. Eggs are few in number and vary in size but do not exceed 350 /A.
In all three species, the number of females in a resting condition is high during
early spring but drops abruptly during early May in Alloophorus and Goodea and
during early April in Ncoophorus (see Figure 1). In general the number of rest-
ing ovaries decreases as the breeding activities start ; thereafter the resting ovaries
constitute a small but variable percentage of the collections until late summer, at
which time breeding ceases. During winter the number of resting ovaries again
rises and reaches a peak in the early spring, at which time breeding is resumed.
Ovaries with growing eggs
Ovaries in this condition resembled resting ovaries but differed in that eggs
exceeded 350 ^ and had grown to a maximum of 1 mm. in Goodca and Alloophorus
and approximately .5 mm. in Ncoophorus. All eggs in this category were still en-
closed in a follicle embedded in the ovarian tissues. Measurement of the eggs was
made with a micrometer eyepiece.
Growing eggs generally appeared during January and February although the
precise time of appearance varied between the three species. If the sampling was
representative, Goodca had a slightly longer period of egg growth than Alloophorus.
Growth of new eggs stopped during June in both species and was not seen in later
collections. Growth of eggs in Ncoophorus, however, started at the same time but
continued until the end of October.
Ovaries unth free eggs
All ovaries with free eggs in the ovarian lumen, regardless of stage of develop-
ment, were arbitrarily placed in this category. The eggs varied from stages near
time of fertilization to stages with young approximately 3 mm. long. Such young
were still enclosed within the egg membranes and were coiled around the yolk-like
mass. At about this time (3 mm.) the young escape from the membranes and
straighten out.
In Goodea and Alloophorus, ovaries with free eggs appeared in collections dur-
ing April, May and June, a period of 2 to 2.5 months; thereafter, free eggs never
appeared in the collections. In Neoophorus, ovaries with free eggs first appeared
REPRODUCTION IN VIVIPAROUS TELEOSTS
ALLOOPHORUS ROBUSTUS
355
embryonic stages.
growing eggs
ll.2mm.
13.7mm.
COLLECTION DATES
FIGURE 2. The appearance throughout the year of ovaries of Alloophorns robustus with
embryos in different stages of development. For any one day, the bars represent the percentage
of ovaries in each stage of gestation.
356 GUILLERMO MENDOZA
during February and thereafter were present continuously until November, at which
time they disappeared.
Ovaries with young 3.5 to 32.5 nun. long
In all ovaries with measureable young the following procedures were employed.
The diameter and length of each ovary were determined with a millimeter ruler.
Following the measurement of the ovary, all young in each gonad were removed and
thoroughly mixed in a petri dish. Four specimens then were chosen at random and
measured with a ruler from the tip of the snout to the posterior edge of the caudal
fin. The average of the four figures was derived and recorded as the size of the
young in that particular ovary.
Development of the young was similar in both Alloophorus and Goodca.
Embryos in the 3.5-5-mm. class appeared either in late April or early May. From
this time on, developing young were found throughout the breeding season ; repro-
duction ceased after August. While cessation was abrupt in Alloophorus, there
were scattered females of Goodea that were late in their cycle as compared to the
bulk of the population. For example, while most young of Goodca had reached
a size of 23.7 mm. by the middle of July, one female was found on December 20
with young 23.7 mm. long. Figure 3 shows this and other similar examples. Most
young of Alloophorus were born after they reached 21.2 mm. although some reached
26.2 mm. before birth. In Goodea, however, many young exceeded 21.2 mm. ; some
even reached a maximum of 31.2 mm. before birth. There is a noticeable temporal
delay in the appearance of the larger sizes, that is, the larger the class size, the
later the appearance of the embryos in the collections.
In Neoophorus, the reproductive period extended over eight or nine months.
Embryos first appeared during the latter part of March and thereafter appeared con-
tinuously until January or February of the following year, at which time reproduc-
tion was suspended for a brief period of one or two months. Figure 4 shows
embryos of maximum size were still present on December 20, 1957, the last collection
of the year. Collections made during 1958 indicate that the 1957 breeding cycle
did not terminate until February of 1958. It is similarly noted that one gravid
female appeared in January 1, 1957; this female no doubt represents the end of the
1956 breeding season.
The collections of the three species made during the years of 1956 and 1958
strongly support the characteristics of the reproductive cycles as expressed during
1957.
Post-partuin ovaries
This category identifies all ovaries from which young have recently been ex-
pelled. Immediately after the birth of young these ovaries appeared thin-walled and
flaccid, the internal folds were thick and swollen and there were few eggs visible.
Later, the ovarian tissues underwent regression but the ovary generally remained
thick. Still later, the gonads again resumed the characteristics of a resting condition.
In Alloophorus the post-partum ovaries appeared on June 21 and thereafter ap-
peared in large numbers in all collections until the end of the year (see Figure 1).
After October 25 the number of these ovaries dropped, concomitant with the rise in
the number of resting ovaries. At this transitional point it became more difficult
REPRODUCTION IN VIVIPAROUS TELEOSTS
357
GO ODE A LUITPOLDII
embryonic stages.-
arowma eoos
ggs
free eggs
1.2 mm.
137 mm.
COLLECT/ON DATES
FIGURE 3. The appearance throughout the year of ovaries of Goodca luitpoldii with
embryos in different stages of development. For any one day, the hars represent the percentage
of ovaries in each stage of gestation.
358
GUILLERMO MENDOZA
NEOOPHORUS DIAZI
embryonic stages.-
growing eggs
COLLECTION DATES
FIGURE 4. The appearance throughout the year of ovaries of Ncoophorus diazi with
embryos in different stages of development. For any one day, the bars represent the percentage
of ovaries in each stage of gestation.
REPRODUCTION IN VIVIPAROUS TELEOSTS 359
to distinguish between post-partum and resting ovaries. The appearance of post-
partum ovaries in Goodca was the same as in Alloophorus. In Neoophorus, these
ovaries appeared throughout most of the year.
CHARACTERISTICS OF THE GESTATION CYCLE
Length of cycle
Estimates of the length of the gestation cycle in the three species can only be
suggestive. In Alloophorus there was approximately a two-month period between
the first appearance of free eggs (April 9) and the first appearance of post-partum
ovaries (June 21). Similarly, the last collection date of free eggs was June 6 and
the last appearance of embryos of maximum size was August 16, a period slightly
in excess of two months. Similar calculations can be made for Goodea and Neo-
ophorus from the data. From this it follows that the length of gestation is approxi-
mately 60-75 days. This admittedly is only an approximation of the length of
gestation but the evidence appears sound.
Number of broods per year
The evidence is conclusive that in Alloophorus and Goodea there is but one
brood per season. In Alloophorus, free eggs disappeared from collections even
before the first young were born ; hence even the females with the first broods could
not start a second brood. In Goodca, the evidence is similar. In Neoophorus, the
conditions are quite different. Young were born continuously between April and
January ; there was no good evidence of periodicity of any kind. Thus the cycle
in Neoophorus could be a single brood with females starting at different times or the
cycle could be a multiple one. If gestation takes approximately two months, these
females could undergo at least three broods in one season, depending on the length
of the brood interval. The fact that young are born over such a prolonged period
during the year explains the difference in the curves for the immature and post-
partum ovaries for NcoopJiorus (Fig. 1 ) as compared to the other two species.
Brood size
Estimates of brood size in the three species are based on sample counts made-
during different periods of development in the three species. In Alloophorus an
average of 23.7 young per ovary was counted in a total of 50 ovaries containing
1186 young. In Goodea, there were 860 young in 44 ovaries for an average of 19.1
young. In Neoophorus, 193 ovaries with 7677 young gave an average of 39.9
young per ovary.
Brood uniformity
All embryos in any one ovary are essentially of the same size. Measurements of
all embryos in many ovaries indicated clearly that embryos seldom differed more
than 2 to 3 mm. in total length in any one ovary. This uniformity of development
is true for all three species.
Abnormal young or runts were very scarce. In Neoophorus, runts comprised
only 0.49% of a total of 7677 sample embryos ; in Alloophorus there were 0.42^ in
1186 embryos ; and in Goodea there were only 0.11% in 860 embryos.
360 GUILLERMO MENDOZA
Life span oj females
There is a general belief among the fishermen that females normally die after
reproducing. If this were true, all or most females caught early in the spring would
tend to be of minimum length ; collections do not support this belief. Measurements
of more than 500 females in the three species indicated that fish caught in the spring
showed a size range typical for females of each species. However, by using a net
with finer mesh unusually small specimens were also caught ; these had the follow-
ing measurements : Alloophorus, 60-90 mm. ; Goodea, 75-85 mm. ; and Neoophorus
50 mm. or less. The commercial fishermen normally do not keep these small speci-
mens ; they form a population with a normal distribution curve at a smaller size-
range than that for normal adults. This was true for Alloophorus and Goodea.
It is suggested here that these are one-year-old specimens and that they probably
attain maturity during the second breeding season following the year of their birth.
Plotting the lengths of all females collected shows a definite bimodal curve ; the two
peaks presumably represent the two populations, the one-year-old specimens and
the normal adults. Neoophorits, on the other hand, does not show this condition.
A comparable curve for this species is a single but skewed curve. It is likely that
these young attain maturity during the breeding season immediately following
their birth ; consequently, one-year-old young merge into the size-range of the adults.
In this species, overlap in size between one-year-old specimens and normal adults
is due in part to the extended breeding period of this species.
Age of female and onset of reproductive activity
Making use of collections involving 513 females of the three species, information
was obtained concerning the relationship between age and size of the female and
brood production. There is no question that the larger females have the larger
gonads ; this is evident in Table I :
TABU-: I
The relationship between size of female and size of ovary in 56 Alloophorus
females collected on May 28, 1956
Average size of ovaries
Size of female Number of females (length X diameter)
90 mm. 8 25.0 X 9.6 mm.
95 12 27.2 X 10.1
100 12 29.0 X 12.0
105 9 37.0 X 15.5
110 8 32.5 X 14.0
115 4 43.5 X 15.2
120 and over 3 43.0 X 19.3
There is a definite correlation between the diameter of an ovary and the size of
the female. It should be noted that at this time of the year most females were in
some stage of gestation. From these same figures it was determined that the
largest young were found in the largest females. This can only mean that the
largest females started their reproductive cycle earlier in the season. Except for
small variations, the collections for Goodea and Alloophorus all confirmed the
data given above : the May 28 collection is representative. In Neoophorus, how-
REPRODUCTION IN VIVIPAROUS TELEOSTS 361
ever, such a progressive relationship does not exist. This can he interpreted only
in the light of the extended, presumably multiple and non-rhythmic reproductive
cycle of this species. Since there is no evidence of synchrony in the cycle of this
species, it means that on any given day any two females of similar size can be in
different stages of reproduction. Under these conditions, there can he no consistent
relationship between size of female and size of developing embryos.
DISCUSSION
Both Allooplwrns and Goodea have a short reproductive cycle with but one brood
per year. In these two species the young were born only during a period of two
months; the females were inactive during the rest of the year. It is likely, how-
ever, that Neoophorus has multiple broods during the breeding season, although
conclusive evidence is not indicated by the data. In Neoophorus the breeding
season extended over a period of 8 to 9 months of the year; this characteristic
clearly distinguished Neoophorus from the other two species. In this respect the
Neoophorus cycle approaches that of Brachyrhaphis cpiscopi in which breeding
occurs throughout the year (Turner, 1938b). This extensive cycle is not sur-
prising since Brachyrhaphis inhabits the area of Barro Colorado Island in the
Panama Canal Zone where the tropical conditions are favorable to prolonged
periods of breeding. In a sense, the multiple cycle of Neoophorus and the single
cycles of Alloophorus and Goodea are antithetic. All forms live in the same lake
(Patzcuaro) and are subject to common physical factors. The long daylight factor
at Patzcuaro, which is in a tropical latitude, could lead to the long breeding cycle of
Neoophorus but it has not had a similar effect on Alloophorus and Goodea. On the
other hand, Patzcuaro is at a high altitude (over 7000 feet) and the weather is cool
to chilly even in the summer. This temperature factor could be conducive to the
single broods in the two larger species but apparently has little effect on Neoophorus.
Ecological factors in the lake could play a role, since there is some evidence of
ecological segregation of species, but the lake is too shallow and probably too
homogeneous to provide great ecological differences. In the final analysis, it ap-
pears that genetic differences between the three species must perforce play an impor-
tant role in determining the different reproductive cycles. Among other goodeids,
it is known that multiple cycles exist in Neotoca bilineata (Mendoza, 1939) and
Xenotoca eiseni (unpublished data). Among fresh-water viviparous forms, multi-
ple broods are common ; this condition is found in forms such as Hctcrandria
jormosa (Seal, 1911 ; Turner, 1937) ; Gambusia affinis (Hildebrand, 1917; Turner,
1937); Lebistes retlculatus (Turner, 1937; Purser, 1938); Anableps anablcps
(Turner, 1938a) and many others. The single breeding cycle per year found in
Alloophorus and Goodea is more commonly found in marine viviparous teleosts
such as Zoarccs vivipants (Stuhlmann, 1887; Wallace, 1903) and Cyiuatogastcr
aggrcgatus (Eigenmann, 1892 ). It has not been reported in any other goodeid.
In the three species, eggs undergo fertilization immediately before or after escape
of the egg from the follicle, since all cleavage and later stages are found only in the
ovarian lumen. This condition has been reported for goodeids in general (Turner,
1933), Neotoca bilineata (Mendoza, 1941), and it also exists in Xenotoca eiseni
(unpublished data). Similar early evacuation of the egg from the follicle has been
well established for species such as Cyinatoc/aster agc/rec/atns (Eigenmann, 1892;
362 GUILLERMO MENDOZA
Turner, 1947), Jenynsia lineata (Scott, 1928; Turner, 1940b) and others. This
condition stands in direct contrast to that found in Anablcps anableps (Turner,
1938a) and poeciliids in general (Turner, 1947), for fertilization and most or all
development takes place in a follicle within the ovarian tissues. The young escape
from the follicle only shortly before birth.
Insemination and fertilization occur in rapid succession in the three goodeid
species, each brood requiring a separate insemination. Sperm have been observed
in the ovaries only about time of fertilization. Although breeding occurs only over
a short period of time in the two species, the males of all three species show abundant
sperm in the testes during the entire year. In Neotoca bilincata (Mendoza, 1941 )
each of the multiple broods also requires a separate insemination. Furthermore, the
phenomenon of sperm storage within the female genital tract does not occur in any
goodeid. Stored sperm are believed to permit fertilization of successive broods
without necessity for further contact between male and female. The phenomenon
of sperm storage and successive fertilization of two or more broods without need
for separate inseminations has been described for many viviparous teleosts, such as
Jenynsia lineata (Scott, 1928; Turner, 1957), Cymatogaster aggregatus (Eigen-
mann, 1892), Gambusia affinis (Hildebrand, 1917), Xiphophorus helleri (van Oordt,
1928) and others.
Another phenomenon which is absent in goodeids but is very common in
poeciliids is the phenomenon of superfetation, a condition in which two or more
broods at different stages of development occupy an ovary at the same time. Ex-
amples among poeciliids that demonstrate an extreme form of superfetation are
Aulophallus and Poeciliopsis (Turner, 1937), in which as many as nine overlapping
broods occur at one time; other poeciliids show varying degrees of superfetation.
Failure to achieve superfetation among goodeids is due, in part, to the failure of
eggs to grow to maximum size before expulsion of a brood and, in part, to the
absence of sperm storage. These two conditions normally occur in many poeciliids
and are requisites for the occurrence of superfetation. In contrast to the writer's
observations, Turner (1940a) states that he has seen aberrant or unsuccessful ex-
amples of superfetation in goodeids such as Xenoophorus crro, Chapalichthys en-
caustus, Skiffia varicyata and others, because he has seen sperm and growing oocytes
in the ovaries, superimposed on another brood. The possibility certainly exists
that occasional eggs may grow, be fertilized, and start development during gestation.
In the goodeids studied by the writer, all abnormal embryos observed were so scarce
and so close to the stage of development of the current brood that they were all
interpreted as abnormalities rather than as younger embryos superimposed on the
normal brood.
In Alloophorus and Goodca, broods average around 20 young but fluctuate under
50. In contrast, broods in Ncoophorus average about 40 young but may on occasion
exceed 100. These brood sizes compare favorably with those in Xenotoca eiseni
(unpublished data) ; Neotoca bilincata (Mendoza, 1939) has much smaller broods,
averaging only six to ten young. Broods numbering under 50 young are very
common among viviparous fresh-water fishes. For example, Gambusia affinis
(Kuntz, 1913) has 40 to 63 young per brood, Jenynsia lineata (Scott, 1928) has 10
to 40 young, and there are 30 to 40 young per brood in Xiphophorus maculatus
(formerly Platypoecilns maculatus) (Tavolga and Rugh, 1947; Tavolga, 1949), etc.
REPRODUCTION IN VIVIPAROUS TELEOSTS 363
In the goodeids studied, the larger and older females have larger broods and, al-
though younger females do have smaller broods, the difference in brood size is not
great. This condition is also true in Neotoca bilincato (Mendoza, 1939) and
Xenotoca eiscni (unpublished data) although in these two forms the size of broods
in the younger females is markedly smaller. This discrepancy of brood size be-
tween younger and older females is very common among other viviparous teleosts.
such as Cyniatogastcr aggregates (Eigenmann, 1892), Gambusia affinis (Hilde-
brand, 1917) Anableps anableps (Turner, 1938a) and others.
The occurrence of much uniformity of development among the young in any one
ovary is not surprising since this is a common phenomenon. Specific reference to
this condition has been reported for viviparous teleosts, such as Neotoca bilineata
(Mendoza 1941), Xiphophorus hellerl (Weyenbergh, 1875), Cymatogaster aggre-
gatus (Eigenmann, 1892), Mollienisia latipinna (Turner, 1937), Anableps anableps
(Turner, 1938a) and others.
Another impressive factor was the occurrence of very few abnormal embryos
during embryonic development. It is likely that if fertilization is successful, the
majority of the embryos will continue through development. Reason for this be-
lief rests on the fact that the free egg counts for both Alloophorus and Goodea agreed
well with the average size of broods. In Neoophorus, however, there is a greater
disparity between the number of free eggs and the number of young in a brood.
Even in this species, however, once the embryos start development mortality appears
to be very low. The writer's observations do not agree with Turner's generaliza-
tion that in the Jenynsiidae and Goodeidae many more eggs are fertilized than
survive till birth (Turner, 1938a). It is important to note that Turner's observations
were not based on the three species in this study.
Finally, the assumption that Alloophorus and Goodea take two years to mature
appears to be unusual among viviparous teleosts. Zoarccs vh'iparus (Wallace,
1903) is one of the few described as maturing at the end of the second year. Spe-
cies such as Cyniatogastcr aggregatus (Eigenmann, 1892) and Jenynsia lineata
(Turner, 1940b) are said to mature by the following season. It is suspected but
cannot be proven that Neoophorus diazi matures by the following year. In other
goodeids, such as Neotoca bilineata (Mendoza, 1939), the young mature within
the same breeding season. The length of time necessary for maturation probably
is related to sheer physical size of adults, along with pertinent ecological and physio-
logical factors, since Alloophorus and Goodea are larger than typical poeciliids and
apparently resemble Zoarces vivipanis, another large species (130-300 mm.), in
taking two years to mature.
Although some factors in the reproductive cycles of these three species have been
demonstrated clearly by the collection of field specimens, it is also evident that
some properties of the cycles, such as the actual length of gestation and the single
or multiple nature of the Neoophorus reproduction cycle, will have to be determined
either by tagged specimens in the field or by a laboratory-controlled study.
SUMMARY
1. The reproductive cycles were determined for three goodeids: Alloophorus
robustus, Goodea luitpoldii, and Neoophorus diazi. The study is based on a year-
long series of collections in the field ; over 3000 females were examined.
364 GUILLERMO MENDOZA
2. Alloophorns and Goodca are shown to have a single cycle ; young are born
from June through August. Neoophorus probably has a multiple cycle and young
are born continuously from April through January or February of the next year.
3. Brood size varies as follows : there are approximately 20 embryos per brood
in both Goodea and Alloophorns but the average is about 40 in Neoophorus.
Younger females have smaller broods although the difference is small.
4. Eggs are discharged from the follicle about time of fertilization and undergo
all development within the ovarian lumen. On birth, young are able to swim
actively.
5. There is no evidence of sperm storage or superfetation.
6. Embryos in any one brood exhibit much uniformity of size.
7. Abnormal development of embryos is at a minimum ; runts constituted less
than \% of all embryos examined.
8. Neoophorus is believed to mature in one year whereas Alloophorns and
Goodea are thought to take two years to develop to sexual maturity.
9. Major differences in the reproductive cycle between the three goodeids are
believed to be primarily genetic in character.
LITERATURE CITED
EIGENMANX, C. H., 1892. Cymatoyastcr aggregatus Gibbons; a contribution to the ontogeny of
viviparous fishes. Bull. U. S. Fish Com., 12 : 401-479.
HILDKBKAND, S. F., 1917. Notes on the life history of the minnows Gambusia affinis and
Cyprinodon varicgatus. Report U. S. Com. Fish., Appendix VI, No. 3, 1918.
KUNTX, A., 1913. Notes on the habits, morphology of the reproductive organs and embryology
of the viviparous fish, Gambusia affinis. Bull. U. S. Bur. Fish., 33 : 181-190.
MEEK, S. E., 1904. The fresh-water fishes of Mexico north of the Isthmus of Tehuantepec.
Field. Col. Mus. PubL, No. 93, Zoo/. Ser., 5: 1-252.
MENDOZA, G., 1939. The reproductive cycle of the viviparous teleost, Neotoca bilineata, a mem-
ber of the family Goodeidae. I. The breeding cycle. Biol. Bull., 76 : 359-370.
MENDOZA, G., 1941. The reproductive cycle of the viviparous telost, Neotoca bilineata, a mem-
ber of the family Goodeidae. III. The germ cell cycle. Biol. Bull., 81 : 70-79.
PURSER, G. L., 1938. Reproduction in Lebistes reticulatus. Quart. J. Micr. Sci., 81 : 150-159.
SCOTT, M. I. H., 1928. Sobre el desarrollo intraovarial de Fitsroyia lineata (Jen.). Berg.
Anal. Mus. Hist. Nat. de Buenos Aires, (Ictiologia, Pub. Num. 13), 34: 361-424.
SEAL, W. T., 1911. Breeding habits of the viviparous fishes Gambusia holbrookii and Hctc-
randria fonnosa. Proc. Biol. Soc. Washington, 24 : 91.
STUHLMANN, F., 1887. Zur Kenntnis des Ovariums der Aalmutter. (Zoarces viviparus Cuv. )
Abliandl. dcs Natunviss., Vercins zu Hamburg, 10: 1.
TAVOLGA, W. N., AND R. RUGH, 1947. Development of the Platyfish, Plat \poecihts maculatus.
Zoologica (New York), 32 (Part 1) : 1-15.
TAVOLGA, W. N., 1949. Embryonic development of the Platyfish (Platypoccilus), the Sword-
tail (Xiphophorus), and their hybrids. Bull. Amcr. Mus. Nat. Hist., 94: 161-230.
TURNER, C. L., 1933. Viviparity superimposed upon ovo-viviparity in the Goodeidae, a family
of cyprinodont teleost fishes of the Mexican Plateau. /. Morph., 55: 207-251.
TURNER, C. L., 1937. Reproductive cycles and superfetation in poeciliid fishes. Biol. Bull., 72 :
145-164.
TURNER, C. L., 1938a. Adaptations for viviparity in embryos and ovary of Anableps anablcps.
J. Morph., 62 : 323-349.
TURNER, C. L., 1938b. The reproductive cycle of Brachyrhapliis cpiscopi, an ovo-viviparous
poeciliid fish, in the natural tropical habitat. Biol. Bull., 75 : 56-65.
TURNER, C. L., 1940a. Superfetation in viviparous Cyprinodont Fishes. Copeia, No. 2 : 88-91.
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REPRODUCTION IX VIVIPAROUS TELEOSTS
TURNER, C. L., 1947. Viviparity in teleost fishes. Sci. Monthly, 65 : 508-518.
TURNER, C. L., 1957. The breeding cycle of the South American fish, Jcnynsia lincata, in the
northern hemisphere. Copcia, No. 3 : 195-203.
VAN OORDT, G. J., 1928. The duration of life of spermatozoa in the fertilized female of
Xiphoplwrus hcllcri Regan. Tijdschr. d. Nederl. Dicrkd. V crccn., 1 : 77-80.
WALLACE, W., 1903. Observations on ovarian ova and follicles in certain Teleostean and
Elasmobranch fishes. Quart. J. Micr. Sci., n.s. 47: 161-213.
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AN ANALYSIS OF THE INITIAL REACTION IN THE SEQUENCE
RESULTING IN HOMOLOGOUS SPLENOMEGALY
IN THE CHICK EMBRYO
A. M. MUN.i P. TARDENT.2 J. ERRICO, J. D. EBERT, L. E. DELANNEY =
AND T. S. ARGYRIS 4
Department of Embryology, Carnegie Institution of Washington, Baltimore 10, Maryland,
and Wabash College, Crazvfordsvillc, Indiana
The splenomegaly induced in the chick embryo by chorioallantoic or intra-
coelomic grafts of homologous adult spleen, or by intravenous inoculations of
homologous spleen cells ( Danchakoff, 1916; Murphy, 1916; Willier, 1924; Ebert,
1951; Simonsen, 1957; reviewed Ebert, 1958, 19591)) is thought to be the conse-
quence of at least two sequential reactions, an initial graft- versus-host reaction (De-
Lanney and Ebert, in Ebert, 1957 ; Ebert and DeLanney, 1960 ; Simonsen, 1957 ;
see also Billingham and Brent, 1957) followed by a tissue-specific growth reaction,
granulocytic proliferation probably being stimulated by products resulting from the
partial necrosis produced by the initial immune reaction (Ebert, 1951, 1954; De-
Lanney, Ebert, Coffman and Mun, 1962 ; see also Weiss, 1960). Evidence has been
advanced also for the involvement of a third process, i.e., a host-versus-graft reac-
tion (Ebert and DeLanney, 1960; Ebert, 1961b) ; cj. Warner and Burnet, 1961),
but it is not clear to what extent this reaction contributes to the splenomegaly. It
is pertinent to inquire whether these processes can be separated experimentally.
The graft-versus-host phenomenon is but one manifestation of the familiar homo-
graft reaction that leads to the rejection of tissue grafts ; in several species, both cold-
and warm-blooded, it has been shown to be a consistent and reproducible immuno-
logical reaction (Ebert and DeLanney, 1960). The immunological character of this
first, destructive phase is widely accepted, being dictated by several lines of evidence.
(1) Recent findings in experiments using grafts of spleen from inbred lines of
fowls have demonstrated that interstrain grafts produce a larger effect than intra-
strain grafts, a finding to be expected if the reaction were an immunological one.
Additional findings to be advanced here agree with those reported by Cock and
Simonsen (1958), Mun, Kosin and Sato (1959), and Jaffe and Payne (1961) who
used inbred strains of white Leghorn chickens.
(2) X-irradiation of a graft of adult chicken spleen removes its ability to affect
the homologous organ of the embryo. According to Mun, Kosin and Sato (1959),
after irradiation at low doses, splenic grafts retain their effectiveness; at moderate
doses, a significant decrease in effectiveness is observed, and at high doses all
activity is lost. Kryukova (1959) also showed that the inoculation of non-irradi-
1 Present address : Department of Zoology, University of Maine, Orono, Maine.
2 Present address : Zoologisches Institut der Universitat, Zurich.
3 Present address : Department of Biology, Wabash College, Crawfordsville, Indiana. L. E.
DeLanney's research is supported in part by a grant (RG-5619) from the United States Public
Health Service.
4 Present address : Department of Zoology, Syracuse University, Syracuse, New York.
366
ANALYSIS OF HOMOLOGOUS SPLENOMEGALY 367
ated homologous spleen cells and cells x-irradiated at low doses caused approxi-
mately six-fold enlargement of the embryo spleen, yet cells irradiated with moderate
doses had little or no effect. Other treatments, e.g., boiling, and freezing and
thawing, also remove all activity (Mun, Kosin and Sato, 1959).
(3) The effectiveness of a tissue in producing the reaction varies directly with
its content of immunologically competent cells ; hence, it appears consistent to state
that the reaction is tissue-specific in the limited sense that the specificity reflects the
proportion of competent cells a tissue contains. It may be premature to attempt to
relate this specificity to a specific cell type, although Terasaki (1959) believes the
large lymphocyte to be the common denominator. Whatever the nature of the cell
for cells) concerned, it is known that plasma cells, indicators of immune reactions,
may be found at the terminal stages of the reaction (Mori, in Ebert, 1961a). All
of the following tissues are effective : bone marrow, liver, spleen, and thymus
(Danchakoff, 1918; Willier, 1924; Ebert, 1951). Moreover, Van Alten and Fennell
(1959) and Billingham and Silvers (1959), respectively, have shown that a graft
of either small intestine or skin also may affect the host's spleen. Although Ebert's
earlier (1954, 1959b) observations of quantitative differences in effectiveness were
based soundly, his generalization that one might expect a hierarchy of decreasing
effectiveness, e.g., spleen, thymus, liver, is difficult to establish. Solomon (1961)
was unable to observe such differences and argued that the ultimate extent of the
splenomegaly is related directly to the number of competent cells in the graft ;
certainly this figure will vary, although it is reasonable to assume that generally,
spleen and thymus will have a larger number of such cells per unit volume or weight
than will other tissues.
The reaction is class-specific ; grafts of spleen of other avian species, such as
duck, turkey, and pheasant, produce some effect but never as much as homologous
spleen ; rat, mouse and guinea pig spleens are completely ineffective in the chick
(Ebert, 1951, 1954; Mun, Kosin and Sato, 1959). Presumably, the ineffectiveness
of mammalian cells is a consequence of their failure to survive the rigors of the
foreign environment long enough to produce an immune response. This explana-
tion is being tested experimentally. If it proves to be correct, then mammalian
immune mechanisms would appear to be unusually sensitive to the avian environ-
ment, for there is evidence for the survival of other kinds of mammalian cells in the
chick embryo (cf. Clarkson and Karnofsky, in Ebert and DeLanney, 1960, p. 97 ).
(4) Finally, the ability of splenic grafts to affect the host varies with the age
of the donor; grafts of spleen from embryonic donors have little or no effect, the
effectiveness of grafts increasing when they are taken from progressively older
donors up to several months after hatching. Additional data are presented herein,
supplementing the comprehensive recent account by Solomon (1961). Although
there are unexplained exceptions to the rule (cf. Ebert, 1951 : Solomon, 1961, pp.
359-363), the effectiveness of grafts is related directly to their immunological ma-
turity. Our perspective of the problem of maturation of the immune response has
been broadened by the findings of Makinodan and Peterson (1962) who have ob-
served that the relative antibody-forming capacity of spleen cells of mice varies with
age from one week to 29 months. A rapid increase in activity was noted from one
week to one month, one less rapid from one to 8 months. A gradual decrease
was then observed from the peak at 8 months through an additional 21 months.
368 MUN, TARDENT, ERRICO, EBERT. DELANNEY AND ARGYRIS
Accepting the argument that a part of the splenic enlargement following a graft
of adult spleen encompasses immune reactions, we may take up next the site of these
reactions. How many cells leave the graft and enter the extraembryonic membranes
and the embryo itself? How many donor cells take up residence in the homologous
organ of the host ? Do they also settle in other organs ? It is clear that when sus-
pensions of adult chicken spleen cells or suspensions of adult chicken lymphocytes
are administered to the embryo intravenously, or when grafts of adult spleen are
made to the chorioallantoic membrane or into the coelom, some of the donor cells
colonize the organs of the host. The evidence is derived from serial transfer
studies by Simonsen (1957) and Ebert and associates (Ebert, 1957; DeLanney,
Ebert, Coffman and Mun, 1962). When a graft of adult chicken spleen is made to
the coelom of a four-day-old chick embryo, the host's spleen is enlarged four- to
five-fold within six days. If fragments of this greatly enlarged ten-day-old em-
bryonic spleen now are transferred to new four-day-old hosts, they elicit a reaction of
the same order of magnitude, whereas fragments of spleen from normal ten-day-old
embryos are ineffective. After nine successive transfers, the effectiveness of the
implant is not reduced markedly below the level attained by the primary graft.
Assuredly, then, there is some colonization. But how much, and to what extent do
these donor cells proliferate? Simonsen (1957) argued that colonization and
proliferation accounted for all the effects of splenic grafts. However, studies by
Ebert and associates (summarized by DeLanney, Ebert, Coffman and Mun, 1962)
of the cellular nature of the response pointed to the host as the principal source of
proliferating cells. Moreover, studies using grafts radioactively labeled, in early
experiments with sulfur35, while not decisive, revealed a predominant localization
of material in the homologous organ, but precluded a massive transfer of cells (Ebert,
1954, 1959b). Biggs and Payne (1959) have presented significant findings in a
study in which they identified proliferating donor cells in chick embryos injected
with adult chicken blood. In the chicken the fifth largest chromosome is paired in
the male, unpaired in the female. Cockerel blood was injected into fourteen-day-old
embryos which were sacrificed at day eighteen. In enlarged spleens taken from
female embryos, male chromosomes could be identified, proving the localization of
some donor cells. The relatively high number of dividing female cells, however,
suggested to Biggs and Payne that an appreciable component of the splenic enlarge-
ment is provided by cells of the host. The evidence available, therefore, suggests
that following the intravenous injection of blood or spleen cell suspensions, some
donor cells colonize the host's spleen. Moreover, such colonization need not result
invariably in splenic enlargement, which may result in whole or in large part from
proliferation of cells of the host.
The fact that splenomegaly is not evoked by noncompetent homologous donor
cells or with competent isologous cells forces the conclusion that the proliferation
of cells of the host is a secondary consequence of a primary immune reaction. The
nature of this secondary reaction must be the principal target of future investigations.
In beginning such a study, it became clear that more information was needed on the
extent of colonization and maintenance of donor cells in the several tissues of
the host.
It is the objective of this report to present findings bearing on that question :
these findings bear importantly also on another question, namely, the ability of the
ANALYSIS OF HOMOLOGOUS SPLENOMEGALY 369
embryonic environment to support an immune reaction. Preliminary accounts of
some of these findings have been published (Mun, Errico and Ebert, 1961 ; Ebert,
1961a, 1961b).
MATERIALS AND METHODS
Non-inbred white Leghorn chickens and eggs were supplied by Elder Farms,
Hyde, Maryland. Hybrid white Leghorn chickens and eggs were obtained from
Truslow Hatchery, Chestertown, Maryland. Chickens and eggs from two inbred
lines (7 and 15) with coefficients of inbreeding of greater than 95 % were supplied
by B. Winton, Director of the Regional Poultry Research Laboratory, East Lansing,
Michigan. New Hampshire red eggs were purchased from Red Gate Farm, New-
port, New Hampshire. Eggs were incubated in a Jamesway incubator at 37.5 to
38.0° C.
The aseptic grafting technique employed was that described by Willier (1924;
see also Hamburger, 1960). A quadrilateral window (1x1 cm.) was cut in the
shell with a fine-toothed hacksaw blade. The shell membrane was punctured and
reflected, and a fragment of tissue measuring approximately 1x1x2 mm., and
weighing 5 to 10 mg., was placed on the chorioallantois. The shell membrane and
shell were replaced and sealed with paraffin. The eggs were placed in the incubator
with the small end down.
The eggs were operated on the ninth or tenth day of incubation. After 7 or 8
additional days of incubation the graft was removed and examined. The size of the
area of implantation (length X width) was recorded and the condition of the
implantation site was graded as follows : ( 1 ) graft enlarged, pink, and larger than
the original; (2) graft pink and as large as the original implant; (3) graft brown
or green, clearly not incorporated and smaller than the original or grafted tissue.
Rarely a graft in category (3) produces a response, but the fact that a reaction did
occur occasionally suggests the movement of viable cells from the graft soon after
implantation. Spleens of recipient embryos were removed and weighed to the
nearest 0.2 mg. The weights of spleens from embryos in group 3 were not
included in the tabulations.
RATE OF COLONIZATION IN THE CHORIOALLANTOIC MEMBRANE ADJACENT TO
SPLEEN GRAFTS, AND IN THE HOSTS' SPLEENS
DeLanney and Ebert (1959a, 1959b), and DeLanney, Ebert, Corrman and Mun
(1962) have followed the cytological changes in the chorioallantois at closely timed
intervals after implantation of homologous adult spleen. Immediately after im-
plantation the epithelium of contact thickens ; the mesenchyme forms spindle cells
and undergoes a shift toward myelogenesis. In the zone of contact between graft
and membrane, the chorionic epithelium is eroded, clusters of granulocytes appear,
spindle cells gather at the border, and tongues of cells, apparently originating in the
graft, invade the membrane. The second set (or third set) chorioallantoic trans-
plantation of fragments of chorioallantois taken from reactive sites surrounding the
original first set spleen implant results in intensified reactions. Further evidence of
colonization of the membrane is provided by the following experiments, in which
fragments of homologous embryonic spleen were placed on the chorioallantois some
distance from grafts of homologous adult spleen. After varying intervals, the em-
bryonic grafts were removed, and their ability to produce splenomegaly determined.
370
MUN, TARDENT, ERRICO, EBERT, DsLANNEY AND ARGYRIS
Two windows, approximately 1 cm. apart, were cut in the shell. A fragment
of adult spleen, kidney, or heart was placed on the chorioallantois through one
window and 17-day-old embryo spleen was implanted through the other. In con-
trol groups embryonic spleen was implanted in both sites. After 7 additional days
of incubation, the adult graft and the embryonic graft were removed, with associ-
ated membrane, and implanted on the chorioallantoic membrane of new 1 0-day -
old hosts. After 7 additional days of incubation the hosts' spleens were removed
TABLE I
Colonization of embryonic spleen grafts adjacent to grafts
of adult spleen and other tissues
Donor
No.
Mean weight of
host spleen (mg.)
SEni
Adult spleen + adult spleen
4
47.4
Graft of adult spleen
3
39.1
Graft of host's spleen
2
118.4
Adult spleen -f- embryo spleen
30
35.9
3.8
Graft of adult spleen
4
38.9
—
Graft of embryo spleen
29
40.3
4.9
Graft of host's spleen
2
36.1
—
Adult kidney + adult kidney
26
17.2
1.7
Graft of adult kidney
14
19.9
3.2
Graft of host's spleen
4
21.7
—
Adult kidney + embryo spleen
22
18.9
2.1
Graft of adult kidney
14
12.1
4.2
Graft of embryo spleen
19
38.8
6.6
Graft of host's spleen
4
42.3
• —
Adult heart + adult heart
9
12.2
0.8
Graft of adult heart
6
18.4
4.9
Graft of host's spleen
6
18.7
4.4
Adult heart + embryo spleen
12
14.9
1.2
Graft of adult heart
3
14.2
—
Graft of embryo spleen
10
16.6
1.6
Graft of host's spleen
9
20.5
3.5
Embryo spleen + embryo spleen
10
13.7
0.9
Graft of embryo spleen
8
12.4
1.0
Graft of host's spleen
3
14.6
~
and weighed. Table I shows that an embryonic spleen graft placed adjacent to an
adult spleen graft can affect the host's spleen to the same extent as an adult spleen
graft. Here, then, is further evidence of movement of cells from the adult spleen
graft to a graft of embryonic spleen on the membrane. How rapid is this movement ?
Homologous spleen and homologous embryonic spleen were implanted approxi-
mately 1 cm. apart as described above. After 2, 3, 5, 6, or 7 additional days of
incubation both grafts and the host's spleen were removed and transferred to new
ANALYSIS OF HOMOLOGOUS SPLENOMEGALY
371
10-day hosts. Table II shows that, as early as tivo days after implantation, both
the embryonic spleen graft and the host's spleen, neither of which show any enlarge-
ment at this time (cf. DeLanney, Kbert. Cofrman and Mun, 1962), are capable of
affecting the spleen after serial transfer.
COLONIZATION OF ADJACENT MEMBRANE BY ADULT SPLEEN CELLS LABELED
WITH TRITIATED THYMIDINE
The suggestion that the use of cells labeled with tritiated thymidine might aid in
resolving the question of migration of adult chicken spleen cells from grafts to the
hosts' membranes and spleens was advanced by Ebert (1959b). However, the
principal limitation of the method, the dilution of label in rapidly dividing cells, is
critical, and in the opinion of the writers the technique is less reliable than the
cytological method, i.e.. recognizing donor cells by sex chromosome differences
(Biggs and Payne, 1959; Ohno, 1961). However, the following experiments do
TABLE II
Rate of colonization of host embryo's spleen (HS) and embryonic spleen graft (GES) adjacent
to adult chicken spleen graft (GAS) on the chorioallantois
Donor
2-3 days
5 days
7-8 days
No.
Mean
weight
of host's
spleen (mg.)
SEm
No.
Mean
weight
of host's
spleen (mg.)
SEm
No.
Mean
weight
of host's
spleen (mg.)
SEm
AS + ES
GAS
13
21.6
6.8
2
47.8
—
4
38.9
•
GES
21
21.8
3.4
5
21.8
6.3
29
40.3
4.9
HS
14
23.1
5.1
6
17.5
2.0
30
35.9
3.8
ES + ES
GES
6
10.8
1.2
8
12.4
1.0
HS
4
11.4
—
3
14.6
—
contribute further to our knowledge of the migration of cells into the adjacent
membranes.
Nineteen experiments were conducted using labeled and non-labeled donor
material. The results of three experiments (XI, XIII, XI I la) will be considered
here.
In experiment XI, the donor tissues were labeled by injecting into the wing
veins of adult white Leghorn chickens 2 millicuries of tritiated thymidine in two
doses, 48 and 24 hours before sacrificing. In experiments XIII and XIHa,
labeled tissue from enlarged embryonic spleen was used as donor tissue. The
cells were labeled by injecting 15 to 25 microcuries of tritiated thymidine into the
yolk sacs of 9-day-old chick embryos. Twenty-four hours later, a piece of un-
labeled adult chicken spleen was implanted on the chorioallantois of each embryo
(Fig. 1). In control series, a piece of homologous embryo spleen was implanted
instead of adult spleen. After 7 or 8 additional days of incubation the enlarged
372
MUN, TARDENT, ERRICO, EBERT, DELANNEY AND ARGYRIS
?,;A«*2
£^#dg**^V*'. '
". . ' m . v^F * *^ ' .. . .mjfr^^* " ,^ %^K
FIGURES 1-3.
&
ANALYSIS OF HOMOLOGOUS SPLENOMEGALY 373
and labeled host spleens were removed, cut into small pieces, and implanted in
9-day-old chick embryos.
The latter approach resulted in at least a doubling of the percentage of labeled
donor spleen cells. The enlarged embryo spleen also elicited a greater increase
in the size of the host spleen than grafts of adult chicken spleen. Large white
nodules and lesions were observed more frequently in foot and head regions.
At recovery a number of tissues, including the membrane containing the graft,
the host's spleen, and a sample of blood, were obtained from each embryo. Rep-
resentative whole embryos also were fixed in Bouin's fluid.
The labeled cells were detected by autoradiography. The host's spleen and
membrane containing the graft were sectioned at 5 microns, and stained with
Mayer's hematoxylin and eosin. The slides were then coated with Kodak NTB-3
photographic emulsion, following in general the procedures developed by Messier
and LeBlond (1957) and Everett and Simmons (1953). Approximately three
drops of a 50% emulsion kept at 40° C. were smeared on the surface of the warmed
glass slide with a wet brush. The smear was slowly rocked to remove the brush
marks. Excess emulsion was shaken off, and the slide was permitted to dry in a
near-vertical position. The coated slides were kept in the refrigerator (4 to
10° C.) and developed in D72 or D19 (Kodak) after 14 days.
In each experiment, more than 30 embryos received grafts of adult chicken
spleen or enlarged embryo spleen on the ninth day of incubation. An equal num-
ber received labeled embryonic spleen, labeled adult chicken kidney, or irradiated
and labeled adult spleen grafts. The different categories of active and inactive,
as well as labeled and nonlabeled donor tissue, also were combined on the
membrane of the same host. A small number was untreated or received three
drops of saline.
A number of embryos from each group, selected at random, were recovered at
postoperative days 1, 2, 3. and 7. Another group, sufficient in numbers to
ascertain the degree of enlargement of the host's spleen elicited by the donor
material, was recovered on the eighth day after the operation (Table III).
In embryos in which donor spleen tissues containing distinctly labeled cells
(see Figure 2) were grafted, labeled cells were still detected in significant numbers
in the graft as well as the adjacent membranes up to the fifth postoperative day
(Fig. 3). By the eighth postoperative day, however, labeled cells were not
readily detected in the 'graft and adjacent tissues. Preliminary examination of
spleens from host embryos, which contained distinctly labeled donor spleen cells
in the CAM graft, revealed few or no distinctly labeled cells. Quantitative evalua-
tion of the autoradiograms is in progress ; however, these preliminary observations
do not suggest a direct large scale migration of donor cells from the CAM graft
to the host's spleen.
FIGURE 1. Chorioallantoic membrane showing the edge of a graft of adult chicken spleen.
Chick embryo injected via the yolk sac with 25 microcuries of initiated thymidine, and the un-
labeled graft implanted on the ninth day of incubation; recovered 24 hours later. X500.
FIGURE 2. Section of spleen from an embryo which received 25 microcuries of tritiatecl
thymidine and a graft of adult chicken spleen on the ninth day of incubation ; recovered after 8
days. X500.
FIGURE 3. Section of chorioallantoic membrane of a 13-day embryo containing cells of a
labeled graft of "second-set" embryonic spleen, implanted 4 days earlier. X500.
374
MUN, TARDENT, ERRICO, EBERT, DsLANNEY AND ARGYRIS
TABLI-: III
Mean weight of host's spleen after chorioallantoic grafting of adult and embryonic spleen
Expt.
(see text)
Day recovered
post-operative
Donor
No.
Average weight
of host spleen
SEm
XI
8
Adult chicken spleen
Saline control
5
7
34.0
10.5
2.6***
0.6
XIII
8
Enlarged embryo spleen
Saline
10
5
86.2
12.4
8.3***
1.4
XIIIa
8
Enlarged embryo spleen
Saline
7
4
29.4
8.8
4.5**
1.0
** Significant at the .01 level.
*** Significant at the .001 level (/ test).
COLONIZATION IN OTHER ORGANS
It might be expected that competent cells from a graft might be lodged in all
tissues of the embryo's body, to some extent, possibly to the same extent to which
the adult tissues normally contain lymphoid and other reticulo-endothelial elements.
This expectation is realized.
Adult chicken spleen was implanted on the chorioallantois of 9-day host
embryos. After 7 more days of incubation the host's spleen, liver, heart, and
kidney were removed and implanted on membranes of new 9-day recipients.
Table IV shows an increase in the weights of such secondary hosts' spleens
receiving grafts of "second set" spleen, liver, heart, and kidney, to approximately
the same extent as that elicited by the corresponding adult organs.
Thus there is a transfer of competent cells not only to the host's spleen but to
other organs as well.
SERIAL PROPAGATION IN CHICK EMBRYOS OF EMBRYONIC SPLEEN CELLS
FROM NON-INBRED AND INBRED CHICKENS
The graft-versus-host reaction has provided unequivocal evidence that, begin-
ning as early as the fourth day of development, the chick embryo provides an
TABLE IV
Effect of grafts of fragments of adult organs and organs from embryos stimulated by seven days'
exposure to adult spleen grafts on the weight of the host embryo's spleen
i
Spleen weight (mg.) following grafts of
Spleen
Liver
Heart
Kidney
Adult organs
47.4 (4)*
10.8 (7)
18.7 (6)
17.2 (26)
Host embryo organs after grafting of adult
42.7 (6)
18.0 (6)
12.1 (7)
23.6 (6)
spleen
Embryonic organs
14.6 (3)
11.4 (5)
12.3
8.0
Host embryo organs after implantation of
16.0 (3)
11.9 (3)
12.4 (3)
embryonic spleen on the membrane
Figures in parentheses indicate number of cases.
ANALYSIS OF HOMOLOGOUS SPLENOMEGALY
375
environment favorable for at least one class of immune reactions, those of trans-
plantation immunity (Ebert, 1961b). Competent cells retain their competence
in the embryonic environment. But would incompetent embryonic spleen cells
which were maintained in an embryonic environment for long periods of time ever
reach a stage of functional maturity? Or, to put the question in more practical
terms, if homologous embryonic cells were transferred from the graft to the
host's spleen where they proliferated, after several transfers the donor embryo cells
thus maintained in this embryonic environment should eventually attain maturity
and be able to elicit rm enlargement of the host spleen. On the other hand, if the
GROUP I
Embryonic
and
hatched chick
SPLEENS
GROUP E
Serially
transferred
embryo
SPLEENS
14 day
embryo
\/
14 day em.
21 day I wk. 10 wk.
embryo hatched chick hatched chick
v
\t
17 day em. 17 day em.
17 day em.
GROUP HE
Untreated
embryo
SPLEENS
1 4 day em.
v
17 day em.
17 day em.
17 day em.
V
I
Time of i mplantation^ 0
7
IN
14
TIME IN DAYS
FIGURE 4. Serial grafting of homologous embryonic spleen.
12
WEEKS
donor embryo cells were incorporated in the host's spleen but did not proliferate,
they would be diluted after only a few passages.
Four groups of embryos have been studied : ( 1 ) Fragments of homologous
spleen from successively older embryos and hatched chicks were grafted to the
membranes of 10-day-old chick embryos at weekly intervals. After 7 additional
days of incubation, the hosts' spleens were removed and weighed to the nearest
0.2 mg.
(2) A fragment of homologous 14-day embryonic spleen was placed on the
membrane of a 10-day host embryo. After 7 additional days of incubation, the
host's spleen was removed, weighed, and transferred to another 10-day embryo.
376
MUN, TARDENT, ERRICO, EBERT, DELANNEY AND ARGYRIS
This procedure was repeated for 7 or 11 weeks. The donor spleens were not
pooled but were kept separate. Thus, for each of the 20 initial donors 20 separate
lines may be traced.
(3) Fourteen- or 17-day-old embryo spleens were placed on the membranes
of 10-day host embryos at weekly intervals. After 7 additional days of incubation
the hosts' spleens were removed and weighed.
These three groups are illustrated graphically in Figure 4.
(4) The weights of the 14- or 17-day embryo donor spleens before grafting-
comprise the fourth group. This group is not included in the final tabulation
because greater variation in spleen weight was observed following implantation
45. 0-1
40.0-
35. 0^
30.0-
Uj
^i
^
25.0-
15.0-
10.0-
5.0-
o I Embrvonic a Hatched Chick Spleens
a H Serially Transferred Embryo Spleens
Control En.bryo Spleens
—I — — I — — I — — r — — I — — I — — r~
4 5 6 7 8 9 10
TIME IN WEEKS
— I
12
FIGURE 5. Splenomegaly after serial grafting of homologous embryonic spleen.
of a piece of embryo spleen. Thus the spleen weights in the third group were
better controls for the second group.
The first experiment was carried out over an 8-week period. Because an
increase in the average weight of the host's spleen in the serial group (2) was
observed in the eighth week, the second experiment was carried out over a longer
12-week period.
The results of the two experiments were consistent, hence the data were pooled.
As may be seen in Figure 5, spleen grafts from successively older embryos and
hatched chicks after a short lag period of one or two weeks produce a progressively
greater enlargement of the host chick embryo spleen. A significant enlargement
of the host embryo spleen is produced by spleen from a 3- week -old hatched chick.
ANALYSIS OF HOMOLOGOUS SPLENOMEGALY
377
These findings are in general agreement with those of Solomon (1961) and the
previously unpublished data of DeLanney (cited in Solomon, 1961) who observed
an approximate doubling in spleen size following grafts of spleen from 28-day-old
juvenile chickens. DeLanney's independent findings are not strictly comparable
to those set forth here, the period of exposure (days 7 through 18) and weighing-
procedure being different, hence they are not included in the tabulation; the
data may be obtained from him upon request.
137.4
I 10-
5 6
TIME
7 8
IN WEEKS
10
12
FIGURE 6. Splenomegaly in individual lines after serial grafting of homologous embryonic spleen.
In the second or serial group, after a lag period of 6 to 7 weeks, a distinct
increase is observed in the average weight of the host's spleen. However, the
differences in the mean weight between the serial group and the third, control,
group even at the peak of 8 weeks is significant only at the 5% level as determined
by Students "t" test.
However, when one follows the changes in spleen weights of subsequent hosts
in each of the individual lines in the serial group, a clearer picture emerges. Of
378 MUN, TARDENT, ERRICO, EBERT, DsLANNEY AND ARGYRIS
the 30 initial donors only 14 lines were successfully transferred for 8 to 12 weeks.
Figure 6 shows fragments of 9 of these 14 surviving lines. The lag period of
6 to 8 weeks is not shown. In 4 of these 14 lines, after the lag period, there is a
definite progressive increase in the weights of the hosts' spleens. Five other lines
show this increase to a lesser extent and 4 lines do not show any change in
weight of the hosts' spleens.
The individual weights of many of the host spleens at the peak of the growth
phase deviate greatly from the distribution of the control (group 3). The 3 s
level (97%) for group 3 is shown (22.3 ing.).
The data show that in 4 out of 14 lines the effects are cumulative, i.e., an in-
crease in spleen weight occurs with each successive transfer. We may interpret
these observations as indicating that in these 4 lines, cells of donor origin are
transferred from the graft to the host's spleen. It would appear that these
embryonic spleen cells proliferate and are maintained in the host embryonic
environment. After several transfers, following the pattern of development of
the normal chick spleen, the cells mature immunologically. The splenomegaly
thus induced is serially propagated.
One may ask next, are the cells which produce this effect truly derived from
the first, or do they stem from the second, or any of the subsequent transferred
spleens? Is a single initial exposure to antigen sufficient to produce splenomegaly
in subsequent hosts of a series? At least a tentative answer to this question
is obtained by the use of embryos from two inbred lines of chicks.
Mun, Kosin and Sato (1959), using two inbred lines of chickens, found that
a greater splenomegaly was obtained when the donor tissue was derived from an
adult chicken of the opposite line than from the same line. Cock and Simonsen
(1958) made similar observations using injection techniques. It should be
possible to determine if a single exposure is sufficient by the following experiment,
illustrated diagrammatically. One need only compare the effectiveness of the
two series :
(1) A->B-»B->B->B-»B->B and (2) B^B->B-*B-*B-»B^B,
where A is the donor spleen from one line and B is the other line.
The effects of spleens from two inbred lines were first compared within and
between lines. Spleen tissue from one-month-old line 7 and line 15 chickens
were implanted, reciprocally and within lines to the membranes of 10-day-old
embryos. As shown in Table V there is a striking difference in the reaction of
these two lines to line 7 donor spleen but not to line 15 donor spleen. However,
the line 7 embryo spleen was affected somewhat more greatly by line 15 donor
spleen than by line 7 donor spleen. These differences are significant on the basis
of the pooled t test.
The serial experiment as outlined above was then carried out on embryos from
these two inbred lines. Four groups of embryos were treated as follows :
(1) A 17-day line 7 embryo spleen was placed on the membrane of a 10-day line
15 embryo. After 7 additional days of incubation, the host's spleen was removed,
weighed to the nearest 0.2 mg. and cut in half. One half of the host's spleen was
transferred to the membrane of another line 15 embryo. After 7 days the host's
spleen was again removed, weighed, and treated in similar fashion. The average
weight of the line 15 hosts' spleens forms the first group (I) in Table VI.
ANALYSIS OF HOMOLOGOUS SPLENOMEGALY
379
TABLE V
Comparison of the ability of spleens from two inbred lines of chickens
to affect spleens of embryos from these two lines
Line of adult donor
Line of host embryo
No.
Average weight of
host's spleen (mg.)
S.E. of mean
7 (4 donors)
7
28
13.0
0.5
15
17
75.5
8.4
15 (3 donors)
7
22
29.3
1.8
15
17
25.2
2.8
(2) The other half of the line 15 embryo spleen was placed on the membrane of
a line 7 embryo. After 7 additional days of incubation, the host's spleen was re-
moved and weighed, but not transferred. The average weight of the line 7
hosts' spleens form the second group (II).
(3) In the third group, a 17-day line 15 embryo spleen was grafted to a 10-day
embryo from the same line. After 7 additional days of incubation, the host's spleen
was removed, weighed, and cut in half, one half being transferred to a new host of
the same line. The average weight of the line 15 hosts' spleens forms the third
(III) group.
(4) The other half of the line 15 spleen was grafted to a line 7 embryo. After
7 additional days of incubation, the spleen was removed and weighed but not trans-
ferred. The average weight of the line 7 hosts' spleens forms the fourth (IV)
group.
(5) As further controls, untreated 17-day-old lines 7 and 15 embryo spleens
were grafted on line 7 embryos each week. The average weight of the donors' and
hosts' spleens formed a fifth (V) group. The data for this group are not included
in the table.
As shown in Table VI a significant increase in the weights of the hosts' spleens
was not observed in any group after 8 transfers. These results suggest that a
TABLE VI
Mean weight of spleens of four groups of inbred embryos serially
transferred at weekly intervals
Week
number
Group I
Group II
Group III
Group IV
1
10.2 (18)*
10.1 (18)
2
11.4 (11)
9.3 (10)
13.9 (13)
10.1 (12)
3
13.3 (15)
10.2 (5)
13.8 (17)
10.7 (9)
4
13.4 (14)
10.5 (10)
13.2 (12)
10.4 (12)
5
13.5 (16)
9.9 (11)
12.4 (15)
10.0 (11)
6
13.8 (14)
11.0 (12)
13.6 (10)
10.0 (8)
7
13.4 (15)
9.4 (12)
14.5 (13)
10.5 (7)
8
14.4 (18)
7.8 (12)
13.5 (17)
9.3 (8)
9
14.9 (15)
8.2 (11)
13.2 (12)
10.8 (ID
Figures in parentheses indicate number of cases.
380
MUN, TARDENT, ERRICO, EBERT, DnLANNEY AND ARGYRIS
single exposure (A — » B) was not sufficient to initiate the reaction. In view of the
observation that a subsequent increase in weight of the hosts' spleens was obtained
in serial transfers of spleens from non-inbred embryos, it must be suggested that
the latter effect is cumulative. Homologous cells of different genetic makeup are
accumulated gradually in the spleen with each transfer, resulting eventually in the
observed reaction.
However, in view of the fact that the homologous hosts and donors involved
were all embryonic, why was a mutual immunological tolerance not developed ? We
were led to inquire then whether "tolerance," as measured by the prevention of
splenomegaly, could be induced by the exposure of 10- to 17-day-old chick embryos
to grafts of embryonic spleen ?
EFFECT OF SPLEENS FROM ADULT NEW HAMPSHIRE RED CHICKENS WHICH HAD
RECEIVED CHORIOALLANTOIC GRAFTS OF WHITE LEGHORN EMBRYO
SPLEEN ON THE NINTH DAY OF INCUBATION
Terasaki, Cannon and Longmire (1958) injected 0.4 ml. of blood intravenously
from 10- to 16-day-old white Leghorn (WL) embryos to New Hampshire red (NH)
embryos and vice versa. Two or 15 days after hatching, skin from chicks other
than the blood donor, but of the same breed as the blood donor, was grafted. A
significant percentage of these homografts survived longer than grafts between con-
trol chicks not previously injected with blood. This observation was extended to
include interbreed differences by Kulangara, Cannon and Longmire (1959). Toler-
ance of skin homografts may be obtained by embryonic injection of blood from a
breed of chicken other than that of the skin donor. The following series of ex-
periments was designed to answer the question, can embryos of one breed (NH)
be made "tolerant" with respect to the ability to affect the spleen of another breed
(WL)?
Embryo spleens pooled from five 19-day-old white Leghorn embryos were
minced and pipetted on the membranes of 10-day New Hampshire red hosts. The
operated eggs were permitted to hatch. On the second, third, and tenth week post-
hatching, spleens from treated and untreated chickens were implanted on the
membranes of 10-dav-old WL hosts. The results are shown in Table VII.
TABLE VII
Effect of spleens from 2-, 3-, and 10-week-ofd New Hampshire red (NH) chickens grafted
with -white Leghorn embryo spleens (WL-ES) on the 10th day of incubation
Donor
No.
Mean weight of
host's spleen (mg.)
SEm
2-week-old NH + WL-ES
21
16.1
1.5
NH not treated
24
16.4
3.9
3-week-old NH + WL-ES
24
14.8
1.2
NH not treated
12
16.4
1.9
10- week-old NH + WL-ES
31
32.2
3.7
NH not treated
28
28.2
3.5
ANALYSIS OF HOMOLOGOUS SPLENOMEGALY 381
The effect on the host spleen of 2- to 3-week-old chicken spleen is not large
(see Figure 5) but there does not appear to be any difference between the groups.
Spleens from 10- week-old chickens produce a four-fold enlargement. Again, there
does not appear to be any difference in the ability of the spleens from treated and
untreated chickens to elicit splenomegaly. Under the conditions employed, there-
fore, tolerance is not induced. Possibly the relative ineffectiveness of the mem-
brane implantation technique, in contrast to intravenous injection, is to be stressed.
In any event, there are insufficient grounds here for questioning the idea of interbreed
tolerance in chickens.
REDUCTION IN EFFECTIVENESS OF ADULT SPLEEN
FOLLOWING PRE-IMMUNIZATION
The availability of inbred lines of chickens made it possible to test further the
possibility of pre-immunizing adult chickens and producing unusually rapid and
severe graft-versus-host reactions. Earlier attempts with non-inbred fowls (Mun,
Kosin and Sato, 1959; Van Alten, 1961) had produced anomalous results. The
specific question to be answered is the following: will spleens from adult chickens
of one line which have rejected skin grafts from another line produce a greater effect
in hosts of the donor line than spleens from animals which had not previously
rejected such skin grafts?
Skin grafts were performed on 10-day-old hatched chicks from inbred lines
(7 and 15), both between and within these two lines. After one month, a great
majority of the skin grafts received from the opposite line (homografts) began to
disintegrate and slough, leaving large open wounds at the site of the graft. Three
chickens (two from line 7 and one from line 15) showing the graft rejection reaction
were sacrificed and fragments of their spleens were implanted on the membranes of
10-day-old line 7 and line 15 embryos. As controls, chickens with intact skin
grafts from chicks from the same line (isografts) , as well as autografts, and untreated
chickens from each line were sacrificed at the same time. The results of these
chorioallantoic grafts are shown in Table VIII. Spleen implants from chicks
showing the graft rejection reaction did not elicit a greater enlargement in the re-
ciprocal line host than spleen implants from the control chicks. In fact, the average
weight of the host spleen was somewhat less than that of the control group in both
lines.
These observations are similar to those reported by Mun, Kosin and Sato
(1959) and Van Alten (1961). In the former experiments, adult chickens were
injected intravenously and intraperitoneally with pooled 15- to 19-day-old chick-
embryo spleens. The spleens from these injected chickens did not produce a
greater increase in the size of the host embryo spleen. Instead, the effect of
spleen from the injected chickens was consistently and significantly less than
that of spleens from non-injected chickens. Terasaki (1959) made similar ob-
servations. Donor chicks were immunized by skin grafting or by injection of
spleen cells intravenously and intraperitoneally. Neither lymphocytes nor spleen
cells from these immunized chickens when injected into embryos isologous with
the immunizing tissue produced marked splenic enlargement or earlier deaths.
Simonsen and Jensen (1959) observed a marked graft-versus-host reaction
(higher spleen indices) in the hybrid mouse (C,H X AKFOF, host when the
382
MUN, TARDENT, ERRICO, EBERT, DELANNEY AND ARGYRIS
TABLE VIII
Effect of spleens from inbred WL adult chickens which had rejected
skin grafts from the opposite line
Line 7 host
Line 15 host
T j J-\p QJ"
T rest men t of donor ind
donor
condition of graft
No.
Mean weight of
host's spleen
SE,,,
No.
Mean weight of
host's spleen
SEm
7
(B3) rejected skin graft
5
12.2
13.3
9
77.1
13.2
from line 15
7
(B8) rejected skin graft
4
19.5
3.9
9
79.0
18.2
from line 1 5
7
(B4) autograph
3
14.5
1.6
8
133.2
24.5
surviving
7
(B19) not operated
5
11.4
1.2
4
55.2
16.6
15
(Y9) rejected skin graft
12
19.2
2.2
3
47.3
—
from line 7
15
(Y2) skin graft from
10
36.1
6.9
2
13.1
—
line 7
15
(Y24) not treated
9
27.0
4.8
3
17.3
—
donor (AKR) was previously immunized with the hybrid cells. The failure to
obtain similar results in the chicken may be due to an insufficient amount of
homogeneity in the two lines used. In preliminary studies, 50% of skin grafts
performed at 10 days post-hatching persisted for at least 5 months in line 7 hosts
and for at least one to two months in line 15 hosts. Studies of the effects of
spleens from chickens which have rejected a number of skin grafts from several
different donors from the opposite line are in progress.
DISCUSSION
The extensive investigations of many laboratories, including our own, have
led to the conclusion that the embryonic splenomegaly induced by grafts or
injections of homologous spleen cells involves at least two major steps : donor
cells actively pervade the host's reticulo-endothelial tissues, there to proliferate
and mount an immunologic reaction against the host. The pattern of this attack
in the host's chorioallantoic membrane and spleen has been followed by DeLannev
and Ebert (1959a, 1959b ; see also DeLanney, Ebert, Coffman and Mun, 1962).
In spleens of hosts receiving grafts of adult chicken spleen, a pronounced shift
toward granulopoiesis is observed by the eleventh day, followed by accumulation
of mucopolysaccharide, breakdown of the vascular bed, and necrotic and
nbrotic foci.
Biggs and Payne (1961) observed similar pathologic changes in the host
spleen following inoculation of chick embryos with competent adult cells : ex-
tensive proliferation of reticulum cell foci, and the formation of blast cells and
granulocytes. This phase is followed by a lymphoid transformation of the
reticulum cell foci.
These observations and others suggest that although some of the cells initially
transferred from the donor graft to the host spleen proliferate, cells of the host
ANALYSIS OF HOMOLOGOUS SPLENOMEGALY
proliferate also. Biggs and Payne observed that mitotic figures of both donor
and host origin were present approximately in the proportion of 1:1 in spleens
enlarged five- to twenty-fold. From cytological studies of changes in the host's
spleen following inoculation of adult chicken blood, they argue that the reticulum
cell foci, together with some of the blast cells, are of donor origin and that the
majority of blast cells and developing granulocytes are of host origin, a conclusion
in good agreement with the observations of the authors (Ebert, 1959; DeLanney,
Ebert, Coffman and Mun, 1962). Although his earlier writings emphasized the
proliferation of donor cells, Burnet (Burnet and Burnet, 1961 ; Warner and Burnet.
1961) now agrees that much of the proliferation is of cells of the host.
Most of the observations presented in the foregoing pages bear directly on
the first phase of the reaction, lending support to the general argument advanced
for its immunologic nature. For these, further discussion would be redundant.
However, a few of the findings depart sufficiently from the expected to open new
questions for discussion.
Earlier, one of us (Ebert, 1961b) had argued that the serial transfer experi-
ments, using embryos of non-inbred lines, supported the idea that not only was
the embryonic environment capable of supporting immune reactions, but also
that a line of cells derived from the very first graft generation matured im-
munologically in that environment. Although the experiments reported herein
with inbred lines do not require a major change in that view, it is necessary to
state that it is not possible to argue for the derivation of the effective cells from
the initial graft. What appears to be necessary is the accumulation of a threshold
number of homologous cells ; under the grafting conditions employed, one transfer
is insufficient (cf. Howard and Michie, 1962).
Although we recognize that tolerance can be induced in adult animals (Rubin,
1959; Shapiro, Martinez, Smith and Good, 1961), it seems unlikely that the
anomalous reduction in effectiveness of spleen taken from pre-immunized animals
could be a degree of unresponsiveness as a consequence of competent cells in-
troduced into an excess of antigen. Possibly, as an alternative explanation, the
concept of allergic death (Boyse, 1959; Gorer and Boyse, 1959) may be ad-
vanced. Pre-immunized cells, exposed to antigen, if not immediately after
implantation, at least upon invading the host tissues, undergo hyperactivityr
resulting in their death. A test of this idea would be the examination of spleens-
of host embryos at time intervals after grafting shorter than the usual 5 days ; if
this argument were correct, one would expect a burst of donor cell proliferation,,
with early death of these lines.
Finally, we may comment briefly on the nature of the host's reaction. It i>
necessary to revive one of the several explanations which Billingham (1959)
described as "ingenious" (p. 951). We do not believe that the development of
the graft-versus-host concept has provided the "final solution" to the problem of
homologous splenomegaly. The emphasis on donor cell proliferation (Billingham.
1959; Burnet and Burnet, 1960; Simonsen, 1957) resulted in a lack of interest
in the host's response (Ebert, 1951, et seq., reviewed 1959b; DeLanney, Ebert,
Coffman and Mun, 1962). Earlier we advanced preliminary findings which sug-
gested an incomplete immune reaction on the part of the host (Ebert and
DeLanney, 1960; DeLanney, Ebert, Coffman and Mun, 1962). Although we
384 MUN, TARDENT, ERRICO, EBERT, DsLAXNEY AND ARGYRIS
have no reason to doubt that argument, it has become increasingly clear that it is
not a sufficient explanation.
Undoubtedly, the sum total of evidence requires that the first step of the
reaction be an immune graft-versus-host reaction. This results in the initiation of
the second step, an intense proliferation of host cells due to the release of growth-
promoting substances from the immunologically damaged host cells. That damage,
irrespective of the mechanism by which it is produced, can lead to growth promo-
tion is now a well-established fact (Abercrombie, 1957; Argyris and Argyris. 1959,
1962; Bullough and Laurence, 1960).
Concomitantly with host cell hyperplasia the donor cells also continue to
proliferate due to the host antigenic stimulus. With increase in the number of
donor cells, a more intense immune attack on the host occurs, leading in turn
to further damage, and to further proliferation of host cells. Tt is apparent that
these two interactions will result in massive growth of the spleen. The relative
contribution of host and donor cells to splenomegaly will vary, and we would
expect a greater contribution from host cells since they are present in much larger
numbers. Thus the wide variations in the relative contributions of host and donor
cells to splenomegaly experimentally observed become understandable, and, in
fact, expected.
This hypothesis helps us to understand another feature of splenomegaly which
so far has remained unexplained, that of fibrosis and its associated metachromasia
(Ebert and DeLanney, 1960). Connective tissue proliferation is to be expected
after damage of an organ, along with parenchymal proliferation, since connective
tissue is stimulated by damage just as parenchymal tissue is (Abercrombie, 1957).
In addition, such connective tissue proliferation is usually associated with increases
in mucopolysaccharides which are responsible for the intense metachromasia
(Washburn, 1960). We do not know if the stimulation of connective tissue
proliferation is due to relatively nonspecific growth-promoting substances released
by damage (Abercrombie, 1957; Swann, 1958), or whether the graft directs a
specific antibody attack on the connective tissue cells which in turn release tissue-
specific growth-promoting substances. That growth-promoting substances re-
leased by damage might be tissue-specific is suggested by the recent work of
Argyris and Argyris (1962), and Bullough and Laurence (1960).
The actual mechanism of growth promotion leading to splenomegaly is un-
known, but it is related clearly to the mechanism advanced by Ebert (1951, 1954).
which was in turn related to Weiss's template-antitemplate theory of growth
regulation. According to this view (reviewed, Weiss. 1960), the introduction of
disintegrating cells should release specific templates which would "combine with,
•or otherwise trap, homologous antitemplates, their presence in the pool will
entail a temporary lowering of antitemplate concentration, hence again a spurt
of growth in the homologous cell strains of the host" (p. 65). Or templates might
be incorporated directly into homologous cells, accelerating the growth rate.
Partial necrosis of an organ (which is precisely what is observed as a con-
sequence of the graft-versus-host reaction) will have the same effect as partial
removal, i.e., compensatory growth. Hence the stimulating effects of tissue-
specific ribonucleoprotein fractions (Ebert and DeLanney, 1960; DeLanney.
Ebert, Coffman and Mun, 1962) and other lines of evidence (reviewed, Ebert and
Wilt. I960) must be re-examined in this light.
ANALYSIS OF HOMOLOGOUS SPLENOMEGALY 385
The authors are pleased to thank William Duncan, Thomas Garnett, Edward
R. Johnson, Virginia LaFleur, and Barbara Trimmier who assisted in these
experiments, and Dr. B. Winton, Director, Regional Poultry Research Lab-
oratory, East Lansing, Michigan, who provided valuable inbred chickens and eggs.
SUMMARY
1. As demonstrated by their capacity to induce splenomegaly and by tritium-
thymidine labeling, some of the cells of chorioallantoic grafts of adult chicken spleen
colonized the chorioallantois, spleen, and other organs of the host embryo within
two days.
2. The capacity of the embryonic environment not only to support immune
reactions but also to permit maturation of mechanisms of immune response was
demonstrated by the serial propagation of embryonic spleen cells in non-inbred
embryos. A cumulative response w7as obtained, beginning with the fifth or sixth
transfer, approximately paralleling the normal development in the chicken of the
ability to elicit splenomegaly.
3. However, stimulation of the host spleen was not obtained by the serial
propagation of embryonic spleen cells in inbred embryos nor in a series in which
the single initial donor was derived from a different inbred line. This suggested
that the accumulation of a threshold number of reactive cells is necessary for the
stimulation.
4. Induction of mutual interbreed "tolerance," as indicated by reduced effective-
ness of adult chicken spleen to induce splenomegaly, was not obtained by previous
chorioallantoic grafts of embryonic spleen.
5. The pre-immunization of adult chickens of one inbred line by skin homografts
from a second line did not render the former's spleen capable of an enhanced
reaction but, instead, reduced its effectiveness to elicit host spleen enlargement.
It was suggested that such hyperimmunized cells undergo allergic death.
6. Attention is redirected to the proliferation of cells of the host following an
initial graft-versus-host reaction. It is again suggested that this granulocytic
response is a tissue-specific growth reaction resulting from the liberation of cell
products in necrotic foci created in the initial immune reaction.
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INFLUENCE OF HOSTS ON THE BEHAVIOR OF THE COMMENSAL
CRAB PINNOTHERES MACULATUS SAY1
A. N. SASTRY AND R. WINSTON MENZEL
Occanoyraphic Institute, Florida State University, Tallahassee, Florida
Analysis of factors binding the complex host-commensal relationship is a prelude
for an understanding of the community integration and interspecific interactions of
marine animals. Extensive accounts of host-commensal partnership have been given
by Caullery (1952), Davenport (1955) and Dales (1957). Davenport and his
collaborators (1950, 1951, 1953a, 1953b, 1957, 1958 and 1960) showed that a
variety of commensals were attracted to their hosts by some diffusible substance
released by the host animals. Lucas (1947) pointed out that external metabolites,
"ectocrines," play a significant role in establishing the commensal symbiotic
relationships.
The commensal crabs, Pinnotheres inaciilatiis Say, are present in bay scallops,
Aequipecten irradians conccntrlcus Say, and penshells, Atrina rigida Solander, in
Alligator Harbor, Franklin County, Florida. Johnson (1952) demonstrated
chemotaxis in pinnotherid crabs with the Dissodactylus-Mellita partnership. In
view of the frequent occurrence of the crabs in bay scallops and penshells it was
decided to examine the host-commensal partnership and to determine to what extent
the host affect the crabs.
The authors thank Dr. S. K. Katti for suggesting the statistical method used in
the study.
MATERIALS AND METHODS
Bay scallops and penshells were collected in Alligator Harbor, Franklin County,
Florida, near the marine laboratory of the Oceanographic Institute, Florida State
University. The host animals were maintained in the Oceanographic Institute
laboratory in running sea water tables and examined for the presence of crabs im-
mediately after being brought in. The crabs were removed from the hosts and kept
in separate running sea water aquaria. In isolation, the crabs were as healthy and
active after a period of time as those just removed from the hosts.
To study the attraction of crabs to the hosts, a circular choice apparatus was
constructed with plastic material. The principle of circular choice apparatus is
well described, with figures indicating the direction of water currents, by Bartel and
Davenport (1956). The only difference in the circular choice apparatus used in
the present study is that it is larger in size to suit the experimental animals under
investigation.
Water circulation in the apparatus was maintained at a steady rate, determined
by preliminary flow tests to eliminate the influence of turbulence on the behavior of
1 Contribution No. 181, Oceanographic Institute, Florida State University.
388
INFLUENCE OF HOSTS ON COMMENSAL CRAB 389
TABLF. I
Infestation of bay scallops by Pinnotheres maculatus in different mouths of a year
Xo. scallops infested with
Montli
Scallops examined
% total infestation
Males
Females
October, 1957
32
1
7
25.0
June, 1958
15
1
2
20.0
July
142
16
51
47.1
August
98
11
29
40.8
September
7
0
2
28.6
October
18
4
3
38.8
November
13
1
2
23.1
crabs. The water in the apparatus was allowed to circulate for some time before
the experimental animals were introduced. A host was introduced first into one
of the radial chambers and the system was allowed to come to equilibrium. Then
the crabs were introduced into the central chamber. (Hosts were introduced before
the crabs because it was found that if both host and commensals were placed at the
same time, the crabs began random choice before the responsible factor from the host
had sufficient time to become equilibrated in the water circulation of the choice ap-
paratus.) The experiments were conducted in subdued light since the crabs were
found to be negatively phototactic ; under brightly lighted conditions they remained
motionless at the margins of the central chamber.
It was found that 12 hours was sufficient for crabs to make their choice and the
number of crabs in each radial chamber was counted after this period. Before and
after each experiment the apparatus was thoroughly cleaned with sea water. The
behavior of the crabs while seeking hosts was noted. The temperature ranged
between 28 and 31° C. during the experimental period. The results were analysed
by x2 formula (Nass, 1959) to test the significance of distribution among the radial
chambers.
TABU: II
Comparison of the average sizes of scallops infested and not infested with crabs
Infested
Not infested
Difference in size
Date of collection
between infested and
uninfested scallops.
No. scallops
Average size, mm.
No. scallops
Average size, mm.
mm.
6/27/58
3
37.4
12
40.1
+2.7
7/7/58
5
44.9
18
42.9
-2.0
7/23/58
27
43.9
38
47.5
+3.6
7/30/58
26
49.8
20
52.0
+2.2
8/5/58
17
50.2
15
51.3
+ 1.1
8/13/58
5
56.2
20
54.7
-1.5
8/23/58
13
54.8
28
57.1
+2.3
9/14/58
2
62.3
5
58.3
-4.0
10/5/58
7
57.9
12
61.8
+3.9
11/14/58
3
66.1
9
64.2
-1.9
300
A. N. SASTRY AND R. WINSTON MENZEL
OBSERVATIONS
The commensal crabs live between the gill folds in the mantle chamber of
scallops and penshells. Rathbun (1917) described the females of this species as
commensals, whereas the young stages of males are free-living. The occurrence of
male and female crabs in 1957-1958 collections of scallops is shown in Table I.
Crabs of both sexes were most abundant during the summer months, and gravid
females were found in the same period.
The feeding behavior of the crabs was similar in general to that described for
other species of Pinnotheridae (Coupin, 1894; Orton, 1921; Stauber, 1954).
Stauber (1945) observed that Pinnotheres ostreum caused gill erosions and thick-
ening of the oyster host gills. Parts of the gills of scallops were broken off by the
movements of the crabs within the mantle chamber. The average sizes of scallops
infested and not infested with crabs are shown in Table II.
EXPERIMENTAL RESULTS
1. Distribution of crabs in choice apparatus in the absence of hosts
The crabs were placed in the central chamber of the choice apparatus without
hosts in the radial chambers and the distribution at the end of the experimental
TABLE III
Distribution of commensal crabs, Pinnotheres maculatus, in the radial chambers
of the choice apparatus in the absence of host influence
Crabs
Distribution in radial chambers
Expt.
\-
Critical *2
value for
Tested
Made
choice
1
2
3
4
5
6
5%
1
13
9
4
0
1
1
f)
-^
9.37
11.98
2
15
13
0
2
2
2
1
6
9.83
11.67
3
14
10
1
5
1
0
1
2
9.5.S
11.98
4
16
16
3
2
5
2
1
3
3.33
11.52
period was noted. The crabs were sluggish and a few remained in the central
chamber at the end of the experiments without making any choice. The x2 analysis
of results (Table III) indicated that the crabs showed no preference for any of the
chambers.
2. Attraction of crabs to host scallops
When a scallop was present in one of the radial chambers, the crabs were very
active and moved freely in the central chamber. At the end of the experiments, the
crabs were not homogeneously distributed in the radial chambers (Table IV). The
crabs showed a statistically significant (P > .05) preference for the chamber con-
taining the host, although in one of the seven tests the distribution was random.
The crabs required considerably less time to make their choice than they did in
the control experiments. They gathered around the host chamber one after an-
other or in groups. Crabs moving towards the non-host chambers sometimes
INFLUENCE OF HOSTS ON COMMENSAL CRAB
391
TABLE IV
Attraction of commensal crabs to bay scallops
Crabs
Distribution in radial chambers
Host vs. non-host
Expt.
no.
Tested
Made
choice
1
2
3
4
5
6
X2
Critical \-
value for 5 ' ',
1
8
8
(5)
1
0
0
1
1
11.25
3.57
2
12
12
0
(9)
3
0
0
0
26.98
3.64
3
9
q
1
(7)
1
0
0
0
22.05
3.57
4
11
11
2
0
(3)
0
3
3
0.82
3.64
5
10
10
0
1
0
0
0
(9)
41.61
3.61
6
20
19
2
1
0
(11)
3
2
21.52
3.57
>-!
1
16
16
1
1
2
1
(8)
3
12.29
3.68
Parentheses indicate chamber containing bay scallop.
reversed their direction of movement and moved directly into the host chamber.
When the crabs moved to an opening leading to a non-host chamber, they re-
mained at the opening for a long time before they made their choice, sometimes
moving away from the openings. After entering the host chamber, the crabs
gathered around and under the scallops. Some climbed on the upper valve of the
scallop and made attempts to enter the host mantle chamber. While some of the
crabs gained immediate entry into the host, others were caught between the valves
of the host when it contracted, gaining entry when the scallops later opened their
valves.
3. Response when the host water is siphoned into one of the radial chambers
These experiments were designed to find out if the crabs would respond to water
coming from an aquarium containing a host scallop. Water from an aquarium con-
taining a host was siphoned into one of the radial chambers of the choice apparatus.
The distance of the host from the crabs in the central chamber was approximately
six times greater than in the previous experiments in which the host was placed in
a radial chamber. The rest of the procedure was the same as described in the
general methods.
Results shown in Table V indicate that the attraction is reduced as compared to
TABLE V
Choice of crabs when the host water is siphoned into one of the radial chambers
Crabs
Distribution in radial chambers
Chamber with host
water vs. non-host
Expt.
no.
Tested
Made
choice
1
2
3
4
5
6
x-
Critical x2^
value for 5','
1
16
14
3
2
(5)
1
1
2
3.47
3.64
2
14
14
(-0
2
2
1
4
1
1.36
3.64
Parentheses indicate chamber into which host water is siphoned.
392
A. N. SASTRY AND R. WINSTON MENZEL
TAUI.I-: \ 1
Response of crabs to scallop shells with attached animals
Chamber with host
Crabs
Distribution in radial chambers
shell vs. those without
host shell
Expt.
Tested
Made
choice
1
2
3
4
5
6
X-
Critical x~
value for 5%
1
14
14
(5)
2
2
3
1
1
3.50
3.68
2
15
12
5
2
0
1
(4)
0
2.28
3.64
3
12
10
0
1
(7)
2
0
0
19.24
3.61
4
15 10
3
0
3
(3)
0
1
0.12
3.61
Parentheses indicate chamber containing the scallop shell.
that of the chambers containing live hosts. The Chi square test showed that crabs
were distributed homogeneously in all six radial chambers ; the distribution of crabs
in the chambers was random.
4. Response of crabs to host shell with attached animals
A variety of sessile animals attach to the outside of the shell of scallops. Scallop
shells with attached animals were washed with sea water after the soft parts of the
scallops were removed and were tested to find whether the crabs would be attracted
to them. The results (Table VI) show that there is no significant attraction of
crabs to the shell with attached organisms, except in one of the experiments there is
a significant attraction of crabs. This could have resulted from insufficient washing
of the shell after removing the soft parts.
5. Response of male commensal crabs to host scallops
Adult males are commensals in their relation with the host, whereas the earlv
male stages are free-living. Experiments were planned to investigate whether the
males, removed from the host scallops, respond to the host in the same manner as
the females. The results are summarized in Table VII. The males were very
active in the presence of the host and occasionally swam within the central chamber
of the choice apparatus.
TABLE VII
Response of male commensal crabs to bay scallops
:\pt.
Crabs
Distribution in radial chambers
Host vs. non-host
chambers
no.
Tested
Made
choice
1
2
3
4
5
6
x-
Critical x"
value for 5",
1
21
21
3
0
0
0
2
(16)
52.18
3.72
2
17
14
3
(8)
0
2
0
1
15.55
3.64
3
13
13
1
1
2
(9)
0
0
24.41
3.64
4
13
13
2
2
0
1
(8)
0
17.89
3.64
Parentheses indicate chamber with host.
INFLUENCE OF HOSTS ON COMMENSAL CRAB
393
TAHLK Mil
Attraction to penshells of crabs removed from bay scallops
Crabs
Distribution in radial chambers
Host chamber vs.
non-host chambers
Expt.
no.
Tested
Made
choice
1
2
3
4
5
6
X2
Critical x~
value for 5%
1
16
16
0
6
(10)
n
0
0
23.23
3.68
2
15
15
0
3
0
0
(11)
1
33.29
3.68
3
16
16
1
0
(11)
i
1
2
30.87
3.68
Parentheses indicate chamber containing penshell.
Chi square analysis of the results indicates that the males were attracted to the
scallops. Since no female crabs were present, the attraction of the male crabs
appears to be entirely to the host.
a. Attraction to penshells of crabs removed from scallops
These experiments were performed to determine whether crabs originally re-
moved from scallops would be attracted to a second host, Atrlna rigida. Crabs
living in the scallops and the penshells are morphologically similar. Penshells of
approximately the same weight as scallops used in the earlier experiments were
placed in the radial chamber of the choice apparatus and the distribution of the
crabs at the end of the experiments was noted. The results (Table VIII) indicate
that the crabs were strongly attracted to the penshells.
7. Preference of crabs between the tzvo hosts, scallops and penshells
The two hosts, penshells and scallops, were placed in two non-adjacent radial
chambers of the choice apparatus and the crabs were introduced in the central
chamber. Crabs obtained from scallops showed no statistically significant preference
for penshells than for the scallops (Table IX).
TABU. IX
Response of crabs, removed from bay scallops, to penshells and bay scallops when both
are present in two separate chambers of the. choice apparatus
Crabs
Distribution in radial chambers
Hosts vs. non-host
chambers
Penshells vs. bay
scallops
Expt.
no.
Tested
Made
choice
1
2
3
4
5
6
*
Critical *-
value for 5%
X2
Critical x*
value for 5 "'t
1
15
15
1
1
(8)
0
3*
2
14.49
5.81
2.50
3.42
2
15
15
(6)
2
1
5*
1
0
10.58
5.81
0.09
3.42
Parentheses indicate chamber with penshell; * indicate? chamber with bay scallop.
394 A. N. SASTRY AND R. WINSTON MENZEL
DISCUSSION
The attraction of commensals to their hosts in response to some diffusible sub-
stance or substances released from hosts was demonstrated by Welsh (1930), Thorpe
and Jones (1937) and Davenport (1950, 1953a). The present experiments showed
that the commensal crab, Pinnotheres inaculatus, is capable of recognizing its hosts,
Aequipecten irradians and Atrina rigida, under the described experimental condi-
tions. The active movements of the commensal crabs in the presence of the hosts
seem to be stimulated by some attractant from the host. The attraction of com-
mensals to the host scallops decreased when the hosts were not directly introduced
in the radial chamber of the choice apparatus. This suggests that perhaps a spatial
proximity of hosts to commensals is necessary for demonstration of extraction under
experimental conditions. The decreased attraction could have resulted either from
a gradient or a highly diffusible nature of the attractant.
The absence of attraction of commensals to empty host shells, with attached
epizooites, indicates that the source of the attractant is the soft parts of the scallops.
The experiments with males of P. inaculatus demonstrated conclusively that
their response to scallops is equal to that of the females of the same species. It is
not known from the present study how the free-living early stages of males change
to commensal habit in their adult stage. Experiments with free-living early-stage
males might reveal the nature of this change.
Crabs removed from scallops were attracted readily to Atrina rigida, another
host which inhabits the same general locality as the scallops. The results of ex-
periments indicate that both scallops and penshells release attractants that stimulate
the crab to seek the hosts. The attraction of crabs from scallops to both the hosts
appears to be equal when both are simultaneously tested for response. Crabs living
in the scallops and those in the penshells are morphologically similar, and crabs
from scallops are not physiologically host-specific. Reciprocal experiments with
crabs obtained from penshells should elucidate the specificity of these commensal
crabs.
SUMMARY
1. Experiments using a circular choice apparatus showed a statistically significant
attraction of commensal crabs, Pinnotheres inaculatus, to bay scallops, Acqnipecten
irradians concentricus, and penshells, Atrina rigida.
2. The adult males of P. inaculatus removed from bay scallops showed a
significant attraction to the host.
3. When tested for preference between the two hosts, crabs removed from bay
scallops showed no preference for one host over the other. The attraction of crabs
to both the hosts was statistically significant. Experiments suggested that the crabs
removed from scallops are not host-specific.
LITERATURE CITED
BARTEL, A. H., AND D. DAVENPORT, 1956. A technique for the investigation of chemical responses
in aquatic animals. Brit. J. Anim. Bchar., 4: 117-119.
CAULLERY, M., 1952. Parasitism and Symbiosis. Sidgwick and Jackson, London.
COUPIN, H., 1894. Sur 1'alimentation de deux commensaux (Ncreilcpas et Pinnotheres).
C. R. Acad. Sci. Paris, 119: 540-543.
DALES, R. P., 1957. Interrelation of organisms. A Commensalism. Treatise on marine ecology
and paleoecology. Gcol. Soc. America., Memoir, 67, 1: 391-412.
INFLUENCE OF HOSTS ON COMMENSAL CRAB 395
DAVENPORT, D., 1950. Studies in the physiology of commensalism. I. The polynoid genus
Arctonoe. Bwl. Bull, 98: 81-93.
DAVENPORT, D., 1953a. Studies in the physiology of commensalism. III. The polynoid genera
Acholoe, Gattyana and Lcpidasthcnia. J. Mar. Biol. Assoc., 32: 161-173.
DAVENPORT, D., 1953b. Studies in the physiology of commensalism. IV. The polynoid genera
Polynoc, Lepidasthenia and Harinothoe. J. Mar. Biol. Assoc., 32: 273-288.
DAVENPORT, D., 1955. Specificity and behavior in symbiosis. Quart. Rev. Biol., 30: 29-46.
DAVENPORT, D., AND J. F. HICKOK, 1951. Studies in the physiology of commensalism. II. The
polynoid genera Arctonoe and Halosydna. Biol. Bull., 100: 71-83.
DAVENPORT, D., AND K. S. NORRIS, 1958. Observations on the symbiosis of the sea anemone
Stoichactis and the pomocentrid fish, Aniphiprion pcrcnla. Biol. Bull., 115: 397-410.
DAVENPORT, D., G. CAMOGUIS AND J. F. HICKOK, 1960. Analysis of the behavior of commensals
in host factor. I. A. Hesionid polychaete and a pinnotherid crab. Brit. J. Anim.
Behav. 8: 209-218.
HICKOK, JOHN F., AND D. DAVENPORT, 1957. Further studies in the behavior of commensal
polychaetes. Biol. Bull, 113: 397-406.
JOHNSON, I. S., 1952. The demonstration of host factor in commensal crab. Trans. Kansas.
Acad. Set., 55: 485-464.
LUCAS, C. E., 1947. The ecological effects of external metabolites. Biol. Rev., 22: 270-295.
NASS, C. E., 1959. The x2 test formula for small expectations in contingency tables, with
special reference to accidents and absenteeism. Biometrica, 46 : 365-385.
ORTON, J. H., 1921. The mode of feeding and sex phenomenon in the pea crab (Pinnotheres
pisuvi). Nature, 106: 533-534.
RATHBUN, M. J., 1917. The grapsoid crabs of America. Bull. U. S. Nat. Mits., No. 97, 461 pp.
161 pi.
STAUBER, L. A., 1945. Pinnotheres ostrcitiu, parasitic on the American oyster, Ostrea (Gryphea~)
rirginica. Biol. Bull., 88: 269-291.
THORPE, W. H., AND F. G. W. JONES, 1937. Olfactory conditioning in parasitic insects and
its relation to host selection. Proc. Roy. Soc. London, Ser. B, 124: 56-81.
WELSH, J. H., 1930. Reversal of phototropism in a parasitic water mite. Biol. Bit!!., 59:
165-169.
REPRODUCTION OF THE POLYCHAETE GLYCERA DIBRANCHIATA
AT SOLOMONS, MARYLAND 1
MARGARET SIMPSON
Department of Biology, Catholic University of America, Washington, D. C., and Chesapeake
Biological Laboratory, Natural Resources Institute of the University
of Maryland, Solomons, Maryland
Remarkably little is known about the life history and the ecology of the
errant polychaete family Glyceridae. Basic information of this nature is needed
not only to allow a synthesis of such isolated data as are presently available, but
also to provide the background necessary for experimental investigations. In
view of this, the work of Klawe and Dickie (1957) on Glycera dibranchiata
Ehlers in the Maritime Provinces is of particular interest, since it appears to be
the only publication dealing with the biology of a glycerid worm. Commonly
known as the "bloodworm" or "beak-thrower," this species is a favorite bait of
salt-water sport fishermen, whose demand has made it of some commercial im-
portance in Maine and the Maritime Provinces. The studies of Klawe and
Dickie were undertaken to obtain information relating to questions of bloodworm
conservation, and their report contains many original observations. But the
chief value of their contribution lies, perhaps, not so much in its extensive data
as in its indication of the numerous problems still requiring considerable study.
Of particular importance are the gaps persisting in our knowledge of
glycerid reproduction, many aspects of which have remained largely a matter of
conjecture. It is with these deficiencies in the case of Glycera dibranchiata that
the present study is concerned. Intended to enlarge upon the work of Klawe and
Dickie, this report deals with the breeding season, swarming and epitoky of
G. dibranchiata in more southern waters, and uses histological findings to supple-
ment field observations. Gametogenesis and early development will be considered
in a separate paper.
METHODS
Most of the information presented here is based on work conducted at the
Chesapeake Biological Laboratory at Solomons, Maryland, from the last week of
June, 1960, to the early part of February, 1961. Unless otherwise stated, all
specimens were collected from the waters immediately surrounding Solomons
Island, situated in the mouth of the Patuxent River, about two miles from its
entrance into Chesapeake Bay (Fig. 1). Some of the hydrographic features of
this area have been described by Nash (1947) and Beaven (I960). Since the
1 Based on portion of a dissertation submitted in partial fulfillment of the requirements for
the degree of Doctor of Philosophy at the Catholic University of America, Washington, D. C.
Contribution No. 200, Chesapeake Biological Laboratory, Natural Resources Institute of the
University of Maryland. This investigation was carried out during the tenure of PHS Pre-
doctoral Fellowship BF-9242-C1 from the National Institute of Neurological Diseases and
Blindness, and GF-9242-C2 from the Division of General Medical Sciences.
396
REPRODUCTION OF GLYCERA
397
FIGURE 1. Location of Solomons Island and other areas mentioned in the text.
Broken line indicates the 12-foot bottom contour.
mean tidal amplitude at Solomons is only 1.2 feet, there is practically no inter-
tidal zone ; all collecting, therefore, was done with a Maryland soft clam
dredge (see Manning, 1959), operated in 6-10 feet of water. This method, using
a wire mesh conveyor belt to bring specimens up from the bottom, proved satis-
factory for obtaining 2-4 dozen uninjured worms in a relatively short period
of time.
Living worms narcotized in magnesium chloride were measured by the method
398
MARGARET SIMPSON
of Klawe and Dickie, who used a watertight trough, V-shaped in cross-section,
with a ruler attached to one side. Some measurements were obtained from fixed
material and will be so indicated. Narcotized animals were fixed in Bouin's fluid
and stored in a preservative consisting of 2 parts ethyl alcohol, 1 part distilled
water and 1 part glycerine. For general examination, paraffin sections 4-7 ^
thick were stained in Ehrlich's hematoxylin and eosin.
Other methods used in connection with specific problems will be described in
the appropriate sections.
BREEDING SEASON
Table I summarizes the available information on reproductive periods in several
species of Glycera. For G. dibranchiata, Klawe and Dickie have indicated mid-
May as the probable time of maximum reproduction in Nova Scotia and Maine.
The present study was begun near the end of June, too late to determine the
presence or absence of a spring spawning. Other records, however, suggested
the possibility of a second breeding season for this species at Solomons, and an
autumn spawning did in fact occur.
Previous reports jor Solomons
Three earlier reports of breeding activity for Glycera were found in records
at the Chesapeake Biological Laboratory. One of these is a brief anonymous note
TABLE I
Breeding seasons of Glycera species
Species
Locality
Time of year
Evidence
Source
G. alba
The Sound
Late autumn?
Larvae in winter
Thorson, 1946
G. americana
Woods Hole
Maryland
Solomons
Summer
Spring— Summer
December
Adults at surface
Swarming
Swarming
Pettibone (in press)
Anonymous, 1948
Beaven (see text)
G. capitals
Norway
May-July
Epitokes at surface
St0p-Bowitz, 1941
G. convoluta
Naples
Algiers
Plymouth
May
April
June- August
Spawning
Epitoke at surface
Ripe gametes
Lo Bianco, 1909
Gravier and Dantan, 1928
Fuchs, 1911
G. dibranchiata
Woods Hole
Maine and Canada
Maryland
Solomons
Solomons
Solomons
August; January
April-June
Spring-Summer
July
November
December
Adults at surface
Ripe gametes
Swarming
Swarming
Swarming
Swarming epitokes
Pettibone (in press)
Klawe and Dickie, 1957
Anonymous, 1948
Myers (see text)
Beaven (see text)
This paper
G. lapidiini
Algiers
Concarneau
Norway and Sweden
February
December-. \iiril
August?
Epitoke at surface
Epitokes at surface
Pre-epitokes in July-
Young in September
Gravier and Dantan, 1928
Fage and Legendre, 1927
Stop-Bowitz, 1941
Arwidsson, 1898
G. nana
Britisli Columbia
Autumn
Swarming
Berkeley and Berkeley. 1948
G. robiisln
Monterey Bay
Spring-Summer
Ripe gametes
MacGinitie, 1935
G. rouxii
Concarneau
Banyuls
Norway and Sweden
October
August
October?
Epitoke at surface
Epitoke at surface
Pre-epitokes in Sep-
tember
Fage and Legendre, 1927
Fage and Legendre, 1927
St0p-Bowitz, 1941
G. siphonostoma
Naples
December-April
Mature adults
Lo Bianco, 1909
G. ?sphyrabrancha
Puerto Rico
October
Ripe adults at surface
Allen, 1957
G. lesselala
Algiers
October-November
Epitokes at surface
Gravier and Dantan, 1928
REPRODUCTION OF GLYCERA 399
appearing in a 1948 issue of the Maryland Tidewater News and describing
swarming of these worms "in the tidal waters of Maryland" during late spring
and early summer of each year. It also states (p. 4) that "there are two species
of Glycera found along our coast, both of which carry on the curious antics re-
ported above." Presumably the species referred to would be G. dibranchiata and
G. americana. The other two reports of swarming were found in a card file of
invertebrates occurring near Solomons. One gives the following information :
"Glycera dibranchiata; large numbers swimming off CBL pier, presumably this
species; one specimen identified; July 15 [no year] ; Marvin Myers." The other
entry reports G. dibranchiata swarming in the same area on the nights of December
4-7, 1944; apparently G. americana was also present the first of these nights. In
none of these cases has it been possible to verify the identity of the worms. The
December, 1944, swarms, however, were witnessed by Mr. Francis Beaven, cur-
rently a member of the Laboratory staff, who was able to provide some further
details concerning the event. There seems little reason to question that in this case,
at least, the generic identification was correct. If the benefit of the doubt is extended
to the other two reports, it must be concluded that Glycera has two breeding periods
a year in this locality.
Rate of se. ntal development
Between late June, 1960. and swarming time in early November, 75% of the
worms examined histologically had gonads in various stages of development.
These specimens, 55 in all, represent ten samples taken the following numbers of
days before the first swarm: 129, 90, 77, 70, 58. 45, 36, 16, 8 and 4. Relaxed
length of the worms ranged from 7 to 26 cm., with the majority falling into the
16-20 cm. group. Although these limited samples allow only tentative generali-
zation, two observations should be mentioned. First, there seemed to be no cor-
relation between specimen length and presence of gonads, though the gonads of
shorter worms were generally smaller. Secondly, contrary to expectation, the
frequency of mature specimens in each sample diminished as swarming time drew
closer. This could, perhaps, indicate a migration of mature worms away from the
usual collecting area into shallower water, where swarming appeared to be more
concentrated. But both of these observations need to be checked by statistical
treatment of larger samples.
The earliest definite sign of approaching maturity occurred in mid-September,
45 days before swarming. Two of the specimens fixed then contained sperm plates
and a third contained eggs in the coelom. The appearance and the small number
of these free gametes suggested that their release from the gonads had just recently
begun. This agrees reasonably well with the observations of Klawe and Dickie, who
found immature eggs free in the coelom of some worms in late August. Evidence
of a more advanced degree of maturity appeared about one month later, in a specimen
fixed 16 days before swarming. This was a female with eggs almost completely
filling the body cavity and with no remaining gonad tissue. The atrophy of the gut
and body wall that accompanies sexual maturity had already begun but was not yet
pronounced. The sample collected four days before swarming included worms in
final stages of maturation. Two females and one male shed gametes when handled
in the laboratory. Although the sperm plates released by the male did not break up
400 MARGARET SIMPSON
into individual sperm, some of the gametes must have been ripe, since an attempt
at fertilization gave a small number of cleaving eggs that developed into swimming
blastulae.
If biannual reproduction is assumed, these observations may be tentatively in-
terpreted as follows : Of the worms collected in summer, those with well developed
gonads probably represented the fall breeders of 1960 ; those with poorly developed
gonads, the spring or summer breeders of 1961 ; and those with no gonads, the fall
breeders of 1961. It would then follow that complete sexual development requires
about one year. The observations also indicate that young gametes of both sexes
are released from the gonads into the coelom at approximately the same time and
ripen within the following 6 to 8 weeks.
Length of breeding season
It is difficult at present to suggest the limits of the reproductive seasons at
Solomons. Both the anonymous article and Myers' notation indicate that the
earlier period may center around June and July. The fact that no mature worms
were found at the beginning of the present study (the end of June) is not incompati-
ble with this suggestion. If actual spawning is of short duration, a matter of a few
days to a week, then it could have taken place in its entirety during the early part
of June. Furthermore, it has already been mentioned that mature worms became
scarcer in samples dredged nearer the time of swarming ; thus it is possible that the
number of mature worms had similarly decreased in the collecting area during late
June. Any swarming activity that might have occurred in June or July could
easily have been overlooked. The information available for the autumn breeding
season is more definite, though still insufficient to form any hard and fast conclu-
sions. In both cases, namely December, 1944, and November, 1960, observed
swarming was limited to four successive days. Whether this coincidence is purely
fortuitous, or whether it reflects a high degree of reproductive synchrony cannot
at present be settled. Nor is there any way to determine that these were the only
spawning events during the fall months of 1944 and 1960.
Environmental factors
Temperature conditions of surface water at Solomons for a 20-year period have
been summarized by Beaven (1960). These records show mean temperatures
ranging from 3.3° C. in February to 26.7° C. in August, with extremes of —0.8 and
31° C. The greatest difference between surface and bottom temperature occurs in
the spring, when readings may be about three degrees lower at 17 feet (Nash, 1947),
and eight degrees lower at 60 feet (Beaven, 1960). During the fall months water
is generally slightly warmer at the bottom than at the surface. There appears to
be little correlation between absolute temperature and the suggested breeding
periods, except that temperature is approaching its maximum during June and July,
and its minimum during November and December. In the two-week period before
swarming, water temperature fell gradually from 10.3 to 5.5° C. in 1944, and from
18.7 to 13.8° C. in 1960. The average daily difference was —0.36° C. in both
years, but this is of questionable significance. Otherwise the data suggest only that
final sexual maturity can be attained within a fairly wide temperature range.
Two attempts were made at Solomons to determine whether temperature change
REPRODUCTION OF GLYCERA 401
can affect gonad development in Gl\cera. The first of these took place in August,
when ten worms were kept at a temperature below the average 26° C. of the water
pumped into the laboratory. The arrangement for maintaining a flow of fresh
water at a constant low temperature proved unstable, allowing irregular variations
within a range of 12° to 20° C. After about three weeks seven of the worms had
died, but all three survivors had gonads approximately twice the size of those in
freshly collected worms fixed at the same time. The second attempt to observe
effect of temperature change was made in January, under somewhat better condi-
tions. Six animals were kept in separate containers placed in a cold-water bath
maintained at 14° C. by a thermostatically controlled heating coil. No attempt
was made in this case to provide running water to the test animals ; rather, the
water in the containers was changed daily. A second group of worms, serving as
a rough control, was kept at about 4° C. in an aquarium supplied with running water
from the pump system. After two weeks both groups were fixed. Of the six
worms exposed to the higher temperature, four were in advanced stages of ma-
turity. In these, the coelom contained well developed eggs or sperm plates, there
was little or no remaining gonad tissue, and atrophy of the gut and body wall was
pronounced, in two cases exceeding by far the degree of atrophy found in any of the
swarming specimens examined. None of the animals kept at 4° C. showed free
gametes or gonads markedly larger than those found in freshly collected worms.
Although inconclusive, these results strongly suggest the importance of temperature
in regulating the rate of sexual maturation in Glycera.
Temperature is generally regarded as a critical factor in determining the repro-
ductive period of many marine invertebrates. Indeed, Orton (1920) concludes that
other environmental conditions are of little significance. There is, however, a grow-
ing body of experimental evidence indicating that while temperature changes may
accelerate gametogenesis and induce spawning, these responses depend to some
degree on the physiological condition of the organism (e.g. Galtsoff, 1940; Loosanoff
and Davis, 1952; Turner and Hanks, 1960). Furthermore, Thorson (1946) has
pointed out that seasonal phytoplankton maxima cannot be excluded from the pos-
sible factors regulating reproductive activity in benthic invertebrates. Large plank-
ton blooms in April and May, and smaller ones in September and October, have
been reported for the waters around Solomons (Nash, 1947). These seasonal
fluctuations occur shortly before the suggested breeding periods and hence, by
augmenting the food supply, may well exert an indirect influence on the timing of
reproduction in Glycera.
Breeding season in Canada and Maine
Klawe and Dickie conclude that bloodworms in Xova Scotia and Maine spawn
only once a year, in the spring. But although they state that no observations were
made in winter, it is not clear how long their observations were continued in
autumn. It cannot be assumed that because two allopatric populations belong to
the same species, their reproductive periods will coincide. The spawning of some
nereids, for example, occurs earlier in southern than in northern latitudes, and even
in the same locality, intertidal populations and those below mean sea level may
spawn at different seasons (Herpin, 1926, 1928; Page and Legendre, 1927). In
addition to affecting the time of reproduction, the habitat apparently can in some
402 MARGARET SIMPSON
polychaetes also influence the method of reproduction (Thorson, 1950). Thus it
is entirely possible that bloodworms in Canada and Maine spawn but once, whereas
those further south spawn twice a year.
Nonetheless there is reason to suspect that spawning may be a biannual phe-
nomenon for the more northern members of G. dibranchiata as well. Two points
reported by Klawe and Dickie tend to support this suggestion. First is their ob-
servation of free oocytes in the coelom of worms examined in late August, which
approximates the present findings. Their report implies that these gametes continue
development through the winter and are not shed until the following spring. Al-
though there is evidence that some invertebrates can often store ripe gametes for
long periods before releasing them (Herpin, 1928; Thorson, 1946), it seems equally
possible that in Nova Scotia, as at Solomons, these germ cells complete growth within
a month or so and are spawned in autumn. Secondly, Klawe and Dickie mention
that swarming worms, caught in a herring fisherman's net off Nova Scotia in
October, 1955, were identified as bloodworms by local worm diggers, who com-
municated this information to the authors. But Klawe and Dickie were unable
to verify this report, and since collections made in the same locality the following
September yielded only swarming nereids, conclude that the diggers' identification
must have been erroneous. In view of the observations from Solomons, this con-
clusion should perhaps be reconsidered. It may finally be mentioned that specimens
originating in Maine and obtained from bait stores at Washington, D. C., in late
September and early October have often contained eggs resembling in size and
appearance those spawned by ripe females at Solomons.
Although final resolution of this question will depend upon confirmatory ob-
servations of spring spawning at Solomons and fall spawning in the Maritime
Provinces, the present evidence permits the suggestion that G. dibranchiata along
the Atlantic coast reproduces twice a year. Should this be correct, it will be
necessary to revise the conclusions on growth rate and life span arrived at by
Klawe and Dickie. These authors find that their size-frequency curves show four
distinct modes, which they interpret as successive age groups. Thus the mode
centering around 5 cm. represents yearling worms, that around 16 cm. represents
two-year-olds, and so 'on, to a maximum of about 31 cm. for four-year-olds. The
sudden decline in frequency of worms three years old, in comparison with the
frequency of two-year-olds, is taken as an indication that most bloodworms spawn
and die as they reach their third year. But as Klawe and Dickie point out, these
conclusions are based on the assumption that spawning occurs only once each year.
If it is in fact a biannual affair, then the modes of the size-frequency curves would
represent two year-classes rather than the four proposed by Klawe and Dickie, and
their consequent deductions would have to be modified accordingly.
SWARMING
Several species of Glycera are reported to take up a brief pelagic existence at the
time of spawning (see Table I). Although Klawe and Dickie found no evidence
for such behavior in G. dibranchiata, they suggest that bloodworms may have a short
nocturnal swarming period, as do many other errant polychaetes. In an effort to
check this possibility, night observations using a 150- watt bulb suspended 18-20
inches above the water were conducted from the end of the Laboratory pier at
REPRODUCTION OF GLYCERA 403
Solomons, about 700 feet from shore, over water 8-9 feet deep. This location was
chosen primarily for its convenience, but also because dredging had indicated a good
concentration of bloodworms in the vicinity. More than 40 such observations were
made between June and November. Most of the first 20 fell within the last half of
the lunar cycle in July, August and September ; the others, in October and Novem-
ber, included all four lunar periods. A single night's session lasted two to three
hours, usually between sunset and midnight, although several observations in October
and early November were conducted at times between midnight and dawn. No
bloodworms appeared at the surface during any of these periods, and the observa-
tions were discontinued after the first week of November.
Dates and areas of observed sivanns
Swarming of Glycera dibranchiaia was first noticed during the afternoon of 5
November 1960 by Mr. Hayes T. Pfitzenmeyer, a member of the Laboratory staff,
who brought it to the author's attention. It was witnessed again on the two fol-
lowing afternoons, and although no personal observations were made on November
8, reports of other staff members indicated that swarming occurred on that day also.
On November 5, swarming took place over the Middleground (Fig. 1), a shoal
area approximately 500 yards east of the pier, in water 1-3 feet deep. The extent
of the swarm could best be gauged by the activity of gulls, large numbers of which
congregated over this area and the north shore, diving toward the water and rising
with worms dangling from their beaks. This swarm lasted from about 3 :00 to
5 :30 P.M. and was investigated from a small boat. One of the Laboratory staff
members later mentioned seeing worms on the surface at approximately 4:30 this
same day while trolling in 6-10 feet of water in the vicinity of Hellen Creek; he
also remarked that the stomachs of striped bass caught by him were filled with
worms. Swarming on November 6 began at 4:10 P.M., in shallow water around
the pier, and was again investigated by boat. Concentrations of gulls were also
noticed along the north shore and to a lesser extent over the Middleground. On
November 7 swarming was indicated by gulls working in the same areas as the
preceding day ; the boat was not used this day, and observations were limited to
activity around the pier.
At the time of swarming on these four days, water temperature ranged from
12.2 to 13.8° C., and the average salinity was 14.5-14.S/£c. Weather conditions
were generally agreeable, except for the first day, which was overcast and rainy.
Composition and density of szvarins
Ten specimens, 14 to 20 cm. long (fixed), were collected during the swarms
of November 5 and 6. Only one of these was a female, but the actual sex ratio
is probably less disparate. In large samples of mature worms, Klawe and Dickie
found a size range of 13 to 36 cm., with males outnumbering females by only 1.3 to 1.
Gravier and Dantan (1928) report lengths of 5 to 18 mm. for swarming Glycera
tesselata and state that males were very predominant.
Although swarming activity at Solomons extended over considerable areas,
individual worms were remarkably dispersed, occurring approximately 3-5 yards
apart, and technically it may be questioned that the term "swarming" applies in
such a case. Whether or not this dispersion is typical of spawning bloodworms.
404 MARGARET SIMPSON
however, remains to be seen. Apparently, pelagic breeders of other Glycera
species have seldom been encountered in great numbers. Gravier and Dantan
(1928) report collecting 25 and 48 specimens of G. tesselata on two different oc-
casions, but this departs from the usual catch of one or two individuals recorded
by other authors and by Gravier and Dantan for other species. The studies of
Gravier and Dantan at Algiers resemble those of Page and Legendre (1927) at
Concarneau, and since in both cases observations were made shortly after sunset,
these authors suggest that swarming maxima probably occurred later at night. Yet
if swarming individuals of other species were as scattered as G. dibranchiata at
Solomons, then it seems entirely possible that observations from a fixed point,
such as the anchored boats used in the Algiers and Concarneau studies, would yield
very few specimens, even during the height of swarming activity.
An increased density could be expected if swarming glycerids were positively
phototropic and concentrated around the lamps used in nocturnal investigations.
But there is little evidence indicating that Glycera shows a positive response to
light. Although primitive epidermal photoreceptor cells have been described for
some species (Stolte, 1932), members of this genus do not possess eyes, nor is
there any sign that such organs develop at maturity. During some observations
made at Woods Hole late in the summer of 1959, several males of G. americana
were collected from the surface at night. These animals did not seem attracted
to the light, but had apparently been carried in by the current. Experimental data
are needed, however, to establish the nature of photosensitive responses in both
mature and immature specimens of this group.
Swimming and shedding behavior
Swarming worms moved slowly, either at or just below the surface. Their
method of swimming was completely different from that of immature animals,
which advance through the water by executing a series of intricate vertical figures-
of-eight, with the tail always leading the way. In contrast, swarming individuals
swam head first, propelled by lateral undulations of the body. These movements
resemble the type of swimming shown by Nereis and Ncphtys, and it is likely
that as in these genera, locomotory waves originate at the posterior end and pass
forward along the body (Gray, 1939; Clark and Clark, 1960). In all observed
instances the proboscis was retracted. There was no indication of any particular
swimming pattern, such as the circling dances of some nereids.
Many of the worms seen at close range were shedding gametes in a steady white
flow from the posterior end. Of the ten swarmers collected, two lack tail segments,
and a third shows a tear in the body wall near the tail. In the intact individuals,
there is a very small rupture on the dorsal surface just anterior to the pygidium.
Evidently this is the avenue for gametal discharge in the majority of cases. At no
time, either during swarming or in the laboratory, were gametes observed to issue
from a rupture in the proboscis or anterior two-thirds of the body. However.
Klawe and Dickie found that shedding in the laboratory, whether spontaneous or
induced by a weak electric current, occurred with about equal frequency through
the tail, the body wall or the proboscis. Judging from observation of animals
spawning in the laboratory, it seems probable that a single individual releases all
its gametes at one time. In a few cases, swimming activity of animals shedding in
REPRODUCTION OF GLYCERA 405
fingerbowls was interrupted by one or two short periods of quiescence during which
gametal discharge ceased. Apparently, the elimination of gametes does not require
the presence of a worm of opposite sex and may well he a mechanical process re-
sulting from the muscular pressures exerted on the coelomic fluid during swimming.
This in turn would bring about a decrease in turgidity, with correspondingly weaker
swimming movements until the spent animal finally sinks.
The only previous account of shedding in Glyccra observed under natural con-
ditions in Allen's (1957) report that pelagic breeders of G. fspliyrabrancha re-
leased their gametes in two streams, apparently from pores in the midbody region.
Genital ducts are not known for this genus, and gametal discharge in other species
is generally presumed to occur through an oral opening left by the dissolution of the
proboscis. There is no record, however, that this has ever been witnessed during
actual swarming. The assumption is based primarily on the occurrence of epitokes
in which the proboscis is either extremely degenerated or totally absent. With few
exceptions, this condition has been found in G. capitata, G. lapidum, G. alba, G.
rou.i'ii and G. tesselata (Arwidsson, 1898; Fage and Legendre, 1927; Gravier and
Dantan, 1928; St^p-Bowitz, 1941). Fragments of mature glycerids have also been
collected at the surface (Fage and Legendre, 1927; Gravier and Dantan, 1928;
Allen, 1957). The indications are, therefore, that shedding in Glyccra is by
dehiscence. The area of rupture probably depends on the degree of muscular
atrophy, which might vary in different species.
Environmental factors
At present it is impossible to indicate the environmental agents that induce
swarming in mature bloodworms or to predict the circumstances under which such
behavior could be expected. Information on temperature, salinity, weather and
tidal conditions was compiled from records maintained at the Chesapeake Biological
Laboratory. A comparison of these data for the reported December, 1944, swarms
and those observed in November, 1960, revealed no remarkable similarities. The
four swarming days in both years fell between full moon and last quarter, but the
significance of this cannot be established on the basis of only two reports. Swarm-
ing in 1960 began somewhat later each day, nearly coinciding with maximum
high water of the second tide, and thus suggesting that tidal influences may be at
least partially responsible for the daily timing of reproductive activity. Although
exact hours are not available, it is known that swarming on December 4—7, 1944, took
place at night (Beaven; personal communication), and hence could have been
similarly associated with the second daily tide, which on those dates reached high
water after dark.
A relationship between tidal conditions and onset of spawning has been reported
for some other polychaetes, and in a few instances may perhaps be involved in day-
time swarming (Herpin, 1926, 1928; Korringa, 1947). The regulation of gametal
discharge to coincide with rising water is not difficult to understand in the case of
intertidal populations. But there seems to be no ready explanation for such timing
in animals not exposed at low water, especially when the tidal amplitude is small,
as at Solomons. It is unlikely, however, that tidal movements alone could be re-
sponsible for stimulating swarming in Glyccra; such behavior is more probably
dependent upon the interaction of a number of factors, both environmental and
physiological.
406
MARGARET SIMPSON
EPITOKY
Almost all polychaetes that become pelagic at sexual maturity undergo some
degree of structural alteration into a specialized reproductive form (see Clark,
1961). This change is known as epitoky (Ehlers, 1864-68), and the transformed
individual, an epitoke. Among the Nereidae this metamorphosis often achieves
remarkable complexity, resulting in the formation of a heteronereis ; but in most
swarming polychaetes the changes are less pronounced and generally comprise
histolysis of the body musculature and the digestive tube, as well as development of
additional or modified setae. Such epitokous alterations have been described for
several G lye era species (see Table I ) and appear in mature specimens of G.
dibranchiata as well.
External appearance of epitokes
In specimens completely or partially spent, the posterior two-thirds of the body
is more collapsed and darker than the anterior portion. This appearance un-
doubtedly results from the retention of the proboscis, which provides more bulk
FIGURE 2. Parapod of (A) an immature specimen and (B) a swarming male. Both
parapods are from the mid-body region and are shown in anterior view.
anteriorly and lacks the dark pigmentation of the gut. Parapodia of epitokes are
elongated and equipped with numerous setae. In the immature parapod shown in
Figure 2A, the notopodial bundle consists of 7 or 8 simple setae, and the neuro-
poclial bundle of about 22 composite setae. Two very short simple bristles are
present just dorsal to the first neuropoclial seta, but since this parapod comes from
a worm with large gonads, these two unarticulated setae probably represent an
initial stage in the development of the mature setal complement. In contrast, the
notopodial bundle of the mature parapod (Fig. 2B) consists of 19 or 20 simple
setae, and the neuropoclial bundle includes, in addition to 37 composite setae, 8
dorsally placed simple ones. All of these bristles are noticeably longer than those
of the atokous specimen but show no structural differences.
Although the epidermis of epitokes is reduced to a very thin layer, there is a
marked increase in the activity of its mucous cells, especially in the parapodial
lobes. A yellow-brown pigment, also present in other tissues of mature animals,
REPRODUCTION OF GLYCERA 407
is particularly abundant in the epidermis, where it often appears in the form of
granular aggregates or minute, needle-like crystals. All the various chromogenic
substances formed at epitoky probably are associated with degenerative phenomena
and contribute to the coloration of mature worms. Klawe and Dickie report that
male bloodworms about to spawn are a creamy color, while females are pale brown,
and attribute these colors to the gametes showing through the thinned body walls.
Though this may be the case in males, the eggs — being colorless — could hardly be
responsible for a brown color in females. It is more likely that these differences
stem rather from alterations of a metabolic nature. In the present study, color
differences could be detected when mature males and females were compared in
the laboratory, but it was very difficult to distinguish the sex of swarming worms in
the field.
Internal changes
Epitoky is characterized by a drastic reduction in the thickness of the body wall
and the diameter of the gut, with a corresponding increase in coelomic volume.
Both muscle layers seem to be equally affected, but as the circular muscles are
relatively thinner to begin with, this layer virtually disappears at epitoky. Since
serpentine movements are executed primarily by longitudinal contractions, the
difference in thickness of the muscle layers may play a role in the altered swimming
behavior previously mentioned. The suspensory muscles of the digestive tube
and the acicular muscles are also attenuated, though to a less striking extent. There
is little apparent structural difference between muscle fibers of epitokous and
atokous individuals ; some vacuolization can be detected in the former, but this has
occasionally been observed in immature worms as well. There is no marked in-
vasion of the musculature by phagocytes, and in general, the epitokous condition
seems to result more from atrophy than true sarcolysis.
The gut of mature animals is much reduced in diameter, with its shrunken
mucosal layer appearing spongy and containing a granular yellow pigmentation.
The columnar cells of this layer are apically disintegrated and have pycnotic nuclei.
A scattered amorphous material occurring in the lumen probably consists of cellular
debris. Despite these degenerative changes, the digestive tube, including proboscis
and jaws, is entire.
The beginning of epitokous modifications in musculature and intestine appear in
an ovigerous female collected 16 days before the first swarm. Since these changes
are not yet visible in specimens that have just started to release gametes into the
coelom, atrophy of the adult tissues may be related to metabolic requirements of the
reproductive cells. Parapodial modification begins considerably earlier, before
gametes appear in the body cavity. According to Sto'p-Bowtiz (1941 ), a similar
sequence is found in G. lapidnm, G. alba and probably G. roH.vii, whereas in G.
capitata it seems to be reversed, with degenerative changes preceding parapodial
modification.
The coelomic epithelium and the septa are not affected by epitoky ; nor do the
segmental organs show any great change, although their protonephridial portions
may be somewhat hypertrophied. The irregular black masses so numerous in the
body fluid of mature worms are generally regarded as products of tissue breakdown,
but their origin remains unknown. They are made up of clumped coelomic cells
408 MARGARET SIMPSON
containing a greenish-brown granular substance that resembles the finer granula-
tion found in red blood cells at earlier stages of maturity. Since these corpuscles
are known to contain hemoglobin (Salomon, 1941), it seems possible that the
greenish-brown pigmentation may be at least partially derived from decomposition
of this molecule. Raphael (1933) proposes that hemoglobin destruction and elimi-
nation takes place in the languettes attached near the jaws and projecting into the
proboscidial coelom, but does not suggest how the compound is transported to these
structures. Since the reduced languettes of epitokes are not particularly pigmented,
it seems unlikely that they play a significant part in hemoglobin destruction during
sexual metamorphosis.
The achievement of maturity in bloodworms is further accompanied by changes
in the saccular apparatus of the brain. It has previously been suggested (Simp-
FIGURE 3. Section through the epidermis (e) on the dorsal prostornial surface of a
swarming epitoke, showing the juncture of the cuticle (c) and the hyaline fiber (hf) from the
saccular apparatus in the brain. The darker material within the hyaline fiber passes through
the cuticle to the exterior.
son, 1959) that this structure performs a secretory function, and present observa-
tions tend to support this hypothesis. In several swarming specimens the hyaline
fiber, at its junction with the cuticle on the dorsal surface of the prostomium, forms
a distinct opening through which an amorphous material passes to the exterior
(Fig. 3). Although this is not clearly shown in all the swarming worms examined,
no sign of such an opening has been found in either the animals studied in the
earlier investigation or the non-swarming specimens of the present study. It seems
probable, therefore, that some type of material elaborated by or stored within the
saccular apparatus is released to the exterior at the time of swarming. Histochemi-
cal tests have not been performed, but the staining reaction of this material resembles
that of mucus in epidermal goblet cells, and it may well be a mucopolysaccharide
similar to that previously demonstrated for the saccular apparatus. The possible
significance of such an external secretion is indicated by Clark's (1961) suggestion
REPRODUCTION OF GLYCERA 409
that neurosecretory hormones released into the water might act as coordinating
factors in the spawning of some nereids.
Other modifications of the saccular apparatus also suggest an enhanced activity
of this structure in worms approaching maturity as well as those fully matured.
Such alterations include an increase (rarely, a decrease) in size or number of sacs,
a dilation and greater convolution of the filaments, and an enlargement of the
vesicles. But since these changes do not occur consistently, and since the original
description of the saccular complex is based on specimens from New England, the
possibility exists that such changes may represent modifications indigenous to the
Solomons population and not necessarily related to reproductive functions.
Epitoky and swarming
There is considerable variation in the degree to which mature specimens are
affected by epitoky, and even among worms swarming at the same time, some indi-
viduals show far less atrophy than others. Furthermore, several animals in
which maturity had been induced by an elevated temperature failed to spawn even
though the gametes seemed to be ripe and epitokous manifestations were more pro-
nounced than in any of the swarming specimens. It appears that of itself epitoky
does not determine the time of swarming, but that such behavior can be elicited
from animals that have undergone varying degrees of metamorphosis.
The epitokous characteristics observed here are of the same type as those de-
scribed for other Glyccra species. Although in the majority of other species de-
generation, especially of the proboscis and gut, is reportedly far more severe, the
degree of atrophy appears to be variable in these cases also. For example, Gravier
and Dantan (1928) note an exceptional swarmer of G. tessclata in which the
proboscis is intact, and Arwidsson (1898) describes a similar instance for G. alba.
It is possible that the November swarming observed at Solomons occurred some-
what earlier than usual, before metamorphosis had reached its peak, or, more likely,
that other species as a rule achieve more pronounced epitoky before swarming than
does G. dibranchiata.
In view of these changes at maturity, it is generally assumed that glycerids do
not survive swarming. For G. dibranchiata, Klawe and Dickie report that the
occurrence of "ghost" worms, i.e. the remains of dead worms, was inversely pro-
portional to the abundance of mature individuals. No "ghosts" were found during
the present study, but since swarming took place in areas not exposed at low tide,
the presence of such remains would be difficult to detect. Although spent worms
placed in running salt water showed movement when handled some four or five
days later, they underwent gradual deterioration and in about a week's time re-
sembled "ghost" worms. However, since the degree of atrophy at swarming ap-
pears to be variable, it is conceivable that some individuals may be able to recover
after shedding.
All of the field work involved in this study was made possible through the
courtesy of the Chesapeake Biological Laboratory of the Natural Resources In-
stitute, University of Maryland. I am greatly indebted to the Laboratory for the
use of its facilities, and to the many staff members who provided valuable information
and assistance during the investigation.
410 MARGARET SIMPSON
SUMMARY
1 . This report is based on field observations made at Solomons, Maryland, be-
tween June, 1960, and February, 1961, and on histological examination of material
collected during this period. It appears that bloodworms breed twice a year at
Solomons : certainly during fall and very likely in late spring or early summer as
well. Gametogenesis probably requires close to a year for completion. Both
temperature and seasonal plankton variation are suggested as factors that may in-
fluence the timing of reproductive activity. There is reason to suspect that blood-
worms also spawn biannually in the Maritime Provinces and Maine, but conclusive
evidence is not available.
2. Swarming occurred mostly over shallow water, during late afternoon on
November 5-8, 1960. It covered a moderately large area, but individual worms
were widely dispersed. Data suggest that the onset of swarming may be co-
ordinated with tidal conditions. Shedding is by dehiscence, through the posterior
end, and is apparently an automatic process initiated by serpentine swimming
movements that differ from the usual locomotion of immature animals.
3. Epitokes are characterized by atrophy of the musculature and alimentary
canal, elongation of the parapods and increase in the number of setae. There are
indications that the saccular apparatus of the brain releases a substance to the ex-
terior during swarming. Although there is variation in the degree of atrophy
attained at spawning, bloodworms apparently undergo less severe degenerative
changes than other Gl\cera species.
LITERATURE CITED
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Biol. Bull, 113: 49-57.
ANONYMOUS, 1948. Those worms again. Maryland Tidcivatcr Ncivs, 5: 4.
ARWIDSSON, I., 1898. Studien iiber die Familien Glyceridae und Goniadidae. Bcrgcns Mus.
Aarb., No. 11, 70 pp.
BEAVEN, G. F., 1960. Temperature and salinity of surface water at Solomons, Maryland.
Chesapeake Sci., 1: 2-11.
BERKELEY, E., AND C. BERKELEY, 1948. Canadian Pacific Fauna: 9. Annelida. 9b. (1). Poly-
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CLARK, R. B., 1961. The origin and formation of the heteronereis. Biol. Rcr., 36: 199-236.
CLARK, R. B., AND M. E. CLARK, 1960. The segmental musculature and the ligamentary system
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EHLERS, E., 1864-68. Die Borstenwurmer nach systematischen und anatomischen Unter-
suchungen dargestellt. Wilhelm Engelmann, Leipzig.
FACE, L., AND R. LEGENDRE, 1927. Peches planctoniques a la lumiere, effectuees a Banyuls-
sur-Mer et a Concarneau. I. Annelides Polychetes. Arch. Zool. Exp. et Gen., 67:
23-222.
FUCHS, H. M., 1911. Note on the early larvae of Ncplith\*s and GIvcera. J. Mar. Biol. Assoc.,
9: 164-170.
GALTSOFF, P. S., 1940. Physiology of reproduction of Ostrca virginica. III. Stimulation of
spawning in the male oyster. Biol. Bull., 78: 117-135.
GRAVIER, C., AND J. L. DANTAN, 1928. Peches nocturnes a la lumiere dans la baie d'Alger.
Ann. Inst. Occanogr., n.s., 5: 1-186.
GRAY, J., 1939. Studies in animal locomotion. VIII. The kinetics of locomotion of Nereis
diversicolor. J. E.vp. Biol., 16: 9-17.
HERPIN, R., 1926. Recherches biologiques sur la reproduction et le developpement de quelques
Annelides Polychetes. Bull. Soc. Sci. Nat. V Quest France, Scr. 4, 5: 1-250.
REPRODUCTION OF GLYCERA 411
HERPIN, R., 1928. Etude sur les essaimages cles Annelides Polychetes. Bull. Biol. France ct
Bclgiquc, 62: 308-377.
KLAWE, W. L., AND L. M. DICKIE, 1957. Biology of the bloodworm Glycera dibranchiata
Ehlers, and its relation to the bloodworm fishery of the Maritime Provinces. Bull. 115,
Fish. Res. Bd. Canada, 37 pp.
KORRINGA, P., 1947. Relations between the moon and periodicity in the breeding of marine
animals. Ecol. Monogr., 17: 347-381.
Lo BIANCO, S., 1909. Notizie biologische riguardanti specialmente il periodo di maturita
sessuale degli animali del golfo di Napoli. Mitthcil. Zool. Stat. Neapcl, 19: 513-761.
LOOSANOFF, V. L., AND H. C. DAVIS, 1952. Temperature requirements for maturation of gonads
of northern oysters. Biol. Bull., 103: 80-96.
MAcGiNlTlE, G. E., 1935. Ecological aspects of a California marine estuary. Amcr. Mid.
Nat., 16: 629-765.
MANNING, J. H., 1959. Commercial and biological uses of the Maryland soft clam dredge.
Proc. Gulf and Caribbean Fisli. Inst., 12th Annual Session, pp. 61-67.
NASH, C. B., 1947. Environmental characteristics of a river estuary. Sears Found. J. Mar.
Res.. 6: 147-174.
ORTON, J. H., 1920. Sea-temperature, breeding and distribution in marine animals. /. Mar.
Biol, Assoc., 12: 339-366.
PETTIBONE, M. H., (In press). Marine polychaete worms of the New England region. I.
Aphroditidae through Trochochaetidae. Bull. U. S. Nat. Mits.
RAPHAEL. C., 1933. Etude de la trompe des Glyceres et de son organe excreteur d'hemoglobine.
Trav. Stat. Biol. Roscoff, 11: 5-18.
SALOMON, K., 1941. Studies on invertebrate hemoglobins (erythrocruorins). /. Gen. Ph\siol.,
24: 367-375.
SIMPSON, M., 1959. The saccular apparatus in the brain of GIvccra dibranchiata. J. Morph.,
104: 561-590.
STOLTE, H. A., 1932. Untersuchungen iiber Ban und Funktion der Sinnesorgane der Poly-
chatengattung Glycera Sav. Zeitschr. zvissen. Zool., 140: 421-538.
Sx0p-BowiTZ, C., 1941. Les Glyceriens de Norvege. Nytt Mag. Naturv., 82: 181-250.
THORSON, G., 1946. Reproduction and larval development of Danish marine bottom inverte-
brates, with special reference to the planktonic larvae in the Sound (0resund). Mcdd.
Koinin. Daninarks Fisk.-Hai'undcrs., Ser. Plankton, 4: 1-523.
THORSON, G., 1950. Reproductive and larval ecology of marine bottom invertebrates. Bio!. Ret'..
25: 1-45.
TURNER, H. J., AND J. E. HANKS, 1960. Experimental stimulation of gametogenesis in
Hydroides dianthus and Pectcn irradians during the winter. Biol. Bull., 119: 145-152.
GAMETOGENESIS AND EARLY DEVELOPMENT OF THE
POLYCHAETE GLYCERA DIBRANCHIATA x
MARGARET SIMPSON
Department of Biolotiy, Catholic University of America, Washington, D. C., and Chesapeake
Biological Laboratory, Natural Resources Institute of the University
of Maryland, Solomons, Maryland
A previous paper (Simpson, 1962) has described fall swarming of the blood-
worm, Glycera dibranchiata Ehlers, observed at Solomons, Maryland, during a
study conducted there from late June, 1960, to early February, 1961. The same
paper also discussed the probable breeding seasons of this species at Solomons, and
presented data on the rate of gonad development, concluding that gametogenesis
very likely requires close to a year for completion. A more detailed account of
gametogenesis and an outline of development to the trochophore stage, obtained in
the course of the same investigation, form the subject of the present report.
An early account of glycerid reproductive organs appears in Ehlers' (1864-68)
original description of G. dibranchiata, and although inaccurate, has remained the
only source of information on gonads in the genus. The embryology of Glycera
is but slightly better known: Allen (1957) gives some data on the rate of early de-
velopment in G. fsphyrabrancha, Fuchs (1911) describes the young larval stages of
G. convoluta, and observations on G. dibranchiata from fertilization to the trocho-
phore are reported by Klawe and Dickie (1957). In the last two cases, as in the
present one, attempts to maintain cultures beyond the trochophore stage were un-
successful. This difficulty has also been alluded to by Wilson (1948), and it
appears that further concentrated effort will be required to determine the complete
developmental history of these annelids.
GAMETOGENESIS
Methods
The following observations are based largely on histological examination of about
70 specimens collected at Solomons (38°19'N., 76°27'W.) between June, 1960, and
February, 1961. Animals were narcotized in magnesium chloride, fixed in Bouin's
fluid and preserved in a mixture of 2 parts ethanol, 1 part distilled water and 1 part
glycerine. Paraffin sections 4—7 p. thick were stained either with Ehrlich's hema-
toxylin and eosin, or by a modified procedure using Gomori's chrome-alum hema-
toxylin, recommended as a chromosome stain by Melander and Wingstrand (1953).
Scruff's reagent, prepared by several different methods, consistently gave either weak
1 Based on portion of a dissertation submitted in partial fulfillment of the requirements for
the Degree of Doctor of Philosophy at the Catholic University of America, Washington, D. C.
Contribution No. 201, Chesapeake Biological Laboratory, Natural Resources Institute of the
University of Maryland. This investigation was carried out during the tenure of PHS Pre-
doctoral Fellowship BF-9242-C1 from the National Institute of Neurological Diseases and
Blindness, and GF-9242-C2 from the Division of General Medical Sciences.
412
GAMETES AND DEVELOPMENT OF GLYCERA
413
or negative Feulgen reactions, and this technique was discontinued. Some at-
tempt was made to study in more detail the chromosomal changes associated with
gametogenesis by making squash preparations of gonads ; results, however, were
entirely unsatisfactory, probably because the material had been fixed and stored
for several months. Gonads of fresh worms purchased at a bait store gave
promising squash preparations with La Cour's (1941) aceto-orcein method, but
this phase of the study was not pursued further.
Gonads
Glycera dibrancliiata is dioecious, with segmentally paired gonads beginning in
the region of segments 45-47 and extending posteriorly for approximately 60 seg-
N
A
VENTRAL
BODY WALL
< — ANTERIOR
FIGURE 1. Diagram of the ventral half of one body segment, viewed from the median plane.
Not to scale. AM, acicular muscles ; CT, connective tissue pad around bases of the acicula ; G,
lobe of gonad ; N, nephridial complex ; PC, parapodial cavity ; S, septum.
ments. Each gonad arises as an outgrowth from connective tissue at the lateral
edge of the ventral longitudinal muscles, where the parapodial cavity opens into the
general coelom. Between the gonad and the longitudinal muscles lies the origin of
the ventral acicular muscle (Fig. 2), which passes dorso-medially from this point
and inserts on a connective tissue pad surrounding the bases of the two para-
podial acicula. From the same connective tissue pad other acicular muscles also
radiate to their various origins on the body wall (Fig. 1) ; thus, the space into
which the gonad grows is limited dorsally by the acicula, and anteriorly, posteriorly
and medially by the acicular muscle bands.
The initial gonial swelling gradually enlarges, extending further toward the
coelom and occasionally a short distance into the parapodial cavity as well, but
414
2
MARGARET SIMPSON
dim
am
vim
FIGURE 2. Transverse section through a mid-body segment, showing the position of the
gonad (g). Part of the intestine appears in the upper left corner, and the dorsal cirrus of the
parapod in the upper right corner, am, acicular muscles ; dim and vim, dorsal and ventral
GAMETES AND DEVELOPMENT OF GLYCERA 415
remains attached to its point of origin by a broad, compact stalk, which penetrates
into the underlying connective tissue (Fig. 2). The nephriclial duct from the
preceding segment usually passes near or through this region of the gonad before
turning laterally to the small nephridial pore just below the parapod. As the gonad
expands toward the coelom, it becomes dorso-ventrally flattened between the acicula
and their muscles, and eventually it pushes out between these muscle bands to form
irregular lobes projecting into the body cavity. Since the degree of tabulation at-
tained is subject to individual variation, no average dimensions can be indicated;
maximum cross-sections range from 70 X 55 ju, for very small gonads to 420 X 360 ^
for large ones.
In dissections, large gonads can at times be confused with the nephridial com-
plexes, which are also segmentally paired. The latter organs have the same
general structure as those described in other Glycera species (Goodrich, 1898 ; Page,
1906), but instead of being attached to the anterior face of the septum, each
nephridial complex is connected only to the posterior acicular muscle (Fig. 1).
The distal end of the ciliated organ makes a turn around this muscle, and the
nephridial duct continues down the muscle to the body wall. Thus the main por-
tion of the nephridial complex, consisting of the phagocytal sac and the proto-
nephridium with its solenocytes, projects freely into the coelom and is occasionally
found lying between the acicular muscles in the region of the gonad. In live speci-
mens, the nephridial complex has a yellow-green tint readily distinguishable from
the light pink of the reproductive organs.
The gonads of Glycera have previously been described by Ehlers ( 1864-68) r
but his account is somewhat confusing, since it is not entirely clear whether the
description pertains wholly to G. dibrancJiiata or also includes G. capitaia. His
description of the ovaries (pp. 697-700) seems to be based on examination of
epitokous females of G. capitata, yet the figures referred to are all labeled G. di-
branchiata. These structures certainly bear little resemblance to the gonads de-
scribed here ; neither the "grape-like clusters" nor the simpler, fiber-like ovaries
that Ehlers described have ever been observed in the present study. Besides, the
females examined by Ehlers must have been far advanced in sexual maturity (as
indicated by their pronounced atrophy and the masses of eggs in their body cavities) „
and according to the present observations, these specimens should have had no re-
maining gonad tissue whatsoever. On the other hand, the description of what
Ehlers believed to be the testes (p. 700) is explicitly based on a specimen of G.
dibrancJiiata and does agree with the present findings.
The matter is further complicated by Lubischev (1924), who states that Ehlers'
illustrations of ovaries represent exactly the multiple nephridial complexes found in
G. capitata. Since these illustrations are indicated as G. dibranchiata, Lubischev
longitudinal muscles ; o, origin of ventral acicular muscle. Melander-Wingstrand's modified
chrome-alum hematoxylin (CAH).
FIGURE 3. Gonadal cells with basophilic material enclosed in separate vesicles (bottom
and center of figure). Two cells at the upper left show the basophilic substance distributed
around the periphery of each vesicle. Melander-Wingstrand's CAH.
FIGURE 4. Young oocytes at the surface of the gonad, shortly before they are released into
the coelom. Gomori's CAH, phloxin.
FIGURE 5. Section through an oocyte shed spontaneously in the laboratory. Ehrlich's
hematoxylin, eosin.
416 MARGARET SIMPSON
concludes that similar multiple nephridia are present in this species also, and that
Ehlers must have mistaken them for ovaries. This conclusion, however, is in-
correct, for G. dibranchiata has only two nephridial complexes in each segment, and
these organs distinctly differ from the structures Lubischev found in G. capitata.
It appears therefore that the "ovaries" described by Ehlers were in fact nephridial
complexes of G. capitata, the legends to his figures notwithstanding. His misin-
terpretation was undoubtedly due to the presence of eggs in these structures, but it
is not at all unusual to find gametes in the sac of the ciliated organ, which is closely
associated with the nephridium proper (see Goodrich, 1945). Ehlers' description
of the segmental organ, although not entirely accurate, indicates that he did
recognize the nephridial complex in G. dibranchiata.
Early gametogenesis
The gonad first appears as an aggregation of cells embedded in connective
tissue near the base of each parapod. The origin of these cells is uncertain, but
they resemble the small, apparently amoeboid, basophilic cells scattered throughout
connective and muscular tissues. The clustered gonial cells enlarge and proliferate
upward, producing a bulge beneath the coelomic epithelium, which forms an invest-
ing membrane around the projection. From this early stage onward, the gonad is
marked by the presence of relatively large (about 20 X 11 /A) oval cells containing
scattered concentrations of chromatin. These cells are somewhat localized toward
the interior of the gametal mass and in larger gonads occasionally seem to form a
core extending from the stalk into the central region of the organ. There is, how-
ever, no sharp division into cortical and medullary zones, and the cells may occur
in other parts of the gonad as well.
The chromatin inclusions within the cell vary in size and density, larger ones
generally being more diffuse, with indistinct outlines, and smaller ones more com-
pact and well defined. Their basophilia also varies with the degree of concentration,
but never becomes very intense. In some cases, each inclusion appears to lie in
a separate vesicle, giving the distinct impression that the larger body consists of a
number of minute cells (Fig. 3). From this condition there follows a series of
stages in which the basophilic material is distributed around the inner surface of
each compartment, the individual vesicles coalesce, and the whole structure takes
on the appearance of a cell with a large nucleus surrounded by a thin layer of
cytoplasm. The significance of these cells in the gametogenetic process is not
clear. In some aspects they strongly resemble stages of orthopteran spermatogonial
divisions, in which individual chromosomes are separately compartmentalized (e.g.
Wenrich, 1916; Rao, 1934), and it is possible that a similar situation occurs in
Glyccra.
The bulk of the gonad consists of developing gametes, which are at first closely
packed and of uniform appearance, each cell containing a large reticulated nucleus
surrounded by a small amount of cytoplasm. After a certain period, during which
the gonad continues to grow, the germ cells enter a phase of nuclear activity and
become more or less segregated into small groups, each containing cells at a similar
stage of development. Although metaphase and anaphase configurations are rela-
tively scarce, the nuclear chromatin shows various prophasic changes, undergoing
condensation into thread-like filaments that thicken, become more basophilic, and
GAMETES AND DEVELOPMENT OF GLYCERA 417
form a compact tangled knot. Chromosome counts indicate that the diploid number
probably lies between 30 and 40. Up to this point, ovaries and testes cannot be
distinguished.
0 agenesis
The first definite sign of sexual difference is the appearance of oocytes in the
peripheral layers of the gonad, especially along its medial border. In these cells
the dense chromatin mass relaxes into a diffuse network, a nucleolus appears, and
the cytoplasmic volume begins to increase (Fig. 4). The stage at which the oocytes
leave the gonad seems to vary. Apparently they are ready to be released upon
reaching a diameter of about 28 p., but occasionally the rupture of the epithelial
covering is delayed, and they may grow larger before escaping into the coelom. As
the oocytes are progressively released, the ovary decreases in size until no sign of
it remains in gravid females. The oocytes complete their growth while circulating
in the coelomic fluid, attaining an average diameter of 140 p, before being shed. No
marked nuclear changes take place during this period, and since both polar bodies
are formed after fertilization, it appears that the egg chromosomes rest in a diffuse
state, having passed through early prophase of meiosis I in the gonad.
The ripe oocyte (Fig. 5) is a flattened sphere, bulging over the centrally placed
germinal vesicle and surrounded by a thin membrane. Discoidal eggs have been
described for other Glyccra species (Fuchs, 1911; Allen, 1957) and are probably
characteristic for the whole family. The colorless cytoplasm of the living egg is
granular, with some spherical, refractile inclusions that may be small oil droplets ;
a thin cortical zone of fine granules is present. Oocytes fixed in Bouin's show a
basophilic, alveolar cytoplasm. The germinal vesicle measures 50 p, in diameter and
contains a diffuse acidophilic reticulum. The nucleolus is about 14 ^ in diameter
and appears to be double, consisting of a weakly basophilic, vacuolated portion
that is cupped around a more homogeneous, acidophilic center.
Sp ermatogcn csis
The spermatocytes similarly appear first in the periphery of the gonad, where
they occur in small clusters of seven or eight cells (Fig. 6), which remain
together after being released into the coelom. As in the female, the male gonad
also becomes smaller and eventually disappears. When first released, individual
spermatocytes have a diameter of 6 /*, and contain a large nucleus with chromatin
evenly distributed in the form of darkly staining condensations. The spermatocyte
clusters develop into oval plates several cells thick, each plate consisting of
approximately 30 or 40 cells and measuring about 16 X 9 ^ (Fig. 7). Although
division figures have not been observed, it seems likely that this increase in cell
number is due to the maturation divisions. The nucleus of each spermatid is a
rounded mass of chromatin, which appears to be honeycombed with minute
vacuoles. The cytoplasm is reduced to a very thin layer and soon disappears
entirely. With increasing condensation of the chromatin, the spermatids assume
a somewhat triangular shape, while the sperm plates begin to loosen and break
up. Apparently, spermiogenesis is completed after the individual spermatids have
separated.
The mature spermatozoon (Fig. 8) is of the primitive type. Its spherical
418
MARGARET SIMPSON
7
.
40
*
FIGURES 6-9.
GAMETES AND DEVELOPMENT OF GLYCERA 419
head is 2.5 ^ across, bears a rounded acrosome, and is slightly flattened at the
posterior border, where the four round elements of the middle piece are arranged
around the base of the flagellum. Sperm remain active for at least six hours
after being shed, but it is not known how long they retain the capacity foi
successful fertilization.
EARLY DEVELOPMENT
Methods
Embryological material was obtained during collection of swarming animals
in November, 1960, when a male and a female were placed together in a jar.
Although mortality due to polyspermy was high, the large number of eggs
fertilized yielded an abundant supply of normally developing embryos. As soon
as they were brought to the laboratory, the eggs were distributed into fingerbowls
and washed five or six times. Thereafter the water in these bowls was changed
every two hours, until swimming larvae appeared. Throughout the observations
the cultures were kept in containers placed in a tray of running water to minimize
temperature changes, and all water added to the cultures was first filtered through
several layers of gravel and sand. Aeration was supplied by a small pump.
Several different methods were tried to maintain the larvae after swimming
stages appeared. Some of the larvae were placed into cages made of small
polyethylene containers of the type used to refrigerate food. The sides and covers
of the containers were cut out to make large windows which were then covered
with bolting silk, using a soldering gun with a smoothing tip to seal the material
to the plastic. Four large corks attached to the corners floated the half-submerged
cages in a large tank of running water. Unfortunately, larvae were able to escape
through No. 18 bolting silk, and No. 20 became clogged so rapidly that within
two days a microbial growth had flourished at the expense of the cultures. The
same difficulty was encountered in using a current rotor device patterned after
the apparatus of Galtsoff and Cable (1933; also Galtsoff, 1959). In this case,
the rotating cylinder designed to keep the larvae in a small aquarium with a
constant change of water was covered with bolting silk, which again clogged up
rapidly. Better results were obtained with larvae placed in gallon jars half full
of water, which was gently agitated by air passed through porous stones. The
jars were covered with cellophane and fresh water was added as necessary to
replace loss through evaporation. The relative success of these cultures sug-
gests that the plunger-jar technique may have much to offer. Attempts to feed
the larvae in different jars included the addition of diatoms (predominantly
Coscinodisc us; also various pennate forms), a fine powder of dried clams, or
substratum obtained from areas over which swarming took place. Despite the
frequent occurrence of larvae with a bolus of reddish-brown matter in the gut
FIGURE 6. Clusters of spermatocytes at the surface of the gonad, shortly before they are
released into the coelom. Ehrlich's hematoxylin, eosin.
FIGURE 7. Two plates of spermatids free in the coelom. The larger plate is cut horizon-
tally, the other vertically. Melander-Wingstrand's CAH.
FIGURE 8. Mature spermatozoa in the coelom of a swarming epitoke. The sperm in the
center shows the acrosome (arrow), two elements of the middle piece and a short portion of
the tail. Melander-Wingstrand's CAH.
FIGURE 9. Section through a fertilized egg in anaphase of the second polar division, show-
ing the sperm head (arrow) penetrating into the center of the egg. Ehrlich's hematoxylin.
420 MARGARET SIMPSON
cavity, development ceased after the sixth day, and all the larvae died by the
seventeenth day.
Rate of development
The observed rate of development at a water temperature of 12.5-14° C. is
shown in Table I. Calculations are based from the time the swarming male and
female were placed together ; since both were shedding freely, it is assumed that
fertilization occurred almost immediately. The times reported by Klawe and
Dickie (1957) differ considerably from the present findings. This disparity arises
mainly from the very slow rate of initial cleavages indicated in their report, and
decreases in the more advanced stages. Thus the period from the 4-cell stage to
the swimming embryo is about 12 hours in their report, and about 91/o hours in
the present schedule, whereas the interval between swimming embryo and
prototroch formation is approximately 10 hours in both cases. A difference in
cleavage rate, however, is not unduly surprising, since Klawe and Dickie worked
with a more northern population of bloodworms and made their observations over
TABLE I
Rate of early development at 12.5-14° C.
Stage Hours after fertilization
Polar divisions <2
First division spindle 1\
Two-cell 3
Four-cell 4|
Eight-cell S\
Swimming embryo 14
Trochophore 25
a wider temperature range (12-20° C.), using eggs shed and fertilized in the
laboratory.
Fertilization and early cleavage
Germinal vesicle breakdown does not normally occur until fertilization. The
nuclear membrane of unfertilized eggs disappears after about 10 hours at room
temperature, but this is a degenerative phenomenon followed by gradual deteriora-
tion of the eggs. Within two hours of fertilization, the oocyte rounds up to a
diameter of about 100 /*, and the vitelline membrane lifts away from the cytoplasm
to form a wrinkled fertilization membrane 1.8 ^ thick. Evidently a block to
polyspermy is established by this stage, and numerous adherent sperm are lifted
by the rising membrane. No external jelly layer is formed. The cortical granules
of the unfertilized ovum have disappeared by this stage, but whether they con-
tribute to the formation of the fertilization membrane is unknown.
Both polar divisions take place while the sperm head is approaching the
center of the egg (Fig. 9). One of the polocytes, and occasionally the second
also, divides again. Male and female pronuclei fuse in the center of the egg,
and the first division spindle is completed by 2l/2 hours after fertilization. The
chromosomes remain vesicular throughout the first cleavage and fuse to form a
GAMETES AND DEVELOPMENT OF GLYCERA
421
tabulated nucleus in each blastomere. The nuclei round out and elongate as
the centrosomes migrate in preparation for the second division, during which
the chromosomes regain their basophilia and typical form. Cleavage is spiral.
The first four blastomeres appear to be equal, and the micromeres of the eight-
cell stage are but slightly smaller than the macromeres.
Pelagic larvae
Thirteen hours after fertilization, perfectly spherical embryos are found
rotating at the bottom of the dish and soon swim up to the surface. Gastrulation,
apparently by epiboly, is in progress within the following three or four hours.
Young trochophores are about 115 ^ in diameter and have a broad prototroch
as well as a patch of long apical cilia. They show no definite reaction to light,
but are strongly geonegative, concentrating at the very surface of the water. This
behavior does not persist, and larvae three days old are evenly distributed
throughout the upper layers of the water.
FIGUKE 10. Ventral view of a six-day-old trochophore, drawn from a live specimen.
Only the lateral portions of the prototroch are shown.
The six-day-old trochophore (Fig. 10) is about 120 p. long. In the legends to
their illustrations of larvae, Klawe and Dickie give a length of 1.3 mm., but
surely this must be a misplaced decimal point. At this stage, the larva has a
complete equatorial ridge bearing two rows of well developed prototrochal cilia.
An akrotroch is located ventrally, between the prototroch and the patch of short
apical cilia. On the lower hemisphere, a band of cilia ( neurotroch?) passes
from the mouth to the posterior surface. The stomodeal opening is strongly
ciliated, as is the whole surface of the gastric cavity, which seems to have in
addition a ventral tract of exceptionally long cilia. When a food bolus is present,
it is usually found in the anterior portion of the gut cavity, rotating counter-
clockwise, as seen in dorsal view under the microscope. The gastric epithelium
consists of large cells, many with vacuoles and granular inclusions. A distinct
anus could not be found. The larva is not pigmented, nor does it possess eyespots.
Except for the lack of pigmentation, these larvae generally resemble the
422 MARGARET SIMPSON
trochophores of G. convoluta described by Fuchs (1911). From Madras, Aiyar
(1933) reports planktonic larvae which, on the basis of their similarity to the
illustrations of Fuchs, he refers to the genus Glyccra. Judging from Aiyar's
figures, however, this similarity is not especially pronounced, and the presence
of eyespots in the Madras larvae makes this generic classification questionable.
Aiyar also describes (as Eonc) nectochaetes of the goniadid Glycindc, in which
eyes are present, and possibly his trochophores should be referred to this group
instead of G lye era. Pelagic larvae of G. alba from Denmark have been described
by Thorson (1946), but the trochophore is 400—450 /x, long, has rudimentary anal
cirri, and thus appears to be a later stage than any of the other trochophores
reported. Thorson's record of metatrochophores and nectochaetes is the only
one known for older larvae of Glycera. Treadwell (1936, p. 55) reports "two
very young specimens of this genus, too immature for identification" taken in a
plankton net at 800 fathoms off Bermuda, but gives no description. The speci-
mens must have been relatively large, since the nets used were of Xo. 2 bolting
silk (Beebe, introduction to Wailes, 1936).
Attempts made at Solomons to collect older larvae and post-larval stages
were unsuccessful. A No. 20 net was used to take plankton hauls, both vertical
and horizontal, at several different locations during the two months following
swarming, but the few polychaete larvae obtained could readily be referred to
Polydora. Examination of bottom samples produced no better results ; all of
these samples, however, were from very shallow areas, and cannot be considered
truly representative. The smallest bloodworms found during this study were
two specimens, 3 and 4 cm. long (fixed), collected in July, 1960. Klawe and
Dickie (1957) report 3 cm. as the minimum for their observations, noting that
these small worms were common in May and June. They also were unsuccessful
in finding pelagic larvae or newly settled juveniles, and are inclined to think that
the larvae spend only a short time in the plankton.
Much of this investigation was conducted at the Chesapeake Biological Lab-
oratory of the Natural Resources Institute, University of Maryland, and I wish
to express my thanks for the generous assistance rendered by members of the
Laboratory staff.
SUMMARY
1. Information on gametogenesis in the bloodworm has been obtained by
histological examination of material collected at Solomons, Maryland, in 1960
and 1961. The paired gonads begin in segments 45-47 and continue to about
segment 110. They arise ventro-laterally, as retroperitoneal outgrowths near
the opening of each parapodial cavity into the general coelom. Young oocytes
and spermatocytes, the latter grouped in small clusters, are released from the gonads
and mature in the coelomic fluid. Ripe oocytes are colorless, discoidal and
about 140 /A in diameter ; sperm are of the primitive type.
2. Insemination is followed by germinal vesicle breakdown, elevation of the
fertilization membrane and both polar divisions. Pelagic stages appear within
14-20 hours and in a few days develop into unspecializecl, apparently plankto-
trophic larvae. The further developmental process, including the period of
metamorphosis and settling, remains completely unknown.
GAMETES AND DEVELOPMENT OF GLYCERA 423
LITERATURE CITED
AIVAR, R. G., 1933. Preliminary observations on some polychaete larvae of the Madras coast
and a note on the occurrence in tow-net water of the larvae of Chaetogordius Moore.
/. Madras Univ., 5: 115-156.
ALLEX, M. J., 1957. The breeding of polychaetous annelids near Parguera, Puerto Rico. Biol.
Bull., 113: 49-57.
EHLERS, E., 1864-68. Die Borstenwiirmer nach systematischen und anatomischen Unter-
Michungen dargestellt. Wilhelm Engelmann, Leipzig.
FACE, L., 1906. Recherches sur les organes segmentaires des Annelides Polychetes. .Inn. Sci.
Nat. ZooL.Ser. 9, 3: 261-410.
FUCHS, H. M., 1911. Note on the early larvae of Ncphthys and Gl\cera. J. Mar. Biol. Assoc..
9: 164-170.
GALTSOFF, P. S., 1959. General methods of collecting, maintaining, and rearing marine in-
vertebrates in the laboratory. In : Culture Methods for Invertebrate Animals, ed. by
P. S. Galtsoff ct al., Dover Publications, Inc., New York, pp. 5^tO.
GALTSOFF, P. S., AND L. CABLE, 1933. The current rotor. Science, 77: 242.
GOODRICH, E. S., 1898. On the nephridia of Polychaeta. Pt. 2: Glvccra and Goniada. (Juart.
J. Micr. Sci., 41: 439-457.
GOODRICH, E. S., 1945. The study of nephridia and genital ducts since 1895. Quart. J. Micr.
Sci., 86: 113-392.
KLAWE, W. L., AND L. M. DICKIE, 1957. Biology of the bloodworm, Glycera dibranchiata
Ehlers, and its relation to the bloodworm fishery of the Maritime Provinces. Bull. 115,
Fish. Res. Bd., Canada, 37 pp.
LA COUR, L. F., 1941. Acetic-orcein : a new stain-fixative for chromosomes. Stain Tech., 16:
169-174.
LUBISCHEV, A. A., 1924. On the nephridial complexes of Ncplitliys ciliata and Glycera capitata
(Polychaeta). [In Russian, English summary]. Rcr. Zool. Russe, 4: 283-301.
MELANDER, Y., AND K. G. WINGSTRAND, 1953. Gomori's hematoxylin as a chromosome stain.
Stain Tech., 28: 217-223.
RAO, T. R., 1934. On the spertnatogonial divisions in Aularchcs niiliaris, L. Proc. Indian
Acad. Sci., B, 1: 19-30.
SIMPSON, M., 1962. Reproduction of the polychaete Glvccra dibranchiata at Solomons. Mary-
land. Biol. Bull., 123: 396-411.
THORSON, G., 1946. Reproduction and larval development of Danish marine bottom inverte-
brates, with special reference to the planktonic larvae in the Sound (0resund). Mcdd.
Koniin. Danniarks Fisk.-Havunders., Ser. Plankton, 4: 1-523.
TREADWELL, A., 1936. Polychaetous annelids from the vicinity of Nonsuch Island, Bermuda.
Zoologica, 21: 49-68.
WAILES, G. H., 1936. Plankton of the Bermuda Oceanographic Expeditions. I. Zoologica,
21 : 75-80.
WENRICH, D. H., 1916. The spermatogenesis of Phrynotettix inagints, with special reference
to synapsis and the individuality of the chromosomes. Bull. Mus. Coinp. Zool. Harvard,
60: 55-133.
WILSON, D. P., 1948. The larval development of Ophelia bicornis Savigny. /. Mar. Biol.
Assoc., 27: 540-553.
CYTOLOGICAL STUDIES DURING GERMINAL VESICLE BREAK-
DOWN OF PECTINARIA GOULDII WITH VITAL DYES,
CENTRIFUGATION AND FLUORESCENCE
MICROSCOPY 1
KENYON S. TWEEDELL
Department of Biology, University of Notre Dame, Notre Dame, Indiana, and
Marine Biological Laboratory, Woods Hole, Massachusetts
It is becoming increasingly evident that subcellular cytoplasmic particles
probably play an important part in the embryonic differentiation of the egg. The
possible role of these entities in differentiation of the marine egg is aptly reviewed
by Brachet (1957), Gustafson (1954), Raven (1958, 1961) and Shaver (1957).
One approach has been to identify and then trace the assignment of certain
participate groups during the early recognizable states in morphogenesis.
In the living egg innumerable investigations have used centrifugal forces to
localize and subsequently identify the cell participates by stratification. The early
experiments by Lillie (1906, 1909) on the annelids Chactoptcrus and Nereis,
and of Morgan (1908, 1909, 1910) on Arbacia indicated that centrifugal force
would stratify granules of yolk, pigment, mitochondria and other cytoplasmic
particles according to their specific gravity.
In his classic paper on the egg of Chaetopterus Lillie (1906) observed the
subcellular participates in the living and fixed egg. With centrifugation of 1500
to 2000 rpm. he found that the endoplasm was stratified into a small grey cap, a
clear band and a yellow hemisphere.
During germinal vesicle breakdown he noted the release of a residual sub-
stance composed of spherules and microsomes which he believed formed the grey
centripetal cap. He also noted that the microsomes changed from an acid to a
basic state as they moved into the cytoplasm and he followed their distribution
during early cleavage.
More recent studies, using sucrose layering and much higher centrifugal forces,
by Harvey (1939) on Chactoptcrus and Costello on Nereis (1939, 1958) have
contributed greatly toward identification of the various lavers stratified in the
annelid egg. Stratification in the egg of Arbacia (Harvey, 1941) was very similar
to the annelid egg; in Sphaerechinus grannlaris the various strata obtained were
almost identical to those obtained in the egg of Chaetoptcrns (Harvey, 1939).
Thus a close comparison between the stratification of these eggs and the closely
related eggs of Pcctinaria seemed warranted.
Other observers have used intravital dyes in combination with centrifugation
to identify cell particles in living eggs (Lucke, 1925; Harvey, 1941; lida, 1942;
Monne, 1944; Tweedell, 1960b).
1 This work was partially supported by a grant from the Allen County Cancer Society,
Ft. Wayne, Indiana.
424
VITAL STAINING OF PECTINARIA 425
Further investigations have demonstrated a close relationship between certain
vitally stained particles, such as the mitochondria, and cleavage activity of the egg.
This linkage has been established in eggs of the sea urchin by lida (1942) and
Kojima (1959a), in Urcchis unicinctus (Kojima, 1959b) and in the mollusc,
Spisula solid issima by Rebhun (1959, 1960).
One role of mitochondria in differentiation of the ascidian eggs, Beroc ovata
and Phallusia mamillata, has been verified. Here segregation of the mitochondria
during development has been traced with Janus green. Subsequently, the
mitochondria were found to be involved in the formation of the ciliary plates of
the larvae. Significantly, the localized activity of the mitochondrial enzymes,
cytochrome oxidase and succinic dehydrogenase, has been correlated with the
Janus green staining of the mitochondria (Reverberi, 1956, 1957a, 1957b).
The pale yellow eggs of Pcctinaria gouldii are extremely useful for cytological
studies of this type because of their unusual transparency and relatively small
amount of yolk. As a result, cytoplasmic and nuclear particles remain visible
during development of the living egg.
In order to recognize and chemically identify the many participates in the egg
protoplasm of Pcctinaria, numerous vital dyes and vital fluorochromes were
applied to the living egg both before and after germinal vesicle breakdown. In
addition to observations on these eggs, other eggs were centrifuged to separate
and identify particles by their stratified position and staining characteristics.
Further cytochemical tests on whole fixed eggs helped verify these findings.
MATERIALS AND METHODS
The marine polychaete annelid, Pcctinaria (Cistcnidcs} gouldii Verrill, after
Hartman (1941), is found in the mud-sand flats beneath shallow water beyond
the low tide mark. This fascinating worm lives in a beautiful cone-shaped tube
constructed of fitted sand grains, one grain in thickness, that are cemented together
with secretions from the cementing organ (Watson, 1928).
Specimens for this study were obtained from the Woods Hole area by the
Marine Biological Laboratory Supply Department. In the laboratory they were
kept either in a large container of rapidly running sea water or in a shallow tray
of sand that was submerged under running sea water. The animals usually
burrowed with the head downward, as in nature, and the tapered end of the tube
extended above the surface, especially at night. The adults did not survive long
in the laboratory once they were removed from their tubes.
Prior to shedding, the eggs of Pectinaria are freely suspended within the
coelomic fluid where they constantly shift back and forth with the pistonlike
movement of the adult female. Many developmental stages of the oocyte are
also present in the fluid. Following mechanical stimulation of the female within
the tube, the coelomic fluid and eggs are vigorously shed into the sea water where
the mature eggs continue maturation with breakdown of the germinal vesicle (GV).
In the male, sperm packets are released in an identical fashion.
Eggs so obtained were studied immediately after shedding, with the bright
field and dark field microscope. Other egg aliquots were shed directly into vital
dyes prepared in sea water to stain various participates. Alternatively, eggs were
dyed with vital fluorochromes and observed under the fluorescence microscope.
426 KENYON S. TWEEDELL
Wherever possible, the specific vital dyes used were designated according to the
revised color index numbers (CI).
Application of a single vital dye to the living egg often resulted in staining of
more than one constituent of the protoplasm. For this reason both the untreated
and dyed eggs were centrifuged in order to stratify and differentiate between
identically dyed particles. Thus particles could be classified from their relative
stratum after centrifugation and from numerous cross-checks with different dyes
or combinations of microscopy.
Because of the preparatory time, the mature eggs had already undergone GV
breakdown at the end of centrifugation but immature eggs were useful for studying
particle distribution in pre-breakdown stages. Immature eggs did not undergo
germinal vesicle breakdown on contact with sea water.
The eggs were layered over fresh sucrose (0.75 molar) in plastic centrifuge
tubes, in the proportion of one-fifth sucrose to four-fifths eggs and sea water.
The tubes were then placed in an ice bath prior to centrifugation. The eggs were
centrifuged either in a refrigerated centrifuge (0° C.) at 3,000 to 4,850 G for
one hour, in a high speed centrifuge at 17,000 G for 30 minutes at 4° C., or in
an insulated preparatory ultracentrifuge (6° C.) at 33,000 G for 12 minutes. This
enabled much sharper separation of granular bands than centrifugation at room
temperatures.
After centrifugation, the eggs were immediately placed in an ice bath imtil
the time of observation since lack of refrigeration allowed rapid "return" of the
stratified components.
For all cytological observations the eggs were transferred in a small drop to
the polished depression of a thin micro culture slide. A coverslip was then placed
over the well so that the drop was in contact with the depression and the coverslip.
The light source for both standard dark field and fluorescence microscopy of
the living eggs was a mercury vapor lamp (G. E. H4AB). The Osram HBO
200 W bulb was found too intense for extended observations of living eggs.
In dark field observations a noviol "O" filter (Corning no. 3060) was used
as an exciter filter at the light source to remove most ultraviolet, and a distilled
water cell or an infrared cut-off filter (Kodak no. 301) for absorbing the infrared.
For blue light fluorescence (dark field) the infrared cut-off filter and a violet
exciter filter (Corning no. 5113) were coupled to one of two barrier filters, a
yellow shade (Corning no. 3486) filter which permits observation of fluorescence
above 5100 A or a noviol "A" filter (Corning no. 3389) that transmits down to
4200 A.
Ultraviolet-induced fluorescence (dark field) was produced with either a blue
purple ultra filter (Corning no. 5850), a red purple corex A (Corning no. 9863)
or a Wratten ISA filter (Kodak). The same barrier and heat filters were used.
Selection of filters for fluorescence depended upon the properties of the particular
fluorochrome used.
A standard binocular microscope was equipped with a front surfaced aluminized
mirror. Special funnel stops were used in the 20 X, 40 X and 97 X objectives
for all standard dark field and fluorescence observations, to reduce haloing from
the relatively thick eggs.
Certain cytochemical reactions cannot be shown with vital dyes. In order to
broaden the chemical tests and substantiate those obtained from the eggs stained
VITAL STAINING OF PECTINARIA 427
/;/ vitro, eggs were fixed at 30-second intervals from the time of shedding to 20
minutes after shedding. Similarly, eggs were centrifuged at 33,000 G , removed
and fixed immediately at 0° C. with Kahle's fixative. The eggs were fixed directly
on coverslips, using the double coverslip sandwich technique, and then stained with
various cytochemical dyes.
Black and white photographs were taken on Panatomic X film with a Micro-
Ibso attachment (Leitz). Color photographs of fluorescence and dark field were
recorded on high speed Ektachrome (daylight) with a Zeiss attachment camera
with movable prism. When the latter film was used for dark field photographs,
a No. 3 gelatin filter (Kodak) was also used in conjunction with an infrared filter
to absorb the low blue illumination.
The author wishes to acknowledge the helpful assistance of Mr. Christopher
Watters whose unbiased suggestions were most useful during this research.
OBSERVATIONS
General appearance of the living egg
Prior to germinal vesicle breakdown the mature egg possesses a large germinal
vesicle about two-thirds the size of the egg diameter. A prominent nucleolus.
often double-lobed, is also present. The cytoplasmic ground color under visible
light is pale yellow-green bounded by a yellow-green cell membrane. The egg
measures 55 microns in diameter and is highly transparent although cytoplasmic
particles are apparent (see Fig. 1).
Under dark field the particles are resolved into varying sizes of constantly
agitated yellow-green granules. Smaller but extremely active silver-grey granules
are more diffusely scattered throughout the cytoplasm. These also occur in ir-
regular clumps around the nuclear membrane. The darker interior of the
germinal vesicle contains a smaller number of similar granules. Sparselv scattered
through the cortical cytoplasm are large yellow particles or clumps of granules.
Minute micro-villi project from the egg surface in a band covering the middle
two-thirds of the egg. They are conspicuously absent in opposite polar regions
and are much shorter in immature eggs. A living egg prior to germinal vesicle
breakdown is seen under dark field in Figure 2.
Germinal vesicle breakdozvn. The eggs of Pectinaria at the time of shedding
are slightly oval, flattened discs which almost invariably settle on their flattened
surface. The germinal vesicle conforms to the oval shape but the outline is
greatly scalloped during the first three minutes after shedding (Fig. 3). If the
eggs are observed laterally, they resemble a dish standing on its edge. At 3.5 to
four minutes post-shedding the germinal vesicle looses its scalloped edge and
takes on a firm elliptical shape. Soon afterwards irregular clumps of hiphly
motile, silver- white particles collect just outside of the membrane. The first
indication of membrane breakdown is a wrinkling of the membrane that resembles
the earlier scalloped appearance. Often, the nucleolus disappears at this time
(Fig. 4). From six to ten minutes after shedding, depending upon the season,
individual animals and temperature, the membrane opens, often on onnosite sides.
The silver particles flow through the gaps and form a temporary bridge, giving a
428
KEN YON S. TWEEDELL
FIGURES 1-12.
VITAL STAINING OF PECTINARIA 429
transitory "hour glass" shape to the degenerating vesicle (Fig. 5). The initial
breakthrough is followed by numerous irregular clumps of silver particles passing
into the interior. The entire process of membrane dissolution lasts for 1.5 to
three minutes (Figs. 6, 7). It is generally completed in all mature eggs 15 to 20
minutes after shedding. The nucleolus, if it has persisted, then gradually fades
after membrane disappearance.
Soon afterwards, an indentation in the cell membrane forms on one side of
the egg and beneath it a clear sac area develops. Here the first polar spindle forms
After disappearance of the germinal vesicle the egg has a small, dark irregular
center surrounded by clumps of silver granules, numerous yellow granules of
various sizes and points of fixed cortical granules (see Fig. 8).
Stratification of cell particles. Since dark field examination of the living egg
had indicated the presence of several types of cytoplasmic particles, they were
stratified by centrifugation. Living eggs were shed and allowed to undergo
germinal vesicle breakdown. After cooling in an ice bath they were centrifuged
for 15 minutes at 4000 G at 4° C. and examined immediately under dark field.
The cytoplasmic particles in Pectinaria consistently separated into three gen-
eral regions, as seen in Figure 9. At the centripetal pole, an oval mass of fine,
highly motile, silver white particles accumulated. This cap, referred to as zone A,
consisted of small oil droplets at the apex and fine lipid particles.
FIGURE 1. Recently shed oocytes prior to GV breakdown. Unstained, reduced natural
light.
FIGURE 2. A full size immature oocyte that did not undergo GV breakdown. Note the
perinuclear ring of granules and the bilobed amphinuceolus. Dark field. 540 X.
FIGURE 3. Newly shed (2.5 minutes) mature oocytes showing typical scalloped germinal
vesicle. Dark field. "375 X.
FIGURE 4. The same oocytes as in Figure 3 at 5.5 minutes after shedding. Germinal
vesicle has become firm. 375 X.
FIGURE 5. Mature oocytes just beginning GV breakdown at 9 minutes after shedding.
The large, bright granular clumps are not involved. An amphiaster is forming on the extreme
right. Dark field. 375 X.
FIGURE 6. The same oocytes as in Figure 5 at 10 minutes after shedding. Notice the
formation of cytoplasmic "bridges" across the old germinal vesicle. Dark field. 375 X.
FIGURE 7. Germinal vesicle breakdown nearing completion at 12 minutes after shedding.
Most of the silver white granules have flowed inward. Dark field. 375 X.
FIGURE 8. Mature oocytes after germinal vesicle breakdown and 15 minutes after shedding.
The oocytes remain in this condition until fertilization. Dark field. 540 X.
FIGURE 9. Oocytes before and after GV breakdown that were centrifuged, showing strati-
fication of cell particulates. In the post-breakdown egg is a crescent-shaped lipid zone (A),
a hyaline zone (B), and a centrifugal granular zone (C). At the extreme centrifugal end is a
flattened vacuolated zone. Dark field. 375 X.
FIGURE 10. Fluorescing oocytes stained with acridine orange. The nucleolus is just dis-
appearing at 10 minutes after shedding. Fluorescence : yellow, yellow green, and orange
cytoplasmic granules; dark green GV ; bright green nucleolus. 375 X.
FIGURE 11. Immature oocytes stained with thionin demonstrating cytoplasmic granular
uptake and slightly separated amphinucleoli, one staining reddish violet, the other remaining
unstained. Mature oocytes are adjacent. Natural light. 750 X.
FIGURE 12. Post-GV breakdown oocytes which were fluorochromed with acridine orange
and centrifuged (15,000 G). The centripetal pole is up in most eggs (dark green fluorescence).
The granular zone (C) consists of a centripetal bright yellow fluorescent band that is slightly
separate from a more centrifugal band of yellow green and orange granules. The centrifugal
pole fluoresces dark green. Filters: Corning 5113, 3486; Kodak 301. 375 X.
430 KENYON S. TWEEDELL
Moving centrifugally a hyaline mid-region, designated as zone B, appeared
black under dark field illumination. In the immature eggs, this area contained
the germinal vesicle or nucleus of the oocyte. In this region also were scattered
isolated clumps of fixed golden particles located in the cortical layer of the egg
since they did not shift their position with centrifugation. These large golden,
twinkling particles extended up into the silver lipid particles of the centripetal
cap and later evidence showed that they reached to the extreme centrifugal end
of the egg.
The lower centrifugal hemisphere of the egg, zone C, contained a heavy con-
centration of irregular sized yellow-green granules. Beginning near the equator
of the centrifuged egg, where a diffuse layer of fine granules, the mitochondria,
were detected, the granules of yolk increased in size and number toward the
centrifugal end of the egg.
At the extreme centrifugal end of the egg, a vacuolated area containing a few
dark granules was discernible.
The position of these stratified layers paralleled that found in the early
investigations on Chaetopterus by Lillie (1906).
These three general zones were also seen in the immature and developing
oocytes which still possessed a germinal vesicle. After centrifugation, a crescent-
shaped cap of silver particles rested on top of the germinal vesicle at the centripetal
pole. The germinal vesicle extended into the middle zone of the egg with the
nucleolus displaced toward the centrifugal end of the vesicle. Sometimes a faint
band of silver particles could be seen displaced halfway down within the vesicle
or displaced around the nucleolus. Centrifugal to the germinal vesicle a U-shaped
zone of mitochondria and yolk granules was found.
Staining the pre-breakdown egg
Earlier it had been found that living marine eggs could be fluorochromed with
vital fluorescent dyes (Tweedell, 1959) and that the fluorescent inclusions persisted
through subsequent cleavage and development. As a means of identifying these
fluorescent cytoplasmic participates, eggs of Pectinaria were freshly shed into a
variety of vital fluorochromes so that the inclusions could be studied before GV
breakdown. The general procedure for these dyes is outlined for acridine orange,
a vital fluorochrome that produces metachromasia in living tissues. Acridine
orange also has a specific affinity for DNA and RNA-proteins under controlled
conditions (von Bertalanffy and Bickis, 1956; Tweedell, 1960a).
Eggs were shed into a 0.001% solution of acridine orange (CI 46005) in
filtered sea water and allowed to stand for three minutes. They were then
centrifuged lightly, the stain decanted off and the eggs washed twice in fresh
filtered sea water to remove background fluorescence. This dye absorbs maxi-
mally at 4100 A and thus most observations were made with a violet exciter filter
that transmits maximally at 4100 A and a yellow shade barrier filter plus an infrared
filter. This combination gave a completely black background but provided green,
yellow and red fluorescence. Substitution of a noviol "A" barrier filter allowed
transmission of visible blue light that also permitted non-fluorescing components
to be seen, a procedure useful when centrifuged eggs were viewed under dark field.
When eggs were fluorochromed as above, the cytoplasm fluoresced pale green
VITAL STAINING OF PECTINARIA 431
and was filled with numerous yellow-green and orange granules of various sizes;
the entire egg was enclosed by an orange fluorescent cell membrane (Fig. 10).
The light yellow-green nucleolus, containing one or more non-fluorescing vesicles,
appeared within the dark green germinal vesicle, which was surrounded by a
perinuclear band of yellow-green granules.
About seven minutes post-shedding, the nucleolus disappeared and distinctive
bright green fluorescent clumps of a constant size and number, presumably
chromosomes, appeared within the germinal vesicle.
Following GV breakdown, the egg cytoplasm contained a scattered mixture of
yellow, orange and yellow-green granules. About 15 minutes after breakdown,
the bright yellow-green chromosomes reappeared in a tight knot near the center
of the egg in the first maturation metaphase. These two observations were the
first ones seen of the chromosomes in the living egg of Pectinaria.
Further substantiation of the identity of the chromosomes before and after
GV" breakdown came from the application of histochemical dyes to the fixed eggs.
The same assemblage of chromosomes could be seen in the intact egg following the
Feulgen reaction, staining with gallocyanin chrome-alum under conditions made
specific for nucleic acid concentration (Lagerstedt, 1949; de Boer and Sarnakcr.
1956, cited in Pearse, 1961) and with Galigher's haematoxylin.
The chromosomes were also very sharply defined in the fixed whole egg after
they were extracted with trichloroacetic acid and stained with Schiff's reagent.
Secondary fluorescence was also induced in the pre-breakdown egg with
neutral acriflavine (National Aniline). Since this dye absorbs at the same wave-
length as acridine orange, the same filters were used. Acriflavine has been shown
to have an in vivo affinity for intranuclear proteins (De Bruyn ct a!., 1953). A
basic dye, it combines with the phosphoric acid groups of the nucleic acids
(Brachet. 1957).
Eggs were shed directly into a 0.001^ solution of the dye in sea water.
In the living egg bright lime-green fluorescent granules were evenly dispersed
through the cytoplasm. Within the nucleus, the nucleolus fluoresced bright
yellow-green. Large vacuolated spheres were also seen within the nucleolus.
With the exception of the nuclear membrane, the interior of the nucleus, strangely,
remained unstained.
TJic nucleolus. The nucleolus in the mature oocyte was a large single body
with one or more eccentrically placed vesicles or vacuoles, differentiated by their
general lack of staining affinity. The body of the nucleolus fluoresced lime green
with acridine orange or acriflavine, dark green with Janus green and blue with
toluidine blue in the living egg. In fixed material, the nucleolus was Feulgen-
positive and stained dark purple with gallocyanin chrome-alum. The vesicles or
vacuoles remained unstained in each case.
In the younger oocytes the vesicles occurred as epinucleolar buds but in the
mature oocytes, they appeared to be intranucleolar vacuoles (Raven, 1958). Both
types existed. The immature oocytes that still possessed nucleoli after high speed
centrifugation (33,000 G) occasionally showed a telescoped chain of three, some-
times four vesicles.
In the less mature oocytes, the nucleoli usually appeared as double-lobed
amphinucleoli (Wilson, 1925), one lobe larger than the other. At times, the
432 KENYON S. TWEEDELL
nucleoli were separated but very often they were united. The principal nucleolus
often had one or more accessory nucleolar buds attached to it.
The chemical differences within the amphinucleoli were apparent after staining
in thionin, a basic vital dye that exhibits metachromasia. The smaller lobe gen-
erally stained deep reddish violet while the larger lobe remained perfectly clear
(Fig. 11). In mature oocytes, the single nucleolus never became stained although
this may have been a function of the short staining time before nucleolar breakdown.
The youngest oocytes, judged by their smaller diameters, generally possessed a
greater number of small separate nucleolar-like bodies. No attempt was made to
study the origin or development of these nucleolar bodies but it was noticed that
the small nucleolar vesicles in the very young oocytes fluoresced bright red in con-
trast to the bright green of the main nucleolar body, after treatment with acridine
orange.
Centrifugation of vitally dyed eggs
With the application of vital fluorochromes and other dyes to the pre-breakdown
egg it became apparent that many granules of diverse shapes and sizes were scattered
randomly throughout the cytoplasm. Many of these consisted of lipid and proteid
yolk granules. However, even the common position of these which stained with
different fluorochromes or dyes did not necessarily indicate that they were identical.
As a step toward resolving the mixture of cytoplasmic granules, the eggs were
vitally dyed or fluorochromed after GV breakdown and centrifuged at 30,000 G for
15 minutes in a precooled head maintained at 4° C. throughout the run.
When the eggs fluorochromed in acridine orange were examined under blue
light and a yellow shade filter, the principal fluorescence came from a heavy con-
centration of yellow or orange granules displaced toward the centrifugal pole in
zone C. The rest of the egg appeared deep green (Fig. 12). The extreme cen-
trifugal pole also contained an irregular agranular area that remained deep green.
In almost the exact center of the centrifuged egg, at the junction of the yolk
granules and the empty mid-region, a tight knot of lime green chromosomes was
often seen.
Substitution of a noviol A barrier filter revealed the silver blue cap of lipid
granules in zone A. The deep green background of the hyaline zone changed to
deep blue and the entire granular area in the centrifugal zone fluoresced violet-
orange.
Proteid yolk and mitochondria. With more extensive centrifugation a second
band of yellow-green granules was slightly separated from the main mass of orange
yolk granules nearer the mid region of the egg. This distinct band appeared at
various levels, depending upon the total centrifugal forces, but always rejoined
with the main centrifugal mass of granules shortly after centrifugation. The
fluorescence of these granules appeared to be identical to the perinuclear band of
granules seen in the pre-breakdown egg.
After centrifugation the apparent homogeneity of the cytoplasmic granules
stained with acriflavine also disappeared. A large concentration of yolk granules
was again located in the heavy centrifugal end (zone C) of the egg. Under U. V.
or blue violet light and a yellow barrier filter they fluoresced yellow green which
became more intense nearer the upper end at the equator of the egg. However,.
VITAL STAINING OF PECTINARIA 433
with blue violet illumination and a noviol filter that permitted blue violet trans-
mission, these granules were quickly differentiated into a more centripetal band
of brilliant, yellow fluorescent granules and a lower centrifugal zone of bluish
granules. Fluorescence in the more centrifugal part of the zone was masked by
the stronger transmitted blue light. Here was another indication that two general
groups of granules made up the large zone at the centrifugal end of the egg
(Fig. 13).
The majority of the granules concentrated in the centrifugal zone of the egg
consisted of proteid yolk granules of different sizes. These granules were gen-
erally readily stained with several fluorochromes and other vital dyes. Centripetal
to and overlapping the yolk granules in the equatorial zone of the egg was another
layer of granules that were often differentiated by color and particularly on the
basis of their fluorescence. The earlier cited centrifugation experiments of Harvey
(1939) and Costello (1939; 1958) on the annelid egg indicated that the fluorescent
band seen with acridine orange and acriflavine, just centripetal to the main mass of
yolk granules in the egg of Pectinaria, corresponded to the position occupied by
the mitochondria.
A separate yellow fluorescent equatorial band was also obtained after the ap-
plication of two other distinctly different cytoplasmic fluorochromes, both used in
the identification of fats (Popper, 1941). The first, thioflavine S (CI 49010)
with a peak absorption at 3650 A, was applied (0.0002^) to the living egg and
observed under U. V. In the germinal vesicle stage the cytoplasm was filled with
a mixture of yellow green granules. A separate perinuclear band of yellow green
granules surrounded the pale green nucleus and a light green nucleolus.
After centrifugation, both the U. V. and blue light illumination showed yellow
green granules confined to zone C in the centrifugal pole of the egg. When U. V.
light was coupled with a noviol O filter permitting full spectrum fluorescence, a
separate yellow green band of granules could be identified across the upper end of
the yolk granules in zone C. The greater mass of proteid yolk granules centrifugal
to the band appeared blue since the complex formed by the dye and the yolk granules
absorbed light at a higher wave-length.
This distinction was not just the result of differences in intensity of fluorescence.
A second fluorochrome, phosphine 3R (CI 46045), produced brilliant yellow fluo-
rescence in a wide band of granules around the germinal vesicle. After GV dis-
appearance, the vitally stained eggs were centrifuged and the whitish yellow granules
were exclusively located in a thick band along the upper margin of the yolk mass
in zone C. The rest of the granules did not fluoresce. This was the same area
demarcated by acridine orange, acriflavine and thioflavine. It suggested that the
difference in fluorescence was a qualitative measure of lipid content as well as
particle size.
The lower centrifugal granular portion of zone C could also be sharply delineated
by its fluorescence from the more centripetal fluorescent band with auramine O
(CI 41000). This fluorochrome was very successfully used for dying the tubercle
capsule (Richards and Miller, 1941 ) which is rich in neutral polysaccharides.
Application of a O.OOl^f solution of auramine O to the pre-breakdown egg
produced fluorescence extending as a broad band of brilliant yellow cytoplasmic
granules around the nuclear membrane.
434
KENYON S. TWEEDELL
15
19
1
16
20
FIGURES 13-24.
24
VITAL STAINING OF PECTINARIA 435
Following centrifugation, the yellow fluorescent yolk granules were only found
in the lower centrifugal region of zone C. The previous fluorescent band that
fluoresced with acriflavine, thioflavine, and phosphine appeared blue from trans-
illumination. Only the proteid yolk granules were activated by the U. V. illumi-
nation (Fig. 14).
The centripetal band of mitochondria in zone C was also clearly denned after
staining with other vital dyes. Both Harvey (1941) and Monne (1944) found
that gentian violet (crystal violet ) gave a very intense stain with mitochondria in
the sea urchin egg.
After staining the eggs with a O.OOOl'/c solution of crystal violet (CI 42555)
for 30 minutes and subsequent centrifugation at 33,000 G for 15 minutes, a sharp
blue band of granules appeared at the centripetal end of zone C. This corresponded
in position to the band seen previously with the fluorescent dyes (Fig. 15).
A temporary light blue ring was also seen just below the lipid cap in zone A
but this diffused rapidly into the lipid cap.
Mature eggs of Pcctinaria were next shed into another vital thiazine dye,
FIGURE 13. Post-GV-breakdown oocytes that were fluorochromed with acriflavine and
centrifuged. The centripetal pole (upper left) contains blue illuminated granules. The lower
centrifugal pole has a bright yellow band of fluorescent granules above the more centrifugal
bluish-yellow granules. Filters: Corning 5113, 3060; Kodak 301. 375 X.
FIGURE 14. Centrifuged oocytes that were fluorochromed with auramine and centrifuged.
The oval centripetal zone appears blue. In the centrifugal zone only the lower proteid yolk
granules fluoresce yellow. Filters: Corning 5850, 3389; Kodak 301. 375 X.
FIGURE 15. Centrifuged oocytes stained with crystal violet. The lipid cap at the
centripetal pole is surrounded by a band of light blue granules. Across the center is a blue
violet granular band, slightly separated from the centrifugal yolk granules. 375 X.
FIGURE 16. Centrifuged oocytes stained with Janus green. An intense layer of deep green
granules lies just below the equator. More diffuse grey green granules extend to the
centrifugal pole. 375 X.
FIGURE 17. Centrifuged oocytes stained with Nile blue sulphate. The wide mass of dark
blue granules at the centrifugal pole is slightly separated from an equatorial band of meta-
chromatic (reddish-purple) granules. 375 X.
FIGURES 18, 19. Post-GV-breakdown oocytes that were stained with Nile blue sulphate,
showing metachromatic astral granules. Deep blue yolk granules are scattered through the
blue cytoplasm. Natural light. Figure 18 shows eggs after recovery from centrifugation.
375 X. Figure 19 is an uncentrifuged egg. 860 X.
FIGURE 20. Centrifuged oocytes after GV breakdown. These eggs were fixed and stained
by the Nile blue test. A deep red lipid drop and red violet lipid cap occur at the centripetal
end. The hyaline layer is light blue. At the equator is a band of metachromatic granules and
scattered purple granules. The centripetal zone contains deep purple granules. 500 X.
FIGURE 21. Centrifuged eggs after GV breakdown, fixed and stained with Sudan black B.
Black lipid droplets and a lipid granular cap located the centripetal end. A faint granular ring
surrounds the lipid cap and a broad band of granules in zone C lies centripetal to the centrifugal
pole. The immature oocyte on the right shows similar stratification plus a thin granular zone
Avithin the germinal vesicle. 500 X.
FIGURE 22. Post-GV-breakdown oocytes stained with rhodamin O and centrifuged. The
pink band of granules centripetal to the lipid cap is faintly detectable. Bright yellow green
granules fill the centrifugal pole. Dark field. 375 X.
FIGURE 23. Pre-breakdown oocytes stained with toluidine blue O. A heavy concentration
of deep blue granules surrounds the nuclear membrane and grades out into the light blue
cytoplasm. 465 X.
FIGURE 24. Post-GV-breakdown oocytes stained with toluidine blue and centrifuged. A
heavy collection of blue granules occurs at the centrifugal end. Just centripetal to the unstained
lipid cap is a thin band of metachromatic granules. The hyaline layer is faint pink. 375 X.
436 KENYON S. TWEEDELL
thionin (CI 52000), also used by Harvey (1941) for the sea urchin egg. This dye
penetrated the living egg extremely slowly at all concentrations but did produce a
faint lavender band of particles just outside the nuclear membrane. During GV
breakdown, these particles collected along the indentations of the degenerating mem-
brane. The particles became diffusely scattered throughout the cytoplasm after GV
breakdown. Centrifugation failed to demonstrate any localized band of particles.
The immature eggs took up the stain readily, which first concentrated in the
perinuclear granules and the nucleoplasm. As mentioned, the most effective con-
centration of the dye occurred in the nucleoli (see Fig. 11).
To further identify and localize the mitochondria, Janus green B (CI 11050)
was applied to the living eggs. The specificity of Janus green for mitochondria has
been well substantiated in a variety of marine eggs. For example, centrifugation
experiments using Janus green as an index of mitochondria in eggs of Ciona in-
testinalis and Phallusia niainillata by Mancuso (1959), and La Spina (1958) all
showed the mitochondria localized in the centrifugal half of the egg between the
yolk and the centrifugal hyaline cap.
After eggs had been shed into a 0.001% solution of Janus green in sea water,
diffusely scattered granules with a rather faint blue green coloration appeared
throughout the cytoplasm. In immature eggs, there was a tendency for these
granules to clump around the nuclear membrane in a perinuclear ring. After GV
breakdown, they remained diffusely scattered in the cytoplasm.
Centrifugation of the eggs stained with Janus green generally showed that the
blue green granules were confined to a slightly concave area in the centrifugal end
of the egg. Rapid cross-comparison of the centrifuged egg under dark field with
the stained egg under bright field demonstrated that the concentration of Janus
green particles overlapped the heavy granular centrifugal portion of the egg. The
cortex around the granular area remained unstained and well defined. In addition,
strongly defined cortical granules were often seen to extend around the circumference
of the egg.
The position of the displaced granules was not always consistent in the cen-
trifuged egg. Often, a sharp, broad band of granules was localized across the
equator of the egg while more grey green granules extended toward the centrifugal
end. This heavy equatorial band always appeared in the centripetal portion of
zone C, and in these cases corresponded to the position where the mitochondria
usually stratified (Fig. 16).
Shaver (1957) found that Janus green was not satisfactory for demonstrating
mitochondria in the intact cells of sea urchin embryos. Under phase contrast he
observed they had a tendency to clump, indicating that a physical change took
place in the granular elements after dye penetration. A similar phenomenon may
have caused the inconsistent localization of mitochondria by Janus green in the
living egg of Pcctmaria after centrifugation.
The previous tests that differentiated the mitochondria from the proteid yolk
suggested the use of Nile blue sulphate on the living eggs. Nile blue sulphate
(CI 51180) is an oxazine dye used for histochemical differentiation of lipids. It
is a commonly used vital dye and exhibits metachromatic properties even in the
living egg. Monne (1944) had found that Nile blue stained the yolk platelets
blue in the sea urchin egg. However, blastulae of the sea urchin were vitally
VITAL STAINING OF PECTINARIA 437
stained with Nile blue sulphate by Gustafson and Lenique ( 1952 ) which enabled
them to follow the number and distribution of mitochondria.
The eggs of Pectinaria were shed into a 0.001 % solution and subsequent to GV
breakdown, the cytoplasm gradually became light blue and the yolk granules became
deep blue. In the immature eggs in which the nucleus had not broken down, an
elliptical band of heavy purple granules eventually appeared around the intact
nuclear membrane.
After centrifugation at 4160 G for 15 minutes, the post-breakdown egg showed
a heavy concentration of deep blue granules at the centrifugal end. Toward the
equatorial region of the egg, the granules of zone C formed a distinct band of reddish
purple granules. This band occupied the same position as the band of granules
seen with the previous fluorochromes. Under prolonged centrifugation at 33,000
G the band was distinctly separated from the yolk and remained in the mid region
of the egg. The centripetal cap in zone A remained unstained (see Fig. 17).
Astral granules. In the mature egg the metachromasia induced by Nile blue
sulphate did not become evident until after germinal vesicle breakdown and the
formation of the first maturation spindle. Upon standing in fresh sea water after
staining, many of the eggs exhibited two crescent-shaped groups of granules that
accumulated around each end of the clear spindle area near the equator of the egg.
These constantly agitated granules appeared about 45 minutes after shedding.
Under visible light they appeared reddish violet, in sharp contrast to the uniform
blue stained yolk granules. Their uniform position in all the eggs (Figs. 18, 19)
suggested that they were associated with the astral rays at each end of the maturation
spindle.
The same phenomenon was observed in the centrifuged eggs after the stratified
granules had begun to return to their original position. Within three hours after
centrifugation, identical groups of reddish violet granules were found in these eggs.
In many instances they were grouped around the first polar body which often
formed after centrifugation.
Extended observations of these eggs revealed that the astral granules originated
from the equatorial band of reddish granules in zone C.
Nile blue test. Fixed centrifuged eggs were next submitted to the Nile blue
histochemical test (Casselman, 1959) in order to verify the metachromatic staining
seen in the living egg. The granules in the lower part of zone C consistently
stained deep blue, the identical result found in the living centrifuged eggs. It also
suggested that these granules were primarily proteid yolk since the oxazine com-
ponent of Nile blue forms blue salts (Cain, 1947, cited in Casselman, 1959) with
fatty acids and phospholipids. The more centripetal band of zone C was again
filled with reddish purple granules. A non-acidic red reaction is created by the
oxazone component with glycolipids and simple lipids. It seemed likely that these
granules were rich in glycolipids.
A strong reddish purple reaction was also produced in the centripetal cap of
zone A. Just above this cap, at the extreme centripetal end of the egg, one or more
wine red lipid droplets collected after centrifugation, just within the cell membrane.
Very often they coalesced into a single red lipid droplet. Scattered between the
lipid cap in zone A and the centrifugal zone were sparsely scattered blue granules,
apparently located in the cortex (see Fig. 20).
438 KENYON S. TWEEDELL
It is well established that fatty substances are found in oocytes of many different
animals (Raven, 1958, 1961). These may occur as free lipids in the cytoplasm, as
fatty yolk globules or they may be contained within cell components such as the
mitochondria, Golgi bodies, etc.
One of the best cytochemical indicators of overall lipid distribution is Sudan
black B. Since the dye is actually dissolved in the lipid, but not in water, it must be
used on fixed material.
Centrifuged eggs were fixed in 10% formalin and 1% CaCL ; the eggs were
dyed with a 1% solution of Sudan black B in 60% triethyl phosphate (Casselman,
1959) and mounted in glycerin jelly (see Fig. 21).
At the centripetal end of the egg in zone A, the lipid cap stained black and was
surrounded centrifugally by a light grey band of fine granules. Either within
the lipid cap or just centripetal to it were one or two large intensely black droplets
formed by free lipids.
The hyaline zone was clear except for prominent black isolated cortical granules
extending the full length of the stratified eggs.
Moving centrifugally, a prominent band of dark granules surrounded the equator
of the egg. The position of this band was identical to the mitochondrial band
previously delineated by the fluorochromes and vital dyes. Very often a clear
indentation in the band indicated the presence of the maturation spindle.
The rest of the centrifugal zone consisted of lighter stained diffusely scattered
granules. At the extreme centrifugal end of the egg, another empty, vacuolated
area appeared overlaid by the prominent peripherally located black cortical granules.
The disposition of the mitochondria in the centrifuged egg was found to coincide
writh many of the granules that consistently fluoresced with lipophilic dyes (Richards,
1955; Metcalf and Patton, 1944). However, not all of the dyes showed that the
mitochondria were concentrated in the equatorial zone of the centrifuged egg. This
was the case after staining with rhodamin O (CI 45170), a vital dye and fluoro-
chrome that has been used in the identification of mitochondria (Lillie, 1948).
Under U. V. illumination, eggs shed and dyed in a 0.001% solution of rhodamin
in sea water revealed small, yellow orange particles evenly distributed throughout
the cytoplasm. These same particles under dark field illumination appeared
brilliant pink and had the additional advantage of being sharply differentiated from
the natural luminescence of the cytoplasmic particles seen under dark field.
After staining, eggs were centrifuged at 33,000 G for 15 minutes in a pre-cooled
head. The pinkish particles always formed a crescent-shaped band around the
centripetal end of the germinal vesicle in the immature eggs.
In the mature eggs after GV breakdown, the pink particles were concentrated
in a ring that was a part of the lipid cap in zone A. This ring formed a border
around the centripetal edge of the cap (Fig. 22).
The appearance of the pink peripheral band around the lipid cap indicated that
certain of the granules were distinctive from the general mass. This was also
suggested from the results after Sudan black application to fixed centrifuged eggs
which showed a thick peripheral band around the lipid cap. The specificity of
rhodamin O for these particles strongly suggested that they represented a second
type of mitochondria.
The ring was transitory in its appearance. A few minutes after the stratified
eggs were removed from the cold, the pink particles of the band would move into the
VITAL STAINING OF PECTINARIA 439
center of the lipid cap. Here they would form a diffuse oval of pink particles within
the center of the silver white particles.
A similar centripetal band of granules lying around the periphery of the lipid
cap was detected after fluorochroming with thioflavine.
Post-breakdown eggs were shed into a 0.0001% solution of thioflavine in sea
water, stained for 10 minutes and centrifuged at 33,000 G for 15 minutes. When
the preparations were excited with U. V. light (filters: 9863, 5970 or ISA) and ob-
served with a noviol O filter, a definite yellow green band appeared centripetal to
the blue violet appearing lipid cap. This band occurred in a position identical to
that taken by the pink granules seen with rhodamin O.
An identical band of granules was demonstrated in living eggs that were dyed
with toluidine blue. Eggs were freshly shed into 0.0001% solutions of toluidine
blue (CI 52040) in sea water. Within ten minutes a heavy concentration of dark
blue granules appeared around the germinal vesicle. This band graded off into
smaller sparsely scattered blue granules throughout the rest of the cytoplasm.
Around the periphery of the cell, just inside the cell membrane, a single row of
dark blue granules was also found ( Fig. 23 ) .
After GV breakdown the blue granules were dispersed toward the periphery of
the egg in the outer two-thirds of the cytoplasm. Upon prolonged staining in a
dilute solution of the dye, smaller red violet metachromatic granules became apparent
in the post-breakdown eggs. These were evenly distributed throughout the
cytoplasm.
The dark blue granules were easily concentrated into the centrifugal hemisphere
of the egg after centrifugation for 15 minutes at 4000 G. Here, the heavier yolk
granules and the more centripetal zone of mitochondria all stained the same. The
dark blue granules then graded off into the equatorial zone of the egg. The loca-
tion of the metachromatic granules was clearly defined in the more heavily cen-
trifuged eggs. Just centripetal to the clear yellow lipicl cap in zone A, a ring of
reddish purple metachromatic granules became concentrated (see Fig. 24). This
sharply defined band was easily seen with natural light and appeared to be identical
to the similar band of pink particles seen with rhodamin O under dark field
illumination.
Since the metachromasia of these particles somewhat resembled that seen after
eggs were stained with Nile blue sulphate, post-breakdown eggs were stained with
each dye and centrifuged concurrently. The position of the metachromatic granules
caused by toluidine blue was always quite distinct from those produced after
application of Nile blue sulphate.
TJic hyaline region
Relativelv few granules were seen in the hvaline region after centrifutration ex-
J O J O O
cept those that drifted in from either of the adjacent stratified layers. The cyto-
plasm did exhibit a deep green fluorescence with acridine orange and acriflavine,
thus suggesting a high nucleic acid concentration. Photographs of the centrifuged
sea urchin egg taken with ultraviolet light showed the greatest absorption in the
hyaline layer, thus indicating nucleic acid compounds in this layer (Harvey and
Lavin, 1944). Similarly, the clear area stained pinkish blue with toluidine blue
and pink with rhodamine B.
440 KENYON S. TWEEDELL
In the fixed egg, Nile blue sulphate colored the area light blue and Schiff's
reagent in the plasmal test gave it a bright pink-violet color.
The presence of extremely small particles, rich in nucleic acid, in this hyaline area
was indicated after the application of gallocyanin chrome-alum. The lower two-
thirds of the hyaline zone contained a diffuse collection of dark blue particles.
The centrifugal pole region
This irregular hyaline area, just centrifugal to the yolk mass, constituted an
enigma because of the apparent lack of particles present. With fluorescent dyes,
it gave the same fluorescence as seen in the more centripetal hyaline band. None
of the vital dyes indicated the presence of any granules.
However, in the Nile blue test on fixed eggs, this area appeared to be formed
from a heap of irregular vesicles, each having dark blue granules around their
borders. On the assumption that this area represented a structural phase of the
cytoplasm, fixed centrifuged eggs were stained with gallocyanin chrome-alum at
pH 0.8. The results indicated that a cone of very dark blue-black basophilic
granules occupied the centrifugal pole, probably embedded in the structural phase
of the cytoplasm.
DISCUSSION AND CONCLUSIONS
Each of the vital dyes and fluorochromes, when combined with centrifugation,
provided specific information about one or more cellular inclusions in the oocytes
of Pectmaria. From these results a composite picture of these inclusions in the
centrifuged cell was projected, based upon their specific staining properties and
stratification pattern.
In general, these investigations showed a stratification pattern as follows, mov-
ing from the centripetal to the centrifugal end of the egg: (1) oil droplets, (2) a
cap of lipid granules, (3) a diffuse layer of fine granules, (4) a hyaline zone with
the germinal vesicle (in immature eggs), (5) a broad layer of heavier granules, (6)
a zone of heavy yolk spheres, (7) an irregular vacuolated area with scattered
basophilic granules. Throughout the cortical region of the entire centrifuged egg
were isolated clumps of cortical granules. A composite diagram of the centrifuged
oocyte after germinal vesicle breakdown is shown in Figure 25.
The first inclusions at the centripetal pole were oil droplets that often fused into
one or two large drops under prolonged centrifugation. Sudan black B turned
them intensely black. These oil drops also stained bright red or pink when the
Nile blue test was applied to the centrifuged fixed egg, which indicated the presence
of neutral lipids.
The centripetal lipid granular cap reacted similarly. Sudan black formed a
black cap, Nile blue sulphate turned it reddish violet in fixed eggs, and it stained an
intense violet with Schiff's reagent in the plasmal test.
While the lipid cap could be easily seen under dark field or blue violet illumi-
nation, few of the fluorochromes used induced fluorescence in the lipid granules. An
exception was thioflavine which induced yellow green fluorescence in the lipid cap.
The granular distribution found by Harvey (1941 ) in the sea urchin egg and in
the annelid egg (1939) following centrifugation showed a mitochondrial band near
the mid-region of the egg, just centripetal to the yolk mass. Monne (1944) and
Monne and Harde (1951) noted that centrifugation of the living sea urchin egg
VITAL STAINING OF PECTINARIA
441
after staining with gentian violet or methylene blue resulted in the mitochondria
concentrating at the extreme centrifugal end of the egg.
However, electron microscope studies of stratified sea urchin eggs by Lansing,
Hillier and Rosenthal (1952) indicated that two layers of mitochondria stratified,
one of high density just above the yolk granule layer and one of low density in the
centripetal lipid layer.
In similar studies, Gross, Philpott and Nass (1956) reported that the mito-
chondria were concentrated in a layer centripetal to the yolk, yet they concluded
••-•::;:-v:M WV/.'- ;••:•••••
— 8
FIGURE 25. A composite diagram of the centrifuged oocyte of Pectinaria after GV break-
down. The stratified particles were stained by different vital dyes, fluorochromes and cyto-
chemical tests. The three zones of the unstained centrifuged egg (A, B and C) are indicated,
beginning with zone A at the centripetal pole. 1, Fat droplets. 2, Lipid granular cap. 3,
Granular band I. 4, Hyaline zone with diffuse granules. 5, Cortical granules. 6, Granular
band II, mitochondria and proteid yolk. 7, Dense proteid yolk. 8, Centrifugal vacuole with
basophilic granules.
that the mitochondria move to new positions both above and below the clear zone
(zone B). Shaver (1957) also reported particles likened to mitochondria lying
in an area just beneath the lipid cap (zone A).
The relative distribution of particles identified as mitochondria in ascidian eggs
seems to follow the same pattern. Reverberi (1956; 1957b) believes there are
probably at least two kinds of mitochondria ; one type is osmiophilic, does not stain
442 KENYON S. TWEEDELL
with Janus green and localizes near the centripetal pole. The second type gathers
above the equator and stains with Janus green. The position of the latter, how-
ever, conflicts with the finding of Mancuso (1959) and La Spina (1958) in eggs of
Phallusia.
This description agrees with the evidence presented here of a second mito-
chondrial band existing just below the lipid cap in the centrifuged eggs of Pectinaria.
The centripetal mitochondrial band is distinctive from the centrifugal band since it
selectively stains bright pink with rhodamin O. Moreover, an identical yellow
green fluorescent band centripetal to a blue lipid cap is also seen with thioflavine.
Both of these acid fluorochromes are lipophilic. The lighter band of granules does
not stain with Janus green B.
It should be noted, however, that a similarly transitory light blue band does
form with crystal violet. Harvey (1941) found that the latter stained the cen-
trifugal mitochondrial band purple (metachromatically) in the sea urchin egg.
A distinct band in the same position, just centripetal to the lipid cap and
separated by a thin, clear band, was also detected following application of Scruff's
reagent in the plasmal reaction.
More significantly, the centripetal band in the egg of Pectinaria reacted se-
lectively with toluidine blue, forming a characteristic metachromatic reddish-violet
band of granules just under the lipid cap.
Toluidine blue, a cationic dye, stains basophilic substances blue and reacts
metachromatically with certain chromotropes as a result of polymerization of the
basic dye (Michaelis, 1947).
The chemical interpretation of metachromasia is very tenuous without parallel
confirmatory tests and controlled reactions. However, the substances that the
metachromatic reaction detects are high molecular weight moieties with free anionic
groups. This includes anionic mucopolysaccharides, both DNA and RNA nucleic
acids and some anionic lipids capable of polymerization (Schubert and Hamerman,
1956). Some substances that cause metachromasia are heparin, chrondroitin
sulphate and hyaluronate.
In a survey of metachromasia with toluidine blue, Kelley (1954) found meta-
chromasia in eggs and ovarian tissue of many animal species, including Arbacia,
Chaetopterus and Spisula. Metachromasia generally occurred in the cytoplasm and
in the jelly around the eggs.
Metachromasia with toluidine blue was also noted by Dalcq (1957) in a study
of the ascidian egg and in the developing rat egg (Dalcq, 1954) where he found a
correlation between the localization of metachromatic granules and distribution of
mucopolysaccharides.
Similar metachromasia occurs in vivo in the molluscan eggs of Borneo, and
Gryphaea, and in the sea urchins, Psammechinus miliaris (Pasteels and Mulnard,
1957) and Paracentrotus lividns (Pasteels, 1958). In these developing eggs
stained with toluidine blue, they note very fine, blue granules (alpha granules)
which are uniformly distributed throughout the cytoplasm. Upon centrifugation,
these granules accumulate at the centrifugal pole. At the appearance of the
sperm aster, new granules (beta granules) appear which are strongly metachro-
matic. The beta granules are believed to derive their metachromasia from the
blue alpha granules when they become associated with the astral rays. With
VITAL STAINING OF PECTINARIA 443
strong centrifugation, the beta granules are stratified beneath the lipid cap. From
this and other evidence of acid phosphatase activity and acid mucopolysaccharides,
Pasteels and Mulnard identify the beta granules as mitochondria.
These metachromatic granules are thus like those seen in Pectinaria in two
respects, their reaction with toluidine blue and their position in the stratified egg.
However, the centripetal band of metachromatic granules in Pectinaria is not seen
associated with the astral rays.
Mulnard (1958) reported identical metachromatic a-granules and /^-granules
in the eggs of Chactoptcrus pergamentaccus after they were stained with brilliant
cresyl blue. He also described the presence of a third granule, material X, which
he believed was the precursor of the ^-granules.
The oocytes of Spisula solidissinia were vitally dyed with methylene blue and
toluidine blue by Rebhun (1959; 1960). Eggs left standing in dilute concentra-
tions of either dye contained metachromatic granules which were extensively
studied and analyzed. Rebhun found two sets of metachromatic particles, both
directly stainable with toluidine blue. These particles measured % to % micron
in diameter initially and were uniformly distributed through the cytoplasm.
At the time of spindle formation, during the formation of the polar bodies
or in the subsequent cleavages, Rebhun found that the metachromatic granules
became associated with the aster. Both sets of granules migrated directly into
the aster. This unusual behavior will be discussed in connection with Nile blue
sulphate.
When the oocytes were centrifuged before GV breakdown (8000 G for I*/*
to 4 minutes), the granules were mainly located in a narrow layer centrifugal to
the germinal vesicle. A few were found in the lipid cap and in the centrifugal
yolk area.
After GV breakdown, centrifugation stratified the metachromatic granules in
two locations, a layer at the centripetal end of the hyaline zone and a layer at the
centrifugal end of the yolk area.
Rebhun equates the centripetal particles with the ^-granules of Pasteels (1958)
on the basis of their location in the centrifuged egg and their migration into the
asters. He also identifies the centrifugal particles with the a-granules of Pasteels
ct al. but these are not astrally located according to Pasteels and Mulnard (1957).
On the basis of their metachromatic staining and stratification in the centrifuged
egg, the centripetal granules in the oocyte of Pectinaria appear to be very similar
to the centripetal layer of metachromatic granules that Rebhun finds in Spisula.
However, the failure to see the metachromatic granules in Pectinaria associated
with the asters during the first polar body formation cannot be explained from
the present findings. It is possible that they were not detected or that they do
not appear until after fertilization.
Demonstration of the proteid yolk particles in the living egg was easily accom-
plished even though the dyes were not always specific. Proteid yolk fluorescence
was orange with acridine orange, yellow green with thioflavine and yellow with
acriflavine. These granules were readily vitally stained pink with neutral red, deep
blue with toluidine blue or Nile blue sulphate.
The reaction of similar dyes in the sea urchin led Monne (1944) to suggest
that the yolk is composed of a phosphoprotein combined with lipids. Later, dif-
444 KENYON S. TWEEDELL
ferential staining of various polysaccharides caused Monne and Slautterback (1950)
to conclude that the yolk probably consisted of aminopolysaccharide combined
with a protein and lipid.
Raven (1958) subdivides the yolk granules of Limnaca into fatty yolk, con-
sisting of free lipids and fat globules, and proteid yolk. The latter, composed of
two granular types, is rich in mucopolysaccharides.
The specificity of auramine for the proteid yolk granules in the eggs of Pectinaria
indicates the presence of mucopolysaccharides, probably in loose combination with
proteins.
Just centrifugal to the hyaline layer in the pre-and post-GV-breakdown egg of
Pectinaria, an equatorial group of granules is concentrated into a band just centrip-
etal to and overlapping with the yolk granules. The identification of a similar
band in centrifuged eggs of the sea urchin (Harvey, 1939, 1941, 1944) and in the
eggs of Chactopterus (1939) and Nereis ( Costello, 1939, 1958) strongly suggests
that the band is rich in mitochondria.
Differential fluorescence of this granular band was produced with thioflavine,
acridine orange, acriflavine and phosphine. Crystal violet also selectively stained
it. Less specific but positive identification was obtained with Janus green and
Sudan black B.
In the sea urchin egg Harvey (1941) was able to stain the mitochondria
with both gentian violet and Janus green. Monne (1944) also reported mito-
chondrial staining with gentian violet.
The specific staining of similar stratified granules in Pectinaria by Janus green
and gentian violet suggests the presence of mitochondria in this band.
Induced fluorescence of granules in the same stratified position by acridine
orange, acriflavine, thioflavine and phosphine suggests that these fluorochromes
are also staining the mitochondria. In particular, thioflavine, which is lipophilic,
also induces fluorescence in the mitochondrial band just centrifugal to the lipid
cap.
The possibility still exists that the induced fluorescence is caused by similarly
localized but not identical granules since the mitochondria do overlap with the yolk
granules. In the centrifuged eggs of Limnaca stagnalis. Raven (1958) also finds
a mixture of mitochondria and y-granules, one type of proteid yolk, in an analogous
position. While the fluorescence of this band of granules with the above fluoro-
chromes under the described conditions appears to be specific, the exact nature
of the granules fluorescing requires further verification.
In the living eggs of Pectinaria Nile blue sulphate produced striking meta-
chromasia of granules that localized in a band corresponding to the position of the
mitochondria. This was also confirmed in whole fixed centrifuged eggs. The
metachromasia produced indicated the granules contained glycolipids.
In Pectinaria the astral granules also have their origin in the metachromatic
granules produced by Nile blue sulphate. It would be tempting to assume that
the metachromatic astral granules were mitochondria. Gustafson and Lenicque
(1952) used Nile blue sulphate to follow the mitochondria in the developing sea
urchin egg. They postulated that the stain adhered to the lipid-rich sheath that
surrounds the mitochondrium. However, they did not report that the granules
were metachromatic.
VITAL STAINING OF PECTINARIA 445
Raven (1958) indicated that the mitochondria in Liinnaca stagnalis gather
around the maturation spindle and often migrated in between the astral rays. He
also noted that the aster was surrounded by the centripetal -/-granules that were
distinct from the centrifugal proteid yolk granules.
Except for their stratified position in the mitochondria! layer, there is not too
much evidence to indicate that the metachromatic astral granules in Pectinaria are
mitochondria. They also differ from the granules stained with mitochondrial dyes
and fluorochromes in that the astral granules are only seen with Nile blue sulphate.
In addition to the metachromatic astral granules in Pectinaria, similar granules
have been seen in related eggs by others.
Taylor (1931) described natural red granules dispersed through the cytoplasm
of the echiuroid, Urechis canpo. In the centrifuged egg they gathered in clusters at
the extreme centrifugal end of the egg. When the amphiaster formed during ma-
turation and cleavage, in the normal or centrifuged egg, the granules migrated along
the astral rays and finally surrounded the nucleus of each daughter cell.
lida (1942) also followed particles stained with neutral red along the astral rays
and along the mitotic spindle in the sea urchin egg. These granules were also
distributed to the daughter cells.
In this connection, Lillie (1906) observed that neutral-red-stained granules col-
lected in a ring around the first maturation spindle in Chaetapterus oocytes. He in-
dicated that they were derived from granules composing the "residual substance" or
the germinal vesicle.
It is fairly certain that this is not the case in oocytes of Pectinaria. However, a
thin layer of granules, perhaps equivalent to the residual substance, does stratify
within the germinal vesicle. It is possible that the latter granules are those which
layer just centrifugal to the lipid cap after GV breakdown in Pectinaria (see
Fig. 26).
Vitally stained granules were observed by Kojima (1959a, 1959b) in several'sea
urchin eggs and in the egg of Urechis iinicinctus. In the fertilized eggs that were
stained with neutral red. toluidine blue, Janus green or Nile blue sulphate, deeply
stained granules appeared around the aster. In the unfertilized egg the granules
collected around the germinal vesicle. After strong centrifugation, the granules
displaced into the centrifugal pole of the egg.
It was difficult to equate the metachromatic astral granules of Pectinaria with
these varied findings. First, the nature of the granules was not clearly established.
The neutral-red-staining granules seen by Lillie were obviously different. Those
reported by lida and Kojima wrere not observed in Pectinaria. The data of Kojima
for Nile blue sulphate and toluidine blue indicated different levels of stratification
and lack of metachromasia.
In Spisitla oocytes Rebhun (1959) found two types of metachromatic particles,
the centripetal ^-particles already referred to and the a-particles which layered in
the centrifugal hemisphere. Both types migrated into the asters and each was
excluded as being mitochondria.
It is not likely that the metachromatic granules produced by Nile blue sulphate
in Pectinaria can be identified with the a-particles seen with toluidine blue in
Spisula. First, the two dyes in question stain different particles in the eggs of
Pectinaria. Secondly, the two sets of particles stratify in different layers upon
446
KEN YON S. TWEEDELL
centrifugation, the astral granules being derived from a layer that is coincident
with the mitochondria in Pectinaria. This is not the case in Spisula.
In electron microscope studies, Rebhun (1960) identified the metachromatic
granules as multivesicular bodies and concluded that the metachromatic granules
were definitely not lipid, mitochondrial, yolk or cortical granules. Two other com-
ponents were present, the Golgi bodies, particularly plentiful in early oocytes, and
annulate lamellae which occasionally orientated with the asters. It is possible that
one of these particles is analogous to the astral granules of Pectinaria.
The nucleolus of the mature oocyte of Pectinaria, characteristically an amphinu-
cleolus, was strongly basophilic in the cortical region but the nucleolar vacuoles re-
mained unstained (Fig. 26). All of the vital dyes and fluorochromes applied to the
oocytes indicated that the body of the amphinucleolus was principally composed of
DNA. This was confirmed after treatment of the fixed eggs with the Feulgen
FIGURE 26. A centrifuged immature oocyte, showing particle distribution in relation to the
intact germinal vesicle. 1, Fat droplets. 2, Lipid granular cap. 3, Hyaline zone. 4, Germinal
vesicle substance. 5, Basophilia. 6, Amphinucleolus. 7, Granular Band I. 8, Granular Band
II, mitochondria and proteid yolk. 9, Dense proteid yolk. 10, Centrifugal vacuole.
reagent. When the eggs were previously extracted with 4% trichloracetic acid at
90° for 15 minutes, the nucleolus remained unstained following the Feulgen test.
Kobayashi (1953, 1954) observed similar amphinucleoli in the eggs of the
oyster (Ostrea lapcrousi} which were Feulgen-positive. He also found that the
karyosome stained with methyl green and the plasmosome with pyronin, indicating
the presence of RNA.
Another type of amphinucleolus was reported by Sawada and Murakami (1959)
in Mactra veuerifonnis. In this form, nucleolus 1 stained deeply with pyronin
while nucleolus 2 gave a weak reaction with pyronin.
Attempts to demonstrate RNA in the non-staining intranucleolar buds of the
mature oocytes in Pectinaria were unsuccessful, while in very young oocytes both
epinucleolar and intranucleolar buds fluoresced bright red with acridine orange and
VITAL STAINING OF PECTINARIA 447
stained metachromatically with thionin. Acridine orange stained RNA nucleoli in
tumor cells bright red (Tweedell, 1960a).
Changes in the amphinucleoli during the growth phase are well known in mol-
luscan eggs; Raven (1958) and Wilson (1925) report on numerous cases of
variation in the amphinucleoli of annelids, molluscs and arthropods. This appears
to be the case in the nucleoli of the developing oocytes of Pectinaria.
SUMMARY
1. Living eggs of Pectinaria gouldii were stained with vital dyes and vital
fluorochromes before and after germinal vesicle breakdown. Observations were
made with the bright field, dark field and fluorescence microscopes. Changes in
the germinal vesicle, nucleolus and chromosomes of the living eggs were followed.
2. Other eggs were vitally dyed and centrifuged in order to stratify the cell
participates. Cell granules were identified, based upon their specific staining and
their stratified position in the centrifuged egg. These included lipid droplets, lipid
granules, mitochondria, proteid yolk, basophilic and cortical granules. Limited
cytochemical tests were made to verify their identity.
3. Two kinds of metachromatic granules were seen. With toluidine blue, the
granules occur at the centripetal pole just beneath the lipid cap. When Nile blue
sulphate was applied, a different metachromatic band stratified just centripetal to
the heavier yolk granules. Astral granules originate from the latter metachromatic
band and became associated with the first maturation spindle.
4. The fluorescent cell components included cell and nuclear membranes,
nucleoli, chromosomes, lipid granules, two types of yolk granules and mitochondria.
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OXYGEN UPTAKE IN SHORT PIECES OF TUBULARIA STEMS
JAMES A. MILLER, JR., LORALEE L. PHILPOTT AND C. LLOYD CLAFF
Department of Anatomy. Tnlane University, New Orleans 18, La., Single Cell
Research Foundation, Woods Hole, Mass., and the Marine
Biological Laboratory, Woods Hole, Mass.
Short pieces (\-ll/2 mm. long) of Tubularia stems reconstitute miniature hy-
dranths or partial hydranths. Unusually long periods are required for reconstitu-
tion of these pieces, and hydrogen ion estimates with indicators show low pH of the
coelenteric fluid of such pieces (Miller and Miller, unpublished data). Since in-
creased O2 in the surrounding sea water increases the number of pieces which re-
constitute and the size of the organ primordia, it became of significance to determine
whether or not the oxygen uptake of 1- and 1^-mm. pieces was depressed as
compared with that of longer stem segments.
In addition, the previously published studies on oxygen uptake in Tubularia
(Hyman, 1926; Earth, 1940b; Sze, 1953) have resulted in a certain degree of con-
fusion regarding the role of oxygen in reconstitution. On the one hand, whether
or not a hydranth develops and the size of the hydranth which reconstitutes at a cut
surface depend upon the oxygen available to the stem (Earth, 1938; Miller, 1937,
1942; Zwiliing, 1939); on the other hand, oxygen uptake measurements during
reconstitution failed to show that reconstituting stems used an appreciable amount
of oxygen more than stems with both ends ligatured (Earth, 1940b). Since recon-
stitution in Tubularia involves cell migrations (Bickford, 1894; Steinberg, 1955),
dedifferentiation and redifferentiation (Bickford, 1894), all processes which require
energy, logic would demand an appreciable increase in O2 uptake during these ac-
tivities. Likewise, there were certain technical problems in both Earth's and Sze's
methods which made it desirable for their results to be checked in another labora-
tory. Therefore, a new study of oxygen requirements in Tubularia was initiated
in the summer of 1961 using the cartesian diver equipment of the Single Cell
Research Foundation.1
MATERIALS AND METHODS
Specimens of Tubularia crocca were collected in the region of Woods Hole,
Mass., and were maintained in aerated running sea water in the laboratory. During
the first half of the summer they came from the warmer waters of the south side
of Cape Cod. During the last half they were collected from the Cape Cod Canal
where the temperature seldom rises above 15° C.
Straight stems of uniform thickness were selected from a single bunch of the
stock supply and placed in filtered sea water to which had been added 16 X 10' ;>
gm./ml. streptomycin to provide bacteriostasis. The stems remained in this solu-
tion in a cold room (18° C.) for at least 12 hours. The hydranth plus 5 mm. of
the stem were then removed and the required length of stem was cut from the
1 Reported by abstract : Biol Bull, 121 : 398, 1961.
450
OXYGEN UPTAKE IN TUBULARIA
451
region immediately proximal. Since the accuracy of the results depended upon the
accuracy of the measurements of length and diameter of the stem segments, the
sizes of the pieces were checked under a microscope with an ocular micrometer, and
stems which varied were discarded.
Oxygen uptake measurements were made in the ClafF modification of the
Holter (1943) and Linderstro'm-Lang (1943) cartesian diver apparatus with 7 flo-
tation tubes suspended in a water bath maintained at 18° C. ±.01° C. For each
series of measurements six of the tubes contained divers with tubularian stems and
the seventh contained an unfilled diver to act as a check on the equipment. A
"braking" pipette (Claff, 1947) was used to insert stem pieces plus 2 mm.3 of in-
cubating solution into the divers. The necks of the divers were sealed with 2 mm.3
NaOH to absorb CO2 and 2 mm.3 paraffin oil to prevent diffusion of gases. After
sufficient time for the divers to equilibrate, manometer readings were taken every
10 minutes for a minimum of two hours.
RESULTS
Since oxygen uptake data obtained on single 3-mm. stems were used as a stand-
ard of reference, in each set of measurements at least one diver contained a 3-mm.
stem, the "control." In the various experiments to be described the oxygen up-
30
26
22
16
14
10
Tubularia From
Warm Water
28.1
868J6
MEAN
20.8
MEAN
10
1mm M)
11010116
1 mm (3)
901110812
3mm(1)
FIGURE 1.
452 JAMES A. MILLER, JR., LORALEE L. THILPOTT AND C. LLOYD CLAFF
22
16
14
- 10
I 2
Tubularia From
Cold Water ies
us
MEAN 118
MEAN
18.1
181
MEAN
~rr
Ul
lt.9
1*J 16.1 MEAN
!
Expf. -* 1517131614
1mm (1)
15151414161716 171516
l/imm (2) 1mm(3)
FIGURE 2.
15151619131417
3mm (1)
10 191919
3 mm (2) 6mm 1 1 ]
takes of the following were compared with that of a single 3 -mm. stem: (1) a
single 1-mm. piece, (2) three 1-mm. pieces, (3) two 1^-mm. pieces, (4) two
3-mm. pieces and (4) one 6-mm. piece (Figs. 1, 2).
Table I summarizes the O2 uptake measurements on 43 stems of various lengths
from 1 mm. to 6 mm. and measured either singly (Columns 1, 4, 6), in pairs
TABLE I
Oa UPTAKE OF 1,1)2. 3 AND 6 MM STEMS OF
TUBULARIA
Source
of Stems
Total Stem Length 3mm
Total
1mm
Total Length 6mm
3mm I'/imm 1mm
1mm
3mm 6mm
o i i i g i
O
« ) D 1
Warm Wafer
(WOODS HOLE]
17.9 20.8* 17.1
6.7
Cold Waler
(C.COO CANAL)
11.1 11.8 11.2
6.2
16.1* 16.1
MEANS
14.2 13.0 14.9
6.4
16.1* 16.1
Column
1 2 3
4
5 6
* Tentative: More data needed
OXYGEN UPTAKE IN TUBULARIA
453
(Columns 2, 5), or in threes (Column 3). As may be seen in the averages little
difference was found between the oxygen uptake of one 3-mm. stem (Column 1),
two ll/2-mm. stems (Column 2) or three 1-mm. stems (Column 3) in the same
diver. However, when only one 1-mm. stem was placed in a diver (Column 4),
it used nearly half as much as three 1-mm. stems. Similarly, although two 3-mm.
stems (Column 5) used the same amount of O2 as one 6-mm. stem (Column 6),
a single 3-mm. stem used f as much as two 3-mm. stems or one 6-mm. stem.
Table I also shows that the stems collected early in the summer (from Woods
Hole) had appreciably higher O2 uptake than those collected from Cape Cod Canal,
although the general relationships between the effects of size of the piece and
crowding appeared to be similar. In order to assess the validity of these im-
pressions the data in each series were calculated as per cent of the O2 uptake of the
T A BLE TJ
UPTAKE IN PERCENT OF OXYGEN USED BY SINGLE 3MM STEM
Source
of Stems
Total Length 3mm
3mm 1}{ mm 1mm
Total Length
1mm
Total Length 6mm
3mm 6mm
a ) o ID ) CDOO
r~)
0)5 ^
Warm
Wafer
(WOODS
HOLE)
100% 125%* 01 %
36%
Cold
Wafer
(C.COO
CANAL)
100% 110% 116%
51 %
152%* 152%
MEANS
100 */0 112% 100%
44'/0
152%* 152%
k Tentative: More data needed
3-mm. stem which served as "control" for that series, and these are summarized in
the next table.
In Table II it is seen that two 1^-mm. stems used 12% more O2 than one
3-mm., and that three 1-mm. stems used the same amount as the one 3-mm. stem.
One 1-mm. stem used 44% of that used by one 3-mm. and two 3-mm. stems or one
6-mm. stem used 152% of that of the 3-mm. control.
Since the data for stems from cold water are more complete than those for
stems from warm water, the former were used for a further analysis of the situ-
ation. If the cut surface were the determining factor in O2 uptake, the 1-mm.,
3-mm., and 6-mm. pieces should have the same uptake since they all have two cut
surfaces; instead, the actual measurements give a 1:2:3 ratio. Likewise, two
1^2-mm. pieces should use two times as much O2 as one 3-mm. piece and three
1-mm. pieces should require three times that of one 1-mm. piece. The actual
findings were 10% and 61% increases, respectively.
454 JAMES A. MILLER, JR., LORALEE L. PHILPOTT AND C. LLOYD CLAFF
If O2 uptake were equal along the entire length of stem, all of the 3-mm. com-
binations should give the same uptake. The observed findings of 110% for two
lj/2-mm. pieces and 116% for three 1-mm. pieces could perhaps be reconciled to
this hypothesis ; however, the observed uptake of the single 1-mm. stem is too high
(51% instead of 33% of the 3-mm. uptake) and that of two 3-mm. stems or one
6-mm. stem is too low (152% of the control uptake rather than 200%).
Table III shows the precise relationship between O2 uptake and length of stem
when calculated per cut surface. In the upper row it is seen that each cut surface
of a 6-mm. stem was associated with an uptake of oxygen which was \l/> times that
of a 3-mm. stem and 2l/2 times that of a 1-mm. stem. In the second row it is seen
that when there were four cut surfaces for 6 mm. of stem (i.e., two 3-mm. pieces)
TABLE IE
*
OXYGEN-UPTAKE PER CUT SURFACE
No. of Cut
Surfaces
Length of Stem
6mm 3mm 1mm
8.1
4.0
5.6
3.0
1.9
3.1
* mm3 x 10" ; per hour
the uptake per surface was reduced, as was the case with a 3-mm. piece cut into two
1/4-mm. pieces. However, oxygen exchange per cut surface again was greater in
the longer than the shorter pieces. That this was the result of a true inhibition of
oxygen consumption was shown when the uptake per cut surface of two 3-mm.
pieces in a diver was compared with that of a single 3-mm. piece (4 as compared
with 5.3 mm.3 X 10~- per hour) and that of two ll/2-mm. pieces or three 1-mm.
pieces compared with that of a single 1-mm. piece in a diver (3.0 or 1.9 as compared
with 3.1).
The influence of total volume of tissue upon oxygen uptake is shown in Table
IV, in which the data on the stems from cold water have been calculated on the
basis of uptake of oxygen per millimeter of stem length. These show that when
the distance between the two cut surfaces is great the average uptake is small, when
the distance is small the average uptake is large. The data are not sufficient to
OXYGEN UPTAKE IN TUBULARIA
455
quantitate the differences in oxygen requirements of the \-\l/2 mm. at the two
ends of the stem which have been activated by exposure to oxygen and the inter-
vening non-activated stem. However, the difference between the uptake of single
6-mm. stems and single 3-mm. stems (16.1 minus 11.1 mm.3 X 10"-) suggests that
under the conditions of the experiment the non-reconstituting parts of the stem con-
sume oxygen at the rate of something in the order of 2 mm.3 X 10~2 per millimeter
length as compared with 5l/> for the ends. Further studies are planned in ordei
to verify this finding.
The lower half of Table IV again shows the inhibitory effects of increasing
the number of cut surfaces per mass of tissue when confined in a small volume of
TABLE IZ
OXYGEN UPTAKE PER MILLIMETER
LENGTH OF STEM*
No. of Cut
Surfaces
Lenglh of Stem
6mm 3mm 1mm
2.7
2.7
3.7
4.7
4.5
6.2
* mm3 x 10"* per hour
fluid. Because oxygen uptake depends on oxygen concentration of the medium
(Earth, 1938), calculations were made to determine the volume of oxygen in the
divers at the beginning of the period of measurement. Since the mean volume of
the divers was 68.85 mm.3, they contained approximately 13.77 mm.3 of O2. Even
using a rate of O2 uptake of 30 mm.3 X 10^2/hr. (higher than any which has been
measured), the oxygen in the diver would suffice for 46 hours. Therefore, hypoxi;i
could not have contributed to the reduction in uptake.
Since CO2 is a potent inhibitor of reconstitution, calculations were made to de-
termine whether the volume of NaOH solution in the necks of the divers was ade-
quate to absorb the CO, liberated. Using the same figure of 30 mm.3 X 10"2/nr-
456 JAMES A. MILLER, JR., LORALEE L. PHILPOTT AND C. LLOYD CLAFF
O2 uptake the calculations showed that the 2 ml. of NaOH could absorb C(X for
75 hours before becoming exhausted. This indicates that some inhibitor other than
CO2 liberated by the cut ends of the stems was responsible for the O2 depression.
Since it has been demonstrated that low pH inhibits reconstitution (Goldin, 1942)
and that pH-lowering substances are released during reconstitution (Miller, 1948;
Miller and Miller, unpublished data), it is suggested that these substances may
depress the O., uptake in the divers containing two or more pieces.
DISCUSSION
1. Oxygen uptake and reconstitution
Barth (1940b) was unable to find any differences in CX uptake of "regenerating"'
stems (with open ends) and "non-regenerating" stems (with both ends ligatured)
and concluded that very little oxygen was used in regeneration, even though his
earlier studies had shown that the process was highly oxygen-dependent. How-
ever, there are aspects of his technique that make his findings difficult to interpret.
His determinations were made in a Warburg apparatus which was shaken during
the measurements. This so greatly increased the oxygen available to the stems
that ligatured stems can hardly be considered as resting stems. Indeed, he re-
ported (p. 372) that 50% of one group of ligatured controls formed hydranths.
Under ordinary circumstances ligatured stems do not show any visible signs of
reconstituting. Therefore, it is possible that the lack of difference in oxygen uptake
between ligatured and non-ligatured stems could be attributed to an artificially
elevated uptake in the ligatured stems, caused by the shaking in the Warburg ap-
paratus. In spite of this possibility Earth's conclusions regarding oxygen uptake
in reconstitution have been widely quoted and have been incorporated in theories of
regeneration (Barth, 1940a, 1944; Spiegelman, 1945; Steinberg, 1954, 1955).
In 1953 Sze reported a study of oxygen uptake in Tubnlaria stems using carte-
sian divers. His technique avoided the problems raised by shaking but encountered
other problems which again complicate the interpretation of the results. He found
it necessary to make his uptake measurements at a temperature of 25° C. even
though the stems had come originally from colder water and had been kept in the
laboratory at 15° C. Tubnlaria colonies from colder water that are brought into
a laboratory at 25° C. lose their hydranths and may even cytolyze (Moore, 1939).
Stem segments are less sensitive than hydranths, but can hardly be considered as
normal under such conditions of temperature stress.
In addition, both Barth and Sze reported their measurements in mm.3/hr./10
mg. dry (or in some cases wet) weight. Although theoretically this should be
the most precise procedure, in the case of Tubnlaria the presence of the chitinous
perisarc introduces a complication which negates its advantages. Since the non-
living perisarc far outweighs the metabolizing tissues of stem, calculations based
on weight will contain a large error if there are differences in thickness in different
parts of the perisarc. Such differences are slight and probably can be safely dis-
regarded in short pieces from adjacent regions. However, the thickness of the
perisarc increases proximad and the differences become appreciable in sections
only a few millimeters apart. To avoid this complication, in the present study the
lengths and diameters of the stems were measured under magnification and O,
uptake comparisons were made on the basis of units of stem length.
OXYGEN UPTAKE IN TUBULARIA 457
Our data on the oxygen uptake of pieces less than 3 mm. long do not offer much
assistance in resolving the question of whether or not reconstitution is accompanied
by an appreciable alteration in oxygen uptake. However, ciliary activity, produc-
tion of pH-lowering substances and the subsequent differentiation of a hydranth all
indicate that under ordinary conditions, from 1 to \l/2 mm. of stem subjacent to the
cut surface is involved in the activation which follows sectioning. On this basis,
the difference in uptake between a 3-mm. and a 6-min. stem was used to compare
the uptake of the peripheral 3 mm. with the interior 3 mm. This showed that the
average oxygen uptake per millimeter of stem at the ends of the stem was two
times that at the middle (10.6/3 or 3.5, as compared with 5.5/3 or 1.8). We have
evidence (unpublished) that there is balance during reconstitution between the
level of oxygen available to the cells and the level of pH-lowering substances which
accumulate in the stem and which are inhibitory (Goldin, 1942). Because of coe-
lenteric circulation, the concentration of these inhibitors is lower in long than in
very short stems (Miller, 1948; Miller and Miller, unpublished data). Therefore,
it is entirely possible that when longer stems are measured, the uptake of the re-
constituting ends will be found to be appreciably greater than two times that of
the resting stem tissue. However, the important fact remains that the measure-
ments reported here bring the changes in oxygen uptake following cutting into a
rational relationship to the well known dependence of reconstitution upon oxygen
which Barth demonstrated so clearly in 1938. The measurements reported here
also accord with studies on the energy requirements in Corymorpha, a related spe-
cies which has a naked stem (Child and Watanabe, 1935), in hydranth development
in Tubularia embryos (Miller, 1946) and in embryological processes and regener-
ative phenomena in general (Child, 1941).
2. Oxygen uptake in 1-inin. pieces
Very short pieces present an interesting complication. Since their length is
less than that of a normal reconstituting hydranth and they have two surfaces for
metabolic exchange, one might expect unusually large hydranth primordia in these
short pieces. Such is not the observed result. They often fail to reconstitute at
all and when reconstitution does occur, they produce the smallest hydranth primordia
or fully formed hydranths that the authors have seen. Other evidence of inhibition
in these short pieces is that instead of completing reconstitution in 48 to 60 hours
they often require 4 to 5 days.
In spite of this, the single 1-mm. pieces gave the highest per millimeter O2 up-
takes of any of the pieces measured. In a parallel study (Miller and Miller, un-
published data) it has been found that the 1-mm. stems have the lowest average pH
of any stems studied. Thus it appears that the antagonism between acid and O2
reported by Goldin (1942) has a counterpart in reverse within the coenosarc of
very short (1-mm.) stems. In spite of increased availability of O2 for the tissues
and increased utilization by them, in the presence of increased acidity reconstitution
is delayed, and when it occurs is inhibited (i.e., the scale of organization is reduced).
If this picture is a correct one, increasing the O2 in the sea water should increase
the scale of organization (i.e., the size of the organ primordia). When tested, this
prediction was verified. Oxygenation so increased the size of the primordia that
the pieces were not long enough to produce complete hydranths. As a result there
458 JAMES A. MILLER, JR., LORALEE L. PHILPOTT AND C. LLOYD CLAFF
was a great increase in the number of partial forms possessing a hypostome, distal
tentacles and gonophore buds or merely a hypostome and distal tentacles. Measure-
ments showed that 10 times as much tissue was included in the distal tentacles of
the latter group as in the distal tentacles of the complete hydranths which developed
in the unoxygenated controls (Miller and Miller, unpublished).
3. Inhibition of re constitution
Reconstitution in Tnbiilaria is initiated by the oxygen which enters the stem
through the cut surfaces. However, this form is extremely sensitive, and reduc-
tion in size or total inhibition of the developing hydranth can be produced by a
wide variety of agents of both exogenous and endogenous origin. The present
discussion will be limited to naturally occurring inhibitors. In 1939 CO2 was
reported to be a powerful inhibitor (Miller, 1939) and later it was shown that effect
was produced by hydrogen ions (Goldin, 1942). Subsequently it was found that
pH-lowering substances accumulate in the coelenteron of reconstituting stems and
especially in the reconstituting hydranth. The concentration in ligatured stems
reaches levels which Goldin found to be inhibitory when externally applied ( Miller,
1948; Miller and Miller, unpublished data). At one time Earth (1940) postulated
competition for nutritive substances circulating within the coelenteron to explain
dominance of the distal over the proximal cut surface. However, his data could
be interpreted equally well on the hypothesis that dominance was maintained by a
differential susceptibility to inhibitors (Child, 1941). When put to a test, the
stems through which fresh filtered sea water flowed throughout the period of re-
generation actually produced slightly more hydranths than did controls from which
no coelenteric fluid was removed (Miller, 1959). In this experiment any nu-
tritive substances liberated into the coelenteron of the experimental stems were
removed before they could reach the distal end, since the flow was from distal to
proximal.
Rose and Rose (1941), Rose (1955), Tardent (1955, 1960) and Tweedell
(1958) have been interested in inhibitors produced by hydranths and stems. Al-
though Fulton (1959) reported that he obtained inhibition from hydranth wrater
only when he could demonstrate bacterial multiplication in the preparations, he
found that preparations either from hydranths or stems made by extraction (Tar-
dent, 1955; Tweedell, 1958) contained inhibitors which were not dependent upon
bacterial action (Fulton, 1959, p. 237). Likewise, Rose (1957) reported polarized
inhibitory effects in grafting experiments which could not be explained on the basis
of contaminants. Also, Beloussov and Geleg (1960) have reported inhibition which
was independent of bacterial action.
Although many authors have reported inhibition of regeneration in Tulndaria
resulting from crowding, the present observations are the first which show that
under these conditions the oxygen uptake is depressed. Calculations showed that
because of the relatively large volume of the air and small volume of sea water in the
divers, no oxygen deficiency could develop in the period of the measurements.
Likewise, the NaOH in the divers was found to be more than adequate. Thus it
was concluded that some other noxious product of metabolism was involved
primarily in this effect.
During the first twelve hours of reconstitution the ends of the stems liberate
OXYGEN UPTAKE IN TUBULARIA 459
substances into the coelenteron which increase the hydrogen ion concentration at
the ends by a factor of 12 (1.2 pH units) and maintain it at this level throughout
the remainder of the reconstitutive period (Miller, 1948; Miller and Miller, un-
published data). This indicates a high rate of production of acidifying substances.
As shown by Goldin (1942 ) a pH of 6.8 in the surrounding sea water will prevent
hydranth formation at ordinary levels of oxygenation (5 cc./l.). It is suggested
that in the small volume of sea water in the clivers, pH may have fallen rather rapidly
to inhibitory levels.
It must be emphasized, however, that these studies were made during the first
6-8 hours after cutting. They give information only during the migratory phase
of reconstitution. Other and organ-specific inhibitors, such as those indicated in
Rose's work (1957), undoubtedly operate during later stages. They may likewise
affect oxygen uptake but as yet there is no information on this question.
SUMMARY
Oxygen uptake measurements were made in cartesian clivers on 43 pieces of
Tnbularia stems between 1 mm. and 6 mm. in length with the following findings:
1. The 1-mm. stems had the highest, 3-mm. stems the next highest and 6-mm.
stems had the lowest uptake when calculated per millimeter of length of stem.
2. By comparing uptake of 3-mm. and 6-mm. stems it was found that the middle
3 mm. of the 6-mm. stems used O2 at less than half the rate of the two ends. This is
in disagreement with the conclusions of Barth and Sze that regeneration does not
involve any appreciable increase in oxygen requirements.
3. When two or more pieces were placed in the same diver their oxygen uptakes
were depressed. Calculations showed that neither hypoxia nor hypercapnia could
have caused this depression. It was suggested from other studies that acid
metabolites liberated through the cut surface may have caused the observed effects.
LITERATURE CITED
BARTH, L. G., 1938. Quantitative studies of the factors governing rate of regeneration in
Tubularia. Biol. Bull., 74: 155-177.
BARTH, L. G., 1940a. The process of regeneration in hydroids. Biol. Rev., IS: 405-420.
BARTH, L. G., 1940b. The relation between oxygen consumption and rate of regeneration.
Biol. Bull., 78 : 366-374.
BARTH, L. G., 1944. The determination of the regenerating hydranth in Tubularia. Physiol.
Zoo/., 17 : 355-366.
BELOUSSOV, L., AND S. GELEG, 1960. Chemical regulation of the morphogenesis of hydroicl
polyps. Doklady-Acad. of Sci., SSSR, 130: 1165-1168.
BICKFORD, E. E., 1894. Notes on regeneration and heteromorphosis of tubularian hydroids. /.
Morphol, 9: 417-430.
CHILD, C. M., 1941. Patterns and Problems of Development. University of Chicago Press,
Chicago, Illinois.
CHILD, C. M., AND Y. WATANABE, 1935. Differential reduction of methylene blue by Cory-
morpha palnta. Phvsiol. Zoo/., 8: 395.
CLAFF, C. L., 1947. "Braking" pipettes. Science, 105: 103-104.
FULTON, C., 1959. Re-examination of an inhibitor of regeneration in Tubularia. Biol. Bull.,
116: 232-238.
GOLDIX, A., 1942. A quantitative study of the interrelationship of oxygen and hydrogen ion
concentration in influencing Tubularia regeneration. Biol. Bull., 82 : 340-346.
HOLTER, H., 1943. Technique of the Cartesian diver. C. R. Lab. Carlsberg. 24: 399-478.
460 JAMES A. MILLER, JR., LORALEE L. PHILPOTT AND C. LLOYD CLAFF
HYMAN, L. H., 1926. The axial gradients in Hydrozoa. VIII. Respiratory differences along
the axis in Tulntlaria with some remarks on regeneration rate. Biol. Bull., 50 : 406-426.
LINDERSTR^M-LANG, K., 1943. On the theory of the Cartesian diver micro respirometer.
C. R. Lab. Carlsberg, 24 : 333-398.
MILLER, J. A., JR., 1937. Some effects of oxygen on polarity in Tubularia crocea. Biol. Bull.,
73: 369.
MILLER, J. A., JR., 1939. Experiments on polarity determination in Tubularia regenerates.
Anat. Rec., 75 : 38-39.
MILLER, J. A., JR., 1940. Oxygenation and ciliary rate in regenerating Tubularia stems. Bull.
Aft. Desert Island Biol. Lab., 41-44.
MILLER, J. A., JR., 1942. Some effects of covering the perisarc upon tubularian regeneration.
Biol. Bull., 83 : 416-427.
MILLER, J. A., JR., 1946. Differential reduction of Janus green in the early development of
Tubularia crocea. Anat. Rec., 94 : 17.
MILLER, J. A., JR., 1948. pH estimations in reconstituting pieces of tubularian stems. Biol.
Bull., 95 : 243.
MILLER, J. A., JR., 1959. Nutritive substances and reconstitution in Tubularia. Proc. Soc.
Exp. Biol. Med., 100 : 186-189.
MILLER, J. A., JR., L. L. PHILPOTT AND C. L. CLAFF, 1961. Oxygen uptake in short pieces
of Tubularia stems. Biol. Bull., 121 : 398.
MOORE, J. A., 1939. The role of temperature in hydranth formation in Tubularia. Biol. Bull.,
76: 104-107.
ROSE, S. M., 1955. Specific inhibition during differentiation. . Inn. A'. }". Acad. Sci., 60:
1136-1159.
ROSE, S. M., 1957. Polarized inhibitory effects during regeneration in Tubularia. J. Morphol.,
100: 187-205.
ROSE, S. M., AND F. C. ROSE, 1941. The role of the cut surface in Tubularia regeneration.
Physiol. Zool., 14: 328-343.
SPIEGELMAN, S., 1945. Physiological competition as a regulatory mechanism in morphogenesis.
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STEINBERG, M. S., 1954. Studies on the mechanism of physiological dominance in Tubularia.
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STEINBERG, M. S., 1955. Cell movement, rate of regeneration, and the axial gradient in
Tubularia. Biol. Bull, 108: 219-234.
SZE, L. C, 1953. Respiratory gradients in Tubularia. Biol. Bull.. 104: 109-113.
TARDENT, P., 1955. Zum Nachweis eines regenerationshemmenden Stoffes in Hydranth von
Tubularia. Rev. Stiisse Zool., 62 : 289-294.
TARDENT, P., 1960. Principles governing the process of regeneration in hydroids. S\mp.
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Biol. Bull, 76: 90-103.
ABSTRACTS OF PAPERS PRESENTED AT
THE MARINE BIOLOGICAL LABORATORY
1962
ABSTRACTS OF SEMINAR PAPERS
JULY 3, 1962
Aniino acid transport in the human erythrocyte: kinetics and mechanism. PETER
RlESER.
The penetration of the human erythrocyte surface by L-valine was studied densimetrically.
"Exit" experiments were done with cells preloaded with 100, 75, and 50 millimolar solutions of
the amino acid. The rates of transfer varied inversely with the differences in valine concen-
tration across the membrane. The results show that valine does not penetrate the cell via
Fickian diffusion. Instead, the same data fit the near-saturation carrier equation with an
experimentally determined value of 4 millimoles for the half-saturation constant. Cells exposed
to a variety of endopeptidases and lipolytic enzymes failed to exhibit altered penetration rate*
for amino acids (valine, leucine). Cells exposed first to trypsin and then to lipose became
completely impermeable to amino acids but retained an intact glucose transport system. This
suggests a binding site of lipid nature with which amino acids temporarily combine in being
transferred across the cell surface.
Some effects of ionizing radiations on the embryo. ROBERTS RUGIT.
The response of the embryo to ionizing radiations indicates that at all stages it is more
radiosensitive than is the adult into which it develops ; its cells cannot recover from irradiation
insult so that they are either eliminated (and phagocytized), resulting in a deficit embryo, or
remain as abnormal cells to interfere with development and cause congenital anomalies ; and,
in contrast with the adult, the embryo has unique powers of realignment of its cells which have
not been differentiated so that a topographically well-balanced organism may result. Cell
deficiencies may be expressed as stunting, microphthalmia, microcephaly, or the actual loss of an
organ. Anomalies appear largely to affect the central nervous system and the sense organs,
probably because there are so many neuroblasts at all times following the initiation of differ-
entiation and until after birth. The primitive neurectoderm is relatively radioresistant (400 r),
and the neuron is very radioresistant (10,000 r) but the neuroblast (as with any -blast stage
for any tissue) is highly radiosensitive, being killed by 25 r. On the basis of examination of
over 20,000 mouse embryos and fetuses, it may be wise to consider establishing a limit of 10 r
to the human embryo during the first 6 weeks following conception.
Electron microscopy of ncurosecrctory cells in ihe preoptic nucleus of the toadfish
(Opsanus tan). ERNST SCHARRER.
The neurosecretory cells of the nucleus praeopticus of the toadfish (Opsanus tau) appear
to contain only one kind of granule when examined with the light microscope. However, in
electron micrographs vesicles and granules showing marked differences in size and density are
seen in the perinuclear zone in much the same manner as in the goldfish (Palay, 1960). The
neurosecretory granules of the toadfish are identifiable by their size (±3000 A) and high
electron density. They are formed by the Golgi apparatus. In addition, there are large (±1 /*)
round vesicles whose content in Epon-embedded material appears finely granular and much less
461
462 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
electron-dense than that of the neurosecretory granules. These large inclusions are frequently
open on one side, and their content mixes with the cytoplasmic matrix. The rupture of the
membrane may occur during the time it takes for the fixing fluid to reach and penetrate the
cell. These and other unclassifiable constituents of similar size with various lamellar and
vesicular inclusions are distributed throughout the perinuclear zone. Such bodies are not
characteristic of secreting neurons and may be found also in non-nervous cells.
Cilia, noted by Palay (1961) in the preoptic nucleus of the goldfish, and by Taxi (1961) in
sympathetic ganglionic cells of the frog and other vertebrates, are prominent in the neurosecre-
tory cells of the toadfish. These cilia are of the 9:0 type which occur, among others, in sensory
cells (Barnes, 1961). Two kinds of sensory perception could plausibly be ascribed to cells of
the preoptic nucleus: (a) As their homologues in mammals (Verney, 1948), they might be
osmoreceptors. (b) They could serve as photoreceptors, in view of the sensitivity to illumina-
tion of the diencephalon of blinded fishes (Scharrer, 1928) and the gonadal response of blinded
ducks to light directed toward the hypothalamus (Benoit and Assenmacher, 1959).
Supported by N.I.H. Grants B-840 and B-2145.
JULY 10, 1962
A common mechanism for temperature adaptation and crossvein deformation in
DrosopJiila. ROGER MILKMAN.
Exposure for 25-40 minutes to 40.5° C. given to D. melanogaster pupae 25 hours (at 23° C.)
after puparium formation causes posterior crossvein defects in the emerging adults. Longer
exposures are lethal. Short exposures, followed by a few minutes at room temperature,
increase resistance to defects and to death.
Kinetic evidence indicates that at 40.5° C. a certain protein passes through the sequence of
tertiary structures '"A," "B," "D," "E," "F," "G." These states have been characterized by
their temperature coefficients of formation, temperatures at which significant formation takes
place, convertibility at room temperature to a heat-resistant ("C") state, and functionality with
respect to crossvein formation.
The lower limit on this type of crossvein defect production, 38.5° C., is set by the "D" to
"E" conversion. At lower temperatures a competing reaction, converting "D" to a resistant
state (C'), prevents the sequence from continuing to a significant degree.
The individual temperature coefficients (Qi = 1.4-1.8, depending upon the reaction) imply
tertiary structure change but cannot account for the overall Qi, which is 2.3. This is explained
by the competition for "D" also, since the rate of formation of "F" depends both on the concen-
tration of "E" and on the "E" to "F" reaction rate. These results relate a form of rapid
temperature adaptation to phenocopying and death via a common path.
The adaptation of Tetrahymena, to a high NaCl environment, PHILIP B.
DUNHAM.
The process of adaptation to a high NaCl environment of Tetrahymena pyrijormis, a fresh-
water ciliate, has been investigated. One per cent of the normal (fresh water) population
survived transfer to 200 ml/ NaCl medium. It was shown that this stress tolerance is a
heritable character which constituted preadaptive variability in the original population.
Average cell volume decreased with adaptation from 16 /ot/al. per cell to 10 /i/ul. However,
the amount of dry material per cell remained constant, as shown by the increase in per cent dry
weight from 19% for normal animals to 29% for adapted animals.
The main feature of the adaptation was an increased ability to maintain a low cellular NaCl
concentration. Cellular Na concentration (Nai) in normal cells in normal medium (Na0,
36 mM) was 12 mM/1. cells. Nai in normal cells in high NaCl medium (Na0, 223 mM) was
105 mM/1. cells. Nai in adapted animals was 43 mAf/1. two weeks after starting the culture,
and fell gradually to 21 mM/1. after 22 months, or 1700 generations. This was selection for
ability to regulate Na. Changes in the Na-regulatory mechanism which accounted for decreased
Nai in adapted cells were: (1) the saturation level of Na extrusion increased; (2) apparent free
Na space decreased.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 463
The electrophoretic patterns of soluble proteins in polyacrylamide gel from adapted and
normal animals were nearly the same qualitatively and quantitatively. However, one protein
was in much greater amounts in the adapted animals. Since the two cell types differed only
with respect to the response of one to an altered environment, the protein difference reflects
adaptive change.
Time-lapse motion pictures of intracellular disturbances induced in Arbacia zygotes
after ultraviolet or .r-n/v irradiation of zygotes, both gametes, or one gamete.
CARL CASKEY SPEIDEL AND RALPH HOLT CHENEY.
Strong irradiation, x-ray or 2537 A ultraviolet, of zygotes, both gametes, or eggs alone,
induced violent internal disturbances during the first cleavage cycle in Arbacia. Rapid move-
ments of pigment granules and other cytoplasmic constituents occurred, heralding approaching
death. The pigment became concentrated in a conspicuous mass, usually centrally located.
The nucleus with membrane intact moved about the cell as part of the general upheaval. In
contrast, strong irradiation of the sperm alone did not elicit a like reaction. In the resultant
zygotes, internal disturbances took place accompanied by sudden gel-sol adjustments, but the
pigment granules were distributed in several small aggregations rather than in a large central
mass. Massive pigment concentration was correlated, therefore, with irradiation of egg or
zygote cytoplasm, but not of sperm.
Ultraviolet effects. Time-lapse motion pictures included : internal upheaval and massive
pigment concentration 4-5 hours after fertilization with normal sperm of eggs given 12-minute
UV (two examples) ; similar scenes with eggs shaken throughout 12-minute UV irradiation,
exposing all sides equally ; sperm sticking to exposed half of unshaken 12-minute eggs with
half fertilization membranes and to entire surface of shaken 12-minute eggs with unelevated
membranes ; highly magnified scenes of UV-induced death throes showing differential viscosity,
pigment concentration, nuclear movements, popping gel-sol reactions, and repeated furrow
obliteration ( two examples ) ; 24-hour delayed development ; exaggerated polygonal gelation at
border two hours after 4-minute UV to sperm alone; death changes, featured by formation of
extra wide hyaline border, 1-2 hours after 12-minute UV to sperm alone.
X-ray effects: adjustments after 3-7 hours, 30-128 kr to zygotes; after 6 hours with furrow
obliteration, 120 kr to sperm alone ; earl}' death throes with massive pigment concentration
within one hour after 60 kr to eggs alone.
Supported by Grant RG-4326(CS) to C.C.S. from the U.S.P.H.S. and by Grant 144 to
R.H.C. from the National Academy of Sciences.
JULY 17, 1962
Organ and ontogenetic patterns of multiple forms of hydrolytic enzymes in Limnaea
palustris. JOHN B. MORRILL AND ELAINE N. Dow.
Soluble electrophoretically mobile hydrolytic enzymes of adult organs and 0- to 9-day-old
larvae were determined by the method of Hunter and Markert (1957). Adult organ tissues,
eggs, embryos or larvae were homogenized, frozen and thawed three times and centrifuged at
10,000 g for three minutes. The supernatants were subjected to starch gel electrophoresis
(borate buffer 0.03 M ; pH 8.6). Mobile enzymes were developed with the following substrates:
a-naphthyl acetate ; a-naphthyl acid phosphate ; leucyl and alanyl-jS-naphthylamide ; 6-bromo-2
naphthyl esters of a-glucoside, /i-glucopyranoside, )3-galactopyranoside, /3-glucoronide.
Forty-two electrophoretically mobile bands were developed with extracts of 16 adult
organs. Several bands were developed with two substrates. Each organ had its own character-
istic enzymatic band patterns with respect to presence, absence and intensity of the bands.
No organ had fewer than 10 bands nor more than 32 bands. The liver or digestive gland had
the maximum number of bands.
In extracts of 0- to 2-day embryos, 5 enzymatic bands were developed. These bands
corresponded to those widely distributed in adult organs. On subsequent days of development
additional bands appeared as follows : third day, 4 bands ; fourth day. 5 bands ; fifth day, 3 bands ;
sixth day, 2 bands ; seventh day, 4 bands : 10-day-old hatched snails, 2 bands. All these bands
464 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
(total, 29 bands) had mobilities similar to those of adult organs. It is not known when the
full complement of adult organ bands is eventually attained. That most of the bands appeared
in the embryo during the period of organogenesis and histogenesis suggests these enzymes are
associated with the functional differentiation of one or more of the organs in which they occur
in the adult snail. The sequential appearance of the several enzyme bands at different stages in
development also reflects the direct type of development of this mollusc egg.
Supported by N.S.F. Grant G-10893.
Structural and control genes regulating dopa ox-Idas c activity in Drosophila.
HERMAN W. LEWIS.
Genetic analysis of dopa oxidase activity in Drosophila melanogaster has revealed that at
least four genes interact to produce this activity. One of the genes is involved with qualitative
aspects of the enzyme system, the others are involved in the determination of quantitative aspects
of the enzyme system. The gene involved in determination of the qualitative nature of the enzyme
has been located on the genetic map at 52.4 of chromosome II. A recessive allele at this locus
when present in the homozygous condition results in an enzyme system that is thermolabile and
has an altered substrate profile relative to the wild type enzyme system. Another gene, located
approximately five map units to the right of the above mentioned gene, has a dominant allelic
form which when present lowers the dopa oxidase activity 50%. A third gene, also dominant
relative to the wild type, has been identified on the right arm of chromosome III. It reduces
dopa oxidase activity approximately 35% and its effect is most readily detected when it interacts
with a fourth gene located on the right arm of chromosome II. These findings indicate that
mechanisms of genetic control of enzyme systems in highly differentiated multicellular organisms
may be analogous, if not identical, to the mechanisms demonstrated in microorganisms. The
following generalization therefore is probably applicable to all genetic systems. Although at
the level of primary events the mode of action of all genes is the same, i.e., the imparting of
information for the primary structure of polypeptide chains, from the point of view of the
interrelation of their products, genes can be divided into two classes : structural genes, which
determine the kind of polypeptide synthesized, and control genes, which are involved in the
determination of how much polypeptide is made.
The A and I bands in contracting Limulus muscle. G. W. DE VILLAFRANCA AND
C. M. MARSCHHAUS.
The dorsal muscles of Liinnliis polypliemns were tied to splints and glycerinated for at
least 10 days. Several bundles were removed at a time, blotted and then blended 45 seconds
in a 0.04 M KC1-0.0067 M phosphate buffer (pH 7.4) solution. The fibrils after two washings
were photographed under phase contrast with a Bolex 16-mm. movie camera during ATP (IQ--
or 10~3 M) induced contraction. Approximately every hundredth frame (18 frames per second)
was enlarged to a final magnification of 1300 or 1700 times. A single, distinctive sarcomere
and its striations were accurately measured on the printed series. Muscle stretched prior to
glycerination gave better, but not different, results than muscle fixed at rest length. Six
complete sequences of different preparations were sharply enough defined to obtain measurements
over the total range of contraction exhibited, while in many other fibrils a single sarcomere
could be measured at the beginning and end of the contraction.
From initial sarcomere lengths as great as 10.8 /* the fibrils shortened as much as 4 /t
(to a minimum length of 5.2 /M, or 3.7 //. when starting from rest length). The A bands
shortened as much as 1.8 p. The major portion of the A band contraction occurred commencing
with and continuing below the rest length (sarcomere = 7.5 ft,). Decreased I band length
accounted for the change in sarcomere size to rest length from the stretched condition. During
contraction the A band first becomes more dense at the junction with the I band. It is as
though an H zone had opened up but in Limit-Ins there is no H zone or M line. Later, during
contraction, the central portion of the A band becomes more dense. If only this portion of the
A band is considered the total A band, the A band would have shortened to 2.5 p in a sarcomere
of 5.0 /j.: that is, the I band would have changed relatively little. Contraction of this muscle
does not, in all probability, occur by sliding of I filaments into the A band.
Supported by U.S.P.H.S. Grant A-2647.
PAPERS PRESENTED AT .MARINE BIOLOGICAL LABORATORY 465
JULY 24, 1962
On the utilisation of C14 from glucose for amino acids and protein synthesis by the
sea urchin embryo. ALBERTO MONROY AND LETIZIA VITTORELLI.
Unfertilized eggs and developmental stages of Paracentrotus Hindus were incubated for 60
minutes in 10 ml. of sea water containing 1 /*C of C14-glucose (U)/ml. (specific activity 10
fj.C/mg.). The eggs were then extracted with 10% trichloroacetic acid (TCA). One aliquot
of the extract was used for the determination of radioactivity while the largest portion was
chromatographed two-dimensionally and the radioactive amino acids identified by radio-
autography. The insoluble residue was extracted with hot TCA and alcohol-ether. Radio-
activity was determined using a liquid scintillation counter. At all stages of development as
well as in the unfertilized eggs glucose is taken up and used for amino acid synthesis. The
following free O4 amino acids have been identified in the TCA-soluble fraction : alanine, serine,
glycine, proline, glutamic and aspartic acid. No C14 peptides have been found. The rate of
uptake in the TCA-soluble fraction rises rapidly following fertilization until the early blastula,
then remains constant until the mesenchyme blastula when it starts rising again, and a new peak
is attained at the midgastrula stage followed by a decline. On the other hand, incorporation in
the proteins only begins after fertilization. The rate of this incorporation increases rapidly until
the 32-64-cell stage, then declines somewhat to rise quite steeply again after the mesenchyme
blastula stage. A decrease is also observed after the midgastrula. The curve of the incorpora-
tion of C14-glucose into the proteins thus duplicates that previously obtained with the administra-
tion of radioactive amino acids (Giudice, G., Vittorelli, M. L. and Monroy, A. — Ada EmbrvoL
Morphol. E.vp. 5: 113 (1962)).
Supported by a Grant (RG-6211) of the U.S.P.H.S.
Reversible enzymatic reduction of insulin. DsWiTT STETTEN, JR., HOWARD M.
KATZEN AND FRANK TIETZE.
The hepatic enzyme first purified by Tomizawa has been further studied and found to be
a transhydrogenase whereby glutathione is oxidized and the disulfide bonds of insulin are re-
duced. Coupling of this enzyme with yeast glutathione reductase gave a system wherein
TPNH reduced insulin. This permitted a ready evaluation of the Km for glutathione (8.9
X 10~3 M) and that for insulin (4.3 X 10~5 M). Oxytocin and pitressin could replace insulin
but lipoate, cystine and homocystine were ineffective. The enzymatic reduction of insulin is
considered to be the initial step in the hepatic destruction of insulin. Its rate may be limited
by two negative feedback mechanisms : the known inhibition of glutathione reductase by the
phenylalanyl chain of insulin, and the failure of TPNH generation in hypoinsulinism.
Reduction of insulin, whether enzymatic or non-enzymatic, results in the complete loss of
detectable physiological and immunological activities of the parent molecule. Reoxidation by
oxidized glutathione yields very slight restoration of these activities. However, when this re-
oxidation is carried out in the presence of hepatic glutathione-insulin transhydrogenase, very
considerable recovery of insulin-like activity is observed. Thus, by our test non-enzymatic
reoxidation yielded 1.7% recovery of physiological activity whereas in the presence of enzyme
32% was recovered.
The possible role of enzyme-directed thiol-disulfide interchange reactions in the biosynthesis
of cystine-containing proteins has been presented.
Golgi apparatus and lysosoines in vertebrate neurons. ALEX B. NOVIKOFF.
In many vertebrate neurons the Golgi apparatus is a large reticular structure as de-
scribed in 1898 by Golgi. This may readily be observed in frozen sections incubated, by a
method developed with S. Goldfischer, for nucleosidediphosphatase or thiaminepyrophosphatase
activity. Together with E. Essner, we have studied incubated sections of rat brain (cerebrum,
hypothalamus, cerebellum) and cord, and cerebellum of barn owl and pigeon, by light and
electron microscopy. In all neurons studied, the reaction product resulting from these phos-
phatase activities is localized exclusively in the Golgi saccules ; none is seen in the ergastoplasm
466 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
(Nissl substance). Intimately associated with the Golgi saccules, and apparently derived from
them, are numerous granules — "dense bodies" in "typical" neurons and "large granules" in
neurosecretory neurons. These granules possess acid phosphatase activity and two other
hydrolase activities shown by lysosomes in incubated sections of liver. A small amount of acid
phosphatase reaction product is sometimes seen in Golgi saccules. The state of development
of ergastoplasm and Golgi apparatus approaches that of neurons only in secretory cells
"packaging" materials within Golgi-derived vacuoles ; those that we have studied possess fewer
Golgi-associated lysosomes than do neurons. The electron microscope evidence and the
lysosome distribution, as seen by light microscopy, in the cell processes of neurons — normal
and following axon section — lead us to the following working hypothesis. "Secretory" ma-
terial, produced by the ergastoplasm, reaches the Golgi saccules, perhaps by smooth-surfaced
vesicles derived from the ergastoplasm. There it is "condensed" into granules or lysosomes.
These move down the cell processes, where their decreasing numbers may reflect release of
their hydrolases, at the surface or within the cytoplasm.
JULY 31, 1962
On the chemistry of the tlivmiis gland. ALBERT SZENT-GYORGYI AND ANDREW
HEGYELI.
The thymus gland contains two biologically active substances, one of which promotes malig-
nant growth, the other which retards it. The former seems to be a specific product of the
gland, while the latter is present in various tissues, although at a considerably lower concen-
tration. The activity of the two substances, being antagonistic, can be demonstrated (in inbred
Swiss albino mice, inoculated with Krebs-2 tumor) only after they have been separated.
Separations can be achieved by paper chromatography.
Thymus glands were extracted with methanol and the active substances precipitated with
Reinecke salt. The two substances seem to contain nitrogen, which induces a basic group with
a low dissociation constant. The two substances have closely related properties which makes
separation difficult. They tend to spread over the various fractions, adhering to any substance
present.
The promoter substance sterilizes both male and female mice reversibly. It seems to
influence the hormonal background, shifting it towards the pre-puberty condition.
Extracts have been purified several thousand-fold and gave strong biological activity with-
out side effects with dry weights of 0.1 mg. The present extracts allow, thus, biological
experimentation.
Effects of heavy water, gl\ccrol and sucrose on glycerol-extracted muscle. BEN-
JAMIN KAMINER.
From previous investigations it was tentatively concluded that the inhibitory effect of
heavy water on muscular contraction is due to retardation of the membrane-contractile coupling-
process. To seek further supporting evidence for this hypothesis, the present investigation was
done on preparations of glycerol-extracted muscle which contained an intrinsic relaxing system.
This particular preparation was chosen since part of the relaxing system, the sarcoplasmic
reticulum, is considered, from work by other investigators, to be involved in the membrane-
•contractile coupling mechanism.
On soaking the glycerol-extracted bundles in normal water for variable periods of time
before the addition of ATP, spontaneous relaxation (and associated responsiveness to calcium)
was lost. In heavy water, on the other hand, this relaxing ability was maintained for longer
periods of time. It is conceivable, therefore, that heavy water retarded the process which
inactivated the intrinsic relaxing system. Conversely, it favored the relaxed state. Whether
in fact the heavy water affected the sarcoplasmic reticulum remains, however, to be elucidated.
The properties of water were then altered by the addition of either glycerol or sucrose in
varying concentrations. It is well known that glycerol will retard the contraction induced by
ATP. In this investigation it was demonstrated, however, that subsequent addition of calcium
augments the contraction. Furthermore, prior addition of deoxycholate also led to an aug-
mented response to ATP. In maximally contracted preparations in water-solutions, replace-
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 467
ment with 25% glycerol led to reversible relaxation. Sucrose had similar effects. In addition,
25% glycerol had a preservative effect on the relaxing property of muscle microsomes (sar-
coplasmic reticulum).
Consideration is being given to what extent the altered properties of normal water are
involved in all the above effects.
AUGUST 7, 1962
Regeneration studies on a brackish-water ciliatc, Trachcloraphis sf>. REUBEN
TORCH.
Tracheloraphis sp. is a large (500-900 M) partially flattened, extremely contractile, worm-
like ciliate living in brackish water (salinity 10%0). The nuclear apparatus consists of four
Feulgen-negative macronuclei clustered around two Feulgen-positive micronuclei, the entire
complex surrounded by a mass of small Feulgen-negative granules. Four regions of the animal
can be distinguished : head, neck, mid-region, and tail. The black head, slightly wider than
the neck, contains the mouth at its anterior tip and is tightly packed with short (2 /*) refractile,
birefringent rods. The narrow, extendible neck widens to form the extensive mid-region, which
terminates posteriorly in a short, pointed, slightly curved tail.
Dissections were made and the regenerates maintained in depression slides containing
small amounts of filtered pond water. The animals were decapitated with glass needles and
then cut into halves or thirds. Regenerates were examined for the presence of nuclei, by phase
microscopy or after fixation in Champy's or Bouin's, followed by staining with dilute (1:3)
Delafield's hematoxylin.
Within 5 minutes after dissection, refractile, 2 /u. rods (head granules) accumulate in the
anterior parts of all fragments. This is followed by extensions of the anterior regions to form
necks. Most fragments (exceptions being anucleate pieces smaller than 150 /*) form necks,
but only nucleate fragments form new tails. Mouth parts are difficult to see, but apparent
mouth regeneration was observed in several anucleate fragments. Complete regeneration by
nucleate fragments occurs within 3-5 hours and is accompanied by a marked increase in body
length (2-3 X). Many anucleate posterior fragments also double in size, but increase in
length was never observed in anucleate anterior fragments.
Some evidence suggests that the small, Feulgen-negative spheres on the periphery of the
nucleus may have some influence on regeneration. The role of the nucleic acids in regeneration
is being investigated by means of radioautography.
"Messenger" RNA and the cell cycle in a fission yeast. PAUL R. GROSS AND
JOHN M. MITCHISON.
When cells of the fission yeast, Schizosaccharouiyccs pombe, are transferred during ex-
ponential growth from a rich malt extract broth medium to a defined minimal medium contain-
ing 4.75% ethanol, there is observed a period of about 30 minutes of no growth, i.e., the optical
density of the "stepped-down" culture does not change. Following this, growth resumes at
the rate characteristic of the new medium (about Vs that in the broth). Following stepdown,.
no net synthesis of RNA can be detected for about one hour, after which it resumes at the
new characteristic rate. By labelling with tritiated, C14-labelled, and P32 tracers, it is possible
to show that both protein and RNA turn over during their respective periods of stasis, with1
respect to net synthesis. Labelling of protein may proceed at almost 30% of the rate found
in controls (i.e., growing normally in the minimal medium). The maximum labelling rate
for RNA is about 7% of that in controls. RNA labelled with P32 in controls is stable, since
no reduction in the radioactivity of a culture sample can be effected by a "chase" of non-
radioactive phosphate. That labelled during a stepdown, i.e., the fraction turning over in the
absence of net synthesis, is unstable, because its radioactivity diminishes rapidly after the
addition of a cold "chase." Base ratio analysis of this RNA shows that its composition is very
different from that of ribosomal RNA and transfer RNA, but approaches that expected for
a DNA. Thus, the RNA being turned over during a stepdown is probably the "messenger"
fraction of this cell, and this may reflect the necessity for reference to the genes concerned
with the production of enzyme systems now needed in the new medium. The growth habit
468 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
and morphology of this yeast permit autoradiographic assessment of the rate of synthesis of
RNA at different times in the cell cycle, this without forcing the culture into synchrony (e.g.,
Mitchison and Wilbur, 1962). This rate rises steadily through the cell cycle during exponential
growth, and when the RNA being made is presumably mostly ribosomal. During the stepdown,
the rate remains constant through most of the cycle, and doubles abruptly at the end, just
prior to the appearance of a cell plate and subsequent fission.
Actin localization in sperm. LEONARD NELSON.
Engelhardt and Burnasheva obtained "spermosin," a substance analogous to myosin, from
homogenized bull sperm on prolonged extractions in solutions of high ionic strength. Spermosin
splits ATP and combines with muscle actin. Since the contractility of muscular systems de-
pends on the interaction of the complex actin-myosin, a number of investigators have been
working on the identification of actin or an analogous protein in sperm. Should the actin-like
material be associated with a specific morphological entity of the spermatozoon, its presence
could be surmised from its nucleotide-binding capacity. Perchloric acid extracts of washed,
sonicated and fractionated bull epididymal sperm flagella show UV absorption maxima at about
260 millimicrons (equivalent to approximately one mole of ATP per 60,000 g. of protein).
Immunocytochemical localization of "spactin" was therefore attempted. Rat skeletal muscle
was extracted according to conventional procedures for myosin and actin. The purified pro-
teins were injected intravenously into two groups of rabbits. Antisera tested against the
antigens yielded single bands in agar gel diffusion tubes. Frozen-dried rat epididymal sperm
blocks were incubated for 30 to 60 minutes in the antisera diluted 1:4 in buffer. Sections were
examined in the electron microscope. The actin antibodies react with the outer ring of nine
longitudinal fibers in the flagellum. The "spactin" seems to be localized in the cortical region
of each of the nine outer fibers, while the spermosin, which reacts antigenically at the EM
level with myosin antibodies, appears to be confined to the core of the same fibers. It thus
appears that in mammalian sperm flagella, there is a differential distribution of substances
responsible for generation of the undulatory wave within each of the nine outer fibers, in
contrast to the situation in vertebrate striated muscle in which the proteins actin and myosin
reportedly occupy separate filaments.
This work has been supported by Senior Research Fellowship SF-193 and Research Grant
RG-6815 of the U.S.P.H.S.
AUGUST 13, 1962
. I possible mechanism jor excitation-contraction coupling in crayfish muscle fibers.
LUCIEN GlRARDIER, JOHN P. REUBEN AND HARRY GRUNDFEST.
Alternating light and dark bands with a 9 /j. periodicity are clearly visible under the light
microscope in living unstained isolated muscle fibers of crayfish. During certain procedures
the fibers darken and the banding is obscured. Electron micrographs then reveal the formation
of vesicles, sometimes > 4 /* in diameter and surrounded by a membrane. Two modes of
vesicle formation were observed, each produced by specific conditions, but only one, which
develops in proximity to the Z lines, is analyzed here.
These vesicles are formed by the swelling of convoluted tubular organelles which originate
j ust under the fiber surface and run radially inward along both sides of the Z lines. They appear
to be comparable to the T-system tubules of other muscles. The diameters of these tubules,
about 200 A, permit free movement of ions, but frictional (Poiseuille) resistance to flow of
water must be high. The membrane of the tubules appears to be permselective for Cl and
positively charged. Since the tubules swell whenever Cl is forced out of the cell the anion
must enter the tubules carrying in water by electroosmosis and the water then must be trapped.
Particularly clear-cut effects were obtained with intracellularly applied currents. Vesicles
developed only with inward currents and only when the intracellular cathode was a KCl-filled
microelectrode. Currents in either direction, but applied through a microelectrode filled with
K propionate, did not produce vesiculation. In muscle fibers loaded with Cl, long-lasting
local currents result when K or Cl in the medium is decreased. Very large vesicles are then
formed.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 469
Depolarization of the cell membrane in any manner also produces a local circuit, with the
tubules forming one branch of the current path and Cl entering the fiber from the tubules.
Thus, during the action potential there will be an outward flow of current through the tubules,
causing depolarization of their membranes and an inward flux of anions. A. F. Huxley has
suggested that depolarization of the triadic membranes might be the stimulant for releasing
an agent that initiates contraction, but no satisfactory means has hitherto been envisaged to
cause the depolarization during the action potential. The properties described here allow
the tubules to mediate excitation-contraction coupling, since an anionic membrane is effectively
in series with the cell membrane.
Water transport and membrane structure in era \fish iniisele fibers. Jonx P.
REUBEN, LUCIEN GIRARDIER AND HARRY GRUNDFEST.
Volume changes under a variety of experimental conditions were determined in isolated
fibers. In correlation with the membrane potentials the data provided information on water
movements initiated by ionic changes under isotonic, hypotonic, or hypertonic conditions, or
movements caused by applied currents. Classical osmosis, anomalous or electroosmosis, and
water movement associated with metabolic processes could be distinguished. Changes in the
structure of the fibers were also observed.
Large volume changes could be produced when the membrane potential was altered while
the activity of water was constant. Swelling was always associated with hyperpolarization,
shrinkage with depolarization. The effects could be produced by intracellularly applied cur-
rents or by altering the ionic environment. Marked swelling occurred when fibers, depolarized
and already swollen in a high-K isotonic medium, were returned to the standard solution. The
transient depolarization that occurs on removing Cl from the medium was accompanied by a
transient shrinkage. The transient hyperpolarization when the Cl was reintroduced was ac-
companied by swelling. Anomalous water movements were also produced under other
conditions that cause large changes in the membrane potential. When ionic changes were made
gradually, so as to diminish the electrical driving force, the volume changes due to anomalous
osmosis were lessened or abolished.
Anomalous osmosis can arise only from flow of current across charged membranes. Thus,
the cell membrane must be so structured as to permit circulation of currents under a large
variety of experimental conditions, and must be heterogeneous in structure. The directions of
net movements of water indicate that the membrane has a net negative charge with channels
of different selectivities. The presence also of sites with positive fixed charges and permselec-
tive for Cl is indicated by electrophysiological and pharmacological evidence. Some, and per-
haps all, of the latter sites appear to be located in tubular organelles associated with the Z
lines and which are probably comparable with the T-system tubules of vertebrate muscle fibers.
AUGUST 21, 1962
Studies on the mechanism hy which allo.ran alters the permeability oj islet eel!
membranes to mannitul. DUDLEY WATKINS. S. J. COOPERSTEIX AND ARNOLD
LAZAROW.
It has been shown that treatment of toadfish islet slices in lilro with 2.5 X 1Q--1 M
alloxan (equivalent to a dose of 40 mg./kg.) increased the permeability to C14-mannitol.
Since alloxan reacts with sulfhydryl groups, other sulfhydryl reagents have been studied.
Tissue slices were pre-incubated in the test solution, washed twice in isotonic saline, and in-
cubated at 0° C. in C14-mannitol. After incubation, the slices were rinsed, weighed and counted.
P-hydroxymercuribenzoate (a monothiol-binding agent) did not affect permeability,
whereas both arsenite and cadmium (which at low concentrations selectively bind dithiols)
increased the permeability of islet cells. The C14 content of control islets was 36% of that in the
medium, whereas the C14 content of islet slices pre-incubated with 1 X 10~5 M arsenite was
62% ; higher arsenite concentrations were less effective. When islets were pretreated with
1 X 10-3 M to IX 1(H4 M cadmium, the C14 content of the tissue was about 50% ; when the
concentration was increased (to 1 X 10"1 ./I/) or decreased (to 1 X 10~:s M) no effect on
470 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
permeability was observed. In contrast to alloxan, which only affected islet tissue (toadfish),
arsenite increased the permeability of rat kidney and heart slices as well as toadfish islet.
Treatment of tissue slices with 2,3-dimercaptopropanol after pretreatment with either alloxan,
arsenite, or cadmium restored the permeability to the control level. Glutathione did not
reverse the effect of these agents. These observations are consistent with the hypothesis that
alloxan may exert its diabetogenic effect by binding dithiol groups in the beta cell membrane.
The arsenite and cadmium effects further suggest that dithiol groups may be of general
importance in maintaining the integrity of many other cell membranes as well.
Supported by Grants A-824 and A-1659 from the National Institute of Arthritis and
Metabolic Diseases, U. S. Public Health Service.
AUGUST 22, 1962
Evidence against participation of a jelly-splitting agent in sperm penetration of
Arbacia eggs. C. R. AUSTIN AND J. PIATIGORSKY.
Dilute suspensions of "dry" Arbacia punctnlata sperm (1 drop in 3 ml. sea water) were
treated with three concentrations (0.02, 0.05 and 0.007%) of the enzyme inhibitor 53 D/k
(Parkes, Rogers and Spensley, 1954). The spermatozoa were washed by centrifugation and
decantation and added to suspensions of eggs. After 10 minutes the eggs were examined; the
proportions found with elevated fertilization membranes were 88% (with 0.007% inhibitor),
64% (with 0.05%) and 11% (with 0.02%). Eggs without fertilization membranes, like those
with these structures, had large numbers of spermatozoa at all levels of the jelly coat. Ex-
amination by electron microscopy of sections of eggs (fixed with 1.5% KMnO4 solution) showed
that the jelly coat had been essentially preserved, and that spermatozoa could be found adjacent
to the vitelline membrane in eggs without fertilization membranes. It is inferred that enzyme
inhibition had prevented sperm passage through the vitelline membrane but not passage through
the jelly coat.
Several attempts to obtain a jelly-dispersing lysin from spermatozoa (as reported by
Hathaway, 1960) were unsuccessful when made with purified fertilizin. Sperm suspensions
were centrifuged after being treated with purified fertilizin (Tyler, 1949) and the supernatants
were added to Arbacia eggs. These eggs had been inseminated so that the existence of the
jelly coat was shown by the presence of trapped sperms. No jelly loss was observed within
one hour (until first cleavage).
In one experiment, spermatozoa were treated with fertilizin containing 100 /otg./cc. of
fucose, the optimum concentration for discharging the acrosome (Piatigorsky and Austin, 19(>J ).
Electron microscopic examination revealed 52% reacted acrosomes (controls 4%). Super-
natants from neither fertilizin-treated nor sea- water-treated sperms dispersed the jelly coat.
Aided by Training Grant 2G-998 from the National Institutes of Health.
Neutralization of the fertilisation inliibitors in anti-Arbacia-sperm serum by sperm
extracts. E. R. BISCHOFF AND C. B. METZ.
Anti-Arbacia-sperm rabbit serum can be converted to the non-sperm-agglutinating form
by treatment with the proteolytic enzyme papain. Pretreatment of sperm with such uni-
valent antiserum markedly reduces the fertilizing capacity of the sperm (Metz and Schuel,
1961). Sea urchin sperm extracts prepared by freeze-thawing contain at least four antigens.
The present study was undertaken to determine whether such extracts neutralize the fertiliza-
tion inhibiting activity of the antiserum.
Frozen-thawed extracts of 25% Arbacia sperm were centrifuged at 10,000 g for 10
minutes and added in excess to papain-digested (Porter, 1958) anti-sperm serum and control
serum. Increasing dilutions of 1% sperm were treated with these sera in the proportions
of one part sperm to two parts serum. The fertilizing capacity of the sperm was measured
by the number of eggs cleaved after 1-2 hours. In a typical experiment 4% of the eggs were
fertilized by sperm pretreated with the unabsorbed serum, while 100% of the eggs were
fertilized when the sperm were pretreated with the extract-absorbed immune serum or control
serum. Thus, sperm extracts appear to contain a substance capable of neutralizing the
fertilization inhibitors present in the anti-sperm serum.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 471
Since high speed centrifugation of sperm extracts removes most of the egg agglutinating
activity (Metz and Kohler, 1960), it seemed of interest to see if the neutralizing substance
could also be removed by centrifugation. Univalent anti-sperm serum was absorbed with the
supernatant of sperm extracts centrifuged at 30,000 g for 30 minutes. The sediment was
resuspended in sea water and also tested. Fertilization tests showed that the neutralizing
substance was present in the sediment but not in the supernatant.
Aided by Training Grant 2G-998 from the National Institutes of Health.
Iininunological identification of an c</g (Kjtjlntinin in Arhacia spcnn extracts. E. R.
BlSCHOFF AND C. B.
Sperm extracts that agglutinate eggs can be prepared from sea urchin sperm by various
procedures. Metz and Kohler (1960) found that egg-jelly precipitating frozen-thawed extracts
of Arbacia sperm contain a minimum of four distinct antigens. This suggests that further
analysis of the extracts might reveal an identity between one of the four soluble antigens and the
egg agglutinin.
When sperm extracts prepared by freeze-thawing are centrifuged at 10,000 g for 10
minutes and absorbed with an excess of Arbacia fertilizin solution, a pink, jelly-like precipitate
forms. Immuno-diffusion of the supernatant in agar against anti-sperm rabbit serum results in
the appearance of only three precipitin bands. Evidently one antigen has been removed by the
fertilizin treatment. As a further test, sperm extracts treated with fertilizin were used to
absorb anti-sperm ^erum. Since the sperm extract now contains only three antigens, a
corresponding number of antibodies should be precipitated from the serum. When such absorbed
serum was diffused against normal sperm extract, a single faint band appeared. It is concluded
that the fertilizin combines with the egg agglutinin in the sperm extract and removes it as a
precipitate.
The control and fertilizin-absorbed sperm extracts were subjected to immuno-electrophoresis
in agar gel at pH 8.6. In parallel runs the component which moves the farthest toward the
anode is always absent or reduced in the fertilizin-absorbed extracts. In concentrated extracts
this arc extends in a continuous band from the origin. This suggests a substance of low
solubility or a heterogeneous collection of molecules or fragments which have the same
antigenic group.
Aided by Training Grant 2G-998 from the National Institutes of Health.
Action of neuraminidase on Arbacia spermatozoa. RALPH L. BRINSTER AND
C. R. AUSTIN.
Arbacia spermatozoa contain a considerable amount of sialic acid, and Hathaway (1961)
has shown that treatment with heat, sodium lauryl sulfate, or fertilizin resulted in the release
of a bound form of the substance. He also demonstrated that the antigen of the sperm extract-
antibody complex contained bound sialic acid. These observations, together with the known
importance of sialic acid as a cell surface component and its role in virus-cell conjunction
( Gottshalk, 1957), suggest that it may have an important function in sperm-egg union.
When 4 X 103 spermatozoa were treated for one hour with one ml. of a neuraminidase
preparation (crude extract, Sigma), the fertilizing capacity was 99% inhibited, the degree of
inhibition showing some variation between different sperm suspensions. In eggs that did
undergo fertilization, the enzyme appeared to inhibit cleavage.
The supernatant fluid of the sperm suspensions after enzyme treatment was analyzed for
free sialic acid by the thiobarbituric acid method (Warren, 1959), but none could be detected.
Furthermore, heating the enzyme to 80° C. for 30 minutes had little effect on its ability to
inhibit the fertilizing capacity of spermatozoa. The possibility that the enzyme might be
covering sialic acid and/or removing larger molecules containing sialic acid was therefore
examined. It was possible to show in preliminary experiments that treatment of spermatozoa
with the enzyme preparation did in fact result in the release of bound sialic acid. In addition,
incubation of the enzyme preparation for 20 minutes with the supernatant fluid of spermatozoa
heated to 60° C. for 5 minutes resulted in a reduction in the ability of the enzyme to decrease the
fertilizing capacity of spermatozoa. This supernatant contains large quantities of a bound sialic
472 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
acid moiety (Hathaway, 1961) and it was confirmed that the substance exercised a competitive
inhibiting action.
These observations are interpreted to mean that neuraminidase is capable of removing
large molecules containing sialic acid from spermatozoa, and that this reaction can prevent sperm
penetration into the egg cytoplasm. A possible general toxic action of the neuraminidase extract
on spermatozoa was, however, not completely excluded.
Aided by Training Grant No. 2G-998 from the National Institutes of Health.
Passage of spermatozoa through the chorion of Ciona eggs. S. D. EZELL, JR.
AND C. R. AUSTIN.
The mature egg of Ciona intcstinalis is enveloped in a tough membrane, the chorion, which
the spermatozoon must penetrate before making contact with the egg proper. Since penetration
could well depend on the action of a lytic agent, attempts were made to extract a chorion-
dissolving lysin from spermatozoa by freezing and thawing, or by treatment with acidified sea
water (pH 4.0-5.0), alkaline sea water (pH 10.5), and "egg water." In no case did the
resulting fluid have the capacity to dissolve the chorion. It is inferred that, if such a lysin is
indeed present in the spermatozoa, it must exist in very small quantity, or be insoluble in
water, or be inactive when extracted.
Study of fertilized and cleaving eggs regularly revealed the presence of many extra sperma-
tozoa within the chorion. These spermatozoa were free within the perivitelline space and active,
though not so active as those outside the chorion. Evidently permeability of the chorion to
sperms does not change after sperm entry ; exclusion of extra sperms from the vitellus
presumably depends on a change in the egg cortex.
As seen with the light microscope, the spermatozoon is asymmetric, with a mass of
protoplasm of indefinite shape associated with the head region. The presence of this laterally
projecting mass posed a problem of special interest in connection with sperm passage through
the chorion. Investigation with the electron microscope showed that the mass contained a
single large mitochondrion. In freshly shed sperms the mitochondrion appeared to be hemi-
cylindrical and ran most of the length of the nucleus. There was no sign of mitochondrial
("midpiece") structures posterior to the nucleus, as displayed by many invertebrate sperms.
The fate of the mitochondrion during chorion penetration has yet to be determined.
Aided by Training Grant 2G-998 from the National Institutes of Health.
Soluble surface and subsurface antigens of the Arbacia sperm. GORDON P. FLAKE
AND C. B. METZ.
Mctz and Kohler (1960a, b) found four antigens in extracts prepared by freeze-thawing
(Tyler, 1939) Arbacia sperm. The present investigation was designed to determine whether
any of these are subsurface antigens. The technique employed was complete absorption of
sperm agglutinins of rabbit anti-Arbacia sperm serum with a 25% suspension of living Arbacia
sperm, followed by diffusion of the absorbed antiserum against frozen-thawed sperm extract in
an Ouchterlony plate. In such experiments two precipitin bands appeared between the extract
and the absorbed antiserum, and four bands between the extract and unabsorbed antiserum.
Two of the latter joined the two bands of absorbed antiserum. Similar experiments involving
urea extracts of Arbacia sperm produced one band against absorbed antiserum and two against
unabsorbed antiserum. These results indicate that the extraction procedures remove antigens
from the Arbacia sperm which are not involved in the agglutination reaction and thus are
probably soluble subsurface substances.
Treatment of sperm with unabsorbed antiserum rendered univalent by papain digestion
(Porter, 1958) prevented their agglutination by egg water, while treatment of sperm with
digested absorbed antiserum did not. In addition, pretreatment of sperm with absorbed, un-
digested antiserum did not prevent their agglutination by egg water. This confirms the fact
that the sperm surface antibodies had been removed from the antiserum by absorption
with sperm.
Finally, it seemed of interest to examine for an effect of digested absorbed and unabsorbed
antisera, as well as undigested absorbed antiserum, on the fertilizing capacity of sperm. Several
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 473
experiments showed that neither the digested nor the undigested form of absorbed antiserum
affected the sperm's fertilizing capacity, while treatment of sperm with the digested unabsorbed
antiserum markedly reduced this capacity.
Aided by Training Grant 2G-998 from the National Institutes of Health.
Electron microscope study of sperm entry into sea urchin oocytes. LUTHER E.
FRANKLIN AND C. B. METZ.
Germinal vesicle stages of sea urchin eggs are especially suitable for sperm penetration
studies at the electron microscope level because they are normally polyspermic and do not elevate
fertilization membranes. Electron micrographs of inseminated Arbacia and Lytechinus oocytes
revealed spermatozoa either just in contact with the egg surface or completely within the
cytoplasm. Spermatozoa in the former situation exhibited reacted acrosomes, whereas those
in the latter lacked plasma membranes and, usually, nuclear membranes. Exaggerated fertiliza-
tion cones, characteristic of inseminated sea urchin oocytes, were totally devoid of cytoplasmic
organelles, as has been reported in classical literature (Wilson, Harvey). Observations to
date have generally agreed with previous studies describing acrosome reactions (Dan) and
sperm entry (Colwin and Colwin).
An exceptional case of sperm entry was found in a Lytechinus oocyte which had been
mildly centrifuged prior to insemination. Serial sections showed a spermatozoon with an intact
acrosomal granule pentrating the egg surface at an acute angle. The gamete plasma membranes
were closely applied in several regions, but fusion did not seem to have occurred. Engulfment
had progressed to the proximal region of the tail.
Aided by Training Grant 2G-998 from the National Institutes of Health.
Relationship of fertilizin to the acrosome reaction in Arbacia. JORAM PIATI-
GORSKY AND C. R. AUSTIN.
Collier (1959) and Haino and Dan (1961) observed that fertilizin discharged sea urchin
acrosomes and that the frequency of reactions was related to agglutinating titer. This inves-
tigation supports these findings and extends the data to Arbacia punctulata. Fucose determina-
tions related frequency of acrosome reactions to actual concentration of fertilizin. Fibers of
precipitated fertilizin (Tyler, 1949) were washed, redissolved in filtered sea water and dialyzed
overnight. Dilute sperm suspensions were treated with serial dilutions of fertilizin of known
fucose concentration. The sperms were examined under the electron microscope (JEM 6A).
Acrosome reactions occurred from 10 to 20% with 2-5 /ig./cc. of fucose. The reaction
frequency increased sharply up to 15 /xg./cc. of fucose. The percentage of reactions continued
to rise, though less steeply, up to a concentration of 100-125 ^g./cc. of fucose, above which there
was no significant increase in reaction frequency. Even at this optimum concentration of
fertilizin, 100% reactions were never observed, the maxima roughly lying between 50 and 60%.
There was large variation between different sperm suspensions, but the relative changes were
consistent.
Fertilizin solution boiled at pH 4 for 30 minutes ceased to agglutinate sperms, reducing
reactions to percentages only slightly higher than the controls. Preliminary experiments
showed that univalent fertilizin, made by gamma irradiation (5000 r/minute) for 5 minutes of a
solution containing 20 Mg-/cc. of fucose, decreased agglutination significantly and lowered the
reaction frequency from 50 to 20%. Irradiation for 6-9 minutes left the fertilizin non-agglutinat-
ing and primarily univalent. Acrosome reactions were reduced to 3-6%.
These results provide evidence that intact agglutinating fertilizin provokes the acrosome
reaction.
Aided by Training Grant 2G-998 from the National Institutes of Health.
Inducement of the "acrosome reaction" by acridine orange. CHARLES A. SHIVERS
AND THOMAS E. EVANS.
Various agents have been shown to be effective in altering the acrosomal region of sperm
(Metz, 1957). In a series of observations on spermatozoa of Echinarachnius parma and
474 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Arbacia punctulata, employing the dark-field microscope, it was noted that acridine orange
(A.O.) caused a morphological change in the acrosomal area that appeared identical with the
change induced by exposure of sperm to homologous egg water (E.W.)- Ultraviolet (UV)
excitation appeared to enhance the reactivity of the A.O.-treated sperm.
Whereas previous reports have dealt with observations of sperm before and after the
acrosomal change, this report deals with observations of the reaction as it occurred in the
living sperm. This change involved a rapid elongation of the sperm nucleus, accompanied by an
apparent disappearance of the acrosomal granule and a slight displacement of the midpiece.
These changes in the sperm cannot be mistaken for the cytolysis of sperm heads which results
from prolonged exposure of sperm to UV light.
Comparison of electron micrographs of whole mounts of untreated, A.O.-treated, and E.W.-
treated sperm of Echinarachnius indicated that the A.O. -induced reaction was comparable to
the E.W.-induced reaction. Although no quantitative data were taken, it was apparent that the
per cent reaction of both the A.O.- and the E.W.-treated sperm was considerably higher than
in the controls. In nearly all cases, sperm that appeared reacted in the electron microscope by
the criteria presented above (especially the elongation of the nucleus) possessed definite acro-
somal filaments.
Aided by Training Grant 2G-998 from the National Institutes of Health.
Localisation of sperm antigens by dissociation of antigen-antibody precipitates. C.
ALEX SHIVERS AND C. B. METZ.
An attempt was made to localize sperm antigens with antibody obtained by reversal of
antigen-antibody precipitates produced in agar-diffusion plates ("echo technique" ; Nace et a!.,
1960). Localization was followed by indirect staining with fluorescein-conjugated sheep anti-
rabbit globulin antiserum.
Anti-Arbacia sperm rabbit serum globulins were used. These produce four precipitin bands
with frozen-thawed sperm extracts in agar-diffusion plates (Metz and Kohler, 1960). Three of
these are strong bands. The weak band was discarded; the strongest of the three remaining (1)
and the other two (2) together were cut from the plate and analyzed in the reversal procedure.
Controls included agar with antibody alone and with control serum alone.
Isolated bands were adjusted to pH 10-11. After dissociation the pH was readjusted to 7-8
and the dissociated complex layered over air-dried sperm smears. These were treated like tissue
sections in the fluorescent technique.
In smears treated with agar and antibody alone, the entire sperm fluoresced intensely.
Smears from the dissociated single band (1) showed an intense fluorescence of sperm heads and
no tail fluorescence. Fresh sperm treated with this dissociated antibody failed to agglutinate.
These observations suggest that the antigen of band 1 is a sub-surface (non-agglutinating)
sperm head antigen.
Smears treated with dissociated bands (2) showed fluorescence over the entire sperm.
This, plus the fact that fresh sperm are agglutinated by the dissociated antigen-antibody complex
of these two bands, suggests that one or both antigens is located on the sperm head or tail or
both, and that at least one of the antigens is a surface antigen.
Aided by Training Grant 2G-998 from the National Institutes of Health.
Axial bod\ and filament formation in oyster sperms. D. H. SPOON, A. FORER
AND C. R. AUSTIN.
The unreacted sperm head of Crassostrea i'ir</inica, seen in dark-field illumination, has a
central light region one-third its diameter and less refractile than the head outline. This
evidently corresponds to the axial body described from phase-contrast observations and electron
micrographs by Galtsoff and Philpott (1960). The axial body lies in a deep anterior depression
in the nucleus, and appears unstained by the Feulgen reagent and by the vital nuclear dye,
methylene blue. The acrosome granule in dark field is a highly refractile cap on the front
of the head.
The acrosome reaction could be observed in individual sperms by introducing under the
coverslip oyster egg water, hen egg albumin, or solutions of high alkalinity (pH 9.4) or con-
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 475
taining divalent ions (Ca++, Mg++, Mn++). The sequence of events began with swelling of the
acrosome granule and loss of its refractility. This was immediately followed by extrusion of
the filament, apparently from the axial body, which now became displaced to the widened front
surface of the sperm head. With slides and coverslips coated with silicone, filaments did not
attach to surfaces and were longer. Depending on the reactant, filaments varied from a stubby
extrusion of less than 0.3 M to a slender rod over 12 /u long.
A high resolution polarizing microscope with rectified lens (A.O. Co.) showed in un-
reacted sperms at the position of the axial body a region of weak birefringence, with alternate
light and dark quadrants at 45° to the angle of the polarizer and analyzer of the microscope.
When distilled water was added, the sperm heads swelled and this region moved to the front
surface. After filament formation, the birefringence of the region became more diffusely
distributed across the front portion of the head. This birefringence seemed to decrease as
filament length increased. The filament itself was either light or dark depending on compensator
angle. Because the observations were made on structures of a size near the limit of resolution
of the light microscope, the interpretation of the birefringence is difficult. However, the
birefringence observed in the axial body region of unreacted sperms is compatible with the
electron microscopic description of a rod-like object within the axial body parallel to the long
axis of the head. The altered birefringence in reacted sperms is possibly attributable to
derivation of the acrosome filament from the axial body by a change involving reorientation of
a preformed molecular structure.
Aided by Training Grant 2G-998 from the National Institutes of Health.
AUGUST 23, 1962
Acetylcholinestera.se in J\I\tiliis spermatozoa. ARTHUR APPLEGATE AND LEONARD
NELSON.
Sperm suspensions from the minced gonads of Mytilns are washed with filtered sea
water, suspended in pH 7.2 phosphate buffer, homogenized, and then tested for cholinesterase
activity by the photometric method of Hestrin (1949). Up to 30% of the substrate is hy-
drolyzed in 10 hours of incubation at 37° C. The crude homogenate shows optimal esterase
activity at 6.0 X 10~3 to 12.0 X 10~3 M substrate concentration, and inhibition at higher con-
centrations of acetylcholine. Vma* of the enzyme at substrate concentrations, 0.1 X 1Q-3 to
10.0 X 10~3 M, is 2.86 X 10~* nM acetylcholine split/minute/mg. protein, and the Km is 5.8
X 10-3 M. The enzyme is competitively inhibited by 1.1 X 10~5 M physostigmine.
A partially purified enzyme has been obtained from the sperm homogenates by precipita-
tion with 28% ammonium sulfate at 22° C. This preparation shows optimal activity at 1.0 X 10~3
to 5.0 X 10-3 M acetylcholine and a Vmax of 4.56 X 1Q-3 pM acetylcholine split/minute/mg.
protein with a Km of 2.58 X 10~3 M. Preliminary studies with various substrate concentrations
indicate that the enzyme splits butyrylcholine at about one-fifth the rate of acetylcholine, and
that benzoylcholine is split extremely slowly at concentrations above 6.0 X 10~3 M.
There is no apparent change in flagellar activity of Mytilus sperm in the presence of 1.25
X 10-3, 10-4, 10-5, or 10-6 M acetylcholine. However as little as 5.0 X 10~5 M physostigmine
causes an increase in the rate of normal flagellation.
Aided by Training Grant No. 2G-998 from the National Institutes of Health.
Uptake of H3-thymidine by eggs of Arbacia punctitlata. HILDEGARD ESPER.
Experiments were undertaken with H3-thymidine in Arbacia punctulata to obtain infor-
mation concerning the presence of a precursor of DNA in the cytoplasm of sea urchin eggs.
Female sea urchins received two injections each of 20 (J.C. of H3-thymidine (specific activity
20 nC./nM}, 12 hours apart; eggs were collected after 36 hours. In preliminary experiments
eggs were centrifuged in a 0.85 M sucrose density gradient, resulting in separation of the egg
into two halves. Data from the liquid scintillation counter showed that the non-nucleated half
contained 41% of the activity of the nucleated half, indicating that H3-thymidine and/or some
metabolic product is present in the cytoplasm. Whole-egg homogenates were then extracted
by the technique of Ogur and Rosen. Total nitrogen determinations were carried out on
476 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
aliquots of the whole egg homogenate by the Nessler procedure. Activity has been calculated
as corrected net counts per minute per milligram total egg nitrogen. Seventy to 80% of the
counts was recovered in the nucleotide fraction, extracted by cold 0.2 N perchloric acid. Only
7% of the counts was found in the DNA fraction, extracted by perchloric acid at 70° C. for 20
minutes. The remaining counts found in the precipitate probably represent unextracted DNA.
These data indicate that considerable H3-thymidine is taken up into the nucleotide pool of the
mature egg. The activity of the nucleotide fraction may suggest a synthesis of oligonu-
cleotides. Whether this fraction corresponds to the cytoplasmic DNA precursor previously
reported remains to be determined. It is impossible to decide whether the activity of the DNA
fraction is due to synthesis by oocytes contaminating the sample or by mature eggs. Pre-
liminary autoradiographic studies have failed to indicate any DNA synthesis by mature eggs.
Aided by Training Grant No. 2G-998 from the National Institutes of Health.
Incorporation of Cl*-glncose into oocytes and ovarian eggs of Arbacia punctnlaia.
HlLDEGARD ESPER.
Experiments indicate that C14-glucose is rapidly metabolized by the developing sea urchin
oocyte. Two female sea urchins were injected with 0.5 ml. of isotonic KC1 to induce shedding
of all mature eggs. On the following day these animals were injected with 10 juC. of uni-
formly labeled C14-glucose (specific activity 20 /uC./mg. ). After 24 hours the ovaries were
removed, fixed in Carney's fluid and paraffin-embedded. Six-micron sections were prepared
for radioautography with Kodak Nuclear Track Emulsion NTB2, and stored for two to three
days before developing. The distribution of tracks indicated different stages of development,
probably correlated with yolk synthesis. Small oocytes which had not yet entered a synthesis
period were unlabeled. Larger oocytes were densely labeled over both nucleus and cytoplasm,
as were a certain number of mature eggs. These latter may have been still in the process
of maturation at the time of labeled glucose injection, and subsequently completed their active
synthesis period. Mature eggs with no tracks had evidently finished their synthesis period
prior to injection of C14-glucose. Extraction by RNase, DNase and hot trichloroacetic acid
did not significantly alter this picture, suggesting that the C14 from glucose is incorporated
primarily into proteins. Numerous grains were found immediately surrounding the eggs,
probably indicating incorporation into the jelly coat material.
Aided by Training Grant No. 2G-998 from the National Institutes of Health.
Free amino acids and peptidcs in unfertilized and fertilised eggs of Arbacia
pnnctulata. THOMAS EVANS, ALBERTO MONROY AND ALFRED SENFT.
Chromatographic analyses of the picric acid-soluble components of eggs of A. punctulata
were carried out, using the Spinco Model 120 ammo acid analyzer. In all cases the jelly coats
were removed prior to analysis. Large amounts of an asparagine-glutamine fraction, glutamic
acid, glycine, arginine, and ammonia were found, as well as lesser amounts of alanine, leucine,
isoleucine, valine, methionine, serine, threonine, ornithine, tyrosine, proline, and hydroxyproline,
and trace amounts of phenylalanine, histidine, and tryptophan. By comparison of unhydrolyzed
and hydrolyzed samples at least five peptides were demonstrated. Calculations of molar ratios
of five amino acids were made with reference to the sum molar quantity of those amino acids.
Accurate calculations were impossible with the other amino acids, due in some cases (notably
the asparagine-glutamine fraction and arginine) to peak asymmetry and in the rest to low
concentration (<0.1 pM). The molar ratios calculated for two runs of unfertilized (UF)
eggs and two runs of fertilized (F) eggs (6 minutes and 13 minutes after fertilization) are:
glycine, 0.34177 (UF) and 0.44790 (F) ; alanine, 0.03646 (UF) and 0.06385 (F) ; glutamic
acid, 0.46840 (UF) and 0.40515 (F) ; isoleucine, 0.10965 (UF) and 0.06046 (F) ; and threonine,
0.04374 (UF) and 0.02264 (F).
Analysis of a sample of 32-cell stage embryos yielded a pattern similar to the UF and
F eggs.
Determinations of total egg N (Ntot) and a-amino N (aN) (Moore and Stein, 1954)
of 10% trichloroacetic acid extractions were made on both 5-minute F and UF eggs. The
ratios obtained of «N to Xt.,t varied somewhat, but indicated a rise in <*N shortly after
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 477
fertilization. Nitrogen determinations on similar amounts of sperm used to fertilize the eggs
indicated that very little if any of this change could be attributed to sperm contamination.
Aided by Training Grant 2G-998 from the National Institutes of Health.
Evidence of a chemotactic substance in the female gonangium of Campanularia.
RICHARD L. MILLER AND LEONARD NELSON.
Fresh sperms released by the male Catnpanularia gonangium were found to swim randomly
in a straight line, although the rotation of the head altered the path into a broad spiral. When
the sperms reached the vicinity of the opening into the female gonangium, a radical change in
the normal mode of swimming was often seen. The frequency of the head rotation increased
noticeably in many cases, and there was often a change from movement in a straight line to
tight circles and sharp turns. Of great significance, however, was the high proportion of
sperms seen to change direction and swim into the female gonangium. Those sperms swimming
out of the gonangium were seen to turn back abruptly, but a few left the area completely. This,
and the fact that neither agglutination nor a decrease in motility were seen, suggests that more
than trap action is involved. In fact, the phenomenon resembles closely chemotaxis as described
in ferns and mosses (Rothschild, 1952).
Different portions of the gonangial tissue, as well as sea water and coenosarc tissue controls,
were placed in glass capillaries and immersed in a sperm suspension. It appeared that the
distal portion of the "funnel" tissue contained the preponderance of the hypothetical attracting
agent. The substance seems quite labile in sea water, is permeable to perisarc, and is not
sensitive to trypsin digestion unless the cells releasing it are damaged.
Supported by Training Grant 2G-998 from the National Institutes of Health.
Changes in some proteins in the course of the development of Arbacia punctulata.
RONALD J. PFOHL AND ALBERTO MONROY.
Modifications of the protein pattern of the egg in the course of development of the sea
urchin, Arbacia punctulata, have been investigated by disc electrophoresis. Eggs or embryos
were extracted in Tris-glycine buffer (0.2 M, pH 8.3), centrifuged at 80,730 g and the su-
pernatant concentrated. The separation of the protein components was effected with a current
of 2 ma per column for If hours in the cold.
In the unfertilized eggs three bands which stained heavily with amido schwarz and seven
to nine fainter bands were observed. Aside from a slight decrease in the intensity of staining
and the variable disappearance of some of the fainter components, there was no change in the
amido schwarz staining band pattern in the course of development.
In the unfertilized eggs, three bands, the fastest one corresponding to the fastest amido
schwarz component, showed esterase activity. In the early pluteus stage, the middle com-
ponent split into two bands, neither of which corresponded to an amido schwarz band. In
the late pluteus, five bands with esterase activity were present. The two new components
corresponded to the two slowest amido schwarz bands. In general the esterase activity was
considerably more intense in the developmental stages than in the unfertilized eggs.
Acid phosphatase activity was distributed in a minimum of four bands. There was no dis-
tinct increase in activity in the developmental stages over the activity in the unfertilized eggs.
The alkaline phosphatase activity in the unfertilized eggs was weak and present in one band
corresponding to the fastest amido schwarz component. At the mesenchyme blastula and later
stages, a substantial increase in activity was apparent, thus confirming the observations of
Mazia et al. (Bio!. Bull., 95: 250). The activity was almost entirely localized in a new, more
slowly moving, component, distinguishable from the former faint band.
Aided by Training Grant No. 2G-998 from the National Institutes of Health.
Electrophoretic and ultracentrifngal analysis of Ihe fractionated extracts of Arbacia
punctulata eggs and early plutci. RONALD J. PFOHL AND ALBERTO MONROY.
By the use of disc electrophoresis, changes have been described in the protein pattern of
the egg in the course of development of Arbacia punctulata (Pfohl and Monro}^ previous
478 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
abstract). Extracts prepared as described have now been fractionated by precipitation at 45%
(45 fract.) and saturation of (NH4)2SO4 (sat. fract.). Precipitates were analyzed, using disc
electrophoresis (DE) and analytical ultracentrifugation (UC).
No differences were detected in the UC patterns of the total extracts and fractions thereof
between unfertilized eggs and early plutei. The 45 fraction exhibited three components with
the following S20 values: (1) 1.22-1.36; (2) 1.98-2.70; (3) 7.08-7.21. The sat. fraction had two
main components with S20 values of (la) 5.32-5.77; and (2) 8.38-9.29. A small component
in the latter formed a shoulder of (la) and seemed to correspond to component (3) of the
45 fraction.
In the DE patterns the slowest, heavily staining (amido schwarz ) components were present
in the saturated fraction, whereas the heavily staining, fastest component was of about equal
intensity in both fractions. All of the esterase activity and most of the acid and alkaline
phosphatase activity corresponding to the latter band was present in the sat. fraction. The
middle esterase band of the unfertilized eggs, which is split into two bands in the early pluteus
stage, is almost entirely present in the 45 fraction.
No correlation is as yet possible between the UC and DE patterns. It seems, however,
worthwhile emphasizing the following points: (1) the esterase and phosphatase activities of
the fast DE band are almost entirely precipitated above 45% of (NH4)2SO4, whereas the part
precipitated at 45% appears to be devoid of such enzymatic activities; (2) the esterase activity
is present in three main bands, showing apparently the same substrate specificity ; by fractional
precipitation, however, they can be separated into two groups.
Aided by Training Grant 2G-998 from the National Institutes of Health.
An actin-like protein isolated from starfish sperm. KENT M. PLOWMAN AND
LEONARD NELSON.
An actin-like protein, "spactin," was prepared from sonicated Astcrias forbcsii sperm from
which about 80% of the heads had been removed by centrifugation. The suspension of flagella
and midpieces was extracted according to the actin isolation of Tsao and Bailey and of
Mommaerts, yielding a clear, gel-like pellet.
Analysis of a 5% perchloric acid extract of the protein by paper chromatography, with
n-butanol-ammonia, yielded a single ultraviolet-absorbing spot which matched the ATP con-
trols in Rt values. The eluate had an absorption spectrum identical with ATP. By assuming
a molecular weight of 60,000, one can estimate from dialyzed preparations that 0.6-1.4 moles
of ATP were bound per mole of protein. The spactin pellet was soluble in distilled water and
"salted-out" at ionic strengths above 0.05 KC1. Water solutions appeared somewhat thixo-
tropic in the Ostwald viscometer. When run in the analytical ultracentrifuge, a fresh sample
in distilled water produced a single major peak, although an older sample gave two peaks of
equal size. When the peak had sedimented nearly to the bottom of the cell, 90% of the
nucleotide was associated with it. When one part of spactin was combined with three parts
of purified rabbit myosin in a final concentration of 0.4 M KC1, a precipitate formed on
addition of 10~3 M ATP. The specific activity of this precipitate, redissolved in 0.5 M KG,
as an ATP-ase, was 50% of that of the rabbit myosin in CaCl2 at pH 8.9 and 7.2 and MgCl2 at
pH 7.2, but was nearly equal in activity to that in MgCl2 at pH 8.9. This solution had two
very sharp peaks in the ultracentrifuge, with Svedberg constants of 4.5 and 6.5 at 0.42rr
protein concentration.
Aided by Training Grant 2G-998 from the National Institutes of Health and by Research
Grant RG-6815.
GENERAL SCIENTIFIC MEETINGS
AUGUST 27-30, 1962
Abstracts in this section (including those of Lalor Fellowship Reports) are
arranged alphabetically by authors under the headings "Papers Read," "Papers
Read by Title," and "Lalor Fellowship Reports." Author and subject references
will also be found in the regular volume index.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 479
PAPERS READ
Differentiation of s\uaptie and GABA inhibitory action in crab ncuroniuscitlar
junctions. EMILIO ALJURE, HAROLD GAINER AND HARRY GRUNDFEST.
Intracellular recording from the adductor muscle of the dactylus of walking legs of Cancer
borcalis, together with selective stimulation of excitatory and inhibitory axons, confirmed that
GABA does not activate inhibitory post-synaptic membrane. At 10~* w/v, GABA blocks epsps
reversibly without decreasing the effective resistance of the muscle membrane, whereas stimu-
lation of the inhibitory axon at 100/sec. decreases the effective resistance by 30%. GABA
does not appear to inactivate the excitatory post-synaptic membrane, since the amplitude and
frequency of spontaneous "miniature" epsps are unaltered by its application. No effect of
GABA on the non-synaptic membrane of the muscle fibers was observed. It did not alter the
rates of movements of K+ and Cl~ as determined from the responses of the membrane potential
to changes in outside concentration of the respective ions. Thus, GABA acts on the pre-
synaptic terminals. Furthermore, combinations of picrotoxin with serotonin or phenylethylamine
(10~* w/v each) cause repetitive antidromic discharge of the axons in response to a single
orthodromic impulse. As in lobster, the firing appears to be due to sustained depolarization
of the axon terminal. This repetitive firing is blocked by GABA (10~* w/v).
Synaptic inhibition of the epsp appears to be greater than can be accounted for by the
conductance increase of the post-synaptic membrane. It seems likely, therefore, that the
inhibitory transmitter has a dual action : ( 1 ) to activate the inhibitory synaptic membrane, and
(2) to block the epsp. The latter might be due to interference with the excitatory transmitter
or with its release.
Studies on thrombocytes of the smooth dogfish, Mustelis canis. FRANK A.
BELAMARICH, RUSSELL F. DOOLITTLE AND DOUGLAS M. SURGENOR.
Thrombocytes of the blood of vertebrates have been implicated in the clotting mechanism,
but only the mammalian thrombocyte (platelet) has been extensively studied. The latter is
not only involved in clot promotion, but is necessary for clot retraction. ATP and serotonin
are released from platelets during the clotting process.
When white cells of the dogfish are added to plasma, clotting activity is increased. No clot
retraction takes place unless white cells are present. Two general populations of white cells
could be achieved by density gradient centrifugation. Granulocytes are the main component
of the layer above 0.75 M sucrose, non-granulocytes collect over 1.0 M sucrose, and the red
cells accumulate at the bottom. When the two populations of white cells were tested for
clot-promoting activity they exhibited no differences.
White cells incubated with plasma containing C14-labeled serotonin exhibit a gradual in-
crease in the uptake of label over a three-hour period. If, after this time, plasma containing
no labeled serotonin is substituted, the labeling decreases at a rate comparable with the rate of
uptake. Since the initial rate of uptake is near the rate of diffusion, it is concluded that
thrombocytes do not actively accumulate serotonin, nor do they retain it against a concentration
gradient.
Whole blood, plasma, serum, red cells, and white cells were tested for smooth muscle-
stimulating activity on a section of dogfish pyloric stomach. Serum had slight activity, while
a crude extract of white cells had a high degree of activity. When the crude extract was
chromatogrammed (n-butanol/acetic/water, 60:15:25) there were a number of ninhydrin-posi-
tive areas as well as one UV-absorbing and one UV-fluorescing area. Only slight activity
could be recovered after chromatogramming, and no conclusion can be made at this time
concerning the chemical nature of the smooth muscle stimulator.
This research \vas supported by N.I.H. Grant H-5828.
On the nature of dogfish trypsinogen and trypsin. DAN C. BRYANT, RONALD S.
WEINSTEIN, DAVID L. KLEIN AND R. F. DOOLITTLE.
The purpose of this study was to find what changes have occurred in the trypsinogen
molecule in the course of evolution. In all experiments tosyl-L-arginine methylester (TAMe)
480 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
colorimetric assays for trypsin activity were used. The pancreas of the spiny dogfish (Squalus
acanthias) was shown to contain an enzyme similar in its specificity to mammalian trypsin.
Spiny dogfish pancreases were homogenized in CaCl2 solutions buffered at pH 8.0 with Tris,
the homogenates centrifuged, and the resulting supernatant fluids kept at 2° C. During the
course of several days, precipitates formed and were removed by centrifugation, leaving a
solution which showed twice as much trypsin activity per gram protein as commercially pre-
pared "purified" hog trypsin. Attempts at further purification indicated that both dogfish and
hog trypsin are salted out in the 40 to 70% range of saturated (NH4)2SO4. Comparison of
dogfish with crystalline bovine trypsin indicated that the dogfish trypsin preparation was far
from crystalline purity. Heat inactivation experiments carried out in the 46° C. to 60° C.
range demonstrated that elasmobranch trypsin is more thermo-labile than hog trypsin. When
incubated for 5 minutes at 56° C., dogfish trypsin retained 10% of its original activity, while
hog trypsin retained 70% original activity. The autocatalytic conversion of dogfish trypsinogen
to trypsin was studied in preparations in which native trypsin was inhibited with diisopropyl
phosphofluoridate (DFP). Aliquots of dogfish trypsinogen were inoculated with dogfish
trypsin and hog trypsin solutions of varying activities. Dogfish trypsin catalyzed the forma-
tion of trypsin from dogfish trypsinogen at a greater rate than did hog trypsin. It is concluded
that dogfish and hog trypsin molecules are similar in that both (1) are inactivated by DFP,
(2) hydrolytically split TAMe, and (3) have similar salting out properties. In contrast,
molecular differences are indicated by differences in temperature stability and the species
specificity of the trypsin-initiated conversion of trypsinogen to trypsin.
Purification and some properties of lipoyl dehydrogenase from dogfish liver. C. P.
CHANNING, A. EBERHARD, A. H. GUINDON, C. KEPLER, V. MASSEY AND C.
VEEGAR.
Lipoyl dehydrogenases isolated previously from other sources (pig heart, beef liver, E. coli,
spinach leaves) are flavoproteins in which catalysis depends not only on the flavin but on another
prosthetic group, a protein disulfide linkage. Lipoyl dehydrogenase from the dogfish (Squalus
acanthias and Mustclus canis) has now been isolated and its properties compared with those
of the pig heart enzyme. A 1000-fold purification was achieved by extraction with dilute salt,
(0.03 M phosphate, pH 6.3, +3 X 10-3 M EDTA + 2% (w/v) (NH4)2SO4), heating to 80° C.
for ten minutes, adsorption on and elution from calcium phosphate gel, (NH4)2SO4 fractionation
between 0.55 and 0.85 saturation, and column fractionation with calcium phosphate gel and
diethylaminoethyl cellulose. The resulting enzyme was yellow, showing absorption maxima at
453 m/j. and 340-360 m/j. and with a pronounced shoulder at 480 m/u. Like the pig enzyme,
the dogfish lipoyl dehydrogenase is also extremely fluorescent, a property almost unique among
flavoproteins where the fluorescence of the flavin is generally quenched on binding to the
protein. The fluorescence excitation spectrum has peaks at 290 m/x, 360 m/x and 460 m/x, and
the emission spectrum is maximal at 518 myu. Evidence has also been obtained indicating the
catalytic functioning of a disulfide prosthetic group. Incubation studies in the presence of
10-3 M arsenite show that inhibition is obtained only when DPNH is included in the incubation
mixture ; inhibition is not obtained on incubation with DPNH or arsenite alone. Thus it ap-
pears that a disulfide is reduced to a dithiol by reducing substrate. A further comparison of
the reaction mechanisms of lipoyl dehydrogenases from various sources is under investigation.
Studies on the dissociation of Loligo pcalei hemocyanin. L. B. COHEN AND K. E.
VAN HOLDE.
The hemocyanin of the squid Loligo pealci has been studied by sedimentation and diffusion
measurements on blood diluted with 0.1 ionic strength buffers. Four main sedimenting
boundaries have been observed between pH 6.1 and 10.7: (A) a homogeneous substance of
molecular weight 3,400,000, as determined by sedimentation and diffusion, and sedimentation
coefficient S,n° = 56.1; (B) and (C), components usually observed together, with 5,0=19 and
13, respectively, and (D) another homogeneous material of molecular weight 270,000, determined
by analysis of boundary spreading in sedimentation, and S,0 = 10. In the absence of added
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 481
Mg*" ion, (A) is the principal component below pH 7.2, at which point dissociation into (B)
and (C) occurs overnight. Between pH 7.6 and 9.1 (B) and (C) are present. At pH 10.0
and above (D) alone is found. A substance apparently identical with (D) was found as the
only component in a solution containing 3 M urea at pH 6.6. In 0.01 M Mg++ concentration
(A) is stable to pH 9.9, at which point dissociation into (D) occurs. This process is
reversible if 0.01 M Mg++ is present.
The apohemocyanin prepared by removing the copper by dialysis against KCN solution
exhibits a sedimentation pattern identical with that of the native protein below pH 7.8 in the
presence of 0.01 M Mg++ ion. At higher pH, dissociation of (A) occurs first with a gradual
decrease of S, and eventually entirely into (B) and (C). Above pH 9 component (D) is
again found. The dissociation at pH 8.4 has been found to follow first order kinetics by
observing the change in boundary area in an ultracentrifuge experiment. Apohemocyanin,
which had been taken to pH 8.4 for 56 minutes, exhibited partial reassociation when stored
overnight at room temperature at pH 7.2. In this reassociated apohemocyanin \ve have seen
a 36S component.
This research was supported in part by a National Science Foundation Cooperative Fellow-
ship to Columbia University, and in part by a grant from the National Institutes of Health,
Council on Arthritis and Metabolic Diseases.
Spectral studies of hemocyanin. H. A. DEPHILLIPS, JR. AND K. E. VAN HOLDE.
An investigation of the spectra of the hemocyanins from Busycon canaliculatum and Loligo
pcalei has been carried out. Blood from Loligo was diluted directly with 0.1 ionic strength
buffers ; the Busycon hemocyanin was purified by dialysis and ultracentrifugation. A pre-
liminary study of the pH-stability diagram of the Busycon hemocyanin was conducted.
Eriksson-Quensel and Svedberg (1936) report that the principal component in diluted Busycon
blood between pH 4.6 and 7.7 was of sedimentation coefficient 100S ; above this pH, partial
dissociation into 60S material was observed. At pH 9 complete dissociation into 13S material
was found. We have been able to confirm these results only if the solutions of purified
hemocyanin were made 0.01 M in Mg++ ion. In the absence of added Mg++ the hemocyanin
undergoes each dissociation at a lower pH value. Also, we have observed dissociation at pH
3.8; when 0.01 M Mg++ is present, boundaries of S20 equal to 96.2, 81.3, and 10.5S are observed.
In the absence of Mg++ 82.8S and 10.5S material is found.
Measurements of the absorption spectra in the range 300 m/x to 700 m^ were carried out with
oxygenated and deoxygenated Bnsycon hemocyanin, and with the oxygenated hemocyanin and
apohemocyanin from Loligo. Both oxygenated materials show maxima at 345 m/n and 570-
580 m/u. The optical densities of the deoxygenated Bnsycon hemocyanin and the apohemocyanin
from Loligo exhibit a smooth increase with decreasing wave-length, presumably due to scatter-
ing. The curve from the deoxygenated Busycon hemocyanin, which was linear in l/\4 be-
tween 330 mtt and 700 m/x, was subtracted from the curve for oxygenated material. The dry
weight of the Busycon material was determined. Extinction coefficients were 593 I/mole-cm.
at 570 m/i, and 14,650 I/mole-cm, at 345 m/t, on the basis of copper molarity, assuming 0.25%
copper. The optical rototory dispersion of the Loligo hemocyanin has been studied between
300 and 700 in/*. Both of the bands exhibit negative Cotton effects.
This research was supported in part by a grant from the Division of General Medical Sci-
ences, National Institutes of Health, and in part by a grant from the National Institutes of
Health, Council on Arthritis and Metabolic Diseases.
Inhibitors of lobster blood clotting. R. F. DOOLITTLE AND L. LORAND.
The plasma of lobsters and certain other crustaceans is known to contain a soluble protein
("fibrinogen") which is capable of forming a clot. This conversion is effected by a thermolabile,
non-dialyzable factor present in various lobster tissues and blood cells and is calcium-dependent.
The finding that lobster clots are insoluble in 5 M urea and in 1% monochloroacetic acid sug-
gested that the crosslinks formed during clotting were covalent in character and possibly
analogous to those formed in vertebrates by the combined action of thrombin and fibrin-
stabilizing factor. The fact that papain can induce similar clots in mammalian systems by
482 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
acting directly on fibrinogen (Biochcm. Binphys. Res. Comm., 7: 457, 1962) strengthened such
a hypothesis. Since crosslinking of mammalian clots can be inhibited by some glycine amides
and esters (Nature, 194: 1148, 1962), a similar study of potential inhibitors of lobster (Homarns
americamts} clotting was undertaken. Lobster blood coagulation induced by the addition of
homologous muscle extract was appreciably inhibited by glycine methylester and glycylglycine
methylester at concentrations of less than 0.2 rrul/. The specificity of the inhibition is apparent
from a quantitative scaling of the degree of retardation based on the concentration of inhibitor
necessary for a five-fold increase in clotting time. The best inhibitors found thus far were
arbitrarily set to 100: glycine methylester (100); glycylglycine methylester (100); L-lysine
ethylester (12) ; glycineamide (6) ; DL-serine methylester (1.7) ; L-tyrosine methylester (0) :
L-isoleucine methylester (0) ; L-histidine methylester (0) ; tosyl-L-arginine methylester (0) ;
glycylglycine (0) ; glycine (0) ; methyl acetate (0) ; lysine (0) ; epsilon-aminocaproic acid CO).
Clotting was also completely inhibited by very low concentrations of p-mercuribenzoate, iodo-
acetate and cupric chloride, indicating the importance of functional sulfhydryl groups. Hy-
droxylamine was also found to be a good inhibitor.
This work was aided by grant H-2212 from the National Institutes of Health.
Electrical activity associated until biolumincscencc in a single cell. ROGER ECKERT.
Single luminescent Noctihica (Eckert and Findlay, this issue) were held in sea water to
the end of a small horizontal pipette by means of slight hydrostatic pressure, and were positioned
over the objective of an inverted-type compound microscope. A selector prism in the base of
the microscope could be used to divert the image of the cell from the oculars to the photo-
cathode of a multiplier tube. Stimulating pulses were passed between the lumen of the
suction pipette and the bath. A recording capillary electrode was inserted through the thin
peripheral cytoplasmic layer into the large internal vacuole. Stimulating current, potentials,
and light flux were simultaneously recorded and displayed.
An all-or-none 40-70 mv negative-going spike was recorded in response to sufficient cur-
rent. It reached its peak in about 7 msec, and subsided in another 4 msec. The emission of light
begins at about the time of peak potential, and is never recorded in its absence. The light flash
reaches its maximum intensity in about 20 msec, and decays to 50% in another 20 msec.
Repetitive stimuli with 100-msec. intervals elicit facilitation of the luminescent flashes, while
stimulation at intervals shorter than the duration of each flash brings about summation of
flashes as well. Neither phenomenon is accompanied by facilitation or summation of the action
potentials.
Further evidence that the intensity of the flash is independent of the potential size per sc
was obtained by the addition of KC1 to the bath in amounts large enough to diminish the ampli-
tude of the action potential. In that case the flash intensity remained unaltered as successive
action potentials became smaller. Only when there was no longer any sign of an active
electrical response did the emission of light suddenly fail.
Supported by U.S.P.H.S. Grant B-3664 and N.S.F. Grant G-21S29.
Nutrient transport in the starfish, Asterias forbcsi, as studied unth isolated diges-
tive glands. JOHN CARRUTHERS FERGUSON.
Individual digestive glands, weighing approximately 0.6 gram, were removed from healthy
starfish and placed in 25 ml. of either filtered sea water or pooled, cell-free coelomic fluid to
which small amounts of high specific activity glucose-C14 or glycine-C14 had been added. The
preparations were gently aerated and maintained at 21° C. Samples of fluid were taken
periodically for 10 hours and activity assayed. Little difference was observed in the mean
rate of removal of glucose-C14 from sea water (half-time, 2.16 hours) and coelomic fluid
(half-time, 2.26 hours). Glycine-C14, however, was removed rapidly from sea water (half-time,
1.76 hours), but its rate of disappearance was somewhat inhibited in coelomic fluid (half-time,
4.73 hours). Assuming exchange rather than simple removal, the total amount of glycine (or
possibly other amino acids) in the coelomic fluid would be turned over in 6.8 hours. Relating
this value to conditions in the animal would indicate a turnover time in vivo of 0.6 to 0.7 hour.
This high rate of movement of amino acids through the coelomic fluid would enable it to
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 483
function effectively as the medium of transport in spite of its low content of nitrogenous sub-
stances, for which a mean value of 63.5 /j.g. N per ml. has been determined. When two
digestive glands were placed in 25 ml. of sea water, nitrogenous substances appeared in the
water at a rate that decreased with time, apparently approaching an equilibrium at a concentra-
tion of 30 jug. N per ml. These results confirm the tracer studies and indicate that nitrogenous
substances are constantly diffusing from the digestive glands at fairly steady rates, but are
being reabsorbed at rates dependent on their concentration in the surrounding medium and on
the physiological state of the tissue. It seems probable that similar phenomena, differing only
in degree, are occurring in all of the tissues of the starfish.
Supported by N.S.F. Grant G-20744 to Cornell University.
Effect (if temperature on polymerisation of G—ADP actin. ROBERT J. GRANT.
Since G-ADP actin polymerizes simply upon the addition of KC1 and Mg, without the
addition of ATP and its subsequent dephosphorylation to ADP, it provides a simplified
material for the study of actin polymerization.
That the polymerization of G-ADP actin to F-ADP by KC1 and Mg has a higher tempera-
ture coefficient than the polymerization of G-ATP by the same reagents is shown by the fact
that at 29° C. these reagents cause both proteins to polymerize in similar fashion, while at 1°
they will cause the polymerization of G-ATP but not that of G-ADP. This suggested that
the polymerization of G-ADP is an endothermic process capable of being reversed simply by a
temperature change. As a prelude to further thermodynamic characterization of the G-ADP
polymerization process, an attempt was made to demonstrate this reversibility.
If salt is added to a G-ADP-Mg solution at 1°, no polymerization occurs, i.e., there is
no viscosity rise. If the solution is transferred to 29° there is a rapid rise in viscosity, de-
noting the formation of polymers. After returning the polymerized material to 1° the high
viscosity falls off. The rate and extent of this reversible depolymerization are dependent on
the pH, the buffer used, and the concentrations of protein, KC1 and MgCL That the protein
is still active, i.e., that true reversal has been effected, is shown by the recovery of high
viscosity upon returning the depolymerized G-ADP to 29°. In this way, 80-90% reversible
depolymerization has been achieved.
Further studies will be undertaken to determine the equilibrium constants of the polymeriza-
tion at various temperatures and the entropy changes involved.
Aided by a National Institutes of Health Pre-doctoral Fellowship and the Muscular
Dystrophy Association.
Influence of aldehyde chain length on the relative qitaiitmn yield of the biolumi-
nescent reaction of Achromobacter fisclieri. ]. W. HASTINGS, J. A. SPUDICH
AND G. MALNIC.
Light emission using highly purified bacterial luciferase requires FMNH2, oxygen, and
long-chain aldehyde, the sequence of reaction being in that order. Added aldehyde is not neces-
sary for the reaction of enzyme with FMNH, or for the subsequent oxidation. However, the
amount of light emitted upon oxidation of the reduced enzyme intermediate is considerably
greater in the presence of aldehyde. Since with aldehyde initial light intensity (I0) is greatly
increased, without a proportionate increase in the decay constant (k), the effect of aldehyde
may be formally described as an effect upon the quantum yield of the oxidation of reduced
enzyme. It was of interest, therefore, to determine both I0 and k for different aldehydes, and
to evaluate the relative quantum yield.
Experiments were carried out at 24° C. with pure luciferase, 0.05 M, pH 6.8 phosphate
buffer, and FMNH2 reduced with H2 with platinized asbestos.
The aldehydes (either obtained commercially or synthesized by a LiAlH4 reduction) were
purified immediately before use by gas chromatography and used as a water-saturated solution,
excess droplets being removed by centrifugation. Measurements at different aldehyde concen-
trations (with 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18 and 20 carbons) showed typical
saturation curves, having characteristic values for both I0 and k with each aldehyde.
I0 and k vary regularly with carbon chain length, showing three maxima, at 4, 9 and 14
carbons. The relationship between I0 and k is such that there occurs a parallel but much less
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
accentuated variation in quantum yield. The highest and approximately equal values for
quantum yield were obtained with aldehydes having 12, 13 and 14 carhons, lower values being
measured with aldehydes having more or fewer carbon atoms. The maximum quantum yields
obtained were approximately 45 times those obtained without added aldehyde.
Hemerythrin: dissociation into subiinits and reconstitution. IRVING M. KLOTZ
AND STEVEN KERESZTES-NAGY.
The molecular weight of hetnerythrin, 107,000, has been evaluated by several hydrodynamic
and thermodynamic methods. This molecule contains 16 Fe atoms, and hence 8 oxygen-bind-
ing sites, since it has been established previously that each OL. is held by 2 Fe. It seemed of
interest, therefore, to see if the native macromolecule was constituted of subunits.
Such subunits, of experimentally determined molecular weight near 14,000, were obtained
first by each of three methods : exposure to high pH in Na2COs solutions ; exposure to an
anionic detergent, sodium dodecyl sulfate ; conversion of cationic lysine side-chains to anionic
ones by reaction with succinic anhydride. These treatments are relatively strong, however,
and irreversible.
It has also been found recently that hemerythrin contains 1 SH group per 2 Fe atoms.
We have now discovered that this SH group plays a vital role in maintaining the size of the
native macromolecule. If a mercurial, salyrgan, which combines with SH groups, is added to
hemerythrin, the macromolecules are dissociated into subunits of 14,000 weight ; at an SH/Fe
ratio of 0.5, dissociation is complete.
The subunits will reaggregate spontaneously into methemerythrin if the mercurial is re-
moved from the protein by addition of a mercaptan. With cysteine ethyl ester, added in 5:1
ratio to the Fe, reconstitution was complete. Furthermore, chemical reduction with NaBH<
plus NaHSO3, followed by admission of oxygen, regenerated the red-violet color of
oxyhemerythrin.
These investigations were assisted in part by a research grant (H-2910) from the National
Heart Institute, United States Public Health Service, and grants from the Graduate School
Research Fund of Northwestern University.
Effects of D2O on the cortical gel structure and cleavage capacity of Arbacia eggs.
DOUGLAS MARSLAND, ARTHUR M. ZIMMERMAN AND HARVEY ASTERITA.
Previous work has shown that a substitution of D2O for H2O in sea water, to the level of
70% or more, stops all activity in the mitotic apparatus, but does not stop the furrowing process
— provided the eggs have approached to within about two minutes of cleavage time when the
treatment is applied. The current work represents an attempt ( 1 ) to evaluate the effects of
lower concentrations of D2O upon the intensity of the furrowing process, as judged by its
resistance to blockage by high hydrostatic pressure, and (2) to relate these observations
to pressure-centrifuge measurements of the gelational state of the cytoplasmic cortex.
Eggs immersed in 5% D2O artificial sea water, starting 40 minutes after insemination,
reached the furrowing stage in synchrony with control eggs in non-deuterated artificial sea
water. The intensity of the furrowing process was slightly but consistently greater in the
deuterated eggs, tested with reference to their capacity to maintain their furrows when exposed
to high pressure (5000 lbs./in.2 at 20° C.). The pressure, applied at the time when 20%
of the eggs displayed incipient furrow and maintained for 20 minutes, caused complete sup-
pression of the furrows in only 47% (±4) of the deuterated eggs, as compared to 64% (±3)
of the controls. Higher concentrations of D2O could not be used, since more heavily deuterated
eggs were retarded and asynchronous in their approach to the cleavage stage.
No measurable effect of 5% deuteration upon the gelational state of the cytoplasmic cortex
could be observed in eggs subjected to pressure-centrifugations (8000 lbs./in.2; 41,000 g ;
20° C.) performed 10 minutes prior to cleavage, after 20 minutes of immersion. In fact, to
obtain a measurable stiffening of the cortical gel, as judged by an increased resistance to the
displacement of the cortical pigment bodies, it was necessary to raise the D2O content of the
sea water to 40%.
Supported by Grants C-807(C13)CB and GM 07157-03, U.S.P.H.S.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 485
Contrasts in activation of the egg of Arbacia punctnlata. ARTHUR K. PARPART
AND THOMAS V. N. BALLANTINE.
In the past, artificial activation of eggs of Arbacia punctulata has been brought about by
non-electrolytes that penetrate the cortical granules, by osmotic shock, by detergents, by acid
and alkali. Each of these, as do sperm, causes cortical granule explosive breakdown.
Present studies show that cysteine in sea water artifically activates eggs without breakdown
of cortical granules. Activation was 100% at 0.005 M and 80% at 0.001 M cysteine. All eggs
were re-exposed to sea water at S minutes. Activation was normal : the subsurface "cortical
gel" gelled in 3 minutes and contained the usual increase in number of motionless echinochrome
granules ; the cytoplasmic changes for centering the nucleus ; the breakdown of the nuclear
membrane and "streak stage" all occurred. The cleaved (25%) and uncleaved eggs had a
good cortical granule layer around them. Glutathione gave less activation.
In contrast to cysteine, the enzyme papain (2 X crystallized) caused normal explosion of
the cortical granules (1 minute), egg activation and cleavage at a concentration of 8 X 10~6 M
in the presence of any one of the following, in 0.001 M concentration: cysteine, glutathione,
EDTA, ascorbic acid and KCN. There was a good tight fertilization membrane and hyaline
layer around eggs activated by papain! EDTA, ascorbic acid and KCN didn't cause break-
down of cortical granules or activation. Trypsin, chymotrypsin or hyaluronidase with EDTA
had no effect. It appears that papain, unlike trypsin, has no effect on the vitelline membrane,
but it does affect the membrane of the cortical granules, leading to the release of the glyco-
protein present inside the granule. It is believed that the action of cysteine is correlated with
its effect on the degree of gelation of the interior of the
Separation of phasic and tonic contractions in Spisnla intestine. C. L. PROSSER,
D. MARTIN AND R. SHA'AFI.
The longitudinal smooth muscle of intestines of bivalveN Mcrccimria and Spisula shows
both phasic and tonic contractions. Spontaneous phasic contractions and those resulting from
electrical stimulation are accompanied by fast action potentials ; no electrical accompaniment
was detected with tonic contractions. Spikes associated with phasic contractions were also
recorded by microelectrodes. The threshold for the phasic response is lower than for the
tonic at short stimulus durations, and tonic threshold is lower at long durations. At high
intensities both responses occur ; phasic chronaxie is 10 msec., tonic 70 msec. Velocity of the
phasic action potential is 2-3 cm./sec. Time for half-relaxation of maximal phasic contraction
is 0.6 second, for tonic 3.0 seconds, for the two together 45.5 seconds. The phasic contractions
facilitate markedly and reach maximum tension at 10/second ; the tonic show less facilitation
and are maximal at 2-3/second. High potassium (100mA/) eliminates the phasic but leaves
the tonic contraction. Omission of Mg++ is without effect but low Ca++ eliminates both con-
tractions. Procaine and tetracaine are ineffective and the muscle is very responsive to mechani-
cal stimulation. It is concluded that conduction is from muscle fiber to fiber, that the phasic
and tonic systems are closely coupled, but that activation of the tonic may occur without
membrane action potentials.
On the phosphatide composition of sen anemones. MAURICE M. RAPPORT AXD
EUGENE L. GOTTFRIED.
It was reported (Bergmann and Landowne, 1958) that the phosphatide composition of
the west coast sea anemone, Anthoplcura elegantisshna, differed remarkably from that of the
anemone of India, Gyrostonia sp., (Rajagopal and Sohonie, 1957). Whereas Gyrostoma con-
tained cephalin, lecithin, and sphingomyelin in the proportion 4:12:1, Antlw pleura contained
only sphingomyelin and a choline plasmalogen in the proportion 20:1. The plasmalogen was
reported by Bergmann and Landowne to have a cyclic glyceryl acetal structure rather than
the novel a,|3-unsaturated ether structure of mammalian plasmalogens. In contrast, Rapport
and Alonzo (1960) showed that lipids of the east coast sea anemone, Mcfridiinn senile, contained
high concentrations of amino plasmalogen having the unsaturated ether structure. The phospha-
486 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
tides of A. clcgantissiiua \vtre therefore reexamined and compared by thin layer chromatog-
raphy with those of M. senile. The findings of Bergmann and Landowne could not be
confirmed. Anthopleura lipids contained both ethanolamine and choline glycerophosphatides
in approximately equal amounts ; the quantity of sphingomyelin was quite small. Plasmalogen
was associated predominantly with ethanolamine phosphatide (cephalin). Both Anthopleura
and Mctridiuni had compositions of complex lipids that were very similar and in accord with
that reported for Gyrostonia, except that the quantity of cephalin relative to lecithin was sub-
stantially higher. Inasmuch as the methods used to analyze Gyrostonia lipids did not take
into account the special properties of the plasmalogen component of the ethanolamine phos-
phatide, it is reasonable to assume that Anthopleura, Mctridiuni, and Gyrostoma have phospha-
tide compositions that are similar ( 1 ) in content of cephalin, lecithin, and sphingomyelin, and
(2) in having phosphatidal ethanolamine as the predominant plasmalogen. The error in the
studies of Bergmann and Landowne was very likely caused by their use of alcohol-preserved
animals. The plasmalogen they found to have a cyclic glyceryl acetal structure was probably
an artifact.
Supported by U.S.P.H.S. Grant B-1570.
Patterns of chemically induced reversions among mutants of Salmonella typJiimu-
rium. J. L. ROSNER.
Freese has made an intensive study of the mutational properties of the rll region of
bacteriophage T4. He found that 98% of the mutants induced by 2-aminopurine (2AP) would
revert to wild type upon treatment with base analogs. In comparison, 87% of nitrous acid
(HNO2) -induced mutants and 14% of spontaneously arising mutants are induced to revert with
base analogs. In an early study with histidineless mutants of Salmonella typhimurium, Kirchner
reported that 4% of the spontaneous and 25% of the 2AP-induced mutants would revert after
treatment with 2AP. Dr. A. Eisenstark and the author have studied the reversion pattern
of nearly 200 cysteineless mutants of Salmonella (manuscript in preparation). Ninety per cent
of the 2AP-induced mutants and about 60% of both spontaneous and HNO2-induced mutants
were found to be revertible with 2AP. In the present study, Kirchner's work was reexamined
utilizing a more sensitive assay for 2AP mutagenicity.
For each mutant, ca. 10"7 cells are spread on minimal plates enriched with 2.5% broth.
Diethyl sulfate (DES) is added to a small paper disc on the plate. The mutagens 2AP and
5-bromdeoxyuridine (5BD) are added directly to the plate. If the disc is used, a majority of
the positive responses are concealed by the disc. The results of these experiments corroborated
the findings with the cysteineless mutants. Of the 11 2AP-induced mutants originally tested
by Kirchner, all were found revertible by DES and 2AP. Eight of eleven were also re-
vertible using 5BD. Of the 20 spontaneous mutants tested, two reverted with all three
mutagens, 10 with DES and 2AP, and 6 with DES alone. In a preliminary study with 13
histidineless mutants induced by HNO^, two were revertible with DES, one with 2AP and
none with 5BD. Further investigation is planned.
Thus, spontaneous mutations in bacteria and bacteriophage respond differently to reversion
by chemical mutagens.
Mechanism of chromatophore control in the common sand flounder Scophthalamus
aquosus. GEORGE T. SCOTT, RICHARD L. CLARK AND JAMES C. HICKMAN.
The great majority of teleost fishes investigated reveal evidence of dineuronic control of
chromatophores, i.e., the presence of both aggregating and dispersing nerve fibers. In our
work on the sand flounder we have found no evidence of dispersing nerve fibers or the necessity
to postulate a dispersing neurohumor. The experiments are summarized as follows: (1)
Sectioning of the sympathetic chain in a light-adapted fish causes rapid dispersion of chro-
matophores on the body posterior to the cut : electrical stimulation produces blanching due to
concentration of the melanin pigment. (2) Sectioning of spinal nerves, or the application of a
pressure block to them, causes dark banding due to melanocyte dispersion. (3) Such a dark
band fades within one to two days when the fish are maintained on a light background. (4)
Recutting distal to the first cut does not produce a second band within the area of the first.
PAPERS PRESENTED AT MARINE BIOLOGICAL LAUORATORY 487
This kind of operation, in fish where a second band is formed, has been used as evidence of
functional dispersing nerve fibers by a number of investigators working on several kinds of
teleosts. Chromatophores in the sand flounder disperse when the aggregating nerve fibers are
separated from the CNS. Stimulation of these fibers causes concentration of pigment. (5)
Acetylcholine or eserine injected in the cord of a dark-adapted fish produces transitory
light banding, presumably by cholinergic facilitation. Some twenty other drugs studied pro-
duce dark banding, presumably by pharmacologic inhibition. Acetylcholine or eserine have
no effect when injected subcutaneously. (6) The following drugs were observed to produce
persistent localized lightening when injected subcutaneously : epinephrine, norepinephrine, iso-
propyl arterenol, serotonin, and the monoamine oxidase inhibitors, pheniprazine and phenelzene.
(7) A large number of depressant drugs including tranquilizers and sedatives (except bar-
biturates, which are inactive) produce localized darkening. The serotonin-blocking agents,
lysergic acid diethylamide and bimaleate, also produce darkening as do the epinephrine-blocking
agents, phenoxybenzamine and N-2-chloroethyl dibenzylamine. Pituitary extracts are inactive
on both normal and denervated chromatophores.
The most active lightening agents are the epinephrines and, on the other hand, epinephrine-
blocking agents are potent drugs causing chromatocyte dispersion ; therefore, it seems likely that
a catechol amine similar to epinephrine is the chemical transmitter of the aggregating nerve
fibers.
The study was aided by a National Institute of Mental Health Grant MY-3903 to
Oberlin College.
Effect of carbon dioxide on gas exchange in Thyone briar CMS. DAVID M. TRAVIS.
Sea cucumbers, Thyone briarens, were exposed to carbon dioxide pressures (pCO2 to 40
mm. Hg) in air at atmospheric pressure and the exchange of oxygen and carbon dioxide
measured. Respiratory chambers consisting of syringes with rubber caps were loaded with
animals, sea water and air containing various concentrations of carbon dioxide and shaken in
a water bath at 22° C. Gas samples were taken at one-hour intervals and analyzed volumet-
rically. Animals were blotted, weighed and their volume determined by weight of displaced
sea water. Oxygen uptake and carbon dioxide output were calculated from changes in gas
concentrations and volumes. Oxygen tensions were allowed to fall only 15-20 mm. Hg.
Initial oxygen concentrations were adjusted to that of air.
Oxygen uptake fell with increasing carbon dioxide tensions. This decrease was 22% less
when the pCO2 was raised from 3 mm. Hg to 12 mm. Hg, 34% at pCO2 of 25 mm. Hg, and
55% at 40 mm. Hg. There was a slow rise of carbon dioxide output from the animal exposed
to air without added carbon dioxide, and a plateau was reached in 4 or 5 hours, then giving
respiratory exchange ratios of around 0.75. Sea cucumbers require longer periods for equi-
libration with higher concentrations of carbon dioxide. Oxygen uptake varied with time in
single animals. The pattern of variation was similar in animals studied simultaneously in
parallel experiments, whether exposed to carbon dioxide or not.
The decrease in oxygen uptake of Thyone briarens on exposure to higher carbon dioxide
tensions is similar to that previously found in another sand- and mud-dwelling marine inverte-
brate, Golfingia gonldii. The pCO2 may be ten- or thirty-fold higher in sand than in sea.
The results suggest a possible physiological function of carbon dioxide in the regulation of
respiratory metabolism of these animals in the natural surroundings.
Carbon dioxide inhibition of growth and respiration in Tetrahymena. DAVID M.
TRAVIS, ALFRED M. ELLIOTT AND IL JIN BAK.
Tetrahymena pyriformis E, growing in broth, was exposed to air with carbon dioxide
tensions varying from 1 to 350 mm. Hg at atmospheric pressure and the changes in respiration
and growth measured. Respiratory vessels consisted of 30-ml. syringes and 350-ml. tonometers
which were immersed in a water bath at 25° C. Glass capillaries placed in the tonometers
permitted recording of pressure changes. The time and volumes of culture and gas were
adjusted so that oxygen concentrations were maintained between 21% and 18% during single
periods of gas sampling. The gases were analyzed by the i-cc. method of Scholander. Oxygen
488 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
uptake and carbon dioxide output were calculated from changes in gas concentrations and the
known volumes and pressures of the vessels. Cells were examined by light microscopy to
determine the number of dead and living ones. Relative changes in number of organisms were
estimated turbidimetrically.
Growth in 24 hours reached a maximum at carbon dioxide tensions below 40 mm. Hg and
is progressively less with higher tensions up to 350 mm. Hg. Fifty per cent inhibition of
growth occurs at 90 mm. Hg, 90% at 220 mm. Hg and above 95% at 350 mm. Hg. Cells
survived for 72 hours in an environment of air with carbon dioxide tensions of 220 mm. Hg but
not with 350 mm. Hg. Changes in oxygen uptake and carbon dioxide output paralleled the
changes in growth.
Logarithmic growth protozoa washed twice in distilled water (resting cells) and placed
in syringes without added buffer for periods of one to two hours showed a decrease in oxygen
uptake of 10% when the carbon dioxide tension was increased from 3 to 25 mm. Hg. Similar
changes were noted in broth cultures studied over short periods in which there was little
change in number of organisms.
PAPERS READ BY TITLE
Correspondence of maximum response of snails to magnetism with the strength of
geomagnetism. FRANKLIN H. BARNWELL AND FRANK A. BROWN, JR.
During a two-month period in the summer of 1960 an attempt was made to determine the
optimal strength of the horizontal vector of magnetism for effecting orientational changes in
the snail, Nassarius. The turning response of south-bound snails to an abrupt experimental
reversal of the field by a bar magnet at each of eight strengths — 0.04, 0.1, 0.2, 0.4, 0.8, 2.0,
5.0, and 10.0 gauss — was assayed. The results indicated the maximum response, left-turning,
to lie at 0.2 gauss with decreasing response to both stronger and weaker fields. Two over-
lapping repetitions of the experiment, a total of three months, were attempted during the sum-
mer of 1961. Again the maximum response lay at 0.2 gauss but the response was ri<7/iMurning.
A third attempt to resolve the problem in the summer of 1962 resulted in June-July in a striking
repetition of the 1960 pattern and in July-August of the inverted pattern. Considering all
seven months of data (involving 51,040 snail paths) analysis of variance revealed no significant
differences among the means. However, the variances themselves for strengths of the series
in increasing order were, respectively, 1.43, 10.03, 10.23, 3.79, 2.79, 3.36, 1.34, and 4.23. Highly
significant F ratios indicate a mirror-imaging response with maximum sensitivity straddling,
remarkably, the earth's horizontal vector, namely 0.17 gauss. Factors determining response
sign remain still obscure. Left-turning response in 1962 was accompanied by return to the
same clear monthly variation reported for the effect of fields within a factor of 4 of the
earth's F (0.6 gauss) relative to ones greater than 4. The comparable variation during right-
turning periods appeared to be semimonthly.
This study was aided by a contract (1228-03) with the Office of Naval Research and by
grants from the National Science Foundation (G- 15008) and the National Institutes of Health
(RG-7405).
Orientational responses in organisms effected b\ very small alterations in gamma
(CV37) radiation. FRANK A. BROWN, JR., H. MARGUERITE WEBB AND LELAND
G. JOHNSON.
Preliminary experiments indicate organismic sensitivity to weak gamma radiation and to
direction of the gamma source. The response varies with time and the animal's geographic
orientation. Plane gamma sources, four inches in diameter, contained 24 (J.C. of Cs1'". The
sources, on outside of apparatus, produced a 6-fold increase in radiation at animals' position.
Sources and dummy sources were enclosed in packages ; the observers were uninformed as to
content. In Woods Hole, snails southbound morning, afternoon and evening were subjected to
shuffled experimental series consisting of 6 groups of 10-path samples under three conditions —
dummy, gamma to left, gamma to right. July 23-30, inclusive, gamma increase, both direc-
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 489
lions, effected right-turning mornings, left-turning afternoons, and no mean turning evenings.
Analysis of variance yielded P < .001. From July 23 through August 24, 1962, analysis of
24,120 paths demonstrated a steady, apparently linear, drift in the difference between morning
and afternoon response, with a change of sign occurring about August 15. Superimposed on tin-
drift was, suggestively, a semimonthly variation with minima just before new and full moon.
Gammap. response minus gammai, response displayed a relatively large amplitude semimonthly
variation with maxima just before new and full moon. Experiments in Evanston, Illinois,
with 15-path samples of planarians, August 7 through 20, 1962, involving 20 groups of shuffled
dummy left, dummy right, gamma right, gamma left, for both North- and West-directed worms
disclosed for the former direction that 32 cases showed turning from the source, 7 cases toward
it, and 1, no response (/:><.001). Corresponding figures for west-directed worms were 17.
20, and 3.
This study was aided by a contract (1228-03) with the Office of Naval Research and by
grants from the National Science Foundation (G-15008) and National Institutes of Health
(RG-7405).
Inductive potencies of the uianubrinin of Tubularia. ALLISON L. BURNETT AND
NORMA A. DlEHL.
Numerous investigators have attempted to interpret form in Tubularia by postulating the
presence of an inhibitor which diffuses proximally along the stem and suppresses the forma-
tion of a hydranth in areas adjacent to the primary or existing hydranth. We have found that
a small piece of manubrium (one-sixteenth the size of the original manubrium or 0.1-0.3 mm. in
diameter), when grafted to the proximal cut surface of a large, healthy 5-mm. stem piece
bearing a hydranth, will induce the formation of a new hydranth within two days. Twenty-
three out of 31 animals treated in the foregoing manner formed normal tentacle ridges, while
24 control animals which had simply been excised showed no signs of hydranth formation
whatsoever.
Within three to five hours after application of the manubrial portion, the coenosarc
projects at least 1 mm. beyond the excised perisarc. Once this shift has occurred, the forma-
tion of a perfectly developed hydranth is invariably the outcome. Without manubrial induc-
tion, the coenosarc retreats back inside the perisarc and remains in this position for several
days.
The grafted manubrium is not contributing directly to hydranth formation, but is furnishing
a diffusible factor which stimulates adjacent tissue to initiate hydranth formation. If manu-
brial portions from Tubularia crorca are grafted to another species of Tubularia (unclassified
at the present time), the crocca fragment induces a hydranth characteristic of the unclassified
species within two days. Grafted stem tissue lacks these inductive potencies.
We feel that it is necessary to interpret form in Tubularia by considering the presence of
a diffusible growth-stimulating factor in the manubrium, and not rely solely for an interpretation
upon the presence of an inhibitor (s) which diffuses proximally along the stem.
The relation between inductive regions and interstitial cell distribution in Hydra
pirardi, Tubularia crocca, and Hydractinia sp. ALLISON L. BURNETT, NORMA
A. DIEHL AND ELLEN MUTTERPERL.
In the previous abstract it was stated that the manubrium of Tubularia contains an induc-
tive principle which is capable of diffusing into the tissues of another species of the same genus,
and initiating hydranth formation. Similar observations have been reported for the common
hydra, and recently it was demonstrated that the manubrial regions of feeding polyps and
gonozooids of Hydractinia also possess inductive capabilities.
Toluidine blue-stained whole mounts of these three forms have revealed that interstitial
cell distribution is intimately linked with inductive areas in the following manner. (1) The
manubrium or primary inductive area contains few interstitial cells. (2) Areas immediately
adjacent to the inductive area contain dense concentrations of interstitial cells. (3) Proximal to
this region is the so-called gastric region which contains about half as many interstitial cells
490 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
per unit area as that observed in sub-manubrial areas. (4) The gastric region is invariably
followed by another region of high interstitial cell population. In hydra this area is the budding
area, in Tubularia it is the region where the gonophore stalks and proximal tentacles arise, in
Hydractinia it is the point from which a stolon begins to grow.
It is postulated that in all three species an inductive principle diffuses proximally from the
manubrium, stimulating interstitial cells and probably other cell types to divide repeatedly, thus
creating a zone of growth. This growtli results in the production of inhibitors which diffuse
proximally to the gastric region, suppressing growth activities. The inhibitors invariably
become dilute or rendered impotent at a precise level along the gastric region, and at this
level a second growth region is initiated.
Xenrul aclii'ity during hypo.via in adult firefly. ALBERT D. CARLSON.
Anoxia in the adult firefly, Plwturis pennsylvanica, inhibits flashing and may elicit a dull,
structureless, hypoxic glow. If oxygen is readmitted after the onset of the hypoxic glow a
brilliant flash, the pseudoflash, occurs, with a duration of approximately 5 times that of the
spontaneous flash. Neural activity in the lantern was monitored to determine whether the
hypoxic glow is initiated by neural activity or by anoxia acting directly on the photocytes or
tracheal end cells.
Tank nitrogen was led into a transparent chamber holding the firefly, ventral side up.
Platinum recording electrodes, penetrating a small opening in the chamber top, were placed in
the photogenic tissue of the sixth abdominal segment. The neural activity was led into a Grass
PS A.C. preamplifier, displayed on one trace of a Tektronix 502 oscilloscope, and stored on
tape. The light response was recorded using a photomultiplier, the output of which was
frequency modulated for storage on the other channel of the tape.
If neural bursts occurred early in hypoxia an hypoxic glow was initiated which increased
stepwise with each burst. Neural bursts were usually not observed during later hypoxia but a
gradual increase in glow intensity accompanied the onset of random neural and muscular activ-
ity. Those parts of the organ glowing in air produced an hypoxic glow apparently without
neural stimulation.
It appears that the hypoxic glow is the result of neural activity during hypoxia which
initiates the formation of light producing complex. These results can explain why the actively
flashing firefly will readily produce an hypoxic glow and pseudoflash while the quiescent, non-
flashing animal will not.
Protein changes in the aycinc/ lobster. ALFRED B. CIIAET AND DAVJD BAU, JR.
Since previous studies have shown a fundamental difference between the blood proteins
of young and old horseshoe crabs, it seemed advantageous to analyze the blood proteins of the
developing lobster, Hoinarns americamis. In the present study both starch gel and disc electro-
phoretic techniques were used. In some experiments, blood was removed by cardiac puncture,
whereas in other instances repetitive freezing-thawing released adequate fluid for electrophoretic
assay. Lobsters representing seven different age groups, ranging from the developing lobster
still in its egg case to the five-year-old animal, were studied. The approximate age of the
various lobsters analyzed was as follows : seven-day pre-hatched, one-day post-hatched, three-
day, seven-day, fourteen-day, two-year and five-year.
The electrophoretic patterns obtained from representative samples of the various groups
differ with age in that only two protein bands found in the younger forms were still present
in the oldest animals. Three or four protein bands disappeared as the animal ages, whereas
six or eight proteins, which were not found in the younger forms, made their appearance in the
two- and five-year-olds. Except for one protein band (egg protein) found in the youngest
form, the disappearance of proteins in the various age groups was a gradual rather than
sudden phenomenon. Similarly the appearance of three new protein bands occurred gradually
with age.
Although previous studies have shown a gamma globulin-like protein in the blood of the
young horseshoe crab, which was absent in the older form, no gamma globulin-like protein bands
were found by either electrophoretic techniques in any of the seven lobster age groups studied.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 491
Supported by grants from the National Science Foundation (G-8718 & G-15895) and the
National Institutes of Health ( A-3362 & B-3269).
Gas absorption from fish su'iinbladdcr. C. LLOYD CLAFF, WITH THE TECHNICAL
ASSISTANCE OF ARTHUR RICHMOND AND GLADYS HARRISON.
A hypodermic needle, the hub of which was fitted with a vaccine pint;, \vas introduced into
the swimbladder, and sutured in place. All gas was removed by needle and syringe through
the vaccine plug, volume noted, and a sample analyzed for CO-> ; O- ; and N2. A volume of gas,
either CO? ; O2 ; N2 : He ; or Ar, equal in volume to the gas removed, was introduced into the
swimbladder. Samples of gas were withdrawn at 30 seconds ; 1 minute ; then every minute to 5
minutes; then at 10-minute intervals to 30 minutes; again at 1 hour; 2 hours; 3 hours; and
4 hours. Samples of venous blood were removed as soon thereafter as possible after each of the
above intervals. Each gas sample was analyzed for CO- ; O2 ; and N2. Each sample of blood
was analyzed for extracted COa and O2; and the pH of each sample was taken.
Absorption of CO2 was remarkably fast. Ninety per cent of the CO2 from the swimbladder
lost in first 10 minutes was replaced by O» and N2. Within six minutes CO2 in blood was
between 32 and 58 volume per cent. (All values not corrected for S.T.P.D. ) The pH of
venous blood dropped to 6.8 and stayed at that value for 40 minutes. Within 10 minutes O2
and N« volume per cent values in swimbladder gas returned to normal values for atmospheric
pressure.
Fish survived 24 hours after the experiment. The experimental fish were northern porgy
(scup), weighing one to one and a half pounds.
Data on other gases will be reported at a later date.
Effects of KI on G-ATP and G-ADP actiu. ELOISE E. CLARK AND RICHARD F.
OLIVO.
Both G-ATP and G-ADP actin can, under appropriate conditions, polymerize to form a
high viscosity product. Since the polymerization of the former is accompanied by dephos-
phorylation of the ATP, while that of the latter is not, it is of interest to know whether the
mechanism of polymerization and the resulting F-actins are the same. An approach to such an
analysis is feasible through the use of KI. Preliminary findings indicate that the processes
(and products) may be distinguishable.
Szent-Gyorgyi and Szentkiralyi have shown that G-ADP formed by the depolymerization
of F-actin by high concentrations of KI does not repolymerize when the ionic strength of the
solution is lowered unless an ATP system is present. Hayashi and Rosenbluth have shown
that when F-actin is depolymerized in water containing Mg++, the resulting G-ADP can be
repolymerized at 29° C. by the addition of .1 M KC1. The present experiments show that when
KI is added to the water-depolymerized F-actin, repolymerization of the G-ADP does not
occur; further, a sample of G-ADP in KI was found to have a sedimentation coefficient of 3.12
X 10-13 S. In addition it has been found that when KI is added to G-ATP solutions at 0° C.,
both the rate and extent of polymerization are markedly lowered. This is in contrast to the
polymerization of G-ATP at 29° C. where both KC1 and KI are almost equally effective in
producing high viscosity products.
Supported by U.S.P.H.S. Grant No. GM-07373.
Influence of brain lesions on melanocyte dispersion. RICHARD L. CLARK AND
GEORGE T. SCOTT.
It has been firmly established that melanocyte stimulating hormone (MSH), secreted by
the pars intermedialis, is the darkening hormone in Rana pipicns. An attempt was made to
elucidate experimentally the neural mechanism by which the pituitary's secretion of MSH is
controlled.
Various semi-micro neuro-surgical techniques were employed systematically to ablate regions
in the brain. The most successful operative procedure was our use of a micro-chemical cautery,
492 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
consisting of bichloracetic acid injected (0.25-0.50 mm.:!) by means of a drawn out glass micro-
pipette (0.1 mm. in tip diameter).
Frogs with discrete lesions in the posterior-ventral wall of the diencephalon showed spon-
taneous and persistent melanocyte dispersion. Animals similarly treated but in different areas
(cerebellum, cerebrum, optic lobes, etc.) showed only a transient post-operative darkening
together with expected behavioral changes. It is probable that a pituitary-mediating suppressor
area exists in the diencephalon, and the removal of this neuro-secretory control center results
in spontaneous hyperactivity in the intermediate lobe.
Recent investigations in our laboratory have shown that many of the clinically potent
tranquilizers (the phenothiazines, reserpine and meprobamate) produce melanocyte dispersion
after intraperitoneal injection. Evidence abounds in the literature concerning the subcortical
target areas in higher vertebrates for tranquilizers. Our work suggests that there is located in
the frog diencephalon homologous target nuclei which control the pituitary's secretion of MSH.
This study was aided by a National Institute of Mental Health grant MY-3903 to
( >b(.Tlin College.
Contraction of the epidermis during tail resorption in the ascidian Amarouciitin
constellatum. RICHARD A. CLONEY.
The larval notochord of Amaroucium is bound by an extracellular membranous sheath.
Squamous notochordal cells form a second membrane within the sheath and the axis of the
notochord is filled with a clear extracellular matrix. The muscle cells of the tail are attached
at their inner surfaces to the sheath. The nerve cord lies adjacent to the notochord on the left
side of the tail. These tail tissues arc closely bound by a thick squamous epithelium comprising
the epidermis. Two layers of tunic are disposed outside the epidermis. The outer layer is lost
at metamorphosis ; the inner layer is retained.
Tail resorption is completed within 6 to 8 minutes after the beginning of metamorphosis.
Early changes are detectable in the notochord. The matrix rapidly disappears, probably passing
into the trunk, and the notochord-muscle-nerve cord (NMN) complex partially collapses.
Simultaneously, the epidermis lifts away from the underlying NMN complex. A fluid-filled
space is formed between these components. The NMN complex buckles and begins to move
into the posterior region of the trunk. The epidermis appears to be under tension at this
time. At the end of tail resorption the epidermis forms a thick cap at the posterior end of the
trunk, enclosing the folded NMN complex.
If the epidermis is ruptured, or if the tail is excised after tail resorption begins, the epi-
dermis in both the anterior and posterior fragments or halves of the tail shorten independently,
forming compact ring-shaped masses at the base and tip of the tail, respectively. Under these
circumstances the NMN complex is never withdrawn and never shortens by itself. After tail
resorption begins it can be reversibly inhibited with 10~2 M KCN (in sea water at pH 8.0).
These observations support the hypothesis (Cloney, 1961) that contraction of the epidermis is
the major motive force involved in ascidian tail resorption.
Fine structure of acrosome and early fertilisation slatjes in Saccoglossus kowalevskii
(Entcropneusta). ARTHUR L. COLWIN AND LAURA HUNTER COLWIN.
The acrosome is a membrane-bounded vesicle with a single shallow tubular invagination in
the region adjoining the nucleus. Within this vesicle a large acrosomal granule adjoins the
invagination but does not extend as far as the acrosomal apex. A layer of fine granular
material lies between the acrosomal granule and the acrosomal membrane, except at the apex.
Dense material widely separates the acrosomal membrane from the plasma membrane except
at the apex, where the two membranes lie very close together. Two envelopes surround the
fertilizable egg but they are not penetrated by egg microvilli.
The early stages of fertilization are as follows. The tip of the spermatozoon attaches to
the outermost envelope ; the acrosome opens or dehisces apically. Around the rim of
dehiscence the acrosomal and sperm plasma membranes are then seen to be fused, constituting
one continuous unit sperm plasma membrane. Then the shallow acrosomal invagination
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 493
lengthens into a long acrosomal tubule (formerly called "filament"), while the acrosomal vesicle
everts. The large acrosomal granule disappears.
The acrosomal tubule penetrates the egg envelopes, fuses with the egg plasma membrane,
and opens apically. Thus the zygote plasma membrane is formed ; although it is clearly one
continuous unit membrane, it arises, then, as a mosaic of the sperm and egg plasma membranes.
The sperm nucleus, mitochondria, etc., pass through the acrosomal tubule and enter the fertiliza-
tion cone. During this passage the nucleus elongates greatly as though squeezing or being
squeezed through the tubule.
The acrosomal structure and the main events of sperm-egg association are basically the
same as those in the annelid Hydroidcs (Colwin and Col win, 1961). Since the same pattern
obtains in species of two such divergent phyla, it is conjectured that this pattern is a fundamental
one, which will be found to occur widely.
Aided by Research Grant RG-4948 from the National Institutes of Health.
Induction <>j spasming in Saccof/lossus kou'ulcrskii (Enter opneusta) at Woods
Hole. LAURA HUNTER COLWIN AND ARTHUR L. COLWIN.
Animals spawned occasionally when one or more were kept in bowls of sand in running
-M'a water, but a routine method was desired for obtaining gametes predictably at convenient
times. Developing embryos collected in the field were compared with timed, artificially in-
-eminated, eggs : natural spawning seemed to occur usually in the middle of the night. Never-
theless, experimental darkening or various changes in the lighting cycle induced no shedding.
Then, since the temperature of the natural habitat rose considerably during low tide on sunny
days, experimental variations in water temperature were investigated. Warming was found
to induce shedding ; the eggs, however, were in best condition when kept cool.
The following procedure finally evolved. Clean animals, in separate bowls of filtered sea
water are kept at ca. 27° C. for 7 to 8 hours, then placed in cool filtered sea water (ca. 22° C.)
and cleaned repeatedly to remove the mucus they secrete. For years this method has succeeded
with some 90% of specimens judged capable of spawning (over 1000 animals). Some animals
spawned while warm. But 70% spawned after cooling, more than half beginning by 10 to
120 minutes, the rest, up to six hours or more. Gametes can be obtained at any desired time
of day.
Animals are most suitable for use when healthy, with no unhealed wounds. Much of the
proboscis, the entire collar, and some of the genital region must be intact. With reflected
light, the usually blue genital regions of ripe females show large discrete eggs through trans-
parent body walls ; male genital regions are ripe when distended and creamy or peach, but not
brown, in color.
Small samples of normal spermatozoa can conveniently be obtained by biopsy of non-heat-
treated males but, thus far, comparable biopsy of untreated females has not yielded eggs which
subsequently could be fertilized.
This study was aided by Research Grant RG-4948 from the National Institutes of Health.
Observations on the gas-secreting epithelium of Physalia. EUGENE COPELAND.
Observations on Physalia collected in the Woods Hole area were made by use of the
electron microscope. The gas gland of Physalia is composed of three layers, ectodermal,
mesogleal and entodermal (gastrodermal) . The ectodermal layer faces the gas cavity and
presumably secretes the specific gas, carbon monoxide (Wittenberg). In osmic-fixed material
the distal ends of the ectodermal layer of cells reveal complicated gatherings of cisternae leading
to the free surface. Orderly rows of vesicles extend from the tips of the cisternae. The bodies
of the cells show a homogeneous cytoplasm with a reticular arrangement of small nondescript
granules. The nuclei are basal and surrounded by a satellite of small vesicles. Just distal
to the nucleus there is a layer of multivesiculate bodies. At the level of the nucleus, the cyto-
plasm becomes quite dense and can be easily traced into the projections which penetrate the
mesoglea and form complicated indigitations with projections from the entoderm cells. Per-
manganate-fixed material shows, in some cases, double membrane profiles in the distal part of
the cell (where osmic fixation revealed only reticular cytoplasm). There were also indications
494 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
of Golgi-type profiles in the multivesiculate layer. It is now interpreted that the fixations are
reasonably good and the variable picture is ascribed to possible degenerative stages of Physalia
which have traveled a long distance in the Gulf current and, after being blown out of that
current into the cold waters off Martha's Vineyard, are near their end. It is planned to check
this by study of animals in the Gulf itself or with more predictable collecting in the Gulf stream
off Florida.
Supported by Grants N.S.F., G-9810 and N.I.H., RG-6836.
Compartmentalization of chloride in lobster muscle. PHILIP B. DUNHAM AND
HAROLD GAINER.
Chloride, potassium, and sodium concentrations in the walking leg bender muscle of
Homarus aincricaints were determined after treatment under various ionic conditions at 5-7° C.
For muscles in normal medium (Na0, 455 m M ; K0, 15 mM ; do, 534 mM), ion concentrations,
determined by elemental analysis and expressed as mM/kg. wet weight of cells (corrected for
extracellular space, determined using C14-inulin) were: Ki, 122 mM/kg. ; Nat, 84 mM/kg.;
Ch, 80 mM/kg.
Results from three kinds of experiments suggested that intracellular Cl is situated in two
distinct compartments. (1) The kinetics of net efflux of Cli were followed for 24 hours from
muscles placed in 15 mM CL medium. The efflux was nearly complete at 14 hours, at which
time Cli was 30 mM/kg. (2) Muscles were equilibrated for 24 hours in media with constant
Ko and changing C10 (534-15 mM). Ch varied linearly with Cl,, over this range at two dif-
ferent concentrations of K0 (15 and 45 mM). The slope and extrapolation to zero C10 was
the same at both K0's. The value at zero CL was about 30 mM/kg. In both cases, Ki and Nai
were constant at all levels of CL. With 45 mM Kn, Nai and Ch were equal to that obtained
in 15 mM K0, whereas Ki was 145 mM/kg. (3) The rate and extent of exchange of Ch with
trace Cl36 added to the medium were determined for a 24-hour period. In 534 mM/CL there
was no further exchange after 14 hours, at which time 65% of Cli had exchanged, leaving
30 mM/kg. not exchangeable. C13G was added to the medium of muscles after 6 hours in
15 mM CL. There was no further exchange after 7 hours, at which time only 5% of Ch was
exchanged, again leaving 30 mM/kg. not exchangeable.
That there is a compartment of immobile Cli, 30 mM/kg. is indicated by two independent
methods. This compartment is constant over a wide range of CL, and is not exchangeable with
CL. There is also a compartment of mobile Cli in equilibrium with CL, with a constant
CLiCli ratio of about 10:1 over a wide range of CL.
Two physiological varieties of Noctilitca niiliaris. ROGER ECKERT AND MARGARET
FlNDLAY.
Noctiluca is widely familiar because of its role in the occurrence of marine bioluminescence
and red tides. In spite of its almost world-wide distribution, only one species, Noctiluca miliaris
(synon. with N. scintillans), has been recognized. However, Sweeney (personal communica-
tion) noted that Noctiluca collected in the Pacific off San Diego did not luminesce and were
smaller than a luminescent variety collected in the Gulf of California.
To extend these observations we are culturing Noctiluca collected from both the North Sea
and from Puget Sound. The North Sea cells luminesce and range in diameter from 400 A
to 850 M, whereas the Puget Sound cells are non-luminescent and range from 200 //. to 450 At.
Although no other morphological differences have been noted, work is in progress to examine
possible ultrastructural differences between the luminescent and non-luminescent varieties.
The bioelectric and bioluminescent behavior of both types of Noctiluca were investigated as
described elsewhere (Eckert, this issue). Both types respond at similar stimulus current
intensities with an all-or-none negative-going action potential as large as 70 mv. This potential
is followed by a distinct movement of the tentacle. In the North Sea culture the action potential
is also invariably followed by a flash of luminescence. On the other hand, even with multiplier
sensitivities three orders of magnitude greater, no light emission could be detected in any of the
Puget Sound cells.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 495
Jn addition to the all-or-none evoked potentials, slow, spontaneous rhythmic potentials were
routinely recorded from the luminescent variety. These potentials are negative-going and of
a magnitude similar to the evoked potentials ; however, they are not all-or-none, are 20-40 times
as long as the evoked spikes, and are never accompanied by luminescence. Spontaneous
potentials were never recorded in the nonluminescent type.
Supported by U.S.P.H.S. Grant B-3664 and N.S.F. Grant G-21S29.
Survival of Tctrahyinena at elevated oxygen pressures. ALFRED M. ELLIOTT,
DAVID M. TRAVIS AND IL JIN BAK.
Tctraliyincihi pyrifonnis K, grown axenically in broth cultures, was subjected to 100%
oxygen under varying pressures from 1 to 2.5 atmospheres. Plastic 10-ml. syringes were
employed as pressure chambers in which glass capillaries were placed to register pressure. The
syringes were maintained at room temperatures (25-27° C.) in a horizontal position to afford
maximal exposure of the cells to the gas. Initial and final numbers of cells were recorded
turbidimetrically. The cultures were examined with the light microscope to determine numbers
of living and dead cells. Hydrogen ion changes during the experiments were noted and the O2
and CO2 analyses were done with the V^-ml. Scholander technique.
Protozoa exposed initially to 2.5 atmospheres of 100% oxygen show a linear increase in
death of the cells with time. Ten per cent are dead at 6 hours, 25% at 9 hours, and over 90%
at 12 hours. At one atmosphere, 100% oxygen is less toxic. Only 10% of the cells are dead
in 9 hours and 25% at 12 hours. During the first 3 hours of exposure at both 1 and 2.5
atmospheres of 100% oxygen cell division appears normal, but thereafter mitosis ceases and the
cells begin to die, preceded by characteristic swelling to form spheres. At 2.5 atmospheres of
oxygen the CO- output is approximately twice that at 1 atmosphere. The O- uptake rises to
a peak in 6 hours at 2.5 atmospheres. At 1 atmosphere the peak appears at 9 hours, thereafter
declining. The pH falls from 6.9 to 6.7 during the 12 hours of exposure at both 1 and 2.5
atmospheres.
Oxygen pressure dose response demonstrates a straight line relationship with CO? output.
Oxygen uptake declines uniformly with increasing oxygen pressure.
Effect of phenothiasine derivatives on the permeability of the dogfish erythrocyte.
ALAN R. FREEMAN AND MORRIS A. SPIRTES.
Three phenothiazine tranquilizers, varying in clinical potency, were tested for their ability
to reduce hemolysis when erythrocytes were exposed to hypotonic, partially hemolytic saline
solutions. Trifluoperazine, clinically the most potent tranquilizer, reduced the control hemolysis
of 40% to 26% at a final concentration of 2.5 X 10~6 M. Chlorpromazine, less active clinically,
produced the same degree of protection at 1 X 10~5 M, and chlorpromazine sulf oxide, a pharma-
cologically almost inactive compound, had no effect at concentrations up to 1 X 10~4 M.
Similarly, chlorpromazine could prevent the swelling of dogfish red cells exposed to hypotonic,
non-hemolytic sodium chloride solutions. Spectrophotometrically, chlorpromazine-treated cells
also appeared to have a smaller average cell volume, even when suspended in hypertonic salt
solutions. Since data using the hypotonic, non-hemolytic technique were obtained by a spectro-
photometric method, further experiments will have to be performed before any definite
conclusions can be drawn from them.
Absorption spectra from 300 to 700 m/* of dogfish hemoglobin and cyanmethemoglobin
showed peaks identical with those of mammalian hemoglobin and cyanmethemoglobin.
The intracellular, ionic pattern of the erythrocytes was determined in eight dogfish.
Values of 11.9 ± 4.0 and 101.9 ± 8.7 were noted for sodium and potassium, respectively, expressed
as milliequivalents of the ion per 3 millimoles of hemoglobin, using mammalian hemoglobin as
the standard in the analyses.
Preliminary experiments indicated that little ion exchange took place in non-hemolytic
hypotonic saline solutions. Furthermore, 1 X 10~5 M chlorpromazine did not change the electro-
lyte balance in these experiments. It thus appears that the phenothiazines primarily affect the
movement of water in the various systems tested.
496 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Intravenous injection of 5 mg. of chlorpromazine into a dogfish two feet in length exerted a
powerful tranquilizing effect.
In conclusion, evidence has been obtained demonstrating that phenothiazine tranquilizers
affect the osmotic movement of water in dogfish erythrocytes to a degree directly related to
clinical potencies. This action appears not to be related to ionic phenomena.
Effects of isolation and denervation of crayfish muscle fibers on their membrane
resistance. LUCIEN GIRARDIER, JOHN P. REUBEN AND HARRY GRUNDFEST.
Currents were applied with one intracellular microelectrode to muscle fibers of crayfish.
Resulting changes in membrane potential were recorded with another electrode. The slope of
the current-voltage curve through the resting potential gives effective resistance, which in
fibers of neurally intact muscles ranged between 7.7 and 29.6-10* ohms. The length constant
(X), measured from the exponential decay of the potential along the fiber, showed much less
variation, its value ranging between 2.73 and 3.95 mm. From these respective values for each
fiber and from its diameter, the specific resistances of the cell membrane and sarcoplasm were
calculated, using the cable equations. There was no significant correlation between diameter
and length constant or sarcoplasmic resistance. A positive correlation was found between the
diameter and membrane resistance (r = 0.90; P < 0.05, n = 7). This finding suggests the
existence of low resistance pathways in parallel with the cell membrane. If they were in
essentially radial distribution, their lengths and resistance would increase with the fiber diameter.
Structures with these properties have been observed and characterized with the aid of electron
microscopy. They are tubules which run inward from the periphery in proximity to the Z
lines, and are probably homologs of the T-system component of the triad. They are not
connected with the sarcoplasmic reticulum.
In muscles that had been denervated for 7 to 19 days the effective resistance of the fibers
did not differ significantly from that of the control muscles of the same animals. The length
constant did decrease significantly with a mean of 2.19 ± 0.16 mm. as compared with 3.41 ± 0.41
mm. in the control preparations. The drop is attributable to a decrease of the membrane
resistance in the fibers of the denervated muscles to about half the value in the controls: e.g.,
3200 ohm cm.2 as against 6700 ohm cm.2 in fibers of 180 /u diameter.
Isolated single fibers have a still smaller length constant, 1.52 ± 0.02 mm. This indicates a
membrane resistance of 1,600 ohm cm.2 in a 180 p. fiber. The lower membrane resistances in
the denervated and isolated fibers are correlated with correspondingly higher rates of movement
of KC1 across the membrane for a given driving force.
Studies on the isolated islet tissue of toad fish: the uptake of injected Cl4-glucosc
by islet arid other tissues. FREDERICK C. GOETZ AND S. J. COOPERSTEIN.
As part of a study of the influence of blood glucose on the secretion of insulin by islet
tissue, we have determined the C14 content of islet and other tissues of the toadfish following
injection of C14-glucose into a gill arch vessel. H3-mannitol was injected simultaneously as
a measure of the extracellular compartment. The samples were counted using the liquid
scintillation spectrometer.
Within five minutes after the injection of C14-glucose, the C14-content of islet reached that
of blood ; this is more rapid equilibration than in any tissue except heart. By 60 minutes the
C1* content of islet was greater than that of any other tissue except brain. The amount of
H3-mannitol in brain was only about one-fifth that in most other tissues, indicating that brain
has a very small extracellular compartment in equilibrium with blood.
The amount of C14 found in heart, liver, kidney and gill following the injection of C14-
glucose was about the same as that following the injection of C14-urea. This suggests that
glucose, like urea, is freely diffusible into these cells. In muscle, there appears to be a barrier
to glucose entry; the C14 content following C14-glucose injection was only one-fourth that
found following injection of C14-urea. Brain seems to have an effective mechanism for
concentrating glucose; the C14 content after glucose injection was four times that after injection
of C14-urea. Islet may also concentrate glucose, since in islet this ratio was 1.6.
When unlabeled glucose was injected to increase the blood sugar level to 350 mg.%
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 497
(compared with 25 mg.% for the control) the uptake of glucose was increased proportionate to
the blood sugar level in all tissues except brain. This suggests that the glucose uptake
mechanism in brain was saturated at a lower blood sugar level.
Supported by Grants A-824, A-1556 and A-1659 from the National Institute of Arthritis
and Metabolic Disease, Public Health Service.
Strontium utilization by Arbacia punctulata. II. LIONEL S. COLORING, HENRY I.
HlRSHFIELD AND IRENE P. GOLDRING.
In order to study the discrimination of marine organisms for calcium over strontium, we
have studied the atomic Ca/Sr ratio of Arbacia punctulata larvae as a function of the Ca/Sr
ratio in the growth medium. As a necessary precursor to this we have studied the development
of pluteal larvae in a variety of saline solutions of sea water ionic strength.
Because of limits imposed by solubility considerations, it was necessary to reduce both
sulfate and calcium concentrations to explore the maximum range of Ca/Sr ratios. At one-
tenth sea water sulfate, plutei with normal or near-normal skeletons were obtained with a
calcium concentration of one-half sea water and with Sr at 10 to 30 times sea water concen-
tration. With one-tenth sea water calcium as well as sulfate, very abnormal plutei were
obtained with little or no deposition of skeletal material, regardless of the Sr content. These
preliminary experiments define limits that will permit us to explore Ca/Sr ratios from 125 to
2.0. Higher ratios can be easily obtained. Lower ratios may be obtained when solubility
limits can be explored in greater detail.
Preliminary attempts to determine the weight of skeletal material deposited showed that
mass cultures were necessary to obtain sufficient material for chemical analysis. As an
alternative to this, radiochemical methods were explored to determine the deposition of both
Ca and Sr in the skeleton. At a Ca/Sr ratio of 125, 0.01 /*C. Ca45 per ml. and 0.10 p.C. Srs»
per ml. gave measurable activity in isolated skeletons obtained from a 20-ml. culture.
Work supported by AEC-MBL-Grant AT- (30-1) -1343 ; AEC-WHOI-Grant AT- (30-1)-
2174, AT- (30-1) -3008 and others; Damon Runyon Grant No. 120; American Cancer Society,
New York University Grant.
Incorporation of C14-thymidine into pool and DNA of deuterated sea urchin eggs.
PAUL R. GROSS AND GILLES H. COUSINEAU.
The report by Gross and Harding (1961) of DNA synthesis blockade by heavy water in
sea urchin eggs has been followed by several descriptions of similar phenomena in mammalian
cells. This is in contrast to the well-established ability of many microorganisms to adapt to
growth and division in D2O. The possibility remained that this difference, at least for the
invertebrate egg, could be accounted for by a reduced permeability of the deuterated cell to-
tracer thymidine. A test of this possibility has been made as follows : eggs were fertilized
normally and divided into two batches. One was transferred to reconstituted normal sea water
and the other to heavy sea water (D = 85%) at ten minutes post-fertilization. These media
contained 0.2 juC./ml. of thymidine-2-14C (25 mC./millimole). When the control cells were at
the second cleavage (50%), the deuterated cells had not divided at all. The suspensions were
each now divided. One half was centrifuged, and the eggs washed quickly but thoroughly with
ordinary filtered sea water. The eggs were then pipetted into detergent-treated planchets
and dried to a thin film for counting of total label taken up. The remainder of each sample
was treated with an equal volume of 10% TCA containing a 500-fold excess of unlabelled
thymidine, stored in the cold overnight, and the eggs collected on Millipore filters with 5 p. pores.
The filters were presoaked in the TCA-thymidine solution. After washing with 5% TCA and
water, the filters were dried and mounted on planchets for counting of the label incorporated
into DNA. Counts were made with a thin-end-window Geiger counter system giving a back-
ground of approximately 3 cpm., and each sample was allowed to accumulate 6400 counts. The
result is that the pool radioactivity in the deuterated cells is as high as, or higher than, that
in the controls. Incorporation of label into DNA was, however, strongly inhibited for the
deuterated eggs, sufficiently so to account easily for the "blockade" observed in the autoradio-
grams. Thus, the inhibition of DNA synthesis by heavy water is exerted at the level of
498 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
nucleoside incorporation or beyond, and the difference between higher cells and microorganisms
in this regard acquires some interest.
Aided by grants from the National Science Foundation and the Anna Fuller Fund.
Electrophysiological concomitants of the shadow reflex in certain barnacles. G. F.
GWILLIAM.
Some electrophysiological concomitants of the shadow reflex in the pedunculate barnacles
Mitella polynicriis and Lcpas anatifcra and in the sessile barnacle Balanus dutnicits have been
investigated. In both pedunculate forms motor output from the supraesophageal ganglion,
recorded externally from stalk nerves and circumesophageal connectives during controlled
shading of the internally located ocelli, is sharply increased at "off" and is directly related to
the degree of shading (controlled with neutral density filters). There is no indication at these
recording sites of the "on" stimulus. The casting of multiple shadows of subthreshold duration
in Alitclla does not lead to a "shadow" response in the motor nerves, nor will as many as 30
rapidly applied threshold shadows completely adapt the response. In Lcpas adaptation occurs
after three to six threshold shadows similarly applied. Recordings from the ocellar nerve
in Lcpas. however, indicate that adaptation is a central phenomenon, for the electroretinogram
is undiminished for at least ten threshold shadows. Cutting the ocellar nerve or destroying the
eye abolishes these responses. Sectioning one of the pair of ocellar nerves in Lcpas leads to
a diminished response in both circumesophageal connectives, but less so in the contralateral than
the homolateral connective.
External recording from the ocellar nerves in Lcpas (second order sensory fibers) and
Inilanus ( presumably primary fibers) results in an electroretinogram of simple form with a
relatively large negative wave at "on" and a much smaller positive wave at "off" when recorded
with one electrode on the nerve and the other in the surrounding sea water medium. As yet no
action potentials have been recorded from the ocellar nerve.
Supported by N.S.F. Grants G5997 and G19209.
Light-induced pigment migration in tJic squid retina. W. A. HAGINS AND P. A.
LIEBMAN.
Migration of black screening pigments in and around photoreceptors in response to light
has often been thought to contribute to light- and dark -adaptation of the retina in arthropods
and lower vertebrates. J. Z. Young has shown histological evidence for the same process in
the eyes of live octopus. Movement of screening pigment has now been observed directly in
isolated slices of living squid retina by infra-red microscopy. In dark-adapted retina, the
black pigment lies concentrated in a thin layer bisecting the photoreceptors at the junction of
their inner and outer segments. After a flash of orange light, sufficient to activate 10-50% of
the photopigment, the black pigment layer divides into a thin part which remains fixed and a
diffuse wide band which advances into the layer of outer segments almost to the internal
limiting membrane. At 10° C., the migration begins in two minutes, reaches its maximum
extent in 20 minutes and recedes in about two hours. At 2° C., migration was not seen after
illumination, but on subsequent warming to 15° C., it occurred without further exposure.
Incubation of the slices in sodium-free water sea water (Na+ replaced by choline+ ) in which
the retinal receptor current is reversibly abolished prevents the pigment migration, even if the
tissue is returned to a normal sodium sea water immediately after illumination. It is suggested
that the pigment response is a local reaction of the photoreceptors to light, depending upon a
change in ionic composition of the cells. When the pigment migrates into the outer segments,
its effect on the function of the retina is probably two-fold. First, it should reduce the overall
light-sensitivity of the photoreceptors by simple shielding. Second, it should markedly restrict
the solid angle from which light entering the pupil can reach the photoreceptors, thus produc-
ing a sort of Stiles-Crawford effect. This latter action may be very important in the squid
retina, since its layer of outer segments is so thick (—250 M.) and its pupil is so large (f3.5)
that the resolution of its receptor mosaic is basically poor despite its receptors being only 4-6 /j.
in cross-section. Pigment migration into the layer of outer segments, however, may improve
resolution of the retina by nearly an order of magnitude.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 499
TJic preparation of sea bass lens epithelial whole-mounts for tritium autoradiotj-
raphv. C. V. HARDING, M. B. NEWMAN, F. E. JONES AND H. ROTHSTEIN.
Whole-mounts of the entire layer of rabbit lens epithelium (a single layer of cells) can
be prepared for tritium autoradiography. Such preparations have proven useful in localizing the
sites of incorporation of tritium-labeled thymidine in normal and injured lens epithelium. It
has been found that a small mechanical injury can induce a large number of the surrounding cells
to undergo thymidine incorporation and division. Attempts have been made to extend this
study to cold-blooded animals. Injury-induced activation of thymidine incorporation has been
demonstrated in the lens epithelium of the sea bass, using the whole-mount technique. How-
ever, the required exposure time was very long, and the epithelial cells were obscured by a
coating of unidentified material (perhaps fragments of lens fibers). Attempts have been made
to make preparations without this coating of material, and the following procedure has
proven effective: (1) Inject eye with 0.2 ml. teleost Ringer (Forster and Taggert), containing
5 (J.C. tritiated thymidine, 3 C./mM spec. act. (2) Two to four hours after injection, fix whole
eye for 24 hours in Carney's solution (3 parts absolute alcohol :1 part glacial acetic acid).
(3) Maintain in 70% alcohol for at least 24 hours. (4) Make whole-mount of lens epithelium
(Arch. Oplitlialiuol., 63, 1960). (5) Treat whole-mount for 6 minutes at room temperature in
0.005% crystalline (salt-free) trypsin made up in teleost Ringer. (6) Wash four times in
teleost Ringer, once in distilled water, and dehydrate. (7) Film with Kodak AR-10 stripping
film, develop at the end of one week's exposure and stain with Harris' hematoxylin. In such
preparations, the cell nuclei were well stained, and radioactive nuclei were evident. In control
preparations, treated in identical fashion except for the absence of the exposure to trypsin, the
nuclei were obscured, and radioactive nuclei were not evident after one week's exposure.
Electron microscopy of the sea gull adrenal. GLADYS HARRISON.
Structures similar to the annulate lamella found in the clam and the snail oocyte by
Rebhun have been observed in the adrenal gland of the sea gull. The lamellae in some instances
are in intimate association with the nuclear membrane, lending support to Swift's theory of the
nuclear membrane being a "mold" on which the lamellae form. When the lamellae are not
closely associated with the nuclear envelope, they may assume a variety of patterns, from straight
parallel arrays to circular configurations. Vesicles have been observed to be continuous with
the ends of the lamellae much in the same manner that the vesicles and membranes of the
Golgi apparatus appear.
Annuli are seen in the nuclear envelope and also in the cytoplasm. These annuli are
often found in association with the lamellae ; some sections show circular lamellae enclosing
groups of annuli. The diameter of these annuli is about 1000 A, which is within the range
reported by Rebhun in his material.
Other basophilic membranes are seen, some arranged in concentric circles, others surround-
ing granular electron-dense bodies. Within the membrane and around these bodies, vesicles
are found.
Cilia have also been found in the adrenal cells of the sea gull.
Supported by N.I.H. Grant H-6214 and an N.S.F. Cooperative Fellowship.
Pharmacology of the radula protractor of Busycon canaUculatnm. ROBERT B. HILL.
The hearts of many molluscs can be excited by high concentrations of acetylcholine and
depressed by lower concentrations. Greenberg has suggested that the demonstration that
the former effect is widespread brings mollusc hearts into line pharmacologically with gastropod
radula muscle, which is also excited to contract by high concentrations of acetylcholine. How-
ever, since isolated radula protractors of Busycon do not show spontaneous rhythmicity, previous
studies would not have revealed a possible depressing effect of lower concentrations of
acetylcholine, which if present, would complete the parallel with cardiac muscle.
Twitches can be elicited from the radula protractor by stimulating the nerve designated 1 by
Herrick. A radula protractor in situ sometimes possesses spontaneous rhymicity at a frequency
of about two per second. Such spontaneous contractions often appeared at the characteristic
500 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
rate, after or during stimulation producing twitches at a higher or lower rate. Application of
1:4000 nicotine, or cutting nerve 1 between the cerebral ganglion and the point of stimulation
abolished spontaneous rhythmicity.
A broad range of concentrations of acetylcholine was tested on radula protractors which
were twitching once per second in response to stimulation of nerve 1. The average effect on
tonus for each concentration follows, expressed as a percentage of the tetanic contraction re-
sulting from stimulation of nerve 1 at 10 per second : 1Q-2 M, 63% ; KH M, 45% ; 1Q-* M, 57% ;
10-5 M, 40% ; 10-6 M, 27% ; 10- M, 5% ; Kh8 M, 1.5%. The average increase in amplitude of
isotonic twitches produced by the lower concentrations was : 1Q-7 M, 33% ; 10~8 M, 35.5% ; 10~9
M, 17%. The effects of 10~10, KH1, and 1Q-1- molar acetylcholine could not be distinguished
from those of an equal quantity of sea water.
Thus it appears that the Busycon canaliciilatutn radula protractor lacks the inhibitory half
of the biphasic response to acetylcholine found in many molluscan hearts.
Factors in the effects of radiation on the growth rate and conidiation in Neurospora
crassa. JOHN KEOSIAN.
Gamma-radiation source : Cs137 irradiator, dose rate 5000 r per minute. X-radiation
source: 182 Kv machine, inherent nitration of 0.15 mm. Cu, dose rate 4860 r per minute.
Incubation temperature: 30° C.
The author stated in a previous abstract that gamma-radiation up to 100,000 r did not
produce the characteristic early conidia formation at the irradiated growing frontier of
Neurospora cultures that could be produced optimally by 9000 r x-radiation. This was
attributed at first to two variables. (1) The relative biological effectiveness (R.B.E.) of
gamma-radiation vs. x-radiation. (2) Falcon plastic vs. Pyrex glass. Falcon plastic petri
dishes (100 mm.) were used in the earlier work with the Cs137 irradiator whose specimen
chamber will not accommodate the long, straight tubes used in Neurospora growth rate
experiments.
The present work revealed the following. (1) A third variable, the age of the culture at
the time of irradiation, is a critical factor. Maximum response occurs in 15-hour cultures or
older. The response is not appreciable in 8-hour cultures or younger. The cultures used
in the earlier work with Cs137 were of sub-optimal age. (2) With cultures of optimal age,
the results obtained previously in Pyrex glass under x-radiation could be repeated in Falcon
plastic dishes under gamma-radiation. (3) The R.B.E. of gamma-radiation for the conidiation
effect is about 0.56, while that for the LD 100 is 0.62 or less. (4) The same post-irradiation
increase in growth rate reported previously with x-radiation occurs also with gamma-radiation.
Studies on growth rate in the present work with the Cs137 irradiator were conducted with
specially constructed spiral tubes which would fit into the specimen chamber. Pyrex tubing
(13 mm.) was bent into a tight spiral having three coils and an over-all diameter not exceeding
4$ inches. The tubes were numbered and calibrated individually for normal growth rate of
unirradiated cultures.
The effect of time of insemination on the development of Fuiiduliis c</</s. EVELYN
KlVY-RoSENBERG.
During a series of experiments concerned with l-itiuiulns, it appeared that embryonic
development beyond blastula formation and hatchability depended on the time lapse between
insemination and egg stripping. Since the experimental design had involved treatment of un-
inseminated eggs for various periods of time prior to insemination, the question whether in-
semination and stripping time were, indeed, intimately related with normal development, arose.
A series of experiments involving samplings of eggs stripped from 25 females between mid-
June and mid-July was investigated. Insemination was carried out at chosen intervals between
1 and 145 minutes after stripping. Repeated samples involved time periods between 1 and 20
minutes : relatively few from thirty minutes up. Data indicate that percentage of fertilization
and cleavage approaches 100 (i.e., 80-100 with few lower percentages) notwithstanding the
time between egg stripping and insemination. The minor differences in fertilizability could
probably be traced to the original condition of egg batches. Blastula formation continued in
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 501
all eggs fertilized. Development to stage 25 (Oppenheimer) was continued only in a small
percentage of eggs which had been inseminated 20-30 minutes after stripping. Not all batches
of eggs were permitted to develop through hatching, since some were fixed or discarded
earlier. However, those which were followed demonstrated that this process was possible
even for eggs which had been inseminated up to 15 minutes after stripping, although a much
greater percentage were hatched from those eggs which had been stripped 1-2 minutes before
insemination.
There appears to be no interference with fertilization and development through blastula
formation if insemination is accomplished within two and a half hours after egg stripping but
further development to hatching requires that insemination take place within fifteen minute*-,
but for best results within several minutes of egg stripping.
Supported by N.I.H. grant and U. S. A. E.G. contract.
Krebs and pentose cycle dehydrogenase systems in the gametes of Asterias as
measured with a tetrazolium salt, INT. EVELYN KIVY-ROSENBERG, FRANCES
RAY AND NATALIE PASCOE.
The quantitative, microchemical study of substrate-dependent dehydrogenase system activity
was continued: sperm of Asterias was compared with eggs (Biol. Bull., 119: 1960). The same
series of 15 substrates was utilized as had been for the egg assays. This includes substrates
which require no cofactor as well as those requiring DPN or TPN. Among the substrates
were four involved in the Krebs cycle (succinate with no cofactor, malate and alpha-keto-
glutarate with DPN as cofactor, isocitrate with TPN as cofactor) and two in the pentose
cycle (glucose-6-phosphate and 6-phosphogluconate with TPN as cofactor). The tetrazolium
salt which acted as hydrogen acceptor was 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetra-
zolium chloride (INT).
"Dry sperm" was brought up to about a 3.5% suspension by volume and kept iced throughout
the pre-incubation period. Incubation was carried out for one hour at 37.5° C. in media con-
taining INT and substrate or appropriate control. Formazan (reduced tetrazolium) wa*.
extracted and the quantity measured spectrophotometrically. The substrate-dependent dehydro-
genase activity was expressed as micrograms of formazan per milligram of protein.
Of the 15 dehydrogenase systems assayed, malate ranked second in activity; alpha-keto-
glutarate possibly third (but not at all consistently) ; succinate, sixth; isocitrate, ninth; glucose-
6-phosphate, seventh; and 6-phosphogluconate among the lowest, i.e., eleventh (possibly due
to the type of purity of the particular batch of salt available). For comparison, the rank
of activity of egg homogenates for parallel substrates were malate, second ; alpha-ketoglutarate
possibly first ; succinate, thirteenth ; isocitrate, third ; glucose-6-phosphate, second-third ; 6-
phosphogluconate, fifth. If rank were considered within the two cycles only, the two obvious
differences between activity of sperm and egg dehydrogenase systems are seen in succinate-
dependent where in sperm the rank is second, in eggs, sixth ; the reverse situation is seen in
6-phosphogluconate (perhaps partially artifact) where sperm ranks fifth and eggs, third.
Supported by N.I.H. grant, U. S. A.E.C. contract and contribution from Saul Sin.ucr
Foundation of Beth Israel Hospital, N. Y.
The incorporation of nicotinaniide-7-C14 into pyridine nitcleotidcs of intact eyc/s
and embryos of Spisula solidissima. STEPHEN M. KRANE AND LEONARD
LASTER.
The level of diphosphopyridine nucleotide (DPN) in unfertilized eggs of Spisula solidissima
increases 3-6-fold by incubation in 10~4 M nicotinamide, whereas levels of DPNH, TPN and
TPNH do not change significantly. To determine whether the nicotinamide is incorporated
into the DPN, eggs were incubated in nicotinamide-7-C14 (1.4-9.0 X 10~r' M} in sea water for
three hours. DPN and TPN were extracted from the washed cells with trichloroacetic acid,
precipitated with acetone, and separated by paper electrophoresis. Labeled pyridine nucleotides
were located with a gas-flow scanner, eluted and specific activities were determined. DPNH
and TPNH were extracted in hot sodium carbonate solution, oxidized with phenazine metho-
502 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
sulfate, isolated and assayed as above. More than 90% of the pyridine nucleotide radioactivity
was in DPN. Ratios of C14 content of DPN: TPN were 84 and 78 in two experiments,
whereas those for DPNH:TPNH were 1.9 and 1.7. Specific activities of DPN:DPNH:TPN
:TPNH were in the ratios 1.11:1.00:0.20:0.32 and 1.03:0.98:0.47:0.38. Carbon1* which mi-
grated on electrophoresis with authentic nicotinamide was recovered in the acetone supernatant
after precipitation of the nucleotides. Its concentration was greater than that in the incubation
medium, suggesting active transport of nicotinamide. Incorporation of nicotinamide-7-C14 into
pyridine nucleotides of three-hour fertilized eggs and 24-hour embryos was also observed, but
specific activities were not determined. These studies demonstrate that intact Spisula eggs
and embryos take up nicotinamide and incorporate it into pyridine nucleotides. The lower
specific activities of TPN and TPNH are consistent with their derivation from DPN. In
addition, the nearly equal specific activities of DPN and DPNH on one hand, and TPN and
TPNH on the other, suggest that only a relatively small fraction of the DPN is converted
to TPN during the time period studied.
Triphosphopyridine nucleotide formation and disappearance in the presence of ex-
tracts of eggs, embryos and adult liver of Spisula solidissima. LEONARD LASTER
AND ROBERT K. CRANE.
To explore whether an increase in DPN kinase activity is associated with the ob>crved
alteration in TPNH concentration of Spisula solidissima eggs after fertilization, this enzyme
has been studied. Activity was assayed by determining TPN formation using a TPN-specific
preparation of glucose-6-phosphate dehydrogenase. Kinase activity of egg homogenates was
linear for one hour and was proportional to enzyme concentration in the range used. Require-
ments for DPN, ATP and Mg++ were demonstrated. Most of the kinase of homogenates
centrifuged with particles recovered at 600 g. Solubilization was achieved by freezing and
thawing. Highly approximate Km values were determined : ATP, 8.5 X \Q~3 M and DPN,
1.5 X 10~4 M. Kinase activity per unit volume of 18-hour embryos and per unit weight (wet)
of adult liver was not greater but somewhat less than kinase activity per unit volume of un-
fertilized eggs. The soluble fractions of embryo and liver homogenates contained a greater
percentage of the whole homogenate's kinase activity than did that of eggs. The assay for
DPN kinase was complicated by the presence of an enzymatic activity in eggs and embryos
that caused the disappearance of TPN added to homogenates. This activity was stimulated
by addition of DPN and Mg++. It remained predominantly in the soluble supernatant of
centrifuged homogenates. In contrast, liver-soluble supernatant caused added TPN to
disappear quite rapidly without added cofactors and this disappearance of TPN was suppressed
by the addition of DPN.
Metabolic pathways in the dogfish and skate lens. SIDNEY LERMAX, JEANNE
FONTAINE AND KENNETH WOODSIDE.
A comprehensive study of carbohydrate, protein and RNA metabolism was performed on
lenses derived from dogfish of various ages ranging from the foetal dogfish lens (approximately
25 mg.) to the mature lens (approximately 1500 mg.). The results of these investigations
indicate that there is a very marked increase in albuminoid RNA of the dogfish lens as it ages,
while microsomal and soluble RNA remain relatively unchanged. The turnover of these
RNA fractions shows a similar pattern and there is a close correlation between these results
and the relative rates of amino acid incorporation into the soluble and insoluble lens protein
fractions. However, there is little if any measurable RNAse activity in any of these lenses.
While protein and RNA metabolism in the dogfish lens show an aging pattern that is
similar to certain mammalian lenses (rat and rabbit), there is quite a marked difference in carbo-
hydrate metabolism. In the latter (rat lens) there is an active hexose monophosphate pathway
of glucose metabolism in the young and rapidly developing lens, which diminishes in activity
and importance as the lens ages. Studies on the dogfish lens indicate that glycolysis is the
major pathway of glucose oxidation in lenses derived from dogfish of any age group, while
glucose oxidation via the direct oxidative pathway occurs to a negligible extent.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 503
Preliminary investigations on the skate lens indicate that carbohydrate as well as RNA
and protein metabolism are more closely akin to the mammalian lens than the dogfish lens.
A comparative stud\ of <lof>a o.rit/asc systems in marine invertebrates. HERMAN
W. LEWIS.
Analysis of the dopa oxidase system of Drosophila has revealed a complex system, one
component of which is an activating enzyme. Similar complexity has not been found in lower
forms, offering the possibility that this enzyme system may be a useful tool for investigating
evolution at the biochemical level. To gain insight into such evolution, dopa oxidase systems
are being surveyed among the invertebrates. This report describes preliminary findings in
representative samples of a few phyla, with respect to the presence or absence of an extractable
activating enzyme of dopa oxidase. Activated dopa oxidase is stable at room temperature but
in -ritro activation occurs only in the cold, presumably because the activating enzyme is attacked
by proteolytic enzymes at room temperature. During incubation in M/IS phosphate buffer,
pH 7.0 at 0° C., aliquots of the crude extract are periodically removed and added to a solution
of 0.67 M dopa. The presence of active dopa oxidase results in the production of dopachrome,
which is measured spectrophotometrically. The increase in optical density at 475 m/i per
minute is used as the measure of dopa oxidase activity. The presence of an activating enzyme
is indicated when an extract has no or little activity shortly after extraction but shows an
increase in activity, following a sigmoid-shaped curve, when activity is plotted against time.
This situation is found in Hcnricia sangninolenta, Callinectcs sapidits, Carcinidcs inaenas, Cancer
irroratus, Paynms pollicaris, Libinia emarginata, Linntlits polyphcunts, Palaemonctes rult/arix.
Loligo pcalcii, Mercenaria inerccnaria, Aequipcctcn irruiiians, Busycon aunilicitlatitiu, Phascol-
osoma gouldii, Mctridiuin sp. and Alicrocionia prolifcru. In Astcrias jorhcsi and Crassnstrca
inrginica the dopa oxidase was fully activated when extracted. In the concentrations used and
the tissues extracted, no dopa oxidase has been found in the following: Ophiodcruia brevispina,
Arbacia pnncfnlata, Strongyloccntrotits drobachicusis, Ecliinarachnius panna, Chnctnplcnni
apicttlata, Crcpiditla fornicnta, Poliniccs duplicate, Polinices licrux, and Thais lupilli/s.
Separation of an insulin-containing fraction from the islet of the </oosefisli. ARNOLD
W. LlNDALL, JR. AND ARNOLD LAZAROW.
Previous investigations have suggested that most of the insulin in a fish islet homogenate
is removed by centrifugation ; it is recovered in the mitochondrial fraction.
Islets (50-100 mg.) were homogenized in 0.25 M sucrose and separated into nuclear (I),
mitochondrial (II), microsomal (III), and supernatant (IV) fractions. Fraction II con-
tained 80% of the cytochrome oxidase activity and more than 75% of the total insulin, as de-
termined by paper chromatography of the purified acid alcohol-soluble protein (ASF), immune-
assay and blood sugar-lowering.
Fraction II was further subfractionated by centrifugation (2 hours at 100,000 </ and 0° C.)
using a continuous linear density gradient (1.0-2.0 .17 sucrose). The gradient was separated
(from the bottom) into 18-20 subfractions of 10 drops each. The protein was distributed into
a bimodal curve with peaks at densities of 1.205 and 1.173 gm./cc. of sucrose. The high density
component (1.205) contained about 80% of the total protein present in fraction II. The cyto-
chrome oxidase activity coincided with the low density protein component (1.173), and was
completely separated from the high density protein component. The distribution of the purified
acid alcohol-soluble protein coincided with the high density component; more than 75% of this
purified ASF migrated with the same Rf as bovine insulin. Furthermore, chromatography
showed that while large quantities of "insulin" were present in the high density component
(1.205), none could be detected in the component containing the cytochrome oxidase activity
(1.173). These findings suggest that the insulin-containing (secretion) granule can be
separated from the cytochrome oxidase granule (mitochondria).
Supported by grant A-1659 from the National Institute of Arthritis and Metabolic
Diseases, Public Health Service.
504 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Intracellular cardiac potentials in Liinulns during ganglionic stimulation. FRANCES
V. McCANN AND DONALD W. MILLER, JR.
The cardiac action potential of Liinulns is distinctive because of the barrage of .small po-
tentials which persist during the prolonged phase of repolarization or "plateau." The neurogenic
nature of the origin of the heart beat in Limulus suggests that the ganglion continues to dis-
charge during the plateau, but its contribution to the maintenance of this phase of the cardiac
action potential remains obscure. Recordings of electrical activity in single cardiac cells with
conventional microelectrodes during spontaneous and driven activity indicate that as the fre-
quency of stimulation increases, the plateau shortens markedly with a consequent shortening of
the diastolic phase. As the frequency is further increased, the plateau is completely obliterated,
and the ganglionic discharge is no longer evident. At stimulation frequencies greater than
three times the normal heart rate, a new level of polarization is established (12 mV), which is
much less than the original level (48 mV), and the action potential duration and magnitude
are markedly diminished.
The nature of tlic pigments in the integument and eye of the hermit crab, Pagunis
pollicaris. JOHN J. MCNAMARA, GEORGE SZABO AND R. T. SIMS.
This investigation was made as part of a project to find ommochromes in the Crustacea.
The hermit crab is a useful animal because of its soft integument. The tissues were ground in
filtered sea water and then extracted consecutively with acetone and acid methanol to remove
carotenoids and ommochromes, respectively. The integument contained a large amount of
carotenoids but no ommochrome. The eyes contained a small amount of carotenoid and a
large amount of red ommochrome. This ommochrome showed the typical redox behavior
characteristic of this pigment and became straw-colored on the addition of sodium thiosulfate.
Histological sections, prepared from paraffin-embedded integument, showed no ommochrome.
Also, no pigment granules were revealed by the hexamine silver and Schmorl's techniques.
The orange carotenoid granules were shown to be in dendritic chromatophores by mounting
pieces of integument whole in glycerine jelly. They were stained by histochemical tests for
lipid. Granules of a blue pigment were seen in the unstained preparations. The blue color
disappeared when the tissue was boiled, so it is suggested that this pigment is a carotenoprotein.
Both eyestalks were removed from the animals and the chromatophores observed over a
period of seven days. No effect was demonstrable.
It is concluded that the eye of Pagurus pollicaris contains ommochrome and carotenoid
pigments and the integument contains only carotenoids.
Action potentials in single cells of a tunicate heart. DONALD W. MILLER. JR. AND
FRANCES V. McCANN.
The tunicate heart is known to exhibit the phenomenon of beat-reversal, i.e., the origin of
the heart beat may occur at either end of the V-shaped heart and thus pump blood alternately
forward or backward. The site of origin of the beat is generally believed to be localized at
either pole of the heart, and thus pacemaker activity is described as "bipolar." Intracellular
recordings of spontaneous electrical activity in single cells of Ciona intestinalis myocardium were
studied with conventional microelectrodes less than 0.5 /u. outside tip diameter. A portion of
the tunic was removed, and a very small incision was made through the pericardium to expose
only the area of electrode impalement. The maximum resting potential recorded was 48 mV,
and action potentials reached a maximum value of 50 mV. There was no significant overshoot
or delayed period of repolarization (plateau) in the polar or interpolar regions of the heart.
At a heart rate of 60 beats/minute, pacemaker depolarization persists for 500 msec., and
threshold for the rapid upstroke of the action potential occurs at 5 mV, or approximately 25 c/c
depolarization. That the origin of the cardiac action potential is myogenic is supported by
this observation.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 505
Experiments on interspecific fertilisation between Ciona, Stycla and Molgula
(ascidians) . A. MINGANTI.
Eggs and sperms of the ascidians Ciona intcs/inalis (Linnaeus), Stycla partita (Stimpson)
and Molgula manhattcnsis (De Kay) have been crossed \vith each other in the six possible
combinations. Mature eggs were obtained from the oviducts or the gonads. They were de-
prived of their membranes with steel needles, and inseminated with a dense sperm suspension
of another species. From 1% to 10% of the eggs, according to the crosses, were activated by
the foreign sperm. No activation ever occurred in eggs still enveloped by their membranes.
Two eggs out of 700 in the cross Ciona $ X Stycla c?, and one egg out of 350 in the cross
Ciona $X Molgula cT, cleaved normally until a pregastrular stage; they did not gastrulate
or differentiate, and cytolyzed after some hours. In all other cases the activation brought
about continuous changes, going on for many hours, in the egg shape and in the distribution
of the pigmented granules. No polar bodies were formed, although many eggs produced
hyaline lobes that were soon reabsorbed. In the activated Ciona eggs which did not develop
as described above, abortive cleavages were also observed. In such eggs a cytological study
revealed an abnormally high number of chromosomes, possibly due to endomitosis. In
the activated Molgula eggs many sperm heads of either Ciona or Stycla, not resolved into
chromosomes, were seen.
Malic dehydrogenases of developing Arbacia embryos. RICHARD O. MOORE AND
CLAUDE A. VILLEE.
Arbacia embryos grown in sea water at 20.5° were harvested by centrifugation after 6,
12, 24, or 48 hours. Any group in which less than 95% of the eggs were fertilized was dis-
carded. The embryos were homogenized in 0.025 M barbital buffer, pH 8.7, and centrifuged at
10,000 g for 20 minutes. The supernatant fraction was mixed with starch granules to a thick
paste, inserted 5 cm. from the cathode in a 1 X 15 cm. slit in a 33 cm. long starch gel block
and subjected to electrophoresis (10 hours, 200 v, 45 mA, 0.025 M barbital, pH 8.7). After
electrophoresis, 1-cm. sections were cut, eluted with artificial sea water and centrifuged. The
supernatant fractions were assayed for malic dehydrogenase activity, using DPN, 3-acetyl
pyridine DPN and thionicotinamide DPN.
Unfertilized eggs have five DPN-malic dehydrogenases, numbered I to V in order of mi-
gration toward the anode. Peak II, the major one representing 60% of the total activity,
migrates about 6 cm. from the origin. In 6-hour embryos only peaks I, II and IV could be
detected. Twelve- and 24-hour embryos have peaks I, II, IV and V ; 48-hour embryos have
the same four, but peak V is relatively larger than at 12 or 24 hours. The ratio of malic
dehydrogenase activity with APDPN and DPN in embryo extracts not subjected to electro-
phoresis changes from 0.68 in the unfertilized egg to 2.2 in the 48-hour embryo. The
TNDPN/DPN ratio decreased from 0.86 in the unfertilized egg to 0.22 at 6 hours and then
increased to 0.55 at 48 hours.
The malic dehydrogenase activity with APDPN migrated differently from the DPN
enzymes and some fractions had only DPN, others had only APDPN, activity. As many as
7 peaks with APDPN activity were observed. The ratio of APDPN: DPN activity differs
among the peaks. Ratios of activity with DPN analogues in simple tissue extracts may be
misleading, for they may represent the sum of several individual enzymes with varying analogue
ratios. In the course of these investigations an enzyme with D-malic dehydrogenase activity
with APDPN, but not DPN, was discovered. This migrates electrophoretically at a different
rate from the L-malic dehydrogenases.
Studies on the isolated islet tissue of the toadfish (Opsanns tan) : aldolasc content
of islet and other tissues. JOSEPH F. MORAN, JR.
As a part of a systematic study of the metabolism of the isolated islet tissue of the toadfish
and because of the known effect of glucose on the release of insulin from the beta cell we have
been investigating the enzymes involved in the metabolism of glucose by islet tissue. Previously
506 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
\vc have measured the glucose-6-phosphate dehydrogenase (G-6-PD) and 6-phosphogluconate
dehydrogenase (6-PGD) contents of toadfish tissues. In the present study the aldolase con-
tent of toadfish tissues was determined by measuring the rate of reduction of diphosphopyridine
nucleotide (DPN) at 340 m/u in a Beckman spectrophotometer under standardized conditions.
Weighed samples of islet (1-5 nig.) and other tissues were homogenized in glycylglycine buffer
and aliquots (10-20 n\. ) were added to the assay system; the final volume was 300 /xl.
The average aldolase content of the islet tissue of 10 animals was 57 pM DPN reduced
per gram wet weight of tissue added per hour ; this was the lowest of any tissue examined. The
aldolase contents of liver, heart, gill and testis were about 1.5 times greater, that of kidney
was twice as great, whereas brain and muscle had 5 times as much aldolase as did islet tissue.
Although islet has about equal amounts of aldolase, G-6-PD, and 6-PGD, other tissues
differ in their relative enzyme contents. For example, muscle has little, if any, G-6-PD or
6-PGD activity, but it has the highest aldolase activity. Similarly, the aldolase content of
brain is 2 to 4 times greater than that of G-6-PD and 6-PGD, whereas the aldolase content of
all other tissues examined was only 25-50% of the G-6-PD content.
Supported by Grants A-824 and A-1659 from the National Institute of Arthritis and
Metabolic Diseases, Public Health Service.
Long rcfractorv periods of branchial sensory ucrrc cndiiif/s in dogfish. RICHARD
W. MURRAY.
Nerve impulses originating at sensory endings in the skin of the pharyngeal face of the gill
bars of Mustehis ranis were recorded in fine strands of a pre-trematic (i.e., pure sensory)
branch of the vagus. Electrical stimuli (E) were applied through wick electrodes, one over
the ending and the other indifferent; the stimulating current was monitored. Mechanical
stimuli (M) were given by a probe attached to a loudspeaker.
The time-course of relative refractoriness was followed, using paired stimuli, either E-E,
E-M or M-E. Paired mechanical stimuli were not used because of possible changes in the
effectiveness of the second stimulus due to the deformation of the skin caused by the first.
Refractoriness following an antidromic impulse (A) was also tested (A-E and A-M).
The E-E combination gave the following time-course for relative refractoriness (the
strength of the second stimulus is expressed as a percentage of the threshold for single shocks,
with its S.D. ; 22 units; temperature 21°-24° C.) : 20 msec., 173 ± 13 ; 30 msec., 162 ± 12 ;
40 msec., 155 ± 11 ; 60 msec., 145 ±10; 80 msec., 139 ±9; 100 msec., 134 ± 9 ; 150 msec.
119±5; 200 msec., 113 ±6; 250 msec., 106 ± 3. All the other combinations gave comparable
values.
The long time-course found here, unlike that in axons, supports the argument that re-
fractoriness in the classical sense (as tested by paired stimuli) can be one of the factors con-
trolling the frequency of the repetitive discharge of a sense organ, even at the low rates of
firing which are commonly found.
Work carried out during tenure of a Rockefeller Foundation Fellowship.
Xitrof/en inliibition of active absorption of D-glncose in fish intestine. X. J.
MUSACCHIA AND D. D. WESTHOFF.
In vitro preparations of everted intestinal sacs from the marine fish, Stciwtonnis versicolor,
and the fresh-water fish, Ameiitrits ncbulosus, were used to measure active absorption of
D-glucose. Seventy A. nehtilosus and 75 S. versicolor preparations were run in the experi-
ments. In order to induce inhibition by anaerobic conditions, nitrogen gas (100%), was
flushed continuously through the incubation medium, either fresh-water or marine teleost
Ringer's. In the intestinal segments (whole intestine, or upper and lower areas) of A.
ncbulosus with both 5 mg.% and 10 mg.% D-glucose, active absorption continued during the
30-minute incubation period. Small amounts of endogenously produced glucose with and
without nitrogen were confirmed with blank tests. Blank tests consisted of complete experi-
ments without D-glucose added to the Ringer's incubation medium. These tests were necessary
to substantiate that under anaerobic conditions, some active absorption occurred.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 507
By way of comparison, intestinal segments from the marine fish, S. vcrsicolor, under
nitrogen anaerobiosis differed considerably. In both upper and lower segments there was up-
take of 10 mg.% D-glucose by both the mucosal and the serosal surfaces. It was concluded,
therefore, that nitrogen was an effective inhibitor of active absorption of glucose.
Comparisons of active absorption in upper ("duodenal") and lower ("ileal") segments
with 5 mg.% and 10 mg.% D-glucose were made. The upper intestinal segments from the
catfish, A. ncbulosus, showed the greater levels of active absorption with 10 mg.% D-glucose
in the medium, in terms of pM glucose/gm. dry wt./30 min. Average values were mucosal
uptake, 21.36 and serosal transfer, 8.45. The upper and lower intestinal segments in the scup,
S. rcrsicolor, were comparable in active absorption of 5 mg.% and 10 mg.% D-glucose.
Phlorizin inhibition of active absorption of D-glucose in fish intestine. X. J.
MuSACCHIA AND D. D. WESTHOFF.
Inhibition of active absorption with phlorizin at the cell surface level of the "lumenal"
intestinal epithelium has been well documented in mammalian preparations.
Phlorizin, 5 X 10~4 M, placed on both sides of intestinal wall in catfish, A. ncbulosns.
preparations resulted in inhibition of active absorption. In fact, endogenously produced glucose
was added to the medium on both sides, from 11.38 to 13.32 pM/gm. dry wt./30 min. on the
serosal side, and from 17.09 to 27.53 /^M/gm. dry wt./30 min. on the mucosal side. The levels
of inhibition were comparable with each of 16 upper segments with 10 mg.% D-glucose. When
phlorizin was only added to the mucosal side, there continued to be endogenously produced
glucose added to the serosal medium but there was no uptake from the mucosal medium. Thus,
phlorizin inhibits active absorption in intestine of catfish.
In the intestinal preparations from scup, S. vcrsicolor, the action of phlorizin, 5 X 10~4 .17,
differed somewhat. For example, when phlorizin was placed on both sides of the intestinal
wall there was uptake from the mucosal and the serosal side as well. Thus, inhibition of
active absorption was primarily in terms of glucose transferred. These differences in transport
mechanisms in the marine and fresh-water fish intestinal preparations suggest the presence of
at least a two-step process in active absorption in fish intestine.
There were 40 experiments with catfish preparations and 36 experiments with scup. Each
experiment consisted of the upper segment under phlorizin inhibition and the lower segment
as controls, without phlorizin in the incubation medium.
A persistent diurnal phototactic rhythm in the fiddler crab, Uca piti/na.r. JOHN D.
PALMER.
In simple tests designed to establish the sign of the phototactic response of fiddler crabs
it was found that the crabs sometimes responded positively and sometimes negatively. These
opposing results suggested the possibility of a rhythmic sign reversal in responsiveness to light.
To test this, small, rectangular, aluminum pans were constructed and the tops covered with
transparent plastic covers. One half of each cover was painted flat black as was the inside of
the pan beneath this area. These pans, each containing a single crab, were centered on a
fulcrum so that they would tip slightly in one direction when the crab moved into the lighted
portion and the other direction when the crab moved into the darkened end. A thread connected
each pan to a kymograph pen, and the position of the crabs within the pans was recorded
continuously during July and August, 1962. Over 11,000 individual, one-hour observations
were made in this manner. The experiment was conducted in a constant temperature room
(18°C.) at a constant light intensity of 250 foot candles. Fresh crabs were substituted
every 15 days.
A persistent daily rhythm was found in the length of time spent in the light and dark
ends of the pans (the "preference" being a measure of phototactic response). Between 5 AM
and 8 AM the crabs spent up to 70% of each hour in the lighted end of their pans. This
response gradually decreased to 50% for the hours between 3 PM and 7 PM. Between
7 PM and the early morning hours the crabs spent the major part of each hour in the darkened
end of the pan.
508 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Extrapolating to the natural habitat, at sunrise and during the cool early morning hours,
tiddler crabs are quite strongly attracted to light and they emerge from their burrows. During
the remaining hotter part of the day, when desiccation problems increase, the attraction to
light becomes less intense. At sunset the crabs become photonegatlve and return to their
burrows.
The persistence of a biological riiytlnn in continuous bright illumination. Jonx D.
PALMER, CHARLES S. YENTSCH AND SUSAN A. DEROPP.
It is well known that most plants do not tolerate uninterrupted strong illumination.
There are, however, a few algae such as Chlorclla, Sccncdesinus, etc., which grow well under
these conditions, and these, therefore, have been extensively studied. It is also well documented
that continuous bright light inhibits the expression of biological rhythms in both plants and
animals. The question was raised as to whether algae which were known to grow well in
continuous light might also maintain rhythmic variations in their growth processes.
The green alga Nannochloris has been found to grow quite well in continuous illumination
and was used in the following experiment. A turbidostat was placed in a constant temperature
cabinet (21 ± 1° C.) and provided with unilateral illumination from a bank of fluorescent lights
(700 foot candles). To monitor the amount of light passing through the culture, a photocell
was attached to the side of the growth chamber away from the light source. When the cell
number surpassed a prescribed density, nutrient automatically flowed into the growth chamber,
flushing out a sufficient number of cells to return the optical density to the prescribed level.
The amount of nutrient added was thus a measure of the rate of cell division per unit time.
Each time new nutrient was added the time of addition was automatically recorded and this
record was used to indicate the rates and times of cell divisions. Data were gathered between
13 July and 31 July, 1962.
A persistent solar-day rhythm was found in the rate of cell division. The division rate
remained relatively constant through the late afternoon, night time and early morning, rose
sharply to a maximum at noon and gradually returned to the preceding constant level by 6 PM.
More than a two-fold increase in the rate of cell division occurred at noon and over half of the
daily cell divisions took place between 9 AM and 5 PM.
Gel-sol transformations in the unfertilised egg of Arbacia punctidata. ARTHUR K.
PARPART AND THOMAS V. N. BALLANTINE.
Gel-sol transformations of the cytoplasm of the unfertilized egg of Arbacia punctulata have
been studied by numerous investigators under a great variety of conditions. The present report
comprises studies on the independent motion of the echinochrome granules and of the fine
cytoplasmic streaming observed by television microscopy. Cysteine, in concentrations of O.OOS
Mi to 0.0005 M dissolved in sea water, was observed to markedly decrease both these motions
in 30 to 180 seconds. By 7 to 15 minutes all cytoplasmic motions, except Brownian, of minute
particles was stopped. By the sucrose flotation method, centrifugation at 18,000 g for 30
seconds of eggs thus exposed to 0.005 M cysteine in sea water presented a picture of complete
immovability of the egg participates, while untreated eggs gave the normal sedimentation
pattern and a number of quarter and half eggs. The gelation of the egg cytoplasm induced by
0.005 M cysteine in sea water invariably leads to activation of the egg, and the ensuing gel-sol
changes follow those of an activated egg, if the egg is re-exposed to sea water within 10 to 15
minutes. Longer exposures produce irreversible gelation. Other agents, e.g., 1 M sucrose and
EDTA in sea water, also produce a gelation of the cytoplasm but this is readily reversible
on re-exposure to sea water, and never produces activation.
It is doubtful that cysteine or sucrose or EDTA penetrate the egg cytoplasm in the short
time required for gelation in these compounds. It is suggested that changes in the plasma
membrane of these eggs induced by these compounds lead to a wave of gelation throughout the
cytoplasm so that particles which are normally moved about the egg by fibers (echinochrome
granules) and cytoplasmic streaming are prevented.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 509
Electron microscopic observations oj secretory t/ra miles in the adhesive surjace o\
Hydra pirardi. DELBERT E. PIIILPOTT AND ALFRED B. CIIAET.
Previous work on the starfish tube foot has revealed a correlation between the ultrastructurc
of the fibrous-ellipsoidal "secretory packets" and the adhesive properties of this organism. The
present report deals with a similar investigation which attempts to observe the ultrastructure of
the basal portion of Hydra pirardi, and to correlate, if possible, these findings with its adhesive
ability.
Electron micrographs reveal small spherical structures, 1-1.5 microns in diameter, which arc
located in the epidermis of the distal portion of the peduncle and are more abundantly found in
the basal disc itself. Many of these structures (secretory granules) appear as loose "balls of
yarn" surrounded by one or two outer shells. Others are more homogeneous in appearance and
are also surrounded by an outer shell. As the matrix of these secretory granules becomes more
homogeneous in nature, the vacuolar space around them, which is bounded by a double membrane,
gradually disappears.
Mitochondria are found interspersed between the granules, and Golgi bodies frequently are
found in association with them. Although the mode of release of these granules has yet to be
conclusively determined, preliminary evidence suggests that the individual structures bulge
against, and are finally extruded through, the cell membrane into the external environment.
Since light microscopy studies have demonstrated that these secretory granules are PAS-
and alcian blue-positive, and since these granules are found mainly on the attaching surface, it
is hypothesized that they are used in the adhesion of Hydra to a substratum. There is an
interesting similarity in the staining properties and in the ultrastructure between the secretory
granules of Hydra and the "secretory packets" previously demonstrated in the starfish tube feet.
Supported by grants from the National Science Foundation (G-8718) and the National
Institutes of Health (A-3362, B-3269, H-6214).
Chromatophores of decapod Crustacea in hypodcnnal organ culture. NANCY
PlANFETTI, JUDITH HlCKMAN AND RlCHARD C. SANBORN.
Organ cultures of the hypodermis of Homants ainericanns. Cancer irroratus, Craiu/on
septemspinosus, Callincctes sapidits. Libinia cinanjinata, and L. dnbia have been prepared. The
media used contained balanced crustacean saline solution, organic acids, carbohydrates, lactal-
bumin hydrolysate, 10 to 20% Callincctes or fetal calf serum, and antibiotics. In certain of these
media, the hypodermis survives without visible morphological change or growth for at least
60 days when the medium is changed at seven- to ten-day intervals.
Chromatophores of such cultured hypodermis behave differently from those of animals from
which the eye-stalks have been removed. For example, following eye-stalk ablation the melano-
phores of Callincctes contract to stage 1 while the erythrophores expand to stage 5. In culture,
both types remain contracted (stages 1 or 2). In vivo, following eye-stalk removal, the
melanophores of Crangon uropods and telson are first expanded (stage 4 or 5) then contract
while the body Chromatophores contract and then expand to stage 4 or 5. In vitro, both groups
of Chromatophores remain in stage 2.
An exception to the contraction of Crangon Chromatophores in vitro is noteworthy. The
melanophores of the hypodermis of whole eye-stalk explants are dispersed rather than con-
tracted. This suggests that under our culture conditions the Chromatophores are susceptible
to humoral factors. Attempts to test this hypothesis using other hypodermis explants have not,
however, shown changes under the influence of extracted, boiled, or alcohol-soluble or
-insoluble preparations of eye-stalks. These same preparations act in the usual fashion on
the Chromatophores of Uca in vivo, both before and after incubation with hypodermal explants.
Aided, in part, by National Science Foundation Grant G-11234.
The chloride permeability oj crayfish muscle fibers. JOHN P. REUBEN, LUCIEN
GlRARDIER AND HARRY GRUNDFEST.
Electrical and volumetric data on single fibers indicate that the membrane of crayfish muscle
is permeable to Cl, and the sites for Cl movement appear to he distinct from those permeable
510 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
for K. Under normal conditions the changes in membrane potential caused by altering the
level of external Cl are much faster than those produced by changing K. Raising the pH to 10
does not affect the membrane potential, but the theoretically expected response (58 mv./decade )
is obtained on changing the external K, while changing the Cl affects the potential little or none.
Thus, it appears that Cl penetrates through positively charged membrane sites whose specificity
for Cl decreases with decrease of the titrable positive charge density.
Crayfish fibers normally exhibit marked hyperpolarizing rectification due to increased
Cl conductance. This rectification is abolished by depleting intracellular Cl, by high pH, or
by applying picrotoxin, the effective resistance to hyperpolarizing currents rising accordingly.
At pH 10 the length constant also rose from 3.15 to 4.22 mm. GAB A, which normally increases
conductance, did not affect the current-voltage curves of fibers with depleted Cl, and there was
no change on applying picrotoxin. Hyperpolarizing rectification is also abolished by removing
the Cl with inward current delivered through a microelectrode filled with 3 M K propionate as
the intracellular cathode. Hyperpolarizing responses, which under normal conditions never
occur in crayfish muscle fibers, are elicited regularly when the Cl is depleted, and applications of
GAB A do not block the response. Since the latter result from hyperpolarizing K inactivatimi.
the K- and Cl-selective channels must have distinctive pharmacological properties.
GABA and picrotoxin affect the rates of water movements that are produced by altering
the ionic environment of the fibers. The swelling of fibers which are transferred from the
standard medium to one containing 153 meq./l. K (isotonic) was 5-fold slower in picrotoxin
than in GABA. Since the electrophysiological data indicate that picrotoxin blocks Cl permea-
bility, this result strongly suggests that local currents are set up by the movement of ions
through specific sites permeable to K and Cl, respectively.
Some properties of stcllarin, the photosensitive pigment of the starfish, dstcrius
forbesi. MORRIS ROCKSTEIN.
Careful control of the temperature of extraction, the pH of the 2% digitonin extracting
solution and the temperature during exposure to light conclusively proved the presence of a
photosensitive pigment in the dorsal skin of the starfish, A. forbesi. When extracted at low
pH Teorell buffer solution of digitonin, the pigment assumed a true violet hue with an
absorption maximum at 567 nifj., but failed to exhibit any photolability. Alkalinization of this
extract with 1 N NaOH (from a pH of 4.8 to 12.4) converted the pigment to a now-
photosensitive, orange-peach pigment with an absorption maximum at 485 to 490 m/t. This
was similar to the extract which could be made of the pigment from aqueous suspension by an
alkaline Teorell buffer solution of digitonin (pH 12.0) as the primary extracting medium.
Intermediate pH values for the digitonin solutions employed in extracting this pigment yielded
intermediate colors, varying from violet-peach to peachy-violet, and possessing intermediate
absorption maxima as well. Pronounced and highly reproducible difference spectra were
obtained for all alkaline digitonin pigment extracts, exposed for short periods of time to white
light of medium to moderate intensity under controlled temperature and buffered pH values
of the extracting medium. This difference spectrum possesses a maximum at 576 and a mini-
mum at about 485 m/u. One can summarize these data by stating that the photosensitive pigment
of this species, stellarin, is a pH indicator-like substance, which, in the alkaline range, is unstable
in light and in the acid range is stable to light effects. This susceptibility to light in the
alkaline range is readily eliminated by acidification and the stable form of the pigment at low
pH is readily converted to the photolabile form upon alkalinization.
Ultraviolet damage to the cortex of the sea urchin egg. RONALD C. RUSTAD.
Various types of radiation interfere with the fertilization reaction. The elevation of the
fertilization membrane and the differentiation of the hyaline layer are known to be suppressed
on the hemisphere of a sea urchin egg which faces an ultraviolet lamp.
Phase contrast observations on U.V. -irradiated cells indicate that the round cortical granules
can be transformed into an irregular splotchy shape. This change often does not occur until
after fertilization, when normal granules yield fine filaments.
The irradiated eggs elevate a highly birefringent fertilization membrane on the cyto-
plasmically-shaded side. At high doses there is no detectable change in the weak birefringence
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 511
at the irradiated surface following insemination. Hence, not even a "tight fertilization
membrane" is formed on the damaged side.
Pigment granules sometimes migrate preferentially to the cortex of the directly-irradiated
hemisphere of the unfertilized egg.
These observations indicate that U.Y. damages some component of the cortex of the sea
urchin egg and prevents the normal breakdown of the cortical granules. The granule material
is not extruded into the perivitelline space to aid in the elevation of the fertilization membrane
or in the formation of the final fertilization membrane structure.
Drugs causing localized lightening and darkening of the common sand dab,
Scophthalamus aqitosits. GEORGE T. SCOTT, RICHARD L. CLARK AND JAMES
C. HlCKMAN.
Preliminary dose-response observations were made by injecting 0.05 ml. of the dilutions
of the drugs subcutaneously in the flank of one or two fish. Finally, three injections adjusted
about the approximate effective dose (ED) were injected into five fish. The amount of drug
required to produce a distinct light or dark patch at least one half inch in diameter in four out
of five fish was taken as the ED-*,.
The EDso in fig. for drugs producing localized chromatophore aggregation were as follows :
epinephrine 1, norepinephrine 1, isopropylarterenol 10, serotonin 100, phenelzine 400, pheniprazine
400.
Drugs producing localized darkening fell into several classes. The EDb0 in M8- were as
follows: (local anesthetic) dibucaine 2; (tranquilizing) eleven phenothiazine ataraxics ranged
from 10 to 150 (the most active was mepazine and the least active was chlorpromazine sulfoxide)
reserpine phosphate 30, isoreserpine phosphate 60, meprobamate 500; (sedatives) meperidine
(Demerol) 40, ethchlorvynol (Placidyl) 200; (serotonin-blocking) lysergic acid diethylamide
500; bimaleate 500; (adrenaline-blocking) phenoxybenzamine 10, N-(2-chloroethyl) dibenzyl-
amine 10, dichloroisopropyl arterenol 100; (antidepressant) imipramine 12. Barbiturates were
found to be inactive.
The high potency of the epinephrines in producing localized lightening, together with the
observation that the epinephrine blocking agents are active chromatophore dispersers, suggests
that a physiologically active amine secreted by the aggregating nerve fibers is the chemical
mediator. Pheniprazine and phenelzine may act by monoamine oxidase inhibition.
No evidence of dispersing nerve fibers or pituitary control of chromatophores has been
found. Therefore, the drugs causing darkening may be acting by an adrenolytic property at
the chromatoneural junction or acting directly on the melanocyte cell.
This study was aided by a National Institute of Mental Health grant MY-3%3 to
Oberlin College.
Resistance to gamma irradiation of fertilized eggs of Arbacia correlated with their
stage of development. CARL CASKEY SPEIDEL AND RALPH HOLT CHENEY.
Fertilized eggs of Arbacia at 13 time-stages of development were subjected to equal
gamma irradiation at 5-minute intervals, as follows : time in minutes after insemination ; 2, 7,
12, 17, 22, 27, 32, 37, 42, 52, 57, 62. In Experiment A, the dose at each time-stage was 4 kr ;
in B, 8 kr ; C, 12 kr; D and E, 16 kr; F, 24 kr ; G. 32 kr. Four experiments included three
later time-stages of 72, 82, and 92 minutes.
Comparative irradiation damage was best estimated from cultures of thousands of embryos
observed at (1) 7-13 hours, for differences in hatching time and motility, (2) 24-48 hours and
longer, for motility, differentiation, injury and viability. Delays in first and later cleavages
were also noted. The most resistant time-stages were those from which embryos developed
that displayed earliest and greatest amount of motility, least degree of injury, and best differen-
tiation and viability. Based on these criteria, 12 time-stages from fertilization to first cleavage
were arranged in order from most radio-resistant to most vulnerable, as follows : 32, 27, 37,
42, 47, 22, 52, 57, 2, 7, 12, 17.
Eggs in the most resistant time-stages (32, 27, 37) were in the streak phase, approximately
512 PAPERS PRESENTED AT MARI.NE BIOLOGICAL LABORATORY
t
midway between early vulnerable time-stages (17, 12), characterized by the monaster, and later
vulnerable time-stages (57, 52), characterized by the end phases of mitosis and the onset of
first cleavage. Eggs in time-stages 7 and 2, also very vulnerable, contained separate pronuclei.
Time-stages 22, 42, and 47 were transitional.
Comparison of unequal radiation dosages showed that a one-unit dose to eggs in vulnerable
stages of monaster (17, 12), separate pronuclei (7, 2), and first cleavage onset (57), induced
more damage in the developing embryos than a two-unit dose to eggs in resistant streak stages
(27, 32).
Supported by Grant RG-4326(C5) to C.C.S. from the U.S.P.H.S. and by Grant 144
to R.H.C. from the National Academy of Sciences.
Ammo acids in the economy of the bamboo worm, Clymenella torquata. GROVER
C. STEPHENS.
An analysis of the amino acids in sea water was made using a Spinco Model 120 amino
acid analyzer. Sea water was obtained at low tide from a mud flat where Clymenella was
abundant. An attempt was made to obtain sub-surface water by digging a few inches below
the surface at the water's edge. Samples were frozen at the time of collection and were later
evaporated to dryness and the resulting salt extracted with 2% HC1 in acetone. Eleven neutral
and acidic amino acids were identified. Total amino acid concentration was 74 micromoles
per liter. Major components were glutamic acid (25.1 micromoles), alanine (15.8), glycine
(9.64), and aspartic acid (8.65). These figures are only approximate since recovery was not
checked for all amino acids. Recovery for valine, glycine, and phenylalanine, using C14-labelled
compounds, was 55.5%, 54.5%, and 55.5% at 5.0 micromoles per liter.
Oxygen consumption of these worms is of the order of 0.1 ml./gm./hr. If amino acids
were the substrate, this is roughly equivalent to 0.1 mg. per hour. At the rate of uptake of
glycine by Clymenella observed using glycine-C14, the* observed concentration of glycine alone in
sea water would supply 30% of this amount. The other amino acids which have been studied
(phenylalanine, valine, and lysine) are also accumulated by Clymenella. Although uptake from
complex mixtures has not been investigated, it may be suggested that the free amino acids of its
habitat offer this organism a significant nutritive source. Since Clymenella is often found in
low oxygen environments, it is of interest to report that their uptake of amino acids occurs
unimpaired in nitrogen-saturated water.
Supported by P.H.S. Grant RG-6378.
Uptake of amino acids by the bamboo worm, Clymenella torquata. GROVER C.
STEPHENS.
The observations to be summarized were made using C14-labelled amino acids. Labelled
phenylalanine, valine, glycine, and lysine were supplied in the ambient sea water. Entry of
amino acids was followed by measuring the radioactivity of the sea water and of 80%
ethanol extracts of the worms. The uptake of phenylalanine will be discussed as typical.
Uptake is linear with time for at least the initial stages of the process. After fifteen
minutes at an ambient concentration of 1Q-6 to 5 X 10~5 molar, the radioactivity of an alcohol
extract of the worms is approximately ten times that of the medium after correction for
volume, self-absorption, and background. Chromatography indicates that the radioactivity of
the extract is in the form of phenylalanine. Further increase in ambient concentration does
not produce a corresponding increase in rate of uptake. The rate of uptake is a function of
surface and occurs across the body wall, independent of the gut. There is some incorporation
after 24 hours but the bulk of the amino acid taken in remains in the alcohol-soluble fraction.
Once accumulated, amino acids are not exchangeable with amino acids in the medium to any
significant extent. By pre-loading the organisms, accumulation can be observed against
gradients of 5000:1 or greater. Uptake is not stereospecific, at least for phenylalanine. The
Q,0 for the process is approximately 1.7 for the temperature range 5-25° C.
Glycine is accumulated at approximately the same rate as phenylalanine. Lysine and
valine enter at about 30% to 40% of this rate.
Supported by P.H.S. Grant RG-6378.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 513
Osmotic pressure relationships in the spiny dogfish (Sqnalus acanthias). WILLIAM
STONE, JR. AND WILLIAM C. DEWEL.
Recent evidence (Science, 132: 36, 1960) indicates that the osmotic pressure of the aqueous
humor is lower than blood plasma in the smooth dogfish (Mitstclus canis). However, other
workers (Comp. Biochcm. Physiol., 5: 193, 1962) have reported no difference in the osmotic
pressure of these two fluids in the spiny dogfish (Squalus acanthias). The present study was
undertaken to resolve the apparent discrepancy. The two species belong to different sub-orders,
and differences in the morphology of the eye as well as the whole fish were numerous and pro-
nounced. The osmolarity of the plasma and aqueous humor from both species were measured,
using the Fiske osmometer. The results for the smooth dogfish were similar to those reported
earlier. Extracting sufficient aqueous humor for measurement from the spiny dogfish was
difficult but adequate samples were obtained from 10 large fish. The osmotic pressure of spiny
dogfish aqueous humor was found to be 963 (S.D. 28.3) milliosmoles while the plasma
measured 983 (S.D. 18.6). The average difference between the two sets of measurements was
20.6 milliosmoles. In only one case was the osmotic pressure of the aqueous humor higher
than the plasma. In the remaining 9 cases it was lower. Statistical analysis, using the t
test, revealed that the lower osmotic pressure in aqueous humor, as compared with plasma,
was significant at 0.01 confidence limits.
Studies of melanin biosynthesis in the ink sac oj the squid (Loligo pealci). II.
(Histology, autoradiograpJiy, tissue culture and in i-i-c'o inhibition oj ink gland ).
GEORGE SZABO AND R. T. SIMS.
The ink gland is unique in many ways as an experimental model for the study of melano-
genesis. Not only does it produce large amounts of tyrosinase and melanin but it is apparently
continuously synthesizing these materials.
There are two types of epithelial cells in the gland. (1) A columnar cell with well defined
polarization, showing a strongly basophilic cytoplasm towards the basement membrane and
large pigment granules towards the lumen. The granules were found to be melanin by histo-
chemical tests. The nucleus is large with a thin chromatin network. There are several
nucleoli. (2) The other type of epithelial cell is in the caudal portion of the gland. It is tall
columnar, has little or no melanin, but contains a single large vacuole at the apical end. This
vacuole does not contain PAS-positive material. The epithelium of the ink gland is tyrosine-
and dopa-positive.
The rate of melanin production was studied by inhibiting tyrosinase activity with phenyl-
thiourea added to sea water (0.004% and 0.008%). The ink gland starts to turn white after
the living newly hatched squid has been in PTU for 48 hours. When returned to normal sea
water they start to regain pigment after 24 hours. Autoradiographic studies of the gland of
newly hatched squid showed uptake of H3-tyrosine at a high rate and indicate a turnover of
melanin of 24 hours or less.
The ink gland was cultured on glass surface in Gatenby's molluscan saline, in Hedon-Fleig
saline with or without horse serum. The epithelial cells migrated either individually or in a
sheet. The cells retained their polarity during migration, as the pigment was concentrated at
one end. Movement of cilia was observed in the explant and isolated cells moved in the
fashion of ciliated cells. Electron microscope pictures confirmed the existence of cilia.
Inhibition oj regeneration in Tubularia b\ tissue extract injection. KENYON S.
TWEEDELL.
Extracts of individual parts of adult hydranths were injected into the coenosarc cavity
of amputated Tnbnlaria stems. The tissues were homogenized in a small amount of filtered
sea water with an iced Teflon homogenizer. The homogenates were then centrifuged at
30,000 g for 20 minutes in a refrigerated centrifuge. The supernatant was removed and
refrigerated. Freshly amputated stems were injected from the proximal end with 0.5 to 1.0 /ul.
of extract, and then placed in standing filtered sea water at 19-21° C. Other stems were in-
514 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
jected at 6, 12, 18 and 24 hours post-amputation. For the latter, a minute opening was made
with a "00" needle at the healed distal end, to eliminate pressure. Controls were amputated
untreated stems and amputated stems injected with filtered sea water.
Hydranths were subdivided into the distal hypostome, including distal tentacles, and the
complete proximal hydranth. A single injection of the proximal hydranth extract (0.04
hydranth/Vl. ) up to 24 hours after amputation caused complete and permanent inhibition in
90% or more of the stems. Single injections of the distal hypostome with tentacles at 0, 6, or
12 hours after amputation retarded development in most stems for 24-30 hours. Thereafter,
many retarded stems (from 40-100%) would recover and regenerate latently. Extracts
injected at 18 hours or later had little effect.
Individual extracts were also made from distal hypostomes, gonophores, proximal tentacles
and the remaining basal hydranth in concentrations from 0.03 to 0.05 part//*l. Each of the
extracts was singly injected from 0 to 18 hours post-amputation (one band stage). The most
effective tissue fractions were the basal hydranth and the gonophore extracts. Total inhibition
of stems by the tissue extracts was not significant over the controls. Instead, development was
often retarded up to 24 hours after injection. Alternatively, stunting of the hydranth occurred
in 22 to 45% of the stems. This often reduced the number and length of the proximal tentacles.
The extracts were effective at 0, 6 and 12 hours after amputation but had little action thereafter.
Analysis of motllit\ in a ncu' species of grci/arinc. CHRISTOPHER D. WAITERS.
The acephaline gregarine, Urospora sp. (after Torch), exhibits characteristic motions
which may be observed through the body wall of its annelid host, Pcctinaria gouldii; this
coelomic parasite has continued such movements up to eight hours after isolation in filtered
sea water.
The movements of specimens 80 to 500 microns long were recorded cinematographically
for detailed analysis : ( 1 ) The most obvious movement is a wave of constriction that is
propagated unidirectionally along the body axis from the more narrow end which attaches
to the substratum; velocity of the wave is ca. 150 microns/second and the period about 2
seconds. (2) Cytoplasmic inclusions are observed to move rapidly in a direction opposite to
that taken by the propagated wave, until the wave has passed midway along the body length:
then, as the wave traverses the rest of the organism, these inclusions reverse their direction,
first in the wave region, and thereby move more slowly with the wave. (3) The entire
cytoplasm, including the nucleus, shuttles back and forth during the propagation of one wave.
(4) When the surface is marked with carmine particles, it can be seen that each wave is ac-
companied by a 90° recoiling torsion of the whole organism. (5) Carmine particles on se-
lected surface regions sometimes are swept to the ends of the cell. While this type of motion
could cause the typical gregarine gliding movement, longitudinal displacements along a sub-
stratum were observed only when the organisms were compressed under a coverglass.
Observations with phase, interference, polarizing, and electron microscopes have so far
revealed only a weak, generalized cortical birefringence (apparently not due to resolvable
myonemes) and at the very surface spirally arranged rows of ridges of ca. 2 microns high;
when the wave is propagated along the cell surface, these ridges move apart and then back
together after the wave has passed. Further studies are in progress in collaboration with
Delbert Philpott (electron microscope) and Robert Allen (polarizing microscope).
Seasonal fluctuations in mean paths of snails (Nassarius) in a uniform light field.
H. MARGUERITE WEBB AND FRANKLIN H. BARNWELL.
The mean paths taken by snails in a uniformly illuminated field were observed under the
following conditions : in the morning, heading south and heading north, in the afternoon heading
south and heading north. The test group in each case consisted of 10 animals which were
observed for three trials in one direction and then three trials in the opposite direction. The
observations were made during the period June 27 through August 24, 1962. When results
were averaged, regardless of direction or time of day, and grouped into 7-day periods it was
found that the mean paths varied throughout the study in the following manner : for the period
beginning June 27 the path was 4.5° ±0.5 to the left; for the period beginning July 4 the path
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 515
was 0.2° ± 0.4 to the left; thereafter for five periods there was a progressive increase in amount
of left-turning until the period beginning August 13 when the path was 8.6° ± 0.5 to the left.
The intervening values were 4.7°, 5.9°, 6.2°, and 7.0°, all with comparable standard errors. For
the period beginning August 20 the path was 1.7° ±0.5 to the left. The coefficient of corre-
lation (r = 0.857 ± 0.107) between the paths of southbound and of northbound animals indicates
that the two groups were similarly affected by whatever brought about the variation in mean
paths.
Supported by contract 1228-03 with O.X.R., Grant G-15008 from the N.S.F. and Grant
RG-7405 from the U.S.P.H.S.
Studies on the structure of the thyiuus. I. Electron microscopic observations on
the cortical vascular barrier. LEON WEISS.
The presence of a vascular barrier in the thymus, similar to the blood-brain barrier, has
been postulated by Marshall and White because antigen injected intravenously induces no anti-
body response in the thymus, whereas antigen injected directly into the thymus does.
In the mouse, fine cortical vessels have an outside diameter of 4 to 8 /M and a lumenal
diameter of 1 to 3 /JL. Endothelial cells form a complete lining. Many lumenal processes are
present and the cytoplasm is rich in vesicles. Basal endothelial processes extend into a broad
basement membrane. Several layers of adventitial cells may be present. Each adventitial cell
is surrounded by extracellular tissue consisting of ground substance and collagenous fibers, and
continuous with the basement membrane. Thus, the vessel wall is often lamellated, layers of
cytoplasm alternating with layers of extracellular tissue.
The most peripheral element in the wall is the epithelial reticular cell. It is a large cell
whose cytoplasm may envelop the vessel. It may also extend cytoplasmic processes which
surround perivascular lymphocytes. Reticular cells have desmosomes, show evidence of marked
reduplications of the plasma membrane, and possess many vesicular processes. Reticular cells
contain granules, suggesting secretion, and phagocytic vacuoles. Both granules and vacuoles arc
stained in the periodic acid-Schiff reaction. Reticular cells form Hassall's corpuscles.
After a single dose of Thorotrast, thorium dioxide is found primarily in the extracellular
tissue of the vessel wall.
The morphological arrangement of these vessels is similar to that in the blood-brain
barrier. They also resemble sheathed arteries in the spleen. It appears that thymic reticular
cells mediate humoral influences upon perivascular lymphocytes.
The incorporation of iododeoxyuridine by the developing Arbacia embryo. M. B.
WHEELER, C. V. HARDING, W. L. HUGHES AND W. L. WILSON.
Under certain conditions, iododeoxyuridine (1UDR), an analogue of thymidine, can be
incorporated into DNA. There is evidence, furthermore, that its incorporation mimics that of
thymidine (Prusoff, 1960; Gitlin, 1961). When labeled with Iiai, the uptake of IUDR can be
detected externally with a scintillation counter. The possibility exists, therefore, that an index
of the rate of DNA synthesis can be determined in the same tissue or group of cells at several
different times. As a preliminary to such determinations, the present study was conducted to
determine the extent of uptake of IUDR into the Arbacia punctulata embryo at different stages
of development. Experiments were performed, in each of which the eggs from a single
female were used ; aliquots of the developing embryos were taken at various times after
fertilization, incubation in IUDR (carrier-free, 0.022-0.006 yuC./ml.) for periods of 0.5 to 1.0
hour, and then their radioactive content was determined. The concentration of IUDR and
the duration of incubation were maintained constant for the aliquots within a given experiment.
After incubation, the eggs were washed 3-4 times in sea water and extracted three times with
cold 5% TCA or Carney's solution. Radioactivity in the sea water washes and in the acid-
soluble and -insoluble fractions was then determined. There was insignificant incorporation
into the acid-insoluble fraction and very little into the acid-soluble fraction of the unfertilized
eggs. Following fertilization, there was a significant incorporation into both fractions. The
rate of incorporation increased with increasing age of development until at the end of 15 to
18 hours, the curve reached a maximum, and decreased thereafter for several hours. The radio-
516 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
active component (s) in the acid-soluble fraction was apparently not in the form of IUDR since
the radioactivity did not crystallize with added carrier IUDR. The incorporation of IUDR
into both fractions was strikingly inhibited in the presence of thymidine (cone., 0.002 M).
Studies on Euplotcs. I. Structure and life cycle of a new species of marine
Euplotcs. RALPH WICHTERMAN.
Characteristics: Body 132x70 /j., somewhat convex dorsally and ventrally with 11 latero-
dorsal kinetics. Anterior end widest and truncated ; posterior end rounded. The adoral zone
of membranelles extends to three-fourths the body length. Cirri: 10 frontal-ventral; 5 anal; 4
caudal. Macronucleus 82 fj., band-like and C-shaped. Micronucleus 3 /a. Location : Bay of
Naples.
From collections, isolations were made and 6 clonal cultures established in filtered sea water
with associated bacteria, to which was added Cerophyl, a powder of dehydrated cereal grass
leaves. At the log-growth phase, the fission rate averaged 1.75 divisions per day. Fission
required one and a quarter to one and a half hours, from the time the vegetative animal was
seen to begin division until the two separated daughters were formed. Mean salinity of sea
water of the Bay of Naples is 38. Organisms are presently being cultivated and studied in
sea water from Woods Hole Harbor, which has a salinity of 31-32.
Attempts were made to determine the existence of mating types for a genetic analysis by
making all possible mixtures of clones. Mating and conjugation did not occur. However,
the processes of encystment and excystment took place and could be controlled.
Normally, in an old culture, animals encyst. For this to occur, the vegetative animal stops
eating, settles to the bottom of the container and undergoes dedifferentiation of cirri and
membranelles. Seen from top view, cysts appear circular and average 78 p. in diameter.
Seen from side view, the upper surface of the cyst is highly convex but the lower surface
only slightly convex. A sticky substance secreted by the encysting animal enables the cyst to
adhere to the substratum.
In a rich culture, encystment can be induced by sharply cutting off the supply of available
food. Excystment can be accomplished by surrounding the cysts with fresh medium. When
single cysts were placed in this medium, no excystment occurred for at least one and a half
hours, but most animals excystecl not later than 5 hours, after which they resumed normal fission.
Part of a project aided by grants from the American Philosophical Society, the Committee
on Research of Temple University, and the American Tables Committee for the Naples
Zoological Station.
Studies on Euplotcs. II. Mating types and conjugation in a marine species of
Euplotes. RALPH WICHTERMAN.
Characteristics : Body ovoid, 50 X 27 /*, slightly convex dorsally and ventrally with 8 latero-
dorsal kinetics. Adoral zone of membranelles extends to four-fifths body length. Cirri : 10
frontal-ventral ; 5 anal ; 4 caudal. Macronucleus 26 n, band-like and C-shaped, with posterior
end consisting of a small knob attached to larger part by a thin strand. • Micronucleus 1.5 M-
Location : Bay of Naples. Investigations at present reveal that the ciliate may be Euplotes
cristatus or a closely related species, if not a new one.
Nine clones, designated A, G, H, J, K, L, M, N, and Q are in cultivation in filtered sea
water from Woods Hole Harbor. The results of all possible mixtures of the nine clones re-
vealed the existence of mating types as follows : clone G mated with all clones except A and N ;
clones H, J, L, and Q mated with all clones except A ; clone K mated with all clones except
A and M ; clone M mated with all clones except A and K ; clone N mated with all clones
except A and G. Obviously, clone A failed to mate with any other clone.
Upon mixing reactive opposite mating types, two ciliates ready to mate spiral rapidly for-
ward and parallel to each other on a longitudinal axis. After this cooperative spiralling,
animals join along their adoral zone of membranelles. They do not join in the sexual union
immediately after being mixed but from 3 to 12 hours later at 26° C. Data thus far suggest
they mate more readily in the morning, beginning with daylight, than at other times. Mating
pairs remain joined for approximately 20 hours.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 517
In the sexual process the macronucleus of each member segments into three, then four, lobes,
tach connected by a thin strand of nuclear material. After the strands break, two of the lobes
become localized at the anterior end of a conjugant and two near the posterior end. Those in
the posterior end are the first to disintegrate.
Concomitant with macronuclear breakdown the micronucleus of each conjugant undergoes
the pregamic divisions. A single spherical anlage is produced in each, which later occupies
about one-third the body length. In Feulgen preparations the anlage appears homogeneous
and does not react to the stain. As long as 80 hours after mating, the anlage is then faintly
Feulgen-positive while the micronucleus adjacent to it is well stained.
Part of a project aided by grants from the American Philosophical Society, the Committee
on Research of Temple University, and the American Tables Committee for the Naples
Zoological Station.
A comparison of methods using Ca*5 as a tracer for calcium activity in Arbacia
eggs. FLOYD J. WIERCINSKT AND CAROL E. WIERCINSKI.
During the past four summers Ca4"', in combination with various methods, has been used
to determine whether or not calcium ion is absorbed or released in the eggs of Arbacia punctu-
lata before and after fertilization. A Geiger tube and a gas flow /3 detector with dried planchet
samples of eggs yielded results in the range of the standard deviation. Autoradiographic study
of sectioned ovary from in -firo experiments with Ca45 indicated the presence of the radioisotope
in and around the immature cells. Studies of sectioned eggs before and after fertilization indi-
cated Ca40 in the jelly coat and on the cortical layer. Filtration of incubated eggs showed
significant amounts of activity related to the mass of cells after fertilization.
.•Irhacia and Laminaria were put into aerated Ca45 sea water for a period of 6-10 days.
The eggs of these animals were shed into 150 cc. of fresh sea water by means of electrical
stimulation and were found to be moderately radioactive. A Geiger tube placed above a dish
of settled or agitated eggs made no significant difference in the count. A Geiger tube was
placed into the sea water 0.5 mm. above the layer of eggs. Also, a glass cylinder was placed
over an inverted tube so that the window formed the bottom of the vessel with eggs resting
on the window. Liquid scintillation counting techniques were used with samples taken from
1 cm. of sea water above the egg layer resting at the bottom of a beaker. Statistically signifi-
cant counting showed no difference of Ca45 activity before and 60 minutes after fertilization.
The above experimental conditions and methods give data to indicate that calcium does not move
and is neither absorbed nor released on fertilization. Other experiments are planned.
The growth of brain in teleosts. CHARLES G. WILBER AND RICHARD SCHNEIDER.
The expression of relative growth of organs with respect to growth of total body is usually
formulated in logarithmic terms. The log-log relationship leads to calculation of "constants of
allometry" and other constants, some of which are biologically meaningful. Nevertheless
logarithmic relationships are not particularly easy to visualize, especially by the non-mathema-
tician. It is our contention that, for certain aspects of relative growth, the rectangular hyperbola
is especially useful because such a curve is easily visualized and presents a generalization which
makes sense biologically over a wide range of values. Measurements of brain weight and total
body weight were made for a number of local marine species of bony fish. The results were
plotted on ordinary coordinate paper. Plotted points fell along a path which by inspection de-
scribed an hyperbola. Curves were fitted to the points by successive approximation. Examples
of the fitted curves are given in the following expressions : for the sea bass, Centropristes
striatus, brain weight in mg. equals 420 minus the quantity 32,000 divided by body weight in
grams ; for the puffer, Sphacroidcs macnlatus, brain weight in mg. equals 380 minus the
quantity 10,800 divided by the body weight in gm. ; for the scup, Stcnotounis rcrsicolor, brain
weight in mg. equals 807 minus the quantity 61,500 divided by body weight in gm. Growth of
brain in tautog, sea robin, and flounder seems to follow the same general pattern. Detailed
analyses of the relative growth of the following organs (in addition to brain) with respect to
body growth are being made : eyes, liver, gut, spleen, heart.
518 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Observations on marine eggs subjected to ultrasonic vibration. WALTER L. WIL-
SON, FLOYD J. WIERCINSKI, WESLEY L. NYBORG AND F. J. SICHEL.
Eggs were subjected to ultrasonic vibration by means of a steel needle applied directly to
the cell surface or inserted into a drop of an egg suspension. The needle was mounted in the
tip of a steel cone fixed at its base to one end of a cylindrical, ceramic transducer. The trans-
ducer was driven by means of an oscillator working through a power amplifier and had a tuned
frequency of about 83,000 cycles per second. The needle, with a shaft diameter of 0.2 mm.,
extended about 3 mm. beyond the tip of the cone and tapered to a blunted tip. Careful
machining and mounting ensured that the vibrations induced in the needle were in the long
axis, with little lateral motion. In some experiments eggs were held stationary on the end of
a micropipette ; in other experiments eggs were allowed to move freely in a drop of sea water.
Observations were carried out with the aid of an inverted microscope, and motion pictures
were taken.
With the needle tip applied directly to the cell surface of unfertilized eggs of Astcrias or
of Sf>isula held with a micropipette, ultrasonic vibration in some cases causes the nucleolus
to move about within the nucleus ; in other cases the nucleolus remains in a given position but
rotates, turning faster as the intensity of the ultrasonic energy is increased. In one Astcrias
egg the nucleolus was observed to break into two parts.
Unfertilized eggs of Astcrias free to move in a sea water drop move close to the vibrating
needle. In this position the surface of the egg closest to the needle undergoes undulations. In
several eggs rather large cone-like projections were formed at the cell surface.
This work was supported by a Grant (RG-8775) from the National Institutes of Health.
DNA synthesis in early niitotic stages: a pressure study. ARTHUR M. ZIMMERMAN.
Fertilized eggs of Arbacia pitnctitlata were placed into tritiated thymidine (1-2 /uC./ml.)
at early stages of the first mitotic cycle. Immediately after immersion in the isotope, the
fertilized cells were subjected to hydrostatic pressure for varying periods of time. The pres-
sure chosen, 5000 lbs/in.L>, has previously been shown to block the formation of the mitotic ap-
paratus as well as the furrowing reaction in cleaving eggs; in eggs with well-formed spindles
and asters this pressure causes drastic disorganization to the mitotic apparatus. Following
pressure treatment, some of the cells were permitted to develop and some were placed into
fixative. The paraffin-imbedded material was sectioned and subjected to autoradiography.
Alternate slides were subjected to DNase digestion prior to autoradiography. The incorporation
of tritiated thymidine was employed as an index of DNA synthesis.
When pressure treatment was initiated at presyngamy (5 minutes after insemination) and
maintained for 60 minutes, incorporation of H3-thymidine into nuclear DNA was established in
both male and female pronuclei prior to their union. Evidently, this pressure-temperature
treatment (5000 lbs/in.2 at 20° C.) blocks the union of the pronuclei, which normally occurs
within 12 minutes after insemination. The incorporation of H3-thymidine is localized in the
pronuclei. Moreover, when the pressure treatment is initiated after syngamy (15 minutes after
insemination) the eggs incorporate H3-thymidine in the zygote nucleus.
The data presented indicate that pressure as high as 5000 lbs/in.-, which may cause ex-
tensive cytoplasmic disorganization, does not block DNA synthesis. Furthermore, the
incorporation of H3-thymidine into chromosomal DNA may occur prior to or after syngamy.
Work supported by grant GM 07157-03 from the Division of General Medical Sciences,
U. S. Public Health Service.
The effects of mercaptoethanol on cleaving eggs of Arbacia pnnctulata. ARTHUR
M. ZIMMERMAN.
The fertilized eggs of Arbacia punctulata were immersed into various concentrations of
mercaptoethanol, and the structural state of the cortical cytoplasm, as well as the "cleavage
potential" of the cells were measured. Previous studies have established that mercaptoethanol
has a marked effect on the mitotic apparatus. Mercaptoethanol blocks the formation as well as
disorganizes the structural integrity of this highly complex structure.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 51(>
Pressure-centrifuge measurements of the structural state of the cortical cytoplasm were
made at various pressures (6000-12,000 lbs/in.2) at 20° C., employing a centrifugal force of
33,000 g. Immersion treatment in mercaptoethanol \vas initiated 20 minutes after insemina-
tion. After 20 minutes' incubation, the eggs were subjected to pressure-centrifugation. Two
concentrations of mercaptoethanol were employed. A blocking concentration of mercapto-
ethanol, 0.075 M , yielded a value for the strength of the cortical gel which was 22-24% lower
than that found in the non-treated controls. At a lower mercaptoethanol concentration, 0.01 M,
division was not blocked and the gel strength curve .was parallel to the curve for the blocking
concentration and the control curve, but lying intermediate between the two.
The decrease in the gel strength was shown to be related to a decrease in the "cleavage
potential." A pressure of 4500 lbs/in.2 applied at the time of furrowing will, in general, block
about 50% of the cells from cleaving. When the eggs were pretreated with 0.01 M mercapto-
ethanol 20 minutes prior to division, there was a 24% lowering in the number of cells which
completed division under pressure treatment as compared to the non-treated pressurized controls.
In general, the data support the hypothesis that interference with the SH <=^ S-S interaction
in protoplasmic gel system is similar in both the mitotic gel system and the cortical gel system
and any interference with the delicate balance may markedly disrupt mitosis and cytokinesis.
Work supported by grant GM 07157-03 from the Division of General Medical Sciences,
U. S. Public Health Service.
LALOR FELLOWSHIP REPORTS
Blood protein changes in Crustacea. HANS LAUFER AND THOMAS MCNAMARA.
The hemolymph of Uca pitc/nax and Uca pitf/ilator, decapod Crustacea, was examined by
zone electrophoresis in starch gels in intermolt, during normal molting, and after the initiation
of molting induced by eyestalk ablation. Histochemical staining of gels revealed serum pro-
teins, hemocyanins, and esterases. The purpose was to determine whether there are changes
in the patterns during molting.
About 300 Uca were studied, of which more than 50 molted in the laboratory. In one
experiment, 50% of the eyestalkless animals and 20% of the controls molted during the first
month. The protein concentration was followed over a period of five days in the same indi-
viduals after eyestalk removal. The average protein content, as measured by the Biuret
reaction, before the operation was approximately 21 mg./ml. (16 animals). The controls lost
approximately 4 mg./ml. after five days ; the experimentals showed a decrease of more than
twice this amount. Newly molted Uca (6) averaged the same low concentration as the
eyestalkless animals.
Analysis for serum proteins, esterases, and hemocyanins in starch gels revealed the fol-
lowing: Two major blood proteins are present in all control and experimental animals, with
no consistent differences between species. A third minor component was observed in some of
the experimentals. The esterases of controls are most commonly either two or three (75%).
Experimentals had fewer esterases. One hemocyanin band is detected in 70% of the control
animals, two bands in 30%. Only one hemocyanin band was displayed by 93% of the experi-
mentals. The hemocyanins, therefore, exist in two forms which often occur together in control
animals, but are rarely together in experimentals. Thus, contrary to the recent report of
Woods ct al. (1958), proteins change during molting in quantity, and in diversity. Considerable
individual differences in the changes of blood proteins during molting suggest that these
changes are influenced by additional, yet uncontrolled variables.
Supported in part by grants from the N.S.F. and the Lalor Foundation.
IntraccUitlar pH in Arbacia eggs. ROBERT W. WINTKRS.
The distribution of the weak acid 5,5-dimethyl-2,4-oxazolidinedione (DMO) has been
studied in unfertilized Arbacia eggs in order to calculate the "aggregate" intracellular pH.
This method is based on the principle that if the total concentration of DMO per unit volume
of egg water is known, "aggregate" intracellular pH can be calculated on the reasonable as-
sumption that the undissociated moiety of the compound achieves equal concentrations on both
520
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
sides of the membrane. A spectrophotometric method of analysis for DMO, based upon the
finding of an absorption peak at 208 m,u, was found to be unsatisfactory because of interfering
substances. A radioactive method was therefore used employing C14-DMO. Binding of DMO
by intracellular components was excluded in prolonged dialysis experiments using egg homog-
enates. Recovery experiments demonstrated that over 96% of added counts could be recovered
in a form showing identical behavior to DMO, with respect to partition between ether and
aqueous phases, as pH of the aqueous phase was altered. DMO in concentrations up to 4 mM
did not interfere with normal fertilization or early cleavage. The major uncertainty of the
method is the volume to be assigned to the trapped medium within the centrifuged pellet. Studies
of this volume using I131 -albumin, C14-carboxyinulin and C14-sucrose demonstrate that the
former may give artifactually high values under certain conditions. Using either of the latter
substances, the calculated intracellular pH was found to be between 6.5 and 6.8 and to be
relatively independent of wide shifts in ambient pH (4.5 to 8.5) produced by addition of strong
acid or base.
Vol. 123, No. 3 December, 1962
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
OBSERVATIONS ON BURROWING IN THE VENERIDAE
(EULAMELLIBRANCHIA)
ALAN D. ANSELLi
Department of Zoology, University of Southampton, England
Among bivalves, the conflicting requirements of maintaining contact with the
Surface for feeding, and retiring into the substratum to avoid disturbance or for
protection against enemies have led to adaptations of the foot and associated
muscles, and the siphons in a number of different ways. In shallow-burrowing
bivalves, mobility is essential, and such animals possess a large wedge-shaped
foot capable of being protruded at any angle ventrally through a large pedal gape.
Such active shallow-burrowing animals represent a relatively unspecialized type
within the bivalves, and it was probably from similar types that the more specialized
deep-burrowing forms evolved, although by many separate, convergent and
divergent routes.
The result of such evolution along separate lines may be seen in the sub-order
Veneracea : members of the Petricolidae are adapted to a greater or lesser extent
to a rock-boring habit (Purchon, 1955; Yonge, 1958). In the Glaucomyidae
mobility has been lost and the animal is embedded permanently in the substratum
at a considerable depth (Owen, 1959). The genus Vcnerupis of the Veneridae
shows affinities with both these groups and with the shallow-burrowing genera
such as Gafrarium and Venus (Ansell, 1961). The genus Dosinia has taken a
course to deeper burrowing : the shell is almost circular and lies normally with the
ligament more or less parallel to the surface, the powerful foot is protruded
ventrally and the anterior and posterior sets of retractor muscles perform equal
work in the digging process, and the flattened lunule area anterior to the umbones
possibly assists burrowing by acting as a pressure plate preventing the animal
from moving upwards as the foot is extended. This paper describes observa-
tions of the burrowing movements of some members of the Veneridae and attempts
to define a generalized time/motion pattern for this type of activity.
Apart from brief references in more general papers, the literature on the
digging movements of bivalves is scanty. Locomotion in members of the Proto-
branchia has been described by Drew (1899), Vies (1904), Morse (1913) and
Stoll (1938). Methods of locomotion in various other species have been described
1 Present address : University of Southampton, Plankton Lab., Poole Generating Station,
Rigler Road, Poole, Dorset.
521
Copyright © 1962, by the Marine Biological Laboratory
522
ALAN D. ANSELL
by Drew (1907), Jordan (1915) and Stoll (1937, 1938). but the only papers
of real significance are those of Fraenkel (1927), which gives a fairly compre-
hensive description of co-ordination of movement in the Solenidae, and of Quayle
(1949), describing movements in rencrupis (-- Paphia) puUastra.
MATERIAL AND METHODS
Recordings were made of the time sequence of movements of Venus striatula,
Venus casina, Dosinia lupinus, Venerupis decussata and Mercenaria mercenaria.
Some of these were made with the animal attached to a lever system writing on
a kymograph (Fig. 1). In others the time sequence only was recorded manually
using a stop watch to time the intervals. In the latter cases it was found possible
to continue recording even after the animal was completely buried, by observing
FIGURE 1. Kymograph records of five consecutive digging-periods for Mercenaria mercenaria.
the characteristic sequences of opening and closing of the siphonal apertures and
extension of the siphons described later. All the observations recorded here were
made with the animals burrowing in clean sand ; for Venus stria-tula, Dosinia
lupinus and Venus casinia from Kames Bay, Millport, Scotland, for Venerupis
decussata from Hamworthy Beach, Poole Harbour, England, and for Mercenaria
mcrcenaria from the shores of Fivers Island, Beaufort, North Carolina, U.S.A.
No attempt was made to use substrata from the normal habitat of the species.
No grade analyses were made but the sands were apparently of similar constitution.
THE NORMAL SERIES OF BURROWING MOVEMENTS
In describing the movements performed in burrowing in the Solenidae,
Fraenkel (1927) used the terms "Grabstufe" and "Grabperiode," English equiva-
lents of which may be given as "digging-sequence" (Quayle, 1949) and digging-
period. These two terms in the English form will be used here in their original
BURROWING IN THE VENERIDAE 523
meanings, the digging-period being the period from the start of burrowing until
the final position is reached, and the digging-sequence one of the number of
separate downward movements of which the digging-period consists.
Ouayle (1949, pp. 32-33) described the digging-sequence in Venerupis
pullastra as follows:
"(1) The valves separate.
"(2) The foot is protruded, pointed with a probing motion. This back and
forth searching motion is continued until the foot is fully extended. The tip of
the foot may extend to a length equal to that of the animal. The degree of vertical
penetration in this phase may vary considerably and is partly dependent on the
type of substratum.
"(3) The heel of the foot is protruded vertically.
"(4) The heel expands both laterally and posteriorly so that, coupled with the
anterior extension of the foot, an anchor is formed. If the substratum is firm
enough, the foot maintains this position and the shell moves.
"(5) The valves open slightly.
"(6) The two siphonal apertures close, and then the adductor muscles con-
tract, reducing the volume of the mantle cavity, the excess water being forced out
in a stream from the anterior end just below the adductor muscle. Presumably
the pallial curtain (velum) maintains a seal around the foot and the remainder
of the mantle edge during the operation. Almost immediately the anterior pedal
retractors contract and the posterior retractors relax, causing the anterior end of
the shell to dip and the posterior end to rise. It is probable, and this description
of the play of the muscles is only conjectural, that contraction of the anterior
retractors is confined to that part near its insertion into the shell.
"(7) The final movement now takes place as the shell moves forward and
down. The foot remains in its position of anchorage and the more distal portion
of the anterior retractors now comes into action and the body is drawn forward.
At the same time elements of the posterior retractors running downward and
forward to the base of the foot contract, so assisting in the movement.''
This description of the digging-sequence in Venerupis pnllastra describes
accurately the events occurring in the other members of the Veneridae studied.
A few further points may be added, however.
At all times during the sequence and for the whole of the digging-period the
tips of the siphons maintain contact with the surface of the substratum. If such
contact is lost, through withdrawal due to disturbance or from some other cause,
there is a break in the sequence of movements. During the digging-sequence the
siphons perform a characteristic series of movements. Quayle (1949) has de-
scribed the closing of the siphonal apertures, which occurs immediately before the
final downward movement of the bivalve after anchorage has been secured, and
which is associated with a jet of water being forced from the mantle cavity. After
the completion of the downward movement, the siphonal apertures reopen. A
short time later, if the shell is completely buried, the apertures close once more
and the siphons are slightly withdrawn and then "stretched." The apertures
then reopen.
The actual downward movement in each digging-sequence is brought about
524 ALAN D. ANSELL
by a complex coordination of all the muscular systems of the animal. A detailed
analysis of the role of each group has not been attempted. In general the active
downward movement is brought about by the contraction of the anterior and
posterior pairs of pedal retractor muscles, and is aided by the liquefying effect on
the substratum of the jet of water ejected from the mantle cavity. Relaxation
of the retractor muscles, as well as the changes in shape of the foot associated
with anchorage, are brought about by means of the intrinsic musculature of the
foot and visceral mass acting on a hydroskeleton provided by the blood filling
the large sinuses.
The downward movement of all members of the Veneridae examined has
associated with it a forward movement, with the result that the animal moves
obliquely downwards. This forward movement is the result of asymmetry in
the protrusion of the foot, the posterior end being anchored more or less immedi-
ately below the posterior margin of the shell by the heel, while the anterior end
of the foot is thrust forward for some distance. In those bivalves where the
protrusion of the foot is symmetrical no such forward component is present and
downwards movement is vertical. Such vertical burrowing, by an exactly similar
method to that described here, is seen in the case of some members of the
Lucinacea — the Thyasiridae and Ungulinidae — where the heel of the foot is poorly
or not at all developed (Allen, 1958).
Should the downward component on the shell be prevented from acting, the
forward component results in horizontal movements. This is the case in adult
bivalves moving on a hard substratum (Quayle, 1949, and personal observation).
The essential similarity of the muscular action involved is indicated by the rocking
movement of the shell which occurs in both cases. Essentially the same actions
are responsible for the horizontal movements of young post-larval bivalves on
hard substrata. The characteristic burrowing movements appear in the pedi-
veliger stage (Ansell, 1962), although for some time the forward movement
and the extension of the foot are aided by the beating of the strong pedal cilia.
Such ciliary-aided movement may be retained in the adult stage of some bivalves,
e.g. KellieUa (Clausen, 1958).
THE DIGGING-PERIOD
The digging-period constitutes the time between the initiation of burrowing
and the attainment of the final position of the substratum, and consists of continued
repetition of the characteristic digging-sequence. Although all digging-sequences
are similar, the time taken to complete individual sequences, within the period,
varies. The variation in time/sequence presents a characteristic appearance on
analysis. Thus, if the time/sequence is plotted graphically against the number of
that sequence in the period analysis curves of the type shown in Figures 2, 3, 4
and 5 are obtained.
Variations in the time/sequence are the result largely of variation in the time
taken for the foot to obtain anchorage in stages (2) to (4) of the digging-sequence.
In the early sequences of the digging-period this time is long in comparison to
that of later sequences. These early sequences may be regarded as comprising
an initial stabilizing period during which the movements of the foot serve to
bring the shell into a vertical position where it is supported by the surrounding
substratum.
BURROWING IN THE VENERIDAE
Further sequences of movements follow rapidly at more or less equal time
intervals. This period, during which the time/sequence remains more or less
constant, ends when the shell reaches a position where the hinge margin is level
with the surface of the substratum. Up to this time only the first siphonal move-
ments of the digging sequence have occurred. The second series of siphonal
movements described earlier are included in subsequent sequences, and these later
sequences occupy progressively longer time intervals (Fig. 2).
The time pattern of repetition of sequences during the digging period was
repeated in whole or in part by all members of the Veneridae examined. The
fullest records were obtained with those animals which burrow more deeply, where
the number of sequences making up the digging-period was greater. Deeper
burrowing is apparently achieved by quicker repetition of the characteristic
sequences and an increase in the number of movement sequences/period, and is
3
Ul
I-
B
NUMBER OF SEQUENCE IN THE DIGGING PERIOD
FIGURE 2. Analysis curves of time/sequence (mins.) for one complete digging-period for
Venerupis decussata (A). The time interval between the final downward movement and the
second extension of the siphons (see text) is also shown for each sequence (B).
associated with large size (Mercenaries) or with the possession of elongated
siphons (Dosinia). In those species such as Venus casina, with short siphons,
where the normal habit is to He near the surface of the substratum with the
posterior end of the shell exposed, the burrowing-period consists of the initial
fixation and downward movements only and ceases when the ligament margin
is more or less level with the surface of the substratum.
If the change-in-depth/sequence is analyzed in the same way, a similar
although opposite pattern appears. Depth/sequence falls off progressively as
the time/sequence increases (Fig. 3). The early fixation sequences (not well
represented in Figure 3) result in little change in the depth reached.
Analysis curves of records of the digging-periods of 17 Mercenaria inercenaria
burrowing in sand are presented in Figure 4. Although there is considerable
individual variation, the pattern for each animal is fairly constant and repeatable
while the general overall pattern is discernible in each record. The most constant
feature of comparison between repeated records from individual animals and
526
ALAN D. ANSELL
-3
I
I-
a
LJ
a
-I
-O
NUMBER OF SEQUENCE IN THE DIGGING PERIOD
FIGURE 3. Analysis curves of (A) depth/sequence (mm.) and (B) time/sequence (mins.)
for one complete digging-period for Dosinia litpinits.
z
UJ
O 10
UJ
2 o
O
o
NUMBER OF SEQUENCE IN THE DIGGING PERIOD
FIGURE 4. Analysis curves of time/sequence (mins.) for five consecutive complete-
digging-periods for each of 17 Mercenaria mercenaries.
BURROWING IN THE VENERIDAE
527
between animals is the time/sequence in the period immediately following fixation,
values for which are presented in Table I.
The total depth reached at the completion of burrowing in 84 cases from 17
Mercenaria mercenaria, together with the number of consecutive movement se-
quences/period, are also shown in Table I. The depth to which any individual
animal will repeatedly burrow is relatively constant, and this is reflected to some
extent in the number of consecutive sequences/period.
To record these observations the animals were attached to a kymograph lever
by means of a thread fastened to the midposterior region of the shell. The depth
recorded in Table I is thus the depth of this attachment point below the surface.
TABLE I
Time /sequence during the period immediately following fixation (see text), total depth
reached and the number of consecutive sequences /period for each of five consecutive
complete digging-periods for Mercenaria mercenaria burrowing in sand
Animal
No.
Length
(cm.)
Time/sequence (min.)
Total depth reached (cm.)
No. of consecutive sequences
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
A
3.76
4.0
4.0
3.5
4.0
6.0
5.0
4.1
4.2
2.4
3.6
53
33
38
18
20
B
3.43
13.0
5.5
4.5
4.5
4.0
7.2
3.1
3.2
3.3
3.0
20
10
10
8
11
C
3.66
5.0
5.0
5.0
6.0
6.5
5.0
3.4
3.6
4.0
3.4
19
16
14
22
10
D
3.46
4.5
5.5
6.5
5.5
5.0
2.8
2.4
2.6
3.3
3.0
24
13
15
22
18
E
3.56
6.0
6.5
5.5
6.5
8.5
1.4
2.3
2.4
2.2
3.8
17
22
16
24
27
F
3.54
5.0
5.5
6.0
8.5
—
3.1
2.3
2.2
2.2
—
45
24
19
18
— •
G
3.54
7.5
6.5
6.0
5.0
5.5
3.3
3.3
3.1
3.3
3.4
15
14
13
14
17
H
6.55
3.0
3.0
3.0
3.5
3.0
5.4
4.9
4.2
5.3
4.4
45
26
22
41
24
I
6.55
5.0
5.0
5.5
6.5
6.0
3.5
3.9
4.1
3.6
3.9
37
36
33
28
32
J
6.30
3.5
4.5
5.0
5.0
6.0
9.2
8.6
6.3
6.5
7.6
62
60
22
38
64
K
6.34
4.0
3.5
4.0
5.0
5.0
4.3
4.0
4.0
3.2
3.9
25
18
16
15
17
L
5.84
4.5
5.0
6.0
7.5
8.5
5.9
5.8
6.8
5.5
5.4
46
47
47
32
46
M
9.37
5.5
7.0
8.0
8.0
5.0
3.7
3.6
3.7
3.4
3.7
32
24
22
28
25
N
9.62
6.5
6.5
5.0
6.5
5.5
5.1
6.6
7.8
7.6
3.4
19
30
29
30
26
O
9.58
5.5
5.5
7.0
6.0
6.0
3.1
3.5
2.7
4.9
5.2
41
35
50
34
49
P
9.65
6.0
5.0
5.5
6.0
6.5
7.0
6.2
5.8
5.0
5.0
48
32
25
29
28
Q
8.50
4.5
4.5
5.0
4.5
5.5
4.6
3.9
4.4
3.6
3.7
22
20
18
19
20
At the completion of burrowing, most of the animals were orientated with the hinge
margin more or less parallel to the surface of the substratum, and were completely
buried.
In animals attempting to burrow in a hard substratum the time, 'sequence is
irregular since the foot does not readily secure anchorage. A typical example of
an analysis curve under such circumstances is shown for Dosinia lupinus in
Figure 5. Also in Figure 5 is shown an analysis of the digging-period of the
same animal in deep sand and in shallow sand over a hard bottom. The change
in time/sequence as the animal reaches hard substratum from soft is readily seen.
DISCUSSION
The observations made here extend those of other workers and confirm that
there is a basic sequence of movements involved in burrowing in bivalves. This
528
ALAN D. ANSELL
basic sequence is common to all the Veneridae examined, and is similar to that
described by Fraenkel (1927) for the Solenidae. Personal observation and scat-
tered references in the literature suggest also that the movements in other groups
conform to this pattern.
The characteristic pattern of time/sequence and depth/sequence may arise
from the intrinsic nervous mechanism controlling the burrowing process, or from
extrinsic environmental factors. These alternatives may be briefly discussed
although further work is needed before the significance of this pattern can be
fully appreciated.
5-
4-
3-
LJ
5
I- 2-
2-
B
NUMBER OF SEQUENCE IN THE DIGGING PERIOD
FIGURE 5. Analysis curves of time/sequence (mins.) for complete digging-periods for
Dosinin lupinus (A) in sand, (B) in shallow sand over glass, and (C) on glass.
The gradual increase in the time/sequence and the decrease in change-of-
depth/sequence noted may arise from a gradual increase in resistance to burrowing
experienced as the animal moves deeper into the substratum. Since the greater
time/sequence involves mainly an increase in the time for the foot to obtain
anchorage in stages 2-4 this increase would suggest that penetration of the foot
into the substratum becomes progressively more difficult, while the decrease in
depth/sequence suggests that the substratum offers increasing resistance to
penetration of the shell.
As an alternative explanation we may consider the possibility that the changes
BURROWING IN THE VENERIDAE 529
in time/sequence and depth, 'sequence indicate a change in the intensity of the
burrowing response, i.e., the digging-sequence, rather than the effect of a changing
environment on a constant response. The observed changes would then indicate a
gradual weakening of the response throughout the digging-period. Such a
weakening could result from a gradual lessening of intensity of the burrowing
stimulus, or alternatively an increase in some inhibitory stimulus opposing the
response to the original stimulus which initiated digging. Such an explanation
suggests a possible mechanism by which the burrowing response in bivalves is
controlled.
In the Solenidae, burrowing may be initiated by stimulation of the siphons
or mantle edge and may be regarded as an escape reaction, the animal retiring
into its burrow in response to danger. In contrast, the escape reaction of most
eulamellibranchs, including the Veneridae, is withdrawal of the siphons followed
by shell closure. Burrowing occurs normally in response to disturbance; an
animal removed from the substratum will, if covered with water, attempt to
re-burrow. The stimulus which results in this reaction is disturbance, and pos-
sibly also exposure of the siphons and mantle edge. Animals which have been
kept for some time in laboratory tanks or aquaria without a soft substratum lose
this response, although they may make weak attempts to burrow at irregular
intervals and often respond vigorously to a rapid change of water by extending the
foot and attempting to burrow.
The cause of cessation of burrowing has not been found. Fatigue has been
suggested (Fraenkel, 1927) but does not appear likely, since animals may be
made to burrow repeatedly by removing them from the substratum immediately
on completion of the digging-period.
The observation that the siphons maintain contact with the surface of the
substratum throughout the digging-period and that extension of the siphons follows
each downward movement suggests that burrowing may cease in response to
stimuli originating from the siphons. Such a stimulus to be effective must give
information on the state of extension of the siphons and hence involve stretch
receptors or other proprioceptors. A gradual extension of the siphons during
the digging-period might then result in a gradual buildup of stimuli from such
receptors acting as a progressive inhibition to the burrowing response. In the
Veneridae such proprioceptors, if they occur, are presumably distributed through-
out the siphonal walls. In other and more active bivalves it is possible that they
might form recognizable sense organs. Thus, for the Tellinacea, Yonge (1949)
has suggested that the sense organ associated with the cruciform muscle may
serve to give information on the state of extension of the siphons. It would be of
interest to observe the digging-periods of members of this and other groups in
more detail.
Thanks are extended to Dr. C. H. Mortimer for the use of facilities at The
Marine Station, Millport, Isle of Cumbrae, Scotland, where some of the observa-
tions recorded here were made, and to Dr. G. Talbot, the Director ; Dr. T. R.
Rice, and other members of the staff of the Beaufort Laboratory of the United
States Fish and Wildlife Service. The work was completed while the author was
in receipt of the John Murray Travelling Studentship of the Royal Society.
530 ALAN D. ANSELL
LITERATURE CITED
ALLEN, J. A., 1958. On the basic form and adaptations to habitat in the Lucinacea (Eulamelli-
branchia). Phil. Trans. Roy. Soc. London, Ser. B, 241: 421-481.
ANSELL, A. D., 1961. The functional morphology of the British species of Veneracea (Eulamelli-
branchia). /. Mar. Biol. Assoc., 41: 489-515.
ANSELL, A. D., 1962. The functional morphology of the larva, and the post-larval development
of Venus striatula (da Costa). /. Mar. Biol. Assoc., 42: 419-443.
CLAUSEN, C., 1958. On the anatomy and histology of the Eulamellibranch Kclliella miliaris
(Philippi) with observations on the ciliary mechanisms in the mantle cavity. N\ft.
Mag. Zool, 6: 144-175.
DREW, G. A., 1899. Some observations on the habits, anatomy and embryology of the Proto-
branchia. Anat. Anz., 15: 493-519.
DREW, G. A., 1907. The habits and movements of the razor-shell clam, Ensis dircctus. Biol.
Bull., 12: 127-140.
FRAENKEL, G. V., 1927. Die Grabbewegung der Soleniden. Zcitschr. vcrgl. Physiol., 6: 167-220.
JORDAN, H., 1915. Liber die Art, wie Mactra inflata sich in den Sand einwiihlt. Zool. Jahrb.,
Abt. Zool. Physiol, 35: 289-300.
MORSE, E. S., 1913. Observations on living Solenomya. Biol. Bull., 25: 261-281.
OWEN, G., 1959. Observations on the Solenacea with reasons for excluding the family Glau-
comyidae. Phil. Trans. Roy. Soc. London, Scr. B, 242: 59-97.
PURCHON, R. D., 1955. The functional morphology of the rock boring lamellibranch, Pctricola
pholadiforinis (Lam.). J. Mar. Biol. Assoc., 34: 257-278.
QUAYLE, D. B., 1949. Movements in Vencrupis ( — Paphia) pitllastra (Montagu). Proc. Malac.
Soc. London, 28: 31-37.
STOLL, E., 1937. Beobachtungen iiber die Fortbewegung bie einigen grabenden Muscheln.
Rev. Suisse Zool., 44: 383-390.
STOLL, E., 1938. Sur le mode de locomotion de quelques mollusques marins. Trav. Sta. Biol.
Roscoff, 16: 5-29.
VLES, F., 1904. Locomotion de la Nucule. Bull. Soc. Zool. France, 29: 191-196.
YONGE, C. M., 1949. On the structure and adaptations of the Tellinacea, deposit-feeding
Eulamellibranchia. Phil. Trans. Roy. Soc. London, Ser. B, 234: 29-76.
YONGE, C. M., 1958. Observations on Pctricola carditoides (Conrad). Proc. Malac. Soc.
London, 33: 25-31.
^
THE VITELLIXE COAT OF THE MYTILUS EGG. I. NORMAL
STRUCTURE AND EFFECT OF ACROSOMAL LYSIN
JEAN C. DAN
Ochanoinizn University. Tokyo, and The Misuki Marine Bi/ilouical Station . Miura-Slu. Japan
The first study of a lysin extractable from Mytilus spermatozoa was made by
Berg (1950), who distinguished two types of lytic activity : a "membrane-dissolving"
action, which was described as attacking an outer layer of the egg surface made
microscopically visible by plasmolyzing the eggs, and a "cement-dissolving" activity,
which causes the first two blastomeres to become partially or completely separated.
Wada, Collier and Dan (1956) showed that the lysin in question is a component
of the intact acrosome which is released into the medium when the acrosome is
induced to react.
Col win and Col win ( 1960a, b) have also investigated the effect of a lysin extracted
from the spermatozoa of Hydroides hexagonus on the egg of this species. Using
thin sections and electron microscopy, they found that the lysin dissolves the middle,
and major, component of the thick vitelline coat investing the cytoplasmic surface,
although it appears not to affect the outer and inner borders of this envelope.
A report concerning the structure of the Mytilus cdulis ''egg membrane" has
recently been published by Mancuso (1960). This author used a fixing solution
which included formalin, acetone, acetic acid and sometimes chromic acid, as well as
osmium tetroxide ; the images observed in the electron microscope after this fixation
led him to certain conclusions which differ considerably from those reached in this
study. None of these differences, however, is so radical that it cannot be attributed
to the effect of the fixative.
The present investigation was undertaken to observe the fine structure of the
Mytilus egg surface and determine in detail how the acrosomal lysin affects it after
fertilization, particularly in connection with the role of the vitelline coat in controlling
the pattern of the first cleavage, and the shape and mutual relations of the first
two blastomeres.
MATERIAL AND METHODS
Mytilus cdulis from the Tokyo area is readily induced to spawn by keeping
freshly collected animals dry in a refrigerator for several hours and then placing
them in sea water at room temperature (20-23°), or by raising the temperature of
the running sea water about 5° (to 18-20°) and administering an electrical stimulus,
according to the method of Iwata (1949). Stimulated animals are returned to sea
water in separate containers, and males which have begun to shed are stood, broad
end downward, in a dry beaker to obtain concentrated sperm suspensions.
Pooled sperm from several males was used as the source of the acrosomal lysin.
If 1 ml. of 0.36 M CaCL is added to 9 ml. of rather concentrated sperm suspension,
most of the spermatozoa undergo a reaction of the acrosome (see Wada ct a/., 1956).
531
532
JEAN C. DAN
The sperm cells were removed by 10 minutes' centrifugation at 12,000 g (0°), and
the clear supernatant was dialyzed against running sea water and used as the lysin.
This solution retains its lytic activity indefinitely if it is kept frozen.
B
H
FIGURE 1. Camera lucida drawings of Mytili/s cditlis eggs cleaving under various conditions.
A-C : in sea water ; D-F : suspended in strong solution of acrosomal lysin ; G, H : membrane
removed by lysin and eggs transferred to calcium-free sea water. A, D : shortly before cleavage ;
B, E, G : mid-cleavage trefoil stage ; C, F, H : interphase between first and second cleavages.
Microvilli are visible with phase contrast as striated "halo."
All fixation was done at room temperature with 1 % OsO4 in sea water. The
egg suspensions were fixed for 30 minutes, washed and post-fixed in 5% formalin-sea
water for several hours, embedded in methacrylate, sectioned with a Porter-Blum
microtome and observed with a JEM-5G electron microscope.
VITELLINE COAT OF MYTILUS EGG 533
RESULTS
Lii'hu/
The unfertilized J\I \tilus egg is irregularly oval, and is surrounded by a conspicu-
ous hyaline zone about 1 p. thick, which is referred to as the vitelline membrane in
Field's original description (1921-1922). Outside of this "membrane" is a rather
thin (7-10 p.) layer of transparent material ("jelly") which can most easily be
detected by adding india ink to the egg suspension (see Wada et a!., 1956; Fig. 6).
On being fertilized, the egg immediately becomes spherical (diameter about 63 /u, ) .
but no change can be observed in the surface layers except that by the time of the
first cleavage, the thickness of the vitelline coat appears to increase slightly.
\Yhen the polar bodies are extruded, they lie under the vitelline coat, flattened
against the cytoplasmic surface (Fig. 1A). At the first cleavage the egg forms a
polar lobe ; as cleavage proceeds, this lobe is compressed against the opposite, AB,
blastomere by the tension exerted by the vitelline coat (Fig. IB). Once cleavage
is complete, the polar lobe material flows back into the CD cell, and the two
blastomeres become closely apposed during the succeeding interphase (Fig. 1C ).
If an egg is treated with acrosomal lysin 10 minutes after fertilization and contin-
uously observed with phase contrast, it is seen that the vitelline coat loses first its
sharp outline and then its hyaline refringency, and finally gives place to a layer of
fine processes which cover the whole surface of the cell. These processes are
clearly longer at the vegetal side of the egg, and also in a restricted area at the animal
pole (Fig. ID).
The first polar body bulges out freely as it is formed, and drifts away from the
egg if the preparation is jarred. The second polar body remains attached to the egg
surface, the first polar body usually dividing as the second is formed. At cleavage,
the polar lobe extends out at right angles to the mitotic spindle ( Fig. IE); the
connection between polar lobe and CD blastomere is narrower than normal, and in
very strong lysin or when the eggs are transferred to calcium-free sea water after
strong lysin, the connection is often severed (Fig. 1G). As Berg has reported
(1950), the two resting blastomeres tend to be more spherical than those of the
controls, especially after extended exposure to strong lysin, although a considerable
degree of contact is more common than complete separation (Fig. IF, H) (see also
Berg, 1950; PL 1, c, d; Wada ct al. Fig. 7).
Electron microscopy
Xonnal egg surface. Thin sections of the unfertilized AI \tilns egg (Fig. 2")
show that its surface is similar to that of the egg of another bivalve mollusc, Spisula,
according to an electron micrograph by Rebhun (Allen, 1958). The cytoplasmic
surface is extended into fine microvilli of a relatively uniform size and regular
distribution, 0.7-1 p, in length, and usually straight, although two may be connected
at their bases to give an effect of branching. These microvilli extend into and
through a rather dense layer, about 0.5 p. thick, of homogeneous material of the sort
described by the Colwins as "felt-like," which is obviously the hyaline component of
the vitelline coat as observed with light microscopy. The tips of the microvilli
protrude slightly beyond the outer surface of this layer; its conspicuously smooth
inner surface is separated by a definite perivitelline space from the outer border of
534
JEAN C. DAN
FIGURES 2-3.
VITELLINE COAT OF MYTILUS EGG 535
the cytoplasmic mass between the bases of the microvilli. From the tips of the
microvilli numerous extremely fine fibrils extend outward, constituting at least one
component of the so-called jelly layer.
As can be seen in Figures 2, 3 and 8, no formation which could be described as
a "membrane" lies outside this layer of hyaline material, although the micrographs
show regions of greater absorption, as at the right of Figure 2, which suggest that
the surface of the vitelline coat is somewhat denser than its interior. A similar but
more pronounced condensation is apparent in Rebhun's micrograph of the Spisulu
egg, and Mancuso's figures of the Mvtilus egg show most of the hyaline substances
concentrated into two layers, corresponding to the inner and outer surfaces of what
appears in this study as a nearly homogeneous matrix.
The cytoplasm is bounded by a plasma membrane, which is continuous with the
walls of the microvilli. Beneath this is a region of cortical cytoplasm 1-1.5 p- thick,
generally free from yolk granules but containing conspicuous spherical membranes
which in Mancuso's micrographs includes a substance having an electron absorbancy
somewhat greater than that of the yolk.
Fertilization induces no changes in any of these structures that can be detected
in the electron micrographs (Fig. 3). In eggs fixed between 20 and 70 minutes
after fertilization, however, it is found that on parts of the egg surface, the bases of
adjacent microvilli have united, while the wider spaces between these villous trunks
have also deepened. The microvilli thus come to present an overall appearance of
branching, but careful observation shows that their dimensions and arrangement
within the vitelline coat are the same as those of the unfertilized eggs ; what has
changed is the intervillous cytoplasmic surface. This new state of affairs can be
observed in the untreated living egg as a thickening of the vitelline coat, as mentioned
above, and the greater length in the polar regions of the processes observed after
lysis of the hyaline layer material is due to an extreme expression of this tendency, in
all probability connected with the special role of these areas in polar lobe and polar
body formation.
Lysin-treated eggs. Sections of eggs fixed after having been exposed to lysin
for one minute (Fig. 4) show that the hyaline material of the vitelline coat has been
evenly attacked by the lysin — i.e., dissolution of the material has taken place rather
uniformly throughout the layer. After an exposure of two minutes (Fig. 5), the
material is virtually all dissolved, except for some vague remnants of it left clinging
to the microvilli ; the latter remain exposed as straight, unbranched processes,
continuous with the main body of the cytoplasm and apparently unaffected by the
lysin. The fine fibrils of the jelly layer are also intact (cf. Wada ct al., 1956;
Fig. 7 ) , and with the hyaline substance removed, it can be seen that there are short
fibrils of the same kind projecting from the sides of the microvilli.
When eggs are exposed to the lytic solution for 10 minutes (Fig. 6), the hyaline
material is completely dissolved, whereas the microvilli and the fine fibrils of the jelly
are quite unaffected. The portion of the egg surface appearing in Figure 6 is
FIGURE 2. Surface of unfertilized Mytilus egg, showing vitelline coat consisting of hyaline
material supported by microvilli. X 16,000.
FIGURE 3. Surface of fertilized Hfytilus egg fixed 10 minutes after insemination. Note fine
fibrils of "jelly layer" and empty membranes of cortical granules which have been extracted
during preparation (see text). < 16,000.
536
JEAN C. DAN
FIGURES 4-6.
VITELLINE COAT OF MYTILUS EGG 537
apparently from the vegetal region, since it represents an extreme case of the
"branching" effect.
Further exposure to the lytic activity, up to 60 minutes, still leaves the microvilli
and their fibrils unaffected (Fig. 7). The cytoplasmic protuberances carrying the
microvilli in this section are coarser than those shown in Figure 6 ; it is not clear
whether this represents a topographical characteristic or is the result of exposing the
cytoplasmic surface without its supporting coat for a long period.
Lysin pins calciiuii-jree sea water. To investigate the effect of lack of calcium
on these surface structures, fertilized eggs were transferred to calcium-free artificial
sea water 1 10 minutes after insemination, as controls for another lot of fertilized eggs
which were first exposed to lysin for 10 minutes and then washed with calcium-free
sea water and left in it for 50 minutes (fixation at 70 minutes after insemination,
shortly before beginning of first cleavage).
The calcium-free controls (Fig. 8) show no differences from the sea water
controls, indicating that the integrity of the vitelline coat in these eggs is not
dependent on the presence of calcium in the medium. The fibrils of the jelly layer
are also found intact, both in the controls and in the sample of eggs exposed to lysin
followed by calcium-free sea water (Fig. 9), and no special effect of the lack of
calcium on the denuded cytoplasmic surface can be observed.
DISCUSSION
The general structure of the Mytilus egg surface, as seen at high magnification,
bears a surprisingly close resemblance to the surface complex of the fertilized sea
urchin egg, in which secondarily-formed microvilli extend into and attach the egg
surface to a hyaline layer similarly consisting of a homogeneous material (Endo,
1961 ). In both these systems, the overlying layer of the hyaline substance supports
the cytoplasmic surface and controls the shape of the embryo as it develops through
the cleavage stages.
Experiments of the sort first performed by A. R. Moore (1940), showing that
sucrose freely penetrates the hyaline layer of echinoderm embryos during the cleav-
age stages, furnish evidence that the osmotic properties of this layer are less exclusive
than those of cytoplasmic membranes. On the other hand, the fact that the width
of the echinoderm hyaline layer is observed to increase just before each of the
early cleavages (Dan, 1952) indicates that some osmotically active substances are
retained within it. The observation presented in this study, that the M \tilns egg
surface proper becomes indented so that the microvilli come to project from the
summits of thicker cytoplasmic protuberances, suggests that a similar osmotic process
is at work in these eggs, causing an increase in the volume of the perivitelline fluid.
In view of the extent to which the dividing egg departs from the spherical shape,
especially as it forms and retracts the polar lobes, the intervention of some such
FIGURE 4. Surface of fertilized Mytilus egg fixed after exposure of one minute to strong
acrosomal lysin. Hyaline material partly dissolved. X 24,000.
FIGURE 5. Hyaline material almost completely dissolved after two-minute exposure to lysin.
Note that microvilli and fibrils of jelly layer are unaffected. X 24,000.
FIGURE 6. Vegetal surface of fertilized Mytilus egg after 10-minute exposure to lysin.
Hyaline material completely removed; microvilli and fibrils unaffected. X 16,000.
1 Dan's (1954) "chloride mixture No. I."
538
JEAN C. DAN
i ' <**
FIGURES 7-9.
VITELLINE COAT OF MYTILUS EGG 539
device to reduce the restraining effect of the vitelline coat on the cytoplasmic surface
would seem to be an essential prerequisite for cleavage.
That the tensile properties of the intact Mytilus egg surface are chiefly due to
the hyaline material of its vitelline coat is suggested by the separation of the polar
bodies and the considerable change in the configuration of the first cleavage following
lysis of this layer (in Figure 1, compare D and E with A and B). On the other
hand, the vitelline coat must be capable not only of expanding to some extent, but
also of being contracted to a comparable extent, since the formation of the polar
lobes involves an increase in surface area, while their retraction causes it to decrease.
When the eggs are not in the best condition, wrinkling of the vitelline coat, or its
complete separation from the plasma membrane as a large blister at the vegetal pole,
attests to the failure of such contraction.
It is clear that the activity of the lysin derived from the sperm acrosome is
specifically directed against the hyaline material of the vitelline coat, and has no
effect, even after 60 minutes, on the plasma membrane. Comparing Figures 6 and 7,
which both show areas of the vegetal surface, it at first appears as though prolonged
exposure to the lysin has weakened the egg surface so that the slender processes
supporting the clusters of microvilli in Figure 6 spread out into the thick, poorly
organized protuberances seen in Figure 7. It is necessary to consider, however,
that during this period the polar lobes associated with first and second polar body
formation have caused the expansion of this surface in the absence of the vitelline
coat, which would normally have held the distal parts of the microvilli in a fixed
arrangement. It therefore seems probable, especially since the microvilli remain
unchanged even after prolonged exposure, that the observed effect is secondarily
produced by the absence of the supporting layer, rather than primarily, by some
action of the lysin on the cytoplasmic surface.
The long microfibrils which arise from the tips of the microvilli and constitute
what has been thought of as the jelly layer are interesting because of their resistance
to the dissolving actions of lysin and of calcium-free sea water, and because they are
fixed by osmium. The two latter characteristics set them in contrast to the
mucopolysaccharide jelly of the sea urchin egg, and suggest that the zone around the
Mytilus egg consisting of these massed fibrils should not be thought of in the same
terms unless evidence can be found to indicate the presence of a more labile
component.
The result of the present investigation supports the doubt wrhich was expressed in
the earlier study (Wada et a!., 1956) concerning Berg's (1950) suggestion that the
AB and CD blastomeres are held together by a cementing substance, presumably
secreted in the furrow region of the cleaving egg. It seems evident that it is rather
the restraint exerted by the encircling vitelline coat which presses the blastomeres
against each other in normal cleavage. That this is not the whole explanation,
FIGURE 7. Surface of Mytilus egg after 60-minute exposure to strong acrosomal lysin.
Microvilli and fibrils still unaffected ; structure of cytoplasmic surface somewhat modified as
result of prolonged absence of supporting layer of hyaline material. X 16,000.
FIGURE 8. Vitelline coat of fertilized Mytilus egg transferred to calcium-free sea water 10
minutes after insemination; fixed at 70 minutes. Note that hyaline material and fibrils of jelly
layer are both resistant to lack of calcium. X 16,000.
FIGURE 9. Surface of fertilized Mytilus egg exposed for 10 minutes to strong lysin, washed
in calcium-free sea water and left in this medium until just before first cleavage ; fixed 70 minutes.
X 16,000.
540 JEAN C. DAN
however, is shown by the observation that even when the hyaline component of
this layer has been dissolved, the blastomeres preserve a considerable degree of
mutual contact (Fig. IF) unless cleavage takes place in calcium-free sea water
(Fig. 1G, H).
If an analogy may be drawn between these cells and sea urchin blastomeres, which
also normally have their outer surfaces attached by cytoplasmic processes to a hyaline
layer (Dan and Ono, 1952), the extreme sphericity of the Mytilns blastomeres in
calcium-free sea water after removal of their vitelline coat can be explained as an
abnormal equalization of the post-cleavage membrane tension involving the whole
weakened (by the absence of calcium) surfaces of the blastomeres, instead of the
usual localization of such stretching in the furrow region (Dan, 1954).
Since electron microscopy shows that the outermost covering of the Mytilus egg
is a single layer of what can be called a cementing substance or matrix material
supporting and fixing in regular arrangement a brush of microvilli, rather than any
structure which conforms with the usual concept of "membrane," it appears that
the two lytic activities suggested by Berg ( membrane- ly tic and cement-lytic) would
be better described as degrees of effectiveness of a single activity, combined with
secondary effects of variable experimental conditions such as the degree of calcium
deficiency and the length of the period during which the cytoplasmic surface is
without its supporting layer.
The author acknowledges with gratitude the cooperation of Mr. A. Kitahara of
the Tokyo Institute of Technology, who performed the electron microscopy.
SUMMARY
1. Electron microscopy shows the egg of M\tilus cdulis to be surrounded by a
vitelline coat consisting of a layer about 0.5 p. thick, which corresponds to the
refringent hyaline zone seen with the light microscope. This layer has a smooth
inner surface, separated from the cytoplasmic surface proper by a space about 0.2 /j,
wide. The plasma membrane forms a brush of regularly arranged, straight micro-
villi 0.7-1 //, in length. These pass through and protrude slightly beyond the outer
surface of the hyaline material, where their tips give rise to numerous extremely
delicate fibrils which constitute at least one component of the "jelly layer." Fertili-
zation does not cause any visible changes in these structures of the egg surface.
2. Exposure of a fertilized egg for one minute to a strong solution of acrosomal
lysin causes an evident dissolution of the hyaline substance, and a two-minute
exposure removes it almost completely, leaving the microvilli exposed but otherwise
unaffected. The fibrils of the jelly layer also resist the lytic action. Exposure to
lysin for 60 minutes induces no further changes in these structures.
3. It is concluded that the acrosomal lysin is specific for the single substance
constituting the hyaline portion of the vitelline coat, and that the layer composed of
this material is chiefly responsible for the configuration of the cleaving egg and the
close contact of the blastomeres after cleavage.
LITERATURE CITED
ALLEN, R. D., 1958. The Initiation of Development. In: A Symposium on the Chemical Basis
of Development, ed. by W. McElroy and B. Glass, Johns Hopkins University Press,
Baltimore, Md., pp. 17-67.
VITELLINE COAT OF MYTILUS EGG 541
BERG, W. E., 1950. Lytic effects of sperm extracts on the eggs of Mvtilus cdulis. Biol. Bull.,
98: 128-138.
COLWIN, A. L., AND L. H. COLWIN, 1960a. Egg membrane lytic activity of sperm extract and its
significance in relation to sperm entry in Hvdrtndcs hc.nu/onus (Annelida). /. B'n>ph\s.
Biochcm. Cytol, 7: 321-328.
COLWIN, L. H., AND A. L. COLWIN, 1960b. Formation of sperm entry holes in the vitelline
membrane of H\droides hcxagonus (Annelida) and evidence of their lytic origin.
/. Biophys. Biochcm. Cytol., 7: 315-320.
DAN, K., 1952. Cyto-embryological studies of sea urchins. II. Blastula stage. Biol. Bull.,
102: 74-89.
DAN, K., 1954. Further study on the formation of the "new membrane" in the eggs of the sea
urchin, Hemieentrotus (Strongylocentrotus) pulcherrimus. Embryologia, 2: 99-114.
DAN, K., AND T. ONO, 1952. Cyto-embryological studies of sea urchins. I. The means of
fixation of the mutual positions among the blastomeres of sea urchin larvae. Biol. Bui/.,
102: 58-73.
ENDO, Y., 1961. Changes in the cortical layer of sea urchin eggs at fertilization as studied with
the electron microscope. I. Clypcastcr japonicus. Exp. Cell Res., 25: 383-397.
FIELD, I. A., 1921-22. Biology and economic value of the sea mussel, Mytilus cdulis. Bull. U. S.
Bur. Fish., 38: 127-259.
IWATA, K. S., 1949. Spawning of J\I\<tilus cdulis. II. Discharge by electrical stimulation.
Bull. Japan Soc. Sci. Fish., 15: 443-446.
MANCUSO, V., 1960. La membrana ovulare di "Mytilus cdulis" studiata al microscopio elet-
tronico. Rend. Inst. Suf>. Sanita — Ronni, 23: 793-796.
MOORE, A. R., 1940. Osmotic and structural properties of the blastular wall in Dcndnistcr
cxccntricus. J. Exp. Zoo/., 84: 73-83.
WADA, S. K., J. R. COLLIER AND J. C. DAN, 1956. Studies on the acrosome. V. An egg-
membrane lysin from the acrosomes of M \tilns cdulis spermatozoa. Exp. Cell Res..
10: 168-180.
MIGRATORY RESTLESSNESS IN CAGED BOBOLINKS
(DOLICHONYX ORYZIVORUS, A TRANS-
EQUATORIAL MIGRANT)1
WILLIAM L. ENGELS
Department nf Zooluyy, University of North Carolina, Chapel Hill, N. C.
It has been known for a very long time that caged migratory birds become
increasingly restless at the onset of the migratory season. This restlessness is
expressed especially by fluttering and hopping on the perches after dark when,
ordinarily, caged birds sit quietly. Most investigators have assumed, at least
tacitly, that the development of such nocturnal restlessness reflects the develop-
ment of a disposition to migrate. It is frequently referred to as Zugunruhe
(migratory unrest). Thus equated with migratory behavior, it could be a useful
indicator in various aspects of the experimental study of migration (e.g., regulation,
physiology, navigation).
In the past three decades there have been a number of studies on the induction
and regulation of this restlessness with respect to such factors as temperature,
food, sex hormones, and photoperiod. Although one may doubt in some cases
that the restlessness truly reflected a migratory state, there is clear evidence for
the photoperiodic induction of migratory behavior in at least a few temperate
zone migrants (e.g.. King, 1961, Zonotrichia leucophrys ganibclii). Transequa-
torial migrants have received little attention although obviously they are of special
interest in relation to photoperiodism. Each of their two annual migrations,
northward and southward, begins during the declining day-lengths of late summer
or early autumn in one hemisphere and ends during the increasingly longer days
of middle or late spring in the other. In his studies on star-navigation by birds
Sauer (1957) has employed restlessness in the European- African transequatorial
migrant Sylvia borin to determine directional orientation. Recently Hamilton
(1962) demonstrated orientation under clear skies in the nocturnal restlessness
of caged bobolinks during both spring and fall migratory periods. Neither of
these was concerned with the induction or regulation of the restlessness per sc,
although an intrinsic rhythm seems to be implied for S. borin (Sauer and Sauer,
1955).
It has already been shown that the annual testicular cycle of caged bobolinks
(Dolichony.Y orvzivorus} is under photoperiodic control (Engels, 1959, 1961).
This species breeds in North America above Lat. 40° N., reaching the nesting area
in late May or very early June after a migration, begun in late March or early
April, from a "wintering" area in South America lying roughly between Lat. 10°
and 30° S. It is then of some interest to determine if caged bobolinks display a
seasonal nocturnal restlessness which may be related to migratory behavior and
controlled or influenced in any way by day-length.
1 This study was supported by a grant (G-6163) from the National Science Foundation.
542
MIGRATORY UNREST IN BOBOLINKS 543
MATERIALS AND METHODS
The bobolinks used in these experiments were captured in Xorth Carolina
at about Lat. 36° N., some in May, others in September, thus near the end of
the northward or shortly after the beginning of the southward migration. These
are referred to hereafter as "spring captures" and "autumn captures," respectively.
Some individuals wrere used as experimental animals in successive years ; after
the first year these are designated "2nd year experimentals." Each such bird had
spent at least six months (June through November) in an outdoor aviary
exposed to natural day-lengths before being used in another experiment.
Over a three-year period four groups of birds were used, confined in outdoor
aviaries exposed to normal outside air temperatures. Of these, two groups
experienced only natural day-lengths (Lat. 36° N.), while two were exposed to
constant 14-hour photoperiods beginning in November. The birds were con-
fined individually in small cages placed on a shelf in the sheltered rear part of
the otherwise open aviaries.
The cages used were "Hendryx finch-breeder" cages measuring about
8 X 9 X 16 inches. Each was fitted with two perches which pivoted at one end
on a horizontal metal rod (% inch diameter) placed about three inches outside
the rear of the cage and attached to it at each end by a frame. At the front each
perch rested on a microswitch attached to the cage. Closure of either switch,
resulting from a depression of the perch as the bird hopped on it, actuated an
electronic counting device,2 each closure advancing a four-digit counter by one.
Every 15 minutes the accumulated count automatically was printed on a clock-
motor-driven, chronologically marked tape, and the counter then returned to zero.
To each cage were attached a food-hopper and two 100-cc. water tubes which
provided at one filling sufficient food and water to supply a bobolink of normal
behavior for more than a week. A small amount of soluble terramycin was added
to the drinking water. At first a chick laying-mash (Purina Layena) was used
as food; we later changed to a mash prepared for game birds (also Purina). The
pan of the cage was covered with a layer of finely crushed granite.
The birds were left undisturbed except for periodic handling for weighing
and inspection of the plumage. This was done usually once a week. On these
occasions the cages and water tubes were cleaned, fresh water and clean granite
supplied and the food-hopper refilled. Body weight was determined on a balance
reading to the nearest 0.01 gram and recorded to the nearest 0.1 gram.
An enormous amount of raw data on perch hopping activity was accumulated
over the three seasons of observation. To reduce this to manageable and mean-
ingful figures, three indices were used, as follows :
Index A: This is simply the number of quarter-hour periods during the night
in which one or more perch movements were recorded. (This is similar to the
index used by Weise, 1956, but his interval was a more satisfactory 0.1 hour).
Such periods may be designated as active night periods. Selection of the quarter-
hour as the basic interval was dictated by the recording device, which automatically
printed out the number of perch movements every 15 minutes. Night here means
the total dark period for birds exposed to artificial illumination, or, for birds
exposed to natural photoperiods, the interval between the end of evening civil
- "Tally- Print," Model AR, Standard Instrument Corp., New York.
544
WILLIAM L. ENGELS
twilight and the beginning of morning civil twilight. Obviously, one hop per
quarter-hour is hardly indicative of restlessness, but the index has usefulness and
value when combined with the next index ( B ) , as giving some information on
the possible maximum duration of unrest during the night.
Indc.\- B: This is the average number of perch movements (hops) recorded
per active night period and thus is an expansion of Index A.
Index C: This is the total number of perch movements (hops) recorded
during the night, the product of the first two indices.
I would like to acknowledge, with thanks, the technical assistance of Donald
E. Kent, especially in the design and construction of the perch-microswitch
arrangements and in maintenance of the electronic recorders, and the help of
Catherine Henley in the preparation of the manuscript. The figures were prepared
by Alary Scroggs.
RESULTS
Experiment 1 (Table I)
During the fall, winter and spring of 1959-60, essentially continuous records
of perch-hopping activity were obtained for four males confined in an outdoor aviary
and thus exposed to essentially natural day-lengths and normal air temperatures.
TABLE I
Summary of nocturnal activity of four male bobolinks in relation to season, natural photo-
period, and ambient temperature, as measured by recorded perch-hopping movements,
January 3 to May 3, 1960, Chapel Hill, N. C., Lai. 36° N. Outdoor aviary,
natural lighting only
Week ending
Air T ° C.
Nocturnal Activity Indices
Aver. H
Aver. L
A
B
C
fan. 10
9
-1
1.4
3.0
4
17
18
+4
9.9
21.2
210
24
6
-6
2.2
7.6
17
31
11
-1
6.0
5.9
35
Feb. 7
11
-1
6.5
13.1
85
14
13
-1
1.9
3.8
7
21
8
-4
<1
1.5
<1
28
9
-3
1.2
5.0
6
Mar. 6
4
-6
<1
5.0
<1
13
2
-8
<1
2.5
<1
20
7
_2
3.0
19.4
58
27
12
-3
17.6
41.4
729
Apr. 3
23
+9
17.3
79.0
1367
9
21
7
25.7
54.2
1393
19
23
8
no records
26
28
12
22.8
66.6
1518
May 3
23
8
24.9
86.1
2144
Nocturnal Activity Index A: average number of active quarter-hour periods per bird per night
(one or more hops recorded during the quarter-hour period) ; B: average number of hops recorded
per active quarter-hour night period; C: average number of hops per bird per night. See text.
MIGRATORY UNREST IN BOBOLINKS 545
The photoperiocl (including twilight) increased from a low of about lO1/^ hours
in December to about 15 hours in May. Two of the four males were "autumn
captures," one was a "spring capture" and one a "2nd year experimental." The
latter two both had experienced natural day-lengths through the summer of 1959.
(These are the birds designated as "Group F" in Engels, 1961, Table II, p. 143.)
Through the autumn these birds displayed bursts of nocturnal activity in a
somewhat sporadic manner, but this eventually declined in intensity, more or less
coincident with the advent of cold weather. At this time diurnal activity also
declined, so that frequently an individual bird did not record more than 100 perch
movements in a 24-hour period. The nocturnal activity indices for these birds,
from January 4 to May 4, are presented as weekly averages in Table I, together
with average high and low air temperatures as recorded by the local station of
the U. S. Weather Bureau. It will be noticed that all indices have low values
from January on through the week ending March 20. The slight elevation of
the indices for the week ending January 17 might be a reflection of the slight
rise, about 6° C, in average ambient temperature. However, the sharp rise in all
indices apparent for the week ending March 27 occurred while the average low
(night) air temperatures were still below the freezing point. Although the
continued maintenance of intensive perch-hopping activity during the nights of suc-
ceeding weeks coincided with the normal spring rise in air temperature, the whole
picture does suggest a nocturnal restlessness induced by something other than an
increase in environmental temperature.
The intensification of nocturnal activity was also more or less coincident with
a photoperiodically induced production of male sex hormone. This latter event is
manifested in bobolinks by a change in the pigmentation of the horny beak, which
eventually becomes a deep glossy black (Engels. 1959, p. 761). With one
exception the onset of nocturnal restlessness was abrupt, occurring in a single
night. The actual dates for the individual birds were the nights of March 19/20,
23/24 and April 5/6. For one bird restlessness began also on March 23/24, then
slacked off after a few nights but was persistent every night after April 3/4. In
two cases this onset of restlessness preceded the initial appearance of beak
pigmentation by at least a week, in one case by at least three days, and in the
fourth bird the two events may have been essentially coincident.
Two of the four birds went through an almost complete prenuptial molt.
In both, molting was intense and general in early February and continued on into
mid-March. During this period body weight diminished by about 10 grams or
by about 20 to 25^- In both, nocturnal restlessness appeared within one or
two weeks of cessation of molt and shortly following an upturn in body weight.
In the other two birds the prenuptial molt was very incomplete, a few new replace-
feathers appearing in middle to late March and early April. However, both lost
weight during this time, approximately to the same extent as the birds described
above. In both, the beginning of nocturnal restlessness occurred more or less
coincidentally with an upturn in body weight.
In view of the varied previous history of these four birds, it should be
mentioned that of the two "autumn captures" one went through an essentially
complete molt resulting in typical cock plumage, the other remained essentially
henny in appearance. Restlessness developed earliest (March 19/20) in the
546
WILLIAM L. ENGELS
male caught in the previous spring; it set in only three days later (March 23/24)
in the "2nd year experimental" and in one of the two "autumn captures."
During this same winter, activity records were being obtained for a number
of bobolinks caged indoors where night air temperatures never fell below about
11° C. Some of these birds were persistently active at night during December,
when the outdoor birds had practically given up all exercise. In January an
exchange was made, transferring an exceptionally active female to the outdoor
aviary in place of one of the males, and putting him into her cage in the laboratory
attic. The change in photoperiod was slight, from a constant 10 hours light-
14 hours dark in the attic to approximately 11 hours light-13 hours dark in the
TABLE 1 1
Apparent effect of ambient temperatures on the nocturnal activity of two bobolinks, in an outdoor aviary
(natural photo periods, Lat. 36° N., outdoor temperatures) and in the laboratory attic (constant
10-hour photoperiod, 14 hours darkness daily, night temperatures from about 11° C. to about
17° C.). Activity Indices as in Table I.
Periods
Outside temp. " C.
Nocturnal Activity Indices
Aver. H
Aver. L
d"81
968
A
B
C
A
B
C
in aviary
in attic
Dec. 27-Jan. 7
Jan. 3-8
14
8
-1
-1
0 0
2 <1
0
2
34.7
26.3
59.0
54.1
2047
1423
in attic
in aviarv
9-15
15
+ 3
0
0
0
6.9
5.0
35
16-23
12
0
32.1
71.2
2286
3.0
4.8
14
24-29
7
-6
27.9
89.2
2489
2.8
5.0
14
Jan. 30-Feb. 3
Feb. 4-10
9
11
+ 2
-2
4.6
42.3
56.3
141.2
259
5973
5.0
3.9
5.0
3.0
25
12
11-17
11
-1
3.4
in aviary
6.2
21
24.6
in attic
60.9
1498
18-24
4
-3
0
0
0
30.0
64.6
1938
aviary. But there was a complete, almost dramatic reversal in the nocturnal
activity performances of the two birds as they were shifted back and forth between
the warmer and colder environments (Table II). Looking at these data, it is
impossible not to suspect that temperature here is playing a decisive role in
regulating the extent and degree of nocturnal activity.
In view of the subsequent development of nocturnal restlessness in the aviary
birds (Table I) while night air temperatures were still regularly dropping below
the freezing point, it seemed logical to test the hypothesis that this activity had
been photoperiodically induced by exposing some birds at low air temperatures to
long, others to short photoperiods. If warmer temperatures promote nocturnal
activity in these birds when caged, and colder temperatures inhibit or suppress
it, could a photoperiodic response "break through" and express itself as persistent
perch-hopping during the night despite low air temperature? In the absence of
suitable low-temperature control facilities, it \vas decided to make use of normal
MIGRATORY UNREST IN BOBOLINKS
547
winter temperatures and to repeat the outdoor aviary experiments, suitably
modified, in the following year.
Experiment 2 (Table III, Figures 1 and 2)
In 1960-61 two aviaries were used and records of activity obtained for six
males, three in each aviary. The aviaries were illuminated only by natural light
until November 27. After this date one aviary continued to receive only natural
light, while in the other white fluorescent lights (minimum intensity about 35 foot-
TABLE III
Summary of nocturnal activity of two groups of bobolinks (3 <? cf each) in relation to season, natural
and lengthened photoperiods , and ambient temperature, as measured by recorded perch-hopping
movements January 3 to May 2, 1961, Chapel Hill, N. C., Lat. 36° N.
Air T ° C.
Nocturnal Activity Indices
Week ending
Group AvN
Group AvL
Aver. H
Aver. L
A
B
C
A
B
C
Jan. 10
11
-5
2.9
9.1
26
1.1
1.6
2
17
12
-2
2.1
3.1
7
1.3
11.2
15
24
7
-6
2.7
2.7
7
1.0
1.3
1
31
3
-9
1.4
1.8
3
<1
2.0
<1
Feb. 7
6
-7
3.5
11.9
42
1.0
3.7
4
14
11
-2
4.6
5.5
25
1.2
8.8
11
21
19
+4
6.6
9.6
63
8.6
25.9
223
28
18
3
2.8
10.2
29
11.5
19.6
225
Mar. 7
20
7
7.7
20.7
159
19.0
35.4
673
14
19
5
8.4
19.7
165
16.5
40.9
675
21
15
1
10.6
18.3
194
18.8
41.0
771
28
14
1
25.3
27.9
706
26.1
50.1
1308
Apr. 4
18
4
29.3
37.4
1096
25.0
46.5
1163
11
16
2
27.8
38.1
1059
24.3
37.1
1273
18
18
4
17.6
30.1
530
22.9
38.5
882
25
21
8
31.0
45.9
1423
24.5
43.4
1066
May 2
22
9
31.1
38.3
1191
16.5*
35.2*
581*
Group AvN, outdoor aviary, natural illumination only; Group AvL, outdoor aviary, extra,
artificial lighting, in addition to natural light, 5:15 A.M. to 7:15 P.M. daily from November 28
to April 20. Activity Indices as in Table I.
* One bird apparently becoming inactive at night this week ; no later records.
candles at perch level) burned daily from 5:15 AM to 7:15 PM. The constant
artificial photoperiod was thus 14 hours, which previously had been shown to
be stimulatory in the testicular photoperiodic response of bobolinks when preceded
by several weeks of shorter photoperiods (Engels, 1961). In the naturally lighted
aviary the photoperiods declined to about 10% hours at the December solstice,
then gradually increased but did not reach 14 hours until mid- April.
None of the bobolinks used was newly captured. However, two of the three
birds in each group were in their second year in the aviary under continuously
natural day-lengths, and of these, one in each group had never been exposed to
artificial photoperiods.
548
WILLIAM L. ENGELS
The results are given in Table III. The winter was much more mild than
the previous one, the weekly average low air temperatures remaining above
freezing after mid-February, whereas in 1960 they remained below freezing
throughout March. Nevertheless, a marked rise in the nocturnal activity indices
for the naturally lighted birds (Group AvN) occurred in the same week, the
last week in March, as it had for the aviary birds in the preceding year (cf.
Table I). On the other hand, in the aviary birds exposed to 14-hour photoperiods
this pronounced increase in nocturnal activity occurred about three weeks earlier.
CMS
52
48
44
40
36
32
DEC
JAN
FEB i
MAR
APR | MAY
FIGURE 1. Variation in body weight of 3 male bobolinks in an outdoor aviary, Lat. 36° N.,
natural photoperiods; group AvN of Table III. Dotted lines indicate periods of molt; P:
approximate time of beginning of nuptial pigmentation in the beak indicative of testicular
recrudescence ; Z : approximate time of onset of pronounced nocturnal restlessness. Body weights
determined (usually) at one-week intervals.
This appears to be a definite acceleration of the cycle, which may be attributed to
the lengthened photoperiod. Considering the long period of exposure to long days,
about three months, before the response occurred, the acceleration seems slight
indeed. Yet this result might have been anticipated from our previous studies
on the testicular cycle of bobolinks, which demonstrated a long delay in the
testicular response to photoperiodic stimulation (Engels, 1961, pp. 144-145).
The lean weight of male bobolinks is 30 grams or less. Birds weighing 40
grams or more are conspicuously fat, especially over the rump and in the
abdominal region. As shown in Figures 1 and 2, all of the birds were very
heavy through December and early January. All but one experienced sharp
MIGRATORY UNREST IN BOBOLINKS
549
losses in body weight between the end of January and the middle of February.
Each of these also went through an almost complete prenuptial molt to the cock
plumage. This molt did not occur in the single bird in which body weight
remained high (no. 30, Fig. 2). The five birds which had lost weight, and which
had molted, developed nocturnal restlessness subsequent to the molt and either
just prior to or shortly after the beginning of increase in body weight. In all
cases but one, the onset of nocturnal restlessness preceded the development of
CMS
56
52
48
44
40
36
32
DEC
JAN
FEB
MAR
APR
_L
MAY
FIGURE 2. Variation in body weight of 3 male bobolinks in an outdoor aviary, as in Figure
1, but exposed to 14-hour photoperiods (5 :15 A.M. to 7:15 P.M.) daily, beginning November 28;
group AvL of Table III. (See also legend of Figure 1.)
beak pigmentation, as in the four aviary birds of the previous year, described
above. One male (no. 28, Fig. 2) did not develop nocturnal restlessness until
about 10 days after the beginning of peak pigmentation.
Experiment 3 (Table IV)
This was essentially a duplication of part of the second experiment, using
only "spring captures." Three males captured in the spring of 1961 were
confined in an outdoor aviary. Beginning November 15, 1961, a 14-hour photo-
period (5:15 AM.-7:15 PM) was superimposed on the natural day-length
550
WILLIAM L. ENGELS
(white fluorescent lights; minimum intensity at perch level about 35 f.c.). A
single perch in each cage was balanced on the tip of the 6-inch actuating arm of
a microswitch. Perch-hopping activity was relatively infrequent at night until
after the middle of March when restlessness became pronounced. There again
appeared to be some correlation with ambient temperature, with flurries of restless-
ness, appearing during the weeks ending January 28 and February 25, associated
with rises of air temperature. Throughout the period of recording, it was only
when the weekly average of the daily mean air temperature exceeded 9° C. that
the average index A was above 16 (= 4 hours), index B above 15 (= 1 hop per
minute), and index C above 400 (hops per bird per night).
TABLE IV
Nocturnal activity of 3 male bobolinks in an outdoor aviary at Chapel Hill, N. C., Jan. 7-Apr. 22,
1962; 14-hour photoperiod (5:15 AM-7-.15 PAT) superimposed on natural day-lengths beginning
Nov. 15, 1961
Week ending
Air T ° C. average
Nocturnal Activity Indices
H
m
L
A
B
C
Jan. 14
3
-3
-9
3.2
10.0
32
21
7
1
-5
6.3
7.4
47
28
16
10
+3
9.8
22.6
221
Feb. 4
9
3
-4
2.1
9.0
19
11
10
3
-J
4.5
6.3
28
18
9
3
-3
2.7
11.7
30
25
16
10
+4
6.9
39.7
274
Mar. 4
11
6
+ 1
8.4
11.5
97
11
6
2
-1
1.8
3.7
7
18
13
7
+ 1
12.5
10.8
135
25
17
10
3
22.6
19.1
432
Apr. 1
20
13
6
19.0
27.3
519
8
18
12
5
20.2
27.0
545
15
19
14
8
20.7
18.3
379
22
24
15
5
18.3
24.2
443
One bird developed the black pigmentation of the beak characteristic of
testicular recrudescence during the week ending February 28. The same bird
abruptly became restless at night about two weeks later on March 11/12. The
other two males both developed black pigment in the beak during the week ending
March 8 and a verv distinct beginning of heightened restlessness on the night of
March 18/19.
DISCUSSION
The primary question is : "Does pronounced nocturnal restlessness in these caged
bobolinks reflect a disposition to migrate?" Questions as to the induction and
regulation of the restlessness must remain secondary, and academic, until the first
is answered. The results of the present experiments are not unequivocal.
The relatively abrupt change in nocturnal behavior in all experiments occurred
during March. Northward migration of free-living birds probably begins in the
MIGRATORY UNREST IN BOBOLINKS 551
latter part of March and early April. (The first flocks of migrating males usually
arrive in the coastal areas of the southernmost U. S. shortly after the middle of
April. I have seen three male specimens taken between Lat. 22° S. and 17° S.,
within the "wintering" area, on March 23, 28 and April 1.) Had the experiments
taken place in South America, the near coincidence of the onset of restlessness with
the migratory season might be interpreted as meaningful.
There was also some degree of correlation between the onset of pronounced
nocturnal restlessness and some other cyclic events which are associated with migra-
tion, namely molt, fattening, and testicular recrudescence. None of the several
museum specimens I have seen from the "wintering" grounds has the black beak
indicative of testicular recrudescence, even though three were collected (between
Lat. 22° S. and 17° S.) as late as the last week of March. Moreover, thirteen South
American April specimens definitely were migrants, and of these, nine (northern
Brazil, Venezuela) likewise had light-colored beaks. In the other four (Colombia)
the beak was darkening at the time (collector's hand- written notes on labels indi-
cated color of "mandible" as "gray," "light gray," and [two] "gray with border and
point black," respectively).3 Evidently migration gets underway before the blacken-
ing of the beak. In our experiments, pronounced nocturnal restlessness set in after
the change in beak pigmentation in only four of thirteen cases, in another one the
two events occurred more or less simultaneously, while in eight the onset of restless-
ness occurred before the change in beak pigmentation, just as in nature the beginning
of migration precedes this event.
A molt from the winter "henny" plumage to the nuptial "cock" plumage may be
complete by the end of January (University of Michigan #90875) but apparently
more usually occurs during February and March. At any rate it is completed before
northward migration begins. Presumably premigratory fattening occurs subsequent
to the molt, but I have no information about this. (Datum on weight is given on
the label of only one of almost 100 museum specimens, known to me, taken south of
the U. S. Spring migrants taken in the southern U. S. are conspicuously fat.)
Our caged birds tended to remain fat throughout the year, except during periods
of molt. Molt in these birds presents a puzzling problem. In some the molt to
nuptial or "cock" plumage was essentially complete, in some it was partial, in some
it was more or less completely suppressed. These differences appeared among birds
with identical previous history of capture and treatment. However, when molt was
complete (or partial), it occurred prior both to the onset of pronounced nocturnal
restlessness and to the development of black beak pigmentation. Following the molt
there was always a sharp rise in body weight, caused by the deposition of fat. The
onset of pronounced nocturnal restlessness was always associated with this rise in
body weight.
All of these observations, associating nocturnal restlessness with season, fattening,
molt, and testicular recrudescence in a general temporal sequence comparable to that
obtaining in nature, at least suggest that the restlessness of the caged birds reflects
a true migratory unrest. Since there is no good reason to suppose that these
3 Grateful acknowledgment is here made to the following for the loan of specimens : Dean
Amadon (American Museum of Natural History) ; Kenneth C. Parkes (Carnegie Museum) ;
Emmet R. Blake (Chicago Natural History Museum) ; Harrison B. Tordoff (Museum of
Zoology, University of Michigan) ; James Bond (Philadelphia Academy of Sciences) ; P. S.
Humphrey and Mary A. Heimerdinger (Peabody Museum of Natural History).
552 WILLIAM L. EXCELS
various events are all causally related, the few discrepancies in sequence in the
experimental birds are not unexpected. The mechanisms responsible for each may
proceed from different thresholds, from different stimuli, at different times and along
different pathways, the natural coordination of which may be upset by the experi-
mental treatment. One may surmise, for example, the existence of an antagonism
such that if the hypothalamic-hypophyseal gonadotropic mechanism gets started a
bit relatively early in a particular captive individual, the mechanism (s) leading to
molt may be partially or fully inhibited.
In studies on temperate-zone migrants, the experimental birds are obtained on
the wintering grounds, after the fall migration has been completed. Our bobolinks
were caught in May and in September, and hence their southward migration was
prohibited. That a nocturnal restlessness tended to persist in them is not surprising
since it is a common observation that caged birds under natural photoperiods remain
in a state of migratory unrest far beyond the natural migratory period, often until
the next molt (cf. Farner, 1960). The restlessness in our bobolinks caged out of
doors was inhibited seemingly by low environmental temperatures (cf. Table II).
Nevertheless (in Experiment 1, Table I) pronounced restlessness set in, during
March, when air temperatures were still below freezing every night and the daily
mean temperature averaged only about 5° C. As Schildmacher (1938) concluded
from rather comparable observations on European robins (Eritha-ciis rnbecula),
". . . one must assume that in these birds the effect of [an inductive mechanism]
overweighed the effect of low temperature" (p. 151, free translation).
In the quoted statement Schildmacher actually specified "lengthened daylight" as
the inductor. That lengthened daylight may play a role in the development of
nocturnal unrest in bobolinks is indicated by the results of our Experiment 2 which
show that this activity appeared earlier in birds exposed to 14-hour photoperiods as
compared to those experiencing only natural (winter) photoperiods (Table III).
Farner (1960) has reported positive results in similar experiments with Zonotrichia
leucophrys gambelii; a chief difference in the data lies in the lapse of time between
the first 14-hour photoperiod and the Zugunruhe response — a few weeks for Z. I.
gambelii (a temperate-zone migrant), more than three months for bobolinks. A
similar delay appears in the photoperiodic induction of testicular development in
bobolinks (Engels, 1961 ; also Fig. 2, above) ; it seems to be an essential part of this
species' adaptation to the long days it experiences in the southern hemisphere between
breeding seasons.
A. J. Marshall, a notable and vigorous protagonist of the idea that photoperiod
is concerned with reproduction of birds only insofar as it may influence an internal
rhythm, has stated (Marshall, 1961, p. 331) that "Spring Zugunruhe is a behavior
pattern that is undeniably activated by photostimulation and probably by testos-
terone." If genuine migratory unrest in bobolinks is in any way activated by
testosterone, either the testosterone must be stimulatory at a threshold concentration
far below that which produces a change in pigmentation of the beak in the male or
the behavior response must occur much more quickly, since, on the evidence of
museum specimens, bobolinks in their northward migration may reach northern
South America without showing this change. Doubt is cast on any testosterone
activation by those experimental cases in which the beak became black as much as
two weeks before the onset of pronounced nocturnal restlessness (Experiment 3).
MIGRATORY UNREST IN BOBOLINKS 553
It should be admitted that, from the evidence obtained thus far, the possibility is
not precluded that annual cycles in this transequatorial migrant may rest funda-
mentally on an internal rhythm as "the primary seasonal initiator" (Marshall, 1961,
p. 309). Actually, the very long delay in response to 14-hour photoperiods, the
slight acceleration as compared to natural (winter) photoperiods (Experiment 2),
might readily be interpreted in terms of this concept.
SUMMARY
1. The bobolink (Dolichonyx oryzivorus} is a transequatorial migrant which
breeds (June- July) above Lat. 40° N. in North America and "winters" (November
through March) below about Lat. 10° S. in South America. The northward
migration occurs during April and May.
2. Observations on nocturnal restlessness, molt, body weight and the testicular
cycle were made on some captive bobolinks at Lat. 36° N., caged out-of-doors and
exposed to natural as well as to lengthened photoperiods, and to normal outdoor air
temperatures. "Restlessness" was recorded every quarter-hour, by an electronic
counting device, as the number of hops made by each bird on the perches of its cage.
3. Restlessness was almost completely suppressed by the low air temperatures
of winter. Nevertheless, intense nocturnal unrest set in rather abruptly in late
March when air temperatures at night were still regularly below the freezing point
(natural photoperiods).
4. When 14-hour photoperiods were superimposed on natural day-lengths, begin-
ning November 28, restlessness set in about three weeks earlier than in the controls
which experienced only natural day-lengths. This was interpreted as evidence at
least of a photoperiodic influence on, if not photoperiodic induction of, nocturnal
restlessness.
5. The onset of pronounced unrest was always associated with a marked rise in
body weight, due to the deposition of subcutaneous and intraperitoneal fat, usually
following a molt. In most cases this onset of restlessness preceded, by an appreciable
interval, the appearance of black beak pigmentation, which indicates testicular
recrudescence. These temporal relationships correspond to a sequence of events in
nature and are tentatively interpreted to mean that the restlessness reflects the
induction of a migratory state.
6. The long delay (about three months) in the response to the presumed
stimulation by long photoperiods is similar to the delay found in the response of
the testicular cycle to photostimulation. An interpretation in terms of an internal
rhythm as a primary seasonal initiator, with other factors (such as photoperiod),
acting as accelerators-inhibitors, is not precluded.
LITERATURE CITED
ENGELS, W. L., 1959. The influence of different daylengths on the testes of a transequatorial
migrant, the Bobolink (Dolichonyx orysivorus). In: Photoperiodism and Related
Phenomena in Plants and Animals (pp. 759-766). R. B. Withrow, Ed. Publ. No. 55,
Amer. Assoc. Adv. Science, Washington, D. C.
ENGELS, W. L., 1961. Photoperiodism and the annual testicular cycle of the bobolink
(Dolichonyx oryzivorus), a transequatorial migrant, as compared with two temperate
zone migrants. Biol. Bull, 120: 140-147.
554 WILLIAM L. ENGELS
FARNER, D. S., 1960. Metabolic adaptations in migration. Proc. Xllth International Ornitho-
logical Congress, Helsinki, 1958, pp. 197-208.
HAMILTON, W. J., Ill, 1962. Bobolink migratory pathways and their experimental analysis
under night skies. Auk, 79: 208-233.
KING, J. R., 1961. On the regulation of vernal premigratory fattening in the white-crowned
sparrow. Physiol. Zool., 34: 145-157.
MARSHALL, A. J., 1961. Breeding season and migration. In: Biology and Comparative Physiol-
ogy of Birds, Vol. 2, (pp. 307-339). A. J. Marshall, Ed. New York and London:
Academic Press.
SAUER, F., 1957. Die Sternenorientierung nachtlich ziehender Grasmikken (Sylvia atricapilla,
borin, und curruca). Zeitschr. f. Tierpsychol., 14: 29-70.
SAUER, F., AND E. SAUER, 1955. Zur Frage der nachtlichen Zugorientierung von Grasmiicken.
Rev. snisse Zool, 62: 250-259.
SCHILDMACHER, H., 1938. Zur Physiologic des Zugtriebes. IV. Weitere Versuche mit kiinstlich
veranderte Belichtungzeit. Vogelsug,9: 146-152.
WEISE, C. M., 1956. Nightly unrest in caged migratory sparrows under outdoor conditions.
Ecology, 37: 274-287.
ANAEROBIC GLYCOLYSIS IN AMPHIBIAN DEVELOPMENT *
JOHN R. GREGG
Department of Zoology, Duke University, Durham, North Carolina
Under anaerobic conditions, the embryos of frogs probably degrade carbo-
hydrate to lactic acid by a sequence of chemical reactions of the Embden-Meyerhof
type. The operation of the sequence (glycolysis) presumably is able to generate
some or all of the energy required for cleavage under anaerobiosis (Brachet,
1934). It may even meet the energetic demands of anaerobic gastrulation
(Gregg and Kahlbrock, 1957), although Brachet (1960) has pointed out that the
evidence is conflicting. In any case, it would appear to be an important device
for sustaining embryos in straitened respiratory circumstances, of frequent occur-
rence in the interior of large clumps of naturally oviposited eggs. It is surprising,
therefore, to find that little attention has been given to systematic study of the
glycolytic capacities of frog embryos. A few papers on the subject exist in the
literature. But some of these (Lennerstrand, 1933; Brachet, 1934) were pub-
lished before the exigencies of rearing embryos under strictly aerobic conditions
were understood or overcome, and are subject to still other criticisms noted by
Cohen (1954). Others, including Cohen's paper and the earlier one of Barth
(1946), cover only rather narrowly circumscribed morphogenetic periods. We
are thus without a complete picture of the glycolytic behavior of pre-hatching
frog embryos.
This paper has two purposes. The first is to take some steps toward filling
the hiatus mentioned above, by surveying the glycolytic activity of developing
Rana pip I ens embryos. The second is to explore more thoroughly than before
the reduced glycolytic activity exhibited by gastrula-arrested hybrid embryos
obtained by fertilizing Rana pipiens eggs with Rana sylvatica sperm. Hybrids of
this type were first studied by Moore (1946), from a morphological point of view.
Later analysis has provided a rough outline of their physiological or biochemical
peculiarities: its current status may be ascertained by consulting Barth and Barth
(1954), Gregg (1957), Gregg and Kahlbrock (1957), Gregg and Ray (1957)
and Gregg (1960).
METHODS
Embryological
Developing embryos were obtained by stripping eggs from pituitary-activated
R. pipiens females into suspensions of active R. pipiens or R. sylvatica sperm.
After not more than one hour, the developing eggs were separated into small
groups of 20-40 members each, and distributed into several large fingerbowls.
Each fingerbowl contained 100-200 ml. of 10% amphibian Ringer's solution,
1 This work has been supported in part by a research grant, No. A-2146, from the Public
Health Service. The assistance of Harry T. Klugel III is gratefully acknowledged.
555
556
JOHN R. GREGG
without phosphate or bicarbonate. Development was allowed to proceed at 10° C.,
the medium being renewed every two days or so. Immediately before using them
as experimental subjects, the embryos were freed of their jelly-coats with the
aid of jeweler's forceps.
Chemical
Anaerobiosis was obtained with the help of the apparatus depicted in Figure 1.
Twenty jelly-free embryos, along with enough rear ing-medium to make a total
volume of 2 ml., were placed in each of several 25-ml. Erlenmeyer flasks. The
ground glass neck of each flask was closed with a No. 1 two-hole rubber stopper
bearing glass inlet and outlet tubes. The flasks were set in the clips of a Dubnof
\
I
\
/
f
f
I
\
FIGURE 1. Apparatus used to obtain anaerobic embryos. See description in section on
chemical methods. Arrows indicate direction of gas flow.
shaking bath running at 24° C., and connected in series with short lengths of
rubber tubing. The shaking mechanism was then set in motion at a rate of
85 cycles per minute. At time zero, a source of washed 95% N2: 5% CO, was
connected to the inlet tube of the first flask, and gassing was allowed to proceed
at a rate of one liter per minute for the duration of the experiment.
At the ends of chosen intervals, flasks were removed from the distal end of the
train and their contents were emptied into 12-ml. graduated centrifuge tubes, each
containing 0.5 ml. of 30% trichloracetic acid. The volumes were adjusted with 6%
trichloracetic acid to values depending on the expected amounts of lactic acid, and the
tubes were placed in a Deepfreeze. After freezing and thawing, the embryos were
homogenized with a ball-tipped glass rod, and protein-free extracts were obtained
by centrifuging.
The amounts of lactic acid in the protein-free extracts were estimated by a modi-
fication of the method of Barker and Summerson (1941). One-mi, aliquots of
protein-free extract were treated with equal volumes of 2.5% CuSO4-5H2O and
EMBRYONIC GLYCOLYSIS
557
125-mg. portions of Ca(OH)2 to remove interfering substances. After centrifuging,
0.5-ml. aliquots of supernatant were combined with 3-ml. aliquots of concentrated
sulfuric acid-copper reagent and heated to convert the lactic acid to acetaldehyde.
Polyindophenol reagent was added, and the resulting color intensities were read at
560 m/j, with the help of a Beckman Spectronic colorimeter. Standards were
prepared with lithium lactate.
Terminological
Developmental stages were determined by reference to the data of Shumway
(1940), which standardize the course of R. pipiens development at 18° C. Regard-
less of their actual temperature histories, embryos in a given Shumway stage have
been assigned the corresponding standard age in hours. Hybrid embryos, even
after the curtailment of morphogenesis at Stage 10, have been assigned the same
stages as R. pipiens control embryos of the same female parentage.
RESULTS
(1) Aerobic embryos contain negligible amounts of lactic acid.
This finding was established directly by a few analyses of R. pipiens embryos
reared under the conditions described in the section on embryological methods. The
results are summarized below :
Clutch
Stage
Standard age
Lactic acid, /jg. per embryo
B
3
3.5
0
A
9
21
0.06
A
12
42
0
C
19
118
0.15
C
20
140
0.20
A similar result is implicit in the data presented in Figure 2 and Figure 3, where
the values for lactic acid production under anaerobiosis extrapolate satisfactorily to
a value of zero at time zero.
These results are in contrast to those of Lennerstrand (1933) and Brachet
(1934), who reported much higher aerobic lactic acid values. Earth (1946), how-
ever, was able to show that the partial anaerobiosis induced by crowding will result
in the production of large amounts of lactic acid, and both he and Cohen (1954)
showed that when care is taken to keep embryos well-aerated, there is little or no
lactic acid produced. It may be, therefore, that the embryos of Lennerstrand and
Brachet were not reared under strictly aerobic conditions.
(2) Embryos begin to produce lactic acid as soon as they are deprived of oxygen,
This result is clearly established by the data of Figure 2 and Figure 3. There is
no indication of a lag in the onset of glycolysis of the sort mentioned by Cohen
(1954). But the apparent discrepancy between his results and ours may be re-
solved by considering the methods used to induce anaerobiosis. In our experiments,
the embryos were gassed while spread out in a very thin layer of medium over a
relatively large surface; therefore, there was probably little delay in the establish-
558
JOHN R. GREGG
meat of anaerobiosis. Cohen's embryos, on the other hand, were gassed in a thicker
layer of medium spread over a much smaller surface, and anaerobiosis may have
developed more slowly. Indeed, Cohen noted that the period of lag was reduced
by increasing the number of embryos per volume of medium : on our interpretation,
this is to be expected. Thus, Cohen may be regarded as having shown not a lag in
the production of lactic acid after anaerobiosis is attained, but merely a lag in the
attainment of anaerobiosis.
(3) For at least four hours of anaerobiosis, embryos younger than 72 hours
produce lactic acid at constant rates, whereas the rates of lactic acid production
by older embryos under similar conditions are decreasing functions of time (Fig. 2,
6-
4-
2-
01234
FIGURE 2. Time course of lactic acid production by anaerobic R. pipiens embryos, Clutch C.
Reading from below to above: Stage 2+, standard age 2 hours; Stages 11* and 15-, standard ages
38 hours and 66 hours ; Stage 18, standard age 96 hours ; Stage 19, standard age 118 hours ; Stage
20, standard age 140 hours. Abscissa, time in hours. Ordinate, fig. lactic acid per embryo.
(Two points near (0, 0) have been omitted. See table in discussion of result (1).)
Fig. 3). But, during the first hour of anaerobiosis, embryos of any age glycolyze
at rates which for all practical purposes may be regarded as constant (Fig. 3).
The latter result has been obtained in experiments on three clutches of control
embryos and two clutches of hybrids, other than that represented by Figure 3.
Among other things, these findings mean that glycolytic rates obtained by
estimating the lactic acid contents of post-neurulae at the beginning and at the
end of a long period of anaerobiosis (periods of lengths up to 30 hours are not
untypical of the sparse literature on the subject) are not strictly comparable with
those similarly obtained on pre-neurulae and neurulae. In establishing the data
for Figure 4, we have avoided misleading comparisons by taking the rates of
glycolysis to be those exhibited during the first hour of anaerobiosis.
EMBRYONIC GLYCOLYSIS
559
(4) The rate of glycolysis of R. pipiens embryos is an increasing function
of age. The graph relating age to glycolytic rate (Fig. 4) shows that the capacity
to glycolyze develops in two phases. The first phase, spanning the interval
between fertilization and the onset of neural fold formation, is characterized by a
constant rate of glycolysis. The second phase, from the onset of neural fold
FIGURE 3. Time course of lactic acid production by anaerobic hybrid embryos (right-hand
section) and normal control embryos (left-hand section), Clutch F. Reading either section from
below to above : Stage 3i, standard age 4 hours ; Stage 10?, standard age 30 hours ; Stage 131,
standard age 59 hours ; Stage 17i, standard age 90 hours ; Stage 19s, standard age 129 hours.
Abscissal units lower three curves either section, hours ; upper two curves either section, quarter-
hours. Ordinal units either section, jug. lactic acid per embryo. (Rates of glycolysis determined
from these curves are plotted in Figure 4.)
formation to hatching, is characterized by an exponentially increasing rate of
glycolysis. More precisely:
g(0=0.44
g(i')=0.44e°'0225a-59)
where t is the standard age in hours and g(0 is the glycolytic rate in /tg. lactic
acid per embryo per hour.
It is interesting to note that the acceleratory change in glycolytic rate at 59
hours is paralleled by a similar change in respiratory acceleration at 56 hours
(Gregg, 1960.) The close temporal coincidence of the two changes suggests a
structural connection of some sort, but details are not yet available.
560
JOHN R. GREGG
Comparison with the results of Lennerstrand (1933) and Brachet (1934) is
made difficult by complications mentioned in the discussion of results (1) and (3).
The data more or less agree with the more circumscribed ones of Barth (1946).
But they are clearly inconsistent with those of Cohen (1954), who found that
the glycolytic activity of pre-neurulae is a linearly increasing function of age,
not a constant function. There is no obvious way to resolve the discrepancy ;
and, pending further investigation, the two sets of results must remain irreconciled.
3H
2-
50
100
150
FIGURE 4. Rate of lactic acid production as a function of age, Clutches F, G, H. Lower
curve, hybrid embryos. Upper curve, normal control embryos. Abscissa, standard age in
hours. Ordinate, /*g. lactic acid per embryo per hour. The three points at 4 hours represent
both normal and hybrid embryos, whose glycolytic rates at this age are indistinguishable.
(5) Developing hybrid embryos undergo changes of glycolytic rate that are
difficult to characterize in any simple way as a mathematical function of age
(Fig. 4). Roughly speaking, their capacity to produce lactic acid under anaero-
biosis declines from the control value at fertilization to about a fifth of the control
value at 59 hours, and then rises slowly until, at 140 hours, the glycolytic rate is
about one-eleventh of the control rate. The acceleration at 59 hours may be of
the same sort as that in control embryos, but of reduced magnitude. There is no
corresponding respiratory acceleration (Barth, 1946). Briefly, our results agree
EMBRYONIC GLYCOLYSIS 561
with those of Earth in showing that hybrid embryos are unable to generate energy
by anaerobic glycolysis except at increasingly sub-normal rates. The reasons why
are yet unknown.
SUMMARY
1. R. pipiens embryos, and gastrula-arrested hybrid embryos obtained by
fertilizing R. pipiens eggs with R. sylvatica sperm, begin to produce lactic acid,
without initial lag, as soon as they are deprived of oxygen.
2. Provided that they are younger than about 72 hours (18° C.), embryos of
both types are able to sustain the initial rate of glycolysis for at least four hours.
Older embryos of both types exhibit a linear production of lactic acid for at least
one hour.
3. The development of glycolytic capacity in R. pipiens embryos occurs in
two phases: one of constant glycolytic rate (0-59 hrs.), the other of exponentially
increasing glycolytic rate (59-140 hrs.).
4. The glycolytic rates of hybrid embryos decline from the normal control
value at fertilization to about one-fifth of the control value at 59 hours, then
increase to about one-eleventh of the control value at 140 hours.
LITERATURE CITED
BARKER, S. B., AND W. H. SUMMERSON, 1941. The colorimetric determination of lactic acid in
biological material. /. Biol. Chem., 138: 535-554.
BARTH, L. G., 1946. Studies on the metabolism of development. /. Exp. Zool, 103: 463-486.
EARTH, L. G., AND L. J. BARTH, 1954. The Energetics of Development. Columbia University
Press, New York.
BRACKET, J., 1934. fitude du metabolisme de 1'oeuf de Grenouille (Rana fusca) au cours du
developpement. I. La respiration et la glycolyse, de la segmentation a 1'eclosion.
Arch, de Biol., 45: 611-727.
BRACKET, J., 1960.The Biochemistry of Development. Pergamon Press, New York.
COHEN, A. L, 1954. Studies on glycolysis during the early development of the Rana pipiens
embryo. Physiol Zool, 27: 128-141.
GREGG, JOHN R., 1957. Morphogenesis and metabolism of gastrula-arrested embryos in the
hybrid Rana pipiens $ X Rana sylvatica d\ In: The Beginnings of Embryonic Develop-
ment, edited by Albert Tyler, R. C. von Borstel and Charles B. Metz, Publication No. 48
of The American Association for the Advancement of Science. Washington, D. C.
GREGG, JOHN R., 1960. Respiratory regulation in amphibian development. Biol. Bull., 119:
428-439.
GREGG, JOHN R., AND MARGIT KAHLBROCK, 1957. The effects of some developmental inhibitors
on the phosphorus balance of amphibian gastrulae. Biol. Bull., 113: 376-381.
GREGG, JOHN R., AND FRANCES L. RAY, 1957. Respiration of homogenized embryos : Rana
pipiens and Rana pipiens ? X Rana sylvatica $. Biol. Bull., 113: 382-387.
LENNERSTRAND, ARE, 1933. Aerobe und anaerobe Glycolyse bei der Entwicklung des Froscheies
(Rana temporaria L.). Zeitschr. vergl. Physiol., 20: 287-290.
MOORE, J. A., 1946. Studies in the development of frog hybrids. I. Embryonic development in
the cross Rana pipiens ? X Rana sylvatica d. J. Exp. Zool., 101 : 173-220.
SHUMWAY, W., 1940. Stages in the normal development of Rana pipiens. I. External form.
Anat. Rcc., 78: 139-147.
SPECTRAL SENSITIVITY AND PHOTOTAXIS IN THE OPOSSSUM
SHRIMP, NEOMYSIS AMERICANA SMITH *• 2
SIDNEY S. HERMAN s
Narragansett Marine Laboratory, University of Rhode Island, Kingston, Rhode Island
Few studies have been conducted on photoreception in the Mysidacea. Hess
(1910) and Beeton (1959) have both worked on the spectral sensitivity of mysids,
but neither has subjected animals to various colors of the spectrum while
controlling the intensity of light.
There is no information available in the literature on photoreception in
Neomysis americana. Hulburt (1957) has shown that their vertical distribution
in Delaware Bay is a direct result of light intensity.
This report presents methods and results of laboratory studies on spectral
sensitivity and phototaxis in the opossum shrimp, Neomysis americana Smith.
The results of a complementary study of the vertical migration of this animal
in Narragansett Bay, Rhode Island, will be reported in a separate publication.
For purposes of this paper it is sufficient to state that the pattern of vertical
migration was similar to that described by Gushing (1951) as typical of many
zooplankton species: (1) ascent towards the surface from the day-depth, (2)
departure from the surface at or before midnight, (3) return to the surface just
before dawn, and (4) sharp descent to the variable day-depth when sunlight starts
to penetrate the water. Light was found to be the most important single factor
responsible for the migration of these animals.
MATERIALS AND METHODS
Spectral sensitivity
Although there are many references to the effect of different parts of the
spectrum on animal distribution, few have been made where light intensity has
been controlled. In order to study the effects of various parts of the spectrum
on TV. americana, it was necessary to keep the light intensity uniform. The pro-
cedure adopted was to calibrate a model #846 Weston photronic cell against an
Eppley thermopile. The thermopile measures light as a linear function, and
therefore is equally sensitive to energy from all parts of the spectrum.
With calibration complete, it was possible to use the photoelectric cell to
measure light intensity. The calibration was carried out at the Eppley Labora-
tories, Newport, Rhode Island, with the following equipment : a Leeds and
Northrup model 983 5- A stabilized DC microvolt amplifier, a Tinsley photocell
1 Contribution No. 49 from the Narragansett Marine Laboratory of the University of Rhode
Island. Based on a thesis submitted in partial fulfillment of the requirements for the Ph.D.
degree, University of Rhode Island, 1962.
2 This study was aided by the Office of Naval Research, Contract NR 104-100.
3 Present address, Marine Science Center, Lehigh University, Bethlehem, Pennsylvania.
562
SPECTRAL SENSITIVITY IN A MVSID
563
galvanometer amplifier type 5214. a Kipp model AL-1 portable galvanometer,
an Kppley thermopile #2427 (X junction, Bi-AS circular, lampblack receiver of
sensitivity 0.111 m/j,, an Kpplev microvolt comparator, and a #846 \Yeston
pbotronic cell.
The photocell and the thermopile were exposed in turn to the radiant tlux
through Corning narrow-band interference niters and the outputs compared. The
energy intensities of the different light beams were then equalized, as far as
TABLE I
Calibration of photometer with Corning glass color filters
Photometer sensi-
Filter
Photometer jtv.
Thermopile yuv.
Energy ^v. cm. -
tivity MV. M"'.
cm. 2
Corning
1-02(546-559 niM)
360
.37
3.3
109
1-05(515 HIM)
500
.63
5.7
88
5-77(610 mM)
360
.51
4.6
78
5-75(460 mM)
40
.43
3.9
10.1
possible, with \V ratten gelatin neutral density niters, and photocell outputs were
determined to insure approximate equality of energy flux.
The photocell was used with a 200-ohm resistor across its terminals and the
voltage drop across the resistor was read as the photometer output.
The source of radiant energy was a Westinghouse 150-watt tungsten flood
lamp maintained at 110 volts plus or minus 0.5 per cent throughout. The results
of the calibration may be seen in Tables I and II.
TABLK II
Calibration of photometer with Corning glass color filters showing equalization of intensity
Filter
Photometer
MV.
Thermopile
Energv
MV. cm."--'
Photometer
sensitivity
MV. M^V. cm.~'-
Corning
1-02 with
Pvrex
315
.32
2.9
108
1-05 with
Wratten
filter .3
260
.31
2.9
84
5-77 with
Wratten
filter .2
147
.30
2.7
54
5-75 with
Wratten
filter .1
32
.30
2.7
11.8
The thermopile outputs were read with an accuracy of 5%:. The calibration
of the reference thermopile reproduces the International Pyroheliometric Scale
of radiation to about \%. The photocell outputs were read to within \%. The
precision of the above readings (i.e., repeatability) is better than 5%.
Recalibration of the photocell at the Eppley Laboratories after completion of
the experiments showed that no significant changes in energy had taken place
during the course of the experiments, and therefore equal light intensity had
been maintained.
564 SIDNEY S. HERMAN
Spectral sensitivity of animals
A special aquarium, 25" X 18", was constructed with a depth of 10 inches.
The bottom of the aquarium was of one-quarter-inch plate glass, making it possible
to measure light at the bottom of the aquarium. One side was also one-quarter-
inch plate glass, to permit observation of the animals during the experiment. This
side of the aquarium was covered with a cloth that excluded all extraneous light
and served as a hood for the observer during experiments.
The wooden lid of the aquarium included a circular piece 15 inches in diameter,
through which holes had been cut in a circular pattern, for the insertion of color
filters. With this arrangement, the experimental lamp (centered above the wheel
at a height of 15% inches and regulated at 110 volts with a variable transformer)
projected down into the water a circular pattern of colored beams, permitting the
animals to choose between any of the colored beams and darkness. The wheel
was movable and the light beams could be rotated.
Four color filters were used: red, blue, and two shades of green (Table I).
Unfortunately, at the time, it was not possible to utilize other filters which trans-
mitted an accurately measurable amount of energy under the experimental con-
ditions. To avoid confusion, green filter 105 (515 m/i) will be referred to as
blue-green and green filter 102 (546-559 mp.) as green.
The experiments were conducted at a constant temperature of 19° C. and
usually 70-75 animals were placed in the aquarium. After the mysids had re-
mained in darkness for measured periods of time, the experimental light was
turned on and every 30 seconds, for ten minutes, the number of individuals con-
gregated in each beam of colored light was counted. Usually another 10-minute
experiment was conducted directly after this, wherein the neutral density filters
were removed from the three filters containing the lowest numbers of mysids,
thereby increasing the energy passing through these three filters. In this way it
was possible to observe whether the animals changed their behavior pattern when
intensities were greater.
During the course of the experiments, the uniformity of the light intensity
was checked by measurement with a Leeds and Northrup K2 potentiometer and
the Weston photronic cell.
Phototaxis
Phototactic response of N. atncricana was studied by the method employed
by Beeton (1959) in his observations of Mysis rclicta. Six individuals were
placed in a 24-inch glass tube of one-inch diameter, lying horizontally to eliminate
any gravitational effects. The experimental light, a 7C7 General Electric lamp,
was suspended one foot above the midpoint of the tube (for spectral distribution
of lamp, see Beeton, 1959; p. 205, Fig. 1). "After the mysids had been subjected
for measured intervals to total darkness or light, one-half of the tube was shaded
and the number of mysids in the unshaded half of the tube were recorded at
30-second intervals for a five-minute period. First the right and then the left
half of the tube was shaded to detect any bias in the mysid distribution. Control
runs with neither half being shaded were made at frequent intervals," Beeton
(1959, p. 206).
SPECTRAL SENSITIVITY IN A MYSII) 565
Biological clocks
Experiments were conducted to determine whether or not N . aincricana would
continue to migrate if light stimulation was removed. The observations were
made in Plexiglas tuhes with an inside diameter of 7.6 cm. Tubes of two lengths
were used (one meter and one-half meter) and these could be joined together
to obtain greater depth with the use of "O" rings and brass ring nuts and bolts.
Mysids were kept in the dark at a constant temperature and observed around
the period of sunset to determine whether or not they would rise to the surface
of the water in the tube, as would be expected if a biological time clock were
functioning. The animals were viewed through a U. S. Army snooper scope
using an infra-red light source (Baylor, 1959). Preliminary observations indi-
cated that mysids were quite insensitive to the red region of the spectrum and
therefore the important prerequisite that the experimental animal be unaffected
by the light source could be fulfilled. "*»
RESULTS
Spectral scnsitii'itv
In each 30-second interval the total number of mysids counted in all four color
beams averaged 10 to 15, the rest remaining in the darkened portion of the tank
or in the periphery of the light beams. Only those considered to be within beams
were counted. Of those animals which were photopositive, a significant number
stationed themselves in the blue-green light beam, the animals in this beam usually
outnumbering those in the next most densely occupied beam by approximately
2 to 1 (Table III). Fewer mysids were attracted to light passing through the
green and the blue filters, while the red beam attracted the least number of
animals. The same order and ratio prevailed regardless of the position of the
projected beams in the tank; even as the wheel containing the filters was rotated,
mysids could be seen following their respective beams. The neutral density filters
were then removed from the green, blue, and red color filters, increasing the
energy of these beams above that of the blue-green (see Tables I and II). When
this was done, the same 2 to 1 preference for the blue-green was maintained,
although there was a slight increase in numbers in the blue beam and a further
decrease in numbers in the red.
The Kolmogorov-Smirnov one-sample test was used to determine the signifi-
cance of the results. This is a test of goodness of fit and is concerned with the
degree of agreement between the distribution of a set of sample values (observed
scores) and some specified theoretical distribution. It determines whether the
scores in the sample can reasonably be thought to have come from a population
having a theoretical distribution (Siegel, 1956). This nonparametric technique
was selected because it is more powerful than the Chi Square test when there is a
continuous variable and the sample is small. D values represent maximum
deviation, and in each experiment they show that the distribution of animals was
non-random and that the animals showed significant preferences for different colors.
Reaction towards the colored lights remained the same regardless of the time
of day or the number of hours the mysids were kept in the dark. In experimental
runs in which animals were kept in the dark for over 12 hours, few could be seen
566
SIDNEY S. HERM \X
TABLI-: 1 1 1
Spectral sensitivity oj \. americana
I-.ST Time
Time in dark
(hr.)
Number counted in each 10-min. period:
Total -
mysids in
tank
5', level 1)
Red
(610 HIM)
Blue
(460 HIM)
Green
(546-559 HIM)
Blue-green
(515 HIM)
0930
2
12
29
28
71
75
.257
*
9
35
32
69
75
.221
1200
3
11
26
34
72
75
.258
*
8
32
30
76
75
.226
1022
1
19
31
49
78
75
.215
*
10
41
43
76
75
.200
1015
U
17
37
40
75
73
.195
*
10
36
35
79
73
.244
1415
H
35
44
36
81
75
.163
1535
U
27
43
46
71
71
.144
*
13
52
44
87
71
.194
1607
H
45
59
61
107
73
.142
2120
2
21
38
44
99
72
.240
*
12
39
41
99
72
.272
1230
2*
15
35
36
70
71
.198
*
11
47
47
75
71
.189
1423
2f
20
27
35
65
70
.193
1 543
4
35
46
58
104
72
.178
2125
5
35
58
48
94
69
.150
1023
6
29
52
50
93
74
.120
*
11
42
48
92
74
.227
1642
7
24
30
41
102
67
.264
*
17
56
37
88
67
.189
2215
8
13
23
34
67
46
.233
*
5
34
29
65
46
.233
0906
10|
16
32
31
64
41
.203
*
8
30
28
66
41
.250
0853
Hi
36
47
51
88
65
.148
1234
14
24
44
50
81
62
.161
1113
171
16
28
29
49
70
.151
1523
18|
24
34
33
53
60
.117
0943
211
15
28
24
55
48
.200
1757
22J
25
34
26
61
69
.168
1602
26
18
33
45
81
53
.209
*
11
36
28
78
53
.255
1100
42^
9
20
19
53
40
.168
* Indicates experiments conducted with neutral density niters removed from red, blue, a
green color filters.
ncl
in the light beams for the first three minutes, indicating good agreement with
phototactic experiments. It is also interesting to note that copepods. which were
present in the tank as food for the mysids. exhibited generally the same behavior
towards the color beams in both types of experiments.
When unfiltered light from the experimental lamp was permitted to enter the
water, all of the mysids in the colored beams were attracted to this white light of
much greater intensity. If, however, this white light was then reduced to 4.5
SPECTRAL SENSITIVITY IX A MYSID
567
microwatts 1>y interposing neutral clensitv filters, the animals again showed a
preference for the blue-green beam, despite the fact that the energy of the
\vhite light \vas greater.
TABI.K IV
Photo/in ti: response of \. americana after periods in l/'^ht and total darkness
KST Time
Dark exposure
(hrs.)
Light exposure
(hrs.)
Numbers in tube
Chi Square
Shaded
1 "ushaded
1253
i
4
22
38
4.27*
2148
1
4
13
47
19.27*
1347
3
1
13
47
19.27*
1500
1
16
44
13.06*
1 555
1
8
52
32.36*
1314
1
21
39
5.40*
2110
11
6
54
38.40*
1629
2
12
48
21.60*
1945
3
23
37
3.27
1122
3
15
45
15.00*
2301
31
31
29
.067
1944
4
26
34
1.067
2400
4
34
26
1.067
1917
5i
18
42
9.60*
1437
5,'
35
25
1.67
2136
81
9
41
20.48*
0856
Hi
30
30
.000
2259
121
38
22
4.27*
1116
13
32
18
3.91*
2206
13
32
18
3.91*
0850
141
39
21
5.40*
1 343
15|
31
19
2.88
0900
17
9
31
12.10*
0902
22
38
22
4.27*
11 11
23i
31
29
.067
1405
23i
33
27
.900
2145
45 i
31
29
.067
Control runs
0859
30
30
.000
1117
30
30
.000
1124
30
30
.000
1320
33
27
.900
1945
35
25
1.67
1952
29
31
.067
2116
35
25
1.67
2142
31
29
.067
2211
28
32
.13
2307
37
23
3.27
0013
28
32
.13
* Indicates significant Chi Square values at 5', level,
possible after each experiment.
Controls were run as frequently
568 SIDNEY S. HERMAN
Photota.ris
Six mysids were placed in the horizontal tube and subjected for measured
intervals to total darkness or light. One-half of the tube was shaded and the
numbers of mysids in the unshaded half were recorded at 30-second intervals for
a five-minute period.
Significant differences in distribution were never found in control runs, but
were found when one-half of the tube was shaded after the mysids had been in
light or dark for a period of time. Mysids were photopositive unless they had
been subjected to total darkness for 12 hours; after longer periods in the dark
they were photonegative (Table IV). The photonegative condition lasted only
for a short time, as they became light-adapted within 3 to 5 minutes of exposure
to light. Beeton (1959) found in his laboratory experiments that Mysis rclicta
could be photonegative in the morning and also in the evening, and he stated
that it was not likely that the photic response had a persistent diurnal rhythm.
The same is true of TV. auicricana, since in both the evening and the morning it
could become photonegative if kept in the dark for over 12 hours.
Experiments also revealed that mysids which were photopositive could be made
to move into the shaded area of the tube if the intensity of the light was increased.
This agrees with the results obtained by other workers (Johnson. 1938; Beeton,
1959).
Biological clocks
Examination of mysids kept in total darkness in Plexiglas tubes revealed no
significant movement towards the surface at the time of day when the animals
in the Bay were ascending. Usually the mysids remained equally distributed
throughout the tube at all times.
DISCUSSION
In spectral sensitivity experimentation on mysids, the experimental animals
have not previously been offered a choice of lights of different wave-lengths of the
same intensity. Hess (1910) worked with Mcsopodopsis slabber I and found that
if these mysids were kept for a time in the dark, and then brought into the light,
all of the animals swam rapidly towards the source of the light. When a spectrum
was passed through the tank, they rapidly congregated in the yellow-green region
and remained there. Since the relative intensities of the different parts of the
spectrum were neither controlled nor measured, the animals' apparent preference
for the yellow-green may have been due to differences in intensity.
Beeton (1959), experimenting with Mysis rclicta, measured the response of
animals in an aquarium to an experimental light which was passed through dif-
ferent combinations of Corning glass color filters and neutral density filters. He
mathematically calculated the total energy output of each filter combination, using
the per cent transmission of the color filter and the distribution curve of spectral
energy of the experimental lamp. He determined that M. rclicta showed greatest
sensitivity at wave-lengths in the vicinity of 515 m^ and 395 m^.
Results of spectral sensitivity experiments on N. americana indicate a distinct
preference for light having a wave-length of 515 m/x. The yellow-green light
SPECTRAL SENSITIVITY IN A MYSID 569
which attracted Hess' Mesopodopsis is closely approximated by the green filter
102 (546-559 HI/A), hut N. aincricana showed no preference for this under con-
trolled intensity conditions.
The experiments were conducted at a temperature of 19° C. and it would have
been desirable to repeat them at lower temperatures, since 19° C. is near the
annual maximum in Xarragansett Bay.
The comparatively high temperatures may have been one of the reasons why
relatively few of the mysids in the experimental tank were attracted to the light
beam. On the other hand, the field results of this study indicate that not all of
the mysids undergo vertical migration, some animals remaining on the bottom
during the night throughout the year. Thus there must be other physiological
mechansims operating both in the field and in the laboratory, which are responsible
for keeping certain members of the population from responding to light by
migrating vertically.
In the spectral sensitivity experiments it was showrn that mysids still preferred
the blue-green light beam even when the intensity of the other colored light beams
was greater. Ar. aincricana is capable of distinguishing between colors differing
in wave-band maxima by only 31-35 m/x., and shows a distinct preference for one
of these. Even where greater intensity is present, the mysids seek out this blue-
green light. When unfiltered light from the experimental lamp was passed into
the aquarium, the mysids quickly congregated in this light, deserting all of the
color beams. However, when this light was adjusted with Wratten neutral
density filters (4.5 microwatts cm.~-), reducing its intensity to a level which was
still above that of the colored beams, the mysids again showed a preference for the
blue-green. According to John Roche of the Eppley Laboratories (personal com-
munication), when the unfiltered light from the experimental lamp was projected
into the tank, more light of the wave-length 515 m^u, was present in this beam
than in the blue-green beam, but when this white light was reduced with neutral
density filters, less light of 515 mp, was present in the white beam than in the
blue-green beam. In each case the mysids congregated in that beam transmitting
the greatest amount of energy of the wave-length 515 m/x.
Experiments indicate that it is not likely that the photic response in A', aincr-
icana is governed by a biological time clock. Experiments also revealed that
12 hours in continuous darkness are required to make this species photonegative.
The significance of these findings in regard to the vertical migration of A7, aincr-
icana will be discussed in a subsequent publication.
Dr. David M. Pratt reviewed the manuscript and Mr. Theodore A. Napora
gave valuable assistance during the experiments. Calibration of equipment and
test runs of the entire experimental apparatus were conducted at the Eppley
Laboratories, Newport, Rhode Island.
SUMMARY
1. Laboratory experiments were conducted to determine the spectral sensitivity
of N. aincricana. The intensity of light beams passing through four Corning glass
color filters was made equal with Wratten neutral density filters.
570 SIDNEY S. HERMAN
2. The positively phototactic animals showed a definite preference for light
passing through a color filter having peak transmission at 515 nip..
3. Increasing the intensity of light passing through the three other color filters
did not alter the mysid preference for the wave-length 515 m/x.
4. Phototactic experiments revealed that N. amcricana was photopositive
unless subjected to total darkness for \1 hours; after longer periods in the dark
they were photonegative.
5. Experiments indicate that it is not likely that the photic response in .V.
amcricana is governed by a biological time clock.
LITERATURE CITED
BAYLOR, E. R., 1959. Infra-red observations and cinematography of Microcrustacea. Liuniol.
Occanogr., 4: 498-409.
BEETON, A. M., 1959. Photoreception in the opossum shrimp, Mvsis rchcta Loven. Biol. Bull..
116: 204-216.
GUSHING, D. H., 1951. The vertical migration of planktonic Crustacea. Biol. Rcr.. 26: 158-192.
HESS, C, 1910. Neue Untersuchungen iiber den Lichtsinn bei wirbellosen Tieren. Pfluci/cr's
Arch., 136: 282-367.
HULBURT, E. M., 1957. The distribution of Neomysis uniencuiut in the estuary of the Delaware
River. Liinnol. Oceanogr., 2: 1-11.
JOHNSON, W. H., 1938. The effect of light on the vertical movements of Acortia cluusii
((iiesbrecht). Bin/. Bull.. 75: 106-118.
SIEGEL, S., 1956. Non Parametric Statistics for tlic Beliavioural Sciences. McGraw-Hill,
New York, 312 pp.
FURTHER STUDIES ON FEEDING AND DIGESTION IX
TRICLAL) TURBFLLARIA
J. B. JKXXINGS
/ >epiirtiuenf of /ni'lin/v. The L'nirersit\ of Leeds, England
Previous accounts ( ' I callings, 1957, 1959) have shown that the triclad Turbel-
laria feed oa a variety of iavertehrate animals, such as annelids, molluscs, crustaceans
and insect larvae, and that the basis of the feeding mechanism is the protrusible
plicate pharynx which is thrust through the integument of the prey to withdraw
body contents and pass them back in a finely divided condition into the flatworm gut.
The penetration of the prey and the subsequent disruption of its tissues appear to lie
achieved largely by direct muscular action, but the possibility that this is supple-
mented by some enzymatic activity has not been investigated, apart from a brief
study by Westblad (1922), who failed to find digestive activity in extracts of
Dendrocoelum or Pol \cclis pharynges. On arrival in the gut the food is phagocy-
tosed by columnar cells of the gastrodermis and digested intracellularly. The
sequence of food vacuole formation and intracellular digestion has been described
in detail (Willier, Hynian and Rifenburgh, 1925; Jennings, 1957, 1959) but little is
known of the enzvmes concerned, other than the fact that the food vacuoles contain
j
acid phosphatase and leucine aminopeptidase (Rosenbaum and Rolon, 1960).
In the present investigation two species of triclad Turbellaria, one aquatic and one
terrestrial, have been investigated by histochemical methods, to locate and identify
any enzymes produced by the pharynx to assist its penetration and disorganization of
the food. The course of digestion has been similarly investigated, in each species,
in an attempt to identify more of the enzymes concerned and to establish the
sequence in which they are produced.
MATERIALS AND METHODS
The two triclad species examined were Polycelis coniuta Schmarda ( fresh-water)
and Ortliodcunis tcrrcstris (terrestrial). The bulk of the work was carried out on
the fresh-water species because of its relative abundance and ease of maintenance in
the laboratory.
Flatworms starved for 7 days to clear the gut of traces of previous meals were fed
oa liver, beef fat or starch paste, the two latter foods being made attractive by
mixing with frog blood. The foods were heated to 100° C. and subsequently cooled
before being presented to the flatworms, to prevent their inherent enzyme activity
being confused with any produced within the flatworm pharynx or gut. The flat-
worms were fixed at progressive intervals up to 48 hours after an observed meal on
one or another of the test foods, and serial sections cut at 8 p. examined for enzyme
activity in the pharynx, gut lumen and gastrodermis. Full details of the methods
used for fixation, preparation of sections and visualization of enzyme activity have
571
572 j. B. JENNINGS
been given in an earlier account of similar studies on digestion in the rhynchocoelan,
Linens ntbcr (Jennings, 1962), and are only summarized here.
Fixation was for 12 hours at 4° C. in 10% formalin buffered to pH 7.0, followed
by rapid dehydration in absolute acetone at the same temperature and subsequent
embedding in either polyester wax (melting point 37° C.) or paraffin wax (42° C. ).
When the latter was used, brief clearing in xylol at room temperature was necessary.
The polyester technique gave a better histological picture but caused a significant
decrease in the demonstrable amount of certain enzymes, notably phosphatases and
aminopeptidase, despite the apparent advantage of the lower melting point of the
wax, and consequently paraffin wax was used almost exclusively for studies on
these particular enzymes. Proteolytic enzymes were demonstrated by the Hess and
Pearse (1958) method for endopeptidases of the cathepsin C type (homologous with
mammalian chymotrypsin), using as controls incubation media containing cysteine
or lead nitrate which act respectively as specific activator or inhibitor, and by the
Burstone and Folk (1956) method for exopeptidases of the leucine aminopeptidase
type, using heat-inactivated sections as controls. Lipolytic activity was demon-
strated after a meal containing beef fat by the Tween 80 method of Gomori (1952),
again with heat-inactivated controls. Attempts were made to demonstrate diastatic
activity by the Billet and McGee-Russell (1955 ) method but this gave unsatisfactory
results and the presence of carbohydrate-splitting enzymes could only be inferred by
tracing progressive digestion of a starch meal by the Lugol's iodine technique. Acid
and alkaline phosphatases were visualized by the glycerophosphate methods of
Gomori (1952), and controls performed by omitting the substrate from the incuba-
tion media and by heat inactivation of sections. The pharynx was examined for
possible carbonic anhydrase activity, often associated with production of acid diges-
tive juices, by Hausler's cobalt method (1958), with control sections incubated in
the presence of Diamox sodium, a specific inhibitor for this enzyme.
Incubation times at 20° C. and the pH values of the various incubation media
can be found in the study on rhynchocoelan digestion referred to earlier.
OBSERVATIONS
The structure of the pharynx and gut
The structure of the triclad pharynx and of the gut and its lining gastrodermis
have been described in detail elsewhere (Hyman, 1951; Jennings, 1957). Briefly,
in both species investigated here, the pharynx is a highly muscular tube which lies
in the pharyngeal chamber in the posterior region of the body. It is directed back-
wards and can be protruded through the ventral mouth by simple muscular
elongation. The pharynx contains along its entire length outer and inner longi-
tudinal and circular muscle layers, a layer of acidophil and basophil gland cells
between these, radial muscles and a well developed nerve plexus. The gut proper,
in each species, is of the typical triclad pattern with one anterior and two posterior
branches, each of which is further subdivided. The gastrodermis consists of a
single layer of cells standing on a thin basement membrane and containing only two
cell types. The larger and more numerous cells are columnar, 35—40 /* in height,
with basal nuclei and granular cytoplasm usually containing phagocytosed food in
various stages of digestion. The second type of cell is the "granular club" (Hyman,
DIGESTION IN TRICLAD TURBELLARIA 573
1951 ) or "sphere cell" (Jennings, 1957) and is pear-shaped, 20-30 //, in height, and
contains numerous homogeneous spheres which in the fully developed cell are
intensely acidophilic and stain strongly with Millon or similar reagents for protein.
The spheres within any one cell are always of the same size and appear to mature
with the cell. Thus, in small sphere cells the spheres are 1 //, or less in diameter
and increase up to 5-6 ^ in the mature cell. During prolonged starvation the
number of sphere cells decreases, relative to the columnar cells, and the spheres of
those persisting show reduced affinity for stains.
Enzymes produced in the pharynx and gut during feeding and digestion
( 1 ) The pharynx
In both Polycclis and Orthodennts a large proportion of the acidophil gland cells
of the pharynx show a strong positive reaction for endopeptidases of the cathepsin
C type, particularly around the free distal end (Fig. 1 ) . The glands are flask-shaped
and open on to the outer surface, only, of the pharynx (Fig. 2), never into the lumen.
Sections of the pharynx prepared immediately after feeding showed that many of
these gland cells were discharged and shrunken, and there can be little doubt that
their secretions are used to supplement the muscular pressure exerted by the
pharynx during the penetration of the prey, by softening or dissolving the tissues
of the body wall. The marked concentration of gland cells around the tip of the
pharynx supports this conclusion.
Penetration of the prey occupies 30 to 60 seconds and once within it the pharynx
moves about and draws up organs, tissues and body fluid. This part of the feeding
process may last for several minutes, and again there can be little doubt that break-up
of the prey's body contents by the muscular activity of the pharynx is supplemented
by proteolysis effected by secretions from the pharyngeal glands. Since these open
on to the outer surface of the pharynx and not into the lumen, their secretions are
presumably poured into the body cavity of the prey to attack and disrupt its contents
whilst tissues already disorganized are being ingested. In this connection it is
significant that the pharynx is always inserted into the prey, even when the latter is
manifestly small enough to be swallowed whole, as when oligochaetes of a smaller
diameter than the resting pharynx are captured. In such cases the pharynx, or its
distal portion, is extended until it is slim enough to enter the prey in the usual
manner (Fig. 3) and so allow the secretions of the pharyngeal glands to attack its
contents. This feeding pattern is followed even with test meals of blood or finely
chopped liver, when the pharynx enters the food mass and withdraws material from
the center rather than merely being applied to the surface layers.
The optimum pH for visualizing the endopeptidase activity was 5.0, and conse-
quently it was thought that the pharynx might produce acid to provide the proteolytic
secretions with the necessary working conditions. The enzyme often concerned
with production of acid digestive juices is carbonic anhydrase, but no trace of this
enzyme could be found in either the Polycclis or Orthodcnius pharynx.
The cytoplasm of the acidophil endopeptidase gland cells shows at all times a
weak reaction for acid phosphatase. No other enzyme activity, proteolytic or
otherwise, could be detected in the pharynx of either species.
574
J. B. JKXXLV.S
:- .r;r r:
/^ rr \
/ * F
i
FIGCKE 1. Longitudinal section of the Polycclis pharynx, showing the distribution of
acidophil endopeptidase-producing gland cells. Hess and Pearse method. The tissue at the
extreme bottom right is body wall and epidermis, and the dark bodies seen here are rhabdites
which have stained strongly with the eosin counterstain. Scale : 1 cm. = 125 fi.
FIGURE 2. A portion of the outer layers of the Polycclis pharynx, showing endopeptidase
gland cells discharging on to the outer ciliated epithelium. Hess and Pearse method. Scale :
1 cm. = 25 p..
FIGURE 3. The Pulycclis pharynx attacking a small oligochaete. Note that the pharynx
( P.) has been inserted into the oligochaete and that only the integument (I.) remains outside the
pharynx. Unstained whole mount. Scale : 1 cm. = 250 /j..
DIGESTION IN TRICLAD TURBELLARIA 575
( 2 ) The gut
Cathepsin C type endopeptidases
The spheres of the gastrodernial sphere cells show in both species an intense
positive reaction for the cathepsin C type endopeptidases (Fig. 4), and a similar
reaction is given by fine granules which occur in the cytoplasm of the columnar
cells when these are cleared of digesting food by 2 to 3 days' starvation.
Flatworms killed immediately after a meal of boiled liver show faint traces of
endopeptidase activity in the material lying in the gut lumen, and this is derived, no
doubt, from secretions poured on to the food by the glands of the pharynx before
ingestion. The amount of endopeptidase activity in the contents of the lumen
increases with time up to a maximum reached 4 hours after feeding (Fig. 5), and
during this time there is a decrease in the number of the large and mature sphere
cells relative to the number of columnar cells. This decrease in the number of
sphere cells is not constant throughout the gastrodermis, however, and some regions
may be quite devoid of them whilst others have the normal complement. Usually the
disappearance of sphere cells from a region of the gut coincides with the presence
of food and the development of maximum endopeptidase activity in that region, but
food showing such activity may be found in parts of the gut lined by the normal
proportions of sphere and columnar cells. Such situations are probably due to
material in the lumen being passed into a region of the gut away from that where
the enzyme activity originated, by the convulsive contractions of the flatworm during
fixation. Individual spheres of the same size and reaction as those within mature
sphere cells are occasionally found either between columnar cells or lying free in the
gut lumen. It would appear from this, and the decrease in sphere cell numbers
noted above, that mature sphere cells discharge their contents when food enters the
gut, and that the lumen endopeptidase activity comes from this source.
The endopeptidase activity developed in the gut lumen does not cause complete
homogenization of the food, and even at the peak of its activity, as shown by the
intensity of the histochemical reaction, distinctive components of the food, such as
erythrocytes, muscle fibers, liver cell nuclei, etc., are often clearly recognizable. The
columnar cells of the gastrodermis commence phagocytosis of the food immediately
it enters the gut, and the smaller food particles pass rapidly into the cells so that they
are not exposed for long to the lumen proteolysis. The function of the latter appears
to be primarily to facilitate phagocytosis by softening and breaking up the larger
pieces of the food, rather than to render it completely soluble. There appears to be
only an initial discharge of endopeptidase when food enters the gut. not a continuous
one for as long as it remains in the lumen, and sections prepared at intervals up to
48 hours after feeding show that any food particles too large for phagocytosis which
survive this initial discharge persist unchanged until eventually expelled from the
gut. This situation is particularly liable to arise if starved flatworms are allowed to
FIGURE 4. The gastrodermis in a Polycclis starved for 7 days, showing sphere cells and
columnar cells. The sphere cells show an intense positive endopeptidase reaction. Hess and
Pearse method. Scale : 1 cm. = 20 p..
FIGURE 5. Transverse section of a portion of the Polycclis gut 4 hours after a meal of
boiled liver. Liver lying in the gut lumen ( top left ) shows a positive endopeptidase reaction,
especially the right-hand portion, and the gastrodermis is loaded with phagocytosed liver showing
a similar but stronger reaction. Hess and Pearse method. Scale : 1 cm. = 40 /u.
576
J. B. JENNINGS
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FIGURE 6. Transverse section of a portion of the Orthodcnnts gut 12 hours after a meal of
boiled liver. The gastrodermis is loaded with food vacuoles, all showing an intense positive
reaction for leucine aminopeptidase. Burstone and Folk method. Scale : 1 cm. — 20 /j,.
FIGURE 7. Transverse section of a branch of the Orthodcmus gut 12 hours after a meal
containing a large proportion of fat. Many of the food vacuoles show lipolytic activity, seen here
as black spheres or granules. Gomori Tween 80 method. Scale : 1 cm. = 20 /u.
FIGURE 8. Transverse section of the Polycclis gut 4 hours after a meal of boiled liver.
The cytoplasm and food vacuoles show acid phosphatase activity (dark areas). Gomori method.
Scale : 1 cm. = 40 /u.
FIGURE 9. Transverse section of the Orthodctuus gut as in Figure 6 but treated here for
alkaline phosphatase. The food vacuoles show intense alkaline phosphatase activity. Gomori
method. Scale : 1 cm. = 20 /x.
DIGESTION IN TRICLAD TURBELLARIA 577
feed until replete, when they often ingest more food than can he adequately dealt
with in the lumen. The persistence of unchanged food elements in the lumen for
up to 48 hours after feeding has heen interpreted previously as showing the complete
absence of intraluminar digestion (Jennings, 1957), hut the present demonstration
of endopeptidase activity in the contents of the lumen leaves little doubt as to the
occurrence of at least a limited amount of intraluminar digestive activity in the two
species investigated here.
Food phagocytosed from the lumen continues to show endopeptidase activity
within the vacuoles of the columnar cells, and since this increases in intensity as
the vacuoles pass back deeper into the cells, endopeptidases must be secreted into
the vacuoles from the surrounding cytoplasm, perhaps from the reactive granules
so prominent when the cells are cleared of other inclusions. As the vacuoles pass
back into the columnar cells, more form distally until the cells are loaded with food
undergoing intracellular digestion and showing intense endopeptidase activity
(Fig. 5 ). Eight to 12 hours after feeding, the contents of the vacuoles are reduced
to compact homogeneous masses, and the endopeptidase activity fades gradually,
first from vacuoles deep within the cells and then from the rest, indicating that the
first stage of digestion affecting breakdown of protein to peptones and polypeptides
is completed. The food then passes into the second stage of digestion, in which
exopeptidases complete proteolysis down to amino acids, and lipases and carbohy-
drases attack fats and carbohydrates exposed by the digestion of cell walls or other
cytoplasmic membranes.
The optimum pH value for visualizing endopeptidase activity in the lumen and
gastrodermis of both species was pH 5.0, and this indication that the first stage of
digestion is carried on in an acid medium agrees with the results obtained by feeding
test foods plus indicators when a pH value of 4.6 was found in the food vacuoles 6
hours after feeding (Jennings, 1957).
Lencinc aminopeptidase (c.vopeptidasc]
Leucine aminopeptidase activity is confined to the columnar cells of the gastro-
dermis in both species and was never found in either the gut lumen or the
sphere cells.
As endopeptidase activity fades from the food vacuoles it is gradually replaced by
leucine aminopeptidase, supplemented, presumably, by other exopeptidases not
demonstrated by the technique used here. The time of onset of the exopeptidase
activity varies with the size of the original meal which influences the amount of food
phagocytosed by the columnar cells. Thus, when only a small meal is taken, and
each columnar cell forms relatively few food vacuoles, exopeptidase activity appears
in the latter as early as two hours after feeding, but when a large meal has allowed
the columnar cells to become packed with food vacuoles, the activity may not appear
for 12 to 18 hours. On the average, endopeptidase activity is replaced by exopep-
tidase 8 to 12 hours after feeding.
The exopeptidase activity overlaps the endopeptidase, to a degree determined
FIGURE 10. Longitudinal section through the anterior end of Poly cells, showing an eye (E.)
and acidophil endopeptidase-producing gland cells ( G.C. ) in the parenchyma, which discharge
through the epidermis. Rhabdites in the latter are stained strongly by the eosin counterstain.
Hess and Pearse method. Scale : 1 cm. = 25 /u.
578 J. B. JENNINGS
by the amount of food ingested, and recently formed vacuoles in the distal region of
the columnar cells may show marked endopeptidase activity whilst those deeper
within the cell give a weak exopeptidase reaction. The latter increases in strength
with time and appears in more and more of the vacuoles until eventually all food
undergoing intracellular digestion gives an intense reaction (Fig. 6). This activity
persists for as long as food remains in the gastrodermis and, depending upon the
size of the meal taken, may still he present 48 hours after feeding.
Columnar cells of starved flatworms show no reaction for leucine aminopeptidase,
and it would appear that exopeptidases, unlike the endopeptidases, are normally
present in an inactive form and are not activated until food vacuoles are present.
The optimum pH for visualizing exopeptidase activity was 7.2 in both species,
indicating that the second and final stage of proteolysis proceeds in a slightly alkaline
medium, as in most other animals.
Lip as e
In both species the gastrodermis shows a small amount of lipolytic activity during
starvation, and this probably represents the utilization of reserve fat which is laid
down in the columnar cells when food is plentiful (Jennings, 1957, 1959).
Both species show lipolytic activity in food vacuoles formed within the columnar
cells after a meal containing beef fat (Fig. 7 ), and this develops as the endopeptidase
activity fades. No lipolysis was found in the gut lumen, and pieces of fat too
large for phagocytosis lay there quite unchanged until expelled from the gut.
The optimum pH value for demonstrating lipolytic activity was 7.2 in both cases,
the same as for the exopeptidase.
Carbohydrascs
No conclusive results were obtained from the Billett and McGee-Russell method
for /3-glucuronidase when applied to sections of flatworms killed at intervals after
meals containing boiled starch. Treatment with Lugol's iodine, however, showed
progressive conversion and disappearance of the starch within food vacuoles, whilst
any remaining in the lumen was quite unchanged. Thus, both Polycelis and
Orthodcmus possess diastatic enzymes but these remain as yet unidentified.
Phosphatases
Both acid and alkaline phosphatases occur in the columnar cells of the gastro-
dermis during intracellular digestion, and there is a marked correlation between the
development of endopeptidase and acid phosphatase, in the first stage, and the
remaining digestive enzymes and alkaline phosphatase, in the second.
In starved individuals the cytoplasm of the columnar cells shows a weak reaction
for acid phosphatase but none for alkaline. As food vacuoles form and endopep-
tidase activity develops within them, there is a simultaneous but less marked increase
in acid phosphatase in both the vacuoles and the surrounding cytoplasm. The peak
of acid phosphatase activity coincides with that of the endopeptidase (Fig. 8), but
at no time is the reaction particularly intense. This is due, perhaps, to loss of
enzyme during preparation of sections, since check sections of mammalian tissue
treated in the same way show less than the expected amount of acid phosphatase.
DIGESTION IN TRICLAD TURBELLARIA 579
The acid phosphatase activity decreases as the endopeptidases fade from the
vacuoles, and is gradually replaced by alkaline phosphatase. This develops in both
cytoplasm and vacuoles simultaneously with the leucine aminopeptidase and lipase,
and at its peak every vacuole shows a most intense reaction (Fig. 9). The activity
persists for as long as food vacuoles are present in the columnar cells.
Neither acid nor alkaline phosphatases are demonstrable in the sphere cells or
gut lumen at any stage of digestion.
The pH optima for demonstration of the two phosphatases were, respectively, pH
5.0 (acid) and pH 9.0 (alkaline).
Enzymes in the parenchyma
During starvation, regions of the parenchyma often show a weak reaction for
endopeptidase, aminopeptidase and lipase activity. This reflects, no doubt, the
utilization of reserve protein and fat which are stored in the parenchyma ( Jennings,
1957).
Certain acidophil gland cells which lie in the parenchyma along the anterior
margin of the body give a marked endopeptidase reaction (Fig. 10). These glands
are of an elongate flask shape and discharge between the epidermal cilia. Their
function is unknown, but it is possible that their secretions are passed over the body
by ciliary action during locomotion to help in keeping the surface free of micro-
organisms or to make the flatworm distasteful to would-be predators.
DISCUSSION
The main features of interest emerging from the present study on triclad feeding
and digestion are, respectively, the demonstration of proteolytic activity in the
acidophil gland cells of the pharynx and the proof that digestion is not exclusively
intracellular as was previously believed.
The presence of cathepsin C type endopeptidases (proteases of the type initiating
proteolysis ) in the pharynx glands, the concentration of such glands around the tip
of the pharynx, and the discharged and shrunken appearance of the glands im-
mediately after feeding leave little doubt that proteolytic secretions are used to
supplement muscular action in the penetration and subsequent internal disorganiza-
tion of the prey by the pharynx. The triclads are not unique amongst the Turbel-
laria in this respect, however, for the acotylean polyclads likewise use proteolytic
secretions from the pharynx or gut to supplement the muscular action of the
pharynx during pre-ingestion break-up of the food (Jennings, 1957). In their case
the pharynx is of the same basic structure as the triclad but is much expanded to
form a ruffled curtain — the ruffled plicate pharynx — which is extended over the prey
to envelope it and act as an external "stomach," rather than being inserted into it
to act as a suction tube.
The belief that digestion in the triclad is exclusively intracellular rested on the
fact that recognizable food elements may persist in the lumen for up to 48 hours
after feeding, but whilst the present work has confirmed that this does occur, for
reasons mentioned in the text, it has also shown beyond reasonable doubt that there
is some intraluminar digestion by endopeptidases.
The endopeptidase responsible for intraluminar digestion is produced by the
580 J. B. JENNINGS
sphere cells of the gastrodermis which in the past have been regarded as protein
reserve cells (Hyman, 1951 ; Jennings, 1957). This conclusion was based on the
progressive reduction in the number of sphere cells during starvation, but in view
of the undoubted glandular nature of these cells this probably represents a simple
regression, such as occurs in the cells of other animal digestive organs during
prolonged starvation, rather than the utilization of specific protein reserves.
Intraluminar digestion is followed by phagocytosis and completion of digestion
intracellularly by exopeptidases, lipase and carbohydrases. This sequence of events
closely resembles that occurring during digestion in the related rhynchocoelan,
Linens rnber (Jennings, 1962). In the rhynchocoelan, however, lumen digestion
is far more extensive and results in the food being completely homogenized before
it enters the gut cells. This is clearly related to the fact that the food is swallowed
whole, whereas in the triclad it is already considerably broken up when it reaches
the gut, and the bulk of it is immediately available for phagocytosis and intracellular
digestion. Consequently there is relatively less intraluminar digestion in the triclad,
and what does occur appears to be aimed at reducing the particle size of the food to
make it available for phagocytosis, rather than at achieving complete breakdown to
simpler substances.
The difference in the amount of intraluminar digestion in the triclad and the
rhynchocoelan, itself the result of differences in the respective feeding mechanisms,
is reflected in the subsequent intracellular processes. In the rhynchocoelan, food
entering the gastrodermis passes almost immediately into the second exopeptidase
stage of digestion. In the triclad, food may enter the gastrodermis only slightly
affected by the lumen-acting endopeptidase, or even completely unaffected, if phago-
cytosed soon after the meal, and consequently it must first be attacked by endo-
peptidases before it is available to the later acting enzymes. As a consequence of
this there is far more intracellular endopeptidase activity in the triclad than in the
rhynchocoelan. This affords a good demonstration of the effect a particular type of
feeding mechanism may have upon subsequent digestive processes.
The two types of phosphatase found in the triclad gut appear to be linked with
formation of the intracellular enzymes. Acid phosphatase is closely linked with
the first or endopeptidase stage, and Rosenbaum and Rolon (1960) suggest that
it may be concerned with food vacuole formation. Alkaline phosphatase is linked
with the appearance of the later acting enzymes, and may well be concerned in the
release of energy needed for secretion of the various enzymes and the absorption of
the products of digestion from the vacuoles.
SUMMARY
1. Feeding and digestion in two species of triclad Turbellaria, one aquatic, the
other terrestrial, have been investigated by histochemical methods to locate and
identify a selection of the enzymes concerned in the two processes.
2. In both species the pharynx possesses acidophil gland cells which produce
endopeptidases of the cathepsin C type, and the available evidence indicates that
these are used to assist the pharynx in its penetration of the prey's body wall, and
the subsequent disruption of the body contents prior to ingestion.
3. Food entering the gut is attacked by extracellularly-acting endopeptidases,
similar to those produced in the pharynx, and originating from the sphere cells of
DIGESTION IN TRICLAD TURBELLARIA 581
the gastrodermis. This intraluminar digestion continues and extends break-up of
the food initiated by the pharynx, and serves to make the bulk of it available for
phagocytosis and intracellular digestion.
4. Columnar cells of the gastrodermis phagocytose food from the gut lumen and
digest it within vacuoles containing enzymes secreted from the cytoplasm in a
definite sequence.
5. The contents of the food vacuoles are attacked first by endopeptidases similar
to those secreted into the gut lumen and acting in an acid medium of pH 5.0.
6. Endopeptidase activity within the vacuoles is eventually replaced by exopep-
tidases, such as leucine aminopeptidase, plus lipase and unidentified carbohydrases,
all acting in a slightly alkaline medium of pH 7.2.
7. Secretion of the various intracellular enzymes involves the appearance of
phosphatases in both the cytoplasm and the vacuoles of the columnar cells. Acid
phosphatase appears to be concerned with the secretion of endopeptidase in the first
stage of intracellular digestion and alkaline phosphatase with the production of the
other digestive enzymes.
LITERATURE CITED
BILLETT, F., AND S. M. McGEE-RussELL, 1955. The histochemical localisation of 0-glucuronidase
in the digestive gland of the Roman snail (Hcli.v poinatia). Quart. J. Micr. Sci., 96:
35-48.
BURSTONE, M. S., AND J. E. FOLK, 1956. Histochemical demonstration of aminopeptidase.
/. Histoclicju. Cytochem., 4: 217-226.
GOMORI, G., 1952. Microscopic Histochemistry. University of Chicago Press, Chicago.
HAUSLER, G., 1958. Zur Technik und Spezifitat des histochemischen Carboanhydrasenachweises
im Modellversuch und in Gewebsschnitten von Rattennieren. Histochemie, 1 : 29-47.
HESS, R., AND A. G. E. PEARSE, 1958. The histochemistry of indoxyl-esterase of rat kidney with
special reference to its cathepsin-like activity. Brit. J. Exp. Path., 39: 292-299.
HYMAN, L. H., 1951. The Invertebrates. Vol. II: Platyhelminthes and Rhynchocoela.
McGraw-Hill Book Co. Inc., New York.
JENNINGS, J. B., 1957. Studies on feeding, digestion and food storage in free-living flatworms
(Platyhelminthes: Turbellaria). Biol Bui!., 112: 63-80.
JENNINGS, J. B., 1959. Observations on the nutrition of the land planarian Orthodcmus
tcrrcstris ( O. F. Miiller). Bwl Bull.. 117: 119-124.
JENNINGS, J. B., 1962. A histochemical study of digestion and digestive enzymes in the
rhynchocoelan Linens rubcr (O. F. Miiller). Biol. Bull, 122: 63-72.
ROSENBAUM, R. M., AND CARMEN I. ROLON, 1960. Intracellular digestion and hydrolytic enzymes
in the phagocytes of planarians. Biol. Bull., 118: 315-323.
WESTBLAD, E., 1922. Zur Physiologic der Turbellarien. I. Die Verdauung. II. Die Exkretion.
Lunds Univ. Arsskrift, nF., Avd. 2 18. 1.
WILLIER, B. H., L. H. HYMAN AND S. A. RIFENBURGH, 1925. A histochemical study of intra-
cellular digestion in triclad flatworms. /. Morph., 40: 299-340.
THE "HERTWIG EFFECT" IN TELEOST DEVELOPMENT
R. LASHER AND R. RUGH 1
Marine Biological Laboratory, Woods Hole, Mass., and Dcpt. of Radiology,
Columbia University, Art'«' York 32, New York
In 1911 O. Hertwig found that when frog sperm were treated to prolonged
exposures of radium they retained their ahility to fertilize eggs but lost their
genetic function. The result was similar to parthenogenetic (gynogenetic) de-
velopment, wherein the egg developed without benefit of sperm chromatin. Since
1911 the study of parthenogenesis produced by this method has been limited,
among the vertebrates, almost exclusively to the Amphibia. It is therefore of
interest to determine whether the exposure of other vertebrate sperm to ionizing
radiations could similarly result in parthenogenetic development.
MATERIALS AND METHOD
Fundulus heteroclitus is a marine teleost common in the Woods Hole area,
readily obtained by the Marine Biological Laboratory Supply Department. They
are kept in the laboratory in running sea water until used. The method for
obtaining eggs and rearing the embryos is that described by Costello ct al. (1957).
Prior to x-irradiation a concentrated suspension of sperm was prepared by
removing the testes from five to six sexually mature males and placing them on
a plastic depression plate and macerating them. A portion of this suspension was
then removed to another and similar plate to be kept and used as control. The
remaining sperm were irradiated in a cesium- 137 irradiator at an output of 5000
r/min. The exposures used were 500 r, 1000 r, 5000 r, 50,000 r, 100,000 r, and
150,000 r. Sperm samples were removed at appropriate intervals for the fertiliza-
tion of normal eggs.
Fertilization was accomplished by placing normal eggs, recently stripped, in
a stender dish with a very small volume of filtered sea water, and adding the sperm
by means of a glass rod dipped into the appropriate suspension. Between each
use the rod was washed in tap water to kill any adherent sperm, and was thoroughly
dried. In the earlier series the fertilization occurred immediately after irradiation
of each portion of sperm, but since the time required for the highest level of
irradiation was brief, all later series were fertilized simultaneously following the
completion of all irradiations. It was found that sperm samples added to the
normal eggs were all active, showing motility even after 150,000 r.
The developing embryos were raised in fingerbowls containing filtered sea
water, to avoid unnecessary contamination. All non-cleaving eggs were removed
after the cleaving eggs had attained the blastodisc stage. At the end of the experi-
ment the embryos were again photographed, and fixed in Bouin's solution and
1 Under contract AT-30-1-2740 for the Atomic Energy Commission and aided by PH
Grant RH 97 administered by the senior author.
582
X-IRRADIATION OF FUNDULUS SPERM
583
prepared for possible cytological study according to the method of Costello
et al. (1957).
EXPERIMENTAL DATA
In all cases, the per cent of eggs which cleaved, following fertilization with
x-irradiated sperm, was half or less than that of the control. The per cent cleavage
did not necessarily decrease proportionately with increased exposure to the sperm
(see Table I). Normal variations in cleavage per cent are such that separate
controls of the same eggs were used for each series. As the season progressed
fewer eggs were available and fewer of the control eggs developed.
Among those eggs fertilized with irradiated sperm there was no observable lag
in the cleavage time for the first division and until the blastula stage, when
compared with the controls. This held true for all irradiation levels, even at
150,000 r to the sperm. Following gastrulation (stage #12; see Oppenheimer,
1937; Solberg, 1938; or Rugh, 1962) most eggs fertilized by sperm which had
TAHLE I
Percentage of eggs developing (cleaving) after fertilization with x-irradiated sperm*
Exposure (r)
Series 1
Series 2
Series 3
Control-0
84.1 (107)
100 (25)
20 (5)
500 r
29.5 (44)
1000 r
47.2 (36)
2000 r
42.0 (50)
5000 r
47.1 (34)
46.6 (30)
50,000 r
53.1 (49)
7.1 (28)
20 (10)
100,000 r
3.4 (29)
7.7 (13)
150,000 r
7.1 (14)
* Note: Total number of eggs examined in parentheses.
been exposed to 5000 r or more showed some slight retardation over the controls,
to the extent of about one full stage of development (Plate I, Figs. 1-8). Those
eggs fertilized with sperm receiving the higher exposures of 50,000 r or more
showed a slightly greater retardation than those fertilized by sperm which had
been irradiated to a lower level. However, the anomalies following fertilization
with 5000 r sperm appeared to be more severe than those arising from sperm
exposed to higher levels of irradiation. Some retardation was seen in eggs ferti-
lized by 2000 r sperm, and this retardation occurred beginning at stage #14,
while the lower exposures delayed the retardation to stage #22. Sperm exposed
to 500 r were unable to adversely affect development.
In eggs fertilized by sperm exposed to 5000 r or 50,000 r, some appeared to
develop equally well with the controls in every respect (Plate I, Figs. 9, 10 and
Plate II, Figs] 11, 12).
More than half of the embryos developing from eggs fertilized by sperm
exposed to 5000 r or more developed pulsating hearts and pigment patterns similar
to those of the controls, but all were stunted or otherwise malformed with the
exception of the few aforementioned (Plate I, Figs. 5-8, Plate II, Figs. 13-16).
Some of these embryos developed corpuscles, and many showed these corpuscles
584
R. LASHER AND R. RUGH
PLATE I
FIGURES 1-4. All embryos are from the same series and all are one day in development.
FIGURE 1. Control, midgastrula. FIGURE 2. From 50,000 r to sperm, early gastrula. FIGURE 3.
From 100,000 r to sperm, early gastrula. FIGURE 4. From 150,000 r to sperm, late blastula.
X-IRRADIATION OF FUNDULUS SPERM 585
circulating. In all embryos not possessing a pulsating heart, edema developed.
The heart beat, even without corpuscles, was found to be of a rate similar to that
of the normal controls with their full complement of corpuscles.
One severe abnormality rather common to both the 5000 r and the 50,000 r
series was the failure of the embryo to form either a neural or body axis. The
blastoderm developed into an amorphous mass of protoplasm devoid of any
recognizable structure, but often possessing pigment cells on the surface. Some
eggs ceased development at late blastula or early gastrula stages.
DISCUSSION
The work of O. Hertwig (1911), Oppermann (1913), Porter (1939), Rugh
(1939) and others has shown that haploid development (either androgenetic or
gynogenetic) generally exhibits a specific set of anomalies. Among the Amphibia,
haploid development appears to be normal until gastrulation, when a delay in
development is noted when comparisons are made with simultaneous controls.
Neurulation is even more delayed and abnormal. In older embryos (six to eight
days), the brain is poorly differentiated and the circulatory system is non-
functional, although heart and corpuscles may have formed. Edema generally
appears under such conditions, probably because of the failure in excretory func-
tion. Tail formation is retarded, giving the haploids a stunted appearance,
accentuated by lordosis. Similar observations have been made by Oppermann
(1913) on parthenogenetic trout.
Some of the eggs developing from eggs fertilized by sperm exposed to 50,000 r
and all embryos from sperm exposed to higher levels of irradiation showed the
same characteristics described above. Since Rugh (1939) found that among the
Amphibia, some 90 % of the embryos developing from eggs fertilized by sperm
exposed to 50,000 r were haploids, it is quite probable that the Fnndnliis embryos
which survived the high levels of irradiation and appeared normal were indeed
haploids and parthenogenetic at the beginning. This is supported by the fact
that the eggs fertilized by sperm which had been exposed to 5000 r were more
severely abnormal than any of those which survived fertilization by sperm
exposed to either 100,000 r or 150,000 r. It thus appears that exposures of
sperm up to 50,000 r are not quite sufficient to completely eliminate the genetic
contribution of the sperm in every case.
There was one embryo from the 5000 r series and two from the 50,000 series
which were indistinguishable from the controls. These were probably recovered
diploid embryos. Tyler (1941) discussed the possible methods of regulation
from haploidy to diploidy in the frog. Since there was no evidence of cleavage
FIGURES 5-8. The same series of embryos seen in Figures 1^4 but 3% days after fertiliza-
tion, each derived from the egg shown to its left. Embryo in Figure 7 is almost normal,
possessing circulation comparable to the control. Note poorly formed central nervous system
in all but the controls.
FIGURE 9. A group of control eggs 7 days after fertilization. Note uniformity in
development.
FIGURE 10. A comparable group of embryos developing from eggs fertilized by sperm
exposed to 5000 r x-rays (Cs-137), showing optimum development among this group. One
embryo appears to closely resemble the controls.
586
R. LASHER AND R. RUGH
16
X-IRRADIATION OF FUNDULUS SPERM 587
delay in these experiments, the other possible explanations might he considered:
(1) Retention of the second polar body; (2) omission of polar divisions; (3)
diploidy of virgin eggs; and (4) progressive regulation during cleavage. Informa-
tion is not available as to the state of the nucleus of Fundulits at the time of
insemination, and it is unlikely that 3 out of 42 eggs would be diploid before
fertilization. Parthenogenetic development in Fitndulus is most likely to occur
from progressive regulation in eggs fertilized by sperm whose genetic complement
has been destroyed by 100,000 r or more.
Man}* of the embryos in these experiments were able to survive for several
days, even with poorly developed central nervous system and circulation. The
heart was seen pulsating in some embryos even after seven days' development,
and some showed muscular movements of the body and tail. It has been found
that in haploid amphibian cells the utilization of yolk is much slower than in the
controls (Porter, 1939). This might also be correlated with the retardation of
carbohydrate metabolism. The amount of oxygen required by haploid cells is
not as great as that of diploid cells, enabling them to survive for an extended
period without a circulatory supply of oxygen.
A cytological study of control and experimental embryos was made in order
to determine whether the parthenogenetic individuals were haploid or diploid,
and whether any of their organs reflected these variables. The cells of the gut
and kidney were most suitable for counting nucleoli, and the eye for organ
development.
The cells of the control embryos in every case possessed paired nucleoli
while the majority of the parthenogenetic embryos, from sperm exposed to 50,000 r
or more, had single nucleoli. The exception was the embryo from 50,000 r sperm
shown in Figure 12, which had two nucleoli per cell. This embryo could not
be distinguished from the parallel control, with regard to organ differentiation
and development, so that it is presumed to be a recovered-diploid embryo. The
chromosomes of this form are almost impossible to count, they are so small
and numerous. Many of the embryos from sperm exposed to less than 50,000 r
were also probably diploid, possessing two nucleoli in each cell. Thus, such
cytological study as was possible corroborated the gross findings of the "Hertwig
Effect" even with the fish embryos.
It is of interest that mature sperm of Fnndulus can tolerate exposures of
150,000 r without impairing their motility or their ability to activate normal eggs.
FIGURE 11. Control embryo at 7 days' development.
FIGURE 12. One of two embryos developing from eggs fertilized with sperm which had
been exposed to 50,000 r, shown at 7 days and to be compared with that in Figure 11. Probably
diploid.
FIGURE 13. Control embryo at three days of development.
FIGURE 14. An embryo developing from an egg fertilized with sperm which had been
exposed to 100,000 r, now seen at three days of age, to be compared with control in Figure 13.
Note malformation of brain vesicles, general retardation including the eyes.
FIGURES 15-16. The same embryos (Figures 13, 14) seen at 7 days of development. The
experimental (from irradiated sperm) embryo in Figure 16 now possessing circulation. Note
poorly developed nervous system, myotomes, and tail, indicating general retardation in
development.
588 R. LASHER AND R. RUGH
The eggs of Fiindulus are irrevocably damaged so that they cannot develop
beyond the stage #15 if they are exposed to as little as 4000 r x-rays.
SUMMARY AND CONCLUSIONS
1. Mature sperm of Fund it! us heteroclitus were exposed to ionizing radiations
from Cs.-137 at the rate of 5000 r/min., for doses ranging from 5000 r to 150.000 r,
and were then used to fertilize normal eggs of the same species. Control eggs
from the same batch were inseminated with unirradiated sperm from the same
source.
2. While variations in normal fertilizability of the control eggs do occur,
associated with the season and breeding activity, in every case some eggs were
fertilized and developed following insemination with sperm which had been
exposed to every dose level. This was riot mere activation since cleavages
followed.
3. The presence of irradiated (sperm) chromatin had no effect on the time
or nature of the early cleavages. The initial adverse effects were noted at the
time of gastrulation.
4. Exposures of sperm to 500 r had no apparent effect on the development
of eggs, while exposures to 5000 r caused high mortality and morbidity, and
above 5000 r (to the sperm) the effect of ionizing radiations appeared to decrease
so that a greater percentage of near-normal embryos resulted from cleaving eggs.
5. The fact that a few specimens from 50,000 r or more sperm could not be
readily distinguished from the controls suggests that complete recovery of diploidy
may sometimes occur after activation. This was substantiated by cytological
examination.
6. While parthenogenesis does not. or need not, occur naturally for this species,
the fact that it can occur is of biological significance, suggesting that all vertebrates
may possess eggs with such regulatory potentialities.
LITERATURE CITED
COSTELLO, D. P., M. E. DAVIDSON, A. EGGERS, M. H. Fox AND C. HENLEY, 1957. Methods for
Obtaining and Handling Marine Eggs and Embryos. Marine Biological Laboratory,
Woods Hole, Mass.
HERTWIG, O., 1911. Die Radiumkrankheit tierischer Keimzellen. Ein Beitrag zur experi-
mentellen Zeugungs- und Vererbungslehre. Arch. f. mikr. Anat., Abt. II, 77: 1-164.
OPPERMANN, K., 1913. Die Entwicklung von Forelleneiern nach Befruchtung mit radiumstrahlen
Samenfaden. Arch. j. mikr. Anat., Abt. II, 83: 141-190.
OPPENHEIMER, J., 1937. The normal stages of Fiindulus heteroclitus. Anat. Rec., 68: 1-15.
PORTER, K. R., 1939. Androgenetic development of the egg of Rana pipicns. Biol. Bui/.. 77:
233-257.
RUGH, R., 1939. Developmental effects resulting from exposure to x-rays. I. Effect on the
embryo of irradiation of frog sperm. Proc. Amcr. Phil. Soc., 81 : 447-471.
RUGH, R., 1962. Experimental Embryology : Techniques and Procedures. Burgess Publ. Co.,
Minneapolis, Minnesota.
SOLBERG, A. N., 1938. The susceptibility of Fuiuhtlits heteroclitus embryos to x-radiation.
/. Exp. Zool, 78: 441-469.
TYLER, A., 1941. Artificial parthenogenesis. Biol. Rev., 16: 291-336.
UPTAKE AND INTRACELLULAR DIGESTION OF PROTEIN'
(PEROXIDASE) IN PLANARIANS 1
PAUL J. OSBORNE2 AND A. T. MILLER, JR.
Department of Physiology, Unii'crsity of North Carolina Medical School, Chapel Hill, N. C.
It is thought (Willier ct a/., 1925; Jennings, 1957; Rosenbaum and Rolon,
1960a) that digestion in aquatic planaria is exclusively intracellular, occurring in
the spherules of the phagocytic gastrodermal cells. There is, however, very little
information concerning the formation of the spherules, the rate of digestion of their
contents, and their ultimate fate. Data based on the rate of disappearance of alkaline
phosphatase from the spherules in planarians which had been fed raw earthworms
(Osborne, 1955) were inconclusive, because of the impossibility of distinguishing
exogenous from endogenous enzyme. Nor was it possible in these studies to
determine whether the uptake of nutrients occurs exclusively by the phagocytic
action of the gastrodermal cells. A new approach to these problems was suggested
by the experiments of Straus (1959) on the intracellular disposition of parenterally
administered horseradish peroxidase in the rat. Peroxidase, which does not
occur in most cells of animal organisms, is readily visualized histochemically and
can be used as a tracer for exogenous protein.
MATERIALS AND METHODS
Specimens of Dugesia tigrina were starved for 10 days before the administration
of peroxidase ; this period of starvation is adequate to induce immediate feeding when
food is offered but is not long enough to cause extensive resorption of the gastro-
dermal cells, which would delay the phagocytic uptake of food. Experiments were
performed on both normal and pharyngectomized worms.
Pharyngeal feeding of peroxidase was carried out in the following manner, which
simulates the conditions of normal feeding. The procedure was suggested by our
observation that raw kidney is superior to other commonly-used foods for growth-
promotion, and by the report of Straus (1959) that the greatest concentration of
peroxidase, following parenteral administration in the rat, occurs in the kidney
tubule cells. Mice were given an intracardiac injection of peroxidase (10 mg. in 1
nil. saline), and one hour allowed for glomerular filtration and tubular reabsorption
of the enzyme (Straus, 1961). The mice were then killed by decapitation and the
kidneys removed and frozen. Thin slices of kidney cortex were placed in jars
containing starved planarians, and removed after the completion of feeding, which
usually required about 30 minutes. At intervals ranging from 30 minutes to 8 days
after the completion of feeding, planarians were removed and fixed for 12-24 hours
in cold (4° C.) formol-calcium, rinsed for one hour in cold distilled water (4° C.)
and embedded in lO^c gelatin. The gelatin blocks were mounted on chilled cryostat
1 Supported by NIH grants C-3996 and A-1699.
2 Present Address : Biology Department, Lynchburg College, Lynchburg, Va.
589
590
PAUL J. OSBORNE AXD A. T. MILLER, JR.
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FIGURES 1-6. Stages in the accumulation and subsequent disappearance of peroxidase
reaction product in the phagocytic cells.
FIGURE 1. Thirty minutes after feeding. Peroxidase activity is localized in diffuse form
in the phagocytic cells. 75 X.
FIGURE 2. Three hours after feeding. Peroxidase activity is predominantly concentrated
in the forming spherules in the phagocytic cells. 75 X.
FIGURE 3. One day after feeding. Peroxidase activity is present entirely within spherules
of varying sizes. 75 X.
FIGURE 4. Three days after feeding. Considerable digestion of the peroxidase has
occurred. 75 X.
FIGURE 5. Five days after feeding. Many of the spherules no longer show peroxidase
activity. 75 X.
FIGURE 6. Six days after feeding. Only an occasional spherule shows peroxidase activity.
75 X.
UPTAKE OF PROTEIN BY PLANARIANS 591
object holders, frozen by contact with dry ice and cut at 8 /A in a Pearse cryostat.
The sections were mounted on chilled slides without adhesive, air-dried for two
hours, and the gelatin removed by gentle rinsing in a stream of warm water. The
preparations were then incubated at 4° C. for three minutes in the medium recom-
mended by Gomori (1952). as modified by Straus (1959), for the visualization of
peroxidase activity. The incubation period was somewhat longer than that used by
Straus, but this was necessary for sharp staining of the forming foodballs. Control
sections were incubated in a similar manner with the omission of peroxide from
the medium.
Permanent preservation of the blue color of the reaction product was achieved
by complete dehydration of the specimens through absolute alcohol, followed by
clearing in xylene and mounting in Permount.
In order to determine whether or not significant ingestion of protein can occur
by extra-pharyngeal routes, planarians were pharyngectomized or transected at the
base of the pharynx (which was removed) and the two halves separated from one
another. One day was allowed for healing, since McWhinnie and Gleason (1957)
have demonstrated that after this interval the cut surface of a transected planarian
is covered with epidermis continuous with that of the rest of the organism. Speci-
mens were then placed in solutions of horseradish peroxidase in filtered pond water,
in concentrations of 10, 50 or 100 mg. per ml., for periods of 15 minutes to 6 hours.
All worms exposed to peroxidase concentrations of 50 mg. per ml. for 6 hours and of
100 mg. per ml. for three hours died, but all survived exposure to 10 mg. peroxidase
per ml. for 6 hours. The cause of death after prolonged exposure to higher con-
centrations of the enzyme is unknown. Control specimens were killed by fixation in
cold formol-calcium before being placed in peroxidase-containing media.
At the end of the exposure periods, the specimens were removed from the
peroxidase solution, rinsed in filtered pond w7ater for 5 minutes, and then chilled
for a few minutes (4° C.) to produce a non-motile, slightly contracted condition.
Cold formol-calcium \vas poured over each specimen individually, with care to
ensure that each was fixed in a smooth, distended state. Fixation and subsequent
treatment of these specimens were the same as for the worms fed mouse kidney.
RESULTS
Pharyngeal feeding
In planarians killed 30 minutes after the cessation of feeding there was a diffuse
coloration of most of the phagocytic cells with the blue peroxidase reaction product
( Fig. 1 ) . Two to three hours following cessation of feeding, the reaction product
formed discrete "droplets" within the phagocytic cells of the gut (Figs. 8, 9).
Distinct spherules were present in specimens killed one day after feeding (Fig. 3),
and gradual disappearance of enzyme activity occurred during subsequent days
(Figs. 4—6). No activity was demonstrable on the eighth day following feeding.
Details of the formation of intracellular protein spherules are shown in Figures
7-12. One hour after feeding, the peroxidase reaction product appeared in the
form of small granules ranging in diameter from about 0.2 /A up to 2.0 p. (Fig. 7).
They were present exclusively within the phagocytic cells ; the larger granules ap-
peared to result from the fusion of many smaller ones, since the number and the
size of the granules within a cell varied inversely. They increased in size
592
4V ^ ' "
PAUL J. OSBORXE AND A. T. MILLER, JR.
8
ii
12
FIGURES 7-12. Stages in the formation of peroxidase-containing spherules.
FIGURE 7. One hour after feeding. Peroxidase reaction product appears as small granules
ranging in size from the limit of visibility up to approximately 2 /j. in diameter. 600 X.
FIGURE 8. Two hours after feeding. Small granules are still present (arrow a), but the
largest now approach the dimensions of spherules (arrow b). 600 X.
FIGURE 9. Three hours after feeding. Note the phagocytic cell, cut longitudinally, con-
taining numerous spherules and several smaller granules. 600 X.
UPTAKE OF PROTEIN BY PLANARIANS 593
progressively (Figs. 8, 9), reaching a maximum diameter of 15-20 ^ by the end of
one day after feeding (Fig. 11). The classical intracellular "spherules" were clearly
seen by the end of the third hour after feeding (Fig. 9). The onset of protein
digestion could not be detected by the peroxidase method, but it was well-advanced
by the second day after feeding, as indicated by the decrease in the numbers of
spherules which gave an enzyme reaction. By the fifth day after feeding, the
phagocytic cells were still filled with spherules, but only an occasional spherule
contained active enzyme (Fig. 12). Three weeks after feeding, the phagocytic cells
contained large numbers of refractile droplets similar to those previously (Osborne,
1955) shown by Nile blue and Sudan III staining to be fat. This suggests a
conversion of protein to fat, as has been reported by Willier ct al. (1925).
Extra-pharyngeal absorption of peroxidase.
\Yhen pharyngectomized planarians were exposed to media containing various
concentrations of peroxidase, a definite penetration of the enzyme through the
intact epidermis could be demonstrated (Figs. 13 and 14), and rows of peroxidase-
positive granules were seen extending into the interior. Most of the penetration
occurred through the ventral surface, but there was definite evidence of absorp-
tion through the dorsal surface as well. In Figure 13, penetration of peroxidase
seems to be taking place via the canals left by the extrusion of rhabdites. A few
phagocytic cells were filled with peroxidase-positive material within 30 minutes
after the beginning of exposure (Fig. 15), and the apparent fusion of smaller
into larger intracellular granules is illustrated in Figure 16. After 3 hours'
exposure to peroxidase, the typical localization of peroxidase-positive material
in the phagocytic cells of the gut was clearly demonstrable (Figs. 17 and 18),
although the concentration of enzyme reaction product was very much less than
in the case of intact planarians fed peroxidase-containing food.
Specimens killed by formol-calcium fixation prior to exposure showed minimal
uptake of peroxidase and absence of intracellular localization.
DISCUSSION
Studies on intracellular digestion in lower organisms are of interest both in
their own right and for the light they may shed on similar processes in higher
animals. Planarians are particularly well-suited for studies of this type because
the phagocytic cells of the gastrodermis are readily accessible to exogenous
materials, they respond to the uptake of these materials by the production of a
variety of hydrolytic enzymes, and digestion is entirely intracelluar. While
changes in enzyme activity associated with intracellular digestion are readily
visualized by standard histochemical methods, changes in the materials undergoing
digestion have been more difficult to demonstrate. The experiments of Straus
FIGURE 10. Three hours after feeding. Note the phagocytic cell, cut in cross-section
(arrow a), containing a number of granules which appear to be coalescing, and other phagocytic
cells (arrow b) which still contain many small granules. 1350 X.
FIGURE 11. One day after feeding. Nearly all the peroxidase reaction product is present
in well-formed spherules of varying sizes. 600 X.
FIGURE 12. Five days after feeding. Large numbers of spherules are now devoid of
peroxidase activity. 600 X.
594
PAUL J. OSBORNE AND A. T. MILLER, JR.
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FIGURES 13-18. Transepidermal uptake of peroxidase in pharyngectomized planarians
exposed to solutions of peroxidase in pond water.
FIGURE 13. Exposure time 15 minutes, peroxidase concentration 100 mg. per ml. Arrows
point to concentration of peroxidase reaction product in canals from which rhabdites had been
extruded. 1000 X.
FIGURE 14. Exposure time 30 minutes, peroxidase concentration 50 mg. per ml. Arrows
point to rows of peroxidase-positive granules extending from the surface toward the interior.
1000 X.
FIGURE 15. Exposure time 30 minutes, peroxidase concentration 10 mg. per ml. Different
stages in the formation of spherules are shown. 600 X.
UPTAKE OF PROTEIN BY PLANARIANS 595
(1959) have shown that horseradish peroxidase is readily taken up hv the cells
of various tissues of the rat, and that its ultimate disposition can he followed by
histochemical methods. This provides a convenient procedure which should be
applicable to studies on the uptake and digestion of exogenous protein by the
phagocytic cells of lower forms; the major uncertainty in the interpretation of
the results concerns the degree of degradation of the enzyme molecule necessary
for the abolition of its enzyme properties. It is interesting to note that the rate
of disappearance of exogenous peroxidase activity from the phagocytic cells in
the present study is in approximate agreement with the rate of disappearance
of food spherules reported by Willier ct al. (1925), based on non-specific histo-
logical staining methods.
The formation of intracellular food spherules appears to involve the progres-
sive fusion of large numbers of very small granules. The smooth, circular profile
of spherules of varying sizes suggests the presence of a limiting membrane of
the type commonly associated with food vacuoles, a supposition which is
supported by the presence of spherules devoid of peroxidase activity some days
after the ingestion of peroxidase-containing food (Fig. 12). The mechanism of
formation of these spherules is obscure and might profitably be studied by electron
microscopy.
The transepidermal uptake of peroxidase in pharyngectomized planarians
suggests a possible route by which nutrients are absorbed from the medium by
planarians undergoing regeneration after transection or binary fission. Further
work will be required before the quantitative importance of transepidermal
absorption can be assessed.
While this material was being prepared for publication, our attention was
drawn to the paper of Rosenbaum and Rolon (1960b), thus far available only
in abstract form, in which the absorption by planarians of peroxidase dissolved
in the medium was reported. Our results are in general agreement with theirs,
although the procedure differed somewhat in the two experiments; the major
difference in results is the much greater toxicity of peroxidase in our experience,
the basis of which is to be investigated.
SUMMARY
1. Stages in the ingestion of protein, and the formation and ultimate dis-
appearance of spherules in the phagocytic cells of Dugcsia tigrina were visualized
histochemically by the peroxidase technique.
2. The formation of spherules involved the coalescence of numerous small
peroxidase-positive granules. Typical spherules were present three hours after
feeding kidney from a mouse previously injected with peroxidase. and maximal
size of the spherules was achieved by the end of 24 hours.
FIGURE 16. Exposure time one hour, peroxidase concentration 100 mg. per ml. Earlier
(b) and later (a) stages in the aggregation of peroxidase-positive granules in the phagocytic
cells. Other phagocytic cells, as yet devoid of enzyme activity, are also present. 1350 X.
FIGURE 17. Exposure time three hours, peroxidase concentration 50 mg. per ml. Typical
spherules are present in the phagocytic cells. 300 X.
FIGURE 18. Exposure time three hours, peroxidase concentration 50 mg. per ml. The
enzyme activity is localized in the phagocytic cells but in much smaller amounts than after
pharyngeal ingestion of peroxidase-containing mouse liver (cf. Fig. 2). 75 X.
596 PAUL J. OSBORNE AND A. T. MILLER, JR.
3. Peroxidase-positive material was confined to the phagocytic cells and
disappeared gradually until none remained 8 days following feeding.
4. Pharyngectomized planarians exposed to a medium containing peroxidase
in solution absorbed the protein through the epidermis, and formed typical
spherules in the phagocytic cells. It is suggested that this may indicate a role
of transepidermal absorption of nutrients in regenerating planarians.
LITERATURE CITED
GOMORI, G., 1952. Microscopic Histochemistry. Chicago, The University of Chicago Press.
JENNINGS, J. B., 1957. Studies on feeding, digestion and food storage in free-living flatworms
(Platyhelminthes: Turbellaria). Biol. Bull., 112: 63-80.
McWniNNiE, M. A., AND M. M. GLEASON, 1957. Histological changes in regenerating pieces of
Dugcsia dorotoccphala treated with colchicine. Biol. Bull., 112: 371-376.
OSBORNE, PAUL J., 1955. Studies on phosphatases and lipases in certain Turbellaria. Unpub-
lished doctoral dissertation, University of Florida.
ROSENBAUM, R. M., AND C. I. RoLON, 1960a. Intracellular digestion and hydrolytic enzymes in
the phagocytes of planarians. Biol. Bull., 118: 315-323.
ROSENBAUM, R. M., AND C. I. ROLON, 1960b. Pinocytosis in phagocytes of planarians. Aunt.
Rec., 137: 389.
STRAUS, W., 1959. Rapid cytochemical identification of phagosomes in various tissues of the
rat and their differentiation from mitochondria by the peroxidase method. /. Biophys.
Biochcm. Cytol., 5: 193-204.
STRAUS, W., 1961. Cytochemical observations on the transport of intravenously injected horse-
radish peroxidase and the development of phagosomes in the cells of the kidney of the
rat. Exp. Cell Res., 22: 282-291.
WILLIER, B. H., L. H. HYMAN AND S. A. RIFENBURGH, 1925. A histochemical study of intra-
cellular digestion in triclad flatworms. /. Morph., 40: 299-340.
FILTER-FEEDING PATTERN AND LOCAL DISTRIBUTION
OF THE BRACHIOPOD, DISCINISCA STRIGATA
ROBERT T. PAINE
Department of Zoology, University »f Washington, Seattle 5, Washington
The recent surge of interest in the functional morphology of living brachiopods
has been led mainly by paleontologists, motivated by an intent to increase the
reliability of their interpretations of fossil structures. Research has been focused
on the mechanics of feeding, since the morphology of the filtering organ, the
lophophore, and, where present, its supports, is basic to understanding the phylum's
systematics. Orton (1914) contributed the first paper in this series and has
been followed by Richards (1952), Atkins (1956-1961; see Rudwick, 1962, for
complete citations), Chuang (1956). Williams (1956, 1960), Rudwick (1960a,
1960b, 1962), Rowell (1961), and Williams and Wright (1961). These works
suggest that, with the possible exception of Discinisca (Rowell, 1961), all Recent
brachiopods show a fair degree of convergence in the characteristics of their
feeding and the fleshy portions of their lophophores. On the other hand, the
functioning of "fossil" feeding mechanisms has not been agreed upon. This paper
describes the filtering mechanism of the inarticulate brachiopod, Discinisca strigata
Broderip, which, because of its uniqueness, will permit some features common
to all known brachiopod filtering mechanisms to be evaluated.
MATERIAL
Large numbers of D. strigata were discovered living in the tidal zone near
Puertecitos, Baja California, Mexico (approximately 30° 17' N.; 114° 40' W.).
Field observations were made from 29 March to 1 April, 1962, principally at a
station 2-3 km. north of Puertecitos, and were facilitated by an estimated 20-foot
tidal range. Living specimens were successfully transported back to the Scripps
Institution of Oceanography where details of the feeding process were worked out.
ECOLOGICAL OBSERVATIONS
At the principal station an extensive sand beach is interrupted at regular
intervals by patches of cobbles and small boulders extending down to the low-
water mark. D. strigata lives under the flatter of these rocks, conspicuously
associated with the sponge, Hyuicniacidon adrcissifonnies Dickinson, the gastro-
pods, Acanthina angelica Oldroyd, Ncrita funiciilata Menke, and Monda fcr-
rnginosa Reeve, and the bivalves, Barbatia rccrcana (Orbigny) and Isognoinon
cliciiiuitzianits (Orbigny). Whether suitable habitats in other areas can be
recognized by the presence of these species is not yet known. The brachiopods
often had settled in small crevices in the rocks, and occurred in microenvironments
probably characterized by reduced current scour. The size distribution of the
597
598
ROBERT T. PAINE
Puertecitos population (Fig. 1), based on measurements of all specimens obtained
from a large (200 X 50 yards) area of the cobble patch, shows no multimodality
indicative of either two or more breedings or a number of year classes. The
position of the normally shaped curve suggests a single prolonged spawning
sometime prior to the date of collection, and that the animals may be annuals.
N = 238
o
z
LJ
O
UJ
tr
T 1 1 1 1 1 1 1 1 1 1 T
0 5 10 15
AVERAGE SHELL DIAMETER (MM.)
FIGURE 1. Diameter-frequency distribution of D. strigata. The diameter of each specimen
is composed of an average of its length and width ; the use of this measure is necessitated by the
extremely variable individual shape.
Like many marine species, D. strigata shows signs of zonation. One cobble
patch was completely exposed on an extreme low tide, permitting a transect to
be made (Table I). Six flattish rocks, each about a foot square on their lower
surface, were overturned at 10-foot intervals from the beginning of the cobble
patch to the high- water mark, and the number of brachiopods seen recorded for
FEEDING PATTERN OF DISCINISCA 599
each interval. Brachiopod distribution was curtailed at both seaward and land-
ward extremes, although seemingly acceptable rocks existed throughout the body
of the reef. The position of this upper limit was further substantiated by two
less complete transects. The lower limit cannot be explained at present, but
desiccation appears to influence the upper extension. \Yhen adjacent flat rocks,
high in the intertidal, were chosen such that one was slightly more elevated than
the other. D. strigata was always under the wetter of the two rocks. Although
G. A. Cooper (in Cloud, 1948) has also taken this species in intertidal water, the
type was dredged from 18 fathoms (Broderip, 1833). Zonation at Puertecitos
may thus be a local phenomenon, or possibly the type was collected at an atypical
depth.
The evidence is substantial that this species, unlike other discinids, never forms
clusters of many individuals. From collections at three Baja California locations,
San Felipe. Puertecitos, and Bahia de Los Angeles (Courtesy of B. N. Kobayashi)
11, 238, and 2 specimens, respectively, were scanned with a microscope. Although
TABLE I
Zonation of D. strigata based on a transect. Numbers indicate the quantity of brachiopods observed
under 6 flat rocks in each 10-foot interval. Distances are given in feet from the high-water mark.
The vertical excursion is approximately 20 feet.
Distance 0-160 170 180 190 200 210 220
Numbers 01 0 5 20 25 12 6
Distance 230 240 250 260 270 280 290 300
X umbers 5 7 11 17 0 3 3 2
Distance 310 320 330 340 350-500
X umbers 21110
all degrees of incrustation existed, no small brachiopods were observed. In
addition, by inference the type specimens from Guatemala were collected singly
(Broderip, 1833) as were those obtained by G. A. Cooper (personal communica-
tion) near Matzatlan, Mexico. On the other hand, clusters of D. lamcllosa and
D. lacvis are well-documented (Davidson. 1888), implying the existence of
distinctive differences in larval settling behavior within the genus. Blochmann
(1908) has suggested that discinid clustering was associated with poor dispersal
powers. It seems as probable, however, in light of an increased knowledge of
the role of site selection exercised by certain larvae (Wilson, 1958), that the
intrageneric variation stems from differences in settling behavior.
FILTER-FEEDING MECHANISM
Hyman (1959) has cited the extensive and varied elaboration of the brachiopod
lophophore as characteristic of the phylum. One diverse group of Recent species,
drawn from both the Inarticulata and Articulata and encompassing four families,
can be described as having a spirolophous lophophore (spirolophes) ; that is,
in the mature individual the lophophore is coiled into two simple arms which,
in all known spirolophes except Discinisca and the closely related Disclna (Rowell.
600
ROBERT T. PAINE
personal communication), point dorsally. Each arm (or brachium) bears a
double row of ciliated filaments separated by the brachial groove from the lip.
The side containing the lip is termed the frontal surface, and lateral cilia on the
filaments beat across the length of the filament from the frontal toward the alt-
frontal surface (Atkins, 1956). Thus, the organism's food is filtered from water
currents drawn perpendicularly to the length of the filament in a frontal to
abfrontal direction. The interested reader is referred to Rudwick (1962) for a
more complete account of the ontogeny and functioning of a variety of brachiopod
lophophores.
FIGURE 2. View of the expanded lophophore of D. strigata after removal of most of the
ventral valve. The arrows represent the observed direction of water flow across the lophophore.
The setae fringing the shell have been omitted.
The above arrangement of spiral arms, filaments, and ciliary beat specifies the
feeding currents of most spirolophes. However, the ventrally-directed Discinisca
spires have paleontological implications and the situation needs clarification.
The ventral valves of a number of D. strigata were partially removed and the
lophophores allowed to relax fully, without narcotization. The water currents
around the lophophore were then drawn as they appeared when viewed from the
ventral aspect (Fig. 2). The apices of the slightly elevated spires are oriented
ventrally (pointed toward the observer) and perhaps canted toward the median
plane. The proximal whorl of each brachium appears greatly expanded, extend-
ing from the median plane almost to the lateral margin of the shell. The remaining
more distal whorls comprise an insignificant portion of the spire, and. in fact,
give rise to less than 20c/f of the total filament length. This figure was arrived
at by measuring scattered filaments on the proximal and more distal whorls, and
then multiplying their mean lengths (1.7 mm. and 0.5 mm., respectively) by their
FEEDING PATTERN OF DISCINISCA 601
numbers. Presumably, as the animals become larger, the disparity in allocation
of filtering surface will continue to increase in favor of the proximal whorl.
In Figure 2 the frontal surface of the anterior filaments of the proximal whorl
faces the observer, and in this region water currents pass normal to the plane
of commissure (into the plane of the paper). These anteriormost filaments tend
to lie against the mantle, forming an exhalant chamber leading to the posterolateral
margins, dorsal to the main body of the lophophore. Water filtered by these
filaments occupies this dorsal chamber. Similarly, if the ventral shell and mantle
were present, those posterior filaments behind the mouth and closest to the
observer, in lying against the mantle, would form another analogous exhalant
chamber ventral to the lophophore. Some unfiltered water is also drawn into
the distal portions of the spire and after being filtered must move dorsally and
then laterally to join the main exhalant currents at the posterolateral margin of
the valves. This previously filtered water inside each spire cannot mix with the
unfiltered water because the filaments on the distal whorls are flexed abfrontally,
touching the frontal surface of the next more proximal whorl. Most of the
unfiltered water, however, passes along the expanded body of the proximal whorl
and is filtered by the long filaments toward its lateral end.
As in most brachiopods, the filaments are incompletely ciliated, a tract being
absent from the abf rental surface. On excised filaments the lateral cilia were
usually still, but the frontal ones continually beat toward the filament tips.
Their normal beat would be toward the base and adjacent food groove, and this
beat reversal can serve as a rejection mechanism.
At least two such rejection mechanisms are functional in the whole organism.
The beat of the frontal cilia appears to be frequently reversed, since bands of
mucus-bound particles were seen to be carried away from the brachial lip. And
heavy particles, once inside the inhalant chamber, are pushed toward the chamber's
margins by a coordinated flexing of the filaments, similar to that illustrated for
Lin gitl a ungiiis by Chuang (1956). Once at the edge these particles are prob-
ably expelled by a gentle clapping shut of the valves, as is known in the in-
articulates, Crania (Orton, 1914) and Glottidia (Paine, unpublished), and some
articulates (Rudwick, 1962).
In the normally feeding intact animal the slight gape of the valves is masked
by a heavy fringe of long, barbed setae. The gross current pattern consists of a
single, median inhalant current and paired, posterolateral exhalant ones. This
pattern was never strongly developed, especially the exhalant currents, though in
20 specimens examined little variation was noticed. Not much current distortion
is caused by the fringing setae which function, aside from being tactile elements,
to catch and hold all but the finest water-drawn particles. In freshly collected
specimens these setae were invariable festooned with detritus, particularly around
the median inhalant aperture where they also reach their maximum length.
The nature of the currents inside the unopened animal can only lie speculated
on. Most likely all filaments of the proximal whorl touch the dorsal or ventral
mantle surfaces. The tips of the longer, laterally placed filaments also probably
intermesh completely in the restricted space of the mantle cavity, and in so doing
form a tunnel trending laterally which encloses the main body of unfiltered water.
The apices of the spires, enclosed within this tunnel, will be canted toward the
median plane and possibly serve to orient the incoming current.
602 ROBERT T. PAINE
DISCUSSION AND CONCLUSION
A revival of interest in brachiopods, apparently focused on their feeding mecha-
nism, has shown that most species can accurately be characterized by certain
generalities. ( 1 ) In adult specimens the unfiltered water enters the mantle cavity
as paired lateral currents and exits as a single median one, although in small
individuals this external current pattern is usually reversed (Atkins, 1956;
Rudwick, 1962). (2) The feeding currents are mainly produced by lateral cilia
on the lophophoral filaments beating in a frontal to abfrontal direction, and the
beat of these is seldom reversed. The report by Atkins (1960) of current
reversal in the Megathyridae represents a momentary phenomenon; the usual
beat is similar to that in other brachiopods and Atkins suggests (p. 471) that
the reversal is elicited "... when strong cleansing action is called for." (3) And
in spirolophous brachiopods the apices of the spires point dorsally.
Rudwick (1960a) has added further considerations. Filtered and unfiltered
water may be kept separate by the arrangement of the lophophoral filaments. If
this is achieved the animal possesses an "efficient" filtration system, if not, an
"inefficient" one. All Recent brachiopods appear to be "efficient." Second, when
unfiltered water occupies the center of the spire, the current system can be called
inhalant, a condition characterizing most living spirolophes. An alternative in-
ferred for Discinisca on topological grounds by Rowell (1961), and potentially
equally efficient, is an exhalant system characterized by previously filtered water
in the spire's center. Finally, Rudwick (1960a) has shown that all spirolophes
can fall into either of two mutually exclusive categories. When the left brachium
is viewed from its base toward its apex, it will coil either clockwise or counter-
clockwise. Among Recent species the latter group includes most spirolophes, the
former only Discinisca (and presumably Discina as well). Both categories are
well represented in fossil forms. Because the frontal surfaces of the brachia
always face the mouth in early lophophoral stages, the counterclockwise group
eventually develops an inhalant system and the clockwise group an exhalant one.
Rudwick's (1960a) contention that extinct spirolophes had only inhalant or
exhalant systems of the construction presently extant has been challenged by
Williams (I960) and Williams and Wright (1961). Much of what has been
learned about Discinisca cannot help to resolve the central issues of this debate,
which involve possible functions of structures only present in articulate brachio-
pods. However, because the left brachium of the Discinisca spire does coil clock-
wise, analogies drawn with extinct spire-bearers also characterized by clockwise
coiling of their left brachium become more reasonable. And, through comparison
with other spirolophes, those characteristics of the brachiopod feeding mechanism
which are independent of lophophore orientation can be specified.
Perhaps the most germane observations are that the basic construction of the
brachium has not been altered, and that the lateral cilia continue to beat in a
frontal to abfrontal direction. However, the filaments of D. striyata are flexed
abfrontally rather than frontally as in the inarticulates, Linyula (Chuang, 1956) and
Crania (Atkins, in Rudwick, 1960a). The result of this alteration in flexure is
that, given a ventrally-oriented spire, the feeding mechanism retains its efficiency.
As Rowell (1961) has shown, if the filaments were flexed frontally, the inhalant
and exhalant chambers could not be effectivelv isolated and the system would be
FEEDING PATTERN OF DISCINISCA 603
inefficient. The filaments, flexed as they are in D. strigata, form a system of
inhalant and exhalant spaces which prevent previously filtered water from being
recycled before it has been pumped from the animal. With the retention of the
usual direction of ciliary beat and the maintenance of an efficient system, the gross
current pattern can only be as it is.
D. strigata, however, shows a mixture of inhalant and exhalant systems. The
water in the central portions of the spire already has been filtered, and on this
basis this species must be considered at least partially exhalant. However, only
20% of the water is processed in this manner, the remainder probably passing
along the tunnels formed by the lateral extension of the proximal whorl. The
water here, in what would normally be the inside of the spire, is unfiltered and
thus fits Rudwick's (1960a) description of inhalant systems. Blochmann's (1900;
plate 8, Fig. 10) illustration of D. laincllosa suggests that a similar extension of
the proximal whorl is found in another Discinisca species, and thus that a combina-
tion of inhalant and exhalant systems may typify the genus. This finding does not
alter the logic of Rowell's (1961) conjecture based on a Discinisca lophophore in
which all the whorls are concentric and similarly shaped, and where the filaments
of the spire touch the next more proximal whorl. It should be emphasized that if
the terms inhalant and exhalant current systems are simply descriptive, then
D. strigata belongs to the exhalant group because only filtered water lies within
the central axis of the spire. However, if total lophophore functioning is also
considered, the dual interpretation of D. strigata is reasonable.
These results suggest that problems associated with gross current pattern,
orientation of the spire, and the presence of inhalant or exhalant filtering systems
can be minimized in typically constructed species, both extant and fossil. Recon-
struction of the lophophores of fossil spirolophes can probably be satisfactorily
based on the consistent properties of efficient filtering and unidirectional ciliary
beat in a frontal to abfrontal direction.
This work was done during the tenure of a Sverdrup Postdoctoral Fellowship
at the Scripps Institution of Oceanography. The author wishes to thank the
following specialists for specific identifications: Dr. G. A. Cooper (brachiopod),
Dr. W. D. Hartman (sponge), and Dr. R. Stohler (Mollusca). The paper has
benefited from the suggestions of and critical reading by Dr. A. J. Rowell. Special
recognition is due Mr. E. P. Chace, of the San Diego Museum of Natural History,
for suggesting the locality where the Discinisca were found.
SUMMARY
1. Ecological observations on the brachiopod, Discinisca sfrigata, suggest that
this species is zoned in shallow water in the northern Gulf of California. It occurs
singly rather than in great clumps, and on the basis of a size-frequency distribution
appears to be an annual.
2. Examination of the filter-feeding apparatus and its operation shows, despite
an exceptional orientation of the lophophore, that there are a number of points
in common with other brachiopods. The lateral lophophoral cilia beat in a frontal
to abfrontal direction, and the current system through the animal is efficient.
604 ROBERT T. PAINE
3. The position of the inhalant aperture, relative direction of coiling of the
left brachium. and orientation of the spire, although differing from those in other
adult spirolophes, do not diminish the efficiency of operation. D. strigata, though
showing a mixture of inhalant (80%) and exhalant (20%) filtering systems, is
able to maintain its filtering efficiency, due to the disposition of filaments within
the organism.
LITERATURE CITED
ATKINS, D., 1956. Ciliary feeding mechanisms in brachiopods. Nature, 177: 706-707.
ATKINS, D., 1960. The ciliary feeding mechanism of the Megathyridae (Brachiopoda), and the
growth stages of the lophophore. /. Afar. Biol. Assoc., 39: 459-479.
BLOCHMANN, F., 1900. Untersuchungen iiber den Bau der Brachiopoden. Die Anatomic von
Disciuisca lamellosa (Broderip) und Lingula anatina Bruguiere. II: 69-124. Jena.
BLOCHMANN, F., 1908. Zur systematik und geographischen Verbeitung der Brachiopoden.
Zcitschr. f. U'iss. Zool, 90: 596-644.
BRODERIP, W. J., 1833. Descriptions of some new species of Cuvier's family of Brachiopoda.
Trans. Zool. Soc. London. 1: 141-144.
CHUANG, S. H., 1956. The ciliary feeding mechanism of Lingula uiu/uis (L.) (Brachiopoda).
Proc. Zool. Soc. London' 127: 167-189.
CLOUD, P. E., 1948. Notes on Recent brachiopods. Amcr. J. Sci.. 246: 241-250.
DAVIDSON, T., 1888. A monograph of Recent Brachiopoda. Trans. Linn. Soc. London, 4: 1-248.
HYMAN, L. H., 1959. The Invertebrates : Smaller Coelomate Groups. McGraw-Hill Book Co.,
Inc. New York.
ORTON, J. H., 1914. On ciliary mechanisms in brachiopods and some polychaetes, with a
comparison of the ciliary mechanisms on the gills of Molluscs, Protochordata, Brachio-
pods, and cryptocephalous Polychaetes, and an account of the endostyle of Crepidula
and its allies. /. Mar. Biol. Assoc., 10: 283-311.
RICHARDS, J. R., 1952. The ciliary feeding mechanism of Ncotlivris Icnticnlaris (Desh.).
J. Morph., 90: 65-91.
ROWELL, A. J., 1961. Inhalant and exhalant feeding current systems in Recent brachiopods.
Gcol. Mag., 98: 261-263.
RUDWICK, M. J. S., 1960a. The feeding mechanisms of spire-bearing fossil hrachiopods.
Geol Mag., 97 : 369-383.
RUDWICK, M. J. S., 1960b. Correspondence. Gcol. Mag., 97: 516-518.
RUDWICK, M. J. S., 1962. Filter-feeding mechanisms in some brachiopods from New Zealand.
/. Linn. Soc. London, 44: 592-615.
WILLIAMS, A., 1956. The calcareous shell of the Brachiopoda and its importance to their
classification. Biol. Rev., 31: 243-287.
WILLIAMS, A., 1960. Correspondence. Gcol. Mag., 97: 514-516.
W'ILLIAMS, A., AND A. D. WRIGHT, 1961. The origin of the loop in articulate brachiopods.
Palcunt., 4: 149-176.
WILSON, D. P., 1958. Some problems in larval ecology related to the localized distribution of
bottom animals. Perspectives in Marine Biology : 87-99. Univ. of California Press,
Berkeley.
THE HEMOGLOBIN OF THE BIVALVED MOLLUSC,
PHACOIDES PECTINATUS GMELIN
KENNETH R. H. READ
77/r Biological Laboratories, Harvard University, Cambridge, Mass.
Hemoglobin has been reported from only a few species of bivalved molluscs ;
it occurs in cells in the blood of Porouiya granitlata, Solcn Icyuincn. Tcllina
planata, Capsa fragilis, Cardita acnlcata. Area tctragona, Area noac, Pcctitnculns
glyciuieris, Astarte jusca (?) Griesbach, 1891), Cardita snlcata (Paladino,
1909), Anadara inflata (Kawamoto, 1928), Area pc.vata (Svedberg and Hedenius,
1934) and Area subcrenata (Kobayashi, 1935). In Tivcla stnltonun hemoglobin
occurs in the brain (sic'), mantle, gills, foot and adductor muscle (Fox, 1953).
Studies of absorption spectra have been carried out on the hemoglobin of Area
subcrenata by Kobayashi (1935). and Svedberg and Hedenius (1934) have run
ultracentrifuge sedimentation studies on the pigment of Area pc.rata; biochemically,
however, the most extensively studied lamellibranch hemoglobin is that of
Anadara inflata.
The study of Anadara inflata hemoglobin began with the work of Kawamoto
(1928), who reported the oxygen dissociation curve of the pigment. Sato (1931)
and Kobayashi (1935) established the absorption spectra of the pigment and its
derivatives. Work on the hemoglobin of Anadara inflata culminated with the
efforts of Yagi ct al. (1955a, 1955b), who purified the hemoglobin, determined its
molecular weight from sedimentation and diffusion studies, its electrophoretic
mobility, iron content, nitrogen content and N- and C-terminal amino acids ; in
addition, molar extinction coefficients were also reported.
The present communication deals with the hemoglobin of the lucinid pelecypod,
Phaeoidcs pcctinatiis Gmelin, which the author chanced on in Puerto Rico. This
animal lives deep (down to 18 inches) in the mud of mangrove swamps, and
when it is opened exhibits dark purplish ctenidia with an appearance reminiscent
of ripe muscatel grapes. The bloom on the surfaces of the ctenidia is due to a
superficial layer of pigment-free cells ; however, when the ctenidium is torn, it
exhibits a bright red interior suggestive of hemoglobin. For illustration of the
appearance of the gills see Figure 1. Although the clam is commonly eaten,
at least in the neighborhood of La Parguera, P. R., and must therefore be some-
what well known, the author can find no mention of its hemoglobin in the
literature.
A four-sided study of the red pigment of the ctenidia of Phaeoidcs pcctinatiis
has been undertaken. First, evidence for the identity of the pigment with hemo-
globin has been obtained from studies of absorption spectra ; second, the oxygen-
combining properties of the pigment have been studied ; third, the behavior of the
pigment in the ultracentrifuge has been examined with a view to gaining some
idea of the size of the molecule ; and fourth, the histology of the pigment has
been studied.
605
606
KENNETH R. H. READ
MATERIALS AND METHODS
General
I'hacoidcs pcctinatus Gmelin was collected in the neighborhood of Point
Pitahaya near La Parguera, Puerto Rico. They were stored at the Marine
Station of the University of Puerto Rico on Magueyes Island, La Parguera, in
running sea water at a temperature ranging between 26 and 28° C. ; they were then
taken back to the Biological Laboratories, Harvard University, where they were
stored in aerated sea water at room temperature.
FIGURE 1. Phacoidcs pcctinatus Gmelin. Length 5 cm. Dorsal side uppermost. The large
dark mass shown by the dissected animal is the right ctenidium ; the dark color is due to
hemoglobin. Note the colorless edge of the demibranch, which is an indication of the hemoglobin-
free, ciliated cells of the exterior of the gill. Members of the family Lucinidae have only the
outer demibranch ( Purchon, 1939).
Absorption spectra
Centrifuged homogenates of ctenidia, in phosphate buffers ranging in pH from
6.8 to 7.5 and molarity from 0.067 to 0.2 M, were examined spectroscopically
with a Beckman Model DB recording spectrophotometer. Deoxygenation was
brought about either by washing the preparation repeatedly with nitrogen or by
adding a small amount of sodium dithionite. Carboxyhemoglobin was made by
bubbling carbon monoxide through the dithionite-treated preparation or through
the untreated preparation of oxyhemoglobin.
Oxygen dissociation curve
Samples of hemoglobin were prepared by homogenization of dissected ctenidia
in a glass homogenizer with pH 7.4, 0.2 M phosphate buffer at 3° C. The
homogenate was centrifuged at 8,000 X g for ten minutes to remove the coarser
cellular debris, and the resulting supernatant was further centrifuged at 100,000
X g in the ultracentrifuge for 30 minutes.
After ultracentrifugation, the clarified preparation was transferred to tonome-
ters of known volume, and the oxygen dissociation curve of the pigment determined
by the method of Riggs (1951), slightly modified. In Riggs' method the solution
HEMOGLOBIN OF PHACOIDES PECTINATUS 607
of hemoglobin in a tonometer, of which a cuvette for spectrophotometry forms an
integral part, is freed of all oxygen hy repeated washing with nitrogen ; measured
amounts of air are then introduced by way of syringes into the tonometer, and
after each addition the absorption spectrum of the hemoglobin solution is measured.
The oxygen tensions resulting from each increment of added air must be computed
with regard being taken for the vapor pressure of water, oxygen dissolved in the
water of the hemoglobin solution and oxygen combined with the hemoglobin
itself; the degree of saturation of the hemoglobin with oxygen is computed from
measurements of the absorption spectra at 560 mp.. The volume of the tonome-
ters employed was about 430 ml. and the amount of hemoglobin solution 8 ml. ;
the amount of hemoglobin in solution was estimated from measurements of its
optical density at the a peak and an assumed millimolar extinction coefficient
of 15. The absorption spectra were measured with Beckman Model DB or Gary
Model 11 MS recording spectrophotometers. At the end of each run a check
against leakage was automatically provided, since the actual pressure within a
tonometer could be compared with the pressure computed from measurements
of the initial pressure and the varying increments of added gas ; the results were
deemed acceptable if the pressures obtained by the two methods agreed within
1 mm. Hg.
Sedimentation constant
A sample of centrifuged homogenate of ctenidia in pH 7.4, 0.2 M phosphate
buffer was placed in the cell of a Spinco Model E analytical ultracentrifuge and
the sedimentation constant determined at 59,780 rpm with a Wratten No. 2412
filter (red) in the optical system.
The sedimentation constant ^ of a protein is the velocity of sedimentation of
the protein molecule in a field of centrifugal force of unit strength under the
particular conditions of medium and temperature at which the determination is
performed. A correction factor can be estimated from tables given by Svedberg
and Pederseu (1940), whereby the experimental value of ^ may be corrected for
viscosity and density effects to a base of 20° C. and water; the value for the
sedimentation constant under these conditions is given the symbol s^.,,-.
As a protein sediments it leaves a volume of solution clear of that particular
protein directly behind it so that a boundary is formed. The concentration gradient
represented by the moving boundary is converted into a peak by an optical system
and the position of this peak is photographed at intervals of time ; this allows
the rate of sedimentation of the protein to be followed. Now there may be several
proteins in the sample, and if one of them is colored, a change of density of
film exposure will be associated with the colored protein's peak, since along with
the peak the contents of the sedimentation cell are also photographed. Thus, in
Figure 3 if it is assumed that the only colored protein present is hemoglobin,
then this will be the protein associated with the slow peak and as such it has
been taken.
Histology
The ctenidial tissue of PJiacoides pcctinatns was treated in two ways. The
first approach was the examination of unstained cryostat sections, while the second
608
KENNETH R. H. READ
UJ
2.0
1.0
0.9
0.8
0.7
0.6
0.5
0.4
LU 0.3
Q
o 0.2
i—
CL
o
O.I
0.0
PHACOIDES PECTINATUS HEMOGLOBIN
i r
_L
I I I I I I I I
I I
600
400
500
WAVELENGTH nyi
FIGURE 2. Absorption spectra of Pliacoidcs pectinatns hemoglobin and derivatives.
included the fixation of the tissue in 10% acrolein in xylene at 0° C., embedding
in wax and staining for occult iron by the methods of Click and Hutchison as
described in Humason (1962), somewhat modified.
The most satisfactory method for the iron stain proved to be the following :
(1) Deceration and hydration of the tissue to water; (2) two minutes in 30%
H2O2 alkalized with one drop concentrated NH3 solution per 100 ml.; (3) thor-
ough rinsing with several changes of distilled water for 5 minutes; (4) 15 minutes
at 56° C. in a freshly made, filtered and heated solution of acidified potassium
ferrocyanide, made by mixing 25 ml. each of solutions containing 2 g. potassium
ferrocyanide to 50 ml. water and 2 ml. concentrated HC1 to 50 ml. water; (5) thor-
ough rinsing with several changes of distilled water for 5 minutes; (6) counter-
staining 4 minutes in \% carminic acid; (7) 5 minutes' differentiation in 4%
potassium aluminum sulfate ; (8) thorough rinsing in several changes of distilled
water for 5 minutes; (9) dehydration, clearing and mounting. The iron-containing
material is stained a bright green and the nuclei red; the remainder of the tissues
stain various shades of pink.
RESULTS
Absorption spectra
The results of the measurement of the absorption spectra of the hemoglobin
of Phacoides pectinotus are shown in Figure 2. In the following description the
numbers in parentheses represent the range of the observations in m/* and the
number of observations, respectively. The Pliacoidcs pectinatns preparation in
HEMOGLOBIN OF PHACOIDES PECTINATUS
600
the oxygenated state shows strong «, ft and Soret bands with peaks at 578.7 (1,6).
543.3 (2,6) and 416.3 m^ (1,3), respectively; upon deoxygenation the a and ft
bands disappear, to be replaced by a single band with a peak at 558.1 m/A (2,6),
while the Soret peak shifts to 433.5 m/j. (1,2) ; treatment of the preparation with
carbon monoxide causes reappearance of the a and ft bands, but shifted towards
the violet compared with oxyhemoglobin, and their peaks lie at 572 (0,2) and
541 m/A (0.2), respectively; the Soret band of the carbon monoxide-treated prepa-
16 MIN
32 MIN
70 MIN.
86 MIN.
102 MIN.
FIGURE 3. Ultracentrifuge run. The hemoglobin is sedimenting from right to left. Note
how the larger schlieren peak is associated with the change in optical density in the cell, indicating
that it is probably the hemoglobin peak. The 16- and 32-minute photographs were taken on a
different plate from those taken during the interval 70 to 102 minutes.
610
KENNETH R. H. READ
100
90
80
70
60
co
o
o
o
50
x 40
o
30
20
10
+ pH = 7.40
A pH - 7.43
T = 25.0°C.
1.0 2.0
OXYGEN TENSION mm Hg
3.0
FIGURE 4. Oxygen dissociation curve for Phacoides pcctinatus hemoglobin. The solid
line is drawn for the Hill equation with p-M equal to 0.19 mm. Hg and H. equal to 1.
ration shifts to 422 in/A (0,2). The Phacoides pectinatus preparation absorbs to a
greater extent at the (3 peak than at the a for both oxy- and carboxyhemoglobin.
Oxygen dissociation curve
The results of this section are summarized by Figure 4. The curve shown
is drawn from the well-known Hill Equation :
(P/P^Y
i + (P/P^Y
with />r,0 equal to 0.19 mm. Hg and n equal to 1 ; y represents the fraction of
hemoglobin in the oxygenated form, /> the partial pressure of oxygen, />50 the
HEMOGLOBIN OF PHACOIDES PECTIXATUS
partial pressure of oxygen at 50 '7v saturation of the pigment and » a constant
which can he considered a measure of heme-heme interaction. A value of H equal
to 1 indicates zero heme-heme interaction and a hyperbolic dissociation curve.
Sedimentation constant
Two very fast-moving peaks rapidly formed and swiftly moved across the
field of view even before the ultracentrifuge had reached full speed. The hemo-
globin peak then formed (see Figure 3) and this in turn split into two peaks,
with the slower moving peak appearing to be associated with the hemoglobin,
as judged by the optical density changes in the cell. The value of the sedimenta-
tion constant s-^,R- was found to be 2.2 X 10~ia seconds from one determination only.
Histology
Microscopic examination of the unstained cryostat sections showed highly
localized patches of yellow-brown pigment scattered throughout the cells of the
interior of the gill. The patches were in the form of dense granular masses about
5-7 /j. in diameter, with the individual granules ranging from about 0.6 to 1.3 ^
in diameter ; this is apparent from the photomicrograph in Figure 5.
Examination of the tissue stained for occult iron showed green granular masses
IQ/i
FIGURE 5. Photomicrograph of unstained cryostat section of the ctenidium of Phacoidex
pectinahis taken without a filter in the optical system. The color of the dark masses was
yellow-brown.
612
KENNETH R. H. READ
IO/x
FIGURE 6. Photomicrograph of section stained for occult iron, taken with a Wratten No. 25
(red) filter in the optical system; the magnification is the same as for Figure 5. The color of
the two dense granular masses was green ; note the shadowy image of the nucleus at the left-hand
side of the picture (arrow).
of approximately the same size and structure as the yellow-brown masses seen in
the unstained cryostat section ; Figure 6 shows a photomicrograph taken with a
Wratten No. 25 (red) filter in the optical system at the same magnification as the
photomicrograph of the unstained cryostat section in Figure 5.
The green-staining masses were confined to the interior cells of the gill and
were not associated with the ciliated cells of the exterior surface of the ctenidium;
this distribution is apparent from examination of Figures 7a, b and c, which are
photomicrographs of the same section of the ctenidium taken without any filter,
with a Wratten No. 58 (green) and with a Wratten No. 25 (red) filter in the
optical system, respectively.
DISCUSSION
Absorption spectra
The behavior of the Phacoides pcctinatus preparation, with respect to its ab-
sorption spectra under various conditions, is very strong evidence in favor of its
containing hemoglobin. Unfortunately, the absorption spectra of other lamellibranch
hemoglobins appear to be known only for Area inflata, Area subcrenata and
Th'cla stultorum.
HEMOGLOBIN OF PHACOIDES PECTINATUS 613
For Area inflata the following wave-lengths for the absorption maxima have
been reported: oxyhemoglobin « peak. 578 m/x (Sato, 1931; Kobayashi, 1935),
576 m/x for the purified preparation of Yagi ct al. (1955a); oxyhemoglobin
ft peak, 540.8 m/i (Sato, 1931), 541 mp. (Kobayashi, 1935), 540 m/i (Yagi et al.,
1955a) ; deoxyhemoglobin, 559 m/x (Sato, 1931), 556 m/x (Kobayashi, 1935), 555
m/x (Yagi ct al., 1955a) ; carboxyhemoglobin a peak, 573.1 m/x (Sato, 1931),
570 m/x (Yagi ct al., 1955a) ; carboxyhemoglobin ft peak, 537.8 m/x (Sato, 1931),
540 m/x (Yagi ct al., 1955a).
For Area subcrenata, Kobayashi (1935) obtained values for the wave-lengths
of the absorption peaks identical with those for Area inflata. Fox (1953) states
that the hemoglobin of Tivcla st it I torn in shows sharp absorption bands at 577 and
540 m/x.
With regard to the extent of the absorption at the a and ft peaks of oxyhemo-
globin, Kobayashi (1935) found that the ratio of the absorption of the « peak to
that at the ft peak was 0.99±0.02 for Area inflata ( ?) and 1.00±0.05 for Area sub-
crenata; Yagi ct al. (1955a) with a purified preparation of Anadara inflata
hemoglobin observed values of the millimolar extinction coefficient, e,,,j/, for the
a peak of 11.1 and for the ft peak of 11.0, thus indicating a slightly greater absorp-
tion at the a peak than at the ft ; this difference was intensified when the carboxy-
hemoglobin was examined, and here the value of e,,,.,/ for the a peak was 11.2 and
for the ft peak 10.8.
The hemoglobin of Anadara inflata thus has absorption peaks at wave-lengths
only slightly different from those obtained for Phaeoides pcctinatus. However,
the two species differ in the values they show for the ratio of absorbency at the
a peak to that at the ft peak, both for oxy- and carboxyhemoglobin; for Phaeoides
pectinatus this value is slightly less than one, while for Anadara inflata the reverse
is true.
Oxygen dissociation curve
Manwell (1960a) and Prosser and Brown (1961) have thoroughly reviewed
the subject of the function of invertebrate respiratory pigments. In order for a
respiratory pigment to play any role in the transport of oxygen from the environ-
ment to the tissues, the oxygen tension at which the pigment becomes saturated
with oxygen must be of at most the same order of size as the partial pressure of
oxygen in the environment ; this statement is supported by the innumerable cases
in which the oxygen affinities of invertebrate respiratory pigments have been corre-
lated with the ecology of the species studied, such that high oxygen affinities are
associated with low environmental oxygen tensions. A state of controversy exists
as to whether the pigments serve as reserve stores of oxygen, which come into
use only when ambient oxygen levels become low, or whether they serve to
facilitate the transfer of oxygen, even when ambient oxygen levels are high
enough to maintain the pigment almost entirely in the saturated condition. Those
who hold the latter view, point out that the total oxygen capacity of the pigment
is too small to constitute an effective reserve. In actuality the pigment probably
serves both purposes, facilitating oxygen transport even when environmental
oxygen tensions are high and acting as a small reserve store when low. except
in the nematodes where the function of their hemoglobin is not understood.
614
KENNETH k. H. READ
100/1
-
loq/u
FIGURE 7a. Photomicrograph of section of gill stained for occult iron, taken with no filter
in the optical system. Note the dense mats of cilia of the ciliated cells of the surface of the
ctenidium.
HEMOGLOBIN OF PHACOlDES PECTINATUS 615
The oxygen affinity of at least one lamellibranch hemoglobin, that of Anadara
inflata, has hitherto been determined ; working with hemolyzed blood of this
species. Kawamoto (1928) found a value for />-,„ equal to 10 mm. Hg at 20± 1° C.
and a value for n of 1.155; the determination was made in the absence of CO2-
Species of Anadara are surface-living forms (as far as this author is aware) ;
Phacoides pectinatus, on the other hand, lives deep in what are most likely
anaerobic muds. The differences in the values of />.-,,) reflect the differences in
ecology of the two species. The hemoglobin of Phacoides pectinatus with its p-,Q
of 0.19 mm. Hg is far more comparable to that of the polychaete worm, Travisia
pupa than to Anadara inflata. This worm was studied by Manwell (1960b), and
its muscle hemoglobin has a /'so of only 0.08 mm. Hg ; coelomic hemoglobin, 0.36
mm. Hg; and vascular hemoglobin, a p->(} varying between 0.53 and 1.10 mm. Hg,
depending on pH ; this worm lives, like Phacoides pectinatus, totally buried in the
mud and apparently dies of oxygen poisoning when removed from its substrate
and kept in aerated sea water. However, in making interpretations such as these
from values of />.-,<) or n, obtained from impure or unphysiological preparations of a
respiratory pigment, it must be borne in mind that the values for these same
parameters obtained for the pure pigment or for the pigment in the intact animal
may be different. This was brought out by Manwell (1960c) who showed how
values of n changed with the degree of manipulation of the pigment.
Sedimentation constant
The results of the sedimentation determination should be taken with certain
reservations, not only because of the impure nature of the preparation, but also
because the results have not been extrapolated to zero protein concentration ; the
possibility also exists that actually it may not have been the hemoglobin peak
which was measured.
Prosser and Brown (1961) have published a table giving values of molecular
weight and s-2o ,„• for a number of respiratory proteins. Vertebrate hemoglobins,
excluding those of the cyclostomes, contain four heme groups and associated
polypeptide chains, and have molecular weights of about 4 X 17,000; values of
jjio.ir obtained for this type of molecule are about 4.5 X 10~13 seconds. The
circulating hemoglobins of the cyclostomes appear to have but one heme group
per molecule, molecular weights ranging from 19,100 to 23,100 and values of Jo0.,r
ranging from 1.87 to 2.3 X 10~13 seconds.
Sedimentation constants have been measured for the hemoglobins of two other
species of lamellibranch. Svedberg and Hedenius (1934) reported values of SOQ,W
ranging between 3.20 and 4.09, with a mean of 3.46 X 10~13 seconds, for the hemo-
globin of Area pexata in an impure preparation. Yagi et al. (1955a) reported a
value of -s'oo.ir of 4.6 X 10~13 seconds at pH 7.45 for their purified preparation of
FIGURE 7b. Same section as Figure 7a, taken with a Wratten No. 58 (green) filter in the
optical system. This filter accentuates the red-staining nuclei and pink background in relation
to the green-staining iron-containing granules.
FIGURE 7c. Same section as in Figures 7a and 7b but taken with a Wratten No. 25 (red)
filter in the optical system. This filter accentuates the green-staining iron-containing granules.
Note, by comparing Figures 7a, 7b and 7c, how the green-staining granules are confined to the
cells of the interior of the ctenidium and are not associated at all with the cells of the surface of
the ctenidium ; compare this with the legend under Figure 1.
616 KENNETH R. H. READ
Anadara inflata hemoglobin ; these data were combined with measurements of the
diffusion constant to give a molecular weight of 71,000; coupled with data on iron
content, which was about 0.31%, this suggests that molecules of Anadara inflata
hemoglobin are of a size and shape similar to those of the hemoglobins of higher
vertebrates.
The value of s^ „, of 2.2 X 10 13 seconds obtained for the hemoglobin of Phacoides
pcctinatns suggests that the molecule consists of one heme-polypeptide unit with a
molecular weight in the neighborhood of 17,000. The apparent absence of heme-
heme interactions in the oxygen dissociation curve is consistent with a molecule that
contains but one heme group ; in addition, the apparently low molecular weight is
consonant with the intracellular location of the pigment ( Prosser and Brown, 1961).
Histology
Two-fold lines of evidence are presented for the occurrence of hemoglobin in the
form of granular masses within the cells of the interior of the ctenidia of Phacoides
pectinatits. First, the color of the granules in the cryostat sections strongly suggests
hemoglobin, and second, particles of similar size, shape, and structure take the
characteristic green color of iron with the ferrocyanide stain.
One of the most striking findings emerging from the histological studies is the
fact that the hemoglobin, if such the yellow- or green-staining particles be, does not
appear to be associated with the energy-using ciliated cells of the surface of the
ctenidium. The localization of the pigment in granules within the cells is similar
to the findings of Griesbach (1891) and Sato (1931) for the erythrocytes of certain
other lamellibranchs.
The localization of the presumptive hemoglobin within the cell is in accord with
its sedimentation characteristics, which indicate that it has low molecular weight,
perhaps of the order of 17,000, and would tend to escape by diffusion unless it were
confined within a membrane.
The author is deeply indebted to all those whose kind advice, cooperation or help
made this work possible. These were: Professor J. H. Welsh, Dr. W. J. Clench,
Dr. Ruth D. Turner, Professor Ned Feder, Professor P. Albersheim, Dr. R. Briehl,
Dr. C. Botticelli, Mrs. B. Gibbons, Miss Norma Currie, Mr. Guy Bush, all of Har-
vard University ; and Dr. Juan A. Rivero and the staff of the Institute of Marine
Biology, University of Puerto Rico. Gratitude is also expressed to the Society of
the Sigma Xi and RESA for a grant-in-aid which covered travelling expenses.
SUMMARY
1 . The ctenidia of Phacoides pectinatus contain hemoglobin.
2. The hemoglobin appears to be located in the inner tissue of the ctenidia ; it
does not appear to be associated with the ciliated cells of the surface of the ctenidia.
3. In the cells in which it occurs, the presumptive hemoglobin is highly localized
in the form of granular masses, of diameter about 5-7 ,0., with the individual granules
having diameters ranging from about 0.6 to 1 .3 /A.
4. Evidence is presented that the hemoglobin has a sedimentation constant
s-^.,,- of 2.2 X 10~13 seconds, but this value needs confirmation.
HEMOGLOBIN OF PHACOIDES PECTINATUS 617
5. The oxygen dissociation curve of the hemoglobin exhibits no evidence of
heme-heme interaction. The pigment has a />.-,<, of 0.19 mm. Hg at 25° C. in pH 7.4,
0.2 M phosphate buffer in an impure preparation: this value is in accord with the
ecology of the species.
LITERATURE CITED
Fox, D. L., 1953. Animal Biochromes and Structural Colours. P. 254. Cambridge at the
University Press. 379 pp.
GRIESBACH, H., 1891. Beitrage zur Histologie des Blutes. Arch, fiir mikros. Anat., 37: 22-99.
HUMASON, G. L., 1962. Animal Tissue Techniques. Pp. 234-235. W. H. Freeman and Com-
pany, San Francisco. 468 pp.
KAWAMOTO, N., 1928. Oxygen capacity of the blood of certain invertebrates which contains
haemoglobin. Sci. Rep. Tohoku. Univ., Ser. 4., 3: 561-575.
KOBAYASHI, S., 1935. The spectral pro])erties of haemoglobin in the molluscs, Area inflata
(Reeve) and Area snherenata (Lischke). Sci. Rep. Tohoku. Unit1., Ser. 4., 10: 257-267.
MAXWELL, C., 1960a. Comparative physiology: blood pigments. Ann. Rcr. Pln'sioL, 22:
191-244.
MAXWELL, C., 1960b. Histological specificity of respiratory pigments — I. Comparisons of the
coelom and muscle hemoglobins of the polychaete worm Trarisia pupa and the
echiuroid worm Arynehite pugcttensis. Conip. Bioclicin. Pliysiol., 1: 267-276.
MAXWELL, C., 1960c. Heme-heme interactions in the oxygen equilibrium of some invertebrate
myoglobins. Arch. Bioclicin. Biophys.. 89: 194-201.
PALADINO, R., 1909. Vergleichung des Hamoglobins einiger Weichtiere mit clem der Wirbeltiere.
Bioclicin. Zcitschr., 22: 495-505.
PROSSER, C. L., AND F. A. BROWN, JR., 1961. Comparative Animal Physiology, 2nd Ed.
Chapter 8. W. B. Saunders Company, Philadelphia. 688 pp.
PURCHON, R. D., 1939. Reduction of the ctenidia in the Lamellibranchia. Nature. 144: 206.
RIGGS, A., 1951. The metamorphosis of hemoglobin in the bullfrog. /. Gen. Pliysiol., 35: 23-40.
SATO, T., 1931. Untersuchungen am Blut der gemeinen japanischen Archemuschel (Area inflata
Rve.). Zcitschr. rcryl. Pliysiol.. 14: 763-783.
SVEDBERG, T., AXD A. HEDENius, 1934. The sedimentation constants of the respiratory proteins.
Biol. Bull., 66: 191-223.
SVEDBERG, T., AND K. O. PEDERSEX, 1940. The Ultracentrifuge. Pp. 273-275. Clarendon Press,
Oxford. 478 pp.
YAGI, Y., T. MISHIMA, T. TSUJIMURA, K. SATO AND F. EGAMI, 1955a. Recherches sur 1'hemo-
globine d'Anadara inflata (Reeve). I. Purification et proprietes. C. R. Sac. Biol.. 149:
2285-2287.
YAGI, Y., T. TSUJIMURA AND K. SATO, 1955b. Recherches sur 1'hemoglobine d'Anadara inflata
(Reeve). II. Acides amines N- et C- terminaux. C. R. Soc. Biol.. 149: 2287-2291.
ON PHOTORECEPTOR MECHANISMS OF RETINULA CELLS
PHILIP RUCK
Department of Zoology, University of Wisconsin, Madison 6, Wisconsin
The neurophysiology of photoreception in arthropods has been studied most
intensively in compound eyes and dorsal ocelli of insects and the compound eye of
the horseshoe crab, Li in nl us. The photoreceptor cells in all of these eyes are quite
similar. They are unipolar neurons provided distally with a specialized region of
cell membrane, the rhabdomere. The basic similarity in structure suggests a
common mechanism of action of these cells. A simple and appealing hypothesis is
that the primary photochemical event in all compound eyes and ocelli triggers the
same sequence of excitatory processes in the photoreceptor cells, and that correspond-
ing parts of these cells in different eyes play the same roles. However, the complete
sequence of excitatory processes has not been worked out for any particular eye.
Granted that the above hypothesis is essentially correct, a common mechanism may
be discoverable by pooling data from a number of different eyes. A comparative
approach will be used here to arrive at a possible common mechanism of photo-
receptor cell action in insects and Limit I us.
PHOTORECEPTOR CELLS AND RKTIXULAS
Photoreceptor cells in insects and Linntlns occur in groups, or retinulas. Two
kinds of retinulas are shown in Figure 1. The one on the left, and all others in this
paper, is arbitrarily drawn with four receptor cells. The number of receptor cells
per retinula varies from species to species, and even from retinula to retinula within
the same eye. For example, retinulas of the dorsal ocellus of the cockroach,
Blaberns craniijer. contain from two to five receptor cells, three being the most com-
mon number ; a two-cell retinula from a Blabents ocellus is shown in Figure 2.
Retinulas in the compound eye of the housefly, Musca domcstica, contain 7 receptor
cells (Fernandez-Moran, 1958), while those of the compound eye of the damselfly,
.li/riocneniis (Naka, 1961) contain four. Retinulas of compound eyes in Hymenop-
tera typically contain 8 receptor cells (Hesse, 1908). Each receptor cell has a
rhabdomere, and such cells will be called retinula cells henceforth. All of the
rhabdomeres occur at cell boundaries inside the retinula.
A rhabdomere consists of tightly packed microvilli formed from a region of
limiting membrane of the retinula cell. Some details of this structure are visible
in Figure 2, and in numerous other studies (e.g.. Goldsmith and Philpott, 1957;
Miller, 1957; Fernandez-Moran, 1958). The rhabdomere appears to be diagnostic
of arthropod photoreceptor cells. It is the most probable site of the visual pigment
for the following reasons: (1 ) the distal ends of the rhabdomeres lie at the focus of
the dioptric apparatus of the ommatidium of apposition-type compound eyes (Exner,
1891 ; de Vries, 1956; de Vries and Kuiper, 1958) ; (2) the rhabdomere has a higher
refractive index than the material surrounding it, and therefore it "traps" incident
618
MECHANISMS OF RETINULA CELLS
619
light by internal reflection (de Vries, 1956) ; (3) the cytoplasm closest to the
rhabdomere is often packed, as in Lhnulus (e.g.. Miller, 1957), with opaque, black
pigment granules; (4) the distal ends of the rhabdomeres in the compound eye of
the backswimmer, Notonecta glauca (Liidtke, 1953), undergo retinomotor move-
ments, and lie closer to the cornea in the dark-adapted state than in the light-
adapted state. In short, the rhabdomeres have a number of properties which seem
X1
•
o|
jo
y
\/
fcA*
FIGURE 1. Diagrams of two kinds of rctinulas. Left: retinula consisting of four retinula cells.
Right : an eccentric cell has been added. Rhabdomeres are stippled.
adaptively appropriate to light-sensitive organelles of the photoreceptor cells, and no
other parts of these cells appear to have such properties. Direct proof that the
rhabdomeres contain visual pigment has not yet been given.
Cells with rhabdomeres are the only kind which have been described in retinulas
of insect dorsal ocelli. This is typical for compound eyes of insects as well, except
perhaps for the occurrence of rudimentary retinula cells in many Diptera (Dietrich,
1909) and certain other insects (Hesse, 1908). In the compound eye of Limulus,
however, retinulas contain an "eccentric cell," which has no rhabdomere, in addition
to typical retinula cells (Demoll, 1914; Miller, 1957). In the diagram to the right
620
PHILIP RUCK
in Figure 1 an "eccentric cell" has been added. The Liuiuhts retinula is similar
to this diagram except that in Limn I us there are ten to twenty (Hartline, Wagner
and Ratliff, 1956) retinula cells per retinula instead of four.
'
IMC.URE 2. Electron micrograph of retinula cells from the ocellus of the cockroach, Blabcn/s
cnuiiifcr. A two-cell retinula in cross-section appears in the center. Part of a three-cell
retinula appears at upper left. Arrows in the two-cell retinula indicate limiting membrane of the
retinula cells. Tracheoblasts fill the interstices between neighboring retinulas. Rhabdomere, r;
nucleus, »; mitochondria, in; tracheoles, t. (From unpublished data of G. A. Edwards and the
author.)
MECHANISMS OF RETINULA CELLS 621
RETINULA CELL RESPONSES IN THE ABSENCE OF ECCENTRIC CELLS
Retinulas without eccentric cells appear to be the simpler kind and their re-
sponses to light will be considered first. The ommatidia in compound eyes of
grasshoppers (Fernandez-Moran, 1958), dragonflies (Goldsmith and Philpott,
1957), damselflies (Naka, 1961), and dipteran flies (Fernandez-Moran, 1958;
Wolken, Capenos and Turano, 1957; Yasuzumi and Deguchi, 1958; Danneel and
130 msec
110 msec
0.5 mv
sec
ERG of surgically isolated,
rhabdomere- bearing ends
of retinula cells of Calli-
phora compound eye .
(Deganglionated prepara-
tion,Autrum and Gallwitz,
1951)
5mv
Intracellularly recorded responses
of retinula cell of damselfly
compound eye. Left, preparation
fresh. Right, deteriorating.
(Naka, 1961)
1
20 mv
ERG of retinula cells
of ocellus of Anox }
poor preparation.
( Ruck, 1961 c)
1/8 sec
1/8 sec
sec
ERG of intact retinula
cells of Lucilia com-
pound eye. (Ruck, 1961 d)
Intracellularly recorded
responses of retinula
cell of Lucilia com-
pound eye. (Naka, I960
ERG of retinula cells
of Blaberus ocellus,
normal preparation.
(Ruck, 1961 a)
FIGURE 3. Responses of insect retinula cells. For ERG's of left and right columns, an
upward deflection signifies negativity of an extracellular electrode at the corneal surface of the
retinula cell layer. For intracellular recordings of the center column, an upward deflection
signifies decreasing negativity of the intracellular electrode, i.e., depolarization of the cell. Of
the two intracellularly recorded responses from Litcilia (bottom center), the upper one was
obtained at 1000 times the stimulus intensity used for the lower.
Zeutzschel, 1957) contain retinulas of this kind. Axons of the retinula cells pass
through a fenestrated basement membrane, which forms the inner boundary of the
ommatidial layer, and enter the optic ganglion. Several investigators have studied
the electroretinograms of compound eyes from which the optic ganglia had been cut
or pulled away from the basement membrane ( Jahn and Wulff, 1942 ; Autrum and
Gallwitz, 1951; Hartline, Wagner and MacNichol, 1952). This procedure of de-
ganglionation must inevitably remove or damage the axons of the retinula cells, so
that the resulting preparation consists essentially of just the ommatidial portions of
622 PHILIP RUCK
the retinula cells. All workers agree on the nature of the electroretinogram evoked
from deganglionated compound eyes. It is a sustained, monophasic, cornea-negative
wave. One of the records of Autrum and Gallwitz (1951), obtained from the
deganglionated compound eye of the fly Calliphora, is shown in Figure 3 (top left).
Similar records have been obtained from the grasshopper, Trimerotropis (Jahn and
Wulff, 1942), and from the fly, Musca (Hartline, Wagner and MacNichol, 1952).
The results all indicate that photic excitation results in increased negativity of the
extracellular medium at the corneal ends of the retinula cells. A simple interpreta-
tion, often made, is that light induces depolarization of the rhabdomere-bearing
ends of the retinula cells.
Deganglionated preparations provide no information about responses of retinula
cell axons. Dorsal ocelli of cockroaches and dragonflies have been helpful in this
regard (Ruck, 1961a, 1961b, 1961c). Retinula cell axons of dorsal ocelli make
synaptic contact deep within the ocellus with dendrites of ocellar nerve fibers. Elec-
troretinograms of these organs are quite complex but only two kinds of cells
contribute components, retinula cells and ocellar nerve fibers. Components originat-
ing in the ocellar nerve fibers can be excluded by recording between the corneal
surface of the retinula cell layer and a point deep within that layer. The responses
of the retinula cells isolated in this way contain two components (Fig. 3, bottom
right). One is a cornea-negative wave essentially like that recorded from de-
ganglionated compound eyes. This event can be isolated in at least two ways, either
by bathing the retinula cells in solutions containing high concentrations of potassium
ion, or by waiting until a preparation deteriorates physiologically (Fig. 3, top right).
The sustained, cornea-negative wave is always the residual component remaining
after all other features of the electroretinogram have disappeared. This event, as in
the compound eyes, is believed to originate as a depolarization of the rhabdomere-
bearing ends of the retinula cells (Ruck, 1961a).
In addition to the sustained, cornea-negative wave, the retinula cell response of
the ocellus includes a transient, cornea-positive wave at "on" (Fig. 3, bottom right).
Several lines of indirect evidence led to the conclusion (Ruck, 1961a) that the
cornea-positive on-component is a depolarizing response of the retinula cell axons.
It appears as a negative wave extracellularly in the vicinity of the retinula cell axons.
An extracellular electrode at the corneal surface is presumably at the "source" of
current flowing to the "sink" in the vicinity of the retinula cell axons. A very
similar and presumably homologous component (Fig. 3, bottom left) has been
recorded across the intact retinula cell layer of the compound eye of the fly, Lucilla
scricata (Ruck, 196kl).
It can be deduced from the analysis of the ocellar electroretinogram (Ruck,
1961a) that an intracellnlar electrode in a retinula cell should record two depolariz-
ing components, one which is sustained throughout the period of illumination, and
one which has a transient phase at "on." As yet there are no intracellular record-
ings from retinula cells of dorsal ocelli, but there are from retinula cells of the
compound eye of Lucilia (Naka, 1961). In Naka's records, two of which were
copied and reproduced in Figure 3 (bottom center), two depolarizing components
appear. One is a sustained potential, the other a transient at "on." Naka (1961)
recorded similar components from retinula cells in compound eyes of damselflies
(Fig. 3, top center), and showed that in retinula cells which are deteriorating
MECHANISMS OF RETINULA CELLS 623
physiologically the sustained depolarizing potential appears in isolation. The
amplitudes of both components in preparations in good condition are graded with
stimulus intensity, and depend upon the state of adaptation (Naka, 1961 ; Naka
and Eguchi, 1962).
Burkhardt and Autrum (1960) have reported intracellular recordings from
retinula cells of compound eyes of Calliphora. Their records are similar to those of
Naka (1961) from Lucilia. However, Burkhardt and Autrum feel that the
transient event at "on" is not a response of the cell inside of which their electrode is
situated. They consider that retinula cells produce only sustained depolarizations,
hut they have offered no decisive data in support of this interpretation.
The nature of insect retinula cell responses may he summarized briefly. Results
from deganglionated compound eyes establish the rhabdomere-bearing ends of the
retinula cells as the source of a sustained, cornea-negative potential in response to
light. Intracellular recordings establish that the sustained potential is a depolar-
ization of the retinula cells. Intracellular recordings also indicate a transient,
depolarizing on-component. Since this latter event appears as a positive wave
extracellularly near the cornea, it cannot originate in the rhabdomere-bearing ends
of the retinula cells. If it did it would have the same polarity (negative) extra-
cellularly as the sustained depolarization. The most plausible inference is that the
transient on-component is a sign of depolarization of the retinula cell axons.
RETINULA CELL RESPONSES IN THE PRESENCE OF ECCENTRIC CELLS
In the compound eye of Limulus, identification of the responses of retinula cells
(those with rhabdomeres) is more difficult than in insects because retinulas of
Lhnitlits contain eccentric cells. The difficulty is mitigated by two circumstances:
( 1 ) the nature of eccentric cell responses is well known because these cells have
been impaled with microelectrodes under direct observation (Hartline, Wagner and
Ratliff, 1956; MacNichol, 1956); (2) intracellular recordings, unlike those from
eccentric cells, but very similar to those from insect retinula cells, have been obtained
from retinulas of Limulus (Fuortes, 1958; MacNichol, 1956; Benolken, 1961).
A quotation from Benolken (1961) may serve to describe the kinds of intra-
cellular records obtained from Limulus ommatidia : "When a micropipette is inserted
into a cell of an ommatidium, a resting potential is recorded such that the micro-
pipette becomes polarized about 55 mv negative with reference to an extracellular
electrode. As the micropipette is probed through an ommatidium, the electrode may
or may not record an electrical response to light. The success or failure of recording
a response to light presumably depends upon the location of the micropipette in the
photoreceptor unit. If the micropipette has been positioned in a region where an
electrical response to light can be recorded, the response takes the form of (a) a
graded receptor potential (generator potential) and (b) nerve impulses propagated
from the optic nerve. Impulses propagated in the optic nerve appear to be generated
near the eccentric cell (MacNichol, 1956). Presumably the electrial activity asso-
ciated with the propagated impulses is recorded via passive conduction through the
various structures of the ommatidium when the micropipette is placed in a location
which is remote from the eccentric cell.
"The relative amplitudes of the generator potential and the amplitudes of
impulse activity which were recorded from the eye were markedly dependent upon
624
PHILIP RUCK
electrode placement. In general, whenever the micropipette was positioned so
that generator potentials of relatively large amplitude (60 to 90 mv) could be
recorded in response to intense illumination, nerve impulse activity of relatively
small amplitude (less than 1 mv) was recorded. Conversely, whenever large-
amplitude (40 to 50 mv) nerve impulses were recorded, the generator potential
amplitude (50 mv or less) was reduced in response to intense illumination."
0 t-
20
40
0
20
40
I sec
V
Intracellularly recorded responses of
one kind of cell in ommatidium of
Limulus . (Fuortes, 1958)
ERG of compound eye
of Limulus .
FIGURE 4. Presumptive retinula cell responses from the compound eye of Linuiliis. Left:
the ordinate in Fuortes' records measures the potential in mv of an intracellular electrode relative
to an indifferent electrode in the external medium. Negativity is measured downward. Right :
(from unpublished records of the author) upward deflection signifies negativity of an extracellular
electrode placed just under the cornea. The first of a series of high intensity flashes, Vs of a
second in duration, elicited the upper response from the dark-adapted eye. Later flashes elicited
the next two responses. Each flash produced an illumination of about 10,000 foot-candles (white
light) at the cornea. Electrodes were condenser-coupled to the amplifier. The film strip on
which the recordings were made moved continuously during the series of flashes.
MacNichol (1956) recognized three types of intracellularly recorded responses
from Limulus ommatidia : large spikes together with small slow potentials, small
spikes with large slow potentials, and small spikes with small slow potentials.
MacNichol stated that he had never seen large spikes and large slow potentials
together.
Fuortes (1958) stated that in most penetrations of ommatidial cells of Liinnlits,
illumination produced large slow potentials and small spikes, spike size ranging
between zero and 15 mv. Penetrations of units giving large spikes of 40 mv or
more were made more rarely, according to Fuortes.
That large spikes are recorded intracellularly from eccentric cells is known
MECHANISMS OF RETINULA CELLS 625
because MacNichol (1956) and Hartline, Wagner and Ratliff (1956) have pene-
trated exposed eccentric cells under direct observation and obtained large spikes.
\Yaterman and Wiersma (1954) presented strong evidence that axons of eccentric
cells are the only ones in the optic nerve of Lhintlits which conduct nerve impulses
in response to illumination.
The nature of the retinula cell response in Lima! us has been more problematical.
On the basis of existing information, a reasonable argument can be made that the
penetrated units (Benolken, 1961; MacNichol, 1956; Fuortes, 1958) which have
given large slow potentials and small spikes, or no spikes at all, in response to
illumination are retinula cells. This is intimated by Fuortes (1958) on grounds
that this kind of unit is encountered much more commonly than the kind of unit
(eccentric cell) which gives large spikes. Two responses of the more commonly
recorded type were copied from Fuortes' 1958 paper and appear in Figure 4 (left).
The similarity of the responses to those recorded from retinula cells of Lucilia and
damselflies (Naka, 1961), and Calliphora (Burkhardt and Autrum, 1960) is strik-
ing. This category of response, in Li in nl us as well as in the insects, includes two
depolarizing components, one which is sustained during illumination, and one which
is transient at "on."
Benolken's paper is apparently the first in the Liinnliis literature to be concerned
entirely with properties of units giving large slow potentials and small spikes.
Benolken reports that the transient on-component and the sustained slow potential
are graded with stimulus intensity, and that the transient on-component may reverse
the membrane potential at very high stimulus intensities. Reversal of membrane
potential is apparent also in the records of Figure 4 copied from Fuortes (1958).
Evidence was presented in the previous section of this paper that the sustained
component (generator potential) of the insect retinula cell originates in the rhabdo-
mere-bearing end of the cell, the transient on-component in the axon. If this is
also the case in Limulus, one would expect the sustained depolarization to appear
as a negative wave extracellularly near the cornea, with the transient on-component
superimposed as a positive wave. That is, one would predict an electroretinogram
similar to those recorded from retinula cell layers of insect dorsal ocelli and of the
compound eye of Lucilia (Fig. 3, bottom right and bottom left). The expectation
is borne out as Figure 4 (right) indicates. The electroretinograms shown there
were recorded from an intact horseshoe crab which measured about 7 inches across
the carapace. One stainless steel electrode was inserted just underneath the cornea,
and another was thrust through the carapace into hemolymph about an inch away
from the experimental eye. Stimuli of high intensity and %-second duration were
presented at rates varying from 1 per second to 1 per 5 seconds for a period of several
minutes. The upper record in Figure 4 (right) is the response of the dark-
adapted eye. It is a simple, cornea-negative wave, with perhaps a slight inflection
at the foot of the ascending limb. The next two responses are those of the partially
light-adapted eye and were selected for greatest prominence of the inflections on the
ascending limb. Under the stated conditions, the electroretinogram of Limulus
becomes quite similar to that of the insect retinula cell layer. Duality of the Limulus
electroretinogram was the subject of a study of Wulff (1950), who suggested that
retinula cells and also eccentric cells contribute to the response. An alternative
suggestion is that the Limulus electroretinogram is generated entirely in the retinula
626 PHILIP RUCK
cells, and consists of a generator potential originating in the rhabdomere-bearing ends
of the cells, together with a retinula cell axon response. The latter is generally much
less conspicuous in Limulus than in certain insect eyes, but it can be made fairly
prominent with repetitive stimulation at high intensity. The suggestion that a
retinula cell axon response occurs in Limulus must be reconciled with the evidence
that the retinula cell axons in Limulus do not conduct nerve impulses (Waterman
and Wiersma, 1954).
THE FUNCTION OF THE RETINULA CELL AXONS
The function of the retinula cell axons has been suggested in the case of the dorsal
ocelli of dragonflies (Ruck, 1961a, 1961b, 1961c) : depolarizing responses of the
retinula cell axons cause the release of inhibitory transmitter substance which
evokes hyperpolarizing postsynaptic potentials in the dendrites of ocellar nerve
fibers ; a spontaneous dark discharge of nerve impulses is inhibited as a consequence.
Inhibition of ocellar nerve impulses has also been observed in Locusta (Hoyle,
1955) and Calliphora (Autrum and Metschl, 1961).
Properties of the presumptive retinula cell axon response are well enough docu-
mented to attest that it is not a conventional propagating nerve impulse. Its ampli-
tude (i.e., that of the transient on-component) in intracellular recordings from
retinula cells of Lucilia and damselflies (Naka, 1961 ; Naka and Eguchi, 1962), and
perhaps from Calliphora (Burkhardt and Autrum, 1960; their Figure 3), is graded,
and depends upon stimulus intensity and the level of adaptation. In Limulus, like-
wise, intracellular recordings from units giving large slow potentials and small
spikes show that the transient on-component is graded in amplitude with stimulus
intensity, and may even reverse the membrane potential at high stimulus intensities
(Benolken, 1961 ; Fuortes, 1958). These data indicate that this component does
not have the properties of propagating nerve impulses.
Tentatively, then, the retinula cell axon response is a local, nonpropagating or
decrementally propagating event. If transmission at the first synapses in the optic
pathway depends upon such a local event, the distance between the origin of the
retinula cell axons and the synapses must be very short. Retinula cell axons in
ocelli and compound eyes of insects are indeed very short, less than a mm. in length.
The first synapses in the ocellus lie at the base of the ocellar cup, a fraction of a mm.
from the cornea (Cajal, 1918; Ruck, 1957). The postsynaptic potential has been
described for the dragonfly ocellus (Ruck, 1961a, 1961b). It is a cornea-positive
wave associated with inhibition of ocellar nerve impulses, and it is easily interpreted
as a hyperpolarizing postsynaptic potential.
In the insect compound eye the first synapses occur in the lamina ganglionaris, a
complex neuropile situated close to the basement membrane of the ommatidial layer
(e.g., Cajal and Sanchez, 1915). Whether nerve impulses occur in second order
neurons of the lamina ganglionaris has not been definitely established, but post-
synaptic potentials have been allocated to the lamina. Autrum and Gallwitz (1951)
found that a sustained cornea-positive component of the electroretinogram of Calli-
phora could be removed completely by surgical means only when the lamina
ganglionaris was cut away from the ommatidia. Surgical removal of the lamina
involves simultaneous removal of the retinula cell axons, as mentioned previously in
this paper and elsewhere (Ruck, 1961a, 1961d), and consequently another kind of
MECHANISMS OF RETINULA CELLS 627
experiment is needed to discriminate between cornea-positive components which may
originate in the retinula cell axons and in the lamina ganglionaris. Such an experi-
ment has been performed (Ruck, 1961d) on the compound eye of Lucilia. There
are indeed two cornea-positive components, one the retinula cell axon response, and
the other a sustained potential from the lamina ganglionaris. The latter event has
the same wave form and polarity as the postsynaptic potential of dragonfly ocellar
nerve fibers, and consequently has been identified tentatively as a hyperpolarizing
postsynaptic potential in second order neurons of the lamina ganglionaris (Ruck,
1961d).
In the Limulus compound eye, as in the insects, a synaptic region lies very close
to the ommatidia. This is the peripheral plexus formed by the intermingling of
collaterals from both retinula cell axons and eccentric cell axons (Ratliff, Miller
and Hartline, 1958). Positive identification of the pre- and postsynaptic units has
not yet been reported. The plexus mediates inhibitory interactions among neighbor-
ing ommatidia. The nature of the interactions has been described very thoroughly
(Hartline. Wagner and Ratliff, 1956; Hartline and Ratliff, 1957, 1958). If A and
B are neighboring ommatidia, illumination of A alone causes an increased frequency
of nerve impulses in the eccentric cell axon from A. Illumination of B alone causes
an increased frequency of impulses in the eccentric cell axon from B. The impulse
frequency in eccentric cell A is reduced if B is simultaneously illuminated, and
vice versa. Interactions among neighboring ommatidia are eliminated when the
axon collaterals which interconnect them are severed.
In a comparative sense it seems significant that lateral interactions in the Limulus
compound eye are of an inhibitory nature. It reminds one of inhibition of nerve
impulses in ocellar nerve fibers, and the evocation of hyperpolarizing postsynaptic
potentials by retinula cells in ocelli, and perhaps also in compound eyes of insects.
It suggests that the retinula cells in Limulus are the presynaptic units in the
peripheral plexus, the eccentric cells the postsynaptic units, and that the retinula
cells are performing a general, perhaps evolutionary primitive, inhibitory pre-
synaptic function in the plexus.
The observations of Tomita (1958) that antidromic electrical stimulation of
the optic nerve of Limulus produces lateral inhibition do not support the sug-
gestion made here that retinula cells initiate the inhibition, but neither do they
refute it. One possible interpretation of Tomita's observations is that antidromic
nerve impulses propagating in a given eccentric cell axon enter the peripheral
plexus and initiate inhibition at junctions between this axon and neighboring
eccentric cell axons, and that orthodromic nerve impulses evoked by illumination
in eccentric cell axons act in the same manner. Another possible interpretation
is that inhibition of nerve impulses in a given eccentric cell axon is initiated by
retinula cells of neighboring ommatidia in two different ways: (1) directly,
through the agency of a local potential, and subsequent release of inhibitory trans-
mitter, evoked by the generator potential in the retinula cell axons and their
collaterals in the plexus; (2) indirectly, through ephaptic excitation of retinula
cell axons and their collaterals by nerve impulses, antidromic or orthodromic,
propagating through the peripheral plexus in eccentric cell axons. In their present
state, the data on antidromically produced lateral inhibition appear to have no
decisive bearing on the suggestion that retinula cells initiate the inhibition.
628
PHILIP RUCK
Major comparative points which have been made are summarized briefly in
Table I. The evidence supporting entries in the table may be found in previous
parts of the text. Information to replace the two question marks in the table
would be most helpful.
RETINULA MORPHOLOGY AND THE ORIGIN OF THE GENERATOR POTENTIAL
1. Rhabdomere membrane and distal, non-rhabdomere membrane
The conclusion that the generator potential originates in the rhabdomere-
bearing end of the retinula cell depends upon the evidence (see above) that this
event occurs in retinulas which contain retinula cells only, and persists in such
retinulas following surgical removal of the retinula cell axons. The rhabdomere-
bearing end of the retinula cell is bounded by two morphologically distinguishable
regions of limiting membrane (Fig. 2), rhabdomere membrane, from which the
microvilli of the rhabdomere are formed, and the remainder which may be desig-
TABLE I
Insect ocellus
Insect compound eye
Limulus compound eye
Retinula cells
rhabdomere-bearing ends
depolarizing generator po-
tential
graded, depolarizing gen-
erator potential
graded, depolarizing gener-
ator potential
axons
depolarizing potential, rec-
ognizable as transient on-
component
graded, depolarizing po-
tential, recognizable as
transient on-component
graded, depolarizing, non-
propagating potential, rec-
ognizable as transient on-
component
Second order neurons
(ocellar nerve fibers)
(lamina ganglionaris units)
(eccentric cell axons of
neighboring omniatidia)
postsynaptic membrane
response
hyperpolarizing postsynap-
tic potential
hyperpolarizing postsynap-
tic potential
?
nerve impulses
inhibited by illumination
?
inhibited by illumination
nated distal, non-rhabdomere membrane. Both of these membrane regions must
be considered as possible sites of origin of the generator potential. A light-
induced change, such as a decrease in membrane resistance, occurring in either
region could cause depolarization of the retinula cell. Thus far, neither intra-
cellular nor extracellular recordings have permitted discrimination between elec-
trical properties of the two kinds of membrane. A significant problem probably
exists here, and the experimental evidence needed to solve it is likely to be difficult
to obtain. There appears to be little doubt that the primary photochemical event
occurs in the rhabdomere, or at least in very close association with it. The problem
is to determine whether the primary electrical event, the generator potential,
occurs in rhabdomere membrane in close proximity with the photochemical event,
or in distal, non-rhabdomere membrane some distance away. In the absence of
direct evidence bearing upon this problem, it may be worthwhile to suggest a
speculative solution based upon indirect evidence. The rest of this section is
frankly speculative in nature.
There are data, both morphological and physiological, which suggest the
hypothesis that the generator potential originates in distal, non-rhabdomere
membrane, and that rhabdomere membrane behaves simply as a fixed resistance
MECHANISMS OF RETINULA CELLS
629
through which an appreciable fraction of the current associated with the generator
potential may flow. Consider that solitary retinula cells do not occur in insects
or Limulus, and that rhabdomeres, almost without exception (e.g., see Fig. 6,
right), occur at cell boundaries inside the retinula. According to the hypothesis,
a solitary retinula cell (Fig. 5, left) undergoing depolarization might be expected
to suffer current "leakage" through rhabdomere membrane. It is conceivable
that the retinula-type organization (Fig. 5, center) is advantageous because, with
rhabdomere facing rhabdomere, current flowing inward through distal, non-
rhabdomere membrane is conserved for outward, excitatory flow through mem-
brane of the retinula cell axons.
A
FIGURE 5. Suggested pattern of current flow produced by the generator potential mechanism
in three different morphological situations. Arrows indicate direction of movement of positive
charge. See text for explanation.
In a retinula (Figs. 1, 2), the rhabdomeres taken collectively are virtually
surrounded by distal, non-rhabdomere membrane which forms the circumference
of the retinulas as a whole. If distal, non-rhabdomere membrane is the site of
origin of the generator potential, while rhabdomere membrane is not directly
involved and behaves simply as a fixed resistance, a test probe inserted between
adjacent rhabdomeres might be expected to "see" the generator potential as a
positive-going wave, much as though the test probe were actually inside a retinula
cell. Manipulating a test probe into position between adjacent rhabdomeres
would be extremely difficult in most retinulas. Perhaps the technical problem
has been simplified somewhat by the existence of a naturally evolved "test probe"
in the form of the distal process of the eccentric cell of Liiunlns (Fig. 5, right).
630 PHILIP RUCK
With this thought in mind, some of the data obtained from the Limulits ommatidium
will be reviewed.
One kind of unit in the Limulus ommatidium gives large, depolarizing slow
potentials and small spikes, or no spikes at all, in response to illumination
(MacNichol, 1956; Fuortes, 1958; Benolken, 1961). It was suggested earlier
that this kind of unit is the retinula cell. Another kind of unit gives much larger
spikes (MacNichol, 1956; Fuortes, 1958; Benolken, 1961), but smaller, de-
polarizing slow potentials (MacNichol, 1956; Benolken, 1961); Fuortes (1958)
makes no comment concerning the relative size of the slow potential. The latter
kind of unit is the eccentric cell, according to the observations of MacNichol
(1956) and Hartline, Wagner and Ratliff (1956). The depolarizing slow poten-
tial (generator potential) coincides in time with a decrease in resistance measured
between an electrode inside an eccentric cell and an electrode in the saline medium
bathing the eye (Fuortes, 1959). The magnitude of potential change of the
electrode inside the eccentric cell is directly proportional to the magnitude of the
resistance change, and both are directly proportional to the frequency of nerve
impulses in the eccentric cell axon (Fuortes, 1959; Rushton, 1959). On the
basis of these data, Fuortes (1959) and Rushton (1959) suggested that the
change in potential of the electrode inside the eccentric cell arises because of a
permeability change of eccentric cell membrane, and that this change is most prob-
ably produced by a chemical substance released during illumination of the photo-
receptor.
The experiments of Fuortes (1959) may be interpreted differently according
to one's assumptions regarding the site of the light-induced decrease in membrane
resistance. Between the inside of the distal process of the eccentric cell and the
external medium (Fig. 5, right) lie eccentric cell membrane, rhabdomere mem-
brane, and distal, non-rhabdomere membrane of the retinula cells. Any one of
these could conceivably be the site of the resistance change measured by Fuortes
(1959). If eccentric cell membrane is the site, it is reasonable to assume that
the retinula cells release a substance which increases the permeability of eccentric
cell membrane, and that the generator potential originates in the eccentric cell.
However, this interpretation forces one to seek another explanation of the origin
of the generator potential in retinulas which lack eccentric cells.
A quite different interpretation emerges if two assumptions are made : ( 1 ) that
the resistance change in Limulus is restricted to distal, non-rhabdomere membrane ;
(2) that there is, in effect, no intercellular space between rhabdomere and distal
process of the eccentric cell. The distal process of the eccentric cell may then be
regarded as a passive structure, so situated that it acts as a pathway for a fraction
of the current which flows inward through distal, non-rhabdomere membrane
during the interval that that membrane is occupied by the generator potential.
In completing the circuit, current flows outward through soma or axon of the
eccentric cell and excites the discharge of nerve impulses there. This idea is
consistent with the observation (MacNichol, 1956; Benolken, 1961) that the
generator potential appears smaller in intracellular recordings from (eccentric) cells
giving large spikes than from cells giving small spikes, or no spikes at all ; if the
generator potential originates in distal, non-rhabdomere membrane of the retinula
cell, it is reasonable to expect that a fraction of it be "dropped" across the resist-
MECHANISMS OF RETINULA CELLS
631
ances represented by rhabdomere membrane and eccentric cell membrane. If tbis
interpretation is correct, no light-induced resistance change should occur between
an electrode in the eccentric cell and an electrode in a retinula cell of the same
retinula. An experiment to test this prediction has not yet been reported.
If the rhabdomere is the site of the primary photochemical event, and distal,
non-rhabdomere membrane is the site of the change in ionic permeability which is
responsible for establishing the generator potential, it is reasonable to suggest that
the rhabdomere releases a substance \vhich traverses the interior of the retinula
cell to reach distal, non-rhabdomere membrane and alter its permeability.
2. Variations in retinula morphology and their possible significance
If it is plausible that the eccentric cell of Limulus is excited by currents generated
in the retinula cells, it is equally plausible that in retinulas without eccentric cells,
currents generated in one cell flow through all of the cells. In other words, func-
tional interactions among retinula cells of the same retinula must be considered a
FIGURE 6. Two variations in retinula morphology found in insects. Left: like Figure 1
(left) except for space in center of retinula. Right : a retinula cell with an "internal" rhabdomere
is added. See text for explanation.
definite possibility. In many insects, for example, in dipteran flies (Dietrich,
1909), the rhabdomeres are separated by a space in the center of the retinula.
A retinula with such a central space is represented diagrammatically in Figure 6
(left). Certainly the separation of the rhabdomeres increases the optical isolation
of the individual retinula cells, and may consequently improve the visual acuity
of the eye (de Vries, 1956), but the space between the rhabdomeres may also act
as a shunt path for currents flowing into it through the rhabdomeres, and thus
may decrease electrical interactions among the retinula cells.
In at least one insect, Notonecta glauca (Liidtke, 1953), one retinula cell differs
from its neighbors in that it has an "internal" rhabdomere and occupies a central
position in the retinula (Fig. 6, right). It would be most interesting to know
whether the "internal" rhabdomere is formed by invagination of outer membrane
of the retinula cell. The internal position of the rhabdomere may have the
significance that it prevents "leakage" of excitatory current (the kind of leakage
suggested for the hypothetical, solitary retinula cell in Figure 5, left), and
reduces electrical interactions between this retinula cell and its more typical
neighbors. The existence of an "internal" rhabdomere incidentally suggests that
632 PHILIP RUCK
the photochemical event, if it occurs in the rhabdomere, leads to excitation of
distal, non-rhabdomere membrane ; in this case the rhabdomere appears to be
surrounded by distal, non-rhabdomere membrane. More information is needed
concerning the structure of the central retinula cell of Notonecta.
Finally, the argument that neighboring retinula cells may interact electrically
induces caution in interpreting intracellular recordings from retinula cells. It is
quite conceivable that a microelectrode situated inside one retinula cell, or even
in the central space of some retinulas, actually records activity of all the cells of
the retinula.
RECAPITULATION
A common mechanism of action has been suggested for retinula cells of insects
and Limulus. The rhabdomere is assumed to be the site of the primary photo-
chemical event in the photoreceptor process, but the generator potential is thought
to originate some distance away in distal, non-rhabdomere membrane of the retinula
cell. The generator potential is considered to evoke a graded, depolarizing, non-
propagating response in the retinula cell axons. The retinula cell axon response is
thought to cause the release of a chemical transmitter substance which mediates syn-
aptic transmission at the junctions between the retinula cells and second order
neurons of the optic pathway. The general, perhaps evolutionarily primitive, synap-
tic relationship between retinula cells and the second order neurons is thought to be
an inhibitory one, exemplified by the dorsal ocellus of the dragonfly (Ruck, 1961a,
1961b), in which spontaneous nerve impulse activity of second order neurons is
inhibited by illumination of the retinula cells. This relationship is thought to be
preserved in the peripheral plexus of the Limulus compound eye where the ec-
centric cell is inhibited by (retinula) cells of neighboring ommatidia. The distal
process of the eccentric cell, a structure which to present knowledge has no
counterpart in insect retinulas, is thought to lead off a fraction of the current
produced by the generator potential mechanism of the retinula cells. This current,
flowing outward through membrane of the soma and/or the proximal portion of
the eccentric cell axon, is thought to excite the discharge of eccentric cell nerve
impulses ; that is, excitation of the eccentric cell is considered to depend, in effect,
upon electrical synaptic transmission. Waterman and Wiersma (1954) pointed
out that retinula cell axons in Limulus do not conduct nerve impulses, and that
therefore the eccentric cell, which is not itself a photoreceptor, is essential to the
transmission of information to the central nervous system. To their discussion
may be added the suggestion that the evolutionary forerunner of the Limulus
eccentric cell might have been a spontaneously active postsynaptic neuron, which
was inhibited by illumination of the retinula cells. A new growth from the soma
of this postulated ancestral neuron could have invaded the retinula to become the
distal process of the eccentric cell. With this addition, the nerve impulse fre-
quency of the eccentric cell axon could be increased by illumination, whereas
without the distal process the impulse frequency could only be decreased. Evolu-
tionary adjustments of threshold of the eccentric cell to its own autoexcitatory
mechanism, to electrical currents generated by the retinula cells, and to inhibitory
transmitter substance released by retinula cell axons, might have combined to
produce the physiological properties of the present eccentric cell of Limitlus.
MECHANISMS OF RETINULA CELLS 633
The experimental work of the author was supported by grants from the National
Science Foundation and the U. S. Public Health Service to Tufts University where
the work was done. Additional support from NSF Grant GB-127 to the University
of Wisconsin is acknowledged. The electron micrograph of Figure 2 originated in
the laboratory of Dr. G. A. Edwards at the New York State Department of
Health, Albany.
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Gen. Physiol., 39: 651-673.
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HARTLINE, H. K., AND F. RATLIFF, 1958. Spatial summation of inhibitory influences in the eye
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HESSE, R., 1908. Das Sehen der niederen Tiere. Jena. Fischer.
HOYLE, G., 1955. Functioning of the insect ocellar nerve. /. Exp. Biol., 32: 397-407.
JAHN, T. L., AND V. J. WULFF, 1942. Allocation of the electrical responses from the compound
eyes of grasshoppers. /. Gen. Physiol., 26: 75-88.
LUDTKE, H., 1953. Retinomotorik und Adaptationsvorgange im Auge des Riickenschwimmers
(Notonecta glauca L.). Zeitschr. vergl. Physiol., 35: 129-152.
MAcNiCHOL, E. F., JR., 1956. Visual receptors as biological transducers. In: Molecular Struc-
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MILLER, W. H., 1957. Morphology of the ommatidia of the compound eye of Limulus. J.
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potential. Science, 136: 877-879.
634 PHILIP RUCK
RATLIFF, F., W. H. MILLER AND H. K. HARTLINE, 1958. Neural interaction in the eye and the
integration of receptor activity. Ann. N. Y. Acad. Sci., 74: 210-222.
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and inhibition of impulses in the ocellar nerve of dragonflies. /. Gen. Physiol., 44:
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RUCK, P., 1961c. Electrophysiology of the insect dorsal ocellus. III. Responses to flickering
light of the dragonfly ocellus. /. Gen. Physiol., 44: 641-657.
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419-429.
DE VRIES, H., 1956. Physical aspects of the sense organs. Progress in Biophysics and Bio-
physical Chemistry, 6: 207-264.
DE VRIES, H., AND J. W. KUIPER, 1958. Optics of the insect eye. Ann. N. Y. Acad. Sci., 74:
196-203.
WATERMAN, T. H., AND C. A. G. WIERSMA, 1954. The functional relation between retinal cells
and optic nerve in Limulus. J. Exp. Zoo!., 126: 59-85.
WOLKEN, J. J., J. CAPENOS AND A. TURANO, 1957. Photoreceptor structures. III. Drosophila
mclanogaster. J. Biophys. Biochem. Cytol., 3: 441-448.
WULFF, V. J., 1950. Duality in the electrical response of the lateral eye of Limulus polyphemus.
Biol. Bull., 98: 258-265.
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THE STRUCTURE AND METABOLISM OF A CRUSTACEAN
INTEGUMENTARY TISSUE DURING A MOLT CYCLE1
DOROTHY M. SKINNER 2
The Biological Laboratories, Harvard University, Cambridge, Massachusetts
"And the body form is moulded by the epidermis. It is the epidermis which
shapes the organism in all its details ; the other tissues, which support and nourish
and connect one part with another, follow the lead which the epidermis gives.
Even the great integrating systems, the endocrine organs and the central nervous
system, are historically a part of the ectoderm, and where they influence the body
form they do so chiefly by the activation of the epidermis."
In these few sentences Wigglesworth (1945, p. 23) outlined one of the great
challenges of arthropod physiology. It is known that during the period preceding
ecdysis the arthropod epidermis undergoes profound changes in structure which
probably reflect the synthesis of a new exocuticle (or exoskeleton) to encompass
the reshaped and enlarged animal (Kuhn and Piepho, 1938; Travis, 1955, 1958;
Wigglesworth, 1933). The preparation for molting is also accompanied by a
50 to 1900% increase in oxygen consumption by the whole animal (Bliss, 1953;
Edwards. 1950, 1953 ; Nyst, 1941 ; Poulson, 1935 ; Schneiderman, 1952 ; Schneider-
man and Williams, 1953; Scudamore, 1947), which means that the metabolism
of some or all of the tissues is vastly increased.
This paper describes the structure and metabolism of the integumentary tissue
of the land crab, Gecarcinus lateralis, during the molt cycle. Integumentary tissue
is comprised of two sheets of epidermal cells separated by a layer of connective
tissue.
As a source of integumentary tissue, the branchiostegites, sheets of tissue
which form the covering of the branchial chambers, were selected for several
reasons. A small piece of tissue, 10 to 20 mm.2, could be excised from each
branchiostegite without affecting the length of the molt cycle. Routinely, two
samples were taken from the same animal at different times in the molt cycle.
At its maximum, the integumentary tissue of the branchiostegites is only 450 p
thick. Without being sliced it should, therefore, permit adequate diffusion of
oxygen to interior cells (Field, 1948). The use of a tissue which did not have
to be sliced reduced to a minimum changes in oxygen consumption due to loss of
coenzymes by diffusion from injured cells or by the action of nucleotidases (Mann
and Ouastel, 1941).
Drach (1939) subdivided the crustacean molt cycle into stages A through D,
1 This report is taken from a thesis presented by the author to the Department of Biology in
partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of
biology. This investigation was supported by predoctoral fellowship 5576 from the United
States Public Health Service.
2 Present address : Department of Physiology and Biophysics, New York University School
of Medicine, New York, New York.
635
636 DOROTHY M. SKINNER
depending on the state of exoskeleton. The exoskeleton was pliable in stages
A and B, 4 to 8 days immediately following ecdysis, whereas stage C, the three-
month intermolt period, was marked by a rigid exoskeleton. The 15- to 30-day
premolt period, during which the two outer layers of the new exoskeleton were
formed, was designated as stage D.
The cytological changes of the epidermis and other cells of the integumentary
tissue of Gecarcinus were correlated with the exoskeletal changes described by
Drach. Metabolic studies of the integumentary tissues were then undertaken.
The rates of oxygen consumption of pieces of integumentary tissues taken from
a series of animals in each stage of the molt cycle were measured. The exact
stage of each animal was determined from inspection of sections of tissue removed
from the animal on the day of the metabolic studies.
Limbs autotomized from crustaceans are regenerated during the premolt period
(Bliss, 1956). In the present study, when the size of regenerating limb buds
was correlated with the structure of the epidermis, it was found that regeneration
of limbs is complete before any morphological changes are detected in the epidermis.
A report on some of these results has appeared previously (Skinner, 1958).
MATERIALS AND METHODS
1. Selection and maintenance of animals
Specimens of the land crab, Gecarcinus lateralis, collected in Bermuda or
Bimini, were maintained in the laboratory as described by Bliss (1953). Animals
ranging in carapace width from 3.5 to 5 cm. were used. At each feeding period
(i.e., every ten days), regenerating limb buds were measured. During the pre-
molt period, they were measured more frequently.
2. Removal of tissue
Animals were anesthetized by chilling at 4° C. for 15 to 20 minutes. A piece
of tissue approximately 3 mm. by 4 mm. was removed from one branchiostegite
and the opening in the branchial chamber was covered by a piece of plastic sealed
in place by melted paraffin. Operated animals were returned to their individual
containers and observed until external signs of an approaching ecdysis were seen
(i.e., growth of regenerating limb buds, swelling of pericardial sacs (Bliss, 1953,
1956) ; depressibility of the exoskeleton (Drach, 1939)). A second piece of tissue
was then removed from the other branchiostegite, the crab being similarly treated
and observed until ecdysis. The time from tissue removal until ecdysis was thus
known. These data, coupled with the histological condition of the tissue, permitted
the determination of the duration of each stage of the premolt period.
3. Histological and histochemical methods
Pieces of tissue were fixed in Bourn's solution, dehydrated in ethanol, im-
bedded in paraffin and sectioned at 7 to 10 p. Sections were stained with either
Mallory's triple stain or phosphotungstic acid.
RNA was visualized by staining with dilute solutions (0.01%) of methylene
blue over a pH range of 3 to 6.2. In that pH range, most basic staining is
A CRUSTACEAN INTEGUMENTARY TISSUE
637
attributed to nucleic acids, since the carboxyl group of proteins has a pK of 2
and is not dissociated (Swift, 1955). Control sides were subjected to RXase3
hydrolysis before methylene blue staining. For contrast, the sections were counter-
stained with dilute eosin.
The periodic acid-Schiff method was used to demonstrate the presence of
glycogen. Control sections in this series were pretreatecl with salivary amylase.
The tissues of 75 animals were studied.
4. Preparation of tissues
Pieces of tissue (50 to 100 mg. wet weight) were cut from the branchiostegites.
During the intermolt period, the epidermis is tightly attached to the innermost
region of the exoskeleton, the membranous layer (Fig. 1), which can be separated
EPICUTICLE
EXOCUTICLE
ENDOCUTICLE
EXOSKELETON
MEMBRANOUS LAYER
EPIDERMIS
CELL OF LEYDIG
TEGUMENTAL GLAND
LIPOPROTEIN CELL
RESERVE CELL
BLOOD SINUS
INNER EPIDERMIS
EXOSKELETON
FIGURE 1. Diagrammatic cross-section of the integumentary tissue of an intermolt
Gecarcinus, drawn to scale. The epidermal layer described is the one adjacent to the thick outer
exoskeleton. 100 X magnification.
as a thin sheet from the outer region of the exoskeleton. To avoid disrupting
the epidermis, intermolt tissues were removed with the membranous layer attached.
During that part of the premolt period (stage Dx and later) when the membranous
layer is being resorbed, the remainder of the old exoskeleton can be lifted away
from the integumentary tissues. Pieces of isolated tissue, with membranous
layer (intermolt), without membranous layer (early premolt), or with newly
synthesized exoskeleton (late premolt and early postmolt) were weighed on a
Roller Smith torsion balance and immersed in 0.5 ml. iced Carcinus perfusion fluid.
They were then blotted on filter paper and placed in the Warburg vessels.
5. O.vygcn consumption measurements
The oxygen consumption of pieces of tissue was determined manometrically.
The main chamber of five-mi. Warburg vessels received buffered (0.02 M Tris,
3 The following abbreviations are used : RNase, ribonuclease ; Tris, trishydroxymethylamino
methane; PAS, periodic acid-Schiff; DNP, dinitrophenol.
638
DOROTHY M. SKINNER
pH 7.7) Carcinus perfusion fluid (Pantin, 1946) containing 15 y streptomycin
and 4 y penicillin per ml. The center well contained 0.1 ml. of a 10% solution
of potassium hydroxide. Dinitrophenol and Krebs substrates (Krebs, 1950),
when added, were placed in the sidearm. To each 0.75 ml. was added 0.25 ml.
of a solution containing the following substrates in milliequivalents/liter : 4.9
pyruvate, 4.9 glutamate, 5.4 fumarate, 9.2 glucose. In experiments testing the
effect of cyanide, potassium hydroxide was replaced by 0.1 ml. of a calcium
TABLE I
Schedule of premolt and early postmolt events in Gecarcinus
Stage
Initiation (days
before ecdysis)
Completion (days
before ecdysis)
Event
Do
25 +
?
Gastrolith formation
Do
25
5 to 10
Regeneration of autotomized limbs
D!
12
10
Resorption of old exoskeleton, beginning w
ith
the membranous layer; increase in height
of
epidermal cells to 10 M-
D,
10
8
Further enlargement of epidermal cells
to
30 fj., separation from old exoskeleton by
re-
sorption of membranous layer.
D2 (early)
7
5
Formation of two-layered epicuticle
Do (late)
4
2
Formation of exocuticle
D3
1
0.5
Slight decrease in size of epidermal cells
D4
0.5
0
Blood pink
Ecdysis
A
0
1
Epidermal cells shrink slightly
B
1
5
Formation of endocuticle, about 7 n each
day
Ci and
5
?
Formation of endocuticle continued, at
the
C2
same rate
hydroxide-potassium cyanide suspension, of the concentration required to saturate
the gas phase at the desired molarity (Robbie, 1948). Calcium hydroxide was
used as the alkali in control vessels.
The total volume, including the tissue, was 1 ml. The flasks were incubated
at 25° C. and shaken at the rate of 130 oscillations per minute.
At the end of the experiment, tissues were rinsed in distilled water, blotted
on filter paper and dried in a 100° oven for 24 hours. They were then weighed
on a Sartorius balance. The rate of oxygen consumption (Qo-j) was expressed
as /*,!. O2/mg. dry weight/hour. At least two aliquots of tissue were taken from
each animal.
A CRUSTACEAN INTEGUMENTARY TISSUE 639
RESULTS
1. The molt cycle
The duration of the molt cycle of a mature Gccarcinus lateral-is (carapace width
3 cm. or greater) is four to six months (Table I). The intermolt period, Q to
C4, comprises all of the cycle except for a 30-day premolt period (D0 through D4)
and a short postmolt period (A through B) when synthesis of the exoskeleton
continues. In the premolt period, animals regenerate autotomized limbs, resorb
more than three-fourths of the old exoskeleton and synthesize an exoskeleton to
replace the one lost at ecdysis.
During the premolt period, the weight of animals increased by 13 to 30%
of the intermolt value, due to the absorption of water. After ecydysis, animals
weighed one-half as much as during the preceding intermolt period. Within 10
days after ecdysis, they had regained the weight lost at ecdysis and an additional
increment due to growth, which occurs only during the early postmolt period when
the exoskeleton is still pliable. After each ecdysis, there was a 1 to 7% increase
in carapace width and a 6 to 22% increase in weight.
2. Cytology of the integumentary tissue
The branchiostegites are bounded on their inner and outer surfaces by single
sheets of epidermis (Fig. 1). The epidermal layer bounding the inner surface
of the branchiostegites synthesizes a 7-^,-thick layer of cuticle with staining charac-
teristics similar to those of the two-layered epicuticle. The outer epidermal layer,
on the other hand, synthesizes the thick exoskeleton. composed of a 7-^ epicuticle.
a 30- p. exocuticle and a 200- to 400-p. endocuticle. Both epicuticle and exocuticle
are synthesized during the premolt period, while the endocuticle is formed during
the postmolt period. In this study, attention has been directed to the structural
changes of the outer epidermal layer.
Between the two epidermal layers there is a layer of connective tissue, the bulk
of which is composed of cells of Leydig (Cuenot, 1893). Among the cells of
Ley dig are scattered reserve cells (Hardy, 1892) and small blood sinuses which
contain lipoprotein cells (Sewell, 1955). At the inner edge of each epidermal
layer there are tegumental glands whose secretory cycle is not correlated with
the molt cycle, since both replete and empty glands are present at all stages of
the molt cycle.
3. Cytological changes of the integumentary tissue
a. Epidermis
Integumentary tissue removed from an animal 16 days before ecdysis (Fig. 2)
is identical to that from an intermolt animal (Fig. 1). Resorption of the mem-
branous layer, the innermost region of the exoskeleton, begins approximately 11
days before ecdysis, and the nuclei of the epidermal cells have enlarged (Fig. 3).
As the membranous layer is digested, its staining characteristics change. Intact
membranous layer is PAS-negative while partially digested membranous layer is
PAS-positive. indicating that a material with adjacent hydroxyl groups is made
640
DOROTHY M. SKINNER
FIGURE 2, Stage D0. Integumentary tissue from an animal 16 days before ecdysis. Only the
nuclei of the epidermal cells (E) are visible beneath the membranous layer (M) of the
exoskeleton. The duct of a tegumental gland (TD) can be seen entering the exoskeleton. Note
that the tissue is identical to tissue from an intermolt animal (Fig. 1).
FIGURE 3, Stage Di. Integumentary tissue from an animal 11 days before ecdysis. The
membranous layer (M) is being resorbed.
A CRUSTACEAN INTEGUMENTARY TISSUE C41
available for oxidation by HIO4 and for consequent reaction with the leucho-
fuchsin dye. The nature of the reactive material is unknown. However, it is
known that the crustacean exoskeleton is composed of approximately equal amounts
of chitin, which is PAS-negative, and protein (Lafon, 1948). Part of the protein
may be a mucoprotein with a carbohydrate component possessing adjacent hydroxyl
groups.
Eight days before ecdysis there is complete separation of the exoskeleton from
the epidermal cells which have enlarged further (Fig. 4). Synthesis of both layers
of the epicuticle has been completed by the fifth day preceding ecdysis (Fig. 5),
and the 30-^,-thick exocuticle is formed during the following two days (Fig. 6).
The endocuticle. whose formation begins on the second day following ecdysis, is
thickened at the rate of 7 ^ per day (Fig. 7) for at least a week (the period of
time during which samples of tissue were taken).
b. Other integumentary cells
Near the end of the intermolt period, the number of lipoprotein cells increases.
The cytoplasm of these cells becomes dotted with acidophilic granules during the
early premolt stages. As ecdysis approaches, the cells increase their granular
contents and move to the epidermis. By the time the epicuticle has been com-
pleted, near the end of the premolt period, the lipoprotein cells have disappeared.
Their disappearance, coupled with the similar staining characteristics of the outer
epicuticle and the lipoprotein cell granules, leads to the speculation that the
granules are incorporated into the epicuticle. The changes of the lipoprotein cells
of Gecarcinus parallel those of the homologous cells of the green crab (Sewell.
1955) to this point. However, in the green crab the small granules coalesce to
form one large droplet immediately preceding ecdysis. In Gecarcinus, the granules
do not coalesce ; rather, large cells with homogeneous cytoplasm, similar to the
reserve cells described by Hardy (1892), are seen at all stages of the molt cycle.
c. Glycogen metabolism of the integumentary tissue
As can be seen in Table II, the glycogen content of the outer epidermal layer
changes markedly during the premolt period. As the old exoskeleton is broken
down and new exoskeleton synthesized, there is an increase in glycogen content
of the epidermis. The glycogen content of the cells of Leydig also increases before
and decreases after ecdysis, suggesting that these cells serve as intermediates in
glycogen metabolism, probably receiving glucose from the blood and releasing it to
the epidermis.
FIGURE 4, Stage Di. Integumentary tissue from an animal 8 days before ecdysis. The
epidermal cells (E) are greatly enlarged and are completely separated from the old exoskeleton.
FIGURE 5, Stage D2 <<..,riy>. Integumentary tissue from an animal 5 days before ecdysis. The
epidermal cells (E) have completed synthesis of both layers of the epicuticle (EP).
FIGURE 6, Stage D2 date). Two-layered epicuticle completed; exocuticle (EX) partially
formed. Hair follicle visible.
FIGURE 7, Stage B. Integumentary tissue from an animal two days after ecdysis. Epicuticle,
exocuticle as above. First layers of endocuticle (EN) seen. Epidermal cells have decreased in
size and their nuclei are no longer visible.
642
DOROTHY M. SKINNER
TABLE II
Glycogen content of cells of the integumentary tissue
Stage
Epidermal cells
Cells of
Ley dig
Lipoprotein cells
Size
Glycogen
content
Glycogen
content
Number
Contents
Height, n
Width, yu
Intermolt C4
4
10-17
+
+
+ + + +
Do
4
10-17
+
+
+ + + + +
D,
10
10-17
+
+ +
+ + +
+
D2 (early)
30
10-17
+ +
+ + +
+ +
+ +
Premolt<
Do ([at,-!
100
10-17
+ + +
+ + +
+
+ + +
D3
80
10-17
+ +
+ + +
+
+ +
.04
80
10-17
+
+ +
+
+ +
[A
20
10
+
+
—
10
10
+
+
4. Formation of gastroliths
Gecarcinus stores calcium resorbed from the old exoskeleton as concretions
(gastroliths) which form in the lining of the stomach. In Gecarcinus, gastrolith
formation begins about 30 days before ecdysis. Within three days after ecdysis,
the gastroliths have disappeared completely.
LIMB REGENERATION
dato from 12 experimental animals
38-49 cm carapace width
~ 1.0
E
u
<r
UJ
z
u
o
0.6
0-4
0.2
40
30 20
DAYS BEFORE ECDYSIS
FIGURE 8. Compilation of growth curves of regenerating limbs of 12 Gecarcinus. Note
plateau until 25 days before ecdysis, when limb buds begin to grow again. Limb bud reaches
maximum size approximately 10 days before ecdysis.
A CRUSTACEAN INTEGUMENTARY TISSUE
643
5. Regeneration oj limbs
\Yithin the first two to three weeks after a limh is autotomized, a small limb
bud, 2—1- mm. long, grows out from the scar tissue which forms over the stump
of the autotomized limb. The limb bud remains in this form until the succeeding
premolt period when it resumes growth (Bliss, 1956).
In Figure 8, the length of regenerating limbs of animals used in this study
is plotted against time. It can be seen that about 30 days before molt, limb
buds begin to elongate, that they grow at a rapid rate for approximately 20 days,
completing their growth about 10 days before ecdysis.
6. Oxygen consumption of the integumentary tissue
a. Rate of oxygen consumption at each stage of the molt cycle
The integumentary tissues of intermolt, early premolt (stages D1? D2 early)
and early postmolt (stage B) consume oxygen at approximately equal rates
(Table III; Fig. 9). The Qo2 of integumentary tissue synthesizing the 30-/A-thick
TABLE III
The mean Qo2 of the integumentary tissue at each stage of the molt cycle
Stage
Number of animals
Mean Qo2
Standard deviation
C4
8
0.53
0.14
Do
5
0.30
0.14
D,
7
0.49
0.19
Da (early)
3
0.46
—
Da (l;,te)
11
0.85
0.28
A
2
0.72
—
B
6
0.38
0.08
exocuticle (D2 lnte) is significantly higher than that of intermolt tissue (Table III;
Fig. 9 ) . The Qo2 of tissue removed from two animals immediately after ecdysis
(stage A) is also significantly higher than that of intermolt tissue.
The mean Qo2 of tissues removed from animals in stages D1} D2 early, and
B has been tested statistically against the mean Qo2 of tissues removed from C4
animals. They have been found not to differ significantly. However, the mean
Qo2 of tissues removed from DO animals is significantly lower than that of the
tissue from intermolt animals. No explanation can be given for this decrease in
respiratory rate at the initiation of the premolt period. The mean Qo of tissues
removed from animals in stage D2 iilte, when the exocuticle is being formed,
is significantly higher than the mean Qo2 of tissues from animals at all other stages.
b. Effect of cyanide and dinitrophenol
Both 10"* and 10~5 M cyanide inhibited oxygen consumption of integumentary
tissues from intermolt and premolt animals by 60 to 95%. As seen in Figure 10,
10~4 and 10~5 M DNP increased the oxygen consumption of the integumentary
tissues.
644
DOROTHY M. SKINNER
c. Effect of endogenous substrates in blood serum and of Krebs substrates
Tissues bathed in Carcinns perfusion fluid (Pantin, 1946), which was 25%
(v/v) in Gecarcinits blood serum, respired at a greater rate than tissues bathed
in the salt solution alone. The increase was in the order of 50 to 200%.
Q0:
g 1.0
1 0.5
i
i • :
^
£
• t •
*
I"
*
3s1
^ 0
•
i
NTERMOLT ^^^ PREMOLT^ ECDYSIS ^OSTMOLT
STAGE C Di^ D2EARLY D2LAT7^ D3.4
A B
DURATION 120+ 15 4 2'3 2~3 1
1 5
(DAYS)
FIGURE 9. The Qo= and cytology of the integumentary tissue of Gecarcinus at each stage of
the molt cycle. In stage D0, the cytology of the integumentary tissue is the same as in the intermolt
period, ep = epicuticle ; ex = exocuticle ; en = endocuticle ; t = tegumental gland ; cl = cell of
Leydig ; s = blood sinus ; Ip = lipoprotein cell.
Attempts at replacing the unknown stimulating components of blood serum
with Krebs' substrates (Krebs, 1950) produced only minor increases in oxygen
consumption (14 to 30%).
DISCUSSION
As can be seen in Table I, morphological evidence indicates that the first 15
days of the 30-day premolt period in Gecarcinits lateralis are devoted to limb
A CRUSTACEAN INTEGUMENTARY TISSUE
645
regeneration and gastrolith formation. However, it is obvious that during this
first portion of the premolt period the integumentary tissues, which retain their
intermolt morphology, are active in resorbing calcium from the exoskeleton and
allowing its passage to the blood for storage as gastroliths in the stomach lining.
Additional evidence of the catabolic activity of the integumentary tissues, preceding
any change in their structure, is seen as the membranous layer of the exoskeleton
is resorbed.
Q02 7
I 4
in
• CONTROL
A 10" 5M DNP
11 10 ~4M DNP
PREMOLT
(D2,lote>
INTERMOLT
(C4)
I 2 3
HOURS AFTER TIPPING DNP
FIGURE 10. The effect of DNP on intermolt and premolt integumentary tissues. After a
three-hour incubation period, DNP was tipped from the sidearm into the main vessel.
The time course of events as found in Gccarchuts lateralis has been fitted into
the stages of Drach (1939) in Table I. Stage D,, marked by the resorption of
the membranous layer of the exoskeleton, occurs 1 1 days before ecdysis. Synthesis
of epicuticle on the seventh day before ecdysis signals the beginning of Stage D2.
On the fourth day before ecdysis, exocuticle formation begins. This stage has
been called D^. 1:1U. to distinguish it from D2. (.;lriy because it is during Do ,ate that
the oxygen consumption of the integumentary tissues increases. D3 and D4, 1.5
days immediately preceding molt, are marked by no further synthesis of exo-
skeleton. There is, however, some reduction in the size of the epidermal cells.
The blood of DS and D4 animals has lost the characteristic blue color of crustacean
646 DOROTHY M. SKINNER
blood and assumed a pink tinge, clue to astaxanthin resorbed from the old exo-
skeleton (Skinner and Krinsky, unpublished observations).
The increased rate of oxygen consumption of integumentary tissues in D2. i;,te
has been attributed to the rapid synthesis of exoskeleton. The 30-/z-thick exo-
cuticle, composed of approximately equal parts of chitin and protein, is synthesized
in this two-day period.
The author would like to express her deep appreciation to her sponsor, Dr.
John H. Welsh, for his helpful discussions during the course of this work.
SUMMARY
1. The morphological changes undergone during the molt cycle by the integu-
mentary tissue of the land crab, Gecarcinus latcralis, have been described.
2. The time course of limb regeneration and gastrolith formation has been
correlated with the morphological changes of the integumentary tissue. The
period of premolt activity during which limb regeneration and gastrolith forma-
tion occurs precedes the changes in the integumentary tissues and has. therefore,
been called D0.
3. The oxygen consumption of the integumentary tissues has been measured at
each stage of the molt cycle. It has been found to increase at the time of synthesis
of the exocuticle. The effects of cyanide, dinitrophenol and added substrates on
the oxygen consumption of the integumentary tissue have been studied.
LITERATURE CITED
BLISS, D. E., 1953. Endocrine control of metabolism in the land crab, Gecarcinus latcralis
(Freminville). I. Differences in the respiratory metabolism of sinusglandless and
eyestalkless crabs. Blol. Bull, 104: 275-296.
BLISS, D. E., 1956. Neurosecretion and the control of growth in a decapod crustacean. Bertil
Hanstrom, Zoological Papers, 56-75.
CUENOT, L., 1893. fitudes physiologiques sur les Crustaces Decapodes. Arch. BioL, 13: 245-303.
DRACH, P., 1939. Mue et cycle d'intermue chez les Crustaces Decapodes. Ann. Inst. Oceanogr.
Monaco, 19: 103-391.
EDWARDS, G. A., 1950. The influence of eyestalk removal on the metabolism of the fiddler crab.
Physiol. Camp. Occologia, 2: 34-50.
EDWARDS, G. A., 1953. Respiratory Metabolism. In: Insect Physiology, K. D. Roeder, editor.
Wiley, New York, pp. 96-146.
FIELD, J., 1948. Respiration of tissue slices. In: Methods in Medical Research, V. R. Potter,
editor, vol. 1, pp. 289-307.
HARDY, W. B., 1892. The blood corpuscles of the Crustacea, together with a suggestion as to
the origin of the crustacean fibrin-ferment. /. Physio!., 13: 165-190.
KREBS, H. A., 1950. Body size and tissue respiration. Biochiiu. Biophys. Acta. 4: 249-269.
KUHN, A., AND H. PIEPHO, 1938. Die Reaction der Hypodermis und der Versonschen Driizen
auf das Verpuppungshormon bei Ephcstia kilhniella Z. Blol. Zbl., 58: 12-51.
LAFON, M., 1948. Nouvelles recherches biochimiques et physiologiques sur le squelette
tegumentaire des Crustaces. Bull. Insf. Oceanogr. Monaco, 45: 1-28.
MANN, P. J. G., AND J. H. QUASTEL, 1941. Nicotinamide, cozymase and tissue metabolism.
Biochcm. J., 35: 502-517.
NYST, R. H., 1941. Contribution a 1'etude de 1'hormone nymphogene. Ann. Soc. Zool Bclg.,
72: 74-104.
PANTIN, C. F. A., 1946. Notes on Microscopical Techniques for Zoologists. University Press,
Cambridge, England ; p. 66.
A CRUSTACEAN INTEGUMENTARY TISSUE 647
POULSON, D. F., 1935. Oxygen consumption of Drosophila pupae. I. Drosophila melanogaster.
Zcitschr. vcnjl Physio!., 22: 466-472.
ROBBIE, W. A., 1948. Use of cyanide in tissue respiration studies. In: Methods in Medical
Research, V. R. Potter, editor, vol. 1, pp. 307-316.
SCHNEIDERMAN, H. A., 1952. Variations in dehydrogenase activity during the metamorphosis of
the Cecropia silkworm. Ph.D. Thesis, Harvard University.
SCHNEIDERMAN, H. A., AND C. M. WILLIAMS, 1953. The physiology of insect diapause. VII.
The respiratory metabolism of the Cecropia silkworm during diapause and development.
Biol. Bull., 105: 320-334.
SCUDAMORE, H. H., 1947. The influence of the sinus gland upon molting and associated changes
in the crayfish. Phys'wl. Zoo!., 20: 187-208.
SEWELL, M. T., 1955. Lipoprotein cells in the blood of Carcinns inucnas, and their cycle of
activity correlated with the molt. Quart. J. Micr. Sci., 96: 73-83.
SKINNER, D. M., 1958. The molt cycle of the land crab, Gecarcinux lutcralis. Anat. Rcc., 132:
507.
SWIFT, H., 1955. Cytochemical techniques for nucleic acids. In: The Nucleic Acids, E.
Chargaff and J. N. Davidson, editors, vol. II, pp. 51-92, Academic Press, New York.
TRAVIS, D., 1955. The molting cycle of the spiny lobster, Panitlints argus Latreille. II. Pre-
ecdysial histological and histochemical changes in the hepatopancreas and integumental
tissues. Biol. Bull., 108: 88-112.
TRAVIS, D., 1958. The molting cycle of the spiny lobster, Punitlints argus Latreille. IV. Post-
ecdysial histological and histochemical changes in the hepatopancreas and integumental
tissues. Biol. Bull., 113: 451-479.
WIGGLESWORTH, V. G., 1933. The physiology of the cuticle and of ecdysis in Rhodnius proli.rus
( Triatomidae, Hemiptera) ; with special reference to the function of the oenocytes and
of the dermal glands. Quart. J. Micr. Sci.. 76: 269-318.
WIGGLESWORTH, V. G., 1945. Growth and form in an insect. In: Essays on Growth and Form
presented to D'Arcy Wentworth Thompson. A. E. Le Gros Clark and P. B. Medawar,
editors, pp. 23-41, Oxford Press.
ZWICKY, K., AND V. B. WIGGLESWORTH, 1956. The course of oxygen consumption during the
moulting cycle of Rhodnius prali.rus Stal (Hemiptera). Proc. Roy. Ent. Soc. London,
31: 153-160.
UPTAKE OF ORGANIC MATERIAL BY AQUATIC INVERTEBRATES.
I. UPTAKE OF GLUCOSE BY THE SOLITARY CORAL,
FUNGIA SCUTARIA1
GROVER C. STEPHENS 2
Zoology Department, University of Minnesota, Minneapolis 14, Minnesota, and
Hawaii Marine Laboratory, Honolulu, Haii'aii
The suggestion that naturally occurring dissolved organic material may contribute
to the nutrition of aquatic animals is usually associated with the name of Putter
(1909 ). This hypothesis was critically reviewed by Krogh in 1931. He concluded
that no firm evidence could be adduced to support it.
In recent years little work has been undertaken which bears directly on this
possibility. Collier et al. (1953) reported the retention of an unidentified carbohy-
drate by oysters. Fox and his co-workers in a series of papers (1952, 1953)
suggested that dissolved organic material may be adsorbed on inorganic particles
of colloidal dimensions and become available to filter-feeding animals in this way.
These workers have suggested that such colloidal micelles may comprise a con-
siderable fraction of the "dissolved" organic material normally occurring in sea
water. Goldacre (1949) and Cheesman (1956) have argued that protein mono-
layers at the air-water interface may be significant as food sources for tadpoles
and snails.
It is apparent that this work (with the possible exception of that of Collier's
group) is not really concerned with dissolved organic material in the sense of small
organic molecules in true solution as a potential source of nutrition for aquatic
animals. Rather, physical processes of adsorption are invoked to produce colloidal
micelles or a denatured monolayer, which then is available to the animal. There is
no reason to doubt that such devices do indeed operate. However, the significance,
if any, of materials in true crystalloid solution remains to be assessed.
Recent work in our laboratory (Stephens and Schinske, 1961) demonstrated
the uptake of several amino acids from dilute solution by a variety of marine
invertebrates. However, the concentration of acids employed for most of this work
was very high compared to concentrations of organic materials in natural waters.
Furthermore, the observations were totally dependent on measuring the rate of
disappearance of materials and attempting by suitable control procedures to implicate
the animal concerned as the agent. The present work was undertaken, using C1"
labelled compounds, in order (a) to permit use of lower concentrations of added
organic material, and (b ) to provide unambiguous evidence that such material was
entering the experimental animal.
1 This work was supported by the Graduate School of the University of Minnesota, PHS
Grant RG-6378, and a Senior Postdoctoral Fellowship of the National Science Foundation. The
author also wishes to express appreciation to the Hawaii Marine Laboratory for their hospitality.
- Hawaii Marine Laboratory Contribution No. 170.
648
UPTAKE OF GLUCOSE BY FUNGIA 649
Any dissolved organic compound which may be available to marine organisms
must be present at very modest concentrations, since the total dissolved organic
material of sea water is reported as a few milligrams per liter. The ability to deal
effectively with at least some species of organic molecules in extremely dilute
solution is a critical consequence of the idea that dissolved material may contribute
to the nutrition of aquatic animals. However, demonstration that a particular
compound is available to an animal from dilute solution is merely consistent with
this hypothesis.
The observations to be reported were undertaken to determine whether the
solitary coral, Fungia, could remove glucose from dilute solution at a significant
rate. They were extended to provide information concerning the mechanisms of
uptake. Preliminary reports of this work have appeared (Stephens, 1960a, 1960b).
MATERIALS AND METHODS
The solitary coral, Fitngia, was selected as an experimental animal for several
reasons: (a) it is locally abundant on coral reef flats fringing Oahu, is easily
collected, and is a hardy organism in the laboratory ; (b) individuals are sufficiently
large to permit easy experimental manipulation; and (c) individuals produce large
quantities of mucus which can be readily collected. Animals were collected in the
vicinity of the Hawaii Marine Laboratory. Individuals were maintained in running
sea water until they were employed in experiments.
The basic procedure involved the addition of measured amounts of uniformly
labelled glucose-C14 (or other labelled material) to a measured volume of sea water.
The movement of this material was followed by monitoring the radioactivity of the
ambient sea water and of suitable extracts. Radioactivity was measured using a
thin- window Geiger tube. Quadruplicate samples of 0.5 ml. were dried on aluminum
planchets and the time for 2000 counts measured. Extracts of the animals were
prepared by boiling with dilute NaOH. In the case of all such extracts and solu-
tions, suitable concentration curves were established to correct for differences in
self-absorption by the dried samples. All values are corrected for background.
Observations were made on animals maintained in 100 to 200 ml. of sea water.
Observations were not continued beyond 24 hours under such conditions. Animals
survived such confinement for at least ten days and there was no mortality in the
course of the observations.
Oxygen consumption was measured using the Winkler method. During such
measurements, the animals were kept in darkness because of the presence of
symbiotic algae.
RESULTS
Figure 1 illustrates the disappearance of radioactivity from the solution in a
typical set of observations. Initial glucose concentration in this case was 10
mg./liter. Wet weight of the animal was 33.0 grams, temperature 25° C., and the
volume of solution was 200 ml. Streptomycin (50 /Ag./ml.) added to the ambient
sea water did not modify this rate of disappearance. Controls, consisting of a blank
container with a similar volume of solution, and the skeleton of Fungia in a glucose
solution, showed no change in radioactivity over a period of 24 hours.
Recovery of radioactivity from a digest of the animals was complete. Digests
650
l-KOVKR C. STKI'HKX:
10-
0>
E
- 8
O
O
8
0
OD
§
3
24
HOURS
FIGURE 1. Decrease in glucose as measured by radioactivity of the ambient sea water.
Volume is 200 ml., weight of animal is 33 grams, temperature 25° C.
were made by boiling in dilute NaOH. After correction for the lower absorption of
radiation by the digest, good agreement was obtained between the loss of radio-
activity from sea water and its appearance in the animal (Table I). A 5-ml. sample
of the ambient sea water solution was acidified. The resulting CO2 was trapped in a
drop of KOH. Counts of this trapped material were not significantly different from
TABLE I
Distribution of radioactive material after four hours. Initial glucose concentration
1.25 mg. /liter, temperature 25° C.
Animal
Weight
Wet
Ash
Total activity in sea water
Initial
Final
Activity of digest (corrected for absorption)
Recoverv
34.2 gin.
26.7
18,700 c.p.m.
4,600
14,900 c.p.m.
104.4%
17.0 gm.
13.3
18,700 c.p.m.
9,100
10,200 c.p.m.
103.1%
UPTAKE OF GLUCOSE BY FUNGIA
651
the background rate. Hence, no significant quantity of C14(X had been produced
during the four hours of observation.
Figure 2 is a graph of the log of wet weight in grams against the log of the
rate of glucose uptake. Uptake is expressed as milligrams/hour/individual. The
open circles represent the uptake of animals in 400-800 ft. candles' illumination at
an initial sugar concentration of 5.0 mg./liter. The regression line calculated for
these points has a slope of 0.54 and a standard error of 0.09.
The crosses represent the uptake of sugar at an initial concentration of 5.0
mg. liter in darkness. Hence, light intensity is not a factor in rate of uptake.
100-
O
O
10-
o
Q.
o
o
o
10 100
wet weight (gms)
1000
FIGURE 2. Log uptake expressed as mg./hr./individual times 100, plotted against the log of
the wet weight. Open circles represent animals in light, crosses are animals in darkness,
triangles are animals whose mouths have been plugged with paraffin.
Table II provides data relevant to the relation between initial concentration and
the rate of uptake. Animals used weighed 30 to 45 grams. Initial concentrations
are listed, together with the amounts of glucose removed in one hour, based on
disappearance of radioactive material from the solution. Concentrations above
10 mg./liter were obtained by adding unlabelled glucose. The lowest initial radio-
activity was employed in the measurements at 1.0 mg./liter, which was 5.3 ± 0.68
counts /minute. The values for 0.37 mg./liter were obtained with glucose of higher
specific activity, so that initial activity exceeded background (21.5 ± 1.1 c.p.m.).
Although expressed as uptake/hour, the uptake at high concentrations of sugar was
measured over a period of four or twenty-four hours. This was necessary at the
highest concentrations because of low percentage rates of upake, and convenient at
other concentrations. From such primary data, two methods of calculation are
652 GROVER C. STEPHENS
apparently appropriate. If the rate of uptake is linearly related to ambient concen-
tration, the concentration would decrease exponentially, a fixed percentage being
removed per hour. This assumption is consistent with the data at ambient concen-
trations below 20 mg./liter. At high concentrations uptake is apparently inde-
pendent of concentration. Hence, hourly uptake would most appropriately be
estimated by simply dividing total uptake by time. This was done for ambient
concentrations greater than 60 mg./liter.
TABLE II
Relation
between ambient
glucose concentration (S) and rate
Weight is 30 to 45 grams.
of uptake (V).
S
(mg./l.)
V
(mg./hr.)
S
(mg./l.)
V
(mg./hr.)
0.37
0.03
30.0
0.83
0.02
0.91
1.0
0.03
0.06
0.05
0.04
40.0
1.28
1.46
0.92
0.84
1.25
0.07
0.92
0.04
50.0
0.79
4.0
0.23
1.17
0.25
0.30
0.20
60.0
1.39
0.96
5.0
0.19
100.0*
1.90
0.22
0.90
0.21
1.93
0.21
2.23
10.0
0.60
110.0*
0.99
0.32
1.19
0.46
0.59
20.0
0.44
0.65
0.50
200.0*
0.83
0.76
0.60
500.0*
1.46
0.68
1.56
* Estimated, assuming uptake was linear.
A regression line, calculated by the least squares method for the concentration
range 0.37 to 20 mg./liter, has a slope of 0.042 with a standard error of 0.008
(Fig. 3). A regression line for the concentration range 40 to 500 mg./liter has a
slope of 0.0012 and a standard error of 0.0009. This is not significantly different
from zero, indicating that uptake is independent of concentration in this range. One
may conclude that the rate of uptake for animals in this size range is 4.2 ± 0.8% of
the ambient glucose per hour at low concentrations and reaches a maximum of
1.20 ± .43 mg./hour (the average of uptake over the range 40 to 500 mg./liter.3
3 The general form of the relationship of velocity of uptake to concentration of ambient
glucose suggests that an adsorptive step is rate-limiting. An alternate form of data presentation
is a plot of the reciprocal of the rate of uptake against the reciprocal of amhient glucose concen-
UPTAKE OF GLUCOSE BY FUNGIA
653
It is of interest to consider the energy requirements of these animals, as indicated
by their oxygen consumption. Table III lists the oxygen consumed per hour for
18 animals. The equivalent amount of glucose is also tabulated. The slope and
standard error of a regression line relating the log of oxygen consumption and
log wet weight is 0.45 ± 0.16. Although the standard error is large, the slope is
significantly different from zero. The slope may be used to calculate mean oxygen
consumptions at weights of 30 to 45 grams. These are, respectively, 0.383 and
0.472 ml. (X/hour. These are in turn equivalent to 0.514 and 0.633 mg. of glucose
10
S ( mg / I )
i
15
20
FIGURE 3.
Rate of uptake as a function of glucose concentration. The line is the least
squares regression line. Weight is 30-45 grams.
oxidized per hour. At the mean rate of uptake calculated for this size range above,
ambient glucose concentrations of 12.2 to 15.0 mg./liter would suffice to provide these
amounts of sugar. Furthermore, since the rate of uptake is related to size in the
same fashion as is oxygen consumption, ambient concentrations of glucose in this
general range should suffice to account for oxygen consumption regardless of size.
The general form of the curve relating concentration and the rate of glucose
tration. This should approximate a straight line. The regression line of such a plot has a slope
of 16.94 ± 0.85 and an intercept of 1.21 ± 0.31. This is formally equivalent to an enzyme-
catalyzed reaction and Vmax and Km (the concentration at which the velocity is half maximal)
can be evaluated. Vma* is 0.83 mg./hr. and Km is 20.5 mg./liter. This is in reasonable agreement
with the rates calculated more directly from the data. Since departures from linearity for double
reciprocal plots are quite common (Neilands and Stumpf, 1958), the presentation in the text
is preferable.
654 GROVER C. STEPHENS
uptake corresponds to a Langmuir isotherm. This form suggests the possibility
that an adsorptive step in the uptake process might be rate-limiting. Evidence was
sought to evaluate two variants of this possibility.
The first hypothetical mechanism considered was the adsorption or binding of
glucose to mucus, with subsequent recovery of mucus by the animal. This was
suggested by Collier ct al. (1953) in connection with the retention of carbohydrate
by oysters, and has been proposed (Rao and Goldberg, 1954) as a mechanism for
uptake of calcium in other invertebrates, including sea anemones. A similar
mechanism was also suggested by Korringa (1952) to account for selective retention
of phytoplankton by oysters. Finally, Stephens and Schinske (1961) allude to this
as a possible but unsupported explanation for the uptake of amino acids they
observed.
TABLE III
Oxygen consumption
of Fungia as ml. O^/hr./ individual
and as equivalent
amounts of glucose
Wet weight
ml. Ch/hr.
mg. sugar/hr.
52
0.575
0.770
20
0.384
0.515
53
0.645
0.865
53
0.660
0.884
85
0.795
1.065
59
0.577
0.774
51
0.550
0.736
33
0.365
0.489
62
0.550
0.736
58
0.412
0.552
47
0.362
0.485
64
0.400
0.536
35
0.362
0.485
64
0.563
0.754
56
0.400
0.536
52
0.613
0.820
53
0.587
0.785
55
0.425
0.570
Fresh mucus was obtained by simply collecting the copious flow from one or
more Fungia in a beaker. The possibility of adsorption was tested directly by
filtering mucus after incubation with labelled material. A Millipore filter, type VM
(pore size 50 m/x), was employed. Fresh mucus samples could be coagulated by
heat, alcohol, and acetic acid, and gave a strongly positive test for carbohydrate with
anthrone. The filtrate was negative to these tests. The radioactivity of the filter
disc then served as a measure of the material bound to the mucus, which was
retained.
Ten ml. of mucus were incubated for 30 minutes with glucose in sea water.
Radioactivity was 665 ±15 c.p.m. After filtering, the material retained was washed
with 20 ml. of sea water. After drying, the filter disc showed 8.7 ± 0.6 c.p.m.
Since the initial glucose concentration was 8.3 mg./liter, the radioactivity of the
filter disc implies the retention of 0.054 micrograms glucose as well as the mucus
in the 10-ml. sample. Total mucus solids were determined by precipitation, washing
in ethanol and weighing the precipitate. This figure is 278/micrograms/ml. Hence,
UPTAKE OF GLUCOSE BY FUNGIA 655
51.5 mg. dry mucus solids bind 1 microgram of glucose. Phrased differently, a
45-gram organism would have to secrete and recover about one-half its weight as dry
mucus solids each hour to account for the rate of uptake observed at this concentra-
tion of glucose. It should be pointed out that the measurement of glucose retained
on the filter is probably high, since less self-absorption of the sample would be
expected. The comparable figure for binding in the case of glycine and mucus is
15.1 mg. mucus solids for each microgram of glycine.
Some check on the technique is provided by using Ca45 and measuring its reten-
tion. Kwart and Shashoua (1957) published a discussion of mucus structure in
Busycon, giving the calcium content of mucus. Their proposed structure implies
that this should exchange so that at least the equivalent of the calcium linking
TABLE IV
Effect of phlorizin on uptake of glucose and glycine
I. Glucose (0.5 mg./l.) plus phlorizin
Phlor. concentration % uptake (4 hours)
0 69
67
1CT5 M 32
33
10~4 M 27
30
10~3 M 0
0
II. Glycine (0.25 mg./l.) plus phlorizin
Phlor. concentration % uptake (4 hours)
0 89
91
10-3 M 87
90
the protein and polysaccharide should be bound. Their figure for calcium in mucus
extracted in NaCl is 10.3 ^.g./mg. solids. Using the technique above, 1 mg. mucus
solids binds 8.4 ^g. calcium, which is in reasonable agreement.
A further test of the hypothesis was undertaken by plugging the mouth of
specimens of Fungia with paraffin. Low-melting-point paraffin was melted and
poured into and around the mouths of four animals. Their uptake of glucose from
a solution of 0.37 mg. /liter is indicated on Figure 2 by dark triangles. The points
are plotted in such a manner as to compensate for the difference in ambient
concentration. The rate of uptake is unaffected.
The preceding experiments rule out mucus binding as a mechanism, and sug-
gest some form of transport across the body wall. Table IV reports the effect
of phlorizin on uptake of glucose. The failure to inhibit uptake of glycine serves
as a control. Glucose uptake is not inhibited by 10~3 molar 2,4-dinitrophenol.
Specificity of the pathway of uptake was investigated in two ways. A number
of sugars were tested as possible competitive inhibitors of the uptake of glucose.
The data are presented in Table V. It is clear that loading the system with these
656 GROVER C. STEPHENS
TABLE V
Glucose uptake in presence of other sugars. Wet weight is 25—30 grams, initial glucose concentration
is 1.25 mg./l., and initial concentration of other sugars is 500 mg./l.
Sugar Uptake (%/hr.)
glucose alone 9.5
11.4
glucose + sucrose 14.3
10.2
glucose + galactose 14.4
13.6
glucose + ribose 11.6
13.6
glucose + arabinose 15.2
15.9
sugars does not affect the uptake of glucose. Observations were also made to
determine whether these same sugars could be removed from solution by Fung la
over a 24-hour period. Carbohydrate was determined using the anthrone tech-
nique as outlined by Lewis and Rakestraw (1955). Initial concentrations were
50 mg. /liter, and animals weighing 30 to 40 grams were used. All of the glucose
in 200 ml. of solution was removed after 24 hours. No uptake of any of the
other sugars occurred. Hence, the pathway of uptake is rather specific for glucose.
Finally, it was of interest to determine the relation between temperature and
rate of transport. Observations were made at several temperatures, ranging from
20° C. to 35° C. The effect of temperature on rate of uptake was modest. The
Q10 for the range 20°-30° was 1.22; that for the range 25°-35° was 1.32. Initial
concentration of glucose in these observations was 0.37 mg. /liter. Another set of
observations was undertaken at 100 mg./liter to determine if the temperature
relations of the system differed at higher ambient concentrations. Measurements
were made at 18°, 25°, and 32° C. The Q1M for the lower range was 1.19, and
was 1.36 for the upper range.
A number of observations were undertaken using the techniques described but
employing other compounds and other experimental animals. Some observations
using glycine have already been mentioned. Table VI lists the percentage uptake
TABLE VI
Uptake of other organic compounds at the concentrations listed. Weight is 20 to 40 grams.
Compound Cone, (mg./l.) ' ', uptake (4 hr.)
Tyrosine 0.15 89
70
Lysine 0.53 77
75
Aspartic acid 0.35 86
80
Glycine 1.2 96
83
Lactate 0.1 48
54
UPTAKE OF (iLUCOSE BY FUNGIA 657
for five small organic compounds4 at the concentrations stipulated. In each case,
specimens of Fungia weighing from 20 to 40 grams were placed in 200 ml. of
solution. Attention may be directed to two points of interest. The isoelectric
point of lysine lies on the base side of the pH of sea water. Hence, both cations
and anions can be taken up. Racemic mixtures of optically active amino acids
were employed. Neither the data presented nor more frequent monitoring of
ambient concentrations provide evidence for differential handling of D and L forms,
though this remains possible.
A concentration curve for glycine differed from that reported above for glucose
primarily in indicating a higher capacity of the system. At 250 and 600 mg./liter,
18 to 23 milligrams of glycine were removed from solution in four hours by animals
weighing 30 to 40 gm. Blocking the mouth with paraffin was without effect on
the rate of glycine uptake.
Observations on the uptake of glycine by several colonial corals indicated
significant rates of uptake in all cases. Corals used included Acropora sp., Favla
speciosa, and Dendrophyllia micranthus. Dendrophyllia is of interest in that it
contains no symbiotic algae. Scattered observations on other phyla of reef-dwelling
invertebrates indicate that the capacity to deal with small organic molecules in
dilute solution is not limited to corals or to the Cnidaria.
DISCUSSION
The data reported strongly support the conclusion that Fungia is capable of
removing several small organic molecules of biological significance from very
dilute solution. Minimum concentrations of glucose and amino acids were dictated
by the analytical technique and the specific activity of the labelled compounds
employed. At these minimum concentrations, there was no apparent decline in
the rate of uptake for the compounds employed, though absolute rate was of course
a function of concentration. Hence, there is no reason to think that such uptake
of small organic molecules does not occur at the very low concentrations one
would expect in natural waters.
Not merely is glucose removed from dilute solution but this process occurs
at a rate which is significant when compared with the energy requirements of the
animal. It has been pointed out in presentation of the data that an ambient
concentration of approximately 15mg. /liter of glucose would provide sufficient
material to support the observed oxygen consumption of the animals.5 Further-
more, the observations concerning uptake of amino acids and lactate indicate that
there is at least a modest spectrum of small organic molecules which can be
effectively manipulated by the organism.
The preceding remarks should be balanced by a quite explicit statement that
4 The compounds used were: DL-lysine-1-C14, DL-aspartic acid-4-C14, DL-tyrosine-2-C14,
Klycine-2-C14, and Zn lactate-1-C14.
•"' The ambient concentration which is calculated as sufficient to support the observed oxygen
consumption is somewhat higher than that previously reported (Stephens, 1960a). This differ-
ence springs primarily from the difference in oxygen consumption exhibited by the animals. The
measurements reported here were made in May at a temperature of approximately 26°, those
reported previously in December at a temperature of approximately 24°. In both cases, the
stipulated concentrations lie in the general range of values reported for total dissolved organic
material present in sea water.
658 GROVER C. STEPHENS
the relation of the present observations to any postulated nutritive significance
of naturally occurring organic material is indirect. It is a prerequisite condition
for utilization of organic material in true solution that an organism possess an
effective collecting mechanism for such material. The present work demonstrates
the existence of such a mechanism for some selected compounds. However,
failing an adequate qualitative analysis of dissolved organic material, one cannot
argue directly for a nutritive significance of these observations.
Membrane transport of glucose is suggested by the present observations.
Inhibition of transport by phlorizin has been classically reported for vertebrate
gut and kidney. The specificity of the transport for glucose is also suggestive.
However, the fact that galactose is not manipulated indicates that the transport
system differs in some respects from that reported for other preparations (Crane
and Mandelstam, 1960).
Active transport, in the sense of transport against a concentration gradient,
cannot be drawn as a conclusion although it seems quite possible. If the data
presented in Table I are interpreted on the basis of the naive assumption that the
radioactivity of an extract is present as glucose, considerable concentration has
clearly occurred. It is also true that Hosoi (1938) reported sugar concentrations
of approximately 0.5% of dry weight in Fungia actinifonnis. However, Hosoi's
sugar was not identified, although glycogen was demonstrated. Neither is there
support for the assumption concerning the form in which radioactive material was
extracted. Even granting this dubious assumption and incomplete data in the
literature, what glucose may be present in the animal is not necessarily osmotically
active. Hence the question remains unresolved.
The low Qu, observed for glucose intake by Fungia contrasts with higher
temperature coefficients typically reported for membrane transport systems. At
low concentrations, this is not surprising, since transport would presumably be
limited by the rate at which diffusion and mixing by flagellar activity could supply
material, rather than by the transport mechanism itself. The failure to obtain
a higher coefficient at the concentration of 100 mg. /liter seems anomalous, however.
Possibly this concentration did not exceed the capacity of the system sufficiently
to overcome diffusion limitations.
The data presented concerning the retention of material adsorbed on samples
of mucus indicate clearly that adsorption on mucus can provide only a trivial
amount of the observed uptake. It is possible that the procedure of washing the
mucus retained on the filter might permit the exchange of some material. How-
ever, the fact that plugging the mouth of the organism had no impact on the
observed rate of uptake provides convincing evidence that this is not a major
pathway of uptake.
While direct conclusions from the present work concerning the possible
nutritive role of dissolved organic matter in natural waters must be eschewed, the
existence of an efficient pathway for uptake of small organic compounds serves to
renew interest in this hypothesis.
LITERATURE CITED
CHEESMAN, D. F., 1956. The snail's foot as a Langmuir trough. Nature, 178: 987-988.
COLLIER, A., S. M. RAY, A. W. MAGNITSKY AND J. O. BELL, 1953. Effect of dissolved organic
substances on oysters. U. S. Fish Wildlife Serr., Fish. Bull., 54: 167-185.
UPTAKE OF GLUCOSE BY FUNGIA 659
CRANE, R. K., AND P. MANDELSTAM, 1960. The active transport of sugars by various prepara-
tions of hamster intestine. Biophys. ct Biochim. Ada, 45: 460-^476.
Fox, D. L., J. D. ISAACS AND E. F. CORCORAN, 1952. Marine leptopel, its recovery, measurement
and distribution. /. Mar. Res., 11: 29^46.
Fox, D. L., C. H. OPPENHEIMER AND J. S. KITTREDGE, 1953. Microfiltration in oceanographic
research. II. Retention of colloidal micelles by adsorptive filters and by filter-feeding
invertebrates ; proportions of dispersed organic to dispersed inorganic matter and to
organic solutes. /. Mar. Res., 12: 233-243.
GOLDACRE, R. J., 1949. Surface films on natural bodies of water. /. Animal Ecology, 18: 36-39.
Hosoi, K., 1938. Contribution to the biochemistry of the coral. I. On the occurrence of glycogen
and its content in the polyp of Fungia actiniformis var. palawcnsis Doderlein. Palao
Trap. Biol. Sta. Studies, 3: 447-451.
KORRINGA, P., 1952. Recent advances in oyster biology. Quart. Rev. Biol., 27 : 266-308 ; 339-365.
KROGH, A., 1931. Dissolved substances as food of aquatic organisms. Biol. Rev., 6: 412-442.
KWART, H., AND V. E. SHASHOUA, 1957. The structure and composition of mucus. Trans.
N. Y. Acad. Sci., Ser. II, 19: 595-612.
LEWIS, G. J., JR., AND N. W. RAKESTRAW, 1955. Carbohydrate in sea water. /. Mar. Res.,
14: 253-258.
NEILANDS, J. B., AND P. K. STUMPF, 1958. Outlines of Enzyme Chemistry. Wiley, New York.
PUTTER, A., 1909. Die Ernahrung der Wassertiere und der Stoffhaushalt der Gewasser.
Fischer, Jena.
RAO, K. P., AND E. D. GOLDBERG, 1954. Utilization of dissolved calcium by a pelecypod. /. Cell.
Comp. Physiol., 43: 283-292.
STEPHENS, G. C., 1960a. Uptake of glucose from solution by the solitary coral, Fungia. Science,
131: 1532.
STEPHENS, G. C., 1960b. The mechanism of glucose uptake by the coral, Fungia. Anat. Rec.,
137: 395.
STEPHENS, G. C., AND R. A. SCHINSKE, 1961. Uptake of amino acids by marine invertebrates.
Limnol. and Oceanog., 6: 175-181.
RESPONSES FROM A PROPRIOCEPTIVE ORGAN OF THE CRAB,
SESARMA RETICULATUM, DURING THE MOLT CYCLE1
RICHARD B. YULES 2
Department of Zoology, Yale University, Nezv Haven, Connecticut
It has long been recognized that the various physiological and morpho-
logical changes associated with molting may occupy a considerable portion of the
life of arthropods. During periods of growth, so-called intermolt stages are
scarcely separated from preparations for, and recovery from, ecdysis. In attempt-
ing to assess the role of sensory receptors in the overall biology of the animal,
there is need of quantitative information on the effect of these changes associated
with molting on sensory input.
In fact, however, no arthropod sense organs have been so investigated. If
sensory input remains essentially unaltered by the morphological changes during
molting, this fact is of value both in extending our understanding of the function-
ing of the central nervous system to all adult stages of these animals, and in posing
the problem of how such constancy is achieved in spite of replication of the exo-
skeleton. On the contrary, if sensory input does change markedly, this fact
must be incorporated into our assessment of the functioning of the central nervous
system.
The need of quantitative assessment of the effects of molting is particularly
evident for the proprioceptive organs which signal position or movement of the
exoskeleton-supported appendages. Several investigators have described such
organs in brachyurans, beginning with Burke (1954), who described the propodite-
dactylopodite joint organ (PDO). He recorded PDO activity both in situ and
from the isolated organ, and using macroelectrodes demonstrated proprioceptive
and "vibration sense" responses. Wiersma and Boettiger (1959) reexamined the
PDO, and found that the organ was not acting as a vibration receptor but con-
tained sense cells responsive unidirectionally to both position and movement. The
PDO contains scolopale cells and is thus a chordotonal organ (Whitear, 1960),
since this term is now used for any structure containing such cells (Pringle, 1960),
in which the axial filaments of two bipolar sense cells end. With the electron
microscope Whitear (1960) showed that one of the axial filaments contains a
ciliary segment and is different from the other axial filament ; the scolopale cell
was thus termed heterodynal. Whitear (personal communication) now believes
this scolopale cell to be a single cell and not a complex of scolopale cell, tube cell,
and sheath cell as she has previously reported.
Burke, Wiersma and Boettiger, and Whitear employed Carcinus macnas as
an experimental animal. Wiersma (1959) subsequently described the PDO in
Mala, Houiarus, and Palinurus, and counted the numbers of fibers responding
1 This investigation was aided by Grant No. 11847 from the National Science Foundation.
- Present address : Yale University Medical School, New Haven, Connecticut.
660
PROPRIOCEPTION AND MOLT CYCLE 661
to movement and position during opening and closing. In carrying out the work
reported here the identification and description of the PDO is extended to another
brachyuran family, the Grapsidae. The experimental approach is to demonstrate
and study the response of the PDO in an intermolt crab, and then to compare
the intermolt condition and response to crabs which have been forced into different
stages of the molt cycle by eyestalk ablation.
MATERIALS AND METHODS
A. Molting
The marsh crab, Sesarma reticulatum (Say), (family Grapsidae) was em-
ployed as the experimental animal. Crabs were dug from tidal mud banks in
New Haven. Connecticut, during mid-winter. These crabs were placed in
individual glass containers, just covered with sea water (salinity 2S%r,), acclimated
for two days at 15° C., and then maintained at 25° C. in a constant-temperature
room. Water was changed every three days; the crabs were not fed. Animals
were manipulated into "forced" proecdysis by ablation of their eyestalks, which
causes an abrupt release of molting hormone (see Passano, 1960; Passano and
Jyssum, unpublished data).
Proecdysis stages were determined by measuring the regeneration (R) of a
walking leg autotomized concurrently with the ablation of the second eyestalk
(Bliss, 1956) :
length of regenerating limb
R = - rr~ r X 100
carapace width
Such R values are precisely related to the stage of the crab in the premolt cycle
(Bliss, 1960). Animals dying were rated as having entered proecdysis (Dt) if
they possessed a mucilaginous membranous layer (Drach, 1939; Passano, 1960).
Of 50 eyestalkless crabs. 25 reached proecdysis and 8 reached exuviation.
PDO response was measured from isolated legs obtained by forced autotomy
from experimental animals of different R values, postecdysial crabs, and intermolt
crabs. Figure 1 shows the point at which a leg was removed to test the PDO.
Since autotomy does not seriously affect the molt sequence (Passano, 1960), the
same crab could be used as a leg donor for two molt stages. No apparently
regenerated limbs were utilized since the PDO in these may possess an abnormal
response (Wiersma, 1959).
B. Histology
The PDO was stained vitally with rongalit methylene blue and examined in
intermolt, premolt, and postecdysial crabs. The organ was exposed, held with
forceps and gently pulled away from its attachment, first distally and then
proximally. If the organ pulled epidermis with it for at least 1 mm., it was
termed "loose" ; if not, the organ was term "intact." The PDO in animals of
different R values and in postecdysial animals was compared to the PDO in
intermolt crabs.
662
RICHARD B. YULES
C. Electrophysiology
All recordings were made from the PDO nerve fibers in the meropodite (see
Figure 2). The propodite and carpodite were immobilized on the side of a
Petri dish in a vertical position with modeling clay (F), the dactylopodite remain-
ing above the clay and thereby moveable, and the meropodite remaining below
the clay. The exoskeleton of the meropodite and the two apodemes and their
attached muscles were removed, allowing the nerve bundle to float free in Pantin's
(1934) crustacean Ringer's solution (R). The nerves were then teased apart
with sharpened tungsten needles and lifted individually or in small bundles into
the air on a platinum wire electrode (E). The reference electrode was a platinum
x
UJ
20-
15-
10-
K TIME OF MOLT
O PDO TEST
10
— I—
15
— i—
20
i
25
— i—
35
DAYS AFTER ES ABLATION
FIGURE 1. Proecdysis in eyestalkless (ES) Sesarma, as determined by limb regeneration
(R; solid black circles). The starred circles represent the points at which a leg was removed
to provide a PDO for testing; see Table I. Note that A-E represent different animals.
wire in the solution. Stimulating the PDO by moving the dactylopodite revealed
a responding nerve bundle, which in preliminary experiments had been visually
traced from the meropodite back to the PDO and which with methylene blue stain-
ing at the site of the organ was shown to contain nerve fibers from all of the
PDO receptors. Stimulating the PDO revealed no other responding nerve
bundles. In some experiments fibers running with the PDO group, but not
coming from the PDO, were found to fire in proecdysis crabs with R values
above 10 : these were separated from the PDO group. The preparation was iso-
lated from vibration by mounting it on a heavy steel plate (V) resting on sawdust.
The signal was amplified (Tektronix 122 preamplifier; AMP), displayed on
an oscilloscope, and recorded on tape. The tape (Scotch 109) moved at 3.75
PROPRIOCEPTION AND MOLT CYCLE
663
inches per second on an Ampex 650 recorder. Output was also monitored
audibly.
The dactylopodite was moved precisely and without vibration by the following
method: A D.C. linear actuator motor (M) drove a steel shaft (S) back and
forth. This shaft pulled a spring-loaded nylon thread (T) which entered a
Faraday cage (C) and circumscribed a grooved aluminum wheel (W), which
rotated on ball bearings on a fixed shaft. This wheel pushed a counterbalanced
lever arm (L) whose end pivoted a 2-mm. diameter aluminum tube (Q) into
which the dactylopodite (D) was fixed with dental cement. The counterbalanced
arm provided a nearly weightless linkage. In order that the arm not displace
the propodite-dactylopodite joint, the preparation was set so that the center of the
V
rs
i
i
/
I
1
II
II
v \
FIGURE 2. A diagram showing the mechanical stimulator and the recording situation :
AMP, preamplifier; B, sliding bolt; C, Faraday cage; E, platinum electrode; F, clay; L, counter-
balanced lever arm ; M, motor ; P, potentiometer ; Q, tube cemented over dactylopodite ; R,
Ringer's solution ; r and r', equal radii ; S, shaft ; T, spring-loaded thread ; V, vibration-free
mounting plate. Insert 2A shows the dactylopodite (D) fully closed.
joint was at the same height as the center of the wheel. The lever arm was then
fixed by means of a sliding bolt (B), so that the radius r equaled r', and the arc
through which the dactylopodite was pushed was always the same and closely
approximated its natural arc. With a two-way spring-loaded switch and a regu-
lated power supply of adjustable output, the dactylopodite could be moved at any
predetermined rate from 1.5°/sec. to 13.5°/sec. in either direction and held at
any point. The total range of movement could be preset and automatically
engaged. The nylon thread also turned a potentiometer (P) which was used
to adjust the vertical position of the second beam on the oscilloscope, thereby
recording the position of the dactylopodite. Since the motor and control apparatus
were isolated from the preparation, and the moving parts were near frictionless,
the movement of the dactylopodite was effectively vibration-free. For example
a vibration-sensitive opening fiber which responded to a gentle stream of air on
664 RICHARD B. YULES
the dactylopodite did not respond while the dactylopodite was closed through 110°.
Preliminary experiments appeared to indicate that all PDO receptors, described
in the results, were stimulated at a speed of movement of 8.58°/sec. through a
range of 109.2°. This speed and range were used to obtain the results reported
here. The fully closed position (where the dactylopodite was at 90° to the
propodite, as indicated in insert, Fig. 2A) was taken as 0°. Fully open was thus
109.2° from the fully closed position.
The smoothness and evenness of the standard stimulus was shown by photo-
graphic analysis of the response of a movement fiber with a constant frequency
of firing (Wiersma and Boettiger. 1959). However, photographic analysis
allowed the determination of only a small number of the total PDO firings. Since
isolation of single or small numbers of fibers leads to destruction of others,
photographic analysis does not allow good quantitative analysis of total PDO
response.
The method finally used was to play back a tape recording of the total opening
and closing responses through a linear amplifier (TMC AL-2A) into a pulse
counter (Berkeley 410) which counted the number of nerve spikes. Since the
tape recorder amplifiers introduce some high frequency interference or "noise,"
the TMC amplifier gain was lowered to eliminate the noise. Thus, the final
quantitative count is suitable for comparing the PDO from crabs of different R
values, to intermolt and postmolt crabs, but the accuracy of the absolute number
of nerve firings coming from the organ is limited by the discrimination of the
counter.
In each experiment the dactylopodite was first closed fully. Starting with the
resulting resting discharge, the experiment was taped while the dactylopodite was
opened, held one second, and returned to the closed position ; the motor switch
artifact indicated when movement began and ceased. The gate-switch of the
pulse counter was manually opened and closed when the tapes were played back
for analysis.
The experimental recordings were played back into the pulse counter and
analyzed in terms of the total number of firings accumulated over 109.2° of
movement, both in opening and in closing. At least two runs were made with
each preparation, but since there was an indication in some cases of slight
diminution in total response with successive trials, the first runs were used for
the quantitative comparisons. Individual runs were counted several times, but
individual countings differed by less than \% (± 30 counts).
RESULTS
A. Histology
A PDO corresponding in location and in dimensions to the PDO of C. macnas
(Burke, 1954) was located in 6". reticulahan. Vital staining of the organ revealed
that in an intermolt crab with a 24.3-mm.-wide carapace, the PDO was 6.54 mm.
long when the dactylopodite made a 90° angle with the propodite, and was 5.10
mm. long when the dactylopodite made a 125° angle with the propodite. The
average diameter of the middle of the organ was 0.11 mm. There are about
40 cell bodies and their processes; 10 of these cell bodies are located more
proximally and are larger than the other and more distal cell bodies. Unlike the
PROPRIOCEPTION AND MOLT CYCLE 665
PDO of Care-inns, the PDO in Scsanna attaches distally without the presence of
an inner protuberance from the exoskeleton of the dactylopodite. Rather, the
organ seems to broaden and send off several strands of connective tissue to the
adjacent epidermis within an area of 1 mm.- from the point at which the main
portion of the organ attaches. The PDO attaches proximally in the propodite
by widening and meeting the epidermis at a point where the apodeme of the
dactylopodite flexor muscle narrows sharply.
The condition of the distal and proximal attachments in crabs of different inter-
molt stages is summarized in Table I. Since the postmolt crab (Ax stage) has such
a soft exoskeleton, no accurate rating of "intact" or "loose" could be assigned to
the organ's attachments during the first 5 hours after exuviation.
B. General organ response
The responses of individual units in the PDO of S. reticulatuni are similar to
the responses of units found in C. maenas by Wiersma and Boettiger (1959). The
same physiological types were found : (a) large unidirectional phasic fibers respond-
ing with a constant frequency only to movement, (b) large unidirectional phasic
fibers responding with an increase in frequency only to movement toward the fully
open or closed positions, (c) large unidirectional phasic fibers responding only to
movement at the extreme open or closed portions of the arc, (d) large adapting
position fibers responding only at the fully open or closed positions, and (e) small
tonic fibers— position fibers — responding at different positions in the arc. Unlike
the situation reported by Wiersma and Boettiger in Carcinus, a position in which
no fibers fire was not found. Occasionally opener and closer "c" category fibers
were isolated as one bundle.
The above types of responses were found in crabs with R values from 10 through
exuviation, in postmolt crabs, and in intermolt crabs. The responses thus do not
vary qualitatively in those stages of the intermolt cycle that were examined.
C. Total response of the PDO
The total response and the state of attachment of the PDO in crabs with different
R values, postmolt crabs, and intermolt crabs are presented in Table I. The source
of the PDO from crabs of different R values is indicated in Figure 1 .
As already noted above, the numbers of counts in Table I are from the first
recorded run at each R value. Preliminary trials with intermolt animals showed an
essentially constant response, within the experimental accuracy achieved here,
between different legs of the same crab and also between legs of different crabs.
The only results which show any significant difference from the mean values
obtained were those from the animal with the two lowest R values, i.e., the earliest
proecdysis stages measured. These values are significantly lower for responses
to both opening and closing. The combination of probabilities gave a x2 of 20.6810,
d.f 4, for an overall P < 0.001.
DISCUSSION AND CONCLUSIONS
The clear-cut conclusion from this study is that the intermolt PDO output does
not vary markedly in crabs of R value 10 to exuviation, and in the immediate post-
666
RICHARD B. YULES
exuvial stages. Since the analysis was limited to the first run of each experiment,
the possibility of organ deterioration or damage to its attachments due to excessive
stimulation was reduced.
It would help in understanding these findings if we knew how the PDO is
attached to the integument. The transfer from the old to the new exoskeleton should
be studied if the organ is attached to the exoskeleton beyond the epidermis. Burke
(1954) in his drawings shows the distal end of the PDO attached to the exo-
skeleton above the epidermis in C. maenas; Whitear (personal communication)
found that the epidermis in Carcinus can be teased away from the distal end, leaving
the organ still connected to the integument. But decisions based on gross optical
TABLE I
Comparison of PDO response and attachments during the molt cycle
Crab R
Counts
Organ connection
Opener
Closer
Proximal
Distal
E 10.0
6128*
4098*
loose
intact
E 11.1
6092*
4045*
loose
intact
A 12.2
6420
4683
intact
intact
A 13.0
6743
4571
intact
intact
B 14.2
6923
4685
intact
intact
B 15.4
6395
4938
intact
intact
D 16.0
6728
4359
intact
intact
C 17.5
6865
4232
intact
intact
D 18.0
6791
4245
intact
intact
C 19.3
6651
4227
intact
intact
« Hours
O 4
r-* 4-
6587
4493
**
**
-t-i C
t/3 *J
6621
4719
**
**
£ 48
6699
4288
intact
intact
Intermolt (normal
6205
4320
intact
intact
control)
* Significantly lower than combined other values.
** Animal too soft to determine.
observations are liable to error. For example, the epidermis can be bound tightly to
the membranous layer (particularly if Carcinus has reached anecdysis), and teasing
the epidermis away from the organ does not prove the organ extends beyond the
epidermis at its point of attachment. Greater resolution, as provided by the electron
microscope, is needed ; but no studies of this type have yet been published. Whitear
(personal communication) has, however, examined the connections of a chordotonal
organ crossing the meropodite-carpopodiate joining (MQ) in Carcinus. Electron
micrographs show this proprioceptive organ attaches onto the epidermis and does
not extend through the "chitin-epidermis" junction, although the epidermis seems
connected to the chitin by fibrous strands. That the PDO is so connected has not
been demonstrated, but since a similar organ has been found to connect onto epi-
PROPRIOCEPTION AND MOLT CYCLE 667
dermis, but not beyond it, it is possible that the PDO is similarly attached solely
to the epidermis.
If both ends of the PDO are connected only to the epidermis, then any change
in response during proecdysis would be due to a difference in tension exerted on
the organ during dactylopodite movement, or to changed mechanical properties
within the organ. A loose peripheral connection would act as an elastic coupling in
series with the stretched organ, and would take up some of the increased tension
which must generate the PDO response. Either some mechanism allows the organ
to respond similarly under different tensions, or the epidermis is never significantly
separated from the remainder of the integument. Since dissections of the PDO
of all stages showed an apparently normal organ without greatly altered connections,
it is likely that the epidermis-exoskeleton separation does not occur in proecdysis
stages corresponding to R values of 12 or more. It is only after excessive manual
pulling that the organ and its attached epidermis pull away from the remainder of
the integument. Since the response of the PDO does not vary markedly within the
portion of the molt cycle investigated here, it seems probable that the tension in the
organ which produces the response does not vary. The PDO would thus be inde-
pendent of the state of the surrounding integument. This would be true whether
the PDO attaches at the epidermis or extends beyond it, but is most easily understood
if the organ is not attached beyond the epidermis.
Premolt growth is associated with crabs having R values greater than 10 (Bliss,
1960). Jyssum and Passano (1957) have shown two separate stages of limb re-
generation : the first, basal limb growth, shows R values up to 10 and is molt-
hormone-independent ; the second stage, premolt growth, shows R values greater
than 10 and is molt-hormone-dependent. But Bliss' results were from normal ani-
mals. Eyestalkless crabs molt in a shorter time than do normal crabs. Although
the precipitous molt of eyestalkless Sesanna can still be halted when both Y-organs
are removed at R value of 10 (Passano and Jyssum, unpublished data), it is clear
that such animals are not precisely comparable to normal proecdysis crabs of the
same R value in Drach stage D0. An eyestalkless crab's R value 10 probably cor-
responds to stage D,, for an eyestalkless crab's new dactylus is formed enough to
be separated from its old exoskeleton at an R value of 12. Thus the range of data
includes stages D, through B, and C4 in Drach's terminology. It is during these
stages that the PDO response has been measured and found to be constant.
Although not appreciated at the time these experiments were conducted, analysis
of these data shows that the R values of the regenerating limbs of the experimental
animals were lower than those from corresponding Drach stages of normal
proecdysis crabs. This may have been due to the precipitous nature of eyestalk-
removal-forced ecdysis, or of the failure to measure R values in regenerating limbs
which had achieved basal limb growth before bilateral eyestalk extirpation, or from
a combination of these two factors. Whatever the cause of this discrepancy, it
resulted in a failure to test the PDO output in the earliest proecdysis stages, D0 and
early D1. This is unfortunate, since there is a slightly smaller output from the
earliest two stages tested, those in which the distal PDO attachment was termed
"loose." But while significantly lower statistically, the reduction is less than 10%
of the total PDO output ; it is difficult to consider this reduction very important to
the animal's total sensory input. These data were obtained from a single crab,
668 RICHARD B. YULES
and any conclusion should remain tentative. It is likely, however, that the loose
distal connection found only at these stages allows some of the organ tension to be
taken up by the displaced epidermis. The PDO response from crabs with R values
lower than 10 should be examined to see if this slightly reduced response is truly
characteristic of early premolt stages. But even if further work demonstrates that
sensory input falls during the early stages of proecdysis, the fact remains that the
sensory input from the PDO remains at a constant intermolt level during most of
proecdysis, including the crucial period immediately around exuviation.
Although separate from the problem under investigation, one interesting finding
should be noted. Several fibers firing with constant frequency were detected running
with the PDO group in crabs of R value 10 or above ; no such firings were noted in
intermolt crabs. It is known that the tendon nerve (Wiersma and Boettiger, 1959)
travels with the PDO bundle. Alexandrowicz (1957) has suggested that these
tendon nerves, whose processes end somewhere in the integument, function to signal
changes in the integument during molting. It is possible that these firings noted
above originated in the tendon nerve cell bodies ; this possibility should be explored.
I am indebted to Dr. L. M. Passano for his constant help and encouragement ; I
also wish to thank R. C. Morrison for his aid in quantitatively analyzing the data.
SUMMARY
1. The identification of a proprioceptive organ (PDO), spanning the propodite-
dactylopodite joint in the crab, Sesarma reticulatum, has extended the study of the
PDO to the family Grapsidae.
2. The appearance and physiological response of the PDO in Sesarma are essen-
tially identical to the appearance and response found by Wiersma and Boettiger
(1959) in the Carcmus PDO.
3. A dactylopodite-moving stimulator is described; it provides a variable and
controlled stimulus to the organ, allowing quantitative comparisons of the PDO out-
put in proecdysis, postexuvial, and in intermolt stage crabs.
4. Sensory input from the PDO does not vary in proecdysis animals from Drach
stage mid Dt to exuviation, and in the immediate postexuvial stages, as compared
to the response from intermolt crabs.
5. The constancy of the PDO output is discussed in terms of the organ's attach-
ment to the integument.
LITERATURE CITED
ALEXANDROWICZ, J. S., 1957. Notes on the nervous system in the Stomatopoda. V. The various
types of sensory nerve cells. Pubbl. Staz. Zoo/. Napoli, 29: 213-225.
BLISS, D. E., 1956. Neurosecretion and the control of growth in a decapod crustacean. In:
Bertil Hanstrom. Zoological Papers in Honour of his Sixty-fifth Birthday, November
20th, 1956, ed. K. G. Wingstrand ; pp. 56-75. Zoological Institute, Lund, Sweden.
BLISS, D. E., 1960. Autotomy and regeneration. In: The Physiology of Crustacea, ed. T. H.
Waterman. Vol. 1, pp. 561-589. New York and London, Academic Press, Inc.
BURKE, W., 1954. An organ for proprioception and vibration sense in Carcinus maenas. J.
Exp. Biol., 31 : 127-138.
DRACH, P., 1939. Mue et cycle d'intermue chez les Crustaces decapodes. Ann. Inst. Oceanog.,
19: 103-391.
PROPRIOCEPTION AND MOLT CYCLE 669
JYSSUM, S., AND L. M. PASSANO, 1957. Endocrine regulation of preliminary limb regeneration
and molting in the crab Scsarina. Atwt. Rcc., 128: 571-572.
PANTIN, C. F. A., 1934. On the excitation of crustacean muscle. /. Exp. Biol, 11: 11-27.
PASSANO, L. M., 1960. Molting and its control. In: The Physiology of Crustacea, ed. T. H.
Waterman. Vol. 1, pp. 473-536. New York and London, Academic Press, Inc.
PRINGLE, J. W. S., 1961. Proprioception in arthropods. In: The Cell and the Organism, eds.
J. A. Ramsay and V. B. Wigglesworth ; pp. 256-282. London and New York, Cam-
bridge University Press.
WHITEAR, M., 1960. Chordotonal organs in Crustacea. Nature, 187: 522-523.
WIERSMA, C. A. G., 1959. Movement receptors in decapod Crustacea. /. Mar. Biol. Assoc.,
38: 143-152.
WIERSMA, C. A. G., AND E. G. BOETTIGER, 1959. Unidirectional movement fibres from a
proprioceptive organ of the crab, Carcinus maenas. J. Exp. Biol., 36: 102-113.
INDEX
ABSTRACTS of papers presented at the
Marine Biological Laboratory, 461.
Acridine dye, effect of on sea urchin develop-
ment, 132.
Acrosomal lysin, effect of on Mytilus egg, 531.
Acrosome reaction, induction of by acridine
orange, 473 (abstract).
Actin, G-ADP, polymerization of, 483 (ab-
stract).
Actin, G-ATP and G-ADP, effects of KI on,
491 (abstract).
Activation of Arbacia egg by cysteine, 485
(abstract).
Adrenal of sea gull, electron microscopy of,
499 (abstract).
Aequipecten, influence of on behavior of com-
mensal Pinnotheres, 388.
Aerobic respiration of oyster embryos, 71.
Age of Rana embryos, in relation to glycolysis,
555.
Alewife, serology of, 330.
Alga, phosphorus uptake by, 134.
Algae, use of in study of sea urchin nutrition,
105.
ALJURE, E., H. GAINER AND H. GRUNDFEST.
Differentiation of synaptic and GABA
inhibitory action in crab neuromuscular
junctions, 479 (abstract).
Alloophorus, reproductive cycle of, 351.
Alosa, serology of, 330.
Amino acid and protein synthesis by sea urchin
embryo, 465 (abstract).
Amino acid transport of human erythrocytes,
461 (abstract).
Amino acid uptake by Clymenella, 512 (ab-
stract).
Amino acids, uptake of by Fungia, 648.
Amino acids and peptides in Arbacia eggs, 476
(abstract).
Amphibian development, anaerobic glycolysis
in, 555.
Amphipod, oxygen consumption of, 225.
Analysis of initial reaction resulting in homol-
ogous splenomegaly in chick, 366.
Analysis of polarized light in eye of Daphnia,
233.
Anatomy of brachiopod, 597.
Anatomy of Calcinus larvae, 179.
Anatomy of Cardisoma, 207.
Anatomy of echiuroid proboscis, 80.
Anatomy of Lychas, 344.
Anatomy of sea urchin digestive system, 105.
Anatomy of triclad turbellarian gut, 571.
Androgenetic hybrids of California newts,
karyoplasmic studies of, 253.
Annelid, development of, 412.
Annelid, reproduction of, 396.
Annelid eggs, cytological studies of, 424.
Annual testicular cycle of bobolink, 94.
Anomuran, larval development of, in labora-
tory, 179.
ANSELL, A. D. Observations on burrowing in
the Veneridae (Eulamellibranchia), 521.
APPLEGATE, A., AND L. NELSON. Acetylcho-
linesterase in Mytilus spermatozoa, 475
(abstract).
Aquatic invertebrates, uptake of organic
material by, 648.
Arbacia development, effect of proflavin on,
132.
ARGYRIS, T. S. See A. M. MUN, 366.
ARMITAGE, K. B. Temperature and oxygen
consumption of Orchomonella, 225.
ARNOLD, J. M. Mating behavior and social
structure in Loligo, 53.
Artemia cysts, glycerol in, 295.
Artemia populations, survival of in radioactive
sea water, 302.
Assay of crustacean retinal pigment hormone,
317.
ASTERITA, H. See D. MARSLAND, 484 (ab-
stract).
Atrina, influence of on behavior of commensal
Pinnotheres, 388.
AUSTIN, C. R., AND J. PIATIGORSKY. Evidence
against participation of a jelly-splitting
agent in sperm penetration of Arbacia
eggs, 470 (abstract).
AUSTIN, C. R. See R. L. BRINSTER, 471 ;
S. D. EZELL, JR., 472; J. PIATIGORSKY,
473 ; D. H. SPOON, 474 (abstracts).
Australian gastropod, intertidal clustering of,
170.
Australian whales, body temperatures of, 154.
Autoradiography, preparation of fish lens
epithelial whole-mounts for, 499 (abstract).
Axons of retinula cells, function of, 618.
, I. J. See D. M. TRAVIS, 487; A. M.
ELLIOTT, 495 (abstracts).
BALLENTINE, T. V. N. See A. K. PARPART,
485, 508 (abstracts).
670
INDEX
671
BARNWELL, F. H., AND F. A. BROWN, JR.
Correspondence of maximum response of
snails to magnetism with the strength of
geomagnetism, 488 (abstract).
BARNWELL, F. H. See H. M. WEBB, 514
(abstract).
BAU, D., JR. See A. B. CHAET, 490 (abstract).
BAYLOR, E. R., AND W. E. HAZEN. The
analysis of polarized light in the eye of
Daphnia, 233.
BAYLOR, E. R. See W. E. HAZEN, 243.
Behavior, mating, in Loligo, 53.
Behavior of Daphnia in polarized light, 243.
BELAMARICH, F. A., R. F. DOOLITTLE AND
D. M. SURGENOR. Studies on throm-
bocytes of the smooth dogfish, Mustelus,
479 (abstract).
Biology of Cardisoma, 207.
Bioluminescence, presence and absence of in
Noctiluca, 494 (abstract).
Bioluminescence of Achromobacter, quantum
yield of, 483 (abstract).
Bioluminescence of Noctiluca, electrophysiol-
ogy of, 482 (abstract).
Bird, migratory restlessness in, 542.
Bird, testicular cycle in, 94.
BISCHOFF, E. R., AND C. B. METZ. Immuno-
logical identification of an egg agglutinin
in Arbacia sperm extracts, 471 (abstract).
BISCHOFF, E. R., AND C. B. METZ. Neu-
tralization of the fertilization inhibitors
in anti-Arbacia sperm serum by sperm
extracts, 470 (abstract).
BLACK, R. E. The concentrations of some
enzymes of the citric acid cycle and elec-
tron transport system in the large granule
fraction of eggs and trochophores of the
oyster Crassostrea, 71.
BLACK, R. E. Respiration, electron-transport
enzymes and Krebs-cycle enzymes in early
developmental stages of the oyster,
Crassostrea, 58.
Blood clotting, lobster, inhibitors of, 481
(abstract).
Blueback herring, serology of, 330.
Bobolink, testicular cycle in, 94.
Bobolinks, migratory restlessness in, 542.
Body temperatures of whales, 154.
Brachiopod, filter-feeding of, 597.
B RANDOM, W. F. Karyoplasmic studies in
haploid, androgenetic hybrids of Cali-
fornia newts, 253.
Breakdown of germinal vesicle in Pectinaria
eggs, 424.
Breeding experiments with Lychas, 344.
Breeding season of Glycera, 396.
Breeding season of Goodeidae, 351.
Brevoortia, serology of, 330.
Brine shrimp, survival of in radioactive sea
water, 302.
Brine shrimp cysts, glycerol in, 295.
BRINSTER, R. L., AND C. R. AUSTIN. Action
of neuraminidase on Arbacia spermatozoa,
471 (abstract).
BROWN, F. A., JR. Response of the planarian,
Dugesia, to very weak horizontal electro-
static fields, 282.
BROWN, F. A., JR. Responses of the planarian,
Dugesia, and the protozoan, Paramecium,
to very weak horizontal magnetic fields,
264.
BROWN, F. A., JR., H. M. WEBB, AND L. G.
JOHNSON. Orientational responses in or-
ganisms effected by very small alterations
in gamma radiation, 488 (abstract).
BROWN, F. A., JR. See F. H. BARNWELL, 488
(abstract).
BRYANT, D. C., R. S. WEINSTEIN, D. L. KLEIN
AND R. F. DOOLITTLE. On the nature of
dogfish trypsinogen and trypsin, 479
(abstract).
BURNETT, A. L., AND N. A. DIEHL. Inductive
potencies of the manubrium of Tubularia,
489 (abstract).
BURNETT, A. L., N. A. DIEHL AND E. MUTTER-
PERL. The relation between inductive
regions and interstitial cell distribution
in Hydra, Tubularia and Hydractinia,
489 (abstract).
Burrowing in Veneridae, 521.
(^ALCINUS, larval development of, 179.
Calcium-lack, effect of on Mytilus egg, 531.
Calcium uptake and release by Arbacia eggs,
method for study of, 517 (abstract).
California newts, karyoplasmic studies of, 253.
Carbohydrases of triclad Turbellaria, 571.
Carbohydrate components in Artemia cysts,
295.
Carcinus, retinal pigment hormone of, 317.
CARLSON, A. D. Neural activity during
hypoxia in adult firefly, 490 (abstract).
Cathepsin-C-type endopeptidases of triclad
Turbellaria, 571.
Cell division, effect of proflavin on, 132.
Cell pH of Arbacia eggs, 519 (abstract).
Cell size in hybrid newts, 253.
Cells, retinula, photoreceptor mechanism of,
618.
Centrifugation of Pectinaria eggs, 424.
Cephalopod, mating behavior of, 53.
Cerithium, intertidal clustering of, 170.
Cetaceans, body temperatures of, 154.
CHAET, A. B., AND D. BAU, JR. Protein
changes in the ageing lobster, 490 (ab-
stract).
672
INDEX
CHAET, A. B. See D. E. PHILPOTT, 509
(abstract).
CHANNING, C. P., A. EBERHARD, A. H. GUIN-
DON, C. KEPLER, V. MASSEY AND C.
VEEGAR. Purification and some proper-
ties of lipoyl dehydrogenase from dogfish
liver, 480 (abstract).
Chemotaxis in Campanularia, 477 (abstract).
CHENEY, R. H. See C. C. SPEIDEL, 463, 511
(abstracts).
Chick embryo, homologous splenomegaly in,
366.
Chorioallantoic grafts in chick embryo, 366.
Chromatophore aggregation in fish, pharma-
cology of, 511 (abstract).
Chromatophore control in sand flounder, 486
(abstract).
Chromatophores of Cardisoma, 207.
Chromatophores of Crustacea, culture of, 509
(abstract).
Chromosome counts in Fundulus blastoderms,
582.
Chromosomes of hybrid newts, 253.
CHUANG, S. H. Feeding mechanism of the
echiuroid, Ochetostoma, 80.
CHUANG, S. H. Sites of oxygen uptake in
Ochetostoma, 86.
Ciliary patterns of brachiopod lophophore, 597.
Cistenides eggs, cytological studies of, 424.
Citric acid cycle enzymes of oyster embryo, 71.
CLAFF, C. L. Gas absorption from fish swim-
bladder, 491 (abstract).
CLAFF, C. L. See J. A. MILLER, JR., 450.
CLARK, E. E., AND R. F. OLIVO. Effects of KI
on G-ATP and G-ADP actin, 491 (ab-
stract).
CLARK, R. L., AND G. T. SCOTT. Influence of
brain lesions on melanocyte dispersion,
491 (abstract).
CLARK, R. L. See G. T. SCOTT, 486, 511
(abstracts).
Cleavage, effects of proflavin on, 132.
Cleavage capacity and cortical gel structure of
Arbacia eggs, effect of D2O on, 484 (ab-
stract).
Cleavage of Fundulus eggs fertilized with
irradiated sperm, 582.
CLEGG, J. S. Free glycerol in dormant cysts
of the brine shrimp, Artemia, and its
disappearance during development, 295.
CLONEY, R. A. Contraction of the epidermis
during tail resorption in the ascidian
Amaroucium, 492 (abstract).
Clupea, serology of, 330.
Coelenterate stem, oxygen uptake of, 450.
COHEN, L. B., AND K. E. VAN HOLDE. Studies
on the dissociation of Loligo hemocyanin,
480 (abstract).
Cold, effect of on movements of flatworm, 146.
Cold, effect of on oxygen consumption of
Orchomonella, 225.
Colored lights, reactions of mysids to, 562.
COLWIN, A. L., AND L. H. COL WIN. Fine
structure of acrosome and early fertiliza-
tion stages in Saccoglossus, 492 (abstract).
COLWIN, L. H., AND A. L. COLWIN. Induction
of spawning in Saccoglossus at Woods
Hole, 493 (abstract).
Commensal crab, influence of hosts on behavior
of, 388.
COOPERSTEIN, S. J. See D. WATKINS, 469;
F. C. GOETZ, 496 (abstracts).
COPELAND, E. Observations on the gas-
secreting epithelium of Physalia, 493 (ab-
stract).
Coral, uptake of glucose by, 648.
COUSINEAU, G. H. See P. R. GROSS, 497
(abstract).
Crab, commensal, influence of hosts on be-
havior of, 388.
Crab, land, biology of, 207.
Crab, larval development of, 179.
Crab, proprioceptor responses of, during molt
cycle, 660.
CRANE, R. K. See L. LASTER, 502 (abstract).
Crassostrea embryos, respiration and enzymes
of, 58, 71.
Crustacea, photomechanical responses of prox-
imal pigment of, 121.
Crustacean, larval development of, in labora-
tory, 179.
Crustacean, proprioceptor responses of during
molt cycle, 660.
Crustacean "cysts," glycerol in, 295.
Crustacean integument, structure and metabo-
lism of, during molt cycle, 635.
Crustacean retinal pigment hormone and
neurosecretion, 317.
Cycle, molt, of crab, proprioceptor responses
during, 660.
Cycle, molt, of crustacean, structure and
metabolism of integument during, 635.
Cycle, testicular, of bobolink, 94.
Cycles, reproductive, of three teleosts, 351.
Cyprinodont fishes, reproductive cycles of, 351.
"Cysts" of Artemia, glycerol in, 295.
Cytological studies of Pectinaria eggs, 424.
Cytology of Gecarcintis integument during
molt cycle, 635.
Cytology of hybrid newts, 253.
Cytoplasm vs. nucleus in hybrid newts, 253.
synthesis in early mitotic stages, pres-
sure study of, 518 (abstract).
DNA synthesis in mature Arbacia eggs, 475
(abstract).
INDEX
673
DAN, J. C. The vitelline coat of the Mytilus
egg. I. ,531.
Daphnia, analysis of polarized light in eye of,
23.3.
Daphnia, behavior of in polarized light, 243.
Dark, role of in phosphorus uptake of Phaeo-
dactylum, 134.
Dark-adaptation in Crustacea, 121.
Darkness, effect of on inysids, 562.
Day-length, role of in photorefractoriness of
bobolinks, 94.
Day-length in relation to Zugunruhe of bobo-
links, 542.
Dehydrogenase system activity in Asterias
gametes, 501 (abstract).
DELANNEY, L. E. See A. M. MUN, 366.
DEPHILLIPS, H. A., JR., AND K. E. VAN HOLDE.
Spectral studies of hemocyanin, 481
(abstract).
Development, amphibian, anaerobic glycolysis
in, 555.
Development of Artemia, disappearance of
glycerol during, 295.
Development of Calcinus, 179.
Development of Glycera, 412.
Development of hybrid newts, 253.
Development of Lychas, 344.
Development of Mytilus egg, 531.
Development of sea urchin, effect of proflavin
on, 132.
Development of splenomegaly in chick embryo,
366.
Development of teleost eggs after x-irradiation
of sperm, 582.
Developmental patterns of enzymes in Lim-
naea, 463 (abstract).
Developmental stages of oyster, respiration
and enzymes of, 58, 71.
DEWEL, W. C. See W. STONE, JR., 513
(abstract).
Diapause in Lychas, 344.
DIEHL, N. A. See A. L. BURNETT, 489 (ab-
stracts).
Digestion of protein in planarians, 571.
Digestion in Strongylocentrotus, 105.
Digestion in triclad Turbellaria, 571.
Digging movements of molluscs, 521.
Discinisca, filter-feeding of, 597.
Distribution of brachiopod, 597.
Diurnal phototactic rhythm in Uca, 507
(abstract).
Dolichonyx, migratory restlessness in, 542.
Dolichonyx, testicular cycle in, 94.
DOOLITTLE, R. F., AND L. LORAND. Inhibitors
of lobster blood clotting, 481 (abstract).
DOOLITTLE, R. F. See F. A. BELAMARICH,
479; D. C. BRYANT, 479 (abstracts).
opa oxidase systems of marine invertebrates,
D 503 (abstract).
Dormant Artemia cysts, glycerol in, 295.
Dosinia, burrowing in, 521.
Dow, E. N. See }. B. MORRILL, 463 (ab-
stract).
Drosophila, common mechanism for tempera-
ture adaptation and crossvein deformation
in, 462 (abstract).
Drosophila, genes regulating dopa oxidase
activity in, 464 (abstract).
Dugesia, digestion of protein by, 589.
Dugesia, response of to electrostatic fields, 282.
Dugesia, response of to very weak horizontal
magnetic fields, 264.
DUNHAM, P. B. The adaptation of Tetra-
hymena to a high NaCl environment, 462
(abstract).
DUNHAM, P. B., AND H. GAINER. Compart-
mentalization of chloride in lobster muscle,
494 (abstract).
JfBERHARD, A. See C. P. CHANNING, 480
(abstract).
EBERT, J. D. See A. M. MUN, 366.
Ecdysis of crabs, proprioceptor responses dur-
ing, 660.
Echinoderm, nutrition of, 105.
Echiuroid, electrical induction of spawning in,
203.
Echiuroid, feeding mechanism of, 80.
Echiuroid, oxygen uptake of, 86.
ECKERT, R. Electrical activity associated with
bioluminescence in a single cell, 482
(abstract).
ECKERT R., AND M. FINDLAY. Two physio-
logical varieties of Noctiluca, 494 (ab-
stract).
Ecology of Australian gastropod, 170.
Ecology of brachiopod, 597.
Ecology of Cardisoma, 207.
Ecology of Ochetostoma, 80.
Egg-laying of squid, 53.
Egg, Mytilus, vitelline coat of, 531.
Eggs, teleost, development of after x-irradia-
tion of sperm, 582.
Eggs of oyster, respiration and enzymes of, 58,
71.
Eggs of Pectinaria, cytological studies of, 424.
Electrical induction of spawning in Urechis
and Mytilus, 203.
Electron microscopy of Mytilus egg, 531.
Electron microscopy of toadfish neurosecretory
cells, 461 (abstract).
Electron-transport enzymes in oyster embryos,
58, 71.
Electrophoresis of clupeoid fish sera, 330.
674
INDEX
Electrophysiological concomitants of shadow
reflex in barnacles, 498 (abstract).
Electrophysiology of crayfish muscle fibers,
468 (abstract).
Electrophysiology of dogfish branchial sensory
nerve endings, 506 (abstract).
Electrophysiology of Limulus eye, 618.
Electrophysiology of Sesarma, 660.
Electrophysiology of Spisula intestine, 485
(abstract).
Electrostatic fields, response of planarian to,
282.
ELLIOTT, A. M., D. M. TRAVIS AND I. J. BAR.
Survival of Tetrahymena at elevated
oxygen pressures, 495 (abstract).
ELLIOTT, A. M. See D. M. TRAVIS, 487
(abstract).
Embryo, chick, splenomegaly in, 366.
Embryology of hybrid newts, 253.
Embryology of Mytilus egg, 531.
Embryology of teleost eggs after x-irradiation
of sperm, 582.
Embryonic diapause in Lychas, 344.
Embryos of oyster, respiration and enzymes of,
58, 71.
Embryos of Rana, anaerobic glycolysis in, 555.
Endocrine control of pigment responses in
Crustacea, 121.
Endopeptidases of triclad Turbellaria, 571.
ENGELS, W. L. Day-length and termination
of photorefractoriness in the annual
testicular cycle of the transequatorial
migrant Dolichonyx, 94.
ENGELS, W. L. Migratory restlessness in
caged bobolinks (Dolichonyx, a trans-
equatorial migrant), 542.
Enzymes of oyster embryo, 58, 71.
Enzymes of triclad Turbellaria, 571.
Epitoky in Glycera, 396.
ERRICO, J. See A. M. MUN, 366.
ESPER, H. Incorporation of C14-glucose into
oocytes and ovarian eggs of Arbacia,
475 (abstract).
ESPER, H. Uptake of H3-thymidine by eggs
of Arbacia, 475 (abstract).
ESPER, H. See L. H. KLEINHOLZ, 317.
Eulamellibranchia, burrowing in, 521.
Euplotes, mating types and conjugation in new
species of, 516 (abstract).
Euplotes, structure and life cycle of new species
of, 516 (abstract).
EVANS, T., A. MONROY and A. SENFT. Free
amino acids and peptides in unfertilized
and fertilized eggs of Arbacia, 476 (ab-
stract).
EVANS, T. E. See C. A. SHIVERS, 473 (ab-
stract).
Exopeptidases of triclad Turbellaria, 571.
Eye, dogfish, osmotic pressure relationships in,
513 (abstract).
Eye of Daphnia, light relations in, 233.
Eyestalk hormone of crustaceans, assay and
properties of, 317.
EZELL, S. D., JR., AND C. R. AUSTIN. Passage
of spermatozoa through the chorion of
Ciona eggs, 472 (abstract).
pARMANFARMAIAN, A., AND J. H.
PHILLIPS. Digestion, storage and trans-
location of nutrients in the purple sea ur-
chin Strongylocentrotus, 105.
Feeding mechanism of echiuroid, 80.
Feeding patterns of brachiopod, 597.
Feeding in triclad Turbellaria, 571.
Female reproductive cycles of three teleosts,
351.
FERGUSON, J. C. Nutrient transport in the
starfish, Asterias, as studied with isolated
digestive glands, 482 (abstract).
Fertilization of Fundulus eggs with x-irradiated
sperm, 582.
Fertilization inhibitors in anti-Arbacia-sperm
serum, 470 (abstract).
Fertilization of lysin-treated Mytilus eggs, 531.
Fertilization in Saccoglossus eggs, 492 (ab-
stract).
Fertilizin, relationship of to acrosome reaction
in Arbacia, 473 (abstract).
Filter-feeding of brachiopod, 597.
FINDLAY, M. See R. ECKERT, 494 (abstract).
FlNGERMAN, M., R. NAGABHUSHANAM AND
L. PHILPOTT. Photomechanical responses
of the proximal pigment in Palaemonetes
and Orconectes, 121.
Fishes, clupeoid, serology of, 330.
FLAKE, G. P., AND C. B. METZ. Soluble sur-
face and subsurface antigens of the
Arbacia sperm, 472 (abstract).
Flatworm, survival and movements of at differ-
ent salinities and temperatures, 146.
Flatworms, feeding and digestion in, 571.
Fluorescence microscopy of Pectinaria eggs,
424.
FONTAINE, J. See S. LERMAN, 502 (abstract).
FORER, A. See D. H. SPOON, 474 (abstract).
FRANKLIN, L. E., AND C. B. METZ. Electron
microscope study of sperm entry into sea
urchin oocytes, 473 (abstract).
FREEMAN, A. R., AND M. A. SPIRTES. Effect
of phenothiazine derivatives on the
permeability of the dogfish erythrocyte,
495 (abstract).
Frog embryos, anaerobic glycolysis in, 555.
Fundulus eggs, development of after x-irradia-
tion of sperm, 582.
Fungia, uptake of glucose by, 648.
INDEX
675
rjAINER, H. See E. ALJURE, 479; P. B.
DUNHAM, 494 (abstracts).
Gametes of marine invertebrates, obtaining
of by electrical stimulation, 203.
Gametogenesis of Glycera, 412.
Gas exchange in Thyone, effects of carbon
dioxide on, 487 (abstract).
Gas-secreting epithelium of Physalia, 493
(abstract).
Gastrolith formation in Gecarcinus, 635.
Gastropod, intertidal clustering of, 170.
Gecarcinus, structure and metabolism of
integument of, during molt cycle, 635.
Gel-sol transformations in Arbacia egg, 508
(abstract).
Geomagnetism, response of snails to, 488
(abstract).
Germinal vesicle breakdown in Pectinaria eggs,
424.
Gestation stages of Goodeidae, 351.
GIFFORD, C. A. Some observations on the
general biology of the land crab, Car-
disoma, in south Florida, 207.
GIRARDIER, L., J. P. REUBEN AND H. GRUND-
FEST. Effects of isolation and denerva-
tion of crayfish muscle fibers on their
membrane resistance, 496 (abstract).
GIRARDIER, L., J. P. REUBEN AND H. GRUND-
FEST. A possible mechanism for excita-
tion-contraction coupling in crayfish
muscle fibers, 468 (abstract).
GIRARDIER, L. See J. P. REUBEN, 469, 509
(abstracts).
Glaucothoe stage of Calcinus, 179.
Glucose incorporation into Arbacia oocytes
and ovarian eggs, 476 (abstract).
Glucose uptake by Fungia, 648.
Glycerol in Artemia cysts, 295.
Glycogen metabolism of Gecarcinus integument
during molt cycle, 635.
Glycolysis, anaerobic, in amphibian develop-
ment, 555.
Glycera, development of, 396, 412.
Glycera, reproduction of, 396.
GOETZ, F. C., AND S. J. COOPERSTEIN. Studies
on the isolated islet tissue of toadfish:
the uptake of injected C14-glucose by islet
and other tissues, 496 (abstract).
COLORING, L. S., H. I. HIRSHFIELD AND I. P.
GOLDRING. Strontium utilization by Ar-
bacia, 497 (abstract).
Golgi apparatus and lysosomes in vertebrate
neurons, 465 (abstract).
Goodea, reproductive cycle of, 351.
GOTTFRIED, E. L. See M. M. RAPPORT, 485
(abstract).
GRANT, R. J. Effect of temperature on poly-
merization of G-ADP actin, 483 (abstract).
Gregarine, motility in, 514 (abstract).
GREGG, J. R. Anaerobic glycolysis in am-
phibian development, 555.
GROSCH, D. S. The survival of Artemia
populations in radioactive sea water, 302.
GROSS, P. R., AND G. H. COUSINEAU. Incor-
poration of C14-thymidine into pool and
DNA of deuterated sea urchin eggs, 497
(abstract).
GROSS, P. R., AND J. M. MITCHISON. "Mes-
senger" RNA and the cell cycle in a fission
yeast, 467 (abstract).
Growth of brain in teleosts, 517 (abstract).
GRUNDFEST, H. See L. GIRARDIER, 468, 469,
496; E. ALJURE, 479; J. P. REUBEN, 509
(abstracts).
GUINDON, A. H. See C. P. CHANNING, 480
(abstract).
GWILLIAM, G. F. Electrophysiological con-
comitants of the shadow reflex in certain
barnacles, 498 (abstract).
"LJ AEMAL system of Strongylocentrotus, 105.
HAGINS, W. A., AND P. A. LIEBMAN. Light-
induced pigment migration in the squid
retina, 498 (abstract).
Haploid California newts, karyoplasmic studies
of, 253.
HARDING, C. V., M. B. NEWMAN, F. E. JONES
AND H. ROTHSTEIN. The preparation of
sea bass lens epithelial whole-mounts for
tritium autoradiography, 499 (abstract).
HARDING, C. V. See M. B. WHEELER, 515
(abstract).
HARRISON, G. Electron microscopy of the
sea gull adrenal, 499 (abstract).
HARVEY, E. B. Proflavin and its influence on
cleavage and development, 132.
HASTINGS, J. W., J. A. SPUDICH AND G. MAL-
NIC. Influence of aldehyde chain length
on the relative quantum yield of the bio-
luminescent reaction of Achromobacter,
483 (abstract).
HAZEN, W. E., AND E. R. BAYLOR. Behavior
of Daphnia in polarized light, 243.
HAZEN, W. E. See E. R. BAYLOR, 233.
Heat-treatment of newt eggs, 253.
HEGYELI, A. See A. SZENT-GYORGYI, 466
(abstract).
Hemerythrin dissociation, 484 (abstract).
Hemocyanin, spectral studies of, 481 (abstract).
Hemocyanin of Loligo, dissociation of, 480
(abstract).
Hemoglobin of Phacoides, 605.
HERMAN, S. S. Spectral sensitivity and photo-
taxis in the opossum shrimp Neomysis, 562.
Hermaphroditic Mytilus, electrical induction
of spawning in, 203.
676
INDEX
Herring, serology of, 330.
"Hertwig Effect" in teleost development, 582.
Heteroploidy in hybrid newts, 253.
HICKMAN, J. See N. PIANFETTI, 509 (ab-
stract).
HICKMAN, J. C. See G. T. SCOTT, 486, 511
(abstracts).
HILL, R. B. Pharmacology of the radula
protractor of Busycon, 499 (abstract).
HIRSHFIELD, H. I. See L. S. COLORING, 497
(abstract).
Histochemistry of feeding and digestion in
triclad Turbellaria, 571.
Histochemistry of Gecarcinus during molt
cycle, 635.
Histology of crab proprioceptors, 660.
Histology of Glycera, 396, 412.
Histology of Phacoides pigment, 605.
Homologous splenomegaly in chick embryo,
366.
Hormone, crustacean retinal pigment, assay
and properties of, 317.
Hormones of Crustacea, role of in light-adapta-
tion, 121.
Hosts, influence of on behavior of commensal
crab Pinnotheres, 388.
HUGHES, W. L. See M. B. WHEELER, 515
(abstract).
Humpback whale, body temperatures of, 154.
Hyaline layer of Mytilus egg, 531.
Hybrid fertilization of ascidians, 505 (abstract).
Hybrid frog embryos, anaerobic glycolysis in,
555.
Hybrid newts, karyoplasmic studies on, 253.
Hydra adhesive surface, electron microscopy
of, 509 (abstract).
TMMUNOLOGICAL responses of chick em-
bryo, 366.
Immunology of clupeoid fishes, 330.
Indian scorpion, reproductive biology of, 344.
Inductive regions in Hydra, Tubularia and
Hydractinia, 489 (abstract).
Inductive potencies of Tubularia manubrium,
489 (abstract).
Influence of hosts on behavior of commensal
crab Pinnotheres, 388.
Inhibition of reconstitution of Tubularia, 450
(abstract).
Insemination of Fundulus eggs, effect of time
of, on development, 500 (abstract).
Insulin, reversible enzymatic reduction of, 465
(abstract).
Insulin-containing fraction separated from
goosefish islet tissue, 503 (abstract).
Integument, crustacean, structure and metabo-
lism of during molt cycle, 635.
Intertidal clustering of Australian gastropod,
170.
Intestinal absorption in fish, nitrogen inhibition
of, 506 (abstract).
Intestinal absorption in fish, phlorizin inhibi-
tion of, 507 (abstract).
Intracellular digestion of protein in planarians,
571.
Invertebrates, uptake of organic material by,
648.
lododeoxyuridine, uptake of by Arbacia em-
bryos, 515 (abstract).
Ionic relations in lobster muscle, 494 (abstract).
Iridaea, use of in study of sea urchin nutrition,
105.
Irradiation, gamma, of Arbacia eggs, 511
(abstract).
Irradiation, ultraviolet, of sea urchin egg, 510
(abstract).
Irradiation of Arbacia zygotes and gametes,
463 (abstract).
Irradiation of Artemia populations, 302.
Irradiation of mouse embryo, 461 (abstract).
"TELLY layer" of Mytilus egg, 531.
JENNINGS, J. B. Further studies on feeding
and digestion in triclad Turbellaria, 571.
JOHNSON, C. See L. H. KLEINHOLZ, 317.
JOHNSON, L. G. See F. A. BROWN, JR., 488
(abstract).
JONES, F. E. See C. V. HARDING, 499 (ab-
stract).
J^AMINER, B. Effects of heavy water,
glycerol and sucrose on glycerol-extracted
muscle, 466 (abstract).
Karyoplasmic studies of California newts, 253.
KATZEN, H. M. See DE\VITT STETTEN, JR.,
465 (abstract).
KEOSIAN, J. Factors in the effects of radiation
on the growth rate and conidiation in
Neurospora, 500 (abstract).
KEPLER, C. See C. P. CHANNING, 480 (ab-
stract).
KERESZTES-NAGY, S. See I. M. KLOTZ, 484
(abstract).
KETCHUM, B. H. See E. J. KUENZLER, 134.
KIMBALL, F. See L. H. KLEINHOLZ, 317.
KIVY-ROSENBERG, E. The effect of time of
insemination on the development of
Fundulus, 500 (abstract).
KIVY-ROSENBERG, E., F. RAY AND N. PASCOE.
Krebs and pentose cycle dehydrogenase
systems in the gametes of Asterias as
measured with a tetrazolium salt, INT,
501 (abstract).
INDEX
677
KLEIN, D. L. See D. C. BRYANT, 479 (ab-
stract).
KI.EINHOLZ, L. H., H. ESPER, C. JOHNSON AND
F. KIMBALL. Neurosecretion and crus-
stacean retinal pigment hormone: assay
and properties of the light-adapting
hormone, 317.
KLOTZ, I. M., AND S. KERESZTES-NAGY.
Hemerythrin: dissociation into subunits
and reconstitution, 484 (abstract).
KRANE, S. M., AND L. LASTER. The incorpora-
tion of nicotinamide-7-C14 into pyridine
nucleotides of intact eggs and embryos of
Spisula, 501 (abstract).
Krebs-cycle enzymes of oyster embryo, 58.
KUENZLER, E. J., AND B. H. IvETCHUM. Rate
of phosphorus uptake by Phaeodactylum,
134.
J^AND crab, biology of, 207.
LANDERS, W. S., AND R. C. TONER. Survival
and movements of the flatworm Stylochus
in different salinities and temperatures,
146.
Larval development of Calcinus, 179.
LASHER, R., AND R. RUGH. The "Hertwig
Effect" in teleost development, 582.
LASTER, L., AND R. K. CRANE. Triphospho-
pyridine nucleotide formation and disap-
pearance in the presence of extracts of eggs,
embryos and adult liver of Spisula, 502
(abstract).
LASTER, L. See S. M. KRANE, 501 (abstract).
LAUFER, H., AND T. MCNAMARA. Blood pro-
tein changes in Crustacea, 519 (abstract).
LAZAROW, A. See D. WATKINS, 469; A. W.
LINDALL, JR., 503 (abstracts).
Length of stem, in relation to oxygen uptake
of Tubularia, 450.
Lens of dogfish and skate, metabolic pathways
in, 502 (abstract).
LERMAN, S., J. FONTAINE AND K. WOODSIDE.
Metabolic pathways in the dogfish and
skate lens, 502 (abstract).
Leucine aminopeptidase activity of triclad
Turbellaria, 571.
LEWIS, H. W. A comparative study of dopa
oxidase systems in marine invertebrates,
503 (abstract).
LEWIS, H. W. Structural and control genes
regulating dopa oxidase activity in Droso-
phila, 464 (abstract).
Libinia, retinal pigment hormone of, 317.
LIEBMAN, P. A. See W. A. HAGINS, 498
(abstract).
Life-cycle of Cardisoma, 207.
Life-history of Calcinus, 179.
Light, effect of on mysids, 562.
Light, polarized, behavior of Daphnia in
243.
Light, role of in migratory restlessness of bobo-
link, 542.
Light, role of in responses of crustacean eye,
121.
Light, role of in testicular cycle of bobolink, 94.
Light, role of in uptake of phosphorus by
Phaeodactylum, 134.
Light-adapting hormone of crustaceans, assay
and properties of, 317.
Light production of firefly, neural activity
during, 490 (abstract).
Light relations in eye of Daphnia, 233.
Limb regeneration of Gecarcinus during molt
cycle, 635.
Limulus eye, electrophysiology of, 618.
LINDALL, A. W., JR., AND A. LAZAROW. Sepa-
ration of an insulin-containing fraction
from the islet of the goosefish, 503 (ab-
stract).
Lipase of triclad Turbellaria, 571.
Lipoyl dehydrogenase from dogfish liver, 480
(abstract).
Locomotion of Stylochus at different salinities
and temperatures, 146.
Loligo, mating behavior of, 53.
Lophophore orientation of Discinisca, 597.
LORAND, L. See R. F. DOOLITTLE, 481 (ab-
stract).
Low temperature, effect of on movements of
flatworm, 146.
Low temperature, effect of on oxygen consump-
tion of Orchomonella, 225.
Lunar periodicity of spawning in crab, 207.
Lychas, reproductive biology of, 344.
Lysin, effect of on Mytilus egg, 531.
Lysine, uptake of by Fungia, 648.
^/JAGNETIC fields, response of Dugesia and
Paramecium to, 264.
MAIRS, D. F., AND C. J. SINDERMANN. A
serological comparison of five species of
Atlantic clupeoid fishes, 330.
Malic dehydrogenases of developing Arbacia
embryos, 505 (abstract).
MALNIC, G. See J. W. HASTINGS, 483 (ab-
stract).
MARSCHHAUS, C. M. See G. W. DE VILLA-
FRANCA, 464 (abstract).
MARSLAND, D., A. M. ZIMMERMAN AND H.
ASTERITA. Effects of D2O on the cortical
gel structure and cleavage capacity of
Arbacia eggs, 484 (abstract).
MARTIN, D. See C. L. PROSSER, 485 (ab-
stract).
MASSEY, V. See C. P. CHANNING, 480 (ab-
stract).
678
INDEX
MATHEW, A. P. Reproductive biology of
Lychas, 344.
Mating behavior in Loligo, 53.
McCANN, F. V., AND D. W. MILLER, JR.
Intracellular cardiac potentials in Limulus
during ganglionic stimulation, 504 (ab-
stract).
McCANN, F. V. See D. W. MILLER, JR., 504
(abstract).
McNAMARA, J. J., G. SZABO AND R. T. SlMS.
The nature of the pigments in the integu-
ment and eye of the hermit crab, Pagurus,
504 (abstract).
McNAMARA, T. See H. LAUFER, 519 (ab-
stract).
Mechanism, photoreceptor, of retinula cells,
618.
Megaptera, body temperatures of, 154.
Melanin biosynthesis in squid ink sac, 513
(abstract).
Melanocyte dispersion, influence of brain
lesions on, 491 (abstract).
Melanophores in hybrid California newts, 253.
Membrane potentials of crayfish muscle fibers,
496 (abstract).
MENDOZA, G. The reproductive cycles of
three viviparous teleosts, Alloophorus,
Goodea and Neoophorus, 351.
Menhaden, serology of, 330.
MENZEL, R. W. See A. N. SASTRY, 388.
Mercaptoethanol, effects of on cleavage in
Arbacia, 518 (abstract).
Mercenaria, burrowing in, 521.
Metabolism of crustacean integument during
molt cycle, 635.
Metabolism of Fungia, 648.
Metabolism of Orchomonella, 225.
Metabolism of Tubularia stems, 450.
Metachromasia of Pectinaria egg, 424.
Metamorphosis of Amaroucium, contraction
of epidermis during, 492 (abstract).
METZ, C. B. See pp. 470-474 (abstracts).
Migrant birds, restlessness in, 542.
Migrations of Cardisoma, 207.
Migrations of Cerithium, 170.
MILKMAN, R. A common mechanism for
temperature adaptation and crossvein de-
formation in Drosophila, 462 (abstract).
MILLER, A. T., JR. See P. J. OSBORNE, 589.
MILLER, D. W., JR., AND F. V. McCANN.
Action potentials in single cells of a
tunicate heart, 504 (abstract).
MILLER, D. W., JR. See F. V. McCANN, 504
(abstract).
MILLER, J. A., JR., L. L. PHILPOTT AND C. L.
CLAFF. Oxygen uptake in short pieces of
Tubularia stems, 450.
MILLER, R. L., AND L. NELSON. Evidence of
a chemotactic substance in the female
gonangium of Campanularia, 477 (ab-
stract).
MINGANTI, A. Experiments on interspecific
fertilization between Ciona, Styela and
Molgula, 505 (abstract).
MITCHISON, J. M. See P. R. GROSS, 467
(abstract).
Mitochondria of Pectinaria egg, 424.
Mitosis, effect of proflavin on, 132.
Models of optics of Daphnia eye, 233.
Molecular size of Phacoides hemoglobin, 605.
Mollusc, electrical induction of spawning in,
203.
Mollusc, hemoglobin in, 605.
Mollusc, intertidal clustering of, 170.
Mollusc, mating behavior of, 53.
Mollusc egg, vitelline coat of, 531.
Mollusc embryos, respiration and enzymes of,
58, 71.
Molluscs, burrowing in, 521.
Molt cycle of crab, proprioceptor responses
during, 660.
Molt cycle, crustacean, structure and metabo-
lism of integument during, 635.
MONROY, A., AND L. VlTTORELLI. On the
utilization of C14 from glucose for amino
acids and protein synthesis by the sea
urchin embryo, 465 (abstract).
MONROY, A. See T. EVANS, 476; R. J. PFOHL,
477 (abstracts).
MOORE, R. O., AND C. A. VILLEE. Malic
dehydrogenases of developing Arbacia
embryos, 505 (abstract).
MORAN, J. F., JR. Studies on the isolated
islet tissue of the toadfish (Opsanus) :
aldolase content of islet and other tissues,
505 (abstract).
Morphology of brachiopod, 597.
Morphology of Calcinus larvae, 179.
Morphology of Cardisoma, 207.
MORRILL, J. B., JR., AND E. N. Dow. Organ
and ontogenetic patterns of multiple forms
of hydrolytic enzymes in Limnaea, 463
(abstract).
MORRISON, P. Body temperatures in some
Australian mammals. III., 154.
MOULTON, J. M. Intertidal clustering of an
Australian gastropod, 170.
Movements of flatworm at different salinities
and temperatures, 146.
MUN, A. M., P. TARDENT, J. ERRICO, J. D.
EBERT, L. E. DELANNEY AND T. S.
ARGYRIS. An analysis of the initial reac-
tion in the sequence resulting in homol-
ogous splenomegaly in the chick embryo,
366.
INDEX
679
MURRAY, R. W. Long refractory periods of
branchial sensory nerve endings in dog-
fish, 506 (abstract).
MUSACCHIA, X. J., AND D. D. WESTHOFF.
Nitrogen inhibition of active absorption
of D-glucose in fish intestine, 506 (ab-
stract).
MUSACCHIA, X. J., AND D. D. WESTHOFF.
Phlorizin inhibition of active absorption
of D-glucose in fish intestine, 507 (ab-
stract).
Muscle, glycerol-extracted, effects of heavy
water, glycerol and sucrose on, 466
(abstract).
Muscle, Limulus, A and I bands in, 464 (ab-
stract).
Mussel egg, vitelline coat of, 531.
Mutants of Salmonella, patterns of chemically
induced reversions among, 486 (abstract).
MUTTERPERL, E. See A. L. BURNETT, 489
(abstract).
Mytilus, electrical induction of spawning in,
203.
Mytilus egg, vitelline coat of, 531.
NAGABHUSHANAM, R. See M. FINGER-
MAN, 121.
NELSON, L. Actin localization in sperm, 468
(abstract).
NELSON, L. See A. APPLEGATE, 475 ; R. L.
MILLER, 477; K. M. PLOWMAN, 478
(abstracts).
Neomysis, spectral sensitivity and phototaxis
in, 562.
Neoophorus, reproductive cycle of, 351.
Nerve physiology of Limulus eye, 618.
Neuromuscular physiology of crab, 479 (ab-
stract).
Neurophysiology of Sesarma, 660.
Neurosecretion and crustacean retinal pigment
hormone, 317.
Neurosecretory cells, toadfish, electron micros-
copy of, 461 (abstract).
Neurospora, factors in effects of radiation on,
500 (abstract).
NEWMAN, M. B. See C. V. HARDING, 499
(abstract).
Newts, karyoplasmic studies of, 253.
Nocturnal activity of caged bobolinks, 542.
NOVIKOFF, A. B. Golgi apparatus and ly-
sosomes in vertebrate neurons, 465 (ab-
stract).
Nucleolus of Pectinaria egg, 424.
Nucleotide formation and disappearance in
presence of Spisula eggs, embryos and
adult liver, 502 (abstract).
Nucleotides of Spisula eggs and embryos, 501
(abstract).
Nucleus vs. cytoplasm in hybrid newts, 253.
Nutrient transport in isolated digestive glands
of Asterias, 482 (abstract).
Nutrition of Strongylocentrotus, 105.
NYBORG, W. L. See W. L. WILSON, 518
(abstract).
QCHETOSTOMA, feeding mechanism of, 80.
Ochetostoma, oxygen uptake of, 86.
OLIVO, R. F. See E. E. CLARK, 491 (abstract).
Opossum shrimp, spectral sensitivity and
phototaxis in, 562.
Optics of Daphnia eye, 233.
Orconectes, photomechanical responses of
proximal pigment of, 121.
Organic material, uptake of by aquatic in-
vertebrates, 648.
Orientation of snails, 514 (abstract).
Orientation of snails, as affected by radiation,
488 (abstract).
Orthodemus, feeding and digestion in, 571.
OSBORNE, P. J., AND A. T. MlLLER, JR. Up-
take and intracellular digestion of protein
(peroxidase) in planarians, 589.
Osmotic relations of Tetrahymena, 462 (ab-
stract).
Ova of Pectinaria, cytological studies of, 424.
Ova, teleost, development of, after x-irradia-
tion of sperm, 582.
Ovum, Mytilus, vitelline coat of, 531.
Oxygen-combining properties of Phacoides
hemoglobin, 605.
Oxygen consumption of Fungia, 648.
Oxygen consumption of Gecarcinus integument,
during molt cycle, 635.
Oxygen consumption of Orchomonella, 225.
Oxygen uptake of Ochetostoma, 86.
Oxygen uptake of Tubularia stems, 450.
Oyster embryos, respiration and enzymes of,
58, 71.
Oyster predator, survival of at different salini-
ties and temperatures, 146.
pAINE, R. T. Filter-feeding pattern and
local distribution of the brachiopod,
Discinisca, 597.
Palaemonetes, photomechanical responses of
proximal pigment of, 121.
Palaemonetes, retinal pigment hormone of, 317.
PALMER, J. D. A persistent diurnal photo-
tactic rhythm in the fiddler crab, Uca, 507
(abstract).
PALMER, J. D., C. S. YENTSCH AND S. A.
DERopp. The persistence of a biological
rhythm in continuous light illumination,
508 (abstract).
Pancreas islet cell membranes, permeability of,
469 (abstract).
680
INDEX
Pancreatic islet tissue of toadfish, aldolase
content of, 505 (abstract).
Pancreatic islet tissue of toadfish, uptake of
glucose by, 496 (abstract).
Paramecium, response of to very weak hori-
zontal magnetic fields, 264.
PARPART, A. K., AND T. V. N. BALLENTINE.
Contrasts in activation of the egg of
Arbacia, 485 (abstract).
PARPART, A. K., AND T. V. N. BALLENTINE.
Gel-sol transformations in the unfertilized
egg of Arbacia, 508 (abstract).
Parthenogenesis in Fundulus eggs, 582.
PASCOE, N. See E. KIVY-ROSENBERG, 501
(abstract).
Pectinaria eggs, cytological studies of, 424.
Penshells, influence of on behavior of com-
mensal Pinnotheres, 388.
Periodicity, lunar, of spawning in crab, 207.
Peristalsis in echiuroid, 86.
Perivisceral fluid of sea urchin, role of in nutri-
tion, 105.
Permeability, chloride, of crayfish muscle fibers,
509 (abstract).
Permeability of dogfish erythrocyte, effect of
phenothiazine derivatives on, 495 (ab-
stract).
Peroxidase, digestion of by planarians, 589.
PFOHL, R. J., AND A. MONROY. Changes in
some proteins in the course of development
of Arbacia, 477 (abstract).
PFOHL, R. J., AND A. MONROY. Electro-
phoretic and ultracentrifugal analysis of
the fractionated extracts of Arbacia eggs
and early plutei, 477 (abstract).
Phacoides, hemoglobin of, 605.
Phaeodactylum, phosphorus uptake by, 134.
Phagocytosis in planarians, 571.
Pharmacology of Busycon radula protractor,
499 (abstract).
Pharyngeal feeding in planarians, 589.
PHILLIPS, J. H. See A. FARMANFARMAIAN, 105.
PHILPOTT, D. E., AND A. B. CHAET. Electron
microscopic observations of secretory
granules in the adhesive surface of Hydra,
509.
PHILPOTT, L. See M. FINGERMAN, 121.
PHILPOTT, L. L. See J. A. MILLER, JR., 450.
Phosphatases of triclad Turbellaria, 571.
Phosphatide composition of sea anemones, 485
(abstract).
Phosphorus uptake of Phaeodactylum, 134.
Photomechanical responses of proximal pigment
in Crustacea, 121.
Photoperiod, role of in migratory restlessness of
bobolinks, 542.
Photoreceptor mechanisms of retinula cells, 618.
Photorefractoriness in bobolinks, 94.
Photosensitive pigment of starfish, 510 (ab-
stract).
Phototaxis in Neomysis, 562.
PlANFETTI, N., J. HlCKMAN AND R. C. SAN-
BORN. Chromatophores of decapod Crus-
tacea in hypodermal organ culture, 509
(abstract).
PIATIGORSKY, J., AND C. R. AUSTIN. Rela-
tionship of fertilizin to the acrosome reac-
tion in Arbacia, 473 (abstract).
PIATIGORSKY, J. See C. R. AUSTIN, 470
(abstract).
Pigment concentration in proflavin-treated
Arbacia eggs, 132.
Pigment of crustacean eye, photomechanical
responses of, 121.
Pigment hormone, crustacean retinal, assay
and properties of, 317.
Pigment patterns in hybrid California newts,
253.
Pigmentation of Cardisoma, 207.
Pigments in Pagurus integument and eye,
nature of, 504 (abstract).
Pigments of Phacoides, 605.
Pinnotheres, influence of hosts on behavior of,
388.
Planarian, response of to electrostatic fields,
282.
Planarian, response of to very weak horizontal
magnetic fields, 264.
Planarians, digestion of protein by, 589.
Plasma membrane of Mytilus egg, 531.
Platyhelminth, digestion of protein by, 589.
Platyhelminths, feeding and digestion in, 571.
PLOWMAN, K. M., AND L. NELSON. An actin-
like protein isolated from starfish sperm,
478 (abstract).
Polarized light, analysis of in eye of Daphnia,
233.
Polarized light, behavior of Daphnia in, 243.
Polycelis, feeding and digestion in, 571.
Polychaete, development of, 412.
Polychaete, reproduction of, 396.
Populations of Artemia, survival of in radio-
active sea water, 302.
Potential change, response of Dugesia to, 282.
Potentials, cardiac action, in tunicate heart,
504 (abstract).
Potentials, intracellular cardiac, of Limulus,
504 (abstract).
Prawn, photomechanical responses of proximal
pigment of, 121.
Precipitin tests of clupeoid fishes, 330.
Proboscis of echiuroid, role of in oxygen uptake,
86.
Proflavin, effects of on development of sea
urchin, 132.
INDEX
681
Properties of crustacean retinal pigment hor-
mone, 317.
Proprioceptor responses during molt cycle in
crab, 660.
PROSSER, C. L., D. MARTIN AND R. SHA'AFI.
Separation of phasic and tonic contractions
in Spisula intestine, 485 (abstract).
Protein, digestion of by planarians, 589.
Protein changes in ageing lobster, 490 (ab-
stract).
Protein changes in crustacean blood, 519
(abstract).
Proteins in Arbacia development, 477 (ab-
stract).
Protozoan, ciliate, regeneration studies on, 467
(abstract).
Protozoan, response of to very weak horizontal
magnetic fields, 264.
PROVENZANO, A. J., JR. The larval develop-
ment of Calcinus in the laboratory, 179.
Proximal pigment of Crustacea, photomechan-
ical responses of, 121.
t> NA, "messenger," and cell cycle in a fission
yeast, 467 (abstract).
Radiocarbon, use of in study of sea urchin
nutrition, 105.
Radiocarbon-labelled glucose, uptake of by
Fungia, 648.
Radiophosphorus, effects of on Artemia popu-
lations, 302.
Radiophosphorus uptake of Phaeodactylum,
134.
Radiozinc, effect of on Artemia populations,
302.
Rana embryos, anaerobic glycolysis in, 555.
RAPPORT, M. M., AND E. L. GOTTFRIED. On
the phosphatide composition of sea
anemones, 485 (abstract).
Rate of phosphorus uptake by Phaeodactylum,
134.
RAY, F. See E. KIVY-ROSENBERG, 501 (ab-
stract).
READ, K. R. H. The hemoglobin of the
bivalved mollusc Phacoides, 605.
Reconstitution in Tubularia, oxygen uptake
during, 450.
Recrudescence of testes in bobolinks, 94.
Regeneration in Tubularia, inhibition of, 513
(abstract).
Reproduction of Glycera, 396.
Reproductive biology of Lychas, 344.
Reproductive cycles of three teleosts, 351.
Reproductive performance of irradiated Ar-
temia populations, 302.
Respiration of Ochetostoma, 86.
Respiration of oyster eggs and embryos, 58.
Response of planarian to electrostatic fields,
282.
Response of planarian and Paramecium to very
weak horizontal magnetic fields, 264.
Responses from proprioceptor of crab during
molt cycle, 660.
Restlessness, migratory, in bobolinks, 542.
Retina of squid, light-induced pigment migra-
tion in, 498 (abstract).
Retinal pigment hormone, assay and proper-
ties of, 317.
Retinal pigments of Palaemonetes and Or-
conectes, 121.
Retinula cells, photoreceptor mechanism of,
618.
REUBEN, J. P., L. GIRARDIER AND H. GRUND-
FEST. The chloride permeability of cray-
fish muscle fibers, 509 (abstract).
REUBEN, J. P., L. GIRARDIER AND H. GRUND-
FEST. Water transport and membrane
structure in crayfish muscle fibers, 469
(abstract).
REUBEN, J. P. See L. GIRARDIER, 468, 496
(abstracts).
Rhabdomeres of Limtilus eye, 618.
Rhythm in alga, persistence of in continuous
light illumination, 508 (abstract).
Rhythmical clustering of Cerithium, 170.
RIESER, P. Amino acid transport in the
human erythrocyte: kinetics and mecha-
nism, 461 (abstract).
ROCKSTEIN, M. Some properties of stellarin,
the photosensitive pigment of the starfish,
Asterias, 510 (abstract).
Roentgen irradiation of Artemia populations,
302.
DERopp, S. A. See J. D. PALMER, 508 (ab-
stract).
ROSNER, J. L. Patterns of chemically induced
reversions among mutants of Salmonella,
486 (abstract).
ROTHSTEIN, H. See C. V. HARDING, 499
(abstract).
RUCK, P. On photoreceptor mechanisms of
retinula cells, 618.
RUGH, R. Some effects of ionizing radiations
on the embryo, 461 (abstract).
RUGH, R. See R. LASHER, 582.
RUSTAD, R. C. Ultraviolet damage to the
cortex of the sea urchin egg, 510 (abstract).
CALINITY, effect of on development of
Calcinus larvae, 179.
Salinity, effect of on survival and movements
of flatworm, 146.
SANBORN, R. C. See X. PIANFETTI, 509
(abstract).
682
INDEX
SASTRY, A. N., AND R. W. MENZEL. Influence
of hosts on the behavior of the commensal
crab Pinnotheres, 388.
.Scallops, influence of on behavior of commensal
Pinnotheres, 388.
SCHARRER, E. Electron microscopy of neuro-
secretory cells in the preoptic nucleus of the
toadfish (Opsanus), 461 (abstract).
SCHNEIDER, R. See C. G. WILBER, 517
(abstract).
Scorpion, reproductive biology of, 344.
SCOTT, G. T., R. L. CLARK AND J. C. HICKMAN.
Drugs causing localized lightening and
darkening of the common sand dab,
Scophthalamus, 511 (abstract).
SCOTT, G. T., R. L. CLARK AND J. C. HICKMAN.
Mechanism of chromatophore control
in the common sand flounder Scophthala-
mus, 486 (abstract).
SCOTT, G. T. See R. L. CLARK, 491 (abstract).
Sea urchin development, effect of proflavin on,
132.
Sea urchin, nutrition of, 105.
Self-fertility of Mytilus hermaphroditic gam-
etes, 203.
SENFT, A. See T. EVANS, 476 (abstract).
Sensitivity, spectral, of Neomysis, 562.
Serology of clupeoid fishes, 330.
Sesarma, proprioceptor responses of, during
molt cycle, 660.
Sexual activities of squid, 53.
Shad, serology of, 330.
SHA'AFI, R. See C. L. PROSSER, 485 (abstract).
SHIVERS, C. A., AND T. E. EVANS. Induce-
ment of the "acrosome reaction" by acri-
dine orange, 473 (abstract).
SHIVERS, C. A., AND C. B. METZ. Localization
of sperm antigens by dissociation of anti-
gen-antibody precipitates, 474 (abstract).
Shrimp, opossum, spectral sensitivity and
phototaxis in, 562.
SICHEL, F. J. See W. L. WILSON, 518 (ab-
stract).
SIMPSON, M. Gametogenesis and early devel-
opment of the polychaete Glycera, 412.
SIMPSON, M. Reproduction of the polychaete
Glycera at Solomons, Maryland, 396.
SIMS, R. T. See J. J. MCNAMARA, 504; G.
SZABO, 513 (abstracts).
SINDERMANN, C. J. See D. F. MAIRS, 330.
Sites of oxygen uptake in Ochetostoma, 86.
Size of stem, in relation to oxygen uptake of
Tubularia stems, 450.
SKINNER, D. M. The structure and metabo-
lism of a crustacean integumentary tissue
during a molt cycle, 635.
Solitary coral, glucose uptake by, 648.
Spawning, electrical induction of, in Urechis
and Mytilus, 203.
Spawning of Cardisoma, 207.
Spawning of Mytilus, induction of, 531.
Spawning of Saccoglossus, induction of, 493
(abstract).
Species differences in California newts, 253.
Spectral sensitivity and phototaxis in the
opossum shrimp, Neomysis, 562.
SPEIDEL, C. C., AND R. H. CHENEY. Resist-
ance to gamma irradiation of fertilized
eggs of Arbacia correlated with their stage
of development, 511 (abstract).
SPEIDEL, C. C., AND R. H. CHENEY. Time-
lapse motion pictures of intracellular dis-
turbances induced in Arbacia zygotes after
ultraviolet or x-ray irradiation of zygotes,
both gametes, or one gamete, 463 (ab-
stract).
Sperm, actin localization in, 468 (abstract).
Sperm, Arbacia, action of neuraminidase on,
471 (abstract).
Sperm, Arbacia, antigens of, 472 (abstract).
Sperm, Fundulus, x-irradiation of, 582.
Sperm, Mytilus, acetylcholinesterase in, 475
(abstract).
Sperm, oyster, axial body and filament forma-
tion in, 474 (abstract).
Sperm, passage of through Ciona egg chorion,
472 (abstract).
Sperm, starfish, actin-like protein from, 478
(abstract).
Sperm antigens, localization of, 474 (abstract).
Sperm entry into sea urchin oocytes, electron
microscopy of, 473 (abstract).
Sperm extracts, Arbacia, immunological iden-
tification of egg agglutinin in, 471 (ab-
stract).
Sperm lysin, effect of on Mytilus egg, 531.
Sperm penetration of Arbacia eggs, role of
jelly-splitting agent in, 470 (abstract).
SPIRTES, M. See A. R. FREEMAN, 495 (ab-
stract).
SPOON, D. H., A. FORER AND C. R. AUSTIN.
Axial body and filament formation in
oyster sperms, 474 (abstract).
Squid, mating behavior of, 53.
Social structure in Loligo, 53.
SPURDICH, J. A. See J. W. HASTINGS, 483
(abstract).
STEPHENS, G. C. Amino acids in the economy
of the bamboo worm, Clymenella, 512
(abstract).
STEPHENS, G. C. Uptake of amino acids by
the bamboo worm, Clymenella, 512 (ab-
stract).
STEPHENS, G. C. Uptake of organic material
by aquatic invertebrates. I., 648.
STETTEN, DEWITT, JR., H. M. KATZEN AND
F. TIETZE. Reversible enzymatic reduc-
tion of insulin, 465 (abstract).
INDEX
683
STONE, \Y., JR., AND W. C. DEWEL. Osmotic
pressure relationships in the spiny dogfish
Squalus, 513 (abstract).
Storage of nutrients in Strongylocentrotus, 105.
Strongylocentrotus, nutrition of, 105.
Strontium utilization by Arbacia larvae, 497
(abstract).
Structure of crustacean integument during
molt cycle, 635.
Structure of Mytilus egg vitelline coat, 531.
Stylochus, survival and movements of, at dif-
ferent salinities and temperatures, 146.
Substratum, role of in burrowing of Veneridae,
521.
SUGIURA, Y. Electrical induction of spawning
in two marine invertebrates, 203.
Surface area, in relation to oxygen uptake of
Tubularia stems, 450.
SURGENOR, D. M. See F. A. BELAMARICH, 479
(abstract).
Survival of Artemia populations in radioactive
sea water, 302.
Survival of flatworm at different salinities and
temperatures, 146.
Swarming of Glycera, 396.
Swimbladder of fish, gas absorption from, 491
(abstract).
SZABO, G., AND R. T. SIMS. Studies of mel-
anin biosynthesis in the ink sac of the
squid (Loligo). II, 513 (abstract).
SzAB6, G. See J. J. MCNAMARA, 504 (ab-
stract).
SZENT-GYORGYI, A., AND A. HEGYELI. On the
chemistry of the thymus gland, 466
(abstract).
'p ARDENT, P. See A. M. MUN, 366.
Taxonomy of clupeoid fishes, 330.
Teleost development, "Hertwig Effect" in,
582.
Teleosts, reproductive cycles of, 351.
Temperature, effect of on survival and move-
ments of flatworm, 146.
Temperature, role of in glucose uptake by
Fungia, 648.
Temperature, role of in migratory restlessness
of bobolinks, 542.
Temperature and oxygen consumption of
Orchomonella, 225.
Temperature-shock treatment of newts, 253.
Temperatures, body, of whales, 154.
Termination of photorefractoriness in bobolink,
94.
Terrestrial magnetism, response of Dugesia and
Paramecium to, 264.
Testicular cycle of bobolink, 94.
Tetrahymena, survival of at elevated oxygen
pressures, 495 (abstract).
Tetrahymena growth and respiration, effect
of carbon dioxide on, 487 (abstract).
Thrombocytes of Mustelus, 479 (abstract).
Thymidine, incorporation of into pool and
DNA of deuterated sea urchin eggs, 497
(abstract).
Thymus, electron microscope study of, 515
(abstract).
Thymus gland, chemistry of, 466 (abstract).
Tide, role of in clustering of Cerithium, 170.
TIETZE, F. See DE\YITT STETTEN, JR., 465
(abstract).
TONER, R. C. See W. S. LANDERS, 146.
TORCH, R. Regeneration studies on a brack-
ish-water ciliate, Tracheloraphis, 467 (ab-
stract).
Transequatorial bird migrant, annual testic-
ular cycle of, 94.
Transequatorial bird migrant, Zugunruhe in,
542.
Translocation of nutrients in Strongylocentro-
tus, 105.
TRAVIS, D. M. Effect of carbon dioxide on
gas exchange in Thyone, 487 (abstract).
TRAVIS, D. M., A. M. ELLIOTT AND I. J. BAK.
Carbon dioxide inhibition of growth and
respiration in Tetrahymena, 487 (ab-
stract).
TRAVIS, D. M. See A. M. ELLIOTT, 495
(abstract).
Triclad Turbellaria, feeding and digestion in,
571.
Tritium-labelled grafts in chick embryo, 366.
Trochophores of oyster, respiration and en-
zymes of, 58, 71.
Trypsin and trypsinogen of dogfish, 479
(abstract).
Tube-dwelling echiuroid, feeding of, 80.
Tubularia stems, oxygen uptake of, 450.
Turbellaria, feeding and digestion in, 571.
TWEEDELL, K. S. Cytological studies during
germinal vesicle breakdown of Pectinaria
with vital dyes, centrifugation and fluores-
cence microscopy, 424.
TWEEDELL, K. S. Inhibition of regeneration
in Tubularia by tissue extract injection,
513 (abstract).
TJLTRASONIC treatment of marine eggs,
518 (abstract).
Uptake and intracellular digestion of protein
in planarians, 589.
Uptake of organic material by aquatic inverte-
brates, 648.
Uptake of oxygen by Tubularia stems, 450.
Uptake of phosphorus by Phaeodactylum, 134.
Urechis, electrical induction of spawning in,
203.
684
INDEX
HOLDE, K. E. See L. B. COHEN, 480;
H. A. DEPHILLIPS, JR., 481 (abstracts).
VEEGAR, C. See C. P. CHANNING, 480 (ab-
stract).
Veneridae, burrowing in, 521.
Venerupis, burrowing in, 521.
Venus, burrowing in, 521.
DE VlLLAFRANCA, G. W., AND C. M. MARSCH-
HAUS. The A and I bands in contracting
Limulus muscle, 464 (abstract).
VILLEE, C. A. See R. O. MOORE, 505 (ab-
stract).
Vital staining of Pectinaria eggs, 424.
Vitelline coat of Mytilus eggs, 531.
VITTORELLI, L. See A. MONROY, 465 (ab-
stract).
Viviparous teleosts, reproductive cycles of,
351.
Volume of tissue, in relation to oxygen uptake of
Tubularia stems, 450.
\A/ATER transport in crayfish muscle fibers,
469 (abstract).
Water vascular system of Strongylocentrotus,
105.
WATKINS, D., S. J. COOPERSTEIN AND A.
LAZAROW. Studies on the mechanism by
which alloxan alters the permeability of
islet cell membranes to mannitol, 469
(abstract).
WAITERS, C. D. Analysis of motility in a new
species of gregarine, 514 (abstract).
WEBB, H. M., AND F. H. BARNWELL. Seasonal
fluctuations in mean paths of snails
(Nassarius) in a uniform light field, 514
(abstract).
WEBB, H. M. See F. A. BROWN, JR., 488
(abstract).
Weight, with respect to oxygen consumption
of Orchomonella, 225.
Weight changes in caged bobolinks, 542.
WEINSTEIN, R. S. See D. C. BRYANT, 479
(abstract).
WEISS, L. Studies on the structure of the
thymus. I., 515 (abstract).
WESTHOFF, D. D. See X. J. MUSACCHIA, 506,
507 (abstracts).
Whales, body temperatures of, 154.
WHEELER, M. B., C. V. HARDING, W. L.
HUGHES AND W. L. WILSON. The incor-
poration of iododeoxyuridine by the
developing Arbacia embryo, 515 (ab-
stract).
WICHTERMAN, R. Studies on Euplotes. I,
II, 516 (abstracts).
\YlERCINSKI, F. J., AND C. E. WlERCINSKI.
A comparison of methods using Ca46 as a
tracer for calcium activity in Arbacia
eggs, 517 (abstract).
WIERCINSKI, F. J. See W. L. WILSON, 518
(abstract).
WlLBER, C. G., AND R. SCHNEIDER. The
growth of brain in teleosts, 517 (abstract).
WILSON, W. L., F. J. WIERCINSKI, W. L.
NYBORG AND F. J. SICHEL. Observations
on marine eggs subjected to ultrasonic
vibration, 518 (abstract).
WILSON, W. L. See M. B. WHEELER, 515
(abstract).
WINTERS, R. W. Intracellular pH in Arbacia
eggs, 519 (abstract).
WOODSIDE, K. SeeS. LERMAN, 502 (abstract).
^-IRRADIATION, effect of on Artemia
populations, 302.
X-irradiation of teleost sperm, effect of on
development of eggs, 582.
yENTSCH, C. S. See J. D. PALMER, 508
(abstract).
YULES, R. B. Responses from a propriocep-
tive organ of the crab, Sesarma, during the
molt cycle, 660.
£ I M MERMAN, A. M. DNA synthesis in
early mitotic stages: a pressure study, 518
(abstract).
ZIMMERMAN, A. M. The effects of mercapto-
ethanol on cleaving eggs of Arbacia, 518
(abstract).
ZIMMERMAN, A. M. See D. MARSLAXD, 484
(abstract).
Zoeal stages of Calcinus, 179.
Zugunruhe in bobolinks, 542.
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