THE
BIOLOGICAL BULLETIN
PUBLISHED BY
*
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
DAVID W. BISHOP, Carnegie Institution of V. L. LOOSANOFF, U. S. Fish and Wildlife
Washington Service
HAROLD C. BOLD, University of Texas C. L. PROSSER, University of Illinois
FRANK A. BROWN, JR., Northwestern University BERTA SCHARRER, Albert Einstein College of
JOHN B. BUCK, National Institutes of Health Medicine
LIBBIE H. HYMAN, American Museum of FRANZ SCHRADER, Duke University
Natural History WM. RANDOLPH TAYLOR, University of Michigan
J. LOGAN IRVIN, University of North Carolina CARROLL M. WILLIAMS, Harvard University
DONALD P. COSTELLO, University of North Carolina
Managing Editor
VOLUME 119
AUGUST TO DECEMBER, i960
Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE £ LEMON STS.
LANCASTER, PA.
ii
THE BIOLOGICAL BULLETIN is issued six times a year at the
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sylvania.
Subscriptions and similar matter should be addressed to The
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Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London,
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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, 1960
PAGE
Annual Report of the Marine Biological Laboratory 1
ALDRICH, DAVID V., AND WILLIAM B. WILSON
The effect of salinity on growth of Gymnodinium breve Davis 57
BROWN, F. A., JR., M. F. BENNETT AND H. M. WEBB
A magnetic compass response of an organism 65
CAMPBELL, JAMES W.
The occurrence of /3-alanine and j3-aminoisobutyric acid in flatworms. . 75
( ".OODBODY, IVAN
The feeding mechanism in the sand dollar Mellita sexiesperforata (Leske) 80
HARVEY, ETHEL BROWNE
Cleavage with nucleus intact in sea urchin eggs 87
MUN, A. M., AND I. L. KOSIN
Developmental stages in the broad breasted bronze turkey embryo. ... 90
NADAKAL, A. M.
Carotenoid and chlorophyllic pigments in the marine snail, Cerithidea
californica Haldeman, intermediate host for several avian trematodes. . 98
PRITCHARD, AUSTIN W., AND AUBREY GORBMAN
Thyroid hormone treatment and oxygen consumption in embryos of the
spiny dogfish 109
READ, C. P., J. E. SIMMONS, JR., J. W. CAMPBELL AND A. H. ROTHMAN
Permeation and membrane transport in parasitism : studies on a tape-
worm-elasmobranch symbiosis 120
RIZKI, M. T. M.
Pigmented fat cells in a mutant of Drosophila melanogaster 134
TURNER, HARRY J., JR., AND JAMES E. HANKS
Experimental stimulation of gametogenesis in Hydroides dianthus and
Pecten irradians during the winter 145
WIGLEY, ROLAND L.
A new species of Chiridotea (Crustacea : Isopoda) from New England
waters 153
Xo. 2. OCTOBER, 1960
FRIZ, CARL T., ARNOLD LAZAROW AND S. J. COOPERSTEIN
Studies on the isolated islet tissue of fish. III. The effect of substrates
and inhibitors on the oxygen uptake of pancreatic islet slices of toadfish 161
7,
iv CONTENTS
HlLGARD, GALEN II()\V.\KI)
A study of reproduction in the intertidal barnacle, Mitella polymerus,
in Monterey Bay, California 169
JENNINGS, J. B.
Observations on the nutrition of the rhynchocoelan Linens ruber ((). F.
Miiller) . . 189
MKRKII.L, ARTHUR S., AND JOHN B. BURCH
Hermaphroditism in the sea scallop, Placopecten magellanicus (Gmelin) 197
METZ, CHARLES B., AND KI:RT KOHLER
Antigens of Arbacia sperm extracts 202
MOULTON, JAMES M.
Swimming sounds and the schooling of fishes 210
PARMENTER, CHARLES L., MARVIN DEREZIN AND HAZELTENE S. PARMENTER
Binucleate and trinucleate oocytes in post-ovulation ovaries of Raua
pipens 224
PITKOW, RONALD B.
Cold death in the guppy 231
SCOTT, ALLAN C.
Furrowing in flattened sea urchin eggs 246
SCOTT, ALLAN C.
Surface changes during cell division 260
TRIPP, M. R.
Mechanisms of removal of injected microorganisms from the American
oyster, Crassostrea virginica (Gmelin) 273
Abstracts of papers presented at the Marine Biological Laboratory 283
No. 3. DECEMBER, 1960
ABBOTT, WALTER, AND J. AXVAPARA
Sulfur metabolism in the lugworm, Arenicola cristata Stimpson 357
ANDERSON, JOHN MAXWELL
Histological studies on the digestive system of a starfish, Henricia, with
notes on Tiedemann's pouches in starfishes 371
CONOVER, ROBERT J.
The feeding behavior and respiration of some marine planktonic
Crustacea 399
GOREAU, THOMAS F., AND NORA I. GOREAU
The physiology of skeleton formation in corals. IV. On isotopic equilib-
rium exchanges of calcium between corallum and environment in living
and dead reef-building corals 416
GREGG, JOHN R.
Respiratory regulation in amphibian development 428
GROSS, WARREN J., AND LEE ANN MARSHALL
The influence of salinity on the magnesium and water fluxes of a crab. 440
GWILLIAM, G. F.
Neuromuscular physiology of a sessile scyphozoan 454
PARKER, JOHNSON
Seasonal changes in cold-hardiness of Fucus vesiculosus 474
CONTENTS v
ROGICK, MARY D.
Studies of marine Bryo/oa. XIII. Two new genera and new species from
Antarctica 478
SCHNEIDERMAN, HOWARD A.
Discontinuous respiration in insects: role of the spiracles. . 494
STUNKARD, HORACE \Y.
Further studies on the trematode genus Himasthla with descriptions of
H. mcintoshi n. sp., H. piscicola n. sp., and stages in the life-history of
1 1. compacta n. sp 529
WELLS, HARRY W., AND I. E. GRAY
The seasonal occurrence of Mytilus edulis on the Carolina coast as a
result of transport around Cape Hatteras 550
WILLIAMS, AUSTIN B.
The influence of temperature on osmotic regulation in two species of
estuarine shrimps (Penaeus) 560
Vol. 119, No. 1 August, 1960
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
THE MARINE BIOLOGICAL LABORATORY
SIXTY-SECOND REPORT, FOR THE YEAR 1959 — SEVENTY-SECOND YEAR
I. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST 15, 1959) ... 1
STANDING COMMITTEES
II. ACT OF INCORPORATION 4
III. BY-LA \vs OF THE CORPORATION 4
IV. REPORT OF THE DIRECTOR 6
Statement 7
Memorials 9
Addenda :
1. The Staff 18
2. Investigators, Lalor and Lillie Fellows, and Students 19
3. Fellowships and Scholarships 30
4. Tabular View of Attendance, 1955-1959 31
5. Institutions Represented 31
6. Evening Lectures 33
7. Shorter Scientific Papers ( Seminars ) 34
8. Members of the Corporation 35
V. REPORT OF THE LIBRARIAN 54
VI. GENERAL BIOLOGICAL SUPPLY HOUSE, INC 55
I. TRUSTEES
EX OFFICIO
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
MARINE BIOLOGICAL LABORATORY
EMERITI
W. C. CURTIS, University of Missouri
PAUL S. GALTSOFF, Woods Hole, Mass.
E. B. HARVEY, Woods Hole, Mass.
M. H. JACOBS, University of Pennsylvania School of Medicine
F. P. KNOWLTON, Syracuse University
CHARLES W. METZ, Woods Hole, Massachusetts
W. J. V. OSTERHOUT, Rockefeller Institute
CHARLES PACKARD, Woods Hole, Mass.
LAWRASON RIGGS, 74 Trinity Place, New York 6, N. Y.
TO SERVE UNTIL 1963
L. G. EARTH, Columhia 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 HAYASHI, 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. MC£LROY, Johns Hopkins University
C. LADD PROSSER, University of Illinois
S. MERYL ROSE, University of Illinois
MARY SEARS, Woods Hole Oceanographic Institution
ALBERT TYLER, California Institute of Technology
TO SERVE UNTIL 1961
ERIC BALL, Harvard University Medical School
D. W. BRONK, Rockefeller Institute
G. FAILLA, Columbia University, College of Physicians & Surgeons
R. T. KEMPTON, Vassar College
L. H. KLEINHOLZ, Reed College
IRVING M. KLOTZ, Northwestern University
ALBERT SZENT-GYORGYI, Marine Biological Laboratory
WM. RANDOLPH TAYLOR, University of Michigan
TO SERVE UNTIL 1960
H. F. BLUM, Princeton University
K. S. COLE, National Institutes of Health
S. W. KUFFLER, Harvard Medical School
C B. METZ, Florida State University
G. T. SCOTT, Oberlin College
A. H. S/TURTEVANT, California Institute of Technology
E. ZWILLING, Brandeis University
TRUSTEES
EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES
GERARD SWOPE, JR., ex officio. Chairman EDGAR ZWILLING, 1960
JAMES H. \\'ICKERSHAM, ex officio \Y. I). MCELROY, 1961
ARTHUR K. PARPART, ex officio F. A. BROWN, JR., 1961
P. B. ARMSTRONG, ex officio JOHN BUCK, 1962
RUDOLF KEMPTON, 1960 ALBERT I. LANSING, 1962
THE LIBRARY COMMITTEE
MARY SEARS, Chairman ANTHONY C. CLEMENT
SEYMOUR S. COHEN C. LADD PROSSER
THE APPARATUS COMMITTEE
ALBERT I. LANSING, Chairman RALPH H. CHENEY
HARRY GRUNDFEST FREDERIK BANG
HOWARD K. SCHACHMAN
THE SUPPLY DEPARTMENT COMMITTEE
RUDOLF T. KEMPTON, Chairman GROVER C. STEPHENS
SEARS CROWELL DAVID BISHOP
THE EVENING LECTURE COMMITTEE
PHILIP B. ARMSTRONG, Chairman DONALD P. COSTELLO
H. BURR STEINBACH S. MERYL ROSE
FRANK A. BROWN, JR.
THE INSTRUCTION COMMITTEE
JOHN B. BUCK. Chairman BOSTWICK KETCHUM
ARNOLD LAZAROW JAMES \V. GREEN
TERU HAYASHI
THE BUILDINGS AND GROUNDS COMMITTEE
EDGAR ZWILLING, Chairman JAMES CASE
MORRIS ROCKSTEIN DANIEL GROSCH
THE RADIATION COMMITTEE
G. FAILLA, Chairman 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 I). MCELROY
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;
Noiv, therefore, I, HENRY B. PIERCE, Secretary of the Commonwealth of Massachu-
setts, do hereby certify that said A. Hyatt, W. S. Stevens, W. T. Sedgwick, E. G. Gardi-
ner, S. Minns,~C. S. Minot, S. Wells, W. G. Farlow, A. D. Phillips, and B. H. Van Vleck,
their associates and successors, are legally organized and established as, and are hereby
made, an existing Corporation, under the name of the MARINE BIOLOGICAL LAB-
ORATORY, with the powers, rights, and privileges, and subject to the limitations, duties,
and restrictions, which by law appertain thereto.
Witness my official signature hereunto subscribed, and the seal of the Commonwealth
of Massachusetts hereunto affixed, this twentieth day of March, in the year of our Lord
One Thousand Eight Hundred and Eighty-Eight.
[SEAL] HENRY B. PIERCE,
Secretary of the Commonwealth.
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, Di-
rector, 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.
BY-LAWS OF THE CORPORATION
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
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 Emeritus 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
6 MARINE BIOLOGICAL LABORATORY
and shall be organized and operated under the general supervision and authority of the
Trustees.
XI. The consent of every Trustee shall be necessary to dissolution of the Marine
Biological Laboratory. In case of dissolution, the property shall be disposed of in such
manner and upon such terms as shall be determined by the affirmative vote of two-thirds
of the Board of Trustees.
XII. The account of the Treasurer shall be audited annually by a certified public
accountant.
XIII. These 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-second session of the Marine
Biological Laboratory.
During the past year the Laboratory made significant progress in rehabilitating
some of its research space and facilities and also funds were obtained for a new
research building and additional housing.
1. Policy
During the past several years there have been about thirty people including
investigators and their co-workers engaged in research on a year-round basis at
the Laboratory. Investigators have made application through the Laboratory to
various granting agencies for support for their various projects. In general, such
investigators have been provided with laboratory space and services for a limited
tenure, usually no more than five years. In addition, there have been other inves-
tigators, either retired or on sabbatical leave, who have availed themselves of the
opportunity to work at the Laboratory continuously for one or more years at the
discretion of the Executive Committee.
There has been a growing interest among the members of the Corporation, par-
ticularly some of those engaged in invertebrate zoological research, in the possibility
of developing year-round research programs at the Laboratory in marine systematics
and ecology. It is felt by many that such programs will serve to strengthen the
summer research programs and will represent the most profitable type of year-
round research. The Board of Trustees concurred in a recommendation from the
Executive Committee that such a combined systematics-ecology program be oper-
ated by the Laboratory, the staff to be selected by the Executive Committee.
Efforts are being made to receive support for this program. Also, a grant has been
obtained from the Office of Naval Research writh which to explore the feasibility
of developing a year-round research program in marine microbiology.
The Laboratory will continue to make research space and facilities available to
REPORT OF THE DIRECTOR 7
retired scientists and those on sabbatical leave on the approval of the Executive
Committee.
2. Research Training Programs
In 1959 the course in Physiology operated as a research training program under
the direction of Dr. \Yilliam McElroy with support from the National Institutes of
Health. The success of this operation strongly supported the desirability of devel-
oping the other courses along similar lines. Research training program support has
been obtained from the National Institutes of Health for Experimental Embryology
starting in 1960 and for Marine Ecology starting in 1961. During this present
winter the Old Lecture Hall has been completely remodeled to house the Experi-
mental Embryology Training Program. Also in 1960 Invertebrate Zoology and
Marine Botany will start operating as research training programs with support from
the National Science Foundation. It is confidently anticipated that these programs
will attract staff members of the same high caliber as have the courses in the past.
At this writing the trainee applicants far exceed the numbers that can be accommo-
dated in the various programs, which permits the selection of highly qualified
individuals.
All the training programs will run for the entire summer season. Each pro-
gram will operate in the manner best calculated in the minds of its director and
his staff to develop background knowledge, technical competence and research
interest in the trainees. It is anticipated that these programs will stimulate in-
creased interest in marine biology since it will introduce the beginning investigator
to marine research material early in his career and emphasize the unique advan-
tages that such material presents for a great variety of problems.
3. Xcw Laboratory Building
The current progress on the new building assures completion for 1960 summer
occupancy barring unforeseen delays in the construction. The schedule is a very
tight one so any interruption of the work will be most serious. Mr. Homer P.
Smith, General Manager of the Laboratory, merits the highest commendation for
the energetic and effective way in which he has promoted the construction of the
building. The original planning called for completion of the building for 1961
summer occupancy so that the present schedule represents the gain of a full year.
4. Grants, Contracts and Contributions
The total income from these sources of support amounted to $181,726.00 in
1959. This represents 29.6% of the total income and is made up of the following
accounts :
American Cancer Soc. — RC4A (+) Studies in Radiobiology $ 3,300.00
AEC--1343 — Program of Research on the Physiology of Marine Or-
ganisms L'sing Radioisotopes 16,165.00
NIH — 1-359 — Biological Research on the Morphology, Ecology, Physi-
ology, Biochemistry and Biophysics of Marine Organisms 40,000.00
NIH — 5143 — Training Program in Nerve Muscle Physiology 21,654.00
MARINE BIOLOGICAL LABORATORY
ONR— 1497— Studies in Marine Biology 1 5,000.00
ONR — 09701 — Studies on Isolated Nerve Fibers 6,072.00
ONR— 09702— Studies in Ecology 3,055.00
NSF — 8295 — Studies in Marine Biology 40,000.00
AEC-MBL-BM-78-59— Equipment . . '. 13,690.00
A1BL Associates 4,170.00
Abbott Laboratories 1 ,000.00
Carter Products 1,000.00
Ciba Pharmaceutical Products, Inc 1,000.00
Josephine B. Crane Foundation 2,000.00
Eli Lilly & Company 5,000.00
Merck Company Foundation 1 ,000.00
Sobering Foundation, Inc 1 ,000.00
Smith, Kline & French Foundation 3,000.00
The Upjohn Company 1 ,000.00
AYveth Laboratories, Inc 1,000.00
Olin Mathieson Chemical Corporation Charitable Trust 500.00
George Frederick Jewett Foundation 1 ,000.00
Miscellaneous Individual 120.00
$181,726.00
5. Future Plans
During the summer of 1959 the Executive Committee in several meetings con-
sidered the immediate future needs of the Laboratory and recommended that steps
be taken to (1) develop plans and obtain funds for a dormitory-dining hall build-
ing, (2) construct an additional 25 cottages on the Devil's Lane Property, and
(3) prepare an application for funds to cover the detailed planning and construc-
tion of a research instruction building.
The concentration of both the research and dormitory buildings on our present
campus creates a serious problem of congestion. The ten old wooden residences
used as dormitories take up space out of proportion to the numbers they accommo-
date. Also they do not adapt satisfactorily to dormitory use. The present dining
hall, constructed for table service, has been modified to cafeteria service but it
leaves much to be desired in fully meeting the needs of the Laboratory. A new
dormitory-dining hall facility is urgently needed in a convenient location off of our
present limited campus. There were sixty applications in 1959 for the twenty-five
new cottages built in the spring in the Devil's Lane Tract. An additional twenty-
five cottages are urgently needed particularly since the training programs will at-
tract a larger number of advanced students. A research training building to
replace the wooden laboratories will provide modern facilities for the training pro-
gram staffs and the trainees. Such a building will permit the consolidation under
one roof of the variety of services which must be provided the present training
programs housed in three wooden buildings. It will also permit certain economies
in operation and maintenance.
Respectfully submitted,
PHILIP B. ARMSTRONG,
Director
REPORT OF THE DIRECTOR 9
MEMORIAL
EDMUND NEWTON HARVEY
. by
Aurin M. Chase
The death of Edmund Newton Harvey from a heart attack on the morning of July
21st at his Penzance Point home was so unexpected as even now hardly to seem possible
to his multitude of friends. Indeed, so difficult is it to realize that for a long time many
of those who knew him will still expect to hear his voice in the corridors of the M.B.L.,
or to see him at their doors asking what's new, or reporting, with that characteristic
enthusiasm, the most recent discovery in bioluminescence or in any one of a dozen other
fields.
It is rare indeed for a man to reach the age ordinarily associated with retirement and
still remain so young. Newton Harvey was equally at home with people of all ages, and
they with him. He will be greatly missed at Woods Hole and the many other places
where he lived, worked and played.
Having graduated from Germantown Academy and then received his Bachelor of
Science at the University of Pennsylvania, Harvey went to Columbia for his graduate
work and was awarded the Ph.D. in 1911. He started teaching immediately at Prince-
ton where, virtually single-handed, he initiated courses in general physiology and bio-
chemistry, subjects not often included in biology curricula at that time. In 1916 he mar-
ried Ethel Nicholson Browne, herself a Columbia Ph.D. in zoology. They shared lab-
oratories at Princeton and Woods Hole over the years, except for the period when it was
necessary for her to devote full time to raising their two sons, Ned and Dick. The Har-
veys had a host of friends and greatly enjoyed interesting company. Many will remem-
ber, for example, the "Harvey Table" at the M.B.L. Mess as a center of good conversa-
tion and congeniality.
At Princeton, Newton Harvey was promoted to full professor in 1919 and, fourteen
years later, became Henry Fairfield Osborn Professor of Biology, occupying that chair
until his retirement in 1956. During his years of teaching he attracted many graduate
students, most of whom based their Ph.D. theses on some aspect of bioluminescence.
Nearly all his summers were spent at the M.B.L., where he had been elected a member
of the Corporation in 1910, when barely out of college, and became a trustee in 1929.
He served as Vice President of the Corporation from 1942 until 1952, and was always
most active and influential in the affairs of the Laboratory. He was elected Trustee
Emeritus in 1958. Although it was only natural that a man of his interests and energy
should become involved in the direction and operation of many organizations — he was,
for example, a trustee of the Bermuda Biological Station — the activities and welfare of
the Marine Biological Laboratory were always closest to his heart.
With as many interests as he had, it is not surprising that Harvey belonged to some
twenty societies covering the fields of physiology, biochemistry and biophysics, as well
as less specialized areas of biology. He was a member of The National Academy of
Sciences and The American Philosophical Society, and had been vice president and
president of The International Society of Cell Biology. In 1953 he was president of The
American Society of Zoologists, as he was also of The American Society of Naturalists
in 1956. He was an associate editor of several journals, and served as Managing Editor
of The Journal of Cellular and Comparative Physiology during the first sixteen years of
its existence.
Among the formal awards made to him were the John Price Wetherill Medal of The
Franklin Institute, in 1934, for his development of the centrifuge-microscope, and the
Rumford Medal, by The American Academy of Arts and Sciences, in 1947, for his work
10 MAKIXK BIOLOGICAL LABORATORY
in bioluminescence. He received the Certificate of Merit of the U. S. Armed Forces for
his services, both experimental and advisory, during- the second world war. More re-
cently, the Johns Hopkins University awarded him an honorary Doctor of Science de-
gree, and Temple University the honorary degree of Doctor of Laws.
HarveyV attitude toward research was alwavs that of the explorer and pioneer. He
opened up new regions for others to develop. His tremendous curiosity and drive were
not satisfied by the sort of routine experimentation required to wrap up completely all
the loose ends of a problem. Without his kind, the discovery of new things would be
slow if it occurred at all. So extensively did he explore in the field of bioluminescence,
for example, that it is difficult to do any experiment involving a luminous organism with-
out finding, sooner or later, that Harvey had had the same idea and tested it — at least in
a preliminary way — years before.
A person of such consuming curiosity could never be satisfied in a single line of re-
search. His 250 or so published papers include such topics as cell permeability and
oxidations, physical measurements at cell surfaces, brain potentials, effects of supersonic
waves and of centrifugal forces on cells and — during the war — decompression sickness
and wound ballistics. He loved instrumentation and was always eager to apply new
apparatus and techniques to biological problems. His vast knowledge of organisms was
most useful in this connection. But his greatest interest, and the one to which he re-
turned again and again, and which was occupying him at the time of his death, was bio-
luminescence. It is for this that he is best known and the acknowledged authority
throughout the world. He wrote four books on the subject, and was working on a fifth.
One who did not know Newton Harvey might get the mistaken impression from a re-
view of his many scientific accomplishments that his time was spent entirely in the lab-
•oratory and the study. Nothing could be farther from the truth ! He enjoyed life to the
full ! He was an excellent tennis player in his time and an experienced mountain climber,
went on numerous scientific expeditions, and at one time was even reported to have been
eaten by cannibals ! At Woods Hole he always found time for swimming and sailing,
and the Harveys' sloop, the "Rip Tide," carried many a happy party across Vineyard
Sound to Edgartown, or down the islands to Tarpaulin Cove or Cuttyhunk.
He loved to surprise his friends in unusual and pleasant ways, such as pulling off the
road into a secluded grove, opening the luggage compartment of his big Buick, and serv-
ing cocktails from the portable bar which nobody had suspected was there. Then would
follow dinner at some nearby restaurant which he had previously tested and found to be
good.
Yet in the midst of a social gathering his scientific interest might suddenly come to
the fore. As when, once, he tossed a corked bottle to the ceiling so that all could observe
that the large air bubble trapped inside did not rise in the bottle while it was in "free fall."
All those who have come in contact with Edmund Newton Harvey, his friends, col-
leagues, graduate students, and those fortunate undergraduates who did their senior theses
under his direction, have absorbed some of his boundless enthusiasm and spirit. Cer-
tainly he will lie sorely missed. But surely he would not have preferred to have gone
otherwise than as he did; in full possession of all his faculties; vigorous, joyful and
active until the last.
MEMORIAL
GEORGE H. A. CLOWES
by
Philip B. Armstrong
Rarely do we encounter in one man such a variety of talents and interests as charac-
terized George Henry Alexander Clowes. With unbounded energy, he used these tal-
ents effectively, following up his interests with persistent determination. He was a
REPORT OF THE DIRECTOR 11
scientist and investigator who sparked many practical applications of basic research.
He was a man of business, a civic leader, and patron of the fine arts and of music.
Dr. Clowes was born in Ipswich, England, in 1877, in a period of developing scien-
tific interest. Through environment and natural bent, he early developed an interest in
science. His family was in the business of producing intermediates for the brewing
industry.
He attended the Royal College of Science in London from 1893-1896 and completed
his studies for a Ph.D. degree in chemistry in 1899 at the University of Gottingen. His
thesis was concerned with the methyl derivatives of sugar. After additional work in
some of the other leading chemical laboratories in France and Germany, he came to
this country in 1901 as chemist at the Institute for the Study of Malignant Diseases at
Buffalo.
For the following period of fifteen years, Dr. Clowes published extensively, using a
variety of approaches to the problem of cell division and growth, particularly as it ap-
plied to cancer. An important contribution concerned the evidence for immunity against
cancer in mice after spontaneous remissions. Other papers dealt with the mechanism of
the action of mustard gas. enzyme action in fermentation, ion antagonisms, and the action
of anesthetics in biological and physical systems. His outstanding scientific contributions
were on the effects of calcium or sodium at oil-water interfaces. Surface phenomena
remained a life-long interest resulting in many practical developments.
Dr. Clowes left Buffalo in 1918 to serve in the Chemical Warfare Service where he
continued his work on mustard gas, particularly its mechanism of action. In order to
study its action under most favorable experimental conditions, he spent the summer of
1918 at the Marine Biological Laboratory, working with Robert Chambers and Ralph
Lillie. Although Dr. Clowes had a highly practical turn of mind, he had a deep appre-
ciation of the potential significance of basic research. The scientific intellectual atmos-
phere of the Marine Biological Laboratory was so attractive to Dr. Clowes that he spent
most of the following 40 summers at Woods Hole. At the Laboratory he found what he
considered ideal biological materials for many of the basic problems in which he was
interested.
After the war he joined the research staff of Eli Lilly and Company and became Di-
rector of Research in 1921. Here he served very effectively in the development of the
commercial production of several products, starting with insulin. His earlier interest
in salt antagonisms and the effects of pH on proteins served him in good stead in the
precipitation and purification of insulin. He also played a prominent role in the develop-
ment of liver extract, protamine insulin and penicillin. From the Marine Biological
Laboratory with collaborative workers Homer Smith, Maurice Krahl, Anna Keltch and
others, Dr. Clowes published a series of papers on the possible control of mitosis by
chemical agents.
In 1918 he was elected a member of the Corporation of the Marine Biological Lab-
oratory, served three terms as a trustee, and was elected a Trustee Emeritus in 1948.
He was active in seeking support for the Laboratory as well as contributing generously
himself. In Indianapolis he played an active role in the development of the Indianapolis
Symphony Orchestra and the John Huron Art Institute.
Dr. Clowes enjoyed a felicitous marriage. He is survived by his wife, Mrs. Edith
Whitehill Clowes, who shared with him many of his civic and philanthropic interests.
Also surviving him are two sons, Dr. G. H. A. Clowes, Jr. and Allen W. Clowes. Dr.
Clowes died in Woods Hole on August 25, 1958. He will be remembered by his Woods
Hole friends for his outstanding generosity and kindness and his active participation in
the scientific and physical development of the Marine Biological Laboratory. He had a
keen interest in the work of others, participated actively in the scientific discussion at the
Laboratory, and was always ready to give help in the development of science.
12 MARINE BIOLOGICAL LABORATORY
MEMORIAL
EUGENE FLOYD Du Bois
by
Paul Reznikoff
Dr. Eugene Floyd Du Bois died on February 12, 1959, at his home at the age of 76.
His death is not only a great loss to medicine and physiology but to his many stu-
dents, house officers and colleagues who were associated with him at Cornell University
Medical College, Bellevue Hospital, the New York Hospital and the Marine Biological
Laboratory, it is a personal tragedy.
Doctor Du Bois was born on June 4. 1882 in West New Brighton, Staten Island,
New York, the son of Eugene and Anna Brooks Du Bois. He attended the Staten Island
Academy and Milton Academy in Massachusetts and received his A.B. degree from Har-
vard in 1903 and his M.D. degree from Columbia in 1906. In 1948 he was awarded an
honorary Doctor of Science degree by Rochester University.
After interning at the Presbyterian Hospital (1907-08) and acting as assistant pathol-
ogist (1909) at this institution he decided to secure a training in bacteriology. But just
before he was about to leave for France he changed his plans at the suggestion of Dr.
John Rowland and he went to Germany to study metabolism. Dr. Graham Lusk visited
the laboratory in Berlin and met Dr. Du Bois there. This was the beginning of a life-
long association of these two pioneers in the field of metabolism. When Dr. Du Bois
returned to the United States he soon became the Medical Director of the Russell Sage
Institute of Pathology, of which Dr. Lusk was the Scientific Director. Thus Dr. Du Bois
became a scientific descendant of Lavoisier. Under Du Bois' guidance the Russell Sage
Institute has had world-wide influence in advancing scientific knowledge in the field of
metabolism and from his "Calorimeter Room" there have gone forth many of our most
important scientists and medical educators.
Doctor Du Bois was Director of the Second (Cornell ) Medical Division of Bellevue
Hospital from 1919 to 1932, Professor of Medicine at the Cornell University Medical
College from 1930 to 1941. Physician-in-Chief at the New York Hospital from 1932 to
1941, and Professor of Physiology at Cornell from 1941 to 1950 when he became Emeritus
Professor.
Some of Dr. Du Bois' most important contributions were concerned with his work for
the United States Navy. Because of his modesty few of his associates knew that he was
an outstanding authority in the fields of submarine warfare and aviation medicine. For
heroism in the conduct of hazardous experiments during World War I he received the
Navy Cross. During the second World War he was recognized by Commendation and
Ribbon Bar. He retired from the Navy with the rank of Captain and continued to work
with the military service until the very day of his death.
He belonged to many societies — the National Academy of Sciences, the Philosophical
Society, the American Physiological Society, the Aero Medical Association, the Society
for Experimental Biology and Medicine, and was president of the American Society for
Clinical Investigation, of the Association of American Physicians, of the Harvey Society
and of the Institute of Nutrition.
He became a member of the Corporation of the Marine Biological Laboratory in 1929
and was elected a Trustee in 1942. He was re-elected in 1944 for an additional full term
to 1948. In 1952 he was elected Trustee Emeritus which position he held until his death.
He was as conscientious in his duties to the Laboratory as he was in all undertakings and
even during the later years of his incapacity he attended the meetings of the Trustees
despite his physical limitations. He planned to be present at the Annual Winter Meet-
ing which took place just one day after his death.
REPORT OF THE DIRECTOR 13
Many honors came to Dr. Du Bois, such as the Kober Medal of the Association of
American Physicians in 1947 and the Academy Medal of the New York Academy of
Medicine in 1956. He was to receive the John Phillips Memorial Award of the Ameri-
can College of Physicians in April.
Doctor Du Bois' accomplishments and honors are of minor importance compared to
the influence he has had upon his students and associates by virtue of his personality and
character. As his life-long- friend and successor as Professor of Medicine, Dr. David P.
Barr. has said, "This extraordinary influence has been attributable only in part to his
mastery of experimental procedure and the intrinsic value of his scientific contributions.
Its essence derives from his own character and personality. Inspiration has come to
others from his abiding faith in principles of scientific and personal conduct, from his
integrity and tolerance, and from his sympathetic understanding of the problems of those
about him. His character has influenced behavior of his colleagues. It has also influ-
enced innumerable students who have learned from him lessons of critical evaluation,
clear expression, unvarying courtesy, and true humility."
Doctor Du Bois was a gentleman, gentle in all his dealings with his fellow men and
with suffering, and a man in his uncompromising attitude toward injustice and dishonesty.
A colleague once asked him why he did not delegate some of his difficult and unpleas-
ant problems to his subordinates. His reply was that such tasks were the duties of the
chief.
The principles which guided him in educating the medical students were described in
an article entitled "The Clinical Clerkship in Medicine," published in the Journal of the
American Medical Association, August 21, 1926. and were these: "The purpose of instruc-
tion is to teach the students to teach themselves ; the manner of instruction is by example
and work ; the spirit of instruction is sympathy for and faith in the students."
All his friends join his widow, his three children and his nine grandchildren in being
proud of their association with a great and good man. His life may be summarized by
the citation on the Academy Medal : "Eugene Floyd Du Bois, physiologist, physician,
educator, patriot. His life and work have brought honor to the profession of medicine"
and to science.
MEMORIAL
JACQUES LOEB
by
W. J. V. Osterhout
This year marks the 100th anniversary of the birth of Jacques Loeb, who contributed
so much to the study of marine biology, and it seems appropriate that in a marine bio-
logical station where he worked, we should recall his activities.
Thirty-five years have passed since his death and yet our memory of him is still fresh
and vivid.
He was above all an idealist. Protected by his devoted wife who knew how to help
him, he lived in a world of ideals. Their inspiration dominated his life and set him apart
from others. Yet he had also a tender heart, and his sympathy was always with those
who were in need of help.
His outstanding feature was his creative imagination, implying prophetic vision, the
intuitive, and emotional urge of ideas.
Fortunately his poetic imagination was associated with a keen critical sense. He
would test his conceptions over and over again and repeat his experiments very carefully.
He published only a small part of his experimental work. It is remarkable that his ob-
servations remain valid without fundamental modifications.
14 MARIXH HIOI.OCICAL LABORATORY
The questions lie put to nature were never dull and the answers he received were al-
ways interesting and at times startlingly so.
He was not content to pursue a special part of a problem without considering its rela-
tion to all the rest. To achieve this, it was necessary both to simplify and to generalize,
and these powers he possessed to an extraordinary degree.
Courage played a great part in his success. He did not select problems because they
were easy but because of their importance. His courage sprang largely from his faith
in the mechanistic conception to which he consecrated his life.
He had a truly lovable and sympathetic personality that drew men irresistibly to him.
His teaching was inspiring and unforgettable, so that it was not strange that young men
gladly followed him.
One felt instinctively that he cared only for truth and that in its quest he would spare
no labor and sacrifice.
The breadth of his knowledge made it natural for him to utilize in his work recent
advances in other fields of science. Thus he took the ideas of tropism and of hetero-
morphosis from botany. He applied to biology theories of dissociation and osmotic pres-
sure which resulted in the discovery of artificial parthenogenesis and antagonistic salt
action. To the very end of his life he kept in touch with recent progress in physics and
chemistry which he applied to his own studies.
Death came while he was actively engaged in what he regarded as the most funda-
mental investigation of his life. In the midst of this research on proteins he was stricken
down.
Here we may pause to ask ourselves, how are we to remember him ? He was an
idealist, sympathizing with all suffering, consecrating his gifts to humanity; a scientist
with an artist's soul, emotional, intuitive, creative; a thinker, strangely original, born to
blaze fresh trails and teach new doctrines; a dreamer, regarding the world of life with
poetic insight and seeking with creative imagination rarely equalled to sweep aside its
mystery and set free the mind of men. His visions, that have made others see visions,
cannot but continue to shed inspiration ; and in shaping the soul of the future he may
serve humanity more than he dared to dream.
MEMORIAL
FRANK M. MACNAUGHT
by
Charles Packard
Long and faithful was the service rendered to this Laboratory by Frank M. Mac-
Naught who died in June of this year at the age of 83. Coming to Woods Hole as an-
accountant in 1913 when the Laboratory began its rapid growth, he was soon made
Registrar, and then, in 1916, the Business Manager, a position which he held for 34
years. For much of this time his only assistant was Miss Polly Crowell. Among his
many responsibilities was the task of assigning laboratory rooms and tables, and the
much more difficult work of apportioning space in the Apartment House and dormitories.
Only those who worked closely with him can appreciate the care which he exercised in
selecting places best adapted to the needs of each applicant. In addition to these duties,
he was responsible for the Mess and its many employees.
From the first he devoted himself to these various tasks, discharging them with great
efficiency. Always in the Office, even on Sundays and holidays, he was quick to help
newcomers unfamiliar with the operation of the Laboratory. His memory was extra-
ordinary. He could at once call by name investigators and students returning after an
REPORT OF THE DIRECTOR 15
absence of many seasons, even recalling the year of their last attendance and the rooms
they occupied. His friendliness endeared him to all. He once remarked that if he
should take a trip across the country he could spend each night at the home of investi-
gators or students who had especially asked him to visit them. In his relations with all
he worked with he showed patience, good judgment, and great tact. Many times he re-
lieved a tense situation with an apt, humorous remark.
He was active in Town affairs, serving on the Finance and other committees, and in
the village, as Treasurer and Trustee of the Woods Hole Public Library, and Clerk of
the Coonamesset Ranch.
The Laboratory has lost an exceptional man, a devoted friend whose outstanding
services and genial personality will long be remembered.
MEMORIAL TO CHARLES R. CRANE ON HIS
HUNDREDTH ANNIVERSARY
by
Lawrason Riggs
On the eighth day of August 100 years ago was born the greatest benefactor and
friend of the Marine Biological Laboratory, Charles R. Crane.
Mr. Crane was most interested in education. I think this was because he had no
formal education beyond grade school. His father, the founder of the Crane Company,
did not believe in colleges, in fact he wrote a book against college education, enlisting in
the writing of the book two employees of the Crane Company, both of whom turned out
to be college graduates.
He went to work at an early age for his father. In 1878 he happened to be in New
York. He wandered down to Front Street where in those days the bowsprits of sailing
vessels projected over the street. Young Crane went aboard one of these ships and on
telegraphic consent from his father arranged to sail on her to Java as the sole passenger.
The only additions he made to his baggage were a set of Herbert Spencer and 12 dozen
bottles of Guiness Stout. On the voyage he read the Spencer, drank a bottle of stout
every day and learned navigation, and on his 21st birthday he furled the main royal in
a gale off the Cape of Good Hope. Toward the end of the voyage the first mate died
and Mr. Crane was offered his position. He did not accept as he wanted to see as much
of Asia as possible. This was fortunate as on the ship's return voyage the captain and
most of the crew died, undoubtedly of beri-beri. Later his doctor informed him that his
health had been preserved by those 144 bottles of Guiness Stout.
So began his informal education, which he pursued with unrelenting vigor so that in
time he became one of the best informed Americans about the Moslem world, Russia and
the Far East. During the Peace Conference after World War I he was appointed on a
Commission with President King of Oberlin to investigate and make recommendations
on the future of Syria. This mission further deepened and extended his contacts with
the Near East. Subsequently he was for a number of years American Ambassador to
China.
He had an extraordinary interest in exotic places and a real flair for people. He was
as much at home in Paris, St. Petersburg, Cairo, Damascus, Constantinople, Samarkand
and Pekin as he was in Chicago, and he numbered among his friends, presidents, espe-
cially President Wilson, cabinet members, educators, judges, Moslem leaders, including
the King of Hejaz and Sherif of Mecca and his son Feisal, later King of Iraq.
He made some 32 visits to Russia, penetrated the most remote parts of Asia, includ-
16 MARINE BIOLOGICAL LABORATORY
ing Bokhara and the Transoxnas, and went with one servant on horseback through Al-
bania after being deserted by the Turkish bodyguard supplied by the Sultan.
The M.B.L. was not the only beneficiary of his interest in education and research.
He was interested in the Near East colleges, especially the American College for Girls
on the Bosphorus and the Sofia-American Schools in Bulgaria and also a school in Al-
bania and in many universities in this country. The main purpose of his Foundation,
The Friendship Fund, was to assist individuals to get an education and the purpose of
the Institute of Current World Affairs, which he also founded, was to train specialists
in critical areas under conditions that would develop their talents and personality.
His first important contact with the Marine Biological Laboratory was his joining
in an offer of assistance with several other persons through President Harper of Chicago
University. This happened in 1901.
When this offer and that of the Carnegie Institution were finally rejected 1>y the Lab-
oratory because the trustees and members did not wish to allow the Laboratory to lose
its independence, Mr. Crane became more interested through his brother-in-law, Dr.
Frank R. Lillie, and before long was contributing about $20,000 a year towards its
expenses.
He had a large part in purchasing real property for the Laboratory and in 1913 pro-
vided the first brick building, the so-called Crane Building. He had a very important
part in the expansion and endowment of the Laboratory between the years 1919 and
1925. He not only capitalized his annual contribution of $20,000 by a gift of $405,000
to endowment, but he guaranteed to pay any cost of the new Rockefeller Building in
excess of $500,000. This guarantee cost him $221,000. He had a large part in inter-
esting Mr. John D. Rockefeller, Jr. and the Rockefeller Foundation, as is clear from
Mr. Rockefeller's letter found at page 75 of Dr. Lillie's book.
While his gifts were important and always timely, his appreciation of the spirit of
the Laboratory and of its democratic and self-governing organization — a group of sci-
entists running their own affairs — was almost more important. He acted as President
from 1904 to 1925.
That the Laboratory came through its early and difficult years and survived to be-
come the great institution that it now is is, I feel, largely due to his help and encourage-
ment.
ZOOLOGY
I. CONSULTANTS
F. A. BROWN, JR., Morrison Professor of Zoology, Northwestern University
LIBBIE H. HYMAN, American Museum of Natural History
A. C. REDFIELD, Woods Hole Oceanographic Institution
II. INSTRUCTORS
GROVER C. STEPHENS, Assistant Professor of Zoology, University of Minnesota ; in
charge of course.
JOHN B. BUCK, Senior Biologist, National Institutes of Health
RALPH I. SMITH, Associate Professor of Zoology, University of California, Berkeley
BERNARD L. STREHLER, Chief, Cellular and Comparative Physiology, Division of Geron-
tology, National Institutes of Health
PAUL P. WEINSTEIN, Laboratory of Tropical Diseases. National Institutes of Health
RICHARD C. SANBORN, Professor of Zoology, Department of Biological Sciences, Purdue
University
REPORT OF THE DIRECTOR 17
MORRIS ROCKSTEIN, Associate Professor of Physiology, New York University College
of Medicine
MILTON FINGERMAN, Assistant Professor of Zoology, Tulane University
III. LABORATORY ASSISTANTS
ROBERT ASHMAN, Wabash College
DONALD HALL, University of Michigan
EMBRYOLOGY
I. INSTRUCTORS
MAC V. EDDS, JR., Professor of Biology, Brown University; in charge of course
PHILIP GRANT, Assistant Professor of Pathobiology, Johns Hopkins University
JOHN W. SAUNDERS, JR., Professor of Zoology, Marquette University
NELSON T. SPRATT, JR., Professor of Zoology, University of Minnesota
MAURICE SUSSMAN, Associate Professor of Biological Sciences, Brandeis University
LIONEL REBHUN, Assistant Professor of Biology, Princeton University
II. LABORATORY ASSISTANTS
CHANDLER M. FULTON, Rockefeller Institute for Medical Research
DAVID S. LOVE, University of Colorado
PHYSIOLOGY
I. CONSULTANTS
MERKEL H. JACOBS, Professor of Physiology, University of Pennsylvania
ARTHUR K. PARPART, Professor of Biology, Princeton University
ALBERT SZENT-GYORGYI, Director, Institute for Muscle Research, Marine Biological
Laboratory
II. INSTRUCTORS
W. D. McELROY, Professor of Biology, Johns Hopkins University ; in charge of
course
FRANCIS D. CARLSON, Associate Professor of Biophysics, Johns Hopkins University
BERNARD D. DAVIS, Professor of Bacteriology, Harvard Medical School
DONALD GRIFFIN, Professor of Zoology, Harvard University
HOWARD SCHACHMAN, Virus Laboratory, University of California
TIMOTHY GOLDSMITH, Fellow, Harvard University
ROBERT LOFTFIELD, Massachusetts General Hospital
III. LABORATORY ASSISTANT
Louis OTERO, University of Puerto Rico, Rio Piedras
BOTANY
I. CONSULTANT
WM. RANDOLPH TAYLOR, Professor of Botany, University of Michigan
18 MARINE BIOLOGICAL LABORATORY
II. INSTRUCTORS
RICHARD C. STARR, Associate Professor of Botany, Indiana University; in charge of
course
JOHN M. KINGSBURY, Assistant Professor of Botany, Cornell University
WALTER R. HERNDON, Assistant Professor of Biology, University of Alabama
III. COLLECTOR
G. BENJAMIN BOUCK, Columbia University
IV. LABORATORY ASSISTANTS
LARRY HOFFMAN, University of Texas
ROBERT W. KORN, Indiana University
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. TENNER, University of North Carolina
HOWARD L. SANDERS, Woods Hole Oceanographic Institution
II. INSTRUCTORS
EUGENE P. ODUM, Alumni Foundation Professor of Zoology, University of Georgia;
in charge of course
HOWARD T. ODUM, University of Texas
HAROLD J. HUMM, Associate Professor of Botany, Duke University
JOHN H. RYTHER, Marine Biologist, Woods Hole Oceanographic Institution
III. LABORATORY ASSISTANT
RICHARD B. WILLIAMS, Harvard University
1. THE LABORATORY STAFF, 1959
HOMER P. SMITH, General Manager
MRS. DEBORAH LAWRENCE HARLOW, Librarian ROBERT KAHLER, Superintendent,
CARL O. SCHWEIDENBACK, Manager of the Buildings and Grounds
Supply Department ROBERT B. MILLS, Manager, De-
IRVINE L. BROADBENT, Office Manager partment of Research Service
GENERAL OFFICE
MRS. LILA S. MYERS MRS. MARION C. CHASE
MRS. VIVIEN R. BROWN MRS. VIVIAN I. MANSON
MRS. VIRGINIA M. MOREHOUSE MRS. SHIRLEY A. ELDER
MRS. LORETTA J. MCCARTNEY
REPORT OF THE DIRECTOR 19
LIBRARY
MRS. M. VERNA HANKS MRS. NAOMI BOTELHO
MRS. GWENDOLYN S. BLOMBERG ALBERT K. NEAL
MAINTENANCE OF BUILDINGS AND GROUNDS
ROBERT ADAMS RALPH H. LEWIS
ELDON P. ALLEN RUSSELL F. LEWIS
ARTHUR D. CALLAHAN ALAN G. LUNN
ROBERT GUNNING ALTON J. PIERCE
WALTER J. JASKUN ROBERT H. WALKER, JR.
DONALD B. LEHY JAMES S. THAYER
DEPARTMENT OF RESEARCH SERVICE
GAIL M. CAVANAUGH SEAVER R. HARLOW
JOHN P. HARLOW MRS. ARLENE BROWN
SUPPLY DEPARTMENT
DONALD P. BURNHAM ROBERT M. PERRY
MILTON B. GRAY BRUNO F. TRAPASSO
GEOFFREY J. LEHY JOHN J. VALOIS
ROBERT O. LEHY JARED L. VINCENT
MRS. MILDRED H. MIXSON HALLETT S. WAGSTAFF
2. INVESTIGATORS, LALOR AND LILLIE FELLOWS, AND STUDENTS
Independent Investigators, 1959
ADELMAN, WILLIAM J., Assistant Professor of Physiology, University of Buffalo
ALLEN, ROBERT D., Assistant Professor of Biology, Princeton University
AMBERSON, WILLIAM R., Investigator, Marine Biological Laboratory
ARMSTRONG, PHILIP B., Professor and Chairman of Anatomy, State University of New York,
College of Aledicine at Syracuse
ARNOLD, WILLIAM, Principal Biologist, Oak Ridge National Laboratory
BALTUS, ELYANE, Charge de Course, University of Brussels, Belgium
BANG, FREDERIK B., Professor of Pathobiology, Johns Hopkins University School of Hygiene
BARTH, L. G., Professor of Zoology, Columbia University
BAYLOR, MARTHA B., Independent Investigator, Marine Biological Laboratory
BELL, EUGENE, Assistant Professor of Biology, Massachusetts Institute of Technology
BENESCH, REINHOLD, Investigator, Marine Biological Laboratory
BENIGNA, SISTER MARIA, Professor of Biology, Saint Joseph College
BENNETT, MICHAEL, Research Associate, Columbia University, College of Physicians and
Surgeons
BENNETT, MIRIAM F., Assistant Professor of Biology, Sweet Briar College
BEXZER, SEYMOUR, Professor of Biophysics, Purdue University
BERGMANN, FELIX, Research Fellow, College of Physicians and Surgeons, Columbia University
BERMAN, MONES, Physicist, National Institutes of Health
BERNARD, GEORGE R., Assistant Professor of Biology, University of Notre Dame
BERNSTEIN, MAURICE H., Assistant Professor of Anatomy, Wayne State University
BISHOP, DAVID W., Staff Member, Carnegie Institution of Washington
BOSLER, ROBERT, Instructor of Physiological Optics, Johns Hopkins Hospital
BRETT, WILLIAM J., Associate Professor of Biology, Indiana State Teachers College
BROWN, FRANK A., JR., Morrison Professor of Biology, Northwestern University
20 MARINE BIOLOGICAL LABORATORY
BRYANT, SHIRLEY H., Assistant Professor of Pharmacology, University of Cincinnati
BUCK, JOHN B., Physiologist, National Institutes of Health, Laboratory of Physical Biology
BURBANCK, W. D., Professor of Biology, Emory University
BURK, REV. JOSEPH A., Assistant Professor, Loyola College
CABRERA, GUILLERMO, Assistant Professor of Biochemistry, New York University College of
Medicine
CARLSON, FRANCIS D., Associate Professor of Biophysics, Johns Hopkins University
CASCARANO, JOSEPH, Instructor of Pathology, New York University College of Medicine
CASE, JAMES, Assistant Professor of Zoology, State University of Iowa
CHAET, ALFRED B., Associate Professor of Biology, American University
CHENEY, RALPH HOLT, Professor of Biology, Brooklyn College
CHILD, FRANK M., Instructor in Zoology, University of Chicago
CLAFF, C. LLOYD, Research Associate in Surgery, Harvard Medical School
COLE, KENNETH S., Chief, Laboratory of Biophysics, National Institutes of Health
COLWIN, ARTHUR L., Professor of Biology, Queens College
COLWIN, LAURA H., Lecturer, Queens College
COOPERSTEIN, SHERWIN J., Associate Professor of Anatomy, Western Reserve University School
of Medicine
COSTELLO, DONALD P., Kenan Professor of Zoology, University of North Carolina
CRANE, ROBERT K., Associate Professor of Biological Chemistry, Washington University Medi-
cal School
CROSTI, NICOLETTA, Foreign Italian Student, Bryn Mawr College
CROWELL, SEARS, Associate Professor of Zoology, Indiana University
CSAPO, ARPAD, Associate Professor, Rockefeller Institute for Medical Research
DAVIS, BERNARD, Professor of Bacteriology, Harvard Medical School
DOOLIN, PAUL F., Assistant Professor of Biology, Washington and Jefferson College
DuBois, ARTHUR B., Associate Professor of Physiology, University of Pennsylvania School of
Medicine
ECHALIER, GUY P. R., Research Fellow in Biology, Harvard College
Eons, MAC V., JR., Professor of Biology, Brown University
EDMONDS, MARY, Research Associate, Montefiore Hospital Research Institute
FAILLA, G., Professor of Radiology, Columbia University
FEIGELSON, PHILIP, Assistant Professor of Biochemistry, College of Physicians and Surgeons
FINGERMAN, MILTON, Assistant Professor of Zoology, Newcomb College of Tulane University
FISHMAN, Louis, Research Associate, New York University College of Dentistry
FORREST, HUGH S., Associate Professor of Zoology, University of Texas
FRANK, ULRICH, Extraordinary Professor, Technical University, Darmstadt, Germany
FRIZ, CARL T., Public Health Fellow, University of Minnesota
FUJIMORI, EIJI, Investigator, Institute for Muscle Research, Marine Biological Laboratory
FUORTES, M. G. F., Physiologist, National Institutes of Health
FURSHPAN, EDWIN, Instructor in Ophthalmic Physiology, Johns Hopkins University
FURUKAWA, TARO, Instructor in Ophthalmic Physiology, Johns Hopkins University
GALTSOFF, PAUL S., Director, Shellfish Laboratory, U. S. Bureau of Commercial Fisheries
GARDELLA, JOSEPH W., Assistant Dean, Harvard Medical School
GLADE, RICHARD W., Assistant Professor of Zoology, University of Vermont
GOLDSMITH, TIMOTHY H., Junior Fellow, Harvard University
GONSE, PIERRE H., Research Fellow, University of Pennsylvania
GOREAU, THOMAS F., Lecturer in Physiology, University College of the West Indies
GORINI, LUIGI, Lecturer, Harvard Medical School
GRANT, PHILIP, Assistant Professor of Pathobiology, Johns Hopkins University School of
Hygiene
GRAY, I. E., Professor of Zoology, Duke University
GREEN, JAMES W., Associate Professor of Physiology, Rutgers, The State University
GREIF, ROGER L., Associate Professor of Physiology, Cornell University Medical College
GRIFFIN, DONALD R., Professor of Zoology, Harvard University
GROSCH, DANIEL S., Professor of Genetics, North Carolina State College
GROSS, PAUL RANDOLPH, Associate Professor of Biology, New York University
GROSS, SAMSON RICHARD, Assistant Professor, Rockefeller Institute for Medical Research
REPORT OF THE DIRECTOR 21
GRUNDFEST, HARRY, Associate Professor of Neurology, College of Physicians and Surgeons,
Columbia University
GUTTMAN, RITA, Associate Professor of Biology, Brooklyn College
HARVEY, ETHEL BROWNE, Investigator in Biology, Princeton University
HARVEY, E. NEWTON, Professor of Physiology, Emeritus, Princeton University
HAY, ELIZABETH D., Assistant Professor of Anatomy, Cornell University Medical College
HAYASHI, TERU, Professor of Zoology, Columbia University
HEGYELI, ANDREW, Investigator, Institute for Muscle Research, Marine Biological Laboratory
HEILBRUNN, L. V., Professor of Zoology, University of Pennsylvania
HENLEY, CATHERINE, Research Associate, University of North Carolina
HERNDON, WALTER R., Assistant Professor of Biology, University of Alabama
HERVEY, JOHN P., Senior Electronic Engineer, Rockefeller Institute
HIATT, HOWARD H., Assistant Professor of Aledicine, Harvard Medical School
HIBBARD, HOPE, Professor, Oberlin College
HILL, ROBERT B., Instructor in Zoology, University of Maine
HOLZ, GEORGE G., JR., Associate Professor of Zoology, Syracuse University
HUMM, HAROLD J., Associate Professor of Botany, Duke University
HURSH, JOHN B., Professor of Radiation Biology, University of Rochester
HURWITZ, JERARD, Assistant Professor of Microbiology, New York University
ISENBERG, IRVIN, Investigator, Institute for Muscle Research, Marine Biological Laboratory
JACOBS, WILLIAM P., Associate Professor of Biology, Princeton University
JENNER, CHARLES E., Associate Professor and Chairman of Zoology, University of North
Carolina
JONES, MARY ELLEN, Assistant Professor of Biochemistry, Brandeis University
JONES, RAYMOND F., Visiting Research Associate, Marine Biological Laboratory
KAMINER, BENJAMIN, Senior Lecturer in Physiology, University of Witwatersrand, Johannes-
burg, South Africa
KANE, ROBERT E., Assistant Professor of Biochemistry, Brandeis University
KAPLAN, NATHAN O., Professor and Chairman of Biochemistry, Brandeis University
KEMPTON, RUDOLF T., Chairman, Department of Zoology. Vassar College
KINGSBURY, JOHN M., Assistant Professor of Botany, Cornell University
KLEIN HOLZ, L. H., Professor of Biology, Reed College
KOHLER, KURT, Research Associate, Florida State University
KUFFLER, STEPHEN W., Investigator, Johns Hopkins Hospital
KURIYAMA, HIROSI, Rockefeller Institute for Medical Research
KURY, LIVIA REV, Investigator, Institute for Muscle Research, Marine Biological Laboratory
LANSING, ALBERT L, Professor and Chairman of Anatomy, University of Pittsburgh
LASH, JAMES W., Associate in Anatomy, University of Pennsylvania, School of Medicine
LAZAROW, ARNOLD, Professor and Head, Dept. of Anatomy, University of Minnesota
LEONE, VINCENZO, Professor, Istituto di Zoologia, Milano, Italy
LEVY, MILTON, Professor and Chairman, Dept. of Biochemistry, New York University College
of Dentistry
LEWIN, RALPH ARNOLD, Investigator, Marine Biological Laboratory
LOCHHEAD, JOHN H., Professor of Zoology, University of Vermont
LOFTFIELD, ROBERT B., Associate in Organic Chemistry, Harvard Medical School
LORAND, L., Associate Professor of Chemistry, Northwestern University
LOVE, W'ARNER E., Assistant Professor of Biophysics, Johns Hopkins University
LOWENHAUPT, BENJAMIN, Research Associate, Rockefeller Institute for Medical Research
MCELROY, WILLIAM D., Head, McCollum-Pratt Institute, Johns Hopkins University
MARKS, PAUL A., Assistant Professor of Medicine, College of Physicians and Surgeons, Co-
lumbia University
MARSH, JULIAN B., Assistant Professor of Biochemistry, University of Pennsylvania
MARSHALL, JEAN M., Assistant Professor of Physiology, Johns Hopkins University School of
Medicine
MARSLAND, DOUGLAS, Professor of Biology, New York University, Washington Square College
MATEYKO, GLADYS M., Assistant Professor of Biology, New York University, Washington
Square College
METZ, CHARLES B., Professor, Florida State University
MARINE BIOLOGICAL LABORATORY
MIDDLEBROOK, W. ROBERT, Research Fellow, Institute for Muscle Research, Marine Biological
Laboratory
MONIER, ROGER, Post-doctoral Associate, University of Paris
MOORE, JOHN W., Associate Chief, Laboratory of Biophysics, National Institutes of Health
NACE, PAUL FOLEY, Associate Professor of Biology, McMaster University
NELSON, LEONARD, Assistant Professor, University of Chicago
NEWTON, JACK W., Research Associate, Brandeis University
ODUM, EUGENE P., Professor of Zoology, University of Georgia
OSTERHOUT, W. J. V., Member Emeritus, Rockefeller Institute for Medical Research
PALINCSAR, EDWARD E., Instructor of Biology, Loyola University
PAPACONSTANTINOU, JOHN, Post-doctoral Research Fellow, Carnegie Institution of Washington
PARPART, ARTHUR K., Chairman, Department of Biology, Princeton University
PATERSON, MABEL C., Assistant Professor of Zoology, Vassar College
PERLMANN, GERTRUDE E., Associate Professor, Rockefeller Institute for Medical Research
PROSSER, C. LADD, Professor of Physiology, University of Illinois
RANZI, SILVIO, Full Professor, Istituto di Zoologia, Milano, Italy
RAPPORT, MAURICE M., Professor of Biochemistry, Albert Einstein College of Medicine
RASMUSSEN, HOWARD, Graduate Fellow, Rockefeller Institute for Medical Research
READ, CLARK P., Associate Professor of Parasitology, Johns Hopkins University
REBHUN, LIONEL I., Assistant Professor of Biology, Princeton University
RIESER, PETER, Research Associate, University of Pennsylvania
ROCKSTEIN, MORRIS, Associate Professor of Physiology, New York University College of
Medicine
ROSE, S. MERYL, Professor of Zoology, University of Illinois
ROSENBERG, EVELYN E., Associate Professor of Pathology, New York University-Bellevue
Medical Center
ROSLANSKY, JOHN D., Research Associate, Princeton University
ROTH, JAY S., Associate Professor of Biochemistry, Hahnemann Medical College
ROTHMAN, ALVIN H., Research Fellow, Johns Hopkins University School of Hygiene
RUDOMIN, P., Research Fellow, College of Physicians and Surgeons, Columbia University
RUSHTON, W. A. H., Reader in Physiology, Trinity College, Cambridge, England
RUSTAD, RONALD C., Instructor in Physiology, Florida State University
RYTHER, JOHN H., Staff, Woods Hole Oceanographic Institution
SANBORN, RICHARD C., Professor of Zoology, Purdue University
SANDEEN, MURIEL I., Assistant Professor of Zoology, Duke University
SANDERS, HOWARD L., Research Associate, Woods Hole Oceanographic Institution
SAUNDERS, JOHN W., Professor of Zoology, Chairman Department of Biology, Marquette Uni-
versity
SCHACHMAN, HOWARD K., Associate Professor of Biochemistry, University of California,
Berkeley
SCHUH, REV. JOSEPH E., Associate Professor of Biology and Chairman of Department, Saint
Peter's College
SCOTT, SISTER FLORENCE MARIE, Professor of Biology, Seton Hill College
SCOTT, GEORGE T., Professor and Chairman, Department of Biology, Oberlin College
SELIGER, HOWARD H., Guggenheim Fellow, Johns Hopkins University
SKOGLUND, CARL RUDOLF, Associate Professor, Karolinska Institutet, Stockholm, Sweden
SMITH, PAUL FERRIS, Electronics Engineer, Rockefeller Institute for Medical Research
SMITH, RALPH I., Associate Professor of Zoology, University of California, Berkeley
SPECTOR, ABRAHAM, Instructor, Harvard Medical School
SPEIDEL, CARL C., Professor and Chairman of Anatomy, University of Virginia
SPIEGEL, MELVIN, Assistant Professor of Biology, Colby College
SPRATT, NELSON T., Chairman, Department of Zoology, University of Minnesota
SPYROPOULOS, CONSTANTINE S., Neurophysiologist, National Institutes of Health
STARR, RICHARD C., Associate Professor of Botany, Indiana University
STEELE, RICHARD, Associate Professor of Biochemistry, Tulane University
STEINBACH, H. BURR, Professor and Chairman, Department of Zoology, University of Chicago
STEINHARDT, JACINTO, Director, Operations Evaluation Group, Massachusetts Institute of Tech-
nology
REPORT OF THE DIRECTOR
STEPHENS, GROVER C, Associate Professor of Zoology, University of Minnesota
STETTEN, DEWixx, Associate Director in Charge of Research, National Institutes of Health
STETTEN, MARJORIE R., Biochemist, National Institutes of Health
STEVENS, CHARLES F., Medical Student, Yale University School of Medicine
STONE, WILLIAM, Director of Ophthalmic Plastics Laboratory, Massachusetts Eye and Ear
Infirmary
STREHLER, BERNARD L., Chief, Comparative Physiology Section, National Institutes of Health
STRITTMATTER, PHILIPP, Assistant Professor of Biochemistry, Washington University
STUNKARD, HORACE W., Research Scientist, U. S. Fish and Wildlife Service
SUDAK, FREDERICK N., Instructor in Physiology, Albert Einstein College of Medicine
SUSSMAN, MAURICE, Associate Professor, Brandeis University
SZENT-GYORGYI, ALBERT, Director, Institute for Muscle Research, Marine Biological Laboratory
SZENT-GYORGYI, ANDREW, Investigator, Institute for Muscle Research, Marine Biological Lab-
oratory
TASAKI, ICHIJI, Chief, Special Senses Section, National Institutes of Health
TAYLOR, ROBERT E., Neurophysiologist, National Institutes of Health
TAYLOR, WM. RANDOLPH, Professor of Botany, University of Michigan
TEORELL, TORSTEN, Professor of Physiology, Uppsala University, Sweden
TORCH, REUBEN, Assistant Professor of Zoology, University of Vermont
TROLL, WALTER, Assistant Professor of Industrial Medicine, New York University-Bellevue
Medical Center
TSUBOI, KENNETH K., Assistant Professor of Biochemistry, Cornell University Medical College
TWEEDELL, KENYON S., Assistant Professor of Biology, University of Notre Dame
DEVILLAFRANCA, GEORGE W., Assistant 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 Univer-
sity of New York
WAINIO, WTALTER W., Associate Professor of Biochemistry, Rutgers University
WATANABE, AKIRA, Research Fellow, College of Physicians and Surgeons, Columbia University
WEBB, H. MARGUERITE, Assistant Professor of Biology, Goucher College
WEINSTEIN, PAUL P., Senior Scientist, National Institutes of Health
WEISS, LEON P., Assistant Professor of Anatomy, Harvard Medical School
WELLS, G. P., Professor of Zoology, University College, London, England
WERMAN, ROBERT, Research Associate, College of Physicians and Surgeons, Columbia University
WHITING, ANNA R., Guest Investigator, University of Pennsylvania
WICHTERMAN, RALPH, Professor of Biology, Temple University
WIERCINSKI, FLOYD J., Research Associate, University of Pennsylvania
WILLEY, C. H., Professor of Biology and Chairman of Department, New York University
WILSON, WALTER L., Assistant Professor of Physiology, University of Vermont College of
Medicine
WITTENBERG, JONATHAN B., Assistant Professor of Physiology, Albert Einstein College of
Medicine
WRIGHT, PAUL A., Associate Professor of Zoology, University of New Hampshire
ZWEIFACH, B. W., Professor of Pathology, New York University-Bellevue Medical Center
ZWILLING, EDGAR, Associate Professor, University of Connecticut
Lalor Fellows, 1959
BERNSTEIN, M. H., Wayne State University
CROSTI, NICOLETTA, Bryn Mawr College
ECHALIER, G. P. R., Harvard College
GONSE, P. H., University of Pennsylvania
LEONE, V., Istituto di Zoologia, Milano, Italy
MARSH, J. B., University of Pennsylvania
NELSON, LEONARD, University of Chicago
PALINCSAR, E. E., Loyola University
RANZI, SILVIO, Istituto di Zoologia, Milano, Italy
ROSLANSKY, J. D., Princeton University
24 MARINE BIOLOGICAL LABORATORY
RUSTAD, R. C, Florida State University
SKOGLUND, CARL, Karolinska Instituted Stockholm, Sweden
STRITTMATTER, P., Washington University
Lillie Fellow
RANZI, SILVIO, Istituto di Zoologia, Milano, Italy
Grass Fellows
LIPICKY, RAYMOND, University of Cincinnati
STEVENS, CHARLES, Yale University, School of Medicine
Beginning Investigators, 1959
BENSAM, BERTRAND J., State University of New York, Upstate Medical Center, Syracuse
BROBERG, PATRICIA L., Brandeis University
BURNSTOCK, G., University of Illinois
BUTTERWORTH, FRANK M., University of Pennsylvania
BYERS, THOMAS J., University of Pennsylvania
CAMPBELL, JAMES WAYNE, Johns Hopkins University
CARLSON, ALBERT D., State University of Iowa
CHERNETSKI, KENT EUGENE, University of California
CURTIS, BRIAN A., Rockefeller Institute for Medical Research
DAVIDSON, MORTON, New York University Medical College
DUBNAU, DAVID, Columbia University
DUDEL, JOSEF, Johns Hopkins University
FAUST, ROBERT GILBERT, Princeton University
FILOSA, MICHAEL, Princeton University
GRAHAM, CHARLES EDWARD, Johns Hopkins University School of Medicine
GRIFFIN, DEAN H., American University
GUMP, DIETER W., Johns Hopkins University
GUTTMAN, BURTON S., Institute of Molecular Biology, University of Oregon
HURWITZ, CHARLES, VA Hospital, Albany
HUVER, CHARLES W., Yale University
JACKSON, JAMES A., Western Reserve University
KUPERMAN, ALBERT S., Cornell University Medical College
LAURIE, JOHN S., Tulane University
LIPICKY, RAYMOND JOHN, University of Cincinnati
DE LORENZO, A. J., Johns Hopkins University Medical School
NAGLER, ARNOLD L., New York University-Bellevue Medical Center
NARBAITZ, ROBERTO, Carnegie Institution of Washington and Universidad de Buenos Aires
ORKAND, RICHARD K., University of Utah
PEPE, FRANK A., University of Pennsylvania
POLGAR, GEORGE, University of Pennsylvania School of Medicine
POTTER, DAVID, Johns Hopkins University
REUBEN, JOHN P., College of Physicians and Surgeons, Columbia University
RUBIN, ARNOLD D., New York University College of Medicine
SCHAFER, DAVID G., New York University College of Medicine
SHEPHARD, DAVID, University of Chicago
SMITH, THOMAS G., JR., College of Physicians and Surgeons, Columbia University
SMYTH, THOMAS, JR., Pennsylvania State University
SUDDUTH, SOLON SCOTT, Johns Hopkins University School of Medicine
SWAMI, KARUMURI S., University of Pennsylvania
THEORELL, KLAS T. G., Karolinska Institute!, Stockholm, Sweden
TOBIN, MICHAEL, New York University Medical Center, Downstate
WARNER, ELDON D., University of Wisconsin
REPORT OF THE DIRECTOR 25
WERTHEIM, GUTA, Hebrew University
WHEELER, JAMES ENGLISH, Johns Hopkins University School of Medicine
WINICK, PAUL, Columbia University
Research Assistants, 1959
ALLEN, CONSTANCE, Massachusetts Eye and Ear Infirmary
ASHMAN, ROBERT F., Wabash College
ASHTON, FRANCIS T., University of Pennsylvania
ASTERITA, HARVEY L., New York University
BAIRD, SPENCER, Marine Biological Laboratory
BARN WELL, FRANKLIN H., Northwestern University
BARRON, EVELYN, Massachusetts Eye and Ear Infirmary
BERMAN, LAWRENCE J., Princeton University
BLUMSTEIN, JOYCE R., Albert Einstein College of Medicine
BOLEYN, BRENDA J., University of Rhode Island
BOUCK, G. BENJAMIN, Columbia University
BRANHAM, JOSEPH, Florida State University
BRAVERMAN, MAXWELL H., University of Illinois
BROOKS, KENNETH H., Indiana University
BUNIM, LESLEY S., Barnard College
CICAK, ANNA, Albert Einstein College of Medicine
CLARK, ELOISE E., University of California
CLARK, LYNNE G., Queens College
CONWAY, DOROTHY M., Rockefeller Institute for Medical Research
CORLETTE, SALLY L., Institute for Cancer Research
COUSINEAU, GILLES, University of New York
DELSON, ROZANNE, Massachusetts Institute of Technology
DINGLE, AL D., University of Illinois
DOOLITTLE, RUSSELL F., Harvard University
Doss, DICKY E., American University
DUNSKY, MILTON H., Rockefeller Institute for Medical Research
EIGNER, ELIZABETH ANN, Massachusetts General Hospital
EIN, DANIEL, New York University-Bellevue Medical Center
ERSKINE, LOUISE, Institute for Muscle Research, Marine Biological Laboratory
ESPER, HILDEGARD, Columbia University
FELDHERR, CARL M., University of Pennsylvania
FIELDEN, ANN, University of Illinois
FINKEL, ARNOLD, New York University College of Medicine
FIORENTINO, EILEEN, Hahnemann Medical College
FISHER, FRANK M., JR., Purdue University
FORAN, ELIZABETH H., Smith College
FRIEDLER, GLADYS, Tufts Medical School
FRIEDMAN, LEONARD, Rutgers University
FULTON, CHANDLER M., Rockefeller Institute for Medical Research
GASSELING, MARY T., Marquette University
GEBHART, JOHN H., National Institutes of Health
GOLDFARB, DAVID, Johns Hopkins University
GOUDSMIT, ESTHER M., University of Michigan
GRIFFIN, JOE L., Princeton University
HAAS, FLORENCE ANNE, Western University Medical School
HALEY, BARBARA, Brandeis University
HALL, DONALD J., University of Michigan
HAMPSON, GEORGE RICHARD, Northeastern University
HANSON, FRANK E., JR., State University of Iowa
HASKELL, JUDITH ANN, Purdue University
HATHAWAY, RALPH R., Florida State University
26 MARINE BIOLOGICAL LABORATORY
HIKE, SALLY JAYNE, Mount Holyoke College
HILLMAN, CELIA A., Harvard University
HIMMELFARB, SYLVIA, University of Maryland School of Medicine
HOFFMAN, LARRY R., University of Texas
HOLSTEN, GEORGE H., Ill, Rutgers University
HOLT, CHARLES E., Ill, Massachusetts Institute of Technology
JACKSON, THOMAS JOHN, Lehigh University
JOHNSON, CHRISTINE A., Wheaton College
KAIGHN, MORRIS E., Massachusetts Institute of Technology
KORN, ROBERT WILLIAM, Indiana University
LAMONT, HAYES C, Columbia University
LEIGHTON, CHARLES, Colby College
LIBBIN, DICK, Bard College
LIPPERT, BYRON E., Indiana University
LONIGRO, NORMA, Seton Hill College
LORING, JANET, Harvard Medical School
LOVE, DAVID S., University of Colorado
McCoNNAUGHY, R. A., American University
McGowAN, BERNARD L., Johns Hopkins University
MCLAUGHLIN, JANE, Institute for Muscle Research, Marine Biological Laboratory
MANGUM, CHARLOTTE PRESTON, Vassar College
MAKINEN, PAULA, University of Minnesota
MALKOFF, DONALD B., University of Pittsburgh Medical School
MAVRIDIS, PARASKEVI J., Purdue University
MERRILL, CHARLOTTE F., Massachusetts Institute of Technology
MERSON, GERALD, New York University
MINGIOLI, ELIZABETH S., Harvard University
MOBBERLY, WILLIAM C., Tulane University
MORRISON, ROBERTA ANNE, Smith College
MOULE, JOHN WILLIAM, McMaster University
MOULE, MARGARET, McMaster University
MUELLER, HELMUT, Institute for Muscle Research, Marine Biological Laboratory
OTERO-VILARDEBO, Luis, University of Puerto Rico
PALUBINKAS, BERTHA, College of Physicians and Surgeons, Columbia University
PERRY, BARBARA, Institute for Muscle Research, Marine Biological Laboratory
PHILPOTT, CHARLES W., Tulane University
REICH, MELVIN, Rutgers University
REUBEN, JOHN PHILLIP, University of Florida
ROBERTS, MARY Lou, Washington University Medical School
ROGERS, ANNETTE, North Carolina State College
ROSE, JEANNETTE, Bates College
ROSENBLUTH, RAJA, Columbia University
ROTHSTEIN, HOWARD, University of Pennsylvania
SATUREN, JANICE, State University of New York Upstate Medical Center at Syracuse
SCHROEDER, PAUL C., St. Peter's College
SCHUEL, HERBERT, University of Pennsylvania
SELLERS, RICHARD LEE, American University
SIEGEL, PAULA, University of Cincinnati
SIGER, ALVIN, Johns Hopkins University
SILKOVSKIS, IZOLDE, McMaster University
SIMMONS, JOHN E., Johns Hopkins University
SPENCER, JOYCE M., Harvard Medical School
STAUB, HERBERT W., Rutgers University
STOLL, LOUISE, Johns Hopkins University School of Hygiene
SWOPE, JULIA, Massachusetts General Hospital
SZENT-GYORGYI, EVA, Institute for Muscle Research, Marine Biological Laboratory
SZENT-GYORGYI, MARTA, Institute for Muscle Research, Marine Biological Laboratory
REPORT OF THE DIRECTOR 27
TEYAN, FRED, Albert Einstein College of Medicine
THOMAS, CYNTHIA, Massachusetts Eye and Ear Infirmary
VANLIEW, HUGH D., U. S. Navy, Bethesda
VANNORMAN, EARL, Princeton University
WAHBE, VERA, Kansas University
WATKINS, DUDLEY T., Oberlin College
YATES, LLOYD AUSTIN, University of Minnesota
YIP, CECIL C, McMaster University
Library Readers 1959
BALL, ERIC G., Professor of Biological Chemistry, Harvard Medical School
BAYLOR, MARTHA B., Investigator, Marine Biological Laboratory
BEIDLER, LLOYD M., Professor of Physiology, Florida State University
BODANSKY, OSCAR, Chief, Division of Metabolism and Enzyme Studies, Sloan-Kettering Institute
BROWN, DUGALD, Professor of Zoology, University of Michigan
BUTLER, ELMER G., Professor of Biology, Princeton University
CHASE, AURIN M., Associate Professor of Biology, Princeton University
CLARK, ELIOT R., University of Pennsylvania
COHEN, SEYMOUR S., Professor of Biochemistry, University of Pennsylvania School of Medicine
COLLIER, JACK R., Marine Biological Laboratory
FOERSTER, THEODOR, Professor of Physical Chemistry, Technische Hochschule, Stuttgart, W.
Germany
FRIES, E. F. B., Associate Professor, City College of New York
GABRIEL, MORDECAI L., Associate Professor of Biology, Brooklyn College
GAFFRON, HANS, Professor of Biochemistry, University of Chicago
GINSBERG, HAROLD S., Associate Professor of Preventive Medicine, Western Reserve University
GOLDTHWAIT, DAVID A., Assistant Professor of Biochemistry, Western Reserve University
HUNTER, F. R., Professor and Head, Dept. of Biology, Univ. de los Andes, Bogota, Colombia
JACOBS, M. H., Professor Emeritus, University of Pennsylvania
KARUSH, FRED, Professor of Immunochemistry, University of Pennsylvania
KASHA, A!ICHAEL, Professor of Chemistry, State University of Florida
KLEIN, MORTON, Professor of Microbiology, Temple University School of Medicine
KOZLOFF, LLOYD M., Associate Professor of Biochemistry, University of Chicago
LEIGHTON, JOSEPH, Associate Professor of Pathology, University of Pittsburgh School of
Medicine
LUBIN, MARTIN, Assistant Professor of Pharmacology, Harvard Medical School
LUDWIG, GEORGE D., Assistant Professor of Medicine, University of Pennsylvania
MCDONALD, SISTER ELIZABETH SETOX, Professor of Biology, College of Mt. St. Joseph on
the Ohio
MINARD, FREDERICK, Research Biochemist, Abbott Laboratories
MOUL, EDWIN T., Associate Professor of Botany, Rutgers University
MUSACCHIA, X. J., Associate Professor in Biology, St. Louis University
NOVIKOFF, ALEX B., Research Professor, Albert Einstein College of Medicine
PULLMAN, BERNARD, Professor of Theoretical Chemistry, University of Paris, France
RHULAND, LIONEL E., Research Section Head, The Upjohn Company
ROCHOVANSKY, OLGA M., Research Assistant, Public Health Research Institute of New York .,
City
ROOT, WALTER S., Professor of Physiology, College of Physicians and Surgeons, Columbia
University
ROTH, FR. OWEN H., Associate Professor of Zoology, St. Vincent College
SCHLAMOWITZ, MAX, Associate Cancer Research Scientist, Roswell Park Memorial Institute
SERBER, BARBARA Jo, Assistant Professor of Anatomy, New York University-Bellevue Medical '
Center
SONNENBLICK, B. P., Professor of Biology, Rutgers University
SULKIN, S. EDWARD, Professor and Chairman, Dept. of Microbiology, University of Texas,
Southwestern Medical School
MARINE BIOLOGICAL LABORATORY
TRURNIT, HANS J., Senior Scientist, Research Institute for Advanced Study
WARNER, ROBERT C, Associate Professor of Biochemistry, New York University College of
Medicine
WEIGLE, WILLIAM O., Assistant Research Professor, University of Pittsburgh School of
Medicine
WHEELER, GEORGE E., Instructor in Biology, Brooklyn College
YNTEMA, CHESTER L., Professor of Anatomy, State University of New York, Upstate Medical
Center
ZINN, DONALD J., Associate Professor of Zoology, University of Rhode Island
Students 1959
BOTANY
BROWN, MALCOLM, University of Texas
CHURCHILL, ALGERNON C., Harvard University
CORRELL, DAVID L., Michigan State University
EDWARDS, JACKIE L., University of Alabama
EHRLICH, DIANA LEE, College of the City of New York
FINDLEY, DAVIS L., University of Alabama
FLACH, MARY E., Vassar College
FOLDATS, ERNESTO, Universidad Central de Venezuela
FREDERICKS, WALTER W., Johns Hopkins University
GOLAS, MARY, Marquette University
KALIL, MILDRED, Wellesley College
KOOB, DERRY DELOS, Cornell University
MASON, CHARLES P., Cornell University
MILES, MARJORIE L., Acadia University
MORRIS, RUTH CAROL, Cornell University
NOLAN, RICHARD A., University of Nebraska
SHOR, BERNICE C., Rollins College
WAGNER, KENNETH A., College of William and Mary
WILLIAMS, RICHARD B., Harvard University
ZACHARIA, KURUVILA, Princeton University
EMBRYOLOGY
ASHMAN, ROBERT F., Wabash College
BAKER, JOHN R., University of Minnesota
BERGMANN, FRED H., Brandeis University
BIRKY, C. WILLIAM, JR., Indiana University
CORDES, EUGENE H., Brandeis University
CURTIS, JOSEPH C., Brown University
GIBLEY, CHARLES W., JR., Iowa State College
GRAND, THEODORE L, Brown University
GRINNELL, ALAN D., Harvard University
,HARRIS, THOMAS M., University of North Carolina
HENNEN, SALLY H., Indiana University
HOLT, CHARLES E., Ill, Massachusetts Institute of Technology
KESSLER, DIETRICH, University of Wisconsin
LAWRENCE, IRVIN E., Kansas University
LESSUPS, ROLAND J., S. J., Johns Hopkins University
"LEVINE, STEPHEN, Brandeis University
MERSON, GERALD, New York University Medical School
PIERCE, GORDON B., University of Pittsburgh
ROSE, IRWIN A., Yale University
SCHULER, MARGERY E., Wesleyan University
REPORT OF THE DIRECTOR 29
STEINBERG, SONIA NAOMI, Northwestern University
WHITTAKER, J. RICHARD, Yale University
VATES, ROBERT D., University of Alabama Medical Center
PHYSIOLOGY
ALVAREDO, FRANCISCO, New York University, College of Medicine
ANGELES, LETICIA, Tulane University
BENJAMIN, THOMAS, Amherst College
EISEN, JAMES, Emory University
GARRICK, MICHAEL, Johns Hopkins University
GILLESPIE, BARBARA, Radcliffe University
GOTTLIEB, ABRAHAM, New York University-Bellevue Medical Center
GREEN, MORRIS, University of Rochester
HAMILTON, MARY, Sloan-Kettering Institute
HANDLER, JOSEPH, University of Pennsylvania
HOMER, Louis, Medical College of Virginia
KALEY, GABOR, New York University
KEAN, EDWARD, University of Pennsylvania
KINSOLVING, CLYDE, Vanderbilt University
LIEBMAN, PAUL, Barnes Hospital
LUCHI, ROBERT, University of Pennsylvania
PLOTZ, PAUL, Harvard Medical School
PURPLE, RICHARD, Rockefeller Institute
ROSENBAUM, JOEL, Syracuse University
RYSER, HUGUES, Massachusetts General Hospital
SLAYMAN, CLIFFORD, Rockefeller Institute
THEORELL, HENNING, Karolinska Inst., Stockholm
TODARO, GEORGE, New York University College of Medicine
TOWNSEND, EDITH, McGill University
WALCH, CAROLYN, Johns Hopkins University
WEISS, CHARLES, Harvard University
WRITTEN BURY, GUILLERMO, Harvard Medical School
INVERTEBRATE ZOOLOGY
ANDREW, OLIVER T., Franklin and Marshall College
BERCHMANS, SISTER ANN, St. Mary of the Woods College
BREBBIA, DANTE R., Fordham University Graduate School
BRENOWITZ, HARRY, Adelphi College
BUCKLEY, BROTHER WILLIAM, Fordham University
CHURCHILL, ALGERNON, Harvard University
COXROW, MARY M., Wilson College
CORRELL, DAVID, Michigan State University
DELONG, KARL T., Oberlin College
EDDY, JANE, Tufts University
EDWARD, BROTHER C, Fordham University
ELLISON, ESTHER, University of Minnesota
ENGLUND, PAUL, Hamilton College
EPEL, DAVID, University of California, Berkeley
FEIR, DOROTHY J., University of Wisconsin
FERGUSON, JOHN, Cornell University
GAGE, ELIZABETH M., Gushing Academy
GATES, DAVID A., Clark University
GOLDMAN, LAWRENCE, University of California, Los Angeles
GREENE, LAUREL E., Goucher College
GUTKNECHT, JOHN, University of North Carolina
30 MARINE BIOLOGICAL LABORATORY
HAYES, WILLIAM, University of Michigan
HENDERSON, OLIVER, JR., The Citadel
HUBER, SALLY A., Mt. Holyoke College
IZOWER, JACK, City College of New York
JONES, LYNNE A., Connecticut College
KRAUSE, HELEN, University of Massachusetts
LAFAUCI, GRACE, Wilson College
MANGUM, CHARLOTTE, Vassar College
MARZOLF, GEORGE, University of Michigan
MCDOWELL, SISTER MARGARET ANN, College of St. Mary of the Springs
AIcWniNNiE, DOLORES J., DePaul University
MESCHER, SISTER ALMA L., University of Notre Dame
AIoFFEY, ELIZABETH S., University of Michigan
MOULTON, JOHN, Hastings College and Clark University
NORBECK, BETTY, University of Minnesota
NORDLIE, FRANK, University of Minnesota
PROSSER, JANE ELLEN, Earlham College
RAPPAPORT, LUCINDA, Brandeis University
SEECK, MARGARET A., Oberlin College
SHAW, WILLIAM N., Bureau of Commercial Fisheries
SHOR, BERNICE, Rollins College
SIMPSON, MARGARET, Catholic University of America
STERNS, CAROL W., Peekskill, New York
STONG, CYNTHIA C, Wellesley College
THOMAS, CAROLINE, University of Vermont
VERRUSIO, A. CARL, Drew University
WILLIAMS, JUNARDEN, Northwestern University
ZIMMERMAN, WILLIAM, Princeton University
ZOTTOLI, ROBERT, Bowdoin College
ECOLOGY
ABBIATE, LORRAINE M., Douglass College
BACHMAN, ROGER W., University of Michigan
BIANCHI, CARLA F., Chatham College
BURKHOLDER, K. M., Emory University
DAVEY, TESSA, Mount Holyoke College
HAYWARD, GEORGE E., Drew University
PALMER, JOHN D., Northwestern University
PINCHOT, GIFFORD B., Johns Hopkins University
MCLAUGHLIN, ELLEN, University of North Carolina
SWEENEY, EDWARD F., Boston University
SWIFT, ELIJAH, Swarthmore College
TAYLOR, WALTER R., Johns Hopkins University
WATT, WALTON D., Dalhousie University
WHITELEY, GEORGE Co., The Hill School
WILLIAMS, ELSIE LOUISE, Goucher College
3. FELLOWSHIPS AND SCHOLARSHIPS, 1959
Lucretia Crocker Scholarships :
CHARLES P. MASON, Botany Course
JOHN D. PALMER, Ecology Course
Conklin Scholarship :
STEPHEN LEVINE, Embryology Course
Bio Club Scholarships :
DIANA LEE EHRLICH, Botany Course
JACK IZOWER, Invertebrate Zoology Course
REPORT OF THE DIRECTOR
31
4. TABULAR VIEW OF ATTENDANCE, 1955-1959
1955 1956 1957 1958 1959
INVESTIGATORS— TOTAL 250 304 326 410 427
Independent 162 184 186 203 215
Under Instruction 9 20 23 39 45
Library Readers 54 50 42 54 51
Research Assistants 25 50 75 114 116
STUDENTS— TOTAL 148 140 139 138 134
Invertebrate Zoology 56 55 55 55 49
Embryology 30 28 27 22 23
Physiology 30 30 30 27 27
Botany 19 18 18 18 20
Ecology 13 9 9 16 15
TOTAL ATTENDANCE 398 444 465 548 561
Less persons represented as both investigators and
students 2 3 4
398 442 462 543 557
INSTITUTIONS REPRESENTED — TOTAI 129 130 129 142 143
By Investigators 95 97 94 110 98
By Students 34 33 35 74 73
SCHOOLS AND ACADEMIES REPRESENTED
By Investigators 3 3 5 12
By Students 2 1 1 2 8
FOREIGN INSTITUTIONS REPRESENTED
By Investigators 8 9 11 20 29
By Students 6 6 5 6 9
5. INSTITUTIONS REPRESENTED, 1959
Abbott Laboratories
A & M College of Texas
Adelphi College
Agricultural Research Center
Alabama, University of
Albert Einstein College of Medicine
American Heart Association
American University
Amherst College
Barnes Hospital
Bowdoin College
Brandeis University
Brooklyn College
Brown University
Bryn Mawr College
Buffalo, University of
California, University of
Carnegie Institution of Washington
Catholic University
Chatham College
Chicago, University of
Cincinnati, University of
City College of New York
Colby College
College of St. Mary of the Springs
College of William and Mary
Columbia University
Columbia University, College of Physicians
and Surgeons
Connecticut College
Connecticut, University of
Cornell University
Cornell University Medical School
Cushing Academy
Department of the Interior
DePaul University
Drew University
Duke University
Earlham College
Emory University
Florida State University
Fordham University
Franklin and Marshall College
Georgia, University of
Goucher College
Hahnemann Medical School
Hamilton College
Harvard University
Harvard University Medical School
Hastings College
Illinois, University of
Indiana State Teachers College
Indiana University
Institute for Muscle Research
32
MARINE BIOLOGICAL LABORATORY
Iowa State University
Johns Hopkins University
Kansas University
Louisiana State University
Loyola College
Maine, University of
Manhattan College
Marquette University
Maryland, University of
Massachusetts Eye and Ear Infirmary
Massachusetts General Hospital
Massachusetts Institute of Technology
Medical College of Virginia
Michigan State University
Michigan, University of
Minnesota, University of
Montefiore Hospital Research Institute
Mount Holyoke College
Mt. St. Joseph, College of
National Institutes of Health
Nebraska, University of
New Hampshire, University of
New York State University College of Medi-
cine at Syracuse
New York University
New York University, Bellevue Medical
Center
New York University School of Dentistry
New York University, Washington Square
College
North Carolina State College
North Carolina, University of
Northwestern University
Notre Dame University
Oak Ridge National Laboratory
Oberlin College
Ohio Wesleyan University
Oklahoma, University of
Oregon, University of
Orleans High School
Pennsylvania, University of
Pennsylvania Medical School, University of
Pittsburgh, University of
Princeton University
Purdue University
Queens College
Radcliffe College
Reed College
Research Institute for Advanced Studies
Rhode Island, University of
Rochester, University of
Rockefeller Institute for Medical Research
Rollins College
Roswell Park Memorial Institute
Rutgers University
St. Joseph's College
St. Louis University
St. Mary of the Woods College.
St. Peter's College
St. Vincent College
Seton Hill College
Single Cell Research Foundation
Sloan-Kettering Institute
Smith College
Swarthmore College
Sweet Briar College
Syracuse University
Temple University
Texas, University of
Texas, University of, Southwestern Medical
School
The Hill School
Tufts University
Tulane University
Upjohn Company
Utah, University of
U. S. Fish and Wildlife Service
U. S. Public Health Service
Vanderbilt University
Vassar College
Vermont, University of
Veterans Administration Hospital
Virginia, University of
Washington University
Washington University Medical School
Washington and Jefferson College
Wayne State University
Wellesley College
Wesleyan University
Western Reserve University
Wilson College
Wisconsin, University of
Woods Hole Oceanographic Institution
Yale University
FOREIGN INSTITUTIONS REPRESENTED, 1959
Institute de Anatomica y Embriologia, Uni-
versidad de Buenos Aires, Argentina
University of Brussels, Belgium
Arcadia University, Canada
Dalhousie University, Canada
McGill University, Canada
McMaster University, Canada
University de los Andes, Bogota, Colombia
King's College, England
Trinity College, England
University College, England
Sorbonne, Paris, France
University of Paris, France
Max-Plank Institiit fur Virusforschung, Ger-
many
Technical University, Darmtstadt, Germany
REPORT OF THE DIRECTOR
Technische Hochschule, Germany Karolinska Institutet, Stockholm, Sweden
Madras Christian College, Madras, India Uppsala University, Sweden
Hebrew University, Israel Clinique Medicale Universitaire, Switzerland
University of Milan, Milan, Italy University of Witwatersrand, Johannesburg,
University of Tokyo, Japan South Africa
University of Puerto Rico, Puerto Rico Universidad Central de Venezuela, Venezuela
University of the Philippines, Philippines University College of the West Indies, Ja-
Centro Investigaciones Biologicas, Madrid, maica, West Indies
Spain
SUPPORTING INSTITUTIONS AND AGENCIES, 1959
American Cancer Society Olin Mathieson Chem. Corporation, Charitable
Associates of the Marine Biological Labora- Trust
tory National Institutes of Health
Atomic Energy Commission National Science Foundation
Josephine B. Crane Foundation Office of Naval Research
The Grass Foundation The Rockefeller Foundation
The Lalor Foundation Smith, Kline and French Foundation
F. R. Lillie Fellowship
CORPORATE ASSOCIATES
Abbott Laboratories Merck Company Foundation
Ciba Pharmaceutical Products, Inc. Schering Corporation
Carter Products, Inc. The Upjohn Company
Eli Lilly and Company Wyeth Laboratories
6. EVENING LECTURES, 1959
June 26
R. E. BILLINGHAM "Studies on the Y chromosome antigen in
rodents"
July 3
ALEXANDER FORBES "The growth of physiology"
July 6
ALEXANDER FORBES "Electrophysiology of color vision"
July 10
I. M. KLOTZ "Protein hydration and behavior"
July 17
C. LADD PROSSER •' 'The origin' after a century; prospects for
the future"
July 24
SILVIO RANZI "Protein differentiation during embryonic
and larval development"
July 31
V. G. DETHIER "Chemical sense of the blowfly and hunger''
August 7
CLAUDE A. VILLEE "Interrelations of hormones and enzymes"
August 14
HUGO THEORELL "Mode of action of enzyme-coenzyme com-
plexes"
34 MARINE BIOLOGICAL LABORATORY
August 21
W. A. H. RUSHTON "The retina is the net of a fisherman who
catches quanta and barters them for infor-
mation"
August 28
EUGENE P. ODUM "The energy flow to the study of populations
in nature"
7. TUESDAY EVENING SEMINARS, 1959
July 7
J. L. GRIFFIN "Isolation and chemical identification of the
crystalline cytoplasmic inclusions in the
large, free-living amebae"
MILTON FINGERMAN "Physicochemical characterization of chro-
matophorotropins in the crayfish, Cam-
barellus shujeldti"
WILLIAM H. JOHNSON AND
ANDREW G. SZENT GYORGYI "The molecular basis for the 'catch mech-
anism' in molluscan muscles"
July 14
JAMES D. EISEN "A study on the physiology of the predator-
prey relationship existing between Para-
mecium aurelia and Didinium nasututn"
WOLFGANG WIESER "Growth, metabolism and coexistence in ma-
rine nematodes"
RALPH A. LEWIN "Uptake of strontium by Syracosphaera"
July 21
A. B. NOVIKOFF "Lysosomes in the physiology and pathology
of cells"
J. R. COLLIER "Localization and synthesis of ribonucleic
acid in the development of Ilyanassa ob-
soleta"
G. G. HOLZ, JR. AND
C. C. SPEIDEL "Mating behavior of x-rayed Tetrahymena
pyriformis." Motion pictures
C. FULTON "Polarized tissue movement in hydroid re-
generation." Motion pictures
July 28
PHILIP PERSON, JAY W. LASH AND
ALBERT FINE "Myoglobin and cytochrome oxidase in odon-
tophore cartilage of Busycon"
W. TROLL, S. BELMAN AND
N. NELSON . ."Aromatic amine metabolism and bladder
cancer"
PAUL S. GALTSOF'F AND
D. E. PHILPOTT "Ultra structure of the spermatozoon of the
oyster"
August 4
VINCENZO LEONE "Some structures found in electron micro-
scopic pictures of an amphibian tumour"
ALFRED W. SENFT "Ultrastructure of the human parasite, Schis-
tosoina mansoni"
REPORT OF THE DIRECTOR 35
GEORGE W. DE VILLAFRANCA AND
DELBERT E. PIIILPOTT "A study of the fine structure of skeletal
muscle from Limuhis polyphemus"
August 11
MAURICE M. RAPPORT "Present status of the problem of plasmalo-
gen structure"
ERIC G. BALL "On the mode of action of insulin"
WALTER S. VINCENT AND
ELYANE BALTIS "Incorporation of isotopic label into RNA :
synthesis or terminal addition?"
August 18
L. V. HEILBRUNX "The action of glycerol on protoplasm"
WALTER L. WILSON AND
K. S. SWAMI "Electrophoretic studies on protoplasm"
FRANCIS T. ASIITON "Germinal vesicle breakdown in the eggs of
Spisula and Hydroides"
R. D. ALLEN "Polarized optical studies on Ameba"
F. CHILD "Isolation and analysis of cilia"
8. MEMBERS OF THE CORPORATION, 1959
1. LIFE MEMBERS
BRODIE, MR. DONALD M., 522 Fifth Avenue, New York 18, New York
CALVERT, DR. PHILIP P., University of Pennsylvania, Philadelphia, Pennsylvania
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
DEDERER, DR. PAULINE H., Connecticut College, New London, Connecticut
GOLDFARB, DR. A. J., College of the City of New York, New York City, New York
KNOWLTON, DR. F. P., 1356 Westmoreland Avenue, Syracuse, New York
LEWIS, DR. W. H., Johns Hopkins 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
MOORE, DR. J. PERCY, University of Pennsylvania, Philadelphia, Pennsylvania
PAYNE, DR. FERNANDUS, Indiana University, Bloomington, Indiana
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
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
YOUNG, DR. B. P., Cornell University, Ithaca, New York
2. REGULAR MEMBERS
ABELL, DR. RICHARD G., 7 Cooper Road, New York City, New York
ADAMS, DR. A. ELIZABETH, Mount Holyoke College, South Hadley, Massachusetts
36 MARINE BIOLOGICAL LABORATORY
ADDISON, DR. W. H. F., 286 East Sidney Avenue, Mount Vernon, New York
ADOLPH, DR. EDWARD F., University of Rochester School of Medicine and
Dentistry, 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
AMBERSON, DR. WILLIAM R., Department of Physiology, University of Maryland
School of Medicine, Baltimore, Maryland
ANDERSON, DR. J. M., Department of Zoology, Cornell University, Ithaca, New
York
ANDERSON, DR. RUBERT S., Medical Laboratories, Army Chemical Center, Mary-
land (Box 632, Edgewood, Maryland)
ANDERSON, DR. T. F., Institute for Cancer Research, Fox Chase, Philadelphia,
Pennsylvania
ARMSTRONG, DR. PHILIP B., 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., Department of Pediatrics, University of Chicago, Chi-
cago, 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
BAKER, DR. H. B., Department of Zoology, University of Pennsylvania, Philadel-
phia 4, Pennsylvania
BALL, DR. ERIC G., Department of Biological Chemistry, Harvard University
Medical School, Boston 15, Massachusetts
BALLARD, DR. WILLIAM W., Dartmouth College, Hanover, New Hampshire
BANG, DR. F. B., Department of Pathobiology, Johns Hopkins University School
of Hygiene, Baltimore 5, Maryland
BARD, DR. PHILIP, Johns Hopkins Medical School, Baltimore, Maryland
EARTH, DR. L. G., Department of Zoology, Columbia University, New York 27,
New York
BARTLETT, DR. JAMES H., Department of Physics, University of Illinois, Urbana,
Illinois
BEAMS, DR. HAROLD W., Department of Zoology, State University of Iowa, Iowa
City, Iowa
BECK, DR. L. V., Department of Physiology and Pharmacology, University of Pitts-
burgh School of Medicine, Pittsburgh 13, Pennsylvania
BEERS, DR. C. D., Department of Zoology, University of North Carolina, Chapel
Hill, North Carolina
REPORT OF THE DIRECTOR 37
BEHRE, DR. ELINOR H., Black Mountain, North Carolina
BENESCH, DR. REINHOLD, Marine Biological Laboratory, Woods Hole, Massa-
chusetts
BENESCH, DR. RUTH, Marine Biological Laboratory, Woods Hole, Massachusetts
BENNETT, DR. MIRIAM F., Department of Biology, Sweet Briar College, Sweet
Briar, Virginia
BERG, DR. WILLIAM E., Department of Zoology, University of California, Berke-
ley 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 University College of
Medicine, Detroit 7, Michigan
BERTHOLF, DR. LLOYD, Illinois Wesleyan University, Bloomington, Illinois
BEVELANDER, DR. GERRIT, New York University School of Medicine, 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, Baltimore 5, Maryland
BLANCHARD, DR. K. C, Johns Hopkins Medical School, Baltimore, Maryland
BLOCK, DR. ROBERT, 518 South 42nd Street, Apt. C 7, 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, Department of Zoology, University of Pennsylvania, Philadel-
phia 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
University, Evanston, Illinois
BROWNELL, DR. KATHERINE 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 An-
geles 24, California
BURBANCK, DR. WILLIAM D., Box 834, Emory University, 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. GIULIO, 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 City, New York
CATTELL, MR. WARE, Cosmos Club, Washington 5, D. C.
CHAET, DR. ALFRED B., Department of Biology, American University, Washing-
ton 16, D. C.
CHAMBERS, DR. EDWARD, Department of Physiology, University of Miami Medical
School, Coral Gables, Florida
CHANG, DR. JOSEPH J., Akademiestr. 3, Physiologiscb.es Inst., Postfach 201,
Heidelberg, Germany
CHASE, DR. AURIN M., Department of Biology, Princeton University, Princeton,
New Jersey
CHENEY, DR. RALPH H., Biology Department, Brooklyn College, Brooklyn 10,
New York
CLAFF, DR. C. LLOYD, 5 Van Beal Road, Randolph, Massachusetts
CLARK, DR. A. M., Department of Biology, University of Delaware, Newark,
Delaware
CLARK, DR. E. R., The Wistar Institute, Woodland Avenue and 36th Street, Phila-
delphia 4, Pennsylvania
CLARK, DR. LEONARD B., Department of Biology, Union College, Schenectady,
New York
CLARKE, DR. GEORGE L., Harvard University, Biological Laboratories, Cambridge
38, Massachusetts
REPORT OF THE DIRECTOR 39
CLELAND, DR. RALPH E., Indiana University, Bloomington, Indiana
CLEMENT, DR. A. C, Department of Biology, Emory University, Atlanta 22,
Georgia
COE, DR. W. R., 183 Third Avenue, Chula Vista, California
COHEN, DR. SEYMOUR S., Department of Biochemistry, University of Pennsyl-
vania School of Medicine. Philadelphia 4, Pennsylvania
COLE, DR. KENNETH S., National Institutes of Health (NINDB), Bethesda 14,
Maryland
COLLETT, DR. MARY E., 34 Weston Road, Wellesley 81, Massachusetts
COLLIER, DR. JACK R., Department of Zoology, Louisiana State University, Baton
Rouge, Louisiana
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 Zoology, University of Florida, Gaines-
ville, Florida
COOPERSTEIN, DR. SHERWIN J., Department of Anatomy, Western Reserve Uni-
versity Medical School, Cleveland, Ohio
COPELAND, DR. D. E., 8705 Susanna Lane, Chevy Chase 15, Maryland
COPELAND, DR. MANTON, Bowdoin College, Brunswick, Maine
COPLEY, DR. A. L., Medical Research Laboratories, Charing Cross Hospital, 8 Ex-
change Ct., Strand, London, W. C. 2
CORN MAN, DR. IVOR, Hazleton 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 Univer-
sity Medical School, St. Louis, Missouri
CROASDALE, DR. HANNAH T., Dartmouth College, Hanover, New Hampshire
CROUSE, DR. HELEN V., Goucher College, Towson, Baltimore 4, Maryland
CROWELL, DR. P. S., JR., Department of Zoology, Indiana University, Blooming-
ton, Indiana
CSAPO, DR. ARPAD I., Rockefeller Institute for Medical Research, 66th Street and
York Avenue, New York 21, New York
CURTIS, DR. MAYNIE R., University of Miami, Box 1015, 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
40 MARINE BIOLOGICAL LABORATORY
DAWSON, DR. A. J., College of the City of New York, New York City, New York
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,
Pennsylvania
DILLER, DR. WILLIAM F., 2417 Fairhill Avenue, Glenside, Pennsylvania
DIXON, DR. FRANK J., Department of Pathology, University of Pittsburgh School
of Medicine, Pittsburgh 13, Pennsylvania
DODDS, DR. G. S., West Virginia University School of Medicine, Morgantown,
West Virginia
DOLLEY, DR. WILLIAM L., Department of Biology, Randolph- Macon College,
Ashland, Virginia
DONALDSON, DR. JOHN C., University of Pittsburgh School of Medicine, Pitts-
burgh, Pennsylvania
DOTY, DR. MAXWELL S., Department of Biology, University of Hawaii, Honolulu,
Hawaii
DURYEE, DR. WILLIAM R., George Washington University School of Medicine,
Department of Physiology, Washington 5, D. C.
EDDS, DR. MAC V., JR., Department of Biology, Brown University, Providence 12,
Rhode Island
EDWARDS, DR. CHARLES, University of Utah, Salt Lake City, Utah
EICHEL, DR. HERBERT J., Hahnemann Medical College, Philadelphia, Pennsylvania
EISEN, DR. HERMAN, Department of Medicine, Washington University, St. Louis,
Missouri
ELLIOT, 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, Iowa City, Iowa
FAILLA, DR. G., Columbia University, College of Physicians and Surgeons, New
York 32, New York
FAURE-FREMIET, DR. EMMANUEL, College de France, Paris, France
FERGUSON, DR. F. P., Department of Physiology, University of Maryland Medical
School, Baltimore 1, 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 19, 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
REPORT OF THE DIRECTOR 41
FRAENKEL, DR. GOTTFRIED S., Department of Entomology, University of Illinois,
Urbana, Illinois
FREYGANG, DR. WALTER H., JR., Box 516, Essex Fells, New Jersey
FRIES, DR. ERIK F. B., Box 605, Woods Hole, Massachusetts
FRISCH, DR. JOHN A., Canisius College, Buffalo, New York
FURTH, DR. JACOB, 18 Springdale Road, Wellesley Farms, Massachusetts
FYE, DR. PAUL M., Director, Woods Hole Oceanographic Institution, Woods
Hole, Massachusetts
GABRIEL, DR. MORDECAI, Department of Biology, Brooklyn College, Brooklyn 10,
New York
GAFFRON, DR. HANS, Research Institutes, University of Chicago, 5650 Ellis
Avenue, Chicago 37, Illinois
GALL, DR. JOSEPH G., Department of Zoology, University of Minnesota, Minneapolis
14, Minnesota
GALTSOFF, DR. PAUL S., Woods Hole, Massachusetts
GASSER, DR. HERBERT S., Rockefeller Institute, 66th Street and York Avenue,
New York 21, New York
GILMAN, DR. LAUREN C, Department of Zoology, University of Miami, Coral
Gables, Florida
GINSBERG, DR. HAROLD S., Western Reserve University School of Medicine, Cleve-
land, Ohio
GOLDSTEIN, DR. LESTER, Department of Zoology, University of Pennsylvania, Phila-
delphia, Pennsylvania
GOODCHILD, DR. CHAUNCEY G., Department of Biology, Emory University, Atlanta
22, Georgia
GOODRICH, DR. H. B., 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, Massachu-
setts
GRAND, MR. C. G., Dade County Cancer Institute, 1155 N. W. 15th Street, Miami,
Florida
GRANT, DR. M. P., Sarah Lawrence College, Bronxville, New York
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, Microbiology Department, 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
42 MARINE BIOLOGICAL LABORATORY
GRIFFIN, DR. DONALD R., Biological Laboratories, Harvard University, Cam-
bridge 38, Massachusetts
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 City, New York
GUDERNATSCH, DR. FREDERICK, 41 Fifth Avenue, New York 3, New York
GUTHRIE, DR. MARY J., Detroit Institute for Cancer Research, 4811 John R. Street,
Detroit, Michigan
GUTTMAN, DR. RITA, Department of Physiology, Brooklyn College, Brooklyn 10,
New York
HAJDU, DR. STEPHEN, U. S. Public Health Institute, 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., Box R.R. #3, 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
HARRISON, DR. Ross G., Osborn Zoological Laboratories, Yale University, New
Haven, Connecticut
HARTLINE, DR. H. KEFFER, Rockefeller Institute for Medical Research, 66th Street
and York Avenue, New York 21, New York
HARTMAN, DR. FRANK A., Hamilton Hall, Ohio State University, Columbus, Ohio
HARVEY, DR. ETHEL BROWNE, 48 Cleveland Lane, Princeton, New Jersey
HAUSCHKA, DR. T. S., Roswell Park Memorial Institute, 666 Elm Street, Buffalo
3, New York
HAXO, DR. FRANCIS T., Division of Marine Botany, Scripps Institute of Ocean-
ography, University of California, La Jolla, California
HAYASHI, DR. TERU, Department of Zoology, Columbia University, New York
27, New York
HAYDEN, DR. MARGARET A., 34 Weston Road, Wellesly 81, Massachusetts
HAYWOOD, DR. CHARLOTTE, Mount Holyoke College, South Hadley, Massachusetts
HEILBRUNN, DR. L. V., Department of Zoology, University of Pennsylvania,
Philadelphia 4, Pennsylvania
HENDLEY, DR. CHARLES D., 615 South Second Avenue, Highland Park, New
Jersey
HENLEY, DR. CATHERINE, Department of Zoology, University of North Carolina,
Chapel Hill, North Carolina
HERVEY, DR. JOHN P., Box 735, Woods Hole, Massachusetts
HESS, DR. WALTER N.. Hamilton College, Clinton, New York
HTATT, DR. HOWARD H., Department of Medicine, Harvard Medical School, Boston
15. Massachusetts
REPORT OF THE DIRECTOR 43
HIBBARD, DR. HOPE, Department of Zoology, Oberlin College, Oberlin, Ohio
HILL, DR. SAMUEL E., 135 Brunswick Road, Troy, New York
HISAW, DR. F. L., Biological Laboratories, Harvard University, Cambridge 38,
Massachusetts
HOADLEY, DR. LEIGH, Biological Laboratories, Harvard University, Cambridge
38, Massachusetts
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,
Pennsylvania
HOLLAENDER, DR. ALEXANDER, Biology Division, O.R.N.L., Oak Ridge, Tennessee
HOLZ, DR. GEORGE G., JR., Department of Zoology, Syracuse University, Syracuse,
New York
HOPKINS, DR. HOYT S., New York University College of Dentistry, New York
City, New York
HUNTER, DR. FRANCIS R., University of the Andes, Calle 18-a Carreral-E, Bogota,
Colombia, South America
HUTCHENS, DR. JOHN O., Department of Physiology, University of Chicago,
Chicago 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
ISELIN, MR. COLUMBUS O'D., Woods Hole, Massachusetts
JACOBS, DR. M. H., University of Pennsylvania School of Medicine, Philadelphia 4,
Pennsvlvania
^
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., Biology Department, Princeton University, Princeton,
New Jersey
JONES, DR. E. RUFFIN, JR., Department of Biological Sciences, University of
Florida, Gainesville, Florida
KAAN, DR. HELEN W., Marine Biological Laboratory, Woods Hole, Massachu-
setts
RABAT, DR. E. A., Neurological Institute, College of Physicians and Surgeons,
New- York City, New York
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
44 MARINE BIOLOGICAL LABORATORY
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, 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 Chemistry, University of Connecticut, Storrs,
Connecticut
KINDRED, DR. J. E., University of Virginia, Charlottesville, Virginia
KING, DR. JOHN W., Morgan State College, Baltimore 12, Maryland
KING, DR. ROBERT L., State University of Iowa, Iowa City, Iowa
KISCH, DR. BRUNO, 845 West End Avenue, New York City, New York
KLEIN, DR. MORTON, Department of Microbiology, Temple University, Philadel-
phia, Pennsylvania
KLEIN HOLZ, DR. LEWIS H., Department of Biology, Reed College, Portland. 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. I. M., Department of Physiology, Kirksville College of Osteopathy,
Kirksville, Missouri
KRAHL, DR. M. E., Department of Physiology, University of Chicago, Chicago 37,
Illinois
KRAUSS, DR. ROBERT, Department of Botany, University of Maryland, Baltimore 5,
Maryland
KREIG, DR. WENDELL J. S., 303 East Chicago Avenue, Chicago, Illinois
KUFFLER, DR. STEPHEN, Department of Ophthalmology, Johns Hopkins Univer-
sity, Baltimore 5, Maryland
KUNITZ, DR. MOSES, Rockefeller Institute, 66th Street and York Avenue, New
York 21, New York
LACKEY, DR. JAMES B., Box 497, Melrose, Florida
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
LANDIS, DR. E. M., Harvard Medical School, Boston 15, Massachusetts
LANSING, DR. ALBERT L, Department of Anatomy, University of Pittsburgh Medical
School, Pittsburgh 13, Pennsylvania
LAUFFER, DR. MAX A., Department of Biophysics, University of Pittsburgh, Pitts-
burgh, Pennsylvania
LAVIN, DR. GEORGE L, 3714 Springdale Avenue, Baltimore, Maryland
LAZAROW, DR. ARNOLD, Department of Anatomy, University of Minnesota Medical
School, Minneapolis 14, Minnesota
LEDERBERG, DR. JOSHUA, Department of Genetics, Stanford University Medical
School, Stanford, California
LEE, DR. RICHARD E., Cornell University College of Medicine, New York City,
New York
LEFEVRE, DR. PAUL G., Brookhaven Apartments, Upton. Long Island, New York
REPORT OF THE DIRECTOR 45
LEHMANN, DR. FRITZ, Zoologische Institut, University of Berne, Berne, Switzerland
LEVINE, DR. RACHMIEL, Michael Rees 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., Marine Biological Laboratory, Woods Hole, Massachusetts
LEWIS, DR. IVEY F., 1110 Rugby Road, Charlottesville, Virginia
LING, DR. GILBERT, 307 Berkely 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. LEO, 40 Crestwood Drive, St. Louis 5, Missouri
LOEB, DR. R. F., 950 Park Avenue, New York 28, New York
LOEWI, DR. OTTO, 155 East 93rd Street, New York City, New York
LORAND, DR. LASZLO, Department of Chemistry, Northwestern University, Evans-
ton, Illinois
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
LYNCH, DR. RUTH STOCKING, Department of Botany, University of California, Los
Angeles 24, California
LYNCH, DR. WILLIAM, Department of Biology, St. Ambrose College, Davenport,
Iowa
LYNN, DR. W. GARDNER, Department of Biology, Catholic University of America,
Washington, D. C.
McCoucH, DR. MARGARET SUMWALT, University of Pennsylvania Medical School,
Philadelphia, Pennsylvania
MCDONALD, SISTER ELIZABETH SETON, Department of Biology, 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
MACDOUGALL, DR. MARY STUART, Mt. Vernon Apartments, 423 Clairmont Avenue,
Decatur, Georgia
MAGRUDER, DR. SAMUEL R., Department of Anatomy, Tufts Medical School, 136
Harrison Avenue, Boston, Massachusetts
MANWELL, DR. REGINALD D., Syracuse University, Syracuse, New York
MARSHAK, DR. ALFRED, Department of Biology, University of Notre Dame, Notre
Dame, Indiana
MARSLAND, DR. DOUGLAS A., New York University, Washington Square College,
New York 3, New York
46 MARINE BIOLOGICAL LABORATORY
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
MEDES, DR. GRACE, Lankenau Research Institute, Philadelphia, Pennsylvania
MEINKOTH, DR. Norman A., Department of Biology, Swarthmore College, Swarth-
more, Pennsylvania
MENKIN, DR. VALY, Agnes Barr Chase Foundation for Cancer Research, Temple
University Medical School, Philadelphia, 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
Laboratory, Woods Hole, Massachusetts
MILLER, DR. J. A., JR., Department of Anatomy, Emory University, Atlanta 22,
Georgia
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
MOORE, DR. JOHN W., Laboratory of Biophysics, NINDB, National Institutes of
Health, Bethesda 14, Maryland
MOUL, DR. E. T., Department of Botany, Rutgers University, New Brunswick,
New Jersey
MOUNTAIN, MRS. J. D., 8 Coolidge Avenue, White Plains, New York
MULLER, DR. H. J., Department of Zoology, Indiana University, Bloomington,
Indiana
MULLINS, DR. LORIN J., Biophysical Laboratory, Purdue University, Lafayette,
Indiana
MUSACCHIA, DR. XAVIER J., 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, Canada
NACHMANSOHN, DR. DAVID, Columbia University, College of Physicians and Sur-
geons, New York City, New York
NAVEZ, DR. ALBERT E., 206 Churchill's Lane, Milton 86, Massachusetts
REPORT OF THE DIRECTOR 47
NELSON, DR. LEONARD, Department of Anatomy, University of Chicago, Chicago,
Illinois
NEURATH, DR. H., Department of Biochemistry, University of Washington, Seattle
5, Washington
NICOLL, DR. PAUL A., Indiana Contract, Box K, A.P.O. 474, San Francisco,
California
Niu, DR. MAN-CHIANG, Rockefeller Institute for Medical Research, 66th Street
and York Avenue, New York 21, 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
OSTER, DR. ROBERT H., University of Maryland School of Medicine, Baltimore 1,
Maryland
OSTERHOUT, MRS. MARION IRWIN, Rockefeller Institute, 66th Street and York
Avenue, New York 21, New York
OSTERHOUT, DR. W. J. V., 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
PERSON, DR. PHILIP, Chief, Special Dental Research Program, Veterans Adminis-
tration 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
Medical Center, New York City, New York
PIERCE, DR. MADELENE E., Vassar College, Poughkeepsie, New York
PLOUGH, DR. HAROLD H., Department of Biology, Amherst College, Amherst,
Massachusetts
POLLISTER, DR. A. W., Department of Zoology, Columbia University, New York
27, New York
POND, DR. SAMUEL E., 53 Alexander Street, Manchester, Connecticut
PROCTOR, DR. NATHANIEL, Department of Biology, Morgan State College, Balti-
more 12, Maryland
PROSSER, DR. C. LADD, 401 Natural History Building, University of Illinois, Urbana,
Illinois
48 MARINE BIOLOGICAL LABORATORY
PROVASOLI, DR. LUIGI, Raskins Laboratories, 305 E. 43rd Street, New York 17,
New York
RAMSEY, DR. ROBERT W., Medical College of Virginia, Richmond, Virginia
RAND, DR. HERBERT W., 7 Siders Pond Road, Falmouth, Massachusetts
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 East 15th Street, New York 9, New York
RAY, DR. CHARLES, JR., Department of Biology, Emory University, Atlanta 22,
Georgia
READ, DR. CLARK P., Johns Hopkins University, Baltimore, Maryland
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
REINER, DR. J. M., V. A. Hospital, Albany, New York
RENN, DR. CHARLES E., 509 Ames Hall, Johns Hopkins University, Baltimore 18,
Maryland
REZNIKOFF, DR. PAUL, Cornell University Medical College, 1300 York Avenue,
New York City, New York
RICE, DR. E. L., 2241 Seneca Avenue, Alliance. Ohio
RICHARDS, DR. A., 2950E 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
ROGICK, DR. MARY D., College of New Rochelle, New Rochelle, New York
ROMER, DR. ALFRED S., Harvard University, Museum of Comparative Zoology,
Cambridge, 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., Columbia University, College of Physicians and Surgeons, De-
partment of Physiology, New York City, New York
ROSE, DR. S. MERYL, Department of Zoology, University of Illinois, Champaign,
Illinois
ROSENBERG, DR. EVELYN K., Department of Pathology, New York University,
Bellevue Medical Center, New York 16, New York
ROSENTHAL, DR. THEODORE B., Department of Anatomy, University of Pittsburgh
Medical School, Pittsburgh 13, Pennsylvania
Rossi, DR. HAROLD H., Department of Radiology, Columbia University, 630 West
168th Street, New York 32, New York
REPORT OF THE DIRECTOR 49
ROTH, DR. JAY S., Department of Biochemistry, Hahnemann Medical College
Philadelphia 2, Pennsylvania
ROTHENBERG, DR. M. A., Scientific Director, Dugway Proving Ground, Dugway,
Utah
RUGH, DR. ROBERTS, Radiological Research Laboratory, College of Physicians and
Surgeons, 630 West 168th Street, New York 32, New York
RUNNSTROM, DR. JOHN, Wenner-Grens Institute, Stockholm, Sweden
RUTMAN, DR. ROBERT J., General Laboratory Bldg., 215 S. 34th Street, Philadel-
phia 4, 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, MR. LAWRENCE, R. D. 7, Bryn Mawr, Pennsylvania
SCHACHMAN, DR. HOWARD K., Department of Biochemistry, University of Cali-
fornia, Berkeley 4, California
SCHARRER, DR. ERNST A., Albert Einstein College of Medicine, 1710 Newport
Avenue, 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
SCHMITT, DR. O. H., Department of Physics, University of Minnesota, Minneapolis
14, Minnesota
SCHNEIDERMAN, DR. HOWARD A., Department of Zoology, Cornell University,
Ithaca, New York
SCHOLANDER, DR. P. F., Scripps Institution of Oceanography, La Jolla, California
SCHOTTE, DR. OSCAR E., Department of Biology, Amherst College, Amherst,
Massachusetts
SCHRADER, DR. FRANZ, Department of Zoology, Duke University, Durham, North
Carolina
SCHRADER, DR. SALLY HUGHES, Department of Zoology, Duke University, Dur-
ham, North Carolina
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, Pennsylvania
SCOTT, SISTER FLORENCE MARIE, Seton Hill College, Greensburg, Pennsylvania
SCOTT, DR. GEORGE T., Department of Zoology, Oberlin College, Oberlin, Ohio
SEARS. DR. MARY, Woods Hole Oceanographic Institution, Woods Hole, Massachu-
setts
SENFT, DR. ALFRED W., Woods Hole, Massachusetts
SEVERINGHAUS, DR. AURA E., Department of Anatomy, College of Physicians and
Surgeons, New York City, New York
50 MARINE BIOLOGICAL LABORATORY
SHANES, DR. ABRAHAM M., Experimental Biology and Medicine Institute, National
Institutes of Health, Bethesda 14, Maryland
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., 35 Henderson Terrace, Burlington, Vermont
SILVA, DR. PAUL, Department of Botany, University of Illinois, Urbana, Illinois
SLIFER, DR. ELEANOR H., Department of Zoology, State University of Iowa, Iowa
City, Iowa
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, Massachu-
setts
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
SPEIDEL, DR. CARL C., Department of Anatomy, University of Virginia, University,
Virginia
SPIEGEL, DR. MELVIN, Old Waterville, Maine
SPRATT, DR. NELSON T., JR., Department of Zoology, University of Minnesota,
Minneapolis 14, Minnesota
SPYROPOULOS, DR. C. S., Department of Neurophysiology, National Institutes of
Health, Bethesda 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
STEIN HARDT, DR. JACINTO, Director of Operations Evaluation Group, Massachu-
setts Institute of Technology, Cambridge, Massachusetts
STEPHENS, DR. GROVER C., Department of Zoology, University of Minnesota,
Minneapolis 14, Minnesota
STEWART, DR. DOROTHY, Rockford College, Rockford, Illinois
STOREY, DR. ALMA G., Department of Botany, Mount Holyoke College, South
Hadley, Massachusetts
STONE, DR. WILLIAM, 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, Marvland
REPORT OF THE DIRECTOR 51
STUNKARD, DR. HORACE W., American Museum of Natural History, 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, Marine Biological Laboratory, Woods Hole, Massa-
chusetts
SZENT-GYORGYI, DR. ANDREW G., Marine Biological Laboratory, Woods Hole,
Massachusetts
TASAKI, DR. ICHIJI, Laboratory of Neurophysiology, National Institute of
Neurological Diseases and Blindness, Bethesda 14, Maryland
TASHIRO, DR. SHIRO, University of Cincinnati, Medical College, Cincinnati, Ohio
TAYLOR, DR. ROBERT E., Laboratory of Neurophysiology, National Institute of
Neurological Diseases and Blindness, Bethesda 14, Maryland
TAYLOR, DR. WM. RANDOLPH, Department of Botany, University of Michigan,
Ann Arbor, Michigan
TEWINKEL, 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, Osborn Zoological Laboratories, Yale University, New
Haven, Connecticut
TROLL, DR. WALTER, Department of Industrial Medicine, New York University
College of Medicine, New York City, 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
UHLENHUTH, DR. EDWARD, University of Maryland School of Medicine, Baltimore,
Maryland
URETZ, DR. ROBERT B., Department of Biophysics, University of Chicago, Chicago,
Illinois
DEViLLAFRANCA, DR. GEORGE M., Department of Zoology, Smith College, North-
ampton, Massachusetts
VILLEE, DR. CLAUDE A., Department of Biological Chemistry, Harvard Medical
School. Boston 15, 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 Reserch, Rutgers University, New
Brunswick, New Jersey
52 MARINE BIOLOGICAL LABORATORY
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., Osborn Zoological Laboratories, 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 Street and York Avenue, New York 21, New York
WENRICH, DR. D. H., University of Pennsylvania, Philadelphia 4, Pennsylvania
WHEDON, DR. A. D., 21 Lawncrest, Danbury, Connecticut
WHITAKER, DR. DOUGLAS M., Rockefeller Institute for Medical Research, 66th
Street and York Avenue, New York 21, New York
WHITE, DR. E. GRACE, Wilson College, Chambersburg, Pennsylvania
WHITING, DR. ANNA R., University of Pennsylvania, Philadelphia 4, Pennsylvania
WHITING, DR. PHINEAS W., Zoological Laboratory, University of Pennsylvania,
Philadelphia 4, Pennsylvania
WICHTERMAN, DR. RALPH, Biology Department, Temple University, Philadelphia,
Pennsylvania
WICKERSHAM, MR. JAMES H., 530 Fifth Avenue, New York 36, New York
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
WILBER, DR. C. G., Medical Laboratories, Applied Physiology Branch, Army
Chemical Center, Maryland
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,
Iowra
WITTENBERG, DR. JONATHAN B., Department of Physiology and Biochemistry,
Albert Einstein College of Medicine, New York 61, New York
WOLF, DR. ERNST, Pendleton Hall, Wellesley College, Wellesley, Massachusetts
WOODWARD, DR. ARTHUR A., Army Chemical Center, Maryland (Applied Physi-
ology Branch, Army Chemical Corps, Medical Laboratory)
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 York
College of Medicine, Syracuse 10, New York
YOUNG, DR. D. B., Main Street, North Hanover, Massachusetts
ZINN, DR. DONALD J., Department of Zoology, University of Rhode Island, Kings-
ton, Rhode Island
REPORT OF THE DIRECTOR
53
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 Medical Center, New
York City, New York
ZWILLING, DR. EDGAR, Department of Biology, Brandeis University, Waltham 54,
Massachusetts
3. ASSOCIATE MEMBERS
ALDRICH, Miss AMY
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
CRANE, MR. AND MRS. BRUCE
CRANE, MR. JOHN
CRANE, Miss LOUISE
CRANE, MRS. MURRAY
CRANE, MR. STEPHEN
CRANE, MRS. W. CAREY
COWDRY, DR. AND MRS. E. V.
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, MR. AND MRS. FREDERICK
EWING, MR. AND MRS. FREDERIC
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.
GlLCHRIST, MR. AND MRS. JOHN M.
GlLDEA, DR. AND MRS. E. F.
GREEN, Miss GLADYS M.
HAIG, MRS. R. H.
HAMLEN, MR. AND MRS. J. MONROE
HARRELL, MR. AND MRS. JOEL E.
HARRINGTON, MR. AND MRS. ROBERT
HERRINGTON, MRS. A. W. S.
HERVEY, DR. AND MRS. JOHN P.
HlRSCHFELD, MRS. NATHAN B.
HOUSTON, MR. AND MRS. HOWARD
JEWETT. MRS. G. F.
KEITH. MR. AND MRS. HAROLD C.
KING, MR. AND MRS. FRANKLIN
KOLLER, MR. AND MRS. LEWIS
LEMANN, MRS. BENJAMIN
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
54
MARINE BIOLOGICAL LABORATORY
MITCHELL, MRS. JAMES McC.
MIXTER, MRS. W. JASON
MOSSER, MRS. BENJAMIN D.
MOTLEY, MRS. THOMAS
NEWTON, Miss HELEN
NICHOLS, MRS. GEORGE
NICHOLSON, REV. ROBERT W.
NIMS, MRS. E. D.
NORMAN FUND, INC., AARON E.
PACKARD, DR. AND MRS. CHARLES
PARK, MR. AND MRS. M. S.
PENNINGTON, Miss ANNE H.
REDFIELD, DR. AND MRS. ALFRED C.
REZNIKOFF, DR. AND MRS. PAUL
RIGGS, MR. AND MRS. LAWRASON
RIVINUS, MRS. F. M., JR.
ROOT, MRS. WALTER S.
ROZENDAAL, DR. H. M.
RUDD, MR. AND MRS. H. W. DWIGHT
SANDS, Miss ADELAIDE G.
SAUNDERS, MR. AND MRS. LAWRENCE
SHIVERICK, MRS. ARTHUR
SINCLAIR, MR. AND MRS. W. RICHARD-
SON
SPEIDEL, DR. AND MRS. CARL
STOCKARD, MRS. CHARLES R.
STONE, MR. AND MRS. LEO
STONE, MR. AND MRS. S. M.
STRAUS, DR. AND MRS. DONALD B.
SWIFT, MR. E. KENT
SWOPE, MR. AND MRS. GERARD, JR.
SWOPE, 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, Miss EMILY
WILSON, MRS. EDMUND B.
WOLFINSOHN, MRS. WOLFE
V. REPORT OF THE LIBRARIAN
During 1959, forty-eight new journals were acquired making a total of 1665
currently-received titles. Of these, there were 484 (12 new) Marine Biological
Laboratory subscriptions, 638 (9 new) exchanges and 184 (5 new) gifts; 100
(7 new) were Woods Hole Oceanographic Institution subscriptions; 199 (12 new)
were exchanges and 60 (3 new) were gifts. Between the years 1950 and 1959,
439 new journals were obtained with initial date of publication, in each case, falling
within that period.
The Laboratory purchased 92 books (15 of these from the Montgomery Memo-
rial Fund), received 119 complimentary copies (7 from authors and 112 from
publishers) and accepted 43 miscellaneous gifts. The Institution purchased 50
books and received 6 as gifts. The total number of books accessioned amounted
to 310.
Through purchase, exchange and gift the Laboratory completed 10 journal sets
and partially completed 15. The Institution completed 6 sets and partially com-
pleted 6. There were 5,629 reprints added to the collection, of which 1772 were
of current issue.
At the close of the year there were 76,073 bound volumes and 212,627 reprints.
The Library mailed out on inter-library loan 384 volumes and borrowed 72.
About 900 volumes were bound, as well as 85 pamphlets.
Dr. E. V. Cowdry presented his large collection of reprints to the Library, of
which 2000 were added to the shelves. Among his collection there were several
journal numbers which filled in gaps of long standing. Dr. F. A. Hartman pre-
sented a collection which will be processed in 1960. Also, gifts of reprints and
books were received from Dr. H. W. Kaan, Dr. P. W. Whiting and Mrs. A. R.
REPORT OF THE LIBRARIAN 55
Memhard. Dr. W. R. Amberson donated a long series of the serial entitled
"Onderzoekingen gedaan in het Physiologisch Laboratorium der Rijksuniversiteit
te Utrecht," as well as several books. To each of these generous friends the Lab-
oratory wishes to extend grateful thanks for the valuable literature acquired by the
Library.
Two foreign institutions benefited from the collection of duplicates, namely, the
National Institution of Oceanography in England and the Marine Biological Lab-
oratory at Helsingor, Denmark.
During 1959, the Staff noticed a considerable increase in the use of the Library
during the winter months. This is very gratifying, as it indicates further year-
round use of the Library facilities, as Woods Hole becomes more and more a
scientific research center.
Respectfully submitted,
DEBORAH L. HARLOW,
Librarian
VI. GENERAL BIOLOGICAL SUPPLY HOUSE, INC.
It would seem that a short resume of the history of "Turtox" would be of
interest to the members of the Corporation at this time.
In 1913, Morris Wells was a graduate student in the Department of Zoology
of the University of Chicago. Dr. Frank R. Lillie was Chairman of the Depart-
ment, and also President of the Marine Biological Laboratory. Prior to this, Mr.
Wells had taught biology for one year in a high school in Kansas. This experience
made him realize that biology teachers needed aid in obtaining material for instruc-
tion. In 1914, he and his wife prepared a one-page mimeographed sheet, listing
slides and other material, which was mailed to a list of biology teachers. Several
orders for $1.00 each were received, which were processed in the cellar of Mrs.
Wells' parents' home. Mr. Wells received his Doctor's degree in 1915, and
accepted a position as Instructor under Dr. Frank R. Lillie, at the University of
Chicago. He was promoted to the rank of Assistant Professor three years later.
By 1918, Dr. Wells realized he could no longer do justice to his teaching, and take
care of his growing business. He discussed this with Dr. Frank Lillie. Dr. Lillie
suggested that if Dr. Wells wished to devote all his time to furnishing biological
supplies to teachers, he might interest his brother-in-law, Mr. Charles Crane, in
the business. A corporation was formed, under the laws of the State of New York ;
Dr. Frank Lillie was Chairman of its Board of Directors for many years. Mr.
Crane purchased 51% of the stock of the new company, and turned it over to the
Marine Biological Laboratory as a gift. The amount involved was $10,000.
In 1920, due to the growth of the business, the General Biological Supply House
increased their stock by $5000, and the M. B. L. purchased one-half of the new
issue for $2500.
In 1921, the Treasurer's report of the M. B. L. shows the entire holdings of
General Biological Supply House stock held by M. B. L. listed at $12,700. This
figure includes 51% of the voting stock. The purpose of this arrangement was to
keep the business in the control of a scientific institution, which would prevent any
56 MARINE BIOLOGICAL LABORATORY
possible future hazards of the business being run just for the profit of the private
owners of the stock. Dr. Wells insisted from the start that the most important
functions of the new company were to help biology teachers get material, and pass
on to them information concerning new techniques.
The new company was called "The General Biological Supply House Inc." and
Dr. Wells coined the word "Turtox," with a design of a turtle holding up the world.
Information about the new company during the first five years is not available,
except that 8% of the par value of the stock was paid in dividends during that
period. From 1923 to the present date, a period of 36 years, the M. B. L. has
received $476,144 in dividends and its stock interest in the company at this time
is worth well over $500,000.
In 1919 Dr. Wells had a student in one of his courses who seemed to have
potentialities. Dr. Wells suggested that he continue his courses in fundamental
biology, and take courses in commerce. This student followed Dr. Wells' sugges-
tion, and took his degree in Business Administration. He worked part time, as a
student, for "Turtox," and became a full time employee when he graduated. Dr.
Wells was ill for long periods, and this man was elected Vice President. When
Dr. Wells died, he became President, a position he still holds. He is, of course.
Mr. C. Blair Coursen. Part of Blair Coursen's work until recently was to edit
the magazine, "Turtox News" ; Mrs. Shepherd, who worked with Mr. Coursen
for several years, is the present editor. "Turtox News" devotes more than half its
pages to non-advertising material. Secondary school biology teachers find it
"good reading."
In 1955, General Biological Supply House moved to a new building, especially
designed for their operation. This building has proved to have been a wise
investment.
In 1957, the Board of Directors established the "Turtox Scholarship." Any
American citizen who is currently, or has been, enrolled in a graduate school of
biology is eligible. The award is based upon evidence bearing upon the promise
of the applicant as a prospective teacher and research scholar. The stipend is
$5000 per year — one of the largest scholarships available. The Scholarship Com-
mittee consists of Dr. Frank A. Brown, Jr., Chairman, Dr. Philip B. Armstrong,
Dr. C. E. Olmstead, Dr. D. P. Rogers and Dr. S. Meryl Rose.
In preparing this report, I wish to acknowledge help from Mr. C. Blair Coursen,
Mrs. Edith Wells, Dr. Winterton C. Curtis, Mr. Homer P. Smith and the
Librarian, Mrs. Deborah L. Harlow\
The successful operation of the General Biological Supply House Inc. reflects
the management of its President, C. Blair Coursen, ably assisted by the Vice Presi-
dent, Arold Blaufuss, the Export Manager, Charles Coursen, Jr., and the As-
sistant to the President and Editor of "Turtox News," Mrs. Ruth L. Shepherd.
They, together with a group of well trained and loyal employees, have rendered a
unique service to teachers of biology throughout the world. Thus, the original
purpose of its founder. Dr. Morris Wells — to give aid and assistance to biology
teachers — is being carried out.
Respectfully submitted,
C. LLOYD CLAFF
THE EFFECT OF SALINITY ON GROWTH OF
GYMNODINIUM BREVE DAVIS
DAVID V. ALDRICH AND WILLIAM B. WILSON
Biological Laboratory, U. S. Bureau of Commercial Fisheries, Galveston, Texas
Field observations have established the close physical association of mass
mortalities of marine animals in the Gulf of Mexico with high concentrations of
the non-thecate dinoflagellate Gymnodiniuw breve Davis (Davis, 1948; Galtsoff,"
1948, 1949; Gunter ct al, 1948"; Wilson and Ray, 1956, among others). With
the development of satisfactory culture media (Wilson and Collier, 1955) and
successful methods for growing the organism in the absence of bacteria (Ray and
Wilson, unpublished results), more definitive study of this association became
possible. Subsequently Ray and Wilson (1957), and Starr (1958) conclusively
demonstrated the toxicity of G. breve to fishes.
The catastrophic manifestations of naturally-occurring G. breve blooms have
attracted considerable attention. The sporadic nature of the outbreaks has stimu-
lated particular interest in possible relationships between environmental factors
and these "red tides." In this regard, various investigators, drawing from rela-
tively sparse field data, have postulated the importance of salinity, dissolved
nutrients, and meteorological conditions (see Ryther, 1955, for review). This
report deals with the effect of salinity on the growth in vitro of G. breve and
compares these findings with the field observations of other workers.
The technical assistance of Mrs. Alice Kitchel is gratefully acknowledged.
MATERIALS AND METHODS
Bacteria-free cultures (10-ml. aliquots in 16 X 125 mm. screw-capped Pyrex
tubes) were employed throughout this work. These tubes, together with the
flasks and pipettes used in medium preparation or inoculation, were rigorously
cleaned before each use. The cleaning routine, found by Ray and Wilson (un-
published results) to be an important factor in the successful culturing of this
organism, included the use of a detergent, hot lO^o nitric acid, and repeated
rinses in tap water and distilled water. In an additional step, culture tubes were
filled with, and inoculation micropipettes immersed in, triple-distilled water and
autoclaved for 15 minutes at 15 p.s.i.
All control tubes contained a completely synthetic medium (Table I), com-
pounded by one of us (W.B.W.) which supported good growth of G. breve.
The medium in experimental tubes differed from control medium only in major
salt content (NaCl, MgSO4, MgCl,, CaCL and KC1). In varying salinity these
major constituents were varied proportionally, thus producing no change in the
ion ratios.
57
DAVID V. ALDRICH AND WILLIAM B. WILSON
Pasteur capillary pipettes were used to inoculate each of a series of tubes
of medium with 100-200 cells from well-established cultures of G. breve. Two or
three cultures were used to inoculate each experiment. Equal numbers of replicates
from each salinity group were inoculated with a given culture so that physiological
differences between inocula would not bias results. After inoculation, the new
cultures were maintained at a temperature of 26-27° C., and illuminated by two
30-watt "standard cool white" fluorescent lights two to three inches from the
culture tubes.
Glassware and media were sterilized by autoclaving at 15 p.s.i. for 15
minutes. After inoculations were completed, two bacterial sterility tests were
conducted for each culture used as inoculum. These tests involved pipetting
1 ml. of the inoculum culture into each of two culture tubes, one containing 10 ml.
TABLE I
Gymnodinium breve culture medium
NaCl* 29.0 gm. KNO2 1.0 mg.
MgSO4-7H2O 6.0 gm. KNO3 1.0 mg.
MgCl2-6H2O 4.5 gm. Thiaminef 1.0 mg.
CaCl2 0.7 gm. Vitamin BJ2t 1.0 Mg-
KC1 0.6 gm. Biotinf 0.5 /ig.
Tris(hydroxymethyl)- 20.0 mg. Sulfide solution! 5.0 ml.
aminomethane**
Na2S-9H2O 3.0 mg. Metals solution§ 20.0ml.
K2HPO4 1.0 mg. Triple-distilled water 1000 ml.
* A. R. grade, recrystallized from triple-distilled water by the addition of C. P. HC1. All
other inorganic compounds were C. P. or A. R. grade.
** Fisher Scientific Co. Added as 50 ml. of a stock solution adjusted to pH 8.2 by the
addition of HC1.
t Nutritional Biochemicals Corp.
J Five ml. of this solution (derived from van Niel, 1931) contains: NH4C1, 1.0 mg. ; NaHCOs,
1.0 mg.; Na2S-9H2O, 0.8 mg. ; KH2PO4, 0.5 mg. ; MgCl2-6H2O, 0.2 mg.
§ Twenty ml. of this solution contains: (Ethylenedinitrilo)tetraacetic acid disodium salt
(Eastman Kodak Co.), 3.0 mg.; Mn as MnCl2-4H2O, 0.2 mg. ; Rb as RbCl, 0.2 mg. ; Alas A1C13-
6H2O, 0. 1 mg. ; Co as CoCl2 • 6H 2O, 0. 1 mg. ; Cs as CsCl, 0. 1 mg. ; B as H3BO3, 0. 1 mg. ; Se as H2SeO3,
0.1 mg.; Cr as K2CrO7, 0.1 mg. ; Mo as Na2MoO4-2H2O, 0.1 mg. ; Sr as SrCl2-6H2O, 0.1 mg. ;
Ti as TiO2, 0.1 mg. ; Zn as ZnCl2, 0.1 mg.; Zr as ZrOCl2-8H2O, 0.1 mg.; Ba as BaCl2, 0.02 mg. ;
Cd as CdCl2-2iH2O, 0.02 mg.; Cu as CuCl2, 0.02 mg.; Fe as FeCl2-4H2O, 0.02 mg. ; Ce
as (NH4)2Ce(NO3)6, 0.02 mg. ; V as NH4VO3, 0.02 mg. ; Ni as NiCl2-6H2O, 0.02 mg. ; Rh as RhCl3,
0.02 mg. ; Ru as RuCl3, 0.02 mg. ; Sn as SnCl2-2H2O, 0.02 mg.
of peptone sea water broth, the other a 10 ml. peptone sea water agar slant
(Spencer, 1952).
Growth of the dinoflagellate was estimated by visual examination of the tubed
cultures with the aid of a stereoscopic miscroscope, using 9x magnification for
most cultures, and 18 X or 27 X when populations were low. Eleven graded
population categories were adopted and "peak population" arbitrarily defined to
include the top three. A rough calibration of this method was carried out by
making estimates and actual cell counts from the same cultures, and comparing
results. This check was conducted on four occasions, and, in all, 99 test cultures
were examined by both methods. Cultures showing peak population by visual
SALINITY AND GYMNODINIUM BREVE
59
100
50
2 o
< 100
i|
Q.
o
Q.
UJ
Q.
50
t 0
£ too
CO
UJ
oe
• • • •••{ s
• • • 1
• • • • • •
•
• •
1 *l 1 1 1 1 1 1
35 DAYS
•
•
1
i
• • ••• s 1
t •
• • • •
• •
• •
•
,MI 1 1 1 1 1 1 *
25 DAYS
•
•
i !i i
i
50
o
u.
o
18 DAYS
[. I I |-L
50
10 DAYS
22 24 26 28 30 32 34 36 38 40 42 44 46
SALINITY %o
FIGURE 1. Rate of peak population development of Gymnodinium breve at various salinities.
Each point is based on 10 replicate cultures.
60 DAVID V. ALDRICH AND WILLIAM B. WILSON
estimate never proved to have fewer than 750 cells per ml., and usually contained
from one thousand to several thousand cells per ml. Although sacrificing a degree
of quantitative accuracy, the visual estimate method was selected because of the
ease, speed, and lack of bacterial contamination with which it could be performed.
All cultures were examined by this method at 4, 10, 18, 25, and 35 days after
inoculation. The five-week period was considered adequate, since cultures seldom
grow after five weeks post-inoculation.
Five experiments were conducted, each having nine salinity levels and ten
replicate tubes at each level. The first experiment had the widest salinity range
(6.3, 8.4, 11.9, 13.7, 17.8, 18.3, 25.8, 31.7, and 41.1%0). The four later'experi-
ments were designed for a salinity range of 22.5 to 4l.l%o with intervals of ap-
proximately 2.3%c. However, due to technical error, the last two experiments
had ranges of 25.4 to 36.3%c and 23.8 to 46.0%0 and somewhat irregular intervals.
RESULTS
Cultures of G. breve grew well throughout a salinity range of 27 to 37%c
(Fig. 1). Within this range, at least 80% of replicate cultures reached popula-
tions of 750 cells or more per ml. during the five-week experimental period.
More variable results and generally poorer growth occurred at salinity levels
immediately adjacent to the optimal range. No instances of optimum growth
occurred at less than 24%c or more than 44%c. Some indication of comparative
growth rates may also be obtained from Figure 1. It is apparent that cultures
reach high populations more rapidly within the optimal range (27 to 37%o).
Some organisms survived throughout a salinity range of 22.5 to 46.0%o. From
24.8 to 46.0%c, 91% of cultures contained living cells at the end of the five-week
observation period. There was no indication of reduced survival at the extremes
of this range. Below this range the incidence of survival was lower; at 23.8%o
the organism survived in only one of 10 replicate cultures, and at 22.5%c 10 of
20 replicates contained surviving cells. No instances of five-week survival were
noted at any of the tested salinity levels below 22.5%c. At 18.3 and 17.8%0 three
and two, respectively, of the 10 tubes in each group contained a few live organisms
10 days after inoculation, but no survivors were found eight days later. Media
with salt concentrations of 13.7/ansa, Thysanosoma actinioidcs, and Cittotaenia
per pie xa. Exp. Parasit., 9: 1-8.
CAMPBELL, J. W., 1960b. Pyrimidine metabolism in parasitic flatworms. Biochem. J., in press.
CANELLAKIS, E. S., J. J. JAFFE, J. R. MANTSAVINOS AND J. S. KRAKOW, 1959. Pyrimidine me-
tabolism. IV. A comparison of normal and regenerating rat liver. /. Biol. Chan.,
234: 2096-2099.
CRUMPLER, H. R., AND C. E. DENT, 1949. Distinctive test for a-amino acids in paper chroma-
tography. Nature, 164 : 441-442.
CRUMPLER, H. R., C. E. DENT, H. HARRIS AND R. G. WESTALL, 1951. /3-Aminoisobutyric acid
(a-methyl-jtf-alanine) : A new amino acid obtained from human urine. Nature, 167:
307-308.
FINK, R. M., C. MCGAUGHEV, R. E. CLINE AND KAY FINK, 1956. Metabolism of intermediate
pyrimidine reduction products in vitro. J. Biol. Chcm., 218 : 1-7.
FOWDEN, L., 1951. The quantitative recovery and color imetric estimation of amino acids
separated by paper chromatography. Biochem. J., 48 : 327-333.
HAUSMANN, W., 1952. Amino acid composition of crystalline inorganic pyrophosphatase
isolated from bakers yeast. /. Amer. Chcm. Soc., 74: 3181-3182.
HULME, A. C., AND W. ARTHINGTON, 1950. 7-Amino-butyric acid and /3-alanine in plant
tissues. Nature, 165: 716-717.
LANG, C. A., 1958. Simple microdetermination of Kjeldahl nitrogen in biological materials.
Analyt. Chem., 30: 1692-1694.
LEVY, A. L., AND D. CHUNG, 1953. Two-dimensional chromatography of amino acids on
buffered papers. Analyt. Chcm., 25 : 396-399.
PoCHEDLEY, D. S., 1956. II. The chromatographic separation of amino acids from insect
blood. Trans. N. Y. Acad. Sci., 19: 19-22.
p-AMINO ACIDS IN FLATWORMS 79
ROBERTS, E., S. FRANKEL AND J. H. PINCKNEY, 1950. Amino acids of nervous tissue. Proc.
Soc. Exp. Biol Med., 74: 383-387.
SIMPSON, J. W., K. ALLEN AND J. AWAPARA, 1959. Free amino acids in some aquatic inverte-
brates. Biol. Bull.. 117: 371-381.
SYNGK. R. L. M., 1951. Methods for isolating omega-ammo acids: gamma-aminobutyric acid
from rye grass. Bioclicin. J., 48 : 429-435.
TALLEN, H. H., S. MOORE AND W. H. STEIN, 1954. Studies on the free amino acids and re-
lated compounds in the tissues of the cat. /. Biol. Chcm., 211: 927-939.
VIRTANEN, A. L, AND T. LAINE, 1937. The decarboxylations of d-lysine and 1-aspartic acid
Enzymologia, 3 : 266-270.
THE FEEDING MECHANISM IN THE SAND DOLLAR MELLITA
SEXIESPERFORATA (LESKE)
IVAN GOODBODY
Department of Zoology, University College of the West Indies, Mono, Jamaica
MacGinitie and MacGinitie (1949) give the following account of feeding in
the scutellid echinoderm Dendraster excentricus (Eschscholtz) (p. 237) : "The
spines on the upper side of the sand dollar are club shaped and are covered by
cilia. These cilia create currents that flow from the direction in which the animal
is moving toward what could be called the posterior edge. ... As the currents
flow through these spines, little eddies are created at the posterior sides of the
spines. These eddies allow tiny particles and organisms to become trapped in
mucus that is secreted onto the surface of the spines. This mucus goes downward
and is led into tiny tracts to unite with others. These in turn unite again, passing
around the edge to the underside, until near the mouth five tracts or strings of
mucus feed directly into the mouth of the sand dollar." This appears to be the
only available account of feeding in the Clypeasteroida. Hyman (1958) studied
the five-lunuled sand dollar Mellita quinquiesperforata (Leske), but was unable
to elucidate the feeding mechanism. The present paper describes observations
made on another of the lunuled sand dollars, Mellita (Lcodia) sexiesperjorata
(Leske), and shows that it is a ciliary mucus feeder collecting particles on the
aboral surface and transporting them through the lunules and around the margin
of the test to food tracts on the oral surface.
M. sexiesperjorata is common in certain shallow water sandy areas around
Jamaica and normally lives either in the surface layer of the sand, so that its
outline is discernible from above, or else buried in the sand very close to the surface.
The animals used for these observations were collected on the Port Royal Cays
and observations were made both in the aquarium and in dishes under a microscope.
MORPHOLOGY
Figure 1 shows an individual in both oral and aboral views. The size of the
animals varies considerably, fully grown animals being about 70 to 80 mm. in
diameter. In surface view the animal has a roughly pentagonal outline and its
surface is pierced by six slit-like lunules : five of these are in the ambulacral areas,
the sixth or anal lunule is interambulacral and marks the posterior side of the
animal. The anterior side of the test is markedly pointed. Both aboral and oral
surfaces are densely clothed with short spines which are described below. On
the aboral side of the only other structures visible are lunules, petaloicls, and gono-
pores. On the oral surface the mouth is central and the anus lies posterior to it,
just on the edge of the anal lunule. Leading away from the oral margins of the
five ambulacral lunules are a number of broad food tracts: one of these, the radial
tract, leads straight from the inner tip of the lunule to the mouth, the remainder
80
FEEDING IN MELLITA 81
run laterally from the lunule and terminate in the ambulacra! or food grooves.
These are deep hut narrow grooves, a pair to each ambulacral area running into
the mouth; the two members of a pair form a petaloid outline and unite just before
reaching the mouth. The food tracts of the anal lunule are less well developed
and discharge laterally to the ambulacral grooves of the two neighboring lunules.
Figure 2 shows the profile of a sagittal section through the test and shows
that it is thin around the margin and dome-shaped in the center ; the anterior margin
is thicker than the posterior margin. The mean measurements for the ten indi-
viduals of 60 to 65 mm. diameter are :
Anterior margin : 1.8mm. ±0.04
Central dome: 5.9mm. ±0.49
Posterior margin : 1.0mm. ±0.01
It is significant that the animal progresses through the sand with the anterior and
thicker margin foremost.
Podia
On the oral surface there are dense concentrations of podia around the periphery
of the test, around the margins of the food tracts and in the lunules. They are
less dense on the remainder of the ambulacral areas and are absent from areas
FIGURE 1. a. The aboral surface of a living Mcllita sexiesperforata. The anterior end is
towards the top of the picture. The club-shaped spines appear as minute spots all over the
surface of the animal. Notice that the peripheral ( ambulatory ) spines are more dense
anteriorly than posteriorly. The ring of protective spines can be seen clearly around the
margin of each lunule.
82
IVAN GOODBODY
FIGURE 1, b. The oral surface of a living McUita sexiesperforata stained for two minutes
in toluidine blue. Orientation as in Figure 1, a. The stain is taken up by podia and other
structures, and was used to improve contrast. The mouth is central and the anal papilla
appears as a black spot between it and the anal lunule. The anal spines between anus and
mouth are just visible. A — Ambulatory spine; F = Food tracts; G = Food (ambulacra!)
grooves.
with ambulatory spines. On the aboral surface there are only a few scattered
podia. When extended the podia are very long and thin with a slightly swollen
tip and poorly developed sucking disc. They function exclusively as an accessory
food collecting device (see below) and do not appear to play any part in locomotion.
Spines
There are four principal types of spine. On the oral surface ambulatory and
non-ambulatory spines can be distinguished. Ambulatory spines are confined to five
locomotory areas in the interambulacra of the oral surface. In the anal inter-
ambulacrum they form a transverse group behind the anal lunule, in the other
four interambulacra they form a radially disposed wedge-shaped group (Fig. Ib).
These spines, which are long and thick (1300//, X 85 /A) normally have a rounded
tip but in many spines the tip is abraded into a roughened end (Fig. 3, A) ; their
FIGURE 2. Profile of McUita sexiesperforata. The anterior end
is toward the right of the picture.
FEEDING IN MELLITA
83
function appears to be entirely that of locomotion. Similar spines are found all
around the margin of the test hut they are almost twice as thick (1300 /A X 160 /A) ;
they are densely arranged anteriorly and more scattered posteriorly. Their extra
thickness is probably correlated with stresses set up during movement through
the sand in a horizontal direction. Non-ambulatory or protective spines of the
oral surface are shorter and thinner (430 /AX 30 /A) than ambulatory ones and
are often bent near the middle of their length (Fig. 3, B) ; they cover all the re-
maining areas of the oral surface. Similar spines are found in the lunule and on
the aboral margin of the lunule they form a protective ring projecting up higher
than the other spines of the aboral surface ; here they prevent very large sand
particles from entering the lunule and blocking it.
On the aboral surface there are two types of spine distributed together all
over the animal. The larger of these are club-shaped spines which move the
A/
FIGURE 3. The principal types of spine found in Mcllita sexiesj>erforata. A = Ambulatory
spine with abraded tip. B = protective spine of oral surface. C : - Two views of club-shaped
spines of aboral surface. D = Aboral miliary spine.
sand grains posteriorly over the aboral surface (Fig. 3, C). The club-shaped
head of these spines is set at a slight angle to the stem and its tip is oriented
toward the margin of the test. The others or miliary spines (Fig. 3, D) are
shorter than the club-shaped spines and are characterized by having a sac-like
swelling on the tip ; this sac contains yellowish granules which stain darkly with
toluidine blue. The epithelium at the base of the cilia on these spines stains
purple with the same stain and it seems plausible therefore to suggest that these
spines are the principal site of mucus secretion. Miliary spines also occur on
the inner walls of the lunules and along the food tracts of the oral surface ; the
latter have a smaller sac than those of the aboral surface or lunule.
As well as these four main types of spine there is a group of very large spines
forming a circle between the mouth and anus and with their tips overarching the
anus. They play an important part in preventing feces from entering the mouth.
84 IVAN GOODBODY
These spines are in three groups: seven or eight very long ones (up to 5 nun. in
length) form a group nearest to the anus, outside these there are ten to twelve
of intermediate size, then a group of many smaller ones forming an outer ring
next to the mouth. (Fig. Ib).
Cilia
Cilia are confined to the epithelia of the spines and are found on only one
side of the stem. This side of the stem is here referred to as its back and the
cilia beat in such a way as to drive a horizontal current from it in a backward
direction. On the aboral surface the club-shaped spines have dense ciliation at
the base while the miliary spines have dense ciliation along the whole length of
the stem. The club-shaped and miliary spines are oriented in such a way as to
produce centrifugal currents across the aboral surface leading away from the
center and towards the margins and lunules. On the oral surface the ambulatory
spines have a thin ciliation at the base while the non-ambulatory spines have dense
ciliation at the base producing a strong current. The ciliation of the miliary spines
of the food tracts is similar to aboral miliary spines. At the margins of the
oral surface the spines are oriented so as to drive currents in towards the center.
In the food tracts they produce currents towards the mouth in the radial tracts and
laterally towards the ambulacral grooves in the remainder. Over the rest of the
ambulacral areas the currents drive towards the adjacent ambulacral grooves.
FEEDING BEHAVIOR
M. sexiesperjorata is a microphagous feeder. Removal and microscopal exami-
nation of the food cord passing along the ambulacral groove shows that most
particles are less than 20 p. in diameter and in a high proportion of them are of the
order of 1 /A in diameter. Within these size limits there appears to lie no selection
of different types of food — algal cells, detrital particles and sand grains are all
collected. Larger particles are sometimes collected and sand grains which only
just fit in the groove have been seen passing along it and entering the mouth.
An active animal ploughs slowly through the surface sand always keeping the
anterior end (Loven's Ray III) forward. As it moves it builds up a small wall
of sand in front of itself, the sand grains of which constantly fall down on the aboral
surface where they are carried slowly backwards on the tips of the club-shaped
spines. This movement of sand appears to be oriented posteriorly across the
aboral surface without special reference to the lunules, but small sand grains which
reach the margins of the lunules are carried down through them ; larger particles
are carried off the posterier margin of the test. Particles entering the lunule
usually do not drop through it, but are lowered slowly down by means of the
spines and then deposited back in the substratum ; during this time they may
be actively probed by podia in the lunule. suggesting that the latter may remove
from it minute absorbed particles.
If carmine particles instead of sand are placed on the aboral surface of the
animal a further process of selection may be observed. Larger particles are treated
exactly as sand and it can also be seen that the very small particles (visible only
under the microscope) drop down between the club-shaped spines and are carried
away by the ciliary currents around the spines. At this lower level some particles
FEEDING IN MELLITA 85
travel to the margin of the test and then around it to the oral surface, other particles
are carried to the lunules and pass down through them and onto the food tracts
on the oral surface. By placing carmine particles in the food tracts of an inverted
animal it can he shown that the ciliary currents lead to the ambulacral grooves,
except in the radial tract where they lead directly to the mouth. Carmine particles
placed on the aboral surface of an animal appear in the ambulacral grooves in a
few moments. In the ambulacral groove food can be seen to be loosely aggregated
in mucus but the precise point at which the mucus is secreted is still in doubt.
As pointed out above the miliary spines of the aboral surface are the most probable
site of mucus secretion. In the ambulacral groove the food is carried along by
podia and not by cilia.
In summary, then, an animal ploughing through the sand pushes sand onto the
aboral surface where it is crudely sorted, large particles being carried along on
the tips of the club-shaped spines and ultimately deposited back in the sand either
through the lunules or off the posterior edge of the test. Fine particles fall down
between the bases of the spines where they are carried away by ciliary currents
through the lunules to food tracts on the oral surface. From the food tracts cilia
carry them to the ambulacral groove thence to the mouth in a mucus aggregation
carried along by podia.
There remains the question of food collection by the podia. Because they are
confined to the oral surface it is difficult to see them in normal function, but
following the method of Nichols (1959) I have examined them in a perspex
box with the aid of a prism and a binocular microscope. Under these circum-
stances the podia at the margin are seen to be constantly extending and contracting
and probing the surroundings of the animal. If carmine particles or yeast stained
in congo red are pipetted around the margin, particles may sometimes be seen
to be picked up by the podia and drawn in to the margin of the animal where they
are released and carried inwards by the ciliary currents. At the same time
man}- podia are seen to extend and contract with no visible particles attached.
However, only relatively small magnifications ( X 30) can be used successfully
in examining with the prism and 1 believe that the principal function of these
podia is to probe the surrounding sand for very small particles of food, i.e., particles
of about 1 p. diameter. Mention has already been made of the manner in which
sand grains are probed by podia as they pass down through the lunules. In the
perspex box only a few scattered grains of sand can be included with the animal ;
otherwise they obscure it from view. The animal thus rests directly on the
bottom of the box and only the marginal podia, which extend laterally, can be
extended effectively. It seems certain, however, that under normal conditions
the podia of the oral surface all probe the sand in the same way, collecting small
food particles.
DEFECATION
In an animal such as M. sexiesperjorata in which the anus is in close proximity
to the mouth, special precautions are required to ensure that feces do not re-enter
the mouth. Although many animals have been examined from time to time
defecation has only been observed on a very fewr occasions and it appears that it
must be an intermittent and not a continuous process.
Defecation commences with a pumping action of the anal papilla followed by
86 IVAN GOODBODY
cessation of feeding activity; passage of the mucus cord in the ambulacra! groove
stops completely. The spines between the mouth and the anus beat gently away
from the mouth and over the anus, and all around this area pedicellariae become
intensely active. The tip of the anus is directed towards the anal lunule and
feces are ejected in intermittent puffs of loose particles and not in a mucus string.
Examination of a defecating animal from below shows that feces are not, as might
1)e expected, carried up through the anal lunule and so removed in water currents.
They fall down from the animal and must in normal circumstances be deposited
back in the sand in which the sand dollar community is feeding.
Grateful acknowledgment is made to the Nuffield Foundation from whom the
author was in receipt of a research grant when these observations were made.
SUMMARY
1. A brief description of Mellita sc.riespcrforata is given and the process of
food collection and defecation are described.
2. Sand pushed onto the aboral surface is sorted by club-shaped spines. Fine
particles drop down between the spines and are carried round to the oral surface
by ciliary currents, thence to the ambulacra! grooves. Mucus is probably secreted
by the miliary spines on the aboral surface ; podia play an accessory role in food
gathering.
3. Defecation is an intermittent process and feeding stops while it is in progress.
Spines and pedicellariae prevent feces from reaching the mouth.
LITERATURE CITED
HYMAN, L. H., 1958. Notes on the biology of the five-lunuled sand dollar. Biol. Bull., 114:
54-56.
MAcGiNixiE, G. E., AND N. MAcGiNixiE, 1949. Natural History of Marine Animals. New
York. McGraw-Hill Book Co.
NICHOLS, D., 1959. Changes in the chalk heart urchin Micrastcr interpreted in relation to
living forms. Phil. Trans. Roy. Soc. Scr. B. 242 : 347-437.
CLEAVAGE WITH NUCLEUS INTACT IN SEA URCHIN EGGS
ETHEL BROWNE HARVEY
Marine Biological Laboratory, Woods Hole, Massachusetts, and the Biological Laboratory,
Princeton University, Princeton, Nezv Jersey
Though, in general, nuclear changes associated with mitosis precede the
cleavage of a cell, there are some rare cases in which this is not true. It has been
found that in some cases in developing sea urchin eggs, the nucleus may remain
as it is in a resting cell, hut nevertheless the cell may cleave as it does after
mitosis, and produce two quite normal "resting" cells. This has been found to
occur in centrifuged eggs which have been stimulated to develop parthenogenetically
by treatment with hypertonic sea water (30 grams of NaCl per liter of sea water)
for 5 to 15 minutes. After one half to one hour, the egg nucleus remains un-
changed but a cleavage plane may come in between the nucleate part of the cell
and the non-nucleate part, as it does in such eggs when fertilized (see E. B.
Harvey, 1932), resulting in two cells. This has been found to occur in Sphae-
rcchinus granularis, Psaininccltiniis (Parccliinus) inicrotuberculatus and more
recently in Arbacia punctulata and A. pustulasa (photographs of living eggs are
reproduced in Figs. 1-8). These occasional cases have been observed over a
period of twenty years. No further development or change has been observed.
It has not been possible to produce such a cleavage lacking nuclear change, with any
of many chemical substances tried.
There are in the literature a few references indicating that the nucleus may
be removed experimentally. Mazia and Dan (1952) succeeded in removing the
mitotic apparatus in an isolated condition from the "fixed" Strongylocentrotus
franciscanns egg, and later Dan and Nakajima (1956) removed it "fixed" from
other sea urchins, Pscudoccntrotits depressus and Hcmicentrotus pulcherrimus,
with observations also on Arbacia punctulata. According to Swann and Mitchison
(1953), the eggs of the heart urchin, Clypeaster japonicus, may be treated with
concentrated colchicine at mid-anaphase, completely abolishing the asters and
spindle, and still the egg will divide. There is, of course, the possibility that
some part of these structures still remains. To make the experiment more decisive,
Hiramoto (1956) sucked out the spindle and asters with a micropipette inserted
into the egg, and he found that cleavage still took place.
Some years ago (1938), I made a reference in one of my papers to "cleavage
planes coming in independently of any nuclear changes" (p. 182) in sea urchin
eggs, and Holtfreter called attention to this in his 1948 paper (p. 723). My paper
was accompanied by photographs (44 and 57, 58).
SUMMARY
There now seems no doubt that cleavage can take place without any visible
change in the nucleus.
87
88
ETHEL BROWNE HARVEY
j '
FIGURES 1-8. Living eggs.
CLEAVAGE WITH NUCLEUS INTACT 89
LITERATURE CITED
DAN, K., AND T. NAKAJIMA, 1956. On the morphology of the mitotic apparatus isolated from
echinoderm eggs. Embryologia, 3 : 187-200.
HARVEY, E. B., 1932. The development of half and quarter eggs of Arbacia pnnctulata and of
strongly centrifuged whole eggs. Biol. Bull.. 62 : 155-167.
HARVEY, E. B., 1938. Parthenogenetic merogony or development without nuclei of the eggs
of sea urchins from Naples. Biol. Bull. 75: 170-188.
HIRAMOTO, Y., 1956. Cell division without mitotic apparatus in sea urchin eggs. £.r/>. Cell
Res.. 11: 630-636.
HOLTFRETER, J., 1948. Significance of the cell membrane in embryonic processes. Ann. N. ) .
A cad. Sci.. 49: 709-760.
MAZIA, D., AND K. DAN, 1952. The isolation and biochemical characterization of the mitotic
apparatus of dividing cells. Proc. Nat. Acad. Sci., 38 : 826-838.
SWANN, M. M., AND J. M. MITCHISON, 1953. Cleavage of sea-urchin eggs in colchicine.
/. Exp. Biol., 30: 506-514.
FIGURES 1-3. Stratification of unfertilized eggs of Arhacia piinctulnta with centrifugal
force.
FIGURES 4-6. Cleavage of centrifuged, parthenogenetic eggs of Arbacia pnnctulata. without
the nucleus taking part. Notice the nucleus in Figure 4, slightly enlarged. It becomes some-
times, not always, slightly enlarged.
FIGURE 7. Cleavage of parthenogenetic, centrifuged eggs of Sphacrcchimts grannlaris,
without the nucleus taking part.
FIGURE 8. Cleavage of parthenogenetic, centrifuged eggs of Psammechinus (Parcchinus)
microtuberculatus without the nucleus taking part.
DEVELOPMENTAL STAGES OF THE BROAD BREASTED
BRONZE TURKEY EMBRYO '• -
A. M. MUN * AND I. L. KOSIN *
Department of Poultry Science, Washington State University, Pullman, Washington
In studying early mortality in the turkey embryo, it became necessary to
determine with considerable accuracy the extent of development of the embryo.
Phillips and Williams (1944) described the Black and the Beltsville Small
White turkey embryos after different durations of incubation. However, chrono-
logical age, Lc., incubation time, per sc, is not a reliable expression of the extent
of morphological differentiation of the embryo. Such factors as temperature
and humidity during incubation, genetic composition, and size of the egg. have
been shown to affect the rate of growth of the avian embryo (for review, see
Landauer, 1951). It has previously been shown in this laboratory (Kosin and St.
Pierre, 1956) that storage of Broad Breasted Bronze hatching eggs for 8 to 14
days results in a lowered mean somite count after 60 hours of incubation, as
compared with eggs held for 1 to 7 days.
Hamburger and Hamilton (1951) established a series of normal stages of
development for the chick embryo, based on various morphological criteria. These
criteria were found to be useful in this laboratory in estimating the extent of
development of the turkey embryo, although the turkey embryo takes approximately
28 days to hatch as compared with 21 days in the chicken. Thus, the major
objective of the study reported in this paper was to determine for turkey embryos
the period of incubation necessary to obtain the different stages of embryonic
development described for the chicken by Hamburger and Hamilton (1951 ).
Similar studies on "staging" of embryonic development in Aves have previously
been reported by Rempel and Eastlick (1957) and Koecke (1958) for the White
Silkie bantam chicken and the Khaki Campbell and White Indian Runner ducks,
respectively.
MATERIALS AND METHODS
All embryos used in this study were obtained from eggs produced by a flock
of Broad Breasted Bronze (BBB) turkeys maintained at the Station. The birds
1 Scientific Paper No. 1958, Washington Agricultural Experiment Stations, Pullman.
Project No. 717.
2 Supported, in part, by Federal Funds for Regional Research (W-7) under the Hatch
Amended Act.
3 Present address : Department of Embryology, Carnegie Institution of Washington, Balti-
more 5, Maryland.
4 We wish to acknowledge the advice and help of Dr. Thomas J. Russell, Washington Agri-
cultural Experiment Stations Statistician, in the analysis of the data on which this study
is based. We are also indebted to Mrs. Lynne Frutiger, Mrs. Jewell Keeney, Mrs. Mary
Ellen Schy, and Mrs. Jeannette Wright for their technical assistance in the collection of these
data.
90
EMBRYONIC STAGES IN TURKKVS
91
were trapnested and the eggs were collected three times a day, after which they
were placed in the holding room at 50° F. and 85c/c relative humidity for not more
than three to four days. The eggs were incubated for a desired length of time
in a forced draft incubator at 99.5° F. The earlier embryos (1 to 7 days) were
removed from the yolk, placed in chick Ringer's solution and then measured and
staged. Each embryo was staged separately according to (1) the development
of the mesodermal derivatives, e.g., somites; (2) the development of the ectodermal
derivatives, e.g.. optic vesicles, neuromeres ; and (3) the development of the heart.
The "average" stage of development of the embryo was then obtained from these
three separate stagings. Although there were individual differences, no striking
and consistent differences between the turkey and the chicken were observed in
terms of rate of development in these three groups of morphological criteria. Older
embryos ( 17 to 28 days) were fixed in Benin's fluid or Baker's calcium formol
before staging.
In the later stages of development (Hamburger-Hamilton, stage 36 to stage 40),
the turkey embryo has a distinct structure, the "snood" or "leader," which was
included among the criteria used for describing the stage of development of the
embryo.
Measurements of the beak and toe, which are the main criteria for identifying
chick embryos from stage 40 to 44. were obtained for the turkey embryo. How-
ever, owing to the relatively small increments of increase in length in these
structures, measurements of the foot, i.e., from the outer edge of the tarsal joint
to the tip of the claw of the third toe, were used to characterize the development
from seventeenth to the twenty-seventh day of incubation.
This study is based on the observation of more than 4000 embryos collected
over a period of three years.
FIGURE 1. Stage (Hamburger and Hamilton, 1951) of development of the turkey embryo
after various periods of incubation. The figures in boxes indicate the number of specimens
for each point on the coordinate.
\. M. MUN AND 1. L. KOSIN
TABLE I
Anteroposterior (AP) lengths of the area pellucida and of the BRB turkey embryo at
different stages of development
Stage
No.
cases
AP length
(mm.)
Sd
Embryo length
(mm.)
Sd
4
11
3.3
.375
. —
— _
5
132
3.9
.423
—
6
233
4.3
.400
1.4*
.390
7
101
4.7
.458
2.3*
.242
8
52
5.3
.454
3.1**
.341
9
2
5.6
—
3.8
—
10
5
6.3
—
4.6
— .
11
9
7.0
.508
5.6
.225
12
12
7.0
.673
5.8
.310
* From the head fold to Hensen's node.
** From the tip of the head to Hensen's node.
RESULTS AND DISCUSSION
The approximate periods of incubation to obtain stages 1 to 39 can be obtained
from Figure 1. There is a wide range of variation in the stage of development
in the turkey embryo after a definite period of incubation. This becomes particu-
larly apparent in the early stages.
In the earlier stages (stages 4 to 12) the stage of the embryo can also be
estimated from measurements of the anteroposterior lengths of the area pellucida
or of the embryo, i.e., from the tip of the head, or in stage 6, from the head fold
to Henson's node (Table I). These measurements are highly correlated with the
stage and somite number ( Mun and Kosin, 1958).
The development of the snood ("leader") is summarized in the following
tabulation :
Day of
incubation
13th day
12 to 14
15
16 to 17
17
18
19th day
Hamburger
& Hamilton
Stage
36
37
38
39
40
Snood characteristics
Snood appears (Fig. 2).
Snood is as high as it is wide at the base (Fig. 3).
Snood is higher than wide and distinctly columnar in appear-
ance (Fig. 4).
Snood is columnar and almost twice as tall as it is wide. Papil-
lae may be seen at the base of the snood (Fig. 5).
The snood is covered with papillae (Fig. 6).
The snood is larger and conical feather germs at base of snood
are colored (Fig. 7).
The feather germs are as long as the snood and may cover it
completely (Fig. 8).
The growth of the beak, toe, and foot from the seventeenth to the twenty-seventh
day of incubation is presented, graphically, in Figure 9. Each point represents
the average measurements of 10 to 21 embryos by three different individuals on
FIGURE 2. Snood at 13th day of incubation.
FIGURE 3. Snood at 14th day of incubation.
FIGURE 4. Snood at 15th day of incubation.
93
FIGURE 5. Snood at 16th day of incubation.
FIGURE 6. Snood at 17th day of incubation.
FIGURE 7. Snood at 18th day of incubation.
94
EMBRYONIC STAGES IN TURKEYS
95
FIGURE 8. Snood at 19th day of incubation.
•bU
o
A
Q
FOOT
TOE
BEAK
> {
oc
/i r\
i
i :
M
r J
)^^^
i
>
\
METERS
•'fU —
c
c
j ^^--*"
I
<
^^
i
-~
i
_i
d <
2
-2.
? ^"— ""*
^ — "*i
p
j
i i
, <
X
O
z
1 /-\ <
k <
! '
^ !
» :
1 !
^ !
i 1
3
1
LU t
_l
[
5 \ U
J C
XD |
| '
I '
r '
r '
3D I
P f
1
1
7 1
8 1
9 2
0 2
1 2
2 2
3 2
4 2
5 2
6 2
PERIOD OF INCUBATION IN DAYS
FIGURE 9. Length of beak, toe, and foot after different periods of incubation.
96 A. M. MUN AND I. L. KOSIN
different groups of embryos at different times. The measurements were made
with a pair of vernier calipers.
As may be seen from Figure 9, the rate of growth of the foot is greater than
the rate of growth of the beak and toe. A straight line obtained from the regression
of the length of the foot on time was then constructed. The equation of this line
(solid line in Figure 9) is as follows:
Y -- - 25.3 + 2.9.v
where Y - length of the foot in mm. and .v = period of incubation in days.
The standard deviation of the regression line is 3.3 and the standard error of
the slope is 0.051.
The 95% confidence interval in age of the embryo, for any particular length
of the foot, can be calculated from the following equation :
= 36.6 + 0.35 To ± 0.7 o.00024(K0 - 40.0)2 + 8.342
where Lx = upper limit of age of the embryo in days, Lx = lower limit of age of
the embryo in days and Fo — observed length of the foot in millimeters.
Similarly, the 95% confidence interval for the length of the foot following a
definite interval of incubation can be calculated from the following equation :
= - 25.3 + 2.9J\r0 ± 6.47 l.002 + 0.00024(X0 - 22. 6)2
Ly\
where Ly = upper limit of the length in mm., Ly = lower limit of the length
in mm. and *o = observed period of incubation in days.
This information has been used in our laboratory to approximate the time of
death of the embryo, whether it was accidental, e.g., due to incubation failures,
or due to causes associated with the problem of hatchability, and to compare the
growth rates of embryos from different lines of BBB turkeys cultivated in vitro.
SUMMARY
1. The period of incubation of Broad Breasted Bronze turkey eggs necessary
to obtain the various normal stages of development established by Hamburger and
Hamilton for the chick embryo is presented.
2. Data have been submitted on the development of the snood ("leader") in
the turkey embryo.
3. Measurements of the beak, toe, and foot were obtained from the seventeenth
to the twenty-seventh day of incubation. From these measurements, a straight
line obtained from the regression of the length of the foot on time was constructed.
The equation of this line is presented, as well as equations for determining the
approximate age of the embryo from measurements of the foot, or the approximate
length of the foot.
LITERATURE CITED
HAMBURGER, V., AND H. L. HAMILTON, 1951. A series of normal stages in the development
of the chick embryo. /. Morphul., 88: 49-92.
EMBRYONIC STAGES IX TURKEYS 97
KOECKE, H., 1958. Normalstadien der Embryonalentwicklung bei der Hausentee (Anas Boschas
domestica). Embryologia, 4 : 55-78.
KOSIN, I. L., AND E. ST. PIERRE, 1956. Studies on pre-incubation warming of chicken and
turkey eggs. Poultry Sci., 35 : 1384-1392.
LANDAUER, W., 1951. The hatchability of chicken eggs as influenced by environment and
heredity. Bull. 262, Storrs Agric. Exper. Sta. ; 223 pp.
MUN, A. M., AND I. L. KOSIN, 1958. The early turkey embryo, on media prepared from eggs
of "high" and "low" hatchability hens. Growth, 22: 9-15.
PHILLIPS, R. E., AND C. S. WILLIAMS, 1944. External morphology of the turkey during the
incubation period. Poultry Sci., 23 : 270-277.
REMPEL, A. G., AND H. L. EASTLICK, 1957. Developmental stages of normal White Silkie
fowl embryos. Northzvest Science, 31 : 1-13.
CAROTENOIDS AND CHLOROPHYLLIC PIGMENTS IN THE MARINE
SNAIL, CERITHIDEA CALIFORNICA HALDEMAN, INTERMEDIATE
HOST FOR SEVERAL AVIAN TREMATODES *• 2
A. M. NADAKALs
Department of Biology, University of Southern California, Los Angeles, California
The marine snail, Cerithidea californica Haldeman, is a favorable host for
more than twenty species of larval trematodes (Martin, 1955; Hunter, 1942).
These larvae occupy different regions of the body of the snail such as the digestive
gland, mantle, gills and part of the digestive tract. The digestive gland of the
snail, which is the main organ of infection, presents a variety of coloration in
different specimens. It may be green, brown, yellow, orange or creamy white.
The visceral part including the digestive tract is frequently dark blue. The mantle
and the integument are usually greenish-blue or blue and yellow intermingled.
A striking similarity exists between the coloration of the snail tissues and that
of the parasitic larvae harbored by them.
A considerable amount of information is available concerning the occurrence
and distribution of pigments, particularly carotenoids in various species of gastropod
molluscs. Earlier work has been reviewed by Fox (1953) and Goodwin (1954).
Although several species of snails are known to be hosts for pigmented larval
trematodes, no critical study so far has been made concerning their pigments with
a view to understanding the host-parasite relationship of pigmentation. In the
snail, Littorina littorea, pigmented foot has been reported to be a means of recog-
nizing infection with larval trematodes (Willey and Gross, 1957). Spectro-
photometric absorption studies of L. littorea extracts indicated the presence of
carotenoids ; however, chromatographic methods were not used for the separation
of various pigments. The author was interested to study the chemical nature and
origin of pigments found in certain species of larval trematodes harbored by the
snail. Cerithidea (Nadakal, 1960a, 1960b). In order to trace the host-parasite
relationship of pigments, it was necessary to analyze the pigments of the snail.
The present paper describes the pigments found in Cerithidea with special reference
to carotenoids.
MATERIALS AND METHODS
Specimens of Cerithidea and four species of algae, including three green and
one red algae which serve as food for the snails, were collected from the mud flats
of Newport Bay, California. The green algae were identified as Ulva sp., Chacto-
1 From a thesis submitted in partial fulfillment of the requirements for the Degree of Doctor
of Philosophy, University of Southern California.
- The author wishes to express his deep appreciation to Prof. W. E. Martin for the
guidance, interest, and criticisms throughout the course of this work.
3 Present address : Department of Biology, Immaculate Heart College, Los Angeles 27,
California.
98
PIGMENTS IN A MARINE SNAIL
99
morpha torta, and Enteronwrpha clathrata ; the red alga as Hypnca johnstonii.
The algal pigments were studied by the methods of the following workers : Strain
(1942) for chlorophyll a and b; Manning and Strain (1943) for chlorophyll d;
and Haxo et al. (1955) for phycobilins. After sorting out the various snail
tissues such as the digestive gland, mantle and foot, and visceral mass, they were
lyophilized separately and ground in an ordinary mortar. The methods outlined
by Fox and Pantin (1941) were followed with necessary modifications for the
extraction and analysis of pigments from the snail tissues. Chromatographic
separation of pigments was carried out according to the directions given by
Karrer and Jucker (1950). A cylindrical glass tube measuring 20 cm. X 12 mm.
was used for preparing the adsorption column. Among several adsorbents tried
for the separation of various pigments, such as calcium hydroxide for epiphasic
carotenoids, calcium carbonate and zinc carbonate for hypophasic carotenoids, and
powdered sugar (C & H Confectioner's) for chlorophyll derivatives, activated
alumina was found most satisfactory. Various pigment fractions obtained by
chromatographic separation were eluted in appropriate solvents like petroleum-
ether, methanol, etc. for determining the absorption spectra, with a Beckman
Spectrophotometer. Efforts were made to identify the pigments by spectro-
photometric absorption analyses, partition tests, color reactions, solubility,
fluorescence, and chromatographic behavior.
Figures 1-3 include spectral curves of the pigments A-E in petroleum-ether (b.p. 50-70° C.)
and F and G in methanol.
0-4
03
LJ
o
L±J
DQ
gO-2
CO
00
--A
400
560
440 48O 520
WAVELENGTH (M|J)
FIGURE 1. A. Light orange pigment HIII (Table II). B. Yellow pigment HII (Table II).
100
A. M. NADAKAL
0-4
400
560
440 480 52O
WAVELENGTH (M|J)
FIGURE 2. C. Orange pigment EIV (Table I). D. Orange pigment EIII (Table III),
E. Violet pigment EIII (Table I).
o
£0-3
z
UJ
00
CC
§02
CD
<
o
—
- -F
— G
400
800
500 GOO 700
WAVELENGTH (Mp)
FIGURE 3. F. Yellowish-brown pigment EII (Table I). G. Pale green pigment Ella
(Table I).
PIGMENTS IN A MARINE SNAIL
101
RESULTS
The pigment fractions obtained by chromatographic separation of the epiphasic
and hypophasic portions of the pigment extracts of the various snail tissues and
their characteristics are listed in Tables I-V. The pigment fractions in the case
of epiphasic portions of the extracts are numbered in order of decreasing adsorption
and hypophasic portions in order of increasing adsorption on the columns.
Absorption maxima and the forms of the spectral curve (Fig. 3, G) of the
pigments El and Ella (Table I) indicate that these pigments resemble chlorophyll
or pigments derived from chlorophyll. However, the absorption maxima in the
violet region of the spectrum are different from that of the chlorophyll reported
from plant sources. The Gmelin reaction (Pearse. 1953) was negative for these
pigments, suggesting that they are not open-ring tetrapyrrole compounds.
The brown pigment Ellb (Table I) showed a maximum at 450 m^. This
fraction could not be made hypophasic even after prolonged saponification. This
may be a carotenoid acid.
TABLE I
Ep! 'phasic portion of the digestive gland extract. Adsorbent: activated alumina.
Developing solvent: petroleum ether (50-70° C.) with 1-5% methanol
Band No.
Color of band
Percentage of solvent
required for elution
Absorption
maxima mju
Solvent
El
EII
EIII
Greenish-yellow
Yellowish-brown
Violet
2-3 methanol
2-3 methanol
3-4 methanol
410, 667
410, 450, 667
454
Ethanol
Ethanol
Petroleum ether
EIV
Orange
0
452, 482
Petroleum ether
Band EII was chromatographed again on alumina column and the two
fractions obtained are given below:
Ella
Pale green
2-3 methanol
416, 665
Methanol
Ellb
Brown
3-4 methanol with a few
450
Petroleum ether
drops of glacial acetic acid
The violet pigment EIII (Table I) showed a maximum at 454 m/x. The
form of the spectral curve (Fig. 2, E) and the single absorption maximum are
suggestive of a keto-carotenoid (Vevers and Millott, 1957).
The orange pigment EIV (Table I) has been found to possess properties
similar to those of /^-carotene (Fig. 2, C). The absorption maxima are in good
agreement with the figures given by Karrer and Jucker (1950), and Lederer
(1938). Besides, solubility, behavior on partition test, fluorescence (bluish-green
in ultra violet light), color reactions (pigment in chloroform solution turned
bluish-green on addition of concentrated sulfuric acid), color in solutions, and
chromatographic behavior also indicate that this is /^-carotene.
The absorption maxima of the pink pigment HI (Table II) in petroleum ether
and benzene are in good agreement with the figures given by Karrer and Jucker
(1950), and Goodwin (1953) for zeaxanthin. Its hypophasic behavior on parti-
tion test and chromatographic behavior also lend support to the conclusion that
102
A. M. NADAKAL
TABLE 11
Hypo phasic portion of the digestive gland extract. Adsorbent: activated alumina.
Developing solvent: petroleum ether with 1-5% methanol
Band No.
Color of band
Percentage of solvent
required for elution
Absorption
maxima m/i
Solvent
HI
Pink
2-3 ethanol
420, 450, 482
Petroleum ether
462, 490
Benzene
HII
Yellow
1-2 methanol
426, 448, 478
Petroleum ether
429, 457, 487
Chloroform
Hill
Light orange
1-2 methanol with a few
424, 454
Petroleum ether
drops of glacial acetic acid
HIV
Blue-green
2—3 ethanol
416, 670
Methanol
this is zeaxanthin. The fact that this pigment fraction stayed hypophasic even
after continued saponification and could not he made epiphasic with acid treatment,
showed that this pigment occurs in the snail tissues in the free state.
The yellow pigment HII (Tahle II) showed maxima in petroleum ether and
chloroform which are in good agreement with the figures quoted by Karrer and
Jucker (1950) for the xanthophyllic pigment lutein (Fig. 1, B). This pigment
turned bluish-green with concentrated sulfuric acid. These properties, coupled
with the chromatographic behavior, hypophasic nature on partition test, and
color in solutions, prove that this is lutein.
A yellow pigment fraction, separated from the epiphasic portion of the un-
saponified pigment extract, showed maxima in petroleum ether at 424, 445, and
475 niju. This pigment became hypophasic on saponification and could be made
epiphasic again with acetic acid treatment. This indicated that some of the lutein
in the snail's tissues is esterified. After saponification the pigment showed the
maxima at 446 and 478 m/x in petroleum ether.
The light orange pigment HIII (Table II) could be removed from the
column with a few drops of acetic acid in the eluting solvent. It is difficult to
identify this pigment conclusively ; it may be a carotenoid acid or some pigment
derived from hypophasic carotenoids (Fig. 1, A).
The blue-green pigment HIV (Table II) is characterized by absorption maxima
and spectral curve suggestive of chlorophyll or a pigment derived from it.
The brown pigment El (Table III) showed only one absorption maximum.
TABLE III
Pigment extracts from the mantle, branchial, and pedal tissues. No hypophasic fraction was obtained
on partition of the original extracts between 90% methanol and petroleum ether systems.
Pigments chromatographed as the epiphasic portion of the digestive gland extract
Band No.
Color of band
Percentage of solvent
required for elution
Absorption
maxima m/*
Solvent
El
Brown
2—3 methanol
450
Petroleum ether
EII
Pink
1-2 methanol with a few
452
Petroleum ether
EIII
Orange
drops of glacial acetic acid
0
424, 452, 480
Petroleum ether
PIGMENTS IN A MARINE SNAIL
103
This fraction could not be made hypophasic even after prolonged saponification
in 90% methanol-petroleum ether systems. Identification of this pigment fraction
was difficult.
The pink pigment El I (Table III) could be eluted with a few drops of glacial
acetic acid. It showed a maximum at 452 m/x. On saponification this became
hypophasic and could be made epiphasic again by treating with acetic acid. This
behavior indicated that this pigment could be an esterified carotenoid acid.
The orange pigment EIII (Table III) showed all characteristics of /3-carotene.
However, a small shoulder at 424 m/x in the spectral curve of this pigment is
remarkable (Fig. 2, D).
The khaki pigment El (Table IV) showed an absorption maximum only in
the violet region of the visible spectrum. This may be some breakdown product
TABLE IV
Pigment extract from the visceral mass. EpipJiasic portion chromatographed on activated alumina.
Developing solvent: petroleum ether with 1-5% methanol
Band NTo.
Color of band
Percentage of solvent
required for elution
Absorption
maxima m/i
Solvent
El
Khaki
1-2 methanol
425
Methanol
EII
Greenish-yellow
2-5 methanol
416, 667
Methanol
EIII
Yellowish-brown
2-4 methanol
420, 452, 667
Methanol
EIV
Violet
2-3 methanol
452
Petroleum ether
EV
Orange
0
450, 482
Petroleum ether
Band EIII was chromatographed again on alumina column and the two
fractions obtained are given below:
EHIa
Pale green
1-2 methanol
416, 665
Methanol
EHIb
Pale orange
1-2 methanol with a few
450
Petroleum ether
drops of glacial acetic acid
of chlorophyll or carotenoid pigments. Such breakdown products are known to
be adsorbed at the top of the columns (Fox, 1953).
The spectral properties of the pigments EII and Ellla (Table IV) indicated
that they are chlorophyll derivatives.
The pale orange pigment EHIb (Table IV) is considered to be a carotenoid
acid because of its single absorption band and acidic properties.
The violet pigment EIV (Table IV) was more or less similar to the violet
pigment EIII (Table I) extracted from the digestive gland and may be a keto-
carotenoid.
The orange pigment EV (Table IV) was identical with the pigments EIV
(Table I) and EIII (Table III) extracted from the digestive gland and mantle,
respectively. It is therefore concluded to be /^-carotene.
The pink pigment HI (Table V) was similar in properties to the one HI
(Table II) recovered from the hypophasic portion of the digestive gland extract
and is concluded to be zeaxanthin.
104
A. M. NADAKAL
The yellow pigment HI I (Table V) has been identified as lutein, as its
properties resemble those of the yellow pigment HII (Table II) extracted from
the hypophasic portion of the digestive gland extract.
The light orange pigment HIII (Table V) with its acidic properties and
two absorption maxima in the blue-violet region of the visible spectrum may be
considered as a carotenoid acid. No trace of chlorophyll derivatives could be
detected in the hypophasic portion of the visceral extracts.
A blue-green residue was left in the methanol-water fraction of the original
extracts of the digestive gland and visceral tissues. Part of the colored substance
could be taken up in ether after addition of a few drops of glacial acetic acid. It
was then washed with water, evaporated to dryness under vacuum and finally
taken up in methanol. The absorption maxima of this pigment at 416 and 667 m/*
and the form of the spectral curve was characteristic of chlorophyll a (Atkins and
Jenkins, 1953; Green, 1957). Even after extraction of the chlorophyllic pigments
by acidified ether, a bluish residue was left behind in the aqueous methanolic
solution. This was not extractable by any of the solvents tried. Attempts to
TABLE V
Hypophasic portion of the visceral extract. Chromatographed as the hypophasic
portion of the digestive gland extract
Band No.
Color of band
Percentage of solvent
required for elution
Absorption
maxima m^
Solvent
HI
Pink
2-3 methanol
420, 450, 482
Petroleum ether
HII
Yellow
1-2 methanol
422, 448, 478
Petroleum ether
HIII
Light orange
1-3 methanol with a few
drops of glacial acetic acid
424, 454
Petroleum ether
separate the pigment on adsorption columns also failed. This bluish residue
probably contained haemocyanin which is common in molluscan body fluids.
PIGMENTS FOUND IN THE ALGAE
The three species of green algae, Ulva sp.. Enter omorpha, clathrata, and
Chaetomorpha torta, have been found to contain chlorophyll a and b, /3-carotene,
and the xanthophyllic pigment lutein. No trace of a-carotene or any other pig-
ments related to carotenoids could be detected. The red alga, Hypnea johnstonii,
contained chlorophyll a and d, /3-carotene, lutein, phycoerythrin, and phycocyanin.
The principal carotenoid pigment extracted from the algae was /2-carotene.
DISCUSSION
The occurrence of carotenoids and chlorophyll derivatives in Ccrithidca is in
agreement with the previous findings in various species of molluscs. /3-Carotene and
lutein have been reported from several gastropods (Fox, 1953; Goodwin, 1954).
Zeaxanthin is known to occur in Patella rulgata and P. dcpressa (Goodwin, 1954;
Goodwin and Taha, 1950), and Mytilus californianus (Scheer, 1940). Carotenoid
acids with single absorption bands in the visible spectrum have also been reported
PIGMENTS IN A MARINE SNAIL 105
from many invertebrates such as sponges and molluscs (Fox, 1953). Demonstra-
tion of chlorophyll derivatives in molluscs has been made by MacMunn (1886a,
1886b), Dhere and Vegezzi (1916), and many others.
A survey of the occurrence and distribution of carotenoid pigments in in-
vertebrates reveals the fact that the digestive gland plays an important role in the
storage of these pigments. Crane (1949) found that in the cephalopods, Octopus
bimaculatus and Loligo opalescens, the liver-pancreas accumulated relatively large
amounts of carotenoid pigments. In Cerithidea, undoubtedly the digestive gland
functions as the chief organ for the storage of carotenoids. The preferential
accumulation of carotenoids in the lipid-rich digestive gland is not surprising
since these pigments have a tendency to be associated with lipids.
The visceral extracts of Cerithidea have also been found rich in carotenoid and
other pigments. This might be due to the presence in the gut of plant materials
ingested as food. The mantle and branchial tissues contained relatively small
amounts of carotenoids and no traces of chlorophyll derivatives could be detected
in these tissues. A similar situation is described by Brooks and Paulais (1939)
in the lamellibranchs, Ostrca edulis and Gryphaea angulata.
In Cerithidea, the principal pigment found in various tissues is ^-carotene.
Next in importance, on a quantitative basis, are the chlorophyll derivatives ; third
comes the lutein and fourth only the zeaxanthin. Carotenoid acids and keto-
carotenoids occur in traces only. The snail apparently shows a preference for
storing /2-carotene in its tissues. This is perhaps due to the preponderance of
/^-carotene in the algae which serve as food for the snail. Since Cerithidea assimi-
lates and stores both hydrocarbons and xanthophylls as well as chlorophyll deriva-
tives, it may be regarded as non-selective in its chromatic storage. However, the
preferential accumulation of carotenes in the mantle and the branchial tissues is
remarkable indeed.
There is ample evidence for the elementary origin of carotenoids and chlorophyll
derivatives in animals. The crustacean, Daphnia, builds up its carotenoid supply
from the algae upon which it feeds (Green, 1957). The sea mussel, Mytilus
calijornianus, is known to absorb and store carotenoid pigments "from a very
plentiful and widely varied diet" (Fox and Coe, 1943). Dhere and Vegazzi
(1916) concluded from experimental evidence that the greenish and grayish
hepatic pigments of Helix pomatia were derived from the chlorophyll of its diet.
The dark green pigment "chaetopterin" found in the intestinal epithelium of the
polychaete worm, Chaetopterus, is derived from chlorophyll by the elimination of
magnesium and the phytol chain (Lederer, 1940). Similarly, Lederer and Huttrer
(1942) and Winkler (1957) showed that the sea slug, Aplysia, accumulates the
pigment "aplysioviolin" in its ink-gland, which is derived from red algae consumed
as part of its diet.
As regards Cerithidea, sources of pigments could also be attributed to nutritional
factors. Examination of four species of algae which serve as food for the snail
indicated the presence of chlorophylls and carotenoids, in addition to the phycobilins
and chlorophyll d in the red alga. The snail probably builds up its pigment supply
from these algal sources. /3-Carotene seems to have been accumulated in the
snail tissues without any metabolic alteration. However, the bluish-green color
of mantle tissues is an indication that carotenoids may exist in them as a carotenoid-
106 A. M. NADAKAL
protein complex. The fact that no chlorophyll derivatives could be detected from
these tissues also lends support to this conclusion. Zeaxanthin, carotenoid acids,
and keto-carotenoids were not observed in the algae studied ; they may be con-
sidered as products of metabolic activities of the snail. Moreover, some of the
lutein and carotenoid acids were found esterified in the tissues of the snail. The
spectral properties of chlorophyll derivatives indicate that these pigments must
have also undergone some kind of metabolic change, possibly oxidation. The
absorption maxima of the chlorophyll derivatives in the red region of the spectrum
suggest that these pigments are derived from chlorophyll a of the algae. There
was no indication of the presence of chlorophyll d or phycobilins in the tissues of
the snail, negating the possibility that the snail absorbs these pigments from the
red alga, Hypnea.
Several examples can be cited to prove that the accumulation of pigments
in the body tissues of animals frequently results from catabolic activities. Such
accumulation of pigments may or may not be significant in the functional economy
of these organisms. It has been reported that the large pigment cells found in
the deeper layer of connective tissues adjoining the intestinal caeca of the leech,
Glossiphonia complanata, represent a kidney for the storage of waste products
derived from haemoglobin metabolism (Bradbury, 1957). Wigglesworth (1943)
found that in the blood-sucking bug, Rhodnius proli.vus, some of the ingested
blood is denatured to form biliverdin which is subsequently either excreted through
the gut or stored in the pericardial cells. Stephenson (1947) noticed that the
pigment in the gut epithelium of Fasciola hepatica is derived from the haemolysis
of the ingested blood. The occurrence of chlorophyll derivatives in Cerithidca
may not have any functional significance ; they simply happen to be deposited in
the tissues as metabolic wastes resulting from the digestion of algal food. Never-
theless, the storage of carotenoids in various tissues, particularly in the digestive
gland of the snail, may be beneficial since there are indications that certain
carotenoids may serve to prevent autoxidation of lipids in animal tissues (Verne,
1936a, 1936b). It is yet to be found out whether the snail needs vitamin A for
its metabolic activities, and, in case it does, it might make use of /3-carotene as
a potential source. Although in certain invertebrates carotenoids are known to
be utilized in sexual reproduction (Scheer, 1940) and in maintenance of mucous
surfaces, nothing is known about their roles in similar processes in Cerithidea.
SUMMARY
1. Evidences obtained from chromatography, spectrophotometric absorption
analyses, partition tests, etc. suggested the occurrence of the following pigments
in the marine snail, Cerithidea californica : /3-carotene, carotenoid acids, keto-
carotenoids, lutein, and chlorophyll derivatives.
2. In an attempt to understand the dietary relationship of pigmentation in the
snail, four species of algae were studied for their pigment contents. The three
green algae were found to contain chlorophyll a and b, /3-carotene, and lutein ; the
red alga, chlorophyll a and d, /3-carotene, lutein, and phycobilins.
3. The spectral properties of the chlorophyll derivatives recovered from the
snail suggested that they are derived from chlorophyll a of the algae and that the
molecular structure is still intact with the magnesium atom atached to it. However,
PIGMENTS IN A MARINE SNAIL 107
absorption maxima in the violet region of the spectrum are shifted toward shorter,
and in the red toward longer wave lengths, indicating some metabolic change
in these pigments, possibly oxidation.
4. Part of the lutein and carotenoid acids were found to be esterified in the
digestive gland and mantle tissues. No metabolic alteration has been noticed
in the case of /3-carotene. All available evidence suggests that zeaxanthin,
carotenoid acids, and keto-carotenoids are products of the snail's metabolic
activities.
5. Apparently the snails do not absorb phycobilins or chlorophyll d from
the red alga.
6. The snail has been found to be non-selective in its chromatic storage.
7. The nutritional relationship and biological significance of pigments in the
snail have been discussed.
LITERATURE CITED
ATKINS, W. R. G., AND P. G. JENKINS, 1953. Seasonal changes in the phytoplankton during
the year 1951-52 as indicated by spectrophotometric chlorophyll estimations. 7. Mar.
Biol. Assoc., 31 : 495-507.
BRADBURY, S., 1957. A histochemical study of the pigment cells of the leech, Glossiphonia
complanata. Quart. J. Micr. Sci., 98: 301-314.
BROOKS, G., AND R. PAULAIS, 1939. Repartition et localisation des carotenoides, des flavin
et de l'acide-L-ascorbique chez les mollusques lamellibranches : cas des huitres et des
gryphees vertes et blanches. C. R. Acad. Sci., Paris, 208 : 833-835.
CRANE, S. C., 1949. Studies of hepatopancreatic function and carotenoid metabolism of the
octopus, Octopus biiuaculatus. Dissertation, University of California.
DHERE, C., AND G. VEGEZZI, 1916. Sur la composition pigmentaire de I'hepato-chlorophylle.
C. R. Acad. Sci. Paris, 163: 399-401.
Fox, D. L., 1953. Animal Biochromes and Structural Colors. Cambridge Univ. Press.
Fox, D. L., AND W. R. COE, 1943. Biology of the California sea mussel (Mytilus calif ornianus).
II. Nutrition, metabolism, growth, and calcium deposition. /. E.vp. Zoo!., 93: 205-249.
Fox, D. L., AND C. F. A. PANTIN, 1941. The colors of the plumose anemone, Metridium senile.
Philos. Trans. Roy. Soc.. Ser. B, 230: 415-450.
GOODWIN, T. W., 1954. Carotenoids, Their Comparative Biochemistry. Chemical Publishing
Company, New York.
GOODWIN, T. W., AND M. M. TAHA, 1950. The carotenoids of the gonads of the limpets. Patella
vulgata and P. depressa. Biochem. J., 47 : 249-251.
GREEN, J., 1957. Carotenoids in Daphnia. Proc. Roy. Soc. London. Scr. B, 147 : 392-401.
HAXO, F., COLM O'nEocHA AND P. NORRIS, 1955. Comparative studies of chromatographically
separated phycoerythrins and phycocyanins. Arch. Biochem. Biophys., 54: 162-173.
HUNTER, W. S., 1942. Studies on cercariae of the common mud-flat snail, Cerithidea californica.
Ph.D. Thesis. University of California.
KARRER, P., AND E. JUCKER, 1950. Carotenoids. Elsevier Publishing Co., Inc., New York.
LEDERER, E., 1938. Recherches sur les carotenoides des Invertebres. Bull. Soc. On;;;. Biol..
Paris. 20: 567-610.
LEDERER, E., 1940. Les pigments des Invertebres. Biol. Rei1.. 15: 273-306.
LEDERER, E., AND C. HUTTRER, 1942. Pigments from the secretion of Aplvsia (sea hare or sea
slug). Trans. Mem. Soc. Chim. Biol., 1055-1061.
MACMUNN, C. A., 1886a. Further observations on enterochlorophyll and allied pigments.
Philos. Trans. Roy. Soc., 177: 267-298.
MACMUNN, C. A., 1886b. Further observations on some of the applications of the spectro-
scope in biology, with special reference to the presence of chlorophyll in animals.
Proc. Birmingham Nat. Hist. Soc., 5: 177-218.
MANNING, W. M., AND H. H. STRAIN, 1943. Chlorophyll d, a green pigment of red algae.
/. Bwl. Chetii., 151 : 1-19.
108 A. M. NADAKAL
MARTIN, W. E., 1955. Seasonal infections of the snail, Ccrithidca californica Haldeman with
larval trematodes. Hancock Commemoration Volume, pp. 203-210.
NADAKAL, A. M., 1960a. Chemical nature of cercarial eye-spot and other tissue pigments.
/. Parasitol. (in press).
NADAKAL, A. M., 1960b. Types and sources of pigments in certain species of larval trematodes.
/. Parasitol. (in press).
PEARSE, A. G. E., 1953. Histo-chemistry, Theoretical and Applied. London. Churchill.
SCHEER, B. T., 1940. Some features of the metabolism of the carotenoid pigments of the Cali-
fornia sea mussel (Mytilus calif orniamts) . J. Biol. Chem., 136: 275-299.
STEPHENSON, W., 1947. Physiological and histo-chemical observations on the adult liver fluke,
Fasciola hcpatica L. II. Feeding. Parasitol., 38 : 123-127.
STRAIN, H. H., 1942. Chromatographic Adsorption Analysis. Interscience Publishers, New
York.
VERNE, J., 1936a. Carotenoides et oxydation des lipides. C. R. Soc. Biol., Paris, 121 : 609-610.
VERNE, J., 1936b. Observations histochemiques sur 1'oxydation des lipides et ses rapports avec
les carotenoides. Bull. Histol. Tech. Micr., 13: 433-440.
VEVERS, H. G., AND N. MILLOTT, 1957. Carotenoid pigments in the integument of the starfish,
Marthasterias glacialis (L.) Proc. Mar. Biol. Assoc., 30: 569-574.
WIGGLES WORTH, V. B., 1943. The fate of haemoglobin in Rhodnins prolixus (Hemiptera) and
other blood-sucking arthropods. Proc. Roy. Soc. London, Scr. B, 131 : 313-339.
WILLEY, C. H., AND P. R. GROSS, 1957. Pigmentation in the foot of Littorina littorca as a
means of recognition of infection with trematode larvae. /. Parasitol., 43 : 324—327.
WINKLER, L. R., 1957. The biology of California sea hares of the genus Aplysia. Doctoral
thesis. University of Southern California.
THYROID HORMONE TREATMENT AND OXYGEN CONSUMPTION
IN EMBRYOS OF THE SPINY DOGFISH 1
AUSTIN W. PRITCHARD AND AUBREY GORBMAN
Department of Zoology, Oregon State College^ Corvallis, Oregon, and the Department of
Zoology, Columbia University, New York 27 , N. Y.
The inability of adult cold-blooded vertebrates to respond to thyroxine
treatment with an increased oxygen consumption rate is now a well documented
finding (Hoar, 1957; Gorbman, 1959). The two often cited exceptions to this
general experience are the thyroxine-induced increases in oxygen consumption in
adult goldfish observed by Miiller (1953), and in parrot fish of certain sizes, as
described by Smith and Matthews (1948). Both of these claims have been
denied by opposite results in the same species (Etkin, Root and Mofshin, 1940;
Chavin and Rossmore, 1956; Matty, 1957). Measurements of metabolic rate in
fishes are subject to numerous variables which are not as easily controlled as they
are in mammals (responses to handling, previous temperature history, illumination,
endogenous activity cycles) (Fry, 1957), so that it is not surprising that con-
flicting claims may exist for some species. Of the factors which may contribute
misleading information in measurements of oxygen consumption in fishes, among
the most significant is muscular activity. Hoar (1958) has shown clearly that
treatment of fishes with thyroid hormone induces behavioral changes, expressed
primarily by an increased spontaneous motor activity. If this is so, then any valid
test for basal metabolic stimulation by thyroxine must exclude the variable of
locomotor muscular work. Although testing systems are available which make
this possible (Fry, 1957), neither of the two exceptional claims mentioned above
utilized them.
While working with near-term embryos of the spiny dogfish, Sqitalus sucklevi,
removed from the uterus and kept in flowing sea water, we noticed a behavioral
feature which makes this animal useful in respiratory studies. When kept in
subdued light they remain still, even after treatment with thyroid hormone. Since
under these circumstances, spontaneous muscular movements are rare, then respira-
tory measurements can be taken to reflect "basal" requirements (or at least
"standard" metabolism as defined by Fry, 1957), not a thyroxine-induced increase
in swimming. In these experiments oxygen consumption of such exteriorized
dogfish pups was measured after treatment with thyroxine, or two of its analogues,
or propyl thiouracil. To our knowledge the only other studies of the metabolic
responsiveness to thyroid hormone in larval vertebrates have concerned anuran
tadpoles. In this regard, too, the published literature is in disagreement (Etkin,
1955; Lewis and Frieden, 1959). Accordingly, it was hoped that the experiments
1 Supported by grants from the National Science Foundation. We would like to express
our appreciation to the Friday Harbor Laboratories of the University of Washington for
generous assistance rendered during this investigation. We also thank Dr. Frederick L. Hisaw,
Jr., who contributed time, experience, and materials towards collecting the dogfish, and Mr.
Robert Lasher, who aided in capturing the dogfish and in running oxygen analyses.
109
110
AUSTIN W. PRITCHARD AND AUBREY GORBMAN
with dogfish embryos would prove enlightening, both with regard to the metabolic
action of thyroid hormone in poikilotherms and its action in differentiating forms
of such animals.
MATERIAL AND METHODS
Oxygen consumption was measured in a continuous-flow apparatus of the type
used by Job (1955) in measuring the "standard" metabolism of trout. Four
respirometer flasks (2.5-liter Fernbach culture jars) were used at any one time,
one of these being used as a "blank." The four flasks were immersed in a large
wooden tank in which the water level was maintained constant by means of an
overflow. The flow of water through the flasks was so adjusted that Squalus
embryos in groups of three removed about 0.5 to 1.0 ml. of oxygen. Rate of
oxygen consumption was calculated from the flow rate, the difference in oxygen
content between incur rent and excurrent water, and the wet weight of the animals
2
Q.
O en
--.
UJ <->
>
X
o
25
0800
1200
1600
2000
0800
1200
JUNE 30
JULY I
CLOCK TIME
FIGURE 1. Serial determination of metabolic rate in untreated embryos of Squalus sitcklcyi
over a 27-hour period. Two respirometers used with three pups in each.
tested. Oxygen content of the water was determined by the unmodified Winkler
technique.
Water temperature never varied more than 1° C. during a single series of
tests, and usually it did not change at all measurably. During most measure-
ments of oxygen consumption the water temperature ranged from 13° to 14° C.
However, extremes of 12° C. and 16° C. (on one unusually hot day) were recorded.
The water in the large water bath was continuously aerated through a stone "air
breaker," and preliminary tests showed that the oxygen content of the water was
uniform at all points in the bath.
Embryos were tested in groups of three, being placed in the respirometers
one hour before the first measurement of oxygen consumption. The bath con-
taining the respirometers was covered to shield the animals from most of the
light and other extraneous factors. Frequent observation indicated that under
these conditions spontaneous muscular movements were rare. Several prelimi-
THYROID TREATMENT OF DOGFISH
111
TABLE I
Metabolic rates of non-treated dogfish pups showing normal day-to-day variability in the laboratory.
All runs made bet-ween 1:00 and 3:00 PM
Date
July3
July 4
July 6
July 8
July 10
No. of
trials
4
4
3
3
3
Mean Oi consumption
(cc./kg./hr.)
28.8 ± 1.4 (std. dev.)
31.4 ± 1.9
29.5 db .89
31.3 ± 1.4
33.7 ± 1.7
nary measurements were made on untreated pups, to ascertain the variability in
oxygen consumption under laboratory conditions. Figure 1 illustrates the results
of serial determinations on two groups of embryos, all taken from the same female,
over a H-day period. The period of steadiest metabolic rate was in the afternoon;
accordingly all measurements reported here were made in the afternoon only.
In another preliminary test, metabolic rates were determined on pups from one
female dogfish, over a period of one week immediately following transfer of the
pups to the laboratory. The results (Table I) show that the average metabolic
rate remained at about the same level over this period and that the variability in
oxygen uptake (as indicated by the standard deviations) was quite small.
TABLE II
Experimental protocol for each injection group. Numbers in parentheses
indicate the number of pups from that female
Experiment
Total no. of
injections
Females from which
pups were taken
Substances
tested*
Dose per
injection
1
10
A(15)
triac
10 Mg.
1-Tx
10 Mg.
2
9
B(11),C(9)
triac
10 Mg-
1-Tx
10 /ig-
PTU
50 Mg.
3
9
D(10), E(10)
triac
10 Mg-
100 Mg-
1-Tx
10 Mg-
PTU
50 Mg.
4
8
F(11),G(4), H(4),
triac
10 Mg-
I(4),J(5), K(7)
100 Mg.
1-Tx
10 Mg.
T3
100 Mg.
PTU
50 Mg-
5
5
L(19)
1-Tx
100 Mg-
T3
100 Mg-
PTU
50 Mg.
6
4
M(2S), N(10)
triac
10 Mg.
1-Tx
10 Mg.
* Abbreviations : triac, triiodothyroacetic acid; 1-Tx, 1-thyroxine; PTU, propylthiouracil ;
T3, triiodothyronine.
112
AUSTIN W. PRITCHARD AND AUBRKY GORBMAN
TABLE III
Effects of repeated injections of thyroid hormones on oxygen consumption (cc./kg./hr.) of embryos of
Squalus suckleyi.* Values in the table are means of 3 to 6 consecutive determinations,
taken at 15-minute intervals. E = experimental. C = control
Experi-
ment
No. of
injections
Triac
10 Mg.
Triac
100 MS.
1-Tx
10 Mg-
1-Tx
100 Mg.
T3
100 Mg-
PTU
50 Mg.
1
E
C
E/C X 100
32.40
30.79
105
33.52
32.52
103
1
4
E
C
E/C X 100
52.25
39.28
133
44.78
39.28
114
9
E
C
E/C X 100
46.82
44.60
105
2
E
C
E/C X 100
32.25
31.52
102
32.15
31.52
102
28.78
31.52
91
2
4
E
C
E/C X 100
41.90
35.80
117
40.90
35.80
114
36.00
35.80
101
9
E
C
E/C X 100
33.80
34.16
99
41.40
34.16
121
31.27
34.16
91
1
E
C
E/C X 100
33.10
33.20
99
33.40
33.20
101
33.30
33.20
100
32.70
33.20
98
3
4
E
C
E/C X 100
36.70
30.40
121
33.90
30.40
108
29.20
30.40
96
29.00
30.40
98
9
E
C
E/C X 100
35.87
32.96
109
35.50
32.96
108
33.62
32.96
102
38.09
32.96
115
2
E
C
E/C X 100
34.13
29.86
114
32.56
29.86
109
30.82
29.86
103
30.12
29.86
101
30.30
29.86
102
4
5
E
C
E/C X 100
37.02
30.41
122
33.45
30.41
110
33.97
30.41
112
34.63
30.41
114
32.51
30.41
107
8
E
C
E/C X 100
32.33
2749
118
33.66
27.49
122
31.86
27.49
116
32.28
27.49
118
29.44
27.49
107
* This table includes absolute values for oxygen consumption at the beginning (after 1 or 2
injections), the middle (after 4 or 5 injections), and end after 8 or 9 injections in each experiment.
The complete course of each experiment is shown in Figures 4A, 4B and 4C, but these do not show
absolute values. To tabulate all the absolute values would require an impractically long table.
THYROID TREATMENT OF DOGFISH
TABLE III — Continued
113
Experi-
No. of
Triac
Triac
1-Tx
l-Tx
T3
PTU
ment
injections
10 Mg.
100 ng.
10 Mg.
100 Mg.
100 Mg.
50 Mg.
E
30.01
25.78
26.96
1
C
26.86
22.82
22.82
E/C X 100
112
113
118
5
E
39.92
42.96
37.07
5
C
35.78
35.78
35.78
E/C X 100
112
120
104
E
28.49
30.37
1
C
26.18
26.18
E/C X 100
109
110
6
E
35.34
32.82
4
C
29.78
29.78
E/C X 100
119
110
The animals used were "pups" removed from the uteri of Squalus sitckleyi
females caught during July and August, 1958, at Friday Harbor, Washington,
within 200 yards of the laboratory. The ovoviviparous young of this species
remain in the uterus for two years. It could be estimated from the sizes of the
yolk sacs that the embryos we used were approximately 19-23 months of age.
Occasional "spontaneous" birth of pups of captive females was observed in late
August. Embryos removed from the uterus were kept in apparently good con-
dition for periods as long as several weeks in large, covered glass dishes (2-liter
capacity) in groups of 4 or 5, in slowly flowing sea water. Whenever possible,
all pups used in an experiment were taken from the same mother. In experiments
requiring large numbers of pups it was necessary to combine litters from several
mothers and these were distributed as evenly as possible into the different experi-
mental groups (Table II). The limited number of embryos of equivalent de-
velopment available at one time, and the limited capacity of the respirometers
made it impossible to test all hormones at the same time. For this reason, six
different experiments were performed during an eight-week period.
In each experiment groups of embryos were injected intraperitoneally on
alternate days with various doses (Table II) of hormones, propylthiouracil, or
0.7% NaCl solution, always in a volume of 0.05 cc. Oxygen consumption was
determined on the clay after injection to avoid possible responses to handling.
The compounds tested for their effect on oxygen consumption were 1-thyroxine
(Tx). 1-triiodothyronine (3 : 5 : 3'-triiodo-l-thyronine, To), triiodothyroacetic acid
(3:5:3' triiodothyroacetic acid, Triac) in doses of 10 micrograms or 100 micro-
grams, and propylthiouracil in doses of 50 micrograms.
EXPERIMENTS AND RESULTS
Six experiments (Table III) were completed. The total number of injections,
given at 2-day intervals, was as few as 4 or 5, but was usually (in four of the six
experiments) 8 to 10. The shorter experiments were ended when accidental
114
AUSTIN W. PRITCHARD AND AUBREY GORBMAN
blockage of the sea water occurred. Since such occurrences were obviously
harmful, and their effects difficult to assay, respiratory measurements were
accordingly not continued. In experiment 1 such a blockage killed all Triac-
injected animals after the fifth injection (Fig. 2). The Tx-injected animals in
this experiment showed an extreme but temporary respiratory depression (Fig. 2)
at the same time, so may have had a brief experience of the same nature. Only
one other extremely variant datum is seen in Figure 3, which shows the results
of experiment 2. Here an exceptionally high respiratory rate was observed in
saline solution-injected controls after three injections. Since these same animals
in succeeding measurements showed relatively little variation in oxygen consump-
2
3456
NUMBER OF INJECTIONS
8
10
FIGURE 2. The effects of repeated injections of thyroid compounds on metabolic rate of
dogfish embryos — injection group 1. Solid line, control; dashed line, triiodothyroacetic acid
(10/ug.); dotted line, 1-thyroxine (10/xg. ).
tion, it is felt that the exceptional figure may have been due to some limited
experience, possibly a brief interruption in water supply. The remaining data,
summarized in the figures, and Table III, appear to show consistent trends of
response, or lack of response, to the various forms of treatment.
It may be seen in Figures 1 and 2, and Table III, that the oxygen consumption
of control embryos varied throughout the periods of respiratory measurement,
but not in any particular pattern. The nature of this variation, whether due to
maturational or environmental factors, is not clear. However, whatever the
basis for the variation in control respiratory metabolism, the changes were generally
gradual. In the first experiment (Fig. 2) oxygen consumption increased gradu-
THYROID TREATMENT OF DOGFISH
115
ally through most of the three-week period of observation ; in the second experiment
(Fig. 3) this variation was, in general, less and showed no such constant trend.
Fortunately, the general variation of the controls was paralleled by the hormone-
injected embryos, and in addition, a relative difference from the controls was
usually maintained, if it occurred at all. Accordingly, when the results are ex-
pressed as per cent of the control oxygen consumption some conclusions appear
to be offered (Table III, Fig. 4).
The most potent stimulator of oxygen consumption in these tests was triiodo-
thyroacetic acid (Fig. 4A). Despite all the variations to which such experiments
seem to be prone, in all four experiments in which Triac was injected in 10-
microgram quantities, it clearly induced an increase in oxygen consumption to
maxima 17% to 33% above the control. These maxima were achieved 8 to 10
days after beginning the injections and thereafter oxygen consumption progressively
45
o
u
O 40
Q.
CO
O 35
o
z
UJ
o
O 30
o
34567
NUMBER OF INJECTIONS
8
FIGURE 3. The effects of repeated injections of thyroid compounds on metabolic rate of
dogfish embryos — injection group 2. Solid line, control; dashed line, triiodothyroacetic acid
(10/ttg.) ; dotted line, 1-thyroxine (10/ug.).
decreased. The larger dose of Triac (100 micrograms) was less effective than
the smaller one (Table III).
Thyroxine, in 10-microgram doses, was not as clearly a stimulator of oxygen
consumption in the Sqitahts pups as was Triac. It consistently raised oxygen
consumption in two of four experiments (Table III, Fig. 4B) above that of
controls, but failed to do so in one, and in another did not produce a significant
stimulation until the very end. However, in all thyroxine experiments oxygen
consumption was rising at the end of the period of treatment, in comparison
with the controls. In one test with 100-microgram quantities of thyroxine a 12%
increase above the controls in respiratory metabolism was noted.
Triiodothyronine, in 100-microgram quantities, in all instances stimulated
oxygen consumption to levels as high as 18% to 20% above the controls (Table
III).
116
AUSTIN W. PRITCHARD AND AUBREY GORBMAN
UJ
130
0120
z
o
o
110
zlOO
UJ
o
£90
a
A TRIAC, lOpgm
34567
NUMBER OF INJECTIONS
8
UJ
£130
^120
ac
| no
O
^100
UJ
B Thyroxine,
-" >
_._.X- -8-'
4567
NUMBER OF INJECTIONS
8
10
lil
C PTU
120
110
...o
..•••
0100
« "2"' A.... '~
£
* - ~—^.-:-i.:..o:^-^ ^ ^^^^
890
•*' x^^ / Ne *
or
9
u
34567
NUMBER OF INJECTIONS
8
FIGURE 4; A, B, and C. The effects of repeated injections of thyroid compounds on
metabolic rate of dogfish embryos. A, triiodothyroacetic acid, 10 /xg. each injection; B, 1-
thyroxine, 10 /ug. ; C, propylthiouracil, 50 ^g- Points represent per cent of the control rate.
Open circles, injection group 1; horizontal-barred circles, injection group 2; vertical-barred
circles, injection group 3; filled circles, injection group 4.
THYROID TREATMENT OF DOGFISH 117
Propylthiouracil had no particular effect on oxygen consumption relative to the
saline-injected controls (Table III, Fig. 4C).
DISCUSSION
Results of this investigation indicate that, under the conditions of these experi-
ments, 10 micrograms of triiodothyroacetic acid (Triac), given on alternate days,
have acted as a stimulant of oxygen consumption in the near-term shark embryo
(Fig. 4A). The unusual feature of this response is its diminution after 8 to 10
days despite continued injections of the hormone. In laboratory mammals (Foster,
Palmer and Leland, 1936) and in clinical use (Means, Lerman and Salter, 1933)
the continued administration of thyroxine or crude thyroid preparations is usually
accompanied by a sustained increased respiratory rate. However, even in clinical
experience it has been reported (Eppinger and Salter, 1935) that an initial rise in
metabolism following a week of treatment with thyroid hormone may be followed
by a sharp drop, even though treatment is continued. Thus, this pattern of
metabolic response is not unprecedented, and may depend on particular physio-
logical factors involved in the response to thyroid hormone. It is of especial
interest that Triac has been found to be about 10 to 25 times as active as thyroxine
in stimulating amphibian metamorphosis (Pitt-Rivers and Tata, 1959). A ten-fold
larger dose of Triac was no more active than the 10-microgram quantity, and
appeared, in fact, less active (Table III).
Thyroxine, in either the 10- or 100-microgram dosage, was less clearly a
metabolic stimulant. In two of four experiments animals receiving the 10-
microgram dose remained consistently higher than controls in oxygen consumption
by some 10% to 20%. In the other two experiments this superiority was either
lacking or irregularly variable. However, in all four instances oxygen consump-
tion was rising (relative to the saline-injected controls) at the ends of the experi-
ments (Fig. 4B). This was the most variably effective metabolic stimulant and
no explanation can be offered for this variability. Larger doses of thyroxine
and triiodothyronine (Table III) produce a 12 to 20% increase in oxygen con-
sumption by 10 to 16 days after beginning the injections of hormone.
Propylthiouracil was neither a stimulant nor depressant for oxygen consumption
in four different experiments which lasted about 18 days each. Almost all
measures of oxygen consumption in dogfish pups treated with this antithyroid drug
were within 10% of the control. It has been reported by Zaks and Zamkova
(1952) that thiourea consistently reduces the oxygen consumption of young
salmon and sturgeons below that of controls. Chavin and Rossmore (1956),
working with thiouracil-treated young goldfish, found no effect on oxygen con-
sumption. Interpretation of results of treatment with antithyroid drugs is
always complicated by the fact that they are known to be toxic, even in small doses.
Since the absence of a metabolic response to propylthiouracil might mean merely
that no hormone is yet produced by the thyroid of these embryonic animals, five
of them were injected with radioiodide (5 microcuries) and the rest were fixed
for histological examination to investigate this possibility. The 24-hour thyroidal
radioiodine uptake varied from 0.25% to 2%, a small but significant degree of
accumulation. This compares favorably with thyroid uptakes of about 1.5%
found by Gorbman and \Yaterman (unpublished) in pups of the Atlantic spiny
118 AUSTIN W. PRITCHARD AND AUBREY GORBMAN
dogfish, Squalus acanthias. Vivien and Rechenmann (1954) who also treated
shark pups (Scyliorhinns canicula) with I131, observed by radioautographic tech-
niques that it is deposited in the thyroid, presumably in protein-bound form. The
thyroid tissue examined histologically showed a slight increase in average cell
height (about 25% ) and "vacuolization" of the colloid. This would appear to
indicate that the pituitary-thyroid axis of mutual responsiveness is differentiated
in these animals, and that it had responded in the PTU-treated animals to a change
in thyroid hormone output by TSH secretion. However, despite this apparent
decrease in endogenous thyroid hormone production there was no detectable
change in oxygen consumption. It is possible that this decrease in endogenous
thyroid hormone, if real, was much smaller in size than the 10 micrograms in
the injected dose.
In summary, it may be said that triiodothyroacetic acid has been shown in
these experiments to be a temporary stimulant of oxygen consumption in near-
term embryos of Squalus sitckleyi. Triiodothyronine proved slightly less active,
and the metabolic stimulation by thyroxine was irregular. The thyroid glands
of these animals appeared to be functioning at a low rate, and interruption of this
function by propylthiouracil had no demonstrable effect on oxygen consumption.
SUMMARY
1. The oxygen consumption rate of "near-term" pups of the dogfish, Squalus
suckleyi, was determined at regular intervals during the course of repeated in-
jections of physiological saline solution, thyroid hormones, or of anti-thyroid sub-
stances. Up to 10 injections were given on alternate days.
2. Of the compounds tested, triiodothyroacetic acid at a dosage level of 10
micrograms per injection was the most consistent in raising the level of oxygen
consumption. The effect, however, was transitory with oxygen consumption
rising to a maximum (17% to 33%) level above the saline-injected controls after
four injections, thereafter declining slowly to control levels.
3. L-thyroxine at a dosage level of 10 micrograms had a variable effect on
oxygen consumption. In two of four experiments the oxygen consumption rate
rose irregularly, reaching a level about 2Gc/( above the controls after 9-10
injections. In the remaining experiments, there was no clear tendency to remain
above the controls.
4. Propylthiouracil, after 9 injections, had no consistent effect on metabolic
rate in four experiments.
5. The results are discussed with reference to the possible level of thyroid
function in these animals.
LITERATURE CITED
CHAVIX, W., AND H. W. ROSSMORE, 1956. Pituitary-thyroid regulation of respiration in the
goldfish, Carassins auratus L. Anat. Rcc., 125: 599.
EPPINGER, E. C., AND W. T. SALTER, 1935. The daily requirement in human hypothyroidism of
purified human thyroid hormone at various metabolic levels. Aincr. J. Alcd. Sci.,
190: 649-655.
ETKIN, W., 1955. In: Analysis of Development, edited by B. H. Willier, P. \Yeiss, and V.
Hamburger, Philadelphia : W. B. Saunders Co.
ETKIN, W., R. W. ROOT AND B. P. MOFSHIN, 1940. The effect of thyroid feeding on oxygen
consumption of the goldfish. Physiol. Zool., 13: 415-429.
THYROID TREATMENT OF DOGFISH 119
FOSTER, G. L., W. W. PALMER AND J. P. LELAND, 1936. A comparison of the calorigenic
potencies of /-thyroxine, rfMhyroxine, and thyroid gland. /. Biol. Client., 115: 467-478.
FRY, F. E. J., 1957. In : The Physiology of Fishes, M. E. Brown, ed. New York, Academic
Press.
GORBMAN, A. (editor), 1959. Comparative Endocrinology. New York, John Wiley and Sons.
HOAR, W. S., 1957. In : The Physiology of Fishes, M. E. Brown, ed. New York, Academic
Press.
HOAR, W. S., 1958. Effects of synthetic thyroxine and gonadal steroids on the metabolism of
goldfish. Canad. J. Zooi, 36: 113-121.
TOB, S. V., 1955. The oxygen consumption of Salvclinus fontinalis. Univ. of Toronto Biol.
Ser. No. 61 : 33 pp.
LEWIS, E. J. C, AND E. FRIEDEN, 1959. Biochemistry of amphibian metamorphosis : effect of
triiodothyronine, thyroxine, and dinitrophenol on the respiration of the tadpole.
Endocrinol., 65: 273-282.
MATTY, A. J., 1957. Thyroidectomy and its effect upon oxygen consumption of the teleost fish,
Pscudoscarus guacamaia. J. Endocrinol., 15: 1-8.
MEANS, J. H., J. LERMAN AND W. T. SALTER, 1933. The role of thyroxin iodine and total
organic iodine in the calorigenic action of whole thyroid gland. /. Clin. Investig., 12 :
683-688.
MULLER, J., 1953. t'ber die Wirkung von Thyroxin und Thyreotropem Hormon auf den
Stoffwechsel und die Farbung der Goldfisches. Zeitschr. vcri/l. Physiol., 35:1-12.
PITT-RIVERS, R., AND J. R. TATA, 1959. The Thyroid Hormone. New York, Pergamon Press.
SMITH, D. C., AND S. A. MATTHEWS, 1948. Parrot fish thyroid extract and its effect on
oxygen consumption in the fish, Bathystoina. Amcr. J. Physiol., 153: 215-221.
VIVIEN, JEAN, AND ROGER RECHENMANN, 1954. fitude sur la fonction thyroidienne de 1'embryon
de selacien. C. R. Soc. Biol., 148: 170-172.
ZAKS, M. G., AND M. A. ZAMKOVA, 1952. On the effect of thiourea on gaseous exchange in
larval salmon and sturgeon. Dokladv Akad. Nank S.S.S.R., 48: 1101-1103.
PERMEATION AND MEMBRANE TRANSPORT IN PARASITISM :
STUDIES ON A TAPEWORM-ELASMOBRANCH SYMBIOSIS 1
C. P. READ,? J. E. SIMMONS, JR.,- J. W. CAMPBELL 2, 3
AND A. H. ROTHMAN -• *
Marine Biological Laboratory, Woods Hole, Massachusetts; Department of Pathobiology,
School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland;
and Department of Biology, The Rice Institute, Houston, Texas
It was shown by Read, Simmons and Rothman (1960) that the amino acids
L-valine and L-leucine enter the tapeworm, Calliobothrium verticillatum, by a
process showing adsorption kinetics. The data obtained ruled out simple diffusion
but did not permit a definite conclusion as to whether the permeation is a process
of active transport. L-valine and L-leucine were each shown to competitively
inhibit the entry of the other into the worm. Several other amino acids were
shown to inhibit L-valine and L-leucine permeation, but it was not established that
these inhibitions were competitive. The studies to be reported have shown that
L-valine is actively transported, although further investigation has revealed that
when Calliobothrium is treated as a member of a symbiotic relationship, the
operation of the amino acid entry systems is indeed complex. Appreciation is
expressed to Dr. G. Wertheim who lent technical assistance during part of this
study, and to the gentlemen of the Supply Department of the Marine Biological
Laboratory, Woods Hole, Massachausetts, who furnished many living dogfish.
MATERIALS AND METHODS
The methods of collection and handling of Calliobothrium from the dogfish,
Mustelus canis, were similar to those used in a previous study (Read, Simmons
and Rothman, 1960). The salt solution used in handling both worms and host
tissues had the following composition: NaCl, 250 mM. ; KC1, 4.4 mM. ; CaGU,
5.1 mM.; MgCL, 2.9 mM.; urea 300 mM.; and tris (hydroxy methyl) amino
methane-maleate buffer, 10 mM. (pH 7.2). All incubations were carried out
in this medium, with appropriate experimental additions, at 10° C. In preparing
tissues for experimental incubation, the worms were washed in several changes
of the salt solution and incubated for 60 minutes at 10° C. before an experiment.
Host tissues were removed in ice-cold salt solution and, before the experiment, were
incubated at 10° C. for 30 minutes in a large volume of salt solution containing
10 mM. glucose. Throughout this period, the tissue was vigorously aerated
with a sintered bubbler attached to a small air pump. For experimental incuba-
1 This work was supported by grants from the National Institutes of Health, U. S. Public
Health Service (E-1508 and E-1384) and Smith, Kline and French Foundation.
2 Present address : Department of Biology, The Rice Institute, Houston, Texas.
3 Research Fellow, National Academy of Sciences-National Research Council.
4 U. S. Public Health Service Postdoctoral Fellow.
120
MEMBRANE TRANSPORT IN PARASITISM 121
tion, the worms or host tissues were lightly blotted on hard filter paper and
transferred to the experimental medium. At the end of an experimental incubation,
the worms or host tissues were rinsed by dipping twice in large volumes of the
salt solution, blotted quickly on hard filter paper, and placed in a measured
volume of 70% ethyl alcohol. It was previously shown that, with occasional
shaking, free amino acids are extracted from worm tissues in 50 to 70 per cent
alcohol in less than 24 hours (Read, Simmons and Rothman, 1960). In most
cases extraction was carried out for over 48 hours but in no case for less than
24 hours. Actually, the concentration in the fluid of the tissues probably comes
to equilibrium with the alcohol external to the tissues. The quantity of worm
tissue with respect to the volume of the extracting fluid was kept sufficiently low
so that an error of less than \% was introduced by the addition of the worm
volume to that of the extracting fluid. Aliquots of alcoholic extracts were used
for determination of radioactivity or analysis of amino acids.
Many of the data are expressed in terms of the alcohol-extracted dry weight
of tissue ; this was determined by heating the extracted tissue for 5 to 6 hours
at 100° C. in tared foil pans. Drying for longer periods produced no significant
change in dry weight values. Determinations of the alcohol-extracted dry
weight/wet weight for 16 worms at the end of a 60-minute incubation in the salt
solution at 10° C. gave a value of 0.302 ± 0.016. Nineteen dogfish gut samples,
identical with those used in experiments, gave an alcohol-extracted dry weight/wet
weight value of 0.135 ± 0.011 after a 30-minute, aerated incubation in salt solution
with glucose at 10° C. Plating of extract samples and determinations of radio-
activity were carried out as previously described (Read, Simmons and Rothman,
1960).
Two-dimensional chromatography was carried out by a modification of the
method of Levy and Chung (1953), as described by Campbell (1960). One-
dimensional chromatograms were prepared on Whatman No. 52 paper using
sec-butyl alcohol, formic acid, and water (75:15:10) as the solvent system.
Amino acids were quantitatively estimated using the methods of Fowden (1951).
Radioautographs of chromatograms wrere prepared by exposing Eastman "no-
screen" x-ray film to the chromatograms after removing the solvent. Histidine
was determined by the method of Macpherson (1946). Nitrogen was determined
by the micro-Kjeldahl method described by Lang (1958). Other details of
methods will be described in context.
EXPERIMENTAL
Further characterization oj the ammo acid entry systems of Calliobothrium
Several amino acids have been shown to inhibit the penetration of L-valine
and L-leucine into Calliobothrium (Read, Simmons and Rothman, 1960). In the
present study a number of experiments were carried out to determine whether
these inhibitions are competitive in nature, and whether there is a reciprocal
inhibitory effect of L-valine on penetration of certain of the inhibitory amino
acids. An analysis of the inhibitory effects of L-serine, L-threonine, and L-alanine
on valine entry showed that the inhibitions indeed are competitive in nature
(Table I). Conversely, experimental analysis of the effect of L-valine on the
122
READ, SIMMONS, JR., CAMPBELL AND ROTHMAN
TABLE I
Effect of L-serine, L-threonine, and L-alanine on the entry of L-valine into Calliobothrium.
5 = concentration of L-valine; V = counts per minute per gram of alcohol-extracted
dry tissue; N = number of samples
Amino acid
Inhibitor
N
l/S
V
4
200
59,575 ± 3673
5 X 10-W
4
500
36,200 ± 2876
L-serine
4
1000
18,212 ± 1004
4
2000
11,515 ± 1529
4
200
96,900 ± 4300
L-valine-C14
2 X 10-W
L-serine
4
4
500
1000
41,850 ± 1947
24,300 ± 571
4
2000
16,812 ± 857
4
200
116,600 ± 10,148
None
4
500
61,222 ± 5793
4
1000
46,700 ± 2200
4
2000
31,775 ± 1520
4
200
65,666 ± 4843
5 X 10~W
4
500
36,368 ± 6319
L-threonine
4
1000
18,769 ± 852
4
2000
10,042 ± 1102
4
200
73,450 ± 5576
L-valine-C14
2 X 10-W
L-threonine
4
4
500
1000
45,300 ± 4529
27,937 ± 1971
4
2000
16,875 ± 1639
4
200
93,725 ± 7925
None
4
500
64,550 ± 5794
4
1000
40,920 ± 3415
4
2000
28,300 ± 3706
4
200
53,825 ± 3594
5 X 10-W
4
500
31,070 ± 2400
L-alanine
4
1000
22,400 ± 842
4
2000
10,100 ± 597
L-valine-Cl4
4
200
91,550 ±4132
None
4
500
75,902 ± 2926
4
1000
41,850 ±4903
4
2000
28,475 ± 2685
penetration of L-serine demonstrated that valine competitively inhibits the entry
of this amino acid (Table II).
Previous studies showed that at a concentration ratio of 2:1, glutamic acid
did not inhibit the entry of L-valine. Therefore, it was surprising to find that
aspartic acid is an effective inhibitor of L-valine entry. With L-valine at a
concentration of 2 X 10~n M, aspartic acid or proline at a concentration of
5 X 10~3 M, inhibited L-valine entry an average of 33 and 45%, respectively, in
five experiments. A shortage of experimental material prevented a determination
of whether or not aspartic acid is a competitive inhibitor.
MEMBRANE TRANSPORT IN PARASITISM
123
L-lysine-C14 penetrates Calliobothriimi at a very low rate. A series of experi-
ments were performed to determine whether L-lysine or L-valine affect the entry
of the other. L-lysine entry was not significantly affected by L-valine and L-valine
entry was not affected by L-lysine (Table II). The apparent stimulation of
L-valine entry by L-lysine at a lysine/valine ratio of 2, previously reported (Read,
Simmons and Rothman, 1960), is not considered to be significant in view of the
results obtained when this broader range of concentration ratios was examined.
When Calliobothrium was incubated for two minutes in 5 X 10~3 M L-valine-
C14 and subsequently incubated for additional periods in salt medium without
TABLE II
Effects of certain amino acids on entry of L-serine, L-valine, and L-lysine.
Data presented as in Table I
Amino acid
Inhibitor
N
l/S
V
4
200
45,600 ±6111
5 X 10-W
4
500
29,500 ± 8201
L-valine
4
1000
19,100 ± 1124
4
2000
11,460 ± 823
L-serine-C14
4
200
57,433 d= 2441
None
4
500
42,150 ± 3300
4
1000
29,100 ± 629
4
2000
20,200 ± 4540
4
200
105,975 ± 13,192
5 X 10-W
4
500
73,050 ± 2563
L-lysine
4
1000
47,300 ± 3379
4
2000
32,750 ± 2958
4
200
97,025 ± 12,604
L-valine-C14
2 X 10~3M
L-lysine
4
4
500
1000
72,025 ± 3620
46,250 ± 2418
4
2000
31,433 ± 2240
4
200
103,775 ± 14,202
None
4
500
61,300 ± 963
4
1000
47,300 ± 2588
4
2000
34,050 ± 2952
4
200
14,970 ± 1207
5 X 10- W
4
500
7552 ± 1965
L-valine
4
1000
6247 ± 1406
4
2000
5410 ± 441
4
200
15,115 ± 3924
L-lysine-C14
2 X 10~3M
L-valine
4
4
500
1000
7997 ± 1534
6713 ± 1408
4
2000
5290 ± 805
4
200
15,358 ± 1665
None
4
500
7930 ± 1262
4
1000
6232 ± 1836
4
2000
5142 ± 909
124
READ, SIMMONS, JR., CAMPBELL AND ROTHMAN
CPB
FIGURE 2, Chromatogram of free amino acids extracted from Calliobothrium after 40
minutes' incubation in presence of L-valine-C14. Solvent systems were sec-butyl alcohol,
formic acid, water (75:15:10) = BFW and meta-cresol, phenol, borate buffer, ph 8.3
(60 : 30 : 15) = CPB. Black areas show presence of radioactivity, X being a non-ninhydrin-
positive, unidentified metabolite.
Table III. It is apparent that valine is concentrated against a gradient. Radio-
autographs prepared from the chromatograms of tissue extract from the 40-minute
incubations revealed that some L-valine is metabolized in this period. However,
L-valine was the only radioactive ninhydrin-positive compound on the chromato-
grams. A second radioactive spot was present but did not react with ninhydrin.
A representative chromatogram is shown in Figure 2. It was not feasible to
characterize the unknown metabolite further. On the other hand, radioautographs
prepared from 2-dimensional chromatograms of worm extracts from 2-minute
incubations in L-valine-C14 revealed a single radioactive spot which proved to be
identical with valine by the "fingerprint" method.
The amino acid entry systems of the host gut
Agar et al. (1956) differentiated and studied (1) the absorption of amino
acids by the rat intestine in vivo; (2) the transfer of amino acids from inner to
126
READ, SIMMONS, JR., CAMPBELL AND ROTHMAN
TABLE IV
Uptake of L-valine-C14 by spiral valve tissue of Mustelus spiral valve. Incubation in 5 X 10~3 M
L-valine for 2 minutes at 10° C. Values are counts per minute per gram
alcohol-extracted dry -weight
Spiral No. 2
80,300
85,900
93,500
74,700
79,000
87,000
88,800
82,000
Spiral No. 6
79,300
89,600
65,100
86,500
88,900
80,600
90,000
Mean 83,900
82,857
outer fluids using loops of rat intestine; and (3) the uptake of amino acids by
intestinal tissue. In short experiments, the kinetics of the latter two showed rather
good agreement, although, as might be expected, a lag was observed in transfer
experiments. Since the removal of amino acids from the lumen of the gut by
tapeworms and by the host mucosa, rather than transport of amino acids in the
UJ
OC
UJ
0.
(0
H
o
o
MILLIGRAMS DRY WEIGHT
FIGURE 3. The quantity of labeled valine taken up by various amounts of spiral valve tissue
of Mustelus. Tissue weight is alcohol-extracted dry weight.
MEMBRANE TRANSPORT IN PARASITISM
127
extra-intestinal host tissues, is the primary biological aspect to be considered
here, experiments to study the uptake of amino acids by dogfish intestinal tissues
were carried out. The preparations used were pieces from the lamina of the
spiral valve cut approximately 1 cm. square. As many as 30 such pieces can be
obtained from the lamina of a single spiral turn in the intestine of a sexually
mature dogfish. Most of the experiments to be described were carried out
with tissues from the fourth spiral posterior to the pyloric valve, although, as
will be shown below, preparations from other spirals would probably yield quite
comparable data. Calliobothrinm is found predominantly in the region of the
fourth and fifth spirals. The methods of handling the dogfish tissue were de-
TABLE V
Reciprocal inhibition of L-serine and L-valine entry and L-lysine entry into spiral valve tissue of
Mustelus canis. Data presented as in Table I. For experimental details, see text
Amino acid
Inhibitor
No.
l/S
V
200
42,000 ± 3725
None
4
500
28,700 ± 4340
4
1000
14,900 ± 1859
L-serine-C14
4
2000
7695 ± 1236
4
500
10,666 ± 1231
5 X 10-W
4
1000
6056 ± 586
L-valine
4
2000
2966 ± 561
4
200
68,980 db 8271
4
500
35,630 ± 6643
None
4
1000
21,996 ± 5036
L-valine-Cl4
4
2000
13,286 ± 1306
4
200
32,666 ± 2714
5 X lO"3,!/
4
1000
15,860 ± 2286
L-serine
4
2000
9760 ± 1699
4
200
27,293 ± 646
L-lysine-C14
None
4
4
500
1000
17,551 ± 3409
10,800 ± 1184
4
2000
6575 ± 1197
scribed earlier. It was reasoned that if preparations from different parts of single
or separate spiral lamina of the intestine showed consistency in rate of amino
acid uptake, with respect to weight of tissue, replicate samples from a single fish
could be used in amino acid uptake studies. Initially, therefore, a determination
of amino acid uptake was made with tissue samples removed from the second
and sixth spirals. Data obtained with tissues from a single fish are presented
in Table IV and show remarkably that there is no significant difference in the
entry of L-valine into tissues from these two regions of the intestine nor into
tissues from different parts of the same lamina. Further, the amount taken up
is proportional to the dry weight of tissue used (Fig. 3). These findings showed
that multiple sampling for kinetic studies is feasible with these preparations.
128
READ, SIMMONS, JR., CAMPBELL AND ROTHMAN
TABLE VI
Effects of other amino acids on entry of L-valine into spiral valve tissue. V = counts /min.f gram
alcohol-extracted dry tissue. N = Number of experiments
Inhibitor
Glycine
L-leucine
L-isoleucine
L-methionine
L-threonine
L-lysine
None
N
4
4
4
4
4
4
4
98,771
57,692
44,326
40,253
71,655
79,363
91,799
6129
3406
1267
3294
4029
2326
1079
When the concentration of L-valine was varied, it was found that the entry
of the amino acid into mucosal tissues follows an adsorption isotherm, and the
apparent Michaelis constant for valine entry is about 5 X 10~3. The addition of
unlabeled L-serine at a concentration of 5 X 10~3 produced an inhibition of L-valine
entry which was competitive in nature (Table V.) Conversely, L-valine com-
petitively inhibits L-serine entry (Table V) which has an apparent Michaelis
constant of about 4.5 X 10'".
A number of other amino acids were tested as inhibitors of L-valine entry.
Data obtained are summarized in Table VI. It was found that "preloading"
the mucosa, by incubating for 40 minutes in non-radioactive L-valine, produced
no effect on the subsequent entry rate of L-valine-C14. In balanced salt solution
without added non-radioactive amino acid, L-valine does not leak out of the
intestinal tissue to a significant extent.
TABLE VII
Relative concentrations of free amino acids in the fluid contents of the spiral valve of Mustelus canis.
All values are related to a valine concentration of 1.00. mg. N = Mg.
of alcohol-soluble nitrogen in the sample
Amino acid
Dogfish No.
X ± S.E.
1
2
3
4
5
6
7
8
9
KI
11
12
Leucine
1.43
1.46
1.20
1.53
1.43
1.67
1.44
1.15
1.18
1.38
1.61
1.41
1.41 ± 0.047
Phenylalanine
Valine
0.77
1.00
0.77
1.00
0.53
1.00
0.71
1.00
0.47
1.00
0.58
1.00
0.62
1.00
0.54
1.00
0.84
1.00
0.57
1.00
0.75
1.00
0.59
1.00
0.64 ± 0.034
1.00
Tyrosine
Alanine
0.76
1.35
0.91
0.78
0.55
0.87
0.94
1.94
0.43
1.50
0.65
1.42
0.82
1.65
0.63
1.21
0.90
1.13
0.50
1.25
0.75
1.66
0.70
1.51
0.71 ± 0.049
1.36 ± 0.096
Threonine
0.58
0.42
0.35
0.65
0.78
0.49
0.88
0.75
0.83
0.72
1.07
0.84
0.70 ± 0.060
Glutamic
1.98
1.34
1.61
4.24
2.66
3.63
7.65
2.70
2.42
1.99
2.87
2.92
3.00 ±0.49
Glycine & serine
Aspartic
Lysine
Histidine
1.61
0.95
1.37
0.51
1.18
0.67
1.00
0.48
1.72
1.11
1.70
0.73
5.41
2.47
5.77
2.90
1.52
7.05
3.12
1.43
2.27
0.77
4.61
3.06
1.85
1.01
2.41
1.15
1.78
0.69
6.26
2.60
2.04
1.09
4.13
3.50
3.32
0.33
3.44
1.38
6.97
1.61
3.14
3.46
2.44
0.51
3.33 ± 0.45
1.94 ± 0.29
3.13 ± 0.63
0.64 ± 0.095
Arginine
Cysteine
Beta aminoiso-
0.46
0.57
0.53
—
0.84
0.18
0.39
0.70
0.19
0.82
0.60
0.52
0.34
0.86
0.35
0.26
0.59
0.33
0.21
0.46 ± 0.078
0.19 ± 0.090
0.081 ±0.037
butyric
Beta alanine
0.24
1.94
2.21
0.99
0.55
1.44
0.62 ± 0.23
mg. N
4.70
1.43
2.27
4.20
7.40
3.35
4.84
3.28
2.60
5.80
3.00
9.40
MEMBRANE TRANSPORT IN PARASITISM 129
Using dogfish gut preparations, attempts were made to show the inhibition
of L-histidine uptake by other amino acids in experiments of 30-minute duration.
L-histidine was taken up by the tissues and, during the experimental period,
molar ratios of histidine in the tissue water/histidine in the external fluid of more
than 2 developed. Initial concentration of histidine in external fluid was 1 mM.
Addition of L-alanine, L-proline, L-valine, L-serine, or L-aspartic acid at a
concentration of 2.5 mM. did not affect L-histidine uptake significantly. Histidine
was removed from the medium at the rate of 20 to 40 micromoles per gram dry
weight per 30 minutes.
Free amino acids of the intestinal lumen
Samples of the fluid contents of the spiral intestine were collected from a
number of dogfish. There was great variation in the nutritional state of these
animals. Some were freshly captured and some had been held in captivity for
as long as 8 days without food. The samples were taken with a calibrated pipette
from the middle portion of the spiral valve of living fish. The measured volume
was immediately added to 10 volumes of 70% ethyl alcohol, mixed, and allowed
to settle for several days. Free amino acids in the supernatant liquid were quanti-
tatively determined. The analyses are summarized in Table VII. It is evident
that there is great variation in the absolute quantity of alcohol-soluble nitrogen
and of single amino acid components. However, study of the data shows that,
for the most part, the molar ratios of one amino acid to another are strikingly
constant. The dicarboxylic acids, aspartic and glutamic acid, show considerable
variation, but it may be seen that the ratio aspartic/glutamic is relatively stable.
The lysine/valine ratio also showed considerable variation. This may be associ-
ated with the relatively low rate at which lysine penetrates the mucosa. The
methods used did not allow a clear separation of glycine and serine and these
two amino acids were estimated together. However, it was roughly estimated
that serine made up about 75 per cent of this value.
DISCUSSION
The demonstration that certain amino acids, which inhibit the entry of L-valine
into CaUiobothriiim, do so competitively, as previously shown with L-leucine
(Read. Simmons and Rothman, 1960) suggests that this may also be the case
with other inhibitory amino acids. Limitations of time and material have not
allowed a complete analysis of the inhibitions of valine entry produced by cysteine,
methionine. glycine, or proline.
Furthermore, some of the amino acids which do not inhibit the entry of L-valine
or L-leucine at a concentration ratio of 2 : 1 may very well inhibit at higher
concentration ratios. D-valine was found to inhibit L-valine entry at a very high
D/L ratio but had no significant effect when D-valine/L-valine was 2 (Read.
Simmons and Rothman, 1960). Of great interest is the failure of mutual com-
petition between L-lysine and L-valine over a wide range of concentration ratios.
Since L-lysine entry appears to follow adsorption kinetics, the lack of interaction
with L-valine entry suggests that the two compounds enter at different sites.
The inhibition bv other amino acids of L-valine and L-serine entrv into the
130
READ, SIMMONS, JR., CAMPBELL AND ROTHMAN
intestinal tissue of the dogfish, shown to be mutually competitive in the case of
L-serine and L-valine, is consistent with the observations of others who have
reported inhibition of intestinal absorption of single amino acids by other amino
acid species in warm-blooded vertebrates (Wiseman, 1955, 1956; Agar et al., 1956).
A difference, however, is observed in the case of L-histidine uptake by dogfish
and rat intestinal tissues. Agar ct al. (1956) found that L-histidine uptake was
markedly inhibited by addition of equimolar concentrations of a number of single
amino acids. Inhibition was not observed with dogfish tissues when several amino
acids were singly added at twice the histidine concentration. Christensen and his
co-workers (reviewed by Christensen, 1959) have described reciprocal inhibitions
of amino acid uptake with Ehrlich ascites tumor cells and such relationships between
amino acids are known to occur with Ncurospora (Mathieson and Catchside, 1955)
CALLIOBOTHRIUM
-600
ISO
MUSTELUS SPIRAL VALVE
-200
200
500
1000 -400 -200
-I
(MOLAR)
200
500
1000
I/S
FIGURE 4. A kinetic comparison of the uptake of three amino acids by
Calliobothrhun and Mustclus spiral valve tissue.
and certain bacteria (Cohen and Monod, 1957). It appears to be a very general
phenomenon, perhaps as general as the distribution of special membrane mecha-
nisms for amino acid entry. It seems unfortunate that some of the bacterial
physiologists have chosen to refer to such entry systems as "permeases," a term
implying that there is an entry enzyme. It is to be hoped that a term with such
specific connotations is not widely adopted until considerably more understanding
of mechanism is attained.
There are definite differences in the amino acid entry systems of Calliobotliriinn
and the dogfish intestinal tissue. In Figure 4 the worm and host tissue are
compared with regard to their affinities for three amino acids.
Several years ago the senior author reviewed the literature on the physiology
of the small intestine with special reference to its peculiarities as a habitat (Read,
MEMBRANE TRANSPORT IN PARASITISM 131
1950). It was concluded that there is a flow of organic compounds, with the
notable exception of carbohydrates, from the tissues into the gut lumen and that
much of this material is resorbed in areas of the gut distal to the point of secretion.
It seemed obvious to infer that many of these materials are available to lumen-
dwelling parasites. The present data on the free amino acids in the gut lumen of
dogfish in highly variable states of nutrition, and the great stability of molar
ratios of these amino acids lend weight to the above concept. As a part of a
study of mammalian nutrition, Nasset and his colleagues (1955) demonstrated
that molar ratios of free amino acids, one to another, in the small intestine of the
dog are astonishingly constant and independent of the composition of protein
ingested. The concentration ratios were essentially unchanged in dogs receiving
no protein by mouth. This has broad implications in considering intestinal para-
sitism. It reinforces the senior author's argument that in many chemical charac-
teristics, the small intestine represents a relatively stable environment in a particular
host. More specifically, it invites inquiry as to what effects constancy of the
relative amounts of different amino acids might have on a particular intestinal
parasite. It seems plain that if the environment offers a mixture of amino acids
or other compounds of nutritional significance, and if these compounds compete
with or otherwise affect the entry of one another into the tissues of the parasite,
the ratios of amino acids in the mixture will be extremely important in determin-
ing whether the nutritional requirements of the parasite can be maintained in a
balanced state. The concept emerges that the ratios of nutrients may be critical
in determining whether or not a given mixture of amino acids will represent a
satisfactory food for a parasitic organism such as a tapeworm. Thus, ratios of
nutrient concentration may be critical limiting parameters at the interface between
host and parasite. Further, the ratios of rates of entry of amino acids may be
manifestations of an important regulatory system governing the make-up of the
amino acid pool in a given worm.
As a homeostatic mechanism of importance in the physiology of the vertebrate,
the competitions between amino acids would seem to represent part of a mecha-
nism for regulating the composition of the amino acid mixture entering the portal
system and hence the liver. Nasset (1957) has presented evidence that the
relative concentrations of amino acids in the small gut are maintained by the
secretion of endogenous nitrogenous material which is mixed with the ingesta.
It would seem that the regulation of the composition of the amino acid pool for
protein synthesis in the vertebrate begins at the mucosa.
While it would appear that the host and the parasite are competing with each
other as whole organisms and, from this standpoint, the competition of worm and
host should be considered in terms of total absorption by worm and intestine,
we may consider highly localized competition in terms of the entry systems for a
particular amino acid. If other amino acids affect entry of this amino acid into
the mucosa and the worm to differing extents, it is apparent that the concentration
ratios of amino acids may undergo alteration in the immediate vicinity of the
worm-mucosa system. If the rates involved do not undergo marked change, a
new set of concentration ratios should be established. If this is indeed true, it may
have wider implications in considering parasitisms in which the most obvious effects
on the host are general unthriftiness or ill defined interferences with nutrition.
It has not been feasible to study this experimentally in the dogfish-cestode system
132 READ, SIMMONS, JR., CAMPBELL AND ROTHMAN
hut it may be practical with other host-parasite combinations more amenable to
lal (oratory control.
It becomes increasingly apparent that the gut should not be considered a space
outside the vertebrate body. The rapid changes in the properties of intestinal
mucus indicating hydrolysis of components (Hartiala and Grossman. 1952) suggest
that secretion, hydrolysis, and resorption must occur constantly. Lumen parasites
are in a position to remove from this exocrine-enteric circulation compounds of
nutritional value. If the data on rate of entry of individual amino acids into
CaUiobuthriuin and host intestinal tissue are calculated on the basis of water content,
making the assumption that the amino acids are in solution in this water, the
tapeworm takes up the amino acids studied at a much higher rate than host
intestinal tissue. However, the data on competitions indicate that rates of
absorption for single amino acids are not directly applicable to complex mixtures.
Stud}' of entry of single components from complex mixtures is obviously required.
SUMMARY
1. The entry of CT4-L-valine into the tapeworm, CalliobotJiriitiu verticillatum.,
is competitively inhibited by L-serine, L-threonine, and L-alanine. Conversely,
L-valine competitively inhibits the entry of C14-L-serine.
2. The entry of C14-L-valine is not significantly affected by L-lysine and,
conversely. L-lysine entry is not affected by L-valine.
3. L-valine is concentrated against a gradient by Calliobothriuin in experi-
ments of 40-minute duration.
4. The entry of C14-L-valine into mucosal tissues of the dogfish host, Must el us
canis, is competitively inhibited by L-serine and, conversely, C14-L-serine entry
is competitively inhibited by L-valine. L-leucine, L-isoleucine, L-methionine,
L-threonine, and L-lysine also inhibit C14-L-valine entry but it has not been
shown that inhibition is competitive.
5. In experiments of 30-minute duration, L-histidine uptake by dogfish mucosa
was not affected by L-alanine, L-proline, L-valine. L-serine, or L-aspartic acid
at the concentrations tested.
6. Quantitative analyses of free amino acids of the dogfish intestinal lumen
showed variability in the absolute concentrations but great stability in the relative
concentrations.
7. The data are discussed in terms of differences in amino acid entry systems
of host and parasite, the significance of stability of amino acid ratios in the nutrition
of host and parasite, and the necessity for evaluating host-parasite competitions in
terms of entry of single components from complex mixtures.
LITERATURE CITED
AGAR, W. T., F. J. R. HIRD AND G. S. SIDHU, 1956. The absorption, transfer, and uptake of
amino acids by intestinal tissue. Biochiin. Biopliys. Acta, 22: 21-30.
CAMPBELL, J. W., 1960. The nitrogen and amino acid composition of three species of anoplo-
cephalid cestodes : Monicsia cxpansa, Thysanosoina actinoides, and Cittotaenia perplexa.
E.vp. Parasitol. (in press).
CHRISTENSEN, H. N., 1959. Active transport, with special references to the amino acids.
Perspectives in Biol. Mcd., 2: 228-242.
MEMBRANE TRANSPORT IN PARASITISM 133
COHEN, G. N., AND J. MONOD, 1957. Bacterial permeases. Bact. Rev., 21 : 169-194.
FOWDEX, L., 1951. The quantitative recovery and colorimetric estimation of amino-acids sepa-
rated by paper chromatography. Biochcut. J., 48: 327-333.
HARTIALA, K., AND M. I. GROSSMAN, 1952. Studies on chemical and physical changes in
duodenal mucus. /. Biol. Chcin., 195 : 251-256.
LAM;, C. A., 1958. Simple microdetermination of Kjeldahl nitrogen in biological materials.
Anal. Chan.. 30: 1692-1697.
LEVY, A. L., AND D. CHUXG, 1953. Two-dimensional chromatography of amino acids on
buffered papers. Anal. Chew.. 25: 296-299.
MACPHERSON, H. T., 1946. The basic amino acid contents of proteins. Biochcm. J., 40:
470-480.
M ATHIESON, M. J., AND D. G. CATCHsiDE, 1955. Inhibition of histidine uptake in Ncnrospora
crassa. J. Gen. Microhiol.. 13: 72-83.
X ASSET, E. S., 1957. Role of the digestive tract in the utilization of protein and amino acids.
J. Amcr. Mcd. Assoc., 164: 172-177.
NASSET, E. S., P. SCHWARTZ AND H. V. WEISS, 1955. The digestion of proteins in rivo.
J. Xiitr.. 56: 83-94.
READ, C. P., 1950. The vertebrate small intestine as an environment for parasitic helminths.
Rice I nst. P am phi.. 37(2) : 1-94.
READ. C. P., J. E. SIMMONS, JR. AND A. H. ROTH MAX, 1960. Permeation and membrane
transport in animal parasites : Amino acid permeation in cestodes from elasmobrachs.
/. Parasit., 46: 33-41.
WISEMAN, G., 1955. Preferential transference of amino-acids from amino-acid mixtures by
sacs of everted small intestine of the golden hamster (Mesocricctns itnratns) . J.
Parasit., 127 : 414-422.
WISEMAN, G., 1956. Active transport of amino acids by sacs of everted small intestine of the
golden hamster ( Mesocricctus tinnitus). J. Plivsiol.. 133: 626-630.
PIGMENTED FAT CELLS IN A MUTANT OF
DROSOPHILA MELANOGASTFR '
M. T. M. RIZKi
Department oj Biology, Rccd College, Portland 2, Oregon
The "red cell" mutant of Drosophila melanog aster is characterized by the
presence of scattered pigmented cells in the thorax and head of the adult fly. This
recessive mutant factor, re, is located on the second chromosome, and histological
examination of the re phenotype by Jones and Lewis ( 1957) revealed that the red
pigment was localized in granules in some of the pupal fat cells. The expression
of the re factor was suppressed in the presence of mutant genes which interrupt
the synthesis of the brown pigment of the eye: vermilion, scarlet, and cinnabar.
On the other hand, the mutant gene brown which blocks the synthesis of the red
component of the normal eye color did not interfere with the expression of the re
gene as red cells in the thorax and head of the adult fly. Therefore Jones and
Lewis (1957) concluded that the pigment in the red fat cells of re flies is related
to the brown pigment component of the eye.
This proposed relationship to insect eye pigments enhances the usefulness of
the re mutant as a tool in probing the pigmentation process at a cellular level. The
large pupal fat cells offer excellent material for experimental manipulations of
individual cells. In addition, the implantation experiments of Beadle (1937a,
1937b) with eye discs and various tissues of Drosophila established the fatbody
as a source of pigment precursors for the synthesis of brown eye pigment. Prelimi-
nary observations of interaction between the re mutant and a mutant strain which
develops melanotic masses in the fatbody suggested the present study.
MATERIALS AND METHODS
A stock homozygous for both the re and tit"' factors was made, and all experi-
mental procedures utilized material from this stock. The wild type strain. Orc-R,
was used to verify the morphological relationships of the various fat masses in the
larval stages. The re mutant stock was kindly provided by Dr. E. B. Lewis in
1955, and the re gene has been maintained in our laboratory in combination with
the tuw factor since that time. Melanotic tumorous masses occur in larvae homo-
zygous for the recessive factor, tuw, located on the second chromosome. Detailed
studies of this mutant stock have been reported previously (Wilson et al., 1955;
Rizki, 1957).
The tuwrc stock has been raised on Cream of Wheat medium with Fleischmann's
yeast. Timed material for the experiments was collected in the following manner.
Adult flies were placed in a half-pint bottle containing a paper teaspoon with
Cream of Wheat medium heavily coated with a yeast-honey suspension. A fresh
1 This investigation was supported by a research grant RG 5285 from the National
Institutes of Health, Public Health Service.
134
PIGMENTED FAT CELLS IN DROSOPHILA 135
food spoon was placed in the bottle twice daily, and after removal from the bottle
the spoons were stored in an incubator at 23°-25° C. Beginning at approximately
20 hours after a spoon had first been placed in the collection bottle, the newly
emerged larvae were collected at intervals of one or two hours. These larvae were
raised on Cream of Wheat-Fleischmann's yeast medium in crystallizing dishes in
the incubator at 23°-25° C. All ages of larvae and pupae are thus counted from
the time of eclosion from the egg.
In the starvation experiments, larvae were removed from the food dishes at
65 hours of age, rinsed in a saturated solution of NaCl, 2% solution of NaOCl,
followed by repeated washing in six changes of distilled water. This procedure
removed a considerable proportion of the adhering yeast and food particles.
\Yashed larvae were placed on tissue paper strips (Kleenex brand) moistened
with distilled water in petri dishes ; paper was also placed under the cover of the
dish to prevent larvae from crawling out of the dishes. Care was taken to maintain
the paper strip moist without excessive wetting. With each starvation experiment,
a group of washed larvae from the same collection period was placed in petri dishes
on paper strips to which a thin layer of Cream of Wheat medium and Fleischmann's
yeast was added. These larvae served as the control fed material. The larvae
removed from food at 65 hours of age generally pupated several hours before those
which were left on food. No difference between the percentages of adults emerging
in the two groups was noted.
RESULTS
No "red cells" appeared in the tit"' re homozygotes in the stock bottles or in
the control fed series. However, the expression of the re factor was 100 per cent
if the tu"' re larvae were removed from food at approximately 65 hours of age, that
is, during the early third instar. In this case, the mutant pattern appearing in the
adult flies was the same as that described by Jones and Lewis (1957) for the re
stock. Red-pigmented cells were most abundant in the thorax and head, a few
were found in the abdomen, and an occasional pigmented cell was seen in the
appendages. In pupae shortly before hatching the red pattern in the thorax was
striking. The red-pigmented cells occupied the haemocoel spaces between the
flight muscles, and a dorsal striped pattern in the thorax was the result of alter-
nating accumulations of red cells and regions of the muscle insertions (Figs. 1
and 2).
The re factor exhibited no apparent influence on the development of melanotic
masses in the posterior fatbody, the phenotypic character of the tit11' factor. The
penetrance of the tu"' factor varied between 90% and 95% during the course of
the present investigation; however, the re phenotype was expressed in tuli're flies
which had been starved during development whether melanotic tumors were
visible or not.
Control fed larvae and starved larvae were examined at timed intervals after
the beginning of the starvation period at age 65 hours. Starved larvae generally
pupated two or three hours earlier than larvae which were feeding. No morpho-
logical differences were noted between the two groups of larvae until after pupa-
tion. During the early period of tanning of the puparium. a slightly yellow tinge
was noticeable in the anteriormost fat masses of starved tit"' re pupae. This color
136
M. T. M. RIXKI
PLATE I
PIGMENTED FAT CELLS IN DROSOPHILA 137
was visible through the puparium which was still very light, but removal of this
covering was necessary to reveal the extent of the coloring. Figure 9 is a
camera lucicla drawing of a starved tit"' re pupa showing the location of the yellow
masses underneath the puparium. and a photograph of a specimen removed from
the puparium shortly after the pupal molt is labelled Figure 3. Dissection of
pupae showed that the yellow color was localized in the most anterior pair of
dorsal fat masses just posterior to the cerebral hemisphere, as well as those fat
masses which are lateral to this first region and extend ventrally where they join
in a commissure. From the ventral side of each of these extensions, a strand of
fat cells passes anteriorly where they adhere to the paired salivary glands. The
fat masses just described are the only fat cells which developed the yellow pigment.
Larvae of the Ore-R strain have been dissected to establish morphological correla-
tions of the fat masses in the mutant strain with the fat masses in the normal mate-
rial. Dissection of the larvae of the wild type strain was considerably easier since
manipulation of the fat masses in the starved tit "'re larvae tended to loosen the
fat cells which did not seem to be held together so compactly as in the normal
strain. Starvation also decreased the size of the fatbody cells as compared to fed
material. In order to demonstrate the relationship of the intact fat masses,
camera lucida drawings of the Orc-R strain are given in Figure 10. The common
feature of these anterior fat masses is their proximity to the distal ends of the
anterior pair of Malpighian tubules.
As pupation progressed the cells of the fatbody became separated from one
another, and were thus involved in the extensive reorganization of the body tissues
which takes place during metamorphosis. The pigmented cells of the anterior
fatbody were redistributed during this process and were found primarily in the
head and thorax of the developing pupa. The color of the fat cells in the intact
fatbody of starved tit"-' re pupae gradually changed from yellow to yellow-brown,
and as the scattering of the separated cells occurred, the color deepened, and
finally red pigment was apparent. The yellow color in the fatbody appeared
before the imaginal discs had everted, and the red pigment in the fat cells
preceded the appearance of the red eye color.
FIGURE 1. Photomicrograph of a tit "'re pupa (starved) removed from the puparium.
Red cells, RC, are distributed between the longitudinal muscles, M, of the thorax. A typical
melanotic tumor, T, is apparent in the abdomen and two re fat cells are visible on the left
margin of the tumor. (Darkfield illumination with a green filter. < 63.)
FIGURE 2. A fully formed imago removed from the puparium to show the further
dispersion of the red-pigmented fat cells (arrows). Note the retention of re cells in the mid-
dorsal region and the further change in the distribution of re cells in the areas not occupied
by the insertions of the dorsoventral muscles of flight. Very few re cells are seen among
the fat cells of the abdominal region. The eyes are fully pigmented. ( Darkfield illumination,
green filter, magnification X 60.)
FIGURE 3. A tuwre (starved) prepupa removed from the pupal case, showing the position
of the intact pigmented masses of fat, RCF. Note the intimate association of the anterior pair
of Malpighian tubes, MP, with the RCF. The photograph represents the left dorsolateral
view of the prepupa so that the left tracheal trunk is apparent and the right tracheal trunk
can be visualized as a white band at the lower right margin of the photograph. The dark
area under the left tracheal trunk is a melanotic tumor in the caudal fatbody, T. (Transmitted
light from a Corning filter #CS 7-59. Magnification X 63.)
FIGURE 4. The intact fat masses removed from a tn"-'rc prepupa (starved) showing the
difference between pigmented fat masses, RCF, and unpigmented fat masses, F. This fat was
treated with potassium metabisulfite to intensify the color. (Corning filter #CS 7-59; X 63.)
138
M. T. M. RIXKI
PLATE II
FIGURE 5. A pigmented fat cell isolated from an early prepupa (tit"' re starved) showing
the intracellular distribution of pigment globules, PG. (Corning filter #CS 7-59.)
FIGURE 6. A pigment globule removed from the cytoplasm of a red cell from a tit"' re
prepupa (starved) showing the threadlike, DT, internal structures characteristic of these
globules. A fat droplet is indicated at FG. (Corning filter #CS 7-59.)
FIGURE 7. The re fat cells as seen through the body wall of a tit11' re starved pupa corre-
sponding in age to that given in Figure 1. Note the appearance of pigment in granular form.
(Darkfield illumination, green filter.)
FIGURE 8. Granular appearance of pigmented inclusions in a tit "'re starved imago corre-
sponding in age to that in Figure 2. Bristles, B, and setae, S, are visible in this photograph
of a red cell as seen through the body wall. ( Darkfield illumination, green filter. )
PIGMENTED FAT CELLS IN DROSOPHILA
139
Both the red and hrmvn pigment extracts from the eye of Drosophila are
altered by oxidation and reduction (Ephrussi, 1942). It seemed desirable to
determine whether the yellow pigment in the fat cells would undergo any changes
/;; I'ltro. and a search was undertaken for conditions which might cause such an
alteration. Anterior fat masses which had become yellow were dissected from
starved pupae in \\addington Ringer- 109^ glucose. The various reagents to be
tested were then added to this medium. The reducing rinse which had been pre-
pared for use in the Feulgen reaction proved most satisfactory. It was then found
•
•t, ^ .••-.:•:••• -7t ••;'
/:' ' • ij .. ::'..r *..•'.• .''•$• :'
t ' k '. ••?" • .v-h Is' :-
£•• • -a . ••".. JV: ^ :•••
FIGURE 9. Camera lucida drawing of a dorsal view of a tit ""re (starved) prepupa within
the puparium illustrating the position of the brownish yellow anterior fat mass, RCF.
convenient to add a few crystals of potassium metabisulfite to the drop of glucose-
Ringer containing the isolated fat masses while they were under microscopic ob-
servation on a white porcelain plate. The color of the fat cells showed a change
from yellow to red within a minute. Figure 4 is a photograph of the isolated fat
mass in which the re pigment had been intensified in this manner.
The development of pigment in the starved tit"' re flies has been followed at the
cellular level. Isolated cells from the anterior fatbodies of young pupae contained
140
M. T. M. RIZKI
numerous yellow cytoplasniic globules. These globules were distinguishable from
the fat droplets of the cells which are highly refractile and always spherical in
fresh preparations. Isolated cells have been examined with darkfield illumination
as well as brightfield illumination, and in addition, the use of a blue Corning
* - , ''•.-' ' '"• " " • * - r'-
'•• • '• ' ' j^-** \"i*> • ''V
;^^%^ff...
^/f^f0l^ '. ^^l^S^T^X
•-jX^Jhv-v.'^ ;-•:, "•••:*? k« '--i-iijfc" -'•«>
.»*" --^v-. '.-v-^-.f'.'-.' ' • •' ••* : -*tev *
: / -i\ •&&•••: ^-IKMte
^•t^lil
' -<•':./'' -^ "^^^-3? ' -
'• • 1:''. "'-..' •-^/<'iv!i:-- ' , 'A''.*:-' iKl'ji ^.,..-'
BR
FIGURE 10. Camera lucida drawings of a dissection of a late third instar larva of the
Ore-R strain showing the relative positions of the fat masses ; the salivary glands, SG ; brain,
BR; Malpighian tubes, MP. This specimen has been stained with Oil Red O. The diagram
on the left is the ventrolateral view, and the same dissection has been turned over to show the
lateral view of the fat masses in the drawing on the right. A, B, C, D, E are the regions
of the fatbody which become pigmented in starved tn"'rc pupae. A, dorsal pair of fat masses;
B, C, lateral fat masses ; D, fat mass attached to salivary gland ; E, fat cells forming the ventral
commissure between the right and left masses of fat. Only the anterior region of C becomes
pigmented. The club-shaped structures with concentric rings are the imaginal discs and the
long tubular structure with branches is the tracheal trunk.
Filter No. CS 7-59 proved most satisfactory for studying the cytoplasmic inclusions
in the fat cells. With this filter, all yellow objects appeared bright red. In early
tuwrc pupae (after starvation), this included the lightly tanned cuticle, isolated
cells from melanotic masses, the granular structures in the Malpighian tubules,
PIGMENTED FAT CELLS IX DROSOPHILA 141
and the yellow globules in the re fat cells. The yellow globules showed a definite
threadlike internal structure when examined with this filter, while no structure
was discernible in the fat droplets (Figs. 5 and 6). Fat cells from the more
posterior regions of the pupa, i.e., cells other than the fatbody cells involved in
the expression of the re phenotype. contained similar cytoplasmic inclusions in
addition to the fat droplets. Whether these inclusions are structurally and func-
tionally the same as those globules which become pigmented in the re cells remains
to be examined. The cells containing the yellow globules were placed on a slide
in \Yaddington- lO'/r glucose solution and several crystals of potassium meta-
bisulfite were added under the coverslip in the vicinity of the cells. The yellow
inclusions became red in color under these conditions.
After the pigmented fat cells had become scattered throughout the thorax and
head during the developmental processes occurring in pupal life, the color in the
fat cells appeared more intense. In the late pupae and young adults, the pigmented
structures in these cells were more granular in appearance (Figs. 7 and 8).
DISCUSSIOX
Two types of pigment are found in the eye of Drosophila, one brown and the
other red. and many mutants are known which affect the production of these
pigment components in the eye. Interference with brown pigment production
results in a bright red eye color of the type found in the mutants vermilion,
cinnabar, and scarlet while the phenotype of the brown eye mutant represents an
interruption in the biochemical pathways leading to red pigment. The absence
of both pigments occurs in the mutant, white eye. The literature on brown eye
pigments in insects has been reviewed by Ephrussi (1942) Xolte (1952), and
Kikkawa (1953). The synthesis of brown pigment proceeds through a pathway
involving tryptophan, formylkynurenine, kynurenine, and hydroxykynurenine, and
the known eye color mutants are blocks at successive stages in this synthetic chain.
Transplantation of imaginal discs of mutant larvae into hosts of different geno-
types has shown that in some cases the eye color is autonomous, whereas some
mutant eyes do not themselves produce the prerequisites for brown pigment and
are dependent upon other sources in the body for these precursors (Beadle and
Ephrussi. 1936; Ephrussi, 1942; Ephrussi and Beadle, 1937). The presence
of these pigment precursors has been demonstrated in the fatbody and the Mal-
pighian tubules by transplantation experiments (Beadle, 1937a, 1937b). The
time during which each of these tissues produced the pigment precursors was
dependent upon the stage of development of the donor. Malpighian tubes showed
activity through larval life from the earliest stages tested, appearance of active
substances in the fatbody was not detected until after pupation, and in the eyes
much later during pupal development (Beadle, 1937b; Clancy, 1940).
The pigment granules in the re cells have been shown to be related to the brown
eye pigment of Drosophila. Jones and Lewis (1957) found that the mutant
factors, vermilion, cinnabar and scarlet, which interfere with brown pigment de-
velopment in the eye, also prevent the formation of pigment in the re cells when
each of these mutant factors is combined with the re gene. The mutant factor,
brown, which blocks the synthesis of red pigment, does not interfere with the
expression of the re gene in the fat cells.
142 M. T. M. RIZKI
The explanation for the suppression of pigment in the fat cells when the re
gene is combined with the tit"' factor may not he so direct. In the starvation
experiments the penetrance of the tu'r factor was 90%— 95%, and no difference in
the expression of re was noted between the pupae with melanotic tumors and those
that did not develop black masses. One point of comparison is the fact that both
mutants have a common domain of expression, i.e., fat cells: re, the anterior fat
mass and tit"', the posterior region of the fatbody. Tryptophan metabolism is not
only related to the development of brown eye pigment and protein metabolism,
but it also influences the expressivity and the penetrance of various melanotic
tumor genes in nrosophila. Addition of tryptophan to the medium increases the
frequency of melanotic tumors in strains carrying tumor genes (Hartung and
Hartnett. 1951; Plaine and Glass, 1955), and Kanehisa ( 1956a, 1956b) reported
an increase in tumor incidence by combining a tumorous factor with eye color
genes. The appearance of pigmented fat cells in tn"'re pupae after starvation
parallels the behavior of the vermilion mutants which develop brown eye pigment
after the larvae have been starved (Beadle ct a/., 1938). Starvation of Drosophila
adults results in a reduction in the size of the fatbody, and the reserves of fat and
glycogen are rapidly depleted from the fat cells (Wiggles worth, 1949). In many
insects the fatbodies may serve as storage sites for excretory products as well
as food reserves. Wigglesworth (1942) has shown that starvation of Aedes
larvae causes an increase in uric acid vacuoles in the fat cells and these deposits
disappear from the cells after the feeding has resumed. The conditions imposed
by starvation in the tit"' re larvae alter the metabolic pattern of the fat cell such
that it differentiates as a pigmented cell. A similar effect, of course, is produced
in the re mutant under normal feeding conditions. The presence of the tuw
factor may restore the normal metabolic balance in the fat cell such that their
phenotype resembles that of the wild type. The expression of the re phenotype
is also dependent upon the action of another recessive gene, I\s, which causes an
accumulation of the amino acid, lysine (Grell, 1958). It is thus obvious that
the expression of the red cell phenotype is influenced by the interaction of a number
of non-allelic genes. One suggestion may be made which will encompass the
various aspects of the problem known at the present time. Any modification,
genetic or environmental, which influences the normal pattern of protein synthesis
will also alter the metabolic pool of various amino acids. Such changes which
affect the availability of tryptophan may be reflected in the phenotypic expression
of the re pigment.
The larval fat cells of Drosophila form organized tissue masses, whereas soon
after pupation the fatbody becomes separated into single cells or small clusters
of cells. The cells of the caudal fat masses in tit"' larvae which are involved in
the production of melanotic tumors in this strain resemble pupal fat cells in their
tendency toward smaller cell aggregations and a loss of adherence to neighboring
cells (Rizki, 1957). This precocious change in the structure of the caudal fat-
body of tit"' larvae, as well as precocious changes in the blood cells, are processes
which lead to tumor formation in the caudal fatbody prior to pupation. Therefore
the hypothesis was presented that the melanotic masses in this tumorous strain
of Drosophila represented an upset in the normal timed pattern of events occurring
(luring metamorphosis. Under conditions favoring expression of the re gene, the
PIGMENTED FAT CELLS IN DROSOPHILA 143
tit"' factor influences an earlier appearance of red pigment in the anterior fat cells.
This pigmentation in the re mutant is not apparent until after the fat cells become
isolated and scattered during the pupal stage (Jones and Lewis, 1957). However,
the combination of re with the tn"' factor has shifted the time of development of
the red pigment to a stage preceding this dispersion of the fat cells. Although no
obvious explanation for the distribution of the red fat cells among other non-
pigmented fat cells existed in the re strain, the morphological relationship of all
the red cells in larval development becomes apparent in the tit"' re starved material.
A measure of the dispersion of the cells of the anterior fat masses during early
pupation is provided in this case by a mutant cytoplasmic marker. The localiza-
tion of mutant characteristics in the tn"' caudal fat masses and the anterior re fat
cells suggests that the cells of various regions of the fatbody may differ in their
developmental physiology. It is interesting to note that the cellular ecology of
the anterior and the posterior fatbody includes a common feature : the re fat cells
are intimately associated with the distal ends of the anterior pair of Malpighian
tubules, and the tn"' fat cells encircle the distal ends of the posterior Malpighian
tubules.
SUMMARY
1. Cytoplasmic pigment granules are found in some of the fat cells of the
recessive mutant, re, of Drosophila melanogaster. These scattered red fat cells are
located chiefly in the thorax and head, but a few occur in the abdomen and ap-
pendages of the adult. Using genetic methods it had been shown previously that
these pigment accumulations are related to the synthesis of the brown eye pigment
of this insect (Jones and Lewis, 1957).
2. The pigmentation in the re fat cells is suppressed when the re gene is
combined with the recessive factor, tit"'. This combination, however, in no way
alters the expression of the characteristic pattern of tn"' as revealed by the pres-
ence of melanotic tumors in the caudal fat masses of the homozygous tu'rre flies.
After a period of larval starvation, the tnlcre flies develop both the red-pigmented
fat cells and melanotic tumors. The time of appearance of the re pigment has
been shifted under these nutritional and genetic conditions. The cytoplasmic pig-
ment granules appear in the cells originating from the anteriormost section of the
larval fatbody which is closely associated with the anterior Malpighian tubules.
During the reorganization accompanying metamorphosis from the larval to the
adult stage, these cells are redistributed mostly to the thoracic and cephalic regions
while a few are found in the abdomen and appendages. An explanation is thus
provided for the cytodifferentiation of pigmented and nonpigmented fat cells found
side by side in the adult fly.
3. The nature of the pigment granules has been examined in in vitro prepara-
tions at each of these periods of development, and of particular interest is the
internal threadlike structure of these cytoplasmic inclusions during the early stages
of pigment formation.
LITERATURE CITED
BEADLE, G. W., 1937a. Development of eye colors in Drosophila : fat bodies and Malpighian
tubes as sources of diffusible substances. Proc. Nat. Acad. Sci., 23: 146-152.
144 M. T. M. RIZKI
BEADLE, G. W., 19371). Development of eye colors in Drosophila: fat bodies and Malpighian
tubes in relation to diffusible substances. Genetics, 22: 587-611.
BEADLE, G. W., AND B. EPHRUSSI, 1936. The differentiation of eye pigments in Drosophila as
studied by transplantation, Genetics, 21 : 225-247.
BEADLE, G. W., E. L. TATUM AND C. W. CLANCY, 1938. Food level in relation to rate of
development and eye pigmentation in Drosophilti melanogaster. Biol. Bull.. 75:
447-462.
CLANCY, E. B., 1940. Production of eye color hormone by the eyes of Drosophila melanogaster.
Biol. Bull., 78: 217-225.
EPHRUSSI, B., 1942. Analysis of eye color differentiation in Drosophila. Cold Sprint/ Harbor
Symp. Quant. Biol., 10: 40-48.
EPHRUSSI, B., AND G. W. BEADLE, 1937. Development of eye colors in Drosophila : transplanta-
tion experiments on the interaction of vermilion with other eye colors. Genetics, 22 :
65-75.
GRELL, E. H., 1958. Genetics and biochemistry of "red cell." Proc. X Int. Cong. Genetics, 2:
104-105.
HARTUNG, E., AND W. HARTNETT, 1951. A study of the relation of various dietary factors to
tumor incidence in Drosophila melanogaster. Anat. Rcc., Ill : 3.
JONES, J. C., AND E. B. LEWIS, 1957. The nature of certain red cells in Drosophila melano-
gaster. Biol. Bull., 112: 220-224.
KANEHISA, T., 1956a. Eye-colour genes and tumor incidence. Jap. J . Genetics. 31 : 144-146.
KANEHISA, T., 1956b. Relation between the formation of melanotic tumors and tryptophane
metabolism involving eye-colour in Drosophila. Annot. Zool. Jap.. 29: 97-100.
KIKKAWA, H., 1953. Biochemical genetics of Boinb\.i- inori (Silkworm). Adi', w Genetics.
5: 107-140.
NOLTE, D. J., 1952. The eye-pigmentary system of Drosophila III. The action of eye-colour
genes. /. Genetics. 51 : 142-186.
PLAINE, H. L., AND B. GLASS, 1955. Influence of tryptophan and related compounds upon the
action of a specific gene and the induction of melanotic tumors in Drosophila melano-
gaster. J. Genetics. 53: 244-261.
RIZKI, M. T. M., 1957. Tumor formation in relation to metamorphosis in Drosophila melano-
gaster. J. Morph., 100: 459-472.
WIGGLESWORTH, V. B., 1942. The storage of protein, fat, glycogen and uric acid in the fat
body and other tissues of mosquito larvae. /. Ex p. Biol., 19 : 56-77.
WIGGLESWORTH, V. B., 1949. The utilization of reserve substances in Drosophila during flight.
/. Exp. Biol., 26: 150-163.
WILSON, L. P., R. C. KING AND J. L. LOWRY, 1955. Studies on the tu"' strain of Drosophila
melanogaster: Phenotypic and genotypic characterization. Growth, 19: 215-244.
EXPERIMENTAL STIMULATION OF GAMETOGEXESIS
HYDROIDES DIANTHUS AXD PECTEX IRRADIAXS
DURIXG THE WIXTER 1
HARRY J. TURNER, JR. AND JAMES E. HANKS
ll'oods Hole ( >ccunognipliic Institution, Woods Hole. Massachusetts
There is a long list of benthic marine invertebrates found in the Woods Hole,
Mass, region that reproduce during the summer months. The majority of the
species of organisms suitable for embryological experiments, listed by Costello
et al. (1957), fall into this category. In addition, the extensive investigations of
Redfield and Deevey (1952) have shown that most members of the fouling com-
munity attach during the summer months at Woods Hole and in other localities
where there is a considerable difference between the summer and winter tempera-
ture extremes.
The question arises as to which of the various environmental variables controls
reproduction. Hutchins (1947) studied the world-wide distribution of a variety
of benthic marine forms and came to the conclusion that the northward extension
of the ranges of a number of organisms is regulated by the minimal summer
temperature that permits propagation, thus suggesting that elevated temperatures
either stimulate gametogenesis or induce spawning.
Experimentally, Townsend (1940) obtained ripe gametes from the sea urchin
Arbacia by holding specimens in aquaria at a temperature of 18° to 19° C. for one
to two months in the late fall and winter. An ample supply of food was provided.
Subsequently, Loosanoff and his co-workers have contributed greatly to the field
by demonstrating the stimulating effects of elevated temperatures on the develop-
ment of the gonads of certain commercial mollusks. Loosanoff and Davis ( 1950)
succeeded in bringing the hard clam. I'enns incrccnaria, into reproducing condition
during the winter months by gradually raising the temperature over a period of
three weeks from that of the natural environment (5.0 to 7.0° C.) to 20° C.
They mentioned that the same could be accomplished by placing the clams directly
in water at 20° C. but some mortality occurred. Later (1952) these investigators
studied the influence of temperature on the maturation of the gonad of the eastern
oyster, Crassostrca I'irt/inica, and demonstrated a logarithmic increase in the rate
of ripening of the gametes as temperatures were elevated from 15° C. to 30° C.
They also determined that physiologically ripe gametes were formed earlier in
the males than in the females under the same conditions. The importance of
nutrition on gonad development was indicated by the failure of oysters in "poor"
condition, i.e., containing little glycogen, to respond satisfactorily to the thermal
stimulus.
Experimental work on the effect of temperature on gonad development of
1 Contribution No. 1099 from the Woods Hole Oceanographic Institution. This investiga-
tion was supported by the National Science Foundation NSF-G8905.
145
146 HARRY J. TURNER, JR. AND JAMKS K. HANKS
other henthic marine invertebrates appears to lie lacking. The investigations to
be* described were undertaken to determine if certain organisms occupying ap-
proximately the same geographical range as that of the oyster and the hard clam
would respond in a similar fashion. The serpulid polychaete, Hydroidcs dianthus
(-- H. Itc.nn/onns) (Verrill), ranges from Cape Cod to Florida (Pratt, 1948) and
is found from the low tide mark to depths of several fathoms. It reproduces from
the middle of June to the end of October (Grave, 1933) and the ripeness of the
gametes may be easily determined by removing the worms from their tubes and
observing the products issuing from the nephridiopores (Grave, 1933). The bay
scallop, Pectcn irradians Lamarck, ranges from Cape Cod to Texas (Turner,
1953). It is a hermaphroditic species that spawns from mid-June to mid-August
in the Woods Hole region (Belding. 1931). The ripening of the reproductive
products may be determined on macroscopic examination by the development of
a bright orange color in the ovary at the distal extremity of the visceral mass.
MATERIAL AND METHODS
Specimens of H. dianthus were obtained from the supply department of the
Marine Biological Laboratory in the middle of December. They had been freshly
collected from the adjacent waters where the temperature was approximately 8C C.
The specimens were attached to old J'cniis and Pectcn shells in a community which
included Astrangia, sulphur sponges and dead barnacle shells. The tubes ranged
between 5 and 7 cm. in length, well beyond the size of earliest sexual maturity
(Grave, 1933).
Bay scallops were collected from the Eel Pond, Woods Hole, in December
and January, in shallow water at low^ tide. They averaged a little over 3 cm.
in the longest dimension and were the young of the year (Belding. 1931). There
were no year-and-one-half old specimens in the Eel Pond and those usually found
in nearby localities had been almost completely depleted by commercial fishing.
Consequently, larger specimens were not available in adequate numbers for experi-
mental work.
At the beginning of the experiments, a number of specimens of H. dianthus
were removed from their tubes and placed in warm (23° C.) sea water to deter-
mine if they would extrude ripe gametes. No gametes were extruded and the
worms were preserved in saturated aqueous mercuric chloride with 5r/c acetic
acid for histological study. Similarly a number of scallops were opened and ex-
amined for the characteristic orange color of the ripe ovary. None showed this
character and a number of appropriate anatomical portions were preserved in the
same manner as were the worms.
The worms were subjected to an elevated temperature in aerated still water.
Several tube-encrusted shells, containing about two dozen worms, were placed in
a non-toxic five-gallon polyethylene container of warmed sea water held at 23° C.
by a constant temperature bath. The water was changed every three days and
continuously aerated. Suspensions of the planktonic alga, Phacodahctylluin
tricornutum ( -- Nitzschia clostcrinin, fonua ininiitissinia), wrere added daily for
food. The amount added was adjusted to the quantity that the worms would just
consume in 24 hours. A control was set up and maintained in an identical manner
except that the container was placed in a tank supplied by the laboratory sea water
STIMULATION OF GAMETOGENESIS 147
system in which the temperature declined slowly from an initial level of 8° C.
The illumination of the experimental group and the control group was practically
identical. Growth of the tubes was determined in most cases by measuring the
new white addition at the mouth beyond the discolored and fouled original tubes.
In cases where the tubes were not sufficiently discolored to make new growth
clearly distinguishable, a gram of powdered alizarin was added to the sea water
and kept in suspension for 24 hours. The worms readily took up the alizarin and
combined it with the calcareous material of the new growth, forming a prominent
purple ring of alizarin lake. This treatment provided a very satisfactory method
of marking the tubes and apparently had no deleterious effect on the worms.
Specimens were removed on the third, fifth, seventh, and tenth days, tested for
extrusion of gametes and preserved for histological examination. The experiment
was repeated four times during December, January and early February.
Preliminary experiments indicated that bay scallops could not be maintained
successfully in still water so they were treated in a different manner. In late
January a number of specimens were placed in a 3' X 3' X 8" Fiberglas tank
supplied with running warmed sea water at a rate of -J gallon per minute. The
apparatus used to warm the water was similar to that described by Loosanoff
(1949) but modified to use electric power instead of gas. The temperature was
maintained at 23° C. No food was added because the laboratory facilities for
rearing phytoplankton were inadequate to supply the vast quantities required in
running sea water systems. Consequently the scallops had to subsist on such
quantities of food materials as remained in the water after it had passed through
the intake line, reserve tank, and lengthy distributing system. A control was
set up in a similar manner except that it was supplied with sea water at the
ambient temperature which was holding steady at approximately 3° C. The light-
ing conditions over both groups were identical. Specimens were removed weekly,
examined grossly for gonad development and preserved for sectioning.
RESULTS
Hydroides diaiitJuis
Xone of the worms shed gametes when first obtained, nor did any taken from
the control group which was maintained at a temperature approximating that of
the natural environment at any subsequent time. In the experimental group
which was maintained at 23° C., gametes were first obtained after seven days of
warming. Males shed copious quantities of reproductive material consisting largely
of spermatocytes and spermatids with a few tailed spermatozoa. By this time,
females shed numerous eggs which were very much smaller than normal size and
could not be fertilized. On the tenth day all males tested produced normal
spermatozoa and the females shed eggs, the majority of which were of normal
size. These eggs were successfully fertilized, underwent normal cleavage ac-
cording to the usual time schedule (Costello ct ol., 1957), and produced vigorous
trochophores. During the ten days of warming all the worms increased the length
of their tubes from three to five millimeters, indicating that they were vigorous
and healthy. Worms in the control group showed no measurable growth.
The sequence of events during gametogenesis of the tube worm was followed
148
HARRY J. TURNER, JR. AND JAMES E. HANKS
in histological sections. The winter gonad consists of a series of pairs of syncytial
masses of germ cells, one pair to each of the abdominal segments. Each segment
contains a mass of reproductive material on either side at the ventro-lateral aspect
of the wall of the coelom. Figure 1, A is a typical cross-section of a worm pre-
c
.
E
•1mm
FIGURE 1. Transverse sections of H. diaiitlnts. Delafield's haematoxylin and eosin stain.
A, Specimen collected in December. Germinal masses indicated by arrows. B, Female held
at 23° C. for five days. A few ovocytes free in the coelom. C, Male held at 23° C. for three
days. Numerous spermatocytes free in the coelom. D, Female held at 23° C. for seven days.
Numerous ovocytes partially grown free in the coelom. E, Male held at 23° C. for ten days.
Mature spermatozoa in the coelom. F, Female held at 23° C. for ten days. Mature ova in
the coelom.
STIMULATION OF GAMETOGENESIS 149
served in mid-winter. There was little evidence of sexual differentiation at this
time although some specimens contained masses with slightly enlarged nuclei,
suggestive of primitive ovocytes. All specimens examined contained a few mitotic
figures in the germinal masses, indicating that the germinal material was pro-
liferating slowly.
Sexual differentiation became apparent in males after three days of warming.
Cells broke away from the germinal masses into the coelom where the maturation
divisions took place (Fig. 1, C). This process continued for the next seven days
and by the tenth day the coelom wras packed with mature spermatozoa, as shown
in Figure 1, E.
No significant changes in the female gonads were observed until the fifth day
of warming. At this time the nuclei began to enlarge as typical germinal vesicles
and a few ovocytes broke free into the coelom (Fig. 1, B). By the seventh day,
there were more free ovocytes in the coelom and some had shown appreciable
growth (Fig. 1, D). By the tenth day, the coelom was completely filled with
ovocytes, the majority of which were of mature size (Fig. 1. F).
There was remarkable uniformity in the rate of development of the gonads in
all specimens examined and this was repeated in each of the four experiments
run in sequence. At no time did any of the animals taken from the low temperature
controls show any evidence of gametogenesis.
Pcctcn irradians
The reaction of the bay scallops to the elevated temperature was slower and
more erratic than that of the tube worm. Approximately 5^ died during the
course of the experiment. A few specimens failed to show any development of
the gametes, and in many, gametogenesis proceeded for a short time and then
ceased. The majority of the specimens examined eventually produced tailed
spermatozoa and ova which appeared to be structurally mature on histological
examination. However, normal spawning did not occur so that physiological
maturity could not be determined.
The bay scallop is hermaphroditic. The gonad consists of cylindrical, branch-
ing, tubular follicles ramifying through the visceral mass in the blood space sur-
rounding the digestive tract. The follicle wall consists of a single layer of
squamous epithelium with germ cells scattered along the inside. Male and female
germ cells develop in separate follicles.
Sections of specimens taken from the natural environment in January showed
no significant gametogenesis. The follicles of the male gonad as they appeared
in sections are shown in Figure 2, A. The follicle walls of the female gonad
(Fig. 2, B) appeared to be thicker because of the larger sizes of the ovogonia but
there was no evidence of significant development.
Marked changes occurred after the scallops had been subjected to a tempera-
ture of 23° C. for one week. Rapid proliferation of the germinal material in the
male resulted in a multi-layered lining of the follicles, consisting of spermatogonia
and spermatocytes (Fig. 2, C), while the germ cells of the female gonad (Fig. 2, D)
enlarged considerably and develop characteristic germinal vesicles with prominent
nucleoli (Fig. 2, D.)
150
HARRY J. TURNER, JR. AND JAMES E. HANKS
At the end of the third week of warming', the ovarian portion of the gonad
acquired the orange pigmentation characteristic of the ripe ovary and the testicular
portion took on a light, cream color with a plump appearance. Histological sections
showed that the male follicles were lined with many layers' of spermatozoa with
the tails projecting out into the much reduced lumens (Fig. 2, K). The ovarian
follicles were much enlarged and were completely filled with ova of mature size
-'4 5«a»sS UEfc*"^
1mm
FIGURE 2. Sections of gonads of P. irradians. Delafield's haematoxylin and eosin stain.
A, Male gonad of specimen collected in January. B, Female gonad of same specimen. C, Male
gonad of specimen held at 23° C. for one week. D, Female gonad of the same specimen.
E, Male gonad of specimen held at 23° C. for three weeks. F, Female gonad of same specimen.
STIMULATION OF GAMETOGENESIS 151
(Fig. 2. F). None of the specimens taken from the control group showed any
evidence of gametogenesis during January and February.
DISCUSSION
It is clear from the foregoing that the tube worm, Hydroidcs dianthus, and the
hay scallop, Pcctcn irradians, respond to artificially elevated temperatures during
the winter in a manner similar to that shown by many commercial molluscs, as
described by Loosanoff, by developing their gametes out of season. All of the
tube worms subjected to a temperature approximating that of the natural environ-
ment during the normal reproductive period developed ripe gametes within ten
days. These underwent normal fertilization, cleavage, and developed into viable
larvae.
The response of the bay scallop was somewhat erratic and the development
of the gametes was much slower. It is quite probable that the experimental con-
ditions were not entirely satisfactory, particularly in regard to nutrition. Oysters
and clams maintained in the laboratory sea water system grow thin and watery
after a time, indicating that suspended nutrient material is sparse. Consequently,
the scallops held in running sea water at the elevated temperature undoubtedly
had to synthesize the reproductive materials from stored substances which may
have varied considerably among individuals at the time they were collected.
Individuals in which gametogenesis failed to go on to completion were probably
those with inadequate supplies of reserve nutrients, similar to the oysters in "poor"
condition described by Loosanoff and Davis (1952). In any event it is clear
that temperatures approximating those existing during the normal spawning
period will stimulate gametogenesis in both P. irradians and H. dianthus if imposed
experimentally during the winter months when the temperature of the natural
environment is approaching the seasonal minimum.
SUMMARY
1 . Temperatures approximating those existing during the normal summer
reproductive period will stimulate gametogenesis in Hydroidcs dianthus and Pcctcn
irradians if artificially imposed during the coldest winter months.
2. H. dianthus will produce mature gametes in ten days if held in aerated
sea water at 23° C. and fed adequate quantities of Phaeodactyllum ti-icoruiitiini.
3. P. irradians may produce gametes that appear to ,be mature on histological
examination in three weeks if held in running sea water warmed to 23° C. but in
many cases development fails to go to completion.
4. Failure of gametogenesis to reach completion in some" specimens of P.
irradians may be due to inadequate food supply under the conditions of the
experiment.
LITERATURE CITED
(BELDING, D. L., 1931. The Scallop Fishery of Massachusetts. Marine Fisheries Series — No. 3.
Commonwealth of Mass., Dept. of Cons., Div. of Fish, and Game, Marine Fish. Sec.
COSTELLO, D. P., M. E. DAVIDSON, A. EGGERS, M. H. Fox AND C. HENLEY, 1957. Alethods for
Obtaining and Handling Marine Eggs and Embryos. Marine Biological Laboratory,
\Yoods Hole, Mass.
152 HARRY J. TURNER, JR. AND JAMES E. HANKS
GRAVE, B. H., 1933. Rate of growth, age at sexual maturity, and duration of life of certain
sessile organisms, at Woods Hole, Massachusetts. Biol. Bull., 65: 375-386.
HUTCHINS, L. W., 1947. The bases for temperature zonation in geographical distribution.
licol. Monofir., 17: 325-335.
LOOSANOFF, V. L., 1949. Method for supplying a laboratory with warm sea water in winter.
Science, 110: 192-193.
LOOSANOFF, V. L., AND H. C. DAVIS, 1950. Conditioning /'. nierccnaria for spawning in
winter and breeding its larvae in the laboratory. Biol. Bull., 98: 60-65.
LOOSANOFF, V. L., AND H. C. DAVIS, 1952. Temperature requirements for maturation of
gonads of northern oysters. Biol. Bull.. 103: 80-96.
PRATT, H. S., 1948. A Manual of the Common Invertebrate Animals, Revised Edition. The
Blakiston Co., Philadelphia.
REDFIELD, A. C., AND E. S. DEEVEY, JR., 1952. Pt. II. The Biology of Fouling. In: Marine
Fouling and its Prevention. United States Naval Institute, Annapolis. Chap. 5.
The Seasonal Sequence, 48-76.
TOWNSEND, G., 1940. Laboratory ripening of Arbacia in winter. Biol. Bull., 79: 363.
TURNER, H. J., JR., 1953. A review of the biology of some commercial molluscs of the East
Coast of North America. In : Sixth Report on Investigations of the Shellfisheries of
Mass., Dept. of Nat. Res., Div. of Marine Fisheries, 39-74.
A NEW SPECIES OF CHIRIDOTEA (CRUSTACEA: ISOPODA)
FROM NEW ENGLAND WATERS
ROLAND L. WIGLEY
r. S. Department of the Interior, I:islj and Wildlife Service, Bureau of Commercial fisheries,
Biological Laboratory, Woods Hole, Alassachusctts
The genus Chiridotca is unique to the eastern coastal region of the United
States and southeastern Canada. Members of this genus are small (usually
< 1 cm.), broad, depressed valviferous forms that inhabit sandy areas and charac-
teristically burrow just beneath the sediment surface. The species described herein
constitutes the fourth known species of this genus. Those previously described
are: C. cocca (Say, 1818); C. tuj'tsi ( Stimpson, 1853); and C. alnivra Bowman,
1955. Included in Bowman's paper is a revision of the generic characteristics of
this group. The description of this new form is based on specimens collected on
Georges Bank, a relatively shallow portion of the continental shelf east of Massa-
chusetts, U. S. A. These specimens were encountered while processing collections
of benthic invertebrates taken by the R/V Albatross III and the R/V Delaware
for the Woods Hole Laboratory, Bureau of Commercial Fisheries, U. S. Fish and
Wildlife Service.
Material E.raniined. — Holotype, adult female with oostegites developed, 7.5
mm. in length, deposited in the U. S. National Museum (Catalogue No. 104282) ;
allotype, adult male 6.0 mm. in length (U.S.N.M. Catalogue No. 104280) ; para-
types, 1 male 5.0 mm. in length and 1 ovigerous female 6.5 mm. in length
(U.S.N.M. Catalogue No. 104281 ) ; all type specimens were collected August 6,
1959, by means of a grab-type bottom sampler on Georges Bank at lat. 41° 48' N.,
long. 67° 53' \Y. ; water depth 15 fathoms, sand substrate, bottom water tempera-
ture 57.0° F. (R/V Delaware, cruise number 59-9, station 21).
One female, 7.5 mm. in length, collected December 7, 1955, at lat. 40° 51' N.,
long. 68° 55' Wr. ; substrate coarse sand, water depth 36 fathoms, bottom water
temperature 47.2° F. (R/V Albatross III, cruise number 70, collection 3).
One male, 6.5 mm. in length, collected December 14, 1955. at lat. 41° 40' N.,
long. 67° 36' W. ; substrate gravelly sand, water depth 28 fathoms, bottom water
temperature 45.7° F. (R/V Albatross III, cruise number 70, collection 38).
Five females, body lengths 4.0, 5.0, 6.5, 7.5, 7.5 mm., and two males, body
lengths 7.0 and 7.0. collected August 24, 1957, at lat. 41° 22' N.. long. 68° 20' W.;
substrate coarse sand, water depth 24 fathoms (R/V Albatross III, cruise number
101, station 64).
Three specimens, 4.0, 4.5, 4.5 mm. in length, collected August 24, 1957, at lat.
41° 34' N., long.. 67° 28' W. ; substrate coarse sand, water depth 23 fathoms
(R/V Albatross III, cruise number 101, station 90).
Diagnosis. — Medium-sized Chiridotca with short antennae; flagellum of
antenna 2 is much shorter than the peduncle ; antenna 1 usually not reaching
beyond the peduncle of antenna 2 ; outer margin of pereion epimeral plates 5-7
153
ROLAND L. WKiLEY
Fic.rkKS 1-9. Chiridotea urcnifolu n. sp.
1
FIGURE 1. Dorsal view of female holotype.
NEW CHIRIDOTEA FROM NEW ENGLAND
155
E
£
in
6
E
E
m
d
FIGURE 2.
FIGURE 3.
Pereiopod 1, <$.
Pereiopod 2, <$.
156
ROLAND L. WIGLEY
FIGURE 4. Antenna 1, d.
with relatively few or no setae. This species most closely resembles C. cocca (Say)
but differs in that it is smaller, the pereiopods are more slender, the body is thinner
and less convex dorso-ventrally, the pleotelson is narrower and more evenly
tapered, and the anterior lobes of the antero-lateral sections of the head are shorter
and have margins devoid of setae. Other differences are mentioned later in this
paper under the heading Discussion.
Description. — Anterior margin of head broadly and shallowly excavated each
side of the rostrum. Lateral anterior projections on the head are rounded or
obtusely pointed. Pronounced, but comparatively shallow, V-shaped notch in
each antero-lateral margin of the head ; margin anterior to the notch is without
FIGURE 5. Antenna 2,
NEW CHIRIDOTEA FROM NEW ENGLAND
157
E
E
OJ
O
E
E
CVJ
6
£
E
CVJ
6
E
E
C\J
6
FIGURE 6.
FIGURE 7.
FIGURE 8.
FIGURE 9.
Left mandible,
Maxilla 1, rf-
Maxilla 2, .
Maxilliped, J1-
setae, the margin posterior to the notch has approximately 6 setae. The anterior
lobe does not extend outwardly as far as the posterior lobe. Rather small, irregu-
larly rounded compound eyes are located dorsally on the head near the base of
the postero-lateral lobe ; they are only faintly visible in many of the specimens
examined. Epimeral plates distinct on pereion somites 2-7, their postero-
lateral margins acutely produced posteriorly; this projection is more pronounced
158 ROLAND L. WKJLEY
(in plates 5-7 than on plates 2-4. Stout setae fringe the lateral margins of pereion
somite 1 and epimeral plates 2-4, and are sparse or absent on plates 5-7. Pleotel-
son elongate, its length is about l/> times its width at the base; sides of the pleo-
telson taper somewhat irregularly from the base to the apex; lateral margins near
the apex are very finely denticulate and beset with setae. General body proportions
are : body width 0.43 times body length ; abdomen length 0.45 times body length ;
head length about 0.17 times body length.
Antenna 1 is short, extending only to about the end of the peduncle of antenna 2 ;
the flagellum consists of 1 segment and it usually bears 4 pairs of inflated setae on
the anterior margin. Antenna 2 is only slightly longer than antenna 1, its flagellum
is made up of 3-5 segments. First segment of the peduncle is expanded ; second
segment with the distal two-thirds expanded ; third segment elongate and slender,
approximately equal in length to the flagellum. Maxilliped palp is composed of
3 segments. Maxilla 1 with the inner branch possessing a large, plumose seta
and a minute seta. Mandible is without a molar process. Propodus of
pereiopod 1 is 1.2 times as long as broad; posterior margin of dactyl 1 bears 4—6
fine setae ; a few small setae are present on the external lateral surface of the pro-
podus, near the posterior margin. The posterior (ventral) margin of the carpus of
pereiopod 1 is armed with only one stout spine. Pereiopods 1. 2, and 3 are
generally similar in conformation and embellishment ; likewise, pereiopods 4, 5, 6,
and 7 resemble one another.
Color. — Basic color of the body and appendages ranges from light tan or a
pinkish hue to nearly white with the integument partially covered with dark
chromatophores. The chromatophores are black or deep violet and are distributed
somewhat unevenly over the body and the larger exposed appendages. Chromato-
phores are not evident on the pleopods or the inner mouth parts. In the material
studied, much variation was observed in the pigmentation pattern from one
specimen to another ; however, in all specimens examined the chromatophores are
consistently more densely concentrated on the uropods and pleotelson than on other
parts of the body. Differences in pigmentation appear to be unrelated to size,
sex. or season of capture.
Range. — Georges Bank (east of Massachusetts. U. S. A.) in water depths
15 to 36 fathoms and water temperature 45.7° to 57.0° F.
Discussion.- — -Shape of the head in C. arcnicola is quite like that found in
C. aluiyra. The posterior portion is comparatively long and somewhat narrowed,
and the antero-lateral lobes are rather short. The notch separating the antero-
lateral margin into two lobes is shallow, as compared to related species.
C. arcnicola is distinguished from all other species in this genus by the absence
of setae on the anterior lobe of the antero-lateral margin of the head.
The propodus of pereiopod 1 in C. arcnicola is relatively short and wide, similar
to that of C. cocca, but it lacks the setation on the outer central, lateral surface
found in that species. The dactyl of pereiopod 1 has only a few thin setae on
the occluded margin ; in this feature it resembles C. alinyra and C. cocca.
In C. arenicola the size and number of setae on the outer margin of epimeral
plates 5-7 varies considerably from one specimen to another ; however, these
setae are generally shorter and much less numerous in this species than in other
members of the genus. A slight sexual dimorphism in this feature is apparent
XF.VY CHIRIDOTEA FROM NEW ENGLAND 159
in the few specimens available for study. The setae appear to be longer and more
numerous in large males than in adult females and small males.
The shape of the pleotelson of C. arcnicola is intermediate between that of
C. cocca, which is broad and tapers irregularly, and that of C. titftsi, which is rather
narrow and evenly tapered.
The eye is dark and distinct in some specimens and faint in others. Variation
in this characteristic occurs at random among the sexes, specimens of various sizes,
and in different collections (some of which have been in preservative for two
months and others nearly two years). In the specimens at hand, the eye appears
to be proportionately much smaller in C. arenicola than that depicted for C. cocca
by Bousfield (1956).
To my knowledge neither C. alinyra nor C. cocca have been reported from
offshore waters, and they have not been observed in our collections of benthic
invertebrates from the Georges Bank area. C. arcnicola and C. tnftsi are the only
representatives of this genus found in our samples taken during the past few years.
Of these two species the latter is far more numerous than arcnicola ; a ratio of
approximately 20:1, based on the total number captured. Our records indicate
that C. tnftsi is rather widely distributed over Georges Bank and nearly always
within the 50-fathom isobath. C. arcnicola seems to be more restricted, having
been taken only near the shoals on the north-central part of Georges Bank and
on the western end near Great South Channel.
A factor which appears to be important in affecting the distribution of these
two species is the particle size composition of the substrate. C. arcnicola has been
taken most frequently from areas where the predominant sediment fraction is a
coarse sand (0.5-1.0 mm.). Conversely, C. tnftsi is most commonly found where
the predominant sediment fraction is a medium sand (0.25-0.5 mm.). Also, there
is some evidence that C. arcnicola either burrows more deeply into the sand or for
some other reason is more difficult to evict from the substrate than C. tnftsi. This
inference is based on two factors : ( 1 ) in dredges that normally scrape only the
surface of the sea bed, hundreds of tnftsi have been caught, compared to only two
specimens of arcnicola; (2) in grab-samplers that usually dig 3 to 6 inches into
the sea floor, 10 specimens of arcnicola have been taken, versus 7 specimens of
tuftsi.
Some of the more common Crustacea with which C. arcnicola has been found
associated are the amphipods : Acginina longicornis (Kroyer), Ampclisca spinipes
Boeck, Auiphiporcia rirginiana Shoemaker, Ericthonins rnbricornis Stimpson.
Hanstorins arcnarins (Slabber), Leptocheirus pinguis (Stimpson), Plwtis dentata
Shoemaker, Pontogoicia incnnis (Kroyer). Podoccropsis nitida (Stimpson).
Sviuplcnstcs glabcr (Boeck), and Tiuctony.r nobilis (Stimpson); the mysid :
\coin\sis aincricana (Smith); and the decapods: Cancer borcalis, Stimpson.
Cancer irroratns Say, Crangon septemspinosa Say, Dichelopandahts Icpto-
ccrns (Smith), and Pagnrns acadianns Benedict. These species are the most
abundant crustaceans that were taken in the same bottom-grab samples and dredge
hauls with C. arcnicola. It will be recognized that many of these associates are
infauna forms ; however, all of the larger species except one are epibenthic. Some
species listed above are exceedingly tolerant and are adaptable to diverse environ-
mental features, but others listed here are restricted to very specific types of habitats.
160 ROLAND L. WIGLEY
Judging from the information available at this time, it seems likely that C. arcnicola
will be found most closely associated with some of the burrowing amphipods listed
above, such as Amphiporeia, Hanstorius, and Tinctony.v.
KEY TO THE SPECIES OF CmRiooTEA1
1 . Flagellum of antenna 2 much shorter than peduncle, segments 5 or less ;
antenna 1 nearly as long as antenna 2 2
Flagellum of antenna 2 longer than peduncle, 8—12 segmented ; antenna 1 much
shorter than antenna 2 3
2. Antenna 1 extends beyond peduncle of antenna 2 ; margin of anterior lobe of
the antero-lateral margin of head is setose C. coeca
Antenna 1 does not extend beyond peduncle of antenna 2 ; margin of anterior
lobe of the antero-lateral margin of head is not setose C. arenicola, n. sp.
3. Posterior margin of dactyl of pereiopod 1 armed with strong spines ; pleotelson
narrow, tapering evenly from base to apex C. tiiftsi
Posterior margin of dactyl of pereiopod 1 armed with a few fine setae ; pleotelson
broad, sides nearly parallel at the basal half C. alinyra
LITERATURE CITED
BOUSFIELD, E. L., 1956. Malacostracan crustaceans from the shores of western Nova Scotia.
Proc. Nova Scotiaii hist. Sci., 24: 25-38.
BOWMAN, T. E., 1955. The isopod genus Chiridotea Harger, with a description of a new
species from brackish waters. /. Wash. Acad. Sci., 45 : 224-229.
SAY, T., 1818. An account of the Crustacea of the United States. /. .lead. Nut. Sci. Phila..
1 : 424-425.
STIMPSON, W., 1853. Synopsis of the marine Invertebrata of Grand Manan. Smithsonian
Contr. Knsunns tan ) was used for this metabolic study
of islet tissue because of the accessibility of the islets and the general availability
and hardiness of this species.
In a previous study ( Lazarow, Cooperstein, Bloomfield and Friz, 1957), the
oxygen uptake of islet tissue was measured under varying conditions of pH,
tonicity, and electrolyte composition, and the baseline conditions under which
maximal respiration of islet tissue occurs were defined. The present paper reports
further characterization of the over-all metabolic pathways of islet tissue achieved
by studying the effects of specific exogenous substrates and inhibitors on the
respiration of islet slices.
M. \TKRIAL AND AlKTIIODS
After the toadfish were killed by a blow on the head, the principal islets were
dissected from the mesentery and the capsules of the islets were removed. The
latter is an important step in the procedure, since any acinar tissue that may be
present underneath the capsule, as well as any acinar tissue that may be present
1 This investigation \vas supported by research grants A-l'o1' and A-1887 from the National
Institute of Arthritis and Metabolic Diseases, Public Health Service.
-Present Address: Carlsberg Laboratories, Copenhagen, Denmark.
161
162 CARL T. FRIZ, ARNOLD I.AZAROW AND S. J. COOPERSTEIX
within the islet but attached to the capsule by trabeculae, is removed when the
capsule is stripped.
In each experiment a single islet was cut into eight slices and these slices were
distributed between the experimental and control groups. The Cartesian diver
microrespirometer was used to measure the oxygen uptake of each toadfish slice,
in a manner previously described (Lazarow ct a/., 1957). A modification ( Laza-
row ct al., 1957) of the method of Lowry, Rosebrough, Farr and Randall (1951)
was used to measure the protein content of each slice. The metabolic activity
was expressed as millimicroliters (m/J.) of oxygen per microgram (ju.g.) of pro-
tein per hour.
The metabolic activity of toadfish liver tissue was also studied in order to
compare it with that of toadfish islet tissue. The Warburg manometer was used
to make these measurements, and the oxygen uptakes were also expressed in
millimicroliters per microgram of protein per hour.
A constant amount of phosphate buffer at pH 7.4 (0.054 Jl/) was present in
all of the experiments in this study. The respiration of islet tissue was studied
in a medium containing phosphate plus substrate or inhibitor, and the oxygen
uptakes thus obtained were compared to those observed in either a medium con-
taining phosphate plus saline or a medium containing phosphate alone.3
The various solutions of substrates and inhibitors were prepared at twice the
desired final concentration and mixed with an equal volume of 0.108 M phosphate
buffer so that the final phosphate concentration was 0.054 M .
RESULTS
Effect of substrates
The substrates studied included glucose, pyruvate (lithium salt), a-ketogluta-
rate, glutamate, succinate, and isocitrate.
When glucose was added to the medium /;; I'itro (Table I ) neither islet
(/> -- 0.21 ) nor liver ( f> -- 0.9) respiration was altered. It should be noted that
the endogenous metabolic activity of liver tissue slices was considerably higher
than that of islet tissue.
In a similar series of experiments using pyruvate as the substrate, respiration
of islet slices was compared in the following media: (a) a hypotonic medium
containing phosphate ; ( b) an isotonic medium containing phosphate plus NaCl ;
and (c) a medium containing phosphate plus pyruvate (Table II ). If one assumes
that the added pyruvate readily enters the islet cells, then the respiration of islet
slices in the pyruvate-containing medium should be compared with that in the
hypotonic medium because (a) and (c) would be of equal tonicity. When these
media are in fact compared, it is noted that pyruvate has no effect on the respira-
tion of islet tissue. If, on the other hand, one assumes that the added pyruvate
does not enter the islet cells, then media (b) and (c) should be compared since
3 Whereas Lazarow ct ol. (1957) reported that optimal respiration of islet slices was
observed in a hypotonic medium (0.054 M phosphate), in the present study optimal respiration
of islet slices was sometimes observed in an isotonic medium whereas at other times no differ-
ence was noted between respiration in hypotonic and isotonic media. Evidence indicates that
part of this discrepancy may be due to the varying temperature of the water in which the fish
were kept. Further work is now in progress in order to clarify these differences.
METABOLISM OF TOADFISH ISLET TISSl I.
163
TAFJLE I
Effect of glucose on the respiration of toad fish islet and liver s
Tonicitv of
Islet
Li\ ci
medium
Medium
(equiv. XaCl
cone.*
M /liter)
No. of
deter.
Ave. mjil. ( )•_'.
Hg. prot. hr.
) was calculated
using the following equation :
difference
/> =
\ Nl
N2 -
where a\ and <72 are the standard deviations of the control (without substrate) and experimental
(uith substrate) groups, respectively; Ni and A\> are the corresponding number of determinations
in each group.
they would be of approximately equal tonicity. Comparison of the respiration in
these media indicates that pyruvate addition inhibits by 23% (p -- 0.019).
The addition of a-ketoglutarate (Table III ) increased the oxygen uptake of islet
slices by approximately 33 % when compared with either a hypotonic (p -- 0.07)
or isotonic (/> -- .008) medium.
Table IV shows that a 4470 increase in oxygen uptake was observed when islet
slices were placed in a phosphate-glutamate medium (/> == 0.016). This is to be
compared to a 30-36^/c increase observed when toadfish liver slices were placed
in the same medium (/> = <0.001 ).
Oxygen uptake was also increased when succinate ( Table Y ) was added either
to toadfish islet or to toadfish liver tissue. The increase in islet tissue respiration
in an isotonic phosphate-succinate medium was 47rr ( f> -- 0.01 ) when compared
to respiration in an isotonic phosphate-saline medium and 100'v ( [> -- 0.001) when
TABLI-; II
Effect of pyruvate on the respiration of toadfish islet slices
Tonicitv of
N-i if-
Medium
medium (equiv.
XaCl cone.
Xo. of
deter.
Ave. mjil. Oi/
Mg. prot. hr.
a
.\f liter)
a
0.054 ,17 PO4
0.0755
43
1.7
0.70
1)
0.054 M PO4 + 0.0645 .17
0.140
14
2.2
0.69
XaCl
c
0.054 .17PO4 + 0.040-
0.1155-0.140
28
1.7
0.70
0.0645 .17 pyruvate
164
CARL T. FRIZ, ARNOLD LA/AKOW AND S. J. COOPHRSTKIN
TABU-: III
/'Iff fit of a-ketoglutarate on the respiration of load fish islet slices
Medium
Tonicitv of
medium (equiv.
NaCl cone.
No. of
deter.
Ave. niAil. < >
MK- prot. lir.
a
M liter)
0.054 .17 PO4
0.0755
13
2.1
0,83
0.054 .17 PO4
+ 0.0645 M Nad
0.140
13
2.0 0.60
0.054 M PO4
+ 0.0645 .17 fv-kftnijiit. irate
0.140
14
2.7
0.84
compared to respiration in the hypotonic medium. The addition of succinate
increased the respiration of toadfish liver slices by about 75 r/f (/> =: < 0.001).
In contrast to a-ketoglutarate, glutamate, and succinate, another Krebs cycle
intermediate, isocitrate, did not stimulate islet respiration (Table Yl).
I'.fjcct oj inhibitors
The use of inhibitors on whole cells, extracts, homogenates, and tissue slices
has provided much detailed information concerning- the occurrence and components
of complex enzyme systems, such as the glycolytic and tricarboxylic acid systems.
\ arious inhibitors were used in this study in order to assess the importance of
these enzyme systems for the metabolism of islet tissue.
TABU- IV
Effect of glutamate on the respiration of toadfish islet mid liver slices
Tonicitv of
Islet
Liver
medium
Medium
(equiv. NaCl
cone.
No. of
Ave. m/il. O"
No. of
Ave. m/al. O*/
.U liter)
deter.
Aig. prot., hr.
deter.
Mg. prot./hr.
a
0.054 M PO4
0.0755
14
1.6
0.66
9
i ~>
0.40
0.054 M PO4 +
0.140 11
1.6
0.64
9
2.3 0.23
0.0645 .17 NaCl
0.054 .17 PO4 +
0.140 13
2.3
0.78
9
3.0 0.30
0.043 .17
glutamate
Table YII shows the per cent inhibition produced by the various inhibitors.
Fluoride added to a phosphate medium was found to inhibit respiration of islet
slices by 35f/r ( /> == 0.0063), iocloacetate inhibited by 53% (p -- <0.001 ). malonate
by 43% (/> = <0.001 ), and azide by 50rv ( /> == 0.001 ).
DISCUSSION
Although the addition of glucose did not stimulate islet tissue respiration, this
does not mean that glucose can not be utilized by islet tissue. There are a number
of reasons why an added substrate might not stimulate respiration, such as its
failure to readily enter the cell or the presence of sufficient endogenous substrate
to saturate the enzyme system concerned. This is emphasized by our finding that
MKTABOLISM OF TOADFISH ISLET TISSUE
165
TABLK \'
Effect of siiccinatc on the respiration of toad fish islet and lii'er slices
[slel
Liver
Tonicitv of
Tonicity of
Medium
medium
(equiv.
XaCl
Xo. of
deter.
Ave. m/jl.
02/Mg.
prot. hr.
(7
medium
(equiv.
XaCl
No. of
deter.
A\ r. rn/nl.
( ).. Mg.
pint. hr.
a
cone.
cone.
M. liter)
M liter)
0.054 M PO4
0.0755
14
1.1
0.61
0.0755
9
2.9
0.37
0.054 M PO4 +
0.140 14
1.5
0.64
0.140
9
2.7
0.37
0.0645 M XaCl
0.054 M PO4 +
0.172 13 2.2
0.77
0.140*
9
4.9
0.89
0.0645 .1/surrinate
* 0.043 M surrinate used in this case.
the addition of glucose did not stimulate liver respiration, despite the known
occurrence of the glycolytic system in the liver of most species. Our studies with
fluoride and iodoacetate provide suggestive evidence that at least two enzymes
of the glycolytic scheme (enolase and phosphoglyceraldehyde dehydrogenase) are
also present in islet tissue. Neither of the inhibitors can be considered specific,
but phosphoglyceraldehyde dehydrogenase is extremely sensitive to iodoacetate
( MeyerhofT and Kiessling, 1933; Adler, Euler and dumber, 1938) and at least
one of the actions of fluoride is to inhibit enolase ( Warburg and Christian, 1941 ).
Of the enzymes involved in the tricarboxylic acid cycle, the presence of
a-ketoglutarate oxidase and succinoxidase are indicated bv the stimulation of oxy-
gen uptake observed following the addition of their respective substrates. The
presence of succinoxidase is further indicated by the observed inhibition by malonate
(43rr ). This inhibition can be compared with the 50-6CK4 inhibition of rat liver
homogenates reported by Holtkamp and Hill ( 1951 ) and Pardee and Potter (1949)
as well as the 70cr inhibition of rat brain homogenates (using 0.02 M malonate )
reported by Pardee and Potter (1949). Finally, the participation of succinoxidase
in islet tissue respiration is also consistent with the previous demonstration of the
presence of succinate-cytochrome c reductase in this tissue ( Lazarow and Cooper-
stein, 1951 ).
Neither pvruvate nor isocitrate stimulated islet respiration but this could be-
due to failure of these substrates to penetrate to their site of utilization. The
TABLE VI
Effect of isocitrate on the respiration of toad fish islet slices
Medium
Tonicitv of
medium (equiv.
XaCl cone.
Xo. of
deter.
A vi'. nifil. (>.'
Mg. prot./hr.
a
M liter)
0.054 M PO4
0.0755
12
2.2
0.58
0.054 M PO4
+ 0.645 .17 XaCl
0.140
12
2.2
0.57
0.054 M PO4
+ 0.032 M isocitrate
0.140
12
2.3
0.45
166
CARL T. FRIZ, ARNOLD LAZAROW AND S. L COOPERSTEIN
Glucose
Glycogen
II
Glucose -I- phosphate
It
Glucose - 6 - phosphate
If
Fructose - 6 - phosphate
it
Fructose- 1,6 - diphosphate
I t
3 Phosphoglyceraldehyde ^=^ Dihydroxyacetone phosphate
53% inhibition by
0.01 M lodoacetate
1,3 dip/iosphoglycenc acid
It
3 phosphoglyceric acid
it
2 phosphoglyceric acid
1 35% inhibition by 001 M fluoride
Phosphoenol pyruvate
It
Pyruvate
Oxalacetate +Acetyl-Co A
Malate
Fumarate
47% stimulation
by succmate,
43% inhibition
by 001 M malonate
glutamate ~ — " proteins
44% stimulation
by glutamate
Succinate
35% stimulation
by a-ketoglutarate
A = Electron transport chain
[50% inhibition by 0001 M azidel
FIGURE 1. A simplified scheme of carbohydrate metabolism, which indicates the evidence
obtained for its operation in islet tissue metabolism.
MKTABOLISM OF TOADFISH ISLET TISSUE
167
TARI.K VII
Effect of inhibitors on the respiration of toadfish islet tissue. (Each inhibitor was studied in
a separate series of experiments and compared with controls run at the same time)
Medium
No. of
deter.
Ave. ni/jl. < >••
^g. prot., hr.
a
%
inhibition
0.054 .17 PO4
13
2.0
0.85
0.054 .17 PO4 + 0.01 ,17 fluoride
14
I..?
0.37
35
0.054 .17 PO4
16
1.9
0.74
0.054 .17 PO4 + 0.01 .17 iodoacetatL-
13
0.9
0.45
53
0.054 .17 PO4
12
2.1
0.43
0.054 .17 PO4 + 0.01 .17 malonatt-
12
1.2
0.37
43
0.054 .17 PO4
8
2.0
0.79
0.054 .17 PO4 + 0.001 J7a/ide
8
1.0
0.24
50
enzymes of the tricarboxylic acid cycle are localized within the mitochondria, and
it has heen reported (Schneider, Striebich and Hogeboom, 1956) that the mito-
chondrial membrane may not be permeable to citrate.
The ability of islet to utilize glutamate is of considerable interest since this
compound is a key link between the tricarboxylic acid cycle and protein synthesis.
The existence of cytochrome oxidase in islet tissue has been reported previously
( Lazarow and Cooperstein, 1951) and its participation in the electron transport
system is indicated by the inhibition of respiration produced by azide,4 a known
inhibitor of cytochrome oxidase (Keilin, 1936).
Figure 1 depicts a simplified scheme of carbohydrate metabolism, and indicates
the evidence obtained for its operation in islet tissue metabolism.
SUMMARY AND CONCLUSIONS
The effects of substrates and inhibitors on the metabolic activitv of toadfish
*>
islet slices were measured in the Cartesian diver microrespirometer under varying
experimental conditions. Of the substrates added, a-ketoglutarate, glutamate, and
succinate increased islet respiration while glucose, pyruvate, and isocitrate were
ineffective. lodoacetate, fluoride, malonate, and azide inhibited islet respiration.
LITERATURE CITED
ADLKK, E., H. Y. EULER AND G. GUNTHER, 1938. Dehydrasen und Jodessigsaure. Skand.
Arch. f. Physiol, 80: 1-15.
DIAMARE, V., 1899. Studii comparative sulle isole di Langerhans del pancreas. Intern.
Monatschr. f. Anat. u. Physiology, 16: 155-209.
HOI.TKAMP, D. E., AND R. M. HILL, 1951. Comparison of malonate and malondialdehyde in
in vitro oxygen uptake studies. Arch. Biochcm. Biophys.. 34: 216-218.
KF.II.IX, D., 1936. The action of sodium azide on cellular respiration and on some catalytic-
oxidation reactions. Proc. Roy. Soc. London, Scr. B. 121: 165-173.
4 Studies with cyanide have not been carried out to date because these are very difficult
to perform in the Cartesian diver.
168 CARL T. FRIZ. ARNOLD LAZAROW AND S. J. COOPERSTEIN
I.A/Ako\v, ARNOLD. AMI S. J. Cooi'KKSTi-.i \, 1951. Studies on the isolated islet tissue of fish.
1. The cytochrome oxidase and sticcinic dehydrogenase c< intents of normal toadfish
(Opsanus tan}. />';«/. />»//.. 100: 101-198.
'.. \XAKO\V, ARNOLD, S. J. COOPKKSTEIN, I). K. IILOOMFIKLD AND C. T. EKIX, 1957. Studies on
the isolated islet tissue of fish. II. The effect of electrolytes and other factors on the
oxygen uptake of pancreatic islet slices of toadfish, using the Cartesian diver micro-
respirometer. />/<)/. Hull., 113: 414-425.
l.mvKY, O. H., N. J. ROSEBROUGH, A. L. FAKK AMI R . J. RANDALL, 1951. Protein measurement
with the Folin phenol reagent. /. Hiol. L'licin., 193: 205-275.
MEVEKHOFF, O., AND \\". KIKSSLINI;, 1933. tvher die phosphorylierten Zwischenprodukte und
die letzten Phasen der alkoholischen Garung. Biochein. Zcitschr.. 267: 313-348.
I'AKDKK, A. B., AND Y. R. POTTER, 1949. Malonate inhibition of oxidations in the Krehs tri-
carboxylic acid cycle. /. Hiol. Chan.. 178: 241-250.
SCHNEIDER, W. C., M. J. STRIKBUH AND (i. H. HOGEBOOM, 1956. Cytochemical studies Yll.
Localization of endogenous citrate in rat liver tractions. ./. Hiol. Chan.. 222: 969-977.
\\'.\RBn<(;, O., AND \\ . CHRISTIAN, 1°-41. Isolierung und Kristallisation des Garungsferments
Enolasc. Hioclicm. /.citschr., 310: 384-421.
A STUDY OF REPRODUCTION IX THE LXTERTIDAL BARXACLE,
MITELLA POLYMERUS, IX MONTEREY BAY, CALIFORNIA1
GALEX HOWARD H1LGARI) :!
Hopkins Marine Station of Stanford ['inrersily. California
The goose barnacle, Mitclla polymerus (Sowerby, 1S33), sometimes referred
to as Pollicipes polymerus or the leaf barnacle, is distributed along the exposed
rocky coast of Western North America from British Columbia to the middle of
Baja California (Cornwall, 1925). It is generally abundant here in the upper
two-thirds of the intertidal belt ( Ricketts and Calvin, 1952), though it is occa-
sionally found below this level where there is considerable surging wave action.
Along the central California coast, clusters of Mitclla patch the exposed rocky
regions, the barnacles usually attaching themselves to rock, to Mytilus calijornianus,
or to other individuals of Mitclla. Individuals are seldom isolated and one sec>
among the mussel beds or on the rocks rosette-shaped clusters in which large
barnacles are at the center and smaller barnacles grade toward the periphery. In
other aggregations, individuals of nearly the same size are packed closely together,
frequently with such uniformity in size and orientation that their valves form a
geometric pattern, and their closely packed bodies make a strong but resilient mat
against the pounding surf. Often where the animals are attached to rock beneath
the mussel beds, their stalks extend up eight inches or more to the surface. Occa-
sionally, solitary animals occur on tables of rock exposed to strong wave action ;
in these the stalks remain short and stubby while the shells and bodies grow.
While Mitclla polymerus is abundant, conspicuous, and well-known taxonomi-
cally, almost nothing is known of its reproductive biology. Xussbaum ( 1890) has
described the anatomy of M. polymerus in some detail. The only published studv
of reproduction and development in this genus is that of Batham (1944-45), on
the New Zealand species, Mitclla sphwsus. Since Batham's study of the repro-
ductive cycle was carried out at latitude 44° 52' South, a comparison of her results
with the situation occurring in M . polvmcnis at Monterey Bay ( 36°40' North i
seemed particularly interesting. The following study of reproduction in M. poly-
merus includes: the anatomy of the reproductive system, the relationship between
size and sexual reproduction, the seasonal reproductive cycle, the rate of egg and
embryo development, an estimate of fecundity, and evidence concerning self-
fertilization.
1 This investigation was carried out as partial fulfillment for the degree. Master of Arts.
- The author wishes to express her gratitude to Dr. Donald P. Abbott, under whose
guidance this work was carried out ; and to acknowledge with appreciation his generous help
with arrangement and discussion of the data.
•'•Author's current address: Department of Biology. Stanford University, Stanford. Cali-
fornia.
169
170
GALEN HOWARD HILGARD
TIIK REPRODUCTIVE SYSTEM
The gross reproductive anatomy of Mitclla polymerus is shown in Figure 1.
Ovarian tissue is found in the upper portion of the peduncle. From the ovaries,
a pair of oviducts lead up into the body proper, emptying into the glandular oviducal
atria in the bases of the first thoracic cirri. Fggs pass clown the oviducts to the
FIGURE 1. Gross anatomy of the reproductive system of M. polymcrns, exposed by dis-
section from the left side. ad. muse. = adductor muscle; dig. gl. = digestive gland; fil. ap.
-filamentary appendages; gut = gut ; m. c. = mantle cavity; o. ap. — oviducal aperture; o. at.
— oviducal atrium ; o. duct = oviduct ; ov. — ovaries ; ped. — peduncle ; pen. — penis ; test. —
testes ; s. ves. = seminal vesicle ; v. def. = vas deferens.
atria, where they receive a protective coating which glues them together into
masses. The egg masses are then extruded through the oviducal apertures and
come to lie in the mantle cavity on either side, where they are pressed flat to
form the two ovigerous lamellae.
REPRODUCTION IN MITELLA POI.YMKRl'S 171
The numerous small testes are found on either side of the gut, extending
ventrally into the coxopodites of the first four pairs of thoracic appendages and
dorsally into the numerous paired filamentary appendages. Fine efferent ducts
connect the testes to the paired vasa deferentia, which lead to the paired storage
organs or seminal vesicles. These in turn join posteriorly at the base of the penis,
and form a single duct extending to its tip.
Copulation was not observed, but sperm are deposited in the mantle cavity.
The embryos are brooded in the ovigerous lamellae in the mantle cavity until they
are hatched out as nauplius larvae.
The gross reproductive anatomy of .17. polymer us is similar to that of M.
spinosus; but M. spinosus has no filamentary appendages and the testis is de-
scribed as (p. 370) "a median structure lying closely in the U-bend of the gut,"
with a pair of ducts leading from either side of it which expand to form the
seminal vesicles (Batham, 1944 — 1-5).
[MATERIALS AND METHODS FOR THE STUDY OF REPRODUCTION
Nearly all living material used in the present study was collected in or very
near Monterey Bay, California, and studies were carried out at the Hopkins Marine
Station of Stanford University, Pacific Grove.
For the study of the reproductive cycle, a population of Mitella polynients was
sampled at approximately monthly intervals for a period of fifteen months. All
individuals were collected within an area approximately fifteen feet square on
granite rocks and adjacent beds of Alytilus californianus on the northern shores
of Mussel Point, Pacific Grove, California, at an intertidal level corresponding to
the upper middle horizon of Zone Three of Ricketts and Calvin ( 1952). Care was
taken to insure that all barnacles large enough to be reproductively mature were
collected in close proximity to other individuals of a reproductive size, and to avoid
taking isolated individuals which had lacked the opportunity for cross-fertilization.
Animals were anaesthetized for four hours in a solution of magnesium chloride
isotonic with sea water. This was sufficient to relax the cirri and peduncle. The
animals were then preserved in 10% formalin buffered with borax.
Individuals of all sizes were collected and examined. For most of the repro-
ductive data, the largest common animals available were used. All of these were
of a reproductive size, and for purposes of consistency, large animals which ranged
in breadth (distance from rostrum to carina ) from 27.5 mm. to 32.5 mm. were
used (occasionally larger animals are found).
When maturing, the ovarian tissue in the peduncle (Fig. 1 ) undergoes marked
and visible changes. Tiny eggs appear and grow, accumulating yolk until they
are passed up through the oviducts and are extruded into the mantle cavity.
Preliminary observations showed that throughout the ovarian tissue, eggs are
generally of about the same size and stage of development at any one time ; an
exception to this is provided where a new batch of tiny eggs appears in the ovary
before the previous batch of very much larger eggs is extruded. With a compound
microscope and calibrated ocular micrometer, the greatest diameters of five to ten
eggs were measured (in each animal), and an average ovarian egg size was re-
corded for the individual. Where two batches of eggs were present at once in
17J GALEN HO\VARI> HIMiAUI)
an ovary (i.e.. large and small), this situation was quite evident, and the two were
treated separately. The average egg sizes were then grouped into three useful
classes for purposes of comparison : small eggs (diameter 0.016 mm. to 0.065 mm. ),
medium eggs (diameter 0.066 mm. to diameter 0.09C' mm.), and large eggs
(diameter 0.100 mm. to diameter 0.127 mm.). The few individuals in which no
eggs could he seen were treated separately.
As the male gonads of Mitclla mature, they also show easily visible changes:
testes in the filamentary appendages and throughout the body lose their trans-
lucency and become opaque white with sperm. Sperm then travel through efferent
tubules and vasa deferentia to the paired seminal vesicles, where they are stored.
As more and more sperm accumulate in the seminal vesicles, these, too, change
from translucent to opaque white and increase in diameter. Diameter of the
seminal vesicles affords a fairly good index for the maturation of the male repro-
ductive organs. \Yidth was measured with a pair of dividers at a point just back
of the anterior sharp bend in the seminal vesicle ( Fig. 1 ) . Conditions of the male
organs were finally grouped into three categories : ( 1 ) seminal vesicles translucent
and apparently empty of sperm; (2) seminal vesicles ranging in width from one
to two mm.; and (3) seminal vesicles more than two mm. in width (ranging up
to a maximum observed width of 3.S mm. ).
Fertilized eggs and developmental stages present in ovigerous lamellae were
also studied. Embryos were examined in the ovigerous lamellae from animals
taken in the monthly samples. In a pair of ovigerous lamellae taken from any one
parent, embryos are all at about the same stage of development. As the fertilized
egg develops, it increases slightly in size, but not enough for size to yield a good
criterion for stage of development. Major morphological changes can be studied
fairly readily, and it proved practicable for the purpose of the present problem
to divide embryonic development into three stages : ( 1) early stages, with neither
limb buds nor nauplius eye; (2) middle stages, with limb buds developing but no
nauplius eye ; and ( 3 ) late stages, with well-formed limbs and median eye present.
Studies were also made of lamellae removed from the parent and raised in vitro.
Ovigerous lamellae were isolated from their parent barnacles, placed in clear glass
dishes of filtered sea water, and kept at a constant 13° C. in a water bath or a
refrigerated room. Sea water was changed approximately daily, at which times
the embryos were examined in small sections of the ovigerous lamellae plucked
off with glass needles. Many embryos raised in vitro were observed through
development to hatching, and many larvae were raised beyond this through several
naupliar molts.
SIZE AND SEXUAL REPRODUCTION
Figure 2 shows the occurrence of ovigerous lamellae in animals of different
sizes at selected times during the breeding season (May through December).
Animals with a breadth of more than 27.5 mm. were always found to be sexually
mature during the reproductive season ; all individuals examined bore ovigerous
lamellae, or enlarging ova in the ovaries, or both. In the individuals below 27.5
mm. breadth, the percentage of animals with ovigerous lamellae can be seen to
decrease more or less directly with decreasing size. For individual months, the
tendency is not always clear, and this may be due to sampling deficiencies (c.l
O O .
rt ot
•
rt .- —
V
s°-
V
V
FK.CKE 2. Variation in reproductive activity with size of animals. Vertical bars indicate
percentage of individuals of each size class which contained ovigerous lamellae. Size classes
are designated as follows: I, breadth less than 17.2 mm.; II, breadth 17.2 to 22.5 mm.:
Ill, breadth 22.(> to 27.5 mm. ; and IV. breadth 27.5 to 32.5 mm.
THE REPRODUCTIVE CYCLE ix THE MITELLA POLYMERUS POPULATION
Each month, 10 to 25 large animals were collected, measured, and examined
for reproductive condition. The number of individuals carrying ovigerous lamellae
was noted ; conditions of the female and male gonads, and stage of development
of the embryos (where present) were studied.
The distribution of egg sizes and larval stages is shown in Table I and Fig-
ure 3. Table I shows for each sample the number and per cent of individuals which
contained given egg size classes and which brooded particular embryonic stages.
However, in Figure 3, each separate mass of eggs or embryos is treated as a sepa-
rate unit. For example, in cases where a given individual contained simultaneously
small eggs, large eggs, and late embryos, these appear in Figure 3 in three different
horizons in the same date column.
It is apparent in Figure 3 that between November, 1956. and January, 1957,
breeding waned and was discontinued until the spring of 1957. Then, during
March, eggs began to enlarge, but did not exceed the upper limits of the class of
small eggs. l»y April, the first medium and large ovarian eggs were present, and
174
CAI.KX HOWARD HII.(,.\K1)
the first fertilized egg mass appeared. Egg production continued through the year
to January, 195S, when the number of ovigerous lamellae present in the population
dropped significantly. The season of reproductive activity for the population cov-
ers three-quarters of the year, and it is of interest that all stages of egg and
embryo development were found throughout the season. On the average, during
the height of the breeding season, between 50r/r and 6Qc/f of the population of large
animals are carrying ovigerous lamellae at any one time.
Data for the male gonads are plotted in Table I and in Figure 4. Sperm is
present in at least some members of the population throughout the year. While
there is seasonal variation in the condition of the male gonads, this is less well
defined than is that of the female gonads. In the fall of 1956 and through January,
Dare oj collection
3IOCT
IO56
3 1 NOV.
1957
8 JAN.
1956
M
--O <*
S^
U-l 3
u O
00
oo
g
vg
o
Late
Intermeaiate
IvnbryoS
Earty
Embryos
large eggs
(0 1 am.. 100 -.127 nun )
Mecuum c^s
(Diam,. 066- 099mm)
Smaff eggs
(Diam., 0)6 -065mm)
absent
sampfe)
14
10
10
o/o oj- total no of eua masses.
°A> of individuals examined..
Sin of sample
12
14
IO
15
IO
25
13
15
15
12
IO
FIGURE 3. Distribution of egg and embryo masses of various stages
of development in large M. pulyiiicrns.
1957, some seminal vesicles were very thick with sperm, while others appeared
spent. In February and March, 1957, most of the vesicles had a meager amount
of sperm, presumably building up, and some were empty. In April, most vesicles
were quite full with sperm, others had a meager amount, and none were empty,
and from this point on through the summer and fall of 1957, the mean vesicle width
was high (well over two mm.). Empty vesicles were not observed again until
January, 1958. Thus during the winter months, the quantity of sperm present in
the seminal vesicles of most of the population was considerably less than the amount
present during the rest of the year.
There is a suggestion of waves of reproductive activity during the breeding sea-
son, in the data presented in Figure 3 and Table I. This appears most clearly in
Table I in the column showing the percentage of animals which are carrying
REPRODUCTION IX MITELLA POLYMERUS
175
TABI.K I
variation in egg size, seminal vesicle width, and stage of development of
brooded embryos in M. polymer us
Dates
1956
Oct.
Nov.
30
1957
Jan.
Feb.
11
Mar.
11
Apr.
18
May
18
Jun.
28
Jul.
24
Sept.
5
Nov.
7
Dec.
5
I95X
Jan.
31
19
8
Xo. of animals examined :
12
14
10
15
10
22
25
13
15
15
12
10
18
Xo. and per cent of
animals \vith :
Eggs absent
No.
3
2
1
4
1
—
—
—
—
—
—
—
—
, %
25
14
10
27
10
—
—
—
—
—
—
Small eggs
Xo.
3
9
9
11
9
11
5
1
1
2
—
4
9
%
25
65
90
73
90
50
20
8
7
13
—
40
SO
Medium eggs
No.
—
3
—
—
—
9
10
6
3
2
8
4
9
%
—
21
—
—
—
41
40
46
20
13
67
40
50
Large eggs
Xo.
6
—
—
—
—
—
6
5
3
1
—
1
—
%
50
—
—
—
—
—
24
38
20
7
—
10
—
Both small
and
Large eggs
Xo.
—
—
—
—
—
2
4
1
8
10
4
1
—
%
—
—
—
—
—
9
16
8
53
67
33
10
—
Xo. and per cent of ani-
mals with ovigerous
lamellae :
Xo.
4
6
—
—
—
1
13
9
8
10
5
7
2
%
33
43
—
—
—
5
52
69
53
67
42
70
11
X'o. and per cent of ani-
mals with ovigerous
lamellae bearing :
Early embryos
No.
1
—
—
—
—
1
5
3
1
2
—
—
—
%
8
—
—
—
—
5
20
23
7
13.5
—
—
—
Middle embrvos
No.
—
3
—
—
—
—
5
4
—
2
2
5
1
%
—
21.5
—
—
—
—
20
31
—
13.5
17
50
5.5
Late embrvos
No.
3
3
—
—
—
—
3
2
7
6
3
2
1
Of
/o
25
21.5
—
—
—
—
12
15
46
40
25
20
5.5
Conditions of seminal
vesicles present in
the population
XTo. of animals examined :
12
13
18
15
10
21
23
13
14
14
12
10
18
X'o. of animals with
seminal vesicles in
following conditions :
Apparently empty
1
1
1
4
3
—
—
—
—
—
—
—
2
Width 1-2 mm.
5
6
5
9
7
4
1
2
2
5
4
9
7
\\ 'idth 2-3.8 mm.
6
6
12
2
17 22
11 12 i ')
8 1
9
176
GALEN HOWARD HILGARD
MONTEREY BAY, CALIF, o
1956 '957 1958
Oct. Nov. Deo, Jan. Felj. Mar. Apr. May Tun. JuC. Au.g, Sept. Oct, Nov. Dec. Jem
17
Sdore temperatures,
average everj 72,c£ay5
at: 14
Hopkins Marine Station,
Mean(o)nnc( extreme (.)
wi'cttks oj seminal vestcUs
in 5a.-m.ptes of farae
taken at intervals
tkrouodoat tke jecu%
(•mm., or empty)
No, of ani-ma.ls examined •-
10
io
'3 H-
14-
11 10
Percentages of ianipies
erf Carge M,po(ymerixs
700
ovujerows Caweffae
at" various times
ctxnVtq t At year
No. of anima
10 15
'J '5
li (0
M.SPINOSU5)
NEWZEAiAND
Numfcer of Reproductive
siie barnacles ^
(M, spinosus)
carrying ovigemvis (ameffae JQ
out of 5 ampte oj1 25
(Data from Batfumij 1945) 5
r '
Surjace sea. temperatu.re5^
naont^i(v averages from
ii> j.OJ
•weekly readmos on
the eastern coast of
Otaqo Penninsuta-j
(C?)
Oct Nov. Dec, Jan. Feb. Mar Apr. May Jun, Ju[, Aiy. Sept, Oct- Nov. Dec,
Dcxte
FIGURE 4. Temperature and reproductive activity in M. polymerus
in Monterey Bay and in M. spinosus in New Zealand.
REPRODUCTION IX MITELLA POLYMERUS
177
ovigerous lamellae at different times during the year. The highest values, i.e.,
those for June, September, and December (69%, 67%, and 70%, respectively),
alternate with the lower values shown for May, July, and November (53%, 53%,
and 42%, respectively), and therefore suggest that the population as a whole may
be reproducing in waves, more or less simultaneously. As will be indicated later,
this phenomenon is very likely related to the successive waves of reproductive
activity shown by individual barnacles during the breeding season. However, not
all individuals start to reproduce at precisely the same time, and waves of activity
do not proceed in all individuals at exactly the same rate. The differences between
alternate highs and lows and their deviations from the mean for the population
are not statistically significant ; and a larger sample might be expected to yield a
smoother plateau for reproductive activity of the population.
THE REPRODUCTIVE CYCLE IN INDIVIDUALS
The reproductive cycle in the population of M. [>oly merits represents a summa-
tion of the reproductive processes in individual barnacles. The animals are
hermaphroditic and ovoviviparous, brooding their young for a period of time in
the mantle cavity. It is accordingly of interest to examine the relative degrees
of development of the two gonads in individual animals, and to relate the ovarian
egg size with the stage of development of brooded embryos present simultaneously
in individual barnacles.
Ovcm'cm egg size classes'
small -medium large
55.
Percent of rndivtdixaLs
less titan
1 mm,
in cC{a.nt.
sperm.
1-itnw./
in cCt'am,
Sjjerm
Z-3.Xrn.ru
m cCiarvt .
julC of
sperm.
10
FIGURE 5. Egg sizes and seminal vesicle widths found simultaneously in large individuals.
Left-hand side : numbers within the blocks represent numbers of individuals examined ; right-
hand side : numbers above blocks represent percentages of individuals with seminal vesicles
of given sizes.
178
GALKX HOWARD HIUIAKD
For studies of tluj two gonads, the large animals taken at intervals throughout
the study period were examined. Animals were grouped into four categories
according to egg size : animals with small eggs, those with medium eggs, those with
large eggs, and those with hoth small and large eggs. The seminal vesicle width
of each animal of a given egg category was noted. The data for all animals
examined are shown in Figure 5.
In Figure 5, the bars on the right hand side represent the frequency of occur-
rence of the various conditions of the seminal vesicle which may be found in indi-
TAHI.K II
Cm-relation of size of ovarian egg with developmental stage of brooded embryos present in the
same individual. Numbers of individuals showing each combination of conditions are
indicated bv numbers within the squares. Heavy vertical lines enclose
the "peak" months of the breeding season.
Kgg
size
Embryos
31
Oct.
30
Nov.
14
Jan.
1 1
Feb.
11
Mar.
18
Apr.
IX
May
28
June
24
July
5
Sept.
7
Nov.
5
Dec.
8
Jan.
Absent
2
4
1
4
6
3
1
Small &
Early
Large
Middle
Late
5
4
1
Absent
2
5
9
11
9
11
1
o
Small
Early
Middle
2
4
1
1
1
1
3
Late
]
2
1
1
Absent
1
9
3
4
1
8
Medium
Early
Middle
1
1
4
1
4
1
1
2
2
1
Late
1
2
1
1
1
2
1
Absent
4
5
4
2
1
Large
Early
Middle
Late
2
1
1
1
1
Absent
3
2
1
3
1
Eggs
Early
Absent
Middle
Late
12
14
10
14
10
22
25
13
15
15
12
10
18
viduals with eggs of any given size category. We see from the graph that sperm
is absent only in animals where eggs are small or absent. Under all other ovarian
conditions, sperm is always present, and in the majority of barnacles with medium
or large eggs, the seminal vesicles are full.
Table II contains data illustrating the conditions which may occur simul-
taneously in the ovary and in the brooded ovigerous lamellae, in the same individual
at different times of the year. From these data, the information on large animals
collected during the breeding season (October to November, 1956; and May, 1957
REPRODUCTION IX MITHU.A POLYMERUS
179
to January, 1958) and containing both ovarian eggs and ovigerous lamellae, has
been selected and is shown graphically in Figure 6.
It is evident from Figure 6 that there is a general relationship between ovarian
egg size and brooded embryo stage in the same individual, particularly during the
height of the breeding season. In general, where eggs are small in the ovaries,
embryos are in an early stage of development in the mantle cavity ; where eggs
are medium-sized in the ovaries, embryos are in the middle stage of development in
the mantle cavity: and where eggs in the ovaries are large (nearly ready to be
extruded), embryos in the mantle cavity are advanced (nearlv ready to be lib-
erated). The parallel diagonal lines drawn through successive peak conditions
show clearly the synchronized pattern of development of ovaries and embryos
during the peak months of the reproductive season. Certainly such a condition,
in which eggs and embryos show more or less parallel development, appears to
No.of i'ndi-
victuals ex-
«.7fnrud •.
Developmental stage
, of, e-m&rjos
in ovtcjerous (ameuac ;
FIGURE 6. Developmental stages, within individuals, of ovarian eggs and brooded embryos,
where both are present simultaneously. Numbers pointing to black bars show percentages of
given stages present during the "peak" months of the reproductive season ; numbers within the
larger bars show percentages of given stages present during the total reproductive season.
represent the most efficient timing pattern for an ovoviviparous organism which
produces successive broods of young in a single breeding season. However, it
is clear from the graph and the table that precise coordination between rates of
egg enlargement and larval development does not exist for all animals examined
during the reproductive season. For example, some animals with small eggs in
the ovaries were found brooding middle or late stage embryos. In such cases, it
appears that the interval between successive broods of larvae is greater than in
cases where eggs and embryos develop in phase, and that ovarian egg development
is retarded with respect to embryonic development. Table II shows clearly that
these animals with relatively retarded ovarian development tend to be localized
in time, predominating during the later months and not the peak months of the
reproductive season. Thus, the five cases in which late embryos accompany small
180
GALEN HOWARD HILGARD
ovarian eggs occurred in October and November of 1956, December of 1957, and
January of 1958, suggesting that at this time development of the ovaries had
slowed down or stopped for the season, and that the final batch of nauplii would
soon have been shed. Also, the combination of small eggs with middle embryonic
stages occurred, with one exception, either at the beginning (May, 1957) or near
the end (November, 1956 and December, 1957) of the breeding season. Animals
with medium eggs and no ovigerous lamellae occurred in April and May, 1957
(when they were presumably giving rise to their first batch of eggs of the season)
and in November, 1956 and 1957 (when they may represent individuals in which
the ovarian growth has slowed down toward the close of the season). In con-
trast, a few animals contained medium-sized eggs along with early embryos in the
ovigerous lamellae, suggesting a relative acceleration of egg development. How-
ever, in all three cases, the eggs measured fell close to the lower size limits of the
medium egg size class, and the apparent acceleration is exaggerated by the position-
ing of size-class limits in the grouping of data.
TABLE III
Apparent conditions of ovaries (ovarian eggs) with relation to embryos
(found simultaneously in an individual)
Dates
Number of times ovaries appeared in following conditions:
Accelerated
Synchronized
Retarded
31 Oct. 1956
—
2
1
30 Nov. 1956
—
1
5
14 Jan. 1957
11 Feb. 1957
11 Mar. 1957
no reproductive activity
18 Apr. 1957
18 Mav 1957
1
9
3
28 fun. 1957
1
6
1
24Jul. 1957
5 Sept. 1957
7 Nov. 1957
1
6
7
3
1
2
2
5 Dec. 1957
—
2
5
8 Jan. 1958
1
1
Table III summarizes the condition of the ovaries with relation to embryonic
stages. We see that during the early and middle months of the reproductive sea-
son, most of the animals examined appeared to show synchrony in rates of ovarian
and embryonic development. Toward the end of the reproductive season, a
relative lag in ovarian egg development became noticeable. By combining data
for the months of May through September (the main reproductive period ) and for
the period of October through December (the end of the breeding season), we
can show that the prevalence of animals with synchronized ovaries during the
former months, and of animals with retarded ovaries during the latter months,
is indeed statistically significant.
REPRODUCTION IX MITELLA POLYMERUS 181
While animals with apparently well synchronized brood development are prob-
ably producing batches of eggs at a relatively efficient rate, there is evidence that
even in such animals some time does elapse between broods. We might expect,
in a perfectly synchronized animal, that immediately following the liberation of
a batch of larvae, a second batch of eggs would be extruded, and that such an
animal would be carrying ovigerous lamellae virtually all of the time. However,
at no time during the reproductive period were all reproductively mature members
of the population carrying ovigerous lamellae ; even during the height of the
breeding season, only 50% to 60% of the population were brooding embryos.
The occurrence of a very few slow developers during this time could hardly account
for such a discrepancy ; and it must be assumed that even during the height of
reproduction, some time does elapse between the hatching of a batch of larvae and
the extrusion of the next batch of eggs from the same individual.
NUMBER OF BROODS AND LARVAE LIBERATED BY MITELLA POLYMERUS,
AND ITS RELATIVE FECUNDITY
No direct observations are available on the number of broods produced by an
adult barnacle per year. However, by combining all the lines of evidence at hand,
we can arrive at a hypothetical figure for the number of batches of larvae liberated
by a large individual during one reproductive season. Three lines of evidence are
considered here : the duration of the reproductive season, the rate of development
of embryos in the ovigerous lamellae, and the developmental stages of eggs and
embryos present at any one time in an individual during the reproductive season.
We have already seen that the reproductive period for most barnacles extends
through about eight months or about 240 days (Fig. 3). The approximate dura-
tion of the lamellar brood period was determined in the laboratory. Several
batches of larvae were raised /;/ vitro at 13° C., starting with what appeared to be
the two-cell stage, and continuing to hatching. Periodic examination of these
ovigerous lamellae showed that embryos remain in the "early" stage for the first
nine days, are in the "middle" stage from the tenth through the fifteenth day, and
remain in the "late" stage from the sixteenth day to hatching, which occurs on the
twenty-ninth to the thirty-first day. Development from fertilization to hatching
of the nauplius thus averages about thirty days. This rate is substantiated for field
conditions in Figure 3, where we find the first large eggs being extruded in April
and the first late embryo just one month later. It is interesting to note that the
brood period for "normal" M. spinosus, raised under conditions where tempera-
ture was not controlled but averaged 14° C. to 15° C., was thirty to thirty-two
days (Batham, 1946).
The developmental stages present simultaneously in individuals have been shown
in Figure 6 and Table II. Here, the simultaneous presence in single animals of
three stages (small eggs, large eggs, and late embryos, especially in animals taken
in July and September) strongly suggests that an animal may give rise to at least
three batches of embryos during one reproductive season. Tables II and III show
that during the first six months of the reproductive season, egg and embryo stages
tended to be more or less synchronized in individuals ; and during the last two
months of the breeding season, the majority of animals carried broods which tended
to be out of phase by two stages. In a perfectly synchronized animal, in which
GALEN HOWARD HILGARD
ovarian eggs and lamellar embryos develop in phase, we might assume that the
interval between the average small egg and the average early embryo is equal to
the lamellar brood period, or thirty days. In animals one step out of phase, that
is, in animals in which medium embryos accompany small eggs, or late embryos
accompany medium eggs, we might assume that the interval between broods is
thirty-eight days (or thirty days plus the difference between the average age of an
early embryo and the average age of a middle stage embryo — eight days). In
animals two steps out of phase, in which late embryos accompany small eggs, or
(specifically in November, 1957) no embryos accompany medium eggs, we might
assume that the interval between successive broods is at least 48.5 days (or thirty
days plus the difference between the average age of an early embryo and the average
age of a late embryo — 18.5 days).
Through the breeding season (eight months), these various intervals were
found to be more or less localized in time. That is, for approximately the first
six months, most of the population seemed to be producing broods either in phase
or one stage out of phase. Hence, at the assumed rate of one brood for every
thirty to thirty-eight days, a minimum of 4.7 broods and a maximum of six broods
could have been extruded by an individual during this time. For the last two
reproductive months, a majority of the population carried broods two steps out of
phase. At the assumed rate, then, of one brood for every 48.5 days, one to 1.2
broods may have been extruded by an individual during these two months. During
the TOTAL reproductive period, then, it appears that a single large barnacle could
have liberated from five to 7.2 broods of larvae.
If an average animal gives rise to six broods in a season, each brood developing
in the mantle cavity for thirty days, we would expect such an animal to be carrying-
embryos for approximately 180 days out of the total 240 days, or approximately
7S% of the total time. Since no single individual was followed during the breed-
ing season, no data are available to provide a direct means of checking this figure.
However, 59% of the large individuals collected during the eight-month repro-
ductive season bore ovigerous lamellae. This suggests that perhaps large indi-
viduals contain ovigerous lamellae for only about 59c/c of the total reproductive
period, or 142 out of 240 days. On this basis, then, the average animal probably
produced only four or five broods during the season.
There seem to be two good reasons for the discrepancy between the two esti-
mates for average number of broods per season. The first, already mentioned,
is that even in well-synchronized animals, some time did elapse between successive
lamellar broods. There was no direct measurement of the duration of this mini-
mum interval, and thirty days was used as the minimum time for the enlargement
and extrusion of eggs ; but actually, the period during which eggs remain in the
<>vary is probably a few days longer. A suggestion providing some independent
support for this is seen in the data in Figure 3, where on March 11, 1957, all
ovarian eggs were in the small size class (though some approached the upper
limits of this class), and on April 18, 1957, 36 days later, one individual was just
extruding eggs from the oviduct. The other possible reason for the discrepancy be-
tween the two estimates of number of broods produced per year is shown by the fact
that many stages of development were found in the population at any one time, and
that the whole population did not produce broods synchronously. Ft is probable
REPRODUCTION IX MITELLA POLYMERUS
1ol\inerus may
liberate from 104.000 to 240,000 larvae from a single brood contained in one pair
of ovigerous lamellae ; the slightly smaller M. spinosus liberates approximately
3000 larvae per brood (Batham, 1944-45). It appears that a single adult M.
polyments may produce roughly 52 to 280 times as many larvae per year as a
single adult M. s
100
§>
TptaC no,
in eac k
sarnpie
excLJan. '.
Con,tro(5
(adjacent)
Distance from nearest neighbor of a.
reproductive sizx :
2."-4" 4.1- fe" fc.7-8" 8.J-/0" 70.f"-74"
55
29
30
32.
25
27
FIGURE 7. Summary graph showing percentages of large ,17. pnlyincnis containing ovigerous
lamellae when separated from other large specimens by given distances.
SELF-FERTILIZATION
For a study of self-fertilization, a series of large and relatively isolated barnacles,
situated at various measured distances from their nearest neighbors of reproduc-
tive size, were collected and inspected for the presence of ovigerous lamellae.
These samples were collected at different times throughout the reproductive sea-
son. The data, grouped for the months of collection, and for the distances by
which the sexually mature barnacles were separated, are presented in Table IV
and Figure 7. It can be seen that over 509r of the control animals for each month
(except January, 1960) carried ovigerous lamellae; animals separated by two to
four inches appeared to carry ovigerous lamellae less frequently, but the differences
are not statistically significant. There is a statistically significant drop in the
presence of embryos in animals separated from their nearest neighbor of a repro-
ductive size by over four inches, and an absence of embryos in animals separated
by more than eight inches. Thus it appears that eight inches was the maximum
distance over which copulation of large animals collected could take place, and
that animals separated by greater distances failed to receive sperm. Despite the
184
GALEN HOWARD HILGARD
TABLE IV
Numbers of barnacles of reproductive size isolated from other such barnacles by given distan<-< \,
showing presence ( +) and absence ( — ) of ovigerous lamellae
Date
Distance from nearest neighbor of reproductive size:
Controls
(adjacent)
2"-4"
4"-6"
6"-8"
S"-10"
10"-14"
+
+
!
+
+
+
1958
12 8
3 2
1 3
0 5
0 2
0 8
1959
20 July
7 Aug.
2 Nov.
10 5
6 4
4 (i
3 3
2 8
2 6
1 6
0 10
1 8
1 6
0 12
0 8
0 8
0 7
0 6
0 3
0 6
0 10
1960
2 Jan.
0 30
0 8
0 6
0 5
0 8
— —
Summary,
excluding
Jan. I960
32 23
10 19
3 27
1 31
0 23
0 27
simultaneous maturity of male and female gonads, and despite the evidence for
self-fertilization in other barnacle species (Barnes and Crisp, 1956), self-fertiliza-
tion apparently does not take place in M. polymerus. It also appears that formation
of ovigerous lamellae does not occur in the absence of fertilization.
DISCUSSION
On the subject of reproduction in cirripeds, a good deal has been written.
Some accounts, such as those of Darwin (1851), Broch (1922), and others are
concerned with the questions of hermaphroditism, the existence of complemental
males, etc., rather than reproductive anatomy, reproductive cycles, and fecundity.
Comparison of the seasonal reproductive cycle of M . polywierus in Monterey
Bay with that of M. spinosits on the New Zealand coast (Batham, 1944—45) shows
some interesting features. Figure 4 summarizes reproductive and temperature
data for the two species. In Monterey Bay, from November, 1956, through the
greater part of April, 1957, shore temperatures remained between 11.2° C. and
12.4° C., but showed a definite rise toward 13° C. during February and March.
From May, 1957, through about one-half of November, shore temperatures re-
mained above 13° C., rising to over 14° C. in May, July, and August, and to above
16° C. during September and October. Thus, the year may be roughly divided
into the colder winter months and the warmer spring, summer, and fall months.
In M. polymerus, the increase in mean seminal vesicle width during the early
spring roughly parallels the rise in shore temperature ; the decrease in mean seminal
vesicle width in the winter follows roughly the winter decrease in temperature.
Likewise, the earliest occurrence of embryos follows closely behind the seasonal
rise in temperature, and embryos continue to be present in the population until just
after temperatures begin to drop during the winter. There is thus a clear corre-
spondence between reproduction and shore temperature. The three peaks seen
REPRODUCTION IN MITELLA POLYMERUS 1S5
in the occurrence of embryos in the latter half of 1957, while not statistically sig-
nificant, show a relationship with the temperature peaks of May, July, and Sep-
tember which is perhaps suggestive.
The data given by Batham (1944-45) for M. spinosns, from the open coast
of Otago Peninsula, New Zealand, and the corresponding temperature curve (from
two later years and a neighboring vicinity ; Batham, 1958) show a similar relation-
ship between temperature and reproductive activity. As might be expected for
a related species occurring at a slightly higher latitude but in the southern hemi-
sphere, the reproductive cycle is a rough mirror image of that of M. polymer us in
Monterey Bay, California, though the breeding season is somewhat shorter.
Other barnacle species have been observed to be reproductively active pri-
marily during the months of warmer water temperatures : Balanus crcnatus, studied
on the Atlantic coast of Canada (Bousfield, 1952-53) and in San Francisco Bay.
California (Herz, 1933) ; Balanus improrisus, studied on the Atlantic coast of
Canada (Bousfield. 1952-53) ; and Chthainalus stellatus, studied in Great Britain
(Crisp, 1950).
While such a correlation between temperature and reproductive activity might
seem obvious and only reasonable, a number of barnacles reproduce primarily when
water temperatures are low or at a minimum. This group includes the following :
Balanus balanoides, studied on the Atlantic coast of Canada (Bousfield, 1952-53),
at Woods Hole, Massachusetts (Fish, 1925), and in Great Britain (Moore, 1935;
Crisp and Patel, 1960) ; Balanus haincri, studied on the Atlantic coast of Canada
(Bousfield, 1952-53), and in Great Britain (Crisp, 1954) ; Balanus porcatus,
studied in Great Britain (Crisp, 1954) ; and Balanus glandula, studied at Van-
couver, B. C., and La Jolla, California (Barnes and Barnes, 1956) and in San
Francisco Bay, California (Herz. 1933).
There is evidence that still another group of barnacles reproduce (perhaps with
some variation in rate) throughout the entire year. These include Elminius
modest us, studied in Great Britain (Crisp and Davies, 1955), Verruca stroemia,
studied in Great Britain ( Pyefinch. 1948), and Balanus tintinabulum, observed at
La Jolla, California (Coe, 1932 J.
Bousfield (1952-53) has studied the distribution and spawning seasons of the
barnacles of the Atlantic coast of Canada, and reviewed the evidence supporting
temperature as a principal factor governing reproduction in cirripeds. He clearly
indicated that there is variability in reproductive period within a given species at
different latitudes within its geographic range, and showed that reproductive
activity tended to occur at times when temperatures were similar, regardless of
latitude.
The relationship of temperature and food supply to rate of reproduction has
been studied by Crisp and Davies (1955) in the barnacle Elminius modestus.
By growing these barnacles on glass slides and observing development through the
translucent bases, these workers were able to follow, in i'n'0, the development of
both ovarian eggs and lamellar embryos. They found that (p. 379) "the time
interval occupied by successive broods varies among individuals, and with the
season of the year. Rate of development of embryos seems to be a function of
temperature alone, but regeneration of the ovary depends on nutrition and food
supply." Crisp (1959), working with Balanus balanoides, showed that the rates
(iAI.KX HOWARD HIi.CARb
of development of the early embryonic stages (through the limb bud stage) are
temperature-dependent up to 12°-14° C., but that the later stages vary little in
rate of development between 3 C. and 12° C.
Further points of comparison may be brought out between M. polymer us, M.
sphwsus, and other barnacles. The present study indicated that sexual maturity
is not necessarily a function of size of the animals alone. Results of studies on
.17. spinosus and litininnts modestus showed similarly that in populations of
Miialler barnacles, sexually mature individuals are found, but less frequently than
in populations of larger barnacles.
Self-fertilization apparently does not occur in large isolated individuals of M.
polynicrns, and cross-fertilization appears to be the rule. Crisp (1954) and Crisp
and Patel (I960) pointed out that cross-fertilization also appears obligatory in
B. crenatiis, Elmiiihts inodcstns, and in B. balanoidcs. However, self-fertilization
very probably can occur in at least three species of acorn barnacles. Barnes and
Crisp (1956) experimentally isolated individuals of Chthamalns stellatus, Verruca
strucnria, and Balaniis pcrjoratns and found that they frequently produced ovigerous
lamellae. They also observed that fertilized eggs found in such isolated individuals
are frequently less viable than cross-fertilized eggs. The genetic advantage of
cross-fertilization is well known, and it appears that the Mitclla polymerus popu-
lation, composed usually of closely-packed individuals, is well adapted for cross-
fertilization.
SUMMARY
1. The gross structure of the reproductive system of M. poly merits is de-
scribed and compared with that of the southern hemisphere species, M, spinosus
(studied by Batham, 1944-45).
2. Size and reproductive activity in M. polymerus are related. All animals
over 27.5 mm. in breadth (distance from rostrum to carinaj are found to repro-
duce ; smaller animals arc- found to contain developing embryos less frequently.
Xo sexually mature animals less than 17.2 mm. in breadth were found.
3. A fifteen-month study of the reproductive cycle in the population is de-
scribed. Reproductive activity is evident during the greater part of the year.
For the year of 1957, developing embryos were present in the population for a
period of eight months during which time the shore temperature ranged from
12.3° C. to 17° C. The reproductive season for the southern hemisphere species
likewise occurs during the warmest months ; thus the yearly cycle of M. polymerus
shows a perhaps expected mirror image of the situation occurring in the southern
hemisphere.
4. Within individuals, male and female gonads mature at approximately the
same time during the year, and during the greater part of the year, an individual
contains both developing eggs and seminal vesicles full of sperm.
5. Stages of development of ovarian eggs and brooded embryos found simul-
taneously in individuals are compared. During the early and middle months of the
reproductive season, ovarian eggs and brooded embryos tend to be in similar stages
of development (that is, small eggs are found with early stage embryos, large eggs
with late stage embryos, etc.). During the later reproductive months, a relative
lag in ovarian egg development is evident.
REPRODUCTION* IX MITEI.LA POLVMERUS 1itro under controlled temperatures. The em-
bryos took an average time of thirty days for development from fertilized egg to
free-swimming larva.
7. Estimates are given of the number of broods of young and the number
of larvae liberated by a large individual during a year, and these are compared
with the results of Batham (1944-45) for <\L spinosus. Studies of the larval
brood period, the stages of eggs and embryos found simultaneously in individuals,
and the length of the reproductive season allow an hypothesis that five to seven
broods may be liberated by a large individual during a year. Three to four broods
appears more probable for an average large animal. A pair of ovigerous lamellae
may contain from 104,000 to 240,000 larvae. Batham's data (1944-45) showed
that probably two broods, each containing approximately 3000 larvae, are liberated
by M. spinosus during a year. Thus M . polymer us may liberate from 52 to 280
times as many larvae per year as a single large M. spinosus.
8. The possibility of self-fertilization in M. polymerus is studied. Relatively
isolated large animals are found to carry ovigerous lamellae less frequently than
those adjacent to each other ; and large animals isolated from each other by over
eight inches were never found carrying embryos. From this evidence, it appears
that self-fertilization does not occur in this species, and that cross-fertilization is
necessary for the formation of ovigerous lamellae.
LITERATURE CITED
BARNES, H., AND M. BARNES, 1956. The general biology of Balanus f/Iandula Darwin. Pac.
Set., 10(4) : 415-430.
BARNES, H., AND D. J. CRISP, 1956. Evidence of self-fertilization in certain species of bar-
nacles. /. Mar. Biol. Assoc., 35: 631-639.
BATHAM, E. J., 1944-45. Pollicipcs spinosus Quoy and Gaimard. I. Notes on biology and
anatomy of the adult barnacle. Trans. Roy. Soc. Nczi1 Zealand, 74: 359-374.
BATHAM, E. J., 1946. Pollicipcs spinosus Quoy and Gaimard, II. Embryonic and larval
development. Trans. Roy. Soc. JVYrc1 Zealand, 75: 405-418.
BATHAM, E. J., 1958. Ecology of Southern New Zealand exposed rocky shore at Little
Papanui, Otago Peninsula. Trans. Roy Soc. Nezv Zealand, 85 (Part 4) : 647-658.
BOUSFIELD, E. L., 1952-53. The distribution and spawning seasons of barnacles on the Atlantic
coast of Canada. Ann. Rep. Nat. Mus. Canada, Bull. No. 132: 112-154.
BROCH, H., 1922. Studies on Pacific cirripeds. I' id. Mccld. Dansk Naturli. Foren. Kobenhavn.,
73: 215-358.
COE, W. R., 1932. Season of attachment and rate of growth of sedentary marine organisms
at the pier of the Scripps Institution of Oceanography, La Jolla, California. Univ.
of California Press, Berkeley.
CORNWALL, I. E., 1925. A review of the Cirripedia of the coast of British Columbia with a
glossary, and key to genera and species. Contr. Lanad. Biol., 2: 469-502.
CRISP, D. J., 1950. Breeding and distribution of Chthainalns stcllatus. Nature, 166: 311-312.
CRIST, D. J., 1954. The breeding of Balannx porcatus (da Costa) in the Irish Sea. /. Mar.
Biol. Assoc.. 33: 473-494.
CRISP, D. J., 1959. The rate of development of Balanus balanoidcs (L.) embryos in ritro.
J. Anini. Ecol, 28: 119-132.
CRISP, D. J., AND P. A. DAVIES, 1955. Observations in rit'n on the breeding of Elininins
modcstns grown on glass slides. /. Mar. Biol. Assoc., 34: 357-380.
CRISP, D. J., AND B. S. PATEL, 1960. The moulting cycle in Balanus balanoides (L.). Biol.
Bull., 118: 31-47.
INS GALEN HOWARD HILGARD
DARWIX, C., 1851. A Monograph on the Subclass Cirripedia. I. Lcpaclidae. Ray Society,
London.
FISH, C. J., 1925. -Seasonal distribution of the plankton of the Woods Hole region. Bull.
U. S. Bur. Fish.. 41: 91-179.
i IKKZ, L. E., 1933. The culture and morphology of the later stages of Balanus crenatus. Ph.D.
thesis, Stanford University. (Published in part, in Biol. Bui!., 64: 432-442, 1933.)
MOORE, H. B., 1935. The biology of Balanus balanoides. III. The soft parts. /. Mar.
Biol. Assoc., 20: 263-274.
NUSSBAUM, M., 1890. Anatomische Studien an Californischen Cirripedien. Bonn, Germany.
PYEFINCH, K. A., 1948. Notes on the biology of cirripedes. /. Afar. Biol. Assoc., 27: 464-503.
RICKETTS, E. F., AND J. CALVIN, 1952. Between Pacific Tides. Stanford Univ. Press, Stan-
ford, Calif.
OBSERVATIONS ON THE NUTRITION OF THE RHYNCHOCOELAN
LINEUS RUBER (O. F. MULLER)
J. B. JENNINGS
Department of Zoology, University of Leeds, England
Feeding and digestion in the Rhynchocoela have received relatively little
attention apart from brief accounts by \Yilson (1900), Reisinger (1926), Coe
(1943) and Hyman (1951). These indicate that the group is carnivorous, preying
upon a variety of invertebrates which are captured by means of the extensible
proboscis and swallowed whole, and that digestion may be either extracellular or
partially intracellular. No further details of rhynchocoel nutrition are available
and to remedy this deficiency, that of the common British species Linens rnber
(O. F. Miiller) has been investigated.
MATERIALS AND METHODS
Specimens of Linens ntbcr were collected from beneath rocks embedded in sand
at mid-tide level at Cremyll, Plymouth. After starvation for two days to induce
a readiness to feed and to clear the gut of remnants of previous meals, individuals
were presented with representatives of the fauna of their habitat, and the methods
of capture and ingestion of the selected prey observed.
The course of digestion was followed by histological examination of individuals
fixed at progressive intervals after feeding upon either the natural food or easily
identifiable test foods such as frog erythrocytes and raw, optically active starch
grains. Fixation was in Susa at 30° C. and sections cut at 8 /* were stained \vith
the haematoxylin and eosin. Feulgen, periodic acid-Schiff (P.A.S.), Alcian blue
(for mucin), benzidine-peroxide (for haemoglobin), and Lugol's iodine techniques.
Changes in the pH of the gut contents during digestion were followed by feeding
particles of fish muscle stained with 0.5% sea water solutions of various indicators
and observing subsequent color changes by periodically flattening the fed indi-
viduals and examining by both reflected and transmitted light.
Food reserves were studied after fixation in Flamming (for fats) and 90%
alcohol containing \% picric acid (for carbohydrates and proteins). Sections of
individuals fixed in the latter reagent were stained by the Best's carmine, P.A.S.
and modified Millon's methods.
OBSERVATIONS
The food and feeding mechanism
Linens ruber feeds mainly on small annelids and crustaceans but particles of
any dead organic material will be taken, providing it is not too decomposed. The
oligochaete Clitellio arcnarins was particularly common in the habitat and at the
time of collection (July-August) appeared to form the bulk of the food.
189
J. 15. JKXXIXCS
Living prey is detected by the eye-spots and a starved Linens will respond to
animals moving within 2 cm. of the head. Dead or injured animals emitting de-
composition products or body fluids can be located at greater- distances and here
it is presumably chemoreceptors in the cephalic ciliated grooves which are stimu-
lated. Detection of living prey is followed by immediate everMon of the proboscis1
through the proboscis pore at the anterior tip of the bod}-, and this occurs with such
explosive force that as the proboscis strikes the prey it becomes coiled around it
in a tight spiral grip. It does not penetrate the prey, since it lacks stylets or
similar piercing organs, but the tightness of its grip may rupture the integument
and cause loss of body fluids or gut contents. The grip is aided by sticky secretions
from the proboscis epithelium and immediately it is secured the proboscis begins
to retract and draws the prey, usually struggling violently, back towards the mouth.
This lies ventrally 2-3 mm. behind the proboscis pore, and as the prey is drawn
towards it the body anterior to the mouth is raised and extended until it can grasp
the prey by curling downwards over it. This movement of the anterior tip of the
body continues downwards and backwards and forces the prey into the mouth
which gapes open to receive it. The proboscis then gradually relinquishes its
grip and withdraws into the rhynchocoel as movements of the mouth, aided by
contractions of the anterior body musculature, force the prey into the gut. Inges-
tion of small animals is completed in 15 to 20 seconds but with larger prey, such
as annelids one-third to one-half the length of the feeding individual, it may take as
long as 30 minutes, and in such cases the first part to be swallowed is partially
disintegrated before ingestion is complete. Small animals usually die within sec-
onds of entering the gut, but active errant polychaetes with armored jaws may
survive long enough either to force their way to the exterior through the gut and
body walls or propel themselves down the length of the gut to emerge unharmed
at the anus. This is particularly liable to happen when the prey is ingested head
first, but in the majority of cases the proboscis seizes an animal about its middle
and consequently draws it back to the mouth bent upon itself in the shape of
a U. It is then ingested in this form and is unable to escape from the gut before
being killed.
During capture and ingestion Linens extends to its fullest length and produces
copious sticky mucoid secretions from the ventral surface. This enables a firm
hold to be retained upon the substratum whilst dealing with the prey,, and even if
the latter is partially buried it can be drawn from its retreat and swallowed without
causing the Linens to shift position.
Inert masses of food, such as animal remains or the test foods used in this
investigation, do not stimulate eversion of the proboscis but are seized directly
by the mouth and swallowed piecemeal.
The structure of the c/nt
The gut consists of three histologically distinct regions, namely the mouth and
buccal cavity, the foregut, and the intestine. It runs the length of the body from
mouth to anus without coiling, is ciliated throughout and lacks both multicellular
lands and musculature.
1 Details of the proboscis and the mechanism of its eversion are given by Hyinan (1951)
and are not included here.
NUTRITION OF LINEUS RUBER 191
The mouth consists of a ventral subterminal invagination of the epidermis
some 200 //, deep and opening directly into the buccal cavity. It is fringed with
large cilia and externally has a lobed appearance due to folds in its walls which
allow expansion during ingestion. The invaginated epidermis contains acidophil,
P.A.S.- and Alcian blue-positive gland cells whose secretions probably facilitate
passage of food, and the entire mouth region is surrounded by concentrations of
similar sub-epidermal gland cells. The buccal cavity is lined by ciliated cuboidal
cells lO-12/i tall and these are backed by masses of acidophil and basophil gland
cells, the majority of which stain with Alcian blue and appear to have the same
function as those around the mouth. The walls of the cavity are much folded
and ascend diagonally backwards to become continuous with those of the foregut
beneath the proboscis sheath.
The foregut (Fig. 1) runs posteriorly for one-tenth the length of the animal
and its walls are considerably thickened, especially ventrally where the wall may
be up to 300 fj. in depth. They are throw-n into small simple folds and consist of
a single layer of ciliated cuboidal cells, 10-12 /i tall, lining the lumen and lying upon
acidophil syncytial tissue containing numerous gland cells, free nuclei and occasional
large lacunae. In the anterior portion the gland cells consist of P.A.S.- and Alcian
blue-positive acidophils and basophils in approximately equal amounts, together
with a number of others which are intensely basophilic but give no reaction to
Alcian blue. The proportion of the latter increases along the length of the fore-
gut to the median portion where all the gland cells are of this type. The gut wall
then gradually decreases in thickness and gland cell content as it nears the intestine
and terminates in a constriction (Fig. 1) separating the latter from the foregut.
The intestine is the longest part of the gut and runs from its junction with the
foregut in the anterior part of the body direct to the anus at the extreme posterior
end. It bears paired and serially repeated lateral pouches or caeca throughout its
length, apart from a short unpouched region immediately before the anus. The
intestinal wall or gastrodermis (Fig. 3) is made up of two types of cells arranged
in a single layer upon a thin basement membrane. The larger and more numerous
of these are attenuated columnar cells, 50-55 ^ tall and 8 /A wide, with granular
basophilic cytoplasm containing various acidophil inclusions and basal vesicular
nuclei. The free distal borders of the cells bear cilia which in unfed individuals
are of uniform appearance and size, but in the presence of digesting food the cilia
lose their uniformity and coalesce into pseudopodia-like processes which extend
out into the lumen (Fig. 5). This peculiar modification of the cilia is correlated
with entry of food material into the cells and is dealt with later.
The second type of cell found in the gastrodermis is glandular and occurs
between the bases of the columnar cells. These gland cells (Figs. 2 and 3) are
40-50 p. tall and 5-6 /A wide, unciliated and contain up to thirty acidophil pro-
teinaceous spheres, each 0.5 /A in diameter, which are discharged into the intestinal
lumen when food enters from the foregut. They are most numerous in the anterior
part of the intestine, where there may be as many as one gland cell to every three
columnar, but this ratio is graded down the length of the intestine to approximately
one in twenty in the middle region and one in fifty or more beyond until the gland
cells disappear, finally, in the short unpouched region before the anus.
192
T. B. 1ENN1N',-
* ^>^R
f&
"*^
FIGURE 1. Longitudinal section of Linens showing the posterior portion of the foregut
(left) and the constriction which separates this from the intestine (right). Haematoxylin and
eosin. Scale : 1 cm. = 50 AI.
FIGURE 2. ' Longitudinal section of the intestine in Linens showing part of a newly in-
gested Clitcllio lying intact and undamaged in the lumen. Acidophil gland cells are prominent
in the gastrodermis in the lower portion of the figure. Haematoxylin and eosin. Scale :
1 cm. = 100 M-
FIGURE 3. A portion of the gastrodermis in Linens showing ciliated columnar cells inter-
spersed with darker acidophil gland cells. Intestine empty. Haematoxylin and eosin. Scale :
1 cm. = 50 fj..
FIGURE 4. The gastrodermis in Linens 30 minutes after a meal of frog erythrocytes. The
intestinal lumen (top) contains a homogeneous mass of digested haemolyzed erythrocytes
which stains heavily with Feulgen and almost obscures the ciliary processes. The columnar
cells are loaded with engulfed spherical masses identical in nature with the contents of the
lumen. Feulgen and light green. Scale : 1 cm. = 50 /*.
FIGURE 5. The gastrodermis in Linens 30 minutes after a meal of raw starch grains.
The cilia have coalesced into pseudopodia-like processes and a few starch grains already engulfed
are ranged along the distal margins of the columnar cells. Lugol. Scale : 1 cm. = 50 ,u.
FIGURE 6. The gastrodermis in Linens 60 minutes after a meal of raw starch grains.
The columnar cells are loaded with grains, many of which are as yet unchanged and still
exhibit the characteristic black cross in polarized light. The lumen contains occasional free
grains and on the left portions of two gregarine trophozoites with prominent nuclei. Section
stained with haematoxylin and eosin and photographed by polarized light. Scale: 1 cm.
= 40^.
NUTRITION OF LINEUS RUBER I'M
The lateral pouches have the same structure as the rest of the intestine and arc
merely simple extensions to increase its area, not specialized digestive caeca.
Tlic course of digestion
Ingested food passes rapidly through the buccal cavity into the foregut where
it is held for a few seconds before its passage intact into the intestine (Fig. 2).
Living food usually dies during the brief pause in the foregut and this is due, no
doubt, to acid secretions from the numerous basophilic gland cells present here,
for when particles of fish muscle stained with bromo-cresol purple or chlor-phenol
red were fed, their pH value fell from 7.0 to 5.5 as they passed through the fore-
gut, and sections of newly fed individuals showed the majority of the glands to
be discharged.
There is no trituration or break-up of the food in the foregut but digestion
begins immediately it enters the intestine. A series of individuals fixed at inter-
vals after ingestion of the oligochaete Clitellio showed that as early as fifteen
minutes after feeding the gland cells had discharged their spheres and digestion
was well advanced. The Clitellio lay in the main median portion of the intestine
with the epidermis deeply eroded and the entire body starting to fragment. The
cilia of the columnar cells were still uniform in appearance and apparently creating
currents in the gut contents to distribute the fragmenting food, for pieces of tissue
were already passing into the lateral pouches. Digestion progressed rapidly with
time and 30 minutes after feeding the intestine contained a heterogeneous mass
of heavily eroded pieces of tissue, intact and fragmented setae, nephridia (which
resisted digestion for longer than other tissues and stood out from these with sur-
prising clarity) and diatoms, algal chains, etc. released from the oligochaete gut.
The gastrodermal cilia were now beginning to lose their uniformity and coales.ce
into pseudopodia-like processes stretching into the lumen of the intestine, whilst
semidigested material from the latter was appearing as acidophil spheres in the
distal portions of the columnar cells bearing these structures. These spheres passed
back deeper into the cells and their number increased rapidly with time. Sixty
minutes after feeding, the material in the lumen was almost homogeneous, with
setal fragments and diatom cases being the only recognizable elements in it, whilst
the columnar cells of the gastrodermis were packed with spheres of food under-
going intracellular digestion. The spheres decreased in size and affinity for stains
( especially the Millon reagent for protein) as they passed down the cells to dis-
appear finally in the basal region, the cells presumably then passing the products
of digestion to other tissues whilst taking up more semidigested material distally
until the lumen was emptied. This occurred some six hours after feeding and
the amount of intracellular material then rapidly decreased. After a further three
hours the columnar cells contained only a few acidophil inclusions, their cilia had
resumed their normal shape and size, and the gland cells were again full of en-
zymatic spheres. Indigestible residues were collected in the short unpouched region
of the intestine near the anus, being swept there, probably, by the reconstituted
cilia, and observations on living specimens showed that they were expelled even-
tually by a sudden contraction of the posterior body musculature.
A parallel series of feeding experiments, using fish muscle stained with indi-
cators, showed that the initial drop in pH as the food passes through the foregut
194 J. B. JENNINGS
is maintained in the intestine during digestion. In some cases it fell even further,
to pH 5.0, and when sufficient indicator-stained material entered the columnar cells
to be visible in neutral saline squashes, the intracellular digestion was seen to be
progressing in a similarly acid medium of pH 5.0-5.5.
It was not clear from the Clitellio-ied series how semidigested material enters
the columnar cells, but the pseudopodia-like appearance of the coalesced cilia and
spherical compact form of the material when within the cells suggested a form of
phagocytosis. This possibility was investigated by feeding Linens on frog eryth-
rocytes and raw, optically active starch grains made palatable by mixing with frog
plasma, to ascertain whether such discrete particles were in fact taken into the
columnar cells. In the series fed on erythrocytes, however, haemolysis occurred as
they entered the intestine, the break-up including the majority of the nuclei, and
30 minutes after feeding the lumen contained a semidigested mass which gave a
strong reaction with Feulgen, due to released nuclear materials, and with the
benzidine-peroxide reaction for haemoglobin. The cilia had coalesced into the
usual processes and many of the cells contained spherical masses with the same
staining properties as the material in the lumen (Fig. 4). These apparently
phagocytosed masses passed back into the cells as more appeared distally, and
gradually decreased in size and their reaction to Feulgen and benzidine-peroxide
as intracellular digestion progressed. Digestion of the blood meal was completed
in six hours and the intracellular spheres disappeared without leaving residues of
haematin or other insoluble pigments from the degradation of the haemoglobin.
Final confirmation of the occurrence of phagocytosis came from the series fed
on starch grains. Thirty minutes after feeding the cilia had formed filamentous
processes extending into the lumen, and a few intact grains, staining blue with
Lugol and still exhibiting the characteristic black cross in polarized light, had al-
ready been taken into the cells and were ranged along their distal margins (Fig. 5).
The number of such grains increased rapidly and 60 minutes after feeding packed
the columnar cells (Fig. 6). Only grains 5 ^ or less in diameter were engulfed
and larger ones remained in the lumen where they gradually lost their optical
activity, fragmented and stained brown with Lugol. The fragments then passed
into the cells and joined the previously engulfed grains which were undergoing
intracellular digestion, losing their identity and disappearing towards the bases
of the cells.
Parasites of tJie gut
Approximately 75 % of the Linens examined contained in the intestine an
acephaline eugregarine identified as Urospora nenicrtes (Koelliker). Trophozoites
(Fig. 6) 150-180 /j. long and 15-20 ^ wide, with basophil, P. A. S. -positive cyto-
plasm and prominent nuclei, occurred in all parts of the lumen, and the intracellular
stages, strikingly prominent with P.A.S., were common in the columnar cells. The
gregarine did not appear to harm Linens in any way, apart from a few occasions
when infected columnar cells reacted against developing intracellular stages and
caused them to degenerate into masses of yellowish brown crystals. Such cells
then burst, either hi situ or after being shed into the lumen, and the crystals were
eliminated with the faeces.
NUTRITION OF LINEUS RUBER 195
The food reserves
Fat forms the principal food reserve in Linens and occurs as intracellular glob-
ules up to 5 /x in diameter in the parenchyma and, to a lesser extent, in the columnar
cells of the intestine. There are no specific protein reserves and the only demon-
strable carbohydrate reserve is in the form of tiny granules of glycogen scattered
throughout the parenchyma, musculature, and columnar cells.
DISCUSSION
The main points of interest in the nutrition of Linens ruber lie in the feeding
mechanism and the digestive processes. In the case of the former a simple but
effective method of capturing the food, supplemented by slight modification of the
anterior portion of the alimentary canal into a thick-walled glandular foregut for its
reception and killing, enables this rhynchocoelan to prey successfully upon animals
far more active and elaborate than itself. In this respect Linens resembles the tur-
bellarian flatworms where similarly simple feeding mechanisms make available prey
ranging from protozoa to molluscs and tunicates (Jennings, 1957; 1959a). In the
Turbellaria it is the pharynx which forms the principal element of the feeding
mechanism and this organ is thus analogous in function to the rhynchocoelan pro-
boscis as seen in Linens. The only disadvantage apparent in the type of feeding
found in Linens is the possibility of escape by the prey before the secretions of the
foregut can take effect but this is overcome, no doubt, in those fhynchocoelans which
possess a proboscis armed with stylets and poison glands by killing or paralyzing
the prey at the moment of capture.
Digestion in Linens follows a pattern observed in other Acoelomata (Jennings,
1957; 1959b) in that both extracellular and intracellular processes are concerned,
but here the intestinal wall is ciliated and consequently the semidigested food would
be expected to enter by absorption. In fact, however, it enters by a form of
phagocytosis, as is proved beyond doubt by the appearance in the columnar cells
of starch grains which retain their form and optical activity after entry and so
must have passed into the cells as solid discrete particles. This method of taking-
material into the columnar cells involves temporary modifications in the form and
behavior of the cilia during the digestion of each meal, and the protoplasmic
pseudopodia-like processes formed from the coalescence of neighboring cilia are
probably concerned in the engulfing of semidigested food, although this has not been
observed histologically. The need for the intestine to be ciliated probably arises
from its length and the absence of musculature, which together create a need for
some method of distributing fragmenting food in the early stages of digestion and
collecting residues near the anus at the end. Contractions of the body musculature
appear to be insufficient for anything but the final expulsion of the collected resi-
dues and hence ciliary currents are used. The reason for the retention of phago-
cytic uptake of food under these conditions is unknown, for there is no apparent
reason why extracellular digestion should not be carried to a point where the semi-
digested food is soluble enough for absorption, and this presents an interesting
subject for further investigations.
196 J. B. JENNINGS
SUMMARY
1. The rhynchocoelan Linens ruhcr feeds on small annelids and crustaceans
which are captured by the unarmed proboscis and swallowed whole.
2. The alimentary canal is differentiated into three regions : a buccal cavity,
a foregut where the prey is killed by 'acid secretions, and an intestine where it
is digested.
3. Digestion is the result of both extracellular and intracellular processes and
occurs in an acid medium of pH 5.0-5.5. The enzymes responsible for the initial
extracellular breakdown come from gland cells in the intestinal wall and digestion
is completed within the columnar cells of the latter. Semidigested food enters
these columnar cells by a phagocytic process and this involves temporary modifi-
cations in the form and function of their cilia.
4. The food reserves consist of fat deposits in the parenchyma and, to a lesser
extent, in the columnar cells of the intestine.
LITERATURE CITED
COE, W. R., 1943. Biology of the nemerteans of the Atlantic coast of North America. Trans.
Conn. Acad. Arts Sci., 35: 129-328.
HVMAN, L. H., 1951. The Invertebrates. Vol. II: Platyhelminthes and Rhynchocoela. Mc-
Graw-Hill Book Co., Inc., New York.
JENNINGS, J. B., 1957. Studies on feeding, digestion and food storage in free-living flatworms
(Platyhelminthes: Turbellaria). Biol. Bull., 112: 63-80.
JENNINGS, J. B., 1959a. Observations on the nutrition of the land planarian Orthodcnuis
tcrrcstris (O. F. Miiller). Biol. Bull., 117: 119-124.
JENNINGS, J. B., 1959b. Studies on digestion in the monogenetic trematode Polystoma
intcgerriinuin. J. Helminth., 33: 197-204.
REISINGER, E., 1926. Nemertini. /;;: P. Schulze (ed.), Biologic der Ticrc Deutschlands,
Lief. 17.
WILSON, C. B., 1900. The habits and early development of Cercbratulus lactcus (Verrill).
Quart. J. Micr. Sci,, 43: 97-198.
HERMAPHRODITISM IN THE SEA SCALLOP, PLACOPECTEX
MAGELLANICUS (GMELIX)
ARTHUR S. MERRILL AND JOHN B. BURCH 1
U. S. Department of the Interim-, Fish and U'ildlifc Scnicc, Bureau of Commercial
Fisheries, Biological Laboratory, Woods Hole, Mass.; and Museum of
Zoology, University of Michigan, Ann Arbor, Midi.
While hermaphroditism is of common occurrence among normally dioecious
mollusks, a careful search of the literature failed to reveal any published record
of this condition for the commercially important sea scallop, Placopecten inageUani-
cns. Consequently, observations on the occurrence of the phenomenon in this
species are of some interest and importance.
Our first hermaphroditic sea scallop was collected on September 17, 1959,
while doing routine investigatory work relative to the scallop fishery aboard the
chartered scalloper Whaling City in the western part of Georges Bank (68° 45' AY.
Long., 43° 03' N. Lat.). September is within the spawning season for the sea
scallop on Georges Bank (Posgay and Norman, 1958). However, spawning had
not yet started at this locality and all specimens, including the hermaphrodite,
were ripe and full.
After close macroscopic examination the hermaphroditic gland was fixed for
histological study in Newcomer's (1953) fluid and stained either by means of the
Feulgen reaction or with Harris' hematoxylin and eosin. Some Feulgen-stained
sections were counterstained with light green (yellowish). Pieces of tissue were
cleared in chloroform, embedded in paraffin, and sectioned at 10 micra. For com-
parison normal male and female gonads were likewise treated except that in these
Benin's fluid was the fixative.
In the normal gonad the size and shape are similar in both sexes. The single
gonad is tongue-shaped and occupies most of that portion of the body ventral
to the foot. It extends dorsally to form a thin layer over the surface of a portion
of the digestive gland. The genital organ is large and plump when ripe but after
spawning it becomes much smaller, shriveled, and quite flaccid. The sexual prod-
ucts are easily seen through the tissue of the gonads, the ova giving the female
gonad a bright coral red appearance at maturity, the sperm producing a whitish-
cream coloration in the male gonad.
The hermaphrodite mentioned above has the male and female parts located in
different regions of the same gonad. The proximal part forms the ovary while
the testis lies distal to it. The boundary between the two regions is indefinite
with quite irregular islets of one tissue occurring within the" tissue of the other
(Fig. la). This is even more pronounced histologically (Fig. 2a). The gonad
is unspent and, relative to the degree of development, compares favorably with
normal unspent gonads.
1 Grant 2E-41, National Institute of Allergy and Infectious Diseases, U. S. Public Health
Service (in part).
197
I OS
ARTHUR S. MERRILL AND JOHN B. BURCH
The ovarian follicles are closely crowded and are filled with mature ova which
take the shape of polyhedrons because of their tightly packed condition. The
lumina of the follicles are completely filled. Kach follicle is lined on the outside
by a single cell layer of squamous epithelium. Inside the follicles the individual
ova are separated by a non-granular intracellular substance sandwiched between
and separating the cell membranes. This matrix is rather uniformly distributed
and of little variation in thickness. Actually, it may not be intracellular in the
strict sense but a component of the cell membranes. Mature ova average about
45-50 micra in diameter in fixed material. Their germinal vesicles are large and
clear, and contain a fine network of chromatin and several conspicuous nucleoli
(Fig. 2b). Connective tissue or follicular cells are rarely found between either
male or female follicles in this individual.
The testicular follicles are tightly packed and are covered by the same kind
of squamous cell layer as found covering the ovarian follicles. Inside the follicle
and adjacent to the covering epithelium is a layer of spermatogonial cells. This
layer of primordial germ cells is one to several cells thick (usually two or three,
but sometimes up to ten or more). Progressing inward toward the lumen of the
follicle are the primary and secondary spermatocytes, followed by spermatids, then
mature sperm at or near the center. The tails of the spermatids are usually located
in the most central part of the follicle. In portions of the testis where spermato-
IMGURK la. Hermaphroditic I'lacopcclcn magellanicus. Mantle and gill tissue folded back
to show the large bisexual gonad (X2/3). Ib. Hermaphroditic gonad of P. magellanicits.
Male portion has spawned, female (bulbous) part still unspawned (X2). (Note: gonad fixed
in Bouin's and preserved in alcohol before photo taken, this resulting in loss of color.)
HERMAPHRODITISM IX THE SEA SCALLOP
199
L1 \1 L? I*
b b b b
b7
a b
FIGURE 2a. Histological section through central portion of the hermaphroditic gland of
specimen shown in Figure la (X 50). a1, Portions of female follicles filled with ova. a2, Por-
tions of male follicles containing all stages of spermatogenesis. 2b. Enlarged portion of Fig-
ure 2a showing part of a male and a female follicle and their boundary (X 250). b1, Mature
sperm, b-, Spermatids. b3. Secondary spermatocytes. b4. Primary spermatocytes. b5, Sper-
matogonia. b6. Epithelial lining, b7. Portion of a mature ovum.
genesis is completed, the follicles are completely filled, from epithelial lining to
epithelial lining, with mature sperm. However, even in such follicles, there are
occasional cells at the epithelium which we take to be primordial germ cells but
which may perhaps be follicle cells.
The number of spermatogonial divisions before synapsis can not be determined,
but there is no evidence in this specimen, or in normal unisexual males, to suggest
that there are not more than two (e.g., see Coe and Turner, 1938, Fig. 17, p. 103).
Since whole bunches of spermatogonia, ten or more cells deep, are sometimes seen
to project out toward the lumen of the follicle, they probably go through a series
of divisions before synapsis. In pulmonate gastropods, where the number of
spermatogonial divisions can be accurately determined, there are normally six such
divisions prior to spermatocyte formation (Burch, 1959).
Spermatogonia average about 5 micra in diameter in sectioned material, primary
spermatocytes about 2.5 micra in diameter, and the width of each sperm head is
about 1.7 micra.
The individual follicles in this hermaphroditic gland are either all male or all
female ; there are no ambisexual follicles. In the proximal end of the gland most
follicles are female, and in the distal end most are male. But, in a wide central
area both male and female follicles are widely and indiscriminately dispersed
(Fig. 2a). The size and appearance of the cells, as far as can be ascertained,
are identical to similar stages in normal individuals of this species. Atypical
spermatogenesis as found by Coe and Turner ( 1938) was not observed.
A second hermaphrodite was found on November 21, 1959, in a sample from
the eastern part of Georges Bank (66° 45' W. Long., 41° 23' N. Lat.). The
ARTHUR S. MKRRII.L AXU JOHX I',. MURCH
arrangement of gonadal tissue in this specimen was similar to that of the first.
However, the spermary \vas mostly spent while the ovary still remained large and
plump (Fig. 11)) with no sign of having started to spawn. This differs from the
observed spawning habit of the normally monoecious great scallop, Pec ten ina.v units.
In this species the products are released within a few hours of each other, with
either the eggs or the sperm being shed first (Mason, 1958). Also, the arrange-
ment of the genital tissues within the gonad of /'. nw.viinus is the reverse of that
of the two hermaphroditic P. magellanicus (e.g., the testis proximal and the ovary
distal).
Of colleagues who have investigated various aspects of the scallop fishery,
only Dickie- (personal communication) has advised of having observed this phe-
nomenon. He remembered seeing hermaphroditic sea scallops on two separate
occasions. Once during the summer of 1956 he was shown several preserved
mature hermaphroditic gonads from Georges Bank by one of the scallop-vessel
skippers. Another time (July 22, 1949) he found a 95-mm. predominantly female
hermaphrodite from "Hour Ground" off Digby, Nova Scotia.
Hermaphroditism is the usual condition in most of the Pectinidae (Coe, 1945) ;
P. magellanicus is an exception. Hermaphrodites in other unisexual genera of
pelecypods are not uncommon and it seems likely that the occasional deviations
in the developmental processes which produce these hermaphrodites are due to the
failure of the sex-differentiating mechanism to function normally, as has been
suggested by Coe and others. According to the classification of Coe (1942) these
abnormalities would be termed accidental functional ambisexuals, in which the
primary sex factors go astray and fail to activate or suppress either the male or
female hereditary mechanism at some early stage of development. This results
in various amounts of both kinds of tissue being produced.
Young's (1941) suggestion that hermaphroditism in normally bisexual species
may be due to aberrant chromosomal behavior during gametogenesis has not yet
been corroborated by cytological or experimental evidence. Similar suppositions
were advanced to account for production of male and female cells in the ovotestis
of the pulmonate gastropod L\mnaca stognalis apf>rcssa ( =- L. s. JHfjnlaris) by
Crabb (1927) and in Physa gyrina by Mahoney (1940). Perrot (1930), how-
ever, showed that this was not the case for L. stagnalis and Burch and Bush (1960)
have shown the observations on P. gyrina to be in error.
In the commercially important bivalves in which sex has been extensively
studied, and which are dioecious, occasional occurrence of hermaphrodites is nor-
mal. Thus, to list a few examples common to the North Atlantic coastline,
Thorson (1936) remarked that Mytilus c dulls has a considerable percentage of
hermaphrodites. Loosanoff (1936) in his sexual studies of the quahog (Jl/rr-
cenaria mercenaria) found two cases of functional hermaphroditism among several
hundred mature clams. Also, Turner11 (personal communication) mentioned
having seen a functional hermaphrodite in this species. He observed it to release
the sperm first, and then the eggs. Coe and Turner (1938) found three cases of
hermaphroditism on examining about a thousand soft-shelled clams (Mya arenaria).
In the case of P. magellanicus about 3000 gonads were inspected after the first and
- Lloyd M. Dickie, Biologist, Biological Station, St. Andrews, New Brunswick, Canada.
3 Harry J. Turner, Jr., Marine Biologist, Woods Hole Oceanographic Institution.
HERMAPHRODITISM IN THE SKA SCALLOP 201
before the second hermaphrodite was found. The low frequency of occurrence
accounts for this condition seldom being observed in the sea scallop.
LITERATURE CITKU
BURCH, JOHN B., 1959. Chromosomes of aquatic pulmonate snails (Bassommatophora). Ph.D.
Dissert., Univ. Michigan, Ann Arbor, Pp. 97.
BURCH, JOHN B., AND LINDA L. BUSH, I960. Chromosomes of I'hysa i/yrina Say (Mollusca:
Pulmonata). /. dc Couch.. 100: 49-54.
COE, \V. R., 1942. Sexual differentiation in mollusks. 1. Pelecypods. Quart. }\cr. Biol., 18:
154-164.
COE, W. R., 1945. Development of the reproductive system and variations in sexuality in
Pccten and other pelecypod mollusks. Trans. Connecticut Acad. Arts Sci., 36: 673-700.
COE, WESLEY ROSWELL, AND HARRY J. TURNER, JR., 1938. Development of the gonads and
gametes in the soft-shell clam (Mya arcnaria). J. Morph., 62: 91-111.
CRABB, E. D., 1927. The fertilization process in the snail Lvuniacn stin/nalis apprcssa Say.
Biol. Bull., 53: 67-97.
LOOSANOFF, VICTOR, 1936. Sexual phases in the quohog. Science, 83: 287-288.
MAHONEY, F. J., 1940. Spermatogenesis with special reference to certain extranuclear struc-
tures in the pulmonate Physa (/yrina Say. Unir. Colorado Stud.. 26: 81-83.
MAMIX, JAMES, 1958. The breeding of the scallop, Pccten ina.rinnis (L.), in Manx waters.
/. Mar. Biol. Assoc.. 37: 653-671.
NEWCOMER, E. H., 1953. A new cytological and histological fixing fluid. Science. 118: 161.
PERROT, J.-L., 1930. A propos du nombre des chromosomes dans les deux lignees germinales
du Gasteropode hermaphrodite Limnca stpgnalis ( variete rhodani). Rev. Smssc Zool.,
41: 693-697.
POSGAY, J. A., AND K. DUANE NORMAN, 1958. An observation on the spawning of the sea
scallop, Placopecten magellanicus (Gmelin), on Georges Bank. Limnol. Oceanogr.,
3: 478.
THORSON, GUNNAR, 1936. The larval development, growth, and metabolism of Arctic marine
bottom invertebrates compared with those of other seas. Mcdd. Gronland. 100(6) :
1-155.
YOUNG, R. T.. 1941. An hermaphroditic Mytilus. Xau/ilus, 54: 90-91.
ANTIGENS OF ARBACIA SI'KKM EXTRACTS L
CHARLES B. METZ AND KURT KOHLER -
Oceano graphic Institute, Plorida State University, Tallahassee, Florida,
and Marine Biological Laboratory, }\'oodx Hole, Mass.
The initial steps in fertilization appear to involve interactions of the sperm
and egg surfaces at the molecular level (see Tyler, 1948, 1949; Metz, 1957a,
1957b). Most of the present information concerning such interaction has been
obtained from studies of egg and sperm extracts. Among sperm extracts those
with action on eggs have commanded most interest. In the sea urchin and certain
other forms, extracts prepared by a variety of methods precipitate the egg jelly
layer, agglutinate eggs and neutralize the sperm agglutinating action of the fertili-
zin obtained from eggs. These effects of the extracts may result from the action
of the sperm-surface receptor substance, antifertilizin, with which fertilizin com-
bines in the sperm agglutination reaction (e.g. Tyler, 1948; Metz, 1957b).
Whether or not these effects are to be identified with antifertilizin, absorption
experiments have shown that such extracts contain some antigens in common with
those of the sperm surface (Kohler and Metz, 1959a, 1959b, 1960). Further
examination to reveal a more complete spectrum of antigens in such extracts seemed
desirable. Accordingly, in the present investigation, Arbacia sperm extracts were
examined for antigens by means of agar diffusion and immunoelectrophoretic tech-
niques. The study revealed a maximum of four antigens in extracts prepared
by f feeze-thawing sperm.
MATERIAL AND METHODS
Arbacia f>iuictulatii from the vicinity of the Florida State University Marine
Laboratory, Alligator Point, Florida, and from Woods Hole, Massachusetts, were
used in the study. Semen was usually obtained from the animals by electrical
stimulation (Harvey, 1956). The spermatozoa were separated from the seminal
plasma by centrifugation (approximately 3000 X g ; 20 minutes) at 4° C. The
packed sperm were resuspended once in sea water and settled again by low speed
centrifugation. The final supernatants following such washing regularly failed to
give precipitation bands when diffused against anti-sperm serum. Standard sperm
suspensions were prepared by diluting the packed sperm with three volumes of
sea water.
Sperm extracts were prepared from such suspensions of washed sperm by a
variety of methods. These included the established methods for preparing agents
1 Contribution from the ( )ceanographic Institute, Florida State University. Aided by grants
from the National Science Foundation, the National Institutes of Health and the Research
Council of Florida State University.
- Fulbright Fellow. Present address: Max-Planck-Institut fur Virusforschung, Tubingen,
Germany.
202
ANTIGENS OF ARBACIA SPERM EXTRACTS 203
which act upon the egg jelly layer, namely, heating sperm to 100° C. (Frank,
1939) and freeze-thawing sperm (Tyler, 1939). The latter extracts are called
"frozen-thawed extracts" below. Other methods are described with the individual
experiments.
Antisera were prepared by injecting rabbits with sperm (25% washed sperm
in sea water). The immunizing antigens were administered through intravenous,
intraperitoneal and subscapular injections. In the last instance the antigen was
injected as an emulsion in Freund's adjuvant (Difco). Several axiti-Arbacia sera
were examined. With the exceptions noted in the text the experiments reported
here were performed with serum from the hyper-immune rabbit "#33." This
rabbit received three injections of antigen in Freund's adjuvant (Difco) over a
period of five months and was bled two weeks subsequent to the final injection.
The immune serum obtained regularly agglutinated sperm to dilutions of 2~8 to
2~10. No sera were pooled.
Agar diffusion and electrophoresis. Agar diffusion experiments using the
technique of Ouchterlony (1948) were performed in 2% agar containing Mer-
thiolate (0.01%) as a preservative. The reaction plates were incubated at room
temperature for several weeks. Immunoelectrophoretic analysis (Wunderly,
1957 ) was performed using 2% agar blocks prepared in 0.05 ionic strength veronal
buffer, pH 8.5 and containing 0.01% Merthlolate. Wells in the agar block were
filled with antigen prepared as follows : after dialysis against 0.05 ionic strength
veronal, the antigen was heated to 45° C. and mixed with an equal volume of
melted (45° C.) 4% agar, also in veronal buffer. The mixture was then pipetted
into the antigen wells of the agar slab and allowed to solidify. Agar slabs meas-
uring approximately 20 X 6.05 cm. with antigen wells of 0.3-0.4 ml. capacity were
used in these experiments. To achieve electrophoretic migration the preparations
were subjected to a current of 25 ma for about six hours.
To improve the resolution of precipitin bands the agar blocks were fixed in
2% acetic acid, stained with Amidoschwarz (0.1% in acetate buffer, pH 4.0 solu-
tion) and destained in methanol-water-acetic acid (45:45: 10).
RESULTS
Agar diffusion precipitin tests were performed on extracts prepared by freeze-
thawing washed Arbacid sperm and subsequently centrifuging the extracts in a
clinical centrifuge (approximately 3000 X g) . When diffused against anti-whole
sperm serum such extracts yielded a maximum of four precipitin bands. Proceed-
ing from the antigen to the antibody well in the agar plate, these four bands are
designated a, b, c, and d. As seen in Figure 1, some variation in band number
was found in repeated tests (e.g. two bands in Figures Ia5, Ib2, Ib3; three bands
in Ib4, Ic6; four bands in Icl, Id3). Antisera other than #33 gave one to two
bands. These differences in tests with different "frozen-thawed extracts" using
serum #33 are attributed to differences in antigen concentration. However, the
possibility of qualitative differences has not been eliminated. Differences in the
band number using other antisera may reflect differences in concentration of anti-
body as well as antigen in the tests. In view of the fact that the sperm were
washed sufficiently before extraction to remove seminal plasma antigens, the pre-
204
CHARLES B. METZ AND KURT KOHLEK
cipitin bands in the extracts must have arisen from antigens extracted from the
sperm cells.
In this connection it is of interest that undiluted seminal plasma forms three
hands when diffused against anti-whole sperm serum (Fig. Ia4, Ibl). Two of
these seminal plasma hands join but do not cross two of the "frozen-thawed extract"
bands. It appears, then, that the seminal plasma shares at least two antigens with
the extract. In fact these seminal plasma antigens may have diffused from the
FIGURE 1. Ouchterlony agar diffusion tests. The center wells of all plates contain anti-
Arbacia sperm serum No. 33. All surrounding wells were filled with extract and fluid of Ar-
bacia punctulata. Each well received 0.5 ml. of the sample : ( a ) ( 1 ) supernatant over frozen-
thawed sperm after centrifugation at 26,000 X g for 20 minutes; (2) residue of No. (1),
resuspended in sea water and centrifuged at low speed; (3) supernatant over Mickle-clisinte-
grated sperm; (4) seminal plasma; (5) "frozen-thawed extract" of sperm, low speed centri-
fuged; (6) body fluid; (b) (1) seminal plasma; (2) "frozen-thawed extract" of whole sperm,
low speed centrifugation; (3) "frozen-thawed extract" of whole sperm, low speed centrifuga-
tion; (4) "frozen-thawed extract," low speed centrifugation; (5) supernatant over Mickle-
disintegrated sperm, low speed centrifugation; (6) supernatant over Mickle-disintegrated
sperm, low speed centrifugation; (c) (1) "frozen-thawed extract," low speed centrifugation;
(2) basic protein, pH 0.9 extract; (3) supernatant over washed sperm after standing (aging),
low speed centrifugation ; (4) supernatant over citric acid-extracted sperm, low speed centrifu-
gation; (5) supernatant over urea-treated sperm; (6) frozen-thawed extract, low speed cen-
trifugation; (d) (1) isolated heads, "frozen-thawed extract," low speed centrifugation;
(2) isolated tails, "frozen-thawed extract," low speed centrifugation; (3) whole sperm, "frozen-
thawed extract," low speed centrifugation ; (4) isolated heads, "frozen-thawed extract," low
speed centrifugation; (5) acid extract (pH 3) of sperm, low speed centrifugation; (6) acid
extract (pH 1.9) of sperm, low speed centrifugation.
sperm. However, the one band that has been clearly demonstrated in the super-
natant of aging sperm is band a of "frozen-thawed extract" (Fig. Ic3). It should
be noted that the three bands just described do not constitute the complete antigenic
spectrum of Arbacia seminal plasma. As seen in Figure 2cl, immunoelectrophoresis
resolved seven bands in this material. Three of these correspond in position to
three precipitin bands in the sperm extract (Fig. 2c ) .
It should be clear from these results that extracts prepared by freeze-thawing
ANTIGENS OF ARBACIA SPERM EXTRACTS
205
sperm are not solutions of a single macromolecule. On the assumption that each
precipitin hand represents a single antigen, the extracts can contain up to four
different antigens. Extracts of sperm prepared by other procedures also contained
antigens. As seen in Figure Id5, extraction of Arhacia sperm at pH 3 yielded a
preparation which produced four precipitin bands. Evidently two of these antigens
are labile to or are insoluble in stronger acids for, as seen in Figure Id6, an aliquot
of the same sperm suspension extracted at pH 1.9 yielded but two precipitin bands.
Extraction at even lower pH (pH 0.9) yielded preparations that failed to form
precipitin bands. v The extracts prepared by extracting Arbacia sperm at pH 3
failed to precipitate egg jellies.
czi
a
FIGURE 2. Immunoelectrophoresis in agar of various fluids from Arbacia. The central
channel contains anti-Arbacia sperm serum No. 33, lateral channels contain preinjection serum
No. 33. (a) Heated sperm extract; (b) "frozen-thawed extract," low speed centrifugation ;
(c) "frozen-thawed extract," low speed centrifugation; (d) seminal plasma.
Likewise, extracts prepared by treatment of sperm in the Mickle disintegrator
(20 minutes, 20° C.) yielded three precipitin bands (Fig. Ib5 and Ib6). These have
not been homologized with the antigens of "frozen-thawed extract." However,
t\vo ( Fig. 11)4 and Ib5) of the bands correspond in position to two of the bands
from "frozen-thawed extract." The third band crosses one of the bands of the
"frozen-thawed extract" (probably the d band) and may represent an antigen not
present in "frozen-thawed extract." < )ther extracting agents used were 1/18 M
Na-citrate and 4 M urea. As seen in Figures Ic4 and Ic5, citrate extract pro-
206 CHARLES B. METZ AND KURT KOHLER
duced two bands and urea extract three. The two citrate extract bands join two
of the three urea extract bands. The third band of the urea extract is probably
identical with the a band of "frozen-thawed extract."
As might be expected, extracts prepared by heating (100°C. ) .-Irhacia sperm
for one to five minutes do not have the full complement of soluble antigens. Such
extracts at most yield a single band when diffused against anti- whole sperm serum.
This antigen is evidently present in low concentration in extracts prepared by
heating 25% sperm suspensions, for most such preparations fail to produce any
precipitin bands in Ouchterlony or immunoelectrophoretic tests (e.g. Fig. 3a3j.
The band that does appear corresponds in position to one of the immunoelectro-
phoresis bands of "frozen-thawed extract" (see Figure 2 and below).
As seen above, extracts of whole sperm can contain at least four precipitating
antigens. As in the case of the sperm agglutination antigens (Kohler and Metz,
1960), it seemed of interest to attempt to determine if these are present in both
sperm heads and tails. Accordingly, sperm were broken into heads and tails by
Mickle disintegration, these structures were separated by differential centrifugation
and finally extracted by freeze-thawing (for details, see Kohler and Metz, 1960).
When examined for precipitating antigens by agar diffusion, the frozen-thawed
whole sperm extract produced four bands (Fig. Id3). Head and tail extracts,
adjusted to corresponding concentration, each produced two bands. One of these
was common to both extracts and joined the d band of whole sperm extract. The
second band in the head and tail extracts also joined bands produced by whole
sperm extracts. However, these bands were not (Fig. Id4) identical. The sec-
ond band from the head extract joined the b band of whole sperm extract whereas
the second band of tail extract joined the e band of the whole sperm extract
(Fig. Id2).
From the foregoing, it appears that both head and tail extracts lack band a of
whole sperm extract. The absence of band a in the head and tail extracts is
attributed to loss of this readily extracted antigen in the isolation process. Band
b is present in extracts of isolated heads, but not tails ; band c is present in tails
but not heads and band d is present in both head and tail extracts.
Immunoclectrophoresis. In attempts to further resolve the antigenic composi-
tion of sperm extracts and seminal plasma these were subjected to imrnunoelectro-
phoresis in agar blocks (2c/r agar in 0.05 ionic strength veronal, pH 8.6). This
method resolved three precipitin arcs (in three separate experiments ) in frozen-
thawed sperm extracts. As seen in Figure 2, b and c, one of these moved rapidly
toward the anode, a second moved with intermediate speed and the third did not
move appreciably. In comparative studies seminal plasma produced seven definite
arcs (Fig. 2d). Three of these corresponded in position to the three arcs of the
frozen-thawed sperm extract. Heat-extracted sperm ( 100° C., one minute) never
produced more than a single arc (Fig. 2a). This corresponded in position to the
fastest migrating antigen of unheated extract and seminal plasma.
Experiments unth extracts centrijuged at higii speed. In the studies presented
above (Figs. 1 and 2), the extracts of frozen-thawed sperm were centrifuged at
low speed (e.g. 3000 X g) only. Upon high speed centrifugation (e.g. 26,000
X g) of such extracts a pink, semi-gelatinous pellet is obtained and much, if not
all of the egg jelly precipitating activity of the extracts is associated with this sedi-
ANTIGENS OF ARBACIA SPERM EXTRACTS 207
mentable material ( Kohler and Metz, 1959b). Therefore, a comparative study of
extracts before and after such high speed centrifugation was undertaken. This
showed that three of the bands (b, c, d) remained in the supernatant of "frozen-
thawed extract" after centrifugation at 26,000 X g (Fig. 3al and 3a4). The a
band was absent from such preparations. The pink pellet obtained after the high
speed centrifugation was homogenized in a small amount of sea water, and gave
only the a band when diffused against anti-sperm serum (Fig. 3a2 and 3a6). This
result suggests that the a band (e.g. in Fig. Ia3, Id, Ic3 and Id3) represents
antigenic material, that is readily sedimented by high speed centrifugation.
In comparative studies with extracts prepared by other means and centrifuged
at high speed, frozen-thawed and acid extracts (pH 3.2) showed one band (prob-
ably the c band ) in common (Fig. 3a5 ). In a second experiment (Fig. 3c and 3d)
only two bands were resolved in the high speed supernatant of "frozen-thawed
extract." One of these (probably the b band) is common to the high speed super-
natants of seminal plasma (Fig. 3c3 ) and heat extract (Fig. 3c6) and the low
speed sediment of the original "frozen-thawed extract." Histone prepared by pH
0.8 extraction of sperm and nucleoprotein prepared according to Mirsky and Pol-
lister (1942) gave no precipitation bands with the antiserum after high speed cen-
trifugation (Fig. 3d). This is not surprising since histone and nucleoprotein are
generally found to be poor antigens (Gushing and Campbell, 1957).
The supernatant obtained after high speed centrifugation of "frozen-thawed
extracts'' was also compared with extracts obtained by aging sperm (48 hours, in
sea water), extraction with 4 M urea and with 1/18 M citrate for 48 hours (Fig.
3b). One band, possibly the c band, is common to all of these extracts.
DISCUSSION
The results reported here show that extracts of frozen-thawed Arbacia sperm
can yield four distinct precipitation bands when diffused against anti-whole sperm
serum. These four bands are interpreted as four separate antigens. Detection
of all of these antigens in agar diffusion precipitin tests appears to depend upon
having high titer antisera and concentrated solutions of extract. Indeed these
antigens may be only slightly soluble substances. Other extraction procedures
(e.g. heating, acid, citric acid, and urea extraction) have not clearly revealed addi-
tional antigens. Likewise immunoelectrophoretic analysis revealed three antigens
in frozen-thawed extracts. It appears, then, that only a few sea water-soluble
antigens are obtained from Arbacia sperm in appreciable concentration. Possibly
immunization of additional rabbits or other animals might yield an antiserum of
unusual resolving power and reveal additional sperm antigens. However, the
experience so far suggests that but few additional antigens would be discovered.
The relatively small number of antigens in sperm extracts is in sharp contrast
to Arbacia seminal plasma and egg extracts prepared by freeze-thawing. Parallel
immunoelectrophoretic studies using anti- Arbacia egg sera and frozen-thawed ex-
tracts of Arbacia eggs readily resolved nine antigens. Likewise Perlmann (1953)
found at least ten precipitin bands in agar diffusion tests using 0.15 M NaCl ex-
tracts of Paracentrotus lli'idus eggs. In preliminary tests no cross precipitin re-
actions were obtained between sperm extracts and anti-egg sera or the reverse.
208
CHARLES B. METZ AND KURT KOHLER
FIGURE 3. Ouchterlony agar diffusion tests. The center wells of all plates contain anti-
Arbacia sperm serum No. 33. All surrounding wells were filled with extracts or fluids of
Arbacia punctulata. Each well received 0.5 ml. of sample: (a) (1) "frozen-thawed extract"
immediately subjected to centrifugation at 26,000 X /y; (2) the pellet from No. 1 and No. 4,
washed and resuspended; (3) heat extract, centrifuged at 26,000 X g; (4) "frozen-thawed
extract" immediately subjected to centrifugation at 26,000 X g; (5) acid extract, centrifuged
at 26,000 X g; (6) the pellet from No. 1 and No. 4, washed and resuspended. (b) (1) A very
active egg jelly precipitating preparation obtained by resuspending the cake of frozen-thawed
sperm followed by centrifugation at low7 speed; (2) 4 M urea extract of sperm, centrifuged
at 26,000 X g; (3) 1/18 M Na-citrate extract of sperm, centrifuged at 26,000 X g; (4) super-
natant over aged sperm centrifuged at 26,000 X g; (5) 4 M urea extract of sperm centrifuged
at 26,000 X g; (6) 1/18 M Na-citrate extract of sperm, centrifuged at 26,000 X g. (c) (1) and
(4) Supernatant over frozen-thawed sperm after high speed centrifugation; (2) and (5) the
cake of frozen-thawed sperm was resuspended ( same as Figure 3bl ) in sea water for addi-
tional extraction (wells contain the supernatant after low speed centrifugation) ; (3) seminal
plasma centrifuged at 26,000 X g; (6) heat extract, centrifuged at 26,000X0. (d) (1) and
(4) Supernatant over frozen-thawed sperm after high speed centrifugation; (2) and (5) the
cake of frozen-thawed sperm was resuspended (same as Figure 3bl ) in sea water for addi-
tional extraction (wells contain the supernatant after low speed centrifugation) ; (3) nucleo-
protein centrifuged at 26,000 X g; (6) historic centrifuged at 26,000 X g.
Perlmann (1953), however, obtained one precipitin band when saline (0.14 M)
extract of sperm was diffused against anti-egg serum.
The relationship, if any, of the four soluble sperm antigens to the sperm surface
and to the egg jelly precipitating activity of the extracts has not been examined in
detail. However, it is likely that the three antigens, b, c. and d, of "frozen-thawed
extracts" are not related to the egg jelly precipitating activity because the latter
activity is removed by high speed centrifugation whereas the b, c, d antigens are
not sedimented by such centrifugation (Kohler and Metz, 1959b). With regard
to the relationship of the soluble antigens to sperm surface antigens it should be
recalled that absorption of anti-sperm serum with "frozen-thawed extracts" lowers
the titer but does not completely neutralize the sperm agglutinating action of such
antiserum (Kohler and Metz, 1960). This shows that the extracts contain some
but not all of the sperm surface antigens. Agar diffusion experiments employing
sera absorbed with whole sperm might reveal whether the soluble antigens are
surface or subsurface material.
ANTIGENS OF ARBACIA SPERM ENTRACTS 209
SUMMARY
1. Extracts were prepared from Arbacia sperm In several procedures. These
were tested for antigenic composition by diffusion against &\\\\- Arbacia sperm rabbit
serum on Ouchterlony plates and by means of immunoelectrophoresis in agar gel.
2. Extracts prepared by freeze-tbawing the sperm followed by low speed cen-
trifugation produced a maximum of four precipitin bands. It is concluded that
such extracts contain at least four soluble antigens.
3. Seminal plasma revealed seven arcs in an immunoelectrophoretic experi-
ment, and a maximum of three bands on Ouchterlony plates. Two such bands
join bands from "frozen-thawed extracts."
4. Extracts prepared by heating sperm at 100° C. yielded at the most one
band. This antigen seemed to be common to several other extracts.
5. Xucleoprotein ( Mir sky and Pollister) and histone failed to form precipitin
bands.
6. One of the four bands in frozen-thawed extract is associated with material
sedimented at 26,000 X and 15 inches/sec., and analyzed on a Kay Vibralyzer vibration
frequency analyzer. Recordings were played back into the water with an Ekotape
tape recorder Model 205, a Craftsman C550 amplifier and a QBG transducer.
Recordings at sea were made from the motor launch ABUDEFDUF of the Ber-
muda Biological Station, Mr. Brunell Spurling, Captain.
1 Contribution No. 1126 from the Woods Hole Oceanographic Institution.
- Contribution No. 271 from the Bermuda Biological Station.
3 The work was performed at the Bermuda Biological Station and at Bowdoin College
under Grant NSF-G4403 of the National Science Foundation, and through assistance of the
Woods Hole Oceanographic Institution and of the Bowdoin College Faculty Research Fund
established by the Class of 1928.
210
SWIMMING SOUNDS OF FISHES
211
SWIMMING SOUNDS OF SOME BERMUDA FISHES
Anchoi'iclla choerostoma (Goode) — Hog-mouth fry. This small engraulid oc-
curred in large and small schools on Bermuda shores during the summer of 1958.
The schools were most commonly seen in shallow bays along the north shore of
St. George's and Hamilton Islands where they are said to he driven by the com-
monly observed activity of predators (chiefly carangids and lutianids ) feeding on
the schools (B. Spurling, personal communication). Menidia (Atherinidae) is
probably similarly driven on Long Island shores (Butner and Brattstrom, 1960).
E. L. Mark (1905) described some Bermuda fishes as gathering schools of smaller
fishes to lure prey for the former.
Once established in a bay, a school may remain for several days and even for
weeks, gradually reducing in size under peripheral predation and the depredations
of fishermen obtaining bait for which the fry are highly valued. A large school
darkens the water so that it may be seen over a considerable distance, and the
experienced Bermuda boatman can readily distinguish between that discoloration
caused by a fish school and that due to rock formation and weed patches.
Observations on the behavior of a large school at sea, estimated to contain
several million individuals, of A. choerostoma were obtained mainly in Bailey's
Bay on August 7, 1958. The launch was repeatedly brought over the school which
moved in and out relative to shore during recording. Recordings were obtained
when the sea surface was calm and the only extraneous noise stemmed from the
system and from snapping shrimp.
Xo predators were seen feeding during recording ; activity around the borders
of A. choerostoma schools on other occasions consisted of feeding by jacks (Caran.v
spp. ) and pompano (Trachinotus palometa}. Small Caran.r latus (2 to 6 inches)
were frequently netted with A. choerostoma and individuals were tolerated as mem-
bers of small schools of the latter (up to 1000) maintained in the laboratory; the
jacks fed on the host schools. Jcnkinsio and Sardinella (Clupeidae) were also
taken with Anchoriella on occasion.
For recording, the launch drifted over the Bailey's Bay school, engine off ;
although not obviously so, the behavior of the school was probably modified to some
extent by the presence of the boat. During recording, the school was either lying
at rest with individual fish turning slowly within a narrow circumference, or the
TABLE I
Characteristics of swimming sounds
Species
Size
Sound
Maximum
frequency
Frequency of
greatest intensity
Time
duration
. 1 nchni'iflla
choerostoma
3 inches
Streaming
1.6 kc.
Below .5 kc.
Variable
Car any, I at us
2-6 inches
Veering of school
Veering of
individual
2 kc.
1.7 kc.
Below .8 kc.
•x Below 1 kc.
A
.2-. 6 sec.
.03 sec.
Ciirnnx r uber
1 foot
Veering of
individual
1.6 kc.
Below .7 kc.
.05 sec.
Trachinotus
palometa
1 foot
Veering of
individual
.7 kc.
Below .25 kc.
.06 sec.
212
JAMES M. MOULTON
•
•
1.0-
5 10
SECONDS
20 ?5 30 35 40 4S so 55
1
il
1
'" Yl lliaiAlki. • • i
2 4
5 .1
i
•w
12
1,6
2 .4 6
12 14 16
12 "14 16
,L
246
F ••*: '
6 8
12 14 16
i ' !• f1
ii * .
5 10 15 20 Z5 30 35 40
FIGURE 1. Streaming, resting and veering of Anchoviella choerostoma.
FIGURE 2. Beginning of a streaming movement of A. choerostoma.
FIGURE 3. Two veerings during streaming of A. choerostoma.
FIGURE 4. Background and system noise during recording at sea in Bailey's Bay, August
7, 1958.
SWIMMING SOUNDS OF FISHES 213
school had assumed a single direction and the fish were streaming around and by
the hydrophone, or the school in the process of streaming suddenly veered in direc-
tion with a resultant sharp increase in sound intensity.
Sound could not be recorded from the school at rest ; streaming and veering
both produced considerable sound (Table I). Sounds similar to these are aptly
described by Shishkova (1958a) for T. trachurus as being like a pouring or a
splash and like a rise and fall in system noise. The greater the degree of veering,
the greater the intensity of its noise. The school streamed slowly and rapidly past
the hydrophone; sound intensity fluctuated with variations in speed of movement
of the school.
Suitcase amplifier and Magnecorder settings varied from 0 + 4-10 and 20-3
db, respectively, to 0 + 14-7 and 30-3 db during recording; at all settings, some
veerings of the school overloaded the system.
The entire school covered approximately half an acre ; during the observation
period, the school broke occasionally into two or three smaller groups. The school
and its subdivisions tended to disperse somewhat and gather closely again, so that
concentration of fish varied. At times, the school appeared layered, a thinner
upper stratum moving in a different direction than the lower bulk of the school.
All recordings were done over sandy bottom while the school lay in 2 to 4
fathoms of water ; the hydrophone was lowered to mid-depth of the school. When
streaming, the school moved past the hydrophone leaving a circular area clear of
fish, extending about 6 inches from the casing. With a casing of different color,
this interval might be expected to vary (Breder, 1951 ).
When the school was at rest, it could be stimulated to move by the slightest
flexing and stiffening of the observer's knees aboard the 21 -foot launch when
visual clues were excluded. The casting of a shadow over the school by hand and
body movements did not cause this behavior. If the school were at rest when
pressure was applied to the deck of the boat by the knee-bend method, it would
begin to stream ; if it were streaming, it would veer and usually alter the speed of
swimming to some extent.
The lower portion (up to 1.5 kc.) of a frequency analysis of sounds stemming
from a typical sequence of movements of the Anchovlclla school is shown in Fig-
ure 1. During the 6 seconds of recording illustrated, the school came nearly to
rest, began moving again at 2 seconds, veered a few times between 2 and 5 seconds,
then quieted again. Abbreviated vertical streaks in the background are due to
snapping shrimp ; some system noise is indicated below .25 kc.
A similar sequence of events is shown in Figure 2 over a broader frequency
scale (5 kc.) and briefer time interval (1.7 seconds). Here the beginning of a
streaming movement is shown, again with a few snapping shrimp spectra in the
background. Figure 3 shows the spectra of two veerings of the school during a
streaming movement. In each of these records, there has been sufficient attenua-
FIGURE 5. Sections through spectrum of recording of A. choerostoma school while at rest
and while streaming.
FIGURE 6. Veering of Carau.r lutus school.
FIGURE 7. Feeding and swimming sounds of Canni.r Ititits.
FIGURE 8. Veering of Caran.r nii>cr.
FIGURE 9. Veering of Trachinotus fi
214 JAMKS M. MOULTON
tion to bring the schooling movements clearly into the foreground. In Figure 4,
the ambient underwater sound of the area (mainly snapping shrimp) and system
noise is shown after the Anchoviella school had moved inshore from the launch
on one occasion.
Figure 5 shows sound intensity spectra (sections) during a recording made
from the school at rest and after it had begun to stream. The first section was
taken at .6 second of the time scale (school at rest), and the other at 1.3 seconds
(school streaming). These sections provide a qualitative indication of the amount
of sound stemming from a streaming movement at various frequencies at a point
in time. Swimming sound of another Anchoviella school of considerably smaller
dimensions could not be separated from background noise, due mainly to wave
action during a moderate sea state with the equipment employed. In summary,
Anchoviella swimming sound lies mainly below 2 kc. (Table I). Veering of the
school introduces sharp intensity increases of between .2 and .6 second duration.
Attempts failed to record the swimming sound of Anchoviella in the laboratory
from groups of 500 to 1000 individuals; the sound did not rise above extraneous
noise. These experiments were performed in a cement tank, the fish being con-
lined by a wood and plastic screen to an area 8 feet X 2 feet X 1 foot deep. The
. \X-58-C hydrophone was placed across the tank and fish driven around it. This
paper presents evidence that the swimming activity of even small groups of Anclio-
viclla is probably important in maintaining the coherence of the school ; the swim-
ming sound of a large school is one component of this activity.
A school of blue-fry (Athcrina harringtoriensis, Atherinidae ) passing slowly
under the launch on August 7 near Bay Island in Bailey's Bay did not respond to
the knee-bend method successful in stimulating streaming and veering in Anchovi-
clla. The blue-fry school was in the form of a compact ball of perhaps 1000 fish,
each turning in its own particular pathway within the school as the latter passed
beneath the boat. No swimming sound could be detected.
Caran.r latns Agassiz and C. ruhcr ( Bloch) — Yellow Jack and Skip Jack.
These carangids, common in Bermuda, feed voraciously on Anchoviella as well as
on other fishes. The C. lafns, from 2 to 6 inches in length, were captured in small
numbers with Anchoviella and separately off the dock of the Bermuda Biological
Station. Observations are based mainly on aquarium experiments. Two large
impounded schools of adult Carau.v ruhcr were recorded on the west side of Coney
Island on June 26 and August 6, 1958, through the kindness of a commercial
fisherman (Mr. Spurling, Senior), and a third school was recorded during under-
water listening in Castle Roads on July 11.
Since pharyngeal tooth stridulation may accompany the swimming sound of
Caran.v spp., this habit bears comment. Pharyngeal tooth stridulation of Caran.v
hippos has been described (Burkenroad, 1931; Fish, 1948; Moulton, 1958), and
Fish (1948, 1954) describes the stridulating sounds of a number of other
•carangids. The pharyngeal tooth stridulation is produced more readily by young
jacks hand-held gently under water ( C. latns, C. hippos) than by adult jacks so
treated (Moulton, 1958, p. 364) ; it was recorded at Bermuda from adult C. ruhcr
held next to the hydroprone in an impounded school, and by C. crysos speared
off Nonsuch Island on August 17.
Several attempts failed to elicit pharyngeal tooth stridulation from adult pom-
SWIMMING SOUNDS OF FISHES 215
pano (Trachinotus palowieta, Carangidae) and Calamus bajanado ( 1)lue-bone porgy,
Sparidae), both of which are equipped with formidable pharyngeal teeth. Adult
grunts (Hacinulon sciurns, Pomadasyidae) of some species, on the other hand,
stridulate readily when similarly treated.
The present account deals mainly with the thumping sound produced by veering
of Caron.v spp., but pharyngeal tooth stridulation is also produced at times during
this maneuver.
Several small groups (from 12 to 20) of young C. latns were confined in
aquaria l1^ feet X 1% feet X 1 foot deep with the hydrophone. Undisturbed,
these fish milled about the aquarium equidistant from each other, each on its own
pathway. When a hand was flicked at the aquarium or a stick poked into the
water, the small group of jacks became at once a tightly coherent school, streaming
about the aquarium and veering on repetitive stimuli as had the Anchoviella at sea.
Streaming did not introduce sound detectable above background ; veering did.
This movement even of individual fish resulted in distinct thumps similar to those
described by Fish (1954) from shocked C. crysos (7.75 mm.?). When stemming
from a group of C. latus, the thumps occurred in volleys ( Fig. 6, Table I ) .
During feeding on bits of meat, the C. latns dispersed through the aquarium.
The thumps then became scattered as individuals darted for food, and were accom-
panied by occasional sounds of the stridulation of teeth (Fig. 7).
During the Coney Island recordings, the hydrophone was hung in the midst
of schools of C. rubcr before and during raising of the net. Veering of the fish
about the hydrophone caused volleys of thump-like sounds similar to those of C.
latus, but somewhat deeper in predominant tone (Fig. 8, Table I). Swimming
sound engendered by streaming movements was also detected in these recordings
and that obtained at Castle Roads.
Trachinotus palomcta Regan — Gaff-topsail Pompano. The swimming sound of
two pompano approximately 1 foot long was recorded in the cement tank. The
animals were not fed during recording. Only thumps coincident with veering
occasioned by sudden hand movements in the water were recorded. Sound spec-
trograms of two flurries of activity are illustrated ( Fig. 9, Table I).
Diplodns anjcntcns (C. and V. ) — Bream. Attempts failed to record a swim-
ming sound from 6 of these fish (Sparidae) approximately 8 inches long in a
Station aquarium. The only sound detected was made by the fish bumping against
the sides and bottom of the tank as a probe was moved gently about the aquarium.
These sounds are not clear and sharp like carangid thumps ; they are variable and
random and are not clearly correlated with describable behavioral phenomena.
Various spear fishermen at Bermuda during 1958, including reliable observers
from the Station, reported hearing sounds underwater produced during evasive
swimming of grouper (Serranidae) and parrotfish (Scaridae), "any large fish,"
and hogfish (Lachnolaiunis nia.viinus, Labridae), as well as grunts of the hamlet
or Nassau grouper (Epinephalus striatus) and volleys of squirrelfish (Holoccntnis
ascensionis) sound (Moulton, 1958).
THE PLAYBACK OF UNDERWATER SOUXD TO FISH SCHOOLS
The playback of underwater sound has been useful in modifying fish behavior
(Moulton, 1956a, 19561); Tavolga, 1956, 1958). The habit of Caranx of feeding
216 JAMES M. MOULTON
on Anchoviella led to playing hack to the Anchoviella school recorded on August
7 recordings of the striclulation sound of C. latns and of the swimming sound which
accompanies its feeding. The transducer was lowered to mid-school level. The
Anchoviella cleared the area beneath the launch more quickly during this play-
hack than during recording. Further, during sound playback, the school showed
a greater tendency to divide than during recording.
A collateral observation is of interest : When playback began of the stridulation
sound at sea, an adult barracuda (Sphyraena barracuda, Sphyraenidae) came
abruptly to a spot about 8 feet from the suspended transducer and lay quietly facing
it for about three minutes. This behavior was unique to all observers, including
Mr. Spurling, an experienced Bermuda fisherman and boat captain. (Mr. Spur-
ling's comment: "I've never seen a barracuda act like that before.")
Stridulating sounds and thumps of young C. latns played back to about 500
Anchoviella in the Station cement tank were accompanied by quickened swimming
and milling which gradually subsided. Approximately one-fourth of the fry moved
to the opposite end of the tank over and under a horizontally suspended hydrophone
which they did not pass spontaneously before sound transmission, and during
hand movements in the tank.
Playback to the impounded school of C. rnhcr recorded at Coney Island on
August 6, as it milled within the net, of a series of pharyngeal tooth stridulations
of C. latns recorded in Station aquaria, initially was accompanied by an immediate
movement of the school to the far end of the net. After some minutes of playback,
the school failed to respond to these sounds and to feeding sounds. These sounds
were listened to by surface divers and sounded like the amplified sounds heard
during recording.
After the swimming sound of the large school of Anchoviella had been recorded
at sea on August 7, the recording was played back into an aquarium containing
young C. latns. The latter showed quickened swimming movements of a non-
directional type. It has already been mentioned that small C. latns may join
Anchoviella schools, feeding on the members of the school; they may be attracted
to the school by its swimming sound.
Pharyngeal tooth rasps played back to the source species (C. latns) in a Sta-
tion aquarium on August 1 appeared to initiate feeding reaction. The fish became
exceedingly active, swimming about furiously, and facing the transducer to nibble
at its rubber surface. After several minutes the activity subsided and the fish
swam about as usual.
SWIMMING MOVEMENTS AND SCHOOLING BEHAVIOR
OF CARANX AND ANCHOVIELLA
The normal behavior of schooling fishes. Caran.v, Trachinotns, and Anchovi-
ella are truly schooling fishes, and whether at sea or in aquaria, groups of these
fishes form cohesive schools. Their behavior bears examination as it relates to
factors maintaining the school at sea.
When adult pompano attacked Anchoviella schools at sea, the former were in
compact school formation, and usually swam directly through the school of fry ;
feeding of Caran.v cr\sos at the periphery of a school was characterized by irregu-
lar darting movements and swift swimming of individual jacks.
SWIMMING SOUNDS OF FISHES 217
At rest in aquaria and at sea, the Anchoviclla school is clearly outlined, although
individuals take independent pathways within the school in daytime as at night.
When the school is streaming, and when a schooling fish (Mcnidia nienidia) of
the Woods Hole area is subjected to a tidal current in a live car, these fishes line
up parallel to each other and veer together upon appropriate stimulus.
The importance of water currents in mediating behavior of fishes is further
underlined by the behavior of small amberjack (2-inch Scriola sp., Carangidae)
lurking under sargassum weed clumps southeast of Bermuda on August 18. The
fish abandoned each clump as the 65-foot Panulirus drifted down upon it. and
swam directly to another clump, often some yards away but always along the same
line of weed and thus within the same convergent zone (Woodcock, 1944, 1950).
Due to a combination of factors, significantly wind and Coriolis force, sargassum
weed in the open sea tends to arrange itself in parallel rows, the rows being spaced
at quite regular intervals under a given set of conditions, and parallel to wind
direction (Woodcock, 1944, 1950). Water currents, probably even micro-currents,
are of great importance in the open sea in determining the distribution of fishes
(see Hasler, 1956) ; persistent sensory orientation to a particular current or moving
body of water may be of considerable importance in maintaining not only the
position but also the cohesiveness of fish schools, particularly through hours of
darkness (Moulton, 1957a).
Members of a single school vary in their behavior. Schools of 500 to 1000
Anchoi'iclla in the cement tank tended to divide at rest into two or three smaller
groups; one group was usually markedly larger than the other (s). These groups,
treated alike from capture, differed in sensitivity to stimuli ; some were easily
frightened, others held their position in the face of a variety of stimuli ( e.g., a
prodding stick or hand).
In a study of variation in reactions to stimuli, a plastic screen confined a single
school of about 1000 Anchoviella to an 8-foot section of the cement tank during
attempts to record swimming sound. The hydrophone was placed across the tank,
3l/2 feet from the end containing the fish. During transmission of jack stridulation
recordings in the area containing the fish, approximately one-fourth of the school
moved past the hydrophone. Pushing down on a small board floating over the
school (in simulation of the knee-bend method) caused approximately one-half
the. remaining fish to pass the hydrophone. Then a hand was waved vigorously
in the water with the balance of the school ; a recalcitrant two dozen fish still
remained in the smaller portion of the tank, gathered in a cohesive school.
Young Caran.r lotus tend to school with Anchoviclla; in an aquarium, a school
of 12 of the former formed a tight formation immediately above the back of a
10-inch squirrelfish (Holoccntnis ascensionis, Holocentridae). They turned with
the squirrelfish as it moved slowly about the tank ; the latter made no move toward
the jacks. Another group of 20 C. latits was confined for several hours in an
aquarium with an adult spiny lobster (Patiulints argus). So long as they were
contained together, night and day, the C. latits remained at the top of the tank
in a tight school. The spiny lobster was removed one night under artificial light,
and at once the 20 jacks dispersed over the bottom of the aquarium and assumed
a striking striped pattern completely unlike their usual drab coloration.
Experiments on blinded fish. In order to examine factors other than vision
218 JAMES M. MOULTON
mediating the schooling of fish, adult Anchoviella were blinded by l)ilateral eye
removal under anaesthesia (MS222 1 : 3000, Sandoz Pharmaceuticals) and allowed
to recover, or became blinded through eye loss in a syndrome developing within
hours of capture in a school held in the cement tank. On successive trials, a blind
individual was placed in an aquarium with 12 normal individuals. When the
latter were at rest, individuals milling slowly about, the blinded individuals swam
through the group and about the aquarium generally until reaching the side of the
tank, then took up new headings ; they did not orient to the normal fish.
If the normal fish were startled by a hand movement next to the aquarium,
they streamed and veered as did large schools at sea ; at such times, the blinded
individual immediately joined the normal fish in school formation and behaved as
did the latter. Similarly, in the cement tank, blinded Anchoviella moved with the
larger group of normal fish during streaming movements. When the normal fish
came to rest again, blinded individuals returned to random movements, while even
at rest the normal fish maintained approximately equal intervals between indi-
viduals. Isolated blinded fish did not respond to hand movements adjacent to
the aquarium.
Unilaterally blinded Anchoviella maintained position with the normal fish when
the latter were within the field of vision. This latter observation is in agreement
with observations obtained on Menidia behavior at Woods Hole in 1957, where
the experiments were performed in a live car suspended from a raft in Great
Harbor ; bilaterally blinded fish swam directly through groups of normal fish at rest
and oriented independently of normal fish in currents flowing through the live
car, while unilaterally blinded specimens maintained position with schools located
at least in part on the side of the unoperated eye (Moulton, 1957a).
While movements of the individual members of the fish school are important
in maintaining the remarkable integrity of the school, the members of the school
are probably sensitive to pressure waves created by the school and probably orient
to these waves ; the movements of the school as a whole are probably important
in maintaining its own integration.
Anchoviella did not disperse through the cement tank after dark as Jenkinsia
(Breder, 1951) and chub mackerel — Scomber colias, Scombridae ( Shlaifer, 1942)
—do in aquaria. Schools of 500 to 1000 were in tight formation in the center of
the cement tank in resting behavior during several examination periods on dark
nights when an electric light was turned on, and behavior did not change notably
with the light ; the basement-located tank was too dark on moonless nights for me
to see the fish in it.
C. latns in small numbers confined alone in darkness for several hours did not
noticeably change their distribution when a light was turned on. Neither C. latns
nor Anchoviella seems to depend so markedly on light for schooling formation as
apparently do Jenkinsia and Scomber.
A GHANA FISHERY DEPENDING ON SWIMMING SOUND
A. P. Brown, in an anthropological account of the fishing industry of the Labadi
District of the Gold Coast (Irvine, 1947, p. 25), mentioned the three-pronged
paddles found in the sea-going canoes of the Coast, without indication as to their
SWIMMING SOUNDS OF FISHES 219
use. Inquiry was made subsequent to a United Nations news release suggesting
an application to the fisheries.
The paddle, for possession of one of which I am indebted to Mr. D. A.. Ham-
mond of the Fisheries Department, Accra, is in typical form 4 feet 9 inches in
length with a handle 1% inches in diameter. The foot-long flattened blade termi-
nates in three broad, blunt teeth, comprising 4H inches of the blade length, each
approximately 2 inches in width at the base, the center one tapering slightly more
than the other two. Mr. Hammond describes the use of this paddle as follows :
'The use of the paddle as (a) hearing aid in fishing is well-known to our
fishermen from Ada to Takoradi. It is particularly used by the herring fishermen.
'Herring' here refers to Sardinella aurita and S. camera onensis. It is also used,
however, to detect shoals of shad (Ethmalosa dorsalis), long-firmed herring (Ilislia
inclanota ) and cassava fish (Cynoscion senegalla). (Identifications from Irvine,
1947).
"Where there are no surface fish visible, the method is to place the broad part
of the paddle in the water over the stern of the canoe, place the ear to the top of
the handle, and rotate the paddle very slowly. Only skillful and experienced
fishermen are appointed to do it. The face of the paddle acts as a sounding board
and receives the vibrations which it transmits to the hearer. By rotating the
paddle, the fisherman is able to get the direction of movement of the fish and from
the intensity of the sound, he can judge how (distant) the fish (are). Those fish
which move in large shoals — for example, herring or shad, as well as the large
rocks in the sea, make distinct sounds. From the sound, the fisherman can tell
whether it is a fish or rock. The sound of the herring is characteristic and never
mistaken. This method is particularly successful in waters of 7 to 10 fathoms
and between 11 P.M. and 3 A.M. Those paddles which are particularly good are
treasured. Some of them are very old." A variety of the Ghana-type paddle
is said to be used for a similar purpose in Liberia ( Mr. William Watkins, personal
communication) .
Underwater listening occurs in various parts of the world as an important aspect
of commercial fisheries (Cousteau, 1953; Kesteven. 1949; Marshall. 1954; Parry.
1954; Westenberg, 1953). It is likely that in the fishery described by Mr. Ham-
mond, the swimming sound of the clupeids (sciaenids may produce other sounds)
is conveyed by the three-pronged paddle to the fisherman's ear.
DISCUSSION
The schooling behavior of teleosts probably depends primarily on vision in
the daytime (Shlaifer, 1942; Breder. 1951). Yet schools of Anchoviella, Clupea
harciif/Hs and other schooling fishes maintain their schools at sea through hours
of darkness ; herring fishermen depend on this fact in "lighting up" herring schools
at night ( Moulton and Backus, 1955). While it may be that light stemming from
luminescent organisms and that of moon and stars on clear nights may afford suf-
ficient stimulus to abet schooling at night, some conditions of darkness at sea
undoubtedly preclude vision and require other stimuli as predominant in providing
for maintenance of the fish school at night. Certainly other factors than vision
enable blinded fish (Anchoi'iclhi, Mcuidia) to move with their respective schools.
220 JAMES M. MOULTON
The possible role of water currents in maintaining fish schools at sea has been
briefly discussed.
Significant factors in low frequency sensitivity of teleosts are the lateral line
and isolated cutaneous receptors (von Frisch, 1938; Griffin, 1950; Lowenstein,
1957). Tracy ( 1920a, 19201)) and others have suggested that the connection
between air bladder and inner ear of clupeids (engraulids possess a similar con-
nection— Berg, 1947) may be more important as a pressure control device related
to gas physiology of the air 1 (ladder than to hearing facility. While it is not
unlikely that the paired passages may serve in both capacities, the remarkable
sensitivity of Anclwciclla, as well as of some clupeids (Moulton and Backus, 1955;
Aloulton, 1956a), to pressure waves in the water is suggestive of special adaptations
to reception of pressure changes.
Anchoi'iclhi are sensitive to very slight pressure waves in the water; the swim-
ming sounds described fall within the frequency range of sensitivity of all fishes
studied in this connection. It is now simply conjectural, although some supporting
evidence has been presented, to suggest that the swimming sounds of various
carangids and of Anchoviella furnish a mechanism in addition to water currents
and visual sensitivity by which schools of fish at sea are maintained under a variety
of circumstances. Either these sounds or the body movements from which they
stem must be a primary factor.
The swimming sounds described are to a degree species-specific. Shlaifer
(1942) observed that chub mackerel, pairs of which will school together, will not
school with abnormally moving fish of the same species. Normal Menidia and
Anchoviella do not arrange themselves in schooling formation with blinded indi-
viduals ; the latter will school with the former. It is suggested that the fish school
at sea is maintained by a number of factors, given the proper amounts of food and
minimum predation : sensitivity to particular water currents and masses, vision,
and the behavior of the school as a whole which acts an an acoustical core and as
a wave pressure source to which individuals of the schooling species orient. It is
suggested that the swimming sound is not simply a mechanical or accidental sound,
but that it possesses biological significance. It is demonstrated that source species
and other species may react to amplified swimming sound.
Another factor influencing the maintenance of the fish school is reflected in dif-
ferential sensitivity of Anchoviella to increasingly dispersive stimuli. After a series
of such stimuli, a core of recalcitrant individuals remains in the test area in an
experimental tank, and may furnish the nucleus around which more sensitive
members of the school fluctuate at sea ; streaming and veering may orient around
a group of relatively insensitive individuals forming an "anchor" to the school.
It is clear that the swimming sound of different species of schooling fishes
(Anchoviella, Carau.v, Trachinotus) varies in its characteristics with species as well
as with size of the fishes concerned. Large schools of fishes as small as Anclwri-
clla (about 3 inches) and as large as Carau.v nibcr (about 1 foot) introduce con-
siderable sound into the water. This may in future be utilized in experiments on
schooling fishes in waters less clear than those studied, to test influence of various
stimuli on schooling fishes and to explore sensitivity to these stimuli. Although
Shishkova (1958a) abandoned the hope of stemming acoustically the flow of an-
chovies from the Russian Sea of Azov into the Black Sea, and of masking ship
SWIMMING SOUNDS OF FISHES 21
noise with swimming sound, other possible uses of swimming sound exist; a like-
lihood is present that fishermen can control acoustically to some extent the move-
ments of clupeid and engraulid schools.
The characteristics of these sounds are more like those of sounds produced
under circumstances related to defensive behavior of fishes than of sounds produced
under other circumstances (Moulton, 19571)). The sound attendant on veering-
ma}- have some protective value. If this were true, large schools of herring and
anchovies would be more resistant to predation by other fishes than would small
schools. The joining of Anchoviella schools by young C. latits may be primarily
protective for the latter ; their feeding on the host school would simply indicate
poor "guestmanship."
The swimming sounds described, as well as sound stemming from the move-
ment of any large fish in the sea, are probably due to a number of factors ; hydro-
dynamic noise, to which Shishkova relates all of the sound, skeletal movements, and
contraction of axial musculature during strong swimming movements against a
gas-filled, resonating air bladder. None of the sounds described are continuous
even during steady streaming movements, indicating that they stem from the bodies
of individual fishes during muscle contraction, rather than from the hydrodynamic
effects of continuous water flow around fish bodies.
All of the swimming sounds studied lie below 2 kc., and thus are of lower
frequencies than many sounds created by fishes by organs apparently specialized for
sound production (Moulton, 1958). These results differ from those of Shishkova
(19581)) who found components of T. trachurus swimming sound at 16 kc. (It
seems likely that her fish were chewing or stridulating during recording.) The
frequency span of greatest intensity of swimming sound, as indicated by darkening
of vibragrams, is lower for large fish than for small (Table I).
After submission of the manuscript of this paper for publication, the author's
attention was called to a recent extensive study of social groupings in fishes by
Breder (1959). Of special pertinence to the present work is Breder's caution that
the responses of members of a fish school to light and darkness will vary with indi-
viduals, and that light exposure which fish have received earlier will be of great
importance.
The author is grateful to Dr. Richard H. Backus for constructive criticism of
the manuscript of this paper.
SUMMARY
1. The swimming sounds of four schooling species of Bermuda fishes are
described. The sounds stem from streaming and veering of schools at sea.
2. Responses of schooling species to playback of sounds stemming from schools
are described. Exploitation of these responses in experimental and commercial
fishing is suggested.
3. On the basis of observation at sea and of laboratory experiments, mecha-
nisms useful in maintaining formation of schools at sea are discussed. These include
vision, water currents and pressure waves initiated by the movements of fish bodies
and the sounds they engender.
JAMES M. MOULTON
4. An African acoustical fishery, probably relying on the swimming sounds
of fishes, is described.
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Part 2.
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trawler noise. ) Ryhnoye Khozyayst-i'o, 34, No. 3 : 33-39.
SHISHKOVA, E. V., 1958b. (Recording and analysis of noise made by fish.) Trudy J'NIRO,
36 : 280-294.
SHLAIFER, A., 1942. The schooling behavior of mackerel. Zoologica, 27: 75-80.
SWIMMING SOUNDS OF FISHES
TAVOLGA, W. X., 1956. Visual, chemical and sound stimuli as cues in the sex discriminatory
behavior of the gobiid fish Bathygobius soporator. Zoologica, 41, Part 2: 49-64.
TAVOLGA, W. N., 1958. The significance of underwater sounds produced by males of the
gobiid fish. Bathygobius soporator. Physiol. Zool., 31 : 259-271.
TRACY, H. C., 1920a. The clupeoid cranium in its relation to the swimbladder diverticulum
and the membranous labyrinth. /. Alorph., 33 : 439-483.
TRACY, H. C., 1920b. The membranous labyrinth and its relation to the precoelomic diver-
ticulum in clupeoids. /. Coinp. Neural., 31: 219-257.
WKSTEXBERG, J., 1953. Acoustical aspects of some Indonesian fisheries. /. du Conscil pour
I' Exploration dc la Mcr, 18: 311-325.
WOODCOCK, A. H., 1944. A theory of surface water motion deduced from the wind-induced
motion of the Physalia. J. Mar. Res., 5 : 196-205.
WOODCOCK, A. H., 1950. Subsurface pelagic sargassum. /. Mar. Res., 9: 77-92.
BINUCLEATE AND TRINUCLEATE OOCYTES IN POST-OVULATION
OVARIES OF RANA PIPIENS x
CHARLES L. PARMENTER. MARVIN DEREZIN AND
HAZELTENE S. PARMENTER 2
Di-i-isiaii af Biology, l^nirersity of Pennsylvania. Philadelphia 4. Pcniia.
The senior author had long been interested in the means by which diploidy can
be produced in parthenogenetically developed frogs and larvae (Parmenter, 1920,
1925, 1933, 1940, 1952). He demonstrated (1952) the existence of the diploid
chromosome number in three mature virgin eggs and considered this to be one
source of diploidy with a known history of no delay in cleavage. He had found
one instance (1940) of a diploid many-celled embryo in which, by direct observa-
tion, he knew that both polar bodies were given off and that it developed with
no delayed cleavage. The discovery of a small number of binucleate ovarian
eggs suggested another important possible source of such diploid parthenogenetic
individuals.
In the course of undergraduate research in which nuclei were dissected from
o
ovarian oocytes of Rano pipicns, several eggs were found which possessed two
completely separate nuclei, and it was established that the binucleate condition
was not an artifact of manipulation. The eggs used were in those stages where
the nuclei had reached their fullest growth (stages 4, 5, and 6 of Duryee, 1950).
Subsequently, a method was devised for scanning in ovarian tissue a large number
of transparent oocytes in such early stages (stages 1 to 3 of Duryee, 1950) that
the yolk would not interfere with direct observation of the cells /';/ situ. Tt was
hoped eventually that the chromosome content could be determined.
MATERIAL AND METHODS
Technique. The sac-like ovaries of female frogs in the post-ovulated condition
were opened along their edges in Ringer solution by means of fine-pointed jeweler's
forceps. The two halves, or sometimes individual pieces only, were floated onto
a slide. They were scanned under low power ( 16 mm. objective ) in the living
condition, or were fixed in Bouin's fluid (which served as a stain), dehydrated,
and mounted in damar. In all cases the slides were systematically surveyed and
./ *> *
all eggs counted in which there were visible nuclei. Some cells were examined
and measured first unfixed and then fixed.
1 Mrs. Parmenter acknowledges with sincere appreciation the encouragement and helpful
advice given her in the writing of this paper by Professor D. H. Wenrich ; also the kind sug-
gestions of Professors D. R. Goddard, W. R. Duryee, Gerhard Fankhauser, and others.
Supported by USPHS Grant RG-S482.
2 All of the data herewith presented were collected and organized by Dr. Parmenter,
assisted by Mr. Derezin. Unfortunately Dr. Parmenter died before the writing of the paper,
which subsequently was undertaken by his wife.
224
BINUCLEATE AND TKt NUCLEATE OOCYTKS
..
225
FIGURE 1. Binucleate #2 photographed hi sifit in unfixed ovarian tissue. Adjacent is
the mononucleate used as a control for comparative measurements. Included also are younger
oocytes and an older yolk-filled one. 430 X
Measurements. Three different methods of measurement of egg and nucleus
diameters were employed, depending on the exigencies of the moment.
Fixed eggs : When an egg with two nuclei was found, it was measured with an
o o oo
ocular micrometer in at least three or four diameters to correct for any variation
from the spherical condition. For comparison as a "control" a single-nucleated
cell of approximately the same size was located as close in the field as possible
and its diameter measured. Similarly the nuclear diameters of both eggs were
determined. From these data the volumes of the eggs and of their nuclei were
calculated, assuming them to be spherical.
Fresh unfixed material was treated in one of two quite different ways : Usually
the living binucleate was photographed in situ, together with a nearby mono-
nucleate. In two cases both were fortunately in the same field. (For one, see
Figure 1.) \Yithout changing the setting of the microscope or camera, a stage
micrometer was also photographed. The films were processed together and en-
larged to the same magnification. Measurements were made by means of these
photographs. Occasionally, when a camera was not available, camera lucida draw-
ings were carefully made of the living binucleate, its control, and the stage mi-
crometer, and measurements obtained from these drawings.
Extreme care was taken with all calibrations so that the measurements as finally
presented in the tables are comparable with each other.
OBSERVATIONS
Fifty-six binucleate and two tnnucleate eggs were found among the 249,616
small transparent primary oocytes observed. This total represented all the eggs
226 C. L. PARMENTER, M. DEREZIN AND H. S. PARMENTER
with visihk' nuclei found in 25 frogs. Thus the binucleates constituted a percent-
age of 0.022/f or a ratio of 1 to 4,457 ova.
The percentages of multinucleates in the frogs deviated significantly from one
another. Fifteen of the 25 females possessed no multinucleated eggs at all. Of the
10 with multinucleated oocytes, 8 contained very few each, only one to four. How-
ever, in the ovaries of two individuals a large number of ova with two nuclei
were found, namely 14 or 0.093^ in one (#44), and 24 or 0.175% in the other
( #39). This indicates that certain females are more prone to produce the multi-
nucleate condition than others where it is determined by chance. This situation,
while certainly of physiological significance, is not unusual. The literature abounds
with similar cases. Indeed, Parmenter's ( 1952) rare diploid metaphases were
found in three virgin eggs, all of which came from the same female, whereas none
appeared in eggs from 11 other frogs. He discussed the literature in some detail
(pp. 253-254).
There was considerable variation in the number of eggs found in each frog.
The range was from 5,426 to 16,066. There did not appear to be any relationship
between the abundance of ova in a female and her size as indicated by the length,
measured from nose tip to anus.
Three of the 25 frogs contained ovaries with considerably more eggs than the
others, 13,687-16,066. The two most productive of multinucleates were among
these, but a frog with 13,717 eggs possessed only one binucleate. Moreover, the
first three eggs which were found with two nuclei were in an individual with small
ovaries containing a total of only 5,632 eggs.
One could not predict, therefore, from external conditions such as the size of the
animal, or abundance of eggs, whether multinucleate oocytes were likely to be
present.
The sizes of those oocytes containing either two or three nuclei varied in
diameter from very small, 0.099 mm., to 0.350 mm. (Table I). Nine ova were
smaller than 0.200 mm., i.e., in "stages 1 or 2" (Duryee, 1950). The rest, in-
cluding the two trinucleates, were all in "stage 3." The majority of the oocytes
( 20 ) were found to measure between 0.200 and 0.300 mm. Even in one female
the multinucleated eggs varied markedly in size. In female #39 which produced
the 24 binucleates the diameters of the oocytes ranged from 0.163 to 0.323 mm.
with most from 0.200 to 0.300 mm. Also in frog #44 (14 hi- and 1 trinucleate)
the variation included the tiniest egg of all (0.099 mm.) and extended to 0.292 mm.
It is recognized that within an ovary a condition of egg growth is in progress
with the various stages of growth distributed indiscriminately throughout the
structure. A suggestion of this is seen in Figure 1 where one can observe a yolk-
filled egg of a later stage adjacent to the mono- and binucleates which were com-
pared, and nearby much smaller eggs. It is thus easy to see how the oocytes to
be compared were chosen. The senior author was much concerned with the
possibility that, due to the extended growth period of primary oocytes, the cells
compared, although of the same size, might not have been growing for the same
length of time. This difference in the age of the oocytes would not affect the
validity of the observations, merely the conjectures as to interpretation.
In an attempt to find a clue to the chromosome content of each nucleus, the
nuclear volumes of the hi- or trinucleated cells were determined and compared
with that of the nucleus of the normal mononucleate which would serve as a "con-
BINUCLEATE AND TRINUCLEATE OOCYTES
TABU-: I
Occurrence of biniicleate and trhnicleate oocytes in frogs (Rana pipiensi
A. Biniicleate oocytes
Biniicleate
oocyte
number
Frog
number
Nuclear volume
s (mm. 3 X ID-4)
Cell diameters (mm.)
Binucleates
Mono-
nucleate
control
Biniicleate
Mono-
nucleate
control
Nucleus Nucleus
a b
Sum of
a and b
Binurleates with the volume of both nuclei approximately equal
1
36
4.94
4.94
9.88 12.3 0.350
0.334
2
36
5.24
5.24
10.5 10.2 0.275 0.260
3
36
6.83
6.83
13.7 10.8
0.303 0.291
8
39
14.2
13.0
27.2 36.0
0.339
0.343
12
39
7.05
6.28
13.3 17.7
0.290 0.290
13
39
5.11
5.72 10.8
7.59 0.215 0.220
14
39
10.4 8.64 19.0
15.3 0.289 0.266
15
39
7.27
6.59 13.9
11.9 0.263 0.265
18
39
2.35
2.04 4.39
3.79 0.162 0.161
19
39
7.16
7.43
14.6
14.0
0.272
0.271
21
39
3.97
4.18
8.15
6.74 0.209 0.208
23
39
5.89
5.89
14.2 15.7 0.280 0.256
24
39
6.59
7.59
14.2 11.5 0.239
0.234
27
39
5.89
5.75
11.6 11.5
0.248
0.276
28
39
10.0
12.5
22.5
23.5
0.323 0.321
29
42
3.79
4.08
7.87
6.09
0.191
0.190
31
44
5.59
5.36
11.0
7.59
0.215
0.225
35
44
1.75
1.65
3.40
3.52
0.137 0.137
36
44
9.14
8.04 17.2 13.1 0.258 0.247
37
44
0.662
0.593 1.26 1.75 0.099 0.102
38
44
5.02
5.46 10.5 21.7
0.292 0.305
39
44
2.86
3.17
6.03 7.00
0.193
0.199
41
44
3.62
4.49
8.11
11.9 0.256 0.251
44
44
3.52
3.52
7.04
5.11
0.183 0.177
45
44
1.03 0.98
2.01
1.81
0.114
0.113
48
48
3.42 4.49 7.91
9.32 0.207 0.215
49
48
7.01
6.89 13.9
15.8
0.245 0.245
51
51 6.09 5.02 11.1
9.07
0.229
0.223
57
59
0.95
0.95
1.90
2.35
0.119
0.120
Binucleates with markedly unequal volumes
30
44
1.19
0.39
1.58
1.25
0.113
0.112
43
44
8.04
4.90
12.9
8.04
0.237 0.237
B. Tritiucleate oocvtes
Trinucleate
oocyte
number
Frog
number
Nuclear volumes (mm3 X 10 4)
Cell diameters (mm.)
Trinucleates
Mono-
nucleate
control
Tri-
nucleate
Mono-
nucleate
control
Nucleus
a
Nucleus
b
Nucleus
c
Sum of
a. b. and c
1
44
5.96
6.09
6.34
18.4
15.1
0.271
0.266
2
51
3.01
4.37
4.26
11.6
9.32
0.212 0.214
228 C. L. PAKMKXTKR. M. DEREZIN AXI) H. S. PARMENTER
trol." The sum of the volumes of the twin nuclei was approximately equal to that
of the single one chosen for comparison in 29 measurable cells, and markedly
unequal in 2 (Table IA). Four others possessed what appeared to be unequal
nuclei, but due to misshapen cells or nuclei, or visible shrinkage in one or two
cases, meaningful measurements seemed impossible. These were omitted from the
table, as were three ova with two nuclei of apparently the same size where the con-
ditions were such that the measurements were questionable. The other twenty
eggs, although clearly binucleate, could not properly be measured.
In none of the binucleates did the volume of either of the two nuclei approach
that of the selected "control," except in the case of egg #43. It will be seen from
Table I that the larger nucleus had exactly the same volume as that of the mono-
nucleate control, and the smaller was somewhat more than half that volume. One
other difference between the two nuclei of egg #43 besides size should be men-
tioned. There is a conspicuous dissimilarity in the granular appearance. The
smaller one resembles the usual binucleate in the peripheral arrangement of the
large chromatic granules (nucleoli), whereas in the larger nucleus these bodies
tend to be smaller and distributed more uniformly throughout the nucleoplasm.
The total volume of the twin nuclei that were of the same size approximately
equalled that of the single nucleus of the cell chosen for comparison (Table IA).
There was one exception also to this statement in the case of egg #38. In this
interesting cell the sum of the volumes of the two nuclei was only about one-half
the volume of the nucleus of the mononucleate. Measurements of additional control
cells confirmed this relationship.
While searching for the cells with two nuclei, unexpectedly the two trinucleates
were found. These conformed to the general pattern for binucleates in that the
diameters of the cells were in the same size range, and the sum of the volumes
of the three nuclei approximated that of a "control" mononucleate. Both were
located in ovaries containing cells with two nuclei also. Trinucleate #1 was found
in the same lobe as binucleate #43 which possessed the large nucleus equal in size
to that of the control. The three nuclei of trinucleate #1 (Table IB) were of
about the same size. Since in both cases the three nuclei were at different levels
within the cell a photograph was not feasible. In trinucleate #2 two of the nuclei
were larger in size than the other, and equal in volume to each other. Interestingly,
a similar difference in granular appearance existed between the two large nuclei
and the smallest one as was described for binucleate #43. The small nucleus had
fewer and larger chromatic granules whereas the two larger nuclei possessed
smaller and more numerous ones.
DISCUSSION
The following ideas were found among Doctor Parmenter's notes. No further
discussion will be attempted.
The unequal-sized binucleates and the trinucleates suggest that a possible ori-
gin may have been from separated groups of chromosome vesicles. But such an
origin does not seem probable for the large proportion of binucleated oocytes, the
nuclei of which were of equal volume. More likely this condition arose by a
nuclear division followed by a failure of cytosomic division. These binucleated cells
may have been produced during the last oogonial division.
BIXUCLKATK AND TRIXUC1.KATK OOCYTES 22°
Should further information demonstrate that in some cases each of the two
equal-sized nuclei contains the full set of 13 tetrads, hoth the normal behavior and
the possible failure of one of these nuclei to give off its polar body in either meiotic
division would produce various chromosome numbers in mature eggs and in em-
bryos resulting either from parthenogenetic stimulation or from fertilization.
LITERATURE
Information in the literature concerning multinucleate oocytes is sparse. In-
deed Humphries (1956) in discussing the origin of spontaneous polyploidy in
Trititnts 1'iricicsccns hesitated to "assume" (p. 120) the existence of a binucleate
oocyte as a source of diploidy in such embryos. He preferred the explanation of
(p. 120) "direct pathways actually seen to exist," one of which he describes in his
paper on the effects of heat shock on the first meiotic division. We have now
shown that binucleate primary oocytes actually do exist, at least in Ratio pipicns.
Beatty (1957) who reviewed the literature on polynuclear ovarian eggs, states
that such are (p. 81) "rare but widely spread." He mentions twelve species of
mammals (including man), reptiles, birds, insects, but no amphibians. Sentein
(1958) was able to produce multinucleated eggs of Tritiirus and Pleurodelcs by
treatment with phenol. He saw cytoplasmic division inhibited. This treatment
constitutes, of course, an unnatural source of the poly nucleate condition.
In embryonic tissue Parmenter (1937, 1940) reported multinucleated erythro-
cytes in parthenogenetic frog larvae, also five epithelial cells each with two nuclei
plus one with three. Moore (1957) has presented evidence that chromosomal
vesicles constitute a basis for the origin of what she refers to as "double nuclei"
(p. 209) in early embryos of diploid frog hybrids. She reviewed the literature
extensively. Of interest here is her discussion of the distribution of peripheral
coarse vs. diffuse fine chromatin in some of the cell nuclei of her material. She
states that similar conditions were also found by King and Briggs, by Brachet,
and by others. She wonders if some of the (p. 222) "so-called nuclear anomalies"
found in hybrids are not really of (p. 222) "normal occurrence in the development
of amphibian eggs."
The occurrence of the hi- and multinucleate condition normally in liver tissue
of mammals including human is well known and has been reviewed recently by
Inamdar (1958). By microspectrophotometric measurements of DXA in resting
nuclei of mouse liver, he was able to confirm the conclusions of Beams and King
(1942) and others that the origin of the binucleates is best explained by division
of the nucleus without division of the cell.
No discussion will be undertaken on the often-noted polynucleate condition in
tissue cultures, or in pathological material.
SUM MARY
1. Fifty-six binucleated and two trinucleated cells were found among 249,616
young transparent primary oocytes in post-ovulation ovaries of 25 females of
Rana />//>/>».<• (0.022%).
2. Multinucleated oocytes were absent in 15 females, present in 10. Eight
of these 10 produced only one to four binucleates each; one female was the source
230 C. L. PARMENTER, M. DEREZIN AND H. S. PARMENTER
of 1 tri- and 14 binucleates; and another gave the remarkahle number of 24
(0.175%), all binucleates.
3. The multiple nucleated condition did not seem to be correlated in any way
with the size of the female, the abundance of her eggs, nor the size of the egg.
4. In none of the binucleates did the volume of either of the two nuclei ap-
proach that of the nucleus of a mononucleate of the same size. The one exception
was egg #43 where one nucleus did have exactly the volume of the control, the
other about half that.
In 29 of the 31 measurable binucleates the two nuclei were of approximately
the same size and the sum of the two volumes equalled that of the mononucleate,
except in one case where it was half.
The origin of binucleate oocytes remains uncertain ; it may be connected with
a final division of an oogonial nucleus that was not followed by cell division. In
this case the two nuclei would both be diploid.
5. The two trinucleates conformed in general to the same pattern as the bi-
nucleates as to their distribution, size of oocyte, and the volumes of their nuclei.
The sum of the three nuclear volumes approximated that of the mononucleate.
6. In two cases where the nuclei were markedly unequal in size, there was
a definite difference in the appearance of their chromatic granules. These bodies
were more abundant and finer in the larger nuclei, peripheral, larger and more
distinct in the smaller nucleus.
LITERATURE CITED
BEATTY, R. A., 1957. Parthenogenesis and polyploidy in mammalian development. Cam-
bridge Monographs in Experimental Biology 7. Cambridge University Press.
BEAMS, H. W., AND R. L. KING, 1942. The origin of binucleate and large mononucleate cells
in the liver of the rat. Anat. Rcc., 83: 281-297.
DURYEE, W. R., 1950. Chromosomal physiology in relation to nuclear structure. Ann. N. Y.
Acad. Scl., 50: 920-953.
HUMPHRIES, A. A., JR., 1956. A study of meiosis in coelomic and oviducal oocytes of Triturus
I'iridesccns, with particular emphasis on the origin of spontaneous polyploidy and the
effects of heat shock on the first meiotic division. /. Morph., 99: 97-136.
INAMDAR, N. B., 1958. Development of polyploidy in mouse liver. /. Morph., 103: 65-86.
MOORE, B. C., 1957. Chromosomal vesicles and double nuclei in amphibian embryos. J . Morph.,
101: 209-225.
PARMENTER, C. L., 1920. The chromosomes of parthenogenetic frogs. /. Gen. I'hysiol.. 2:
205-206.
PARMENTER, C. L., 1925. The chromosomes of parthenogenetic frogs and tadpoles. /. Gen.
Physiol., 8: 1-20.
PARMENTER, C. L., 1933. Haploid, diploid, triploid, and tetraploid chromosome numbers, and
their origin in parthenogenetically developed larvae and frogs of Ra>ia pipicns and
Rana palustris. J. £.r/>. Zoot., 66: 409-453.
PARMENTER, C. L., 1937. Mono-,di-,tri- and tetra-nucleate blood cells in parthenogenetically
developed Rana fusca tadpoles. Anat. Rcc., 70 (Suppl. 1) : 129.
PARMENTER, C. L., 1940. Chromosome numbers in Rana fusca parthenogenetically developed
from eggs with known polar body and cleavage histories. /. Morph., 66: 241-260.
PARMENTER, C. L., 1952. Diploid virgin frog eggs ; a possible origin of diploid partheno-
genetically developed frog larvae without delay in cleavage and of triploid larvae
developed from fertilized eggs. /. Morph., 90 : 243-261.
SENTEIN, P., 1958. Action du phenol sur les mitoses des segmentation des oeufs d'amphibiens.
.Ich, Anat., 34: 201-234.
COLD DEATH IX THE GUPPY l
RONALD B. PITKOW^
Department of Physiology, Scliool of Medicine.
I'nircrsity of Pennsylvania, Philadelphia, Pennsylvania
There are numerous reports describing the death of vast numbers of fish subse-
quent to cold waves, often where the water temperature remained well above 0° C.
(Wilcox, 1887; Verrill, 1901; Vatova, 1929; Storey and Gudger, 1936; Storey,
1937; Miller, 1940; Gunter, 1941; Gunter, 1947; Mosevich, 1944).
Maurel and Lagriffe (1899) divided the changes induced by placing fish into
colder and colder water into five stages. Stage five consisted of convulsions, loss
of equilibrium, complete paralysis, and a state of apparent death; the fish would
revive only if warmed within a few minutes. If not warmed, they would go on to
die without showing any visible signs of life. Doudoroff (1942) called this
"primary chill coma."
At less extreme temperatures a different sequence of events occurred. The fish
would recover from the initial shock, resume respiration, if it had stopped, and
take on a relatively normal appearance. If kept at such a temperature for some
hours or days, the fish would show increasing distress and finally cease to respond
to stimuli and to respire. This characteristic response to less extreme tempera-
tures, Doudoroff (1942) called "secondary chill coma." In "primary chill coma"
the fish die without a return of vital signs, unless rewarmed ; in "secondary chill
coma." vital signs, if they disappear, return for a time and the fish then gradually
die unless rewarmed.
Virtually nothing is known of the mechanism of death due to "primary chill
coma." Brett (1952) experimented with cold tolerance in young Pacific salmon
and concluded that death occurring in the first hour of a cold exposure was prob-
ably due to a disturbance of the central nervous system. It has been shown that
with decreased acclimation temperature a lower temperature is required to induce
"primary chill coma" (Samochvalova. 1938) and the fish can withstand longer
exposures to a lethal cold temperature (Fry, Brett and Clawson, 1942; Doudoroff,
1942; Brett, 1956).
Only Doudoroff (1945) has proposed a mechanism of death due to "secondary
chill coma." He showed that the death of Fiindnliis, a salt-water fish, at slowly
lethal temperatures in sea water was preceded by osmotic dehydration of tissues,
and was delayed in diluted sea water. He concluded that osmoregulative failure
was one of the causes of slow death in cold sea water.
The characteristics of cold tolerance in the guppy, Lcbistcs rcticulatits, have
never been thoroughly explored. The guppy is an ideal subject for inquiry into
1 Supported by a National Foundation for Infantile Paralysis Research Fellowship, and
Public Health Service Training Grants to the author, and by a National Science Foundation
grant to Dr. John R. Brobeck.
- Present address : Abington Memorial Hospital, Abington, Pennsylvania.
231
232
RONALD B. PITKOW
the effects of cold on fish. It is hardv, inexpensive, and easily procured. Afore
than 4500 guppies were used in the past three and one-half years to secure the
information presented in this paper.
This paper will propose for the first time a possible mechanism of death due
to "primary chill coma" in fish.
METHODS AND MATERIAL
Approximately 1000 guppies were obtained from aquatic plant tubs in the
greenhouses of the University of Pennsylvania, Department of Botany. The
remainder were bought from Florida fish farms. The fish were acclimated for
two weeks to five-, ten-, or fifteen-gallon stock aquaria, with up to 40 fish per
gallon of w?ater. The fish were fed various commercial brands of dry fish food
and Dash dog food once daily.
Randomly chosen fish were taken from stock aquaria by means of a soft net
and quickly placed into the test water which was maintained at uniform tempera-
ture (0.1° C.) by gentle shaking of the container unless otherwise stated, by the
gradual addition of ice to a constant temperature bath into which the test container
FIGURE 1. Comatose fish secured to a notched glass slide
with a soft cotton thread.
was immersed. Temperatures were read from standardized mercury thermometers
graduated to 0.1° C. or 0.05° C. At the completion of a test exposure or a series
of stepwise test exposures the fish were quickly removed and placed in water from
the stock tank.
Once a fish regained normal respiration and was able to swim normally after
a test exposure, it almost invariably lived. Those fish alive 24 hours after test
exposures were considered as having survived the insult. Deaths after this 24-hour
period were few, always less than 5%, and seemed to depend mostly on the general
condition of the fish prior to the cold exposure.
In the experiments in which fish were subjected to cold in oxygen-depleted
water or oxygen-enriched water, the water was vigorously bubbled and equilibrated
with 100% nitrogen or 100% oxygen for one hour immediately prior to its use.
In approximately one-half of these experiments a sample of test water was siphoned
directly into a sampling jar at the midpoint of the cold exposure and immediately
subjected to a Winkler oxygen determination. In all the experiments where a
Winkler oxygen determination was performed, distilled water was used in the
COLD DEATH IX THE GUPPY 233
test container, rather than stock tank water, so as to minimize the amount of
organic material which might interfere with the accuracy of the oxygen determina-
tion. The substitution of distilled water for stock tank water did not alter the
results. In those experiments where an oxygen determination was not performed,
the oxygen content was estimated and rounded off to the nearest 5 mg./L.
Some of the data to be presented are observations on individual fish of the
relationship of respiration to the presence of a functioning circulation. Gill and
mouth respiratory movements were easily seen with the naked eye. The criterion
for the presence of a functioning circulation was the presence of blood flow in the
arteries and veins at the base of the fish's tail. Thirty-five or 100 diameters mag-
nification of these vessels rendered blood flow clearly visible. A fish after being
paralyzed in the cold test water was quickly placed on a notched glass slide and
gently secured with soft cotton thread as diagrammed in Figure 1. This necessi-
tated removing the fish from the test water for only a few seconds. The slide was
replaced into the test container on a microscope stage and the tail vessels brought
into focus. The time intervals required for respiration and circulation, respec-
tively, to stop after chilling and to start after warming, were measured by means
of a stop-watch. The microscope and other equipment \vere set up in a constant
temperature cold room set at the temperature of the test \vater. The temperature
of the test water remained constant to within 0.2° C. during each experiment.
All data were analyzed statistically by a special method of computing chi square
in an Rx2 table ( Snedecor, 1956, p. 227).
RESULTS
The guppy's normal temperature range, according to Innes (1955), is 16° C.
to 40° C. ; Gibson (1954), however limited the upper temperature to 32° C. When
a guppy was exposed suddenly to water below 10° C.. it exhibited the character-
istics of "primary chill coma." Often after a few minutes at the low temperature,
the fish's chromatophores expanded, causing the fish's color to darken. Exposures
to cold temperatures above 10° C. resulted in "secondary chill coma."
Upon being replaced in a stock tank after "primary chill coma," the fish would
usually begin to respire within eight minutes and almost invariably within twenty
minutes, if they were to survive. Occasionally a fish would revive, show respira-
tory movements for several minutes to a few hours and then die. In "secondary
chill coma," if a fish had gradually ceased to respire during the cold exposure, it
usually did not revive when replaced in stock water of normal temperature, but
if it was still respiring when taken from the cold, it usually survived.
I. Primary Chill Coma
A. Study of a single population
Approximately 700 guppies of all sizes were taken from a large aquatic
plant tub in a University of Pennsylvania Botany Department greenhouse and
acclimated to 23° C. ±1° C. for 10 days. Batches of 10 adults, 5 males
(80-130 mg.) and 5 females (100-900 mg.), and batches of 10 young guppies
(6-18 mg.) were randomly picked and subjected to specific cold exposures.
The rest of the fish were then acclimated to 30° C. ±1° C. for 10 davs ; batches
234
RONALD B. PITKOW
Q
23 °C ADULT
(80-900mg)
23° C. YOUNG
(6-l8mg)
30° C. ADULT
(80-900mg)
I
30 °C YOUNG
(6 - 18 mg)
(lOO/o) 10
•
•
•
•
Q 7
Ul
J 6
•
•
•
•
•
•
•
•
X
•
•
•
|
U. 4
•
•
•
•
•
•
•
•
•
J
•
•
•
•
•
D|
K-
•
•
•
•
•
•
H
•
••
•
•
•
•
|
•
TEST TEMP
("0
DURATION 0
EXPOSURE
(min )
F
1
>
r
-
i
!
e
>
i
5
l
t
i
l
i
>
2
I
1
3
l
2
3
0
f
2
3
4
f
2
5
8
FIGURE 2. Death due to "primary chill coma" in the guppy. The black bar
separates the females above from the males below.
of these adults and young were then subjected to the same cold exposures as
those fish acclimated to 23° C. The results are summarized in Figures 2 and 5 ;
Figure 2 shows the results of exposures to temperatures causing primary chill
coma, and Figure 5 the results of exposures to temperatures causing secondary
chill coma.
1. Effect of the temperature, duration of cold exposure, sex, size, and acclima-
tion temperature on mortality
Combining all the data in Figure 2 it is seen that : ( 1 ) the lower the
temperature, the more rapidly lethal effects occurred ; (2) each increase in
(100%) 40
35
30
Q 25
LU
20
15
10
5
I
(O
2°C
8°C
2 46 8 10 12 14 16 18 20 22 24 26 28
DURATION OF EXPOSURE (min.)
FIGURE 3. Total number of fish killed by each cold exposure at 2° C.
O< 0.005), 5° C. O < C 0.001). and 8° C. (/> < 0.005).
COLD DEATH IN THE GUPPY
235
B.
the duration of exposure to a specific cold temperature was associated with
an increase in the total number killed (Fig. 3) ; (3) males are less cold-
tolerant than females (/> < 0.005) ; (4) young are less cold-tolerant than
adults (> == 0.005) ; (5) in both young (/>"< 0.001) and adults (p < 0.001)
tolerance to "primary chill coma" is inversely related to acclimation tem-
perature.
2. Effect of acclimation temperature on tolerance to anoxia at normal tem-
peratures
When placed in 21° C. oxygen-depleted water (less than 0.25 mg.
O2/L.) for 15 minutes, none out of 20 fish acclimated to 23° C. succumbed,
yet 10 out of 20 of fish acclimated to 30° C. died (/> == 0.001). Tolerance
to oxygen lack is thus inversely proportional to acclimation temperature.
Sumner and Doudoroff (1938), using boiled sea water and 0.001 molar
potassium cyanide in sea water, likewise demonstrated that in the gobie,
Gillichthys mirabilis Cooper, tolerance to anoxia was inversely related to
acclimation temperature.
Lethal roles of the rapidness and repetition of chilling
1. Rapid versus slow chilling
In experiments where the water temperature was gradually reduced from
the acclimation temperature to 2° C. in 15 minutes or to 8° C. in 7 minutes,
13 out of 16 fish died, whereas with rapid chilling to the same temperatures
13 out of 16 fish also died.
2. Lethal role of repetitive chilling
A batch of 20 adult guppies, 10 males and 10 females, acclimated to
23° C. was placed at 5° C. for one minute and subsequently at 23° C. for
TABLE I
Relationship of circulatory and respiratory functions in death due to "primary chill coma"
in the gitppy. Fish were acclimated to 29° C. for ten days
Fish
No.
When chilled
When placed at 25° C.
Resp.
started at
Condition
24 hrs. later
Resp.
stopped at
Circ.
stopped at
Circ.
started at
Circ.
stopped at
#1
5° C. for 3 min.
less than
30 to 80 sec.
Alive
($ LD50)
10 sec.
#2
5° C. for 3 min.
less than
60 sec.
Alive
(i LD50)
10 sec.
#3
5° C. for 5 min.
3.6 sec.
15 sec.
75 sec.
Alive
#4
(1 LD50)
5° C. for 5 min.
4.7 sec.
10 sec.
50 sec.
Alive
#5
(1 LD50)
2° C. for 3 min.
2.3 sec.
15 sec.
2 to 11 min.
_
Dead
(3 LD50)
#6
2°C. for 10 min.
3.5 sec.
210 sec.
7 to 10 min.
—
Dead
#7
(10 LD50)
2° C. for 65 min.
2.6 sec.
_
Dead
#8
(65 LD50)
2° C. for 75 min.
4.0 sec.
_
_
Dead
(75 LD50)
236 RONALD B. PITKOW
one minute. These exposures were repeated six times in succession. Each
time the fish were chilled they lost all respiratory and swimming movement :
but when they were placed at 23° C.. most of them regained respiratory
movements. None of these fish died.
C. Relationship of respiratory movements to the presence of a functioning cir-
culation (Table I).
Careful individual observations were made on 30 fish subjected to 2° C.,
5° C., or 8° C. Twenty fish received an LD50 exposure, 8 received 3 to 12
times the LD50 exposure, and 2 received over 60 times the LD50 exposure.
In each of these 30 fish a complete cessation of all respiratory and swimming
movements occurred within 10 seconds. The results were consistent; the data
from 8 typical fish of this group are presented in Table I. The circulation, as
judged by blood flow in the tail vessels of two fish (Fish #1 and #2, Table I).
ceased approximately one minute after the fish were placed at 5° C.
If the heart beat returns it does so before there are any respiratory move-
ments, as described by Britton (1924) ; in the present experiments if the circu-
lation returned it did so before the respiratory movements, and the respiratory
movements never returned unless a functioning circulation was already present.
Fish #3 and #4 (Table I) were subjected to an LD50 cold exposure and
only transient respiratory and circulatory depression resulted. In all of the 20
fish subjected to an LD50 cold exposure the circulation returned. Respiration
returned in 13 out of the 20 and they survived ; in 4 others out of the 20. respira-
tion returned for a brief period, subsequently stopped and the fish died ; in the 3
others remaining out of the 20, respiration did not return and they died.
Fish #5 and #6 (Table I) were exposed to cold long enough to permit
permanent respiratory depression but only transient circulatory depression.
This resulted in the 8 fish exposed to 3 to 12 times the LD50 cold exposure as
well as 3 of the 20 fish which received approximately an LD50 cold exposure.
Only 2 of the 30 fish (#7 and #8, Table I) received cold exposures of
greater than 12 times the LD50 exposure. Neither the circulation nor the
respiration returned in either fish and they of course did not survive.
D. Effect of increasing the tonicity of the chilling water
Fish were chilled at 5° C. for 8 minutes in distilled water, 0.32 molar so-
dium chloride (P.P. - - 1.2° C.), or 0.65 molar glucose (P.P. - - 1.2° C.) (the
freezing point of teleost body fluid is between -- 0.5° C. and •- 0.9° C. ; Brett,
1956). Increasing the tonicity of the chilling water exerted no significant effect
on the guppy's cold tolerance, as shown by the fact that 21 out of 30 fish chilled in
distilled water, 21 out of 30 fish chilled in 0.32 molar sodium chloride, and 20 out
of 30 fish chilled in 0.65 molar glucose succumbed.
E. Effect of varying the gaseous content of the test water
1 . Effect on mortality of varying the oxygen content and carbon dioxide content
of the chilling water (Table II)
Increased (X + CO2 or increased O2 alone, during a cold exposure,
lessened the lethality of the exposure (Exps. #1 and #3 Table II). In-
creased CO2 alone had no such effect (Exp. #2 Table II). Exp. #4
(Table II) demonstrated that decreased O., during a cold exposure increases
the lethality of the exposure.
COLD DEATH IX THK (.iUITY
237
TABLE II
Effect of increased oxygen mid carbon dioxide (Exf>. #/), increased carbon dioxide (Exp. #2),
increased oxygen (Exp. #3], and decreased oxygen (Exp. #4) during cold
exposures on tolerance to "primary chill coma"
Treatment
Test water
equilibrated with
Oz content
mg./L.
Number
killed
Level of
significance
Exp. # 1
5°C. for
8 niin.
Air
95% 0, + 5% CO,
14.1
51.1
49 of 59 (83%)
8 of 55 (15%)
p « 0.001
Exp. #2
5°C. for
10 niin.
Air
21 %0, + 5% CO,
14
14
39 of 40 (98%)
38 of 40 (95%)
p = 0.7
Exp. #3
5°C. for
10 niin.
Air
100% O:
13.6
51.5
49 of 50 (98%)
31 of 46 (67%)
p « 0.001
Exp. #4
8°C. for
10 inin.
Air
100% N2
13.3
<0.25
6 of 40 (15%)
15 to 40 (38%)
p = 0.02
2. Effect on mortality of increased oxygen before, during, or after a cold ex-
posure (Table III)
Increased environmental oxygen during or after a cold exposure de-
creased the lethality of the exposure, but increased environmental oxygen
before the exposure did not significantly alter this lethality (Table III).
There is no significant difference ( [> —0.1) between increased oxygen dur-
ing or after a cold exposure (Batches #3 and #4, Table III).
3. Effect of increased oxygen on mortality when given during or after ex-
posures to 2° C.
Increased oxygen during one-minute cold exposures to 2° C. did not
lessen mortality significantly (/> == 0.2) ; 20 out of 30 young guppies (6-18
mg.) died when chilled in oxygen-depleted water (< 0.30 ing. O../L.). and
20 out of 40 died in O, -enriched water (50 mg. O,/L. ).
Twenty minutes in oxygen-enriched water after a three-minute exposure
to 2° C. lessened mortality significantly ( /» - 0.005), for 24 out of 25 adult
guppies died when replaced in a stock tank (10 mg. O,/L.) while only 8
TABLK III
Effect of increased oxygen before, during, or after a cold exposure. Batches of 24 young fish
(8 to 20 mg.} were chilled at 5° C. for 8 minutes. Batches #1, #2, and #4 were chilled in oxygen-
depleted water (less than 0.30 mg. 0->/L.). Batch #3 was chilled in oxygen-enriched water (50 wg.
Oz/L.). (Batch #2 was placed in oxygen-enriched water 40 mg.O-2/L.) for 20 minutes prior to the
cold exposure and batch #4 for 20 minutes afier the cold exposure.
Batch
No
Treatment
Per cent
L-illoH
P Value as
compared to
the control
1
CONTROL (
Xormal O> before and after)
100
2
Increased O2
BEFORE cold exposure
96
0.4 1
3
Increased O2
DURING cold exposure
21
<0.001
4
Increased Oo
AFTER cold exposure
46
0.001
238 RONALD B. PITKOW
out of 25 died when they were placed in oxygen-enriched water (40 mg. O,/
L.) of normal temperature prior to return to the stock tank.
F. Significance of anoxia and cold in causing death
1. Tolerance to anoxia at normal temperatures
Three randomly chosen batches of 10 adult guppies each, acclimated to
27 ± 2° C., were placed in oxygen-depleted water at 26° C. ( < 0.30 mg.
Oo/L.) for different periods of time. Eight minutes of exposure killed none ;
15" minutes killed 50% ; and 20 minutes killed 100^ (/> < 0.001').
2. Interaction of cold and anoxia (Table IV)
There is a statistically significant difference in number killed between
batch #3 and batches #1, #2, and #4 (Table IV). Nine minutes of
anoxia prior to 5° C. for one minute (#3) killed significantly more than
5° C. for one minute alone (#1) (p -- 0.02), 10 minutes of anoxia alone
(#2) (p ~ 0.005), or 9 minutes of anoxia after 5° C. for one minute (#4)
(/>==0.05). This indicated that anoxia before a cold exposure is more
lethal than anoxia after a cold exposure.
TABLE IV
Interaction of cold and anoxia in producing death in "primary chill coma."
Fish acclimated to 23 ± 1.0° C. for ten days
Batch No. Treatment Number killed
Decreased oxygen ( <0.25 mg./L.)
1 5°C. for 1 min. 2 of 20 (10%)
2 25° C. for 10 min. 1 of 20 (5%)
3 25° C. for 9 min. followed by 5° C for 1 min. 9 of 20 (45%)
4 5° C. for 1 niin. followed by 25° C. for 9 min. 3 of 20 (15%)
5 5° C. for 10 min. 18 of 20 (90%)
Increased oxygen (50 mg./L.)
6 5 °C. for 10 min. 2 of 20 (10%)
When batch #5 (Table IV) was exposed to 5° C. for 10 minutes, a
significantly higher number ( f> -- 0.005) died than in batch #3 where fish
were exposed to anoxia for 9 minutes and then to 5° C. for one minute.
There is a highly significant difference between batch #5 and all the other
batches. Batch #6 was exposed to 5° C. for 10 minutes in oxygen-enriched
water (50 mg. O2/L.) ; but only 2 out of 20 died as compared to 18 out of
20 in batch #5 which was exposed to 5° C. for 10 minutes in oxygen-
depleted water (< 0.25 mg. O,/L.) (/> « 0.001).
G. Effect of prior anesthesia with \°/c urethan (Fig. 4)
Prior anesthesia with 1% urethan decreases the lethal effect of "primary
chill coma" (/> == 0.005) (Fig. 4).
H. Effect of varying the sequence of two cold exposures (Table V)
Comparing batches #1 and #2 (Table V), a significantly higher number
died (p = 0.001 ) among the fish exposed to the colder temperature first.
A suggestive decrease in mortality (/> = 0.1) to an exposure to 2° C. for
one minute occurred if the fish wrere first exposed to 8° C. for 4 minutes (Batches
#2 and #3. Table V).
There is no statistically significant difference (/> = 0.25) between 2° C. for
COLD DEATH IN THE GUPPY
239
Exp.*L
Exp*3
CONTROLS
IUU
90
10/10
80-
70-
Q
UJ
7/10
6/10
h 50-
2
UJ
o 40-
UJ
Q.
30-
9/21
4/10
20-
10-
DURATION OF
ANESTHESIA (min
3/20
0/10 0/10
0 10 07 05 10 8
TIME CHILLED
AT 5DC. (min.)
GUPPY TYPE (80_A9d^mg) (6_Y°-mg
™™ »«
Young Adult Young
(6 -I8mg.
23 ±2 26 ±2 26 ±2
FIGURE 4. Protective effect of anesthesia with \% urethan immediately
before a cold exposure (p — 0.005).
5 minutes (Batch #4), and 2° C. for 1 minute when followed by 8° C. for 4
minutes (Batch #1).
There is a profound difference (p « 0.001 ) in lethal effect between 2° C. for
5 minutes (Batch #4), and 8° C. for 5 minutes (Batch #5).
II. Secondary Chill Coma
A. Study of a single population (Fig. 5)
1. Effect of the duration of a cold exposure on mortality
At 10.5° C., 20 out of 40 fish exposed for one hour died while 29 out
of 40 exposed for one and one quarter hours died (/> ==0.05). Only two
TABLE V
Effect of varying the sequence of two cold exposures. Three groups of ten
adult guppies were used for each treatment
Batch No. Treatment
1 2° C. for 1 min. followed by 8C
2 8° C. for 4 min. followed by 2C
3 2° C. for 1 min.
4 2° C. for 5 min.
5 8° C. for 5 min.
C. for 4 min.
C. for 1 min.
Number killed
25 of 30 (83%)
12 of 30 (40%)
19 of 30 (63%)
30 of 30 (100%)
2 of 30 (7%)
240
RONALD B. PITKOW
23° C. ADULT
(80-900mg)
23°C. YOUNG
(6-l8mg)
I
30° C. ADULT
(80-900mg)
30° C. YOUNG
(6-l8mg)
TEST TEMP.
DURATION OF
EXPOSURE
(hr.)
FIGURE 5.
18
2.7
Death due to "secondary chill coma" in the guppy.
bar separates females above from males beknv.
The black
out of 20 young guppies exposed to 12.5° C. for 3 hours died while 15 out
of 20 exposed for 18 hours died (p < 0.001 ). Thirteen out of 20 adults ex-
posed to 12.5° C. for 18 hours died while 17 out of 20 exposed for 27 hours
died (p -- 0.25). (This last comparison is the only one of the three where
the difference is not statistically significant.) These data indicate that in
"secondary chill coma," as in "primary chill coma," the duration of exposure
determines the mortality.
COLD DEATH IN THE GUPPY 241
TABLE VI
Effect of increased oxygen content of the chilling icater on tolerance to "secondary chill coma."
Fish acclimated to 27 ± 2° C. Combining the results from the three experiments, p = 1.0
Number killed
Treatment 10 ing. O2/L. 45 mg. O2/L.
Adult fish
i 100-800 mg.)
Exp. #1 11.4 db(U°C. for 15.5 hrs. 24 of 25 (96%) 25 of 25 (100%)
Exp. #2 12.1 ± 0.2° C. for 8.0 hrs. 15 of 20 (75%) 15 of 20 (75%)
Juvenile hsh
(20-40 mg.)
Exp. sft 12.1 ± 0.2° C. for 12.0 hrs. ' 20 of 25 (80%) 19 of 25 (76%)
1. Effect of sex, size, and acclimation temperature on mortality
Comparing data in Figure 5, males do not seem less cold-tolerant than
females (/i — 0.3); but young are much less cold-tolerant than adults
( p == 0.005 ') . Cold-tolerance is inversely related to the acclimation tempera-
ture in both young (/> < 0.001) and adults (/> == 0.005).
B. Effect of increased oxygen during the cold exposure (Table VI)
Increased oxygen during secondary chill coma did not lessen mortality
(Table VI).
C. Effect of isosmotic chilling medium on mortality
Two batches of young guppies (8-18 mg.) were subjected to 12.0° C. for
5.5 hours. Batch #1 was chilled in distilled water and batch #2 in 0.16 molar
sodium chloride ( F.P. - 0.6° C. This is approximately isotonic with teleost
body fluids (Brett, 1956). Nine out of 23 fish in batch #1 succumbed; 10 out
of 24 in batch #2 died. There is no significant difference between these two
batches ( /> == 0.75).
DISCUSSION
Among the possible causes of death inherent in cold exposure, two factors may
be excluded. The suddeness of a cold exposure is not of itself lethal, for sudden ex-
posures did not cause more mortality than gradual exposures (TB1)3 (Britten,
1924). Moreover, the cooling process per sc is not lethal since even repetitive
chilling into "primary chill coma" caused no mortality (IB2). At a specific cold
temperature, the duration of cold exposure is the decisive determinant of lethality
rather than the abruptness or repetition of the temperature change.
"Primary Chill Coma"
Taken altogether, the data suggest that "primary chill coma" kills by causing
anoxic damage to a cold-depressed respirator}- center. The permanent re-estab-
lishment of respiration after "primary chill coma" is the critical event in determin-
ing survival (1C). Even after prolonged lethal exposures to cold, the fish's cir-
culation often returns within a few minutes (1C) and then, depending on one major
variable, permanent respiration does or does not return. This variable is the
:; This refers to pertinent experimental data listed under results.
242 RONALD B. PITKOW
amount of oxygen which the circulation can bring to the respiratory center to fore-
stall anoxia damage while the cold depression of respiration subsides.
There appear to be several factors limiting the availability of oxygen. Simmer
and Doudoroff (1938) showed that fish have a considerable oxygen reserve in their
tissues. Consequently, in the present experiments the relative depletion or satura-
tion of the fish's oxygen reserve partially determines the amount of oxygen that
the circulation can carry to the fish's respiratory center. Normally the oxygen re-
serve is probably maximal, since increased oxygen before "primary chill coma" did
not lessen mortality (IE2). If the oxygen reserve had been submaximal, then
the extra oxygen should have diffused into the fish's superficial tissues ( Privol'nev,
1956; Breder. 1941) and protected the animal against anoxia. Any process which
depletes the oxygen reserve increases mortality. The oxygen reserve may be de-
pleted by a prior anoxic period (IF2), decreased environmental oxygen during a
cold exposure (IE1), or the extra oxygen consumption which accompanies the
initial convulsive paroxysm. Anesthesia prior to a cold exposure lowered mortality
(IG), probably by lessening the initial convulsive paroxysm and so lessening this
initial depletion of the oxygen reserve.
The protective value of increased oxygen during and after chilling (IE) may be
explained by the hypothesis that when the fish's oxygen reserve is depleted, more
oxygen can diffuse into the fish and replenish that portion of the depleted oxygen
reserve located in the cutaneous and subcutaneous tissues. Ultimately the oxygen
probably protects by acting centrally. Nervous tissue is the most sensitive to
anoxia of any vital tissue, and so it is probable that the increased oxygen benefits
the respiratory center directly. This effect of increased environmental oxygen
in promoting respiratory return and survival does not seem to be related to any
reflex mechanism for at least two reasons : ( 1 ) Increased environmental oxygen
would lessen rather than increase reflex stimulation of the respiratory center ; ( 2 ) in-
creased oxygen protected even when given only during the cold exposure and not
when respiration actually restarted (IE).
The observation that four of the thirty individually observed fish died after res-
piration was re-established (1C) may have either of two alternative explanations:
(1) There is an additional mechanism or mechanisms for death due to primary chill
coma; (2) when rewarmed the cold depression of the respiratory center was relieved
and it began functioning while still anoxic ; this rendered it more anoxic and per-
mitted irreversible damage to occur.
In experiments where oxygen was unaltered, mortality was increased by lower-
ing the temperature of a cold exposure of a specific duration (IA1) (IH) and also
by subjecting fish first to the lower of two sequential cold exposures (IH). The
lower chilling temperature probably induces a more profound respiratory depres-
sion and a more vigorous initial convulsive paroxysm which more completely de-
pletes the oxygen reserve.
Tolerance to "primary chill coma" is inversely related to acclimation temperature
(IA1). Perhaps a higher acclimation temperature lessens the fish's oxygen reserve
since there is less oxygen dissolved in body water of higher temperatures ; however,
this is partially offset by an increase in diffusion ( Krogh, 1919). A higher ac-
climation temperature increases the overall metabolic rate so that the fish's oxygen
reserve is depleted more rapidly. With an increase in the acclimation temperature
COLD DEATH IX THK (ilTTY 243
the central nervous system would have an increased oxygen need (Freeman, 1950)
and the respiratory center would become anoxic more rapidly.
Oxygen may be important also in the observations that young are less tolerant
to "primary chill coma" than adults ( IA1 ) and males are less tolerant than females
( IA1 ). These less tolerant groups are smaller, have a proportionately higher meta-
bolic rate ( Muller. 1942), and so upon chilling might become anoxic more rapidly.
Perhaps they would experience also a more profound respiratory depression, for
their smaller size would permit the cold to penetrate more rapidly to the respiratory
center. Their smaller size should permit a more rapid replenishment of oxygen
reserve via cutaneous diffusion, but evidently this advantage is overshadowed by
the higher metabolic rate and perhaps by the more rapid penetration of the cold
in the smaller fish. It is possible that young guppies and, or male guppies may have
a respiratory center which is innately more susceptible to cold depression or anoxic
damage or both.
"Secondary Chill Coma"
The experimental data presented in this paper give no indication of the cause
of death due to "secondary chill coma." Osmoregulative failure (Doudoroff, 1945)
does not seem to occur in the guppy, for chilling in isosmotic sodium chloride had
no protective effect (IIC). Although increased oxygen protects against "primary
chill coma." it does not protect against "secondary chill coma" (IIB ).
Certain observations are perhaps significant. Fish acclimated to higher tem-
peratures are more sensitive to "secondary chill coma" (IIA2) ; young are more
sensitive than adults (IIA2). These more sensitive groups have a significantly
higher metabolic rate and would exhaust their energy stores more rapidly so that
death due to "secondary chill coma" might be some sort of exhaustion phenomenon.
There does not seem to be a sexual difference in sensitivity to "secondary chill
coma." The difference in metabolic rate between male and female guppies is per-
haps not enough to produce significant differences in the limited number of fish
used.
Despite the present inability to explain the mechanism or mechanisms of death
due to "secondary chill coma." all the data that have been obtained to the present
time can be reconciled with the proposition that death due to "primary chill coma"
is caused by anoxic damage to a cold-depressed respiratory center.
I am indebted to the late Dr. L. Y. Heilbrunn for first stimulating my interest
in this problem and for providing laboratory space. I wish to thank Dr. John R.
Brobeck for sponsoring this project and for his many valuable suggestions and en-
couragement, and Dr. Harold T. Hammel for his advice and aid.
SUMMARY
A. Observations concerning both "primary chill coma" and "secondary chill coma"
1 . In guppies acclimated to 23 to 30° C., exposures to temperatures below 10° C.
produced "primary chill coma" while exposures to lethal temperatures above 10° C.
caused "secondary chill coma."
2. The duration of a cold exposure at a specific temperature is the decisive lethal
244 RONALD R. PITKOW
determinant rather than the chilling temperature per se. An increase in the dura-
tion of a cold exposure causes an increase in mortality.
3. Tolerance to a cold exposure is inversely related to acclimation temperature.
Tolerance to oxygen lack at normal temperatures also is inversely related to ac-
climation temperature.
4. Males are less cold-tolerant than females in the temperature range of "pri-
mary chill coma." There does not seem to be a sexual difference in cold tolerance
in the temperature range of "secondary chill coma."
5. Young guppies (6-18 mg.) are less cold-tolerant than adults (80-900 mg.).
B. Observations on "primary chill coma"
1. Respiration did not return in any fish subjected to approximately three or
more times the LD50 cold exposure, despite the fact that the circulation returned in
all fish subjected to approximately twelve times the LD50 exposure or less.
2. The lethality of a cold exposure was increased by decreased oxygen before
or during the exposure.
3. The lethality of a cold exposure was decreased by prior anesthesia with \%
urethan, increased oxygen during or after the exposure, or with two sequential
cold exposures by exposing the fish to the less extreme temperature first.
4. The lethality of a cold exposure was unaltered by increased oxygen before the
exposure, decreased oxygen (of a duration which was not lethal of itself) after
the exposure, increased tonicity of the chilling water, or equilibrating the chilling wa-
ter with 5% CO,.
C. Observations on "secondary chill coma"
1. The lethality of a cold exposure was unaltered by increased oxygen during the
exposure, or by the use of isotonic sodium chloride as the chilling medium.
D. The following conclusions may be drawn from the observations made :
1 . Death due to "primary chill coma" in the guppy may be due to anoxic damage
to a cold-depressed respiratory center.
2. The lethal effect of "primary chill coma" is related to the profoundness of
respiratory depression and by the degree of depletion of the fish's oxygen reserve.
3. Osmoregulative failure does not seem to be a cause of death in "secondary
chill coma" in the guppy.
4. Oxygen lack is not a lethal determinant in "secondary chill coma" in the
guppy.
LITERATURE CITED
BREDER, C. M., JR., 1941. Respiratory behavior in fishes not especially modified for breathing
air under conditions of depleted oxygen. Zoologica, 26 : 243-244.
BRETT, J. R., 1952. Temperature tolerance in young Pacific Salmon, genus Oncorhynchus.
/. Fish. Res. Canad.. 9 : 265-323.
BRETT, J. R., 1956. Some principles in thermal requirements of fishes. Quart. Rev. Biol., 31 :
75-87.
BRITTON, S. W., 1924. The effects of extreme temperatures on fishes. Aincr. J. Physio!., 67:
411-421.
DOUDOROFF, P., 1942. The resistance and acclimation of marine fishes to temperature changes.
I. Experiments with GircUa nii or near abscission of polar segments. Figures 5—8, 9—12,
and 49-55:
It is possible to cut away one or both poles at the cylinder stage, removing as
much as two-thirds of the egg substance, and still get successful furrowing. Neither
the position of the furrow nor its course is altered by the operation. This experi-
FURROWING IX FLATTENED EGGS
249
;
•
f46
-,',
57
FIGURES 44-58. Photographs illustrating Operations I-III. Operation I, Figures 44-48.
hemisection of entire egg with continued furrowing. Operation II, Figures 49-51, continued
furrowing after near abscission of one pole. Operation II, Figures 52-55, continued furrowing
after near abscission of both poles. Operation III, Figures 56-58, rapid opening of slit perfora-
tions (see text). Figure 57 is a few seconds after Figure 56; Figure 58 is 5 minutes later.
250
ALLAN C. SCOTT
OPERATION I!
2O
OPERATION IV
23
OPERATION V
FIGURES 13-23. Operation III, Figures 13-16, longitudinal slits in the furrow showing the
immediate opening-out of the slits and the completion of three internal furrows. Operation IV,
Figures 17-20, near isolation of a furrow segment. Figure 18, followed by shrinkage of the
segment, Figures 19 and 20. Operation V, Figures 21-23, cleavage of a flattened diastral egg-
to four cells ; two cells with, and two without, aster and nucleus.
FURROWING IN FLATTENED EGGS
251
inent separates the expanding polar surface from the rest of the egg, during a
considerable part of the time that it would be expanding in the unoperated egg,
without interfering with cleavage.
Operation III, longitudinal slits i)i the jitrrow, Figures 13—1 6 and 56—58:
During this operation, the egg is flattened in the cylinder stage and one or more
slit perforations are made across the furrow with the long axis of the slit parallel
OPERATION VI
OPERATION VII
CASE I
OPERATION VII CASE II
FIGURES 24-33. Operation VI, Figures 24-26, transection of the spindle : development of
four furrows and cleavage to four cells, two cells without asters and without nucleus. Operation
VII. Case I, Figures 27-29, slit perforations lateral to the spindle; five furrows produce four
cells, two without aster or nucleus. Operation VII, Case II, Figures 30-33, slit perforations
lateral to the spindle ; three cells are formed, one without aster or nucleus.
252
ALLAX C. SCOTT
7O
FURROW IX(; IX FLATTKXED EGGS 25.?
to the long axis of the egg. The slits immediately open in a way that gives evidence
that the furrow ring is a region of tension in flattened eggs. Within seconds
the slits open and multiple furrows produced gradually cut through the narrowing
stalks. This operation makes cleavage possible in eggs that would otherwise be
stalled at the cylinder stage; it is apparently easier for an egg to furrow through
several small stalks than through one big one.
Operation II', near isolation of a furrow sec/incut, Fiyitres 17-20 and 5V-61 :
In this operation four perforating cuts are made as shown in Figure 18, so
that a central plug of egg substance, the surface of which is virtually all furrow
cortex, is isolated from the rest of the egg. Only slender stalks connect the plug
with the sub-furrow region. During the next few minutes the whole semi-isolated
plug shrinks in volume; meanwhile its contained endoplasm flows through the
attachments into the main part of the cell. We consider this to result from an
active contraction of the entire surface of the plug; it appears to be incompatible
with the membrane expansion theory.
Operation I', flattening without inierodisseetion, Fiynrcs 21—23 and ()2—64:
\\ hen eggs in the middle diaster are swollen, flattened but otherwise unoperated,
they may cleave directly to four cells, two of which contain asters and nuclei and
two that do not. The expected single equatorial furrow does not develop ; instead,
four areas of furrowing move in from the periphery following the tips of the astral
rays. In two cases the four-celled embryo was followed through a second cleavage
during which the nucleated cells divided again while the anucleate ones failed to
divide; the latter did. however, each develop a large monaster during the "division
phase." which is the more interesting since they were patently devoid of any part
of the amphiaster during the first division.
(Deration I'l, equatorial transeetion of the spindle I. Fiyures 24—26 and 65— o. •••• :*r.
* *.'*
» W J» * » - *
#, * * *" -109 microorganisms, de-
pending on the species of organism and the nature of the experiment. Withdrawal
of the needle allowed the wound to close and little of the inoculum was lost.
Animals from which excessive leakage occurred were discarded. Each inoculated
oyster was passed through six changes of fresh sea water, during which the area
over the injection site was flushed gently with a pipette to remove extraneous micro-
organisms. The oysters were then returned to aquaria and maintained as
described above.
4. Sampling procedures
Two types of experiments were performed in order to determine the fate of
injected microorganisms. In one group of experiments oysters which had received
intracardial injections of B. mycoides or S. cerevisiae were relaxed, fixed, and
prepared sections were examined microscopically. In the second group of experi-
ments tissues from experimental oysters were cultured (at intervals) to recover
microorganisms.
a. Sections of inoculated oysters
Preliminary experiments showed that tissue shrinkage due to fixation and de-
hydration could be minimized by relaxing intact oysters in 0.4 M magnesium sulfate
at 4° C. for several hours prior to fixation of the whole oyster in Benin's solution
overnight. Ten oysters were injected intracardially with a suspension of B.
nivcoides and one animal was sacrified at each of the following intervals after
injection: 1 hour, and 1. 2, 4, 5, 8, 9, 12, 16 and 20 days. Similarly six oysters
were injected intracardially with a suspension of S. cerevisiae and one animal was
sacrificed at each of the following intervals thereafter: 1 hour, and 1, 2, 3, 4 and
12 days. Two or three control oysters were injected with equal volumes of sterile
sea water and sacrificed at suitable intervals in each experiment. Examination
of control animals disclosed no histological abnormalities or microorganisms.
Following initial fixation the soft parts were removed from the shell, divided
transversely into six approximately equal parts and placed in fresh Benin's solution
for 1-4 hours. These blocks of tissue were then washed, dehydrated, cleared,
276
M. R. TRIPP
imbedded in paraffin, sectioned at 10 p. and stained with Gram's tissue stain. Two
sections were taken from each block, one representing the anterior edge of the
tissue and one from the center of the tissue. Thus, for each animal 12 transverse
sections were obtained at regular intervals along the anteroposterior axis.
Certain vessels, sinuses and tissues were regularly examined for microorganisms
under high dry and oil immersion objectives. These included : heart, anterior and
posterior aortae, large arteries of the visceral mass, subepithelial blood sinuses of
the gut, sinuses between digestive diverticula, dorsal and ventral circumpallial
arteries, proximal mantle vessels, medial and lateral gill axis sinuses, vertical gill
vessels, palp arteries and the blood spaces of the adductor muscle and kidney. In
addition, the following epithelial areas were examined for microorganisms : walls
of the alimentary canal at all levels, walls of twenty of the digestive diverticula,
inner and outer aspects of the palps, outer aspect of the mantle, nephridial tubules,
gonaducts, epicardium and pericardium.
1). Cultivation of microorganisms from tissues
Groups of 20-25 oysters received injections of microorganisms suspended in
sterile sea water, while 6-8 controls were injected with sterile sea water only.
Estimates of the numbers of microorganisms in heart blood, mantle, visceral mass
and in aliquots of homogenized whole oysters were made at intervals of one hour,
one day, two days, and irregularly thereafter for 6-50 days.
At each interval three experimental and one control oysters were tested for
microorganisms according to the following scheme. Each heart yielded 0.1-0.5
ml. of blood which was added to 4.5 ml. of sterile distilled water. Portions of
tissue, approximately 0.1 ml. in volume, were removed from the mantle and visceral
mass and ground separately in Ten Broeck tissue grinders containing 5.0 ml. of
sterile distilled water. The remaining soft parts were homogenized in a Waring
Blendor containing 20 ml. of sterile distilled water, and the actual volume of the
soft parts was determined by displacement. One-tenth ml. of the material from
each tissue or homogenate sampled was placed in each of three Petri dishes, melted
agar added and the plates incubated at the optimum temperature for the species
TABLE II
Media, incubation times and temperatures for bacteriologic tests of oyster tissues
Organism
Medium
Incubation
Temp. (°C.) Time (Hours)
Bacillus cereus var. mycoides
spores
vegetative cells
Staphylococcus aureus
Eschcrichia coli
Pseudomonas fluorcscens
Flarobacterium invisibile
Saccharomyces ccrevisiae
Nutrient agar (dist.)*
Nutrient agar (dist.)
Nutrient agar (dist.)
Nutrient agar (dist.)
Nutrient agar (sea)
Nutrient agar (sea)
Sabouraud agar (dist.)
30
30
37
37
25
30
30
24
24
24
24
48-72
18
18-24
* Difco Bacto-nutrient agar or Sabouraud agar rehydrated with distilled or sea water.
MICROBES IXJECTED INTO OYSTERS
of microorganism being studied. Individual colonies were counted, averaged and
the number of viable microorganisms per ml. of sample was calculated. Using
these data, and estimates of the volumes of mantle and visceral mass (respectively
30% and 40 % of the soft parts) derived from previous experiments, it was possible
to estimate the total number of viable microorganisms in these organs. Results are
expressed as the mean values from three oysters. In general, values obtained were
in close agreement and only rarely were extreme variations noted. Since there
was no suitable method of estimating the total volume of oyster blood, the number
of microorganisms recovered from blood samples is expressed per ml. of heart
blood sampled.
RESULTS
1. Observations based on tissue seetions following injection of yeast cells
Masses of yeast cells seemed to occlude main arteries immediately after intra-
cardial injection. Phagocytosis began immediately and by 24 hours phagocytes
contained approximately 95% of yeast cells visible in sampled areas of tissue sec-
tions. Tissue sections from the ovster fixed at 24 hours showed that most leuko-
I00
80
S 60
£T
UJ
O
O
0)
40
§ 20
hagocvtosis, and Ivsosouics: cytocJieinical and electron microscopic
studies. ALEX B. NOVIKOFF.
Cytochemical stains and electron microscopy permit the study of lysosomes, characterized
by: (1) acid phosphatase activity in cell smears or frozen sections (fixed in cold formal-
calcium) incubated in Gomori glycerophosphate medium, (2) a single ("unit") outer mem-
brane. In erythrophagocytes, Kupffer cells, lung macrophages, cells of proximal convolution
and glomerular epithelium, phagocytosis-pinocytosis vacuoles have been studied in both pro-
cedures and have both properties. Acid phosphatase activity is also present in Chaos chaos
pinocytosis vacuoles, Amelia proteus food vacuoles, and vacuoles of spleen macrophages. The
distinction between phagocytosis and pinocytosis seems less rigid : the vacuoles are bounded
by former cell membrane and appear to have high acid phosphatase (and presumably other
acid hydrolase) activities ; as light microscopy visualized colloidal particles, electron micros-
copy now visualizes ferritin molecules in solution. Tracers (ferritin [glomerulus, Farquhar
and Palade; Trypanosoma mega, Steinert and Novikoff] : colloidal particles [liver, Hampton:
proximal convolution, Burgos] ; enzymatic proteins — peroxidase, acid and alkaline phosphatase
[kidney, liver, Novikoff et a/.]) indicate that micropinocytosis vacuoles transport materials
into larger vacuoles or lysosomes. To determine whether the small vacuoles also are lyso-
somes will require visualization of low levels of hydrolase activity in well-preserved electron
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
microscope preparations. Awaiting future study are the origin of acid hydrolases, the rela-
tionships of "cytotic" vacuoles and Golgi apparatus, and the quantitative and qualitative
significance of "cytosis" in the cell's economy. From their vital dye uptake and acid phospha-
tase activity (Pasteels, Mulnard, Dalcq), metachromatic granules appear to be lysosomes ;
from their fine structure (Rebhun) they appear to be "multivesicular bodies" (Sotelo and
Porter). These bodies are seen in many cells; their unit vesicles might be formed by
micropinocytosis or a membrane-generating process involving the Golgi apparatus.
AUGUST 1, 1960
Electrical inc.vcitability of the frog neuromuscular synapse. ROBERT WERMAN.
Frog muscle endplates were explored with an extracellular microelectrode. An intra-
cellular electrode simultaneously monitored invasion into the endplate region of a spike which
was directly evoked by a distant microelectrode in the same fiber. In the external recordings
from sites generating miniature endplate potentials, an external positivity was invariably seen
in association with the rising phase of the intracellular spike. Frequently, a subsequent small
prolonged negativity terminated the potential. In contrast, when the exploring microelectrode
was moved as little as 5 /*, the external recording became a positive-negative-positive sequence
which is characteristic of the approach, local development and onward movement of propagated
responses. The negativity w-as associated with the rising phase of the intracellular spike and
its amplitude increased as the exploring electrode was moved further from sites of externally
recorded miniature activity. It is generally agreed that the external potential closely follows
the time course of the membrane current. Thus, the absence of negativity associated with the
rise of the invading spike indicates absence of inward current at sites of externally recorded
miniature activity. The late negativity can be accounted for by capacitative current through
passive membrane. Since the sites at which the miniature endplate potentials are generated
are presumably also the sites of synaptic activation, the synaptic membrane appears not to be
excited by an electric stimulus of the magnitude of an action potential.
AUGUST 2, 1960
Free radical formation during indophenol blue synthesis by heart muscle respiratory
enzymes. PHILIP PERSON AND ALBERT FINE.
A study of indophenol blue synthesis from dimethylparaphenylenediamine and alpha-
naphthol, as mediated by Keilin and Hartree type beef heart muscle preparations, has revealed
that of the above reagents only the diamine is enzymatically oxidized by the heart muscle
particulates. Enzymatic oxidation of the naphthol could not be detected using manometric and
spectrophotometric methods capable of measuring such oxidations. The first step in the
sequence of events leading to indophenol blue synthesis is the oxidation, by the heart muscle
particulates, of the diamine to a free radical. The latter is stable and may be identified by its
absorption spectrum which contains maxima at 550 and 520 m/*. The free radical forms a
dimer, also identifiable by an absorption maximum at 640-650 m^. Indophenol blue is formed
as a result of a non-enzymatic oxidative coupling of the free radical and its dimer with the
alpha-naphthol.
AUGUST 9, 1960
Cation permeability in muscle. R. A. SJODIN.
If the influx of potassium ions in frog sartorius muscle is determined at various external
concentrations of potassium, the results can be fit to the "constant field" equation with a nearly
constant permeability coefficient, as long as the system is close to the steady-state. When
rubidium or cesium ions are employed, the influxes decline more with rising concentration than
predicted by theory. When the potassium content of the muscle is not in the steady-state, the
calculated permeability coefficients vary up to 15-fold, depending on the sign and magnitude
of the driving force.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
In general, the theory that ions in muscle are following simple electrochemical driving
forces across the cell membrane appears inadequate and leaves one with the problem of
accounting for the variations in measured permeability coefficients. One situation which could
provide a basis for the observed departures from the flux equations is the case in which the
number of membrane sites for a particular ion is limiting. A collision between an ion and an
available membrane site seems a likely prerequisite for membrane entrance. The product of the
external concentration and the number of sites available at the outer membrane surface should
give a measure of the probability for membrane entrance from the external solution. This
probability also involves a factor which represents the interaction energy (affinity) between
the particular ion and the site.
It has been possible to demonstrate that the limited site model adequately describes the
entry of potassium, rubidium, and cesium ions in frog muscle, either individually or competi-
tively in mixtures. The affinity constant required for each ion remains invarient over a 50-fold
range of concentration and flux. The order of decreasing affinity was found to be Rb > K > Cs.
Effects of ions on potential in lepidopteran muscle fibers. PETER BELTON.
Nerve-muscle preparations were made from the prothoracic leg of saturniid moths and
the longitudinal locomotor muscles of pierid larvae. The effects of changes in external concen-
trations of K, Na, Ca and Mg on resting and neurally-evoked action potentials (r.p., a.p.)
were described. The latter are graded events and are compounded from synaptically and
electrically excited components. Changes in r.p. were evoked more readily by variation of
K0 than by the other three cations. A KC1 solution isosmotic with the control saline did not
depolarize muscle fibers immediately but prolonged the falling phase of the synaptic component.
Longer soaking produced a neural block or repetitive firing of the muscle as a result of depolar-
ization of the nerve terminals. K-free salines initially shortened the falling phase of the a.p.
but later caused a plateau similar to that found in frog muscle with K- and Cl-free salines.
Omission of Na greatly reduced the electrically excitable and synaptic components of the
response. When Na was replaced by sucrose, an initial enhancement of r.p. and a.p. was seen.
Seventy mM/1. Mg did not usually delay the take-off of the electrically excitable com-
ponent, but decreased the size and increased the duration of the a.p. Neither did the omission
of Ca block neuromuscuiar transmission. When responses were evoked at the rate of 0.5/sec.
slight increases in r.p. and a.p. were evident in solutions with 30 mM/1. Ca. When paired
stimuli were used, the relative refractory period for the electrically excitable component was
found to have increased from 10 to about 500 msec. Bursts of stimuli at 50/sec. decreased the
size of the responses more rapidly than stimuli at 25/sec. The effect of Can increase was more
rapid than that of the other changes described.
It is concluded that other factors can, to some extent, override the K and Xa electrode
properties of the excitable membrane of these cells.
AUGUST 16, 1960
A scheme for the mechanochcmisty of muscle. FRAXCIS D. CARLSON.
Our studies on frog muscle have shown that 0.286 /umole/gm. of phosphocreatine are
dephosphorylated in an isotonic twitch with a time constant less than thirty seconds. This
corresponds to approximately one molecule of phosphocreatine split for each molecule of G-actin
in the muscle. Chance and Connelly found only 0.009 ^mole/gin, of adenosine diphosphate
produced per twitch, and the half time of ADP rephosphorylation was of the order of minutes.
The following scheme which reconciles these results assumes, as the stoichiometry cited sug-
gests, that the reversible transformation of G-ATP-actin to F-ADP-actin occurs in the
muscular contraction-relaxation cycle. Reaction one : G-ATP-actin is dephosphorylated during
the contraction-relaxation cycle to give F-ADP-actin and orthophosphate. Reaction two : F-ADP-
actin forms G-ADP-actin during depolymerization. Reaction three : G-ADP-actin is re-
phosphorylated directly by the creatinephosphokinase system as reported by Strohman, or else
indirectly by exchange of traces of free ATP for the bound ADP and subsequent phosphoryla-
tion of the latter by the creatinephosphokinase system to give more ATP. These reactions
involve nucleotide bound to actin and hence not available to exchange readily with free nucleo-
290 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
tide. Reaction four: Phosphocreatine and ADP are formed in the sarcoplasm from the creatine-
phosphokinase reaction as a result of the displacement of equilibrium by the creatine produced
in reaction three. The equilibrium of reaction four favors creatine and ATP formation; hence
only small amounts of ADP and phosphocreatine will be produced. Reaction five: Mitochondria!
oxidative phosphorylation, stimulated by the ADP from reaction four, produces ATP which
results in the restoration of creatine, phosphocreatine, and ADP concentrations. The slight
increase in sarcoplasmic ADP produced by reaction four would correspond to that seen by
Chance and Connelly. In the absence of glycolysis and oxidative phosphorylation the creatine
and phosphocreatine concentrations would not be restored and our results would obtain.
Reversibility of actin depolymerisation in presence of KI. ANDREW G. SZKNT-
GYORGYI AND EVA M. SZENTKIRALYI.
In presence of 1 mM MgCL and 0.1 mM ATP or phosphocreatine (PC) a relatively high
concentration of KI (0.8 M} is required for the complete depolymerization of purified F-actin.
At lower KI concentrations the viscosity of actin has intermediate values. Depolymerization
performed in presence of Mg and phosphagens can always be reversed by reducing the ionic
strength. If Mg or the phosphagens are omitted, depolymerization is complete already at
0.3 M KI and is irreversible. The effect of PC and ATP can be explained by assuming the
simultaneous polymerization and depolymerization of actin, a process similar to the findings
of Oosawa ct al. (J . Polymer Sci. 37: 1323, 1959) in low concentrations of MgCL. In
accordance with such a picture, the steady formation of ADP, exceeding the stoichiometric
ratio, can be measured at the intermediate KI concentrations, but not at 0.8 M KI where
depolymerization is complete, or at 0.1 M KI where depolymerization is very slow. The
maximum rate of ADP formation at room temperature occurs at 0.4 to 0.5 M KI, and 1 mole
of ADP is formed in about 100 minutes by 57,000 grams of actin. The role of Mg in these
reactions is specific and cannot be replaced by Ca, neither do ATP and PC act without Mg.
During depolymerization GADr-actin is formed which requires Mg for its stability, possibly by
facilitating the binding of the nucleotide to actin. When F-actin is depolymerized in the
presence of Mg only, the resulting GAi>r-actin will not polymerize when the ionic strength is
reduced, even though it is not denatured. Polymerization can be induced, though, by the
addition of ATP or PC. The polymerization process thus can be separated into at least two
steps. In the first step ATP or PC alters actin in such a fashion that it can polymerize,
provided the ionic strength is favorable for the polymerization proper. During the process
ATP is dephosphorylated. The nature of the intermediate is not known.
Studies on actin. II. Polymerisation and the bound nucleotide. TERU HAYASHI
AND RAJA ROSENBLUTH.
The necessity of actin for actomyosin contraction has been established, and the possible
role of actin in muscle contraction as a localized, fixed site of phosphate turnover has been
revived by Strohman and, more recently, Carlson. A crucial point in these speculations centers
on the question — is the actin-associated nucleotide amenable to enzymatic attack while bound
to the actin protein? The answer was sought in a study of the properties of G-ADP actin,
since theoretically it was the form most likely to be the focal point for phosphate turnover.
At 1° C, G-ADP actin loses ca. 20% of its bound nucleotide in an hour, considerably
faster at 29°. This loss is strongly inhibited by 1 mM Mg++. It was found, surprisingly,
that G-ADP actin is capable of polymerization upon the addition of salt; that is, it is not
necessary to convert G-ADP to G-ATP for polymerization. However, at 1° C., G-ADP poly-
merizes slowly, whereas G-ATP polymerizes rapidly. In all cases, the extent of polymerization
is directly proportional to the bound nucleotide. Thus, the facts have been established for the
test of the principal question.
G-ADP actin in 1 mM Mg'1"1" (where only traces of bound nucleotide are lost) is incubated
with CP and CPase. The resulting solution is tested for polymerizability at 1° C. If the
actin polymerizes slowly, it is still G-ADP, and no enzymatic transphosphorylation has occurred.
If it polymerizes rapidly, it is G-ATP, and the bound nucleotide has been transphosphorylated.
The latter is found to be the case, and it is concluded that the bound nucleotide of G-ADP
is capable of enzymatic transphosphorylation.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 2(M
AUGUST 23, 1960
The tlircc hearts of the oyster. PAUL S. GALTSOFF.
The open circulatory system of the oysters and other bivalves is characterized by the
presence of large sinuses and open lacunae or spaces which receive blood from the arteries.
It is obvious that under such a condition effective circulation is impossible. This deficiency is
compensated by several mechanisms : the pulsation of the radial vessels of the mantle ; the
squeezing- of blood in the vessels of the gill lamellae by the contraction of gill muscles ; the
contraction of the wall of the common afferent vein ; and by the contractions of the two acces-
sory hearts located on the wall of the cloaca on each side of the adductor muscle. The
accessor}- heart is not a simple tubular structure, as it had been heretofore described : it
consists of three large branches, joined at a center in the form of a letter Y. Injection of
dyes shows that the accessory hearts are in communication with the anterior pallial arteries,
the renal sinus, the efferent veins and the circumpallial artery. It is interesting that the blood
from the accessory heart enters this vessel against the ventricular pressure of the principal
heart. The contraction wave starts from the center of the accessory heart and goes into two
opposite directions. The rate of contraction is independent of the beating of the principal
heart and is much slower. The function of the accessory hearts is to oscillate the blood
within the pallium.
GENERAL SCIENTIFIC MEETINGS
AUGUST 29-31, SEPTEMBER 1. 1960
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 READ
The pupillary niecJianisin in the toad. PHILIP B. ARMSTRONG.
The isolated sphincter pupillae of the toad (Bnfo marinits) can be stimulated to contract
by the direct action of light. If maximal light constriction is produced and the illumination
of the pupil continued, there is a slight adaptation to the light, as manifested by a slow con-
tinuing dilatation of the pupil. The constriction of the pupil produced by light is not antagonized
by atropine even in high concentrations (IfT3). The threshold for acetylcholine is 10"9 with
the dilated dark-adapted pupil. Acetylcholine 10~5 produces constriction about equal to that
produced by maximally effective light. A further increase in the acetylcholine concentration
(10~4) results in some dilatation of the pupil but the sensitivity of the constricter to light appears
to be unchanged. Acetylcholine is antagonized by atropine. Pilocarpine produced a slight
constriction of the pupil but only in high concentrations, the threshold being 10~4. Serotonin
produces some slight pupillary constriction also only at high concentrations (10~3). DL
arterenol produces a striking dilatation in both the light-adapted and dark-adapted pupils. The
threshold is 10'9 for the dark-adapted pupil.
Stimulation of the sympathetic trunk at the level of the most anterior vertebrae produces a
striking pupillary dilatation, as does also the stimulation of a pair of nerves entering the eyeball
at its posterior inferior aspect. It may be that this is a mixed nerve since occasionally a very
slight constriction of the pupil can be induced by stimulation of these same nerves when the
preparation is dark-adapted.
It appears that the sphincter pupillae "has little if any motor innervation, that the dilator
is sympathetically innervated probably by adrenergic fibers.
Aided by a grant from the National Institutes of Health, B-643.
2{)2 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
A method for obtaining gametes from Asterias jorbesi. A. B. CHART AND R. S.
MUSICK, JR.
A new method of obtaining starfish gametes has been developed, enabling one to obtain
substantial quantities of fertilizable eggs (and sperm ) even during the latter part of August.
Animals were injected with an extract prepared from isolated radial nerves of large unripe
starfish. The nerves, after washing in sea water, were lysed in distilled water for 10 minutes
(1 cc. per nerve), and the supernatant was dialyzed against running sea water for 12 hours
(or at 6° C. with frequent changes of sea water). A second treatment with distilled water
yielded another fraction which also contained substantial concentrations of "shedding substance."
Fifteen-hundredths cc. per gram of the heat-stable extract (s), which may be stored frozen,
was injected through an arm into the coelomic cavity of a small, 10-30-gram (about 3-inch)
ripe starfish. The animals were kept in dry iced fingerbowls (approximately 17° C.) until
shedding was initiated, usually within 45 minutes. The eggs were collected in beakers by
suspending the shedding females (oral side up) over beakers of sea water. Fifty to 70% of
the animals injected in this manner shed, depending upon the state of the gonads. The eggs
of 82% of the females shedding after an injection of the "shedding substance" exhibited 90%
or better germinal vesicle breakdown. Preliminary evidence indicated that the eggs were
highly fertilizable (88% or above). Yields of as much as 3-5 cc. of eggs were obtained from
one 20-40-gram starfish. These small ripe starfish were obtained from cold water and were
available until at least the latter part of August. Since the stock animals were stored in refrig-
erated sea water tanks, shedding could be induced when required. Further studies are being
carried out on the characteristics and physiologic function of the shedding substance obtained
from the starfish nerve.
This work was supported by National Science Foundation Grants No. G-8718 and G-12045,
and National Institutes of Health Grant No. A-3362.
The modification of life span by x-rays for Jiaploids and diploids of tlie wasp,
Habrobracon sp. ARNOLD M. CLARK.
Haploid males, diploid males and diploid females of the wasp, Habrobracon sp. (an Indian
species related to Habrobracon juglandis), show a decrease in adult life-span following exposure
to x-rays as larvae-in-cocoons, white pupae or adults. The median life span for non-irradiated
adults was 62 days for haploid males and diploid males fed on honey-water, 92 days for diploid
females fed on honey-water and 40 days for diploid females fed on larvae of the Mediterranean
flour moth, Ephestia. This difference in life-span related to sex, but not to genome number,
indicates that the aging process is not due to an accumulation of somatic mutations during adult
life. Haploid males exposed as adults to 10,000-50,000 r have a shorter life span than com-
parable diploid males. Diploid males and diploid females show similar decreases in life span
relative to their controls. Pupae after exposure to 10,000 and 15,000 r and larvae after exposure
to 2,000 r are equal to controls in post-embryonic survival and in ability to develop into struc-
turally normal adults. These adults, however, show a decrease in life-span. Diploid males
resulting from irradiated larvae or pupae show a decrease in life-span that is smaller than
comparable hapleid males but similar to diploid females. Radiation-induced decrease in life-span
is markedly influenced by genome number but not by sex. This indicates that in contrast to the
normal aging process, the decrease in life-span by x-rays is due to damage to the genetic
material.
Distribution and substrate specificity of sea urchin egg phosphatases. GILLES
COUSINEAU AND PAUL R. GROSS.
Phosphoesterase activities of sea urchin egg homogenates have been assayed against a
variety of substrates and at three pH values. The substrates employed were glucose-6-phosphate
(G6P), a-glycerophosphate (GP), nitrophenyl phosphate (NPP), fructose-l,6-diphosphate
(F1.6P), and glucose-1-phosphate (G1P). These substrates were incubated with the enzyme
sources at pH's of 5.4, 6.4, and 8.4. Fractions were obtained by differential centrifugation of
the homogenates, which were prepared in isotonic KC1 from unfertilized eggs that had been
PAPERS PRESENTED AT .MARINE BIOLOGICAL LABORATORY
\vashed, successively, in filtered sea water, Ca-Mg-free artificial sea water, isotonic sodium
citrate, and isotonic KC1. The homogenization medium contained 10~3 M EDTA. The ex-
pected activities for acid and alkaline phosphatases were observed. At pH 5.4, setting the
hydrolysis rate for glucose-6-phosphate at 100, phosphate release rates for the other substrates
were computed as%: F1,6P, 132; NPP, 119; GP, 47; GIF, zero. At pH 8.4, with G6P again
set at 100, release rates for the other phosphate esters were: F1,6P, 97: GP, 47; NPP and
G1P, zero. At pH 6.4, where the activities of the acid and alkaline phosphatases could be
expected to be minimized, significantly high hydrolysis rates were obtained for two substrates,
G6P and NPP. The rate pattern was as follows: NPP, 415; G6P, 100; GP, 17; F1.6P, 17;
(ilP, zero. Although the behavior of these systems toward NPP at pH 6.4 constitutes a diffi-
culty, the patterns of activity of the preparations are consistent with the existence, in the sea
urchin egg, of a distinct glucose-6-phosphatase, possessing substrate specificities similar to
those of the enzyme obtained from mammalian tissues. The homogenate fractions (pigment
vacuoles, yolk particles, mitochondria, "microsomes," and soluble phase) showed different but
characteristic patterns of activity against the substrates at the different pH values. The essen-
tial conclusions of this part of the study are as follows : the distribution of phosphatase activity
among the fractions is different at the three pH values chosen, supporting the possibility that
the catalysts are distinct from one another. Most of the glucose-6-phosphatase activity, so
characteristically a "microsomal" function in mammalian cells, is localized in particles identical
with, or sedimenting along with, the yolk granules.
Supported by grant #A-2302 (Cl) from the National Institutes of Health, U.S.P.H.S.
Osmolarities of some body fluids in the elasmobranch and teleost. H. DAVSOX AND
CYNTHIA THOMAS GRANT.
Earlier work has shown that, in the mammal, the cerebrospinal fluid (CSF) is hyper-
osmolar to the blood plasma whilst the aqueous humour is approximately iso-osmolar ; in the
dog-fish (Mustelus canis) the aqueous humour is markedly hypo-osmolar to the plasma. In
the present study on the dog-fish the osmolarities of the plasma, aqueous humour, CSF and
the "subdural fluid" lying immediately below the cartilage of the skull were found to be 970,
947, 964 and 944 milliosmoles per liter, respectively. Thus, the CSF is approximately iso-
osmolar with plasma whilst the "subdural fluid" is similar to aqueous humour. The chloride
concentrations followed the same pattern, being 275, 245, 282 and 273 millimoles/kg. ILO,
respectively. In the scup (Stenotomus versicolor) the osmolarities of plasma and eye-fluids
(a mixture of aqueous and vitreous humours) were 397 and 364 milliosmoles per liter: thus
there is a high difference of osmolarity across the cornea of the teleost, but in spite of this
the eye-fluids remain hypo-osmolar to plasma. It is likely that the "spectacle" represents an
adaptation that slows down the osmotic withdrawal of water from the eye-fluids since removal
of this skin from one eye caused a progressive increase in the osmolarity of its eye-fluids : thus,
1, 4, 6 and 10 hours after removal the differences between the two eyes were 10, 17, 31 and 40
milliosmoles per liter. The chloride concentrations followed the same pattern. In one fish it
was found that its spectacle was opaque, presumably through damage to its epithelium, and it
is interesting that the fluids of this eye were stronger than those of the other by 130 milliosmoles
per liter. In Tautoga onitis plasma and eye-fluids contained 360 and 321 milliosmoles per liter,
respectively. The intraocular pressure of dog-fish and scup was 10 cm. H2O.
This investigation was carried out in part under a Fight for Sight Fellowship of the
National Council to Combat Blindness, Inc., New York.
The relationship between oxygen tension and light production before and after
grinding up the light organs of fireflies. ARTHUR B. DuBois.
Fireflies of the local variety of Photinus and Photuris. when studied in rh'n. require
approximately 100% O2 to achieve a maximal steady glow. Extracts of their light organs are
said to require only \% O2 for a maximal steady glow. This difference may be due to a
lower rate of reaction in vitro, requiring less oxygen, or to a diffusion gradient for O, in vii'o,
yielding a low O= tension at the site of reaction. The present experiment was an attempt to
estimate the intensity of glow at different O. tensions before and after grinding up the light
2(H PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
nrean. A phototnultiplier system was used to measure the light emission. A small glass tissue
grinder was fitted with an air-tight rubber stopper and two side arms, to flush through different
concentrations of O2 in N2 before or after grinding. Before grinding, the light intensity was
easily measured, and increased up to a maximum of 20 scale divisions on pure O=. After
grinding, the light intensity was less than half a scale division (unreadable), even after adding
ATP. The maximum intensity occurred at a concentration of approximately 10% O-. It was
concluded that the reaction occurred more intensely in the organized cell than in its homogenate,
and hence more oxygen was used up in the intact cells, resulting in a larger diffusion gradient
for oxygen. The reason for the decrease in light production after disruption of the cell
structure was not determined.
Studies on actin. I. The properties of G-ADP actin. TERU HAVASHI AND RAJA
ROSENBLUTH.
The changes of state of the muscle protein actin can be described as a cycle of events to
regenerate actin; vis., G-ATP polymerizes to F-ADP with KC1 and Mg, F-ADP depolymer-
izes to G-ADP in water, G-ADP converts to G-ATP by two methods: (a) Incubation in
water with ATP, or (b) incubation in small amounts of Mg with CP and CPase. Method (a)
is known to be a whole nuleotide replacement; method (b) is of interest because of the possibility
of transphosphorylation of the bound ADP. As a start toward a study of this possibility, a
study of the properties of G-ADP as a key substance in the cycle was undertaken.
G-ADP polymerizes upon the addition of KC1, MgCL and ATP. The extent of the
polymerizability is a measure of the activity of the protein. At 1° C., 20% of this activity
is lost in an hour, and is found to be due to the loss of bound nucleotide. At 29° the loss is
faster. The continuous presence of Dowex-1, which quantitatively removes free nucleotide, also
hastens the loss of bound nucleotide. Mg ion inhibits this loss to varying degrees, depending
on the concentration of the Mg.
G-ADP also polymerizes without ATP, simply upon the addition of KC1 and Mg. At 29°,
this polymerization is similar to that produced with ATP, both in rate and extent, but at 1°
it is much slower. A tentative explanation for this difference is given. Of considerable
interest is the fact that it is apparently unnecessary to dephosphorylate the nucleotide for
polymerization per sc unless, in this case, the ADP is being dephosphorylated to AMP.
A procedure for obtaining completely dissociated sponge cells. TOM HUMPHREYS.
SUSIE HUMPHREYS AND A. A. MOSCONA.
When sponges are pressed through bolting cloth, most cells remain in clumps. Further
straining excludes larger clumps, but small clusters remain. This, as well as the likelihood of
cell-type selection, and the variability inherent in this procedure, restricts its usefulness in studies
requiring dispersed cells. An effective method, based on the removal of calcium and magnesium,
was therefore developed for complete dissociation of sponge tissue into viable cells. It has
been extensively applied to aggregation studies on Microciona prolifcra, but is effective also for
Cliona celata and Haliclona permollis.
One gram of blotted sponge lobe tips was immersed in 80 ml. calcium- and magnesium-free
sea water (CMF-SW) at 0° C. (NaCl, 27.0; KC1. 8.0; Na«SO.,, 1.0; and NaHCO,, 0.04
gm./l. Hj.O, pH 7.2), cut into 2-mm. fragments, soaked for 30 minutes, and squeezed through
#25 standard quality bolting cloth into 80 ml. fresh, cold CMF-SW. The resulting suspension
contained single cells and small clumps at an approximate concentration of 15 X 10" cells/ml.
The material was centrifuged at 1000 rpm and resuspended at a concentration of 20 X 10a
cells/ml, in CMF-SW containing 0.1% trypsin (crystalline-lyophilized, Worthington) , at 0° C.
Fifteen-mi, aliquots of this suspension in 100-ml. beakers were rotated on a gyratory shaker at
80 rpm for 6 hours at 5° C. Three ml. of the resulting suspension were then transferred into
a conical centrifuge tube, vigorously squirted 15 times through a pipette (•"••4-mm. orifice, 2-ml.
bulb), spun down and resuspended in 1.5 ml. of sea water by pipetting.
The resulting suspension consisted of single cells. Maintained in stationary vessels, the
cells formed aggregates which developed into sponges in a manner similar to that of conven-
tionally dispersed sponge tissue.
It should be noted that trypsin did not noticeably assist in dispersion of the cells ; however,
for reasons yet unclear, trypsin-treated cells gave better aggregation results.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 295
Rotation-mediated aggregation of dissociated sponge cells. TOM HUMPHREYS,
SUSIE HUMPHREYS AND A. A. MOSCONA.
Aggregation of dissociated sponge cells provides a useful means for studying various cellular
activities and interactions. Conventional "self-aggregation," in depending largely on cell
migration and cell-sheet contraction, is susceptible to variables indirectly related to the pertinent
problems, and is therefore inadequate for analysis of cell interactions. In the present procedure,
cellular material is continuously maintained in suspension by rotation on a shaker and the cells
brought in contact by the motion of the liquid. All experimental parameters are readily con-
trollable and, under standard conditions, the results are consistent and amenable to quantitative
assessment.
Aliquots of Microciona prolifera cell suspension (30 X 106 cells in 3 ml. sea water)
dispensed into 25-ml. Erlenmeyer flasks are rotated for 24 hours on a shaker, %-inch diameter
of rotation, 80 rpm at 25° C. The cells rapidly accumulate in the center of flasks and within
minutes begin to cohere. After one hour all cells capable of cohesion are in small, irregular,
loose clusters ; these tend to fuse and reach final size at about 8 hours. By 24 hours the
aggregates are round, compact, smooth, average 0.12 mm. in diameter, contain 1 to 2 X 105 cells,
and are capable of development. Under these standard conditions, consistent, reproducible
"aggregation patterns," defined by size distribution, shape and compactness of aggregates, are
obtained. These are highly sensitive to experimental conditions, thus providing useful baselines
for testing selected variables.
Using this procedure it has been found that : ( 1 ) aggregates are obtainable across a wide
range of cell concentrations, (2) cells from different species have distinct aggregation patterns,
(3) variations in dissociation procedure affect aggregation, (4) aggregation is temperature-
sensitive in that cells remain dispersed at 5° C., although brought in contact by rotation, and
form only small clusters at 10° C., (5) calcium is the only divalent cation sufficient for normal
aggregation, (6) no cell cohesion occurs in the absence of divalent cations.
Delayed cleavage of fertilised Arbacia eggs after treatment zvith a "nitrogen
mustard" or with formaldehyde. MILTON LEVY AND PEDDRICK WEIS.
Bis (beta chloroethyl) ethylamine (a nitrogen mustard gas) inhibits mitosis. The chemical
action of the agent results in "alkylation" by replacement of active hydrogen by the alkylamine
group with elimination of hydrogen chloride. The presence of two reactive groups allows the
formation of cross-links between molecules, and seems to be essential for the biological effect
of nitrogen mustards. Formaldehyde can also form cross-links.
Experiments (initiated in 1944) were done to define the actions of these agents on Arbacia
eggs and sperm by following the progress of the first and sometimes the second cleavage. Egg
sets and sperm were obtained by electrical stimulation, washed, and divided into batches. At
appropriate times a reagent was added in I/SO of the final volume. Treatments were terminated
by sucking the supernatant off the settled eggs and washing to a nominal dilution of 2500-fold.
Eggs were fixed and counted at intervals from each batch. To compensate for the variability
among sets of eggs, the normal first cleavage time (55-68 minutes at 21°) of each set is taken
as 100% and delays are given in terms of this time. For constant time of exposure the delay
increases with concentration, but at 0.0009 M formaldehyde and 60-minute exposure the eggs
cytolyze. The delay increases with time of exposure. Thus, using 0.00044 M formaldehyde
for 10 minutes before fertilization produces no delay, but 15 minutes produces 13% delay and
60 minutes produces 75% delay.
Time of treatment in the cleavage cycle is very important. For the same treatment
(5 minutes, 0.00072 M nitrogen mustard) the following data were obtained. Treatment before
fertilization 75% delay, started just after fertilization 150% delay, 3 minutes later 115%, 12
minutes later 106%, 20 minutes, 40%, 24, 30, or 45 minutes no delay in the first cleavage but
considerable delays in the second cleavage. The same treatment of sperm caused a 60% delay
in the first cleavage of eggs fertilized with them.
Constancy of the pC02 in the ocean. W. F. LOOMIS AND W. F. LOOMIS, JR.
The carbon dioxide tension or pCO= of the ocean was studied under a variety of conditions.
Using scuba, syringes were visually filled with bubble-free ocean samples taken at various
296 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
depths down to 60 feet. After collection, all samples were placed on ice and taken directly
to the laboratory where their oxygen tension was determined by the Winkler method and
their pCO. determined by a new and direct method that does not involve pH. Duplicate samples
were taken at various depths and localities from April to August, sites including the ocean,
fresh- and brackish-water lakes and a salt marsh. The diurnal cycle was studied by taking
measurements at 3 PM, 9 PM, and 3 AM, in both fresh-water and ocean sites. The findings
were compared to oxygen determinations made by the Oceanographic Institution at stations
between Woods Hole and Bermuda at all depths down to the bottom of the ocean (5140 meters).
The highest pCO? found was 1.43% atm. This occurred in a 9° C. exolimnotic layer 15
feet down in a fresh-water lake in April. Since fully aerated water has a pCO2 of 0.03% atm.,
this finding contrasts with the fact that no pCO... above 0.13% atm. was ever found in the
ocean. Oceanographic data indicate a layer 1000 meters down that is about 55% saturated
with air and hence has a calculated pCO: of around 0.3-0.4% atm. This would appear to be
the maximum pCO? of the ocean as the per cent saturation with air rises both above and below
this deep sounding layer. Since the diurnal cycle was found to be less than 0.1% atm. at 10
feet of depth in the ocean, it can be concluded that pCO2 is an ecological variable that varies
widely in fresh-water lakes but is held extremely constant in the ocean. This finding confirms
the view that the oceans form a remarkably constant milieu c.rtcricnrc.
Carbonic anhydrasc in ihc female reproductive tract of clasmobranch fishes.
CECILIA LUTWAK-MANN.
Some years ago the author discovered the presence of carbonic anhydrase in the endometrial
mucosa and placental tissue of various mammals, and in the uterine deciduoma of the rat. The
occurrence of carbonic anhydrase was also established in certain parts of the oviduct of the
egg-laying hen. These studies clearly demonstrated that carbonic anhydrase in the female
reproductive tract depends upon hormones which control the function of the genital tract.
It was now decided to extend the study of the genital tract of certain elasmobranch fishes,
at different stages of reproductive activity. The investigation was done on the vivparous-
placental smooth dogfish (Mustclits canis), the viviparous-aplacental spiny dogfish (Squalits
acanthias), and the oviparous skate (Raja crinacca). The genital tract of immature, or mature
but non-pregnant, dogfishes showed none or negligible carbonic anhydrase activity. On the
other hand, carbonic anhydrase was markedly active during early, and slightly less so in
advanced gestation. The enzyme was localized in the lining and the wall of the uterus, the
degree of activity being roughly equal to that found in the gills of these fishes. The oviducts
and shell-glands were less active. In mature egg-laying skates the oviducts were the main site
of carbonic anhydrase activity; the shell-glands were also active, and slight activity was
demonstrable in the lining of the uterine folds. Practically no activity was detected in these
organs in immature skates. Early dogfish embryos (4-7 cm.), yolk-sacs, or mature eggs of
either dogfishes or skates were inactive. The ovarian tissue of these elasmobranchs was devoid
of activity. In general, carbonic anhydrase values w-ere highest in the tissues of the skate, and
lowest in the spiny dogfish.
Reducing substances in blood of toad fish and catfish. PAUL FOLEY XACE.
It was apparent early in the investigation of alloxan diabetes in fish that pancreatic beta
cell changes and blood Folin values followed a similar course. The use of the enzymatic
Glucostat determination for glucose, in conjunction with the Folin-Malmros procedure for total
reducing substances, confirmed this relationship for glucose but showed that another substance
or substances participated even more markedly. Because the major concern of the investiga-
tion was the post-alloxan regeneration of the beta cells of the toadfish as compared to the
non-regeneration in the catfish, it appeared necessary to examine these substances as possible
factors in this species difference. In both species of fish, these substances accounted for most
of the early change in Folin value. In toadfish, 60 minutes after alloxan injection, the Glucostat
glucose had increased only 10 mg.% above control levels, while the total Folin reducing
substances had increased nearly 100 mg.%. In man, Saifer found only 10% differences in
non-diabetic patients and 20% differences in diabetics. The suggestion that alloxan or its
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 297
compounds were important components was not supported by the course of the blood changes
or by the observation that egg-bearing females failed to show the post-alloxan increase in
Folin value. The relationship of alloxan to uric acid led to assay of this compound in catfish
blood after alloxan, with failure to demonstrate a significant contribution. Since Fashena had
shown the importance of glutathione and glucuronic acid in the "saccharoid" fraction of human
blood, both test tube and chromatographic studies of these substances were made, using catfish
blood. No significant increases were found. In the toadfish, blood fructose appeared constant
through post-alloxan hyperglycemia. N.I.H. A-1129 (C3).
Measurements of volume and composition of flic swim bladder gas of toadfisli
(Opsanus tan}. GEORGE POLGAR.
A pair of narrow lumen plastic tubes were sewn into both compartments of the swim
bladder of unanesthetised toadfish through an abdominal incision. Their distal ends, sealed
with caps, were left outside the abdomen after suturing the skin. Following this operation, and
later at regular intervals, the caps were removed and. through a hypodermic needle, gas samples
were withdrawn into syringes, the plungers of which were a Mylar-covered oxygen electrode
and a Teflon-covered carbon dioxide electrode, respectively, for measuring the partial pressure
of these gases. First the bladder was allowed to deflate to induce gas production. Subse-
quently the gas samples were reinjected into the bladder. The gas volume was measured before
each analysis, with the fish in a water-filled pressure cooker serving as a plethysmograph.
The volumes were calculated from Boyle's law. In seven fish, the initial oxygen concentration
was between atmospheric (20.9%) and 49%, being higher in the anterior than in the posterior
chamber in all but one fish. After deflation, a less concentrated oxygen was found in a small
volume at first, but 18-48 hours after the operation, a peak was reached with a single maximum
value of 86%. The changes of oxygen concentration were generally parallel in the two
chambers. In one fish the carbon dioxide concentration rose in the anterior chamber prior
to the rise in oxygen concentration. In another one, there was no such early peak. The
concentration of carbon dioxide did not exceed 6% in any determination made on 7 fish, and
the direction of its changes was usually the same as for oxygen.
These experiments indicate ( 1 ) that it is technically possible to sample both chambers of
the swim bladder repeatedly under nearly physiological conditions; (2) some trends have been
observed in the changes of composition and volume of the gases after deflation; and (3) a
consistent difference was found between the concentrations in the two chambers.
An addition reaction of flic a,(3-unsaturated ether linkage of f>lasina!oc/etis.
MAURICE M. RAPPORT.
It was unexpectedly found that lipids containing high concentrations of plasmalogen
(derived from Asterias eggs and Mytilus (whole animal) ) lose their unsaturated ether linkage
on standing for several days at 20-24° in chloroform-niethanol solution. This instability was
not seen when the original chloroform-methanol extract was exhaustively washed with water.
Lipids which had lost the unsaturated ether linkage fully retained their capacity to generate
higher fatty aldehyde as determined by p-nitrophenylhydrazone formation. These properties
indicate an addition reaction of the unsaturated ether producing a mixed acetal (or hemi-acetal
if the addiict is water). The reaction was observed in only one of two extracts of starfish
eggs but in all four of the extracts of mussel that were studied. In unwashed chloroform-
methanol extracts, the unsaturated ether was relatively stable. It has been suggested that the
function of plasmalogen may be related to its capacity to add water-soluble compounds at the
activated double bond. \Yhile the observations made are consistent with this hypothesis,
interpretation must await a more complete knowledge of the reactants.
Inhibition of fertilization of Asterias, Spisitla and Chaetopterus eggs by Arbacia
dermal secretion. HERBERT SCHUEL AND CHARLES B. METZ.
Upon brief (60-second) exposure to tap water the adult Arbacia releases a yellowish-
green fluid called dermal secretion (DS). which inhibits fertilization of Arbacia eggs. The
298 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
DS appears to inactivate some egg substance that is essential for fertilization. In order to
determine if this were a highly specific effect, the Arbacia DS was tested for action on
fertilization in other species.
Eggs of the starfish Asterias jorbesi did not fertilize in the presence of DS. Eggs washed
from DS exhibited an almost complete loss of fecundity; /.£'., even when very high sperm
concentrations (0.2%) fertilized few of the eggs. Exposure of eggs to DS subsequent to
elevation of the fertilization membrane did not inhibit cleavage. Sperm washed from DS
fertilized eggs as readily as sperm washed from sea water. These results are similar to those
that have been obtained with Arbacia eggs and sperm.
In the presence of DS, fertilization of the eggs of the clam Sfisula solidissima was inhibited
because the sperm were killed. Eggs washed out of DS fertilized as well as the controls
(the germinal vesicle broke down), but the DS-treated eggs did not divide. Exposure of
eggs to DS after fertilization resulted in an inhibition of cleavage that could be partly reversed
upon return to sea water. High concentrations of Spisula sperm (10%) absorbed and removed
from solution the fertilization and cleavage inhibitors.
Preliminary experiments indicate that DS inhibited the fertilization of Chaetopterus
pcryamentaccits eggs. This may be the result of action on both the eggs and the sperm. Eggs
exposed to DS after fertilization did not divide.
This work was supported by grant RG-6234 from the National Institutes of Health to
Dr. Charles B. Metz.
Further studies on the antimitotic action of heavy water. WILLIAM SPINDEL AND
P. R. GROSS.
The immersion of fertilized sea urchin eggs in sea waters reconstituted with excess
deuterium results in delays in, or blockade of, mitosis and cytokinesis. D2O concentrations
up to 30-40% cause relatively simple delays in the cleavage cycle, although the incremental
periods may be very long. Higher concentrations change the kinetics of cleavage as well,
and permit smaller total numbers of eggs to enter mitosis. At concentrations higher than
65-70%, cell division is totally blocked. These effects are entirely reversible upon removal
of the deuterium-enriched medium and replacement with sea water, except in cases where
fertilized eggs have remained blocked in D2O for several hours. The blockade is related to the
mitotic cycle in general and not to nuclear events unique to the first cleavage cycle, for eggs
can be blocked in the same way during cleavage cycles subsequent to the first. Reproduction
and division of the cell centers, and perhaps of the genetic material, appear to continue during
the blockade of karyo- and cytokinesis, since cells which have remained arrested in D^O for
long periods divide directly into multiple-celled forms when washed with normal water. The
inhibition of fertilization itself is an effect upon the egg, not the sperm, since motility and
fertilizing power are retained for significantly long periods after immersion of spermatozoa
in DjO. Current notions concerning the mechanism of antimitotic action by D2O invoke
changes in the normal hydrogen-bond cross-linking of cytoplasmic structure-forming macro-
molecules and particulates. In this connection, it has been found that the cytoplasmic "viscosity,"
measured by particle sedimentation, rises exponentially with increasing deuteration of the
medium, reaching, in 95% D.O, values more than sixteen times as great as those characteristic
of sea water controls. This type of effect is also observed in fertilized eggs. The simple
cleavage delay in 30% DL,O falls sharply in absolute length as the temperature is raised from
15 to 29° C. The effect of this is to make the delay a constant fraction of the cleavage interval
in the temperature range indicated.
Supported by grant #A-2302(C1) from the National Institute of Arthritis and Metabolic
Diseases, and by grant # AT (30-1) -2250 from the U. S. Atomic Energy Commission.
Comparative studies of malic and glutamic dehydrogenases. CLAUDE A. VILLEE.
The studies of Kaplan ct al. (Science 131 : 392, 1960) have shown that lactic dehydrogenases
from different tissues of the same animal may react at different rates with the several pyridine
nucleotide analogs. In addition, lactic dehydrogenases from different animals have reaction
rates with the several analogs in patterns which indicate evolutionary relationships : closely
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 299
related animals have lactic dehydrogenases with similar patterns of reaction rates with these
analogs, whereas unrelated animals have lactic dehydrogenases with markedly different patterns.
To determine whether other dehydrogenases show comparable differences, and give patterns
of reactivity that correspond to those for lactic dehydrogenases, tissues from a variety of
invertebrate and vertebrate animals were homogenized and centrifuged in the cold, to sediment
particulate matter. Aliquots of the supernatant fluid were tested for malic and glutamic
dehydrogenase activity with diphosphopyridine nucleotide (DPN), 3-acetyl pyridine DPN
(APDPN). 3-pyridine aldehyde DPN (PADPN), deamino DPN, and thionicotinamide DPN
(TNDPN). Reaction rates were measured spectrophotometrically at the wave-length of
maximum absorption of the reduced pyridine nucleotide or analog. Control cuvettes contained
the complete system minus the substrate (malate or glutamate), to correct for reduction of
pyridine nucleotides due to other enzymes and endogenous substrates. The patterns of relative
reaction rates with the different analogs for malic and glutamic dehydrogenases are quite
different. The rates with DPN and deamino DPN are generally similar but the enzymes
tested reacted much more rapidly with the other three analogs than with DPN. The differences
which characterize each tissue are based primarily on the rates with APDPN, PADPN, and
TNDPN. Malic dehydrogenases of comparable tissues, e.g.. ovary, of related animals, e.g.,
Arbacia, Strongylocentrotus, Astcrias. Echinarachnius and Thyone, were more similar than
those of different tissues, e.g., ovary, testis, gut, and muscle, from the same species. Although
the absolute patterns of reactivity are different for malic, glutamic and lactic dehydrogenase,
evolutionary relationships are discernible on the basis of any of these.
Experiments with Ca4'' in marine egg cells. FLOYD J. WIERCINSKI AND JAMES K.
TAYLOR.
The egg cells of Arbacia punctulata and Spisula solidissiina were used in various experi-
ments with Ca45. Egg cells were exposed to sodium citrate and EDTA, then Ca45 was applied.
With ultraviolet light and x-ray Ca45 was present in the medium during the exposure. Also,
cells were incubated in calcium-free sea water with known amounts of Ca45. Samples of dried
eggs and dried medium on planchets counted with a beta detector gave data that were in the
range of the standard deviation.
Observations were made with the cells crowded in Ca45 solutions of 0.8 mm. thickness by
means of a beta gas flow detector coupled to a rate meter and a continuous recording device.
Under normal conditions there was very little, if any, uptake of Ca45. Cell calcium is in
equilibrium with that found in sea water. Long exposure to ultraviolet light, 150 Kr of x-ray,
sodium citrate and EDTA showed varied uptake of Ca45 by a decrease in the counting rate.
Autoradiographic stripping film technique on whole mounts, sectioned eggs and ovaries
of Arbacia showed the localization of Ca43. Arbacia were grown in Ca45 sea water for ten
days. Sections of the ovary showed that the immature eggs were rich in Ca45. These data
indicate that calcium is incorporated into the cell structure before the maturity of the egg cell.
Silver grain counts were made on whole mounts of Arbacia eggs from the unfertilized stage to
the blastula stage. A progressive increase in the number of grain counts per unit area on the
jelly coat and the cortex had been observed during development. Staining techniques indicate
that the cortex is probably a layer of calcium-nucleic acid complexes. Many slides have been
prepared for further analysis.
Acknowledgment is due to Smith, Kline and French for equipment, and to the National
Science Foundation for support. Technical assistance was aided by an AEC grant administered
by the MBL.
A junction for the nuclcohis. W. S. VINCENT AND E. BALTUS.
The nucleolus has long been associated with concepts of protein synthesis. Caspersson
and Brachet recognized that the basophilia of nucleoli was due to RNA, and postulated that
the RNA portion of the nucleolus might direct protein synthesis. Recent biochemical studies
have shown that at least two types of RNA are required in the synthesis of protein. One of
these is bound to the RNP granules of the cytoplasm (granule RNA) ; the other of the RNA's
is "soluble," and has been shown to act as an activation agent for amino acids through the
addition of terminal nucleotides.
300 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Our analyses have shown that the starfish oocyte nucleolus contains an RNA fraction
which adds (terminally) t\vo cytidylic acid residues. This fraction, when exposed in situ to
C'Meucine, binds the leucine with an alkali labile bond. Attempts to extract this RNA by the
cold M NaCl technique of Goldthwait resulted in the solubilization of 80-85% of the nucleolus.
Part of the RNA contained in this fraction is tightly bound to the protein, the rest remains
in solution after the protein is precipitated by dilution to 0.1 M NaCl. The leucine appears
to be initially bound to the free RNA ; upon further incubation the specific activity of the
protein-bound RNA increases, while that of the soluble RNA decreases.
Analytical ultracentrifugation of the cold salt extract reveals the presence of a large
homogeneous peak with a sedimentation coefficient of 4.5 S. The Cu-leucine is associated
with the RNA attached to this protein.
The data reported here, and many other observations in the literature, are consistent with
the following hypothesis: A major function of the nucleolus is the provision of "soluble" or
"activation" RNA for use in the synthesis of cellular proteins. The "activation" RNA pro-
vided by the nucleolus is bound to a carrier protein, likewise nucleolar in origin ; this protein
is considered to be essential for the integrity of the RNA.
Senior Research Fellow, USPHS, and Chargee de Recherches du Fonds National de la
Recherche Scientifique, Beige, respectively.
The "black" eye colors in Monnoniello. P. W. WHITING.
Many mutations have occurred changing dark brown wild-type eyes to brighter colors —
red, orange, yellow, peach, white. Very few mutants have been found with eyes darker than
wild-type. These "blacks" undoubtedly occur much more frequently than the records show,
possibly as frequently as the brighter colors, but are difficult to detect. They have been
divided into two groups — the bk blacks and the eb (ebony) blacks. The latter have no effect on
scarlet (st) or on vermilion (?'»;). st cb and 77;; cb being scarlet and vermilion, respectively.
Dahlia, a dark red, appears black in combination with ebony (da eb). The action of the bk
blacks appears to be to prevent the development of the bright pigments, leaving the black
pigments unaffected. Thus scarlet, having no (or very little) black pigment, is changed to
white (st bk) ; vermilion, with intermediate amount of black pigment, becomes light lavender
(vm bk) and dahlia, with much black pigment, becomes dark lavender (da bk). It is con-
sidered that the lavender chroma is due to a structural condition overlying black pigment, as
in the feathers of Blue Andalusian fowl. Both the bk blacks and the eb blacks change orange
and peach to white (or bk, or eb, pc bk, pc eb). It is expected that certain genetics tests now
under way clarify the seemingly contradictory effects in the ebony combinations with the bright
color genes. Irradiation of dahlia, orange or peach stocks should show more "black" mutants
than irradiation of wild type, scarlet or vermilion. Homologies and linkage relationships are
being determined. There are at least three loci, with multiple alleles at one of them. ( Work
supported under contract AT (30-1) -1471 with the U. S. Atomic Energy Commission.)
PAPERS READ BY TITLE
The ionic composition of squid photoreceptors. R. G. ADAMS AND W. A. HAGINS.
Though the electrical activity of many retinas has been studied, little seems known of the
electrochemical basis of the currents and voltages so frequently seen. The squid retina is a
favorable tissue for use in estimating the chemical composition of photoreceptors, since at least
90% of the retinal cellular bulk consists of the receptors and their cell processes. Opened
squid eyes were dark-adapted and equilibrated at 10° C. in oxygenated, sulfate-free artificial
sea water for 2-3 hours in the dark. The retinas were then freed from their scleras in dim red
light, blotted and analyzed. They contained 79% water, 35% of which was accessible to inulin
and is thus considered to be "extracellular." Allowing for the extracellular fluid, the mean
ionic composition of the retinal cells was: K+, 300 mM ; Na+, 110 mM ; Cr, 140 mM. Since
the artificial sea water contained 10 mM K+, 430 mM Na+, and 571 mM Cl", the Nernst poten-
tials for the ions were : Na+, 34 mV ; Cl", —35 mV ; K+, —93 mV. The ionic composition of the
cells of the squid retina is thus grossly similar to that of many other types of cells. However.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 301
the failure of agreement between the K+ and Cl~ potentials implies that the cell membranes are
not equally permeable to both ions, or else the receptors are not fully equilibrated after 3 hours
in ASW. Nevertheless it is clear that electrochemical batteries are available in the photo-
receptor membranes for production of electrolytic currents of the type seen in other cells, such
as nerve and muscle.
Transport of nutrients in the holothnrian Leptosynapta inhaerens. A. S. D'Aoos-
TIKO AXD A. FARMANFARMAIAN.
Several investigators have reported that the digestive tube of holothurians, although per-
meable to water in both directions, is completely impermeable to a variety of substances tested.
These experiments, in addition to histological observations, have given rise to the hypothesis
that the transport of nutrients in these organisms is largely or entirely mediated by amebocytes
which occur abundantly in various tissues. The mode of nutrient transport in the echinoderms
in general is, furthermore, in confusion.
To elucidate some aspects of this problem a number of experiments were carried out,
using the holothurian Lcf>tosynaf>fa inhaercns.
The body wall was cut from a point just posterior to the mouth to a point just anterior
to the anus. The anus was ligatured and the mouth cannulated. The preparation was then
rinsed in filtered sea water and suspended in 5 ml. of the desirable isotonic solution. Under
these conditions both the body wall and the digestive tube continue to exhibit peristalsis for
8 hours in sea water. Punctures in the gut wall could readily be detected and the preparation
discarded when a suspension of neutral red in sea water was introduced into the digestive tube.
Experiments involving the use of various indicators showed the pH of the esophagus, stomach
and the intestine to be 6.8, 6.8, and 8.0, respectively. In a number of experiments 2000-3000
fig. of dextrose in sea water solutions were introduced into the digestive tract, and at time
intervals the outside fluid tested for dextrose. The results of these experiments show that
there is a passage of dextrose from the gut to the outside fluid at a rate of 16-17 ^g./min. at 20°-
23° C. Under these conditions 70-80% of the sugar appears in the outside fluid within two
hours. When parallel experiments were carried out in isotonic MgCL> solution, in which the
animal was relaxed, the rate dropped to 8 /^g./min. These results indicate that contrary to
earlier reports for other holothurians, the digestive tube of Leptosynapta inhaerens does allow
direct passage of dextrose to the outside, and that mechanisms other than simple diffusion
may be involved.
This work was supported by the National Science Foundation, NSF G-11595.
Directional movements of the intertidal snail, Littorina littorea. DOUGLAS G.
ALEXANDER.
Individuals of Littnrina littorea are located within a specific intertidal region. When they
are displaced above or below this region they move back into the preferred zone. Through
a marking and recapture program the intertidal movements were recorded. Individual snails
were marked with quick drying paint during low tide. After varying lengths of time sub-
sequent movements were determined by triangulation.
With the exception of the forms displaced above the spring high tide level, all displaced
forms moved in a direction which compensated for their displacement and brought them back
into the normal zone of distribution. Individuals released within the zone of normal distri-
bution displayed random movement at a reduced rate compared to the directional movements of
displaced forms. This rate of movement within the zone of normal distribution became further
reduced after a series of tidal cycles. The initial movement may have been due to the abnormal
concentration of marked snails at the release point, and their subsequent reduction in movement
may have been the result of their integration into the naturally existing population. Snails
moved only when covered with water. Animals displaced above the tide level failed to move,
indicating that the snails are inactive when exposed to the drying conditions of low tide. The
forms displaced below normal distribution were placed upon a different type of sediment, con-
sisting of fine silt. The trails indicated a rather direct movement toward the region of normal
occurrence.
302 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
This work was part of the student training program of the Marine Ecology Course and
supported in part by the Department of Zoology, University of North Carolina.
Observations on the ribosomes of sea urchins. WILLIAM S. ALLISON AND ELOISE
E. CLARK.
Preliminary studies, conducted primarily by ultracentrifugal methods, were undertaken in
order to determine conditions for isolating and stabilizing the ribosomes of the sea urchin.
Unfertilized eggs and embryos in various stages of development (mid-gastrulae, 48-, 60- and 65-
hour plutei) were chosen as the material to be used. Examination of both the crude extracts of
cells and partially purified preparations of the fast sedimenting components revealed that the
conditions required for the stabilization of the larger ribosomes of the sea urchin were not
the same as those reported for other organisms. It has been found that the addition of mag-
nesium to the extracting solvent is necessary for maintaining the structural integrity of the
so-called 70S particles of E. coli or the SOS particles found in yeast and various mammalian
tissues. This does not appear to be the case for extracts prepared from sea urchin eggs or
embryos. When extracts of either unfertilized eggs or plutei were prepared in the absence of
added magnesium (using 0.01 M phosphate buffer at pH 7.0, or with this buffer containing
0.002 M ethylene diamine tetraacetate), the schlieren pattern in the analytical ultracentrifuge
showed a major component which had a sedimentation coefficient of 70-75S. When comparable
extracts were prepared using either Tris or phosphate buffer and with varying concentrations
of magnesium (from 0.001 to 0.1 M}, the fastest component observed sedimented at 23-26S,
indicating that the larger components had been degraded. Partial purification of the 75S com-
ponent by means bf cycles of alternate low- and high-speed centrifugation, followed by suspend-
ing the particles in a buffer containing 0.1 M KC1, led to the disappearance of the 75S compo-
nent and the appearance of components which were observed to sediment at 43 and 24S. It is
of interest that whereas the classes of particles from this organism were similar to those
observed in other forms, the properties of stability were found to be somewhat different.
Regeneration of the cardiac stomach in Asterias forbesi. JOHN MAXWELL ANDER-
SON.
Starfishes that feed by everting the cardiac stomach continually place this delicate and
essential feeding organ in hazardous situations ; it would seem that ability to repair or regen-
erate the stomach in case of loss or damage would be of great survival value to the starfish.
In Asterias, eversion can be induced by soaking the animal in MgClr; solution and then squeezing
one or more of the rays. The everted stomach can be excised by cutting it across at the
peristome and just below the pyloric stomach and severing its connections with the retractor
harness. Study of a series of animals thus prepared and sacrificed at weekly intervals has
provided the following information: (1) within three weeks, the integrity of the gut is re-
established and the wall of the regenerated organ contains all the normal tissue layers; (2) the
new stomach forms by upward growth of a sleeve of tissue from the peristome, making contact
with the retractor nodule in each ray and eventually joining the pyloric stomach; (3) establish-
ment of contact with the retractor nodule apparently induces formation of a pouch-like en-
largement in the adjacent sector of the stomach — where contact is missed, as sometimes
happens, no pouch is formed; (4) at three weeks, the wall of each pouch has begun to form
a small but normal pattern of gutters and folds, with normal histological differentiation in the
epithelium — multiflagellated cells with huge, dense, spindle-shaped nuclei over the folds, single-
flagellated cells with granular, ovoid nuclei in the gutters; (5) between three and five weeks,
the intrinsic retractor fibers invade the connective-tissue layer, growing downward beneath the
branching gutters to form the normal corresponding pattern; (6) although the stomach is small
and its normal specializations are present in only rudimentary condition, the starfish is capable
of ingesting and digesting prey (amphipods, small snails) within the fourth to fifth post-
operative week. The regenerating cardiac stomach provides valuable material for study of
early stages in histological differentiation.
Supported by NSF Grant #G6007 to Cornell University.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 303
A day-to-day relationship between o.ridative metabolism and world-wide geo-
magnetic activity. FRANKLIN H. BARNWELL.
Os-consumptiorj of mud snails, Nassarius obsoletns. was measured continuously with Brown
recording respirometers from June 19 to July 19, 1959, in Woods Hole, Mass. There was
found to be a remarkable similarity between the day-to-day changes in mean daily metabolic
rate and the form of the day-to-day changes in magnetic activity as measured by the inter-
national magnetic character figure, C. Throughout the period of study the metabolic activity on
any given day, n, was clearly related to the intensity of magnetic disturbance on the first and
second days thereafter, n + 1 and n + 2. This is indicated by the coefficients of correlation,
—0.550 for the relation .-.hip to n + 1 and —0.539, to n + 2. Examination of other lag or lead
relationships revealed little or no agreement between the two series. Twenty-nine coefficients
of correlation for other temporal relationships of metabolic activity on day n to magnetic
activity, from day n — 15 to day n + 15, had a mean value of —0.068 and standard error of
±0.028. The coefficient of correlation for each day n + 1 and day n + 2 was significantly dif-
ferent from this population, p < .005. Such a correlation suggests that in rigorously controlled,
constant conditions metabolic rate of the snails was reflecting a response to a pervasive factor
related to solar, and subsequent world-wide geomagnetic, activity.
This study was aided by a contract between the Office of Naval Research, Department of
Navy, and Northwestern University, NONR-122803.
Comparative electrophysiology of supramedullary neurons. M. V. L. BENNETT.
The supramedullary neurons (SMC) of Lophlus piscatorius (angler fish) and Tauto-
golabrus adiposus (cunner) have been studied with microelectrode recordings. In both forms,
a number of similarities were found to the SMC of Spheroides maculatus (puffer; cf. Bennett
ct al., J. Gen. Physiol., 1959, 43: 159). Spikes could be evoked by brief stimuli to the spinal
cord or cranial nerves, or by tactile stimulation of the skin. These indirectly evoked impulses
arise in the axons, some distance from the soma, but subsequently invade the latter. An in-
flection on the rising phase of the spike recorded in the soma indicates delay at the axon hillock.
The impulses are initiated by two kinds of depolarization arising in the axon. One is long-
lasting, graded, and presumably of synaptic origin. The other is due to reciprocal excitatory
connections between the cells. These connections are electrotonic, since hyperpolarization as
well as depolarization is transmitted during intracellularly applied testing currents. As a
result, when one cell fires, it depolarizes adjacent cells and all cells tend to fire synchronously.
Thus, on indirect stimulation, with a near-threshold stimulus, all cells tend to fire, or not to
fire, and in multiple discharges to a single stimulus all cells tend to produce the same number
of spikes. However, the synchronization is less effective than in Spheroides, and frequently
the number of spikes differed in different cells. The electrophysiological similarities found in
the three forms indicate that the supramedullary neurons are homologous in all. However, the
function of the SMC remains unknown. In Lophins the impulses of the SMC were shown to
pass out the dorsal roots, but not the ventral. Thus, whatever their function, it is probably
similar in Lophius and Spheroides.
Electron microscopic studies of the reflecting structures of clasmobranch and telcost
eyes. M. H. BERNSTEIN AND T. S. DIETRICH.
Comparative observations have been made of the elasmobranch tapetum and teleost argentea
to correlate their fine structure and reflecting functions. Electron microscopic preparations of
three elasmobranchs : smooth dogfish (Mustclus cams'), sting ray (Dasyatis centntra) , and
basking shark (Cetorhinus maxime) , and a teleost (Tautoga onitis) were used. Histological
observations were correlated with the electron microscopy.
All of the elasmobranchs examined were similar. The tapetum is formed by modified
choroidal cells and covers the whole outer surface of the retina. Cellular processes containing
melanin granules interdigitate in regular alternation with elongate crystals (presumably
guanine) and their associated cytoplasmic extensions. In the light-adapted eye, the inner
aspect of the crystalline plates is covered by one or more layers of melanin granules. In
304 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
specimens dark-adapted for 12 hours, the melanin granules have migrated away from the
retina so as to expose more of the crystalline material to the incoming light.
The angle between the crystalline plates and the optical axis approximates 45 degrees over
much of the eye. In the area of the optic pit the plates are perpendicular to the axis. Toward
the anterior portions of the eye, the crystals remain parallel to one another, but also become
parallel to the incoming light. That is, a change is observed between the angle of the plates
and the light axis. The orientation of the plates with respect to the light axis changes from
90 degrees at the optic pit to 0 degrees near the ciliary body. It has also been observed that
the relative amount of crystalline material appears to be greater in the posterior pole of the eye.
The argentea of Tautoga on-itis lies between the choroid and cartilagenous sclera. Bundles
of parallel crystals lie in a connective tissue net. They lack the orderly relationship seen in the
tapetum. This might be expected since the camouflaging function of the argentea does not re-
quire optically true reflection.
In addition to a confirmation of the above observations, work is now being done on the
migration of elongated pigment granules in the retinal epithelium of T. onitis during dark
adaptation.
The morphology of starfish spermatozoa. M. H. BERNSTEIN AND L. G. FEHREN-
BAKER.
Electron microscopic examination of thin sections of the starfish (Asterias forbesi)
spermatozoa shows a roughly spherical, slightly flattened head composed of an acrosome,
nucleus and mid-piece. The acrosome is a complex mushroom-shaped structure contained
within a bowl-shaped depression, as demonstrated by J. C. Dan, with the inferior portion
elongating into a sac. The acrosomal depression is defined by a strong osmiophilic membrane.
The acrosome body is faintly osmiophilic and contains a central electron-dense star. The star
has a spherical central region with seven projections lying in a common plane. A crescentic
electron-dense mass surrounds the acrosome body in its equatorial region. Posteriorly, the
limiting membrane of the acrosome is thickened into a plate which separates the narrow caudal
extension from the main mass of the acrosome body. A dense osmiophilic knob is attached
to the plate and hangs down into the posterior sac.
The genetic or chromosomal material of the sperm head forms a cup around the posterior
aspect of the acrosome. The nuclear material is granular and electron-dense in character.
The crescent-shaped mid-piece in turn holds the nuclear material of the spermatozoan on
its superior concave surface. The mid-piece appears to be a single ring mitrochondrion with
a central hole for the insertion of the tail. The mid-piece is separated from the head by a
thin line of moderately electron-dense amorphous material, which thickens to a large mass
at the periphery of the head mid-piece connection.
The sperm tail is composed of a central core of two filaments surrounded by nine pairs
of filaments. The central core is lost at the entry of the tail into the opening of the mid-piece,
as the remaining filaments extend into the mid-piece in the region of the tail attachment to
form the proximal or ring centriole of the mid-piece.
A common membrane encloses the whole spermatozoan. It fuses anteriorly with the
acrosome membrane, which in turn splits to form two membranes on either side of the outer
dense crescent of the acrosome. The sperm membrane continues over the nucleus and mid-
piece and is reflected onto the tail.
He.vosc and pentosc utilization by mackerel crythrocytes. T. A. BORGESE AND
JAMES W. GREEN.
The object of the present study was to examine the anaerobic metabolism of the mackerel
red blood cell. A glycolytic pathway exists in these nucleated erythrocytes, as measured
by the disappearance of glucose and the production of lactic acid at 15° C. and pH 7.6. It
was demonstrated that the glycolytic quotient was dependent upon the level of phosphate in
the suspension medium. When the phosphate concentration ranged from 13 to 80 /iM/ml.
suspension, with an hematocrit of 13%, the glycolytic quotient dropped from 0.47 to 0.30.
respectively, suggesting that an increasingly greater dependence was being placed on respiration.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 305
On the addition of IAA or KCN there resulted a decrease in the rate of glucose disappear-
ance. Lactate production was virtually abolished with 10 /uM KCN/ml. suspension (hematocrit
15%). IAA resulted in a reduction in lactic acid formation of about 64% relative to control
suspensions.
The production of lactate from ribose appears to rival, and in at least one experiment
exceeded, the production of lactate from glucose. The use of KCN in ribose supplemented
suspensions also decreased the rate of lactate formation to approximately 70% of the control.
\Yith the control cells the rate of ribose utilization was best represented by a biphasic curve.
The more rapid initial phase had a rate of 0.61 ^M ribose used/ml, suspension/hr. The second
phase was considerably slower, 0.12 /uM/ml./hr. The hematocrit in this experiment was 15%.
Ribose disappearance in the presence of KCN was not measured since additional experiments
showed that cyanide interfered with the orcinol reaction for pentoses. The apparent stimula-
tion of ribose used was not a real effect but simply a linear function of the KCN concentration.
The overall results suggest that a glycolytic pathway exists in mackerel erythrocytes,
and that pentose is metabolized to lactate via a path comparable to the hexose monophosphate
shunt, which is the main alternative to the glycolytic pathway of carbohydrate metabolism in
animal tissues.
Nature and action of the fertilisation inhibitor from Fuciis. }. M. BRANHAM AND
C. B. METZ.
Certain extracts of the brown alga, Fucus vesiculosus, inhibit fertilization and the fertilizin
agglutination of echinoderm sperm. The chemical nature and mode of action of the inhibitor (s)
have been further investigated.
Activity was found in the ethanol-soluble fraction of water extracts of the alga. The
inhibiting material was precipitated with lead acetate and recovered from the precipitate.
Esping (1957) concluded that such preparations contained polyphenolic substances. Color tests
(F+++, KaFe (Cn)6, la. OH-), precipitation reactions (heavy metals, Cr2O7=, (NHi), Mod, CaO,
streptomycin, gelatin, agar, and in salt solutions), solubilities (soluble in methanol, ethanol,
butanol, insoluble in xylene, acetone, ether), and nondializability suggest that the activity may
be associated with tannins. Tannic acid (Merck) showed similar biological activity when
tested on Arbacia punctulata gametes.
Fertilization inhibition resulted from an action on eggs, for sperm washed from inhibiting
concentrations of the extracts were capable of fertilizing eggs, whereas membrane formation
and cleavage (i.e., fertilization) were irreversibly inhibited by exposure of eggs to Fucus
extracts. If fertilized eggs were exposed during membrane elevation, incomplete membranes
resulted and cleavage was greatly altered. Eggs with complete membranes cleaved normally
in the extracts. Trypsin digestion (one hour in 0.05%) did not restore fertilizability (cleavage)
to extract treated eggs, although trypsin treatment prior to exposure to Fucus extracts rendered
eggs insensitive to the inhibiting action of the Fucus extracts.
Fucus extracts inhibited both fertilization and fertilizin agglutination of sperm ( where
applicable) in all species tested. Thus, the action of the agents is not specific.
Aided by grant RG-6234 from the National Institutes of Health to Dr. C. B. Metz.
Endocrine control of the chromatophorcs of the zoeae of the prawn, Palaemonetes
vnlgaris. EDMUND S. BROCH.
Work done on the color adaptation of the zoeae of decapod crustaceans has indicated that
the color responses differ substantially from those of the adults : specifically that the chromato-
phores respond directly to light and show no background response. An exception to this
was a slight albedo response noted by Keeble and Gamble in second zoea of Palaemonetes.
This was attributed to the extension of the eyestalk at that stage. It has been reported that
chromatophores of first zoea do not respond to adult chromatophorotropins. There is no
mention in the literature concerning retinal pigment movements in zoeae, as has been reported
in adult decapods. Larvae of Palaemonetes vnlgaris were reared in the laboratory through
the eight zoea stages. Animals from one brood were used in each experiment, to insure
similar stages of development and minimal genetic variation. The study was made only of the
306 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
monochromatic red chromatophores which make up the secondary system, as denned by Keeble
and Gamble. In all experiments conducted, the chromatophores were in a concentrated state
when animals were on a white background and completely expanded on a black background,
thus displaying a true albedo response. This response occurred from the time of hatching to
the eighth zoea stage. First stage zoeae with sessile eyes responded as completely and rapidly
to different backgrounds as did second stage zoeae with eyestalks. The red chromatophores
on an excised thoracic section of a second stage zoea, kept in crustacean tissue culture medium,
contracted upon addition of brain extract, expanded upon addition of abdominal nerve cord
extract, and contracted when eyestalk extract was added. The distal retinal pigment of animals
kept in darkness did not move to a dark-adapted state. These observations indicate that
endocrine control of the chromatophores in the zoea of Palaemonetes vitlgaris is comparable
to that of the adult.
Magnetic field strength and organismic orientation. FRANK A. BROWN, JR. AND
FRANKLIN H. BARNWELL.
Snails, Nassarhis obsolctus, were observed as they emerged from a magnetic-south-directed
corridor into a symmetrically illuminated field under alternating conditions of the earth's
magnetic field alone and experimentally modified magnetic fields. The characteristics of
orientation of the snails were observed during their first 3 cm. of free movement. The experi-
mental magnetic fields consisted of a reversed direction of the horizontal component of the
field, and with a series of 8 horizontal strengths: 0.04, 0.1, 0.2, 0.4, 0.8, 2, 5, and 10 gausses.
For each experiment 5 snails emerged alternately in the earth's horizontal field of 0.2 gauss
and in an experimental field, first in an ascending and then in a descending order of strength.
Four such experiments were always run contemporaneously, and always between 9 AM and
noon to obviate influence of the known daily rhythm in orientation. Fifty groups of four
experiments were conducted, uniformly distributed over a period of exactly two synodic months.
The experimentally reversed magnetic fields resulted in an altered mean path of the snails.
When the strength of the horizontal component (H) of the reversed field differed by no more
than a factor of 3 to 4 (0.2, 0.4, 0.8 and 2 gausses) from the earth's total field, F (ca. 0.6 gauss),
there was a synodic monthly cycle of response with right-turning over full moon and left-turning
the remainder of the month. For reversed fields with H differing by a factor of 4 to 15 from
the earth's F (0.04, 0.1. 5 and 10 gausses), there was similarly a synodic monthly cycle of
response but now 180° out of phase, with right-turning over new moon and left-turning the
rest of the month.
This study was aided by a contract between the Office of Naval Research, Department of
Navy, and Northwestern University, N ON R- 1228-03.
A relationship between photic and magnetic response in snails. FRANK A. BROWN,
JR. AND ANNICK HUTTRER.
Mud-snails, Nassarius obsoletus, were permitted to emerge from a magnetic-south-directed
corridor over a polar grid such that the mean angle of right or left turning could be ascer-
tained during their first 3 cm. of free movement. The conditions under which the snails emerged
were the following, in order: (1) in the earth's magnetic field into a symmetrically illuminated
white field, (2) in a field black to left and white to right, (3) same as the preceding but in a
S-gauss horizontal magnetic field oriented in reverse to the earth's, (4) same as (1), (5) in a
field black to right and white to left, (6) same as 5 but in a 5-gauss horizontal magnetic field
oriented as in (3). For each experimental run the series was repeated, with 5 snails in each
condition, four times. Such experiments were carried out 18 times, 6 during mornings
8-10 AM, 6 during afternoons 1 :30-3 :30 PM, and 6 during evenings 6-9 PM. Incident
illumination in all cases was 150 lux from above, 20 lux from the black side and 60 lux from
the white. The snails showed a distinct positive phototaxis ; when the black field was to the
left the mean path was +15.9 ±1.96°, and when to the right, - 18.4 ± 2.52°. The deviation
in path in response to the asymmetrical light field varied from experiment to experiment. There
was, however, a distinct correlation between the strength of the response to the asymmetrical
light field and the effect of the 5-gauss magnetic field. For weaker light responses (< 15°),
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 307
either + or —/the magnet produced left-turning and for stronger light responses (15° to 42°),
right-turning. The coefficient of correlation between strength of light response and strength of
magnet response was + 0.40 (t = 3.6, N = 72, P < 0.001).
This study was aided by a contract between the Office of Naval Research, Department
of Navy, and Northwestern University, NONR-1228-03.
A "compass-direction effect" for snails in constant conditions, and its lunar modula-
tion. FRANK A. BROWN, JR. AND H. MARGUERITE WEBB.
At the same time each day, to avoid the daily rhythm of mean path of orientation,
Nassariiis were allowed to emerge from narrow corridors directed in four compass directions,
North, East, South and West, into a symmetrically illuminated field. In four contemporane-
ously conducted experiments a total of 40 snails emerged in each direction between the hours
1 and 4 PM. For each of two synodic months there was minimum left turning, or maximum
right turning when the snails were emerging northward. An analysis of the data indicated,
however, that there was a synodic monthly modulation in the form of the change in mean
path with compass direction. For both months, from first to third lunar quarter the relation-
ship was bimodal with two peaks of greater right turning, one for the north- and the other
for the south-directed snails, and for the semimonth from third to first quarter there was a
unimodal pattern of fluctuation with maximum right turning in northerly directed snails and
maximum left turning in southerly directed ones. The ranges of differences for the two
semimonths following new moon (3.4° and 3.8°) were only about half those observed for the
two semimonths following full moon (7.2° and 7.4°). That magnetic field clearly plays some
role in this organismic "direction-effect" was evident in comparing the relative effects of re-
versing the field of the north- and south-bound snails with, consecutively, 0.4 and 2 gauss
horizontal fields. For the north-bound snails the response to the weaker field lay 2.54°
to the left of that for the stronger field, and, to the contrary, for the south-bound snails the
response to the weaker field lay 2.92° to the right of that of the stronger field.
This study was aided by a contract between the Office of Naval Research, Department
of Navy, and Northwestern University, NONR-1228-03.
Some effects of lysergic acid diethylamide and related agents on embryonic heart
rate in Fitndulus. JOSEPH A. BURKE.
In an attempt to ascertain the earliest measurable effect of lysergic acid diethylamide
(LSD-25) and related psychotomimetic drugs on some specific organ, 25 Funduhis eggs were
grown in 50-ml. solutions of lysergic acid diethylamide, 1-methyl-lysergic acid butanolamide
(UML-491), yohimbine and serotonin, respectively, in sea water held in finger bowls at 21° C.
In concentrations of 100, 50, 10 and 1 /ug./ml., LSD-25 and yohimbine depressed heart rate.
In LSD-25, development of a functional heart was delayed at least 48 hours. When a pulse
was obtained in test embryos it varied inversely with concentration of drug. For example, with
LSD-25, the first rates, after a functional heart was present in test embryos, were : control,
122 beats/minute; 100 ng./ml LSD-25, 80/minute; 50 /ug./rnl. LSD-25, 97/minute; 10 ng./ml.
LSD-25, 115/minute; l/ig./ml. LSD-25, 140/minute. Mean heart rates for all days up to
hatching were: control, 147 beats/minute; 79, 93, 115 and 141 beats/minute for the 100, 50,
10 and 1 /tg./ml. concentrations of drug, respectively. LSD data plotted as logarithm of dose
against heart rate form a straight line. Using the least squares method, the resulting prediction
equation is : heart rate equals 144 minus 32 times logarithm of dose. With yohimbine, mean
atrial heart rates for 100, 50, 10 and 1 /xg./ml. concentrations were, respectively, 102, 122, 131
and 152 beats/minute; control, 147. In yohimbine concentrations above 10 fig. /ml., frequent
atrioventricular dissociation, with a ratio of one ventricular beat to from 2 to 8 atrial beats,
occurred. UML-491 had little effect on pulse rate. The effect of LSD-25 and UML-491 on
embryonic heart rate apparently is unconnected with their antiserotonin activity: UML-491
has an antiserotonin activity over four times greater than that of LSD-25. Serotonin itself
had no effect on pulse rate. The Sandoz Pharmaceutical Company kindly supplied the LSD-25
and UML-491.
Supported by National Institute of Mental Health grant MY-3235.
308 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Some morphological effects of lysergic acid dicthylamidc and related agents on
early embryonic development in Fimdttlus. JOSEPH A. BURKE.
To ascertain whether representative psychotropic drugs have an effect on development,
fertilized eggs of Funditlus were exposed at 2- to 4-cell stage (Oppenheimer, stages 3 and 4)
to 100, 50, 10 and 1 /ug./ml. sea water solutions of lysergic acid diethylamide (LSD-25),
methysergide (UML-491 Sandoz), yohimbine and serotonin. Twenty-five eggs per SO ml.
solution were grown in finger bowls at 21° C. The concentrations were low enough to obviate
changes in osmotic pressure or pH. No inhibition was noted in stages from Oppenheimer 4
to 16: up to the stage when the forebrain expands to form the optic vesicle. At this stage all
four substances exerted some degree of growth inhibition. LSD-25 was the most potent.
In embryos in the 100 fj,g./m\. LSD-25 solution, heart formation was delayed for about 48
hours. No melanophores or xanthophores were visible ; optic vesicles were small and often
solid. The embryo was undersized and underdeveloped. The same effects were evident but
to a lesser degree in the 50, 10 and 1 ng./ml. solutions. Hatching was inhibited in the higher
concentrations; the embryos finally died if not removed from the LSD-25 solutions. In the
UML-491 test embryos, there was similiar inhibition but a lesser degree : the embryos were
smaller than controls ; there were fewer melanophores and those were dispersed. Eventually
the embryos hatched with controls, but such embryos remained slightly undersized. Heart
formation was delayed about 24 hours. Serotonin in the concentrations used showed slight
inhibitory action. Heart formation was not delayed ; embryos in general were smaller. Yo-
himbine concentrations above 10 fig. /ml. induced abnormal heart formation. Embryos in this
substance, if hatched, soon died. Rated in order of potency for inhibition : LSD-25, yohimbine,
UML-491 and serotonin. The Sandoz Pharmaceutical Company kindly supplied the LSD-25
and UML-491.
Supported by NIH grant MY-3235.
Dactyl chcjnorcccptors of brachynrans. J. CASE, G. F. GWILLIAM AND F. HANSON.
The presence of chemoreceptors sensitive to amino acids is demonstrated on all limbs of
Libinia cmarginata, Callincctcs sapidns and Corcinidcs macnas by recording from small bundles
dissected from the leg nerves during application of localized stimuli to various parts of the
limb. These receptors, whose end organs are not known with certainty, respond well to boiled
or dialyzed-aqueous extract of Mytilus, are but slightly stimulated by 25% and not at all by
200% sea water, and are unaffected by dilute hydrochloric and acetic acids. Methyl, ethyl,
n-butyl and n-propyl alcohols at concentrations as high as 0.2 M are non-stimulatory, as are
maltose at 0.5 M and lactose at 0.25 M. The polypeptides glutathione and tryptamine are non-
stimulatory at 0.1 M. All amino acids tested were stimulatory, but of these dl-methionine,
1-glycine, 1-arginine, ^-alanine, 1-proline and 1-lysine were active only at concentrations of
0.2 M or greater. The most active substance tested was 1-glutamic acid with a response
threshold approximating 0.001 M, either unbuffered or at pH 7.8, approximately that of sea
water. Considerable specificity of the glutamic acid response is suggested by the poor stimu-
latory action of a-methyl glutamic acid and glutamine. Behavioral experiments in which these
substances were applied to mouthparts and legs of Carcinidcs confirm these observations. An
active compound applied to the mouthparts produces feeding movements or, if applied to a
cheliped, causes immediate feeding movements followed by touching of the cheliped to the mouth.
Supported by NSF grant G-5997 and NIH grant B-2083.
Secretory structures in the lube foot of starfish. A. B. CHAET AND D. E. PHIL-
POTT.
The means by which starfish manage to climb up the vertical side of a glass aquarium
is usually explained by the suction cup theory. However, if tube feet adhered to a glass wall
are cut, they will continue to remain in place even though the vacuum has been released. It
has also been observed that the tube feet leave behind a stainable substance, as well as particles
which can be observed under the electron microscope. With these preliminary observations in
mind an attempt was made to study the ultra structure of starfish tube feet.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 309
Tube feet of Asterias forhcsi were obtained by allowing them to adhere to a glass surface,
and then cutting the foot at its proximal end. They were fixed in ostnic acid sea water and,
after dehydration, embedded in methacrylate or vestopal. Observing both longitudinal and cross-
sections in the electron microscope disclosed that the side of the tube foot, which contains at
least two types of secretory glands and is covered with micro villi, is completely different from
the base of the tube foot — the area which adheres to surfaces. The base of the tube foot
contains many elongated tubes, each one possessing dozens of ellipsoidal structures appearing
to be "secretory packets." Each "secretory packet," about ^ X •% microns in size, is sur-
rounded by a layer of fine granules, inside of which may be found 50 or so fibers (200 A
diameter), arranged much like the continuous fibers of mitotic spindles. It has been shown
that the "secretory packets" are secreted through the walking surface of the tube foot. It is
possible that they function in "gluing" the tube foot to either rough or smooth surfaces, thus
aiding in locomotion. Identical structures have been found in Asterias vulgaris.
Supported by grants from the National Science Foundation (0-8718) and the National
Institutes of Health (A-3362).
Recovery of uncase activity in concentrated urea solutions. AURIN M. CHASE
AND JEAN M. BUSSARD.
The oxidation of uric acid by uricase is inhibited in the presence of urea (Chase, 1957,
Blol. Bull., 113: 320), although the experimental results can be greatly influenced by the kind
and concentration of buffer used to stabilize the pH (Chase, 1956, Biol. Bull., Ill: 299).
In addition to this immediate effect of urea, which (in the case of lower concentrations)
is reversible upon dilution, a slower — relatively irreversible — inactivation of the enzyme (Worth-
ington purified uricase) can also be demonstrated. This was observed when the enzyme was
allowed to stand in 3 to 8 M urea solutions in 0.1 M, pH 9 borate buffer. After the uricase
had been exposed to the experimental urea solutions for the desired time, samples were diluted
ten-fold with buffer containing uric acid, and the initial rate of decrease of absorbance of the
reaction mixture was measured at 290 m//- The temperature of all solutions and of the spectro-
photometer's cell compartment was maintained at 2o° C. The rate of irreversible inactivation
of the uricase was greater, the higher the concentration of urea to which the enzyme was
exposed before dilution.
In addition to the reversible and irreversible inactivations just described, another effect
of urea upon the enzyme was observed. At the highest urea concentrations studied (6 to 8 A/),
the uricase activity — as measured after the ten-fold dilution — increased rapidly during the first
fifteen minutes or so in the undiluted urea solutions, and then decreased again slowly. This
initial recovery of activity was relatively slight in 6 M urea solution but very pronounced in
7.5 or 8 M concentrations.
So far, such experiments have been performed only at 26° C., using borate buffer of pH 9.
The effects of temperature, of pH, and of other buffers should obviously be investigated.
Aided in part by a National Science Foundation grant.
Effect of non-static conditions during gamete irradiation on Arbacia fertilization
and injury. RALPH HOLT CHENEY AND CARL CASKEY SPEIDEL.
Fertilization membrane characteristics and injury expressed by developmental stages are
determined to a deciree by the state of motion during gamete irradiation. Gametes were exposed
to 2537 A ultraviolet (UV) rays at a constant distance while in a static state or while agi-
tated by two-per-second oscillation excursions over one-half inch right and left from the
stationary center. Shaken or non-shaken eggs after irradiation (4 seconds to 16 minutes)
were inseminated with normal sperm. Membrane characteristics, developmental injury, and
death were noted comparatively over a three-day period.
Four-minute UV-rayed shaken eggs produced concentric tight fertilization membranes.
Non-shaken UV-rayed eggs, as a result of differential cytoplasmic damage, developed eccentric
membranes. Such membranes usually assumed normal form some time prior to first cleavage.
Normal eggs, fertilized by shaken or non-shaken sperm, showed no membrane eccentricity.
Later abnormal stages were similar whether derived from irradiated sperm or eggs. Cultures
310 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
from irradiated shaken eggs exhibited earlier and greater percentages of death than cultures
from irradiated non-shaken eggs. This greater damage under non-static conditions probably
was associated with the fact that injury depended upon depth of nucleus from the surface
during irradiation. Shaking assured more uniform damage by bringing more egg nuclei into
position for maximal reception of the UV rays.
Shaken or non-shaken x-rayed (2 to 120 kr) eggs did not give rise typically to eccentric
membranes, but tight membranes resulted from strong doses. Deep x-ray penetration caused
equivalent effects regardless of motion, whereas shallow UV-ray penetration induced differ-
ential effects.
Significant sequences of fertilization membrane formation and adjustments were recorded
by time-lapse cinephotomicrography.
This investigation was supported by a researcli grant (PHS RG-4326 C3) to C. C. S.
from the National Institutes of Health, Public Health Service.
Monoauxenic culture of Arcclla vulgaris. ANNA CICAK AND J. B. WITTENBERG.
At the turn of the century Engelmann captured a few individual protozoans which formed
indubitable gas bubbles in their cytoplasm. Bles, 20 years later, made an extended study of
gas bubble formation in one of these forms, Arcclla vulgaris. The nature of the gas is not
known. In order to carry further the study of gas secretion by the organism, viable, densely
populated cultures are essential. Although Arcella is commonly maintained in infusion cul-
tures, the population density is low and the majority of the animals are in an unsatisfactory
physiological state. We have found that Arcella maintained in a simple salt solution will
reproduce vigorously when fed a suspension of washed Acrobactcr aerogcnes every few days.
The Arcella remain attached to the bottom of the Pyrex dish. Spent medium is poured off
and replaced once a week. The cultures have not as yet been obtained free of other organisms.
However, at this stage the cultures contain a very dense population of vigorous individuals,
many of which are seen to be dividing at any one time.
Development of the Ilyanassa embryo after removal of the mesentoblast cell.
ANTHONY C. CLEMENT.
The mesentoblast cell (4d) of the molluscan egg gives rise to the primary mesoblast bands
and a portion of the entoderm. Within a period of about an hour after its appearance, the 4d
cell of the Ilyanassa egg can be experimentally removed by puncturing or tearing it with a
glass needle; following injury the cell swells and separates from the embryo, without damage
to neighboring cells. The development of about a dozen embryos of Ilyanassa obsolcla has
been followed after removal of the 4d cell. In the better cases, normal or nearly normal
development has been observed of velum, eyes, cerebral ganglia and commissure, foot, operculum,
statocysts, stomodeum, esophagus, and shell. The larval retractor muscle was seen in one case.
The shell may give the appearance of being empty, except for the mantle and occasional masses
of yolk spherules. The esophagus leads into an entodermal complex which is situated in front
of the shell, and in which components of the liver and stomach can be recognized. Neither
intestine nor heart lias been observed to develop after removal of the mesentoblast cell.
Absence of the intestine accords with Conklin's conclusion that this structure arises (in
Crepidula) from the entodermal derivative of 4d. The larval heart of prosobranchs has been
described as an ectodermal vesicle which pulsates by means of mesodermal muscle cells. The
present experiments suggest that the heart musculature is derived from the 4d cell.
Rectification in skate electroplaques and its abolition by barium ions. B. COHEN,
M. V. L. BENNETT AND H. GRUNDFEST.
The electroplaques of Raja crinacca and R. ocellata exhibit a marked increase in conduct-
ance when they are depolarized by more than 10 mV. The conductance change tends to
restore the membrane potential to about 5 mV positive with respect to the resting value.
The change requires one to several msec, to develop. It decays over several hundred msec,
after a brief initiating pulse, and it is graded, depending on stimulus strength and duration.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 311
The conductance increase occurs predominantly in the uninnervated, caudal face. It is quali-
tatively similar to the "delayed rectification" in squid axons, which has been attributed to
increased K+-conductance, and is a mechanism which increases the external current flow that
is produced by the discharge of the electrically inexcitable innervated membrane. Though it
is potential-determined, i.e., electrically excitable, delayed rectification is not regenerative.
Presumably, it lacks the component of inward flux of positive charge which provides the
regenerative property of spike-generating membrane.
On applying Ba++, the rectification was eliminated with little change in resting resistance,
or in the responses to ionophoretically applied acetylcholine. In addition, there was a small
depolarization. The neurally evoked responses were elicited for some time after the delayed
rectification was eliminated, but the presynaptic fibers soon became inexcitable, often passing
through a stage of repetitive firing, as indicated by "spontaneous" depolarizing potentials in
the electroplaques.
Thus, the electrically inexcitable membrane component which produces the neurally and
chemically evoked depolarization of the electroplaque was not affected by Ba++, while the
electrically initiated processes that produced the delayed rectification were blocked. The
effect on potential-determined processes may be related to the block of K+-conductance by
Ba++ and other agents in various electrically excitable cells (Grundfest, this issue).
Permeability of red blood cells to allo.van. S. J. COOPERSTEIN, DUDLEY W ATKINS,
EVELYN HALPERN AND ARNOLD LAZAROW.
Previous studies on the comparative distribution of injected radioactive alloxan, urea,
mannitol and inulin in the various tissues of the toadfish suggested that alloxan may not enter
the cell ; it is possible, therefore, that alloxan may exert its diabetogenic effect by acting on
the beta cell membrane. These studies have been extended by determining the degree of lysis
of washed red blood cells in alloxan solutions of varying tonicities.
Since alloxan rapidly decomposes at body temperature and pH, these studies were carried
out at 0° and pH 7.0. It was shown that under these conditions the half-life of alloxan was
over two hours. A stock solution of alloxan was neutralized to pH 7.0 by adding NaOH to
give a final concentration of 0.308 M alloxan and 0.085 M sodium ion ; the total tonicity was
0.393 M. The concentration of ionized alloxan is presumed to be 0.085 M ; that of un-ionized
alloxan would then be 0.223 M.
One hundred microliters of washed human red blood cells were added to 3 ml. of various
dilutions of the stock alloxan solution. The degree of hemolysis was measured spectropho-
tometrically at 650 m/a- The results were compared with those obtained using various dilutions
of NaCl and of phosphate buffer (Na,HPO4-KH:PO4, pH 7.0). With alloxan, 50% hemolysis
was observed at a final tonicity of 0.118 M; the final alloxan concentration was 0.092 M.
When NaCl was used 50% hemolysis was observed at a tonicity of 0.15 M (0.075 M NaCl).
Phosphate buffer produced 50% hemolysis at a tonicity of 0.113 M (0.423 M phosphate).
Thus, at equal tonicities alloxan and phosphate buffer produce a similar degree of hemolysis,
suggesting that at 0° and pH 7.0 the red cell membrane is impermeable to alloxan.
Supported by grants A-1659 and A-824 from the National Institute of Arthritis and
Metabolic Diseases, Public Health Service.
Secretory epithelium of the szviiu bladder in Fundulits. EUGENE COPELAND.
Preparations of the swimbladder epithelium from Fundulus were studied with the aid
of the electron microscope. Earlier studies of Regaud preparations had revealed no obvious
mitochondria under the light microscope. The electron microscope demonstrates fine fila-
mentous mitochondria with tubular or bleb-like cristae. The nuclei show a characteristically
deep infolding on one side. The free surface of the cells (gas facing) is smooth and un-
interrupted. The basal surface (capillary facing) possesses deep infoldings similar to those
described in the cells of the distal tubule of the kidney. Vesicles of varying density, frequently
in rows, are associated with the folds. Within the double membranes of the folds, i.e., exterior
to the cell, can be seen density-free, vacuole-like spaces. These are assumed to contain oxygen
mobilized by the bladder mechanism. Larger bubbles have been observed in the space between
epithelial cells. Desmosomes connect the free ends of the cells at intervals but the impression
312 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
is gained that gas escapes through areas or slots not so guarded. Patches of Golgi structure
are seen. They have no obvious orientation in reference to the nucleus and the rest of the cell.
Granules of varying size and density (lipid bodies?) are also seen. Correlation of their size
and density with positional relationship to other structures has not been completed. All endo-
plasmic reticulum observed to date has been of the smooth variety. A very few "granular"
strands were seen but they may well have been artifactual.
Oxygen to.vicity among arthropods; in particular, modification of respiratory be-
havior and o.vidative metabolism. F. EUGENE CORRIDEN.
Organisms across the entire taxonomic spectrum are known to be vulnerable to hyperoxia.
Sensitivity is a variable of not only organism character but developmental stage and tissue
type. Symptomatology has been rather well described. Commonly, the syndrome manifested
includes an attenuation in respiration, suggesting an impairment of aerobic metabolism.
The provisional hypothesis adopted is that oxygen toxicity is mediated via an impedance
of tricarboxylic acid cycle activity.
Insect forms frequently exhibit a depression in oxygen consumption subsequent to exposure
to high tensions of oxygen (100%). Insects are known to possess a functional TCA cycle.
Investigation has revealed that among pupal specimens of Tcncbrio, pyruvic acid content (whole
body) varies reciprocally with depression of oxygen consumption ; the correlation is quite
dramatic. Similarly, among pupal specimens of Anagasta and Prodeiiia, depression of oxygen
consumption is accompanied by an accumulation of pyruvic acid and furthermore by a con-
current depletion of alpha-ketoglutaric acid. Pupal specimens of Musca, which did not exhibit
any appreciable disparity in oxygen consumption, failed to show any modification in keto acid
content. Pupal specimens of Habrobracon, in spite of a considerable retardation in oxygen
consumption, maintained extreme stability in keto acid content. Extensions of experimental
design to a study of Uca pitc/iw.r demonstrated toxicologic response to be paralleled by a rise
in pyruvic acid content (muscle).
The tentative conclusion is that oxygen causes a block in the TCA cycle between the level
of pyruvate and alpha-ketoglutarate. The speculation is that, specifically, the pyruvic oxidase
system is the site of inhibition since it has a number of essential components known to be
labile to oxygen in ritro.
Observations on the feeding activity of the isopod, IdotJiea baltica (Pallas').
ARMANDO A. DE LA CRUZ.
Observations both in the laboratory and in the field were made on food preference, feeding
behavior, rate and amount of feeding in the isopod, IdotJica baltica (Pallas). These observa-
tions demonstrated that /. baltica is an omnivore that feeds readily on both the I'licits that
harbors it and on living and dead animals. Values obtained from several experiments showed
that on the average, an individual consumed about one-filth of its body weight in half an hour
when feeding on Fitcits. When preying on a member of its own species, it consumed approxi-
mately half of its body weight in four hours.
It was demonstrated that the animals fed preferentially on the filiform processes on the
surface of the Fncits thailus and on the younger and more tender portions of the plant. When
feeding on other crustaceans, including individuals of their own species, they attacked living
animals of up to one-half their own length, and dead or molting animals of almost their own
size. They grasped their prey with all their pereiopods and usually feeding was initiated on
the soft ventral parts. Eventually, the prey, including the exoskeleton, was totally consumed.
This work was undertaken as part of the student training program of the Marine Ecology
Course and was partially supported by an ICA-NEC grant (Project No. 92-66-012-1-90197)
and sponsored bv the U. S. Office of Education. Department of Health, Education and
Welfare.
Inhibitory action of a tissue e.vtract on regeneration of Tubularia. AL. D. DIXCLK.
Inhibition of regeneration of Tubularia has been obtained using supernatants of mature
hydranth tissue breis at concentrations variously reported as V?. to l/> hydranth/ml. and \Ys to
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 313
2% hydranths/ml. Wet weight determinations gave values ranging from 3 to 22 mg./hydranth,
indicating that further quantitative studies should be based on hydranth weight rather than
number. Supernatants, prepared by homegenizing washed mature hydranths in filtered sea
water containing 125 jtg./ml. streptomycin, and centrifuging the brei for 15 minutes at 10,300 ,
were found to be inhibitory at concentrations between 15 and 30 mg./ml. The inhibitor was
not species-specific, did not alter the pH of sea water, and its action could be simulated by
equivalent concentrations of stem tissue (minus perisarc which contributes 20-40% of the wet
weight).
When freshly cut stems were placed in the inhibitor, cellular protrusions usually capped
the open ends, and further development stopped. Stems inhibited as long as 60 hours were
capable of completely normal regeneration when returned to sea water. Application of the
inhibitor to developing stems caused regression of any primordium already differentiated, and
regenerated hydranths cytolyzed within 12 hours of treatment. Degeneration proceeded to the
level of the neck in hydranths, and to the proximal end of the primordium in differentiating
stems, then stopped— the stem did not cytolyze and remained capable of regeneration.
If the inhibitory supernatant is applied to a 2-mm. block of coenosarc left in the middle
of a 10-mm. perisarc, the cells spread to fill the stem. This indicates that the extract does
not act by stopping the migration of cells, and thereby preventing the build-up of a cell
density critical for regeneration. It also demonstrates that the inhibitor does not act simply as
a non-specific poison since the cells are able to undergo their normal movements.
Permeability studies on ground hog red blood cells. R. G. FAUST AND A. K.
PARPART.
The relative rates of penetration of various monosaccharides and polyhydric alcohols into
the ground hog red cell were studied by means of a photoelectric densimeter. Isosmotic con-
centrations (0.3 M) of these compounds were prepared in an isotonic NaCl-phosphate buffer
which was adjusted to a desired pH. At pH 7 and 37° C, the rates of penetration to one-half
diffusion equilibrium of the completely mutarotated sugars were as follows: for the pentoses ;
D-ribose 9 seconds, D-lyxose 16 seconds, D-xylose 22 seconds, L-xylose 24 seconds, D-arabinose
24 seconds, L-arabinose 29 seconds : for the hexoses : L-sorbose 33 seconds, D-galactose 57
seconds, D-fructose 125 seconds, and D-glucose 160 seconds. The disaccharide, sucrose, did
not penetrate. There was no appreciable difference in the rates of penetration of the a- and
^-isomers of D-glucose. Also at pH 7 and 37° C., the rates of penetration of the polyhydric
alcohols are approximately linearly related to the number of carbon atoms in their molecule
through 5 carbons. The rates were as follows : glycerol 1 second, i-erythritol 2.1 seconds,
D-arabitol 3.5 seconds, L-arabitol 3.5 seconds, D-xylitol 3.5 seconds, ribitol 4.0 seconds,
sorbitol 6 seconds, galactitol 9 seconds, and D-mannitol 11.5 seconds. The cyclic polyhydric
alcohol, i-inositol, which contains 6 carbon atoms in its molecule, did not penetrate. The
temperature coefficients (Q10) which were calculated from the rates of penetration at 37°C. and
27° C. (at pH 7) were between 2.0 and 2.8 for all the sugars and between 1.2 and 1.6 for all
the polyhydric alcohols that were tested. However, the influence of pH on the rates of penetra-
tion of the monosaccharides and the polyhydric alcohols was similar. Although pH variation
affected their rates of penetration slightly, it was obvious that these compounds penetrated more
slowly as the pH of their medium became more acid. Their rates of penetration were more
rapid at pH 8 than at pH 6. This pH effect was reversible.
Comparison of the oxidation of C-l and C-6 labeled glucose by islet tissue. JAMES
B. FIELD AND ARNOLD LAZAROW.
The amounts of C14O: produced from glucose labeled in the one and six positions, respec-
tively, were measured ; a ratio greater than one suggests the presence of the hexose mono-
phosphate shunt. Islet, liver and heart were removed from toadfish and goosefish ; these tissues
were incubated with radioactive glucose at 22° C. for t\vo hours. The carbon dioxide produced
was collected in hyamine and the radioactivity was measured in a liquid scintillation counter.
The amounts of glucose oxidized to C14O» by islet tissue were 3- to 10-fold greater than that
observed for liver and heart. With islet tissue the amount of C"O; produced from glucose
314 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
labeled in the one position was several-fold greater than that produced from glucose labeled
in the six position ; this indicates the presence of the hexose monophosphate shunt in islet tissue.
Preliminary experiments were carried out in which varying amounts of either C-l or C-6
labeled glucose were added to the islet tissue ; the final glucose concentrations were 12.5, 25,
50, 100 and 200 mg. per 100 cc., respectively. The amount of C-6 labeled glucose oxidized by
goosefish islet tissue increased progressively with increasing glucose concentration in the
media ; a maximal value was not reached at the highest glucose concentration (200 mg. per
100 cc.). By contrast, the amount of C-l labeled glucose oxidized by islet tissue reached a
maximum value at a glucose concentration between 25 and 50 mg. per 100 cc. At all glucose
concentrations the ratio of CO2 produced from C-l vs. C-6 labeled glucose was greater than
2.0 ; at the lowest glucose levels, it was greater than 5.0. With liver the amounts of both
C-l and C-6 labeled glucose oxidized increased progressively with increasing glucose
concentrations.
Supported by grant A-1659 from the National Institute of Arthritis and Metabolic Diseases,
Public Health Service.
Metachromatic granules in eggs of Hvdroides, M. FILOSA.
Metachromatic granules in the cytoplasm of a number of marine eggs have been reported
(Rebhun, Pasteels) to show specific movements during cleavage, in that they follow the
centrioles. When unfertilized eggs of Hydroidcs are stained with toluidine blue (1:125,000)
for five minutes, pink granules are found at random in the cortex of the egg. At no time
during first or second cleavage of this egg do these metachroinatic particles become associated
with the mitotic spindle, but rather remain in the surface of the egg throughout. Eggs stained
at first cleavage show the same localization of metachromatic granules as do eggs at the same
stage that have been stained before fertilization. Eggs that were stained before fertilization
were centrifuged at 8550 X g for both one and three minutes. When the germinal vesicle is
intact, the metachromatic granules become localized near its centrifugal end. After the
germinal vesicle has broken down, the location of the granules, up until first cleavage, differs,
depending on the batch of eggs. In most cases the granules are moved internally and are
located in the clear layer just below the lipid layer, with some granules scattered about in the
clear layer. In some instances the granules form a layer in the yolk at the border of the
clear layer.
Under oil immersion the metachromatic granules in these centrifuged eggs are seen to
consist of two or three intensely stained small spheres on a light pink larger sphere. When
unstained eggs were centrifuged at any stage between germinal vesicle breakdown and first
cleavage, and then stained for five minutes, the metachromatic granules are always seen to lie
in the clear layer just below the lipid cap. In these cases the particles are smaller and less
intense in color than those of eggs that were stained before fertilization and centrifugation.
The influences of light and endoerines on the chromatophores of the mud shrimp,
Upogebia affinis. MILTON FINGERMAN, R. NAGABHUSHANAM AND LORALEE
PHILPOTT.
The anomuran Upogebia affinis possesses red chromatophores. Those on the telson and
uropods can be readily observed and staged. Intact specimens on black and white backgrounds
were placed under a series of light intensities ranging from 2 to 560 ft.c. No response to
shade of backgound was observed. However, a response to intensity of incident illumination
was apparent. The red pigment of specimens under an illumination of 2 ft.c. was maximally
dispersed. With increased illumination (560 ft.c.) a slight decrease in the degree of pigment
dispersion to stage 4 of the Hogben and Slome scale was noted in specimens on both back-
grounds. To determine whether Upoyebia displays a rhythm of color change, specimens were
kept in darkness and in black pans under a series of light intensities ranging from 2 to 560 ft.c.,
and observed during the daytime and about midnight. No cycle was evident. However, the
response to intensity of incident illumination was apparent at night as well as during the day.
The red pigment was maximally dispersed in eyestalkless individuals and in intact specimens
in darkness. The sinus gland of Upogebia lies on the dorsal surface of the supraesophageal
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 315
ganglia rather than in the eyestalk. Extracts were prepared of the eyestalks, sinus glands,
and supraesophageal ganglia, and injected into eyestalkless Upogebia. In all cases concentra-
tion of red pigment was observed. The least effect was produced by the eyestalk extracts.
The responses to the extracts of sinus glands and supraesophageal ganglia were large and
approximately equal.
This investigation was supported in part by Grant No. B-838 from the National Institutes
of Health.
The responses of the inelanophores of eyestalkless specimens of Sesarma reticulatiim
to illumination and endocrines. MILTON FINGERMAN, R. NAGABHUSHANAM
AND LORALEE PHILPOTT.
Eyestalkless specimens of Sesarma were exposed for two hours to a series of incident light
intensities ranging from 2 to 1110 ft.c. The degree of melanin dispersion in these eyestalkless
crabs was a function of the incident light intensity. The melanin was maximally concentrated
at 2 and 20 ft.c. With increased light intensity the melanin began to disperse. This pigment
was almost maximally dispersed in specimens exposed to an illumination of 1110 ft.c. Melano-
phores on isolated legs showed a similar direct response to incident light, although the amplitude
of the response was not as great as was observed when the legs were attached to the body.
To determine the role of endocrines in pigment migration in Sesarma, extracts were prepared
of the sinus gland, optic ganglia, supraesophageal ganglia, circumesophageal connectives, and
thoracic nerve cord. These extracts were injected into eyestalkless Sesarma whose melanin
was maximally concentrated, and into specimens maintained under a high intensity of illumina-
tion so that the melanin was in a state intermediate between the fully dispersed and fully
concentrated conditions. Each extract dispersed melanin. Extracts of sinus glands produced
the least dispersion. In the case of the sinus glands, after the dispersing action of the extract,
pigment concentration was noticed in the specimens whose pigment had been in an intermediate
state at the time of injection of the extracts. Although the amount of melanin was not large,
it was statistically highly significant.
This investigation was supported in part by Grant No. B-838 from the National Institutes
of Health.
Responses of the melanophores of the grapsoid crab Sesarma reticulatiim to light
and temperature. MILTON FINGERMAN, R. NAGABHUSHANAM AND LORALEE
PHILPOTT.
Sesarma exhibits a cycle of color change whose frequency closely approximates 24 hours.
Crabs were placed in darkness and observed daily at 8-8:30 A.M., noon-1 P.M., 5 P.M., and
12 P.M. for one week. The pigment was less dispersed at midnight than at other times of
day or night. Crabs were then tested to determine whether differences in incident light intensity
and in shade of background would have an influence on the degree of melanin dispersion.
During the daytime the melanin was maximally dispersed in animals kept in black pans under
a series of light intensities from 2 to 1110 ft.c. At 2 ft.c. the melanin of animals in white
pans was in an intermediate stage of dispersion. With increase in intensity of incident illu-
mination a smoothly graded response was noted ; the melanin became maximally dispersed in
specimens in white pans under an illumination of 1110 ft.c. The responses to black and to white
backgrounds and to incident light intensities between 2 and 1110 ft.c. were then determined
between 11 P.M. and 3 A.M. The melanin of crabs in white pans was more concentrated than
that of crabs in black pans at all light intensities. A response to intensity of incident illumi-
nation was also apparent ; the melanin was more dispersed in crabs under the high intensities
than under the lower ones, whether the crabs were in black or in white containers. However,
at night the melanin did not disperse maximally in crabs on a black or a white background
under any of the light intensities used. Responses to temperature were determined during the
daytime with crabs in white pans under an illumination of 2 ft.c. At 3° C. the melanin was
more dispersed than at 36° C.
This investigation was supported in part by Grant No. B-838 from the National Institutes
of Health.
316 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Effects of Blepharisma pigment on marine invertebrate development. B. A. FUM,
AND H. I. HlRSHFIELD.
The fresh-water ciliate, Blepharisma unditlans, contains a red pigment that is photolytic
and cytotoxic to a variety of cells and organisms. Initial studies were made on the effects
of the pigment on gametes and developmental stages of the marine annelid, Chaetopterus per-
(lamentaccus, and the sea urchin, Arbacla punctulata. These two species were selected since
they are members of two major superphyla. Accordingly, differential responses to the pigment
of these two species may have possible evolutionary significance.
The alcohol-extracted pigment was dried and a 1 mg./ml. concentration was prepared.
This concentration and serial dilutions in sea water of 0.1, 0.01 and 0.001 mg./ml. were used.
The effects of the pigment in an intense light field and in the dark were noted on the
gametes and developmental stages of the two species. A significant decrease in egg fertility
was obtained, whereas the inseminated eggs showed less developmental delay and cytolysis.
Exposure to light enhanced the cytolytic damage considerably.
Chaetopterus eggs exposed to the maximal concentration of pigment for 90 minutes, prior
to insemination with untreated sperm, showed large numbers of abnormal and unhatched
blastulae. In striking contrast, Arbacia showed abnormal fertilization membranes and 99%
mortality. Those eggs of Arbacia that survived showed either abnormal cleavage patterns or
developed normally through blastulation and hatching. Large numbers of abnormal larvae
of both species were seen in the pigment-exposed groups.
Light and dark controls were kept for each series.
Supported by AEC-MBL grant, AT 30-1-1343.
Transplantation of distal limb tissues to upper arm stumps in adult Triturus
riridcscens. RICHARD W. GLADE.
Pertaining to the problem of the establishment of pattern during limb regeneration, the
following experimental series were performed: (1) implantation of three ulnae per stump
(9 cases); (2) implantation of the phalanges of three digits per stump (12 cases); and
(3) transplantation of a sheath of forearm skin to encircle the stump (10 cases). The bone
transplants carried some adherent connective tissue and muscle. The skin grafts, however,
rarely included muscle, and then only as an occasional fiber. Two control series involved
the implantation of the distal halves of two humeri (15 cases), and the implantation of the
proximal halves of two mandibles (11 cases) per stump. In all series, mock operations were
performed on the opposite forelimbs, which served as controls.
Regeneration of the experimental stump was slightly delayed, beginning with the blastema
or cone stage in the ulna, phalanges, and mandible series (6/9, 8/12 and 11/11 cases, respectively)
as compared with its control. Delay occurred in 7/15 cases in the series in which humeri
were implanted. In all of the remaining animals, the experimentals and controls regenerated
at equal rates. The forearm skin series differed from the above in that in only 2/10 cases were
the experimental regenerates delayed, while in 3/10 cases the controls were delayed. In 5/10
of the cases regeneration of the two limbs occurred at an equal rate.
Concerning the morphology of the regenerates, a variable degree of shortening in the
length of the forearm, without significant inhibition of the hand, was observed in the ulna,
phalanges and skin series. In no animal was the control forearm regenerate shorter than the
experimental. An histological examination of the regenerates is being carried out.
This work was done with the technical assistance of Miss Nancy J. Scott.
Supported by Grant G-9846 from the National Science Foundation.
Movements of 2iNa and *-K in the squid retina. W. A. HAGINS AND R. G.
ADAMS.
Exchange of MNa and 42K with sodium and potassium in the isolated squid retina has been
measured. The retinas were isolated in darkness under an infra-red image converter and
mounted in a thin chamber between two rapidly flowing layers of artificial sea water. Isotope
solutions were flo\ved past the retina and then washed away with non-radioactive ASW and
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 317
the activity remaining in the tissue monitored with a counter placed against the chamber.
The responses of the retina to light were measured with electrodes fixed in the chamber. ^Na
moved into 30% of the retinal water within one minute and could be washed out with similar
speed. Long exposure to ^Na revealed an additional slow uptake into a further 10% of the
retinal water in two hours. This latter compartment lost its ^Na slowly with an exponential
time course whose half-time was 30 minutes at 10° C. 42K uptake by the retina was slow
and roughly exponential, with 70% of the intracellular K+ exchanged in 3 hours. "K outflux
was also slow and precisely exponential. It was not affected by raising the external K+ from
10 to 30 mM even though the retinal photocurrent was reduced 30%. Similarly, 2 mM procaine
left the outflux unaffected while increasing the photocurrent by 60%. In procaine- A SW, light
which caused a total flow of 0.02 micromole of ions per cm.2 of retina produced no change in
the ^K outflux, though an increased total outflux of this size would have been just detectable.
In the absence of procaine, light did produce a small increase in 4-'K outflux lasting for 10 min-
utes after cessation of illumination. Light adaptation which reduced the retinal sensitivity
30-fold left the fluxes unaltered and the K content of the cells essentially undisturbed. Since
the exposure used isomerized less than 1% of the retinal photopigment, light adaptation must
involve changes other than simple destruction of rhodopsin or gross changes in the Na and K
content of the receptors.
Light-induced current from t/ie receptors of the squid retina. W. A. HAGINS, R.
G. ADAMS AND H. G. WAGNER.
Isolated, dark-adapted squid retinas at 10° C. bathed in flowing oxygenated artificial sea
water (ASW) maintain their sensitivity to light for many hours. Characteristically, the
retina responds to the onset of illumination with a transretinal voltage rising sigmoidally from
nearly zero to a steady level. The value reached is directly proportional to light intensity be-
tween 106 and 1011 photons cm.'2 sec."1 (500 m^ . When the light is turned off the voltage
returns rapidly to zero. The rise and fall of the voltage show a latency of about 80 msec, and
a transition time of about 100 msec.; the exact values depend on temperature. Since the elec-
trical impedance of the retina is essentially an ohmic resistance of 20-30 ohm cm. between 5
and 5000 sec."1, a light-induced transretinal voltage implies the flow of current within the
retina. With the two surfaces of the retina short-circuited through a feedback amplifier,
measurement of the current shows it is of the order of 103 electronic charges per incident
photon (500 HIM). The origin of the photocurrent is attributed to the photoreceptors since
(1) these cells are the bulk of the retina and the principal nervous elements, and (2) the action
spectrum of the photovoltage closely resembles the absorption spectrum of squid rhodopsin,
and (3) the amplitude of the photovoltage varies with depth as recorded by micro-electrode,
consistent with the presence of a positive current arising in the region of the receptor cell
bodies, flowing longitudinally between them and ending near their photo-pigment-bearing ends.
The electro-chemical nature of the current source has been studied by ionic substitution in the
solutions bathing the retina. Replacement of Na+ by choline* reversibly reduces the photo-
current by 30%, as does increasing the external [K ']. Conversely, removal of K* from ASW
increased the current slightly while 2.0 mM procaine increased it 50%. It is thus suggested
that the photocurrent depends in some way on the membrane potential of the receptors but
not on the presence of electrical excitability in the receptor axons.
The effect of polyvinylpyrroHdonc on volume of the isolated rat lens. CLIFFORD
V. HARDING.
When isolated rat lenses are maintained in Ringer-Locke solution at low temperature,
they" slowly increase in volume and lose transparency. It has been suggested that the increase
in volume is due to the colloid osmotic pressure of the lens, which would become effective
following the gradual inactivation of ion transport mechanisms under the in vitro conditions
employed. Other explanations for the swelling are possible. This suggests, however, that
lenses maintained in a solution of proper colloid osmotic pressure would not undergo an in-
crease in volume. The earlier work of Pau with both lens and cornea demonstrated this to be
the case. In the present studies, rat lenses were maintained at approximately 5° C. in Ringer-
318 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Locke containing various concentrations of polyvinylpyrrolidone (PVP), a synthetic polymer
which can be obtained at different molecular weights. The two molecular weights used were
approximately 40,000 (K-30) and 360,000 (K-90). It was found possible to control lens
volume by adjusting the concentration of PVP in the medium. For example, in low concen-
trations of K-30, the lens swells; in high concentrations, it shrinks, and at 15%, approximately
normal volume is maintained and transparency is not lost over long periods of time (48 hours
and longer). Regardless of the mechanism whereby PVP controls the volume of the lens,
it is suggestive that a proper adjustment of the colloid osmotic pressure of the medium can
prevent the swelling which normally follows lens isolation. This could conceivably avoid
damage to cells and fibers, which might accompany lens swelling, and thus maintain the lens
in a viable state in vitro for longer periods of time.
It is planned to check this by measuring the extent of cellular proliferation in the lens
epithelium, a factor which is apparently a sensitive index of the viability of the lens.
Colloid osmotic pressure might also be .the effective characteristic of solutions of PVP
which have been shown to prolong the time that rabbit corneas can be maintained i'» vitro
without losing their capacity for transplantation (La Tessa).
Thymidine incorporation in epithelium of fish lens maintained in vitro. CLIFFORD
V. HARDING AND B. D. SRINIVASAN.
Tritium has been localized autoradiographically in whole-mount preparations of lens
epithelium, a single layer of cells, from the rabbit and rat. The use of whole-mounts for this
purpose makes possible a comparison of the extent of incorporation of tritium-labelled com-
pounds in one portion of the epithelium with any other portion, in one preparation. This
procedure has proven useful in studies on the incorporation of tritium-labelled thymidine in
normal and injured epithelium.
It was thought of interest to extend this study to the lenses of cold-blooded animals.
These would have the advantage of enabling a study of temperature effects in the intact
animal under physiological conditions ; and, they would also, perhaps, furnish a more favorable
material for in vitro studies on cell division. Results of the present investigation have shown
that the whole-mount method can be applied to the lens epithelium of dogfish, tautog, and
sea bass. Freshly isolated dogfish lenses were incubated for two hours in a solution of elasmo-
branch Ringer (made up in glass-distilled water, pH approximately 7.5) plus tritium-labelled
thymidine at 2.5 to 5 /xc/ml. (Spec. act. 1.9 curies/mM), after which they were fixed in 3 parts
absolute alcohol : 1 part glacial acetic acid. Whole-mounts were then prepared for autoradi-
ography. The autoradiograms were exposed for 7 to 17 days before photographic development.
The results show incorporation by many of the epithelial cells. The cells showing incorpora-
tion are relatively more scattered than in the rabbit lens, where the germinative zone is
relatively localized.
Experiments were also carried out in which dogfish lenses were incubated in elasmobranch
Ringer (with dextrose) for various periods of time before exposure to thymidine. Lenses
maintained in elasmobranch Ringer at 21° C., for at least up to 22 hours before a two-hour
exposure to tritium-labelled thymidine, showed incorporation of thymidine. Similar results
were obtained with the epithelium of tautog lenses, maintained in teleost Ringer at 16° C. for
22 hours. The results suggest the possible use of this tissue for in vitro studies on cell division.
This work supported in part by U. S. Atomic Energy Commission contract AT (30-1) 2456
and National Society for the Prevention of Blindness.
Stimulation of Arbacia sperm respiration by egg substances. RALPH R. HATH-
AWAY.
Arhacia fertilizin is generally thought to depress the respiration of specific sperm. In a
reinvestigation of this question, Arbacia sperm respiration and motility were markedly in-
creased by a substance which freely diffuses from eggs. This Arbacia sperm-activating sub-
stance was prepared as the sea water supernatant of jellyless eggs. Such a sperm-activating
preparation has no sperm-agglutinating properties, is diffusible in dialysis, soluble in alcohol,
heat-stable, and non-volatile. The activator is evidenly a substance distinct from fertilizin.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 319
When Arbacia sperm activator is mixed with an excess of specific sperm in glycyl-glycine
buffered sea water, there is a four- to 20-fold increase in respiration rate. The high rate of
Oi> consumption rapidly decreases as a power function of time. When the activator is in
excess, the elevated respiration rate slowly decreases as a linear function of time. The pH
is essentially constant throughout. When activator-sperm mixtures are centrifuged after one-
half hour, the supernatants no longer contain activating properties. Conversely, sperm washed
from the activator after one hour do not respond to fresh activator preparation. These facts
suggest a mutual exhaustion of the activator and a sperm substance essential for sperm response
to the activator.
Sperm activator occurs in Arbacia eggs from Woods Hole, Mass., and Alligator Point,
Florida. Sperm activators were also found in Lytechimis variegatiis and Mclllta quinquies-
perforata from Alligator Point. Asterias forbcsi eggs from Woods Hole yielded no sperm
activator. In specificity studies, Arbacia activator failed to stimulate sperm of Lyt echinus,
Mellita and Asterias. Lytechinus sperm activator did not affect Arbacia sperm, but Mellita
activator caused a moderate increase in Arbacia sperm respiration.
Sperm of Arbacia, Lytechinus and Melita have invariably displayed an increased oxygen
consumption in the presence of homologous fertilizin. This response may be due to the
presence in fertilizin preparations of small quantities of active sperm activator substance (s).
Aided by a National Science Foundation predoctoral fellowship, and by National Institutes
of Health (RG-6234), to Dr. Charles B. Metz.
Egg jelly dispersal by Arbacia sperm extracts. RALPH R. HATHAWAY, LEONARD
\YARREN AND JOEL G. FLAKS.
Various substances, some containing sialic acid, are released by Arbacia sperm when they
are treated with either Arbacia fertilizin or ca. 10~4 M sodium lauryl sulphate (see separate
abstract). These sperm extracts affect Arbacia eggs by causing the disappearance of the egg
jellies in 15 to 60 minutes at room temperature. Eggs which have lost their jelly coats are
easily recognized by their close contact with each other. In addition, when Arbacia eggs are
exposed to Asterias sperm, the egg jellies become impregnated with trapped non-fertilizing
sperm. Subsequent exposure of these eggs to Arbacia sperm extracts results in the disappear-
ance of the jelly coat and the release of the starfish sperm. This observation suggests that the
disappearance of the egg jelly is caused by a true jelly dispersal rather than by a precipitation
and contraction of the jelly, as is sometimes reported when other sperm extracts, e.g., anti-
fertilizin, are mixed with eggs. Furthermore, in preliminary tests supernatants from eggs
treated with the sperm extracts contained more fucose than supernatants of control eggs. Since
fucose is a major constituent of the egg jelly, this result indicates solubilization of the egg-
jelly coat.
The jelly "dispersing" activity is destroyed by heat (80° C. for 5 minutes), and acid (pH
3.0 for 5 minutes), and is inactive at 0°. These properties distinguish this agent from anti-
fertilizin, and suggest an enzymatic activity.
The sodium lauryl sulfate extracts of sperm contain antifertilizin which precipitates a
membrane around the jelly coat. Observation of the jelly "dispersing" activity of these
preparations can be made only after the antifertilizin is removed. The jelly precipitating
activity can usually be removed by centrifugation at 33,000 g for 35 minutes. The pellet
contains the jelly precipitating activity and all of the sialic acid which was extracted from the
sperm by the sodium lauryl sulfate. The 33,000 g supernatant contains a strong jelly coat
"dispersing" activity.
Aided by National Institutes of Health (RG-6234), to Dr. Charles B. Metz.
The psendocilia of Tctraspora. WALTER R. HERNDOX AND DELBERT E. PHILPOTT.
The immobile, paired, pseudocilia of the vegetative cells of the chlorophycean alga Tetraspora
lubrica Roth strongly resemble the flagella of its motile cells when examined microscopically,
especially with lower magnifications. However, with oil immersion and phase contrast, it is
clear that these protoplasmic strands have a structure different from that of typical flagella ;
they are comprised of an axial portion surrounded by a relatively broad sheath. The axial
320 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
portion is of greater optical density than the sheath and is beaded ; the sheath stains readily
with methylene blue. Pseudocilia of colonies fixed in \% osmic acid or 5% potassium permanga-
nate, embedded in methacrylate and sectioned show the same structure as seen in the living mate-
rial when examined with the light microscope, except there is in some a slight shrinkage of the
sheath and in these the axial portion may be somewhat coiled. In sections thus prepared and ex-
amined with the electron microscope, the sheath and axis are clearly distinguished. The sheath is
about one micron in diameter and relatively uniform along its length. The axial portion appears
to be basically tubular but has numerous constrictions which delimit spherical and elongate
segments of varying sizes (mostly 0.2-0.4 micron in diameter). At its insertion, the axial
portion appears to be an extension of the anterior cytoplasm peripherally disposed about a
vacuolate interior. The beaded structure of the pseudocilia was found in colonies of different
ages, in collections from different localities, and in a different species of the genus. The
position of the pseudocilia of vegetative cells and some features of their morphology suggest
that they might be produced by a process which is a modification of that producing the typical
flagellar structure of motile cells also examined with the electron microscope, for comparative
purposes, in this study.
The stage at fertilization of the egg of Fiindulus hetcroditns. CHARLES W. HUVER.
According to Costello, Davidson, Eggers, Fox and Henley (1957) in Methods lot-
Obtaining and Handling Marine Eggs and Embryos, the stage at which the egg of Fundulns
hcteroclitus is fertilized has not been determined. This is due to the fact that the polar bodies
of this popular embryological material have not been previously described.
While examining time-lapse movies of fertilization and cytoplasmic streaming during
blastodisc formation, both polar bodies were observed, apparently for the first time. The
polocytes have now been observed in a total of 15 eggs. They project into the perivitelline
space from the rim of the funnel-shaped mouth of the micropyle. The first and second polar
bodies are of equal size (0.04 mm. -diameter). They are in close contact with each other and
are incompletely elevated from the egg surface.
The reasons why students of early Fitndnltts development have previously failed to observe
the polar bodies are: (1) the mature egg is very difficult material for proper fixation and
sectioning, (2) the extreme transparency of the polocytes, (3) the relatively small size of the
polar bodies in relation to the size of the egg, (4) the resemblance of the polar bodies in size
and in shape to cortical alveoli, and (5) the masking effect of the thick chorion which encloses
the egg.
The first polar body becomes clearly visible upon the elevation of the chorion at approxi-
mately three minutes after insemination. The time of emergence of the first polar body is
unknown. However, it appears to be present in freshly stripped unactivated eggs where it is
closely compressed to the egg surface. The actual emergence of the second polar body can
be seen in fertilized eggs. It arises next to its predecessor in a smooth but quick movement
at 4.5-5.0 minutes after insemination. The presence of the first polocyte in the ripe unfertilized
egg, and the rapid formation of the second after sperm entrance, indicate that the egg is a
secondary oocyte when fertilized. The emergence of the second polar body so soon after
insemination suggests that the second maturation division proceeds to an advanced stage before
it reaches the pre-fertilization arrest.
X.I.H. Predoctoral Fellow.
Effects of temperature on charge transfer through a receptor membrane. NOKU-
SADA ISHIKO AND WERNER R. LOEWENSTEIN.
Mechanical stimulation of the nerve ending of Pacinian corpuscles produces transfer of
charges through its receptor membrane. The energy requirements for the transfer are markedly
influenced by temperature. For example, the mechanical stimulus strength necessary to
produce a given generator potential at 25° C. may be reduced to one-third at 35° C. A tempera-
ture change alone, however, elicits no detectable transfer.
The main effect of a temperature change is to vary the rate of rise and the amplitude of
the mechanically elicited generator potential. Both increase linearly with temperature, with
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 321
Qio of 2.5 and 2.0, respectively (15° C.-350 C. ). The primary event here appears to be the
effect on the rate : the active phase is shorter than the rise time of the generator potential and,
hence, the total charge transferred increases as the transfer rate augments with temperature.
The rate of fall of the generator potential is not appreciably affected by temperature.
The first Ranvier node, the site of nerve impulse initiation, behaves quite differently. The
amplitude of the nodal action potential remains practically unchanged between 20° and 40° C.,
and its duration increases considerably with temperature (Q,0, 3-4). The nodal threshold
varies inversely with temperature; impulse initiation fails completely below 12° C., although
the receptor membrane still produces generator potentials.
A simple model for receptor excitation is that of ions diffusing simply along their gradients
through mechanically stretched pores of the receptor membrane. This model is now no longer
tenable without modification. The high activation energy of the rate-limiting process in excita-
tion, 16,300 cal./mole (as derived from the temperature dependence of the rate of rise of the
generator potential), forces at least one additional element upon this or any other model,
namely an energy barrier for charge transfer. To surmount the barrier, energy may be
supplied directly by heat transfer, or indirectly by a chemical reaction.
Stimulation of conidia formation at the growing tips of Neurospora crassa by
.r-irradiation. J. KEOSIAN AND B. P. SONNENBLICK.
-Y. crassa grows with a sharp frontier in an agar minimal medium in growth tubes.
When this frontier is x-irradiated (9000 r at 182 kvp) the following are noted:
1. Formation of a dense band of conidia, about 5 mm. wide, at the site of the original
irradiated frontier, visible in 6-8 hours. This band persists as to location, width and relative
density, even after the tube is overgrown with conidia.
2. The conidial band forms when the frontier alone is irradiated and the rest of the growth
is lead-shielded, but the band does not form when the frontier is shielded and the remainder
exposed. This suggests that the radiation influence is limited to the growing tips.
3. Age appears to have no significant effect on the formation of the conidial band since
irradiation of 24-, 48- and 72-hour cultures produced comparable results.
4. The threshold for the formation of the band is between 1000 r and 2000 r, resulting in
a definite but sparse band. With increasing dose the band becomes denser with conidia.
Experiments performed with a previous machine (85 kvp) indicated an optimum effect at
about 9000 r.
5. Retardation of growth rate for the first six hours, followed by an acceleration of growth
rate over normal. This increase is small but is consistently found in the irradiated cultures.
6. Conidial band formation at the original frontier, with visibly different characteristics
from that induced by radiation, occurs after exposure for given periods of time at certain
temperatures (10° C. and 42° C).
Histochemical and biochemical analyses are projected in an attempt to ascertain whether
the localized conidial band formation described above is a generalized stress response to an
unusual environment, or whether there are differences in the response to radiation per sc and
to other agents.
Observations on tJic submicroscopic cytology of the epithelial cells of the cardia
of a dipteran insect, Hypoderma bovis. RICHARD G. KESSEL.
The cardia of dipterous insects is the most anterior portion of the midgut. It is known
to be highly active in secretion and involved in the production of the peritrophic membrane.
The lumen surface of the epithelial cells possesses a multitude of microvilli, often arranged
in hexagonal array. Numerous blebs of the apical portion of the plasma membrane can be
observed. A budding or blebbing of the distal portion of the microvilli is also apparent, but on
a smaller scale. The most abundant cellular organelle is the endoplasmic reticulum. In the
basal portion of the cells, the reticulum consists of numerous, parallel, interbranching, rough-
surfaced lamellae. In the central portion of the cell, the lamellae of the endoplasmic reticulum
are observed to be greatly expanded at their end region (forming- ergastoplasmic sacs) or
along their length. In the most apical portion of the cell, numerous smooth- and rough-surfaced
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
vesicles are characteristically present. The latter may also be observed within the large blebs
of the apical plasma membrane. It appears that the secretory vesicles have their origin from
the endoplasmic reticulum. Mitochondria and parallel, smooth-surfaced lamellae of the Golgi
apparatus are also present, but both are extremely small. The Golgi material was never
observed to be conspicuous. The cells abut upon a basement membrane approximately 0.75
micron thick. The nuclear membrane is porous and a prominent nucleolus is present. Ob-
servations at various stages in the formation of the peritrophic membrane suggest that the
microvilli contribute, at least in part, to its formation. (Supported by a grant, RG-6942,
from the National Institutes of Health, U.S.P.H.S.
An electron microscope study of the mitochondria-rich "chloride cells" from the
gill filaments of fresh water- and sea water-adapted Ftindiilus hctcroclitus.
RICHARD G. KESSEL AND H. W. BEAMS.
The ultrastructure of the gill filaments of Finidiilns hctcroclitus \vas studied with special
reference to the mitochondria-rich chloride cells which were earlier described with the light
microscope by Copeland (1948, 1950). Filaments of animals from sea water were examined,
as well as those from animals adapted to fresh water for twelve hours.
In sea water-adapted animals, the chloride cells appear very dense, due to the numerous,
filamentous mitochondria. The cristae of the mitochondria are mainly oriented parallel to its
long axis. Located among the mitochondria and closely associated with them is a prominent,
tubular network of an agranular type. This structure is similar to a specialized form of
endoplasmic reticulum described in the pseudobranch gland (Copeland, 1959). The nucleus is
characterized by two areas of different electron densities, each of which appears lobated.
In fresh water-adapted animals, the chloride cells do not appear to be as densely populated
with mitochondria. In addition, the mitochondria appear shorter and thicker than those in
sea water and the cristae in many cases are oriented transversely. The network of smooth-
surfaced, branching tubules (thought to represent a form of the endoplasmic reticulum)
appears more abundant, but smaller in size, than is the case in sea water. A continuity of the
tubular endoplasmic reticulum and the plasma membrane of the cells is observed under both
sea water and fresh water conditions. A small amount of rough-surfaced lamellae of the endo-
plasmic reticulum is also present. The nucleus appears homogeneous, is of moderate electron
density and often contains a nucleolus. ( Supported by grants from the National Institutes of
Health, U.S.P.H.S.)
The effect of waves in influencing the composition of a flora of attached sea-zveeds.
JOHN M. KINGSBURY.
The granite jetty projecting across the harbor entrance at West Falmouth, Mass., lies
parallel to the general shore outline of Buzzards Bay. Its western, bay exposure (with 69%
of total wind over open water fetch of 61/! miles or more) receives constant wash of waves
or swells, usually producing spray. Its eastern, harbor side (maximum fetch less than ^ mile,
wind exposure about 2%) rarely experiences waves and never has significant spray. Tidal
effects, water temperature, light, currents, and rock substrate were observed or measured and
found identical or nearly so at the six collecting stations. Stations were paired, leeward and
windward members opposite, the first pair 10 feet from the projecting end, second and third
pairs each 12 feet further to landward. At each station all manually removable vegetation
was collected from 6-inch quadrants forming a vertical transect from highest growth to jetty
bottom.
A Calothrix zone, above lowest high water, was present only to windward. A 3-foot
(vertical) zone of Fucus vesiculosus, present on both sides, was a/£ to 1 foot lower on the
leeward side. Its lower boundary leeward corresponded with extreme low tide level ; windward
it was bounded by mean low and lowest high tide levels. The leeward side showed fewer
germlings, presence of bladders and larger plants in comparison with windward. A zone,
between highest and lowest low tide levels, dominated by Entcronwrpha linsa, occurred only
on the windward side. Of 35 species found on windward, 8 were not found on leeward ;
similarly, 2 of 29 leeward species were not found on windward. Clwndrus crispits, the principal
non-epiphytic member below the Fucus and Enteromorplia zones, was displaced upwards 6 to
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
9 inches on the leeward side. On the leeward side the vertical level equivalent to the windward
Entcromorpha zone was occupied chiefly by the downward FUCKS and upward Chondrus
displacements.
Centrifugal influence on the electroretinogram of the frog. STEPHEN T. KITAI.
The existence of centrifugal fibers in the optic nerve was investigated by studying the effects
of Nembutal on the electroretinogram b-wave responses of frogs under the following conditions :
(1) spinal pithing, (2) spinal pithing and unilateral optic nerve sectioning before the injection
of the Nembutal, and (3) section of the optic nerve after the Nembutal effect on the electro-
retinogram responses appeared.
The electroretinogram responses were recorded from the cornea of frogs, using sodium
chloride-plated silver electrodes. The signals from the electrodes were fed into Grass P6 d.c.
pre-amplifier and finally recorded by an oscilloscope. The electroretinogram records were
taken before and after the Nembutal injection under spinal pithed condition, and under spinal
pithed unilateral optic nerve-sectioned condition.
Changes in the magnitude of the electroretinogram responses due to extraneous variables,
such as changes in the retinal area stimulated or in the amount of stimulus light, were con-
trolled. Optic nerve sectioning did not damage the retinal artery. The electroretinogram
responses from the unoperated contralateral eye were taken as a control.
The effect of Nembutal on frogs under spinal pithed condition was to decrease the electro-
retinogram responses. The Nembutal injection failed to decrease the electroretinogram re-
sponses when the optic nerve was sectioned before the injection. The decreased electroretino-
gram responses by Nembutal injection were eliminated when the optic nerve was sectioned.
Sectioning of the optic nerve abolished the effects of Nembutal on the electroretinogram
b-wave responses. This is interpreted as an evidence for the existence of the centrifugal fibers
in the optic nerve, since the higher center upon which the drug acts no longer connected with
the retina.
Krcbs cycle dchydrogcnase systems in eggs of Asterias as measured with a tctra-
solimn salt. EVELYN KIVY-ROSENBERG, FRANCES RAY AND HELENE ELEFANT.
The quantitative study of substrate-dependent dehydrogenase activity has been in progress
for several years (Kivy-Rosenberg, Cascarano and Merson, 1959; Biol. Bull. 117). Work
was continued on Asterias uninseminated and inseminated eggs. An extensive series of sub-
strates was utilized, including those which require DPN or TPN as cofactors and succinate
which requires none. Included in this series were four which are involved in the Krebs cycle:
alpha-ketoglutarate, and malate, each with DPN, isocitrate with TPN, and succinate. The
tetrazolium salt used as a hydrogen acceptor was 2-(p-iodophenyl)-3-(p-nitrophenyl ) -S-phenyl
tetrazolium chloride (TNT).
Homogenates of uninseminated and inseminated eggs of the same female (of a large series
of starfish) were incubated aerobically for one hour at 37.5° C. in media containing INT and
substrate or appropriate controls. Formazan was extracted and the quantity of reduced tetra-
zolium measured photocolorimetrically. The substrate-dependent dehydrogenase activity was
expressed as micrograms of formazan per milligram of protein.
Of the fifteen hydrogenase systems measured, three in the Krebs cycle are among the
most active : alpha-ketoglutarate ranking first ; isocitrate, second ; malate, third, in practically
all cases. Occasionally ranks of alpha-ketoglutarate and isocitrate were interchanged, and
malate ranked fourth. The activity of the other member of the cycle, i.e., succinic dehydro-
genase, was among the lowest of the entire series. The ranks mentioned were similar for both
uninseminated and inseminated eggs.
Insemination resulted, in general, in a rise of dehydrogenase activity in homogenates ( as
we had reported earlier also for Spisitla). In a few cases the reverse was true. On the whole,
the activity of the Krebs cycle dehydrogenases followed the general trend : i.e., a rise ( when
there was a general rise with insemination), and a fall (when the reverse was true). This was
true for the one which showed relatively low activity as well as the three highly active
dehydrogenases.
Further consideration and study w:ill be given to the surprising relationship between the
lo\v activity of succinic and high malic dehydrogenase systems.
324 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
The iron requirement of some marine plankton algae. DANA D. KRAMER AND
JOHN H. RYTHER.
The iron requirement of some marine phytoplankton was investigated by determining final
populations attained in media containing different concentrations of Fe-EDTA. The organisms
studied were three oceanic species (Coccolithus huxleyi, Chaetoceros sp. and an unidentified
centric diatom "13-1") and four inshore species (Skeletonema costatuin, Isochrysis galbana,
Amphidinum carteri and an unidentified flagellate "3C"), all maintained in pure culture at the
Woods Hole Oceanographic Institution.
The algae were grown in offshore ocean water enriched with all essential nutrients except
iron, until further growth was obviously iron-limited. Iron-deficient cells were then inoculated
into media enriched respectively with 0, 2.6, 6.5, 13, 26, 65 and 130 ,ug. Fe/L. These cultures
were incubated (1000 foot candles, 20° C.) until maximum growth was attained (ca. 7 days),
after which population density was determined by cell counts.
Coccolithus could not be made iron-deficient. After several transfers, more growth
occurred in media without than with iron added. All other species became iron-deficient after
2-3 transfers, with no growth in control cultures containing no added iron. The two other
offshore species (Chaetoceros sp. and "13-1") reached maximum densities at 26 and 2.6 ng.
Fe/L, respectively; growth was depressed at higher levels. Of the inshore species, Isochrysis
and Amphidinum reached maximum growth at 65 /*g. Fe/L; populations of Skeletonema and
"3C" were still increasing at the highest iron concentration employed.
Skeletonema, an unbiquitous shallow-water diatom never found in the open sea, does not grow
at iron concentrations supporting maximum growth of the oceanic species Coccolithus and
"13-1." It is suggested that the higher level of iron in coastal waters associated with land
drainage may influence or control the distribution of this and other neritic species. • This work
was carried out as part of the student training program of the Marine Ecology Course.
Effect of nicotinamide on p\'ndine nude o tide levels and cleavage rate of eggs of
Spisula solidissima. STEPHEN M. KRANE AND ROBERT K. CRANE.
It was previously demonstrated in this laboratory that the major change in the concentra-
tion of pyridine nucleotides in early cleavage stages of marine eggs was a net increase in
triphosphopyridine nucleotide in its reduced form (TPNH). In the present stud}', eggs
of Spisn-la solidissima were incubated with nicotinamide in an attempt to modify the levels of
pyridine nucleotides as well as the rate of cleavage. Pyridine nucleotides were measured
fluorimetrically. The concentration of diphosphopyridine nucleotide (DPN) in unfertilized
eggs incubated in nicotinamide ( 1 X 10~2 M) in sea water increased linearly from 210 milli-
micromoles/ml. cells to 535 millimicromoles/ml. cells in three hours. With lower concentra-
tions of nicotinamide the increase was more striking (1010 millimicromoles/ml. at 1 X 10~4 M
nicotinamide; 781 millimicromoles/ml. at 1 X 10"" M nicotinamide). In eggs transferred to
nicotinamide (1 X 10'-' M) in sea water 40-60 minutes after fertilization, the DPN levels were
doubled, compared to controls three hours later, and cell division completely but reversibly
blocked. Cleavage was blocked in concentrations as low as 5 X 10"* M but only delayed at
1 X 10~4 M. Unfertilized eggs incubated three hours in nicotinamide (1 X 10"4 M) showed
no change in TPNH levels compared to controls (5 versus 4 millimicromoles/ml.) despite
marked increase in DPN (820 versus 194 millimicromoles/ml.). In fertilized eggs in nicotin-
amide, TPNH was 72 millimicromoles/ml. versus 34 millimicromoles/ml. in controls, whereas
DPN was 1280 millimicromoles/ml. versus 185 millimicromoles/ml. in controls.
Effects of nicotinamide in Spisula fggs were therefore as follows: (1) Reversible inhibition
of cell division; (2) Increase in levels of DPN in fertilized and unfertilized eggs; (3) Increase
in TPNH only if cell division had proceeded.
Mechanisms of nerve impulse generation in a Lorenzinian ampulla. WERNER R.
LOEWENSTEIN.
Single isolated ampullae of dogfish (Mustelus canis) were enclosed in a pressure chamber.
The ampulla tubule was cannulated and connected to a pump. Thus, the internal and/or
external pressure of the ampulla could be varied over a wide range. Single axons were dis-
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 325
sected up to their point of emergence from the ampulla, and electrical activity was led off that
point. Generator and action potentials were thus recordable.
At normal atmospheric pressure only a few endings produce generator potentials of sufficient
amplitude to fire nerve impulses. But as the pressure of the ampulla is increased, the generator
potential is driven progressively to the critical firing level, and impulses are discharged
rhythmically and continuously. The main effect of a pressure increment is to increase the
rate of rise and the amplitude of the generator potential, and, thereby, to increase the frequency
of discharged impulses.
Progressive ablation of ampulla cell structures shows that the generator potential arises
in, or close to, the pyramidal cells. The generator structures are excited effectively by either
(1) an increase in internal ampulla pressure, that involves stretching; but not by (2) increasing
equally and simultaneously the internal and external ampulla pressure, i.e., by a pure pressure
stimulus.
The Lorenzinian ampulla seems thus well suited to detect hydrodynamic pressure changes,
caused by the fish's own movement or by external objects. In tine dynamic phase, its sensitivity
is about 11 impulses/second per meter.
Electron microscopy of electrical synapses in the crayfish. A. ]. DE LORENZO.
Synaptic junctions in the abdominal ganglia of the crayfish were examined with the
electron microscope. Particular attention was directed to axo-axonal junctions which exhibit
physiological properties of electrical transmission. The lateral giant to motor junction is
characterized by many post-junctional processes which invaginate into the lateral giant or
prejunctional fiber. The appositional membranes are separated by a small space approximately
50-75 A wide. This space is much smaller than that described in other synapses. Each of the
appositional membranes is composed of a structural unit consisting of two electron-dense lines,
about 25 A thick, separated by a light zone about 30 A wide. Occasionally, the unit membranes
appear to fuse into a single electron-dense zone about 50 A thick. Thus, in certain regions,
the synaptic membranes exhibit structural modifications at variance with the unit membrane
concept. Throughout the region of synapse, however, the synaptic cleft is uniformly 50-75 A
wide.
The axoplasm of the prefiber at the junction is structurally unremarkable, but occasionally
rows of mitochrondria are evident. The post-fiber, however, contains large clusters of
"synaptic vesicles" which are typically found in the presynaptic terminals of other synapses.
Since these synapses have been shown to permit the flow of electrical current only in one
direction, it is surprising to find an abundance of synaptic vesicles in the post-synaptic processes.
This observation is particularly interesting in view of the current speculation regarding the
role of synaptic vesicles in chemical transmission. The role of "synaptic vesicles" in a strictly
electrical synapse remains unresolved.
Segmental or septal junctions were also examined. In this junction the synaptic cleft is
quite large, often on the order of 400 A. However, in regions where the pre- and post-fibers
interdigitate, the fine processes are separated by clefts about 100 A wide. It appears that the
synaptic cleft in this junction is not uniform in dimensions. Thus, preliminary observations
of two electrical junctions suggest differences in ultrastructural organization.
Reversible inhibition of metamorphosis in tadpoles of Amaroecium constellatum
by calcium-free sea ivater. WILLIAM F. LYNCH.
Tadpoles of Amaroeciuin constellatum were taken through four washes of Moore's calcium-
tree sea water at a pH of 6.2 and were deposited in Stender dishes of the same medium win-re
they became quiescent in a few minutes. All remained in the larval state during seven-hour
periods of observation. (The normal maximum natatory period is one hundred minutes.)
At the end of seven hours the tadpoles were washed in natural sea water and placed in Stender
dishes of this medium. Twenty-four hours later they had formed ascidiozoids with well-
developed pharyngeal regions and siphons. The internal organs had assumed the adult axial
relationship. Tadpoles that were not removed from the first bath of calcium-free sea water
had begun metamorphosis by seven hours, but those in the other baths had not. In the first
washing dish metamorphosis had proceeded to the stage in which rotation of the larval axis
326 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
had occurred, but the process ceased while the larvae remained in a contracted state. At a
pH of 8.1 a similar inhibition of metamorphosis was noted, but this inhibition was no longer
reversible when the exposure to calcium-free water lasted for nine hours.
Silicoflagellate populations in the plankton of Cape Cod area, past and present.
RAMON MARGALEF.
In the superior layers of the sediment of Cape Cod and Buzzards Bays, skeletons of silico-
flagellates are common. The following species of Siphonotestales have been recognized :
Dictyocha fibula Ehrenb., including var. pentagona Schulz, Distephanus crux (Ehrenb.)
Haeck., Distephanus speculum (Ehrenb.) Haeck., including vars. pcntaaonus Lemm., regularis
Lemm. and septenarius (Ehrenb.) Joerg., and Cannopilits binoculus (Ehrenb.) Lemm. (prob-
ably a simple form of Distephanus speculum).
Skeletons increase in abundance from around 16 cm. below the top of all sediment cores
towards the surface. Typical D. fibula and D. speculum make together 99% of the populations.
As we approach present time, a shift, similar in both bays, is observed in the composition of
populations. Dictyocha fibula is almost exclusively represented in the inferior levels. Di-
stephanus speculum increases in a regular way from a negligible representation at 8-16 cm.
below the surface of sediments up to make 50-60% of the skeletons of silicoflagellates present
in the uppermost, soft, layer of the cores.
Dictyocha fibula is a species perhaps of more oceanic character and of rather warm
environments ; Distephanus speculum is more commonly reported from colder water with lower
salinity. A differential dissolution of skeletons is unlikely, so that change in the representa-
tion of both species may reflect changes in the average composition of plankton accomplished
over many years.
The size of the marine diatom Melosira sulcata (Ehrenb.) Kuetz. in the Cape Cod
area. RAMON MARGALEF.
In natural populations of several species of Melosira, distribution of cells according to
diameter shows definite peaks. Such species, all thick-walled, do not experience a noticeable
reduction of diameter along successive cellular divisions. Their size distribution suggests the
manifestation of a polymorphism.
Different morphs (peaks) seem to have a different selection value under definite environ-
mental conditions and their proportions change in space and in time. Observations in Vigo
(NW Spain) suggest that in Melosira sulcata low salinity and colder water are, in an
independent way, favorable to the high diameter classes.
Forty-two hundred frustules of Melosira sulcata have been measured in 5 bottom cores
taken in Buzzards Bay and 4000 in two cores obtained in Cape Cod Bay, at depths of 15-29
meters. Diameter ranges from 7 to 50 p and distribution of cells by diameter offers an image
similar to European populations, with peaks at 10, 12-13, 16-17, 20-21, 27, 36-37 /j.. Differences
exist between bays, between stations and between layers. Shifting of sediments by bottom
dwellers does not disturb completely the historical record.
The superior sections of cores show notable parallelism. Considering three levels :
a) about 8 cm. below the surface of sediments; b) top of consolidated sediment, ordinarily
3 to 4 cm. below surface; c) unconsolidated materials about 1 cm. below surface, we get the
following average diameters: Cape Cod Bay: a) 18 /*, b) 16.7 /*, c) 17 M: Buzzards Bay:
a) 20 fi, b) 17 n, c) 17.3 /u. The decrease of size, starting at an indeterminate time, is followed
by a recent and slight increase. A tentative explanation may be acceptance of an increase
of temperature followed by a recent cooling (or decrease of salinity). Salinity rather than
temperature may explain the differences between bays.
Pigment composition and productivity as related to succession in experimental
populations of phytoplankton. RAMON MARGALEF AND JOHN H. RYTHER.
A type of compound chemostat, made with serially-connected culture vessels, has been em-
ployed for the laboratory study of phytoplankton succession. After initial inoculation, fresh
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 327
medium is flowed slowly through the system. Soon a steady-state is reached where cell density
is independent of the initial state. Then flow can be discontinued and the following measure-
ments made on each vessel: productivity (C14 assimilation), cell counts and pigment analyses.
The rate of increase, r, in a given vessel is obtained by the expression r = In [1+ (V/C)
(C-B)]. where C is the cell concentration in the vessel, B the concentration in the previous
vessel and V the volumes of the vessel exchanged per unit of time.
In experiments with uni-algal (Ainphidinum) and mixed (Syracosphacra, Skeletonema
and Chactoceros) cultures there was consistently observed a decrease in productivity and an
increase in the ratio of optical density, 430/665, of acetone extracts of the pigments along the
successive elements of the system representing stages of a succession.
Typical results with Amphidinum (22° C, 450 foot candles) in a series of three vessels
are shown below. In this experiment pH reached 9.5 in some vessels and productivity was
based on available bicarbonate only, there being a suspicion that tagged carbonate was not
exchanged rapidly with bicarbonate or actually taken up by the algae.
(a) continuous illumination. D430/D665 : 3.25, 3.80, 3.85; chlorophyll a Og./106 cells):
3.67, 3.65, 2.25; r: 0.88, 0.39, 0.27; productivity Og. C/10a cells/hr.) : 1.06, 0.78, 0.37.
0» 13 hours light/day. D430/D665 : 3.55, 3.95, 4.74; chlorophyll a: 3.75, 2.70, 1.80;
;-: 0.78, 0.33, 0.16; productivity: 2.16, 1.86, 1.39.
Note the greater activity of cells subjected to the day-night rhythm.
Purification and properties of Linntlus arginine phosphokinase. R. A. MORRISON,
A. M. MORGAN AND G. W. DE VILLAFRANCA.
A method for the partial purification of arginine phosphokinase (APK) from the skeletal
muscle of Limultts polyphemus was investigated. The bulk of the enzyme was precipitated
from 50-100% ammonium sulfate saturation of the initial water extract. Dialysis of the residue
against 0.001 M arginine, pH 8.7 and refractionation with ammonium sulfate between 65-90%
saturation resulted in a fraction with a 6.6-fold purification and a 69% recovery. Further
fractionation did not increase the specific activity but led only to a decrease in the recovery.
The reaction catalyzed by arginine phosphokinase \vas followed by assaying for free
arginine and was run in the direction :
ATP 4- arginine — >• ADP + phosphoarginine
Activity was found to be directly proportional to time, up to 10 minutes, and also to the protein
concentration over a 5 -fold range with the substrate concentrations used. The pH optimum
is 8.6 and the temperature optimum is in the range of 15-18° C. When the reaction was
allowed to proceed at 37° C., the activity was reduced to 50% of the maximum. Although a
slight residual activity was found in the absence of added cations, activation occurred in the
presence of the following cations in decreasing order : Mn > Mg » Fe > Ca > Hg > Co ;
Cd inhibited. Optimal activity was noted when the ratio of arginine: ATP: Mg+2 was 1:1:1.
When the Mg : ATP ratio was greater or less than one, APK activity decreased. The Km
value for arginine at pH 8.7, 25° C. is 2.52 X 10~3 M. Substrate specificity was tested with
respect to creatine, glycocyamine, taurocyamine, lombricine, and arginine ; phosphorylation
occurred only with arginine. Limuhis APK is very labile ; it is easily denatured by alcohol,
dilution, heat, and by standing at 5° C. Freezing, both in the presence and absence of cysteine,
led to a complete loss of activity, while the same treatment in arginine buffer, pH 8.4, showed
a 67.7% decrease in activity.
Work supported by USPHS grant A-2647 and an ONR grant administered by the M.B.L.
Comparative aspects of active transport of D-glncose by in vitro preparations of
fish intestine. X. J. MUSACCHIA AND S. S. FISHER.
hi vitro preparations of everted intestinal sacs were used to measure absorption and trans-
port of D-glucose in segments of intestine from a variety of fish; 33 scup, Stenotomus
rcrsicolor; 2 sea bass, Centropristes striatits; 3 tautog, Tantoga onitis; 6 toadfish, Opsanns tau;
8 eel, Anguilla chrysypa; 71 bullhead catfish, Ameinrns nebnlosns; 2 perch, Perca flavescens.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Active transport of D-glucose was obtained in all the preparations from the fresh-\vater
fishes, in four preparations from eels and in none of the preparations from any of the marine
fish.
In a typical experiment, the everted segment of mid-intestine \vas filled and suspended in
teleost Ringer's containing a specific concentration of sugar (5 and 10 mg.%). Preparations
were incubated at fish body temperatures (22° to 24° C.) for 20, 30 or 40 minutes. The
starting concentrations in the mucosal and serosal fluids were equal. During absorption and
transport, sugar was moved against a concentration gradient and increased in concentration in
the serosal fluid and concommitantly decreased on the mucosal side. Active transport was
uniformly evident in the preparations made from catfish; starting with 10 mg.% glucose,
approximately three-fold differences were obtained after 30 minutes. The mean values for 41
preparations changed to 23 mg.% glucose in the serosal fluid and 6.5% in the mucosal fluid.
Comparable results were obtained where S mg.% glucose was the starting concentration and
after 30 minutes, for an average of 30 preparations, glucose in the serosal fluid increased to
13 mg.% and decreased to 2.6 mg.% in the mucosal fluid.
A series of 33 preparations (5 and 10 mg.% glucose at 22° C.) made from scup showed
mucosal absorption but no transport of D-glucose. Incubation at elevated temperatures, 37° C.,
showed very little enhancement of absorption.
Histological preparations showed that the intestinal mucosa retained its integrity during
the experimental procedures.
Aided by grant funds from Saint Louis L'niversity, Cancer Research Institutional Com-
mittee and a Grant-in-aid from the Sigma Xi-RESA Research Fund.
Some characteristics of active transport of sugars />v intestinal segments of
.-Inieiitnis nehiilosiis. X. J. MUSACCHIA.
Absorption and active transport of D-glucose in vitro preparations of intestinal segments
from the catfish, Amciunis nebulosus, have been reported. In the present study the upper
region of the midgut intestine was found to be more efficient in D-glucose transport. Average
serosal/mucosal ratios (15 preparations) in 5 mg.% D-glucose were: upper segments, 8.37
± 1.66; lower segments, 4.93 ± 1.69. At 10 mg.% D-glucose average S/M ratios were: upper
segments (19 preparations), 4. 74 ±0.79; lower segments (21 preparations), 2.80 ± 0.40.
Routine histological examinations showed shorter and more compact villi in the lower midgut
regions, indicating a decrease in total mucosal surface area. Other differences, vis. cytological
architecture of the mucosal epithelium, distribution of goblet cells, etc., were noted. Such
features may have some bearing on the differences in absorption and transport of D-glucose.
A control series of 15 "blank" preparations, wherein the segment was incubated in teleost
Ringer's minus the sugar, showed that the amount of glucose endogenously produced ranged
from a non-detectable amount in the mucosal fluid to a mean fraction of 2 mg.% in the
serosal fluid. These values had no effect in the ultimate determination of glucose.
Low temperatures, 0° to 2° C., inhibit absorption and transport of D-glucose. Prepara-
tions were incubated in 10 mg.% D-glucose at low temperatures for 20 minutes and S/M
ratios were 1.18 ±0.04. The same preparations were then incubated at fish body temperature,
22° to 24° C., and active transport was obtained, the S/M ratios being 2.64 ± 0.24. Four
preparations were made anoxic with 100% nitrogen for 30 minutes and little or no inhibition
of D-glucose transport resulted.
Other sugars, D-xylose, 10 mg.% and 20 mg.%, and D-fructose, 30 mg.% and 50 mg.%,
were not transported.
Aided by grant funds from Saint Louis University, Cancer Research Institutional Com-
mittee and a Grant-in-aid from the Sigma Xi-RESA Research Fund.
Further studies on the protoplasmic contraction of tlie marine alga, Chaetomorpha
Litntm Kiltziny. \\ . J. Y. OSTERHOUT.
The following experiments form a continuation of the reports published in this Bulletin
in 1955 and 1959 and the present Chaetomorpha resemble those described in 1959. The purpose
is to determine what substances cause the protoplasmic contraction of dead Chaetomorpha cells.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 329
A string of living cells was placed in filtered sea water at 50° C. for five minutes. It \\as
then transferred to filtered sea water at 25° C. for one-half hour, after which it was rinsed
in distilled water, wiped, and placed in a test solution at 25° C. The cells were at once
examined under the microscope and at the start no contraction was detected. But subsequently
the protoplasm began to contract. The rate and the degree of contraction in various solutions
\\ ere compared as follows.
The following solutions are arranged in the decreasing order of their effective action on
protoplasmic contraction. HC1, H.PO^, both at pH 1.6>KH,PO,, NaH,PO4, both at pH
4.5 > phosphate buffer mixture at pH 6 > glass-distilled water at pH 6 > phosphate buffer
mixture at pH 8 > borate buffer mixture at pH 8 > Na2HPO,, K.HPO4, both at pH 9.5 >
borax at pH 9.5. Control in filtered sea water showed no contraction, even after 40 hours.
Very rapid and severe contraction occurred in acid solutions at pH 1.6, but much slower
and less severe contraction occurred in alkaline solutions at pH 9.5, showing a great difference
between the acid and the alkaline solutions. There was less difference between the phosphate
and borate buffer solutions at the same pH value. Five-hundredths M and 0.01 M buffer
solutions behaved alike. Sodium and potassium behaved alike.
The contraction was reversed in NaOH solutions at pH 13.
These results indicate that the protoplasmic contraction was brought about primarily by
H+ and secondarily by PO4~" and that the contraction was reversed by OH~. They may suggest
that in a living cell the acids and phosphates formed by metabolism may cause protoplasm to
contract, which is reversed by the production of an alkaline substance. This may partly account
for the protoplasmic movement.
Experiments with triphosphates will be reported later.
The effects of chloramphenicol on cleavage of Arbacia eggs. EDWARD E. PAL-
TXCSAR.
The effects of chloramphenicol on early cleavage of Arbacia were examined to determine
the effect of possible inhibition of protein synthesis on the structures and dynamics of cell
division. Arbacia eggs and sperm were treated with various concentrations (0.2-2.0 mg./ml.)
of chloramphenicol in sea water. Cleavage was delayed up to four hours depending upon the
concentration, temperature and time of immersion into the chemical. The concentration for
blockage of division was 1.5-2.0 mg./ml. Temperatures were experimentally varied from
20-30° C.
Thirty-minute pretreatments of unfertilized eggs and sperm in 0.5-1.0 mg./ml. yielded up
to a 34-minute delay in the first cleavage. Fertilization was not affected, but if both treated
eggs and sperm were used in fertilization, there was a marked inhibition of cleavage rate and
failure to survive past the morula. Five- and 10-minute treatments of the zygotes in 1.0
mg./ml. of chloramphenicol suggested that the period most sensitive to subcritical concentra-
tions is prior to prophase. Blockage in 1-2 mg./ml. is reversible and the inhibition appears
to be a freezing of cleavage at the stage when treated. Once the block is reversed, the eggs
proceed with the stage at the time of arrest. Using a Warburg respirometer, preliminary
data were obtained which indicate no effect of inhibitory concentrations (0.2-0.5 mg./ml.)
on oxygen consumption. Subcritical concentrations (0.2-0.5 mg./ml.) did not appear to affect
the microscopic appearance, solubility, response to enzymes (trypsin, pepsin, chymotrypsin,
ribonuclease) or RNA distribution (azure-B, methyl green-pyronin) of the mitotic apparatus,
whicli was isolated using the dithiodiglycol method. Cytokinesis is unaffected in subcritical
concentrations, but there is a marked inhibition of membrane elevation. These preliminary
results suggest that the proteins of the mitotic apparatus might be synthesized prior to prophase,
since that is where this depressant of protein synthesis has its greatest effect. This possibility
is being investigated with isotopes.
The effects of 8-azaguanine and chloramphenicol on the regression-replacement
cycle of hydranths. EDWARD E. PALINCSAR AND JOAN S. PALINCSAR.
Substances believed to inhibit protein synthesis and nucleic acid metabolism were used to
treat Obelia colonies, to determine the phase of the hydranth cycle which might be most affected
330 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
and thereby to gain some insight into the cycle transformation. Obclia colonies were cultured
in aerated sea water (Crowell's method) containing concentrations of 8-azaguanine ranging
from 10~4 M to 10~3 M, or chloramphenicol concentrations ranging from 0.1 mg./ml. to 1.0
mg./ml. Temperature was constant and observations and counts were made up to 14 days.
Concentrations of chloramphenicol above 0.3 mg./ml. and concentrations of 8-azaguanine above
4 X 10~4 M were toxic, bringing about regression of the hydranth to the bud stage and eventual
disintegration. Eliminating the chemicals resulted in recovery by new upgrowths from the
stolon, which thus appeared to be the most resistant. At the lowest concentrations of both
chemicals, peculiar, large, elongated, free stolon-like structures grew from the ends of the
uprights and occasionally from the sides. These growths were twice the width of a bud, and
from 4 to 10 times the length by the second day. The perisarc was not annulated and there
was no visible differentiation except a narrowing of the stalk. After four days, small, but
otherwise normal hydranths differentiated on the ends and sides of the growth. These pre-
liminary results suggest that stages involving differentiation and organization are most sensitive
to the chemicals, and that proliferation phases are least inhibited. The possibility of un-
coupling growth and differentiation is being further investigated. The chemicals also delayed
the regeneration time of cut stems of Titbularia. Histochemical studies showed no changes in
distribution of RNA and DNA, increased concentrations of succinic dehydrogenase in active
hydranths and areas of growth, and increase in calcium concentration during late regression.
The role of moisture and illumination on tlie expression of tJie rhythmic behavior
of the diatom, Hantzschia amphio.rys. JOHN D. PALMER.
The diatom, H. amphioxys, surfaces on the sand flats in Barnstable Harbor, Mass., at the
time of low tide and migrates downward into the sand when the area is reflooded. In 1951
Faure-Fremiet reported this rhythmic behavior to persist for six days away from tides in
laboratory cultures.
Experiments were conducted during the summer of 1960 to see if any environmental influ-
ences on this rhythm could be detected. At high tide sheet metal dams were positioned over
previously delimited patches of cells, to retain water in these areas as the tide receded. The
resulting cover of water prevented the appearance of algae on the surface, the algae appearing,
however, within 30 minutes following draining of the dammed area. The natural flooding
tide, or artificial flooding, caused the organisms to migrate downward. In the laboratory, the
rhythmic migrations failed to occur under conditions of either constant flooding or drying
beyond some critical degree.
Darkness also inhibited the rhythm. Opaque shields placed on the sand before the diatoms
normally come up prevent the vertical migration during low tide, and similarly, shields placed
at low tide induce the return of the organisms into the sand. That light is the factor involved
here is evident from the observation that movements proceed normally under a transparent
covering.
Diatoms observed in "preference-chambers" in the field, but isolated from the tidal
change, move into the illuminated portion at low tide and into the darkened portion in synchrony
with the downward migration of the natural population.
These studies indicate that there is a tidal rhythm in the sign of the phototactic response,
it being positive during the time of low tide, and that the expression of the rhythm is influenced
by tidal water movements.
Electron microscopic studies of Fucus vcsiculosus cytoplasm in summer and
winter. JOHNSON PARKER AND DELBERT E. PHILPOTT.
Longitudinal hand sections of Fucus midrib 2 inches from the tip were made in January,
fixed in buffered OsO4 at 0° C, dehydrated, embedded in 1:3 methyl and butyl methacrylate,
and sectioned longitudinally with a diamond knife. Chloroplasts were found clumped together
in winter, but lamellae were clearly visible with six layers each (not four as reported in the
literature). In cells with slightly shrunken contents, cytoplasm tended to adhere in small
strands to the wall, and as a result of this the wall was sometimes slightly expanded, revealing
about 25 layers. Good OsO4 fixation in June was difficult, possibly because of the state of
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 331
hydration of the cytoplasm or because of its permeability, a situation which might be related
to the lower resistance of Fitcus to sub-zero temperatures in summer than in winter. KMnO4
at 5% for 10 minutes at 22° C. by Mollenhauer's method gave better results for summer
material ; chloroplasts were observed resembling those of winter except that they were not
clumped. Vacuoles in cytoplasm were more evident in summer and the cytoplasm as a whole
was more electron-dense in winter. Cytoplasm clearly passed from cell to cell in medullary
elongated cells, as has long been suspected. These connections arc relatively massive intrusions
of cytoplasm in comparison to the fine strands penetrating the pores of Macrocystis pyrifera
(brought from the West Coast) sieve plates. Strands in Macrocystis appeared to be extensions
of endoplasmic reticulum.
Histological investigation of the central nervous system of Clyuienella torquata.
MABEL C. PATERSON.
In order to understand the significance of regeneration studies in Clymenclla torquata a
more complete knowledge of the histology was considered necessary ; consequently an investiga-
tion of the central nervous system was undertaken. Anterior regions were serially cut, frontal ly
and sagittally, and stained with a modification of the Holme's buffered silver nitrate technique.
The central nervous system is similar to previously described polychaete systems, and consists
of a ventral nerve cord which terminates in an enlargement, the subpharyngeal ganglion. This
ganglion is united with the paired suprapharyngeal ganglia, located on the dorsal aspect of
the pharnyx, by a pair of circumpharyngeal commissures which encircle the pharynx. A
number of branches • arise from the circumpharyngeal commissures. Each suprapharyngeal
ganglion consists of two lobes and receives two major fiber tracts. One of these enters
antero-medially and connects with a group of special sensory cells located on the dorsal head
plaque, the other enters ventro-laterally and innervates the stomadeal region. The two supra-
pharyngeal ganglia are connected midway by a bridge of fibers.
The nerve cells within each suprapharyngeal ganglion are peripherally located and appear
to be collected in two main groups, anteroventral and dorsal. The fibers of the circum-
pharyngeal commissure enter the lateral side of each ganglion and therefore separate the two
groups of cells. The majority of the cells are of medium size and appear to be bipolar. Their
nuclei are round and centrally located. They usually contain one, or occasionally two, eccen-
trically placed nucleoli. The cytoplasm is granular and some indication of neurofibrils has
been observed.
Currently the cellular nature of the subpharyngeal ganglion and the ventral nerve cord is
being investigated, as well as possible neurosecretory activity.
Histological investigation of the nephridia of Clymenella torquata. MABEL C.
PATERSON AND CARRIE R. KREWSON.
The anatomy and histology of the nephridia of the polychaete Clymenclla torquata were
studied. Four pairs of large nephridia are located laterally in the coelom in segments seven
through eleven. Each nephridium occupies portions of two segments, the nephrostome lying
in front of the segmental septum and the remainder of the organ immediately posterior.
Nephridial openings are observable ventrally at the base of each parapodium.
A plastic reconstruction of a nephridium showed that the organ consists of a funnel-
shaped nephrostome opening to the coelom, a long, slightly-coiled, unbranched tubule extending
from the nephrostome, a bladder, and a short, straight neck region leading to a nephropore.
The nephrostome is composed of a single layer of ciliated columnar epithelium with a
distinct basement membrane. The everted lips of the nephrostome appear in close association
with a branch of the sub-intestinal blood vessel. The tubular portion consists of a single
layer of irregularly-shaped, ciliated columnar epithelium with a basement membrane ; the
cytoplasm is densely granulated and highly vacuolated. The bladder and neck regions are
formed of a single layer of ciliated squamous epithelium. At the tubule-bladder junction a
large tuft of cilia was noted. Ova and sperm packets were observed in the tubule and bladder,
suggesting the organ functions in gamete release.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Preliminary histochemical studies indicated alkaline phosphatase localizations in large,
sharply-defined granules packed uniformly throughout the cytoplasm in the tubule. No
positive alkaline phosphatase reaction was observed in the nephrostome; the bladder showed a
positive reaction only at its junction with the tubule. The ciliated inner border of the entire
organ is PAS-positive. PAS-positive material was also observed in the basement membrane and
in small granules scattered throughout the cytoplasm, particularly in the tubular area. These
PAS-positive granules are not comparable to the alkaline phosphatase-positive granules observed
in the tubule.
Further electron microscopic observations on the sperm of Limulus polyphemus.
DELBERT E. PHILPOTT.
Previous description of the ultrastructure of the sperm of Limulus polyphemus reported
the head of the sperm as having an acrosome cap and a small "axial" body at the anterior end.
An axial core was also seen, traversing the head centrally from the small axial body to the
region of the centriole in the distal end of the head. At the time of the first investigation it
was not possible to determine if the posterior spiral, consisting of six closely adhering turns,
continued up the axial core to the axial body or if the axial core was a process unto itself.
Embedding the sperm in epoxy resin and making numerous sections revealed that the posterior
spiral does continue up the axial core to the axial body and is thus a continuous structure.
The use of carbon for single molecule visualisation. DELBERT E. PHILPOTT.
The method of Cecil Hall for the visualization of individual molecules has been modified
in the following way. After the specimen has been sprayed on mica and shadowed with
platinum according to his method, carbon is evaporated down onto the specimen from 90° in
the place of silicon monoxide. This film is now sufficiently strong to eliminate the next step
of flooding the surface with collodion. This saves the time involved in the last step, but more
importantly the replacement of silicon monoxide plus collodion as a supporting film increases
the brilliance and contrast of the specimen image in the electron microscope. In fact, in some
instances the latex particles have been left out of the specimen to be sprayed, both because it
was felt advantageous to keep the specimen pure, and because all of the material which is
visible on the final print is also visible on the fluorescent screen during specimen scanning.
After the carbon evaporation the mica must be placed at once into distilled water in order
to float off the prepared specimen. This results in a silver film which easily floats off without
the aid of score marks or any other help. However, waiting 24 hours nearly always results
in unstrippable films. This stripped film is picked up on 200-mesh copper sheet cut to the size
of the floating specimen film, and brought up from underneath, thus mounting the specimen.
After blotting the grid material on filter paper, grids are punched out in the usual way for
examination. The final result will not be a great deal different from the original and clever
method referred to above, but the increased ease and simplicity with which the method can
be applied should prove an asset to others, as it has in this laboratory.
Electron microscopic observations of the starfish eyespot. D. E. PHILPOTT AND
A. B. CHAET.
A preliminary investigation of the red eyespot located on the short sensory tentacle at
the tip of the arm of the starfish, Asterias forbesi, was undertaken by means of electron
microscopy. The eyespots were dissected from the animal, fixed in osmic acid sea water,
embedded in methacrylate and sectioned.
Each of the many eye cups found in the eyespot consists of a cone-shaped layer of red
granules, the opening of which is directed towards the outer surface. Electron micrographs
indicate two different-sized granules. The smaller, more osmiophilic pigment granules measure
about 0.2 X 0.1 micron in diameter, whereas the larger ones are 1.3 X 0.8 microns in size.
The pigments of the eyespot, B carotene and esterfied astaxanthine, may be represented by
the two sizes of granules.
PAPERS PRESENTED AT MARINE BI< (LOGICAL LABORATORY
Within the cup two different structures are visible. The one filling the posterior four-
fifths of the pigment cup appears quite uniform in nature and is connected at intervals to the
lateral walls. The second structure, which is found in the anterior portion, as a layer lying
at right angles to the cup's axis, appears to he identical to the outer ectodermal surface of
the eyespot itself. This layer as well as the outer surface is about one micron in thickness,
and both are covered with micro villi. The layer within the pigment cup may have arisen
as part of an invagination of the outer surface, thus explaining the presence of the micro villi
which are pointed into the cup. Future attempts will be made to determine if this ectodermal-
appearing layer represents a potential lens, and whether the posterior four-fifths of the cup
represents neural conducting material.
This work was supported by National Science Foundation Grant No. G-8718 and National
Institutes of Health Grant No. A-3362.
Comparative ecology of two species of intertidal amphipods: Talorchestia incga-
lopthama and Orchcstia agilis. SARA J. PLATZMAN.
In an intertidal environment humidity is obviously a central factor in determining the
distribution of organisms. The intertidal animals would therefore be expected to show adapta-
tions for reduced humidity. Experiments were performed on Talorchestia megalopthama and
Orchcstia agilis, two species of intertidal amphipods found near Woods Hole, Massachusetts,
at 26.5° C., the approximate summer temperature of this area. At 0% R.H. Talorchestia
survived for an average of 44 and Orchestia 33 minutes. By comparison the members of the
wholly marine genus Amphithoe survived an average of 13.5 minutes and Armidillidium rulgare,
a terrestrial isopod, was found by Waloff to live for 429 minutes in 0% R.H. Therefore the
R.H. tolerances of Orchcstia and Talorchestia represent a modification toward a land habitat.
The slight difference in humidity tolerance between Talorchestia and Orchestia is not
large enough alone to restrict them to different intertidal habitats. Orchcstia is found under
moist sea-weed at the high water level and is seldom, if ever, present on the surface of the sand.
In contrast, the adults of Talorchestia live, during the day, beneath 1-5 inches of sand; the
lower limit of distribution is 3-4 feet above the water mark of all phases of the tide. Unlike
Orchestia, they are not confined to the high-water mark. At least part of the populations of
Orchestia are active day and night. Talorchestia, however, is only active on the surface, near
the water, at night, although a few juveniles can be found hopping on the surface near the
water or in the sea-weed during the day. These observations indicate that behavioral and
distributional differences between these two closely related species seem more important than
humidity in differentiating their niches.
This work was done as part of the student training program of the Marine Ecology Course.
Analysis of plwtndynamic effects in lobster nenroniuscitlar preparations. J. P.
REUBEN.
Muscle fibers soaked for brief periods in 10~* M acridine red, neutral red, or sevron blue
responded to illumination with white light by 2 to 10 mV changes in membrane potential.
The potentials were of either sign and, when hyperpolarizing, they were reversed by increasing
the negative resting potential somewhat more than 15 mV with intracellularly applied hyper-
polarizing currents. The membrane resistance, which was increased in the dark after applica-
tion of the dyes, was lowered during the period of illumination and for some seconds thereafter.
The directly evoked responses of the muscle fibers, which were converted from graded responses
to spikes by the dye, were reduced during the illumination. Both the excitatory and inhibitory
postsynaptic potentials (p.s.p.'s) were also reduced during the irradiation. Return of the
amplitudes of the responses to their initial values occurred in the tempo of the return of the
membrane resistance.
The frequency and amplitudes of both excitatory and inhibitory miniature p.s.p.'s were,
however, greatly enhanced during illumination of the dye-treated preparations. Picrotoxin,
which blocked the inhibitory miniature p.s.p.'s, did not block hyperpolarization of the fibers
by irradiation. The increased miniature activity appears to be caused by photodynamic effects
on the presynaptic nerve fibers. In the muscle fibers, however, the dyes appear to sensitize a
334 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
photodynamic conductance change in a component of the membrane that is independent from
the electrically excitable or the synaptically activated membrane components. This effect may
be analogous to the conversion of previously inert membrane to a chemically excited one in
denervated vertebrate muscle fibers. The increased conductance of the photodynamically
activated membrane shunts and decreases both the directly evoked responses and the p.s.p.'s.
Electrotonic connections between lobster muscle fibers. J. P. REUBEN.
Depolarizing or hyperpolarizing currents that are applied intracellularly to one (the
"proximal") muscle fiber produce changes of the same sign, but of lower amplitude and changed
form, in the membrane potential of other ("distal") fibers. The electrophysiological data on
the size and form of these electrotonic potentials indicate the occurrence of anatomical structures
which form high resistance connections between the two approximately symmetrical resistance-
capacity networks of the two muscle fibers. The electrotonic potentials in the distal fiber
were attenuated as much as 10-fold in many fibers, but in some there was only 2- to 3-fold
attenuation. The greater the attenuation, the more marked was a phase shift between the
applied current and the electrotonic potential in the distal fiber. With the larger phase shifts,
the potential in the distal fiber continued to grow after the cessation of a brief pulse of current.
All-or-none spikes, which were directly evoked in one fiber of procaine-treated preparations,
also caused potentials in other fibers, the degree of their attenuation being related to the
degree of attenuation of applied pulses. The connections did not exhibit rectification, flow of
current apparently being symmetrical in the two directions.
These data and experiments on a model equivalent circuit indicate that the muscle fibers
are connected by a link which has a resistance some 3 to 10 times higher than that of the
individual fibers. Whether the interconnections are few or numerous between a pair of fibers
could not be determined because lobster muscle fibers have a large length constant. There
appear to be mutual interconnections in groups of adjacent fibers. However, the electro-
physiological evidence of electrotonic connections cannot resolve whether they are formed by
syncytial or ephaptic links or by both. In some cases, no electrotonic effects were observed
between immediately adjacent fibers, indicating the absence of connections between these pairs.
In Carc'mus and Cancer muscles the findings are different.
Actions of cesium ions on the electrically excitable membrane of lobster muscle
fibers. ]. P. REUBEN AND H. GRUNDFEST.
As is also the case for neuromuscular transmission, short-term exposure of the fibers to Cs+,
with or without removal of K+, did not affect the electrically excitable membrane. Soaking
the preparation for 10 hours or more in K+-free Ringer's solutions containing 15 to 50 meq./l.
of Cs^ did not markedly change the membrane potential or resistance from their values in K+-
free solutions without Cs*. Direct stimulation still produced small graded responses, although
the large excitatory postsynaptic potential of the Cs+-soaked preparations caused a large spike-
like response. Profound changes in the ionic conductance mechanisms of the electrically
excitable membrane could nevertheless be shown on additional treatment of the preparations
with various agents. Thus, while increasing external K+ caused depolarization, the change
was only about 30 mV/decade change in K+. On changing the medium from zero K+ to
30 meq./l., the resistance fell only 3- to 4-fold, while in untreated muscle fibers the change
is about 100-fold. Unlike untreated preparations, those soaked in Cs+ tolerated exposure to a
Ca++-free medium for long periods. On diminishing the external Ca++, the directly evoked
responses increased in size. However, there was also a large, reversible decrease in membrane
resistance, though only a small decrease in resting potential.
The addition of procaine (10~3 to 10~2 w/v) to normal preparations converts their graded
responses to brief, all-or-none spikes. In Cs+-soaked preparations, application of the drug
caused indefinitely prolonged spikes, that were followed in some experiments for more than
30 minutes and terminated only by applying strong hyperpolarizing currents. The conductance
during the spike was up to 20 times higher than that of the resting membrane.
The overt effects of Cs+ in general resemble and are synergistic with those of Ca+% but
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 335
they are more extreme under some experimental conditions and less so under others. These
similarities and differences indicate that Cs+, like the alkali earth ions, has several modes of
action. They also indicate that the several molecular mechanisms which are presumed to
underly these effects are independent to some degree.
Further analysis of the conversion of graded to all-or-none responsiveness in the
electrically excitable membrane of lobster muscle fibers. J. P. REUBEN AND
H. GRUNDFEST.
The normal graded activity of lobster muscle fibers can be converted to all-or-none by
various agents. These effects can be analyzed in terms of the various changes of ionic con-
ductances and electrode properties of the electrically excitable membrane (see Grundfest,
this issue).
The conversion by cocaine (10"e to 10~5 \v/v) and procaine CIO'3 to 10~2 w/v) occurs with
only small changes in resting membrane potential and resistance. The spikes are brief com-
pared with those produced by alkali earth or onium ions. The drugs therefore probably only
decrease the outward current during the response, by inactivation of K+-conductance. Cocaine
in higher concentrations abolishes activity, probably by also blocking inward current. In
contrast, the conversion of the membrane to all-or-none responsiveness by d-tubocurarine is
associated with hyperpolarization and increased resting membrane resistance. Thus, this drug
also exerts another of the actions of the divalent alkali earth ions, block of resting K+-conduct-
ance. However, enhanced Na+-conductance does not develop, and the spikes are brief.
Methylene blue, sevron blue, acridine red and neutral red appear to act in the same way as
does d-tubocurarine. Procaine, in combination with other agents (Ba++, Cs+, phenethylamine
derivatives, or serotonin-like compounds), causes spikes that may last for 30 minutes or more.
Thus, the combined action of the agents also blocks NaMnactivation to an extreme degree.
Procaine and d-tubocurarine convert all-or-none responses of other electrically excitable
membranes to graded activity. Thus, in their actions on different cells, various agents may
have "excitant" or "depressant" effects, depending upon the conditions that prevail in the
specific membrane. The nature of the effects probably derives from the degree and the critical
ratios to which the different factors of electrical excitability participate in each response, and
from the degree in which each component is affected by the various agents. The latter variable
is probably determined by different details of molecular structure that are as yet unknown.
Inhibitory and excitatory miniature postsynaptic potentials in lobster muscle fibers.
]. P. REUBEN AND H. GRUNDFEST.
Miniature potentials of synaptic origin occur spontaneously in lobster muscle fibers, due
to activation of the inhibitory as well as the excitatory synaptic membranes. The inhibitory
miniatures may be of either sign, depending on electrochemical conditions. Both activities are
probably due to summation of transmitter actions of many presynaptic terminals. Inhibitory
miniatures of up to 1 mV have been observed and the excitatory tend to be still larger. The
inhibitory activity is abolished by picrotoxin and the amplitudes of the excitatory miniature
potentials are then greatly augmented. The frequency and amplitudes of the miniature poten-
tials are modified by applying various agents to the preparation. NH4C1 (15 meq./l. or more)
at first causes an increase in the frequency of both types of miniature potentials. Later, both
are few in number, but large and prolonged. When the inhibitory are eliminated by picro-
toxin, the excitatory potentials grow to very large amplitudes. Large, spontaneous potentials,
principally or exclusively of the excitatory variety, are induced after serotonin. The poten-
tials occur independently in different fibers of the same muscle, though all are innervated by
the same axon. Thus, the independent origins of the spontaneous potentials indicate that they
probably occurred independently of an axonal spike, and that the transmitter actions occurred
independently in different presynaptic terminals.
Presence or absence of miniature potentials provides a criterion for the mode of action
of various agents on the different components of the neuromuscular system. Thus, a very
powerful blockader of lobster neuromuscular transmission, the purified dinoflagellate "clam
336 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
poison,'' does not eliminate either variety of miniature potentials, nor the larger potentials
induced by NH4C1. Therefore, the poison must block arrival of the spike at the presynaptic
terminals, while activity of the latter is unimpaired.
The action of cesium ions on neuromnscnlar transmission in lobster. J. P. REUBEN
AND H. GRUNDFEST.
Addition of Cs+ (15 to 50 meq./l.), with or without removal of K+ in Homarus Ringer's
solution, had no apparent effect on short-term exposure of the preparation to this ion. Pro-
found alterations were produced, however, on soaking the preparations for 10 to 20 hours in
K+-free solutions containing Cs+. The excitatory and inhibitory postsynaptic potentials
Cp.s.p.'s) of the muscle fibers were greatly augmented when recorded in a K*-free medium,
with or without Cs+. A single stimulus to the excitatory axon evoked a large, spike-like
potential and a vigorous twitch. The i.p.s.p. evoked by a single stimulus to the inhibitory axon
was maximal, or nearly so. The degree of facilitation of the i.p.s.p.'s was correspondingly
small.
The sensitivity of Cs+-treated fibers to GABA was about like that of untreated prepara-
tions. Thus, the inhibitory synaptic membrane had not become more sensitive to this drug.
The reversal potential was unchanged and the augmented i.p.s.p.'s could not have resulted from
changed electrode properties of the synaptic membrane. The reversal potential of the e.p.s.p.'s
was between 20 and 30 mV, inside negative, and probably had not been changed by the ion,
but a direct test for the chemical sensitivity of the excitatory synaptic membrane was not
available. It is unlikely, however, that the augmentation of both p.s.p.'s by Cs+ was due to
actions on the presynaptic terminals, increasing the transmitter action of both.
When K+ was present in the medium, the p.s.p.'s were markedly reduced in size. The
decrease of the i.p.s.p. was directly related to the decreased membrane resistance and the changed
membrane potential of the muscle fibers. The reduction in the e.p.s.p.'s was much greater.
When the external K" was double that of Homanis Ringer's solution the membrane resistance
had decreased 3- to 4-fold, but the e.p.s.p.'s were diminished about 10-fold. The effect of K+
on the e.p.s.p. therefore indicates that K+ blocks competitively the Xa+ channels opened during
activity of the synaptic membrane.
Properties oj indefinitely prolonged spikes of lobster muscle fibers. ]. P. REUBEN,
R. WERMAN AND H. GRUNDFEST.
When treated with various agents (Reuben and Grundfest, this issue), lobster muscle
fibers produce directly evoked responses that are spikes with amplitudes up to 120 mV and
durations of more than 30 minutes. The membrane conductance during the response was
increased 3- to 20-fold. Responses with conductances in the lower part of this range could
be terminated by applying strong inward currents. The termination was associated with an
increase in membrane resistance, and the change in potential had a time course resembling
that of hyperpolarizing responses. In spikes with high membrane conductance, the potential
returned to its plateau value even after strong inward currents had hyperpolarized the membrane
for several seconds. This return may have been a manifestation of anodal break excitation.
Spontaneous termination of the spike was frequently associated with up to 15 mV hyper-
polarization, lasting 15 seconds or more. In other cases it occurred through stages of inter-
mediate plateaus, each lasting several seconds. Low frequency oscillations between metastable
states often occurred and sometimes lasted more than 10 minutes. The durations of the
individual oscillations varied. At different times during a single sequence their forms resembled
those of vertebrate cardiac potentials, "upside-down" potentials, or hyperpolarizing responses.
Thej- ranged in amplitude from less than 1 mV to about 50 m\~.
These different effects are ascribable to the operation of different combinations of the
various ionic conductances and the consequent electrode properties of the electrically excitable
membrane (Grundfest, this issue). Local changes in these relations, which probably reflect
non-uniformities in different regions of the membrane, add further complications. Their
various manifestations in lobster muscle fibers suggest a possible mechanism for "pacemaker"
activity in other types of cells.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Further studies on the biochemical effects of .\--radiation on Tetrahyjncna /ivn-
formis. JAY S. ROTH, ELEANOR HENRY AND CAROL WIERCINSKI.
Three-day cultures of Tetrahymena pyrijormis W, grown in proteose-peptone plus yeast
extract, were collected by gentle centrifugation and thoroughly washed. Cells were suspended
in water or buffer and irradiated with from 300,000-600,000 r. In some cases cells were
irradiated in the presence of metabolites, or metabolites were added immediately after
irradiation.
Growth studies using an inoculum of approximately 2 X 103 cells indicated that no cells
survived 600,000 r. With lower doses there was a lag period in the growrth of irradiated cells,
the length of the lag being roughly proportional to the dose, but thereafter irradiated cells grew
at the same rate as controls. When cells were irradiated with up to 600,000 r in 0.05 M
phosphate buffer, pH 7.4, there was either a slight stimulation of respiration or no change.
However, irradiation in water resulted in the depression of respiration from 50-90% at 600,000
r. Addition of thioctic acid (0.5 mg./ml.) to cells during or immediately after irradiation
with 300,000 r had no significant effect on respiration, nor did it appreciably affect the increase
in respiration observed when irradiated cells were treated with 0.05 M pyruvate. Lactate
(0.05-0.2 A/) stimulated the respiration of control cells, and the oxidation of lactate by irradiated
cells was increased over that of controls by from 20-100% as long as two hours post-
irradiation with 300,000 r. The amount of increase was dependent on the time post-irradiation
and the concentration of lactate. The oxidation of lactate was depressed, nevertheless, in a
cell-free homogenate of Tetrahymena irradiated with 300,000 r, as well as in a homogenate
prepared from irradiated cells.
The effects observed appear to be explained best on the basis of an increase in permeability
of the cell membrane, and possibly mitochondrial membranes to lactate and pyruvate after
irradiation. (Supported by a grant from the U. S. Atomic Energy Commission.)
X-ray-induced initotic delay in the Arbacia egg. RONALD C. RUSTAD.
Since multipolar divisions appear to define damage to a system controlling the multiplica-
tion of asters, further studies on the parallelism between the induction of multipolar spindles
and mitotic delay have been conducted. A pre-fertilization recovery period reduces both effects
in irradiated eggs. The same mitotic stage (early streak) is maximally sensitive to the
induction of both effects. Direct tri- or quadripolar second divisions indicate that the effect is
general and not a special property of fusion nuclei.
The preparation for a multipolar division includes a previously undescribed mitotic stage :
a double streak.
The experimental induction of polyspermy has been used to analyze the additivity of x-ray-
induced mitotic delay. The presence of several irradiated sperm neither lengthens nor shortens
the period of mitotic delay. When unfertilized eggs are irradiated and then made polyspermic,
some of the eggs divide sooner than monospermic ones.
The division cycle of Arbacia eggs becomes insensitive to the induction of mitotic delay
by either acridine orange or irradiation at approximately the same time. Treatment of sperm
with either acridine or x-rays extends the x-ray-sensitive portion of the mitotic cycle, which
corresponds cytologically to early streak, the period of multiplication and separation of the
centrioles.
Low dose ^.--irradiation and the possibility of accelerated root growth. B. P.
SONNENBLICK AND JOHN KEOSIAN.
It would be of theoretical and practical significance to determine definitively the existence
of, and then induce as desired, the so-called "stimulating'' effect of radiation on intact living
organisms. An influence has been claimed by some earlier investigators and sporadically by
recent ones, but others deny its existence. Would exposure of bulbs to doses circa 100 r influ-
ence subsequent rooth growth? Allium bulbs of heterogeneous origin (heterogeneity was
desired) were exposed in exploratory tests to x-rays of 85 and 182 kvp, dose rates of 45 r
and 350 r/minute. Germination began 12 hours later and test temperatures recorded from
338 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
24.1-26.3° C. Periodic observations were made and after 14 days all roots removed and
their lengths measured. Almost 11,000 roots from approximately 400 irradiated and unexposed
bulbs have been measured.
The results are equivocal. It is possible to record with doses of 50-100 r an accelerative
influence, or the absence of such, according to the criterion used. Criteria may be (a) total
number of roots, (b) average root length, (c) percentage of roots surpassing an arbitrarily
chosen length, (d) percentage of bulbs germinating. In a given experiment an effect may
be indicated by one or more of the criteria but not by others. Employing criteria (b) and (c)
above, it would appear that low dose irradiation could have stimulated root growth relative
to controls in two of six tests. It will be interesting to note future results if some variables
in our complex system, or with other projected biological material, are eliminated.
Duration of the experiment is an important consideration. Roots from exposed bulbs may,
during the first week, manifest an accelerated growth rate which might be regarded as
stimulation were the test ended at that time. Control roots can often bypass the others in
length during the second week. Early acceleration may thus be a spurious phenomenon.
Comparative effects of x-ray and ultraviolet radiation of gametes on the develop-
ing sea urchin Arbacia. CARL CASKEY SPEIDEL AND RALPH HOLT CHENEY.
Effects of x-ray and 2537 A ultraviolet (UV) radiation of Arbacia gametes were ana-
lyzed by observing the subsequent stages of development after mixing various combinations
of radiated gametes. In addition to fertilization of normal eggs with normal sperm, 8 gamete
combinations were studied: (1) x-rayed eggs with normal sperm, (2) UV-rayed eggs with
normal sperm, (3) normal eggs with x-rayed sperm, (4) normal eggs with UV-rayed sperm,
(5) x-rayed eggs with x-rayed sperm, (6) UV-rayed eggs with UV-rayed sperm, (7) x-rayed
eggs with UV-rayed sperm, (8) UV-rayed eggs with x-rayed sperm. Radiation doses ranged
from mild to severe. Fertilization occurred with all gamete combinations. Development was
watched from the moment of insemination to the larval pluteus stage. Time-lapse cinephoto-
micrography recorded early viscosity changes and the sequence of protoplasmic movements
culminating in irregular cleavages.
Certain differences in the progeny resulting from the 8 combinations of radiated gametes
were ascribed to differences in penetration of the two kinds of radiation, deep with x-rays and
shallow with UV-rays. Graded quantitative results, based upon both amount of develop-
mental retardation and degree of injury, were more uniform after graded x-ray dosages than
after graded UV exposures. Greater variation in UV-rayed egg experiments resulted from
the different amounts of radiation that reached the near (to UV source) and far sides of the
exposed eggs, combined with the variable position of the nucleus in individual eggs. Differ-
ences in radiation penetration also affected comparative radiosensitivity. Thus a short ex-
posure of sperm (small-sized cells) to UV-rays caused developmental retardation and injury
equal to a longer exposure of eggs (large-sized cells), 4-16 times as long. In contrast an x-ray
dose to sperm was equal to a dose of only 1.5-2 times as much to eggs.
This investigation was supported by a research grant (PHS RG-4326 C3) to C. C. S. from
the National Institutes of Health, Public Health Service.
Developmental potential of the chick blastida (unincubated blastoderm). NELSON
SPRATT AND HERMANN HAAS.
Typical development (through 10± somite stages) occurs in almost all unincubated
blastoderms explanted upper surface against a new yolk-albumen extract agar medium. This
method has made possible for the first time an extensive analysis of the blastula stage (24—28
hours post-fertilization) in the freshly-laid chick egg.
The bilaterally symmetrical organization of the unincubated blastoderm, although quite
evident in a cell density gradient and pattern of lower layer morphogenetic movements, is in
no sense irrevocably determined either in its position or direction. Complete transection and
longitudinal section, partial longitudinal section with a sizable connecting bridge of pellucid
and opaque area cells, separation of the prospective posterior quadrant from the remaining
three-fourths of the blastoderm, etc., in short, any cut through or into the system stimulates
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 339
regenerative growth which tends to transform the outline of a fragment into a circle, e.g., like
that of the intact whole blastoderm. This in turn may bring about either: (1) a change in
direction of the median plane of lower layer movement (and consequent position of the
embryonic axis upon the blastoderm), (2) complete reversal in polarity of the movement
pattern (and of the direction of the embryonic axis), (3) induction of one or two new sec-
ondary bilateral patterns of movements (one or two complete secondary embryos) or (4)
division of the normal axial pattern into two bilaterally symmetrical movement patterns (two
complete embryos).
The unincubated blastoderm is thus capable of great regulation and regeneration and
consequently exhibits the properties of an embryonic field. Any part of the system large enough
(exceeding about }4 of the original mass) is able to form a complete, bilaterally symmetrical
embryo morphologically and functionally equivalent to a normal 10± somite stage. About 80
twins and 7 triplets have been obtained to date.
Whole-mount preparations of fish lens epithelium from various species. B. DOBLI
SRINIVASAN.
Whole-mount preparations of epithelium from the crystalline lens of rabbit, rat and frog
have proven useful in studies on cellular proliferation in normal and injured tissues (Harding
ct a!.. 1960). The unique characteristics of the lens (e.g., its avascularity, lack of a nerve
supply, enclosure within a membrane, presence of epithelium as a single layer of cells) have
been shown to be of advantage in studies on the relationship between injury, and DNA synthesis
and mitosis in the rabbit lens (Harding ct al., 1959). It would be of interest, for a number
of reasons, to extend this study to include the lenses of cold-blooded animals, and the present
report indicates that whole-mounts of the entire layer of lens epithelium can be prepared
from the following species: sea bass (Centropristes striatus), dogfish (Musteltts canis), dusky
shark (Carcharhinus obscurus) , goose fish (Lophius americanus), sea robin (Prionotns
carolinus), tautog (Taiitoga onitis), scup (Stenotomus versicolor), and skate (Raja erinacea).
A procedure previously described (Harding, 1960) was followed in making the whole-
mounts. In all cases essentially complete layers of epithelium could be prepared. They
consisted of a single layer of cells. Stainability with Harris' hematoxylin varied from species
to species. Good results were obtained with dogfish, dusky shark, goose fish and skate.
Mitotic figures were found in preparations from dogfish, sea bass, scup, tautog and skate.
Mitotic figures, in these species, were found scattered over a relatively wide area as compared
with the peripheral band seen in the rabbit and rat previously described. Lenses from goose
fish and dusky shark were not fixed immediately after death of the animals and mitotic
figures were not seen in these specimens.
It is concluded that the entire layer of lens epithelium, in the form of a whole-mount,
can be prepared from a number of species of fish, and that a few of these species, namely
dogfish and skate, appear to be very suitable for studies on cellular proliferation.
Extraction of chlorophyll from marine plankton algae with acetone and methanol.
CAROL STERNS.
Standard spectrophotometric methods for the quantitative determination of chlorophyll
are based upon the specific absorption of pure acetone solutions of the pigment. This tech-
nique has been widely adapted in ecological research for the measurement of chlorophyll in
natural plankton populations. There are many indications, however, that chlorophyll extrac-
tion of marine plankton algae with cold acetone as a solvent is far from complete. This is
particularly true of certain species (i.e., many of the green algae) with tough, resistant cell
walls. Higher chlorophyll values are consistently obtained if the cells (millipore-filtered or
centrifuged from suspension) are subjected to mechanical grinding or Bonification. Neither
of these latter techniques is practical for field analyses or for measurement of large numbers
of samples.
On the other hand, hot methanol, long used for chlorophyll extraction of higher plants,
appears to be considerably more effective than acetone for pigment extraction of marine
phytoplankton. While some modification of the standard formulae for relating optical den-
340 PAPERS PRESENTED AT MARIXK UK >!.()< ilCAL LABORATORY
sitics at various wave-lengths to concentrations of the different pigments may be necessary,
it is noteworthy that the red absorption peaks of methanol and acetone solutions of chloro-
phyll (664 vs. 661 m/u) are nearly the same, and that relative absorption at the respective
peaks is only about 3% higher for methanol than for acetone.
A comparison of the optical densities at 665 ni/u of cold 90% acetone and hot methanol
extracts (methanol hot initially, extraction for 24 hours in dark and refrigeration) of a
variety of organisms gave the following results (shown as O.D. methanol/acetone) : Nanno-
chloris atotmts (green) 1.96; Syncchococcus sp. (bluegreen) 1.60; unidentified diatom 1.18;
Dnnaliella ciichlora (green) 1.00; Ainphidiniiim cartcri (dinoflagellate) 1.00; Pyramimonas
sp. (green) 0.94; natural plankton from Vineyard Sound, 1.29. This work was carried out
as a part of the student training program of the Marine Ecology Course.
Further inquiry into the mechanisms of aqueous humor formation in dogfish.
WILLIAM STONE, JR., DANA FELDSHUH, JUDITH LEINING AND R. F. Doo-
LITTLE.
The osmotic pressure of the aqueous humor of the smooth dogfish (Mustelus can is) is
lower than the osmotic pressure of its blood plasma. The osmotic pressures of both plasma
(960 milliosmoles) and aqueous humor (935) are higher than that of the environmental sea
water. It has been suggested that water moves across the cornea from the sea. thereby
diluting the aqueous humor with respect to the plasma from which it is derived. If this were
so, differences in the osmotic pressure of the sea water bathing the cornea should be reflected
in the degree of dilution of aqueous humor. Smooth dogfish were subjected to sea waters of
varying tonicities for times ranging from one to six hours. When the osmolarity was lowered
as much as 100 milliosmoles below normal, neither aqueous humor nor plasma exhibited any
significant change. When the osmolarity was raised, the plasma and aqueous humor osmo-
larities increased in parallel, the difference between them remaining constant. Furthermore,
when the formation of aqueous humor was arrested by intravenous administration of Diamox
(acetazoleamide), the aqueous humor osmotic pressure increased, in the limit reaching that
of the plasma. These phenomena were interpreted as meaning that the difference in the
osmotic pressures of dogfish aqueous humor and its plasma is independent of any water
entering the eye across the cornea. A preliminary investigation of the enzymatic profile of
the dogfish ciliary body confirmed a high level of carbonic anhydrase, inhibition of which could
be effected by both in vivo and in vitro Diamox administration. The Qo= of dogfish ciliary
body is higher than that of its retina, when compared on a dry weight basis. In addition,
the two tissues respond differently to the presence of substrate (glucose) during respiration
studies, the ciliary body Qo2 being higher with glucose added (3.08) than the ciliary body
endogenous (1.86), whereas retina endogenous is higher (1.13) than when added glucose is
present (0.95).
Separation and isolation of Spisnla emJ'ryo cells. CORNELIUS F. STRITTMATTKR
AND PHILIPP STKITTMATTKR.
A technique has been developed for separation and isolation in mass of individual cell
types from embryos of Spisitln snlidissium, in order to permit studies on enzyme components
of various cell types during early embryonic development. The technique involves dissocia-
tion of embryo cells in calcium-free medium and subsequent isolation of cell types of different
sizes by density gradient centrifugation.
Spisnla embryos are harvested at a selected stage of development, washed twice with cold
0.52 M NaCl-0.02 M Tris buffer. pH 8, (NaCl) to remove calcium, then suspended in cold
0.07 M sucrose-0.02 .17 Tris buffer, pH 8 (SM). On prolonged standing in this medium, the
cells of the embryo dissociate slowly, but complete dissociation is rapidly achieved by adding
cit. 0.0005 M Versene and and gently agitating the suspension. The separated cells retain a
normal appearance and are capable of further cleavage.
The subsequent density gradient centrifugation may be illustrated by the isolation from
4-cell Spisula embryos of the large D cell and a mixture of the small A, B and C ceils. The
suspension of dissociated cells in sucrose medium is diluted \\ith NaL'l medium to give a
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 341
60 SM : 40 NaCl mixture. A 2-ml. aliquot of this mixture is layered over 10 ml. of 75 SM : 25
NaCl in a 15-ml. tube and centrifuged at ca. 70 g for three minutes. A 2-ml. fraction drawn
off from the gradient boundary contains an 80-90% pure mixture of A, B and C cells in
ro. 20% yield, while a 2-ml. fraction from near the bottom of the tube contains 90% D cells
in ca. 20% yield. The purity of each fraction may be increased by a second centrifugation.
The density gradient centrifugation is applicable to isolation of cell types from dissociated
embryos of other developmental stages by appropriate modifications of medium densities and
of the duration and force of centrifugation.
Electron transport in eggs, developing embryos and adult tissues of Spisula
solidissiina. CORNELIUS F. STRITTMATTER, PHILIPP STRITTMATTER AND
CAROLYN BURDICK.
Enzymes of electron transport in unfertilized eggs and in adult heart and liver of
Spisitla solidissima have been studied by spectrophotometric assay of homogenates and frac-
tions obtained by differential centrifugation. Unfertilized eggs showed a high activity of
DPNH-cytochrome r reductase (10-12 /uinoles electrons/min./ml. eggs), DPNH-ferricyanide
reduction and cytochrome oxidase (2-4 yumoles electrons/min./ml. eggs), appreciable activities
of succinic dehydrogenase (0.13-.20 ^moles electrons/min./ml. eggs) and TPNH-cytochrome
c reductase, and a lower DPNH oxidase activity. Adult tissues showed somewhat similar
but distinctive patterns. Unfertilized eggs, heart and liver each contained a participate frac-
tion resembling mammalian mitochondria in (1) centrifugal behavior, (2) absorption spectra,
which indicated the presence of cytochromes a (and (is), b and c. (3) oxidative activities,
including essentially the total cell content of cytochrome oxidase and antimycin A-sensitive
succinic dehydrogenase and a large portion of the DPNH-cytochrome r reductase activity, and
(4) staining reactions, being the major site of cyanide-sensitive Nadi and Janus green
reactions.
Tests on homogenates from fertilized eggs and embryonic stages up to 22-hour swimming
forms showed no marked changes from unfertilized eggs in cytochrome oxidase, DPNH-
cytochrome c reductase and DPNH-ferricyanide reduction. As these activities were in excess
of values reported in the literature for oxygen consumption of Spisnlci eggs and early embryos,
it appears that these terminal electron transport capacities are not limiting factors involved in
the reported increase of oxygen consumption rate during early stages of Spisnla development.
The ratio of succinic dehydrogenase to cytochrome oxidase activity was markedly higher
in adult heart than in unfertilized eggs or early embryos. This change may possibly reflect
a differentiation of mitochondria.
Cardiovascular and respiratory activity in dog fish. FREDERICK X. SUDAK AND
CHARLES G. WILBER.
Squalus acantliias and Mitstehts canis were pithed anteriorly to the mesencephalic region
and posteriorly from the 14th to 18th vertebrae. Blood pressures were measured with pressure
transducers. Respiratory activity was recorded directly from the branchial muscles. Electro-
cardiograms were recorded using bipolar electrodes. Deep body temperatures were maintained
at 23.0° C. ± 0.5° C. Average heart rates at this temperature were 46.8 beats/minute in
Miistclns, 28.5 beats/minute in Squalus. The heart rate Q,n (23° C. to 33° C.) in Mustclns
was 3. EKG's in Mnsteliis showed a P-R interval of 0.25 sec., QRS of 0.07 sec. and QT
interval of 0.58 sec. The ST segment was isoelectric and the T wave biphasic. Cardiac
rhythmicity bore a phasic relationship to respiratory activity in both species. RR intervals
were constant when the ratio between respiration rate and heart rate was 1 in Mustelus and
2 in Squalus. Arrhythmias of various degrees were observed whenever the two cycles fell out
of phase. Respiratory activity occurring during the middle third of a given cardiac cycle
consistently prolonged the diastolic period of that cycle. Respiration rate in Mustelus was
48.7/'minute, in Sijnolus 69.0/minute.
The average mean perfusion pressure from the ventral aorta to the venous side of the
circulation was 22.4 mm. Hg. End diastolic intraventricular pressure varied from minus 0.7
to 0 mm. Hg with the pericardium intact. Average blood pressures in the ventral aorta were :
342 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
systolic 29.5 mm. Hg, diastolic 21.2 mm. Hg and pulse pressure 8.2 mm. Hg. The mean
blood pressure showed periodic variations, resembling Meyer waves described in mammals.
The periodicity was 2.8 cycles/minute in Mustelus and 0.8 cycles/minute in Sqnalns. The
magnitude of the pressure change was 1.4 mm. Hg.
Supported in part by NIH grants H-3563 and MY-3235.
Cardiovascular responses to hemorrhage in the dog fish. FREDERICK N. SUDAK
AND CHARLES G. WILBER.
Cardiovascular and respiratory responses to acute hemorrhage were studied in Mustelus
canis and Squalus acanthias. All animals were pithed anterior to the mesencephalon and
posteriorly from the 14th to 16th vertebrae. Blood pressures were recorded directly from the
ventral aorta. Electrograms obtained from the branchial muscles were used to record respira-
tory activity. Blood pressures and respiration were recorded before, during and after the
removal of blood from the caudal vein, and following reinfusions of blood into the ventral aorta.
Systolic and diastolic pressures decreased linearly with the increase in blood deficit.
Systolic blood pressure decreased an average of 1.8 mm. Hg/nil. of blood lost, while
the diastolic pressure decreased an average of 1.0 mm. Hg/ml. of blood lost. A similar
pressure-volume relationship was obtained when blood was reinfused. Mean blood pressures
were reduced approximately 50% after removal of 5-7 ml. of blood (10% of estimated total
blood volume) from a 700-gram fish. Changes in heart rate and respiration rate were in-
consistent. In general, the cardiovascular system in both species behaved like a simple
pressure-volume system in response to blood loss. No evidence was found to indicate the
presence of a compensatory mechanism against decreases in blood pressure resulting from
reduction of blood volume.
Supported in part by NIH grants H-3563 and MY-3235.
Uptake arid release of calcimn-45 by Fiicus vcsiciilosus. ELIJAH SWIFT AND W.
ROWLAND TAYLOR.
Five to seven grams (dry weight) of whole young thalli of Fucus vcsiculosus, without
macroscopic epiphytes, were incubated in sea water in 260-ml. bottles maintained at 22° C.
and under constant illumination (700 to 1300 foot candles, depending on the experiment). The
bottles contained about 1 nC. of Ca15 CL. The uptake or release of isotope was followed by
removing aliquots of sea water, plating in the usual manner, and assaying in a gas flow counter.
The algae appeared to take up radiocalcium rapidly, most of the uptake occurring in the
first hour. The isotope did not appear to be concentrated since calculations from Vinagradov's
tables indicate the amount of isotope taken up was of the order of magnitude that would be
expected if the radiocalcium in the sea water came into equilibrium with the calcium in the
algae.
Fucus vcsiculosus maintained in sea water containing calcium-45 was rinsed, and placed
in millipore-filtered sea water to which no isotope had been added. In 24-hour experiments,
90% of the radiocalcium appearing in the medium \vas released in the first two hours. Similar
studies were done with thalli that had been used in uptake experiments. If it is assumed that
the radiocalcium in the thalli came into equilibrium with the medium in both the uptake and
release experiments, the calculated and observed values check within 10%. These techniques
did not show a significant difference between Fucus maintained in the dark or illuminated, in
either the uptake or release experiments. These data indicate there is a rapid turnover of
calcium by Fucus vesiculosus. (Partly supported by contracts between the Chesapeake Bay
Institute, The Johns Hopkins University and the A.E.C. and O.N.R.)
The effect of photosynthesis by bentliic inocroalgae on the titration alkalinity of
sea wafer. W. ROWLAND TAYLOR.
Six species of benthic macroalgae have been examined to determine the effect of their
photosynthetic and respiratory activity on the alkalinity and pH of the sea water medium.
The algae, 0.5 to 1 gram wet weight depending on species, were incubated in series of 125-ml.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 343
light and dark bottles at constant temperature (21° C.) and illumination (1800 foot candles).
Changes in the titration alkalinity and pH were noted and rates of photosynthesis and respira-
tion were followed by oxygen measurements.
When Uh'a lactuca or Entcromorpha intestinalis was incubated in this manner, the pH of
the medium increased from 8.0 to 9.0 in about six hours but the alkalinity did not change.
On continued incubation, the pH rose to 10.0 and the titration alkalinity decreased from initial
values of 2.20 to 1.50 milliequivalents/liter. Oxygen determinations indicated continued
photosynthesis at these high pH levels. Photosynthesis by Ceramium rubrum, Chondrus crispus,
Chorda filum and Fitcns vesiculosus resulted in an increase in pH to maximum values of
9.0-9.2. With one exception, there was no change in the alkalinity. In the case of the Fucus
there was a small decrease in the alkalinity during the latter part of extended runs. Respiration
by all of these algae resulted in a decrease in pH but no change in alkalinity. Preliminary
experiments, using sea water in which the partial pressure of CO2 had been lowered, indicated
the rate of oxygen production by the Fucus and Entcromorpha species may be dependent on
the concentration of dissolved CO;..
These findings suggest that these algae probably use dissolved CO2 as their carbon
source under their usual environmental conditions. However, the two Chlorophyta can use
bicarbonate (or carbonate) when growing at high pH. (Partially supported by contracts
between The Johns Hopkins University and the A.E.C. and O.N.R.)
Uptake of iron-59 by marine benthic algae. W. ROLAND TAYLOR AND EUGENE
P. ODUM.
Exploratory experiments have been undertaken to investigate the uptake of iron-59 by
marine benthic algae. A typical experiment consisted of placing a weighed amount of an alga
in each of a series of stoppered bottles or flasks filled with sea water. Suitable amounts of
Fe59Cl~ were added to the containers ; they were maintained at constant temperature and either
illuminated or darkened as required. The uptake of the isotope was followed by removing
aliquots of the water and counting with a scintillation detector. In some experiments, pH
and oxygen production were also measured. Such studies were complicated by adsorption
of the isotope on the walls of the container, and it was necessary to apply corrections obtained
from blanks or add chelating agents to the medium.
The isotope was taken up by Ceramium rubrum and Enteromorpha intestinalis rapidly
and there was little loss of the isotope when algae were removed from the radioactive medium
and placed in fresh sea water. A rough estimate of the concentration factor would be about
100 to 300. Addition of 30 mg./ liter of the chelating agent ethylenediaminetetraacetic acid
was found to prevent the loss to the surface of the container. In experiments lasting three
to four hours, both the amount and rate of uptake was less in the presence of EDTA. There
appeared to be no appreciable difference of uptake in light or dark bottles. In the case of
Ceramium, addition of buffer ("Tris") to minimize the pH changes resulting from photo-
synthesis indicated pH does not effect the uptake.
In an experiment using Fucus vcsiculosus it was found that the radioactivity in the
medium decreased at a rate less than that in a blank containing no algae. It is speculated
that the Fucus may release a chelating substance into the medium. (Partially supported by
contracts with the A.E.C. and O.N.R.)
Nuclear division in Holosticha lacazei Maupas. REUBEN TORCH.
Holosticha lacazei, a relatively obscure, multinucleate, free-living, hypotrichous, marine
ciliate, was collected on June 23 at Woods Hole and has been cultured in the laboratory for
two months.
Nuclear division was studied in animals fixed in Champy's fluid, followed by bleaching in
either 3% hydrogen peroxide or dioxan, and stained by the Feulgen reaction or with Heiden-
hain's hematoxylin. Champy's fixative was used since the animals dissolved in all fixatives
except those containing osmium tetroxicle.
The number of macronuclei in non-dividing individuals varies from 32 to 71, with most
individuals having 40-50. The spherical macronuclei measure 4-8 /j. and contain a central
344 TAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
chromatin mass surrounded by numerous small, achromatic spheres. The homogeneous Feulgen-
positive spherical micronuclei measure 2 /j, and number from 5-13 in non-dividing individuals.
There is no apparent correlation between the numbers of macronuclei and micronuclei.
Prior to nuclear division, the macronuclei fuse into an elongate, intensely Feulgen-positive
mass in the center of the cell along the longitudinal axis. Concurrently, the daughter adoral
membranellae are formed and the future division plane is demarcated. The micronuclei cluster
around the fused macronucleus and proceed to divide. Micronuclear division is not synchro-
nized, since no more than half the micronuclei divide at one time. Division of the micronuclei
was not observed at any other stage in the life cycle. The micronuclei are distributed more
or less equally among the daughters, but in one exceptional case, one daughter contained 6
micronuclei while its sister contained 15.
The macronuclear mass divides approximately in half, one product going to each daughter.
Macronuclear divisions continue until each daughter contains 20-30 macronuclei, whereupon
the daughters separate. The earlier macronuclear divisions are generally synchronized, but
later divisions are sporadic. After separation of the daughters, many of the macronuclei
continue to divide. Macronuclear reorganization was not observed at any stage in the life cycle.
Ammonia in Golfingia gouldii and ambient air. DAVID M. TRAVIS.
This work was begun with determination of normal levels of ammonia in ambient air
and in coelomic fluid of Goldfingia c/oitldii, in order to devise approaches to a study of effects
of increased environmental ammonia gas concentration on metabolic processes. For analysis
of air, a large volume, greater than 400 liters, was passed through a cotton filter and two acid
traps in series, in order to collect the ammonia. Coelomic fluid was analyzed directly and by
use of a trichloracetic acid filtrate prepared immediately after withdrawal from the body
cavity. Ammonia was determined by microdiffusion and colorimetric measurement of the
reaction with ninhydrin-hydrindantin reagent.
Results were as follows: (1) The ammonia fraction in ambient air was 1.74 X 10~8 (the
mean of 14 determinations ranging from 0.55 to 3.47 X 10~"). (2) Total ammonia of coelomic
fluid, using trichloracetic acid filtrate, was 230 micromoles per liter (the mean of 18 determina-
tions, ranging from 115 to 560 micromoles per liter). (3) Direct determinations of ammonia
on coelomic fluid at timed intervals after fluid was drawn from the animal showed a rising
curve, the flat portion of which was used for extrapolation back to zero time. Values obtained
by this extrapolation were about 25% lower than those obtained on the same specimen using
trichloracetic acid filtrates.
The results of this work have led to the following approaches to further study of ammonia
in physiologic processes: (1) large volumes of ammonia gas may be prepared, analyzed, and
used in experimental situations; (2) serial samples of 1 ml. of coelomic fluid may be deter-
mined for ammonia in triplicate: (3) release of ammonia into the closed space, containing the
animal and suitable reagents, of a respiratory flask may allow precise measurement of
metabolic processes.
Respiratory changes induced in Golfingia gouldii by alteration in environmental
carbon dio.vidc. DAVID M. TRAVIS.
Respiration of Golfingia (imtldii at environmental carbon dioxide concentrations below and
above approximately normal levels was studied in order to learn whether this gas exerts
effects on the rate of oxygen consumption in the whole animal. For essentially zero carbon
dioxide in surrounding air, a volumetric respirometer was used in which carbon dioxide was
removed by alkali. For normally small and larger concentrations of environmental carbon
dioxide, a syringe method was adopted which allowed measurement of changes in oxygen
concentration of a known volume of gas from which oxygen consumption could be calculated.
Actual oxygen concentrations, kept between 19.7 and 20%, changed less than 0.5% during
periods of one hour.
At zero carbon dioxide tension, oxygen was consumed by the animal at a rate of 33
microliters/hour and gram of wet weight to the 2/3 power (Ws/3) (mean value for 13 observa-
tions on 4 worms, ranging from 20-53). At low concentrations of 0.36-0.99% carbon dioxide.
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 345
obtained by allowing equilibration for one hour with room air, the rate was 45% lower, or
15 microliters/W-/3-hour (mean of 9 observations on 6 worms, range 9-19). In simultaneous
experiments using pairs of worms crossed alternately between low concentrations of 0.49,
0.91 and 0.48% carbon dioxide and higher concentrations, the oxygen consumption was reduced
by 27, 35 and 70% when the concentration was raised by 1.0, 3.5 and 8.0% above the lower
levels. Oxygen concentrations were approximately the same at the two levels.
From these results it is suggested that carbon dioxide depresses aerobic respiration in
Golfingia ciouldii in approximately physiologic as well as higher pharmacologic concentrations,
and, indeed, appears to accelerate respiration when removed from the environment. Studies
of mechanisms involved and of the actual role carbon dioxide may play in natural surroundings
are indicated.
Studies of cannibal giant Blepharisma undulans. N. TULCHIX AND H. I. HIRSH-
FIELD.
Previous studies have shown that selected feeding will induce formation of cannibal giant
types of the pink, heterotrichous, fresh-water ciliate, Blepharisma. The ingestion of smaller
blepharisma by the giant forms occurs readily. The fate of the ingested forms can be easily
followed in the food vacuole. The digestion of the ingested forms and egestion of non-absorbed
substances occurs within twelve hours.
Feulgen and methyl green-pyronin preparations show the DNA and RNA of the cannibal
and of the ingested forms within the food vacuole. During digestion, the DNA in the food
vacuole is progressively hydrolyzed, as shown by decreasing affinity for methyl green and
Feulgen stains. However, pyroninophilia of the mass within the vacuole fluctuates during
digestion. RNA-ase and perchloric acid treatments indicate that RNA may persist or accumu-
late in the vacuole during digestion and that some is present in the egested residues.
The cannibal giant forms have appreciably larger amounts of macronuclear DNA than
bacteria-fed controls. The large quantities of DNA in the food vacuoles of the cannibals may
thus contribute to the increased polyploidy of the cannibal giant forms.
Supported by grants from the National Science Foundation and from the Damon Runyon
Memorial Fund for Cancer Research.
Differential staining of cytoplasniic inclusions in eggs of Pectinaria. KEXYOX S.
TWEEDELL.
Constituents of the egg cytoplasm were studied in the germinal vesicle stage, during
germinal vesicle breakdown and in the primary oocyte. Inclusions of living eggs were observed
before and after centrifugation. using vital fluorochromes. other specific vital dyes and dark-
field microscopy. In addition, cytochemical studies were made on fixed eggs before and after
centrifugation.
Besides germinal vesicle, nucleolus, chromosomes and spindle, many highly motile inclusions
were observed vitally. The latter included yolk, mitochondria, astral granules, germinal vesicle
substance, oil droplets and chromidia. Additional stationary, peripherally located granules
were seen. By specific cytochemical staining reactions (vital and non-vital) these inclusions
could be further differentiated.
Of the vital fluorochromes utilized, acriflavine, thioflavine and acridine orange gave poly- »
chromatic staining. Others dyed specific particulates. The fluorescent cell components include
cell and nuclear membranes, nucleus, nucleolus, yolk, mitochondria and other unidentified
granules.
Two of the vital dyes, toluidine blue and nile blue sulphate, stain several cell components
in the germinal vesicle stage. These include nucleolus, peripheral cell granules, yolk and a
heavy zone of granules surrounding the germinal vesicle. Both dyes stain the yolk particles
blue and show metachromasia with other granules. After centrifugation the metachromatic
granules become stratified into separate bands. With toluidine blue, a thin red-violet band
of particles occurs just beneath a large cap of unstained granules and droplets at the centripetal
pole. When nile blue sulphate is applied, a different red-violet band of small particles stratifies
out, just centripetal to the yolk. The astral granules, which become associated with the asters of
the first maturation spindle, are derived from this latter band.
346 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
Reduction and delay of cleavage in Chaetopterus eggs by acridine orange. K. S.
TWEEDELL AND C. D. WATTERS.
Recently, acridine orange was shown to have an inhibitory action on mitosis in Arbacia
(Rustad, 1959). Additional effects on fertilization and early cleavages were observed when
acridine orange was utilized as a vital fluorochrome to follow early development in Chaetopterus.
The staining period, concentrations of A.O. in sea water, choice of gametes and time of
application all affect the number of eggs cleaved.
Acridine orange was applied in concentrations of 10~3 (15 minutes), 10~4 (30 minutes) and
10~5 (60 minutes) in each experiment. When sperm were stained prior to fertilization, the
percentage of eggs reaching first cleavage increased (72 to 81%) as the stain intensity de-
creased. Previously stained eggs in the same concentrations showed an increase in cleavage
from 50 to 60% as stain concentration decreased. When both sperm and eggs were dyed prior
to fertilization, the percentage of eggs cleaved did not vary significantly from eggs stained
alone. Average cleavages in controls for all groups remained constant between 80 and 82%.
This suggests that the inhibitory effect on cleavage was principally mediated through the egg.
When eggs and sperm were fertilized in 10"3 A.O. and removed after 15 minutes, cleavages
were sharply (10%) curtailed. In 10'* A.O., 27% of the eggs cleaved but in 10~B A.O. cleavage
recovered (84%). This suggests the factors here either act during fertilization or affect some
phase of the cleavage process.
Application of the threshold concentration (10~5) to the egg alone also caused a slight
delay (3 minutes) in the first cleavage. When sperm or sperm and eggs are dyed prior to
fertilization, an identical delay of 10 minutes occurs. This suggests a sperm-induced cleavage
delay. In each case, the rate of development remained constant with the controls. Eggs and
sperm fertilized in the stain (then removed) showed no delay in first cleavage, but the rate of
succeeding cleavages decreased.
Phosphagen and nnclcotides of Limulus muscle. G. W. DE VILLAFRANCA, J. E.
ROSS AND A. M. MoRGANr
Horseshoe crab muscle was extracted with trichloracetic acid and precipitated as the
alcohol-insoluble barium salt. It was hydrolyzed and the free base chromatographed with
arginine, taurocyamine, glycocyamine and creatine standards. The average Rf values in 3
solvents (propanol-ammonia-water, butanol-acetic acid-water, and butanol-ethanol-water) were:
arginine, 0.43, 0.14 and 0.07, respectively, and closest to the values found for the Limulus base
of 0.43, 0.13 and 0.07. The amount of phosphagen, arginine phosphate, was determined by
colorimetric assay of the total arginine and the free arginine, the difference being due to
bound arginine and assumed to be arginine phosphate. The average values for 6 different
animals were 6.26 mM of arginine and 3.10 mM of bound arginine per 100 grams of muscle,
wet weight. It is of some interest that the values were always greater than zero, and relatively
constant, once it was realized that the crabs must be used fresh. The amount of AP probably
decreases once the animal has been removed from its natural habitat ; it apparently decreases
rapidly and to zero in the running sea water tables.
The nucleotide composition was determined by ion-exchange chromatography (Dowex in
the formate cycle) on both barium salts obtained by TCA extraction and, later, on neutralized
perchloric acid extracts. In the former case, ATP was the major component with an average
of about 100 /tiM per 100 grams of muscle, fresh weight. ATP was recovered from perchloric
acid extracts in concentrations ranging from 163 nM to 338 /u.M, with an average of 265 nM
per 100 grams of muscle. Traces of ADP (43 ^M), AMP (26 (J.M) and other, as yet, uniden-
tified, minor nucleotides were also found.
Work supported by USPHS grant A-2647 and an ONR grant administered by the M.B.L.
Oxygen consumption in Uca pugnax: seasonal variations. H. MARGUERITE WEBB.
Oo-consumption of the fiddler crab, Uca pugna.r. was recorded continuously from July 25,
1959, through October 6 and for six semi-monthly periods between October 11, 1959, and
February 18, 1960; a further lunar period was recorded from June 25 to July 23, 1960. The
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 347
mean hourly rate of O,-consumption \vas found to be highest during the months of July and
August, and to be lowest (about 55% of summer values) during the period from November
to February. No gross changes in the form of the diurnal rhythm were observed during the
period of the investigation nor were there any consistent variations in the amplitude, which
ranged from 28% to 43% of the mean hourly rate. When the data were analyzed for lunar-day
rhythms it was seen that from July 25 through October 25 the rhythm exhibited a symmetrically
bimodal form with maxima at lunar zenith and nadir. During the period from November 19
through January 23, although the lunar-day rhythm was less regular than for the earlier periods,
it was clearly uni-modal with a maximum about an hour after lunar zenith. During the period
from February 2 to 18 the lunar rhythm resumed its bimodal character, but with minima at
lunar zenith and nadir. It thus appeared to be inverted with respect to the "summer" rhythm.
Like the diurnal rhythm, the lunar rhythm exhibited no systematic changes in percentage
amplitude throughout the period studied. During the period from June 25 to July 23, 1960,
the lunar rhythm was found to have the "summer" form and phase relations — bimodal, with
maxima at lunar zenith and nadir.
This study was aided by a contract between the Office of Naval Research, Department
of Navy, and Northwestern University, NONR-122803.
Effects of environmental changes on indefinitely prolonged action potentials of
lobster muscle fibers. R. WERMAN, J. P. REUBEN AND H. GRUNDFEST.
Prolonged spikes evoked in muscle fibers treated with a combination of procaine and
some other agent (Reuben and Grundfest, this issue) were affected very little by drastic
changes in the environment, made during the plateau of the response. The stability of the
membrane potential and conductance during the plateau were particularly marked with the
responses that had a high membrane conductance (Reuben, Werman and Grundfest, this issue).
These spikes were not affected by replacement of all the ions in the bathing medium with
sucrose or urea ; by replacement of sodium with alkali earth or organic cations ; by 20-fold
increase in potassium ; by substitution of chloride with various anions ; or by maximal activation
of the inhibitory synapses with 7-aminobutyric acid (GABA).
The spikes with lower-conductance plateaus could be affected to some degree. Bathing
the fibers in sucrose or urea induced oscillations, between the plateau and more negative values.
Substitution of SCN ions for chloride, which hyperpolarized and increased the membrane
resistance of resting muscle fibers, abolished the spikes. Occasionally, the addition of GABA
also abolished the spikes.
These findings suggest that in its "upper stable state" the membrane does not behave as a
simple electrode for ionic changes. This is particularly the case for conditions in which the
"upper stable state" is associated with high membrane conductance.
Relative effectiveness of inhibitory membrane in different fibers of a lobster muscle.
R. WERMAN, J. P. REUBEN AND H. GRUNDFEST.
During its maximum activity, the inhibitory membrane of the fibers operates as a high
conductance Cl~-electrode, tending to set the membrane at the Cl'-equilibrium potential. The
degree to which the synaptic activity prevents changes in the membrane potential that are
normally produced by various means is a measure of the relative area of the synaptic membrane
in different fibers.
The inhibitory synapses were maximally activated with GABA, in Ringer's solutions con-
taining various amounts of K", or none. In the absence of GABA, the membrane potential
changed nearly linearly with log K+, with a slope of 54 mV/decade change in K+. In zero 1C1"
there was hyperpolarization of 10 to 15 mV. When the synaptic membrane was activated by
GABA, the membrane potential changed relatively little on removal of K+ in most fibers and
not at all in many. On raising K+ above 15 meq./l. (the value in Homants saline) the slope
of the change was again 54 mV/decade change in K+, but with a different origin. Removal
of K* during tetanic activation of the inhibitory synapses also produced less hyperpolarization,
but abolition of all hyperpolarization, as with GABA, was not seen.
The variation of the clamping action of inhibitory synaptic activity indicates that the con-
348 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
ductancc of the synaptic membrane was less in some fibers than in others, presumably because
different fibers have different areas of innervated membrane. Block of propagation is readily
produced during high frequency activity of the inhibitory axon, and accounts for the lower
effectiveness of synaptic clamping as compared with that of GABA. The identical slopes of
the depolarization curves in high K+, with and without GABA, reflect the increased conductance
of depolarized membrane, and they provide further evidence that the activity of the inhibitory
membrane does not involve K+-conductance.
In situ measurements of the community metabolism of Quohog Pond. GEORGE C.
WHITELY, JR.
In situ estimates of oxygen production in Quohog Pond, West Falmouth. were obtained
by means of a polarographic oxygen electrode during September, 1959. In July, 1960, the
water column at one station was analyzed by Winkler technique for dissolved oxygen every
three hours for a duration of 24 hours. Rates of respiration, diffusion, and total photosynthesis
were computed graphically for both the September and July data.
Total photosynthesis in a water column one square meter in area was 7 grams
oxygen/nr/day or 2.9 grams carbon/nr/day for September, 1959 ; 24 grams oxygen/nr/day
or 10 grams carbon/nr/day for July, 1960.
During June, 1960, the Marine Ecology Course class study of Quohog Pond determined
primary production of the phytoplankton in vitro (light and dark bottle method) to be 0.5
gram carbon : benthic algae by the same method averaged 10 grams carbon. Low value for
the phytoplankton suggests that bottle measurements alone may have some of the same dis-
advantages in Quohog Pond as have been found elsewhere; i.e., shallow Texas bays, where
such measurements do not take into account the metabolism of benthic organisms.
Production of viable races of Paramecium caiidatum after micronuclear elimination
with x-rays. RALPH WICHTERMAN.
X-irradiation has been shown to affect micronuclear number in several species of ciliate
Protozoa. As a result, there is a diminution of the number of micronuclei and. with reasonably
high or repeated dosages, their complete disappearance.
Specimens from clonal cultures of opposite mating types of Paramecium caudatitm (9 and
10 of variety 5) were treated as follows: 200 counted paramecia were placed in each of four
Nylon syringes (2-ml.) and irradiated simultaneously with dosages of 150, 200. or 250 kr, all
of which were below the LD 50 value. Immediately after irradiation, some specimens were
examined and the remainder expressed from the syringes into flasks of lettuce medium which
contained Aerobacter aerogenes as the food source. Upon regaining reproductive ability,
progeny of x-rayed survivors were then harvested, placed again in syringes and irradiated as
before, while others were killed and stained for further cytological study. In this manner seven,
approximately evenly spaced irradiation exposures were given over a 52-day period, to bring
the cumulative clonal dosage to 1100 kr.
When specimens of opposite mating type from cultures which had received the cumulative
clonal dosage of 1100 kr regained reproductive ability and later were mixed, mating and
conjugation followed.
Paramecium caudatum has but one compact micronucleus, measuring approximately 8 /*.
Repeated and successive x-irradiation of paramecia and their progeny in the manner reported
here results in a gradual disappearance of the micronucleus or the production of a "ghost-like"
condition of this structure as revealed in stained preparations, whereas the micronucleus from
unirradiated controls stains intensely. At the highest cumulative clonal dosage, dividing speci-
mens show either no micronuclear spindle (since such paramecia are now amicronucleate) ,
or else a dividing "ghost-like" micronuclear spindle in contrast to the conspicuous, dividing
micronucleus of the unirradiated controls. At least in appearance, the structure of the macro-
nucleus appears little affected by the irradiation.
Part of a project aided by a grant from the Committee on Research, Temple University,
and a contract between the Office of Naval Research, Department of the Navy and Temple
Universitv CNR 104-475).
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 349
The effect of LSD 25 on heart rate in Cistenides. CHARLES G. WILBER.
In previous work, it was shown that the pulsation of the dorsal blood vessel in the trumpet
worm is temperature-dependent. The rate response to temperature may be described as
V = 3.5 .r — 49, where v is beats per minute and .r is degrees Centigrade. Approximately 50
worms were immersed in different concentrations of lysergic acid diethylamide (LSD 25) in
sea water at 22° C. The effect of this treatment on pulsation of the dorsal vessel was recorded.
By 30 minutes after immersion in 20 micrograms LSD 25 per ml. sea water, the rate of pulsa-
tion of the dorsal vessel was increased to 130% of control. In more concentrated solutions,
the worms became flaccid, extended, and the dorsal vessel ceased to beat. In sea water con-
taining 10 micrograms per ml. LSD 25, there was no change in rate, or possibly a depression
to 10% of control. By way of comparison, worms immersed for 30 minutes in 10 micrograms
per ml. atropine sulfate in sea water showed a decrease to 59% of control rate. In 20 micro-
grams per ml. the rate increased to 120% of control. After two hours in the above solutions,
the rates were essentially the same as those indicated above. The LSD 25 \vas kindly supplied
by the Sandoz Pharmaceutical Company.
Supported in part by National Science Foundation Grant 4005.
Cardiovascular and respiratory refle.ves in elasniobranclis. CHARLES G. WILBER
AND FREDERICK X. SUDAK.
During a simulated dive, certain mammals, birds and reptiles show a profound bradycardia
with accompanying changes in blood pressure. Fish are said to show similar changes if
removed from the water. To ascertain in some detail the nature of such responses in elasmo-
branchs, we used specimens of Squalus acanthias and of Mustelus canis. The unanesthetized
fish wrere immobilized in a plastic trough. Fresh sea water was run into the mouth through a
plastic hose. Blood pressure was recorded directly from the ventral aorta with a pressure
transducer. Heart rate was read from the pressure record. Respiration was recorded through
electrodes placed directly into the branchial muscles. Control records were made with
full water flow. The water was then shut off ; heart rate, blood pressure and respiratory rate
were monitored for several minutes. Within 6 to 20 seconds after water flow was stopped,
the following physiological responses (expressed as change to per cent of control) occurred:
Squalus heart rate 45%, respiratory rate 51%, pulse pressure 144%, mean pressure 82%;
Mustelus heart rate 42%, respiratory rate 19%, pulse pressure 214%, mean pressure 90%.
The heart rate response in both species was abolished by atropinization of the fish. Atropine
abolished the pulse pressure response in Squalus but not in Mustelus. The observed brady-
cardia is apparently mediated through the vagus nerve. Atropinization did not modify the
respiratory response in either species. The small changes in mean blood pressure, despite
relatively large decreases in heart rate, after interruption of water flow, do not confirm the
view that this response serves to protect delicate gill structures.
Supported in part by research grants MY-3235 and H-3563 from the National Institutes
of Health.
Some effects of lysergic acid diethylamide on circulation in elasuwbranchs.
CHARLES G. WILBER AND FREDERICK X. SUDAK.
Lysergic acid diethylamide (LSD 25), a psychotogenic agent in man, is known to provoke
bradycardia in mammals by central vagal stimulation. The agent induces a drop in blood
pressure as a result of depression of the vasomotor center, despite the vasoconstrictive action
which the drug exhibits in perfused blood vessels or in spinal animals. We made some
experiments with LSD 25 on Mustelus couis. Blood pressure and heart rate were measured
before and at different times after various doses of LSD 25 were administered via the ventral
aorta. Doses from 0.01 to 1.3 micromol per kg. body weight were given. After all doses there
was a rise in systolic, diastolic, mean, and pulse pressures. Heart rate, which decreased by 40%
at a dose of 1.3 micromol per kg., showed no decrease at doses less than 0.01 micromol. The
rise in mean pressure amounted to 15% of control value at a dose of 0.01 micromol; 45% at 0.06
micromol; 81% at 0.56 micromol. Various methods of plotting dose against blood pressure
350 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
response were tried. At the moment, the data seem to fall along a curve in the most orderly
fashion if dose in micromol per kg. is plotted against the reciprocal of the per cent change in
blood pressure. The results are now being processed through a computer to arrive at the best
mathematical expression of the dose-response curve. The LSD 25 was kindly supplied by Sandoz
Pharmaceutical Company.
Supported in part by research grants MY-3235 and H-3563 from the National Institutes
of Health.
An autoradiogrdphic study of the regenerating limb in the adult newt, Triturus
viridcscens. RUTH T. WILF.
The source of cells for the blastema of the regenerating limb of the adult newt, Triturus
viridcscens, has been investigated, using autoradiography. In this two-year study the radio-
isotopes used were tritiated thymidine (principally), and C" isotopes of glycine, leucine, and
methionine. With tritiated thymidine, we assume takeup of radioisotope only in DNA-
synthesizing, premitotic cells. In limbs with high epidermal cell labeling compared to control
non-regenerating limbs, or with positive internal cells, regenerative activity of these cells is
assumed.
With tritiated thymidine the approaches were : (1) time course regeneration study from
third to twenty-eighth day post-amputation; injection and fixation on same day to give im-
mediate picture of new cell formation; (2) newts injected from third to twenty-first day;
fixation on subsequent days to study cellular divisions and migrations ; (3) following the
discovery that prior to the fifth day, only epidermal cells were labeled, tritiated thymidine was
injected four hours pre-amputation or three days post-amputation, to test a possible epidermal
cell contribution to blastema.
It was found that internal mesodermal components contribute to the blastema. From the
fifth day many dedifferentiating muscle nuclei, muscle sheath nuclei, and some subcutaneous
nuclei are synthesizing DNA. The periosteum is strikingly labeled. Schwann cells are
generally negative. These conditions persist until after the blastema is well formed. The
labeled dedifferentiated mesodermal cells divide and contribute extensively to the blastema. The
blastema itself has a high rate of synthetic activity, which falls off as the blastema lengthens.
The epidermis is usually particularly heavily labeled in a band at the level of the amputa-
tion. These cells migrate distally continuously until about the eighteenth day, to form the
bulk of the apical cap of epidermis, although some DNA synthesis is almost always taking
place across the top of the cap. There is no evidence that epidermal cells contribute to the
blastema.
Heat tolerance and thermoregulation in the fiddler crab, Uca pugilaior. JERREL
L. WILKENS.
Fiddler crabs often feed in their natural habitat during the hottest part of a day in direct
sunlight. The following experiments were undertaken to determine the upper extremes of
temperature fiddler crabs can tolerate, and, under these conditions, if the effects of evaporation
and radiation would be of any survival value. To determine environmental heat tolerances,
freshly collected animals were subjected to temperature increases for one-hour periods over
sea water and in dry air. In saturated air, 50% of the population died at 40.5° C. and 100%
at 42° C. In dry air, 50% died at 45.5° C. and 100% at 47° C. The crabs, therefore, have a
thermal tolerance of as much as 10 to 15° above average daily temperature (about 30° C.)
for the Woods Hole area. However, temperatures approaching 45° C. were occasionally
recorded on the surface of the sand; therefore, some thermoregulatory mechanism would be
an advantage.
Through the use of thermocouples, internal temperatures of light-adapted and of dark-
adapted specimens were determined during exposure to infra-red radiation in still air. The
body temperatures approached equilibrium about 6° C. below ambient air temperatures in light
crabs and 3° C. below air temperatures in dark crabs, for periods up to 15 minutes. These
plateaus are due primarily to radiation and evaporation. To determine the effects of evapora-
tion alone, crabs were subjected to slowly moving air at 37.5° C. Body temperatures leveled
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 351
off at 33.5° C. in dry air, at 35.5° C. in air at 50% relative humidity, and at 37.5° C. in
saturated air. The rate of evaporation of water was essentially proportional to increasing
temperature.
This work was undertaken as part of the student training program of the Marine Ecology
Course, and was partially supported by the Graduate School of Tulane University.
Experiments with ovulation induced in vitro by means of steroids in frogs and
marine fishes. PAUL A. WRIGHT.
Certain androgenic, progestational, and cortical steroids have potent ovulatory capacity
on isolated lobes of frog ovary, Rana pipiens, when added in amounts ranging from 2 to 0.001
mg. in 50 ml. of Ringer's solution and in the absence of pituitary factors. Several compounds
(e.g., progesterone, corticosterone. cortisone-21-alcohol) induced responses equaling or exceed-
ing those brought about by optimal concentrations of frog pituitary extract. These same
classes of steroids in moderate dosage enhanced ovarian response to pituitary factors in vitro.
but overdoses often inhibited ovulatory responses. Estrone, estradiol, and diethylstilbestrol were
incapable of inducing ovulation by themselves, and inhibited pituitary-induced ovulation,
diethylstilbestrol > estradiol > estrone. Interestingly, these same estrogens did not inhibit
ovulation in vitro induced by progesterone, testosterone, or hydrocortisone, but instead aug-
mented the ovarian response to these ovulatory steroids.
Attempts to induce ovulation in excised ovaries of marine fishes (Lophius, Fundnlus,
Tauto(ia') by means of either steroids or pituitary extracts were not successful. A few eggs
were released from a ripe ovary of Fundiilus inajalis with both progesterone and Fundulus
pituitary extract, but controls in Ringer's solution also released ova spontaneously.
Aided by research grant A-2986 from the National Institutes of Health, USPHS. Thanks
are due to Dr. Preston L. Perlman of Schering Corporation for supplying a variety of steroids
for these experiments.
Metabolism of three species of the gastropod genus Littorina in and out of water.
ROGER G. ZIEG.
Results of the experiments conducted on three intertidal snails of the Woods Hole region
demonstrated that the respiration rate of these animals varied significantly when in water and
when exposed. The three forms considered are dispersed, in more or less definite zones, on
the shore. Littorina palliata is primarily in an aquatic habitat among the sea-weeds, Littorina
rudis is confined to the upper regions of the shore exposed during low tide. Littorina littorea
appears to be intermediate, being neither restricted to the exposed nor the submerged zones.
Rate of oxygen consumption was determined by Winkler method for measurements "in
water," and a manometric method for measurements "out of water." Snails were collected
at random and respiration measurements obtained for periods not exceeding three hours.
Results obtained by these methods, from about 500 snails of each species, indicate that :
L. palliata gave mean values of 1.60 ml. O2/gm./hr. in air and 1.01 ml. O2/gm./hr. in water
(P < .001). Results indicate that L. littorea consumes .54 ml. On/gm./hr. in air and .34 ml.
Oo/gm./hr. in water (P<.001). On the other hand, L. rudis has lower respiration rates in
air than in water, mean values of .59 ml. O2/gm./hr. in air and .91 ml. Oi/gm./hr. in water
fP = .01) being obtained.
The metabolic rates measured in air and in water tend to reflect the differential distribution
of the three gastropods in the intertidal environment.
This work was carried out as a part of the student training program of the Marine
Ecology Course.
A study of the nucleic acids of the dogfish cornea. SEYMOUR ZIGMAN AND
WILLIAM P. AREND.
Our objective is to determine whether nucleic acids, due to their influence over protein
synthesis, have a function in the maintenance of tissue transparency. Initial efforts were
352 TAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
directed toward the development of a procedure for measuring the nucleic acid phosphorus
content of smooth dogfish (Music/us cunis) corneas.
A modification of the Schmidt and Thannhauser method for the fractionation of acid-
soluble, lipid, ribonucleic acid (RNA), deoxyribonucleic (DNA), and protein phosphorus
components of the cornea was employed. All fractions contained appreciable amounts of
phosphorus, both in the epithelium and stroma, except the protein phosphorus, which was
nearly absent. As was expected, the highly cellular epithelium was far richer in RXA and
DNA phosphorus than the very fibrous stroma.
These results led us to investigate the uptake of P3~ by the compounds mentioned. Several
incubations of whole corneas or of separated epithelia and stromas were attempted, incubating
with elasmobranch Ringer's solutions containing 1 /uc. of P32 for two and four hours. Since
little incorporation of radioactivity into the nucleic acids was observed, it seems that more
suitable systems must be sought.
Further work was carried out to develop a method for the quantitative analysis of the
nucleotides of corneal RNA by paper electrophoresis. Although complete separation of 50 fj.g.
of pure nucleotide solutions or of yeast RNA hydrolysates could be accomplished, corneal
nucleic acid hydrolysates have not as yet given clear-cut separations of cytidylic, adenylic,
guanylic and uridylic acids.
Future studies will include similar experiments on such transparent tissues as teleost
corneas, avian nictitating membranes and squid integument.
Isolation of mitotic apparatus from pressurized Arbacia eggs. ARTHUR M. ZIM-
MERMAN AND DOUGLAS MARSLAND.
Mitotic apparatus (spindle-aster-chromosome complex) were isolated from the eggs of
- Irbacia punctulata immediately subsequent to a short exposure to high (up to 10,000 lbs./in.2)
pressure, applied at metaphase of first cleavage. The isolation procedure consisted of selective
solubilization with digitonin after treatment with cold 35% ethanol.
In general it was found that high pressures had marked disorganizing effects upon the
structure of the mitotic apparatus. However, the disorientation of the spindle-aster configura-
tion decreased progressively as the magnitude and duration of the treatment were decreased.
A pressure of 10,000 lbs./in.2 applied for only one minute yielded spindle-aster remnants
completely devoid of any detectable linear or radial structure, although the centriole areas and
chromosomes were clearly visible. The chromosomes were clumped very irregularly near
the center of the spindle area. Often the spindle area was abnormally short and sometimes
curved.
In contrast to the foregoing result, seemingly normal spindle-aster complexes were
recovered from eggs exposed to 4000 lbs./in.2 for one minute. The fibrillar structure of the
spindle and asters was clear, the chromosomes and centriole areas appeared normal, and
judging from the large number of anaphases found, movement of the chromosomes had not
been abolished.
At intermediate pressure (8000 lbs./in.2) a longer treatment (5 minutes) was required to
approximate the effects previously described, and a still longer treatment (10 minutes) pro-
duced even greater disorganization.
Presumably the observed disorganization of the spindle and asters results from a pressure-
induced solation of these intracellular gel structures. Moreover, the evidence suggests that the
spindle is slightly more susceptible than the asters to such solation.
Supported by grant RG-7157 from the Division of General Medical Sciences, U. S. Public
Health Service (A. M. Z. ), and by grant C-807 from the National Cancer Institute, U. S. Public
Health Service (D. M.).
Uptake of adcnosine nucleotides in Arbacia eggs. ARTHUR M. ZIMMERMAN AND
SELMA B. ZIMMERMAN.
Studies were conducted to determine the mode of penetration of adenosine triphosphate
(ATP) and related adenine nucleotides into the eggs of Arbacia punctulata. Arbacia eggs
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 353
were immersed into Cu labeled adenosine triphosphate, adenosine diphosphate and adenosine
monophosphate, and subsequently prepared for autoradiographic analysis.
Experiments with unfertilized eggs immersed into C14ATP (at 20-22° C.) for varying
periods of time show that effective uptake was obtained after 3—4 hours immersion at relatively
low ATP concentrations (5 X 10~4 to 10~s M). Shorter immersion times of 60 and 120 minutes
resulted in negligible amounts of C14 uptake. The uptake of labeled ATP in fertilized eggs
was greater than that in unfertilized eggs. When eggs were placed into 5 X 10"4 M C14ATP
(0.12 fie/ml.') 5 minutes after insemination, an appreciable uptake of radioactivity was observed
at the following metaphase. Qualitatively, the amount of uptake after 30-35 minutes incubation
in the nucleotides was comparable to that found in unfertilized eggs incubated for a duration
of 3-4 hours.
Preliminary observations of ATP-treated eggs (fertilized and unfertilized), centrifuged just
prior to fixation, indicate that the radioactivity within the eggs is associated with the mito-
chondrial and yolk zones. Further studies are being conducted in order to determine the
mechanism of uptake of ATP. ADP, and AMP.
This work was done with the technical assistance of Miss Patricia Rodgers.
Supported by grant G-6416 from the National Science Foundation.
A report of hermaphroditism in Arbacia punctulata. ARTHUR M. ZIMMERMAN,
SELMA B. ZIMMERMAN AND ETHEL BROWNE HARVEY.
Hermaphroditism is not frequently found in the sea urchin, Arbacia punctulata. To date
there have been, only seven reported instances in this species. The hermaphrodite described
in this report was given to the investigators at the Marine Biological Laboratory, Woods Hole,
by Dr. E. E. Palincsar on the 17th of June, 1960.
Viable eggs and sperm were obtained simultaneously from the hermaphrodite upon electrical
stimulation. Four of the gonads yielded eggs and the fifth gave sperm. Controlled electrical
stimulation made it possible to collect unfertilized eggs. These eggs could then be fertilized
with sperm from the same animal. Furthermore, cross-fertilization was possible, using sperm
from another animal and eggs from the hermaphrodite. The unfertilized eggs averaged 71 fj.
in diameter. The fertilized eggs in the two- and four-cell stage measured 78 ju (without
fertilization membrane). This is within the range of normal eggs.
Developmental studies under carefully controlled temperature conditions showed that the
duration of time from insemination to 50% first cleavage furrows was 58-60 minutes at 20° C.
This is similar to that found in normal control eggs. Both the cross-fertilized and the self-
fertilized eggs were normal with respect to their morphology and rates of development to the
pluteus stage.
The work was supported in part by grant G-6416 from the National Science Foundation
and grant RG-7157 from the Division of General Medical Sciences, U. S. Public Health Service.
LALOR FELLOWSHIP REPORTS
On the natural inhibitors of ghicuronosyl transferase present in serum of pregnant
women. ROBERT M. DOWBEN.
The enzyme glucuronosyl transferase catalyzes the transfer of the glucuronide moiety from
uridine diphosphoglucuronic acid (UDPGA) to a large number of hormones, metabolic products
and drugs which are excreted as glucuronide conjugates. Previous studies disclosed the
presence of inhibitors of this enzyme in the serum of pregnant women ; one inhibitor was
tentatively identified as pregnanediol-3a,20a, a metabolite of progesterone.
Ten liters of pooled serum obtained from pregnant women in the last trimester of pregnancy
were acidified with sulfuric acid to pH 1.6 and extracted in batches with ethyl acetate for 12
hours by means of a continuous liquid-liquid extractor. All of the inhibitory activity of the
serum was extracted by this means. The combined extracts were chromatographed on an
alumina column, using a benzene-ethanol developer. A number of fractions possessing in-
hibitory activity have been obtained and assessed by measuring the decreased conjugation of
354 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY
phenolphthalein by a rat liver microsomal system in the presence of UPDGA. Aliquots of
several of the more polar fractions lost most of their inhibitory activity after hydrolysis
with /3-glucuronidase. Similarly, the inhibitory activity of the pooled serum \vas greatly
diminished by treatment with /3-glucuronidase, indicating that some of the inhibitors may be
glucuronides. The chemical identification of the various fractions is to be pursued.
The membrane surrounding milk fat globules. ROBERT M. DOWBEN AND DELBERT
E. PHILPOTT.
The fat globules in cream were observed in the electron microscope. Fat in milk occurs
as rather uniform globules about 3-4 microns in diameter. These fat globules do not coalesce
to form butter unless the cream is churned, sonicated or frozen. Heat, treatment with fat
solvents such as ethyl ether, and dilution with distilled water are much less effective in
disrupting the fat globules. Calcium salts appear to stabilize the fat globules.
The cream of raw cow's milk was separated by centrifugation and washed three times
with five volumes of 0.25 M sucrose containing 0.01 M CaCL The third washings contained
no detectable protein by the biuret reaction. Analysis of the washed cream revealed about
2.4 mg. protein/g. cream. The washed cream was examined in the electron microscope, using
neutral mammalian isotonic osmic acid solution or neutral isotonic osmic acid dichromate
solution for fixation.
The protein surrounding the fat globules appears to be a true membrane rather than
adsorbed protein. The globules could be lysed by freezing and thawing, giving rise to discoid
ghosts which resembled in many ways erythrocyte ghosts.
Serotonin (5-hydro.vytryptamine) in the male reproductive tract of the spiny dog-
fish. THADDEUS MANN.
The male copulatory organs or "claspers" of the spiny dogfish, Squalus acanthias, by means
of which semen is transferred from the male to the female, are associated with accessory
abdominal organs known as "clasper siphons" which were believed for a long time to act
merely as a reservoir of sea-water. In the course of an investigation on the function of these
organs it was observed that the fluid obtained by flushing the siphon sacs with water gives
a strong reaction with diazo reagents. Fluids obtained in this manner from five males were
pooled, deproteinized, and the protein-free extract, representing a dry weight of 175 mg.,
subjected to further analyses. These have shown that the diazo-reactive substance is identical
with serotonin (5-hydroxytryptamine). On the basis of optical activity measured at 275 m,u,
the serotonin content of the protein-free extract was found to represent 6.2% of the dry weight.
Determination based on absorption measurement of the purple-colored reaction product with
l-nitroso-2-naphthol gave a closely similar result, namely, 6.4%. Paper chromatograms sprayed
with either diazobenzenesulfonic acid, para-dimethylaminobenzaldehyde, or ninhydrine, yielded
spots with the same RP value and color as pure serotonin.
Serotonin is known to possess the ability to stimulate uterine contractions. The participa-
tion of the fluid derived from the clasper siphons in the formation of fish seminal plasma,
and the abundance of serotonin in that fluid suggest that, in the spiny dogfish at any rate,
serotonin may play a role in the process of reproduction, either by influencing contractions
of the female reproductive tract, thus facilitating sperm passage and fertilization, or by
affecting the mechanism of copulation and ejaculation in the male.
Lipid-soluble sialic acid containing material in Arbacia eggs. L. WARREN AND
R. HATHAWAY.
An Arbacia egg contains 3.8 X 10"" /ugm. bound sialic acid which can be completely
released by hydrolysis for 2 hours at 80° C. in 0.1 N H,SO4. None of the sialic acid is
normally free (i.e., reactive in the thiobarbituric acid assay before hydrolysis) and there is
none in the jelly coat.
At least two forms of sialic acid-containing substances are present in the egg. one of
PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 355
which is soluble in organic solvents. Approximately 25% of the total sialic acid can be
readily extracted into n-butanol from a sea urchin egg homegenate. The lipid sialic acid
complex is soluble in chloroform-methariol (2:1) and in ether. The ether solubility suggests
that the carboxyl group of the sialic acid residue may be blocked. Free sialic acids
(N-acetylneuraminic acid and N-glycolylneuraminic acid) are insoluble in ether.
The sialic acid is released from the complex in one hour in 0.1 N H,SO4 at 80°. It is
also released enzymatically by sialidase of Vibrio cholcrac. The freed sialic acid has been
purified by chromatography on a column of Dowex-1 -acetate and has the following properties:
It demonstrates the same color reactions as authentic material in the orcinol, Ehrlich, and
thiobarbituric acid tests. It co-chromatographs on paper in two different solvent systems
with N-glycolylneuraminic acid and separates completely from N-acetylneuraminic acid.
Other components of the lipid-soluble sialic acid-containing complex of Arbacia eggs
described here are not known. The evidence obtained to date suggests that it differs from the
gangliosides and other mucolipoproteins of animal brain.
Aided by RG-6234, National Institutes of Health, to Dr. Charles B. Metz.
Sialic acid in semen of Arbacia punctnlata. L. WARREN, R. HATHAWAY AND J.
G. FLAKS.
Sialic acid is present in bound form in both the seminal plasma and sperm of Arbacia.
Sialic acid appears to be present in at least two forms in seminal plasma : a readily hydrolyzable
type (67%) which is cleaved within 30 minutes at 80° in 0.1 N H..SO4, and another form
(33%) which takes four hours to hydrolyze under the same conditions. In one experiment
50% of seminal plasma sialic acid was enzymatically removed by the sialidase of Vibrio cholerac.
There are approximately 9.5 X 10"6 fj.gm. sialic acid in an Arbacia sperm. When sea
water-washed sperm are treated with fertilizin. a soluble non-dialyzable sialic acid-containing
material is removed from the sperm. The reaction appears to be complete within one minute
and there is no further solubilization of sperm material unless more fertilizin is added. In one
experiment four successive fertilizin treatments of a preparation of sperm led to the removal
of 20% (12.0, 3.6, 3.2, 1.2%) of the sperm sialic acid. Heated fertilizin (80°, 5 minutes) was
also active. Sialic acid substances in sperm may also be removed by detergents, such as sodium
lauryl sulfate or cetyltrimethylammonium chloride. Four successive extractions of sperm by
a 7.5 X 10"4 M sea water solution of sodium lauryl sulfate led to the removal of 82% (55.0,
18.8, 9.2, 5.9) of the sperm sialic acid, and to a loss of 25% of sperm dry weight. Approxi-
mately 12% of the material removed from the sperm could be accounted for as sialic acid. The
sperm were still motile after the second extraction. Sperm also releases sialic acid-containing
substances when suspended in distilled water and when incubated at 45° or higher, for 5 minutes.
Incubation of the solubilized sperm material at 80° for four hours in 0.1 N H.SO, is
required to cleave the sialic acid residue from the nondialyzable part. Only 5% of the sialic
acid can be removed enzymatically. The sialic acid obtained by acid hydrolysis has been
purified on a column of Dowex-1-acetate and gives the thiobarbituric acid, orcinol, and Ehrlich
tests for sialic acid. On paper it co-chromatographed with N-glycolylneuraminic acid. The
selective removal of materials from sperm by fertilizin appears to take place in a rapid
non-enzymatic manner. The biological activities of the substances removed from sperm are
described in an adjoining abstract.
Aided by RG-6234, National Institutes of Health, to Dr. Charles B. Metz.
Vol. 119, No. 3 December, 1960
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
SULFUR METABOLISM IN THE LUGWORM, ARENICOLA
CRISTATA STIMPSON1'2
WALTER ABBOTT 3 AND J. AWAPARA
Department of Biology, Rice University, Houston, Texas
Among the published reports on invertebrate biochemistry is a study by Robin
(1954) using Arcnicola marina L. Robin hypothesized that taurocyamine, a
sulfur-containing guanidine derivative, was formed from taurine. Although the
hypothesis has not been demonstrated experimentally, it constitutes a major part
of the information presently available on sulfur metabolism in invertebrates.
Taurine in invertebrates generally has been regarded as a metabolically inactive
compound. In Arcnicola, however, since taurocyamine appears to be the phospha-
gen involved in energy transfers related to muscular activity, Robin's hypothesis
requires an active metabolic role for taurine. Relatively high levels of taurine
have been reported from a diversity of marine invertebrates. The fact that no
definite role has been demonstrated for these high levels suggests that an organism
such as Arcnicola, which presumably both synthesizes and metabolizes taurine,
might be a useful experimental tool for studying sulfur metabolism in invertebrates.
The present study was undertaken to determine the biochemical pathway
leading to taurine formation in Arenicola cristata Stimpson, and to determine
whether taurocyamine is synthesized by the route suggested by Robin or by some
other mechanism.
MATERIALS AND METHODS
Animals
The specimens of A. cristata employed in this study were obtained from two
sources. The Marine Biological Laboratory, Woods Hole, Massachusetts, sup-
plied large specimens (50 to 200 gm. fresh weight). Worms of this size apparently
are typical of the cold water environment in the Cape Cod area. Smaller indi-
viduals (5 to 20 gm. fresh weight) were obtained from the vicinity of St. Peters-
burg, Florida, through the courtesy of Dr. Victor G. Springer of the Florida State
Board of Conservation Marine Laboratory.
1 This study was supported by a grant from the Robert A. Welch Foundation, Houston,
Texas.
- The data presented here are extracted from the Ph.D. thesis of Walter Abbott.
3 National Science Foundation Predoctoral Fellow in Biology, 1957-60.
357
Copyright © 1961 by the Marine Biological Laboratory
358 WALTER ABBOTT AND J. AWAPARA
Compounds used
Taurocyamine was synthesized by reacting taurine with S-methylisothiourea
in ammoniacal medium, according to the general method of Schiitte (1943) as
modified by Thoai and Robin (1954). Traces of taurine in the product were
removed by chromatography on a column of Dowex 50 (H+).
Hypotaurine ( 2-aminoethanesulfinic acid) was prepared by the method of
Cavallini ct al, (1955).
S35-taurine was prepared from S35-sodium sulfite (Abbott Laboratories) and
/2-bromoethylamine hydrobromide (Cortese, 1943). The product was purified by
chromatography on a column of Dowex 50 (H+).
S3r'-methionine, S35-cystine, and 3-C14-serine were obtained from various com-
mercial sources. SS5-cysteine was prepared by reduction of S35-cystine (Lucas
and Beveridge, 1940).
Extract preparation
Enzymatically active preparations were made by homogenizing living A.
cristata with 10 volumes of 0.01 M, pH 7.3 phosphate buffer at temperatures of
0° to 4° C. Choice of pH was based on values given by Spector (1956) for
Arenicola sp. The homogenates were centrifuged in the cold for 15 minutes at
1500 X g to remove large amounts of fibrous debris, then, after decantation, for
30 minutes at 25,000 X g to remove very small participate components. The clear
supernatant from the second centrifugation was quick-frozen immediately in Erlen-
meyer flasks, and was lyophilized. A light, flesh-colored powder resulted. This
powder was totally soluble in the phosphate buffer.
For determination of amino acids and related compounds, water-soluble fractions
were prepared by homogenizing living worms in 80% ethanol and extracting by
the method of Awapara (1948).
Before worms were used to make either type of extract, they were placed in
enamel trays containing small amounts of clean sea water, in order to allow time
for the worms to empty their digestive tracts. Attainment of this condition was
assumed when fecal casts were not extruded for a period of at least 30 minutes.
Separation and identification of compounds
Dowex 50 (H+), 200- to 400-mesh, a sulfonic acid-type cation exchanger,
was used to separate polar compounds into two groups. Those compounds not
retained on a column of the resin during elution with distilled water were termed
"acidic compounds" in this study. Those compounds retained on the resin under
these conditions were designated "basic compounds." Acidic compounds of in-
terest here were taurine, taurocyamine, cysteinesulfinic acid, and cysteic acid.
Basic compounds were displaced with 4 N NH4OH.
For group separation, 3 gm. of Dowex 50 (H+) were sufficient to treat an
amount of water extract equivalent to at least 1 gm. fresh weight of tissue. In
a small column were placed 1.5 gm. of resin. Another 1.5 gm. were slurried with
the sample in a small beaker for 5 to 10 minutes. Contents of the beaker then
were poured into the column. Beaker and column were rinsed liberally with
distilled water.
Quantitative recovery of the acidic compounds was demonstrated by treating
SULFUR METABOLISM IN ARENICOLA
359
individual standards of these compounds in the manner just described. Recoveries
in excess of 95% were obtained in all cases when 15 standard samples were
treated as just outlined.
Thompson and Morris (1959) reported the chromatographic separation of
mixtures of amino acids on a column of Dowex 50 (Na+), using water or
aqueous ethanol as solvent. This suggested separation of acidic compounds from
Arenicola by chromatography on a column of Dowex 50 (H+), using distilled
water for elution. The column, 60 X 0.9 cm., was prepared from a slurry of
reagent grade Dowex 50 (H+) which had previously been washed to remove
excess acidity. A column was used only once. The sample to be resolved was
placed in the column in a volume of 1 ml. Elution was carried out at a rate of
5 to 6 ml. per hour. One-milliliter fractions were collected. Mixtures of standards
could not be resolved until a soluble inorganic salt was added to the sample
(25 to 50 mg./ml. NaCl), whereupon resolution became excellent (Fig. 1). The
4.0
3.0
E
>
•£ 2.0
1.0
TAURINE
CYSTEIC ACID
TAUROCYAMINE
CYSTEINE
SULFINIC ACID
15
25
35 45
FRACTION NUMBER
55
65
FIGURE 1. Elution of standards from 60X0.9 cm. Dowex 50 (H+) column.
worm extracts contained sufficient inorganic material to obviate addition of salt.
Schram and Crokaert (1957) obtained similar resolution of taurine and tauro-
cyamine by eluting Dowex 50 (H+) columns with 0.1 M, pH 2.5 citrate buffer.
The four acidic components could be resolved by one-dimensional paper
chromatography with phenol: water (72% v/v) as solvent. The basic components
were separated by two-dimensional paper chromatography with phenol : water and
2,4-lutidine : water (65% v/v) as solvents. Papers used were Whatman Nos.
1, 4, or 3 MM.
Quantitative determinations of amino compounds were carried out by the
method of Landua and Awapara (1949). Guanidylated entities were determined
colorimetrically with diacetyl and a-naphthol (Rosenberg, Ennor and Morrison,
1956). Location of spots on papers was accomplished by two methods. Amino
compounds were found by treatment of papers with 0.025% ninhydrin in acetone.
Guanidino compounds were detected with the diacetyl reagent of Smith (1958),
using the reagent as a spray. Table I shows the limits of detectability for various
guanidine compounds when this reagent is used.
360
WALTER ABBOTT AND J. AWAPARA
I
TABLE I
Detection limits for various guanidine compounds on paper chromatograms with the
diacetyl spot reagent (Smith, 1958)
Compound
Taurocyamine
Glycocyamine
Creatine
Arginine
Guanidine
Agmatine
of paper*
0.75
1.06
1.30
2.00
1.59
2.00
* Values represent the smallest amount of a compound which was found to give a definite
positive test on Whatman 3MM paper.
Hypotaurine was determined either by eltition from Dowex 50 (H+) columns
with 2 X 10~3 N HC1 (Bergeret and Chatagner, 1954), or by oxidation to taurine
with hydrogen peroxide and determination as taurine. In the latter procedure,
preliminary removal of endogenous taurine by Dowex 50 (H+) was necessary.
Inorganic ions were determined as follows : ( I ) sulfites by the distillation-
iodine titration method (Fromageot, Chatagner and Bergeret, 1948) ; (2) sulfates
by the Versene technique (Welcher, 1958) ; and (3) chlorides by the Mohr
technique.
Determination of endogenous levels of various substances was performed on
a composite homogenate of 25 worms.
Experimental techniques
In vivo tracer studies were made by injecting worms intracoelomically with
labeled metabolites. Leakage of compounds to the environment was found to be
negligible during the time periods (12 to 36 hours) involved in these studies.
Counting of aliquots of whole homogenates of injected worms showed recoveries
of radioactivity which averaged 93.4% of the amount injected (Table II). Self-
absorption was found to be negligible if the amount of material on the planchet
was kept lower than 200 fj.g. per square centimeter. Hilchey, Cotty and Henry
(1957) arrived at a value of 130 p.g. per square centimeter, in similar studies.
The distribution of radioactivity into various compounds was determined in
several ways- — preparation of radioautographs from paper chromatograms, count-
ing of material eluted from paper chromatograms, cocrystallization of metabolites
TABLE II
Recovery of radioactivity 24 hours after injection
Compound injected
Counts/minute
% recovery
Injected
Found
S35-taurine
S35-cysteine
S35-methionine
3-C14-serine
1.17 X 106
8.00 X 104
2.20 X 107
2.20 X 106
1.04 X 106
7.50 X 104
2.19 X 107
1.98 X 106
89.9
94.0
99.5
90.0
SULFUR METABOLISM IN ARENICOLA 361
from extracts by addition of unlabeled carrier. A Nuclear-Chicago, thin window,
gas flow counter was used for all counting.
In vitro studies were made with both fresh homogenates and with the lyophilized
enzymatic preparation described previously. Enzyme preparations were dialyzed
only when the reaction product of interest was present at a high endogenous
level. All samples were prepared and adjusted to proper volume before addition
of the enzyme preparation. Incubations usually were for one hour, and were
performed at room temperature (22° to 23° C.).
TABLE III
Concentrations of free amino acids and related compounds in Arenicola cristata
Compound Leucine equivalents/gin, of fresh weight*
Cysteine 3.92 ±0.21
Homocysteine 0.52 ± 0.10
Aspartic acid 1.05 ±0.05
Glutamic acid 1.73 ±0.19
Serine 0.22 ± 0.05
Glycine 17.28 ±0.09
Arginine 0.50 ± 0.07
Alanine 6.00 ± 0.22
\ "aline plus methionine 0.55 ± 0.03
Leucine 0.75 ± 0.03
Tyrosine 0.025 ± 0.020
Methionine sulfone 0.32 ± 0.08
Hypotaurine 0.48 ± 0.08**
Cysteinesulfinic acid 0.48 ± 0.05**
Taurine 3.28 ± 0.36**
Taurocyamine 5.06 ± 0.21*'
* Values represent equivalent /orioles of leucine, except where otherwise indicated. All
values represent mean values of four determinations, with standard errors, on aliquots of a homog-
enate of 25 worms.
* This value represents actual jumoles rather than leucine equivalents.
THE CHEMICAL COMPOSITION OF ARENICOLA
Vinogradov (1953) has summarized information on the chemical composition
of Arenicola marina and A. piscatontm. These data are assumed to be applicable
to A. cristata.
During the present study, a few additional determinations of inorganic com-
ponents were made. Moisture content was determined on two worms. An
average of 80.6% H2O was found. Sulfate, determined on six worms, was
84.1 ±5.3 //.moles per gram of fresh weight. Chloride was measured on three
aliquots of the pooled extract of 25 worms. A level of 254 ± 1.9 //.moles per
gram of fresh weight was established. Determinations on four aliquots of the
same pooled extract by the colorimetric persulfate method showed total manganese
to be less than 3.6 X 10~3 /zmoles per gram of fresh weight.
Analyses for amino acids and other related materials were performed on the
25-worm extract. Results are given in Table III. Levels of basic compounds
are reported as leucine equivalents. Hypotaurine and acidic compounds are
reported directely in micromoles.
362
WALTER ABBOTT AND J. AWAPARA
The status of cysteic acid as a metabolite is in question. It appears that in
most cases where cysteic acid is found in extracts of normal tissues, it represents
an artifact created by the extraction and isolation procedures. In the present
work with Arenicola, it was found that cysteic acid always occurred when extracts
were prepared at room temperature and were evaporated on the steam bath.
When, however, extraction was carried out in the cold, and evaporation was done
in vacua without heat, no cysteic acid was detected. For this reason, cysteic acid
is regarded as an artifact in Arenicola cristata.
TABLE IV
Qualitative studies in vivo using radioactive compounds
Methods by which radioactivity was detected after injection of
Compound found to be
radioactive
S35-
s«-
S35-
S35_
3-C14-
taurine
methionine
cysteine
cystine
serine
Sulfate
1
1
1
Homocysteine
2,3
Cystathionine
2
2
2
2, 4
Cysteine
2, 3
3
2
4
Cysteinesulhnic acid
2,3,4
3
Cysteic acid
2,3,4
3
Hypota urine
5
5
Taurine
3,4
2,3,4
3
3
Methods:
1. Fifty mg. nonradioactive NaoSO4 added/ml, of sample. Sample acidified with HC1.
Sulfate precipitated with slight excess of 10% BaCl2 solution. Precipitate filtered, washed, and
ignited for one hour at 1000° C. Ignited precipitate pulverized, distributed on planchet, and
counted.
2. Compound extracted from sample by crystallization with nonradioactive carrier. Product
recrystallized twice. Crystals dissolved. Solution evaporated on planchet and counted. Purity
of final product checked by chromatography in four solvents.
3. Compound isolated by paper chromatography in several solvents. Spots eluted from
papers. Eluates evaporated on planchets and counted.
4. Compounds isolated by paper chromatography. Radioautographs prepared from
chromatograms.
5. Acidic compounds removed by Dowex 50 (H+) treatment. Basic fraction oxidized with
30% HoOo. Oxidized material again separated with Dowex 50 (H+). Taurine isolated from
acidic fraction by paper chromatography in four solvents. Taurine spots eluted. Eluates
evaporated on planchets and counted.
Robin's (1954) report of high arginase activity in homogenates of A. marina
suggested a need for determination of the endogenous urea level in this worm.
Manometric estimations with urease (Umbreit, Burris and Stauffer, 1957) showed,
for the 25-worm extract, a urea level of 0.96 ± 0.013 //.moles per gram of fresh
weight (four replications). Three standard urea samples were analyzed concur-
rently. The average value obtained for the standards was 101.6% (range: 97.2%
to 105.3%) of the urea added.
SULFUR METABOLISM IN ARENICOLA 363
RESULTS AND DISCUSSION
Qualitative studies
These studies were conducted with the relatively large specimens of A. cristtitti
obtained from Woods Hole Marine Biological Laboratory.
S35-taurine was administered to a worm by intracoelomic injection. The worm
was dissected after 24 hours, and all organs were shown to contain some radio-
activity. Radioautographs prepared from chromatograms, and direct counting of
material eluted from chromatograms, revealed no radioactivity in any compound
other than taurine. This experiment indicated that taurocyamine, although known
to contain sulfur, apparently was not formed from taurine. Therefore, a tracer
study of sulfur metabolism in A. cristata was undertaken.
Radioactive compounds were given by injection and after 12 to 36 hours the
animals were homogenized and extracts made. The results of the tracer studies
and the methods used to obtain these results are given in Table IV. The labeled
intermediates found did not suggest any qualitative departure from the basic
sulfur catabolism scheme known for vertebrates. Taurine was formed from
methionine or cystine, presumably via decarboxylation of cysteinesulfmic acid to
hypotaurine with subsequent oxidation of the latter. Cysteine and cystine were
metabolically interconvertible, although the rate and nature of this conversion
are not known. A cystathionine pathway from methionine to cysteine exists and, as
is also known in the vertebrate, involves serine. In no case was any radioactivity
detected in taurocyamine, even 36 hours after injection of cystine.
Quantitative studies
These studies were conducted with the small specimens of A. cristata obtained
from the St. Petersburg, Florida, area. In all studies using labeled compounds,
total recovery of radioactivity was assumed. With this assumption, data regarding
distribution of radioactivity may be regarded as representing the distribution of
the entire metabolic pool of the injected compound. Because of the complete
overlap between elution peaks from Dowex 50 (H+) columns for sulfate and
for cysteic acid, radioactivity levels for these compounds are pooled in all values
given for distribution of radioactivity after injection of S35-labeled compounds.
Tracer studies showed that A. cristata converted approximately 50% of in-
jected S33-cysteine into oxidized, strongly acidic entities within 12 hours. With
cystine, the process was much slower, only about 18% being oxidized in 12 hours.
The 50% level was attained with cystine by 36 hours.
When 3-C14-serine was injected into the worms, almost 60% of the total radio-
activity appeared within 24 hours in strongly acidic compounds, i.e., those which
passed through the Dowex 50 (H+) column. Most of these components showed
no reaction with ninhydrin. An estimated 12% of the total radioactivity was
associated with strongly acidic sulfur-containing compounds (Table V). This
\vas about the same amount of incorporation as was obtained in 12 hours with
S35-cysteine, excluding inorganic sulfate.
With methionine, only 17.8% of the total radioactivity appeared in the acidic
fraction in 24 hours. Of this, 13.5% was in sulfate plus cysteic acid, 2.7% in
taurine plus an unidentified compound, and 1.6% in cysteinesulfinic acid. Pre-
364
WALTER ABBOTT AND J. AWAPARA
sumably, the rate of demethylation of methionine to form homocysteine was the
limiting factor in this system.
Relatively high percentages of radioactivity were incorporated into sulfate,
as compared with other sulfur compounds, after injection of S35-cysteine or
TABLE V
Distribution of radioactivity among acidic compounds in Arenicola cristata following
injections of labeled compounds
Counts/minute/
Distribution of
Injected compound
Fraction
gm. of fresh
total radioactivity
weight
%
S35-cysteine
Whole extract
21,360
100
(extraction 12 hours
Acidic portion
11,070
51.83
after injection)
Sulfate + cysteic acid
8,460
39.61
Ta urine
490
2.29
Cysteinesulfinic acid
1,208
5.66
Compounds Xi and X->
912
4.27
S35-cystine
Whole extract
23,925
100
(extraction 12 hours
Acidic portion
4,250
17.77
after injection)
Sulfate + cysteic acid
3,938
16.46
Taurine
155
0.65
Cysteinesulfinic acid
79
0.33
Compounds Xi and Xa
78
0.33
S35-cystine
Whole extract
21,510
100
(extraction 36 hours
Acidic portion
10,823
50.32
after injection)
Sulfate + cysteic acid
8,708
40.48
Taurine
1,462
6.80
Remainder
653
3.04
S35-methionine
Whole extract
438,000
100
(extraction 24 hours
Acidic portion
78,000
17.8
after injection)
Sulfate + cysteic acid
59,300
13.5
Taurine +X:
11,500
2.7
Cysteinesulfinic acid
7,200
1.6
3-C14-serine
Whole extract
14,710
100
(extraction 24 hours
Acidic portion
8,600
58.5
after injection)
Taurine*
1,860
12.6
Compounds not containing
6,740
45.9
sulfur
* Since many highly acidic carbon compounds emerge at the front during elution of the
Dowex 50 (H+) columns, radioactivity associated with cysteic acid cannot be isolated. Cysteine-
sulfinic acid shows no radioactivity. Therefore, it is assumed that cysteic acid also is not radio-
active and that all radioactivity appearing in sulfur-containing strongly acidic compounds is
associated with taurine.
S35-cystine (Table V, Fig. 2). These percentages are of the same order as those
reported from other sources (Kay and Entenman, 1959; Skarzynski, Szczepkowski
and Weber, 1959; Eldjarn, Pihl and Svedrup, 1956; Tarver and Schmidt, 1942).
Fromageot (1953), Kearney and Singer (1953a, 1953b), Chatagner et al.
(1952) and others have all emphasized that the most important pathway of sulfur
SULFUR METABOLISM IN ARENICOLA
365
from cysteine to sulfate is through transamination between cysteinesulfinic acid
and a-ketoglutaric acid to form glutamic acid and an unstable intermediate,
/8-sulfinylpyruvic acid. The latter decomposes into sulfite, which is oxidized to
sulfate, and into pyruvic acid, which by transamination with glutamic acid re-
generates a-ketoglutaric acid and forms alanine.
It has been demonstrated that both enzymatic and non-enzymatic oxidation of
sulfite to sulfate occur in mammals (Heimberg, Fridovich and Handler, 1953;
Fridovich and Handler, 1956). The enzymatic process is heat-labile. When
sodium sulfite solutions were incubated with the soluble enzyme preparations from
A. cristata, the results in terms of sulfite remaining after one hour, although some-
what erratic, indicated that no decrease in activity was effected by boiling the
enzyme preparation. Thus, the enzymatic mechanism may be of little importance
in Arcnicola, or mar be absent.
3000-30
i.
3i
1
52000-20^
o
o
1000
o RADIOACTIVITY
• ACIDIC COMPOUNDS
CYSTEINE
SULFINIC ACID o
- 1.0
20
40
FRACTION NUMBER
50
60
FIGURE 2. Acidic components from 1 gm. of Arcnicola cristata 12 hours after injection
of S^-cysteine. Fractions are 1-ml. portions of eluate from 60 X 0.9 cm. Dowex 50 (H+)
column.
The activity of the cysteinesulfinic acid-a-ketoglutaric acid transaminase system
in vitro may be evaluated from Table VI, which presents the most consistent series
of experimental results obtained in several trials of this system. The values
reported for glutamic acid represent increases above the endogenous level for
this substance. There seems little reason to doubt the formation of sulfate via
the transaminase-^-sulfinylpyruvate pathway.
The formation of hypotaurine in the mammal by decarboxylation of cysteine-
sulfinic acid has been established for some time (Bergeret and Chatagner, 1952;
Bergeret, Chatagner and Fromageot, 1952 ; Awapara and Wingo, 1953). Pyridoxal
phosphate has been shown to act as coenzyme for this reaction and to resist dialysis,
indicating that it is tightly bound to the apoenzyme (Bergeret and Chatagner,
1952; Blaschko and Hope, 1954; Hope, 1955). In the present study, hypotaurine
was found to occur in A. cristata at an endogenous level of 0.5 /xmole per gram of
fresh weight. Attempts to show decarboxylation of cysteine-sulfinic acid in vitro
gave highly variable results. Whole homogenates and solutions of the lyophilized
soluble fraction of the worm were tested. Pyridoxal phosphate was added to
366
WALTER ABBOTT AND J. AWAPARA
samples at levels of 4 to 8 /ug. per milliliter. In all manometric experiments carbon
dioxide was released. Chromatography of reaction mixtures after 60-minute in-
cubation periods revealed distinctly more hypotaurine in reactive samples than in
controls, based on comparison of colors and sizes of spots on ninhydrin-developed
papers. Colorimetric estimation showed that an erratic increase in hypotaurine
occurred in samples as compared with controls. The reaction was heat-labile.
TABLE VI
Cysteinesidfinic acid-a-ketoglutamic acid transamination*
Sample number
ml. of extract added
/jmoles of
cysteinesulfinic
acid added
yumoles of
a-ketoglutaric
acid added
^moles of
glutamic acid
formed/gm. of
fresh weight/hour
1
1.0**
25
25
0.0
2
1.0**
25
25
0.0
3
0.0
25
25
0.0
4
0.0
25
25
0.0
5
1.0
25
0
0.0
6
1.0
25
0
0.0
7
1.0
0
25
0.0
8
1.0
0
25
0.0
9
1.0
25
25
12.1
10
1.0
25
25
6.0
11
1.0
25
25
7.2
12
1.0
50
25
10.5
13
1.0
50
25
9.9
14
1.0
50
25
10.5
15
1.0
75
25
11.0
16
1.0
75
25
11.0
17
1.0
75
25
11.6
18
1.0
100
25
11.0
19
1.0
100
25
10.5
20
1.0
25
50
8.3
21
1.0
25
50
8.3
22
1.0
25
75
9.9
23
1.0
25
75
9.3
24
1.0
25
100
9.3
25
1.0
25
100
9.9
26
1.0
50
50
12.7
27
1.0
50
50
12.7
28
1.0
100
100
15.0
29
1.0
100
100
17.7
30
2.0
25
25
15.0
31
2.0
25
25
12.2
32
2.0
25
25
12.7
33
3.0
25
25
19.4
34
3.0
25
25
17.2
35
3.0
25
25
17.2
* Conditions: All solutions prepared in 0.01 M phosphate buffer, pH 7.3. Total volume:
5.0 ml. Each ml. of extract contained 50 mg. of lyophilized soluble fraction of A. cristata. Aero-
bic incubation of samples for 120 minutes at temperatures of 22° to 23° C. Acids neutralized
with NaOH prior to making stock solutions.
** Extract boiled for 5 minutes before addition to sample.
SULFUR METABOLISM IN ARENICOLA
367
TABLE VII
Oxidation of hypotaurine to taurine*
Sample number
ml. of homogenate added
Molarity of hypotaurine
/irnoles of taurine
formed/hour/gm. of
fresh weight
1
1.0
0.0040
12.0
2
1.0
0.0040
12.5
3
1.0
0.0040
13.0
4
1.0
0.0035
11.5
5
1.0
0.0035
11.5
6
1.0
0.0035
12.0
7
1.0
0.0030
10.0
8
1.0
0.0030
11.5
9
1.0
0.0030
11.0
10
1.0
0.0025
8.5
11
1.0
0.0025
10.5
12
1.0
0.0025
9.0
13
1.0**
0.0025
8.0
14
1.0**
0.0025
8.5
15
2.0
0.0025
9.0
16
3.0
0.0025
9.0
17
0.0
0.0025
0.0
* Conditions: All solutions prepared in 0.01 M phosphate buffer, pH 7.3. Total volume:
11.0 ml. Fresh homogenate of A. cristata: 1 ml. equivalent to 200 mg. of fresh weight. Aerobic
incubation of samples for 60 minutes at temperatures of 22° to 23° C. Hypotaurine neutralized
with NaOH before stock solution was prepared. Taurine determined by separation on Dowex
50 (H+), columns followed by ninhydrin treatment of aliquots of filtrates.
** Homogenates boiled for 5 minutes before addition to sample.
The oxidation of hypotaurine to taurine was studied with fresh whole homog-
enates and with the soluble enzyme extract. The reaction appeared to be non-
enzymatic ; it was not hindered by prolonged boiling (up to 20 minutes) of
homogenates and extracts. Table VII shows the most consistent results obtained
from the series of experiments on this reaction. Values for taurine formation are
based on increases above the endogenous level. A reciprocal plot of this experi-
ment was derived by least squares analysis. Statistical testing indicates that the
line does not pass through the origin (0.02 >p> 0.01). Other less extensive
experiments gave regression lines which were not significantly different from
lines passing through the origin. It is postulated that the reaction is non-enzymatic
because of its total insensitivity to prolonged heating. Metal ion catalysis may
be involved here since the presence of the worm homogenate or extract is necessary
to the reaction. It is further suggested that the reciprocal plot should not pass
through the origin, as there is undoubtedly some substrate level at which catalyst
saturation occurs and the reaction becomes zero order.
Repeated attempts were made to demonstrate transamidination between arginine
and a variety of acceptor compounds — taurine, hypotaurine, cysteine-sulfinic acid,
cysteic acid, cysteine, cystathionine, and mercaptoethylamine. All results were
negative. Care was taken to add sufficient arginine (20 or more ^moles per
milliliter of sample) so that arginase activity could not seriously deplete the supply
of the amidine group donor during incubation.
368
WALTER ABBOTT AND J. AWAPARA
The experiment with the S35-taurine injection was repeated with taurine of
higher specific activity. Twelve hours after injection, the animal was homogenized
and the homogenate was fractionated by chromatography on the Dowex 50 (H+)
column. When the fractions were checked for radioactivity, a slight amount of
radioactive sulfur was detected in the taurocyamine. On the assumption of no
leakage of compounds to the environment, and with the endogenous concentration
of taurine known, it was calculated that only 0.60 ^mole of taurocyamine — 0.10
^.rnole per gram of fresh weight — had been formed in 12 hours. The correlation
of taurocyamine content with radioactivity in 1-ml. fractions eluted from the
column is shown in Figure 3.
120
100
80
fee
c
~ 40
o
o
20
3.0-
2.0
o>
c
"E
o
o
o
1.0 *>
o
E
RADIOACTIVITY O
fi moles •
20 30 40 50 60
FRACTION NUMBER
70
FIGURE 3. Taurocyamine fractions from 1 gm. of Arenicola cristata 12 hours after injec-
tion with S^-taurine. Fractions are 1-ml. portions of eluate from 60 X 0.9 cm. Dowex 50
( H+) column.
As soon as this information was obtained, a solution of moderately radioactive
S35-taurine was prepared for use in in vitro studies. Three experiments — two
with fresh homogenates of A. cristata and one with a lyophilized soluble fraction-
were performed in an attempt to obtain transamidination between arginine and
taurine. Success was achieved with only one sample, which was part of the
experiment using the lyophilized preparation. After incubation for 60 minutes,
0.004 /xmole of taurocyamine was formed by this sample, which contained 100
^.moles of arginine, 5 /xmoles of taurine (representing 2335 counts per minute),
and an amount of extract equivalent to 0.61 gm. of fresh weight of worm tissue.
Total volume was 5.0 ml.
From the in I'iro results, it might be assumed that the slow rate of tauro-
SULFUR METABOLISM IN ARENICOLA 369
cyamine formation resulted from competition between arginase (Robin, 1954) and
transamidinase for the limited arginine supply. The over-all endogenous level of
arginine was about 0.5 ^mole per gram of fresh weight, all of which seemed to
occur in the wall of the digestive tract. However, the assumption of competition
was not supported by in vitro results, as sufficient arginine was present to supply
both systems adequately. Dr. James B. Walker (personal communication) has
speculated that his transamidinase preparations from mammalian tissues and from
Streptomyces griscus probably would show similar low levels of activity toward
almost any suitable amidine group acceptor.
SUMMARY AND CONCLUSIONS
The pattern of sulfur metabolism in Arenicola cristata was examined. Quali-
tatively, it differs little from the pathway known in mammals, except in one of the
terminal reactions. This reaction leads to incorporation of cysteine sulfur and,
hence, of methionine sulfur into the compound taurocyamine by transamidination
between taurine and arginine, as suggested by Robin (1954).
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HISTOLOGICAL STUDIES ON THE DIGESTIVE SYSTEM
OF A STARFISH, HEXRICIA, WITH NOTES ON
TIEDEMANN'S POUCHES IN STARFISHES1
JOHN MAXWELL ANDERSON
Department of Zoology, Cornell University, Ithaca, N. Y.
In starfishes of the genus Hcnricia (Family Echinasteridae), the digestive tract
is a complex system presenting many highly specialized features and contrasting
markedly with the much simpler digestive organs of forms such as Asterias or
Pisaster. In consequence of a number of descriptive and experimental studies,
structural and functional aspects of feeding and digestion in Asteriidae are reason-
ably well understood ; in contrast, very little is known of the details of structure
and function in the alimentary tract of Hcnricia. From the functional standpoint,
for instance, we remain in ignorance of the normal food of Henricia (Mortensen,
1927, p. 120), and of its mode of feeding. Anatomy and histology have received
somewhat more attention: Cuenot (1887) devotes some descriptive remarks to
the digestive system in Cribdla oculata ( = Henricia sanguinolenta} , and Hayashi
(1935) includes sections on the structure and histology of the digestive tract in
his article on the anatomy of a Japanese variety of H. sangitinolenta which later
(1940) he considers a separate species, H. ohshimai. Hayashi's account is reason-
ably accurate as far as it goes, but in terms of the digestive tract it is disappointingly
superficial, omitting to mention several conspicuous features and relationships
that are both interesting and significant. The present study was undertaken
originally as a necessary foundation for contemplated experimental studies on the
digestive tract. It presents a more nearly complete description of anatomical and
histological details, seeks to combine these with histochemical characteristics of
parts of the digestive system as possible clues to function, and draws attention
to unique features of the system unaccountably omitted from previous descriptions.
Prominent among these are the structures associated with the pyloric caeca
which Ludwig and Hamann (1899) named "Tiedemann's pouches" in recognition
of their discoverer. In his epochal monograph of 1816, which laid the foundations
of our modern knowledge of echinoderm morphology, Friedrich Tiedemann briefly
described a detail of structure in Astropectcn aurandacus, as follows (p. 48) :
"On the lower surface of each caecum there is a small, elongate, and hollow ap-
pendage, which commonly contains a yellowish fluid. Perhaps this appendage
is a kind of secretory organ." Cuenot (1887) called attention to the singular
development of these structures in starfishes of the families Asterinidae and Echin-
asteridae (p. 34) : ". . . the median sac is considerably elongated and forms a
voluminous reservoir, which, in the natural position of the animal, is situated
below the radial caecum ; this reservoir runs about half or three-quarters the
1 Supported by funds from USPHS Grant No. RG-5755 and NSF Grant No. G6007 to
Cornell University.
371
372 JOHN MAXWELL ANDERSON
length of the caecum and is marked by regularly spaced oblique folds ; it opens
widely into the gastric sac, of which it is nothing more than a continuation."
(See also Cuenot's Plate II, Figures 11 and 18.)
Considering the conspicuous and distinctive nature of these structures in the
starfishes that possess them, it is surprising that Tiedemann's pouches (or
Tiedemann's diverticula, as they are also called) have attracted so little attention
with regard to either structural details or functional possibilities. Vogt and Yung
(1888), referring to the relatively small pouches in Astropcctcn, mention that
solid food has been observed in the pouches but not in the pyloric caeca them-
selves. Irving (1924), in his discussion of ciliary currents in Patina miniata,
describes Tiedemann's pouches as the median ducts of the caeca which bear the
glandular pockets on their aboral walls. Misled by this description, I later
(Anderson, 1953) used the same terminology in discussing the pyloric caeca of
Astcrias forbcsi. It must be noted that the caeca of the family Asteriidae do not
possess Tiedemann's pouches and that where they do occur, these pouches are
not the median ducts themselves but form extensive appendages on the oral sides
of the median ducts. Finally, Hayashi (1935) pays practically no attention to
these conspicuous structures in his anatomical study of Henricia, saying only
(p. 9), "Each caecum has a spacious median canal which is elongated dorso-
ventrally in cross-section, and the wall is thin and folded."
We thus appear to lack altogether any detailed information on structure and
function in Tiedemann's pouches. Accordingly, in the present study some em-
phasis is placed upon the anatomical and histological features of Tiedemann's
pouches in Henricia, and upon the very considerable differences that exist between
the pouches in Henricia and those in Patiria, a member of the other family
(Asterinidae) in which such pouches typically occur, according to Cuenot (1887).
Where pertinent, brief remarks are included concerning preliminary studies of the
pouches in Astcrina gibbosa, Astropcctcn irrcgularis and annata, and Linckia
guildingi.
These studies were begun during tenure of a John Simon Guggenheim Memorial
Fellowship in 1958-59, at the Hopkins Marine Station of Stanford University,
Pacific Grove, Calif., and continued at the Marine Biological Laboratory, Woods
Hole, Mass. The generous support of the Guggenheim Memorial Foundation,
and the hospitality and cooperation of the Director and Staff of the Hopkins
Marine Station, are hereby gratefully acknowledged. I also acknowledge with
thanks the aid of the following, who provided specimens of starfishes from the
indicated areas: Dr. F. S. Russell, Plymouth; Dr. R. A. Boolootian, Los Angeles;
Aage Moller Christensen, Helsingor ; and Jonathan Green, Hawaii.
MATERIALS AND METHODS
Small to moderate-sized specimens of Henricia leviuscula were collected beneath
stones in the intertidal zone at Point Pinos, Pacific Grove, Calif. They were
maintained in running sea- water in one-gallon jars provided with escape-proof
collars of plastic screening. The specimens could not be induced to feed upon
any of the variety of items readily accepted by Patiria and other species, but as they
remained in apparently vigorous condition for periods of two months or more,
DIGESTIVE SYSTEM OF HENRICIA 373
the possibility exists that they were feeding unobserved upon suspended particulate
material in the water, or upon the algal and bacterial film on the walls of their
containers. Comparative and confirmatory observations were made at Woods Hole
on locally-collected specimens of Hcnricia sanguinolcnta maintained under similar
conditions. It is apparent that the two species do not differ significantly in the
details of their internal anatomy, and the results to be reported are composite.
Gross anatomical studies involved specimens dissected after treatment with
MgClo (8% in tap water) to prevent movement or autotomy. For histological
examination, tissues were fixed in Helly's fluid, washed, dehydrated, imbedded in
paraffin, and sectioned serially at 7 to 10 //,; or fixed in Baker's formol-saline,
post-chromed 24 hours in potassium bichromate, imbedded in gelatin, and sectioned
on the freezing microtome. Tissues that included parts of the body wall were
decalcified by soaking for approximately a week in 5% aqueous disodium ethylene-
dinitrilo-tetraacetate (EDTA), a chelating agent, between fixation and dehydration
steps.
For general study and orientation, paraffin sections were stained writh Harris'
hematoxylin and eosin. For the demonstration of muscle fibers, connective
tissue, cell membranes, flagella and their basal bodies, and secretion granules,
excellent results were obtained by the use of Mallory's phosphotungstic acid
hematoxylin. Glycogen and other polysaccharide compounds were demonstrated
by a periodic-acid-Schiff technic, controlled by salivary digestion, and meta-
chromatic substances were stained by overnight exposure to very dilute solutions
of tolnidine blue. Steedman's Alcian Blue technic. as given by Pearse (1953),
was employed for staining acid mucopolysaccharides. The frozen sections of
gelatin-imbedded material were colored with Sudan black, counterstained with
carmalum, to reveal lipid deposits.
OBSERVATIONS
A. Anatomy
As in starfishes generally, the digestive tract of Hcnricia is essentially a tube,
running from mouth to anus in the short vertical axis of the body, divided into
specialized successive regions termed cardiac stomach, pyloric stomach, and in-
testine (esophagus and rectum are additionally distinguished by many authors).
The pyloric stomach gives off a pair of glandular appendages, the pyloric caeca,
into each ray, and the intestine (or rectum) bears an exceptionally well-developed
group of sacculate appendages, of unknown function, termed rectal caeca or
intestinal caeca.
Mewed from below, as in Figure 1, the mouth appears as a stellate opening
bounded by 5 peristomial lobes which approach each other centrally. When the
animal is undisturbed, the mouth is usually partially open, as in this photograph.
Hayashi (1935) terms the cardiac stomach, into which the mouth opens,
"rudimental." It is relatively small, compared with that of Astcrias. for example,
and lies wholly within the circular frame bounded by the proximal ambulacral
ossicles (Fig. 2). The stomach consists largely of a series of structures termed
"esophageal pouches" by Cuenot (1887). There are 10 of these, 5 radial and
5 interradial in position; each radial pouch lies just medial to the large proximal
ambulacral ossicle in its rav and is movablv bound to this ossicle by a pair of
374
JOHN MAXWELL ANDERSON
Figures 1 through 27 illustrate features of the digestive system in Henricia; Figures 28
through 31 show details of Tiedemann's pouches in Patiria ininiata.
FIGURE 1. Oral view of living specimen, showing partially open mouth surrounded by
peristomial membrane. Approximately 20 X.
FIGURE 2. Cardiac stomach from above, after removal of all aboral parts by transecting
the pyloric stomach at the line indicated by the arrow. Note radial pouches (r) ; interradial
DIGESTIVE SYSTEM OF HENRICIA 375
stout, fibrous retractor strands. From the same origins a pair of longer strands
pass upward to insertions higher on the wall of the stomach. Hayashi remarks
on the thick and compact appearance of these retractor strands in contrast to the
highly branched fibers in Astcrias ; in Hcnricia, although short strands do extend
to the outer surface of the adjacent esophageal pouches, these are neither so
numerous nor so extensive as those forming what has been termed the intrinsic
retractor mechanism in Asterias and Patina (Anderson, 1954, 1959).
Above the interradial pouches the wall of the stomach folds inward longitudi-
nally to form 5 or 6 (usually 5) large, centrally-directed bulges that almost occlude
the lumen of the stomach but leave a narrow, stellate central passageway (Fig. 2).
These swollen vesicles are separated from one another by deep, radiating folds
which are restricted lateral extensions from the angles of the central passage and
which, in consequence of their radial positions, form conspicuous gutters leading
upward from the cavities of the radial esophageal pouches. Above the level of
these alternating vesicles and gutters the stomach is encircled by a slender
fibrous girdle, into which the longer retractor strands from each ray insert. There
is no real constriction marking the separation between cardiac and pyloric portions
of the stomach, and in the absence of other landmarks the regions above this
circumferential girdle will be considered as belonging to the pyloric stomach.
This is in agreement with the situation in Patiria, where a similar fibrous girdle
has been taken to mark the upper limit of the cardiac stomach (Anderson, 1959).
Just above the level of the girdle, the pyloric stomach tapers somewhat,
and its walls are perforated by a marginal opening at each of its radial angles.
Each of these 5 openings leads into a duct which immediately bifurcates, giving
rise to pale, translucent, cylindrical branches forming the tubular proximal channels
that expand vertically into the two Tiedemann's pouches in its ray (Figs. 3, 4, 5).
It should be noted that each marginal passage constitutes, in effect, a continuation
of one of the radial gutters leading upward between the vesicles of the cardiac
stomach, from a radial esophageal pouch.
Above the marginal openings, the pyloric stomach forms 10 radiating branches,
rather deep in the vertical dimension, two leading towards each ray. In each of
these branches the side walls evaginate and are thrown into coarse folds, forming
what I shall call radial reservoirs, with creamy-white, opaque walls. The radial
reservoir tapers upward and outward, narrowing as the underlying Tiedemann's
pouch expands, and becomes the median duct of one of the paired pyloric caeca
pouches (t) ; vesicles (v) surrounding the central aperture; and the stout retractor harness
sending strands over the radial pouches to attach on the circumferential girdle (arroiv) marking
the boundary between cardiac and pyloric stomach. Approximately 15 X.
FIGURE 3. Aboral parts of the digestive system viewed from below. Arrow indicates the
central lumen of the pyloric stomach, from which the caecum-pouch complexes radiate in pairs
(for details of these parts, see following figures). Note the well-developed rectal caeca (re).
Approximately 15 X.
FIGURE 4. Basal parts of one pair of caecum-pouch complexes, cut off from central attach-
ments and spread apart to show components. Tiedemann's duct widens into Tiedemann's pouch
(Tp), with gutter (g) forming its oral margin; above, the median duct (nid) of the caecum
widens into the thick-walled radial reservoir (rr) by which it connects to the pyloric stomach.
Approximately 15 X.
FIGURE 5. A single caecum-pouch complex, cut off at left from its attachment to the pyloric
stomach, showing relationship between Tiedemann's pouch (note parallel channels traversing its
walls), radial reservoir, median duct, and lateral diverticula. Approximately 5 X.
376
1OHN MAXWELL ANDERSON
FIGURE 6. Extreme lower end of cardiac stomach ("esophagus") ; note the heavy partitions
extending upward from the connective-tissue layer ; the tall epithelial cells with apparently
empty distal ends ; and the strands of secretory granules marking locations of the few zymogen
cells in this area. Phosphotungstic acid hematoxylin ; 470 X.
DIGESTIVE SYSTEM OF HENRICIA 377
(Figs. 4, 5). The first of the lateral diverticula of the pyloric caecum leaves the
median duct somewhat beyond the end of the radial reservoir.
The figures clearly demonstrate that the pyloric caecum-Tiedemann pouch
complex is dual in nature. The aboral part, consisting of the median duct with
its many lateral diverticula, has its affinities with the radial reservoir, of which it
appears to he a continuation. Tiedemann's pouch, on the other hand, altogether
different in gross appearance, originates almost at the junction between cardiac
and pyloric portions of the stomach and is evidently closely related to the lower
region. The two ducts, the median duct and what we may call Tiedemann's duct,
accompany each other and are closely bound together, but their cavities are com-
pletely separated by a continuous partition. Passing outward, the lower duct
becomes a gutter that forms the oral margin of the deep, narrow pouch, and the
body of the pouch itself extends between this gutter and the median duct of the
pyloric caecum above. Throughout its length, the side walls of the pouch are
traversed by a series of diagonal, parallel lines, giving the external appearance of
the "regularly spaced oblique folds" remarked by Cuenot (1887). Proceeding
outward, the depth of the pouch decreases gradually, and at its outer end (about
two-thirds the length of the caecum from its base) the oral gutter enters the floor
of the median duct of the caecum (Fig. 5).
The roof of the pyloric stomach shows folds corresponding to the radial
branches of the walls ; these converge centrally upon the opening of the intestine.
I can add nothing to Hayashi's description of the anatomy of this portion of the
digestive tract, or of the rectal caeca which are extraordinarily well developed in
Hcnricia (Fig. 3).
B. Histology
Histologically, the gut wall of Hcnricia presents the general features that appear
to be standard throughout the Asteroidea. The usual tissue layers are present-
peritoneum, muscular layers, connective-tissue layer, nerve plexus layer, and
lining epithelium — and occupy the same relative positions as in other starfishes.
The chief inadequacies of Hayashi's otherwise good account describing these
layers involve his omission of significant details concerning the lining epithelium.
This is basically a very tall layer, composed of what may be termed "typical"
cells — long, slender cells, crowded together, \vith their ovoid nuclei lying at
varying levels in the epithelium ; each cell is provided, usually, with a single
flagellum, springing from an apical basal body that sends a tapering fibril down-
ward towards the nucleus. Better developed in some parts of the stomach than
in others, longitudinal supporting fibrils also run through the basal portions of the
FIGURE 7. Adjacent section, showing intense metachromatic staining with toluidine blue
in the distal areas that do not stain with PTAH. The faint coarse granules scattered in the
epithelial cells are also metachromatic, as are the contents of the mucous goblets. 470 X.
FIGURE 8. Section across a radial pouch, low in the cardiac stomach, showing tall, flagel-
lated epithelium and relative scarcity of secretory cells in the pouch itself. Note also the rela-
tionship of the retractor strands to the connective-tissue layer in this region. Periodic-acid-
Schiff, Weigert hematoxylin, fast green; 235 X.
FIGURE 9. Detail of epithelium in a radial pouch. Note the tall, crowded, flagellated cells
with their conspicuous brush border, and the well-developed nerve plexus layer through which
bases of the epithelial cells extend to attach on the connective-tissue layer. Alcian Blue-
carmalum ; 470 X.
378
JOHN MAXWELL ANDERSON
12
FIGURE 10. An interradial pouch area, showing abundance of zymogen cells; note also the
scanty nature of brush border development and flagella. PTAH ; 235 X,
DIGESTIVE SYSTEM OF HENRICIA 379
cells and enter the roots that attach to the surface of the underlying connective-
tissue layer. The apical ends of typical cells are provided with a brush border,
sometimes rather scanty, about the base of the flagellum. Variations in such
typical cells, and in the distribution of different types of secretory cells among
them, form the basis of the marked regional specializations characteristic of the
digestive tract in Hcnn'cia.
At the extreme oral end of the cardiac stomach, in the region termed
"esophagus" by Hayashi and others, the epithelium lies in numerous folds, sup-
ported by lamellar inward extensions from the basal connective-tissue layer.
Scattered among the typical epithelial cells here are occasional clumps or strings
of secretory granules, and somewhat more numerous tall, flask-shaped mucous
goblets with distorted, deeply staining basal nuclei. These features are brought
out by staining with phosphotungstic acid hematoxylin (PTAH) and are illustrated
in Figure 6. By far the most conspicuous aspect of the epithelium in this region,
however, concerns the clear and empty appearance of the apical ends in all of the
otherwise typical cells here; PTAH stains nothing (other than flagellary basal
bodies) in the distal quarter or so of these cells. In contrast (Fig. 7), treatment
of adjacent sections with dilute toluidine blue elicits a most vivid gamma meta-
chromasia in precisely those parts of the cells that fail to stain with PTAH. The
same regions are stained by Harris' hematoxylin, selectively colored by Alcian
Blue, and even after salivary digestion give a positive reaction with the periodic-
acid- SchifF technic. Such staining behavior indicates that the apical portions
of the typical cells in this portion of the stomach are filled with masses of an acid
mucopolysaccharide, and this is the only place in the entire digestive tract where
such materials are found to be so intimately and so copiously associated with
"typical" epithelial cells. Metachromatic staining, along with the other reactions
described for these cells, is also exhibited by the globular or flocculent contents of
the mucous goblets, by material associated with the brush border, and by numerous
rather coarse granules or deposits randomly scattered in the deeper parts of the
epithelial cells (Fig. 7).
Above this region the metachromatic staining of the typical cells gradually
disappears, except for that associated with the brush border, and the wall of the
stomach differentiates into its radial and interradial pouches. A clear distinction
can usually be made between these two series of pouches on the basis of their
epithelial characteristics. In the relatively narrow and deep radial pouches, the
epithelium consists almost entirely of typical cells, very crowded, with conspicuous,
long flagella and well-developed apical brush borders (Figs. 8. 9) ; in these areas
there are few secretory cells, but some zymogen cells are present and some mucous
cells containing flocculent secretion. By contrast, the larger and broader inter-
radial pouch areas exhibit an epithelium very rich in mucous goblets and with
relatively large numbers of zymogen cells (Figs. 10, 11). Although the typical
FIGURE 11. Mucous gland cells in an interradial pouch area comparable with that shown in
Figure 10. Periodic-acid-Schiff after salivary digestion, Weigert hematoxylin, fast green.
Note the tall, slender, deeply-staining mucous goblets, and the diffuse positive stain in the distal
parts of the epithelium after removal of all glycogen. 470 X.
FIGURE 12. Radial gutter in cardiac stomach, upward extension of a radial pouch. Tall,
crowded, conspicuously-flagellated cells, well-developed nerve plexus layer ; note also the large,
darkly-stained mucous goblet. PTAH; 235 X.
380
JOHN MAXWELL ANDERSON
15
FIGURE 13. Detail of a region similar to that shown in Figure 12. Note bulbous mucous
goblets, with scattered spindle-shaped bodies deep in the epithelium (cf. Fig. 16). PTAH ;
470 X.
FIGURE 14. Frontal section through the central aperture leading into the pyloric stomach,
showing secretory type of epithelium with numerous mucous glands, scanty brush borders and
flagella. PAS-Weigert-fast green, salivary digestion; 235 X.
FIGURE 15. Frontal section through portion of radial reservoir of the pyloric stomach,
stained to reveal the abundance of zymogen cells in this region. PTAH; 235 X.
DIGESTIVE SYSTEM OF HENRICIA 381
cells that are found here show flagella on close examination, these are sparse and
relatively short, and the brush borders are scanty. These general distinctions
in epithelial characteristics follow consistently upward through the cardiac stomach ;
the crowded, flagellated, conspicuously brush-bordered cells of the radial pouches
continue upward through the radial gutters, crossing into the pyloric stomach as
tracts leading to the marginal openings in its walls, with the addition of a large,
dense, bulbous type of mucous gland cell (Figs. 12, 13). The richly secretory
epithelium of the interradial pouch areas, with its short, scanty flagella and brush
borders, extends to clothe the centrally-directed vesicles of the cardiac stomach
and even to furnish the characteristic lining of the floor and walls of the branching
pyloric stomach (Fig. 14). Zymogen cells are numerous throughout, but par-
ticularly in the radial reservoirs (Fig. 15). All of these areas also contain very
abundant mucous cells, varying from tall, slender goblets with fine granular
secretion to large, flask-shaped glands filled with coarse, homogeneous globules,
resembling those encountered in the radial gutters. These may represent different
secretory phases of a single basic type of mucous gland. In the central folds
between radial branches of the pyloric stomach the epithelium exhibits an interest-
ing feature ; interspersed among the upper ends of the tall, crowded epithelial cells
lie small, spindle-shaped bodies staining deeply with the hematoxylins or with
carmalum. They increase in abundance in the folds of the radial reservoirs and
are particularly numerous and conspicuous in areas where opposite side walls
are closely apposed (Figs. 16, 17a. 17b). They are, at least in these areas, almost
entirely limited to the upper third or quarter of the epithelium and so are in
reasonable proximity to the lumen. These elements resemble most strongly the
bodies described by Smith (1937) as primary sense-cells in Marthasterias; they
are very much like the sense cells Smith found in the radial nerve cord and bear
a less striking resemblance to the cigar-shaped nuclei of the cells he interprets
as representing the same elements in the lining of the stomach (see also Anderson,
1954). The deeply-staining spindles in my preparations of Henncia are evidently
nuclei, sometimes granular but most frequently dense and homogeneous. The
cytoplasm to which they belong cannot be distinguished, crowded as these bodies
are in the great profusion of secretory cells (Figs. 17a, 17b) ; it probably consists,
as Smith's Figure 5 suggests (1937, p. 123), of slender, fibrous processes running
downward from the free border of the epithelium into the nerve plexus layer below.
Diligent search for similar bodies in the cardiac stomach reveals a number of
spindle-shaped nuclei deep in the epithelium of the flagellated tracts, usually in
association with large mucous glands (Fig. 13).
In each radial reservoir, an abrupt line of transition is evident where the richly
secretory side- wall epithelium gives way to the conspicuously- flagellated typical-
cell epithelium (Fig. 18) that covers the roof of the pyloric stomach and continues
as the lining of the intestine.
The "granulated gland cells well-stained by eosin" mentioned by Hayashi
(1935, p. 10) as a component of the epithelium in the cardiac stomach, and as
being present in suddenly increasing numbers in the pyloric stomach, cannot be
identified, unless they correspond to what I have called zymogen cells. In my
preparations the zymogen granules stain neither with eosin nor with Harris'
hematoxylin ; they appear as clear, refractile spherules crowrded in an eosinophilic
ground cytoplasm. This, it will be noted, may offer an answer to the puzzling
382
JOHN MAXWELL ANDERSON
19
FIGURE 16. Side wall of a radial reservoir, showing abundance and localization of spindle-
shaped nuclei interpreted as pertaining to sensory cells. PTAH ; 470 X.
DIGESTIVE SYSTEM OF HENRICIA 383
problem : the cells, as Hayashi says, are granulated and do stain well with eosin ;
but the granules themselves are not eosinophilic.
The duality so evident in the gross anatomy of the pyloric caecum-Tiedemann
pouch complex is revealed also in the histology of the organs. The caecum and
its median duct share epithelial characteristics with the central pyloric stomach
and its radial reservoirs, while the histological affinities of the pouch are, not
surprisingly, with the lower part of the pyloric stomach and even with the radial
pouches and gutters of the cardiac stomach. It will be recalled that these areas
form continuous flagellated tracts in the stomach, leading directly into the open-
ings forming the roots of the paired Tiedemann pouches. Figure 19 illustrates
the striking histological contrast between the upper and lower parts of the caecum-
pouch complex.
As each radial reservoir of the pyloric stomach tapers to form the median
duct of its caecum, the proportion of zymogen cells in its epithelium increases
to a maximum. The roof and side-walls of this median duct present a greater
concentration of zymogen cells than any other part of the caecum ; the folded walls
of the lateral diverticula branching from the duct always contain large numbers of
such cells (Figs. 19, 20), but nowhere in these lateral areas do they occur in such
profusion as in the median duct. In the scattered zymogen cells of the diverticula,
but not in the crowded ones of the duct, the masses or strings of granules are
commonly accompanied by clear vacuoles (Fig. 21), a condition previously noted
in the caecal zymogen cells of Asterias forbesi (Anderson, 1953) and still without
explanation. The epithelium of the median duct, and to a lesser extent that of
the lateral diverticula, also contains numerous mucous goblets like those found
in the pyloric stomach, as well as a considerable representation of the dense
spindle-shaped bodies interpreted as pertaining to sense-cells. These last are
very difficult to find in the lateral diverticula and particularly so in the outer parts.
Beyond these special features, the pyloric caeca present little to distinguish
them histologically from those of Asterias forbesi (Anderson, 1953). Most of the
cells making up the epithelium of the lateral diverticula are relatively undifferen-
tiated. with flagella and apical brush borders, and evidently function chiefly as
absorptive and storage cells. Most of them contain several to many coarse,
granular bodies, usually lying below the level of the nucleus (Fig. 21). These
are highly basophilic and are Schiff -positive after periodic acid oxidation, resist-
ing salivary digestion ; they are not metachromatic and do not stain with Alcian
Blue. Although this is insufficient for specific characterization, the staining
behavior of these bodies suggests that they consist of some type of mucopoly-
saccharide that may be serving as a nutritional reserve. In contrast to the storage
cells in the caeca of Asterias, those of Henricia appear to contain very little glycogen
FIGURES 17a, 17b. Details of sense-cell nuclei. PTAH ; 1100 X.
FIGURE 18. Sharp transition zone (arrow) between richly secretory side-wall epithelium
of radial reservoir (bclozc) and current-producing, flagellary epithelium of roof (above).
PTAH ; 470 X.
FIGURE 19. Overall cross-section of a caecum-pouch complex; the pouch (belozv) normally
hangs straight down (cf. Fig. 5) but has been bent over in histological processing. Note the
marked localization of deeply-staining zymogen cells in the median caecal duct (upper right),
and the arrangement of the parallel flagellated channels traversing the pouch from the gutter
(lower left) to the lower part of the median duct. Seam-cell adhesions separate adjacent
channels. PTAH ; approximately 75 X.
JOHN MAXWELL ANDERSON
22
FIGURE 20. Frontal section through side wall of median duct, showing relationship between
its lumen (md) and the lateral diverticula branching from it. Note also distribution of zymogen
cells (cf. Fig. 19). PTAH ; approximately 75 X.
DIGESTIVE SYSTEM OF HENRICIA 385
or lipicl. This condition is perhaps related to the fact that my specimens of
Hcnricia Ici'iiiscnla, from which tissues were taken for the periodic-acid-Schiff
and Sudan black technics, were collected just as they entered their breeding season.
Although detailed studies involving Hcnricia have not been made, the work of
Greenfield, Giese, Farmanfarmaian and Boolootian ( 1958) has conclusively demon-
strated for two other starfishes that glycogen and particularly lipids are transferred
from storage in the caeca for utilization in the ripening gonads as the breeding
season approaches.
Along a line at the lower edge of the median duct, below the origins of the
lateral diverticula, the epithelium changes abruptly (Figs. 19, 23). This line
marks the junction between Tiedemann's pouch and the caecum proper. Within
the pouch, the epithelium of the side walls is almost a pure population of "typical
cells" ; zymogen cells are few and widely scattered, and mucous cells, as will be
seen, are strictly localized according to a definite pattern. The typical cells are
extremely crowded and laterally compressed ; the nuclei lie in a wide band of
varying levels, a brush border is well developed, and each cell bears one or two
(difficult to determine) long and powerful flagella, with conspicuous, elongate
basal bodies and intracellular fibrils.
The floor of Tiedemann's pouch is narrow, formed by a gutter leading outward
from one of the marginal openings low in the pyloric stomach. The epithelium
lining this gutter, contains an abundance of large mucous gland cells of the dense,
bulbous type, their swollen cavities packed with globular secretion masses distort-
ing their own nuclei and crowding the neighboring epithelial cells in all directions
(Figs. 22, 24). Deep in the epithelium, between the mucous goblets, lie a
considerable number of spindles, like those seen in similar locations in the cardiac
stomach.
At regular intervals, bands of the crowded mucous-epithelial cells pass diago-
nally upward from the gutter to the low^er margin of the caecum above, traversing
the normal epithelium of the side walls of the pouch as a series of parallel
striations clearly visible through the wall of the organ (see Figure 5). A trans-
verse section of the caecum-pouch complex, such as that shown in Figure 19,
cuts across several of these striations ; examination reveals that each is a line
of adhesion between corresponding bands of mucous-epithelial cells on facing
walls of the pouch. These seams divide the pouch into approximately 30 parallel
channels (in an average-sized animal), running diagonally upward from the gutter
to join the median duct of the caecum, each channel lined on both its side walls
by a type of epithelium evidently highly specialized for the production of powerful
flagellary currents. The seams or lines of adhesion separating adjacent channels
involve bands of tissue several to many cells wide, in which the nuclei in both of
FIGURE 21. Detail of epithelium in a lateral diverticulum. Note vacuoles associated with
zymogen granules, and the abundant coarse granules below the nuclear level, interpreted as
mucopolysaccharide deposits. PTAH ; 470 X.
FIGURE 22. Cross-section of the oral gutter of Tiedemann's pouch, bounded at top by an
adhesion-seam. Note the crowded current-producing cells in the side wall, the few and
scattered zymogen cells, the abundant mucous gland cells in the floor of the gutter (cf. Fig. 24),
and the thickened tracts (clear basal circles) of the nerve plexus layer in the floor of the
gutter. PTAH; 470 X.
FIGURE 23. Transition zone marking the junction between Tiedemann's pouch and the
pyloric caecum, where the current-producing typical-cell epithelium of the pouch gives way to
the secretory and storage-cell epithelium of the caecum. PTAH; 470 X.
386
JOHN MAXWELL ANDERSON
I
26
|
FIGURE 24. Frontal section, side wall of oral gutter in Tiedemann's pouch, to show the
abundance of huge, bulbous mucous glands characteristic of this area. Note also spindle-
shaped nuclei scattered deep in the epithelium. PTAH ; 470 X.
DIGESTIVE SYSTEM OF HENRICIA 387
the apposed epithelia lie exceptionally high in the tissue. In section (Fig. 25)
they give the impression of having heen heaved or herniated outward from the
basement membrane by pressure from the swelling bands of mucous gland cells
between which they are confined.
The localization of the herniated seam-cells and their flanking bands of mucous
cells, and the regular alternation of these zones of adhesion and the flagellated
channels which they separate, are clearly shown in Figure 19. Although their
nuclei have been raised almost to the level of the free edge of the epithelium, the
seam-cells retain their attachment to the basal connective-tissue layer by way of
elongated, compressed cytoplasmic stalks in which supporting fibrils are strongly
developed. These intracellular fibrils continue upward past the nuclear level and
form a characteristic and conspicuous feature of the distal ends of the joined cells
(Figs. 25. 26). They do not, however, cross the plane of fusion to join the
fibrils of cells in the opposite walls, and I have seen no other evidence of actual
cytoplasmic continuity between the adherent cells ; in favorable preparations a
distinct double membrane can be observed at the seam-line. Nevertheless, the
adhesion-seams are firm, close, and evidently permanent ; when the tissue is broken
or torn at a seam in the course of histological manipulations, it is noteworthy that
the seam almost never tears at the line of fusion but rather just below the nuclei
of the seam-cells of one side or the other.
The distal ends of the seam-cells contain a number of moderately coarse
granular deposits which in their staining behavior resemble the Schiff-positive
globules of the storage cells in the pyloric caecum. As in the caecum, very little
glycogen or lipid appears in any of the cells of Tiedemann's pouch. Oddly, the
only conspicuous sudanophile inclusions in the pouch areas lie in the distal ends
of the seam-cells, appearing as discrete droplets scattered in the supranuclear
cytoplasm (Fig. 27). The significance of lipid localization in these highly
specialized and aberrant cells is problematic.
The sense-cell spindles identified deep in the epithelium of the gutter accompany
the adhesion-bands also. Here they lie among the swollen mucous goblets (Fig.
25) and are often compressed against the elongate, fibrous stalks of the seam-cells.
Smith has noted that in Marthasterias the sense-cells occur principally in those
areas of the gut where the subepithelial nerve plexus layer is particularly well
developed. In this connection it is of interest that in Henricia the floor of the
gutter is underlain by several notably thickened tracts of the plexus layer (Fig. 22),
and that similar thickenings accompany the specialized adhesion-tracts and unite
with conspicuously well-developed areas of the plexus layer along the line of
attachment between Tiedemann's pouch and the caecum (Fig. 23). The corre-
spondence in distribution between the thickenings of the plexus layer and the
FIGURE 25. Detail of a seam-cell adhesion-band in Tiedemann's pouch. Note the apparent
herniation of the seam-cell nuclei upward, and the attachment of these cells to those opposite
in the epithelium across the lumen. The seam-cells maintain attachment to the basal connective-
tissue layer ; note the band of bulbous mucous goblets flanking the seam-cell zone on both
sides, scattered sense-cell nuclei, and crowded typical-cell epithelium in channels to left and
right. PTAH; 1100X.
FIGURE 26. Distal ends of apposed seam-cells. Note that the intracellular fibrils are not
continuous across the line of fusion. PTAH ; 2100 X.
FIGURE 27. Sudanophilic droplets in distal ends of seam-cells. Sudan black-carmalum ;
470 X.
388
JOHN MAXWELL ANDERSON
i
Ik
31
FIGURE 28. A pair of caecum-pouch units in Patiria, from below. Note the separate
origins from the pyloric stomach, and the broad, bag-like nature of the Tiedemann's pouches.
Dissection of a living specimen, approximately 5 X.
DIGESTIVE SYSTEM OF HENRICIA 389
concentrations of sense-cell spindles in Tiedemann's pouch seems unlikely to be
merely coincidental.
C. Function
The relatively small size of the cardiac stomach, and the absence of voluminous
gastric pouches, make it appear improbable that feeding in Henricia involves
aversion of the stomach to the extent characteristic of Asterias or Patiria. Living
specimens at rest are frequently observed with the mouth widely open, with folds
of the lower cardiac stomach lying over the peristomial lips. Within the stomach
the lumen is largely occupied by the swelling vesicles of the upper cardiac stomach,
framing the narrow, stellate opening into the pyloric stomach. In some specimens,
these vesicles appear partially everted through the mouth, where they form lobe-
like structures extending only about a millimeter. It is to be noted that the
stomach is provided with a stout muscular and fibrous retractor harness which,
while differing markedly from that described for Asterias and Patiria (Anderson,
1954, 1959), still appears much too well-developed to be merely an anchor for
the resting stomach. The action of the stomach suggests that the retractors
function in securing the vesicular lobes, adjusting their degree of eversion, and
maintaining the folds or gutters between them.
The organization of the various flagellated tracts, ducts, and channels suggests
that the digestive tract in Henricia is particularly well adapted for the production
of currents and the transport of suspended particles. The existence of such
currents, and their intricate interrelationships within the digestive tract, can be
demonstrated by the use of suspensions of India ink or stained yeast cells.
Particles move rapidly upward through the radial pouches and gutters of the
cardiac stomach, into the pyloric stomach, and out through the marginal openings
into Tiedemann's ducts. In dissected specimens, suspensions introduced into
one of these ducts are observed to pass distally with astonishing rapidity through
the gutter of the pouch, from which successive streams are diverted and directed
upward through all the diagonal flagellated channels into the median duct of the
pyloric caecum above. Reaching this, particles move into the lateral diverticula
of the caecum, up their side walls, and back across their roofs into the aboral part
of the median duct. Here, a strong centrally-directed current streams inward,
moving the suspension across the folded surfaces of the radial reservoir and into
the narrow central lumen of the pyloric stomach. Currents converging here from
all the radial reservoirs pass downward into the lower part of the pyloric stomach,
where particles again come under the influence of the radial flagellated gutters
and the currents setting towards the marginal openings and into Tiedemann's
FIGURE 29. Detail of similar structures. Note the oral gutter on the margin of Tiede-
mann's pouch, and the fold-patterns traversing the side walls. Approximately 10 X.
FIGURE 30. Cross-section of a caecum-pouch complex. Note the absence of flagellated
channels or adhesion seams, the relationship between the broad lumen of the pouch and the
median duct (md) of the caecum above, and the crowded band of typical cells (arroiu) at the
junction between pouch and caecum. Zymogen cells are also visible in the epithelium of the
lateral diverticula. PTAH ; 75 X.
FIGURE 31. Sudanophilia in side-wall epithelium of Tiedemann's pouch, Patiria. Note
that the mucous-gland patch (clear center area) is flanked on each side by a band of highly
sudanophilic cells which gradually fades into a typical-cell epithelium. These alternating bands
are responsible for the appearance of parallel folds in the side walls. Sudan black-carmalum ;
470 X.
390 JOHN MAXWELL ANDERSON
ducts. A complete and very active circulation through the digestive tract is thus
maintained, featuring one-way transport of solutions or suspensions by specifically-
directed flagellary currents. Sorting mechanisms undoubtedly occur; in the radial
reservoirs, for example, selected particles are probably carried across the roof of
the pyloric stomach and into the intestine for elimination. But it is clear that a
given particle, until thus eliminated or until completely digested, can make in-
numerable circuits through the secretory regions of the digestive system. There
are regional differences in transport velocity — currents in the lateral diverticula of
the caeca, for instance, move much more slowly than those in Tiedemann's ducts
and pouches — but there appear to be no static areas and no chambers in which
large masses of ingested food can lie while undergoing dissolution.
D. Tiedemann's poucJies in other starfishes
As pointed out by Cuenot (1887), members of the Family Echinasteridae
(Echinaster, Henricia} share with those of the Family Asterinidae (Asterina,
Patiria} conspicuous development of the organs we are calling Tiedemann's
pouches. In Patina nriniata, for example, the paired caeca of a single ray originate
separately from the pyloric stomach, and a well-developed, elongate pouch hangs
from the oral midline of each caecum (Figs. 28, 29). The lower margin of the
pouch is formed by a tubular gutter, taking its origin, like the similar structure in
Henricia, from an opening in the wall of the stomach just above the circumferential
fibrous girdle. As in Henricia also, the walls of the pouch are traversed by a
regular series of parallel folds, visible externally, that run diagonally upward
from the gutter to the median duct of the pyloric caecum above. The lateral
diverticula of the caecum form complexly folded pockets arising at intervals from
the lateral-aboral walls of the median duct. Although superficially similar, the
caecum-pouch complexes in Patiria and Henricia present many fundamental dif-
ferences. Patiria lacks, for example, the specialized portions of the pyloric stomach
termed radial reservoirs in Henricia; Tiedemann's pouch and the median duct of
the caecum open into the pyloric stomach by a common aperture, tall and narrow
but not divided. Histological comparison reveals further contrasts (Fig. 30).
Within the caecum, zymogen cells, mucous gland cells, and storage cells are more
or less uniformly distributed, without the marked segregation of zymogen cells
in the median duct so conspicuous in Henricia. It will be noted also that Tiede-
mann's pouch is more broadly attached below the caecum, and that the pouch
itself is broader and more bag-like. This is explained by the fact that, as Figure
30 shows, the opposite walls of the pouch are not held together by mucous-
epithelial adhesions as in Henricia but are only approximately parallel and not
closely apposed. The conspicuous striations in the walls, superficially so similar
to those of Henricia, are produced by parallel bands of large, crowded mucous
gland cells alternating with bands of extremely crowded, very tall "typical" cells.
A notable concentration of these non-secretory cells is always found in the zone of
attachment where Tiedemann's pouch joins the median duct of the caecum. No
zymogen cells are found in the pouch. The distribution of lipids is significantly
different from that in the pouch of Henricia. All of the crowded typical cells
show accumulations of sudanophile droplets in their basal portions, even extend-
ing through the thin strands that penetrate the thick nerve plexus layer and
DIGESTIVE SYSTEM OF HENRICIA 391
insert on the connective-tissue sheet. (Fig. 31). The mucous gland cells contain
no notable lipid deposits, but each of the mucous bands is flanked by strips in which
the typical cells contain exceptionally high concentrations of lipid droplets. These
fade gradually into the normal typical cells in the areas between the mucous bands.
There are no cells here comparable to the herniated seam-cells of Henricia with
their apical concentrations of sudanophile droplets.
Suspensions of stained yeast cells introduced at the opening leading into one
of the pouches are carried through a pattern of circulation very similar to that
observed in Henricia. Even though the walls of the pouch do not adhere to form
separate channels, the concentrated flagellary bands are capable of producing rapid
currents that distribute materials along the entire length of the median duct.
There are no quiet or stagnant areas ; whatever enters the pouch moves immediately
into the caecum, then back to the pyloric stomach and outward through the pouch
again. The general pattern described by Irving (1924) in the caecum-pouch
complex is verified, although the anatomical relationships shown in his figure
(p. 117) are inaccurate.
Without going into detail, brief anatomical and histological examination of
the digestive tract of Asterina gibbosa shows that the features of its pyloric
caecum-Tiedemann pouch complex are very similar to those of its close relative
Patiria and differ in the same ways from those of Henricia.
A figure sketched by Richters (1912) in his study of regeneration in Linckia
led me to suspect that the pyloric caeca of this starfish might also bear Tiedemann's
pouches, although this has not been reported in the literature and Linckia is placed
in a different order (Phanerozonia) from that to which the asterinids and echin-
asterids belong (Spinulosa) (see Hyman, 1955, pp. 334, 336-337). Preliminary
studies on preserved specimens of Linckia guildingi reveal that Tiedemann's
pouches are indeed present and are larger and better-developed than those of any
other species examined so far. They extend almost to the distal ends of the long
pyloric caeca, and their proximal ducts are separated by partitions from specialized
portions of the pyloric stomach resembling the radial reservoirs of Henricia.
Transverse sections of the pouches show that adhesion-bands similar to those of
Henricia divide the pouch into separate flagellated channels. The gutter forming
the oral margin of the pouch is divided by a fold arising from its floor, which
externally encloses a stout band of muscle fibers. Altogether, although details
have not been exhaustively studied, it is evident that the caecum-pouch complex
in Linckia is strikingly similar in its fundamental characteristics to that described
in Henricia. This is the more interesting in view of the wide taxonomic gap that
apparently separates the two genera.
Astropecten, in which Tiedemann (1816) originally described the pouches that
now bear his name, has very small ones extending a centimeter or less along the
oral side of the median duct of the caecum. Their side walls show a few moder-
ately developed parallel striations, but in the preserved material on which I have
made a preliminary study these do not form firm adhesions across the lumen.
DISCUSSION
There are mam- anatomical and histological differences between the organs
of the digestive tract in Henricia and those of other starfishes in which details
JOHN MAXWELL ANDERSON
have been described. The small size of the cardiac portion of the stomach, the
large size and peculiarly branched structure of the pyloric portion, the conspicuous
development of the rectal caeca, and other features, were noted by Hayashi (1935).
Further, the cardiac stomach of Hcnricia lacks any hint of the elaborately branch-
ing patterns of flagellated gutters, with accompanying ramifications of the retractor
strands and corresponding localizations of specialized epithelial cells, characteristic
of the cardiac stomach in Astcrias, Pisaster, Pycnopodia, and Patiria (Anderson,
1954, 1959). Lacking these, the stomach of Hcnricia is not, however, without
specializations of its own, such as the so-called esophageal pouches and the alter-
nating gutters and vesicles at higher levels, with their marked histological dis-
tinctions. Other histological peculiarities are common in various parts of the
digestive tract. For instance, areas in the cardiac stomach and almost the whole
of the pyloric stomach are lined with zymogen cells ; in contrast, the zymogen
cells of Asterias jorbesi and Patiria miniata are found only in the pyloric caeca.
Within the caeca of Hcnricia, the startling concentrations of zymogen cells in
the walls of the median ducts are unique ; in Astcrias, this area is given over to a
population of tall, crowded, current-producing cells interspersed with mucous
goblets, and the zymogen cells are restricted to the lateral diverticula.
Such instances of structural contrast could be multiplied, all leading to the
conclusion that in starfishes, as in other animals, corresponding parts of the
digestive system, clearly homologous, may be adapted to diverse functions in
relation to differences in food and in feeding methods. The food habits and feed-
ing mechanisms of Asterias and similar forms are reasonably well understood ;
that they are probably not at all like those of Henricia, which are unknown, is
indicated by the conspicuous structural differences revealed by detailed study.
In general, the specializations noted in Hcnricia seem to involve a tendency to
segregate tissues with predominantly secretory functions in areas more or less
distinct from those serving primarily for current production. Further, while the
transport of materials through the gut by flagellary currents is common to all star-
fishes, in Henricia this function has been raised to a peak of efficiency by the
development of the special modifications of the pyloric stomach called Tiedemann's
pouches. By means of these elaborate organs, suspensions or solutions brought
to them by the flagellary tracts of the stomach are very rapidly distributed to
the secretory and absorptive areas of the pyloric caeca and pyloric stomach, and
maintained in repeated circulation through these areas until digested, absorbed,
or eliminated.
Studies elucidating the current-patterns maintained within the digestive tract
have been made on a variety of asteroids, including Astcrias rnbens, Solaster
papposus, Porania pulvillus, and Astropcctcn irrcgularis (by Gemmill, 1915) ;
Patiria miniata (by Irving, 1924) ; and Asterias jorbesi (by Budington, 1942).
It is noteworthy that in all of these species, of diverse taxonomic affinities, the
patterns are reasonably similar in general outline, with minor variations related
to anatomical differences. In all, currents flow in the oral-aboral direction from
cardiac to pyloric stomach; radially across the floor of the pyloric stomach and
outward along the oral sides of the pyloric ducts, continuing on the oral side of
the median duct in each caecum ; in a circular fashion through the lateral diverticula
and back to the median duct, this time aborally ; and finally returning toward the
DIGESTIVE SYSTEM OF HENRICIA 393
pyloric stomach again in the aboral part of this duct. In the Asteriidae, particularly,
the pyloric duct is a restricted passage ; within it, however, currents must be main-
tained both centrifugally and centripetally, the one current running on its oral side,
the other on its aboral side.
It is obvious that the feeding and digestive activities of the predatory,
carnivorous starfishes in this family place no demands upon their transport mecha-
nisms that are not satisfied by the system as it exists. Yet, if one speculates upon
ways in which this system might have developed to operate more efficiently for
the maintenance of directed currents in the standard pattern, one is likely to be
struck first by the existence of the bottle-neck at the pyloric duct. For increased
efficiency, the pyloric duct should be larger, it would seem, or at least taller, so
that the centrifugal and centripetal streams might be more widely separated.
Alternatively, a continuous partition should be provided, set horizontally between
the floor and the roof of the duct, for complete isolation of the two currents.
In effect, it will be noted, the pyloric ducts of Patina embody the former principle,
while those of Henricia follow the latter. These considerations seem to justify
the conclusion that in both Patiria and Henricia the functions of the digestive
system have placed a higher premium upon efficiency of the flagellary transport
mechanisms than is the case in Asterias and its close relatives.
Below and distal to the partition that divides the pyloric duct in Henricia,
Tiedemann's pouch has evolved into a hydrodynamic organ or flagellary pump
of prodigious effectiveness. Its design involves close-set, strongly-flagellated side
walls, strengthened, reinforced, and held at what we may assume to be an optimal
distance from one another by the unique mucous-epithelial seams, which also
break up the otherwise broad epithelial expanse into channels set in regular
parallel array. These features make it possible for the organ to develop what
must be a considerable suction, as the force developed in all of the 30 or so flagel-
lated channels is transmitted to the upper cardiac and lower pyloric stomach by
way of the restricted Tiedemann's duct. Fluids and suspensions are drawn
rapidly from the stomach through this duct and are ejected at reduced velocity
along almost the entire length of the median caecal duct, into which the channels
of Tiedemann's pouch lead. It is reasonable to assume that the development of
the 10 Tiedemann's pouches as organs furnishing the principal motive power for
circulation through the entire digestive tract brought about the emancipation of
areas in the pyloric stomach, pyloric ducts, and median caecal ducts from the
primarily flagellary functions they must serve in the Asteriidae, and their de-
velopment of the richly secretory epithelium that is their chief characteristic in
Henricia.
In contrast, Tiedemann's pouches in Patiria are, as we have seen, broad and
bag-like, with no adhesion-seams to hold their side walls in apposition and only their
similarities in origin and anatomical relationships to suggest that they are related
to the pouches of Henricia. The alternating diagonal bands of mucous glands and
typical flagellated cells undoubtedly approach in functional significance the parallel
channels of Henricia, to which they show a superficial resemblance in external ap-
pearance. Irving (1924) concluded that Tiedemann's pouches in Patiria represent
regions specialized for distribution, and all observational evidence bears out this
supposition. It is to be doubted, however, that these relatively crudely-developed
394 JOHN MAXWELL ANDERSON
organs even approach in efficiency the more elaborate ones of Henricia. The
enlargement of the pyloric duct and the median duct of the caecum resulting from
the development of the pouch in Patiria must represent an advance over the condi-
tion that exists in Astcrlas, but one is led to the conclusion that as organs of dis-
tribution, or flagellary pumping organs maintaining circulation within the digestive
tract, the pouches of Henricia are far superior to those of Patiria.'2
Many features of anatomy and histology in the digestive system of Henricia—
the small size and doubtful eversibility of the cardiac stomach, the absence of any
sizable chambers in which masses of food might lie while undergoing digestion, and
the obviously high degree of specialization of mechanisms for maintaining flagellary
circulation, among others — suggest most strongly that this animal is dependent in
its nutrition upon suspended participate matter rather than upon predation or any
other form of macrophagy. The following experiment was performed as a pre-
liminary test of this hypothesis. Two specimens of Henricia sangninolenta, which
had been maintained in an aquarium for 6 weeks, apparently without feeding, were
placed in a fingerbowl containing a cloudy suspension of Mytilus sperm in sea-water,
to which had been added a quantity of minute particles of Nile blue sulfate (spar-
ingly soluble in sea- water). Externally, streams of particles could be observed
converging upon the mouth, most rapidly and conspicuously in the ambulacra!
grooves. The mouths of both specimens remained open, much as in Figure 1, and
while coarse clumps or large particles were rejected, smaller particles were either
swept directly through the radial peristomial grooves at the angles of the mouth or
entangled in strands or sheets of mucus which also moved into the stomach. After
12 hours' exposure to the suspension the animals were carefully washed to remove
adherent particles and then dissected for examination of the digestive tract. In
both specimens, concentrations of dye particles were found at various places in
several of the Tiedemann's pouches.
Such a crude experiment should not be interpreted as having established the fact
that in its normal nutrition Henricia is dependent upon a flagellary-mucous feeding
mechanism or that this suffices for all its needs. The experiment does, however,
provide a demonstration that at least under certain conditions participate matter
suspended in the surrounding water finds its way into the digestive tract. Further,
- I take this opportunity of clearing up a misunderstanding involving functional relation-
ships between the pyloric caeca and Tiedemann's pouches in Patiria. Irving (1926) performed
digestion experiments with isolated surviving caeca of this species and concluded that these
organs are capable of digesting gelatin and absorbing amino acids. Hyman (1955, p. 385)
questions this interpretation on the ground that the pyloric caecum of Patiria ". . . is underlain
on the oral side by an extensive stomach diverticulum (so-called Tiedemann's diverticulum . . .),
so that in Irving's experiments the gelatin solution went into this stomach diverticulum and the
resulting digestion is probably to be attributed to the stomach." It must be pointed out that in
Patiria the cavity of the pouch is everywhere completely open to, and in free communication
with, the median duct of the caecum, so that anything going into the one is very soon distributed
and thoroughly mixed in all parts of the other. The stomach of Patina contains a vanishingly
small proportion of zymogen cells, and it has been experimentally established that digestion
does not occur in the stomach of an animal that has been deprived of all its caecum-pouch
complexes (Anderson, 1959). Zymogen cells do not occur in Tiedemann's pouches in Patiria, and
the only apparent source of digestive enzymes in this species is in the epithelium of the pyloric
caeca themselves. Therefore, while Hyman's criticism was certainly justified in the light of
information then available, the facts that have subsequently been established make it clear that
Irving's conclusions cannot properly be questioned for the reasons advanced by Hyman.
DIGESTIVE SYSTEM OF HENRICIA 395
it shows that in the intact animal particles so ingested pass into the radial parts of
the system by pathways previously deduced on anatomical and histological grounds
and on the basis of experiments with excised organs. In the absence of any other
information on food and feeding in Hcnricia, all the indirect evidence that can be
brought to bear seems consistent with the conclusion that finely-divided participate
matter in suspension, exploited by a flagellary-mucous mechanism, is significant in
the nutrition of this starfish. If this is valid, then the very close and detailed struc-
tural correspondence between the digestive system of Hcnricia and that of Linckia,
particularly in relation to the highly specialized Tiedemann's pouches, makes it
reasonable to extend this conclusion to Linckia also. I can find no information on
the food or feeding habits of Linckia guildingi.
Flagellary-mucous feeding of the type suggested for Hcnricia (and Linckia} is
not unknown among starfishes. The experiments and extensive observations of
Gemmill (1915) on Porania puh'illus demonstrate that alone among the starfishes
tested, individuals of this species are capable of maintaining themselves for long
periods on a diet of suspended particles only. The external and internal currents
by which this diet is manipulated are apparently similar to those described in
Henricia. Gemmill makes no mention of Tiedemann's pouches in Porania, and I
have been unable to determine from the literature whether they are present. The
powerful currents produced by this starfish suggest the presence of some type of
specialization of the general nature of the Tiedemann's pouches in Henricia.
Several species of starfishes known to be chiefly macrophagous have been sus-
pected of utilizing particulate matter, also, to some extent. It is interesting that
these same forms possess more or less well-developed Tiedemann's pouches. For
example, Gemmill (1915) finds that flagellary currents carry considerable amounts
of particulate food into the digestive tract of Astropcctcn, a sand-dwelling starfish
well known as a voracious carnivore. The behavior of Patiria miniata, particularly
with regard to its habitual exposure of the huge, everted cardiac stomach in the
external environment (Anderson, 1959), strongly suggests that it may in this way
collect significant amounts of particulate matter to add to its extremely varied diet.
Astropecten, it will be recalled, has relatively small Tiedemann's pouches, while
those of Patiria are much larger but of simple construction compared with the
elaborate pouches of Henricia and Linckia. The significance of these structures
may lie in the enhancement of circulatory efficiency which even small or simple
pouches may contribute, in relation to feeding currents. The marked structural
similarity between the digestive tract of Patiria and that of Aster ina gibbosa (which
according to MacBride, 1906, subsists principally upon sponges and ascidians) is
strongly suggestive of extensive functional correspondences, perhaps including par-
ticulate feeding.
Resemblances involving visceral organs, and particularly those of the digestive
tract, are usually considered less reliable as indices of taxonomic relationship than,
for example, similarities in skeletal structures or other hard parts. Nevertheless,
it is interesting to consider from this standpoint the fragmentary information avail-
able concerning the distribution of Tiedemann's pouches in starfishes. The similar-
ities between the pouches of Patina and those of Asterina are to be expected in
genera belonging to the same family. Although the flagellary pumping organs of
the Patiria type superficially resemble those of Henricia and both are called Tiede-
396 JOHN MAXWELL ANDERSON
mann's pouches, they are fundamentally very different structures. The resem-
blances are so superficial, and the structural differences so profound, that one is led
to conclude that in the Asterinidae and Echinasteridae these organs have evolved
independently and convergently as solutions to the problem of increasing circulatory
efficiency within the digestive tract. These two families, it may be noted, are by-
all accounts closely related members of the Order Spinulosa.
In contrast, Tiedemann's pouches and other features of the digestive tract in
Linckia are astonishingly similar, at least in the material available to me for pre-
liminary study, to those of Henricia. The similarities are so striking and extend
to such details as to make it appear unlikely that they could have arisen convergently
in unrelated lines of starfishes. Yet the Linckiidae, chiefly on the basis of skeletal
features that are fundamental in asteroid taxonomy, are placed nowhere near the
Echinasteridae but among the valvate Phanerozonia. However, MacBride (1906,
p. 471) makes the following statement about the Linckiidae, interesting in the
present context : "It is possible that these forms, so different in many respects from
the other families of the order, may have been directly derived from the long-armed
Echinasteridae." My observations on structural details of the digestive system
seem strongly to support the idea of a closer relationship between these families
than is indicated by the criteria commonly used in starfish systematics.
It is obvious that any attempt to survey similarities and differences in organ
systems, and particularly to trace the distribution of anatomically and histologically
specialized visceral structures, among the various families and orders of starfishes
will be meaningless until it can be based upon vastly augmented data. These data
accumulate slowly, requiring careful and detailed study of the internal anatomy of
many species on which the literature to date offers only descriptions of external
appearance, skeletal features, geographical distribution, and the like. The present
attempt at a comparative study of Tiedemann's pouches in a small group of star-
fishes is inadequate, based as it is upon insufficient information. At least, however,
it provides a tantalizing glimpse of the surprisingly broad areas of knowledge still
awaiting exploration and interpretation in the Asteroidea.
SUMMARY
1. Detailed study of the digestive system in Henricia reveals that as a result of
regional differentiation, particularly involving the lining epithelium, areas specialized
for zymogenic and mucous secretion are segregated from other areas adapted for
current-production. Secretory areas include the five interradial pouches and ves-
icles of the cardiac stomach, the pyloric stomach generally but particularly its radial
reservoirs, and the median ducts and lateral diverticula of the pyloric caeca. Cur-
rent-producing areas include the five radial pouches of the cardiac stomach and the
gutters leading upward to marginal openings low in the pyloric stomach, and
especially the very elaborate Tiedemann's pouches which spring in pairs from these
openings and extend along the oral midlines of the pyloric caeca. Other starfishes,
such as Asterias, which lack Tiedemann's pouches, restrict their zymogen cells to
the lateral diverticula of the caeca and crowd current-producing cells into the median
caecal ducts, an area which in Henricia contains an extremely high concentration of
zymogen cells.
DIGESTIVE SYSTEM OF HENRICIA
2. Tiedemann's pouches in Henricia are divided into numerous parallel flagel-
lated channels leading diagonally upward into the pyloric caeca. These channels
are separated by unique partitions formed by adhesion-seams between opposite
side-walls of the pouch. It is evident from their structure and anatomical relation-
ships, and has been experimentally demonstrated, that Tiedemann's pouches are
flagellary pumping organs of great effectiveness. They produce currents capable
of drawing suspensions or solutions from the stomach and delivering them rapidly
along almost the entire length of the pyloric caeca. Centripetal currents stream back
into the stomach, and thus a constant circulation of materials can be maintained
through the radial secretory and absorptive areas, depending chiefly upon currents
generated in the close-set channels of the ten Tiedemann's pouches.
3. The customary food and the feeding habits of Henricia are unknown, but
several lines of evidence, anatomical and experimental, combine to suggest that this
starfish, like at least one other (Porania), may subsist either wholly or in part upon
suspended particles gathered by a flagellary-mucous mechanism.
4. In Patina miniata, a species not distantly related to Henricia (same order,
different family), Tiedemann's pouches are present and lie in about the same rela-
tionship to the pyloric caeca but are fundamentally dissimilar ; they lack separate
flagellated channels, the side walls being traversed only by parallel stripes of mucous
cells alternating with bands of typical cells, and thus are more bag-like in general
structure. Although they function similarly to those of Henricia, these much
simpler pouches are probably less effective in current production. Asterina gibbosa,
closely related to Patiria, has Patiria-type Tiedemann's pouches. Astropcctcn, in
which these pouches were originally described (1816), has relatively small ones of
simple construction. Both Patiria and Astropecten have been suspected of supple-
menting their macrophagous diet by flagellary-mucous particle-feeding, and although
their pouches are far less elaborate than those of Henricia they are probably of
significance in this connection.
5. It is disconcerting to find that Linckia, not at all closely related to Henricia
(different order), nevertheless has Tiedemann's pouches and other specializations
of the digestive system similar in most respects to those of Henricia. The differ-
ences between the pouches in Henricia and Patiria are so fundamental as to suggest
that they represent independently evolved solutions to the problem of increasing-
circulatory efficiency within the digestive tract. In contrast, the pouches of Henricia
and Linckia resemble each other so strikingly that it is difficult to conceive of them
as having been produced by convergent evolution.
6. It is pointed out that detailed studies on many species of starfishes now known
only from external anatomy and skeletal features of preserved specimens will provide
information upon which to base broader and more meaningful comparative surveys
of internal specializations.
LITERATURE CITED
ANDERSON, J. M., 1953. Structure and function in the pyloric caeca of Asterias forbesi. Biol.
Bull, 105 : 47-61.
ANDERSON, J. M., 1954. Studies on the cardiac stomach of the starfish, Asterias forbesi. Biol.
Bull., 107 : 157-173.
ANDERSON, J. M., 1959. Studies on the cardiac stomach of a starfish, Patiria miniata (Brandt).
Biol. Bull, 117: 185-201.
398 JOHN MAXWELL ANDERSON
BUDINGTON, R. A., 1942. The ciliary transport-system of Asterias jorbesi. Biol. Bull., 83:
438-450.
CUENOT, L., 1887. Contribution a 1'etude anatomique des Asterides. Arch. Zool. ex p. ct gen,,
Ser. 2, T. 5, bis, Supp. Mem. 2: 1-144.
GEMMILL, J. F., 1915. On the ciliation of asterids, and on the question of ciliary nutrition in
certain species. Proc. Zool. Soc. Loud., 1 : 1-19.
GREENFIELD, L., A. C. GIESE, A. FARMANFARMAIAN AND R. A. BOOLOOTIAN, 1958. Cyclic
biochemical changes in several echinoderms. /. E.rp. Zool.. 139: 507-524.
HAYASHI, R., 1935. Studies on the morphology of Japanese sea-stars, I. Anatomy of Henricia
sanguinolenta var. ohshimai, n. var. /. Fac. Sci., Hokkaido Imp. Univ., Ser. VI,
Zool., 4 : 1-26.
HAYASHI, R., 1940. Contributions to the classification of the sea-stars of Japan, I. Spinulosa.
/. Fac. Sci., Hokkaido Imp. Univ., So: VI, Zool.. 1 : 107-204.
HYMAN, L. H., 1955. The Invertebrates : Vol. IV, Echinodermata. McGraw-Hill, New York.
IRVING, L., 1924. Ciliary currents in starfish. /. E.rp. Zool.. 41 : 115-124.
IRVING, L., 1926. Regulation of the hydrogen ion concentration and its relation to metabolism
and respiration in the starfish. /. Gen. Physiol., 10: 345-358.
LUDWIG, H., AND O. HAMANN, 1899. Echinodermen, II. Buch. Die Seesterne. In: Klassen
und Ordnungen des Thierreichs, H. G. Bronn, Ed. Bd. 2, Abt. 3. Winter, Leipzig.
MACBRIDE, E. W., 1906. Echinodermata. /;;: Cambridge Natural History, S. F. Harmer and
A. E. Shipley, Eds. Vol. I. Macmillan, London.
MORTENSEN, TH., 1927. Handbook of the Echinoderms of the British Isles. Oxford, London.
PEARSE, A. G. E., 1953. Histochemistry — Theoretical and Applied. Little, Brown, Boston.
RICHTERS, C., 1912. Zur Kenntnis der Regenerationsvorgange bei Linckia. Zeitschr. wissen.
Zool., 100: 116-175.
SMITH, J. E., 1937. On the nervous system of the starfish Marthasterias glacialis (L.).
Phil. Trans. Roy. Soc., Ser. B, 227: 111-173.
TIEDEMANN, F., 1816. Anatomic der Rohrenholothurie, des pommeranzfarbigen Seesterne, und
Stein-Seeigels. Thomann, Landshut.
VOGT, C., AND E. YUNG, 1888. Lehrbuch der praktischen vergleichenden Anatomic, Bd. I.
Vieweg, Braunschweig.
THE FEEDING BEHAVIOR AND RESPIRATION OF SOME MARINE
PLANKTONIC CRUSTACEA 1
ROBERT J. CONOVER
Woods Hole Oceanographic Institution, Woods Hole. Mass.
Interest in the respiratory rate and food requirements of the marine zooplankton
probably dates from the studies of Putter (1907, 1909), but data are presently avail-
able for only a dozen or so species of neritic copepods and for Euphausia pacifica
(Lasker, 1960). Paraeuchaeta norvcgica, studied by Raymont and Gauld (1951),
is the only marine copepod for which respiratory measurements are available, which
commonly occurs at depths greater than 100 meters.
In the present work a number of shipboard and laboratory experiments were
performed with oceanic species of copepods, amphipods and euphausids, some from
deep water, to learn if such organisms were amenable to artificially controlled condi-
tions, and to obtain additional respiratory data for a very important but little-studied
group of animals. A few species previously investigated from other localities (i.e.,
Calanus finmarchicus) were also included for comparative purposes.
In addition to the respiratory measurements, representatives of most species
investigated were also kept in laboratory culture vessels where observations were
made on their behavior and food habits. Some individuals of the copepod, Calanus
hyperboreus, have been maintained in the laboratory for approximately one year
which would seem to be the life span for it. Because of its large size, long life span,
and ability to adapt to laboratory conditions, this species is currently the subject of
intensive experimental investigation.
The author would like to express his appreciation to Dr. G. L. Clarke, Dr.
Herbert Curl, Michael Mullin, \Yilliam Dawson, Henry Fuller, John Clarke,
Thomas Renshaw and Masateru Anraku, all of whom gave field or laboratory
assistance during different phases of the work reported here. Dr. Robert Guillard
supplied the phytoplankton cultures and Dr. Rudolf Scheltema supplied Artemia
eggs and certain planktonic larvae used in feeding experiments. Dr. Robert Hessler
supplied the Artemia used in the respiratory experiments.
MATERIALS AND METHODS
The zooplankton organisms used in the present program included the copepods :
Calanus finmarchicus , C. hyperboreus, Paraeuchaeta norvegica, Pleuroinamma ro-
busta, Bathycalanus sp.,- Rhincalanus nasntns, Euchirella rostrata; the amphipods
Phronima sp., Euthemisto comprcssa. and Hypcria galba, and an unidentified
euphausid probably belonging to the genus Thysanoessa. All these animals are of
1 Contribution No. 1115 from the Woods Hole Oceanographic Institution. This research
was supported by the National Science Foundation under Research Grants 3838, 8913 and 8339.
399
400
ROBERT J. CONOVER
large size and easily recognizable with the naked eye, even on shipboard. For the
graphic analyses of respiratory rates use was also made of some data, hitherto un-
published in the present form, for the small neritic copepod Acartia clans i.
The animals were captured with a %-meter or 1 -meter net of mesh size #00
or #000, with a glass jar of one quart capacity secured in the cod-end. Tows were
generally 15 to 30 minutes at depth, but in the case of the deeper tows the time
required to reach the desired depth and to recover the net again increased the total
towing time appreciably. Before the net was brought aboard after fishing, care was
TABLE I
Summary of all experimental data on the respiratory rates of zooplankton
Respiration
rate:
Copepods
Location of experi-
mental tow and
estimated depth
Date
No. &
stage
Experi-
mental
temp.
0 C.
Dry
weight/
animal
mg.
Ml./
animal
lt\./mg.
dry
& day
wt.&
day
Calanus
Georges Bank
Aug. 18, 1958
4V
7.5
0.188
4.4
23.0
finmarchicus
41°00'N
Aug. 18, 1958
4V
7.5
0.188
5.4
28.8
67°35' W
Aug. 18, 1958
15V
7.5
0.232
5.5
23.3
25 m.
Aug. 18, 1958
4 9
7.5
0.211
9.5
44.1
Aug. 18, 1958
15 9
7.5
0.214
9.0
42.0
Aug. 18, 1958
4V
18.5
0.188
9.0
47.3
Aug. 18, 1958
4V
18.5
0.188
8.9
51.9
Aug. 18, 1958
15V
18.5
0.199
8.7
42.0
Aug. 18, 1958
49*
18.5
0.211
15.7
74.2
Calanus
Gulf of Maine
Aug. 20, 1958
15V
8
0.294
4.7
16.2
finmarchicus
42°2S' N
Aug. 20, 1958
15V
8
0.264
3.9
14.8
69°47' \Y
Aug. 20, 1958
15V
8
0.334
3.6
10.9
200 m.
Aug. 20, 1958
4V
8
0.277
5.4
19.5
Aug. 20, 1958
4V
8
0.277
4.7
16.9
Calanus
Slope water
Aug. 19, 1958
7V
<10
1.55
18.3
11.8
hyperboreus
41°46' N
5 9
<10
3.68
24.8
5.5
65°28' W
1 9
<10
3.62
26.0
7.1
1000 m.
Paraeuchaeta
Slope water
Aug. 19, 1958
5 9
<10
3.88
47.6
12.2
norvegica
41°46' N
5 9
<10
3.80
51.5
13.8
•
65°28' W
1 9
<10
4.59
48.7
10.5
1000 m.
Pleuromamma
Slope water
Aug. 15, 1958
3 9
8
0.283
19.0
66.5
robusta
38°28' N
70°59' W
3 9
8
0.283
15.5
54.3
450 m.
Bathycalanus sp.
Slope water
Aug. 19, 1958
1 9
5.1
17.9
163.4
9.1
41°46' N
65°28' W
1000 m.
* 3 animals died.
FEEDING AND RESPIRATION OF PLANKTON
401
TABLE I — Continued
Respiration
rate:
Copepods
Location of experi-
mental tow and
estimated depth
Date
No. &
stage
Experi-
mental
temp.
0 C.
Dry
weight/
animal
mg.
Ml-/
animal
/il./mg.
dry
& day
wt. &
day
Rhincalanus
Slope water
April 22, 1959
2 9
6.5
0.826
13.0
15.7
nasutus
39°48' N
2 9
6.5
1.079
15.3
14.2
71°12' \V
2 9
6.5
1.019
15.0
14.7
400 m.
Enchirella
Slope water
April 22, 1959
3 9
6.5
0.890
22.4
25.2
rostrata
39°48' N
3 9
6.5
0.947
28.6
34.9
71°12' W
3 9
6.5
0.820
25.6
27.0
400 m.
Acartia clausi
Long Island
July 22, 1953
119 9
5.0
0.0041
0.30
73.1
Sound
Jan. 6, 1954
47 9
5.0
0.0047
0.37
78.7
5-10 m.
Jan. 26, 1954
156 9
5.6
0.0073
0.67
91.8
Feb. 2, 1954
49 9
5.0
0.0072
0.69
95.8
July 3, 1954
100 9
5.9
0.0044
0.40
90.9
July 10, 1954
52 9
5.9
0.0049
0.22
42.6
Euphausids
Slope water
Aug. 19, 1958
3 **
5.1
2.67
43.1
16.1
(unidentified)
41°46' N
1
5.1
3.39
105.7
31.0
65°28' W
1000 m.
Amphipods
Phronima sp.
Slope water
Aug. 15, 1958
1
8
27.3
284.9
10.4
38°28' N
70°59' W
450 m.
Euthemisto
Gulf of Maine
Aug. 20, 1958
4
4
4.97
92.7
18.6
compressa
42°25' N
4
4
4.87
87.5
18.0
69°47' W
4
4
4.35
72.3
16.6
200 m.
Hyperia galba
Gulf of Maine
Dec. 4, 1959
1
5.6
3.52
51.2
14.5
42°35' N
1
5.6
8.24
81.0
9.8
69°35' W
1
5.6
4.62
70.7
15.3
250 m.
1
5.6
10.15
114.2
11.2
** 1 animal in poor condition.
taken to have on hand fresh sea water pre-cooled to a temperature at or near that
of the depth fished. The cod-end was immersed in this water and the contents of
the glass jar only were retained. It was assumed that animals crushed against the
meshes of the net were likely to be damaged, so that the net was never washed down
and the entire process of getting the organisms from their natural temperature condi-
tions to conditions simulating them was carried out as rapidly as possible. If the
temperature difference between the surface water and the depth fished was great,
402 ROBERT J. CONOVER
the mortality among the deep-water forms was high despite the precautions taken.
However, the animals were sorted immediately and only the healthy specimens kept
for experimental work.
On shipboard, a portable refrigerator which opened from the top was used as
an experimental laboratory. In the earlier studies, some difficulty was encountered
in regulating the temperature of this box when it was opened frequently. A larger
expansion valve, enabling higher refrigerant pressures, eliminated this difficulty,
and in its present form the box can be kept open for long periods even in summer
without excessive temperature change. On return to land facilities, a constant
temperature room and conventional refrigerated water baths provided supplemen-
tary, controlled-temperature conditions when required.
Since most of the animals used in this study came from water deeper than 200
meters, 5—6° C. was chosen as the temperature at which the experimental and
observational studies would be run, although for reasons mentioned above this was
not attained in every case (see Table I). Initially it was hoped to compare the
respiration of some of the animals at 5° and at surface temperature, to enable the
computation of a rough Q10. However, prolonged exposure to warmer surface
waters proved lethal to most of the experimental material so that except for the
relatively eurythermal Calanns finmarchicus, such a comparative study was not
possible.
Respiration was measured using glass-stoppered bottles of appropriate size.
Animals were placed in a small quantity of water in a bottle, and then the bottle
and contents were flushed several times with water of known oxygen content, using
a siphon arrangement with a bolting cloth screen to prevent loss of the experimental
material. Control bottles were prepared in precisely the same manner except that
the animals were omitted. All bottles were filled completely, taking care to insure
that no air bubbles were included. As Marshall et al. (1935) demonstrated an
increase in respiration for Calanus finmarchicus on exposure to light, all bottles were
placed in black cloth bags during the run, regardless of the experimental conditions.
At the end of a suitable period of time (8 to 48 hours), single samples from the
experimental and the control bottles were siphoned into smaller glass-stoppered
bottles and the oxygen content determined by the Winkler method. The oxygen
utilization was determined from the difference between the bottles containing animals
and those without. (For a detailed description of the method, with a discussion
of its advantages and disadvantages, see Conover, 1956, 1959.)
The animals themselves were generally dried for weighing while in fresh condi-
tion, or in some instances a sample of the same species and stage of animal was dried
as representative of the experimental animals. Animals were weighed on a suitable
quartz helix microbalance made by the Microchemical Specialties Company, Berke-
ley, California. In the case of the smaller organisms such as Calanus finmarchicus
a balance with working sensitivity of 2 mg. ± 1 /j,g. was used. For larger forms a
20 mg. ±10 fig. balance was employed. Aside from the accuracy of weighing in
this manner, the process is extremely rapid and largely free from errors due to
sudden temperature change, varying humidity, etc. In a few instances, the animals
were too large for either helix and they were weighed on a conventional ana-
lytical balance.
In the respiration experiments, as well as in laboratory culture studies, antibiotics
FEEDING AND RESPIRATION OF PLANKTON 403
were used to control bacterial growth and respiration. Dihydrostreptomycin sulfate
in concentration 50 mg./L. was generally used in respiratory studies, sometimes
supplemented with 10 mg./L. of chloromycetin. Chloromycetin was found to have
an inhibitory effect on the feeding of Calanus fininarchicns when used at 50 mg./L.
(Conover, Marshall and Orr, 1959). Since oxygen utilization attributable to
bacteria was generally less than 0.1 ml./L., even in 48- hour experiments, with
streptomycin alone, chloromycetin was eventually eliminated altogether from the
experimental procedure.
For laboratory culture experiments, the organisms were kept in polyethylene
"freezer containers" of pint, pint and a half, quart, or gallon size, depending on the
size and number of animals to be cultured. These containers proved non-toxic to
all animals tested, and, when fitted with their plastic tops, were safe from spillage or
breakage in a rough sea. For most organisms, one or two animals in the pint-sized
container proved most satisfactory ; the containers then could be conveniently stacked
four or five high without danger of upsetting, and in this size the sides are low
enough to permit easy observation with a dissecting microscope.
Sea water for cultures was passed through a type AA Millipore filter (pore size
0.80 fj.), cooled, and aerated before use. In the earlier studies, the culture water
was generally taken from the same area that produced the animals, but it was found
that local water from Vineyard Sound was also satisfactory. Streptomycin and
penicillin "G" potassium 50 mg./L. were used together at first, but later alternated
at each change of culture medium to lessen the possibility of "antibiotic resistance"
developing among the contaminating bacteria.
The phytoplankton organisms tried as food for the animals included Skeletonema
costatinn, Thalassiosira decipiens, T. fluriatilis, Chaetoceros affinis, Rhisosolenia
setigcra, and Coscinodiscus aster omphalus, all from laboratory cultures. Living
Artemia nauplii and Pinnotheres zoeae, fresh-caught harbor copepods including
Acartia clausi, A. tonsa, Temora longicornis, Centropages hamatns, Eurytemora
hlntndoides, and Labidocera acstira, as well as various invertebrate larvae in the
plankton were given as animal food. Bits of mussel, clam, and living and dead
offshore zooplankton were also given to some of the larger carnivorous forms. The
number of fecal pellets produced was used as a criterion for the amount of feeding
although in a few7 instances change in the number of food organisms was also
determined.
No attempt was made to determine the food in nature by examination of gut
contents. Food passes through the animal's gut very rapidly and particularly in
the case of offshore forms the gut is usually empty.
OBSERVATIONS ON FEEDING AND BEHAVIOR OF ANIMALS IN THE LABORATORY
Copepods
Calanus finmarchicus. Marshall and Orr (1955a, 1955b) have summarized
what is known concerning the feeding and behavior of this species, and little can be
added by this investigation. Calanus finmarchicus is knowrn to eat a wide variety
of diatoms, dinoflagellates, and other flagellated forms. Nannoplankton is eaten
but the animal showed a decided preference for larger food (Marshall and Orr.
1955b). In addition, radiolarians, tintinnids, and crustacean remains have been
404 ROBERT J. CONOVER
found in the gut (Marshall and Orr, 1955aj. In the present study, C. finmarchicus
ate Skeletonema, Rhizosolenia, and both species of Thalassiosira. Although it may
take in animal food inadvertently, it would seem that this species is primarily a
herbivore.
Calamis hyperboreus. This species is generally regarded as an arctic form,
where it commonly occurs in the surface waters. It is moderately abundant in the
Gulf of Maine and has been observed in the slope water at depths greater than 400
meters as far south as about 38° N. Juveniles were captured a mile south of Gay
Head on Martha's Vineyard, Massachusetts, in March and April, 1958.
When first returned to the laboratory, C. hyperboreus generally did not eat any
plant or animal food readily, but after one to two weeks healthy specimens ate all
phytoplankton species presented to them, including Skeletonema costatwn, Thalas-
siosira decipiens, T. ftwviatilis, Chaetoceros affinis and Coscinodiscus aster omphalus.
Many large fecal pellets were produced, often two or three millimeters long, which
on microscopic examination contained some green material and abundant smashed
tests of the species of diatom fed.
This species is anatomically very similar to C. finmarchicus and is almost cer-
tainly herbivorous in northern seas. However, it is difficult to understand how it
can obtain plant food in sufficient abundance to sustain it in the slope waters south
of Woods Hole, Massachusetts, in summer when the waters of the euphotic zone are
too warm for it. Possibly the animal goes into a state of "quiescence" when food
is scarce, which may explain why no food is taken when it is first brought into the
laboratory. Recent unpublished experiments suggest that the increase in activity
after some days in the laboratory is also accompanied by an increase in respir-
atory rate.
Sp'mme (1934) observed that breeding animals frequently ate their own eggs.
This observation has been confirmed in the present study, but it was found that
egg-eating was much greater when animals had no other food available than when
abundant phytoplankton was present.
Paraeuchaeta norvegica. Lowndes (1935) examined living and preserved
specimens of this large copepod and concluded that it was entirely carnivorous.
The animal refused all plant food presented to it in the current work. On the other
hand, some of the laboratory specimens fed on small neritic copepods readily as long
as they were alive. Acartia tonsa and Centropages hamatus were taken frequently,
although it is questionable whether Paraeuchaeta would encounter either in nature
commonly. It did not eat Artemia, though some decapod larvae were consumed.
During feeding, fecal pellets were produced which contained obvious animal frag-
ments. If both plant and animal material were fed simultaneously, the fecal pellets
produced contained only animal remains.
The maxillipeds in this animal are large, prehensile and carried far forward in
"praying mantis" fashion. The actual capture of the prey was not observed but
the animal would seize the end of a needle or micropipette when irritated in a
manner which must closely duplicate the process of food getting. The strength of
the animal was surprising and a smaller copepod would have little chance of escape
once it was grasped by the maxillipeds. Curiously, some of the laboratory speci-
mens hooked their maxillipeds over their first antennae. In this position they
seemed quite helpless and unable to get free. When the maxillipeds were released
FEEDING AND RESPIRATION OF PLANKTON 405
by the investigator, the animal seemed quite healthy, but usually caught the maxil-
lipeds again in a few hours.
Bathycalanus sp. There can be little doubt that these large red copepods from
deep water must be carnivorous, although no food, plant or animal, was taken during
the three weeks they were kept in the laboratory. The female whose respiration
was measured was over 13 mm. long with antennae reaching over 2.5 cm. from
tip to tip. In swimming, the movements were generally unhurried, almost de-
liberate, but when disturbed the animal dashed about the culture vessel in a frantic
effort to escape, frequently sustained for some seconds.
Rliincalanus nasutus. This species survived well under laboratory conditions
and ate any species of phytoplankton offered (Skeletonema costatuni, Thalassiosira
decipiens, Rliizosolenia sctigera). One female lived for three months before being
accidentally killed, during which time she matured and laid numerous fertile eggs.
Attempts to raise the nauplii were unsuccessful.
Euchirclla rostrata. These robust-appearing copepods did not survive partic-
ularly well in the laboratory although a few fecal pellets were produced when the
animals were fed on Skeletonema, Thalassiosira decipiens and Rhizosolcnia. All
were dead within five weeks of capture, but during this period several females
matured and laid. The eggs, which were a deep purple, were remarkable for their
size relative to the animal which produced them. The lengths of the cephalothorax
for the captive animals ranged from about 2.9 to 3.1 mm., while the eggs produced
measured over 0.4 mm. in diameter. In contrast, Rhincalanus nasutus, size range
4.2-4.7 mm., laid eggs about 230^ in diameter, and in the still larger Calanus
hyperboreus, cephalothorax 5.5-6.0 mm., the eggs ranged from 190-210 ju,. Euchi-
rclla laid only a few eggs at a time and they were very buoyant. Unfortunately none
of the eggs developed.
The density of this animal in the adult stage was remarkably high in contrast
to its eggs. The animals did not swim continuously in the laboratory containers
and when swimming ceased, they sank rapidly to the bottom. The carapace seemed
unusually sclerosed for so small an animal, being hard and smooth to the point of
a dissecting needle. Their movements were exceedingly rapid and they leaped
about vigorously when out of water. One individual traveled a measured distance
of 20 cm. in a single leap from the shallow dish, containing water several millimeters
deep, in which it was being examined. Although it was not demonstrated that
Euchirella rostrata does prefer animal food, the generally poor feeding on phyto-
plankton, robust anatomy and prehensile head appendages make it virtually certain
that the animal is largely carnivorous.
Other copepods. Only a few specimens of Pleuromamma robusta were taken in
near-surface tows over the continental slope and no observations on the organisms
in captivity were made. The structure of the mouth parts and general morphology
would suggest that it is largely herbivorous (George Grice, personal commu-
nication ) .
No attempts were made to culture Acartia clausi at this time but earlier studies
(Conover, 1956) leave little doubt that it is primarily a herbivore. However, two
observations of considerable interest were made on the closely related species
A. tons a.
On one occasion, some A. tonsa taken from Woods Hole harbor were given as
406 ROBERT J. CONOVER
food to a larger carnivorous copepod (Paraeuchaeta norvegica) in the company of
some Arteinia nauplii. During the experiment, an Acartia male was found which
had firmly grasped with its head appendages an Artemia nauplius. When both
animals were transferred to another dish for observation the male released the
nauplius which was seen to have its abdomen almost totally eaten. The Acartia was
given additional Artemia nauplii but died shortly thereafter without any further
predation.
On another occasion, A. tonsa was observed in the act of feeding on a culture
of large Tlialassiosira deci pi-ens (cells 70-85 ^ in diameter). A female (cephalo-
thorax length 0.87 mm.), lying ventral side up on the bottom of the dish, was seen
to grasp single Thalassiosira cells with rapid movements of the maxillipeds, bringing
them to the mouth region. Several times the individual cell could be seen poised
on the edge of the labrum for an instant before it passed inside the animal without
being broken or apparently damaged in any way. Once inside, the cells could be
seen through the transparent carapace like beads on a string lined up along the
foregut. The cells were carried posteriorly by a series of peristaltic movements
during which they continued to be discrete, undamaged cells until quite suddenly
they lost their distinct outline and seemed to fuse into a mass which soon became
noticeably darker in color. The material was passed posteriorly and eventually
extruded as a fecal pellet. The entire process took about 30-45 minutes, depending
on which cell was timed. On examination the pellet was seen to contain only
shattered frustules of T. decipiens and some unidentified organic matter.
Amphipods
Only two Phronima were taken during the program and one was dried after the
respiration experiment. Despite their transparent, somewhat delicate appearance,
the organisms were quite dense with a tough, sclerosed exoskeleton. The specimens
studied here were found free-living in the plankton but the animal is frequently
found "living" in an empty test of a salp. Most probably the organism eats the
salp in whose test it is found, for it would seem poorly equipped for filter feeding.
Euthemisto comprcssa was found to produce an occasional fecal pellet when
given phytoplankton (Skeletonema) but was obviously much more successful with
animal food. For instance, between October 3 and October 6, 1958, a single female
was observed to eat four harbor copepods and two zoeae, one nearly as large as
itself. Gravid females were taken in the plankton on several occasions, and even
the newly hatched young seemed carnivorous, swarming all over a piece of dead
euphausid given them. Hyperia galba likewise is largely carnivorous and was ob-
served to eat bits of mussel (Mytilus edulis) , smashed snail (Littorina littorea) as
well as living and dead copepods. This species is frequently associated with Aurelia
aurita or other large medusae and may share the food captured by its larger host,
but it can be a free-living member of the plankton community as well (Bige-
low, 1925).
Both Euthemisto and Hyperia are quite dense, heavily sclerosed, and strong
swimmers. Curiously, their carapaces are strongly hydrophobia and despite their
density and obvious strength, they are very prone to become caught in the sur-
face film.
FEEDING AND RESPIRATION OF PLANKTON 407
Euphausids
Of all the oceanic organisms tried, the euphausids seemed to be the most difficult
to keep in the laboratory. Specimens taken on August 19, 1958, were all dead by
September 4, before anything could be ascertained about their food habits. On other
occasions. Thysanoessa, Mcganyctiphanes, and Nematoscelis have been observed
to eat phytoplankton (Skeletonema costatum, Thalassiosira flitviatilis ) , but the rate
of consumption would seem to be much too low to meet food requirements. These
animals did not survive appreciably better than the first group.
Several workers have noted that euphausids consume a variety of foods.
Nyctiphanes couchii, a neritic species, eats diatoms and organic detritus predom-
inantly but also catches Sagitta, smaller crustaceans, and is cannibalistic in the
laboratory (Lebour, 1925). Similarly, Meganyctiphanes noruegica consumed plant
detritus when it was present in the water, but also fed on Calanns finmarchicus,
Paraeuchaeta norvegica and smaller copepods ( MacDonald, 1927 ). Very probably
most euphausids are omnivorous, but it is difficult to explain their extreme sensitivity
to deficiencies of the laboratory environment.
RESPIRATION IN RELATION TO SIZE OF PLANKTONIC ORGANISM
It is generally believed that the respiratory rate of poikilothermal animals is
related to some power of body weight by the expression
R----kW* (1)
where R is the volume of oxygen consumed, W the body weight. A' a constant for a
given set of conditions, and .r is the exponent, generally between 0.66 and 1.00.
When oxygen consumption is plotted against weight on log log paper the data
should give a straight line with a regression coefficient equivalent to x in equa-
tion (1).
Raymont and Gauld (1951 ) obtained a regression coefficient of 2.19 (or 2.30
with a single aberrant value removed ) when log respiration was plotted against
log length of the cephalothorax for four species of marine copepods ranging in length
over a size range of nearly an order of magnitude. If it is assumed that weight
varies as the cube of the length, then the coefficient obtained by Raymont and Gauld
becomes 0.73 (or 0.77) in the form of equation (1).
In the case of nine species of small neritic copepods, Conover (1959) computed
an overall regression coefficient of 0.86 for log respiration against log dry weight.
For these copepods with a total size range considerably less than an order of
magnitude, weight was found to vary as the power 3.17 of cephalothorax length.
The range of variation in weight of the organisms included in the current
investigation is nearly four orders of magnitude and the variation in respiratory
rate per organism is likewise considerable (Table I '). A log log plot of respiration
against weight would be expected to give a straight line relationship with a positive
regression coefficient. However, if the respiration rate is first divided by the weight
of the animal, the log log plot of this value R' against weight W should yield a
negative regression coefficient. Of the two methods, the second shows more clearly
the decrease in metabolic rate with increasing size, and its use should save a step
in the calculation of energy flow and production rates.
408
ROBERT J. CONOVER
The least squares regression on double logarithmic coordinates of the respiratory
rates as jul. oxygen/mg. dry weight and day plotted against dry weight in /x,g.,
excluding only the values for Calamts finmarchicus at 18.5° C. and those for Plcnro-
niaiiuua robust a, gives the equation
log R' -0.35 log fF + 2.2888.
In exponential form equation (2) becomes
R' ---- 194 W-°-™.
(2)
(3)
For comparative purposes, equation (3) may be converted to the form of equation
(1) by replacing R' with its equivalent R/1V,
R ---- 194 W-70-65. (4)
It can be readily seen that the exponential constant in (4) is decidedly lower
than that observed by Conover (1959), and also is lower than the probable exponent
computed from Raymont and Gauld (1951 ). Weymouth ct al. (1944) obtained a
coefficient of 0.798 for Pugcttia producta and Vinberg (1950) recorded 0.81 for
3.5
o
oB
- 3.0
Z
LJ
O
X
O
cc
LJ
H
cc
rr
o
h-
5.87, reject null hypothesis.
Gammarus lacustris. Assuming that surface area is proportional to the square of
length and that weight is proportional to the cube of length, von Bertalanffy (1951 )
suggested that an exponential constant of 0.667 indicates direct proportionality of
metabolism to the relative surface area of the organisms. The exponential constant
from equation (4) might, therefore, suggest that the surface rule applied in the case
of the zooplankton investigated in the current study but not for the earlier work.
However, there is also the possibility that the lower coefficient of proportionality
observed here may result from the different temperature at which the experiments
were run. In contrast with the temperatures of 4-8° C. used in the present work,
Raymont and Gauld (1951) performed their experiments at about 17° C. and
Conover (1959) used 20° as the experimental temperature. Vinberg (1950) and
Weymouth ct al. (1944) also used temperatures appreciably higher than those in
the present work.
To test the hypothesis that the temperature at which a series of experiments is
performed with different sized organisms might affect the proportionality of respir-
atory rate to size, an initial experiment with three species of calanoid copepods,
ranging in weight from 0.017 to 5.45 mg., was set up at two temperatures, 5° and
13° C. ; however, the scatter around the least squares regression lines was too great
to permit disproof of the null hypothesis that there was no difference in the log log
regression coefficient of respiration against weight at the chosen temperatures.
A second experiment at the same two temperatures was then performed with a
single species, Artemia salina, which can be cultured in the laboratory so as to supply
a number of different size classes. The least square regression lines for log respira-
tion against log weight for Artemia at 5 and 13° C. are shown in Figure 1. The
regression coefficients, 0.67 at 5° C. and 0.93 at 13° C., can be demonstrated to be
statistically different at P =0.025 (Table II). According to von Bertalanffy
(1951 ) the two regression coefficients obtained here for the same animal would be
indicative of two very different metabolic types.
410
ROBERT J. CONOVER
So long as the Q10 for any temperature change is the same for an animal over its
entire size range, the regression of respiration against weight should give the same
coefficient of proportionality regardless of the experimental temperature. In this
regard, Rao and Bullock (1954) reviewed data from several sources, and concluded
that the O10 of various measures of activity commonly increases with increasing
size over the range of ordinary physiological temperatures, although there were
several cases in which the trend was reversed. Artemia was not one of the animals
considered by Rao and Bullock, but the present data would seem to suggest that
this animal does have a Q10 w7hich varies with size.
3.0
it
O
(0
t-
X
O
UJ
>-
o:
o
2.0
K
.
M
UJ
K
o
S
0.0
, log R'= 2.41-0.441 logW
A'
CALANUS FINMARCHICUS
CALAMUS HYPERBOREUS
PARAEUCHAETA NORVESICA
PLEUROMAMMA R08USTA
BATHYCALANUS SP.
RHINCALANUS NASUTUS
EUCHIRELLA ROSTRATA
ACARTIA CLAUSI
EUPHAUSID
PHRONIMA SP.
EUTHEMISTO COMPRESSA
HYPERIA GALBA
logR'= 2.15-0.348 logW
A
log R'* 2. 47- 0.351 log W -
B
1.0
2.0 3.0
log DRY WEIGHT W/ANIMAL IN
4.0
5.0
6.0
FIGURE 2. Scatter diagram and regression lines showing the relation between log respira-
tion in jul. O2/mg. dry weight and day and log dry weight in [j.g. Line A is fitted to data for
suspected herbivores. Line A' is fitted to data for herbivores, omitting values for Acartia clausi.
Line B is fitted to data for suspected carnivores. See text for further explanation.
In the case of studies involving several different organisms taken from nature,
there is the additional complexity of species differences in Q10. Rao and Bullock
(1954) also showed that the habitat temperatures of the animal prior to examination
could affect Q10. In this regard. Berg and Ockelmann (1959) observed a seasonal
shift in the size-respiration relationship for the fresh-water snail Lymnaea palustris.
Other factors, such as nutritional status, reproductive activity or some endogenous
rhythm, might lead to increased variability in the observations. The resultant of
one or several factors of the sort described here might be to increase or decrease the
spread of values at one end of the temperature scale while having the opposite effect
at the other end, regardless of the size of the animals being studied. Both the slope
of the regression line and the scatter of points around it would be affected.
RESPIRATION IN RELATION TO FOOD HABITS
It was noted early in the study that Calanus hypcrboreus and Paraeuchaeta
norvegica taken in the same tow and studied under the same conditions had different
FEEDING AND RESPIRATION OF PLANKTON
411
respiratory rates despite their similarity in size and weight (see Table I). Both
animals were kept in the laboratory for a lengthy period, and it became obvious that
one, Calanus hyperboreus, was principally herbivorous while the other was entirely
carnivorous. Raymont (1959) found that Tortamis discaudatus, also believed to
be a carnivore, appeared to have a higher metabolic rate in relation to its body size
than other copepods inhabiting the same type of environment.
In Figure 2 the respiratory rate at low temperatures for all the animals studied
has been plotted against their weight on double log paper. On examination of the
scatter diagram it seemed that the suspected carnivores in general had higher rates
than the known herbivores. To test this hypothesis, separate regression lines were
TABLE III
Analysis of covariance for respiratory rates of herbivores (Acartia clausi included)
and non-herbivores
Null hypothesis: That there is no difference in the respiration rate per nig. of dry body
weight for herbivorous and non-herbivorous zoo plankton
Krrors of Estimate
Sums of
Sums of
Sums of
Sources of variation
of
freedom
squares
for res-
piration
products for
respiration
and weight
squares
for
weights
Sums of
Degrees
of
Mean
squares
freedom
square
Total
38
3.76580
-10.74861
43.19223
1.09095
37
Between animals having dif-
ferent food habits
1
0.38487
-2.80085
20.38336
Within each type
37
0.38093
-7.94776
22.80887
0.61153
36
0.01699
For test of significance of mean respiration with effect of
weight removed
0.47942
1
0.47942
0 47942
Variance ratio F = - - = 28.2; F0.9«(l, 40) = 12.61. Since F(l, 36) > 12.61, reject
0.01699
null hypothesis.
fitted to the data for the herbivores, Calanus fininarchicus, C. hyperboreus, Rhincal-
anus nasutus, and Acartia clausi (line A), and a second line fitted for the suspected
carnivores, Paraeuchacta norvegica, Bath\calanus, Euchirclla rostrata, Phronima,
Euthemisto comprcssa, Hypcria galba, and the euphausids (B ). For the herbivores
an additional regression line was also computed, omitting the data for Acartia since
it had been acquired originally for different reasons (A'). Obviously the slope of
the regression lines for the herbivores (including Acartia data) and the non-
herbivores is not significantly different (0.348 and 0.350, respectively). With the
Acartia data omitted the slope of the regression line fitted to the herbivore data
becomes 0.441, but it can be shown statistically (Student's t test) that the slopes
are still not significantly different.
To test the null hypothesis that there is no difference between the regression
lines for herbivores and non-herbivores, an analysis of covariance was employed as
shown in Table III. Since the slope for the herbivorous animals was not affected
412 ROBERT J. CONOVER
by removing the Acartia data, it was decided to include this in the overall analysis.
It is one of the advantages of co variance analysis that differences between groups
of measurements can be tested in a single operation without the effect of other
measurements that would normally complicate the interpretation. Thus, errors of
estimate are calculated from the sums of squares and sums of products in such a
way that the variance ratio tests only the respiratory rates after adjustment to
remove the effect of the variable weight. Since the F test suggests that the
distribution of respiration rates in relation to feeding type observed here would
occur by chance much less than one time in a thousand, clearly the reason for the
distribution bears some consideration.
Certain of the non-herbivorous zooplankton organisms studied were observed to
be quite dense, with a hardened exoskeleton. Thus, organisms such as Euchirella
rostrata, Euthcniisto compressa, and Hypcria galba are presumably carrying around
a considerable weight of inert organic material. More muscle protein, also quite
dense, and more energy would be required to maintain the organism in the water
column against its negative buoyancy.
There is little doubt that many herbivorous copepods also carry a high portion
of their body weight as inert organic material, but in this case the substance may be
oil with a positive buoyancy. In the case of the large Calanus hyperboreus, cope-
pods of approximately the same external dimensions may differ in dry weight by
several hundred per cent because of differences in the amount of stored oil.
It is also probable that a carnivorous animal is normally more active than an
herbivorous one, regardless of their basal metabolic rates. The predator has to
move about actively in search of the prey and then must overcome the natural
reluctance of the prey to be caught by using its greater physical strength and
swiftness. On the other hand, the prey organism in this association is more often
than not the herbivorous copepod which can feed while it swims with a more or
less continuous expenditure of a smaller amount of energy, since its food has
at best extremely feeble power of escape.
Finally the possibility remains that a real difference could exist in the form of
the organic matter oxidized by the herbivore and non-herbivore. Thus, an
organism which burns carbohydrate exclusively would use decidedly less oxygen
per unit of carbon oxidized than one which metabolizes oxygen-poor fats.
Before leaving the subject of respiration, a few remarks should be made con-
cerning Plcuromamina robusta and its somewhat enigmatic position metabolically.
As remarked earlier, it would appear that this species belongs in the herbivorous
group, but so far as its respiration is concerned the organism would seem to be
more closely allied to the non-herbivores. Clearly, with only two points on the
graph it is not certain that any real difference exists between this and other herbivo-
rous forms so far as respiration is concerned, and yet it remains possible that the
very high metabolic rate observed for this form may be in some way related to
the fact that it has a powerful bioluminescent organ. The species should certainly
be given further attention.
DISCUSSION
There is a persistent tendency for the biologist working with the most complex
of organized systems to seek a simple solution or approximation which holds for
the majority of cases. Thus certain scientific "laws" as Ou, -- 2, metabolism equals
FEEDING AND RESPIRATION OF PLANKTON 413
a constant times weight to some power between 0.7 and 0.8, weight equals cube
of length, and so on have come into common usage in environmental studies. Most
of these "laws" had their origin in laboratory studies on mammals and they need re-
examination before being applied to many ecological situations. It would be a
great advantage to the biological oceanographer to have a single expression which
w-ould predict the metabolism of a population of organisms with a given size
distribution under any temperature conditions, but as more data become available
the evidence would suggest that such a prediction equation, if it is ever formulated,
will give a high-speed computer a good workout.
The relationship between metabolism and size in the warm-blooded organisms
on the average has the form
M -_= 70W °-Ti cal./day, (5)
but as Kleiber (1947) admits, the basis for this relationship is not really under-
stood for the best known group of mammals. In the case of warm-blooded organ-
isms metabolism is usually measured at or near the same temperature and the
necessity to maintain this temperature against external environmental temperatures
often very different would seem to give some importance to the relative surface area
across which heat would be lost or gained.
In the sea, among the cold-blooded organisms there is tremendous variety in
body form, biochemical mechanism and chemical composition. The surface of the
marine organism is important for several reasons. Besides heat, nutrient sub-
stances, excretory substances, and gases pass back and forth across body membranes,
and the frictional resistance of the surface area with the surrounding medium is
critical for all organisms which move from place to place, whether they swim or
are carried by their environment. It would seem very coincidental indeed should
all the myriad forms of life be governed by a lawr derived from the warm-blooded
organisms.
Among the marine invertebrates these problems have been examined in detail
only for the Crustacea, and here it is true that there are relationships between
metabolism and size very similar to those derived for homeothermal organisms.
However, the effect of environmental temperatures on these relationships has not
been given much prior consideration.
That other factors besides size and the direct effect of temperature influence
respiratory rates of zooplankton forms has also been demonstrated recently.
Conover (1956) observed a possible seasonal adaptation to the changing tempera-
ture regime for Acartla. clansi and A. tonsa. Subsequently Conover (1959) and
Marshall and Orr (1958) demonstrated seasonal differences in the respiration
rate of several neritic species. Conover (1959) also demonstrated a significant
difference between the respiratory rate of Acartla clausi in Long Island Sound and
in Southampton Water. It can be seen from Table I of this study that Calanus
finmarchicus had a decidedly higher respiratory rate on Georges Bank than in the
Gulf of Maine. In this case, the populations are separated by not more than a
hundred miles of water. To this list can now be added the difference in metabo-
lism between organisms belonging to the same community but differing in their
position within the food chain.
As a final note of complexity to the already confused picture of metabolic rela-
414 ROBERT J. CONOVER
tionships for the planktonic organisms, it must be emphasized that even though
reliable data were available for respiration per unit mass for a group of organisms,
conversion of this information to food requirements or energy flow might introduce
an appreciable error, due to insufficient knowledge regarding the nature of the
food substance oxidized. It is well known that an animal requires more oxygen
to oxidize fat than to oxidize carbohydrate. Although more energy is produced
per volume of oxygen used in the case of carbohydrate metabolism, there is also
more energy available per gram of fat than per gram of carbohydrate. Raymont
and Conover (unpublished data) observed that several zooplankton organisms
oxidized carbon in some form at a rate of at least ten times greater than changes
in their carbohydrate reserves would predict. For instance, in the case of Calanus
liyperboreus, the respiration rate was equivalent to an oxidation of 10 to 20 jug. of
carbohydrate per day, an amount somewhat greater than the total carbohydrate
content of the animals, and yet there was no detectable change in the total sugars.
It is becoming increasingly popular among environmental biologists to think
of production as a single dynamic process which can be made to conform to some
idealized mathematical model. Such an approach has had useful application,
for instance in the North Sea pelagic fishery studies by Gushing (1955, 1959).
Yet, as Steele (1960) points out, herring eat Calanus but they do not eat salps,
'even though each has a similar position in the food chain. Perhaps it is fortunate
that salps are not particularly abundant in the North Sea ! In any event, there
would seem to be a good argument in favor of an increased emphasis on certain
qualitative aspects of energy dynamics and food relations in the marine environment
as a supplement to the purely quantitative approach.
SUMMARY
1. The respiration rate of twelve species of zooplankton, the majority from
oceanic waters and from depths greater than 100 meters, has been measured at
temperatures close to that of their environment (4-8° C.). In most cases healthy
specimens were brought to the laboratory and their food habits and behavior
studied.
2. The following species seemed largely herbivorous : Calanns finmarchicus, C.
hyperboreus, and Rhincalamis nasutus.
3. The copepod Paraeuchacta norvegica, and the amphipods Euthcinisto covn-
pressa and Hypcria galbct all took animal food readily. Bathycalanus sp., Euchirella
rostrata and the euphausids also are believed to be at least partially carnivorous
although they demonstrated little or no feeding.
4. The animals studied had a total range in dry weight of nearly four orders
of magnitude. When log respiration was correlated with log weight, a positive
linear regression coefficient of 0.65 was obtained. This value, which is lower
than most previously determined regression coefficients relating size and metabo-
lism in the Crustacea, may result from the lower temperatures used in these
experiments compared with those used in the earlier work.
5. As confirmatory evidence, size and metabolism were related by a coefficient
of 0.67 at 5° and 0.93 at 13° in the case of Artemia saiina.
6. Those zooplankton animals which seemed to be largely carnivorous on the
basis of the behavioral studies had a significantly higher respiratory rate than
those which seemed to be predominantly herbivorous.
FEEDING AND RESPIRATION OF PLANKTON 415
7. Some of the possible explanations and ecological implications of the above-
mentioned observations are discussed.
LITERATURE CITED
BERG, K., AND K. W. OCKELMAXX, 1959. The respiration of freshwater snails. /. Exp. Biol.,
36: 690-708.
vox BERTALANFFY, L.. 1951. Metabolic types and growth types. Aincr. Nat., 85: 111-117.
BIGELOW, H. B., 1925. Plankton of the offshore waters of the Gulf of Maine. Bull. U. S. Bur.
Fish., 40, pt. II, 509 pp.
COXOVER, R. J., 1956. Oceanography of Long Island Sound, 1952-1954. VI. Biology of Acartia
claitsi and A. tonsa. Bull. Bingliiun Occanogr. Coll., 56: 156—233.
COXOVER, R. J., 1959. Regional and seasonal variation in the respiratory rate of marine cope-
pods. Limnol. and Occanogr., 4 : 259-268.
COXOVER, R. J., S. M. MARSHALL AXD A. P. ORR, 1959. (unpublished report) Feeding and
excretion of Calanus finmarchiciis with reference to the possible role of the zooplankton
in the mineralization of organic matter. WHOI Ref. no. 59-32.
CUSHIXG, D. H., 1955. Production and a pelagic fishery. Fish. Invest., Lond., ser. II, 18 :
104 pp.
CUSHIXG, D. H., 1959. On the nature of production in the sea. Fish. Invest. Lond., ser. II,
22 : 40 pp.
KLEIBER, MAX, 1947. Body size and metabolic rate. Physiol. Rev., 27: 511-541.
LASKER, R., 1960. Utilization of organic carbon by a marine crustacean : analysis with
carbon-14. Science. 131 : 1098-1100.
LEBOUR, M. V., 1925. The Euphausiidae in the neighbourhood of Plymouth and their importance
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MAcDoxALD, RODERICK, 1927. Food and habits of Meganyctiphanes norvcgica. J. Mar. Biol.
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MARSHALL, S. M., AXD A. P. ORR, 1955b. On the biology of Calanus finmarchiciis. VIII. Food
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MARSHALL, S. M., AXD A. P. ORR, 1958. On the biology of Calanus finmarchicus. X. Seasonal
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PUTTER, AUGUST, 1907. Die Ernahrung der Wassertiere. Zcitschr. allg. Physiol, 7 : 283-320.
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RAO, K. P., AXD T. H. BULLOCK, 1954. Qw as a function of size and habitat temperature in
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RAYMOXT, J. E. G., 1959. The respiration of some planktonic copepods. III. The oxygen
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RAYMOXT, J. E. G., AXD D. T. GAULD. 1951. The respiration of some planktonic copepods.
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S0MME, J. D., 1934. Animal plankton of the Norwegian coast waters and the open sea. Rcpt.
Nonveg. Fish. Invest.. 4(9) : 163 pp.
STEELE, J. H., 1960. Plant-animal food cycles in the sea. Nature. 185: 218-219.
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THE PHYSIOLOGY OF SKELETON FORMATION IN CORALS.
IV. ON ISOTOPIC EQUILIBRIUM EXCHANGES OF CALCIUM
BETWEEN CORALLUM AND ENVIRONMENT IN LIVING
AND DEAD REEF-BUILDING CORALS
THOMAS F. GOREAU1 AND NORA I. GOREAU
Physiology Department, University College of the West Indies and the Laboratory
of Marine Biochemistry and Ecology, New York Zoological Society
The experiments reported here were carried out to determine the extent to
which the polyp layer covering the outside of a coral colony can act as a barrier
cutting down or preventing altogether the equilibrium exchange of calcium between
the corallum and the environment.
The occurrence of a calcium equilibrium exchange process between the skeletal
mineral of corals and the sea water was described in a previous paper (Goreau,
1959a), but in comparing the calcium-45 uptake of living corals with that of dead
macerated pieces of corallum, we observed that the incorporation of the radioactive
calcium by exchange was in most cases much slower than that deposited by active
secretion in living corals.
The total rate of exchange between the solid and dissolved phases in a system
at equilibrium depends on the temperature and on the surface area of the solid
phase in free contact with the medium. The former variable will not be con-
sidered further in this study since the temperature of most West Indian coral reefs
remains stable within the range of 25° C. to 29° C. On the other hand, the
total surface area that is potentially available for exchange in an average coral
colony is enormous, due to the fact that the corrallum is not a solid mass of mineral
matter, but a complex framework of interlocking calcareous lamellae with water-
filled spaces in between. In the Scleractinia, the mineral structure is composed
of more or less trabeculate spherulitic aggregates of aragonite crystals (Wells,
1956), with the living polyps located only on the external surface of the skeletal
mass. In eight Indo-Pacific reef-building corals Odum and Odum (1955) esti-
mated that the pores occupied between 7% and 3S
a 100
75
50
. 25
P FURCATA
A
k--* LIVING PARTS
B
Q£AD PARTS
M. ALCICORNIS
C
» B- B
0.5
5 10
TIME IN DAYS
50
100
500
FIGURE 1. Changes of specific activity as a function of the time in the skeletons of living
and dead corals, plotted on semi-logarithmic coordinates. Curves A and B represent the activity
due to calcium-45 over a period of 23 days in living and dead Poritcs furcata, respectively.
Curve C shows the same in Millepora alcicornis over a period of 156 days. The regression
lines were fitted by the method of least squares. Note that no significant loss of calcium-45
activity occurs in the living corals, but in the dead parts of Poritcs fnrcata the slope of the
curve shows that the exchangeable calcium-45 activity has a half-life of approximately five days.
data the fit of the regression curve to the points is quite good. The line yields the
equation
A = 200 r-°-136 T,
(1)
for the relation between the specific activity of the corallum (A) and the time (T).
The slope of the curve shows that one-half of the original activity on the surface of
the dead parts of P. furcata colonies is lost in about five days, and nearly all in
about 110 days.
CALCIUM EXCHANGE IN DEAD CORALS WITH AND WITHOUT COENOSARC
The effect of the mechanical presence of a tissue barrier on the calcium exchange
in corals was tested by comparing isotopic equilibration rates in killed corals, in
EQUILIBRIUM EXCHANGES IN CORALS 421
which the coenosarc was still present, with that in corals from which the fleshy
parts had been removed by maceration.
Corals of several different species were killed and preserved in sea water
containing 4% formaldehyde buffered to pH 8 with bicarbonate. Each specimen
was split into two pieces, one being preserved in the formaldehyde while the other
was macerated in sea water until the tissue layer covering the outside of the
corallum was completely removed. The clean macerated halves were then placed
together with the preserved halves into the buffered sea water-formaldehyde
solution, and neutralized Ca45Cl2 was added to a final activity of approximately
0.05 /AC. /ml. The medium was stirred with a stream of saturated air pumped
through an airstone. The corals were allowed to soak in the radioactive sea water
for 48 hours, the temperature being maintained at 28 ± 1 ° C. during this time.
Samples of water were withdrawn for measurement of the specific activity at the
beginning and at the end of the runs. The pieces of coral were drained on filter
paper to remove the most of the adherent radioactive water, rinsed in six changes
of large volumes of inactive sea water over a period of about 15 minutes and
allowed to drain for eight hours on blotting paper. All specimens were treated
exactly the same way.
TABLE II
Exchange uptake of Ca45 by killed corals with and without coenosarc. Average specific
activities in counts min.~l mg. Ca~l ± S. D. Number of colonies
sampled in brackets
Species Macerated colonies without coenosarc Colonies with coenosarc
Acropora cervicornia 11. 20 ±1.00 (4) 10.10 ± 0.80 (4)
Manicina areolata 0.82 ± 0.10 (5) 1.78 ± 0.10 (5)
Montastrea annularis 1.70 ± 0.20 (4) 1.70 ±0.10 (4)
Montastrea cavernosa 1.00 ± 0.20 (4) 1.10 ±0.05 (4)
Diploria strigosa 0.60 ± 0.05 (4) 1.50 ±0.06 (4)
Sampling was carried out by cutting off 3-cm.-long pieces from the terminal parts
of branching corals, or taking core samples with a hollow punch from the massive
corals (cj. Goreau and Goreau, 1959). To make the data from macerated and
unmacerated specimens as comparable as possible, great care was taken to sample
only strictly corresponding areas of the colonies. Three pieces were removed from
each test specimen. These were once more rinsed in fresh sea water for 30 seconds,
drained on filter paper, and dissolved in individual flasks containing concentrated
HC1. The samples were homogenized, boiled, cooled, made up to a standard
volume and the radioactivity determined by the oxalate method on replicate 1-ml.
aliquots. Due to the low activities of the same samples, the time taken for 2000
counts was used as a basis for our measurements, and the results expressed as
counts per minute per milligram calcium.
The results of these experiments are given in Table II for five species of reef-
building corals. The numbers represent the specific activities ; each value is the
mean of three measurements. The numbers in brackets correspond to the number
of separate colonies tested.
In three of the five species tested, there were no significant differences in the
amount of calcium-45 taken up by the macerated specimens as compared with those
still having a mechanically intact layer of tissue. The brain corals, Diploria strigosa
422 THOMAS F. GOREAU AND NORA I. GOREAU
and Manicina arcolata, were the exceptions to this ; significantly higher specific
activities were observed in the preserved colonies which still had the intact coenosarc.
As there is no evidence that coral tissues contain substances which bind inorganic
calcium, we believe that the increased activity was due to occlusion in the coral
of a small amount of radioactive sea water that was not washed out completely.
The rinsing procedure was kept as short as possible to prevent loss of calcium-45
from the specimens by exchange with the wash water.
These data indicate that the mere mechanical presence of an intact, but dead,
tissue layer does not appear to hinder significantly the isotopic exchange of calcium
between the corallum and medium over a period of 48 hours.
A notable feature of these results is that much more calcium-45 was exchanged
by the skeleton of Acropora palmata than by any of the other corals tested. This
is due to the greater porosity and surface area in the corallum of the Acroporidae
as compared with that of the Faviidae, the family to which all the rest of the corals
listed in Table II belong. The isotope exchange method may eventually become
useful as a quantitative measure of the skeletal surface area and porosity in
different corals if suitable reference standards can be developed.
CALCIUM EXCHANGE IN CORALS WITH EXTREMELY Low GROWTH RATE
The experiments described in this section were conducted on colonies of Oculina
diffusa in which the active calcium uptake rate was depressed to extremely low
levels through the combined removal of the zooxanthellae and treatment with
acetazolamide (2-acetylamino-l,3.4-thiazole-5-sulfonamide ) to inhibit the enzyme
carbonic anhydrase (rf. Goreau, 1959a). The calcium-45 deposited by these corals
was compared with that taken up through isotopic exchange by control colonies of
the same species which had first been killed and macerated to remove the tissue
layer covering the skeleton.
Zooxanthellae were removed from the corals by keeping the colonies in darkened
aquaria for two months. The corals fed on zooplankton brought in from the out-
side by the circulating fresh sea water supply showed no signs of starvation from
this treatment. Complete extrusion of the zooxanthellae caused the coenosarc to
change from a translucent yellowish brown to a transparent colorless appearance.
The "bleached" corals were healthy and fully expanded at all times, but their
calcium deposition rates were extremely low in comparison with normal colonies
of the same species containing a full complement of zooxanthellae (Goreau, 1959a;
Goreau and Goreau, 1959, 1960).
The zooxanthella-less corals and the macerated controls were distributed into
four glass jars, two of which were painted black to exclude light. One light-dark
pair contained only fresh sea water, the other contained sea water with 10"3 M
acetazolamide.3 For the 48-hour duration of the experiment, the jars were kept
under a twin bank of 80-watt fluorescent tubes, the temperature was maintained at
28 ±0.5° C. and the contents of the jars were stirred and aerated with a fine
stream of saturated air.
After the corals were exposed to the action of 1O3 M acetazolamide for 24
3 Kindly supplied as "Diamox" by the Lederle Laboratories Division of the American
Cyanamid Company.
EQUILIBRIUM EXCHANGES IN CORALS
423
hours, neutral Ca45Clo was added to a final concentration of about 0.1 /AC. /ml.
Multiple samples of the living experimental colonies and the dead controls were
taken at 3-, /-, 12- and 15-hour intervals after addition of the isotope. Water
aliquots were taken at the same times to measure the specific activity of the medium.
The coral samples, taken by breaking off terminal portions of the branches, were
immediately drained on filter paper, rinsed in six changes of fresh inactive sea
water, drained again and dissolved in dilute HC1 (1:1) in individual volumetric
tubes. Analysis for calcium-45 activity was done by the oxalate method and the
results calculated in terms of the specific activity, e.g. counts per minute per milli-
TABLE III
Calcium-45 activities in living zooxanthella-less Oculina diffusa compared with dead control
colonies, in light and darkness, and in presence or absence of acetazolamide .
Figures represent the specific activities in counts min.~l mg. Ca~l ± S.D.
Number of samples in brackets
Plain sea water
Time in
hours
Light
Dark
Living corals
Dead corals
Living corals
Dead corals
3
1.9
±0
1
(4)
2.6 ±
0.3
(3)
1.4 ± 0.2
(4)
1.6 ±0.2
(3)
7
5.3
± 1
0
(5)
3.5 ±
0.2
(3)
3.0 ± 0.8
(3)
2.7 ±0.1
(3)
12
9.4
±0
6
(3)
3.6 ±
0.2
(3)
3.1 ±0.3
(5)
4.0 ± 0.1
(3)
15
3.8 ± 0.5
' (4)
4.2 ± 0.05
(3)
10 3 M acetazolamide
3
1.4 ±0.1
(4)
1.6
±0.2
(3)
1.2
± 0.01
(4)
3.7
±0.1
(3)
7
0.95 ± 0.3
(6)
3.0
±0.3
(3)
1.4
±0.1
(4)
4.4
±0.1
(3)
12
1.9 ±0.6
(5)
3.3
±0.05
(3)
1.8
±0.2
(3)
5.0
±0.05
(3)
15
1.3 ±0.6
(4)
3.8
±0.2
(3)
2.0
±0.5
(4)
4.9
±0.2
(3)
gram calcium. At the end of the experiment the remaining corals which had
been in the acetazolamide solution for a total of nearly 40 hours appeared as
healthy as the controls kept in plain sea water. The colonies \vere fully expanded
and able to ingest small pieces of clam meat. When returned to fresh sea wrater,
they lived for a further two weeks until sacrificed for histological purposes.
The results of these experiments are given in Table III. In the absence of
acetazolamide, the zooxanthella-less corals which had been kept in darkness (Fig.
2B) contained about the same specific activity as the dead controls, whereas those
growing in light had more than twice the activity of the controls (Fig. 2A).
The reason for this is not clear since zooxanthellae appeared to be completely
absent from these colonies. Some potentiation of the growth rate might have
occurred if boring algae were still present, but no chlorophyll tests were made on
these specimens at the time. In previous experiments on this and other corals
from which zooxanthellae had been removed, we found no significant differences of
the calcium uptake rates in light and darkness (Goreau, 1959a).
424
THOMAS F. GOREAU AND NORA I. GOREAU
In the presence of 10 3 M acetazolamide, the calcification rate of the experi-
mental colonies was so reduced that they contained less than half of the specific
activity of the dead controls. The effect was the same in light as in darkness
(Figs. 2C and 2D). Whereas the activity of the dead controls increased con-
tinuously for the first 12 hours, the activity of the living test corals showed little
t-
X
e>
CO
CO
LU
cr
<
O
PLAIN SEA WATER
A.
LIVING CORALS
--C
DEAD CORALS
-B.
10 M ACETAZOLAMIDE
DEAD CORALS
\
-- D
6
I -o
m
o
T]
0 o
5 §
12 15 0 3
TIME IN HOURS
12 15
FIGURE 2. The effect of the inhibition of carbonic anhydrase by 10~3 M acetazolamide on
the rate of calcium-45 incorporation by zooxanthella-less Oculina diffusa, compared with the
rate of isotopic exchange in dead macerated control colonies, in light and in darkness.
Calcium-45 incorporation by living and dead corals in plain sea water is shown in Figures 2A
and 2B. Figures 2C and 2D show the same in the presence of 10~3 M acetazolamide. Note
that in the presence of the carbonic anhydrase inhibitor the specific activity of the living corals
is less than half that of the dead exchange controls.
EQUILIBRIUM EXCHANGES IN CORALS 425
increase beyond that incorporated during the initial three hours of the experiment.
We consider it probable that most of this activity was due to a small amount of
calcium taken up into the tissues but not deposited into the skeleton. It is clear
from these results that the living zooxanthella-less colonies in which the enzyme
carbonic anhydrase was also inhibited probably did not grow at all. However, due
to the presence of the living coenosarc, the process of isotopic exchange between
the skeleton and medium was very much slower than in the dead controls in which
the skeletal surface was in direct contact with the medium and so free to exchange
with it.
DISCUSSION
The equilibrium exchange rate of calcium between the coral skeleton and the
surrounding sea water is very different in dead corals as compared to living corals.
We have found earlier that isotopic equilibration between corallum and sea water
enriched with calcium-45 is more or less complete in about 48 hours at a tempera-
ture of 28° C. (Goreau, 1959a). However, corals in which the calcification rate
was reduced to very low levels by the simultaneous removal of zooxanthellae and
inhibition of carbonic anhydrase with acetazolamide contained less radioactivity
than macerated "exchange controls." Our data indicate that the mechanical
presence of the coenosarc does not hinder calcium equilibrium exchange if the
tissues have been killed. On the other hand, the living coenosarc is comparatively
calcium-proof, thus preventing the underlying skeletal mass from exchanging with
the surrounding environment. This is supported by our previous observation
that the living polyps of Astrangia danac, a cold-water coral, tend to exclude calcium
relative to the sea water in which they are growing (Goreau and Bowen, 1955).
In corals growing under natural conditions on a reef the exchange rate of calcium
between the skeleton and the medium is much higher in the dead parts of the colonies
which are unprotected by the polyparium than in those parts which are covered
by living coenosarc. The extremely low rate of exchange in this case is not
entirely due to the isolating action of the living tissue sheet covering the skeleton :
after the colonies were labelled with calcium-45 they were returned to the sea
where they continued to grow, so that the radioactive layer eventually became
buried under successive new layers of inactive skeletal material. Under these con-
ditions the radioactive material was well protected from isotopic exchange which
can occur only at the skeletal surface, in consequence of which no significant loss
of calcium-45 could be observed.
The low exchangeability of already deposited calcium indicates that it does not
significantly take part in physiological interactions with the overlying coral tissue.
This is very different from what is observed in vertebrates where 25% to 35% of the
bone calcium remains exchangeable with the calcium dissolved in the medium (r/.,
for example, Dawson, 1955). The much greater exchangeability of the skeletal
calcium in the vertebrates as compared with that of the corals is due to the fact that
the bones of the former constitute an endogenous tissue system in dynamic equi-
librium with the rest of the body constituents (Armstrong, 1955; Neuman and
Neuman, 1953; Marshall, 1960), whereas the scleractinian corallum is an exoskele-
ton which, once formed, is entirely outside of the body proper, and its mineral
contituents do not appear to be in steady-state equilibrium with the "milieu
interieur" of the polyps.
426 THOMAS F. GOREAU AND NORA I. GOREAU
SUMMARY AND CONCLUSIONS
1. Equilibrium exchange of calcium between the skeleton and the medium was
measured in dead and living corals under various conditions, using calcium-45 as
isotopic tracer.
2. The calcium which is deposited as aragonitic carbonate into the skeleton of
living corals is not in contact with the environment. Under the conditions of
our experiments, no significant isotopic calcium exchange occurred in Porites
furcata and Millepora alcicornis over a period of 23 days and 156 days, respectively.
3. In the dead parts of Porites furcata colonies where the corallum was exposed
to direct contact with the medium, rapid isotopic exchange occurred so that the
half-life of calcium-45 activity on the skeletal surface was only about five days.
4. In reef corals killed with formaldehyde, the amount of calcium-45 activity
picked up by the skeleton from the medium in 48 hours by equilibrium exchange
was the same in colonies in which the coenosarc had been preserved as in those
from which all tissues had been removed, so that the naked corallum was in direct
contact with the radioactive sea water. This indicates that, under the conditions
of our experiments, the mechanical presence of the non-living coenosarc did not
constitute a barrier to equilibrium exchange between the corallum and the en-
vironment.
5. In living corals in which the calcification rate was depressed to extremely
low levels by simultaneous removal of the zooxanthellae and inhibition of carbonic
anhydrase with 10~3 M acetazolamide, the amount of calcium-45 activity incorpo-
rated was 50% to 70% less than that exchanged by the dead control colonies from
which all tissues had been removed by maceration. This indicates that the living
coenosarc is an effective barrier to equilibrium exchange of calcium between the
skeleton and the environment, even when the calcium deposition rate is almost zero.
6. Our experimental evidence indicates that the living coenosarc is fairly
calcium-proof and prevents the skeleton from exchanging with the environment.
Unlike vertebrates, in which a large fraction of the bone calcium remains in
dynamic equilibrium with the dissolved calcium of the body fluids, the skeletal
calcium of corals, once it is deposited into the corallum, probably does not take
part in steady-state or equilibrium exchanges with the overlying tissues.
LITERATURE CITED
ARMSTRONG, W. D., 1955. Radiotracer studies of hard tissue. Ann. N. Y. Acad. Sci., 60 :
670-684.
CHAVE, K. E., 1954. Aspects of the biogeochemistry of magnesium. I. Calcareous marine
organisms. /. Gcol., 62 : 266—283.
DAWSON, K. B., 1955. Calcium exchange in bone. Biochan. /., 60 : 389-391.
GOREAU, T. F., 1959a. The physiology of skeleton formation in corals. I. A method for
measuring the rate of calcium deposition by corals under different conditions. Biol.
Bull., 116: 59-75.
GOREAU, T. F., 1959b. The coral reefs of Jamaica. I. Species composition and zonation.
Ecology, 40 : 67-90.
GOREAU, T. F., AND V. T. BOWEN, 1955. Calcium uptake by a coral. Science, 122: 1188-1189.
GOREAU, T. F., AND N. I. GOREAU, 1959. The physiology of skeleton formation in corals.
II. Calcium deposition by hermatypic corals under various conditions in the reef. Biol.
Bull, 117: 239-250.
EQUILIBRIUM EXCHANGES IN CORALS 427
GOREAU, T. F., AXD N. I. GOREAU, 1960. The physiology of skeleton formation in corals.
III. Calcification as a function of colony weight and total nitrogen content in the reef
coral Manicina areolata (Linnaeus). Biol. Bull., 118: 419-429.
HYMAN, L. H., 1940. The Invertebrates. Volume 1. Protozoa through Ctenophora. New
York. McGraw-Hill Book Co., Inc., xii + 726 pp.
LOWENSTAM, H. A., 1954. Factors affecting the aragonite-calcite ratios in carbonate secreting
marine organisms. /. Geol., 62 : 284-322.
MARSHALL, J. H., 1960. Microscopic metabolism in bone. Chapter 8 in Rodahl, K., J. T.
Nicholson and E. M. Brown, Eds., Bone as a Tissue. New York. McGraw-Hill Book
Co., Inc. ix + 358 pp.
MEIGEN, W., 1903. Beitrage zur Kenntnis des Kohlensauren Kalk. Xatnrzviss. Ges. Freiburg.
Ber., 13 : 1-55.
NEUMAN, W. F., AND M. W. NEUMAN, 1953. The nature of the mineral phase of bone.
Chem Rev., 53 : 1-45.
ODUM, H. T., AND E. P. ODUM, 1955. Trophic structure and productivity of a windward coral
reef community on Eniwetok Atoll. Ecol. Monogr., 25 : 291-320.
REVELLE, R., AND R. FAIRBRIDGE, 1957. Carbonates and carbon dioxide. Gcol. Soc. Amcr.
Mem., 67(1) : 239-285.
'\YELLS. I. W., 1956. The Scleractinia. Chapter in Treatise on Invertebrate Paleontology.
Ed. by R. C. Moore. Part F, pp. 328-444.
RESPIRATORY REGULATION IN AMPHIBIAN DEVELOPMENT 1
JOHN R. GREGG
Zoology Department, Duke University, Durham, North Carolina
There is now considerable evidence (reviewed by Slater and Hulsmann, 1959)
that the respiratory rate of a cell abundantly supplied with oxidizable substrates is
a function of the rate at which it metabolizes labile phosphorus compounds in re-
sponse to energy demand. Consider, for example, the oxidation of glucose :
C6H12O,i + 6 O2 -* 6 CO2 + 6 H2O.
Normally, this process is coupled to the esterification of inorganic phosphorus :
C6H12OtJ + 6O2 + 38 ADP + 38 H3PO4 -» 6 CO, + 44 H2O + 38 ATP.
When work is to be done, stored ATP is utilized :
38 ATP + 38 JLO -» 38 ADP + 38 H3PO4 + Energy.
As a result, the levels of ADP and inorganic phosphorus are raised momentarily,
and respiration is quickened until ATP is restored. Thus, under normal conditions,
the respiratory rate of a cell depends upon its rate of energy expenditure.
In the presence of uncoupling agents, e.g., 2,4-dinitrophenol (DNP). the link
between oxidation and phosphorylation is severed. When this happens, respiration
proceeds at a rate limited only by the availability of oxidizable substrates, and with-
out concurrent formation of ATP. At the same time, ATP-stores are depleted by
destructive catalysis, and the ability to perform work deteriorates rapidly.
The burgeoning respiratory rates of developing embryos surely reflect ever-
increasing expenditures of energy. Therefore, a study of the respiratory responses
of developing embryos to uncoupling agents should yield important information
about the energetics of development. Along these lines, a recent study of sea
urchin embryos by Immers and Runnstrom (1960) has provided interesting data,
and there is reason to believe that amphibian embryos are amenable to similar
analysis. In the presence of DNP, explants of the tissues of frog gastrulae respire
at twice the normal rate (Ornstein and Gregg, 1952 ) ; and, under similar conditions,
intact gastrulae are partially depleted of their stores of esterified phosphorus and are
prevented from undergoing further morphological change (Gregg and Kahl-
brock, 1957).
In the work about to be reported, an uncoupling agent (DNP) has been used to
study the development of respiratory regulation in Rana pif>icns embryos, and also
in hybrid embryos obtained by fertilizing Rana [>ipicns eggs with Rana sylvatica
sperm. First studied by Moore (1946), these hybrids are incapable of developing
1 This work has been supported in part by a research grant, No. A-2146, from the Public
Health Service. The assistance of James T. Love and B. YV. Ruffner is gratefully ac-
knowledged.
428
RESPIRATORY REGULATION 429
beyond the early gastrula stage, and exhibit numerous other morphogenetic or
metabolic anomalies (Gregg, 1957).
METHODS
Embryological
Developing embryos were obtained by stripping eggs from pituitary-injected
Rana pipicns females into suspensions of active Rana pipiens or Rana syh'atica
sperm. After two hours, they were dispersed thinly among fingerbowls and al-
lowed to develop at temperatures ranging from 10° C. to 25° C. The medium,
10% Ringer's solution without phosphate or bicarbonate, was changed every two
days, or more often. Before manometric measurements were made, watchmaker's
forceps were used to free the embryos of their jelly coats.
Manometric
Respiratory rates were determined with a refrigerated Warburg respirometer
equipped with 7-ml. single-side-arm center-well flasks. Carbon dioxide was ab-
sorbed on filter paper rolls placed in the center wells and saturated with 10% KOH.
The flasks were shaken 75 complete cycles per minute at an amplitude of 6 cm.
The temperature of the water bath was held constant at 24° C. Further details
will be cited as the need arises.
Terminological
Developmental stages were determined by reference to the charts of Shumway
(1940), which standardize the course of Rana pipiens development at 18° C.
Therefore, regardless of their actual temperature histories, embryos in a given
Shumway stage have been assigned the corresponding standard age at 18° C.
Hybrid embryos have been assigned the same stages, and ages, as simultaneously
developing Rana pipiens controls.
Respiratory rates are expressed in the following units : microliters of oxygen per
hour per 50 embryos.
The respiratory rate exhibited by intact embryos at a given stage, and under
standard conditions, is called the respiratory norm of embryos at that stage.
The respiratory rate exhibited by intact embryos at a given stage, and under
maximal stimulation by DNP, is called the respiratory potential of embryos at
that stage.
The quotient obtained by dividing the respiratory potential by the corresponding
respiratory norm is called the respiratory control quotient.
RESULTS
The results obtained in the present work are now listed without commentary.
They will be discussed in the next section.
(1) The respiratory norm of Rana pipiens embryos is a strongly increasing
function of developmental age (Table I, Table III).
(2) The relation between the developmental age and respiratory norm of Rana
pipiens embryos is best characterized as the exponential function consisting of all
pairs (t,y) satisfying the following equations :
y == 5 e 0.026* (0 ^ /< 56)
v- 21 e°-017 ('-56)
430
JOHN R. GREGG
TABLE I
Influence of 2,4-dinitrophenol on the respiratory rate of Rana pipiens embryos
DNP-concentration, molar
Egg
clutch
Stage
Age
18° C.
Control
5 X ID-6
10-5
5 X 10-5
10^
10-3
A
11
34
11
19
24
33
31
12f
46
18
28
35
40
35
1
16
72
29
42
50
45
39
4
19
118
56
93
104
85
79
25
B
6
7
6
6
13
24
23
3
10|
30
12
16
22
32
28
2
15
67
24
33
36
40
35
9
10!
90
38
54
71
53
51
6
19
118
56
85
102
71
64
26
20
140
84
112
118
104
78
19
Main compartment of each flask: 20-50 embryos in 1 ml. 10% Ringer's. Side-arm of each
flask: 0.5 ml. DNP in 10% Ringer's adjusted to give final concentration shown. Side-arm
contents delivered to main compartment immediately after first reading. Readings were made
for four or five hours (usually five) at half-hour intervals. Respiratory rates were constant after
first hour. Entries designate average rates for last three (four) hours of four (five) hour runs.
FIGURE 1. Respiratory norm (lower curve) and potential (upper curve) of Rana pipiens
embryos. Abscissa, developmental age. Ordinate, respiratory rate.
RESPIRATORY REGULATION
431
4-
2-
35
70
105
140
FIGURE 2. Respiratory control quotient, Rana pipiens. Abscissa, developmental age.
Ordinate, respiratory control quotient.
TABLE II
Influence of 2,4-dinitrophenol on the respiratory rate of Rana
pipiens 9 X Rana sylvatica cf embryos
DNP-concentration, molar
Egg
clutch
Stage
Age
18° C.
Control
5 X ICr6
10-5
5 X 10-5
10-"
5 X 10-4
c
n
11
5
13
20
22
18
20
10$
30
3
10
18
31
30
17
13|
56
3
13
21
28
26
15
17*
90
12
18
22
29
—
20
19
118
11
19
21
35
32
32
D
7+
9
8
13
20
26
28
15
HI
38
9
18
25
30
28
12
15
67
13 17
22
35
37
19
18
96
10
18
26
38
38
28
Fifty embryos per flask. DNP-treated embryos were equilibrated in DNP-solutions in
10% Ringer's for two hours preceding measurements. Readings were taken for one hour at 5-
minute intervals.
Hybrid embryos do not develop beyond Stage 10: entries in the stage and age columns
designate average developmental stages and corresponding ages of Rana pipiens control embryos.
432
JOHN R. GREGG
where t is the developmental age and y is the respiratory norm (Fig. 1, lower curve) .
(3) The respiratory activity of Rana pipiens embryos at any stage is stimulated
by DNP in concentrations ranging from 5 X 1O6 M to 1 X 1O4 M (Table I,
Table III).
(4) The respiratory potential of Rana pipiens pre-neurulae is exhibited under
treatment with DNP at concentrations near 5 X 10~5 M ; that of neurulae and older
embryos is exhibited under treatment with DNP at concentrations near 10~5 M
(Table I, Table III).
30-
20*
0
80
120
FIGURE 3. Respiratory norm (lower curve) and potential (upper curve) of Rana pipiens
$ X Rana sylvatica cT embryos. Abscissa, developmental age. Ordinate, respiratory rate.
(24° C.)
(5) The relation between the developmental age and respiratory potential of
Rana pipiens embryos is best characterized as the exponential function consisting of
all pairs (t,y) satisfying the following equations :
y= 19eo.om
v == 41 e °-016 (*~67)
(0
(67
46)
140
where t is the developmental age and y is the respiratory potential (Fig. 1, upper
curve). For reasons explained later, the respiratory potentials corresponding to
some values of t (46 < t < 67 ) are left undefined.
(6) The respiratory control quotient of Rana pipiens embryos decreases rapidly
from 3.8 at fertilization to 2.5 at 46 hours, and slowly from 1.6 at 67 hours to 1.5
at 140 hours (Fig. 2). For reasons explained later, the respiratory control
quotients of embryos between the ages of 46 hours and 67 hours are left undefined.
RESPIRATORY REGULATION
433
(7) The respiratory norm of hybrid embryos is a weakly increasing function of
developmental age (Table II j.
(8) The relation between the developmental age and respiratory norm of hybrid
embryos is best characterized as the linear function consisting of all pairs (t,y)
satisfying the following equation :
(iii)
0.045 f
where t is the developmental age and 3; is the respiratory norm (Fig. 3, lower curve).
(9) The respiratory activity of hybrid embryos at any stage is stimulated by
DNP in concentrations ranging from 5 X 10'6 M to 5 X 10~4 M (Table II).
7
5-
3-
1
40
80
120
'•' wv i ^.v
FIGURE 4. Respiratory control quotient, Rana pipicns $ X Rana sylvatica <$. Abscissa,
developmental age. Ordinate, respiratory control quotient.
(10) The respiratory potential of hybrid embryos is exhibited under treatment
with DNP at concentrations near 5 X lO"5 M (Table II).
(11) The relation between the developmental age and respiratory potential of
hybrid embryos is best characterized as the linear function consisting of all pairs
(t,y) satisfying the following equation :
(iv)
3' = 24 + 0.10;
where t is the developmental age and 3- is the respiratory potential (Fig. 3, upper
curve ) .
(12) The respiratory control quotient of hybrid embryos decreases from 4 at
fertilization to 3.3 at 118 hours (Fig. 4).
434
JOHN R. GREGG
(13) Homogenization in buffer-saline solution has little effect upon the respira-
tory activity of young Rana pipiens embryos, but it powerfully stimulates the
respiratory activity of older ones (Table III).
(14) The respiratory rates exhibited by homogenized Rana pipiens embryos are
not affected by DNP in concentrations ranging from 10~5 M to 5 X 10~5 M
(Table III).
TABLE III
Influence of 2, 4-dinitro phenol on the respiratory rate of intact embryos and
cell-free homogenates (Rana pipiens}
DNP-concentration, molar
Egg clutch
Stage
Age 18° C.
Control
lO'5
5 x 10-5
E
7§
11
7
—
20
Intact
lOf
30
9
—
35
16
72
28
31
—
18
96
44
68
—
19*
129
84
99
—
E
1\
11
8
6
9
Homogenized
io§
30
8
10
7
16
72
132
130
129
18
96
132
128
124
19*
129
146
136
137
Intact embryos. Fifty embryos per flask. DNP-treated embryos were equilibrated in DNP
solutions (in 10% Ringer's) for one to two hours preceding measurements. Readings were taken
for one hour at 5-minute intervals.
Homogenates. Embryos suspended in ice-cold 10~2 M phosphate buffer made up in 0.065%
NaCl (50 embryos per ml.) were homogenized with a Lourdes homogenizer. Aliquots (1 ml.) were
transferred to respirometer flasks. Immediately after first reading, DNP solutions (0.5 ml. in
buffer-saline, "pH 7.48") were tipped into main compartments to give final DNP-concentra-
tions desired. Readings were taken for one hour at 5-minute intervals.
Entries in the stage and age columns designate average developmental stages and corre-
sponding ages.
DISCUSSION
The results listed in the previous section will now be discussed, in sequence.
(1) Embryologists have known for a long time that developing amphibian
embryos are characterized by waxing respiratory norms. Our first result, then, is
not new, and our chief concern will be to find an explanation for it.
One explanation is provided by the assumption that developmentally increasing
respiratory rate is the direct result of the synthesis of respiratory machinery. But
recent experiments (Spiegelman and Steinbach, 1945; Gregg and Ray, 1957) have
established that homogenates of newly fertilized eggs can be made to respire
endogenous substrates at rates exceeding those of intact embryos at any stage of
development. From the outset, therefore, there is more than enough respiratory
apparatus to support any rate of respiration normally exhibited by a developing
embryo, and the suggested explanation cannot be correct.
RESPIRATORY REGULATION 435
Another explanation, sponsored by Spiegelman and Steinbach, and also by
Gregg and Ray, assumes that increasing respiratory norms are the direct result
of processes progressively facilitating effective contact between respiratory enzymes
and their substrates, i.e., that the respiratory rate at any stage is limited simply by
the rate at which some respiratory enzyme is able to combine with its substrate.
But, under this assumption, we are left without good reason to expect an elevation
of respiratory rate in the presence of uncoupling agents, and it is best abandoned.
Still another explanation (the one we shall adopt here) is provided by the
theory outlined in the introduction. According to this theory, the respiratory norm
is a function of the rate of energy expenditure ; and waxing respiratory norms thus
are to be ascribed to waxing rates of energy expenditure. In this connection it is
known that developing Rana pipiens embryos are characterized by increasing rates
of carbohydrate utilization (Gregg, 1948), and by increasing rates of turnover of
labile phosphorus (Kutsky, 1950).
(2) Our mathematical analysis of the relation between developmental age and
respiratory norm (Rana pipiens) has been made following the precedent established
by Atlas (1938) and by Moog (1944), and the results of it are in considerable
agreement with theirs. The following version of their equations is obtained by
changing units to correspond with those used in the present investigation and by
confining values of t to the interval ( 0-140 ) :
3,= 16 e°-019 (t-62)
v=:5eo.or^ t)
y = 21 e °-031 (t'50) ( 50 ^ f ^ 140 )
Considering the nature of the supporting data, (i), (v) and (vi) are in good agree-
ment. For Rana pipiens embryos, therefore, it appears to be established that
respiratory norm is an exponential function of developmental age, and that respira-
tory acceleration decreases at some point in the age interval (50-62). For possible
explanations of the accelerator}- change, readers are referred to the papers of Atlas
and Moog.
(3) We have already mentioned that Rana pipiens embryos are well-provisioned
with respiratory substrates ; therefore, on the basis of the theory outlined in the
introduction, the stimulatory effect of DNP is to be expected.
(4) What is unexpected is the finding that the concentration of DNP eliciting
the respiratory potential of Rana pipiens embryos is five times as great for the age
interval (0-67) as for the age interval (72-140). There is no parallel for it in the
development of sea urchin embryos, whose respiration is maximally stimulated at any
stage by 5 X 10~5 M DNP (Immers and Runnstrom, 1960) ; and, pending further
investigation, it remains unexplained.
(5) There is no precedent to guide mathematical analysis of the data relating
developmental age to respiratory potential (Rana pipiens) ; and subsequent investi-
gation may necessitate revision of equations (iv), which have been obtained by
taking the data at face value. For what it is worth, Figure 1 (upper curve) shows
that the development of the respiratory potential of Rana pipiens embryos proceeds
in three phases : two of exponentially increasing potential, separated by one whose
characteristics are not known. The second phase may be one of constant potential,
436 JOHN R. GREGG
as the data suggest ; or, if the first and third phases are of greater duration than
shown, it may be one of abruptly decreasing potential ; or, although there is no reason
for so believing, later work may show that the first and third phases actually intersect
in the age interval (46-67), thus abolishing the second phase entirely. We shall
leave the question open, after noting that Immers and Runnstrom (1960) observed
a transient decline of respiratory potential in sea urchin embryos entering the
mesenchyme blastula stage. But their result, also, was reported in a mood of
skepticism.
In any case, it is clear that the respiratory potential of Rana pipiens embryos
increases with age, remaining Avell above the respiratory norm, and thus maintaining
a considerable margin of safety for energy expenditure. We have accounted for
the developmental increase of respiratory norm by supposing that it is a function
of increasing rate of energy expenditure. To account for increasing respiratory
potential, it is necessary to assume the occurrence of intracellular structural changes
progressively enhancing contact between respiratory enzymes and substrates. Data
bearing upon this assumption are neither crucial nor consistent. Weber and Boell
(1955) have found that the specific activity of mitochondrial cytochrome oxidase
is an increasing function of developmental age (Xenopus laevis), thus indicating-
some process of mitochondrial differentiation ; on the other hand, Spiegelman and
Steinbach (1945) were unable to observe any developmental increase of the cyto-
chrome oxidase activity of homogenates (Rana pipiens). Nevertheless, our as-
sumption is supported by the electron microscopical study of Eakin and Lehmann
(1957), who discovered profound developmental alterations of structural com-
plexity and localization of the intracellular components, including mitochondria, of
the ectoderm of neurulating amphibian embryos (Xenopus laevis, Triton alpcstris).
Therefore, until better assumptions are available, we shall adhere to our present one.
(6) The respiratory control quotient is a convenient measure of the degree to
which the rate of energy expenditure holds the respiratory norm below the respira-
tory potential. Figure 1 shows that, during the first 46 hours of development,
energy expenditure in Rana pipiens embryos is such as to permit a rapid approach
of the respiratory norm to the respiratory potential ; from 67 hours on, the relation
is nearly stabilized, and respiratory norm is practically a constant fraction of respira-
tory potential. For reasons stated in the discussion of result (5), respiratory
control quotients corresponding to the age interval (46-67) are left undefined. It
is worth noting that a similar relation between respiratory norm and respiratory
potential characterizes the development of sea urchin embryos ( Immers and Runn-
strom, 1960).
(7) We come now to the respiration of hybrid embryos. In a general way the
data agree with those of Barth ( 1946) in showing that the respiratory norms of such
embryos become increasingly subnormal as time goes on, and the same may be said
of the rates at which they utilize carbohydrate reserves (Gregg, 1948). It appears,
therefore, that they expend energy at increasingly subnormal rates ; and this, on the
theory of respiratory control we are adopting, is the reason for their progressively
subnormal respiratory norms. On this basis, we should expect to find increasingly
subnormal rates of turnover in their pools of labile phosphorus, but data are not yet
available. There may be nothing wrong with their respiratory machinery, for their
RESPIRATORY REGULATION 437
homogenates respire at rates quantitatively similar to those of homogenates of Rana
pipiens control embryos (Gregg and Ray, 1957).
(8) Our mathematical analysis of the relation between the developmental age
and respiratory norm of hybrid embryos is based upon the assumption of linearity.
The more precise data of Earth (1946) suggest that this assumption is not quite
correct, but it is a useful approximation to the exact state of affairs. It should be
noted that the intercept 6 of equation (iii ) is in good agreement with the intercept
5 of equation (i).
(9) The view that the respiratory machinery of hybrid embryos may be entirely
normal is supported by the finding that their respiratory rates are stimulated from
300 to 400% by 5 X 10~5 M DNP ; for no better response is obtainable from Rana
pipiens control embryos.
(TO) This result need not be elaborated, except by pointing out that the con-
centration of DNP eliciting the respiratory potential of hybrid embryos at any stage
is the same as that eliciting the respiratory potential of Rana pipiens pre-neurulae :
there is no developmental shift in sensitivity to DNP like that exhibited by neurulat-
ing Rana pipiens embryos.
(11) On the assumption of linearity, the rate at which the respiratory potential
of hybrid embryos increases is given by equation (iv). The increase of respiratory
potential, though slower than normal, still is unnecessary; for the respiratory norms
of hybrid embryos never overtake their initial respiratory potential (Fig. 2).
Nevertheless, an increase of respiratory potential occurs ; and to explain it we shall
assume, in accordance with the discussion of result (5), that intracellular changes
facilitating respiratory enzyme-substrate union take place in hybrid embryos as well
as in normal ones, though at a much slower rate. Electron microscopical and bio-
chemical studies of the intracellular particulates of hybrid embryos are much needed.
(12) This result does not require further commentary: reference to the discus-
sion of result (6) will make its interpretation perfectly evident.
(13 ) This finding confirms the work of Gregg and Ray (1957) : unless they are
treated with a detergent (e.g., deoxycholic acid), homogenates of very young Rana
pipiens embryos do not respire at rates much different from the respiratory norm;
but the respiratory rates of homogenates of progressively older embryos rapidly
exceed the respiratory norm. We shall return to this topic in the discussion of
the last result.
(14) The failure of DNP to elevate the respiratory activity of homogenates of
Rana pipiens embryos at any stage of development is extremely puzzling, and no
adequate explanation for it is now at hand.
Homogenates of adult tissues frequently do not exhibit a respiratory response
to the presence of uncoupling agents. In such cases, respiratory rate appears to be
limited, not by the rate of turnover of labile phosphorus, but by the low availability
of readily oxidizable substrates ; generally, this limitation is overcome by adding
pyruvate, succinate or other respiratory metabolites (Krebs, 1959). This account
of the matter is not applicable to homogenates of amphibian embryos (see the dis-
cussion of result (1)).
An arbitrary explanation, for which there is little independent support, may be
constructed along the following lines. First, let us suppose that homogenization, In-
activating ATP-ase, results in some maximum elevation of the levels of ADP and
438 JOHN R. GREGG
inorganic phosphorus. Second, let us suppose that homogenization is accompanied
by the envelopment of respiratory particulates in lipo-protein or other envelopes.
Third, let us suppose that the degree of envelopment decreases with developmental
age. The first assumption guarantees a high rate of respiratory activity, other
conditions permitting; and explains why DNP is without effect, the levels of ADP
and inorganic phosphorus already being maximum. The second assumption ex-
plains why the respiratory activity of homogenates of young embryos is low, for we
may expect that respiration under these conditions will be limited by the rate at
which respiratory substrates are able to penetrate lipo-protein barriers. It also
explains why the respiratory rate of homogenates of young embryos is elevated by
detergents, for these may be expected to disperse lipo-protein deposits around
respiratory particulates; or, even, to fragment those particulates (Siekevitz and
Watson, 1956). The third assumption explains why the respiratory activity of
homogenates is an increasing function of developmental age.
SUMMARY
1. The respiratory rate of Rana pipiens control embryos is an increasing function
of developmental stage, with an acceleratory change at the onset of the formation of
the neural folds.
2. At any stage of development, the respiratory rate of Rana pipiens embryos is
elevated by the presence of 2,4-dinitrophenol (DNP). The degree of stimulation
obtainable ranges from about 400% of the control rate at the beginning of develop-
ment to about 150% of the control rate at the gill-circulation stage.
3. The respiratory rate of Rana pipiens § x Rana sylvatica <$ embryos is an
increasing function of time, but the rate of increase is very much lower than that of
the respiratory rate of Rana pipiens controls.
4. At any stage, the respiratory rate of hybrid embryos is elevated by DNP.
The degree of stimulation obtainable ranges from about 400% at the beginning of
development to about 300% at 118 hours after fertilization (18° C).
5. The respiratory activity of homogenates of Rana pipiens embryos at any stage
is not altered by the addition of DNP.
6. The relevance of these findings to the question of embryonic respiratory con-
trol is discussed. It is concluded that, within the capacity to respire, respiration is
governed by energy expenditure, and that the capacity to respire increases with age
as the result of intracellular changes facilitating contact between respiratory
enzymes and substrates.
LITERATURE CITED
ATLAS, M., 1938. The rate of oxygen consumption of frogs during embryonic development.
Physiol. Zoo/., 11 : 278-291.
EARTH, L. G., 1946. Studies on the metabolism of development. /. Exp. Zoo/., 103 : 463-486.
EAKIN, R. M., AND F. E. LEHMANN, 1957. An electronmicroscopic study of developing
amphibian ectoderm. Arch. f. Entiv., 150: 177-198.
GREGG, JOHN R., 1948. Carbohydrate metabolism of normal and of hybrid embryos. /. Exp.
Zool, 109: 119-134.
GREGG, JOHN R., 1957. Morphogenesis and metabolism of gastrula-arrested embryos in the
hybrid Rana pipiens $ X Rana sylvatica <$. 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.
RESPIRATORY REGULATION 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 syh'atica J1. Biol. Bull., 113 : 382-387.
IMMERS, J., AND J. RUNNSTROM, 1960. Release of respiratory control by 2,4 dinitrophenol in
different stages of sea-urchin development. Dev. Biology, 2 : 90-104.
KREBS, HANS, 1959. Chairman's introductory address. Rate-limiting factors in cell-respiration.
Ciba Foundation Symposium on the Regulation of Cell Metabolism, pp. 1-10.
KUTSKY, PHYLLIS B., 1950. Phosphate metabolism in the early development of Rana pipiens.
J. E.rp. Zool., 115: 424-460.
MOOG, FLORENCE, 1944. The chloretone sensitivity of frog's eggs in relation to respiration and
development. /. Cell. Comp. PhysioL, 23 : 131-155.
MOORE, J. A., 1946. Studies in the development of frog hybrids. I. Embryonic development in
the cross Rana pipiens $ X Rana sylvatica <$. J. Exp. Zool., 101 : 173-220.
ORNSTEIN, NORMA, AND JOHN R. GREGG, 1952. Respiratory metabolism of amphibian gastrula
explants. Biol. Bull., 103 : 407-420.
SHUMWAY, W., 1940. Stages in the normal development of Rana pipiens I. External form.
Anat. Rec., 78: 139-147.
SIEKEVITZ, P., AND M. WATSON, 1956. Cytochemical studies of mitochondria. I. The separa-
tion and identification of a membrane fraction from isolated mitochondria. /. Biophys.
Biochem. Res., 2 : 639-652.
SLATER, E. C, AND W. C. HULSMANN, 1959. Control of rate of intracellular respiration.
Ciba Foundation Symposium on the Regulation of Cell Metabolism, pp. 58-83.
SPIEGELMAN, S., AND H. B. STEINBACH, 1945. Substrate-enzyme orientation during embryonic
development. Biol. Bull., 88 : 254-268.
WEBER, R., AND E. J. BOELL, 1955. tJber die Cytochromoxydaseaktivitat der Mitochondrien
von friihen Entwicklungsstadien des Krallenfrosches (Xenopus laevis Baud.). Rev.
Suisse de Zool., 62 : 260-268.
THE INFLUENCE OF SALINITY ON THE MAGNESIUM
AND WATER FLUXES OF A CRAB
WARREN J. GROSS AND LEE ANN MARSHALL 1
Division of Life Sciences, University of California, Riverside, California
The shore crab Pachygrapsus crassipcs is known to be an osmotic regulator in
both dilute and concentrated sea water, but its antennary glands are ineffective as
organs of osmo-regulation inasmuch as the urine remains essentially isotonic to the
blood, regardless of the salinity of the external medium (Jones, 1941 ; Prosser ct al.,
1955 ; Gross, 1957a). On the other hand, Prosser ct al. ( 1955 ) demonstrated Mg
concentrations in the urine of this species when it is exposed to osmotic stresses,
which suggests that the antennary glands are effective regulators of Mg. Gross
( 1959) confirmed these observations, but pointed out that the volume of urine
produced, as well as the urine concentration of Mg, must be known before the
antennary glands could be considered certain regulators of this cation. Prosser
ct al. (1955) also observed that while the urine concentration of Mg increased tre-
mendously when the crab was immersed in increasingly saline media, the urine Na,
contrary to expectation, decreased. They suggested that Na and Mg compete for
transport across the membranes of the antennary gland with Mg predominating.
When the crab was immersed in artificial Mg-free sea water equivalent to 170% of
normal salinities, the observed concentration of Na in the urine was much higher
than it was when the crab was immersed in 170% natural sea water, thus supporting
the suggestion. However, the effects of such a treatment on the urine Mg con-
centration were not reported.
Even though Pachygrapsus is a strong regulator in large osmotic stresses, its
blood tends toward concentrations which are intermediate between those it has in
normal sea water and the concentration of the external medium (Jones, 1941;
Prosser ct al., 1955; Gross, 1957a). Gross (1957a) demonstrated that such
changes in the concentration of the blood are brought about by salts and not water ;
that is, the volume changes of the animal were insignificant. This must mean that
either the formed tissues when bathed by such altered blood concentrations also
remain unchanged in volume or that their volumes change at the expense of the blood
space. Shaw (1955) demonstrated that the volume of muscle tissue in the hyper-
regulating crab Card mis increased when the animal was immersed in dilute
sea water.
The present investigation will show that the efflux of Mg from Pachygrapsus is
principally a function of water turnover and not due immediately to the Mg gradient
between blood and external medium. It also will be shown that muscle tissue
increases in volume when the crab is immersed in dilute sea water, and that it
decreases in volume when the crab is immersed in concentrated sea water. This
results in volume alterations in the blood space.
1 Present address : Department of Zoology, University of Michigan, Ann Arbor.
440
MAGNESIUM AND WATER FLUXES OF A CRAB 441
MATERIAL AND METHODS
The lined shore crab, Pachygrapsus crassipes Randall, was collected at Dana
Point, Laguna, and Ballona Creek, California. Only mature crabs of more than
15 grams were used. Care was taken that none was undergoing moult.
Blood and urine were sampled as previously described; analyses of Na and K
were made by flame photometry and Ca and Mg by titration with ethylene diamine
tetra acetic acid (EDTA) (Gross, 1959).
Artificial sea water was prepared according to the tables of Barnes (1954).
normal sea water being considered to contain the following concentrations (meq./l.)
of the major ions: Na, 460; K, 10; Ca, 20; Mg, 104; Cl, 538; SO4, 56. The pH
was adjusted to 8.0. In the Mg-free media Na was substituted for the deleted Mg
and where Mg was increased above normal, relative to the other ions, Na was
deleted accordingly to attain the desired osmotic pressure.
The effect of abnormal concentrations of medium Mg on blood and urine ionic
concentrations was studied in two ways: (1) Groups of crabs from 100% natural
sea water were immersed into small volumes of Mg-free artificial sea water of
salinities equivalent to 50%, 100% and 150% of natural sea water, and into media
equivalent to 50% in salinity but containing Mg equal to that of 100% natural sea
water (104 meq./l.). Also, one group was immersed in a medium equivalent to
100% sea water in total salinity, but which contained half again as much Mg (156
meq./l. ) . All animals first were rinsed in the respective test medium before immer-
sion. After 24 hours, the blood and urine of the experimental animals were
analyzed for Na, K, Ca and Mg ; the media which were originally Mg-free were
analyzed for Mg. In the small volumes of media used for this group, the crabs
could rise partially out of the water. This kept the mortality rate low over the
24-hour period of immersion, thus permitting a study of ionic alterations in the blood
and urine in the artificial media.
(2) The second group of experiments was conducted primarily to measure rates
of Mg excretion. Here animals were placed in large volumes of medium to assure
complete and uniform immersion throughout the test period. Crabs previously
acclimatized for 24 hours in natural sea water of salinities respective to their subse-
quent test media were immersed in large volumes of Mg-free 50%, 100% and 150%
artificial sea water, these, again, having been rinsed first in the test media. During
acclimatization in natural sea water, the crabs could rise out of the water. Again
after a period of immersion ranging from one tc six hours the blood and urine of the
animals were analyzed for Na, K, Ca and Mg and the medium \vas analyzed for Mg.
All experiments were conducted in a temperature-controlled room at 15° C.
In order to determine the volume changes in the muscle tissue of Pachygrapsus
under different osmotic stresses, leg muscle from animals which had been immersed
for three days in 50%, 100% and 150% natural sea water was rinsed in isotonic
glucose and blotted uniformly. (Glucose concentrations were calculated from the
tables in Gross, 1959. ) These were then weighed and dried to constant weight in
a drying oven at 95° C. The difference between dry weight and wet weight was
considered to be the water content of the tissue.
Changes in the blood space volume were shown by calculating the volume of
distribution of C14-tagged sucrose in the blood space one minute after injection.
Crabs removed from immersion for three days in 50%, 100% and 150% natural sea
442
WARREN J. GROSS AND LEE ANN MARSHALL
water were injected at the base of the fourth walking leg with 0.3 to 0.5 ml. (de-
pending on the size of the crab) of 1 M sucrose which had been tagged with C14.
About one minute after the injection, a sample of blood was taken from the
opposite side of the animal and diluted with 5 ml. of 1 M untagged sucrose. The
quantity of blood was determined by weight and averaged about 0.3 gram. Then a
0.10-ml. aliquot of the diluted blood was absorbed onto a filter paper disc, allowed
to dry and counted on a Nuclear- Chicago scaling unit. Mean counts thus obtained
for three discs made from each diluted blood sample were compared with the mean
counts of three 0.10-ml. aliquots of the dose which were plated in the same manner.
Since the blood was diluted in 1 M sucrose and the dose was 1 M sucrose, the error
TABLE I
Ion concentration in the urine of Pachygrapsus immersed in small volumes of
artificial sea water
50%
100%
150%
Mean
S.D.
No.
Mean
S.D.
No.
Mean
S.D.
No.
N
380
60
37
378
64
15
353
106
30
Na (meq./l.) 0
400
28
12
332
96
12
430
81
15
+
401
33
25
319
80
8
N
10.0
3.5
37
7.8
1.4
15
9.6
1.1
30
K (meq./l.) 0
14.3
8.7
12
15.9
8.0
12
16.0
4.2
15
+
11.0
3.8
25
7.0
2.0
8
N
32.7
7.1
31
36.0
6.3
15
47.9
5.2
20
Ca (meq./l.) 0
34.5
4.5
13
34.5
4.5
13
47.6
18.6
15
+
31.1
8.7
19
37.0
7.8
14
N
70.5
41
31
305
130
15
408
122
29
Mg (meq./l.) 0
85.0
42
16
298
104
13
281
85
15
+
65.5
23
18
368
101
14
N = crabs immersed in concentrations of natural sea water.
0 = crabs immersed in Mg-free sea water.
+ = crabs immersed in artificial sea water containing abnormally high Mg: in 50% sea
water = 104 meq./l. ; in 100% sea water = 156 meq./l.
due to self-absorption should be essentially the same in radio-assays of both blood
and dose. Care was taken to assure uniform geometry. At least 1000 counts were
observed for each sample, and maximum rates did not allow significant coincidence.
The volume of distribution for sucrose in one minute was, therefore, calculated
d/b
from the equation : V
X 100
where V - volume of distribution of sucrose in one minute (% body weight) ;
d -- observed total activity (counts/min.) injected into the crab;
b -- observed activity (counts/min.) per gram of blood ;
TC' -- weight of crab (g. ) .
Also, a correction was applied for the volume of the dose.
After the blood samples were taken, the crabs were placed in a closed chamber
MAGNESIUM AND WATER FLUXES OF A CRAB
443
containing a Ba(OH)2 trap designed to absorb CO,. Radio-assay of the total
precipitate thus collected in 24 hours for ten crabs demonstrated no significant
activity, suggesting that the sucrose was not metabolized. It is possible that some
was fixed in the body, but it seems more likely that it remained in solution in an
unchanged state at least for the brief period (one minute) during which the dilution
was being observed.
RESULTS
Table I compares the urine Mg of crabs immersed in small volumes of artificial
sea water with the urine Mg of crabs immersed for 24 hours in small volumes of
natural sea water. The value for crabs in 100% natural sea water is higher than
previously reported, but is believed to be more reliable because of improved tech-
nique in sampling the urine ; the values for crabs in 50% and 150% natural sea water
have been reported previously (Gross, 1959). It is clear from these data that when
Pachygrapsus is immersed in 50% and 100% Mg-free artificial sea water, the con-
TABLE II
Excretion of Mg by Pachygrapsus in stress media
Test medium
Medium volume*
Mean
urine Mg
meq./l.
S.D.
Xo.
Mean Mg
excreted
meq./day/g.
S.D.
No.
150% sea water
without Mg
small (24 hrs.)
large (6 hrs.)
281
471
85
152
15
17
0.0021
0.0073
0.001
0.0054
14
18
100% sea water
without Mg
small (24 hrs.)
large (6 hrs.)
298
243
104
103
13
15
0.0070
0.0095
.0033
0.0084
14
15
50% sea water
without Mg
small (24 hrs.)
large (1 hr.)
85.0
72.0
42
44
16
12
0.0066
0.0433
0.0030
0.022
20
12
k Time in parentheses = period of immersion.
centration of Mg in its urine is close to the concentration of Mg in the urine of crabs
immersed in the same salinities of natural sea water. When a crab is immersed in
50% sea water for 24 hovirs, the Mg concentration of its urine is about the same
whether the medium Mg is 0, 52 or 104 meq./l. In salinities of 100% normal, the
observed mean concentration for urine Mg was about the same for animals immersed
in Mg-free water as for those from natural sea water ; for animals immersed in 100%
artificial sea water containing abnormally high Mg, the urine Mg was slightly higher
than for crabs from 100% natural sea water (368 meq./l. to 314 meq./l., respec-
tively), but these means are not significantly different (Table I). The urine Mg
of crabs immersed in small volumes of Mg-free 150% sea water was less than that
for crabs from 100% or 150% natural sea water. This is difficult to interpret, but
possibly could be explained as a reflection of the crab's greate/ tendency to remain
out of the artificial medium. Such an argument is supported' by the fact that the
urine Mg for crabs immersed in large volumes of Mg-free 150% sea water averaged
about the same (470 meq./l.) as did crabs from small volumes of 150% natural sea
water (408 meq./l.). Also, w'hen the crabs were immersed in large volumes of
100% Mg-free sea water, the urine Mg was about the same as for crabs immersed
444
WARREN J. GROSS AND LEE ANN MARSHALL
in 100%. natural sea water (Tables I and II). Table III presents probability values
for analyses of Mg excretion.
It might be argued that the animals immersed in the larger volumes of artificial
sea water were previously acclimatized to the respective salinities, and that sufficient
time had not been given to permit alteration of the urine concentrations. However,
when 13 crabs were transferred directly from 100% natural sea water and immersed
for six hours in a large volume of 150% Mg-free sea water, the mean urine Mg was
713 meq./l., indicating not only that considerable changes can occur in the urine Mg
concentration in six hours, but also that the greater hyperosmotic stress due to trans-
ferring the animals directly from 100% natural sea water to 150% artificial sea
water probably made the urine even more concentrated with respect to Mg, again
independently of the influx of this ion from the medium. Thus, for the periods
TABLE III
Probability values for analyses of Mg excretion
A. Comparison of urine Mg concentrations: crabs immersed in artificial
sea water vs. crabs immersed in natural sea water
50% Sea water
100% Sea water
150% Sea water
Large volume
Small volume
Large volume
Small volume
Large volume
Small volume
Mg-free
>0.50
>0.20
= 0.20
>0.50
>0.10
<0.001
Excess Mg
>0.50
>0.20
B. Comparison of rates of Mg loss by crabs completely immersed in
Mg-free artificial sea water
100% SAY. vs. 150% SAY.
100% SAY. vs. 50% S.W.
150% SAY. vs. 50% S.W.
>0.30
<0.001
<0.001
indicated the concentration of Mg in the urine is not determined by the influx of
this ion from medium, but rather, at least indirectly, by the osmotic pressure of the
external medium.
Table I also compares the urine concentrations of the other three major cations
of the crabs immersed in small volumes of artificial sea water with those immersed in
the respective salinities of natural sea water. Contrary to the findings of Prosser
ct al. (1955), there is no dramatic increase in urine Na when Mg is deleted from
the medium. Although in 100% artificial sea water with high Mg the urine Na of
Pachygrapsus was somewhat low, this could be accounted for by the low Na in the
medium rather than the high Mg. Then in 150% Mg-free sea water the urine Na
Avas high, but again this was likely due to the high Na in the medium, substituting
for the deleted Mg. The concentration of Ca in the urine also seems unaffected by
the absence or relative increase of Mg. With regard to K, the urine concentrations
of this ion are significantly higher when the animal is immersed in 100% and 150%
Mg-free sea water, than when immersed in the same concentrations of natural sea
water (P < 0.01 ) . The mean urine K for animals immersed in 50% Mg-free sea
water also was higher than for crabs immersed in 50% natural sea water, but the
difference cannot be shown to be significant. Neither is the urine K of animals
MAGNESIUM AND WATER FLUXES OF A CRAB 445
immersed in 50% sea water with normal Mg concentrations (104 meq./l. ) signifi-
cantly different from that of crabs from 50% natural sea water.
Table II reveals the rate of Mg excretion in the different Mg-free salinities.
Thus, considering only those crabs immersed in the large volume where they could
not rise out of the water, it can be seen that the mean Mg excreted in 100% sea
water is greater than the mean Mg excretion in 150% sea water, but the difference
between these means is not significant (Table III ). On the other hand the rate
of Mg loss is four times as great in 50% sea water as it is in normal sea water ;
this difference is highly significant, P < 0.001 (Table III). Table II also shows
that less Mg is lost to a small medium than to a large one. This, of course, would
be expected because the animals could rise out of the small volume. Among all
test media the difference in rates of Mg excretion between large and small volume
treatments was smallest for 100% sea water, suggesting that in this salinity there
is a minimum attempt to rise out of the water. The tendency for this crab to
avoid an osmotic stress has been noted previously (Gross, 1957b).
The concentration of Mg in the urine of crabs immersed in large volumes of arti-
ficial sea water also is given in Table II. It will be recalled that these crabs first
were acclimatized to the respective salinities of natural sea water before treatment in
artificial sea water. Thus, there was no large change in the osmotic gradient to
which the animal was subjected following acclimatization. The indicated periods
of immersion were chosen because after such time in the test media, the urine Mg
did not differ significantly from that of crabs removed from the respective acclima-
tizing salinities of natural sea water. Immersion periods of more than one hour
in large volumes of Mg-free 50% sea water apparently deplete the Mg supply of
the crab and the urine Mg becomes greatly reduced in concentration. Mg excretion
of Pachygrapsus immersed for one hour in all three salinities of Mg-free sea water
could not be compared because in this brief time insufficient amounts of the ion
were released in 100% or 150% sea water to be detected with precision by the
methods available.
If, then, the consistent concentration of Mg in the urine were known throughout
a period of immersion and the amount of Mg lost to the medium in that period also
were known, then assuming that the antennary glands are the sole pathways of
Mg efflux, the volume of urine necessary to excrete the observed loss of Mg can
be calculated. Estimations of urine flow from mean rates of Mg loss and mean
Mg concentrations in the urine follow: in 150% sea water the volume of urine
production was calculated to be 1.5% body weight/day; in 100% sea water, 3.9%
body weight/day and in 50% sea water, 58% body weight/day.
The value for Pachygrapsus immersed in 100% sea water compares favorably
with values reported by Webb (1940) on Carchuts and by Robertson (1939) for
Cancer when the crabs were immersed in normal sea water, but only half the value
reported by Nagel (1934) for Carcimts immersed in normal sea water. These
workers, however, plugged the nephropores and assumed the gain in weight was
due to urine which could not escape. Values obtained by three different methods
on the prawn, Palaemonetes, in 100% sea water were more than twice as large
as the above rate for Pachygrapsus (Parry. 1955).
As might be expected, the calculated rate of urine flow for Pachygrapsus was
less in 150% sea water than in 100% sea water and the rate in 50% sea water
446 WARREN J. GROSS AND LEE ANN MARSHALL
greater than in normal sea water, but of such magnitude (58% body weight/day)
that it is subject to question. This rate was determined on the basis of Mg
excretion after one hour total immersion, but since the crabs had been acclimatized
to 50% natural sea water before treatment in the Mg-free medium, there was no
large increase in the osmotic gradient between external medium and the blood of
the animal. However, forced total immersion, which did not take place in the
acclimatizing procedure, caused more surface of the crab to be exposed to stress
and this probably caused an increase in the water influx and the consequent increase
in urine flow. It should be emphasized again that the concentration of Mg in the
urine following immersion in large volumes of 50% Mg-free sea water averaged
about the same as for crabs immersed for 24 hours in 50% natural sea water.
It is thus likely that the urine Mg concentration for this group of crabs did not
change during the one-hour immersion period. Either, then, the rate of urine
flow for crabs thus treated is as calculated (58% body weight/day) during that
period of immersion, or in such a hypotonic medium, mechanisms of Mg loss
are different from those in crabs immersed in 100% and 150% Mg-free sea water.
The Mg gradient between blood and external medium (Mg-free) would be about
the same for all three conditions ; yet Table II demonstrates the greatest mean
loss to 50% and the smallest mean loss to 150% sea water. It is our opinion that
if the principal pathway for Mg loss in all the above conditions were the antennary
glands, then the above value for the rate of urine flow is a fair approximation for
the conditions described. It would follow that such a rate could not be sus-
tained, and it is interesting that the number of fatalities for crabs immersed for
one hour in the large volume of 50% sea water was twice as large as the combined
number of fatalities for crabs totally immersed for six hours in 100% and 150% sea
water. Also, urine Mg in a few crabs which survived for six hours in 50% Mg-free
sea water was essentially nil, and the actual amount of Mg lost to the medium was
about twice that lost by crabs immersed in large volumes of Mg-free 100% sea
water for the same period. This indicates exhaustion of the Mg supply in the
crabs. When nine crabs which had been acclimated for 24 hours in small volumes
of 50% natural sea water were transferred to large volumes of the same medium,
for six hours, the mean urine Mg dropped to 47.2 meq./l., S.D. = 13.4. This is
significantly less than the urine Mg of crabs from small volumes of 50% natural
sea water (Table I) : P = 0.01. It is suggested that sudden total immersion in
natural 50% sea water causes a depletion of Mg reserves more rapidly than they
can be replenished from a medium of this salinity. The low mortality rate in
this group of crabs also suggests that Mg depletion is a cause of death in the crabs
immersed in Mg-free 50% sea water, but also raises the question as to how long
Pachygrapsus can survive totally immersed in 50% sea water in nature.
In order to estimate the urine flow when Pachygrapsus was removed from
water, ten crabs were taken from normal sea water, rinsed in distilled water to
wash away residual salts, then blotted dry. The crabs then were placed in dry
containers and kept in a relatively humid temperature-controlled room at 15° C.
for 72 hours. Then the crabs and their containers were rinsed with distilled water
and the washings saved for Mg analysis. Also, the urine from these animals was
sampled and analyzed for Mg. The urine concentration for Mg and the total
excretion of Mg into the container then should yield the volume of urine flow
MAGNESIUM AND WATER FLUXES OF A CRAB
447
during the 72-hour period. The average calculated rate of urine produced, thus
determined, was 0.02% body weight/day, which is hardly significant.
Table IV demonstrates the effects of altered Mg in the medium on the blood
concentration of the four major cations in Pachygrapsus. As would be expected,
the blood Mg concentrations of crabs immersed in 100% and 150% Mg-free sea
water are less than those of crabs from the same salinities of natural sea water.
However, the blood Mg of crabs immersed in Mg-free 50% sea water was not
TABLE IV
Ion concentrations in the blood of Pachygrapsus immersed in small volumes of
artificial sea water
50%
100%
150%
Mean
S.D.
No.
Mean
S.D.
No.
Mean
S.D.
No.
N
397
24
37
483
17.3
36
582
34
30
Na (meq./l.) 0
410
410
30
30
15
21
493
478
9.6
15.1
14
8
562
40
15
N/0
N/ +
0.97
0.97
0.98
1.01
1.04
N
7.36
1.4
37
7.43
0.72
36
10.2
1.5
30
0
5.78
1.0
15
9.19
0.82
14
8.62
1.4
15
K (meq./l.) +
N/0
N/+
6.53
1.27*
1.13
1.0
21
7.88
0.81*
0.94
1.4
8
1.19*
N
34.8
7.9
24
29.6
5.9
44
36.4
4.8
30
0
33.1
9.6
14
32.0
8.1
14
40.5
6.3
15
Ca (meq./l.) +
N/0
N/ +
29.7
1.05
1.17
6.4
16
38.1
0.93
0.78*
8.4
17
0.90
N
13.6
5.4
24
20.0
6.1
44
27.1
4.2
30
0
13.2
5.1
14
11.7
5.7
14
16.7
7.5
15
Mg (meq./l.) +
N/0
N/+
19.7
1.03
0.69*
7.4
16
33.2
1.71*
0.60*
6.1
15
1.62*
N = crabs immersed in natural sea water.
0 = crabs immersed in Mg-free artificial sea water.
+ = crabs immersed in artificial sea water containing abnormally high Mg: in 50% sea
water = 104 meq./l.; in 100% sea water = 156 meq./l.
* = significantly different from unity: P < 0.01.
significantly less than the blood Mg for crabs from 50% natural sea water. On
the other hand, blood Mg for crabs immersed in 50% artificial sea water which
contained normal Mg (104 meq./l.) was about equal to the blood Mg of crabs
from 100% natural sea water (20 meq./l. ). Also, the mean blood Mg of crabs
immersed in 100% artificial sea water with high Mg (156 meq./l.) was 33.2 meq./l.,
which is significantly higher (P < 0.01) than the concentration of this ion for
crabs which had been immersed in either 100% or 150% of natural sea water
(20.0 and 27.1 meq./l.. respectively). This is particularly interesting because as
448
WARREN J. GROSS AND LEE ANN MARSHALL
indicated above, the urine Mg of crabs from this artificial medium is about equal
in concentration to that of crabs from 100% natural sea water. It is apparent
that the blood concentration of Mg is influenced by the influx of Mg from the
medium, even though the Mg concentration in the urine is not directly affected.
Table IV also shows that blood Na is essentially unaltered by abnormal con-
centrations of Mg in the medium. It is interesting, however, that when immersed
in 100% artificial sea water containing high Mg, the blood Na of Pachygrapsus
remains normal, even though the concentration of this ion in the medium was re-
duced because of the high Mg. Blood Ca is unaltered in all conditions except when
the crab is immersed in 100% sea water with high Mg. Here the blood Ca is
significantly higher (P < 0.01 ) than for crabs from 100% natural sea water.
There seems to be an interdependence between the regulation of Ca and Mg under
these conditions. In Mg-free 50%, 100% and 150% sea water the blood K differs
from the blood K of crabs from natural sea water. There is no consistent trend,
TABLE V
Apparent volume of distribution of sucrose* in the blood space and water content for
muscle in Pachygrapsus following immersion in different salinities
Test medium
(% sea water)
Mean volume of
distribution
(% body wt.)
S.D.
No.
Mean water content
of muscle
(% wet wt.)
S.D.
No.
50
15.4
1.39
10
76.58
1.77
18
100
18.7
3.45
11
75.00
1.40
25
150
26.7
5.34
14
71.70
1.71
34
* One minute after injection.
but it can be suggested that there also is an interdependence between the regulation
of Mg and K. Such a suggestion is supported by the above mentioned differences
in urine K between animals from Mg-free sea water and those from natural sea
water.
Table V demonstrates that the muscle tissue of Pachygrapsus gains water when
the animal is immersed in 50% sea water and loses water when it is immersed in
150% sea water. That is, the muscle is permeable to water in both directions.
This is particularly interesting inasmuch as the animal itself shows no significant
weight changes during such treatments (Gross, 1957a). Table V also shows
that the calculated apparent volume of distribution for sucrose one minute after
injection into the blood space is smallest when the crab is in 50% sea water and
largest when the crab is immersed in 150% sea water. This is interpreted to
mean that the blood space volume of Pachygrapsus is altered when the animal is
transferred from one salinity to another by the changing volume of the formed
tissues. There may be objections to the use of only one concentration of sucrose
for the injected dose (1 M), but the volume of the dose was no greater than 0.5
ml. and, if anything, would be expected to cause an increase in blood volume for
crabs from dilute sea water and a reduced blood volume for crabs from concentrated
sea water.
MAGNESIUM AND WATER FLUXES OF A CRAB
449
DISCUSSION
The diagram presented in Figure 1 suggests the course of events with respect
to Mg and water fluxes when Pachygrapsus is immersed in different concentrations
of sea water. Since the indicated Mg values are based on the crab's response to
Mg-free media, it can be seen that the concentration of Mg in the urine of
150%
sea water
47Omeq/l Mg
1-5% body wt /day
urine flow
TOTAL
Mg Excreted =7meq
100%
sea water
a
o a:
ui
I
240meq/l Mg
\
3-9 % body wt/day
urine flow
TOTAL
Mg Excreted = lOmeq
50%
sea water
^^
(O
_l
^^^^;
<
ww^
=>
;^^^^\
O
UJ
ui cc
5
uj u)
3 i
w 5
§
T^Omeq/l Mg 58% body wt/day V
« *
f|
_l (D
Ss
O
_i
W 0
3 f-
Z
»
TOTAL
Mg Excreted =40meq
FIGURE 1. Scheme of urine flow, water shifts and Mg effluxes in Pachygrapsus when
exposed to different osmotic situations (indicated by the concentration of the external medium
in % sea water on left side of diagram). Arrows which represent urine flow and Mg efflux
are based on mean values for Mg concentrations in the urine and Mg losses to the medium when
the crab is completely immersed in 50%, 100% and 150% Mg-free artificial sea water (see
Table II). Length of arrow = volume of urine flow (% body weight/day); width = urine
concentration of Mg (meq./l.) ; arrow area = relative amount of Mg excreted (meq.). Rec-
tangles which are not drawn to scale represent crabs and illustrate the differences in blood
volume (hatched area) in different osmotic situations, as suggested by the calculated volume of
distribution for sucrose (Table V), and the volume of formed tissue (blank area), as suggested
by the water content of muscle for crabs from different osmotic situations (Table V).
450 WARREN J. GROSS AND LEE ANN MARSHALL
Pachygrapsus is directly related to the salinity of the external medium and not to
the concentration of Mg in the medium or its influx into the animal. Likewise, it is
shown that the efflux of Mg is inversely related to the concentration of this ion
in the urine. (While the mean Mg loss in 150% was less than the mean Mg loss
in 100% sea water, these values are not significantly different. However, the
mean Mg loss in 50% sea water was more than four times the loss in the other
two media.) Assuming that the antennary glands are the principal pathways for
Mg efflux for all conditions, then small volumes of urine are produced in concen-
trated sea water and large volumes of urine are produced in dilute sea water.
Figure 1 likewise shows that when Pachygrapsus is immersed in dilute sea water
the water content of formed tissues is higher than when it is immersed in normal
sea water, and conversely when the crab is immersed in concentrated sea water
the water content of its formed tissues is less than when it is immersed in normal
sea water. Such changes in water content effect volume alterations in the tissues
at the expense of the blood space. As shown in Figure 1, then, the concentration
of urine Mg is apparently determined by the water flux and is immediately inde-
pendent of the concentration of this ion in the medium. Also, it is relatively
independent of the Mg concentration in the blood. It will be observed (Table IV)
that the blood Mg of crabs immersed in Mg-free sea water can be reduced below-
normal, yet the urine Mg (Table I) remains high (e.g. in 100% sea water).
Again, in crabs immersed in 100% artificial sea water with high Mg the blood Mg
becomes elevated above normal, but the urine Mg remains essentially the same
as it is in crabs from 100% natural sea water. This response is contrary to the
findings of Webb (1940), who demonstrated in Carcinus in a similar experiment
that increases in the concentration of Mg in the external medium were reflected
in the urine, but very little in the blood. Thus the mechanism of Mg regulation
in Pachygrapsus may be fundamentally different from that of Carcinus. In the
latter case the concentration of Mg in the urine increased even though the total
salinity of the medium was normal ; yet the blood Mg remained close to the
concentration it attains in crabs from 100% natural sea water. As proposed by
Webb (1940), excretion of Mg in Carcinus by way of the antennary glands does
depend on the influx of this ion from the medium and consequently on the concentra-
tion of Mg in the blood. On the other hand, in Pachygrapsus, as shown above,
the concentration of Mg in the urine is relatively independent of both the influx of
this ion from the external medium and the concentration of Mg in the blood per se.
It seems, then, that in Pachygrapsus the concentration of urine Mg depends
inversely upon the rate at which the antennary glands form urine or the rate at
which the Mg-containing fluid is transported across the membranes of the antennary
glands. This, in turn, depends on the magnitude and direction of water flux
between the animal and its external medium.
The volume of urine flow calculated from the rates of Mg excretion seems
reasonable for crabs immersed in 100% sea water. At least this value (3.9% body
weight/day) agrees favorably with other values in the literature on other species,
suggesting that this may be a valid method for estimating urine production. It is
interesting that in a crab completely immersed in 150% sea water the calculated
urine flow is 1.5% body weight/day, because there is a tendency for the crab to
lose water to the hypertonic medium, yet there is no significant weight change
MAGNESIUM AND WATER FLUXES OF A CRAB 451
even though fluid is being lost by way of the urine and probably by diffusion.
The animal, therefore, must possess a mechanism for actively taking up water.
Drinking is suggested as the principal method for replacing lost fluid. Burger
(1957) has demonstrated drinking in Homarus; Green ct al. (1959) have produced
evidence that the gut takes part in the hypo-osmotic regulatory mechanism of Uca.
The excessive calculated urine flow for crabs completely immersed in 50% sea
water (58% body weight/day) is difficult to interpret. It does not seem that such
a rate could be sustained for long. This must mean that should this species inhabit
water of such low salinities for prolonged periods, it must either alter its perme-
ability or perhaps, being free to come out of the water, limit its period of immersion.
The calculated rate of urine flow in the small volume of 50% sea water where the
crab could rise out of the water was less than 10% body weight/day.
Again it becomes clear that the concentration alone of a given ion in the urine
does not reveal the relative rate of excretion of that ion or, in the case of crabs,
the relative quantitative role of the antennary glands in regulation of a particular
substance. Table II reveals that the greatest mean loss of Mg was by crabs
immersed in 50% sea water; yet these same crabs possessed the lowest concentra-
tions of Mg in the urine. Conversely, the smallest mean loss of Mg was by the
crabs immersed in 150% sea water which in turn had the highest Mg concentrations
in the urine (Table II, Figure 1 ). In the case of crabs kept out of the water,
the concentration of urine Mg was high (300 meq./l.), yet the amount excreted
in three days could hardly be measured.
It is suggested from the lack of Mg loss when the crab is out of the water that
Pachygrapsus must depend upon an uptake of water in order to excrete urine.
This, then, is another physiological limitation binding this semi-terrestrial species
to the sea. Gross (1955) discusses other characters which limit the terrestrial
life of this crab.
It has long been recognized that when an aquatic animal enters a medium of a
different salinity, it must undergo certain physiological adjustments which include
the mechanisms of tolerance permitting adequate functioning of the cells and tissues
despite changes in the salt concentration of the surrounding body fluids, or regula-
tion which keeps those changes in blood concentrations at a minimum. Evidence
has been produced by the present investigation that Pachygrapsus is capable of
regulating the total water content of its body by expelling the excessive influx
when in a hypotonic medium by a rapid flow of urine and, conversely, compensating
in some way for the physical efflux of water when immersed in a hypertonic medium
(perhaps by drinking). Table V, however, shows that the muscle tissue cannot
regulate its volume, at least not during a three-day exposure to osmotic stress, the
result being that the anatomy of the vascular system also becomes altered. It does
not seem that the changes in the volume of the blood space suggested by data in
Table V would not affect the efficiency of the animal.
It would seem, therefore, that should Pachygrapsus inhabit salinities which vary
much from those of normal sea water for prolonged periods, it would be obliged to
control the volume of its formed tissues so that a normal and efficient anatomy
could be assured for the vascular system.
These studies were aided by a contract between the Office of Naval Research,
Department of the Navy, and the University of California. NR 104-309.
452 WARREN J. GROSS AND LEE ANN MARSHALL
We wish to thank Mr. John Bristow and Miss Mary Conlee for their able
technical assistance. Also, we wish to express our gratitude to all those students
who assisted in collecting the experimental animals.
SUMMARY
1. The concentration of Mg in the urine of Pachygrapsus is dictated by the.
salinity of the external medium and not by the Mg concentration in that medium
or by the rate of Mg influx from the medium into the animal. Thus, during brief
periods of immersion in 50%, 100% or 150% sea water the urine Mg concentration
will reflect the salinity of the medium, irrespective of whether Mg is absent or in
abnormally high concentrations.
2. The Na concentration in both blood and urine is not drastically altered by
abnormal Mg levels in the external medium of any salinity tested.
3. After immersion in Mg-free 100% and 150% sea water the urine K of
Pachygrapsus is higher than it is after immersion in the respective concentrations
of natural sea water. Urine K is not influenced by the Mg concentration of 50%
artificial sea water or by abnormally high Mg in 100% sea water. Blood K con-
centrations are affected by varying concentrations of Mg in the external medium
of both dilute and concentrated salinities, but there is no definite trend.
4. The concentration of Ca in the urine of Pacliygrapsus is unaffected by the
Mg levels of all salinities tested. Blood Ca was not observed to be altered by
abnormally high or low Mg levels in all media tested except in 100% artificial sea
water with high Mg (156 meq./l. ), where the blood Ca was significantly
higher than for animals from 100% natural sea water.
5. While the concentration of urine Mg is not determined immediately by the
influx of this ion into the animal, the blood Mg concentration is lowered when the
crab is immersed in a Mg-free medium and raised when the medium Mg is
abnormally high. The concentration of urine Mg is relatively independent of
the levels of Mg in the blood.
6. Pachygrapsus excretes more Mg in 50% sea water than in 100% sea water
and perhaps less in 150% sea water than in 100% sea water, even though the con-
centration of Mg in the urine is in the reverse order (i.e., 150% > 100% > 50% ).
7. Calculated rates of urine production for Pachygrapsns completely immersed
in different salinities follow: in 150% sea water, 1.5% body weight/day; in 100%
sea water, 3.9% body weight/day; in 50% sea water, 58% body weight/day. The
observed rate for crabs immersed in 50% sea water is not believed to be sustained
for long, as suggested by the high mortality rate. When removed from water, the
volume of urine excreted by Pachygrapsus is insignificant.
8. The concentration of urine Mg in Pachygrapsus thus is inversely related to
the rate at which urine is produced by the antennary glands and this is dependent
on the magnitude and direction of the water flux, imposed by the physical gradient
between the crab and its external medium.
9. The volume for muscle tissue in Pachygrapsus increases when the crab is
transferred from normal sea water to dilute sea water and decreases when it is
transferred to concentrated sea water. Such volume changes take place at the
expense of the blood space.
MAGNESIUM AND WATER FLUXES OF A CRAB 453
10. It is suggested that alterations in the volume of the blood space caused by
osmotic stress likely would reduce the efficiency of the vascular system which in
turn would impose further ecological limitations on this species.
LITERATURE CITED
BARNES, A., 1954. Some tables for ionic composition of sea water. /. E.vp. Bio!., 31 : 582-588.
BURGER, J. W., 1957. The general form of excretion in the lobster Honianis. Biol. Bull., 113:
207-223.
GREEN, J. \Y., M. HARSCH, L. BARK AND C. L. PROSSER, 1959. The regulation of water and salt
by the fiddler crabs, L"ca pituna.i' and Ucct pin/ilatur. Biol. Bull.. 116: 76-87.
GROSS, W. L, 1955. Aspects of osmotic regulation in crabs showing the terrestrial habit.
.liner. Nat., 89: 205-222.
GROSS, W. J., 1957a. An analysis of response to osmotic stress in selected decapod Crustacea.
Biol. Bull., 112: 43-62.
GROSS, W. L, 1957b. A behavioral mechanism for osmotic regulation in a semi-terrestrial crab.
Biol. Bull.. 113: 268-274.
GROSS, W. I., 1959. The effect of osmotic stress on the ionic exchange of a shore crab. Biol.
Bull.. 116: 248-257.
JONES, L. L.. 1941. Osmotic regulation in several crabs of the Pacific Coast of North America.
/. Cell. Comp. Physiol.,18: 79-91.
NAGEL, H., 1934. Die Aufgaben der Excretionesorgane und der Kiemen bei der Osmoregulation
von Carciinis inacnas. Zcitschr. f. Pliysiol.. 21 : 468-491.
PARRY, G., 1955. LTrine production by the antennal glands of Palaemonetes various (Leach).
/. Exp. Biol.. 32: 408-422.
PROSSER, C. L., J. W. GREEN AND T. J. CHOW, 1955. Ionic and osmotic concentrations in blood
and urine of Pachvgrapsus crassipcs acclimated to different salinities. Biol. Bull.,
109: 99-107.
ROBERTSON, I. D., 1939. The inorganic composition of body fluids of three marine invertebrates.
/. Exp. Biol.. 16: 387-397.
SHAW, J., 1955. Ionic regulation in the muscle fibres of Carciinis mamas. II. The effect of
reduced blood concentration. /. £.r/>. Biol.. 32: 664-680.
WEBB, D. A., 1940. Ionic regulation in Carciinis inacnas. Proc. Rov. Soc. London, Scr. B,
129: 107-136.
XEUROMUSCULAR PHYSIOLOGY OF A SESSILE SCYPHOZOAN
G. F. GWILLIAM 1
Department of Zoology, University of California, Berkeley 4, Calif., and
Friday Harbor Laboratories, University of Washington, Seattle 5, Wash.
Interest in the coelenterate neuromuscular system has persisted for many years,
and information gained by a variety of approaches. Pantin (1935a, 1935b, 1935c,
1935d) first introduced the nse of controlled electrical stimuli and the observation
of subsequent muscular contractions as a means of analysis of the properties of
the conducting system in Actiniaria. Bullock (1943) has shown that the propaga-
tion of the swimming pulse in Scyphozoa differs in no fundamental way from
excitation in anemones, although there are certain quantitative differences that
result in the remarkable dissimilarity of behavior of the two groups of organisms.
In anemones, contraction of the muscles is relatively rapid, but relaxation is slow,
and because of neuromuscular facilitation each impulse after the first arriving at
the muscle excites further contraction so that a typical "staircase" is recorded from
the contraction of the sphincter muscle in Calliactis. In scyphozoan medusae the
first impulse is effective in eliciting a response. Each succeeding impulse, if de-
livered within a well defined time limit, enhances the response for several contrac-
tions until a plateau is reached which may be maintained for long periods of time.
After each contraction the muscle relaxes completely (or almost so) so that the
typical kymograph record appears as in Figure 1, a recording of Cyanca bell
contractions. This is the result of rapid contraction, rapid relaxation, the restoring
force of the mesoglea, and an extraordinarily long absolute refractory period
(0.7 second, according to Bullock, 1943 ) of the muscles of the medusa bell.
As indicated above, all critical work using controlled electrical stimulation has
been confined to Anthozoa and free-swimming jellyfish. This raises the question
of the relationship of response to mode of life. Are the phenomena described
sharply divided along systematic lines, or are there some definite and demonstrable
properties that may be associated with the sessile habit or the free-swimming habit?
The Stauromedusae, about which very little is known, offer ideal material for such
a study.
Except for a brief creeping larval phase, Stauromedusae are sessile throughout
their existence, but there is no doubt of their affinities with the Scyphozoa. Do they
retain the neuromuscular patterns of their nearest relatives, or has the sessile habit
led to modifications (or retentions from the scyphistoma larva) that make the
Stauromedusae functionally more closely associated with the anemones? Although
Stauromedusae lack the ability to perform pulsating swimming motions, one might
wonder if the system is capable of such contractions under artificial stimulation.
Is the absence of a pacemaker the only essential factor here?
The comparative aspect of the problem would be ideally developed by studying:
1 Present address : Dept. of Biology, Reed College, Portland 2, Oregon.
454
PHYSIOLOGY OF A SESSILE SCYPHOZOAN
455
( a ) the responses of anemones ; ( b ) the responses of free-swimming Scyphozoa :
( c ) the responses of the scyphistoma larva of the Scyphozoa ; and ( d ) the responses
of Stauromedusae. A knowledge of all of these might enahle one to assess the
consequences of the sessile habit and give some insight into the evolution of their
neuromuscular mechanisms and behavior. The present stud}- deals only with the
last of these.
MATERIALS AND METHODS
Haliclyshis auricula ( Rathke ) was used because of its abundance near the
Friday Harbor Laboratories of the University of Washington. Large individuals
were collected, along with a portion of the blade of Zostcra to which they are
normally attached. The animals were maintained in the laboratory in running
FIGURK 1. Cyaiica cupillata. Record of bell contractions.
Electrical stimuli, 0.5 per second.
sea water at a temperature close to that of their natural environment ( 10-13° C.).
Reactions were first explored by means of manually controlled mechanical stimuli
and later by controlled condenser discharge shocks. Recordings were made with
very light isotonic levers on a smoked drum.
Electrical stimuli were delivered under sea water to the exumbrellar surface,
usually at the base of the stalk or at the stalk-calyx junction, by means of silver
electrodes insulated to the tip, with Ag-AgCl-sea water electrodes or non-polarizable
calomel electrodes. A student "Electrodyne" stimulator, which delivers brief con-
denser discharge shocks, and for some experiments a Grass model S4B stimulator,
were used. All experiments were made on fresh animals at temperatures of
11-13° C. At temperatures above 16-18° C. responses became very erratic,
probably due to the rapid decay of facilitation ( Hall and Pantin. 1937; Pantin and
Vianna Dias, 1952). Mechanical stimuli were delivered with a clean, blunted
glass rod or with a glass rod tipped with a short length of silver wire.
Anatomical studies to determine the details of the musculature were carried
out by observation on entire living and preserved animals under reflected and
transmitted light and between crossed polaroids. To confirm the results obtained
from a study of entire animals, portions of several were sectioned and stained
456
G. F. GWILLIAM
with picro-indigo-carmine following bulk staining in Grenacher's borax carmine,
or with alum hematoxylin and eosin.
I would like to express my gratitude to the Director and Staff of the Friday
Harbor Laboratories of the University of Washington for a grant which made
residence at the Friday Harbor Laboratories possible for a period of seven months.
My thanks are also due to the Department of Zoology, University of California,
Berkeley, for support and encouragement over a period of several years. Dr.
Ralph I. Smith and Dr. Cadet Hand have aided in many important ways, not
the least of which was a critical reading of the manuscript. Their interest and
criticism is deeply appreciated. Dr. T. H. Bullock has been kind enough to
read the manuscript. His criticisms and suggestions have been very helpful and
are gratefully acknowledged.
I MM
MS
FIGURE 2. Cross-section through the stalk of Haliclystns auricula. SC, stalk canal ;
MS, stalk muscle; M. mesoglea.
RESULTS
Musculature of Haliclystns
It is possible to divide the muscles of Haliclystns into three categories associated
with particular regions of the body. The stalk possesses four interradial muscular
bundles (Fig. 2) embedded in the mesoglea which continue distally into the sub-
umbrellar ectoderm as eight perradial sheets that fan out over the subumbrellar
surface (Fig. 3). Proximally. the stalk muscles are inserted at the pedal disc
over a rather wide area. There is also a marginal muscle (subumbrellar) which
borders the margin of the calyx ( Fig. 3 ) and is only partially embedded in the
mesoglea. This latter muscle is interrupted at the arms and so consists of four
perradial and four interradial segments. In addition, arm tentacles, gastric tenta-
cles, manubrium, and anchors are provided with muscle fibers. The principal
PHYSIOLOGY OF A SESSILE SCYPHOZOAN
457
muscles involved in the reactions studied, however, are those of the stalk, sub-
umbrella, and margin. "With the possible exception of the stalk musculature, all
the muscles of Haliclystns are confined to the subumbrellar surface, just as in
free-swimming Scyphozoa. Thiel (1936) states that there are probably fine muscle
fibrils distributed all over the body, although their existence has not been estab-
lished, and no particular function has been ascribed to them. I have been unable
to determine the presence of exumbrellar muscle fibers in Haliclyshts and believe
that all of the gross actions of the organism can be explained on the basis of inter-
actions of the well-defined muscle fields and the mesoglea.
i I CM i
MP
FIGURE 3. Diagram of H. auricula subumbrella to illustrate musculature. T, tentacle
group; G, gonad ; MR, radial muscle; A, anchor; MM, marginal muscle; MO, mouth; MP,
perradial muscle.
Spontaneous activity
A considerable amount of apparently spontaneous activity may be observed in
Halicl\stiis under both field and laboratory conditions. The arms are flicked in
toward the mouth, the calyx is rotated, the stalk alone or the whole animal may
contract without any noticeable external stimulation. No well-defined rhythm is
apparent from visual observations, nor is any particular order of contracting parts
consistently manifested.
Kymograph records of this activity were made on slow drums. There is no
orderly progression of height of contraction as in free-swimming scyphozoan jelly-
fish. The contractions recorded are due primarily to the stalk muscles and do
not show the contractions of individual arms. However, such recordings serve to
illustrate the arrhythmic nature of spontaneous activity of unmolested animals
(Fig. 4, A).
It was apparent from the outset that difficulty would be encountered in attempts
to record responses to electrical stimuli in such an active animal. With this in
mind, various parts of the body were isolated, to ascertain whether or not any
45S
G. F. GWILL1AM
structure or structures exercised a "pacemaker" control over any other part.
Considering the systematic position of the animal, the most obvious place to look
for such control was the umbrellar margin, which, in free-swimming scyphomedusae,
bears the rhopalia, known to be the seat of pacemaker control of the swimming
pulsations. In the majority of medusae (both Hydrozoa and Scyphozoa ) removal
of the bell margin results in a more or less complete paralysis of the swimming
movements (Romanes, 1885; Bullock, 1943). X<> such pacemaker region has
DEMARGINATE
FIGURE 4. Records of spontaneous activity. A. Intact animal. B. Stalk only. Drum
speed increased in central portion for 15 minutes. C. Stalk only. Hour marker applies to
entire record except central part of B.
been demonstrated in anemones or in hydrozoan polyps, but in the case of anemones,
the spontaneous activity is of such a slow nature as to allow investigation of neuro-
muscular responses with considerable confidence. In Haliclystus this activity occurs
with sufficient frequency to be extremely troublesome.
In order to determine whether or not "surgical paralysis" was possible, a large
Haliclystus was set up in such a manner that stalk and calyx contractions could
be recorded independently and simultaneously. Pins were placed at the stalk-
calyx junction, fastening the animal to a wax-bottomed dish so that the two parts
could be separated without disturbing the mechanical recording devices. Contrac-
tions of the intact animal were recorded for several hours, after which a sharp
knife was drawn across the animal at the stalk-calyx junction, isolating each part
from the other, and the recording continued for several m.ire hours. Examination
PHYSIOLOGY OF A SESSILE SCYPHOZOAN 459
of the record shows that no difference can be observed in the nature of the contrac-
tions before and after separation. Further recordings were made of intact animals
and isolated stalks, and no difference was noted (Fig. 4).
Removal of arm tips (tentacles), anchors, bell margin, and, as has been seen,
the whole calyx, has no effect on the nature of spontaneous activity. From the
above, it is apparent that there is no physiological centralization of the exciting
system that manifests an}' pacemaker control over any other part. The mechanism
of this type of activity has not been established directly, but may be inferred from
evidence to be presented later.
Responses to mechanical stimulation
The stalk and exumbrella: Stimulation of the surface of the stalk and of the
exumbrella in attached and unattached Haliclystus seldom elicited a response.
Gentle stroking of the exumbrellar surface, stimulation by light touch, and fairly
vigorous prodding almost invariably failed to call forth a response. If, however,
firm pressure were maintained with the glass rod, e.g. on the edge of the pedal disc,
a sudden shortening of the stalk and closure of the bell resulted. On several
occasions the response was elicited only upon injury to the animal, and continued
pressure did not cause a maintained calyx contraction. Shortly after the sudden
closure the animal would relax, the calyx relaxing first, even if pressure sufficient
to cause injury were maintained. This response could be obtained from any
point on the exumbrellar surface and did not appear to be restricted to the pedal
disc. The pedal disc does not appear to be any more sensitive than other areas,
although the methods used would not give information on any slight gradient of
sensitivity. After numerous attempts at mechanical stimulation of the exumbrella,
it was concluded that the exumbrellar surface was very nearly insensitive to ordinary
mechanical stimulation, responding only to very vigorous stimuli.
This is not surprising when one considers the conditions under which Haliclvstiis
normally lives. The population from which experimental animals were taken is
almost completely confined to rather dense beds of Zostcra marina, with the animals
usually attached to the blades in a pendant attitude. The Zostcra is exposed only
at the lowest tides, which means that for the greater part of the life-cycle of the
animal it is hanging in the water among constantly moving blades of the plant,
and is therefore subjected to a considerable amount of buffeting. The exumbrella
would be exposed to repeated stimulation by blades of Zostcra, debris in the
surrounding water, and perhaps fairly frequent rubbing over the bottom. If these
medusae were very sensitive to exumbrellar stimulation, they would lie in the
contracted state for a major part of their existence.
Subumbrellar surface and associated structures: The tentacles are extremely
sensitive to weak mechanical stimulation. The slightest touch to a single tentacle
usually brings about an arm-bending response. Sometimes only a few tentacles
react, but more often there is a rapid bending of the whole arm toward the mouth.
The arm tips at the base of the tentacles on the exumbrellar side are quite in-
sensitive if the stimulus is applied outside the area of the tentacles, but are quite
sensitive on the subumbrellar side.
The entire stibumbrella is very sensitive to mechanical stimulation. If the
stimulus is applied on the adradius, the arm in the position responds by bending
460 G. F. GWILLTAM
toward the stimulated spot, and frequently the arm on either side also responds.
If the stimulus is applied between the two arms (i.e., in the inter- or perradius),
the two adjacent arms respond by bending to the stimulated spot, and frequently,
the next adjacent arm on either side also bends. In addition it was noted that
the manubrium moved toward the stimulated area whenever the arm-bending
response was obtained.
Repeated stimulation of a single spot on the subumbrella may either bring about
a total calyx closure, or local insensitivity develops and the animal fails to respond.
If a slight stroking movement covering several millimeters is made with a glass
rod or silver wire, the total closure response results. It was noted on several
occasions that repeated light touches in one spot would bring about total calyx
closure in a fairly regular fashion. First the adjacent arms would bend toward
the stimulated spot, then on the next stimulus the next adjacent pair, and so on
until all eight arms were bent inwards. The exact relationship between stimulus
and response in such cases was difficult to assess. If the stimulating object were
not removed quickly, it would be caught by the tentacles and the stimulus of this
contact would almost invariably cause total calyx closure. Quick removal of the
glass rod, however, may have caused sufficient local disturbance to provide addi-
tional sources of stimuli, so that accurate appraisal of the stimulus-response
relationship under these conditions seems impossible.
If the total calyx closure response were elicited by the stimulus of a linear
stroke, stalk contraction and a characteristic rotation of the calyx usually occurred
as well. The calyx rotation appeared simultaneously with the stalk contraction.
It was noted that any adequate mechanical stimulus delivered at the edge of
the pedal disc always brought about a calyx closure reaction in addition to the
stalk contraction, but that adequate calyx (subumbrellar) stimulation did not always
elicit stalk contraction. This might be considered evidence of polarization in the
conducting system, in the sense that it is "easier'' to drive an impulse in one
direction than in the other, but due to the methods of stimulation and the extreme
difference in the intensity of "adequate" stimulation of the two areas concerned,
it would seem unwarranted to accept this observation as evidence of polarity.
Conduction of excitation may be described as diffuse. A single touch elicits
the arm-bending response in two to four arms, as well as a shift of the manubrium
toward the stimulated point. The so-called "decremental" nature of conduction
is also noted in the spread of contraction around the calyx from repeated or more
vigorous stimulation. That conduction of excitation is rather slow is quite evident
from observations made during mechanical stimulation. The arm nearest the site
of stimulation always responds first, the other two following in order depending
on whether the stimulated point is nearer one than the other. If they are equi-
distant they react simultaneously. The reaction is sufficiently slow so that the
above is readily observed. Thus, the special properties of the nerve net as outlined
by Pantin (1935a) are apparently fulfilled in Haliclystus.
Response to electrical stimulation
It has been indicated previously that the normal activity of H. auricula presents
an unpredictable variable when one attempts to determine the responses of the
organism to artificial stimulation. I found no convenient method of suppressing
PHYSIOLOGY OF A SESSILE SCYPHOZOAN 461
this activity. Further difficulty was encountered in determining the area to be
stimulated. The demonstrated insensitivity of the exumbrellar surface offered the
advantage that placement of the electrodes on it caused little disturbance, but this
insensitivity may have been the result of absence of nervous elements in the
exumbrellar ectoderm. If the electrodes were placed on the subumbrellar surface
there was an immediate response to the tactile stimulation, and after a time local
insensitivity developed.
The site finally selected was the exumbrella. Kassianow (1901 ) demonstrated
what appears to be a nerve plexus in the exumbrellar ectoderm of Stauromedusae,
and experiments to be described support this. Insensitivity to mechanical stimula-
tion may be due to rapid sensory adaptation or to high sensory thresholds and
paucity of mechano-receptors in the exumbrella. Neither of these possibilities has
been directly demonstrated, but the responses suggest such explanations.
It was necessary to exercise caution in setting up the animals for recording.
When possible, the animals were left attached to the Zostcra on which they were
collected and the plant pinned to the bottom of a wax- or clay-lined container.
Small glass hooks were used to attach the animal to the recording lever. These
were inserted through the bell margin beneath the marginal muscle at one side of
an anchor. If the hooks were placed through the arm tips the animals remained
unduly agitated for some time, presumably due to stimulation of the tentacles.
Each preparation had to be observed carefully to eliminate the possibility of addi-
tional stimulation from the recording connections. Animals were not used until
three to four hours after having been set up. to allow recovery from the effects of
injury. Tests indicated that the responses did not differ appreciably after one-half
hour from those after twenty-four hours following injury. All recordings were
made from animals in standing sea water.
Response to single-impulse stimulation: As indicated previously, a single
condenser-discharge shock results in a contraction of small amplitude in free-
swimming Scyphozoa (Bullock. 1943). In the anemones that have been studied
(Pantin. 1935a; Hall and Pantin, 1937; Pantin and Vianna Bias, 1952), single-
shock stimulation does not result in a recorded response. To determine the re-
sponse of HaMclystus to this type of stimulation, a large specimen was set up for
recording and the electrodes applied to the stalk ectoderm.
As might be expected, the results were variable. Stimulation was being ap-
plied against a background of spontaneous activity. Contractions were recorded
during the course of these experiments, but they were not always associated with
the stimulus. Responses occurred before, after, and at stimulation in an apparently,
random manner. There was no consistent contraction associated with the delivery
j
of the stimulus until high intensities were reached. Examination of preparations
receiving such shocks revealed an injured area at the site of the electrodes, and
these responses were interpreted as a result of multiple stimuli resulting from
injury. Because of the fact that contractions occurred at random with this type of
stimulation except at high voltages, it was concluded that Haliclystits does not
characteristically respond to single-impulse exumbrellar stimulation.
The failure to respond to single-impulse stimulation also characterizes reflex
closure of the sphincter of the anemone Calliactis (Pantin. 1935a) and the response
of the longitudinal mesenteric muscles of Mctridiniu (Hall and Pantin, 1937).
462
G. F. GWILLIAM
0.5
0.3
0.2
FIGURE 5. Tracings of records of stalk response to pairs of shocks. Upper trace of each pair,
contraction record ; lower trace, signal marker. Interval between shocks in seconds.
Following single-shock stimulation in Haliclystus, a twitching of the tentacles was
often observed, as has been reported for Calliactis by Pantin (1935a), who con-
sidered it an indication that the impulse was being transmitted. In scyphozoan
medusae there is a consistent measurable response to a single stimulus, although
the magnitude of the response is very much less than that of which the preparation
is capable (Bullock, 1943).
Response to pairs of stimuli : After having established the failure of Haliclystus
to respond in a consistent manner to single-impulse stimulation, the response to
pairs of stimuli was investigated. Pairs of shocks at a frequency of one in two
seconds, and one, two, three, and five per second were delivered to the preparation.
A standard five-minute rest period was observed between the delivery of each pair
of stimuli, but because of spontaneous activity this interval was frequently extended.
At least one minute of observed inactivity was allowed before the delivery of stimuli.
A record of a typical experiment of this sort is shown in Figure 5. It will be noted
that a response does not occur until the members of the pair of stimuli are separated
by only 0.2 second, i.e., at a frequency of five per second. Occasionally a response
occurred at three per second, but the illustrated case is more typical.
FIGURE 6. Diagram of experimental set-up for simultaneous
stalk-calyx recordings.
PHYSIOLOGY OF A SESSILE SCYPHOZOAX 463
During the course of the investigation described above, it was noted that total
calyx closure regularly occurred at a lower frequency stimulation than stalk
contraction. Because of the recording method, such calyx contractions were not
recorded. Accordingly, animals were set up in such a way that independent
and simultaneous records could be obtained of stalk and calyx contractions (Fig. 6).
Stimuli were delivered on the exumbrellar surface at the stalk-calyx junction in the
>ame manner as in the previous experiments. These were repeated on individuals
without the encumbrance of the recording and holding devices, and in all cases the
results corresponded to those shown in Figure 7. The calyx contracts weakly at
a frequency of two per second, more strongly at three and five per second ; at the
latter interval the stalk contracts, as would be predicted from the previous deter-
mination. In contrast to the observed reactions to single stimuli, the responses to
pairs of stimuli under the conditions described above are remarkably consistent.
Calyx
•I— t-
Slalk
Calyx
02
FIGURE 7. Tracings of records obtained as illustrated in Figure 6. Lower trace, signal marker.
Interval between shocks in seconds. Unrecorded contraction observed at arrow.
Characteristic response frequencies for different parts of the responding system
have been demonstrated in anemones by Pantin ( 1935b ) and can be related to the
function of the parts. In Haliclystus the calyx is the feeding organ of the animal
and as such must be sensitive to very light mechanical disturbances if it is to
fulfill its function.
Responses to trains of stimuli: The response to pairs of stimuli is essentially a
total response. It is evident on observation of unmolested animals that natural
responses are graded, i.e., by no means all responses to naturally occurring stimuli
involve all of the responding muscles to the full extent. It is also apparent that each
of the three categories of muscles described earlier is capable of at least some inde-
pendent action. The calyx is able to contract (as has been shown) without stalk
contraction, and the stalk under certain conditions may show slow, partial, and
frequently asymmetrical responses without the calyx being involved. This is ap-
parent in the characteristic movements by which the expanded calyx is swept
through the water, rotated, and inclined at various angles. The marginal muscle
is also apparently capable of independent activity, as indicated by certain postures
5 V, 3 PER SECOND
20 V, 3 PER SECOND
50 V, 3 PER SECOND
25 V, 10 PER SECOND
FIGURE 8. Records of stalk responses to various voltages
and frequencies of electrical stimulation.
464
PHYSIOLOGY OF A SESSILE SCYPHOZOAN 465
the animal assumes. In the expanded individual it is noted that the eight adraclial
arms are most frequently equidistantly arranged. At times, however, the arms are
drawn together so that they are associated in four pairs, and such pairs may lie in
the perradii or, more frequently, in the interradii. Examination of such animals
reveals slight folding of the mesogiea over the marginal muscle between the pairs
of arms and the corresponding radial muscles of the suhumhrella. The intervening
muscles show no sign of contraction.
In order to investigate the underlying mechanism of the graded response, the
reactions to a variety of stimuli were examined. No information concerning the
mechanism of the postural response has been obtained, but certain inferences con-
cerning symmetrical graded responses may be made from reactions to trains of
stimuli at different frequencies. Because of the spontaneous activity of the animals,
it was impossible to interpret the results when long trains of stimuli at very low
frequencies were administered. Any observed contraction could just as well be
spontaneous as a reaction to the applied stimuli. At intermediate frequencies, how-
ever, i.e., lower than that required for a response to a single pair, but higher than
one shock every two seconds, a consistent pattern emerged.
Figure 8 illustrates a possible mechanism of graded response. It will be noted
that there is a well-marked threshold and that increasing the intensity of the stimula-
tion does not appreciably affect the height of contraction. At voltages just above
threshold, however, increased frequency of stimulation has a marked effect on
contraction amplitude.
A similar experiment was conducted on an animal set up for simultaneous stalk
and calyx recording, as in Figure 6. The results shown in Figure 9 were recorded
from the same animal in the space of less than one-half hour. It will be noted that
there is no evidence of a well-marked "staircase" in any of these responses, as has
been observed in the anemone Calliactis by Pantin ( 1935a ), but that the contractions
are essentially continuous. This may be interpreted as the result of the relationship
between speed of contraction and decay of facilitation if factors similar to those in
other coelenterates are operative. There is none of the machine-like precision in
Haliclystiis that is demonstrated by anemones and free-swimming Scyphozoa. This
is probably because of the complicating factors of frequent spontaneous activity,
speed of contraction, and the rapid decay of facilitation.
Responses to subumbrellar stimulation: The responses to electrical stimuli re-
ported above are from stimulation of the exumbrellar surface, and are essentially
only symmetrical responses. Asymmetrical responses are clearly possible, as indi-
cated by mechanical stimulation and by observation of the undisturbed animal.
Certain of these reactions may be examined by subumbrellar electrical stimulation
despite the disadvantages pointed out earlier. Such responses have not been re-
corded because of the weakness of the muscles involved. In some cases, isolated
calyces were used, while in others the observations were made on intact animals.
Upon placement of the electrodes there wras an immediate and sometimes re-
peated response, either asymmetrical or total in nature. Electrodes were placed as
lightly as possible, but the response ensued upon contact, however light. The
animal was then left undisturbed for at least fifteen minutes.
Because of the extreme sensitivity of the subumbrella, precise analysis of the
neuromuscular properties was very difficult. Single-impulse stimulation frequently
elicited a localized response involving only one or two arms, as was observed follow-
466
G. F. GWILLIAM
ing light mechanical stimulation.
Single-impulse stimulation was never observed
to cause a generalized response, but pairs of stimuli at 0.5- to 0.1-second intervals
would cause a generalized contraction. Pairs at greater intervals would cause either
two localized contractions coincident with stimulus delivery or would spread to the
adjacent arms, depending on the interval. If several stimuli were delivered at a
frequency of one per second, the contraction would spread in both directions around
FIGURE 9. Simultaneous stalk-calyx contractions recorded as for Figure 7. Contraction
records inked to increase contrast. A. Response to a train of shocks at 2/second. B. Response
to a train of shocks at 10/second. Each preparation received 25 shocks.
the calyx, bringing successive arms into contraction coincident with each successive
stimulus. Those already contracted would contract again. Thus, the total calyx
response could be elicited in a stepwise manner.
A possible explanation of these results is that there is an immediate spread of
the impulse to the neuromuscular junctions on the first stimulus and then neuro-
muscular facilitation governs the excitation of the muscle. A single pair of stimuli
at a sufficiently short interval excites the whole system, but it takes several stimuli
at lower frequencies to accomplish this. This is probably related to the rapid decay
of facilitation, and suggests through-conducting tracts in the subumbrella which, on
the basis of this evidence, might be syncytial. Evidence to be presented later, how-
ever, suggests that there is interneural facilitation as well.
PHYSIOLOGY OF A SESSILE SCYPHOZOAN 467
Conduction speed in the "nerrc net"
A number of attempts to measure speed of conduction in the system were carried
out. Each determination was repeated several times before any consistency was
obtained, and even then there was considerable variation. A strip of tissue was
cut from the calyx, leaving one end of this tongue attached to the animal, which was
then pinned to a wax-bottomed dish with the stalk projecting upwards. The stalk
was connected to a recording lever in the usual way. A pair of stimuli was de-
livered at an appropriate interval distally on the tongue of tissue, and after five to
ten minutes' rest, a similar pair was delivered at a measured distance proximal to
the original site. The time from the second stimulus to contraction was carefully
measured for each pair of stimuli, and the speed of conduction calculated from the
difference in time and the distance between the stimulating electrodes. This was
done on several animals, and the values obtained were 7-15 cm. /sec. at 11-12° C,
comparable to those of 10-20 cm. /sec. obtained for Calliactis body wall by Pantin
(1935b).
Other attempts were made by placing the electrodes at two different points on
a line with the reacting muscles on the exumbrella in the intact animal. The results
of such determinations varied so radically that they have not been included. Orig-
inally the assumption was made in these experiments that no conducting tissue
crossed the mesoglea. and that the route of the impulse from stalk stimulation was
up the stalk ectoderm, over the bell margin and down the subumbrella to the stalk
muscles. The results suggested that there was transmission through the mesoglea
at some point along the route.
The form of the "nen-e net"
The most careful study of the nervous system of the Stauromedusae is thtit of
Kassianow (1901 ). In a very thorough investigation using various staining tech-
niques, including vital methylene blue, Kassianow studied the histology and cytology
of the nervous system and associated structures in Liicenwria ( — Cali'adosia )
campanulata, Halielystits oetoradiatus (--H. auricula?}, and Craterlophus tetliys.
He describes and figures a diffuse exumbrellar plexus of hi- and tripolar ganglion
cells. The experiments to be described largely confirm Kassianow's morphological
findings.
Exumbrellar transmission of excitation: In order to test for the presence of
exumbrellar nervous elements, several large specimens of Haliclystns auricula were
selected and a circular incision was made through the stalk ectoderm at a level four
to six millimeters above the pedal disc (Fig. 10, A). Following the operation the
animals were given two to four hours in which to recover and were then stimulated
on the stalk ectoderm aboral to the cut. Under these conditions a contraction could
not be elicited.
Stimulus intensities up to 100 volts failed to cause excitation at frequencies at
and above threshold for normal animals. Simply transferring the electrodes to the
oral side of the cut resulted in excitation at normal frequencies and voltages.
Animals were examined for muscle and endodermal continuity before and after
stimulation. All animals showed continuity, although there was some damage to
stalk canals in all specimens except one which gave the same results as the others.
468 G. F. GWILLIAM
The stalk of another individual was split from the base up toward the calyx and a
cut made from the inside out toward the ectoderm, leaving only the ectoderm in
continuity, and stimuli applied basal to the cut (Fig. 10, B). Under these condi-
tions excitation occurred at normal voltages and frequencies. Subsequent examina-
tion revealed that a strip of ectoderm two to three millimeters wide wras the only
continuous tissue except for the very superficial mesoglea underlying the strip.
This indicates that conduction of excitation is diffuse. If the conducting tissue is
in the form of fibers, these are probably diffuse ; but if conduction is a general
property of the ectoderm or other superficial layers, one would also expect the
results obtained. The latter possibility seems unlikely, but is not excluded by
the evidence.
It might then be assumed that the normal path of exumbrellar transmission is
over the exumbrellar surface to the bell margin, over the marginal edge to the
subumbrellar muscles and to the stalk muscles. To test this assumption, the entire
subumbrellar surface, including the oral region, was removed from an animal
(Fig. 10, C). The ring muscle was left intact, as were the distal ends of the
gonads, the arm tips, and tentacles. Under these conditions there is no ectodermal
continuity from any point on the exumbrella to the stalk muscles, and since the
radial muscles of the calyx have also been removed, transmission cannot take place
through them. Stimuli were then delivered at the base of the stalk, and at normal
voltages and frequencies a response of the stalk muscles was elicited. Such a
preparation does not respond to a pair of stimuli at 0.2-second interval as does an
intact animal, which may indicate that the normal pathway of excitation has been
interrupted. Also, this preparation still had some septal tissue, which may have
given continuity.
To insure no marginal continuity, another animal had the entire upper portion
of the calyx removed as well as the subumbrellar tissue (Fig. 10, D). Under these
conditions, a contraction could also be obtained at normal voltages and frequencies,
although it took four shocks at five per second to elicit the response.
Still another animal was completely demarginated, the stalk split from the base
up to within one millimeter of the cut edge, and stimuli applied at the base of one-
half of the stalk (Fig. 10, E ). This meant that if excitation were transmitted to the
other half, it would be forced to traverse the mesoglea at some point in the non-
stimulated half after having gone through the one-millimeter strip of ectoderm at
the cut margin. The four groups of stalk muscles are separated by mesoglea at
this level, and there is no opportunity for transmission through the split base of the
stalk. Under these conditions, the non-stimulated half responds to shocks at normal
voltages and frequencies. In this preparation there was some continuous endo-
dermal tissue at the oral end, but the previous demonstration of the necessity of
ectodermal continuity for transmission in the stalk reduces the significance of this.
Furthermore, it does not affect the contention of mesogleal transmission, because
neither the endoderm nor the ectoderm is in contact with the muscles.
Wietrzykowski (1912) clearly shows the development of the stalk muscles from
the "cordon cellulaire de la taeniole" which has its point of proliferation in a sub-
umbrellar position. This means that the direct innervation of the stalk musculature
from the exumbrellar ectoderm is a result of conducting tissue arising in the
exumbrellar ectoderm and becoming associated with the musculature, or of con-
PHYSIOLOGY OF A SESSILE SCYPHOZOAN
469
B
FIGURE 10. Diagrams to illustrate operations to ascertain the form of the conducting
system. See text for explanation. E, ectoderm; EN, endoderm; M, mesoglea ; SP, septum;
T, tentacles. Position of stimulating electrodes indicated by arrows.
470 G. F. GWILLIAM
ducting tissue migrating out from the muscle to the ectoderm. Horridge (1956),
however, has demonstrated a diffuse nerve net on the exumbrellar surface of the
ephyra of Aurellia aitrita, and it is possible that the exumbrellar conducting system
of H. auricula is the homologue of the diffuse system in other scyphozoans.
Subumbrcllar transmission of e.v citation: To ascertain the nature of subumbrellar
conduction, the stalk and oral structures were removed from a specimen of
Haliclystiis. Four incomplete cuts were made from the margin to within a few
millimeters of the cut inner surface between alternate pairs of arms, and similar cuts
were made from the inside out toward the margin, alternating with the first cuts
described (Fig. 10, F). This gave a ring preparation similar to those used by
Romanes (1885) in which the impulse is forced to take a devious path to circle the
bell. The electrodes were applied in the position indicated and trains of impulses
were delivered to the preparation.
Under these conditions a train of impulses at normal voltage and at a frequency
of five per second elicited a local contraction involving only the nearest two or four
arms. At ten per second, the whole calyx could be brought into contraction in the
following particular manner. First the arms nearest the electrodes respond, then
those on each side in sequence and by pairs until all contract. There is a noticeable
delay between the contractions of successive parts, as if junctions were being facil-
itated in turn. This preparation emphasizes the functional autonomy noticed in
the intact animal. It also establishes the diffuseness of conduction and proves that
no one pathway is essential for conduction around the bell. The expected pathway
would be around the bell margin in the region of the marginal muscle, but the
preparation described above shows that continuity of the muscle, and hence of any
accompanying nerve tracts, is not essential to conduction. With arms and anchors
removed, the preparation reacts in the same way as when these structures are present.
The above indicates that the arms are functionally associated into pairs. This
functional pairing appears to depend on the integrity of the intervening marginal
muscle and may indicate a normal through-conducting mechanism around the bell
from arm to arm. Furthermore, if the stimulus is applied in the interradius, the
contraction progresses by interradial pairs of arms. If applied in the perradius,
the first reaction involves the adjacent arms only, or may involve the interradial
pair on either side. This indicates that an interradial pair of arms, with its asso-
ciated radial musculature (see description of musculature) and marginal muscle,
has a degree of autonomy as a pair that separates it functionally from the other
interradial pairs.
If the above results are examined in the light of stimulation of the intact calyx,
the evidence for a through-conducting system is supported. That it is probably
associated with the marginal muscle is indicated by the failure to respond to a pair
of stimuli when this muscle is cut. It appears that under these conditions of mutila-
tion, several junctions must be facilitated in order to bring about a generalized
contraction.
DISCUSSION
The results of this study enable one to construct a coherent picture of the normal
behavior and responses of Haliclystus auricula. The pulsating contractions of the
free-swimming Scyphozoa are lacking in Haliclystus. It has been demonstrated
that there is no pacemaker present, and also that the requisite degree of specialization
PHYSIOLOGY OF A SESSILE SCYPHOZOAN 471
of the conducting system is lacking. Thus, the calyx of Haliciystus lacks the ca-
pability of responding in the repetitive fashion of its free-swimming relatives to
repeated shocks at regular intervals. However, the available evidence indicates that
a through-conducting system is normally operative in the subumbrella of the calyx.
No evidence for higher conduction speed in that part of the animal, as compared to
other parts, has been obtained. The evidence is primarily the different responses
of experimentally incised and intact animals. The through-conducting system is
brought into play only by relatively high frequency stimulation. At low frequencies
the radial parts of the calyx exhibit considerable autonomy. In addition to this
through-conducting system there also appears to be a diffuse "nerve net" which
operates to correlate the subumbrellar structures, such as the manubrium, with the
action of the principal food-gathering devices, the arms and the tentacles. There is
no clear evidence of more than one nerve net as has been reported in AurcUia by
Horridge (1956).
Inherent spontaneous activity may be interpreted in the following way : no part
of the animal exercises control over any other part. From this we may assume that
impulses are spontaneously generated in many, if not all, ganglion cells. Such
impulses spread over the entire nerve net, but do not activate the muscles concerned
unless two or more arrive at any one junction within an appropriate time interval.
The nature of the spontaneous activity seems to indicate that each ganglion cell
discharges at its own rate, each independently of the others. Activity may or may
not be rhythmical. The graded and asymmetrical nature of the spontaneous activ-
ity, together with the lack of a regular pattern supports such an explanation. A
pattern of endogenous activation of the muscles could lead to the observed sweeping,
"seeking" motions of the unstimulated animal. Exogenous stimuli impinging upon
the exumbrella must be of sufficient intensity to generate relatively high-frequency
impulses if the protective closure-contraction response is to ensue, as indicated by
the relatively high frequency of electrical stimulation required to bring about a
response. The subumbrella, on the other hand, responds as a whole to lower fre-
quency stimulation in a local fashion. Thus, if a food organism brushes very lightly
over the subumbrellar surface a suitable reaction will be forthcoming in the arm-
bending response to secure the prey. If the prey struggles, further impulses are
generated, and the entire calyx responds to ensure capture and ingestion. The role
of chemical stimuli in the predation process has not been investigated, but is no
doubt of some importance — especially in the discharge of nematocysts and gland
cell secretions.
If, as has been assumed, Haliciystus possesses a simple plexiform nerve net, the
reactions reported in this study are subject to two possible interpretations. The
high degree of autonomy noted in different regions of the subumbrellar structures
is most easily explained on the basis of neuroneural facilitation at synapses between
strategic areas of the net, or a hierarchy of thresholds of neuromuscular junctions
to frequency and number of arriving impulses. Siich a system, along with the
endogenous activation of muscles, is admirably adapted to sweeping through the
water in a "seeking" motion, and to catching prey on contact.
The exumbrellar nerve net shows a high degree of facilitation with rapid decay
characteristics not noted in either anemones or free-swimming Scyphozoa, and this
occurs at comparatively low temperatures (11-13° C.). The relationship of decay
472 G. F. GWILLIAM
of facilitation (rapid) to contraction time (comparatively rapid) to relaxation time
(relatively slow) is such that a fused contraction results at frequencies sufficiently
high to elicit a contraction.
The subumbrellar nerve net exhibits the properties of through-conduction, neuro-
muscular facilitation and, probably, interneural facilitation. It responds locally to
single-impulse stimulation, indicating a partial permanently facilitated state.
The properties of the neuromuscular system of Haliclystus do not appear to
differ in any fundamental way from those noted in anemones (Pantin, 1935a,
1935d) and free-swimming Scyphozoa (Bullock, 1943). Modifications of the char-
acteristics of the nerve net with regard to the time sequence of decay of facilitation
and certain time characteristics of the muscles would appear to be sufficient to
explain the differences in behavior noted in these animals. No new properties need
be postulated to account for the reactions that do not fall in the range of variation
already noted in the coelenterates.
Bearing in mind the presumed derivation of the Stauromedusae from free-
swimming Scyphozoa, we may speculate upon the differences involved with the
sessile habit. The most obvious of these is the loss of the pacemaker mechanism of
swimming, and the loss of (or failure to develop) the specialized through-conducting
mechanism that makes the swimming pulsations possible. Secondly, the stalk mus-
cles are able to maintain a prolonged contraction not possible in free-swimming
Scyphozoa. This is not as pronounced in the calyx musculature, which relaxes
rather quickly even under sustained stimulation (see Figure 9). The properties
of the mesoglea indicate that contraction is maintained against a mesogleal restoring
force, hence the muscle must possess a refractor}' period of shorter duration than
that found in free-swimming forms. Since sustained contraction differs in the stalk
and calyx muscle systems, it is probable that each system has a muscle refractory
period different from the other. Neither system is able to maintain a contraction
for the length of time noted in anemones, so it may be reasoned that the physiological
properties of both systems lie somewhere between those of free-swimming scypho-
zoans and those of anemones.
SUMMARY
1. Previous work on the neuromuscular systems of coelenterates has been con-
fined to Anthozoa and free-swimming medusae, and certain differences in the re-
sponse to electrical stimulation have been noted. It was thus of interest to inves-
tigate the responses of a sessile scyphozoan, Haliclystus auricula (Rathke), to
determine the characteristics of the response mechanism.
2. Spontaneous activity of H. auricula is arrhythmic, and there is no demonstra-
ble "pacemaker." The exumbrellar surface of the organism is relatively insensitive
to mechanical stimuli, but the tentacles and subumbrellar surfaces are very sensitive.
The conducting system possesses properties of a "nerve net." Conduction is slow,
diffuse, and with an apparent decrement.
3. H. auricula does not respond to single-impulse electrical stimulation of the
exumbrella. A pair of stimuli elicits stalk contraction at an interval of 0.2 second,
and calyx contraction at 0.5 second. On the other hand, single-impulse stimulation
of the subumbrella will elicit a response, but this response is local, involving only a
part of the calyx near the position of the electrodes. A possible mechanism of the
graded response is suggested.
PHYSIOLOGY OF A SESSILE SCYPHOZOAN 473
4. Physiological evidence indicates that the conducting system is diffuse, with
some evidence of a through-conducting pathway at the bell margin that operates at
relatively high-frequency stimulation. Conduction speed in the subumbrellar nerve
net is calculated at 7-15 cm. per second at 11-13° C.
5. The physiological properties of the neuromuscular system of Haliclystus ap-
pear to be intermediate between Calliactis and Cyanea. There is a demonstrable
difference in the response of stalk and calyx which shows emphasis on sequential
elicitation by mechanical stimulation of progressively greater intensity of tentacle,
local marginal, essentially whole calyx, and finally symmetrical stalk involvement.
The significance of this series of events in relation to the normal behavior of the
animal is pointed out.
LITERATURE CITED
BULLOCK, T. H., 1943. Neuromuscular facilitation in Scyphomedusae. /. Cell. Coinp. Physiol.,
22 : 251-272.
HALL, D. M., AND C. F. A. PANTIN, 1937. The nerve net of the Actinozoa : V. Temperature
and facilitation in Metridium senile. J. Exp. Biol., 14 : 71-78.
HORRIDGE, G. A., 1956. The nervous system of the ephyra larva of Aurellia aitrita. Quart. J.
Micr. Sci., 97 : 59-74.
KASSIANOW, N., 1901. Studien iiber des Nervensystem der Lucernariden, nebst sonstigen histo-
logischen Beobachtungen iiber diese Gruppe. Zcitschr. zviss. Zool., 69 : 1-93.
PANTIN, C. F. A., 1935a. The nerve net of the Actinozoa. I. Facilitation. /. Exp. Biol., 12 :
119-138.
PANTIN, C. F. A., 1935b. The nerve net of the Actinozoa. II. Plan of the nerve net. /. Exp.
Biol., 12 : 139-155.
PANTIN, C. F. A., 1935c. The nerve net of the Actinozoa. III. Polarity and after-discharge.
/. Exp. Biol., 12 : 156-164.
PANTIN, C. F. A., 1935d. The nerve net of the Actinozoa. IV. Facilitation and the "stair-
case." /. Exp. Biol., 12 : 389-396.
PANTIN, C. F. A., AND M. VIANNA BIAS, 1952. Excitation phenomena in an actinian
(Bunodactis sp. ?) from Guanabara Bay. Anales Acad. Bras, de Cienc., 24: 335-349.
ROMANES, G. J., 1885. Jelly-fish, Star-fish, and Sea-urchins. The international scientific series,
New York, D. Appleton and Co. 323 pp.
THIEL, M. E., 1936. Scyphomedusae. In: Klassen und Ordnungen des Tierreichs, by H. G.
Bronn, Bd. II, 2. Buch, Lfr. 1 and 2. Leipzig, Akademische Verlagsgesellschaft m.b. H.
WIETRZYKOWSKI, W., 1912. Rechcrches sur le developpement des Lucernaires. Arch. Zool.
Exp. Gen., Ser. 5, 10 : 1-95.
SEASONAL CHANGES IN COLD-HARDINESS OF
FUCUS VESICULOSUS
JOHNSON PARKER
William B. Greelcy Memorial Laboratory, Yale University, New Haven, Connecticut,
and the Marine Biological Laboratory, Woods Hole, Massachusetts
Some marine algae, such as the fucoids, grow attached above mean low water and
are, therefore, subjected at ebb tide to rather severe atmospheric conditions in some
climates. In winter in the arctic, species of Fit ens may be subjected to air tempera-
tures down to -- 40° C., yet do not seem to be at all injured (Scholander, Flagg,
Hock and Irving, 1953; Kanwisher, 1957). The drying resistance of marine algae
'has been studied frequently in the past and it is apparent that those growing highest
in the littoral zone are generally the most desiccation-resistant (Muenscher, 1915;
Pringsheim, 1923; Isaac, 1935; Stocker and Holdheide, 1937; Biebl, 1939). The
fact that they are desiccation-resistant suggests that they might also be fairly cold-
resistant. This idea is borne out by findings of Kylin (1917) with Fncns and by
those of Biebl (1958) with various marine algae.
In spite of this work, there appear to have been no seasonal studies of cold-
hardiness of the common littoral marine algae. A number of such studies have been
made with land plants (for example, Parker, 1955, 1959), and these have shown
that there are wide fluctuations in cold-hardiness with the season in all woody plants
of relatively cold climates. It therefore seemed of general biological interest to
determine whether a marine plant such as Fncits fcsicnlosits L. would also go
through such fluctuations.
METHODS
From June of 1958 to late May of 1959 Fncns plants were collected from con-
venient locations along the Long Island shore, near New Haven, as a preliminary
study. Although there appeared to be a definite trend in hardiness from about
- 20° C. in August to about - 60° C. in February, results were so irregular that
it was suspected that some other environmental factor or factors besides air tempera-
ture were involved. It was therefore decided to repeat these experiments another
year but to obtain the plants from a single location, both as regards the harbor and
the position above low tide. It was found that hardiness varied as much as 15° C.
in May between plants brought from about the mean low tide level and those
brought from the highest fucoid zone, the higher ones being more resistant. This
latter zone was used in the 1959-1960 experiments which are reported in this paper.
It was decided that the stone jetty at Hammonasset beach, near Madison, Con-
necticut, was the best location, since it was accessible and the water relatively clean.
An attempt was also made to obtain the plants as near to 2 :00 P.M. as possible, in
spite of variable tide levels for a particular time of day at different times of the year.
Fncns plants about eight inches long were plucked from the rock, put in
474
COLD-HARDINESS IN FUCUS
475
stoppered jars of fresh sea water, and taken to the laboratory within 30 minutes.
Fronds three inches long, including reproductive tips as well as vegetative ones, were
cut from these plants, blotted on filter paper, and put in 500-ml. Dewar flasks in a
damp condition. These were stoppered with a cork and cooled by an ethane com-
pression system in a low temperature apparatus having a two-foot square compart-
ment (Parker, 1959). The ethane, in turn, was cooled by a Freon-22 compressor
system (Cincinnati Sub-Zero Products, Ohio). After being cooled at a rate of
20
10
-10
-20
-30
-40
-50
-60
_L
f •
• o
I i O
_L
N
M
M
FIGURE 1. Upper curves represent maximum and minimum temperatures at the New Haven
municipal airport, averaged for each month for the 1959-1960 season. Lower curve (dashed)
represents the relative hardiness of Fucus, drawn rather freely as the approximate point at which
50% of the frond was alive (reducing tetrazolium chloride). Data are given in Table I.
Blacked-in circles represent temperatures to which a group of plants were cooled on a
particular day.
4° C. per hour to one of the temperatures indicated by the blacked-in circles in the
results (Fig. 1), flasks were removed to an ordinary refrigerator for three hours and
then to room air to obtain a warming rate of about 8° C. per hour.
Determination of viability by ordinary means, for example by leaf color, is very
difficult in these marine algae and may account for the lack of research in this field.
But by means of the tetrazolium test, very clear and reliable results could be ob-
tained. The fact that reduction of this compound by dehydrogenases to its red
formazan derivative is a good indication of cellular viability has been previously
476
JOHNSON PARKER
discussed (Parker, 1953a, 1953b). One-inch-long cuttings from the tops of the
cold-treated frond material were placed in 8-ml. test tubes and 6 ml. of a solution
added, consisting of a 1 : 1 mixture of sea water and 0.6% 2,3,5-triphenyl tetrazolium
chloride in tap water. Tubes were stoppered and placed in the dark for 4 to 18
hours at 23° C. Although some results can be observed in 4 hours, it was found
best to wait 18 hours. After about 24 hours, bacterial action commonly interferes
with results and clouds up the water with red precipitate. Reliability of the tetra-
TABLE I
Data shown in the lower part of Figure 1, together with effects of tetrazolium (TTC) test
June 15
Temp.*
TTC test**
-19
100
-29
100
-42
0
Aug. 15
Temp.
TTC test
-10
100
-19
100
-24
100
-36
40
-58
0
Aug. 29
Temp.
TTC test
-26
100
-31
50
-36
40
-48
5
Sept. 10
Temp.
TTC test
-14
100
-23
100
-32
40
-57
0
Oct. 5
Temp.
TTC test
-8
100
-17
100
-25
100
-34
100
-46
5
-57
0
-67
0
Xov. 15
Temp.
TTC test
-24
100
-43
5
-62
0
Dec. 4
Temp.
TTC test
-25
100
-35
100
-42
10
-59
5
Jan. 15
Temp.
TTC test
-24
100
-35
100
-43
80
-59
10
Feb. 12
Temp.
TTC test
-25
100
-35
100
-44
80
-59
15
Mar. 18
Temp.
TTC test
-25
100
-37
100
-44
60
-57
50
Apr. 10
Temp.
TTC test
-25
95
-35
100
-47
10
-67
5
May 9
Temp.
TTC test
-24
100
-35
70
-45
5
-61
3
May 31
Temp.
TTC test
-24
100
-32
5
-44
0
-65
0
June 6
Temp.
TTC test
-24
100
-34
5
-44
0
-60
0
* Temperature in degrees centigrade to which fronds were cooled.
** Frond surface in % showing positive tetrazolium test.
COLD-HARDINESS IN FUCUS 477
zolium test was also supported by the fact that Fucus failing to reduce the compound
emitted a rotten odor in 48 hours while those reducing it did not.
RESULTS
Cold-hardiness was at a minimum during the summer at about -- 30° C. (Fig.
1 ) . Hardiness increased by October and by late winter reached about • - 50° C.
Since there were frequently borderline cases of damage, it was necessary to express
results in terms of percentage tissue area reducing tetrazolium chloride (Table I).
The dashed line in Figure 1 is drawn at approximately the point where there was
50% of the tissue not injured (reducing tetrazolium chloride). But even this
system was somewhat complicated by the fact that in late summer, growing tips were
more sensitive to cold than the rest of the plant, whereas in winter and early spring,
tips appeared to be more hardy than the rest of the plant.
In spring, dehardening began to appear by mid- April when woody land plants
were showing rapid dehardening. This was apparently related to the higher air
temperatures of spring, beginning near the end of March. Surface water tempera-
tures measured at the western end of Long Island Sound (Anon., 1947) and plotted
as monthly means for a year showed little deviation from the air temperature data
shown in Figure 1. It is therefore impossible to say whether air or water tempera-
ture is better related to changes in hardiness. There is, of course, no proof that
seasonal changes in hardiness are not controlled endogenonsly or by some other
environmental factor such as day-length.
SUMMARY
A seasonal study of the changes in cold-hardiness of Fucus vesiculosus L. was
made over a two-year period. Plants in summer could withstand about -- 30° C.
(the lowest temperature at which 50% of the frond was still alive after treatment),
whereas in January and February, plants could withstand — 45° C. to nearly
- 60° C. Changes in hardiness with the season appeared to be related to air
temperature variations, but also to mean monthly surface water temperatures plotted
for a year. In May, Fucus taken from the low tide level was 15° C. more sensitive
to the cold treatment than those from the highest level of the fucoid zone. Growing
tips were the hardiest part of the plants in winter and early spring, but the least
hardy in late summer.
LITERATURE CITED
ANON., 1947. Surface water temperatures at Coast and Geodetic Survey tide stations. Dept.
Comm., U. S. Coast and Geodetic Survey, Wash., D. C., pp. 1-39 .
BIEBL, R., 1939. Protoplasmatische Okologie der Meeresalgen. Bcr. d. deutsch. hot. Ges., 57:
(78)-(92).
BIEBL, R., 1958. Temperatur- und osmotische Resistenz von Meeresalgen der bretonischen
Kiiste. Protoplasma, 50: 217-242.
ISAAC, W. E., 1935. A preliminary study of the water loss of Laminaria digitata during inter-
tidal exposure. Ann. Bot., 49: 109-117.
KANWISHER, J., 1957. Freezing and drying in intertidal algae. Biol. Bull., 113: 275-285.
KYLIN, H., 1917. t)ber die Kalteresistenz der Meeresalgen. Ber. d. dciitsch. bot. Ges., 35:
370-384.
478 JOHNSON PARKER
MUENSCHER, W. L. C, 1915. Ability of seaweeds to withstand desiccation. Puc/ct Sound Alar.
Sfa. Pub., 1 : 19-23.
PARKER, J., 1953a. Some applications and limitations of tetrazolium chloride. Science, 118:
77-79.
PARKER, J., 19S3b. Criteria of life: some methods of measuring viability. Amcr. Sci.. 41:
614-618.
PARKER, J., 1955. Annual trends in cold hardiness of ponderosa pine and grand fir. Ecology,
36 : 377-380.
PARKER, J., 1959. Seasonal changes in white pine leaves: a comparison of cold resistance and
free sugar fluctuations. Bot. Gaz., 121 : 46-50.
PRINGSHEIM, E. G., 1923. t)ber die Transpiration bei Fucns. Jahrb. f. iviss. Bot., 62: 244-257.
SCHOLAXDER, P. F., W. F. FLAGG, R. J. HOCK AND L. IRVING, 1953. Studies on the physiology
of frozen plants and animals in the arctic. /. Cell. Coinp. Physio!., 42: 1-56.
SrorKER, ()., AND W. HOLDHEIDE, 1937. Die Assimilation Holgolander Gezeitenalgen wahrend
der Ebbezeit. Zcitschr. f. Bot., 32: 1-59.
STUDIES ON MARINE BRYOZOA.
XIII. TWO NEW GENERA AND NEW SPECIES FROM ANTARCTICA
MARY D. ROGICK
College of Nciv Rochcllc, ATnv Rochcllc, Nciv York
The purpose of the present study is to describe two new genera, Toretocheilum
and Isoschizoporella, and a new species, T. ab sidatum, from Marguerite Bay,
Antarctica, and to elevate a previously known variety. Sclusoporella tumida var.
tricitspis Calvet 1909, to species rank: Isoschisoporella tricuspis (Calvet) 1909.
The two species were collected for the Smithsonian Institution by the U. S. Navy's
1947-1948 Antarctic Expedition (hereafter referred to as USN) by Comdr. D. C.
Nutt. The writer wishes to express her very grateful appreciation to the Smith-
sonian, U .S. National Museum, for the loan of these specimens and to the National
Science Foundation for grants so generously supporting this and related researches.
Both species belong to the family Hippoporinidae as redefined by Osburn (1952.
pp. 316 and 343). He limited the family to include those schizoporellid species
which have a pleurocyst or olocyst type of frontal wall with marginal areolar pores
(areolae). avicularia. usually strong cardelles, orifice and operculum constricted at
sides, in some species at least.
GENUS TORETOCHEILUM, NEW GENUS
Diagnosis. Colony encrusting. Zooecia entirely adherent. Dietellae present
(Fig. 9). Zooecial front wall convex, an areolate and moderately costate pleuro-
cyst.' Frontal not otherwise porous. Large pointed adventitious avicularia on
front, over areolar pores (Fig. 1 ). Globose ovicell with areolae around border in
ectooecium or where ectooecium and entooecium meet (Figs. 2, 3, 5). Ovicell
partly immersed in next distal zooecium (Fig. 5). Zooecial operculum does not
close ovicell aperture. Low spine-bearing peristome surrounds zooecial orifice
(Figs. 1, 6, 10). Orifice suborbicular, laterally and distally bounded by a C-shaped
vestibular arch which supports the operculum (Figs. 6, 7). Vestibular arch ends
proximally on each side in thick ledge-like cardelle (Figs. 8, 10). Between the
cardelles is a gap or inner sinus closed by the tab of the operculum (Fig. 8). In
front of this, externally, the low peristome rises to form two curved cusps which
encircle the rounded median peristomial sinus (Fig. 10).
Name derivation. The genus was named Toretocheilum, meaning "pierced
lip," because of the sinus-pierced proximal peristome which forms the lower "lip"
of the orifice. It is of Greek derivation (see Brown's lexicon ) from toretos, bored
or pierced, and cheilos (neuter noun), lip or rim.
Type species: Toretocheilum absidatum, n. sp.
Type locality: Marguerite Bay, Antarctica, Sta. 240.
479
480
MARY D. ROGICK
PLATE I.
MARINE BRYOZOA, XIII
481
Remarks. The new species absidatum presented a problem in identification
and relationships. It had features strongly allying it to both genus Chiastosella
and genus Stephanosella.
Chiastosella is known only from the southern hemisphere and is represented by
both fossil and Recent species. Stephanosella has a wider distribution.
Chiastosella is classified in different families by different authors. Stach (1937)
and Brown (1952) allocate it to the family Schizoporellidae, while Bassler (1953)
allocates it to the family Hippoporinidae, which was once a subfamily of Schizo-
porellidae but which is now a separate family. Bassler (1953) and Osburn (1952)
Torctocheilmn absidatwn, new7 species
LIST OF ABBREVIATIONS USED ON THE PLATES
A Areolae or areolar pores
B Areolar costae
Avicularial chamber
Avicularial pivot
Avicularium
Ectooecium of ovicell
Entooecium of ovicell
Frontal wall
Mural rim
K Opercular sclerite
L Operculum
M Spines or spine bases
C
D
E
F
G
H
J
N Orifice of zooecium
O Ovicell
P Paraoral areolar pores
R Peristome
S Peristomial cusps
T Peristomial sinus
U Vestibular arch or collar
V Vestibular ledge or cardelles
W Distal part of zooecium
X Proximal part of zooecium
Y Distal wall
Z Lateral wall
PLATE I
All figures on this plate are of Torctocheilitui absidatum, new genus and new species, and are
drawn with the aid of a camera lucida.
FIGURE 1. Seven calcined non-ovicelled zooecia, some with one or two avicularia, some
without any.
FIGURE 2. An ovicell drawn directly from the rock under low power (dissecting micro-
scope). Above the two spine bases are the spaces ("pores") between ectooecium and entooecium.
FIGURE 3. A calcined ovicelled zooecium with an avicularial chamber in which the under-
lying areolar pore is visible. The floor of the avicularial chamber is the frontal wall of the
zooecium.
FIGURE 4. Mandible of an avicularium.
FIGURE 5. Two young calcined zooecia, the one at left ovicelled, the one at right non-
ovicelled. At lower right, imbedded in the proximal front wall of the latter, is a damaged ovicell
which belongs to a zooecium below those shown. The front wall of the damaged ovicell is
broken off, exposing the inner wall of the ovicell. The ovicell rim shown over the orifice later
becomes reduced as calcification proceeds.
FIGURE 6. External view of orifice of a non-ovicelled zoid. One cardelle of vestibular arch
is hidden by a peristomial cusp.
FIGURE 7. Operculum with curved sclerites for muscle attachment. The median tab below
fits the space between the cardelles and the back of the cusps.
FIGURE 8. Interior of the frontal wall, operculum and half the orifice. The thick ends
(V, ledges or cardelles) of the vestibular arch hold the operculum in place. Through the
opercular tab can be seen the peristomial cusps and peristomial sinus. These peristomial struc-
tures are external to the operculum. Drawn to the 0.2 mm. -long scale at right.
FIGURE 9. The attached back or basal wall of two zooecia, showing the three distal darkly
shaded dietellae of each.
FIGURE 10. Zooecial orifice tipped forward to show the exact relation of the C-shaped
vestibular arch and vestibular sinus to the peristomial cusps and peristomial sinus.
482
MARY D. ROGICK
place Stephanusclla in the family Hippoporinidae. The difference between the
two families is based on the nature of the orifice and the frontal wall.
The new USN species absidatnin is closer to Cliiastosclla daedala (Mac-
Gillivray) 1887, type species of Chiastosella Canu and Bassler 1934 (see Bassler,
1934, p. 407) as regards the distinctive orifice, peristome, spines, and avicularia
but differs in the type of frontal wall and ovicell porosity and sculpturing. In the
latter features (frontal wall and ovicell) absidatnin is closer to Stephanosella Canu
and Bassler 1917, but the orifices are quite different.
Chiastosella at present contains a diversity of species (cf. Stach, 1937; D. A.
Brown, 1952, 1954) that could split the genus between two families (Osburn's
limited families Hippoporinidae and Schizoporellidae ) . Therefore, it was thought
more sensible to erect a new genus Toretochcilum for the dissident and complicating
USN absidatum than to further diversify the genus Chiastosella by including a
new species of such divergence from the type C. daedala.
Diagnosis. Colony encrusting, well calcified. Zooecia convex and approxi-
mately hexagonal. Frontal a pleurocyst with one row of areolae, the paraoral pair
often more pronounced. Areolar grooves and costae prominent. None, one or
two large transverse, pointed avicularia over areolar pores on mid-frontal corners.
Five to 7 stout oral spines on peristome. Orifice suborbicular, lined by a C-shaped
vestibular arch whose ends form prominent blunt cardelles proximally. Proximal
border of orifice straight to concave, with round, median sinus inserted between
or flanked by two arched cusps. Ovicell with row of areolae where ectooecium
and entooecium meet peripherally, but otherwise non-porous. Ovicell has faint
proximal rim, bordered by two oral spines. Three or more large basal dietellae.
Name derivation. The species T. absidatnin was named for the arched proxi-
mal peristomial cusps which outline the median orificial sinus, and also for the
remainder of the arched orifice. The trivial name is derived from the Latin apsis
(absis), arch; absidatus, arched, vaulted. (Cf. Brown's lexicon.)
Measurements. Given below are minimum, maximum and the average of a
number of readings, usually 10, in millimeters. L is for length, W for width, D for
diameter.
0.965-1.267
0.432-0.763
0.576-0.605
0.547-0.576
0.202-0.245
0.216-0.245
0.014-0.058
0.259-0.331
0.130-0.173
0.216-0.346
0.029-0.058
0.163-0.202
0.124-0.130
(1.081 ) L zooecia
(0.634) W zooecia
(0.586) L ovicell, 4 readings
(0.566) W ovicell, 4 readings
(0.217) L orifice, including sinus
(0.230) W orifice
(0.033) D sinus
(0.297) L avicularium
(0.151) W avicularium
(0.250) L oral spines, 6 readings
(0.045 ) D oral spines
(0.182) L mandible, 4 readings
(0.128) W mandible, 4 readings
Colony. Four small patches of colony were found encrusting a thick flat hand-
sized rock from Marguerite Bay. Pieces had to be scraped or burned off for study
MARINE BRYOZOA, XIII
because they were on the sides of the rock in areas that could not be maneuvered
for study under the compound microscope.
Zooccia. Zooecia are box-like, their fronts 4- to 6-sided. The side walls
are vertical, low and about the same height all around. Three or so large dietellae
outline the distal half of the basal wall (Fig. 9).
The frontal wall is very convex, mound-like, with thin, raised mural rim.
A row of deeply sunk oval areolae outlines it. Grooves radiate centerward from
the areolae. Short costal ridges separate them.
The frontal wall consists of a translucent calcareous olocyst fast overgrown by
an opaque granular or roughened pleurocyst. The central part of the pleurocyst
is non-porous except for an occasional pore puncturing the avicularial chamber
near its base. The areolar pores are not large but the grooves and ridges make
them more conspicuous than wrould otherwise be the case. The two areolae nearest
the orifice corners are often slightly larger than the others (Figs. 5, 6). In young,
less heavily calcified zooecia the grooves leading from these two paraorificial areolae
pass just proximal to the orifice, leaving the orifice elevated slightly above the
immediate frontal wall (Fig. 5).
Avicularia. Avicularia are all of one type and of approximately the same size.
Usually one, sometimes two, large pointed avicularia occur on some zooecia. Other
zooecia may be without any. If two avicularia are present on one zoid they are
placed bilaterally, opposite each other. Avicularia are located at the corners over
areolar pores, midway up the side of the front wall. They are transversely oriented
with mandible pointing out. Sometimes they slant a bit obliquely (Figs. 1,3).
Each avicularium is mounted on a prominent avicularial chamber which tips
the avicularial beak to a slightly upward-directed or oblique position. The avicu-
larial chamber is large at the base and covers one or two areolae. As is the
custom in calcified species with large adventitious avicularial chambers, there is an
occasional small pore perforating the chamber wall near its base, probably for
hydrostatic reasons.
The avicularial back area is hemispherical. The beak is pointed. Separating
them is a straight pivot bar on which the base of the triangular mandible rests.
The mandible is a tall narrow triangle, with curved tip and a lucida near its base
(Fig. 4).
Orifice. The unusual orifice of T. absidatitin made it difficult to classify be-
cause either few species have this type or else if they have it the illustrations
generally do not show the orifice in very great detail.
The orifice is tipped forward a bit distally so that it seems horizontal. In
front of it the frontal wall soon rises like a mound. The orifice is placed at the
very extremity of the frontal wall.
It is lined by a C-shaped inner collar, the \-estibular arch, whose ends curve
around proximally to form the cardelles (Figs. 8, 10). The cardelles are sturdy,
blunt, smooth usually and separated from each other by a median gap approxi-
mately one-third the width of the orifice.
In front of the gap is the rounded spout-like sinus whose wralls are formed by
the two curving cusps which arise from the proximal wall of the peristome (Figs.
6, 8, 10). The sinus and gap form an inward slanting channel. In young zooecia
the orifice with the frontal peristomial tabs and sinus is set off from the zooecial
484
MARY D. ROGICK
Isoschisoporella tricuspis (Calvet) 1909
PLATE II.
MARINE BRYOZOA, XIII 485
front by a crease or groove that extends between the two paraorincial areolae (Fig.
5 ) but this later is obliterated. In C. daedala the cusps are transverse. In T. absi-
datum they are more vertical or obliquely arched.
Operculum. The operculum is rather delicate but reinforced by a long curved
chitinous sclerite or reinforcement at each side (Figs. 7, 8). The sclerite is for
muscle attachment. The extent or length of the sclerite depends on the degree of
chitinization of the operculum. The operculum is shaped to fit the distal semi-
circular anter of the orifice and the intercardellar gap rather than the more
external sinus.
Spines. Non-ovicelled zoids have 5 to 7 peristomial spines around the hemi-
spherical part of the orifice. On ovicelled zoids the distal part of the orifice is
not visible, so whether spine bases are present distally cannot at present be deter-
mined, but there is a thick spine at each side of the orifice just proximal to the side
of the ovicell (Figs. 2, 5).
The spines are coarse and jointed at the thick base. The bases of the proximal
pair are a bit sturdier or bigger on some zoids.
Ovicells. In general appearance the Toretocheilum ab si datum ovicells resemble
those of the genus Stephanosella and of Stach's Chaestosella gabricli and Brown's
C. enigma.
The T. absidatum ovicell is deeply immersed in the frontal of the next distal
zoid. Also, it hides the distal part of the orifice of its own zoid. Its very own
aperture cannot be seen from the front because the ovicell overhangs so. The
peristome does not encroach upon the ovicell.
The ovicell has two calcareous layers, the ectooecium and the entooecium.
Brown's (1952, p. 36) interpretation of these two layers is here followed. The
layers are separated by a very narrow space. The entooecium is globose, rough-
PLATE II
All figures on this plate are of Isoschizoporclla tricuspis (Calvet) 1909. All except Figures
14 and 19 are drawn with a camera lucida. Figure 13 is from Sta. 226 material, the rest are
from Sta. 234 specimens.
FIGURE 11. Detail of distal third of zooecium, showing well calcified beaded frontal wall
and a characteristic uncalcified space above the orifice. The avicularium is prominent enough
but the avicularial chamber is immersed and the heavy calcification makes it inconspicuous.
FIGURE 12. Operculum. Tendon fibers are attached to the two distal muscle dots.
FIGURE 13. Three ovicells flanked by aviculiferous spines.
FIGURE 14. Diagram of a suboral avicularial mandible.
FIGURE 15. Mandible of a spinal avicularium.
FIGURE 16. Portion of a colony showing both ovicelled and non-ovicelled zooecia. Some
zooecia have the large frontal spines. One of the spines and one ovicell are broken, in the
fourth row from the left. In the row at extreme right the two lower zooecia show degrees of
occlusion of orifice by secondary calcification. The orifice of the upper one is completely calci-
fied and overgrown. That of the lower right is partly so. The upper zooecium has given rise
to two new rows of zooecia.
FIGURE 17. Cross-section through 15 zooecia of a bilaminate colony. Zooecial cavities are
in black, zooecial walls are in white. The two layers of zooecia are back-to-back. The three
filled in compartments are the end walls (cf. Fig. 18).
FIGURE 18. End or distal wall of a zooecium, showing the interzoidal communication pores
or sieve plate in bottom half and the pattern or lines of calcification in the upper half.
FIGURE 19. Piece of a bilaminate blade or frond of a colony, drawn to the 2 cm. scale at
left. The three darker patches on the lower half are spined and ovicelled areas.
MARY D. ROG1CK
30
PLATE III.
MARINE BRYOZOA, XIII 487
ened and complete. The ectooecium forms a partial incomplete shallow shell or
band about the lateral and distal periphery of the entooecium.
Areolae occur in the ectooecium. Faint depressions emanate from these
areolae. The ectooecium grows upward from around and between them to form
the band about the entooecium. At the advancing border of the ectooecium the
entooecial surface appears tucked or crowded because of the peculiar growth
method or encroachment of the ectooecium. Not enough material was available
for a more detailed study of the ovicell.
Distribution and ecology. A flat rock, measuring roughly about 17 cm. long,
10 cm. wide and 3 cm. thick, well encrusted with a dozen species of bryozoa and
sponge, contained four small patches of Toretocheilum absidatum. These were on
the side of the rock and colony fragments had to be chipped off or calcined off for
study under the compound microscope.
The rock came from a depth of 40 fathoms, from USN Sta. 240 of Marguerite
Bay, Antarctica, Comdr. D. C. Nutt collector. The specimens will be deposited
PLATE III
All figures on this plate are of Isoschisoporclla tricuspis. All except Figures 21 through 25
are drawn with a camera lucida. Figures 20, 26, 29 and 32 are from Sta. 226 material. The rest
are from Sta. 234.
FIGURE 20. Portion of zooecium, showing aviculiferous spine, frontal and lateral walls.
The lateral wall has two pore plates (the two lower porous discs) and the "corresponding open-
ings" (the two upper doughnut-shaped discs). The pore plates of one zooecium line up with
the corresponding openings of its neighbor zooecia and vice versa.
FIGURES 21 THROUGH 25. Diagrams of external surface of frontal wall, depicting stages
in the formation of the suboral avicularial chamber.
FIGURE 21. Zooecial front before an avicularial chamber has begun to form.
FIGURE 22. The avicularial chamber is outlined by a growing calcareous rim. The border
is still incomplete distally.
FIGURE 23. Avicularial chamber now completely outlined.
FIGURE 24. Borders of the avicularial chamber growing and approximating.
FIGURE 25. Roofing over and fusion stage of avicularial chamber formation. Three open
spaces still remain but these will be reduced in time to two areolar pores and an avicularium.
FIGURE 26. Side view of a spiny patch of colony with hooded ovicells between the spines.
The spines at extreme right are either low or broken off and the ovicells can be seen just
beyond them.
FIGURE 27. Back wall removed to show the interior of the front wall and the relative
position of the distal wall. The distal wall with its sieve plate hides half the orifice. Cardelles
and avicularial chamber also are visible from this side.
FIGURE 28. Enlargement of inner surface of orifice, cardelles, peristomial and vestibular
sinuses and avicularial chamber. The avicularium shows through the translucent wall.
FIGURE 29. Early stage in the development of an aviculiferous spine. At this stage the
avicularium and its chamber are present and the spine will result from the growth of the tip
of the mound.
FIGURE 30. A more advanced stage in the growth of an aviculiferous spine. Two such
spines flank a broken ovicell. Between their base lies a suboral avicularium. In the foreground
is an exposed avicularial chamber of another aviculiferous spine (broken off).
FIGURE 31. Sagittal section through a zooecium. The darkest areas represent the zooecial
and avicularial cavities. The upper three circles in the gray lateral wall are the pore plates.
The two lower circles are the "corresponding openings" which would fit next to the pore plates
of a neighbor zoid.
FIGURE 32. An ovicelled, spined, well calcified section of a colony. The upper tier of
zooecia shows a curved spine and a double spine. The four spines of the middle and lower tiers
are broken off. Ovicell shapes are variable. Two left ovicells are broken off and reveal the
double calcareous wall.
488 MARY D. ROGICK
with the Smithsonian Institution, U. S. National Museum, after the other species
on the rock have been thoroughly studied.
GENUS ISOSCHIZOPORELLA, NEW GENUS
Diagnosis. Orifice suborbicular, with V-shaped sinus in its proximal lip. The
two ends of the C-shaped vestibular arch stop at the sinus and form ledges
(cardelles) to support the operculum. Operculum has two muscle dots distally
and a proximal median tab to fit the sinus. Frontal wall smooth to beaded,
flattened except for avicularial chamber and bordered by faint mural rim. Median
suboral avicularium with mandible usually proximally directed present some dis-
tance below orifice. The reniform, bilaterally symmetrical avicularial chamber ex-
tends across the entire front wall and has a lateral pore on each side usually.
Other avicularia are present in association with stout umbonate spines or tumid
mounds. Frontal wall a pleurocyst, punctured by only a few peripheral pores
spaced far apart and with bilateral symmetry. Ovicell globose to hood-like,
smooth to granulated, imperforate, with double calcareous wall. Ovicells may
grow downward over orifice.
Name derivation. The name Isoschisoporclla was coined from the Greek word
isos meaning like or equal and the already long existing generic name ScJiisoporella
which was derived from schiso (to divide), poros (pore) and clla (diminutive).
Genotype. The genotype, here chosen for the new genus Isoschisoporella, is
Schizoporella tumida var. tricuspis Calvet 1909, here elevated to species rank:
Isoschisoporella tricuspis (Calvet).
Remarks. The reason for the erection of this new genus was two-fold. Firstly,
there was need to set up a taxon for a troublesome species which had a schizoporellicl
orifice but which possessed other features which disqualified it from the presently
restricted family Schizoporellidae. Secondly, the troublesome species seemed to
belong to the family Hippoporinidae but possessed a totality of characters that
would not permit its uncontested inclusion in any presently known hippoporinid
genus. So it was thought best to erect a new genus for it, since this is a very
rare form restricted to the Antarctic and sub-Antarctic regions.
The character by which Isoschisoporella differs from the family Schizoporellidae
and resembles family Hippoporinidae is the nature of its frontal wall. Isoschiso-
porella has a pleurocyst, as does the family Hippoporinidae, while the Schizopo-
rellidae have a tremocyst. A pleurocyst is a frontal wall that is granular, im-
perforate over the central area and perforated only around the periphery by
areolar pores. A tremocyst is a frontal wall usually liberally sprinkled all over
with pores.
The Isoschisoporella pleurocyst has a few elongate inconspicuous peripheral
pores, very widely spaced, usually placed with bilateral symmetry at zooecial
corners, and some elsewhere en route, like the paired pores of some Reteporidae as
lodictyum (Harmer, 1934; pp. 515, 522) in particular. The Hippadenella
carsonac (Rogick, 1957b; Plate I) frontal is almost identical with that of Iso-
schisoporella but the orifice, operculum and ovicells are different.
MARINE BRYOZOA, XIII
489
Synonymy and previous records:
1909. Scliizoporella tuniida Hincks 1881, var. tricuspis. Calvet, pp. 28—30;
PI. Ill, Figs. 1-3. Excellent description and figures showing
ovicelled and non-ovicelled zooecia, operculum and suboral avicu-
larial mandible. No measurements given. From 30 meters'
depth at Booth Wandel Isle and 110 meters' depth in Biscoe Bay.
1924. Schizoporella twnida var. tricuspis. Thornely, p. 12. Common-
wealth Bay, Sta. 1, Lat. 66°50' S., Long. 142°6' E. at 354 fathoms.
1928. Schizoporella tuniida var. tricuspis. Livingstone, pp. 7, 52.
1957a. Schizoporella tuniida var. tricuspis. Rogick, p. 8, USN Sta. 226,
Marguerite Bay.
Ecological note.
The above are the only records of this species up to date.
Diagnosis. Colony bilaminate, foliaceous ; smooth except for spiny or ovicelled
patches. Zooecia long, narrow, flattened, bracket-shaped. Mural rims thin, faintly
salient. Frontal a granulated or beaded pleurocyst with a few paired peripheral
pores. A small median oval to pointed suboral avicularium sits atop the wide
immersed reniform avicularial chamber. A stout tusklike aviculiferous process
("spine") occvirs on the midfrontal of zooecia adjacent to an ovicelled zoid. Some
zoids without spines or ovicells. Ovicell imperf orate, granular, shaped like an
elephant's head, overhanging the zooecial orifice, usually flanked on each side by
its neighbors' aviculiferous frontal spines. Zooecial orifice and operculum as in
genus. The median V-shaped peristomial sinus is less than half the width of the
proximal lip. Zooecial orifice is at distal end of frontal and is touched by the
f rentals of the three adjacent zoids. Distal to orifice, at the beginning of the next
zoid is a membranous area, crescent-shaped. Zooecial end wall a sieve plate. Four
or five multiporous pore plates or corresponding openings present in each lateral
wall.
Measurements. All readings are in millimeters. H is for height; D is for
diameter at base.
1.181-1.991
0.272-0.432
0.144-0.173
0.173-0.202
0.143-0.176
0.166-0.195
0.44^0.619
0.317-0.461
0.072-0.115
0.043-0.072
0.156-0.234
0.091-0.137
0.760-1.177
0.377-0.514
0.260-0.358
0.046-0.078
(1.506) L zooecia, 20 readings
(0.351) W zooecia, 20 readings
(0.158) L orifice, including sinus
(0.184) W orifice
(0.163) L operculum
(0.182) W operculum
(0.531) L ovicell, including its beak
(0.413) W ovicell
(0.093) L suboral avicularium
(0.055) W suboral avicularium
(0.190) L spinal avicularium, 8 readings
(0.107) W spinal avicularium, 8 readings
(1.039) H frontal spine
(0.456) D at widest part of spine base avicularial chamber
(0.311) D at narrowest part of spine base avicularial chamber
(0.061 ) L mandible of suboral avicularium
490 MARY D. ROGICK
0.039-0.065 (0.051) W mandible of suboral avicularium
0.117—0.130 (0.124) L mandible of spinal avicularium, 2 readings
(0.130) W mandible of spinal avicularium, 1 reading
Remarks. Calvet's hesitancy (1909, p. 28) about the identity of his tricuspis
and its exact relationship to Schizoporclla tumida Hincks (1881, p. 13, PI. I, Fig. 3)
is understandable in view of the brevity and incompleteness of Hincks' description
and illustration. Hincks' figure shows four non-ovicelled zooecia and a mound
bearing a special avicularium. This mound is described by Hincks as "fre-
quently an ovate rising on the side of the cell extending from the orifice down a
considerable portion of its length, bearing on its upper extremity an immersed
avicularium, with pointed mandible directed downwards." The JT. tumida ovicell
is described as "globose and prominent, with a smooth surface."
Hincks neither mentions nor figures the frontal pores or frontal spines and the
ovicell is apparently without the downward orifice-covering beak. Also, the
position of the suboral avicularium is much closer to the orifice in 6". tumida than
in Isoschisoporella tricuspis. Schisoporella tumida Hincks was collected in the
Bass Straits of Australia, while Isoschizoporella tricuspis is known only from the
Antarctic. In these w^ays then does S. tumida differ from Calvet's var. tricuspis,
so it is best to elevate Calvet's variety to species rank.
Colony. Colony color is light ecru. Judging from the fragments the colony should
be good sized, foliaceous or fan-shaped, with crinkled edges. In the USN collec-
tion were a number of chips or fragments, none of them a complete colony. The
largest piece was 67 mm. long by 60 mm. wide, and probably represented less than
half a colony because it was a portion of the growing edge, apparently some distance
away from the base of the colony, judging by its width.
Some of the pieces have occasional faint markings, like growth rings (Fig. 19).
New linear or radial rows of zooecia are added in such zones, or when needed, to
make possible the ever widening periphery or ruffled edge of the colony.
Colonies are bilaminate and solid, with zooecia back to back, and mostly smooth.
Occasional bristly patches occur on either face (Figs. 13, 16, 19, 26). These
patches represent areas where ovicells and aviculiferous spines have arisen. One
such patch was 12 mm. long by 10 mm. wide.
Sometimes secondary calcification closes over the zooecial orifices (Fig. 16) but
the suboral avicularia and frontal pores seem much more resistant to secondary
filling-in. The secondary calcification seems to begin with the operculum itself.
It fills in or frosts over lightly, then more solidly till the whole orifice is like the
surrounding f rentals.
Colonies are relatively clean, living zoids apparently being resistant to encrusta-
tion with extraneous material, but dead parts show some settlers.
Soft parts. Many zoids had opercula and mandibles. Far fewer contained
polypides (gut, tentacles, musculature). These polypide remains, when present,
were very slim, in keeping with the slenderness and elongation of the zooecia and
also perhaps the food supply was insufficient. Some of the zoids had the opercula
fouled or rimmed about by a border of scummy orange-colored material, as if
wastes had accumulated about the orifice and killed the zooecia. It was not possible
to determine if embryos were present in ovicells because the ovicells are so opaque.
MARINE BRYOZOA, XIII 491
Suboral avicularium. A suboral avicularium develops on nearly every zoid.
It is median, oval, with a triangular mandible that is proximally directed (cf. Figs.
11, 14). Most of the suboral avicularia are so oriented longitudinally but oc-
casional ones do occur a bit off course. That is. their slant may be slightly oblique,
to right or left. One colony fragment from USN Sta. 234 contained a zoid whose
suboral avicularium pointed transversely while the rest of the suboral avicularia
in that fragment were oriented in the usual manner.
Sometimes the mandibular area tip (rostrum) is flat against the frontal
("chest"), other times its tip is more elevated.
The suboral avicularium forms after its reniform chamber is complete (Figs. 11,
21—25, 27, 28, 31). The floor of the chamber is the zooecial frontal wall. The
chamber stretches from lateral wall to lateral wall. The chamber walls grow
upward from the zooecial frontal, converge in a transverse line that has three
diminishing gaps in it (Figs. 22-25). These gaps are the two lateral pores and
the median avicularium. The mandibular area, back area and pivot develop over
the median gap. At the base of the chamber, particularly on the lateral side, can
be found the aforementioned pore and perhaps another one, puncturing the chamber,
Aviculijerous spines. The big tusk-like frontal spines are present on some
zoids, either ovicelled or non-ovicelled, and absent from others. They are avicu-
liferous, i.e., house an avicularium and avicularial chamber in their base (Figs.
13, 20, 30, 31). When present, there is generally only one spine per zoid. An
occasional spine may be forked (Fig. 32) but most of them are not. Some of the
spines are straight, some curved. They are tall, thick-walled, wide at the base
and taper to a blunt tip and sometimes look corrugated. They are hollow, the
basal part being especially large (Figs. 20, 31). The cavity of the spine is not
continuous with the cavity of the zooecium. The basal part of the spine cavity
is the avicularial chamber.
These spines arise as calcareous blisters on the midf rental wall of a zoid (Figs.
31, 32). They sprout close to a neighbor's orifice and ovicell as if to protect
both (Figs. 16, 32). One wonders what influence, if any, the developing ovicell
or developing embryo of a zoid exerts on neighboring zoids to cause them to
produce the protective aviculiferous spines.
When still in the first stage of formation the avicularial spine base is very
extensive. One was 0.247 mm. wide by 0.52 mm. long, as wide as some of the
zooecial fronts. The base is oval or reniform in outline. Strictly speaking the
spine base represents the avicularial chamber.
The avicularium at first is oblique in position like a door leaning against a
mound. But, with the continued upward growth of the mound to form a hollow
spine the position of the avicularium shifts until it is roughly vertical, i.e., at right
angles to the zooecial plane. The avicularium appears as if propped up against
or incorporated into the proximo-lateral side of the spine. Its duckbill-shaped
mandible (Fig. 15) is larger than the triangular mandible of the suboral avicularium.
Also, the suboral avicularium itself is smaller than the spine avicularium. The
mandible of the spine avicularium is directed toward the tip of the spine (Figs.
20,26).
In the USN material these aviculiferous spines develop in the vicinity of ovicells
or orifices of ovicelligerous zoids, leaning toward the ovicells. Generally an ovicell
492 MARY D. ROGICK
has an aviculiferous spine on each side of it (Figs. 13, 16) but there can be excep-
tions. Occasional ovicells may share a spine between them. Still others may
have but a single spine beside them.
Interzoidal communications. In /. tricuspis the end walls of zooecia are gener-
ally single partitions or transverse septa between succeeding zoids in a linear or
radial series, i.e., two zoids share the same single end wall between them. The
lateral walls are double, each neighboring zoid having its own individual lateral
wall. Therefore it is not surprising that the interzoidal communications between
zoids should be different in the two different kinds of walls. Silen (1944) made
an extensive study of this situation in a number of different species.
In /. tricuspis the transverse and the lateral walls are perforated, but differ-
ently. The end wall is truncated, i.e., divided into two sections, one with the
porous sieve plate, the other without pores (Figs. 17, 18, 31). The sieve plate
is nearer the basal wall than is the non-porous section and slants away from the
basal wall at about a 45° angle. The other section is more nearly vertical with
respect to the basal wall. The number of closely set pores in the slanting sieve
plate is hard to count but one zoid had about 16, more or less.
The lateral wall has 4 or 5 multiporous pore chambers or corresponding open-
ings (Figs. 20, 31). These blister-like pore chambers have fewer pores, about
3 to 8, than do the sieve plates.
Distribution and ecology. A small number of fragments, about 18, and none
of them a complete colony, of Isoschizoporella tricuspis, were collected by the 1947-
1948 USN Expedition from two stations in Marguerite Bay, Antarctica, Sta. 226
and 234, from depths of 40 fathoms. These localities are near that from which
Calvet's specimens came. The Thornely specimens were collected at opposite
sides of the Antarctic continent and from a considerably greater depth, 354 fathoms.
So, although the species showed up in very small quantity in the collections of only
three Antarctic expeditions, its occurrence at opposite sides of the pole would
suggest a circumpolar distribution.
The USN specimens were relatively clean of encrustation by other forms but a
few bryozoans did gain a foothold on some of the fragments. One Beania erecta
zoid and a lichenoporoid cyclostomatous bryozoan colony, to be identified later,
grew on the Sta. 226 fragment. Species found on the Sta. 234 fragments included
the bryozoans Osthimosia mill cp oroides (Calvet) 1909, Phylactellipora lyrulata
(Calvet) 1909 and Raniphonotus inermis (Kluge) 1914 and foraminifera.
The /. tricuspis specimens will be deposited with the Smithsonian Institution,
U. S. National Museum.
SUMMARY
1. Two new genera, Toretocheilum and Isoschizoporella, of the family Hippo-
porinidae (Bryozoa, Cheilostomata) have been erected on the basis of their orifices
and areolated frontal walls.
2. One new species, Toretocheilum absidatum, was described. Another, Schizo-
porella tumid a var. tricuspis Calvet 1909, was elevated to species rank and trans-
ferred to a new genus : Isoschizoporella tricuspis.
3. Both species are amply illustrated, measurements given and the description
for /. tricuspis has been amplified. Attention is given to the range of variation
of each species.
MARINE BRYOZOA, XIII 493
4. Each species is designated as the genotype of its new genus. Both species
are from Marguerite Bay, Antarctica.
LITERATURE CITED
BASSLER, R. S., 1934. Notes on fossil and recent Bryozoa. /. Washington Acad. Sci., 24:
404-408.
BASSLER, R. S., 1953. Bryozoa. In: Treatise on Invertebrate Paleontology, R. C. Moore, Ed.,
Geol. Soc. Amer., Univ. of Kansas Press, Lawrence, Kansas, 253 pp.
BROWN, D. A., 1952. The Tertiary Cheilostomatous Polyzoa of New Zealand. British Museum
(Nat. Hist.), London, 405 pp.
BROWN, D. A., 1954. A new species of polyzoan, and notes on taxonomy. Trans. Roy. Soc.
New Zealand, 81 : 557-561.
BROWN, R. W., 1954. Composition of Scientific Words. G. W. King Printing Co., Baltimore,
Md., 885 pp.
CALVET, L., 1909. Bryozoaires. Exped. Antarct. Frang. 1903-05. Charcot. Masson et Cie.,
Paris. 50 pp.
CANU, F., AND R. S. BASSLER, 1917. A synopsis of American Early Tertiary Cheilostome
Bryozoa. Smithson. hist. USNM. Bull, 96 : 1-81.
CANU, F., AND R. S. BASSLER, 1934. (Part of Bassler, 1934, above.)
HARMER, S. F., 1934. The Polyzoa of the Siboga Expedition. Part III. Cheilostomata
Ascophora. I. Family Reteporidae. Siboga Expcd. Monogr., 28c : 502-640.
HINCKS, T., 1881. Polyzoa from Bass's Straits. Ann. Mag. Nat. Hist., (5) 8: 1-14.
KLUGE, H., 1914. Die Bryozoen der Deutschen Siidpoiar-Expedition I. Die Familien Aetidae,
Cellularidae, Bicellaridae, Farciminaridae, Flustridae, Membraniporidae und Cribili-
nidae. Deutsche Sildpolar-Expcd. 1901-1903 von Dryyalski, Band xv; ZooL Band
vii : 599-675.
LIVINGSTONE, A., 1928. The Bryozoa. In: Mawson's Australasian Antarct. Exped., 1911-14.
Sec. C, Zool. Botany, 9: 1-93.
MACGILLIVRAY, P. H., 1887a. Descriptions of new or little known Polyzoa, Part XII.
Trans. Proc. Roy. Soc. Victoria, 23: 179-186.
MAcGiLLivRAv, P. H., 1887b. A catalog of the marine Polyzoa of Victoria. Trans. Proc.
Roy Soc. Victoria, 23 : 187-224.
MACGILLIVRAY, P. H., 1887c. Bryozoa. In: McCoy's Prodromus of the Zool. of Victoria,
Decade XIV : 137-150.
OSBURN, R. C., 1952. Bryozoa of the Pacific Coast, Part 2. Allan Hancock Exped., 14: 271-
611.
ROGICK, M., 1957a. Studies on marine Bryozoa. IX. Phylactellipora. Ohio J. Sci., 57 : 1-9.
ROGICK, M., 1957b. Studies on marine Bryozoa. X. Hippadcnella. Biol. Bull, 112: 120-131.
SILEN, L., 1944. On the formation of interzoidal communications of the Bryozoa. Zool. Bidrag
frdn Uppsala, 22 : 433-488.
STACH, L., 1937. The species of Chiastosella (Bryozoa). The Australian Zoologist, 8: 334-
340.
THORNELY, L., 1924. Polyzoa. /;;: Mawson's Australasian Antarct. Exped., 1911-14, Sec. C,
Zool. Botany. VI : 1-23.
DISCONTINUOUS RESPIRATION IN INSECTS:
ROLE OF THE SPIRACLES 1
HOWARD A. SCHNEIDERMAN
Department of Zoology, Cornell University, Ithaca, New York
In many groups of insects 2 metabolic carbon dioxide is retained within the insect
and released during brief periods in "bursts" (Punt, 1944, 1948, 1950a, 1950b, 1956;
Schneiderman, 1953; Schneiderman and Williams, 1953-1955; Buck et al., 1953;
Buck and Keister. 1955; Ito, 1954). In diapausing pupae of the Cecropia silk-
worm, for example, more than nine-tenths of the metabolic carbon dioxide is stored
and then released in brief bursts, which occur from once every week to many times
per hour, depending on the temperature and metabolic rate. The remaining carbon
dioxide escapes during the interburst period. When measured by usual manometric
procedures, the uptake of oxygen, unlike the release of carbon dioxide, appears con-
tinuous and almost steady (Schneiderman and Williams, 1953a, 1955; Buck and
Keister, 1954, 1955). If the spiracles are sealed with paraffin, virtually all the
respiratory exchange ceases (Ito, 1953; Schneiderman and Williams, 1955); the
spiracles are, therefore, the site of both the discontinuous release of carbon dioxide
and the simultaneous continuous uptake of oxygen. When metabolic rate is low,
the bursts are accentuated. Thus at 10° C. carbon dioxide may be given off only
once a week and the interburst rate of carbon dioxide may be but l/100th the rate
of oxygen uptake. This indicates the true dimensions of the respiratory paradox :
oxygen enters the spiracles during the interburst period at many times the rate
at which carbon dioxide leaves and, furthermore, the insect releas'es its carbon
dioxide periodically.
The central importance of spiracular behavior in the discontinuous release of
carbon dioxide was suggested by the observation of Buck and his co-workers (1953,
1955) that intubating the spiracles of Agapema pupae eliminated the bursts of carbon
dioxide. Subsequently, we observed that excision of the valve from one of the
fourteen functional spiracles of a Cecropia pupa caused carbon dioxide output to
become continuous (Schneiderman and Beckel, 1954; Schneiderman, 1956). The
only maneuver that restored the discontinuous release of carbon dioxide was the
sealing of the open spiracle. It appears from this experiment that all the spiracles
1 This research was aided by a research grant, H-1887, from the National Heart Institute of
the United States Public Health Service.
2 Groups include larval and adult Hemiptera (Rhodnius proli.rus; Triatoma rubrofasciata,
Cimex Icctularins; adult Dictyoptera (Pcriplancta amcricana) ; larval and adult Orthoptera
(Locusta mif/ratoria) : adult Coleoptera (Carabus nemoralis, Meloe proscarabaens, Hadro-
carabus problematicns) ; diapausing larvae of Lepidoptera (Arctia sp.) ; diapausing pupae of
Lepidoptera (Hyalophora cccropia, Anthcraca polypheinus, Samia cynthia, Rothschildia oryzaba,
Agapema yalbina, Bomby.r mori ("dauer pupae''), Sphinx ligustri, A gratis sp., Papilio
macheon) ; non-diapausing pupae of Bomby.r mori, diapausing adults of Lepidoptera (Vanessa
iirticac).
494
SPIRACLES IN CYCLICAL RESPIRATION 495
remain nearly closed during the interburst, preventing carbon dioxide from diffusing
out in substantial quantities, and that one or more of the spiracles opens during a
burst. The spiracular valves thus hold an important key to the discontinuous re-
lease of carbon dioxide.
Several theories have been proposed to explain the disparate rates of oxygen and
carbon dioxide transfer during the interburst period, as well as the bursts them-
selves (Punt, 1944, 1950a; Punt el a!., 1957; Zeuthen, 1955; Buck ct al., 1953;
Buck and Keister, 1954, 1955; Schneiderman, 1956). The most recent and com-
prehensive theoretical analysis of the entire phenomenon is that of Buck (1958a,
1958b). This theory and all of the others depended for the most part upon (a) in-
direct estimations of tracheal carbon dioxide and oxygen tensions during the "burst
cycle," (b) postulated behavior of the valves that regulate the opening of the
spiracles, (c) assumed changes in intratracheal barometric pressure for which there
was no empirical evidence whatever, (d) hypothetical changes in the volume of the
tracheal system, (e) cataclysmic biochemical changes. To test these theories and
to resolve the paradox, it proved necessary : (a) to continuously record the behavior
of the spiracular valves during the burst cycle, (b) to measure directly the changing
composition of tracheal gases during the cycle, (c) to measure the changing intra-
tracheal barometric pressure, and (d) the changing tracheal volume during the cycle.
The present report initiates a series in which methods will be described that accom-
plish these objectives and provide fairly precise pictures of both the partial and
absolute pressure gradients driving oxygen into the insect, the gradients driving
carbon dioxide out of the insect, and cyclic variations in the aperture of the spiracles
and in the volume of the tracheal system. The results to be reported confirm that
the breathing of silkworm pupae involves processes other than physical diffusion,
and also bear out many of the theoretical predictions of Buck (1958b). However,
in some essential points, they do not support his theory, but instead provide evidence
for another kind of insect breathing which seems different from any previously
proposed or demonstrated and which may account for discontinuous respiration. In
addition, they define the stimuli that cause the cyclical activity of the spiracles.
The first paper focusses on the role of the spiracles in discontinuous respiration.
A preliminary account of some of these results has been given elsewhere (Schneider-
man and Beckel, 1954; Schneiderman, 1956).
MATERIALS AND METHODS
1. Experimental animals
Experiments were performed on diapausing pupae and developing adults of the
giant Saturniid silkworms Hyalophora cccropia, Antheraea polyphemus and Samia
cynthia. In our experience these species of closely related moths behave in virtually
identical fashion in the sorts of experiments that were undertaken. Animals were
reared on net-covered trees or purchased from dealers and stored at 25° C. and 60%
to 70% relative humidity. All experiments were conducted at 25° ± 1° C.
2. Respiratory measurements
Respiratory exchange was determined manometrically by techniques previously
described (Schneiderman and Williams, 1953a, 1955). Measurements were per-
496
HOWARD A. SCHNEIDERMAN
CUT
STP FA
PTR
B
FIGURE 1. (A) Pupal spiracular regulatory apparatus of Cecropia, as seen from inside
the animal. No tracheae are shown. ATR, atrium; BW ' , body wall; cl, closer muscle; CLB,
closing bow; CLL, closing lever; CLN, nerve of closer muscle; FA, filter apparatus or stigmal
plate ; OP, elastic opener ; SCO, scolopophorous organ ; SCON, nerve of scolopophorous organ ;
X, dorsolateral closing bar; Y, dorsomedian closing bar; Z, ventral closing bar. (B) Frontal
sections through the spiracular region, above the closing lever, and cutting the two dorsal
closing bars. These show how the spiracle closes when the closing bars are pushed against
the closing bow or atrium. CUT, cuticle; HYP, hypodermis ; PTR, peritreme ; STP, stigmal
plate, STR, spiracular tracheal manifold; TM, tracheal membrane. (C) Apparatus viewed
from the inside with the scolopophorous organ and part of the elastic opener removed. Only
part of the spiracular tracheal manifold is shown. The valve is open. (D) Same as (C) only
the valve is closed.
SPIRACLES IN CYCLICAL RESPIRATION 497
formed by the "direct manometric method" (Umbreit ct a!., 1958) in 45-cc. cylin-
drical vessels equipped with venting plugs and adapters, for use with standard
Warburg manometers.
3. Recording of spiracular movement
(a) Anatomy of the spiracle
These silkworm pupae have a pair of thoracic spiracles and six pairs of functional
abdominal spiracles. Each spiracle is surrounded by a chitinous peritreme, and
covered by a stigmal plate, or filter apparatus, which communicates by a thin slit to
a chamber below — the atrium — which contains the spiracular valve. Gas exchange
between the atrial chamber (and hence the atmosphere) and the interconnecting
tracheal system, which lies just below it, is regulated by this valve. The morphol-
ogy' of the spiracular apparatus and some of the physiological properties of the
spiracular muscle of pupae of Saturniid moths have been described most recently
by Beckel (Beckel. 1955, 1958; Beckel and Schneiderman, 1956, 1957). For our
present purposes, it is sufficient to note that the spiracular valve is an epithelial
membrane which is firmly attached to a chitinous frame consisting of a bow and
three bars which unite in the middle to give rise to a lever (Fig. 1). A closer-
muscle stretches from the ventral tip of the lever to the ventral corner of the valve.
It is opposed by an elastic ligament which extends from the dorsal tip of the lever
to the body wall. When the closer muscle contracts, it pulls on the lever and closes
the valve. When it relaxes, the valve opens because of the elasticity of the chitinous
frame and the tension of the opposing elastic ligament. The muscle is innervated by
a nerve from the corresponding segmental ganglion and by a branch of the median
nerve of the next anterior segment.
(b) Recording valve movements
To expose the valves in the living insect it was necessary to remove the overlying
chitinous stigmal plate. This could easily be done under the dissecting microscope
after the animal had been anaesthetized with carbon dioxide. Insofar as could be
judged, this operation in no way interfered with the normal functioning of the valves,
which were now clearly visible. When the valves were so exposed for a week or
more, they occasionally dried out and ceased functioning normally. To prevent this,
after each period of observation, the spiracular opening was sealed with Tackiwax.3
Because of their greater accessibility, the abdominal spiracles were examined in
preference to the thoracic pair. Of these the first, second, and third abdominal were
the easiest to study because they served non-collapsible segments and were not
obscured when the animal moved its abdomen.
Several methods for recording spiracular movements of intact insects have been
employed in the past and there have been several descriptions of spiracular behavior
in the cockroach Periplaneta americana (Hazelhoff, 1926a. 1926b), the phasmid
Di.vippus morosns (Stahn. 1929) ; the rat flea Xenopsylla chcopis (Wigglesworth,
1935; Herford. 1938). the bedbug Cimc.r Icctular'ms (Wigglesworth, 1941), the
3 After the present experiments were completed, we discovered that a valve can be pre-
vented from drying out by sealing a small transparent plastic window over the exposed spiracle.
Although the window prevents gas exchange through the spiracle, it allows convenient observa-
tion of spiracular movements, and the valve functions for manv weeks.
498
HOWARD A. SCHNEIDERMAN
grasshoppers Dissosteira Carolina (McCutcheon, 1940), Schistocerca obscura
(Watts, 1951), and Schistocerca gr eg aria (Hoyle, 1959), the larva of the com-
mercial silkworm (Shimizu and Ono, 1942), the flies Musca domestica and Calli-
troga macelleria (Case, 1956a) and the cockroaches P. amcricana and Blaberus
craniifer (Case, 1957b). The older literature has been summarized by Hazelhoff
(1926a). The present experiments employed a modification of the ocular microm-
eter system of McCutcheon. A pupa with one or more of its spiracles exposed to
view was placed beneath a binocular dissecting microscope furnished with an ocular
micrometer and examined under 60 X. The hairline of the micrometer was fo-
cussed on the leading edge of the spiracular valve and was adjusted to follow the
valve as it opened and closed (Fig. 2). The crosshairs on the micrometer eye-
piece indicated the position of the hairline when the valve was closed. The hairline
was moved by a rotating knob whose action was translated into a kymograph trace
by means of a system of pulleys and levers and an ink-writing pen recording on a
ANTERIOR
POSTERIOR
leading edge of valve
chitmous bar
hairline
leading edge of valve
chitinous bar
OPEN
spiracular
opening
width of valve opening
FIGURE 2. Spiracle as seen through ocular micrometer. The filter apparatus and most of the
stigmal plate have been removed and the valve is visible. For further details see text.
SPIRACLES IN CYCLICAL RESPIRATION
499
diomber
FIGURE 3. Apparatus used for recording spiracular valve movements. The observation
chamber shown is of the sort also employed in studies with intubated pupae. For further
details see text.
continuous feed kymograph (Fig. 3 ). The tracings of the ink-writing pen provided
a record of the movements of the spiracular valve, each deflection on the record
representing a valve movement. The ordinate of the trace shows the width of
valve opening in arbitrary units, while the abscissa denotes the duration of the
opening. Directly beneath the record of valve movements the time was recorded
by an ink-writing timer.
In most of the experiments spiracular behavior was viewed through a 20-cc.
glass chamber in which the insect was held secure in a plasticene support, or in a
250-cc. lucite chamber with a flat surface for optical convenience. Air and gas
mixtures were flushed through the chamber as desired. The rate of gas flow varied
from 25 to about 500 cc./min. in different experiments and appeared to have no
effect on the phenomena under investigation. The time required to change the
atmosphere in the small chamber was rarely longer than 10 seconds, but for the large
chamber it took about two minutes.
Gas mixtures were either pre-mixed in pressure cylinders or made up by propor-
tional flow. All mixtures were analyzed periodically.
A. EXPERIMENTS WITH INTACT PUPAE
1 . Normal behavior of spiracles
Figure 4 is a portion of a typical record of the activity of the third abdominal
spiracle of a Cecropia pupa over a period of four hours. A consistent pattern of
500 HOWARD A. SCHNEIDERMAN
valve movement occurred in cycles lasting 45 minutes to 2 hours. After a period
of about 10 minutes, during which it remained closed and motionless, the valve
fluttered for between 15 and 40 minutes and then, within a minute, the valve move-
ments progressively increased in amplitude until the valve opened fully. Swaying
slightly, it remained open for several minutes. Then it alternately opened and
closed for several minutes, and the valve movements gradually decreased in ampli-
tude and duration, until the valve closed altogether. Following this, the valve
remained motionless until the fluttering preceding the next period of wide openings.
This cycle was repeated over and over, and apparently represented the normal
behavior of the spiracle.
A^J (|i
FIGURE 4. Record of the valve movements of a third abdominal spiracle of a diapausing
Cecropia pupa over a 4-hour period in air at 25° C. Each mark above the baseline represents
a valve opening. Occasional marks below the baseline are artifacts caused by vibration of
the apparatus.
Manometric observations of the carbon dioxide output of this pupa revealed
bursts of carbon dioxide at intervals of 30 minutes to two hours. A similar correla-
tion between the frequency of carbon dioxide bursts and periods when the valve
was widely open has been consistently observed in all other silkworm pupae studied.
Moreover, by utilizing techniques which permit simultaneous recording of both valve
movements and gas exchange, it has been possible to show the correlation directly
(Schechter and Schneiderman, unpublished observations). Hence it seems evident
that the periodic bursts of carbon dioxide result from the periodical prolonged
openings of the spiracles, which we have termed "spiracular bursts." The term
"burst" seems appropriate for both the spiracular and the manometric events, since
they coincide. The spiracular burst can conveniently be partitioned into a period
of wide openings — the "open phase"- —which is followed by a period of rapid
closures — the "decline phase." The end of the decline period and of the spiracular
burst is marked bv the moment the valve constricts tightly. The "interburst"
SPIRACLES IN CYCLICAL RESPIRATION
501
consists of a "constriction period" after the burst, when the valve appears closed
and motionless, and a "flutter period" prior to the next burst.
The flutter period was usually quite irregular in terms of both the frequency
and amplitude of the valve movements. As Figure 4 reveals, the flutters usually
TABLE I
Duration of variotis phases of spiracular burst cycles in a typical series of diapausing
Cecropia pupae and developing adults of Polyphemus
Duration of various phases (minutes)*
Experiment
No.***
Cycle length
(minutes)
Interburst
Spiracular burst
Constriction
Flutter
Open
Decline
Cecropia
827
114
13 (11)
91.5 (80)
5.5 (4.8)
4.0 (3.5)
8316
74
10 (14)
61 (81)
1.0 (1.4)
2.0 (2.7)
938
73
26 (36)
42 (57)
1.5 (2.1)
3.5 (4.8)
739
68
26 (38)
37.5 (55)
1.0 (1.5)
3.5 (5.1)
8315
65
12 (19)
49 (75)
1.0 (1.6)
3.0 (4.7)
625 a
63
10 (16)
43 (68)
3.0 (4.8)
7.0 (11.1)
8314
50
16 (32)
29.5 (59)
1.5 (3.0)
3.0 (6.0)
625 b
44
16 (36)
19 (43)
2.5 (5.7)
6.5 (14.8)
725 c
43
12 (28)
26 (61)
2.0 (4.7)
3.0 (6.9)
8311 b
39
12.5 (32)
23.5 (60)
1.0 (2.6)
2.0 (5.1)
439
38
17 (45)
16 (42)
1.0 (2.6)
4.0 (10.5)
8311 c
32
18 (56)
12.5 (39)
1.0 (3.1)
0.5 (1.6)
8311 a
29.5
7.5 (25)
19 (65)
1.5 (5.1)
1.5 (5.1)
Average**
56.35 ± 23.25
15.08 ± 5.71
36.12 ± 21.96
1.81 ± 1.28
3.35 ± 1.82
Average %
100
29.85 ± 12.94
60.38 ± 13.67
3.31 ± 1.52
6.30 ± 3.71
Polyphemus
522 a
16.5
0
15.0 (91)
—
—
522 b
15.4
0
12.3 (80)
—
—
527 a
10.7
0
6.4 (60)
—
—
527 c
9.7
0
7.0 (72)
—
—
527 d
9.1
0
6.2 (68)
—
—
527 f
8.1
0
5.5 (68)
—
—
5218
7.0
0
4.6 (66)
—
—
113 a
6.0
0
3.5 (58)
1.5 (25)
1.0 (17)
113 b
6.0
0
3.0 (50)
2.0 (33)
1.0 (17)
114
6.0
0
3.6 (60)
1.7 (28)
0.7 (12)
115
5.5
0
2.5 (45)
2.3 (42)
0.7 (13)
521 c
5.5
0
3.5 (64)
— •
—
117
4.3
0
2.8 (65)
1.0 (23)
0.5 (12)
521 c
2.7
0
1.4 (52)
—
—
Average
8.04 ± 3.98
0
5.52 ± 3.84
1.70
0.78
Average %
100
0
64.21 ±11.94
30.2
14.2
* Figures in parentheses record the average per cent of the cycle length.
** ± standard deviation.
*** In this and subsequent experiments, the first two digits refer to the particular animal
employed, i.e., all experiments beginning with "82" refer to pupa No. 82.
502
HOWARD A. SCHNEIDERMAN
came in volleys which lasted from 10 seconds to 8 minutes and which were punc-
tuated by closed periods which lasted from 20 seconds to several minutes. Less
commonly, flutters occurred singly. Frequency varied from short volleys of about
one per second to long volleys of one opening every two to ten seconds. The
openings were invariably brief, commonly lasting less than a second. Amplitude
also varied somewhat and an occasional wide opening punctuated a series of smaller
ones. There appeared to be no systematic variation in amplitude, however, until
a minute or two before the burst.
Several hundred hours of records of such cycles have been obtained from about
30 individual pupae. Table I records the duration of the various phases of several
typical cycles recorded from diapausing Cecropia pupae and developing adults of
Polyphemus. Although the cycles that were studied varied in frequency from
20 per hour to one every two hours, the pattern of valve movements in all cases
was remarkably similar. The only variation occurred in pupae with cycles of 15
minutes or less. Here the constriction period was lacking and the spiracular valve
was in constant slight motion (see Figures 6 and 7). Also, in pupae with brief
cycles the frequency of flutters was considerably greater than in pupae with long
cycles, and the spiracular bursts occupied a larger and larger proportion of the cycle.
Simultaneous observations of two or three spiracles on the same side indicated
that the spiracles were coordinated. When the valves opened in a burst or closed
at the end of a burst, they did so within a minute of each other, though when they
fluttered, the pulsations were not in exact synchrony. For our present purposes,
these observations indicate that recording the behavior of one spiracle provides an
accurate picture of the behavior of all the spiracles of the animal, except possibly the
thoracic spiracles whose behavior we have never succeeded in observing.
Previous experiments (Punt, 1950a; Schneiderman and Williams, 1955; Buck
and Keister, 1955) pointed out that various factors such as metabolic rate, oxygen
and carbon dioxide tensions, etc., profoundly affected the cyclical release of carbon
TABLE II
Burst cycle of a Polyphemus during adult development
Days of adult
development
Cycle length
(minutes)
Spiracular burst duration
(minutes)
Amount of interburst
fluttering*
-13
120.0
6.8
+
0
9.8
3.3
+ +
2
7.0
2.4
+ +
3
5.0
2.7
+ +
4
5.4
2.0
+ +
5
4.4
1.7
+ +
6
4.7
2.0
+ + +
9
4.0
1.7
+ + +
10
4.3
2.0
+ + +
15
2.7
1.3
+ + + +
16
0
0
+ + + +
20 (emergence)
0
0
+ + + +
+ : Normal amplitude, moderate frequency; ++: normal amplitude, high frequency;
+ + + : greater than normal amplitude, high frequency; + + + + : fluttering about half-open
position, high frequency.
SPIRACLES IN CYCLICAL RESPIRATION 503
dioxide. Recognizing the key role of the spiracular valves, it was reasonable to
anticipate that these several factors would influence the movement of the spiracular
valves. This is clearly shown in the following experiments.
2. Effects of metabolic rate on the behavior of the spiracular valves
During the pupal-adult transformation, oxygen uptake rises markedly, the cycles
of carbon dioxide release become more frequent and the interburst rate of carbon
dioxide output increases until the cycles disappear and carbon dioxide is released
continuously (Punt, 1950a; Schneiderman and Williams, 1955). Table II sum-
marizes the spiracular behavior of a Polyphemus during adult development, and
reveals that the spiracular bursts increased in frequency and that the amount of
interburst fluttering also increased, first in frequency and then in amplitude, until
eventually on the sixteenth day of the 21 -day period of adult development, the
spiracular bursts disappeared and the spiracles fluttered continuously about a half-
open position. It is important to note that in insects with very high metabolic rates
the flutters had far greater amplitude than normal interburst flutters and resembled
the spiracular movements encountered in the decline period after a spiracular burst.
In any case, by using developing adults, it was possible to obtain animals with brief
burst cycles. Another useful means of securing animals with brief cycles for con-
venient study was to injure pupae. Injury provokes a prompt increase in metabolic
rate, which persists for days, and a tremendous increase in burst frequency
(Schneiderman and Williams, 1955). In a typical experiment excision of the
facial region from a Polyphemus pupa increased cycle frequency from one spiracular
burst every two hours before injury, to 12 per hour four days after injury.
The explanation for these effects of metabolic rate on the spiracular burst cycle
seems clear. One of the probable consequences of increased metabolic rate is to
lower tracheal PO2 and increase tracheal PCo2- It seems likely that these factors are
directly responsible for the acceleration of the burst cycle. This is confirmed in the
following experiments on spiracular occlusion.
3. Effects of spiracular occlusion
In pupal silkworms, the spiracles are the sole gateways to the tracheal system
and their occlusion affords a simple method of lowering internal Po2 and raising
internal Pco2- The effects of spiracular occlusion on the cycle of valve activity of
a Polyphemus pupa are seen in Figure 5. Sealing five pairs of abdominal spiracles
with paraffin called forth an immediate increase in spiracular burst frequency from
about one burst per hour to 6 or 8 per hour. After 12 or 13 of the 14 spiracles were
sealed, the spiracular bursts disappeared completely. Unsealing the spiracles ini-
tiated the return to substantially normal behavior.
In pupae with five pairs of abdominal spiracles sealed and a spiracular burst
frequency of 5 or 10 per hour, it was occasionally possible to restore normal burst
frequencies of one to two per hour by exposing the pupa to pure oxygen. The
increased oxygen also decreased interburst fluttering and led to wider openings at
the time of the spiracular burst. The implications of these observations will be
examined in the Discussion. For our present purposes suffice it to say that these
spiracular occlusion experiments indicate that low oxygen combined with high
carbon dioxide increases spiracular valve movements and speeds up the burst cycle.
504
HOWARD A. SCHNEIDERMAN
A
"o'
1MIN.
10
20
30
B
"TEN"
I WIN.
10
20
30
"TWELVE"
IMIN.
30
90
100
80
'•'THIRTEEN"
IMIM
10
20
T
•J
40
30
FIGURE 5.
SPIRACLES IN CYCLICAL RESPIRATION
505
TABLE III
Effects of Po2 on the various phases of the spiracular burst cycle of two diapausing
Cecropia pupae (S-82 and S-93)
Expt. No.
Ambient
Po2
Average
cycle length
(minutes)
Mean duration of various phases (minutes)
Interburst*
Spiracular burst
Constric-
tion
Percentage
change in
constriction
period**
Flutter
Percentage
change in
flutter
period**
Open
Decline
827
827a
21
100
114
75
13 (11)
73 (97)
\ +462
91.5 (80)
0 (0)
-100
5.5 (4.8)
2.1 (2.8)
4.0 (3.5)
0 (0)
938a
939a
930 (Av.)
9310
9311
5
15
21
35
75
Continuous
fluttering
73
66
50
61
11.5 (16)
18.0 (28)
28 (56)
58 (95)
-36
0
+56
+220
58 (79)
44 (65)
18 (36)
0 (0)
+32
0
-59
-100
1.4 (1.9)
1.2 (1.9)
2.0 (4.0)
1.0 (1.6)
1.5 (2.1)
3.0 (4.7)
2.0 (4.0)
2.0 (3.3)
* Figures in parentheses record the average percentage of the cycle length occupied by the particular phase. Most
of the results represent the average of two cycles except the results in air (930) which represent the average of five cycles
of 50 to 74 minutes duration.
** Compared with air controls.
To separate the action of these gases on spiracular behavior, we tested the effects
of specific gas mixtures on valve activity.
4. Effects of oxygen tension on the spiracles
The effects of decreased oxygen tension on spiracular behavior are evident in
Figure 6. This pupa had a high metabolic rate and a brief burst cycle. As Figure
6A shows, within six minutes of lowering the ambient oxygen tension to 15%,
interburst fluttering increased so much that only a semblance of a burst cycle re-
mained. The cycle returned to normal after about five minutes.
As Figure 6B shows, 7% oxygen completely obliterated the burst cycle. Within
one minute the valve opened widely without fluttering, remained open for five
minutes with only an occasional closure, and then closed partially five to ten times
each minute. Manifestly, this gradual return of spiracular fluttering looks like
adaptation, but in our opinion it is more likely the result of a lowered Pco2 within
the insect as a result of prolonged spiracular opening (cf. Discussion). Within one
minute of return to air the valve began to flutter closed and soon thereafter the
burst cycle reappeared.
FIGURE 5. Effects of spiracular occlusion on the behavior of the second right abdominal
spiracle of a Polyphemus pupa. Air at 25° C. (A) No spiracles sealed; 14 functional. A
burst occurred just before the recording was begun. For 63 minutes thereafter no bursts
occurred. (B) Ten spiracles sealed: first, third, fourth, fifth and sixth pairs of abdominal
spiracles ; four functional. Burst frequency increased from less than one per hour to one
every 8 or 10 minutes. The pattern persisted essentially unchanged for 20 hours. (C) Twelve
spiracles sealed : all of above plus both thoracic spiracles ; two functional. Fluttering of valve
in open position for periods of 8 to 20 minutes punctuated by brief one-half- to two-minute
periods of normal fluttering about the closed position. (D) Thirteen spiracles sealed: all of
above plus left second abdominal spiracle ; one functional. Only the exposed spiracle was avail-
able for gas exchange. The result was intermittent closures from a wide-open position, first
rapidly and then more slowly.
506
HOWARD A. SCHNEIDERMAN
AIR
IITL ITV.
./ILJH
IMIN.
10
20
I5%02
riVj^r^^jTi^r-f^^s^A.
40
50
60
\ r*
70
80
90
AIR
-jlL..»lnL fV iffV nk. fk... rti, .^vVi
IMIN.
10
20
30
jUJ
7%02
,' II HI11*!!!!!"!!!
JJ I I
'"' Mr ii fi i NI i mini'. i{M|i|i[j!j,i||'n|;i! r
50
60
'l'Tni"'riiii||i"i|iliip[ii!i!i|(ii[f: i nil
70
80
90
f
if i
\._ ,r^R ___ (*\ ...^.
^ u_ Ky A. A >,.A
100
no
120
130
C
AIR
5\02
\\\
IMIN. 10
T 111"
20
30
40
50
AIR 60
70
80
90
y^«N^^
100
110
120
130
140
ISO
160
FIGURE 6.
SPIRACLES IN CYCLICAL RESPIRATION 507
-azro *
oojq, . For details see text.
Figure 6C reveals that within 30 seconds of exposure to 5% oxygen, the valve
opened widely with no initial fluttering. It gaped open for 70 minutes, closing at
irregular intervals for about 40 minutes, and finally stayed wide open and motion-
less. Within one minute of return to air, fluttering began and continued for 40
minutes, whereupon the burst cycle reasserted itself. Recovery from 5% oxygen
thus took nearly forty times longer than recovery from 7% oxygen, suggesting that
this low oxygen tension was inadequate for respiration and caused the pupa to
become anoxic and build up an oxygen debt. In pure nitrogen, a record similar to
the 5% oxygen record was obtained (not illustrated). The spiracle closed occa-
FIGURE 6. Effects of P0_. on the behavior of the second right abdominal spiracle of a
diapausing Polyphemus pupa. Three records were obtained in studying each gas mixture :
in air, in the specfic gas mixture, and again in air, to appraise the recovery of the spiracle.
The time required to change the atmosphere in the chamber was less than 15 seconds. In this
experiment and in succeeding ones, the reaction time of the spiracle depended very much on
the phase of the burst cycle in which the gas was administered. As one would expect, the most
rapid responses occurred when gases were admitted during a spiracular burst. In experiments
illustrated the gases were added midway in the interburst period. (A) 15% Oi. Record of
valve movements in air, in 15% Oz + 85% N2> and in air. (B) 7% On. Record of valve move-
ments in air, in 7% O2 + 93% Ni, and in air. (C) 5% O2. Record of valve movements in air,
in 5% Ot + 93% N2, and in air.
508
HOWARD A. SCHNEIDERMAN
sionally for about 10 minutes, but then gaped widely, closing only once during thirty
minutes of observation. From these results it appears that decreased oxygen ten-
sion can cause valve opening.
A clearer picture of the effects of oxygen tension is seen in Table III, which
records the effects of several oxygen tensions on the spiracular behavior of two
pupae with longer burst cycles than the pupa just described. These pupae took
somewhat longer to respond to both high and low oxygen tensions, often more than
10 minutes except when the gas was administered during a spiracular burst.
Figure 7 illustrates portions of several representative records. In 5% to 10%
oxygen the cycles were absent and the valves were continuously in motion. The
openings were larger than normal interburst openings, but smaller than normal
burst openings. The frequency of flutters, however, was not very different from
typical interburst flutters and remained irregular. The period between volleys,
however, was much decreased and rarely exceeded 20 seconds, W7hereas in air,
two-minute intervals between volleys of flutters were common.
In 15% oxygen the bursts reappeared. As the oxygen concentration increased
above 15%, the width of valve openings in the interburst decreased, while the fre-
quency remained essentially the same. Also, as oxygen tension increased, the
fluttering period got progressively shorter and the constriction period progressively
longer. Finally, in pure oxygen all the fluttering before and after the spiracular
burst was completely eliminated : the valve remained closed until it abruptly shot
open in the burst, and then almost as abruptly closed until the next burst. During
the spiracular burst itself, at oxygen tensions above 15%, the higher the oxygen
tension, the wider was the valve opening. From these results it appears that oxygen
tension affects primarily interburst fluttering but also has some minor effects on
the burst itself. It is also noteworthy that increasing or decreasing oxygen tension
failed to have any predictable effect on burst frequencies : sometimes frequencies
wrere lowered, other times increased.
TABLE IV
Effects of Pco-2 on various phases of the spiracular burst cycle of a diapausing
Cecropia pupa (No. S-83)*
Expt.
No.
Ambient
Pco2
Average
cycle
length
(minutes)
Average duration of various phases (minutes)**
Percentage
change in
spiracular
burst
Open
Decline
Con-
striction
Percentage
change in
constric-
tion period
Flutter
Percentage
change in
flutter
period
Spiracular
burst
8311
Air
5%
34
38
10 (29)
13 (36)
| +30
21 (62)
20 (50)
I -
3.1 (9.2)
5.0 (13.7)
} +62
1.4 (4.1)
1.8 (5.1)
1.7 (5.1)
3.2 (8.6)
8314
Air
8%
50
31
16 (32)
18 (59)
+ 13
29.5 (59)
5.5 (18)
} -81
4.5 (9.0)
7.5 (24.0)
} +67
1.5 (3.0)
3.0 (9.7)
3.0 (6.0)
4.5 (14.3)
8315
Air
10%
65
26
12 (19)
12 (45)
I «
49 (75)
4.5 (17)
} -
4 (6.2)
10 (38)
+ 150
1.0 (1.6)
4.0 (15.0)
3.0 (4.7)
6.0 (22.5)
8316
Air
15%
74
Continuous
flutter
10 (14)
—
61 (81)
—
3 (4.1)
—
1.0 (1.4)
2.0 (2.7)
* Experiments were performed on successive days and the normal and experimental records for each day are
paired. Most of the results represent the average of two cycles.
** Figures in parentheses record the average percentage of the cycle length occupied by the particular phase.
SPIRACLES IN CYCLICAL RESPIRATION 509
A AIR
IMIN. 10
!..,>«- „ . f li--.. -
50 60
... .
90 100 110 120 130
140 150 160 170
B
IMIN. 10 20 30 I5%C02 40
A
i • »• "** »!!•«>• n » ^ », I., ,^- „ - - V1 -r r i- .»«%. •.•.». • • i .».!.•.. .. . ffl'H^^ini . * »».«(1m. n ^ r-nl *•
50 60 70 80 90
,,, ^. .• - ... ^— ~ i- .... r,.^n ,, , liaii ..j,!! '"^iVSiii __^ ... ,_ ,_^i b
'20
_ I,
140 ISO 160 170 180
190
FIGURE 8. Records of spiracular movements of a Cecropia pupa with a moderately long burst
cycle in air, followed by 10% or 15% CO.. (A) 10% CO2; (B) 15% CO..
In occasional experiments, particularly with pupae with high metabolic rates
and frequent bursts, pure oxygen failed to suppress interburst fluttering. Instead
the spiracular bursts disappeared and the spiracles fluttered continuously. Possible
reasons for this curious behavior will be offered in the Discussion.
5. Effects of carbon dioxide tension
Increasing the PCo2 increased burst frequency and lengthened both "open" and
"decline" phases of the cycle (Table IV and Fig. 8). Besides the effect on burst
frequency, the table and figure reveal that high tensions of carbon dioxide also
lengthened both the open phase of the spiracular burst and the decline phase after
the burst. Thus, in 10% carbon dioxide, the open phases were nearly three times
as long as in air (compare Figure 7D with 7B ) . In addition, although increasing
PCo2 nad little effect on the duration of the constriction period, it greatly shortened
the flutter period until, in 10% carbon dioxide, fluttering was reduced to only a few
minutes (Fig. 8). Between 10 and 15% carbon dioxide the burst cycle broke down
completely, so that in 15% carbon dioxide the valves fluttered continuously (Fig. 8),
510 HOWARD A. SCHNEIDERMAN
and after an initial spiracular burst, no further bursts occurred. The openings were
a bit larger than normal interburst flutterings, but much smaller than normal open-
ings in a burst.
The reaction times for a carbon dioxide response never appeared as long as the
reaction times for a response to oxygen. Also, pupae with high metabolic rates,
and hence frequent bursts, appeared far more sensitive to carbon dioxide than pupae
with low metabolic rates. Thus, in a pupa which had a 4- to 5-minute burst cycle,
as little as 2% carbon dioxide had a detectable effect on spiracular behavior (pro-
longed the burst), while 3% eliminated spiracular bursts and provoked continuous
wide flutters. Furthermore, when the carbon dioxide tension was raised to 15%
in a pupa with such a brief cycle, the valves opened widely, and nearly a half hour
AIR
IMIN. I0 20 30
I5%C02
40 50 60
90
100 110
FIGURE 9. Records of spiracular movements of a Polyphemus pupa with frequent bursts in air,
in 15% CO» + 21% O2, and in air.
was required for recovery upon return to air (Fig. 9). Such sensitivity to carbon
dioxide contrasts markedly with the more modest responses recorded in Figure 8,
shown by a pupa with a longer burst cycle. A further observation important to our
analysis is that in a pupa in which 3% carbon dioxide eliminated the spiracular
bursts, bursts could be restored in 3% CO2 by raising the oxygen tension to about
90%. This observation suggests that high oxygen tensions decrease the sensitivity
of the spiracle to carbon dioxide, a fact to which we shall return.
Although these results convey a general picture of the effects of carbon dioxide
and oxygen on the pupal spiracles, they suffer from a conspicuous defect : they
provide only little information about the actual gas concentrations within the
tracheal system that produce a particular effect. In other words, when the insect
SPIRACLES IN CYCLICAL RESPIRATION 511
is exposed to 15% oxygen or 10% carbon dioxide what are the actual intratracheal
oxygen and carbon dioxide tensions? A partial solution to this problem was pro-
vided by experiments with intubated pupae which are described below.
B. EXPERIMENTS WITH INTUBATED PUPAE
1. Effects of spiracular intubation
Just as spiracular occlusion decreases the Po2 and increases the Pco2 within the
pupal tracheal system, so spiracular intubation has the opposite effect. In the fol-
lowing experiment, the spiracular valves were exposed in four or five abdominal
spiracles on one side of a series of diapausing Cecropia pupae. Into two, three or
four of these spiracles short fire-polished glass tubes, about 1 mm. in diameter, were
inserted beyond the spiracular valves, placing the tracheal system in free com-
munion writh the air. This maneuver caused the Pco2 within the insect to fall and
the Po2 to rise, as the gaseous composition of the tracheal system approached that
of the outside.
One or two days after the operation, and periodically thereafter for about a week,
the unintubated spiracles were examined (usually the second and third abdominal).
In every case the spiracular valves appeared to stay tightly constricted for many
days. They could be opened, however, by exposing the insect to low oxygen or
high carbon dioxide. Thus, although the untouched spiracles of the intubated pupa
could still function, their cyclical activity had disappeared, presumably because the
normal triggering stimuli for spiracular activity were absent.
When intubations were performed on animals with very high metabolic rates
(e.g. developing adults), despite four intubated spiracles some spiracular fluttering
occurred. Apparently, intubating even four spiracles in animals with high metabolic
rates does not permit equilibration of internal and external gases.
These experiments, coupled with the other experiments considered thus far, are
consistent with the view that the triggering stimulus for spiracular opening is either
low internal tension of oxygen, high internal tension of carbon dioxide, or a com-
bination of the two. This matter is considered in further detail below.
2. Effects of oxygen tension on the spiracles of intubated animals
Intubation eliminates the spiracular burst cycle and the spiracles exhibit a per-
sistent pattern of behavior in various gas mixtures. Thus, the intubated pupae
provide a simple means of determining the approximate concentrations of oxygen
and carbon dioxide that produce a particular kind of spiracular behavior, e.g. valve
fluttering, bursts etc., and also a means of examining the interaction between carbon
dioxide and oxygen in controlling spiracular movement. To conduct these experi-
ments, pupae with two or three intubated spiracles were exposed to various gas
mixtures in a flow system, and the movements of two normal spiracles observed
until they exhibited a constant behavior. It took anywhere from 5 to 35 minutes for
the constant response to assert itself, except in very low oxygen tensions or very
high carbon dioxide tensions where the response appeared more rapidly. The
decision to study the final response of the spiracles rather than their initial response
was taken for several reasons. The principal one was that, in these large insects,
it very likely takes many minutes for gases to reach a steady-state. A second
512
HOWARD A. SCHNEIDERMAN
TABLE V
Determination of the "0-i-open" threshold of two spiracles of an intubated Cynthia pupa*
Per cent Response of spiracle 2R
oxygen
21 Closed
15.5 Active fluttering
8.5 Partly open with rapid closures
5.0 Wide open with occasional flutters
4.5 Wide open with occasional flutters
4.0 Wide open with occasional flutters
3.5 Wide open with occasional flutters
3.0 Wide open with no closures
2.5 Wide open with no closures
2.0 Wide open with no closures
1.5 Wide open with no closures
Response of spiracle 3R
Closed
Closed
Slight fluttering (pulsation)
Active fluttering
Active fluttering
Wide open with occasional flutters
Wide open with occasional flutters
Wide open with occasional flutters
Wide open with occasional flutters
Wide open with no closures
Wide open with no closures
* Almost identical results were obtained when the gas mixtures were flushed through the
chamber at random instead of in order, provided adequate recovery periods were allowed. Hence,
under the conditions of our experiments, previous exposure to a test gas mixture had no effect on
subsequent tests.
reason was that, in the normal intact insect, gas changes are ordinarily gradual and
not cataclysmic, and hence it seemed a sensible procedure to allow time for adjust-
ment to each change of gas.
Table V illustrates the data recorded to determine the effects of oxygen on two
spiracles of a typical diapausing pupa. Progressive lowering of the ambient oxygen
tension caused in sequence: pulsation of the valve (no visible opening), fluttering
(small openings), partial openings and closings, full opening and occasional closure,
and finally full opening with no movement, i.e., complete relaxation of the spiracular
closer muscle.
TABLE VI
"O2-flutter" thresholds and "02-open" thresholds of abdominal spiracles of five
intubated Cecropia pupae
Experiment No.
Spiracle observed
"O2-flutter" threshold*
(per cent)
"O2-open" threshold**
(per cent)
21 b
1 L
3 L
12
8.5
3
3
22 b
1 L
2 L
12
12
1.5
1.5
23 b
2 L
3 L
8.5
5.5
1.5
1.5
24 b
1 L
2 L
12
8.5
1.5
1.5
25 b
2 L
3 L
5.5
5.5
1.5
1.5
* Po2 which caused first pulsations.
** Po2 which caused full spiracular opening for one minute.
SPIRACLES IN CYCLICAL RESPIRATION
513
Two oxygen tensions are of special interest : the tension that initiates spiracular
movement, i.e., pulsations or fluttering, and the tension that causes a spiracle to open
fully, as in a burst. A typical set of values for these two oxygen tensions for a
series of diapausing pupae is recorded in Table VI. From the table it can be seen
that the five pupae studied varied considerably in their sensitivity to oxygen.
Spiracular movements began at oxygen tensions ranging from 12 to 5.5%. The
tensions required for full opening were more uniform. Unfortunately, the oxygen
tension that induced pulsations proved to be exceedingly difficult to determine since
it was not reproducible in successive experiments. The oxygen tension that pro-
duced full opening of the spiracle was far simpler to ascertain. Therefore, to facil-
itate quantitative measurements we concentrated on one spiracular response : full
opening of the valve for one minute. This response occurs normally during a burst
cycle, was easily judged, and was altered by changes in oxygen or carbon dioxide
tensions of ±0.5%. Moreover, it was easily reproducible.
The tension of oxygen necessary to open the spiracles widely was determined for
33 spiracles of more than a dozen Cynthia pupae that had several spiracles intubated.
This tension of oxygen, hereafter termed the "O2-open threshold," averaged 2.58
± 1.12 (s.d.) % with a range of 1% to 5.5%. Two-thirds of the spiracles responded
to between 2% and 3.5% oxygen. Within any pupa the O2-open threshold for
100
CECROPIA
A
B
C02
FIGURE 10. (A) The effect of Po= on the Pco2 necessary to open a spiracle of an intubated
Cynthia pupa widely for one minute. Partial pressures are expressed as % atm. For details
see text. (B) Same curve as above for an intubated Cecropia pupa.
514 HOWARD A. SCHNEIDERMAN
different spiracles did not vary by more than 1.5%. For a given spiracle, at all
oxygen tensions below threshold the spiracle was wide open and the spiracular
muscle remained relaxed; at tensions 0.5% or more above the O2-open threshold,
intermittent to prolonged contractions of the muscle occurred, which closed the valve
(cj. Table V). The O2-open threshold was also determined for a few developing
adults of Polyphemus and found to average 3.1% oxygen with a range of 2.0-4.5%.
3. The interaction between oxygen and carbon dioxide in the spiracles of intubated
animals
The carbon dioxide tension necessary to open the spiracle widely for one minute
varied with the oxygen tension. This is seen in Figure 10A which records the
behavior of a Cynthia pupa. The first point (A) on the curve gives the highest
partial pressure of oxygen in a nitrogen-oxygen mixture that kept the spiracle open,
i.e., the O2-open threshold which we considered in the previous section. The third
point (B) on the curve was determined by adding a mixture of 4% carbon dioxide
to air, a mixture which kept the spiracle wide open, and progressively increasing
the oxygen tension until the spiracle began to close. In this experiment, in the
presence of 4% carbon dioxide, oxygen tensions above 35% were necessary to
close the spiracle. In other words at any point along the curve for a given partial
pressure of carbon dioxide there was a critical tension of oxygen, below which the
valve would remain fully open and motionless ; oxygen tensions above this critical
tension caused closure of the valve. The end-point of this curve is (C) . At carbon
dioxide tensions above this value the valve remains open and motionless and no
partial pressure of oxygen at atmospheric pressure will cause fluttering. This
point, which is designated the "CO2-open threshold," was determined by starting
in pure oxygen where the valve was closed and increasing the carbon dioxide tension
until a tension was reached at which the spiracle remained open for one minute.
This CO2-open threshold was simple to determine and reproducible within about
1%. Several curves of this sort were recorded for a number of pupae and all
looked essentially the same. One typical of a Cecropia pupa is presented in
Figure 10B.
Although such curves are difficult to obtain, by measuring only the O2-open
threshold and the CO2-open threshold of a spiracle it was possible to assess in a
general way the sensitivity of the spiracle and to compare spiracles of pupae under
various conditions. This was done for a series of Cynthia spiracles and the O2-open
thresholds have already been discussed. The CO2-open thresholds of this same
series of spiracles averaged 17.63 ± 5.42(s.d.)% and covered a far greater range
(13.5% to 31%) than the O2-open thresholds. Notwithstanding, two-thirds of the
spiracles had a CO,-open threshold between 14% and 16%. The CO2-open
threshold appeared quite uniform for different spiracles of any individual. A few
determinations were also made on the spiracles of developing adults of Poly-
phemus. Whereas the O2-open thresholds for these insects with high metabolic
rates did not differ significantly from the thresholds for diapausing pupae, the
CO2-open thresholds of the developing adults were lower than for the diapausing
pupae and averaged only 10.5% with a range of 9.5-12.0%. Furthermore, the
CO2-open thresholds decreased on successive days of adult development as the
metabolic rate increased.
SPIRACLES IN CYCLICAL RESPIRATION 515
These results demonstrate the influence of oxygen on the response of the spiracle
to carbon dioxide, and indicate that the sensitivity to carbon dioxide decreases as
oxygen tension increases. Similar results were obtained when the carbon dioxide
tension that caused fluttering was determined : the sensitivity to carbon dioxide
decreased as the oxygen tension increased.
DISCUSSION
1. Scope of the analysis
The spiracular behavior of giant silkworms just described represents one of the
most extreme kinds of spiracular behavior recorded for any insect, with a complex
cycle of activity which commonly lasts for hours and occasionally for days. The
present experiments confirm and amplify earlier opinions that the cyclical behavior
of the spiracles is somehow responsible for discontinuous respiration. They may
have, in addition, some intrinsic interest for insect respiration as a whole. For,
in a sense, these giant pupae breathe in "slow motion," and events which may-
occupy only a second or two in the respiratory cycles of other insects may take
many minutes or even hours. This enables the experimenter to analyze complex
events in gas exchange and spiracular behavior which are not readily separable in
insects with brief cycles. The present experiments have exploited this property
in an effort to define the kinds of behavior that spiracles can show, and to clarify
the manner in which oxygen and carbon dioxide interact to provoke various modes
of spiracular activity.
2. The behavior of the spiracular muscle
Interpretation of the present experiments is simplified by the fact that the
movements of each pupal spiracle are controlled by a single muscle. Thus, the
records of spiracular activity are records of the behavior of this muscle. As we
have seen, the muscle displays three sorts of behavior : sustained contraction which
causes valve closure ; brief partial relaxation which causes valve fluttering ; and
almost complete relaxation which causes the valve to gape open.
In the normal pupa these different kinds of behavior occur in two characteristic
patterns: (1) the first is the "flutter," in which the muscle relaxes slightly and
then quickly re-contracts fully. This occurs many times each minute in irregular
volleys and may continue for hours. It is important to note that the relaxations
are very brief and that during most of the flutter period the valves are constricted.
Indeed, as far as one can tell, they are as tightly constricted between flutters as they
are during the constriction period after a burst. (2) The second pattern, the
"burst" cycle, is more dramatic and far more regular. It is compounded of numer-
ous flutters and periods of prolonged valve opening and prolonged constriction.
The initiation of each of these patterns of spiracular behavior does not appear
to depend upon an endogenous nervous rhythm as is true, for example, in the
respiratory movements of Schistocerca and Di.rippus (cf. review by Wiggles worth,
1953a, and also Hoyle, 1959) or of vertebrates, but is the result of cyclical changes
in the composition of the tracheal gases. The mechanism of this extrinsic control
is described below.
516 HOWARD A. SCHNEIDERMAN
3. An explanation for the burst cycle
All events in the normal burst cycle, as well as the experimental observations,
can be explained in terms of the following three properties of the spiracular muscle
and its associated nerves : (a) the muscle is ordinarily continuously stimulated to
contract; (b) when tracheal P02 falls below a critical value, the flutter threshold,
the muscle repeatedly relaxes slightly and re-contracts; (c) when tracheal Pco2 rises
above a critical value, the burst threshold, the muscle relaxes fully. These asser-
tions permit us to frame the following hypothesis which appears to be consistent
with all of our observations and accounts for the burst cycle.
Immediately after a burst the valves are constricted, tracheal PO2 is far above
the flutter threshold, and tracheal PCo2 is ^ar below the burst threshold. This is
the constriction period. As the insect respires, oxygen inflow fails to match oxygen
utilization and the Po2 within the tracheal system falls. When the tracheal Po2
falls to about 3%, the flutter threshold, spiracular fluttering begins, permitting
oxygen to enter. This increases the tracheal Po2 and the valve promptly closes.
This pattern of rapid fluttering, punctuated by short periods of constriction, repre-
sents the flutter period. In high oxygen tensions tracheal Po2 never reaches
triggering concentration and hence no fluttering occurs.
What about the spiracular burst itself? This involves carbon dioxide. During
the interburst period some carbon dioxide escapes, but at a rate considerably less
than that of its production. As a consequence, internal Pco2 ultimately reaches
the spiracular burst threshold, and the spiracular muscle relaxes. The valve opens
widely in a burst ; carbon dioxide and nitrogen flow out and oxygen flows in, as
the composition of the tracheal gas approaches that of the atmosphere. When the
internal Pco2 decreases below a certain value, the valve begins to open and close
periodically, with diminishing width and duration of opening, until it finally remains
constricted and the cyle is completed.
Detailed support for this hypothesis and for the assertions regarding the effect
of Po2 and Pco2 on the spiracular muscle are examined below.
4. Oxygen effects: the constriction and flutter periods
A number of experiments prove that fluttering is caused by low tracheal Po2
and is little affected by tracheal PCo2- Firstly, in a normal pupa, lowering ambient
Po2 to about 10% evokes continuous valve fluttering (presumably because tracheal
Po2 is lowered). Secondly, raising ambient Po2 suppresses entirely the valve
fluttering of a normal pupa : apparently under this condition tracheal PO2 never
falls to the flutter threshold. Thirdly, when several spiracles are intubated, thereby
raising the internal Po2, fluttering ceases. Finally, in such an intubated pupa,
when ambient Po2 is lowered to about 2% fluttering continues indefinitely.
Under this analysis, in a normal pupa the duration of the constriction period
after a burst is determined primarily by the rate at which Po2 falls within the
tracheal system. This in turn depends upon three factors : ( 1 ) the tracheal volume
of the pupa, (2) the rate of oxygen utilization by the pupa, and (3) the rate at
which oxygen leaks in through the constricted spiracles. In general, large pupae
with low metabolic rates have long constriction periods. Also, as one would pre-
dict, the constriction period is prolonged by increasing ambient Po2 (cf. Table III),
SPIRACLES IN CYCLICAL RESPIRATION 517
for at the end of a burst, not only is there a higher O2 content in the tracheal
system, but also more O.2 leaks inward.
In occasional experiments, pure oxygen failed to suppress valve fluttering but
instead abolished the burst cycle. Similar results were found by Buck et al. (1955)
in manometric experiments with Agapema pupae. Neuromuscular mechanisms of
insects are particularly susceptible to oxygen poisoning (Goldsmith and Schneider-
man, 1960) and this may account for these results.
What determines the frequency of fluttering and the duration of the closed
period between each volley of flutters ? Both of these appear to be governed, as
is the duration of the constriction period, by the rate at which Po2 falls within the
tracheal system, and therefore depend in part upon metabolic rate and upon ambient
PO-. As one would expect, the higher the metabolic rate, the more frequent the
flutters and the more frequent the volleys of flutters (rf. Figures 4 and 6). More-
over, since the frequency of the flutters and the frequency of volleys depend primarily
upon tracheal PQO, it is not surprising that they do not change systematically during
the course of the flutter period, for once fluttering begins, trachea! P0.2 presumably
varies very little since oxygen continuously enters. This prediction has been con-
firmed by analyses of tracheal gas composition (Levy and Schneiderman, 1957,
1958). which will be discussed in detail in a subsequent report. For our present
purposes this constant P02 is of singular importance, for it tells us that the termi-
nation of the flutter period by a spiracular burst depends not upon a change in
tracheal PQO, but upon the accumulation of carbon dioxide. This is considered
below.
5. Carbon dioxide effects: "triggering" and termination of the spiracular burst
The most conspicuous effect of an increase in ambient Pco2 was the shortening
of the burst cycle, which confirms previous manometric findings (Schneiderman and
Williams, 1955). This reduction in cycle length was largely at the expense of the
flutter period, which was greatly shortened, whereas the duration of the constriction
period was unchanged. In short, increasing ambient Pro- led to the premature
triggering of the burst early in the flutter period. Moreover, once triggered, the
burst was prolonged. These observations can be understood in the following terms :
( 1 ) The absence of an effect of carbon dioxide on the duration of the constric-
tion period supports the opinion offered above, that in the intact pupa the onset
of fluttering is triggered primarily by low tracheal P0.j. Further support comes
from the fact that the P0l> which triggered oft" the fluttering was not significantly
affected by modest increases in tracheal PCOL- Indeed, in pupae with long cycles,
until ambient Pco2 rose above lO^f. it did not appear to affect the amplitude or
frequency of flutters.
(2) The premature triggering of spiracular bursts by ambient PCo->- A variety
of experiments revealed that in a normal burst cycle, the spiracular bursts them-
selves are triggered by tracheal P, •<>,, and not by tracheal P02. Thus exposing
both normal and intubated pupae to physiological and non-anaesthetic concentrations
of carbon dioxide causes the spiracular valves to open widely. In a normal cycle,
tracheal Pro., gradually increases after a burst as a result of metabolism and, when
it reaches a certain level, the spiracular muscle fully relaxes and a burst occurs.
At all concentrations of carbon dioxide, tracheal Pro- more quickly reaches the
518 HOWARD A. SCHNEIDERMAN
triggering concentration, because the partial pressure gradient driving carbon
dioxide out of the insect is decreased, with a consequent decrease in the rate of
carbon dioxide leakage. In addition, high ambient concentrations of carbon
dioxide (i.e., above 5%) prevent tracheal carbon dioxide from falling to its
normal low level after a burst. This constant high level of tracheal carbon dioxide
also shortens the time required to reach a trigger value. Thus, for these two
reasons, the higher the ambient carbon dioxide concentration, the shorter the time
between bursts. The second reason is by far the more important in pupae with
low metabolic rates. In such insects ambient carbon dioxide tensions below 5%
had no detectable effect on burst frequency, whereas increasing carbon dioxide
from 5% to 10% often increased the burst frequency 10 to 20 times (Schneider-
man and Williams, 1955). However, in pupae with high metabolic rates and high
burst frequencies, the reduction in carbon dioxide escape caused by the presence
of even 1% ambient carbon dioxide was often sufficient to abolish the bursts
entirely.
A different kind of evidence for carbon dioxide triggering spiracular bursts
comes from experiments on Agapema pupae, in which there was demonstrated an
inverse relation between ambient Pco2 and the delay in the first subsequent spiracu-
lar opening (Buck and Keister, 1958). Pupae in 9% carbon dioxide opened their
spiracles 2f hours after exposure, whereas in 24% carbon dioxide they took only
one hour. As the authors point out, this is what would be expected "if the carbon
dioxide were leaking into the tracheal system and accelerating the rise to triggering
concentration" (p. 335).
(3) Further evidence for the triggering of spiracular bursts by tracheal PCOZ-
The triggering of bursts by tracheal Pco2 is Avell seen m 100% oxygen, where
internal Po2 might be expected to remain relatively high throughout the cycle.
Here it seems clear that increasing tracheal PCo2 was the trigger that caused the
spiracular muscle to relax. The converse result is seen in intubated pupae, where
one prevents carbon dioxide from reaching a concentration high enough to trigger
a spiracular burst and the valves remain permanently closed.
(4) Termination of the spiracular burst. Once triggered to open in a burst,
the spiracle remains open and carbon dioxide diffuses out until tracheal Pco2 falls
to some critical level, whereupon the spiracle closes again. Increased ambient
Pco2 prolonged bursts, presumably because it delayed the diffusive outflow of
carbon dioxide. It is worth recalling that it takes the blood and tissues some
time to unload carbon dioxide. In most cases, long before this is done the tracheal
Po2 has risen to its maximum value as oxygen diffuses in at the burst. Hence,
the closing of the spiracle at the end of a burst has much less to do with tracheal
P0o than with tracheal Pco2-
It is puzzling that immediately the carbon dioxide tension falls somewhat, the
muscle does not contract again, but instead remains relaxed for many minutes,
even when carbon dioxide tension has fallen well below the triggering threshold.
In some way, high Pco2 provokes a response in the muscle which is sustained
for a considerable period (cf. discussion of this point by Buck, 1957). This sharply
contrasts with the partial relaxations induced by low Po2, which are very transitory,
and suggests that flutters and spiracular bursts are fundamentally different re-
sponses.
SPIRACLES IN CYCLICAL RESPIRATION 519
6. Effects of metabolic rate
The effects of metabolic rate on spiracular activity appear to be similar in
most insects: high metabolism leads to increased spiracular movement (cf. review
by Wigglesworth, 1953a). For example, Nunome (1944) has reported that the
spiracles of Bombyx mori open more frequently and more widely when larvae were
active than when they were inactive. In the present experiments, spiracular burst
frequency increased with metabolic rate, confirming manometric studies. This
increase in frequency is largely the result of more rapid production of carbon
dioxide which shortens the time required for tracheal PCO2 to reach triggering
concentration. The more rapid decrease in tracheal P02 which attends high
metabolism probably has only a limited impact, inasmuch as once fluttering begins,
tracheal P02 remains constant.
When metabolic rate gets very high and bursts occur every 5 or 6 minutes
(see for example Figure 6), the constriction period is obliterated, and between
bursts the valves are in continuous motion, i.e., fluttering. In effect, the decline
period after the burst fuses with the flutter period before the next burst. This
suggests that oxygen utilization is so rapid that even during the decline period
tracheal P0l. falls rapidly to the flutter threshold. When metabolic rate gets very
high and the cycles last for less than four minutes, as is the case with late developing
adults, the interburst flutterings increase markedly in amplitude until the burst
cycles disappear entirely and are replaced by continuous wide flutters. As men-
tioned previously, these flutters are far larger than oxygen-induced flutters. It
appears likely that when metabolism gets high enough to cause continuous flutter-
ing, carbon dioxide may be viewed as the principal stimulus. Further support
for this opinion comes from the observation that in intubated pupae the O2-open
threshold did not vary with metabolic rate, whereas the CCX-open threshold de-
creased as metabolic rate increased. This suggests that in pupae with high
metabolic rates, there is a considerable accumulation of carbon dioxide even when
several spiracles are open. This high tracheal PCo2 probably accounts for the fact
that intubated pupae with very high metabolic rates showed continuous valve
fluttering.
7. Interaction between carbon dioxide and oxygen
In the above analysis we have considered oxygen and carbon dioxide as acting
essentially independently. This is not the case, but, as we shall see, this fact
does not impair the analysis. Evidence that oxygen tension affects the carbon
dioxide threshold was provided by the observation that high oxygen tension
markedly reduced the burst frequency from 5 to 10 bursts per hour to one to two
per hour in a pupa in which 5 pairs of spiracles were sealed. This finding suggests
that the high oxygen tension raised the carbon dioxide threshold so that more
carbon dioxide had to accumulate to cause a spiracular burst. A further piece of
circumstantial evidence was the discovery that very high oxygen tensions often
restored bursts in pupae in which carbon dioxide had eliminated the bursts.
However, interpretation of these experiments was hampered by the fact that it
was difficult to be certain of the tracheal gas composition, which varied continuously.
Part of this difficulty was by-passed in experiments with intubated pupae. To be
520 HOWARD A. SCHNEIDERMAN
sure, even in intubated pupae the tracheal gas composition is not known with
certainty, but at least it can be kept constant and the oxygen and carbon dioxide
tensions can be varied independently.
The intubation experiments reveal that the concentration of carbon dioxide
necessary to open the spiracles wide varies with the oxygen tension. In the
presence of 2.3% oxygen, 5% carbon dioxide opened the spiracles of a typical
Cecropia pupa, whereas in 4.7% oxygen, 10% carbon dioxide was required. This
result demonstrates clearly the interaction between PO2 and PCo2 m controlling
the wide opening of spiracles, and it finds several parallels in the literature. Thus,
in the cockroach (Hazelhoff, 1926a, 1926b), the flea (Wiggles worth, 1935), and
in various muscid flies (Case, 1956a), the opening of spiracles in response to
carbon dioxide is favored by low oxygen and depressed by high oxygen, just as in
Cynthia and Cecropia pupae.
In the present experiments it is of some interest that, although PO2 influenced
the PCo2 necessary to cause a spiracular burst, there did not seem to be a similar
interaction in the triggering of flutters. Thus, as discussed in Section 5 above,
increasing ambient PCo2 UP to 10% nad no significant effects on the oxygen flutter
threshold. Hence, it seems unlikely that during a normal burst cycle internal
PCo2 affects the oxygen flutter threshold, inasmuch as tracheal PCO2 in these pupae
rarely rises above 6.5% (Buck and Keister, 1958; Levy and Schneiderman, 1958).
It is not our present purpose to explore the mechanism of the interaction between
oxygen and carbon dioxide in triggering bursts. However, recognition of this
phenomenon enables us to interpret several events in the burst cycle to which we
have not yet attended, notably the onset of the decline period. Why does the
relaxed spiracular muscle start contracting again about one-third of the way
through a burst? This might be the result of falling tracheal PCO^, rising tracheal
P02, or both. Evidence that it is largely rising PO2 comes from records of pupae
in pure oxygen. Here there is no decline period and the spiracle simply shuts.
This suggests that under normal conditions the decline period is related to tracheal
Po2. A possible mechanism might be the following.
During a spiracular burst, tracheal Po2 equilibrates with the atmosphere far
more rapidly than does tracheal PC02 because of the long time required to unload
dissolved carbon dioxide. In a relatively short time, tracheal P02 rises to a
concentration that increases the carbon dioxide burst threshold. The valve shuts
briefly, to reopen promptly as carbon dioxide continues to accumulate in the tracheal
system. Under this analysis, the onset of the decline period marks the point in
a spiracular burst where tracheal P02 has risen high enough to depress appreciably
the sensitivity of the spiracle to carbon dioxide. As mentioned in Section 5 above,
the duration of the decline period is determined by the time necessary to unload
carbon dioxide.
A second instance of O2-CO2 interaction appears to occur in the period just
before a spiracular burst. Ordinarily, 20 to 120 seconds before a burst, flutters
are larger than normal and gradually build up to the burst. This brief build-up
period resembles the decline period and is also absent in pure oxygen, suggesting
that, like the decline period, it reflects O2-CO2 interaction. The mechanism of
this interaction will be considered subsequently, but for the present we can con-
clude that at the low P02 which obtains in a normal pupa just prior to a burst, the
SPIRACLES IN CYCLICAL RESPIRATION
521
carbon dioxide threshold of different excitatory elements is not as uniform as in
high P02.
Interaction between oxygen and carbon dioxide also explains why variations
in ambient P0o have no predictable effect on burst frequency. High ambient Po2
raises the carbon dioxide threshold of the spiracles, and this tends to decrease the
burst frequency. But. simultaneously, high oxygen decreases interburst fluttering
and this, in turn, decreases the rate of interburst carbon dioxide release, and
increases the rate of carbon dioxide accumulation. This tends to increase the
burst frequency. The ultimate outcome, as we know from manometric experi-
ments ( Schneiderman and Williams, 1955) and from the present study, is
sometimes increased frequency, sometimes decreased frequency, and in other cases
no change.
Table VII summarizes the conclusions reached in this discussion relating to
the effects of tracheal P02 and Pro2 on the behavior of the spiracular valves during
the burst cycle.
TABLE VII
Summary of the effects of trachea! Po-2 and Pco* on the behavior of the spiracular -valves
during a burst cycle
Constriction period
Flutter period
Burst
Tracheal
composition
Initiate
Terminate
Initiate
Terminate
Initiate
Terminate
Low Pcoo
+
0
0
0
0
+
High PC02
0
(+)
( + )
+
+
0
Low PC-,
0
+
+
(+)
(+)
0
High P02
±
0
0
0
0
±
+ Principal agency responsible.
± Secondary factor.
( + ) These conditions do not normally prevail in the phase of the burst cycle indicated.
8. The existence of an independent oxygen-sensitive mechanism
It is not a principal purpose of the present report to discuss the mechanisms
whereby oxygen and carbon dioxide exert their effects, nor to consider the sites
of action of these gases. Earlier experiments on these pupae ruled out the existence
of any indispensable respiratory center (Schneiderman and Williams, 1953b;
Schneiderman and Beckel. 1954; Schneiderman. 1956). They also showed that
although nervous control was involved in the normal stimulation and coordination
of the spiracular muscles (Schneiderman, 1956), the immediate response to
carbon dioxide resided in the spiracular muscle itself (Beckel and Schneiderman,
1956, 1957). However, the present studies appear to clarify one point which
has hitherto not been resolved for any insect, namely that, at least in silkworm
pupae, oxygen and carbon dioxide affect the spiracle via rcr\ different mechanisms.
It has been suggested by various workers that oxygen-lack produces acidity, and
this might be its mode of action in stimulating spiracles. Wigglesworth (1935,
19531)) argues that in the flea both O,-lack and CCX-excess act in virtue of the
522 HOWARD A. SCHNEIDERMAN
acidity they produce. Case (1957 a) and Punt ct al. (1957), on the other hand,
raise the possibility that the acidity produced by oxygen-lack might release bound
carbon dioxide from the blood and that CO., is the effective agent to which
spiracles respond in both (X-lack and CCX-excess. In the case of the silkworm,
the experimental results forbid either interpretation, as the following considerations
show.
During the course of a burst cycle, when PO2 falls to a critical threshold,
fluttering begins and may continue uniformly for many hours. This sustained
pattern of behavior in response to low P02 indicates that the primary response
to oxygen lack is not acidity itself, nor acidity releasing bound carbon dioxide.
For, during the entire course of the flutter period, acidity increases steadily as
carbon dioxide accumulates. Notwithstanding, the flutter response did not change
appreciably, nor did it change after ambient PCo2 was raised to 10% (cf. Figure 8
and Section 5 above ) , a maneuver which certainly increased acidity. These ob-
servations suggest that, in the silkworm, increasing acidity is probably not the
means whereby oxygen lack makes itself felt. They also seem to prove that in the
silkworm pupa there is a specific mechanism sensitive to oxygen lack that may
act independently of any carbon dioxide-sensitive mechanism.4 Evidence will
be presented in a subsequent paper that P02 may act on the central nervous system
in contrast to Pco2 which acts peripherally.
9. Comparison zvitli the flea
In concluding, it is fruitful to compare the spiracular behavior of these silkworm
pupae with the behavior of the spiracles of the flea described by Wigglesworth
(1935) in his fundamental study. The flea spiracle is operated by a single closer
muscle and shows a simple pattern of opening and closing. In air at 20 to 22° C.
cycles are 12 to 16 seconds long, 6 to 8 seconds during which the spiracle stays
closed followed by about an equal time during which the spiracle is open. PO2 and
Pco2 affected this cycle in a predictable way. As Po2 diminished from 100% there
was an increase in cycle frequency ; the closed period was considerably shortened,
whereas the open period was less affected. As POO- increased, the open period
was lengthened considerably, whereas the closed period was slightly shortened.
It was concluded that the spiracles were caused to open chiefly by falling P02,
rising PCo2 being less important.
In the pupal burst cycle, the constriction period corresponds to the closed period
in the flea, and the spiracular burst corresponds to the open period in the flea.
Because the flea has no flutter period we cannot compare the effects of various
gas mixtures on the length of an entire cycle (i.e., on the frequency of the cycles),
but we can compare the periods of valve closure and of valve opening in both
insects.
(a) Oxygen effects
In both the flea and the pupa the duration of the constricted period is propor-
tional to P02, and in both, the spiracle is caused to open by low Po2. However.
4 Very recently Hoyle (1960) has provided convincing evidence that in the spiracular
muscle of the locust, CO» acts directly on the neuromuscular junction and blocks transmission
of nervous excitation. This important discovery supports the view outlined above that CO2
acts peripherally.
SPIRACLES IN CYCLICAL RESPIRATION 523
unlike the flea spiracle, which opens widely and stays open for a number of seconds
after a constriction period, the pupal spiracular muscle relaxes only slightly when
a critical PU2 is reached and then re-contracts immediately. This behavior is
repeated over and over during the flutter period, for which there is no counterpart
in the flea. In the pupa, for reasons already discussed in Section 7, Po2 has no
predictable effect on cycle frequency.
(b) Carbon dio.vide effects
In both the flea and the pupa the duration of the spiracular burst or open period
is proportional to Pco2- And in both, Pro2 has only small effects on the duration
of the constriction period. It is noteworthy that in the flea, carbon dioxide fails
to affect the frequency of openings and in the pupa it fails to affect the frequency
of flutters or volleys of flutters in the flutter period. These observations support
the view that the open period of the flea and the flutter period of the pupa are
largely triggered by low tracheal Po2, and that tracheal Pco2 is ordinarily of much
less importance in this connection. It is nonetheless of some significance that, in
the flea, raising the Pco2 decreased the closed period, albeit only slightly. In
commenting upon this, Wigglesworth (1935) remarks (p. 405) that the closed
period of the flea is terminated "chiefly by oxygen want (carbon dioxide con-
tributing to a small extent)." The pupal spiracular burst cycle enables us to
separate clearly the oxygen-want effect from the carbon dioxide effect, for in the
pupa the oxygen-want effect — fluttering — not only appears first, but is qualitatively
different from the carbon dioxide effect — the initiation of a spiracular burst.
In only one place in the flea are these effects separable, namely, in pure oxygen.
Here, in the flea as in the pupa, the spiracular bursts seem to have been triggered
mainly by Pco- and not by oxygen lack. It is also noteworthy that in both the
flea and the pupa, the effects of carbon dioxide varied directly with the intensity
of metabolism.
A further point of similarity between the flea and the pupa is that in both,
the denervated spiracular muscle remains contracted (Wigglesworth, 1935;
Schneiderman, 1956). In the case of the pupal silkworm the muscle remains
contracted for many weeks, and this appears to be true also of the bed-bug
(Wigglesworth, 1941 ) and the roach (Case, 1956b, 1957b). These are all insects
without nervous-controlled ventilation movements under ordinary conditions. By
contrast, the spiracular muscles of insects like Schistocerca, which have ventilation
movements, relax when denervated ( Fraenkel, 1932; Hoyle, 1959). Indeed, it
ma}- be that in man}- insects where the rhythmical activity of spiracles is under
nervous control, denervation leads to relaxation, whereas in others, like the flea
and silkworm pupae, where the spiracles are under the extrinsic control of respira-
tory gases, the denervated spiracular muscle continues to contract.
In sum. it appears that the behavior of the spiracles of both the flea and the
pupa is remarkably similar. A crucial difference between the two respiratory
cycles is the flutter period of the pupa. This cannot stem simply from differences
in the intensity of metabolism, for when fleas are placed at temperatures as low
as 4° C, which reduces their metabolism to levels characteristic of diapausing
pupae, they do not show flutters. Similarly, raising the metabolism of pupae to
that of fleas by injury or by other means fails to produce the flea pattern of pro-
524 HOWARD A. SCHNEIDERMAN
longed openings. The reason for the different modes of spiracular behavior is
probably to be found, at least in part, in the size of the insects and the attendant
differences in the lengths of both fluid and gaseous diffusion paths. Contributing
also to the differences in spiracular behavior is the relative insensitivity of the
(X- and COo-sensitive mechanisms. This was recognized at the outset by Wiggles-
worth (personal communication), who pointed out that among the preconditions
for discontinuous respiration was that the responding system "must have an ex-
tremelv high threshold of stimulation bv carbon dioxide . . . and this means that
-' r>
there must be a very large accumulation of CO., before it will cause the spiracles
to open." This is surely so, and the CO2 threshold of the flea is likely much
lower than that of the pupa.
However, the differences between the spiracular behavior of the pupa and the
flea are modest when weighed against the similarities. In summarizing his find-
ings on the flea. Wigglesworth (1935) concluded (p. 402) that each respiratory
act was "determined by an immediate stimulus of a chemical nature." The present
experiments extend these conclusions to the silkworm and indicate that carbon
dioxide bursts result in large measure from an exaggeration of a basically simple
sort of spiracular behavior.
10. Comparison with other insects
When the spiracles of other insect groups are examined, it becomes immediately
clear that fluttering, although not unique to silkworms, may be less common than
the simple closing and opening observed in the flea. For example, Hazelhoff
(1926a) reported no fluttering in any of the Diptera, Trichoptera, Neuroptera and
Odonata that he examined. However, he says that in P eriplaneta atnericana
". . . All the stigmata are almost closed. . . . The movable stigmal valve per-
forms usually quick vibrating movements'* (flutters) "whereby nevertheless the
opening width of the stigmata remains generally quite small" (p. 70). He adds
that ". . . the stigmata in pure O.2 are more closed than in ordinary air," which
suggests that here, as in the silkworm, fluttering is controlled by POL>- Fluttering
also occurs in locusts (Hoyle, 1959). How widespread fluttering is in other orders
and whether it is usually controlled by P0o remains to be seen.
11. Prospects
The main function of spiracular closing mechanisms is water conservation.
From an evolutionary viewpoint, we may imagine that natural selection has favored
the development of mechanisms that keep spiracles open just enough to permit the
exchange of respiratory gases but otherwise restrict their aperture. Such a
water-conserving mechanism might be expected to be best developed in insects
with severe \vater problems, like diapausing pupae which live for long periods
without imbibing (cj. Imms, 1957, p. 145). As Buck points out (1957, p. 77)
discontinuous respiration is an example of such a well-developed spiracular mecha-
nism. And, bursts of carbon dioxide are easily understandable in terms of the
spiracular movements, which have been analyzed in earlier sections of this dis-
cussion. But what of the disparate rates of gas exchange between bursts, which
are the real enigma of discontinuous respiration? Somehow during the interburst
the spiracles manage to let oxygen enter, yet simultaneously prevent carbon dioxide
SPIRACLES IN CYCLICAL RESPIRATION 525
and water vapor from leaving. Whilst the present experiments have not pene-
trated this secret, they nonetheless provide an experimental framework upon which
a theory of mechanism must he based. For the heart of the problem surely lies
in the flutter period, where hour after hour the insect practices the "trick" of
filtering in oxygen, while retaining carbon dioxide and water. It is our belief that
the real significance of the flutter period may have been overlooked in previous
explanations of this peculiar one-way transfer of gases. The importance of the
flutters in the kinetics of gas exchange will be discussed in a succeeding com-
munication.
The author wishes to express his sincere thanks to Air. Rishon Stembler and
Miss Diana Yeit who undertook many of the recordings of spiracular movement
and to Dr. W. E. Beckel who was kind enough to prepare drawings of the spiracular
apparatus. Special thanks are also due Professor V. B. Wiggles worth, Dr. A. D.
Lees and Dr. John B. Buck for their helpful comments on the typescript.
SUMMARY
1 . Experiments were conducted to examine the role of the spiracles in dis-
continuous respiration, to define the kinds of behavior that spiracles can show, and
to clarify the manner in which tracheal P,,, and Pro, interact to provoke various
modes of spiracular activity. To accomplish this, records were made of the move-
ments of the spiracular valves of diapausing pupae and developing adults of the
Cecropia. Polyphemus and Cynthia silkworms, in. air and in gas mixtures.
2. Spiracular valve movements in these silkworms occur in repeated cycles,
with periods of from a few minutes to many hours. Each cycle consists of an
open period or spiracular burst ( which corresponds to the CO, burst ) , a closed
or constriction period, and a flutter period which ordinarily occupies most of the
cycle. In pupae with long cycles, the respiratory events occur virtually in slow
motion when compared with other insects, and this permits careful analysis of
complex events in gas exchange and spiracular behavior which are not readily
separable in other insects. Evidence is presented that each spiracular act ( flutter-
ing, burst, valve closure) is a response to a specific chemical stimulus: the gaseous
composition of the tracheal system.
3. In pure O.,. the flutter period is ordinarily eliminated. As ambient Po2
decreases, fluttering reappears and the flutter period progressively lengthens, until,
in Po,'s below 15^, the spiracular bursts disappear and fluttering is continuous.
4. Ambient PC,,, ordinarily has no effect until it increases above 5%, where-
upon the cycles shorten. This shortening occurs at the expense of the flutter
period, which progressively diminishes as Pco2 increases. "When Pro., rises above
about I5c/c , the cycles break down completely and the valves flutter continuously.
5. Intubating even one pupal spiracle eliminates the cycles in the remaining 13.
and the valves stay permanently constricted, presumably because normal triggering
stimuli for spiracular activity are absent.
6. The spiracles of intubated pupae can be caused to open by lowering the
P0., or raising the Pro- The Pro, which opens the spiracles varies with the
ambient P0, : in a typical pupa, in 2.3r/> O,, 5% CO, opened the spiracles, whereas
in 4.7r/f O',, 10% CO, was required.
526 HOWARD A. SCHNEIDERMAN
7. From these and other data, it is concluded that the cyclical movements of
the spiracles result from cyclical changes in tracheal composition. In particular,
fluttering is initiated by low P02, while spiracular bursts are caused by high PCo2-
8. Evidence is presented to prove that the pupa possesses an independent
Oo-sensitive mechanism, which is quite separate from any CO2-sensitive mecha-
nism. It is also argued that low Po2 does not affect spiracular behavior by virtue
of anoxia-produced acidity, nor by virtue of acidity releasing bound carbon dioxide.
9. The spiracular behavior of these silkworms is compared in detail with the
picture of spiracular behavior in the flea provided by Wigglesworth, and it is
concluded that there is no fundamental difference between the two, except for
the flutter period which seems peculiar to the pupa. Evidence is presented that the
flutter period holds the key to the disparate rates of gas exchange between bursts,
which remains the central problem of discontinuous respiration.
LITERATURE CITED
BECKEL, W. E., 195S. The morphology, histology, and physiology of the spiracular regulatory
apparatus of Hyalophora cccropia (L.) (Lepidoptera). Doctoral thesis, Cornell Uni-
versity.
BECKEL, W. E., 1958. The morphology, histology and physiology of the spiracular regulatory
apparatus of Hyalophora cccropia ( L. ) (Lepidoptera). Proc. 10th Int. Congress of
Entouwl.. 2: 87-115.
BECKEL, W. E., AND H. A. SCHNEIDERMAN, 1956. The spiracle of the Cecropia moth as an
independent effector. Anat. Rcc., 125: 559-560.
BECKEL, W. E., AND H. A. SCHNEIDERMAN, 1957. The insect spiracle as an independent effector.
Science, 126: 352-353.
BUCK, J. B., 1957. Triggering of insect spiracular valves. Pp. 72-79 in "Physiological Trig-
gers," ed. by T. H. Bullock, Amer. Physiol. Soc., Washington, D. C.
BUCK, J. B., 1958a. Possible mechanism and rationale of cyclic CCX retention by insects.
Proc. 10th Int. Congress of Entouwl., 2: 339-342.
BUCK, J. B., 1958b. Cyclic CCK release in insects. IV. A theory of mechanism. Biol. Bull.,
114: 118-140. '
BUCK, J. B., M. KEISTER AND H. SPECHT, 1953. Discontinuous respiration in diapausing
Agapcma pupae. Anat. Rcc.. 117: 541.
BUCK, J. B., AND M. KEISTER, 1954. Spiracular retention of CO2 without CX limitation.
Anat. Rcc., 120: 731.
BUCK, J. B., AND M. KEISTER, 1955. Cyclic CO., release in diapausing Agapcuia pupa. Biol.
Bull.. 109: 144-163.
BUCK, J. B., AND M. KEISTER, 1958. Cyclic CO2 release in diapausing pupae — II. Tracheal
anatomy, volume and Pco ; blood volume; interburst CCX release rate. /. Ins. Physiol.,
1 : 327-340.
CASE, J. F., 1956a. Carbon dioxide and oxygen effects on the spiracles of flies. Physiol.
ZooL, 29: 163-171.
CASE, J. F., 1956b. Spontaneous activity in denervated insect muscle. Science, 124 : 1079-1080.
CASE, J. F., 1957a. Differentiation of the effects of pH and CO2 on spiracular function of
insects. /. Cell. Comp. Physiol., 49 : 103-114.
CASE, J. F., 1957b. The median nerves and cockroach spiracular function. /. Ins. Physiol.,
1 : 85-94.
FRAENKEL, G., 1932. Untersuchungen iiber die Koordination von Reflexen und automatisch-
nervosen Rhythmen bei Insekten III. Das Problem des gerichteten Atemstromes in
den Tracheen der Insekten. Zeitschr. i
a
PLATE IV
FIGURE 15. Himastlila piscicolu, type specimen, length 9.80 mm.
FIGURE 16. Himastlila incintoslii, type specimen, length 8.00 mm.
FIGURE 17. Spines at the ventro-lateral corners of the collar of worm shown in Figure 16.
FIGURE 18. Himasthlu cmnpacta, drawing of specimen shown in Figure 1.
STUDIES ON HIMASTHLA 537
and one from Nycticora.v nycticorax which had been identified by Linton as H.
elongata. It contained, also, the type material of H. incisa Linton, 1928 and H.
tensa Linton, 1940. Examination of the specimens of H. tensa discloses that the
number of collar-spines is 29, not "about 32" as reported by Linton (1940) . In the
three specimens (Fig. 7), the vitelline follicles terminate a short distance posterior
to the caudal end of the cirrus-sac. In the specimens of H. elongata shown in
Figures 4 and 5, the vitellaria extend anteriad slightly past the caudal end of the
cirrus-sac but in other individuals, especially younger ones, the vitellaria do not
extend to the level of the cirrus-sac. No constant differences were found between
these worms and, accordingly, the name H. tensa is suppressed as a synonym of
H. elongata. The Museum Collection also contains four slides bearing the number
54,721, with seven specimens from the "long-billed curlew," Numenius americanus
americanus, collected by J. Bushman at Orr's Ranch, Tooele Co., Utah, April 21,
1954. These worms have 35 collar-spines and are described as a new species,
Himasthla mcintoshi. Subsequently, Dr. George R. La Rue of the Research Center,
U. S. Department of Agriculture, Beltsville, Maryland, sent two specimens taken
from the digestive tract of a South American fish, Arapaima gigas, that died in the
Toledo, Ohio, zoo and that had been referred to him for identification. Grateful
acknowledgment is made also to Dr. La Rue for the privilege of examining and
describing these worms.
To locate the asexual generations of the metacercariae which occur in the gills
and palps of M. arenaria, a survey of the mollusks in the Boothbay Harbor area was
begun. As noted above, N. obsoletus had been identified as the first intermediate
host of H. quissetensis. The first intermediate hosts of H. elongata and H. alincia
are yet unknown, but there is strong and almost conclusive evidence that an
echinostome cercaria from Hydrobia mi nut a is the larval stage of one of the meta-
cercariae in M. arenaria and that this metacercaria develops in L. argentatus to
adults described in this paper as Himasthla compacta n. sp. Hydrobia minuta harbors
at least six different species of cercariae. Examination of 5000 snails, isolated 100
per dish, yielded three specimens shedding an echinostome cercaria and examination
of 5000 snails in another series, in which an individual count was kept as the snails
were crushed and examined under the microscope, disclosed six infections by this
echinostome cercaria. It has 29 collar-spines and is the only echinostome cercaria,
other than that of H. quissetensis which has 31 spines, found so far in the region.
Very small specimens of M. arenaria collected in the Woods Hole, Massachusetts,
area were added to the dishes containing the cercariae from H. minuta and metacer-
cariae, presumably of experimental infection, were recovered from them. Owing
to the low incidence of infection and the small size of the snails, and the resulting
scarcity of cercariae, it has not been possible to produce massive infections which
are easily obtained with the abundant cercariae of H. quissetensis. When using
clams collected in the field, there is the possibility that they may be carrying meta-
cercariae of natural infection, but examination of 200 specimens from the area where
those used in the experiment were taken, revealed no infection. Since laboratory-
reared clams were not available, very small clams from the Woods Hole area were
employed. These clams, exposed to the cercariae, were fed subsequently to a young,
laboratory-reared gull, L. argentatus. The bird began to pass trematode eggs some
four weeks after the first feeding and later, on examination, it contained 26 small
538
HORACE W. STUNKARD
o
21
II
PLATE V.
STUDIES ON HIMASTHLA 539
echinostomes, identified as H. compacta n. sp. This species has 29 collar-spines and
identification of the larval stages is not easy. The sizes of the cercariae and of their
metacercariae are not significantly different from those of H. qiiissetensis and do
not provide certain criteria for specific determination of the larvae. The adults are
distinguished easily. Consequently, it appears that one of the best means of positive
identification is to expose small clams to the cercariae and then feed the metacer-
cariae to previously unexposed birds.
Additional data on the life-cycle are provided by results of attempts to infect
the snail host. Eggs of H. compacta, taken from worms that had developed in a
laboratory-reared gull, were incubated in sea-water at room temperature. At the
end of four weeks they contained fully formed miracidia and were added to a finger-
bowl containing 20 juvenile H. ininuta. Four weeks later the number of living
snails was reduced to 13. Snails that died were not examined, since the tissues
decomposed quickly and trematode larvae would probably not be recognizable. One
snail, dissected four weeks after exposure, contained three small rediae (Fig. 22 )
but whether they were mother or daughter rediae was not determined. No cer-
cariae were obtained and the experiment was disappointing ; however, it illustrates
the difficulties inherent in this type of research. The number of eggs was limited,
development seemed to proceed at variable and inconstant rates, and actual hatching
was not observed. Since individual exposure of a snail to one or more miracidia
could not be made, the mass exposure method was undertaken to determine whether
or not certain of the miracidia on emergence could find and infect snails. With such
"shotgun" technique, it is impossible to know how many, if any, miracidia penetrated
a particular snail and it is possible that the death of certain snails was caused by
superinfection. The one experimental infection, however, supplements other data
and strongly supports the postulated life-cycle.
DESCRIPTIONS
Himasthla elongata (Mehlis, 1831)
Linton (1928) described trematodes from Larus argentatus at Woods Hole,
Massachusetts, which he identified as Himasthla elongata. Stunkard (1938)
pointed out that the material of Linton comprised two distinct species, one of which
was identified as Himasthla- quissetensis, whose cercarial stage had been described
PLATE V
FIGURE 19. Himasthla compacta, young cercaria from Hydrobia minuta, natural infection,
specimen fixed and stained ; body 0.50 mm. long ; tail 0.37 mm. long ; to show digestive, nervous
and excretory structures.
FIGURE 20. Himasthla compacta, normally emerged cercaria, sketch of living specimen to
show glands and excretory ducts.
FIGURE 21. Himasthla compacta, redia, natural infection, fixed and stained; specimen 1.12
mm. long.
FIGURE 22. Himasthla compacta, redia, experimental infection, fixed and stained ; specimen
0.47 mm. long.
FIGURE 23. Himasthla compacta, young specimen from gull, fixed and stained; 0.46 mm.
long.
FIGURE 24. Himasthla compacta, young specimen from gull, fixed and stained; 0.76 mm.
long.
540 HORACE W. STUNKARD
and named by Miller and Northup (1926). The members of the other species were
not identified although Stunkard noted that measurements of 12 representative
specimens are intermediate between or overlap the figures given by Dietz (1910)
as characteristic for H. clongata and H . inilitaris.
Certain of the adult worms recovered from the intestine of L. argentatus after
feeding metacercariae encysted in the tissues of M. arenaria from Maine are iden-
tified as H. clongata. These worms unquestionably are specifically identical with
those identified as H. clongata by Linton (1928). In most of the specimens the
collar-spines are slightly larger and the testes considerably larger than the figures
given by Dietz (1910) for H. elongata but there is considerable variation, and the
location of the testes and ovary near the posterior end of the body clearly differen-
tiates them from H. inilitaris. One specimen, killed in a very extended condition
(Figs. 12, 13, 14), exhibits the pseudosegmentation characteristic of the postacetab-
ular region of the body and the annular arrangement of the flattened, scale-like
spines. In this specimen, the two median members of the corner-spines (Fig. 13)
on the collar are very small, recalling the condition shown in Figure Bl of Dietz.
The worm measures 12.5 mm. in length and 0.45 mm. in greatest width. In Figure
12, 6 mm. and 110 annulations are omitted from the middle of the body. Five of
the annuli are portrayed in Figure 14. Another, younger specimen, with a few
eggs in the uterus gave the following measurements : length, 4.4 mm. ; width, 0.5
mm.; width at collar, 0.3 mm.; spines, 0.058 mm. long; acetabulum, 0.31 mm. long
and 0.26 mm. wide; oral sucker, diameter 0.11 mm.; pharynx, 0.098 mm. long and
0.056 mm. wide ; vitellaria do not extend forward to the level of the cirrus-sac ;
ovary 0.156 mm. wide and 0.125 mm. long; anterior testis, 0.25 mm. long and 0.155
mm. wide; posterior testis, 0.28 mm. long and 0.15 mm. wide; eggs 0.090 to 0.100
mm. long and 0.060 to 0.068 mm. wide. Gravid worms vary in size from these
measurements to older individuals that are twice as large with correspondingly
larger organs, but the structural pattern of the species is always evident.
The single specimen from the night heron, N. nycticora.v, shown in Figures 6
and 1 1 , has 29 collar-spines but manifests certain differences from the worms found
in L. argentatus. It is only slightly smaller than the worm shown in Figure 5 ;
however, the collar-spines and suckers are smaller, the vitellaria do not extend as far
anteriad, and the gonads are smaller and slightly farther forward. In this specimen
the spines are 0.050 mm. long; the oral sucker measures 0.114 by 0.107 mm.; the
acetabulum is 0.325 mm. long and 0.290 mm. wide; the ovary is spherical, 0.143
mm. in diameter ; the anterior testis is 0.40 mm. long and 0.23 mm. wide ; the
posterior testis is 0.42 mm. long and 0.25 mm. wide; the eggs average 0.097 by
0.062 mm. The measurements are almost identical with those given by Nicoll for
HiinastJila sccmida, and the small size of the gonads agrees well with the description
of H. clongata as given by Dietz. The similarity suggests possible identity of H.
sccunda and H. clongata. Since the material at hand consists of a single specimen,
it is referred for the present to H. elongata. When the life-history of that species
is known and cercariae are available for experimental infections, it will be possible
to determine definitely whether the present worm belongs in H . clongata or is a
member of some other Species.
The specimen from Lams dclau'arcnsis (Fig. 3) is referred to H. clongata. but
it is juvenile and possibly members of that species do not attain sexual maturity
in L. dclawarensis.
STUDIES ON HIMASTHLA 541
Himasthla compacta n. sp.
Adult
Material of this species consists of worms recovered from laboratory-reared L.
argcntatiis fed M. ar en aria collected near Boothbay Harbor, Maine, and others from
another laboratory-reared L. argentatns, five weeks after the beginning of an experi-
ment in which the bird was fed metacercariae encysted in small M. arenaria. These
clams had been exposed to echinostome cercariae from H. minuta collected in
Sagadahoc Bay, near Boothbay Harbor, Maine, and the metacercariae were pre-
sumed to be encysted stages of the same cercariae. Most of the worms were sex-
ually mature but several were juveniles. The bird had been fed five weeks, twc
weeks and one week before it was killed and the small worms shown in Figures 23
and 24 are probably from the last two feedings. A specimen 2.130 mm. long and
0.275 mm. wide does not have eggs in the uterus, although there are spermatozoa
in the testes.
Gravid specimens, fixed and stained, measure 3.00 to 4.30 mm. in length and
0.35 to 0.44 mm. in width. For such a small species of Himasthla, the organs are
large and compactly disposed. The sides of the body are almost parallel; the
acetabulum protrudes ; in the preacetabular region the lateral edges are often turned
ventracl and mediad, forming a ventral depression. The anterior end has a reniform
collar, open ventrally. which bears 29 spines, 25 arranged in a linear row and two
smaller corner-spines on each side behind the terminal ones of the row. The lineal
spines are 0.054 to 0.062 mm. in length and 0.012 to 0.014 mm. wide; the smaller
corner ones are 0.026 to 0.032 mm. long and 0.009 mm. wide. In mature specimens
the acetabulum is about one-seventh of the body length from the anterior end
whereas in juvenile worms it is relatively farther back and in young worms it is near
the middle of the body. The shift in relative position of the acetabulum results from
development of the reproductive organs in the postacetabular portion of the body.
The acetabulum is usually longer than broad, oriented with the opening at the
antero-ventral face, just behind the common genital pore. The sucker measures
0.20 to 0.26 mm. in length and 0.18 to 0.22 mm. in width. The cuticula in the
preacetabular area bears scale-like spines arranged in an imbricate pattern, while
behind the sucker the spines are smaller and are arranged in the annular fashion
characteristic of the genus.
The mouth is subterminal ; the oral sucker measures 0.075 to 0.090 mm. in diam-
eter. There is a short prepharynx ; the pharynx is oval to pyriform, usually wider
posteriorly, it measures 0.060 to 0.075 mm. in length and 0.040 to 0.050 mm. in
width. The esophagus extends almost to the acetabulum and the ceca end blindly
near the posterior end of the body.
The testes are almost contiguous, one behind the other, in the caudal one-third
of the body. They are oval, with notched but not lobed surfaces. The posterior
testis is usually somewhat larger than the anterior one. The anterior testis is 0.36
to 0.49 mm. in length and 0.18 to 0.21 mm. in width; the posterior testis is 0.40 to
0.58 mm. in length and 0.18 to 0.21 mm. in width. From each testis a sperm duct
passes forward and the two unite just before reaching the cirrus-sac. The common
duct, on entering the sac. expands into a coiled seminal vesicle, which fills the
posterior one-half or more of the cirrus-sac. The vesicle is followed by a shorter
prostatic portion of the duct and then by a protrusible cirrus armed with very small
542 HORACE W. STUNKARD
spines. The cirrus-sac extends behind the acetabulum more than the diameter of
that sucker and terminates between the anterior ends of the vitelline glands.
The ovary is spherical to oval, usually broader than long, situated from two-
sevenths to three-eighths of the body length from the posterior end. It is about its
diameter in front of the anterior testis. It measures 0.06 to 0.13 mm. in length and
0.08 to 0.16 mm. in width. The oviduct arises at the posterior face and passes
backward where it enters the ootype region. It expands somewhat and gives off
Laurer's canal, which passes to the dorsal surface of the body, after which it receives
the common vitelline duct. This portion is partially enclosed in the cells of Mehlis'
gland, which surrounds the ootype. The initial portion of the uterus is filled with
masses of spermatozoa. The uterus coils about and passes forward to the level of
the caudal end of the cirrus-sac where it joins the metraterm. Both metraterm and
cirrus-sac pass forward above the acetabulum to open into a shallow genital sinus,
and the genital pore is on the median ventral surface, immediately anterior to the
acetabulum. The vitelline follicles are lateral to and partially overlap the digestive
ceca ; they extend from the caudal end of the body to a level about the length of the
acetabulum behind that sucker. They are not interrupted at the levels of the testes.
Longitudinal ducts connect the follicles and transverse ducts pass mediad at the level
of the ootype, where they unite to form a vitelline receptacle from which a common
duct leads to the initial portion of the ootype. The eggs are large, oval, thin-shelled,
collapsed in the preserved specimens, 0.085 to 0.090 mm. in length and 0.050 to
0.058 mm. in width. Each egg contains an ovum and several vitelline cells. The
ovum is situated toward the opercular end of the egg. Cleavage begins in the
uterus, but development there does not go much beyond the four-cell stage.
Himasthla compacta differs from all other species of the genus in its smaller
size, its compact structure and relatively larger gonads. The ovary is situated about
one-third of the body length from the posterior end and the testes extend through
most of the postovarian distance, a condition not found in any other species. Since
H . compacta has not been reported previously from L. argentatus, some other avian
species may be its normal host.
The type specimen (Figs. 1, 18) is deposited in the U. S. National Museum
Helminthological Collection under the number 39.444.
Rcdia
In structure and behavior, the rediae are very similar to those of H. quissetensis
as described by Stunkard (1938). Figure 21 is a drawing of a large, gravid redia
of natural infection. It is 1.12 mm. long and 0.28 mm. wide; the pharynx is 0.028
mm. in diameter. Figure 22 shows a young redia recovered from one of the small
specimens of H. mimtta which had been exposed for four weeks to embryonated eggs
of H. compacta taken from worms that had developed in the intestine of a laboratory-
reared specimen of L. argentatus. Whether this is a first or second generation redia
could not be determined, since the germ balls in it could be embryos of either rediae
or cercariae. At this stage they are quite indistinguishable. The specimen meas-
ures, fixed, stained and mounted, 0.47 mm. long, 0.12 mm. wide, and the pharynx is
0.027 mm. in diameter. The young rediae have collars which become visible as the
larvae move ; they progress in a lumbricid manner, with temporary protrusions of
foot-like projections to anchor one region while advancing another. In young rediae
STUDIES ON HIMASTHLA 543
the gut is conspicuous, filled with yellowish-green material, obviously the residue
from digestion of snail tissue. The intestine does not increase in size and becomes
proportionately smaller as the cavity of the redia becomes filled with larvae of the
next generation. With increase in size and accumulation of growing progeny, the
rediae become less and less active. Meanwhile, with advancing maturity, the
cercariae become more vigorous in their movements until they emerge through the
birth-pore, situated near the pharynx.
Cercaria
The incidence of infection of H. minuta is low, about one in five hundred, and
cercariae emerged from less than one-half of the infected snails. The snails are
small and few cercariae are produced. They leave the rediae and complete develop-
ment in the haemocoele of the snail. In a crushed snail with mature infection,
usually there are two to four or five cercariae free in the sinuses and others still in
rediae. Some of those in the rediae swim actively when released, but the small
number of cercariae restricts experimental procedure. In swimming, the body is
contracted until it is almost spherical while the tail is extended and lashes vigorously.
The body in living cercariae measures 0.30 to 0.60 mm. in length and 0.08 to
0.19 mm. in width; it is elongate oval in outline although the shape varies with the
degree of elongation and retraction. The collar gives the anterior end a character-
istic form and when the larva is fully extended, this may be the widest part of the
body. The tail is shorter than the body and capable of great extension and retrac-
tion by contraction of the circular or longitudinal muscles which form its wall. In
naturally emerged cercariae the oral sucker measures 0.057 to 0.065 mm. in diam-
eter; the ventral sucker 0.072 to 0.086 mm.; and the pharynx 0.014 to 0.020 mm.
Figure 19 is of a well extended young cercaria from a crushed snail, fixed with hot
AFAG (alcohol-formalin-acetic acid-glycerin) fixing fluid added to a small amount
of sea-water in a beaker in which the larvae were being whirled. Stained and
mounted, it has a total length of 0.87 mm. ; the bodv is 0.50 mm. long and 0.13 mm.
o ^ o
wide at the collar ; the acetabulum is 0.080 mm., the oral sucker is 0.057 mm., and
the pharynx is 0.020 mm. in diameter. The tail is 0.037 mm. wide at the base. The
body of the cercaria contains three types of glandular cells. There are three pairs
of penetration glands situated in the preacetabular area, and four pairs of glands in
the oral sucker, all of \vhich open through pores at the anterior end of the body.
The entire dorsal area of the body is occupied by cystogenous cells whose cytoplasm
is filled with bacilliform granules. These granules do not stain with vital dyes.
Other glandular cells, more ventral in location, stain faintly with eosin and
erythrosin.
The reproductive organs are represented by two groups of deeply staining cells,
one near the posterior end of the body and the other at the anterior margin of the
acetabulum, and a strand of cells extending between the larger groups. The poste-
rior group is the rudiment of the gonads and the anterior one of the copulatory
organs. Only the major portion of the excretory system was observed. In im-
mature larvae the tissues are fragile and disintegrate before the tubules are visible
and in older ones the excretory ducts are obscured by the masses of glandular cells.
Certain observations indicate that the flame cells are arranged in groups of three,
but the complete pattern was not worked out. The system forms as separate left
544 HORACE W. STUNKARD
and right components which meet and fuse at the posterior end of the body and the
anterior portion of the tail. The two excretory pores are located on the sides of
the tail, as shown in Figure 19. The vesicle is spherical to oval, thin-walled, and
from either side a collecting duct passes forward, median to the digestive cecum,
below the cecum at the level of the acetabulum, and then forward to the level of the
oral sucker, where it recurves and passes posteriad. Near the middle of the body
the recurrent duct divides into anterior and posterior branches. In the preace-
tabular area the collecting ducts have median and lateral branches and these
branches subdivide in turn to form a ramified pattern as shown in Figure 20. In
mature cercariae the collecting ducts contain spherical to oval concretions ; in the
postacetabular area the granules are larger, 0.005 to 0.01 mm. in diameter and more
numerous, four or five at any level of the duct, whereas in the preacetabular ducts
and their branches the granules are smaller, 0.003 to 0.005 mm. in diameter, and are
arranged in single rows.
Himasthla incintoshi n. sp.
This name is proposed for the seven specimens from the long-billed curlew,
Xunicnius americanus americanus, collected by J. Bushman in Tooele County, Utah,
on April 21, 1954 and deposited in the Helminthological Collection of the U. S. Na-
tional Museum under the number 54,721. The species is named in honor of Dr.
Allen Mclntosh, Parasitologist in the Agricultural Research Service of the U. S.
Department of Agriculture, in recognition of his contributions to helminthology and
of the generous aid he has provided for other workers.
The worms are all sexually mature, with eggs in the uteri, chiefly in the initial
one-half of the organ, with the terminal portion almost if not quite empty. Five of
the worms are much bent or coiled and the one shown in Figure 16 is the only
straight-bodied specimen. They vary from 6 to 11 mm. in length and from 0.5 to
0.7 mm. in greatest width. The anterior end bears a reniform collar which, in the
two specimens in which they could be counted, is armed with 35 spines. There is a
single row, interrupted ventrally, with 29 large spines, each 0.078 to 0.084 mm. in
length and 0.02 mm. wide at the base, and at either end of the row, on the ventral
side, there are three smaller corner-spines, about 0.055 mm. in length and 0.016 mm.
wide at their bases. The lateral edges of the preacetabular region are curved
ventrad and mediad, creating a median ventral depression (Fig. 17). When the
body is contracted, the lateral walls are crenated and each annulus in the post-
acetabular region bears a circlet of small cuticular spines. In the preacetabular
region the spines are closer together and arranged in an imbricated pattern, a
cuticular arrangement characteristic of the genus Himasthla. The acetabulum is
slightly less than twice its diameter from the anterior end of the body ; it is directed
antero-ventrad, protrudes slightly, and measures from 0.33 to 0.39 mm. in length
and 0.31 to 0.35 mm. in width.
The mouth is subterminal, the oral sucker is 0.13 to 0.16 mm. in diameter,
followed almost immediately by the pharynx, about 0.14 mm. long and 0.10 mm.
wide. The esophagus extends almost to the acetabulum and the digestive ceca
terminate blindly near the posterior end of the body.
The excretory pore is terminal, the vesicle is short and divides behind the
posterior testis, with the collecting ducts passing forward just median to the
STUDIES ON HIMASTHLA 545
digestive ceca. They are clearly visible in the region between the anterior ends
of the vitellaria and the acetabulum. Anterior to the acetabulum they are lateral
in position and extend forward to the level of the oral sucker where they turn back-
ward. Further details of the excretory system could not be observed.
The testes are oval, elongate, the posterior testis about its length from the pos-
terior end of the body. There may be a short interval between the testes or they
may be almost contiguous. The posterior testis is slightly larger than the anterior
one and measures from 0.56 to 0.65 mm. in length and 0.22 to 0.28 mm. in width.
Sperm ducts are not visible in the whole mounts ; in most of the specimens the
cirrus-sac extends about the diameter of the acetabulum behind that sucker ; in the
one shown in Figure 16, the cirrus-sac is coiled and consequently does not extend as
far posteriad. The posterior one-half to two-thirds of the cirrus-sac is filled with
a seminal vesicle and the anterior portion contains the eversible male duct, sur-
rounded by secretory cells. The cirrus bears small recurved spines and the genital
pore is median at the anterior margin of the acetabulum.
The ovary is spherical, 0.18 to 0.20 mm. in diameter, situated a short distance
in front of the anterior testis. The oviduct arises at the posterior margin and the
ootype and Mehlis' gland are posterior to the ovary. The vitellaria are lateral to
and somewhat overlap the digestive ceca dorsally and ventrally. The follicles are
spherical to oval, 0.04 to 0.065 mm. in diameter, continuous on both sides of the
body, although somewhat reduced in one specimen at the level of the posterior testis.
They extend from the posterior end of the body about two-thirds of the distance to
the anterior end, terminating about two-thirds of the distance from the ovary to
the acetabulum. Transverse ducts at the level of the ootype pass mediad to form
a vitelline receptacle which discharges into the oviduct immediately before the
ootype. The initial portion of the uterus is filled with spermatozoa. The eggs are
broadly oval, those near the ovary average 0.100 by 0.076 mm., those farther along
in the uterus are rounded, often collapsed, and may be slightly longer.
Himasthla mcintoshi agrees most closely with H. rhigcdana, type of genus.
Both are from species of X it matins and they are the only described species with
more than 31 spines on the collar. Dietz reported a total of 34 to 38 spines in H.
rhigedana, with 2, 3, or 4 corner-spines at each end of the row. In both of these
species the corner-spines are very close together and often superimposed on those
of the lineal row. Contractions of muscles in these locations produce variable
orientation of the spines, so determination of their number and disposition is dif-
ficult. In Figure T of Dietz, the upper corner-spine on the left side could be inter-
preted either as a corner-spine or as the terminal spine in the collar-row. The two
species differ in geographical distribution; H. rhigcdana is from Arabia and H.
mcintoslii from northwestern United States. Although the suckers do not differ
greatly in size, H. rliigedana is more than twice as large as H. mcintoshi, the repro-
ductive organs are much larger, although the eggs are smaller. The most obvious
difference is in the disposition of the vitellaria ; in H ' . rhigcdana the vitellaria
are interrupted at the testicular levels whereas in H. mcintoshi the follicles are
continuous.
The type specimen is deposited in the U. S. National Museum Helminthological
Collection under number 54,721.
546 HORACE W. STUNKARD
Himasthla piscicola n. sp.
This species is based on specimens found by Dr. H. O. Ewert, veterinarian of
the Zoological Society, Toledo, Ohio, in the alimentary canal of a fish, Arapainia
gigas, from the Amazon River, Brazil. They were sent for determination to Dr.
Leonard Allison of the Institute for Fisheries Research at the State Fish Hatchery,
Grayling, Michigan. With the specimens there was the following information :
"HISTORY; the 24 inch specimen arrived here in September. The fish ate in
the first weeks, four to five goldfishes three inches long, daily, and came gradually
down to one fish a day until he stopped eating around the 18th of December. In
this week he vomited several small balls of mucus. Under the microscope, these
balls appeared to consist of cells, mucus and many flagellates, Octomitus intestinalis.
AUTOPSY ; the abdominal investigation showed inflammation of the intestinal
tract as well as the abdominal lining (peritonitis). The stomach lining was highly
inflamed and congested. The stomach cavity was filled with a tenacious mucus and
a certain parasite, which will be found separated on the accompanying slide."
According to Allison (in lift.), the parasite was a trematode which had been
mounted in water under a cover-glass and arrived perfectly dry. Other specimens
were removed from the contents of the stomach which was preserved in water.
Allison identified the worms as members of the genus Himasthla. Professor S.
Yamaguti examined certain of the specimens and agreed with the generic determi-
nation, noting differences between these specimens and H. tensa Linton, 1940.
Subsequently, Allison wrote Dr. G. R. La Rue, at the Animal Parasite Research
Laboratory, Beltsville, Maryland, and sent him two of the worms in the belief
that La Rue would write the description. But Dr. La Rue suggested that the
writer examine the specimens and make the report. They are here described as
a new species, Himasthla piscicola.
The two specimens measure 8.2 and 9.8 mm., respectively, in length. The
larger one, shown in Figure 15, is designated as type. The organs of the smaller
worm are almost as large as those of the type specimen. In the smaller one there
are masses of spermatozoa in the initial portion of the uterus but no eggs. There
are two eggs, one of them collapsed, in the uterus of the larger worm. In these
specimens the reniform collar, open ventrally, delimits a short, flattened area at
the anterior end of the body. The collar-spines are intact but other spines have
been lost. There are 29 spines on the collar, 25 in the lineal row and two on
either side behind the terminal ones. Those at the ventral corners are as large
as those in the lineal row ; they measure 0.085 mm. in length and 0.025 in maximum
width. Behind the collar there is a short, neck-like constriction. The specimens
are much extended, a result of their protracted immersion in water, and the uterine
region between the cirrus-sac and the ovary is especially narrow. In the larger
worm the width at the collar is 0.875 mm. The acetabulum is 0.44 mm. long and
0.50 mm. wide; it is about its diameter behind the collar. The oral sucker pro-
trudes slightly and measures 0.18 by 0.19 mm. The pharynx is large, 0.24 mm.
long and 0.11 mm. wide. The esophagus extends to the level of the acetabulum
and the digestive ceca end blindly near the posterior end of the body. The
testes are situated much nearer the middle than the posterior end of the body.
STUDIES ON HIMASTHLA 547
They partially overlap, the posterior third of the anterior testis is in the same
zone as the anterior third of the posterior testis. They are elongate, slightly
notched ; the anterior one is 0.875 mm. long and 0.20 mm. wide, the posterior
one is 0.89 mm. long and 0.20 mm. wide. The cirrus-sac extends posterior to the
acetabulum more than twice the diameter of that sucker. A large seminal vesicle
occupies the posterior half of the sac ; the anterior portion of the vesicle and the
succeeding duct are enclosed in a large, many-celled prostate gland. The cirrus
is not protruded and no spines were observed. The ovary is situated near the
middle of the body, only a short distance in front of the anterior testis, but would
be relatively more posteriad if the uterus were filled with eggs. It measures
0.18 by 0.20 mm. The ootype complex is large, situated immediately posterior to
the ovary ; the initial portion of the uterus is filled with spermatozoa and there are
two eggs in the uterus. One is collapsed, the other measures 0.114 by 0.064 mm.
The vitelline follicles are continuous on each side of the body and extend from the
posterior ends of the digestive ceca about three-fourths of the distance from the
ovary to the posterior end of the cirrus-sac. The follicles would probably extend
farther forward in more mature individuals.
Although the specimens are not mature, H. piscicola differs from all other
adequately described species with 29 collar-spines in the position of the gonads,
the shape and overlapping arrangement of the testes, and in the relative length
of the posttesticular region of the body. Himasthla piscicola and H. annulata were
found in the digestive tract of fishes from the Amazon River ; it is possible that
the two are identical, that some avian species is the natural host, and that the
discovery of these worms in fish hosts is entirely incidental. The worms are similar
in size, but the description and figures of Diesing give no information concerning
internal morphology of H. annulata and it is quite impossible to determine whether
the two are identical.
The type specimen of H. piscicola is deposited in the Helminthological Collection
of the U. S. National Museum under the number 39,445.
DISCUSSION
An investigation conducted by the U. S. Bureau of Commercial Fisheries is
attempting to determine the causes for the decline in populations of Mya arenaria
along the coast of New England and possible biological measures for control of
the principal predators, the green crab (Carcinus maenas} and the horseshoe crab
( Limulus polyphemus). Mya arenaria harbors the sporocysts and cercariae of
Cercaria inyae Uzmann, 1952; the larval stages of an as yet undetermined species
of Gymnophallus (Stunkard and Uzmann, 1958). The palps and gills contain
metacercariae of digenetic trematodes. Since the asexual generations of these
metacercariae must occur in mollusks which live in the immediate vicinity of the
infected clams, a survey of the more abundant species and those most likely to carry
the trematode infections, is in progress. Furthermore, since the definitive hosts
of these metacercariae are animals that feed on M. arenaria, examination of shore-
birds has been started. To obtain precise information under controlled conditions,
metacercariae from M. arenaria have been fed to laboratory-reared eider ducks,
548 HORACE W. STUNKARD
herring gulls, common terns, white mice, and golden hamsters. The results,,
together with other pertinent information, are presented in this paper.
The metacercariae from M. arenaria proved to be larvae of three different
species, all in the genus Himasthla, and the adults recovered from L. argent at us
have been of value in resolving taxonomic problems in the genus. Typically,
echinostomes are parasites of warm-blooded vertebrates and members of Himasthla
have metacercarial stages in marine mollusks and mature in avian hosts. Himasthla
ambigua Palombi, 1934 was described from metacercariae found in Tapes decussatus
from the Gulf of Naples, but the adult stage is yet unknown. Palombi reported that
the worms have 32 cephalic spines, that the infection is seasonal, and suggested
that a bird, perhaps a migrant, is the final host. Elucidation of the life-cycle of
H. couipacta supports previous belief that species of Himasthla are parasites of
birds and that the larvae occur in marine mollusks. Although adults of H. compacta
are less than one-half the size of those of H. quissctcnsis, the cercariae of the two
species are almost identical in size.
Since members of Himasthla typically are parasites of shore-birds, it is sur-
prising that four species have been recorded from abnormal hosts, three from fishes
and the fourth from a fruit-eating pigeon. Two species, H. multilccithosa from the
pigeon and H. piscicola from Arapaima gigas, were taken from captive hosts and
the time and place of infection are unknown. The finding of specimens of Himasthla
in the digestive tract of marine and fresh-water fishes presents a biological anomaly.
The fishes could have ingested a bird or its entrails that had fallen in the water
and the proposed identity of H. tensa and H. clongata suggests such an explanation.
H. annulata and H. piscicola were taken from the alimentary tract of fishes from
the Amazon River of Brazil, and if the worms found in A. gigas were acquired in
South America, they must have persisted for an unusually long time in the fish.
The specimens were still juvenile, which indicates that they were recently ingested
or that they fail to attain sexual maturity in the cold-blooded host. Since H.
annulata (Diesing, 1850) and H. piscicola are from Amazonian fishes, it is pos-
sible that the two are identical, that some avian species is the natural host, and that
the discovery of these worms in the digestive tract of fishes is entirely incidental.
SUMMARY
The validity of species in the genus Himasthla is discussed; Echinostoma annu-
latuui (Diesing, 1850) is transferred to Himasthla and H. tcnsa Linton, 1940 is
suppressed as a synonym of H. clongata ( Mehlis, 1831). A specimen from
\\cticora.\- nycticora.v, tentatively assigned to H. clongata. is very similar to H.
secunda (Nicoll, 1906), which suggests the possibility that H. sccnnda may be a
not-fully mature form of H. clongata. Three new species are described ; H.
mcintoshi from Nnmcniiis americanus amcricanns taken in Tooele County, Utah;
H. piscicola, probably an accidental infection, from the South American fish,
Arapaima gigas; and H. couipacta from experimental infection of the herring gull,
Lams argentatus. The life cycle of H. couipacta has been traced ; the asexual
generations occur in H \drobia minuta. the cercariae encyst in Mya arenaria and
probably other mollusks.
STUDIES ON HIMASTHLA 549
LITERATURE CITED
BEAVER, P. C, 1937. Experimental studies on Echinostonia rcrolntitm (Froelich) a fluke from
birds and mammals. Illinois Biol. Moiwur.. 15: 1-96.
COBBOLD, T. S., 1860. Sypnosis of the Distomidae. J. Proc. Linn. Soc. London. Zool. (17)
5: 1-56.
CUEXOT, L., 1892. Commensaux et parasites des Echinodermes. Rev. Biol. Nord. France,
5: 1-23.
DAWES, B., 1946. The Trematoda. Cambridge University Press.
DIESING, K. M., 1850. Systema helminthum I. 679 pp. Vindobonnae.
DIESIXG, K. M., 1855. Neunzehn Arten von Trematoden. Denkschr. K. Akad. Wisscnsch.
ll'icn. Math.-Naturzi'. Cl. V, 10: 59-70.
DIETZ, E., 1909. Die Echinostomiden der Vogel. Zool Ans., 34: 180-192.
DIETZ, E.. 1910. Die Echinostomiden der Vogel. Zool. Jahrb.. SuppL, 12: 256-572.
LINTON, E., 1928. Notes on trematode parasites of birds. Proc. U. S. Nat. Museum, 73 :
1-36.
LIXTON, E., 1940. Trematodes from fishes mainly from the Woods Hole region, Alassachusetts.
Proc. U. S. Nat. Museum. 88: 1-72.
MENDHEIM, H., 1940. Beitrage zur Systematik und Biologic der Familie Echinostomidae.
Nova Acta Leo. Carol. Halle, N.F., 8: 489-588.
MENDHEIM, H., 1943. Beitrage zur Systematik und Biologic der Familie Echinostomidae.
Arch. Naturgcsch., Leipzig, N.F., 12 : 175-302.
MILLER. H. M., AND F. E. NORTHUP. 1926. The seasonal infestation of Nassa obsolcta with
larval trematodes. Biol. Bull., 50: 490-509.
PALOMBI, A., 1925. Di un nuovo ospitatore della cercaria dell' Echinostoinuin sccunduni Nicoll,
1906: Mytilns t/alloprovincialis Lamk. Boll. Soc. Nat. Napoli (1924), 36: 49-51.
PALOMBI, A., 1934. Gli stadi larvali dei Trematodi del Golfo di Napoli. Pubbl. Staz. Zoo!.
Napoli, 14: 51-94.
PRUDHOE, S., 1944. On two echinostome trematodes from Ceylon. Ann. May. Nat. Hist., (11)
11: 1-13.
SKRJABIX, K. I., 1956. Trematodes of animals and man. vol. XII. Akad. Nauk SSSR,
Moscow.
SPREHX, C. E. W., 1932. Lehrbuch der Helminthologie. Gebr. Borntrager, Berlin.
STUNKARD, H. W., 1934. The life-history of Hiniasthla qiiissefcnsis (Miller and Northup,
1926). /. ParasitoL. 20: 336.
STUNKARD, H. W., 1937. The life-cycle of Hiniasthla quissctcnsis (Miller and Northup, 1926)
Stunkard, 1934 (Trematoda). Papers on Hchninthol.. 30 year Jubil., K. I. Skrjabin,
pp. 689-698.
STUXKARD, H. W., 1938. The morphology and life-cycle of the trematode, Himasthla quis-
sctcnsis (Miller and Northup, 1926). Biol. Bull., 75: 145-164.
STUXKARD, H. W., 1939. Determination of species in the trematode genus Hiniasthla. Zeitschr.
Parasitenk., 10: 719-721.
STUNKARD, H. W., AND J. R. UZMAXX, 1958. Studies on digenetic trematodes of the genera
Gytnnophallus and Parvatrema. Biol. Bull., 115: 276-302.
VILLOT, F. C. A., 1878. Organization et developpement de quelques especes de trematodes endo-
parasites marins. Ann. Sci. Nat. Zool., 49 (6 ser) 8: 40 pp.
VOGEL, H., 1933. Hiniasthla muchlcnsi n. sp., ein neuer menschlicher Trematode der Familie
Echinostomidae. Zcntralbl. Baktcriol. 1 AM., Ori '"
1.2
1.0
.9
Exp.aContr. at28.4°C.
48 Hr. Exp. •• 17.8
48Hr. Contr. •• 28.4
96Hr. Exp. 8.75
96Hr. Contr. •• 28.1
O
»
7
.6
.7
1.0 I.I 1.2 1.3 1.4
Melting Point of Water in— "C
1.5
1.6
1.7
1.8 1.9
FIGURE 4. Pcnaeus duorarum, 35-100 mm. total length. Regression lines drawn through
average melting points of blood at average melting points of water of —0.77. --1.01, —1.22,
— 1.47 and —1.74° C. Key to lines on figure.
568
AUSTIN B. WILLIAMS
when immersed in an array of 10-30/£c sea water mixtures (b =- .182). Within
three hours those in dilute media have more dilute blood than those in the less dilute.
At the 48-hour point, with temperature gradually depressed to 17.8° C., the slope
of this line has changed materially (b -- .376). Though animals in 20%0 water have
changed little, those in lower salinities have more dilute blood than in the beginning
and those in high concentrations have more concentrated blood. Controls at
28.4° C. are essentially unchanged from conditions at the start, but those in higher
salinities have somewhat more concentrated blood (b -- .210). By the end of 96
hours at a temperature of 8.75° C. a marked change in the slope of the line for blood
concentrations is again seen, with a trend toward isotonicity (b = .525). This
trend is not so pronounced as in aztecus of comparable size (Fig. 2). In controls
at 28.1° C. there has been a material dilution of blood at lower salinities (b = .343 ).
TABLE 1 1
Per cent survival of shrimp at 48 and 96 hours. Forty-eight and 96 hours indicated
as mean temperatures for those points. Significant figures in columns for
Chi-squares are marked with an asterisk
Per cent survival
Salinity
Xa2
Xb'
10«/oo
15 »/oo
20 o/oo
25 »/oo
30 »/oo
P. aztecus
Exp.
48 Hrs.
16.2° C.
58.8
83.6
90.0
92.5
88.8
17.98*
4.37
42-100 mm.
96
8.8°
37.5
77.5
85.0
87.5
80.0
41.55*
10.30*
Contr.
48
28.6°
70.8
79.2
95.8
91.7
100.0
3.29
1.25
96
28.8°
45.8
66.6
70.8
83.3
95.8
12.46*
5.42
P. aztecus
Exp.
48
16.2°
28.6
64.3
92.9
96.4
96.4
18.08*
3.79
120-150 mm.
96
8.8°
7.1
46.4
89.3
78.6
92.9
38.11*
13.97*
P. duorarmn
Exp.
48
17.8°
67.7
92.5
98.9
98.9
97.8
10.27*
0.59
35-100 mm.
96
8.75°
63.4
89.2
97.8
94.6
96.7
13.82*
1.39
Contr.
48
28.4°
66.7
86.4
91.7
100.0
100.0
3.88
1.21
96
28.1°
62.5
77.3
91.7
95.8
100.0
5.63
2.25
Analyses of covariance of the data for initial experimental and control animals
paired with 48-hour controls and 96-hour controls show no significant difference in
slope or elevation (P = 0.05) ; however, the lines representing data for experimental
animals at 17.8° C. and 8.75° C. both differ significantly in slope from the line
representing the initial animals (P — 0.05).
It is instructive to compare survival of shrimp in the array of salinities and
temperatures with the results of melting point determinations as expressed in regres-
sion lines (Table II, Figs. 2, 3, 4). Some deaths were due to injury, but if we
examine the percentages (Table II) it is apparent that poorest survival was in
10%o salinity. Chi-square (xa2) values for differences in survival among salinities
are significant in seven of the ten series (P -- 0.05 ) , indicating a high influence of
salinity on survival. However, if we eliminate the animals so obviously affected by
OSMOTIC REGULATION IN SHRIMPS 569
W%c salinity from the Chi-square totals (xi>~). it is apparent that higher salinities
have little effect on survival. The only significant (xb~ ) values (P - 0.05) are for
aztecus young and adults at 96 hours in 8.8° C. water. This indicates a response
to temperature such as suggested by the regression lines in Figures 2 and 3. Fur-
ther, it is apparent that survival of diwrarum is on the whole better than young
astecus, indicating greater tolerance to lowered salinity and temperature.
DISCUSSION
Melting point determinations show a high degree of individual variation, as has
been noted by Gross (T957b) for P achy grap sits crassipcs. Such variations may be
attributed to stage in the molting cycle, age, sex, and in the present case perhaps to
changes in salinity and non-uniformity of sample size. Replications tend to smooth
these inequalities.
Each regression coefficient, except one, is significant at the 5% level. The ex-
ception is that for P. dnoranini experimental and control at 28.4° C. and this in-
stance nearly attains significance at the 5% level. The reason for this exception
remains unexplained, though it is thought that the average blood melting point value
of - - 1.48° C. in water with a melting point of •- 0.77° C. is low and perhaps not a
good approximation.
Both of these species demonstrate possession of osmoregulatory powers. It is
apparent that in the size ranges studied both species are hypotonic to sea water, but
that in water under 30%o they are hypertonic. This is in accord with Verwey
(1957), who has suggested that it may be a general rule that Crustacea which
regulate their internal environments are hypertonic in water of low salinity and
hypotonic in water of high salinity.
Under the experimental conditions imposed, both species maintained themselves
fairly well for limited periods in a range of 10-30/{e sea water, but with the lowering
of temperature the regulatory powers meet more resistance. In such circumstances
there appears to be a species difference. Juveniles and subadults of P. aztecus
demonstrate a loss of osmoregulatory ability with lowering temperature which is
statistically demonstrable after 96 hours, though a trend toward lowered activity is
apparent even at 48 hours. In ditorantin a significant difference was manifest in
both 48- and 96-hour experimentally cooled samples. From the standpoint of per
cent survival, though, indications are that P. aztecus, normally only a summer
resident in North Carolina, does not regulate in lowered salinities at lowered tem-
peratures as well as does P. duorarum, which is normally resident in North Carolina
in winter in the juvenile state. The results provide experimental confirmation of
information gained from field observations.
The results do not corroborate those of Broekema (1941 ) that salinity of blood
increases with a fall in temperature, with the exception of shrimp in the 30/^ water,
but it is possible that longer exposure to lowered temperature in a constant salinity
might give different results. Likewise, the results are not in accord with those of
Verwey (1957) that though an animal may not maintain a constant internal environ-
ment, it does tend to maintain a constant difference between internal and external
environment in terms of osmotic pressure expressed in atmospheres at different
570 AUSTIN B. WILLIAMS
temperatures. Again, longer exposure to a given temperature might alter this
picture.
In both species it is shown that in all salinities the blood tends to approach
isotonicity with the surrounding medium as temperature is lowered.
SUMMARY
1. Melting point determinations on blood of two species of shrimp, Pcnaeus
aztecus and P. duorarum, were made in an array of salinities of 10.06, 15.11, 20.17,
25.09, and 30.50^. at 28.1-28.8, 16.2-17.8 and 8.75-8.8° C.
2. These shrimp are hypotonic to sea water at room temperature and hypertonic
to dilutions of sea water below 30%c.
3. These shrimp regulate moderately well in experimental dilutions at room
temperature, though the blood is diluted somewhat in lowered salinities.
4. At lowered temperatures, 8.75-8.8° C., regulatory ability is impaired and
blood tends toward isotonicity.
5. P. duorarum is a better regulator at low temperatures than P. aztccus.
6. Survival of these shrimp is better in higher salinities at low temperatures.
LITERATURE CITED
BROEKEMA, M. M. M., 1941. Seasonal movements and the osmotic behaviour of the shrimp
Crangon crangon L. Arch. Neerl. Zoo/., 6: 1-100.
BURKENROAD, M. D., 1934. The Penaeidae of Louisiana. Bull. Amcr. Mus. Nat. Hist., 68:
61-143.
BURKENROAD, M. D., 1939. Further observations on Penaeidae of the northern Gulf of Mexico.
Bull. Bingham Occanog. Coll., 6 : 1-62.
EDMONDS, E., 1935. The relation between the internal fluid of marine invertebrates and the
water of the environment, with special reference to Australian Crustacea. Proc. Linn.
Soc. New S. Wales, 60 : 233-247.
GROSS, W. J., 1954. Osmotic responses in the sipunculid Dendrostomwn zostcricolitni. J. Exp.
Biol, 31 : 402-423.
GROSS, W. J., 1955. Aspects of osmotic regulation in crabs showing the terrestrial habit.
Amcr. Nat., 89: 205-222.
GROSS, W. J., 1957a. An analysis of response to osmotic stress in selected decapod Crustacea.
Bio I. Bull., 112: 43-62.
GROSS, W. J., 1957b. A behavioral mechanism for osmotic regulation in a semi-terrestrial crab.
Biol. Bull, 113: 269-274.
GUNTER, G., 1950. Seasonal population changes and distribution as related to salinity of certain
invertebrates of the Texas coast, including the commercial shrimp. Pub. Jnst. Mar.
Sci., Texas, 1 : 7-51.
HEEGAARD, P. E., 1953. Observations on spawning and larval history of the shrimp, Penaeus
setiferus (L.). Pub. Inst. Mar. Sci., Texas, 3: 73-105.
HICKMAN, C. P., 1959. The osmoregulatory role of the thyroid gland in the starry flounder,
Platichthys stcllatus. Canadian J. Zoo/., 37 : 997-1060.
JONES, L. L., 1941. Osmotic regulation in several crabs of the Pacific coast of North America.
/. Cell Comp. Physiol, 18: 179-192.
LINDNER, M. J., AND W. W. ANDERSON, 1956. Growth, migrations, spawning and size distribu-
tions of shrimp Pcnaeus setiferus. Fish. Bull. U. S. Fish & Wildl. Serv., 56(106) :
555-645.
MIYAKE, Y., 1939. Chemical studies of the Western Pacific Ocean. III. Freezing point,
osmotic pressure, boiling point and vapour pressure of sea water. Bull. Chan. Soc.
Japan. 14: 58-62.
OSMOTIC REGULATION IN SHRIMPS 571
PANIKKAR, N. K., 1941. Osmoregulation in some Palaemonid prawns. /. Mar. Biol. Assoc.,
25 : 317-359.
PANIKKAR, N. K., 1951. Physiological aspects of adaptation to estuarine conditions. Proc.
Indo-Pacific Fish. Council, 2nd Meeting, 17th-18th April 1950, Cronulla, N.S.W.
Australia, Sect. 3, pp. 168-175. Bangkok.
PAXIKKAR, N. K., AND R. VISWANATHAN, 1948. Active regulation of chloride in Mctapcnaeus
monoceros Fabricius. Nature, 161 : 137-138.
PEARSON, J. C, 1939. The early life histories of some American Penaeidae, chiefly the com-
merical shrimp, Penacus setifcnts (Linn.). Bull. U. S. Bur. Fish., 49 (for 1950)
(30) : 1-73.
SNEDECOR, G. W., 1956. Statistical Methods. 5th ed., Iowa State College Press, Ames, Iowa,
ix + 534 pp.
VERWEY, J., 1957. A plea for the study of temperature influence on osmotic regulation.
L'Annee Biologique, 33 : 129-149.
WILLIAMS, A. B., 1955. A contribution to the life histories of commercial shrimps (Penaeidae)
in North Carolina. Bull. Mar. Sci. Gulf Caribbean, 5: 116-146.
INDEX
ABBOTT, W., AND J. AWAPARA. Sulfur
metabolism in the lugworm, Arenicola,
357.
Abstracts of papers presented at the Marine
Biological Laboratory, 283.
Acartia, feeding and respiration of, 399.
Acclimation of guppy to cold, 231.
Acetazolamide, effects of on skeleton forma-
tion in corals, 416.
Acetylmyosin, action of trypsin on, 286.
Actin, studies on, 290, 294.
Actin depolymerization in presence of KI, 290.
ADAMS, R. G., AND W. A. HAGINS. The ionic
composition of squid photoreceptors, 300.
ADAMS, R. G. See W. A. HAGINS, 316, 317.
Age in relation to cold death in guppy, 231.
Age in relation to osmotic regulation of Pe-
naeus, 560.
Age in relation to respiration of Rana em-
bryos, 428.
D'AGOSTINO, A. S., AND A. FARMANFARMAIAN.
Transport of nutrients in the holothurian
Leptosynapta, 301.
ALDRICH, D. V., AND W. B. WILSON. The
effect of salinity on growth of Gymno-
dinium, 57.
ALEXANDER, D. G. Directional movements of
the intertidal snail, Littorina, 301.
Alga, cold-hardiness of, 474.
Alga, pseudocilia of, 319.
Algae, plankton, iron requirements of. 324.
ALLISON, W. S., AND E. E. CLARK. Observa-
tions on the ribosomes of sea urchins, 302.
Alloxan, permeability of red blood cells to, 311.
Amino acids in Calliobothrium, 120.
Amino acids in flatworms, 75.
Amino compounds in Arenicola, 357.
Ammonia in Golfingia and ambient air, 344.
Amoebocytes of oysters, 273.
Amphibian, oocytes of, 224.
Amphibian development, respiratory regulation
in, 428.
Amphipods, feeding and respiration of, 399.
Anatomy of bryozoa, 479.
Anatomy of Chiridotea, 153.
Anatomy of digestive system of Henricia, 371.
Anatomy of Himasthla, 529.
Anatomy of Lineus gut, 189.
Anatomy of Mitella, 169.
Anatomy of moth spiracles, 494.
Anchoviella, swimming sounds and schooling
of, 210.
ANDERSON, J. M. Histological studies on the
digestive system of a starfish, Henricia,
with notes on Tiedemann's pouches in
starfishes, 371.
ANDERSON, J. M. Regeneration of the cardiac
stomach in Asterias, 302.
Annelid, gametogenesis in, 145.
Annelid, sulfur metabolism of, 357.
Annelids as food for Lineus, 189.
Annual Report of the Marine Biological Lab-
oratory, 1.
Antarctic bryozoa, 479.
Antherea, discontinuous respiration of, 494.
Antigens of Arbacia sperm extract, 202.
Antimitotic action of heavy water, 298.
Aqueous humor formation in dogfish, 340.
Arbacia eggs, cleavage in, with nucleus intact,
87.
Arbacia eggs, surface changes in, 260.
Arbacia sperm extract, antigens of, 202.
Arcella, monauxenic culture of, 310.
AREND, W. P. See S. ZIGMAN, 351.
Arenicola, sulfur metabolism in, 357.
ARMSTRONG, P. B. The pupillary mechanism
in the toad, 291.
Artemia, size in relation to metabolism of, 399.
Asterias, digestive system of, 371.
Asterina, digestive system of, 371.
Astropecten, digestive system of, 371.
Autoradiography of Arenicola extracts, 357.
AWAPARA, J. Sec W. ABBOTT, 357.
BACTERIA, injected, removal of from
oyster, 273.
BALTUS, E. See W. S. VINCENT, 299.
Barnacle, reproduction in, 169.
BARNWELL, F. H. A day-to-day relationship
between oxidative metabolism and world-
wide geomagnetic activity, 303.
BARNWELL, F. H. See F. A. BROWN, JR., 306.
Bathycalanus, feeding and respiration of, 399.
BEAMS, H. W. See R. G. KESSEL, 322.
Behavior of schooling fishes, 210.
BELTON, P. Effects of ions on potential in
lepidopteran muscle fibers, 289.
BENNETT, M. F. See F. A. BROWN, JR., 65.
BENNETT, M. V. L. Comparative electrophys-
iology of supramedullary neurons, 303.
572
INDEX
573
BENNETT, M. V. L. See B. COHEN, 310.
BERENDSEN, H. J. C. The structure of water
in tissue, as studied by nuclear magnetic
resonance, 287.
Bermuda fishes, swimming sounds and school-
ing of, 210.
BERNSTEIN, M. H., AND T. S. DIETRICH.
Electron microscope studies of the reflect-
ing structures of elasmobranch and tele-
ost eyes, 303.
BERNSTEIN, M. H., AND L. G. FEHRENBAKER.
The morphology of starfish spermatozoa,
304.
Beta amino acids in flatworms, 75.
Binucleate oocytes of Rana, 224.
Bioluminescence, in relation to oxygen tension
in fireflies, 293.
Bird, developmental stages of, 90.
Blepharisma, cannibal giant, 345.
Blood of shrimps, salinity determinations on,
560.
Body fluids of elasmobranchs and teleosts, os-
molarities of, 293.
BORGESE, T. A., AND J. W. GREEN. HexOSC
and pentose utilization by mackerel eryth-
rocytes, 304.
BRANHAM, J. M., AND C. B. METZ. Nature
and action of the fertilization inhibitor
from Fucus, 305.
Breeding of Mitella, 169.
BROCH, E. S. Endocrine control of the chro-
matophores of the zoeae of the prawn
Palaemonetes, 305.
BROWN, F. A., JR., AND F. H. BARNWELL.
Magnetic field strength and organismic
orientation, 306.
BROWN, F. A., JR., AND A. HUTTRER. A re-
lationship between photic and magnetic re-
sponse in snails, 306.
BROWN, F. A., JR., AND H. M. WEBB. A
"compass-direction" effect for snails in
constant conditions, and its lunar modu-
lation, 307.
BROWN, F. A., JR., M. F. BENNETT AND H.
M. WEBB. A magnetic compass response
of an organism, 65.
Bryozoa from Antarctica, 479.
BURCH, J. B. See A. S. MERRILL, 197.
BURDICK, C. See C. F. STRITTMATTER, 341.
BURKE, J. A. Some effects of lysergic acid
diethylamide and related agents on embry-
onic heart rate in Fundulus, 307.
BURKE, J. A. Some morphological effects of
lysergic acid diethylamide and related
agents on early embryonic development in
Fundulus, 308.'
BUSSARD, J. M. See A. M. CHASE, 309.
(^)ALANUS, feeding and respiration of, 399.
Calcium, equilibrium exchanges of in corals,
416.
Calcium content of crab blood and urine, 440.
California barnacle, reproduction in, 169.
Calliobothrium-elasmobranch symbiosis, 120.
CAMPBELL, J. W. The occurrence of beta-
alanine and beta-aminoisobutyric acid in
flatworms, 75.
CAMPBELL, J. W. See C. P. READ, 120.
Caranx, swimming sounds and schooling of,
210.
Carbohydrate metabolism of islet tissue, 313.
Carbohydrate metabolism of mackerel erythro-
cytes, 304.
Carbohydrate utilization by Rana embryos, 428.
Carbon-14 as label for amino acids, 120.
Carbon dioxide, role of in oxygen uptake of
insects, 494.
Carbon dioxide tension of ocean, 295.
Carbonic anhydrase in reproductive tracts of
elasmobranchs, 296.
Cardiovascular and respiratory activity of dog-
fish, 341.
CARLSON, F. D. A scheme for the mechano-
chemistry of muscle, 289.
Carolina coast, seasonal occurrence of Mytilus
on, 550.
Carotenoids in Cerithidea, 98.
CASE, J., G. F. GWILLIAM AND F. HANSON.
Dactyl chemoreceptors of brachyurans,
308.
Cell division, surface changes during, 260.
Centrifuged sea urchin eggs, cleavage with
nucleus intact in, 87.
Cerithidea, pigments in, 98.
Cestode-shark symbiosis, 120.
Cestodes, beta-amino acids in, 75.
CHAET, A. B., AND R. S. MUSICK, JR. A
method for obtaining gametes from As-
terias, 292.
CHAET, A. B., AND D. E. PHILPOTT. Secretory
structures in the tube foot of starfish, 308.
CHAET, A. B. See D. E. PHILPOTT, 332.
Chaetomorpha, pigments in, 98.
CHASE, A. M.t AND J. M. BUSSARD. Recovery
of uricase activity in concentrated urea so-
lutions, 309.
Chemoreceptors, dactyl, of brachyurans, 308.
CHENEY, R. H., AND C. C. SPEIDEL. Effect of
non-static conditions during gamete irradi-
ation on Arbacia fertilization and injury,
309.
CHENEY, R. H. See C. C. SPEIDEL, 285, 338.
Chick blastoderm, developmental potential of,
338.
Chiridotea, new species of, 153.
574
INDEX
Chloride cells of Fundulus, electron microscopy
of, 322.
Chloride content of Arenicola, 357.
Chlorophyll, extraction of from marine algae
with acetone and methanol, 339.
Chlorophyllic pigments in Cerithidea, 98.
Chromatography of Arenicola extracts, 357.
Chromatography of Calliobothrium amino
acids, 120.
Chromatography of flatworms, 75.
Chromatography of snail and algal pigments,
98.
Chromatophores of Palaemonetes, endocrine
control of, 305.
Chromatophores of Sesarma, endocrine con-
trol of, 315.
Chromatophores of Sesarma, response of to
light and temperature, 315.
Chromatophores of Upogebia, 314.
CICAK, A., AND J. B. WITTENBERG. Mon-
auxenic culture of Arcella, 310.
Cirripedes, reproduction in, 169.
CLARK, A. M. The modification of life span
by x-rays for haploids and diploids of the
wasp Habrobracon, 292.
CLARK, E. E. See W. S. ALLISON, 302.
Cleavage of Arbacia eggs, effects of chloram-
phenicol on, 329.
Cleavage delay in Chaetopterus eggs, induced
by acridine orange, 346.
Cleavage with intact nucleus, in sea urchin
eggs, 87.
Cleavage rates and nucleotide levels in Spisula
eggs, 324.
Cleavage of swollen sea urchin eggs, 246.
CLEMENT, A. C. Development of the Ilyanassa
embryo after removal of the mesentoblast
cell, 310.
Clitellio as food for Lineus, 189.
"Clock," biological, in Arbacia eggs, 284.
"Clock-compass" of snails, 65.
Coelenterate, neuromuscular physiology of, 454.
Coelenterate, skeleton formation in, 416.
COHEN, B., M. V. L. BENNETT AND H. GRUND-
FEST. Rectification in skate electroplaques
and its abolition by barium ions, 310.
Cold death in the guppy, 231.
Cold as a factor in gametogenesis of Hydroides
and Pecten, 145.
Cold-hardiness of Fucus, 474.
Competitive inhibition of entry of amino acids
into Calliobothrium, 120.
Connective tissue, biochemistry of, 283.
CONOVER,_ R. J. The feeding behavior and
respiration of some marine planktonic
Crustacea, 399.
Contractile responses in presence of electron
donors and acceptors, 286.
COOPERSTEIN, S. J., D. WATKINS, E. HALPERN
AND A. LAZAROW. Permeability of red
blood cells to alloxan, 311.
COOPERSTEIN, S. J. See C. T. FRIZ, 161.
COPELAND, E. Secretory epithelium of the
swim bladder in Fundulus, 311.
Copepods, feeding and respiration of, 399.
Corals, skeleton formation in, 416.
CORRIDEN, E. Oxygen toxicity among arthro-
pods, 312.
Cortex, possible role of in cell division, 246.
Cortical changes in Arbacia eggs, 260.
COSTELLO, D. P. The giant cleavage spindle
of the egg of Polychoerus, 285.
COUSINEAU, G., AND P. R. GROSS. Distribu-
tion and substrate specificity of sea urchin
egg phosphatases, 292.
Crab, magnesium and water fluxes of, 440.
CRANE, R. K. Sec S. M. KRANE, 324.
Crassostrea, removal of injected microorgan-
isms from, 273.
Crustacea, feeding and respiration of, 399.
Crustacean, magnesium and water fluxes of,
440.
Crustacean, new species of, 153.
Crustacean, osmotic regulation in, 560.
Crustaceans as food for Lineus, 189.
DE LA CRUZ, A. Observations on the feeding
activity of the isopod, Idothea, 312.
Cycles of reproduction in Mitella, 169.
Cycles in responses of snails to magnetic force,
65.
Cyclic respiration of saturniid moth pupae, 494.
Cytokinesis, theories of, 246, 260.
Cytology of epithelial cells of dipteran insect,
321.
Cytoplasmic inclusions in egg of Pectinaria,
345.
F)NP, effect of on respiration of Rana em-
bryos, 428.
DAVSON, H., AND C. T. GRANT. Osmolarities
of some body fluids in the elasmobranch
and teleost, 293.
Death in the guppy, caused by low tempera-
ture, 231.
Dehydrogenase systems in Asterias eggs, 323.
Dehydrogenases, malic and glutamic, compara-
tive studies of, 298.
Delayed cleavage in Arbacia eggs, 295.
DEREZIN, M. Sec C. L. PARMENTER, 224.
Developing amphibians, respiratory regulation
in, 428.
Developing sea urchin eggs, cleavage with nu-
cleus intact in, 87.
Development of Hydroides and Pecten during
winter, 145.
INDEX
575
Developmental stages in Mitella, 169.
Developmental stages of turkey, 90.
Diatom, size of, 326.
DIETRICH, T. S. See M. H. BERNSTEIN, 303.
Digestion in Lineus, 189.
Digestive system of Henricia, 371.
DINGLE, A. D. Inhibitory action of a tissue
extract on regeneration of Tubularia, 312.
Dinitrophenol, effects of on Rana embryo res-
spiration, 428.
Dinorlagellate, effect of salinity on growth of,
57.
Diplodus, swimming sounds and schooling of,
210.
Discontinuous respiration in insects, 494.
Distribution of Mytilus, 550.
Division, cell, study of mechanism of, 246, 260.
Dogfish embryos, thyroid treatment of, 109.
DOOLITTLE, R. F. Sec W. STONE, JR., 340.
DOWBEX, R. M. On the natural inhibitors of
glucuronosyl transferase present in serum
of pregnant women, 353.
DOWBEN, R. M., AND D. E. PHILPOTT. The
membrane surrounding milk fat globules,
354.
Drosophila mutant, pigmented fat cells in, 134.
DuBois, A. B. The relationship between oxy-
gen tension and light production before
and after grinding of the light organs of
fireflies, 293.
JTCHINOCHROME granules of Arbacia
eggs, 260.
Echinoderm, digestive system of, 371.
Echinoderm eggs, cleavage of, with nucleus
intact, 87.
Echinoderm eggs, flattened, furrowing in, 246.
Echinoderm eggs, surface changes in, 246, 260.
Echinoderm sperm extract antigens, 202.
Ecology of amphipods, 333.
Ecology of Chiridotea, 153.
Ecology of corals, 416.
Ecology of Mytilus, 550.
Ecology of sea-weeds, as affected by waves,
322.
Effect of salinity on growth of Gymnodinium,
57.
Egg jelly dispersal by Arbacia sperm extracts,
319.
Eggs, cleavage with nucleus intact in, 87.
Eggs, sea urchin, furrowing in, 246.
Eggs, sea urchin, surface changes during cell
division of, 246, 260.
Elasmobranch embryos, thyroid treatment of,
109.
Elasmobranch-tapeworm symbiosis, 120.
Electrical synapses in crayfish, electron mi-
croscopy of, 325.
Electrogenesis, spike, four-factor ionic hypoth-
esis of, 284.
Electron donors, detection of, 286.
Electron microscopy, use of carbon for single
molecule visualization in, 332.
Electron spin resonance study of serotonin-
FMN interaction, 286.
Electron transport in Spisula embryos and
adult tissues, 341.
Electrophoresis of Arbacia sperm extracts, 202.
Electrophysiology of fish supramedullary neu-
rons, 303.
Electrophysiology of lepidopterans, 289.
Electrophysiology of Lorenzinian ampulla, 324.
Electrophysiology of Pacinian corpuscles, 320.
Electrophysiology of scyphozoan, 454.
Electrophysiology of skate, 310.
ELEFAXT, H. Sec E. KIVY-ROSENBERG, 323.
Embryo, turkey, developmental stages of, 90.
Embryonic development of Ilyanassa after re-
moval of mesentoblast cell, 310.
Embryonic stages of Mitella, 169.
Embryos, respiratory regulation in, 428.
Embryos of spiny dogfish, thyroid treatment
of, 109.
Endocrine function in dogfish embryos, 109.
Energy relations in Rana development, 428.
Entry of amino acids into Calliobothrium, 120.
Enzyme activity in Arenicola, 357.
Equilibrium exchanges of calcium in corals,
416.
Estuarine shrimps, osmotic regulation in, 560.
Euchirella, feeding and respiration of, 399.
Euphausid, feeding and respiration of, 399.
Euthemisto, feeding and respiration of, 399.
Excretion in Pachygrapsus, 440.
Experimental stimulation of gametogenesis in
Hydroides and Pecten, 145.
Extracts of Arbacia sperm, antigens of, 202.
Eyes, elasmobranch and teleost, electron mi-
croscope studies of, 303.
Eyespot of Asterias, electron microscopy of,
332.
PARMANFARMAIAN, A. see A. s.
D'AGOSTINO, 301.
Fat cells in Drosophila mutant, 134.
FAUST, R. G., AND A. K. PARPART. Perme-
ability studies on ground hog red blood
cells, 313.
Feeding, role of in gametogenesis of Hydroides
and Pecten, 145.
Feeding behavior of isopod, 312.
Feeding behavior of marine Crustacea, 399.
Feeding experiments with mutant Drosophila,
134.
FEHRENBAKER, L. G. Sec M. H. BERNSTEIN,
304.
576
INDEX
FELDSHUH, D. Sec W. STONE, JR., 340.
Fertilization, stage of Fundulus eggs at, 320.
Fertilization inhibition by Arbacia dermal se-
cretion, 297.
Fertilization inhibitor from Fucus, 305.
FIELD, J. B., AND A. LAZAROW. Comparison
of the oxidation of C-l and C-6 labelled
glucose by islet tissue, 313.
FILOSA, M. Metachromatic granules in eggs
of Hydroides, 314.
FINE, A. Sec P. PERSON, 288.
FlNGERMAN, M., R. NAGABHUSHANAM AND L.
PHILPOTT. The influence of light and en-
docrines on the chromatophores of the mud
shrimp, Upogebia, 314.
FlNGERMAN, M., R. NAGABHUSHANAM AND L.
PHILPOTT. Responses of the melanophores
of the grapsoid crab Sesarma to light and
temperature, 315.
FlNGERMAN, M., R. NAGABHUSHANAM AND L.
PHILPOTT. The responses of the melano-
phores of eyestalkless specimens of Se-
sarma to illumination and endocrines, 315.
Fish, cold death in, 231.
Fish islet tissue, respiration of, 161.
FISHER, S. S. See X. J. MUSACCHIA, 327.
Fishes, swimming sounds and schooling of, 210.
FLAKS, J. G. See R. R. HATHAWAY, 319; L.
WARREN, 355.
Flattened sea urchin eggs, furrowing in, 246.
Flatworms, beta amino acids in, 75.
Flea respiration, as compared with saturniid
moth, 494.
Fluxes, water and magnesium, of crab, 440.
FONG, B. A., AND H. I. HIRSHFIELD. Effects
of Blepharisma pigment on marine inver-
tebrate development, 316.
Food of Henricia, 371.
Food of Lineus, 189.
FRIZ, C. T., A. LAZAROW AND S. J. COOPER-
STEIN. Studies on the isolated islet tis-
sue of fish. III., 161.
Frogs, oocytes of, 224.
Fruit fly, pigmented fat cells in mutant of, 134.
Fucus, cold-hardiness of, 474.
Furrowing in flattened sea urchin eggs, 246.
QALTSOFF, P. S. The three hearts of
the oyster, 291.
Gametes of Asterias, method for obtaining, 292.
Gametogenesis in Hydroides and Pecten, 145.
Geographical distribution of Mytilus, 550.
GLADE, R. W. Transplantation of distal limb
tissues to upper arm stumps in adult Tri-
turus, 316.
Glucose levels of catfish and toadfish blood,
296.
Glucuronosyl transferase inhibitors in serum
of pregnant women, 353.
GORBMAN, A. Sec A. W. PRITCHARD, 109.
GOREAU, T. F., AND N. I. GOREAU. Physiology
of skeleton formation in corals. IV., 416.
GRANT, C. T. See H. DAVSON, 293.
GRAY, I. E. Sec H. W. WELLS, 550.
GREEN, J. W. Sec T. A. BORGESE, 304.
Gregarine parasite of Lineus, 189.
GREGG, J. R. Respiratory regulation in am-
phibian development, 428.
GROSS, P. R. Sec G. COUSINEAU, 292 ; W.
SPINDEL, 298.
GROSS, W. J., AND L. A. MARSHALL. The in-
fluence of salinity on the magnesium and
water fluxes of a crab, 440.
Growing tips of Fucus, cold-hardiness of, 474.
Growth of Gymnodinium, effect of salinity on,
57.
Growth and regression of hydranths, effects of
8-azaguanine and chloramphenicol on, 329.
GRUNDFEST, H. A four-factor ionic hypothesis
of spike electrogenesis, 284.
GRUNDFEST, H. Sec B. COHEN, 310; J. P.
REUBEN, 334, 335, 336; R. WERMAN, 347.
Guppy, cold death in, 231.
GWILLIAM, G. F. Neuromuscular physiology
of a sessile scyphozoan, 454.
GWILLIAM, G. F. Sec J. CASE, 308.
Gymnodinium, effect of salinity on growth of.
57.
]-J AAS, H. Sec N. SPRATT, 338.
J-IAGINS, W. A., AND R. G. ADAMS. Move-
ments of 24-Na and 42-K in the squid
retina, 316.
HAGINS, W. A., R. G. ADAMS AND H. G.
WAGNER. Light-induced current from the
receptors of the squid retina, 317.
HAGINS, W. A. Sec R. G. ADAMS, 300.
Haliclystus, neuromuscular physiology of, 454.
HALPERN, E. Sec S. J. COOPERSTEIN, 311.
HANKS, J. E. Sec H. J. TURNER, JR., 145.
HANSON, F. Sec J. CASE, 308.
HARDING, C. V. The effect of polyvinylpyr-
rolidone on volume of the isolated rat lens,
317.
HARDING, C. V., AND B. D. SRINIVASAN.
Thymidine incorporation in epithelium of
fish lens maintained in vitro, 318.
HARVEY, E. B. Cleavage with nucleus intact
in sea urchin eggs, 87.
HARVEY, E. B. See A. M. ZIMMERMAN, 353.
HATHAWAY, R. R. Stimulation of Arbacia
sperm respiration by egg substance, 318.
INDEX
577
HATHAWAY, R. R., L. WARREN AND J. G.
FLAKS. Egg jelly dispersal by Arbacia
sperm extracts, 319.
HATHAWAY, R. R. Sec L. WARREN, 354, 355.
HAYASHI, T., AND R. ROSENBLUTH. Studies
on actin. II., 290; I, 294.
Heat tolerance and thermoregulation in Uca,
350.
HEGYELI, A. Detection of electron donors,
286.
Hemorrhage in dogfish, cardiovascular re-
sponses to, 342.
Henricia, digestive system of, 371.
HENRY, E. Sec J. S. ROTH, 337.
Hermaphroditism in Arbacia, 353.
Hermaphroditism in sea scallop, 197.
HERNDON, W. R., AND D. E. PHILPOTT. The
pseudocilia of Tetraspora, 319.
HILGARD, G. H. A study of reproduction in
the intertidal barnacle, Mitella, in Mon-
terey Bay, California, 169.
Himasthla, taxonomy and morphology of, 529.
Hippoporinid bryozoa, 479.
HIRSHFIELD, H.'l. Sec B. A. PONG, 316; N.
TULCHIN, 345.
Histological studies on Henricia, 371.
Histology of hermaphroditic scallop, 197.
Histology of Hydroides and Pecten, 145.
Histology of Lineus, 189.
Histology of oysters injected with microorgan-
isms, 273.
Holothurian, transport of nutrients in, 301.
Hormone treatment, thyroid, of spiny dogfish
embryos, 109.
HUMPHREYS, T., S. HUMPHREYS AND A. A.
MOSCONA. A procedure for obtaining
completely dissociated sponge cells, 294.
HUMPHREYS, T., S. HUMPHREYS AND A. A.
MOSCONA. Rotation-mediated aggregation
of dissociated sponge cells, 295.
HUTTRER, A. Sec F. A. BROWN, JR., 306.
HUVER, C. W. The stage at fertilization of
the egg of Fundulus, 320.
Hyalophora, discontinuous respiration of, 494.
Hybrid Rana embryos, respiration of, 428.
Hydroides, stimulation of gametogenesis in,
145.
Hyperia, feeding and respiration of, 399.
Hypnea, pigments in, 98.
JLYANASSA (Nassarius), effect of mag-
netic force on, 65.
Immunology of Arbacia sperm extracts, 202.
Influence of salinity on magnesium and water
fluxes of crab, 440.
Influence of temperature on osmotic regula-
tion of shrimps, 560.
Inhibitors, effect of on fish islet tissue respira-
tion, 161.
Injected microorganisms, removal of from
oyster, 273.
Insect respiration, role of spiracles in, 494.
Intertidal barnacle, reproduction in, 169.
Intestinal absorption and transport of D-glu-
cose by fishes, 327, 328.
Invertebrates, sulfur metabolism in, 357.
Irradiation of Arbacia eggs, 284, 285, 309, 337,
338.
ISENBERG, I. Electron spin resonance study of
serotonin-FMN interaction, 286.
ISHIKO, N., AND W. R. LOEWENSTEIN. Effects
of temperature on charge transfer through
a receptor membrane, 320.
Islet tissue of fish, respiration of, 161.
Isolation of Spisula embryo cells, 340.
Isopod, new species of, 153.
Isoschizoporella, studies on, 479.
JENNINGS, J. B. Observations on the nu-
J trition of the rhynchocoelan Lineus, 189.
1^ AMINER, B. Contractile responses in
the presence of electron donors and ac-
ceptors, 286.
KEOSIAN, J., AND B. P. SONNENBLICK. Stim-
ulation of conidia formation at the grow-
ing tips of Neurospora by x-irradiation,
321.
KEOSIAN, J. Sec B. P. SONNENBLICK, 337.
KESSEL, R. G. Observations on the submicro-
scopic cytology of the epithelial cells of
the cardia of a dipteran insect, Hypo-
derma, 321.
KESSEL, R. G., AND H. W. BEAMS. An elec-
tron microscope study of the mitochondria-
rich "chloride cells" from the gill filaments
of fresh water- and salt water-adapted
Fundulus, 322.
KINGSBURY, J. M. The effect of waves in in-
fluencing the composition of a flora of at-
tached sea-weeds, 322.
KITAI, S. T. Centrifugal influence on the
electroretinogram of the frog, 323.
KIVY-ROSENBERG, E., F. RAY AND H. ELE-
FANT. Krebs cycle dehydrogenase systems
in eggs of Asterias as measured with a
tetrazolium salt, 323.
KOHLER, K. Sec C. B. METZ, 202.
KOSIN, I. L. See A. M. MUN, 90.
KRAMER, D. D., AND J. H. RYTHER. The iron
requirement of some marine plankton al-
gae, 324.
578
INDEX
KRANE, S. M., AND R. K. CRANE. Effect of
nicotinamide on pyridine nucleotide levels
and cleavage rate of eggs of Spisula, 324.
KREWSON, C. R. Sec M. C. PATERSON, 331.
LARVAE of Drosophila, fat cells in, 134.
Larvae of Mitella, 169.
Larvae, Mytilus, transport of around Cape
Hatteras, 550.
LAZAROW, A. Sec C. T. FRIZ, 161; S. J.
COOPERSTEIN, 311; J. B. FIELD, 313.
Lebistes, cold death in, 231.
LEINING, J. Sec W. STOXE, JR., 340.
Lens, fish, thymidine incorporation in epi-
thelium of, 318.
Lens, rat, effect of polyvinylpyrrolidone on,
317.
Lens epithelium, whole-mount preparations of,
339.
LEVY, M., AND P. WEIS. Delayed cleavage of
fertilized Arbacia eggs after treatment
with a "nitrogen mustard" or with formal-
dehyde, 295.
Life-history of Himasthla, 529.
Linckia, digestive system of, 371.
Lineus, nutrition of, 189.
LOEWENSTEIN, W. R. Mechanisms of nerve
impulse generation in a Lorenzinian am-
pulla, 324.
LOEWENSTEIN, W. R. Sec N. ISHIKO, 320.
LOOMIS, W. F., AND W. F. LOOMIS, JR. Con-
stancy of the pCOs in the ocean, 295.
DE LORENZO, A. J. Electron microscopy of
electrical synapses in the crayfish, 325.
Low temperature, effect of on guppy, 231.
Low temperature as a factor in gametogenesis
of Hydroides and Pecten, 145.
Low temperature-treatment of Fucus, 474.
Lugworm, sulfur metabolism in, 357.
Lunar-day fluctuations in response of snails to
magnetic force, 65.
Lutein in marine algae, 98.
LUTWAK-MANN, C. Carbonic anhydrase in
the female reproductive tract of elasmo-
branch fishes, 296.
LYNCH, W. F. Reversible inhibition of meta-
morphosis in tadpoles of Amaroecium by
calcium-free sea water, 325.
Lysergic acid diethylamide, effects of on cir-
culation in elasmobranchs, 349.
Lysergic acid diethylamide, effects of on early
embryonic development of Fundulus, 308.
Lysergic acid diethylamide, effects of on Fun-
dulus embryonic heart rate, 307.
Lysergic acid diethylamide, effects of on heart
rate of Cistenides, 349.
AGNESIUM fluxes of crab, 440.
Magnetic forces, effect of on snail, 65.
MANN, T. Serotonin in the male reproductive
tract of the spiny dogfish, 354.
MARGALEF, R. Silicoflagellate populations in
the plankton of the Cape Cod area, past
and present, 326.
MARGALEF, R. The size of the marine diatom
Melosira in the Cape Cod area, 326.
MARGALEF, R., AND J. H. RYTHER. Pigment
composition and productivity as related to
succession in experimental populations of
phytoplankton, 326.
Marine alga, cold-hardiness of, 474.
Marine bryozoa, 479.
Marine crustaceans, feeding of, 399.
MARSHALL, L. A. Sec W. J. GROSS, 440.
MARSLAND, D. Sec A. M. ZIMMERMAN, 352.
MATHEWS, M. B. Comparative biochemistry
of connective tissue ground substance, 283.
Mechanisms of removal of microorganisms
from oyster, 273.
Medusa, neuromuscular physiology of, 454.
Melanotic tumors in Drosophila, 134.
Membrane transport in parasitism, 120.
MERRILL, A. S., AND J. B. BURCH. Hermaph-
roditism in the sea scallop Placopecten,
197.
Metabolism, community, of Quohog Pond, 348.
Metabolism of fish islet tissue, 161.
Metabolism of Littorina, in and out of water
351.
Metabolism of marine planktonic Crustacea
399.
Metabolism of Nassarius, in relation to geo-
magnetic activity, 303.
Metabolism of Rana embryos, 428.
Metabolism of saturniid moths, 494.
Metabolism of spiny dogfish embryos, 109.
Metabolism of sulfur in Arenicola, 357.
Metachromasy in Hydroides eggs, 314.
Metachromasy in starfish digestive system
371.
Metamorphosis of Amaroucium tadpoles, in-
hibition of, 325.
METZ, C. B., AND K. KOHLER. Antigens of
Arbacia sperm extracts, 202.
METZ, C. B. See H. SCHUEL, 297; J. M.
BRANHAM, 305.
Microorganisms, removal of from oyster, 273.
MIDDLEBROOK, W. R. The action of trypsin
on acetylmyosin, 286.
Milk fat globules, electron microscopy of, 354.
Millepora, skeleton formation in, 416.
Mitella, reproduction in, 169.
Mitosis, study on mechanism of, 246, 260.
INDEX
579
Mitosis with intact nucleus in sea urchin eggs,
87.
Mitotic apparatus, isolation of from pressur-
ized Arbacia eggs, 352.
Moisture content of Arenicola, 357.
Mollusc, effect of magnetic force on, 65.
Mollusc, hermaphroditism in, 197.
Mollusc, phagocytosis in, 273.
Mollusc, pigments in, 98.
Mollusc, seasonal occurrence of, 550.
Mollusc, winter gametogenesis of, 145.
MOMMAERTS, W. F. H. M. See L. NELSON,
283.
Monterey Bay barnacle, reproduction in, 169.
MORGAN, A. M. Sec R. A. MORRISON, 327 ; G.
W. DE VlLLAFRANCA, 346.
MORRISON, R. A., A. M. MORGAN AND G. W.
VILLAFRANCA. Purification and properties
of Limulus arginine phosphokinase, 327.
Mormoniella, "black" eye colors in, 300.
Morphogenesis of turkey, 90.
Morphology of bryozoa, 479.
Morphology of Chiridotea, 153.
Morphology of digestive system of Henricia,
371.
Morphology of Himasthla, 529.
Morphology of Mitella, 169.
MOSCONA, A. A. Sec T. HUMPHREYS, 294,
295.
Moths, discontinuous respiration in, 494.
MOULTON, J. M. Swimming sounds and the
schooling of fishes, 210.
Mucous secretion in Henricia, 371.
Mud-snail, effect of magnetic forces on, 65.
MUN, A. M., AND I. L. KOSIN. Develop-
mental stages of the broad breasted bronze
turkey embryo, 90.
MUSACCHIA, X. J. Some characteristics of
active transport of sugars by intestinal
segments of Ameiurus, 328.
MUSACCHIA, X. J., AND S. S. FISHER. Com-
parative aspects of active transport of
D-glucose by in vitro preparations of fish
intestine, 327.
Muscle, cation permeability in, 288.
Muscle, Limulus, arginine phosphokinase in,
327.
Muscle, Limulus, phosphagen and nucleotides
of, 346.
Muscle, mechanochemistry of, 289.
Muscle extracts, physical properties of, 283.
Musculature of sessile scyphozoan, 454.
MUSICK, R. S., JR. See A. B. CHAET, 292.
Mussel, seasonal occurrence of, 550.
Mustelus-Calliobothrium symbiosis, 120.
Mutant, Drosophila, pigmented fat cells in,
134.
Mya, as intermediate host for Himasthla, 529.
Mytilus, seasonal occurrence of on Carolina
coast, 550.
E, P. F. Reducing substances in blood
of toadfish and catfish, 296.
NADAKAL, A. M. Carotenoids and chlorophyl-
lic pigments in the marine snail, Ceri-
thidea, intermediate host for several avian
trematodes, 98.
NAGABHUSHANAM, R. See M. FINGERMAN,
314, 315.
Nassarius, effect of magnetic response on, 65.
NELSON, L., AND W. F. H. M. MOMMAERTS.
Physical properties of muscle extracts,
283.
Nephridia of Clymenella, histology of, 331.
Nerve net of scyphozoan, 454.
Neuromuscular physiology of lobster, 333, 334,
335, 336, 347. "
Neuromuscular physiology of sessile scypho-
zoan, 454.
Neuromuscular synapse of frog, electrical in-
excitability of, 288.
Nervous system of Clymenella, histology of,
331.
New England Chiridotea, 153.
New genera and species of bryozoa, 479.
New species of Chiridotea, 153.
New species of Himasthla, 529.
Noises of fish, in relation to schooling, 210.
NOVIKOFF, A. B. Pinocytosis, phagocytosis,
and lysosomes : cytochemical and electron
microscopic studies, 287.
Nuclear division in marine ciliate, 343.
Nuclei of Rana oocytes, 224.
Nucleic acids in dogfish cornea, 351.
Nucleolus, function of, 299.
Nucleotides, adenosine, in Arbacia eggs, 352.
Nucleus, role of in cleavage of sea urchin egg,
87.
Nutrition of dogfish in relation to parasitism,
120.
Nutrition of Linens, 189.
Nutrition in relation to gametogenesis of Hy-
droides and Pecten, 145.
O CCURRENCE of ammo acids in flatworm,
75.
Occurrence of Mytilus on Carolina coast, 550.
Ocean currents as factor in distribution of My-
tilus, 550.
ODUM, E. P. Sec W. R. TAYLOR, 343.
Oocytes of Rana, 224.
Opsanus, respiration of islet tissue of, 161.
Optic nerve of frog, centrifugal fibers in, 323.
Orientation of Littorina, 301.
580
INDEX
Orientation of snails, 65.
Orientation of snails, lunar modulation of, 307.
Orientation of snails, in relation to light, 306.
Orientation of snails, in relation to magnetic
force, 306.
Osmoregulation in relation to cold death in
guppy, 231.
Osmotic regulation in shrimps, 560.
Osmotic relations in Pachygrapsus, 440.
OSTERHOUT, W. J. V. Further studies on the
protoplasmic contraction of the marine
alga Chaetomorpha, 328.
Ovaries of Rana, multinucleate oocytes of, 224.
Ovulation induced by steroids in frogs and
fishes, 351.
Oxygen, role of in cold death of guppy, 231.
Oxygen consumption of marine planktonic
Crustacea, 399.
Oxygen consumption of Rana embryos, 428.
Oxygen consumption of spiny dogfish embryos,
109.
Oxygen consumption of Uca, seasonal varia-
tions in, 346.
Oxygen toxicity among arthropods, 312.
Oxygen uptake of fish islet tissue, 161.
Oxygen uptake in insects, 494.
Oyster, hearts of, 291.
Oyster, removal of injected microorganisms
from, 273.
pACHYGRAPSUS, magnesium and water
fluxes of, 440.
PALINSCAR, E. E. The effects of chloram-
phenicol on cleavage of Arbacia eggs, 329.
PALINSCAR, E. E., AND J. S. PALINSCAR. The
effects of 8-azaguanine and chlorampheni-
col on the regression-replacement cycle of
hydranths, 329.
PALMER, J. D. The role of moisture and illu-
mination on the expression of the rhythmic
behavior of the diatom, Hantzschia, 330.
Pancreatic islet tissue, fish, respiration of, 161.
Paracentrotus eggs, flattened, furrowing in,
246.
Paraeuchaeta, feeding and respiration of, 399.
Parasite intermediate host, pigments in, 98.
Parasites of Lineus, 189.
Parasitic trematode, new species of, 529.
Parasitism, permeation and membrane trans-
port in, 120.
PARKER, J. Seasonal changes in cold-hardi-
ness of Fucus, 474.
PARKER, J., AND D. E. PHILPOTT. Electron
microscope studies of Fucus cytoplasm in
summer and winter, 330.
PARMENTER, C. L., M. DEREZIN AND H. S.
PARMENTER. Binucleate and trinucleate
oocytes in post-ovulation ovaries of Rana,
224"
PARPART, A. K. Sec R. G. FAUST, 313.
Parthenogenetic sea urchin eggs, cleavage with
nucleus intact in, 87.
PATERSON, M. C. Histological investigation
of the central nervous system of Cly-
menella, 331.
PATERSON, M. C., AND C. R. KREWSON. His-
tological investigation of the nephridia of
Clymenella, 331.
Patiria, digestive system of, 371.
Pecten, stimulation of gametogenesis in, 145.
Penaeus, osmotic regulation in, 560.
Periodism in response of snails to magnetic
force, 65.
Permeability of ground hog red blood cells,
313.
Permeation in parasitism, 120.
PERSON, P., AND A. FINE. Free radical forma-
tion during indophenol blue synthesis by
heart muscle respiratory enzymes, 288.
Phagocytosis in oyster, 273.
PHILPOTT, D. E. Further electron microscopic
observations on the sperm of Limulus, 332.
PHILPOTT, D. E. The use of carbon for single
molecule visualization, 332.
PHILPOTT, D. E., AND A. B. CHAET. Electron
microscope observations of the starfish
eyespot, 332.
PHILPOTT, D. E. See A. B. CHAET, 308; W.
R. HERNDON, 319 ; J. PARKER, 330 ; R. M.
DOWBEN, 354.
PHILPOTT, L. See M. FINGERMAN, 314, 315.
Phosphatases of sea urchin egg, 292.
Photoreceptors, squid, ionic composition of,
300.
Photosynthesis of benthic macroalgae, 342.
Phronima, feeding and respiration of, 399.
Phycobilins in marine algae, 98.
Physiology of skeleton formation in corals, 416.
Phytoplankton succession, 326.
Pigment, Blepharisma, effect of on invertebrate
development, 316.
Pigmented fat cells in Drosophila mutant, 134.
Pigments in Cerithidea, 98.
Pinocytosis, phagocytosis and lysosomes : cyto-
chemistry of, 287.
PITKOW, R. B. Cold death in the guppy, 231.
Placopecten, hermaphroditism in, 197.
Plankton, silicoflagellate populations in, 326.
Plankton crustaceans, feeding and respiration
of, 399.
Plasmalogens, ether linkages of, 297.
Platyhelminth, beta amino acids in, 75.
Platyhelminth-shark symbiosis, 120.
Platyhelminthes, new species of, 529.
INDEX
581
PLATZMAN, S. J. Comparative ecology of two
species in intertidal amphipods, 333.
Pleuromamma, feeding and respiration of, 399.
POLGAR, G. Measurements of volume and
composition of the swim bladder gas of
toadfish, 297.
Polyspermic Arbacia eggs, surface changes in,
"260.
Population fluctuations in Gymnodinium, 57.
Porites, skeleton formation in, 416.
Potassium content of crab blood and urine, 440.
PRITCHARD, A. W., AND A. GORBMAN. Thy-
roid hormone treatment and oxygen con-
sumption in embryos of the spiny dogfish,
109.
Propylthiouracil treatment of dogfish embryos,
109.
Protein content of fish islet tissue, 161.
Protoplasmic contraction in Chaetomorpha,
328.
Psammechinus eggs, cleavage with nucleus in-
tact in, 87.
Pupae, moth, discontinuous respiration of, 494.
Pupillary mechanism in toad, 291.
DADIOCALCIUM, effects of on marine
' eggs, 299.
Radiocalcium, use of in studies of coral skele-
ton formation, 416.
Radiocalcium uptake and release by Fucus, 342.
Radiocarbon as label for amino acids, 120.
Radiocarbon, use of in experiments on osmo-
regulation in crab, 440.
Radioiron, uptake of by marine benthic algae,
343.
Radiosulfur, use of in study of sulfur metabo-
lism of Arenicola, 357.
Rana, oocytes of, 224.
Rana, respiratory regulation in embryos of,
428.
RAPPORT, M. H. An addition reaction of the
alpha, beta-unsaturated ether linkage of
plasmalogens, 297.
RAY, F. See E. KIVY-ROSENBERG, 323.
READ, C. P., J. E. SIMMONS, JR., J. W. CAMP-
BELL AND A. H. ROTH MAN. Permeation
and membrane transport in parasitism:
studies on a tapeworm-elasmobranch sym-
biosis, 120.
"Red cells" of Drosophila, 134.
"Red tide" in relation to Gymnodinium, 57.
Reef-building corals, skeleton formation in,
416.
Reflexes, cardiovascular and respiratory, in
elasmobranchs, 349.
Regeneration, limb, in Triturus, 316.
Regeneration of cardiac stomach in Asterias,
302.
Regeneration in Triturus, autoradiographic
study of, 350.
Regeneration of Tubularia, inhibitory action
of tissue extract on, 312.
Regulation of respiratory metabolism in am-
phibian development, 428.
Removal of injected microorganisms from
oyster, 273.
Reproduction in Mitella, 169.
Respiration of Arbacia sperm, effects of egg
substances on, 318.
Respiration of dogfish embryos, 109.
Respiration of fish islet tissue. 161.
Respiration of Golfingia in environmental car-
bon dioxide, 344.
Respiration of insects, role of spiracles in, 494.
Respiration of marine crustaceans, 399.
Respiration in relation to cold death of guppy,
231.
Respiratory enzymes, free radical formation
during indophenol blue synthesis by, 288.
Respiratory regulation in amphibian develop-
ment, 428.
Response of organism to magnetic forces, 65.
Retina, squid, light-induced current from re-
ceptors of, 317.
Retina, squid, movements of Na and K in, 316.
REUBEN, J. P. Analysis of photodynamic ef-
fects in lobster neuromuscular prepara-
tions, 333.
REUBEN, J. P. Electrotonic connections be-
tween lobster muscle fibers, 334.
REUBEN, J. P., AND H. GRUNDFEST. The ac-
tion of cesium ions on neuromuscular
transmission in lobster, 336.
REUBEN, J. P., AND H. GRUNDFEST. Actions
of cesium ions on the electrically excitable
membrane of lobster muscle fibers, 334.
REUBEN, J. P., AND H. GRUNDFEST. Further
analysis of the conversion of graded to
all-or-none responsiveness in the elec-
trically excitable membrane of lobster
muscle fibers, 335.
REUBEN, J. P., AND H. GRUNDFEST. Inhibitory
and excitatory miniature post-synaptic po-
tentials in lobster muscle fibers, 335.
REUBEN, J. P., R. WERMAN AND H. GRUND-
FEST. Properties of indefinitely prolonged
spikes of lobster muscle fibers, 336.
REUBEN, J. P. Sec R. WERMAN, 347.
Rhincalanus, feeding and respiration of, 399.
Rhynchocoelan, nutrition of, 189.
Rhythm of response of snails to magnetic force,
65.
Rhythmicity of diatom, effects of humidity and
illumination on. 330.
Ribosomes of sea urchin eggs and embryos,
302.
582
INDEX
RIZKI, M. T. M. Pigmented fat cells in a
mutant of Drosophila, 134.
ROGICK, M. D. Studies on marine bryozoa.
XIII., 479.
Role of spiracles in insect respiration, 494.
ROSENBLUTH, R. Sec T. HAYASHI, 290, 294.
ROSS, J. E. S'CC G. W. DE VlLLAFRANCA, 346.
ROTH, J. S., E. HENRY AND C. WIERCINSKI.
Further studies on the biochemical effects
of x-irradiation on Tetrahymena, 337.
ROTHMAN, A. H. Sec C. P. READ, 120.
RUSTAD, R. C. X-ray-induced dissociation of
the mitotic and micromere "clocks," 284.
RUSTAD, R. C. X-ray-induced mitotic delay
in the Arbacia egg, 337.
RYTHER, J. H. Sec D. D. KRAMER, 324; R.
MARGALEF, 326.
gALINITY, effect of on growth of Gym-
nodinium, 57.
Salinity, influence of on magnesium and water
fluxes of crab, 440.
Salinity relations in Penaeus, 560.
Samia, discontinuous respiration of, 494.
Saturniid moths, discontinuous respiration of,
494.
Scallop, gametogenesis in, 145.
Scallop, hermaphroditism in, 197.
SCHNEIDERMAN, H. A. Discontinuous respira-
tion in insects : role of the spiracles, 494.
Schooling of fishes, 210.
SCHUEL, H., AND C. B. METz. Inhibition of
fertilization of Asterias, Spisula and
Chaetopterus eggs by Arbacia dermal se-
cretion, 297.
SCOTT, A. C. Furrowing in flattened sea
urchin eggs, 246.
SCOTT, A. C. Surface changes during cell di-
vision, 260.
Scyphozoan, neuromuscular physiology of, 454.
Sea scallop, hermaphroditism in, 197.
Sea urchin eggs, cleavage of with nucleus in-
tact, 87.
Sea urchin eggs, flattened, furrowing in, 246.
Sea urchin eggs, surface changes in, 260.
Sea urchin sperm extract antigens, 202.
Seasonal changes in cold-hardiness of Fucus,
474.
Seasonal differences in Fucus cytoplasm, elec-
tron microscope study of, 330.
Seasonal occurrence of Mytilus on Carolina
coast, 550.
Secretory structures in starfish tubefeet, 308.
Self-fertilization in Mitella, 169.
Serotonin in reproductive tract of male dog-
fish, 354.
Sessile scyphozoan, neuromuscular physiology
of, 454.
Sex in relation to cold death in guppy, 231.
Sexual activities of Mitella, 169.
Sexuality in sea scallop, 197.
Shark embryo, thyroid hormone treatment of,
109.
Shark-tapeworm symbiosis, 120.
Shrimps, osmotic regulation in, 560.
Sialic acid in Arbacia eggs, 354.
Sialic acid in Arbacia semen, 355.
Silkworm pupae, discontinuous respiration in,
494.
SIMMONS, J. E. Sec C. P. READ, 120.
Size of Rana oocytes, 224.
SJODIN, R. A. Cation permeability in muscle,
288.
Skeleton formation in corals, 416.
Snail, effect of magnetic forces on, 65.
Snail, pigments in, 98.
Sodium concentrations in blood and urine of
crab, 440.
Solar-day fluctuations in response of snails to
magnetic force, 65.
SONNENBLICK, B. P., AND J. KEOSIAN. Low
dose x-irradiation and the possibility of
accelerated root growth, 337.
SONNENBLICK, B. P. See J. KEOSIAN, 321.
Sounds, swimming, and schooling of fishes,
210.
SPEIDEL, C. C., AND R. H. CHENEY. Com-
parative effects of x-ray and ultraviolet
radiation of gametes on the developing sea
urchin Arbacia, 338.
SPEIDEL, C. C, AND R. H. CHENEY. Motion
pictures of radiation-induced modifications
in the early development of Arbacia, 285.
SPEIDEL, C. C. Sec R. H. CHENEY, 309.
Sperm, Limulus, electron microscopy of, 332.
Sperm, starfish, morphology of, 304.
Sperm extracts, Arbacia, antigens of, 202.
Sphaerechinus, cleavage with nucleus intact in
eggs of, 87.
SPINDEL, W., AND P. R. GROSS. Further stud-
ies on the antimitotic action of heavy wa-
ter, 298.
Spindle of Polychoerus, 285.
Spiny dogfish, thyroid treatment of embryos
of, 109.
Spiracles, role of in insect respiration, 494.
Sponge cells, dissociated, rotation-induced ag-
gregation of, 295.
Sponge cells, method for dissociating, 294.
SPRATT, N., AND H. HAAS. Developmental po-
tential of the chick blastula (unincubated
blastoderm), 338.
Squalus embryos, thyroid treatment of, 109.
SRINIVASAN, B. D. Whole-mount prepara-
tions of fish lens epithelium from various
species, 339.
INDEX
583
SRIXIVASAN, B. D. Sec C. V. HARDING, 318.
Starfish, digestive system of, 371.
Stauromedusa, neuromuscular physiology of,
454.
Stenohaline character of Gymnodinium, 57.
STERNS, C. Extraction of chlorophyll from
marine plankton algae with acetone and
methanol, 339.
Stimulation, electrical, of sessile scyphozoan,
454.
Stimulation of gametogenesis in Hydroides
and Pecten, 145.
STONE, W., JR., D. FELDSHUH, J. LEINING AND
R. F. DOOLITTLE. Further inquiry into
the mechanisms of aqueous humor forma-
tion in dogfish, 340.
Storms, in relation to distribution of Mytilus,
550.
STRITTMATTER, C. F., AND P. STRITTMATTER.
Separation and isolation of Spisula em-
bryo cells, 340.
STRITTMATTER, C. F., P. STRITTMATTER AND
C. BURDICK. Electron transport in eggs,
developing embryos and adult tissues of
Spisula, 341.
Studies on isolated fish islet tissue, 161.
Studies on marine bryozoa, 479.
Studies on trematode genus Himasthla, 529.
Study of reproduction in Mitella, 169.
STUNKARD, H. W. Further studies on the
trematode genus Himasthla, 529.
Substrates, effect of on fish islet tissue respira-
tion, 161.
SUDAK, F. N., AND C. G. WILDER. Cardiovas-
cular and respiratory activity in dogfish,
341.
SUDAK, F. N., AND C. G. WILBER. Cardiovas-
cular responses to hemorrhage in the dog-
fish, 342.
SUDAK, F. N. Sec C. G. WILBER, 349.
Sulfur metabolism in Arenicola, 357.
Surface changes during cell division, 260.
SWIFT, E., AND W. R. TAYLOR. Uptake and
release of Ca-45 by Fucus, 342.
Swimbladder of Fundulus, secretory epithelium
of, 311.
Swimbladder gas of toadfish, composition of,
297.
Swimming sounds and the schooling of fishes,
210.
Symbiosis of tapeworm and elasmobranch, 120.
Symbiotic flatworms, beta amino acids in, 75.
SZENT-GYORGYI, A. G., AND E. M. SzENT-
KIRALYI. Reversibility of actin depoly-
merization in presence of KI, 290.
SZENTKIRALYI, E. M. See A. G. SzENT-
GYORGYI, 290.
rpAPEWORM-elasmobranch symbiosis, 120.
Taxonomy of bryozoa, 479.
Taxonomy of Chiridotea, 153.
Taxonomy of Himasthla, 529.
TAYLOR, J. K. Sec F. J. WIERCINSKI, 299.
TAYLOR, W. R. The effect of photosynthesis
by benthic macroalgae on the titration al-
kalinity of sea water, 342.
TAYLOR, W. R., AND E. P. ODUM. Uptake of
iron-59 by marine benthic algae, 343.
TAYLOR, W. R. Sec E. SWIFT, 342.
Teleost, cold death in, 231.
Teleosts, swimming sounds and schooling of,
210.
Temperature, influence of on osmotic regula-
tion of shrimps, 560.
Temperature as a factor in gametogenesis of
Hydroides and Pecten, 145.
Temperature-hardiness of Fucus, 474.
Temperature in relation to distribution of My-
tilus, 550.
Temperature in relation to reproduction of
Mitella, 169.
Tetrazolium test for viability of Fucus, 474.
Thyroid treatment of spiny dogfish embryos,
109.
Thyroxine treatment of dogfish embryos, 109.
Tiedemann's pouches in Henricia, 371.
Toadfish islet tissue, respiration of, 161.
TORCH, R. Nuclear division in Holosticha,
343.
Toretocheilum, studies on, 479.
Tracheae of moth pupae, role of in discontinu-
ous respiration, 494.
Trachinotus, swimming sounds and schooling
of, 210.
Transport, membrane, in parasitism, 120.
Transport of Mytilus larvae around Cape Hat-
teras, 550.
TRAVIS, D. M. Ammonia in Golfingia and am-
bient air, 344.
TRAVIS, D. M. Respiratory changes induced
in Golfingia by alteration in environmen-
tal carbon dioxide, 344.
Trematode genus Himasthla, studies on, 529.
Trematodes, beta amino acids in, 75.
Trematodes, pigments in intermediate hosts
of, 98.
Triac treatment of dogfish embryos, 109.
Triiodothyroacetic acid treatment of dogfish
embryos, 109.
Trinucleate oocytes of Rana, 224.
TRIPP, M. R. Mechanism of removal of in-
jected microorganisms from the American
oyster, Crassostrea, 273.
TULCHIN, N., AND H. I. HlRSHFIELD. Studies
of cannibal giant Blepharisma, 345.
584
INDEX
Tumors in Drosophila, 134.
Turbellaria, beta amino acids in, 75.
Turkey, developmental stages of, 90.
TURNER, H. J., JR., AND J. E. HANKS. Ex-
perimental stimulation of gametogenesis in
Hydroides and Pecten during the winter,
145.
T\VEEDELL, K. S. Differential staining of cy-
toplasmic inclusions in eggs of Pectinaria,
345.
TWEEDELL, K. S., AND C. D. WATTERS. Re-
duction and delay of cleavage in Chaetop-
terus eggs by acridine orange, 346.
\JLVA, pigments of, 98.
Uptake, oxygen, of fish islet tissue, 161.
Urea content of Arenicola, 357.
Urethan as antidote to cold death in guppy,
231.
Uricase activity in concentrated urea solutions,
309.
Urine of crabs, magnesium of, 440.
Urospora as parasite of Lineus, 189.
DE yiLLAFRANCA, G. W., J. E. Ross
AND A. M. MORGAN. Phosphagen
and nucleotides of Limulus muscle, 346.
DE VILLAFRANCA, G. W. See R. A. MORRISON,
327.
VILLEE, C. A. Comparative studies of malic
and glutamic dehydrogenases, 298.
VINCENT, W. S., AND E. BALTUS. A function
for the nucleolus, 299.
Vision, in relation to schooling of fishes, 210.
WAGNER, H. G. See W. A. HAGINS, 317.
Warmth as a factor in gametogenesis of Hy-
droides and Pecten, 145.
WARREN, L., AND R. R. HATHAWAY. Lipid-
soluble sialic acid-containing material in
Arbacia eggs, 354.
WARREN, L., R. HATHAWAY AND J. G. FLAKS.
Sialic acid in semen of Arbacia, 355.
WARREN, L. See R. R. HATHAWAY, 319.
Water fluxes of crab, 440.
Water in tissue, structure of, 287.
WATKINS, D. See S. J. COOPERSTEIN, 311.
WATTERS, C. D. See K. S. TWEEDELL, 346.
Weather records, correlation of with distribu-
tion of Mytilus, 550.
WEBB, H. M. Oxygen consumption in Uca:
seasonal variations, 346.
WEBB, H. M. See F. A. BROWN, JR., 65, 307.
WEIS, P. See M. LEVY, 295.
WELLS, H. W., AND I. E. GRAY. The sea-
sonal occurrence of Mytilus on the Caro-
lina coast as a result of transport around
Cape Hatteras, 550.
WEKMAN, R. Electrical inexcitability of the
frog neuromuscular synapse, 288.
WERMAX, R., J. P. REUBEN AND H. GRUND-
FEST. Effects of environmental changes
on indefinitely prolonged action potentials
of lobster muscle, 347.
WERMAN, R., J. P. REUBEN AND H. GRUND-
FEST. Relative effectiveness of inhibitory
membrane in different fibers of a lobster
muscle, 347.
WERMAN, R. See J. P. REUBEN, 336.
WHITELEY, G. C., JR. In situ measurement
of the community metabolism of Quohog
Pond, 348.
WHITING, P. W. The "black" eye colors in
Mormoniella, 300.
WICHTERMAN, R. Production of viable races
of Paramecium after micronuclear elim-
ination with x-rays, 348.
WIERCINSKI, C. See J. S. ROTH, 337.
WIERCINSKI, F. J., AND J. K. TAYLOR. Ex-
periments with Ca-45 in marine eggs, 299.
WIGLEY, R. L. A new species of Chiridotea
from New England waters, 153.
WILBER, C. G. The effect of LSD 25 on heart
rate in Cistenides, 349.
WILBER, C. G., AND F. N. SUDAK. Cardio-
vascular and respiratory reflexes in elas-
mobranchs, 349.
WILBER, C. G., AND F. N. SUDAK. Some ef-
fects of lysergic acid diethylamide on cir-
culation in elasmobranchs, 349.
WILBER, C. G. See F. N. SUDAK, 341, 342.
WILF, R. T. An autoradiographic study of
the regenerating limb in the adult newt,
Triturus, 350.
WILKENS, J. L. Heat tolerance and thermo-
regulation in the fiddler crab, Uca, 350.
WILLIAMS, A. B. The influence of tempera-
ture on osmotic regulation in two species
of estuarine shrimps, 560.
WILSON, W. B. See D. V. ALDRICH, 57.
Winter gametogenesis of Hydroides and Pec-
ten, 145.
WITTENBERG, J. B. See A. CICAK, 310.
Worm, gametogenesis in, 145.
Worm, sulfur metabolism of, 357.
WRIGHT, P. A. Experiments with ovulation
induced in vitro by means of steroids in
frogs and marine fishes, 351.
INDEX
585
X-IRRADIATION of Aiiium bulbs, low
dose, 337.
X-irradiation of Habrobracon, 292.
X-irradiation of Neurospora, 321.
X-irradiation of Paramecium, 348.
X-irradiation of Tetrahymena, 337.
Xanthophyllic pigment in marine algae, 98.
VEAST, injected, removal of from oyster,
273.
£EAXANTHIN in marine snail, 98.
ZIEG, R. G. Metabolism of three species of
the gastropod genus Littorina in and out
of water, 351.
ZIGMAX, S., AND W. P. AREND. A study of
the nucleic acids of the dogfish cornea,
351.
ZIMMERMAN, A. M., AND D. MARSLAND. Iso-
lation of mitotic apparatus from pressur-
ized Arbacia eggs, 352.
ZIMMERMAN, A. M., AND S. B. ZIMMERMAN.
Uptake of adenosine nucleotides in Ar-
bacia eggs, 352.
ZIMMERMAN, A. M., S. B. ZIMMERMAN AND
E. B. HARVEY. A report of hermaphro-
ditism in Arbacia, 353.
Zoids of Antarctic bryozoa, 479.
Zooplankton, feeding and respiration of, 399.
Zooxanthellae, role of in coral skeleton forma-
tion, 416.
070
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